Steroidogenic Acute Regulatory Gene Transcription Research Articles

A STAR for the ages: a 30-year historical perspective of the role of transcription factors in the regulation of steroidogenic acute regulatory gene expression.

The steroidogenic acute regulatory (STAR) protein is an essential cholesterol transporter that shuttles cholesterol from the outer to the inner mitochondrial membrane in the major steroidogenic endocrine organs. It is a key player in the acute regulation of steroid hormone biosynthesis in response to tropic hormone stimulation. Its discovery 30 years ago sparked immediate interest in understanding how STAR action is controlled. Since increased STAR gene expression is a classic feature of the acute regulation of steroidogenesis, a special emphasis was placed on defining the transcriptional regulatory mechanisms that underlie its rapid induction in response to tropic hormone stimulation. These mechanisms include the effects of enhancers, the regulation of chromatin accessibility, the impact of epigenetic factors, and the role of transcription factors. Over the past three decades, understanding the transcription factors that regulate STAR gene expression has been the subject of more than 170 independent scientific publications, making it one of, and if not the best, studied genes in the steroidogenic pathway. This intense research effort has led to the identification of dozens of transcription factors and their related binding sites in STAR 5' flanking (promoter) sequences across multiple species. STAR gene transcription appears to be complex in that a large number of transcription factors have been proposed to interact with either isolated or overlapping regulatory sequences that are tightly clustered over a relatively short promoter region upstream of the STAR transcription start site. Many of these transcription factors appear to work in unique combinatorial codes and are impacted by diverse hormonal and intracellular signaling pathways. This review provides a retrospective overview of the transcription factors proposed to regulate both basal and acute (hormonal) STAR gene expression, and how insights in this area have evolved over the past 30 years.

Involvement of the Thromboxane A2 Receptor in the Regulation of Steroidogenic Acute Regulatory Gene Expression in Murine Leydig Cells

Recent studies suggested an involvement of thromboxane A2 in cyclooxygenase-2-dependent inhibition of steroidogenic acute regulatory (StAR) gene expression. The present study further investigated the role of thromboxane A2 receptor in StAR gene expression and steroidogenesis in testicular Leydig cells. The thromboxane A2 receptor was detected in several Leydig cell lines. Blocking thromboxane A2 binding to the receptor using specific antagonist SQ29548 or BM567 resulted in dose-dependent increases in StAR protein and steroid production in MA-10 mouse Leydig cells. The results were confirmed with Leydig cells isolated from rats. StAR promoter activity and StAR mRNA level in the cells were also increased after the treatments, suggesting an involvement of the thromboxane A2 receptor in StAR gene transcription. Furthermore study indicated that blocking the thromboxane A2 receptor reduced dosage sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1 protein, a transcriptional repressor of StAR gene expression. Specific binding of the antagonists to the receptors on cellular membrane was demonstrated by binding assays using (3)H-SQ29548 and binding competition between (3)H-SQ29548 and BM567. Whereas SQ29548 enhanced cAMP-induced StAR gene expression, in the absence of cAMP, it was unable to increase StAR protein and steroidogenesis. However, when the receptor was blocked by the antagonist, subthreshold levels of cAMP were able to induce maximal levels of StAR protein expression, suggesting that blocking the thromboxane A2 receptor increase sensitivity of MA-10 cells to cAMP stimulation. Taken together, the results from the present and previous studies suggest an autocrine loop, involving cyclooxygenase-2, thromboxane A synthase, and thromboxane A2 and its receptor, in cyclooxygenase-2-dependent inhibition of StAR gene expression.

