We subsequently performed immunohistochemistry on sections o

We subsequently performed immunohistochemistry on sections obtained from mice at 16 weeks after surgery. Type II collagen expression was detected in all articular cartilage zones, but was weak in the upper zone in the vehicle-treated group. MMP-13 and type X collagen expression were increased in the mid and deep zones of osteoarthritic cartilage and below the tidemark in the vehicle-treated group but not in the T-5224-treated group (Fig. 3). These results suggest that T-5224 inhibited MMP-13 expression in cartilage and blocked the pathological hypertrophic differentiation of chondrocytes expressing type X collagen. In addition, to visualize bony changes, we used micro-CT to examine the knee joints of mice at 24 weeks after surgery. Mild but apparent osteophyte development in the femoral condyle and antero-medial aspect of the tibia was observed in the vehicle-treated group but not in the T-5224-treated group (Fig. 4a). Osteophyte score by 3D reconstructions of micro-CT was significantly higher in the vehicle-treated group than in the T-5224-treated group at 24 weeks after surgery (Fig. 4b). These results suggest that T-5224 inhibits the progression of osteoarthritic changes for at least 24 weeks.
Discussion We showed that a selective c-Fos/AP-1 inhibitor effectively prevented articular cartilage damage by suppressing, at least in part, the expression of MMP-13, which is a major initiator of cartilage matrix degradation in OA. This finding is supported by previous reports that surgical OA mice with MMP-13 gene AG 013736 are resistant to cartilage erosion [24]. In addition, selective MMP-13 inhibitors have been showed to be efficacious in several mammalian models of OA [[7], [8], [9]]. However, the effects of such selective inhibitors seemed to be mild compared with those observed in the present study. We showed that the progression of cartilage destruction was almost completely halted following treatment with the c-Fos/AP-1 inhibitor T-5224. T-5224 inhibited the expressions of most of the MMPs examined, including MMP-13, and inflammatory cytokines such as IL-1β in human chondrocytes. Additionally, T-5224 increased TIMP-3 expression. These results suggest that T-5224 may protect articular cartilage from joint damage by inhibiting the RNA expression of MMPs including MMP-13 and inflammatory cytokines in human articular cartilage, and that protection of articular cartilage may occur by blocking the IL-1b loop [19]. A number of mouse models with surgically-, enzymatically- or chemically-induced OA have been developed to investigate the pathogenesis of OA. OA mouse models induced by knee joint instability, using a microsurgical technique, have been used to evaluate gene deletions and gene over-expression in vivo [22,25]. Among these, the DMM model provides extremely good reproducibility and a slower disease progression, making it suitable for evaluating the potential of disease-modifying drugs. We therefore chose to use the DMM model for our in vivo study. As in previous studies, we found that OA changes were predominantly located in the anterior-central portion of the medial tibia plateau, but these were more slowly developed in our DMM model. Differences in disease progression may be due to the use of different background mouse strains and feeding procedures. T-5224 at a dose of 100 mg/kg was orally administered once a day for 24 weeks following surgery. This dose was determined as the no-observed adverse effect level in toxicity tests [16,19]. T-5224-treated mice did not exhibit weight loss or musculoskeletal syndromes associated with broad-spectrum MMP inhibitors [6]. T-5224 specifically inhibited the DNA binding activity of c-Fos/c-Jun without affecting those of other transcription factors including NF-κB, which regulates the upstream transcription of several MMPs [16]. For these reasons, T-5224 may exhibit efficacy for halting the progression of OA without the associated joint toxicity. Some studies have reported that the early stage of OA is characterized by hypertrophic differentiation of chondrocytes, as determined by type X collagen and MMP-13 expression in the cartilage above the tidemark [26,27]. Overexpression of MMP-13 in articular cartilage leads to increased type X collagen expression, while inhibition of MMP-13 reduces chondrocyte hypertrophy and the expression of type X collagen and RUNX2 in vitro [5,28]. In contrast, studies have also demonstrated c-Fos mRNA expression in the hypertrophic zone of mouse mandibular condyles and c-Jun mRNA expression in the proliferative zone and partly in the upper layer of the hypertrophic zone of rat growth plates by in situ hybridization [29,30]. It is clear that c-Fos and c-Jun are involved in driving chondrocytes to hypertrophy during enchodral ossification. In the present study, we showed that a c-Fos/AP-1 inhibitor suppressed type X collagen and MMP-13 expression in articular cartilage, resulting in osteophyte formation. This suggests that c-Fos/AP-1 might also play a crucial role in the regulation of pathological hypertrophic chondrocyte formation and osteophyte development in OA.