Oxysterol-binding proteins: Sterol and phosphoinositide sensors coordinating transport,signaling and metabolism

Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) constitute a family of sterol and phosphoinositide binding proteins conserved in eukaryotes. The mechanisms of ORP function have remained incompletely understood. However, several ORPs are present at membrane contact sites and control the activity of enzymatic effectors or assembly of protein complexes, with impacts on signaling, vesicle transport, and lipid metabolism. An increasing number of protein interaction partners of ORPs have been identified, providing clues of their involvement in multiple aspects of cell regulation.The functions assigned for mammalian ORPs include coordination of sterol and sphingolipid metabolism and mitogenic signaling (OSBP), control of ER-late endosome (LE) contacts and LE motility (ORP1L), neutral lipid metabolism (ORP2), cell adhesion (ORP3), cholesterol eggress from LE (ORP5), macrophage lipid homeostasis, migration and high-density lipoprotein metabolism (ORP8), apolipoprotein B-100 secretion (ORP10), and adipogenesis (ORP11). The anti-proliferative ORPphilin compounds target OSBP and ORP4, revealing a function of ORPs in cell proliferation and survival. The Saccharomyces cerevisiae OSBP homologue (Osh) proteins execute multifaceted functions in sterol and sphingolipid homeostasis, post-Golgi vesicle transport, as well as phosphatidylinositol-4-phosphate and target of rapamycin complex 1 (TORC1) signaling. These observations identify ORPs as coordinators of lipid signals with an unforeseen variety of cellular processes.     

OSBP-related protein 8 (ORP8) interacts with Homo sapiens sperm associated antigen 5 (SPAG5) and mediates oxysterol interference of HepG2 cell cycle

We earlier identified OSBP-related protein 8 (ORP8) as an endoplasmic reticulum/nuclear envelope oxysterol-binding protein implicated in cellular lipid homeostasis, migration, and organization of the microtubule cytoskeleton. Here, a yeast two-hybrid screen identified Homo sapiens sperm associated antigen 5 (SPAG5)/Astrin as interaction partner of ORP8. The putative interaction was further confirmed by pull-down and co-immunoprecipitation assays. ORP8 did not colocalize with kinetochore-associated SPAG5 in mitotic HepG2 or HuH7 cells, but overexpressed ORP8 was capable of recruiting SPAG5 onto endoplasmic reticulum membranes in interphase cells. In our experiments, 25-hydroxycholesterol (25OHC) retarded the HepG2 cell cycle, causing accumulation in G2/M phase; ORP8 overexpression resulted in the same phenotype. Importantly, ORP8 knock-down dramatically inhibited the oxysterol effect on HepG2 cell cycle, suggesting a mediating role of ORP8. Furthermore, knock-down of SPAG5 significantly reduced the effects of both ORP8 overexpression and 25OHC on the cell cycle, placing SPAG5 downstream of the two cell-cycle interfering factors. Taken together, the present results suggest that ORP8 may via SPAG5 mediate oxysterol interference of the HepG2 cell cycle.     

Oxysterol binding protein-related protein 8 mediates the cytotoxicity of 25-hydroxycholesterol

Oxysterols are 27-carbon oxidized derivatives of cholesterol or by-products of cholesterol biosynthesis that can induce cell apoptosis in addition to a number of other bioactions. However, the mechanisms underlying this cytotoxicity are not completely understood. ORP8 is a member of the oxysterol binding protein-related protein (ORP) family, implicated in cellular lipid homeostasis, migration, and organization of the microtubule cytoskeleton. Here, we report that 25-hydroxycholesterol (OHC) induced apoptosis of the hepatoma cell lines, HepG2 and Huh7, via the endoplasmic reticulum (ER) stress response pathway, and ORP8 overexpression resulted in a similar cell response as 25-OHC, indicating a putative functional relationship between oxysterol cytotoxicity and ORP8. Further experiments demonstrated that ORP8 overexpression significantly enhanced the 25-OHC effect on ER stress and apoptosis in HepG2 cells. A truncated ORP8 construct lacking the ligand-binding domain or a closely related protein, ORP5, was devoid of this activity, evidencing for specificity of the observed effects. Importantly, ORP8 knockdown markedly dampened such responses to 25-OHC. Taken together, the present study suggests that ORP8 may mediate the cytotoxicity of 25-OHC.     

