scholarly journals Assessment of the Role of Activator Protein-1 on Transcription of the Mouse Steroidogenic Acute Regulatory Protein Gene

2004 ◽  
Vol 18 (3) ◽  
pp. 558-573 ◽  
Author(s):  
Pulak R. Manna ◽  
Darrell W. Eubank ◽  
Douglas M. Stocco
Keyword(s):  
Binding Site ◽  
Gene Transcription ◽  
Dna Sequences ◽  
Regulatory Protein ◽  
Activator Protein 1 ◽  
Transcriptional Responses ◽  
Activator Protein ◽  
Steroidogenic Acute Regulatory ◽  
Star Gene

Abstract 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-β. 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.

scholarly journals Sp1 and SF-1 Interact and Cooperate in the Regulation of Human Steroidogenic Acute Regulatory Protein Gene Expression*

Endocrinology ◽  
2000 ◽  
Vol 141 (8) ◽  
pp. 2895-2903 ◽  
Author(s):  
Teruo Sugawara ◽  
Masaki Saito ◽  
Seiichiro Fujimoto
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Promoter Activity ◽  
Critical Role ◽  
Regulatory Protein ◽  
Inner Mitochondrial Membrane ◽  
Site Mutation ◽  
Steroidogenic Acute Regulatory ◽  
Star Gene

Steroidogenic acute regulatory (StAR) protein plays a critical role in the movement of cholesterol from the outer to the inner mitochondrial membrane. Steroidogenic factor 1 (SF-1) controls basal and cAMP-stimulated transcription of the StAR gene. The 1.3-kb StAR promoter has three SF-1 binding sites, and two consensus transcription factor Sp1 binding sequences near the two most distal SF-1 binding sites. Sp1 mediates cAMP-dependent transcription of steroidogenic P450 enzyme genes, raising the possibility of Sp1 involvement in cAMP regulation of the StAR gene. However, the mechanism of Sp1-mediated, cAMP-stimulated responsiveness is not known. In this study, we elucidated the roles of Sp1 and SF-1 in the regulation of the human StAR gene promoter. We found that there was negligible promoter activity in a pGL2 StAR construct (−235 to +39) in which Sp1 and SF-1 binding sites were mutated in Y-1 adrenal tumor cells. An Sp1 binding site mutation (pGL2Sp1M) did not support promoter activity, suggesting that Sp1 cooperates with SF-1 in regulating StAR promoter function. In gel shift assays, the SF-1 binding site formed a complex with an SF-1-GST fusion protein and Sp1. Coimmunoprecipitation cross-linking experiments indicated that SF-1 physically interacts with Sp1 in vitro. Finally, a mammalian two-hybrid system was employed to demonstrate that Sp1 and SF-1 associate in vivo. In conclusion, our data indicate that Sp1 and SF-1 physically interact and cooperate in the regulation of human StAR promoter activity.

Acute regulation of the bovine gene for the steroidogenic acute regulatory protein in ovarian theca and adrenocortical cells

2000 ◽  
Vol 24 (1) ◽  
pp. 109-118 ◽  
Author(s):  
R Ivell ◽  
G Tillmann ◽  
H Wang ◽  
M Nicol ◽  
PM Stewart ◽  
...  
Keyword(s):  
Gene Transcription ◽  
Cell Cultures ◽  
Protein Biosynthesis ◽  
Regulatory Protein ◽  
Steroidogenic Acute Regulatory Protein ◽  
Mrna Levels ◽  
Adrenocortical Cells ◽  
Theca Cells ◽  
Steroidogenic Acute Regulatory ◽  
Star Gene

Upregulation of the steroidogenic acute regulatory protein (StAR) is implicated in the rapid synthesis and secretion of steroidogenic cells to produce steroids in response to stimulation by trophic hormones of the gonadal and stress axes. In the present study, we have assessed the kinetics of both StAR gene transcription and protein biosynthesis in primary cell cultures of bovine adrenocortical and ovarian theca cells, under conditions of acute stimulation by corticotrophin (ACTH) and luteinizing hormone (LH), respectively. In both cell systems, detectable upregulation of StAR gene transcription occurred within 1-2 h, reaching maxima at 4 h (theca cells) or 6 h (adrenocortical cells). mRNA levels returned rapidly to baseline, by 12 h or 24 h, respectively. Specific StAR protein levels were assessed by western blotting using a novel antibody raised against a bovine StAR peptide, and showed a similar fast upregulation, albeit delayed by 1-2 h compared with the mRNA. The response of the cultured theca cells was more acute than that of the adrenocortical cells, possibly reflecting the propensity of the LH receptor to desensitize rapidly, unlike the ACTH receptor. The primary bovine theca cell cultures were also used for fully homologous transfection studies using various deletion promoter-reporter constructs of the bovine StAR gene. Kinetic analysis of the results indicated that the acute transcriptional response resides within the proximal (-315 bp) promoter region, which includes two putative responsive elements for the steroidogenic factor-1. More distal promoter regions may be involved in modulating the specificity of expression by combining enhancer and inhibitory functions.

