Steroidogenic Acute Regulatory Gene Research Articles

A STAR for the ages: a 30-year historical perspective of the role of transcription factors in the regulation of steroidogenic acute regulatory gene expression.

The steroidogenic acute regulatory (STAR) protein is an essential cholesterol transporter that shuttles cholesterol from the outer to the inner mitochondrial membrane in the major steroidogenic endocrine organs. It is a key player in the acute regulation of steroid hormone biosynthesis in response to tropic hormone stimulation. Its discovery 30 years ago sparked immediate interest in understanding how STAR action is controlled. Since increased STAR gene expression is a classic feature of the acute regulation of steroidogenesis, a special emphasis was placed on defining the transcriptional regulatory mechanisms that underlie its rapid induction in response to tropic hormone stimulation. These mechanisms include the effects of enhancers, the regulation of chromatin accessibility, the impact of epigenetic factors, and the role of transcription factors. Over the past three decades, understanding the transcription factors that regulate STAR gene expression has been the subject of more than 170 independent scientific publications, making it one of, and if not the best, studied genes in the steroidogenic pathway. This intense research effort has led to the identification of dozens of transcription factors and their related binding sites in STAR 5' flanking (promoter) sequences across multiple species. STAR gene transcription appears to be complex in that a large number of transcription factors have been proposed to interact with either isolated or overlapping regulatory sequences that are tightly clustered over a relatively short promoter region upstream of the STAR transcription start site. Many of these transcription factors appear to work in unique combinatorial codes and are impacted by diverse hormonal and intracellular signaling pathways. This review provides a retrospective overview of the transcription factors proposed to regulate both basal and acute (hormonal) STAR gene expression, and how insights in this area have evolved over the past 30 years.

Melatonin administration during the first half of pregnancy improves the reproductive performance of rabbits: Emphasis on ovarian and placental functions

This study was designed to investigate the roles of melatonin administration during different sensitive windows of the first half of pregnancy in the function and gene expression of the ovary and placenta, hormone profile, and pregnancy outcomes in rabbits. Four equal experimental groups of 20 rabbits each were used. The first (FW), second (SW), and third (F + SW) groups comprised rabbits that orally received 0.7-mg melatonin/kg body weight during the first week, second weeks, and during both weeks of pregnancy; and the fourth group served as the control group (C). The total number of visible follicles significantly increased in all melatonin-treated groups compared with that in the C group. In all melatonin-treated groups, the number of absorbed fetuses was significantly reduced, whereas the weights of embryonic sacs and fetuses were higher than in the C group. The placenta efficiency was significantly increased in the F + SW group compared with that in the C group, followed by the SW group, whereas no significant difference in the placenta efficiency was found between the FW and C groups. Melatonin treatments significantly improved the expression of antioxidants, gonadotropin receptors, and cell cycle regulatory genes in the ovary, whereas only FW treatment upregulated steroidogenic acute regulatory gene. Compared with the C and FW groups, melatonin treatments during the SW and F + SW significantly upregulated the expression of most genes in the placenta. The concentrations of estradiol were significantly higher in the SW and F + SW groups than in the FW and C groups. The concentrations of progesterone were significantly increased in the FW group compared with those in the C and SW groups, whereas the F + SW group showed intermediate values. The litter size and weight at birth significantly increased in all melatonin-treated groups compared with those in the C group. The second week of pregnancy seems to be a sensitive window for melatonin actions during pregnancy. Thus, melatonin administration during the second week of pregnancy can be effective in improving pregnancy outcomes in rabbits.

L-carnitine-loaded nanoparticle ameliorates cypermethrin-induced reproductive toxicity in adult male rats.

The objective of this investigation was to find out whether L-carnitine-loaded nanoparticle (LCn) could reduce the reproductive toxicity of cypermethrin (CYP), the widely used insecticide in veterinary medicine in male rats. Twenty male Wistar rats that weighed between 210 and 240 g were split into four groups and treated daily for 2 months. The control group was given 0.9% normal saline solution daily. The second group received CYP (3.83 mg/kg b. w. p. o.) daily. The third group was administered with LCn and CYP (50 mg/kg b. wt. p. o. and 3.83 mg/kg b. wt. p. o., respectively) daily, whereas the fourth group received LCn alone (50 mg/kg b. wt. p. o.) daily. On day 60, all rats were sacrificed and samples were collected. CYP-treated animals exhibited inhibition of testicular anti-oxidative stress mechanisms, testicular steroidogenesis enzyme activity (3β-hydroxysteroid dehydrogenase [3β-HSD] and 17β-HSD), and downregulation of steroidogenic acute regulatory (StAR) gene expression. In addition, it decreased testosterone, follicle-stimulating hormone, and LH levels and had detrimental consequences for sperm quality. LCn attenuated CYP-induced reproductive toxicity via the alleviation of testicular oxidative stress status, improvement of steroidogenic enzyme activity, and upregulation of StAR gene expression, which are probably responsible for the concomitant improvement in testicular hormonal levels and improvement in sperm properties. Intriguingly, LCn treatment alone could enhance the functions of the male reproductive system.

