Lipid Raft Clustering Research Articles

Formation of N-type (Ca v2.2) voltage-gated calcium channel membrane microdomains: Lipid raft association and clustering

Voltage-gated calcium channels (Ca vs) comprise a pore-forming α 1 with auxiliary α 2δ and β subunits which modulate Ca v function and surface expression. Ca vα 1 and α 2δ are present in signalling complexes termed lipid rafts but it is unclear whether α 2δ is obligatory for targeting Ca vs to rafts or to what extent this influences cell surface organisation of Ca vs. Here, we have used imaging, biochemistry and electrophysiology to determine localisation and raft-partitioning of WT and functionally active HA-epitope tagged α 2δ-1 and Ca v2.2 subunits expressed in COS-7 cells. We show that α 2δ-1 not only partitions into lipid rafts itself but also mediates raft-partitioning of Ca v2.2/β 1b complexes. Ca vα 2δ-1, Ca v2.2/β 1b and Ca v2.2/β 1b/α 2δ-1 complexes are all organised into cell surface clusters although only in the presence of α 2δ-1 do they co-localise with raft markers, caveolin and flotillin. Such clusters persist in the presence of 3-methyl-β-cyclodextrin even though the raft markers disperse. However, clustering is profoundly sensitive to disruption of the actin-based cytoskeleton by cytochalasin-D. We conclude that α 2δ-1, and likely other α 2δ subunits, is necessary and sufficient for targeting Ca vs to lipid rafts. However, formation of clusters supporting “hotspots” of Ca v activity requires aggregation of macromolecular complexes containing raft components, stabilised by interactions with the cytoskeleton.

Visfatin-induced lipid raft redox signaling platforms and dysfunction in glomerular endothelial cells

Adipokines have been reported to contribute to glomerular injury during obesity or diabetes mellitus. However, the mechanisms mediating the actions of various adipokines on the kidney remained elusive. The present study was performed to determine whether acid sphingomyelinase (ASM)-ceramide associated lipid raft (LR) clustering is involved in local oxidative stress in glomerular endothelial cells (GECs) induced by adipokines such as visfatin and adiponectin. Using confocal microscopy, visfatin but not adiponectin was found to increase LRs clustering in the membrane of GECs in a dose and time dependent manner. Upon visfatin stimulation ASMase activity was increased, and an aggregation of ASMase product, ceramide and NADPH oxidase subunits, gp91 phox and p47 phox was observed in the LR clusters, forming a LR redox signaling platform. The formation of this signaling platform was blocked by prior treatment with LR disruptor filipin, ASMase inhibitor amitriptyline, ASMase siRNA, gp91 phox siRNA and adiponectin. Corresponding to LR clustering and aggregation of NADPH subunits, superoxide (O 2 −) production was significantly increased (2.7 folds) upon visfatin stimulation, as measured by electron spin resonance (ESR) spectrometry. Functionally, visfatin significantly increased the permeability of GEC layer in culture and disrupted microtubular networks, which were blocked by inhibition of LR redox signaling platform formation. In conclusion, the injurious effect of visfatin, but not adiponectin on the glomerular endothelium is associated with the formation of LR redox signaling platforms via LR clustering, which produces local oxidative stress resulting in the disruption of microtubular networks in GECs and increases the glomerular permeability.

The epidermal growth factor receptor (EGFR) promotes uptake of influenza A viruses (IAV) into host cells.

Influenza A viruses (IAV) bind to sialic-acids at cellular surfaces and enter cells by using endocytotic routes. There is evidence that this process does not occur constitutively but requires induction of specific cellular signals, including activation of PI3K that promotes virus internalization. This implies engagement of cellular signaling receptors during viral entry. Here, we present first indications for an interplay of IAV with receptor tyrosine kinases (RTKs). As representative RTK family-members the epidermal growth factor receptor (EGFR) and the c-Met receptor were studied. Modulation of expression or activity of both RTKs resulted in altered uptake of IAV, showing that these receptors transmit entry relevant signals upon virus binding. More detailed studies on EGFR function revealed that virus binding lead to clustering of lipid-rafts, suggesting that multivalent binding of IAV to cells induces a signaling platform leading to activation of EGFR and other RTKs that in turn facilitates IAV uptake.

