Steroidogenic factor-1 interacts with a gonadotrope-specific element within the first exon of the human gonadotropin-releasing hormone receptor gene to mediate gonadotrope-specific expression

GnRH plays a pivotal role in regulating human reproductive functions. This hypothalamic peptide interacts with its receptor (GnRHR) on the pituitary g onadotropes to trigger the secretion of gonadotropins, which, in turn, regu lates the release of sex steroids from the gonads. In light of the importan ce of GnRHR, the molecular mechanisms underlying the transcriptional regula tion of the human GnRHR (hGnRHR) gene become a key issue in understanding h uman reproduction. In this report, the possible involvement of steriodogeni c factor-1 (SF-1) as a key cell-specific regulator for hGnRHR gene expressi on was examined. By the transient luciferase reporter gene assays, the wild -type promoter, containing 2.3 kb of the hGnRHR gene 5'-flanking region rel ative to the ATG codon, was able to drive a 3.6 +/- 0.2-fold (P < 0.05) inc rease in luciferase activity in the mouse alpha T3-1 gonadotropes. Subseque nt deletion analysis indicated that the most proximal 173 bp within the fir st exon of the gene, although not a promoter itself, contains a critical re gulatory element(s) essential for the basal expression of the hGnRHR gene. The functional roles of the putative gonadotrope-specific elements (GSE; co nsensus 5'-CTG(A)/(T)CCTTG-3') residing at positions -5, -134, and -396 wer e studied by site-directed mutagenesis, and it was found that only the muta tion at position -134 significantly reduced the promoter activity (80% redu ction; P < 0.05). The attenuation effect of this GSE mutant was cell specif ic, as it was restricted to alpha T3-1 cells, but not to COS-7 and human ov arian adenocarcinoma (SKOV-3) cells. Competitive mobility shift assays usin g either alpha T3-1 nuclear extract or recombinant SF-1 protein clearly ind icated that SF-1 able to interact specifically with this GSE element positi oned at -134. Using a SF-1 antibody that completely abrogated complex forma tion in the gel shift assays, the involvement of endogenous nuclear SF-1 wa s further evidenced. By competitive gel shift assays using oligoprimers wit h 2-bp scanning mutations, the sequences essential for the interaction with SF-1 were identified (5'-TTG(A)/(T)CCCTG-3', underlined sequences were imp ortant). To study the in vivo function of SF-1, vector directing expression of sense or antisense SF-1 messenger RNA (mRNA) was cotransfected with the hGnRHR promoter-luciferase construct into alpha T3-1, SKOV-3, and COS-7 ce lls. Overexpression of the SF-1 mRNA was able to enhance promoter activitie s in all of the cells tested. On the contrary, expression of the antisense SF-1 mRNA reduced the hGnRHR promoter activity only in alpha T3-1 cells, no t in COS-7 or SKOV-3 cells. In summary, the data reported here provide conc lusive evidence that SF-1 interacts with the GSE motif at position -134 wit hin the first exon of the hGnRHR gene to mediate its cell-specific expressi on.