Studies in adrenocortical cells have implicated the orphan nuclear receptor SF-1 in the gene regulation of the steroid hydroxylases. Targeted disruption of the Ftz-F1 gene, which encodes SF-1, was used to examine its role in intact mice. Despite normal survival in utero, all Ftz-F1 null animals die by postnatal day 8; these animals lack adrenal glands and gonads and are severely deficient in corticosterone, supporting adrenocortical insufficiency as the probable cause of death. Male and female Ftz-F1 null mice have female internal genitalia, despite complete gonadal agenesis. These studies establish that the Ftz-F1 gene is essential for sexual differentiation and formation of the primary steroidogenic tissues (Luo, 1994).

Associated with these dramatic developmental abnormalities, all Ftz-F1-disrupted mice die in the immediate postnatal period and have very low glucocorticoid levels. Treatment with corticosteroids markedly prolongs survival of the Ftz-F1-disrupted mice, proving that steroid hormone deficiency causes their death. SF-1-specific knockout mice were generated with a targeting construct that specifically disrupts the SF-1 coding sequence without impairing the ELP protein. The phenotype of the SF-1-specific knockout mice is indistinguishable from that observed in Ftz-F1-disrupted mice that lack both SF-1 and ELP. Taken together, these results indicate that SF-1 is the Ftz-F1-encoded protein that is required for multiple aspects of endocrine development and for postnatal survival (Luo, 1995).

While serum levels of corticosterone in SF-1-deficient mice are diminished, levels of adrenocorticotropic hormone (ACTH) are elevated, consistent with intact pituitary corticotrophs. Intrauterine survival of SF-1-deficient mice appears normal, and they have normal serum level of corticosterone and ACTH, probably reflecting transplacental passage of maternal steroids. SF-1 was examined for whether it is required for P450 side-chain-cleavage enzyme (P450scc) expression in the placenta, which expresses both SF-1 and P450scc. In contrast to its strong activation of the P450scc gene promoter in vitro, the absence of SF-1 has no effect on P450scc mRNA levels in vivo. Although the region targeted by disruption is shared by SF-1 and by ELP ( a hypothesized alternatively spliced product), it is thought that the observed phenotype reflects absent SF-1 alone, as PCR analysis fails to detect ELP transcripts in any mouse tissue, and sequences corresponding to ELP are not conserved across species. These results confirm that SF-1 is an important regulator of adrenal and gonadal development, but its regulation of steroid hydroxylase expression in vivo remains to be established (Sadovsky, 1995).

The spleen has two main functions. The first is to provide a proper microenvironment to lymphoid and myeloid cells, whereas the second involves clearance of abnormal erythrocytes. Ad4BP/SF-1, a product of the mammalian FTZ-F1 gene (mFTZ-F1), was originally identified as a steroidogenic, tissue-specific transcription factor. Immunohistochemical examination of the mammalian spleens confirms the expression of Ad4BP/SF-1 in endothelial cells of the splenic venous sinuses and pulp vein. In mFtz-F1 gene-disrupted (KO) mice, several structural abnormalities are detected in the spleen, including underdevelopment and nonuniform distribution of erythrocytes. Examination of the spleen of KO fetuses shows failure of development of certain tubular structures during embryogenesis. These structures are normally assembled by Ad4BP/SF-1 immunoreactive cells, and most likely form the vascular system during later stages of development. Other structural abnormalities in the spleen of the KO mice include defects in the tissue distribution of type-IV collagen, laminin, c-kit, and vimentin. These morphologic defects in the vascular system are associated with a decrease in the proportion of hematopoietic cells, although differentiation of these cells is not affected significantly. A high number of abnormal red blood cells containing Howell-Jolly bodies are noted in the KO mice, indicating impaired clearance by the splenic vascular system. The presence of an mRNA-encoding cholesterol side-chain cleavage P450 was detected in the spleen, resembling the findings in steroidogenic tissues such as the gonads and adrenal cortex. The mRNA transcript is not involved in splenic structural defects, since it is detected in the spleens of both normal and KO mice, indicating that the regulatory mechanism of the P450 gene in the spleen is different from that in steroidogenic tissues. These results indicate that a lack of the mFtz-F1 gene in mice is associated with structural and functional abnormalities of the splenic vascular system (Morohashi, 1999).

In Ftz-F1-disrupted mice, immunohistochemical analyses with antibodies against pituitary trophic hormones show a selective loss of gonadotrope-specific markers, supporting the role of SF-1 in gonadotrope function. Pituitaries from Ftz-F1-disrupted mice lack transcripts for three gonadotrope-specific markers (LH beta, FSH beta, and the receptor for gonadotropin-releasing hormone), whereas they exhibit decreased but detectable expression of the alpha-subunit of glycoprotein hormones. SF-1 transcripts in the developing mouse pituitary, which first become detectable at embryonic day 13.5-14.5, precede the appearance of FSH beta and LH beta transcripts. In adult rat pituitary cells, SF-1 transcripts colocalize with immunoreactivity for the gonadotrope-specific LH. Finally, SF-1 interacts with a previously defined promoter element in the glycoprotein hormone alpha-subunit gene, providing a possible mechanism for the impaired gonadotropin expression in Ftz-F1-disrupted mice (Ingraham, 1994).

