Effect of increasing the expression of cholesterol transporters (StAR, MLN64, and SCP-2) on bile acid synthesis

There are two major pathways of bile acid synthesis: the "neutral" pathway, initiated by highly regulated microsomal cholesterol 7alpha-hydroxylase (CYP7A1), and an "alternative" pathway, initiated by mitochondrial sterol 27-hydroxylase (CYP27A1). In hepatocyte cultures, overexpression of CYP7A1 increases bile acid synthesis by >8-fold. However, overexpression of CYP27A1 in hepatocytes only increases it by 1.5-fold, suggesting that additional rate-limiting steps must be involved in the regulation of this pathway. The effects of intracellular cholesterol transport proteins on bile acid synthesis have been investigated in the current study. Under culture conditions in which the neutral pathway was inactive, selective overexpression of the gene encoding steroidogenic acute regulatory protein (StAR), MLN64 (StAR homolog protein), and sterol carrier protein-2 (SCP-2) led to 5.7-, 1.2-, and 1.7-fold increases, respectively, in the rates of bile acid synthesis in primary rat hepatocytes. Surprisingly, co-overexpression of MLN64 with StAR, SCP-2, or CYP7A1 blunted the upregulated bile acid synthesis by 48, 47, and 45%, respectively. These results suggest that MLN64, in its full-length form, is not responsible for the transport of cholesterol to the mitochondria or the endoplasmic reticulum, where CYP27A1 or CYP7A1 is located, respectively.     

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.     

Enzyme activity assay for cholesterol 27-hydroxylase in mitochondria

Mitochondrial cholesterol 27-hydroxylase (CYP27A1) plays an important role in the maintenance of intracellular cholesterol homeostasis. Cholesterol delivery to the mitochondrial inner membrane is believed to be a rate-limiting step for the "acidic" pathway of bile acid synthesis. This work reports that proteinase K treatment of mitochondria markedly increases CYP27A1 specific activity. With endogenous mitochondrial cholesterol, treatment with proteinase K increased CYP27A1 specific activity by 5-fold. Moreover, the addition of the exogenous cholesterol in beta-cyclodextrin plus proteinase K treatment increased the specific activity by 7-fold. Kinetic studies showed that the increased activity was time-, proteinase K-, and substrate concentration-dependent. Proteinase K treatment decreased the apparent K(m) of CYP27A1 for cholesterol from 400 to 150 microM. Using this new assay, we found that during rat hepatocyte preparation and cell culture, mitochondria gradually lose CYP27A1 activity compared with mitochondria freshly isolated from rat liver tissue.     

Human sterol 27-hydroxylase (CYP27) overexpressor transgenic mouse model. Evidence against 27-hydroxycholesterol as a critical regulator of cholesterol homeostasis

CYP27-overexpressed transgenic mice were generated with the use of a human full-length CYP27 coding region cloned into a ubiquitous expression vector. Positive transgenic mice were identified by tail DNA genotyping and high fecal 27-hydroxycholesterol content. The levels of 27-hydroxycholesterol were found to be 3-5 times higher in the circulation and the tissues of the overexpressed mice when compared with littermate controls. There were no gross morphological differences between the overexpressed mice and their controls. Total cholesterol and triglyceride levels were not affected by overexpression of CYP27. Serum lathosterol was also normal, suggesting a normal rate of cholesterol synthesis. Serum levels of 7alpha-hydroxycholesterol were unaffected, suggesting a normal rate of bile acid formation in the pathway involving cholesterol 7alpha-hydroxylase. Biliary bile acid composition was slightly affected by CYP27 overexpression in female but not in male mice. Fecal levels of neutral steroids were slightly but significantly increased in overexpressor female mice but not in male mice. Levels of 24-hydroxycholesterol in the circulation were significantly reduced in the overexpressed mice, probably as a consequence of a recently described catabolic pathway involving CYP27. Combined with the results of our previous work on mice with a disruption of the CYP27 gene, the present results suggest that the levels of 27-hydroxycholesterol are not of critical importance for cholesterol homeostasis in mice.     

