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During Experimental Osteoarthritis Prevents Osteophyte Formation and Impairs Cartilage Repair
Rheumatology Research Laboratory, Department of Rheumatology, University Medical Center, Nijmegen, The Netherlands
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Abstract |
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1
and -3 was detected in developing osteophytes and articular
cartilage during murine experimental osteoarthritis. To determine the
role of endogenous TGF- on osteophyte formation and articular
cartilage, TGF- activity was blocked via a scavenging soluble
TGF--RII. Our results clearly show that inhibition of endogenous
TGF- nearly completely prevented osteophyte formation. In contrast,
treatment with recombinant soluble TGF--RII markedly enhanced
articular cartilage proteoglycan loss and reduced the thickness of
articular cartilage. In conclusion, we show for the first time that
endogenous TGF- is a crucial factor in the process of osteophyte
formation and has an important function in protection against cartilage
loss. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TGF- is a multifunctional cytokine involved in crucial biological
processes such as development, extracellular matrix synthesis, cell
proliferation/differentiation, and tissue repair. So far, three
mammalian isoforms have been described: TGF-1, -2, and -3.
TGF- is produced in a latent form, associated with the
latency-associated peptide. TGF- can be activated after dissociation
of latency-associated peptide from the mature protein (1).
TGF- signaling starts with binding of TGF- to TGF--RII, a
constitutively active serine/threonine kinase (2). After
transphosphorylation of TGF--RI by TGF--RII the signal is further
propagated involving Smad proteins (3, 4).
TGF- has been suggested as an important factor in the pathogenesis
of OA. One indication is the significant levels of active TGF- in
the synovial fluid of OA patients (5, 6). Indeed, OA
changes of the cartilage have been described after exposure of knee
joints to TGF-, supporting a role for TGF- in the pathogenesis of
OA (7, 8). Local administration of TGF- in the knee
joint also induced inflammation and fibrosis (7, 8, 9).
Another remarkable finding was the formation of osteophytes after
multiple intra-articular (i.a.) injections of TGF- protein or
adenoviral overexpression of TGF-1 in the knee joint (7, 8, 10). Osteophytes are newly formed bony structures located at the
joint margins, and their occurrence is strongly associated with OA.
Osteophytes originate from activated periosteum leading to new
cartilaginous outgrowths that eventually ossify into osteophytes via
the process of endochondral ossification. In developing osteophytes,
mRNAs for TGF- and TGF- protein expression are strongly
up-regulated (11, 12, 13). These data suggest that TGF-
induces osteophyte formation.
Although TGF- seems implicated in pathology, TGF- has also been
suggested as a beneficial factor in cartilage repair. We have
previously shown that injection of TGF- in naive murine knee joints
results in an increase in PTG synthesis and PTG content of articular
cartilage (14). Moreover, i.a. injection of TGF- during
experimental arthritis resulted in protection from PTG loss
(15). In addition, effects of IL-1, such as inhibition of
cartilage PTG synthesis and release of cartilage PTG content, could be
blocked by local application of TGF- (16, 17). This
demonstrates that TGF- is able to counteract the deleterious effects
of IL-1, a cytokine considered to be a key mediator during erosive
joint diseases. Taken together, these experiments suggest a protective
function for TGF- in articular cartilage.