Regulation of retinoid mediated StAR transcription and steroidogenesis in hippocampal neuronal cells: Implications for StAR in protecting Alzheimer's disease

Retinoids (vitamin A and its derivatives) play pivotal roles in diverse processes, ranging from homeostasis to neurodegeneration, which are also influenced by steroid hormones. The rate-limiting step in steroid biosynthesis is mediated by the steroidogenic acute regulatory (StAR) protein. In the present study, we demonstrate that retinoids enhanced StAR expression and pregnenolone biosynthesis, and these parameters were markedly augmented by activation of the PKA pathway in mouse hippocampal neuronal HT22 cells. Deletion and mutational analyses of the 5′-flanking regions of the StAR gene revealed the importance of a retinoic acid receptor (RAR)/retinoid X receptor (RXR)-liver X receptor (LXR) heterodimeric motif at −200/−185 bp region in retinoid responsiveness. The RAR/RXR-LXR sequence motif can bind RARα and RXRα, and retinoid regulated transcription of the StAR gene was found to be influenced by the LXR pathway, representing signaling cross-talk in hippocampal neurosteroid biosynthesis. Steroidogenesis decreases during senescence due to declines in the central nervous system and the endocrine system, and results in hormone deficiencies, inferring the need for hormonal balance for healthy aging. Loss of neuronal cells, involving accumulation of amyloid beta (Aβ) and/or phosphorylated Tau within the brain, is the pathological hallmark of Alzheimer's disease (AD). HT22 cells overexpressing either mutant APP (mAPP) or mutant Tau (mTau), conditions mimetic to AD, enhanced toxicities, and resulted in attenuation of both basal and retinoid-responsive StAR and pregnenolone levels. Co-expression of StAR with either mAPP or mTau diminished cytotoxicity, and concomitantly elevated neurosteroid biosynthesis, pointing to a protective role of StAR in AD. These findings provide insights into the molecular events by which retinoid signaling upregulates StAR and steroid levels in hippocampal neuronal cells, and StAR, by rescuing mAPP and/or mTau-induced toxicities, modulates neurosteroidogenesis and restores hormonal balance, which may have important implications in protecting AD and age-related complications and diseases.

Hormonal and Genetic Regulatory Events in Breast Cancer and Its Therapeutics: Importance of the Steroidogenic Acute Regulatory Protein.

Estrogen promotes the development and survival of the majority of breast cancers (BCs). Aromatase is the rate-limiting enzyme in estrogen biosynthesis, and it is immensely expressed in both cancerous and non-cancerous breast tissues. Endocrine therapy based on estrogen blockade, by aromatase inhibitors, has been the mainstay of BC treatment in post-menopausal women; however, resistance to hormone therapy is the leading cause of cancer death. An improved understanding of the molecular underpinnings is the key to develop therapeutic strategies for countering the most prevalent hormone receptor positive BCs. Of note, cholesterol is the precursor of all steroid hormones that are synthesized in a variety of tissues and play crucial roles in diverse processes, ranging from organogenesis to homeostasis to carcinogenesis. The rate-limiting step in steroid biosynthesis is the transport of cholesterol from the outer to the inner mitochondrial membrane, a process that is primarily mediated by the steroidogenic acute regulatory (StAR) protein. Advances in genomic and proteomic technologies have revealed a dynamic link between histone deacetylases (HDACs) and StAR, aromatase, and estrogen regulation. We were the first to report that StAR is abundantly expressed, along with large amounts of 17β-estradiol (E2), in hormone-dependent, but not hormone-independent, BCs, in which StAR was also identified as a novel acetylated protein. Our in-silico analyses of The Cancer Genome Atlas (TCGA) datasets, for StAR and steroidogenic enzyme genes, revealed an inverse correlation between the amplification of the StAR gene and the poor survival of BC patients. Additionally, we reported that a number of HDAC inhibitors, by altering StAR acetylation patterns, repress E2 synthesis in hormone-sensitive BC cells. This review highlights the current understanding of molecular pathogenesis of BCs, especially for luminal subtypes, and their therapeutics, underlining that StAR could serve not only as a prognostic marker, but also as a therapeutic candidate, in the prevention and treatment of this life-threatening disease.

