1. family and parenting
2. motherhood
3. pregnancy

The FASEB Journal express article 10.1096/fj.03-1120fje. Published online August 2, 2004.
Identification of the pregnancy hormone relaxin as
glucocorticoid receptor agonist
Thomas Dschietzig, Cornelia Bartsch, Verena Stangl, Gert Baumann, and Karl Stangl
Medizinische Klinik m. S. Kardiologie, Angiologie, Pulmologie Charité Berlin, Campus Mitte,
Schumannstr. 20/21, 10117 Berlin, Germany
Corresponding author: Thomas Dschietzig, Charité Berlin, Campus Mitte Medizinische Klinik
m. S. Kardiologie, Angiologie und Pulmologie, Schumannstr. 20/21, 10117 Berlin, Germany. Email: [email protected]
ABSTRACT
The insulin-like peptide relaxin is a central hormone of pregnancy, but it also produces antifibrotic, myocardial, renal, central-nervous, and vascular effects. Recently, two G proteincoupled receptors, LGR7 and LGR8, have been identified as relaxin receptors. Prompted by
reports on immunoregulatory effects of relaxin, we investigated possible interactions with the
human glucocorticoid receptor (GR). Relaxin blunted the endotoxin-induced production of
inflammatory cytokines (IL-1, IL-6, TNF-α) by human macrophages—an effect that was
suppressed by the GR antagonist RU-486. In three different cell lines, relaxin induced GR
activation, nuclear translocation, and DNA binding as assessed in GRE-luciferase assays. Coimmunoprecipitation experiments revealed physical interaction of endogenous and exogenous
relaxin with cytoplasmic and nuclear GR. Relaxin competed with GR agonists for GR binding,
both in vivo, in whole-cell assays, and in vitro, in fluorescence polarization assays. Relaxin was
shown to up-regulate GR protein expression as well as the number of functionally active GR
sites. In LGR7/8-free cells, the relaxin-mediated activation of GR was preserved. In conclusion,
relaxin acts as GR agonist—a pathway pivotal to its effects on cytokine secretion by human
macrophages. These findings may deepen our understanding of relaxin’s abundant physiological
actions, as well as our insights into general principles of hormone signaling.
Key words: signal transduction • macrophages • cytokines
T
he peptide hormone relaxin belongs to the insulin family of structurally related molecules,
which include insulin, insulin-like growth factors, relaxin, the relaxin-like factor [also
designated insulin-like factor-3 (INSL3)], placentin (also designated INSL4), INSL5, and
INSL6. Mature relaxin has a molecular weight of ~6000 Da and consists of two chains, termed A
and B, which are covalently linked by two interchain disulfide bonds with an intradisulfide bond
in the A chain (1). Whereas in several mammalian species, only a single relaxin gene has been
found (2), the great apes (chimpanzee, gorilla, and orangutan; 3), as well as rats and mice (4),
possess two relaxin genes. Three different relaxin peptides originating from three different genes
are currently known in humans: H1 and H2 [the amino acid sequences of which were deduced
from the nucleotide sequences by Hudson et al. (5, 6)], and H3 [recently identified by Bathgate
et al. (4)].
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In 1926, the peptide received its name from its particular property of elongating the interpubic
ligament of non-pregnant guinea pigs (7). The role of relaxin has by now become well
established as a central hormone of human pregnancy, in its contribution to changes in
connective-tissue composition and to the regulation of implantation, myometrial activity, and
labor (8). It was not until the 1980s, however, that researchers began to recognize the astonishing
pleiotropy of relaxin: e.g., anti-fibrotic actions, regulation of pituitary oxytocin and vasopressin
release, vasodilation, renal hyperfiltration, promotion of angiogenesis, and versatile myocardial
actions (1, 9). We have recently established that relaxin is constitutively expressed in human
cardiovascular tissues and that the hormone—owing to up-regulation of its myocardial gene
expression—plays a compensatory role in human congestive heart failure (10).
In 2002, Hsu et al. (11) identified two G protein-coupled seven-transmembrane domain orphan
receptors as relaxin receptors: LGR7 and LGR8; LGR8 is the receptor for insulin-like peptide-3
(12), which can also be activated by relaxin. These receptors are coupled by mechanisms that are
in part still obscure to at least two major signaling cascades: the nitric-oxide and the cyclic AMP
pathways (9).
Prompted by reports on the immunoregulatory effects of relaxin (13, 14, 15), we speculated that
it might interact with the glucocorticoid system—in particular, with the glucocorticoid receptor
(GR). The GR belongs to the superfamily of steroid/thyroid/retinoic acid receptor proteins that
function as ligand-dependent transcription factors, i.e., as nuclear receptors (16). Upon hormone
binding, the cytoplasmic GR becomes activated and translocates to the nucleus, where it binds to
specific glucocorticoid response elements (GRE) of the DNA, thereby stimulating transcription
of responsive genes (transactivation). Activated GR can also capable interact with other
transcription factors such as NF-κB and AP-1, which may indirectly influence gene expression
(transrepression; 17). Cytoplasmic GR occurs as a complex with different proteins, of which at
least the molecular chaperones Hsp70 and Hsp90 seem essential for ligand binding and receptor
activation (18). Two human isoforms exist, GR-α and GR-β, which originate from the same gene
by alternative splicing, with GR-α abundantly expressed and GR-β hardly detectable at the
protein level (19). Whereas GR-α fulfills all the classical GR criteria, the proposed function of
GR-β—dominant negative inhibition of GR-α activity (20)—is not generally accepted and still
represents a matter of debate.
In our study, we present experimental evidence that relaxin binds to, activates, and regulates
human GR, and reach the novel conclusion that this interaction is functionally relevant to its
effects on stimulated cytokine secretion in human macrophages.
MATERIALS AND METHODS
Cultured cells
HeLa cells [epithelial cells derived from human cervix carcinoma, which demonstrate significant
endogenous synthesis of relaxin (8)]; spleen fibroblasts (cells lacking expression of the relaxin
membrane receptors LGR7 and LGR8 [11]); and 293 cells derived from human embryonic
kidney were obtained from American Type Culture Collection (ATCC, Rockville, MD). The
cells were grown in RPMI (Gibco, Berlin, Germany) supplemented with 10% FCS, 100 U/ml
penicillin, and 100 µg/ml streptomycin in a humidified 5% CO2 atmosphere. At subconfluence,
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we split cells at a 1:6 ratio using trypsin/EDTA. For the experiments, cells were also used at
subconfluence.