T-Cell Receptor Microclusters Critical for T-Cell Activation Are Formed Independently of Lipid Raft Clustering

We studied the function of lipid rafts in generation and signaling of T-cell receptor microclusters (TCR-MCs) and central supramolecular activation clusters (cSMACs) at immunological synapse (IS). It has been suggested that lipid raft accumulation creates a platform for recruitment of signaling molecules upon T-cell activation. However, several lipid raft probes did not accumulate at TCR-MCs or cSMACs even with costimulation and the fluorescence resonance energy transfer (FRET) between TCR or LAT and lipid raft probes was not induced at TCR-MCs under the condition of positive induction of FRET between CD3 zeta and ZAP-70. The analysis of LAT mutants revealed that raft association is essential for the membrane localization but dispensable for TCR-MC formation. Careful analysis of the accumulation of raft probes in the cell interface revealed that their accumulation occurred after cSMAC formation, probably due to membrane ruffling and/or endocytosis. These results suggest that lipid rafts control protein translocation to the membrane but are not involved in the clustering of raft-associated molecules and therefore that the lipid rafts do not serve as a platform for T-cell activation.

Visfatin‐Induced Lipid Raft Redox Signaling Platforms and Hyperpermeability in Glomerular Endothelial Cells

Adipocytokines have been reported to contribute to the glomerular injury during obesity or diabetes mellitus. However, the mechanisms mediating the actions of various adipocytokines on the kidney remained elusive. The present study was performed to determine whether acid sphingomyelinase (ASM)‐ceramide signaling pathway is involved in local oxidative stress in glomerular endothelial cells (GECs) induced by adipocytokines such as visfatin and adiponectin. Using confocal microscope, visfatin but not adiponectin was found to increase lipid rafts (LRs) clustering on the membrane of GECs in a dose‐dependent manner. Upon visfatin stimulation ASM activity was increased, an aggregation of ceramide or NADPH oxidase subunits, gp91phox and p47phox was observed in LR clusters, which was blocked by prior treatment with LR disruptor filipin, adiponectin, ASM inhibitor amitriptyline or ASM siRNA. Corresponding to aggregation of LR clustering with NADPH subunits in GECs upon stimulation with visfatin, superoxide (O2·−) production was significantly increased (2.7 folds) as measured by ESR. Functionally, visfatin treatment significantly increased the GEC permeability and this action of visfatin was blocked by inhibition of LR redox signaling platform formation. In conclusion, the injurious effect of visfatin, but not adiponection on the glomerular endothelium is associated with the formation of LR redox signaling platforms via LR clustering, which increases the glomerular permeability by disruption of microtubule network in GECs (supported by NIH grants HL‐091464, HL‐75316 and DK54927).

Docosahexaenoic acid disrupts lipid raft clustering and enhances proliferation and survival of EL4 lymphomas

An emerging molecular mechanism by which docosahexaenoic acid (DHA) acyl chains modify cellular function is through changes in lipid raft organization. Biophysical studies in liposomes show that DHA does not directly modify rafts; instead, it incorporates into non‐rafts to modify the lateral organization of proteins such as the major histocompatibility complex (MHC) class I. Here we tested predictions of the model at a cellular level by incorporating DHA and eicosapentaenoic acid (EPA) into the membranes of EL4 lymphomas. Quantification of fluorescence microscopy images showed that DHA, but not EPA, diminished lipid raft clustering by incorporating into rafts and changed MHC I lateral organization by increasing the fraction of the non‐raft protein into rafts. We next addressed the functional consequences of treating EL4 cells given changes in raft organization are associated with suppression of cellular proliferation. Both EPA and DHA enhanced the proliferation and survival of EL4 cells relative to controls. Taken together, our findings are not in agreement with biophysical studies; therefore, we propose a new model, which reconciles contradictory viewpoints from biophysical and cellular studies, to explain how DHA‐containing phospholipids modify rafts. Furthermore, our data highlight the notion that n‐3 PUFAs exert differential functional effects depending on the cell type.

