1. health and fitness
2. disease

Journal of Molecular Medicine
Volume 85, Number 8 / August, 2007,pp. 783-906
Cachexia has only one meaning
783-785
Authors Friedrich C. Luft
Connexin37: a potential modifier gene of inflammatory
disease
787-795
Authors Marc Chanson and Brenda R. Kwak
Natriuretic peptide receptor B signaling in the cardiovascular
system: protection from cardiac hypertrophy
797-810
Authors Ines Pagel-Langenickel, Jens
Buttgereit, Michael Bader and Thomas H.
Langenickel
Involvement of autophagy in viral infections: antiviral function
and subversion by viruses
811-823
Authors Lucile Espert, Patrice Codogno and
Martine Biard-Piechaczyk
Laminin isoforms in development and disease
825-836
Authors Susanne Schéele, Alexander Nyström,
Madeleine Durbeej, Jan F. Talts, Marja Ekblom
and Peter Ekblom
Adoptive precursor cell therapy to enhance immune
reconstitution after hematopoietic stem cell transplantation
837-843
Authors J. L. Zakrzewski, A. M. Holland and M. R. M. van den Brink
A key player in biomedical sciences and clinical service in
China, Chinese Academy of Medical Sciences (CAMS) and
Peking Union Medical College (PUMC)
Authors Qimin Zhan and Depei Liu
845-850
Protein kinase C-ζ regulation of GLUT4 translocation through
actin remodeling in CHO cells
851-861
Authors Xiao-Jun Liu, Chang Yang, Nishith
Gupta, Jin Zuo, Yong-Sheng Chang and Fu-De
Fang
Proteomic profiling of proteins dysregulted in Chinese
esophageal squamous cell carcinoma
863-875
Authors Xiao-Li Du, Hai Hu, De-Chen Lin, ShuHua Xia, Xiao-Ming Shen, Yu Zhang, Man-Li
Luo, Yan-Bin Feng, Yan Cai, Xin Xu, Ya-Ling
Han, Qi-Min Zhan and Ming-Rong Wang
Renalase gene is a novel susceptibility gene for essential
hypertension: a two-stage association study in northern Han
Chinese population
877-885
Authors Qi Zhao, Zhongjie Fan, Jiang He,
Shufeng Chen, Hongfan Li, Penghua Zhang,
Laiyuan Wang, Dongsheng Hu, Jianfeng
Huang, Boqin Qiang and Dongfeng Gu
Myoglobin plasma level related to muscle mass and fiber
composition – a clinical marker of muscle wasting?
887-896
Authors Marc-André Weber, Ralf Kinscherf,
Holger Krakowski-Roosen, Michael Aulmann,
Hanna Renk, Annette Künkele, Lutz Edler,
Hans-Ulrich Kauczor and Wulf Hildebrandt
Targeting of human renal tumor-derived endothelial cells with
peptides obtained by phage display
Authors Benedetta Bussolati, Cristina Grange,
Lorenzo Tei, Maria Chiara Deregibus, Mauro
Ercolani, Silvio Aime and Giovanni Camussi
897-906
J Mol Med (2007) 85:783–785
DOI 10.1007/s00109-007-0231-0
CLINICAL IMPLICATIONS
Cachexia has only one meaning
Friedrich C. Luft
Received: 31 May 2007 / Accepted: 31 May 2007 / Published online: 3 July 2007
# Springer-Verlag 2007
Cachexia means any general reduction in vitality and
strength of body and mind resulting from any debilitating
chronic disease. Cachexia is defined as loss of weight,
muscle atrophy, fatigue, weakness, and loss of appetite in
someone who is not actively trying to lose weight. These
features markedly distinguish cachexia from starvation.
Cachexia can be a sign of various underlying disorders.
Physicians confronted with cachexia generally consider the
possibility of cancer, certain infectious diseases such as
tuberculosis or AIDS, parasitic diseases, autoimmune
disorders, or chronic heart failure. Cachexia physically
weakens patients to a state of immobility stemming from
loss of appetite, asthenia, and anemia. The response to
standard treatments is poor [1]. The above sounds straightforward enough. However, in PubMed, about 4,500 papers
have been published on cachexia; interestingly, >1,000 of
these papers are reviews. Such a relationship raises
suspicion that little is known about the subject. Weber
et al. [2], in this issue, introduce a novel inverse marker of
clinical muscle wasting, namely, the measurement of
plasma myoglobin concentration. Their report is immediately disconcerting, as the title of their paper ends in a
question mark, implying a lack of self-confidence on behalf
of the authors. However, the entire cachexia field is riddled
with question marks.
Weber et al. studied 17 cancer patients, the prototype
patients exhibiting cachexia. The patients had lost >20% of
their body mass without intending to do so. A suitable
matched control group of 27 subjects was recruited who
F. C. Luft (*)
HELIOS Kliniken-Berlin, Franz Volhard Clinic at the Max
Delbrück Center Medical Faculty of the Charité,
13122 Berlin, Germany
e-mail: [email protected]
had lost no weight. Plasma myoglobin, creatine kinase,
quadriceps muscle cross-sectional area (by magnetic resonance imaging), muscle morphology from biopsies of the
Vastus lateralis, body cell mass by impedance, and maximal
oxygen uptake (VO2 max) were all measured in these
patients and control subjects. To no surprise, myoglobin,
muscle cross-sectional area, body cell mass, and VO2 max
were all lower in cachexic cancer patients than in healthy
controls. However, in a multiple-regression analysis, myoglobin (hypomyoglobinemia) won out over other indicators
(27 μg/dl vs 42 μg/dl); or did it? The authors found that
myoglobin was directly correlated with cross-sectional
muscle mass and was better than reduced creatine kinase
in this regard, although the magnitude of the creatine kinase
reduction was greater. The fact that creatine kinase and
myoglobin were not elevated is of some mechanistic
interest, since active muscle destruction would have
featured elevation of both myoglobin and creatine kinase.
Weber et al. [2] performed muscle biopsies on 11 patients
and 15 control subjects. A marked size reduction in type 1
and 2 fibers occurred in the cachexic patients that
corresponded to the reduction in cross-sectional area and
correlated significantly with the reduced myoglobin levels.
What causes cachexia anyway? Are the diverse chronic
(or not so chronic) cachexia conditions related? The cachexic
patients presented by Weber et al. [2] all had cancer.
Skipworth et al. [3] recently reviewed cancer cachexia.
They stressed the role of host–tumor interaction, particularly pro-inflammatory cytokines. Tumor cells release
cytokines and other factors locally to promote inflammation
and thereby activate a local response. For instance,
proteolysis-inducing factor (PIF) is a sulphated glycoprotein that produces muscle wasting in tumor-free mice when
injected. The material has been identified in patients with
pancreatic, breast, ovarian, lung, colon, rectum, and liver
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J Mol Med (2007) 85:783–785
cancers. PIF may activate the ubiquitin–proteasome pathway by a nuclear factor kappaB (NF-κB) related mechanism. Lipid-mobilizing factor (LMF) is another cachexia
protein produced by tumors. LMF increases lipid oxidation
via induction of uncoupling protein expression, perhaps by
interacting with the β3 adrenoceptor.
Tumor products activate host cells. The activated host
cells then initiate their own cytokine cascade, thereby
inducing the hepatic acute phase protein response (APPR).
APPR induction appears to be primarily an interleukin-6
(IL-6)-driven mechanism; however, tumor necrosis factoralpha (TNF-α), IL-2, IL-8, interferon-gamma (IFN-γ),
parathyroid hormone-related peptide (PTHrP), and macrophage migratory inhibitory factor (MIF) also participate.
Other cytokines such as IL-4, IL-10, and IL-13 have antiinflammatory effects and are thought to repress cachexia.
IL-15 has anabolic effects on skeletal muscle through the
direct inhibition of muscle proteolysis.
The hepatic APPR includes a large number of “positive”
acute-phase proteins, such as C-reactive protein (CRP) and
fibrinogen. CRP correlates with weight loss, hypermetabolism, anorexia, and reduced survival. On the other hand, the
circulating levels of “negative” acute-phase proteins such as
albumin decrease. Cancer cachexia is also associated with a
neuroendocrine stress response. Weight-losing cancer patients
exhibit insulin resistance, dysregulation of the autonomic
nervous system, and sympathetic activation. A schematic
suggested by Skipworth et al. [3] is given in Fig. 1.
Another chronic cachexia condition is provoked by chronic
heart failure (CHF). CHF patients who involuntarily lose nonedematous weight >6% of their previous normal weight are at
substantially greater risk for subsequent mortality [4]. The
Anorexia
Systemic
inflammation
Acute phase
proteins
Reduced
Cytokines
substrate
supply Direct
catabolism
Increased
substrate
demands
Skeletal
muscle
wasting
Urinary nitrogen loss
Fig. 1 Pro-inflammatory cytokines induce muscle wasting either
directly or indirectly. The process involves a cytokine storm, anorexia,
metabolic disturbances, and enhanced catabolism, as well as acutephase response proteins and their effects [3]
parameter has a very high sensitivity–specificity product. CHF
cachexia correlates rather poorly with ejection fraction or the
New York Heart Association functional cardiac classification.
Proinflammatory cytokines, TNF-α, IL-1, IL-6, and others
again appear, as does the hepatic APPR. In CHF, there are no
invading tumor cells that release mediators, although catecholamines, cortisol, natriuretic peptides, and heat-shock proteins
have been implicated. An imbalance between anabolic and
catabolic pathways becomes established that involves numerous hormone systems, including neuropeptide Y, insulin,
cortisol, leptin, and ghrelin. A decrease in food intake alone
does not trigger the wasting process. The initial triggers are
unknown.
A related, more short-term condition developing over
days to weeks is critical illness myopathy (CIM). This
condition was first identified in intensive care unit patients
who had developed the systemic inflammatory response
syndrome (SIRS) usually in the framework of sepsis [5].
CIM patients commonly develop encephalopathy and
neuropathy, as well as a diffuse, flaccid weakness of limbs
and diaphragm. CIM is associated with SIRS, multiple
organ failure, and corticosteroid and neuromuscular blocking agent administration. CIM features bioenergetic failure,
mitochondrial dysfunction, inflammatory mediator-related
proteolysis, hormonal dysregulation with glucose toxicity,
myosin loss, and markedly increased total muscle catabolism. The triggers are unknown. The ubiquitin–proteosome
pathway has been implicated along with cyclooxygenase
activation, altered glucose transporter expression, MyoD
suppression, impaired respiratory chain enzymes, ATP
depletion, and insulin resistance. As mediators, TNF-α,
IFN-γ, IL-1, IL-6, CRP, and the regulatory transcription
factor NF-κB again prominently appear.
The cachexia syndromes are diverse diseases with
certain features that suggest a final common pathway. The
pathway leads to marked muscle wasting, systemic inflammation, hepatic APPR, and disturbed metabolism. The
pathophysiological changes are tightly regulated by the
same cytokines. Thus far, the final common pathway has
not proved to be a therapeutic avenue. Elucidation of the
triggers would appear more promising. The muscle biopsies
performed by Weber et al. [2] could be helpful in this
regard. Possibly, very early changes that could be identified
by serial biopsy might be helpful. Such biopsies could be
coupled with metabolic studies that can be discerned at the
tissue level by microdialysis and similar techniques.
Magnetic resonance spectroscopy coupled with imaging
could be applied. The area is ripe for novel patient-oriented
translational research.
Respectfully,
Friedrich C. Luft
J Mol Med (2007) 85:783–785
References
1. Kotler DP (2000) Cachexia. Ann Intern Med 133:622–634
2. Weber MA, Kinscherf R, Krakowski-Roosen H, Aulmann M, Renk
H, Künkele A, Edler L, Kauczor HU, Hildebrant W (2007)
Myoglobin plasma level related to muscle mass and fiber
composition—a clinical marker of muscle wasting? J Mol Med
DOI 10.1007/s00109-007-0220-3
785
3. Skipworth RJE, Stewart GD, Dejong CHC, Preston T, Fearon KCH
(2007) Pathophysiology of cancer cachexia: much more than a
host–tumour interaction? Clin Nutr DOI 10.1016/jclnu.2007.03.01
4. Von Haehling S, Doehner W, Anker SD (2007) Nutrition,
metabolism, and the complex pathophysiology of cachexia in
chronic heart failure. Cardiovasc Res 73:298–309
5. Friedrich O (2006) Critical illness myopathy: what is happening?
Curr Opin Clin Nutr Metab Care 9:403–409
J Mol Med (2007) 85:787–795
DOI 10.1007/s00109-007-0169-2
REVIEW
Connexin37: a potential modifier gene
of inflammatory disease
Marc Chanson & Brenda R. Kwak
Received: 21 December 2006 / Revised: 31 January 2007 / Accepted: 1 February 2007 / Published online: 22 February 2007
# Springer-Verlag 2007
Abstract There is an increasing appreciation of the
importance of gap junction proteins (connexins) in modulating the severity of inflammatory diseases. Multiple
epidemiological gene association studies have detected a
link between a single nucleotide polymorphism in the
human connexin37 (Cx37) gene and coronary artery
disease or myocardial infarction in various populations.
This C1019T polymorphism causes a proline-to-serine
substitution (P319S) in the regulatory C terminal tail of
Cx37, a protein that is expressed in the vascular endothelium as well as in monocytes and macrophages. Indeed,
these three cell types are key players in atherogenesis. In
the early phases of atherosclerosis, blood monocytes are
recruited to the sites of injury in response to chemotactic
factors. Monocytes adhere to the dysfunctional endothelium
and transmigrate across endothelial cells to penetrate the
arterial intima. In the intima, monocytes proliferate, mature,
and accumulate lipids to progress into macrophage foam
cells. This review focuses on Cx37 and its impact on the
cellular and molecular events underlying tissue function,
with particular emphasis of the contribution of the C1019T
polymorphism in atherosclerosis. We will also discuss
evidence for a potential mechanism by which allelic
variants of Cx37 are differentially predictive of increased
risk for inflammatory diseases.
MARC CHANSON
received his PhD in Biology
from the University of Geneva,
Switzerland in 1991. He
currently holds a faculty
position at the Department of
Pediatrics of the Geneva
University Hospitals. His
research interests include the
contribution of connexin-made
channels during the inflammatory process, especially in the
context of cystic fibrosis.
BRENDA R. KWAK
received her PhD in 1993 from
the University of Amsterdam
(Department of Physiology), the
Netherlands. She holds presently
a professorship of the Swiss
National Science Foundation.
Her research at the Division of
Cardiology of the Geneva University Hospitals (Switzerland)
focuses on the role of connexins
in cardiovascular disease, in
particular, in the pathogenesis of
atherosclerosis and restenosis.
Keywords Connexin . Hemichannels . Atherosclerosis .
Monocytes . Inflammation . Polymorphism
M. Chanson
Department of Pediatrics, Geneva University Hospitals,
1211 Geneva 14, Switzerland
B. R. Kwak (*)
Division of Cardiology, Department of Medicine,
Geneva University Hospitals, Foundation for Medical Research,
64 Avenue de la Roseraie,
1211 Geneva 4, Switzerland
e-mail: [email protected]
Connexins, connexons, and gap junctions
Connexins (Cx) are members of a family of proteins
encoded by at least 20 different mammalian genes that are
expressed in a wide variety of tissues [1, 2]. These genes
show 40% sequence identity and a common structure, the
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exon being interrupted by introns in only a few exceptions
[3]. Accordingly, the amino acid sequences of Cx proteins
are highly conserved. A connexin exhibits four α-helical
transmembrane domains (M1–M4), two extracellular loops
(E1 and E2), a short cytoplasmic loop (CL), and cytoplasmic NH2- and COOH-termini (NT and CT, respectively).
Connexins are classified in three-to-four groups, and the
most used nomenclature distinguishes Cx by their molecular mass deduced from their respective cDNAs. The CT,
which varies significantly in both length and composition,
is nearly unique to each Cx type. For most Cx studied so
far, the CT is a substrate for specific kinases and/or protein
partners, acting as a regulatory domain to modulate activity
of Cx channels in response to appropriate biochemical
stimuli [4–6].
The life cycle of connexins begins with the non-covalent
oligomerization of 6 Cx monomers into annular structures
called connexons [7, 8]. Connexons can be made of one
(homomeric) or several (heteromeric) Cx types. After their
assembly, connexons are delivered in vesicular carriers
traveling along microtubules from the Golgi to the plasma
membrane. These connexons at the plasma membrane
move laterally to reach the margins of channel clusters
and dock with their counterparts in the neighboring cells to
form intercellular channels, the gap junctions [9]. Thus, gap
junctions grow by accretion at their outer margins from
connexons to form plaques that can be resolved by electron
microscopy [10].
Connexins, connexons, and gap junctions are involved
in numerous processes contributing to the maintenance of
normal cell growth and differentiation [1, 11]. Particularly,
connexons can function as hemichannels in transmembrane
signaling, whereas gap junctions mediate the direct exchange of ions and small molecules (second messengers,
metabolites, linear peptides, mRNA) between cells in
contact [12, 13]. Experiments of functional replacement of
one connexin gene with another have revealed that cellular
homeostasis depends on the correct types of Cx expressed
[14]. This implies that the specific trafficking, permeability,
and interaction with protein partners and transduction
networks of each Cx type are contributing to tissue
response. Connexons and gap junctions are membrane
channels that are gated by chemicals and by membrane
potential (Vm). Whereas gap junction channels remain open
when Vm is identical between cells (Vm in cell 1 is equal to
Vm in cell 2, Vm1 = Vm2), they close with increasing
differences in transjunctional potential (Vj = Vm1 − Vm2). In
contrast, hemichannels seem to open with long Vm
depolarization [15, 16]. It is therefore not surprising that
mutations and polymorphisms of connexin genes would
affect Cx-made channel functions and, thus, are associated
with a variety of pathological conditions [17]. In this paper,
we will review the current knowledge on Cx37 function
J Mol Med (2007) 85:787–795
and discuss evidence for a potential mechanism by which
allelic variants of Cx37 are differentially predictive of
increased risk for inflammatory diseases.
Specific expression of Cx37 and its role in tissue
physiology
Some Cx display a rather ubiquitous expression pattern,
whereas others show a more restricted expression to
certain organs or cell types where they exert a unique
role in tissue function. Cx37, which belongs to the latter
group, has been found in the ovary, the vasculature, and
inflammatory cells.
Ovary
In the developing ovarian follicle, the oocyte is separated
from the local blood supply by an increasing number of
granulosa cell layers. These cells, which form the theca
externa, are the only ones in direct contact with ovarian
capillaries [18]. In this avascular system, intercellular
communication via gap junctions between the oocyte and
the surrounding somatic cells is essential for correct
functioning and development of the follicle [19, 20]. Gap
junctions mediate metabolic cooperation between granulosa cells and the oocyte by transmitting endocrine,
paracrine, and growth factor effects [21, 22]. Consequently, it has been hypothesized that gap junctional intercellular communication (GJIC) may play a role in the
coordination of follicular growth and steroid hormone
production [23] as well as in the maturation of the oocyte
[24]. Immunohistochemistry has revealed Cx37 in the gap
junctions between the oocyte and the granulosa cells of the
follicle [25, 26]. In addition, Cx43 has been identified as
the major component of gap junctions between granulosa
cells. Targeted disruption of the gene encoding Cx37 in
mice (Gja4) results in female infertility [25]. In fact,
Cx37-deficient mice lack mature Graaf follicles, fail to
ovulate, and develop numerous inappropriate corpora
lutea. These results suggest that in the normal Cx37expressing follicle, GJIC allows for bidirectional signaling. On the one hand, the GJIC between the oocyte and
surrounding granulose cells are required for oocyte growth
and development during the pre-antral stages of the
follicle. On the other hand, an inhibitory signal is
transferred through gap junctions from the oocyte to the
granulosa cells that results in the prevention of luteinization until ovulation has occurred [27]. An additional role
that has been proposed for follicular gap junctions is the
maintenance of meiotic arrest of the oocyte in a follicle via
low tonic amounts of cAMP signaling from the granulosa
to the oocyte [24, 28–30].
J Mol Med (2007) 85:787–795
Blood vessels
The vascular endothelium consists of a continuous monolayer of cells, lining the luminal surface of the entire
cardiovascular system, providing a non-thrombogenic barrier between the blood and the underlying tissues. Four
connexins, namely Cx37, Cx40, Cx43, and Cx45, have
been described in the vascular wall, a tissue that contains
not only endothelial–endothelial and smooth muscle–
smooth muscle gap junctions but also endothelial–smooth
muscle transmembrane channels [31–36]. Although connexin expression profiles have not yet been completely
described for all parts of the vascular tree, it is already clear
that Cx expression is not uniform in all blood vessels [37].
In addition, differences in Cx expression have been
reported in some vessels, like coronary arteries, when
comparing different species [38]. Most commonly, endothelial cells (ECs) express Cx37 and Cx40, whereas smooth
muscle cells (SMCs) express Cx43 and Cx45. Cx43 has
also been found in a subset of ECs near branch points of
arteries and in other localizations subjected to oscillatory
flow [39, 40]. The replacement of the Cx43 gene by a LacZ
reporter gene has revealed the expression of this connexin
in ECs of capillaries [41]. Others have reported the
expression of Cx37 or Cx40 in SMCs of specific blood
vessels [42–44] or under specific conditions [40, 45–47].
Of note, Cx37 might be excluded form myoendothelial
junctions, as recently reported in an in vitro model [48].
Several physiological roles have been proposed for
vascular gap junctions. Arterioles within the microcirculation span considerable distances, and coordination of
cellular behavior is required to allow for the synchronous
diameter changes over the entire length of the vessel that
are necessary for drastic changes in blood flow. GJIC
appeared crucial for the conduction of vasomotor responses
along arterioles and small arteries [49–51]. Moreover, ECs
are induced to migrate during the process of new capillary
sprout formation and during repair of the endothelial lining
after injury in large vessels. In a microvascular cell line in
which the expression of endothelial Cx was altered by
dominant negative connexin inhibitors, wound-induced
migration of ECs was found to be dependent on temporary
switches in Cx expression [52].
Connexin45, Cx43, Cx40, and Cx37 gene-targeted mice
have been created, each having a different vascular
phenotype. The complete deletion of Cx45 causes striking
abnormalities in vascular development, and mouse embryos
die early, between days 9.5 and 10.5 [53]. The deletion of
Cx43 causes dramatic cardiac defects, and these homozygous knockout mice (Cx43−/−) die in the early postnatal
period [54]. To circumvent this problem, the Cre/loxP
system was used to inactivate Cx43 expression exclusively
in ECs. These conditional Cx43 knockout mice display
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hypotension and bradycardia [55]. However, this observation remains to be confirmed because similar mice that
were developed by another laboratory do not display a
vascular phenotype [41]. Although the deletion of Cx37
(Cx37−/−) leads to female infertility, these animals survive
and do not show an obvious vascular phenotype [25, 56].
The removal of Cx40 (Cx40−/−) results in abnormal cardiac
conduction [57, 58] as well as in hypertension [59, 60].
More recently, connexin-deficient mice have been interbred
to enhance our understanding on the unique and redundant
roles of the Cx vascular genes. In contrast to the single
knockout animals, mice that completely lack both Cx37 and
Cx40 (Cx37−/−Cx40−/− double knockout mice) are not
viable beyond the first postnatal day and display severe
vascular abnormalities [61]. However, Cx37+/−Cx40−/−
mice appeared viable and may be used for studies towards
vascular function [62]. In contrast, Cx43+/−Cx40−/− mice
exhibit cardiac malformations and die neonatally [63].
Inflammatory cells
The establishment of GJIC between macrophages, based on
electrical coupling of adherent murine macrophages, was
first reported by Levy et al. [64]. Subsequently, gap
junctions were morphologically detected between various
types of macrophages and between macrophages and other
cell types by freeze fracture electron microscopy [65–68].
Further support for GJIC between macrophages and other
cells has come from dye transfer assays. Dye coupling was
observed between murine peritoneal macrophages as well
as between murine macrophages and intestinal epithelial
cells [69]. A low dye coupling was also observed at brain
stab wounds and in primary culture of murine microglia
[70]. This coupling was dramatically increased with the
treatment of IFN-γ and LPS or IFN-γ and TNF-α as well
as inhibited by a gap junction blocker. In addition, freshly
isolated human monocytes treated with LPS or TNF-α and
IFN-γ exhibited dye coupling [71]. However, these studies
are in conflict with others reporting lack of GJIC between
monocytes/macrophages and other cells. For example, dye
transfer was not observed in untreated human or mouse
monocytes/macrophages [72, 73], between human monocytes/macrophages and ECs, or between human monocytes/
macrophages and SMCs [71, 72].
To date, the expression of two Cxs has been reported in
monocytes/macrophages. Cx43 was found in the mouse
macrophage cell line J774 [74], activated peritoneal macrophages from hamsters and mice [66, 73, 75], brain stab
wound and primary cultures of murine microglia [70], and
human monocytes/macrophages stimulated with TNF-α
and INF-γ or LPS and INF-γ [71]. Moreover, Cx43 mRNA
was detected in macrophage foam cells of human atherosclerotic carotid arteries [72]. In addition, we observed this
790
connexin in peritoneal macrophages and in macrophages of
late atheromas [40, 75]. Finally, Cx37 was also detected in
peripheral blood monocytes from human or mice [76]. As
described in detail below, Cx37 plays a pivotal role in the
recruitment of monocytes and macrophages to atherosclerotic lesions [76].
Epidemiology of Cx37 association with human
pathologies
GJIC is often impaired in cancers. When genes coding for
Cxs are transfected into cancerous cells, this restores not
only their GJIC, but normal growth control is often restored
as well [77], thus, identifying connexins as possible ‘tumor
suppressor genes’. Mutations in Cx proteins can have major
effects on GJIC. Interestingly, mutated Cx37 has been
reported to be a tumor-associated antigen in the murine
Lewis lung carcinoma (3LL-D122) cell line [78]. Moreover, vaccination with a synthetic peptide corresponding to
the mutated domain of Cx37 induced effective anti-tumor
cytotoxic T lymphocytes and protected mice from spontaneous metastases of 3LL-D122 tumors [79]. In addition,
these peptide vaccines reduced metastatic loads in mice
carrying pre-established micrometastases [79]. However,
genome screening of a set of human lung and breast cancers
revealed no somatic mutations in Cx37 in these samples.
Interestingly, these studies revealed polymorphisms in the
Cx37 gene, but the majority of these polymorphisms reside
outside of the open reading frame of the protein [80].
Genetic linkage studies in erythrokeratodermias (EKV),
a clinically heterogeneous group of rare autosomal dominant disorders of cornification with hyperkeratosis and
erythema, revealed that these diseases map to the chromosomal region 1p34-35 [81]. Human Cx37 gene (GJA4)
maps to chromosome 1p35.1 by fluorescence in situ
hybridization and was thus considered an attractive candidate gene. By direct sequence analysis of GJA4 in control
samples, the authors detected a sequence variant (cytosineto-thymine) at position 1019, causing a substitution of
serine for proline at codon 319 in the regulatory cytoplasmic tail of Cx37. This point mutation creates a unique Sau
IIIA cleavage sequence that was used to screen all EKV
families and a series of unaffected controls for this
polymorphism. The serine variant was found in both
affected and unaffected EKV family members as well as
in a control group of unrelated Caucasians. Moreover,
extensive further screening of the EKV families for
mutations in GJA4 did not reveal a pathologic sequence
aberration in the coding region, thus, excluding Cx37 as a
candidate for this disease.
A few years later, a genome-wide linkage analysis for
premature myocardial infarction (MI) identified an almost
J Mol Med (2007) 85:787–795
identical region on chromosome 1, i.e., 1p34-36, as novel
susceptibility locus for this disease [82]. Coronary artery
disease (CAD) is the most common cause of ischemic heart
disease resulting primarily from atherosclerosis. The development and outcome of this progressive inflammatory
disease are known to depend on the interactions between
genetic, behavior, and environmental factors [83]. There are
ongoing searches for genes and proteins that influence the
development of CAD, with the aim to use these markers
along with established risk factors in screening tests for
patient risk stratification [84, 85]. These searches have
identified genetic polymorphisms in a number of human
genes that are associated with CAD and/or MI, including
the Cx37 gene.
To date, several gene polymorphism-association studies
have detected a link between the C1019T single nucleotide
polymorphism (SNP) in the human Cx37 gene and CAD as
well as MI in various populations. Surprisingly, the
published association studies appear contradictory, which
might have arisen in part from comparing different clinical
statuses, CAD versus MI. Whereas atherosclerotic plaque
development in carotid and coronary arteries seems
associated with the 1019C SNP coding for Cx37-319P
[86–88], increased risk for MI appeared associated to the
1019T SNP coding for Cx37-319S [89, 90]. This far, only
one study could not reveal an association between the
C1019T polymorphism in the Cx37 gene and the presence
of either CAD or MI [91]. The association between CAD
and the Cx37 polymorphism appeared particularly strong in
men with type 2 diabetes [92]. In contrast, the polymorphism appeared not associated with other vascular diseases
such as hypertension [93] and restenosis after balloon
angioplasty [94]. The relevance of Cx37 for MI is further
underlined by a report describing an association between
this condition and another polymorphism in the 3′-untranslated region of the gene. This I1297D polymorphism may
be related to the stability of the mRNA [95].
Although the development of CAD and MI is dependent
on many of the same risk factors, the two clinical
conditions are considerably different especially regarding
features of the atherosclerotic plaques. The key process
underlying acute MI is atherothrombosis, which is the
rupturing of an unstable or “vulnerable” atherosclerotic
plaque followed by acute coronary thrombosis [96, 97].
Plaques that are most likely to break exhibit a thin fibrous
cap, a large lipid pool, and many macrophages. This plaque
phenotype is partially dependent on the activities of
macrophages. Macrophage foam cells secrete pro-inflammatory cytokines that amplify the local inflammatory
response in the lesion as well as reactive oxygen species
that further induce macrophage proliferation and lipid
uptake. In addition, the activated macrophages produce
matrix metalloproteinases that can degrade the extracel-
J Mol Med (2007) 85:787–795
lular matrix, thus, further weakening the plaque’s fibrous
cap.
Cx37 polymorphism modulates the severity
of atherosclerosis: possible mechanisms
The identification of the Cx37 C1019T polymorphism as a
prognostic marker for atherosclerosis suggests that sequence differences between the two Cx37 proteins (Cx37319S and Cx37-319P) account for the phenotype. How can
the two forms of Cx37 differently modulate the severity of
atherosclerosis? To address this question, we have first
evaluated the contribution of Cx37 in the development of
atherosclerosis in a mouse model of the disease. Thus,
Cx37-deficient mice were crossed with apoliprotein Edeficient (ApoE−/−) mice to obtain double knockout
animals that were subjected to a high-cholesterol diet [76].
In these mice, the expression of Cx40 was not significantly
altered. Deletion of Cx37 accelerated atherogenesis in
Cx37−/−ApoE−/− mice as compared to the control group
(Cx37+/+ApoE−/−). This was demonstrated by the twofold
increase of Sudan IV-stained lipids in thoracic abdominal
aortas and in aortic sinuses after a 10-week diet. These
observations are indicative that Cx37 plays a protective role
against atherosclerosis in ApoE−/− mice.
Cx37 is normally expressed in endothelial and macrophage foam cells [40, 98], two cell types that are key
players in atherogenesis. In the early phases of atherosclerosis, blood monocytes are recruited to the sites of injury in
response to chemotactic factors. Monocytes adhere to the
dysfunctional endothelium and transmigrate across ECs to
penetrate the arterial intima. In the intima, monocytes
proliferate, mature, and accumulate lipids to progress into
macrophage foam cells. Because monocytes appeared to
express Cx37, the possibility that Cx37 contributes to the
interaction between monocytes and endothelial cells was
investigated [76]. Indeed, there is evidence in the literature
for gap junction-mediated heterocellular communication
between leukocytes and ECs [98, 99]. To test for this
possibility, Cx37-deficient monocytes or macrophages were
introduced in hypercholesterolemic recipient mice by adoptive transfer and the number of adherent leukocytes to or
within atherosclerotic plaques determined. This was compared with the number of normal leukocytes introduced to
Cx37-deficient recipient mice with atherosclerotic lesions.
Interestingly, these experiments revealed that deletion of
Cx37 in monocyte/macrophages, but not in ECs, did account
for higher number of leukocytes associated with atherosclerotic plaques. These results indicate that heterocellular GJIC
does not contribute to the increased recruitment of leukocytes to the atherosclerotic lesions but rather suggest a role
of Cx37 in monocytes/macrophage function.
791
Monocyte migration and accumulation of lipid-filled
macrophages are critical events in the progression of
atherosclerosis. It is currently unclear why macrophages that
enter atherosclerotic lesions do not depart with their lipid
loads. During their transmigration across the endothelium,
monocytes are subject to profound reorganization of their
actin cytoskeleton and plasma membrane receptors and
adhesion molecules [100]. These modifications enhanced
their adhesion properties and ability to migrate on a
substrate. In this context, we observed that adhesion of
Cx37-deficient monocyte/macrophages to either EC monolayers, plastic, or glass was enhanced as compared to
leukocytes normally expressing Cx37 [76]. The implication
of Cx37 in the regulation of monocyte/macrophage adhesion
was indicated by that connexin-channel blockers, including
α-glycyrrhetinic acid and connexin mimetic peptides, increased leukocyte adhesion, and that expression of Cx37 in a
Cx-deficient macrophage cell line decreased its adhesiveness
to substrates. Because these assays were performed using
isolated leukocytes, it is likely that connexons, and not gap
junctions, are involved in the process of cell adhesion.
Extracellular purines (ATP, ADP, adenosine) are important signaling molecules that mediate both inflammatory
and anti-inflammatory effects. ATP is also known to pass
through various types of gap junctions and hemichannels
[101]. Interestingly, a causal relationship was observed
between extracellular ATP release and decreased adhesion
in monocyte/macrophages expressing Cx37. Conversely,
absence of Cx37 or blockade of Cx37 hemichannels
reduced the release of ATP out of the cells and increased
their adhesion to substrates. Furthermore, the use of an
extracellular ATP scavenger increased adhesion of normal
monocyte/macrophages, whereas addition of extracellular
ATP equalized the adhesive properties of Cx37-deficient
leukocytes to that of Cx37-expressing leukocytes. Altogether, these observations suggest that extracellular ATP
provides a link between Cx37 hemichannel activity and
leukocyte adhesiveness. It is hypothesized that Cx37
hemichannels release ATP, which in turn interferes with
leukocyte adhesion by a mechanism that remains to be
demonstrated (Fig. 1). According to this hypothesis,
absence of Cx37 would be associated with increased
adhesion of monocyte/macrophages to and within the
atherosclerotic plaques. A change in the adhesion properties
of these cells will also likely favor their accumulation in the
atherosclerotic lesions and worsen the phenotype. In this
context, the observation that expression of Cx37-319S or
Cx37-319P by transfection of a human macrophage cell
line revealed differential adhesiveness to substrates is of
particular importance [76]. This may be caused by
increased permeability of the Cx37-319P hemichannels
for ATP, thus, providing a potential mechanism by which
the Cx37-1019C variant protects against atherosclerosis.
792
J Mol Med (2007) 85:787–795
Anti-inflammatory
CD73
Pro-inflammatory
Endothelial cells
Ad
Monocytes
ATP
Reduced adhesion
Increased adhesion
Fig. 1 Hypothetical model of the anti-adhesive function of Cx37 in
mouse monocytes. Rolling monocytes at the surface of the vessel slow
down and firmly adhere to ECs before extravasation. Cx37-hemichannels at the surface of monocytes allow for the release to the
extracellular space of ATP. Extracellular ATP negatively regulates the
adhesion of monocytes to ECs by a yet undetermined mechanism. In
the absence of functional Cx37 (by hemichannel blockade or Cx37
gene deletion), ATP is not released out of the cell, resulting in
enhanced adhesiveness of monocytes to the endothelium. Possibly,
ATP released by monocytes can be sequentially degraded by
ectoenzymes to AMP and then nucleosides and inosine. For instance,
ecto-5′ -nucleotidase (CD73) is up-regulated at the endothelium
surface during inflammation to convert AMP into adenosine (Ad).
Adenosine has an anti-inflammatory and cell-protective effect through
its binding to receptors localized on the cell surface of endothelial and
some inflammatory cells [108]. Absence of production of ATP by
Cx37-deficient monocytes may reduce adenosine production, which in
turn would accelerate atherogenesis in mice by favoring a proinflammatory environment
Speculative remarks and conclusion
sensitivity of Cx37-319S and Cx37-319P to cholesterol
increase may also account for the enhanced ATP leakage
through Cx37-319P hemichannels. Hence, Cx37-319P, by
releasing ATP, may reduce the adhesion of macrophages
and allow them to egress from the affected area. The
decreased adhesiveness of Cx37-319P-expressing leukocytes may therefore serve as a “protector” mechanism that
prevents excessive monocyte recruitment in atherosclerosis.
Because the rupture of vulnerable atherosclerotic plaques, a
key process underlying acute MI, strongly depends on the
presence and the activity of macrophages in the lesions, our
study may provide a rationale for the epidemiological
association between increased risk for acute MI and the
Cx37-319S polymorphism. The generation of knock-in
mice for either Cx37 polymorph may help to resolve these
issues. Our improved understanding of the role of the Cx37
C1019T polymorphism may not only lead to the use of this
genetic variant in risk stratification for MI, but may also
have implications for other chronic inflammatory diseases
where monocytes and/or macrophages are involved.
Additional experiments are needed to determine whether
Cx37-319S and Cx37-319P hemichannels exhibit differential biophysical and permeability properties. However, one
can speculate on the mechanism underlying the regulation
of Cx37-319S and Cx37-319P hemichannels. One consequence of the study by Wong et al. [76] is that leukocytes
may need to close Cx37 hemichannels to increase their
adhesive properties. It is long known that adherent macrophages showed a more negative membrane potential as
compared to macrophages in suspension [102–104]. One
consequence of more negative Vm would be to turn off
hemichannel activity. Thus, differences in Vm sensitivity
between Cx37-319S and Cx37-319P hemichannels could
account for the differential ATP transport by these
connexons. An alternative possibility is that elevated
macrophage plasma membrane cholesterol content may
differentially affect the regulation of Cx37-319S and Cx37319P hemichannels. There is indeed increasing evidence
that high cholesterol levels may alter plasma membrane and
actin cytoskeleton organization of macrophages during
atherosclerosis [105]. The presence of cholesterol in plasma
membranes is also known to affect the chemical regulation
of gap junction channels [106, 107]. Thus, a differential
Acknowledgments We thank Suzanne Duperret for secretarial help.
This work was supported by grants from the Swiss National Science
Foundation (310000-107846/1 to MC; PPOOA-68883 and 3100067777 to BRK).
J Mol Med (2007) 85:787–795
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J Mol Med (2007) 85:797–810
DOI 10.1007/s00109-007-0183-4
REVIEW
Natriuretic peptide receptor B signaling
in the cardiovascular system: protection
from cardiac hypertrophy
Ines Pagel-Langenickel & Jens Buttgereit &
Michael Bader & Thomas H. Langenickel
Received: 10 October 2006 / Revised: 6 February 2007 / Accepted: 27 February 2007 / Published online: 12 April 2007
# Springer-Verlag 2007
Abstract Natriuretic peptides (NP) represent a family of
structurally homologous but genetically distinct peptide
hormones involved in regulation of fluid and electrolyte
balance, blood pressure, fat metabolism, cell proliferation,
and long bone growth. Recent work suggests a role for
natriuretic peptide receptor B (NPR-B) signaling in regulation of cardiac growth by either a direct effect on
cardiomyocytes or by modulation of other signaling pathways including the autonomic nervous system. The
research links NPR-B for the first time to a cardiac
phenotype in vivo and underlines the importance of the
NP in the cardiovascular system. This manuscript will focus
on the role of NPR-B and its ligand C-type natriuretic
peptide in cardiovascular physiology and disease and will
evaluate these new findings in the context of the known
function of this receptor, with a perspective on how future
research might further elucidate NPR-B function.
Keywords Cardiac hypertrophy . Natriuretic peptide
receptor B . C-type natriuretic peptide . Cardiovascular
I. Pagel-Langenickel
National Heart, Lung, and Blood Institute,
National Institutes of Health,
Bethesda, MD, USA
J. Buttgereit : M. Bader
Max Delbrueck Center for Molecular Medicine Berlin-Buch,
Berlin, Germany
T. H. Langenickel (*)
Vascular Biology Section, Genome Technology Branch, National
Human Genome Research Institute, National Institutes of Health,
50 South Drive, Building 50, Room 4529,
Bethesda, MD 20892, USA
e-mail: [email protected]
INES PAGEL LANGENICKEL
received her M.D. and doctorate
from the Medical Faculty of the
Humboldt University, Berlin,
Germany. She is currently visiting fellow at the National Heart,
Lung, and Blood Institute,
Bethesda, USA. Her research
interests include natriuretic peptide receptor signaling in heart
failure and mitochondrial biology in diabetes and diabetic
cardiomyopathy.
THOMAS H. LANGENICKEL
received his M.D. and doctorate
from the Medical Faculty of the
Humboldt University, Berlin,
Germany. He is currently visiting fellow at the National Human Genome Research Institute,
Bethesda, USA. His research
interests include natriuretic peptide receptor signaling in the
vasculature and the role of cell
cycle regulating proteins in vascular wound repair and pulmonary hypertension.
Abbreviations
CNP
C-type natriuretic peptide
NPR-B natriuretic peptide receptor B
Natriuretic peptide system
de Bold et al. [1] demonstrated for the first time that atrial
extracts exerted a long lasting natriuretic and diuretic response
798
J Mol Med (2007) 85:797–810
after intravenous injection into rats. The peptide responsible
for this observed effect was identified as atrial natriuretic
peptide, ANP [2]. ANP was able to increase cyclic guanosine
monophosphate (cGMP) concentration in tissue culture and
in tissues and urine of rats, suggesting that cGMP is the
second messenger of ANP [3]. Subsequently, other peptides
with structural and functional homologies to ANP were
purified and named brain natriuretic peptide, BNP, and Ctype natriuretic peptide, CNP [4, 5]. Although CNP does not
share the diuretic and natriuretic properties of ANP and BNP,
it is highly conserved among species and considered the
most ancient member of the natriuretic peptide family.
Evolutionary studies suggest that ANP and BNP have
diverged from CNP by gene duplication events most likely
reflecting changes in osmoregulatory systems [6]. A key
feature of all three peptides is a 17 amino acid (aa) ring
structure that is formed by an intramolecular disulfide bridge
[7]. Differences in the primary structure of natriuretic
peptides (NP) are predominantly located at the C- and Nterminal end. Studies employing photoaffinity labeling and
cross-linking studies of ANP led to identification of the
natriuretic peptide receptors A (NPR-A) and C (NPR-C) [8,
9]. Chinkers et al. [10] reported the complementary DNA
(cDNA) sequence of NPR-A derived from rat brain and
showed ANP binding and generation of cGMP when NPR-A
was overexpressed in mammalian cells. The second guanylyl
cyclase (GC)-coupled receptor, natriuretic peptide receptor B
(NPR-B), was discovered by cloning and sequencing from a
human or rat cDNA library, respectively [11, 12]. Binding
studies revealed different receptor binding properties of NP,
with ANP and BNP having the highest affinity to NPR-A,
whereas CNP binds preferentially to NPR-B [13]. All three
peptides bind NPR-C with comparable affinity. A detailed
insight into the complex interaction between the NP and
NPR was recently provided by crystallographic analysis of
the various receptor ligand combinations. Here, it was
demonstrated that two pockets on the receptor surfaces
function as anchoring sites for the hormone side chains and
determine receptor selectivity of the NP [14].
It is important to note that the different receptor affinities
for NP do not exclude binding of ANP and BNP to NPR-B
or CNP to NPR-A. As supraphysiological concentrations
were used in most in vitro experiments or were administered intravenously into intact animals, it is difficult to
attribute the respective findings to the signaling and
physiological function of one single NPR. To our knowledge, there is only one NPR-B selective peptide antagonist
described in the recent literature [15]. It has, however, not
been widely used yet to study NPR-B function. HS-142-1,
the most used antagonist, blocks NPR-A and -B signaling
through an allotropic mechanism [16, 17]. Genetic models
have contributed to our understanding of the many
physiological functions of NP and their receptors (Table 1).
Experiments in mice with targeted disruption of ANP, BNP,
or NPR-A suggested a role of this signaling pathway in
regulation of blood pressure, cardiac hypertrophy, and
fibrosis [18–20], while CNP and NPR-B knockout mice
displayed severe dwarfism resulting in early death due to
impaired endochondral ossification [21, 22]. Interestingly,
transgenic rats expressing a dominant negative NPR-B
mutant have a normal life span probably due to a minimally
remaining receptor activity that is sufficient to blunt the
skeletal phenotype. Cardiovascular phenotyping of these
animals has suggested a role of NPR-B in growth
modulation of cardiac myocytes and regulation of sympathetic
nerve activity [23].
Table 1 Animal models resulting from genetic alterations of natriuretic peptides or their receptors
Genetic alteration
Cardiovascular phenotype
References
ANP deletion
ANP overexpression
BNP deletion
BNP overexpression
CNP deletion
NPR-A deletion
Salt-sensitive hypertension, pulmonary hypertension
Arterial hypotension
Cardiac fibrosis
Arterial hypotension, Skeletal overgrowth
None (altered endochondral ossification, dwarfism, early death)
Salt-resistant hypertension, cardiac hypertrophy and fibrosis, enhanced
cardiac remodeling after myocardial infarction
Arterial hypotension, protection against dietary salt
None (dwarfism, accumulation of white adipose tissue,
seizure attacks, infertility)
Concentric cardiac hypertrophy, normotension, increased
sympathic nerve activity
Hypotension, reduced ability to concentrate urine, mild
diuresis, volume depletion
[18]
[19]
[19]
[120, 121]
[122]
[20, 123, 124]
NPR-A overexpression
NPR-B deletion
NPR-BΔKC overexpression
NPR-C deletion
NPR Natriuretic peptide receptors; NPR-BΔKC dominant negative mutant of the catalytical receptor subunit.
