The acute regulation of steroid hormone biosynthesis requires the de novo synthesis of a protein whose function is to effect the transfer of cholesterol from the outer to the inner mitochondrial membrane where it is cleaved to... more
The acute regulation of steroid hormone biosynthesis requires the de novo synthesis of a protein whose function is to effect the transfer of cholesterol from the outer to the inner mitochondrial membrane where it is cleaved to pregnenolone. This review attempts to summarize the list of this protein's characteristics, which have emerged as a result of experimental observations, and to make the case that the Steroidogenic Acute Regulatory (StAR) Protein is the acute regulator of steroid hormone synthesis.
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A key regulatory point in fine tuning of steroidogenesis is the synthesis of steroidogenic acute regulatory protein, which transfers cholesterol into mitochondria. Heat shock and toxic insults reduce steroidogenic acute regulatory... more
A key regulatory point in fine tuning of steroidogenesis is the synthesis of steroidogenic acute regulatory protein, which transfers cholesterol into mitochondria. Heat shock and toxic insults reduce steroidogenic acute regulatory protein, severely compromising steroid synthesis. As the molecular mechanisms for this reduction remain elusive, we tested the hypothesis that heat shock directly interferes with transcription of the steroidogenic acute regulatory protein gene. We show that, in mouse MA-10 Leydig tumor cells, heat shock caused drastic declines in (Bu)(2)cAMP-induced progesterone accumulation and steroidogenic acute regulatory protein transcript abundance. A proximal steroidogenic acute regulatory protein promoter fragment (-85 to +39) is sufficient to direct both cAMP inducibility and heat shock inhibition. Nuclear extracts from MA-10 cells displayed binding to this proximal promoter fragment as a low mobility complex in gel shift experiments. This complex disappeared in nuclear extracts taken at 5 and 10 min after initiation of heat shock and reappeared in extracts taken at 2 and 8 h. Similar low- mobility complexes formed on oligonucleotides representing the overlapping subfragments of the minimal steroidogenic acute regulatory protein promoter fragment sensitive to the heat shock effect. Extracts from heat-shocked MA-10 cells displayed reduced complex formation to each of the subfragments. We conclude that heat shock reduces progesterone synthesis, steroidogenic acute regulatory protein mRNA abundance, and steroidogenic acute regulatory protein promoter activity and disrupts binding of nuclear proteins to the proximal region of the steroidogenic acute regulatory protein promoter. Together these observations provide strong evidence for a mechanism of transcriptional inhibition in the down-regulation of steroidogenic acute regulatory protein expression by heat shock.
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How rapid induction of steroid hormone biosynthesis occurs in response to trophic hormone stimulation of steroidogenic cells has been a subject of intensive investigation for approximately six decades. A key observation made very early... more
How rapid induction of steroid hormone biosynthesis occurs in response to trophic hormone stimulation of steroidogenic cells has been a subject of intensive investigation for approximately six decades. A key observation made very early was that acute regulation of steroid biosynthesis required swift and timely synthesis of a new protein whose role appeared to be involved in the delivery of the substrate for all steroid hormones, cholesterol, from the outer to the inner mitochondrial membrane where the process of steroidogenesis begins. It was quickly learned that this transfer of cholesterol to the inner mitochondrial membrane was the regulated and rate limiting step in steroidogenesis. Following this observation, the quest for this putative regulator protein(s) began in earnest in the late 1950s. This review provides a history of this quest, the candidate proteins that arose over the years, and facts surrounding their rise or decline. Only two have persisted-Translocator Protein (T...
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The synthesis of steroid hormones occurs in specific cells and tissues in the body in response to trophic hormones and other signals. In order to synthesize steroids de novo, cholesterol, the precursor of all steroid hormones, must be... more
The synthesis of steroid hormones occurs in specific cells and tissues in the body in response to trophic hormones and other signals. In order to synthesize steroids de novo, cholesterol, the precursor of all steroid hormones, must be mobilized from cellular stores to the inner mitochondrial membrane (IMM) to be converted into the first steroid formed, pregnenolone. This delivery of cholesterol to the IMM is the rate-limiting step in this process, and has long been known to require the rapid synthesis of a new protein(s) in response to stimulation. Although several possibilities for this protein have arisen over the past few decades, most of the recent attention to fill this role has centered on the candidacies of the proteins the Translocator Protein (TSPO) and the Steroidogenic Acute Regulatory Protein (StAR). In this review, the process of regulating steroidogenesis is briefly described, the characteristics of the candidate proteins and the data supporting their candidacies summa...
