Aster Proteins Facilitate Nonvesicular Plasma Membrane to ER Cholesterol Transport in Mammalian Cells.

The mechanisms underlying sterol transport in mammalian cells are poorly understood. In particular, how cholesterol internalized from HDL is made available to the cell for storage or modification is unknown. Here, we describe three ER-resident proteins (Aster-A, -B, -C) that bind cholesterol and facilitate its removal from the plasma membrane. The crystal structure of the central domain of Aster-A broadly resembles the sterol-binding fold of mammalian StARD proteins, but sequence differences in the Aster pocket result in a distinct mode of ligand binding. The Aster N-terminal GRAM domain binds phosphatidylserine and mediates Aster recruitment to plasma membrane-ER contact sites in response to cholesterol accumulation in the plasma membrane. Mice lacking Aster-B are deficient in adrenal cholesterol ester storage and steroidogenesis because of an inability to transport cholesterol from SR-BI to the ER. These findings identify a nonvesicular pathway for plasma membrane to ER sterol trafficking in mammals.

Scavenger Receptor Class B Type I Modulates Epileptic Seizures and Receptor α2δ-1 Expression.

Scavenger receptor class B type I (SR-BI) is abundant in adult mouse and human brains, but its function in the central nervous system (CNS) remains unclear. This study explored the role of SR-BI in epilepsy and its possible underlying mechanism. Expression patterns of SR-BI in the brains of mice with kainic acid (KA)-induced epilepsy were detected using immunofluorescence staining, quantitative real-time polymerase chain reaction (qPCR), and Western blotting(WB). Behavioral analysis was performed by 24-hour video monitoring and hippocampal local field potential (LFP) recordings were employed to verify the role of SR-BI in epileptogenesis. RNA sequencing (RNA-seq) was used to obtain biological information on SR-BI in the CNS. WB, qPCR, and co-immunoprecipitation (Co-IP) were performed to identify the relationship between SR-BI and the gabapentin receptor α2δ-1.The results showed that SR-BI was primarily co-localized with astrocytes and its expression was down-regulated in the hippocampus of KA mice. Notably, overexpressing SR-BI alleviated the epileptic behavioral phenotype in KA mice. Hippocampal transcriptomic analysis revealed 1043 differentially expressed genes (DEGs) in the SR-BI-overexpressing group. Most DEGs confirmed by RNA-seq analysis were associated with synapses, neuronal projections, neuron development, and ion binding. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis demonstrated that the DEGs were enriched in the glutamatergic synapse pathway. Furthermore, the gabapentin receptor α2δ-1 decreased with SR-BI overexpression in epileptic mice. Overall, these findings highlight the important role of SR-BI in regulating epileptogenesis and that the gabapentin receptor α2δ-1 is a potential downstream target of SR-BI.

Is reverse cholesterol transport regulated by active cholesterol?

This review considers the hypothesis that a small portion of plasma membrane cholesterol regulates reverse cholesterol transport in coordination with overall cellular homeostasis. It appears that almost all of the plasma membrane cholesterol is held in stoichiometric complexes with bilayer phospholipids. The minor fraction of cholesterol that exceeds the complexation capacity of the phospholipids is called active cholesterol. It has an elevated chemical activity and circulates among the organelles. It also moves down its chemical activity gradient to plasma HDL, facilitated by the activity of ABCA1, ABCG1, and SR-BI. ABCA1 initiates this process by perturbing the organization of the plasma membrane bilayer, thereby priming its phospholipids for translocation to apoA-I to form nascent HDL. The active excess sterol and that activated by ABCA1 itself follow the phospholipids to the nascent HDL. ABCG1 similarly rearranges the bilayer and sends additional active cholesterol to nascent HDL, while SR-BI simply facilitates the equilibration of the active sterol between plasma membranes and plasma proteins. Active cholesterol also flows downhill to cytoplasmic membranes where it serves both as a feedback signal to homeostatic ER proteins and as the substrate for the synthesis of mitochondrial 27-hydroxycholesterol (27HC). 27HC binds the LXR and promotes the expression of the aforementioned transport proteins. 27HC-LXR also activates ABCA1 by competitively displacing its inhibitor, unliganded LXR. § Considerable indirect evidence suggests that active cholesterol serves as both a substrate and a feedback signal for reverse cholesterol transport. Direct tests of this novel hypothesis are proposed.