As can be concluded from the discussion above, TGF- appears to have
a dualistic role in OA: protection against cartilage damage but
induction of osteophyte formation. Experiments conducted to investigate
the role of TGF- in joint diseases are mainly based on TGF-
supplementation. In this study, we aimed to determine the role of
endogenous TGF- during experimental OA. We selectively blocked
endogenous TGF- via systemic treatment with soluble TGF--RII
(solRII), the cloned scavenging extracellular domain of the
TGF--RII, an approach that to our knowledge has never been
previously used in OA. solRII has a very high affinity for
TGF-1 and -3, the two most abundant isoforms of TGF- in the
knee joint. Moreover, due to the small size of solRII it can penetrate
the articular cartilage and affect chondrocytes. We show for the first
time that inhibition of endogenous TGF- during experimental OA
dramatically decreases osteophyte size but enhances PTG loss. Our study
implies a crucial role for endogenous TGF- in osteophyte formation
and cartilage maintenance.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The expression of TGF-1, -2, and -3 was studied during
papain-induced OA. The papain model is characterized by PTG depletion
of articular cartilage, which is followed by attempted replenishment of
the articular cartilage with PTGs at approximately day 10
(18). The right knee joints of mice were injected with 1 U
papain solution; the left knee joints served as internal controls. Knee
joints were dissected at days 7 and 14. Immunohistochemistry was
performed on cryosections with specific Ab against TGF-1, -2, and
-3 (R&D Systems, Abingdon, U.K.). As a negative control, the primary
Ab was replaced with chicken IgYs or goat IgGs. Biotin-labeled
secondary Ab were used (Vector Laboratories, Burlingame, CA) followed
by a biotin-streptavidin detection system (Vectra elite kit; Vector
Laboratories). Bound complexes were visualized via reaction with
3',3'-diaminobenzidine (Sigma-Aldrich, St. Louis, MO) and
H2O2. Sections were briefly
counterstained with hematoxylin and mounted with permount.
Quantification of TGF-1 and TGF-3 isoforms
Because TGF-1 and -3 were detected by
immunohistochemistry, TGF-1 and -3 levels in knee joints were
quantified via ELISA. Therefore, 1 U papain was injected in the right
knee joints of mice (n = 6); uninjected left knee
joints served as internal controls. After 3 days, patellae were
isolated and placed in RPMI medium/0.1% BSA. After 2 or 10 h of
culturing, the medium was analyzed for the presence of TGF-1 and
-3 via ELISA. In short, ELISA plates were coated with solRII (R&D
Systems). TGF-1 or -3 standards (R&D Systems) or samples were
applied and subsequently incubated with anti-TGF-1 or -3 Ab.
The appropriate secondary biotinylated Ab were used, followed by
incubation with streptavidin-polyperoxidase conjugate. Bound complexes
were detected by reaction with orthophenylenediamine (Sigma-Aldrich)
and H2O2. Absorbance was
read at 492 nm using an ELISA plate reader (Multiscan MCC/340;
Titertek, Huntsville, AL).
Construction of the yeast expression vector
Construction of the yeast expression vector and screening of
solRII-expressing clones are described elsewhere (19). In
short, the complete extracellular domain of human TGF- type II
receptor was cloned in the pPic-9 expression plasmid (Invitrogen, San
Diego, CA). During the cloning procedure the cleavage signal of the
Saccharomyces cerevisiae 
-factor secretion signal peptide
and a sequence coding for six consecutive histidine residues (6xHis
tag) were introduced via PCR.
Production and purification of solRII
For the production of solRII the Pichia pastoris
expression system (Invitrogen) was used. P. pastoris was
cultured in the Bioflow 3000 tabletop fermenter (New Brunswick
Scientific, Edison, NJ). In essence, the fermentation process was
conducted as described elsewhere (20). Yeast culture
supernatant was filtered through a 0.2-µm filter (Schleicher &
Schull, Dassel, Germany). solRII was purified from the supernatant by
means of a 6xHis tag and a nickel-nitrilotriacetic acid column
(Qiagen, Leusden, The Netherlands). Bound protein was eluted from the
column with 300 mM imidazole in wash buffer. Imidazole was removed via
a HiPrep 26/10 desalting column (Amersham Pharmacia Biotech, Little
Chalfont, U.K.). The protein was subsequently further purified and
concentrated using m.w. cut-off 100,000 and 10,000 filters (Millipore,
Etten-Leur, The Netherlands) to a final concentration of 
60 mg/ml.
Although yeast cells do not contain LPS, the endotoxin level of
purified protein was analyzed via the endosafe kinetic turbidimetric
assay (Charles River Endosafe, Kent, U.K.). However, yeast supernatant
contained an unknown interfering substance that confounded test results
(data not shown). Therefore, an endotoxin inhibitor, polymyxin B (PMB;
Sigma-Aldrich), was added before implantation as a precautionary
measure.
SDS-PAGE and Western blotting procedures
SDS-PAGE analysis of recombinant solRII was performed on a 12% gel under denaturating conditions. Proteins were visualized via a standard silverstaining procedure.
