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Biomedical subjects

D M Stocco

Publications and source records attributed to D M Stocco.

At least 55 records · Page 3Linked to original sources
PubMed1999

Growth factor modulation of steroidogenic acute regulatory protein and luteinization in the pig ovary.

In vivo and in vitro luteinization were investigated in the porcine ovary, with emphasis on expression of steroidogenic acute regulatory protein (StAR). StAR mRNA and protein as well as cytochrome P450 side-chain cleavage mRNA (P450scc) increased during the luteal phase in the corpus luteum (CL) and were absent in regressed CL. Cytochrome P450 aromatase mRNA (P450arom) was not detectable at any time in CL. In vitro luteinization of granulosa cells occurred over 96 h in culture, during which P450arom mRNA was present at 1 h after cell isolation but not detectable at 6 h; and P450scc and StAR mRNAs were first detectable at 6 h and 48 h, respectively. Incubation of cultures with insulin-like growth factor I (IGF-I, 10 ng/ml), dibutyryl cAMP (cAMP, 300 microM), or their combination, induced measurable StAR mRNA at 24 h (p < 0.05), increased progesterone accumulation at 48 h, and elevated both StAR and P450scc expression through 96 h. Incubation of luteinized granulosa cells with epidermal growth factor (EGF, 10 nM) changed their phenotype from epithelioid to fibroblastic, eliminated steady-state StAR expression, and interfered with cAMP induction of StAR mRNA and progesterone accumulation. EGF had little apparent effect on P450scc mRNA abundance. It is concluded that StAR expression characterizes luteinization, and early luteinization is induced by cAMP and IGF-I in vitro. Further, EGF induces a morphological and functional phenotype that appears similar to an earlier stage of granulosa cell function.

Animals↗
PubMed1999

SF-1 (steroidogenic factor-1) and C/EBP beta (CCAAT/enhancer binding protein-beta) cooperate to regulate the murine StAR (steroidogenic acute regulatory) promoter.

The steroidogenic acute regulatory (StAR) protein mediates the rate-limiting step of steroidogenesis, which is the transfer of cholesterol to the inner mitochondrial membrane. In steroidogenic tissues, StAR expression is acutely regulated by trophic hormones through a cAMP second messenger pathway, leading to increased StAR mRNA levels within 30 min, reaching maximal levels after 4-6 h of stimulation. The molecular mechanisms underlying such regulation remain unknown. We have examined the StAR promoter for putative transcription factor-binding sites that may regulate transcription in a developmental and/or hormone-induced context. Through sequence analysis, deoxyribonuclease I (DNAse I) footprinting and electrophoretic mobility shift assays (EMSAs), we have identified two putative CCAAT/enhancer binding protein (C/EBP) DNA elements at -113 (C1) and -87 (C2) in the mouse StAR promoter. Characterization of these sites by EMSA indicated that C/EBP beta bound with high affinity to C1 and C2 was a low-affinity C/EBP site. Functional analysis of these sites in the murine StAR promoter showed that mutation of one or both of these binding sites decreases both basal and (Bu)2cAMP-stimulated StAR promoter activity in MA-10 Leydig tumor cells, without affecting the fold activation [(Bu)2cAMP-stimulated/basal] of the promoter. Furthermore, we have demonstrated that these two C/EBP binding sites are required for steroidogenic factor-1 (SF-1)-dependent transactivation of the StAR promoter in a nonsteroidogenic cell line. These data indicate that in addition to SF-1, C/EBP beta is involved in the transcriptional regulation of the StAR gene and may play an important role in developmental and hormone-responsive regulation of steroidogenesis.

Animals↗
PubMed1999

The role of arachidonic acid on LH-stimulated steroidogenesis and steroidogenic acute regulatory protein accumulation in MA-10 mouse Leydig tumor cells.

Metabolic pathways leading to the production of arachidonic acid (AA) and its metabolites have been reported to have modulatory effects on steroidogenesis in a number of cell types. To examine the importance of the arachidonic acid pathway in steroid production and steroidogenic acute regulatory (StAR) protein expression, luteinizing hormones (LH) or N6-2-o-dibutyryl-adenosine-3:5-cyclic monophosphate-(Bt2cAMP) stimulated MA-10 mouse Leydig tumor cells were treated with various concentrations of quinacrine (an inhibitor of arachidonic acid production). Incubation of the cells with quinacrine resulted in dose-dependent decreases in steroid production and StAR protein. Twenty micromolars quinacrine inhibited 92 and 91% of LH-induced progesterone and StAR protein, respectively, and 98 and 90% of Bt2cAMP-induced progesterone and StAR protein. Reversal of this inhibition was obtained by incubation of quinacrine-treated cells with various levels of AA, which resulted in a dose-dependent increase in both steroid and StAR protein levels. Two hundred micromolars of AA rescued 57 and 60% of the LH-induced steroid production and StAR protein, respectively, and 52 and 89% of Bt2cAMP-induced steroid production and StAR protein. These results suggest that the effect of AA on LH- and cAMP-stimulated steroidogenesis is associated with the modulation of StAR protein expression.