Alternate pathways of bile acid synthesis in the cholesterol 7alpha-hydroxylase knockout mouse are not upregulated by either cholesterol or cholestyramine feeding

Bile acids are synthesized via the classic pathway initiated by cholesterol 7alpha-hydroxylase (CYP7A1), and via alternate pathways, one of which is initiated by sterol 27-hydroxylase (CYP27). These studies used mice lacking cholesterol 7alpha-hydroxylase (Cyp7a1(-/-)) to establish whether the loss of the classic pathway affected cholesterol homeostasis differently in males and females, and to determine if the rate of bile acid synthesis via alternate pathways was responsive to changes in the enterohepatic flux of cholesterol and bile acids. In both the Cyp7a1(-/-) males and females, the basal rate of bile acid synthesis was only half of that in matching Cyp7a1(+/+) animals. Although bile acid pool size contracted markedly in all the Cyp7a1(-/-) mice, the female Cyp7a1(-/-) mice maintained a larger, more cholic acid-rich pool than their male counterparts. Intestinal cholesterol absorption in the Cyp7a1(-/-) males fell from 46% to 3%, and in the matching females from 58% to 17%. Bile acid synthesis in Cyp7a1(+/+) males and females was increased 2-fold by cholesterol feeding, and 4-fold by cholestyramine treatment, but was not changed in matching Cyp7a1(-/-) mice by either of these manipulations. In the Cyp7a1(-/-) mice fed cholesterol, hepatic cholesterol concentrations increased only marginally in the males, but rose almost 3-fold in the females. CYP7A1 activity and mRNA levels were greater in females than in males, and were increased by cholesterol feeding in both sexes. CYP27 activity and mRNA levels did not vary as a function of CYP7A1 genotype, gender, or dietary cholesterol intake. We conclude that in the mouse the rate of bile acid synthesis via alternative pathways is unresponsive to changes in the enterohepatic flux of cholesterol and bile acid, and that factors governing gender-related differences in bile acid synthesis, pool size, and pool composition play an important role in determining the impact of CYP7A1 deficiency on cholesterol homeostasis in this species.     

25R,26-Hydroxycholesterol revisited: synthesis, metabolism, and biologic roles

The CYP27 gene is expressed in arterial endothelium, macrophages, and other tissues. The gene product generates sterol intermediates that function as ligands for nuclear receptors prior to their transport to the liver for metabolism, mostly to bile acids. Most attention has been given to 27-hydroxycholesterol as a ligand for LXR activated receptors and to chenodeoxycholic acid as a ligand for farnesoid X activated receptors (FXRs). Expression of the pathway in macrophages is essential for normal reverse cholesterol transport. Thus, ABC transporter activity is upregulated, which enhances cholesterol efflux. Absence of these mechanisms probably accounts for the accelerated atherosclerosis that occurs in cerebrotendinous xanthomatosis. Accumulation of 27-hydroxycholesterol in human atheroma is puzzling and may reflect low levels of oxysterol 7alpha-hydroxylase activity in human macrophages. The same enzyme determines the proportion of mono-, di-, and tri-hydroxy bile acids synthesized in the liver. Oxysterol 7alpha-hydroxylase deficiency is a molecular basis for cholestatic liver disease. Chenodeoxycholic acid, the major normal end product, downregulates expression of cholesterol 7alpha-hydroxylase via the FXR/short heterodimer protein nuclear receptor and thus limits total bile acid production. The challenge is to quantify in a physiologic setting the magnitude of the pathway in different tissues and to further evaluate the biologic roles of all the intermediates that may function as ligands for orphan nuclear receptors or via other regulatory mechanisms.     

Regulation of sterol 27-hydroxylase and an alternative pathway of bile acid biosynthesis in primary cultures of rat hepatocytes