Cardiac Arrest in a Child with Non-classic Lipoid Congenital Adrenal Hyperplasia Associated with a New STAR Gene Mutation

Abstract Introduction: Steroidogenic acute regulatory (STAR) protein regulates steroid hormone synthesis by transporting cholesterol into mitochondria. STAR gene mutations lead to lipoid congenital adrenal hyperplasia (LCAH), the rare but most severe form of congenital adrenal hyperplasia in children. We present an unusual case with an episode of cardiac arrest in a young girl during an acute febrile illness and later she was diagnosed with adrenal insufficiency secondary to a non-classic LCAH. Case: 2-year 11-month-old previously healthy white female was brought to an urgent care clinic due to severe lethargy and a seizure-like activity during a fever illness. She was found to have an undetectable blood glucose level and went into cardiac arrest shortly after arrival. CPR was performed for approximately 11 minutes. She then developed sever respiratory distress and was intubated. She was transferred to the PICU with IV sodium bicarbonate given en route. On admission, her body weight was 13.26 kg (36.80th percentile), height 90 cm (17.56th percentile), and BMI 16.17 (62.88th percentile). Her physical exam revealed normal external female genitalia and normal skin pigmentation. Lab evaluation revealed normal sodium and potassium but elevated anion gap, hyperuricemia, elevated creatinine kinase, abnormal liver function tests and abnormal coagulation profile. Brain MRI revealed findings consistent with hypoxic-ischemic encephalopathy. Renal function improved within 24 hours and hepatic function returned to normal after 20 days. Due to her severe hypoglycemic event, a high-dose ACTH stimulation test was performed. The results were consistent with adrenal insufficiency: baseline cortisol level, 7.3 μg/dL; 30 minutes cortisol, 7.8 μg/dL; 60 minutes cortisol, 9 μg/dL (normal response, ≥18 mcg/mL at 30 or 60 minutes). The baseline ACTH level was significantly elevated, 1688 pg/mL (0–46) as well as the renin activity, 24.3 ng/hour (1.7–11.2). Genetic testing revealed a 46 XX karyotype. STAR gene analysis identified compound heterozygosity; a novel deletion (c.811delC, p.Leu271Cysfs*50) and a previously reported missense mutation (c.661G>A, p.Gly221Ser). The girl is now 11 years old and exhibits normal growth, normal cognitive development, and she has developed early signs of puberty (Tanner stage 2 for breast). She takes daily hydrocortisone, fludrocortisone and stress dose hydrocortisone as needed. Conclusion: In non-classic LACH, the onset is generally late or not acute. Initial clinical features are variable and nonspecific. For this reason, non-classic LCAH may be overlooked. Adrenal crisis is a life-threatening complication, and it is important that clinicians are aware of the clinical features of non-classic LCAH and consider it in the differential diagnoses. Genetic testing for STAR should be considered in individuals with non-autoimmune primary adrenocortical insufficiency.

Novel StAR Gene Mutation Identified in a Moroccan Patient with Lipoid Congenital Adrenal Hyperplasia

Congenital Adrenal Hyperplasia (CAH) is an autosomal recessive condition that results from the deficiency of one of the steroidogenesis enzymes responsible for cortisol biosynthesis. In the majority of cases, CAH is caused by 21-hydroxylase deficiency. More rarely, the deficiency concerns 11b-hydroxylase, 3b-hydroxysteroid dehydrogenase, 17hydroxylase, or exceptionally StAR and P450 oxydoreductase. Here, we report the case of a 3 year and 4 months old male child, born from a consanguineous marriage who presented at 15 months old with the salt-loss syndrome. Physical examination found generalized melanoderma, micropenis and bilateral cryptorchidism. Biological assessment at the time of diagnosis revealed hyponatremia, hyperkalemia, functional renal failure, hypoglycemia, low blood cortisol level, and high blood level of ACTH, suggesting primary adrenal insufficiency. The patient presented also with the abnormality of sexual differentiation with a 46 XY karyotype, testosteronemia level was low at the baseline and after HCG stimulation, pelvic ultrasound and MRI showed bilateral testicular atrophy in the inguinal position. The genetic study revealed a likely pathogenic homozygous variant in the StAR (steroidogenic acute regulatory) gene. Therapeutically, our patient was hydrated by saline solution and treated with hydrocortisone and fludrocortisone, then benefited from a surgical testicular correction marked by a favorable evolution. Although mutations in StAR gene are rare, they can be responsible for the defect in the early stage of steroidogenesis and therefore cause a deficiency in adrenal and sexual hormones biosynthesis.