Animals↗
PubMed1999

Effect of leptin on ACTH-stimulated secretion of cortisol in rhesus macaques and on human adrenal carcinoma cells.

OBJECTIVE: Because glucocorticoids stimulate leptin release and, at least in vitro, leptin inhibits cortisol secretion, a feedback system between glucocorticoids and leptin has been proposed. However, in humans and non-human primates there are no in vivo studies to support any role for leptin in the control of the hypothalamic-pituitary-adrenal axis. In this study, we investigated the effect of leptin on (i) ACTH-stimulated secretion of cortisol in six male rhesus monkeys and (ii) basal and forskolin (FSK)-stimulated cortisol secretion by the human adrenal carcinoma cell H295R in vitro. DESIGN AND METHODS: In vivo studies: after suppression of endogenous ACTH with either dexamethasone (n=6) or a corticotropin-releasing factor (CRF) antagonist (d-Phe CRF(12-41)) (n=3), 1 microg bolus of human ACTH(1-24) was administered to stimulate adrenal cortisol release. Blood samples were collected every 15 min for 3 h. Leptin (1 mg) was infused over 4 h, starting 1 h before ACTH bolus. IN VITRO STUDIES: NCI-H295R cells were incubated for 6, 12, 24 and 48 h in the absence or presence of 20 micromol/l FSK in combination with leptin (100 ng/ml medium). Cortisol levels in serum and medium were measured by solid phase radioimmunoassay. RESULTS: Acute leptin infusion to rhesus monkeys did not change basal cortisol levels, peak cortisol levels after ACTH(1-24) or the area under the curve when compared with studies in which leptin was not given. FSK increased cortisol levels in medium at 24 and 48 h, but leptin did not change cortisol release in either control or FSK-stimulated cells. CONCLUSIONS: Short-term leptin infusion affected neither the cortisol response to ACTH in non-human primates in vivo nor cortisol release (basal or FSK stimulated) by H295R cells, in vitro. These data suggest that leptin may not be an acute regulator of primate adrenal cortisol secretion.

Adrenal Cortex↗
PubMed1999

GnRH agonist treatment decreases progesterone synthesis, luteal peripheral benzodiazepine receptor mRNA, ligand binding and steroidogenic acute regulatory protein expression during pregnancy.

We have demonstrated that continuous administration of a gonadotropin-releasing hormone agonist (GnRH-Ag) suppresses luteal steroidogenesis in the pregnant rat. We further demonstrated that the peripheral-type benzodiazepine receptor (PBR) and the steroidogenic acute regulatory protein (StAR) play key roles in cholesterol transport leading to steroidogenesis. The purpose of this study was to understand the cellular and molecular mechanisms involved in the suppression of luteal steroidogenesis leading to a fall in serum progesterone levels in GnRH-Ag-treated rats during early pregnancy. Pregnant rats were treated individually starting on day 8 of pregnancy with 5 microgram/day GnRH-Ag using an osmotic minipump. Sham-operated control rats received no treatment. At 0, 4, 8 and 24 h after initiation of the treatment, rats were killed and corpora lutea (CL) were removed for PBR mRNA, protein and radioligand binding analyses, immunoblot 1-D gel analysis of StAR, P450 scc and 3beta-hydroxysteroid dehydrogenase as well as 2-D gel analysis of StAR. The treatment decreased the luteal PBR mRNA expression at all time periods starting at 4 h compared with that in corresponding sham controls. GnRH-Ag also reduced, in the CL, the PBR protein/ligand binding, the StAR protein and P450 scc protein and its activity as early as 8 h after the treatment and they remained low compared with those in corresponding sham controls. The data from 2-D gel studies suggest that the majority of the decrease in StAR protein appears to be in the phosphorylated forms of StAR. Thus, we have demonstrated, for the first time, the presence of PBR and StAR in the pregnant rat CL and that the coordinated suppression of these proteins involved in the mitochondrial cholesterol transport along with P450 scc by GnRH-Ag leads to reduced ovarian steroidogenesis.

