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.     

The expression of steroidogenic acute regulatory protein (StAR) in bovine adrenocortical cells

StAR protein may facilitate rapid transfer of cholesterol from the outer to the inner mitochondrial membrane, the site at which cholesterol is converted to pregnenolone by the cholesterol side chain cleavage complex. We have studied the effect of ACTH treatment on StAR mRNA and protein levels in bovine adrenocortical cells in primary culture. Cells were initially cultured for 3 days after isolation, and then treated with ACTH (10(-8) M) for various times up to 24 hours. Northern analysis of total BAC mRNA, using a [alpha32P]-labelled cDNA probe encoding a 5' region of bovine StAR mRNA, revealed two principal hybridising species of 1.6 and 3.0 kb. Western immunoblot analysis revealed a principal band at 30 kDa. Levels of both StAR mRNA and protein showed an increase at 1 hour, reached a maximum at around 6 hours and declined to basal levels at 24 hours. Cortisol secretion (measured by RIA) showed a similar change over the same period. From these results it appears that StAR mRNA and protein levels in BAC are acutely regulated in concert with ACTH-stimulated cortisol secretion.     

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.     

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.     

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.     

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.     

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

This study compared levels of referential communication disturbance in speech samples from 41 stable schizophrenia outpatients, 46 parents of patients, and 23 nonpsychiatric control participants in affectively positive versus affectively negative conditions. The speech of the patients and parents showed elevated frequencies of reference failures in the affectively positive condition compared with control participants: the speech of the patients became more disordered in the affectively negative condition, whereas the speech of the parents did not. These results support the idea that referential communication disturbances reflect vulnerability, as well as overt illness, but that affective reactivity of these disturbances is associated mainly with the manifest illness. These findings are consistent with biological, cognitive, and psychological theories about the processes underlying stress responsiveness of schizophrenic symptoms more generally.     

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.     

Ethane dimethane sulfonate and NNN'N'-tetrakis-(2-pyridylmethyl)ethylenediamine inhibit steroidogenic acute regulatory (StAR) protein expression in MA-10 Leydig cells and rat Sertoli cells

Apoptosis inhibits steroid biosynthesis, but it is not clear how the Steroidogenic Acute Regulatory (StAR) protein, is affected. To characterize StAR expression during apoptosis, mouse MA-10 Leydig tumor cells were treated with ethane dimethane sulfonate (EDS), an inducer of apoptosis, and the metal ion chelator NNN'N'-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN), an inducer of cell death. Both chemicals induced cell death and similarly inhibited dbcAMP-stimulated steroidogenesis and accumulation of the 30 kDa form of StAR. Utilizing the dye JC-1, it was found that TPEN and EDS also impaired the mitochondrial electrochemical potential (delta psi). In Sertoli cells, which also express StAR, EDS induced cell death and attenuated StAR expression. We conclude 1) steroidogenesis and accumulation of mature StAR protein are inhibited as a consequence of the induction of apoptosis; 2) reduced levels of StAR may be partially attributed to inhibition of import because of the loss of delta psi; 3) loss of steroidogenesis is probably due to loss of StAR synthesis and disruption of delta psi.     

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.     

A family of secreted proteins contains homology to the cysteine-rich ligand-binding domain of frizzled receptors

This paper describes the identification of a new family of mammalian genes that encode secreted proteins containing homology to the cysteine-rich ligand-binding domain found in the frizzled family of transmembrane receptors. The secreted frizzled-related proteins (sFRPs) are approximately 30 kDa in size, and each contains a putative signal sequence, a frizzled-like cysteine-rich domain, and a conserved hydrophilic carboxy-terminal domain. The sFRPs are not the products of differential splicing of the known frizzled genes. Glycosylphosphatidylinositol-anchored derivatives of sFRP-2 and sFRP-3 produced in transfected human embryonic kidney cells confer cell-surface binding by the Drosophila Wingless protein. These observations suggest that sFRPs may function in vivo to modulate Wnt signaling, or, alternatively, as novel ligands for as yet unidentified receptors.     

