Role Of Lipid Rafts Research Articles

MβCD inhibits SFTSV entry by disrupting lipid raft structure of the host cells

Severe fever with thrombocytopenia syndrome virus (SFTSV), recently named as Dabie bandavirus, belongs to the family Phenuiviridae of the order Bunyavirales, is a newly-identified bunyavirus with a case fatality rate of up to 30%, posing a serious threat to public health. Lipid rafts on plasm membranes are important for the entry of enveloped viruses; however, the role of lipid rafts in bunyavirus entry remains unclear. In this study, we found that methyl-beta-cyclodextrin (MβCD), a drug that disrupts cholesterol in lipid rafts of cell membranes, inhibits SFTSV infection. Additionally, there is a back-complementary effect of SFTSV infection upon the addition of cholesterol. Moreover, the concentration of SFTSV particles in lipid rafts during entry directly indicated the role of lipid rafts as a gateway, whereas MβCD could inhibit SFTSV entry by affecting the structure of lipid rafts. In an in vivo study, MβCD also reduced the susceptibility of mice to SFTSV infection. Our results suggest that SFTSV can interact with Talin1 proteins on lipid rafts to enter host cells by endocytosis of lipid rafts and reveal the potential therapeutic value of MβCD for SFTSV infection.

Role of Lipid Rafts on LRP8 Signaling Triggered by Anti-β2-GPI Antibodies in Endothelial Cells.

Antiphospholipid antibody syndrome is an autoimmune disease characterized by thrombosis and/or pregnancy morbidity in association with circulating antiphospholipid antibodies, mainly anti-β2 glycoprotein 1 antibodies (anti-β2-GPI antibodies). Previous studies demonstrated that the signaling pathway may involve lipid rafts, plasma membrane microdomains enriched in glycosphingolipid and cholesterol. In this study, we analyzed the signaling pathway of LRP8/ApoER2, a putative receptor of anti-β2-GPI antibodies, through lipid rafts in human endothelial cells. LRP8, Dab2 and endothelial nitric oxide synthase (e-NOS) phosphorylation were evaluated using Western blot, Nitric Oxide (NO) production with cytofluorimetric analysis, LRP8 enrichment in lipid rafts via sucrose gradient fractionation, and scanning confocal microscopy analysis of its association with ganglioside GM1 was also conducted. The analyses demonstrated that affinity-purified anti-β2-GPI antibodies induced LRP8 and Dab-2 phosphorylation, together with a significant decrease in e-NOS phosphorylation, with consequent decrease in NO intracellular production. These effects were almost completely prevented by Methyl-β-cyclodextrin (MβCD), indicating the involvement of lipid rafts. It was supported with the observation of LRP8 enrichment in lipid raft fractions and its association with ganglioside GM1, detected with scanning confocal microscopy. These findings demonstrate that LRP8 signaling triggered by anti-β2-GPI antibodies in endothelial cells occurs through lipid rafts. It represents a new task for valuable therapeutic approaches, such as raft-targeted therapy, including cyclodextrins and statins.

Unveiling the Role of Cholesterol in Subnanomolar Ouabain Rescue of Cortical Neurons from Calcium Overload Caused by Excitotoxic Insults.

Na/K-ATPase maintains transmembrane ionic gradients and acts as a signal transducer when bound to endogenous cardiotonic steroids. At subnanomolar concentrations, ouabain induces neuroprotection against calcium overload and apoptosis of neurons during excitotoxic stress. Here, the role of lipid rafts in interactions between Na/K-ATPase, sodium-calcium exchanger (NCX), and N-methy-D-aspartate receptors (NMDARs) was investigated. We analyzed 0.5-1-nanometer ouabain's effects on calcium responses and miniature post-synaptic current (mEPSCs) frequencies of cortical neurons during the action of NMDA in rat primary culture and brain slices. In both objects, ouabain attenuated NMDA-evoked calcium responses and prevented an increase in mEPSC frequency, while the cholesterol extraction by methyl-β-cyclodextrin prevented the effects. The data support the conclusions that (i) ouabain-induced inhibition of NMDA-elicited calcium response involves both pre- and post-synapse, (ii) the presence of astrocytes in the tripartite synapse is not critical for the ouabain effects, which are found to be similar in cell cultures and brain slices, and (iii) ouabain action requires the integrity of cholesterol-rich membrane microdomains in which the colocalization and functional interaction of NMDAR-transferred calcium influx, calcium extrusion by NCX, and Na/K-ATPase modulation of the exchanger occur. This regulation of the molecules by cardiotonic steroids may influence synaptic transmission, prevent excitotoxic neuronal death, and interfere with the pharmacological actions of neurological medicines.

