Annexin-mediated Ca²⁺ influx regulates growth plate chondrocyte maturation and apoptosis

Maturation of epiphyseal growth plate chondrocytes plays an important role in endochondral bone formation. Previously, we demonstrated that retinoic acid (RA) treatment stimulated annexin-mediated Ca²⁺ influx into growth plate chondrocytes leading to a significant increase in cytosolic Ca²⁺, whereas K-201, a specific annexin Ca²⁺ channel blocker, inhibited this increase markedly. The present study addressed the hypothesis that annexin-mediated Ca²⁺ influx into growth plate chondrocytes is a major regulator of terminal differentiation, mineralization, and apoptosis of these cells. We found that K-201 significantly reduced up-regulation of expression of terminal differentiation marker genes, such as cbfal, alkaline phosphatase (APase), osteocalcin, and type I collagen in RA-treated cultures. Furthermore, K-201 inhibited up-regulation of annexin II, V, and VI gene expression in these cells. RA-treated chondrocytes released mineralization-competent matrix vesicles, which contained significantly higher amounts of annexins II, V, and VI as well as APase activity than vesicles isolated from untreated or RA/K-201-treated cultures. Consistently, only RA-treated cultures showed significant mineralization. RA treatment stimulated the whole sequence of terminal differentiation events, including apoptosis as the final event. After a 6-day treatment gene expression of bcl-2, an anti-apoptotic protein, was down-regulated, whereas caspase-3 activity and the percentage of TUNEL-positive cells were significantly increased in RA-treated cultures compared with untreated cultures. Interestingly, the cy-. tosolic calcium chelator BAPTA-AM and K-201 protected RA-treated chondrocytes from undergoing apoptotic changes, as indicated by higher bcl-2 gene expression, reduced caspase-3 activity, and the percentage of TUNEL-positive cells. In conclusion, annexin-mediated Ca²⁺ influx into growth plate chondrocytes is a positive regulator of terminal differentiation, mineralization, and apoptosis events in growth plate chondrocytes.