Matrix Gla Protein Is a Developmental Regulator of Chondrocyte Mineralization And, When Constitutively Expressed, Blocks Endochondral and Intramembranous Ossification in the Limb

Abstract

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.

Indian hedgehog and syndecans‐3 coregulate chondrocyte proliferation and function during chick limb skeletogenesis

“…Syndecan-3-encoding viral particles were microinjected in the vicinity of incipient cartilaginous humerus, radius, and/or ulna in wing buds of day 4 -5 chick embryos in ovo; contralateral wing buds served as control (Yagami et al, 1999). Embryos were re-incubated for 2 to 3 days, injected with BrdU 2 hr before harvesting, and processed for detection of incorporated BrdU and syndecan-3 gene expression.…”

“…Fibroblasts were grown for a total of approximately 10 days with two passages at which point 100% of the cells were infected. Recombinant viral particles present in the medium were concentrated by ultracentrifugation at 25,000 rpm for 3 hr and used to infect freshly isolated chondrocytes (Yagami et al, 1999). Insert-less control viral particles were produced, isolated, and used in the same manner.…”

“…Protocols to achieve virally driven gene misexpression were as described (Yagami et al, 1999). Concentrated syndecan-3-encoding RCAS viral particles prepared as above or a small fragment of confluent fibroblast cell layer producing the virus were microinjected or implanted by microsurgical procedures in the vicinity of incipient cartilaginous humerus, radius, and/or ulna in wing buds of day 4.5-5 chick embryos in ovo; contralateral wing buds served as controls.…”

“…These procedures have all been detailed in previous studies (Koyama et al, 1995;Nakamura et al, 1997;Yagami et al, 1999). Briefly, RNA was isolated from chondrocyte cultures with TRIZOL (Life Technologies, Inc.) following manufacturer's protocol.…”

“…After staining with hematoxylin and eosin, slides were analyzed with a Nikon microscope by using brightand darkfield optics. The cDNA clones encoding portions of chick syndecan-3, Ihh, type X collagen, Patched and osteopontin were described previously (Koyama et al, 1995(Koyama et al, , 1996aYagami et al, 1999).…”