Increased Membrane Cholesterol Research Articles

Cholesterol-dependent dynamic changes in the conformation of the type 1 cholecystokinin receptor affect ligand binding and G protein coupling.

Development of optimal therapeutics for disease states that can be associated with increased membrane cholesterol requires better molecular understanding of lipid modulation of the drug target. Type 1 cholecystokinin receptor (CCK1R) agonist actions are affected by increased membrane cholesterol, enhancing ligand binding and reducing calcium signaling, while agonist actions of the closely related CCK2R are not. In this work, we identified a set of chimeric human CCK1R/CCK2R mutations that exchange the cholesterol sensitivity of these 2 receptors, providing powerful tools when expressed in CHO and HEK-293 model cell lines to explore mechanisms. Static, low energy, high-resolution structures of the mutant CCK1R constructs, stabilized in complex with G protein, were not substantially different, suggesting that alterations to receptor dynamics were key to altered function. We reveal that cholesterol-dependent dynamic changes in the conformation of the helical bundle of CCK receptors affects both ligand binding at the extracellular surface and G protein coupling at the cytosolic surface, as well as their interrelationships involved in stimulus-response coupling. This provides an ideal setting for potential allosteric modulators to correct the negative impact of membrane cholesterol on CCK1R.

Cholesterol-induced suppression of Kir2 channels is mediated by decoupling at the inter-subunit interfaces

SummaryCholesterol is a major regulator of multiple types of ion channels. Although there is increasing information about cholesterol binding sites, the molecular mechanisms through which cholesterol binding alters channel function are virtually unknown. In this study, we used a combination of Martini coarse-grained simulations, a network theory-based analysis, and electrophysiology to determine the effect of cholesterol on the dynamic structure of the Kir2.2 channel. We found that increasing membrane cholesterol reduced the likelihood of contact between specific regions of the cytoplasmic and transmembrane domains of the channel, most prominently at the subunit-subunit interfaces of the cytosolic domains. This decrease in contact was mediated by pairwise interactions of specific residues and correlated to the stoichiometry of cholesterol binding events. The predictions of the model were tested by site-directed mutagenesis of two identified residues—V265 and H222—and high throughput electrophysiology.

Cholesterol in Synaptic Vesicle Membranes Regulates the Vesicle-Binding, Function, and Aggregation of α-Synuclein.

Loss of function and aggregation of the neuronal protein α-Synuclein (A-Syn) underlies the pathogenesis of Parkinson's disease (PD), and both the function and aggregation of this protein happen to be mediated via its binding to the synaptic vesicles (SVs) at the presynaptic termini. An essential constituent of SV membranes is cholesterol, with which A-Syn directly interacts while binding to membranes. Thus, cholesterol content in SV membranes is likely to affect the binding of A-Syn to these vesicles and consequently its functional and pathogenic behaviors. Interestingly, the dyshomeostasis of cholesterol has often been associated with PD, with reports linking both high and low cholesterol levels to an increased risk of neurodegeneration. Herein, using SV-mimicking liposomes containing increasing percentages of membrane cholesterol, we show (with mathematical interpretation) that the binding of A-Syn to synaptic-like vesicles is strongest in the presence of an optimum cholesterol content, which correlates to its maximum function and minimum aggregation. This implicates a minimum risk of neurodegeneration at optimum cholesterol levels and rationalizes the existing controversial relationship between cholesterol levels and PD. Increased membrane cholesterol was, however, found to protect against damage caused by aggregated A-Syn, complementing previous reports and portraying one advantage of high cholesterol over low.

