Cholesterol-enriched Membrane Microdomains Research Articles

Cholesterol-enriched microdomains regulate pseudopod extension in the MSP-based cytoskeleton of amoeboid sperm

In the amoeboid spermatozoa from Caenorhabditis elegans, motility acquisition is preceded by substantial rearrangement of the plasma membrane. The current genetic model posits a multicomponent complex of membrane and cytoplasmic proteins responsible for pseudopod extension. This model can be translated into a physiological context through the involvement of cholesterol-enriched signaling platforms. We show that discrete cholesterol-enriched microdomains are present in C. elegans spermatids. These microdomains redistributed towards the cell body upon pseudopod extension resulting in a phospholipid-enriched pseudopod. Cholesterol saturation in the spermatids prevented pseudopod extension and motility acquisition, whereas cholesterol depletion increased the rate of in vitro pseudopod extension. This work suggests that plasma membrane cholesterol plays an important role in regulating the membrane dynamics that precede pseudopod extension and motility acquisition.

Proteomics Annotation of Lipid Rafts Modified by Virus Infection

Lipid rafts are sphingolipid- and cholesterol-enriched membrane microdomains, which are involved in entry, assembly and budding of various types of viruses. Identification of rafts associated proteins modified by virus infection could therefore provide novel insights into the mechanisms of virus infection as well as the development of new biomarkers for diagnosis and drug development. Proteomic approaches, such as LC-ESI-MS/MS, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), two-dimensional difference gel electrophoresis (2D-DIGE), isotope-coded affinity tags (ICAT), Proteolytic ¹⁸O Labeling, isotope Tags for Relative and Absolute Quantification (iTRAQ), Stable Isotope Labeling with Amino acids in Cell culture (SILAC) and Multidimensional Protein Identification Technology (MudPIT), provide the large scale and unbiased platform to determine the dynamic profiles of the lipid rafts proteome. In this review, we summarized the research advance regarding modern proteomics analysis of host lipid rafts alterations in response to diverse virus infection.

Uncommon membrane distribution of Shiga toxin glycosphingolipid receptors in toxin-sensitive human glomerular microvascular endothelial cells

Membrane microdomain association of the glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), the highly and less effective receptors, respectively, for Shiga toxins (Stxs), is assumed as a functional requirement for Stx-mediated cytotoxicity. In a previous study, we demonstrated predominant localization of Stx receptors in cholesterol-enriched membrane microdomains of moderately Stx-sensitive human brain microvascular endothelial cells (HBMECs) by means of detergent-resistant membranes (DRMs). Here we report a different preferential distribution of Stx receptors in non-DRM fractions of human glomerular microvascular endothelial cells (GMVECs), the major targets of Stxs in the human kidney. Full structural characterization of Stx receptors using electrospray ionization (ESI) mass spectrometry revealed Gb3Cer and Gb4Cer lipoforms with ceramide moieties mainly composed of C24:0/C24:1 or C16:0 fatty acid and sphingosine (d18:1) in GMVECs comparable to those previously found in HBMECs. Thin-layer chromatography immunostaining demonstrated an approximately 2-fold higher content of Gb3Cer and a 1.4-fold higher content of Gb4Cer in GMVECs than in HBMECs. However, this does not explain the remarkable higher cytotoxic action of Stx1 and Stx2 toward GMVECs as compared with HBMECs. Our finding opens new questions on the microdomain association of Stx receptors and the functional role of GSLs in the membrane assembly of GMVECs.

