scholarly journals Involvement of Cholesterol-Rich Lipid Rafts in Interleukin-6-Induced Neuroendocrine Differentiation of LNCaP Prostate Cancer Cells

Endocrinology ◽  
2004 ◽  
Vol 145 (2) ◽  
pp. 613-619 ◽  
Author(s):  
Jayoung Kim ◽  
Rosalyn M. Adam ◽  
Keith R. Solomon ◽  
Michael R. Freeman
Keyword(s):  
Prostate Cancer ◽  
Signal Transduction ◽  
Lipid Rafts ◽  
Lipid Raft ◽  
Neuron Specific Enolase ◽  
Membrane Microdomains ◽  
Membrane Rafts ◽  
Lncap Cells ◽  
Discontinuous Sucrose Gradient ◽  
Triton X

Abstract IL-6 is an inflammatory cytokine that has been linked to aggressive prostate cancer (PCa). Previous studies have demonstrated that IL-6 can enhance the differentiation of PCa cells toward a neuroendocrine (NE) phenotype, a possible indicator of hormone-refractory disease. In this report, we present evidence that the mechanism of IL-6-stimulated NE differentiation employs a detergent-resistant (lipid raft) membrane compartment for signal transduction in LNCaP PCa cells. Signal transducer and activator of transcription (STAT)3, a mediator of IL-6 signaling, was rapidly phosphorylated and translocated to the nucleus in LNCaP cells treated with IL-6. Both processes were inhibited by filipin, a cholesterol-binding compound that disrupts plasma membrane lipid rafts. Isolation of Triton X-100-insoluble raft fractions from LNCaP cells by discontinuous sucrose gradient centrifugation demonstrated that the 80-kDa IL-6 receptor localized almost exclusively to the raft compartment. Although STAT3 was located predominantly in the Triton X-100-soluble subcellular fraction in exponentially growing cells, abundant phosphorylated STAT3 was detected in the raft fraction after stimulation with IL-6. Increases in expression of the NE marker, neuron-specific enolase, and neuron-specific enolase promoter activity after IL-6 treatment were reduced after membrane rafts were disrupted by filipin treatment. LNCaP cells expressed the raft-resident proteins flotillin-2 and Giα2, but notably not caveolins, the predominant structural protein present in caveolar membrane rafts in many tissues and tumor cells. These results are the first to define a role for lipid raft membrane microdomains in signal transduction mechanisms capable of promoting the NE phenotype in PCa cells, and they demonstrate that the raft compartment is capable of mediating such signals in the absence of caveolins. Our results also suggest a mechanistic role for membrane cholesterol in cell signaling events relevant to PCa progression.

scholarly journals Lipid raft integrity affects GABAA receptor, but not NMDA receptor modulation by psychopharmacological compounds

2013 ◽  
Vol 16 (6) ◽  
pp. 1361-1371 ◽  
Author(s):  
Caroline Nothdurfter ◽  
Sascha Tanasic ◽  
Barbara Di Benedetto ◽  
Manfred Uhr ◽  
Eva-Maria Wagner ◽  
...  
Keyword(s):  
Nmda Receptor ◽  
Lipid Rafts ◽  
Gabaa Receptor ◽  
Lipid Raft ◽  
Tricyclic Antidepressant ◽  
Membrane Microdomains ◽  
Cholesterol Depletion ◽  
Receptor Modulation ◽  
Gabaa Receptor Subunits ◽  
Triton X