mRNA coding for SF-1 is detected in the hypothalamus and pituitary. The transcription factor is expressed in nuclei of the dorsomedial part of the ventromedial hypothalamus (dmVMH) and in some subpopulation of the adenohypophysial cells. Staining for SF-1 and trophic peptide hormones (FSH, TSH, and ACTH), indicates a restricted localization of SF-1 to the gonadotroph. Disruption of the mouse Ftz-F1 gene induces severe defects in the organization of the dmVMH and the function of the pituitary gonadotroph. However, some of the dm VMH neurons and pituitary gonadotrophs persist, providing a sharp contrast to complete agenesis of the peripheral steroidogenic tissues (adrenal and gonads) in the mutant mouse. Additional abnormalities are seen in the ventrolateral part of the VMH and the dorsomedial hypothalamic nucleus, neither of which expresses SF-1, but both have strong reciprocal fiber-connections with the dmVMH. Aromatase P450-containing cells in the medial preoptico-amygdaloid region, which are devoid of SF-1, persist even in the brain of the gene disrupted mice. The hypothalamic and pituitary SF-1 are clearly seen to be essential for normal development of the functional VMH and gonadotroph through some mechanism distinct from that in the peripheral steroidogenic tissues (Shinoda, 1995).

The nuclear receptor steroidogenic factor 1 (SF-1) regulates the biosynthesis of the two essential mediators of male sexual differentiation: androgens and Mullerian-inhibiting substance. SF-1 is required for adrenal and gonadal development and gonadotropin expression. SF-1 is also expressed in the embryonic ventral diencephalon, subsequently localizing to the ventromedial hypothalamic nucleus, a region important for reproductive behavior. Mice lacking SF-1 secondary to targeted disruption of the Ftz-F1 gene have normal numbers and location of GnRH neurons but exhibit grossly impaired ventromedial hypothalamic nucleus structure. Despite their apparently normal GnRH neurons, treatment of Ftz-F1-disrupted mice with GnRH restores pituitary gonadotropin expression. These studies define SF-1's essential role within a discrete hypothalamic nucleus previously linked to reproduction (Ikeda, 1995).

The Ptx1 (pituitary homeobox 1) homeobox transcription factor is a transcription factor of the pituitary POMC gene. In corticotrope cells that express POMC, cell-specific transcription is conferred in part by the synergistic action of Ptx1 with the basic helix-loop-helix factor NeuroD1. Since Ptx1 expression precedes pituitary development and differentiation, its expression and function was examined in other pituitary lineages. Ptx1 is expressed in most pituitary-derived cell lines as is the related Ptx2 (Rieger) gene. However, Ptx1 appears to be the only Ptx protein in corticotropes and the predominant one in gonadotrope cells. Most pituitary hormone-coding gene promoters are activated by Ptx1. Thus, Ptx1 appears to be a general regulator of pituitary-specific transcription. In addition, Ptx1 action is synergized by cell-restricted transcription factors to confer promoter-specific expression. Indeed, in the somatolactotrope lineage, synergism between Ptx1 and Pit1 is observed on the PRL promoter, and strong synergism between Ptx1 and SF-1 is observed in gonadotrope cells on the betaLH promoter but not on the alphaGSU (glycoprotein hormone alpha-subunit gene) and betaFSH promoters. Synergism between these two classes of factors is reminiscent of the interaction between the products of the Drosophila genes ftz (fushi tarazu) and ftz-F1. Antisense RNA experiments performed in alphaT3-1 cells that express the alphaGSU gene show that expression of endogenous alphaGSU is highly dependent on Ptx1, whereas many other genes are not affected. Interestingly, the only other gene found to be highly dependent on Ptx1 for expression is the gene for the Lim3/Lhx3 transcription factor. Thus, these experiments place Ptx1 upstream of Lim3/Lhx3 in a cascade of regulators that appear to work in a combinatorial code to direct pituitary-, lineage-, and promoter-specific transcription (Tremblay, 1998).

Pituitary gonadotropins are critical regulators of gonadal development and function. Expression and secretion of the mature hormones are regulated by gonadotropin-releasing hormone (GnRH), which is itself secreted from the hypothalamus. GnRH stimulation of gonadotropin expression and secretion occurs through the G-protein-linked phospholipase C/inositol triphosphate intracellular signaling pathway, which ultimately leads to protein kinase C (PKC) activation and increased intracellular calcium levels. Transcription factors mediating the effects of GnRH-induced signals on transcription of gonadotropin genes have not yet been identified. Recent studies have identified three key factors involved in luteinizing hormone beta (LHbeta) gonadotropin gene transcription: the nuclear receptor SF-1, the bicoid-related homeoprotein Ptx1 (Pitx1), and the immediate-early Egr-1 gene. GnRH is a potent stimulator of Egr-1, but not Ptx1 or SF-1, expression. Further, Egr-1 activation of the LHbeta promoter is specifically enhanced by PKC, in agreement with a role for Egr-1 in mediating a GnRH effect on transcription. Egr-1 interacts directly with Ptx1 and with SF-1, leading to an enhancement of Ptx1- and SF-1-induced LHbeta transcription. Thus, Egr-1 is a likely transcriptional mediator of GnRH-induced signals for activation of the LHbeta gene (Tremblay, 1999).