Indications that the Cgamma2 homology region is not a regular domain

The Cgamma2 homology region of rabbit IgG does not behave like a domain. Thus, there is no trans-interaction between the two Cgamma2 regions; instead there is an unusual cis-interaction between Cgamma2 and Cgamma3 regions. The observations were made on the plasmin digestion products Facb (IgG minus the Cgamma3 region) and pFc' (Cgamma3 region), which did not dissociate under neutral conditions but dissociated in 3M guanidine solution (that is, cis-interaction between Cgamma2 and Cgamma3). The Facb fragment split into subunits with equal molecular weights under neutral conditions on partial reduction and alkylation (that is, lack of trans-interaction between the two Cgamma2 in the molecule).     

Isolation of a brefeldin A-inhibited guanine nucleotide-exchange protein for ADP ribosylation factor (ARF) 1 and ARF3 that contains a Sec7-like domain

Brefeldin A (BFA) inhibited the exchange of ADP ribosylation factor (ARF)-bound GDP for GTP by a Golgi-associated guanine nucleotide-exchange protein (GEP) [Helms, J.B. & Rothman, J.E. (1992) Nature (London) 360, 352-354; Donaldson, J.G., Finazzi, D. & Klausner, R.D. (1992) Nature (London) 360, 350-352]. Cytosolic ARF GEP was also inhibited by BFA, but after purification from bovine brain and rat spleen, it was no longer BFA-sensitive [Tsai, S.-C., Adamik, R., Moss, J. & Vaughan, M. (1996) Proc. Natl. Acad. Sci. USA 93, 305-309]. We describe here purification from bovine brain cytosol of a BFA-inhibited GEP. After chromatography on DEAE-Sephacel, hydroxylapatite, and Mono Q and precipitation at pH 5.8, GEP was eluted from Superose 6 as a large molecular weight complex at the position of thyroglobulin (approximately 670 kDa). After SDS/PAGE of samples from column fractions, silver-stained protein bands of approximately 190 and 200 kDa correlated with activity. BFA-inhibited GEP activity of the 200-kDa protein was demonstrated following electroelution from the gel and renaturation by dialysis. Four tryptic peptides from the 200-kDa protein had amino acid sequences that were 47% identical to sequences in Sec7 from Saccharomyces cerevisiae (total of 51 amino acids), consistent with the view that the BFA-sensitive 200-kDa protein may be a mammalian counterpart of Sec7 that plays a similar role in cellular vesicular transport and Sec7 may be a GEP for one or more yeast ARFs.     

The pleckstrin homology domain of oxysterol-binding protein recognises a determinant specific to Golgi membranes

BACKGROUND: Peripheral membrane proteins are targeted to the cytoplasmic face of specific intracellular membranes. The organelle-specific ligands recognised by peripheral proteins include other proteins and lipids. Oxysterol-binding protein (OSBP) translocates from the cytoplasm to the Golgi apparatus on binding oxygenated derivatives of cholesterol. The mechanism by which OSBP recognises the Golgi is unknown. It does, however, contain a pleckstrin homology (PH) domain, which in other proteins has been found to mediate regulated membrane binding, although in all previously studied examples the binding is to the plasma membrane. RESULTS: The PH domain of OSBP and of a yeast homologue, Osh1p, were sufficient to target proteins specifically to mammalian Golgi membranes. In addition, high level expression disrupted Golgi architecture and prevented forward traffic of cargo protein. In vitro, the OSBP PH domain bound to Golgi membranes in a manner apparently dependent on phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) or a related phosphatidylinositide. The OSBP PH domain bound to PI(4,5)P2 in liposomes with a submicromolar dissociation constant. CONCLUSIONS: The PH domains of OSBP and its yeast homologue recognise a determinant which is specific to Golgi membranes and important for Golgi function. The determinant appears to be a combination or a phosphatidylinositol polyphosphate and a second, Golgi-specific feature.     

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.