Steroidogenic Acute Regulatory Promoter Activity Research Articles

Regulation of retinoid mediated cholesterol efflux involves liver X receptor activation in mouse macrophages

Removal of cholesterol from macrophage-derived foam cells is a critical step to the prevention of atherosclerotic lesions. We have recently demonstrated the functional importance of retinoids in the regulation of the steroidogenic acute regulatory (StAR) protein that predominantly mediates the intramitochondrial transport of cholesterol in target tissues. In the present study, treatment of mouse macrophages with retinoids, particularly all-trans retinoic acid (atRA) and 9-cis RA, resulted in increases in cholesterol efflux to apolipoprotein AI (Apo-A1). Activation of the PKA pathway by a cAMP analog, (Bu)2cAMP, markedly augmented retinoid mediated cholesterol efflux. Macrophages overexpressing hormone-sensitive lipase increased the hydrolysis of cholesteryl esters and concomitantly enhanced the efficacy of retinoic acid receptor and liver X receptor (LXR) ligands on StAR and ATP-binding cassette transporter A1 (ABCA1) protein levels. RAs elevated StAR promoter activity in macrophages, and an increase in StAR levels augmented cholesterol efflux to Apo-A1, suggesting retinoid-mediated efflux of cholesterol involves enhanced oxysterol production. Further studies revealed that retinoids activate the LXR regulated genes, sterol receptor-element binding protein-1c and ABCA1. These findings provide insights into the regulatory events in which retinoid signaling effectively enhances macrophage cholesterol efflux and indicate that retinoid therapy may have important implications in limiting and/or regressing atherosclerotic cardiovascular disease.

Valproic acid alters mitochondrial cholesterol transport in Y1 adrenocortical cells

Several reports suggest putative interactions between valproic acid (VPA) treatment and the hypothalamus–pituitary–adrenal axis. Given that VPA alters mitochondrial functions, an action of this drug on a mitochondrial process such as steroid synthesis in adrenal cells should be expected. In order to disclose a putative action of VPA on the adrenocortical cell itself we evaluated VPA effects on regulatory steps of the acute stimulation of steroidogenesis in Y1 adrenocortical cells. This study demonstrates that VPA increases progesterone production in non-stimulated cells without inducing the levels of Steroidogenic Acute Regulatory (StAR) protein, which facilitates cholesterol transport. This result suggests that VPA increases mitochondrial cholesterol transport through a StAR-independent mechanism and is further supported by the fact that in isolated mitochondria VPA stimulates exogenous cholesterol metabolization to progesterone. VPA also reduces the cAMP-mediated increase of the StAR protein, mRNA levels, promoter activity and progesterone production. In summary, the present data show that VPA can alter steroid production in adrenal cells by a complex mechanism that mainly involves an action on cholesterol access to the inner mitochondrial membrane. The VPA-mediated increase of basal steroidogenesis could be linked to the increase of basal cortisolemia described in patients under VPA treatment.

Effects of apigenin on steroidogenesis and steroidogenic acute regulatory gene expression in mouse Leydig cells

Previous studies reported that the age-related decline in testosterone biosynthesis is associated with a decrease in the steroidogenic acute regulatory (StAR) protein which regulates the rate-limiting step of testosterone biosynthesis. To explore the possibility of delaying this decline using a dietary approach, we have examined the effect of a natural flavonoid, apigenin, on StAR gene expression in mouse Leydig cells. Incubation of these cells with the flavonoid enhanced cyclic adenosine monophosphate (cAMP)-induced steroidogenesis and StAR protein expression. The results from the analyses of StAR mRNA by reverse transcription-polymerase chain reaction and the luciferase assays of StAR promoter activity indicated that this flavonoid enhanced StAR gene expression at the level of transcription. Further studies showed that apigenin blocked the thromboxane A2 receptor and interrupted the signaling through the cyclooxygenase-2-thromboxane A synthase-thromboxane A2-receptor pathway, resulting in a reduction of DAX-1 (dosage sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene-1) protein, a transcriptional repressor of StAR gene expression. When DAX-1 protein was reduced, the sensitivity of the Leydig cells was dramatically enhanced, with sub-threshold level of cAMP being able to induce maximal levels of StAR protein expression and steroid hormone production. The present study suggests a potential application of apigenin to improve StAR protein expression and steroidogenic sensitivity of aging Leydig cells.