In man, hepatic mitochondrial sterol 27-hydroxylase and microsomal cholesterol 7-hydroxylase initiate distinct pathways of bile acid biosynthesis from cholesterol, the “acidic” and “neutral” pathways, respectively. A similar acidic pathway in the rat has been hypothesized, but its quantitative importance and ability to be regulated at the level of sterol 27-hydroxylase are uncertain. In this study, we explored the molecular regulation of sterol 27-hydroxylase and the acidic pathway of bile acid biosynthesis in primary cultures of adult rat hepatocytes. mRNA and protein turnover rates were approximately 10-fold slower for sterol 27-hydroxylase than for cholesterol 7-hydroxylase. Sterol 27-hydroxylase mRNA was not spontaneously expressed in culture. The sole requirement for preserving sterol 27-hydroxylase mRNA at the level of freshly isolated hepatocytes (0 h) after 72 h was the addition of dexamethasone (0.1 μM; > 7-fold induction). Sterol 27-hydroxylase mRNA, mass and specific activity were not affected by thyroxine (1.0 μM), dibutyryl-cAMP (50 μM), nor squalestatin 1 (150 nM-1.0 μM), an inhibitor of cholesterol biosynthesis. Taurocholate (50 μM), however, repressed sterol 27-hydroxylase mRNA levels by 55%. Sterol 27-hydroxylase specific activity in isolated mitochondria was increased > 10-fold by the addition of 2-hydroxypropyl-β-cyclodextrin. Under culture conditions designed to maximally repress cholesterol 7-hydroxylase and bile acid synthesis from the neutral pathway but maintain sterol 27-hydroxylase mRNA and activity near 0 h levels, bile acid synthesis from [¹⁴C]cholesterol remained relatively high and consisted of β-muricholate, the product of chenodeoxycholate in the rat. We conclude that rat liver harbors a quantitatively important alternative pathway of bile acid biosynthesis and that its initiating enzyme, sterol 27-hydroxylase, may be slowly regulated by glucocorticoids and bile acids.     

27-hydroxycholesterol: production rates in normal human subjects.

We attempted to quantitate production of bile acid via the 27-hydroxylation pathway in six human subjects. After bolus intravenous injection of known amounts of [24-14C]cholic acid and [24-14C]chenodeoxycholic acid, each subject underwent a constant intravenous infusion of a mixture of [22, 23-3H]-27-hydroxycholesterol and [2H]-27-hydroxycholesterol for 6;-10 h. Production rate of 27-hydroxycholesterol was calculated from the infusion rate of [2H]-27-hydroxycholesterol and the serum ratio of deuterated/protium 27-hydroxycholesterol, which reached a plateau level by 4 h of infusion. Conversion of 27-hydroxycholesterol to cholic and chenodeoxycholic acids was determined from the 3H/14C ratio of these two bile acids in bile samples obtained the day after infusion. In five of the six subjects, independent measurement of bile acid synthesis by fecal acidic sterol output was available from previous studies. Endogenous production of 27-hydroxycholesterol averaged 17.6 mg/day and ranged from 5.0 to 28.2 mg/day, which amounted to 8.7% (range 3.0;-17.9%) of total bile acid synthesis. On average 66% of infused 27-hydroxycholesterol was converted to bile acid, of which 72.6% was chenodeoxycholic acid.These data suggest that relatively little bile acid synthesis takes place via the 27-hydroxylation pathway in healthy humans. Nevertheless, even this amount, occurring predominantly in vascular endothelium and macrophages, could represent an important means for removal of cholesterol deposited in endothelium.     

26-Hydroxycholesterol: synthesis, metabolism, and biologic activities

Cholest-5-ene-3 beta,26-diol (26-hydroxycholesterol) is synthesized by a mitochondrial P-450 enzyme that appears to be widely distributed in tissues. Together with other C-27 steroid intermediates it is transported to the liver and metabolized to bile acids. Although 26-hydroxycholesterol is transported in plasma lipoproteins mostly as the fatty acid ester, neither its assembly and orientation within lipoproteins nor its mechanism of transport across the sinusoidal liver membrane is known. Cell culture studies indicate that 26-hydroxycholesterol can inhibit both cholesterol synthesis and low density lipoprotein (LDL) receptor activity. Inhibition of DNA synthesis also occurs and may not be related to the reduction in HMG-CoA reductase activity. The relationship of these in vitro activities to the physiologic role(s) of 26-hydroxycholesterol remains to be clarified. A clue to its biologic role is the knowledge that markedly decreased 26-hydroxylase activity appears to be the molecular basis of cerebrotendinous xanthomatosis, an inborn error of metabolism characterized by a significant decrease in 26-hydroxycholesterol and bile acid synthesis and an increase in cholesterol synthesis.     