Sterol-phospholipid interactions in model membranes Effect of polar group substitutions in the cholesterol side-chain at C20 and C22

The interactions of phospholipids with four different cholesterol derivatives substituted with one OH or one keto group at position C₂₀ or C₂₂ of the side-chain were studied. The derivatives were the 22,R-hydroxy; 22,S-hydroxy; 22-keto- and 20,S-hydroxycholesterol. Two aspects of the interactions were investigated: (1) the effect of the cholesterol derivatives on the gel → liquid crystalline phase transition of dipalmitoylphosphatidylcholine (DPPC) and of dielaidoylphosphatidylethanolamine (DEPE) monitored by differential scanning calorimetry and (2) The effect on the lamellar → hexagonal H_II phase transition of DEPE monitored by DSC and by ³¹P-NMR to determine structural changes. The gel → liquid crystalline phase transition was affected by the cholesterol derivatives to a much larger extent in the case of DPPC than of DEPE. In both cases, there was a differential effect of the four derivatives, the 22,R-hydroxycholesterol being the less effective. In DPPC-sterol 1:1 systems, 22,R-hydroxycholesterol does not suppress the melting transition, the ΔH values becomes 7.1 kcal · mol^?1 as compared to 8.2 kcal · mol^?1 for the pure lipid. 22,S-OH cholesterol has a much stronger effect (ΔH = 3.1 kcal · mol^?1) and 22-ketocholesterol suppresses the transition completely. In DEPE mixtures of all these compounds, the melting transition of the phospholipid is still observable. The transition temperature was shifted to lower values (?13.5°C in the presence of 20,S-OH cholesterol). The ΔH of the transition was lowered by these compounds except in DEPE-22,R-OH cholesterol mixtures and the cooperativity of the transition (reflected by the width at half peak height) was reduced. The lamellar → hexagonal H_II phase transition was also affected by the presence of these cholesterol derivatives. The transition temperature value was depressed with all these compounds. 20,S-OH cholesterol was the most effective followed by 22,R-OH cholesterol. The ΔH of the transition was not strongly affected. The molecular interfacial properties of these derivatives were studied by the monomolecular film technique. It is most likely that 22,R-OH cholesterol due to the hydroxyl groups at the 3β- and 22,R-positions orients with the sterol nucleus lying flat at the air/water interface, since the compression isotherm of either the pure sterol or the DOPC-sterol mixture (molar ration, 1:1) monomolecular film exhibits a transition at approx. 103 Ų, corresponding to the area of revolution of the sterol nucleus. This remarkable property, due probably to the existence of a kink between the side-chain and the long axis of the steroid nucleus, might explain the smaller effect of this sterol on the melting transition of either PC or PE systems.     

Hepatic SR-BI, not endothelial lipase, expression determines biliary cholesterol secretion in mice

High density lipoprotein cholesterol is thought to represent a preferred source of sterols secreted into bile following hepatic uptake by scavenger receptor class B type I (SR-BI). The present study aimed to determine the metabolic effects of an endothelial lipase (EL)–mediated stimulation of HDL cholesterol uptake on liver lipid metabolism and biliary cholesterol secretion in wild-type, SR-BI knockout, and SR-BI overexpressing mice. In each model, injection of an EL expressing adenovirus decreased plasma HDL cholesterol (P < 0.001) whereas hepatic cholesterol content increased (P < 0.05), translating into decreased expression of sterol-regulatory element binding protein 2 (SREBP2) and its target genes HMG-CoA reductase and LDL receptor (each P < 0.01). Biliary cholesterol secretion was dependent on hepatic SR-BI expression, being decreased in SR-BI knockouts (P < 0.001) and increased following hepatic SR-BI overexpression (P < 0.001). However, in each model, biliary secretion of cholesterol, bile acids, and phospholipids as well as fecal bile acid and neutral sterol content, remained unchanged in response to EL overexpression. Importantly, hepatic ABCG5/G8 expression did not correlate with biliary cholesterol secretion rates under these conditions. These results demonstrate that an acute decrease of plasma HDL cholesterol levels by overexpressing EL increases hepatic cholesterol content but leaves biliary sterol secretion unaltered. Instead, biliary cholesterol secretion rates are related to the hepatic expression level of SR-BI. These data stress the importance of SR-BI for biliary cholesterol secretion and might have relevance for concepts of reverse cholesterol transport.     