Antagonistic Regulation of StAR Gene Expression and Synergistic Regulation of Mc2R Gene Expression by FoxL2 and SF-1.

FoxL2, a gene encoding a forkhead transcription factor, is one of the earliest ovarian markers and plays an important role in regulation of cholesterol and steroid metabolism, inflammation, apoptosis, and ovarian development and function. Mutations and deficiencies of FoxL2 have been shown to cause blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) as well as ovarian defects. Previous reports have shown that FoxL2 represses the promoter activity of the steroidogenic acute regulatory (StAR) gene by binding to StAR promoter. SF-1, an orphan nuclear receptor, is expressed throughout adrenal cortex and gonads where it regulates a number of genes involved in steroid hormone biosynthesis, including StAR and Mc2R genes. It is defined that FoxL2 interacts with SF1 in fish and up-regulates aromatase gene transcription. However, this interaction has not been studied in mammals. Therefore, we are interested in the interplay between FoxL2 and SF-1 in regulating StAR and Mc2R gene expression in mammalian systems. First, we find that FoxL2 interacts with SF-1 in mammalian two-hybrid system. While both FoxL2 and SF-1 are expressed in both ovary and adrenal gland tissues, FoxL2 is predominantly expressed in the ovary. Interestingly, we find that FoxL2 and SF-1 are highly expressed in day 12 and day18 of mouse embryonic tissues. As expected, FoxL2 represses and SF-1 enhances StAR promoter activity. Interestingly, we find that SF-1 cannot rescue FoxL2's repressive activity even at very high doses. This indicates that repressive activity of FoxL2 on StAR promoter may be due to competition binding to SF-1's response element. Numerous genes required for adrenocortical steroidogenesis are also activated by SF-1. Interestingly, Mc2R promoter activity is activated by FoxL2 in a dose-dependent manner. Surprisingly, we find that FoxL2 and SF-1 synergistically up-regulate the luciferase activity of Mc2R promoter. Because the -2000-bp mouse Mc2R promoter contains two SF-1 and multiple forkhead-responsive consensus sites, the Mc2R promoter was truncated to determine the minimal region that is important for synergistically transcriptional up-regulation by FoxL2 and SF-1. The results suggest that the first 1320 bp upstream of the Mc2R transcriptional start site are sufficient for synergistically transcriptional up-regulation by FoxL2 and SF-1. In summary, these results indicate that FoxL2 and SF-1 may play important and divergent roles in regulating adrenal and gonadal tissue differentiation and development as well as steroidogenesis. (poster)

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.

Cyclic Adenosine 3′,5′-Monophosphate Response Element-Binding Protein and CCAAT/Enhancer-Binding Protein Mediate Prostaglandin E 2-Induced Steroidogenic Acute Regulatory Protein Expression in Endometriotic Stromal Cells

