p38 MAPK regulates steroidogenesis through transcriptional repression of STAR gene.

STAR/StarD1, part of a protein complex, mediates the transport of cholesterol from the outer to inner mitochondrial membrane, which is the rate-limiting step for steroidogenesis, and where steroid hormone synthesis begins. Herein, we examined the role of oxidant-sensitive p38 MAPKs in the regulation of STAR gene transcription, using model steroidogenic cell lines. Our data indicate that oxidant activation of p38 MAPK exhibits a negative regulatory role in the induction of functional expression of STAR, as evidenced by enhanced induction of STAR (mRNA/protein) expression and increased steroidogenesis during pharmacological inhibition of p38 MAPK or in cells with increased transient overexpression of a dominant-negative (dn) form of p38 MAPKα or p38 MAPKß. Studies with rat Star-promoter demonstrated that overexpression of p38 MAPKα-wt, -ß, or -γ significantly reduced both basal and cAMP-sensitive promoter activity. In contrast, overexpression of p38 MAPKα-dn, -ß, or -γ enhanced the Star promoter activity under basal conditions and in response to cAMP stimulation. Use of various constitutively active and dn constructs and designer knock-out cell lines demonstrated that MKK3 and MKK6, the upstream activators of p38 MAPKs, play a role in p38 MAPKα-mediated inhibition of Star promoter activity. In addition, our studies raised the possibility of CREB being a potential target of the p38 MAPK inhibitory effect on Star promoter activity. Collectively, these data provide novel mechanistic information about how oxidant-sensitive p38 MAPKs, particularly p38 MAPKα, contribute to the negative regulation of Star gene expression and inhibit steroidogenesis.

Telomere shortening and DNA damage of embryonic stem cells induced by cigarette smoke.

Embryonic stem cells (ESCs) provide a valuable in vitro model for testing toxicity of chemicals and environmental contaminants including cigarette smoke. Mouse ESCs were acutely or chronically exposed to smoke components, cigarette smoke condensate (CSC), or cadmium, an abundant component of CSC, and then evaluated for their self-renewal, apoptosis, DNA damage and telomere function. Acute exposure of ESCs to high dose of CSC or cadmium increased DNA damage and apoptosis. Yet, ESCs exhibited a remarkable capacity to recover following absence of exposure. Chronic exposure of ESCs to low dose of CSC or cadmium resulted in shorter telomeres and DNA damage. Together, acute exposure of ESCs to CSC or cadmium causes immediate cell death and reduces pluripotency, while chronic exposure of ESCs to CSC or cadmium leads to DNA damage and telomere shortening. Notably, a sub-proportion of ESCs during passages is selected to resist to smoke-induced oxidative damage to telomeres.

Characterization of regulatory intronic and exonic sequences involved in alternative splicing of scavenger receptor class B gene.

Scavenger receptor class B type I (SR-BI) is a major receptor of the high-density lipoprotein that mediates cholesterol efflux and reverse cholesterol transport. Alternative splicing of the scavenger receptor class B (SR-B) gene is observed and different splice forms, SR-BI and scavenger receptor class B type II (SR-BII), have been shown to function and localize in distinct ways. We have previously shown that SR-B alternative splicing regulation is associated with splicing factor ASF/SF2. In this study, using a SR-B minigene as a model, we determined the critical regulatory regions in the upstream intron, intron 11, by serial deletion and mutation analyses. We also further characterized the regulatory elements in intron 11 as well as in the skipped exon, exon 12. Moreover, we studied the interactions of these elements with the splicing factor ASF/SF2. This study provides new insights into the mechanism of SR-B splicing and it is important for further study on the mechanism of SR-B alternative splicing regulation, such as its regulation by estrogen.

Lauric acid dependent enhancement in hepatic SCPx protein requires an insulin deficient environment.

Sterol carrier protein X (SCPx) is a peroxisomal protein with both lipid transfer and thiolase activity. Treating with the fatty acid, lauric acid, induced SCPx mRNA levels in rat liver and in rat hepatoma H4IIE cells but enhanced protein levels of SCPx and the thiolase produced as a post-translational modification of SCPx were only seen in H4IIE cells. Further investigation revealed that the presence of insulin can mask lauric acid effects on the SCPx gene especially at the protein level. These data are in agreement with the findings that diabetes, a medical condition characterized by high levels of fatty acids in an insulin deficient environment, enhances the hepatic expression of SCPx.

Prostaglandin F2alpha 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.

Regulation of alternative splicing of liver scavenger receptor class B gene by estrogen and the involved regulatory splicing factors.