Rapid Lysosome Fusion to Lipid Raft Clusters and Endothelial Dysfunction Mediated by VAMP‐2 in Coronary Arterial Endothelial Cells

Lysosome fusion mediates acid sphingomyelinase translocation to and activation in plasma membrane to form membrane lipid raft (LR) signaling in coronary arterial endothelial cells (CAECs). The molecular mechanism mediating this lysosome fusion to cell plasma, however, remains poorly understood. The present study attempted to evaluate whether vesicles associated membrane proteins‐2 (VAMP‐2) mediates lysosome fusion and LR clustering, leading to endothelial dysfunction in CAECs. By immunohistochemistry, VAMP‐2 was found to be abundantly expressed in the endothelium of bovine coronary arteries. Fluorescent confocal microscopy showed that VAMP‐2 was significantly aggregated with lysosome marker lamp‐1 and LRs when the CAECs were stimulated by FasL, a well‐known LR clustering stimulator. Using FM1‐43 quenching and dequenching technique, lysosome fusion to plasma membrane in response to FasL was blocked by VAMP‐2 inhibitor, tetanus toxin. In addition, fluorescence resonance energy transfer (FRET) detection demonstrated that Fas L‐induced FRET between LRs marker and VAMP‐2 increased by 21.2 ± 3.3 during FasL stimulation. Functionally, inhibition of VAMP‐2 significantly restored FasL‐induced endothelial dysfunction in coronary arterial rings. In conclusion, VAMP‐2 on lysosome membrane in CAECs is critical to the lysosome fusion to LR areas of plasma membrane in CAECs and this VAMP‐2‐mediated lysosome fusion may contributes to FasL‐induced endothelial dysfunction (Supported by NIH grants HL057244, HL091464 and HL075316).

Activation of Membrane NADPH Oxidase Associated with Lysosome-Targeted Acid Sphingomyelinase in Coronary Endothelial Cells

This study explored the mechanism mediating the aggregation of membrane NADPH oxidase (NOX) subunits and subsequent activation of this enzyme in bovine coronary arterial endothelial cells (CAECs). With confocal microscopy, we found that FasL stimulated lipid rafts (LRs) clustering with NOX subunit aggregation and acid sphingomyelinase (ASM) gathering, which was blocked by the siRNA of sortilin, an intracellular protein responsible for the binding and targeting of ASM to lysosomes. Correspondingly, FasL-induced O(2)(.-) production through NOX in LRs fractions was abolished by sortilin siRNA. Further, with flow-cytometry and fluorescence resonance energy transfer (FRET) analysis, we surprisingly demonstrated that after FasL stimulation, sortilin was exposed to cell membranes from lysosomes together with Lamp-1 and ASM, and these lysosomal components were aggregated and form a signaling complex in cell membranes. With co-immunoprecipitation, lysosomal sortilin and ASM were found to interact more strongly when CAECs were stimulated by FasL. Functionally, inhibition of either sortilin expression, lysosome function, LRs clustering, or NOX activity significantly attenuated FasL-induced decrease in nitric oxide (NO) levels. It is concluded that lysosome-targeted ASM, through sortilin, is able to traffic to and expose to cell-membrane surface, which may lead to LRs clustering and NOX activation in CAECs.

Triggering role of acid sphingomyelinase in endothelial lysosome-membrane fusion and dysfunction in coronary arteries

The present study determined whether activation of acid sphingomyelinase (ASM) drives membrane proximal lysosomes to fuse to the cell surface, facilitating membrane lipid rafts (LRs) clustering in coronary arterial endothelial cells (CAECs) and leading to endothelial dysfunction. By confocal microscopy, the activators of ASM, phosphatidylinositol (PI), and bis (monoacylglyceryl) phosphate (Bis), and an inducer of ASM, butyrate, were found to increase LRs clustering in bovine CAECs, which was blocked by lysosome fusion inhibitor vacuolin-1. However, arsenic trioxide (Ars), an inducer of de novo synthesis of ceramide, had no such effect. Similarly, vacuolin-1-blockable effects were observed using fluorescence resonance energy transfer detection. Liquid chromatography-electrospray ionization-tandem mass spectrometry analysis demonstrated that all of these treatments, even Ars, increased ceramide production in CAECs. When ASM gene was silenced, all treatments except Ars no longer increased ceramide levels. Furthermore, dynamic fluorescence monitoring in live cells showed that PI and Bis stimulated lysosome-membrane fusion in CAECs. Functionally, PI and Bis impaired endothelium-dependent vasodilation in perfused coronary arteries, which was blocked by vacuolin-1 and a lysosome function inhibitor, bafilomycine. FasL (Fas ligand), a previously confirmed lysosome fusion stimulator as a comparison, also produced a similar effect. It is concluded that ASM activation serves as a triggering mechanism and driving force, leading to fusion of membrane proximal lysosomes into LR clusters on the cell membrane of CAECs, which represents a novel mechanism mediating endothelial dysfunction during death receptor activation or other pathological situation.

