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Early Elevation of Transforming Growth Factor-6,
Decorin, and Biglycan mRNA Levels During Cartilage
Matrix Restoration After Mild Proteoglycan Depletion
PETER JVL van der KRAAN, HARRIE L. GLANSBEEK, ELLY L. VITTERS, and WIM B. van den BERG
ABSTRACT Objective. To elucidate the role of transforming growth factor-(TGF-fi) and the small proteogly­
cans biglycan and decorin in the repair of articular cartilage after proteoglycan depletion.
Methods. Limited and reversible proteoglycan depletion was induced by injection of murine knee
joints with 0.5% papain. Proteoglycan content of patellar cartilage was examined by safranin O
staining on histological sections and overall proteoglycan synthesis was measured by incorporation
of 35S sulfate. Changes in mRNA expression of TGF-B, aggrecan, decorin, and biglycan were deter­
mined by semiquantitative reverse transcription polymerase chain reaction.
Results. Papain injection led to rapid depletion of proteoglycans, which was partly overcome 7 days
after injection, while total replenishment of the cartilage matrix with proteoglycans was observed on
Day 24. The incorporation of radiolabeled sulfate in patellar proteoglycans was initially decreased
(up to Day 3), but significantly enhanced on Days 4 and 7 after papain injection. Upregulation of
TGF-8, decorin, and biglycan mRNA in patellar cartilage was observed on Day 2, markedly before
elevation of overall proteoglycan synthesis. mRNA levels were less augmented on Day 7, and on
Day 24 all messenger RNA levels had returned to control values. As well, in the soft tissue adjoin­
ing the patella swift upregulation of TGF-fi mRNA was observed,
Conclusion. mRNA of both TGF-B and the small proteoglycans decorin and biglycan are elevated
at an early phase during cartilage repair after moderate proteoglycan depletion, implying a func­
tional role for these molecules in this repair process. (./ Rheumatol I997;24:543-9)
Key Indexing Terms:
CARTILAGE
DECORIN
REPAIR
TRANSFORMING GROWTH FACTOR-B
BIGLYCAN
AGGRECAN
Articular cartilage covers the ends of the long bones and
makes smooth and nearly frictionless movement of the artic­
ulating surfaces possible under normal conditions.
However, during pathology such as rheumatoid arthritis
(RA) or osteoarthritis articular cartilage is degraded, result­
ing in loss of joint function 1,2. One factor contributing to the
irreversible damage of articular cartilage in these diseases is
the limited capability of articular cartilage for repair of the
extracellular matrix3. The extracellular matrix of articular
cartilage consists mainly of collagen (e.g., type II, IX, XI)
and proteoglycans such as aggrecan and to a lesser extent
decorin, biglycan, and fibromodulin4“6. All these matrix
molecules are synthesized and degraded by the articular
chondrocytes.
Factors suggested to be important in repair processes in
From the Department of Rheumatology, University Hospital Nijmegen,
Nijmegen, The Netherlands.
Supported by the Nationaal Reumafonds (Dutch League Against
Rheumatism).
P.M. van der Kraan, PhD; H.L Glansbeek, MS; E.L. Vitters; W. van den
Berg, PhD, Department o f Rheumatology.
Address reprint requests to Dr. P.M. van der Kraan, Department o f
Rheumatology, University Hospital Nijmegen, Geert Grooteplein 8, 6525
GA Nijmegen, The Netherlands.
Submitted March 27, 1996 revision accepted September 4, 1996.
van der Kraan, et al: Cartilage repair
miscellaneous tissues are the various transforming growth
factor-13 (TGF-B) subtypes7. In mice, TGF-B is upregulated
in an early phase during adult and embryonic wound repair,
but temporal and spatial differences between the 3 mam­
malian isoforms were observed8,9. Application of additional
TGF-fi appears to lead in several systems to stimulation of
the repair process. Closure of skull defects, repair of macu­
lar holes, recovery of damaged heart tissue, and repair of
incisional wounds can be accelerated by application of
TGF-fl,(M3. As well, it has been suggested that the presence
of TGF-61 in articular cartilage lesions as a i*esult of RA is
an indication for a role for TGF-B 1 in tissue repair at these
sites14. In addition, stimulation of murine articular cartilage
repair after a simulated arthritic insult, induced by intraarticular interleukin 1 injection, was found by oar group15,16.
Both the large aggregating proteoglycan of cartilage,
aggrecan, and the so-called small proteoglycans like
decorin, biglycan, and fibromodulin are structural and regu­
latory components of articular cartilage5. The major func­
tion of aggrecan is hydration of the extracellular matrix,
while the functions of the small proteoglycans in cartilage
are largely unknown. Immunohistochemical studies have
shown that biglycan has a different localization from
decorin in articular cartilage, suggesting different func­
tions17*18. Decorin was distributed throughout the matrix,
543
whereas biglycan was found mainly in the pericellular space
of chondrocytes.
