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The Glycosphingolipid, Lactosylceramide, Regulates B1-Integrin Clustering and Endocytosis

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The Glycosphingolipid, Lactosylceramide, Regulates B1-Integrin Clustering and Endocytosis Research Article The Glycosphingolipid, Lactosylceramide, Regulates B1-Integrin Clustering and Endocytosis Deepak K. Sharma, Jennifer C. Brown, Zhijie Cheng, Eileen L. Holicky, David L. Marks, and Richard E. Pagano Department of Biochemistry and Molecular Biology, Thoracic Diseases Research Unit, and Mayo Clinic Cancer Center, Mayo Clinic College of Medicine, Rochester, Minnesota Abstract signaling complexes (3, 5) and are also initiation sites for clathrin- independent endocytic events (3, 5, 6). Glycosphingolipids are known to play roles in integrin- mediated cell adhesion and migration; however, the mecha- One mechanism by which glycosphingolipids could affect cell nisms by which glycosphingolipids affect integrins are adhesion and migration is via their interaction with integrins. ah unknown. Here, we show that addition of the glycosphingolipid, Integrins are a family of heterodimeric, integral membrane C8-lactosylceramide (C8-LacCer), or free cholesterol to human proteins at the plasma membrane, which bind to extracellular fibroblasts at 10°C causes the formation of glycosphingolipid- matrix (ECM) proteins and cell surface ligands, and are responsible enriched plasma membrane domains as shown by visualizing a for many types of cell adhesion events (7, 8). Glycosphingolipids have fluorescent glycosphingolipid probe, BODIPY-LacCer, incorpo- been shown to directly modulate integrin-based cell attachment. For rated into the plasma membrane of living cells. Addition of C8- example, gangliosides (sialic acid–terminated glycosphingolipids) LacCer or cholesterol to cells initiated the clustering of extracted from neuroblastoma cells or atherosclerotic plaques enhance platelet adhesion via integrin binding to collagen (9–11). B -integrins within these glycosphingolipid-enriched domains 1 Gangliosides also enhance binding of integrins to the ECM in mouse and the activation of the B1-integrins as assessed using a HUTS antibody that only binds activated integrin. On warming to mammary carcinoma, melanoma, and neuroblastoma cells (12–14). Several models have been proposed for the mechanisms 37°C, B1-integrins were rapidly internalized via caveolar endocytosis in cells treated with C8-LacCer or cholesterol, by which glycosphingolipids or glycosphingolipid-enriched microdomains may regulate integrin function (11, 15, 16). whereas little B1-integrin was endocytosed in untreated fibroblasts. Incubation of cells with C8-LacCer or cholesterol First, glycosphingolipids could initiate signaling events, which followed by warm-up caused src activation, a reorganization of cause downstream activation of integrins. Indeed, addition of the actin cytoskeleton, translocation of RhoA GTPase away exogenous glycosphingolipids to cells has been shown to have from the plasma membrane as visualized using total internal significant effects on signaling cascades. Another possibility is reflection fluorescence microscopy, and transient cell detach- that glycosphingolipids promote the clustering of integrins in ment. These studies show that LacCer can regulate integrin glycosphingolipid-enriched microdomains, thus increasing their function both by modulating integrin clustering in micro- avidity for ligand. The cross-linking of integrins with certain integrin antibodies is an established method for integrin activation domains and by regulating integrin endocytosis via caveolae. Our findings suggest the possibility that aberrant levels of (17, 18). Similarly, integrin function can be modulated by antibody glycosphingolipids found in cancer cells may influence cell cross-linking of cholera toxin B subunit bound to GM1 ganglioside attachment events by direct effects on integrin clustering and or glycophosphatidylinositol-linked proteins (15, 16). However, no studies have provided direct evidence that glycosphingolipids internalization. (Cancer Res 2005; 65(18): 8233-41) modulate integrin clustering in glycosphingolipid-enriched micro- domains in the absence of cross-linking agents. Introduction An additional mechanism by which glycosphingolipids could Glycosphingolipids are plasma membrane constituents in mam- regulate integrins is by affecting their endocytosis from the plasma malian cells, which play important roles in normal cell adhesion, membrane. Recent studies have shown that some integrins can be migration, and proliferation as well as in pathologic conditions, internalized via caveolae (18, 19), a subset of glycosphingolipid- such as tumorigenesis and atherosclerosis (1–3). Glycosphingolipids enriched microdomains defined as invaginations at the plasma are present mainly in the outer leaflet of the plasma