Cholesterol-enriched Lipid Rafts Research Articles

Cholesterol Allosterically Modulates the Structure and Dynamics of the Taurocholate Export Pump (ABCB11).

The BSEP/ABCB11 transmembrane protein translocates taurine- and glycine-conjugated bile salts across the hepatocyte bilayer driven by ATP-hydrolysis. Direct inhibition of BSEP/ABCB11 leads to idiosyncratic drug-induced liver injury. ABCB11 is localized within the cholesterol-enriched lipid raft, and membrane cholesterol depletion leads to impaired taurocholate transport. However, structural insight into the mechanism of the cholesterol-mediated regulation of ABCB11 activity remains elusive. We used extensive molecular dynamics simulation coupled with well-tempered metadynamics to elucidate the role of membrane cholesterol in the structure and dynamics of ABCB11. We identified specific high-residence binding sites for cholesterol within the transmembrane domain. The free-energy simulations have elucidated that the bound cholesterol stabilizes the "inward-open" conformation of the protein. Cholesterol-ABCB11 interactions trigger allosteric communications between the transmembrane and nucleotide-binding domains through the linker region. Cholesterol depletion destabilizes the allosteric network of the protein. As a result, it adopts a more collapsed form with a reduced volume of the taurocholate-binding pocket.

Lipidation states orchestrate CLICK-III/CaMKIγ's stepwise association with Golgi and rafts-enriched membranes and specify its functional coupling to STEF-Rac1-dependent neurite extension.

CLICK-III/CaMKIγ is a lipid-anchored neuronal isoform of multifunctional Ca2+/calmodulin-dependent protein kinases, which mediates BDNF-dependent dendritogenesis in cultured cortical neurons. We found that two distinct lipidation states of CaMKIγ, namely, prenylation and palmitoylation, controlled its association with detergent-resistant microdomains in the dendrites and were essential for its dendritogenic activity. However, the impact of each lipid modification on membrane targeting/trafficking and how it specifies functional coupling leading to polarized changes in neuronal morphology are not clear. Here, we show that prenylation induces membrane anchoring of CaMKIγ, permitting access to the Golgi apparatus, and a subsequent palmitoylation facilitates association with cholesterol-enriched lipid microdomains or lipid rafts, in particular at the Golgi. To specifically test the role of palmitoylated CaMKγ in neurite extension, we identified and took advantage of a cell system, PC12, which, unlike neurons, conveniently lacked CaMKIγ and was deficient in the activity-dependent release of a neuritogenic growth factor while possessing the ability to activate polarized rafts signaling for morphogenesis. This system allowed us to rigorously demonstrate that an activity-dependent, lipid rafts-restricted Rac activation leading to neuritogenesis could be functionally rescued by dually lipidated CaMKIγ expression, revealing that not only prenylation but also palmitoylation is essential for CaMKIγ to activate a compartmentalized STEF-Rac1 pathway. These results shed light on the significance of recruiting prenylated and palmitoylated CaMKIγ into the coalescing signalosomes at lipid rafts together with Rac1 and its specific GEF and STEF and forming a compartmentalized Ca2+ signaling pathway that underlies activity-dependent neuritogenesis and morphogenesis during axodendritic polarization critical for brain development and circuitogenesis.

Lipid Raft Integrity and Cellular Cholesterol Homeostasis Are Critical for SARS-CoV-2 Entry into Cells.

Lipid rafts in cell plasma membranes play a critical role in the life cycle of many viruses. However, the involvement of membrane cholesterol-rich lipid rafts in the entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into target cells is not well known. In this study, we investigated whether the presence of cholesterol-rich microdomains is required for the entry of SARS-CoV-2 into host cells. Our results show that depletion of cholesterol in the rafts by methyl-beta-cyclodextrin (MβCD) treatment impaired the expression of the cell surface receptor angiotensin-converting enzyme 2 (ACE2), resulting in a significant increase in SARS-CoV-2 entry into cells. The effects exerted by MβCD could be substantially reversed by exogenous cholesterol replenishment. In contrast, disturbance of intracellular cholesterol homeostasis by statins or siRNA knockdown of key genes involved in the cholesterol biosynthesis and transport pathways reduced SARS-CoV-2 entry into cells. Our study also reveals that SREBP2-mediated cholesterol biosynthesis is involved in the process of SARS-CoV-2 entry in target cells. These results suggest that the host membrane cholesterol-enriched lipid rafts and cellular cholesterol homeostasis are essential for SARS-CoV-2 entry into cells. Pharmacological manipulation of intracellular cholesterol might provide new therapeutic strategies to alleviate SARS-CoV-2 entry into cells.