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The metabolism of acid-soluble and acid-insoluble material was investigated in starvation–refed synchronized Tetrahymena pyriformis. RNA synthesis was initiated 90 min after the addition of enriched nutrient medium, whereas DNA synthesis... more
The metabolism of acid-soluble and acid-insoluble material was investigated in starvation–refed synchronized Tetrahymena pyriformis. RNA synthesis was initiated 90 min after the addition of enriched nutrient medium, whereas DNA synthesis was not detectable until 180 min after refeeding. Studies on the acid-soluble pool indicated that the incorporation of radioactive precursors into the soluble nucleotide pool increased with time following refeeding of the cells; the increased incorporation of radioactive precursors in the acid-soluble nucleotide pool parallels the increase in nucleic acid synthesis.Further investigations indicated that starvation-synchronized cells were able to convert almost 80% of adenine ribonucleotide to adenine deoxyribonucleotide in a 30 min period during DNA synthesis. This conversion apparently served as an additional source of precursors for the synchronous burst of DNA synthesis observed in these cells. In contrast logarithmic cells and heat-synchronized cells converted less than 10% of this ribonucleotide to the deoxyribonucleotide during a 30 min period.
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Interferon-gamma (IFNgamma) is an immunomodulating cytokine that has profound effects on reproductive function. IFNgamma inhibits steroidogenesis both in vivo and in vitro. The mechanism by which IFNgamma inhibits Leydig cell... more
Interferon-gamma (IFNgamma) is an immunomodulating cytokine that has profound effects on reproductive function. IFNgamma inhibits steroidogenesis both in vivo and in vitro. The mechanism by which IFNgamma inhibits Leydig cell steroidogenesis remains unclear. In the present study, we evaluated the effect of IFNgamma on the expression and regulation of the steroidogenic acute regulatory protein (StAR) gene in primary cultures of rat Leydig cells. StAR facilitates the efficient production of steroid hormone by regulating the translocation of cholesterol from the outer to the inner mitochondrial membrane, the site of the cytochrome P450 side-chain cleavage (P450scc) enzyme system that converts cholesterol to pregnenolone. IFNgamma inhibited hCG-induced StAR messenger RNA (mRNA) levels in a dose-dependent manner. The addition of IFNgamma in a concentration of 500 U/ml decreased hCG-induced 3.8- and 1.7-kilobase StAR mRNA by 78% and 70%, respectively. IFNgamma also reduced hCG-stimulated P450scc mRNA levels by 69%. The inhibitory effects of IFNgamma on StAR mRNA levels were confirmed by ribonuclease protection assay. As early as 12 h after the addition of IFNgamma, hCG-induced StAR mRNA levels decreased by more than 44%. To evaluate the effects of IFNgamma on StAR protein levels, Western blot analyses were performed. hCG in a concentration of 10 ng/ml increased StAR protein by 5.6-fold. Treatment of Leydig cells with IFNgamma (500 U/ml) decreased hCG-induced StAR protein by 44%. In contrast, interleukin-1 and murine tumor necrosis factor-alpha reduced hCG-induced P450scc mRNA expression without inhibiting StAR mRNA or protein levels. In conclusion, IFNgamma inhibits Leydig cell steroidogenesis by down-regulating StAR gene expression and protein production.