Proteoglycan synthesis in connective tissues can be mod­
ulated by TGF-B. Incubation of human fibroblast with TGFB results in upregulation of biglycan mRNA, while decorin
expression is reduced or unaltered19,20. Also, in isolated
human articular chondrocytes biglycan mRNA expression
was increased, while decorin mRNA was diminished under
the influence of TGF-B21. In mesangial cells both decorin
and biglycan synthesis is reported to be upregulated up to
50-fold by TGF-B22. Moreover, TGF-B not only interferes
with the synthesis of the small proteoglycans, but these mol­
ecules also interact on the protein level23,24. Both decorin
and biglycan are able to bind TGF-B, which can result in
either inhibition of TGF-B biological activity25,26 or stimula­
tion of TGF-B action by these molecules27. In this respect
biglycan as well as decorin can be considered as both nega­
tive and/or positive modifiers of TGF-B activity, regulating
the potential repair stimulating ability of TGF-B.
For insight into the roles of TGF-B and the cartilage pro­
teoglycans aggrecan, decorin, and biglycan during restora­
tion of the articular cartilage matrix after moderate proteo­
glycan depletion we studied temporal expression of mRNA
of these molecules during in vivo cartilage repair.
MATERIALS AND METHODS
Animals. Male C57BI/6 mice at 10-12 weeks of age were used in all exper­
iments. The animals were kept in cages with wood chip bedding in air con­
ditioned rooms at constant temperature. They were fed a standard laborato­
ry diet and had access to tap water ad libitum.
Induction of mild proteoglycan depletion. Proteoglycan depletion was
induced as described by van der Kraan, et a/28. The right knee joint of mice
was injected once, intraarticularly along the patellar ligament, with 6 pi of
0.5% papain solution (type IV, 15 units/mg; Sigma, St. Louis, MO, USA).
To activate the papain, the solution was supplemented with 0.03 M L-cysteine HC1 (Sigma). The left (control) knees were injected with physiologi­
cal saline. The papain injection in the patellar cartilage leads to limited,
reversible proteoglycan depletion and inhibition of proteoglycan synthesis
one and 2 days after the injection, followed by supranormal proteoglycan
synthesis later28. At several time points after papain injection, patellae were
isolated and used for measurement of proteoglycan synthesis and determi­
nation of mRNA levels. Surrounding tissue was used for detection of
mRNA levels only.
Determination of patellar proteoglycan synthesis. Patellar cartilage proteo­
glycan synthesis was measured ex vivo according to the method of van den
Berg» et aP9. Whole patellae, with a standard amount of surrounding tissue,
were dissected from the knee joints. Patellae were labeled immediately
after isolation with 35S sulfate (LI MBq/ml) for 2 h at 37°C in R P M I1640
DM medium (Flow Laboratories, Irvine, UK). After labeling, the patellae
were washed, fixed, decalcified, and punched out of the surrounding tissue.
The patellae were dissolved in lumasolve (Lumac, Groningen, The
Netherlands) and the quantity of incorporated 35S sulfate was detenuined
by liquid scintillation counting.
Whole knee joint histology. Mice were killed by cervical dislocation, and
carefully dissected knee joints were fixed in phosphate buffered formalin
(pH 7.4) for 5 days and subsequently decalcified in 5% formic acid for 4
days, Standard processing of the knee joints in an automatic tissue pro­
cessing apparatus was followed by embedding of the knee joints in paraf­
fin wax. Frontal knee joint sections were prepared (6 pm) and stained with
safranin O and fast green.
544
Isolation o f patellar cartilage and synovial tissue. Patellae were dissected
from the murine knee joints and immediately decalcified for 4 h at 4°C in
3.5% EDTA (Sigma). After décalcification the complete articular cartilage
layer was stripped from the underlying bone. The isolated cartilage was
instantly put in TRIzol reagent (Life Technologies) for RNA extraction. In
control experiments it was shown that this procedure did not affect the
RNA isolation or reverse transcription polymerase chain reaction (RT-PCR)
performance negatively (data not shown).
The patellar tendon and synovium of knee joints were dissected and 2
pieces of synovial tissue were punched out from the tissue on a standard
location (area 7 mm2). Directly after punching out, the synovial tissue was
quickly frozen in liquid nitrogen.