The Role of Lipid Rafts in Virus Assembly. Identification and Characterization of Microdomain Partitioning Factors of the HIV-1 Glycoprotein gp41 using Flim-FRET and Fluorescence Anisotropy Microscopy

Recent experimental results indicate that host cell invasion as well as assembly and budding of the Human Immunodeficiency Virus (HIV) are highly cholesterol dependent. Supposably, cholesterol enriched plasma membrane microdomains, so called rafts, play an important role in different steps of the virus lifecycle. However, the exact function and molecular background of this sensitivity to bilayer composition remains unknown.We produced different variants of the HIV transmembrane protein gp41 labelled with a yellow fluorescent protein. Fluorescence lifetime imaging microscopy was used to report Forster Resonance Energy Transfer (FRET) between a raft marker labelled with a cyan fluorescent protein and gp41 chimeras in living cells. Since it is highly distance dependent, occurring FRET reflects a co-clustering of both fluorescent protein species in microdomains. By comparison of FRET efficiencies from different truncation and mutation variants of gp41, the Cholesterol Recognition Amino Acid Consensus (CRAC) was identified as main determinant of the protein's raft partitioning. Whereas localization and trafficking of the fusion proteins resembled reported wildtype behaviour, FACS experiments revealed a remarkable influence of CRAC mutations on plasma membrane perturbation properties of gp41. Furthermore, using fluorescence polarization anisotropy microscopy it could be shown, that wildtype gp41 oligomerization occurs at the plasma membrane. Oligomerization of CRAC mutants was found to be significantly impaired. This suggests a pooling function of lipid rafts not only for interactions with other viral components but also for assemblies of functional homo-oligomers.This study is to our knowledge the first live cell approach characterizing gp41 raft partitioning factors and relating lateral plasma membrane sorting to distinct protein functions and properties.The reported raft dependent oligomerization might be representative for general mechanisms of microdomain-facilitated protein interactions.

Functional Aspects of Membrane Association of Reggie/Flotillin Proteins

Flotillin-2 and flotillin-1, also called reggie-1 and reggie-2, are ubiquitously expressed and highly conserved proteins. Originally, they were described as neuronal regeneration proteins, but they appear to function in a wide variety of cellular processes, such as membrane receptor signaling, endocytosis, phagocytosis and cell adhesion. The molecular details of the function of flotillins in these processes have only been partially clarified. Flotillins are associated with cholesterol and sphingolipid enriched membrane microdomains known as rafts, and some findings even suggest that they define their own kind of a microdomain. The mechanism of the membrane association of flotillins appears to rely mainly on acylation (myristoylation and/or palmitoylation), localizing flotillins onto the cytosolic side of the membranes, whereas no transmembrane domains are present. In addition, flotillins show a strong tendency to form homo- and hetero-oligomers with each other. In this review, we will summarize the recent findings on the function of flotillins and discuss the mechanisms that might regulate their function, such as membrane association, oligomerization and phosphorylation.

Lipid Raft Redox Signaling: Molecular Mechanisms in Health and Disease

Lipid rafts, the sphingolipid and cholesterol-enriched membrane microdomains, are able to form different membrane macrodomains or platforms upon stimulations, including redox signaling platforms, which serve as a critical signaling mechanism to mediate or regulate cellular activities or functions. In particular, this raft platform formation provides an important driving force for the assembling of NADPH oxidase subunits and the recruitment of other related receptors, effectors, and regulatory components, resulting, in turn, in the activation of NADPH oxidase and downstream redox regulation of cell functions. This comprehensive review attempts to summarize all basic and advanced information about the formation, regulation, and functions of lipid raft redox signaling platforms as well as their physiological and pathophysiological relevance. Several molecular mechanisms involving the formation of lipid raft redox signaling platforms and the related therapeutic strategies targeting them are discussed. It is hoped that all information and thoughts included in this review could provide more comprehensive insights into the understanding of lipid raft redox signaling, in particular, of their molecular mechanisms, spatial-temporal regulations, and physiological, pathophysiological relevances to human health and diseases.