The scavenger receptor class B isoforms (SR-B) type I and type II mediate the selective uptake of high-density lipoprotein cholesterol and promote reverse cholesterol transport, an important atherosclerosis protection mechanism, in the liver. Previously it was shown that the hepatic expression of SR-BI and SR-BII is regulated by estrogen. In the present study, we demonstrate that estrogen differentially regulates expression of the glycosylated and nonglycosylated forms of SR-BI and SR-BII in rat liver and hepatic cells. We report that estrogen mainly induces the down-regulation of glycosylated SR-BI and the up-regulation of nonglycosylated SR-BII. To study how estrogen regulates expression of the SR-B isoforms, we constructed a SR-B minigene containing minimal genomic sequences and were able to demonstrate that estrogen directly regulates the pre-mRNA alternative splicing of the exogenously expressed SR-B minigene in hepatic cells. Furthermore, we showed that the overexpression of splicing factors alternative splicing factor/splicing factor 2, Transformer (Tra)-2alpha, and Tra2beta changes the splicing pattern of SR-B dramatically, whereas other splicing factors, such as heterogeneous nuclear ribonucleoprotein-G, SC-35, and arginine/serine-rich p40, had no effect. We also demonstrate that estrogen regulates Tra2beta expression levels in liver cells. These studies suggest that estrogen may regulate SR-B isoform expression at both the RNA splicing and posttranslational modification levels and that, for alternative splicing regulation, estrogen may function by regulating the expression of the splicing factors alternative splicing factor/splicing factor 2, Tra2alpha, and especially Tra2beta.

Activation of the SCPx promoter in mouse adrenocortical Y1 cells.

Sterol carrier protein X (SCPx) is a peroxisomal protein with both lipid transfer and thiolase activity. Treatment of mouse adrenal Y1 cells with cAMP for 24h caused a significant induction of SCPx mRNA levels. Reporter gene studies demonstrated that treatment with cAMP and SF-1 was able to activate the SCPx promoter. Sequence analysis revealed the presence of three putative steroidogenic factor-1 (SF-1) binding motifs (designated SFB1, SFB2, and SFB3) and one CRE. Only SFB1 and SFB3 were able to bind recombinant SF-1 protein in electrophoretic mobility shift assays. The CRE was able to form a DNA/protein complex in the presence of Y1 nuclear extracts. Mutational analysis studies demonstrated that SFB3 is required for full activation of the SCPx promoter by cAMP treatment. Regulation of the SCPx gene by SF-1 and cAMP is similar to the regulatory mechanisms observed for other steroidogenic genes.

Activation of the rat scavenger receptor class B type I gene by PPARalpha.

Peroxisomal proliferator activated receptor alpha (PPARalpha) is activated by fibrate drugs which are known to protect against atherosclerosis. The present study examines the effects of PPARalpha on SR-BI expression. For this study, a rat SR-BI promoter-luciferase reporter gene construct was co-transfected into different cell lines with expression vectors that encode for PPARalpha+/-retinoic X receptor alpha (RXRalpha). PPARalpha/RXR increased the activity of the SR-BI promoter, an effect that was enhanced by clofibrate. Sequence analysis of the rat SR-BI promoter revealed the presence of a putative peroxisomal proliferator response element (PPRE) at bp -1,622. Electrophoretic mobility shift assays demonstrated that PPARalpha and RXRalpha are able to bind to the SR-BI PPRE motif. In addition, mutational analysis studies confirmed that this PPRE motif is responsible for the PPARalpha/RXRalpha-dependent activation of the rat SR-BI promoter in the cell lines examined.

Estrogen regulation of the scavenger receptor class B gene: Anti-atherogenic or steroidogenic, is there a priority?

High density lipoprotein (HDL) participates in reverse cholesterol transport and in the delivery of cholesterol to the liver and steroidogenic tissues by a mechanism called "selective lipid uptake" which is mediated by the HDL receptor, scavenger receptor B type I (SR-BI). Overexpression of SR-BI suppresses atherosclerosis by increasing reverse cholesterol transport. In contrast, genetic ablation of SR-BI has a negative effect on cardiovascular physiology in both males and females and a gender specific negative impact on female fertility. Cholesterol is essential for mammalian embryonic development as a necessary component of cell membranes and as a substrate for steroidogenesis. The SR-BI receptor is highly expressed in the human placenta allowing the growing fetus to obtain a considerable portion of cholesterol from maternal lipoproteins. Estrogen, which plays an important role in maintaining pregnancy, has been shown to enhance plasma HDL levels and promote reverse cholesterol transport. Since SR-BI is the major determinant of serum HDL levels, direct regulation of the SR-BI gene by estrogen is theorized. The objective of this manuscript is to summarize the current information related to estrogen regulation of the gene that codes for the SR-BI receptor.