N-3 Polyunsaturated Fatty Acid Incorporates Directly into Lipid Rafts to Disrupt Domain Clustering and MHC Lateral Organization of Antigen Presenting Cells

N-3 polyunsaturated fatty acids (PUFA) are under clinical testing for the treatment of symptoms associated with inflammatory disorders such as cardiovascular disease. However, effective use n-3 PUFAs as nutraceuticals has been limited by a poor understanding of their molecular mechanisms. Here we addressed how the n-3 PUFAs eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids modified lipid raft and protein lateral organization of antigen presenting cells, whose function is suppressed by n-3 PUFAs. Quantitative fluorescence microscopy showed that DHA, but not EPA, relative to controls, diminished lipid raft clustering and increased their size. A significant amount of DHA incorporated directly into rafts without changing cholesterol distribution between rafts and non-rafts. Quantification of fluorescence co-localization images showed that DHA selectively altered the lateral organization of the major histocompatibility complex (MHC) class I protein. FRET microscopy measurements showed that DHA modified MHC class I clustering on a nanometer scale. Taken together, our findings are not in agreement with studies in model membranes on unsaturated fatty acids and lipid rafts. Therefore, we propose a new model, which reconciles contradictory viewpoints from biophysical and cellular studies, to explain how an unsaturated fatty acid modifies lipid rafts on several length scales. Our study provides mechanistic details by which DHA suppresses antigen presenting cell function, which allows us to more effectively use these fatty acids in the clinic.

Redox signaling via lipid raft clustering in homocysteine-induced injury of podocytes

Our recent studies have indicated that hyperhomocysteinemia (hHcys) may induce podocyte damage, resulting in glomerulosclerosis. However, the molecular mechanisms mediating hHcys-induced podocyte injury are still poorly understood. In the present study, we first demonstrated that an intact NADPH oxidase system is present in podocytes as shown by detection of its membrane subunit (gp91 phox ) and cytosolic subunit (p47 phox ). Then, confocal microscopy showed that gp91 phox and p47 phox could be aggregated in lipid raft (LR) clusters in podocytes treated with homocysteine (Hcys), which were illustrated by their colocalization with cholera toxin B, a common LR marker. Different mechanistic LR disruptors, either methyl-β-cyclodextrin (MCD) or filipin abolished such Hcys-induced formation of LR-gp91 phox or LR-p47 phox transmembrane signaling complexes. By flotation of detergent-resistant membrane fractions we found that gp91 phox and p47 phox were enriched in LR fractions upon Hcys stimulation, and such enrichment of NADPH oxidase subunits and increase in its enzyme activity were blocked by MCD or filipin. Functionally, disruption of LR clustering significantly attenuated Hcys-induced podocyte injury, as shown by their inhibitory effects on Hcys-decreased expression of slit diaphragm molecules such as nephrin and podocin. Similarly, Hcys-increased expression of desmin was also reduced by disruption of LR clustering. In addition, inhibition of such LR-associated redox signaling prevented cytoskeleton disarrangement and apoptosis induced by Hcys. It is concluded that NADPH oxidase subunits aggregation and consequent activation of this enzyme through LR clustering is an important molecular mechanism triggering oxidative injury of podocytes induced by Hcys.

