WT1 and DAX-1 regulate SF-1-mediated human P450arom gene expression in gonadal cells

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Abstract

Binding activity of steroidogenic factor-1 (SF-1) to promoters of the majority of steroidogenic genes in response to gonadotropins is a critical mechanism that regulates steroidogenesis in gonads. Thus, the modulation of SF-1 action may be essential for the differential regulation of formation of sex steroids in the ovary. Aromatase P450 (P450arom) is the rate-limiting enzyme for estrogen formation. In this study, we characterize another nuclear receptor half site in the gonadal aromatase promoter which we show to be important for aromatase regulation. We also show herein that the stimulation of P450arom promoter activity by SF-1 in ovarian granulosa, testicular Sertoli and JEG-3 choriocarcinoma cells is inhibited by two transcription factors, Wilms’ tumor suppressor gene (WT1) and dosage sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome gene 1 (DAX-1). Given the characterized roles of these transcription factors in gonadal development and function, modulation of SF-1 action by WT1 and DAX-1 may represent an important key mechanism in steroidogenesis.

Introduction

Estradiol (E2) plays essential roles in the development and physiology of the female and male reproductive system (Couse et al., 1999). Estrogen synthesis is catalyzed by a microsomal member of the P450 superfamily, aromatase P450 (P450arom, the product of the CYP19 gene). The expression of the P450arom gene is regulated in various human tissues via the use of tissue-specific promoters (Simpson et al., 1994, Sebastian and Bulun, 2001). P450arom expression in the ovary and testis is regulated via the proximal promoter, promoter II (Jenkins et al., 1993, Bulun et al., 1993). Promoter II activity in the gonads is regulated by FSH and a cyclic AMP (cAMP) dependent signaling mechanism giving rise to an interaction of the gonadal promoter II with the transcription factors cAMP-responsive element-binding protein (CREB) and steroidogenic factor-1 (SF-1) (Michael et al., 1997, Carlone and Richards, 1997, Lynch et al., 1993).

SF-1 belongs to the nuclear receptor superfamily (NR5A1) and regulates gene transcription through its interactions with a number of co-regulators (e.g. steroid receptor coactivator-1, CREB-binding protein, dosage sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome gene 1 (DAX-1), early growth response-1, sex determining region Y-box 9, and Wilms’ tumor suppressor gene (WT1)) (Ito et al., 1997, Halvorson et al., 1998, De Santa Barbara et al., 1998, Nachtigal et al., 1998, Monte et al., 1998). In general, members of the nuclear receptor superfamily bind to their respective DNA elements as dimers. Each monomer within these dimer pairs interacts with a nuclear half site sequence, AGNNCA (Luisi et al., 1991, Schwabe et al., 1993). In contrast to these dimeric receptors, small groups of orphan nuclear hormone receptors bind to DNA as monomers. These factors recognize the estrogen response element-type half site, AGGTCA, also known as nuclear receptor half site (NRHS), and include SF-1, fushi tarazu F1 (Ftz-F1), NGFI-B, Rev-ErbA, and RORα (Tsukiyama et al., 1992, Ueda et al., 1992, Wilson et al., 1992, Wilson et al., 1993, Harding and Lazar, 1993, Giguere et al., 1995). SF-1 plays an essential role in the development of the adrenal gland, testis, ovary, pituitary gonadotropes, and hypothalamus (Luo et al., 1994, Luo et al., 1995). In the ovary, activation of multiple steroidogenic genes by LH and FSH in both theca and granulosa cells is mediated by common signaling molecules including cAMP and SF-1.

DAX-1 is also an orphan member of the nuclear hormone receptor superfamily of transcription factors (Bardoni et al., 1994, Swain et al., 1996, Swain and Lovell-Badge, 1997). Duplication of the region on the X chromosome containing the DAX-1 gene is associated with male-to-female sex reversal in XY individuals (Bardoni et al., 1994). Loss-of-function mutations in DAX-1 are responsible for X-linked AHC, a disorder characterized by primary adrenal insufficiency (Habiby et al., 1996). The levels of Cyp19 mRNA is increased by fourfold in Leydig cells of DAX-1-deficient mice compared with wild type (Wang et al., 2001). The colocalization of Sf-1 and DAX-1 led to the suggestion of a functional interaction between these two orphan nuclear receptors (Guo et al., 1995, Ikeda et al., 1996). DAX-1 has been shown to repress SF-1-mediated transactivation of steroidogenic acute regulatory protein (StAR), 3β-hydroxysteroid dehydrogenase (3β-HSD) type II, Müllerian inhibiting substance (MIS), P450c17 (Cyp17) and P450arom (Cyp19) genes (Nachtigal et al., 1998, Wang et al., 2001, Zazopoulos et al., 1997, Lalli et al., 1998, Hanley et al., 2001).

