Matrix GLA protein (MGP), a γ-carboxyglutamic acid (GLA)–rich, vitamin K–dependent and apatite-binding protein, is a regulator of hypertrophic cartilage mineralization during development. However, MGP is produced by both hypertrophic and immature chondrocytes, suggesting that MGP's role in mineralization is cell stage–dependent, and that MGP may have other roles in immature cells. It is also unclear whether MGP regulates the quantity of mineral or mineral nature and quality as well. To address these issues, we determined the effects of manipulations of MGP synthesis and expression in (a) immature and hypertrophic chondrocyte cultures and (b) the chick limb bud in vivo. The two chondrocyte cultures displayed comparable levels of MGP gene expression. Yet, treatment with warfarin, a γ-carboxylase inhibitor and vitamin K antagonist, triggered mineralization in hypertrophic but not immature cultures. Warfarin effects on mineralization were highly selective, were accompanied by no appreciable changes in MGP expression, alkaline phosphatase activity, or cell number, and were counteracted by vitamin K cotreatment. Scanning electron microscopy, x-ray microanalysis, and Fourier-transform infrared spectroscopy revealed that mineral forming in control and warfarin-treated hypertrophic cell cultures was similar and represented stoichiometric apatite. Virally driven MGP overexpression in cultured chondrocytes greatly decreased mineralization. Surprisingly, MGP overexpression in the developing limb not only inhibited cartilage mineralization, but also delayed chondrocyte maturation and blocked endochondral ossification and formation of a diaphyseal intramembranous bone collar. The results show that MGP is a powerful but developmentally regulated inhibitor of cartilage mineralization, controls mineral quantity but not type, and appears to have a previously unsuspected role in regulating chondrocyte maturation and ossification processes.

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During the process of endochondral bone formation, chondrocytes undergo a series of complex maturational changes. Our recent studies indicate that this maturational process is influenced by the vitamin A derivative retinoic acid (RA). To learn how this agent regulates chondrocyte development, we characterized matrix gene expression during maturation of cartilage cells in chick sternum. RNAs were isolated from the cephalic portion of day 13, 14, 16, 18, and 20 chick embryo sternum and analyzed via northern blots. Type II collagen RNA levels remained fairly constant during this developmental period. In contrast, expression of type X collagen and alkaline phosphatase (APase) genes was first detected at day 16, followed by that of osteonectin (ON) and osteopontin (OP). To explore the mechanisms triggering these changes, chondrocytes were isolated from the cephalic portion of day 17-18 sternum (US cells) and grown in monolayer in standard serum-containing medium. After 3 weeks in culture, most of the cells enlarged and became type X collagen-positive, but they exhibited low APase activity and contained only trace amounts of ON and OP mRNAs. Treatment of parallel 3-week-old cultures with RA (10-100 nM) rapidly increased expression of the APase, ON, and OP genes severalfold. In concert with a significant increase in APase activity, there was abundant calcium accumulation in the RA-treated cultures. Electron microscopy confirmed the formation of large matrix-associated mineral crystals and the presence of numerous matrix vesicles. The effects of RA were also studied in cultures of immature chondrocytes isolated from the caudal portion of sternum (LS cells).(ABSTRACT TRUNCATED AT 250 WORDS)