[125]
[22]
[23]
[126]
J Mol Med (2007) 85:797–810
799
C-type natriuretic peptide
Natriuretic peptide receptor B
In contrast to ANP and BNP, which are most abundant in the
heart, CNP is predominantly expressed in the brain, chondrocytes, cytokine-stimulated endothelial cells, uterus, and
ovaries [24]. CNP is released upon stimulation of endothelial
cells with tumor necrosis factor alpha (TNF-α), transforming
growth factor beta, interleukin-1, and shear stress [25–27].
Human CNP is synthesized as a 126 aa pre-pro peptide.
After a signal peptide is removed by the signal peptidase, the
resulting 103 aa pro-CNP is further cleaved by the
endoprotease furin into the biologically active CNP-53 and
a 50 aa pro peptide [28]. Both peptides are released from the
cells and have been detected in several tissues. Another form
of CNP, CNP-22, is similar to CNP-53 in terms of biological
activity, although it shows different tissue distribution. CNP53 is present in brain, heart, and endothelial cells, whereas
CNP-22 is the predominant form in plasma and cerebral
spinal fluid [29–31]. The mechanism by which CNP-22 is
processed from the precursor protein has not yet been
identified, but the cleavage of pro-CNP by furin is assumed
to be the critical step in converting the precursor to the active
hormone. Therefore, a differential regulation of furin
expression in various tissues could effect the availability of
CNP and NPR-B stimulation. Because the plasma concentration is very low as compared to ANP and BNP, CNP is
thought to act mainly in a paracrine fashion [29]. CNP has
been demonstrated to have potent vasorelaxing properties
[32]. It negatively regulates smooth muscle cell proliferation
[33, 34], but does not share the diuretic and natriuretic
features of ANP and BNP. Several studies revealed that CNP
exerts its vasodilating effects independent of endothelium via
hyperpolarization of smooth muscle cell membranes [35,
36]. In fact, CNP, released from endothelial cells, accounts
for the biological activity of the earlier described endotheliumderived hyperpolarizing factor [37]. The proposed mechanism
involves binding of CNP to NPR-C, Gi protein activation,
and Gi coupling to a smooth muscle potassium channel.
In pathophysiological conditions such as congestive heart
failure, where circulating ANP and BNP are highly upregulated, data on the regulation of CNP are conflicting. Unlike
early reports, recent studies have described increased myocardial production and elevated plasma concentration of CNP
in patients with heart failure [38, 39]. Interestingly, increased
CNP secretion into the coronary circulation of heart failure
patients has been found [40], suggesting an involvement of
CNP in pathophysiological cardiac remodeling. In addition, a
mild increase in circulating CNP levels was detected in
patients with chronic renal failure, and marked CNP
elevation was found in patients with septic shock [41],
possibly due to the presence of high levels of TNF-α and
lipopolysaccharides that are known to modulate CNP
secretion from endothelial cells.
NPR-B is expressed in the brain, chondrocytes, lung, vascular
smooth muscle cells, fibroblasts, and uterus [12, 42] and
represents the main natriuretic peptide receptor in the brain
[43, 44]. NPR-A and -B share structural similarities. Both
have an extracellular ligand-binding domain characterized by
three loops formed by intramolecular disulfide bridges [45,
46]. A short transmembrane region anchors the receptor in
the cell membrane. The intracellular domain consists of a
regulatory kinase homology domain (KHD), a hinge region,
and a GC. The KHD is able to bind ATP and is phosphorylated, thus, allowing ligand dependent activation of the GC
[47]. Nonactivated NPR-B is present as an oligomer with
very low GC activity [48]. Ligand binding to the receptor
dimer leads to a rotational shift of the transmembrane region
[49], which is translated into a conformational change of the
intracellular receptor domain, reversing the inhibitory effect
of the KHD on the C-terminal GC and subsequently leading
to formation of 3′,5′-cGMP [10, 50]. The detailed crystal
structure of NPR-B is not known, but a model of the NPR-B/
CNP complex was recently proposed by He et al. [14] based
on crystallographic analyses of interactions between the NP
and NPR-A or NPR-C, respectively. Similar to the topology
of NPR-A and -B, CNP binds to a NPR-B dimer as
demonstrated for ANP and NPR-A [49, 51].
To further understand the biology of NPR signaling in
vitro and in vivo and given that dimerization is a
prerequisite for receptor activation, dominant negative
receptors have been developed and studied in detail.
Chinkers and Wilson [48] demonstrated that extracellular
NPR domains were necessary and sufficient to form
receptor heterodimers, including receptor mutants lacking
either part of or the entire intracellular domain. An NPR-A
mutant (NPR-AΔKC) lacking KHD and the GC itself was
identified as a dominant negative mutant specifically
interacting with NPR-A, not NPR-B. This concept has
been applied to NPR-B by our group to generate transgenic
rats with a functional downregulation of NPR-B signaling
[23]. We demonstrated unaffected NPR-A signaling in cells
derived from NPR-BΔKC transgenic rats to prove that the
phenotype can be attributed to reduced NPR-B signaling.
Cardiovascular functions of CNP and NPR-B signaling
While the role of NPR-A on cardiac hypertrophy and blood
pressure regulation has been extensively characterized, fewer
data are available to provide insight into the function of
NPR-B signaling in the cardiovascular system. Until recently,
neither genetic models nor NPR-B specific pharmacological
inhibitor existed that allowed the discrimination between
NPR-A and NPR-B signaling. Because CNP possesses
800
relative receptor specificity [13], several CNP effects have
been attributed to NPR-B stimulation. Furthermore, it has
been revealed that also NPR-C plays a significant role in
mediating CNP effects in several cell types [52]. Despite
controversial data, recent research suggests a role for CNP/
NPR-B signaling in regulation of cardiac growth, vascular
remodeling, and autonomic nervous activity.
Effects on vascular tone
NPR-B is highly abundant in vascular endothelial cells
and smooth muscle cells [33], implying its role in the
regulation of vascular tone and blood pressure. Intravenous administration of CNP in dogs has resulted in a
reduction in blood pressure, with a greater effect from
CNP than ANP [53]. The effect of CNP was not associated
with increased natriuresis as observed for ANP administration, indicating direct modulation of vascular tone,
rather than a reduction in the circulating blood volume as
the underlying mechanism. Several ex vivo models have
strengthened this hypothesis.
CNP induced a dose-dependent relaxation in precontracted porcine coronary arteries and guinea pig aortas that
was mediated by cGMP [32, 35]. The vasorelaxation was
endothelium-independent and accompanied by hyperpolarization of smooth muscle cell membranes. Furthermore, it
involved the activation of voltage-dependent and calciumsensitive potassium (BKCa) channels [54]. A similar
mechanism has been demonstrated for the vasorelaxation
of isolated canine femoral veins [55]. The effects of CNP
on arterial vasodilation are further modulated by the
presence of nitric oxide (NO). As Wennberg et al. [56]
showed, CNP release is involved in endothelium-dependent
relaxation due to acetylcholine or bradykinin in isolated
canine coronary arteries. Similarly, the inhibition of GCcoupled NPR with HS-142-1 blunted bradykinin-induced
vasodilatation in porcine coronary arteries [35]. Another
hint at a possible interaction between CNP and NO-induced
vasorelaxation came from a study in endothelial NO
synthase (eNOS) knockout mice. Here, CNP caused a
dose-dependent vasorelaxation in aortas from wild-type
mice that could be blocked by HS-142-1. The effect was
enhanced in mice lacking eNOS or in the presence of the
eNOS inhibitor L-NAME, suggesting a cross talk of the NO
and NP system in regulating vascular tone [57]. In contrast,
a study using porcine coronary rings revealed that vasorelaxation, upon CNP stimulation, was not affected by
inhibition of NOS with L-NAME [35], raising the possibility that the degree of interaction between both systems is
tissue and/or species specific.
Despite the relative specificity of CNP for the stimulation
of NPR-B, the vasorelaxing effect of CNP in isolated renal
arteries of NPR-A knockout mice was blunted compared to
J Mol Med (2007) 85:797–810
wild-type mice. Meanwhile, this response was unaffected in
aortic rings [58]. This effect could neither be attributed to a
change in NPR-B or -C expression levels nor to impaired
downstream signaling. Differential expression of receptor
subtypes in different sections of the vasculature provides a
possible explanation for this phenomenon.
In animal models as well as in humans, CNP infusion
lowered arterial blood pressure [53, 59, 60]. Igaki et al. [59]
have shown that intravenously administered CNP in humans
decreased systolic and diastolic blood pressure. In healthy
volunteers, intra-arterial infusion of CNP led to increased
forearm blood flow that was not dependent on NOS activity
but attenuated by inhibition of calcium-sensitive potassium
channels [36]. These data suggest that CNP, in accordance
with in vitro effects, mediates arterial vasodilatation in vivo
via potassium channel opening and hyperpolarization of
vascular smooth muscle cells. In rat renal microvessels,
CNP was able to dilate pre- and postglomerular vessels,
while stimulation with the NPR-C specific ligand C-ANP
was devoid of any vascular effect [61]. However, several
studies suggest that the arterial vasodilating properties of
CNP are at least partially mediated through NPR-C binding
[62]. Thus, the precise role of NPR-B in the regulation of
the arterial vascular tone is not entirely clear.
In contrast, CNP/NPR-B seems to be the responsible
signaling pathway for venodilatory effects of CNP. Furthermore, when compared to ANP, CNP was more potent in
dilating pulmonary veins but less capable of relaxing
pulmonary arteries, suggesting distinct mechanisms for
NPR-A and NPR-B in mediating vasodilation in arteries
and veins [63]. NPR-B could, therefore, have a predominant role for the reduction in cardiac preload.
In most of these studies, however, CNP was applied at
supraphysiological doses, and the observed effects cannot
entirely be attributed to exclusive NPR-B stimulation.
Hence, when NPR-B receptors in healthy volunteers were
stimulated by a low-dose CNP infusion that led to a
fourfold and tenfold increase in circulating CNP levels,
respectively, neither cardiac output nor systemic blood
pressure was affected [64]. In a similar study, CNP infusion
did not have any additional effect on arterial response when
coadministered with ANP [65]. Another limitation of
supraphysiological doses is a possible competition for
NPR-C leading to an increased efficiency of circulating
ANP and BNP due to reduced clearance from circulation.
The generation of a mouse line lacking NPR-B by
Tamura et al. [22] partially overcame this problem and
promised a better insight into the role of NPR-B in the
chronic regulation of blood pressure and vascular tone.
These NPR-B null mice showed a significantly blunted
cGMP response upon CNP stimulation on a cellular level.
Main phenotypic findings included dwarfism due to
impaired enchondral ossification and female infertility
J Mol Med (2007) 85:797–810
resulting from abnormal uterine and ovarial development.
Unexpectedly, blood pressure assessment under either low
or high salt diet did not show any differences between
NPR-B null and wild-type control mice. In addition, the
suppression of aldosterone secretion by high salt diet was
preserved in both genotypes. However, these data only
reflect the blood pressure regulation in young animals
because the majority of the NPR-B null mice died
prematurely (before 100 days of age) as a consequence of
skull deformation with compression of the medulla oblongata or seizures. Likewise, an earlier model of targeted
disruption of CNP demonstrated a similar skeletal phenotype along with a reduced survival rate, and no evident
cardiovascular phenotype was reported [21]. Therefore, a
role of NPR-B for the long-term blood pressure regulation
cannot be concluded from these studies.
A different approach was employed by our group using
transgenic rats expressing a dominant negative mutant of
the NPR-B mutant (NPR-BΔKC) that resulted in a
functional knockdown of the receptor [23]. These rats were
viable, had a normal life span, and only modest skeletal
abnormalities. Under normal conditions, the arterial blood
pressure, as assessed telemetrically, in conscious rats did
not differ between NPR-BΔKC and wild-type animals.
Given the increase in circulating levels of both ANP and
BNP in the NPR-BΔKC rats, it is possible although
unlikely that a hypertensive phenotype, resulting from a
disrupted NPR-B signaling, is masked.
In conclusion, despite mediating a vasodilatory response
upon CNP stimulation, NPR-B does not seem to play a
significant role in long-term blood pressure regulation
under physiological conditions. Its signaling may, however,
prevent the manifestation of hypertension under pathophysiological conditions such as an activated renin–angiotensin–
aldosterone system.
Effects on vascular remodeling
In vitro studies have shown that CNP mediates antiproliferative and antihypertrophic actions in several cell types
including vascular smooth muscle cells and cardiac fibroblasts [33, 66]. Interestingly, while the vasorelaxing
properties of CNP where inferior to ANP, its antiproliferative effects were superior. CNP was shown to be the most
potent among the three NP regarding the inhibition of
smooth muscle cell migration after stimulation with
platelet-derived growth factor [67]. This effect was accompanied by an increase in cellular cGMP levels and
mimicked by the cGMP analog 8-Br-cGMP, suggesting a
NPR-B-dependent mechanism. These antiproliferative and
antimigratory effects could play a role in vascular remodeling. In fact, CNP has reduced neointima formation and
thrombosis in different vascular injury models [68, 69].
801
When CNP was administered locally, it prevented intima
proliferation and endothelial dysfunction in rabbits without
affecting endothelium-independent relaxation [70]. As
observed for the vasodilatory effects, the involvement of
NOS seems to play an important role in this process, as
CNP caused a significant iNOS induction after carotid
artery balloon injury in rabbits. Conversely, the antiproliferative effect could be blunted by inhibiting NOS with LNAME [69]. Furthermore, CNP-mediated suppression of
the antifibrinolytic enzyme plasminogen activator inhibitor1 transcription, regulation of its release from VSMC in
vitro, and its expression in neointima, media, and adventitia
could have a beneficial effect in the pathogenesis of
atherosclerosis [71, 72]. Similar results were obtained in a
model of bleomycin-induced pulmonary fibrosis in mice
[73]. Here, CNP attenuated the infiltration of monocytes
and macrophages in the lung and reduced the collagen
content and the cell proliferation in fibrotic lesions. In
addition, a recent study by Scotland et al. [74] demonstrated
that CNP was able to suppress basal and inflammationstimulated leukocyte activation, inhibited platelet–leukocyte interaction, prevented thrombin-induced platelet
aggregation in human blood, and reduced endothelial
expression of P-selectin, an important regulator of leukocyte recruitment into tissue. Thus, due to the beneficial
combination of antiproliferative, anti-inflammatory, and
vasoactive properties and the lack of side effects on systemic
hemodynamics, CNP might have therapeutic potential for
the management of diseases involving pathological vascular
remodeling, such as primary pulmonary hypertension.
To date, it has not been completely elucidated if the
stimulation of NPR-C by CNP also contributes to the observed effects on vascular remodeling. While the expression
of CNP and NPR-C were upregulated in parallel in the
neointima after vascular injury in rabbits, NPR-B was not
detectable in the neointima and did not significantly change
in the media [75, 76]. A similar observation has been made
in patients undergoing percutaneous coronary intervention.
CNP was strongly upregulated in the neointima, but NPR-B
expression was not regulated [77]. Of note, a recent study
showed NPR-B messenger RNA (mRNA) in intermediate
plaques and advanced atherosclerotic lesions in human
coronary arteries, together with upregulated CNP expression that was localized in endothelial cells, smooth muscle
cells, macrophages, and microvessels [78]. Hence, the
possibility of NPR-B involvement in the process of
vascular remodeling and development of atherosclerosis
has to be considered despite inconsistent data regarding
receptor regulation. Studies employing animals lacking a
functional receptor are necessary to further clarify this
issue. The antiproliferative effects of NP on smooth muscle
cells raise the question of whether the CNP/NPR-B
signaling could also prevent angiogenesis. On the contrary,
802
the vascular regeneration in a mouse model of hindlimb
ischemia was improved as demonstrated by increased
capillary density and perfusion rate when either BNP or
the downstream target of the NP, guanylyl cyclase I (cGKI),
was overexpressed [79]. This effect was mimicked by
adenoviral gene transfer of CNP. Conversely, the recovery
after surgery was blunted in cGKI knockout mice. The in
vivo data were further complemented by the observation that
ANP, BNP, and CNP stimulate the capillary network
formation of human endothelial cells, with CNP being the
most potent of the three NP. These data suggest that the CNP/
NPR-B signaling pathway could play an important role in
ischemia-induced angiogenesis. However, vascular endothelial growth factor (VEGF), a potent angiogenetic factor, has
been demonstrated to suppress CNP mRNA expression and
CNP secretion [80]. The interaction between VEGF and
CNP-signaling has not been verified yet.
Effects on pathological cardiac hypertrophy and remodeling
The normal cardiac response to an increase in workload, as
seen during exercise, involves the stimulation of insulin-like
growth factor 1 and results in hypertrophy of cardiomyocytes
and increased contractility as an adaptive mechanism [81].
By contrast, the presence of chronic overload as in arterial
hypertension or after loss of viable myocardium due to
infarction leads to pathological remodeling that is characterized by proliferation of fibroblasts with subsequent
fibrosis, loss of cardiomyocytes by apoptosis or necrosis,
Fig. 1 Signaling pathways involved in antihypertrophic
actions of CNP. CNP binds to
NPR-B and activates the GC,
leading to intracellular cGMP
formation and subsequent activation of the cGMP-dependent
protein kinase (PKG). PKG
phosphorylates intracellular target proteins, inhibits activation
of transcription factors facilitating cardiac growth such as
MEF-2 and GATA-4, and
impairs DNA as well as protein
synthesis. In addition, PKG
inhibits endothelin-1 (ET-1) and
angiotensin II (Ang II) mediated
cardiac hypertrophy. Another
relevant pathway of CNP signaling involves binding to NPRC that leads to activation of
inhibitory G protein (Gi) and
subsequent inhibition of adenylate cyclase (AC) at the Gprotein coupled receptor
(GPCR)
J Mol Med (2007) 85:797–810
and cardiac dysfunction. These effects are mediated by an
activated renin–angiotensin–system, endothelin-1 (ET-1),
and enhanced sympathetic tone.
The NP play an important role in counterbalancing the
effects of hypertrophic stimuli such as angiotensin II, ET-1,
vasopressin, and aldosterone on pathological cardiac growth.
While the role of NPR-A has been analyzed in several in vitro
as well as animal models, fewer data are available linking
CNP/NPR-B signaling to the regulation of cardiac growth and
hypertrophy (Fig. 1). A recent study by Tokudome et al. [82]
demonstrated that CNP was able to reduce basal as well as
ET-1 stimulated protein synthesis in cardiomyocytes. In
parallel, a significant reduction in the expression levels of
hypertrophy-associated genes, transcriptional activity of
MEF2 and GATA4, diminished ANP secretion and Ca2+/
calmodulin-dependent kinase II activity were found. Conversely, the antihypertrophic properties of CNP were blunted
by ET-1 involving a protein kinase C (PKC)-depending
mechanism [66]. In isolated adult rat cardiomyocytes, the
stimulation of NPR-B with CNP was as potent as ANP or
BNP in inhibiting angiotensin II-induced hypertrophy [83].
This antihypertrophic action was mediated via cGMP, as the
effect was abolished by either blocking the GC-coupled NPR
with HS-142-1 or inhibiting the cGMP-dependent PK.
Despite conflicting data regarding a possible role of NPR-C
in mediating antiproliferative effects of CNP, specific NPR-C
ligands did not mimic the CNP effect in these cells [33, 84].
It seems that, similar to its mode of action in the
vasculature, CNP/NPR-B signaling plays a paracrine role in
J Mol Med (2007) 85:797–810
the heart, where endothelial cells and fibroblasts secrete
CNP that targets NPR on cardiomyocytes, fibroblasts, and
vascular smooth muscle cells [85]. In fact, synthesis and
secretion of CNP from cardiac fibroblasts was recently
demonstrated in cultured rat fibroblasts [66]. Here, the
production of CNP was stimulated by TNF-β1, basic
fibroblast growth factor, and ET-1, and CNP was able to
suppress hypertrophy and DNA synthesis in the fibroblasts
in a cGMP-dependent mode.
In addition to its antihypertrophic properties, NPR-B has
been shown to mediate pro-apoptotic effects in neonatal
cardiomyocytes [86]. The stimulation of apoptosis in these
cells was also cGMP-dependent and antagonized by ET-1.
In contrast to mice lacking NPR-A, which are characterized
by cardiac hypertrophy, NPR-B knockout mice did not
show any signs of heart hypertrophy or fibrosis. As
mentioned above, these mice have a reduced life expectancy that limits a more comprehensive cardiovascular
phenotyping; thus, it cannot be truly inferred from this
model that NPR-B does not possess antihypertrophic effects
in the heart in vivo.
Despite the fact that the nonmyocyte population in the heart
constitutes the predominant localization of NPR-B [87],
transgenic rats expressing the dominant negative receptor
mutant (NPR-BΔKC) developed significant cardiac hypertrophy that could be attributed to an increased size of single
cardiomyocytes without evident fibrosis (Fig. 2) [23].
Echocardiographic and histological analysis in these animals
revealed concentric left ventricular hypertrophy which
aggravated with age. However, no left ventricular dysfunction was observed in the transgenic rats up to 1 year of age.
Furthermore, when cardiac hypertrophy was challenged by
chronic volume overload, the hypertrophy was enhanced in
the transgenic rats, and deterioration of left ventricular
contractility was blunted in these animals. Because the blood
pressure was unchanged in the NPR-BΔKC rats compared
to wild-type rats, these results suggest a role for NPR-B in
cardiac growth under both physiological and pathophysiological conditions. The lack of significant fibrosis was
surprising, as CNP has been demonstrated to have an
antiproliferative effect on fibroblasts, NPR-B is the predominant natriuretic receptor in cardiac fibroblasts, and cardiomyocytes mainly express NPR-A. A possible explanation for
this phenomenon is the lack of complete disruption of NPRB signaling in the transgenic rats. The remaining baseline
receptor activity might be sufficient to prevent cardiac
fibrosis in these animals and could also limit the extent of
cardiac hypertrophy, meaning the antihypertrophic effect of
NPR-B signaling might be underestimated in this model.
Given that antihypertrophic and antiproliferative effects
of CNP via activation of NPR-B have been demonstrated in
several in vitro experiments and in NPR-BΔKC transgenic
rats, these features could be of potential clinical relevance
803
in the context of cardiac remodeling. As a study by Soeki et
al. [88] demonstrated, when infused for 2 weeks after an
experimental myocardial infarction in rats, CNP prevented
cardiac hypertrophy and dysfunction as determined by
attenuated left ventricular enlargement and preserved left
ventricular contractility and relaxation. These effects were
observed using a CNP dose that did not affect the blood
pressure. The effect on both cardiomyocytes and cardiac
fibroblasts contributed to this phenotype, as the hearts were
characterized by a reduced cardiomyocyte cross-sectional
area, reduced levels of fibrosis markers collagen I, collagen
III, and fibronectin along with lower collagen volume
fraction compared to the vehicle-treated animals. Interestingly, the left ventricular CNP mRNA levels showed a
dramatic upregulation 3 days after coronary ligation and
returned to baseline levels within 3 weeks, suggesting a
paracrine compensatory role for CNP/NPR-B signaling
under these pathophysiological conditions.
In addition to cardiac remodeling after myocardial
infarction, a possible involvement of NPR-B has been
analyzed in the pathogenesis of diabetic cardiomyopathy
[89]. The regulation of cardiac NPR-B was assessed in two
different models of diabetes in mice resembling either type
I (streptozotocin-treated mice) or type II diabetes (ob/ob
mice). In both models, cardiac NPR-B mRNA was
increased compared to control mice, while NPR-A and
NPR-C mRNA levels did not change, suggesting that
upregulation of NPR-B may counteract the development
of cardiac fibrosis under these conditions. In addition,
chronic CNP infusion ameliorated the development of
pulmonary hypertension in monocrotaline-treated rats
without affecting systemic blood pressure [90]. The
reduction in right ventricular pressure and hypertrophy
was accompanied by suppressed inflammatory response,
blunted vascular remodeling, and improved survival
compared to vehicle-treated animals. The possible compensatory role for CNP/NPR-B signaling in states of
cardiac hypertrophy and heart failure is further underlined
by a recent clinical report demonstrating that the myocardium is a site of CNP production in chronic heart failure
[91]. Taken together, the close proximity of ligand and
receptor-expressing cells points towards a paracrine role of
the CNP/NPR-B signaling mechanism in controlling
cellular growth and hypertrophy in the heart.
Effects on myocardial function
Positive effects of CNP on cardiac contractility, lusitropy,
chronotropy, and dromotropy have been described in several
studies using intact animals, isolated hearts, cardiac muscle
preparations, and isolated cardiomyocytes [92], although
conflicting data characterizing the effect of CNP on cardiac
function have been published. When CNP was adminis-
804
J Mol Med (2007) 85:797–810
Fig. 2 Morphological analysis
of cardiac hypertrophy and fibrosis in 6-month-old NPRBΔKC transgenic animals. a
Exemplary hearts and hematoxylin and eosin-stained cardiac
cross and longitudinal sections
of wild-type (WT) and transgenic (TG) rats. b Quantification of
cardiomyocyte (CMC) diameter
and c cross-sectional area
revealed hypertrophy of cardiomyocytes in NPR-BΔKC transgenic animals. **p<0.01 vs WT
control; n=5 hearts, and 200
images per genotype for statistical analysis. d Masson’s trichrome staining of cross and
longitudinal left ventricular sections showed no increase in
interstitial or perivascular fibrosis in NPR-BΔKC transgenic
(TG) rats compared to wild-type
(WT) controls ([23], Copyright
(2006) National Academy of
Sciences, U.S.A.)
tered intravenously in dogs, it slightly reduced end-diastolic
volume and pressure along with an approximately 15-fold
increase in circulating CNP levels [93]. In addition, cGMP
levels increased significantly, suggesting a NPR-B mediated effect. These cardiac actions were abolished, however, in
dogs with pacing-induced heart failure, in parallel with
blunted cGMP response, suggesting an impaired receptor
signaling under the conditions of heart failure.
CNP perfusion of isolated mouse hearts showed a
biphasic regulation with an initial increase in contraction
and relaxation rates, increased ventricular pressure, and
reduced relaxation time, followed by a slow decline of
these hemodynamic parameters to baseline levels [94, 95].
The positive inotropic and lusitropic effect in this model
involved the generation of cGMP and the subsequent
activation of cGMP-dependent protein kinase-I (PKG I),
phosphorylation of phospholamban and intracellular Ca2+
release from the sarcoplasmatic reticulum. Concurrently,
CNP caused a dose-dependent increase in atrial and
ventricular contractility and slightly enhanced sinus rate in
J Mol Med (2007) 85:797–810
isolated blood perfused dog heart preparations, involving
the activation of guanylyl cyclase-coupled receptors [96].
Although it could be speculated that at higher concentrations CNP binds to and stimulates NPR-A, the cardiac
responsiveness to CNP was even enhanced in NPR-A
knockout mice [95], possibly due to enhanced expression of
PKG I in these mice. In isolated mouse cardiomyocytes,
CNP stimulation increased cell shortening and systolic Ca2+
levels in parallel with an accelerated Ca2+ decay [94]. In
contrast, negative inotropic properties of CNP, mediated via
NPR-B/PKG activation, have been described in rat papillary muscle [97] despite an increase in relaxation rate, in
neonatal rat [98] and mouse cardiomyocytes [99], and
rabbit ventricular cardiomyocytes [100]. Furthermore, the
cGMP analog 8Br-cGMP reduced contractile force in
mouse atrial and ventricular myocardial preparations
[101]. In addition, in intact animals such as conscious
sheep, a significant reduction in cardiac output after CNP
infusion has been shown, while the arterial blood pressure
remained unchanged in this experimental design [102]. In
accordance with a possible negative inotropic CNP effect,
NPR-BΔKC transgenic rats had enhanced ventricular
contractility and showed a blunted left ventricular dysfunction after chronic volume overload [23]. However, the
activation of NPR-C might also contribute to the observed
negative inotropic effect of CNP. In two recent studies using
either mouse sinoatrial or bullfrog atrial cells, CNP inhibited
2+
L-type Ca
current, thereby, slowing the pacemaker
activity of these cells [103]. This effect was mimicked by
either cANP, a NPR-C specific ligand, or a Gi protein
activator but was not blocked by the NPR-A/-B antagonist
HS-142-1, suggesting that NPR-C is the predominant
receptor mediating CNP effects on cardiac chronotropy.
Effects on the autonomic nervous system
CNP is the major natriuretic peptide in the central nervous
system and cerebrospinal fluid [104]. Large or intermediate
NPR-B expression was observed in limbic cortex, neocortex olfactory bulb, hippocampus, amygdala, preoptic–
hypothalamic neuroendocrine circuits, motor nuclei of
cranial nerves, and in brainstem nuclei controlling autonomic function, suggesting a prominent physiological role
of CNP and NPR-B in the central control of cardiovascular
homeostasis [43, 105]. Several studies demonstrated the
regulatory properties of NP by facilitating vagal or
suppressing sympathetic neurotransmission. Upon injection
into the lateral cerebroventricular area of the brain, CNP
enhanced pancreatic fluid and protein secretion through a
vagal pathway in rats [106]. Although injection of supraphysiological dosages of CNP may not exclude coactivation of NPR-A, this study suggests involvement of NPR-B
in central regulation of autonomic nerve activity. In
805
addition, ANP was able to mediate sympatho-inhibitory
responses when injected locally into the anterior hypothalamic nucleus of conscious mice; however, CNP was not
applied in this study [107]. When administered intravenously into conscious sheep, CNP, along with ANP and
BNP, has been reported to enhance reflex bradycardia as
induced by repetitive intravenous injection of a serotonin
agonist [108]. Local positive chronotropic effects were
found when CNP was injected directly into the sinus node
artery of anesthetized dogs [109]. This was supported by
another study on isolated, blood-perfused canine right atrial
and left ventricular preparations, where CNP was found to
increase myocardial contractile force along with sinus rate
in a cGMP-dependent manner [96]. In isolated guinea pig
right atrial vagal nerve preparations, both BNP and CNP
facilitated heart rate response to vagal-induced bradycardia
in a cGMP-dependent manner, further suggesting a local
effect of CNP on cardiac vagal neurotransmission [110].
Because regulatory effects on autonomic nerve activity
have been observed for ANP, BNP, and CNP, effects
mediated by either NPR-A or -B were difficult to
distinguish. Interestingly, NPR-BΔKC transgenic rats demonstrated an increased heart rate in parallel with enhanced
sympathetic nerve activity [23], suggesting that the cardiac
phenotype of NPR-BΔKC transgenic rats might also be a
result of reduced CNP-mediated inhibition of the autonomic
nervous system. In addition, NBR-BΔKC rats displayed
elevated renin levels (own unpublished data), most likely as
a result of enhanced sympathetic nerve activity in this
model. This function seems to be specific for NPR-B, as it
has not been found in genetic mouse models with altered
ANP or NPR-A signaling [111, 112]. Further studies are
necessary to determine whether CNP-mediated and NPR-B
play an exclusive role in controlling cardiovascular functions by regulating the autonomic nervous system, including regulation of renin secretion, and to distinguish direct
effects on cardiomyocytes from indirect effects involving
modulation of vagal and sympathetic nerve activity.
Contribution of genetic alteration of CNP and NPR-B
to cardiovascular disease
Loss-of-function mutations in the human NPR-B have been
reported in patients with acromesomelic dysplasia, type
Maroteaux [113]. Given the cardiovascular effects described in this review, however, information on genetic
alterations of CNP and NPR-B and their association with
cardiovascular diseases in humans are of great interest.
Ono et al. [114] performed an association study linking
mutations in the CNP gene to essential hypertension in a
Japanese population using data from 2006 subjects. Out of
four genetic variants found in the CNP gene, a G2628A
806
polymorphism in the 3′-untranslated region was significantly associated with essential hypertension. The odds ratio
was even higher when only a subpopulation of subjects age
65 and younger was studied. This study suggests that this
CNP gene variant may contribute to development of
hypertension. Rehemudula et al. [115] unraveled the
structure of NPR-B and were able to find a CA/GT
microsatellite repeat localized to intron 2 approximately
150 bp downstream of the exon-intron junction. Although
only 103 patients with hypertension and 101 normotensive
control subjects were enrolled in this study, a GT(11)
microsatellite repeat distribution was significantly associated with essential hypertension in this Japanese population.
Other mutations found in the NPR-B gene, such as a
C2077T polymorphism in exon 11 or a 9-bp insertion/
deletion in intron 18, were neither associated with essential
hypertension nor myocardial or cerebral infarction [116–
118]. These data show that mutations in CNP or NPR-B
genes might be related to essential hypertension, however,
are limited to some extent by sample size and ethnic
background of the study population. To draw a more
general conclusion, it would be of great interest to confirm
these findings in larger study populations and diverse ethnic
groups. The findings from these genetic studies also seem
to contradict findings from the genetic mouse and rat
models altering NPR-B signaling, where no effect on blood
pressure could be demonstrated. This raises the question of
whether compensatory mechanisms are activated in genetic
animal models counteracting the loss of NPR-B signaling,
such as increased NPR-A signaling by increased ANP and
Fig. 3 Summary of cardiovascular functions of C-type natriuretic peptide (CNP) and
natriuretic peptide receptor B
(NPR-B). Recent findings link
NPR-B signaling to control of
cardiomyocyte growth, involving either direct cardiac effects
or regulatory effects on the
autonomic nervous system. Asterisk CNP possesses affinity to
NPR-B and -C. Some of the
described effects cannot be
clearly attributed to signaling of
either receptor, or there are
conflicting data reported in the
literature
J Mol Med (2007) 85:797–810
BNP plasma concentrations, or whether the finding from
the genetic studies cannot be generalized due to methodical
restriction. Further studies are necessary to fully elucidate
and understand the role of genetic alterations of CNP or
NPR-B in cardiovascular diseases.
Conclusion
CNP and NPR-B signaling play an important regulatory
role in the cardiovascular system (Fig. 3). Significant
progress has been made to understand underlying mechanisms, accelerated by recent publications of genetic mouse
and rat models. Although CNP and NPR-B null mice
suggest a major role of NPR-B signaling in endochondral
ossification and development of female reproductive organs,
the first in vivo evidence of cardiovascular functions of
NPR-B was derived from transgenic rats overexpressing a
dominant negative mutant of the receptor. These rats
demonstrated cardiac hypertrophy, along with increased
heart rate and sympathetic nerve activity. In contrast to ex
vivo data, none of the genetically engineered animal models
had elevated blood pressure, suggesting that CNP and NPRB are not involved in long-term blood pressure regulation
and that the observed cardiac hypertrophy is not a result of
elevated blood pressure. Further research is needed to
distinguish direct effects on cardiomyocytes from effects
mediated by modulation of heart rate and autonomic nervous
activity. Pharmacological approaches employing available
genetic mouse and rat models as well as organ specific
J Mol Med (2007) 85:797–810
deletion of NPR-B signaling in the heart and brain will be
necessary. Moreover, other cardiovascular functions of CNP
may be mediated in part by coactivation of NPR-C and will
need to be further elucidated employing animal models
lacking NPR-B signaling, including regulation of vascular
smooth muscle cell proliferation during vascular wound
repair and atherosclerosis, cardiac performance and remodeling, and the beneficial anti-inflammatory and antiproliferative effects of CNP in primary pulmonary hypertension.
These questions are of fundamental interest not only to
further understand the pathophysiological significance of
CNP and NPR-B, but also to identify NPR-B as potential
pharmacological target for treatment of cardiovascular
diseases.
Acknowledgements This work was supported by a grant from
Deutsche Forschungsgemeinschaft (BA 1374/14-1). The authors
would like to acknowledge the critical review and discussion of this
manuscript by Dr. Daniel Schwartz (NHLBI, NIH).
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DOI 10.1007/s00109-007-0173-6
REVIEW
Involvement of autophagy in viral infections: antiviral
function and subversion by viruses
Lucile Espert & Patrice Codogno &
Martine Biard-Piechaczyk
Received: 19 December 2006 / Revised: 31 January 2007 / Accepted: 12 February 2007 / Published online: 6 March 2007
# Springer-Verlag 2007
Abstract Autophagy is a cellular process involved in the
degradation and turn-over of long-lived proteins and organelles,
which can be subjected to suppression or further induction in
response to different stimuli. According to its essential role in
cellular homeostasis, autophagy has been implicated in several
pathologies including cancer, neurodegeneration and myopathies. More recently, autophagy has been described as a
mechanism of both innate and adaptive immunity against
intracellular bacteria and viruses. In this context, autophagy has
been proposed as a protective mechanism against viral infection
by degrading the pathogens into autolysosomes. This is
strengthened by the fact that several proteins involved in
interferon (IFN) signalling pathways are linked to autophagy
regulation. However, several viruses have evolved strategies to
divert IFN-mediated pathways and autophagy to their own
benefit. This review provides an overview of the autophagic
process and its involvement in the infection by different viral
pathogens and of the connections existing between autophagy
and proteins involved in IFN signalling pathways.
Keywords Autophagy . Virus . Interferon . Virophagy
LUCILE ESPERT
received her Ph.D. in molecular
and cellular biology from the
University of Sciences in
Montpellier, France. She is
currently a postdoctoral research
fellow in the laboratory UMR
5236 CNRS UM1, UM2 at the
Institute of Biology
(Montpellier). Her research
interests include the characterization of the role of autophagy
in viral infections.
MARTINE BIARD-PIECHACZYK
received her Ph.D. in immunology from the University of
Montpellier 1, France. She is
presently a senior researcher in
the laboratory UMR 5236
CNRS UM1, UM2 at the Institute of Biology (Montpellier).
Her research interests currently
focus on the mechanisms
leading to immunodeficiency
during HIV-1 infection.
Introduction
Cellular homeostasis requires a highly regulated equilibrium
between protein synthesis and proteolysis, and its deregulation
L. Espert : P. Codogno : M. Biard-Piechaczyk (*)
CPBS, UM1, UM2, CNRS, Institut de Biologie,
4, Bd Henri IV, CS69033,
34965 Montpellier Cedex 2, France
e-mail: [email protected]
P. Codogno
INSERM U756, Faculté de Pharmacie, Université Paris-Sud XI,
92296 Châtenay-Malabry, France
has been implicated in several pathologies. Two major pathways are involved in the degradation of macromolecules in
eukaryotes: the ubiquitin/proteasome pathway and autophagy.
The former is involved in the constitutive degradation of shortlived proteins, maintaining a continuous protein turn-over in the
cell [1]. Autophagy, which literally means “to eat oneself”, is
necessary for the lysosomal degradation and recycling of
long-lived proteins and entire organelles [2]. This process has
been observed in all eukaryotes and is morphologically
identical in plants, yeasts and animals. Autophagy can be
classified into at least three different types: macroautophagy,
812
microautophagy and chaperone-mediated autophagy (CMA).
The last one is the only autophagic process that is not
conserved through evolution and exits only in higher
eukaryotes. The first CMA substrate identified was ribonuclase A and a particular motif in this protein, KFERQ, was
shown necessary for its selective degradation. This motif is
recognised by the chaperone protein Hsc70 and targeted to
lysosomal membranes where Hsc70 interacts with the
lysosomal membrane protein (lamp) type 2a. The substrates
are then translocated to the lysosomal lumen where they are
degraded. It is now known that all the CMA substrates contain
a KFERK-like motif, which is recognised by the hsc70. This
motif is composed of a basic (K, R), an acidic (D, E), a
hydrophobic (F, I, L, V), and another basic or hydrophobic
amino acid and is flanked by a Q on either side [3].
Microautophagy is characterised by the invagination of the
lysosomal membrane that sequesters cytoplasmic constituents
and, subsequently, degradation of the sequestered material.
Macroautophagy is the major lysosomal route for the turnover
of cytoplasmic constituents, and will hereafter be referred as
autophagy. This process begins with an engulfment event of
portions of the cytosol into a characteristic double-membrane
vacuole, called the autophagosome. After maturation, autophagosomes fuse with lysosomes for the degradation of the
sequestered material by lysosomal hydrolases. This last event
allows the recycling of the degraded constituents [4, 5].
Autophagy is a highly regulated mechanism involving
specific genes called Atg (autophagy-related genes). Although this process has been identified for more than
40 years, the first understandings of its molecular mechanisms dated from about 10 years, with the discovery of Atg
genes in yeast and orthologs in humans [6]. Interestingly, it
has been shown recently that the ubiquitin/proteasome
pathway and autophagy are connected. Indeed, Atg7
knock-out mice present an accumulation of ubiquitinated
aggregates. This observation suggests that, in addition to
the proteasome pathway, ubiquitinated proteins can be
removed by autophagy [7]. Moreover, it may also reflect
a compensatory mechanism in the absence of autophagy.
Even if the origin of the lipids that compose autophagosomes has been an aspect extensively studied since the
discovery of autophagy, this question is still hotly debated
[8]. The first studies provided evidence that the source of
autophagosomal membranes was both the Golgi and the
endoplasmic reticulum and, at present, the endoplasmic
reticulum is the most probable one [9]. Another possibility
is that the membranes are unique or formed de novo [10].
Thus, it has been proposed that, in mammals, autophagosomes derived from a unique membrane of unknown origin,
called the phagophore. Furthermore, a novel structure
necessary for autophagosome formation, called the preautophagosomal structure (PAS), has been identified in
yeast [11].
J Mol Med (2007) 85:811–823
According to its essential role in cellular homeostasis,
autophagy has been implicated in several pathologies
including cancer, neurodegeneration and myopathies [12].
More recently, autophagy has been described as a mechanism of innate immunity against intracellular pathogens
[13], and its implication in major histocompatibility
complex (MHC) class II antigen presentation extends its
function in adaptive immunity [13–15]. The term immunophagy has recently been proposed by Deretic [13] for the
specialised role of autophagy in immunity. A new field of
investigation is now emerging about the role of autophagy
in viral infections. Besides its role as an intracellular host
defence pathway against viruses, autophagy can also be
used by the virus for its own profit to replicate more efficiently
in the cells, or to control cell survival [16]. The term
virophagy could be used to define the relationships that
exist between autophagy and viral infections. In this paper,
presented are the different molecular steps of the autophagic
process, its implication in both cell survival and cell death
and the relationships that are known between autophagy and
viral infections.
Overview
Molecular mechanisms of the autophagic process
In all eucaryotes, autophagy occurs as a house-keeping
mechanism in normal growing conditions. In animal cells,
it is subjected to suppression or further induction in
response to different stresses, starvation, specific hormones
or other stimuli [17–20]. The discovery of Atg genes has
been essential in the molecular understanding of this
process. A major challenge in the autophagy field is now
to decipher the signalling pathways that act downstream the
initial autophagy induction signals. Basically, the autophagic process can be divided into different steps: activation,
autophagosome formation, targeting to and fusion with
lysosomes and breakdown [21] (Fig. 1).
Activation
In mammalian cells, the autophagic Beclin 1 protein
(ortholog of yeast Atg6) functions as part of a class III
phosphatidylinositol 3-kinase (PI3K) complex and plays a
crucial role in the early steps of autophagosome formation
[22, 23]. The mammalian class III PI3K/Beclin 1 complex
has its yeast counterpart that consists in the association of
Vps34 (the ortholog of class III PI3K) with the autophagic
proteins Atg6 and Atg14 [22]. The mammalian counterpart
of Atg14 has not been identified so far. Recent data suggest
that other proteins belong to this complex and thus regulate
the outcome of the signalling via class III PI3K. Indeed,
J Mol Med (2007) 85:811–823
813
Fig. 1 The autophagic process
and its regulation. Here, presented are two major pathways
that regulate autophagy triggering. The mTOR pathway inhibits autophagy in response to
Class I PI3K and amino acids.
The eIF-2α kinases are positive
regulators of autophagy in response to starvation, ER stress
and viral infection. Autophagy
triggering is dependent on the
Class III PI3K signalling and on
two conjugation systems. First, a
pre-autophagosome is formed
and sequesters cytoplasmic material. Its completion leads to
autophagosome formation that
fuses with lysosome to form
autolysosome where the sequestered material is degraded
Bcl-2 can negatively regulate the activation of autophagy
through its interaction with Beclin 1 [24]. Recently, an
activator of autophagy, called UVRAG (UV irradiation
resistance-associated gene), has been identified to be part of
the PI3K/Beclin 1 complex and to represent an important
signalling checkpoint in autophagy and tumour-cell growth
[25].
Autophagosome formation
The autophagic process depends on two ubiquitin-like
conjugation systems essential for autophagosome formation
[26] (Fig. 2). In general, three different enzymes are
required for the process of conjugation: an E1-activating
enzyme, an E2-conjugating enzyme and an E3 ligase
enzyme. The first conjugation system mediates formation
of the conjugate Atg12/Atg5. Atg12 is activated by the E1like enzyme Atg7, and then Atg12 is transferred to the E2like enzyme Atg10. Finally, Atg12 is covalently linked to a
specific lysine of Atg5. No E3-like enzyme has been
discovered to date. Then, the conjugate Atg12/Atg5
interacts non-covalently with Atg16L (Atg16 in yeast) to
trigger homo-oligomerisation, leading to the formation of a
macromolecular complex of approximately 800 kDa
(350 kDa in yeast) necessary for the formation of
autophagosomes. This structure is associated with the outer
side of the autophagosomes in formation and dissociates
from membranes before the autophagosome completion.
The second conjugation system is original because it
consists in the conjugation of a protein to a lipid, resulting
in the formation of the Atg8/PE (phosphatidyl-ethanolamin)
conjugate. Atg8 is a soluble cytoplasmic protein that must
be first proteolysed by Atg4 to enter the conjugation system.