Scavenger receptor class B type I genetic variants associated with disease severity in chronic hepatitis C virus infection.

Analysis of host genetic polymorphisms is an increasingly important tool for understanding and predicting pathogenesis and treatment response of viral diseases. The gene locus of scavenger receptor class B type I (SR-BI), encoding a cell entry factor and receptor for hepatitis C virus (HCV), contains several genetic polymorphisms. We applied a probe extension assay to determine the frequency of six single nucleotide polymorphisms (SNPs) within the SR-BI gene locus in 374 individuals with history of HCV infection. In addition, SR-BI messenger RNA (mRNA) levels were analyzed in liver biopsy specimens of chronically infected HCV subjects. The rs5888 variant allele T was present at a higher frequency in subjects with advanced fibrosis (χ2 , p = 0.016) and after adjusting for age, duration of infection and alcohol intake as confounding factors. Haplotype analysis of SNP frequencies showed that a haplotype consisting of rs61932577 variant allele C and rs5888 variant allele T was associated with an increased risk of advanced liver fibrosis (defined by an Ishak score 4-6) (adjusted odds ratio 2.81; 95% confidence interval 1.06-7.46. p = 0.038). Carriers of the rs5888 variant allele T displayed reduced SR-BI mRNA expression in liver biopsy specimens. In conclusion the rs5888 polymorphism variant is associated with decreased SR-BI expression and an increased risk of development of advanced fibrosis in chronic HCV infection. These findings provide further evidence for a role of SR-BI in HCV pathogenesis and provides a genetic marker for prediction of those infected individuals at greater risk of developing severe disease.

Scavenger receptor BI attenuates oxidized phospholipid-induced pulmonary inflammation.

Damage associated molecular patterns (DAMPs) are molecules released from dead/dying cells following toxicant and/or environmental exposures that activate the immune response through binding of pattern recognition receptors (PRRs). Excessive production of DAMPs or failed clearance leads to chronic inflammation and delayed inflammation resolution. One category of DAMPs are oxidized phospholipids (oxPLs) produced upon exposure to high levels of oxidative stress, such as following ozone (O3) induced inflammation. OxPLs are bound by multiple classes of PRRs that include scavenger receptors (SRs) such as SR class B-1 (SR-BI) and toll-like receptors (TLRs). Interactions between oxPLs and PRRs appear to regulate inflammation; however, the role of SR-BI in oxPL-induced lung inflammation has not been defined. Therefore, we hypothesize that SR-BI is critical in protecting the lung from oxPL-induced pulmonary inflammation/injury. To test this hypothesis, C57BL/6J (WT) female mice were dosed with oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine (oxPAPC) by oropharyngeal aspiration which increased pulmonary SR-BI expression. Following oxPAPC exposure, SR-BI deficient (SR-BI-/-) mice exhibited increased lung pathology and inflammatory cytokine/chemokine production. Lipidomic analysis revealed that SR-BI-/- mice had an altered pulmonary lipidome prior to and following oxPAPC exposure, which correlated with increased oxidized phosphatidylcholines (PCs). Finally, we characterized TLR4-mediated activation of NF-κB following oxPAPC exposure and discovered that SR-BI-/- mice had increased TLR4 mRNA expression in lung tissue and macrophages, increased nuclear p65, and decreased cytoplasmic IκBα. Overall, we conclude that SR-BI is required for limiting oxPAPC-induced lung pathology by maintaining lipid homeostasis, reducing oxidized PCs, and attenuating TLR4-NF-κB activation, thereby preventing excessive and persistent inflammation.

Mechanism for the selective uptake of macular carotenoids mediated by the HDL cholesterol receptor SR-BI.

