Deleterious Cholesterol Hydroperoxide Trafficking in Steroidogenic Acute Regulatory (StAR) Protein-expressing MA-10 Leydig Cells

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

Endocrinology ◽

10.1210/en.2007-0470 ◽

2007 ◽

Vol 149 (2) ◽

pp. 851-857 ◽

Cited By ~ 6

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.

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Interleukin-1alpha stimulates steroidogenic acute regulatory protein expression via p38 MAP kinase in immature rat Leydig cells

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0300059 ◽

2003 ◽

Vol 30 (1) ◽

pp. 59-67 ◽

Cited By ~ 21

Author(s):

K Svechnikov ◽

DM Stocco ◽

O Soder

Keyword(s):

Protein Expression ◽

Map Kinase ◽

Leydig Cell ◽

Leydig Cells ◽

Regulatory Protein ◽

P38 Map Kinase ◽

Dependent Manner ◽

Star Protein ◽

Adult Leydig Cells ◽

Steroidogenic Acute Regulatory

We have investigated the involvement of the steroidogenic acute regulatory (StAR) protein in interleukin-1alpha (IL-1alpha)-induced steroidogenesis in immature (40-day-old) and adult Leydig cells in vitro. Further, IL-1alpha-mediated signaling pathway(s) controlling StAR expression in immature Leydig cells were also studied. IL-1alpha stimulated both androgen production and StAR protein expression in a dose- and time-dependent manner in immature but not adult Leydig cells. These effects of IL-1alpha were prevented by pretreatment of the cells with the specific inhibitors of the p38 MAP kinase, SB203580 and PD169316, suggesting that this kinase is an important part of IL-1alpha signaling in the immature Leydig cell. The present results suggest that IL-1alpha, which is constitutively produced by the rat testis from postnatal day 25, is an important paracrine regulator of postnatal Leydig cell maturation. Regulation of StAR protein expression is one of the possible mechanisms by which IL-1alpha contributes to the differentiation of immature Leydig cells into adult cells.

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Diametric Effects of Bacterial Endotoxin Lipopolysaccharide on Adrenal and Leydig Cell Steroidogenic Acute Regulatory Protein

Endocrinology ◽

10.1210/endo.141.11.7780 ◽

2000 ◽

Vol 141 (11) ◽

pp. 4000-4012 ◽

Cited By ~ 36

Author(s):

Karen Held Hales ◽

Thorsten Diemer ◽

Salil Ginde ◽

Birinder K. Shankar ◽

Maretha Roberts ◽

...

Keyword(s):

Messenger Rna ◽

Leydig Cells ◽

Regulatory Protein ◽

Serum Testosterone ◽

Northern Analysis ◽

Dependent Manner ◽

Star Protein ◽

Adrenal Steroidogenesis ◽

Serum Lh ◽

Steroidogenic Acute Regulatory

Abstract Immune activation results in the activation of adrenal steroidogenesis and inhibition of gonadal steroidogenesis. Previous studies indicated that these effects were caused primarily by activation and suppression of the secretion of ACTH and LH, respectively. However, other evidence indicated a direct effect of the immune system on the gonads. In this study, serum testosterone, quantitated by RIA after lipopolysaccharide injection, showed a significant decrease within 2 h. Parallel measurement of serum LH showed no change. There were no differences in LH receptor or cAMP produced in Leydig cells between vehicle- and lipopolysaccharide-injected mice. The 30-kDa form of the steroidogenic acute regulatory (StAR) protein was quantitated, by Western blot, in Leydig cells and was found to decrease in a time-dependent manner. No change in StAR protein messenger RNA (mRNA) was detected by Northern analysis during this time, nor were any changes found in the levels of mRNA for the steroidogenic enzymes P450scc, 3β-hydroxysteroid dehydrogenaseΔ 4-Δ5-isomerase, or P450c17. In the adrenal, StAR protein was increased, as was StAR protein mRNA. No changes were observed in the levels of mRNA for P450scc, 3β-hydroxysteroid dehydrogenaseΔ 4-Δ5-isomerase, or P450c21. Thus, although the mechanisms of regulation differ, changes in the levels of StAR protein are a sensitive indicator of the steroidogenic capacity of these two tissues.

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Reactive Oxygen Disrupts Mitochondria in MA-10 Tumor Leydig Cells and Inhibits Steroidogenic Acute Regulatory (StAR) Protein and Steroidogenesis

Endocrinology ◽

10.1210/en.2002-0090 ◽

2003 ◽

Vol 144 (7) ◽

pp. 2882-2891 ◽

Cited By ~ 211

Author(s):

Thorsten Diemer ◽

John A. Allen ◽

Karen Held Hales ◽

Dale Buchanan Hales

Keyword(s):

Leydig Cells ◽

Reactive Oxygen ◽

Star Protein ◽

Steroidogenic Acute Regulatory

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Acute in vivo inhibition of testosterone by endotoxin parallels loss of steroidogenic acute regulatory (StAR) protein in Leydig cells.

Endocrinology ◽

10.1210/endo.137.10.8828518 ◽

1996 ◽

Vol 137 (10) ◽

pp. 4522-4525 ◽

Cited By ~ 32

Author(s):

H B Bosmann ◽

K H Hales ◽

X Li ◽

Z Liu ◽

D M Stocco ◽

...

Keyword(s):

Leydig Cells ◽

Star Protein ◽

Steroidogenic Acute Regulatory

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Down-regulation of steroidogenic acute regulatory (StAR) protein in rat Leydig cells: implications for regulation of testosterone production during aging

Mechanisms of Ageing and Development ◽

10.1016/s0047-6374(98)00149-3 ◽

1999 ◽

Vol 107 (2) ◽

pp. 197-203 ◽

Cited By ~ 25

Author(s):

Susan Leers-Sucheta ◽

Douglas M. Stocco ◽

Salman Azhar

Keyword(s):

Leydig Cells ◽

Down Regulation ◽

Star Protein ◽

Testosterone Production ◽

Steroidogenic Acute Regulatory

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

Journal of Endocrinology ◽

10.1677/joe-09-0206 ◽

2009 ◽

Vol 204 (1) ◽

pp. 67-74 ◽

Cited By ~ 12

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.

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