Dehydroepiandrosterone and its sulfate (DHEA(S)) show a cycle of large changes in secretion from the adrenal cortex over the life span in humans. The secretion of these steroids is very high in the fetus, low in childhood, high again in young adulthood, followed by a progressive age- related decline. Dependent on age, 40-100% of local tissue androgens and estrogens in both men and women are derived from DHEAS. The critical regulatory point in the biosynthesis of DHEAS is 3beta- hydroxysteroid/delta4 '5-isomerase (3beta-HSD). The low level of activity of this enzyme in the human adrenal cortex, but not in animal glands such as that of the cow, limits the flux of pregnenolone to A steroids, such as cortisol, and maintains the synthesis of delta5 steroids, principally DHEA(S). The proposed experiments will analyze the differences between the type Il human 3beta-HSD gene, which is expressed in the adrenal cortex and other steroidogenic tissues, and the 3beta-HSD gene expressed in the bovine adrenal. Transcription rates and mRNA stability will be assessed as possible causes of the differences in 3beta-HSD expression between the bovine and human adrenal cortex, and between the different zones of the human adrenal cortex. The bovine 3beta-HSD gene (or genes) expressed in the adrenal cortex will be isolated, its structure analyzed, and the tissue distribution of its transcript determined. The difference in expression levels between the human type Il and the bovine gene will be tested using reporter constructs from the two genes transfected into primary human and bovine adrenal cells. The elements in the type II human and the bovine 3beta-HSD genes responsible for tissue-specific expression and second messenger regulation, and nuclear proteins from the adrenal cortex that bind to these elements, will be characterized. The possible identity of such proteins to known transcription factors will be tested. Cell culture experiments will examine whether the cycle of DHEAS synthesis over the life span in humans results from intrinsic changes in expression of 3beta-HSD. The abundance in adrenocortical tissue of nuclear proteins that bind to the regulatory elements of the type II 3beta-HSD gene will be correlated with the cycle. These experiments will elucidate the molecular basis for the regulation of the human 3beta-HSD type II gene and thereby the regulation of DHEAS synthesis. This information will provide a basis for understanding the significance of the unique secretion of this hormone in humans and the significance of its age-related decline for aging and age-related diseases.
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