Abstract Lipid rafts have been shown to play an important role for G-protein mediated signal transduction and the function of ligand-gated ion channels including their modulation by psychopharmacological compounds. In this study, we investigated the functional significance of the membrane distribution of NMDA and GABAA receptor subunits in relation to the accumulation of the tricyclic antidepressant desipramine (DMI) and the benzodiazepine diazepam (Diaz). In the presence of Triton X-100, which allowed proper separation of the lipid raft marker proteins caveolin-1 and flotillin-1 from the transferrin receptor, all receptor subunits were shifted to the non-raft fractions. In contrast, under detergent-free conditions, NMDA and GABAA receptor subunits were detected both in raft and non-raft fractions. Diaz was enriched in non-raft fractions without Triton X-100 in contrast to DMI, which preferentially accumulated in lipid rafts. Impairment of lipid raft integrity by methyl-β-cyclodextrine (MβCD)-induced cholesterol depletion did not change the inhibitory effect of DMI at the NMDA receptor, whereas it enhanced the potentiating effect of Diaz at the GABAA receptor at non-saturating concentrations of GABA. These results support the hypothesis that the interaction of benzodiazepines with the GABAA receptor likely occurs outside of lipid rafts while the antidepressant DMI acts on ionotropic receptors both within and outside these membrane microdomains.

scholarly journals RaftProt V2: understanding membrane microdomain function through lipid raft proteomes

2018 ◽  
Author(s):  
Ahmed Mohamed ◽  
Anup Shah ◽  
David Chen ◽  
Michelle M. Hill
Keyword(s):  
Experimental Evidence ◽  
Lipid Rafts ◽  
Lipid Raft ◽  
Protein Composition ◽  
Biological Data ◽  
Tissue Type ◽  
Membrane Microdomains ◽  
Membrane Rafts ◽  
Link Type ◽  
Membrane Microdomain

ABSTRACTCellular membranes feature dynamic submicrometer-scale lateral membrane domainsvariously referred to as lipid rafts, membrane rafts or glycosphingolipid-enriched microdomains (GEM). In order to understand the molecular functions of lipid rafts, numerous studies have utilized various biochemical methods to isolate and examine the protein composition of membrane rafts. However, interpretation of individual raft proteomics studies are confounded by the limitations of isolation methods and the dynamic nature of rafts. Knowledge-based approaches can facilitate biological data interpretation by integrating experimental evidence from existing studies. To this end, we previously developed RaftProt (http://lipid-raft-database.di.uq.edu.au/), a searchable database of mammalian lipid raft-associated proteins. Despite being a valuable and highly used resource, improvements in search capabilities and visualisation were still needed. Here, we present RaftProt V2 (http://raftprot.org), an improved update of RaftProt, enabling interrogation and integration of datasets at the cell/tissue type and UniRef/Gene level. Besides the addition of new datasets and re-mapping of all entries to both UniProt and UniRef IDs, we have annotated the level of experimental evidence for each protein entry. The search engine now allows for multiple protein or experiment searches where correlations, interactions or overlaps can be investigated. The web-interface has been completely re-designed and offers new interactive tools for data and subset selection, correlation analysis and network visualization. Overall, RaftProt aims to advance our understanding of lipid raft function by revealing the proteomes and pathways that are associated with membrane microdomains in diverse tissue and conditions.Database URL: http://raftprot.org

scholarly journals Monosialoganglioside GM1 Deficiency Inhibits the Neurotrophic Effects of GDNF by Disrupting Lipid Raft Assembly

2021 ◽  
Author(s):  
Shimin Jiang ◽  
Tai Zhou ◽  
Kejia Zhang ◽  
Yao Zhou ◽  
Zhongcheng Wang ◽  
...  
Keyword(s):  
Lipid Rafts ◽  
Lipid Raft ◽  
Gradient Centrifugation ◽  
Membrane Microdomains ◽  
Western Blot Assay ◽  
Caveolin 1 ◽  
Downstream Signaling ◽  
Proliferation And Differentiation ◽  
Lipid Raft Microdomains ◽  
Triton X