Tissue-specific expression of the mammalian FTZ-F1 gene is essential for adrenal and gonadal development and sexual differentiation. The FTZ-F1 gene encodes an orphan nuclear receptor, termed SF-1 (steroidogenic factor-1) or Ad4BP, which is a primary transcriptional regulator of several hormone and steroidogenic enzyme genes that are critical for normal physiological function of the hypothalamic-pituitary-gonadal axis in reproduction. The objective of the current study was to understand the molecular mechanisms underlying transcriptional regulation of SF-1 gene expression in the pituitary. A series of deletion and point mutations in the SF-1 promoter region was studied for transcriptional activity in alphaT3-1 and L/betaT2 (pituitary gonadotrope), CV-1, JEG-3, and Y1 (adrenocortical) cell lines. Maximal expression of the SF-1 promoter in all cell types requires an E box element at -82/-77. This E box sequence (CACGTG) is identical to the binding element for USF (upstream stimulatory factor), a member of the helix-loop-helix family of transcription factors. Studies of the SF-1 gene E box element using gel mobility shift and antibody supershift assays indicate that USF may be a key transcriptional regulator of SF-1 gene expression (Harris, 1998).

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 means of 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 indicates 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 reduces the promoter activity (80% reduction; P < 0.05). The attenuation effect of this GSE mutant is cell specific, since it is restricted to alphaT3-1 cells, but not to COS-7 and human ovarian adenocarcinoma (SKOV-3) cells. Competitive mobility shift assays indicates that SF-1 is able to interact specifically with this GSE element positioned at -134. A SF-1 antibody completely abrogates complex formation in the gel shift assays. The sequences essential for the interaction with SF-1 have been identified [5'-TTG(A)/(T)CCCTG-3']. 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 reduces 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 (Ngan, 1999).

The hypothalamic neuropeptide, GnRH, regulates the synthesis and secretion of LH from pituitary gonadotropes. Furthermore, it has been shown that the LH beta-subunit gene is regulated by the transcription factors steroidogenic factor-1 (SF-1) and early growth response protein 1 (Egr1) in vitro and in vivo. The present study investigated the roles played by Egr1 and SF-1 in regulating activity of the equine LH beta-subunit promoter in the gonadotrope cell line, alpha T3-1, and the importance of these factors and cis-acting elements in regulation of the promoter by GnRH. All four members of the Egr family induce activity of the equine promoter. The region responsible for induction by Egr was localized to the proximal 185 bp of the promoter, which contains two Egr response elements. Coexpression of Egr1 and SF-1 leads to a synergistic activation of the equine (e)LH beta promoter. Mutation of any of the Egr or SF-1 response elements attenuate this synergism. Endogenous expression of Egr1 in alpha T3-1 cells is not detectable under basal conditions, but is rapidly induced after GnRH stimulation. Reexamination of the promoter constructs harboring mutant Egr or SF-1 sites indicates that these sites are required for GnRH induction. Mutation of both Egr sites within the eLH beta promoter completely attenuates its induction by GnRH. Thus, GnRH induces expression of Egr1, which subsequently activates the eLH beta promoter. Finally, GnRH not only induces expression of Egr1, but also its corepressor, NGFI-A (Egr1) binding protein (Nab1), which can repress Egr1-induced transcription of the eLH beta promoter (Wolfe, 1999).

Early growth response (Egr) 1-deficient mice exhibit female infertility, reflecting a luteinizing hormone (LH) beta deficiency. Egr-1 activates the LHbeta gene in vitro through synergy with steroidogenic factor-1 (SF-1), a protein required for gonadotrope function. To test if this synergy is essential for stimulation of LHbeta by gonadotropin-releasing hormone (GnRH), the activity of the LHbeta promoter was examined in the gonadotrope cell line LbetaT2. GnRH markedly stimulates the LHbeta promoter (15-fold). Mutation of either Egr-1 or SF-1 elements within the LHbeta promoter attenuates this stimulation, whereas mutation of both promoter elements abrogates GnRH induction of the LHbeta promoter. Furthermore, GnRH stimulates Egr-1 but not SF-1 expression in LbetaT2 cells. Importantly, overexpression of Egr-1 alone is sufficient to enhance LHbeta expression. Although other Egr proteins are expressed in LbetaT2 cells and are capable of interacting with SF-1, GnRH stimulation of Egr-1 is the most robust. The nuclear receptor DAX-1, a repressor of SF-1 activity, reduces Egr-1-SF-1 synergy and diminishes GnRH stimulation of the LHbeta promoter. It is concluded that the synergy between Egr-1 and SF-1 is essential for GnRH stimulation of the LHbeta gene and plays a central role in the dynamic regulation of LHbeta expression (Dorn, 1999).