The impact of TiO2 nanoparticle exposure on transmembrane cholesterol transport and enhanced bacterial infectivity in HeLa cells

We have previously shown that exposure to TiO2 nanoparticles (NPs) reduces the resistance of HeLa cells to bacterial infection. Here we demonstrate that the increased infectivity is associated with enhanced asymmetry in the cholesterol distribution. We applied a live cell imaging method which uses tunable orthogonal cholesterol sensors to visualize and quantify in-situ cholesterol distribution between the two leaflets of the plasma membrane (PM). In the control culture, we found marked transbilayer asymmetry of cholesterol, with the concentration in the outer plasma membrane (OPM) being 13 ± 2-fold higher than that in the inner plasma membrane (IPM). Exposure of the culture to 0.1 mg/mL of rutile TiO2 NPs increased the asymmetry such that the concentration in the OPM was 51 ± 10 times higher, while the total cholesterol content increased only 21 ± 2%. This change in cholesterol gradient may explain the increase in bacterial infectivity in HeLa cells exposed to TiO2 NPs since many pathogens, including Staphylococcus aureus used in the present study, require cholesterol for proper membrane attachment and virulence. RT-PCR indicated that exposure to TiO2 was responsible for upregulation of the ABCA1 and ABCG1 mRNAs, which are responsible for the production of the cholesterol transporter proteins that facilitate cholesterol transport across cellular membranes. This was confirmed by the observation of an overall decrease in bacterial infection in ABCA1 knockout or methyl-β-cyclodextrin-treated HeLa cells, as regardless of TiO2 NP exposure. Hence rather than preventing bacterial infection, TiO2 nanoparticles upregulate genes associated with membrane cholesterol production and distribution, hence increasing infectivity. Statement of significanceA great deal of work has been done regarding the toxicology of the particles, especially focusing on detrimental outcomes associated with reactive oxygen species (ROS) production. In this paper we show unambiguously a very surprising result, namely the ability of these particles to enhance bacterial infection even at very small exposure levels, where none of the deleterious effects of ROS products can yet be detected. Using a new imaging technique, we are able to demonstrate, in operando, the effect of the particles on cholesterol generation and distribution in live HeLa cells. This paper also represents the first in a series where we explore other consequences of increased membrane cholesterol, due to particle exposure, which are known to have multiple other consequences on human tissue function and development.

Perspective: What Should I Eat to Maintain Membrane Lipid Rafts and Avoid Metabolic Disease?

Discerning meaningful patterns in lipidomic studies of metabolic diseases is daunting. Consideration of changes in dietary lipids over the last 150 years and the effects of this variance on membrane raft formation provides a tool to better understand lipidomic data. In vitro, mixtures of low and high melting point lipids and cholesterol spontaneously form bilayers with heterologous areas of differing order, composition, and physical properties. Through dozens of studies, the general behavior of different lipid mixtures in phase separation has been mapped and displayed as ternary lipid mixture phase diagrams; areas that show both liquid order (Lo) and liquid disorder (Ld) allow raft formation (Figure 1). In vivo, ordered membrane areas composed of sterols, sphingolipids and proteins provide platforms necessary for a complex network of cellular signaling. Although there is controversy regarding the primacy of lipids versus proteins in organizing these signaling rafts, this perspective focuses on the role that variations in dietary lipids might play in raft formation. Comparison of dietary lipids in humans living before the agricultural revolution to those living in 21st century developed areas reveals major differences. Modern diets have: increased polyunsaturated fatty acids (PUFA) from vegetable oils, an increased ratio of omega 6:3 PUFA, increased trans fatty acids (TFA), and decreased cholesterol. Plotting these variations in dietary lipids on a ternary graph provides a framework to understand the theoretical effect of dietary lipid variance on phase separation and membrane behavior. For example, how might increased dietary TFA, a widely recognized risk factor in cardiovascular disease, disrupt raft formation? Substitution of TFA for PUFA at sn-2 in low melting point phospholipids would raise the melting point; substitution at sn-1 on sphingolipids would lower the melting point. Because disparity in the order of membrane domains is critical in the maintenance of phase separation, convergence of melting points of these compounds would thus impede raft formation (Figure 2). To compensate, incorporation of highly unsaturated PUFA to lower the melting point of non raft phospholipids, or incorporation of longer chain saturated fatty acids to raise the melting point of raft associated lipids would restore order differential and allow raft formation, but would require other changes to maintain optimal membrane viscosity. Elevated membrane PUFA increases oxidative damage and an elevated omega 6:3 ratio increases thrombosis and inflammation. Compared to cis forms, TFA phospholipids require eight times more cholesterol to be incorporated into a membrane; with reduced cholesterol diets, accommodation of TFAs into membranes would require stimulation of endogenous cholesterol synthesis. Increased membrane cholesterol also decreases permeability to oxygen. Because most fatty acids are incorporated into cell membranes unmodified from the form in which they were absorbed from the GI tract, changes in dietary lipids critically impact cell membrane composition. This example illustrates the theoretical impact of dietary lipids on raft formation and behavior as a means to understand lipidomic data and the pathophysiologic elements of dyslipidemia, oxidative stress, inflammation and cellular hypoxia.