THP-1 cells (ATCC)—a cell line derived from human monocyte leukemia—are widely used for
endotoxin studies, since they closely mimic the characteristics observed in leucocytes of sepsis
patients (21). Cells of passage 5 to 10 were grown in suspension in RPMI supplemented with 1.5
g/l NaHCO3, 4.5 g/l glucose, 10 mM HEPES, 1 mM sodium pyruvate, 0.05 mM 2mercaptoethanol, 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin in a humidified
5% CO2 atmosphere. At a concentration of 2–5 × 105 cells/ml, cells were split at 1:2. For
differentiation into macrophages, cells were treated with 20 ng/ml PMA over 72 h, washed three
times with medium, and used for experiments after 24 h of rest.
Cytokine secretion of THP-1 cells
THP-1 (3 × 105 cells in 1.5 ml) were seeded in 24-well plates, and the cells were differentiated as
described above. Subsequent to differentiation, cells were maintained in RPMI medium
(composition as described above) that was phenol-free and contained charcoal/dextran-treated
FCS to avoid any bias caused by endogenous steroids. Interleukin-1 (IL-1), interleukin-6 (IL-6),
and tumor necrosis factor-α (TNF-α) were determined using ELISAs (R&D Systems, Munich,
Germany) with mouse monoclonal capture antibodies and biotinylated detection antibodies
according to the manufacturer’s instructions. Detection limits were 20 pg/ml for IL-1 and IL-6,
and 30 pg/ml for TNF-α. All samples were measured as duplicates.
Preparation of cytoplasmic and nuclear extracts
We applied the method described by Haber and co-workers (22). In brief, HeLa cells were
treated with trypsin/EDTA and centrifuged, and the cell pellet was washed and then resuspended
in 150 µl of buffer A [10% glycerol, 10 mM Tris (pH 7.9), 10 mM KCl, 10 mM NaF, 10 mM
K2HPO4, 1.5 mM MgCl2, 1 mM NaVO3, 0.5 mM DTT, and 0.5 mM PefaBloc SC (Roche,
Cologne, Germany)]. We obtained cytoplasmic extracts by treatment with 1 µl of the detergent
Nonidet P-40 (Roche) over 2 min, subsequent centrifugation (4°C, 10 min, 1000 rpm), and
removal of the cytoplasmic supernatant. The remaining nuclear pellet was lysed using 40 µl of
buffer C [10% glycerol, 0.42 M NaCl, 10 mM Tris (pH 7.9), 10 mM NaF, 10 mM K2HPO4, 1.5
mM MgCl2, 1 mM NaVO3, 0.2 mM EDTA, 0.5 mM DTT, and 0.5 mM PefaBloc SC], and the
nuclear extract removed after centrifugation (4°C, 10 min, 13,000 rpm). Both extracts were
stored at –70°C for further analysis.
Western blots
This analysis has been described elsewhere (10). As primary antibodies (dilution 1:2500), we
used a rabbit anti-GR-α/β polyclonal antibody (Santa Cruz Biotechnologies, Santa Cruz, CA), a
mouse anti-actin antibody (Research Diagnostics, San Diego, CA), a mouse monoclonal
antibody against Hsp 70 (Biomol, Palo Alto, CA), a mouse monoclonal antibody against the
human endothelin Type-B receptor (Research Diagnostics), a mouse monoclonal antibody
against Hsp 90 (Biomol), mouse monoclonal antibodies specific for the estrogen receptor-α (ERα) and the ER-β (Biomol), and a rabbit polyclonal antibody specific for human relaxin H1 and
H2 (10).
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Immunoprecipitation
Cytoplasmic or nuclear extracts (see above) obtained from subconfluent HeLa cells (10 cm
plates) were incubated with 50 µl protein A sepharose (Pharmacia Biotech, Munich, Germany)
for 30 min to saturate nonspecific binding sites. We subsequently incubated extracts with 50 µl
of the antibody-protein A sepharose complex at 4°C overnight. After centrifugation, the resulting
pellet was washed 10 times in 300 µl RIPA buffer, transferred into SDS buffer (60 mM Tris [pH
6.8], 25% glycerol, 5% β-mercaptoethanol, 2% SDS, and 0.1% bromophenol blue), and heated to
94°C for 5 min to release the precipitated proteins. After centrifugation, supernatants were used
for further analysis in Western blots. As precipitation antibodies, we used a rabbit polyclonal
antibody specific for human relaxin H1 and H2 (10), a rabbit anti-GR-α/β polyclonal antibody,
and a rabbit anti-grp94 polyclonal antibody (both from Santa Cruz). In the Western blots, we
also used a rabbit anti-insulin polyclonal antibody (Santa Cruz).
Fluorescence microscopy tracking of labeled relaxin
Fluoroisothiocyanate-(FITC-) labeled human H2 relaxin obtained from Immundiagnostik was
diluted in RPMI, and HeLa cells were incubated at 37°C for 30 min with 0.1, 2, or 10 nmol/l
labeled relaxin. To avoid any artificial intracellular accumulation of labeled relaxin, these cells
were neither fixed nor permeabilized. A subset of HeLa cells, however, was used to visualize
localization of the nucleus. These cells were fixed in paraformaldehyde (4% w/v in PBS) for 5
min at room temperature and then permeabilized using Triton X-100 (Sigma) (0.1% w/v in PBS)
for 10 min. After washing with PBS, cells were stained with propidium iodide (1µg/ml in PBS)
for 5 min at room temperature. Finally, both relaxin-treated and propidium-stained cells were
washed three times with PBS and mounted onto glass microscope slides. Distribution of
fluorescence was analyzed on a Zeiss Axiovert 100TV microscope; images were captured with
BioRad Laser Sharp and Adobe Photoshop 5.0 software.
RNA analysis
To yield total RNA, we performed extraction with Trizol (Gibco) as already described (10).