scholarly journals NF-κB and Activator Protein 1 Response Elements and the Role of Histone Modifications in IL-1β-Induced TGF-β1 Gene Transcription

2005 ◽  
Vol 176 (1) ◽  
pp. 603-615 ◽  
Author(s):  
Kang-Yun Lee ◽  
Kazuhiro Ito ◽  
Ryuji Hayashi ◽  
Elen P. I. Jazrawi ◽  
Peter J. Barnes ◽  
...  
Keyword(s):  
Gene Transcription ◽  
Histone Modifications ◽  
Activator Protein 1 ◽  
Response Elements ◽  
Activator Protein ◽  
Tgf Β1

Activator protein-1 is necessary for angiotensin-II stimulation of human adrenocorticotropin receptor gene transcription

2001 ◽  
Vol 268 (6) ◽  
pp. 1802-1810
Author(s):  
Danielle Naville ◽  
Estelle Bordet ◽  
Marie-Claude Berthelon ◽  
Philippe Durand ◽  
Martine Begeot
Keyword(s):  
Angiotensin Ii ◽  
Gene Transcription ◽  
Receptor Gene ◽  
Activator Protein 1 ◽  
Activator Protein ◽  
Stimulation Of

scholarly journals DAX-1 Expression in Human Adrenocortical Neoplasms: Implications for Steroidogenesis

1998 ◽  
Vol 83 (7) ◽  
pp. 2597-2600 ◽  
Author(s):  
M. Reincke ◽  
F. Beuschlein ◽  
E. Lalli ◽  
W. Arlt ◽  
S. Vay ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mrna Expression ◽  
Protein Gene ◽  
Regulatory Protein ◽  
Nuclear Hormone Receptor ◽  
Adrenocortical Tumors ◽  
Steroidogenic Acute Regulatory ◽  
Adrenocortical Carcinomas ◽  
Star Gene ◽  
Functional Phenotype

The DAX-1 gene encodes an orphan nuclear hormone receptor essential for normal fetal development of the adrenal cortex. Recently, DAX-1 has been shown to act as a transcriptional repressor of steroidogenic acute regulatory protein gene expression (StAR), suppressing steroidogenesis. We, therefore, investigated the expression of DAX-1 in a variety of adrenocortical tumors and compared the results with StAR mRNA expression. We found low or absent DAX-1 expression in aldosterone-producing adenomas (n=11: 35±11%; normal adrenals: 100±17%) and in aldosterone-producing adrenocortical carcinomas (n=2: 24 and 36%). Cortisol-producing adenomas showed intermediate DAX-1 expression (n=8; 92±16), as did 3 non-aldosterone-producing carcinomas (72, 132 and 132%). High DAX-1 expression was present in nonfunctional adenomas (n=3; 160±17%). In contrast to DAX-1, StAR mRNA expression did not show significant variations between groups. We did not detect the expected negative correlation between DAX-1 and StAR mRNA in adrenocortical tumors. These data suggest that high DAX-1 expression in adrenocortical tumors is associated with a non-functional phenotype whereas low DAX-1 expression favors mineralo-corticoid secretion. These effects on steroidogenesis are mediated by mechanisms other than repression of StAR gene expression. Our results indicate that DAX-1 may be one of the factors influencing the steroid biosynthesis of adrenocortical neoplasms.

scholarly journals Role of Intramitochondrial Arachidonic Acid and Acyl-CoA Synthetase 4 in Angiotensin II-Regulated Aldosterone Synthesis in NCI-H295R Adrenocortical Cell Line

Endocrinology ◽  
2012 ◽  
Vol 153 (7) ◽  
pp. 3284-3294 ◽  
Author(s):  
Pablo G. Mele ◽  
Alejandra Duarte ◽  
Cristina Paz ◽  
Alessandro Capponi ◽  
Ernesto J. Podestá
Keyword(s):  
Arachidonic Acid ◽  
Angiotensin Ii ◽  
Regulatory Protein ◽  
Steroidogenic Acute Regulatory Protein ◽  
Ang Ii ◽  
Steroid Production ◽  
Glomerulosa Cells ◽  
Steroidogenic Acute Regulatory ◽  
Export System

Although the role of arachidonic acid (AA) in angiotensin II (ANG II)- and potassium-stimulated steroid production in zona glomerulosa cells is well documented, the mechanism responsible for AA release is not fully described. In this study we evaluated the mechanism involved in the release of intramitochondrial AA and its role in the regulation of aldosterone synthesis by ANG II in glomerulosa cells. We show that ANG II and potassium induce the expression of acyl-coenzyme A (CoA) thioesterase 2 and acyl-CoA synthetase 4, two enzymes involved in intramitochondrial AA generation/export system well characterized in other steroidogenic systems. We demonstrate that mitochondrial ATP is required for AA generation/export system, steroid production, and steroidogenic acute regulatory protein induction. We also demonstrate the role of protein tyrosine phosphatases regulating acyl-CoA synthetase 4 and steroidogenic acute regulatory protein induction, and hence ANG II-stimulated aldosterone synthesis.

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