The Regulatory Roles of Cerebellar Glycosphingolipid Microdomains/Lipid Rafts.

Lipid rafts are dynamic assemblies of glycosphingolipids, sphingomyelin, cholesterol, and specific proteins which are stabilized into platforms involved in the regulation of vital cellular processes. Cerebellar lipid rafts are cell surface ganglioside microdomains for the attachment of GPI-anchored neural adhesion molecules and downstream signaling molecules such as Src-family kinases and heterotrimeric G proteins. In this review, we summarize our recent findings on signaling in ganglioside GD3 rafts of cerebellar granule cells and several findings by other groups on the roles of lipid rafts in the cerebellum. TAG-1, of the contactin group of immunoglobulin superfamily cell adhesion molecules, is a phosphacan receptor. Phosphacan regulates the radial migration signaling of cerebellar granule cells, via Src-family kinase Lyn, by binding to TAG-1 on ganglioside GD3 rafts. Chemokine SDF-1α, which induces the tangential migration of cerebellar granule cells, causes heterotrimeric G protein Goα translocation to GD3 rafts. Furthermore, the functional roles of cerebellar raft-binding proteins including cell adhesion molecule L1, heterotrimeric G protein Gsα, and L-type voltage-dependent calcium channels are discussed.

The Role of Lipid Rafts and Membrane Androgen Receptors in Androgen's Neurotoxic Effects.

Sex differences have been observed in multiple oxidative stress–associated neurodegenerative diseases. Androgens, such as testosterone, can exacerbate oxidative stress through a membrane androgen receptor (mAR), AR45, localized to lipid rafts in the plasma membrane. The goal of this study is to determine if interfering with mAR localization to cholesterol-rich lipid rafts decreases androgen induced neurotoxicity under oxidative stress environments. We hypothesize that cholesterol-rich caveolar lipid rafts are necessary for androgens to induce oxidative stress generation in neurons via the mAR localized within the plasma membrane. Nystatin was used to sequester cholesterol and thus decrease cholesterol-rich caveolar lipid rafts in a neuronal cell line (N27 cells). Nystatin was applied prior to testosterone exposure in oxidatively stressed N27 cells. Cell viability, endocytosis, and protein analysis of oxidative stress, apoptosis, and mAR localization were conducted. Our results show that the loss of lipid rafts via cholesterol sequestering blocked androgen-induced oxidative stress in cells by decreasing the localization of mAR to caveolar lipid rafts.

Role of LipidRafts in Pathogen-Host Interaction -A Mini Review.

Lipid rafts, also known as microdomains, are important components of cell membranes and are enriched in cholesterol, glycophospholipids and receptors. They are involved in various essential cellular processes, including endocytosis, exocytosis and cellular signaling. Receptors are concentrated at lipid rafts, through which cellular signaling can be transmitted. Pathogens exploit these signaling mechanisms to enter cells, proliferate and egress. However, lipid rafts also play an important role in initiating antimicrobial responses by sensing pathogens via clustered pathogen-sensing receptors and triggering downstream signaling events such as programmed cell death or cytokine production for pathogen clearance. In this review, we discuss how both host and pathogens use lipid rafts and associated proteins in an arms race to survive. Special attention is given to the involvement of the major vault protein, the main constituent of a ribonucleoprotein complex, which is enriched in lipid rafts upon infection with vaccinia virus.

Cholesterol metabolism and its implication in glioblastoma therapy.

