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

2007 ◽  
Vol 195 (3) ◽  
pp. 451-458 ◽  
Author(s):  
Teruo Sugawara ◽  
Eiji Nomura ◽  
Nobuhiko Hoshi
Keyword(s):  
Gene Expression ◽  
Steroid Hormones ◽  
Protein Level ◽  
Promoter Activity ◽  
Adrenocortical Cells ◽  
Star Protein ◽  
Cell Extracts ◽  
H295r Cells ◽  
Steroidogenic Acute Regulatory ◽  
In 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 μg/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 μg/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 pGL2 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.

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

2006 ◽  
Vol 191 (1) ◽  
pp. 327-337 ◽  
Author(s):  
Teruo Sugawara ◽  
Noriaki Sakuragi ◽  
Hisanori Minakami
Keyword(s):  
Promoter Activity ◽  
Activity Level ◽  
Expression Vectors ◽  
Camp Response Element ◽  
Star Protein ◽  
Protein Levels ◽  
Sirna Treatment ◽  
H295r Cells ◽  
Steroidogenic Acute Regulatory ◽  
Camp Induction

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.

scholarly journals Inhibition of Cyclooxygenase-2 Activity Enhances Steroidogenesis and Steroidogenic Acute Regulatory Gene Expression in MA-10 Mouse Leydig Cells

Endocrinology ◽  
2003 ◽  
Vol 144 (8) ◽  
pp. 3368-3375 ◽  
Author(s):  
XingJia Wang ◽  
Matthew T. Dyson ◽  
Youngah Jo ◽  
Douglas M. Stocco
Keyword(s):  
Gene Expression ◽  
Protein Expression ◽  
Leydig Cells ◽  
Promoter Activity ◽  
Regulatory Gene ◽  
Steroid Production ◽  
Star Protein ◽  
Cox Inhibitor ◽  
Steroidogenic Acute Regulatory ◽  
Star Gene

Abstract 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 (Bt2cAMP) did not increase StAR protein or progesterone in MA-10 mouse Leydig cells, the addition of 1 μm 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 Bt2cAMP 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 Bt2cAMP-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.

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

Endocrinology ◽  
2009 ◽  
Vol 150 (7) ◽  
pp. 3267-3273 ◽  
Author(s):  
Akhilesh K. Pandey ◽  
Xiangling Yin ◽  
Randolph B. Schiffer ◽  
James C. Hutson ◽  
Douglas M. Stocco ◽  
...  
Keyword(s):  
Gene Expression ◽  
Leydig Cells ◽  
Regulatory Gene ◽  
Thromboxane A2 ◽  
Cyclooxygenase 2 ◽  
Thromboxane A2 Receptor ◽  
Star Protein ◽  
Dependent Inhibition ◽  
Steroidogenic Acute Regulatory ◽  
Star Gene

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 3H-SQ29548 and binding competition between 3H-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.

scholarly journals cAMP-Dependent Posttranscriptional Regulation of Steroidogenic Acute Regulatory (STAR) Protein by the Zinc Finger Protein ZFP36L1/TIS11b

2009 ◽  
Vol 23 (4) ◽  
pp. 497-509 ◽  
Author(s):  
Haichuan Duan ◽  
Nadia Cherradi ◽  
Jean-Jacques Feige ◽  
Colin Jefcoate
Keyword(s):  
Zinc Finger ◽  
Posttranscriptional Regulation ◽  
Zinc Finger Protein ◽  
Cholesterol Metabolism ◽  
Adrenocortical Cells ◽  
Star Protein ◽  
Finger Protein ◽  
Steroidogenic Cells ◽  
Steroidogenic Acute Regulatory ◽  
Stimulation Of

Abstract Star is expressed in steroidogenic cells as 3.5- and 1.6-kb transcripts that differ only in their 3′-untranslated regions (3′-UTR). In mouse MA10 testis and Y-1 adrenal lines, Br-cAMP preferentially stimulates 3.5-kb mRNA. ACTH is similarly selective in primary bovine adrenocortical cells. The 3.5-kb form harbors AU-rich elements (AURE) in the extended 3′-UTR, which enhance turnover. After peak stimulation of 3.5-kb mRNA, degradation is seen. Star mRNA turnover is enhanced by the zinc finger protein ZFP36L1/TIS11b, which binds to UAUUUAUU repeats in the extended 3′-UTR. TIS11b is rapidly stimulated in each cell type in parallel with Star mRNA. Cotransfection of TIS11b selectively decreases cytomegalovirus-promoted Star mRNA and luciferase-Star 3′-UTR reporters harboring the extended 3′-UTR. Direct complex formation was demonstrated between TIS11b and the extended 3′-UTR of the 3.5-kb Star. AURE mutations revealed that TIS11b-mediated destabilization required the first two UAUUUAUU motifs. HuR, which also binds AURE, did not affect Star expression. Targeted small interfering RNA knockdown of TIS11b specifically enhanced stimulation of 3.5-kb Star mRNA in bovine adrenocortical cells, MA-10, and Y-1 cells but did not affect the reversals seen after peak stimulation. Direct transfection of Star mRNA demonstrated that Br-cAMP stimulated a selective turnover of 3.5-kb mRNA independent of AURE, which may correspond to these reversal processes. Steroidogenic acute regulatory (STAR) protein induction was halved by TIS11b knockdown, concomitant with decreased cholesterol metabolism. TIS11b suppression of 3.5-kb mRNA is therefore surprisingly coupled to enhanced Star translation leading to increased cholesterol metabolism.