Aberrant expression of the steroidogenic acute regulatory (StAR) protein in human endometriotic stromal cells plays an important role in the development of endometriosis. Prostaglandin E(2) (PGE(2)) is a potent inducer of StAR expression in these cells; however, the mechanisms responsible for the transcriptional regulation of StAR remain to be elucidated. Herein we report that PGE(2)-induced StAR expression is independent of the transcriptional suppressor DAX-1 but is regulated by the transcriptional activator cyclic adenosine 3',5'-monophosphate (cAMP) response element-binding protein (CREB). A promoter activity assay revealed that the cis-element needed for the binding of the CCAAT/enhancer-binding protein (C/EBP) was critical for PGE(2)-induced StAR expression. Electrophoretic mobility shift assay demonstrated that this region of the StAR promoter was bound by C/EBPalpha, C/EBPbeta, and CREB. Forced expression of either C/EBPalpha or C/EBPbeta alone was sufficient to up-regulate StAR promoter activity whereas PGE(2) was needed to induce StAR promoter activity in CREB-overexpressed cells. Results from a chromatin immunoprecipitation assay demonstrated that the binding of C/EBPbeta to the StAR promoter was increased whereas CREB binding was unchanged after PGE(2) treatment. Taken together, PGE(2)-induced StAR promoter activity appears to be regulated by CREB and C/EBPbeta in a cooperative manner in ectopic human endometriotic stromal cells, providing a molecular framework for the etiology of endometriosis.

The Orphan Nuclear Receptor NUR77 Regulates Hormone-Induced StAR Transcription in Leydig Cells through Cooperation with Ca2+/Calmodulin-Dependent Protein Kinase I

Cholesterol transport in the mitochondrial membrane, an essential step of steroid biosynthesis, is mediated by a protein complex containing the steroidogenic acute regulatory (StAR) protein. The importance of this transporter is underscored by mutations in the human StAR gene that cause lipoid congenital adrenal hyperplasia, male pseudohermaphroditism, and adrenal insufficiency. StAR transcription in steroidogenic cells is hormonally regulated and involves several transcription factors. The nuclear receptor NUR77 is present in steroidogenic cells, and its expression is induced by hormones known to activate StAR expression. We have now established that StAR transcription in cAMP-stimulated Leydig cells requires de novo protein synthesis and involves NUR77. We found that cAMP-induced NUR77 expression precedes that of StAR both at the mRNA and protein levels in Leydig cells. In these cells, small interfering RNA-mediated NUR77 knockdown reduces cAMP-induced StAR expression. Chromatin immunoprecipitation assays revealed a cAMP-dependent increase in NUR77 recruitment to the proximal StAR promoter, whereas transient transfections in MA-10 Leydig cells confirmed that NUR77 can activate the StAR promoter and that this requires an element located at -95 bp. cAMP-induced StAR and NUR77 expression in Leydig cells was found to require a Ca2+/calmodulin-dependent protein kinase (CaMK)-dependent signaling pathway. Consistent with this, we show that within the testis, CaMKI is specifically expressed in Leydig cells. Finally, we report that CaMKI transcriptionally cooperates with NUR77, but not steroidogenic factor 1, to further enhance StAR promoter activity in Leydig cells. All together, our results implicate NUR77 as a mediator of cAMP action on StAR transcription in steroidogenic Leydig cells and identify a role for CaMKI in this process.

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.

Cholesterol sulphate affects production of steroid hormones by reducing steroidogenic acute regulatory protein level in adrenocortical cells

Steroidogenic acute regulatory (StAR) protein plays a crucial role in the intramitochondrial movement of cholesterol, where P450 side chain cleavage enzyme resides. Cholesterol sulphate (CS), which is present ubiquitously in mammalian tissues, is not only a precursor of sulphated adrenal steroids but also an inhibitor of cholesterol biosynthesis. This study was designed to examine the biological roles of CS in steroidogenesis in adrenocortical cells. Human adrenocortical carcinoma H295R cells were cultured with various amounts of CS. To evaluate steroid hormone synthesis, pregnenolone production in cells was assayed. The amount of pregnenolone produced by H295R cells in culture medium, to which over 50 mug/ml CS was added, was significantly (P<0.05) decreased compared with that produced by control cells. Western blot analysis was performed to determine StAR protein level using whole cell extracts from cells. StAR protein level decreased when the concentration of CS in the medium was 50 mug/ml, whereas the level of glyceraldehyde-3-phosphate dehydrogenase did not change. To examine the mechanism by which StAR gene expression is controlled, we performed RT-PCR and measured promoter activity in cells transfected with pGL(2) StAR reporter constructs. StAR mRNA level and promoter activity were decreased in cells. The decrease in StAR protein level is a result of the low StAR gene expression level. In conclusion, CS affects the production of steroid hormones by reducing StAR protein level in adrenocortical cells.

