Lutropin (LH), a pituitary glycoprotein hormone, plays a critical role in gonadal development and function, and regulates the production of sex steroids such as estradiol (E2). LH consists of two subunits, the alpha subunit common to all glycoprotein hormones, and the unique LHBeta subunit which is under more stringent physiological regulation. LH production in vivo is under both positive and negative regulation by E2, but it is unclear whether this feedback occurs at the hypothalamic or pituitary level, or both. We have shown that the transcription rate of the kLHBeta gene is negatively regulated by E2 in vivo, but preliminary experiments indicate E2 stimulation of transcription in pituitary fragments and in transient expression assays.
The first aim i s to define the direct actions of E2 on the LHBeta gene by measuring E2- regulated transcription free of hypothalamic influences. The time and dose dependence of the E2 response will be assessed in isolated pituitary fragments and in cell culture in the absence and presence of gonadotropin releasing hormone (GnRH).
The second aim i s to identify regions of the LHBeta gene which confer an E2 response. Filter-binding assays with labeled gene fragments and purified E2 receptor complex indicate at least one putative receptor binding site in the 5'-flanking region of the LHBeta gene. This region and other potential receptor binding sites will be studied by Exonuclease III and DNAse I footprinting studies, as well as by mutational analysis, to identify the sequence for receptor binding. Gene regions necessary for the biological E2 response will be identified by transient expression assays, in which upstream LHBeta gene regions inserted next to a thymidine kinase or prolactin promoter controlling expression of a reporter gene are transfected into GH3 cells or normal pituitary cells. It is anticipated that the receptor binding region will be required for the response, but other DNA elements and binding proteins may be necessary, and will be identified. The ability of specific gene regions to confer the E2 response to the reporter gene, and the sequence and positioning of these elements will be determined by deletion analysis. The importance of specific nucleotides within these sequences will be evaluated by site-directed mutagenesis. The ability of other hormones to modulate the E2 response will be tested. The interaction of the antiestrogen tamoxifen with LHBeta gene E2 regulatory regions will be measured by tamoxifen-E2 receptor footprint analysis, and tamoxifen and progesterone modulation of the E2 effects will be investigated by transcription, and in transient expression assays. Reporter gene constructs containing the homologous LHBeta gene promoter and 5'flanking regions will be transfected into pituitary cells and tested for expression and E2 regulation in the absence or presence of GnRH. These studies will add to our understanding of the direct E2 effects on LH production and clarify some of the complex hormonal regulation in the reproductive process.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD025719-04
Application #
3326910
Study Section
Endocrinology Study Section (END)
Project Start
1989-06-01
Project End
1994-05-31
Budget Start
1992-06-01
Budget End
1993-05-31
Support Year
4
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of Virginia
Department
Type
Schools of Medicine
DUNS #
001910777
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
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Schreihofer, D A; Resnick, E M; Soh, A Y et al. (1999) Transcriptional regulation by a naturally occurring truncated rat estrogen receptor (ER), truncated ER product-1 (TERP-1). Mol Endocrinol 13:320-9
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Weck, J; Fallest, P C; Pitt, L K et al. (1998) Differential gonadotropin-releasing hormone stimulation of rat luteinizing hormone subunit gene transcription by calcium influx and mitogen-activated protein kinase-signaling pathways. Mol Endocrinol 12:451-7
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Rice, L W; Jazaeri, A A; Shupnik, M A (1997) Estrogen receptor mRNA splice variants in pre- and postmenopausal human endometrium and endometrial carcinoma. Gynecol Oncol 65:149-57

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