Steroidogenic acute regulatory protein (StAR), a novel mitochondrial cholesterol transporter

Cholesterol is a vital component of cellular membranes, and is the substrate for biosynthesis of steroids, oxysterols and bile acids. The mechanisms directing the intracellular trafficking of this nearly insoluble molecule have received increased attention through the discovery of the steroidogenic acute regulatory protein (StAR) and similar proteins containing StAR-related lipid transfer (START) domains. StAR can transfer cholesterol between synthetic liposomes in vitro, an activity which appears to correspond to the trans-cytoplasmic transport of cholesterol to mitochondria. However, trans-cytoplasmic cholesterol transport in vivo appears to involve the recently-described protein StarD4, which is expressed in most cells. Steroidogenic cells must also move large amounts of cholesterol from the outer mitochondrial membrane to the first steroidogenic enzyme, which lies on the matrix side of the inner membrane; this action requires StAR. Congenital lipoid adrenal hyperplasia, a rare and severe disorder of human steroidogenesis, results from mutations in StAR, providing a StAR knockout of nature that has provided key insights into its activity. Cell biology experiments show that StAR moves large amounts of cholesterol from the outer to inner mitochondrial membrane, but acts exclusively on the outer membrane. Biophysical data show that only the carboxyl-terminal alpha-helix of StAR interacts with the outer membrane. Spectroscopic data and molecular dynamics simulations show that StAR's interactions with protonated phospholipid head groups on the outer mitochondrial membrane induce a conformational change (molten globule transition) needed for StAR's activity. StAR appears to act in concert with the peripheral benzodiazepine receptor, but the precise itinerary of a cholesterol molecule entering the mitochondrion remains unclear.     

StAR search--what we know about how the steroidogenic acute regulatory protein mediates mitochondrial cholesterol import

Cholesterol is the starting point for biosynthesis of steroids, oxysterols and bile acids, and is also an essential component of cellular membranes. The mechanisms directing the intracellular trafficking of this insoluble molecule have received attention through the discovery of the steroidogenic acute regulatory protein (StAR) and related proteins containing StAR-related lipid transfer domains. Much of our understanding of the physiology of StAR derives from studies of congenital lipoid adrenal hyperplasia, which is caused by StAR mutations. Multiple lines of evidence show that StAR moves cholesterol from the outer to inner mitochondrial membrane, but acts exclusively on the outer membrane. The precise mechanism by which StAR's action on the outer mitochondrial membrane stimulates the flow of cholesterol to the inner membrane remains unclear. When StAR interacts with protonated phospholipid head groups on the outer mitochondrial membrane, it undergoes a conformational change (molten globule transition) that opens and closes StAR's cholesterol-binding pocket; this conformational change is required for cholesterol binding, which is required for StAR activity. The action of StAR probably requires interaction with the peripheral benzodiazepine receptor.     

Detection of the steroidogenic acute regulatory protein, StAR, in human liver cells

Overexpressing StAR (a mitochondrial cholesterol transporter) increases (>5-fold) the rate of 27-hydroxylation of cholesterol and the rates of bile acid synthesis in primary rat hepatocytes; suggesting that the transport of cholesterol into mitochondria is rate-limiting for bile acid biosynthesis via the CYP27A1 initiated 'acidic' pathway. Our objective was to determine the level of StAR expression in human liver and whether changes in StAR would correlate with changes in CYP27A1 activity/bile acid synthesis rates in human liver tissues. StAR mRNA and protein were detected in primary human hepatocytes and HepG2 cells by RT-PCR/Northern analysis and by Western analysis, respectively. In immunocompetition assays, liver StAR was competed away with the addition of purified human adrenal StAR. Overexpressing CYP27A1 in both cell types led to >2-fold increases in liver StAR concentration. StAR protein levels also increased approximately 2-fold with the addition of 27-hydroxycholesterol to HepG2 cell culture medium. Overexpressing StAR increased the rates of 27-hydroxylation of cholesterol/bile acid synthesis in both cell lines and increased intracellular levels of 27-hydroxycholesterol. In conclusion, human liver cells contain regulable StAR protein whose level of expression appears capable of regulating cellular cholesterol homeostasis, representing a potential therapeutic target in the management of hyperlipidemia.     