Targeting ERK/COX-2 signaling pathway in permethrin-induced testicular toxicity: a possible modulating effect of matrine.

Permethrin (PER), the prevalent synthetic pyrethroid, was reported to have genotoxic effects along with male reproductive organs impairment. Matrine, the Chinese herb chief alkaloid constituent, is used extensively owing to its recognized pharmacological properties. The study included 30 rats allocated equally into three groups; Group I: Control group, Group II: PER group and Group III: Matrine treated PER group. All groups were subjected to the measurement of Steroidogenic acute regulatory (StAR) gene expression by PCR technique while testosterone, phosphorylated Extracellular signal-regulated Kinase 1/2 (p-ERK1/2) and Cyclooxygenase 2 (COX-2) levels were assessed by ELISA technique. Malondialdehyde (MDA), total antioxidant capacity (TAC) and glutathione peroxidase (GPx) were also detected spectrophotometrically in addition to assessment of DNA fragmentation. Testicular histological structure as well as sperm count and morphology were studied. Matrine improved testicular toxicity evidenced by significant upregulation of StAR gene expression, elevation of testosterone level and significant decrease of p-ERK1/2 and COX-2 levels. Moreover, enhancements of the antioxidant status together with improvement of the histological findings were observed. These findings could pave the way for matrine to be used as a promising therapeutic agent in treatment of PER toxicity.

Genomic Profiling of the Steroidogenic Acute Regulatory Protein in Breast Cancer: In Silico Assessments and a Mechanistic Perspective.

Cancer is a multifactorial condition with aberrant growth of cells. A substantial number of cancers, breast in particular, are hormone sensitive and evolve due to malfunction in the steroidogenic machinery. Breast cancer, one of the most prevalent form of cancers in women, is primarily stimulated by estrogens. Steroid hormones are made from cholesterol, and regulation of steroid/estrogen biosynthesis is essentially influenced by the steroidogenic acute regulatory (StAR) protein. Although the impact of StAR in breast cancer remains a mystery, we recently reported that StAR protein is abundantly expressed in hormone sensitive breast cancer, but not in its non-cancerous counterpart. Herein, we analyzed genomic profiles, hormone receptor expression, mutation, and survival for StAR and steroidogenic enzyme genes in a variety of hormone sensitive cancers. These profiles were specifically assessed in breast cancer, exploiting The Cancer Genome Atlas (TCGA) datasets. Whereas StAR and key steroidogenic enzyme genes evaluated (CYP11A1, HSD3B, CYP17A1, CYP19A1, and HSD17B) were altered to varying levels in these hormone responsive cancers, amplification of the StAR gene was correlated with poor overall survival of patients afflicted with breast cancer. Amplification of the StAR gene and its correlation to survival was also verified in a number of breast cancer studies. Additionally, TCGA breast cancer tumors associated with aberrant high expression of StAR mRNA were found to be an unfavorable risk factor for survival of patients with breast cancer. Further analyses of tumors, nodal status, and metastases of breast cancer tumors expressing StAR mRNA displayed cancer deaths in stage specific manners. The majority of these tumors were found to express estrogen and progesterone receptors, signifying a link between StAR and luminal subtype breast cancer. Collectively, analyses of genomic and molecular profiles of key steroidogenic factors provide novel insights that StAR plays an important role in the biologic behavior and/or pathogenesis of hormone sensitive breast cancer.

Protective Effect of Resveratrol on Benzo(a)Pyrene Induced Dysfunctions of Steroidogenesis and Steroidogenic Acute Regulatory Gene Expression in Leydig Cells.

Benzo(a)pyrene [B(a)P] is the toxic environmental Polycyclic Aromatic Hydrocarbon (PAH), that exerts male reproductive dysfunctions. In this study the molecular mechanism of B(a)P induced Leydig cell steroidogenic dysfunctions and its protective mechanism of action with a natural Aryl hydrocarbon receptor (AhR) antagonist and anti-oxidant, Resveratrol (Res) has been investigated. B(a)P exposure induced ROS mediated steroidogenic imbalance via activation of p38MAPK and repression of testosterone level as well as other steroidogenic enzymes like CYPIIA1, 3β-HSD, 17β-HSD expressions. B(a)P exposure decreased StAR protein expression along with increased DAX-1, a transcriptional repressor of StAR gene. Along with that B(a)P decreased the expression of SF-1 that acts as a transcriptional inducer of StAR gene expression. The study has established Resveratrol as a potential agent combating the deleterious effect of B(a)P on Leydig cell steroidogenesis. Resveratrol treatment resulted significant protection against B(a)P by scavenging ROS and modulating the transcriptional regulation of anti-oxidant enzymes. Furthermore, Resveratrol also prevented stress kinase like p38 MAPK activation and increased StAR protein expression through the reduction of DAX-1 expression. Moreover, the testosterone production was efficiently restored with Resveratrol treatment. ChIP assay also revealed that resveratrol improved SF-1expression which further increased the StAR gene expression. Resveratrol acted efficiently against B(a)P, through its anti-oxidative properties as well as inhibits p38MAPK and increased steroidogenesis and StAR expression through the modulation of SF-1 gene expression.