Total RNA (2 µg) was reverse-transcribed using avian myeloblastosis virus reverse transcriptase
and dT15 primers according to the manufacturer’s instructions (First Strand cDNA Synthesis Kit,
Boehringer Mannheim, Mannheim, Germany). We then performed PCR amplification of singlestranded cDNA by using primer pairs specific for human GR-α (5′ primer: 5′ GAA TGA CTC
TAC CCT GCA TG 3′, 3′ primer: 5′ TTT CCA TTT GAA TAT TTT GG 3′), GR-β (5′ primer:
5′ gAA TGA CTC TAC CCT GCA Tg 3′, 3′ primer: 5′ GCT TTC TGG TTT TAA CCA CA 3′),
and GAPDH (5′ primer: 5′ TGA AGG TCG GAG TCA ACG GAT TTG GT 3′; 3′ primer: 5′
CAT GTG GGC CAT GAG GTC CAC CAC 3′) (TIB MOLBIOL). Primer specific for the
relaxin receptors LGR7 and LGR 8 and the respective PCR conditions were chosen as described
in (11). Southern blot hybridization was conducted for semi-quantitation of the amplified
sequences. PCR products were separated for this purpose on 2% agarose gels, blotted onto nylon
membranes (Hybond N, Amersham) and were hybridized using radioactively labeled oligos
specific for human GR-α, GR-β, and GAPDH. Finally, autoradiography was performed, and
autoradiographs were quantified by use of the ImageMaster 1D Prime software (Pharmacia
Biotech). All data were normalized to GAPDH mRNA expression.
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Transient transfections and GRE-luciferase assays
Using cationic liposomes (Qiagen, San Diego, CA), we cotransfected cells with the GREluciferase reporter gene construct (pGRE-luc, Clontech, Palo Alto, CA) containing the GR
recognition motif. According to the manufacturer’s instructions (Qiagen), we transfected 0.5 µg
cDNA in HeLa/ 293 cells and 1 µg cDNA in THP-1 cells. After 6 h of incubation at 37°C, cells
were maintained in RPMI (phenol-free, 1% dextran/charcoal-treated FCS) over 24 h. Thereafter,
experiments were performed, cells were harvested for measurement of transactivation activity,
and luciferase activity was assayed in a β-counter as recommended in the manufacturer’s
instructions (Promega, Madison, WI).
3
H-Dexamethasone and 3HCorticosterone whole cell assays
We applied [1,2,4,6,7-3H]-dexamethasone (3.5 TBq/mmol; Amersham) as well as [1,2,6,7-3H]corticosterone (2.8 TBq/mmol; Amersham). HeLa cells (at subconfluence) and THP-1 cells (1 ×
106/ml) were cultured in 12-well plates in RPMI medium supplemented with 0.5% FCS, 0.02 M
NaOH, 0.01 M HEPES, and 0.075% NaHCO3 over 16 h. After various forms of stimulations (see
below), the medium was completely exchanged and cells were incubated for 60 min (HeLa) and
45 min (THP-1) with labeled dexamethasone or corticosterone. Thereafter, cells were washed 10
times with cold PBS and lysed using 100 µl of 100 nM NaOH. The lysates were then measured
in a scintillation counter (Wallac 1409, PerkinElmer Wallac). Non-specific binding was
determined by incubation with 10 µM cold dexamethasone. We performed saturation binding—
for determination of the apparent dissociation constant KD and the maximum number of binding
sites Bmax—as well as competition binding experiments.
Fluorescence polarization
We used the commercially available Glucocorticoid Receptor Competitor Assay (PanVera,
Madison, WI) together with a GENios device (Tecan, San Francisco, CA) for fluorescence
analysis at 485 nm excitation and 530 nm emission wavelength. According to the manufacturer’s
protocol, we prepared GR binding buffer by diluting 1 ml of 10 × GR Screening Buffer (100 mM
K2HPO4/KH2PO4, pH 7.4; 200 mM Na2MoO4; 1 mM EDTA; and 20% DMSO) with 7.95 ml
H2O, 1 ml of a 10 × stabilizing peptide (1 mM), and 50 µl of 1 M DTT. In the binding assay, 1
nM of the fluorescent glucosteroid Fluormone GS Red reacted over 45 min with 4 nM of human
recombinant GR (KD 0.3 ± 0.1 nmol/l according to the manufacturer) on a 100-well microplate,
which resulted in ~80% saturation of the GR. Negative controls (Fluormone GS Red and GR
only) and positive controls (Fluormone GS Red, GR, and 1 mM of dexamethasone) were
included to determine the fluorescence polarization window. Eventually, fluorescence
polarization was measured in the presence of rising concentrations of dexamethasone or relaxin.
Statistic analysis
All values are given as mean ± SEM. An error probability of P < 0.05 was regarded as significant.
Cytokine data, GRE-luciferase experiments, and the binding parameters (KD, Bmax) obtained
from 3H-dexamethasone assays were analyzed using the Kruskal-Wallis ANOVA on ranks. RNA
data, GR protein data, as well as saturation and competition binding curves, were compared with
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a two-way non-parametric ANOVA (23). A multiple-comparison procedure with BonferroniHolm adjustment of P (24) was performed after global testing.
Drugs
Salmonella abortus equii endotoxin, dexamethasone, corticosterone, insulin, estradiol, and RU486 were obtained from Sigma. Recombinant human relaxin H2—purified by using highperformance liquid chromatography—was from Immundiagnostik. We also used porcine relaxin,
which was generously provided by O. D. Sherwood (University of Illinois, Urbana). Judging
from the purification procedure (25)—acetone precipitation, gel filtration, and ion exchange
chromatography—any contamination of porcine relaxin with steroids can be ruled out.
RESULTS
Relaxin blunts endotoxin-induced production of inflammatory cytokines by THP-1 cells
We initially analyzed the immunomodulatory properties of relaxin in THP-1 cells differentiated
into macrophages. Endotoxin was used at a concentration of 10 ng/ml, which had been found to
represent a submaximum stimulatory level in our experimental setting (data not shown). This
endotoxin dose evoked a marked increase in the secretion of IL-1, IL-6, and TNF-α (Fig. 1) over
8, 24, and 48 h. Relaxin suppressed stimulated secretion of all cytokines—an effect that
plateaued between 5 and 10 nmol/l, showed an estimated EC50 of 0.8 nmol/l, and weakened at
higher concentrations, although it was still present at 100 nmol/l. At maximum, stimulated IL-1,
IL-6, and TNF-α were decreased to ~40% of the values in the presence of endotoxin alone.
