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. Author manuscript; available in PMC: 2011 Sep 1.
Published in final edited form as: Arthritis Rheum. 2010 Sep;62(9):2736–2744. doi: 10.1002/art.27582

Attenuation of osteoarthritis progression by reduction of the discoidin domain receptor 2 in mice

Lin Xu 1, Jacqueline Servais 1, Ilona Polur 1, Doil Kim 1, Peter L Lee 1, Kimberly Chung 1, Yefu Li 1
PMCID: PMC2946478  NIHMSID: NIHMS210881  PMID: 20518074

Abstract

Objective

To investigate whether the reduction of discoidin domain receptor 2 (DDR2), a cell membrane tyrosine kinase receptor for native type II collagen, attenuates the progression of articular cartilage degeneration in mouse OA models.

Methods

Double-heterozygous mutant, type XI collagen- and Ddr2-deficient (cho/+ and Ddr2+/−), mice were generated. Knee joints of Ddr2+/− mice were subjected to microsurgery - destabilization of the medial meniscus (DMM). Conditions of the articular cartilage from the knee joints of the double-heterozygous mutant and surgical mice were examined by histology, evaluated by a modified Mankin scoring system and characterized by immunohistochemistry.

Results

The rate of degenerative progression in knee joints was dramatically reduced in the double-heterozygous mutant mice compared with that in the type XI collagen-deficient mice. The progression in the double-heterozygous mutant mice was delayed by approximately 6 months. Following DMM surgery, the degenerative progression towards OA was dramatically delayed in the Ddr2+/− mice, compared with that in their wild-type littermates. The articular cartilage damage present in the knee joints of the mice was directly correlated with the expression profiles of Ddr2 and Mmp-13.

Conclusion

Reduction of Ddr2 expression attenuates the articular cartilage degeneration of knee joints, induced either by type XI collagen deficiency or DMM surgery.

Osteoarthritis (OA) is a debilitating disease that results from the progressive loss of articular cartilage (articular cartilage degeneration), a hallmark of the disease (1). Since mechanisms by which OA ensues are largely unknown, there are no therapeutic targets that effectively prevent and treat the disease. Existing drugs, such as disease-modifying OA drug (DMOAD), provide, at best, symptomatic relief from the pain and inflammation (2). However, a consistent pathologic pattern of the articular cartilage degeneration, regardless of the complicated etiology of OA, indicates that there may be a common chain of events underlying the cartilage degeneration. Thus, a molecular understanding of this chain of events will provide invaluable information towards the search for novel therapeutic targets for the prevention and treatment of OA. One of the earliest indications of articular cartilage degeneration is the degradation of proteoglycans and type II collagen at the surface of joints. Aggrecan and type II collagen, the two major components of articular cartilage, have been shown to be the degradative targets of the matrix metalloproteinase 13 (MMP13) (3). Data from numerous studies indicate that the up-regulated expression of MMP13 may be one of the common molecular events in OA progression (4–6). A very recent study demonstrates that deletion of this enzyme in mice delays the progression of OA (7). However, broad biological effects of MMP13 limit its application as a target enzyme of inhibitor drugs in the treatment of OA (8). This does raise the possibility that a molecule/molecules responsible for inducing expression of MMP13, in articular cartilage, may be used as therapeutic targets for the treatment of OA. During the past few years, through the use of mouse models of OA combined with in vitro experiments, we have identified discoidin domain receptor 2 (DDR2) as, possibly, such a molecule - implicating it in the induction of MMP13 in chondrocytes (9–11).