RNA isolation and RT-PCR. Total RNA was isolated from patellar cartilage
and synovial tissue by TRIzol extraction. RNA was directly extracted from
cartilage but synovial tissue was first homogenized and then put in TRIzol
reagent. Cartilage and synovial tissue of 5 mice were pooled. Before
reverse transcription the isolated RNA was treated with DNAse I (Life
Technologies). The reverse transcription reaction was performed with
Moloney murine leukemia virus (M-MLV) reverse transcriptase (Life
Technologies) using an oligo(Dt)ls primer (Eurogentec, Liege, Belgium).
Amplification of DNA was accomplished by using Taq DNA polymerase
(Life Technologies) up to a cycle number of 35. To determine the relative
mRNA levels samples were taken at different cycle numbers and the
amount of amplified product was calculated as described below. All RTPCR reactions were performed in duplicate.
The amplification products were labeled by digoxigenin labeled
nucleotides (Boehringer, Mannheim, Germany). After separation on a 1.5%
agarose gel the products were blotted on positively charged nylon mem­
branes (Boehringer). Amplification products were detected by chemiluminescence using alkaline phosphatase labeled antidigoxigenin antibodies in
combination with lumigen PPD according to the manufacturer
(Boehringer). Spots were scanned on the photographic film using an image
analyzer. Only spots lying within the linear part of the PCR were used for
scanning. The results are presented as relative mRNA levels using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) levels as an internal con­
trol. Due to the limited amount of tissue and quantities of RNA, measure­
ment of the amount of total RNA was not possible.
The following nucleotide primers were used in the amplification reac­
tions. Aggrecan primers were used as described by Grover and Roughly30
(product 501 bp), while decorin primers were used according to Asundi and
Dreher31 (product 400 bp). The biglycan upper primer had the following
sequence: S'-AGAAGGCCTTTAGCCCTCTG-S', and the lower primer 5'ACTTTGCGGATACGGTTGTC-3' (product 130 bp). Murine TGF-B 1
primers were derived from Clontech (Palo Alto, CA, USA) (product 525
bp), whereas TGF-B3 primers were used as described by Mulheron, et aP2
(product 380 bp). To detect GAPDH, the primers 5'-AACTCCCTCAAGATTGTCAGCA-3' and 5'-TCCACCACCCTGTTG CTGTA-3 ' were used
(product 553 bp). Primer sequences were selected with the computer pro­
grams Primer (Whitehead Institute, Cambridge, MA, USA) and Oligo 4.0
(National Biosciences, Plymouth, MN, USA),
RESULTS
To obtain reversible depletion of proteoglycans from the
patellar cartilage 0.5% papain was injected into the right
knee joints of the mice. Papain injection resulted after one
day in obvious depletion of proteoglycans, measured by
safranin O staining, in the patellar cartilage (Figure 1).
Moderate inflammation of the joint, characterized by granu­
locyte infiltration, was seen at this time. Three days after
papain injection the inflammation had largely vanished,i but
the amount of proteoglycan depletion was not different from
one day after papain injection. On Day 7 the proteoglycan
The Journal of Rheumatology 1997; 24:3
Figure I. Effect of papain injection on proteoglycan content of patellar car­
tilage. These are frontal sections of the murine knee joint stained with
safranin 0 and fast green (original magnification x 100). Depletion of pro-
depletion of the patellar cartilage was partly overcome,
while in the patellaris femoris minor replenishment of the
articular cartilage matrix was still observable. The cartilage
matrix of both patella and opposing femur obtained a nor­
mal appearance 24 days after papain injection.
At several times after papain injection patellar proteogly­
can synthesis was measured ex vivo by incorporation of
radiolabeled sulfate. Inhibition of proteoglycan synthesis
was measured up to 3 days after the injection of papain
(Figure 2). It is possible that part of the newly synthesized
proteoglycan is degraded by papain remaining in the carti­
lage. However, patellar proteoglycan content is restored
within 10 days, making it unlikely that active papain persists
in the patellar cartilage. On Days 4 and 7 proteoglycan syn­
thesis was supranormal, roughly doubled compared to the
control knee joints. Twenty-four days after papain injection
radiolabel incorporation into the papain injected knee joints
had reached control levels.