N-butylparaben exposure during perinatal period impairs fertility of the F1 generation female rats

Parabens are a class of preservatives widely used in the majority of personal care products, cosmetics, medicines, and food products. However, current literature suggests its plausible role as an endocrine disruptor, hence the present study was undertaken to delineate the effects of n-butyl paraben on perinatally exposed F1 female rats. F0 dams were exposed subcutaneously to n-butylparaben from gestation day 6 (GD 6) to postnatal day (PND) 21 with doses of 10, 100, and 1000 mg/kg Bw/day in corn oil. The F1 female rats were monitored for pubertal development and sexual maturation through PND 30, 45, and 75; which were subsequently subjected to fertility assessment at PND 75. Perinatal exposure to n-butylparaben resulted in-a.Delayed age of vaginal opening with disturbed estrus cycle in F1 female rats.b.A significant reduction in fertility with increased pre-and post-implantation loss; impaired steroidogenesis with decreased estradiol, progesterone, and increased testosterone levels.c.An impairment in folliculogenesis with increased number of primordial follicle maturation and reduced number of corpora leutea (CL). The morphological analysis showed degenerated follicles in the ovaries and inadequate development of the myometrium in the uterus.d.Perturbed expression levels of estrogen receptors alpha (ER α), beta (ER β) and Steroidogenic acute regulatory gene (StAR) in the ovary at PND 75.This study documents impaired steroidogenesis and folliculogenesis might be the prime reason for the reduced fertility of F1 female rats. Hence, our study suggests that health monitors need to counsel potential females planning for pregnancy to avoid exposure to parabens.

Diazinon Interrupts Ovarian Steroidogenic Acute Regulatory (StAR) Gene Transcription in Gonadotropin-Stimulated Rat Model.

Organophosphate pesticides are considered as endocrine disruptors that interfere with reproductive functions. The corpus luteum (CL) is a transient endocrine gland that produces progesterone, a crucial hormone for a successful beginning and maintenance of pregnancy. Steroidogenic acute regulatory protein (StAR) facilitates the rate-limiting transfer of cholesterol from the outer mitochondrial membrane to the inner organelle membranes. We investigated the effect of Diazinon (DZN), an organophosphate, on StAR mRNA expression by Sybergreen Real Time-PCR in a time-dependent manner in luteal phase. Fifty immature female Wistar rats (24-day-old) were injected with a single injection of Pregnant mare's Serum Gonadotropin (PMSG) followed by a single injection of human Chorionic Gonadotropin (hCG), 48 h later. Then, DZN was administered in a single dose (70 mg/kg bw, I.P), controls received only the vehicle, 12 h post-hCG injection. Ovaries were collected in 4 h. intervals from 8 to 24 h post-hCG injection. Then, hCG stimulation transcript levels of StAR gene were significantly altered in the hormone-stimulated rats following DZN treatment. In addition, histological study showed that the CL diameter in DZN-treated group was smaller than control group (p = 0.000). Our findings suggest that the critical step in the function of CL is disrupted by DZN and may correlates with female reproductive damage.

Molecular mechanisms of luteinization

Dynamic changes in steroidogenesis occur in ovarian granulosa cells during ovulation after the LH surge. The ovulatory LH surge induces rapid up-regulation of steroidogenic acute regulatory (StAR) protein and rapid down-regulation of aromatase (Cyp19a1) in granulosa cells undergoing luteinization during ovulation. These rapid changes in StAR and Cyp19a1 gene expression after the LH surge efficiently facilitate progesterone production, which plays a crucial role in ovulation and the following luteinization. Recently, it has become clear that epigenetic regulation such as histone modifications and DNA methylation play a key role in gene expression through the chromatin remodeling of the promoter region. This study reports the in vivo evidence that epigenetic mechanisms including histone modifications, DNA methylation and chromatin remodeling are involved in the rapid changes of StAR and Cyp19a1 gene expression in granulosa cells undergoing luteinization during ovulation.

