Lipid Raft Pathway Research Articles

Hypoxic Induction of Exosome Uptake through Proteoglycan-Dependent Endocytosis Fuels the Lipid Droplet Phenotype in Glioma.

As an adaptive response to hypoxic stress, aggressive tumors rewire their metabolic phenotype into increased malignant behavior through extracellular lipid scavenging and storage in lipid droplets (LD). However, the underlying mechanisms and potential lipid source retrieved in the hypoxic tumor microenvironment remain poorly understood. Here, we show that exosome-like extracellular vesicles (EV), known as influential messengers in the tumor microenvironment, may also serve anabolic functions by transforming hypoxic, patient-derived human glioblastoma cell lines into the LD+ phenotype. EVs were internalized via a hypoxia-sensitive, endocytic mechanism that fueled LD formation through direct lipid transfer, and independently of fatty acid synthase activity. EVs can enter cells through multiple and yet ill-defined pathways. On a mechanistic level, we found that hypoxia-mediated EV uptake depends on increased heparan sulfate proteoglycan (HSPG) endocytosis that preferentially followed the lipid raft pathway. The functional relevance of HSPG was evidenced by the reversal of EV-mediated LD loading by targeting of HSPG receptor function. IMPLICATIONS: Together, our data extend the multifaceted role of EVs in cancer biology by showing their LD-inducing capacity in hypoxic glioma cells. Moreover, these findings highlight a potential function for HSPG-mediated endocytosis as a salvage pathway for EV retrieval during tumor stress conditions.

The involvement of lipid raft pathway in suppression of TGFβ-mediated metastasis by tolfenamic acid in hepatocellular carcinoma cells

TGFβ signaling plays an important role in orchestrating a favorable microenvironment for tumor cell growth and promoting epithelial–mesenchymal transition. As a conventional nonsteroidal anti-inflammation drugs, tolfenamic acid (TA) has been previously reported to exhibit anti-cancer activity. Herein, we investigated the effect of TA on TGFβ-mediated pro-metastatic activity and the underlying mechanisms in hepatocellular carcinoma (HCC). As a result, TA suppresses TGFβ-induced migration and glycolysis in HCC cells, which is accompanied with reduced Smad phosphorylation and subsequent nuclear transcription activity. Mechanistically, TA promotes lipid raft-caveolar internalization pathway of TGFβ receptor, therefore leading to its rapid turnover. Consistently, TA inhibits constitutively active TGFβ type I receptor induced Smad phosphorylation and EMT markers, whereas ectopic expression of TGFβ type II receptor could partially rescue TGFβ-mediated Smad2 phosphorylation and downstream genes expression in the presence of TA. Furthermore, TA inhibited HCC cells invasion in nude mice, associated with the alteration of characteristics related with EMT and glycolysis of cancer cells. Our study suggests TA could activate lipid raft pathway and modulate TGFβ mediated metastasis, implicating the potential application of TA as a modulator of tumor microenvironment in HCC.

Internalization and intracellular trafficking of an antitumor molecule

Amblyomin‐X is a proteasome inhibitor that was identified through the transcriptome analysis of the salivary gland from Amblyomma sculptum tick. This recombinant protein triggers apoptosis in different tumor cell lines, and promotes regression of tumor growth and reduction of metastasis. Recent studies have indicated endocytosis mechanism could be associated with pro‐apoptotic activity of this protein. Here, by using confocal microscopy and high content analysis (ImageXpress Micro Confocal High‐Content, Molecular Devices), we found that Amblyomin‐X (Alexa Fluor 488 conjuaged) preferentially co‐localize with Alexa Fluor 555 Cholera Toxin Subunit B, caveolin‐1 and 19S proteasome subunit in human tumor cells (SK‐MEL‐28 and MIA PaCa‐2). Remarkably, we identified Amblyomin‐X co‐immunoprecipitates with caveolin‐1 and Rab‐11. Also, we observed an alteration in Ambyomin‐X trafficking in human tumor cells transfected with GFP‐Rab5 mutants (Rab5:Q79L and Rab5:N133I). Taken all results together, we can suggest that Amblyomin‐X internalization occurs via lipid raft pathway and intracellular trafficking involved of caveosomes and early and recycling endosomes. Then, Amblyomin‐X reaches proteasome system, a possible intracellular destination.Support or Funding InformationSupported by CAPES, Fapesp, BNDES and União Química FarmacêuticaThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Transport Mechanism of Coumarin 6 Nanocrystals with Two Particle Sizes in MDCKII Monolayer and Larval Zebrafish.