Chrysin, a natural flavonoid enhances steroidogenesis and steroidogenic acute regulatory protein gene expression in mouse Leydig cells

During the aging process of males, testosterone biosynthesis declines in testicular Leydig cells resulting in decreases in various physiological functions. To explore the possibility of delaying the decline using food supplements, we have studied steroidogenic effects of a natural flavonoid, chrysin, in mouse Leydig cells. Chrysin dramatically increased cyclic AMP (cAMP)-induced steroidogenesis in MA-10 mouse Leydig tumor cells. This result was confirmed using Leydig cells isolated from mouse testes. The steroidogenic effect of chrysin is not associated with an increase in expression of the P450 side-chain cleavage enzyme, required for the conversion of cholesterol to pregnenolone. In addition, when 22(R)hydroxylcholesterol was used as a substrate, chrysin induced a non-significant increase in steroid hormone, suggesting that the majority of the observed increase in steroidogenesis was due to the increased supply of substrate cholesterol. These observations were corroborated by showing that chrysin induced a marked increase in the expression of steroidogenic acute regulatory (StAR) protein, the factor that controls mitochondrial cholesterol transfer. Also, chrysin significantly increased StAR promoter activity and StAR mRNA level. Further studies indicated that this compound depressed expression of DAX-1, a repressor in StAR gene transcription. In the absence of cAMP, chrysin did not increase steroidogenesis. However, when a sub-threshold level of cAMP was used, StAR protein and steroid hormone were increased by chrysin to the levels seen with maximal stimulation of cAMP. These results suggest that while chrysin itself is unable to induce StAR gene expression and steroidogenesis, it appears to function by increasing the sensitivity of Leydig cells to cAMP stimulation.

Inhibition of Thromboxane A Synthase Activity Enhances Steroidogenesis and Steroidogenic Acute Regulatory Gene Expression in MA-10 Mouse Leydig Cells

The cyclooxygenase-2 (COX2)-dependent inhibition of Leydig cell steroidogenesis has been demonstrated. To understand the mechanism for this effect of COX2, the present study examined the role of an enzyme downstream of COX2, namely thromboxane A synthase (TBXAS), in steroidogenesis. Inhibition of TBXAS activity with the inhibitor furegrelate induced a concentration-dependent increase in cAMP-induced steroidogenic acute regulatory (StAR) protein in MA-10 mouse Leydig cells. The increase in StAR protein occurred concomitantly with a significant increase in steroid hormone production. Similar results were obtained in StAR promoter activity assays and RT-PCR analyses of StAR mRNA levels, suggesting that inhibition of TBXAS activity enhanced StAR gene transcription. These observations were corroborated when TBXAS expression was specifically inhibited by RNA interference. Although the RNA interference reduced mRNA levels of TBXAS, it increased StAR mRNA levels, StAR protein, and steroidogenesis. Additional studies indicated that inhibition of TBXAS activity reduced DAX-1 protein, a repressor in StAR gene transcription. In the absence of cAMP, inhibition of TBXAS activity did not induce a significant increase in steroid hormone and StAR protein. However, addition of a low level of cAMP analogs dramatically increased steroidogenesis. Lastly, inhibition of protein kinase A activity essentially abolished the steroidogenic effect of the TBXAS inhibitor. Thus, the results from the present study suggest that a minimal level of protein kinase A activity is required for the steroidogenic effect of the TBXAS inhibitor and that inhibition of TBXAS activity or its expression increase the steroidogenic sensitivity of MA-10 mouse Leydig cells to cAMP stimulation.

The involvement of epoxygenase metabolites of arachidonic acid in cAMP-stimulated steroidogenesis and steroidogenic acute regulatory protein gene expression

The essential role of arachidonic acid (AA) in steroidogenesis has been previously demonstrated. The present study continues the investigation into how AA regulates steroidogenesis by examining the effects of epoxygenase-derived AA metabolites on cAMP-stimulated steroidogenic acute regulatory (StAR) gene expression and steroid hormone production in MA-10 mouse Leydig cells. The HPLC analysis of cell extracts from MA-10 cells treated with the cAMP analog dibutyryl cAMP (dbcAMP) demonstrated an increase in three epoxygenase-generated AA metabolites: 5,6-epoxyeicosatrienoic acid (EET), 8,9-EET, and 11,12-EET. Incubating MA-10 cells with each of the EETs induced a dose-dependent increase in StAR protein expression and steroid hormone production in the presence of dbcAMP. These metabolites also significantly enhanced StAR gene transcription as determined by luciferase assays of StAR promoter activity and reverse transcriptase-PCR analysis of StAR mRNA levels. While the EETs enhanced steroidogenesis, inhibiting the activity of protein kinase A (PKA) abolished the stimulatory effects of these AA metabolites on StAR expression and steroid hormone production. This study suggests that cAMP stimulation of MA-10 cells increases epoxygenase-generated AA metabolites and the co-action of these metabolites with PKA significantly increases StAR gene expression and steroid hormone production.

