The BMP antagonists follistatin and gremlin in normal and early osteoarthritic cartilage: an immunohistochemical study

Osteoarthritis and Cartilage (2009) 17, 263e270 ª 2008 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.joca.2008.06.022

International Cartilage Repair Society

The BMP antagonists follistatin and gremlin in normal and early osteoarthritic cartilage: an immunohistochemical study G. Tardif Ph.D., J.-P. Pelletier M.D., C. Boileau Ph.D. and J. Martel-Pelletier Ph.D.* Osteoarthritis Research Unit, University of Montreal Hospital Centre, Notre-Dame Hospital, Montreal, Quebec, Canada Summary Objective: Bone morphogenic protein (BMP) activities are controlled in part by antagonists. In human osteoarthritic (OA) cartilage, the BMP antagonists follistatin and gremlin are increased but differentially regulated. Using the OA dog model, we determined if these BMP antagonists were produced at different stages during the disease process by comparing their in situ temporal and spatial distribution. Methods: Dogs were sacrificed at 4, 8, 10 and 12 weeks after surgery; normal dogs served as control. Cartilage was removed, differentiating fibrillated and non-fibrillated areas. Immunohistochemistry and morphometric analyses were performed for follistatin, gremlin, BMP-2/4 and IL-1b. Growth factor-induced gremlin expression was assessed in dog chondrocytes. Results: Follistatin and gremlin production were very low in normal cartilage. Gremlin was significantly up-regulated in both non-fibrillated and fibrillated areas at 4 weeks, and only slightly increased with disease progression. Follistatin showed a time-dependent increased level in the non-fibrillated areas with significance reached at 8e12 weeks; in the fibrillated areas significant high levels were seen as early as 4 weeks. In the whole cartilage, follistatin and IL-1b temporal production showed similar patterns; this was also true for gremlin and BMP-2/4, though BMP2/4 production was already high in the normal dogs. Interestingly, data revealed that basic fibroblast growth factor (bFGF) could be another factor increasing gremlin expression early in the disease process. Comparison between superficial and deep zones revealed similar patterns for follistatin and IL-1b in the superficial zone only; gremlin and BMP-2/4 had similar patterns in both zones. Conclusion: Data show, for the first time, different spatial and temporal production of gremlin and follistatin in cartilage during OA progression. These findings may reflect different roles for each antagonist in this disease. ª 2008 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved. Key words: BMP antagonists, Follistatin, Gremlin, Osteoarthritis, Animal model.

and OA adult human cartilage10e12. BMP-13 (cartilage derived morphogenic protein (CDMP)-2, growth and differentiation factor (GDF)-6) and BMP-14 (CDMP-1, GDF-5) were also found in both normal and OA adult cartilage11. Despite increased BMP levels in the OA cartilage, overall anabolic activities are decreased with disease progression, suggesting the presence of some factor(s) interfering with their activity. BMP activities are controlled in part by specific antagonists13,14, which prevent BMP interaction with their specific receptors. Many BMP antagonists have been identified and they differ in their specificity and affinity for the BMPs. For example, gremlin binds BMP-2, -4 and -7 with similar affinity15,16, whereas follistatin also binds these BMPs, but with a higher affinity for BMP-717,18. The levels at which BMP antagonists are expressed in pathological tissues may lead to decreased BMP anabolic activity, consequently affecting tissue repair and remodeling. We previously reported the involvement of three BMP antagonists, follistatin, gremlin and chordin, in human OA3,4. From these previous studies, we hypothesized that follistatin and gremlin might be produced in the cartilage at different stages during the OA process. It is not possible to monitor the early stages of primary human OA, not only because of the slow course of the degenerative process, but also because of ethical considerations that preclude taking biopsies at different stages of the disease. However, the use of animal models allows for such