Prostaglandin F2α Suppresses Rat Steroidogenic Acute Regulatory Protein Expression via Induction of Yin Yang 1 Protein and Recruitment of Histone Deacetylase 1 Protein

Prostaglandin F2alpha (PGF2alpha) plays a pivotal role in ovarian luteolysis by inhibiting the expression of steroidogenic acute regulatory (StAR) protein, leading to a decrease in intracellular cholesterol transport and luteal steroid production. Previously we have demonstrated that the transcription factor Yin Yang 1 (YY1) bound to three regions in the StAR promoter in vitro and repressed promoter activity. This study further defined the YY1-mediated PGF2alpha effect on the inhibition of StAR protein expression through YY1 interaction with a single region in the StAR promoter in vivo. PGF2alpha consistently suppressed StAR mRNA and protein expression in cultured luteal cells in a dose-dependent manner. PGF2alpha also enhanced YY1 protein expression and binding to its cis-element in a time-dependent pattern that preceded the decline in StAR protein levels. The StAR promoter region bound by YY1 was also associated with histone deacetylase 1 (HDAC1). PGF2alpha treatment promoted HDAC1 binding to and suppressed the histone H3 acetylation in this region. On the contrary, YY1 knockdown decreased HDAC1 binding, increased histone H3 acetylation, enhanced StAR protein expression, and negated PGF2alpha effect on StAR protein expression. Luciferase assays showed that YY1 overexpression inhibited StAR promoter activity and the addition of a HDAC inhibitor, trichostatin A, abrogated the effect of YY1. Trichostatin A-treated luteal cells displayed increased StAR protein expression. These data indicate that PGF2alpha enhances a direct YY1/StAR promoter interaction and the recruitment of HDAC1 to the promoter, thereby preventing transcriptional activation of the StAR gene.

Effects of genistein, resveratrol, and quercetin on steroidogenesis and proliferation of MA-10 mouse Leydig tumor cells

This study was performed to compare the effects of three well-known phytoestrogens such as genistein, resveratrol, and quercetin on steroidogenesis in MA-10 mouse tumor Leydig cells. Addition of genistein or resveratrol to MA-10 cells resulted in decreases in the cAMP-stimulated progesterone secretion, but quercetin had an opposite response. Steroidogenic acute regulatory (StAR) mRNA expression and StAR promoter activity in transiently transfected MA-10 cells were significantly reduced by genistein or resveratrol, but increased by quercetin. Genistein was found to inhibit MA-10 cell proliferation, while resveratrol and quercetin had no effect. Quercetin-induced increase in cAMP-stimulated progesterone secretion was reversed by ICI 182,780, an estrogen receptor (ER) antagonist. However, ICI 182,780 had no effect on cAMP plus quercetin-stimulated StAR promoter activity. To examine whether non-ER factors are associated with quercetin-stimulated progesterone production, we treated MA-10 cells with EGTA to deprive them of extracellular Ca(2+). We found that EGTA inhibited quercetin-plus cAMP-stimulated progesterone secretion and StAR promoter activity. Blocking of Ca(2+) influx through L- or T-type voltage-gated Ca(2+) channels with verapamil or mibefradil respectively, attenuated quercetin-stimulated progesterone secretion, while they had no effect on quercetin-plus cAMP-stimulated StAR promoter activity. Blocking of intracellular Ca(2+) efflux by sodium orthovanadate, a Ca(2+)-pump inhibitor, blocked quercetin- plus cAMP-stimulated progesterone secretion and StAR promoter activity in MA-10 cells. Finally, EGTA or vanadate reduced quercetin and cAMP-increased in StAR mRNA expression in MA-10 cells, while ICI 182,780 had no effect. Taken together, these results indicate that phytoestrogens have differential effects on steroidogenesis in MA-10 cells.