Peripheral-type benzodiazepine receptor-mediated action of steroidogenic acute regulatory protein on cholesterol entry into leydig cell mitochondria

Hormone-induced steroid biosynthesis begins with the transfer of cholesterol from intracellular stores into mitochondria. Steroidogenic acute regulatory protein (StAR) and peripheral-type benzodiazepine receptor (PBR) have been implicated in this rate-determining step of steroidogenesis. MA-10 mouse Leydig tumor cells were treated with and without oligodeoxynucleotides (ODNs) antisense to PBR and StAR followed by treatment with saturating concentrations of human choriogonadotropin. Treatment with ODNs antisense but not missense for both proteins inhibited the respective protein expression and the ability of the cells to synthesize steroids in response to human choriogonadotropin. Treatment of the cells with either ODNs antisense to PBR or a transducible peptide antagonist to PBR resulted in inhibition of the accumulation of the mature mitochondrial 30-kDa StAR protein, suggesting that the presence of PBR is required for StAR import into mitochondria. Addition of in vitro transcribed/translated 37-kDa StAR or a fusion protein of Tom20 (translocase of outer membrane) and StAR (Tom/StAR) to mitochondria isolated from control cells increased pregnenolone formation. Mitochondria isolated from cells treated with ODNs antisense, but not missense, to PBR failed to form pregnenolone and respond to either StAR or Tom/StAR proteins. Reincorporation of in vitro transcribed/translated PBR, but not PBR missing the cholesterol-binding domain, into MA-10 mitochondria rescued the ability of the mitochondria to form steroids and the ability of the mitochondria to respond to StAR and Tom/StAR proteins. These data suggest that both StAR and PBR proteins are indispensable elements of the steroidogenic machinery and function in a coordinated manner to transfer cholesterol into mitochondria.     

The hypocholesterolemic agent LY295427 reverses suppression of sterol regulatory element-binding protein processing mediated by oxysterols

The sterol LY295427 reduces plasma cholesterol levels in animals by increasing the expression of hepatic low density lipoprotein (LDL) receptors. Here we trace the hypocholesterolemic activity of LY295427 to an ability to reverse oxysterol-mediated suppression of sterol regulatory element-binding protein (SREBP) processing. Micromolar concentrations of LY295427 induced the metabolism of LDL in oxysterol-treated cultured cells and inhibited the stimulation of cholesteryl ester synthesis mediated by oxysterols. cDNA microarray and RNA blotting experiments revealed that LY295427 increased levels of the LDL receptor mRNA and those of other SREBP target genes. The compound stimulated the accumulation of SREBPs in the nuclei of cells grown in the presence of oxysterols within 4-6 h of addition to the medium. Induction required components of the normal SREBP-processing pathway, including the SREBP cleavage-activating protein and the Site 1 protease. LY295427 overcame the suppression of SREBP processing mediated by several oxysterols but not by LDL-derived cholesterol. We conclude that LY295427 achieves a therapeutically desirable end point by an unique mechanism of action.     

Isolation of a somatic cell mutant resistant to the induction of apoptosis by oxidized low density lipoprotein

Oxidized low density lipoprotein (oxLDL) induces apoptosis in macrophages, smooth muscle cells, and endothelial cells. To elucidate the molecular mechanism of oxLDL-induced cytotoxicity and determine its tissue specificity, we have used Chinese hamster ovary (CHO)-K1 cells expressing human CD36 (CHO/CD36). Expression of CD36 rendered these cells susceptible to killing by oxLDL. This cytotoxicity was due to the induction of apoptosis. Therefore, CD36 expression is the only requirement for oxLDL-induced apoptosis. Oxysterols apparently mediate the cytotoxicity of oxLDL in macrophage foam cells and endothelial cells. 25-Hydroxycholesterol, at concentrations higher than 1 microg/ml, killed CHO-K1 cells, by apoptosis, in medium supplemented with serum as a source of cholesterol. These effects were not seen in a 25-hydroxycholesterol-resistant CHO/CD36 mutant (OX(R)), which was otherwise capable of undergoing apoptosis in response to staurosporine. This mutant was also resistant to killing by oxLDL, suggesting that oxysterols are at least partially responsible for the toxic effects of oxLDL. Oxysterol-induced apoptosis did not involve regulation of sterol regulatory element-binding protein proteolysis or the cholesterol biosynthetic pathway. 25-Hydroxycholesterol stimulated calcium uptake by CHO-K1 cells within 2 min after addition. Treatment of CHO or THP-1 (macrophage) cells with the calcium channel blocker nifedipine prevented 25-hydroxycholesterol induction of apoptosis. OX(R) showed no enhanced calcium uptake in response to 25-hydroxycholesterol.     

