Roles of the nucleational core complex and collagens (types II and X) in calcification of growth plate cartilage matrix vesicles

Abstract

Matrix vesicles (MV) were shown to initiate mineralization in cartilage and other vertebrate tissues. However, the factors that drive this process remain to be fully elucidated. Recent studies have shown that a preformed nucleational core consisting mainly of a Ca(2+)-phosphatidylserine-Pi complex, is necessary for the accumulation of Ca2+ by MV. In addition, the collagens attached to the MV surface were shown to play an important role in stimulating Ca2+ uptake. In this study, we extend this knowledge by showing that both, the nucleational core and the collagens (types II and X), are co-requirements for rapid influx of Ca2+ into intact MV. MV to which collagen fragments were attached were released from hypertrophic chicken cartilage by trypsin and collagenase digestion (trypsin/collagenase-released MV (TCRMV), while "collagen-free" MV were released by hyaluronidase and collagenase digestion (hyaluronidase/collagenase-released MV (HCRMV). In contrast to TCRMV which showed active uptake of Ca2+, HCRMV showed only little uptake. However, binding of native type II collagen to HCRMV stimulated uptake of Ca2+. Sucrose gradients separated TCRMV and HCRMV into three different density fractions: a low density top fraction (SI), an intermediate density middle fraction (SII), and a high density pellet fraction (SIII). The SIII fractions of TCRMV and HCRMV contained significantly higher levels of mineral ions than did the SI and SII fractions. Only the SIII fraction of TCRMV which contained a stable nucleational core and surface-attached collagens, showed active Ca2+ uptake; all other sucrose fractions of TCRMV and HCRMV showed little or no uptake. Detergent treatment to purposely rupture the membrane greatly enhanced Ca2+ uptake by the SIII fraction of HCRMV, presumably by exposing the internal nucleational core. Addition of either native type II or type X collagen to the intact SIII fraction of HCRMV stimulated Ca2+ uptake to a level similar to that of the SIII fraction of TCRMV; however, incubation of the SI and SII fractions of either TCRMV or HCRMV with type II or X collagen did not activate Ca2+ uptake. These findings indicate that both a functional nucleational core and surface-attached collagens need to be present to support active mineralization of MV.