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The tumor-promoting phorbol ester, phorbol-12-myristate-13-acetate (PMA) markedly stimulated progesterone production in MA-10 Leydig tumor cells. A slight but significant increase (35%) in the activity of the cholesterol side-chain... more
The tumor-promoting phorbol ester, phorbol-12-myristate-13-acetate (PMA) markedly stimulated progesterone production in MA-10 Leydig tumor cells. A slight but significant increase (35%) in the activity of the cholesterol side-chain cleavage (CSCC) enzyme was observed in mitochondria isolated from the PMA-treated MA-10 Leydig cells when compared to mitochondria isolated from non-treated cells. However, this stimulation of CSCC activity appears to be of limited importance when compared to the 240-fold increase observed in progesterone production following PMA stimulation. In contrast, the inactive phorbol ester 4 alpha-phorbol-12,13-didecanoate (alpha-PD) had no effect on either progesterone production or CSCC activity. PMA had no effect on the conversion of 25-hydroxycholesterol and 22R-hydroxycholesterol into progesterone suggesting that one of the mechanism(s) of PMA action may involve the delivery of cholesterol to the mitochondria and/or the affinity of cholesterol with cytochrome P-450scc. Stimulation of steroidogenesis by PMA was also shown to be inhibited by cycloheximide. When PMA was added together with a submaximal dose of hCG, hCG-stimulated steroidogenesis was inhibited. However, at a maximal dose of human chorionic gonadotropin (hCG), PMA inhibited steroid synthesis at 1 and 2 h but had no significant effect at 3 h. Conversely, PMA had an additive effect on cAMP induced steroidogenesis. It was further demonstrated that PMA resulted in a decrease in the hCG-induced accumulation of cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)
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The Steroidogenic Acute Regulatory (StAR) protein is a mitochondrial protein required for the transport of cholesterol substrate to the P450scc enzyme located in the inner mitochondrial membranes of steroid producing cells. This study... more
The Steroidogenic Acute Regulatory (StAR) protein is a mitochondrial protein required for the transport of cholesterol substrate to the P450scc enzyme located in the inner mitochondrial membranes of steroid producing cells. This study suggests that the acute regulation of the rodent StAR gene in the ovary is mediated by two factors, C/EBPbeta and GATA-4. Once translated, the StAR precursor protein is either imported into the mitochondria, or it is rapidly degraded in the cytosol. We predicted that in order to perpetuate StAR activity cycles, imported StAR should turn over rapidly to avoid a potentially harmful accumulation of the protein in sub-mitochondrial compartments. Pulse-chase experiments in metabolically labeled cells showed that: (a) the turnover rate of mature mitochondrial StAR protein (30 kDa) is much faster (t(1/2) = 4-5 h) than that of other mitochondrial proteins; (b) dissipation of the inner membrane potential (-delta psi) by carbonyl cyanide m-chlorophenylhydrazone (mCCCP) accelerates the mitochondrial degradation of StAR; (c) unexpectedly, the mitochondrial degradation of StAR is inhibited by MG132 and lactacystin, but not by epoxomicin. Furthermore, StAR degradation becomes inhibitor-resistant two hours after import. Therefore, these studies suggest a bi-phasic route of StAR turnover in the mitochondria. Shortly after import, StAR is degraded by inhibitor-sensitive protease(s) (phase I), whereas at later times, StAR turnover proceeds to completion through an MG132-resistant proteolytic activity (phase II). Collectively, this study defines StAR as a unique protein that can authentically be used to probe multiple proteolytic activities in mammalian mitochondria.
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The present studies demonstrate that the murine adrenocortical tumor cell line Y-1 releases a digoxin-like immunoreactive material into both serum-supplemented nutrient medium and minimal Krebs-Ringer bicarbonate medium. Release of... more
The present studies demonstrate that the murine adrenocortical tumor cell line Y-1 releases a digoxin-like immunoreactive material into both serum-supplemented nutrient medium and minimal Krebs-Ringer bicarbonate medium. Release of pregnenolone into minimal medium from these cells was consistently inhibited by addition of the cholesterol side-chain cleavage inhibitor aminoglutethimide. However, release of digoxin-like immunoreactivity (DLI) was not similarly affected. To exclude the possibility that DLI could be accounted for by cross-reaction with another known adrenal steroid, aminoglutethimide inhibition was accompanied by inhibition of 17 alpha-hydroxylase with SU-10603 and inhibition of 3 beta-hydroxysteroid dehydrogenase with cyanoketone. Once again, pregnenolone release was effectively inhibited, but no similar pattern of inhibition of DLI release was observed. Increasing the time of the incubation periods from 1 to 2 h did not change the pattern of secretion of pregnenolone or DLI. HPLC analysis of DLI released over prolonged culture periods into serum-supplemented nutrient medium showed high levels of DLI in a single major and several adjacent peaks. Analysis of the ability of extracts of Y-1-conditioned medium to compete with tritiated ouabain for binding to erythrocytes indicates that conditioned medium contained highly enriched levels of ouabain-like activity. On HPLC analysis, the distribution of this activity showed partial correlation with the distribution of DLI. These observations indicate that Y-1 cells produce and release significant quantities of a material with cardiac glycoside-like properties reflected in the cross-reactivity with antidigoxin antibodies and the ability to compete with ouabain for binding to erythrocytes. In substantiation of previous findings in chopped adrenal cultures, the cardiac glycoside-like activity does not appear to result from cholesterol side-chain cleavage or pregnenolone production, since inhibition of side-chain cleavage as well as subsequent 17 alpha-hydroxylation and 3 beta-dehydrogenation did not result in consistent inhibition of DLI release.