Mitochondrial processing of newly synthesized steroidogenic acute regulatory protein (StAR), but not total StAR, mediates cholesterol transfer to cytochrome P450 side chain cleavage enzyme in adrenal cells

The metabolism of cholesterol by cytochrome P450 side chain cleavage enzyme is hormonally regulated in steroidogenic tissues via intramitochondrial cholesterol transport. The mediating steroidogenic acute regulatory protein (StAR) is synthesized as a 37-kDa (p37) precursor that is phosphorylated by protein kinase A and cleaved within the mitochondria to generate 30-kDa forms (p30, pp30). The effectiveness of modified recombinant StAR forms in COS-1 cells without mitochondrial import has led to a prevailing view that cholesterol transport is mediated by p37 StAR via activity on the outer mitochondrial membrane. The present study of the activation of cholesterol metabolism by bromo-cAMP in adrenal cells in relation to (35)S-StAR turnover indicates that targeting of pp30 to the inner membrane provides the dominant cholesterol transport mechanism. We show that 1) only newly synthesized StAR is functional, 2) phosphorylation and processing of p37 to pp30 occurs rapidly and stoichiometrically, 3) both steps are necessary for optimum transport, and 4) newly synthesized pp30 exhibits very high activity (400 molecules of cholesterol/StAR/min). Segregation of cAMP activation and synthesis of StAR from cholesterol metabolism showed that very low levels of newly synthesized StAR (1 fmol/min/10(6) cells) sustained activated cholesterol metabolism (0.4 pmol/min/10(6) cells, t(1/2) = 70 min) long after complete removal of p37 (t(1/2) = 5 min). This activity was highly sensitive to inhibition of processing by CCCP only until sufficient pp30 was formed. Maximum activation preceded bromo-cAMP-induced StAR expression, indicating other limiting steps in cholesterol metabolism.     

Steroidogenic acute regulatory protein binds cholesterol and modulates mitochondrial membrane sterol domain dynamics

The steroidogenic acute regulatory protein (StAR) mediates the rate-limiting step of steroidogenesis, delivery of cholesterol to the inner mitochondrial membrane. However, the mechanism whereby cholesterol translocation is accomplished has not been resolved. Recombinant StAR proteins lacking the first N-terminal 62 amino acids comprising the mitochondrial-targeting sequence were used to determine if StAR binds cholesterol and alters mitochondrial membrane cholesterol domains to enhance sterol transfer. First, a fluorescent NBD-cholesterol binding assay revealed 2 sterol binding sites (K(d) values near 32 nm), whereas the inactive A218V N-62 StAR mutant had only a single binding site with 8-fold lower affinity. Second, NBD-cholesterol spectral shifts and fluorescence resonance energy transfer from StAR Trp residues to NBD-cholesterol showed (i) close molecular interaction between these molecules (R(2/3) = 33 A) and (ii) sensitized NBD-cholesterol emission from only one of the two sterol binding sites. Third, circular dichroism showed that cholesterol binding induced a change in StAR secondary structure. Fourth, a fluorescent sterol transfer assay that did not require separation of donor and acceptor mitochondrial membranes demonstrated that StAR enhanced mitochondrial sterol transfer as much as 100-fold and induced/increased the formation of rapidly transferable cholesterol domains in isolated mitochondrial membranes. StAR was 67-fold more effective in transferring cholesterol from mitochondria of steroidogenic MA-10 cells than from human fibroblast mitochondria. In contrast, sterol carrier protein-2 (SCP-2) was only 2.2-fold more effective in mediating sterol transfer from steroidogenic cell mitochondria. Taken together these data showed that StAR is a cholesterol-binding protein, preferentially enhances sterol transfer from steroidogenic cell mitochondria, and interacts with mitochondrial membranes to alter their sterol domain structure and dynamics.     

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.     