The macular carotenoids lutein and zeaxanthin are taken up from the bloodstream into the human retina through a selective process, for which the HDL cholesterol receptor scavenger receptor BI (SR-BI) in the cells of retinal pigment epithelium (RPE) is thought to be a key mediator. However, the mechanism of SR-BI-mediated selective uptake of macular carotenoids is still not fully understood. Here, we investigate possible mechanisms using biological assays and cultured HEK293 cells, a cell line without endogenous SR-BI expression. Binding affinities between SR-BI and various carotenoids were measured by surface plasmon resonance (SPR) spectroscopy, which shows that SR-BI cannot bind lutein or zeaxanthin specifically. Overexpression of SR-BI in HEK293 cells results in more lutein and zeaxanthin taken up than ß-carotene, and this effect can be eliminated by an SR-BI mutant (C384Y) whose cholesterol uptake tunnel is blocked. Next, we determined the effects of HDL and hepatic lipase (LIPC), SR-BI's partners in HDL cholesterol transport, on SR-BI-mediated carotenoid uptake. HDL addition dramatically reduced lutein, zeaxanthin, and ß-carotene in HEK293 cells expressing SR-BI, but the cellular lutein and zeaxanthin are higher than ß-carotene. LIPC addition increases the uptake of all three carotenoids in HDL-treated cells, and promotes the transport of lutein and zeaxanthin better than ß-carotene. Our results suggest that SR-BI and its HDL cholesterol partner HDL and LIPC may be involved in the selective uptake of macular carotenoids.

LDL Transcytosis by the Arterial Endothelium-Atherosclerosis by a Thousand Cuts?

PURPOSE OF REVIEW: The accumulation of LDL in the arterial intima is an initiating event in atherosclerosis. After decades of controversy, it is now clear that transcytosis of LDL across an intact endothelial monolayer contributes to its intimal deposition. We review recent observations in this field and address the question of whether LDL transcytosis can be manipulated therapeutically. RECENT FINDINGS: The development of a live-cell imaging method for studying transcytosis using total internal reflection fluorescence (TIRF) microscopy has catalyzed recent discoveries. LDL transcytosis is mediated by SR-BI and ALK1. Estrogen down-regulates SR-BI and inhibits LDL transcytosis, while the nuclear structural protein HMGB1 promotes LDL transcytosis. LDL transcytosis by ALK1 is independent of the receptor's kinase activity and is antagonized by BMP9, ALK1's canonical ligand. Inflammation stimulates LDL transcytosis. Identifying the function and mechanisms of LDL transcytosis may ultimately permit its therapeutic manipulation.

Exendin-4 Increases Scavenger Receptor Class BI Expression via Activation of AMPK/FoxO1 in Human Vascular Endothelial Cells.

Glucagon-like peptide-1 receptor agonist (GLP-1RA) has been clinically proven to protect endothelial function. Previously, we demonstrated that endothelial NO synthase (eNOS) was activated by high-density lipoprotein (HDL) via its scavenger receptor of the B class/human homologue of SR-BI, CD36 and LIMPII analogous-1(hSR-BI/CLA-1). Here, we investigated the effect of GLP-1RA and exendin-4 on the expression of hSR-BI/CLA-1 in HUVECs. Our results confirmed that GLP-1R was expressed in HUVECs by PCR and exendin-4 significantly enhanced HDL-induced eNOS activation. Next, exendin-4 increased the expression of hSR-BI/CLA-1 and a blockade of GLP-1R cancelled this effect. Further, the hSR-BI/CLA-1 transcriptional activity was enhanced by exendin-4, which was diminished by the inhibition of AMPK or dominant-negative AMPK-α-subunit. Moreover, AMPK was phosphorylated by the activation of GLP-1R. Next, ChIP assay demonstrated that exendin-4 increased the FoxO1-binding in the hSR-BI/CLA-1 promoter by upregulation of FoxO1. Mutation of FoxO1-binding or silencing of FoxO1 cancelled the effect of exendin-4 on hSR-BI/CLA-1 expression. Exendin-4 reduced FoxO1 phosphorylation and induced its nuclear accumulation, while this effect was altered by the blocking of GLP-1R or inhibition of AMPK pathway. In summary, our results proved that exendin-4 increased hSR-BI/CLA-1 expression via the AMPK/FoxO1 pathway to activate eNOS, providing a basic mechanism underlining the protective effect of GLP-1RA on endothelial function.

Blocking cholesterol efflux mechanism is a potential target for antilymphoma therapy.