Molecular Mechanisms of Insulin-like Growth Factor-I Mediated Regulation of the Steroidogenic Acute Regulatory Protein in Mouse Leydig Cells

Growth factors are known to play diverse roles in steroidogenesis, a process regulated by the mitochondrial steroidogenic acute regulatory (StAR) protein. The mechanism of action of one such growth factor, IGF-I, was investigated in mouse Leydig tumor (mLTC-1) cells to determine its potential role in the regulation of StAR expression. mLTC-1 cells treated with IGF-I demonstrated temporal and concentration-dependent increases in StAR expression and steroid synthesis. However, IGF-I had no effect on cytochrome P450 side-chain cleavage or 3beta-hydroxysteroid dehydrogenase protein levels. IGF-I was capable of augmenting N,O'-dibutyrl-cAMP-stimulated steroidogenic responsiveness in these cells. The steroidogenic potential of IGF-I was also confirmed in primary cultures of isolated mouse Leydig cells. IGF-I increased phosphorylation of ERK1/2, an event inhibited by the MAPK/ERK inhibitors, PD98059 and U0126. Interestingly, inhibition of ERK activity enhanced IGF-I-mediated StAR protein expression, but phosphorylation of StAR was undetectable, an observation in contrast to that seen with N,O'-dibutyrl-cAMP signaling. Further studies demonstrated that these events were tightly correlated with the expression of dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene 1 and scavenger receptor class B type 1. Whereas both protein kinase A and protein kinase C signaling were involved in the IGF-I-mediated steroidogenic response, the majority of the effects of IGF-I were found to be mediated by the protein kinase C pathway. Transcriptional activation of the StAR gene by IGF-I was influenced by several transcription factors, its up-regulation being dependent on phosphorylation of the cAMP response element-binding protein (CREB) and the activator protein 1 family member, c-Jun. Conversely, StAR gene transcription was markedly inhibited by expression of nonphosphorylatable CREB (Ser(133)Ala), dominant negative A-CREB, and dominant negative c-Jun (TAM-67) mutants. Collectively, the present studies identify molecular events in IGF-I signaling that may influence testicular growth, development, and the Leydig cell steroidogenic machinery through autocrine/paracrine regulation.

Assessment of the role of activator protein-1 on transcription of the mouse steroidogenic acute regulatory protein gene.

cAMP-dependent mechanisms regulate the steroidogenic acute regulatory (StAR) protein even though its promoter lacks a consensus cAMP response-element (CRE, TGACGTCA). Transcriptional regulation of the StAR gene has been demonstrated to involve combinations of DNA sequences that provide recognition motifs for sequence-specific transcription factors. We recently identified and characterized three canonical 5'-CRE half-sites within the cAMP-responsive region (-151/-1 bp) of the mouse StAR gene. Among these CRE elements, the CRE2 half-site is analogous (TGACTGA) to an activator protein-1 (AP-1) sequence [TGA(C/G)TCA]; therefore, the role of the AP-1 transcription factor was explored in StAR gene transcription. Mutation in the AP-1 element demonstrated an approximately 50% decrease in StAR reporter activity. Using EMSA, oligonucleotide probes containing an AP-1 binding site were found to specifically bind to nuclear proteins obtained from mouse MA-10 Leydig and Y-1 adrenocortical tumor cells. The integrity of the sequence-specific AP-1 element in StAR gene transcription was assessed using the AP-1 family members, Fos (c-Fos, Fra-1, Fra-2, and Fos B) and Jun (c-Jun, Jun B, and Jun D), which demonstrated the involvement of Fos and Jun in StAR gene transcription to varying degrees. Disruption of the AP-1 binding site reversed the transcriptional responses seen with Fos and Jun. EMSA studies utilizing antibodies specific to Fos and Jun demonstrated the involvement of several AP-1 family proteins. Functional assessment of Fos and Jun was further demonstrated by transfecting antisense c-Fos, Fra-1, and dominant negative forms of Fos (A-Fos) and c-Jun (TAM-67) into MA-10 cells, which significantly (P < 0.01) repressed transcription of the StAR gene. Mutation of the AP-1 site in combination with mutations in other cis-elements resulted in a further decrease of StAR promoter activity, demonstrating a functional cooperation between these factors. Mammalian two-hybrid assays revealed high-affinity protein-protein interactions between c-Fos and c-Jun with steroidogenic factor 1, GATA-4, and CCAAT/enhancer binding protein-beta. These findings demonstrate that Fos and Jun can bind to the TGACTGA element in the StAR promoter and provide novel insights into the mechanisms regulating StAR gene transcription.