Insulin signaling in retinal neurons is regulated within cholesterol-enriched membrane microdomains

Neuronal cell death is an early pathological feature of diabetic retinopathy. We showed previously that insulin receptor signaling is diminished in retinas of animal models of diabetes and that downstream Akt signaling is involved in insulin-mediated retinal neuronal survival. Therefore, further understanding of the mechanisms by which retinal insulin receptor signaling is regulated could have therapeutic implications for neuronal cell death in diabetes. Here, we investigate the role of cholesterol-enriched membrane microdomains to regulate PKC-mediated inhibition of Akt-dependent insulin signaling in R28 retinal neurons. We demonstrate that PKC activation with either a phorbol ester or exogenous application of diacylglycerides impairs insulin-induced Akt activation, whereas PKC inhibition augments insulin-induced Akt activation. To investigate the mechanism by which PKC impairs insulin-stimulated Akt activity, we assessed various upstream mediators of Akt signaling. PKC activation did not alter the tyrosine phosphorylation of the insulin receptor or IRS-2. Additionally, PKC activation did not impair phosphatidylinositol 3-kinase activity, phosphoinositide-dependent kinase phosphorylation, lipid phosphatase (PTEN), or protein phosphatase 2A activities. Thus, we next investigated a biophysical mechanism by which insulin signaling could be disrupted and found that disruption of lipid microdomains via cholesterol depletion blocks insulin-induced Akt activation and reduces insulin receptor tyrosine phosphorylation. We also demonstrated that insulin localizes phosphorylated Akt to lipid microdomains and that PMA reduces phosphorylated Akt. In addition, PMA localizes and recruits PKC isotypes to these cholesterol-enriched microdomains. Taken together, these results demonstrate that both insulin-stimulated Akt signaling and PKC-induced inhibition of Akt signaling depend on cholesterol-enriched membrane microdomains, thus suggesting a putative biophysical mechanism underlying insulin resistance in diabetic retinopathy.

Flotillins play an essential role in Niemann-Pick C1-like 1-mediated cholesterol uptake

Dietary absorption is a major way for mammals to obtain cholesterol, which is mediated by Niemann-Pick C1-like 1 (NPC1L1) via vesicular endocytosis. One fundamental question in this process is how free cholesterol is efficiently taken up through the internalization of NPC1L1. Using exogenously expressed NPC1L1-EGFP, we show that the lipid raft proteins flotillins associate with NPC1L1 and their localization is regulated by NPC1L1 during intracellular trafficking. Furthermore, flotillins are essential for NPC1L1-mediated cellular cholesterol uptake, biliary cholesterol reabsorption, and the regulation of lipid levels in mice. Together with NPC1L1, they form cholesterol-enriched membrane microdomains, which function as carriers for bulk of cholesterol. The hypocholesterolemic drug ezetimibe disrupts the association between NPC1L1 and flotillins, which blocks the formation of the cholesterol-enriched microdomains. Our findings reveal a functional role of flotillins in NPC1L1-mediated cholesterol uptake and elucidate the formation of NPC1L1-flotillins-postive cholesterol-enriched membrane microdomains as a mechanism for efficient cholesterol absorption.

Impact of KCNE subunits on KCNQ1 (Kv7.1) channel membrane surface targeting

The KCNQ1 (Kv7.1) channel plays an important role in cardiovascular physiology. Cardiomyocytes co-express KCNQ1 with KCNE1-5 proteins. KCNQ1 may co-associate with multiple KCNE regulatory subunits to generate different biophysically and pharmacologically distinct channels. Increasing evidence indicates that the location and targeting of channels are important determinants of their function. In this context, the presence of K(+) channels in sphingolipid-cholesterol-enriched membrane microdomains (lipid rafts) is under investigation. Lipid rafts are important for cardiovascular functioning. We aimed to determine whether KCNE subunits modify the localization and targeting of KCNQ1 channels in lipid rafts microdomains. HEK-293 cells were transiently transfected with KCNQ1 and KCNE1-5, and their traffic and presence in lipid rafts were analyzed. Only KCNQ1 and KCNE3, when expressed alone, co-localized in raft fractions. In addition, while KCNE2 and KCNE5 notably stained the cell surface, KCNQ1 and the rest of the KCNEs showed strong intracellular retention. KCNQ1 targets multiple membrane surface microdomains upon association with KCNE peptides. Thus, while KCNQ1/KCNE1 and KCNQ1/KCNE2 channels target lipid rafts, KCNQ1 associated with KCNE3-5 did not. Channel membrane dynamics, analyzed by fluorescence recovery after photobleaching (FRAP) experiments, further supported these results. In conclusion, the trafficking and targeting pattern of KCNQ1 can be influenced by its association with KCNEs. Since KCNQ1 is crucial for cardiovascular physiology, the temporal and spatial regulations that different KCNE subunits may confer to the channels could have a dramatic impact on membrane electrical activity and putative endocrine regulation.

