Localization of StarD5 cholesterol binding protein

Human StarD5 belongs to the StarD4 subfamily of START (for steroidogenic acute regulatory lipid transfer) domain proteins. We previously reported that StarD5 is located in the cytosolic fraction of human liver and binds cholesterol and 25-hydroxycholesterol. After overexpression of the gene encoding StarD5 in primary rat hepatocytes, free cholesterol accumulated in intracellular membranes. These findings suggested StarD5 to be a directional cytosolic sterol transporter. The objective of this study was to determine the localization of StarD5 in human liver. Western blot analysis confirmed StarD5's presence in the liver but not in human hepatocytes. Immunohistochemistry studies showed StarD5 localized within sinusoidal lining cells in the human liver and colocalized with CD68, a marker for Kupffer cells. Western blot analyses identified the presence of StarD5 in monocytes and macrophages as well as mast cells, basophils, and promyelocytic cells, but not in human hepatocytes, endothelial cells, fibroblasts, osteocytes, astrocytes, or brain tissue. Cell fractionation and immunocytochemistry studies on THP-1 macrophages localized StarD5 to the cytosol and supported an association with the Golgi. The presence of this cholesterol/25-hydroxycholesterol-binding protein in cells related to inflammatory processes provides new clues to the role of this protein in free sterol transport in the cells and in lipid-mediated atherogenesis.     

Intracellular cholesterol transporter StarD4 binds free cholesterol and increases cholesteryl ester formation

StarD4 protein is a member of the StarD4 subfamily of steroidogenic acute regulatory-related lipid transfer (START) domain proteins that includes StarD5 and StarD6, proteins whose functions remain poorly defined. The objective of this study was to isolate and characterize StarD4's sterol binding and to determine in a hepatocyte culture model its sterol transport capabilities. Utilizing purified full-length StarD4, in vitro binding assays demonstrated a concentration-dependent binding of [(14)C]cholesterol by StarD4 similar to that of the cholesterol binding START domain proteins StarD1 and StarD5. Other tested sterols showed no detectable binding to StarD4, except for 7alpha-hydroxycholesterol, for which StarD4 demonstrated weak binding on lipid protein overlay assays. Subsequently, an isolated mouse hepatocyte model was used to study the ability of StarD4 to bind/mobilize/distribute cellular cholesterol. Increased expression of StarD4 in primary mouse hepatocytes led to a marked increase in the intracellular cholesteryl ester concentration and in the rates of bile acid synthesis. The ability and specificity of StarD4 to bind cholesterol and, as a function of its level of expression, to direct endogenous cellular cholesterol suggest that StarD4 plays an important role as a directional cholesterol transporter in the maintenance of cellular cholesterol homeostasis.     

Sulfated oxysterol, 25HC3S, is a potent regulator of lipid metabolism in human hepatocytes

Recently, a novel oxysterol, 5-cholesten-3beta, 25-diol 3-sulfate (25HC3S) was identified in primary rat hepatocytes following overexpression of the cholesterol transport protein, StarD1. This oxysterol was also detected in human liver nuclei. In the present study, 25HC3S was chemically synthesized. Addition of 25HC3S (6 microM) to human hepatocytes markedly inhibited cholesterol biosynthesis. Quantitative RT-PCR and Western blot analysis showed that 25HC3S markedly decreased HMG-CoA reductase mRNA and protein levels. Coincidently, 25HC3S inhibited the activation of sterol regulatory element binding proteins (SREBPs), suggesting that inhibition of cholesterol biosynthesis occurred via blocking SREBP-1 activation, and subsequently by inhibiting the expression of HMG CoA reductase. 25HC3S also decreased SREBP-1 mRNA levels and inhibited the expression of target genes encoding acetyl CoA carboxylase-1 (ACC-1) and fatty acid synthase (FAS). In contrast, 25-hydroxycholesterol increased SREBP1 and FAS mRNA levels in primary human hepatocytes. The results imply that 25HC3S is a potent regulator of SREBP mediated lipid metabolism.     