Alectinib induces marked red cell spheroacanthocytosis in a near-ubiquitous fashion and is associated with reduced eosin-5-maleimide binding

We reviewed haematological investigations for 43 patients treated at a single centre with alectinib, an inhibitor of anaplastic lymphoma kinase (ALK) which is considered standard first-line treatment for patients with ALK-rearranged advanced non-small cell lung cancer. Ninety-five percent of patients developed marked acanthocytosis, echinocytosis and/or spheroacanthocytosis, not observable with prior treatment with other ALK-inhibitors. Anaemia developed in 73% of patients (38% <100 g/L, 8% <80 g/L), though definite new haemolysis was present in only 11%. Eosin-5-maleimide binding was reduced in all assessed patients, and increased membrane cholesterol was identified in one patient assessed with lattice light sheet microscopy. We have identified a previously undescribed phenomenon whereby alectinib induces red cell membrane abnormalities in nearly all patients through an unclear, but likely ALK-independent, mechanism, resulting in mild anaemia without universal haemolysis.

Effect of Cholesterol on the Structural Dynamics of Metabotropic Glutamate Receptor (mGluR1): A Molecular Dynamics Study

Metabotropic glutamate receptors (mGluRs) are class C G protein-coupled receptors (GPCRs) which regulate neurotransmission and are prevalently expressed in the central nervous system. The mGluR subfamily has eight members divided into three subgroups (I (mGluR1,5), II (mGluR2,3) and III (mGluR4,6,7,8)) based on their sequence homology, agonist selectivity and signaling function. The group I mGluRs (mGluR1 and mGluR5) are useful for treating diseases such as cancer, pain, schizophrenia, Alzheimer's disease, anxiety, and autism. Unlike most GPCRs which have the orthosteric binding pocket located in the transmembrane helices, the extracellular VFT domain of the mGluRs serves as the orthosteric ligand binding site while the 7-transmembrane (7TM) domain has allosteric binding sites. Using all-atom equilibrium MD simulations, we have studied the effect of different amounts of membrane cholesterol on the local structural dynamics of the mGlur1 7TM region in the inactive state. In addition, we have also studied the effect of thermostabilized apocytochrome b562RIL (BRIL) on the 7TM region. We observed that BRIL and cholesterol had contrasting effects on the 7TM domain of mGluR1. While the presence of BRIL was found to maintain the inactive conformation of the protein, increasing the amount of membrane cholesterol was found to trigger a conformational transition towards the active state. A salt bridge (E783-K834) present in the inactive state was found to break when membrane cholesterol was increased to 25%. We also observed similar protein fluctuations in the absence of BRIL and with the increased membrane cholesterol. This study thus sheds light on conformational changes observed in mGluR1 and the effect of cholesterol within the 7TM region, providing a framework for determining the activation process of mGluRs.

An Increase in Membrane Cholesterol by Graphene Oxide Disrupts Calcium Homeostasis in Primary Astrocytes.

The use of graphene nanomaterials (GNMs) for biomedical applications targeted to the central nervous system is exponentially increasing, although precise information on their effects on brain cells is lacking. In this work, the molecular changes induced in cortical astrocytes by few-layer graphene (FLG) and graphene oxide (GO) flakes are addressed. The results show that exposure to FLG/GO does not affect cell viability or proliferation. However, proteomic and lipidomic analyses unveil alterations in several cellular processes, including intracellular Ca2+ ([Ca2+ ]i ) homeostasis and cholesterol metabolism, which are particularly intense in cells exposed to GO. Indeed, GO exposure impairs spontaneous and evoked astrocyte [Ca2+ ]i signals and induces a marked increase in membrane cholesterol levels. Importantly, cholesterol depletion fully rescues [Ca2+ ]i dynamics in GO-treated cells, indicating a causal relationship between these GO-mediated effects. The results indicate that exposure to GNMs alters intracellular signaling in astrocytes and may impact astrocyte-neuron interactions.

