Glucocorticoid- induced osteoporosis is a serious bone disorder caused by direct and indirect actions of glucocorticoids in bone tissue. Glucocorticoids inhibit bone formation, but the mechanisms responsible for this effect are poorly understood. Cortisol inhibits type I collagen synthesis at the transcriptional level in cultures of osteoblast-enriched cells from 22 day fetal rat calvariae (Ob cells), probably due to direct actions of cortisol on the alpha-l (I) collagen gene. Some effects of glucocorticoids in bone appear to be secondary to their actions on the synthesis or activity of skeletal growth factors and their binding proteins. The applicant group demonstrated that cortisol inhibits transcription of insulin-like growth factor (IGF)-l in Ob cells, but the gene regulatory elements involved are not known. They also found that, in bone cells, cortisol decreases IGF binding protein (IGFBP)-5 transcription, and IGFBP-5 was consistently reported to increase bone cell growth and enhance the actions of IGF-l. The investigators postulate that the inhibitory actions on IGF-l and IGFBP-5 by cortisol are central to its actions on bone cell metabolism. They have demonstrated that cortisol increases interstitial collagenase transcripts and protease levels in osteoblasts. This effect is secondary to post-transcriptional mechanisms. Changes in collagenase are postulated to be important not only in the regulation of bone matrix degradation by glucocorticoids, but also in the regulation of the IGF/IGFBP-5 axis since collagenase degrades IGFBP-5. The goal of this proposal is to extend these studies and understand the mechanisms involved in the actions of cortisol in bone. The project's Specific Aims are to: 1) study the regulation of the IGF-l gene by cortisol in bone cells, and identify and characterize glucocorticoid-responsive regions of the IGF-l promoter. For this purpose, the investigators obtained appropriate rat IGF-l gene promoter constructs and plan to make mutations and to identify protein binding sites in the IGF I gene; 2) study the regulation of the IGFBP-5 gene by cortisol in Ob cells and identify appropriate gene elements involved in its regulation. For this purpose, necessary IGFBP-5 gene promoter constructs have been obtained, and appropriate mutations are planned. These experiments will be complemented by limited studies of the effects of cortisol on IGFBP-5 degradation in Ob cells; and 3) characterize the actions of cortisol on collagen degradation and interstitial collagenase expression in osteoblasts. For this latter purpose, the applicants plan to study the mechanism involved in collagenase transcript stabilization and study appropriate regions of the rat collagenase gene involved in this process, particularly the 3' untranslated region. The investigations are intended to provide important information on the mechanisms involved in the actions of glucocorticoids in bone and increase our understanding of the inhibitory effects of these steroids on bone formation and skeletal mass.
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