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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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Scanning electron micrographs of chondrocyte cultures (A and B) and x-ray element microanalysis of mineral present (C and D). Confluent hypertrophic cultures in medium B were treated with 10 μM warfarin for 4 d (B and D) or were left untreated (A and C), and were then processed for SEM and x-ray analyses. Note the numerous mineral crystal deposits present in both cultures (A and B). Inset in B is a higher magnification view of one such deposit revealing its multibead structure. X-ray analysis of crystals (C and D) shows that they contain prominent phosphorus (P) and calcium (Ca) peaks in ratios typical of biologic apatite. The position of sulfur (S) and iron (Fe) is indicated.
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Figure 5: Scanning electron micrographs of chondrocyte cultures (A and B) and x-ray element microanalysis of mineral present (C and D). Confluent hypertrophic cultures in medium B were treated with 10 μM warfarin for 4 d (B and D) or were left untreated (A and C), and were then processed for SEM and x-ray analyses. Note the numerous mineral crystal deposits present in both cultures (A and B). Inset in B is a higher magnification view of one such deposit revealing its multibead structure. X-ray analysis of crystals (C and D) shows that they contain prominent phosphorus (P) and calcium (Ca) peaks in ratios typical of biologic apatite. The position of sulfur (S) and iron (Fe) is indicated.

Mentions: We determined next whether MGP regulates only the quantity of mineral deposited by cells in their matrix or may also affect mineral nature and overall quality. Thus, we compared the mineral deposited by control and warfarin-treated hypertrophic chondrocytes by SEM, x-ray microanalysis, and FT-IR spectroscopy. Cells used for these experiments were grown in medium B because they formed more mineral than cells in medium A (Fig. 1). When viewed by SEM, many crystals were observed on, and to be associated with, the cell surface in both control and warfarin-treated cells (Fig. 5A and Fig. B). When examined at a higher magnification, each crystal displayed a typical multibead organization, with beads averaging 200–400 nm in diameter (Fig. 5 B, inset). After SEM, multiple microscopic fields were subjected to x-ray microanalysis. Prominent phosphorus and calcium peaks were produced by crystals in both cultures (Fig. 5C and Fig. D). Integration of the peaks showed that the calcium/phosphorus molar ratio was ∼1.3–1.4, thus, resembling that of apatite in hypertrophic cartilage (Boyde and Shapiro 1980; Boskey et al. 1992).


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)

Scanning electron micrographs of chondrocyte cultures (A and B) and x-ray element microanalysis of mineral present (C and D). Confluent hypertrophic cultures in medium B were treated with 10 μM warfarin for 4 d (B and D) or were left untreated (A and C), and were then processed for SEM and x-ray analyses. Note the numerous mineral crystal deposits present in both cultures (A and B). Inset in B is a higher magnification view of one such deposit revealing its multibead structure. X-ray analysis of crystals (C and D) shows that they contain prominent phosphorus (P) and calcium (Ca) peaks in ratios typical of biologic apatite. The position of sulfur (S) and iron (Fe) is indicated.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Scanning electron micrographs of chondrocyte cultures (A and B) and x-ray element microanalysis of mineral present (C and D). Confluent hypertrophic cultures in medium B were treated with 10 μM warfarin for 4 d (B and D) or were left untreated (A and C), and were then processed for SEM and x-ray analyses. Note the numerous mineral crystal deposits present in both cultures (A and B). Inset in B is a higher magnification view of one such deposit revealing its multibead structure. X-ray analysis of crystals (C and D) shows that they contain prominent phosphorus (P) and calcium (Ca) peaks in ratios typical of biologic apatite. The position of sulfur (S) and iron (Fe) is indicated.
Mentions: We determined next whether MGP regulates only the quantity of mineral deposited by cells in their matrix or may also affect mineral nature and overall quality. Thus, we compared the mineral deposited by control and warfarin-treated hypertrophic chondrocytes by SEM, x-ray microanalysis, and FT-IR spectroscopy. Cells used for these experiments were grown in medium B because they formed more mineral than cells in medium A (Fig. 1). When viewed by SEM, many crystals were observed on, and to be associated with, the cell surface in both control and warfarin-treated cells (Fig. 5A and Fig. B). When examined at a higher magnification, each crystal displayed a typical multibead organization, with beads averaging 200–400 nm in diameter (Fig. 5 B, inset). After SEM, multiple microscopic fields were subjected to x-ray microanalysis. Prominent phosphorus and calcium peaks were produced by crystals in both cultures (Fig. 5C and Fig. D). Integration of the peaks showed that the calcium/phosphorus molar ratio was ∼1.3–1.4, thus, resembling that of apatite in hypertrophic cartilage (Boyde and Shapiro 1980; Boskey et al. 1992).

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