Modulation of adrenal cell functions by cadmium salts: 2. Sites affected by CdCl2 during unstimulated steroid synthesis

In previous studies cadmium chloride (CdCl₂) nonlethally inhibited Y-1 adrenal mouse adrenal tumour cell 20-dihydroxyprogesterone (20DHP) secretion, affecting unstimulated and stimulated steroidogenic pathway sites differently. We studied CdCl₂ effects on unstimulated steroidogenesis using Y-1 cells incubated 0.5 h in medium with or without cadmium (using the concentration that inhibited ACTH-stimulated steroid secretion by 50%). Exogenously added 20-hydroxycholesterol (20OHC), 22(R)-hydroxycholesterol (22OHC), 25-hydroxycholesterol (25OHC), pregnenolone (PREG), or progesterone (PROG) were used to bypass any rate-limited steroidogenic pathway sites that CdCl₂ might inhibit. 25OHC is a biologically active nonpathway steroid, while 20OHC, 22OHC, PREG, and PROG are pathway steroids; each increased unstimulated 20DHP secretion nearly 10-fold. Although CdCl₂ could not reduce dibutyryl cyclic AMP- (dbcAMP)-stimulated 20DHP secretion significantly, it did significantly reduce basal and 25OHC-induced 20DHP secretion 25% below untreated levels. When 20OHC, 22OHC, PREG, or PROG were incubated with unstimulated Y-1 cells, their synthesis into 20DHP was unaffected by cadmium. dbcAMP bypasses the plasma membrane enzyme complex that synthesizes intracellular cAMP during exogenous ACTH stimulation; dbcAMP was not inhibited by CdCl₂. The rate-limited step accelerated by cAMP involves plasma membrane and/or cytoplasmic cholesterol transport to and through outer and inner mitochondrial membranes before the cholesterol is synthesized into pregnenolone by side-chain cleavage enzymes on the inner membrane matrix face. Little is known regarding the mechanisms controlling unstimulated steroidogenesis. Under unstimulated conditions the 25-, 20- and 22(R)-monohydroxyls of cholesterol facilitate plasma membrane, cytoplasm and inner and outer mitochondrial solubility, diffusion and/or transport to bypass rate-limited steps and augment unstimulated steroid synthesis. Since conversion of endogenous mitochondrial cholesterol and 25OHC, but not dbcAMP-mobilized cytoplasmic cholesterol, 20OHC or 22OHC conversion, to 20DHP is inhibited by CdCl₂, this suggests that (a) control of mitochondrial cholesterol supplies is independent of the cAMP-regulated mitochondrial steps in the 20DHP steroid synthetic pathway, (b) CdCl₂ specifically inhibited endogenous mitochondrial cholesterol and 25OHC utilization, (c) CdCl₂ toxicity may affect adrenal, testicular, ovarian, and placental basal steroidogenic functions, and (d) 25OHC may be a useful compound to examine unstimulated steroid synthesisAbbreviations ACTH adrenocorticotropin - ANOVA analysis of variance - CdCl₂ cadmium chloride - cAMP cyclic 3,5-adenosine monophosphate - DMSO dimethylsulfoxide - DNA deoxyribonucleic acid - FMEM serum-free Eagle's Minimum Essential Medium - Hepes N-2-hydroxyethyl-piperazine-N-1,2-ethanesulfonic acid - 20OHC 20-hydroxycholesterol - 22OHC 22(R)-hydroxycholesterol - 25OHC 25-hydroxycholesterol - IC_50' concentration inhibiting stimulated steroid secretion by 50% - IU international unit - MEM Eagle's Minimum Essential Medium - P450scc cytochrome P450 side-chain cleavage enzyme - PREG pregnenolone - PROG progesterone - RNA ribonucleic acid - SEM standard error of the mean - SMEM serum-containing Eagle's Minimum Essential Medium - 20DHP 20-hydroxy-4-pregnen-3-one     