Introduction Osteoarthritis (OA) is a degenerative disease of the articular joints, characterized by cartilage degradation. The breakdown of the cartilage matrix leads to the development of fibrillation and fissures, the appearance of gross ulcerations, and fullthickness loss. Alterations in the collagen network and proteoglycans are observed early during the disease process1,2. Among the factors up-regulated in human OA are the bone morphogenic proteins (BMPs) and some of their antagonists3e6. BMPs belong to the transforming growth factor (TGF)-ß super-family of secreted signaling molecules7,8. They play important roles during embryogenesis, and promote the formation of cartilage and bone. They also contribute to the tissue homeostasis of adult cartilage by promoting the production of some cartilage macromolecules. Several BMPs are present in the adult human cartilage9 and some are found elevated during OA. For example, BMP-2/4 was shown to be increased in OA cartilage and present in osteophytes6. BMP-6 and BMP-7 appear to be present at similar levels in both normal *Address correspondence and reprint requests to: Dr Johanne Martel-Pelletier, Ph.D., Osteoarthritis Research Unit, University of Montreal Hospital Centre, Notre-Dame Hospital, 1560 Sherbrooke Street East, Montreal, Quebec, Canada H2L 4M1. Tel: 1-514890-8000x26658; Fax: 1-514-412-7582; E-mail: jm@ martelpelletier.ca Received 23 April 2008; revision accepted 29 June 2008.

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time-oriented studies. One of the well-characterized types of surgically induced OA models is the sectioning of the anterior cruciate ligament (ACL) in dogs, as the pathological structural changes seen in this model reproduce those of human OA. These changes include cartilage destruction, synovial inflammation, subchondral bone alterations, and the presence of osteophytes19e22. In this model, the earliest time point reported for site-specific gene expression is 2 weeks post-surgery, with no evidence of OA-related changes in gross histologic (cell morphology, density and distribution and matrix architecture and composition) or biochemical (proteoglycan and collagen levels) assessments23. However, noticeable changes are detected by 4 weeks and major cartilage lesions occur within 12 weeks post-surgery20,24,25. In addition, the OA dog model also shows parameters indicative of the involvement of proinflammatory and catabolic factors similar to those seen in human OA at the clinical stage of the disease22,26. Therefore, because this model displays the characteristics of human OA, it is suitable for the study of OA progress. The aim of this study was to investigate the production of the two BMP antagonists, follistatin and gremlin, using the experimental OA dog model, to examine their temporal and spatial changes in the cartilage, and to correlate their levels with those of anabolic (BMP-2/4) and catabolic (IL-1b) factors involved in OA cartilage. To this end, immunohistochemistry combined with morphometric analysis was used as it is the most appropriate methodology to investigate the production of these factors at specific locations and to follow their presence throughout the evolution of the disease.

(3) substitution of the primary antibody with an autologous pre-immune serum. These controls showed only background staining. Each section was examined under a light microscope (Leitz Diaplan; Wild Leitz, St. Laurent, QC, Canada) and photographed with a Retiga OEM Fast camera (QImaging, Surrey, BC, Canada).

MORPHOMETRIC ANALYSIS Antigen levels were quantified as previously described4 by determining the number of chondrocytes that stained positive. The total number of cells and the number of cells that stained positive for the specific antigen were first counted for the whole cartilage and then separately for each zone of the cartilage, at 40 magnification. Each slide was examined from 3 fields of the superficial (superficial and upper intermediate layers) and 3 fields of the deep (lower intermediate and deep layers) zones. The results were expressed as the percentage of cells that stained positive for the antigen, the maximum being 100%. Each slide was subjected to a double-blind evaluation; the final score was determined by consensus of the 2 readers.

CELL CULTURE Chondrocytes were released from full-thickness strips of whole normal dog cartilage by sequential enzymatic digestion at 37 C28. The cells were seeded at high density (105/cm2) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco BRL, Burlington, ON, Canada) supplemented with 10% heat-inactivated fetal calf serum (FCS; Gibco BRL) and an antibiotic mixture (100 units/ml penicillin base and 100 mg/ml streptomycin base; Gibco BRL) at 37 C in a humidified atmosphere of 5% CO2/95% air. The effects of growth factors on gremlin expression were assessed by pre-incubating first-passage cultured chondrocytes in DMEM/0.5% FCS for 24 h followed by another 24 h incubation with fresh DMEM/0.5% FCS containing bFGF (100 ng/ml; R&D Systems), GDF-9 (100 ng/ml; Peprotech Inc., Rocky Hill, NJ, USA) or BMP-2 (100 ng/ml; R&D Systems). Total ribonucleic acid (RNA) was extracted and processed for real-time polymerase chain reaction (PCR).