DDR2 was originally cloned as cell surface receptor tyrosine kinases (RTKs). In 1997, two research groups reported that native fibrillar collagens, including collagens type I, II and III, were the ligands of DDR2 (12,13). Further analysis of the interaction of DDR2 with these collagens indicates that DDR2 interacts preferentially with type II collagen (14,15). One intriguing finding is that over-expression of DDR2 in fibroblast-like cell lines enhances the expression of MMP-1. This prompted us to investigate if DDR2 controlled the synthesis of matrix-degrading enzymes in chondrocytes. The results from our studies demonstrated that the expression of DDR2 and MMP-13 was increased in human OA cartilages and knee joints of genetic mutant and surgically induced mouse models of OA (16,17). This indicates that the increased expression of these genes is not unique to cases of genetic OA, but is also seen in cases where the initiating event is joint instability or some other non-genetic events. We also performed a series of in vitro experiments (9,10) to determine whether or not the increased expression of DDR2 and MMP-13 in articular cartilage were dependent events. Results from our previous experiments demonstrated that: 1) the levels of DDR2 and MMP-13 mRNA were elevated in human and mouse chondrocytes cultured on native type II collagen. The levels of MMP-1, MMP-3, and MMP-8 mRNAs were not increased in the cells. 2) The overexpression of DDR2 induced the expression of MMP-13 in chondrocytes. 3) Ras/Raf/MEK/ERK and p38 signaling pathways were involved in the induction of MMP-13 in chondrocytes through the activation of DDR2. Based on these observations, we hypothesize that disruption of the pericellular matrix of chondrocytes enhances interaction of the cells with native type II collagen, leading to the activation of DDR2. The activation of DDR2 increases expression of MMP-13 and the receptor itself, ultimately resulting in the progressive destruction of the cartilage. Therefore, reduction of DDR2 may be able to attenuate OA progression via the down-regulated expression of MMP13 in joints.

In this study, we generated double-heterozygous mutant mice, type XI collagen- and Ddr2-haploinsufficient mice. Our previous study indicated that reduced expression of type XI collagen resulted in early onset OA in mice. We then evaluated the articular cartilages of the double-heterozygous mutant mice and their wild-type littermates for evidence of changes in histology and protein expressions for Ddr2 and Mmp-13. We also examined morphologic changes and protein expressions of Ddr2 and Mmp-13 in the articular cartilages of knee joints from Ddr2-deficient mice and their wild-type littermates following surgical destabilization of the medial meniscus (DMM).

Materials and Methods

Generation of double heterozygous Ddr2+/− and cho/+ mice

A Ddr2 null mutation mouse strain was provided by Regeneron at Tarrytown, NY. Briefly, the endogenous Ddr2 genomic DNA, about 2.2 kb spanning exon 10 and exon 17, was replaced by the LacZ (3.1 kb) and a PGK-neo cassette (1.9 kb) flanked by LoxP sites. The targeted ES cells were selected by PCR to identify the junction of the inserted DNA with the Ddr2 flanking sequences. Between the Ddr2 and LacZ cassette, a DNA fragment of 507 bp was amplified by PCR with the forward primer 5’-tgcgtatgaaaaccgctcac-3’ and the reverse primer 5’-gtctgtcctagcttcctcactg-3’. Between the neo cassette and the Ddr2, a DNA fragment of 258 bp was amplified by PCR with the forward primer 5’-tcattctcagtattgttttgcc-3’ and the reverse primer 5’-aggtgctgatgcatcatcac-3’. The heterozygous, Ddr2+/− mice were generated by standard protocols. PCR was used to distinguish heterozygous, Ddr2+/−, mice from homozygous, Ddr2−/ −, mice. For the heterozygous mice, a DNA fragment of 554 bp was amplified with the forward primer 5’-tgcgtatgaaaaccgctcac-3’ and the reverse primer 5’-acctccttgagttccagtcctg-3’. There was no PCR product with those primers in the homozygous mutant mice. For the generation of double heterozygous, Ddr2+/− and cho/+, mice, heterozygous Ddr2+/− mice were bred with cho/+ mice, Ddr2+/− and cho/+ mice, cho/+ mice and their wild-type littermates were identified by PCR and maintained under a daily schedule of 12 hours with light and 12 hours without light for further experimentation.

