The long-range objective of this application is to determine cellular and molecular mechanisms responsible for aberrant aromatase P450 (P450arom) expression in uterine leiomyomata. The pathologic significance of this application is underscored by the fact that estrogen is essential for the growth of leiomyomata. P450arom is the key enzyme for estrogen biosynthesis in a number of human tissues. We demonstrated local estrogen biosynthesis via P450arom expression in tissue samples and cultured leiomyoma smooth muscle (LSM) cells but not in normal myometrial tissues or cells. We hypothesize that aberrant expression of stimulatory transcription factors and down regulation of inhibitory transcription factors in LSM cells are critical mechanisms for PGE2-dependent induction of P450arom promoter II activity, mRNA levels and enzyme activity in these cells. The clinical relevance of these findings was recently emphasized by the successful treatment of a leiomyoma in a menopausal woman with an aromatase inhibitor. We propose the following aims: To determine the molecular mechanisms responsible for PGE2-dependent regulation of P450arom enzyme, protein and mRNA levels in LSM cells. We will test the hypothesis that PGE2 acts via EP1 and EP2 receptor subtypes to stimulate both PKC and PKA pathways, which induce P450arom expression. To define the DNA motifs that mediate PGE2-dependent activation of P450arom promoter II in LSM cells. We hypothesize that an LRH-l-binding site, a cAMP response element and a C/EBP-binding site mediate PGE2- dependent promoter activation. To determine the roles of transcription factors in aberrant P450arom expression in LSM cells. The effects of over expression and reduction of co-regulators of LRH-1 on P450arom promoter will be studied. In vivo spatial and temporal association between these transcription factors and P450arom in leiomyoma samples will be determined. To define the basic composition of the multimeric complex that occupies P450arom promoter II and enhances its activity in response to PGE2. We will employ immunoprecipitation-PCR to determine the association of LRH-1, its co-regulators, a C/EBP isoform and p300/CBP showing histone acetyl transferase activity with P450arom promoter in response to PGE2. Protein-protein interactions will be demonstrated by immunoprecipitation-western analyses. We predict that these studies will establish the critical role of P450arom in uterine leiomyomata and lead to identification of novel therapeutic targets.
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