RNA EXTRACTION AND REAL-TIME PCR

Materials and methods ANIMALS Twenty-four adult crossbred dogs (2e3 years of age, 20e25 kg each) were used. OA was induced in 17 dogs by surgically sectioning the ACL of the right knee through a stab wound25. Prior to surgery, the animals were anesthetized intravenously with pentobarbital sodium (25 mg/kg) and intubated. Following surgery, the dogs were kept in animal care facilities for 1 week, and were then sent to a housing farm. Seven non-operated (normal) animals were used as controls. Dogs were housed in a large pen where they could exercise ad libitum. All dogs were sacrificed by intravenous injection. Operated dogs were sacrificed 4 weeks (n ¼ 4), 8 weeks (n ¼ 4), 10 weeks (n ¼ 5), or 12 weeks (n ¼ 4) after surgery. This study was approved by the Institutional Ethics Committee.

Total RNA was extracted and quantified4. Reverse transcription was primed with random hexamers. Real-time PCR was performed as previously described4 in the Rotor-Gene RG-3000A (Corbett Research, Mortlake, Australia) with the SYBR GreenER PCR Master Mix (Invitrogen, Carlsbad, CA, USA) used according to the manufacturer’s specifications. The 18S was used as the housekeeping gene. The data were given as a threshold cycle (Ct). Fold changes in gene expression were calculated as 2DðDCt Þ , where DCt ¼ Ct stimulated  Ct18S, and D(DCt) ¼ DCt stimulated  DCt control. The primer efficiencies for the genes were the same as those for the 18S gene. The dog-specific primer sequences were 50 -GAATCAGGGTT CGATTCCG (sense), 50 -CCAAGATCCAACTACGAGC (antisense) for 18S and 50 -ATGTGACCGAGCGCAAGTAC (sense), 50 -ATATGCATCGACA CTGCTTG (antisense) for gremlin.

STATISTICAL ANALYSIS SPECIMEN SELECTION AND PREPARATION Full-thickness sections of articular cartilage from tibial plateaus of operated dogs were removed from the lesional (fibrillated) and non-lesional (non-fibrillated) areas as previously described27. Specimens from normal dogs obtained from the same anatomical sites presented no macroscopic or microscopic lesions. Cartilage specimens were fixed in TissuFix #2 (Chaptec, Montreal, QC, Canada), embedded in paraffin, and 5 mm sections prepared.

Data are expressed as the mean  S.E.M. Statistical significance was assessed by 2-tailed Student’s t test, and P values < 0.05 were considered significant.

Results MORPHOLOGICAL CHANGES

IMMUNOHISTOCHEMISTRY 27

The specimens were processed as previously described . The primary antibodies were a mouse monoclonal anti-human follistatin (50 mg/ml; R&D Systems, Minneapolis, MN, USA), a goat polyclonal anti-human gremlin (4 mg/ml; Santa Cruz, Santa Cruz, CA, USA), a rabbit polyclonal anti-human BMP-2/4 (4 mg/ml; Santa Cruz), a rabbit antiserum against IL-1b (2.1 mg/ml; Rockland, Gilbertsville, PA, USA), and a rabbit polyclonal anti-bFGF (1/400 dilution; Chemicon, Temecula, CA, USA). Preliminary experiments showed that each of the antibodies recognized the specific proteins. Staining specificity was determined by: (1) immunoadsorption for 1 h at 37 C with the corresponding protein or peptide: 20 molar excess recombinant follistatin (R&D Systems), gremlin immunizing peptide (Santa Cruz), BMP-2/4 peptide (Santa Cruz), recombinant IL-1b protein (R&D Systems), recombinant bFGF (R&D Systems); (2) omission of the primary antibody;

The monitoring of specific factors during the OA process was done with cartilage obtained from non-operated (normal) or operated dogs sacrificed at 4, 8, 10 and 12 weeks post-surgery. Specimens were taken from the non-lesional (non-fibrillated) and lesional (fibrillated) areas to localize the temporal and spatial production of the factors. Cartilage histology of the normal dogs (Fig. 1) showed normal structure, cellularity, and matrix staining. OA changes over time were similar to those described previously24,26,29: few lesions were detected at 4 weeks post-surgery, more lesions appeared between the 4th and 8th weeks and progressed over time, being most severe at 10 and 12 weeks. OA