After maturation by Atg4, Atg8 is activated by the E1-like
enzyme Atg7, and then transferred to the E2-like enzyme
Atg3. Finally, Atg8 is covalently linked to PE. This
conjugate is present in both sides of the autophagosomes
and seems fundamental for its completion. After autophagosome completion, the Atg8 molecules present at the
cytoplasmic face of this structure are recycled after cleavage
by Atg4 and the remaining Atg8 proteins present in the inner
membrane of autophagosomes are degraded after fusion
with lysosomes. There are several orthologs of Atg8 in
mammalian cells: MAP-LC3, GATE-16 and GABARAP,
each representing a different subfamily [27]. Among these
proteins, MAP-LC3 is the best characterised one and
represents the most useful marker for autophagosome
identification [28, 29]. In addition, there are four mammalian orthologs of Atg4, called autophagins, but only
autophagin 1 (also called hAtg4B) cleaves specifically
Atg8 and its different mammalian orthologs [30, 31].
Interestingly, it appears that these two conjugation systems
are connected. Indeed, the Atg5/Atg12-Atg16 complex is
necessary for the formation of the second conjugate [32].
Moreover, overexpression of Atg10 facilitates the maturation of MAP-LC3, overexpression of Atg3 facilitates the
conjugation of Atg12 to Atg5, and excess amount of the
Atg12-Atg5 conjugate inhibits the MAP-LC3 maturation
[33].
In yeast, another complex is involved in autophagosome
formation, which requires the autophagic proteins Atg9 and
Atg2. Atg9, the only transmembrane autophagic protein so
814
J Mol Med (2007) 85:811–823
Fig. 2 Conjugation systems involved in autophagy. The first
conjugation system mediates the formation of the conjugate Atg12/
Atg5. Atg12 is activated by the E1-like enzyme Atg7, and then Atg12
is transferred to the E2-like enzyme Atg10. Finally, Atg12 is
covalently linked to a specific lysine of Atg5. Then, the conjugate
Atg12/Atg5 interacts non-covalently with Atg16 (Atg16L in mammals) to trigger homo-oligomerization leading to a macromolecular
complex necessary for the formation of autophagosomes. This
structure is associated with the outer side of the autophagosomes in
formation and dissociates from the membranes before the autophagosome completion. The second conjugation system results in the
formation of the Atg8/PE (phosphatidyl–ethanolamin) conjugate.
Atg8 (LC3 in mammals) is first proteolysed by Atg4, activated by
the E1-like enzyme Atg7, and then transferred to the E2-like enzyme
Atg3. Finally, Atg8 is covalently linked to PE. This conjugate is
present in both sides of the autophagosomes and seems fundamental
for its completion. The two conjugation systems are connected: The
Atg5/Atg12-Atg16 complex is necessary for the formation of the
second conjugate. Over-expression of Atg10 facilitates the maturation
of MAP-LC3, over-expression of Atg3 facilitates the conjugation of
Atg12 to Atg5, and excess amount of the Atg12-Atg5 conjugate
inhibits the MAP-LC3 maturation
far identified, is necessary for the formation of the PAS but
is absent in the mature autophagosomes [34]. mAtg9, the
human ortholog of the yeast Atg9 has been shown to traffic
between the Golgi and endosomes, suggesting an involvement of the Golgi complex in the autophagic pathway [35].
progression of the autophagic process [37, 38]. Fusion is
driven by assembly of pairs of membrane proteins called
SNAREs on each fusion partner [39]. Using bafilomycin A1,
an inhibitor of vacuolar H+ ATPase (V-ATPase), it has been
shown that acidification of the lumenal space of autophagosomes or lysosomes by V-ATPase is an important step for the
fusion between autophagosomes and lysosomes [40].
Targeting to and fusion with lysosomes
After completion, autophagosomes can fuse in yeast with the
vacuole, or in mammals with the lysosomes to form
autolysosomes, using the same molecular fusion machinery.
Before fusing with lysosomes, autophagosomes can also fuse
with endosomes to form amphisomes, making a direct
connection between the endo-lysosomal and autophagic
pathways [36]. Membrane fusion can be delineated into
different steps: tethering, docking and fusion of the
membrane bilayers. Tethering and docking events are
facilitated by multi-protein complexes, and the specificity
of the events is provided by GTPases from the Rab family. In
particular, the GTPase Rab7 is required for the final
maturation of late autophagic vacuoles and thus for the
Breakdown
After the fusion step with lysosomes, the cytoplasmic
material sequestered in autophagosomes is released in the
lysosomal lumen and subsequently consumed by hydrolases. This event requires the acidic pH of the lysosome to
efficiently break the inner membrane of the autolysosome.
In yeast, the involvement of the lipase Atg15, which is
delivered to the vacuole through the multivesicular bodies,
is required for vesicle breakdown [41]. After degradation of
the sequestered material, the constituents are recycled
through lysosomal transporters toward the cytosol. Recently,
the yeast protein Atg22 has been shown to mediate the
J Mol Med (2007) 85:811–823
efflux of leucine and other amino acids resulting from
autophagic degradation [42].
Signalling pathways involved in the regulation
of autophagy
The mammalian serine/threonine protein kinase target of
rapamycin (MTOR) is a key regulator that controls
autophagy triggering and is often called “the gate keeper
of the autophagic pathway” [43, 44]. This kinase senses
environmental changes to modulate autophagy. Several
proteins act positively or negatively on mTOR and, by this
way, influence the autophagic process. The cascade upstream mTOR includes class I PI3K and Akt, whereas the
phosphatase and tension homolog (PTEN) acts antagonistically to the class I PI3K to induce autophagy. Rapamycin, a
specific inhibitor of mTOR, activates autophagy whereas
amino acids are inhibitors of this process. However, the
precise mechanisms involved in the negative regulation of
autophagy by amino acids remain an open question.
Induction of autophagy by nutrient starvation is better
characterised in yeast than in mammals. Indeed, it has been
shown that under nutrient-rich conditions, the autophagic
protein Atg13 is highly phosphorylated in response to the
TOR kinase activation. This phosphorylation status provides a lower affinity of Atg13 for the autophagic serine/
threonine protein kinase Atg1, repressing autophagy. In
contrast, under nutrient-deficient conditions, or following
treatment with the TOR inhibitor rapamycin, Atg13 is
rapidly and partially dephosphorylated, resulting in an
interaction of high affinity with Atg1. In these conditions,
the autophagic activity is increased [45]. A putative
ortholog of the yeast Atg1, called ULK1, has been
identified in mammals. This protein seems to be involved
in the localization of the mAtg9 [46]. It is worth noting that
Atg13 is not conserved in mammals.
Finally, other signalling pathways independent of mTOR
have been involved in the regulation of autophagy [47, 48].
Other regulators of the autophagic pathway are the
eIF2α kinases. They belong to an evolutionarily conserved
serine/threonine kinase family that regulates stress-induced
translational arrest. It has been demonstrated that the yeast
eIF2α kinase GCN2 and the eIF2α-regulated transcriptional transactivator GCN4 are essential for starvation-induced
autophagy [49]. It is worth noting that GCN2 is conserved
in mammals, suggesting that this protein may have a similar
role in higher eukaryotes. Recently, endoplasmic reticulum
stress induced by poly-glutamine 72 repeat (polyQ72) has
been shown to induce autophagy after activation of the
eIF2α kinase PKR-endoplasmic reticulum-related kinase
(PERK) activation [50]. In contrast, another study has
reported that the autophagic process triggered in response
to endoplasmic reticulum stress is PERK-independent [51].
815
The mammalian interferon (IFN)-inducible eIF2α kinase
PKR (protein kinase RNA-activated) plays also an important role in triggering autophagy [49]. As PKR is a protein
essential in cellular response to viral infections, this point
will be discussed in more details afterwards [52].
Dual role of autophagy in cell survival and cell death
Autophagy is a cellular mechanism essential for homeostasis. It has been extensively studied for its ability to maintain
cells alive in response to starvation. This theory has been
confirmed since then by studies demonstrating increased
cell death in cells or organisms lacking gene products
essential for autophagy [53]. A good example to illustrate
this aspect is the demonstration of the role of autophagy
during the early neonatal starvation period. Indeed, at birth,
the placental nutrient supply is interrupted, and neonates
face starvation until the first feeding by milk. It has been
shown that autophagy is up-regulated in various tissues
during this period, and those mice incompetent for
autophagy (Atg5 knock-out mice) die within 1 day after
birth [54]. In addition, autophagy has been shown to play a
critical role in maintaining cellular bioenergetics and
survival during growth factor deprivation in cells incompetent for apoptosis [55]. Furthermore, it has been shown that
inhibition of autophagy triggers apoptotic cell death under
conditions of nutrient depletion [56].
Despite its role in cell survival, an extensive body of
literature highlights the fact that autophagy can also be
considered as a cell death pathway (type II programmed cell
death, PCD). Autophagic cell death can be distinguished from
apoptosis (type I PCD) by morphological criteria, i.e. the
presence of autophagosomes in dying cells. Cell death with
autophagic features can occur in cells lacking critical apoptosis
executioners, indicating that autophagy can compensate for
defect apoptosis [57]. Autophagic cell death has also been
described after treatment with chemotherapeutic drugs [58].
However, cell death, characterised by hallmarks of both type I
PCD and type II PCD, is frequently observed; thus, making a
clear-cut difference between them is difficult [59–62].
The autophagic protein Beclin 1, which has first been
identified as a Bcl-2 interacting protein, has haplo-insufficient tumour suppressor functions. Its gene mapped to a
tumour susceptibility locus on chromosome 17q21 that is
monoallelically deleted in 40 to 75% of cases of sporadic
breast, ovarian and prostate cancers [63]. It has been
recently shown that Bcl-2 negatively regulates Beclin 1dependent autophagy and Beclin 1-dependent autophagic
cell death, connecting the two types of PCD [24]. A new
link between these death processes has been described by
Crighton et al. [64], as a p53 target gene encoding a
lysosomal protein, called DRAM (damage-regulated
autophagy modulator), is also an inducer of autophagy.
816
As autophagy appears essential in the cell fate between
life and death, it is not surprising that it has been implicated
in numerous pathologies, including neurodegenerative
diseases, infectious diseases and cancer. This dual role has
been very well described in several reviews [44, 65–67].
J Mol Med (2007) 85:811–823
extended to uninfected adjacent tissues where autophagy
protects cells from death. In this case, autophagy would
play a pro-survival role by blocking pathogen spread and
bystander cell death [76]. At present, no other studies
connecting plant viruses and autophagy have been done.
Autophagy and viral infections in mammals
Autophagy and viral infections
Both bacteria and viruses can be targeted to autophagic
degradation [67, 68]. The best-characterised examples for
autophagy of pathogens, also termed xenophagy, are
engulfment of Mycobacterium tuberculosis in phagosomes
[69], trapping of cytosolic group A Streptococci in
autophagosomes [70] and immune escape by Shigella
[71]. Moreover, it has been shown recently that Staphylococcus aureus can use autophagy for its replication before
inducing an autophagic host cell death [72].
In this review, we focus on the role of autophagy in the
replication of different viruses.
Autophagy is now recognised as a mechanism of
immunity against microbes that invade eukaryotic cells.
On one hand, evidences indicate that autophagy is involved
in the delivery of cytosolic antigens to the MHC class II
pathway. Indeed, antigens are processed before binding to
MHC class II molecules within endosomal and lysosomal
compartments of antigen-presenting cells for subsequent
presentation to T cells [73]. Autophagy is also involved to
digest endogenously synthesised viral proteins, allowing
their processing for MHC II presentation and thus connecting autophagy with adaptive immunity [14]. On the other
hand, the autophagic process allows the sequestration of
pathogens inside the autophagosomes, leading to their
destruction by lysosomes.
Beside its important role in the immune response against
pathogens, very well described by Schmid et al. in [15],
autophagy plays also a direct role in the life cycle of
viruses.
Autophagy and viral infection in plants
In plants, the hypersensitive response (HR) is a complex, early
defence response that causes necrosis and cell death at the
infection site to restrict the growth of a pathogen. The HR is
also thought to deprive the pathogens of a supply of food and
thus to confine them to the initial infection site [74]. In
addition, the HR could regulate the defence responses of the
plant in both local and distant tissues [75].
It has been suggested that autophagy acts as an antiviral
mechanism since Beclin 1- or Atg7-silenced plants present
an accumulation of tobacco mosaic virus (TMV) at
infection sites. Interestingly, induction of autophagy is not
only restricted to the TMV infection sites but is also
Autophagy has been first proposed as a protective mechanism against viral infection by degrading the pathogens in
autolysosomes. Its antiviral role is strengthened by the fact
that IFN signalling pathways are involved in autophagy
induction. Indeed, IFNs are a family of multifunctional
secreted cytokines that have been characterised by their
ability to interfere with virus infection and replication [77].
However, several viruses have evolved strategies to divert
IFN-mediated pathways and autophagy to their own benefit
[16].
Table 1 summarised the different data available about the
connections between autophagy and viral infections in
mammals and higher plants.
Herpes simplex virus type I (HSV-1)
Herpes simplex virus type I (HSV-1) belongs to the herpes
virus family. It is a double-stranded DNA virus that resides
in a latent state in sensory neurons. During an attack, the
virus grows down the nerves and out into the skin or
mucous membranes where it multiplies, causing the clinical
lesions [78].
It is well characterised that the HSV-1 neurovirulence
ICP34.5 protein (infected cell protein 34.5) plays a crucial
role in viral infection by inducing the dephosphorylation of
the translation initiation factor eIF-2α, and thus negating
the PKR antiviral activity [79, 80]. In accordance with the
role of the PKR-eIF-2α pathway in the positive regulation
of autophagy, wild type HSV-1-infected cells do not present
any autophagosomes. However, HSV-1 mutants that do not
express ICP34.5 are able to trigger autophagy in infected
cells, leading to viral degradation. Moreover, the same
mutant viruses that infect cells in which PKR is not
expressed exhibit a normal viral replication, demonstrating
that ICP34.5 is able to block autophagy by inhibiting the
PKR antiviral activity in infected cells [81]. These results
demonstrate that HSV-1 has evolved strategies to counteract cellular antiviral functions [82].
Poliovirus and rhinoviruses
Poliovirus and rhinovirus belong to the picornavirus family.
They are lytic non-enveloped viruses whose RNA genome
is translated, replicated, and packaged in the cytoplasm of
infected cells. The poliovirus, responsible for poliomyelitis,
J Mol Med (2007) 85:811–823
817
Table 1 Currently known relationships between viruses and the autophagic process
Virus family
Genome
Virus
Viral
protein
involved
Presence of
autophagosomes
Tobamovirus
ssRNA
TMV
?
+
Herpes virus
dsDNA
HSV-1
ICP34.5
–
Picornavirus
ssRNA
Poliovirus
Rhinovirus
2BC
and 3A
+
Retrovirus
ssRNA
HIV-1
Env
Togavirus
Parvovirus
ssRNA
ssDNA
Sindbis virus
B19
?
?
Coronavirus
ssRNA
MHV
?
Reovirus
Flavivirus
dsRNA
ssRNA
Rotavirus
Bovine Viral
Diarrhea Virus
NSP4
NS3
+ in bystander
CD4 T cells
? in infected
CD4 T cells
?
+
+
+
?
invades the nervous system, and the onset of paralysis can
occur in a matter of hours. Rhinoviruses are the most
common viral infective agents in humans, and a causative
agent of the common cold.
In poliovirus-infected cells, it has been observed, early
after infection, an accumulation of membranous structures
in the cytoplasm [83]. These structures are composed of
double-membranes and contain markers of the entire
secretory pathway, including the rough endoplasmic reticulum, the Golgi apparatus and lysosomes. This suggests
that these double-membrane structures originate from a
process analogous to the formation of autophagic vacuoles
[84]. More recently, MAP-LC3 and LAMP-1 have been
found localised in poliovirus- and rhinovirus-induced
vesicles in infected cells. More precisely, the co-expression
of two poliovirus-encoded proteins called 2BC and 3A
triggers MAP-LC3 and LAMP1 co-localization. Moreover,
inhibition of the autophagic process using siRNA directed
against the autophagic proteins LC3 and Atg12 decreases
both intracellular and extracellular virus yield. The authors
suggested that poliovirus and rhinovirus infection induce
the formation of autophagosome-like structures to serve as
Biological effect related
to autophagy
Effect of autophagy on the
viral replication level
Antiviral mechanism at
the infection site
Bystander uninfected cell
protection from death
Blockade of PKR-eIF-2α
signalling pathway:
inhibition of autophagy
Decreased viral replication
Autophagosomes as
membrane support for
viral RNA replication
Bystander uninfected
CD4 T cell death
Infected cells?
Antiviral role of Beclin 1
Autophagy triggering
resulting in infected cell
survival
Autophagosomes as sites
for viral replication
?
?
Blockade of the antiviral
action of autophagy by
ICP34.5, leading
to an increase in viral replication
Increased viral replication
Nothing is currently known about
the role of autophagy in the
replication of HIV-1
Decreased viral replication
Increased viral replication
Increased viral replication
?
Use of autophagy proteins for
the expression of viral proteins
membrane scaffolds for RNA replication and to inhibit their
maturation into degradative organelles [85]. Because of the
presence of both LC3 and the poliovirus capsid protein VP1
in extracellular structures adjacent to poliovirus-infected
cells, Jackson et al. speculate that viral particles may be
released by a non-lytic pathway using autophagosome-like
vesicles. It has also been suggested that autophagosomes,
which can become single-membraned upon maturation,
provide a mechanism for the non-lytic release of cytoplasmic viruses and possibly other cytoplasmic material [86].
Very recently, the Arf family of small GTPases, which
controls secretory trafficking, has been shown to associate
with the newly formed membrane structures used for viral
RNA replication after poliovirus infection [87].
Human immunodeficiency virus type I (HIV-1)
The human immunodeficiency virus type I (HIV-1) is a
member of the retrovirus family. HIV-1 infection usually
leads to progressive decline in functionality and number of
CD4 T lymphocytes, resulting in AIDS development [88].
As early as 1991, apoptosis has been proposed as a possible
818
mechanism responsible for CD4 T cell depletion in patients
infected with HIV-1, and an extensive body of literature
since then has supported this hypothesis [89]. In HIV-1infected patients, both infected and uninfected cells
undergo accelerated apoptosis, in vitro and in vivo.
However, HIV-1-induced apoptosis in bystander uninfected
immune cells is likely the major event leading to the
depletion of CD4 T lymphocytes since the degree of cell
loss largely exceeds the number of infected cells. Furthermore, the vast majority of T cells undergoing apoptosis in
peripheral blood and lymph nodes of HIV patients are
uninfected [90]. HIV-1 infection is mediated by the binding
of envelope glycoproteins (Env) to the receptor CD4 and a
co-receptor, mainly CCR5 or CXCR4. Notably, binding of
Env, expressed on HIV-1-infected cells, to CXCR4, triggers
apoptosis of uninfected CD4 T cells. Very recently, we have
demonstrated that, independently of HIV-1 replication,
Env-transfected or HIV-1-infected cells that express Env
at the cell surface induce autophagy and accumulation of
Beclin 1 in uninfected CD4 T lymphocytes, process
dependent on the presence of CXCR4. Moreover, autophagy
is a prerequisite to Env-induced apoptosis in uninfected
bystander T cells, and CD4 T cells still undergo an Envmediated cell death with autophagic features when apoptosis
is inhibited [62, 91]. To the best of our knowledge, these
findings represent the first example of autophagy triggered
through binding of viral envelope proteins on target cells,
without viral replication, and leading to apoptosis. At
present, we do not know the significance of Env-induced
Beclin 1 accumulation in CD4 T cell death and if autophagy
has a role in HIV-1-replication, and these points are
currently under investigation in the laboratory.
It has been shown that HIV-1 uses cytoplasmic late
endosomal structures, identified as MVBs, for its budding
step, suggesting that HIV-1 may use components of the
autophagic machinery for its replication cycle [92]. In
contrast, a recent study suggests that the initiating site for
constitutive HIV-1 assembly and release is not the endosomal structure but the plasma membrane [93]. Thus,
observation of HIV-1 particles in MVBs may be considered
as a cellular defence mechanism that restricts the HIV-1
replication cycle by degrading assembled virions.
Sindbis virus
The Sindbis virus, which provokes encephalitis, belongs to
the togavirus family. Its genome is a linear, single-stranded
RNA. Sindbis-infected apoptotic cell death is closely linked
to viral replication. Indeed, the antiapoptotic Bcl-2 protein
protects neurons from virus-induced cell death and
decreases viral replication in the central nervous system
[94]. To explore the role of Bcl-2 in this context, a yeast
two-hybrid screen has been performed and has permitted to
J Mol Med (2007) 85:811–823
identify the autophagic protein Beclin 1 as a Bcl-2
interacting protein in infected cells. The brains of mice
infected with a recombinant virus that expresses Beclin 1
show fewer Sindbis virus RNA-positive cells, fewer
apoptotic cells, and lower viral titers than the brains of
mice infected with recombinant viruses that express a
Beclin 1 protein deleted in its Bcl-2 interacting domain, or
Beclin 1 containing a premature stop codon. These results
provide evidence of an antiviral activity of the autophagic
protein Beclin 1 [95]. However, no further studies have
been done on the role of the entire autophagic process in
the reduction in Sindbis virus infection.
The human B19 parvovirus
B19 belongs to the parvovirus family whose genome is a
single-stranded DNA. It infects erythroid cells and causes
several diseases including aplastic crisis in patients with
haemolytic anemia, erythema infectiosum and hydrops
fetalis [96]. B19 infection induces cell cycle arrest at G1
and G2/M phases and apoptosis mediated by a viral nonstructural protein called NS1 [97]. Recently, it has been
shown that autophagy is induced in human parvovirus B19infected cells arrested in G2 phase, before they are
competent for viral replication, leading to infected-cell
survival. These results suggest that autophagy may benefit
B19 in allowing viral multiplication before cell collapse
[98].
Coronavirus
Coronaviruses are enveloped positive sense RNA viruses
that replicate entirely in the cytoplasm of cells. They are the
cause of many domesticated animal diseases and are
responsible for up to 30% of human colds. A new human
coronavirus has recently been identified as the causative
agent of the severe acute respiratory syndrome (SARS).
Murine hepatitis virus (MHV), which belongs to the
coronavirus family, is frequently used to study the
formation and function of the viral replication complexes.
These complexes present a punctate perinuclear localisation
and their number and size increase over the course of
infection. Moreover, MHV RNA replication occurs on
cytoplasmic double-membrane vesicles whose membranes
are issued from the rough endoplasmic reticulum [99]. The
co-localization of late endosomal proteins and LC3 with
viral RNA-replication proteins on these membranes argues
for their derivation from the autophagic pathway [99].
Importantly, the formation of autophagosome-like structures seems beneficial for MHV viral production. Indeed,
the yield of extracellular virus is diminished 1,000-fold in
clonal isolates of Atg5−/− mouse embryonic stem cells. In
addition, expression of ectopic Atg5 in these cells restores
J Mol Med (2007) 85:811–823
the wild-type yield [99]. These results demonstrate that
Atg5, which is crucial for the autophagic pathway, is
required for the production of infectious MHV virions. In
consequences, the autophagic pathway may be required for
the formation of double-membrane-bound MHV replication
complexes and, by this way, may significantly enhance the
efficiency of replication. However, the exact role of Atg5 in
this process is not fully determined. It would be interesting
to know if Atg5 benefits to MHV only by inducing the
formation of autophagic membranes or if it has a more
specific function in the viral replication cycle.
In SARS-infected cells, the early formation and accumulation of typical double-membrane vesicles, which
probably carry the viral replication, have been observed
by electron microscopy. Opposite to what was described for
MHV, morphological and labelling studies argued against
the previously proposed involvement of the autophagic
pathway as the source for the vesicles, and instead
suggested the endoplasmic reticulum to be the most likely
donor of the membranes that carry the SARS replication
complex [100].
Rotavirus
Rotavirus belongs to the reovirus family whose genome is a
dsRNA. Infection with this virus is the first cause of
infantile gastroenteritis. Rotavirus nonstructural protein 4
(NSP4) has functions in viral morphogenesis and pathogenesis. A recent report shows that inhibition of NSP4
expression by small interfering RNAs leads to alteration of
the production and distribution of other viral proteins and
mRNA synthesis, suggesting that NSP4 also affects virus
replication.
In rotavirus-infected cells, it has been shown that NSP4
co-localises with LC3 in structures associated with the sites
of nascent viral RNA replication. This report suggests that
autophagy may be involved in rotavirus replication [101].
However, the precise role of this process in rotavirus
infection needs further investigations.
Bovine viral diarrhoea virus (BVDV)
Bovine viral diarrhoea virus (BVDV) belongs to the
flavivirus family whose genome is a ssRNA. The genomic
RNA contains one long open reading frame that is
translated into a polyprotein. This polyprotein is processed
by cellular and virus-encoded proteases. BVDV-infection
represents an economically important cause of disease of
farm animals. The mucosal desease (MD) is the most severe
clinical condition resulting from infection by BVDV. Both a
cytopathogenic virus (cp-BVDV) and a non-cytopathogenic
virus (noncp-BVDV) are required for induction of MD.
Infection with noncp-BVDV occurs intra-utero, leading
819
to a specific immunotolerance. The disease is induced
by super-infection with a cp-BVDV or by generation of
a cp-mutant of the persisting noncp-virus. In most cases,
RNA recombination is responsible for the switch from a
noncp- to a cp-virus.
Several types of cellular insertions, which code for
ubiquitin, different ubiquitin-like proteins, a protein of
unknown function or a part of LC3, have been found.
Naturally occurring noncp-viruses express the fusion
protein NS2-3, but further processing gives rise to both
proteins NS2 and NS3, only present in cp-BVDV. Interestingly, it has been shown that LC3 inserted in the
polyprotein of a naturally occurring mutant BVDV served
as a proteolytic processing signal by a cellular protease
related to Atg4, allowing the expression of the viral NS3
protein. These data provide evidence of the use of
autophagy proteins for the expression of viral proteins,
linked to the pathology [102].
Interferon system and autophagy
IFNs are a family of multifunctional secreted cytokines that
have been characterised by their ability to inhibit virus
infection and replication [77]. They can be divided in two
sub-groups: IFN type I (α,β) and IFN type II (γ). After
their secretion, IFNs bind to specific cell surface receptors
and act in paracrine and autocrine ways to induce
expression of more than 200 genes called IFN-stimulated
genes (ISGs). The large majority of them are still not
characterised and their involvement in the IFN system is
very fragmentary. ISGs are the effectors of the IFN
functions, namely antiviral, antiproliferative, immunomodulatory and apoptotic functions [77]. All these effects of
IFNs seem to converge towards the most known IFN
activity that is an antiviral function. In consequence, it is
not surprising that many viruses have evolved strategies to
circumvent the IFN antiviral response and even use IFNinduced processes to replicate more efficiently [103].
Interestingly, beside the fact that IFNγ can induce
autophagic vacuole formation, several proteins known to
be involved in triggering autophagy are involved in the IFN
response [104]. Among them, we can mention the doublestranded RNA-dependent PKR, the death-associated protein
kinase and DAPK-related protein-1 (DAPK and DRP-1),
the TNF-related apoptosis inducing ligand (TRAIL) and the
Fas-associated death domain (FADD).
dsRNA-dependent protein kinase (PKR)
PKR is a serine–threonine protein kinase inactive at a basal
level and whose activity can be stimulated by different
inducers. It has been first associated with the antiviral
action of IFNs [105]. It can be activated by dsRNA, a
820
common intermediate in the replication of many viruses
[106]. After interaction with dsRNA, PKR undergoes
conformational changes that trigger its autophosphorylation
and thus its activation. The major substrate of PKR is the
α-subunit of eukaryotic translation initiation factor eIF2α,
resulting in inhibition of protein synthesis [107]. However,
PKR can be activated without viral dsRNA or viral
infection by different stresses or through protein-protein
interactions. For instance, PKR can be activated by
interacting with the protein PKR activating protein (PACT),
leading to apoptosis [108].
The role of PKR in autophagy triggering is mediated by
its capacity to inhibit protein translation through eIF-2α
phosphorylation. At present, HSV-1 is the only virus
known to inhibit induction of PKR-dependent autophagy,
through action of the viral protein ICP34.5 [82]. As a large
number of viruses are able to inhibit dsRNA-mediated PKR
activation and eIF2α phosphorylation, it would be interesting to analyse their role in autophagy regulation [68].
Death-associated protein kinase: DAPk and DRP-1
DAPk is a calcium–calmodulin (CaM)-regulated serine/
threonine kinase involved in IFNγ-mediated cell death
[109]. Four additional kinases of the family have been
identified according to homologies with DAPk in the
catalytic domain [110]. DRP-1 is one of the closest family
members, as its catalytic domains share approximately 80%
identity to those of DAPk. It has been shown that
expression of DAPk or DRP-1 in cells induces morphological changes associated with membrane blebbing and
mediates a caspase-independent cell death pathway with
formation of autophagic vesicles that are characteristic of
type II programmed cell death [111].
TNF-receptor apoptosis induced ligand (TRAIL)
TRAIL, also called Apo2L, initiates apoptosis of tumour
cells by binding to either of its receptors, DR4 or DR5.
Using an in vitro morphogenesis model, in which MCF10A human mammary epithelial cells form hollow acinilike structures, it has been demonstrated that TRAIL is
required for the induction of autophagy. The authors
demonstrated that both apoptotic cell death and autophagic
cell death are required to eliminate the cells during the
lumen formation [112].
Fas-associated death domain (FADD)
FADD protein belongs to the death-inducing signalling
complex (DISC) that transmits apoptotic signals through
activation of caspase-8 [113]. It has been demonstrated that
FADD is involved in IFNγ-induced autophagic cell death.
J Mol Med (2007) 85:811–823
In this study, Pyo and collaborators have demonstrated that
Atg5 mediates IFNγ-induced vacuole formation and subsequent cell death through interaction with FADD [104].
Moreover, recent data has identified a novel cell death
pathway activated by FADD that combines apoptosis and
autophagy and that is selectively inactivated at the earliest
stages of epithelial cancer development [114].
Conclusion
Considering these data, it becomes clear that autophagy is a
fundamental and general process playing a role in viral
infections. Indeed, this process is involved in both adaptive
and innate immunity, contributing to clearance of intracellular pathogens, and in cell survival or cell death.
Moreover, viruses are able to evolve strategies to counteract
or to exploit autophagy to their own profit. Indeed, several
viruses can block autophagy triggering to avoid their
destruction in autolysosomes. In contrast, other viruses
use autophagosomes to their own replication. Unravelling
the specific role of autophagy in different viral infections
would be a crucial step in the understanding of the
pathogenesis.
Finally, knowledge of the relationships between autophagy
and the antiviral IFNs open new routes of investigation. As a
large number of IFN-inducible proteins have still no known
biological function, it would be very interesting to determine
their role in autophagy triggering.
The interplay between the autophagic pathway and the
viral life cycle is thus complex and needs a better
comprehension to provide new antiviral therapeutics.
Acknowledgments Institutional funds from the Centre National de
la Recherche Scientifique (CNRS) and the University (UM1), and
grants from SIDACTION and the Agence Nationale de Recherches
sur Le SIDA (ANRS) supported this work. L. Espert was the recipient
of a fellowship from SIDACTION.
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DOI 10.1007/s00109-007-0182-5
REVIEW
Laminin isoforms in development and disease
Susanne Schéele & Alexander Nyström &
Madeleine Durbeej & Jan F. Talts & Marja Ekblom &
Peter Ekblom
Received: 1 December 2006 / Revised: 28 February 2007 / Accepted: 28 February 2007 / Published online: 11 April 2007
# Springer-Verlag 2007
Abstract The members of the laminin family of heterotrimers are major constituents of all basement membranes,
sheet-like extracellular structures, present in almost all
organs. The laminins bind to cell surface receptors and
thereby tightly connect the basement membrane to the
adjacent cell layer. This provides for the specific basement
membrane functions to stabilize cellular structures, to serve
as effective physical barriers, and furthermore, to govern
cell fate by inducing intracellular signalling cascades. Many
different types of diseases involve basement membranes
and laminins. Metastasizing solid tumors must pass through
basement membranes to reach the vascular system, and
various microbes and viruses enter the cells through direct
interaction with laminins. Furthermore, whereas mutations
Peter Ekblom, deceased December 2005
S. Schéele : A. Nyström : M. Durbeej : P. Ekblom
Section for Cell and Matrix Biology, BMC B12,
Department of Experimental Medical Science, Lund University,
Sölvegatan 19,
22184 Lund, Sweden
Present address:
S. Schéele (*)
Lund University Stem Cell Center, Cardiovascular Group,
BMC B10, Lund University,
Lund, Sweden
email: [email protected]
J. F. Talts
Department of Basic Animal and Veterinary Sciences,
University of Copenhagen,
1870 Frederiksberg C, Denmark
M. Ekblom
Hematopoetic Stem Cell Laboratory, Stem Cell Center,
BMC B12, Lund University,
Lund, Sweden
SUSANNE SCHÉELE
received her PhD in cell and
molecular biology from the
Department of Experimental
Medical Science at Lund University, Sweden. She is currently pursuing postdoctoral
studies at the Lund University
Stem Cell Center. Her main
research interests include in
vitro differentiation of embryonic stem cells as well as
molecular mechanisms of
laminins during development.
PETER EKBLOM
received his PhD at Helsinki
University, Finland. He subsequently worked as a group
leader at the Max Planck Institute in Tubingen, Germany,
and as a Professor at the
universities of Uppsala and
Lund in Sweden. Peter
Ekblom devoted his research
carrier to the laminins with
focus on their developmental
functions. He passed away in
December 2005.
in one specific laminin chain lead to a muscular disorder,
mutations of other laminin chains cause skin blistering and
kidney defects, respectively. This review summarizes recent
progress concerning the molecular mechanisms of laminins
in development and disease. The current knowledge may
lead to clinical treatment of lamininopathies and may
include stem-cell approaches as well as gene therapy.
Keywords Lamininopathies . Integrin . Dystroglycan .
Basement membranes
826
Introduction
Many diseases involve basement membranes (BMs), extracellular matrix (ECM) sheets adjacent to epithelial, endothelial, fat, peripheral nerve, and muscle cells. BMs appear early
in mammalian embryonic development and are present in all
tissues. The major constituents are the laminin (LM) family
of glycoproteins, the three type IV collagens, the two
nidogens, and the proteoglycans perlecan and agrin. The
composition of BMs varies depending on the developmental
stage and tissue type [1], and some BM components such as
fibulins, collagen XV, and XVIII are only found in certain
BMs [2]. Here, we focus on the LMs, large heterotrimeric
proteins composed of α, β, and γ chains [3].
All LMs have a T- or cruciform shape with one long and two
or three short arms (Fig. 1). The long arm is formed by parts of
the three chains, forming an α-helical coiled coil (LCC)
domain and a C-terminal end, which is composed only of αchain LM globular (LG) domains (Fig. 1). Each chain
contributes with an N-terminal short arm consisting of three
types of globular (LN, L4, and LF) domains and rod-like
spacers formed by LM epidermal growth factor like (LE)
domains. The currently known 5 α, 3 β, and 3 γ chains
assemble into many different heterotrimers (Table 1). The
trimers are named after their chain composition; hence,
α1β1γ1 is called LM-111, and α5β2γ3 is called LM-523 [3].
Fig. 1 Left panel: a schematic
drawing of the LM-111 domain
structure. Abbreviations used: LN,
laminin N-terminal domain; LE,
laminin epidermal growth factorlike repeats; L4, laminin 4 domain;
LF, laminin four domain; and LG,
laminin globular domain. Right
panel: a representation of all described LM trimers arranged by
their LM α chain content. The
existence of either LM-212 or -222
is proposed based on studies of
peripheral nerves in wild-type and
LM α2 chain-deficient mice [51].
LM-333 has been described in rat
testis [113]
J Mol Med (2007) 85:825–836
A major feature of most LMs is their ability to form
independent networks via the globular LN domains. The
LMs together with the other BM components form the
largest polymers in the body, as they form continuous
sheets, for instance skin, with a single basement membrane
polymer reaching from head to toe under the epidermis.
Polymerization is an integral part of the biological function
of the LMs and initiates BM assembly. Other components
then assemble close to the LMs. LM polymerization is
calcium dependent, where the LN domain of each chain
noncovalently binds other LN domains so that three chains
meet [4]. As each BM contains several isoforms, LM sheets
are generally polymers of different isoforms rather than
separate polymers of each isoform.
For cells, LG domains are the main business ends. LG
domains bind several cell surface receptors and some ECM
ligands (Table 1). In each LM α chain, the C terminus
consists of a tandem of five homologous LG domains, each
with approximately 180–210 residues or 20 kDa [5]. The
cell-binding LG tandem is often physiologically shortened
by proteolytic processing, usually leading to loss of LG4-5.
Obviously, this can alter LM binding to cells. A cleavage
site has been identified within α2LG3 in the link regions of
α3 and α4 chains and is predicted for the α5 chain because
of furin-type cleavage sites [5]. Thus, the α1 chain has been
considered unique as no processing of its LG4-5 has been
J Mol Med (2007) 85:825–836
827
Table 1 Laminin isoforms and
their receptors
LM β2 chain has been reported
to directly interact with a Ca2+
ion channel [116].
found from organs or cells; although, α1LG4-5 is one of
the fragments (E3) released when LM-111 is incubated with
elastase. However, limited data suggests that α1LG4-5
might exist without the rest of the trimer in the ectoplacental cone [6]. The rarity of a proteolytic release of
α1LG4-5 suggests that it is important to retain it in the
trimer for proper LM-111 function.
The anchoring of cells to the ECM are, to a large extent,
mediated by integrins, heterodimeric transmembrane receptors composed of noncovalently associated α and β chains.
More than 20 integrins have been identified [7], of which
α3β1, α6β1, α7β1, and α6β4 are major LM receptors
(Table 1; [8]). Cell-binding studies performed with proteolytic elastase (E1–E8) fragments of LM-111 revealed the E8
fragment, consisting of the C-terminal part of the coiled coil
and LG1-3, as the main integrin binding site, as first shown
for integrin α6β1 [9]. Apart from cell adhesion and linkage
of the cytoskeleton to the ECM, integrins act as signalling
receptors, mediating growth, differentiation, and survival
signals from the ECM. They are further believed to be
involved in cell migration and certain types of cell shape
change [10].
Several other cell surface proteins bind to LMs (Table 1).
Various LM α-chain LG domains contain binding sites for
dystroglycan, heparin, and sulfatides. Dystroglycan is composed of α- and β-subunits yielded by posttranslational
cleavage of a single precursor protein. It is widely expressed
and plays an important role in muscles. Furthermore, it may
have some role in epithelial development and synaptogenesis
[11]. Within the LM α1LG tandem, α1LG4 alone is
responsible for binding to α-dystroglycan [12, 13]. Sitedirected mutagenesis mapped the α-dystroglycan binding site
to the same basic region involved in heparin and sulfatide
binding within the LM α1LG4 [14]. In contrast, several
domains of the α2LG tandem can bind dystroglycan. Thus,
α2LG1-3 and α2LG4-5 both bind α-dystroglycan and have a
higher affinity than α1LG4. There is little overlap in the
binding sites for α-dystroglycan and heparin/sulfatides in
α2LG4-5 [15], in line with reports that α-dystroglycan binding
to α1LG4-5 but not to α2LG1-3 or α2LG4-5 can be inhibited
by heparin [12]. None of the α3LG1-3, α4LG1-3, or α4LG4-5
showed affinity for α-dystroglycan [16, 17]; however, the α5
chain, via the α5LG4 domain, was shown to bind dystroglycan
with low affinity [18, 19]. Sulfatide and heparin binding sites
have been identified within several LG domains of different α
chains [2]. Furthermore, sulfatides have been suggested to be
key LM anchors, accumulating and orienting LMs at the cell
surface and thereby allowing BM assembly [20].
Laminins in development
Out of the 11 known LM chains, at least nine are essential
for life based on genetic evidence in mice (Table 2).
Without the ubiquitously expressed LM β1 or LM γ1
chains, no BMs can form, and lack of either of these leads
to early postimplantation lethality at embryonic day (E)5.5
just after implantation [21, 22]. The most prominent LM α
chain during early embryogenesis is LM α1, but also the
LM α5 chain is expressed in fair amounts during this
period. LM α1 chain is primarily expressed in epithelial
tissues during development but can also be found in some
epithelial organs in the adult. LM α5 chain, on the contrary,
exhibits the most widespread distribution pattern of all LM
α chains. Mice lacking the LM α1 chain die at E6.5–7 [21].
However, mice lacking only the LM α1 C-terminal LG4-5
domains exhibit an earlier phenotype and arrest around
E6.0 [6]. Furthermore, LM α1-chain deficient embryos do
828
J Mol Med (2007) 85:825–836
Table 2 Phenotypes of LM deficient mice
LM
chain
Primary location in vivo
Deficiency phenotypes in mouse
References
α1
α4
α5
β1
β2
Vascular BMs
Widespread
Ubiquitous
Wide expression pattern.
β3
γ1
γ2
γ3
Primarily in BMs of stratified epithelia.
Ubiquitous
Primarily in BMs of stratified epithelia.
Low expression in a few epithelial organs.
Early embryonic lethality. Only essential in extra
embryonic tissues.
Severe congenital muscular dystrophy, lethal
5 weeks after birth.
Lethal postnatal skin blistering, dies within 3 days
after birth.
20% lethal, 80% are viable and fertile.
Lethal during midgestation.
Lethal E5.5.
Postnatally lethal because of the defects in the
glomerular filtration and NMJ.
Lethal within 24 h after birth.
Lethal E5.5.
Lethal within 5 days after birth.
Not done
[6, 21, 23]
α3
BM of epithelial tissues during embryogenesis and some
epithelial BM in the adult.
BMs of skeletal and cardiac muscle, peripheral nervous system,
and central nervous system.
Primarily in BMs of stratified epithelia.
α2
form the epiblast, from which the entire fetus will be
derived, whereas α1 LG4-5 deficient embryos do not. This
may depend on a partial rescue by the LM α5 chain, only
possible when the entire LM α1 chain is absent, as the
truncated α1 chain still has the ability to assemble into the
LM-111 trimer. During development, the presence of LM
α1 chain seems to be essential only in extra embryonic
tissues, as embryos deficient in LM α1 chain in the epiblast
only are successfully born [23]. Although LM α5 chain is
incorporated into the BMs already before gastrulation, its
presence does not seem to be essential during the early
stages of embryogenesis. Mice deficient in LM α5 chain
survive until midgestation when development arrests
because of multiorgan failure, including exencephaly,
syndactyly, placentophaly, and defective glomerulogenesis
[24].
During embryogenesis, LM α2 chain is expressed along
developing muscles from E11. Mice deficient in LM α2
chain survive embryonic and early postnatal stages but
develop severe muscular dystrophy and die around 5 weeks
after birth [25]. In addition, LM β2 chain is dispensable for
embryogenesis. In the embryo, it is upregulated during
organ maturation as seen in glomerular BMs, neuromuscular junctions (NMJ), and smooth muscle cells in the aorta
[1, 26, 27], and consequently, mice deficient in LM β2
chain die soon after birth because of defects in the NMJ and
with a defective glomerular filtration [28, 29].
LM α3 chain is expressed in two splice variants, LM
α3A with a truncated N-terminal and LM α3B with an
extended N-terminal. LM α3 chain is primarily expressed
in stratified epithelium [30]. The trimer LM-332 is typically
found in the dermoepidermal junction of the skin where it
induces formation of hemidesmosomes, anchoring epithelial cells to the underlying BM [31]. LM α3 as well as the
[25]
[33]
[38, 41]
[24]
[21]
[28, 29]
[34]
[22]
[35]
–
β3 and γ2 chains are expressed at E10.5 during murine
embryogenesis [32]. Apart from the skin, LM-332 is also
expressed in epithelial tissues such as the lung, intestine,
stomach, and kidney [30]. Mice deficient in any of the three
chains of LM-332 die within 5 days of birth because of
severe skin blistering [33, 34].
LM α4 chain is weakly expressed at E7 of murine
embryogenesis and expression peaks at E15–17. Initial
mRNA expression studies detected LM α4 chain mRNA in
organs and tissues mainly derived from cells of mesenchymal origin [36]. The LM α4 chain can be found in, or in the
vicinity of, the BM of all blood vessels [37]. Apart from
endothelial cells, LM α4 chain is also found in the nerves,
NMJ, extraocular muscles, and adipocytes [38, 39]. The
LM α4 chain is the major and, in many cases, the only LM
α chain in late embryonic and neonatal vascular BMs [37].
Mice lacking the α4 chain are viable and fertile [40, 41].
However, hemorrhages limited to smaller vessels and
capillaries from E11, when the LM α4 chain appears in the
capillaries, caused anemia, and about 20% die within the first
2 days after birth. When the mice had reached 1 week of age,
the vascular phenotype healed out [40]. This may be
explained by the premature introduction of the LM α5 chain
in the capillary BMs seen in the LM α4 chain null mice.
Little is known about the function of the LM γ3 chain. It
is expressed in low amounts in several organs including the
testis, kidney, brain, skin, and hair follicles [42].
The kidney
A role for LM in kidney development was suggested
already 27 years ago [43], and over the past decade, gene
targeting experiments in mice have revealed fundamental
J Mol Med (2007) 85:825–836
829
roles of LMs in the embryonic kidney [1]. Kidney
organogenesis begins when the ureter bud, which is an
outgrowth of the Wolffian duct, invades the metanephric
mesenchyme. The ureter bud, in turn, induces a part of the
metanephric mesenchyme to condense and to convert into a
polarized epithelium surrounded by a BM. This polarized
aggregate will extend and develop into a complete nephron
with the tubular system connected to the collecting duct
system at one end and the glomerulus at the other end.
