Heat shock-induced inhibition of acute steroidogenesis in MA-10 cells is associated with inhibition of the synthesis of the steroidogenic acute regulatory protein

The synthesis of heat shock proteins (HSPs) rapidly increases in cells under a broad range of stress conditions in addition to heat shock. Previous studies have shown that the induction of HSPs severely impairs the ability of steroidogenic cells to synthesize steroids in response to acute stimulation. De novo synthesis of the steroidogenic acute regulatory (StAR) protein has been shown to be indispensable for acute steroid hormone biosynthesis; however, the effect of HSP induction on the synthesis of the StAR protein has not yet been studied. In the present study we investigated whether HSP induction might influence the steroidogenic activity of MA-10 mouse Leydig tumor cells, and whether this effect may involve the synthesis of StAR protein. MA-10 cells exposed to 45 C for 10 min and allowed to recover for 2 h at 37 C displayed a 6-fold increase in HSP-70 at 3 h postrecovery and a 20-fold increase in this protein at 6 h postrecovery. This heat shock regimen also acutely inhibited both progesterone production and StAR protein synthesis in MA-10 cells in response to LH and cAMP analog stimulation. The activity and quantity of cytochrome P450 side-chain cleavage and 3beta-hydroxysteroid dehydrogenase were not affected by this heat shock treatment, indicating that the loss of steroidogenic capacity was not a result of inhibition of the enzymes involved in the conversion of cholesterol to progesterone. The results suggest that the previously observed antisteroidogenic effects of heat shock treatment may be due mainly to the acute inhibition of StAR protein synthesis.     

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.     

Steroidogenic acute regulatory protein (StAR) is a sterol transfer protein

Steroidogenic acute regulatory protein (StAR) plays a critical role in steroidogenesis by enhancing the delivery of substrate cholesterol from the outer mitochondrial membrane to the cholesterol side chain cleavage enzyme system on the inner membrane. A recombinant StAR protein lacking the first N-terminal 62 amino acid residues that includes the mitochondrial targeting sequence was shown to stimulate the transfer of cholesterol and beta-sitosterol from liposomes to heat-treated mitochondria in a dose-, time-, and temperature-dependent manner. A recombinant mutant StAR protein that cannot stimulate steroidogenesis by isolated mitochondria did not promote sterol transfer. Unlike the more promiscuous lipid transfer protein, sterol carrier protein 2 (SCP2), StAR did not stimulate phosphatidylcholine transfer in our assay system. The recombinant StAR protein increased cholesterol transfer to heat-treated microsomes as well as to heat- and trypsin-treated mitochondria. These observations demonstrate that StAR has sterol transfer activity, which may reflect an ability to enhance desorption of cholesterol from sterol-rich donor membranes. We suggest that the ability of StAR to promote sterol transfer explains its steroidogenic activity.     

ATP and a mitochondrial electrochemical gradient are required for functional activity of the steroidogenic acute regulatory (StAR) protein in isolated mitochondria

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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Steroidogenic acute regulatory protein (StAR), a 30-kDa protein involved in the transport of cholesterol to inner mitochondrial membrane during stimulation of steroid hormone biosynthesis, has recently been cloned from human adrenals and MA-10 mouse Leydig tumor cells. We examined the regulation of StAR mRNA accumulation upon induction of steroidogenesis in immortalized rat granulosa cells. Granulosa cells were transfected with SV40 DNA alone (POGS5); with SV40 DNA and Ha-ras oncogene (POGRS1); with SV40 DNA, Ha-ras oncogene and LH/CG receptor (GLHR15) or with FSH receptor (GFSHR17) or with the beta 2-adrenergic receptor (G beta 2AR13) expression plasmids. Cells were cultured to confluency and then stimulated for 24 h with oFSH (4 nM), hCG (2.4 nM), isoproterenol (10 microM) or forskolin (50 microM). By quantitative RT-PCR, StAR mRNA was undetectable in non-steroidogenic cells (transfected with SV40 DNA alone, POGS5) either in the presence or in the absence of forskolin. In contrast, variable amount of the message was detected in all steroidogenic cell lines cotransfected with SV40 DNA and Ha-ras. Moreover, an increase in the StAR mRNA expression was evident in all steroidogenic cells upon stimulation with their respective agonists, concomitantly with enhanced progesterone production. The RT-PCR product was sequenced and the 379 base pairs of rat StAR were found to be 93% and 86% identical to mouse and human cDNA, respectively. The deduced 126 amino acid sequence was 95%, 88% and 88% identical to the mouse, human and bovine deduced protein sequences. We conclude that StAR message is expressed only in the steroidogenic rat granulosa cells and can be upregulated by FSH, hCG, isoproterenol and forskolin in the appropriate cell lines. In addition, we find that the rat StAR cDNA exhibit a high degree of homology with the mouse and human sequences.     