Codetermination of Sphingomyelin and Cholesterol in Cellular Plasma Membrane in Sphingomyelin-Depletion-Induced Cholesterol Efflux.

Quantification of multiple lipids with different contents in plasma membrane in single cells is significant, but challenging, for investigating lipid interactions and the role of dominant protein transporters. In this paper, comonitoring the alteration of low-content sphingomyelin (SM) and high-content cholesterol in plasma membrane of one living cell is realized by use of luminol electrochemiluminescence (ECL) for the first time. Concentrations of SM as low as 0.5 μM are detected, which permits the measurement of low-content membrane SM in single cells. More membrane cholesterol is observed in individual cells after depletion of membrane SM, providing direct evidence about SM-depletion-induced cholesterol efflux. The upregulation of ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1) in SM-depleted cells induces a further increase in membrane cholesterol. These results imply that higher expression of ABCA1/G1 promotes cholesterol trafficking, which offers additional information to solve the debate about ABC transporters in cholesterol efflux. Moreover, the established approach offers a special strategy to investigate the correlation of membrane lipids and the role of transporters in cholesterol trafficking.

Functional expression of breast cancer resistance protein and cholesterol effect in human erythrocyte membranes.

In human erythrocyte membranes, various influx and efflux transporters are functionally expressed. However, their transport characteristics and modulation under disease states are not fully understood. In this study, we first examined the expression and detailed transport characteristics of breast cancer resistance protein (BCRP), an efflux ABC transporter, using inside-out membrane vesicles (IOVs) prepared from human erythrocytes, and then studied the effect of membrane cholesterol on BCRP function. The expression of BCRP was confirmed by western blotting; most of them being homodimers. The uptake of lucifer yellow (LY), a fluorescent BCRP substrate, into IOVs was time-, temperature-, and ATP-dependent, and the concentration of ATP which induced half-maximal stimulation of LY uptake was calculated to be 0.39 mM. The uptake of LY by IOVs was saturable with a Km value of 166 μM, and was inhibited by various BCRP inhibitors and substrates, such as fumitremorgin C and mitoxantrone. When membrane cholesterol content was increased by treating IOVs with cholesteryl hemisuccinate, LY uptake decreased with increasing cholesterol content. These results suggest that transport activity of BCRP in human erythrocyte membranes may be suppressed under disease states, such as hypercholesterolemia, that increase membrane cholesterol content.

Electrochemical measurement of membrane cholesterol correlates with CFTR function and is HDAC6-dependent

BackgroundPrevious studies have demonstrated that CF epithelial cells exhibit increased cholesterol content at the plasma membrane compared to wild type controls as measured by electrochemical methods. Microtubule dysregulation that impacts intracellular transport has also been identified in CF cells and is reversible with histone deacetylase 6 (HDAC6) inhibition, a regulator of tubulin acetylation. The hypothesis of this study is that increased membrane cholesterol content in CF cells is dependent on HDAC6 regulation. MethodsElectrochemical measurement of membrane cholesterol in mouse trachea and in primary human CF bronchial epithelial cells is used to monitor CFTR correction and manipulation of cholesterol processing by HDAC6 inhibition. ResultsData demonstrate that induction of Cftr expression in an inducible CF mouse model restores tubulin acetylation levels and normalizes membrane cholesterol content. To test the relationship between tubulin acetylation, membrane cholesterol levels were measured in a CF mouse model depleted of Hdac6 expression (CF/HDA). CF/HDA mouse trachea have WT membrane cholesterol levels while CF mice have approximately two-fold increase in membrane cholesterol compared to WT consistent with previous studies. Pharmacological inhibition of HDAC6 in primary human CF bronchial epithelial cells also reduces membrane cholesterol levels. ConclusionsThis study demonstrates that elevated membrane cholesterol in CF epithelium is regulated by HDAC6 function and that the electrochemical measure of membrane cholesterol correlates with both genetic and pharmacological CFTR correction.