Early in vitro studies demonstrated a role for LM α1 chain
in the conversion of the condensed kidney mesenchyme to
a polarized epithelium [44], and subsequent studies suggested a similar role for the nidogen-binding site in the LM
γ1 chain [45]. Genetic studies in mice confirmed that the
nidogen binding site in the γ1 chain is crucial for kidney
development [46], whereas it remains to be confirmed by
genetic means that the α1 chain is essential for embryonic
kidney development. Gene targeting studies have also
implicated a role for the LM α5 chain in early kidney
development as approximately 20% of LM α5-chain deficient embryos display uni- or bilateral renal agenesis [47].
LMs are also important for the normal function of the
glomerulus, the tuft of the capillaries that filters waste
products from the blood to form urine. Proteinuria is
defined by excess protein (albumin and other plasma
proteins) in the urine and means that protein is leaking
through the glomeruli. Recent data suggest that the
glomerular BM, rather than the epithelial filtration slits, is
the main molecular barrier to albumin [48]. The mature
glomerular BM is composed of α5, β2, and γ1 chains.
Studies of mice deficient in LM α5 and β2 chains,
respectively, have revealed that the LM α5 chain is
essential for glomerulogenesis [47], whereas the LM β2
chain is important for the proper function of the glomerular
filtration barrier as the lack of LM β2 chain in mice results
in a disorganized glomerular BM [48]. In addition,
mutations in the LAMB2 gene cause Pierson syndrome, a
rare disease that is manifested in the newborn with
congenital nephrotic syndrome and mesangial sclerosis
period followed by end-stage renal disease and early death.
Furthermore, ocular anomalies as well as muscle problems
are seen in patients with Pierson syndrome (see “The
neuromuscular system”) [49].
muscle fibre and in the endoneurial BM surrounding each
Schwann cell/axon unit. In addition, the LM α2 chain is
expressed at the myotendinous and NMJ [27]. In agreement
with the widespread distribution of LM α2 chain in the
neuromuscular system, mutations in the gene encoding the
LM α2 chain cause neuromuscular defects both in man and
mouse. Patients with mutations in the LAMA2 gene develop
congenital muscular dystrophy type 1A (MDC1A) that
often leads to death in early childhood (Table 3). The
disease is characterized by severe muscle weakness,
hypotonia, joint contractures, white matter abnormalities,
peripheral neuropathy, respiratory compromise, and failure
to thrive (because of feeding difficulties; [50]). Several
mouse models for the LM α2 chain deficiency are
available, and they also display muscular dystrophy and
peripheral and central nervous system myelination defects.
How the absence of LM α2 chain leads to neuromuscular
defects is not fully understood, but patient studies and
detailed analyses of animal models have begun to shed
some light on the disease mechanisms. BMs are disrupted,
and the expression of LM α2-chain receptors and some BM
associated proteins are altered in the LM α2-chain deficient
muscles, and both structural and signaling defects may be
detrimental for normal muscle function [51–54]. Furthermore, critical roles for LM α2 chain inducing Schwann cell
proliferation and oligodendrocyte spreading, as well as
myelination in the peripheral nervous system and central
nervous system, respectively, have been demonstrated [55,
56]. The analyses of LM α2-chain deficient animals have
furthermore revealed nonneuromuscular defects [57, 58].
To test novel therapeutic strategies for MDC 1A, mouse
models constitute valuable tools. LM α1 and α2 transgenes
have been demonstrated to successfully compensate for the
lack of the LM α2 chain in muscles (Fig. 2) [59, 60] and
later also in the peripheral nerve [50]. Other molecular
routes including the overexpression of the mini-agrin [61–
63] and reduction in apoptosis [64] in the LM α2 chain
deficient animals also effectively improved the muscular
dystrophy phenotype.
The one other LM chain associated with a human
neuromuscular disease is the LM β2 chain, which is
prominently expressed at the NMJ. Human LM β2 chain
deficiency causes Pierson syndrome (Table 3), a rare lethal
disease with congenital nephrotic syndrome (see “The
The neuromuscular system
Table 3 Lamininopathies
Several LM chains (α2, α4, α5, β1, β2, γ1, γ2, and γ3)
are expressed in the various BMs of the neuromuscular
system, which is composed of the muscles of the body
together with the nerves supplying them. The most
ubiquitous α chain in the neuromuscular system is the α2
chain, which is expressed in the muscle BM covering the
Disease
Congenital muscular dystrophy type
I A (MDC1A)
Junctional epidermolysis bullosa
Pierson syndrome
Laminin chain
deficiency
Reference
α2
[25]
α3, β3, γ3
β2
[69, 70]
[49]
830
kidney”), distinct eye abnormalities, and impaired neurodevelopment with severe muscular hypotonia, psychomotor
delay, hemiparesis, and abnormal movements [49]. However,
it remains to be demonstrated whether muscle abnormalities
in patients stem from skeletal NMJ defects. Nevertheless,
LM β2-chain deficient animals fail to thrive and die early,
and it was recently demonstrated that the muscle defects in
these mice are responsible for the severe phenotype [65].
Multiple sclerosis is an inflammatory disorder of the
central nervous system in which gradual destruction of
myelin in the brain or spinal cord interferes with the nerve
pathways and causes muscular weakness. Murine experimental autoimmune encephalomyelitis is a relevant mouse
model for multiple sclerosis where inflammatory cells are
recruited from the circulation into the central nervous
system [66]. To penetrate the blood-brain barrier, infiltrating leukocytes must extravasate the endothelial monolayer,
transmigrate across the underlying endothelial BM (containing LM α4 and α5 chains), and finally penetrate the
parenchymal BMs (containing LM α1 and α2 chains) and
glia limitans whereafter clinical symptoms become apparent
[67]. Recent studies revealed distinct molecular mechanisms behind the extravasation of leukocytes across the two
BMs. Leukocyte extravasation through the endothelial BM
occurs only at sites characterized by the sole presence of
LM-411, and this is an integrin β1-mediated process [67],
whereas extravasation through the parenchymal BM
involves MMP-2 and MMP-9 cleavage of dystroglycan
[68].
The skin
Epidermolysis bullosa is a group of inherited disorders of
the dermoepidermal region in the skin where blistering
occurs as a result of slight mechanical trauma. It is
classified based on the site where the skin blistering occurs,
heredity, clinical features, as well as the causative genes or
proteins [69, 70]. LM-332 is prominent at the dermoepidermal junction in the skin. The lack of any of its chains
causes lethal Herlitz’s junctional epidermiolysis bullosa
(Table 3). Milder forms of junctional epidermolysis bullosa
are caused by mutations causing a perturbed function of the
LM proteins. Other forms of epidermolysis bullosa are
caused by defects in LM-332 binding partners. Examples
include integrin α6β4 in hemidesmosomes or collagen type
VII in the deeper dermal layers. Recent results with ex vivo
cultured, patient-derived keratinocyte stem cells that were
genetically modified, grown into epidermal grafts, and
subsequently transplanted into the patient’s skin have
proven to be successful [71]. In the specific case described,
the patient had a defective gene encoding for the LM β3
chain. Hence, the cells were transduced with a retroviral
J Mol Med (2007) 85:825–836
vector LAMB3 cDNA. The transplanted, genetically
modified epidermis remained stable for the entire followup time (1 year) with no signs of blisters or infections [70].
Most mutations in the LM α3 chain encoding gene that
cause junctional epidermolysis bullosa prevent the expression of both splice variants. However, a disease in which
only the α3A variant is affected was recently described. In
laryngo-onycho-cutaneous syndrome, an α3A-specific exon contains a premature stop codon, but the expression of a
truncated α3A lacking the N-terminal epidermal growth
factor repeats can be initiated at a downstream in-frame
methionine. In these patients, LM-332 trimers form, hemidesmosomes are organized, and the patients do not have
junctional epidermiolysis bullosa. They do have cutaneous
erosions, nail dystrophy, and development of granulation
tissue in the eyes and larynx. Thus, the α3A LE repeats
appear to be important for granulation tissue response [72].
The hematopoietic system
In the bone marrow, extensive proliferation and differentiation of primitive hematopoietic cells and egress of mature
blood cells into circulation continue throughout life. During
steady-state hematopoiesis, small numbers of primitive
hematopoietic stem cells continuously migrate between
the blood stream and the bone marrow extravascular
hematopoietic niches [73]. LM-411 and -511 are expressed
in adult mouse and human bone marrow in subendothelial
sinusoidal BMs at sites of hematopoietic cell trafficking
between the bone marrow and the circulatory system. In
addition, LM-411 is expressed in bone marrow extravascular hematopoietic spaces and in sinusoidal linings of early
postnatal bone marrow. Consequently, these LMs may have
a role in the regulation of hematopoietic cell development
and migration [74, 75], and studies on hematopoietic cell
interactions with LMs have mainly focused on these
isoforms. The expression of other LM chains, including
α3, β2, and γ2, have been reported in bone marrow
stromal cells or stromal-derived cell lines, but their
localization and assembly is still largely unclear [76]. In
addition to stromal cells, synthesis or expression of LMs
have been reported in lineage-differentiated hematopoietic
cells, including platelets [77].
The integrin α6β1 receptor is ubiquitously expressed in
hematopoietic stem and progenitor cells as well as mature
cells of several lineages. In vitro studies have indicated a
role for the integrin α6 receptor and LM-411 and -511/-521
for adhesion, migration, and proliferation of hematopoietic
progenitors [74, 75, 78]. In agreement with this, functional
inhibition of the integrin α6 receptor in mouse bone
marrow cells by blocking antibodies prevented bone
marrow homing of transplanted bone marrow stem and
J Mol Med (2007) 85:825–836
progenitor cells [79]. In vitro, both α4 and α5 LMs support
migration of bone marrow progenitors and mature neutrophils [78, 80]. Accordingly, in vivo studies using LM α4chain deficient mice have shown impaired neutrophil
recruitment in response to inflammation [80]. In contrast,
migration of lymphocytes and monocytes are stimulated by
LM α4 but inhibited by α5 LMs [81], indicating
developmental stage- and lineage-specific differences in
hematopoietic cell interactions with distinct LM isoforms.
Another LM receptor, the Lutheran glycoprotein (Lu
GP) is a blood group, active protein found on erythrocytes.
It is a member of the immunoglobulin superfamily and
binds in particular to α5 LMs. It is suggested that Lu GP
might have a role in the trafficking of mature erythroid
cells through the sinusoidal endothelium. The levels of Lu
GP are increased in sickle cell erythrocytes, and the
adhesion of sickle cells to endothelial α5 LMs may
contribute to the vasoocclusive events associated with
acute episodes [82].
831
α3, β2, and γ2 chains were upregulated in allergic asthma
[84].
Cancer
Early histological observations on carcinomas, which
accounts for 90% of human neoplasia, revealed that a
disrupted BM is a sign of more malignant tumors, whereas
an intact linear BM indicates more benign tumors [85].
Studies on human carcinomas show that an overwhelming
majority of tumors express LMs. In investigated mammary
carcinomas, however, LM expression was strongly reduced or
absent [86]. The LM chains expressed by the carcinomas are,
to a large extent, the same isoforms found in normal
epithelium, that is, LM α5 and α3 [86].
In vivo studies of exposed vascular BMs in pulmonary
capillaries implicate the attachment of tumor cells to the
LM α3 via integrin α3β1 in the arrest of metastasizing
tumor cells [87]. Another study suggests an increased LM332 expression or, more frequently, an excessive LM γ2
Vascular and pulmonary diseases
Atherosclerosis is a major disease in western societies.
Changes in the LM expression from healthy vessels to
atherosclerotic plaques have been reported. During development, the vascular smooth muscle cells (VSMC) of the
vessel wall express LM β1 containing LMs; however, as
the vessels mature, the LM β2 chain becomes the major β
chain of the vessel wall [26]. In a study of human
atherosclerotic plaques, the LM β1 chain was heavily
upregulated, and the LM β2 chain was subsequently
reduced. In this study, also the LM α5 chain was reduced
in the plaques [26]. The reversion of the expression to LM
β1 chain in plaques fits with the notion that during
atherosclerosis, the VSMC revert to a more primitive, less
differentiated, more fibroblast-like phenotype. In a recent
study, it was shown that mice lacking the LM α4 chain
have partially malfunctional hearts, which may cause
premature death, and furthermore, it was suggested that
deficiency in the LM α4 chain could account for cases of
sudden deaths in humans [83]. An impaired capillary
patterning leads to hypoxia, which causes the heart defect
in LM α4 chain null mice. It is likely that other organs
also experience this vascular disorganization; however, as
the heart is the most oxygen-demanding organ in the
body, it is in this organ the defect makes itself known
[83].
Both allergic and nonallergic asthma leads to a thickening of the subepithelial BM (Fig. 3). In a study comparing
the LM expression in two types of asthmas, an increase in
LM α5, β1, and γ1 chains were seen for both types
compared with healthy individuals. In addition, the LM α2,
Fig. 2 Laminin α1 chain reduces muscular dystrophy in laminin α2
chain-deficient mice. Upper panel: Dy3K heterozygous mice were
mated with mice overexpressing laminin α1 chain to generate mice
lacking laminin α2 chain but instead expressing laminin α1 chain in
skeletal muscles (dy3KLNα1TG mice). Dy3K/dy3K animals, completely
deficient in laminin α2 chain, are small, weak, and die at about
5 weeks of age, whereas dy3KLNα1TG mice are as large as wild-type
mice, alert, and lively with good muscle tone and have a near normal
life span. Lower panel: cross-sections of skeletal muscles of
dy3KLNα1TG and dy3K/dy3K mice were stained with antibodies
against laminin α1 chain. Dy3KLNα1TG muscles are rich in laminin
α1 chain, whereas dy3K/dy3K (and also wild type) muscles are devoid
of laminin α1 chain [51]
832
J Mol Med (2007) 85:825–836
Fig. 3 Thickened subepithelial BM in
asthma. a shows sectioned healthy
bronchial tissue. Arrows indicate the
BM. b shows sectioned bronchial tissue
from an asthmatic patient. Note the
extremely thickened BM (courtesy of
Dr. Gunilla Westergren-Thorsson)
synthesis alone, as prognostic markers for aggressive
carcinomas and their invasion [88] and in vitro studies of
human gastric carcinoma cells showed production and
release of the monomeric LM γ2 chain [89]. Furthermore,
there are reports that the cleaved-off LM γ2 short arm
stimulates migration as a soluble factor [89].
Other LM chains have also been implicated in tumor
progression. The LM α4 chain was shown to be upregulated in peritoneal carcinomas and glial tumors [90, 91],
and in glial tumors, there is a shift from normal LM-421
expression to a strong expression of LM-411 in glioma
microvessels, where high LM-411 expression indicates
more malignant tumors, whereas LM-421 indicates lower
graded and benign tumors [92]. In breast cancers, the same
switch from LM β2 to LM β1 containing LMs in the
capillaries of aggressive tumours occur [93]. Studies of
tumor invasion in rat brain show that anti-LM α4 or antiLM β1 si-RNA could reduce invasiveness both in vitro and
ex vivo [94, 95] and reduce tumor vascularization in vivo
[96]. Despite this, experimentally implanted tumors grow
faster and have an increased metastasis in LM α4 chain null
mice as compared to wild-type mice because of an
increased neovascularization [97].
Microbial and viral diseases
A critical first step in the establishment of infection by
many pathogenic organisms is the attachment to and
colonization of epithelial surfaces. In many cases, this is
accomplished via the interaction of specific surface structures on the pathogens, known in bacteria as adhesins, to
cell adhesion receptors [98] or their extracellular ligands
[99]. Recent studies have demonstrated proteins or other
macromolecules with strong LM-binding activity in many
invasive pathogens (Table 4).
In some cases, LMs bound to the surface of the pathogen
is utilized to facilitate entry into host cells via LM
receptors. Examples include the protozoan Toxoplasma
gondii, which through the host cell LM bound on its
surface, it can bind to the ubiquitously expressed receptor
integrin α6β1 [100], which fits with the capacity of T.
gondii to invade almost every cell of the body. Mycoplasma
leprae, on the other hand, show a specific nerve predilection leading to neuropathies. The invasive mechanism of M.
leprae starts with its binding to the LG domain of the LM
α2 chain [101] expressed on the muscle and peripheral
nerve. This LM-pathogen complex then binds α-dystrogly-
Table 4 Some invasive pathogens with laminin binding activity
Pathogen
Disease
Reference
Aspergillus fumigatus
Helicobacter pylori
Histoplasma capsulatum
Mycobacterium leprae
Paracoccidioides
brasiliensis
Rotavirus
Streptococcus pyogenes
Treponema pallidum
Trypanosoma cruzi
Opportunistic infection in immunocompromised patients.
The primary cause of active chronic gastritis.
Opportunistic infection in immunocompromised patients.
Leprosy
Systemic mucosis
[106]
[107]
[106]
[101, 103, 108]
[106]
The most important cause of viral gastroenteritis and dehydrating diarrhea in young children.
Pharyngitis, impetigo, scarlet fever, and streptococcal toxic shock-like syndrome.
Syphilis
Chagas disease
[109]
[110]
[111]
[112]
J Mol Med (2007) 85:825–836
can, which is present in high density on Schwann cell
surfaces [102]. Upon binding, large clusters of α-dystroglycan are formed on the cell surface, and bacteria are
internalized. Internalization occurs also with γ-irradiated
bacteria or beads coated with the M. leprae-specific LM
binding phenolic glycolipid-1 [103], indicating that invasion is not a bacterial-driven phenomenon. α-Dystroglycan
is connected to the cytoskeleton via β-dystroglycan, and
the invasion process is likely dependent on cell cytoskeleton dynamics initiated by receptor clustering.
Arenaviruses are known for their hemorrhagic syndromes
in humans. Several members of the arenavirus family bind
the LM α2-chain receptor α-dystroglycan [104]. These
include the Lassa fever virus and the lymphocytic choriomeningitis virus (LCMV), both shown to cause neurological
abnormalities. Moreover, LCMV is considered an underdiagnosed teratogen as congenital LCMV infection may
affect the developing nervous system. In fact, in an in vitro
model of Schwann cell differentiation, LCMV competed
with LM-211 for binding to α-dystroglycan, downregulated
α-dystroglycan expression, and disrupted organization of the
LM-211 network but not synthesis of this LM [105].
Concluding remarks
The LMs exhibit a large diversity concerning their roles in
various diseases. Considering their positioning within all
BMs together with their multiple functions, it is not
surprising that mutations, over or underexpression, as well
as isoform shifts can lead to pathological conditions. The
mechanisms for LM involvement in some conditions, such
as overexpression of certain LM chains in asthma, remain
poorly understood. For other LM-related diseases, such as
muscular dystrophy or epidermolysis bullosa, the molecular
mechanisms are, to some extent, characterized, and
advancements concerning therapeutics have already been
made. Advancements within the areas of stem-cell biology
and gene therapy, together with an increased understanding
of how LM isoforms are regulated during pathological
conditions, will provide for new ways of treatment of
diseases that today are incurable.
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DOI 10.1007/s00109-007-0175-4
REVIEW
Adoptive precursor cell therapy to enhance immune
reconstitution after hematopoietic stem cell transplantation
J. L. Zakrzewski & A. M. Holland &
M. R. M. van den Brink
Received: 28 December 2006 / Revised: 6 February 2007 / Accepted: 7 February 2007 / Published online: 28 February 2007
# Springer-Verlag 2007
Abstract Strategies to enhance post-transplant immune
reconstitution without aggravating graft-vs-host disease
(GVHD) can improve the outcome of allogeneic hematopoietic stem cell transplantation. Recent preclinical studies
demonstrated that the use of T cell depleted allografts
supplemented with committed progenitor cells (vs stem
cells only) allows enhanced immune reconstitution of
specific hematopoietic lineages including myeloid, B, T,
and natural killer lineages in the absence of GVHD. This
novel adoptive therapy resulted in significantly improved
resistance to microbial pathogens and could, in some cases,
even mediate tumor immunity. Clinical protocols using
adoptive transfer of committed hematopoietic progenitor
cells are currently being evaluated.
Keywords Hematopoietic stem cell transplantation .
Immunodeficiency . Adoptive cell transfer
Hematopoietic stem cell transplantation (HSCT) is an
important therapy for a variety of malignant and nonmalignant diseases. Immune deficiency after HSCT is a major
cause of post-transplant morbidity and mortality. In the
early post-transplant phase, neutropenia is a significant risk
factor for opportunistic infections. B and particularly T cell
reconstitution are delayed resulting in a prolonged period of
post-transplant immunodeficiency, which can persist up to
1–2 years after transplantation [1, 2]. This delay is
associated with various complications including an increased risk of infections [1–4], malignant relapse [5–12],
J. L. Zakrzewski (*) : A. M. Holland : M. R. M. van den Brink
Department of Medicine and Immunology,
Memorial Sloan–Kettering Cancer Center,
1275 York Avenue,
New York, NY 10021, USA
e-mail: [email protected]
JOHANNES L. ZAKRZEWSKI
received his M.D. from the
University of Erlangen–
Nuremberg, Germany. He is
presently a research fellow in
the Laboratory of Allogeneic
Bone Marrow Transplantation
at Memorial Sloan–Kettering
Cancer Center. His research
interests include strategies to
enhance immune reconstitution
and antitumor activity after
hematopoietic stem cell transplantation. His main focus is
the development of precursor
cell therapies to enhance T and
NK cell reconstitution.
MARCEL R. M. VAN DEN BRINK
received his M.D. and Ph.D.
(Immunology) from the
University of Leiden, The
Netherlands. He is presently
the chief of the Adult
Allogeneic Bone Marrow
Transplantation Service at
Memorial Sloan–Kettering
Cancer Center. His laboratory
is devoted to the development
of strategies to enhance
immune reconstitution and
graft-vs-tumor activity and
suppress graft-vs-host disease
after hematopoietic stem cell
transplantation.
and mixed chimerism and graft rejection due to failure to
eliminate residual host T cells [13]. The presence of alloreactive T cells in the graft helps to overcome host-vs-graft
activity but is associated with an increased risk for graft-vshost disease (GVHD), a major complication of allogeneic
HSCT. Important factors contributing to B cell deficiency
after HSCT include decreased B lymphopoiesis, (chronic)
GVHD, and lack of CD4 help [14, 15]. The severity and
838
duration of post-transplant immune deficiency is determined by a number of variables, including previous chemo/
radiation therapy, a lack of sustained transfer of donor
immunity, thymic involution post-puberty, donor/host
histoincompatibility, GVHD, and graft rejection [16].
Strategies to enhance post-transplant immune reconstitution (without enhancing GVH activity) could decrease the
incidence of fatal infectious complications, enhance graftvs-tumor (GVT) activity, and significantly improve the
overall survival of HSCT recipients.
Strategies to enhance immune reconstitution after
HSCT
In recent years, a number of preclinical and clinical studies
have demonstrated the efficacy of various approaches to
enhance immune reconstitution in HSCT recipients. Using
mouse models of allogeneic HSCT, recently established
strategies include (a) hormonal therapies such as sex steroid
ablation [17], gonadotropin-releasing hormone (GnRH)
agonist treatment [18] insulin-like growth factor 1 treatment [19, 20], growth hormone treatment [21], or prolactin
treatment [22], (b) growth factor therapies such as
keratinocyte growth factor (KGF) [23–26], stem cell
factor, fms-like tyrosine kinase 3 (FLT3) ligand, granulocyte colony stimulating factor (G-CSF), or granulocyte/
macrophage colony stimulating factor (GM-CSF) [27–29],
(c) cytokine therapies such as interleukin-7 (IL-7) treatment [30–33] or IL-15 treatment [34], (d) cellular
therapies such as adoptive transfer of mature T or natural
killer (NK) cells to enhance virus or malignancy specific
immunity [35–38] or adoptive transfer of progenitor cells
to enhance T, B, NK, myeloid, and overall immune
reconstitution [39–44]. Several studies in mouse and man
demonstrated that the administration of donor T cells
expressing the herpes simplex–thymidine kinase allows
selective in vivo depletion of these T cells by the use of
ganciclovir [45–47]. This strategy can be employed to
enhance immune reconstitution after HSCT while reducing
the risk for GVHD.
Kinetics of immune reconstitution are related
to the cellular composition of the graft
One of the most important factors influencing the kinetics
of immune reconstitution after HSCT is the cellular
composition of the (allo)graft. The use of T cell depleted
(TCD) instead of whole bone marrow (BM) as stem cell
source results in less GVHD, however at the expense of a
prolonged profound T cell deficiency associated with a
higher incidence of opportunistic infections and a higher
J Mol Med (2007) 85:837–843
risk for mixed chimerism and graft failure [7]. As the speed
of immune reconstitution after HSCT correlates with the
stem cell dose [48], the disadvantages of T cell depletion
can, to some extent, be counteracted by using CD34+
enriched high dose stem cell grafts.
Zhao et al. [27] compared the efficacy of immune
reconstitution in BM populations commonly used for
experimental HSCT. They determined the number of
progenitors giving rise to splenic colonies (hematopoietic
clonogenic progenitor capacity) in TCD but otherwise
unfractionated BM, lineage marker-negative (lin−) BM,
and purified HSCs. Using this information, they transferred
equivalent numbers of repopulating progenitors along with
the respective accompanying cellular milieus into transplant
recipients to compare engraftment capacity and kinetics.
Recipients of unfractionated BM grafts had faster reconstitution, better donor chimerism, and increased cellularity in
the lymphoid organs compared to the lin− BM and HSC
recipients. T, dendritic, and especially B cell reconstitution
were significantly enhanced in the whole BM group.
Addition of GM-CSF-producing bystander cells posttransplant further enhanced DC and overall immune
reconstitution in all groups, although the most pronounced
effect was again in the unfractionated BM group. Later and
lesser effects were seen with treatment with FLT3 ligand
post-transplant. The findings suggest that while purified
HSCs are capable of repopulating an immunodeficient host,
the cellular environment found in the BM augments that
ability and leads to faster, more complete reconstitution.
Several recent preclinical studies, therefore, explored the
possibility of supplementing purified HSCs with committed
precursor cells to enhance immune reconstitution in a
defined, lineage-specific fashion and without increasing
the risk for GVHD.
Adoptive transfer of myeloid progenitors
BitMansour et al. [40] reported that congenic cotransplantation of BM-derived lin−CD16/32loCD34+c-kithiSca-1−
common myeloid progenitors (CMP) or lin − CD16/
32+CD34+c-kithiSca-1− granulocyte–monocyte progenitors
(GMP) resulted in enhanced myeloid reconstitution with
significantly increased splenic neutrophil counts by day 7
after HSCT. Two thirds of those cells were derived from
adoptively transferred myeloid progenitors, but the number
of host neutrophils was also increased compared to
recipients of HSCs alone. Animal models of invasive
aspergillosis and Pseudomonas aeruginosa infection were
used to assess if this enhanced recovery of innate immunity
translated into a functional benefit. In both models,
cotransplantation of CMP/GMP resulted in shortening of
the period of functional neutropenia and subsequently
J Mol Med (2007) 85:837–843
increased resistance to Aspergillus fumigatus and P.
aeruginosa. Combination of CMP/GMP transfer and posttransplant G-CSF administration further improved protection against A. fumigatus. In addition to its use in lethally
irradiated HSCT recipients, this therapy was also shown to
increase resistance to A. fumigatus in a mouse model of
chemotherapy-induced neutropenia [41].
839
on thymic engraftment, cellularity, and chimerism when
combined with KGF, and (e) improved NK cell reconstitution [39]. Moreover, adoptive transfer of T cell precursors
translated into increased resistance to Listeria monocytogenes and significant GVT activity after allogeneic HSCT
[39]. GVHD was not associated with T cell precursor
administration suggesting that these donor cells were
subject to effective selection processes in the host thymus
resulting in host tolerance [39].
Adoptive transfer of common lymphoid progenitors
Common lymphoid progenitor cells (CLP) are found in
adult BM and are characterized by a lin−IL-7Rα+Thy1.2−ckitloSca-1lo phenotype. Arber et al. [42] adoptively transferred these cells into congenic and allogeneic murine
HSCT recipients and evaluated their effect on lymphoid
recovery after HSCT. Cotransplantation of CLPs compared
to transplantation of HCSs alone resulted in enhanced early
immune reconstitution (including T, NK, and B cell
lineages) and enhanced resistance to murine cytomegalovirus infection. After day 14, the beneficial effect on
lymphoid reconstitution was, however, restricted to mainly
the B cell lineage. GHVD was not induced by cotransplantation of allogeneic CLPs.
Adoptive transfer of T cell precursors
Due to the recent establishment of Notch1-based culture
systems [43, 49], ex vivo generation and expansion of
committed T cell progenitors has become feasible. Notch
signaling is involved in various cell fate decisions during
the development of a multicellular organism, including
survival, proliferation, lineage commitment, and tissue
architecture. Notch1 is essential for T cell lineage commitment and differentiation and is activated by its ligand Deltalike 1 (DL1). Tissue culture systems using Notch1 signaling
allow the in vitro development of T (and NK) lineage cells
from murine and human hematopoietic (and embryonic)
stem cells [49–52]. Currently, two Notch1-based culture systems are available: (1) coculture of HSCs with OP9 BM
stromal cells expressing DL1 [46] and (2) culture of HSCs
with immobilized DL1–hIgG fusion protein (DL1ext-IgG) [43].
The OP9-DL1 system allows for the generation of large
numbers of T/NK cell precursors from murine HSCs for
adoptive therapy [39]. Infusion of those cells with TCD BM
or purified HSCs into lethally irradiated allogeneic recipients (Fig. 1) resulted in several highly significant beneficial
effects: (a) enhanced thymic engraftment, cellularity, and
chimerism, (b) increased numbers of donor T cells and
improved chimerism in the periphery, (c) enhanced cytokine
response by donor T cells even 2 months after transplant
and in tumor-bearing HSCT recipients, (d) additive effects
Transfer of heterogenous human progenitors
in a xenograft model
Ohishi et al. [43] and Bernstein et al. [44] utilized Notch1
signaling in a stromal cell-free culture system using
DL1ext-IgG to generate heterogenous populations of human
hematopoietic progenitor cells (the resulting phenotypes
depended on the DL1 density and cytokine/growth factor
cocktail). Progenitor cells originating from CD34+CD38−
human cord blood-derived HSCs cultured with DL1ext-IgG
(including committed myeloid progenitors, lymphoid progenitors, and CD34+CD38low/− hematopoietic progenitors)
were transplanted into sublethally irradiated non-obese
diabetic/severe combined immunodeficiency mice. Recipients of the DL1ext-IgG-cultured progenitors had increased
myeloid and B cell engraftment in the BM than either
uncultured or control cultured cells. Thymic engraftment
of T cell precursors was also achieved in recipients of
DL1ext-IgG-derived cells, while control mice had only
extremely low human engraftment in the thymus. The
immobilized DL1ext-IgG system, therefore, allows for
expansion of a variety of committed human hematopoietic
progenitors with enhanced engraftment capability.
Clinical potential of adoptive precursor cell therapy
The kinetics of immune reconstitution after HSCT depend,
amongst others, on the stem cell dose and the presence of
committed progenitors in the administered cell product. The
supplementation of the graft with (committed) progenitor
cells such as CMP/GMP, CLP, or T cell precursors could
represent a promising new approach to support engraftment
and enhance early immune reconstitution after HSCT
without increasing the risk for GVHD.
Currently, two clinical studies are underway: Weissman
and coworkers are evaluating a myeloid progenitor cell
product (CLT-008, Cellerant Therapeutics, San Carlos, CA)
to treat neutropenia in HSCT recipients and cancer patients.
The administered cells do not have to be human leukocyte
antigen matched with the recipient and give rise to a single
generation of granulocytes, macrophages, platelets, and
840
J Mol Med (2007) 85:837–843
Fig. 1 Adoptive transfer of ex vivo generated T cell precursors into
allogeneic HSCT recipients. C57BL/6 BM-derived HSCs were
cultured on OP9-DL1 cells in the presence of IL-7 and FLT3-L to
generate T cell precursors for adoptive therapy (scanning electron
microscopy image on day 5 of coculture. Credit: Andrea Tuckett,
Polytechnic University). Lethally irradiated allogeneic recipients
(LP, BALB/c, or C3FeB6F1) were transplanted with purified
C57BL/6-derived HSC; control mice received HSCs only, the
treatment group received additional OP9-DL1-derived T cell
precursors
erythrocytes. Current strategies using BM-derived progenitor cells such as CMP/GMP and CLP have, however, an
important disadvantage: These phenotypes constitute very
rare populations of cells in the BM whose isolation is rather
complicated and results in a low yield. In contrast to this,
Notch1-based ex vivo culture systems allow the use of
relatively small numbers of HSCs to generate and expand a
variety of precursor cell phenotypes including myeloid,
NK, B, and T cell progenitors. The use of Notch1-based
culture systems in clinical trials requires the establishment
of Food and Drug Administration-approved animal prod-
uct-free culture systems to generate human progenitor cell
populations in therapeutic quantities. This goal has recently
been achieved by Bernstein and coworkers, who are
conducting a clinical study at Fred Hutchinson Cancer
Research Center, evaluating adoptive transfer of heterogenous progenitors originating from cord blood-derived
CD34+ HSCs cultured with DL1ext-IgG.
Adoptive T cell precursor cell therapies could be
particularly effective in the TCD HSCT setting, as this
approach minimizes the risk for GVHD while promoting
early T (and NK) cell reconstitution. Importantly, several
Fig. 2 Design for a clinical study to evaluate the effect of T/NK cell precursor administration on immune reconstitution after TCD allogeneic HSCT
J Mol Med (2007) 85:837–843
clinical studies indicate that improved early lymphocyte
recovery after TCD HSCT reduces the risk for malignant
relapse [8–12], underlining the significance of the abovedescribed preclinical finding of GVT activity promoted by
adoptive T/NK cell precursor transfer [39]. Clinical
precursor cell therapy could therefore translate into significantly improved overall survival after TCD allogeneic
HSCT due to the combination of increased donor chimerism, antimicrobial resistance, and antitumor activity in the
absence of GVHD. A possible design for a clinical study of
adoptive precursor cell therapy to enhance immune reconstitution after TCD allogeneic HSCT is presented in Fig. 2.
In elderly patients with involuted thymi, combination
therapies with GnRH agonists or KGF could help to expand
thymic stroma before HSCT to facilitate thymic engraftment of lymphoid precursor cells. Other possible adjuvant
strategies include the use of IL-7 to enhance peripheral T
cell reconstitution or the use of G-CSF in combination with
myeloid progenitors to enhance myeloid reconstitution.
In conclusion, the systematic utilization of selected (and
expanded) progenitor cell populations offers new options to
modify the cellular composition of the allograft and brings
us one step closer towards optimal graft engineering to
enhance GVT and antimicrobial activity, prevent graft
failure and minimize the risk for GVHD.
841
7.
8.
9.
10.
11.
12.
13.
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DOI 10.1007/s00109-007-0187-0
EDITORIAL
A key player in biomedical sciences and clinical service
in China, Chinese Academy of Medical Sciences (CAMS)
and Peking Union Medical College (PUMC)
Qimin Zhan & Depei Liu
Received: 24 November 2006 / Revised: 8 February 2007 / Accepted: 22 February 2007 / Published online: 17 April 2007
# Springer-Verlag 2007
Abstract The Chinese Academy of Medical Sciences
(CAMS) and Peking Union Medical College (PUMC) is the
largest medical institution in China and has a leading high-level
multidisciplinary medical research and medial service. Under
the CAMS and PUMC infrastructure, there are 17 biomedical
institutes and 6 large hospitals, which cover most fields of the
human disease-related research. CAMS and PUMC has always
attached great emphasis on the control and cure of severe
diseases, as well as a series of innovative drug researches, and
has made significant progress in those fields. The long-term
goals for CAMS and PUMC in the future development are:
reaching the international advanced level in the areas of severe
disease prediction, prevention, control, diagnosis, and research
on drug innovation; establishing theoretical and technological
system for explanation of the mechanism of severe diseases,
which possesses Chinese style and represents the frontier level
in the world, and at the same time, providing scientific support
for the prevention and treatment of severe disease and making
contribution to the establishment and development of a
harmonious society in China.
History of PUMC and CAMS
Founded by the Rockefeller Foundation in 1917 (Fig. 1),
Peking Union Medical College (PUMC) has grown to its
Q. Zhan : D. Liu (*)
Chinese Academy of Medical Sciences and Peking Union
Medical College,
9# Dong Dan San Tiao,
Beijing 100730, People’s Republic of China
e-mail: [email protected]
Q. Zhan
e-mail: [email protected]
present full-fledged scale after nine decades. In 1915, the
Rockefeller Foundation decided, based on its studies of
Asian countries, to establish a first-rate medical college in
Beijing. The Foundation acquired all the assets of the
Union Medical College from the London Missionary
Society and then purchased all the real estates of a royal
palace in Dong Dan San Tiao hutong in downtown Beijing.
It took USD 7.5 million to build this complex of 22.6-ha
coverage, comprising 14 buildings of the college, hospital,
offices, an auditorium, power room, etc. The complex was
designed and built as a classic Chinese architecture
featuring glazed tiles, upturned eaves, carved beams, and
painted rafters to add to the bright coloring (Fig. 2). All
these were in harmony with the constructing style of
Beijing as an ancient capital city of China. The interior
was designed and decorated at the highest standards of the
time to meet medical education, medical services, and
medical research needs. PUMC was unparalleled in Asia
for its library collection at the time. Most noteworthy of all
was its elites of entrepreneurship recruited as executives,
managers, and discipline leaders, from not only the USA
but also UK, Canada, and within China. They were mostly
young and vigorous people; for example, the first dean
Franklin McLeam was only 28 years old when he took
office. For these young people born in industrialized
countries, it challenged their courage and dedication to
come to China, a nation in the east so mysterious and
remote at that time. September 15–22, 1921 witnessed a
solemn opening ceremony of Peking Union Medical
College (PUMC), attended by principals and professors
from China, other Asian countries, Europe, and America, as
well as 50 delegates from academic associations and
international health organizations.
PUMC was founded in early twentieth century, modeling
on the pacemaker of reforms in medical education and
DO00187; No of Pages
846
Fig. 1 Founding ceremony of PUMC in 1917
hospital management in North American, the John Hopkins
Medical School. The guidelines for PUMC were established in view of the conditions and requirements specific
of China: build a first-rate medical college to educate firstclass medical professionals of clinicians, medical educationists, medical scientists, and public health administrators,
contributing to medical science in China and medicine of
the world. In this regard, the capacity of PUMC was
designed as 30 students per class (50 at maximum), a scale
matched by its classrooms and laboratories. Peking Union
Medical College’s hospital (PUMCH), a hospital designed
as 250 beds, was open to admit in-patients since June 1921.
Founded by the Rockefeller Foundation, the nursing school
of PUMC was also the best in China, which is known for its
advanced teaching methodology, education quality, especially its courses on public healthcare, which are followed
by counterparts in the country. To ensure the command of
natural sciences and English proficiency of the students,
PUMC established pre-medical programs in a number of
universities in China. Other characteristics of PUMC
include the “knock-out” mechanism in the entrance exam,
teaching in English, emphasis of hands-on practice, and
public health courses, with which PUMC became the
Fig. 2 Left Auditorium and,
right, back yard of PUMC/
CAMS buildings in classical
Chinese architecture, organization and different units of the
CAMS and PUMC
J Mol Med (2007) 85:845–850
pioneer of an 8-year curriculum on medical education and
undergraduate nursing programs in China.
In the three decades before the founding of the People’s
Republic of China and the takeover of PUMC, its medical
educationists made numerous contributions to medical
research. As early as PUMC was founded, scientific research
had been earmarked as one of its key commitments. A
recruitment policy was clearly laid down that senior teachers
must be capable of both education and scientific research, a
policy adhered to as of now. Another policy for research
subjects insisted on the emphasis of significant and pressing
issues in medicine and healthcare specific of China. PUMC
was a major contributor of first-class academic publications
carried on prestigious medical magazines such as Chinese
Medical Journal (Chinese/English edition) and Chinese
Journal of Physiology at that time. Papers and reports were
also frequently published on overseas medical journals. For
example, the world-known discovery and studies of “Peking
Man” skulls made by Professor Black Davidson of anthropology of the Department of Anatomy based on the fossil
teeth excavated from Zhoukoudian in Beijing. Invention and
studies of ephedrine, translation and study of the Compendium of Materia Medica (the most famous work on
Traditional Chinese Medicine in Ming Dynasty), biochemical inspection, and study of blood, all of which effectively
propelled the development of medical research in China.
Current CAMS and PUMC
Being the only university directly under the Ministry of
Health (MOH), PUMC is under the management of the same
executives as the Chinese Academy of Medical Sciences
(CAMS). CAMS was founded in 1956 to function as the only
state-level academic center in medical sciences and a highlevel multidisciplinary medical research institution in China.
PUMC played a major role in founding the two key medical
institutions of the new republic. In 1983, a number of research
institutes were separated from CAMS to establish the Chinese
Academy of Preventive Medicine (the Chinese Center for
J Mol Med (2007) 85:845–850
847
PUMC with rich resources of qualified faculty and expertise,
while PUMC trains high-quality graduates for CAMS. PUMC
and CAMS are mutual supportive and interdependent, share
each other’s strengths, and develop from teaching and research.
PUMC&CAMS
Education
system
Research
system
Medical
system
Basic Medicine
School
Basic Medicine Institute
Clinical School
Clinical Medicine Institute
Nursing School
Experimental Animal
Institute
Public Health School
Oncology Institute
Oncology Hospital
Cardiovascular Institute
Cardiovascular Diseases
Hospital
Plastic Surgery Institute
Plastic Surgery Hospital
Hematology Institute
Blood Diseases Hospital
Dermatology Institute
Skin Diseases Hospital
Continuing Education
School
PUMC Hospital
Medicinal Biotechnology
Institute
Materia Medica Institute
Biomedical Engineering
Institute
Medical Information
Institute
Medical Biology Institute
Microcirculation Institute
Establishments under PUMC/CAMS
CAMS and PUMC has under it 17 research institutes, 6
hospitals, 5 schools, 1 graduate school, 2 presses, 1 medical
industry groups, and 2 pharmaceutical enterprises, totaling a
workforce of 11,075 people. Thanks to the ceaseless efforts in
the past decades, a number of R&D bases have taken shape
under CAMS and PUMC, namely 4 state key laboratories, 9
ministry-level key laboratories, 16 state-level industrial experiment bases and centers, 18 state-level key disciplines of
medical sciences, 10 WHO cooperation centers, and 40
national-level academic institutions and associations. In addition, it is the sponsor of 15 and undertaker of 17 national-level
academic periodicals. See Fig. 3 for names of these units.
These hospitals and institutes are located in Beijing,
Tianjin, Nanjing, Chengdu, Kunming, and Hainan. Under
the unified management system of the hospitals and institutes,
the mechanism of shared head office and leaders is followed
in the Basic Medicine College and Basic Medicine Institute,
Fuwai Cardiovascular Hospital and Cardiovascular Institute
(Fig. 4), Plastic Surgery Hospital, and Plastic Surgery
Institute, Blood Diseases Hospital and Hematology Institute,
as well as the Skin Diseases Hospital and Dermatology
Institute. PUMC Hospital is characteristic of the unified
management system comprising the clinical medical school,
clinical teaching hospital, and research institute.
Radiation Medicine Institute
Blood Transfusion Institute
Yunnan Branch of MPI
Institute of Medicinal Plants
(MPI)
Hainan Branch of MPI
Pathogen Biology
Institute
Fig. 3 Units under CAMS and PUMC
Disease Control and Prevention—China CDC) along with
other institutions. China CDC is the state center for science
research center and technology guidance on preventive
medicine. In 1950, many senior medical professionals were
transferred from PUMC to establish the Academy of Military
Medical Sciences. Researches conducted by these institutions
stand for the highest academic level in the medical and public
health sector in China. In 1998, when the Chinese Academy of
Sciences and the Chinese Academy of Engineering released
respectively their first senior academicians, scientists from
PUMC amounted to half of those from medical science sector.
There are more academicians in CAMS and PUMC than any
other medical institutions in the country. CAMS provides
Fig. 4 Cardiovascular Institute and FUWAI Hospital
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Scientific research
In the time before 1954, science subjects were mostly
established to resolve clinical diagnosis or teaching demands
and confined to medical science in general, for such factors
as the changing management structure and teaching assignments, heavy workload of clinical medicine, and slow pace
in science research. Years after 1954 witnessed gradual
adjustment of science research planning made by the
departments and their inclusion into the state science and
technology plan. As China is rapidly becoming an economic
power in the world, thanks to its successful reforms and open
policy, state grants on science research are escalating year by
year as guided by the state administration policy “Science
and technology are the top productivity.” Against this
background, scientific research of CAMS and PUMC is
much more powerful than before.
PUMC maintains six disciplines with doctorate programs,
basic medicine, clinical medicine, biology, pharmacy,
combination of traditional Chinese medicine and western
medicine, and biomedical engineering. Under its divisions/
class-2 disciplines are 49 doctorate programs and 51 master
programs. There are six post-doctorate workstations of basic
medicine, clinical medicine, biology, biomedical engineering, public health, and preventive medicine.
PUMC is renowned for its 18 key national disciplines:
Genetics, Cell Biology, Biochemistry and Molecular Biology, Immunology, Pathology and Pathophysiology, Internal
Medicine (cardiovascular diseases), Internal Medicine
(blood diseases), Internal Medicine (digestive diseases),
Internal Medicine (endocrinology and metabolism diseases),
Dermatology and Venereology, Image Medicine and Nuclear
Medicine, Surgery (thoracic), Obstetrics and Gynecology,
Oncology, Anesthesiology, Pharmacochemistry, Microbiological and Biochemical Pharmacy, and Pharmacology.
These national ones are supplemented by three key disciplines of Beijing municipal level: Surgery (General),
Epidemiology and Health Statistics, and Pharmacognosy.