The glucocorticoid dexamethasone proved less potent than relaxin in decreasing this stimulated
cytokine secretion (EC50 ~10 nmol/l), but showed a higher maximum effect, which amounted to
approximately one-third of the levels measured in the presence of endotoxin alone (data not
shown). We thereafter investigated (Fig. 2) whether the inhibitory effect of both dexamethasone
and relaxin was sensitive to treatment with RU-486 (0.5 µmol/l), a well-established GR
antagonist that shows high affinity to the ligand binding domain of GR and reduces nuclear
transfer and DNA binding capacity of the GR complex (dissociation constant KD = 3 nmol/l; 26).
As shown in Fig. 2, RU-486 not only prevented the inhibitory effect of dexamethasone on the
endotoxin-induced secretion of IL-1, IL-6, and TNF-α, but also abolished the effect of relaxin.
All experimental results regarding the effects of human relaxin on cytokine production by THP-1
cells were confirmed with porcine relaxin (n=4 for each group; data not shown).
These results indicated that relaxin exerted its inhibitory effect on stimulated cytokine secretion
by interacting with the GR in a manner independent of endogenous glucocorticosteroids, since
the charcoal-treated medium was steroid-free. The subsequent experiments were accordingly
designed to investigate whether relaxin (i) activated the GR and/or (ii) modulated its expression.
GRE-luciferase assays
In HeLa, 293 cells, and THP-1 cells transiently transfected with the GRE-luciferase reporter
gene construct, we established that relaxin could activate GR and, consequently, enhance gene
expression of the luciferase reporter in a time- and concentration-dependent fashion. Figure 3A
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shows that, in HeLa, 293 cells, and THP-1, relative luciferase activity reached its maximum after
4 h of stimulation with 10 nmol/l of relaxin. Figure 3B depicts data on concentration
dependency: Relaxin significantly activated gene expression of the reporter at concentrations as
low as 500 pmol/l; the calculated EC50 was ~0.8 nmol/l. Maximum values amounted to 600–
650% of baseline values and were already reached at 5 nmol/l of the peptide. At concentrations
higher than 10 nmol/l, this effect of relaxin weakened, which resulted in a bell-shaped curve. In
all cell types, the GR antagonist RU-486 had no effect on baseline luciferase activity, but
significantly and concentration-dependently suppressed activity stimulated by dexamethasone or
by relaxin (Fig. 3C). As with the cytokine experiments, the glucocorticoid dexamethasone was
less potent than relaxin (EC50 ~8 nmol/l), but it showed a higher maximum effect (data not
shown). Insulin, from 1 nmol/l to 1 µmol/l, never evoked GR activation in this experimental
setting (data not shown).
As negative control, we also transfected HeLa cells with a luciferase reporter gene construct
containing the estrogen response element (Fig. 3D). Whereas estradiol (100 nmol/l) significantly
increased luciferase activity—thereby indicating reliability of the system—relaxin did not
modulate baseline activity of luciferase.
All experimental results regarding the effects of human relaxin in GRE-luciferase assays were
confirmed with porcine relaxin (n = 4 for each group; data not shown).
Co-immunoprecipitation of relaxin and GR
We analyzed cytoplasmic extracts obtained from HeLa cells that were stimulated over 30 min
with solvent (control) or 10 nmol/l relaxin (Fig. 4A). Following immuno-precipitation with the
GR antibody, Western blot analysis clearly demonstrated co-precipitation of Hsp 70, Hsp 90, and
relaxin: both under control conditions and after relaxin treatment. Correspondingly, treatment
with the relaxin antibody co-precipitated Hsp 70, Hsp 90, and the GR—both in controls and after
relaxin administration. In contrast, the relaxin-related peptide insulin and grp94, a chaperon of
the endoplasmic reticulum, were not co-precipitated. Moreover, precipitation with the grp94
antibody likewise failed to yield bands for GR, relaxin, insulin, Hsp 70, or Hsp 90.
In nuclear extracts (Fig. 4B), a certain amount of co-precipitating GR and relaxin was always
detectable under control conditions, both after GR and relaxin precipitation. After relaxin
treatment of the cells over 30 min, we observed a marked increase in these signals. Again,
insulin and grp94 were not co-precipitated, and grp94 precipitation did not reveal any interaction
with GR, relaxin, or insulin.
Cytoplasmic and nuclear kinetics of relaxin and GR
Fig. 5 provides supplementary data concerning the cytoplasmic and nuclear kinetics of GR and
relaxin in response to relaxin stimulation (10 nmol/l). Thirty minutes after exposition to relaxin
of the cells, marked increase in the nuclear GR and relaxin signals occurred, which corresponded
satisfactorily with the data obtained from the immunoprecipitation experiments. Dexamethasone,
at 100 nmol/l, served as a positive control and induced the expected massive rise of nuclear GR
at 30 min of exposition.
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Tracking of fluorescence-labeled relaxin. Figure 6 demonstrates that labeled relaxin entered
intact (i.e., non-permeabilized) HeLa cells. Whereas no fluorescence signal could be observed at
0.1 nmol/l relaxin, a clear intracellular signal was detectable at 2 nmol/l. The highest
concentration used, 10 nmol/l, yielded a very intense fluorescence signal. This signal may reflect
both intracellular distribution of relaxin, as well as sustained binding to membrane receptors,
despite of the extensive washing procedure. Moreover, nuclear staining with propidium iodide
performed in a subset of permeabilized cells resulted in fluorescence distribution identical to that
seen with relaxin. This result gives rise to the notion that relaxin really accumulated in the
nucleus.
Displacement of GR agonists in fluorescence polarization and whole cell assays
As depicted in Fig. 7A, relaxin, at concentrations between 0.5 and 5 nmol/l, proved to be a highly
potent competitor at the human GR (estimated IC50 = 0.4 nmol/l) in the fluorescence polarization
assay. At concentrations of 10 nmol/l and higher, this competition was almost completely
reversed, and again became present at high nanomolar levels. Dexamethasone concentrationdependently competed with the binding of the fluorescent glucosteroid to hGR, with significant
displacement being evident at concentrations higher than 5 nmol/l. Nearly complete
displacement occurred at dexamethasone concentrations higher than 50 nmol/l.