3-Hydroxysteroid Dehydrogenases↗
PubMed1998

Changes in testicular steroidogenic acute regulatory (STAR) protein, steroidogenic enzymes and testicular morphology associated with increased testosterone secretion in bulls receiving the luteinizing hormone releasing hormone agonist deslorelin.

Testosterone secretion and the expression and relative contents of steroidogenic acute regulatory (StAR) protein and steroidogenic enzymes cholesterol side-chain cleavage cytochrome P450 (P450SCC), 3beta-hydroxysteroid dehydrogenase/delta(5)-->delta(4)-isomerase (3 beta-HSD), and (17)alpha-hydroxylase cytochrome P450/C17-20 lyase (P450(17)alpha) were determined in testicular tissues of bulls treated with a LHRH agonist. Testis morphology and spermatogenesis were also examined. In Experiment 1, bulls (30-mo-old) received no treatment (control, n = 7) or were implanted for 10 days with the LHRH agonist deslorelin (n = 7). Bulls were castrated on Day 10 and testis tissues prepared for Western and Northern blotting. At castration, bulls implanted with deslorelin had greater plasma testosterone (5-fold) and testis content of testosterone (10-fold) compared with control bulls. Relative content (per micrograms total testis protein or RNA) of StAR protein, 3beta-HSD, P450SCC, and mRNA for P450(17)alpha in bulls treated with deslorelin ranged from 3- to 6-fold that of control bulls. In Experiment 2, bulls (20-mo-old) were left untreated (control, n = 6) or implanted with deslorelin (n = 12) for 120 days. On Day 120, bulls were castrated and right testis tissues prepared for morphology. Testis volume and weight were increased (P < 0.01) in bulls treated with deslorelin compared with control bulls. Stereological analysis revealed that this increase occurred in all compartments (seminiferous epithelium, lumen and interstitium) studied, but was significant (P < 0.01) only for the seminiferous epithelium. Absolute numbers of round spermatids per testis were increased (P < 0.05) in bulls treated with deslorelin compared with control bulls. Increased testosterone secretion in bulls treated with deslorelin was associated with increased testicular StAR protein and steroidogenic enzymes. Bulls treated long-term with deslorelin had a faster rate of testis growth and increased daily sperm production at the end of the experiment.

17-Hydroxysteroid Dehydrogenases↗
PubMed1998

Leydig cell protein synthesis and steroidogenesis in response to acute stimulation by luteinizing hormone in rats.

We examined the temporal relationship between protein synthesis and testosterone production by rat Leydig cells in primary culture. Leydig cells were isolated from adult control Sprague-Dawley rats and from rats that had received LH-suppressive testosterone and estradiol (TE) implants in vivo for 10 days. The cells were incubated for 1-4 h with [35S]methionine in the presence or absence of maximally stimulating ovine LH, and newly synthesized proteins were examined by two-dimensional PAGE autoradiography. Approximately 800-900 newly synthesized polypeptides were readily visible on all autoradiograms, most of which did not differ in the cells from intact control and TE-treated rats. Incubation of cells from the control and treated rats with maximally stimulating LH for 4 h in both cases resulted in significant increases in testosterone production and in three newly synthesized polypeptides. These polypeptides, along with two others that changed little in response to LH, were similar in apparent molecular mass, 30 kDa, but differed in isoelectric point. Time-course studies revealed a temporal relationship between stimulation of the three 30-kDa proteins and of testosterone production. Western blot analysis identified the 30-kDa proteins as steroidogenic acute regulatory protein (StAR). The results of these studies, for the first time utilizing primary cultures of highly purified, testosterone-producing Leydig cells, provide further correlative evidence of a role for StAR protein in the acute regulation of Leydig cell testosterone biosynthesis by LH.

Animals↗
PubMed1998

Luteinizing hormone-dependent gene regulation in Leydig cells may be mediated by CCAAT/enhancer-binding protein-beta.