Microsurgery of mouse knee joint

Ddr2+/− mice and their wild-type littermates at the age of three months were anesthetized with Ketamine (90 μg/g BW) and Xylazine (25 μg/g BW) intra-peritoneally and their right knees were prepared for aseptic surgery. Buprenorphine were provided peri-operatively at 0.09 μg/kg. The joint capsule immediately medial to the patellar tendon was opened. The intercondylar region was exposed to provide visualization of the medial meniscotibial ligament (MMTL). The MMTL was sectioned with microsurgical scissors, resulting in the destabilization of the medial meniscus (DMM). After sectioning of the MMTL, the joint capsule was closed with a continuous 8-0 tapered Vicryl suture and the subcutaneous layer with 7-0 cutting Vicryl. The skin was closed by the application of tissue adhesive. The mice were maintained under a daily schedule of 12 hours with light and 12 hours without light for further experiments. Eight knee joints were harvested from surgical Ddr2+/− mice and their wild-type littermates at 2, 4, 8, 12 and 16 weeks following the surgery. Sham surgery on wild-type littermates and Ddr2+/− mice was performed as a control.

Histology and immunohistostaining of mouse knee joints

For the histology study, eight knee joints were harvested from Ddr2+/− and cho/+ mice and cho/+ mice and their wild-type littermates at the ages of 3, 6, 9 and 15 months. All joints were fixed and processed for paraffin embedding. For each knee joint, 6 μm thick serial sections were cut from the lateral to the medial side of the joints. About 200 sections covered an entire joint. For knee joints from surgery animals, approximately 150 sections representing the entirety of a mouse knee joint from its anterior to posterior side were collected. Every fifteenth section was collected for Safranine O/Fast green staining. The morphology of the joints was examined under light microscopy. The pathological condition of the knee joints was evaluated by a modified Mankin score system (18). In this system, the condition of the joint was ranked on the basis of intensity in staining of the pericellular matrix (0–2), staining intensity of the territorial and interterritorial matrix (0–3), spatial arrangement of chondrocytes (0–2) and overall cartilage structure (0–3). The score for the articular cartilage of knee joints of wild-type mice at the age of 3 months is 0 and the maximum score for the degenerative articular cartilage is 10. For statistical analyses, 8 scores representing 8 animals from each group were obtained. An average of the 8 scores was used as a statistical comparison, with the Mann-Whitney U-test indicating a significance between the two groups (the P value was less than 0.001). Meniscus, subchondral bone, osteophyte formation were also examined to evaluate the overall condition of the joints.

For immunohistochemistry, paraffin sections were used for immunostaining. Four knee joints were randomly selected from double-heterozygous mutant mice, cho/+ mice and their wild-type littermates at the ages of 3 and 6 months. Eight sections that were evenly distributed throughout each joint were collected for immunostaining. The sections were de-paraffinized and quenched for endogenous peroxidase activity and then were incubated with primary polyclonal antibodies, a polyclonal antibody against Ddr2 (1:200 dilution, Cat. No. sc-7554, Santa Cruz Biotechnology, Inc. CA) or a polyclonal antibody against Mmp-13 (1:200 dilution, Cat. No. MAB3321, Chemicon, Temecula, CA) at 4°C overnight. After washing with PBS three times, the samples were incubated with a biotinylated secondary antibody (goat anti-rabbit IgG-B). Coloring was developed with the use of a peroxidase substrate (VECTOR NovaRED Substrate, Cat. No. SK-4800, Laboratories, Burlingame, CA) following treatment of the sections with a mixture of avidin and biotinylated horseradish peroxidase (VECTASTAIN ABC Kit, Cat.No. PK-4000, Vector Laboratories, Burlingame, CA). Sections were counterstained with 0.2% Fast Green solution. Staining with isotype-matched normal IgG (Vector Laboratories) and staining without primary antibody were also performed as negative controls.

Results

Delay of the progression of articular cartilage degeneration in cho/+ mice by reduction of Ddr2