Figure 7B depicts competition-binding experiments that used labeled corticosterone (48 nmol/l)
and increasing concentrations of cold corticosterone or relaxin in HeLa cells (whole cell assays).
As in the polarization assay, relaxin exhibited high potency of displacing the tracer, with an
estimated IC50 value of 1.2 nmol/l, but this displacement was abolished at higher concentrations.
Rising cold corticosterone led to complete displacement of the tracer.
Relaxin up-regulates GR gene expression
As summarized in Fig. 8 for HeLa cells, relaxin significantly elevated GR-α mRNA, at 30 min;
1, 2, and 4 h of exposure; as well as GR-β mRNA, at 2 and 4 h. Treatment with 100 nmol/l
dexamethasone over 6 h induced down-regulation of GR-α mRNA. All these effects proved
sensitive to the GR antagonist RU-486.
In THP-1 (Fig. 9), relaxin increased levels of GR-α mRNA at 1, 2, and 4 h after initiation of
treatment, whereas, in contrast to HeLa cells, GR-β mRNA remained unaffected. It was possible
to prevent up-regulation of the gene expression of GR-α by application of the GR antagonist
RU-486. Again, dexamethasone administration resulted in a decline of GR-α mRNA without
modulating GR-β gene expression.
Similar results reflecting stimulation of GR-α and GR-β mRNA after 2 and 4 h of relaxin
exposure were obtained in 293 cells (n = 3 for each group; data not shown).
Relaxin elevates GR protein levels
As shown in Fig. 10, relaxin exposition over 4 and 24 h remarkably increased GR protein levels
in HeLa and THP-1 cells. In HeLa, GR protein levels rose to ~500% of control values, whereas
in THP-1, relaxin caused an increase in GR protein to ~280% of controls. In contrast, treatment
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with dexamethasone over 24 h markedly decreased the expression of GR protein in HeLa and
THP-1, to 61 and 56% of control values, respectively. The actions of relaxin and dexamethasone
were found to be sensitive to administration of the GR antagonist RU-486 both in HeLa and
THP-1 cells. Similar results were obtained in 293 cells (n = 3 for each group; data not shown). In
contrast to the relaxin-induced up-regulation of GR, relaxin exposition over 4 and 24 h did not
increase protein expression of ER-α and ER-β in HeLa (n = 3 for each group; data not shown).
Relaxin pretreatment increases binding sites for 3H-dexamethasone and 3H-corticosterone
Finally, we attempted to determine whether relaxin could heighten the expression of functionally
active GR receptors in HeLa and THP-1 cells. Toward this objective, we performed whole cell
assays with labeled dexamethasone and corticosterone after pretreatment of the cells with 10
nmol/l relaxin over 4 or 24 h (Fig. 11A–C and Tables 1 and 2). In both cell types, exposition to
relaxin doubled the maximum number of glucocorticosteroid binding sites Bmax without altering
the apparent dissociation constant KD. In control experiments, we proved that 1 µmol/l RU-486
completely inhibited 3H-dexamethasone binding in HeLa and in THP-1 cells.
Experiments in LGR7/8-free spleen fibroblasts
We initially confirmed that spleen fibroblasts, in contrast to HeLa and THP-1, did not express
relevant amounts of the relaxin binding membrane receptors LGR7 and LGR8 (Fig. 12A). In
these cells, activation of GR by relaxin and dexamethasone as determined in the GRE-luciferase
assay was well preserved (Fig. 12B). Similarly, relaxin evoked elevation of GR protein
comparable with that observed in HeLa and THP-1 cells (Fig. 12C). In contrast, relaxin-induced
up-regulation of endothelin type-B receptors (27)—an ERK-1/2-mediated effect that is not
sensitive to RU-486—was completely abrogated (Fig. 12D).
DISCUSSION
The insulin-related peptide relaxin, discovered as pregnancy hormone at the beginning of the last
century (8), is presently being recognized as one of the central mediators of body fluid and
circulation homeostasis (1, 2, 9). In this study, we demonstrate another surprising facet of
relaxin: the peptide (i) binds to and activates the human GR; (ii) up-regulates, by using this
pathway, GR expression at mRNA, protein, and functional levels; and (iii) influences stimulated
cytokine secretion in human macrophages in glucocorticoid-like fashion.
Glucocorticoids, either endogenously produced by the adrenal gland or therapeutically
administered, affect and regulate a great variety of metabolic, behavioral, cardiovascular, and
immune functions. Among these effects, their anti-inflammatory and immunosuppressive profile
has attracted most intensive attention and has rendered them the most widely used drugs in
treating chronic inflammatory and autoimmune diseases (17). Inhibition of cytokine production,
e.g., IL-1, -2, -4, -6, -8; interferon-γ; TNF-α; and colony-stimulating factors, by various
immunologically competent cells represents one of the major immunosuppressive mechanisms of
glucocorticoids (28). In the present study, we chose the model of endotoxin-stimulated secretion
of IL-1, IL-6, and TNF-α by human macrophages to investigate possible immunomodulatory
actions of relaxin. These cytokines are known to represent key mediators of the so-called acutephase response of inflammation, and they are involved in a vast number of acute and chronic
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inflammatory diseases (17). In our experiments, relaxin potently inhibited stimulated secretion of
IL-1, IL-6, and TNF-α. Similar to the action of the synthetic glucocorticoid dexamethasone, this
effect was significantly reduced by the GR antagonist RU-486.
Encouraged by these results, we attempted to determine whether relaxin activated the human GR
and, if so, to clarify the principal mode of action. In three different cell lines, relaxin increased
luciferase activity in the GRE-luciferase assay, in a time- and concentration-dependent fashion,
which implicated that the peptide leads to GR activation, nuclear translocation, and DNA
binding. As with the control substance dexamethasone, it was possible to terminate the effect by
the GR antagonist RU-486. By means of co-immunoprecipitation techniques, we then
demonstrated physical interaction of relaxin with both the cytoplasmic and the nuclear GR
protein complexes. We would like to emphasize our finding that this interaction is likewise
detectable in the complete absence of exogenously administered relaxin, which proves the
relevance of endogenous relaxin for GR targeting. By sequential analysis of cytoplasmic and
nuclear extracts, we further established that, in close temporal correlation to relaxin application,
concomitant and marked increases occur in the nuclear content of relaxin and GR protein. In
addition, fluorescence-labeled relaxin was shown to enter the nuclear compartment of intact cells
at concentrations identical to those employed for the functional experiments.
