Crosstalk of CREB and Fos/Jun on a single cis-element: transcriptional repression of the steroidogenic acute regulatory protein gene

AbstractTranscriptional regulation of the steroidogenic acute regulatory (StAR) protein gene by cAMP-dependent mechanisms occurs in the absence of a consensus cAMP-response element (CRE; TGACGTCA) and is mediated by several sequence-specific transcription factors. We previously identified three CRE-like sites (within the −151/−1 bp cAMP-responsive region of the mouse StAR gene), of which the CRE2 site overlaps with an activator protein-1 (AP-1) motif (TGACTGA, designated as CRE2/AP-1) that can bind both CRE and AP-1 DNA-binding proteins. The present studies were aimed at exploring the functional crosstalk between CREB (CRE-binding protein) and cFos/cJun (AP-1 family members) on the CRE2/AP-1 element and its role in regulating transcription of the StAR gene. Using MA-10 mouse Leydig tumor cells, we demonstrate that the CRE and AP-1 families of proteins interact with the CRE2/AP-1 sequence. CREB, cFos, and cJun proteins were found to bind to the CRE2/AP-1 motif but not the CRE1 and CRE3 sites. Treatment with the cAMP analog (Bu)2cAMP augmented phosphorylation of CREB (Ser133), cFos (Thr325), and cJun (ser73). Chromatin immunoprecipitation studies revealed that the induction of CREB, cFos, and cJun by (Bu)2cAMP was correlated with protein–DNA interactions and recruitment of the coactivator CREB-binding protein (CBP) to the StAR promoter. EMSA studies employing CREB and cFos/cJun proteins demonstrated competition between these factors for binding to the CRE2/AP-1 motif. Transfection of cells containing the −151/−1 StAR reporter with CREB and cFos/cJun resulted in trans-repression of the StAR gene, an event tightly associated with CBP, demonstrating that both CREB and Fos/Jun compete with each other for binding with limited amounts of intracellular CBP. Overexpression of adenovirus E1A, which binds and inactivates CBP, markedly suppressed StAR gene expression. Ectopic expression of CBP eliminated the repression of the StAR gene by E1A and potentiated the activity of CREB and cFos/cJun on StAR promoter responsiveness. These findings identify molecular events involved in crosstalk between CREB and cFos/cJun, which confer both gain and loss of function on a single cis-element in fine-tuning of the regulatory events involved in transcription of the StAR gene.

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The Involvement of Specific PKC Isoenzymes in Phorbol Ester-Mediated Regulation of Steroidogenic Acute Regulatory Protein Expression and Steroid Synthesis in Mouse Leydig Cells

Endocrinology ◽

10.1210/en.2010-0874 ◽

2011 ◽

Vol 152 (1) ◽

pp. 313-325 ◽

Cited By ~ 35

Author(s):

Pulak R. Manna ◽

Jae-Won Soh ◽

Douglas M. Stocco

Keyword(s):

Protein Kinase ◽

Leydig Cells ◽

Binding Protein ◽

Ectopic Expression ◽

Creb Binding Protein ◽

Steroid Synthesis ◽

Pkc Isoforms ◽

Pkc Signaling ◽

Steroidogenic Acute Regulatory ◽

Star Gene

Abstract Protein kinase C (PKC) is a multigene family of serine/threonine kinases. PKC is involved in regulating adrenal and gonadal steroidogenesis; however, the functional relevance of the different PKC isoenzymes remains obscure. In this study, we demonstrate that MA-10 mouse Leydig tumor cells express several PKC isoforms to varying levels and that the activation of PKC signaling, by phorbol 12-myristate 13-acetate (PMA) elevated the expression and phosphorylation of PKCα, -δ, -ε, and -μ/protein kinase D (PKD). These responses coincided with the expression of the steroidogenic acute regulatory (StAR) protein and progesterone synthesis. Targeted silencing of PKCα, δ, and ε and PKD, using small interfering RNAs, resulted in deceases in basal and PMA-mediated StAR and steroid levels and demonstrated the importance of PKD in steroidogenesis. PKD was capable of controlling PMA and cAMP/PKA-mediated synergism involved in the steroidogenic response. Further studies pointed out that the regulatory events effected by PKD are associated with cAMP response element-binding protein (CREB) and c-Jun/c-Fos-mediated transcription of the StAR gene. Chromatin immunoprecipitation studies revealed that the activation of phosphorylated CREB, c-Jun, and c-Fos by PMA was correlated with in vivo protein-DNA interactions and the recruitment of CREB-binding protein, whereas knockdown of PKD suppressed the association of these factors with the StAR promoter. Ectopic expression of CREB-binding protein enhanced the trans-activation potential of CREB and c-Jun/c-Fos in StAR gene expression. Using EMSA, a −83/−67-bp region of the StAR promoter was shown to bind PKD-transfected MA-10 nuclear extract in a PMA-responsive manner, targeting CREB and c-Jun/c-Fos proteins. These findings provide evidence for the presence of multiple PKC isoforms and demonstrate the molecular events by which selective isozymes, especially PKD, influence PMA/PKC signaling involved in the regulation of the steroidogenic machinery in mouse Leydig cells.

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Sterol Regulatory Element Binding Protein-1a Regulation of the Steroidogenic Acute Regulatory Protein Gene*

Endocrinology ◽

10.1210/endo.142.4.8075 ◽

2001 ◽

Vol 142 (4) ◽

pp. 1525-1533 ◽

Cited By ~ 33

Author(s):

Wendy K. Shea-Eaton ◽

Marie Joy Trinidad ◽

Dayami Lopez ◽

Anna Nackley ◽

Mark P. McLean

Keyword(s):

Protein Gene ◽

Binding Protein ◽

Regulatory Element ◽

Regulatory Protein ◽

Steroidogenic Acute Regulatory Protein ◽

Element Binding Protein ◽

Sterol Regulatory Element ◽

Steroidogenic Acute Regulatory

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Conditional Response of the Human Steroidogenic Acute Regulatory Protein Gene Promoter to Sterol Regulatory Element Binding Protein-1a*

Endocrinology ◽

10.1210/endo.142.1.7867 ◽

2001 ◽

Vol 142 (1) ◽

pp. 28-36 ◽

Cited By ~ 39

Author(s):

Lane K. Christenson ◽

Tim F. Osborne ◽

Jan M. McAllister ◽

Jerome F. Strauss

Keyword(s):

Protein Gene ◽

Binding Protein ◽

Regulatory Element ◽

Regulatory Protein ◽

Gene Promoter ◽

Steroidogenic Acute Regulatory Protein ◽

Conditional Response ◽

Element Binding Protein ◽

Sterol Regulatory Element ◽

Steroidogenic Acute Regulatory

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

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jcem.83.7.5095 ◽

1998 ◽

Vol 83 (7) ◽

pp. 2597-2600 ◽

Cited By ~ 17

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.

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