Nanocrystals (NCs) were utilized as oral formulations in commercial products to deliver lipophilic drug, but their transport mechanisms are not fully understood. This study aimed to explore the transport mechanism of NCs using in vitro Madin-Darby canine kidney II (MDCK II) cells and in vivo larval zebrafish models. Coumarin 6 (C6) was formulated into NCs with particle size of 67.5 ± 5.2 and 190 ± 9.2 nm. In vitro studies showed that 70 nm NCs accumulated in lysosome and endoplasmic reticulum (ER) as destinations. Lipid raft pathways mediated the endocytosis, while lipid raft, ER/Golgi, and Golgi/plasma membrane pathways were involved in exocytosis and transcytosis process. However, 200 nm NCs accumulated more in a lysosome, where lipid raft pathways were also involved in the endocytosis process. In vivo studies in larval zebrafish model further confirmed that the above network plays an important role in the absorption and distribution of C6-NCs.

Transport of Pru p 3 across gastrointestinal epithelium – an essential step towards the induction of food allergy?

Since intestinal absorption of food protein can trigger an allergic reaction, the effect of plant food allergen on intestinal epithelial cell permeability and its ability to cross the epithelial monolayer was evaluated. To study the interaction of Pru p 3 with intestinal epithelium, its natural entrance, analyzing transport kinetics and cellular responses that trigger. This was achieved using Pru p 3, the peach LTP, as a model. Enterocytic monolayers were established by culturing Caco 2 cells, as a model of enterocytes, on permeable supports that separate the apical and basal compartments. Pru p 3 was added to the apical compartment, the transepithelial resistance (TEER) was measured, and the transport was quantified. The peach allergen that crossed the cell monolayer was detected in the cell fraction and in the basal medium by immunodetection with specific antibodies and the quantity was measured by ELISA assay. Pru p 3 was able to cross the monolayer without disturbing the integrity of the tight junctions. This transport was significantly higher than that of a non-allergenic peach LTP, LTP1, and occurred via lipid raft pathway. The incubation of Caco 2 cells with Pru p 3 and LTP1 produced the expression of epithelial-specific cytokines TSLP, IL33 and IL25. These results suggest that Pru p 3 was able to cross the cell monolayer by the transcellular route and then induce the production of Th2 cytokines. The results of the present study represent a step towards clarifying the importance of Pru p 3 as a sensitizer. The capacity of food allergens to cross the intestinal monolayer could explain their high allergenic capacity and its fast diffusion through the body associating to severe symptoms.

Ferristatin II Promotes Degradation of Transferrin Receptor-1 In Vitro and In Vivo

Previous studies have shown that the small molecule iron transport inhibitor ferristatin (NSC30611) acts by down-regulating transferrin receptor-1 (TfR1) via receptor degradation. In this investigation, we show that another small molecule, ferristatin II (NSC8679), acts in a similar manner to degrade the receptor through a nystatin-sensitive lipid raft pathway. Structural domains of the receptor necessary for interactions with the clathrin pathway do not appear to be necessary for ferristatin II induced degradation of TfR1. While TfR1 constitutively traffics through clathrin-mediated endocytosis, with or without ligand, the presence of Tf blocked ferristatin II induced degradation of TfR1. This effect of Tf was lost in a ligand binding receptor mutant G647A TfR1, suggesting that Tf binding to its receptor interferes with the drug’s activity. Rats treated with ferristatin II have lower TfR1 in liver. These effects are associated with reduced intestinal 59Fe uptake, lower serum iron and transferrin saturation, but no change in liver non-heme iron stores. The observed hypoferremia promoted by degradation of TfR1 by ferristatin II appears to be due to induced hepcidin gene expression.