For Western blotting analysis, solRII protein was blotted on a
polyvinylidene difluoride membrane (Amersham Pharmacia Biotech). solRII
protein was detected by an Ab against the extracellular part of the
TGF--RII (R&D Systems) in combination with the appropriate secondary
biotin-labeled Ab (Vector Laboratories). Bound complexes were detected
by the ECL Plus detection reagents (Amersham Pharmacia Biotech).
Activity of solRII
The blocking capacity of the recombinant solRII was determined
via a TGF- competition ELISA using solRII as a capture agent. To
assure the stability of the solRII for the duration of the implantation
period, the used osmotic pumps were retrieved at the end of the
experiment and flushed with 200 µl PBS. The diluted samples were also
tested via ELISA. Samples of recombinant solRII were mixed with
TGF-1 in a molar ratio of 1000:1–8000:1. The TGF-1 ELISA was
performed as described in Quantification of TGF-1 and TGF-3
isoforms.
Animals
Male C57BL/6 mice aged 12 wk were used. Animals were kept in filter-top cages with a wood chip bedding. They were fed a standard diet and tap water ad libitum.
Experimental design and histology
Alzet osmotic pumps (models 1007D and 2002; ALZA, Palo Alto, CA) were filled with the solution containing solRII (60 mg/ml)/PMB (2.63 mg/ml) or PMB alone and as a control empty pumps were used. One day after i.p. implantation of the osmotic pumps, 1 U papain solution was injected into the right knee joint. We aimed to induce a relatively mild OA to allow for diminished and increased PTG loss of the cartilage due to the treatment with solRII. Left knee joints served as internal controls for the systemic treatment with solRII/PMB and PMB. To administer solRII for 3 wk, first an osmotic pump model 2002 was implanted i.p., which after 14 days was replaced by a model 1007D pump. After 7, 14, or 21 days mice were sacrificed and whole knee joints were dissected and fixed for 4 days in 4% formalin. Subsequently, knee joints were decalcified in 5% formic acid and processed for paraffin embedding. Tissue sections (7 µm) were stained with Safranin O (Saf-O)/fast red.
[35S]Sulfate incorporation was studied by i.p. injection of radiolabeled sulfate (75 µCi) 4 h before dissection of the knee joints. After histological processing, 7-µm tissue sections were dipped in K5 emulsion (Ilford, Basildon, Essex, U.K.) and exposed for 3–5 wk. Then the slides were developed and stained with H&E. All findings are the result of two independent experiments.
Image analysis of histology
The surface area of the osteophytes was measured using the Qwin
image analysis system (Leica Imaging Systems, Cambridge, U.K.), using a
JVC 3-CCD color video camera. The size of the osteophytes was
determined by manual selection of the surface area of the osteophyte in
five semiserial sections per knee, spaced 50 µm apart.
Measurements of eight knees per group were averaged.
The PTG content of articular cartilage was also measured using an image analysis system as described elsewhere (21). The noncalcified layer of the patellar and femoral cartilage and cartilage of both the lateral and medial sides of the tibia-femur plateau was selected by hand. Loss of red staining (loss of PTGs) was calculated by measuring the amount of blue light passing through the tissue section. Of each knee joint, three sections per cartilage compartment were measured and the average was taken. Of each compartment the most severe depletion was considered as maximal PTG loss. The noncalcified cartilage of the control left knees were also measured and averaged and the PTG content was stated as being 100%. All measurements were expressed on a scale of 0–100% PTG content as measured by Saf-O staining.
We also noted a thinning of the articular cartilage, which resulted in
a decrease in the thickness of the cartilage. This loss of noncalcified
articular cartilage of the patella and femur was measured in three
sections, spaced 50 µm apart by manual selection of the
noncalcified cartilage. Then the surface area was determined and values
were averaged.
