scholarly journals Blocking L-type calcium channels reduced the threshold of cAMP-induced steroidogenic acute regulatory gene expression in MA-10 mouse Leydig cells

2009 ◽  
Vol 204 (1) ◽  
pp. 67-74 ◽  
Author(s):  
Akhilesh K Pandey ◽  
Wei Li ◽  
Xiangling Yin ◽  
Douglas M Stocco ◽  
Paula Grammas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Transcription ◽  
Leydig Cells ◽  
Channel Blocker ◽  
Regulatory Gene ◽  
Inhibitory Effect ◽  
Channel Blockers ◽  
Inner Mitochondrial Membrane ◽  
Star Protein ◽  
Steroidogenic Acute Regulatory

Previous studies have reported the roles of Ca2+ in steroidogenesis. The present study has investigated an inhibitory effect of Ca2+ influx through L-type Ca2+ channels on gene expression of steroidogenic acute regulatory (STAR) protein that regulates the transfer of substrate cholesterol to the inner mitochondrial membrane for steroidogenesis. Blocking Ca2+ influx through L-type Ca2+ channels using the selective Ca2+ channel blocker, nifedipine, markedly enhanced cAMP-induced STAR protein expression and progesterone production in MA-10 mouse Leydig cells. This was confirmed by utilization of different L-type Ca2+ channel blockers. Reverse transcription-PCR analyses of Star mRNA and luciferase assays of Star promoter activity indicated that blocking Ca2+ influx through L-type Ca2+ channels acted at the level of Star gene transcription. Further studies showed that blocking the Ca2+ channel enhanced Star gene transcription by depressing the expression of DAX-1 (NR0B1 as listed in the MGI Database) protein, a transcriptional repressor of Star gene expression. It was also observed that there is a synergistic interaction between nifedipine and cAMP. Normally, sub-threshold levels of cAMP are unable to induce steroidogenesis, but in the presence of the L-type Ca2+ channel blocker, they increased STAR protein and steroid hormone to the maximal levels. However, in the absence of minimal levels of cAMP, none of the L-type Ca2+ channel blockers are able to induce Star gene expression. These observations indicate that Ca2+ influx through L-type Ca2+ channels is involved in an inhibitory effect on Star gene expression. Blocking L-type Ca2+ channel attenuated the inhibition and reduced the threshold of cAMP-induced Star gene expression in Leydig cells.

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 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 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 Effects of apigenin on steroidogenesis and steroidogenic acute regulatory gene expression in mouse Leydig cells☆

2011 ◽  
Vol 22 (3) ◽  
pp. 212-218 ◽  
Author(s):  
Wei Li ◽  
Akhilesh K. Pandey ◽  
Xiangling Yin ◽  
Jau-Jiin Chen ◽  
Douglas M. Stocco ◽  
...  
Keyword(s):  
Gene Expression ◽  
Leydig Cells ◽  
Regulatory Gene ◽  
Steroidogenic Acute Regulatory

Rat Prl and TSH secretion are regulated differently by K(+)-channel blockers

1994 ◽  
Vol 266 (1) ◽  
pp. E39-E43 ◽  
Author(s):  
X. Wang ◽  
T. Inukai ◽  
M. A. Greer ◽  
S. E. Greer
Keyword(s):  
Channel Blocker ◽  
Cell Types ◽  
Inhibitory Effect ◽  
Thyroid Stimulating Hormone ◽  
K Channel ◽  
Channel Blockers ◽  
Pituitary Cells ◽  
Dependent Manner ◽  
K Channels ◽  
Tsh Secretion

All four different K(+)-channel blockers [tetraethylammonium (TEA), a nonselective K(+)-channel blocker; tolbutamide, an ATP-sensitive K(+)-channel blocker; quinine and 4-aminopyridine, both primarily voltage-dependent K(+)-channel blockers] stimulated prolactin (Prl) secretion by acutely dispersed anterior pituitary cells but had no effect on thyroid-stimulating hormone (TSH) secretion. TEA stimulated Prl secretion in a dose-dependent manner between 1 microM and 20 mM, but even as high as 20 mM, TEA did not induce TSH secretion. Valinomycin (2 microM), a K+ ionophore, inhibited both basal and TEA-induced Prl secretion. TEA-stimulated Prl secretion was abolished by using a Ca(2+)-depleted medium or adding 10 microM dopamine. TEA did not reverse the inhibitory effect of dopamine on thyrotropin-releasing hormone-induced Prl secretion. Our data indicate that K+ channels may play a role in the secretion of adenohypophysial hormones that is idiosyncratic for each hormone. Differences in the role of K+ channels may reflect differences between the various pituitary cell types in plasma membrane ion channel composition, membrane potential, or the mechanism of exocytosis.