Inhibition of cyclooxygenase-2 activity enhances steroidogenesis and steroidogenic acute regulatory gene expression in MA-10 mouse Leydig cells.

To study the mechanism for the regulatory effect of arachidonic acid (AA) on steroidogenesis, the role of cyclooxygenase (COX) in steroid production and steroidogenic acute regulatory (StAR) gene expression was investigated. Although stimulation with 0.05 mM dibutyryl cAMP (Bt(2)cAMP) did not increase StAR protein or progesterone in MA-10 mouse Leydig cells, the addition of 1 microM of the COX inhibitor indomethacin increased StAR protein expression and progesterone production by 5.7-fold and 34.3-fold, respectively. In the presence of indomethacin, the level of Bt(2)cAMP required for maximal steroidogenesis was reduced from 1.0 mM to 0.25 mM. Similar results were obtained in studies on StAR promoter activity and in Northern blot analyses of StAR mRNA expression, suggesting that inhibition of COX activity enhanced StAR gene transcription. COX2 (an inducible isoform of COX) was constitutively detected in MA-10 cells. Although SC560, a selective COX1 inhibitor, did not affect steroidogenesis, the COX2 inhibitor NS398 significantly enhanced Bt(2)cAMP-stimulated StAR protein expression and steroid production. Overexpression of the COX2 gene in COS-1 cells significantly inhibited StAR promoter activity. The results of the present study suggest that inhibition of COX2 activity increases the sensitivity of steroidogenesis to cAMP stimulation in MA-10 Leydig cells.

Involvement of 5-lipoxygenase metabolites of arachidonic acid in cyclic AMP-stimulated steroidogenesis and steroidogenic acute regulatory protein gene expression

To understand the mechanism for the role of arachidonic acid (AA) in steroidogenic acute regulatory (StAR) gene transcription, sections of the −1/−966 StAR promoter were deleted to produce constructs of −1/−426, −1/−211, −1/−151, and −1/−110 and inserted into the PGL3 vector to drive luciferase expression. Results indicated that −1/−151 StAR promoter contains the elements that are most responsive to AA. Electrophoretic mobility shift assays using nuclear extracts from AA-treated MA-10 Leydig tumor cells showed that AA enhanced specific binding of the nuclear extract to a 30 bp (−67/−96) sequence of the StAR promoter. Also, HPLC was used to identify AA metabolites involved in StAR gene transcription. It was found that 1 mM N6,2-O-dibutyryladenosine 3:5-cyclic monophosphate (dbcAMP) significantly increased the 5-lipoxygenase metabolites, 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and 5-hydroxyeicosatetraenoic acid (5-HETE). Moreover, in the presence of 0.2 mM dbcAMP addition of 20 μM 5-HPETE or 5-HETE significantly enhanced StAR protein expression and progesterone production ( P<0.05). Similar results were obtained for StAR gene transcription with StAR mRNA levels and StAR promoter activities being significantly increased ( P<0.05) when 5-HPETE was added to MA-10 cell cultures. In summary, the present studies demonstrated that cyclic AMP (cAMP) stimulated the production of the AA metabolites, 5-HPETE and 5-HETE, and showed that these metabolites enhanced StAR gene expression and steroid hormone production. The results further suggested that the AA-responsive element resides in the −67/−96 region of the StAR promoter.

