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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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RT-PCR and immunocytological analyses of chondrocyte cultures. Freshly isolated cephalic sternal chondrocytes infected with control viral particles (RCAS) or viral particles encoding MGP (RCAS-MGP) were grown for ∼1 wk in medium A. Cultures were processed for RT-PCR analysis of MGP (A and B) and β-actin (C and D) expression or for immunocytochemical determination of virally infected cells (E and F), using an antibody to the viral protein p19. Note that infected cells are dark/black, whereas uninfected cells are light gray.
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Figure 7: RT-PCR and immunocytological analyses of chondrocyte cultures. Freshly isolated cephalic sternal chondrocytes infected with control viral particles (RCAS) or viral particles encoding MGP (RCAS-MGP) were grown for ∼1 wk in medium A. Cultures were processed for RT-PCR analysis of MGP (A and B) and β-actin (C and D) expression or for immunocytochemical determination of virally infected cells (E and F), using an antibody to the viral protein p19. Note that infected cells are dark/black, whereas uninfected cells are light gray.

Mentions: We asked next whether the amount of MGP expressed by hypertrophic chondrocytes is a factor influencing the mineralization potential of the cells. To address this question, we constructed an RCAS-based expression vector (Hughes et al. 1987) encoding full-length chick MGP and used the resulting viral particles (RCAS-MGP) to infect freshly isolated hypertrophic chondrocyte populations. As a control, companion cells were infected with insertless RCAS viral particles. Cultures were grown for ∼1 wk until confluent, and were processed for RT-PCR to estimate the levels of MGP gene expression and for immunocytochemistry to determine the percentage of virally infected cells in control and RCAS-MGP cultures, using an antibody to the viral structural protein p19. We found that MGP gene expression was significantly higher in RCAS-MGP cultures (expressing both endogenous and virally encoded MGP) (Fig. 7 B) than in control cultures (expressing endogenous MGP only) (Fig. 7 A). Housekeeping actin gene expression was identical in both cell populations (Fig. 7C and Fig. D). Immunocytochemistry revealed that ∼60% of the cells in both control and RCAS-MGP cultures were virally infected (Fig. 7E and Fig. F, respectively).


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)

RT-PCR and immunocytological analyses of chondrocyte cultures. Freshly isolated cephalic sternal chondrocytes infected with control viral particles (RCAS) or viral particles encoding MGP (RCAS-MGP) were grown for ∼1 wk in medium A. Cultures were processed for RT-PCR analysis of MGP (A and B) and β-actin (C and D) expression or for immunocytochemical determination of virally infected cells (E and F), using an antibody to the viral protein p19. Note that infected cells are dark/black, whereas uninfected cells are light gray.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169349&req=5

Figure 7: RT-PCR and immunocytological analyses of chondrocyte cultures. Freshly isolated cephalic sternal chondrocytes infected with control viral particles (RCAS) or viral particles encoding MGP (RCAS-MGP) were grown for ∼1 wk in medium A. Cultures were processed for RT-PCR analysis of MGP (A and B) and β-actin (C and D) expression or for immunocytochemical determination of virally infected cells (E and F), using an antibody to the viral protein p19. Note that infected cells are dark/black, whereas uninfected cells are light gray.
Mentions: We asked next whether the amount of MGP expressed by hypertrophic chondrocytes is a factor influencing the mineralization potential of the cells. To address this question, we constructed an RCAS-based expression vector (Hughes et al. 1987) encoding full-length chick MGP and used the resulting viral particles (RCAS-MGP) to infect freshly isolated hypertrophic chondrocyte populations. As a control, companion cells were infected with insertless RCAS viral particles. Cultures were grown for ∼1 wk until confluent, and were processed for RT-PCR to estimate the levels of MGP gene expression and for immunocytochemistry to determine the percentage of virally infected cells in control and RCAS-MGP cultures, using an antibody to the viral structural protein p19. We found that MGP gene expression was significantly higher in RCAS-MGP cultures (expressing both endogenous and virally encoded MGP) (Fig. 7 B) than in control cultures (expressing endogenous MGP only) (Fig. 7 A). Housekeeping actin gene expression was identical in both cell populations (Fig. 7C and Fig. D). Immunocytochemistry revealed that ∼60% of the cells in both control and RCAS-MGP cultures were virally infected (Fig. 7E and Fig. F, respectively).

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