Orphan Nuclear Receptor Steroidogenic Factor-1 Research Articles

Steroidogenic factor-1 is essential for compensatory adrenal growth following unilateral adrenalectomy.

While the orphan nuclear receptor steroidogenic factor-1 (SF-1) has been shown to function as an induction factor to define adrenocortical cell lineage, it remains unclear whether SF-1 plays an additional role as a growth promoting agent in the adult adrenal cortex. The proliferative potential of the adrenal cortex in adult SF-1(+/-) mice was examined using the model of compensatory adrenal growth following unilateral adrenalectomy (uADX). While the right adrenal gland of wild-type (wt) mice grew significantly after uADX, the adrenal of SF-1(+/-) mice exhibited a blunted, statistically nonsignificant weight increase. Accordingly, a profound increase in the proliferation marker proliferating cell nuclear antigen could be detected only in wt mice after uADX but not in the SF-1(+/-) mice. The proposed key regulator in adrenal compensatory growth, the recently cloned adrenal secretory serine protease was up-regulated in the remaining adrenal of wt mice, whereas this increase was blunted in SF-1(+/-) mice. While no differences in preadipocyte factor-1, the presumed marker of primitive adrenocortical cells, were detectable in the adrenals of wt and SF-1(+/-) mice, an increase in the ACTH receptor as well as agouti-related protein was observed only in wt animals but not in the SF-1(+/-) mice following uADX. Taken together, these results reflect a primary inability of adrenal cortical cells of SF-1(+/-) mice to undergo compensatory adrenal growth in response to uADX.

Gonadal Determination and Adrenal Development Are Regulated by the Orphan Nuclear Receptor Steroidogenic Factor-1, in a Dose-Dependent Manner

Gonadal Determination and Adrenal Development Are Regulated by the Orphan Nuclear Receptor Steroidogenic Factor-1, in a Dose-Dependent Manner

DIFFERENTIAL REGULATION OF SF-1-COFACTOR INTERACTIONS

The orphan nuclear receptor steroidogenic factor-1 (SF-1) plays pivotal roles in the development and function of steroidogenic organs. Here we describe the differential effect of protein kinase A (PKA) on coregulation of SF-1 dependent transcription by two p160 family members, p300/CBP co-integrator-associated protein (p/CIP) and transcription intermediary factor-2 (TIF2). Thus, whereas p/CIP-stimulated SF-1 dependent transcription is further potentiated by PKA, we show that activation of PKA leads to selective downregulation of TIF2 protein and a subsequent repression of TIF2 coactivator function. Using a yeast two-hybrid screen we also identified a novel zinc finger containing protein, which interacts with SF-1 via the AF-2 domain.

A Novel Zinc Finger Protein TReP-132 Interacts with CBP/p300 to Regulate Human CYP11A1 Gene Expression

The human CYP11A1 gene is expressed specifically in steroidogenic tissues and encodes cytochrome P450scc, which catalyzes the first step in steroid synthesis. A region of the 5'-flanking DNA of the gene from nucleotides -155 to -131 (-155/-131) is shown to activate transcription in steroidogenic human placental JEG-3 (1) and adrenal NCI-H295 cells. Using this region of the gene as probe, a cDNA clone of 4.4 kilobase pairs was isolated by screening JEG-3 cell and human placental cDNA expression libraries. The open reading frame encodes three zinc fingers of the C(2)H(2) subtype, and separate regions rich in glutamate, proline, and glutamine, which are indicative of a DNA-binding protein involved in gene transcription. Expression of the cDNA in vitro and in HeLa cells yields a protein of 132 kDa, which concurs with the predicted size. Northern blot analysis demonstrate expression of two TReP-132 transcripts of 4.4 and 7.5 kilobase pairs in the thymus, adrenal cortex, and testis; and expression is also found in the steroidogenic JEG-3, NCI-H295, and MCF-7 cell lines. Immunocytochemistry analysis demonstrates localization of the HA-tagged TReP-132 protein in the nucleus. The expression of exogenous TReP-132 in HeLa cells was demonstrated to interact with the -155/-131 region in bandshift analysis. Transfection of the cDNA in placental JEG-3 and adrenal NCI-H295 cells increases expression of a reporter construct controlled by the P450scc gene 5'-flanking region from nucleotides -1676 to +49. Moreover, a chimeric protein generated by fusion of TReP-132 with the Gal4 DNA-binding domain was able to significantly increase promoter activity of a reporter construct via Gal4-binding sites upstream of the E1b minimal promoter. Coexpression of CREB-binding protein (CBP)/p300 with TReP-132 has an additive effect on promoter activity, and the proteins were demonstrated to interact physically. Thus, these results together indicate the isolation of a novel zinc-finger transcriptional regulating protein of 132 kDa (TReP-132) involved in the regulation of P450scc gene expression.