MMP plus ADAMTS plus TIMP mRNA expression
Treatment with solRII during experimental OA increases PTG loss compared with untreated animals. We investigated whether solRII had an effect on the mRNA expression of matrix metalloproteinase (MMP)-3, -13, and -14, a disintegrin and metalloproteinase with thrombospondin motif (ADAMTS)-4, and -5, and tissue inhibitors of MMPs (TIMP)-1 and -3. Therefore, papain (1 U) was injected in the right knee joints of mice (n = 6 per group). Uninjected left knee joints served as internal controls. After 3 days, patellae were isolated and placed in 200 µl RPMI 1640 (Dutch modification) medium (Life Technologies, Breda, The Netherlands) for 24 h as described previously (22, 23). Patellae were treated with solRII (10 µg/ml) or IL-1 (10 ng/ml) as a positive control or were not treated. Next, RNA was isolated and treated with reverse transcriptase (Life Technologies). The RT-PCR protocol started with a 1-min denaturation at 92°C followed by annealing at 60°C for 1 min (58°C for TIMP-3) and a 1-min extension at 72°C. PCR products were subjected to electrophoresis in a 1% low-melting agarose gel (Seaplaque; SanverTech, Boechout, Belgium) containing ethidium bromide. For each primer set, the cycle number at which the PCR product was first detected on the agarose gel was identified. It was determined that in this phase the PCR still performs linearly. The PCR products were excised from the gel. PCR products were quantified using PicoGreen (Molecular Probes, Eugene, OR), a fluorescent dye that selectively stains dsDNA, according to the manufacturer’s recommendations. GAPDH levels were used to correct for the amount of template added to the PCR mix. For control and papain-injected knee joints the mRNA levels in the nontreated group were stated as 100%. Each PCR was performed in triplicate and results were averaged.
Statistical analysis
Results were analyzed via a Student t test. Results were considered significant at p < 0.05.
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Results |
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The expression of TGF- isoforms was studied in papain-injected
joints and compared with normal knee joints, using immunohistochemical
methods on cryosections of whole knee joints.
In the periosteum of normal knee joints, hardly any expression of
TGF-1 and TGF-3 was observed. After injection of papain the
expression of both TGF- isoforms was enhanced in the periosteum,
where the expression of TGF-3 seemed more intense than TGF-1
expression (Fig. 1
, A–D). The expression was present at locations
known to develop osteophytes. TGF-2 expression was not detected in
normal periosteum or in periosteum after papain injection. Thus,
TGF-1 and -3 seem to be up-regulated after papain injection at
locations in the periosteum linked to osteophyte formation.
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1 and -3 isoforms. The expression was
mostly confined to the noncalcified layer of the cartilage. As in the
periosteum, this expression was up-regulated after papain injection
compared with noninjected left knee joints (Fig. 1
, E–H). In addition, in the cartilage the staining
for TGF-3 seemed more intense than TGF-1 staining. Again, no
staining for TGF-2 was observed. Negative controls, such as
substitution of the primary Ab with chicken IgYs or goat IgGs, did not
result in a color reaction (Fig. 1, I and J).
The results show that TGF-1 and TGF-3 isoforms are expressed in
periosteum and articular cartilage and are up-regulated after papain
injection. Next, we tried to quantify TGF-1 and -3 levels in knee
joints and we studied the effect of papain injection.
Quantification of TGF-1 and TGF-3 isoforms
The effect of papain injection on TGF-1 and TGF-3 protein
levels in knee joints was compared with noninjected knee joints via
ELISA. TGF-1 was detected in 2-h patellae wash-outs from control
left knee joints (Table I). Part of this
released TGF- is probably the result of the patella isolation
procedure itself. Nevertheless, injection of papain increased TGF-1
protein expression significantly from 10 pg/ml in control patellae to
30 pg/ml (p < 0.05). TGF-3 protein was also
detected in wash-outs from control patellae (200 pg/ml, Table I).
Injection of papain increased the expression of TGF-3 2-fold in 2-h
wash-outs (p < 0.001). Approximately 20 times
more TGF-3 than TGF-1 was detected, which supports the
observation from the immunohistochemistry experiment that murine knee
joints seem to contain more TGF-3 than TGF-1. The up-regulation
of TGF-1 and -3 protein was also found in 10-h wash-outs with
similar results (data not shown). We proceeded to study the effect of
TGF- inhibition during experimental OA on these joint structures. We
made use of solRII, the extracellular part of the TGF--RII, as a
specific TGF- inhibitor. solRII has a high affinity for TGF-1 and
-3 and only low affinity for TGF-2.