Steroidogenic acute regulatory protein (StAR) acts on the outside of mitochondria to stimulate steroidogenesis

Steroidogenic acute regulatory protein (StAR) facilitates delivery of cholesterol to the inner mitochondrial membranes. StAR is imported into mitochondria and processed to a mature form by cleavage of the N-terminal mitochondrial targeting sequence. We produced His-tagged (His-tag StAR) constructs lacking the N-terminal 62 amino acids that encode the mitochondrial targeting sequence and examined their steroidogenic activity in intact cells and on isolated mitochondria. His-tag StAR proteins stimulated pregnenolone synthesis to the same extent as wild-type StAR when expressed in COS-1 cells transfected with the cholesterol side-chain cleavage system. His-tag StAR was diffusely distributed in the cytoplasm of transfected COS-1 cells, whereas wild-type StAR was localized to mitochondria. There was no evidence at the light or electron microscope levels for selective localization of His-tag StAR protein to mitochondrial membranes. We established an assay system using mitochondria isolated from bovine corpora lutea and purified recombinant His-tag StAR proteins expressed in E. coli. Recombinant His-tag StAR stimulated pregnenolone production in a dose- and time-dependent manner, functioning at nanomolar concentrations. A point mutant of StAR (A218V) that causes lipoid congenital adrenal hyperplasia was incorporated into the His-tag protein. This mutant was steroidogenically inactive in COS-1 cells and on isolated mitochondria. Our observations conclusively document that StAR acts on the outside of mitochondria, independent of mitochondrial import.     

Smad3 is involved in the intracellular signaling pathways that mediate the inhibitory effects of transforming growth factor-beta on StAR expression

Transforming growth factor betas (TGFbetas) constitute a family of dimeric proteins that regulate growth and differentiation of many cell types. TGFbeta1 is also a potent autocrine regulator of adrenocortical steroidogenesis. We have recently shown that in primary cultures of bovine fasciculo-reticularis cells, the main target of TGFbeta is the steroidogenic acute relay protein (StAR), a key protein necessary for intramitochondrial cholesterol transport. Here, we show that StAR expression is also inhibited by TGFbeta1 in the human adrenocortical carcinoma cell line NCI-H295R. This inhibitory effect is mediated by Smad proteins. Indeed, we found that overexpression of wild-type Smad3 inhibited endogenous StAR mRNA expression while overexpression of a dominant negative Smad3 protein reversed the inhibitory effect of TGFbeta1 on StAR mRNA expression. Taken together, these results demonstrate that the Smad3 protein is involved in TGFbeta-dependent regulation of steroidogenesis.     

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.     

Targeted disruption of the peripheral-type benzodiazepine receptor gene inhibits steroidogenesis in the R2C Leydig tumor cell line

To evaluate the role of the mitochondrial peripheral-type benzodiazepine receptor (PBR) in steroidogenesis, we developed a molecular approach based on the disruption of the PBR gene, by homologous recombination, in the constitutive steroid producing R2C rat Leydig tumor cell line. Inactivation of one allele of the PBR gene resulted in the suppression of PBR mRNA and ligand binding expression. Immunoblot and electron microscopic immunogold labeling analyses confirmed the absence of the 18-kDa PBR protein in the selected clone. Although mitochondria from the PBR-negative cells contained high levels of the constitutively expressed 30-kDa steroidogenic activity regulator protein, these cells produced minimal amounts of steroids compared with normal cells (5%). Moreover, mitochondria from PBR-negative cells failed to produce pregnenolone when supplied with exogenous cholesterol. Addition of the hydrosoluble cholesterol derivative, 22R-hydroxycholesterol, increased steroid production by the PBR-negative R2C cells, indicating that the cholesterol transport mechanism was impaired. Stable transfection of the PBR-negative R2C Leydig cells with a vector containing the PBR cDNA resulted in the recovery of the steroidogenic function of the cells. These data demonstrate that PBR is an indispensable element of the steroidogenic machinery, where it mediates the delivery of the substrate cholesterol to the inner mitochondrial side chain cleavage cytochrome P-450.     