Lithocholic Acid Alleviates Deoxynivalenol-Induced Lethal Cholesterol Metabolic Abnormalities in IPI-2I Cells.

Deoxynivalenol (DON) is a secondary metabolite of fungi. Ingestion of feed containing DON causes severe intestinal damage in humans and animals, possibly due to cholesterol-enriched lipid raft abnormalities. Cholic acid (CA) and lithocholic acid (LCA) are metabolites of cholesterol transformation, which have been proven to benefit epithelial cell proliferation and reduce intestinal inflammation and lesions. Therefore, we aimed to study the protective roles of CA and LCA administration on the DON-exposed intestinal epithelial cells (IPI-2I) and the underlying mechanisms involved in cholesterol metabolism. We found that LCA pretreatment, but not CA, alleviated the reduction of cell numbers caused by DON exposure. Furthermore, we demonstrate that LCA restored the DON-induced cell apoptosis by reducing the cleaved caspase 3 and cleaved PARP-1 expression. DON-increased cellular cholesterol and bile acid contents were significantly reduced when LCA was co-treated. Further transcriptomic analysis revealed that the aberrant cholesterol homeostasis genes profile was observed in the cells exposed to DON or pretreated with LCA. We also validated that the key genes involved in cholesterol biosynthesis and transformation (cholesterol to bile acids) were strongly inhibited by the LCA treatment in the DON-exposed cells. Together, this study demonstrated that LCA ameliorated DON-caused toxic apoptosis in IPI-2I cells by maintaining cholesterol metabolism. We suggest that as an endogenous metabolite, LCA may be used as a therapeutic and/or integrated into a dietary intervention against mycotoxin toxicity.

Normalization of cholesterol metabolism in spinal microglia alleviates neuropathic pain.

Neuroinflammation is a major component in the transition to and perpetuation of neuropathic pain states. Spinal neuroinflammation involves activation of TLR4, localized to enlarged, cholesterol-enriched lipid rafts, designated here as inflammarafts. Conditional deletion of cholesterol transporters ABCA1 and ABCG1 in microglia, leading to inflammaraft formation, induced tactile allodynia in naive mice. The apoA-I binding protein (AIBP) facilitated cholesterol depletion from inflammarafts and reversed neuropathic pain in a model of chemotherapy-induced peripheral neuropathy (CIPN) in wild-type mice, but AIBP failed to reverse allodynia in mice with ABCA1/ABCG1-deficient microglia, suggesting a cholesterol-dependent mechanism. An AIBP mutant lacking the TLR4-binding domain did not bind microglia or reverse CIPN allodynia. The long-lasting therapeutic effect of a single AIBP dose in CIPN was associated with anti-inflammatory and cholesterol metabolism reprogramming and reduced accumulation of lipid droplets in microglia. These results suggest a cholesterol-driven mechanism of regulation of neuropathic pain by controlling the TLR4 inflammarafts and gene expression program in microglia and blocking the perpetuation of neuroinflammation.

Preferred Endocytosis of Amyloid Precursor Protein from Cholesterol-Enriched Lipid Raft Microdomains.