Cholesterol is an essential plasma membrane lipid for the maintenance of cellular homeostasis and cancer cell proliferation. Free cholesterol is harmful to cells; therefore, excessive free cholesterol must be quickly esterified by acetyl-coenzyme A:cholesterol acetyltransferase (ACAT) and exported by scavenger receptor class B member I (SR-BI) or ATP-binding cassette protein A1 from specific cells such as macrophage foam cells, which contain cholesteryl ester-derived vacuoles. Many vacuoles are present in the cytoplasm of Burkitt lymphoma cells. In this study, we observed that these vacuoles are often seen in high-grade lymphomas. Cell culture study using lymphoma cell lines found that esterified cholesterol is the main component of these vacuoles and the expression of cholesterol metabolism-related molecules was significantly upregulated in lymphoma cell lines, with SR-BI and ACAT inhibitors (BLT-1 and CI-976, respectively) impeding lymphoma cell proliferation. Cytoplasmic free cholesterol was increased by ACAT and SR-BI inhibitors, and the accumulation of free cholesterol induced lymphoma cell apoptosis by inducing endoplasmic reticulum stress. Furthermore, synergistic effects of SR-BI and ACAT inhibitors were observed in a preclinical study. Treatment with SR-BI inhibitor suppressed lymphoma progression in a tumor-bearing mouse model, whereas ACAT inhibitor did not. Therefore, SR-BI inhibitors are potential new antilymphoma therapeutics that target cholesterol metabolism.

HDL and Scavenger Receptor Class B Type I (SRBI).

The scavenger receptor class B type I (SR-BI) is a versatile HDL receptor protein. It is highly expressed in liver and steroidogenic tissues. SR-BI regulates selective uptake of cholesterol ester (CE) from HDL, revealing its role in mediating reverse cholesterol transport (RCT) and steroid hormone synthesis. In addition, SR-BI is involved in cholesterol transport, cellular inflammatory response, platelet reactivity, and HDL-initiated signaling in the vascular system in several mouse models. Mutations in the human SR-BI gene (SCARB1) have been found to be associated with abnormally high plasma HDL-C levels and an increased risk of atherosclerotic cardiovascular disease. At present, the key regions of SR-BI transmembrane structure and the regulatory mechanisms of SR-BI expression still need to be further studied. In this chapter, the structural, functional, and regulatory characteristics of SR-BI are reviewed, and the importance of SR-BI in related metabolic diseases was expounded.

Xestospongia muta Fraction-7 and Linoleic Acid: Effects on SR-BI Gene Expression and HDL Cholesterol Uptake.

Xestospongia muta is a marine sponge belonging to the family Petrosiidae. It is an important source of biologically active marine natural products, with different kinds of essential fatty acids. Scavenger receptor class B type I (SR-BI) is the main receptor for high-density lipoprotein (HDL) cholesterol, which plays a pivotal role in preventing atherosclerosis. It removes cholesterol from HDL cholesterol, returning lipid-poor lipoprotein into blood circulation. The present study investigated the effects of X. muta Fraction-7 and linoleic acid on SR-BI gene expression and HDL cholesterol uptake. In vitro studies of the activity of X. muta and linoleic acid against the therapeutic target for hypercholesterolemia were conducted using the HDL receptor SR-BI via luciferase assay and HepG2 cells. In the present study, Fraction-7 of X. muta showed the highest expression level of the SR-BI gene via luciferase assay. Profiling of Fraction-7 of X. muta by GC-MS revealed 58 compounds, comprising various fatty acids, particularly linoleic acid. The in vitro study in HepG2 cells showed that the Fraction-7 of X. muta and linoleic acid (an active compound in X. muta) increased SR-BI mRNA expression by 129% and 85%, respectively, compared to the negative control. Linoleic acid increased HDL uptake by 3.21-fold compared to the negative control. Thus, the Fraction-7 of X. muta and linoleic acid have the potential to be explored as adjuncts in the treatment of hypercholesterolemia to prevent or reduce the severity of atherosclerosis development.

The Distinct Role of the HDL Receptor SR-BI in Cholesterol Homeostasis of Human Placental Arterial and Venous Endothelial Cells.

