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 microdomains 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

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, Rochester, MN); GFP-RhoA was generated by subcloning into the treatments on the cell surface distribution of 1-integrin. Human EGFP-C3 vector (BD Biosciences Clontech, Palo Alto, CA). GFP-actin was also skin fibroblasts were treated with C8-LacCer or cholesterol for 30 from BD Biosciences Clontech. minutes at low temperature (10jC) to prevent endocytosis from h 14 Anti- 1-integrin (IgG) and anti-phospho-Tyr Cav1 antibodies were from occurring. The cells were then fixed without permeabilization and h h BD Biosciences PharMingen (San Diego, CA). Fab anti- 1-integrin fragments immunostained to detect 1-integrin. Cells treated with 20 Amol/L h were generated from this IgG using immobilized ficin, purified with protein C8-LacCer showed extensive clustering of 1-integrin compared A-Sepharose, and labeled with Alexa Fluor 647 (AF647) succinimidyl ester with untreated cells (Fig. 1A, 1 versus 2). This clustering was similar (Molecular Probes). The HUTS-4 antibody, which recognizes activated h - 1 to that observed when cells were incubated with h1-integrin integrin (31), was from Chemicon (Temecula, CA). Fluorescent secondary antibody (Fig. 1A, 3). The clustering of h -integrin induced by antibodies were from Molecular Probes and Jackson ImmunoResearch 1 antibody was not sensitive to cholesterol depletion (Fig. 1A, 3 versus (West Grove, PA). Unless otherwise indicated, all other reagents were from Sigma Chemical Co. (St. Louis, MO). 4). Similar results to those obtained with C8-LacCer (Fig. 1A, 2) were h Cell culture, transfection, and adenoviral infection. Normal human also observed when cells were incubated with m CD/cholesterol skin fibroblasts (GM-5659D; Coriell Institute, Camden, NJ) were grown in to increase plasma membrane cholesterol (data not shown). EMEM with 10% fetal bovine serum (FBS); HeLa cells (American Type Previous studies from our laboratory have used fluorescent Culture Collection, Rockville, MD) were grown in DMEM with 10% FBS. (BODIPY-labeled) SL analogues to study lipid trafficking in cells Transfections were carried out by electroporation using a GenePulser Xcell (30, 32, 33). These analogues exhibit a shift in their fluorescence (Bio-Rad Laboratories, Hercules, CA) as described previously (25). Treatment emission from green to red wavelengths with increasing of cells with Cav1 siRNA was done using FuGene6 (Roche Diagnostics, concentrations in membranes (32, 33). We took advantage of Indianapolis, IN). Cells were infected with Ad-KI-src, Ad-Dyn1K44A, or this property to probe the domain organization of BODIPY- ‘‘empty virus’’ (Ad-empty) in culture medium for 4 hours followed by washing LacCer at the plasma membrane under different conditions. In with fresh medium and further incubation for 24 hours before use (25). Incubation with inhibitors. Inhibitors of endocytosis, src kinase, and untreated cells, the plasma membrane was uniformly labeled and PKC were used as described (24–26). Briefly, cells were preincubated in emitted only green fluorescence (Fig. 1B, 1). In contrast, when HEPES-buffered MEM (HMEM) containing inhibitors for 1 hour at 37jC; cells were treated with nonfluorescent C8-LacCer or cholesterol, inhibitors were also present in all subsequent steps of the experiments. For small ‘‘patches’’ of BODIPY-LacCer with increased red emission mhCD, cells were pretreated with 5 mmol/L drug for 30 minutes at 37jC. (shown as yellow/orange areas in green/red overlays) appeared at Incubation with fluorescent lipids and proteins. Incubations with the plasma membrane (Fig. 1B, 2 and 3), suggesting the formation BODIPY-LacCer were done as described (25). Briefly, cells were incubated of plasma membrane ‘‘domains’’ that were enriched in j for 30 minutes at 10 C with 0.5 to 2.5 Amol/L BODIPY-LacCer/BSA, washed BODIPY-LacCer. Polyvalent antibody (IgG)–induced clustering of j twice with HMEM, and further incubated for 5 minutes at 37 C followed by h -integrin also induced similar yellow/orange patches (Fig. 1B, 4); back exchange with 5% DF-BSA (6 Â 10 minutes at 10jC) to remove any 1 however, when monovalent anti-h -integrin Fab fragments were fluorescent lipid remaining at the plasma membrane after endocytosis 1 (30, 32). For labeled proteins, cells were preincubated with 5 Ag/mL AF594 used, no clustering was detected (data not shown). transferrin or 50 Ag/mL AF594 albumin for 30 minutes at 10jC, further Induction of yellow/orange plasma membrane microdomains by h incubated for 5 minutes at 37jC, and acid stripped (26) to remove labeled antibody cross-linking of 1-integrin (Fig. 1B, 5) or C8-LacCer protein remaining at the cell surface. (data not shown) was inhibited by pretreatment of cells with