Amyloid precursor protein (APP) at the plasma membrane is internalized via endocytosis and delivered to endo/lysosomes, where neurotoxic amyloid-β (Aβ) is produced via β-, γ-secretases. Hence, endocytosis plays a key role in the processing of APP and subsequent Aβ generation. β-, γ-secretases as well as APP are localized in cholesterol-enriched lipid raft microdomains. However, it is still unclear whether lipid rafts are the site where APP undergoes endocytosis and whether cholesterol levels affect this process. In this study, we found that localization of APP in lipid rafts was increased by elevated cholesterol level. We also showed that increasing or decreasing cholesterol levels increased or decreased APP endocytosis, respectively. When we labeled cell surface APP, APP localized in lipid rafts preferentially underwent endocytosis compared to nonraft-localized APP. In addition, APP endocytosis from lipid rafts was regulated by cholesterol levels. Our results demonstrate for the first time that cholesterol levels regulate the localization of APP in lipid rafts affecting raft-dependent APP endocytosis. Thus, regulating the microdomain localization of APP could offer a new therapeutic strategy for Alzheimer’s disease.

Role of Death Receptors-associated Lipid Rafts in Oxaliplatin-induced Death Mode Regulation of HepG2 Cells.

We previously showed that oxaliplatin induces necrotic-like cell death in hepatocarcinomas, and combination with ursodexoycholic acid (UDCA) significantly shifts the necrotic-like death to apoptosis. Since cell death mode is crucial on inflammatory responses and chemotherapeutic efficacy, the mechanism underlying determination of cell death mode by UDCA was investigated in this study. Apoptosis or necrosis was determined by apoptotic body formation, caspase-8 activity, LDH release and PI inclusion. The involvement of lipid rafts and death receptors was examined by rafts fractionation, confocal microscopy and gene silencing assays. UDCA combination enhanced recruitment of death receptors and adaptors into cholesterol-enriched lipid rafts, and induced a stronger raft clustering. Lipid raft disruption decreased the UDCA/oxaliplatin-mediated apoptosis and increased necrotic-like death. UDCA promotes lipid raft localization of multiple death receptors, thereby contributing to a shift of cell death mode from oxaliplatin-induced necrotic death to apoptosis in HepG2 cells.

TRPV4 expresses in bone cell lineages and TRPV4-R616Q mutant causing Brachyolmia in human reveals “loss-of-interaction” with cholesterol

Transient receptor potential Vanilloid ion channel sub type 4 (TRPV4) is involved in complex Ca2+-signaling. At least one copy of TRPV4 is present in all vertebrates and is involved in several physiological processes including sensory process and point mutations in TRPV4 leads to development of different pathophysiological disorders in human. R616Q mutant of TRPV4 has been referred as “gain-of-function” mutant causing abnormality in bone cells and develop pathophysiological condition known as “Brachyolmia”. In this work, we demonstrated that R616Q mutation is located in a very critical position of TRPV4 containing a cholesterol-binding motif sequence which is highly conserved in all vertebrates. Accordingly, TRPV4-Wt but not the TRPV4-R616Q localizes preferably in cholesterol-enriched lipid rafts in osteogenic cell line Saos2 and in DRG-neuron derived F11 cell line. Further, FRAP experiment suggest TRPV4-Wt but not the TRPV4-R616Q mutant is more mobile especially in cholesterol-reduced lipid membrane. GST-tagged TM4-Loop-TM5 fragment containing TRPV4-Wt but not R616Q sequence interacts with cholesterol, forms high-molecular weight complex and also show band shift in SDS-PAGE. TRPV4 is expressed in Mesenchymal stem cells and the localization of TRPV4 in lipid raft is dependent on temperature and cholesterol. Our data suggests that TRPV4-R616Q mutant behaves as a “loss-of-interaction” with cholesterol.

Elevated cellular cholesterol in Familial Alzheimer's presenilin 1 mutation is associated with lipid raft localization of β-amyloid precursor protein.