The iron transport inhibitor ferristatin II induces degradation of transferrin receptor‐1

Ferristatin II, a polyaromatic compound previously proposed to be an iron uptake inhibitor, induces degradation of transferrin (Tf) receptor‐1 (TfR1) in HeLa cells within 4 h of treatment. Dose response experiments established an IC50 of 12 μM for inhibition of 55Fe‐Tf uptake, consistent with receptor degradation. Ferristatin II‐induced TfR1 degradation was blocked by nystatin, a cholesterol‐depleting agent that disrupts lipid raft membrane microdomains. siRNA knockdown of flotillin, a protein component of lipid rafts, also blocked TfR1 degradation by ferristatin II. Constitutive trafficking of TfR1 through clathrin‐mediated endocytosis occurs with or without ligand. In contrast, Tf antagonized TfR1 degradation by ferristatin II, an effect that was abrogated for the Tf binding mutant G647A TfR1. Combined, these data indicate that ferristatin II‐induced trafficking and degradation of TfR1 occurs through a lipid raft pathway gated in a ligand dependent manner.This work is supported by NIH grant DK064750 to M.W‐R., DK054488 to C.E., S.L.B is supported by NHGRI/NIEHS Training Grant T32ES016645.

135 Dramatic enhancement of uptake and trafficking of HIV-1 Tat protein via modulation of endocytosis pathways

HIV-1 Tat protein can be secreted from infected cells and taken up by other target cells. Tat uptake and trafficking has been widely investigated. This property is also exploited for drug delivery. We initially confirmed that Tat mainly entered Hela cells by clathrin-dependent endocytosis and was degraded in lysosomes, whereas chloroquine could inhibit the degradation and enhanced its trafficking to the nucleus. Interestingly, a similar effect was noted in presence of chlorpromazine (CPZ). Most likely, the uptake of Tat was shifted to caveolar endocytosis after the clathrin pathway was blocked by CPZ. However, the LTR transactivation of Tat could be enhanced by at least 50 fold when a liposome reagent was introduced into the Tat delivery system. Further evidence showed that liposome reagents could switch the Tat uptake from clathrin-dependent endocytosis to lipid raft pathway and bypass the degradation in lysosomes. This was supported by both pharmacological assays using inhibitors of caveolae-mediated endocytosis (MβCD, etc.) or microtubules (cytochalasin D & lutrunculin A) and co-localization using Tat101venus and immunstaining with anti-clathrin1, anti-caveolin1, anti-EEA1, anti-Lamp1 or anti-Rab7. Subsequently, chemical molecules carrying “+” or “−” charges were tested for blocking the Tat uptake facilitated with the liposome reagent, including macromolecules (e.g., heparin, polybrene) and small molecules (e.g., spermine). Both “+” and “-” charged molecules could inhibit the uptake of Tat, but the “+” molecules actually attached to the membrane carrying “-” charges and the “-” molecules bound to the Tat-liposome complex with “+” charges. However, the Tat-liposome complex was probably formed by hydrophobic bonds linking liposome's lipid to Tat hydrophobic domain because both Tat and the liposome reagent carried “+”charges. A peptide containing partial hydrophobic domain of Tat (CFITKGLGISYGRKK) could competitively inhibit the uptake of Tat-liposome complex. In conclusion, the liposome reagent could facilitate Tat protein to traverse the membrane by changing endocytosis pathways and significantly enhance its delivery to the nucleus in a lysosome-independent manner. These observations have important implications for elucidating the mechanism of Tat uptake and trafficking and exploring drug delivery.

Endocytosis of the Type III Transforming Growth Factor-β (TGF-β) Receptor through the Clathrin-independent/Lipid Raft Pathway Regulates TGF-β Signaling and Receptor Down-regulation