Critical role of lipid rafts in virus entry and activation of phosphoinositide 3′ kinase/Akt signaling during early stages of Japanese encephalitis virus infection in neural stem/progenitor cells

Japanese encephalitis virus (JEV), the leading cause of acute encephalitis in South-East Asia is a neurotropic virus infecting various CNS cell types. Most Flaviviruses including JEV get internalised into cells by receptor-mediated endocytosis, which involve clathrin and membrane cholesterol. The cholesterol-enriched membrane microdomains referred to as lipid rafts act as portals for virus entry in a number of enveloped viruses, including Flavivirus. However, the precise role played by membrane lipid rafts in JEV internalisation into neural stem cells is still unknown. We have established neural stem/progenitor cells and C17.2 cell line as models of productive JEV infection. Increase in membrane fluidity and clustering of viral envelope proteins in lipid rafts was observed in early time points of infection. Localisation of non-structural proteins to rafts at later infection stages was also observed. Co-localisation of JEV glycoprotein with Cholera toxin B confirmed that JEV internalisation occurs in a lipid-raft dependent manner. Though JEV entry is raft dependent, however, there is requirement of functional clathrin during endocytosis inside the cells. Besides virus entry, the lipid rafts act as signalling platforms for Src tyrosine kinases and result in activation of phosphoinositìde 3'-kinase/Akt signalling during early JEV infection. Disruption of lipid raft formation by cholesterol depletion using Methyl β-cyclodextrin, reduced JEV RNA levels and production of infectious virus particles as well as impaired phosphoinositìde 3'-kinase/Akt signalling during initial infection. Overall, our results implicate the importance of host membrane lipid rafts in JEV entry and life cycle, besides maintaining survival of neural stem/progenitor cells during early infection.

Modulation of the Cardiac Transient Outward Potassium Current by CaMKII is Dependent on Lipid Rafts Integrity

The Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylates the Kv4.2/Kv4.3 channel and slows down the cardiac Ito current inactivation. Thereby the regulation of the Ito channel by CaMKII regulates the duration of the plateau phase of the action potential and the calcium entry into the cell. The expression of the CaMKII is very high in the heart, therefore the compartmentalization is essential to get its specificity. We hypothesized that the Ito channel forming proteins Kv4.2/Kv4.3 and CaMKII colocalized within the cholesterol enriched membrane microdomains named lipid rafts. We used freshly isolated ventricular myocytes isolated from Sprague-Dawley rats. Ito current recordings were made by the Patch-Clamp technique. Membrane rafts were isolated by centrifugation in a discontinuous sucrose density gradient. Protein-protein interactions were determined by co-immunoprecipitation. The different proteins were visualized by western blot. The Kv4.2, Kv4.3 and CaMKII proteins were localized by immunohistochemistry. Pach-Clamp recordings show that cholesterol depleting agent metil-β-cyclodextrine, eliminates the CaMKII effect on Ito. This result indicates that the Ito channel and CaMKII are localized in lipid rafts. In contrast, when we incubate the cells with colchicine, a microtubule disrupting agent that internalizes caveolae, the CaMKII effect on Ito is not modified. Separation in density gradients show that the CaMKII is localized in lipid rafts as well as the Kv4.2/Kv4.3 channels. In the co-immunoprecipitation experiments we observe that CaMKII is pulled down with Kv4.2/Kv4.3, but not with caveolin. Immunocitochemistry experiments show that there are two populations of Kv4.2/Kv4.3 channels. The CaMKII regulates the population localized in non-caveolar lipid rafts, whereas a different population is localized in caveolae and is not regulated by CaMKII. Supported by a MEC grant (SAF2007-61159).