Abstract Recent studies have shown that monosialoganglioside GM1 deficiency can inhibit the signal transduction process of glial cell line-derived neurotrophic factor (GDNF), which plays an important role in the pathogenesis of Parkinson's disease (PD). However, its specific mechanism still needs to be explored. We inhibited the expression of GM1 by treating cells with D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP). CCK-8 assay, EdU cell proliferation assay and Western blot assay were used to evaluate the effect of GM1 deficiency on the proliferation and differentiation of SH-SY5Y cells induced by GDNF and on the GDNF-RET signaling pathway. Lipid rafts were isolated by Triton X-100 solubilization and OptiPrepTM density gradient centrifugation. The alterations of lipid raft assembly and the translocation of RET into lipid rafts were evaluated after PDMP treatment. We found that PDMP treatment inhibited the proliferation and differentiation of SH-SY5Y cells induced by GDNF and reduced the phosphorylation of RET and its downstream signaling molecules Erk and Akt. In addition, after PDMP treatment, caveolin-1 and flotillin-1, the prototypical markers of lipid rafts, diffused from lipid rafts to non-lipid raft microdomains, and GDNF-induced RET translocation into lipid rafts was also reduced. These alterations could be partially reversed by adding exogenous GM1. Our results suggest that ganglioside GM1 deficiency could compromise the neurotrophic effects and signals downstream of GDNF by altering the assembly of lipid raft membrane microdomains.

Mediators of innate immune recognition of bacteria concentrate in lipid rafts and facilitate lipopolysaccharide-induced cell activation

2002 ◽  
Vol 115 (12) ◽  
pp. 2603-2611 ◽  
Author(s):  
Martha Triantafilou ◽  
Kensuke Miyake ◽  
Douglas T. Golenbock ◽  
Kathy Triantafilou
Keyword(s):  
Lipid Rafts ◽  
Lipid Raft ◽  
Cell Activation ◽  
Imaging Techniques ◽  
Recognition System ◽  
Innate Immune ◽  
Membrane Microdomains ◽  
Immune Recognition ◽  
Cellular Activation ◽  
Signalling Molecules

The plasma membrane of cells is composed of lateral heterogeneities,patches and microdomains. These membrane microdomains or lipid rafts are enriched in glycosphingolipids and cholesterol and have been implicated in cellular processes such as membrane sorting and signal transduction. In this study we investigated the importance of lipid raft formation in the innate immune recognition of bacteria using biochemical and fluorescence imaging techniques. We found that receptor molecules that are implicated in lipopolysaccharide (LPS)-cellular activation, such as CD14, heat shock protein(hsp) 70, 90, Chemokine receptor 4 (CXCR4), growth differentiation factor 5(GDF5) and Toll-like receptor 4 (TLR4), are present in microdomains following LPS stimulation. Lipid raft integrity is essential for LPS-cellular activation, since raft-disrupting drugs, such as nystatin or MCD, inhibit LPS-induced TNF-α secretion. Our results suggest that the entire bacterial recognition system is based around the ligation of CD14 by bacterial components and the recruitment of multiple signalling molecules, such as hsp70, hsp90, CXCR4, GDF5 and TLR4, at the site of CD14-LPS ligation, within the lipid rafts.

scholarly journals Palmitoylation of the Autographa californica Multicapsid Nucleopolyhedrovirus Envelope Glycoprotein GP64: Mapping, Functional Studies, and Lipid Rafts

2003 ◽  
Vol 77 (11) ◽  
pp. 6265-6273 ◽  
Author(s):  
Sandy Xiaoxin Zhang ◽  
Yu Han ◽  
Gary W. Blissard
Keyword(s):  
Cell Surface ◽  
Lipid Rafts ◽  
Lipid Raft ◽  
Envelope Glycoprotein ◽  
Cysteine Residue ◽  
Autographa Californica ◽  
Membrane Microdomains ◽  
Sf9 Cells ◽  
Wild Type ◽  
Lipid Raft Microdomains