Leydig cells are located in the interstitium of the testis and function as the primary site for testosterone biosynthesis. Leydig cell development and steroidogenic function are dependent upon pituitary-derived LH. Circulating LH levels in prepubertal mammals are low but rise sharply during puberty, inducing terminal differentiation of immature Leydig cells into adult Leydig cells. The molecular mechanisms involved in LH action on differentiation specific gene expression and initiation of steroidogenic function in immature Leydig cells are poorly understood. Members of the CCAAT/enhancer-binding protein (C/EBP) family of basic region/leucine zipper transcription factors have previously been implicated as regulators of terminal differentiation in several cell types. In the present study we have investigated the possible involvement of C/EBP proteins in regulating LH-dependent gene expression in Leydig cells. We have detected the expression of one family member, C/EBPbeta, in Leydig cells. C/EBPbeta messenger RNA and protein levels were significantly higher in mature adult Leydig cells than in immature cells, displaying an expression pattern similar to those of other developmentally regulated genes in Leydig cells such as steroidogenesis acute regulatory (StAR) protein and 3beta-hydroxysteroid dehydrogenase. C/EBPbeta messenger RNA and protein levels also increased when immature Leydig cells were treated with either hCG, a functional analog of LH (hCG/LH), or (Bu)2cAMP. To confirm that hCG/LH and (Bu)2cAMP were acting specifically on Leydig cells, we studied their effects on C/EBPbeta expression in an established Leydig cell line (MA-10). hCG and (Bu)2cAMP treatment also induced the expression of C/EBPbeta and StAR in MA-10 cells, coincident with stimulation of steroid production in these cells. (Bu)2cAMP treatment did not alter the subcellular localization of C/EBPbeta protein in MA-10 cells, suggesting that the increase is due to stimulation of C/EBPbeta expression. We conclude that expression of C/EBPbeta is regulated by hCG/LH in Leydig cells and that C/EBPbeta may play a significant role in LH-regulated Leydig cell differentiation and function.

Animals↗
PubMed1998

Spatio-temporal expression patterns of steroidogenic acute regulatory protein (StAR) during follicular development in the rat ovary.

The steroidogenic acute regulatory protein (StAR) is a vital mitochondrial protein that is indispensable for the synthesis of steroid hormones in the steroidogenic cells of the adrenal cortex and the gonads. Recent studies have shown that StAR enhances the conversion of the substrate for all steroid hormones, cholesterol, into pregnenolone, probably by facilitating cholesterol entry into the inner compartment of the mitochondria where the steroidogenic cytochrome P450scc complex resides. To study the potential of StAR to affect ovarian steroidogenesis during follicular development, we examined the time-dependent expression of StAR protein and messenger RNA in PMSG/human CG (hCG)-treated immature rats. Western blot analyses and immunohistochemical and RT-PCR methodologies have revealed a biphasic expression of StAR in the ovaries responding to hormones. The first peak of StAR expression was generated by PMSG administration and lasted for 24 h. Furthermore, it was restricted to the entire network of the ovarian secondary interstitial tissue, as well as to a fewer scattered theca-interna cells. The second burst of StAR expression was observed in response to the LH surge, as simulated by hCG. This time, StAR was expressed in the entire theca-interna and interstitial tissue, as well as in those granulosa cells that were confined to periovulatory follicles. Immunoelectron microscopy studies revealed the over 90% of StAR antigenic sites are localized in the inner compartments of the mitochondrion, suggesting a rapid removal of StAR precursor from the mitochondrial surface, where it is believed to exert its activity. Altogether, our observations portray dynamic acute alterations of StAR expression during the process of follicular maturation in this animal model. Furthermore, if StAR indeed determines steroidogenic capacities in the ovary, our findings imply that, in immature rats undergoing hormonally induced first ovulation: 1) the early phases of follicular development are supported by androgen production originating from nonfollicular cells; 2) estrogen production in the granulosa cells of Graafian follicles is nourished by a submaximal androgenic output in the theca-interstitial compartments of the ovary.

Animals↗
PubMed1998

DAX-1 blocks steroid production at multiple levels.

DAX-1 is an unusual member of the nuclear hormone receptor superfamily whose expression is mainly, but not uniquely, restricted to steroidogenic tissues. We have recently shown that DAX-1 can block the first and rate-limiting step in steroid biosynthesis by repressing StAR (steroidogenic acute regulatory protein) expression. Here we show that DAX-1 blocks steroid production at multiple levels in the Y-1 mouse adrenocortical tumor cell line. Expression of DAX-1 in Y-1 cells significantly impairs both basal and cAMP-stimulated steroid production, without affecting the functionality of the cAMP-responsive PKA pathway. Experiments using an hydroxylated cholesterol derivative show that biochemical steps in steroidogenesis subsequent to cholesterol delivery to mitochondria are also impaired in Y-1 cells expressing DAX-1. This is explained by the repression of P450scc and 3beta-HSD expression, in addition to StAR. DAX-1 expression in Y-1 cells results in the inhibition of the activity of the StAR, P450scc and 3beta-HSD promoters. An inappropriate steroidogenic block in the male fetus might have an important role in the pathogenesis of sex reversal syndromes caused by a duplication of the genomic region of the X chromosome containing the DAX-1 gene.