As opposed to adults, high-density lipoprotein (HDL) is the main cholesterol carrying lipoprotein in fetal circulation. The major HDL receptor, scavenger receptor class B type I (SR-BI), contributes to local cholesterol homeostasis. Arterial endothelial cells (ECA) from human placenta are enriched with cholesterol compared to venous endothelial cells (ECV). Moreover, umbilical venous and arterial plasma cholesterol levels differ markedly. We tested the hypothesis that the uptake of HDL-cholesteryl esters differs between ECA and ECV because of the differential expression of SR-BI. We aimed to identify the key regulators underlying these differences and the functional consequences. Immunohistochemistry was used for visualization of SR-BI in situ. ECA and ECV were isolated from the chorionic plate of human placenta and used for RT-qPCR, Western Blot, and HDL uptake assays with 3H- and 125I-labeled HDL. DNA was extracted for the methylation profiling of the SR-BI promoter. SR-BI regulation was studied by exposing ECA and ECV to differential oxygen concentrations or shear stress. Our results show elevated SR-BI expression and protein abundance in ECA compared to ECV in situ and in vitro. Immunohistochemistry demonstrated that SR-BI is mainly expressed on the apical side of placental endothelial cells in situ, allowing interaction with mature HDL circulating in the fetal blood. This was functionally linked to a higher increase of selective cholesterol ester uptake from fetal HDL in ECA than in ECV, and resulted in increased cholesterol availability in ECA. SR-BI expression on ECV tended to decrease with shear stress, which, together with heterogeneous immunostaining, suggests that SR-BI expression is locally regulated in the placental vasculature. In addition, hypomethylation of several CpG sites within the SR-BI promoter region might contribute to differential expression of SR-BI between chorionic arteries and veins. Therefore, SR-BI contributes to a local cholesterol homeostasis in ECA and ECV of the human feto-placental vasculature.

Exercise Ameliorates Atherosclerosis via Up-Regulating Serum ß-Hydroxybutyrate Levels.

Atherosclerosis, accompanied by inflammation and metabolic disorders, is the primary cause of clinical cardiovascular death. In recent years, unhealthy lifestyles (e.g., sedentary lifestyles) have contributed to a worldwide epidemic of atherosclerosis. Exercise is a known treatment of atherosclerosis, but the precise mechanisms are still unknown. Here, we show that 12 weeks of regular exercise training on a treadmill significantly decreased lipid accumulation and foam cell formation in ApoE-/- mice fed with a Western diet, which plays a critical role in the process of atherosclerosis. This was associated with an increase in ß-hydroxybutyric acid (BHB) levels in the serum. We provide evidence that BHB treatment in vivo or in vitro increases the protein levels of cholesterol transporters, including ABCA1, ABCG1, and SR-BI, and is capable of reducing lipid accumulation. It also ameliorated autophagy in macrophages and atherosclerosis plaques, which play an important role in the step of cholesterol efflux. Altogether, an increase in serum BHB levels after regular exercise is an important mechanism of exercise inhibiting the development of atherosclerosis. This provides a novel treatment for atherosclerotic patients who are unable to undertake regular exercise for whatever reason. They will gain a benefit from receiving additional BHB.

Scavenger Receptor BI Attenuates IL-17A-Dependent Neutrophilic Inflammation in Asthma.

Asthma is a common respiratory disease currently affecting more than 300 million worldwide and is characterized by airway inflammation, hyperreactivity, and remodeling. It is a heterogeneous disease consisting of corticosteroid-sensitive T-helper cell type 2-driven eosinophilic and corticosteroid-resistant, T-helper cell type 17-driven neutrophilic phenotypes. One pathway recently described to regulate asthma pathogenesis is cholesterol trafficking. Scavenger receptors, in particular SR-BI (scavenger receptor class B type I), are known to direct cellular cholesterol uptake and efflux. We recently defined SR-BI functions in pulmonary host defense; however, the function of SR-BI in asthma pathogenesis is unknown. To elucidate the role of SR-BI in allergic asthma, SR-BI-sufficient (SR-BI+/+) and SR-BI-deficient (SR-BI-/-) mice were sensitized (Days 0 and 7) and then challenged (Days 14, 15, and 16) with a house dust mite (HDM) preparation administered through oropharyngeal aspiration. Airway inflammation and cytokine production were quantified on Day 17. When compared with SR-BI+/+ mice, the HDM-challenged SR-BI-/- mice had increased neutrophils and pulmonary IL-17A production in BAL fluid. This augmented IL-17A production in SR-BI-/- mice originated from a non-T-cell source that included neutrophils and alveolar macrophages. Given that SR-BI regulates adrenal steroid hormone production, we tested whether the changes in SR-BI-/- mice were glucocorticoid dependent. Indeed, SR-BI-/- mice were adrenally insufficient during the HDM challenge, and corticosterone replacement decreased pulmonary neutrophilia and IL-17A production in SR-BI-/- mice. Taken together, these data indicate that SR-BI dampens pulmonary neutrophilic inflammation and IL-17A production in allergic asthma at least in part by maintaining adrenal function.

Class B Scavenger Receptors BI and BII Protect against LPS-Induced Acute Lung Injury in Mice by Mediating LPS.