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Figure 1. C8-LacCer and cholesterol induce h1-integrin clustering within glycosphingolipid-enriched microdomains. Human skin fibroblasts were incubated in buffer (untreated) alone or with C8-LacCer (20 Amol/L), mhCD/cholesterol complex, or h1-integrin IgG for 30 minutes at 10jC. In some cases, cells were preincubated with 5 mmol/L mhCD to deplete cholesterol. A, distribution of h1-integrins at the plasma membrane. After treatments as above, cell samples were washed and fixed without permeabilization. Samples were then incubated with h1-integrin antibody followed by fluorescent secondary antibody (1 and 2) or with fluorescent secondary antibody to detect previously added h1-integrin IgG (3 and 4). Bar, 10 Am. B, visualization of plasma membrane domains after induction by various treatments. Cells were incubated with BODIPY-LacCer for 30 minutes at 10jC, washed, and then treated as above. The samples were then observed by fluorescence microscopy at green and red BODIPY emission wavelengths. Samples were maintained at 10jC at all times to prevent endocytosis. Images shown are overlays of red and green fluorescence. Areas outlined with white rectangles are further magnified in insets. Yellow orange patches indicate regions with the highest red signal, indicating enrichment of BODIPY-LacCer in these regions of the plasma membrane. Bar, 5 Am. C, h1-integrin clusters are localized to plasma membrane lipid domains. Cells were colabeled with BODIPY-LacCer and AF647 anti-h1-integrin Fab fragments for 30 minutes at 10jC. Samples were then further incubated for 30 minutes at 10jC F C8-LacCer, washed, and viewed by fluorescence microscopy. Images were acquired at red and green wavelengths (for BODIPY) and at far red wavelengths (for AF647) fluorescence. Note the overlap of integrin staining (blue) with the enriched (red/orange) plasma membrane domains of BODIPY-LacCer. Bar, 2 Am. mhCD to reduce plasma membrane cholesterol. Treatment of cells enriched microdomains in a process that requires cholesterol with the src kinase inhibitor, PP2, or expression of kinase-inactive but not src kinase. src did not prevent yellow/orange patch formation (data not C8-lactosylceramide stimulates B1-integrin internalization shown). Low temperature treatment with C8-LacCer not only via caveolae. Because we showed previously that C8-LacCer stimulated the formation of yellow/orange patches of BODIPY- stimulates caveolar endocytosis and some integrins are reported LacCer at the plasma membrane but also induced the clustering of to internalize via caveolae (25), we investigated the possibility that h1-integrin (visualized with fluorescent Fab fragments) within h1-integrin endocytosis is also stimulated by C8-LacCer. In these these patches (Fig. 1C). These studies suggest that C8-LacCer experiments, cells were treated with C8-LacCer and AF647-h1- h j j causes the movement of 1-integrins into glycosphingolipid- integrin Fab and labeled at 10 C, then warmed to 37 C for 5 minutes www.aacrjournals.org 8235 Cancer Res 2005; 65: (18). September 15, 2005

h j to allow endocytosis, and finally acid stripped to remove fluores- AF-647 1-integrin Fab and BODIPY-LacCer before warming to 37 C, cence remaining on the outer leaflet of the plasma membrane. In the there was extensive colocalization of h1-integrin Fab with BODIPY- absence of C8-LacCer, little h1-integrin internalization was ob- LacCer in these punctate endosomal structures (Fig. 2B). h h served; however, on treatment with C8-LacCer, 1-integrin was Conversely, antibody-induced clustering of the 1-integrins internalized by fibroblasts into punctate endocytic vesicles (Fig. 2A). significantly (P < 0.001) stimulated BODIPY-LacCer uptake f2-fold When cells were pretreated with C8-LacCer and colabeled with both (Fig. 2C), similar to the extent of stimulation reported previously