Influence of cholesterol/caveolin-1/caveolae homeostasis on membrane properties and substrate adhesion characteristics of adult human mesenchymal stem cells

BackgroundAdult mesenchymal stem cells (MSCs) are an important resource for tissue growth, repair, and regeneration. To utilize MSCs more effectively, a clear understanding of how they react to environmental cues is essential. Currently, relatively little is known about how the composition of the plasma membranes affects stem cell phenotype and properties. The presence of lipid molecules, including cholesterol in particular, in the plasma membrane plays a crucial role in regulating a variety of physiological processes in cells. In this study, we examined the effects of perturbations in cholesterol/caveolin-1 (CAV-1)/caveolae homeostasis on the membrane properties and adhesive characteristics of MSCs. Findings from this study will contribute to the understanding of how cholesterol/CAV-1/caveolae regulates aspects of the cell membrane important to cell adhesion, substrate sensing, and microenvironment interaction.MethodsWe generated five experimental MSC groups: 1) untreated MSCs; 2) cholesterol-depleted MSCs; 3) cholesterol-supplemented MSCs; 4) MSCs transfected with control, nonspecific small interfering (si)RNA; and 5) MSCs transfected with CAV-1 siRNA. Each cell group was analyzed for perturbation of cholesterol status and CAV-1 expression by performing Amplex Red cholesterol assay, filipin fluorescence staining, and real-time polymerase chain reaction (PCR). The membrane fluidity in the five experimental cell groups were measured using pyrene fluorescence probe staining followed by FACS analysis. Cell adhesion to collagen and fibronectin as well as cell surface integrin expression were examined.ResultsCholesterol supplementation to MSCs increased membrane cholesterol, and resulted in decreased membrane fluidity and localization of elevated numbers of caveolae and CAV-1 to the cell membrane. These cells showed increased expression of α1, α4, and β1 integrins, and exhibited higher adhesion rates to fibronectin and collagen. Conversely, knockdown of CAV-1 expression or cholesterol depletion on MSCs caused a parallel decrease in caveolae content and an increase in membrane fluidity due to decreased delivery of cholesterol to the cell membrane. Cells with depleted CAV-1 expression showed decreased cell surface integrin expression and slower adhesion to different substrates.ConclusionsOur results demonstrate that perturbations in cholesterol/CAV-1 levels significantly affect the membrane properties of MSCs. These findings suggest that modification of membrane cholesterol and/or CAV-1 and caveolae may be used to manipulate the biological activities of MSCs.

Membrane cholesterol removal and replenishment affect rat and monkey brain monoamine transporters

The dopamine transporter (DAT) is abundantly expressed in the striatum where it removes extracellular dopamine into the cytosol of presynaptic nerve terminals. It is the target of drugs of abuse and antidepressants. There is a loss of the DAT in Parkinson's disease affecting release of levodopa implicated in levodopa-induced dyskinesias. This study investigated the effect of cholesterol on DAT, serotonin transporter (SERT) and vesicular monoamine transporter 2 (VMAT2) in monkey and rat brains in vitro. DAT protein levels measured by Western blot remained unchanged with in vitro methyl-β-cyclodextrin (MCD) incubations to remove membrane cholesterol or with incubations to increase membrane cholesterol content. By contrast, striatal DAT specific binding labelled with [125I]RTI-121 or with [125I]RTI-55 decreased with increasing concentrations of MCD and increased with cholesterol loading. Moreover, [125I]RTI-121 specific binding of striatal membranes depleted of cholesterol with MCD was restored to initial DAT content with addition of cholesterol showing its rapid and reversible effect. By contrast, striatal VMAT2 and SERT specific binding showed no or limited changes by cholesterol manipulations. Similar results were obtained for monkey caudate nucleus, putamen and nucleus accumbens. Membrane microviscosity was assessed by fluorescence polarization spectroscopy, using the probe 1,6-diphenyl-1,3,5-hexatriene. DAT changes positively correlated with changes of membrane microviscosity in rat and monkey brain regions investigated and with membrane cholesterol contents. Similar findings were observed with desmosterol but to a lower extent than with cholesterol. These results show an important effect of cholesterol on the DAT associated with microviscosity changes that should be considered in drug therapies.

