In vitro functional characterization of prostaglandin-endoperoxide synthase 2 during chondrocyte hypertrophic differentiation

Cyclooxygenase 2 (Cox-2) has been implicated an essential role during bone repair, but the mechanisms remain elusive. Bone repair healing is known to include processes similar to endochondral ossification. In this study, we investigated the in vitro effect of Cox-2 on Col10a1 expression and chondrocyte hypertrophy, two critical components of endochondral ossification. Using quantitative RT-PCR, we detected increased mRNA levels of Cox-2 and Col10a1 in hypertrophic MCT cells, while cells treated with Cox-2 inhibitor, NS398, showed decreased mRNA and protein levels of Cox-2 and Col10a1. Increased Cox-2 also correlated with significantly upregulated Col10a1 in hypertrophic ATDC5 cells, whereas inhibition of Cox-2 significantly decreased Col10a1 expression. We further generated a Cox-2-expressing ATDC5 stable cell line. Compared with the controls, Cox-2 over-expression significantly increased Col10a1 as early as day 7 of continuous culturing, but not at days 14 and 21. Enhanced Alp staining was also observed in day 7 stable cell line. Correspondingly, we detected significantly increased levels of Runx2, Alp, Bcl-2, Bax, Col1a1, Osterix, and Bsp in day 7 stable line. Most of these genes have been associated with chondrocyte maturation and apoptosis. Together, our results support that Cox-2 promotes Col10a1 expression and chondrocyte hypertrophy in vitro, possibly through upregulation of Runx2 and other relevant transcription factors.