Regulatory roles of zinc in matrix vesicle-mediated mineralization of growth plate cartilage

Zinc (Zn²⁺) has long been known to play important roles in mineralization and ossification of skeletal tissues, but the mechanisms of Zn²⁺ action are not well understood. In this study we investigated the effects of Zn²⁺ on mineralization in a cell culture system in which terminal differentiation and mineralization of hypertrophic growth plate chondrocytes was induced by retinoic acid (RA) treatment. Addition of Zn²⁺ to RA-treated cultures decreased mineralization in a dose-dependent manner without affecting alkaline phosphatase (APase) activity. Characterization of matrix vesicles (MVs), particles that initiate the mineralization process, revealed that vesicles isolated from RA-treated and RA/Zn²⁺-treated cultures showed similar APase activity, but vesicles from RA/Zn²⁺-treated cultures contained significantly less Ca²⁺ and P(i). MVs isolated from RA- treated cultures were able to take up Ca²⁺ and mineralize in vitro, whereas vesicles isolated from RA/Zn²⁺-treated cultures were not able to do so. Detergent treatment, which ruptures the MV membrane and exposes preformed intravesicular Ca²⁺-P(i)-phospholipid complexes, did not restore the Ca²⁺ uptake abilities of MVs isolated from RA/Zn²⁺treated cultures, suggesting that vesicles from RA/Zn²⁺-treated cultures did not contain functional Ca²⁺-P(i)phospholipid complexes. Zn²⁺ treatment did not affect the content of annexins II, V, and VI in MVs or the Ca²⁺-dependent, EDTA- reversible binding of these molecules to the membrane surface. However, Zn²⁺ treatment did affect the EDTA-nonreversible binding of these molecules to the MV membrane, suggesting that Zn²⁺ interferes with the assembly of annexins in the MV membrane. In addition, Zn²⁺ inhibited annexin II-, V-, and VI-mediated Ca²⁺ influx into liposomes. In conclusion, Zn²⁺ inhibits the mineralizing competence of intravesicular Ca²⁺-P(i)-phospholipid complexes and function of annexin channels, thereby controlling Ca²⁺ influx into MVs, the formation of the first crystal phase inside the vesicles and initiation of mineralization.