The overall goal of this project is to elucidate and characterize pivotal events in the differentiation of osteoblasts from mesenchymal stem cells. A model is developed which stresses the importance of 1,25-dihydroxyvitamin D3 (vitamin D), ascorbic acid. and the extracellular matrix in osteoblast formation. It is proposed that early commitment steps to the osteoblast lineage are controlled by vitamin D and bone-derived growth factors,. while expression of osteoblast marker proteins requires ascorbic aciddependent synthesis and accumulation of a Type I collagen containing matrix. Among the early changes in gene expression induced by vitamin D are a selective increase in Type I vs Type III collagen synthesis and increased synthesis of fibronectin and integrins. Interaction of collagen with cells via specific integrins is viewed as being crucial for expression of the osteoblast phenotype. The first specific aim will determine the mechanism used by vitamin D to regulate fibronectin, collagen and integrin synthesis. Experiments will identify vitamin D regulated integrins and determine whether this hormone stimulates transcriptional activity. If evidence for. transcriptional control is obtained, studies will determine whether actions of vitamin D are direct or require the synthesis of a protein intermediate and test for the presence of functional vitamin D regulated cis acting sequences in 5'regulatory regions of the alpha2(I) collagen gene.
The second aim i s to define the role of Type I collagen synthesis and cell:collagen interactions in expression of the osteoblast phenotype in committed osteoblast precursors. Studies will determine whether effects of ascorbic acid on osteoblast marker expression require synthesis of a Type I collagen-containing matrix. If so, the role of integrin mediated cell:matrix interactions in osteoblast marker expression will be defined.
In aim three, the mechanism used by ascorbic acid to stimulate the gene expression of collagen and osteoblast markers will be studied. If evidence for transcriptional control is obtained, the mechanism of this regulation will be explored using plasmids containing. 5' regulatory regions of the alpha2(I) collagen and osteocalcin genes. This project will increase our understanding of factors regulating osteogenesis knowledge which is necessary for the eventual treatment of degenerative bone disorders such as periodontal disease, osteoporosis and non-union fractures.
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