Cloning of Factors Related to HIV-inducible LBP Proteins That Regulate Steroidogenic Factor-1-independent Human Placental Transcription of the Cholesterol Side-chain Cleavage Enzyme, P450scc

The cholesterol side-chain cleavage enzyme, cytochrome P450scc, initiates the biosynthesis of all steroid hormones. Adrenal and gonadal strategies for P450scc gene transcription are essentially identical and depend on the orphan nuclear receptor steroidogenic factor-1, but the placental strategy for transcription of P450scc employs cis-acting elements different from those used in the adrenal strategy and is independent of steroidogenic factor-1. Because placental expression of P450scc is required for human pregnancy, we sought factors that bind to the -155/-131 region of the human P450scc promoter, which participates in its placental but not adrenal or gonadal transcription. A yeast one-hybrid screen of 2.4 x 10(6) cDNA clones from human placental JEG-3 cells yielded two unique clones; one is the previously described transcription factor LBP-1b, which is induced by HIV, type I infection of lymphocytes, and the other is a new factor, termed LBP-9, that shares 83% amino acid sequence identity with LBP-1b. When expressed in transfected yeast, both factors bound specifically to the -155/-131 DNA; antisera to LBP proteins supershifted the LBP-9.DNA complex and inhibited formation of the LBP-1b.DNA complex. Reverse transcriptase-polymerase chain reaction detected LBP-1b in human placental JEG-3, adrenal NCI-H295A, liver HepG2, cervical HeLa, and monkey kidney COS-1 cells, but LBP-9 was detected only in JEG-3 cells. When the -155/-131 fragment was linked to a minimal promoter, co-expression of LBP-1b increased transcription 21-fold in a dose-dependent fashion, but addition of LBP-9 suppressed the stimulatory effect of LBP-1b. The roles of LBP transcription factors in normal human physiology have been unclear. Their modulation of placental but not adrenal P450scc transcription underscores the distinctiveness of placental strategies for steroidogenic enzyme gene transcription.

Transcriptional regulation of the rat Müllerian inhibiting substance type II receptor in rodent Leydig cells.

Müllerian inhibiting substance (MIS) causes regression of the fetal Müllerian duct on binding a heteromeric complex of types I and II cell-surface receptors in the fetal urogenital ridge. The MIS type II receptor (MISRII), which provides specificity for MIS, is also expressed in the adult testis, ovary, and uterus. The rat MISRII promoter was cloned to study the molecular mechanisms underlying its temporal and cell-specific expression. The 1.6-kilobase (kb) promoter contained no recognizable TATA or CAAT box, but there was a consensus Sp1 site upstream of the transcription initiation site. Two binding sites for the orphan nuclear receptor steroidogenic factor-1 (SF-1) are occupied in vitro by using nuclear extracts from R2C cells, an MIS-responsive rat Leydig cell line that expresses endogenous MISRII, with differing affinities, indicating that the distal SF-1 site is bound more avidly than is the proximal SF-1 site. R2C cells transfected with MISRII promoter/luciferase reporter constructs show a 12-fold induction with the 1.6-kb fragment and deletion of sequences upstream of -282-bp lowered luciferase expression to one-third. Mutation of both SF-1 sites greatly inhibited luciferase expression, whereas mutation of either site alone resulted in continuing activation by endogenous SF-1, indicating redundancy. In vitro binding and transcriptional analyses suggest that a proximal potential Smad-responsive element and an uncharacterized element also contribute to activation of the MISRII gene. R2C cells and MISRII promoter regulation can now be used to uncover endogenous transcription factors responsible for receptor expression or repression.

25-Hydroxycholesterol is not a ligand for the orphan nuclear receptor steroidogenic factor-1 (SF-1).