Formation of lipid raft redox signalling platforms in glomerular endothelial cells: an early event of homocysteine‐induced glomerular injury

The present study tested the hypothesis that homocysteine (Hcys)-induced ceramide production stimulates lipid rafts (LRs) clustering on the membrane of glomerular endothelial cells (GECs) to form redox signalling platforms by aggregation and activation of NADPH oxidase subunits and thereby enhances superoxide (O2.−) production, leading to glomerular endothelial dysfunction and ultimate injury or sclerosis. Using confocal microscopy, we first demonstrated a co-localization of LR clusters with NADPH oxidase subunits, gp91phox and p47phox in the GECs membrane upon Hcys stimulation. Immunoblot analysis of floated detergent-resistant membrane fractions found that in LR fractions NADPH oxidase subunits gp91phox and p47phox are enriched and that the activity of this enzyme dramatically increased. We also examined the effect of elevated Hcys on the cell monolayer permeability in GECs. It was found that Hcys significantly increased GEC permeability, which was blocked by inhibition of LR redox signalling platform formation. Finally, we found that Hcys-induced enhancement of GEC permeability is associated with the regulation of microtubule stability through these LR-redox platforms. It is concluded that the early injurious effect of Hcys on the glomerular endothelium is associated with the formation of redox signalling platforms via LR clustering, which may lead to increases in glomerular permeability by disruption of microtubule network in GECs.

Docosahexaenoic Acid Modifies the Clustering and Size of Lipid Rafts and the Lateral Organization and Surface Expression of MHC Class I of EL4 Cells

An emerging molecular mechanism by which docosahexaenoic acid (DHA) exerts its effects is modification of lipid raft organization. The biophysical model, based on studies with liposomes, shows that DHA avoids lipid rafts because of steric incompatibility between DHA and cholesterol. The model predicts that DHA does not directly modify rafts; rather, it incorporates into nonrafts to modify the lateral organization and/or conformation of membrane proteins, such as the major histocompatibility complex (MHC) class I. Here, we tested predictions of the model at a cellular level by incorporating oleic acid, eicosapentaenoic acid (EPA), and DHA, compared with a bovine serum albumin (BSA) control, into the membranes of EL4 cells. Quantitative microscopy showed that DHA, but not EPA, treatment, relative to the BSA control diminished lipid raft clustering and increased their size. Approximately 30% of DHA was incorporated directly into rafts without changing the distribution of cholesterol between rafts and nonrafts. Quantification of fluorescence colocalization images showed that DHA selectively altered MHC class I lateral organization by increasing the fraction of the nonraft protein into rafts compared with BSA. Both DHA and EPA treatments increased antibody binding to MHC class I compared with BSA. Antibody titration showed that DHA and EPA did not change MHC I conformation but increased total surface levels relative to BSA. Taken together, our findings are not in agreement with the biophysical model. Therefore, we propose a model that reconciles contradictory viewpoints from biophysical and cellular studies to explain how DHA modifies lipid rafts on several length scales. Our study supports the notion that rafts are an important target of DHA's mode of action.

Dynamic clustering and dispersion of lipid rafts contribute to fusion competence of myogenic cells

Recent research indicates that the leading edge of lamellipodia of myogenic cells (myoblasts and myotubes) contains presumptive fusion sites, yet the mechanisms that render the plasma membrane fusion-competent remain largely unknown. Here we show that dynamic clustering and dispersion of lipid rafts contribute to both cell adhesion and plasma membrane union during myogenic cell fusion. Adhesion-complex proteins including M-cadherin, β-catenin, and p120-catenin accumulated at the leading edge of lamellipodia, which contains the presumptive fusion sites of the plasma membrane, in a lipid raft-dependent fashion prior to cell contact. In addition, disruption of lipid rafts by cholesterol depletion directly prevented the membrane union of myogenic cell fusion. Time-lapse recording showed that lipid rafts were laterally dispersed from the center of the lamellipodia prior to membrane fusion. Adhesion proteins that had accumulated at lipid rafts were also removed from the presumptive fusion sites when lipid rafts were laterally dispersed. The resultant lipid raft- and adhesion complex-free area at the leading edge fused with the opposing plasma membrane. These results demonstrate a key role for dynamic clustering/dispersion of lipid rafts in establishing fusion-competent sites of the myogenic cell membrane, providing a novel mechanistic insight into the regulation of myogenic cell fusion.