Familial Alzheimer’s disease (FAD)-associated presenilin 1 (PS1) serves as a catalytic subunit of γ-secretase complex, which mediates the proteolytic liberation of β-amyloid (Aβ) from β-amyloid precursor protein (APP). In addition to its proteolytic role, PS1 is involved in non-proteolytic functions such as protein trafficking and ion channel regulation. Furthermore, postmortem AD brains as well as AD patients showed dysregulation of cholesterol metabolism. Since cholesterol has been implicated in regulating Aβ production, we investigated whether the FAD PS1-associated cholesterol elevation could influence APP processing. We found that in CHO cells stably expressing FAD-associated PS1 ΔE9, total cholesterol levels are elevated compared to cells expressing wild-type PS1. We also found that localization of APP in cholesterol-enriched lipid rafts is substantially increased in the mutant cells. Reducing the cholesterol levels by either methyl-β-cyclodextrin or an inhibitor of CYP51, an enzyme mediating the elevated cholesterol in PS1 ΔE9-expressing cells, significantly reduced lipid raft-associated APP. In contrast, exogenous cholesterol increased lipid raft-associated APP. These data suggest that in the FAD PS1 ΔE9 cells, the elevated cellular cholesterol level contributes to the altered APP processing by increasing APP localized in lipid rafts.

Molecular Structure and Permeability at the Interface between Phase-Separated Membrane Domains.

Phase-separated membrane domains, also known as lipid rafts, are believed to play an important role in cell function. Although most rafts are sterol-enriched membrane regions, evidence suggests that living cells may also contain gel-like rafts. Interactions between gel and fluid domains have a large impact on membrane properties, as is the case with permeability. The membrane permeability may reach a peak at the main phase transition temperature, by far exceeding the values recorded at the fluid phase. It has been proposed that gel-fluid interfaces are leaky, but the effect has not yet been demonstrated at the molecular level. Here, we performed atomistic molecular dynamics simulations of phospholipid bilayers with coexisting gel-like and fluid domains. We found that the thickness mismatch between both phases, the membrane elasticity, and the lipid packing acted together to promote the formation of a thickness minimum at the gel-fluid interface. Free energy calculations showed that pore-mediated ionic permeation was strongly facilitated at the constriction region, whereas water permeation by simple diffusion was only marginally affected. Long-lived, peristaltic undulations were recorded at the bulk fluid phase near the main transition temperature. They gave rise to thickness minima that, although shallower than the interface constrictions, could also enhance permeability. Finally, we demonstrated that an interface constriction was also formed at the boundaries of regular, cholesterol-enriched lipid rafts. Our simulation results will hopefully contribute to a better understanding of biological processes such as transport, signaling, and cellular damage promoted by low temperature and dehydration.

Cholesterol's location in lipid bilayers

It is well known that cholesterol modifies the physical properties of lipid bilayers. For example, the much studied liquid-ordered Lo phase contains rapidly diffusing lipids with their acyl chains in the all trans configuration, similar to gel phase bilayers. Moreover, the Lo phase is commonly associated with cholesterol-enriched lipid rafts, which are thought to serve as platforms for signaling proteins in the plasma membrane. Cholesterol's location in lipid bilayers has been studied extensively, and it has been shown – at least in some bilayers – to align differently from its canonical upright orientation, where its hydroxyl group is in the vicinity of the lipid–water interface. In this article we review recent works describing cholesterol's location in different model membrane systems with emphasis on results obtained from scattering, spectroscopic and molecular dynamics studies.

Effect of Cyclodextrins on Membrane Biophysical Properties

Ordered domains enriched in cholesterol and sphingolipids, commonly known as lipid rafts, have been linked to the activity of certain membrane receptors involved, for example, on inflammation and cell death processes. Consequently, the characterization of cell membrane properties and drug/membrane interactions has become increasingly relevant. Cyclodextrins (CDs) are biocompatible hydrophilic molecules with a hydrophobic core able to form inclusion complexes with poorly soluble molecules. As a result, β-CDs, safer for pharmaceutical applications, are currently used for solubilizing and safely administer poorly soluble drugs. Recent evidence indicating a possible anti-inflammatory effect of β-CDs in vivo has prompted the study of the mechanism of action of β-CDs. Knowing that β-CDs have long been used to both extract and insert cholesterol in membranes in vitro, this study is aimed at the effect of β-CDs on the biophysical properties of cell membranes, specifically on the cholesterol-enriched lipid raft domains.This study focused on the β-CD mediated extraction of membrane cholesterol and the consequent effects in global and localised membrane order. The interaction of β-CDs, both free and complexed with a drug, with model membrane systems and cellular membranes was evaluated using fluorescence spectroscopy and microscopy techniques. The results showed that β-CDs effectively removed cholesterol from both model membrane systems and cellular membranes. In model systems, a concomitant increase in overall membrane fluidity and permeability and decrease in the cholesterol-enriched raft-like domains was observed. Furthermore, β-CD complexation with a drug did not limit its effect. The results showed the potential synergistic action of β-CD as both a drug delivery system and disruptor of the lipid raft domains. In this way, we aim to further elucidate the effect of β-CDs on membrane biophysical properties, namely the destabilization of lipid raft domains and cellular processes dependent thereof.

