The proposed studies seek to understand the mechanism(s) involved in steroid regulation of the gap junction connexin43 gene (cx43) expression in human myometrium and uterine leiomyomas. Steroid regulation of cx43 can be used as a paradigm to elucidate differences in the response of leiomyomas to E2 and P4 as compared to normal myometrium. Gap junctions play an important role in the biology of the myometrium. Their formation may be abnormally regulated during premature labor and in uterine leiomyomas which may predispose women to premature labor. Connexin43 (Cx43) gap junctions have been detected through indirect immunofluorescence in clustered areas of uterine leiomyoma tumor tissue, but not in homologous myometrial tissue from non-pregnant women. One hypothesis of this grant is that E2-dependent leiomyomas have abnormal cx43 expression due to altered sensitivity to steroids. Myometrial gap junction formation in animals has been shown to be influenced in part by the ratio of serum levels of progesterone (P4) to 17beta-estradiol (E2), however, the mechanism(s) which leads to the formation of gap junctions in humans is not well understood and the roles of E2 and P4 have not been defined. A second hypothesis is that E2 and P4 modulate cx43 expression through regulating transcription of the cx43 gene and/or half-life of cx43 mRNA. A system for culturing primary human myometrial and leiomyoma cells has been developed. Cx43 protein and cx43 mRNA levels appear to be higher in the cultured leiomyoma cells compared to the homologous myometrial cells. Therefore, the relative extent of cx43 expression in the cell culture system seems to reflect this seen in the tissues. Also cx43 mRNA levels increase if the cultured cells are treated with E2 and that increase is inhibited by P4. The goal of this project is to determine the molecular mechanisms by which E2 and P4 regulate cx43 gene expression in human myometrium and to determine which of these mechanisms are altered in leiomyomas. We plan to: 1) characterize the mechanisms by which E2 and P4 regulate cx43 gene expression in myometrial cells; 2) compare the steroid regulation of the cx43 gene in cultured myometrial and leiomyoma cells with each other and with their respective tissues; 3) define putative sequences in the cx43 gene which may be hormone response elements or, alternatively, in the cx43 mRNA which regulate its half-life in response to E2 and P4.