Transcriptional regulation of the mouse steroidogenic acute regulatory protein gene by the cAMP response-element binding protein and steroidogenic factor 1.

Transcriptional induction by cAMP is mediated through the interaction of the cAMP response-element binding protein (CREB) with a cAMP response element (CRE) in the promoter of target genes. The steroidogenic acute regulatory (StAR) protein gene is regulated by cAMP-mediated signaling in steroidogenic cells even though its promoter lacks a consensus CRE. Previously, we have identified three highly conserved 5'-CRE half-sites within the -96/-67 bp region of the mouse StAR gene, and a member of the CREB family (CREB/CRE modulator (CREM)) was shown to be involved in its expression and regulation. Here we show that CREB and CREMtau (but not CREMalpha and CREMbeta) have qualitatively similar effects on StAR promoter activity in response to (Bu)(2)cAMP. Studies on the effects of the functional integrity of the CRE half-sites on CREB-dependent (Bu)(2)cAMP-mediated StAR gene transcription demonstrated the greater importance of the CRE2 site in comparison with the CRE1 and CRE3 sites. The CRE2 sequence was also found to bind specifically to recombinant CREB protein and nuclear extract from MA-10 mouse Leydig tumor cells. The cAMP and CREB/CREM responsive region (-151/-1 bp) of the mouse StAR promoter also contains three recognition motifs for steroidogenic factor 1 (SF-1). Electrophoretic mobility shift assays and reporter gene analyses demonstrated the involvement of different SF-1 elements in StAR gene expression with the order of importance being SF-1/3>SF-1/1>SF-1/2. Specific mutations that eliminated the binding sites of CRE and SF-1 elements, either alone or in combination, resulted in an attenuation of StAR promoter activity, indicating that CREB and SF-1 can regulate StAR gene transcription in a cooperative fashion. In addition, mammalian two-hybrid assays revealed a high affinity protein-protein interaction between CREB/CREMtau and SF-1 which appeared to be dependent upon CREB protein phosphorylation. These findings further demonstrate CREB's role in StAR gene transcription and also provide evidence that the combined action of CREB/CREMtau and SF-1 results in enhanced activation of the StAR promoter.

Transcriptional repression of the rat steroidogenic acute regulatory (StAR) protein gene by the AP-1 family member c-Fos

PGF2α, working via protein kinase C, may inhibit transcription of the StAR gene through negative regulatory factors. Administration of PGF2α increased c-Fos mRNA with a corresponding reduction in StAR mRNA. A search of the rat StAR promoter revealed three putative AP-1 elements at bp positions −85, −187 and −1561, which demonstrated specific binding of c-Fos by mobility shift assays. Co-transfection of c-Fos with the p-1862 StAR promoter caused a reduction in luciferase activity in the presence or absence of cAMP. Mutation of all three AP-1 sites in the p-1862 StAR promoter abolished c-Fos repression. Mutation of the proximal AP-1 site in the p-1862 StAR promoter reduced SF-1 mediated induction. This study is the first to demonstrate that c-Fos represses StAR gene transcription and adds to the current knowledge on the complex relationship that exists between SF-1 and c-Fos in the regulation of StAR activity.

ERKs Regulate Cyclic AMP-induced Steroid Synthesis through Transcription of the Steroidogenic Acute Regulatory (StAR) Gene

Cyclic AMP-dependent expression of the steroidogenic acute regulatory (StAR) protein is thought to be the controlling step for steroid production, but the mechanisms through which external signals are translated into increased transcription of the StAR gene are unknown. We demonstrate that cyclic AMP-induced steroid synthesis is dependent upon the phosphorylation and activation of ERKs and that ERK activation results in enhanced phosphorylation of SF-1 and increased steroid production through increased transcription of the StAR gene. Adenylate cyclase activation with forskolin (FSK) caused a time-dependent increase in ERK activity and translocation from cytoplasm to nucleus, which correlated with an increase in StAR mRNA levels, StAR protein accumulation, and steroidogenesis. Similarly, ERK inhibition led to a reduction in the levels of FSK-stimulated StAR mRNA, StAR protein, and steroid secretion. These effects were attributed to the finding that ERK activity is required for SF-1 phosphorylation, a transcription factor required for the regulation of StAR gene transcription. This conclusion was supported by our demonstration of an ERK-dependent increase in the binding of SF-1 from FSK-treated Y1 nuclei to three consensus double-stranded DNA sequences from the StAR promoter region. These observations suggest that the activation of ERK2/1 by increasing cAMP is an obligatory and regulated stage in the stimulation of steroid synthesis by cyclic AMP-generating stimuli.