3-Hydroxysteroid Dehydrogenases↗
PubMed1998

Interferon-gamma inhibits the steroidogenic acute regulatory protein messenger ribonucleic acid expression and protein levels in primary cultures of rat Leydig cells.

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.

Animals↗
PubMed1998

Tumor necrosis factor-alpha inhibits leydig cell steroidogenesis through a decrease in steroidogenic acute regulatory protein expression.

The aim of the present study was to identify the sites of the inhibitory action of TNFalpha (tumor necrosis factor alpha) on LH/hCG-stimulated testosterone formation. By using cultured porcine Leydig cells as a model, TNFalpha was shown to inhibit testosterone secretion when testicular cells were stimulated with hCG but not when incubated with 22R-hydroxycholesterol (a cholesterol substrate derivative that readily passes through cell and mitochondrial membranes). Such an observation suggested that the cytokine may affect cholesterol transport and/or availability to cytochrome P450scc in the mitochondria. Specifically, we report here that TNFalpha reduced in a dose- and time-dependent manner hCG-induced StAR (steroidogenic acute regulatory protein) levels. The maximal and half-maximal effects were obtained with 20 ng/ml (1.2 nM) and 1.6 ng/ml (0.09 nM) of TNFalpha, respectively. Maximal inhibitory effects of TNFalpha on StAR messenger RNA and protein levels were obtained after 48 h of treatment. Additionally, the presence of TNFalpha receptors P55 in terms of protein (identified through cross-linking experiments) and messenger RNA (identified through RT-PCR analysis) suggested that the effects of the cytokine are directly exerted on the testicular steroidogenic cell type.

Animals↗
PubMed1998

Effect of truncated forms of the steroidogenic acute regulatory protein on intramitochondrial cholesterol transfer.

It has been proposed that the steroidogenic acute regulatory (StAR) protein controls hormone-stimulated steroid production by mediating cholesterol transfer to the mitochondrial inner membrane. This study was conducted to determine the effect of wild-type StAR and several modified forms of StAR on intramitochondrial cholesterol transfer. Forty-seven N-terminal or 28 C-terminal amino acids of the StAR protein were removed, and COS-1 cells were transfected with pCMV vector only, wild-type StAR, N-47, or the C-28 constructs. Lysates from the transfected COS-1 cells were then incubated with mitochondria from MA-10 mouse Leydig tumor cells that were preloaded with [3H]cholesterol. After incubation, mitochondria were collected and fractionated on sucrose gradients into outer membranes, inner membranes, and membrane contact sites, and [3H]cholesterol content was determined in each membrane fraction. Incubation of MA-10 mitochondria with wild-type StAR containing cell lysate resulted in a significant 34.9% increase in [3H]cholesterol content in contact sites and a significant 32.8% increase in inner mitochondrial membranes. Incubations with cell lysate containing N-47 StAR protein also resulted in a 16.4% increase in [3H]cholesterol in contact sites and a significant 26.1% increase in the inner membrane fraction. In contrast, incubation with the C-28 StAR protein had no effect on cholesterol transfer. The cholesterol-transferring activity of the N-47 truncation, in contrast to that of the C-28 mutant, was corroborated when COS-1 cells were cotransfected with F2 vector (containing cytochrome P450 side-chain cleavage enzyme, ferridoxin, and ferridoxin reductase) and either pCMV empty vector or the complementary DNAs of wild-type StAR, N-47 StAR, or C-28 StAR. Pregnenolone production was significantly increased in both wild-type and N-47-transfected cells, whereas that in C-28-transfected cells was similar to the control value. Finally, immunolocalization studies with confocal image and electron microscopy were performed to determine the cellular location of StAR and its truncated forms in transfected COS-1 cells. The results showed that wild-type and most of the C-28 StAR protein were imported into the mitochondria, whereas most of N-47 protein remained in the cytosol. These studies demonstrate a direct effect of StAR protein on cholesterol transfer to the inner mitochondrial membrane, that StAR need not enter the mitochondria to produce this transfer, and the importance of the C-terminus of StAR in this process.