Homozygous Ddr2-deficient mice were phenotypically similar to the two other Ddr2-null mutation mouse strains that had been previously described. This phenotype includes short stature (about 70% of that of a normal wild-type littermate) and decreased chondrocyte proliferation in the cartilage growth plates. There are no obvious abnormalities observed in heterozygous Ddr2-deficient mice. To investigate whether reduction of Ddr2 could attenuate the degenerative progression of knee joints in cho/+ mice, we generated double-heterozygous (cho/+ and Ddr2+/−) mutant mice. In cho/+ mice (see the middle column in Fig. 1) at 3 months of age, a number of chondrocyte clusters (see the inset) were seen in the superficial layer of the articular cartilage. While the location of the chondrocyte clusters was variable, all eight knee joints in this age group from cho/+ mice had such clusters present. The clusters were also seen in all eight knee joints of the double-heterozygous mutant mice (see the inset in the right column). At the age of 6 months, the red staining (indicating proteoglycan presence) appears to be absent from some regions, in particular the superficial layer, of the articular cartilage of cho/+ mice. At the age of 9 months, the regions lacking in red staining extend from the superficial layer to the deeper layers of cartilages in the cho/+ mice. At the age of 15 months, cho/+ mice reveal several features of a typical OA knee joint, including loss of articular cartilage and misshapen menisci. A progression in articular cartilage degeneration was also seen in the double-heterozygous mutant mice, but with a reduced rate. In particular, degenerative damage to the knee joints of the double-heterozygous mutant mice at the age of 15 months was similar to that of cho/+ mice at the age of 9 months, suggesting a 6-month degenerative delay in the double-heterozygous mutant mice. Evidence for mild proteoglycan degradation was also seen in knee joints of wild-type mice with aging.

Figure 1. Histology of the articular cartilage of knee joints from double-heterozygous (cho/+ and Ddr2+/−) mice, cho/+ mice and their wild-type littermates.

Figure 1

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Each image shown is one representative section selected from 96 sections of an experimental group (12 sections from each knee joint and 8 knee joints in each experimental group). The representative section was selected based on the average score from the each experimental group. There were no obvious morphological differences in knee joint degeneration among the double-heterozygous mutant mice, cho/+ mice and their wild-type littermates at 3 months old. However, the degenerative condition of the articular cartilage in the cho/+ mice was far more advanced when compared with that of their wild-type littermates in each of the age groups - 6, 9 and 15 months old. The degenerative process was also observed in the double-heterozygous mutant mice with increasing age, but the rate of progression was dramatically reduced in the double-heterozygous mutant mice compared with that in cho/+ mice. (Bar=100 μm).

The condition of the articular cartilage was also evaluated by the use of a modified Mankin scoring system (Fig. 2) (18,19). The knee joints of wild-type littermates at the age of 3 months were considered as a reference point for normal articular cartilage, with a score of zero (0). At the age of 3 months, scores for the double-heterozygous mutant mice and cho/+ mice were the same, 0.74. At the age of 6 months, scores for the double-heterozygous mutant mice, cho/+ mice and their wild-type littermates were 0.75, 2.22 and 0.31 respectively. There was significance between the cho/+ mice and the wild-type littermates’ scores, but there was no significance between the double-heterozygous mutant mice and the wild-type littermates’ scores. At the age of 9 months, the scores were 3.77 for the cho/+ mice, 2.45 for the double-heterozygous mutant mice and 0.59 for the wild-type littermates. There were significances between the scores of the mutant mice and their wild-type littermates and between the double-heterozygous mutant mice and cho/+ mice. At 15 months of age, the scores were 6.5 for cho/+ mice, 3.6 for the double-heterozygous mutant mice and 1.46 for the wild-type mice. There were significances among the scores of those three groups. The scores did increase in the double-heterozygous mutant mice with age, but were not as dramatically increased as they were in the cho/+ mice.

Figure 2. Average modified Mankin scores of double-heterozygous mutant mice, cho/+ mice and their wild-type littermates.

Figure 2

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The morphologic condition of the articular cartilage from the knee joints was evaluated. An average score representing 8 animals from each experimental group was obtained. A statistical comparison of the scores using the Mann-Whitney U-test indicated a significant difference among the groups at each age and the P value was less than 0.001 (see *). We found that the degenerative progression in the double-heterozygous mutant mice was delayed by approximately 6 months. Interestingly, the scores of the double-heterozygous mutant and cho/+ mice were similar at the age of 3 months old.