Candida albicans Targets a Lipid Raft/Dectin-1 Platform to Enter Human Monocytes and Induce Antigen Specific T Cell Responses.

Several pathogens have been described to enter host cells via cholesterol-enriched membrane lipid raft microdomains. We found that disruption of lipid rafts by the cholesterol-extracting agent methyl-β-cyclodextrin or by the cholesterol-binding antifungal drug Amphotericin B strongly impairs the uptake of the fungal pathogen Candida albicans by human monocytes, suggesting a role of raft microdomains in the phagocytosis of the fungus. Time lapse confocal imaging indicated that Dectin-1, the C-type lectin receptor that recognizes Candida albicans cell wall-associated β-glucan, is recruited to lipid rafts upon Candida albicans uptake by monocytes, supporting the notion that lipid rafts act as an entry platform. Interestingly disruption of lipid raft integrity and interference with fungus uptake do not alter cytokine production by monocytes in response to Candida albicans but drastically dampen fungus specific T cell response. In conclusion, these data suggest that monocyte lipid rafts play a crucial role in the innate and adaptive immune responses to Candida albicans in humans and highlight a new and unexpected immunomodulatory function of the antifungal drug Amphotericin B.

Lipid Membrane Deformation Accompanied by Disk-to-Ring Shape Transition of Cholesterol-Rich Domains.

During vesicle budding or endocytosis, biomembranes undergo a series of lipid- and protein-mediated deformations involving cholesterol-enriched lipid rafts. If lipid rafts of high bending rigidities become confined to the incipient curved membrane topology such as a bud-neck interface, they can be expected to reform as ring-shaped rafts. Here, we report on the observation of a disk-to-ring shape morpho-chemical transition of a model membrane in the absence of geometric constraints. The raft shape transition is triggered by lateral compositional heterogeneity and is accompanied by membrane deformation in the vertical direction, which is detected by height-sensitive fluorescence interference contrast microscopy. Our results suggest that a flat membrane can become curved simply by dynamic changes in local chemical composition and shape transformation of cholesterol-rich domains.

The assembly of GM1 glycolipid- and cholesterol-enriched raft-like membrane microdomains is important for giardial encystation.

Although encystation (or cyst formation) is an important step of the life cycle of Giardia, the cellular events that trigger encystation are poorly understood. Because membrane microdomains are involved in inducing growth and differentiation in many eukaryotes, we wondered if these raft-like domains are assembled by this parasite and participate in the encystation process. Since the GM1 ganglioside is a major constituent of mammalian lipid rafts (LRs) and known to react with cholera toxin B (CTXB), we used Alexa Fluor-conjugated CTXB and GM1 antibodies to detect giardial LRs. Raft-like structures in trophozoites are located in the plasma membranes and on the periphery of ventral discs. In cysts, however, they are localized in the membranes beneath the cyst wall. Nystatin and filipin III, two cholesterol-binding agents, and oseltamivir (Tamiflu), a viral neuraminidase inhibitor, disassembled the microdomains, as evidenced by reduced staining of trophozoites with CTXB and GM1 antibodies. GM1- and cholesterol-enriched LRs were isolated from Giardia by density gradient centrifugation and found to be sensitive to nystatin and oseltamivir. The involvement of LRs in encystation could be supported by the observation that raft inhibitors interrupted the biogenesis of encystation-specific vesicles and cyst production. Furthermore, culturing of trophozoites in dialyzed medium containing fetal bovine serum (which is low in cholesterol) reduced raft assembly and encystation, which could be rescued by adding cholesterol from the outside. Our results suggest that Giardia is able to form GM1- and cholesterol-enriched lipid rafts and these raft domains are important for encystation.