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Fig. 1. Representative histological sections of dog tibial plateaus from normal (non-operated) and OA cartilage 8 weeks post-surgery from nonfibrillated and fibrillated areas. Cartilage from normal dogs shows normal structure, cellularity, and matrix staining. The non-fibrillated area of the OA cartilage presents surface irregularity (dotted arrows), hypercellularity and cloning (arrows) and slight discoloration of the matrix at the superficial zone. The fibrillated area of the OA cartilage is characterized by fibrillation (arrowheads), hypocellularity, and matrix discoloration extending to the lower layers, as well as loss of matrix integrity and cartilage thickness. Magnification: 100.

non-lesional areas showed surface irregularities, hypercellularity and cloning, and slight discoloration of the matrix at the superficial layer. The lesional areas showed fibrillations, hypocellularity and matrix discoloration at the superficial and intermediate layers, reflecting loss of matrix integrity and cartilage thickness. IMMUNOHISTOMORPHOMETRIC ANALYSIS

Immunohistomorphometry was chosen to determine the relative amounts of follistatin, gremlin, BMP-2/4 and IL-1b over time during OA progression and their locations in the cartilage. There was a time-dependent increase of follistatin production in the non-fibrillated OA cartilage (Fig. 2). This is in contrast to the fibrillated areas, where follistatin levels were already high at week 4 and remained relatively stable over time. Gremlin levels (Fig. 2) were already statistically higher at 4 weeks in both the non-fibrillated (P < 0.008) and fibrillated (P < 0.0001) areas compared to the normal cartilage. Levels in the non-fibrillated and fibrillated areas of OA cartilage only slightly increased over time. Furthermore, gremlin levels were not significantly different in the non-fibrillated and fibrillated areas for each given week. BMP-2/4 levels (Fig. 2) were high in the normal dogs, and slightly increased over time in the non-fibrillated cartilage to reach statistical significance at 10 and 12 weeks post-surgery. Levels in the fibrillated cartilage at 4 weeks post-surgery were significantly higher than in the normal and nonfibrillated cartilage for the same week and remained stable over time. The IL-1b production profile followed a pattern similar to that of follistatin (Fig. 2); a significant increase occurred over time in the non-fibrillated cartilage, while levels at week 4 in the fibrillated cartilage were significantly higher

(P < 0.02) than in the normal cartilage and only slightly increased over time. The spatial production was monitored by evaluating protein levels in the superficial (superficial and upper intermediate layers) and deep (lower intermediate and deep layers) zones of the cartilage. As illustrated in Figs. 3 and 4, differences were found in the distribution of the antagonists between the superficial and deep zones.

Superficial zone (Fig. 3) Each factor studied was present in the superficial zones of the normal dogs, but at different levels: follistatin and gremlin at low levels of about 4%, IL-1b and BMP-2/4 at 13% and 22%, respectively. There was a progressive production of follistatin over time in the non-fibrillated areas of OA cartilage that reached statistical difference from week 8. In contrast, follistatin levels in the fibrillated areas were already significantly higher at week 4 compared to the normal (P < 0.001) and the non-fibrillated areas at 12 weeks (P < 0.02). The levels in the fibrillated areas did not vary from 4 to 10 weeks (21e24%), and a slight decrease was found at 12 weeks. Gremlin levels were significantly higher at 4 weeks in the non-fibrillated areas (P < 0.03) and the levels slightly increased with disease progression, but not significantly compared to week 4. In the fibrillated areas, levels were already statistically higher at 4 weeks (P < 0.001) and remained elevated and stable until the end of the study at week 12. BMP-2/4 levels were relatively high in the normal cartilage, and did not vary significantly in the OA non-fibrillated cartilage, although a small increase was found at 10 and 12 weeks. The levels at these times were similar to those seen at 4 weeks in the fibrillated areas. A small but significant increase was noted at week 10 in the fibrillated areas.