New connections in the regulation of PEPCK gene expression by insulin

1996 ◽  
Vol 351 (1336) ◽  
pp. 191-199 ◽  
Keyword(s):  
Gene Expression ◽  
Map Kinase ◽  
Ribosomal Protein ◽  
Gene Transcription ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Phorbol Esters ◽  
Inhibitory Effect ◽  
S6 Kinase ◽  
Ribosomal Protein S6 ◽  
Ribosomal Protein S6 Kinase

Phosphoenolpyruvate carboxykinase (PEPCK) catalyses the rate-limiting step in hepatic gluconeogenesis. Glucagon (via the second messenger cAMP) and glucocorticoids stimulate transcription of the PEPCK gene whereas insulin and phorbol esters have a dominant inhibitory effect. Wortmannin, an inhibitor of 1-phosphatidylinositol 3-kinase (PI 3-kinase), blocks the inhibition of glucocorticoid- and cAMP stimulated PEPCK gene transcription by insulin. By contrast, although phorbol esters mimic the action of insulin on the regulation of PEPCK gene transcription, wortmannin does not block the effect of these agents. Thus PI 3-kinase is required for the regulation of PEPCK gene expression by insulin but not by phorbol esters. In liver cells, insulin administration stimulates the activity of multiple protein kinases, including the p42/p44 Mitogen Activated Protein (MAP) kinase and the p70/p85 ribosomal protein S6 kinase. Selective inhibition of the activation of either kinase, utilizing the compounds PD98059 and rapamycin respectively, does not affect insulin regulation of PEPCK gene transcription. Thus regulation of PEPCK gene transcription requires PI 3-kinase but does not require the activation of either p42/p44 MAP kinase or p70/p85 ribosomal protein S6 kinase.

scholarly journals Gigantol Improves Cholesterol Metabolism and Progesterone Biosynthesis in MA-10 Leydig Cells

2021 ◽  
Vol 44 (1) ◽  
pp. 73-93
Author(s):  
Audrey Basque ◽  
Ha Tuyen Nguyen ◽  
Mohamed Touaibia ◽  
Luc J. Martin
Keyword(s):  
Gene Expression ◽  
Ferulic Acid ◽  
Leydig Cells ◽  
Cholesterol Metabolism ◽  
Biological Activities ◽  
Cholesterol Biosynthesis ◽  
Male Hypogonadism ◽  
Star Protein ◽  
Progesterone Production ◽  
Isoferulic Acid

In aging males, androgen production by testicular Leydig cells decreases at a rate of approximately 1% per year. Phenolic compounds may enhance testosterone biosynthesis and delay the onset of male hypogonadism. Gigantol is a bibenzyl compound isolated from several types of orchids of the genus Dendrobium. This compound has various biological activities, including antioxidant activity. However, its capacity to regulate gene expression and steroid production in testicular Leydig cells has never been evaluated. We investigated the effect of gigantol on MA-10 Leydig cells’ gene expression using an RNA-Seq approach. To further investigate the structure-function relationship of the hydroxy-methoxyphenyl moiety of gigantol, experiments were also performed with ferulic acid and isoferulic acid. According to transcriptomic analysis, all genes coding for cholesterol biosynthesis-related enzymes are increased in response to gigantol treatment, resulting in increased lipid droplets accumulation. Moreover, treatments with 10 μM gigantol increased StAR protein levels and progesterone production from MA-10 Leydig cells. However, neither ferulic acid nor isoferulic acid influenced StAR protein synthesis and progesterone production in MA-10 Leydig cells. Thus, our findings indicate that gigantol improves cholesterol and steroid biosynthesis within testicular Leydig cells.

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.

Sign in / Sign up

Export Citation Format

Share Document