The Role of Arachidonic Acid in Steroidogenesis and Steroidogenic Acute Regulatory (StAR) Gene and Protein Expression

This study was conducted to examine the mechanism for arachidonic acid (AA) regulation of steroidogenic acute regulatory (StAR) protein expression and the relationship between AA and cAMP in hormone-induced steroidogenesis. Dibutyryl cyclic AMP (Bt(2)cAMP)-stimulated MA-10 Leydig cells were treated with AA and/or the phospholipase A(2) inhibitor, dexamethasone. Dexamethasone significantly reduced Bt(2)cAMP-stimulated progesterone production, StAR promoter activity, StAR mRNA, and StAR protein. The inhibitory effects of dexamethasone were reversed by the addition of 150 microm AA to MA-10 cells. In addition, MA-10 cells were treated with the lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA), the 5-lipoxygenase inhibitor, AA861, the epoxygenase inhibitor, miconazole, and the cyclooxygenase inhibitor, indomethacin. Both NDGA and AA861 inhibited progesterone production and StAR protein expression. AA861-inhibited progesterone synthesis and StAR protein were partially reversed by addition of the 5- lipoxygenase metabolite, 5(S)-hydroperoxy-(6E,8Z,11Z, 14Z)-eicosatetraenoic acid. Inhibition of epoxygenase activity inhibited progesterone production significantly, but StAR protein was only slightly reduced. Indomethacin enhanced StAR protein expression and significantly increased progesterone production. Inhibition of AA release or lipoxygenase activities did not affect protein kinase A activity, whereas inhibition of protein kinase A activity using H89 reduced Bt(2)cAMP-induced StAR protein. AA alone did not induce StAR protein expression nor steroid production. These results demonstrate the essential role of AA in steroid biosynthesis and StAR gene transcription and suggest the possible involvement of the lipoxygenase pathway in steroidogenesis. This study further indicates that AA and cAMP transduce signals from trophic hormone receptors to the nucleus through two separate pathways and act to co-regulate steroid production and StAR gene expression and indicates that both pathways are required for trophic hormone-stimulated steroidogenesis.

Phosphoprotein Phosphatases Regulate Steroidogenesis by Influencing StAR Gene Transcription

The rate-limiting step in steroidogenesis is the transport of cholesterol into the mitochondria, and this is controlled by the steroidogenic acute regulatory (StAR) protein. We have previously shown that inhibition of phosphoprotein phosphatase 1 and 2A (PP1/2A) activities with the PP1/2A inhibitor calyculin A selectively reduces StAR protein expression and thus inhibits the synthesis of steroid hormones. The aim of this study was to determine whether this inhibition of StAR protein expression occurs at the level of transcription of StAR mRNA. We have used a competitive reverse transcription-polymerase chain reaction (RT-PCR) technique to determine whether inhibition of PP1/2A activities has any effect on the levels of StAR mRNA. Exposure of Y1 cells to forskolin significantly increased the expression of StAR mRNA and this forskolin-induced increase was reduced after exposure to Cal A at levels similar to those seen in the controls. These results suggest that cyclic AMP-induced increases in StAR mRNA levels are dependent upon phosphoprotein phosphatase activities.

Repression of the rat steroidogenic acute regulatory (StAR) protein gene by PGF2alpha is modulated by the negative transcription factor DAX-1.