Differential gene regulation of StarD4 and StarD5 cholesterol transfer proteins. Activation of StarD4 by sterol regulatory element-binding protein-2 and StarD5 by endoplasmic reticulum stress

The StarD4 and StarD5 proteins share approximately 30% identity, and each is a steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain. We previously showed StarD4 expression is sterol-repressed, consistent with regulation by sterol regulatory element-binding proteins (SREBPs), whereas StarD5 is not sterol-regulated. Here we further address the regulation and function of StarD4 and StarD5. Unlike StAR, the START family prototype, StarD4 and StarD5 were not induced by steroidogenic stimuli in Leydig cells. However, StarD4 and StarD5 showed StAR-like activity in a cell culture steroidogenesis assay, indicating cholesterol transfer. In transgenic mice expressing active SREBPs, StarD4 was predominantly activated by SREBP-2 rather than SREBP-1a. The mouse and human StarD4 proximal promoters share approximately 70% identity, including several potential sterol regulatory elements (SREs). Reporters driven by the StarD4 promoter from either species were transfected into NIH-3T3 cells, and reporter activity was highly repressed by sterols. Site-directed mutagenesis of potential SREs identified a conserved functional SRE in the mouse (TCGGTCCAT) and human (TCATTCCAT) promoters. StarD5 was not sterol-repressed via SREBPs nor was it sterol-activated via liver X receptors (LXRs). Even though StarD4 and StarD5 were not LXR targets, their overexpression stimulated LXR reporter activity, suggesting roles in cholesterol metabolism. StarD5 expression increased 3-fold in free cholesterol-loaded macrophages, which activate the endoplasmic reticulum (ER) stress response. When NIH-3T3 cells were treated with agents to induce ER stress, StarD5 expression increased 6-8-fold. Because StarD4 is regulated by sterols via SREBP-2, whereas StarD5 is activated by ER stress, they likely serve distinct functions in cholesterol metabolism.     

Identification of a novel sulfonated oxysterol, 5-cholesten-3beta,25-diol 3-sulfonate, in hepatocyte nuclei and mitochondria

This study reports the discovery of a novel sulfonated oxysterol found at high levels in the mitochondria and nuclei of primary rat hepatocytes after overexpression of the gene encoding steroidogenic acute regulatory protein (StarD1). Forty-eight hours after infection of primary rat hepatocytes with recombinant adenovirus encoding StarD1, rates of bile acid synthesis increased by 4-fold. Concurrently, [(14)C]cholesterol metabolites (oxysterols) were increased dramatically in both the mitochondria and nuclei of StarD1-overexpressing cells, but not in culture medium. A water-soluble [(14)C]oxysterol product was isolated and purified by chemical extraction and reverse-phase HPLC. Enzymatic digestion, HPLC, and tandem mass spectrometry analysis identified the water-soluble oxysterol as 5-cholesten-3beta,25-diol 3-sulfonate. Further experiments detected this cholesterol metabolite in the nuclei of normal human liver tissues. Based upon these observations, we hypothesized a new pathway by which cholesterol is metabolized in the mitochondrion.     

Cholesterol 25-hydroxylation activity of CYP3A

To date, many studies have been conducted using 25-hydroxycholesterol, which is a potent regulator of lipid metabolism. However, the origins of this oxysterol have not been entirely elucidated. Cholesterol 25-hydroxylase is one of the enzymes responsible for the metabolism of 25-hydroxycholesterol, but the expression of this enzyme is very low in humans. This oxysterol is also synthesized by sterol 27-hydroxylase (CYP27A1) and cholesterol 24-hydroxylase(CYP46A1), but it is only a minor product of these enzymes. We now report that CYP3A synthesizes a significant amount of 25-hydroxycholesterol and may participate in the regulation of lipid metabolism. Induction of CYP3A by pregnenolone-16α-carbonitrile caused the accumulation of 25-hydroxycholesterol in a cell line derived from mouse liver. Furthermore, treatment of the cells with troleandomycin, a specific inhibitor of CYP3A, significantly reduced cellular 25-hydroxycholesterol concentrations. In cells that overexpressed human recombinant CYP3A4, the activity of cholesterol 25-hydroxylation was found to be higher than that of cholesterol 4β-hydroxylation, a known marker activity of CYP3A4. In addition, 25-hydroxycholesterol concentrations in normal human sera correlated positively with the levels of 4β-hydroxycholesterol (r = 0.650, P < 0.0001, n = 78), but did not significantly correlate with the levels of 27-hydroxycholesterol or 24S-hydroxycholesterol. These results demonstrate the significance of CYP3A on the production of 25-hydroxycholesterol.     