Changes in Histone Modification and DNA Methylation of the StAR and Cyp19a1 Promoter Regions in Granulosa Cells Undergoing Luteinization during Ovulation In Rats

The ovulatory LH surge induces rapid up-regulation of steroidogenic acute regulatory (StAR) protein and rapid down-regulation of aromatase (Cyp19a1) in granulosa cells (GCs) undergoing luteinization during ovulation. This study investigated in vivo whether epigenetic mechanisms including histone modifications are involved in the rapid changes of StAR and Cyp19a1 gene expression. GCs were obtained from rats treated with equine chorionic gonadotropin (CG) before (0 h) and after human (h)CG injection. StAR mRNA levels rapidly increased after hCG injection, reached a peak at 4 h, and then remained higher compared with 0 h until 12 h. Cyp19a1 mRNA levels gradually decreased after hCG injection and reached their lowest level at 12 h. A chromatin immunoprecipitation assay revealed that levels of histone-H4 acetylation (Ac-H4) and trimethylation of histone-H3 lysine-4 (H3K4me3) increased whereas H3K9me3 and H3K27me3 decreased in the StAR promoter after hCG injection. On the other hand, the levels of Ac-H3 and -H4 and H3K4me3 decreased, and H3K27me3 increased in the Cyp19a1 promoter after hCG injection. Chromatin condensation, which was analyzed using deoxyribonuclease I, decreased in the StAR promoter and increased in the Cyp19a1 promoter after hCG injection. A chromatin immunoprecipitation assay also showed that binding activities of CAATT/enhancer-binding protein β to the StAR promoter increased and binding activities of phosphorylated-cAMP response element binding protein to the Cyp19a1 promoter decreased after hCG injection. These results provide in vivo evidence that histone modifications are involved in the rapid changes of StAR and Cyp19a1 gene expression by altering chromatin structure of the promoters in GCs undergoing luteinization during ovulation.

Inhibitory effect of bufalin and cinobufagin on steroidogenesis via the activation of ERK in human adrenocortical cells

Bufalin and cinobufagin exhibit cardiotonic and natriuretic activities. The aim of this study was to evaluate the effects of bufalin and cinobufagin on aldosterone and cortisol secretion and their mechanisms of action in human adrenocortical cells (NCI-H295). H295 cells were incubated with bufalin or cinobufagin in the presence or absence of angiotensin II (Ang II), forskolin, 8-Br-cAMP, corticosterone or deoxycortisol. The role of ERK1/2 was studied by use of the inhibitor of MEK (U0126). The binding of transcription factor steroidogenic factor 1 (SF-1) to steroidogenic acute regulatory (StAR) gene promoter was analysed by EMSA. Bufalin and cinobufagin markedly inhibited basal, Ang II-, forskolin- or 8-Br-cAMP-stimulated aldosterone and cortisol secretion, and the conversions of corticosterone to aldosterone and deoxycortisol to cortisol. Bufalin and cinobufagin also inhibited StAR protein expression and SF-1 binding to StAR gene promoter. They both increased phosphorylation of ERK1/2, and U0126 fully abolished these effects on ERK1/2 in H295 cells. Furthermore, U0126 reversed the inhibitory effects of bufalin and cinobufagin on StAR protein expression and the binding of SF-1 to StAR gene promoter. However, U0126 did not completely reverse their inhibitory effects on aldosterone and cortisol release. The inhibitory effects of bufalin and cinobufagin on steroidogenesis of aldosterone and cortisol were associated with inhibition of aldosterone synthase and 11β-hydroxylase, as well as the suppression of StAR protein expression and SF-1 binding to StAR promoter via the phosphorylation of ERK1/2 in H295 cells.

Changes in Epigenetic Status in the Promoter Region of Steroidogenic Acute Regulatory (StAR) Gene in Rat Granulosa Cells During Luteinization.

