Limits...
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.

Show MeSH

Related in: MedlinePlus

FT-IR analysis of mineral. Confluent hypertrophic chondrocyte cultures were left untreated (A, C, and E) or were treated for 4 d with 10 μM warfarin for 4 d (B, D, and F) in medium B. After treatment, cell layers were processed for mineral harvest and samples were analyzed by FT-IR.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2169349&req=5

Figure 6: FT-IR analysis of mineral. Confluent hypertrophic chondrocyte cultures were left untreated (A, C, and E) or were treated for 4 d with 10 μM warfarin for 4 d (B, D, and F) in medium B. After treatment, cell layers were processed for mineral harvest and samples were analyzed by FT-IR.

Mentions: To examine the mineral composition in greater detail, mineral samples from control and warfarin-treated cultures were examined by FT-IR spectroscopy (Fig. 6). The samples elicited similar second derivative spectra in the v1 v3 phosphate region 1,200–950 cm−1 (Fig. 6A and Fig. B), with clear peaks at 1,160, 1,102, 1,028, 962, and 906 cm−1 that are characteristic of stoichiometric hydroxyapatite. Likewise, similar second derivative spectra were observed in the v4 phosphate domain 650–500 cm−1 (Fig. 6C and Fig. D) with strong peaks at 606 and 565 cm−1 attributable to phosphate ions in apatite, and in the v2 carbonate region 890–850 cm−1 (Fig. 6E and Fig. F). Bands at 880, 875, and 872 cm−1 in the carbonate region indicated that CO3 had at least in part substituted for OH and PO4, and that carbonate apatite was present in the mineralized cultures. Thus, qualitatively similar mineral is deposited by hypertrophic chondrocytes in the absence or presence of warfarin. In both cases, the mineral has morphological, organizational, and biochemical characteristics of apatite crystals displaying relatively low crystallinity, typical of mineralizing cells in culture as well as young bone tissue in vivo (Roufosse et al. 1984; Rey et al. 1991; Iwamoto et al. 1993b; Rey et al. 1995).


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)

FT-IR analysis of mineral. Confluent hypertrophic chondrocyte cultures were left untreated (A, C, and E) or were treated for 4 d with 10 μM warfarin for 4 d (B, D, and F) in medium B. After treatment, cell layers were processed for mineral harvest and samples were analyzed by FT-IR.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: FT-IR analysis of mineral. Confluent hypertrophic chondrocyte cultures were left untreated (A, C, and E) or were treated for 4 d with 10 μM warfarin for 4 d (B, D, and F) in medium B. After treatment, cell layers were processed for mineral harvest and samples were analyzed by FT-IR.
Mentions: To examine the mineral composition in greater detail, mineral samples from control and warfarin-treated cultures were examined by FT-IR spectroscopy (Fig. 6). The samples elicited similar second derivative spectra in the v1 v3 phosphate region 1,200–950 cm−1 (Fig. 6A and Fig. B), with clear peaks at 1,160, 1,102, 1,028, 962, and 906 cm−1 that are characteristic of stoichiometric hydroxyapatite. Likewise, similar second derivative spectra were observed in the v4 phosphate domain 650–500 cm−1 (Fig. 6C and Fig. D) with strong peaks at 606 and 565 cm−1 attributable to phosphate ions in apatite, and in the v2 carbonate region 890–850 cm−1 (Fig. 6E and Fig. F). Bands at 880, 875, and 872 cm−1 in the carbonate region indicated that CO3 had at least in part substituted for OH and PO4, and that carbonate apatite was present in the mineralized cultures. Thus, qualitatively similar mineral is deposited by hypertrophic chondrocytes in the absence or presence of warfarin. In both cases, the mineral has morphological, organizational, and biochemical characteristics of apatite crystals displaying relatively low crystallinity, typical of mineralizing cells in culture as well as young bone tissue in vivo (Roufosse et al. 1984; Rey et al. 1991; Iwamoto et al. 1993b; Rey et al. 1995).

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