Figure 2. C8-LacCer stimulates h1-integrin internalization via caveolar endocytosis. A, C8-LacCer stimulates h1-integrin endocytosis. Fibroblasts were incubated with AF647-h1-integrin Fab fragments for 30 minutes at 10jC, washed, and then incubated F C8-LacCer for another 30 minutes at 10jC. Cells were acid stripped to remove plasma membrane fluorescence and then viewed by fluorescence microscopy at far red wavelengths. Dotted line (left) indicates the perimeter of a cell. Arrows (right) indicate some of the punctate endocytic structures containing h1-integrin Fab fragments. B, h1-integrin internalizes with BODIPY-LacCer under stimulated conditions. Cells were coincubated with BODIPY-LacCer and AF647-labeled h1-integrin Fab fragments for 30 minutes at 10jC, washed, and then incubated F C8-LacCer for another 30 minutes at 10jC. Samples were then warmed for 5 minutes at 37jC and then acid stripped and back exchanged to remove plasma membrane fluorescence. The cells were viewed by fluorescence microscopy at red and green (BODIPY) and far red (h1-integrin Fab) wavelengths. AF647 fluorescence is shown in blue pseudocolor. Note the high extent of colocalization of BODIPY-LacCer and internalized h1-integrin (e.g., see arrowheads). Asterisks denote region of images that are shown at higher magnification in the insets. Bar, 2.5 Am. C and D, quantitation of BODIPY-LacCer uptake after stimulation by C8-LacCer or h1-integrin IgG treatment. Fibroblasts were incubated with BODIPY-LacCer and then treated as in Fig. 1. Cells were then warmed for 5 minutes at 37jC and back exchanged. Note that the effects of h1-integrin IgG and C8-LacCer on BODIPY-LacCer uptake are not additive (C) and that peak stimulation by C8-LacCer is similar in magnitude to that with h1-integrin IgG (D). Data are results of image analysis of fluorescence micrographs (24, 25, 27) and are means F SD of at least 30 cells per condition. Means of all treatments were significantly different (P < 0.002) from those of untreated controls. E, h1-integrin IgG stimulate BODIPY-LacCer endocytosis via caveolae. Cells were pretreated with chlorpromazine (CPZ), nystatin, mhCD, transfected with DN proteins or siRNA for Cav1, or treated with tyrosine kinase (PP2) or PKC (Go¨6976) inhibitors as described (24–27). The cells were then treated F h1-integrin IgG, and BODIPY-LacCer endocytosis was studied as in (C and D). Means of all treatments were significantly different (P < 0.0001) than those of the untreated control and means of all treatments, except chlorpromazine, were significantly lower (P < 0.0001) than the sample treated with h1-integrin IgG alone.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2005 American Association for Cancer Research. Glycosphingolipid-Dependent Integrin Clustering using C8-LacCer or cholesterol (25). Integrin cross-linking also 30 minutes at 10jC resulted in increased levels of phospho-Tyr14 stimulated the endocytosis of fluorescent albumin, another marker Cav1 (P-Cav1) as assessed by immunoblotting (Fig. 3C). This phos- for caveolar uptake in fibroblasts (data not shown). Interestingly, phorylation was significantly inhibited in cells that were infected the stimulation of BODIPY-LacCer uptake induced by incubation with KI-src (Fig. 3C; data not shown). This finding is in agreement 14 with h1-integrin IgG clustering and C8-LacCer were not additive, with previous studies showing that Tyr phosphorylation of Cav1 suggesting that these treatments may stimulate caveolar endocytosis occurs via the activity of a src kinase (41, 42). The increase in by similar mechanisms (Fig. 2D). The stimulation of BODIPY-LacCer P-Cav1 levels in treated cells was even more pronounced (f65%) h j uptake by 1-integrin cross-linking was further characterized using after a 5-minute incubation at 37 C (data not shown). In parallel inhibitors of clathrin-mediated endocytosis (chloropromazine; experiments, we found that total Cav1 levels were relatively DN Eps15; refs. 24, 34), inhibitors of caveolar uptake (mhCD; unchanged during each of these treatments (data not shown). nystatin; ref. 24), Cav1 siRNA (35), and DN-Dyn1K44A, which blocks We then examined the effects of integrin clustering on the multiple endocytic mechanisms (Fig. 2E). The inhibition profile was intracellular distribution of P-Cav1 using immunofluorescence consistent with caveolar endocytosis. Moreover, the stimulated microscopy. In control samples, P-Cav1 was localized along actin h uptake of BODIPY-LacCer that was induced by 1-integrin cross- filaments (Fig. 3D, 1) as reported