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Before using solRII to inhibit TGF- in vivo we characterized
the recombinant solRII as produced by P. pastoris.
Therefore, purified solRII was analyzed via SDS-PAGE and Western blot
analysis. Recombinant solRII showed a pattern of protein bands ranging
from 25 to 40 kDa (data not shown). The different bands are the
result of heterogeneous glycosylation (19). The Ab used
did not cross-react with BSA. Our recombinant solRII was estimated to
be >95% pure.
Neutralizing activity of solRII
We proceeded to determine the blocking activity of freshly
prepared solRII with an ELISA. To fully neutralize TGF-, a 4000-fold
excess of solRII was required (data not shown). solRII in excess of
2000- and 1000-fold prevented detection of TGF- in the order of 84
and 68%, respectively.
To apply the TGF- inhibitor in vivo, we first checked the stability
of the solRII in osmotic pumps, implanted i.p. in mice. Therefore,
after 7 and 14 days the remainder of the protein was flushed from the
retrieved osmotic pumps. Comparable concentrations of freshly and
retrieved solRII were equally effective in blocking TGF-, indicating
no loss of blocking activity of the retrieved solRII protein (data not
shown). This implies that, during the complete period, active solRII
was administered.
Osteophytes
To determine the role of endogenous TGF- in osteophyte
formation we treated mice with solRII during experimental OA. Papain
injection in the knee joint was used to cause the formation of
osteophytes at specific locations. Osteophytes developed on the femur
close to the medial collateral ligament and on the tibia between the
articular cartilage and where the growth plate meets the joint space.
Early osteophytes consisted of large round chondrocytes, which stained
with Saf-O and had not yet undergone the process of endochondral
ossification (Fig. 2, A and
E). Autoradiographic analysis showed that these chondrocytes
had incorporated radioactive sulfate in high amounts, suggesting a very
active cell metabolism (Fig. 2, B and F).
Treatment with recombinant solRII reduced the average size of the
osteophytes located on the femur significantly
(p < 0.02) from 9616
µm2 in the solvent-treated animals to 2746
µm2 in the solRII-treated mice (Fig. 3). On the tibia, the average size of the
osteophytes was reduced from 9849 µm2 in
solvent-treated mice to 2653 µm2 in
solRII-treated animals (p = 0.007).
Autoradiographic analysis showed a less-active cell metabolism in the
solRII group than in the solvent-treated group. These results strongly
implicate endogenous TGF- as an important factor in osteophyte
development.
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The role of endogenous TGF- on articular cartilage PTG
content after papain injection was studied using recombinant solRII.
Inhibition of TGF- activity for 7 days with solRII decreased PTG
staining in five of six cartilage compartments compared
with treatment with solvent (Fig. 4, C–F, and Fig. 5).
The effect of TGF- inhibition was most striking in patella and
femoral cartilage. In these cartilage compartments a significant
reduction (p < 0.001) in relative PTG content
of 43 and 38%, respectively, was observed (Fig. 4, C and
D, and Fig. 5). Also, the lateral femur and lateral tibia,
as well as the medial femur, showed a significant decrease in PTG
content (p < 0.05). No significant effects
after 7 days of solRII treatment were observed in medial tibia
cartilage. However, after 14 days of treatment with solRII, the
cartilage PTG content of the medial tibia was significantly less than
in the solvent-treated animals (p < 0.05)
(Fig. 4, G and H). The other cartilage
compartments did not significantly differ between treatments at day 14
(data not shown). After 21 days of treatment with solRII, the overall
PTG content of the articular cartilage of the tibia/femur plateau was
significantly lower than in the control-treated mice
(p < 0.001) (data not shown).
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, A and B), indicating that systemic
treatment with solRII itself did not induce pathological effects in the
knee joint.
Besides a decrease in PTG content, solRII treatment also had an effect
on the thickness of uncalcified cartilage (Fig. 4, C and
D). Measurements of patellar cartilage revealed a
significant decrease in surface area between solvent- and
solRII-treated animals, 22,610 and 16,351 µm2,
respectively, and p < 0.001 (Table II). The femoral uncalcified cartilage
also showed a significant decrease (p < 0.05)
after solRII treatment (22,463 vs 26,077 µm2 in
solvent-treated animals). These findings further imply a role for
endogenous TGF- in cartilage maintenance.