Ovulatory LH surge rapidly induces gene expression of StAR, which is a rate-limiting step for progesterone synthesis in luteal cells. Recent evidence has shown that epigenetic mechanisms such as histone modification and DNA methylation are essentially involved in the regulation of gene expression. The present study investigated the involvement of epigenetic mechanisms in the rapid increase in StAR mRNA expression induced by ovulatory LH surge in luteinized granulosa cells during luteinization. To induce ovulation, 21-days-old immature rats were injected with PMSG (15 IU) followed by hCG (15 IU) injection 48 h later. The ovaries were removed and luteinized granulosa cells were collected before hCG (0 h), and 1, 2, 4, 8, and 12 h after hCG injection. StAR mRNA levels gradually increased after hCG injection, reached the peak at 4h, and decreased thereafter. Histone acetylation status and histone methylation status in the StAR promoter region (-492 bp ~ -42 bp) were analyzed by chromatin immunoprecipitation assay among cells collected at 0, 4 and 12 h after hCG injection. Histone H4 acetylation levels were significantly increased at 4 h compared with 0 h, and then decreased to the basal level at 12 h, whereas histone H3 acetylation status was not changed at any times studied. The levels of tri-methyl histone H3K4, which promotes transcription, were gradually increased after hCG injection and significantly higher at 12 h than at 0 h. The levels of tri-methyl histone H3K9, which suppresses transcription, were gradually decreased and significantly lower at 12 h than at 0 h. DNA methylation status was analyzed in the StAR promoter region including the distal region (17 CpG sites, -1675 bp ~ -42bp) by sodium bisulfite genomic sequencing method. Six CpG sites in the promoter region (-503 bp ~ -42 bp) were demethylated and the other 11 CpG sites in the distal region (-1675 bp ~ -754 bp) were methylated. This DNA methylation profile did not change at any times studied during luteinization induced by hCG injection. These results led us to study whether histone acetylation of the promoter region is involved in StAR mRNA expression. We used RLC-16 cells, which show low histone acetylation status of the StAR promoter and do not express StAR gene. To experimentally induce histone acetylation, cells were treated with histone deacetylase (HDAC) inhibitors. Treatment with HDAC inhibitors induced StAR mRNA expression with induction of histone acetylation of the StAR promoter region. In conclusion, our results show that increases in histone acetylation levels and changes in histone methylation status in the promoter region by ovulatory LH surge in addition to DNA hypomethylation status of the promoter are closely associated with the rapid increase in StAR mRNA expression in luteinized granulosa cells during luteinization. The changes in epigenetic status in the StAR promoter region may be involved in StAR gene expression through the change in chromatin structure in rat granulosa cells during luteinization. (poster)

MAPK Phosphatase-1 (MKP-1) Expression Is Up-Regulated by hCG/cAMP and Modulates Steroidogenesis in MA-10 Leydig Cells

MAP kinases (MAPKs), such as ERK1/2, exert profound effects on a variety of physiological processes. In steroidogenic cells, ERK1/2 are involved in the expression and activation of steroidogenic acute regulatory protein, which plays a central role in the regulation of steroidogenesis. In MA-10 Leydig cells, LH and chorionic gonadotropin (CG) trigger transient ERK1/2 activation via protein kinase A, although the events that lead to ERK1/2 inactivation are not fully described. Here, we describe the hormonal regulation of MAPK phosphatase-1 (MKP-1), an enzyme that inactivates MAPKs, in MA-10 cells. In our experiments, human CG (hCG)/cAMP stimulation rapidly and transiently increased MKP-1 mRNA levels by a transcriptional action. This effect was accompanied by an increase in protein levels in both nuclear and mitochondrial compartments. In cells transiently expressing flag-MKP-1 protein, hCG/cAMP promoted the accumulation of the recombinant protein in a time-dependent manner (10-fold at 1 h). Moreover, hCG/cAMP triggered ERK1/2-dependent MKP-1 phosphorylation. The blockade of cAMP-induced MAPK kinase/ERK activation abated MKP-1 phosphorylation but only partially reduced flag-MKP-1 protein accumulation. Together, these results suggest that hCG regulates MKP-1 at transcriptional and posttranslational level, protein phosphorylation being one of the mechanisms involved in this regulation. Our study also demonstrates that MKP-1 overexpression reduces the effects of cAMP on ERK1/2 phosphorylation, steroidogenic acute regulatory gene promoter activity, mRNA levels, and steroidogenesis, whereas MKP-1 down-regulation by small interfering RNA produces opposite effects. In summary, our data demonstrate that hCG regulates MKP-1 expression at multiple stages as a negative feedback regulatory mechanism to modulate the hormonal action on ERK1/2 activity and steroidogenesis.

The Involvement of Specific PKC Isoenzymes in Phorbol Ester-Mediated Regulation of Steroidogenic Acute Regulatory Protein Expression and Steroid Synthesis in Mouse Leydig Cells