The steroidogenic acute regulatory protein (StAR) is thought to mediate the rapid increase in steroid hormone biosynthesis by facilitating cholesterol transport to the inner mitochondrial membrane. Recent studies indicate that StAR gene expression is enhanced by gonadotropins, whereas prostaglandin F2alpha (PGF2alpha) appears to suppress both basal and gonadotropin-stimulated StAR mRNA levels. While studies have demonstrated that steroidogenic factor 1 (SF-1) mediates transcriptional activation of the StAR gene, the mechanism for the reduction in StAR expression requires analysis. Recent studies have shown that DAX-1 (Dosage-sensitive sex reversal adrenal hypoplasia congenita critical region on the X-chromosome, gene-1), a negative transcription factor, inhibits transcription of reporter genes in vitro. To determine whether DAX-1 could negatively regulate expression of the StAR gene, approx 2 kb of the rat StAR promoter was linked to a luciferase reporter gene (creating p-1862 StAR) and cotransfected into Y1 adrenal tumor cells and HTB9 human bladder carcinoma cells with vectors which encode DAX-1 and SF-1. Luciferase levels were significantly increased in both cell types when SF-1 was present. In contrast, when DAX-1 was cotransfected with the StAR promoter, Y1 adrenal and HTB9 cell luciferase activities were reduced to levels that were 57% and 24% of basal promoter levels, respectively. Furthermore, when dibutyryl-cAMP (dbcAMP) was added to the DAX-1 expressing cells, cAMP responsiveness was repressed 50% and 75% in Y1s and HTB9s respectively, relative to the non-DAX-1 expressing dbcAMP-treated cells. The inhibition of StAR gene transcription by DAX-1 was dose-dependent reducing transcription to 6% of control levels. Consistent with the possibility that PGF2alpha regulates ovarian StAR expression via DAX-1, Western blot analysis indicated a three- and fivefold increase in rat ovarian DAX-1 levels at 2 and 4 h following PGF2alpha injection (250 microg). The increase in DAX-1 protein corresponded to a 50% reduction in StAR mRNA levels concomitant with a 39% reduction in serum progesterone levels. Truncation of the DAX-1 protein at the C-terminal end caused a loss of inhibition of transcriptional activity. Deletion of bp -95 to -50 within the StAR promoter, a proposed DAX-1 binding site, did not alter the ability of wild-type DAX-1 to inhibit transcription. In a mammalian two-hybrid system, cotransfection of DAX-1 and SF-1 caused a 25-fold induction in luciferase activity demonstrating that these proteins interact in the two-hybrid assay. This study is the first to demonstrate that the rat StAR promoter is regulated by DAX-1 and that DAX-1 reduces StAR promoter responsiveness to cAMP. The enhanced level of DAX-1 following PGF2alpha administration is consistent with DAX-1 having a role in controlling both basal, gonadotropin-stimulated, and PGF2alpha-mediated StAR gene expression. These results imply that DAX-1 has an important role in regulating ovarian steroidogenesis by repressing StAR transcription.

Transcriptional regulation of the rat steroidogenic acute regulatory protein gene by steroidogenic factor 1.

Steroidogenic acute regulatory (StAR) protein is synthesized in response to tropic hormones to facilitate cholesterol transport to the inner mitochondrial membrane-bound P450 side-chain cleavage enzyme (P450scc), the first enzymatic step in the steroid hormone biosynthetic pathway. Gonadotropins activate expression of their target genes via the cAMP second messenger system. We have demonstrated that cAMP administration to rat luteal cells stimulates expression of both StAR messenger RNA and protein. Because cholesterol delivery is the first regulated step in steroidogenesis, and because StAR messenger RNA levels are increased in response to tropic hormone and cAMP stimulation, the mechanism by which tropic hormones/cAMP stimulate transcription needs to be elucidated. To this end, approximately 2.7 kb of the rat StAR promoter was isolated and sequenced. Sequence analysis revealed the presence of a TATA-like element as well as multiple regulatory motifs including steroidogenic factor 1 (SF-1) binding sites, an estrogen receptor half-site, and two AP-1 sites within the promoter region. 5'-RACE experiments determined the transcription start site to be located 82 bp upstream of the ATG translation start codon. Electrophoretic mobility shift assays and supershift analysis demonstrated SF-1 binding to three SF-1 binding sites in the rat StAR promoter with high affinity and two SF-1 binding sites with low affinity. Transfection of mouse Y1 adrenal tumor cells and human bladder carcinoma cells (HTB9s) with the rat StAR promoter demonstrated that SF-1 was able to activate transcription of the luciferase reporter gene and that the rat StAR promoter was responsive to cAMP. Nested deletions of the rat StAR promoter (1.9 kb) identified a region between -1413 and -998 that is essential for maximal activation of the rat StAR gene in HTB9 cells; however, deletion of this region does not affect responsiveness to cAMP. 5'-Deletion and site-directed mutagenesis experiments demonstrated that the SF-1 motifs identified within the rat StAR promoter (located at positions -764, -455, and -106) were sufficient to activate transcription as well as confer cAMP responsiveness to the rat StAR gene. Site-directed mutagenesis studies using the smallest promoter fragment demonstrated that the two proximal SF-1 binding sites are crucial for StAR gene transcription, both at a basal level and in response to cAMP stimulation. These studies provide novel insights into the regulation of the rat StAR gene at the transcriptional level by SF-1.