Application of displacement chromatography for the analysis of a lipid raft proteome

Defining membrane proteomes is fundamental to understand the role of membrane proteins in biological processes and to find new targets for drug development. Usually multidimensional chromatography using step or gradient elution is applied for the separation of tryptic peptides of membrane proteins prior to their mass spectrometric analysis. Displacement chromatography (DC) offers several advantages that are helpful for proteome analysis. However, DC has so far been applied for proteomic investigations only in few cases. In this study we therefore applied DC in a multidimensional LC-MS approach for the separation and identification of membrane proteins located in cholesterol-enriched membrane microdomains (lipid rafts) obtained from rat kidney by density gradient centrifugation. The tryptic peptides were separated on a cation-exchange column in the displacement mode with spermine used as displacer. Fractions obtained from DC were analyzed using an HPLC-chip system coupled to an electrospray-ionization ion-trap mass spectrometer. This procedure yielded more than 400 highly significant peptide spectrum matches and led to the identification of more than 140 reliable protein hits within an established rat kidney lipid raft proteome. The majority of identified proteins were membrane proteins. In sum, our results demonstrate that DC is a suitable alternative to gradient elution separations for the identification of proteins via a multidimensional LC-MS approach.

Lipid Rafts and Caveolin-1 Are Required for Invadopodia Formation and Extracellular Matrix Degradation by Human Breast Cancer Cells

Invadopodia are ventral membrane protrusions through which invasive cancer cells degrade the extracellular matrix. They are thought to function in the migration of cancer cells through tissue barriers, which is necessary for cancer invasion and metastasis. Although many protein components of invadopodia have been identified, the organization and the role of membrane lipids in invadopodia are not well understood. In this study, the role of lipid rafts, which are cholesterol-enriched membrane microdomains, in the assembly and function of invadopodia in human breast cancer cells was investigated. Lipid rafts are enriched, internalized, and dynamically trafficked at invadopodia sites. Perturbation of lipid raft formation due to depleting or sequestering membrane cholesterol blocked the invadopodia-mediated degradation of the gelatin matrix. Caveolin-1 (Cav-1), a resident protein of lipid rafts and caveolae, accumulates at invadopodia and colocalizes with the internalized lipid raft membranes. Membrane type 1 matrix metalloproteinase (MT1-MMP), a matrix proteinase associated with invadopodia, is localized at lipid raft-enriched membrane fractions and cotrafficked and colocalized with Cav-1 at invadopodia. The small interfering RNA-mediated silencing of Cav-1 inhibited the invadopodia-mediated and MT1-MMP-dependent degradation of the gelatin matrix. Furthermore, Cav-1 and MT1-MMP are coexpressed in invasive human breast cancer cell lines that have an ability to form invadopodia. These results indicate that invadopodia are the sites where enrichment and trafficking of lipid rafts occur and that Cav-1 is an essential regulator of MT1-MMP function and invadopodia-mediated breast cancer cell invasion.

