scholarly journals Chrysin, a natural flavonoid enhances steroidogenesis and steroidogenic acute regulatory protein gene expression in mouse Leydig cells

2008 ◽  
Vol 197 (2) ◽  
pp. 315-323 ◽  
Author(s):  
Kuladip Jana ◽  
Xiangling Yin ◽  
Randolph B Schiffer ◽  
Jau-Jiin Chen ◽  
Akhilesh K Pandey ◽  
...  
Keyword(s):  
Gene Expression ◽  
Leydig Cells ◽  
Steroid Hormone ◽  
Mrna Level ◽  
Regulatory Protein ◽  
Threshold Level ◽  
Star Protein ◽  
Chain Cleavage ◽  
Steroidogenic Acute Regulatory ◽  
Star Gene

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.

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 Inhibition of Thromboxane A Synthase Activity Enhances Steroidogenesis and Steroidogenic Acute Regulatory Gene Expression in MA-10 Mouse Leydig Cells

Endocrinology ◽  
2007 ◽  
Vol 149 (2) ◽  
pp. 851-857 ◽  
Author(s):  
XingJia Wang ◽  
Xiangling Yin ◽  
Randolph B. Schiffer ◽  
Steven R. King ◽  
Douglas M. Stocco ◽  
...  
Keyword(s):  
Rna Interference ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Leydig Cells ◽  
Steroid Hormone ◽  
Mrna Levels ◽  
Star Protein ◽  
Steroidogenic Acute Regulatory ◽  
Star Gene ◽  
Kinase A

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.

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 MEF2 Cooperates With Forskolin/cAMP and GATA4 to Regulate Star Gene Expression in Mouse MA-10 Leydig Cells

Endocrinology ◽  
2015 ◽  
Vol 156 (7) ◽  
pp. 2693-2703 ◽  
Author(s):  
Caroline Daems ◽  
Mickaël Di-Luoffo ◽  
Élise Paradis ◽  
Jacques J. Tremblay
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Leydig Cells ◽  
Regulatory Protein ◽  
Mrna Levels ◽  
Small Interfering Rnas ◽  
Inner Mitochondrial Membrane ◽  
Protein Levels ◽  
Steroidogenic Acute Regulatory ◽  
Star Gene

In Leydig cells, steroidogenic acute regulatory protein (STAR) participates in cholesterol shuttling from the outer to the inner mitochondrial membrane, the rate-limiting step in steroidogenesis. Steroid hormone biosynthesis and steroidogenic gene expression are regulated by LH, which activates various signaling pathways and transcription factors, including cAMP/Ca2+/CAMK (Ca2+/calmodulin-dependent kinase)–myocyte enhancer factor 2 (MEF2). The 4 MEF2 transcription factors are essential regulators of cell differentiation and organogenesis in numerous tissues. Recently, MEF2 was identified in Sertoli and Leydig cells of the testis. Here, we report that MEF2 regulates steroidogenesis in mouse MA-10 Leydig cells by acting on the Star gene. In MA-10 cells depleted of MEF2 using siRNAs (small interfering RNAs), STAR protein levels, Star mRNA levels, and promoter activity were significantly decreased. On its own, MEF2 did not activate the mouse Star promoter but was found to cooperate with forskolin/cAMP. By chromatin immunoprecipitation and DNA precipitation assays, we confirmed MEF2 binding to a consensus element located at −232 bp of the Star promoter. Mutation or deletion of the MEF2 element reduced but did not abrogate the MEF2/cAMP cooperation, indicating that MEF2 cooperates with other DNA-bound transcription factor(s). We identified GATA4 (GATA binding protein 4) as a partner for MEF2 in Leydig cells, because mutation of the GATA element abrogated the MEF2/cAMP cooperation on a reporter lacking a MEF2 element. MEF2 and GATA4 interact as revealed by coimmunoprecipitation, and MEF2 and GATA4 transcriptionally cooperate on the Star promoter. Altogether, our results define MEF2 as a novel regulator of steroidogenesis and Star transcription in Leydig cells and identify GATA4 as a key partner for MEF2-mediated action.

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 Porcine and Bovine Steroidogenic Acute Regulatory Protein (StAR) Gene Expression during Gestation1

Endocrinology ◽  
1997 ◽  
Vol 138 (3) ◽  
pp. 1085-1091 ◽  
Author(s):  
Nicolas Pilon ◽  
Isabelle Daneau ◽  
Chantal Brisson ◽  
Jean-François Ethier ◽  
Jacques G. Lussier ◽  
...  
Keyword(s):  
Gene Expression ◽  
Regulatory Protein ◽  
Steroidogenic Acute Regulatory Protein ◽  
Steroidogenic Acute Regulatory ◽  
Star Gene

scholarly journals Synergistic Activation of Steroidogenic Acute Regulatory Protein Expression and Steroid Biosynthesis by Retinoids: Involvement of cAMP/PKA Signaling

Endocrinology ◽  
2014 ◽  
Vol 155 (2) ◽  
pp. 576-591 ◽  
Author(s):  
Pulak R. Manna ◽  
Andrzej T. Slominski ◽  
Steven R. King ◽  
Cloyce L. Stetson ◽  
Douglas M. Stocco
Keyword(s):  
Retinoic Acid ◽  
Leydig Cells ◽  
Binding Protein ◽  
Regulatory Protein ◽  
Liver X Receptor ◽  
Steroidogenic Acute Regulatory Protein ◽  
Type I ◽  
Steroid Biosynthesis ◽  
Steroidogenic Acute Regulatory ◽  
Star Gene

Both retinoic acid receptors (RARs) and retinoid X receptors (RXRs) mediate the action of retinoids that play important roles in reproductive development and function, as well as steroidogenesis. Regulation of steroid biosynthesis is principally mediated by the steroidogenic acute regulatory protein (StAR); however, the modes of action of retinoids in the regulation of steroidogenesis remain obscure. In this study we demonstrate that all-trans retinoic acid (atRA) enhances StAR expression, but not its phosphorylation (P-StAR), and progesterone production in MA-10 mouse Leydig cells. Activation of the protein kinase A (PKA) cascade, by dibutyrl-cAMP or type I/II PKA analogs, markedly increased retinoid-responsive StAR, P-StAR, and steroid levels. Targeted silencing of endogenous RARα and RXRα, with small interfering RNAs, resulted in decreases in 9-cis RA-stimulated StAR and progesterone levels. Truncation of and mutational alterations in the 5′-flanking region of the StAR gene demonstrated the importance of the −254/−1-bp region in retinoid responsiveness. An oligonucleotide probe encompassing an RXR/liver X receptor recognition motif, located within the −254/−1-bp region, specifically bound MA-10 nuclear proteins and in vitro transcribed/translated RXRα and RARα in EMSAs. Transcription of the StAR gene in response to atRA and dibutyrl-cAMP was influenced by several factors, its up-regulation being dependent on phosphorylation of cAMP response-element binding protein (CREB). Chromatin immunoprecipitation studies revealed the association of phosphorylation of CREB, CREB binding protein, RXRα, and RARα to the StAR promoter. Further studies elucidated that hormone-sensitive lipase plays an important role in atRA-mediated regulation of the steroidogenic response that involves liver X receptor signaling. These findings delineate the molecular events by which retinoids influence cAMP/PKA signaling and provide additional and novel insight into the regulation of StAR expression and steroidogenesis in mouse Leydig cells.

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