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NEW PERSPECTIVES ON ENDOCRINE SIGNALLING Steroidogenic factor-1: a key regulator of gonadotroph gene expression R C Fowkes and J M Burrin Department of Endocrinology, Barts & the London School of Medicine & Dentistry, Queen Mary, University of London, 59 Bartholomew Close, London EC1A 7BE, UK (Requests for offprints should be addressed to R C Fowkes who is now at Department of Physiology, S-1479, 513 Parnassus Avenue, University of California, San Francisco, California 94143-0444, USA; Email:
[email protected])
Introduction Steroidogenic factor-1 (SF-1) is a member of the orphan nuclear receptor superfamily, also known as Ad4 BP or NR5A1. It was first isolated from an adrenal cDNA library, and subsequently shown to be expressed predominantly within the major steroidogenic organs (adrenals and gonads), but also in the ventromedial hypothalamus and anterior pituitary gonadotrophs (Ikeda et al. 1993, Morohashi et al. 1993, Barnhart & Mellon 1994, Ingraham et al. 1994). SF-1 is the mammalian homologue of the Fushi taruzu (ftz-f1 ) gene in D. melanogaster, and is structurally similar to the group of embryonal long-term repeat proteins (ELPs). The expression profile of SF-1 indicates the major role of SF-1: a key regulator of steroidogenic and gonadotrophic gene expression. This review highlights current understanding of SF-1 expression and action, and focuses on the role of SF-1 in the pituitary as a pivotal regulator of gonadotroph gene expression and the potential mechanisms involved in regulating SF-1 activity.
SF-1 expression SF-1 is encoded by the ftz-f1 gene in mammals, which in humans is located on chromosome 9q33. This gene encodes a nuclear protein of approximately 54 kDa, which binds to target gene promoters and recognises variations of the DNA sequence PyCAAGGTCA as a monomer (Wilson et al. 1993, Parker & Schimmer 1997, Meinke & Sigler 1999). Targeted disruption of the ftz-f1 gene in mice (to create SF-1 / ) revealed the critical role of SF-1 in adrenal and gonadal development. These animals died of glucocorticoid insufficiency within 7 days postpartum, due to poorly developed adrenal glands. Furthermore, ftz-f1 null mice exhibited 46XY sex reversal, abnormal development of the ventromedial hypothalamus
and an absence of pituitary gonadotrophins (luteinizing hormone (LH) and follicle-stimulating hormone (FSH)) (Luo et al. 1994, Sadovsky et al. 1995, Shinoda et al. 1995). More recent studies, using the heterozygous ftz- f1 /+ as a model, have implicated a role for SF-1 in the onset of hypothalamic obesity and response to traumatic stress (Bland et al. 2000, Majdic et al. 2002). These latter studies suggest an important gene–dosage effect of SF-1, as is implicated by naturally occuring mutations identified in humans (Achermann et al. 2002). The 5 untranslated region of the SF-1 gene contains several putative transcription factor response elements, including E and CCAAT boxes, Sp1 and Sox9 sites (Woodson et al. 1997, Shen & Ingraham 2002). However, although these response elements may contribute to basal expression of SF-1, to date there is no evidence for hormone-mediated transcriptional regulation of SF-1 expression. More recent studies have investigated the control of SF-1 translation, and suggest that activation of the protein kinase A (PKA) pathway may prolong the half-life of SF-1 protein in its target tissues (Aesoy et al. 2002). The control of SF-1 expression in the pituitary is poorly elucidated. Ovariectomised rats were found to express enhanced levels of SF-1 mRNA, suggesting either an inhibitory role of gonadal steroids (in particular oestrogen) or a stimulatory role of gonadotrophin-releasing hormone (GnRH) (Haisenleder et al. 1996). However, this increase in SF-1 mRNA could also be attributable to the pronounced gonadotroph hyperplasia which is observed in gonadectomised animals, and subsequent studies in gonadotroph-derived cell lines have failed to observe similar effects of GnRH on SF-1 expression (Dorn et al. 1999, Tremblay & Drouin 1999). It is apparent from SF-1 knock-out mice studies that the pituitary requires SF-1 expression for the proper development of the gonadotroph cell lineage and the expression of the phenotypic markers of these cells, the gonadotrophin hormones LH and FSH. These, and others, are major SF-1 target genes in the gonadotroph.
Journal of Endocrinology (2003) 177, 345–350 0022–0795/03/0177–345 2003 Society for Endocrinology Printed in Great Britain
Online version via http://www.endocrinology.org
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SF-1 activity in gonadotrophs
Figure 1 Comparison of SF-1 binding elements in gonadotroph genes. The aligned ‘GSE’ sequences (5 to 3 ) from promoter regions of the GSU, LH, GnRH-R, nNOS, DAX-1 and inhibin genes. DAX-1, dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X-chromosome gene 1.
SF-1 target genes The glycoprotein hormones LH and FSH are gonadotroph-derived, heterodimeric proteins, consisting of a common -subunit (GSU) and specific -subunits (LH and FSH). SF-1 regulates the expression of GSU and LH (Barnhart & Mellon 1994, Ingraham et al. 1994, Halvorson et al. 1996) along with the GnRH receptor (GnRH-R) (Duval et al. 1997), neuronal nitric oxide synthase (nNOS) (Wei et al. 2002) and the inhibin gene (Ito et al. 2000). Highly conserved SF-1 response elements reside within the promoter regions of these genes, termed gonadotroph-specific elements (GSEs) (Horn et al. 1992). The GSE of the GSU gene was found to specifically bind SF-1 by the Ingraham and Mellon groups in 1994 (Ingraham et al. 1994, Barnhart & Mellon 1994). Since then, other genes have been shown to contain GSEs (see Fig. 1), variant response elements that may modulate SF-1 transcriptional activity (Ito et al. 2000). We have focused our research into the regulation of the human GSU gene by SF-1. Using a series of 5 deletion constructs, we initially observed that basal transcriptional activity of the GSU promoter was lost upon deletion from 244 to 195 bp (Holdstock et al. 1996). Furthermore, the response to GnRH and pituitary adenylate cyclaseactivating polypeptide (PACAP) was also blocked by this mutation, suggesting the element(s) within 244 and 195 may mediate basal and hormone-stimulated GSU gene transcription (Holdstock et al. 1996, Burrin et al. 1998). The only recognised response element within this region of the promoter is the GSE. Mutation of the central CC to TT residues in the GSE inhibited basal promoter activity by at least 50% in transiently transfected gonadotroph-derived T3–1 cells (Barnhart & Mellon 1994, Heckert et al. 1997) and almost obliterated basal promoter activity in the more developed LT2 gonadotroph cell line (Fowkes et al. 2002). Furthermore, when we stimulated wild-type and mutant GSE-transfected LT2 cells with GnRH or activators of the protein kinase C (PKC)/mitogen-activated protein kinase (MAPK) pathway (with PMA, a phorbol ester), we observed a dramatic Journal of Endocrinology (2003) 177, 345–350
Figure 2 The GSE mediates basal and PKC-stimulated GSU transcription in LT2 cells. LT2 cells were transfected with GSU-luciferase reporter genes with wild-type (517LUC) or mutant (517MUT) GSE sequences. Cells were incubated subsequently with culture medium containing 0 (control) or 100 nM GnRH or PMA for 8 h. Reproduced with permission from Fowkes et al. (2002). pA3LUC, empty vector. *P
