STIMULATION OF CALCIFICATION OF GROWTH-PLATE CARTILAGE MATRIX VESICLES BY BINDING TO TYPE-II AND TYPE-X COLLAGENS

Matrix vesicles (MV), microstructures which rapidly accumulate Ca2+ an d induce mineral formation in vitro, are linked to type II and X colla gens and proteoglycans in the hypertrophic cartilage. However, the rol es of these matrix proteins on MV function are not known. This led us to investigate the influence of type II and X collagen binding on Ca2 uptake by MV. MV isolated from chicken growth plate cartilage were tr eated with pure bacterial collagenase and 1 M NaCl in synthetic cartil age lymph to selectively and completely remove associated type II and X collagens. Uptake of Ca-45(2+) by these collagen-depleted vesicles w as markedly reduced. Further treatment with detergent, which disrupted the membrane, restored Ca2+ uptake, indicating that the vesicle membr ane structure and the nucleational core inside the vesicle lumen were still intact after the collagenase and 1 M NaCl treatments. Readdition of either native type II or X collagen to the collagenase, 1 M NaCl-t reated MV stimulated their Ca2+ uptake to levels similar to those of u ntreated vesicles. Pepsin-treated type II and X collagens were less ef fective in stimulating Ca2+ uptake, indicating that non-triple helical domains of these collagens were involved. The pepsin treatment of the se collagens also decreased their binding to annexin V (anchorin CII), one of three annexins found in MV, suggesting that annexin V is invol ved in mediating the binding of type II and X collagens to the MV surf ace. Furthermore, treatment of collagenase, 1 M NaCl-treated MV with c hymotrypsin, which damaged annexin V as well as many other MV proteins , prevented the stimulation of Ca2+ uptake by these collagens. Thus, t he interaction between type II and X collagens with MV activates the i nflux of Ca2+ into MV and may play an important role in calcification of the vesicles.