Peroxisome proliferator-activated receptor alpha induces rat sterol carrier protein x promoter activity through two peroxisome proliferator-response elements.

Sterol carrier protein x (SCPx) plays a critical role in the peroxisomal oxidation of fatty acids. It has been previously demonstrated in streptozotocin-induced diabetic rats that SCPx expression is induced in association with an elevation in serum fatty acid and triglyceride levels. To elucidate the mechanisms underlying the expression of this gene during diabetes, the rat SCPx promoter was cloned and analyzed for regulatory motifs. Sequence analysis of this TATA-less promoter revealed two putative peroxisomal-proliferator-response element (PPRE) binding motifs at positions -134 and -869 relative to the translation start site. To examine peroxisomal-proliferator-activated receptor alpha (PPARalpha) effects on this gene, 935 bp of the SCPx promoter containing both PPRE motifs was cloned in front of the chloramphenicol acetyl-transferase gene or the luciferase gene and co-transfected into HTB-9 cells with vectors that encoded for PPARalpha and retinoid X receptor (RXR). The results indicate that PPARalpha was able to induce SCPx promoter activity in both cases, an effect that was enhanced by RXR and clofibrate. In addition, mutational analysis studies demonstrated that both PPREs contributed to the PPARalpha/RXRalpha-dependent activation of the SCPx promoter. Mobility shift assays and supershift analysis showed that nuclear extracts containing PPARalpha bound to the two PPRE motifs. This investigation indicates that similar to other genes involved in beta-oxidation, SCPx transcription may be controlled by fatty acid levels via PPARalpha.

Estrogen activates the high-density lipoprotein receptor gene via binding to estrogen response elements and interaction with sterol regulatory element binding protein-1A.

The effects of E2 on the high-density lipoprotein receptor (HDL-R) scavenger receptor class B type I (SR-BI) gene were examined. Four putative estrogen response element half-site motifs (ERE(1/2)) (-2176, -1726, -1622, and -1211, designated ERE(1/2)-1, 2, 3, and 4, respectively) were identified in the HDL-R SR-BI promoter. Transfection studies and mutation analysis demonstrated that E2 significantly increased HDL-R SR-BI promoter activity and that mutating ERE(1/2)-1, 2, and 4 resulted in a loss of E2 responsiveness. Both ER alpha and ER beta formed specific complexes with ERE(1/2)-1, 2, and 4 but did not bind ERE(1/2)-3 in vitro. Interestingly, ERE(1/2)-3 was the motif shown not to be important for E2-activation of the HDL-R SR-BI promoter in the mutational analysis studies. The influence of SREBP-1a (sterol regulatory element binding protein-1a) on E2 regulation of the HDL-R SR-BI gene was also examined. SREBP-1a was able to bind directly to the ERE(1/2) motifs and enhanced ER binding when both ER subtypes were present. ER alpha and beta also bound to a sterol response element motif, but they did not enhance SREBP-1a binding. Cotransfection studies demonstrated that the presence of the three factors, ER alpha, ER beta, and SREBP-1a, enhanced the overall luciferase activity produced from the HDL-R SR-BI promoter construct in the presence of only one of the factors. Interaction of SREBP-1a with both ERs was demonstrated using a mammalian two-hybrid assay. The data confirmed that E2 through the ERs can positively regulate the HDL-R SR-BI through binding and activation of three ERE(1/2) motifs and identified SREBP-1a as a potential coactivator of the E2-ER-dependent effects on the HDL-R SR-BI gene.

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

PGF2alpha, working via protein kinase C, may inhibit transcription of the StAR gene through negative regulatory factors. Administration of PGF2alpha 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.

Repression of the steroidogenic acute regulatory gene by the multifunctional transcription factor Yin Yang 1.

The transport of cholesterol to the inner mitochondrial membrane by the steroidogenic acute regulatory (StAR) protein is a critical step in steroidogenesis. The current study was designed to examine whether the multifunctional transcription factor Yin Yang 1 (YY1) was able to repress activation of the StAR gene. YY1 bound to three putative YY1-binding sites in the rat StAR promoter. Cotransfection of the StAR promoter linked to a luciferase reporter gene and YY1 in the presence or absence of sterol regulatory element binding protein-1a (SREBP-1a) resulted in transcriptional repression. YY1 was found to colocalize in the nucleus with SREPB-1a. YY1 inhibited SREBP-1a/nuclear factor Y (NF-Y) enhancement of StAR activation and YY1 decreased SREBP-1a binding to a sterol regulatory element in the presence or absence of NF-Y. YY1 also decreased NF-Y binding to a nonconsensus NF-Y-binding motif in the StAR promoter. These studies provide novel information on the mechanisms of YY1-mediated repression of the StAR gene.