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

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.

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

Journal of Endocrinology ◽

10.1677/joe.1.06933 ◽

2006 ◽

Vol 190 (3) ◽

pp. 871-878 ◽

Cited By ~ 30

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.

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Stimulation of steroidogenic acute regulatory protein (STAR) gene expression by D-aspartate in rat Leydig cells

FEBS Letters ◽

10.1016/s0014-5793(99)00840-6 ◽

1999 ◽

Vol 454 (3) ◽

pp. 317-320 ◽

Cited By ~ 70

Keyword(s):

Gene Expression ◽

Leydig Cells ◽

Regulatory Protein ◽

Steroidogenic Acute Regulatory Protein ◽

Steroidogenic Acute Regulatory ◽

Star Gene ◽

Stimulation Of

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

Journal of Endocrinology ◽

10.1677/joe-07-0282 ◽

2008 ◽

Vol 197 (2) ◽

pp. 315-323 ◽

Cited By ~ 35

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.

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Effects of heme oxygenase isozymes on Leydig cells steroidogenesis

Journal of Endocrinology ◽

10.1677/joe-09-0061 ◽

2009 ◽

Vol 203 (1) ◽

pp. 155-165 ◽

Cited By ~ 5

Author(s):

Barbara Piotrkowski ◽

Casandra M Monzón ◽

Romina M Pagotto ◽

Cecilia G Reche ◽

Marcos Besio ◽

...

Keyword(s):

Heme Oxygenase ◽

Leydig Cells ◽

Specific Activity ◽

Regulatory Protein ◽

Enzymatic System ◽

Cytochrome P450 Enzymes ◽

Inner Mitochondrial Membrane ◽

Steroid Synthesis ◽

Protein Levels ◽

Steroidogenic Acute Regulatory

In the present study, we demonstrate the expression of heme oxygenase (HO) isozymes, HO-1 and HO-2 (listed as HMOX1 and HMOX2 in the MGI Database), in MA-10 Leydig tumor cells and its effect on steroidogenesis. The well-known HO inducer, hemin, increased both HO-1 and HO-2 protein levels and HO-specific activity. Induction of HO by hemin inhibited basal, hCG-, and dibutyryl cAMP (db-cAMP)-induced steroidogenesis in a reversible way. When we studied the effect of HO isozymes along the steroid synthesis, we found that steroidogenic acute regulatory protein levels were decreased, and the conversion of cholesterol to pregnenolone was inhibited by hemin treatment, with no changes in the content of cholesterol side-chain cleavage enzyme (P450scc). hCG and db-cAMP also stimulated the expression of HO-1 and HO-2, and HO enzymatic activity in MA-10 cells. Basal and hCG-stimulated testosterone synthesis was also inhibited by hemin in rat normal Leydig cells. Taken together, these results suggest that: i) at least one of HO products (presumably carbon monoxide) inhibits cholesterol transport to the inner mitochondrial membrane and Leydig cell steroidogenesis by binding to the heme group of the cytochrome P450 enzymes, in a similar way as we described for nitric oxide, and ii) hCG stimulation results in the induction of an antioxidant enzymatic system (HO) acting as a cytoprotective mechanism in Leydig cells, as already demonstrated in the adrenal gland.

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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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BMP15 Suppresses Progesterone Production by Down-Regulating StAR via ALK3 in Human Granulosa Cells

Molecular Endocrinology ◽

10.1210/me.2013-1233 ◽

2013 ◽

Vol 27 (12) ◽

pp. 2093-2104 ◽

Cited By ~ 47

Author(s):

Hsun-Ming Chang ◽

Jung-Chien Cheng ◽

Christian Klausen ◽

Peter C. K. Leung

Keyword(s):

Granulosa Cells ◽

Critical Role ◽

Regulatory Protein ◽

Granulosa Cell Tumor ◽

Type I ◽

Mrna Levels ◽

Protein Levels ◽

Human Granulosa Cells ◽

Progesterone Production ◽

Steroidogenic Acute Regulatory

In addition to somatic cell-derived growth factors, oocyte-derived growth differentiation factor (GDF)9 and bone morphogenetic protein (BMP)15 play essential roles in female fertility. However, few studies have investigated their effects on human ovarian steroidogenesis, and fewer still have examined their differential effects or underlying molecular determinants. In the present study, we used immortalized human granulosa cells (SVOG) and human granulosa cell tumor cells (KGN) to compare the effects of GDF9 and BMP15 on steroidogenic enzyme expression and investigate potential mechanisms of action. In SVOG cells, neither GDF9 nor BMP15 affects the mRNA levels of P450 side-chain cleavage enzyme or 3β-hydroxysteroid dehydrogenase. However, treatment with BMP15, but not GDF9, significantly decreases steroidogenic acute regulatory protein (StAR) mRNA and protein levels as well as progesterone production. These suppressive effects, along with the induction of Sma and Mad-related protein (SMAD)1/5/8 phosphorylation, are attenuated by cotreatment with 2 different BMP type I receptor inhibitors (dorsomorphin and DMH-1). Furthermore, depletion of activin receptor-like kinase (ALK)3 using small interfering RNA reverses the effects of BMP15 on SMAD1/5/8 phosphorylation and StAR expression. Similarly, knockdown of ALK3 abolishes BMP15-induced SMAD1/5/8 phosphorylation in KGN cells. These results provide evidence that oocyte-derived BMP15 down-regulates StAR expression and decreases progesterone production in human granulosa cells, likely via ALK3-mediated SMAD1/5/8 signaling. Our findings suggest that oocyte may play a critical role in the regulation of progesterone to prevent premature luteinization during the late stage of follicle development.

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

Endocrinology ◽

10.1210/en.2002-0081 ◽

2003 ◽

Vol 144 (8) ◽

pp. 3368-3375 ◽

Cited By ~ 45

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.

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

Endocrinology ◽

10.1210/en.2008-1425 ◽

2009 ◽

Vol 150 (7) ◽

pp. 3267-3273 ◽

Cited By ~ 3

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.

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