Regulation of porcine granulosa cell steroidogenic acute regulatory protein (StAR) by insulin-like growth factor I: synergism with follicle-stimulating hormone or protein kinase A agonist

The transfer of cholesterol from the outer to the inner mitochondrial membrane, where side-chain cleavage occurs to form pregnenolone, is a crucial event in the regulation of steroidogenesis and recently has been demonstrated to be mediated by steroidogenic acute regulatory protein (StAR). We generated a partial porcine StAR complementary DNA (280 bp) by RT-PCR and used the corresponding antisense riboprobe to quantify the control of StAR gene expression by FSH and insulin-like growth factor I (IGF-I) in hormonally responsive swine granulosa cells, which typically manifest synergistic steroidogenic stimulation by these two dominant intrafollicular regulators. RNase protection assays were implemented to investigate the time course of the actions of FSH (100 ng/ml), IGF-I (100 ng/ml), and FSH plus IGF-I on StAR messenger RNA accumulation in serum-free cultures granulosa cells. Treatment with FSH (1.6-fold) or IGF-I (2.7-fold) alone had a small but consistent stimulatory effect on StAR message accumulation (corrected for 18S ribosomal RNA in each lane) at 48 h, whereas only IGF-I stimulated StAR protein expression (at least 6-fold as assessed by Western blot). Notably, the combined effect of FSH plus IGF-I was strongly synergistic and already significant by 24 h and maximal at 48 h (P < 0.001). Protein kinase A agonist, 8-bromoadenosine 3',5'-cAMP (8-bromo-cAMP) (1 mM) alone elicited a 3.5-fold increase in StAR message and more than 3.7-fold increase in StAR protein expression by 48 h. The combination of IGF-I and FSH or 8-bromo-cAMP evoked a 26- to 40-fold (P < 0.001) synergistic rise in StAR message accumulation. StAR protein also showed a similar synergistic pattern of expression driven by IGF-I and FSH or 8-bromo-cAMP, namely a greater than 56- to 60-fold increase. In summary, two distinct first messenger regulatory molecules, FSH and IGF-I, interact synergistically to induce amplification of StAR messenger RNA and protein expression in serum-free monolayer cultures of immature (swine) granulosa cells.     

The acute and chronic effects of adrenocorticotropin on the levels of messenger ribonucleic acid and protein of steroidogenic enzymes in rat adrenal in vivo

The purpose of this study was to evaluate the effects of acute (a single injection) and chronic stimulation (twice daily injection for 9 days) by ACTH on changes occurring in the temporal expression of steroidogenic enzymes in the rat adrenal in vivo. Under acute ACTH stimulation, the level of steroidogenic acute regulatory protein (StAR) messenger RNA (mRNA) was increased within 0.5 h in both zona glomerulosa (ZG) and zona fasciculata-reticularis (ZFR), with maximal increases of 220-370% and 300-350% in the ZG and ZFR, respectively. Increases in the levels of StAR protein in homogenates were also found in the ZG (700%) and the ZFR (300%), but were delayed compared with those of their mRNA. Furthermore, the increase in mitochondrial StAR protein was concomitant with that in the homogenate, indicating that the entry of StAR into mitochondria might not be necessary to increase steroidogenesis during the early stimulatory phase. The levels of c-jun, c-fos, junB, and fosB mRNA in ZG and ZFR were also rapidly maximally elevated within 0.5-1 h after ACTH administration and fell to near control levels 5 h posttreatment. The levels of c-jun protein were already increased in both zones at 1 h, reached 200% at 3 h, and remained elevated 5 h post-ACTH treatment. The levels of c-Fos protein were maximally increased by 240% in both zones after 1 h and decreased thereafter to control values at 5 h. Few changes were observed in the adrenal protein contents of cholesterol side-chain cleavage cytochrome P450 (P450scc), cytochrome P450 11beta-hydroxylase (P450C11), cytochrome P450 21-hydroxylase (P450C21), and 3beta-hydroxysteroid dehydrogenase (3betaHSD). Under chronic stimulation by ACTH, we observed elevations in the levels of plasma corticosteroids and changes in the mRNA and protein levels of many adrenal steroidogenic enzymes in both zones. In the ZG, administration of ACTH for 9 days provoked an increase in the level of StAR mRNA (210-270%) and a decrease in the levels of 3betaHSD, cytochrome P450 aldosterone synthase (P450aldo), and AT1 receptor mRNA (by 40%, 70%, and 90%, respectively), whereas the levels of P450scc and P450C21 mRNA did not differ significantly from the control values. Western blotting analysis showed that the adrenal ZG protein levels of StAR and P450scc were increased (150%), 3betaHSD was not changed, and P450C21 was decreased by 70%. In the ZFR, the levels of P450scc and StAR mRNAs were increased (260% and 570-870%, respectively). The levels of 3betaHSD, P450C21, and P450C11 mRNA did not differ from control values in that zone. Western blotting analysis showed that the ZFR protein level of 3betaHSD was not changed, P450scc and P450C21 were decreased by 40% and 60%, respectively, and StAR was increased by 160%. Although c-fos and fosB mRNAs were undetectable after 9 days of chronic ACTH treatment, c-jun mRNA and its protein were still detectable, suggesting a basic role for this protooncogene in maintaining the integrity and function of the adrenal cortex. When dexamethasone was administered to rats for 5 days to inhibit their ACTH secretion, the mRNA levels of many steroidogenic enzymes were decreased, with the exception of StAR, 3betaHSD, and P450aldo. These results confirm the importance of physiological concentrations of ACTH in maintaining normal levels of adrenocortical enzymes and also indicate that in addition to ACTH, other factors are involved in controlling the expression of StAR, 3betaHSD, and P450aldo. In conclusion, we showed that ACTH acutely increases StAR mRNA followed, after a delay, by an increase in the level of StAR protein; this suggests that posttranslational modifications of the StAR precursor occurred during the early stimulatory phase and before the apparent translation of the newly formed mRNA. The rapid induction of protooncogenes suggests their participation in the action of ACTH to stimulate steroidogenesis. (ABSTRACT TRUNCATED)     