DAMP, an acidotropic pH indicator, can be used as a tool to visualize non-esterified cholesterol in cells.

Cholesterol-rich regions are attractive targets for studying metabolic disorders that involve accumulation of cholesterol. Despite efforts to develop probes for labelling cholesterol-rich regions in cells, few of these reagents have a low molecular weight. Previous studies have shown that the acidotropic pH indicator, N-{3-[(2,4-dinitrophenyl)amino]propyl}-N-(3-aminopropyl)methylamine dihydrochloride (DAMP), reacts with cholesterol-rich organelles, such as endocrine secretary granules from endocrine cells. In this study, we demonstrated that DAMP could react with free cholesterol in a dose-dependent manner, and DAMP was able to detect cholesterol-rich subcellular organelles. DAMP was sufficiently potent to detect free cholesterol-enriched organs, but was unable to detect atherosclerotic plaques primarily composed of esterified cholesterol. Taken together, these results demonstrate that DAMP facilitates the study of cholesterol-enriched lipid rafts and disorders which involve cholesterol accumulation.

Loss of functional MYO1C/myosin 1c, a motor protein involved in lipid raft trafficking, disrupts autophagosome-lysosome fusion

MYO1C, a single-headed class I myosin, associates with cholesterol-enriched lipid rafts and facilitates their recycling from intracellular compartments to the cell surface. Absence of functional MYO1C disturbs the cellular distribution of lipid rafts, causes the accumulation of cholesterol-enriched membranes in the perinuclear recycling compartment, and leads to enlargement of endolysosomal membranes. Several feeder pathways, including classical endocytosis but also the autophagy pathway, maintain the health of the cell by selective degradation of cargo through fusion with the lysosome. Here we show that loss of functional MYO1C leads to an increase in total cellular cholesterol and its disrupted subcellular distribution. We observe an accumulation of autophagic structures caused by a block in fusion with the lysosome and a defect in autophagic cargo degradation. Interestingly, the loss of MYO1C has no effect on degradation of endocytic cargo such as EGFR, illustrating that although the endolysosomal compartment is enlarged in size, it is functional, contains active hydrolases, and the correct pH. Our results highlight the importance of correct lipid composition in autophagosomes and lysosomes to enable them to fuse. Ablating MYO1C function causes abnormal cholesterol distribution, which has a major selective impact on the autophagy pathway.

Amplified lipid rafts of malignant cells constitute a target for inhibition of aberrantly active NFAT and melanoma tumor growth by the aminobisphosphonate zoledronic acid.

Nuclear factors of activated T cells (NFAT) are critical modulators of cancer cell growth and survival. However, the mechanisms of their oncogenic dysregulation and strategies for targeting in tumors remain elusive. Here, we report coupling of anti- apoptotic NFAT (NFAT2) activation to cholesterol-enriched lipid raft microdomains of malignant melanoma cells and interruption of this pathway by the aminobisphosphonate zoledronic acid (Zol). The pathway was indicated by capability of Zol to promote apoptosis and to retard in vivo outgrowth of tumorigenic melanoma cell variants through inhibition of permanently active NFAT2. NFAT2 inhibition resulted from disintegration of cholesterol-enriched rafts due to reduction of cellular cholesterol by Zol. Mechanistically, raft disruption abolished raft-localized robust store-operated Ca(2+) (SOC) entry, blocking constitutive activation of protein kinase B/Akt (PKB) and thereby reactivating the NFAT repressor glycogen synthase kinase 3β (GSK3β). Pro-apoptotic inactivation of NFAT2 also followed reactivation of GSK3β by direct inhibition of PKB or SOC, whereas GSK3β blockade prevented Zol-induced NFAT2 inhibition and cell death. The rescuing effect of GSK3β blockade was reproduced by recovery of entire SOC/PKB/GSK3β cascade after reconstitution of rafts by cholesterol replenishment of Zol-treated tumorigenic cells. Remarkably, these malignant cells displayed higher cholesterol and lipid raft content than non-tumorigenic cells, which expressed weak SOC, PKB and NFAT2 activities and resisted raft-ablating action of Zol. Together, the results underscore the functional relevance of amplified melanoma rafts for tumor-promoting NFAT2 signaling and reveal these distinctive microdomains as a target for in vitro and in vivo demise of tumorigenic cells through NFAT2 inhibition by the clinical agent Zol.