Atrial natriuretic peptide inhibits calcium-induced steroidogenic acute regulatory protein gene transcription in adrenal glomerulosa cells.

Atrial natriuretic peptide (ANP) is a potent inhibitor of mineralocorticoid synthesis induced in adrenal glomerulosa cells by physiological agonists activating the calcium messenger system, such as angiotensin II (Ang II) and potassium ion (K+). While the role of calcium in mediating Ang II- and K(+)-induced aldosterone production is clearly established, the mechanisms leading to blockade of this steroidogenic response by ANP remain obscure. We have used bovine adrenal zona glomerulosa cells in primary culture, in which an activation of the calcium messenger system was mimicked by a 2-h exposure to an intracellular high-calcium clamp. The effect of ANP was studied on the following parameters of the steroidogenic pathway: 1) pregnenolone and aldosterone production; 2) changes in cytosolic ([Ca2+]c) and mitochondrial ([Ca2+]m) Ca2+ concentrations, as assessed with targeted recombinant aequorin; 3) cholesterol content in outer mitochondrial membranes (OM), contact sites (CS), and inner membranes (IM); 4) steroidogenic acute regulatory (StAR) protein import into mitochondria by Western blot analysis; 5) StAR protein synthesis, as determined by [35S]methionine incorporation, immunoprecipitation, and SDS-PAGE; 6) StAR mRNA levels by Northern blot analysis with a StAR cDNA; 7) StAR gene transcription by nuclear run-on analysis. While clamping Ca2+ at 950 nM raised pregnenolone output 3.5-fold and aldosterone output 3-fold, ANP prevented these responses with an IC50 of 1 nM and a maximal effect of 90% inhibition at 10 nM. In contrast, ANP did not affect the [Ca2+]c or [Ca2+]m changes occurring under Ca2+ clamp or Ang II stimulation in glomerulosa cells. The accumulation of cholesterol content in CS (139.7 +/- 10.7% of control) observed under high-Ca2+ clamp was prevented by 10 nM ANP (92.4 +/- 4% of control). Similarly, while Ca2+ induced a marked accumulation of StAR protein in mitochondria of glomerulosa cells to 218 +/- 44% (n = 3) of controls, the presence of ANP led to a blockade of StAR protein mitochondrial import (113.3 +/- 15.0%). This effect was due to a complete suppression of the increased [35S]methionine incorporation into StAR protein that occurred under Ca2+ clamp (94.5 +/- 12.8% vs. 167.5 +/- 17.3%, n = 3). Furthermore, while the high-Ca2+ clamp significantly increased StAR mRNA levels to 188.5 +/- 8.4 of controls (n = 4), ANP completely prevented this response. Nuclear run-on analysis showed that increases in intracellular Ca2+ resulted in transcriptional induction of the StAR gene and that ANP inhibited this process. These results demonstrate that Ca2+ exerts a transcriptional control on StAR protein expression and that ANP appears to elicit its inhibitory effect on aldosterone biosynthesis by acting as a negative physiological regulator of StAR gene expression.