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Matrix GLA protein is a developmental regulator of chondrocyte mineralization and, when constitutively expressed, blocks endochondral and intramembranous ossification in the limb.

Yagami K, Suh JY, Enomoto-Iwamoto M, Koyama E, Abrams WR, Shapiro IM, Pacifici M, Iwamoto M - J. Cell Biol. (1999)

Bottom Line: 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.Virally driven MGP overexpression in cultured chondrocytes greatly decreased mineralization.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Surgery, Showa University, Dental School, Ohta-Ku, Tokyo 145, Japan.

ABSTRACT
Matrix GLA protein (MGP), a gamma-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 gamma-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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Histological analyses of the effects of MGP overexpression on limb long bone development. In the control limb, femur (fe), tibia (ti), and tarsal (ta) elements display normal elongated morphology (A). Their diaphyses stain positively with alizarin red (A), are surrounded by an intramembranous bone collar (C and E, arrow), and display fully hypertrophic chondrocytes (E, arrowhead) and invading marrow cells (E, double arrow). In RCAS-MGP limb, tibia is completely cartilaginous and does not stain with alizarin red (B); its diaphysis lacks both a bone collar (D, arrow) and fully hypertrophic chondrocytes (F, arrowheads). Bars (A and B) 2.5 mm; (C–F) 80 μm.
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Figure 9: Histological analyses of the effects of MGP overexpression on limb long bone development. In the control limb, femur (fe), tibia (ti), and tarsal (ta) elements display normal elongated morphology (A). Their diaphyses stain positively with alizarin red (A), are surrounded by an intramembranous bone collar (C and E, arrow), and display fully hypertrophic chondrocytes (E, arrowhead) and invading marrow cells (E, double arrow). In RCAS-MGP limb, tibia is completely cartilaginous and does not stain with alizarin red (B); its diaphysis lacks both a bone collar (D, arrow) and fully hypertrophic chondrocytes (F, arrowheads). Bars (A and B) 2.5 mm; (C–F) 80 μm.

Mentions: We asked next whether MGP overexpression in the developing chick limb would have similar inhibitory effects on the mineralization process of long bones. A small pellet of fibroblasts producing RCAS-MGP or control RCAS viruses was implanted in the vicinity of the mesenchymal condensations of tibia and fibula present in stage 22–23 (day 3.5–4.0) chick embryo hindlimb buds. We aimed to infect only these skeletal elements and to use uninfected femur and tarsal elements as an internal control. Embryos were reincubated until day 10–12, and longitudinal sections through the entire legs were prepared and processed for histology, histochemistry, and in situ hybridization. Analysis of alizarin red–stained sections from control day 12 embryo showed that the femur (fe), tibia (ti), and tarsal (ta) elements displayed their typical elongated morphology, and that their alizarin red staining diaphyses were undergoing mineralization and ossification (Fig. 9 A). In contrast, in RCAS-MGP–infected embryos, the tibia failed to stain with alizarin red (Fig. 9 B); unexpectedly, the tibia was also significantly shorter and broader than the control. These changes were limited to the tibia since femur and tarsal elements in the RCAS-MGP leg were normal and stained with alizarin red (Fig. 9 B). Higher magnification analysis of alizarin red–stained or hematoxylin/eosin–stained sections showed that the diaphysis of control tibia contained hypertrophic mineralizing cartilage (Fig. 9 E, arrowhead) and endochondral bone (not shown), was surrounded by a mineralized intramembranous bone collar (Fig. 9C and Fig. E, arrow), and was being invaded by marrow cells (Fig. 9 E, double arrow). In contrast, the diaphysis of RCAS-MGP tibia was entirely cartilaginous (Fig. 9D and Fig. F). There was no evidence of erosion or invasion by marrow cells, and there was a lack of a bone collar (Fig. 9 D, arrow). In addition, the diaphyseal chondrocytes were not fully hypertrophic (Fig. 9 F, arrowheads) and displayed average cell diameters smaller than those in the control (Fig. 9 E).


