Estradiol modulates thyroid hormone regulation of the human glycoprotein hormone alpha subunit gene

We have examined mechanisms of regulation of the human glycoprotein hormone alpha subunit gene by thyroid hormone (T3) and estradiol. Pituitary-derived GH3 cells were transiently transfected with chimeric constructs comprising between 1,500 and 98 base pairs of human alpha subunit gene 5'-flanking sequence fused to the bacterial gene encoding chloramphenicol acetyltransferase (h alpha CAT) and treated with T3 and estradiol, alone and in combination. In pituitary cells, 98 base pairs of alpha gene 5'-flanking sequence were sufficient to mediate both inhibition of alpha gene promoter activity by T3 and stimulation by estradiol; inhibition of the alpha promoter by T3 was antagonized by estradiol. Mutation of nucleotides essential for T3 receptor binding to the alpha gene thyroid hormone response element abolished the response of h alpha CAT expression to estradiol as well as T3. In contrast to pituitary GH3 cells, estradiol treatment alone had no effect on expression of either h alpha CAT or the endogenous alpha gene in JEG-3 choriocarcinoma cells cotransfected with a human thyroid hormone receptor expression vector, but estradiol antagonized suppression of both endogenous and transfected alpha promoter activity by T3. Gel mobility shift assays demonstrated specific binding of in vitro synthesized human estrogen receptor (ER) to the alpha gene thyroid hormone response element. These findings suggest that estradiol modulates expression of the human alpha subunit gene in pituitary and choriocarcinoma cells by direct binding of ER to the alpha gene promoter, and that interaction of ER with the alpha gene negative TRE accounts for the antagonistic effects of estradiol and T3.