IntroductionOsteoarthritis (OA) is a degenerative joint disease affecting a large population of people. The mechanism of this highly prevalent disease is not fully understood. Currently there is no effective disease-modifying treatment for OA. The purpose of this study was two-fold: 1) to investigate the role of MMP13 in the development of OA; and 2) to evaluate the efficacy of the MMP13 inhibitor CL82198 as a pharmacologic treatment for preventing OA progression.MethodsTo investigate the role of the endogenous Mmp13 gene in OA development, tamoxifen was administered to two-week-old Col2CreER;Mmp13fx/fx (Mmp13Col2ER) and Cre-negative control mice for five days. OA was induced by meniscal-ligamentous injury (MLI) when the mice were 10 weeks old and MLI or sham-operated joints were harvested 4, 8, 12, or 16 weeks after surgery. To evaluate the efficacy of CL82198, MLI surgery was performed on 10-week-old wild type mice. CL82198 or saline was administered to the mice daily beginning immediately after the surgery for up to 16 weeks. The joint tissues collected from both experiments were evaluated by cartilage grading, histology/histomorphometry, immunohistochemistry (IHC), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The ability of CL82198 to inhibit MMP13 activity in vitro was confirmed by ELISA.ResultsThe OA progression was decelerated in Mmp13Col2ER mice 8, 12, and 16 weeks post-surgery. Cartilage grading by blinded observers confirmed decreased articular cartilage degeneration in Mmp13Col2ER mice at 8, 12 and 16 weeks compared to Cre-negative mice. Histomorphometric analysis demonstrated that Mmp13Col2ER mice had a higher articular cartilage area and thickness at 12 and 16 weeks post-surgery compared to the control mice. Results of IHC revealed greater type II collagen and proteoglycan expression in Mmp13Col2ER mice. Chondrocyte apoptosis, as determined by TUNEL staining, was higher in control mice compared to Mmp13Col2ER mice. CL82198 inhibited MMP13 activity in conditioned media from vehicle (> 85%) or bone morphogenetic protein 2 (BMP2)-treated (> 90%) primary murine sternal chondrocytes. Intraperitoneal injection of CL82198 decelerated MLI-induced OA progression, increased type II collagen and proteoglycan levels, and inhibited chondrocyte apoptosis compared to saline treatment as determined by OA grading, histology, histomorphometry, IHC, and TUNEL staining, respectively.ConclusionsMmp13 is critical for OA progression and pharmacologic inhibition of MMP13 is an effective strategy to decelerate articular cartilage loss in a murine model of injury-induced knee OA.

Osteoarthritis (OA), the most prevalent chronic joint disease, increases in prevalence with age, and affects majority of individuals over the age of 65 and is a leading musculoskeletal cause of impaired mobility in the elderly. Because the precise molecular mechanisms which are involved in the degradation of cartilage matrix and development of OA are poorly understood and there are currently no effective interventions to decelerate the progression of OA or retard the irreversible degradation of cartilage except for total joint replacement surgery. In this paper, the important molecular mechanisms related to OA pathogenesis will be summarized and new insights into potential molecular targets for the prevention and treatment of OA will be provided.

Objective While transforming growth factor β (TGFβ) signaling plays a critical role in chondrocyte metabolism, the TGFβ signaling pathways and target genes involved in cartilage homeostasis and the development of osteoarthritis (OA) remain unclear. Using an in vitro cell culture method and an in vivo mouse genetic approach, we undertook this study to investigate TGFβ signaling in chondrocytes and to determine whether Mmp13 and Adamts5 are critical downstream target genes of TGFβ signaling. Methods TGFβ receptor type II (TGFβRII)–conditional knockout (KO) (TGFβRIICol2ER) mice were generated by breeding TGFβRIIflox/flox mice with Col2-CreER–transgenic mice. Histologic, histomorphometric, and gene expression analyses were performed. In vitro TGFβ signaling studies were performed using chondro-genic rat chondrosarcoma cells. To determine whether Mmp13 and Adamts5 are critical downstream target genes of TGFβ signaling, TGFβRII/matrix metalloproteinase 13 (MMP-13)– and TGFβRII/ADAMTS-5–double-KO mice were generated and analyzed. Results Inhibition of TGFβ signaling (deletion of the Tgfbr2 gene in chondrocytes) resulted in up-regulation of Runx2, Mmp13, and Adamts5 expression in articular cartilage tissue and progressive OA development in TGFβRIICol2ER mice. Deletion of the Mmp13 or Adamts5 gene significantly ameliorated the OA-like phenotype induced by the loss of TGFβ signaling. Treatment of TGFβRIICol2ER mice with an MMP-13 inhibitor also slowed OA progression. Conclusion Mmp13 and Adamts5 are critical downstream target genes involved in the TGFβ signaling pathway during the development of OA.

SummaryThe BMP signaling pathway has a crucial role in chondrocyte proliferation and maturation during endochondral bone development. To investigate the specific function of the Bmp2 and Bmp4 genes in growth plate chondrocytes during cartilage development, we generated chondrocyte-specific Bmp2 and Bmp4 conditional knockout (cKO) mice and Bmp2,Bmp4 double knockout (dKO) mice. We found that deletion of Bmp2 and Bmp4 genes or the Bmp2 gene alone results in a severe chondrodysplasia phenotype, whereas deletion of the Bmp4 gene alone produces a minor cartilage phenotype. Both dKO and Bmp2 cKO mice exhibit severe disorganization of chondrocytes within the growth plate region and display profound defects in chondrocyte proliferation, differentiation and apoptosis. To understand the mechanism by which BMP2 regulates these processes, we explored the specific relationship between BMP2 and Runx2, a key regulator of chondrocyte differentiation. We found that BMP2 induces Runx2 expression at both the transcriptional and post-transcriptional levels. BMP2 enhances Runx2 protein levels through inhibition of CDK4 and subsequent prevention of Runx2 ubiquitylation and proteasomal degradation. Our studies provide novel insights into the genetic control and molecular mechanism of BMP signaling during cartilage development.Key words: Bmp2, Bmp4, Chondrocyte, Endochondral bone formation Introduction During skeletal development, the majority of the bones in the body are established by the endochondral bone formation process, which is initiated by mesenchymal cell condensation and subsequent mesenchymal cell differentiation into chondrocytes and surrounding perichondrial cells. The committed chondrocytes proliferate rapidly forming the cartilage growth plate where cells are arranged in columns of proliferating, differentiating and terminally hypertrophic chondrocytes. Chondrocytes near the center of the cartilage elements exit the cell cycle initiating the process of hypertrophic differentiation to generate a calcified cartilage matrix. Eventually, the local vasculature, perichondrial osteoblasts and various other types of cells invade the calcified cartilage, replacing the terminally mature chondrocytes with marrow components and trabecular bone matrix. Primary ossification occurs with osteoblast-mediated bone formation, which initially occurs on the calcified cartilage template. Chondrocyte maturation and the endochondral bone development process is tightly regulated by a series of growth factors and transcription factors, including bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), indian hedgehog (Ihh),