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activity resulted in a
decreased PTG content and decreased cartilage thickness, indicating a
pivotal protective role for endogenous TGF- in cartilage. MMP plus ADAMTS plus TIMP mRNA expression
Inhibition of endogenous TGF- during experimental OA reduces
articular cartilage PTG content. To gain a better understanding of how
TGF- inhibition leads to enhanced PTG loss, we investigated the
effect of solRII on the expression of cartilage-degrading proteinases
MMP-3, -13, and -14, and ADAMTS-4 and -5, or inhibitors of degradation
TIMP-1 and -3.
Treatment with solRII had no significant effect on the MMP-3 expression
in patellae of noninjected knee joints. However, after papain
injection, solRII treatment resulted in an 4-fold up-regulation of
MMP-3 mRNA (Table III). Although no
effect of solRII was seen on MMP-13 expression in control knee joints,
treatment with solRII after papain injection resulted in an 4-fold
up-regulation of MMP-13 mRNA (Table III). We could not identify
significant effects of solRII treatment on MMP-14, ADAMTS-4 and -5, or
TIMP-1 and -3 mRNA expression in patellae from normal and
papain-injected joints (data not shown).
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From these data we conclude that the observed enhanced PTG loss in articular cartilage after solRII treatment is probably the result of up-regulated MMP-3 and -13 expression.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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during experimental OA. Via administration of the
solRII we inhibited endogenous TGF-. This enabled us to study the
impact of neutralization TGF- on osteophyte formation and articular
cartilage degradation during experimental OA. Blocking of endogenous
TGF- in the knee joint resulted in an almost complete inhibition of
osteophyte formation. Furthermore, systemic delivery of solRII not only
decreased articular cartilage PTG content but also resulted in
cartilage loss, probably via the up-regulation of MMP expression.
We chose the extracellular part of TGF--RII as a TGF- antagonist
because it has a very high affinity for TGF-1 and -3 (24, 25), which are both abundantly present in the joint (12, 26, 27). Moreover, because of its small size of 25 kDa, solRII
can penetrate cartilage and directly affect chondrocytes
(28). Chondrocytes are difficult cells to target due to
the dense network of collagen fibrils and PTGs that make up articular
cartilage and render it virtually impenetrable for large molecules such
as Abs.
The solRII we produced via the P. pastoris expression system
is a biologically active protein. A 4000-fold excess of recombinant
solRII to TGF- was able to completely neutralize TGF- activity.
This is well in range with other studies in which a 1,600- to
20,000-fold excess of solRII was needed to fully inhibit TGF-
activity (25, 29). The solRII obtained from the osmotic
pumps retrieved from the mice after implantation showed no loss of
blocking activity compared with freshly produced solRII. This indicates
that during the complete duration of the experiment we were able to
inhibit TGF- activity.
One of the main characteristics of OA is the formation of new cartilage
and bone on the joint edges, so-called osteophytes. Osteophytes are the
principal cause of pain in OA patients and thus an unwanted feature of
the disease. Osteophytes are believed to originate from the periosteum.
Periosteal cells have the potential to undergo chondrogenesis and
osteogenesis in vivo and in vitro (30, 31, 32). In developing
osteophytes, not only is TGF- mRNA strongly expressed but also
TGF--R expression can be found (12, 13). Chondrogenesis
of periosteal cells can be enhanced by TGF- in a dose-dependent
manner (33, 34, 35). We previously showed that already 1 day
after i.a. injection of TGF- increased PTG synthesis was observed in
periosteal cells at sites that later show massive osteophyte
development (14). However, these studies did not prove a
role of endogenous TGF- during osteophyte formation. In this study
we show expression of TGF-1 and -3 in periosteum after papain
injection. Blocking of TGF-1 and -3 activity by use of the solRII
resulted in a dramatic reduction in osteophyte formation. So, here we
show for the first time that endogenous TGF- is essential in
osteophyte formation during experimental OA.