Transforming growth factor-beta (TGF-beta) signals through three highly conserved cell surface receptors, the type III TGF-beta receptor (T beta RIII), the type II TGF-beta receptor (T beta RII), and the type I TGF-beta receptor (T beta RI) to regulate diverse cellular processes including cell proliferation, differentiation, migration, and apoptosis. Although T beta RI and T beta RII undergo ligand-independent endocytosis by both clathrin-mediated endocytosis, resulting in enhanced signaling, and clathrin-independent endocytosis, resulting in receptor degradation, the mechanism and function of T beta RIII endocytosis is poorly understood. T beta RIII is a heparan sulfate proteoglycan with a short cytoplasmic tail that functions as a TGF-beta superfamily co-receptor, contributing to TGF-beta signaling through mechanisms yet to be fully defined. We have reported previously that T beta RIII endocytosis, mediated by a novel interaction with beta arrestin-2, results in decreased TGF-beta signaling. Here we demonstrate that T beta RIII undergoes endocytosis in a ligand and glycosaminoglycan modification-independent and cytoplasmic domain-dependent manner, with the interaction of Thr-841 in the cytoplasmic domain of T beta RIII with beta-arrestin2 enhancing T beta RIII endocytosis. T beta RIII undergoes both clathrin-mediated and clathrin-independent endocytosis. Importantly, inhibition of the clathrin-independent, lipid raft pathway, but not of the clathrin-dependent pathway, results in decreased TGF-beta1 induced Smad2 and p38 phosphorylation, supporting a specific role for clathrin-independent endocytosis of T beta RIII in regulating both Smad-dependent and Smad-independent TGF-beta signaling.

Resistance to alkyl-lysophospholipid-induced apoptosis due to downregulated sphingomyelin synthase 1 expression with consequent sphingomyelin- and cholesterol-deficiency in lipid rafts

The ALP (alkyl-lysophospholipid) edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine; Et-18-OCH3) induces apoptosis in S49 mouse lymphoma cells. To this end, ALP is internalized by lipid raft-dependent endocytosis and inhibits phosphatidylcholine synthesis. A variant cell-line, S49AR, which is resistant to ALP, was shown previously to be unable to internalize ALP via this lipid raft pathway. The reason for this uptake failure is not understood. In the present study, we show that S49AR cells are unable to synthesize SM (sphingomyelin) due to down-regulated SMS1 (SM synthase 1) expression. In parental S49 cells, resistance to ALP could be mimicked by small interfering RNA-induced SMS1 suppression, resulting in SM deficiency and blockage of raft-dependent internalization of ALP and induction of apoptosis. Similar results were obtained by treatment of the cells with myriocin/ISP-1, an inhibitor of general sphingolipid synthesis, or with U18666A, a cholesterol homoeostasis perturbing agent. U18666A is known to inhibit Niemann-Pick C1 protein-dependent vesicular transport of cholesterol from endosomal compartments to the trans-Golgi network and the plasma membrane. U18666A reduced cholesterol partitioning in detergent-resistant lipid rafts and inhibited SM synthesis in S49 cells, causing ALP resistance similar to that observed in S49AR cells. The results are explained by the strong physical interaction between (newly synthesized) SM and available cholesterol at the Golgi, where they facilitate lipid raft formation. We propose that ALP internalization by lipid-raft-dependent endocytosis represents the retrograde route of a constitutive SMS1- and lipid-raft-dependent membrane vesicular recycling process.

Macrophages Rapidly Transfer Pathogens from Lipid Raft Vacuoles to Autophagosomes

Macrophages activate autophagy as an immediate response to Legionella pneumophila infection, but what marks the pathogen phagosome as a target for the autophagy machinery is not known. Because a variety of bacteria, parasites, viruses, and toxins that associate with the endoplasmic reticulum enter host cells by a cholesterol-dependent route, we tested the hypothesis that autophagy is triggered when microbes engage components of lipid raft domains. As the intracellular respiratory pathogen L. pneumophila or the extracellular uropathogen FimH+ Escherichia coli entered macrophages by a cholesterol-sensitive mechanism, they immediately resided in vacuoles rich in glycosylphosphatidylinositol moieties and the autophagy enzyme Atg7. As expected for autophagosomes, the vacuoles sequentially acquired the endoplasmic reticulum protein BiP, the autophagy markers Atg8 and monodansyl-cadaverine, and the lysosomal protein LAMP-1. A robust macrophage response to the pathogens was cholesterol-dependent, since fewer Atg7-rich vacuoles were observed when macrophages were pre-treated with methyl-beta-cyclodextrin or filipin. A model in which macrophages exploit autophagy to capture pathogens within the lipid raft pathway for antigen presentation prior to disposal in lysosomes is discussed.