Implications of cerebrovascular ATP-binding cassette transporter G1 (ABCG1) and apolipoprotein M in cholesterol transport at the blood-brain barrier

Impaired cholesterol/lipoprotein metabolism is linked to neurodegenerative diseases such as Alzheimer's disease (AD). Cerebral cholesterol homeostasis is maintained by the highly efficient blood-brain barrier (BBB) and flux of the oxysterols 24(S)-hydroxycholesterol and 27-hydroxycholesterol, potent liver-X-receptor (LXR) activators. HDL and their apolipoproteins are crucial for cerebral lipid transfer, and loss of ATP binding cassette transporters (ABC)G1 and G4 results in toxic accumulation of oxysterols in the brain. The HDL-associated apolipoprotein (apo)M is positively correlated with pre-β HDL formation in plasma; its presence and function in the brain was thus far unknown. Using an in vitro model of the BBB, we examined expression, regulation, and functions of ABCG1, ABCG4, and apoM in primary porcine brain capillary endothelial cells (pBCEC). RT Q-PCR analyses and immunoblotting revealed that in addition to ABCA1 and scavenger receptor, class B, type I (SR-BI), pBCEC express high levels of ABCG1, which was up-regulated by LXR activation. Immunofluorescent staining, site-specific biotinylation and immunoprecipitation revealed that ABCG1 is localized both to early and late endosomes and on apical and basolateral plasma membranes. Using siRNA interference to silence ABCG1 (by 50%) reduced HDL-mediated [³H]-cholesterol efflux (by 50%) but did not reduce [³H]-24(S)-hydroxycholesterol efflux. In addition to apoA-I, pBCEC express and secrete apoM mainly to the basolateral (brain) compartment. HDL enhanced expression and secretion of apoM by pBCEC, apoM-enriched HDL promoted cellular cholesterol efflux more efficiently than apoM-free HDL, while apoM-silencing diminished cellular cholesterol release. We suggest that ABCG1 and apoM are centrally involved in regulation of cholesterol metabolism/turnover at the BBB.     

Bis(monoacylglycero)phosphate regulates oxysterol binding protein-related protein 11 dependent sterol trafficking

Bis(Monoacylglycero) Phosphate (BMP) is a unique phospholipid localized in late endosomes, a critical cellular compartment in low density lipoprotein (LDL)-cholesterol metabolism. In previous work, we demonstrated the important role of BMP in the regulation of macrophage cholesterol homeostasis. BMP exerts a protective role against the pro-apoptotic effect of oxidized LDL (oxLDL) by reducing the production of deleterious oxysterols. As the intracellular sterol traffic in macrophages is in part regulated by oxysterol binding protein (OSBP) and OSBP-related proteins (ORPs), we investigated the role of ORP11, localized at the Golgi-late endosomes interface, in the BMP-mediated protection from oxLDL/oxysterol cytotoxicity. Stably silencing of ORP11 in mouse RAW264.7 macrophages via a shRNA lentiviruses system had no effect on BMP production. However, ORP11 knockdown abrogated the protective action of BMP against oxLDL induced apoptosis. In oxLDL treated control cells, BMP enrichment was associated with reduced generation of 7-oxysterols, while these oxysterol species were abundant in the ORP11 knock-down cells. Of note, BMP enrichment in ORP11 knock-down cells was associated with a drastic increase in free cholesterol and linked to a decrease of cholesterol efflux. The expression of ATP-binding cassette-transporter G1 (ABCG1) was also reduced in the ORP11 knock-down cells. These observations demonstrate a cooperative function of OPR11 and BMP, in intracellular cholesterol trafficking in cultured macrophages. We suggest that BMP favors the egress of cholesterol from late endosomes via an ORP11-dependent mechanism, resulting in a reduced production of cytotoxic 7-oxysterols.     