Molecular cloning, characterization and cellular distribution of rat steroidogenic acute regulatory protein (StAR) in the ovary

Rat ovarian genes induced by the treatment of immature rats with pregnant mare serum gonadotropin (PMSG) were isolated by a subtraction cloning method. Amongst them was obtained a probable rat homologue of steroidogenic acute regulatory protein (StAR), which has been recently identified as a protein that is an acute regulator of the rate limiting transfer of cholesterol from the outer to the inner mitochondrial membrane. Structure of rat StAR was determined by nucleotide sequence analysis. Northern blot analysis revealed that StAR mRNA levels were rapidly and strongly increased by PMSG/hCG but not by FSH. In situ hybridization revealed that the expression of StAR mRNA was strongly induced by PMSG in theca interna cells as well as in corpora lutea. These findings indicate that expression of StAR mRNA is restricted to and induced in the ovarian steroidogenic cell types where cholesterol is used as a substrate for synthesis of steroid hormones.     

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Sterol carrier protein 2 (SCP2) has been implicated in adrenal steroidogenesis by in vitro studies. In order to clarify the clinical significance of SCP2 in human steroidogenesis, we investigated the expression of SCP2 mRNA in various types of adrenocortical tissue and one testis and examined the correlation between the amounts of SCP2 and other values such as the free cholesterol content and the cholesterol side-chain cleavage (SCC) activity in the tissue mitochondria. The types of adrenocortical tissue examined included adrenocortical carcinomas (N = 3), adrenocortical adenomas from patients with Conn's syndrome (N = 3) and from patients with Cushing's syndrome (N = 3), non-functioning adrenocortical adenomas (N = 2) and normal adrenal glands (N = 2). Northern blot hybridization predominantly revealed a 1.8-kb SCP2 mRNA in all tissue specimens examined. The mRNA concentrations of SCP2 in two out of three adrenocortical carcinomas were relatively lower than those in other types of tissue. No other special tendency was observed regarding the mRNA expression levels in various tissue specimens. The mRNA concentrations of SCP2 correlated significantly with mitochondrial contents of free cholesterol (r = 0.67, p < 0.01), but was not correlated with the SCC activities in mitochondria measured by an in vitro enzyme assay. The mitochondrial SCC activities, however, were correlated significantly with the protein levels of mitochondrial P-450 scc determined by a Western blot analysis (r = 0.79, p < 0.01). The significant positive correlation between mRNA concentrations of SCP2 and the mitochondrial content of free cholesterol suggests that the central role of SCP2 in human steroidogenic tissues may be in part a translocation of cytoplasmic free cholesterol to the mitochondria, as demonstrated previously by in vitro studies.     