Urokinase-receptor-mediated phenotypic changes in vascular smooth muscle cells require the involvement of membrane rafts

The cholesterol-enriched membrane microdomains lipid rafts play a key role in cell activation by recruiting and excluding specific signalling components of cell-surface receptors upon receptor engagement. Our previous studies have demonstrated that the GPI (glycosylphosphatidylinositol)-linked uPAR [uPA (urokinase-type plasminogen activator) receptor], which can be found in lipid rafts and in non-raft fractions, can mediate the differentiation of VSMCs (vascular smooth muscle cells) towards a pathophysiological de-differentiated phenotype. However, the mechanism by which uPAR and its ligand uPA regulate VSMC phenotypic changes is not known. In the present study, we provide evidence that the molecular machinery of uPAR-mediated VSMC differentiation employs lipid rafts. We show that the disruption of rafts in VSMCs by membrane cholesterol depletion using MCD (methyl-beta-cyclodextrin) or filipin leads to the up-regulation of uPAR and cell de-differentiation. uPAR silencing by means of interfering RNA resulted in an increased expression of contractile proteins. Consequently, disruption of lipid rafts impaired the expression of these proteins and transcriptional activity of related genes. We provide evidence that this effect was mediated by uPAR. Similar effects were observed in VSMCs isolated from Cav1Z(-/-) (caveolin-1-deficient) mice. Despite the level of uPAR being significantly higher after the disruption of the rafts, uPA/uPAR-dependent cell migration was impaired. However, caveolin-1 deficiency impaired only uPAR-dependent cell proliferation, whereas cell migration was strongly up-regulated in these cells. Our results provide evidence that rafts are required in the regulation of uPAR-mediated VSMC phenotypic modulations. These findings suggest further that, in the context of uPA/uPAR-dependent processes, caveolae-associated and non-associated rafts represent different signalling membrane domains.

Mycobacterium tuberculosis Entry into Mast Cells Through Cholesterol‐rich Membrane Microdomains

Cholesterol-enriched membrane microdomains (lipid rafts) play a role in the uptake of many pathogens. Mycobacteria are one of the intracellular pathogens that utilize lipid rafts in order to invade both phagocytic and non-phagocytic cells. However, the mechanism of Mycobacterium tuberculosis uptake by mast cell is not known. To address this issue, we investigated the interaction of M. tuberculosis (H37Rv strain) with mast cells. Confocal microscopy showed that interaction of mycobacterium with mast cell resulted in changes in the mast cell surface, with formation of pseudopod-like structure and activation with visibly extruded granules. Moreover, infection of mast cells with Mycobacteria induced cholesterol accumulation at the site of bacterial entry and around intracellular mycobacteria. Disruption of mast cells lipid rafts by cholesterol depletion markedly inhibited the mycobacterium entry. Intracellular multiplication of M. tuberculosis within mast cells was also observed. Overall, our results indicate that M. tuberculosis employs a cholesterol-dependent pathway to infect mast cells, which leads to degranulation and mast cell morphological changes. These results suggest that although mast cells are capable to respond to M. tuberculosis infection, entry of mycobacterium through lipid rafts may allow replication within mast cells.

Modulation of the Cardiac Transient Outward Potassium Current by Alpha1-Adrenoceptors Requires Caveolae Integrity

The alpha1-adrenoceptor is critically involved in controlling cardiac muscle contraction and excitability. In ventricular myocytes, alpha1-adrenoceptors stimulate Gs proteins and reduce the transient outward K+ current (Ito) via a cAMP/PKA-mediated pathway. This alpha1/cAMP response seems to be compartmentalized as adrenoceptor stimulation increases cAMP levels only in localized membrane regions. Moreover, alpha1-agonists have no effect on Ito amplitude when myocytes are pre-treated with the microtubule-disrupting agent colchicine.We tested the possibility that the Ito channel forming proteins Kv4.2 and Kv4.3, as well as the components of the alpha1/cAMP pathway colocalize within the cholesterol enriched membrane microdomains named lipid rafts.We used freshly isolated ventricular myocytes from Sprague Dawley rats. Ito current recordings were made by the whole-cell patch-clamp technique. Membrane rafts were isolated by centrifugation in a discontinuous sucrose density gradient. The presence of the different proteins was visualized by western blot techniques, and protein-protein interactions were determined by coimmunoprecipitation experiments.Patch-Clamp recordings show that cyclodextrine, colchicine and Ht31 block the alpha1-adrenoceptor effect on the Ito current. These results indicate that the Ito channel is locked to the PKA by an A Kinase Anchoring Protein (AKAP), and that the signalling complex is localized in a specific subtype of lipid rafts named caveolae. Separation in density gradients and coimmunoprecipitation experiments show that the components of the alpha1/Ito pathway organize into two separated groups within the lipid rafts. AKAPm, PKA and the Kv4.2/Kv4.3 channel form a supramolecular complex that interacts with caveolin-3, whereas adrenoceptor, Gs protein and AC gather in a second group also connected to the caveolin. Caveolin-3, therefore, maintains both groups of assembled proteins in close proximity, allowing the functional response of the channel to the neurotransmitter.