Research Interests: Endocrinology, Kinetics, Saponins, Cell Culture, Biological Sciences, and 15 moreCell line, Humans, Mice, Animals, Adrenal Gland, Erythrocyte Membrane, Digoxin, Secretion, Blood Proteins, Pregnenolone, Radioimmunoassay, Aminoglutethimide, Ouabain, Medical and Health Sciences, and HPLC Chromatography
Translocator protein (TSPO), also known as the peripheral benzodiazepine receptor (PBR), is a highly conserved outer mitochondrial membrane protein present in specific sub-populations of cells within different tissues. In recent studies,... more
Translocator protein (TSPO), also known as the peripheral benzodiazepine receptor (PBR), is a highly conserved outer mitochondrial membrane protein present in specific sub-populations of cells within different tissues. In recent studies, the presumptive model depicting mammalian TSPO as a critical cholesterol transporter for steroidogenesis has been refuted by studies examining effects of Tspo gene deletion in vivo and in vitro, biochemical testing of TSPO cholesterol transport function, and specificity of TSPO-mediated pharmacological responses. Nevertheless, high TSPO expression in steroid-producing cells seemed to indicate an alternate function for this protein in steroidogenic mitochondria. To seek an explanation, we used CRISPR/Cas9-mediated TSPO knockout steroidogenic MA-10 Leydig cell (MA-10:TspoΔ/Δ) clones to examine changes to core mitochondrial functions resulting from TSPO deficiency. We observed that, 1) MA-10:TspoΔ/Δ cells had a shift in substrate utilization for energy...
Research Interests: Endocrinology, Mitochondria, Ion Channels, Biological Sciences, Progesterone, and 13 moreFatty acids, Reactive Oxygen Species, Mice, GABA receptors, Animals, Male, Lipid metabolism, Leydig cells, Oxidation-Reduction, Gene Expression Regulation, Mitochondrial Proteins, reverse transcriptase polymerase chain reaction, and Medical and Health Sciences
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Research Interests: Aging, STEROIDS, Vitamin A, Humans, Mice, and 7 moreFemale, Animals, Male, Clinical Sciences, Aged, Middle Aged, and Skin Aging
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The present work seeks to determine the parameters for approval, through ultrasonic techniques, of spot welded joints of low carbon steel sheets. For such experience the mechanical behavior of a spotwelded joint was characterized under... more
The present work seeks to determine the parameters for approval, through ultrasonic techniques, of spot welded joints of low carbon steel sheets. For such experience the mechanical behavior of a spotwelded joint was characterized under fatigue, in load cycles ranging from zero to 14 kN, on sheets of 1.5 mm thickness, joined by three spot welds of 7.5 mm of medium diameter, being the distance among spot weld centers 15 mm. The welding parameters were modified to generate indentations of up to 40% of the nominal thickness of the joints, measured by ultrasonic techniques. It was verified that fatigue cracks were nucleated in the areas of maximum equivalent von Mises stress, calculated by Finite Element Method (FEM) analysis, and their propagation occurred in the direction of the maximum main stress also calculated by FEM, together to the action of residual stresses, which were confirmed by X-Ray diffraction. The fatigue tests demonstrated that samples with indentation among 20 and 40% ...