Association of articular cartilage damages with the expression levels of Ddr2 and Mmp-13 in the double-heterozygous mutant and cho/+ mice

To know whether the attenuation of OA progression in the double-heterozygous mutant mice was indeed due to the reduced expression of Ddr2, we examined the expression of Ddr2 and Mmp-13 in the knee joints of double-heterozygous mutant mice, cho/+ mice and their wild-type littermates, at the ages of 3 and 6 months old. We selected those ages of mice based on the initial appearance of the degradation of proteoglycans and type II collagen in cho/+ mice. We found (Fig. 3A and B) that the lesser damage of the articular cartilage was associated with reduced expressions of Ddr2 and Mmp-13 in the knee joints of the double-heterozygous mutant mice, compared with that in the knee joints of cho/+ mice at the age of 6 months.

Figure 3. Expressions of Ddr2 and Mmp13 in knee joints of mice at the age of 6 months old.

Figure 3

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Each image (see panel A) is one representative section selected from 32 sections of an experimental group (8 sections from each knee and 4 knees in each group). More reddish-brown staining cells (Ddr2 or Mmp13 positive) were observed in the superficial layer of the cho/+ mice. In the double-heterozygous mutant mice, there were less Ddr2 or Mmp13 positive cells. There were hardly any positively staining cells detected in wild-type littermates at the age of 6 months and any of the three groups at the 3 months of age (data not shown). (Bar = 50 μm). The percentage of the positive staining cells in knees of mice at the age of 6 months old was obtained (see panel B). There were 23% of Ddr2 positive cells and 21% of Mmp13 positive cells in cho/+ mice and 13% of Ddr2 positive cells and 11% of Mmp13 positive cells in the double-heterozygous mutant mice. There were significant differences between two groups, p<0.05 (t-test). There were hardly Ddr2 positive and Mmp13 positive cells detected in wild-type littermates.

Delay of the progression of articular cartilage degeneration in surgically induced OA mice by reduction of Ddr2

To determine the effect of a null mutation of Ddr2 on the progression of OA induced by a non-genetic factor, we performed microsurgery on the knee joints of heterozygous Ddr2-deficient (Ddr2+/−) mice to destabilize the medial meniscus (DMM) by cutting ligaments between the medial meniscus and the anterior tibial plateau. We found (Fig. 4) that the morphology of the knee joints remained similar among the sham surgery groups of wild-type littermates (and Ddr2+/− mice, data not shown) throughout the aging process. However, following DMM surgery, the Ddr2+/− mice and their wild-type littermates exhibited significant disparities in the progressive process of the articular cartilage degeneration of their knee joints. The progression towards OA was dramatically delayed in the Ddr2+/− mice following surgery.

Figure 4. Histology of the articular cartilage of knee joints from mice following microsurgery.

Figure 4

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In the wild-type littermates, at 4 weeks following surgery, regional reduced Safranine O staining (see the arrow) was observed. At 8 weeks following surgery, fibrillation was seen (see the arrow). At 12 weeks following surgery, the articular cartilage appears thinner and the fibrillation extends to the deep layer of the cartilage. At 16 weeks following surgery, a complete loss of articular cartilage was evident. A delay of the degenerative process was seen in the Ddr2+/− mice following surgery. A mild degradation of proteoglycans was also seen in the knee joints of sham-surgery mice with aging. (Bar=100 μm).

The condition of the knee joint cartilage was also evaluated with a modified Mankin scoring system (Fig. 5). Mice at 4 weeks following sham surgery were used as a normal control (score=0). At 4 weeks following DMM surgery, scores for Ddr2+/− mice and their wild-type littermates were 0.71 and 2.58, respectively. At 8 weeks following surgery, scores were 1.23 for Ddr2+/− mice and 2.88 for the wild-type littermates. At 12 weeks following the surgery, scores were 2.33 for Ddr2+/− mice and 5.71 for their wild-type littermates. At 16 weeks following surgery, scores were 3.1 for Ddr2+/− mice and 7.21 for the wild-type littermates. There were significant differences among the scores of these three groups at each time point after surgery.

Figure 5. Average modified Mankin scores of mice with the DMM microsurgery.

Figure 5

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To evaluate the morphologic condition of the articular cartilage of knee joints, an average score representing 8 animals from each group at each time point was obtained. A statistical comparison of the scores using the Mann-Whitney U-test indicated a significant difference between the two groups at each time point and the P value was less than 0.001 (see *). As the length of time following the surgery increased, the modified Mankin scores also increased, significantly, in the wild-type littermates with DMM surgery. However, the scores were not as dramatically increased in the Ddr2+/− mice with DMM surgery, particularly after 8 weeks post-surgery.