membrane membrane enriched in caveolin-1 (Cav1; refs. 20, 21). Caveolae are where they interact closely with cholesterol to form lipid micro- sites for clathrin-independent endocytosis of glycosphingolipids as domains (4, 5). These glycosphingolipid-enriched microdomains well as some viruses and bacterial toxins (22–27). We reported have been shown to act as organizing centers for some cellular recently that the addition of glycosphingolipids or cholesterol to the plasma membrane of cells stimulates caveolar endocytosis via activation of src kinase (25). During these studies, we noted that on Note: Joint first authors: D.K. Sharma and J.C. Brown. treatment with exogenous sphingolipids the cells began to 1 D.K. Sharma is currently at Photometrics, Inc., 3440 East Britannia Drive, Tucson, reorganize their actin cytoskeleton and retract, suggesting a link AZ 85706. between plasma membrane glycosphingolipid and cholesterol Supplementary data for this article are available at Cancer Research Online (http:// cancerres.aacrjournals.org/). composition and cell adhesion via integrins. Requests for reprints: Richard E. Pagano, Mayo Clinic and Foundation, Stabile 8, 200 First Street, Southwest, Rochester, MN 55905-0001. Phone: 507-284-8754; Fax: 507- 266-4413; E-mail: [email protected]. I2005 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-05-0803 1 Unpublished data. www.aacrjournals.org 8233 Cancer Res 2005; 65: (18). September 15, 2005 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2005 American Association for Cancer Research. Cancer Research Here, we investigated the possibility that addition of glyco- Incubation with C8-lactosylceramide, cholesterol, and integrin antibody. Cells were incubated with 20 Amol/L C8-LacCer/BSA (30), sphingolipid or cholesterol to cells might affect the function of h1- h h h integrin, a key protein that mediates adhesion in many cell types methyl- -cyclodextrin (m CD)/cholesterol complex (25), or anti- 1- A j (28, 29). These studies revealed that exogenously added C8- integrin antibody (2.5 g/mL) for 30 minutes at 10 C in HMEM. In some experiments, samples were subsequently incubated for 5 minutes at lactosylceramide (C8-LacCer) or cholesterol induced the clustering j h 37 C to allow endocytosis to occur. of 1-integrins together with BODIPY-LacCer in glycosphingolipid- Microscopy. Fluorescence microscopy was carried out using an enriched microdomains that were visible by fluorescence micros- Olympus (Melville, NY) IX70 fluorescence microscope and the ‘‘Meta- h copy in living cells. In addition, the endocytosis of 1-integrin was morph’’ image-processing program (Universal Imaging Corp., Downing- significantly stimulated by both C8-LacCer and cholesterol town, PA). Total internal reflection fluorescence (TIRF) microscopy was addition. Finally, the clustering and internalization of h1-integrin carried out using an Olympus attachment for the IX70 microscope. In by C8-LacCer and cholesterol initiated downstream signaling any given experiment, all photomicrographs were exposed and processed events that resulted in changes in cytoskeletal organization and identically for a given fluorophore. Intracellular fluorescence was z adhesion consistent with the modulation of integrin function. quantified by analyzing images of 10 cells in at least three independent These studies have important implications for understanding how experiments. Miscellaneous procedures. SDS-PAGE and immunoblotting (27) and plasma membrane lipid composition may affect integrin-mediated immunofluorescence of formaldehyde-fixed cells (25) were done as cellular processes. described previously. In vitro studies of src kinase activity were determined in cell lysates using a kit from Upstate (Charlottesville, VA), which measures phosphorylation of a model src peptide substrate. Materials and Methods Statistical significance of quantified differences in cell fluorescence and Lipids, fluorescent probes, and miscellaneous reagents. BODIPY- attachment was assessed by unpaired two-tailed t tests using the Prism4 h LacCer (30) or nonfluorescent C8-LacCer (D-lactosyl- 1-1V-N-octanoyl-D- program (GraphPad, San Diego, CA). erythro-sphingosine; Avanti Polar Lipids, Alabaster, AL) were complexed with defatted bovine serum albumin (BSA; Serologicals Corp., Norcross, GA) and used as described (25). Alexa Fluor 594 (AF594)–labeled transferrin and Results albumin were from Molecular Probes (Eugene, OR). Tyrosine kinase (PP2) and Addition of C8-lactosylceramide or cholesterol induces protein kinase C (PKC) inhibitors (Go¨6976) were from Calbiochem (San Diego, B1-integrin clustering into lipid domains. We first examined the CA). Cav1 small interfering RNA (siRNA) was from Super Array (Frederick, effect of C8-LacCer, a glycosphingolipid, or cholesterol/mhCD MD). A RhoA GTPase plasmid was kindly provided by Dr. D. Billadeau (Mayo h Clinic,
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