Recent studies suggest an anti-inflammatory protective role for class B scavenger receptor BI (SR-BI) in endotoxin-induced inflammation and sepsis. Other data, including ours, provide evidence for an alternative role of SR-BI, facilitating bacterial and endotoxin uptake and contributing to inflammation and bacterial infection. Enhanced endotoxin susceptibility of SR-BI-deficient mice due to their anti-inflammatory glucocorticoid deficiency complicates the understanding of SR-BI's role in endotoxemia/sepsis, calling for the use of alternative models. In this study, using human SR-BI (hSR-BI) and hSR-BII transgenic mice, we found that SR-BI and, to a lesser extent, its splicing variant SR-BII protect against LPS-induced lung damage. At 20 h after intratracheal LPS instillation, the extent of pulmonary inflammation and vascular leakage was significantly lower in hSR-BI and hSR-BII transgenic mice than in wild-type mice. Higher bronchoalveolar lavage fluid (BALF) inflammatory cell count and protein content and lung tissue neutrophil infiltration found in wild-type mice were associated with markedly (2 to 3 times) increased proinflammatory cytokine production compared to these parameters in transgenic mice following LPS administration. The markedly lower endotoxin levels detected in BALF of transgenic versus wild-type mice and the significantly increased BODIPY-LPS uptake observed in lungs of hSR-BI and hSR-BII mice 20 h after the i.t. LPS injection suggest that hSR-BI- and hSR-BII-mediated enhanced LPS clearance in the airways could represent the mechanism of their protective role against LPS-induced acute lung injury.

Connecting Cholesterol Efflux Factors to Lung Cancer Biology and Therapeutics.

Cholesterol is a foundational molecule of biology. There is a long-standing interest in understanding how cholesterol metabolism is intertwined with cancer biology. In this review, we focus on the known connections between lung cancer and molecules mediating cholesterol efflux. A major take-home lesson is that the roles of many cholesterol efflux factors remain underexplored. It is our hope that this article would motivate others to investigate how cholesterol efflux factors contribute to lung cancer biology.

Identification of scavenger receptor BI as a potential screening candidate for congenital primary adrenal insufficiency in humans.

Glucocorticoids belong to the superfamily of steroid hormones that are synthesized from the common precursor cholesterol. Adrenal gland-derived glucocorticoids, e.g., cortisol in humans and corticosterone in rodents, contribute to various processes essential for normal daily life. Glucocorticoid deficiency, also referred to as primary adrenal insufficiency, therefore, often becomes evident early in life and can be present with hypoglycemia, a failure to thrive, recurrent development of infections, and neurological problems, such as seizures and coma. The majority of congenital primary adrenal insufficiency cases are caused by deleterious mutations in genes involved in the intracellular mobilization of cholesterol and the subsequent conversion of cholesterol into glucocorticoids. A significant number of glucocorticoid deficiency cases, however, cannot be explained by known genetic variations. This perspective highlights existing literature regarding the importance of lipoprotein-derived cholesterol acquisition through scavenger receptor class B, type I (SR-BI/SCARB1) for the maintenance of an optimal adrenal glucocorticoid function in mice and humans. On the basis of the reviewed findings, it is suggested that the SCARB1 gene should be included in the standard glucocorticoid deficiency genetic screening panel to 1) facilitate knowledge development on the relative contribution of SR-BI-mediated cholesterol acquisition to steroid hormone synthesis in humans and 2) open up the possibility to reclassify glucocorticoid deficiency patients without a currently known genetic cause for concomitant treatment optimization.

Proline residues in scavenger receptor-BI's C-terminal region support efficient cholesterol transport.