Glioblastoma (GBM) is the most lethal malignant tumor in the central nervous system, with a median survival of only 14 months. Cholesterol, which is the main component of cell membrane and the precursor of many hormones, is one of the most important lipid components in human body. Since reprogramming of the cholesterol metabolic profile has been discovered in many cancers including GBM, cholesterol metabolism becomes a promising potential target for therapy. Since GBM cells rely on external cholesterol to survive and accumulate lipid droplets to meet their rapid growth needs, targeting the metabolism of cholesterol by different strategies including inhibition of cholesterol uptake and promotion of cholesterol efflux by activating LXRs and disruption of cellular cholesterol trafficking, inhibition of SREBP signaling, inhibition of cholesterol esterification, could potentially oppose the growth of glial tumors. In this review, we discussed the above findings and describe cholesterol synthesis and homeostatic feedback pathways in normal brain tissues and brain tumors, statin use in GBM and the role of lipid rafts and cholesterol precursors and oxysterols in the treatment and pathogenesis of GBM are also summarized.

Cholesterol and Sphingolipid Enriched Lipid Rafts as Therapeutic Targets in Cancer.

Lipid rafts are critical cell membrane lipid platforms enriched in sphingolipid and cholesterol content involved in diverse cellular processes. They have been proposed to influence membrane properties and to accommodate receptors within themselves by facilitating their interaction with ligands. Over the past decade, technical advances have improved our understanding of lipid rafts as bioactive structures. In this review, we will cover the more recent findings about cholesterol, sphingolipids and lipid rafts located in cellular and nuclear membranes in cancer. Collectively, the data provide insights on the role of lipid rafts as biomolecular targets in cancer with good perspectives for the development of innovative therapeutic strategies.

Abstract A25: Differentiation of human naive T cells to various effector memory cell subtypes correlates with increased binding of the B subunit of Type-IIb heat-labile enterotoxin (LT-IIb-B5) to its cognate ganglioside receptors

Abstract The initial antigenic activation of T cells involves localization of the T-cell receptor to lipid rafts, of which gangliosides are major components. To understand the role of lipid rafts in the activation and differentiation of CD4+ and CD8+ T cells, it is necessary to dissect the ganglioside composition in each respective T-cell subset. In the present study, we used the B subunits of type-II heat-labile enterotoxins (LT-IIa-B5, LT-IIb-B5 and LT-IIc-B5), which possess defined ganglioside-binding specificities, as molecular probes to dissect the membrane ganglioside composition of human T cell subsets. Peripheral blood mononuclear cells isolated from leukocyte reduction filters by Ficoll density gradient centrifugation were stained with recombinant LT-IIa-B5, LT-IIb-B5, mutant LT-IIb-B5(T13I) or LT-IIc-B5 and with monoclonal antibodies to CD4, CD8, CD27, CD45RA, CD45RO and CD62L followed by flow cytometric analysis. Our results show that ligands for LT-IIa (gangliosides GD1b, GM1, and GT-1b), LT-IIb (gangliosides GD1a, GT1b and GM2), and LT-IIc (gangliosides GM1, GM2, and GM3) are increasingly expressed during differentiation from naïve (CD4+ or CD8+CD45RO-CD27+) to memory (CD4+ or CD8+CD45RO+CD27+) T cells, and that discrete subsets of central memory (CD4+ or CD8+CD45RA-CD62L+), effector memory (CD4+ or CD8+CD45RA-CD62L-), and terminally differentiated effector memory (CD4+ or CD8+CD45RO+CD27-) T cells express the highest ganglioside levels, particularly of LT-IIb-ligands. On the other hand, mutant LT-IIb-B5(T13I), which had negligible binding in vitro to gangliosides GD1a, GT1b, and GM2, exhibited no detectable binding to naive or central memory T cells, but significantly bound subsets of terminally differentiated effector memory (CD4+ and CD8+) T cells. Our results suggest an important role for gangliosides GD1b and GM1 in naive T-cell responses, while central memory, effector memory, and terminally differentiated effector memory T-cell responses may be more dependent upon gangliosides GD1a and GT1b. Furthermore, the lymphocyte-binding activities of LT-IIb-B5(T13I) confirm that its parental B-pentameter essentially recognizes gangliosides GD1a, GT1b, and GM2 on T cells and suggest that LT-IIb-B5 (T13I) may bind to one or more unknown receptor(s) expressed on subsets of terminally differentiated effector memory (CD4+ and CD8+) T cells. Citation Format: Taylor A. Johnson, Madison K. Ritter, Mary-Peyton A. Knapp, Robert O. Rainer, Nathalie D. King-Lyons, Terry D. Connell, Sergio Arce. Differentiation of human naive T cells to various effector memory cell subtypes correlates with increased binding of the B subunit of Type-IIb heat-labile enterotoxin (LT-IIb-B5) to its cognate ganglioside receptors [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A25.