The orphan nuclear receptor steroidogenic factor-1 (SF-1) is involved in the transcriptional regulation of all the steroid hydroxylase genes, and also regulates the transcription of the genes for Müllerian Inhibitory substance (MIS), alpha subunit of glycoprotein hormone, LHbeta, oxytocin, GnRH receptor, ACTH receptor, prolactin receptor, DAX-1, and steroidogenic acute regulatory protein. Other members of the nuclear receptor gene family, including steroid hormone, thyroid hormone, retinoic acid, PPAR, and vitamin D receptors must bind ligand to activate transcription, but SF-1 has been considered to be an orphan nuclear receptor because, when identified, it had no known ligand. A recent publication suggested that transcriptional regulation by SF-1, expressed in a non-steroidogenic CV-1 cells, could be activated by oxysterols suggesting that these compounds could serve as natural ligands for SF-1. We now demonstrate that 25-hydroxycholesterol, either added exogenously or synthesized endogenously in steroidogenic mouse Leydig MA-10 cells, did not act as a ligand for SF-1, as it did not increase transcription from six different SF-1-dependent DNA sequences. Furthermore, the abundance of these oxysterols in MA-10 cells was much less than concentrations needed for activation of SF-1 in CV-1 cells, indicating that SF-1 is not constitutively bound by ligand in MA-10 cells. Thus, in steroidogenic cells, transcriptional regulation of the steroid hydroxylase genes by SF-1 does not depend upon the presence of 25-hydroxycholesterol, and is not modified by its presence.

Wilms' Tumor 1 and Dax-1 Modulate the Orphan Nuclear Receptor SF-1 in Sex-Specific Gene Expression

Products of steroidogenic factor 1 (SF-1) and Wilms' tumor 1 ( WT1) genes are essential for mammalian gonadogenesis prior to sexual differentiation. In males, SF-1 participates in sexual development by regulating expression of the polypeptide hormone Müllerian inhibiting substance (MIS). Here, we show that WT1 −KTS isoforms associate and synergize with SF-1 to promote MIS expression. In contrast, WT1 missense mutations, associated with male pseudohermaphroditism in Denys-Drash syndrome, fail to synergize with SF-1. Additionally, the X-linked, candidate dosage-sensitive sex-reversal gene, Dax-1, antagonizes synergy between SF-1 and WT1, most likely through a direct interaction with SF-1. We propose that WT1 and Dax-1 functionally oppose each other in testis development by modulating SF-1–mediated transactivation.

A new step understood in the cascade of tissue-specific regulators orchestrating pituitary lineage determination: the Prophet of Pit-1 (Prop-1).

An early step in generating specific cell types in embryos involves the commitment of multipotent stem cells to specific lineages. The anterior pituitary gland, with its five cell types originating from a common precursor, provides an instructive model to approach the mechanisms that control mammalian organ development and cell differentiation. Using the well-known model of the Ames dwarf mouse, which has a hypoplastic pituitary due to a dramatic reduction of somatotroph, lactotroph and thyrotroph cells, M Rosenfeld’s group has been able to clone the defective gene responsible for Ames’ dwarfism and has therefore clarified a new step in the cascade of tissue-specific regulators orchestrating the development of the anterior pituitary. They identified a novel pituitary paired-like homeodomain factor which seemed to be an important prerequisite for the expression of the transcription factor Pit-1. They named it Prophet of Pit-1 (Prop-1) (1). Pit-1 had been discovered in another dwarf mouse, the Snell and Jackson mouse. These animals also lack growth hormone, prolactin and thyrotrophin (TSH) and it has been shown that this results from a mutation in the Pit-1 gene resulting in a non-functional Pit-1 protein (2). Now the work of Rosenfeld and colleagues (1) indicates that a mutation of Prop-1 induces a specific defect in the determination of the Pit-1 lineages in the Ames dwarf mouse. The development of the pituitary depends on many factors. In short, Rathke’s pouch, the primordium of the anterior pituitary, appears as an invagination of an ectodermal layer of cells that contacts the neuroectoderm of the primordium of the ventral hypothalamus at embryonic day (E) 8.5 in the mouse. Before the formation of Rathke’s pouch, some genes have been identified allowing certain cell compartments to develop into an anterior pituitary and expressing three homeodomain factors, Pax6, Rpx (Rathke’s pouch homeobox) and Six3 (3). Concurrently to the invagination of the Rathke’s pouch, the expression of two homeodomain factors, the LIM homeobox gene Lhx3 (also known as P-Lim and mLim-3) and an OTX-related factor (Ptx1/ P-OTX) are seen. Lhx3 is required for the commitment of gonadotrophs, thyrotrophs, somatotrophs and lactotrophs. The further differentiation of the Lhx3/Ptx1 cells is dependent upon the appearance of two transcription factors: Pit-1 and the orphan nuclear receptor SF-1 (steroidogenic-factor 1). They are both

Characterization of the promoter region of the mouse gene encoding the steroidogenic acute regulatory protein.