High-density lipoproteins (HDLs) facilitate reverse cholesterol transport, a process in which HDL removes cholesterol from circulation and carries it to the liver for biliary excretion. Reverse cholesterol transport is also facilitated by HDL's high-affinity receptor, scavenger receptor-BI (SR-BI), by mechanisms that are not fully understood. To improve our understanding of SR-BI function, we previously solved the NMR (nuclear magnetic resonance) structure of a peptide encompassing amino acids 405-475 of SR-BI. This segment of SR-BI, that includes the functionally critical C-terminal transmembrane domain and part of the extracellular domain, also contains four conserved proline (Pro) residues. We hypothesized that these proline residues support SR-BI in a conformation that allows for efficient cholesterol transport. To test this, we generated individual Pro-to-alanine mutations in full-length SR-BI and transiently expressed the mutant receptors in COS-7 cells to measure the effects on SR-BI-mediated cholesterol transport functions. Our findings reveal that HDL cell association and uptake of HDL-cholesteryl esters are impaired by mutation of Pro-412, Pro-438, or the transmembrane proline kink residue (Pro-459). In addition, SR-BI-mediated cholesterol efflux and membrane cholesterol distribution are impaired by mutation of Pro-412 or Pro-438, indicating that these residues are essential for a fully functional SR-BI receptor. Furthermore, we demonstrate that Pro-408 is necessary for proper SR-BI expression, but mutation of Pro-408 does not cause SR-BI to become misfolded or rapidly degraded by the proteasome or the lysosome. We conclude that key proline residues play an important role in SR-BI function by allowing for the efficient transport of cholesterol between cells and HDL.

High-Density Lipoprotein Therapy in Stroke: Evaluation of Endothelial SR-BI-Dependent Neuroprotective Effects.

High-density lipoproteins (HDLs) display endothelial protective effects. We tested the role of SR-BI, an HDL receptor expressed by endothelial cells, in the neuroprotective effects of HDLs using an experimental model of acute ischemic stroke. After transient intraluminal middle cerebral artery occlusion (tMCAO), control and endothelial SR-BI deficient mice were intravenously injected by HDLs or saline. Infarct volume and blood-brain barrier (BBB) breakdown were assessed 24 h post tMCAO. The potential of HDLs and the role of SR-BI to maintain the BBB integrity was assessed by using a human cellular model of BBB (hCMEC/D3 cell line) subjected to oxygen-glucose deprivation (OGD). HDL therapy limited the infarct volume and the BBB leakage in control mice relative to saline injection. Interestingly, these neuroprotective effects were thwarted by the deletion of SR-BI in endothelial cells and preserved in mice deficient for SR-BI in myeloid cells. In vitro studies revealed that HDLs can preserve the integrity of the BBB in OGD conditions, and that this effect was reduced by the SR-BI inhibitor, BLT-1. The protection of BBB integrity plays a pivotal role in HDL therapy of acute ischemic stroke. Our results show that this effect is partially mediated by the HDL receptor, SR-BI expressed by endothelial cells.

Insulin increases cholesterol uptake, lipid droplet content, and apolipoprotein B secretion in CaCo-2 cells by upregulating SR-BI via a PI3K, AKT, and mTOR-dependent pathway.

The actions of insulin on intestinal cholesterol absorption and lipoprotein secretion are not well understood. Herein, we determined the effects of insulin on the levels of cholesterol transporter scavenger receptor, class B, type I (SR-BI), cellular cholesterol uptake, intracellular lipid accumulation, and lipoprotein secretion in a cellular model of human intestinal epithelium. METHODS: CaCo-2 cells were cultured to postconfluency in Transwell filters and stimulated with glucose (25 mM) in the presence or absence of insulin (100 nM) at their basolateral surface. SR-BI mRNA and protein levels were quantified by quantitative reverse transcription-PCR and immunoblot, respectively. Polarized localization of SR-BI was determined by cell surface proteins biotinylation and streptavidin precipitation. Activities of PI3K, AKT, mTOR, and SR-BI were pharmacologically antagonized. Cholesterol uptake was assessed by NBD-cholesterol incorporation. Apolipoprotein (apo) B concentration was quantified by ELISA. Subcellular localization of neutral lipids (BODIPY) and SR-BI (immunofluorescence) was determined by confocal microscopy. RESULTS: In polarized CaCo-2 cells, insulin increased SR-BI at the mRNA and protein levels. SR-BI was exclusively present at apical cell surface, as indicated by biotinylation and confocal microscopy analysis. Glucose did not modify SR-BI abundance or subcellular localization. Effects of insulin on SR-BI levels were abrogated by PI3K, AKT, or mTOR pharmacological antagonism. Cholesterol uptake, neutral lipid abundance, and apo B secretion were increased by insulin in CaCo-2 cells, and these effects were prevented by SR-BI pharmacological antagonism with block lipid transport-1. CONCLUSIONS: insulin promotes cholesterol uptake, intracellular lipid store, and apo B-containing lipoproteins secretion by SR-BI-dependent mechanisms in a model of human intestinal epithelium.