Abstract 353: Distinct cGMP Compartmentalization by Membrane Guanylate Cyclases

Introduction: In adult cardiomyocytes (CM), cGMP produced by two membrane guanylate cyclases (GC-A and GC-B) play distinct roles. Using Förster Resonance Energy Transfer (FRET) and Scanning Ion Conductance Microscopy (SICM)-based approaches, cGMP compartmentation by GCs and phosphodiesterases (PDEs) were assessed. Methods: cGMP was measured in CM isolated from transgenic mice expressing membrane targeted cGMP sensor (red cGES-DE5). SICM was used to define the t-tubules across sarcolemma and its nano-pipette filled with natriuretic peptides (CNP and ANP) was used to locally stimulate GC in t-tubules and crest. Results: CNP activating GC-B induced maximum cGMP increase, while ANP activating GC-A did not increase cGMP. Pre-inhibiting PDE2 before ANP stimulation massively increased cGMP production to functionally significant levels as measured by PLN phosphorylation in western blot. cGMP in unstimulated cells was regulated mostly by PDE3. Local ANP stimulation in t-tubules, but not in crest, induced cGMP increase and CNP induced cGMP increase in both t-tubule and crest. After methyl-β-cyclodextrin treatment, ANP induced cGMP in both t-tubules and crest suggesting a role for lipid rafts in trapping GC-A in t-tubules. In transfected HEK293 cells, Fluorescence Recovery After Photobleeching (FRAP) showed that membrane diffusion of YFP-tagged GC-A was decreased after methyl-β-cyclodextrin treatment. Conclusions: GC-A exclusively located in T-tubules produce cGMP locally, which is tightly regulated by PDE2, while GC-B is expressed across the sarcolemma and produces global cGMP.

Role of Lipid Rafts in Hematopoietic Stem Cells Homing, Mobilization, Hibernation, and Differentiation.

Hematopoietic stem cells (HSCs) are multipotent, self-renewing cells that can differentiate into myeloid or lymphoid cells. The mobilization and differentiation processes are affected by the external environment, such as extracellular matrix and soluble molecules in the niche, where the lipid rafts (LRs) of the HSCs act as the receptors and control platforms for these effectors. LRs are membrane microdomains that are enriched in cholesterol, sphingolipid, and proteins. They are involved in diverse cellular processes including morphogenesis, cytokinesis, signaling, endocytic events, and response to the environment. They are also involved in different types of diseases, such as cancer, Alzheimer’s, and prion disease. LR clustering and disruption contribute directly to the differentiation, homing, hibernation, or mobilization of HSCs. Thus, characterization of LR integrity may provide a promising approach to controlling the fate of stem cells for clinical applications. In this review, we show the critical role of LR modification (clustering, disruption, protein incorporation, and signal responding) in deciding the fate of HSCs, under the effect of soluble cytokines such as stem cell factor (SCF), transforming growth factor- β (TGF-β), hematopoietic-specific phospholipase Cβ2 (PLC-β2), and granulocyte colony-stimulating factor (G-CSF).

The role of lipid rafts in cell entry of human metapneumovirus.