Adipocyte-derived products induce the transcription of the StAR promoter and stimulate aldosterone and cortisol secretion from adrenocortical cells through the Wnt-signaling pathway

Obesity is associated with hypersecretion of cortisol and aldosterone and a high prevalence of arterial hypertension. At the cellular level, a direct effect of adipocytes on the expression of the steroidogenic acute regulatory (StAR) protein, a regulator of cortisol and aldosterone synthesis, and on aldosterone and cortisol secretion has been shown. However, the molecular mechanisms mediating this effect are not known. Wnt-signaling molecules are secreted by adipocytes and regulate the activity of SF-1, a key transcription factor in adrenal steroidogenesis. Therefore, we investigated whether adipocytes stimulate adrenal steroidogenesis through the activation of Wnt-signaling. Using immunohistochemistry, we detected the expression of frizzled and beta-catenin in the adult human adrenal cortex. Transient transfection of a Wnt-dependent reporter-gene into adrenal NCI-H295R cells showed an induction of Wnt-mediated transcription to 308% after treatment with human fat cell-conditioned medium (FCCM). This finding was paralleled by an induction of StAR promoter activity (420%) by FCCM. The induction of StAR promoter activity by FCCM was inhibited by 49% when Wnt-signaling was blocked by the soluble Wnt-antagonist secreted Frizzled-Related-Protein-1 (sFRP-1). Overexpression of a constitutively active mutant of beta-catenin induced the transcription of the StAR promoter (440%). beta-Catenin and FCCM induced SF-1-mediated transcription at a SF-1-driven reporter gene (420 and 402%, respectively). Furthermore, the secretion of aldosterone and cortisol by NCI-H295R cells induced by FCCM was significantly inhibited by the Wnt-antagonist sFRP-1. These data indicate that the Wnt-signaling pathway is one of the mechanisms mediating the effects of fat cells on adrenal StAR transcription and aldosterone and cortisol secretion.

CREM confers cAMP responsiveness in human steroidogenic acute regulatory protein expression in NCI-H295R cells rather than SF-1/Ad4BP

Steroidogenic acute regulatory (StAR) protein plays a critical role in steroid hormone synthesis. Tropic hormones induce human StAR gene expression by a cAMP-dependent pathway. Steroidogenic factor-1/adrenal-4-binding protein (SF-1/Ad4BP) plays an important role in the expression of human StAR gene. We investigated the mechanism of cAMP responsiveness in human StAR gene expression in NCI-H295R cells. The StAR promoter activity and protein levels in cells subjected to various treatments were examined. Anti-SF-1/Ad4BP IgG transfection treatment resulted in decreases in the basal StAR promoter activity and StAR protein levels, but did not affect cAMP-stimulated promoter activity and protein levels. The basal and cAMP-stimulated StAR promoter activity levels were reduced in SF-1/Ad4BP mutant (G35E)-transfected cells, but the cAMP induction of StAR promoter activity in response to 1 mM 8-Br-cAMP was not inhibited when G35E SF-1/Ad4BP mutant expression vectors were co-transfected with cAMP-response element-binding (CREB) expression vectors. Although the basal StAR mRNA expression and protein levels were decreased by SF-1/Ad4BP-siRNA treatment, the cAMP-stimulated StAR mRNA expression and protein levels did not change. The basal StAR promoter activity level was not decreased by cAMP-response element modulator (CREM)-siRNA treatment, but the cAMP-stimulated StAR promoter activity level, the magnitude of cAMP induction of StAR promoter, and the cAMP-stimulated StAR protein level were decreased. The cAMP induction of StAR promoter activity in cells was inhibited when S117ACREM mutant expressionvectors were transfected. We conclude that inhibition of the function of SF-1/Ad4BP does not reduce the cAMP induction of StAR promoter activity and protein level. CREM is needed to confer cAMP responsiveness in human StAR protein expression.

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.