Eventually, relaxin was shown to compete with GR agonists for GR binding, both in vivo in the
whole cell assay with labeled corticosterone (IC50 ~1.2 nM), and in vitro using a fluorescent
glucosteroid in the fluorescence polarization assay (IC50 ~0.4 nM). These binding data
corresponded well to those concentrations of relaxin that can induce luciferase activation or of
inhibiting stimulated cytokine secretion when administered exogenously to whole cells.
However, we observed that in the GRE-luciferase and cytokine experiments, the effects of
relaxin were only decreased but not abolished at concentrations > 5 nM, which seems to
contradict the nearly complete lack of relaxin binding at these higher concentrations as
demonstrated in the competition binding assays. We explain this as follows: The functional
experiments reflect changes in the gene and protein expression of luciferase and inflammatory
cytokines. In contrast, our binding data indicate GR-relaxin interaction under equilibrium
conditions (to approach such conditions experimentally, the optimum incubation times had to be
identified in the whole cell and polarization assays). In other words, if (in the initial “preequilibrium” period) higher concentrations of exogenous relaxin (> 5 nmol/l) gradually distribute
across the cell membrane into the cytosol this may transiently result in GR-activating
concentrations of the peptide, followed by GR translocation and alteration of gene expression.
This fact, in turn, is not reflected in the corresponding (equilibrium) binding data, but it may
cause gene and protein effects, which extend the dose-effect curve beyond the dose-binding
curve.
The question arises, however, as to the precise mode of GR binding of relaxin and, in this
context, as to the mechanism responsible for the weakening of GR-relaxin binding and of the
corresponding functional effects: luciferase activation and cytokine inhibition. The modes of
relaxin and GR binding to their “classical” interaction targets are well defined. With regard to the
relaxin membrane receptors, LGR7 and LGR8, the structural components essential for receptor
binding of relaxin reside in the B chain: two charged arginine residues in positions 13 and 17
project like fingers from the helix opposed by the hydrophobic isoleucine in position B20, thus
generating a trivalent interaction mechanism (29). With respect to glucocorticoid binding to GR,
Page 10 of 33
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the recent crystallization of the ligand-binding domain of GR by Bledsoe and co-workers (30)
has revealed the presence of a ligand binding pocket consisting of 11 α-helices and 4 β-strands
folded into a three-layer helical sandwich. As a result of sequence-based changes in the positions
of helices 6 and 7, the GR ligand-binding domain is distinct from the steroid pockets of the
estrogen, progesteron, and androgen receptors but shares common features with the
mineralocorticoid receptor. It is, however, unclear which mechanism accounts for relaxin
binding to GR; this question is currently a matter of investigation. The bell-shaped curve of
relaxin binding to GR, in turn, may be caused by the tendency of relaxin to form dimers at higher
concentrations (authors’ unpublished observation), or by the recruitment of additional binding
sites that could induce negative cooperativity.
Because glucocorticoids have been shown to provide feedback to the expression of their own
receptor, we also investigated whether relaxin could regulate gene and protein expression of GR,
as well as the number of functionally active GR sites. Whereas dexamethasone application led to
down-regulation of GR protein – as could be expected after a number of previous publications
(31, 32, 33) – exposure of HeLa, THP-1, and 293 cells to relaxin uniformly increased the protein
levels of GR, after both 4 and 24 h. This elevation of GR protein is at least partly attributable to a
rise in mRNA levels: in all cell types under investigation, relaxin induced an increase in GR-α
mRNA, with GR-β transcripts being also heightened in HeLa and 293 cells, but not in THP-1.
On the basis of recent reports (19, 34) on the expression of GR isoforms in human tissues – i.e.,
studies that detected hardly any GR-β protein – most if not all GR protein should represent GRα. This is corroborated by our finding that relaxin pretreatment in HeLa and THP-1 also elevated
the number of functionally active GR binding sites, as determined in whole cell assays using hot
dexamethasone and corticosterone. Since GR-β has repeatedly been reported not to bind
glucocorticoids (34, 35, 36), this rise of glucocorticoid binding sites in all likelihood reflected
GR-α protein. We can only speculate here about the reason for the opposite effects of relaxin and
dexamethasone on GR expression. This phenomenon is certainly attributable to a different mode
of action, which may reside in the distinct GR-relaxin binding mechanism or in the recruitment
by relaxin of distinct transcription factors.
Disclosure of the dual use of relaxin’s own membrane receptors (LGR7 and LGR8) and a
nuclear receptor (the GR) for relaxin signal transduction represents an unprecedented finding in
terms of hormone signaling and opens a new field of investigation. To commence unraveling this
signal network, we undertook initial experiments in spleen cells, which were known to express
no relaxin binding membrane receptors (11). We confirmed this finding by Hsu and co-workers
and furthermore established that essential characteristics of the relaxin-GR pathway—relaxininduced GRE activation in the luciferase assay and GR up-regulation— were well preserved in
these cells. On the other hand, relaxin-mediated stimulation of endothelin type-B receptors (27)
was not detectable. This up-regulation of endothelin type-B receptors depends on the raf-MEK1/2-ERK-1/2 kinase cascade (27) – a signaling pathway usually driven by membrane receptors
or, at least, by membrane-related processes. It is therefore tempting to speculate that this new
relaxin-GR pathway does not critically depend on the existence and involvement of relaxin
binding membrane receptors. The precise mode of relaxin access to the GR, as well as possible
interactions with the classical LGR7/8 pathway remain to be investigated.
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The experimental finding that relaxin, apart from acting via its membrane receptors, has GRagonistic properties may significantly influence the understanding of its pleiotropic physiological
and pathophysiological role. A number of relaxin effects may well involve GR signaling. First,
relaxin appears to expedite maternal immunotolerance to fetal allograft during implantation and
early pregnancy (13). A glucocorticoid-like mode of action would easily fit into this scheme.