A reappraisal of the mechanism by which plant sterols promote neutral sterol loss in mice

Dietary plant sterols (PS) reduce serum total and LDL-cholesterol in hyperlipidemic animal models and in humans. This hypocholesterolemic effect is generally ascribed to inhibition of cholesterol absorption. However, whether this effect fully explains the reported strong induction of neutral sterol excretion upon plant sterol feeding is not known. Recent data demonstrate that the intestine directly mediates plasma cholesterol excretion into feces, i.e., without involvement of the hepato-biliary route.

Objective

Aim of this study was to determine whether stimulation of fecal neutral sterol loss during PS feeding is (partly) explained by increased intestinal cholesterol excretion and to assess the role of the cholesterol transporter Abcg5/Abcg8 herein.

Methods and Results

Wild-type mice were fed a control diet or diets enriched with increasing amounts of PS (1%, 2%, 4% or 8%, wt/wt) for two weeks. In addition, Abcg5^-/- mice were fed either control or 8% PS diet. PS feeding resulted in a dose-dependent decrease of fractional cholesterol absorption (∼2–7-fold reduction) in wild-type mice and ∼80% reduction in Abcg5^-/- mice. Furthermore, PS feeding led to a strong, dose-independent induction of neutral sterol excretion (3.4-fold in wild-types and 2.7-fold in Abcg5^-/- mice) without changes in biliary cholesterol secretion. It was calculated that PS feeding stimulated intestinal cholesterol excretion by ∼500% in wild-type mice and by ∼250% in Abcg5^-/-.

Conclusions

Our data indicate that in mice the cholesterol-lowering effects of PS are to a large extent attributable to stimulation of intestinal, non-bile derived, cholesterol excretion. The Abcg5/Abcg8 heterodimer is involved in facilitating this PS-induced flux of cholesterol.     

22(R)-hydroxycholesterol and pioglitazone synergistically decrease cholesterol ester via the PPARγ–LXRα–ABCA1 pathway in cholesterosis of the gallbladder

Cholesterosis is a disease of cholesterol metabolism characterized by the presence of excessive lipid droplets in the cytoplasm. These lipid droplets are mainly composed of cholesterol esters derived from free cholesterol. The removal of excess cholesterol from gallbladder epithelial cells (GBECs) is very important for the maintenance of intracellular cholesterol homeostasis and the preservation of gallbladder function. Several lines of evidence have indicated that the activation of either peroxisome proliferator-activated receptor gamma (PPARγ) or liver X receptor α (LXRα) relates to cholesterol efflux. While pioglitazone can regulate the activation of PPARγ, 22(R)-hydroxycholesterol can activate LXRα and is a metabolic intermediate in the biosynthesis of steroid hormones. However, the effect of 22(R)-hydroxycholesterol in combination with pioglitazone on cholesterosis of the gallbladder is unclear. GBECs were treated with pioglitazone, 22(R)-hydroxycholesterol or PPARγ siRNA followed by Western blot analysis for ATP-binding cassette transporter A1 (ABCA1), PPARγ and LXRα. Cholesterol efflux to apoA-I was determined, and Oil Red O staining was performed to monitor variations in lipid levels in treated GBECs. Our data showed that 22(R)-hydroxycholesterol can modestly up-regulate LXRα while simultaneously increasing ABCA1 by 56%. The combination of 22(R)-hydroxycholesterol and pioglitazone resulted in a 3.64-fold increase in ABCA1 expression and a high rate of cholesterol efflux. Oil Red O staining showed an obvious reduction in the lipid droplets associated with cholesterosis in GBECs. In conclusion, the present findings indicate that the anti-lipid deposition action of 22(R)-hydroxycholesterol combined with pioglitazone involves the activation of the PPARγ–LXRα–ABCA1 pathway, increased ABCA1 expression and the efflux of cholesterol from GBECs. Thus, 22(R)-hydroxycholesterol synergistically combined with pioglitazone to produce a remarkable effect on lipid deposition in cholesterosis GBECs.     