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Fig. 2. Temporal production levels of follistatin, gremlin, BMP-2/4, and IL-1b in the full thickness of cartilage from normal and operated dogs at 4, 8, 10 and 12 weeks in non-fibrillated (NF) and fibrillated (F) areas. Protein production was determined by immunomorphometric analysis as described in Materials and methods. P values were calculated by 2-tailed Student’s t test.

IL-1b levels increased over time in the non-fibrillated areas of the cartilage; statistical significance was reached for weeks 8e12 compared to the normal cartilage. Levels decreased slightly at week 12, suggesting attenuation in the inflammatory process. IL-1b levels in the fibrillated areas were significantly higher at week 4 (P < 0.02), remained relatively constant at weeks 8 and 10, and as seen in the non-fibrillated cartilage, decreased slightly at week 12. In summary, follistatin and IL-1b levels in the superficial zone showed similar profiles in both fibrillated and nonfibrillated areas; gremlin and BMP-2/4 also showed similar profiles, the only difference being a high BMP-2/4 level in the normal cartilage.

Deep zone (Fig. 4) The levels of the factors studied were lower in the deep (Fig. 4) than in the superficial zones (Fig. 3). There were very few cells showing immunoreactivity toward follistatin in the deep zone for both non-fibrillated and fibrillated areas. Slightly higher levels were found in the fibrillated areas compared to the non-fibrillated, which reached significance only after week 8. Gremlin production in the superficial zone increased with disease progression in both the non-fibrillated and fibrillated areas. BMP-2/4 production in the deep zone differed from that seen in the superficial zones: normal cartilage levels were lower (6%), production increased over time in the non-fibrillated areas from week 4 (12%) to week 12 (26%), and levels were already significantly higher in the fibrillated

areas at week 4 (P < 0.0007). These levels decreased at 8e10 weeks but reverted at 12 weeks. Of note, these values were not significantly different within the weeks. IL1b levels were low in the deep layers of normal cartilage (3%) and increased in a time-dependent fashion in both non-fibrillated and fibrillated areas. Levels were maximal at week 10, and as in the superficial zone, decreased slightly at week 12 in the non-fibrillated areas. In summary, gremlin and BMP-2/4 levels in the deep zone followed a similar production pattern especially in the non-fibrillated areas, IL-1b increased with disease progression, and follistatin levels remained very low. FACTORS INVOLVED IN THE UP-REGULATION OF GREMLIN

Our previous studies have shown that BMP-2/4 was the only factor tested that could up-regulate gremlin expression in human chondrocytes4. However, in the normal dog cartilage, the high levels of BMP-2/4 contrasted with the low levels of gremlin and its statistical up-regulation early during the disease process. In order to identify which factor could have contributed to the increased gremlin production in the OA cartilage, we determined the effects of some factors either related to the OA process or known up-regulators of gremlin expression, BMP-2, bFGF and GDF-9. Chondrocytes (n ¼ 5) were treated for 24 h with these factors and the expression level of gremlin was determined by real-time PCR. The results showed that GDF-9 did not significantly affect gremlin expression but that BMP-2 and bFGF markedly induced gremlin expression by 1.6  0.1

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Fig. 3. Temporal production levels of follistatin, gremlin, BMP-2/4, and IL-1b in the superficial zone of cartilage from normal and operated dogs at 4, 8, 10 and 12 weeks in non-fibrillated (NF) and fibrillated (F) areas. Protein production was determined by immunomorphometric analysis as described in Materials and methods. P values were calculated by 2-tailed Student’s t test.

fold (P < 0.0001) and 3.2  0.9 fold (P < 0.03), respectively. Hence, we looked for bFGF production in normal and OA dog cartilage (8 weeks) to verify whether the presence of this factor could correlate with the up-regulation of gremlin production with time. Results showed (Fig. 5) that although similar levels of bFGF (20e22%) were produced in both normal and OA cartilage and positive-staining cells were mainly localized in the superficial zones of both, a very important difference was the presence of marked positive staining throughout the extracellular matrix in the OA cartilage only.