Human metapneumovirus (hMPV) is a crucial pathogen in children. A cell entry is the first step for infection. Our previous study indicated that there was an endocytosis pathway for hMPV cell entry. Lipid raft is a specific structure at the cell surface and it has been demonstrated to play an important role in endocytosis process of many viruses. In this study, we investigated whether and how lipid raft can take part in the hMPV entry. The confocal microscope was used to detect colocalization of hMPV and lipid raft marker. We demonstrated that colocalizations were increased along with the viral infection and hMPV particles transferred to the perinuclear region with lipid raft. When specific lipid raft inhibitors: methyl‐β cyclodextrin and nystatin were used, hMPV cell entry was inhibited and viral titer decreased dramatically. With the replenishment of exogenous cholesterol, hMPV recovered quickly. These data suggest that lipid raft plays an important role in hMPV endocytosis and maybe one of the pathways for hMPV cell entry.

Abstract 3550: Lipid rafts contribute to ligand-induced signaling and survival in pediatric AML

Abstract Pediatric acute myeloid leukemia (pAML) is an aggressive malignancy with high mortality due to therapy resistance and relapse. We previously reported that the failure of pAML cells to phosphorylate STAT3 upon G-CSF or IL-6 stimulation was associated with inferior survival. The defect in STAT3 signaling was not due to low expression of receptors or of STAT3. We hypothesized that the signaling failure seen in some pAML samples is not specific for STAT3, but rather a generalized dysfunction due to abnormal lipid raft function. Lipid rafts (LR) are highly ordered plasma membrane microdomains rich in glycosphingolipids and cholesterol. Two types of LR - flat planar and invaginated caveolar - contain different membrane proteins and serve distinct but overlapping cellular functions. Both types of LR organize receptors, downstream mediators, and repressors for efficient signaling. The role of LRs in signaling and clinical outcome is not yet established in pAML. Primary samples (n=95) from the Children's Oncology Group (COG) were stimulated with HS5 stromal cell conditioned medium (HS5CM), which contains IL-6 and dozens of physiological ligands. Basal and induced phospho-STAT3 (pSTAT3), pSTAT5, pAKT and pERK1/2 were measured by intracellular flow cytometry, and the fold change in the mean fluorescence intensity (ΔMFI) was calculated. The HS5CM-induced ΔMFI for pSTAT3 was positively correlated with that for pSTAT5 (p<0.0001, r=0.5), and pAKT (p=0.0002, r=0.4). Samples that failed to increase pSTAT3 failed to activate other pathways, suggesting a generalized signaling dysfunction. In AML cells with intact signaling, disruption of LRs with methylβcyclodextrin markedly reduced HS5CM-induced pSTATs, recapitulating the dysfunctional phenotype. LRs were assessed by immunocytochemistry in 29 primary samples with and without HS5CM stimulation. Choleratoxin B, which binds ganglioside GM-1, stained LRs. Antibodies to caveolin-1 (cav-1) marked caveolar LRs. We also stained for gp130, the signaling subunit of the IL-6 receptor. Images were captured by deconvolution fluorescence microscopy. Expression was quantified by pixel intensity for average of 30 cells per condition. Colocalization of gp130 with LR markers was determined by the Manders Overlap Coefficient (MOC). Intensities and MOCs varied between samples. The MOC of gp130 with cav-1 correlated significantly with pSTAT3 ΔMFI (p=0.02, r=0.43). MOC of gp130 with GM-1 did not correlate with pSTAT3 ΔMFI (p=0.85, r=0.03), suggesting that cav-1 facilitates IL-6/STAT3 signaling. Interestingly, patients whose samples demonstrated basal cav-1 and GM-1 intensities below the median had inferior 5-year event-free survival compared to those with intensities above the median (log-rank p=0.079 for cav-1; p=0.013 for GM-1). Our results suggest that LRs contribute to ligand-induced signaling and possibly to chemotherapy response. Further studies of LR biology in AML are warranted. Citation Format: Padmini Narayanan, Julien Dubrulle, Robert B. Gerbing, Todd A. Alonzo, Fabio Stossi, Michele S. Redell. Lipid rafts contribute to ligand-induced signaling and survival in pediatric AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3550.

HIV Associated Neurodegenerative Disorders: A New Perspective on the Role of Lipid Rafts in Gp120-Mediated Neurotoxicity.