The leading position of PUMC in China is augmented by its
three national key labs, namely the State Key Laboratory of
Molecular Oncology, the State Key Laboratory of Medical
Molecular Biology, and the State Key Laboratory of
Experimental Hematology, and nine ministerial key laboratories, and 17 national-level research bases and centers.
Preliminary statistics of the research projects undertaken
by CAMS and PUMC in the recent 5 years are as follows:
Of the 3,514 projects undertaken at or above CAMS and
PUMC level, there are 1,969 national-level projects,
accounting for 56% of the total projects. Importance of
these projects is evidenced by the year-by-year grants
increase for the subjects, rising from RMB 78.1 million in
2001 to 189.9 million in 2005. Research grants to CAMS
and PUMC in the 5-year period total RMB 760 million.
J Mol Med (2007) 85:845–850
The following are facts and figures for the innovation
power of CAMS and PUMC: 83 patents and 73 certificates
of New Medicine inclusive of six class-one medicines. To
name a few, the first class-one new medicine of independent IPR, Biocyclol, and a new medicine for cerebral
ischemia, 3-n-butyphthalide (NBP). In the past five years,
178 research results of CAMS and PUMC have been
certified by competent academic authorities and received
160 awards for science research, including a first-prize
winner of the National Scientific and Technological
Progress Award—Studies for the Strategy, Prevention and
Treatment Technique and Measures for control and elimination nationwide of leprosy. In the past five years, 10,409
papers have been published, of which 1,224 were carried on
SCI. The following studies have been published on nature,
nature medicine, and Lancert, respectively. The infectious
diseases research has discovered that angiotensin-converting enzyme 2 (ACE2) is the in vivo receptor of severe
acute respiratory syndrome associated coronavirus (SARSCoV), interpreting the mechanism of lung failure caused by
SARS virus at molecular level; major corrections made to
the incidence of Parkinson’s disease among the Chinese
people in the epidemiological survey of the said disease;
finding this incidence as misleading in the past two
decades; studies of Goldthread as used in traditional
Chinese medicine found the blood-fat reducing mechanism
of its ingredient Berberine and important molecular targets;
positional cloning has successfully positioned the genes
associated with hereditary dentinogenesis imperfecta.
CAMS and PUMC made significant contributions to the
nationwide campaign against SARS outbreak in 2003, scoring
major research breakthroughs in its research work to be highly
commended by the state and the society, namely the
“Screening and Establishment of SARS-CoV Sensitive Animal Model”, “Studies of Inactivated Vaccine for SARS”, and
“Studies of SARS Pathogenic Mechanism”. Especially noteworthy are the studies on vaccines and animal models, which
are acknowledged by the Ministry of Science and Technology
as a major breakthrough in China’s science research. The
research results of or above ministry-level as contributed by
CAMS and PUMC account for 18–30% of the medicine and
pharmaceutical system of the country each year. Since its
founding, 2,000 research results of CAMS and PUMC have
been certified, winning 200 some national-level science/
technology prizes and 800 ministry/provincial-level prizes.
Medical care
CAMS and PUMC has under it six hospitals—PUMC
Hospital, Fu Wai Cardiovascular Disease Hospital (Fig. 4),
Cancer Hospital, Plastic Surgery Hospital, Blood Disease
Hospital (Tianjin), and Skin Disease Hospital (Nanjing).
These hospitals constitute a comprehensive medical care
J Mol Med (2007) 85:845–850
system known both at home and abroad, integrating medical
care, teaching and research, and offering new therapies and
new techniques from time to time. Ceaseless efforts of CAMS
and PUMC keep escalating the scale and capabilities of these
hospitals to the present level of 4,301 beds, averaging 2.80
million outpatients, 89,013 inpatients, and 43,715 patients for
operations on yearly basis. Of these hospitals, PUMC
Hospital (PUMCH) is designated as one of the “National
Technical Guidance Center for Rare and Severe Diseases” by
the Ministry of Health for its advantageous expertise,
technology, and resource. In 2004, PUMCH was designated
as a health care hospital for senior leaders of the state. Its
medical records section is named one of the “Three PUMC
Treasures.” Fuwai Hospital is the largest third-level grade-A
hospital for cardiovascular diseases in China; the Cancer
Hospital is the largest cancer hospital in Asia, and one of
the WHO collaboration centers; Plastic Surgery Hospital is
the largest academic authority of its kind in the world;
and the Blood Disease Hospital in Tianjin is the only nationallevel hematology clinical and research establishment in
China, which integrating clinical study and basic research.
This hospital maintains a leading position in the diagnosis
and treatment of hematologic malignancies, and the Skin
Disease Hospital in Nanjing is the largest and most
advanced in dermatology research, which offers academic
guidance to its counterparts in China for their diagnosis
and treatment of difficult skin disease including STDs and
leprosy.
Medical education
PUMC has an 8-year curriculum program in medicine and
an undergraduate nursing program, adhering to the education doctrine of “Small in scale, Elite in quality.” PUMC
has land coverage of 1.082 million square meters, including
a nursing school covering 23,000 square meters. PUMC has
33,000 full-time students.
The 8-year curriculum of PUMC was founded since 1917,
featuring its unique development model of by-section
education and mentor mechanism as always. After their
enrollment, the students have to take 2.5 years of pre-medical
courses and 5.5 years of medical courses, as only the best
graduates are granted with M.D. degree. The year 1995
witnessed the commencement of the dual-degree program
comprising M.D. and Ph.D. courses. Elite graduates from the
8-year curriculum with M.D. degree will proceed with a 3year curriculum, during which they will be receiving
intensified training on their scientific research capabilities.
When they graduate with required credits and qualified exam
scores, in addition to success in papers dissertation defense,
they will be granted with a Ph.D. degree.
The faculty of PUMC is what makes the difference. There
are 643 full-time teachers, of whom 229 with doctoral degree
849
and 160 with master degree. PUMC has 302 mentors for
doctorate candidates and 557 mentors for master candidates.
Prestigious professors make one of the “Three PUMC
Treasures.” Of the noted experts, professors and discipline
leaders of rich experience, outstanding academic achievements, and contributions, there are 11 academicians of the
Chinese Academy of Sciences and 15 academicians of the
Chinese Academy of Engineering (one of whom is an
academician for both academies), one member of the
Academic Degree Committee of the State Council. There
are also 77 middle-aged and young experts of outstanding
contributions as recognized at national or ministry level, nine
winners of the Professorship for Cheung Kong Scholar’s
Program (sponsored by the Ministry of Education), and four
“Excellent Talents in the New Century” as recognized by the
Ministry of Education. Recent years also see the return of
many excellent young scientists to China, further empowering
the research strength of CAMS and PUMC.
PUMC Library has been known as “Top in Asia,” being a
library of the longest history and largest collections in China.
Regarded as one of the “Three PUMC Treasures,” the library
has been cherished in PUMC. At present, the library has
been approved by the State Council as a national-level
central library and a medical branch of the state center for
science/technology books and documentation and a WHOcollaborating center for health and biomedicine information.
Its collections amount to 490,000 copies of books and 5,000
kinds of periodicals. The Chinese medical documentation
analysis and retrieval system provides medical information
to its counterparts in the country and maintains a medical
information network nationwide, contributing important
information for medical teaching, research, and medical care.
International collaborations
CAMS and PUMC attaches great importance to international
academic exchange and collaboration, as it maintains with
medical schools and research institutions in 20 countries
(regions) ties for academic exchange and science, education,
and medical care cooperation. Its nine WHO-collaborating
centers work with WHO to introduce intellectuals and funding
for furthering research, medical care, and education. PUMC
has conferred honorable titles of Honorary Professorship or
Guest Professorship to over 200 professors from other
countries (regions), namely, Dr. John Walker, the Nobel Prize
winner and director of Medical Research Council Dunn
Human Nutrition Unit, Samuel Chao Chung Ting, MIT
professor of Physics, and Dr. Tim Hunt, a UK MRC scientist
and Nobel Prize winner of Physiology or Medicine.
PUMC maintains interschool exchange agreements with
such prestigious medical schools as Harvard Medical
School, University of California School of Medicine, and
Chinese University of Hong Kong Faculty of Medicine,
850
under which PUMC exchanges senior students with the
schools for short-term clinical studies.
In adherence to its doctrine of elite education since its
founding, PUMC also endeavors to be an outstanding
organizational citizen in the country. For example, it takes
initiatives to adapt to the structural and strategy readjustments
of the state, to meet the social development and high-tech
development needs, and to meet the ever-rising demand of the
people for healthcare. On the basis of the small-scale and elite
education of its prestigious 8-year curriculum of medicine and
nursing courses, PUMC takes appropriate and steady steps to
expand its scale of graduate courses with reasonable adjustment of its levels and composition of the professions and with
reasonable setup and makeup of professions, relying on its
key disciplines and feature disciplines, in compliance with the
basic of medical education. These efforts have paid off with
the graduate courses shaped into a system centering on
medicine, with cross-disciplinary pattern combining science,
engineering, management, and philosophy.
To adapt to challenges and opportunities in the new
century, medical science has to develop in an integrated
J Mol Med (2007) 85:845–850
pattern featuring complete systems, reasonable makeup,
multi-regional, multi-sectional, multi-industry, multi-discipline, social, networked, internationalized pattern. Centering on the national key labs and state research centers,
PUMC will establish a medical science innovation system
characteristic of the assurance of resources and technology
sharing platform and international exchange platform. In
addition, CAMS and PUMC will join its resources to
develop a number of advantageous disciplines, research
bases and high-caliber teams and engage in systematic
researches for major diseases prevention and treatment and
leverage the role of national and ministry-level key labs to
launch basic and pioneering studies, provide clinical bases
for prevention and treatment studies based on national-level
research hospitals, and provide demonstration for comprehensive prevention and treatment of diseases based on
national-level community medical centers. In the future,
CAMS and PUMC will continue to center on research
subjects on human health and disease prevention and
treatment and work hand in hand with our international
counterparts to build the world a better place to live.
J Mol Med (2007) 85:851–861
DOI 10.1007/s00109-007-0232-z
ORIGINAL ARTICLE
Protein kinase C-ζ regulation of GLUT4 translocation
through actin remodeling in CHO cells
Xiao-Jun Liu & Chang Yang & Nishith Gupta & Jin Zuo &
Yong-Sheng Chang & Fu-De Fang
Received: 11 January 2007 / Revised: 25 April 2007 / Accepted: 31 May 2007 / Published online: 10 July 2007
# Springer-Verlag 2007
Abstract Actin remodeling plays a crucial role in insulininduced translocation of glucose transporter 4 (GLUT4)
from the cytoplasm to the plasma membrane and subsequent glucose transport. Protein kinase C (PKC) ζ has been
implicated in this translocation process, although the exact
mechanism remains unknown. In this study, we investigated the effect of PKCζ on actin cytoskeleton and translocation of GLUT4 in CHO-K1 cells expressing myc-tagged
GLUT4. Insulin stimulated the phosphorylation of PKCζ at
Thr410 with no apparent effect on its protein expression.
Moreover, insulin promoted colocalization of PKCζ and
actin that could be abolished by Latrunculin B. The
overexpression of PKCζ mimicked the insulin-induced
change in actin cytoskeleton and translocation of GLUT4.
These effects were also completely abrogated by Latrunculin B treatment. Using cell-permeable pseudosubstrate (PS)
inhibitor of PKCζ, the response to insulin could be
alleviated. Our results strongly suggest that PKCζ mediates
the stimulatory effect of insulin on GLUT4 translocation
through its interaction with actin cytoskeleton.
X.-J. Liu : C. Yang : J. Zuo : Y.-S. Chang (*) : F.-D. Fang (*)
National Laboratory of Medical Molecular Biology,
Institute of Basic Medical Sciences,
Chinese Academy of Medical Sciences & School of Basic
Medicine Peking Union Medical College,
Beijing 100005, China
e-mail: [email protected]
e-mail: [email protected]
N. Gupta
Department of Molecular Parasitology,
Humboldt University,
Berlin 10115, Germany
XIAO-JUN LIU
received PhD degree in Biochemistry and Medical Molecular Biology of Chinese Academy
of Medical Sciences & Peking
Union Medical College, Beijing,
China. His research interests
concentrate on the molecular
biological mechanism of type II
diabetes and gene transcriptional
regulation and function.
FU-DE FANG
is Professor of Biochemistry and
Medical Molecular Biology at
the Institute of Basic Medical
Sciences, Chinese Academy of
Medical Sciences & Peking
Union Medical College, Beijing,
China. His research interests are
in the molecular biological and
genetic mechanism of type II
diabetes.
Keywords Protein kinase C-ζ . Insulin . Actin cytoskeleton .
Glucose transporter 4
Abbreviations
PKCζ
protein kinase C-ζ
GLUT4
glucose transporter 4
Lat B
Latrunculin B
PS
PKCζ pseudosubstrate
CHO
Chinese hamster ovary
PI3K
phosphatidylinositol 3-kinase
CAP
Cbl-associated protein
852
IR
IRS
Akt/PKB
J Mol Med (2007) 85:851–861
insulin receptor
insulin receptor substrate
protein kinase B
Introduction
Glucose transporter 4 (GLUT4) protein, a major glucose
transporter with 12 transmembrane domains, is primarily
responsible for elevated glucose uptake in response to
insulin stimulation. During the basal state, GLUT4 predominantly localizes in poorly defined intracellular compartments including the trans-Golgi-network and recycling
endosomes, with minimal presence on cell surface. However, after insulin exposure, GLUT4 relocates to the plasma
membrane [1, 2]. The crucial role of GLUT4 on wholebody glucose homeostasis has been quite well established.
Understanding the regulation of GLUT4 and glucose
transport has proved to be extremely challenging, mainly
because it involves multiple and overlapping signaltransduction pathways governing vesicle transport. The
cytoskeletal network is also proposed to play an important
role in the regulation of GLUT4 translocation [3].
Cytoskeletal proteins are organized as the filamentous
networks distributed throughout the entire cell extending
from the plasma membrane to the interior of the nucleus.
These proteins account for a large fraction of total cellular
protein and are involved in all structural and dynamic
aspects of the living cells, including maintenance of cell
morphology, cell adhesion, motility, replication, apoptosis,
differentiation, and cell signaling [4]. In addition, cytoskeleton also plays an important role in insulin-induced
translocation of GLUT4 and glucose transport. The redistribution of GLUT4 in adipocytes requires insulin-dependent
dynamic remodeling of cortical and perinuclear actin [2] that
causes ruffling of the plasma membrane. Treatment with
actin-disrupting pharmacological agents completely abolishes insulin-mediated redeployment of GLUT4 to the
plasma membrane and subsequent glucose import [5].
The members of protein kinase C (PKC) are Ca2+dependent Ser/Thr protein kinases that are involved in
signaling pathway. So far, 12 PKC isoforms have been
identified in mammalian cells. These isoforms are categorized in three distinct subgroups: classical or conventional
PKCs (PKCα, βI, βII, and γ), novel PKCs (PKCδ, ɛ, η,
and θ), and atypical PKC (PKCζ and λ/ι). PKC isoforms,
being the regulator of numerous cell processes, are central
to intracellular signaling [6]. It has long been known that, in
most cell types, one or more PKC isoforms influence the
morphology of F-actin cytoskeleton and thereby regulate
processes that are dependent on remodeling of the microfilaments. These include, but are not limited to, cellular
migration and neurite outgrowth [7]. Considerable evidence
suggests that atypical protein kinase C isoforms (aPKCs),
serving downstream to insulin receptor substrates and
phosphatidylinositol (PI) 3-kinase, are required for insulinstimulated glucose transport [8]. Our recent transfection
experiments using L6-GLUT4myc cells and pEGFP-N1PKCζ construct indicate the redistribution of PKCζ along
with actin and their colocalization in response to insulin [9].
Furthermore, insulin is reported to trigger GLUT4
translocation to the cell membrane in Chinese hamster
ovary (CHO) cells [10–12]. In addition, the studies on
insulin-responsive aminopeptidase, a protein of unknown
physiological function that cotraffics with GLUT4, demonstrate the retention of this protein in the endosomal
systems of CHO cells and 3T3-L1 adipocytes [13]. Bogan
et al. observed that CHO cells exhibit several adipocytelike features and display insulin-responsive trafficking in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with high glucose. Both, 3T3-L1 as well as the
CHO cells harbor peripheral, vesicular insulin-responsive
compartments involved in GLUT4 trafficking [10]. In this
work, we investigated if PKCζ can modulate GLUT4
translocation through restructuring of cytoskeleton in
CHO cells. We found that the overexpression of PKCζ
resembles insulin-induced remodeling of F-actin stress
fibers and redistribution of GLUT4 to cell surface in CHO
cells. An actin-disrupting drug, Latrunculin B, is able to
reverse these effects. Using a cell-permeable pseudosubstrate (PS) of PKCζ, which is a potent inhibitor of PKCζ
enzyme activity, the effect of insulin could be annulled.
Finally, the overexpression of PKCζ stimulated the
translocation of GLUT4. Taken together, we demonstrate
that PKCζ has the ability to mediate insulin-induced
redistribution of GLUT4 to the cell surface by means of
cytoskeleton remodeling.
Materials and methods
Antibodies and reagents
The cell culture media and supplements were purchased
from Invitrogen (Carlsbad, CA). Anti-HA-monoclonal
antibody, antiactin antibody, soluble insulin, cell-permeable
PKCζ PS “myristoyl trifluoroacetate,” and phalloidin-TRITC
(tetramethylrhodamine isothiocyanate) were purchased from
Sigma-Aldrich (St. Louis, MO). The antibody against
Thr410 PKCζ/λ was obtained from Cell Signaling Technology (Beverly, MA). Polyclonal antibody against PKCζ (C20),
anti-myc-monoclonal antibody (9E10), and all secondary
antibodies were procured from Santa Cruz Biotechnology
(Santa Cruz, CA). Latrunculin B (Calbiochem) was dissolved
in dimethyl sulfoxide at a concentration of 2 mM.
J Mol Med (2007) 85:851–861
Cell culture and plasmid construction and transfection
The CHO-K1 cells were cultured as described elsewhere
[10]. Briefly, CHO-K1 cells maintained in Ham’s F12
medium supplemented with fetal bovine serum (10%),
glutamine (2 mM), penicillin (100 U), and streptomycin
(0.1 mg/ml). For experiments, cells were subcultured in
DMEM (high glucose) instead of Ham’s F12 medium.
pcDNA3-HA-PKCζ construct was kindly provided by Prof.
Farese (University of South Florida College of Medicine).
Myc-GLUT4-EGFP was constructed as follows: rat
GLUT4 cDNA containing a 14-residues c-myc epitope tag
in the first ectodomain [14] was kindly provided by
Yousuke Ebina (University of Tokushima). GLUT4myc
cDNA fragment was introduced into pEGFP-C1 vector (BD
Biosciences Clontech) between the HindIII and BamHI
restriction sites. The forward and reverse PCR primers were
5′-GACAAGCTTCGATGCCGTCGGGTTTCCAG-3′ and
5′-GACGGATCCTCAGTCATTCTCATCTGGC-3′, respectively. The CHO cells were transfected with pcDNA3-HAPKCζ or pEGFP-C1-GLUT4 or pcDNA3 using effectene
transfection reagent (Qiagen).
853
(TBST) containing 5% milk powder and 0.05% Tween 20.
They were incubated overnight at 4°C with indicated
primary antibodies, washed three times in TBST (10 min
each), and incubated with horseradish peroxidaseconjugated IgG for 1 h at room temperature. The membrane
was subjected to three additional TBST washes. Proteins
were visualized by enhanced chemiluminescence and
quantified by densitometry [9].
Fluorescence and confocal microscope
The CHO cells were grown on glass coverslips (24-mm
diameter) placed in six-well plates. Cells were cultured in
Cell stimulation and extraction of PKCζ
(cytoskeletal fraction)
Confluent CHO cells were preincubated in serum-free
medium for 3 h and then in the absence or the presence
of 100-nM insulin for the indicated times. The subcellular
fractions were obtained as described previously [15].
Briefly, cells were lysed and scraped into a protein lysis
buffer (20-mM Tris-HCl, pH 7.5, 250-mM sucrose, 1-mM
ethylene glycol tetraacetic acid, 2-mM ethylenediamine
tetraacetic acid, 50-mM mercaptoethanol, 2-mM phenylmethylsulphonyl fluoride, 5% glycerol, and 40-μg/ml
leupeptin), sonicated, and centrifuged at 100,000 g for
60 min. The supernatant was designated as the cytosolic
fraction. The pellet was resuspended in protein lysis buffer
containing 1% Nonidet P-40 and ultracentrifuged at
100,000 g for 30 min. The resulting supernatant contained
the solubilized particulate membrane fraction. The remaining Nonidet P-40 insoluble pellet was suspended in the
lysis buffer and designated as cytoskeletal fraction. Protein
concentrations were determined using the bicinchoninic
acid assay reagents (Pierce).
SDS-PAGE and immunoblotting
Proteins were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE; 10%
polyacrylamide) and electrophoretically transferred to polyvinylidine difluoride membrane. The membrane was
blocked for 2 h at room temperature in Tris-buffered saline
Fig. 1 Insulin-mediated PKCζ phosphorylation. a Cells were treated
with or without 100-nM insulin for different time intervals at 37°C.
Equal aliquots of total cell extract were subjected to 10% SDSPAGE and immunoblotted with indicated antibodies. b The amounts
of PKCζ Thr410-P and PKCζ proteins were quantified by densitometric
scanning using UVISoft-UVIB and Application V97.04. The pixel
intensity was normalized and a value of 1 was assigned to the basal
condition. The data are representative of three experiments. *P value<0.05
854
serum-free DMEM for 3 h and then with or without 2-μM
Latrunculin B for 2 h. Cells were treated with 100-nmol/l
insulin for indicated minutes at 37°C, fixed with 3% (v/v)
paraformaldehyde in phosphate buffered saline (PBS) for
20 min, washed with 0.1-mol/l glycine in PBS for 10 min,
permeabilized with 0.1% (v/v) Triton X-100 in PBS for
3 min, and then washed with PBS. For labeling of actin
filaments, fixed and permeabilized cells were incubated for
1 h at room temperature with TIRTC-labeled phalloidin
(0.01 U/coverslip). To assess autofluorescence, additional
samples were treated for 1 h in PBS but without phalloidin.
For immunostaining, fixed and permeabilized cells were
first incubated for 1 h at room temperature with primary
antibodies (dilution factors were: 1:100 for myc; 1:100 for
PKCζ; 1:100 for HA, in 0.1% (w/v) BSA (bovine serum
albumin)/PBS). Cells were washed with PBS and subsequently incubated either with flourescein isothiocyanate
(FITC)-conjugated goat antirabbit or antimouse secondary
antibodies, at a dilution of 1:250 for 1 h at room
temperature. Cells were washed by PBS and labeled with
600-nmol/l DAPI in PBS for 5 min at room temperature.
Samples were washed further with PBS and mounted onto
glass slides in ProLong Antifade solution. For fluorescence
and confocal laser microscopy, the stained cells were
examined with Leica confocal microscope [9].
Fig. 2 Insulin induces remodeling of actin cytoskeleton. Cells
were serum starved for 3 h,
treated (optional) with 2-μM Lat
B for 2 h, and then stimulated
with or without insulin (100 nM,
15 min, 37°C). They were
washed, fixed with formaldehyde, and stained with
rhodamine-phalloidin to visualize F-actin as described in
“Materials and methods.” Bar:
20 μm
J Mol Med (2007) 85:851–861
GLUT4 translocation assay
GLUT4 translocation assay was done as described
previously [16]. The CHO cells were cotransfected with
myc-GLUT4-EGFP and HA-PKCζ or control plasmid.
Cells were starved for 3 h in serum-free DMEM and
subjected to an optional treatment with 2-μM Latrunculin
B (2 h at 37°C) or 5-μM PS (45 min at 37°C) followed by
exposure to 100-nM insulin (37°C for 15 min). Cells were
washed twice in PBS and fixed with 3% paraformaldehyde
in PBS for 20 min. Samples were quenched for 10 min in
PBS supplemented with 0.05-M ammonium chloride.
Cells were incubated with antimyc antibody and detected
using antimouse TRITC-conjugated IgG. They were
analyzed by Leica confocal microscope. Images were
compiled from sections representing the entire z-axis of
the cells (12 sections of 0.5- to 1.5-μm separation). Image
analysis and quantification of the EGFP and myc were
performed using the Leica LCS Lite software.
Statistical analysis
Data are expressed as mean ± SEM. For Western blot, X-ray
films were quantified in the linear range by densitometry
using UVISoft-UVIB and Application V97.04. Differences
J Mol Med (2007) 85:851–861
Fig. 3 The overexpression of
PKCζ mimics insulin-induced
remodeling of actin cytoskeleton. Twenty-four-hour posttransfection cells were washed,
fixed, stained, and scanned as
described in “Materials and
methods.” a Cells transfected
with pcDNA3-HA-PKCζ; b
cells transfected with pcDNA3HA-PKCζ and treated with Lat
B; c cells transfected with
pcDNA3; d cells transfected
with pcDNA3 and treated with
Lat B. Bar: 20 μm
855
856
among means were analyzed by one-way analysis of
variance (ANOVA) (SPSS). P value < 0.05 was considered
to be significant.
Results
Insulin increases PKC-zeta phosphorylation at Thr410
It is well known that insulin induces the change in PKCζ
phosphorylation at Thr410 and its activity in adipocytes and
L6 skeletal muscle cells [9, 17, 18]. We questioned if insulin
would have the similar effect in CHO cells. Our results
indicate that insulin increased the PKCζ phosphorylation at
Thr410 by 1.2-fold in 5 min and 1.4-fold in 20 min (Fig. 1).
However, in contrast to L6 skeletal muscle cells, insulin
treatment did not affect the protein expression of PKCζ
(Fig. 1). In L6 cells, insulin not only promotes the phosphorylation of PKCζ, it also increases the expression of PKCζ [9].
Overexpression of PKCζ mimics insulin-induced
remodeling of actin cytoskeleton in CHO cells
A rapid and distinct change in F-actin stress fiber and an
increase in membrane ruffling (lamellipodia) have been
readily observed in insulin-stimulated L6 skeletal muscle
cells, 3T3-L1 fibroblasts, and CHO cells [9, 11, 19, 20].
The CHO cells were treated with 100-nmol/l insulin for
15 min, fixed, and stained with rhodamine phalloidin.
Similar to the previous reports, in the absence of insulin,
Fig. 4 Insulin promotes PKCζ
association with cytoskeleton. a
The Western blot of immunoreactive PKCζ in particulate and
cytoskeletal fractions of
CHO-K1 cells treated with
insulin. Cells were incubated
without or with 100-nM insulin
for 2, 5, 20 min. b The amounts
of PKCζ proteins were quantified by densitometric scanning
using UVISoft-UVIB and
Application V97.04. The pixel
intensity was normalized, and a
value of 1 was assigned to the
basal condition. The data are
representative of three
experiments. *P value<0.05
J Mol Med (2007) 85:851–861
actin stress fibers were distributed in parallel arrays along
the length of the cells. Insulin treatment provoked actin
reorganization and increased the formation of stress fibers
and ilopodia. Furthermore, treatment of cells with Latrunculin B caused a marked disassembly of the actin
cytoskeleton regardless of the presence of insulin (Fig. 2).
Similar to insulin, PKCζ also affects the actin cytoskeleton in various cells including NIH 3T3, myometrial, and
mesangial cells [15, 21, 22]. We have previously reported
that the overexpression of PKCζ in L6 skeletal muscle cells
triggered actin remodeling [9]; in this paper, we extended
our study to CHO cells to determine if PKCζ is indeed
involved in insulin-induced remodeling of F-actin. In fact,
as anticipated, the overexpression of PKCζ imitated
insulin-induced restructuring of F-actin cytoskeleton, and
Latrunculin B reversed this effect of PKCζ (Fig. 3).
Insulin promotes PKCζ association with cytoskeleton
Next, we investigated whether PKCζ participates in insulindependent reorganization of actin in CHO cells. Firstly,
5 min of insulin (100 nM) treatment caused a reduction in
immunoreactivity of PKCζ in the membrane fraction of
CHO cells as concluded by immunoblot analyses. In
contrary, the cytoskeleton-associated immunoreactivity of
PKCζ was enhanced (Fig. 4). In addition, we also detected
insulin-induced colocalization of PKCζ and actin by laser
confocal microscopy. PKCζ and actin showed moderate
colocalization in the absence of insulin; however, within
15 min of insulin exposure, an obvious colocalization of
J Mol Med (2007) 85:851–861
857
Fig. 5 Insulin-mediated colocalization of PKCζ with actin
as shown by laser confocal
microscopy. Cells were serum
starved for 3 h in DMEM. They
were treated with or without Lat
B (2 h at 37°C) followed by
exposure to insulin (100 nM,
37°C, 15 min). Cells were fixed,
permeabilized, and double
stained for PKCζ (anti-PKCζ
antibody followed by FITCconjugated secondary antibody)
and actin (TRITC-phalloidin) as
described in “Materials and
methods.” a Untreated control
cells; b treated with insulin
for 15 min; c treated with Lat B
for 2 h; d treated with insulin for
15 min and with Lat B for 2 h.
The arrows show the colocalization of PKCζ and actin in (b).
Bar: 10 μm
PKCζ and actin was detected (Fig. 5a,b). Latrunculin B
(2 μM) abolished the effect of insulin on colocalization of
PKCζ and actin (Fig. 5c,d). The binding of EGFP-PKCζ
and actin in CHO is not resistant to extraction with the
nonionic detergent Triton X-100 (data not shown) suggesting a weak association between these proteins.
Elevated PKCζ demonstrate insulin-like effects
on GLUT4 translocation
Progressive evidence supports the hypothesis that the actinbased cytoskeleton assists the translocation of GLUT4 to
the plasma membrane in response to insulin. In fact, insulin
has been shown to be the major trigger for F-actin
remodeling in a number of cell types regardless of GLUT4
expression [9, 11, 19, 20, 23–25]. In these studies, insulin
stimulated the phosphorylation of PKCζ and the association
of PKCζ with actin. To determine if PKCζ also participates
in GLUT4 translocation, we employed immunofluorescence microscopy. The CHO-K1 cells overexpressing
PKCζ or control plasmid were treated with Latrunculin B
to depolymerize the actin network or with PS to block the
PKCζ effect. GLUT4 translocation was measured using a
myc-GLUT4-GFP reporter molecule that exposes the mycepitope after the protein has fused with the plasma membrane
[14]. The treatment of control cells with 100-nM insulin for
15 min as well as the overexpression of PKCζ both
enhanced myc staining on the cell surface. To quantify
translocation, the level of exposed myc-epitope was
normalized to the total amount of expressed reporter protein
by measuring the fluorescence intensity of GFP. Insulin and
PKCζ promoted GLUT4 translocation to cell surface by
858
J Mol Med (2007) 85:851–861
Fig.
Fig. 66 The
The effect
effect of
of PKCζ
PKCζ on
on Myc-GLUT4-EGFP
Myc-GLUT4-EGFP translocation
translocation in
in
CHO
cells.
Cells
were
cotransfected
with
pEGFP-C1-Myc-GLUT4
CHO cells. Cells were cotransfected with pEGFP-C1-Myc-GLUT4
reporter
reporter along
along with
with pcDNA3
pcDNA3 or
or pcDNA3-HA-PKCζ.
pcDNA3-HA-PKCζ. They
They were
were
serum
starved
for
3
h
in
DMEM,
treated
with
or
without
Lat
serum starved for 3 h in DMEM, treated with or without Lat B
B for
for 2
2h
h
or
or 5-μM
5-μM PS
PS for
for 45
45 min
min at
at 37°C,
37°C, and
and exposed
exposed to
to 100-nM
100-nM insulin
insulin
(15
(15 min,
min, 37°C).
37°C). All
All samples
samples were
were fixed
fixed and
and stained
stained as
as described
described under
under
“Materials
“Materials and
and methods.”
methods.” GFP
GFP (green)
(green) fluorescence
fluorescence was
was used
used to
to
quantify the total reporter expression, and myc-epitope staining (red)
represented the reporter level at the cell surface. The quantification of
the fluorescence intensity ratio (myc/GFP) is based on an average
value of three independent datasets, involving at least 20 cells in each
experiments. The data were analyzed using one-way ANOVA. The
surface-to-total ratio normalizes the data for the level of reporter
expression. *P value<0.05
4.4-fold and 4.7-fold of the basal value, respectively
(Fig. 6). To further investigate the contribution of PKCζ
to insulin-mediated GLUT4 translocation through actin
remodeling, serum-starved cells were treated with 2-μM
Latrunculin B or 5-μM PS, followed by the addition of 100-nM
insulin and incubation at 37°C for 15 min. In the presence of
Latrunculin B or PS, the fluorescence value of surface
GLUT4 induced by insulin and by the overexpression of
PKCζ was distinctly inhibited (Fig. 6). These data are in
accordance with our previous studies revealing that the
overexpression of PKCζ as well as the insulin exposure
cause increases in surface GLUT4 and glucose uptake in
L6myc cells, and PS can inhibit these effects [9].
Collectively, these data implicate the role of PKCζ-induced
remodeling of actin cytoskeleton as a mechanism of
GLUT4 translocation to the cell surface.
exist in a folded state in which an autoinhibitory PS
sequence at the NH2-terminal regulatory domain aligns
with the substrate-binding site at the COOH-terminal
catalytic domain, and prevents the phosphorylation of its
extrinsic substrates as well as of its own threonine residues,
required for its activation. Thus, aPKCs are activated in two
steps: the unfolding of the folded state and the phosphorylation of specific threonine residue of aPKCs (PKCζ
Thr410/PKCλ Thr402). Furthermore, the autophosphorylation or transphosphorylation of other critical threonine
residues within the catalytic domain of aPKCs has also
been reported [8].
Atypical PKCs have both positive as well as the negative
effect on GLUT4 translocation. For positive regulation,
there are two distinct insulin signaling pathways, IRS-PI3K
(phosphatidylinositol 3-kinase) and Cbl-TC10-Par6 signaling cascades, which function in concert to mediate GLUT4
translocation and subsequent glucose uptake. aPKCs
function as a convergent downstream target in these two
signaling pathways [26]. Contradictorily, for insulin-CblTC10 signaling cascades, Czech group showed that siRNAmediated gene silencing of both Cbl isoforms (CAP
(Cbl-associated protein) or CrkII) in 3T3-L1 adipocytes
failed to attenuate insulin-stimulated deoxyglucose transport or myc-tagged GLUT4-GFP translocation at either
submaximal or maximal concentrations of insulin. The
dose–response relationship for insulin-induced deoxyglucose transport in primary adipocytes derived from c-Cbl
knockout mice was also identical to insulin action on
adipocytes from wild-type mice. These data suggested that
Discussion
Our results demonstrate that the insulin-induced GLUT4
translocation through cytoskeleton remodeling is mediated
by PKCζ. Insulin promoted the phosphorylation of PKCζ
at Thr410 from 1.2-fold to 1.4-fold in CHO cells. The
expression of PKCζ did not exhibit an apparent change,
however. This is in agreement with our previous studies on
L6 skeletal muscle cells except that PKCζ protein level was
also increased in L6 cells after insulin treatment. PKCζ
belongs to the class of aPKC, and the exact mechanism of
its activation remains unclear. Like other PKCs, aPKCs
J Mol Med (2007) 85:851–861
CAP and CrkII are not essential components of insulin
signaling to GLUT4 transporters [27]. R.V. Farese and other
researchers observed that adenovirus-mediated expression
of kinase-inactive aPKCs and RNA interference (RNAi)
targeting of aPKCs can inhibit insulin-triggered glucose
transport in L6 myotubes and 3T3/L1 adipocytes that could
be rescued by the expression of wild-type aPKCs [28]. In
addition, in vivo adenoviral delivery of recombinant PKCζ
stimulates insulin-mediated glucose transport in rat skeletal
muscle [29]. These are the most convincing evidences for
aPKCs involvement in insulin-stimulated glucose transport.
As a negative modulator, aPKCs can phosphorylate some
Ser/Thr residues of IRS1, and provide an autoregulatory
negative feedback mechanism for PI3K-mediated aPKCactivation and insulin-induced glucose transport [22]. Some
studies suggest that aPKCs can also negatively regulate
Akt/PKB activity through its interaction with Akt and
phosphorylation of Akt/PKB at Thr308/Ser473-independent
sites [30]. In contrary, Farese’s group found that the RNAi
on PKCζ/λ did not affect the PKB levels and the Ser473
phosphorylation/activation of PKB [28].
Emerging evidences support the hypothesis that cytoskeletal reorganization connects the event of GLUT4
translocation with the downstream molecules of insulin
signaling pathway [2, 3]. Insulin stimulates actin remodeling at the inner surface of the plasma membrane (cortical
actin) and in the perinuclear region that eventually contributes to the redeployment of GLUT4 to the cell surface [2].
This remodeling of cortical actin filaments promotes
membrane ruffling in cells as diverse as myotubes,
adipocytes, and CHO cells [9, 11, 19, 20]. Besides the
involvement of cortical actin in GLUT4 sorting, recent
studies have also implicated actin remodeling in the
regulation of Golgi transport and vesicle sorting leading to
the translocation of GLUT4 from the storage compartments
[2, 25]. In this study, we demonstrate that in the absence of
insulin, the actin stress fibers are normally distributed in
parallel arrays along the length of the cells. Treatment with
insulin elicited actin organization and increased the formation of stress fibers and ilopodia. Latrunculin B inflicted a
marked disassembly of the actin cytoskeleton regardless of
insulin exposure. Moreover, insulin-induced remodeling of
F-actin cytoskeleton in CHO cells was also triggered by the
overexpression of PKCζ, and the phenomenon could be
reversed by Latrunculin B. Previously, we transfected L6GLUT4myc cells with pEGFP-N1-PKCζ and observed the
redistribution of PKCζ and its colocalization with the
newly organized actin structures in response to insulin [9].
Indeed, insulin caused the decrease in the association of
PKCζ with the membrane fraction and promoted its
interaction with cytoskeleton, which was further corroborated by immunofluorescence analyses. The colocalization
of PKCζ and actin was also evident by laser confocal
859
microscopy. The data show that PKCζ and actin have a
insignificant overlap in the absence of insulin; however, the
colocalization of PKCζ and actin was increased considerably
within 5 min of insulin treatment. The actin-disrupting drug,
Latrunculin B, abolished this effect regardless of insulin.
The association of PKCζ and cytoskeleton appears to be
weak, as it is susceptible to treatment with Triton X-100.
All these data on L6 skeletal muscle and CHO cells
strongly advocate that PKCζ participates in insulin-induced
actin remodeling. Our previous studies have indicated that
PKCζ, similar to insulin, promotes GLUT4 translocation
and glucose import in L6 skeletal muscle, and PS inhibited
their effects. To further test if PKCζ would also affect
GLUT4 translocation in CHO cells that is dependent on
actin remodeling, GLUT4-myc-GFP was transfected into
CHO-K1 cells. As anticipated, PKCζ enhanced GLUT4
relocation, resembling the insulin effect, and it can be
completely disrupted by Latrunculin B or PS. This data
confirms that insulin-stimulated GLUT4 translocation
through actin remodeling is mediated by PKCζ despite its
apparently weak association with cytoskeleton. In most cell
types, one or more PKC isoforms influence the morphology
of F-actin cytoskeleton and thereby regulate processes that
are modulated by remodeling of the microfilaments [7].
Some studies also reveal the participation of aPKCs in the
reorganization of actin cytoskeleton [22].
Actin remodeling plays a crucial role in insulin-induced
GLUT4 translocation from the cytoplasm to the plasma
membrane and glucose transport [2, 3]. The silencing of
actin-based motor Myo1c by RNAi abrogated insulinstimulated translocation of GLUT4 [31]. Actin-regulatory
neural Wiskott-Aldrich syndrome protein (N-WASP) is
believed to transmit insulin signals to the actin network
and facilitate GLUT4 translocation, wherein Arp2/3 relay
signaling from TC10 to actin rearrangement [32]. Actindisrupting or -stabilizing pharmacological agents almost
completely abolished this insulin-mediated gain in GLUT4
at the plasma membrane and glucose uptake [5]. Collectively, these data support the notion that cortical F-actin is
required for insulin-stimulated GLUT4 sorting and glucose
transport. In addition to the active participation of actin,
microtubules and intermediate filament along with accessory cytoskeletal proteins may also have discernible effects
on GLUT4 translocation. However, the counteracting
evidences show that disruption of microtubules in rat
skeletal muscle cells does not inhibit insulin-stimulated
glucose transport [3]. Hence, it is conceived that PKCζinduced remodeling of actin cytoskeleton modulate GLUT4
translocation and glucose transport in insulin signaling
pathway. The mechanism of this aPKCs-induced cytoskeletal remodeling is not clear. One potential explanation is
that aPKCs would phosphorylate proteins that may regulate
cytoskeletal remodeling. Calponin, an actin-binding pro-
860
tein, can bind to actin and inhibit Mg-ATPase of myosin in
its unphosphorylated state. Upon phosphorylation of calponin by PKC, its inhibitory activity is lost. These studies also
reveal the physical association of PKC with calponin [33,
34]. Conversely, calponin can regulate PKC activity by
facilitating its phosphorylation [33]. Finally, a recent study
suggests that PKCζ can phosphorylate myosin II-B on a
specific serine residue in an EGF-dependent manner [35]. It
also indicates that PKCζ induces change in actin cytoskeleton by phosphorylation of accessory cytoskeletal proteins.
Further studies are needed to ascertain how aPKCs would
affect the remodeling of cytoskeleton and if aPKCs directly
participate in translocation of GLUT4 through Golgi
transport and vesicle sorting.
Acknowledgements We are grateful to Dr. Robert Farese for
providing us with PKCζ gene and to Dr. Yousuke Ebina for GLUT4.
We thank Xu Liu for the technical assistance with confocal laser
microscopy. This study was supported by collective grants from the
Major State Basic Research Development Program of China (973
Program 2004CB518602 and 2006CB503909) and the National
Natural Science Foundation of China (30471930). Xiao-Jun Liu and
Chang Yang contributed equally to this work.
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J Mol Med (2007) 85:863–875
DOI 10.1007/s00109-007-0159-4
ORIGINAL ARTICLE
Proteomic profiling of proteins dysregulted in Chinese
esophageal squamous cell carcinoma
Xiao-Li Du & Hai Hu & De-Chen Lin & Shu-Hua Xia &
Xiao-Ming Shen & Yu Zhang & Man-Li Luo &
Yan-Bin Feng & Yan Cai & Xin Xu & Ya-Ling Han &
Qi-Min Zhan & Ming-Rong Wang
Received: 23 July 2006 / Revised: 18 November 2006 / Accepted: 20 December 2006 / Published online: 22 February 2007
# Springer-Verlag 2007
Abstract Esophageal squamous cell carcinoma (ESCC) is
one of the leading causes of cancer death in China. In the
present study, proteins in tumors and adjacent normal
esophageal tissues from 41 patients with ESCC were
extracted, and two-dimensional electrophoresis (2-DE)
was performed using the pH 3–10 and 4–7 immobilized
pH gradient strips. The protein spots expressed differentially between tumors and normal tissues were identified by
matrix-assisted laser desorption/ionization and liquid chromatography electrospray/ionization ion trap mass spectrometry. A total of 22 proteins differentially expressed between
ESCC and normal esophageal tissues were identified, in
which 17 proteins were upregulated and 5 downregulated in
tumors. Biological functions of these proteins are related to
cell signal transduction, cell proliferation, cell motility,
glycolysis, regulation of transcription, oxidative stress
processes, and protein folding. Some of the proteins
obtained were confirmed by Western blotting and immunohistochemical staining. We showed that high expression
of calreticulin and 78-kDa glucose-regulated protein
(GRP78) were correlated with poor prognosis by Kaplan–
Meier analysis and log rank analysis. Zinc finger protein
410, annexin V, similar to the ubiquitin-conjugating
enzyme E2 variant 1 isoform c, mutant hemoglobin beta
chain, TPM4–ALK fusion oncoprotein type 2, similar to
heat shock congnate 71-kDa protein, GRP78, and pyruvate
kinase M2 (M2–PK) were for the first time observed to be
X.-L. Du : H. Hu : D.-C. Lin : S.-H. Xia : X.-M. Shen :
Y. Zhang : M.-L. Luo : Y.-B. Feng : Y. Cai : X. Xu : Y.-L. Han :
Q.-M. Zhan : M.-R. Wang (*)
State Key Laboratory of Molecular Oncology,
Cancer Institute (Hospital), Peking Union Medical College,
Chinese Academy of Medical Sciences,
P.O. Box 2258, Beijing 100021, People’s Republic of China
e-mail: [email protected]
MING-RONG WANG
received his Ph.D. in Histology–
Embryology–Cytogenetics from
the University of Clermont I,
France. He is presently Professor of Cell Biology in State Key
Laboratory of Molecular
Oncology and Deputy President
of Cancer Institute (Hospital),
Peking Union Medical College
and Chinese Academy of
Medical Sciences. His research
interests include biomarkers for
cancer diagnosis and molecular
mechanisms of carcinogenesis.
XIAO-LI DU
is a Ph.D. student in the State
Key Laboratory of Molecular
Oncology, Cancer Institute
(Hospital), Peking Union
Medical College and Chinese
Academy of Medical Sciences.
Her research focuses on protein
alterations associated with the
development and progression
of esophageal cancer.
dysregulated in human ESCC tissues. The proteins here
identified will contribute to the understanding of the
tumorigenesis and progression of Chinese ESCC and may
potentially provide useful markers for diagnosis or targets
for therapeutic intervention and drug development.
Keywords Esophageal squamous cell carcinoma .
Proteomics . Immunohistochemistry . Two-dimensional
electrophoresis (2-DE) . Mass spectrometry (MS)
864
Introduction
Esophageal cancer is one of the most common cancers
worldwide. Esophageal squamous cell carcinoma (ESCC)
is the most prevalent type of esophageal cancer in China
compared to adenocarcinoma in Western countries. Some
southern regions of the Taihang Mountains on the borders
of Henan, Shanxi, and Hebei provinces of China have
significantly higher mortality rates of esophageal cancer.