Quantitative Proteomics Analysis of Macrophage Rafts Reveals Compartmentalized Activation of the Proteasome and of Proteasome-mediated ERK Activation in Response to Lipopolysaccharide

Lipopolysaccharide (LPS), a glycolipid component of the outer membrane of Gram-negative bacteria, is a potent initiator of the innate immune response of the macrophage. LPS triggers downstream signaling by selectively recruiting and activating proteins in cholesterol-rich membrane microdomains called lipid rafts. We applied proteomics analysis to macrophage detergent-resistant membranes (DRMs) during an LPS exposure time course in an effort to identify and validate novel events occurring in macrophage rafts. Following metabolic incorporation in cell culture of heavy isotopes of amino acids arginine and lysine ([(13)C(6)]Arg and [(13)C(6)]Lys) or their light counterparts, a SILAC (stable isotope labeling with amino acids in cell culture)-based quantitative, liquid chromatography-tandem mass spectrometry proteomics approach was used to profile LPS-induced changes in the lipid raft proteome of RAW 264.7 macrophages. Unsupervised network analysis of the proteomics data set revealed a marked representation of the ubiquitin-proteasome system as well as changes in proteasome subunit composition following LPS challenge. Functional analysis of DRMs confirmed that LPS causes selective activation of the proteasome in macrophage rafts and proteasome inactivation outside of rafts. Given previous reports of an essential role for proteasomal degradation of IkappaB kinase-phosphorylated p105 in LPS activation of ERK mitogen-activated protein kinase, we tested for a role of rafts in compartmentalization of these events. Immunoblotting of DRMs revealed proteasome-dependent activation of MEK and ERK specifically occurring in lipid rafts as well as proteasomal activity upon raft-localized p105 that was enhanced by LPS. Cholesterol extraction from the intact macrophage with methyl-beta-cyclodextrin was sufficient to activate ERK, recapitulating the LPS-IkappaB kinase-p105-MEK-ERK cascade, whereas both it and the alternate raft-disrupting agent nystatin blocked subsequent LPS activation of the ERK cascade. Taken together, our findings indicate a critical, selective role for raft compartmentalization and regulation of proteasome activity in activation of the MEK-ERK pathway.

Cholesterol and Cholesterol-Rich Membranes in Prostate Cancer: An Update

Cells maintain normal structure and function by responding appropriately to cues from the surrounding milieu. Extracellular stimuli are transduced from the surface through the plasma membrane by a complex series of interactions between ligands, their receptors and intracellular signaling partners (e.g., kinases, G proteins). Cholesterol-enriched membrane microdomains, generally referred to as "lipid rafts", exist within the lipid bilayer of all mammalian cells and play an important role in signaling from the cell surface to various subcellular compartments. Lipid rafts have also been implicated in tumor growth and aggressiveness. Epidemiological evidence suggests that the modern Western diet, which contains substantial levels of cholesterol and other fatty substances, promotes prostate cancer progression. Consistent with this idea, prolonged inhibition of the cholesterol synthesis pathway by pharmacologic intervention in men has recently been associated with reduction in risk of advanced prostate cancer. In this review, we discuss the possibility that membrane cholesterol promotes prostate cancer progression by a mechanism that involves dysregulation of lipid raft-resident signaling complexes. This hypothesis provides new avenues for mechanistic studies as well as therapeutic intervention.