Possible protective effect of membrane lipid rafts against interleukin-1β-mediated anti-proliferative effect in INS-1 cells.

We recently reported that pancreatic islets from pre-diabetic rats undergo an inflammatory process in which IL-1β takes part and controls β-cell function. In the present study, using the INS-1 rat pancreatic β-cell line, we investigated the potential involvement of membrane-associated cholesterol-enriched lipid rafts in IL-1β signaling and biological effects on insulin secretion, β-cell proliferation and apoptosis. We show that, INS-1 cells exposure to increasing concentrations of IL-1β leads to a progressive inhibition of insulin release, an increase in the number of apoptotic cells and a dose-dependent decrease in pancreatic β-cell proliferation. Disruption of membrane lipid rafts markedly reduced glucose-stimulated insulin secretion but did not affect either cell apoptosis or proliferation rate, demonstrating that membrane lipid raft integrity is essential for β-cell secretory function. In the same conditions, IL-1β treatment of INS-1 cells led to a slight further decrease in insulin secretion for low concentrations of the cytokine, and a more marked one, similar to that observed in normal cells for higher concentrations. These effects occurred together with an increase in iNOS expression and surprisingly with an upregulation of tryptophane hydroxylase and protein Kinase C in membrane lipid rafts suggesting that compensatory mechanisms develop to counteract IL-1β inhibitory effects. We also demonstrate that disruption of membrane lipid rafts did not prevent cytokine-induced cell death recorded after exposure to high IL-1β concentrations. Finally, concerning cell proliferation, we bring strong evidence that membrane lipid rafts exert a protective effect against IL-1β anti-proliferative effect, possibly mediated at least partly by modifications in ERK and PKB expression/activities. Our results 1) demonstrate that IL-1β deleterious effects do not require a cholesterol-dependent plasma membrane compartmentalization of IL-1R1 signaling and 2) confer to membrane lipid rafts integrity a possible protective function that deserves to be considered in the context of inflammation and especially T2D pathogenesis.

The Basic Domain of HIV-Tat Transactivating Protein Is Essential for Its Targeting to Lipid Rafts and Regulating Fibroblast Growth Factor-2 Signaling in Podocytes Isolated from Children with HIV-1–Associated Nephropathy

Podocyte injury has a critical role in the pathogenesis of HIV-associated nephropathy (HIVAN). The HIV-1 transactivator of transcription (Tat), combined with fibroblast growth factor-2 (FGF-2), can induce the dedifferentiation and proliferation of cultured human podocytes. Cellular internalization of Tat requires interactions with heparan sulfate proteoglycans and cholesterol-enriched lipid rafts (LRs). However, the specific distribution of Tat in human podocytes and its ability to associate with LRs have not been documented. Here, we found that Tat is preferentially recruited to LRs in podocytes isolated from children with HIVAN. Furthermore, we identified arginines in the basic domain (RKKRRQRRR) of Tat as essential for (1) targeting Tat to LRs, (2) Tat-mediated increases in the expression of Rho-A and matrix metalloproteinase-9 in LRs, and (3) Tat-mediated enhancement of FGF-2 signaling in human podocytes and HIV-transgenic mouse kidneys and the exacerbation of renal lesions in these mice. Tat carrying alanine substitutions in the basic domain (AKKAAQAAA) remained localized in the cytosol and did not associate with LRs or enhance FGF-2 signaling in cultured podocytes. These results show the specific association of Tat with LRs in podocytes isolated from children with HIVAN, confirm Tat as a regulator of FGF-2 signaling in LRs, and identify the key domain of Tat responsible for promoting these effects and aggravating renal injury in HIV-transgenic mice. Moreover, these results provide a molecular framework for developing novel therapies to improve the clinical outcome of children with HIVAN.