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The 18 kDa translocator protein (TSPO), also known as the peripheral benzodiazepine receptor (PBR), is a transmembrane protein in the outer mitochondrial membrane. TSPO has long been described as being indispensable for mitochondrial... more
The 18 kDa translocator protein (TSPO), also known as the peripheral benzodiazepine receptor (PBR), is a transmembrane protein in the outer mitochondrial membrane. TSPO has long been described as being indispensable for mitochondrial cholesterol import that is essential for steroid hormone production. In contrast to this initial proposition, recent experiments reexamining TSPO function have demonstrated that it is not involved in steroidogenesis. This fundamental change has forced a reexamination of the functional interpretations made for TSPO that broadly impacts both basic and clinical research across multiple fields. In this mini-review, we recapitulate the key studies from 25 years of TSPO research, and concurrently examine their limitations that perhaps led towards the incorrect association of TSPO and steroid hormone production. Although this shift in understanding raises new questions regarding the molecular function of TSPO, these recent developments are poised to have a sig...
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Translocator protein (TSPO), previously known as the peripheral benzodiazepine receptor (PBR), is an outer mitochondrial membrane protein. TSPO has been shown to cooperate with steroidogenic acute regulatory protein (StAR) and function in... more
Translocator protein (TSPO), previously known as the peripheral benzodiazepine receptor (PBR), is an outer mitochondrial membrane protein. TSPO has been shown to cooperate with steroidogenic acute regulatory protein (StAR) and function in the transport of cholesterol into mitochondria. TSPO has also been considered as a structural component of the mitochondrial permeability transition pore (MPTP). However, recent advances have changed these views of TSPO's functions and have prompted a re-evaluation of established concepts. This review summarizes the history of TSPO, key elements of the debate, and functional experiments that have changed our understanding. Moving forward, we examine how this fundamental change impacts our understanding of TSPO and affects the future of TSPO as a therapeutic and diagnostic target.
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Mitogen-activated protein kinases (MAPKs) comprise a family of serine/threonine kinases that are activated by a large variety of extracellular stimuli and play integral roles in controlling many cellular processes, from the cell surface... more
Mitogen-activated protein kinases (MAPKs) comprise a family of serine/threonine kinases that are activated by a large variety of extracellular stimuli and play integral roles in controlling many cellular processes, from the cell surface to the nucleus. The MAPK family includes four distinct MAPK cascades, that is, extracellular signal-regulated kinase 1/2 (ERK1/2), p38 MAPK, c-Jun N-terminal kinase or stress-activated protein kinase, and ERK5. These MAPKs are essentially operated through three-tiered consecutive phosphorylation events catalyzed by a MAPK kinase kinase, a MAPK kinase, and a MAPK. MAPKs lie in protein kinase cascades. The MAPK signaling pathways have been demonstrated to be associated with events regulating the expression of the steroidogenic acute regulatory protein (StAR) and steroidogenesis in steroidogenic tissues. However, it has become clear that the regulation of MAPK-dependent StAR expression and steroid synthesis is a complex process and is context dependent....
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The steroidogenic acute regulatory (StAR) protein is essential for all hormone-stimulated steroid biosynthesis. Accordingly, its absence gives rise to the most severe form of congenital adrenal hyperplasia (CAH), lipoid CAH. This... more
The steroidogenic acute regulatory (StAR) protein is essential for all hormone-stimulated steroid biosynthesis. Accordingly, its absence gives rise to the most severe form of congenital adrenal hyperplasia (CAH), lipoid CAH. This life-threatening condition typically manifests itself in the perinatal period. Partial loss-of-function StAR mutations incompletely manifest the condition later in life and are a cause of familial glucocorticoid deficiency type 3. Here, we discuss StAR, its expression pattern and the clinical consequences of the loss of its activity.
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Following tropic hormone challenge, steroidogenic tissues utilize PKA to phosphorylate unique subsets of proteins necessary to facilitate steroidogenesis. This includes the PKA-dependent expression and activation of the steroidogenic... more
Following tropic hormone challenge, steroidogenic tissues utilize PKA to phosphorylate unique subsets of proteins necessary to facilitate steroidogenesis. This includes the PKA-dependent expression and activation of the steroidogenic acute regulatory protein (STAR), which mediates the rate-limiting step of steroidogenesis by inducing the transfer of cholesterol from the outer to the inner mitochondrial membrane. Since both type I and type II PKA are present in steroidogenic tissues, we have utilized cAMP analog pairs that preferentially activate each PKA subtype in order to examine their impact on STAR synthesis and activity. In MA-10 mouse Leydig tumor cells Star gene expression is more dependent upon type I PKA, while the post-transcriptional regulation of STAR appears subject to type II PKA. These experiments delineate the discrete effects that type I and type II PKA exert on STAR-mediated steroidogenesis, and suggest complimentary roles for each subtype in coordinating steroidog...