Oxysterols from oxidized LDL are cytotoxic but fail to induce hsp70 expression in endothelial cells

Oxidized low density lipoprotein (OxLDL) possesses several proatherogenic characteristics, among which a marked cytotoxicity. In vitro, cytotoxicity of OxLDL to endothelial cells is associated with an increase in the expression of the inducible form of heat shock protein 70 (hsp70), generally regarded as a cytoprotective protein. Oxidized derivatives of cholesterol which form upon LDL oxidation are cytotoxic. Moreover, most of the OxLDL cytotoxicity is due to its lipid moiety, in particular to oxysterols. In this report we demonstrate that although oxysterols identified in OxLDL are cytotoxic, they cannot trigger the increase in hsp70 expression observed with intact oxidized lipoproteins. We speculate therefore that oxysterols may represent the most toxic form of oxidized lipids in LDL because they cannot activate a rescue mechanism (i.e. the hsp response) and may contribute significantly to cell death within atherosclerotic plaques.     

Expression of steroidogenic acute regulatory protein mRNA in rat placenta during mid-late pregnancy

The production of a steroid hormone in the placenta is essential for maintaining the pregnancy and developing the fetus during gestation. In various steroidogenic tissues (including gonads and adrenal cortex), the steroidogenic-acute-regulatory protein (StAR) acutely transfers cholesterol from the outer to the inner mitochondrial membrane for rapid steroidogenesis. Although steroid hormones were synthesized in the rat placenta, the developmental expression of StAR has been poorly understood in the rat placenta during mid-late pregnancy. Therefore, the aim of the present study was to establish the expression and localization of StAR mRNA in the rat placenta during mid-late pregnancy using Northern blots and in situ hybridization. The Northern blot analysis showed that the StAR mRNA expression significantly changed as the gestation day (GD) progressed. The placental expression of StAR mRNA increased between GD 11 and 13, and then slightly decreased until term. In situ hybridization showed a strong StAR expression in giant trophoblast cells on GD 11 and 13, and a moderate expression in trophoblast and stroma cells within the villi of the labyrinth zone throughout the pregnancy. In this study, we reveal for the first time the existence of StAR mRNA in steroidogenic cells of the placenta during mid-late pregnancy. In conclusion, our results suggest that StAR may regulate steroidogenesis in the rat placenta to maintain the pregnancy and developing the fetus.     

HepG2 cell LDL receptor activity and the accumulation of apolipoprotein B and E in response to docosahexaenoic acid and cholesterol.

In the present study, the accumulation of apolipoproteins (apo) A-I, B, and E in culture medium was measured after 0, 3, 6, 12, and 24 h of incubation with 150 microM docosahexaenoic acid complexed to 75 microM bovine serum albumin (BSA-22:6), either in the presence or absence of 50 micrograms/ml cholesterol and 4 micrograms/ml 25-hydroxycholesterol (C/25-OH). HepG2 cells incubated with BSA + C/25-OH for 24 h accumulated approximately 2.0-fold greater apoE and B as compared to BSA-treated cells. Moreover, HepG2 cell apoB accumulation after 24 h of BSA-22:6 treatment was approximately 2.0-fold greater than apoB accumulation from cells treated with BSA alone. When BSA-22:6 and C/25-OH were both included in the incubation, apoB accumulation was approximately 5.0-fold greater than BSA-treated cells. Comparative studies using BSA-18:1 were carried out for 24 h and showed similar levels of apoA-I, B, and E accumulation in culture medium as compared to BSA-22:6-treated cells. In addition, apoA-I, B, and E mRNA abundance were found to be unaffected by type of fatty acid treatment or length of incubation, averaging 48.2 +/- 7.5, 222 +/- 33.6, and 17.1 +/- 0.7 pg mRNA/micrograms RNA (mean +/- SEM), respectively. As the accumulation of apoB and apoE in culture medium may be modified by HepG2 cell LDL receptor expression, LDL receptor mRNA abundance and LDL receptor activity were quantified at various times over the course of the study. By 6 h of BSA + C/25-OH treatment, LDL receptor mRNA was reduced approximately 2.3-fold, while receptor activity was reduced approximately 1.5-fold, as compared to BSA controls. In an experiment designed to determine uptake of HepG2 cell lipoproteins, 3H-labeled apoB-containing lipoproteins derived from HepG2 cells were prepared. The 3H-labeled lipoproteins were 1.25-fold more likely to be removed from the media of HepG2 cells treated with BSA than from cells treated with BSA + C/25-OH. From these results, we postulate that HepG2 cell LDL receptor activity mediates the removal of apoB, E-containing lipoproteins from culture medium and contributes to the lower accumulation of apoB and E observed in culture medium from cells treated with BSA as compared to cells treated with C/25-OH.     

