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Estradiol Sensitization of Rat Pituitary Cells to Gonadotropin-Releasing Hormone: Involvement of Protein Kinase C- and Calcium-Dependent Signaling Pathways¹

Division of Endocrinology, Metabolism, and Molecular Medicine, Northwestern University Medical School, Chicago, Illinois 60611

Address all correspondence and requests for reprints to: J. Larry Jameson, M.D., Ph.D., Division of Endocrinology, Metabolism, and Molecular Medicine, Northwestern University Medical School, Tarry 15-709, 303 East Chicago Avenue, Chicago, Illinois 60611-3008. E-mail: ljameson{at}nwu.edu

During the female reproductive cycle, estrogen enhances the actions of GnRH on the gonadotrope cell. Recently, we reported that in vivo exposure to estradiol causes a marked enhancement GnRH-induced transcription of the gene promoter in primary cultures of pituitary cells. In the present study, we analyzed the GnRH signaling pathways that mediate the sensitizing effects of estradiol on the promoter. Primary cultures of male and female rat pituitary cells were transfected with the -420LUC reporter gene and treated with agonists or antagonists for 24 h. As found previously, the degree of GnRH (1 nM) stimulation was 15-fold greater in females (157-fold) than in males (9-fold). When cells were treated with phorbol esters [phorbol 12-myristate 13-acetate (PMA); 10 nM], the level of stimulation was half that observed with GnRH, but the sexual dimorphism was preserved. When protein kinase C (PKC) activity was either depleted by long term treatment with phorbol esters (1 µM PMA for 24 h) or inhibited with staurosporine, the stimulatory effect of GnRH was minimally affected in males, but was markedly reduced in females. The reduced threshold of GnRH responsiveness after inhibition of PKC suggests that the actions of estrogen involve this pathway. Coexpression of c-jun and c-fos, which are increased by GnRH and PMA, suppressed basal LUC activity, but did not alter the sensitivity to GnRH in a sexually dimorphic manner. Dominant negative mutants of the mitogen-activated protein kinase pathway, which is also activated by GnRH and PMA, failed to reveal sexually dimorphic alterations in GnRH responsiveness. These findings indicate that the mitogen-activated protein kinase pathway and activating protein-1 are probably not involved in estrogen sensitization of transcriptional responses to GnRH. The involvement of Ca²⁺-dependent pathways was analyzed either by chelating extracellular Ca²⁺ with EGTA (5 mM) or by using a Ca²⁺ channel blocker, methoxyverapamil (D600; 1 µM). Depletion of extracellular Ca²⁺ markedly reduced GnRH action in females, but not in males. Treatment with the Ca²⁺ channel blocker D600 did not alter GnRH-induced stimulation of -420LUC in males, but in females, GnRH stimulation was significantly impaired (208- vs. 23-fold). Estrogen replacement in ovariectomized females reconstituted GnRH sensitivity and the inhibitory effect of methoxyverapamil (84- vs. 13-fold). We conclude that both PKC- and Ca²⁺-dependent signaling pathways are involved in estradiol-induced sensitization of female pituitary cells to GnRH.

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