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The decline in blood testosterone concentration during the course of male aging results in decreases in many physiological functions. However, the mechanism(s) responsible for this decline is not clear. Previous observations have... more
The decline in blood testosterone concentration during the course of male aging results in decreases in many physiological functions. However, the mechanism(s) responsible for this decline is not clear. Previous observations have suggested the involvement of multiple alterations or defects that inhibit the activities of proteins involved in steroidogenesis and result in reduced testosterone biosynthesis. Recent studies have demonstrated an age-related increase in cyclooxygenase-2 (COX2) activity and its tonic inhibition of steroidogenic acute regulatory gene expression and steroidogenesis in rat Leydig cells. These findings indicate the presence of a novel mechanism in male aging involving COX2 and suggest the potential application of COX2 inhibitors or other interventions in this mechanism to delay the decline in testosterone biosynthesis in aged males.
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The transport of cholesterol to the inner mitochondrial membrane of steroidogenic cells constitutes the rate-limiting step in trophic hormone regulated steroid biosynthesis and requires de novo protein synthesis. Several years ago a... more
The transport of cholesterol to the inner mitochondrial membrane of steroidogenic cells constitutes the rate-limiting step in trophic hormone regulated steroid biosynthesis and requires de novo protein synthesis. Several years ago a candidate regulator protein was purified and its cDNA cloned from MA-10 mouse Leydig tumor cells. Expression of this protein resulted in an increase in steroidogenesis in unstimulated cells and it was named the Steroidogenic Acute Regulatory protein or StAR. Mutations in the StAR gene were found to be the cause of the potentially lethal disease in humans known as congenital lipoid adrenal hyperplasia (lipoid CAH), a condition characterized by an almost complete inability of the newborn to synthesize steroids. The defect in steroid synthesis in lipoid CAH is caused by the failure of affected individuals to transport cholesterol to the inner mitochondria membrane, thus proving the essential role of StAR in cholesterol transport. StAR null mice display a ph...
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The steroidogenic acute regulatory (StAR) protein, a novel phosphoprotein, is a crucial factor involved in intramitochondrial cholesterol transportation, the rate-limiting step in steroidogenesis. The present investigations were... more
The steroidogenic acute regulatory (StAR) protein, a novel phosphoprotein, is a crucial factor involved in intramitochondrial cholesterol transportation, the rate-limiting step in steroidogenesis. The present investigations were undertaken to elucidate involvement of thyroid hormone and StAR protein in the regulation of steroidogenesis in mouse Leydig cells. Treatment of cells with triiodothyronine (T(3)) coordinately augmented the levels of StAR protein, StAR mRNA, and steroid production, and these responses were progressively dependent on expression of steroidogenic factor 1 (SF-1). With regard to steroidogenesis and StAR expression, the T(3) response requires both on-going mRNA and protein synthesis. In addition, the effects of T(3) were acutely modulated at the steroidogenic machinery and luteinizing hormone receptor (LHR) function, while these levels were suppressed following longer periods of exposure to T(3). Furthermore, the inhibition of SF-1 expression by DAX-1 markedly ab...