Although the prognosis of esophageal cancer has been
slowly improved over the past three decades, the 5-year
survival for ESCC patients who underwent operation
remains as low as 10% or even less [1]. The key reasons
for this disappointingly low survival rate are the lack of
effective screening markers for early detection and effective
targets for treatment. Additional markers are needed to
detect esophageal cancer earlier and to improve the
systemic treatment.
Over the past years, the molecular biology of esophageal
cancer has been extensively studied. Multiple genetic
alterations, such as loss of tumor suppressor genes and
activation of oncogenes, are associated with the development of esophageal cancers [2, 3]. DNA and complementary DNA (cDNA) microarray approaches have been used
to screen for important molecular changes in the ESCC
carcinogenesis [4–6]. A number of molecules have been
analyzed as possible prognostic factors in patients with
esophageal cancer. For example, epidermal growth factor
receptor overexpression has been shown to predict poor
prognosis in ESCC [7]. However, more effective markers
for early detection, prognosis, and more effective targets for
therapy need to be identified.
Proteins are responsible for the functional execution in a
diversity of cells. Many regulatory processes and disease
processes occur at the protein level, and most drug targets
are found through protein studies [8]. Two-dimensional
polyacrylamide gel electrophoresis (2-DE) is a highly
versatile technique for separating proteins according to
their charge and size. It has been used in comparative
studies of protein expression levels between healthy and
diseased states. For example, 2-DE coupled with mass
spectrometry (MS) have been successfully applied to
identify a part of tumor-associated proteins in various
cancers including squamous cervical cancer [9], prostate
cancer [10], renal cell carcinoma [11], gastric carcinoma
[12], and lung squamous carcinoma [13]. In the present
study, we analyzed protein profiles in 41 Chinese ESCC
compared to normal esophageal tissues adjacent to tumors.
By using an 18-cm pH-3 to pH-10 gradient and pH-4 to
pH-7 range immobilized pH gradient (IPG) strips, a total of
22 differentially expressed proteins were revealed and
identified by matrix-assisted laser desorption/ionization–
time of flight (MALDI–TOF) or liquid chromatography
J Mol Med (2007) 85:863–875
electrospray/ionization ion trap (LC–ESI–IT) MS. Confirmatory studies by Western blotting and immunohistochemistry validated the data obtained by proteomic method. The
proteins here identified are expected to lead to some clues
for further study of carcinogenic mechanisms and of
molecular markers for diagnosis or prognosis of ESCC.
Materials and methods
Surgical specimens
Fresh tissues including ESCCs and matched adjacent,
histologically normal tissues from a total of 41 patients were
procured from surgical resection specimens collected by the
Department of Pathology in the Cancer Hospital, Chinese
Academy of Medical Sciences, Beijing, China. All the
patients received no treatment before surgery and signed
separate informed consent forms for sampling. Primary
tumor regions and the corresponding normal tissues from
the same patients were separately excised by experienced
pathologists and immediately stored at −70°C until used.
Preparation of tissue protein samples
The frozen tissues were washed three times with chilled
phosphate-buffered saline (PBS). A total of 100 mg tissues
were ground into power in liquid nitrogen and lysed in 800μl lysis buffer (7-M urea, 2-M thiourea, 4% CHAPS, 40mM Tris, 0.5% parmalyte, 0.1 μg/ml PMSF, 2 μg/ml
aprotinin, DNase I, Dnase, and RNase), and the lysates
were placed on ice for 1 h. The mixture was centrifuged at
12,000 rpm at 4°C for 30 min to remove tissue and cell
debris. The protein concentration was determined by 2-D
quant kitTM (GE Healthcare). Aliquots of protein samples
were kept at a −70°C deep freezer until further use.
Two-dimensional electrophoresis
Isoelectric focusing electrophoresis (IEF) was performed
using 18-cm IPG drystrips (pI range, 3–10, 4–7; Amersham
Biosciences) on an IPGphor isoelectric focusing cell
(Amersham Biosciences) at 20°C with a maximum current
setting of 50 μA/strip. Soluble proteins were hydrated into a
different length of IEF strips for 13 h with the use of 100–
150 μg of protein lysates for silver staining and 1 mg of
protein for micropreparative 2-DE followed by Coomassie
brilliant blue staining. After rehydration, IEF run was carried
out using the following conditions: (1) 30 V, 13 h; (2) 200 V,
2 h; and (3) 1,000 V, 1 h; (4) 3,000 V, 1 h; (5) 8,000 V, 9 h.
Then the IPG strip gels were subjected to a two-step
equilibration. The first was with an equilibration buffer
consisting of 6-M urea, 30% glycerol, 2% sodium dodecyl
J Mol Med (2007) 85:863–875
sulfate (SDS), 50-mM Tris-HCl (pH 6.8), and 1% DTT (w/v)
for 15 min. The second step was with a buffer consisting of
6-M urea, 30% glycerol, 2% SDS, 50-mM Tris-HCl
(pH 6.8), and 2.5% iodoacetamide (w/v) for 15 min. After
dipping in SDS electrophoresis buffer, the strips were
transferred onto the second-dimensional SDS–PAGE
(PAGE, polyacrylamide gel electrophoresis) gel, sealed in
place with 1% agarose. Proteins were separated in the second
dimension with the use of a 1.0-mm thick 12.5% SDS–
PAGE polyacrylamide gels under running conditions of 16°
C, 20 mA per gel for 30 min, followed by 40 mA per gel for
4 h using Bio-Rad protean II vertical slab gradient
electrophoresis unit (Bio-Rad). Molecular weight markers
were also run in the second-dimensional gels to facilitate
molecular weight calibration of protein spots.
Silver staining and image analysis
Following the second-dimensional resolution, the gels were
immersed in 40% ethanol, 10% acetic acid at least for 1 h.
The gels were subsequently stained with silver nitrate and/or
Coomassie blue for image analysis and/or protein spot
identification by standard procedures. To evaluate the 2D
PAGE protein gel protein pattern, the stained gels were
analyzed by Imagemaster 2D elite software (Amersham
Pharmacia Biotechnology). After the auto-detection of
protein spots, gel images were carefully edited including
the quantification of protein spots, as well as the resizing,
alignment, and matching between different 2-DE gels and
between tumor and the corresponding epithelium cells. 2-DE
maps were then matched in a main matchset, and additional
small matchsets were constructed for pair-wise comparison
between duplicate analyses. The criteria to determine a
dysregulated protein spot was as follows: (1) The spot can be
observed on the 2-DE silver staining images at least in two
samples; (2) the average of the spot fold-change was not less
than 1.5 between the tumor and the normal tissues with
p<0.05 by Student’s t test.
In-gel digestion
To analyze and identify the protein spots of interest, alteredexpression protein spots from the preparative Coomassieblue-stained gels were digested by the procedure as
described by Beranova-Giorgianni and Desiderio [14].
Briefly, the protein spots were excised from the gels,
destained in 50-mM NH4HCO3/acetonitrile (60/40), and
dried by vacuum centrifugation. Digestion was carried out
with 12 μg/ml sequencing grade-modified trypsin (Roche)
in 50-mM NH4HCO3 buffer at 37°C for 12–16 h. Peptides
were recovered by extraction with 50% acetonitrile/5%
trifluoroacetic acid (TFA), dried in a speed vac and
resuspended with 15 μl of solution containing 50% CAN
865
and 2.5% TFA. The peptides were desalted by ZiptipTM C18 (Bio-Rad) and cocrystallized in a matrix of a-cyna-4hydroxycinnamic acid.
MALDI–TOF and LC–ESI–IT MS identification
of differentially expressed proteins
MS analysis was carried out using Bruker reflex III
MALDI–TOF equipped with the SCOUT source. All
MALDI spectra were externally and internally calibrated
using two standard peptide mixtures. The databases for
protein identification were searched at the websites http://
www.expacy.ch/tools/ and http://www.matrixscience.com
against the NCBInr protein database. Up to one missed
tryptic cleavage was considered, and a mass accuracy of
100 ppm was used for all the tryptic-mass searches. The
species of origin was restricted to Homo sapiens. A
capillary column LC–ESIion trap (IT) MS (LCQ–DECA
Xpplus; ThermoFinnigan, San Jose, CA, USA) was used to
obtain an amino acid sequence in the MS/MS mode. Ions
were detected in a survey scan from 400 to 1,500 amu
(3 μscans) followed by three data-dependent MS/MS scans
(5 μscans each; isolation width, 3 amu; 35% normalized
collision energy, dynamic exclusion for 3 min) in a
completely automated fashion. All MS/MS spectra were
searched against the NCBInr database with enzyme
constraints and with a static modification of +57 Da on
cysteine residue. The precursor-ion mass tolerance was
1.40 Da, and the fragment-ion mass tolerance was 1.50 Da.
We used Xcorr≥2.0 as Sequest criteria, and proteins with
two or more peptides sequenced were accepted as positive
identifications.
Western blotting analysis
Cell extracts were prepared (see above), and the total
protein (20–80 μg) was run on 12% SDS–polyacrylamide
gels transferred to the polyvinylidene fluoride membrane.
The membranes were blocked with 5% dry milk in TBST
(100-mM Tris-HCl [tris(hydroxymethyl)aminomethane]HCl, pH 8.0; 150-mM NaCl; and 0.05% Tween-20) and
probed with primary antibody for 2 h at room temperature.
After washing, the membranes were incubated with
peroxidase-conjugated secondary antibodies at 1:5,000
dilution for 1 h (Zhongshan Bio). The protein bands were
visualized by enhanced chemiluminescence (Applygen
Technologies). Sources of primary antibodies were as
follows: calreticulin (1:1,000; StressGen), 78-kDa glucoseregulated protein (GRP78; 1:500; Santa Cruz Biotechnology), Hsp27 (1:1,000; Santa Cruz Biotechnology), annexin
V (1:500; MBL Company), M2–PK (1:250; Abgent), and
Beta-actin (1:5,000; Sigma). All other antibodies in this
study were from Zhongshan Company.
866
Immunohistochemistry
Immunohistochemistry was carried out on the 5-μm
sections of paraffin-embedded specimens. All paraffinembedded sections were prepared by the Department of
Pathology, Cancer Hospital, Chinese Academy of Medical
Sciences. For immunohistochemical analysis, the slides
were deparaffinized, rehydrated, dripped in 3% hydrogen
peroxide solution for 10 min, heated in citrate buffer at
pH 6.0 and 95°C for 25 min, and cooled at room
temperature for 50 min. The slides were pretreated by
normal goat serum at 37°C for 30 min, and then incubated
with anti-calreticulin (1:400 dilution), anti-GRP78 (1:50
dilution), anti-Hsp27 (1:100 dilution), anti-annexin V
Fig. 1 Representative images of
2D gel for normal esophageal
tissues (a, c, e) and ESCC
samples (b, d, f). Isoelectric
focusing at pH 3–10 was carried
out at the first dimension using
18-cm IPG strips with low
loading of proteins (a, b) or high
amounts of proteins (c, d). Isoelectric focusing at pH 4–7 was
also carried out to improve the
solubility of the proteins (e, f).
The second-dimensional electrophoresis was performed by
SDS–PAGE on a 12.5% gradient gel. Numbers on the maps
indicate protein spots identified
by mass spectrometry and peptide mass fingerprinting
J Mol Med (2007) 85:863–875
(1:50) or anti-M2–PK (1:50) antibody at 37°C for 2 h.
After washing with PBS, the slides were incubated with
biotinylated second antibody (goat anti-mouse IgG/goat
anti-rabbit/IgG rabbit anti-goat IgG, 1:100 dilution) for
30 min at 37°C followed by streptavidin–peroxidase (1:100
dilution). The development of the slides was carried out
using diaminobenzidin solution. Counterstaining was carried out with hematoxylin. The immunoreactivity of the
proteins selected were recorded by staining intensity and
immunoreactive cell percentage as follows: tissues with no
staining were rated as 0, with a faint or moderate staining to
strong staining in ≤25% of cells as 1, or strong staining in
25 to 50% of cells as 2, and strong staining in ≥50% of
cells as 3.
J Mol Med (2007) 85:863–875
867
Statistical analysis
Results
Two-tailed Student’s t test was used for statistically
analyzing the data extracted from the comparison window
of the ImageMaster software that normalized volumes for
each protein spot. Associations between protein expression
alteration and clinical–pathological parameters were
assessed by Kruskal–Wallis test. Survival probabilities
were estimated using the Kaplan–Meier method and
analyzed by log-rank test. All the statistical analysis was
carried out with Statistical Package for the Social Sciences
13.0 for Windows software.
2-DE analysis of proteins from ESCC and normal
esophageal tissues
We investigated the proteomic profiles of tumor tissues and
the corresponding normal esophageal tissues from 41
patients with ESCC. The analysis was first carried out on
an 18-cm pH-3 to pH-10 gradient IPG strips for 18 samples
with 100-μg proteins loading (Fig. 1a,b). Polyacrylamide
gel with 12.5% SDS–PAGE was used in the seconddimension electrophoresis to separate proteins with molec-
Fig. 2 Representative images of upregulated spots 4 (a) and 11 (b) of 2-DE gel in multiple tumors. These two spots were MS-identified as zinc
finger protein APA-1 and calreticulin
868
J Mol Med (2007) 85:863–875
ular masses from 10 to 220 kDa. To display the proteins in
low abundance, other 15 samples were then detected on the
same range IPG strips (pH 3-10, 18 cm) with 150-μg
protein loading (Fig. 1c,d). Wide protein expression patterns
in 2-DE gel images were analyzed by ImageMaster version
5.0 software. In normal esophageal tissues, 917±76 spots
were detected, and in ESCC, 941±72 spots were observed.
To achieve the best separation of acidic proteins in firstdimensional IEF, 18-cm pH-4 to pH-7 range of IPG strips
were used for more than eight ESCC and normal tissues. The
spots 867±45 and 885±58 were detected on 2-DE gels of
normal and ESCC tissues, respectively (Fig. 1e,f). The
Table 1 Summary of statistical differences for proteins dysregulated between Chinese ESCC tissues and adjacent normal esophageal tissues
Spot Accession
no.a no.b
Protein name
1
2
3
4
gi|4502101
gi|4502101
gi|4502101
gi|37538029
5
6
7
8
gi|11036357
gi|999937
gi|12654345
gi|51467014
9
10
gi|13273496
gi|10441386
11
12
13
14
gi|4757900
gi|4503571
gi|30584049
gi|34707
15
gi|34707
16
gi|4505641
17
18
19
gi|7959791
gi|7959791
gi|2119712
20
gi|37550676
21
gi|2982114
22
gi|125604
23
24
25
26
27
gi|12314197
gi|12314197
gi|16507237
gi|28336
gi|4505763
Annexin I
Annexin I
Annexin I
Zinc finger protein 410 (Zinc
finger protein APA-1)
HSP27
Annexin V
Stratifin
Similar to ubiquitin –conjugating
enzyme E2 variant 1 Isoform
c; DNA-binding protein
Mutant hemoglobin beta chain
TPM4-ALK fusion
oncoprotein type 2
Calreticulin
Alpha enolase
Keratin 6A
Manganese superoxide
dismutase (MnSOD)
Manganese superoxide
dismutase (MnSOD)
Proliferating cell nuclear
antigen (PCNA)
PRO1708
PRO1708
Dank-type molecular
chaperone HSPA1L
Similar to heat shock
congnate 71-kDa protein
Crystal structure of human
recombinant procathepsin B
Pyruvate kinase, M2 isozyme
(M2–PK)
Annexin A2
Annexin A2
Heat shock 70-kDa protein 5 (GRP78)
Mutant beta-actin
Phosphoglycerate kinase 1
a
MW pId
(KD)c
Identification Peptide Protein
Fold
SDf
f
method
no.
coverage change
(%)e
p valuef Number
of
tissuesg
38.7
38.7
38.7
31.0
6.57
6.57
6.57
5.27
MALDI
MALDI
MALDI
MALDI
10
7
10
11
50.0
37.0
42.0
57.6
−3.41
−3.39
−3.31
+1.81
0.60
0.87
1.04
0.53
22.3
35.8
27.9
12.7
7.83
4.98
4.68
9.90
MALDI
MALDI
MALDI
MALDI
4
7
8
5
29.6
33.0
38.0
41.0
−2.88
+2.15
−4.39
−4.80
0.49 0.033
0.68 0.001
1.49 0.027
1.45 0.006
3:18
4:18
3:18
3:18
11.5
27.5
5.00 MALDI
4.77 LC–ESI–IT
6
3
77.0
20.3
−4.20
+1.75
0.58 0.018
0.26 0.029
4:18
4:18
48.1
47.5
60.2
24.7
4.29
7.01
8.09
8.35
6
16
2
3
26.1
45.0
5.1
5.1
+1.60
+4.57
+2.26
+4.27
0.18
0.85
0.63
1.17
0.023
0.012
0.037
0.020
5:15
7:15
7:15
7:15
24.7
8.35 LC–ESI–IT
3
7.2
+5.10
2.00 0.003
7:15
28.8
4.57 LC–ESI–IT
3
14.2
+2.48
0.05 0.026
2:8
32.2
32.2
69.9
6.61 LC–ESI–IT
6.61 LC–ESI–IT
5.42 LC–ESI–IT
7
5
4
32.5
24.7
7.8
+5.40
+4.65
+2.64
1.32 0.037
1.62 0.015
0.51 0.019
4:8
4:8
3:8
72.1
6.27 LC–ESI–IT
3
7.7
+4.22
1.06 0.028
2:8
35.2
5.89 LC–ESI–IT
2
8.8
+5.10
2.05 0.011
2:8
57.9
7.95 LC–ESI–IT
8
26.9
+2.52
0.08 0.027
2:8
38.6
38.7
72.3
41.8
44.6
7.57
7.57
5.07
5.22
8.3
6
7
6
6
5
44.0
25.7
19.3
26.7
24.5
+1.93
+2.37
+2.73
+2.20
+4.20
0.03
0.06
0.15
0.45
0.48
5:8
5:8
2:8
2:8
2:8
LC–ESI–IT
MALDI
LC–ESI–IT
LC–ESI–IT
MALDI
LC–ESI–IT
LC–ESI–IT
LC–ESI–IT
LC–ESI–IT
0.029
0.003
0.019
0.045
0.022
0.008
0.047
0.024
0.023
10:18
10:18
10:18
5:18
Protein spots described in Fig 1.
NCBI database accession number
c
Theoretical molecular mass (kDa)
d
Theoretical pI
e
Sequence coverage of MALDI and LC–ESI–IT MS analysis
f
Vol.% of spots in 2D gel of matched samples was measured by ImageMaster software. The “+” and “−” indicate the upregulated and
downregulated proteins in tumors, respectively, as compared with the normal tissues. The standard deviation and p values were calculated.
g
The number of samples containing protein alteration was listed.
b
J Mol Med (2007) 85:863–875
numbers on the 2-DE gel images indicated 27 protein spots
identified by MS and peptide mass fingerprinting, which
were differentially expressed between tumors and the
corresponding normal esophageal tissues. Figure 2 showed
the representative 2-DE gel zoom images of spots 4 and 11
identified as the protein zinc finger protein APA-1 and
calreticulin.
Identification of protein spots by MALDI–TOF
and LC–ESI–IT MS
The protein spots that differed prominently between ESCC
and the paired normal tissues were excised from the
Coomassie blue staining gel, then subjected to in-gel
trypsin digestion and identified by MALDI–TOF or LC–
ESI–IT MS. Twenty-seven spots representing 22 proteins
were revealed to be differentially expressed between the
cancer and adjacent normal esophageal tissues with p
values less than 0.05 (Table 1). Among the above 27 spots,
MALDI–TOF MS identified 11 spots including 4 upregulated (4 proteins) and 7 downregulated spots (5 proteins) in
ESCC. Figure 3 showed a typical PMF map of spot 6. The
amino acid sequence coverage for this spot was 33%, and
the protein score was greater than the significant score of 64
(p<0.05). The other 16 spots that represented 14 upregulated proteins were identified by LC–ESI–IT MS. For
example, the peptides sequenced by LC–ESI–IT MS were
matched to the calreticulin of spot 11. The amino acid
sequence FYGDEEKD is one of the peptides sequenced by
MS/MS, which is identical to the amino acid sequence 56–
64 of the calreticulin protein (Fig. 4). The score of the spot
Fig. 3 Peptide mass fingerprinting of protein from spot 6
(annexin V) in 2-DE map
869
was 26.1%, and a total of six peptides were sequenced,
significantly higher than the other candidates. Table 1 listed
all the 27 protein spots identified, protein accession
number, protein name, fold change, p value, etc. The
proteins upregulated in ESCC tissues were calreticulin,
alpha enolase, keratin 6A, manganese superoxide dismutase
(MnSOD), zinc finger protein 410 (APA-1), annexin V,
TPM4–ALK fusion oncoprotein type 2, proliferating cell
nuclear antigen, PRO1708, dank-type molecular chaperone
HSPA1L, similar to heat shock congnate 71-kDa protein,
crystal structure of human recombinant procathepsin, M2–
PK isozyme, annexin A2, heat shock 70-kDa protein 5
(GRP78), and mutant beta-actin and phosphoglycerate
kinase 1. The downregulated proteins in tumors included
annexin I, HSP27, Stratifin, similar to the ubiquitinconjugating enzyme E2 variant 1 isoform c, DNA-binding,
and mutant hemoglobin beta chain.
Western blotting and immunohistochemical analysis
of proteins dysregulated in ESCC
Five proteins including calreticulin, heat shock 70-kDa
protein 5 (GRP78), HSP27, annexin V, and M2–PK,
considering their possible functional role in tumorigenesis,
were selected for Western blotting analysis to verify the
results obtained by 2-DE and MS. Four cases of cancerous
tissues and adjacent normal esophageal tissues were
analyzed by Western blotting. The expression of calreticulin, GRP78, annexin V, and M2–PK were obviously
elevated in almost all four ESCC tissues (except for
870
J Mol Med (2007) 85:863–875
Fig. 4 Identification of spot 11
as calreticulin by LC–ESI–IT
MS. The amino acid sequence
FYGDEEKD is one of the peptides sequenced by MS/MS,
which is identical to the amino
acid sequence 56–64 of the
calreticulin protein
GRP78 in case 4), whereas the expression of HSP27 was
downregulated in tumors (Fig. 5).
Furthermore, immunohistochemical analyses were performed in paraffin-embedded tissues of 89 ESCC samples.
Figure 6 displayed the representative images of the
immunoreaction staining for the five proteins mentioned
above. For calreticulin and GRP78, normal esophageal
epithelial cells showed negative cytoplasm immunoreactions in majority cases, whereas the corresponding tumor
cells displayed strong positive immunoreactions in cytoplasm. The elevation of calreticulin expression was found
in 65 out of 89 tumors (p<0.01; Fig. 6a,b). An increased
expression of GRP78 was observed in 58 out of 89 tumors
as compared with the corresponding normal esophageal
epithelia (p<0.01; Fig. 6c,d). The Kaplan–Meier analysis
Fig. 5 Western blotting validation for calreticulin, GRP78,
Hsp27, annexin V, and M2–PK
expression in four ESCC tissues
(T) compared with the
corresponding normal esophageal tissues (N). Equal protein
loading was evidenced by detection of the beta-actin level
using a monoclonal anti-actin
antibody. The quality of each
band was detected by Glyko
BandScan version 4.3 software.
The T/N ratio of calreticulin,
GRP78, annexin V, and M2–PK;
the N/T ratio of Hsp27 for each
case was calculated. The graph
reflects an increase of calreticulin and GRP78, and decrease of
Hsp27 in ESCC tissues
revealed that the overexpression of calreticulin correlated
with a poor prognosis (p=0.002; Fig. 7a). Additionally,
the high expression of GRP78 predicted poor prognosis
(p=0.007; Fig. 7b). HSP27 was positive in all normal
esophageal epithelia, but drastically decreased in 57%
tumors (p<0.01). Besides, the increased positive expression
of M2–PK correlated to the tumor grade (p<0.05). The
staining pattern of the five proteins in cancer cells and the
frequency of alteration were summarized in Table 2.
Discussion
Carcinogenesis of ESCC is a complex process involving
multiple molecular events. Although some studies on
J Mol Med (2007) 85:863–875
Fig. 6 Representative immunohistologic features of proteins
showing significant differences
between the tumor and the
corresponding normal epithelia.
Normal esophageal epithelia
showed negative or weak expression of calreticulin, GRP78,
annexin V, and M2–PK, respectively (a, c, g, i), whereas strong
staining can be observed in
majority of the ESCC cells
(b, d, h, j). The expression of
Hsp27 drastically decreased in
ESCC cells (f) compared with
the normal epithelia (e)
871
872
J Mol Med (2007) 85:863–875
Fig. 7 Kaplan–Meier survival
analysis according to the “low”
or “high” expression of calreticulin (a) and GRP78 (b) in
ESCC patients
ESCC have been undertaken on the gene and transcription
levels, the underlying mechanism is still unclear. Proteomic
analysis gives a portrait of the proteins expressed in tissues
or cells and offers an understanding of cellular behavior.
Concerning human cancer, differential proteome analysis of
tumor and normal tissues allows the identification of
interesting proteins that might provide key information in
understanding carcinogenesis as well as finding biomarkers
useful for diagnosis and treatment of the disease. We
analyzed 41 pairs of ESCC and adjacent normal esophageal
tissues by 2-DE. LC–ESI–IT MS was used to sequence
peptides of the spots excised from the gel. A total of 17
proteins were observed to be up-expressed in ESCC tissues,
whereas five proteins were down-regulated. The biological
functions of these proteins are related to cell signal
transduction, cell proliferation, cell motility, glycolysis,
regulation of transcription, oxidative stress processes, and
protein folding. Our data showed that different isoforms for
some proteins could be observed on a 2-DE map. For
example, two isoforms of MnSOD (spot 14, 15) were
elevated in ESCC as compared to normal esophageal tissues.
Also, the present work demonstrated that the combined use
of narrow IPG (pH 4–7) for the first dimension and LC–
ESI–IT MS helps separate and detect the proteins with a
relative low abundance. More importantly, the proteins here
identified are expected to lead to some clues for explaining
the carcinogenic mechanism of ESCC.
Alpha enolase is a multi-functional enzyme in the glycolytic
pathway catalyzing the formation of phosphoenolpyruvate
from 2-phosphoglycerate. Downregulation of the enzyme was
Table 2 Expression pattern
of five proteins analyzed
by immunohistochemistry
reported in 26% of the primary non-small cell lung cancer and
was associated with a poor clinical outcome [15]. Decreased
expression of alpha enolase was also observed in squamous
cervical cancer [9], whereas overexpression of alpha enolase
was reported in squamous cell carcinoma of the lung [16,
17], head and neck [18], and esophagus [19]. We observed
that the expression of alpha enolase in ESCC tissues was
elevated in 47% (7:15) of the cases compared to adjacent
normal tissues, suggesting that alpha enolase might be one of
the candidate biomarkers for ESCC.
APA-1, a zinc finger protein, showed an overexpression in
ESCC (Fig. 2a). It is a transcription factor that activated the
transcription of matrix-remodeling genes such as MMP-1
during fibroblast senescence [20]. There were no previously
described associations between this protein and esophageal
cancer. Altered MMP-1 expression has been reported in a
wide variety of advanced cancers including colorectal cancer,
pancreatic cancer, gastric cancer, breast cancer, ovarian
cancer, and endometrial carcinomas [21–26]. In esophageal
cancer, the presence of MMP-1 was reported to be associated
with poor prognosis [27, 28]. It will be important to illustrate
whether the elevated expression of APA-1 would lead to the
enhancement of MMP-1 transcription, and whether APA-1
played a role in ESCC carcinogenesis.
Heat shock proteins, highly conserved cytoprotective
proteins in all species, have essential functions in protein
folding, transport, translocation, degradation, and assembly,
even under unstressed conditions. In this paper, we identified
several dysregulated heat shock proteins and chaperones
including heat shock 70-kDa protein 5 (GRP78), calreticulin,
Protein name
Staining pattern
Alteration in ESCC
Frequency of alteration (%)
Calreticulin
GRP78
Hsp27
Annexin V
M2–PK
Cytoplasm staining
Cytoplasm staining
Cytoplasm staining
Cytoplasm staining
Nuclear/cytoplasm staining
Upregulated
Upregulated
Downregulated
Upregulated
Upregulated
65:89
58:89
51:89
29:89
57:82
(73)
(65)
(57)
(33)
(70)
J Mol Med (2007) 85:863–875
proteins similar to heat shock congnate 71-kDa and danktype molecular chaperone HSPA1L.
GRP78 is an endoplasmic reticulum (ER) chaperone
protein and involved in many cellular processes including
the translocation of newly synthesized polypeptides across
the ER membrane, facilitation of the folding and assembly
of newly synthesized proteins, degradation of misfolded
proteins for proteasome, and regulation of calcium homeostasis. The expression level of GRP78 is highly elevated in
a variety of solid tumors, including breast cancer [29, 30],
lung cancer [31], gastric cancer [32], hepatocellular cancer
[33], and prostate cancer [34, 35]. It was reported that
GRP78 was overexpressed in a rat surgical esophageal
adenocarcinoma model induced by esophagogastroduodenal anastomosis compared to normal tissues by 2D protein
gel electrophoresis [36]. We showed that GRP78 expression
in ESCC was elevated compared to normal tissues. ESCC
patients with high expression of GRP78 showed a shorter
survival time than with low or no expression of GRP78
(p=0.007). However, in a study on the GRP78 expression
of lung cancer, patients with a positive GRP78 expression
showed a better prognosis than that without GRP78
reaction [37]. On the other hand, recent studies showed
that downregulation of GRP78 or inhibition of GRP78
activity by small molecules can offer a new therapeutic
approach for cancer. For example, a compound named
versipelostatin can specifically inhibit the induction of the
GRP78 promoter by glucose deprivation and disrupt other
components of the unfolded protein response. The compound selectively kills glucosedeprived cancer cells, and in
combination with cisplatin, it inhibits tumor growth in
xenografts [38]. These suggest that GRP78 might be a
potential molecular marker for prognosis and potential
target for gene therapy of some tumors including ESCC.
Another important upregulated chaperone protein found
in our observation was calreticulin. It is involved in the
regulation of intracellular Ca2+ homeostasis and endoplasmic reticulum Ca2+ storage capacity. Although calreticulin
expression was reported to decrease 5.31-folds in colorectal
cancer tissues [39], this protein was demonstrated to be
upregulated in breast ductal carcinoma [40, 41], prostate
adenocarcinoma [42], and hepatocellular carcinoma [38].
Elevation of calreticulin was found in primary ESCC by
using agarose 2-DE and agarose 2D difference gel
electrophoresis [43]. We showed that calreticulin was
overexpressed in 33% (5 out of 15) of the esophageal
tumors. Both Western blotting and immunohistochemistry
confirmed the dramatically increased expression of calreticulin in tumor tissues (Figs. 5 and 6). It has been reported
that the diagnostic accuracy of urinary calreticulin for
bladder cancer had a sensitivity of 73% and a specificity of
86% [44]. Furthermore, the detection of autoantibodies to
calreticulin may have utility for the early diagnosis of
873
pancreatic cancer and hepatocellular carcinoma [45, 46].
Studies in some tumor cell lines revealed that the increase
of calreticulin stability correlated with the resistance to
apoptosis [47, 48]. In this study, we showed that the
overexpression of calreticulin predicted a poor prognosis by
survival analysis (p=0.002). Additionally, we have used a
siRNA-based approach to study the function of calreticulin
in esophageal cancer cell line and showed that the
knockdown of calreticulin expression decreased the migration ability of cancer cells. Ito et al. [22] suggested that
calreticulin was directly involved in activating of the matrix
metalloprotease-2 in rhabdomyosarcoma cells. Further studies are needed to understand the relationship between the
upregulation of calreticulin and the carcinogenesis of ESCC.
M2–PK is an isoform of glycolytic enzyme pyruvate
kinase that is consistently altered during tumorigenesis. In
malignant cells, the dimeric form of M2–PK (tumor M2–
PK) is always predominant and is caused by direct
interaction of M2–PK with certain oncoproteins [49]. The
amount of tumor M2–PK was reported to be increased in
the ethylenediaminetetraacetic acid plasma of patients with
renal cell carcinoma [50], and pancreatic [51], lung [52],
breast [53], cervical [54], and gastrointestinal cancer [55].
Elevated tumor M2–PK was also observed in plasma
samples of patients with colorectal and stomach cancer,
and correlated with tumor size and stages [56, 57].
Immunohistological studies of Barrett’s esophagus with
monoclonal antibodies that specifically recognize the
dimeric form of M2–PK revealed the increased cytoplasmic
expression with progression along the metaplasia–dysplasia–adenocarcinoma sequence [58]. In this study, Western
blotting confirmed the elevation of M2–PK in ESCC.
Moreover, immunohistochemistry analysis revealed the
elevated expression of M2–PK correlating with the grade
of tumor. This may give clues to evaluate the new functional
role of M2–PK in grade-associated features of ESCC.
In summary, we have found 22 proteins differentially
expressed between ESCCs and normal esophageal tissues.
Some of them such as zinc finger protein 410; annexin V,
similar to ubiquitin-conjugating enzyme E2 variant 1
isoform c; mutant hemoglobin beta chain; TPM4–ALK
fusion oncoprotein type 2, similar to heat shock congnate
71 kDa protein; GRP78; and M2–PK have not yet been
reported to be associated to human ESCC before. High
expression of calreticulin and GRP78 have been revealed to
be poor prognostic factors of ESCC. Interestingly, the
upregulated expression of M2–PK was found to correlate
significantly with tumor grade. Further studies are required
to determine the roles of the identified proteins in
tumorigenesis and progression of ESCC. Also, the data
might provide other candidate protein markers for diagnosis
or targets for therapeutic intervention and drug development to human ESCC.
874
Acknowledgment This study was supported by the State Key Basic
Research grant of China (2001CB510208, 2002CB513101,
2004CB518705), Beijing Science Fund (7042038), Specialized
Research Fund for the Doctoral Program of Higher Education of
China (20050023046), and Program for Changjiang Scholars and
Innovative Research Team in University (IRT0416).
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DOI 10.1007/s00109-006-0151-4
ORIGINAL ARTICLE
Renalase gene is a novel susceptibility gene for essential
hypertension: a two-stage association study in northern Han
Chinese population
Qi Zhao & Zhongjie Fan & Jiang He & Shufeng Chen &
Hongfan Li & Penghua Zhang & Laiyuan Wang &
Dongsheng Hu & Jianfeng Huang & Boqin Qiang &
Dongfeng Gu
Received: 24 July 2006 / Revised: 19 September 2006 / Accepted: 2 November 2006 / Published online: 10 January 2007
# Springer-Verlag 2007
Abstract Renalase, a novel flavin adenine dinucleotidedependent amine oxidase, is secreted by the kidney,
degrades circulating catecholamines, and modulates cardiac
function and systemic blood pressure (BP). Its discovery
may provide novel insights into the mechanisms of BP
regulation and the pathogenesis of essential hypertension
(EH). We designed a two-stage case-control study to
investigate whether the renalase gene harbored any genetic
variants associated with EH in the northern Han Chinese
population. From the International Collaborative Study of
Cardiovascular Disease in Asia (InterASIA in China), 1,317
Q. Zhao : S. Chen : H. Li : P. Zhang : L. Wang : J. Huang :
D. Gu (*)
Department of Evidence Based Medicine and Division of
Population Genetics, Cardiovascular Institute and Fuwai Hospital,
Chinese Academy of Medical Sciences and Peking Union
Medical College,
No. 167 Beilishi Road,
Beijing 100037, China
e-mail: [email protected]
Q. Zhao : B. Qiang : D. Gu
National Human Genome Center at Beijing,
North Yongchang Rd 3-707,
Beijing 100176, China
Z. Fan
Department of Cardiology,
Peking Union Medical College Hospital,
Beijing 100730, China
J. He
Tulane University Medical Center,
New Orleans, LA 70112-2699, USA
D. Hu
Department of Epidemiology, College of Public Health,
Zhengzhou University,
Zhengzhou, Henan 450052, China
QI ZHAO
received her B.S. degree in
preventive medicine from
Peking University Health
Science Center in Beijing,
China. She is currently a Ph.D.
student of Prof. Gu at
Department of Evidence Based
Medicine and Division of
Population Genetics, Fuwai
Hospital, Chinese Academy of
Medical Sciences and Peking
Union Medical College in
Beijing, China. Her research
interests include identification of
genetic factors for essential
hypertension.
DONGFENG GU
is Professor of Epidemiology
and Medical Genetics, Chair of
Department of Evidence Based
Medicine and Division of
Population Genetics at Fuwai
Hospital, Chinese Academy of
Medical Sciences and Peking
Union Medical College in
Beijing, China. He received his
medical training in Nanjing
Medical University and Peking
Union Medical College in
Beijing and did his postdoctoral
training at University of
Minnesota and University of
Southampton. His research
interests in the genetic field
include identification of genetic
factors and gene–environmental
interaction for major
cardiovascular and related
diseases.
hypertensive cases and 1,269 normotensive controls were
recruited. These total 2,586 subjects were taken as the main
study population in this study. In stage 1, all the eight
selected single nucleotide polymorphisms (SNPs) of the
878
renalase gene were genotyped and tested within a subsample
(503 cases and 490 controls) of the main study population. By
single locus analyses, three SNPs, rs2576178, rs2296545,
and rs2114406, showed significant associations with EH (P<
0.05). In stage 2, these three SNPs were genotyped on the
remaining individuals and analyzed using all the individuals.
After Bonferroni correction for multiple comparisons, the
associations of rs2576178 and rs2296545 with EH were still
significant in stage 2. The cases had higher frequencies of
rs2576178 G allele and rs2296545 C allele than the controls
(0.55 versus 0.49, P<0.0001; 0.61 versus 0.55, P<0.0001).
Particularly, under the codominant model, the adjusted odds
ratios for rs2576178 GG genotype and rs2296545 CC
genotype were 1.58 (95% CI, 1.25 to 2.00; P=0.0002) and
1.61 (95% CI, 1.26 to 2.04; P=0.0002), respectively. We
also found risk-associated haplotypes and diplotypes, which
further confirmed the significant association between the
renalase gene and EH. These findings may provide novel
genetic susceptibility markers for EH and lead to a better
understanding of EH pathophysiology. In addition, further
replications in other populations and functional studies
would be warranted.
Keywords Case-control studies . Hypertension . Kidney .
Monoamine oxidase . Single nucleotide polymorphism
Introduction
Hypertension is not only a disease but a major risk factor
for cardiac, brain, and kidney pathology as well [1, 2].
Essential hypertension (EH) accounts for approximately
90–95% of patients diagnosed with hypertension. It is
widely believed that EH is a complex disease influenced by
multiple factors. Inherited predisposition combines with
environmental factors to determine the manifestation and
severity of this disorder [3]. The genetic element contribution to blood pressure (BP) variation ranges from 30 to 50%
[4, 5]. The genetic background of EH is complex and
currently not fully understood. Some candidate genes,
which were selected based on known mechanisms of BP
regulation, have shown association with EH [6]. However,
their variants associated with EH cannot fully explain the
genetic component of EH risk. One possibility is that some
biological mechanisms regulating BP level are still unknown to people. Therefore, the discovery of new molecules and pathways involved in BP regulation may lead to
the identification of novel gene sequence variations
participating in the susceptibility of EH.
Renalase is a novel flavin adenine dinucleotide (FAD)dependent amine oxidase and has been recently discovered
by Xu et al. [7]. They found that renalase was secreted by
the kidney, degraded catecholamines, and regulated sys-
J Mol Med (2007) 85:877–885
temic BP. The catecholamines include such compounds as
norepinephrine, epinephrine, and dopamine. It is well
known that catecholamines can regulate heart rate, myocardial contractility, and the tone of resistance vessels and
thus play an important role in controlling BP. Renalase
injection to Sprague-Dawley rats elicited a rapid depressor
response with a maximal decrease in mean arterial pressure
of almost 30%. The BP lowering effect of renalase was
explained by the degradation of circulating catecholamines,
which would be expected to decrease cardiac contractility
and heart rate. Certainly, the possibility could not be
excluded that the hypotensive effect of renalase might be
partly receptor-mediated.
As renalase is produced within the kidney, its major
function possibly resides within the organ [8]. Xu et al. [7]
found that dopamine was the preferred substrate of renalase,
followed by epinephrine and norepinephrine. Dopamine
produced locally is important in the paracrine/autocrine
regulation of renal tubular sodium transport [9–11]. Renal
sodium reabsorption is a critical process serving to maintain
both extracellular fluid volume and arterial BP. Proteins
participating in sodium reabsorption and its regulation are
therefore important candidate proteins whose genes may
contain sequence variations contributing to the inherited
tendency for EH [12]. It is possible that renalase can
regulate salt and water excretion to influence BP level by
directly metabolizing dopamine within the kidney. Therefore, the discovery of renalase may enhance knowledge of
the role of the kidney in BP control and lead to the
identification of novel genetic susceptibility markers for EH.
The main objective of our study was to investigate whether
the renalase coding gene was associated with EH in a northern
Han Chinese population. Recently, Satagopan et al. proposed
a two-stage design for association study that could provide
near-optimal power to detect the true marker conferring
disease risk while substantially reducing the total number of
marker evaluations [13, 14]. In the present study, we
conducted a two-stage case-control study which was similar
but not identical to the approach proposed by Satagopan et
al. In stage 1, all eight selected single nucleotide polymorphisms (SNPs) of renalase gene were genotyped and
tested in a relatively small case-control subsample of the
main study population. Initial associations found in stage 1
were taken as hypotheses that were further tested and
analyzed in stage 2 using the main study population.
Materials and methods
Subjects and strategy
All the studied subjects were recruited from the International Collaborative Study of Cardiovascular Disease in
J Mol Med (2007) 85:877–885
Asia (InterASIA in China). InterASIA used a four-stage
stratified sampling method to select a nationally representative sample of the general population aged 35 to 74 years
in China. A total of 15,838 persons completed the survey
and examination [15]. InterASIA stratified China into north
and south, as delineated by the Yangtze River. In this study,
we enrolled 1,317 hypertensives (655 men and 662 women)
with systolic BP (SBP)≥150 mmHg, diastolic BP (DBP)≥
95 mmHg, or current use of antihypertensive medication
and 1269 healthy normotensives (658 men and 611 women)
with SBP<140 mmHg and DBP<90 mmHg from the
northern field centers of InterASIA, namely, Beijing, Jilin,
Shandong, and Shanxi. These total 2,586 subjects were
unrelated and taken as the main study population in this
study.
We adopted a two-stage association study strategy. From
the main study population, we selected 993 subjects as a
subsample containing 503 hypertensive patients (SBP≥
160 mmHg and/or DBP≥100 mmHg) and 490 age- and
gender-matched normotensive controls (SBP<140 mmHg
and DBP<90 mmHg). The criterion for case selection of
the subsample was based on the hypothesis that individuals
with higher BP were likely to be enriched for genetic
susceptibility, which might increase the difference in
frequency of susceptibility alleles between cases and
controls to improve power. Under this two-stage approach,
all the selected SNPs were genotyped and tested at stage 1
using the subsample, and the promising SNPs were
genotyped on the remaining individuals and tested using
the main study population at stage 2.
During clinic or home visits, trained research staff
administered a standard questionnaire. They obtained
information on demographic characteristics including age,
gender, ethnicity, education, occupation, and household
income. The interview also included questions related to the
diagnosis and treatment of hypertension [16]. Three BP
measurements were obtained from each participant by
trained and certified observers according to a standard
protocol recommended by the American Heart Association
[17]. BP was measured with the participant in the sitting
position after 5 min of rest. In addition, participants were
advised to avoid alcohol, cigarette smoking, coffee/tea, and
exercise for at least 30 min before their BP measurement.
Subjects with a clinical history of secondary hypertension,
coronary heart disease, diabetes, and chronic kidney disease
were excluded from the study. The protocol was approved
by the local bioethical committee, and informed consent
was obtained from each participant.
879
C10orf59). There were 749 entries of SNPs for the renalase
gene in the public NCBI Single Nucleotide Polymorphism
database (dbSNP, build126; available at http://www.ncbi.
nlm.nih.gov/SNP/; last accessed May 25, 2006), and 389 of
them had available frequency data among Han Chinese in
Beijing, China (CHB) from the International HapMap
project website http://www.hapmap.org/; HapMap Public
Release #20/phaseII; last accessed May 25, 2006). The
renalase gene SNPs were selected based on the following
criteria: minor allele frequency (MAF)≥0.05 in CHB
validated by the HapMap and location in putative functional regions of the gene (e.g., exons, gene flanking regions,
and exon/intron boundaries). Under these criteria, eight
SNPs were selected for genotyping in stage 1 (Table 1), and
their relative physical positions are presented in Fig. 1. SNP
rs2296545 was the only nonsynonymous-coding SNP in the
renalase gene at the time of database research, which
resulted in an amino acid substitution (aspartic acid to
glutamic acid at codon 37, Asp37Glu) and might affect the
function of the gene product. The other SNPs were located
at flanking regions or near exon/intron boundaries where
gene variants might cause variation in gene regulation and
expression or differential splicing. Our goal was not to
investigate all of the renalase gene sequence variation but to
generate a set of markers that could partly represent the
functional region of the gene.
