scholarly journals Effects of heme oxygenase isozymes on Leydig cells steroidogenesis

2009 ◽  
Vol 203 (1) ◽  
pp. 155-165 ◽  
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.

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 Oxysterols regulate expression of the steroidogenic acute regulatory protein

2004 ◽  
Vol 32 (2) ◽  
pp. 507-517 ◽  
Author(s):  
SR King ◽  
AA Matassa ◽  
EK White ◽  
LP Walsh ◽  
Y Jo ◽  
...  
Keyword(s):  
Regulatory Protein ◽  
Direct Conversion ◽  
The Body ◽  
Mrna Abundance ◽  
Mrna Levels ◽  
Steroid Synthesis ◽  
Star Protein ◽  
Protein Levels ◽  
Steroidogenic Cell ◽  
Steroidogenic Acute Regulatory

The steroidogenic acute regulatory (StAR) protein promotes intramitochondrial delivery of cholesterol to the cholesterol side-chain cleavage system, which catalyzes the first enzymatic step in all steroid synthesis. Intriguingly, substrate cholesterol derived from lipoprotein can upregulate StAR gene expression. Moreover, substrate oxysterols have been suggested to also play a role. To investigate whether oxysterols can regulate StAR expression, two steroidogenic cell lines, mouse Y1 adrenocortical and MA-10 Leydig tumor cells, were treated with various oxysterols and steroids, including 25-hydroxycholesterol (25 OHC), 22(R)OHC and 20alphaOHC. The majority of these compounds rapidly increased StAR protein levels within as little as 1 h. The most potent oxysterols were 20alphaOHC for Y1 and 25 OHC for MA-10 cells. After 8 h, StAR mRNA abundance also increased whereas there were no detected changes in promoter activity. Thus, in contrast to lipoprotein, oxysterols acutely increase StAR protein levels independently of mRNA abundance, and later increase mRNA levels independently of new gene transcription. Therefore, we propose that oxysterols modulate steroidogenesis at two levels. First, oxysterols may be important in post-transcriptional regulation of StAR activity and production of steroids for paracrine action. Secondly, through direct conversion to steroid, oxysterols may account in part for StAR-independent steroid production in the body.

scholarly journals Interleukin-1 inhibits Leydig cell steroidogenesis without affecting steroidogenic acute regulatory protein messenger ribonucleic acid or protein levels

1998 ◽  
Vol 156 (3) ◽  
pp. 461-467 ◽  
Author(s):  
T Lin ◽  
D Wang ◽  
DM Stocco
Keyword(s):  
Mrna Expression ◽  
Leydig Cell ◽  
Leydig Cells ◽  
Interleukin 1 ◽  
Mrna Level ◽  
Primary Cultures ◽  
Regulatory Protein ◽  
Steroidogenic Acute Regulatory Protein ◽  
Protein Levels ◽  
Steroidogenic Acute Regulatory

The rate-limiting step of steroidogenesis is the transport of the substrate cholesterol from the outer to the inner mitochondrial membrane which involves a cycloheximide-sensitive newly synthesized protein. A protein believed to carry out this function was recently cloned from MA-10 mouse Leydig tumor cells and named the steroidogenic acute regulatory protein (StAR). In the present study, we evaluated the expression and regulation of StAR in primary cultures of rat Leydig cells. StAR mRNA was expressed in Leydig cells as two major transcripts of 3.8 and 1.7 kb and one minor transcript of 1.2 kb. Induction of StAR mRNA transcripts could be detected as early as 30 min after the addition of human choriogonadotropin (hCG) with peak levels attained between 2 and 4 h. hCG in concentrations of 0.1-10 ng/ml caused a dose-dependent increase in StAR mRNA expression. hCG administered at a dose of 10 ng/ml increased the 3.8 kb StAR mRNA level about 14-fold and the 1.7 kb StAR mRNA level about 13.6-fold. hCG-stimulated StAR mRNA was associated with increased StAR protein levels as determined by immunoblot analysis (a 4.5-fold increase). Murine interleukin-1 alpha (mIL-1 alpha) at a concentration of 100 ng/ml inhibited hCG-induced cytochrome P450 side-chain cleavage (P450 scc) mRNA expression and testosterone formation almost completely. Interestingly, mIL-1 alpha had no effect on hCG-induced StAR mRNA or protein levels. Furthermore, mIL-1 alpha (10 ng/ml) decreased conversion of (22R)-hydroxycholesterol to testosterone while the conversion of pregnenolone, 17-hydroxypregnenolone, dehydroepiandrosterone and androstenedione to testosterone were not affected. These results indicate that the major inhibitory effect of IL-1 on Leydig cell function occurs at the level of P450 scc.