Identification of Novel Regulatory Cholesterol Metabolite, 5-Cholesten, 3β,25-Diol,Disulfate

Oxysterol sulfation plays an important role in regulation of lipid metabolism and inflammatory responses. In the present study, we report the discovery of a novel regulatory sulfated oxysterol in nuclei of primary rat hepatocytes after overexpression of the gene encoding mitochondrial cholesterol delivery protein (StarD1). Forty-eight hours after infection of the hepatocytes with recombinant StarD1 adenovirus, a water-soluble oxysterol product was isolated and purified by chemical extraction and reverse-phase HPLC. Tandem mass spectrometry analysis identified the oxysterol as 5-cholesten-3β, 25-diol, disulfate (25HCDS), and confirmed the structure by comparing with a chemically synthesized compound. Administration of 25HCDS to human THP-1-derived macrophages or HepG2 cells significantly inhibited cholesterol synthesis and markedly decreased lipid levels in vivo in NAFLD mouse models. RT-PCR showed that 25HCDS significantly decreased SREBP-1/2 activities by suppressing expression of their responding genes, including ACC, FAS, and HMG-CoA reductase. Analysis of lipid profiles in the liver tissues showed that administration of 25HCDS significantly decreased cholesterol, free fatty acids, and triglycerides by 30, 25, and 20%, respectively. The results suggest that 25HCDS inhibits lipid biosynthesis via blocking SREBP signaling. We conclude that 25HCDS is a potent regulator of lipid metabolism and propose its biosynthetic pathway.     

High-affinity binding sites for oxygenated sterols in rat liver microsomes: possible identity with antiestrogen binding sites

Oxygenated derivatives of cholesterol are known to exhibit a number of biological activities including the inhibition of cholesterol biosynthesis and of cell proliferation, but their mechanism of action remains unclear. Previous studies have identified a cytosolic protein which binds 25-hydroxycholesterol, as well as several other oxysterols, with high affinity, possibly mediating some of their effects. We now report the existence of a high-affinity oxysterol binding site in rat liver microsomes which is distinct from the cytosolic binding protein. Among the oxygenated sterols examined, 5 alpha-cholestan-3 beta-ol-7-one (7-ketocholestanol) had the highest affinity for this microsomal binding site (Kd = 2.7 nM). Using 7-keto[3H]cholestanol as the radioactive ligand, we found that binding of this oxysterol to the microsomal binding site was saturable and reversible and was displaceable by the following oxysterols in descending order of potency: 7-ketocholestanol greater than 6-ketocholestanol greater than 7 beta-hydroxycholesterol = 7-ketocholesterol greater than cholesten-3 beta,5 alpha, 6 beta-triol = 7 alpha-hydroxycholesterol greater than 4-cholesten-3-one. All other sterols studied, including, notably, 25-hydroxycholesterol, had little or no inhibitory effect on 7-keto[3H]cholestanol binding. Additional studies revealed that the microsomal oxysterol binding site was probably identical to the antiestrogen binding site described by other workers. First, saturation analysis and kinetic studies demonstrated that the antiestrogen tamoxifen competed directly with 7-keto[3H]cholestanol for the same binding site. Second, the ability of different oxysterols and antiestrogens to inhibit 7-keto[3H]cholestanol binding to the microsomal binding site paralleled their ability to inhibit [3H]tamoxifen binding to the antiestrogen binding site. Third, the tissue distribution of binding sites for 7-keto[3H]cholestanol was similar to that of the antiestrogen binding site. We conclude that: (1) in rat liver microsomes there are high-affinity oxysterol binding sites whose ligand specificity is different from that of the cytosolic oxysterol binding protein; and (2) the microsomal oxysterol binding site is probably identical to the antiestrogen binding site. The biological significance of these observations remains to be explored.     

Biosynthesis of the regulatory oxysterol, 5-cholesten-3beta,25-diol 3-sulfate, in hepatocytes