Statins lower the risk of developing Alzheimer’s disease by limiting lipid raft endocytosis and decreasing the neuronal spread of Herpes simplex virus type 1

Many possible risk factors for Alzheimer's disease (AD) have been investigated, with only a very few showing positive associations and none defining the etiology of the neurodegenerative disease. The presence of herpes simplex virus type 1 (HSV-1) DNA in the brain, coupled with apolipoprotein E allele e4 (ApoE e4), has been suggested to confer an increased risk for AD. Studies have shown that pathogens, including viruses, utilize clathrin-independent endocytosis, i.e., lipid rafts that contain cholesterol, as part of their structure. Moreover, cholesterol-lowering statins have recently been linked with a reduced risk of developing Alzheimer's dementia. We, therefore, posit that long-term statin therapy protects individuals from AD by reducing the neuronal spread of HSV-1 via lipid raft domain pathways. Although the mechanism by which statins reduce AD risk is unknown, they reduce the amount of cholesterol in the plasma membrane and, thus, may decrease the availability of lipid raft pathways to spread HSV-1 within the brain.

Endocytosis of Oxidized Low Density Lipoprotein through Scavenger Receptor CD36 Utilizes a Lipid Raft Pathway That Does Not Require Caveolin-1

The scavenger receptor CD36 binds a diverse array of ligands, including thrombospondin-1, oxidized low density lipoprotein (OxLDL), fatty acids, anionic phospholipids, and apoptotic cells. CD36 has been reported to be present in lipid rafts/caveolae, but little is known about the membrane trafficking of this protein at baseline or following ligand binding. Here, we determined that expression of CD36 in Chinese hamster ovary (CHO) cells and endogenous expression of CD36 in C32 cells led to a homogeneous distribution of the protein on the plasma membrane, as judged by confocal fluorescence microscopy. This homogeneous pattern was observed both by anti-CD36 antibody staining and by live cell imaging of CHO cells expressing a chimeric CD36-green fluorescent protein construct. In contrast, caveolin-1 displayed its usual punctate surface distribution. Correspondingly, dual labeling of CD36 and caveolin-1 showed essentially no overlap, neither by immunofluorescence light microscopy nor by immunogold electron microscopy. Furthermore, isolation of lipid rafts by sucrose gradient ultracentrifugation of cold Triton X-100 cell lysates yielded both CD36 and caveolin-1, but immunoprecipitates of caveolin-1 did not contain CD36. Binding of Ox-LDL led to internalization of CD36 and OxLDL into endosomal structures that did not contain caveolin-1 or transferrin but that co-internalized the glycosyl-phosphatidylinositol-anchored protein decay accelerating factor, a lipid raft protein. Furthermore, expression of CD36 in the caveolin-1-negative KB cell line is sufficient for OxLDL-induced internalization of CD36, indicating that caveolin-1 is not required for this endocytic process. Taken together, these data demonstrate that at steady state, CD36 is localized in lipid rafts but not in caveolae, and that binding of OxLDL to CD36 leads to endocytosis through a lipid raft pathway that is distinct from the clathrin-mediated or caveolin internalization pathways.

Physiological regulation of the immunological synapse by agrin.

T cell activation is dependent on both a primary signal delivered through the T cell receptor and a secondary costimulatory signal mediated by coreceptors. Although controversial, costimulation is thought to act through the specific redistribution and clustering of membrane and intracellular kinase-rich lipid raft microdomains at the contact site between T cells and antigen-presenting cells. This site has been termed the immunological synapse. Endogenous mediators of raft clustering in lymphocytes have not been identified, although they are essential for T cell activation. We now demonstrate that agrin, an aggregating protein crucial for formation of the neuromuscular junction, is also expressed in lymphocytes and is important in reorganization of membrane lipid microdomains and setting the threshold for T cell signaling. Our data show that agrin induces the aggregation of signaling proteins and the creation of signaling domains in both immune and nervous systems through a common lipid raft pathway.