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*

Abstract

GnRH plays a pivotal role in regulating human reproductive functions. This hypothalamic peptide interacts with its receptor (GnRHR) on the pituitary gonadotropes to trigger the secretion of gonadotropins, which, in turn, regulates the release of sex steroids from the gonads. In light of the importance of GnRHR, the molecular mechanisms underlying the transcriptional regulation of the human GnRHR (hGnRHR) gene become a key issue in understanding human reproduction. In this report, the possible involvement of steriodogenic factor-1 (SF-1) as a key cell-specific regulator for hGnRHR gene expression was examined. By the transient luciferase reporter gene assays, the wild-type promoter, containing 2.3 kb ofthe hGnRHR gene 5'-flanking region relative to the ATG codon, was able to drive a 3.6 +/- 0.2-fold (P < 0.05) increase in luciferase activity in the mouse alphaT3-1 gonadotropes. Subsequent deletion analysis indicated that the most proximal 173 bp within the first exon of the gene, although not a promoter itself, contains a critical regulatory element(s) essential for the basal expression of the hGnRHR gene. The functional roles of the putative gonadotrope-specific elements (GSE; consensus 5'-CTG(A)/(T)CCTTG-3') residing at positions -5, -134, and -396 were studied by site-directed mutagenesis, and it was found that only the mutation at position -134 significantly reduced the promoter activity (80% reduction; P < 0.05). The attenuation effect of this GSE mutant was cell specific, as it was restricted to alphaT3-1 cells, but not to COS-7 and human ovarian adenocarcinoma (SKOV-3) cells. Competitive mobility shift assays using either alphaT3-1 nuclear extract or recombinant SF-1 protein clearly indicated that SF-1 is able to interact specifically with this GSE element positioned at -134. Using a SF-1 antibody that completely abrogated complex formation in the gel shift assays, the involvement of endogenous nuclear SF-1 was further evidenced. By competitive gel shift assays using oligoprimers with 2-bp scanning mutations, the sequences essential for the interaction with SF-1 were identified (5'-TTG(A)/(T)CCCTG-3', underlined sequences were important). 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 alphaT3-1, SKOV-3, and COS-7 cells. Overexpression of the SF-1 mRNA was able to enhance promoter activities in all of the cells tested. On the contrary, expression of the antisense SF-1 mRNA reduced the hGnRHR promoter activity only in alphaT3-1 cells, not in COS-7 or SKOV-3 cells. In summary, the data reported here provide conclusive evidence that SF-1 interacts with the GSE motif at position -134 within the first exon of the hGnRHR gene to mediate its cell-specific expression.

The Promoter of the Rat Gonadotropin-Releasing Hormone Receptor Gene Directs the Expression of the Human Placental Alkaline Phosphatase Reporter Gene in Gonadotrope Cells in the Anterior Pituitary Gland as well as in Multiple Extrapituitary Tissues

“…In addition, some SF-1 positive cells at E15.5 and E18 do not express the transgene. This contrasts with data derived from transient transfection studies, which have demonstrated a pivotal role for SF-1 in mouse, rat, and human GnRH-R promoter activity (9,11,12,40). Our in vivo data indicate that signals other than SF-1 are necessary for turning on the GnRH-R gene.…”

Pituitary Adenylate Cyclase-activating Polypeptide and Cyclic Adenosine 3′,5′-Monophosphate Stimulate the Promoter Activity of the Rat Gonadotropin-releasing Hormone Receptor Gene via a Bipartite Response Element in Gonadotrope-derived Cells

“…Similarly, the interaction of SF-1 with a gonadotrope-specific element motif in the human gene has been shown to mediate gonadotrope-specific expression (10). Regarding the rat promoter, we and others have reported that full gonadotrope specific activity required a distal regulatory domain in addition to the SF-1 and AP-1 elements present in the proximal domain (7,8,11).…”

Wide spectrum of NR5A1‐related phenotypes in 46,XY and 46,XX individuals

“…NR5A1 is expressed in steroidogenic tissues (Honda et al, 1993; Ikeda et al, 1993; Morohashi et al, 1994; Parker and Schimmer, 1997; Ramayya et al, 1997; Morohashi, 1999), pituitary gonadotrophs (Barnhart and Mellon, 1994; Ingraham et al, 1994; Ngan et al, 1999), and neurons located at the dorsomedial portion of the ventromedial hypothalamus (VMH) (Ramayya et al, 1997; Morohashi et al, 1999). In developing embryos, Nr5a1 is expressed in the urogenital ridge, representing the first marker of gonadal and adrenal differentiation (Ikeda et al, 1994; Morohashi et al, 1995, 1999; Ramayya et al, 1997; Hanley et al, 1999, 2001).…”