Free cholesterol and cholesterol esters in bovine oocytes: Implications in survival and membrane raft organization after cryopreservation.

Part of the damage caused by cryopreservation of mammalian oocytes occurs at the plasma membrane. The addition of cholesterol to cell membranes as a strategy to make it more tolerant to cryopreservation has been little addressed in oocytes. In order to increase the survival of bovine oocytes after cryopreservation, we proposed not only to increase cholesterol level of oocyte membranes before vitrification but also to remove the added cholesterol after warming, thus recovering its original level. Results from our study showed that modulation of membrane cholesterol by methyl-β-cyclodextrin (MβCD) did not affect the apoptotic status of oocytes and improved viability after vitrification yielding levels of apoptosis closer to those of fresh oocytes. Fluorometric measurements based on an enzyme-coupled reaction that detects both free cholesterol (membrane) and cholesteryl esters (stored in lipid droplets), revealed that oocytes and cumulus cells present different levels of cholesterol depending on the seasonal period. Variations at membrane cholesterol level of oocytes were enough to account for the differences found in total cholesterol. Differences found in total cholesterol of cumulus cells were explained by the differences found in both the content of membrane cholesterol and of cholesterol esters. Cholesterol was incorporated into the oocyte plasma membrane as evidenced by comparative labeling of a fluorescent cholesterol. Oocytes and cumulus cells increased membrane cholesterol after incubation with MβCD/cholesterol and recovered their original level after cholesterol removal, regardless of the season. Finally, we evaluated the effect of vitrification on the putative raft molecule GM1. Cholesterol modulation also preserved membrane organization by maintaining ganglioside level at the plasma membrane. Results suggest a distinctive cholesterol metabolic status of cumulus-oocyte complexes (COCs) among seasons and a dynamic organizational structure of cholesterol homeostasis within the COC. Modulation of membrane cholesterol by MβCD improved survival of bovine oocytes and preserved integrity of GM1-related rafts after vitrification.

Membrane Cholesterol in Skeletal Muscle: A Novel Player in Excitation-Contraction Coupling and Insulin Resistance.

Membrane cholesterol is critical for signaling processes in a variety of tissues. We will address here current evidence supporting an emerging role of cholesterol on excitation-contraction coupling and glucose transport in skeletal muscle. We have centered our review on the transverse tubule system, a complex network of narrow plasma membrane invaginations that propagate membrane depolarization into the fiber interior and allow nutrient delivery into the fibers. We will discuss current evidence showing that transverse tubule membranes have remarkably high cholesterol levels and we will address how modifications of cholesterol content influence excitation-contraction coupling. In addition, we will discuss how membrane cholesterol levels affect glucose transport by modulating the insertion into the membrane of the main insulin-sensitive glucose transporter GLUT4. Finally, we will address how the increased membrane cholesterol levels displayed by obese animals, which also present insulin resistance, affect these two particular skeletal muscle functions.

Platelet tissue factor activity and membrane cholesterol are increased in hypercholesterolemia and normalized by rosuvastatin, but not by atorvastatin

Background and aimsHigh plasma LDL-cholesterol (LDL-C) and platelet responses have major pathogenic roles in atherothrombosis. Thus, statins and anti-platelet drugs constitute mainstays in cardiovascular prevention/treatment. However, the role of platelet tissue factor-dependent procoagulant activity (TF-PCA) has remained unexplored in hypercholesterolemia. We aimed to study platelet TF-PCA and its relationship with membrane cholesterol in vitro and in 45 hypercholesterolemic patients (HC-patients) (LDL-C >3.37 mmol/L, 130 mg/dL) and 37 control subjects (LDL-C <3.37 mmol/L). The effect of 1-month administration of 80 mg/day atorvastatin (n = 21) and 20 mg/day rosuvastatin (n = 24) was compared. MethodsPlatelet TF-PCA was induced by GPIbα activation with VWF-ristocetin. ResultsCholesterol-enriched platelets in vitro had augmented aggregation/secretion and platelet FXa generation (1.65-fold increase, p = 0.01). HC-patients had 1.5-, 2.3- and 2.5-fold increases in platelet cholesterol, TF protein and activity, respectively; their platelets had neither hyper-aggregation nor endogenous thrombin generation (ETP). Rosuvastatin, but not atorvastatin, normalized platelet cholesterol, TF protein and FXa generation. It also increased slightly the plasma HDL-C levels, which correlated negatively with TF-PCA. ConclusionsPlatelets from HC-patients were not hyper-responsive to low concentrations of classical agonists and had normal PRP-ETP, before and after statin administration. However, washed platelets from HC-patients had increased membrane cholesterol, TF protein and TF-PCA. The platelet TF-dependent PCA was specifically expressed after VWF-induced GPIbα activation. Rosuvastatin, but not atorvastatin treatment, normalized the membrane cholesterol, TF protein and TF-PCA in HC-patients, possibly unveiling a new pleiotropic effect of rosuvastatin. Modulation of platelet TF-PCA may become a novel target to prevent/treat atherothrombosis without increasing bleeding risks.