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Insulin-like growth factor-I (IGF-I) plays an essential role in reproductive function. Leydig cells express specific IGF-I receptors, and IGF-I enhances human chorionic gonadorphin (hCG)-induced testosterone formation. In the present... more
Insulin-like growth factor-I (IGF-I) plays an essential role in reproductive function. Leydig cells express specific IGF-I receptors, and IGF-I enhances human chorionic gonadorphin (hCG)-induced testosterone formation. In the present study, we evaluate the effect of IGF-I on the gene expression and protein levels of steroidogenic acute regulatory protein (StAR), the rate-limiting step in steroidogenesis. StAR mRNA is expressed in rat Leydig cells as two major transcripts of 3.8 and 1.7 kb. StAR mRNA levels (both 3.8 and 1.7 kb) were markedly induced about 20-fold by hCG (10 ng/mL). Concomitant addition of IGF-I (50 or 100 ng/mL) and hCG (10 ng/mL) resulted in significant increases in StAR and cytochrome P450 side-chain cleavage (P450scc) mRNA levels, whereas lower doses of IGF-I (1 or 10 ng/ mL) had small effects. Synergistic effects of IGF-I and hCG on StAR mRNA levels were confirmed by ribonuclease protection assay (RPA). IGF-I (100 ng/mL) enhanced hCG- and 20 OH-cholesterol + hCG...
Research Interests: Gene expression, Western blotting, Animals, Male, Endocrine, and 12 moreEnzyme, Clinical Sciences, Rats, Western blot, Dehydroepiandrosterone, Leydig cells, Gene Expression Regulation, Synergistic effect, Human Chorionic Gonadotrophin, Rate Limiting, Drug synergism, and Paediatrics and reproductive medicine
Mutations in the DAX-1 gene are responsible for congenital X-linked adrenal hypoplasia, a disease that is associated with hypogonadotropic hypogonadism. DAX-1 expression is tissue-specific and is finely regulated throughout development,... more
Mutations in the DAX-1 gene are responsible for congenital X-linked adrenal hypoplasia, a disease that is associated with hypogonadotropic hypogonadism. DAX-1 expression is tissue-specific and is finely regulated throughout development, suggesting that it has a role in both adrenal and gonadal function. DAX-1 is an unusual member of the nuclear-receptor superfamily of transcription factors which contains no canonical zinc-finger or any other known DNA-binding motif. Binding sites for DAX-1 are found in the promoters of the dax-1 and StAR (for steroidogenic acute regulatory protein) genes. Here we show that DAX-1 binds DNA and acts as a powerful transcriptional repressor of StAR gene expression, leading to a drastic decrease in steroid production. We provide in vitro and in vivo evidence that DAX-1 binds to DNA hairpin structures. Our results establish DAX-1 as the first member of the nuclear receptor superfamily with novel DNA-binding features and reveal that it has regulatory prope...
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Previous reports have demonstrated that corticotropin-releasing hormone (CRH) treatment of primary cultures of mouse Leydig cells and MA-10 mouse Leydig tumor cells results in a dose-dependent stimulation of steroidogenesis, probably by... more
Previous reports have demonstrated that corticotropin-releasing hormone (CRH) treatment of primary cultures of mouse Leydig cells and MA-10 mouse Leydig tumor cells results in a dose-dependent stimulation of steroidogenesis, probably by acting through the cAMP/protein kinase A second messenger pathway. Based on this observation, the mechanism of CRH-stimulated steroidogenesis is now further investigated and compared to trophic hormone stimulation. Both cell types were treated with human chorionic gonadotropin (hCG) or CRH in the absence and presence of the following agents: the translation inhibitor cycloheximide, the transcription inhibitor actinomycin D, the protonophore carbonyl cyanide m-chlorophenylhydrozone (mCCCP), which disrupts the mitochondrial electrochemical gradient or the phorbol ester, phorbol-12-myristate 13-acetate (PMA), a stimulator of protein kinase C. Cortico-releasing hormone-stimulated steroidogenesis was completely blocked by cycloheximide in both cell types,...
Research Interests: Andrology, Kinetics, Comparative Study, Corticotropin Releasing Hormone, Protein synthesis, and 15 moreIn Vitro, Progesterone, Humans, Mice, Animals, Male, Clinical Sciences, Leydig cells, Primary Culture, Luteinizing Hormone, Phorbol ester, Cycloheximide, Protein Kinase C, Paediatrics and reproductive medicine, and Macho
The Steroidogenic Acute Regulatory (StAR) protein has been put forth as the rapidly synthesized, cycloheximide-sensitive protein that is required for the transport of cholesterol to the inner mitochondrial membrane and the P450scc enzyme... more
The Steroidogenic Acute Regulatory (StAR) protein has been put forth as the rapidly synthesized, cycloheximide-sensitive protein that is required for the transport of cholesterol to the inner mitochondrial membrane and the P450scc enzyme and thereby acutely regulates steroidogenesis in steroidogenic tissues. In this study, several of the factors that may be required for StAR activity were examined using an in vitro system. Lysates from StAR-transfected COS-1 cells were added to mitochondria isolated from MA-10 Leydig tumor cells. Results obtained demonstrated that StAR-containing cell lysate increased steroidogenesis in isolated mitochondria, but failed to do so in the presence of m-CCCP, apyrase, or AMP-PNP, suggesting that StAR function requires ATP hydrolysis as well as an electrochemical gradient for maximal steroidogenic activity.