Association of articular cartilage damages with the expression levels of Ddr2 and Mmp-13 in Ddr2+/− mice and their wild-type littermates with DMM surgery

Expressions of Ddr2 and Mmp-13 in Ddr2+/− mice and wild-type littermates at 2 and 4 weeks following surgery were examined. At 4 weeks following surgery, the initial appearance of the degradation of proteoglycans and type II collagen was evident in DMM mice. We found (Fig. 6A and B) that the condition of the articular cartilage damage was mild in Ddr2+/− mice, when compared with that observed in the wild-type littermates at 4 weeks following surgery and the level of Ddr2 and Mmp-13 expressions were reduced in the Ddr2+/− mice.

Figure 6. Expressions of Ddr2 and Mmp-13 in knee joints of mice at 4 weeks following the surgery.

Figure 6

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Each image (see panel A) shown is one representative section selected from 32 sections of an experimental group (8 sections from each knee joint and 4 knee joints in each experimental group). The representative section was selected based upon the percentage of positively staining cells in the section. More reddish-brown staining cells (Ddr2 or Mmp13 positive) were observed in the superficial layer of the wild-type littermates, compared with that of Ddr2+/− mice, following surgery. (Bar = 50 μm) The percentage of positive staining cells in knee joints of mice at 4 weeks following the surgery was obtained (see panel B). There were 19% of Ddr2 positive cells and 21% of Mmp13 positive cells in wild-type mice with DMM surgery and 11% of Ddr2 positive cells and 8% of Mmp13 positive staining cells in Ddr2+/− mice with DMM surgery. There were significant differences in the number of the positive cells between two groups, p<0.05 (t-test). There were hardly Ddr2 positive and Mmp13 positive cells detected in sham-surgery mice.

Discussion

To understand effect of reduction of Ddr2 on articular cartilage degeneration, we generated double-heterozygous mutant mice, cho/+ and Ddr2+/−. Role of reduction of Ddr2 on articular cartilage degeneration, due to type XI collagen haploinsufficiency in cho/+ mice, could be directly tested in the double-heterozygous mutant mice. Currently there are three Ddr2-deficient mouse strains, including the one that we used in our study (20,21). Homozygous (Ddr2−/ −) mutant mice from all three strains exhibit a short stature (dwarfism, 70% of a normal size), whereas heterozygous (Ddr2+/−) mutant mice appear normal in size, without any obvious abnormalities. The dwarfism phenotype in Ddr2−/ − mice is most probably due to a decrease in chondrocyte proliferation during the growth period. Due to the appearance of dwarfism in the homozygous Ddr2-deficient mice, they are not appropriate for the study of OA. Consequently, we used Ddr2+/− mice in this study. Results from our double-heterozygous mutant mice showed that OA progression induced by the type XI collagen haploinsufficiency was delayed in the mouse knee joints. This is consistent with our previous observation that the expression of Ddr2 is regulated in an autocrine manner. Thus removal of one copy of the Ddr2 allele can restrain increased expression of the receptor. The next question is what is effect of the removal of a copy of Ddr2 on OA progression induced by non-genetic factors, which defines the majority of OA cases. Results from our DMM surgery in Ddr2+/− mice indicate that the reduced expression of Ddr2 could attenuate the progression of articular cartilage degeneration induced by non-genetic factors in the mouse knee joints.