Protein kinase C (PKC) is a multigene family of serine/threonine kinases. PKC is involved in regulating adrenal and gonadal steroidogenesis; however, the functional relevance of the different PKC isoenzymes remains obscure. In this study, we demonstrate that MA-10 mouse Leydig tumor cells express several PKC isoforms to varying levels and that the activation of PKC signaling, by phorbol 12-myristate 13-acetate (PMA) elevated the expression and phosphorylation of PKCα, -δ, -ε, and -μ/protein kinase D (PKD). These responses coincided with the expression of the steroidogenic acute regulatory (StAR) protein and progesterone synthesis. Targeted silencing of PKCα, δ, and ε and PKD, using small interfering RNAs, resulted in deceases in basal and PMA-mediated StAR and steroid levels and demonstrated the importance of PKD in steroidogenesis. PKD was capable of controlling PMA and cAMP/PKA-mediated synergism involved in the steroidogenic response. Further studies pointed out that the regulatory events effected by PKD are associated with cAMP response element-binding protein (CREB) and c-Jun/c-Fos-mediated transcription of the StAR gene. Chromatin immunoprecipitation studies revealed that the activation of phosphorylated CREB, c-Jun, and c-Fos by PMA was correlated with in vivo protein-DNA interactions and the recruitment of CREB-binding protein, whereas knockdown of PKD suppressed the association of these factors with the StAR promoter. Ectopic expression of CREB-binding protein enhanced the trans-activation potential of CREB and c-Jun/c-Fos in StAR gene expression. Using EMSA, a -83/-67-bp region of the StAR promoter was shown to bind PKD-transfected MA-10 nuclear extract in a PMA-responsive manner, targeting CREB and c-Jun/c-Fos proteins. These findings provide evidence for the presence of multiple PKC isoforms and demonstrate the molecular events by which selective isozymes, especially PKD, influence PMA/PKC signaling involved in the regulation of the steroidogenic machinery in mouse Leydig cells.

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

Previous studies reported that the age-related decline in testosterone biosynthesis is associated with a decrease in the steroidogenic acute regulatory (StAR) protein which regulates the rate-limiting step of testosterone biosynthesis. To explore the possibility of delaying this decline using a dietary approach, we have examined the effect of a natural flavonoid, apigenin, on StAR gene expression in mouse Leydig cells. Incubation of these cells with the flavonoid enhanced cyclic adenosine monophosphate (cAMP)-induced steroidogenesis and StAR protein expression. The results from the analyses of StAR mRNA by reverse transcription-polymerase chain reaction and the luciferase assays of StAR promoter activity indicated that this flavonoid enhanced StAR gene expression at the level of transcription. Further studies showed that apigenin blocked the thromboxane A2 receptor and interrupted the signaling through the cyclooxygenase-2-thromboxane A synthase-thromboxane A2-receptor pathway, resulting in a reduction of DAX-1 (dosage sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene-1) protein, a transcriptional repressor of StAR gene expression. When DAX-1 protein was reduced, the sensitivity of the Leydig cells was dramatically enhanced, with sub-threshold level of cAMP being able to induce maximal levels of StAR protein expression and steroid hormone production. The present study suggests a potential application of apigenin to improve StAR protein expression and steroidogenic sensitivity of aging Leydig cells.

Nicotine Diminishes Testicular Gametogenesis, Steroidogenesis, and Steroidogenic Acute Regulatory Protein Expression in Adult Albino Rats: Possible Influence on Pituitary Gonadotropins and Alteration of Testicular Antioxidant Status

The present study was done to evaluate the pituitary-testicular activities of rats subjected to chronic nicotine treatment. The testicular key androgenic enzymes activities, plasma and intratesticular testosterone (ITT) concentrations, and plasma concentration of gonadotropin were significantly reduced by nicotine treatment along with the decreased sperm counts and the disruption of spermatogenesis indicated by significant reduction in the number of different generations of germ cells at stage VII of the spermatogenesis cycle with increased sperm head abnormalities. The Western blot and the reverse transcriptase-PCR analysis revealed that the nicotine induced a marked decrease in the expression of testicular steroidogenic acute regulatory (StAR) protein, which helps in the transfer of cholesterol in mitochondria for the testosterone biosynthesis. The increased testicular lipid peroxidation, plasma concentration of corticosterone, with enhanced hydrogen peroxide and hydroxyl radical generations, as well as decreased glutathione level, reduced antioxidant enzymes activities, and mitochondrial membrane potential (Deltapsi(m)) of testis, were noted after nicotine treatment in rats. Human chorionic gonadotropin or taurine supplementation with nicotine prevented the degeneration of germ cells to some extent, restored spermatogenesis moderately with decreased sperm head abnormalities, and enhanced sperm counts, accompanied with increase in plasma and ITT concentrations, testicular StAR gene expression, and key androgenic enzymes activities. Moreover, taurine supplementation to nicotine-treated animals resulted in the diminution of testicular lipid peroxidation, hydrogen peroxide and hydroxyl radical generations, with the elevation in glutathione level as well as different antioxidant enzymes activities and Deltapsi(m) in testis. The results indicated that nicotine caused testicular toxicity by germ cell degeneration, inhibition of StAR gene expression along with androgen production in adult male rats probably by affecting pituitary gonadotropin, and/or modulating the extent of testicular antioxidant status.