The implementation of combination antiretroviral therapy (cART) as the primary means of treatment for HIV infection has achieved a dramatic decline in deaths attributed to AIDS and the re-duced incidence of severe forms of HIV-associated neurocognitive disorders (HAND) in infected in-dividuals. Despite these advances, milder forms of HAND persist and prevalence of these forms of neurocognitive impairment are rising with the aging population of HIV infected individuals. HIV en-ters the CNS early in the pathophysiology establishing persistent infection in resident macrophages and glial cells. These infected cells, in turn, secrete neurotoxic viral proteins, inflammatory cytokines, and small metabolites thought to contribute to neurodegenerative processes. The viral envelope protein gp120 has been identified as a potent neurotoxin affecting neurodegeneration via indirect and direct mechanisms involving interactions with chemokine co-receptors CCR5 and CXCR4. This short re-view focuses on gp120 neurotropism and associated mechanisms of neurotoxicity linked to chemokine receptors CCR5 and CXCR4 with a new perspective on plasma membrane lipid rafts as an active par-ticipant in gp120-mediated neurodegeneration underlying HIV induced CNS pathology.

The mystery of membrane organization: composition, regulation and roles of lipid rafts.

Cellular plasma membranes are laterally heterogeneous, featuring a variety of distinct subcompartments that differ in their biophysical properties and composition. A large number of studies have focused on understanding the basis for this heterogeneity and its physiological relevance. The membrane raft hypothesis formalized a physicochemical principle for a subtype of such lateral membrane heterogeneity, in which the preferential associations between cholesterol and saturated lipids drive the formation of relatively packed (or ordered) membrane domains that selectively recruit certain lipids and proteins. Recent studies have yielded new insights into this mechanism and its relevance in vivo, owing primarily to the development of improved biochemical and biophysical technologies.

HIV Entry: Receptors Cooperate with Membrane Domain Boundaries to form Entry Sites in Host Cells

It has been proposed that lipid rafts of host cell membranes play pivotal roles for cell entry of many enveloped viruses including HIV. However, it remains largely unknown why virions would prefer nanoscopic ordered lipid domains over uniformly fluid membrane regions. Here, we show that HIV does not enter cells from within ordered membrane regions, but rather at the boundaries between raft-like and non-raft-like regions of the plasma membrane. Using cell-derived giant plasma membrane vesicles (GPMVs), which are phase-separated into large-scale liquid-ordered (Lo) and liquid-disordered (Ld) membrane domains, we demonstrate that the HIV receptor CD4 is substantially sequestered into Lo domains while the coreceptor CCR5 localizes preferentially at Lo/Ld domain boundaries. Lo/Ld phase coexistence is not required for HIV attachment, but the recognition of Lo/Ld boundaries is a prerequisite for successful fusion of the viral envelope with the cell membrane. We propose that virions localized to membrane domain boundaries utilize their interfacial energy as an additional driving force for fusion and cell entry. This study provides surprising answers to the long-standing question about the roles of lipid rafts in cell entry of HIV and perhaps other enveloped viruses.

Dual role of cellular prion protein in normal host and Alzheimer’s disease

Using PrPC-knockout cell lines, it has been shown that the inhibition of apoptosis through STI1 is mediated by PrPC-dependent SOD activation. Antioxidant PrPC may contribute to suppression of inflammasome activation. PrPC is functionally involved in copper metabolism, signal transduction, neuroprotection, and cell maturation. Recently several reports have shown that PrPC participates in trans-membrane signaling processes associated with hematopoietic stem cell replication and neuronal differentiation. In another role, PrPC also tends to function as a neurotoxic protein. Aβ oligomer, which is associated with neurodegeneration in Alzheimer’s disease (AD), has also been reported to act as a ligand of PrPC. However, the physiological role of PrPC as an Aβ42-binding protein is not clear. Actually, PrPC is critical in Aβ42-mediated autophagy in neurons. PrPC shows a beneficial role in lipid rafts to promote autophagy. Further search for PrPC-interaction molecules using Prnp−/− mice and various types of Prnp−/− cell lines under various conditions may elucidate other important PrPC important functions.