Interestingly, we found that the reduced expression of Ddr2 could not prevent the molecular events at the very early stages of the disease, such as chondrocyte clustering, which is a consequence of the elevation in chondrocyte activity. This suggests that normal metabolic events in chondrocytes are altered prior to activation of Ddr2. In fact, in one of our other studies, we found that the expression of serine protease HTRA1 (high temperature requirement A1) was up-regulated and co-localized with the increased expression of Ddr2 in mouse OA cartilages (22). The enzyme degrades most of the pericellular matrix components (23–26). This is consistent with our hypothesis that the disappearance of the pericellular matrix is required to expose chondrocytes to native type II collagen fibrils at the early stage of OA progression. This, in turn, elicits the interaction of chondrocytes with type II collagen, resulting in the activation of Ddr2. In normal conditions, type II collagen fibrils are in the interterritorial and territorial extracellular matrix of articular cartilages and there is little to no type II collagen fibril in the pericellular matrix of chondrocytes (27–29). Therefore, protection of the pericellular matrix can prevent chondrocytes from direct contact with type II collagen fibrils. Involvement of the pericellular matrix of chondrocytes in OA progression at the early stages of the disease has been suggested by Poole, et al (30). Based on their observation, they suggest that chondrons, consisting of chondrocytes, a pericellular matrix, and a capsule surrounding the pericellular matrix, are true anatomic and functional entities. The capsule separates chondrocytes and the pericellular matrix from adjacent interterritorial or territorial matrices. The microenvironment of the pericellular matrix is crucial to the chondrocytes’ ability to maintain their normal activity. Results from their study indicate that the pericellular matrix of chondrocytes swells and expands in the degenerative cartilage. The amount of proteoglycans becomes reduced, and a fine collagen network is disrupted in the pericellular matrix. In fact, results from a number of recent studies support this speculation. For instance, deletions of type VI and IX collagens, matrilin 3, biglycan and fibromodulin - all of which are present in the pericellular matrix - result in early onset articular cartilage degeneration (31–36). Clearly, the pericellular matrix is implicated in the initiation and early development of articular cartilage degeneration. Thus maintenance of the pericellular matrix of chondrocytes may be critical in the prevention of OA progression before significant degradation of type II collagen occurs.

Based on results from our and others’ studies, we propose a likely chain of molecular events that underlies the process of articular cartilage degeneration, eventually leading to the development of OA. It is as follows: Normal loading of abnormal joint tissues in the case of cho/+ mice or abnormal loading of normal joint tissues in the case of surgically induced mouse OA model activates chondrocytes to synthesize and release extracellular matrix degrading enzymes, such as HTRA 1. The enzymes can degrade the pericellular matrix of chondrocytes. This, in turn, exposes the chondrocytes to type II collagen fibrils, which are normally separated from the chondrocytes by the pericellular matrix. Interaction of the chondrocytes with type II collagen activates DDR2, resulting in induction of MMP-13 and increased expression of the receptor itself. Fragments of type II collagen and aggrecan generated by MMP-13 stimulate chondrocytes to further increase the synthesis of metalloproteinases, including MMP-13 (37,38). The result is a feedback amplification loop that enhances the degradation of articular cartilage, in the end, leading to the development of OA. Obviously, one legitimate question can be raised as to how newly synthesized type II collagens traverse the pericellular region without causing activation of Ddr2 during the developmental stage or in mature articular cartilages. One possible explanation is that chondrocytes not only synthesize type II collagens, but also produce other extracellular matrix molecules including the components of the pericellular matrix of chondrocytes during the development. The pericellular matrix prevents newly synthesized type II collagen from binding to Ddr2. Another possible explanation is that the interaction of Ddr2 with type II collagen requires the presence of a specific structure on type II collagens. This specific structure can be formed through a post-translational modification process. It is known that type II collagens undergo an extensive post-translational modification process. With regard to the question of type II collagen synthesis in mature articular cartilage, it is unclear how active chondrocytes are able to synthesize type II collagens in mature articular cartilages. A study by Verzijl, et al. demonstrates that the half-life of articular cartilage collagen in human mature articular cartilage is 117 years (39). This suggests that the turnover of the articular cartilage collagen is very slow and that little is needed to produce more collagen once articular cartilage is formed. Several studies report the presence of mRNAs of the embryonic type II collagen isoform (CIIA) in degenerative articular cartilage. However, it is unknown whether the isoform type II collagen is formed and bound to Ddr2.

Taken together, this suggests that the development of antagonists that specifically target DDR2 in chondrocytes may be central to the effort to develop a successful therapeutic agent for the prevention and treatment of OA. It remains to be seen whether inhibiting the activity of DDR2 and its up- or down-stream signaling pathways will be efficacious in treating this crippling disease.

Acknowledgments

R01-AR-051989 from NIH/NIAMS to YL and LX.

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