An analysis of the activity of osteoblasts is central to an understanding of skeletal homeostasis in normal physiology and in disease, since osteoblasts are responsible for synthesizing new bone and for stimulating osteoblasts to resorb bone. The central role of osteoblasts correlates with the wide variety of hormonal and paracrine factors that influence osteoblast activity. 1.25-dihydroxyvitamin D3 [1,25(OH)2D3], a major calcium-regulating hormone, acts directly on osteoblasts to alter expression of the genetic program. An analysis of how 1,25(OH)2D3 modulates osteoblast gene expression will help clarify how the osteoblast's varying roles are regulated and will serve as a useful model of how steroid hormone receptors interact with other regulatory proteins to determine a cell's phenotype. The effects of 2 1,25(OH)2D3 on the rat osteocalcin gene will be analyzed in detail. This gene is expressed virtually only In osteoblasts; osteocalcin gene transcription is stimulated by 1,25(OH)2D3 in cultured osteoblast-like cells. Transfection of cloned genes will be used to define DNA sequences necessary and sufficient to confer responsiveness to 1,25(OH)2D3 to the osteocalcin gene. DNA binding studies, using cell extracts, purified receptors, and genetically produced receptors, will be performed to define DNA sequences that bind the 1,25(OH)2D3 receptor, those that bind other factors required for osteocalcin gene expression, and to define the interactions between the relevant binding proteins. The possibility that other hormones and cellular messengers (thyroxine, retinoic acid, estrogen, gluccorticoids, factors activated by protein kinase C) affect osteocalcin gene transcription will be systematically evaluated. Possible additive, synergistic, and antagonistic effects will be analyzed at the levels of specific DNA binding and activation of transcription. The interaction of hormone-responsive transcription factors and bone-specific transcription factors will similarly be analyzed. The structure for the osteocalcin gene in intact chromatin will be evaluated to define regulatory regions that are hypersensitive to DNAse I. These studies will provide a link between studied of cloned genes and of genes in situ. This project's goal is thus to build a base for a comprehensive understanding of how 1,25(OH)2D3 and other hormones modulate the osteoblast genetic program.
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