One of the first signs of OA is the loss of PTGs from the articular
cartilage, which ultimately leads to cartilage loss. Several lines of
evidence suggest an important role for TGF- in cartilage
maintenance. We and others (14, 15, 36) showed that
TGF- can increase PTG synthesis in normal and OA chondrocytes in
vivo and in vitro. Furthermore, local administration of TGF- can
suppress IL-1-induced matrix degradation (17, 37),
probably via down-regulating MMPs (38, 39) and/or
increasing the level of TIMPs (38, 40). TGF- also
inhibits terminal differentiation of chondrocytes (hypertrophy), which
has been shown to occur during OA. Serra et al. (41) have
shown that blocking of TGF- signaling in transgenic mice expressing
a truncated, kinase-defective TGF--RII in skeletal tissue results in
hypertrophic chondrocytes leading to OA changes in these mice. Recently
it was shown that, in Smad-3-deficient mice, articular chondrocytes
undergo abnormal terminal hypertrophic differentiation, which leads to
OA (42). These studies demonstrate that loss of TGF-
responsiveness in the articular cartilage can lead to OA changes.
Because TGF- has an important regulatory function in articular
cartilage, we studied effects of solRII treatment on articular
chondrocytes. First we determined the expression of TGF- isoforms
during papain-induced OA. The results show that TGF-1 and -3
protein expression occurs in chondrocytes of normal articular
cartilage, which is up-regulated after i.a. papain injection. In
contrast, we were unable to detect TGF-2 in normal knee joints or
during papain-induced OA. So, TGF-1 and -3 are likely to play a
role in cartilage matrix during experimental OA. Next, we show that
inhibition of endogenous TGF- in articular cartilage leads to a
decreased PTG content in nearly all articular cartilage compartments.
The loss of PTGs could be due to impaired synthesis of PTGs, enhanced
breakdown, or poor retention of PTGs in the cartilage. These results
are in line with our earlier studies that show that exogenous TGF-
increases PTG synthesis and content (14, 15). Our present
results illustrate that the solRII is able to penetrate the cartilage
and affect chondrocyte metabolism, leading to reduced PTG content. In
conclusion, endogenous TGF- is a necessary agent to protect
articular cartilage from PTG loss.
Besides effects on PTG content, TGF- deprivation also appeared to
increase cartilage loss. We found that the thickness of cartilage was
decreased after blocking TGF-. Increased cartilage loss might be
related to a decreased resistance to mechanical loading. Because
inhibition of endogenous TGF- decreased PTG content, and because
PTGs are important molecules responsible for the resistance of
articular cartilage (43, 44, 45), loss of PTGs may lead to
loss of cartilage. Therefore, endogenous TGF- appears important in
the prevention of cartilage loss.
We found that inhibition of endogenous TGF- leads to enhanced
cartilage PTG loss. This can be the result of either up-regulation of
cartilage-degrading proteases or down-regulation of the natural
inhibitors of these enzymes. To clarify the mechanistics of TGF-
inhibition on articular cartilage, the mRNA levels of several MMPs,
ADAMTS, and TIMPs in the knee joint were determined after exposure to
solRII. Our results suggest that inhibition of endogenous TGF- via
solRII enhances cartilage PTG loss via up-regulation of MMP-3 and
MMP-13. We observed only a small stimulation on ADAMTS-4 and -5 mRNA
expression after solRII treatment. The expression levels of TIMP-1 and
-3 were unchanged after solRII and IL-1 treatment. Thus, endogenous
TGF- suppresses the expression of several MMPs, such as MMP-3
and -13.
In summary, inhibition of endogenous TGF- leads to an increased loss
of PTGs from the cartilage and enhances cartilage loss during
experimental OA, most likely through the up-regulation of MMPs. This
indicates a protective role for endogenous TGF- on cartilage.
Moreover, with the aid of the soluble form of the TGF--RII as
a TGF- antagonist, we identified endogenous TGF- as the main
contributor to osteophyte development during experimental OA.
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Footnotes |
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2 Abbreviations used in this paper: OA, osteoarthritis; PTG, proteoglycan; solRII, soluble TGF--RII; i.a., intra-articular; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of MMP; ADAMTS, a disintegrin and metalloproteinase with thrombospondin motif; Saf-O, Safranin O; PMB, polymyxin B.
Received for publication February 25, 2002. Accepted for publication April 30, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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