Interdependence of Laminin-mediated Clustering of Lipid Rafts and the Dystrophin Complex in Astrocytes

Astrocyte endfeet surrounding blood vessels are active domains involved in water and potassium ion transport crucial to the maintenance of water and potassium ion homeostasis in brain. A growing body of evidence points to a role for dystroglycan and its interaction with perivascular laminin in the targeting of the dystrophin complex and the water-permeable channel, aquaporin 4 (AQP4), at astrocyte endfeet. However, the mechanisms underlying such compartmentalization remain poorly understood. In the present study we found that AQP4 resided in Triton X-100-insoluble fraction, whereas dystroglycan was recovered in the soluble fraction in astrocytes. Cholesterol depletion resulted in the translocation of a pool of AQP4 to the soluble fraction indicating that its distribution is indeed associated with cholesterol-rich membrane domains. Upon laminin treatment AQP4 and the dystrophin complex, including dystroglycan, reorganized into laminin-associated clusters enriched for the lipid raft markers GM1 and flotillin-1 but not caveolin-1. Reduced diffusion rates of GM1 in the laminin-induced clusters were indicative of the reorganization of raft components in these domains. In addition, both cholesterol depletion and dystroglycan silencing reduced the number and area of laminin-induced clusters of GM1, AQP4, and dystroglycan. These findings demonstrate the interdependence between laminin binding to dystroglycan and GM1-containing lipid raft reorganization and provide novel insight into the dystrophin complex regulation of AQP4 polarization in astrocytes.

Lipid‐raft redox signaling platform and apoptosis of podocytes upon homocysteine stimulation

Lipid raft (LR) redox signaling platform associated with NADPH oxidase (NOX) was reported to mediate the actions of death receptor activation in different cells. It is interesting to know whether this LR redox signaling platform is also involved in podocytes injury during hyperhomocysteinemia. The present study first characterized the presence of the NOX membrane subunit‐gp91phox and it LR clusters in podocytes, and then tested whether this redox signaling platform contributes to homocysteine (Hcys)‐induced podocytes apoptosis. It was found that Hcys markedly increased the expression of gp91phox and stimulated NOX‐dependent superoxide (O2.−) production in a concentration‐dependent manner, as measured by electronic spin resonance (ESR). Using confocal microscopy, gp91phox was found to aggregate in LR clusters upon Hcys stimulation, which was inhibited by lipid‐raft disruptors, methyl‐β ‐cyclodextrin (MCD) and filipin. Functionally, increased O2.− production associated with these LR‐gp91phox platform was also blocked by MCD or filipin. Flow cytometry showed that Hcys induced podocytes apoptosis, which could be attenuated by gp91phox siRNA or LR disruptors. Our results indicate that the formation of gp91phox‐associated LR redox signaling platform importantly contributes to podocytes injury during exposure to high Hcys levels (supported by NIH grants DK54927, HL075316, and HL57244).

ACTIVATION OF ACID SPHINGOMYELINASE DRIVES LYSOSOMAL TRIFFICKING AND FUSION TO MEMBRANE IN CORONARY ENDOTHELIAL CELLS

Recent studies have demonstrated that activation of death receptor results in lysosomal fusion to cell membrane to form a lipid rafts (LRs) signaling platform. However, the driving forces are still unknown. The present study was designed to test a hypothesis that acid sphingomyelinase (ASMase) is firstly activated to produce ceramide, then drives lysosomal fusion and LRs clustering. We first performed confocal microscopy and fluorescence resonance energy transfer (FRET) detection in bovine coronary arterial endothelial cells (BCAECs) and found that FasL significantly led to colocalization of Lamp‐1 and CTX‐B, and their FRET efficiency increased from 5 to 19%. We also found a lysosome‐membrane fusion pattern of fluorescence changes induced by FasL which was blocked by siRNA of ASMase. Both activator and inducer of ASMase markedly increased colocalization of Lamp‐1 with CTX‐B and resulted in lysosome‐membrane fusion. However, the de novo synthesis of ceramide had no such effects. In isolated bovine coronary arteries, endothelium‐dependent vasodilation was impaired after ASMase had been activated, which could be reversed by lysosome functional inhibitor. It is concluded that ASMase activation precedes lysosomal fusion and LRs clustering and may serve as a driving force for lysosome trafficking and fusion to cell membrane in BCAECs. (Supported by NIH Grants HL‐57244, HL‐75316, and DK54927).