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

2013 ◽  
Vol 27 (12) ◽  
pp. 2093-2104 ◽  
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.

scholarly journals The potential function of steroid sulphatase activity in steroid production and steroidogenic acute regulatory protein expression

2004 ◽  
Vol 380 (1) ◽  
pp. 153-160 ◽  
Author(s):  
Teruo SUGAWARA ◽  
Seiichiro FUJIMOTO
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Expression Vector ◽  
Regulatory Protein ◽  
Steroidogenic Acute Regulatory Protein ◽  
Steroid Production ◽  
Star Protein ◽  
Protein Levels ◽  
Steroid Sulphatase ◽  
Steroidogenic Acute Regulatory

The first step in the biosynthesis of steroid hormones is conversion of cholesterol into pregnenolone. StAR (steroidogenic acute regulatory) protein plays a crucial role in the intra-mitochondrial movement of cholesterol. STS (steroid sulphatase), which is present ubiquitously in mammalian tissues, including the placenta, adrenal gland, testis and ovary, desulphates a number of 3β-hydroxysteroid sulphates, including cholesterol sulphate. The present study was designed to examine the effect of STS on StAR protein synthesis and steroidogenesis in cells. Steroidogenic activities of COS-1 cells that had been co-transfected with a vector for the cholesterol P450scc (cytochrome P450 side-chain-cleavage enzyme) system, named F2, a StAR expression vector (pStAR), and an STS expression vector (pSTS) were assayed. Whole-cell extracts were subjected to SDS/PAGE and then to Western blot analysis. pSTS co-expressed in COS-1 cells with F2 and pStAR increased pregnenolone synthesis 2-fold compared with that of co-expression with F2 and pStAR. Western blot analysis using COS-1 cells that had been co-transfected with pSTS, F2 and pStAR revealed that StAR protein levels increased, whereas STS and P450scc protein levels did not change. The amount of StAR protein translation products increased when pSTS was added to an in vitro transcription–translation reaction mixture. Pulse–chase experiments demonstrated that the 37 kDa StAR pre-protein disappeared significantly (P<0.01) more slowly in COS-1 cells that had been transfected with pSTS than in COS-1 cells that had not been transfected with pSTS. The increase in StAR protein level is not a result of an increase in StAR gene expression, but is a result of both an increase in translation and a longer half-life of the 37 kDa pre-StAR protein. In conclusion, STS increases StAR protein expression level and stimulates steroid production.

scholarly journals Cyclooxygenase-2 and Prostaglandin F2α in Syrian Hamster Leydig Cells: Inhibitory Role on Luteinizing Hormone/Human Chorionic Gonadotropin-Stimulated Testosterone Production

Endocrinology ◽  
2006 ◽  
Vol 147 (9) ◽  
pp. 4476-4485 ◽  
Author(s):  
Mónica B. Frungieri ◽  
Silvia I. Gonzalez-Calvar ◽  
Fernanda Parborell ◽  
Martin Albrecht ◽  
Artur Mayerhofer ◽  
...  
Keyword(s):  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Syrian Hamster ◽  
Leydig Cells ◽  
Regulatory Protein ◽  
Hydroxysteroid Dehydrogenase ◽  
Steroidogenic Acute Regulatory Protein ◽  
Testosterone Production ◽  
Cox 2 ◽  
Steroidogenic Acute Regulatory

We have previously found that cyclooxygenase-2 (COX-2), a key enzyme in the biosynthesis of prostaglandins (PGs), is present in the testicular interstitial cells of infertile men, whereas it is absent in human testes with no evident morphological changes or abnormalities. To find an animal model for further investigating COX-2 and its role in testicular steroidogenesis, we screened testes from adult species ranging from mice to monkeys. By using immunohistochemical assays, we found COX-2 expression only in Leydig cells of the reproductively active (peripubertal, pubertal, and adult) seasonal breeder Syrian hamster. COX-2 expression in hamster Leydig cells was confirmed by RT-PCR. In contrast, COX-1 expression was not detected in hamster testes. Because COX-2 expression implies PG synthesis, we investigated the effect of various PGs on testosterone production and found that PGF2α stood out because it significantly reduced human chorionic gonadotropin-stimulated testosterone release from isolated hamster Leydig cells in a dose-dependent manner. This mechanism involves a decreased expression of testicular steroidogenic acute regulatory protein and 17β-hydroxysteroid dehydrogenase. Testicular concentration and content of PGF2α in reproductively active hamsters as well as production of PGF2α from isolated hamster Leydig cells were also determined. Moreover, PGF2α receptors were localized in Leydig cells of hamsters and testicular biopsies from patients with Sertoli cell only and germ arrest syndromes. Thus, in this study, we described a COX-2-initiated pathway that via PGF2α production, PGF2α receptors, steroidogenic acute regulatory protein, and 17β-hydroxysteroid dehydrogenase represents a physiological local inhibitory system of human chorionic gonadotropin-stimulated testosterone production in the Syrian hamster testes.

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