Knockout of the cholesterol 24-hydroxylase gene in mice reveals a brain-specific mechanism of cholesterol turnover

Most cholesterol turnover takes place in the liver and involves the conversion of cholesterol into soluble and readily excreted bile acids. The synthesis of bile acids is limited to the liver, but several enzymes in the bile acid biosynthetic pathway are expressed in extra-hepatic tissues and there also may contribute to cholesterol turnover. An example of the latter type of enzyme is cholesterol 24-hydroxylase, a cytochrome P450 (CYP46A1) that is expressed at 100-fold higher levels in the brain than in the liver. Cholesterol 24-hydroxylase catalyzes the synthesis of the oxysterol 24(S)-hydroxycholesterol. To assess the relative contribution of the 24-hydroxylation pathway to cholesterol turnover, we performed balance studies in mice lacking the cholesterol 24-hydroxylase gene (Cyp46a1-/- mice). Parameters of hepatic cholesterol and bile acid metabolism in the mutant mice remained unchanged relative to wild type controls. In contrast to the liver, the synthesis of new cholesterol was reduced by approximately 40% in the brain, despite steady-state levels of cholesterol being similar in the knockout mice. These data suggest that the synthesis of new cholesterol and the secretion of 24(S)-hydroxycholesterol are closely coupled and that at least 40% of cholesterol turnover in the brain is dependent on the action of cholesterol 24-hydroxylase. We conclude that cholesterol 24-hydroxylase constitutes a major tissue-specific pathway for cholesterol turnover in the brain.     

Cholesterol transport in steroid biosynthesis: Role of protein–protein interactions and implications in disease states

The transfer of cholesterol from the outer to the inner mitochondrial membrane is the rate-limiting step in hormone-induced steroid formation. To ensure that this step is achieved efficiently, free cholesterol must accumulate in excess at the outer mitochondrial membrane and then be transferred to the inner membrane. This is accomplished through a series of steps that involve various intracellular organelles, including lysosomes and lipid droplets, and proteins such as the translocator protein (18 kDa, TSPO) and steroidogenic acute regulatory (StAR) proteins. TSPO, previously known as the peripheral-type benzodiazepine receptor, is a high-affinity drug- and cholesterol-binding mitochondrial protein. StAR is a hormone-induced mitochondria-targeted protein that has been shown to initiate cholesterol transfer into mitochondria. Through the assistance of proteins such as the cAMP-dependent protein kinase regulatory subunit Iα (PKA-RIα) and the PKA-RIα- and TSPO-associated acyl-coenzyme A binding domain containing 3 (ACBD3) protein, PAP7, cholesterol is transferred to and docked at the outer mitochondrial membrane. The TSPO-dependent import of StAR into mitochondria, and the association of TSPO with the outer/inner mitochondrial membrane contact sites, drives the intramitochondrial cholesterol transfer and subsequent steroid formation. The focus of this review is on (i) the intracellular pathways and protein–protein interactions involved in cholesterol transport and steroid biosynthesis and (ii) the roles and interactions of these proteins in endocrine pathologies and neurological diseases where steroid synthesis plays a critical role.     

On the substrate specificity of human CYP27A1: implications for bile acid and cholestanol formation

The mitochondrial sterol 27-hydroxylase (CYP27A1) is required for degradation of the C27-sterol side chain in bile acid biosynthesis. CYP27A1 seems, however, to have roles beyond this, as illustrated by patients with a deficient sterol 27-hydroxylase due to mutations of the CYP27A1 gene [cerebrotendinous xanthomatosis (CTX)]. These subjects have symptoms ranging from accumulation of bile alcohols and cholestanol to accelerated atherosclerosis and progressive neurologic impairment. The present work describes a detailed investigation on the substrate specificity of recombinant human CYP27A1. In accordance with some previous work with rat liver mitochondria, the activity in general increased with the polarity of the substrate. An obvious example was the finding that cholesterol was 27-hydroxylated more efficiently than cholesterol oleate but less efficiently than cholesterol sulfate. The oxysterols 24S-hydroxycholesterol and 25-hydroxycholesterol were 27-hydroxylated less efficiently than cholesterol, possibly due to steric hindrance. Surprisingly, sterols with a 3-oxo-Delta4 structure were found to be hydroxylated at a much higher rate than the corresponding sterols with a 3beta-hydroxy-Delta5 structure. The rates of hydroxylation of the sterols were: 7alpha-hydroxy-4-cholesten-3-one>4-cholesten-3-one>7alpha-hydroxycholesterol>24-hydroxy-4-cholesten-3-one> cholesterol>25-hydroxy-4-cholesten-3-one>24-hydroxycholesterol>or=25-hydroxycholesterol. The possibility is discussed that the findings may have implications for oxysterol-mediated regulation of gene expression. The very high activity of CYP27A1 towards the cholestanol precursor 4-cholesten-3-one may be of importance in connection with the accumulation of cholestanol in patients with CTX.     

