Our goal is to elucidate the mechanisms by which estrogens regulate uterine growth. These are important to understand: (1) the physiological actions of estrogens during the menstrual cycle; (2) the pharmacological actions of the hormone such as replacement therapy during menopause; and (3) the basis for pathological effects such as reproductive dysfunction and endometrial cancer. To produce its effects, the hormone binds to a receptor that interacts with specific nucleotide sequences termed estrogen responsive elements or EREs. These interactions at ERE sites activate target genes. We will study two genes, the nuclear protooncogene c-_fos and the epidermal growth factor (EGF) receptor, that are regulated by estrogens and likely to be involved in uterine growth. The structure of the ERE(s) for growth related genes such as fos and the EGF receptor is unknown. The essence of this project is to precisely determine how estrogens regulate these genes in the physiological setting, and then to determine the ERE(s) and other regulatory sequences that mediate the in vivo uterine responses.
Specific aims are: to define the EREs that activate the selected target genes; to determine the structure-activity relationships for induction of the c-fos and the EGF receptor genes; to determine the mechanisms that limit as well as increase target gene expression in response to estrogens; to determine how other drugs and hormones interact with estrogens to activate target genes and vice versa; to determine if neonatal estrogen exposure, which causes reproductive tract abnormalities, also produces aberrant expression of these target genes; and to determine how selective manipulation of these target genes affects uterine growth. To determine ERE sequences, we will construct hybrid genes containing candidate EREs sequences linked to reporter genes. These hybrid genes win be transfected into cultured cells to determine which sequences confer estrogen responsiveness on reporter genes. This proposal describes parallel sets of studies that: (1) investigate the in vivo regulation of key uterine genes and (2) investigate the regulation of transfected reporter gene constructs that contain regulatory sequences from the same uterine genes. This combined approach will define and characterize the regulatory elements involved more precisely than would be possible from in vivo studies alone, and will assess the physiological significance of the regulatory mechanisms observed more accurately than would be possible from transfection studies alone.