Receptor-Mediated HDL-Lipid Uptake is Regulated by Elastic Properties of the Plasma Membrane

Proper supply of mammalian cells with cholesterol is crucial for membrane function and cellular survival and is regulated by several mechanisms, including cellular uptake, synthesis, storage and export. Transport of excess cholesterol from the periphery back to the liver for excretion into the bile is achieved by high-density lipoproteins (HDL). HDL particles are captured from the blood stream by the scavenger receptor class B type I (SR-BI), the so-called HDL receptor. The subsequent lipid transfer process, however, is highly speculative: it has been proposed to occur after HDL endocytosis or directly at the cell surface across an unstirred water layer or via a hydrophobic channel in the receptor. Here we show via direct imaging techniques that, upon contact with the membrane, HDL is incorporated into the hydrophobic core of the lipid bilayer and amphiphilic cargo is immediately released to the plasma membrane. Particle incorporation and cargo transfer is abolished at increased membrane cholesterol concentrations, as a consequence of the reduced membrane elasticity. Our observations reveal a mechanism for regulation of lipid uptake, which is based on sensing plasma membrane cholesterol levels: HDL-receptors tether particles close to the plasma membrane; once in proximity, elastic membrane properties regulate the particles’ fusion with the bilayer and subsequent cargo transfer. The data indicate that the plasma membrane itself signals a cell's cholesterol demand: high cholesterol levels act repulsive, low cholesterol levels fusogenic.

Abstract 46: Cellular Cholesterol Homeostasis is Altered in Murine Models of Rheumatoid Arthritis and is Linked to Enhanced Myelopoiesis

Rheumatoid arthritis (RA) is associated with a ~2-fold elevated risk of morbidity and mortality from atherosclerotic cardiovascular disease (CVD) compared with the general population. Atherosclerosis in RA patients tends to be more aggressive and therefore more challenging to treat. Identifying CVD in these patients is difficult as traditional CVD risk factors, such as changes in plasma lipid profiles (i.e. elevated LDL, decreased HDL) are not always observed, underscoring the need for better understanding of the reasons contributing to the enhanced atherosclerosis in RA patients. People with RA often have monocytosis and neutrophilia, which we hypothesize to plays causal roles in atherosclerosis. Two mouse models of RA were used, K/BxN serum transfer and collagen induced arthritis (CIA). Flow cytometry was used to quantify the abundance of leukocyte and stem cell subsets. BODIPY-cholesterol was employed to determine the membrane cholesterol status of the various cells. Prominent monocytosis and neutrophilia due to an expansion and increased proliferation of the haematopoietic stem and multipotential progenitor cells (HSPCs) in the bone marrow (BM) was observed in both models of RA. There was also an increase in the mobilisation of HSPCs into the circulation, which homed to the spleen, resulting in extramedullary haematopoiesis. Interestingly, key cholesterol efflux genes, Abca1, Abcg1 and Apoe were down regulated in the BM HSPCs isolated from the K/BxN mice, resulting in increased cell membrane cholesterol levels. We also observed an increase in the expression of the common beta subunit of the interlukin-3 receptor on the HSPCs and the M-CSF receptor in myeloid progenitor cells, likely explaining their increased proliferation and skewing to the myeloid lineage. Moreover, blood monocytes and neutrophils had increased membrane cholesterol content, independent of changes in plasma cholesterol levels. These data suggest that while plasma cholesterol levels may not be increased in RA, cellular cholesterol homeostasis might be increased. We hypothesize that this, together with enhanced monocyte production, underlies the increased risk of CVD in RA.