β-Glycoglycosphingolipid-Induced Alterations of the STAT Signaling Pathways Are Dependent on CD1d and the Lipid Raft Protein Flotillin-2

Beta-glucosylceramide has been shown to affect natural killer T cell function in models of inflammation. We, therefore, investigated the effects of different beta-glycosphingolipids, including beta-glucosylceramide, on STAT (signal transducers and activators of transcription) signaling pathways and determined whether these effects were mediated by lipid raft microdomains and/or CD1d molecules. The effects of alpha- and beta-structured ligands on the lipid raft protein flotillin-2 were studied in both natural killer T hybridoma cells and leptin-deficient mice. To determine whether CD1d was involved in the effects of the beta-glycosphingolipids, an anti-CD1d blocking antibody was used in a cell proliferation assay system. The downstream effects on the protein phosphorylation levels of STAT1, STAT3, and STAT6 were examined in both immune-mediated hepatitis and hepatoma models. The effects of beta-glycosphingolipids on the STAT signaling pathways were found to be dependent on CD1d. Lipid rafts were affected by both the dose and ratio of the beta-glycosphingolipids and the acyl chain length, and these effects were followed by downstream effects on STAT proteins. Our results show that beta-glycosphingolipids have beneficial effects in natural killer T cell-dependent immune-mediated metabolic and malignant animal models in vivo.

Lipid rafts represent a novel therapeutic target in SLE (50.25)

Abstract Lipid rafts play a crucial role in T cell receptor (TCR) signaling. Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by abnormal TCR signaling. We found that lipid rafts are aggregated on the surface membrane of T cells from lupus-prone MRL/lpr mice. High levels of aggregated lipid rafts could also be reproduced on T cells of normal C57BL/6 mice treated with anti-CD3 plus CD28 antibodies. The aggregated lipid rafts peaked at the age of 6-7 weeks prior to the appearance of disease pathology in MRL/lpr mice. Depletion of lipid rafts with methyl-B-cyclodextrin (MBCD), which is known to disrupt lipid rafts, or atorvastatin, which is known to inhibit cholesterol biosynthesis delayed disease progression, whereas acceleration of their formation with cholera toxin B, which is known to induce lipid raft clustering, promoted disease progression in MRL/lpr mice. The aggregated lipid rafts harbor diverse molecules including TCR signaling, inflammatory, costimulatory, adhesion and Toll-like receptor molecules. Recruited diverse molecules have costimulation to enhance TCR-mediated response and production of IFN-gamma through intact lipid rafts. Further, crosslinking of lipid rafts promoted IFN-gamma production in vitro, a cytokine known to contribute to the expression of SLE pathology in mice. We propose that lipid rafts represent a novel therapeutic target in SLE.

Regulation of Gap Junctions in Porcine Cumulus-Oocyte Complexes: Contributions of Granulosa Cell Contact, Gonadotropins, and Lipid Rafts

Gap-junctional communication (GJC) plays a central role in oocyte growth. However, little is known about the regulation of connexin 43 (Cx43)-based gap-junction channels in cumulus-oocyte complexes (COCs) during in vitro maturation. We show that rupture of COCs from mural granulosa cells up-regulates Cx43-mediated GJC and that gonadotropins signal GJC breakdown by recruiting Cx43 to lipid rafts when oocyte meiosis resumes. Oocyte calcein uptake through gap junctions increases during early in vitro oocyte maturation and remains high until 18 h, when it falls simultaneously with the oocyte germinal vesicle breakdown. Immunodetection of Cx43 and fluorescence recovery after photobleaching assays revealed that the increase of GJC is independent of gonadotropins but requires RNA transcription, RNA polyadenylation, and translation. GJC rupture, in contrast, is achieved by a gonadotropin-dependent mechanism involving recruitment of Cx43 to clustered lipid rafts. These results show that GJC up-regulation in COCs in in vitro culture is independent of gonadotropins and transcriptionally regulated. However, GJC breakdown is gonadotropin dependent and mediated by the clustering of Cx43 in lipid raft microdomains. In conclusion, this study supports a functional role of lipid raft clustering of Cx43 in GJC breakdown in the COCs during in vitro maturation.