The goals of this proposal are to understand the molecular basis for the regulation of steroid hormone biosynthesis in the ovary, and in particular for the unique episodic pattern of steroid hormone secretion that is characteristic of the ovarian cycle. This necessitates identifying the elements in the genes encoding steroidogenic enzymes which are involved in this regulation, as well as the factors which interact with these genomic elements to mediate these actions. It also necessitates examining alterations in the structure of chromatin which may underlie changes in the expression of specific genes, and the mechanisms responsible for such changes. Recent studies in this and other laboratories on the regulation of expression of the genes encoding cholesterol side-chain cleavage cytochrome P450 (CYP11A); 17alpha-hydroxylase cytochrome P45O (CYP17 and aromatase cytochrome P450 (CYP19) have indicated that cyclic AMP responsiveness of their expression is not directly mediated by the classical CRE, namely TGACGTCA, nor its associated binding protein, CREB. Rather, in the case of the bovine CYP11A gene, an Sp1-like element is involved, whereas in the case of ovarian expression of the bovine CYP17 and the human CYP19 genes, the hexameric SF1/Ad4 binding element, AGGTCA, is implicated. In the present Application, it is proposed to identify the cell-signalling systems involved in the stimulation of expression of these genes in the ovary by cAMP, as well as the mechanisms involved in inhibition by TGF-beta, FGF, and phorbol esters. The roles of Sp1 and the SF1/Ad4 binding protein in mediating cAMP responsiveness of their respective signalling pathways will be determined by examining the action of cAMP to phosphorylate these proteins in ovarian tissues, as well as the role of cAMP in the regulation of the expression of these proteins and their genes. The proteins with which these factors associate will be defined on the basis of these interactions and the ability of the associated proteins to be phosphorylated in response to cAMP will also be characterized. The possible role of nuclear translocation of factors involved in these signalling pathways in the response to cAMP will also be defined. In this fashion, the signalling pathways involved in these unique cAMP-mediated stimulatory mechanisms will be dissected. Similar studies will be conducted to define the mechanisms whereby TGFbeta, FGF and phorbol esters exert differential inhibition of expression of these genes. We will also examine the cell-specific expression of these steroidogenic genes in bovine and human ovary by determining the role of cis-acting elements in such regulation, as well as changes in expression throughout the cycle of the various transcription factors involved. Particular emphasis will be placed on the role of changes in chromatin structure and DNA methylation as mechanisms which regulate cell-specific expression, in particular, the differential changes in expression of the CYP17 and CYP19 genes which occur following ovulation in the human and bovine ovaries.

National Institute of Health (NIH)
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Research Project (R01)
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Biochemical Endocrinology Study Section (BCE)
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University of Texas Sw Medical Center Dallas
Obstetrics & Gynecology
Schools of Medicine
United States
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Liu, Z; Simpson, E R (1999) Molecular mechanism for cooperation between Sp1 and steroidogenic factor-1 (SF-1) to regulate bovine CYP11A gene expression. Mol Cell Endocrinol 153:183-96
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Fisher, C R; Graves, K H; Parlow, A F et al. (1998) Characterization of mice deficient in aromatase (ArKO) because of targeted disruption of the cyp19 gene. Proc Natl Acad Sci U S A 95:6965-70
Liu, Z; Simpson, E R (1997) Steroidogenic factor 1 (SF-1) and SP1 are required for regulation of bovine CYP11A gene expression in bovine luteal cells and adrenal Y1 cells. Mol Endocrinol 11:127-37
Borroni, R; Liu, Z; Simpson, E R et al. (1997) A putative binding site for Sp1 is involved in transcriptional regulation of CYP17 gene expression in bovine ovary. Endocrinology 138:2011-20
Simpson, E R; Michael, M D; Agarwal, V R et al. (1997) Cytochromes P450 11: expression of the CYP19 (aromatase) gene: an unusual case of alternative promoter usage. FASEB J 11:29-36
Hinshelwood, M M; Dodson Michael, M; Sun, T et al. (1997) Regulation of aromatase expression in the ovary and placenta: a comparison between two species. J Steroid Biochem Mol Biol 61:399-405
Simpson, E R; Zhao, Y; Agarwal, V R et al. (1997) Aromatase expression in health and disease. Recent Prog Horm Res 52:185-213; discussion 213-4
Michael, M D; Michael, L F; Simpson, E R (1997) A CRE-like sequence that binds CREB and contributes to cAMP-dependent regulation of the proximal promoter of the human aromatase P450 (CYP19) gene. Mol Cell Endocrinol 134:147-56
Conley, A J; Corbin, C J; Hinshelwood, M M et al. (1996) Functional aromatase expression in porcine adrenal gland and testis. Biol Reprod 54:497-505

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