Gonadotropin-regulated testicular RNA helicase (GRTH/DDX25), a negative regulator of luteinizing/chorionic gonadotropin hormone-induced steroidogenesis in Leydig cells: central role of steroidogenic acute regulatory protein (StAR)

Gonadotropin-regulated testicular RNA helicase (GRTH/DDX25) is a testis-specific gonadotropin-regulated RNA helicase that is present in Leydig cells (LCs) and germ cells and is essential for spermatid development and completion of spermatogenesis. Normal basal levels of testosterone in serum and LCs were observed in GRTH null (GRTH(-/-)) mice. However, testosterone production was enhanced in LCs of GRTH(-/-) mice compared with WT mice by both in vivo and in vitro human chorionic gonadotropin stimulation. LCs of GRTH(-/-) mice had swollen mitochondria with a significantly increased cholesterol content in the inner mitochondrial membrane. Basal protein levels of SREBP2, HMG-CoA reductase, and steroidogenic acute regulatory protein (StAR; a protein that transports cholesterol to the inner mitochondrial membrane) were markedly increased in LCs of GRTH(-/-) mice compared with WT mice. Gonadotropin stimulation caused an increase in StAR mRNA levels and protein expression in GRTH(-/-) mice versus WT mice, with no further increase in SREBP2 and down-regulation of HMG-CoA reductase protein. The half-life of StAR mRNA was significantly increased in GRTH(-/-) mice. Moreover, association of StAR mRNA with GRTH protein was observed in WT mice. Human chorionic gonadotropin increased GRTH gene expression and its associated StAR protein at cytoplasmic sites. Taken together, these findings indicate that, through its negative role in StAR message stability, GRTH regulates cholesterol availability at the mitochondrial level. The finding of an inhibitory action of GRTH associated with gonadotropin-mediated steroidogenesis has provided insights into a novel negative autocrine molecular control mechanism of this helicase in the regulation of steroid production in the male.     

Oxysterol activation of phosphatidylcholine synthesis involves CTP:phosphocholine cytidylyltransferase alpha translocation to the nuclear envelope

In addition to suppressing cholesterol synthesis and uptake, oxysterols also activate glycerophospholipid and SM (sphingomyelin) synthesis, possibly to buffer cells from excess sterol accumulation. In the present study, we investigated the effects of oxysterols on the CDP-choline pathway for PtdCho (phosphatidylcholine) synthesis using wild-type and sterol-resistant CHO (Chinese-hamster ovary) cells expressing a mutant of SCAP [SREBP (sterol-regulatory-element-binding protein) cleavage-activating protein] (CHO-SCAP D443N). [(3)H]Choline-labelling experiments showed that 25OH (25-hydroxycholesterol), 22OH (22-hydroxycholesterol) and 27OH (27-hydroxycholesterol) increased PtdCho synthesis in CHO cells as a result of CCTalpha (CTP:phosphocholine cytidylyltransferase alpha) translocation and activation at the NE (nuclear envelope). These oxysterols also activate PtdCho synthesis in J774 macrophages. in vitro, CCTalpha activity was stimulated 2- to 2.5-fold by liposomes containing 5 mol% 25OH, 22OH or 27OH. Inclusion of up to 5 mol% cholesterol did not further activate CCTalpha. 25OH activated CCTalpha in CHO-SCAP D443N cells leading to a transient increase in PtdCho synthesis and accumulation of CDP-choline. CCTalpha translocation to the NE and intranuclear tubules in CHO-SCAP D443N cells was complete after 1 h exposure to 25OH compared with only partial translocation by 4-6 h in CHO-Mock cells. These enhanced responses in CHO-D443N cells were sterol-dependent since depletion with cyclodextrin or lovastatin resulted in reduced sensitivity to 25OH. However, the lack of effect of cholesterol on in vitro CCT activity indicates an indirect relationship or involvement of other sterols or oxysterol. We conclude that translocation and activation of CCTalpha at nuclear membranes by side-chain hydroxylated sterols are regulated by the cholesterol status of the cell.     