scholarly journals Regulation of Steroidogenesis and the Steroidogenic Acute Regulatory Protein by a Member of the cAMP Response-Element Binding Protein Family

2002 ◽  
Vol 16 (1) ◽  
pp. 184-199 ◽  
Author(s):  
Pulak R. Manna ◽  
Matthew T. Dyson ◽  
Darrell W. Eubank ◽  
Barbara J. Clark ◽  
Enzo Lalli ◽  
...  
Keyword(s):  
Family Member ◽  
Promoter Activity ◽  
Binding Protein ◽  
Camp Response Element Binding ◽  
Camp Response Element ◽  
Response Element ◽  
Star Protein ◽  
Element Binding Protein ◽  
Steroidogenic Acute Regulatory ◽  
Star Gene

Abstract 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.

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

Endocrinology ◽  
2007 ◽  
Vol 148 (11) ◽  
pp. 5209-5219 ◽  
Author(s):  
Qiyuan Liu ◽  
Kathleen A. Merkler ◽  
Xiaohui Zhang ◽  
Mark P. McLean
Keyword(s):  
Protein Expression ◽  
Promoter Activity ◽  
Trichostatin A ◽  
Histone H3 ◽  
Prostaglandin F2α ◽  
Star Protein ◽  
Histone Deacetylase 1 ◽  
Yin Yang 1 ◽  
Steroidogenic Acute Regulatory ◽  
H3 Acetylation

Prostaglandin F2α (PGF2α) 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 PGF2α effect on the inhibition of StAR protein expression through YY1 interaction with a single region in the StAR promoter in vivo. PGF2α consistently suppressed StAR mRNA and protein expression in cultured luteal cells in a dose-dependent manner. PGF2α 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). PGF2α 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 PGF2α 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 PGF2α enhances a direct YY1/StAR promoter interaction and the recruitment of HDAC1 to the promoter, thereby preventing transcriptional activation of the StAR gene.

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

2006 ◽  
Vol 190 (3) ◽  
pp. 871-878 ◽  
Author(s):  
XingJia Wang ◽  
Chwan-Li Shen ◽  
Matthew T Dyson ◽  
Xianling Yin ◽  
Randolph B Schiffer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Arachidonic Acid ◽  
Steroid Hormone ◽  
Regulatory Protein ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Hormone Production ◽  
Cell Extracts ◽  
Steroidogenic Acute Regulatory ◽  
Star Gene

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.

scholarly journals Structural and functional analysis of the related transcriptional enhancer factor-1 and NF-κB interaction

2014 ◽  
Vol 306 (2) ◽  
pp. H233-H242 ◽  
Author(s):  
Jieliang Ma ◽  
Li Zhang ◽  
Aaron R. Tipton ◽  
Jiaping Wu ◽  
Angela F. Messmer-Blust ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Transcription ◽  
Promoter Activity ◽  
Physical Interaction ◽  
Inflammatory Factor ◽  
Mrna Levels ◽  
Transcriptional Enhancer ◽  
Tnf Α ◽  
Enhancer Factor ◽  
In Cells

The related transcriptional enhancer factor-1 (RTEF-1) increases gene transcription of hypoxia-inducible factor 1α (HIF-1α) and enhances angiogenesis in endothelium. Both hypoxia and inflammatory factor TNF-α regulate gene expression of HIF-1α, but how RTEF-1 and TNF-α coordinately regulate HIF-1α gene transcription is unclear. Here, we found that RTEF-1 interacts with p65 subunit of NF-κB, a primary mediator of TNF-α. RTEF-1 increased HIF-1α promoter activity, whereas expression of p65 subunit inhibited the stimulatory effect. By contrast, knockdown of p65 markedly enhanced RTEF-1 stimulation on the HIF-1α promoter activity (7-fold). A physical interaction between RTEF-1 and p65 was confirmed by coimmunoprecipitation experiments in cells and glutathione S-transferase (GST)-pull-down assays. A computational analysis of RTEF-1 crystal structures revealed that a conserved surface of RTEF-1 potentially interacts with p65 via four amino acid residues located at T347, Y349, R351, and Y352. We performed site-directed mutagenesis and GST-pull-down assays and demonstrated that Tyr352 (Y352) in RTEF-1 is a key site for the formation of RTEF-1 and p65-NF-κB complex. An alanine mutation at Y352 of RTEF-1 disrupted the interaction of RTEF-1 with p65. Moreover, expression of RTEF-1 decreased TNF-α-induced HIF-1α promoter activity, IL-1β, and IL-6 mRNA levels in cells; however, the effect of RTEF-1 was largely lost when Y352 was mutated to alanine. These results indicate that RTEF-1 interacts with p65-NF-κB through Y352 and that they antagonize each other for HIF-1α transcriptional activation, suggesting a novel mechanism by which RTEF-1 regulates gene expression, linking hypoxia to inflammation.

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