Expression of sterol 27-hydroxylase (CYP27A1) enhances cholesterol efflux

Cholesterol efflux from CHOP cells transfected with sterol 27-hydroxylase (CYP27A1) was compared with non-transfected and mock-transfected cells. Transfection caused expression of CYP27A1, formation of 27-hydroxycholesterol, and inhibition of cholesterol biosynthesis. Transfection enhanced cholesterol efflux to apolipoprotein A-I or human plasma by 2-3-fold but did not affect the efflux in the absence of acceptor. The analysis of released sterols revealed that 27-hydroxycholesterol represented only a small proportion of sterols, most of which was non-oxidized cholesterol. Time course and dose dependence studies showed that expression of CYP27A1 in CHOP cells mostly affected the efflux of the "fast" cholesterol pool, and relatively more cholesterol was released with low concentrations of an acceptor. Preincubation of non-transfected cells with exogenous 27-hydroxycholesterol (10(-9) and 10(-7) m) led to the stimulation of cholesterol efflux by 24-60%. Expression of CYP27A1 in CHOP cells did not affect ABCA1 expression and abundance of ABCA1 protein. Thus, introduction of CYP27A1 into cells stimulates cholesterol efflux and therefore may increase protection against atherosclerosis.     

Transfer of cholesterol between phospholipid vesicles mediated by the steroidogenic acute regulatory protein (StAR)

The steroidogenic acute regulatory protein (StAR) mediates the acute stimulation of steroid synthesis by tropic hormones in steroidogenic cells. StAR interacts with the outer mitochondrial membrane and facilitates the rate-limiting transfer of cholesterol to the inner mitochondrial membrane where cytochrome P-450scc converts this cholesterol into pregnenolone. We tested the ability of N-62 StAR to transfer cholesterol from donor vesicles containing cholesterol but no cytochrome P-450scc to acceptor vesicles containing P-450scc but no cholesterol, using P-450scc activity as a reporter of the cholesterol content of synthetic phospholipid vesicles. N-62 StAR stimulated P-450scc activity in acceptor vesicles 5-10-fold following the addition of donor vesicles. Transfer of cholesterol to acceptor vesicles was rapid and sufficient to maintain a linear rate of pregnenolone synthesis for 10 min. The effect of N-62 StAR in stimulating P-450scc activity was specific for cholesterol transfer and was not due to vesicle fusion or P-450scc exchange between vesicles. Maximum stimulation of P-450scc activity in acceptor vesicles required preincubation of N-62 StAR with phospholipid vesicles prior to adding donor vesicles. The amount of N-62 StAR causing half-maximum stimulation of P-450scc activity in acceptor vesicles was 1.9 microm. Half-maximum stimulation required more than a 10-fold higher concentration of R182L N-62 StAR, a mutant associated with congenital lipoid adrenal hyperplasia. N-62 StAR-mediated transfer of cholesterol between vesicles showed low dependence on the cholesterol concentration in the donor vesicles. Thus StAR can transfer cholesterol between synthetic membranes without other protein components found in mitochondria.     

Marked induction of sterol 27-hydroxylase activity and mRNA levels during differentiation of human cultured monocytes into macrophages

Sterol 27-hydroxylase has been suggested to be involved in an alternative pathway for the elimination of cholesterol from macrophages and early atherosclerotic lesions. We have previously shown that human lung macrophages as well as monocyte-derived macrophages have a relatively high activity of sterol 27-hydroxylase (CYP27). This enzyme converts intracellular cholesterol into 27-hydroxycholesterol and cholestenoic acid that flux from cultured cells into the medium. It is shown here that human monocytes have very low CYP27 activity and CYP27 mRNA levels. During differentiation into macrophages, both CYP27 activity and CYP27 mRNA levels increase markedly after 4 days of culture in serum-free medium. Addition of macrophage-colony stimulating factor had no significant effect on the induction and addition of fetal calf serum had an inhibitory effect. Cholesterol synthesis was found to be a critical factor for the production of 27-oxygenated products by the macrophages cultured in serum-free medium. The increased capacity of the differentiated cells to eliminate intracellular cholesterol is of interest and supports the contention that CYP27 is an antiatherogenic factor.