Genotyping
Blood for genotyping was taken into ethylenediamine
tetraacetic acid (EDTA)-containing receptacles; DNA was
isolated according to a standard phenol-chloroform method
and stored at −20°C until required for batch genotyping. All
SNPs were genotyped according to standard polymerase
chain reaction and restriction fragment length polymorphism methods. The primers and related restriction endonuclease can be obtained by request. Ninety-six randomly
Table 1 Selected SNPs in renalase gene region
SNP
dbSNP accession number
Region
Allelesa
SNP selection
1
2
3
4
5
6
7
8
rs2576178
rs2296545
rs2765446
rs11202776
rs1648512
rs10887800
rs1035796
rs2114406
5′ flanking
Exon 2
Intron 4
Intron 5
Intron 6
Intron 6
Intron 7
3′ flanking
G/A
C/G
T/C
C/T
A/G
A/G
C/T
A/G
The renalase gene is located on chromosome 10 at q23.31
and spans 309,388 bp with nine exons (official symbol:
MAF minor allele frequency
a
Major/minor allele
b
MAF in the controls of stage 1 study
MAFb
0.48
0.44
0.46
0.12
0.32
0.50
0.47
0.22
880
J Mol Med (2007) 85:877–885
Fig. 1 Genomic structure of the human renalase gene and location of the eight genotyped SNPs in stage 1 study. Exons are shown as vertical
lines. The locations of SNPs are indicated by arrows and they are denoted numerically with reference to Table 1
selected individuals were genotyped again for quality
control with complete concordance.
Statistical analysis
In stage 1, we used single locus analyses to detect initial
associations between the eight tested SNPs and EH. The
alleles and genotypes of the SNPs were counted, and their
distributions between the case and control groups were
compared by the χ2 test. The criterion used to select SNPs
for stage 2 study was that the SNPs had a P value of <0.05
for the comparisons of allele frequencies or genotype
distributions (under 2-df codominant, 1-df dominant, and
1-df recessive models) between the cases and the controls
of stage 1. Because stage 1 served for hypothesis
generation, correction for multiple comparisons was not
done.
In stage 2, all statistical analyses were performed using
the main study population. In single locus analyses, a
stepwise logistic regression was conducted to adjust for
covariates including age, gender, body mass index (BMI),
glucose (Glu), triglycerides (TG), total cholesterol (TC),
high density lipoprotein cholesterol (HDL-C), creatinine
(Cr), and smoking and drinking status. Multiple testing was
adjusted using Bonferroni correction. Multilocus analyses
including haplotype and diplotype analyses were also
performed in stage 2. To test the associations of statistically
inferred haplotypes with EH, we used the Haplo.score
approach as outlined by Schaid et al. [18]. The method
models an individual’s phenotype as a function of each
inferred haplotype, weighted by their estimated probability,
to account for haplotype ambiguity. To obtain odds ratios
(ORs) of risk haplotypes, the Haplo.glm approach was
performed [19]. Both Haplo.score and Haplo.glm were
implemented in the Haplo.stats software. A diplotype
analysis was then followed by using a weighted logistic
regression, with the weights being the probability for each
possible haplotype pair combination for an individual as
estimated by Haplo.score. Only the haplotypes and diplotypes with frequency >5% were considered for the
haplotype and diplotype analyses, respectively.
Descriptive statistical analyses were performed with
Statistical Analysis Software (SAS; SAS Institute, Cary,
NC, USA). Hardy–Weinberg equilibrium (HWE) of the
SNPs was evaluated by Fisher’s exact test using the
program HWE [20]. The pattern of pairwise linkage
disequilibrium (LD) between the SNPs was measured by
D′ and r2 calculated by the program Haploview [21].
Results
Clinical characteristics
Table 2 shows the characteristics of the subjects included in
the stage 1 and stage 2 studies. Age and the percentage of
men were not significantly different between cases and
controls in both studies. The cases generally had lower
HDL-C and higher BMI, TC, TG, and Glu levels than the
corresponding controls. In both studies, there were no
significant differences in the prevalence of drinking and
smoking between cases and controls. As expected, the SBP
and DBP levels were both significantly higher in the cases
of stage 1 than those of stage 2 (both P values<0.0001).
Single locus analyses in stage 1 study
During stage 1, which served for hypothesis generation, we
evaluated all eight selected SNPs in the subsample. A
graphical representation of pairwise LD as measured by |D′|
is displayed in Fig. 2. Table 3 summarizes the genotype and
allele distributions of the eight SNPs among the cases and
controls involved in stage 1. All SNPs were in HWE in
both cases and controls. We found that the frequencies of
rs2576178 G allele and rs2296545 C allele in the cases
were significantly higher than those in the controls (both P
values<0.05). The differences in allele frequencies of
rs2114406 between the case and control groups showed a
trend toward statistical significance (P=0.073) and a
modest dominant effect of rs2114406 G allele was also
found (P=0.047). According to the criterion defined for
stage 1, these three SNPs, rs2576178, rs2296545, and
J Mol Med (2007) 85:877–885
881
Table 2 Characteristics of the subjects in stage 1 and stage 2 studies
Characteristics
Male (%)
Age (years)
SBP (mmHg)
DBP (mmHg)
BMI (kg/m2)
TC (mmol/l)
HDL-C (mmol/l)
LDL-C (mmol/l)
TG (mmol/l)
Glu (mmol/l)
Cr (μmol/l)
Smokers (%)
Drinkers (%)
Stage 1
Stage 2
Cases (n=503)
Controls (n=490)
Cases (n=1317)
Controls (n=1269)
52.1
53.6±9.3
177.07±28.05*
104.34±12.28*
26.32±3.85*
5.23±0.99****
1.25±0.30***
3.19±0.86
1.70±1.06*
5.93±1.79***
71.22±14.59****
40.6
34.4
52.4
53.5±9.2
117.47±11.64
75.05±8.00
24.30±3.56
5.06±1.05
1.32±0.34
3.09±0.87
1.43±0.86
5.60±1.68
69.20±11.57
43.1
33.5
49.7
54.2±10.2
159.70±26.13*
95.85±12.70*
25.99±3.64*
5.14±0.97*
1.25±0.30**
2.81±1.29****
1.61±0.94*
5.60±1.24*
70.85±14.08
40.0
31.9
51.9
53.5±9.5
115.21±10.72
73.69±7.81
24.02±3.44
4.93±1.04
1.29±0.32
2.71±1.23
1.40±0.89
5.38±1.20
70.18±12.67
43.0
30.5
Mean ± standard deviation values for continuous variables
SBP systolic blood pressure, DBP diastolic blood pressure, BMI body mass index, TC total cholesterol, HDL-C high density lipoprotein
cholesterol, LDL-C low density lipoprotein cholesterol, TG triglyceride, Glu glucose, Cr creatinine
*P<0.0001 for comparison with corresponding controls
**P<0.001 for comparison with corresponding controls
***P< 0.01 for comparison with corresponding controls
****P<0.05 for comparison with corresponding controls
rs2114406, would be taken as hypotheses and enter stage 2
study.
Analyses in stage 2 study
In stage 2 study, the three SNPs (rs2576178, rs2296545,
and rs2114406) associated with EH in stage 1 were
genotyped on the remaining cases and controls and
analyzed using all 2,586 individuals who had been taken
as the main study population. We performed not only single
locus analyses but also multilocus analyses, which included
haplotype and diplotype analyses. Through the single locus
analyses, we found that rs2576178 G allele and rs2296545
C allele were significantly higher in the cases than in the
controls (both P values<0.0001), and the genotype distributions of these two SNPs were also significantly different
between the two groups (P<0.0001 and P=0.0001, respectively). However, the association of rs2114406 with hypertension was not replicated in this stage (Table 4). After
Bonferroni correction, where was set at 0.0063 (0.05 of
8), rs2576178 and rs2296545 were still significantly
associated with EH. Particularly, under the codominant
model, the adjusted ORs for EH associated with
rs2576178 GG genotype (GG versus AA) and rs2296545
CC genotype (CC versus GG) were 1.58 (95% CI, 1.25 to
2.00; P=0.0002) and 1.61 (95% CI, 1.26 to 2.04; P=
0.0002), respectively (Fig. 3).
In the haplotype analyses, we found that five haplotypes
constructed by these three SNPs had the frequencies >0.05
and accounted for 95.5% haplotype variations (Table 5).
The adjusted haplotype global score test was significant
(P=0.0007, 5 df), and the frequencies of Hap2 (G-C-A) and
Hap3 (G-C-G), which consisted of both rs2576178 G and
rs2296545 C risk alleles, were significantly higher in the
cases than in the controls. With Hap1 (A-G-A) used as the
Fig. 2 Linkage disequilibriums among eight SNPs in the controls of
stage 1 study. The numbers inside the squares are D′×100%
882
Table 3 Genotype distributions
and allele frequencies of
the eight SNPs tested in stage 1
Dominant and recessive
models were based on minor
allele of each locus. P
values<0.05 are shown in bold.
a
With degree of freedom 2
With degree of freedom 1
b
J Mol Med (2007) 85:877–885
SNP
Genotype
Allele
frequency
P value (χ2-test)
Codominant
modela
1 rs2576178
Cases
Controls
2 rs2296545
Cases
Controls
3 rs2765446
Cases
Controls
4 rs11202776
Cases
Controls
5 rs1648512
Cases
Controls
6 rs10887800
Cases
Controls
7 rs1035796
Cases
Controls
8 rs2114406
Cases
Controls
GG/GA/AA
158/263/82
130/249/111
CC/CG/GG
182/247/74
156/232/102
TT/TC/CC
149/268/86
136/255/99
CC/CT/TT
390/108/5
375/109/6
AA/AG/GG
225/215/63
221/223/46
AA/AG/GG
117/271/115
115/256/119
CC/CT/TT
149/254/100
133/249/108
AA/AG/GG
283/186/34
306/154/30
reference, the adjusted ORs for EH associated with Hap2
and Hap3 were 1.20 (95% CI, 1.03 to 1.41; P=0.0220) and
1.46 (95% CI, 1.20 to 1.78; P=0.0002), respectively.
Similarly, diplotype analyses showed that compared with
Dip1 (Hap1-Hap1), the ORs for Dip2 (Hap1-Hap2), Dip3
(Hap2-Hap2), and Dip4 (Hap2-Hap3) were 1.56 (95% CI,
1.13 to 2.15; P=0.0071), 1.64 (95% CI, 1.15 to 2.34; P=
0.0066), and 1.88 (95% CI, 1.28 to 2.78; P=0.0014).
G/A
0.58/0.42
0.52/0.48
C/G
0.61/0.39
0.56/0.44
T/C
0.56/0.44
0.54/0.46
C/T
0.88/0.12
0.88/0.12
A/G
0.66/0.34
0.68/0.32
A/G
0.50/0.50
0.50/0.50
C/T
0.55/0.45
0.53/0.47
A/G
0.75/0.25
0.78/0.22
Dominant
modelb
Recessive
modelb
Allele
0.026
0.090
0.012
0.012
0.034
0.148
0.012
0.018
0.436
0.516
0.209
0.265
0.896
0.707
0.729
0.673
0.264
0.907
0.114
0.406
0.843
0.938
0.597
0.787
0.578
0.403
0.386
0.300
0.136
0.047
0.683
0.073
50% of the individuals in stage 1 and evaluating the most
promising 10% of the markers on the remaining individuals
in stage 2 provides a practical cost-effective strategy for
association studies. Although this is only a general
guideline, it is helpful to improve association study design
for disease gene mapping. On the other hand, the correct
Table 4 Genotype distributions and allele frequencies of the three
SNPs tested in stage 2
Discussion
By the two-stage association study, we found that the
renalase coding gene was a novel susceptibility gene for
EH. No other study to date has assessed the relationship
between renalase gene variation and hypertension. These
findings may lead to a novel insight into the mechanisms of
BP regulation and the pathogenesis of hypertension.
Our two-stage case-control study design was similar but
not identical to the approach proposed by Satagopan et al.
[14]. Using simulations, they showed that for a given
sample size, when the markers were independent or
correlated, a two-stage design could provide near-optimal
power to detect the true marker conferring disease risk
while substantially reducing the total number of marker
evaluations. In particular, evaluating all the markers on
rs2576178
GG
GA
AA
G allele frequency
rs2296545
CC
CG
GG
C allele frequency
rs2114406
GG
GA
AA
G allele frequency
P value
Cases
(n=1317)
Controls
(n=1269)
367
702
242
0.55
298
641
321
0.49
<0.0001
<0.0001
481
641
193
0.61
391
619
257
0.55
0.0001
<0.0001
98
475
741
0.26
81
452
734
0.24
0.5059
0.2794
J Mol Med (2007) 85:877–885
883
Fig. 3 Adjusted odd ratios
(ORs) for EH associated with
genotypes of the three SNPs
tested in stage 2. CI indicates
confidence interval. ORs were
adjusted for age, gender, BMI,
TC, HDL-C, TG, Glu, Cr, and
drinking and smoking status.
Dominant and recessive models
were based on minor allele of
each locus as listed in Table 1.
The 95% CI lines crossing the
OR line of 1 indicate no
significance
and powerful strategy depends on disease-specific and
study-specific factors, which are both known (e.g., the
cost) and unknown (e.g., the genetic architecture of the
disease). In our study, the individuals used in stage 1 were
less than half of the total study individuals, which might
decrease the power to some extent. However, the hypertensive cases used in stage 1 had higher BP level and were
likely to be enriched for genetic susceptibility, which might
increase the difference in frequency of susceptibility alleles
between cases and controls to improve the power. In
addition, when we used an uncorrected P<0.05 as the
criterion, three SNPs, more than 10% of the total markers,
were selected for stage 2 study, which might further
maintain the power. Of course, the total number of variants
we tested was much less than that simulated by Satagopan
et al., so the criterion that would be used to evaluate the
most promising 10% of the markers in stage 2 was
inappropriate to our study.
In stage 1, three SNPs (rs2576178, rs2296545, and
rs2114406) showed significant associations with EH in a
Table 5 Associations between
haplotypes, diplotypes, and
EH
a
Loci are arranged in the order
rs2576178–rs2296545–
rs2114406.
b
Haplotype A-G-A (Hap1) was
chosen to be the reference
haplotype.
c
Diplotype Hap1-Hap1 (Dip1)
was chosen to be the reference
diplotype.
NS not significant
*Covariates were adjusted.
Variables
Hap1
Hap2
Hap3
Hap4
Hap5
b
Dip1c
Dip2
Dip3
Dip4
Dip5
Dip6
Dip7
Haplotypea
A-G-A
G-C-A
G-C-G
A-G-G
A-C-A
Diplotype
Hap1-Hap1
Hap1-Hap2
Hap2-Hap2
Hap2-Hap3
Hap1-Hap3
Hap2-Hap4
Hap1-Hap4
subsample containing 503 hypertensive cases and 490
normotensive controls. There is significant but not perfect
LD between rs2576178 and rs2296545 (r2 =0.719). Commonly, pairwise r2 threshold of 0.8 between two markers is
used in selecting tag-SNPs which will be genotyped and
can predict effects of untyped SNPs. With a lower r2
threshold, the power of prediction will decrease rapidly.
Considering this point, we tested both rs2576178 and
rs2296545 in stage 2. In the single locus analyses,
rs2576178 and rs2296545 still showed significant associations with EH in the total 2,586 study subjects, whereas
rs2114406 did not any more. SNPs rs2576178 and
rs2296545 are located in the 5′ flanking region and in the
exon 2 of renalase gene, respectively. There are at least
three possibilities for the associations between these two
SNPs and EH: (1) both SNPs are functional and affect the
expression or activity of renalase protein; (2) one SNP is
nonfunctional, and its association with EH is due to the
tight LD with the other functional SNP; or (3) both SNPs
are nonfunctional and in LD with another functional variant
OR (95% CI)
P value*
0.324
0.344
0.120
0.096
0.068
–
1.20(1.03–1.41)
1.46(1.20–1.78)
NS
NS
–
0.0220
0.0002
NS
NS
0.109
0.229
0.120
0.078
0.075
0.063
0.058
–
1.56(1.13–2.15)
1.64(1.15–2.34)
1.88(1.28–2.78)
NS
NS
NS
–
0.0071
0.0066
0.0014
NS
NS
NS
All
Cases
Controls
0.304
0.361
0.134
0.089
0.067
0.284
0.377
0.147
0.083
0.065
0.090
0.235
0.128
0.090
0.079
0.063
0.050
0.071
0.241
0.135
0.102
0.084
0.063
0.043
884
which was untested in our study. SNP rs2296545 results in
an aspartate to glutamate at codon 37 (Asp37Glu), which is
closely near C terminus of a deduced FAD-binding site of
the renalase protein. Renalase is critically dependent on
FAD for oxidase activity. Whether Asp37Glu variant has
any effect on the FAD-binding properties of renalase should
need further functional study.
To assess the combined effect of SNPs on the EH risk
and find possible risk haplotypes and diplotypes in the
population, we further performed multilocus analyses in
stage 2. The results of haplotype-specific score test showed
that the frequency of Hap1 (A-G-A) was significantly lower
in the cases than in the controls (0.284 versus 0.324, P=
0.0004). In comparison with the Hap1, Hap2 (G-C-A) and
Hap3 (G-C-G) were found to significantly increase the risk
of EH. The only difference between Hap2 and Hap3 was at
the SNP rs2114406. The OR of Hap3 was a little but not
significantly higher than that of Hap2. After we ignored the
rare diplotypes (with estimated frequency <0.05), there
remained three risk diplotypes, Dip2 (Hap1-Hap2), Dip3
(Hap2-Hap2), and Dip4 (Hap2-Hap3). As expected, Dip4,
which contained two risk haplotypes, Hap2 and Hap3, had
the highest risk for EH. However, we failed to observe that
Dip5 (Hap1-Hap3), consisting of a risk haplotype Hap3,
significantly increased the risk of EH. This might be
attributed to the relatively low frequency of Dip5, which
resulted in decreased power to find its effect.
Any reported genetic association should be interpreted
with caution until it is replicated. To control for falsepositive findings, several approaches were considered in
this study. First, we included only Northern Han Chinese
who were ethnically homogeneous, and the subsample of
the main study population has shown no population
stratification by genomic control method in our previous
study [22]. So the positive associations were unlikely to
have resulted from population admixture and stratification.
Second, we used conservative Bonferroni correction to
control the false–positive findings potentially because of
the multiple statistical tests. Finally, after correction for a
range of covariates, significant associations were still noted
between renalase gene variants and EH.
There may be three potential concerns for our study.
First, the renalase gene is not a typical candidate gene for
EH because renalase is a novel protein and its biological
mechanism for BP regulation is not very clear. However,
understanding of molecular mechanisms underlying most
common diseases is still poor, which itself is one of the
main justifications for gene discovery efforts. This point
also makes it imprecise to calculate prior odds associated
with any given candidate gene [23]. In addition, with the
development of large-scale and whole-genome association
study, more disease genes which have not been reported to
be associated with diseases and are even with unclear
J Mol Med (2007) 85:877–885
molecular functions will be discovered [24]. Therefore, the
following issue will focus on how to appropriately interpret
those associations. Second, we only tested limited variants
in the renalase gene and left a relative large region of
introns unexplored. It is possible that, by employing a
denser marker map, we may observe some other significantly associated risk SNPs and risk haplotypes and
diplotypes. Third, although rs2576178 and rs2296545 were
associated with EH, we failed to observe that the BP levels
were significantly different among their genotypes in the
control group, respectively. It is possible that our sample
has not enough power to detect the effects of their
genotypes on BP variation. However, the quantitative trait
association analyses using data of the controls may be
unreliable because the control group was unlikely to be
representative of the general population. To further investigate and confirm the associations between continuous
traits (e.g. BP, BMI, and glucose) and renalase gene
variation, it should be necessary to design new studies that
are based on random samples of the general population.
In conclusion, the present association study suggests that
genetic variations in the renalase gene may influence the
susceptibility of EH in the northern Han Chinese population. These findings will potentially contribute to a better
understanding of the mechanism of BP control and the
pathogenesis of EH. In addition, replications in other
populations and further functional studies are also required
to confirm and interpret the association of renalase gene
with EH.
Acknowledgment This work was supported by the National Basic
Research Program of China (Grant No. 2006CB503805) and the
Beijing Natural Science Foundation (Grant No. 7061006).
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J Mol Med (2007) 85:887–896
DOI 10.1007/s00109-007-0220-3
ORIGINAL ARTICLE
Myoglobin plasma level related to muscle mass and fiber
composition – a clinical marker of muscle wasting?
Marc-André Weber & Ralf Kinscherf &
Holger Krakowski-Roosen & Michael Aulmann &
Hanna Renk & Annette Künkele & Lutz Edler &
Hans-Ulrich Kauczor & Wulf Hildebrandt
Received: 6 December 2006 / Revised: 19 April 2007 / Accepted: 8 May 2007 / Published online: 30 June 2007
# Springer-Verlag 2007
Abstract Progressive muscle wasting is a central feature of
cancer-related cachexia and has been recognized as a
determinant of poor prognosis and quality of life. However,
until now, no easily assessable clinical marker exists that
allows to predict or to track muscle wasting. The present
study evaluated the potential of myoglobin (MG) plasma
levels to indicate wasting of large locomotor muscles and,
moreover, to reflect the loss of MG-rich fiber types, which
are most relevant for daily performance. In 17 cancercachectic patients (weight loss 22%) and 27 age- and
M.-A. Weber : H.-U. Kauczor
Department E010 (Radiology),
Deutsches Krebsforschungszentrum,
Im Neuenheimer Feld 280,
69120 Heidelberg, Germany
R. Kinscherf : H. Renk : A. Künkele
Anatomy and Developmental Biology,
Medical Faculty Mannheim, University of Heidelberg,
Theodor-Kutzer Ufer 1-3,
68167 Mannheim, Germany
H. Krakowski-Roosen
Department G060 (Translational Oncology),
Deutsches Krebsforschungszentrum,
Im Neuenheimer Feld 280,
69120 Heidelberg, Germany
H. Krakowski-Roosen : H. Renk : A. Künkele : W. Hildebrandt
Department D020 (Immunochemistry),
Deutsches Krebsforschungszentrum,
Im Neuenheimer Feld 280,
69120 Heidelberg, Germany
M. Aulmann
Department of Internal Medicine I, Central Laboratory,
University of Heidelberg,
Im Neuenheimer Feld 410,
69120 Heidelberg, Germany
MARC-ANDRÉ WEBER
is presently working as Senior
Physician at the Radiology
Department of the German
Cancer Research Center in
Heidelberg, Germany. His
research interests include
radiological imaging in neurooncological and neuromuscular
diseases.
WULF HILDEBRANDT
studied medicine at the
University of Marburg and
University of Cologne. He has
held a Senior Scientist position in
the Department of Immunochemistry at the German Cancer
Research Center in Heidelberg.
His research interests include
integrative respiratory and muscle physiology with regard to
cardiovascular risks, aging, and
cancer-related wasting.
L. Edler
Department C060 (Biostatistics),
Deutsches Krebsforschungszentrum,
Im Neuenheimer Feld 280,
69120 Heidelberg, Germany
W. Hildebrandt (*)
Rosenbergweg 3,
69121 Heidelberg, Germany
e-mail: [email protected]
DO00220; No of Pages
888
gender-matched healthy controls, we determined plasma
levels of MG and creatine kinase (CK), maximal quadriceps
muscle cross-sectional area (CSA) by magnetic resonance
imaging, muscle morphology and fiber composition in
biopsies from the vastus lateralis muscle, body cell mass
(BCM) by impedance technique as well as maximal oxygen
uptake (VO2max). In cachectic patients, plasma MG,
muscle CSA, BCM, and VO2max were 30–35% below
control levels. MG showed a significant positive correlation
to total muscle CSA (r=0.65, p<0.001) and to the CSA
fraction formed by type 1 and 2a fibers (r=0.80, p<0.001).
However, when adjusted for body height and age by
multiple regression, MG yielded a largely improved
prediction of total CSA (multiple r=0.83, p<0.001) and
of fiber type 1 and 2a CSA (multiple r=0.89, p<0.001).
The correlations between CK and these muscle parameters
were weaker, and elevated CK values were observed in 20% of
control subjects despite a prior abstinence from exercise for
5 days. In conclusion, plasma MG, when adjusted for
anthropometric parameters unaffected by weight, may be
considered as a novel marker of muscle mass (CSA) indicating
best the mass of MG-rich type 1 and 2a fibers as well as
VO2max as an important functional readout. CK plasma
levels appear to be less reliable because prolonged increases
are observed in even subclinical myopathies or after exercise.
Notably, cancer-related muscle wasting was not associated
with increases in plasma MG or CK in this study.
Keywords Cancer cachexia . Muscle wasting .
Muscle biopsy . Muscle morphology . Fiber composition
Introduction
Cachexia has been recognized as a life-threatening syndrome
of progressive weight loss associated with cancer, cardiorespiratory, or inflammatory diseases. It is experienced by up
to 50% of cancer patients and may account for more than
20% of cancer-related deaths [1–5]. The major manifestation
of cachexia, besides lipolysis, is muscle wasting, which in
many cancer patients is only partly attributable to malnutrition, anemia, inactivity, or therapeutic interventions. Muscle
wasting is considered to be critical for impaired mobility,
quality of life, and prognosis. The underlying massive nettoproteolysis, which appears to target especially myosin as the
functional contractile protein, is caused by a metabolic
dysregulation that is just about to be unraveled in animal and
in vitro models [6–9] or humans [10, 11] Progressive
protein loss leads to muscle fiber shrinkage and reduction in
cross-sectional area (CSA), and a wasting-related reduction
in muscle CSA of most relevant locomotor muscles like the
quadriceps femoris muscle together with associated changes
in fiber composition and capillarization explain a major
J Mol Med (2007) 85:887–896
portion of the loss of aerobic capacity (VO2max) in healthy
subjects as well as in cancer patients [12–14] (Hildebrandt
et al., unpublished data). Accordingly, the reduction in
VO2max is most prominent in gastrointestinal cancer types
with the highest incidence of cachexia as opposed to noncachectic breast cancer patients [15–17]. As daily endurance performance required for mobility is best assessed by
VO2max as a functional readout of muscle mass and fiber
composition together with cardiorespiratory functions, it
may be of great clinical value to identify an easily
assessable and reliable marker of reduced muscle mass,
which is more precisely related to muscle mass and fiber
composition than the widely used body weight or body
impedance analysis. At present, no such parameter exists,
and muscle mass determination by dual energy X-ray
absorptiometry (DEXA), magnetic resonance imaging
(MRI), or computed tomography (CT) is limited to studies
and can hardly be routinely established given the high costs
and low availability of these methods.
We therefore addressed the question, to what extent plasma
MG, as an easily measurable blood parameter, could be a
surrogate parameter for relevant muscle mass and fiber
composition. The idea behind this approach was that the
functionally important aerobic slow-twitch type 1 (and 2a)
fibers are the primary source of plasma MG, a rather small,
solely cytoplasmatic protein (17 kDa), which has a very short
half-time (5.5 h) for renal elimination after severe muscle
damage. In comparison, CK, which is less fiber specific, is
present in different cellular compartments, and elevations of
CK plasma levels are often associated with even subclinical
myopathies and are known to be much more prolonged upon
severe exercise or muscle damage than that of plasma MG levels
[18, 19]. Therefore, CK plasma levels were hypothesized to be
less indicative of a functionally relevant muscle mass.
The present study therefore examined the relation of MG
and of CK to the total quadriceps muscle CSA determined
by MRI in severely cachectic patients suffering from
gastrointestinal cancer as well as in healthy controls similar
in age, gender, and body weight at health. Additionally, in
surgically obtained biopsies, we determined each fiber
type’s fractional CSA of the vastus lateralis muscle as the
most important large locomotor muscle and studied their
relation to MG or CK. We found a close positive and highly
significant correlation between MG and total muscle CSA
and the CSA formed by fiber type 1 (or 1 and 2a), which was
considerably strengthened when MG was adjusted for body
height and age by multiple regression (r=0.83 and r=0.89,
respectively). All subjects had abstained from exercise for
at least 5 days to exclude exercise-induced muscle damage
that is a well-known cause of increases in plasma MG [18–
20]. As all MG values were found to be in the normal range—
especially in the cachectic cancer patients—severe muscle
wasting appears not to involve myocellular membrane
J Mol Med (2007) 85:887–896
damage or myolysis. Our explorative, invasive study in a
limited sample size generates the hypothesis that muscle mass
may be predicted from MG and easily assessable anthropometric parameters unaffected by weight loss. Less invasive
studies on larger cohorts are warranted to evaluate whether
MG may be considered as a novel clinically feasible marker to
track muscle wasting and fiber composition.
Materials and methods
889
venous blood sampling for MG, CK, and other parameters.
Exclusion criteria were any known or newly diagnosed
severe neuromuscular, renal, inflammatory, cardiovascular,
respiratory, hepatic, or psychiatric disease. Subjects or
patients were without orthopedic problems of the lower
extremity or spine that would have limited daily mobility and
activity. Informed written and oral consent was obtained from
all patients and control subjects. The study was approved by
the Ethical Committee of the University of Heidelberg and
performed according to the Declaration of Helsinki (1996)
and to good clinical and laboratory practices.
Cancer-cachectic patients and control subjects
Measurements and equipment
Seventeen mobile patients with the diagnosis of a malignoma
and a cancer-related cachexia defined as a progressive
weight loss of >10% within 6 months (inclusion criteria)
were recruited as outpatients of the Medical University
Clinic of Heidelberg (Department of General and Visceral
Surgery or Department of Internal Medicine I or II) as well as
from regional oncological practices. Five types of cancer
associated with a high risk of progressive cachexia were
admitted to the study resulting in the following distribution
among the randomly selected patients: three gastric cancer
(Union internationale contre le cancer (UICC) staging: one
with IIIa and two with IV), seven pancreatic cancer (UICC
staging: one with II and six with III), three colon cancer
(UICC staging: one with II, one with IIIa, and one with IIIc),
one bronchogenic carcinoma (UICC staging IV), and three
chronic lymphatic leukemia (CLL) (Binet-staging: C). The
patients were included into the study irrespective of prior or
simultaneous chemo- or radiotherapy. Nine patients had
completed chemotherapy before inclusion, while six patients
were under chemotherapy at the time of the study. Additional
radiotherapy had been completed by four patients: two with
pancreatic cancer, one with gastric cancer, one with
bronchial cancer. Care was taken that any sampling or
measurement was dated not before at least 8 days after any
radio- or chemotherapeutic intervention.
Twenty-seven healthy controls were recruited by public
announcement such that they were comparable to the
cancer patients with respect to age, gender, and the body
weight reported for health.
Patients and controls, by instruction, had abstained from
any severe exercise at least 5 days before the examinations.
All subjects and patients underwent an initial clinical
examination for exclusion criteria that included routine
laboratory parameters in venous blood samples, blood
pressure measurement in sitting position, and electrocardiogram at rest and during an incremental exercise test for
determination of VO2max. The following main study parameters were obtained within 10 days: maximal quadriceps
muscle CSA by MRI, muscle fiber composition by biopsy,
VO2max, body composition analysis by bioimpedance, and
Venous blood analysis After an overnight, fast blood
samples were drawn from an antecubital vein for clinical
routine laboratory diagnostic in the central laboratory of the
Medical University Clinic of Heidelberg. Additionally,
whole blood samples were drawn and immediately centrifuged at 2,000 rpm for 10 min at 4°C to obtain serum or
ethylenediaminetetraacetic acid (EDTA) plasma, which were
stored at −75°C for further analysis. Interleukin-6 (IL-6)
plasma levels in EDTA plasma were measured in duplicate
using a commercial enzyme-linked immunosorbent assay
(IBL, Hamburg, Germany). Plasma MG levels were determined in serum samples by a two-step sandwich enzyme
immunoassay using the chemoilluminiscence detector device Centaur (Bayer, Leverkusen, Germany). Other blood
parameters, i.e., cholinesterase, hemoglobin, albumin, and
creatinine were obtained by routine laboratory methods.
Magnetic resonance imaging MRI of the right thigh was
performed in the supine position on a 1.5-T clinical MR
system (MAGNETOM Symphony, Siemens AG Medical
Solutions, Erlangen, Germany) using the manufacturer’s
standard phased array coil for signal reception. The imaging
protocol comprised an axial and coronal T1-weighted spinecho sequence (repetition time/echo time in milliseconds,
500/13), an axial (4,000/50) and coronal (5,130/63) T2weighted fat-suppressed short tau inversion recovery sequence, and a fat-suppressed T1-weighted turbo spin-echo
(TSE) sequence (500/13). Muscle CSA of the right quadriceps femoris was determined including the maximal thigh
circumference at 12-, 20-, and 28-cm distances from the
trochanter major. All MR images were displayed as softcopies in a fully electronic, monitored fashion using our
picture archiving and communication system (PACS) with
large-screen, high-resolution cathode-ray tube displays,
which enabled the review of eight images simultaneously
and the individual selection of the different MR sequences,
e.g., to clarify the fascial boundaries of the quadriceps
muscle. CSA was assessed on T1-weighted images using a
computerized digitizer as part of the PACS standard tool. All
890
MR examinations were jointly randomized and shown to the
readers in order of randomization. The reader was blinded to
identifying parameters such as the subject’s name and
clinical data. This analysis of quadriceps muscle CSA
renders precise and reproducible measurement of muscle
CSA and a very high positive correlation between the values
obtained by two independent blinded readers (r>0.98).
Muscle CSA of the whole quadriceps femoris muscles
was determined because, especially in the proximal and
distal axial MRI scans, the fascial boundaries between the
lateral and deep vastus muscles could often not be
identified, as it has been described before [21].
J Mol Med (2007) 85:887–896
Maximal oxygen uptake (VO2max) VO2max was determined breath-by-breath by the spirometric system
ZAN680 (ZAN Ferraris Cardiorespiratory, Oberthulba,
Germany) during an incremental exercise test on a cycle
ergometer type Ergoline 100 (Ergoline, Windhagen, Germany) that increased work load from a starting level of
50 W in steps of 25 W every 2 min until exhaustion.
VO2max was calculated as the maximal mean value
covering 10 s. VO2max could be obtained from 19 controls
and 12 cachectic patients only.
Statistics
Sampling and analysis of muscle biopsy Eleven out of 17
cachectic patients and 15 out of 27 controls gave their
informed consent to muscle biopsy. The biopsies were
taken from the vastus lateralis muscle at about mid-thigh
level by means of the technique of Bergström [22] applied
after careful local anesthesia and disinfection. The muscle
samples were immediately shock-frozen in liquid nitrogencooled isopentane and stored at −80°C. For histochemistry
and morphometric analysis, serial transverse sections
(6 μm) were cut in a cryotome at −20°C. For fiber
population analysis, transverse sections were stained for
myofibrillar ATPase after preincubation at pH 4.35 (5 min,
room temperature), pH 4.6 (5 min, room temperature), and
pH 10.5 (15 min, 37°C) according to [23] as previously
described [21]. The fiber types 1, 2a, 2ax, and 2x were
identified according to their acid-sensitive ATPase-staining
intensity after preincubation at pH 4.6, as well as the fiber
types 1, 2c, and 2 after incubation at pH 10.5. Biopsies with
less than 100 fibers were excluded from the analysis. The
mean fiber number that could be analyzed per sample was
220±142. Furthermore, type 2ax fibers were subsumed in 2a
fibers, as their fraction was below 1%. Microscopic video
recordings of the ATPase-stained cross-sections (pH 4.6)
were digitized by a PC-based image analysis system (VIBAM
0.0-VFG1 frame grabber). Fiber CSAs were determined at a
200-fold magnification. Statistical analysis was performed
including the three main fiber types 1, 2a, and 2x.
Body composition Body composition was analyzed by
measurement of electrical impedance and reactance under
standardized conditions regarding body position, electrode
localization, and overnight fast using the TVI-10 body
composition analyzer purchased from FM Service GmbH
(Leverkusen, Germany) in combination with the BIA-Star
program by RECAL Biomed (Heidelberg, Germany) for
calculation of absolute and percentage values of body fat,
fat-free mass, total body water (TBW), and body cell mass
(BCM) based on body height and actual measured body
weight, as previously described [24]. The theoretical basis
of this method has been described elsewhere [25].
Parameters measured on a continuous quantitative scale were
described statistically using means and their standard error.
Comparisons between cachectic patients and controls were
performed by the Student’s t test for unpaired observations.
Bivariate correlations of MG, CK, or other parameters to
CSA and each fractional fiber type’s CSA, BCM, or
VO2max were assessed using linear regression (Pearson
correlation coefficient r) and graphically presented as scatter
plots including the linear regression line; r2 was used as a
measure of explained variability in linear regression.
In addition to univariate regression, a multiple stepwise
regression analysis was performed between muscle CSA or
fiber type 1 and 2a CSA as dependent variables and MG,
body height, gender, and age as independent variables.
Three of them, i.e., MG, body height, and age, remained in
the multiple regression as independent predictors of muscle
CSA or fiber type 1 and 2a CSA. The respective multiple
regression equation was used to calculate individual predictions for muscle CSA and fiber type 1 and 2a CSA. These
predictions were plotted as x values (based on MG, body
height, and age) against individually corresponding measured values (y values) of both muscle CSA and fiber type 1
and 2a CSA (see Fig. 3a and b). Regression lines together
with r and p values were determined for the total group of
subjects as well as for the two subgroups of cachectic
patients and controls. Moreover, 95% prediction intervals
were calculated according to [26] using the formula
vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
!ffi
u
2
u
1
ð x xÞ
ax þ b 1:96ts2 1 þ þ P
n
ð xi xÞ 2
where a is the regression coefficient, x is the multivariate
predictor (independent variable), b is the y-axis intercept, s2
is the variance, n is the number of observations, and x is the
mean of all individual x. This approach to compare the two
methods of assessing muscle mass was used because both
methods involve different units, which prohibits the application of the Bland–Altman analysis assuming identical units
between the methods to be compared.
J Mol Med (2007) 85:887–896
891
A significance level of p≤0.05 was chosen for all
statistical tests. The program SPSS (version 11.5, SPSS,
Chicago, IL, USA) was used for all statistical tests.
Results
The 17 cachectic cancer patients and the 27 controls were
not significantly different with regard to proportion of
gender, to age, body height, and body weight at health
(Table 1). Cachectic patients had a significantly lower
actual body weight and body mass index due to a cancerrelated mean weight loss of 22.3% and a mean weight loss
rate of 3.45±0.79% per month (Table 1). The bioimpedance
analysis of body composition revealed that the 19-kg
difference in mean body weight was caused by a 10-kg
lower BCM representing mainly muscle mass, a 5-kg lower
fat mass, and lower TBW. Muscle CSA was found to be by
32% lower in cachectic patients compared to controls
(Table 1). These data document the well-known fact that,
besides fat mass, skeletal muscle mass is the primary target
tissue of cancer-related wasting. Muscle CSA and BCM
Table 1 Anthropometric data, body composition, muscle CSA, as
well as venous blood parameters
n (female/male )
Age (years)
Body weight (kg)
Body height (cm)
Body mass index (kg m−2)
Body weight at health (kg)
Weight loss (%)
BCM (kg)
Body fat (%)
TBW (%)
Muscle CSA (cm2)
CK (U l−1)
MG (μg l−1)
Cholinesterase (kU l−1)
Hemoglobin (g dl−1)
IL-6 (pg l−1)
Albumin (g l−1)
Creatinine (mg dl−1)
VO2max (l min−1)
VO2max/body weight
(ml min−1 kg−1)
Control
Cachexia
27 (13/14)
57.9±2.4
78.4±3.0
173.1±1.6
26.0±0.8
78.4±3.0
–
33.8±1.3
22.7±1.2
62.5±1.5
74.4±2.7
131.7±12.9
42.0±6.0
8.91±0.44
15.0±0.4
2.86±0.38
44.8±0.4
0.85±0.03
2.60±0.16a
32.8±1.7a
17 (8/9)
52.5±1.6
59.3±3.0
171.1±1.6
20.1±0.7
75.6±3.8
22.3 ±28
23.4±1.7
17.9±1.5
56.1±1.1
50.8±3.8
52.1±9.9
27.0±2.8
6.24±0.65
12.5±0.4
8.15±3.0
39.4±2.7
0.58±0.08
1.29±0.20b
21.3±2.4b
p value
0.07
0.000***
0.482
0.000***
0.628
0.000***
0.017*
0.001**
0.000***
0.000***
0.000***
0.001**
0.000***
0.092
0.071
0.008**
0.000***
0.001**
Data show the mean±SEM.
Muscle CSA Cross-sectional area (maximum) of the quadriceps
femoris muscle, VO2max maximal oxygen uptake (aerobic capacity)
*p<0.05 by Student’s t test cachexia vs control
**p<0.01 by Student’s t test cachexia vs control
***p<0.001 by Student’s t test cachexia vs control
a
n=19
b
n=12
were significantly positively correlated (r=0.93, p<0.001),
and MRI of the most important and largest locomotor
muscle could thus be considered a reliable measure of
overall muscle wasting.
MG plasma levels were found to be in the normal range
(see Fig. 1a), and mean values in cancer-cachectic patients
were 48% below that of controls (Table 1). CK plasma
levels were about 60% lower in cancer-cachectic patients
compared to the control group (Table 1), which included
supranormal values in at least five control subjects despite
abstinence from exercise 5 days before the study (Fig. 1c).
Plasma creatinine levels were found within the normal
range excluding a relevant renal insufficiency; however,
within this normal range, the significant creatinine reduction in cachectic patients compared to controls should be
noted (Table 1). Moreover, cholinesterase, a plasmatic
indicator of hepatic function, i.e., protein synthesis,
revealed a significant reduction in cachectic patients
occurring within the normal range as well (Table 1). The
same was true for albumin (Table 1), which is known to be
reduced in cancer patients due to vascular escape [27].
Cachectic patients had a slight but significant anemia, with
subnormal values found in the total group (Table 1) as well
as in the female and male subgroup (not shown). Plasma
levels of IL-6, a cytokine predominantly discussed as an
etiologic factor of muscle wasting, were found to be increased
in cachectic patients compared to controls without reaching
the level of statistical significance (Table 1). There was no
significant correlation between IL-6 and MG plasma levels
or muscle CSA. VO2max, in absolute terms, was about 51%
lower in cachectic patients compared to controls, and after
VO2max normalization for body weight, a significant
reduction remained amounting up to 35% (Table 1).
We found a significant positive correlation between
individual MG plasma levels and BCM or muscle CSA in
the total group of subjects (Fig. 1a and b). This correlation of
MG to BCM or muscle CSA was also significant when
analyzing men and women separately (women r=0.45, p=0.05
or r=0.53, p=0.03; men r=0.60, p=0.003 or r=0.68, p<0.001,
respectively). Moreover, MG was found to be significantly
correlated to BCM or CSA within the group of healthy
controls (r=0.61, p=0.001 or r=0.56, p=0.005, respectively),
while the respective correlations within the smaller group of
cachectic patients were r=0.38, p=0.15 or r=0.40, p=0.1.
Plasma levels of CK showed a significant but weaker positive
correlation to BCM or muscle CSA (Fig. 1c and d). There was
a significant positive correlation between MG and CK plasma
levels (r=0.62, p<0.001).
Cholinesterase plasma levels were also positively correlated with BCM (r=0.42, p=0.006) or muscle CSA (r=0.45,
p=0.003). Weaker but still significant correlations were found
between plasma creatinine and BCM (r=0.33, p=0.044) or
muscle CSA (r=0.36, p=0.033).
892
a
b
r = 0.68 p < 0.001
100
80
60
40
20
40
bcm (kg)
60
40
60
40
d
r = 0.49 p = 0.002
300
240
240
180
120
60
80
100
muscle CSA (cm2)
r = 0.55 p < 0.001
300
CK (U l-1)
CK (U l-1)
80
20
20
c
r = 0.65 p < 0.001
100
-1
myoglobin (µg l )
myoglobin (µg l-1)
Fig. 1 Correlation between
plasma MG (a, b) or CK (c, d)
and BCM (a, c) or quadriceps
muscle CSA (b, d). The regression line, the correlation coefficient r, and the p value are given
for the total group of subjects.
Open circles controls, closed
circles patients
J Mol Med (2007) 85:887–896
180
120
60
60
0
0
20
40
bcm (kg)
Table 2 presents fiber size and composition as well as
total fiber type CSA for the three fiber types 1, 2a, and 2x
in 26 biopsies obtained from 11 cachectic patients and 15
controls. We found a markedly reduced size of type 1
(−20%), type 2a (−55%), and type 2x (−45%) fibers in
cachectic patients as compared to control subjects (Table 2).
However, the fractional area of any of the three fiber types
within the biopsies was similar between the two groups
under test (Table 2). Within the given quadriceps CSA as
determined by MRI, the total area contributed by type 1,
type 2a, or type 2x fibers was found to be reduced by 22,
25, and 30% in cachectic patients compared to controls
(Table 2). The overall reduction in fiber size of about 40%
could well account for the 32% decrease in muscle CSA in
cachectic patients below the control level.
