Chondrocyte differentiation is a fundamental process during endochondral ossification

process during endochondral ossification. Retinoic acid (RA) has been shown to regulate this process, however, the mechanisms underlying RA regulation of chondrogenesis are not clearly understood. Chondroprogenitor cells, ATDC5 have been shown to be a useful in vitro model for examining the multiple step differentiation of chondrocytes. The present study investigated the mechanisms underlying RA regulation of chondrogenesis using ATDC5 cell culture. In this study, we show that RA suppresses the cell growth, cartilage nodule formation, accumulation of proteoglycan, alkaline phosphatase (ALPase) activity and mineralization and that RA dose dependently upregulates the levels of type X collagen and matrix metalloproteinase-13 (MMP-13) mRNA which are marker proteins of hypertrophic chondrocytes, in ATDC5 cells. The addition of protein synthesis inhibitor, cycloheximide (CHX), partially inhibits the induction of type X collagen and MMP-13 mRNA by RA. In this system, RA upregulates the mRNA level of Runx2/Cbfa1 (type II), a positive regulator for mineralization, and downregulates the mRNA of Indian hedgehog (Ihh), parathyroid hormone related protein (PTHrP), negative regulators for terminal differentiation. However, RA downregulates ALPase, bone gla protein (BGP) mRNAs and mineralization. These data indicate that RA stimulates cartilage differentiation, however, cell condensation and cartilage nodule formation may be candidates of primary importance in the terminal differentiation of chondrocytes.