Molecular Biological and Immunohistochemical Study of Differentiation of the Osteoblast.

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In the studies supported by this grant, the following results were cleared.1. The expression of BGP and MGP genes in the rat osteoblastic cell lines, C11, C20, C23 and C26 was studied by the method of Northern blot hybridization. All the cell lines expressed MGP gene, but did not express BGP gene. After administration of l,25-dihydroxyvitamin D_3, the levels of MGP gene expression was greatly increased except for C26 cells, which are in more immature stage of osteoblastic differentiation than the other cell lines.2. We found that recombinant human bone morphogenetic protein-2 (BMP-2) stimulates osteoblastic maturation of C26 cells in the collaboration study with Genetics Institute Inc.3. The mouse and rat MGP cDNAs were cloned from the cDNA libraries constructed from mouse osteoblastic cell line MC3T3-El, and the cell line C20, respectively. As a result of the nucleotide sequencing, the cloned mouse MGP cDNAs were found that one of the five glutamic acid residues potentially modified to Gla not conserved.4. To study differentiation of osteoblast in the bone tissue, wo developed the new method for in situ hybridization of non-decalcified bone tissue. Using this method, we studied the expression of BGP and MGP genes in the femur and lumbar of adukt rats. BGP gene was strongly expressed in cuboidal osteoblasts along the mineralized bone traveculars. On the other hand. MGP gene was expressed strongly in hypertrophic chondrocytes. From these results, the expression of BGP and MGP genes in immature bone tissue before mineralization was thought to be different from the expression in mature bone tissue.