Since it has been suggested that TGF-B might play a role
van der Kraan, et al: Cartilage repair
teoglycan can be seen on Day 1 and 7, whereas normal staining is observed
at Day 24. A: control joint, B: Day 1, C: Day 7, D: Day 24 (c: cartilage,
b: bone, f: femur, p: patella).
sulfate incorporation
days after papain injection
Figure 2. Proteoglycan synthesis measured by incorporation of 35S sulfate
in patellar cartilage after injection of 0.5% papain on Day 0. Sulfate incor­
poration is compared to the control knee joint. Values are means ± SD of 6
patella. This is one representative experiment out of 3,
545
in the stimulation of matrix synthesis during repair of con­
nective tissues such as articular cartilage, we studied the
mRNA expression of TGF-B in patellar cartilage and neigh­
boring soft tissue. Both in the surrounding tissue and the
patellar cartilage there was early elevation of messenger
RNA of TGF-ft 1 and TGF-B3 (Figure 3), Even on Day 2
after papain injection, mRNA in the surrounding tissue and
patellar cartilage was elevated 3 to 6-fold. In the surround­
ing tissue the TGF-B1 message appeared to be upregulated
more strongly than the TGF-133. From Day 3 the mRNA lev­
els of both TGF-B1 and TGF-B3 declined, and on Day 7 only
TGF-63 mRNA in the cartilage still appeared to be elevated.
Twenty-four days after papain injection mRNA levels of
TGF-fil and TGF-B3 were normal.
To investigate if there was an association between the
upregulation of TGF-B mRNA and mRNA levels of proteomRNA levels (ratio to control)
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day s after papain Injection
glycans in the recovering patellar cartilage we measured
mRNA levels of aggrecan, decorin, and biglycan in this
structure. Messenger RNA levels of the small proteogly­
cans, decorin and biglycan, were already elevated 2 days
after papain injection, while aggrecan mRNA in the papain
injected knees was comparable to the control knee joints
(Figure 4). Proteoglycan synthesis measured by incorpora­
tion of radiolabeled sulfate was noticeably inhibited up to
Day 3. Also on Days 3 and 4 upregulation of biglycan and
decorin mRNA was observed, whereas on Day 7 the levels
were diminished compared to the levels on Day 2. Aggrecan
mRNA levels were never strikingly altered during the peri­
od of the experiment. On Day 24 all mRNA levels were sim­
ilar to the control levels.
DISCUSSION
Murine knee joints were injected with papain to study the
repair response of articular cartilage after limited proteogly­
can depletion. This model was used since papain injection in
the dosage we used leads to proteoglycan depletion with a
restricted inflammatory response. Our results show that
patellar proteoglycan depletion after papain injection can be
restored to normal by the articular chondrocytes. Even 7
days after the induction of proteoglycan depletion, part of
the matrix was restored, while after 24 days no signs of pro­
teoglycan depletion could be detected by safranin 0 stain of
the matrix. At least one of the mechanisms leading to replen­
ishment of the cartilage matrix with proteoglycans appeared
to be enhanced synthesis of proteoglycans, starting between
3 and 4 days after papain injection.
Even at an early time point during the repair phase, sev­
eral days before increased incorporation of radiolabeled sul-
mRNA levels (relative to control)
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in control knee joints (ratio of 1 is similar to control). For each time point,
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Figure 4. Messenger RNA levels of aggrecan, decorin, and biglycan in
patellar cartilage after injection of 0.5% papain. Values are expressed as a
ratio of the RNA messenger levels in control knee joints (ratio of 1 is sim­
ilar to control). For each time point results of 5 patellae were pooled. This
is one representative experiment out of 3. All RT-PCR were carried out at
least in duplicate. Levels are normalized to GAPDH and control joints
injected with physiological saline.
The Journal o f Rheumatology 1997; 24:3
fate, TGF-fl mRNA levels were augmented, both in patellar
cartilage and the soft tissue adjoining the patellae. This indi­
cates that upregulation of TGF-B mRNA is an early event
during the restoration of the cartilage matrix after proteo­
glycan depletion by papain, suggesting a role for TGF-B in
the repair process. The elevation of TGF-B mRNA was not
very longlasting, and 7 days after papain injection the mes­
sage levels were lower than on Day 2, whereas on Day 24
mRNA levels had reached control values. TGF-B2 was mea­
sured in the surrounding tissue and patellar cartilage on Day
2 only. It also appeared to be elevated in both compartments
at this time (data not shown). These observations are in
accord with the data of Martin, et al , who observed in mouse
embryos rapid induction and clearance of TGF-B after
wounding11.
With respect to the bioavailability of TGF-B, newly syn­
thesized TGF-B protein is only one of the sources of TGF-B.
TGF-B is bound to the extracellular matrix by several bind­
ing proteins33-36. In the matrix of bovine articular cartilage
considerable amounts of TGF-B were detected that appeared
to be inaccessible to the chondrocytes under normal circum­
stances37. However, it can be imagined that as a result of
proteolytic action the majority of matrix bound TGF-B in
cartilage can become available to the chondrocytes after
exposure to papain. Moreover, TGF-B being synthesized as
a latent molecule might be activated by the proteolytic activ­
ity of papain and also in this way TGF-B bioactivity can be
enhanced by papain. On the other hand, papain might
degrade active TGF-B and in this way lower the bioavail­
ability of TGF-B.