Animals↗
PubMed1998

Atrial natriuretic peptide inhibits calcium-induced steroidogenic acute regulatory protein gene transcription in adrenal glomerulosa cells.

Atrial natriuretic peptide (ANP) is a potent inhibitor of mineralocorticoid synthesis induced in adrenal glomerulosa cells by physiological agonists activating the calcium messenger system, such as angiotensin II (Ang II) and potassium ion (K+). While the role of calcium in mediating Ang II- and K(+)-induced aldosterone production is clearly established, the mechanisms leading to blockade of this steroidogenic response by ANP remain obscure. We have used bovine adrenal zona glomerulosa cells in primary culture, in which an activation of the calcium messenger system was mimicked by a 2-h exposure to an intracellular high-calcium clamp. The effect of ANP was studied on the following parameters of the steroidogenic pathway: 1) pregnenolone and aldosterone production; 2) changes in cytosolic ([Ca2+]c) and mitochondrial ([Ca2+]m) Ca2+ concentrations, as assessed with targeted recombinant aequorin; 3) cholesterol content in outer mitochondrial membranes (OM), contact sites (CS), and inner membranes (IM); 4) steroidogenic acute regulatory (StAR) protein import into mitochondria by Western blot analysis; 5) StAR protein synthesis, as determined by [35S]methionine incorporation, immunoprecipitation, and SDS-PAGE; 6) StAR mRNA levels by Northern blot analysis with a StAR cDNA; 7) StAR gene transcription by nuclear run-on analysis. While clamping Ca2+ at 950 nM raised pregnenolone output 3.5-fold and aldosterone output 3-fold, ANP prevented these responses with an IC50 of 1 nM and a maximal effect of 90% inhibition at 10 nM. In contrast, ANP did not affect the [Ca2+]c or [Ca2+]m changes occurring under Ca2+ clamp or Ang II stimulation in glomerulosa cells. The accumulation of cholesterol content in CS (139.7 +/- 10.7% of control) observed under high-Ca2+ clamp was prevented by 10 nM ANP (92.4 +/- 4% of control). Similarly, while Ca2+ induced a marked accumulation of StAR protein in mitochondria of glomerulosa cells to 218 +/- 44% (n = 3) of controls, the presence of ANP led to a blockade of StAR protein mitochondrial import (113.3 +/- 15.0%). This effect was due to a complete suppression of the increased [35S]methionine incorporation into StAR protein that occurred under Ca2+ clamp (94.5 +/- 12.8% vs. 167.5 +/- 17.3%, n = 3). Furthermore, while the high-Ca2+ clamp significantly increased StAR mRNA levels to 188.5 +/- 8.4 of controls (n = 4), ANP completely prevented this response. Nuclear run-on analysis showed that increases in intracellular Ca2+ resulted in transcriptional induction of the StAR gene and that ANP inhibited this process. These results demonstrate that Ca2+ exerts a transcriptional control on StAR protein expression and that ANP appears to elicit its inhibitory effect on aldosterone biosynthesis by acting as a negative physiological regulator of StAR gene expression.

Aldosterone↗
PubMed1998

Upregulation of human chorionic gonadotrophin-induced steroidogenic acute regulatory protein by insulin-like growth factor-I in rat Leydig cells.

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-induced testosterone formation, whereas the conversions of pregnenolone, 17-OH pregnenolone, dehydroepiandrosterone, and androstenedione to testosterone were not affected. This suggests that the major effect of IGF-I is at the steps of StAR and P450scc, whereas other steroidogenic enzymes are not affected. To evaluate whether increased StAR mRNA levels induced by IGF-I and hCG are associated with increased StAR protein levels, we carried out Western blot analyses. Basal StAR protein levels were low after 24 h in culture. hCG (10 ng/mL) increased StAR protein by 4.5-fold. In the presence of IGF-I (100 ng/mL), hCG-induced StAR protein levels were further increased. In conclusion, our present study demonstrated that IGF-I enhances Leydig cell steroidogenesis by upregulating hCG-induced StAR gene expression and protein production.

Animals↗
PubMed1998

Recent advances in the role of StAR.

It has been proposed that the steroidogenic acute regulatory (StAR) protein is the protein responsible for the acute regulation of steroid hormone biosynthesis. Virtually all of the observations made to date are consistent with this proposal. In this short review, background information leading to the discovery and characterization of the StAR protein and several recent and interesting observations concerning this protein are summarized.

Amino Acid Sequence↗
PubMed1998

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

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.

Analysis of Variance↗