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Thymidine resistance in V79 Chinese hamster cells has been investigated. Phenotypically stable variant resistant lines occurred at a high frequency, and the mutation rate (2.67 x 10(-3) per cell per generation) to 400 micrograms/ml... more
Thymidine resistance in V79 Chinese hamster cells has been investigated. Phenotypically stable variant resistant lines occurred at a high frequency, and the mutation rate (2.67 x 10(-3) per cell per generation) to 400 micrograms/ml thymidine resistance as measured by the standard Luria--Delbrück fluctuation analysis was extremely high. Populations of cells maintained for extended periods in F-10 medium spontaneously increased in resistance, possibly as a result of selective pressures due to the thymidine present in F-10 medium since this change was not observed in Dulbecco's medium. The degree of resistance for a given variant was correlated with the amount of thymidine employed in its selection. Metabolic cooperation, resulting in the suppression of the resistant phenotype, was demonstrated in artificial mixtures of sensitive and resistant clonal lines. Clones isolated in high levels of thymidine possessed lowered uptake of [3H]thymidine and the depression in uptake was related...
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The cellular and subcellular distribution of sterol carrier protein 2 (SCP2; nsL-TP) was reinvestigated in rat testicular cells by Western blotting and immunocytochemistry, using the affinity purified antibody against rat liver SCP2.... more
The cellular and subcellular distribution of sterol carrier protein 2 (SCP2; nsL-TP) was reinvestigated in rat testicular cells by Western blotting and immunocytochemistry, using the affinity purified antibody against rat liver SCP2. Western blot analysis revealed high levels of the protein in the somatic cells of the testis, e.g., Leydig and Sertoli cells whereas it could not be detected in germ cells. This cellular localization of SCP2 was confirmed by Northern blotting. Immunocytochemical techniques revealed that in Leydig cells, immunoreactive proteins were concentrated in peroxisomes. Although SCP2 was also detected in Sertoli cells, a specific subcellular localization could not be shown. SCP2 was absent from germ cells. Analysis of subcellular fractions of Leydig cells showed that SCP2 is membrane bound without detectable amounts in the cytosolic fraction. These results are at variance with data published previously which suggested that in Leydig cells a substantial amount of ...
Research Interests: RNA, Immunohistochemistry, Western blotting, Biological Sciences, Germ Cells, and 13 moreAnimals, Nucleic acid hybridization, Male, Physical sciences, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, Sertoli cells, Rats, Rat, Peroxisome, Leydig cells, Sertoli Cell, PLANT PROTEINS, and Germ Cell
Mitochondria from liver whole homogenates of Fischer 344 rats, 3,6, 12 and 24 mo of age were separated by reorienting rate-zonal centrifugation on sucrose gradients. Employing this method, mitochondria can be harvested nearly... more
Mitochondria from liver whole homogenates of Fischer 344 rats, 3,6, 12 and 24 mo of age were separated by reorienting rate-zonal centrifugation on sucrose gradients. Employing this method, mitochondria can be harvested nearly quantitatively and subjected to a number of biochemical analyses. Separation and recovery of the mitochondria from young and old rats was monitored by assaying each fraction from the gradient for cytochrome oxidase and lipoamide dehydrogenase, two mitochondrial specific enzymes. In addition, several regions of the gradient were studied by transmission electron microscopy. In this study, mitochondrial DNA and protein were quantitated from the livers of the animals at each age group. It was demonstrated that between the ages of 12 and 24 mo these macromolecules decreased significantly. These data would seem to indicate that the number of mitochondria is decreased in this tissue in aged animals.