Discussion Animal models of OA allow evaluation of temporal and spatial changes occurring in joint tissues during the progression of the disease. Although no animal model will represent exactly the events leading to human OA, they provide a good basis for understanding the role of specific molecules in the evolution of the disease. We chose the ACL dog model as it mimics changes seen in human OA at the molecular level but also because pro-inflammatory factors play a role in bringing about the structural changes, as they do in human OA. As our study aimed at investigating the production of factors in cartilage in a temporal and spatial manner, immunohistochemisty/morphometric analysis was used as it provides data not only on the relative abundance of a protein, but also its specific location throughout the evolution of the disease. While the etiology of articular cartilage degeneration seen in OA is not fully understood, cytokines and growth factors

play critical mediatory roles. Indeed, it is hypothesized that during the course of the disease insufficient or poor quality of new matrix macromolecules occurs as a consequence of deficient stimulation. Since growth factors of the BMP family are important regulators of matrix production, decreased activity of these factors through increased production of BMP antagonists could lead to the development of an altered matrix. We and others have shown that BMP antagonists are produced in adult cartilage, and that some are found at higher levels in human OA cartilage3e5. We have also shown that in human OA cartilage, follistatin and gremlin are produced in different areas3,4: follistatin is found primarily in the superficial layers of cartilage, while gremlin is mostly detected in the upper intermediate layers. As follistatin is up-regulated by the inflammatory molecules tumor necrosis factor (TNF)-a and interferon (IFN)-g4, we hypothesized that this antagonist would be linked particularly to the inflammatory process of the disease, while gremlin, because of its up-regulation by BMPs, would be associated preferentially with the remodeling phase. Results from this study confirm that the production of both follistatin and gremlin is up-regulated during OA, and that follistatin is located mostly in the superficial zone of the cartilage while gremlin is produced in both the superficial and deeper layers. These data, in addition to the significant up-regulation of gremlin at 4 weeks post-surgery, earlier than follistatin, confirm our hypothesis that gremlin is involved during the early phases. The very early stage of OA has been characterized by a hypertrophic biochemical repair reaction and increased synthesis of extracellular matrix. BMP-2 and -4 are growth factors that are expressed at this stage. Nakase et al.6

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Fig. 4. Temporal production levels of follistatin, gremlin, BMP-2/4, and IL-1b in the deep zone of cartilage from normal and operated dogs at 4, 8, 10 and 12 weeks in non-fibrillated (NF) and fibrillated (F) areas. Protein production was determined by immunomorphometric analysis as described in Materials and methods. P values were calculated by 2-tailed Student’s t test.

have reported that BMP-2 is scarce in normal adult human cartilage, is found in moderately damaged OA cartilage in the upper and middle layers, and in severely damaged cartilage extends to the deep layers. Interestingly, our previous work showed a similar production pattern for gremlin in human OA cartilage4. In this study, similar expression patterns of gremlin and BMP-2/4 were also observed: they are produced in both superficial and deep cartilage zones, in contrast to follistatin which is produced mainly in the superficial zone, strongly suggesting that BMP-2/4 and gremlin, rather

than follistatin, are associated with the remodeling phase of the cartilage matrix. Moreover, the fact that follistatin is involved later during the OA process as well as its similar topographical distribution with IL-1b at the superficial zone, suggests that the two molecules may be regulated by similar pro-inflammatory stimuli, strongly supporting a link between follistatin production and the inflammatory process of this disease. Also supportive of this link is that follistatin preferentially binds activin, a molecule expressed during some inflammatory

Fig. 5. Representative sections of bFGF immunostaining from normal and OA dog cartilage 8 weeks post-surgery. bFGF was found in chondrocytes from both normal and OA cartilage, mainly at the superficial zone. However, diffuse staining in the extracellular matrix was observed only in the OA cartilage throughout the cartilage layers. Magnification: 100.