Oxysterol-binding Protein (OSBP)-related Protein 4 (ORP4) Is Essential for Cell Proliferation and Survival

Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) comprise a large gene family with sterol/lipid transport and regulatory activities. ORP4 (OSBP2) is a closely related paralogue of OSBP, but its function is unknown. Here we show that ORP4 binds similar sterol and lipid ligands as OSBP and other ORPs but is uniquely required for the proliferation and survival of cultured cells. Recombinant ORP4L and a variant without a pleckstrin homology (PH) domain (ORP4S) bind 25-hydroxycholesterol and extract and transfer cholesterol between liposomes. Two conserved histidine residues in the OSBP homology domain ORP4 are essential for binding phosphatidylinositol 4-phosphate but not sterols. The PH domain of ORP4L also binds phosphatidylinositol 4-phosphate in the Golgi apparatus. However, in the context of ORP4L, the PH domain is required for normal organization of the vimentin network. Unlike OSBP, RNAi silencing of all ORP4 variants (including a partial PH domain truncation termed ORP4M) in HEK293 and HeLa cells resulted in growth arrest but not cell death. ORP4 silencing in non-transformed intestinal epithelial cells (IEC)-18 caused apoptosis characterized by caspase 3 and poly(ADP-ribose) polymerase processing, DNA cleavage, and JNK phosphorylation. IEC-18 transformed with oncogenic H-Ras have increased expression of ORP4L and ORP4S proteins and are resistant to the growth-inhibitory effects of ORP4 silencing. Results suggest that ORP4 promotes the survival of rapidly proliferating cells.     

Characterization of the Sterol and Phosphatidylinositol 4-Phosphate Binding Properties of Golgi-Associated OSBP-Related Protein 9 (ORP9)

Oxysterol binding protein (OSBP) and OSBP-related proteins (ORPS) have a conserved lipid-binding fold that accommodates cholesterol, oxysterols and/or phospholipids. The diversity of OSBP/ORPs and their potential ligands has complicated the analysis of transfer and signalling properties of this mammalian gene family. In this study we explored the use of the fluorescent sterol cholestatrienol (CTL) to measure sterol binding by ORP9 and competition by other putative ligands. Relative to cholesterol, CTL and dehydroergosterol (DHE) were poor ligands for OSBP. In contrast, both long (ORP9L) and short (ORP9S) variants of ORP9 rapidly extracted CTL, and to a lesser extent DHE, from liposomes. ORP9L and ORP9S also extracted [³²P]phosphatidylinositol 4-phosphate (PI-4P) from liposomes, which was inhibited by mutating two conserved histidine residues (HH_488,489AA) at the entrance to the binding pocket but not by a mutation in the lid region that inhibited cholesterol binding. Results of direct binding and competition assays showed that phosphatidylserine was poorly extracted from liposomes by ORP9 compared to CTL and PI-4P. ORP9L and PI-4P did not co-localize in the trans-Golgi/TGN of HeLa cells, and siRNA silencing of ORP9L expression did not affect PI-4P distribution in the Golgi apparatus. However, transient overexpression of ORP9L or ORP9S in CHO cells, but not the corresponding PI-4P binding mutants, prevented immunostaining of Golgi-associated PI-4P. The apparent sequestration of Golgi PI-4P by ORP9S was identified as a possible mechanism for its growth inhibitory effects. These studies identify ORP9 as a dual sterol/PI-4P binding protein that could regulate PI-4P in the Golgi apparatus.     