The early upregulation of TGF-B mRNA coincided with
a simultaneous increase of decorin and biglycan mRNA lev­
els. In patellar cartilage chondrocytes, mRNA levels of
decorin and biglycan were elevated about 3 to 4-fold even 2
days after papain injection, when glycosaminoglycan syn­
thesis appeared to be diminished by about 50%. Not before
Day 4 could enhanced synthesis of proteoglycans, measured
by 35S sulfate incorporation, be observed in the patellar car­
tilage after papain injection. During the whole study period
the mRNA levels of aggrecan, the proteoglycan contributing
predominantly to the incorporation of sulfate in cartilage,
did not change strikingly. This indicates that at least part of
the proteoglycan synthesis, as measured by sulfate incorpo­
ration, is not regulated by transcription of the aggrecan core
protein gene but by other factors, such as upregulation of
aggrecan core protein synthesis due to posttranslational
mechanisms. Increased addition of glycosaminoglycan
chains to the aggrecan core protein during the repair phase
seems unlikely, since no changes in the size of aggrecan
could be detected using Sepharose Cl 2B chromatography
(data not shown).
The early elevation of the small proteoglycans decorin
and biglycan can be interpreted in several ways. Since no
upregulation has been found after injection of proteins that
van der Kraan, et al: Cartilage repair
j
cause inflammation similarly to papain but without proteo­
glycan degradation, elevated mRNA levels as a reaction to
the inflammatory response in the joint seem unlikely.
However, TGF-B mRNA levels can be affected by the
inflammatory response in the joint. Upregulation of mRNA
of the small proteoglycans could occur as a result of TGF-B
activity on patellar chondrocytes. This would be a conse­
quence of release and activation of inactive TGF-B by
papain or a result of enhanced TGF-B synthesis. In this way
the small proteoglycans could function as enhancers of
TGF-B bioactivity or function in a negative biofeedback
mechanism to control the TGF-B action on the chondro­
cytes25“27. The observations of Roughly, et cil2\ with incu­
bation of isolated human chondrocytes with TGF-B leading
to increased biglycan mRNA and decreased decorin mRNA
levels, appear to contradict a role for TGF-B in upregulation
of mRNA of both small proteoglycans. Partly in parallel
with the studies of Roughly, et al , Collier and Ghosh
observed increased biglycan synthesis and unaltered decorin
synthesis after exposure of meniscal chondrocytes to TGFB38. However, both studies were performed with chondro­
cytes of different species and with isolated chondrocytes,
instead of an in vivo model as described here.
Nevertheless, TGF-B could play a role in regulation of
cartilage proteoglycan synthesis: the elevation of decorin
and biglycan mRNA we observed could be independent
from TGF-B action. The early elevation o f biglycan and
decorin mRNA suggests an intrinsic role for these proteo­
glycans in assembly of the cartilage matrix during the repair
period, irrespective of their relation to TGF-B. Decorin is
able to influence the fibrillogenesis of type I and type II col­
lagen and has been shown to bind to collagen type VI in
binding assays39,40. In addition, decorin has been reported to
interact with fibronectin and thrombospondin, both extra­
cellular matrix components41'42. Biglycan has a different dis­
tribution than decorin within the cartilage matrix, implicat­
ing different functions for biglycan than decorin in cartilage
physiology17’18. Upregulation of mRNA of decorin and
biglycan might be a functional mechanism in the recon­
struction of damaged cartilage matrix, independent of their
role in control of TGF-B activity. In accord with our results,
Cs-Szabo, et al found 5 to 10-fold higher mRNA levels of
biglycan and decorin in human osteoarthritic cartilage com­
pared to normal cartilage, suggesting a role for these pro­
teoglycans in local cartilage repair foci that can be found in
os teoarthriti c c artil age43.
In conclusion, our results indicate that during restoration
of the patellar cartilage matrix after moderate proteoglycan
depletion, upregulation of TGF-B and decorin and biglycan
messenger RNA in patellar cartilage and TGF-B in adjoining
soft tissue is an early event. Upregulation of mRNA is initi­
ated days before enhancement of proteoglycan synthesis, as
measured by incorporation of radiolabeled sulfate. These
results suggest that both TGF-B and the small proteoglycans
547
decorin and biglycan might play a role in regulation of the
chondrocyte metabolism, while the small proteoglycans
could have a potential structural function in repair of dam­
aged articular cartilage matrix.
21.
22.
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