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

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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Role of Dosage-Sensitive Sex Reversal, Adrenal Hypoplasia Congenita, Critical Region on the X Chromosome, Gene 1 in Protein Kinase A- and Protein Kinase C-Mediated Regulation of the Steroidogenic Acute Regulatory Protein Expression in Mouse Leydig Tumor Cells: Mechanism of Action

Endocrinology ◽

10.1210/en.2008-0368 ◽

2008 ◽

Vol 150 (1) ◽

pp. 187-199 ◽

Cited By ~ 38

Author(s):

Pulak R. Manna ◽

Matthew T. Dyson ◽

Youngah Jo ◽

Douglas M. Stocco

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Protein Kinase A ◽

Tumor Cells ◽

Orphan Nuclear Receptor ◽

Star Protein ◽

Kinase C ◽

Leydig Tumor Cells ◽

Steroidogenic Acute Regulatory ◽

Kinase A

Dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene 1 (DAX-1) is an orphan nuclear receptor that has been demonstrated to be instrumental to the expression of the steroidogenic acute regulatory (StAR) protein that regulates steroid biosynthesis in steroidogenic cells. However, its mechanism of action remains obscure. The present investigation was aimed at exploring the molecular involvement of DAX-1 in protein kinase A (PKA)- and protein kinase C (PKC)-mediated regulation of StAR expression and its concomitant impact on steroid synthesis using MA-10 mouse Leydig tumor cells. We demonstrate that activation of the PKA and PKC pathways, by a cAMP analog dibutyryl (Bu)2cAMP [(Bu)2cAMP] and phorbol 12-myristate 13-acetate (PMA), respectively, markedly decreased DAX-1 expression, an event that was inversely correlated with StAR protein, StAR mRNA, and progesterone levels. Notably, the suppression of DAX-1 requires de novo transcription and translation, suggesting that the effect of DAX-1 in regulating StAR expression is dynamic. Chromatin immunoprecipitation studies revealed the association of DAX-1 with the proximal but not the distal region of the StAR promoter, and both (Bu)2cAMP and PMA decreased in vivo DAX-1-DNA interactions. EMSA and reporter gene analyses demonstrated the functional integrity of this interaction by showing that DAX-1 binds to a DNA hairpin at position −44/−20 bp of the mouse StAR promoter and that the binding of DAX-1 to this region decreases progesterone synthesis by impairing transcription of the StAR gene. In support of this, targeted silencing of endogenous DAX-1 elevated basal, (Bu)2cAMP-, and PMA-stimulated StAR expression and progesterone synthesis. Transrepression of the StAR gene by DAX-1 was tightly associated with expression of the nuclear receptors Nur77 and steroidogenic factor-1, demonstrating these factors negatively modulate the steroidogenic response. These findings provide insight into the molecular events by which DAX-1 influences the PKA and PKC signaling pathways involved in the regulation of the StAR protein and steroidogenesis in mouse Leydig tumor cells. The characterization of protein kinase A- and protein kinase C-mediated steroidogenic acute regulatory (StAR) expression and steroidogenesis suggests that the orphan nuclear receptor DAX-1 is an important regulator of the steroidogenic response in Leydig cells.

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Luteinizing Hormone-Induced Extracellular-Signal Regulated Kinase Activation Differently Modulates Progesterone and Androstenedione Production in Bovine Theca Cells

Endocrinology ◽

10.1210/en.2005-0093 ◽

2005 ◽

Vol 146 (7) ◽

pp. 2903-2910 ◽

Cited By ~ 42

Author(s):

Kimihisa Tajima ◽

Kumiko Yoshii ◽

Shin Fukuda ◽

Makoto Orisaka ◽

Kaoru Miyamoto ◽

...

Keyword(s):

Protein Kinase ◽

Protein Kinase A ◽

Regulatory Protein ◽

Steroidogenic Acute Regulatory Protein ◽

Mrna Levels ◽

Theca Cells ◽

Progesterone Production ◽

Erk Phosphorylation ◽

Steroidogenic Acute Regulatory ◽

Kinase A

Abstract It has been reported that gonadotropins promoted phosphorylation of ERK/MAPK in granulosa cells. However, little is known about the effects of gonadotropin on ERK activity in theca cells. This study explores how LH/forskolin controls ERK phosphorylation in cultured bovine theca cells. Effects of ERK on steroidogenesis were also investigated. Phosphorylation of ERK in bovine theca cells was augmented by LH and forskolin in 5 min; it decreased thereafter below basal levels in 20 min. Nevertheless, phosphorylation of the ERK kinase, MEK, was unaffected. Addition of H89 (a protein kinase A inhibitor) significantly reduced the effect of LH/forskolin on ERK phosphorylation. A potent MEK inhibitor PD98059 eliminated ERK phosphorylation and augmented progesterone production concomitantly with the elevation of intracellular steroidogenic acute regulatory protein mRNA in LH/forskolin-stimulated theca cells. In contrast to progesterone production, androgen production was diminished significantly by inhibition of ERK with decreased intracellular P450c17 mRNA levels. Taking these results together, we conclude that LH/cAMP leads to phosphorylation of ERK in a biphasic manner through MEK-independent pathway in bovine theca cells. Protein kinase A-induced phosphatase could possibly contribute to the phosphorylation process. Furthermore, modulation of ERK phosphorylation involves control of thecal steroidogenesis via modulation of the expression of steroidogenic acute regulatory protein and P450c17.