Second, relaxin has been demonstrated to suppress experimentally induced asthmoid reactions
(14) and cardiac anaphylaxis (37). In addition, relaxin was shown to promote the differentiation
of human activated T cells (15). With regard to central effects of relaxin, the peptide affects
pituitary release of oxytocin, the precise effect depending on the distinct state of pituitary preactivation (38, 39). A similar interaction, differential modulation of neurohypophyseal release of
oxytocin, has been described for glucocorticoids and their influence on stress coping (40). Like
relaxin (38), glucocorticoids are intricately linked to the regulation of the central vasopressin
system (41). Recombinant human relaxin, finally, is among the most promising drugs for
treatment of scleroderma (42), a profile that could also indicate involvement of the relaxin-GR
pathway.
In conclusion, we have shown that the hormone relaxin acts as GR agonist and that this pathway
is pivotal to its effects on cytokine secretion by human macrophages. These findings may impact
on our understanding of the abundant physiological actions that relaxin exerts far beyond
pregnancy. They may, moreover, deepen our insights into the general principles of hormone
signaling.
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Received January 15, 2004; accepted June 17, 2004.
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Table 1
HeLa
4h
HeLa
24 h
THP-1
4h
THP-1
24 h
KD
[nmol/ l]
Bmax
[pmol]
Control
Relaxin
Control
20 ± 7
19 ± 5
19 ± 8
51 ± 6
110 ± 13#
61 ± 8
216%
Relaxin
21 ± 4
135 ± 15#
221%
Control
27 ± 5
161 ± 18
Relaxin
Control
26 ± 7
23 ± 6
301 ± 32#
144 ± 16
187%
Relaxin
24 ± 5
311 ± 33#
216%
Increase in Bmax
Apparent dissociation constant KD and maximum number of binding sites Bmax as determined in whole cell assays using 3H-dexamethasone following
pretreatment of cells with solvent (control) or 10 nmol/l relaxin (n=4 for each group). #P < 0.05 vs. control.
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Table 2
KD [nmol/l]
Bmax [pmol]
Increase in Bmax
4h
Control
Relaxin
50 ± 6
49 ± 5
53 ± 5
119 ± 12#
225%
THP-1
Control
59 ± 7
64 ± 8
4h
Relaxin
61 ± 4
141 ± 15#
HeLa
220%
Apparent dissociation constant KD and maximum number of binding sites Bmax as determined in whole cell assays using 3H-corticosterone following
pretreatment of cells with solvent (control) or 10 nmol/l relaxin (n=4 for each group). #P < 0.05 vs. control.
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Fig. 1
Figure 1. Relaxin concentration-dependently inhibits stimulated cytokine secretion by THP-1 cells. Differentiated
THP-1 cells were treated with 10 ng/ml Salmonella abortus equii endotoxin in the absence or presence of increasing
concentrations of relaxin. Levels of IL-1, IL-6, and TNF-α (in pg/ml) were measured by ELISAs (detection limits: IL-1
and IL-6, 20 pg/ml; TNF-α, 30 pg/ml). Number of experiments is n = 4 for each group. P < 0.05; #, vs. endotoxin alone.
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Fig. 2
Figure 2. Endotoxin-stimulated secretion of IL-1, IL-6, and TNF-α by THP-1 cells is inhibited by dexamethasone
and relaxin in a manner sensitive to treatment with the GR antagonist RU-486. Differentiated THP-1 cells were
treated with solvent (control), 1 nmol/l relaxin (RLX), 100 nmol/l dexamethasone (DX), 0.5 µmol/l of the GR antagonist
RU-486 (RU), 10 ng/ml Salmonella abortus equii endotoxin (Endotoxin), or combinations. Levels of IL-6 and TNF-α (in
pg/ml) were measured by ELISAs (detection limits: IL-1 and IL-6, 20 pg/ml; TNF-α, 30 pg/ml). Number of experiments is
n = 6 for each group. P < 0.05; #, vs. control; *, vs. endotoxin.
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Fig. 3
Figure 3. Relaxin activates the GR and promotes its DNA binding - GRE luciferase assays in HeLa, 293 cells, and
THP-1. A, B) HeLa, 293 cells, and THP-1 transiently transfected with the GRE-luciferase reporter gene construct were
exposed to 10 nmol/l relaxin for different times (A) or to increasing concentrations of relaxin for 4 h (B). Relative
luciferase activity is given as percent of values obtained in unstimulated cells. Number of experiments was n = 5 for each
group. A) P < 0.05; #, vs. baseline; *, vs. 3 h. B) P < 0.05; #, vs. baseline; *, vs. 0.5 nmol/l. C) Cells were treated with
solvent (control), the GR antagonist RU-486 (0.5 µmol/l, RU-0.5; 2.5 µmol/l, RU-2.5), 100 nmol/l dexamethasone (DX),
10 nmol/l relaxin (RLX), or with DX / RLX plus RU-486 over 4 h (n=5 each). P < 0.05; #, vs. baseline; *, vs.
relaxin/dexamethasone alone. D) As negative control, HeLa cells transiently transfected with the estrogen response
element-luciferase reporter gene construct were exposed to estradiol (100 nmol/l; E) and to increasing concentrations of
relaxin (in nmol/l) for 4 h (n=3). P < 0.05; #, vs. baseline.
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Fig. 4
Figure 4. Relaxin co-precipitates with the GR. Representative examples of at least n = 3 independent experiments.
HeLa cells were incubated with solvent (C, control) or 10 nmol/l relaxin (RLX) over 30 min. Immunoprecipitations in
cytoplasmic (A) and nuclear extracts (B) were performed with a rabbit polyclonal antibody specific for human relaxin H1
and H2, with a rabbit anti-grp94 polyclonal antibody, or with a rabbit anti-GR-α/β polyclonal antibody. The precipitated
proteins were separated and analyzed in Western blots.
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Fig. 5
Figure 5. Cytoplasmic and nuclear kinetics of GR and relaxin. Representative example of three independent
experiments. HeLa cells were incubated with solvent (Control) or 10 nmol/l relaxin (RLX). Cytoplasmic (A) and nuclear
extracts (B) were prepared 30 min, 1, 2, and 4 h after relaxin treatment. Extracts were analyzed in Western blots using a
rabbit anti-GR-α/β polyclonal antibody (A) or a rabbit polyclonal antibody specific for human relaxin H1 and H2 (B).