Inhibitory effect of 30-kDa phytoglycoprotein on expression of TNF-α and COX-2 via activation of PKCα and ERK 1/2 in LPS-stimulated RAW 264.7 cells

Endothelial cells may play a potential role in cholesterol efflux from peripheral tissues to liver. Cholesterol efflux from cells is essential for activation of the reverse cholesterol transport pathway and cardiovascular health. One of the cholesterol transporters is steroidogenic acute regulatory protein (StAR) which promotes intramitochondrial delivery of cholesterol to the cholesterol side-chain cleavage system. The aim of the present study was to determine the effects of a niacin–chromium complex on aortas of hyperlipidemic rats and on the cholesterol efflux from aorta endothelial cells by examination under light and transmission electron microscopes and evaluating the StAR immunoreactivity, respectively. Aorta lipid peroxidation (LPO) and glutathione (GSH) levels were determined by spectrophotometric methods. After treating hyperlipidemic animals with the complex, the StAR immunoreactivity in endothelial cells increased to achieve cholesterol homeostasis and efflux. Combined treatment with niacin and chromium resulted in an inhibition in the mast cell secretion and a decrease in lipid vacuole size in unilocular adipose tissue surrounding aorta, as well as in a decrease in morphological degenerations observing in aorta of hyperlipidemic rats. Aorta LPO levels increased and GSH levels decreased in the hyperlipidemic group, whereas treatment with niacin and chromium reversed these effects. In conclusion, this study reveals that combined treatment with niacin and chromium prevents the morphological and biochemical changes observed in thoracic aorta of hyperlipidemic rats, and may regulate effectively cardiovascular diseases inducing an increase in StAR levels on endothelial cells.     

Inhibitory effects of red wine extracts on endothelial-dependent adhesive interactions with monocytes induced by oxysterols

Red wine polyphenolic compounds have been demonstrated to possess antioxidant properties, and several studies have suggested that they might constitute a relevant dietary factor in the protection from coronary heart disease. The aim of the present study is to examine whether red wine extracts (RWE) can ameliorate oxysterol-induced endothelial response, and whether inhibition of adhesion molecule expression is involved in monocyte adhesion to endothelial cells. Surface expression and mRNA levels of adhesion molecules (intercellular adhesion molecule 1 and vascular cell adhesion molecule 1) were determined by ELISA and RT-PCR performed on human aortic endothelial cells (HAEC) monolayers stimulated with 7beta-hydroxycholesterol or 25-hydroxycholesterol. Incubation of HAEC with oxysterols (10 microM) increased expression of adhesion molecules in a time-dependent manner. Pretreatment of HAEC with RWE at final concentrations of 1, 10, and 100 ng/ml significantly inhibited the increase of surface protein expression and mRNA levels. Adherence of monocytes to oxysterol-stimulated HAEC was increased compared to that of unstimulated cells. Treatment of HAEC with RWE significantly inhibited adherence of monocytes. These results suggest that RWE works as an anti-atherogenic agent through the inhibition of endothelial-dependent adhesive interactions with monocytes induced by oxysterols.     

Oxysterol-induced osteoblastic differentiation of pluripotent mesenchymal cells is mediated through a PKC- and PKA-dependent pathway

Oxysterols form a large family of oxygenated derivatives of cholesterol that are present in circulation, and in human and animal tissues. The discovery of osteoinductive molecules that can induce the lineage-specific differentiation of cells into osteoblastic cells and therefore enhance bone formation is crucial for better management of bone fractures and osteoporosis. We previously reported that specific oxysterols have potent osteoinductive properties and induce the osteoblastic differentiation of pluripotent mesenchymal cells. In the present report we demonstrate that the induction of osteoblastic differentiation by oxysterols is mediated through a protein kinase C (PKC)- and protein kinase A (PKA)-dependent mechanism(s). Furthermore, oxysterol-induced-osteoblastic differentiation is marked by the prolonged DNA-binding activity of Runx2 in M2-10B4 bone marrow stromal cells (MSCs) and C3H10T1/2 embryonic fibroblastic cells. This increased activity of Runx2 is almost completely inhibited by PKC inhibitors Bisindolylmaleimide and Rottlerin, and only minimally inhibited by PKA inihibitor H-89. PKC- and PKA-dependent mechanisms appear to also regulate other markers of osteoblastic differentiation including alkaline phosphatase (ALP) activity and osteocalcin mRNA expression in response to oxysterols. Finally, osteogenic oxysterols induce osteoblastic differentiation with BMP7 and BMP14 in a synergistic manner as demonstrated by the enhanced Runx2 DNA-binding activity, ALP activity, and osteocalcin mRNA expression. Since Runx2 is an indispensable factor that regulates the differentiation of osteoblastic cells and bone formation in vitro and in vivo, its increased activity in oxysterol-treated cells further validates the potential role of oxysterols in lineage-specific differentiation of pluripotent mesenchymal cells and their potential therapeutic use as bone anabolic factors.