Within the 11 patients and 15 controls from whom the
muscle biopsies were obtained, we found significant positive
correlations between plasma levels of MG and the total CSA
formed by type 1 and by type 2a fibers (Fig. 2a and b). The
closest correlation (r=0.80) was observed between MG and
60
40
60
80
100
muscle CSA (cm2)
Table 2 Skeletal muscle fiber type composition (vastus lateralis
muscle)
Controls
Samples analyzed
15
Fiber size
4,675±381
Type 1 (μm2)
6,942±225
Type 2a (μm2)
3,765±446
Type 2x (μm2)
Fractional fiber area within the biopsy
Type 1 (%)
44.2±3.2
Type 2a (%)
40.3±3.6
Type 2x (%)
15.5±3.3
Total fiber area within quadriceps CSA
34.7±2.0
Type 1 (cm2)
26.8±2.1
Type 2a (cm2)
11.5±2.7
Type 2x (cm2)
Cachexia
p value
11
3,723±364
3,207±374
2,105±401
0.099
0.195
0.016*
50.2±3.2
35.6±3.4
14.2±4.2
0.316
0.385
0.802
27.1±3.4
19.9±2.7
8.2±2.9
0.068
0.058
0.412
Data show the mean ± SEM
*p<0.05 by Student’s t test cachexia vs control
J Mol Med (2007) 85:887–896
a
893
b
r = 0.68 p = 0.001
r = 0.57 p = 0.006
120
80
80
40
40
myoglobin (µg l-1)
120
0
0
20
40
total type 1 fiber CSA (cm2)
c
60
r = 0.057 ns
d
20
40
total type 2a fiber CSA (cm2)
60
r = 0.80 p < 0.001
120
120
myoglobin (µg l-1)
0
80
80
40
40
0
0
0
20
40
60
total type 2x fiber CSA (cm2)
Fig. 2 Correlation between plasma MG and the total muscle CSA
portion rendered by type 1 fiber (a), type 2a fiber (b), type 2x fiber
(c), as well as by the sum of type 1 and 2a fibers (d). The regression
40
60
80
total type 1 and type 2a fiber CSA (cm2)
line, the correlation coefficient r, and the p value are given for the total
group of subjects. Open circles controls, closed circles patients
the CSA of the sum of type 1 and 2a fibers (Fig. 2d), and this
correlation was also found within each of the two groups
under test (healthy controls: r=0.82, p=0.002; cachectic
cancer patients: r=0.61, p=0.06) and, moreover, within the
subgroup of women (r=0.77, p=0.26) as well as within the
subgroup of men (r=0.76, p=0.002). In contrast, no
significant correlation was found between MG and the total
CSA of type 2x fibers. Moreover, MG was significantly
related to VO2max (r=0.62, p<0.001), which may be
considered to reflect the impact of the MG-containing, i.e.,
the mainly aerobically working, muscle mass on VO2max.
CK plasma levels showed a weaker relation to the total
CSA of type 1 fibers, however, a close relation to the CSA
of type 2a fibers (r=0.59, p=0.002). Moreover, significant
correlations were found between CK and the fiber size of
type 1 and type 2x (not shown).
By multiple stepwise regression analysis, we evaluated
which anthropometric parameters that are easily assessable and
unaffected by weight loss would contribute besides MG to the
variability of muscle CSA and fiber type 1 and 2a CSA. We
identified body height and age as independent factors. The
bivariate correlation of body height to muscle CSA was found to
be significant for all subjects (r=0.59, p<0.001) as well as
separately for cachectic patients (r=0.64, p<0.01) and for
controls (r=0.64, p<0.01). Age revealed its small but significant contribution to muscle CSA only upon multiple regression.
When MG was adjusted for body height and age in the
multiple regression (x=MG×0.539+body height×0.892−
age×0.65−75.409), a larger portion of the measured muscle
CSA variability could be explained (predicted) than by MG
alone (69% vs 42%, multiple r=0.83, p<0.001 vs r=0.64,
p<0.001). Regarding the fiber type 1 and 2a CSA fraction,
the analogue MG adjustment according to the multiple
regression (x=MG×0.558+body height×0.489−age×0.518−
21.59) explained a very large portion (80%, r=0.89, p<0.001)
of MG-containing muscle fraction as compared to a non-
894
J Mol Med (2007) 85:887–896
a
b
total group r = 0.83 p = 0.000
cachexia r = 0.75 p = 0.001
control
r = 0.76 p = 0.001
muscle CSA (cm2)
100
80
60
40
20
0
20
40
60
80
100
myoglobin x 0.539 + body height x 0.892
-age x 0.65 - 75.409
total type1 and 2a fiber CSA (cm2)
120
total group r = 0.89 p = 0.000
cachexia r = 0.84 p = 0.002
r = 0.86 p = 0.000
control
120
100
80
60
40
20
0
20
40
60
80
100
myoglobin x 0.558 + body height x 0.489
-age x 0.518 - 21.59
Fig. 3 Prediction of measured total muscle CSA (a, y-axis) as well as
of the CSA fraction of type 1 and 2a fibers (b, y-axis) by plasma MG
adjusted for body height and age (x-axis). The adjustment of MG for
body height and age was based on the multiple regression (see x-axis
title), which was obtained for individual values of the total group of
subjects with regard to both the measured total CSA (a) and the
measured CSA fraction formed by type 1 and 2a fibers (b). Both the
prediction plots a and b include the upper and lower 95% prediction
limits, i.e., the 95% prediction intervals, the regression lines, the
correlation coefficients (multiple r), and the p value for the total group
of subjects as well as of the subgroups of both the cachectic patients
(closed circles) and the healthy controls (open circles). Each group’s
regression line and 95% prediction interval is indicated by the legend.
For details, see “Materials and methods”/“Statistics”
adjusted MG (42%, r=0.65, p<0.001). In Fig. 3, the measured
individual values of total muscle CSA or fiber type 1 and 2a
CSA (y-axis Fig. 3a or y-axis Fig. 3b, respectively) are plotted
against the such adjusted MG (x-axis), i.e., the individual
predictions as the independent variable. Moreover, Fig. 3a and
b includes the regression lines, r and p values as well as the
upper and lower 95% prediction limits (intervals; for
calculation, see “Materials and methods”) of the total group
as well as both the groups of cachectic patients and of
controls. There was good agreement in regression lines and
the 95% prediction intervals between all groups.
Highly significant correlations of adjusted MG to total
muscle CSA and fiber type 1 and 2a CSA fraction were
also obtained in separately analyzed subgroups of women
(r=0.68, p=0.002 and r=0.90, p=0.002, respectively) and
men (r=0.70, p<0.001 and r=0.83, p<0.001, respectively).
Adjustment of MG for body height and age also
improved its correlation to VO2max (r=0.756, p<0.001)
compared to unadjusted MG alone (r=0.62, p<0.001).
of severely cachectic cancer patients as well as healthy
controls of varying physical activity.
As a first important finding, MG plasma levels were not
only related to total muscle CSA but also revealed a closer
correlation to the type 1 and type 2a fiber CSA, which was
calculated from total CSA by MRI and fractional fiber type
areas in biopsies. The correlation was closest (r=0.80,
p<0.001) when taking the sum of type 1 and type 2a areas,
i.e., the fiber types predominantly containing MG, and was
found to be also significant when analyzing the group of
men, women, cachectic patients, or healthy controls
separately. Through adjustment of MG for body height
and age using multiple regression, the prediction of total
muscle CSA as well as fiber type 1 and 2a CSA
fraction by MG could be considerably improved and
rendered multiple r values of 0.83 and 0.89, i.e.,
explanation of 69 and 80% of the CSA variability,
respectively. Moreover, highly significant and similarly
strong correlations were obtained within the two subgroups of cachectic patients and controls, who in addition
revealed similar regression lines and 95% prediction
intervals (Fig. 3a and b), suggesting that prediction of
muscle mass by the adjusted MG may cover the range of health
as well as of severe wasting as associated with several types of
cancer. Moreover, highly significant correlations between
adjusted MG and muscle CSA were found within the
subgroups of female and male subjects.
Discussion
The present cross-sectional study investigated the relation
of plasma MG and CK to BCM and to the CSA as well as
fiber composition of the quadriceps muscle as the largest
and most relevant locomotor muscle within the total group
J Mol Med (2007) 85:887–896
Our invasive study in a limited number of subjects thus
gives rise to a strong hypothesis toward the usefulness of
MG in non-invasive and clinically feasible assessment of
muscle mass, to be studied in large cohorts including
different wasting conditions and longitudinal designs.
As a second important finding, plasma MG as well as
CK levels were significantly lower in cachectic patients,
whose muscle CSA and BCM were 32% lower than that of
controls. As both, patients and controls, had abstained from
severe exercise 5 days before examination, it is suggested
that cancer-related muscle wasting may not include muscle
damage or even myolysis that would be easily indicated by
rises in MG or CK plasma levels. In fact, our histomorphological screening of more than five transverse sections of all
biopsies revealed no indication for myolysis or necrosis
associated with cachexia. Moreover, upon individual
analyses of muscle biopsies using the TdT-mediated
dUTP-biotin nick end labeling (TUNEL) assay to measure
DNA fragmentation (apoptosis) on transverse sections, we
found no TUNEL-positive nuclei. However, the result
concerning absence of apoptotic nuclei in skeletal muscle
should not be over-interpreted because the time-window to
detect apoptosis may be very small, and one muscle fiber
contains a huge amount of nuclei.
Our findings may be of significance for clinical
management of wasting conditions especially cancer.
Muscle wasting as a major feature of the severe and
complex cachexia syndrome is frequently observed in
cancer as well as in chronic cardiopulmonary or other
diseases and crucially determines performance, quality of
life, and even therapy outcome [1, 3–5]. The performance
state and mass of skeletal muscle are now being recognized
as a new therapeutic target especially because regular
exercise, a stimulus to maintain muscle mass and function
at all ages, appears to have rather large beneficial effects on
survival of cancer patients besides its well-established
cancer-preventing effect [28–30].
However, quantification of muscle mass during progressive wasting, i.e., a loss of up to 50% or more, is still far
from being clinically implemented, and no easily assessable
routine surrogate parameters exist except for the largely
unreliable parameters of body weight and reported performance status. Methods to precisely assess muscle mass like
DEXA, MRI, and CT remain somewhat limited to studies.
Thus, an adequate routine parameter that is clearly related
to muscle mass for longitudinal follow-up is needed, which
can be included into clinical routine at low costs and is less
critically dependent on intraindividual standardization as,
e.g., required for bioimpedance analysis.
The presently found close correlation between MG,
especially when adjusted for the easily assessable anthropometric parameters, and the quadriceps muscle CSA (i.e.,
the mass determined by MRI) or the fiber-type specific
895
muscle mass (determined by MRI and biopsy) may
potentially allow for a clinical use to track muscle mass,
provided the suggested non-invasive prediction model is
confirmed in larger cohorts and longitudinal studies
demonstrate an intraindividual correlation as well. For
clinical routine purposes, of course, another important
well-studied source of plasma MG, i.e., the myocardial
damage, e.g., via infarction, has to be excluded.
The measurement of plasma MG levels may have
important advantages over that of CK, which presently
showed similar but weaker correlations to muscle CSA
(Fig. 1d) or the three fiber type-specific CSA fractions (not
shown): (a) After acutely induced muscle damage, e.g., due
to severe exercise, intramuscular injections, or bagatelle
trauma, normalization of acutely increased plasma MG
levels occurs within 24 h, i.e., quicker than that of CK
levels [18, 31], which may to some extent also explain our
findings of a weaker correlation between CK and CSA; (b)
many, if not most, myopathies are associated with variable
CK elevations at no or marginal MG increases in the
plasma; (c) according to the present analyses, MG is closely
correlated to the fiber 1 and 2a fraction of total CSA or
mass, which represent the main source of skeletal muscle
MG. Thus, MG may be suggested as a novel marker to
render information on muscle fiber composition besides
muscle mass itself. Muscle fiber composition is an
important information about muscle wasting and muscle
performance because a most recent study from our laboratory has shown that there may be substantial fiber transition
at the cost of type 1 fiber, which along with overall muscle
atrophy and reduced capillarization contributes to the low
aerobic capacity (VO2max) in cancer-cachectic patients
compared to age-, gender-, and weight-matched controls
(Hildebrandt et al., unpublished data). In the present study,
we found a significant correlation between plasma MG and
VO2max, which may reflect the impact of fiber 1 and 2a on
both MG release and VO2max.
An obvious limitation of our study is the rather inhomogeneous and small group of cancer-cachectic patients. The
difficult recruitment of cachectic cancer patients with poor
prognosis may at least in part be due to the fact that these
patients, which have to cope with an invasive and mainly noncurative treatment, may not easily agree to an additional
invasive biopsy or hospital-based and time-consuming MRI.
The time for recruitment of the group of cancer patients and
the larger group of controls was 2.5 years and 1 year,
respectively.
In summary, our cross-sectional study shows that plasma
MG is closely related to a quadriceps muscle CSA as a robust
index of locomotor muscle mass, especially to the mass of
MG-containing fiber types that are crucial for daily performance. Adjustment for body height and age as independent
factors of muscle mass considerably improves the prediction
896
of muscle mass, with significant and very close positive
correlations in the total as well as the subgroups of cachectic
cancer patients or healthy controls. Our explorative invasive
study thus suggests adjusted MG as a clinical surrogate
parameter for the tracking of functionally important muscle
mass undergoing wasting. Further studies in larger cohorts
are warranted to evaluate the usefulness of this non-invasive
predictor including intraindividual longitudinal follow-ups
in cancer and in the numerous other important clinical
conditions associated with severe muscle wasting.
Acknowledgment We would like to thank Dr. Werner Rittgen
(Department of Biostatistics, German Cancer Research Center) for
his generous assistance in the statistical analyses. We also gratefully
acknowledge the expert laboratory assistance of Mrs. Ute Winter, Mrs.
Ursula Bollow, Mrs. Silke Vorwald, and Mrs. Ulrike Traut.
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J Mol Med (2007) 85:897–906
DOI 10.1007/s00109-007-0184-3
ORIGINAL ARTICLE
Targeting of human renal tumor-derived endothelial cells
with peptides obtained by phage display
Benedetta Bussolati & Cristina Grange & Lorenzo Tei &
Maria Chiara Deregibus & Mauro Ercolani &
Silvio Aime & Giovanni Camussi
Received: 19 September 2006 / Revised: 14 February 2007 / Accepted: 15 February 2007 / Published online: 24 March 2007
# Springer-Verlag 2007
Abstract The phenotypic and molecular diversity of tumorassociated vasculature provides a basis for the development
of targeted diagnostics and therapeutics. In the present study,
we have developed a peptide-based targeting of human
tumor endothelial cells (TEC) derived from renal carcinomas. We used a murine model of human tumor angiogenesis,
in which TEC injected subcutaneously in severe combined
immunodeficiency (SCID) mice organized in vascular
structures connected with the mouse circulation, to screen
in vivo a phage display library of random peptides. Using
this approach, we identified cyclic peptides showing specific
binding to TEC and not to normal human endothelial cells or
B. Bussolati : C. Grange : M. C. Deregibus : G. Camussi
Cattedra di Nefrologia, Dipartimento di Medicina Interna,
Università di Torino, Turin, Italy
B. Bussolati : C. Grange : M. C. Deregibus : G. Camussi
Centro Ricerca Medicina Sperimentale (CeRMS),
Università di Torino, Turin, Italy
L. Tei
Dipartimento di Scienze dell’Ambiente e della Vita,
Università del Piemonte Orientale,
A. Avogadro, Turin, Italy
M. Ercolani
Tecnobiomedica S.p.A., Pomezia,
Rome, Italy
S. Aime
Dipartimento di Chimica IFM,
Università di Torino, Turin, Italy
G. Camussi (*)
Cattedra di Nefrologia, Dipartimento di Medicina Interna,
Ospedale Maggiore S. Giovanni Battista,
Corso Dogliotti 14,
10126 Turin, Italy
e-mail: [email protected]
GIOVANNI CAMUSSI M.D.,
BENEDETTA BUSSOLATI M.D.,
is a full professor of Nephrology received her PhD in Nephrology
at the University of Torino,
from the University of Parma.
Italy, and director of the Renal She is presently an associate
and Vascular Pathophysiology professor of Nephrology at the
laboratory (http://www.rvplab. University of Torino, Italy. Her
unito.it), Center for Research
main research interests include
in Experimental Medicine and tumor angiogenesis and renal and
Molecular Biotechnology
stem_cell biology.
Center. His research interests
include studies on renal and
cardiovascular immunopathology, inflammatory mediators
and stem
to murine tumor endothelial cells. In particular, the peptide
CVGNDNSSC (BB1) bound to TEC in vitro and in vivo.
Using BB1 peptide conjugated with the ribosome-inactivating toxin saporin, we targeted TEC in vivo. Injection of
BB1-saporin but not saporin alone or control modified BB1ala saporin induced a selective cell apoptosis and disruption
of the TEC vessel network. No increase in cell apoptosis was
found in other murine organs. In conclusion, the identification of peptide sequences able to bind selectively human
tumor-derived endothelial cells may represent a tool to
DO00184; No of Pages
898
deliver antiangiogenic or antitumor agents within the
neoplastic vessels.
Keywords Angiogenesis . Angiogenic therapy .
Phage display . Renal carcinoma . Peptides
J Mol Med (2007) 85:897–906
28]. In the present study, exploiting this unique model of
human tumor angiogenesis in mice, we aimed to screen a
phage display library of random peptides following the
protocol described by Arap et al. [10]. Using this approach,
we identified cyclic peptides showing specific binding to
TEC, and we targeted TEC in vivo using one peptide
conjugated with the ribosome-inactivating toxin saporin.
Introduction
Recent evidences indicate that the cells of the vascular
endothelium are heterogeneous and exhibit specialized
phenotypes, depending on their organs of origin and on their
functional state [1, 2]. In particular, endothelial cells present
in malignant tumors express surface molecules, such as the
receptors for the adhesion to matrix and for circulating
leukocytes, as well as receptors for angiogenic growth
factors, that are absent or barely detectable in normal blood
vessels [3]. Recently, it has been reported by several groups
that tumor endothelial cells possess, at the molecular level,
distinct characteristics from normal endothelial cells [4–7].
The phenotypic and molecular diversity of tumor-associated
vasculature provides a basis for the development of targeted
diagnostics and therapeutics [8]. One of the possible
approaches in the antiangiogenic therapy is to develop
specific pharmacological tools to guide more selectively
chemotherapeutic drugs, toxins, or radio-therapeutics on
tumor endothelial cells [9–11]. Monoclonal antibodies
against surface receptors such as vascular endothelial growth
factor (VEGF) or CD105 [12–14] and peptides generated by
in vivo screening of a phage display random peptide library
[15–20] have been proposed. A critical point in these
approaches is the need of identification of specific markers
for human tumor-derived endothelial cells. For this purpose,
several studies screened peptides or antibodies against
human-activated normal endothelial cells [21, 22] or tumorassociated murine endothelial cells migrated into implanted
tumors [10] or tumor metastases [23]. Another approach
aimed to selectively identify peptides against human
endothelial molecules present in inflamed endothelium has
been the xenograft of synovium from patients with rheumatoid arthritis [24]. Human tumor-derived endothelial cells
have not so far been used to develop specific peptides for
tumor endothelium. As we recently obtained and characterized several endothelial cells lines from human renal carcinomas [5], we intended to develop a peptide-based targeting of
these cells. Tumor-derived endothelial cells (TEC) displayed
a proangiogenic and immature phenotype and expressed
neoantigens that may allow differential targeting in respect to
normal endothelium [25–28]. Using TEC, we generated a
murine model of human tumor angiogenesis in which TEC
injected subcutaneously in severe combined immunodeficiency (SCID) mice into diluted Matrigel organized in
vascular structures connected with the mouse circulation [5,
Materials and methods
Reagents
The disulfide-constrained (seven amino acids with a flanking
cysteine residue at both ends of the peptide) cyclic M13 phage
display library (Ph.D.C7C system; New England Biolabs,
Hitchin, UK) was used. Endothelial cell attachment factor
(ECAF), Dulbecco’s modified Eagle’s medium (DMEM), and
bovine serum albumin (BSA) fraction V (tested for not more
than 1 ng endotoxin per mg) were purchased from Sigma
Chemical (St. Louis, MO, USA). Fetal calf serum (FCS) was
from EuroClone (Wetherby West Yorkshire, UK).
Endothelial cells
TEC were isolated from specimens of clear-cell type renal
cell carcinomas using anti-CD105 Ab coupled to magnetic
beads by magnetic cell sorting using the MACS system
(Miltenyi Biotec, Auburn, CA, USA), as previously described [5]. TEC cell lines were established and maintained
in culture in endothelial basal complete medium (EBM)
supplemented with epidermal growth factor (10 ng/ml),
hydrocortisone (1 mg/ml), bovine brain extract (all from
Cambrex Bioscience, Baltimore, MD, USA) and 10% FCS.
TEC were previously characterized as endothelial cells by
morphology, positive staining for vWF antigen, CD105,
CD146, and vascular endothelial-cadherin and negative
staining for cytokeratin and desmin [5].
A murine line of transformed endothelial cells (H.end) was
previously obtained and shown to form in vivo endothelial
tumors [29]. Human microvascular endothelial cells (HMEC)
and human umbilical vein endothelial cells (HUVEC) were
obtained and cultured as previously described [26].
In vivo model of human tumor angiogenesis
For the in vivo studies, TEC were implanted subcutaneously
into SCID mice (Charles River, Jackson Laboratories, Bar
Harbor, ME, USA) within growth factor reduced Matrigel, as
described [5, 28]. Cells were harvested using nonenzymatic
dissociation solution (Sigma), washed with phosphatebuffered saline (PBS), and resuspended in DMEM (1×106
in 250 μl DMEM). Cells were chilled on ice, added to
J Mol Med (2007) 85:897–906
250 μl of Matrigel at 4°C, and injected subcutaneously into
the back of SCID mice via a 26-gauge needle using a 1-ml
syringe. At day 6, previously reported as the requested time
for connection between mice and human microvessels, mice
were processed for experiments. To study murine tumor
angiogenesis, H.end were subcutaneously injected in Matrigel in the same condition described for TEC.
In vivo selection of TEC-specific phages
TEC homing phages were isolated by three cycles of
enrichment in SCID mice transplanted with TEC. At day 6
after injection, the pep-PDL library [1×1011 plaque-forming
units (PFU) in 200 μl saline] was injected into the tail vein of
anesthetized animals. After 15 min, while under deep/terminal
anesthesia (Sagatal; 5 μg/mouse; Rhone Merieux, Athens,
GA, USA), the mice were perfused via the left ventricle with
50 ml of DMEM within 5 min to ensure phage clearance
from the blood. The inferior vena cava was cut for the outlet.
TEC plugs and mouse organs were then extracted, weighed,
and processed as necessary for phage recovery and histological analysis. The aliquot used for phage recovery was
washed three times in Tris-buffered saline (TBS; 150 mM
NaCl, 50 mM Tris, pH 7.4; Sigma) and then homogenized in
1 ml TBS containing protease inhibitor cocktail (Sigma).
Phages were eluted from the tissues with 1.6 ml of 0.1 M
glycine, pH 2.0, and after 10 min of incubation, were
neutralized with 36 μl of 2 M Tris base. To determine the
number of phages in the eluate, in each round of selection,
titered triplicate samples of the eluate were added with the
Escherichia coli host ER2737 (New England Biolabs) into
melted Luria–Bertani (LB) agar tops (7 g/l agarose, 1 g
MgCl2·6H2O; Sigma), which were then plated onto isopropylbeta-D-thiogalactopyranoside (IPTG)/X-Gal LB agar plates
(Kramel Biotech, Cramlington, UK). After overnight incubation at 37°C, the peptide phages, appearing as blue
plaques, were counted and the yield of phage localizing to
each individual tissue determined. The residual eluate was
amplified by culturing the phages as individual plaques on
IPTG/X-Gal LB agar plates as described above for titering.
The amplified phages in the plaques were recovered from the
agar by homogenizing the agar top layer in LB media,
centrifuging, and then precipitating the supernatant with
3.3% polyethylene glycol 8,000/0.4 M NaCl (Sigma). The
resultant pool of phages was resuspended in TBS and titered,
as described above, for reinjection in subsequent rounds of in
vivo selection. Two further cycles of in vivo selection were
performed to enrich for specificity to TEC.
Identification of TEC-binding peptides
The sequence of the DNA inserts encoding for the peptides
displayed by the phages homing specifically to the TEC-
899
formed vessels in Matrigel was determined in 35-phage
clones selected at random after the last round of in vivo
selection. Single clones were amplified by culturing the
phages as described above, and their single-stranded
DNA were automatically sequenced by MWG Biotech,
(Ebersberger, Germany). The primer used for sequencing
was 5′-HOCCC TCA TAG TTA GCG TAA CG-3′ (−96 gIII
sequencing primer, provided in C7C kit, New England
Biolabs). Alignment by manual comparison of the sequences
was used to identify consensus motifs.
In vitro binding of phage clones to TEC
Tumor-derived or normal endothelial cells were plated on
tissue culture 24-multiwell plates (Nunc, Roskilde, Denmark).
In vitro binding assay of selected clone phages to TEC or
HMEC (1×105 cells) was performed by incubating phage
clones (1×109 PFU) individually with TEC or HMEC for 1 h
in gentle agitation at room temperature. After several washes
with TBS–Tween and with PBS, the phages bound to the cells
were eluted with 1.6 ml of 0.1-M glycine/HCl, pH 2.0, and
after 10 min of incubation, were neutralized with 36 μl of 2 M
Tris base 0.2M, as described above. Phages were rescued by
infection of the Escherichia coli host ER2737 strain. Phages
were titered and expressed as number of PFU.
In vitro synthesis and conjugation of TEC-binding peptides
The BB1 cyclic peptide, containing nine amino acids with a
disulfide bridge connecting the two terminal cysteines
(CVGNDNSSC), and the control peptide BB1-ala, in which
the central Asn-4, Asp-5, and Asn-6 residues were
substituted with alanines were synthesized by Statistical
Package for the Social Sciences (SPSS) using a Rink
Amide resin as solid support and the standard Fmoc
strategy. Amino acids and resin for solid phase synthesis
were purchased from Advanced Biotech, Italia, Seveso
(MI), Italy. For binding studies, all the peptides were
conjugated on solid phase with D-biotin (Sigma) to the Nterminal. After cleavage from the resin and precipitation
with diethyl ether, the disulfide bridge between the two
cysteines was formed by air oxidation in water (10 ml of
H2O for each mg of peptide) with 1% of DMSO. The final
products were obtained in good yields after purification
with semi-preparative high-performance liquid chromatography (HPLC; Amersham AKTA Purifier 10/100 using a
Waters XTerra RPC18 19/50 column, final purity >90%)
and characterized by Maldi mass spectra (Bruker Daltonics,
Bremen, Germany). For cytotoxic studies, the BB1 peptide,
where two serine residues were added (CSSVGNDNSSC),
was modified by reaction (PBS buffer, pH 8) with 2-iminothiolane (Sigma) in a 1:2 molar ratio for 1 h at room temperature. The product was then purified by gel filtration on
900
a Sephadex G25 column (Pharmacia Biotech, Uppsala,
Sweden) using water as eluent and concentrated to 10 g/ml.
Saporin from Saponaria officinalis seeds (Sigma) (10 mg/ml
PBS, pH 7.5) was modified using N-succimidyl-3-(2pyridyldithio) propionylate (SPDP; Sigma) at a 15:1
SPDP/saporin molar ratio [26]. After 30 min at 25°C, the
modified protein was separated from unreacted reagents by
gel filtration on a Sephadex G25 column equilibrated and
eluted with PBS. After concentration to 10 mg/ml, SPDP–
saporin was conjugated to the BB1-iminothiolane-modified
peptide by incubation at room temperature for 18 h. The
number of BB1 peptides conjugated to saporin was determined by the A343, showing the attachment of three BB1 every
saporin molecule. The resulting conjugate was separated from
the unreacted reagents by gel filtration chromatography with a
Superdex 200 column (Pharmacia Biotech) equilibrated and
eluted with PBS.
Cytofluorimetric studies
For cytofluorimetric analysis, cells were detached from
plates with nonenzymatic cell dissociation solution, washed
in PBS containing 2% heat-inactivated human serum, and
incubated for another 15 min with whole heat-inactivated
human serum to block remaining nonspecific sites. For
binding of phage clones, cells were incubated with a
selected phage clone (1×1010 PFU/test) for 1 h at room
temperature in static condition, and after washings with
anti-M13 mAb (Pharmacia, Uppsala, Sweden). Binding
was revealed with the use of antimouse fluorescein
isothiocyanate (FITC) Ab (Dako, Copenhagen, Denmark).
For binding of peptides, cells were incubated for 30 min at
room temperature with biotin-conjugated peptides in different concentrations or with the irrelevant control in PBS
containing 2% heat-inactivated human serum. Cells were
stained by the addition of PE-conjugated streptavidin
incubated for further 30 min at 4°C. Cells were analyzed
on a florescence-activated cell sorting (FACS; Becton
Dickinson, Mountain View, CA, USA). In each experimental point, 10,000 cells were analyzed.
J Mol Med (2007) 85:897–906
peptide. Tissues were subjected to immunofluorescence or
to electron microscopy.
In selected experiments, BB1 labeled with 2 μg saporin
or 2 μg saporin alone were injected intravenously on days
6 and 8 after TEC implantation. Three days later, animals
(n=4 per group) were killed and Matrigel plugs processed
for light microscopy and TUNEL detection.
Immunogold electron microscopy studies
Tissue samples were fixed in 2.5% paraformaldehyde
containing 2% sucrose. Immunogold labeling was performed as described previously in 20 μm sections using the
anti-M13 mAb (1:50) or isotypic control IgG (Dako, Milan,
Italy) and the 5 nm gold-conjugated anti-mouse Ab
(BBInternational, Cardiff, UK), as secondary antibody,
followed by silver enhancement (Silver enhancing kit, BB
International). Samples were postfixed in 2.5% glutaraldehyde, dehydrated in alcohol, dried, and coated with gold by
sputter coating. The specimens were examined in a
scanning Jeol T300 electron microscope. Images were
obtained via secondary electron at a working distance of
15–25 mm and at an accelerating voltage of 20–25 kV.
Assessment of apoptosis and immunofluorescence analysis
Apoptosis was evaluated using the TUNEL assay analysis
(ApopTag Oncor, Gaithersburg, MD, USA), as described in
the protocol of this assay. Sections from paraffin-embedded
blocks of mice kidney or Matrigel plugs were collected onto
poly-lysine-coated slides. After treatment with proteinase K
In vivo injection of clones or BB1 peptide
To evaluate the selective binding of single clones or peptides
to TEC in vivo, single phage clones (1×1011 PFU; 200 μl
saline) or biotin-conjugated peptides (100 μg in 200 μl
saline) were injected intravenously into separate animals
transplanted with human TEC and murine H.end. The
number of phages localizing to human tumor vessels, mouse
tumor vessels, or mouse renal vessels was determined on
homogenized tissues as described above. In selected experiments, FITC-labeled streptavidin (1 μmol/kg; Sigma) was
injected 15 min after administration of biotin-labeled
Fig. 1 Recovery of phages targeting human vessels formed by TEC
injected in Matrigel in SCID mice by in vivo screening of a phagedisplayed peptide library. The library (1×1011 PFU) was injected into
the tail vein of SCID mice. Fifteen minutes after injection, mice were
perfused with DMEM through the hart, and phages were recovered
from different organs. Phages recovered from the TEC-formed vessels
were amplified and reinjected in two consecutive rounds. The number
of PFU is shown. Data are mean ± SD of PFU from triplicate plating
in four different animals. Analysis of variance with Newmann–Keuls
multicomparison test was performed: *p<0.05 TEC in round 3 vs
round 1 and 2; **p<0.05 mouse kidney vs TEC
J Mol Med (2007) 85:897–906
901
Table 1 Peptide inserts, from
35 randomly selected phage
clones homing to TEC
obtained from the final round
of in vivo selection, were
sequenced and analyzed for
consensus motifs
Complete sequence of those
clones displaying consensus
motifs is shown. Two clones
showed repetition of the
sequence and others showed
multiple overlapping motif
regions (underlined amino
acids). Shown in parentheses
is the occurrence of the same
motif in the different clones.
(at 37°C for 10 min), sections were treated with terminal
deoxynucleotidyl transferase (TdT) enzyme and incubated in
an humidified chamber at 37°C for 1 h and then with FITCconjugated anti-digoxigenin for 30 min at room temperature.
In selected experiments, samples were incubated in parallel
with anti-von Willebrand factor Ab (Sigma) and then with
Texas red goat anti-rabbit IgG (Molecular Probes, Leiden, The
Netherlands) as secondary antibody. Confocal microscopy
analysis was performed using a Zeiss LSM 5 Pascal model
Confocal microscope (Carl Zeiss International, Germany).
Hoecst 33258 dye (Sigma) was added for nuclear staining.
M13 coat phage protein was detected on Matrigel plugs
extracted from animals previously injected with single
phage clones on acetone-fixed serial cryostatic sections
using the anti-M13 monoclonal antibody (Pharmacia).
Results
Selection of TEC homing phages using a model of human
tumor angiogenesis in SCID mice
We previously described a model of human tumor angiogenesis in SCID mice using TEC derived from human renal
carcinomas [5, 28]. Using this model, we isolated phages
with homing properties for human TEC by performing three
cycles of in vivo selection. Mice were previously injected
subcutaneously with TEC within Matrigel. After 6 days,
TEC organized in capillary structures of human origin. All
the experiments were performed at this time point when
human tumor vessels were shown to be connected with the
murine vasculature [5, 28]. Mice (n=4 per round) were
Table 2In
Table
2 Invitro
vitrobinding
bindingofofsingle
singlephage
phageclones
clonestotodifferent
differentlines
linesofof human
(H-end)
tumor-derived
was evaluated
endothelial
using cells
cytofluorimetric
(TEC) to normal
analysisendothelial
or recovery
cells
of
(HMECtumor-derived
human
and HUVEC) endothelial
or to murine
cells
transformed
(TEC) to endothelial
normal endothelial
cells (H-end)bound
was evaluated
phages, asusing
described
cytofluorimetric
in “Materials
analysis
and methods”
or recovery of bound
phages,
cells
(HMEC
as described
and HUVEC)
in “Materials
or to murine
and methods”
transformed endothelial cells
In vitro binding (PFU×103/105 cells)
Cytofluorimetric analysis (% positive cells)
30.1
30.3
30.5
30.8
123.7
123.14
123.15
TEC33
57±6
15±3
0
57±8
24±5
0
66±12
TEC25
64±10
66±5
24±3
74±12
88±7
29±5
84±13
TEC28
38±9
25±5
38±7
14±3
49±11
16±6
71±9
HUVEC
0
9±3
0
0
0
0
0
HMEC
12±5
0
0
0
0
0
0
TEC25
15±1
21±1
0
6±5
24±2
0
23±4
TEC28
17±3
25±2
0
0
25±5
0
20±3
HMEC
0
0
0
0
0
0
0
H.end
0
0
0
0
0
0
0
FACS analysis was gated on living cells that represented the 87–92% of the total population based on physical parameters. Data represent the
mean ± 1SD of at least four different experiments.
902
Fig. 2 Recovery of selected phage clones targeting human TECformed vessels. Single amplified phage clones (1×1011 PFU) were
injected into the tail vein of SCID mice carrying human tumor vessels
formed by TEC and murine tumor vessels formed by H.end. Fifteen
minutes after injection, mice were perfused with DMEM through the
J Mol Med (2007) 85:897–906
hart and phages were recovered. A 30.1 phage clone showed the best
binding to TEC as compared to H.end and to murine renal vessels
(kidney). The number of PFU is shown. Data are mean ± SD of PFU
from triplicate plating in three different animals
injected intravenously with 1×1011 PFU of the whole phage
library. After 15 min of circulation time, the animals were
killed, and the number of phages localizing to TEC formed
vessels as well as to murine kidney (control murine tissue)
was determined as described in “Materials and methods”.
Phages recovered from TEC plugs were amplified to 1×
1011 PFU and reinjected into second and third animals (n=4
per group) carrying the TEC-formed vessel network in the
Matrigel plug. A significant increase in the number of
phages recovered from TEC in the Matrigel plug in the third
round was observed (Fig. 1). In contrast, no such enrichment
was seen in the mouse kidneys, suggesting the isolation of
specific phages that preferentially bound to human tumor
vessels rather than to mouse normal vasculature (Fig. 1).
To investigate whether specific consensus sequences
were enriched in the peptides displayed by phages homing
preferentially TEC, the peptide-encoding DNA inserts from
35 clones (selected at random from the last round of
selection) were sequenced. Alignment of the insert sequences identified two peptides that were entirely repeated
in three (30.1) or two (30.3) different phage clones and
others with distinct consensus motifs (Table 1). Serineserine-cysteine (SSC) sequence was identified in 8 clones
out of 35 and could represent a flanking sequence.
In vitro and in vivo screening of phage clones
We next evaluated the binding ability to TEC of seven
phage clones selected because they contain consensus
motifs. In vitro, we compared the binding of individual
phage clones to TEC in respect to normal human
endothelial cells (HMEC and HUVEC) and to tumor
murine endothelial cells (H.end). In static condition,
cytofluorimetric analysis showed a specific binding of
five phage clones to three different lines of TEC and not
Fig. 3 Localization of 30.1 phage clone and of the corresponding
BB1 peptide in TEC-formed vessels. a, b Representative micrographs
showing the presence of gold particle labeled Ab, recognizing the
phage capside protein M13, in vessels formed by TEC (a) but not in
vessels of renal tissue (b). Insets Immunofluorescence detection of the
30.1 phages. c, d Representative micrographs showing fluorescence
detection of the binding of BB1 peptide in TEC-formed vessels (c)
and not in vessels of kidney (d), liver (e), and lung (f ). BB1
biotinylated peptide was i.v. injected followed, after 15 min, by FITC
streptavidin, and the localization was observed by fluorescence on
cryostatic sections. Three animals were performed in each experimental condition with similar results. Original magnification: a and b,
×1,500; inset and c–f, ×250
J Mol Med (2007) 85:897–906
to human normal endothelial cells (Table 2). In dynamic
conditions, in vitro binding experiments confirmed a positive
binding of 30.1, 30.3, 123.7, and 123.15 phage clones to
TEC in respect to HMEC or H.end. Three phage clones,
despite positive binding by cytofluorimetric analysis, were
negative as binding test in dynamic conditions possibly due
to lower affinity. In vivo, we performed experiments in
which animals were double-transplanted with TEC and H.
end, in two distinct sites, to compare the binding of phage
clones to human and mouse tumor vessels. After 6 days,
animals were injected with 1×1011 PFU, and the number of
phages recovered was analyzed. As shown in Fig. 2, the 30.1
clone displayed the highest recruitment of phages within the
tumor vessels. The specific binding to human tumor vessels
was evidenced by comparison with the binding to mouse
renal vessels and to mouse tumor vessels formed by H.end
(Fig. 2).
In animals injected with the 30.1 phage clone, it was
possible to detect 30.1 phage clone by SEM immunogold
and by immunofluorescence within the TEC-formed vessels
using an anti-M13 Ab recognizing a common phage protein
expressed by the phage capside (Fig. 3a). In contrast, the
same animals showed no localization of the 30.1 phages
within the renal vasculature (Fig. 3b).
Binding of BB1 synthetic peptide to TEC
As the clone 30.1 showed the best TEC binding, we
synthesized a biotinylated synthetic cyclic peptide (BB1) of
the sequence expressed by this phage clone (CVGNDNSSC).
A control peptide with three central alanine substituted (BB1ala CVGAAASSC) was also synthesized. BB1 peptide
Fig. 4 Binding of BB1 to TEC.
Cells were incubated with BB1
or with the control peptide BB1ala conjugated to biotin, and the
binding was revealed with PEstreptavidin evaluated by cytofluorimetric analysis. a White
area shows positive binding of
1 μg BB1 (dark line) and of SSelongated BB1 (gray line) to
TEC. b Dose-response binding
of BB1 to TEC (4=10 μg BB1;
3=2 μg BB1; 2=0.1 μg BB1;
1=0.01 μg BB1). Absence of
binding of the control peptide
BB1-ala (10 μg, white area) to
TEC-25 (c) and of BB1 (10 μg,
white area) to HMEC (d) was
observed. In all experiments, the
dark areas represent the binding
of PE-streptavidin alone
903
displayed a good dose-dependent binding to TEC and not to
HMEC or HUVEC as evaluated by cytofluorimetric analysis
(Fig. 4). No binding of BB1-ala to TEC was observed.
In vivo experiments of BB1 binding to TEC-formed
vessels were performed using the biotinylated BB1 peptide
followed by injection of FITC-streptavidin. Fluorescence
analysis of the tissue showed a positive staining for TEC in
the human vessel network developed in Matrigel (Fig. 3c)
but not in mouse tissues, such as muscle (not shown), liver,
lung, and kidney (Fig. 3d–f ).
In vivo targeting
To exploit the selective localization of BB1 to TEC-formed
vessels in vivo, we conjugated BB1 to saporin, a ribosomal
inactivating toxin, from Saponaria officinalis able to induce
cell apoptosis. For this purpose, BB1 was modified by
adding an SS residue. As shown in Fig. 4a, the binding to
TEC was unchanged. BB1 conjugated to saporin (BB1saporin) was injected i.v. at days 6 and 8 after TEC
implantation, and mice were killed 3 days later. As
evaluated by TUNEL assay, the number of apoptotic
endothelial cells was significantly increased in the animals
treated with BB1-saporin in respect to saporin alone or to
BB1-ala saporin used as control (Fig. 5a–c). Moreover, the
number of the vessels formed by TEC was decreased and
the vessel network disrupted in mice injected with BB1saporin (Fig. 5e). Only few apoptotic cells were observed in
control organs such as kidneys, liver, lung, and muscles
(Fig. 5f–i). By light microscopy, nuclear fragmentation and
picnosis, disruption of tumor vessels, and accumulation of
inflammatory cells were observed (Fig. 5k). In mice
904
J Mol Med (2007) 85:897–906
Fig. 5 Targeting of TEC with BB1 saporin. Mice carrying the TECformed vessels were injected with BB1 saporin, BB1-ala saporin, or
saporin alone and tissue analyzed 3 days later (see “Materials and
methods”). a The number of apoptotic cells, as evaluated with
TUNEL, in the TEC-formed Matrigel plug was significantly increased
in mice treated with BB1 saporin in respect to saporin alone or BB1ala saporin. Data are mean ± SD of four different experiments and are
expressed as number of TUNEL positive cells per microscopic field.
Analysis of variance with Dunnet’s multicomparison test was
performed: *p<0.05 BB1 saporin vs saporin alone or vs BB1-ala
saporin. b, c Micrographs representative of the presence of apoptotic
cells in the TEC-formed vessels 3 days after injection of saporin alone
(b) or BB1 saporin (c). d, e Double immunofluorescence staining of
vWF (red) and TUNEL (green) in the TEC-formed vessels 3 days
after injection of saporin alone (d) or BB1 saporin (e). A complete
loss of the endothelial network and the concomitant presence of
apoptotic cells was observed after treatment with BB1 saporin. f–i
Micrographs representative of absence of apoptotic cells in the liver
(f), lung (g), muscle (h), and kidney (i) of mice injected with BB1
saporin. Nuclei were counterstained with Hoecst 33258 dye in b–i.
j–l Representative light microscopy micrographs of the TEC-formed
vessels in the Matrigel plug after treatment with saporin alone (j) or
with BB1 saporin (k) or with BB1-ala saporin (l). The micrograph k
shows nuclear fragmentation, picnosis, and aspects of necrosis. The
inset shows nuclear fragmentation (arrow) and presence of inflammatory cells in a remnant vessel. Original magnification: b–l, ×250;
k inset, ×630
injected with saporin alone or BB1-ala saporin, no
significant increase in the number of apoptotic cells and
no disruption of the TEC vascular network was observed
(Fig 5a,d,j, and l).
Discussion
In the present study, we identified a peptide able to
selectively bind and to target human tumor endothelial
J Mol Med (2007) 85:897–906
cells using an in vivo phage display screening in a model of
tumor angiogenesis. This peptide was exploited to selectively deliver saporin, a ribosome-inactivating toxin, to
human TEC implanted in vivo in SCID mice.
Peptide screening using page display technology has
been previously used to identify selective peptides able to
bind the vasculature of different organs as well as inflamed
or tumor endothelium [10, 11, 15–23]. The novel approach
of the present study was to utilize a model of human tumor
angiogenesis in SCID mice. This approach led to the
identification of a peptide able not only to discriminate
between normal and tumor vasculature but also between
murine and human tumor vessels. In fact, the 30.1 phage
clone and the corresponding BB1 peptide showed in vitro
an increased binding to TEC in respect to human normal
endothelial cells, HMEC and HUVEC. In vivo, the 30.1 phage
clone and the corresponding BB1 showed an enhanced binding
to TEC formed vascular network but not to murine normal
endothelium and to murine tumor vessels formed by H.end.
A selective and potent targeting of the tumor vasculature
is of particular interest in cancer therapy for several different
reasons [9, 30, 31]. First, as the proangiogenic tumor
endothelium may display an autocrine release of several
growth factors, anti-angiogenic therapies aimed to inhibit
one of those, such as VEGF, may not be sufficient per se
[32] and could benefit of associated targeted therapies.
Moreover, tumor endothelial cells are more accessible to
drugs. Thus, the blood vessels in a tumor provide a
potential target for directing a chemotherapeutic agent to
the tumor, thereby reducing the likelihood that the agent
will kill sensitive normal tissues. Biologically active
peptides could affect metastatization per se [33]. In
addition, peptides have been successfully linked to drugs
to increase the therapeutic index and reduce in vivo toxicity
[2, 34]. In addition, the RGD peptide has been successfully
linked to liposomes to deliver genes to tumor vessels [35]
as well as for in vivo imaging in patients [36]. Finally,
incorporation of vasculature-targeting peptides into the
envelope of adeno-associated viruses may allow a selective
delivery of gene therapy [37–39] as well as a combined in
vivo visualization of the transgene [40].
In the present study, we show that the conjugation with
saporin of BB1 peptide induced a strong apoptotic response
in TEC without impairing other organs such as kidney and
liver. The same amount of saporin unconjugated with the
peptide did not induce apoptosis of TEC-formed vessels and
was also insufficient to induce tissue injury in mice.
Therefore, it is conceivable that BB1 concentrated saporin
within tumor vessels favoring their apoptosis.
In conclusion, the identification of peptide sequences
able to bind selectively human tumor-derived endothelial
cells may represent a tool for delivering anti-angiogenic or
antitumor agents within the neoplastic vessels.
905
Acknowledgment This work was supported by Italian Ministry of
University and Research (MIUR): FIRB project (RBNE01HRS5-001)
and COFIN; by the Associazione Italiana per la Ricerca sul Cancro
(AIRC); by Regione Piemonte-Ricerca Scientifica Applicata and by
Progetto S. Paolo Oncologia.
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