Elevated Levels of StAR-Related Lipid Transfer Protein 3 Alter Cholesterol Balance and Adhesiveness of Breast Cancer Cells: Potential Mechanisms Contributing to Progression of HER2-Positive Breast Cancers

The STARD3 gene belongs to the minimal amplicon in HER2-positive breast cancers and encodes a cholesterol-binding membrane protein. To study how elevated StAR-related lipid transfer protein 3 (StARD3) expression affects breast cancer cells, we generated MCF-7 cells stably overexpressing StARD3-green fluorescent protein. We found that StARD3-overexpressing cells exhibited nonadherent morphological features, had increased Src levels, and had altered cholesterol balance, as evidenced by elevated mRNA levels of the cholesterol biosynthesis rate-limiting enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase, and increased plasma membrane cholesterol content. On removal of serum and insulin from the culture medium, the morphological characteristics of the StARD3-overexpressing cells changed, the cells became adherent, and they developed enlarged focal adhesions. Under these conditions, the StARD3-overexpressing cells maintained elevated Src and plasma membrane cholesterol content and showed increased phosphorylation of focal adhesion kinase. In two Finnish nationwide patient cohorts, approximately 10% (212/2220) breast cancers exhibited high StARD3 protein levels, which was strongly associated with HER2 amplification; several factors related to poor disease outcome and poor breast cancer-specific survival. In addition, high StARD3 levels in breast cancers were associated with elevated 3-hydroxy-3-methylglutaryl-coenzyme A reductase mRNA levels and anti-Src-Tyr416 immunoreactivity. These results provide evidence that StARD3 overexpression results in increased cholesterol biosynthesis and Src kinase activity in breast cancer cells and suggest that elevated StARD3 expression may contribute to breast cancer aggressiveness by increasing membrane cholesterol and enhancing oncogenic signaling.

Regulation of Lipid Membrane Trafficking and Transmembrane Signaling by Graphene

Cholesterol, a lipid molecule found ubiquitously in eukaryotic cells, plays a vital role in the integrity, dynamics, and trafficking of the lipid membrane, in addition to influencing many transmembrane proteins. However, the functionality of membrane cholesterol is far from clear, largely due to an inability to manipulate membrane cholesterol with high spatiotemporal precision. Popular tools like statins or methyl-β-cyclodextrin (MβCD) only lead to chronic and indiscriminative cholesterol reduction. Moreover, there is no selective approach to increase membrane cholesterol. Our recent work involving carbon nanomaterials provided an unexpected answer. Graphene, a one-atom thick carbon crystal, has been explored for biomedical applications because of its remarkable chemical and physical properties. Using in vitro and in vivo measurements, we have found that graphene selectively interacts with cholesterol. This enriches cholesterol at the plasma membrane, and thus enhances membrane lipid phase order, likely promoting the formation of cholesterol-rich lipid membrane nanodomains. Neurons grown on graphene exhibited presynaptic potentiation, specifically caused by a larger pool of releasable vesicles and an increase of fast recycling. By addition or depletion of membrane cholesterol, we found that the graphene-induced presynaptic enrichment of membrane cholesterol is necessary and sufficient to promote potentiation. In non-neuronal cells, graphene significantly elevates ATP-induced intracellular Ca2+-signaling by promoting the activation of P2Y receptors, a group of GPCRs which are selectively responsive to extracellular ATP. Furthermore, we found that graphene enhances P2Y receptor signaling on the timescale of seconds, as rapidly as its effect on membrane packing. This then reveals an intriguing interaction between graphene and cholesterol, and its impact on plasma membrane structure, trafficking, and transmembrane proteins. Given the current challenges in manipulating membrane cholesterol, this graphene-cholesterol interaction will accelerate studies of membrane cholesterol function and broaden the biological application of carbon nanomaterials.