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Osteoarthritis and Cartilage Vol. 17, No. 2 arthropathies30, over BMPs. Of note, and according to our previous work with human OA chondrocytes4, IL-1b is not the likely factor responsible for the increased follistatin production, but was used in this study as it is indicative of the inflammatory process occurring in this tissue. One unexpected finding was that the BMP-2/4 levels were relatively high in the normal dog cartilage and were not up-regulated in the superficial zone of the cartilage, whereas gremlin levels were low in the normal cartilage and significantly increased at 4 weeks in both non-fibrillated and fibrillated areas. These data suggest that factors other than BMP-2/4 are able to stimulate gremlin in dog OA cartilage. We looked for factors that might trigger the up-regulation of gremlin in dog chondrocytes. GDF-9 has been shown to increase gremlin expression in mouse granulosa cells31, but our data showed that this factor does not significantly alter gremlin expression in dog chondrocytes. In contrast, bFGF as well as BMP-2 markedly up-regulated gremlin expression in dog chondrocytes; furthermore, bFGF was found at an increased level in the OA cartilage matrix. Although bFGF is one of many factors present in OA cartilage, it may modulate gremlin expression in vivo as it is produced by the chondrocytes, stored in the extracellular matrix and is released from the matrix upon cartilage damage/injury. In addition, bFGF was suggested to contribute to the progression of OA as it interferes with the anabolic activities of insulin-like growth factor (IGF)-1 and BMP-7 in chondrocytes32 and stimulates matrix metalloprotease (MMP)-13 expression33, a metalloprotease capable of degrading many components of the extracellular matrix28,34e36. Our data show that bFGF is present in chondrocytes as well as in the OA cartilage matrix throughout the cartilage layers. The release of this growth factor may then contribute to the up-regulation of gremlin seen in OA dog cartilage. In our previous study of gremlin regulation in OA human chondrocytes4, we found that among all the cytokines and growth factors tested, only BMP-2 and -4 increased gremlin while IL-1b decreased its expression. Data from this study are in agreement with this observation, as they show an inverse correlation between gremlin and IL-1b production: gremlin production in the whole cartilage decreases at 10 weeks, which is also the time when IL-1b increases, and its production is again increased at 12 weeks, when IL-1b is decreased. IL-1b has been reported to increase BMP-2/4 expression in normal and OA human chondrocytes37. This cytokine may also be a regulating factor for BMP-2/4 levels in the dog model. Indeed, although IL-1b basal levels were relatively high in the whole cartilage, production slowly increased with disease progression in the non-fibrillated areas and was already significantly higher at week 4 in the fibrillated areas. This pattern was somewhat similar for BMP-2/4. However, although BMP-2/4 levels increased over time, this was accompanied by increased levels of gremlin and follistatin, thus reducing the anabolic activities of the BMPs. The up-regulation of gremlin and follistatin in OA cartilage is likely the consequence of the actions of different factors, resulting in differential production at different stages of the disease. Gremlin is associated with the remodeling/early phase and follistatin with the inflammatory process. This suggests the possibility that these proteins have functions other than being extracellular BMP antagonists in the OA process. Indeed, different functions were recently discovered for gremlin. In relation to the BMP system, gremlin was recently found to interact with the intracellular BMP-4 precursor and prevent the secretion of mature BMP-438. It

also binds factors other than BMPs, such as the polypeptide YWHAH (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein eta polypeptide)39, a protein that mediates signal transduction by binding to phosphoserine-containing proteins, and the Slit proteins, molecules involved in the regulation of monocyte chemotaxis40. In addition, gremlin was shown to activate the transcription of p21Cip141, involved in the cell cycle. Finally, gremlin was also reported to be a proangiogenic factor42. Although these factors have not yet been related to the OA process, it will be interesting to investigate whether gremlin’s other activities/properties might be involved in the physiological or pathological cartilage processes. In summary, despite the constant presence of BMPs in OA cartilage, these molecules might not be optimally active due to the increased levels of specific antagonists resulting in decreased production of matrix macromolecules such as collagen and proteoglycan, and a shift toward degradation rather than matrix synthesis and repair. The potential interference with BMP activity of different BMP antagonists during OA progression should be taken into consideration in the design of cartilage repair therapies involving BMPs.

Conflict of interest This research was supported by a grant from the CIHR/ IMHA.

Acknowledgments We thank Martin Boily for his technical assistance in immunohistochemistry, David Hum for assistance in the cell culture and real-time PCR experiments, and Virginia Wallis for the manuscript preparation. This research was supported by a grant from the CIHR/IMHA.

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