ABSTRACT Budded virions (BV) of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) contain a major envelope glycoprotein known as GP64, which was previously shown to be palmitoylated. In the present study, we used truncation and amino acid substitution mutations to map the palmitoylation site to cysteine residue 503. Palmitoylation of GP64 was not detected when Cys503 was replaced with alanine or serine. Palmitoylation-minus forms of GP64 were used to replace wild-type GP64 in AcMNPV, and these viruses were used to examine potential functions of GP64 palmitoylation in the context of the infection cycle. Analysis by immunoprecipitation and cell surface studies revealed that palmitoylation of GP64 did not affect GP64 synthesis or its transport to the cell surface in Sf9 cells. GP64 proteins lacking palmitoylation also mediated low-pH-triggered membrane fusion in a manner indistinguishable from that of wild-type GP64. Cells infected with viruses expressing palmitoylation-minus forms of GP64 produced infectious virions at levels similar to those from cells infected with wild-type AcMNPV. In combination, these data suggest that virus entry and exit in Sf9 cells were not significantly affected by GP64 palmitoylation. To determine whether GP64 palmitoylation affected the association of GP64 with membrane microdomains, the potential association of GP64 with lipid raft microdomains was examined. These experiments showed that: (i) AcMNPV-infected Sf9 cell membranes contain lipid raft microdomains, (ii) GP64 association with lipid rafts was not detected in infected Sf9 cells, and (iii) GP64 palmitoylation did not affect the apparent exclusion of GP64 from lipid raft microdomains.

Lipid raft adhesion receptors and Syk regulate selectin-dependent rolling under flow conditions

Blood ◽  
2006 ◽  
Vol 108 (10) ◽  
pp. 3352-3359 ◽  
Author(s):  
Claire Abbal ◽  
Martine Lambelet ◽  
Debora Bertaggia ◽  
Carole Gerbex ◽  
Manuel Martinez ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Lipid Rafts ◽  
Lipid Raft ◽  
Chelating Agents ◽  
Regulatory Mechanisms ◽  
Membrane Microdomains ◽  
Leukocyte Rolling ◽  
Flow Conditions ◽  
Cell Rolling ◽  
Pharmacologic Inhibitors

Abstract Selectins and their ligand P-selectin glycoprotein ligand-1 (PSGL-1) mediate leukocyte rolling along inflamed vessels. Cell rolling is modulated by selectin interactions with their ligands and by topographic requirements including L-selectin and PSGL-1 clustering on tips of leukocyte microvilli. Lipid rafts are cell membrane microdomains reported to function as signaling platforms. Here, we show that disruption of leukocyte lipid rafts with cholesterol chelating agents depleted raft-associated PSGL-1 and L-selectin and strongly reduced L-, P-, and E-selectin–dependent rolling. Cholesterol repletion reversed inhibition of cell rolling. Importantly, leukocyte rolling on P-selectin induced the recruitment of spleen tyrosine kinase (Syk), a tyrosine kinase associated to lipid raft PSGL-1. Furthermore, inhibition of Syk activity or expression, with pharmacologic inhibitors or by RNA interference, strongly reduced leukocyte rolling on P-selectin, but not on E-selectin or PSGL-1. These observations identify novel regulatory mechanisms of leukocyte rolling on selectins with a strong dependency on lipid raft integrity and Syk activity.

scholarly journals Androgen Deprivation Induces Reprogramming of Prostate Cancer Cells to Stem-Like Cells

Cells ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1441 ◽  
Author(s):  
Belén G. Sánchez ◽  
Alicia Bort ◽  
Diana Vara-Ciruelos ◽  
Inés Díaz-Laviada
Keyword(s):  
Prostate Cancer ◽  
Hypoxia Inducible Factor ◽  
Neuron Specific Enolase ◽  
Angiogenic Factor ◽  
Stem Cell Markers ◽  
Lncap Cells ◽  
Androgen Receptor Antagonist ◽  
Enhanced Expression ◽  
Androgen Depletion ◽  
E Cadherin