Ex vivo regulation of adrenal cortical cell steroid and protein synthesis, in response to adrenocorticotropic hormone stimulation, by the Ginkgo biloba extract EGb 761 and isolated ginkgolide B

We previously demonstrated that repeated treatment of rats with the standardized extract of Ginkgo biloba leaves, EGb 761, and its bioactive component ginkgolide B (GKB), specifically reduces the ligand binding, and protein and messenger RNA expression of the adrenal mitochondrial peripheral benzodiazepine receptor (PBR), a key element in the regulation of cholesterol transport, resulting in decreased circulating corticosterone levels. Adrenocortical cells were isolated from rats treated with EGb 761 or GKB and cultured for 2 and 12 days. The effect of ACTH on normal and metabolically labeled cells was examined. Corticosterone levels were measured by RIA, and protein synthesis was analyzed by two-dimensional gel electrophoresis. Ex vivo treatment with EGb 761 and GKB resulted, respectively, in 50% and 80% reductions of ACTH-stimulated corticosterone production by adrenocortical cells cultured for 2 days compared with that by cells isolated from saline-treated rats. Two-dimensional gel electrophoresis analysis revealed that in cells from both control and drug-treated animals, ACTH induced the synthesis, at the same level, of a 29-kDa and pI 6.4-6.7 protein identified as the adrenal steroidogenic acute regulatory protein (StAR). In addition, treatment with EGb 761 and GKB specifically altered the synthesis of seven proteins, including inhibition of synthesis of a 17-kDa, identified as PBR. After 12 days in culture, ACTH-stimulated adrenocortical cell steroid synthesis was maintained, and it was identical among the cells isolated from animals treated with GKB or saline. Under the same conditions, the expression of PBR was recovered, whereas no effect of ACTH on the 29-kDa and 6.4-6.7 pI protein (StAR) or other protein synthesis could be seen. A comparative analysis of the effects of GKB and EGb 761 on adrenocortical steroidogenesis and protein synthesis identified, in addition to the 17-kDa PBR, target proteins of 32 kDa (pI 6.7) and 40 kDa (pI 5.7-6.0) as potential mediators of the effect of EGb 761 and GKB on ACTH-stimulated glucocorticoid synthesis. In conclusion, these results 1) validate and extend our previous in vivo findings on the effect of EGb 761 and GKB on ACTH-stimulated adrenocortical steroidogenesis, 2) demonstrate the specificity and reversibility of EGb 761 and GKB treatment, 3) question the role of the 29-kDa, 6.4-6.7 pI protein (mature StAR) as the sole mediator of ACTH-stimulated steroid production, and 4) demonstrate the obligatory role of PBR in hormone-regulated steroidogenesis.     

Structure and function of the peripheral-type benzodiazepine receptor in steroidogenic cells

Steroidogenesis depends on the rate of cholesterol transport from intracellular stores to the inner mitochondrial membrane cytochrome P-450 side-chain cleavage enzyme. Using steroidogenic cell submitochondrial fractions, mitochondrial preparations, various cell models, and animal models and with the help of pharmacological, biochemical, morphological, and molecular approaches, we provide evidence that the peripheral-type benzodiazepine receptor mediates the intramitochondrial cholesterol transport and the subsequent adrenal, gonadal, placental, and brain steroid biosynthesis.     

Targeted disruption of StAR provides novel insights into congenital adrenal hyperplasia

To explore the function of StAR in a system that can be experimentally manipulated, and to develop a mouse model for the human disorder lipoid congenital adrenal hyperplasia (lipoid CAH), we used targeted gene disruption to produce a mouse line deficient in StAR protein. Initially, StAR knockout mice were indistinguishable from wildtype littermates, except that all had female external genitalia. After birth, they showed signs of either respiratory distress or volume depletion and eventually died. Hormone assays confirmed severe defects in adrenal steroids, whereas hormones constituting the gonadal axis did not differ significantly from levels in wildtype littermates. Histologically, the adrenal cortex of StAR knockout mice contained florid lipid deposits, as visualized with oil red O stain. Lesser lipid deposits were observed in the steroidogenic compartment of the testis and none in the ovary. The sex-specific differences in gonadal involvement provide evidence for a two-stage model of the pathogenesis of StAR deficiency, with trophic hormone stimulation causing progressive accumulation of lipids within the steroidogenic cells which ultimately kills them. These StAR knockout mice provide a novel system in which to study StAR's essential roles in adrenocortical and gonadal steroidogenesis.