561: Role of lipid rafts in human placental nitric oxide production

561: Role of lipid rafts in human placental nitric oxide production

Abstract 12674: LDL Transcytosis by SR-BI Requires Signaling That is Distinct From Reverse Cholesterol Transport

Introduction: How LDL crosses an intact endothelial layer to initiate atherosclerosis is poorly understood, although it is postulated to involve LDL transcytosis. To date, technical limitations have greatly limited the study of its molecular mechanisms. We have recently devised an assay for quantifying LDL transcytosis by primary human coronary artery endothelial cells and have reported an unexpected role for the scavenger receptor SR-BI. SR-BI is best known as the HDL receptor and mediates reverse cholesterol transport; its downstream signaling involves the adaptor PDZK1 and endothelial nitric oxide synthase (eNOS). However, whether SR-BI internalization and its canonical signaling pathway are required for LDL transcytosis is unknown. Hypothesis: We hypothesized that LDL transcytosis and reverse cholesterol transport require different signaling pathways. Methods: Transcytosis of DiI-labeled LDL by primary human coronary artery endothelial cells was studied by total internal reflection fluorescence microscopy. The role of lipid rafts, PDZK1, eNOS and MAP kinases was studied by siRNA and pharmacological inhibitors. Results: Incubation with LDL induced the internalization of SR-BI, which was enriched in lipid rafts. Knockdown of caveolin-1 significantly attenuated LDL transcytosis. Coronary endothelial cells expressed low levels of PDZK1 and knockdown by siRNA had no effect on transcytosis. Similarly, inhibition of eNOS or addition of exogenous nitric oxide (NO) by NO-donors did not affect LDL transcytosis. Over-expression of G420H-SR-BI, a mutant receptor which does not perform reverse cholesterol transport, actually induced LDL transcytosis while the SR-BI inhibitor BLT-1, which prevents reverse cholesterol transport, had no effect on LDL transcytosis. Finally, inhibition of c-Jun N-terminal kinase (JNK) significantly attenuated LDL transcytosis. Conclusions: SR-BI is internalized during LDL transcytosis but PDZK1 and nitric oxide are not required. Instead, JNK kinase is involved. These data raise the possibility that reverse cholesterol transport and LDL transcytosis by SR-BI are regulated separately. These findings suggest that LDL transcytosis by SR-BI may constitute a novel therapeutic target for atherosclerosis.

The Role of Lipid Rafts in the Localization and Function of the Chemosensory TRPA1 Channel

Lipid rafts are highly specialized membrane domains characterized by high concentrations of cholesterol, sphingolipids and gangliosides. This distinctive composition results in lateral phase separation and generation of a liquid-ordered domains accommodating various receptors and channels involved in numerous cellular processes. Recently, several members of the Transient Receptor Potential channels superfamily (TRPCs, TRPM8, and TRPV1) have been shown to locate mainly in the lipid rafts and that this compartmentalization modulates their activation properties. Yet, not much is known about localization and interactions of TRPA1 with the surrounding plasma membrane. Since TRPA1 can be gated by a large number of electrophilic and non-electrophilic compounds, as well as by physiological stimuli such as cold we hypothesize that its localization in the lipid rafts could be a crucial factor influencing the channel activity. Exploiting microscopy and biochemical approaches, this study provides evidence for TRPA1 localization in lipid rafts. TIRF microscopy experiments in HEK293T cells transfected with a mouse or human TRPA1 channel carrying a C-terminal mCherry tag indicate high co-localization rate between channel and the lipid raft maker cholera toxin subunit B. Density gradient centrifugation of Triton-X insoluble fractions confirmed co-expression of TRPA1 and the lipid raft marker flotillin-2 in the low density membrane fractions. Lipid raft disruption experiments further affirmed the cholesterol-related localization of the channel, shifting its localization into higher density gradients. Modification of lipid rafts composition by cholesterol depletion (methyl-b-cyclodextrin) or sphingolipids hydrolysis (sphingomyelinase) decreased the responses of TRPA1 to lipopolysaccharides, thymol, allyl isothiocyanate and cold. Taken together, these results indicate that TRPA1 is present in lipid rafts and this location is essential for its normal activation by different agonists.