Tracking the Subcellular Fate of 20(S)-Hydroxycholesterol with Click Chemistry Reveals a Transport Pathway to the Golgi

Oxysterols, oxidized metabolites of cholesterol, are endogenous small molecules that regulate lipid metabolism, immune function, and developmental signaling. Although the cell biology of cholesterol has been intensively studied, fundamental questions about oxysterols, such as their subcellular distribution and trafficking pathways, remain unanswered. We have therefore developed a useful method to image intracellular 20(S)-hydroxycholesterol with both high sensitivity and spatial resolution using click chemistry and fluorescence microscopy. The metabolic labeling of cells with an alkynyl derivative of 20(S)-hydroxycholesterol has allowed us to directly visualize this oxysterol by attaching an azide fluorophore through cyclo-addition. Unexpectedly, we found that this oxysterol selectively accumulates in the Golgi membrane using a pathway that is sensitive to ATP levels, temperature, and lysosome function. Although previous models have proposed nonvesicular pathways for the rapid equilibration of oxysterols between membranes, direct imaging of oxysterols suggests that a vesicular pathway is responsible for differential accumulation of oxysterols in organelle membranes. More broadly, clickable alkynyl sterols may represent useful tools for sterol cell biology, both to investigate the functions of these important lipids and to decipher the pathways that determine their cellular itineraries.     

Overexpression of sterol carrier protein-2 differentially alters hepatic cholesterol accumulation in cholesterol-fed mice

Although in vitro studies suggest a role for sterol carrier protein-2 (SCP-2) in cholesterol trafficking and metabolism, the physiological significance of these observations remains unclear. This issue was addressed by examining the response of mice overexpressing physiologically relevant levels of SCP-2 to a cholesterol-rich diet. While neither SCP-2 overexpression nor cholesterol-rich diet altered food consumption, increased weight gain, hepatic lipid, and bile acid accumulation were observed in wild-type mice fed the cholesterol-rich diet. SCP-2 overexpression further exacerbated hepatic lipid accumulation in cholesterol-fed females (cholesterol/cholesteryl esters) and males (cholesterol/cholesteryl esters and triacyglycerol). Primarily in female mice, hepatic cholesterol accumulation induced by SCP-2 overexpression was associated with increased levels of LDL-receptor, HDL-receptor scavenger receptor-B1 (SR-B1) (as well as PDZK1 and/or membrane-associated protein 17 kDa), SCP-2, liver fatty acid binding protein (L-FABP), and 3α-hydroxysteroid dehydrogenase, without alteration of other proteins involved in cholesterol uptake (caveolin), esterification (ACAT2), efflux (ATP binding cassette A-1 receptor, ABCG5/8, and apolipoprotein A1), or oxidation/transport of bile salts (cholesterol 7α-hydroxylase, sterol 27α-hydroxylase, Na⁺/taurocholate cotransporter, Oatp1a1, and Oatp1a4). The effects of SCP-2 overexpression and cholesterol-rich diet was downregulation of proteins involved in cholesterol transport (L-FABP and SR-B1), cholesterol synthesis (related to sterol regulatory element binding protein 2 and HMG-CoA reductase), and bile acid oxidation/transport (via Oapt1a1, Oatp1a4, and SCP-x). Levels of serum and hepatic bile acids were decreased in cholesterol-fed SCP-2 overexpression mice, especially in females, while the total bile acid pool was minimally affected. Taken together, these findings support an important role for SCP-2 in hepatic cholesterol homeostasis.     

Growth Retardation, Impaired Triacylglycerol Catabolism, Hepatic Steatosis, and Lethal Skin Barrier Defect in Mice Lacking Comparative Gene Identification-58 (CGI-58)

Comparative gene identification-58 (CGI-58), also designated as α/β-hydrolase domain containing-5 (ABHD-5), is a lipid droplet-associated protein that activates adipose triglyceride lipase (ATGL) and acylates lysophosphatidic acid. Activation of ATGL initiates the hydrolytic catabolism of cellular triacylglycerol (TG) stores to glycerol and nonesterified fatty acids. Mutations in both ATGL and CGI-58 cause “neutral lipid storage disease” characterized by massive accumulation of TG in various tissues. The analysis of CGI-58-deficient (Cgi-58^−/−) mice, presented in this study, reveals a dual function of CGI-58 in lipid metabolism. First, systemic TG accumulation and severe hepatic steatosis in newborn Cgi-58^−/− mice establish a limiting role for CGI-58 in ATGL-mediated TG hydrolysis and supply of nonesterified fatty acids as energy substrate. Second, a severe skin permeability barrier defect uncovers an essential ATGL-independent role of CGI-58 in skin lipid metabolism. The neonatal lethal skin barrier defect is linked to an impaired hydrolysis of epidermal TG. As a consequence, sequestration of fatty acids in TG prevents the synthesis of acylceramides, which are essential lipid precursors for the formation of a functional skin permeability barrier. This mechanism may also underlie the pathogenesis of ichthyosis in neutral lipid storage disease patients lacking functional CGI-58.     