Cellular cholesterol homeostasis is maintained through coordinated regulation of cholesterol synthesis, degradation, and secretion. Nuclear receptors for oxygenated cholesterol derivatives (oxysterols) are known to play key roles in the regulation of cholesterol homeostasis. We recently identified a sulfated oxysterol, 5-cholesten-3beta,25-diol 3-sulfate (25HC3S), that is localized to liver nuclei. The present study reports a biosynthetic pathway for 25HC3S in hepatocytes. Assays using mitochondria isolated from rats and sterol 27-hydroxylase (Cyp27A1) gene knockout mice indicated that 25-hydroxycholesterol (25HC) is synthesized by CYP27A1. Incubation of cholesterol or 25HC with mitochondrial and cytosolic fractions in the presence of 3'-phosphoadenosyl 5'-phosphosulfate resulted in the synthesis of 25HC3S. Real-time RT-PCR and Western blot analysis showed the presence of insulin-regulated hydroxycholesterol sulfotransferase 2B1b (SULT2B1b) in hepatocytes. 25HC3S, but not 25HC, decreased SULT2B1b mRNA and protein levels. Specific small interfering RNA decreased SULT2B1b mRNA, protein, and activity levels. These findings demonstrate that mitochondria synthesize 25HC, which is subsequently 3beta-sulfated to form 25HC3S.     

Disruption of the oxysterol 7alpha-hydroxylase gene in mice

Mice without oxysterol 7alpha-hydroxylase, an enzyme of the alternate bile acid synthesis pathway with a sexually dimorphic expression pattern, were constructed by the introduction of a null mutation at the Cyp7b1 locus. Animals heterozygous (Cyp7b1(+/-)) and homozygous (Cyp7b1(-/-)) for this mutation were grossly indistinguishable from wild-type mice. Plasma and tissue levels of 25- and 27-hydroxycholesterol, two oxysterol substrates of this enzyme with potent regulatory actions in cultured cells, were markedly elevated in Cyp7b1(-/-) knockout animals. Parameters of bile acid metabolism as well as plasma cholesterol and triglyceride levels in male and female Cyp7b1(-/-) mice were normal. The cholesterol contents of major tissues were not altered. In vivo sterol biosynthetic rates were unaffected in multiple tissues with the exception of the male kidney, which showed a approximately 40% decrease in de novo synthesis versus controls. We conclude that the major physiological role of the CYP7B1 oxysterol 7alpha-hydroxylase is to metabolize 25- and 27-hydroxycholesterol and that loss of this enzyme in the liver is compensated for by increases in the synthesis of bile acids by other pathways. A failure to catabolize oxysterols in the male kidney may lead to a decrease in de novo sterol synthesis.     

1H, 13C, and 15N backbone resonance assignments of the L124D mutant of StAR-related lipid transfer domain protein 4 (StARD4)

Protein-mediated cholesterol trafficking is central to maintaining cholesterol homeostasis in cells. START (Steroidogenic acute regulatory protein-related lipid transfer) domains constitute a sterol and lipid binding motif and the START domain protein StARD4 typifies a small family of mammalian sterol transport proteins. StARD4 consists of a single START domain and has been reported to act as a general cholesterol transporter in cells. However, the structural basis of cholesterol uptake and transport is not well understood and no cholesterol-bound START domain structures have been reported. We have undertaken the study of cholesterol binding and transport by StARD4 using solution state NMR spectroscopy. To this end, we report nearly complete ¹H, ¹⁵N, and ¹³C backbone resonance assignments of an inactive but well behaved mutant (L124D) of StARD4.     

Side chain oxygenated cholesterol regulates cellular cholesterol homeostasis through direct sterol-membrane interactions

Side chain oxysterols exert cholesterol homeostatic effects by suppression of sterol regulatory element-binding protein maturation and promoting degradation of hydroxymethylglutaryl-CoA reductase. To examine whether oxysterol-membrane interactions contribute to the regulation of cellular cholesterol homeostasis, we synthesized the enantiomer of 25-hydroxycholesterol. Using this unique oxysterol probe, we provide evidence that oxysterol regulation of cholesterol homeostatic responses is not mediated by enantiospecific oxysterol-protein interactions. We show that side chain oxysterols, but not steroid ring-modified oxysterols, exhibit membrane expansion behavior in phospholipid monolayers and bilayers in vitro. This behavior is non-enantiospecific and is abrogated by increasing the saturation of phospholipid acyl chain constituents. Moreover, we extend these findings into cultured cells by showing that exposure to saturated fatty acids at concentrations that lead to endoplasmic reticulum membrane phospholipid remodeling inhibits oxysterol activity. These studies implicate oxysterol-membrane interactions in acute regulation of sterol homeostatic responses and provide new insights into the mechanism through which oxysterols regulate cellular cholesterol balance.     

