The goals of this grant are to further investigate the mechanisms by which hormones alter collagen synthesis and gene expression in bone. The first goal of this grant is to study the effects of insulin-like growth factor I, protein kinase C agonists and glucocorticoids on collagen synthesis and steady state levels of procollagen mRNA in 21-day fetal rat calvaria, rat osteoblast-like cells and rat osteosarcoma cells. If changes in procollagen mRNA levels are found, the effect of these hormones on transcription and stability of procollagen mRNA will be measured. A second goal is to use in situ hybridization to examine the effects of glucocorticoids on procollagen mRNA levels in osteoblasts of 21- day fetal rat calvaria. Calvaria will be treated with glucocorticoids for 24 and 96 hours and tissue sections hybridized with RNA or cDNA probes for procollagen mRNA. This study should show whether glucocorticoids inhibit bone collagen synthesis by decreasing procollagen mRNA levels per cell or by decreasing the number of osteoblasts on the bone matrix. A third research goal is to transfect bone cells with recombinant plasmids containing type I collagen gene promoters linked to the chloramphenicol gene to examine the hormonal regulation of promoter activity. Site-directed mutagenesis and enzyme cleavage will be used to produce mutations or deletions in collagen gene promoters. Recombinant plasmids containing altered collagen promoters will be transfected into bone cells and the effect of mutations on hormonal regulation of promoter activity will be determined. A gel-migration assay will be used to study proteins in crude nuclear extracts from hormone-treated bone cells which bind and presumably regulate collagen promoter activity. These studies should give a better understanding of the mechanisms by which hormones regulate type I collagen synthesis in bone and identify sequences within type I collagen gene promoters which are modulated by hormones. Knowledge of the hormonal regulation of bone collagen synthesis and gene expression is primary to understanding the pathologic basis of metabolic bone disease.
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