A novel mutation in the steroidogenic acute regulatory protein gene promoter leading to reduced promoter activity

We have identified a novel cytosine/thymidine polymorphism of the human steroidogenic acute regulatory (StAR) gene promoter located 3 bp downstream of the steroidogenic factor-1 (SF-1)-binding site and 9 bp upstream of the TATA box (ATTTAAG). Carriers of this mutation have a high prevalence of primary aldosteronism. In transfection experiments, basal StAR promoter activity was unaltered by the mutation in murine Y-1 cells and human H295R cells. In Y-1 cells, forskolin (25 microM, 6 h) significantly increased wild-type promoter activity to 230+/-33% (P<0.05, n=4). In contrast, forskolin increased mutated promoter activity only to 150+/-27%, with a significant 35% reduction compared to wild type (P<0.05, n=3). In H295R cells, angiotensin II (AngII; 10 nM) increased wild-type StAR promoter activity to 265+/-22% (P<0.01, n=3), while mutated StAR promoter activity in response to AngII only reached 180+/-29% of controls (P< 0.01, n=3). Gel mobility shift assays show the formation of two additional complexes with the mutated promoter: one with the transcription repressor DAX-1 and another with a yet unidentified factor, which strongly binds the SF-1 response element. Thus, this novel mutation in the human StAR promoter is critically involved in the regulation of StAR gene expression and is associated with reduced promoter activity, a finding relevant for adrenal steroid response to physiological stimulators.

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.

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.

Regulation of steroidogenesis and the steroidogenic acute regulatory protein by a member of the cAMP response-element binding protein family.

The mitochondrial phosphoprotein, the steroidogenic acute regulatory (StAR) protein, is an essential component in the regulation of steroid biosynthesis in adrenal and gonadal cells through cAMP-dependent pathways. In many cases transcriptional induction by cAMP is mediated through the interaction of a cAMP response-element binding protein (CREB) family member with a consensus cAMP response element (CRE; 5'-TGACGTCA-3') found in the promoter of target genes. The present investigation was carried out to determine whether a CRE-binding protein (CREB) family member [CREB/CRE modulator (CREM) family] was involved in the regulation of steroidogenesis and StAR protein expression. Transient expression of wild- type CREB in MA-10 mouse Leydig tumor cells further increased the levels of (Bu)2cAMP-induced progesterone synthesis, StAR promoter activity, StAR mRNA, and StAR protein. These responses were significantly inhibited by transfection with a dominant-negative CREB (A-CREB), or with a CREB mutant that cannot be phosphorylated (CREB-M1), the latter observation indicating the importance of phosphorylation of a CREB/CREM family member in steroidogenesis and StAR expression. The CREB/CREM-responsive region in the mouse StAR gene was located between -110 and -67 bp upstream of the transcriptional start site. An oligonucleotide probe (-96/-67 bp) containing three putative half-sites for 5'-canonical CRE sequences (TGAC) demonstrated the formation of protein-DNA complexes in EMSAs with recombinant CREB protein as well as with nuclear extracts from MA-10 or Y-1 mouse adrenal tumor cells. The predominant binding factor observed with EMSA was found to be the CREM protein as demonstrated using specific antibodies and RT-PCR analyses. The CRE elements identified within the -96/-67 bp region were tested for cAMP responsiveness by generating mutations in each of the CRE half-sites either alone or in combination. Although each of the CRE sites contribute in part to the CREM response, the CRE2 appears to be the most important site as determined by EMSA and by reporter gene analyses. Binding specificity was further assessed using specific antibodies to CREB/CREM family members, cold competitors, and mutations in the target sites that resulted in either supershift and/or inhibition of these complexes. We also demonstrate that the inducible cAMP early repressor markedly diminished the endogenous effects of CREM on cAMP-induced StAR promoter activity and on StAR mRNA expression. These are the first observations to provide evidence for the functional involvement of a CREB/CREM family member in the acute regulation of trophic hormone-stimulated steroidogenesis and StAR gene expression.