StarD10, a START domain protein overexpressed in breast cancer, functions as a phospholipid transfer protein

We originally identified StarD10 as a protein overexpressed in breast cancer that cooperates with the ErbB family of receptor tyrosine kinases in cellular transformation. StarD10 contains a steroidogenic acute regulatory protein (StAR/StarD1)-related lipid transfer (START) domain that is thought to mediate binding of lipids. We now provide evidence that StarD10 interacts with phosphatidylcholine (PC) and phosphatidylethanolamine (PE) by electron spin resonance measurement. Interaction with these phospholipids was verified in a fluorescence resonance energy transfer-based assay with 7-nitro-2,1,3-benzoxadiazol-4-yl-labeled lipids. Binding was not restricted to lipid analogs since StarD10 selectively extracted PC and PE from small unilamellar vesicles prepared with endogenous radiolabeled lipids from Vero monkey kidney cells. Mass spectrometry revealed that StarD10 preferentially selects lipid species containing a palmitoyl or stearoyl chain on the sn-1 and an unsaturated fatty acyl chain (18:1 or 18:2) on the sn-2 position. StarD10 was further shown to bind lipids in vivo by cross-linking of protein expressed in transfected HEK-293T cells with photoactivable phosphatidylcholine. In addition to a lipid binding function, StarD10 transferred PC and PE between membranes. Interestingly, these lipid binding and transfer specificities distinguish StarD10 from the related START domain proteins Pctp and CERT, suggesting a distinct biological function.     

Targeted mutation of the MLN64 START domain causes only modest alterations in cellular sterol metabolism

The StAR-related lipid transfer (START) domain, first identified in the steroidogenic acute regulatory protein (StAR), is involved in the intracellular trafficking of lipids. Sixteen mammalian START domain-containing proteins have been identified to date. StAR, a protein targeted to mitochondria, stimulates the movement of cholesterol from the outer to the inner mitochondrial membranes, where it is metabolized into pregnenolone in steroidogenic cells. MLN64, the START domain protein most closely related to StAR, is localized to late endosomes along with other proteins involved in sterol trafficking, including NPC1 and NPC2, where it has been postulated to participate in sterol distribution to intracellular membranes. To investigate the role of MLN64 in sterol metabolism, we created mice with a targeted mutation in the Mln64 START domain, expecting to find a phenotype similar to that in humans and mice lacking NPC1 or NPC2 (progressive neurodegenerative symptoms, free cholesterol accumulation in lysosomes). Unexpectedly, mice homozygous for the Mln64 mutant allele were viable, neurologically intact, and fertile. No significant alterations in plasma lipid levels, liver lipid content and distribution, and expression of genes involved in sterol metabolism were observed, except for an increase in sterol ester storage in mutant mice fed a high fat diet. Embryonic fibroblast cells transfected with the cholesterol side-chain cleavage system and primary cultures of granulosa cells from Mln64 mutant mice showed defects in sterol trafficking as reflected in reduced conversion of endogenous cholesterol to steroid hormones. These observations suggest that the Mln64 START domain is largely dispensable for sterol metabolism in mice.     

Oxysterol-binding protein and vesicle-associated membrane protein-associated protein are required for sterol-dependent activation of the ceramide transport protein

Sphingomyelin (SM) and cholesterol are coregulated metabolically and associate physically in membrane microdomains involved in cargo sorting and signaling. One mechanism for regulation of this metabolic interface involves oxysterol binding protein (OSBP) via high-affinity binding to oxysterol regulators of cholesterol homeostasis and activation of SM synthesis at the Golgi apparatus. Here, we show that OSBP regulation of SM synthesis involves the endoplasmic reticulum (ER)-to-Golgi ceramide transport protein (CERT). RNA interference (RNAi) experiments in Chinese hamster ovary (CHO)-K1 cells revealed that OSBP and vesicle-associated membrane protein-associated protein (VAP) were required for stimulation of CERT-dependent ceramide transport and SM synthesis by 25-hydroxycholesterol and cholesterol depletion in response to cyclodextrin. Additional RNAi experiments in human embryonic kidney 293 cells supported OSBP involvement in oxysterol-activated SM synthesis and also revealed a role for OSBP in basal SM synthesis. Activation of ER-to-Golgi ceramide transport in CHO-K1 cells required interaction of OSBP with the ER and Golgi apparatus, OSBP-dependent Golgi translocation of CERT, and enhanced CERT-VAP interaction. Regulation of CERT by OSBP, sterols, and VAP reveals a novel mechanism for integrating sterol regulatory signals with ceramide transport and SM synthesis in the Golgi apparatus.