title: Regulation of human gonadotropin-releasing hormone receptor gene expression in placental cells.
abstract: GnRH has been suggested to regulate hCG secretion in the placenta. In the present study, we report isolation of full-length GnRH receptor (GnRHR) complementary DNA from human placental cells, including a choriocarcinoma cell line (JEG-3), immortalized extravillous trophoblasts (IEVT), and first trimester cytotrophoblast cells in primary culture. Sequence analysis of the placental GnRHR complementary DNA revealed a 100% similarity to its pituitary counterpart. Northern blot analysis using polyadenylated RNA isolated from JEG-3 and IEVT cells revealed a 2.5- and 1.2-kb GnRHR transcripts. Using semiquantitative RT-PCR, regulation ofplacental GnRHR gene expression was examined. In contrast to pituitary gonadotrope alphaT3-1 cells, down-regulation of GnRHR messenger RNA (mRNA) levels was not observed in placental cells after 24 h of 0.1-microM GnRH agonist (GnRHa) treatment. Instead, a 43% (P < 0.01) and 30% (P < 0.05) increase in GnRHR mRNA levels was observed in JEG-3 and IEVT cells, respectively. In addition, 10 microM phorbol ester or forskolin treatments resulted in a significant increase in GnRHR expression in both JEG-3 and IEVT cells. The GnRHa-induced increase in GnRHR expression was shown to be a receptor-mediated process, as cotreatment of GnRH antagonist abolished the effect. It has also been demonstrated that these stimulatory effects on GnRHR gene expression were regulated at least in part at the transcriptional level. Pretreatment of JEG-3 cells with a specific protein kinase C inhibitor (GF109203X), adenylate cyclase inhibitor (SQ22536), or protein kinase A inhibitor [PKI-(14-22) amide, myristylated] reversed GnRHa-induced GnRHR gene expression, suggesting that the placental GnRHR couples to the protein kinase C (PKC) and cAMP/ protein kinase A (PKA) pathways. By Northern blot analysis, we observed a 100% (P < 0.001) increase in hCGbeta mRNA levels after 0.1 microM GnRHa treatment in JEG-3 cells. Again, this effect was prevented in the presence of either protein kinase C inhibitor or adenylate cyclase inhibitor, further supporting the role of the PKC and PKA pathways in GnRHR-coupled signaling in placental cells. In summary, these data strongly support the idea that 1) GnRH plays an autocrine/paracrine role in regulating placental function through a receptor-mediated mechanism; and 2) the placental GnRHR couples to both the PKC and PKA pathways.
authors.affiliation: Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, Canada.
authors.forename: K W
authors.initials: KW
authors.lastname: Cheng
authors.forename: P S
authors.initials: PS
authors.lastname: Nathwani
authors.forename: P C
authors.initials: PC
authors.lastname: Leung
mesh_terms: D002478:Cells, Cultured; D018997:Chorionic Gonadotropin, beta Subunit, Human / Q000235:genetics; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D005786:Gene Expression Regulation / Q000502:physiology*; D007987:Gonadotropin-Releasing Hormone / Q000494:pharmacology / Q000502:physiology; D006801:Humans; D010920:Placenta / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D011247:Pregnancy; D012333:RNA, Messenger / Q000378:metabolism; D011966:Receptors, LHRH / Q000235:genetics*; D015398:Signal Transduction / Q000502:physiology; D014158:Transcription, Genetic / Q000502:physiology; D014327:Trophoblasts / Q000166:cytology / Q000378:metabolism
chemical_list: D018997:Chorionic Gonadotropin, beta Subunit, Human; D018076:DNA, Complementary; D012333:RNA, Messenger; D011966:Receptors, LHRH; D007987:Gonadotropin-Releasing Hormone