Targeting lipid rafts inhibits protein kinase B by disrupting calcium homeostasis and attenuates malignant properties of melanoma cells

Failure of current therapeutic modalities to treat melanoma remains a challenge for clinical and experimental oncology. The aggressive growth and apoptotic resistance of this tumor are mediated, in part, by aberrantly activated protein kinase B/Akt (PKB). In many cells, PKB signaling depends on integrity of cholesterol-enriched membrane microdomains (rafts). However, it is still unclear if rafts support deregulated PKB activity in melanoma. In this study, ablation of rafts in murine (B16BL6-8, JB/RH1) and human (GA) melanoma lines by cholesterol-chelating methyl-beta-cyclodextrin (MbetaCD) reduced levels of constitutively active PKB in a dose- and time-dependent manner, while reconstitution of microdomains restored PKB activity. PKB was sensitive to the membrane-permeable Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid tetra (acetocymethyl) ester and to the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphtalenesulfonamide (W7) implying the contribution of Ca2+ signaling to PKB deregulation. Indeed, malignant and apoptosis-resistant clone of B16BL6 melanoma (B16BL6-8) displayed significantly higher [Ca2+](i) and store-operated Ca2+ influx (SOC) relative to non-malignant apoptosis-sensitive B16BL6 clone (Kb30) expressing barely detectable basal levels of active PKB. Raft ablation in B16BL6-8 cells robustly inhibited SOC and decreased [Ca2+](i) to levels comparable with those detected in Kb30 cells. Treating cells by PKB-inhibiting doses of M beta CD dramatically impaired their apoptotic resistance and capacity to generate tumors. Furthermore, weekly intraperitoneal injections of M beta CD to mice grafted with melanoma cells at doses of 300 and 800 mg/kg significantly attenuated tumor development. Our data implicate membrane rafts in enhancing the resistance of melanoma to apoptosis and indicate that targeting raft microdomains is a potentially effective strategy to cure this frequently fatal form of cancer.

Suppression of Epidermal Growth Factor Receptor Signaling by Protein Kinase C-α Activation Requires CD82, Caveolin-1, and Ganglioside

Activation of protein kinase C (PKC)-alpha decreases normal and neoplastic cell proliferation by inhibiting epidermal growth factor receptor (EGFR)-related signaling. The molecular interactions upstream to PKC-alpha that influence its suppression of EGFR, however, are poorly understood. We have found that caveolin-1, tetraspanin CD82, and ganglioside GM3 enable the association of EGFR with PKC-alpha, ultimately leading to inhibition of EGFR signaling. GM3- and CD82-induced inhibition of EGFR signaling requires PKC-alpha translocation and serine/threonine phosphorylation, which eventually triggers EGFR Thr654 phosphorylation and receptor internalization. Within this ordered complex of signaling molecules, the ability of CD82 to associate with PKC-alpha requires the presence of caveolin-1, whereas the interaction of caveolin-1 or PKC-alpha with EGFR requires the presence of CD82 and ganglioside GM3. Disruption of the membrane with methyl-beta-cyclodextrin dissociates the EGFR/GM3/caveolin-1/CD82/PKC-alpha complex and prevents the inhibitory effect of PKC-alpha on EGFR phosphorylation, suggesting that caveolin-1, CD82, and ganglioside interact with EGFR and PKC-alpha within intact cholesterol-enriched membrane microdomains. Given the role of these membrane molecules in suppressing EGFR signaling, up-regulation of GM3, caveolin-1, and CD82 function may be an effective adjunctive therapy for treating epithelial cell malignancies.