WT1 encodes a zinc-finger transcription factor, which has been shown to be essential for mammalian gonadal and kidney development. It regulates the expression of several genes involved in cellular proliferation and differentiation. Homozygous inactivation of WT1 in mice results in the absence of both kidneys and gonads (Kreidberg et al., 1993). In addition, various WT1 mutations are associated with three pediatric syndromes: WAGR (Wilms’ tumor, Aniridia, Genitourinary abnormalities, and mental Retardation), Denys–Drash syndrome (DDS), and Frasier syndrome (Little and Wells, 1997). Interestingly, a WT1 mutation has been observed in the juvenile granulosa cell tumor of a patient with DDS (Pelletier et al., 1991a). WT1 mRNA has been detected in granulosa cells of ovarian follicles and Sertoli cells of the testis (Pelletier et al., 1991b, Tilly et al., 1995).

The regulation of the human P450arom gene by WT1 and DAX-1 in endometrial and endometriotic stromal cells was shown in a previous report (Gurates et al., 2002). In this study, we demonstrate that P450arom promoter II is regulated by SF-1, WT1 and DAX-1 in granulosa and Sertoli cells. We also employed the JEG-3 cell line that does not use the gonadal promoter II for aromatase expression to demonstrate these effects in a reconstitutive fashion. Moreover, we characterized a previously unrecognized motif (−263/−251 bp) in P450arom promoter II, which showed 85% homology to a consensus NRHS using TFSearch database on the Internet (Fig. 1). Our findings suggest that two motifs (−263/−251 and −136/−124 bp) showing significant homology to consensus NRHS sequence in promoter II of the P450arom gene (Fig. 1) are important for aromatase expression, and that SF-1-mediated transactivation can be repressed by WT1 and DAX-1 in human ovarian granulosa cells and in mouse Sertoli cells.

Section snippets

Plasmid constructs and site-directed mutagenesis

Human SF-1 cDNA in the expression vector pcDNA2 was a generous gift from Drs Meera Ramayya and Keith L. Parker. Human DAX-1 cDNA in the expression vector pBKCMV was a generous gift from Dr J. Larry Jameson. Mouse WT1 cDNAs (with or without KTS domains) in pcDNA3.1 vectors were generous gifts from Dr Daniel A. Haber. Preparations of the deletion mutants of the P450arom gene promoter II and 5′-flanking sequence in pGL3-basic Luciferase vector have been described previously (Zhou et al., 2001).

Regulatory sequences in promoter II that confer SF-1-induced transcription in JEG-3 cell line

We utilized JEG-3 cells to perform reconstitutive experiments during the first portion of this study. Using transient transfections in JEG-3 choriocarcinoma cells, which lack endogenous SF-1, DAX-1 and WT1 proteins, we sought to identify regions of the P450arom promoter II that are responsible for SF-1-induced transcription (Ito et al., 1997, Nachtigal et al., 1998). Promoter II specific P450arom transcripts are absent in JEG-3 cells probably as a direct consequence of the absence of SF-1.

Discussion

SF-1, WT1 and DAX-1 play important roles in the mammalian gonadogenesis and regulation of the gonadal function. In mice with genetic disruptions of SF-1 or WT1 coding regions, gonads do not develop (Shen et al., 1994, Kreidberg et al., 1993). SF-1 participates further in male sexual differentiation by regulating MIS (Shen et al., 1994). Moreover SF-1 regulates the transcription of an array of human steroidogenic genes including StAR, CYP11A, 3β-HSD type 2, CYP17 and CYP19 (P450arom) (Sugawara

Acknowledgements

We thank Meera Ramayya, Keith L. Parker, J. Larry Jameson and Daniel A. Haber for providing human SF-1 cDNA, human DAX-1 cDNA, and mouse WT1 cDNA. This work was supported by the NIH grant HD38691 (to SEB).

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