Novel members of the human oxysterol-binding protein family bind phospholipids and regulate vesicle transport

Oxysterol-binding proteins (OSBPs) are a family of eukaryotic intracellular lipid receptors. Mammalian OSBP1 binds oxygenated derivatives of cholesterol and mediates sterol and phospholipid synthesis through as yet poorly undefined mechanisms. The precise cellular roles for the remaining members of the oxysterol-binding protein family remain to be elucidated. In yeast, a family of OSBPs has been identified based on primary sequence similarity to the ligand binding domain of mammalian OSBP1. Yeast Kes1p, an oxysterol-binding protein family member that consists of only the ligand binding domain, has been demonstrated to regulate the Sec14p pathway for Golgi-derived vesicle transport. Specifically, inactivation of the KES1 gene resulted in the ability of yeast to survive in the absence of Sec14p, a phosphatidylinositol/phosphatidylcholine transfer protein that is normally required for cell viability due to its essential requirement in transporting vesicles from the Golgi. We cloned the two human members of the OSBP family, ORP1 and ORP2, with the highest degree of similarity to yeast Kes1p. We expressed ORP1 and ORP2 in yeast lacking Sec14p and Kes1p function and found that ORP1 complemented Kes1p function with respect to cell growth and Golgi vesicle transport, whereas ORP2 was unable to do so. Phenotypes associated with overexpression of ORP2 in yeast were a dramatic decrease in cell growth and a block in Golgi-derived vesicle transport distinct from that of ORP1. Purification of ORP1 and ORP2 for ligand binding studies demonstrated ORP1 and ORP2 did not bind 25-hydroxycholesterol but instead bound phospholipids with both proteins exhibiting strong binding to phosphatidic acid and weak binding to phosphatidylinositol 3-phosphate. In Chinese hamster ovary cells, ORP1 localized to a cytosolic location, whereas ORP2 was associated with the Golgi apparatus, consistent with our vesicle transport studies that indicated ORP1 and ORP2 function at different steps in the regulation of vesicle transport.     

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.     

27-hydroxycholesterol is an endogenous ligand for liver X receptor in cholesterol-loaded cells

The nuclear receptors liver X receptor alpha (LXRalpha) (NR1H3) and LXRbeta (NR1H2) are important regulators of genes involved in lipid metabolism, including ABCA1, ABCG1, and sterol regulatory element-binding protein-1c (SREBP-1c). Although it has been demonstrated that oxysterols are LXR ligands, little is known about the identity of the physiological activators of these receptors. Here we confirm earlier studies demonstrating a dose-dependent induction of ABCA1 and ABCG1 in human monocyte-derived macrophages by cholesterol loading. In addition, we show that formation of 27-hydroxycholesterol and cholestenoic acid, products of CYP27 action on cholesterol, is dependent on the dose of cholesterol used to load the cells. Other proposed LXR ligands, including 20(S)-hydroxycholesterol, 22(R)-hydroxycholesterol, and 24(S),25-epoxycholesterol, could not be detected under these conditions. A role for CYP27 in regulation of cholesterol-induced genes was demonstrated by the following findings. 1) Introduction of CYP27 into HEK-293 cells conferred an induction of ABCG1 and SREBP-1c; 2) upon cholesterol loading, CYP27-expressing cells induce these genes to a greater extent than in control cells; 3) in CYP27-deficient human skin fibroblasts, the induction of ABCA1 in response to cholesterol loading was ablated; and 4) in a coactivator association assay, 27-hydroxycholesterol functionally activated LXR. We conclude that 27-hydroxylation of cholesterol is an important pathway for LXR activation in response to cholesterol overload.