Matrix GLA protein is a developmental regulator of chondrocyte mineralization and, when constitutively expressed, blocks endochondral and intramembranous ossification in the limb.

Yagami K, Suh JY, Enomoto-Iwamoto M, Koyama E, Abrams WR, Shapiro IM, Pacifici M, Iwamoto M - J. Cell Biol. (1999)

Histological analyses of the effects of MGP overexpression on limb long bone development. In the control limb, femur (fe), tibia (ti), and tarsal (ta) elements display normal elongated morphology (A). Their diaphyses stain positively with alizarin red (A), are surrounded by an intramembranous bone collar (C and E, arrow), and display fully hypertrophic chondrocytes (E, arrowhead) and invading marrow cells (E, double arrow). In RCAS-MGP limb, tibia is completely cartilaginous and does not stain with alizarin red (B); its diaphysis lacks both a bone collar (D, arrow) and fully hypertrophic chondrocytes (F, arrowheads). Bars (A and B) 2.5 mm; (C–F) 80 μm.
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Related In: Results  -  Collection

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Figure 9: Histological analyses of the effects of MGP overexpression on limb long bone development. In the control limb, femur (fe), tibia (ti), and tarsal (ta) elements display normal elongated morphology (A). Their diaphyses stain positively with alizarin red (A), are surrounded by an intramembranous bone collar (C and E, arrow), and display fully hypertrophic chondrocytes (E, arrowhead) and invading marrow cells (E, double arrow). In RCAS-MGP limb, tibia is completely cartilaginous and does not stain with alizarin red (B); its diaphysis lacks both a bone collar (D, arrow) and fully hypertrophic chondrocytes (F, arrowheads). Bars (A and B) 2.5 mm; (C–F) 80 μm.
Mentions: We asked next whether MGP overexpression in the developing chick limb would have similar inhibitory effects on the mineralization process of long bones. A small pellet of fibroblasts producing RCAS-MGP or control RCAS viruses was implanted in the vicinity of the mesenchymal condensations of tibia and fibula present in stage 22–23 (day 3.5–4.0) chick embryo hindlimb buds. We aimed to infect only these skeletal elements and to use uninfected femur and tarsal elements as an internal control. Embryos were reincubated until day 10–12, and longitudinal sections through the entire legs were prepared and processed for histology, histochemistry, and in situ hybridization. Analysis of alizarin red–stained sections from control day 12 embryo showed that the femur (fe), tibia (ti), and tarsal (ta) elements displayed their typical elongated morphology, and that their alizarin red staining diaphyses were undergoing mineralization and ossification (Fig. 9 A). In contrast, in RCAS-MGP–infected embryos, the tibia failed to stain with alizarin red (Fig. 9 B); unexpectedly, the tibia was also significantly shorter and broader than the control. These changes were limited to the tibia since femur and tarsal elements in the RCAS-MGP leg were normal and stained with alizarin red (Fig. 9 B). Higher magnification analysis of alizarin red–stained or hematoxylin/eosin–stained sections showed that the diaphysis of control tibia contained hypertrophic mineralizing cartilage (Fig. 9 E, arrowhead) and endochondral bone (not shown), was surrounded by a mineralized intramembranous bone collar (Fig. 9C and Fig. E, arrow), and was being invaded by marrow cells (Fig. 9 E, double arrow). In contrast, the diaphysis of RCAS-MGP tibia was entirely cartilaginous (Fig. 9D and Fig. F). There was no evidence of erosion or invasion by marrow cells, and there was a lack of a bone collar (Fig. 9 D, arrow). In addition, the diaphyseal chondrocytes were not fully hypertrophic (Fig. 9 F, arrowheads) and displayed average cell diameters smaller than those in the control (Fig. 9 E).

Bottom Line: 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.Virally driven MGP overexpression in cultured chondrocytes greatly decreased mineralization.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Surgery, Showa University, Dental School, Ohta-Ku, Tokyo 145, Japan.

ABSTRACT
Matrix GLA protein (MGP), a gamma-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 gamma-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.

Show MeSH
Related in: MedlinePlus