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MAPK Phosphatase-2 (MKP-2) Is Induced by hCG and Plays a Role in the Regulation of CYP11A1 Expression in MA-10 Leydig Cells

Endocrinology ◽

10.1210/en.2012-2032 ◽

2013 ◽

Vol 154 (4) ◽

pp. 1488-1500 ◽

Cited By ~ 11

Author(s):

Natalia V. Gómez ◽

Alejandra B. Gorostizaga ◽

María M. Mori Sequeiros García ◽

Laura Brion ◽

Andrea Acquier ◽

...

Keyword(s):

Protein Kinase ◽

Protein Kinase A ◽

Leydig Cells ◽

Late Phase ◽

Receptor Activation ◽

Protein Accumulation ◽

Mrna Levels ◽

Down Regulation ◽

Camp Stimulation ◽

Kinase A

Abstract MAPKs such as ERK1/2 are dephosphorylated, and consequently inactivated, by dual specificity phosphatases (MKPs). In Leydig cells, LH triggers ERK1/2 phosphorylation through the action of protein kinase A. We demonstrate that, in MA-10 Leydig cells, LH receptor activation by human chorionic gonadotropin (hCG) up-regulates MKP-2, a phosphatase that dephosphorylates ERK1/2, among other MAPKs. After 2 hours, hCG and 8-bromo-cAMP (8Br-cAMP) significantly increased MKP-2 mRNA levels (3-fold), which declined to basal levels after 6 hours. MKP-2 protein accumulation exhibited a similar kinetic profile. In cells transiently expressing flag-MKP-2 protein, hCG/8Br-cAMP stimulation promoted the accumulation of the chimera (2.5-fold after 3 h of stimulation). Pharmacologic and biochemical approaches showed that the accumulation of flag-MKP-2 involves a posttranslational modification that increases MKP-2 half-life. MKP-2 down-regulation by a short hairpin RNA (MKP-2 shRNA) raised the levels of phosphorylated ERK1/2 reached by 8Br-cAMP stimulation. This effect was evident after 180 min of stimulation, which suggests that MKP-2 down-regulates the late phase of cAMP-induced ERK1/2 activity. Also, MKP-2 down-regulation by MKP-2 shRNA increased the stimulatory effect of 8Br-cAMP on both promoter activity and messenger levels of CYP11A1, which encodes for the steroidogenic enzyme P450scc and is induced by LH/hCG through protein kinase A and ERK1/2 activities. Our findings demonstrate, for the first time, that LH/hCG tightly regulates MKP-2 expression, which modulates the induction of CYP11A1 by 8Br-cAMP. MKP-2 up-regulation might control ERK1/2 activity in a specific temporal frame to modulate the expression of a finite repertory of ERK-dependent genes.

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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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cAMP has a protein kinase A independent regulatory effect on T cell proliferation, MAP kinase activity and on mRNA levels for IL-2 and INF-gamma

Immunology Letters ◽

10.1016/s0165-2478(97)87084-8 ◽

1997 ◽

Vol 56 (1-3) ◽

pp. 65

Author(s):

B Hofmann

Keyword(s):

Cell Proliferation ◽

T Cell ◽

Protein Kinase ◽

Map Kinase ◽

Protein Kinase A ◽

Kinase Activity ◽

T Cell Proliferation ◽

Mrna Levels ◽

Regulatory Effect ◽

Kinase A

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Curcumin down regulates steroidogenesis in Leydig cells via restriction of protein kinase A activity

Journal of Clinical and Molecular Endocrinology ◽

10.21767/2572-5432-c2-006 ◽

2018 ◽

Vol 03 ◽

Author(s):

Chih Hsien Chiu ◽

Yi Chun Lina ◽

Jyun Yuan Wanga

Keyword(s):

Protein Kinase ◽

Protein Kinase A ◽

Leydig Cells ◽

Kinase A

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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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cAMP-positive regulation of angiotensinogen gene expression and protein secretion in rat adipose tissue

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00188.2003 ◽

2004 ◽

Vol 286 (3) ◽

pp. E434-E438 ◽

Cited By ~ 9

Author(s):

Valérie Serazin ◽

Marie-Noelle Dieudonné ◽

Mireille Morot ◽

Philippe de Mazancourt ◽

Yves Giudicelli

Keyword(s):

Adipose Tissue ◽

Protein Kinase ◽

Protein Kinase A ◽

Protein Secretion ◽

Intracellular Camp ◽

Pathological State ◽

Mrna Levels ◽

Adrenergic Stimulation ◽

Rat Adipose Tissue ◽

Kinase A

The adipose renin-angiotensin system (RAS) has been assigned to participate in the control of adipose tissue development and in the pathogenesis of obesity-related hypertension. In adipose cells, the biological responses to β-adrenergic stimulation are mediated by an increase in intracellular cAMP. Because cAMP is known to promote adipogenesis and because an association exists between body fat mass, hypertension, and increased sympathetic stimulation, we examined the influence of cAMP on angiotensinogen (ATG) expression and secretion in rat adipose tissue. Exposure of primary cultured differentiated preadipocytes to the cAMP analog 8-bromoadenosine 3′,5′-cyclic monophosphate (8-BrcAMP) or cAMP-stimulating agents (forskolin and IBMX) results in a significant increase in ATG mRNA levels. In adipose tissue fragments, 8-BrcAMP also increases ATG mRNA levels and protein secretion, but not in the presence of the protein kinase A inhibitor H89. The addition of isoproterenol, known to stimulate the synthesis of intracellular cAMP via β-adrenoreceptors, had the same stimulatory effect on ATG expression and secretion. These results indicate that cAMP in vitro upregulates ATG expression and secretion in rat adipose tissue via the protein kinase A-dependent pathway. Further studies are required to determine whether this regulatory pathway is activated in human obesity, where increased sympathetic tone is frequently observed, and to elucidate the importance of adipose ATG to the elevated blood pressure observed in this pathological state.

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