Stimulation with 100 nmol/l dexamethasone (DX) served as positive control in (A).
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Fig. 6
Figure 6. Microscopy images of HeLa cells incubated for 30 min at 37°C with the specified concentrations of FITClabeled relaxin. These cells were not permeabilized. Right panel) A subset of permeabilized cells was stained with
propidium iodide to visualize the localization of the nucleus. The bright spots within the nucleus represent nucleoli. The
microscope settings were identical for each image. Bar: 10 µm.
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Fig. 7
Figure 7. Relaxin competes with GR agonists at human GR. A) Relaxin displaces the fluorescent glucocorticoid
Fluormone GS Red from human GR in a fluorescence polarization assay. Binding of Fluormone GS Red to GR in presence
of rising concentrations of relaxin or dexamethasone; data are given as dimensionless polarization values (mP). The
polarization window ranged from 241 ± 23 mP (1 nmol/l Fluormone GS Red and 4 nmol/l GR, without competitor, which
resulted in maximum polarization) to 51 ± 8 mP (Fluormone GS Red, GR, and 1 mM of dexamethasone, which gave
minimum polarization). Incubation time was 2 h; number of experiments was n = 3. #, P < 0.05 vs. polarization in the
absence of competitor. B) Competition binding in HeLa cells incubated for 2 h with 48 nmol/l 3H-corticosterone in the
presence of increasing concentrations of cold corticosterone (Cst) or relaxin. Number of experiments was n = 4 for each
group. #, P < 0.05 vs. control.
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Fig. 8
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Figure 8. Relaxin up-regulates GR gene expression in HeLa cells. Cells were treated with solvent (C, control), 10
nmol/l relaxin (30 min, 1, 2, and 4 h), and with dexamethasone (DX) (6 h) in the presence (A, right panel) or absence of the
GR antagonist RU-486 (0.5 µmol/l). A) Gene expression of GR-α, GR-β, and GAPDH. Representative examples of four
independent RT-PCR experiments. B, C) Semi-quantitative analysis of GR-α and GR-β mRNA expression (n=4 for each
group). All data were normalized to GAPDH expression. #, P < 0.05 vs. control.
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Fig. 9
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Figure 9. Relaxin up-regulates GR gene expression in differentiated THP-1 cells. Cells were treated with solvent (C,
control), 10 nmol/l relaxin (30 min, 1, 2, and 4 h), and with dexamethasone (DX) (6 h) in the presence (A, right panel) or
absence of the GR antagonist RU-486 (0.5 µmol/l). A) Gene expression of GR-α, GR-β, and GAPDH. Representative
examples of four independent RT-PCR experiments. B, C) Semi-quantitative analysis of GR-α and GR-β mRNA
expression (n=4 for each group). All data were normalized to GAPDH expression. #, P < 0.05 vs. control.
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Fig. 10
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Figure 10. Relaxin elevates GR protein levels in HeLa and THP-1 cells. Cells were treated with solvent (C, control),
10 nmol/l relaxin (4, 24 h), and with dexamethasone (DX) (24 h) in the presence or absence of the GR antagonist RU-486
(0.5 µmol/l). A) (HeLa) and (C; THP-1): extracts were analyzed in Western blots using a rabbit anti-GR-α/β polyclonal
antibody and a mouse anti-actin antibody. Representative examples of four independent experiments. B) (HeLa) and
(D; THP-1): Quantitative analysis of GR protein expression (n=4 for each group). All data were normalized to α-actin
expression. #, P < 0.05 vs. control.
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Fig. 11
Figure 11. Relaxin pretreatment elevates 3H-dexamethasone and 3H-corticosterone binding sites—whole cell
assays in HeLa and THP-1 cells. A, B) Cells were pretreated with solvent (control) or 10 nmol/l relaxin (RLX) over 4 h
(HeLa, A) or 24 h (THP-1, B). The medium subsequently was completely exchanged and cells were incubated for 2 h in
HeLa and 1 h in THP-1 with labeled dexamethasone. In subsets of experiments, incubation with hot dexamethasone was
performed in presence of 1 µmol/l RU-486. C) Cells were pretreated as described for (A, B). Then incubation with labeled
corticosterone was performed for 2 h in HeLa and 1 h in THP-1. Number of experiments was n = 4 for each group.
#, P < 0.05 vs. control.
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Fig. 12
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Figure 12. Relaxin-induced GR activation is preserved in LGR7/8-free spleen fibroblasts. A) Expression of LGR7
and LGR8 mRNA in spleen fibroblasts, 293 cells, HeLa cells, and THP-1; representative examples of n = 3 independent
RT-PCR experiments using primer pairs specific for human LGR7 or LGR8. B) Spleen fibroblasts transiently transfected
with the GRE-luciferase reporter gene construct were exposed to increasing concentrations of relaxin for 4 h. Relative
luciferase activity is given as percent of unstimulated baseline values. Number of experiments was n = 3 for each group.
P < 0.05; #, vs. baseline. C) Relaxin elevates GR protein levels in spleen fibroblasts. Cells were treated with solvent
(C, control) or with 10 nmol/l relaxin (4, 6, and 8 h). Extracts were analyzed in Western blots using a rabbit anti-GR-α/β
polyclonal antibody and a mouse anti-actin antibody. Representative examples of three independent experiments.
D) Relaxin-stimulated expression of endothelin type-B receptor (ETB) protein in HeLa cells and in spleen fibroblasts as
analyzed using a mouse monoclonal antibody. Examples of n = 3 independent experiments. HeLa: lane 1, control; lane
2, RU-486 (2.5 µmol/l); lane 3, relaxin (10 nmol/l, 4 h); lane 4, relaxin plus RU-486 (4 h); lane 5, relaxin (10 nmol/l,
8 h); lane 6, relaxin plus RU-486 (8 h). Spleen cells: lane 1, control; lane 2, relaxin (10 nmol/l, 4 h); lane 3, relaxin
(6 h); lane 4, relaxin (8 h).
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