Steroidogenic acute regulatory protein (StAR) delivers cholesterol to the inner mitochondrial membrane, where the cholesterol side-chain cleavage enzyme carries out the first committed step in steroid hormone biosynthesis. StAR expression is restricted to steroidogenic cells and is rapidly induced by treatment with trophic hormones or cAMP. We analyzed the 5'-flanking region of the mouse StAR gene to elucidate the mechanisms that regulate its cell-specific and hormone-induced expression. In transient transfection assays, a luciferase reporter gene driven by the StAR 5'-flanking region was preferentially expressed by steroidogenic Y1 adrenocortical and MA-10 Leydig cells in a cAMP-responsive manner. 5'-Deletion and site-directed mutagenesis studies identified a region between -254 and -113 that is essential for full levels of promoter activity. This region contains a binding site for the orphan nuclear receptor steroidogenic factor-1 (SF-1) that, although not required for hormone induction, is critical for basal promoter activity, thus implicating SF-1 in StAR expression. Analyses of knockout mice deficient in SF-1 further supported an important role for SF-1 in StAR gene expression. These studies provide novel insights into the mechanisms that regulate StAR gene expression and extend our understanding of SF-1's global roles within steroidogenic cells.

The orphan nuclear receptor steroidogenic factor-1 regulates the cyclic adenosine 3',5'-monophosphate-mediated transcriptional activation of rat cytochrome P450c17 (17 alpha-hydroxylase/c17-20 lyase).

The rat steroid cytochrome P450 17 alpha-hydroxylase/c17-20 lyase (rP450c17) gene is transcriptionally regulated in steroidogenic tissues. Previous studies showed that one DNA element located between -75 and -50 base pairs (bp) upstream from the transcriptional initiation site mediated both the basal and cAMP-regulated transcription of rP450c17. Using a series of mutant oligonucleotides in gel mobility shift assays and in functional assays, it is now shown that a core sequence of 12 bp, located at -58/-69 bp, is essential for nuclear protein binding and transcriptional activation. Mutant oligonucleotides cloned into a luciferase reporter gene construct containing a heterologous thymidine kinase promoter, transfected into mouse Leydig MA-10 and adrenocortical Y-1 cells, gave results consistent with those of gel shift assays. Mutants that abolished binding of the nuclear protein to DNA abolished the basal transcription of the gene as well as the responsiveness to cAMP, whereas those mutants that did not abolish binding of the nuclear protein to DNA still showed strong basal transcription as well as responsiveness to cAMP. Comparison of the binding sequence with the consensus binding site for the orphan nuclear receptor steroidogenic factor-1 (SF-1) showed that eight of nine bases were identical. However, the sequence from rP450c17 includes an additional three bases at the 5'-end, not previously demonstrated to be important for SF-1 binding. Recombinant rat SF-1 protein expressed in Escherichia coli binds to this sequence, and antibodies raised against rat SF-1 abolish binding of both recombinant SF-1 and the nuclear protein from Y-1 and MA-10 cells. These observations demonstrate that this region of the rP450c17 gene is responsible for both the basal transcription and cAMP inducibility and is bound by the orphan nuclear receptor SF-1. It is further shown that SF-1 can be phosphorylated in vitro by protein kinase A. This phosphorylation occurs at serine and threonine residues and results in decreased binding to the rP450c17 -58/-69 element. Since SF-1 mediates cAMP-induced transcriptional regulation of the rat P450c17 gene, phosphorylation of SF-1 via protein kinase A is likely to play a regulatory role in transcriptional activation.

The orphan nuclear receptor steroidogenic factor-1 regulates the cyclic adenosine 3',5'-monophosphate-mediated transcriptional activation of rat cytochrome P450c17 (17 alpha-hydroxylase/c17-20 lyase)

The orphan nuclear receptor steroidogenic factor-1 regulates the cyclic adenosine 3',5'-monophosphate-mediated transcriptional activation of rat cytochrome P450c17 (17 alpha-hydroxylase/c17-20 lyase)