In the past few years, cell plasticity has emerged as a mode of targeted therapy evasion in prostate adenocarcinoma. When exposed to anticancer therapies, tumor cells may switch into a different histological subtype, such as the neuroendocrine phenotype which is associated with treatment failure and a poor prognosis. In this study, we demonstrated that long-term androgen signal depletion of prostate LNCaP cells induced a neuroendocrine phenotype followed by re-differentiation towards a “stem-like” state. LNCaP cells incubated for 30 days in charcoal-stripped medium or with the androgen receptor antagonist 2-hydroxyflutamide developed neuroendocrine morphology and increased the expression of the neuroendocrine markers βIII-tubulin and neuron specific enolase (NSE). When cells were incubated for 90 days in androgen-depleted medium, they grew as floating spheres and had enhanced expression of the stem cell markers CD133, ALDH1A1, and the transporter ABCB1A. Additionally, the pluripotent transcription factors Nanog and Oct4 and the angiogenic factor VEGF were up-regulated while the expression of E-cadherin was inhibited. Cell viability revealed that those cells were resistant to docetaxel and 2-hidroxyflutamide. Mechanistically, androgen depletion induced the decrease in AMP-activated kinase (AMPK) expression and activation and stabilization of the hypoxia-inducible factor HIF-1α. Overexpression of AMPK in the stem-like cells decreased the expression of stem markers as well as that of HIF-1α and VEGF while it restored the levels of E-cadherin and PGC-1α. Most importantly, docetaxel sensitivity was restored in stem-like AMPK-transfected cells. Our model provides a new regulatory mechanism of prostate cancer plasticity through AMPK that is worth exploring.

Regulation of ecto-5′-nucleotidase activity via Ca2+-dependent, annexin 2-mediated membrane rearrangement?

2006 ◽  
Vol 34 (3) ◽  
pp. 374-376 ◽  
Author(s):  
E.B. Babiychuk ◽  
A. Draeger
Keyword(s):  
Signal Transduction ◽  
Plasma Membrane ◽  
Lipid Rafts ◽  
Binding Proteins ◽  
Extracellular Atp ◽  
Membrane Rafts ◽  
Nucleotidase Activity ◽  
Annexin 2 ◽  
Associated Proteins ◽  
A Cell

The spatial segregation of the plasma membrane plays a prominent role in distinguishing and sorting a large number of signals a cell receives simultaneously. The plasma membrane comprises regions known as lipid rafts, which serve as signal-transduction hubs and platforms for sorting membrane-associated proteins. Ca2+-binding proteins of the annexin family have been ascribed a role in the regulation of raft dynamics. Glycosylphosphatidylinositol-anchored 5′-nucleotidase is an extracellular, raft-associated enzyme responsible for conversion of extracellular ATP into adenosine. Our results point to a regulation of ecto-5′-nucleotidase activity by Ca2+-dependent, annexin-mediated stabilization of membrane rafts.

scholarly journals Vav1/Rac-dependent actin cytoskeleton reorganization is required for lipid raft clustering in T cells

2001 ◽  
Vol 155 (3) ◽  
pp. 331-338 ◽  
Author(s):  
Martin Villalba ◽  
Kun Bi ◽  
Fernando Rodriguez ◽  
Yoshihiko Tanaka ◽  
Stephen Schoenberger ◽  
...  
Keyword(s):  
T Cells ◽  
Actin Cytoskeleton ◽  
Lipid Rafts ◽  
Lipid Raft ◽  
Large Scale ◽  
Actin Polymerization ◽  
Dominant Negative ◽  
Membrane Microdomains ◽  
Jurkat T Cells ◽  
Cytoskeleton Reorganization

Formation of the immunological synapse (IS) in T cells involves large scale molecular movements that are mediated, at least in part, by reorganization of the actin cytoskeleton. Various signaling proteins accumulate at the IS and are localized in specialized membrane microdomains, known as lipid rafts. We have shown previously that lipid rafts cluster and localize at the IS in antigen-stimulated T cells. Here, we provide evidence that lipid raft polarization to the IS depends on an intracellular pathway that involves Vav1, Rac, and actin cytoskeleton reorganization. Thus, lipid rafts did not translocate to the IS in Vav1-deficient (Vav1−/−) T cells upon antigen stimulation. Similarly, T cell receptor transgenic Jurkat T cells also failed to translocate lipid rafts to the IS when transfected with dominant negative Vav1 mutants. Raft polarization induced by membrane-bound cholera toxin cross-linking was also abolished in Jurkat T cells expressing dominant negative Vav1 or Rac mutants and in cells treated with inhibitors of actin polymerization. However, Vav overexpression that induced F-actin polymerization failed to induce lipid rafts clustering. Therefore, Vav is necessary, but not sufficient, to regulate lipid rafts clustering and polarization at the IS, suggesting that additional signals are required.

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