In accordance with the training objectives of the Physician Scientist Award, the candidate and sponsor have constructed a detailed two phase, five year plan designed to provide intensive post-graduate training in the application of molecular biological techniques to the study of tissue-specific and hormonal regulation of steroidogenesis. Phase I of the plan (2 years) involves graduate-level coursework, seminars and laboratory rotations designed to introduce the candidate to the theory and techniques required to accomplish the goals of Phase III. Phase II (three years) will be directly supervised by the primary sponsor and will focus on the regulation of the rat cytochrome P45Ocl7 gene. The enzyme P45Ocl7 is a single protein molecule encoded by a single gene that has two distinct activities, 17alpha hydroxylation and 17,20 lyase activity. In the human and bovine adrenal gland, absence of both activities results in mineralocorticoid production. Presence of 17alpha hydroxylating activity leads to the synthesis of glucocorticoids and presence of both enzyme activities leads to the synthesis of sex steroids. Therefore, P45Oc17 is a key branchpoint enzyme that determines which class of compounds are produced in different steroid-producing tissues. The rat P45Oc17 gene is of interest because in the rat adrenal, neither activity of the enzyme is present and the rat must depend on corticosterone for glucocorticoid activities. Both activities are present in the rat gonads and testosterone and estradiol are produced. While the physiologic regulation of steroidogenesis in the rat has been well characterized, little is known about the differential regulation of the P45Oc17 gene in rat adrenal and gonads. One reason for this lack of information is that, to this point, there is no stable cell line expressing rat P450c 17. To this end, we propose to clone and sequence the rat P450c 17 gene and todevelop constructions utilizing serial deletions from the 5' flanking DNA region of the P450c 17 gene linked to reporter genes to transfect cultured lines of mouse adrenal and Leydig cells. The gene will also be used to transfect primary cultures of human fetal cells. In this manner, we can gain an understanding of the tissue-specific regulation of P45Ocl7 gene and determine which DNA sequences in the 5' flanking region of the gene are important for expression of the enzyme. Once these regions are identified, further experiments will be carried out to study the regulation of expression by binding of specific nuclear proteins to these regulatory regions. Upon attainment of these specific aims, we can expect to have a better understanding of how identical genes in the adrenal and gonads synthesize different classes of hormones.
| Yamamoto, T; Chapman, B M; Johnson, D C et al. (1996) Cytochrome P450 17 alpha-hydroxylase gene expression in differentiating rat trophoblast cells. J Endocrinol 150:161-8 |
| Givens, C R; Zhang, P; Bair, S R et al. (1994) Transcriptional regulation of rat cytochrome P450c17 expression in mouse Leydig MA-10 and adrenal Y-1 cells: identification of a single protein that mediates both basal and cAMP-induced activities. DNA Cell Biol 13:1087-98 |