previously (42). This distribution linking was also src and PKC dependent (Fig. 2E). Finally, we note was not significantly affected when cells were warmed for 15 j h that transferrin uptake was not stimulated with h1-integrin cross- minutes at 37 C (Fig. 3D, 2). When cells were pretreated with 1- linking or by the addition of transferrin receptor antibody (data not integrin IgG, P-Cav1 levels were enhanced at the tips of actin shown). Addition of h1-integrin Fab fragments or aerolysin filaments (i.e., focal adhesions; Fig. 3D, 3; e.g., at brackets). On (to cluster glycophosphoinositol-anchored proteins at the plasma warming of cells to 37jC, P-Cav1 became relocalized into membrane; ref. 36) also did not stimulate BODIPY-LacCer uptake numerous puncta at the edges of the cells, whereas the actin (data not shown). cytoskeleton in these regions began to depolymerize (Fig. 3D, C8-lactosylceramide promotes B -integrin activation and compare inset of 3 with inset of 4). Similar results to those shown 1 h stimulates src activity. Integrin clustering is a required step in the in Fig. 3D using 1-integrin IgG were also obtained when cells were initiation of signaling by integrins. Thus, we investigated the pretreated with C8-LacCer or mhCD/cholesterol (data not shown). possibility that integrin clustering by glycosphingolipids stimulates Stimulation of caveolar endocytosis by integrin clustering downstream events associated with integrin signaling. First, we induces RhoA translocation from the plasma membrane and determined if glycosphingolipid treatment affected integrin activa- cell detachment. Integrins modulate the distribution and activity tion. C8-LacCer addition led to increased binding to h-integrin by of Rho GTPases (43), and RhoA is also involved in stabilization of cytoskeletal elements at focal adhesions (44, 45). In preliminary the HUTS-4 antibody (Fig. 3A), which only binds h1-integrins in their h studies, we found that incubations with C8-LacCer or cholesterol activated conformation (31), suggesting that 1-integrin was h caused cells to reorganize their actin cytoskeleton and retract,2 activated by these treatments. Because 1-integrin family members have been shown to activate src kinases (37), we examined the effect suggesting that Rho proteins might be affected by these treat- of lipid-induced integrin clustering on src activation (Fig. 3B). ments. We therefore next examined the effects of integrin Treatment with C8-LacCer at 10jC followed by a 5-minute clustering on the distribution of RhoA in live cells under various incubation at 37jC resulted in f10-fold activation of src (P < conditions. Cells were transiently transfected with GFP-RhoA and the extent of plasma membrane localization was determined using 0.005), similar to the activation seen on h1-integrin cross-linking with an antibody (f12-fold activation). Similar src activation was TIRF microscopy. In control cells, the amount of GFP-RhoA j seen when cells were treated with mhCD/cholesterol (25). Acti- present at the plasma membrane was similar at 10 C (Fig. 4A, 1) j vation of src was not significantly reduced in cells infected with an or after a 15-minute incubation at 37 C (Fig. 4A, 4, and B). In h adenovirus encoding DN-Dyn1K44A (Fig. 3B), which inhibits both contrast, when cells were treated with either 1-integrin antibody j j f clathrin and caveolar endocytosis (25, 38, 39), indicating that or C8-LacCer at 10 C and subsequently shifted to 37 C, 50% of the plasma membrane–associated fluorescence were lost (Fig. 4A, internalization was not required for src activation by h1-integrin. However, treatment with mhCD to deplete cholesterol decreased src 2 versus 5 and 3 versus 6 and B). The translocation of GFP-RhoA activation (P < 0.005) by integrin antibody by f60% (Fig. 3B), from the plasma membrane was inhibited in cells expressing suggesting that integrin clustering into cholesterol-enriched plasma Dyn1K44A (data not shown), suggesting that endocytosis is membrane domains is required for src activation. required for RhoA redistribution. Clustering of B -integrin by C8-lactosylceramide causes Due to the known role of RhoA in regulation of actin organization 1 (43), and our observation that some actin depolymerization cytoskeletal alterations. Signaling via integrins is known to h initiate cytoskeletal remodeling (8, 17). Thus, we examined the occurred on stimulation of caveolar endocytosis by 1-integrin cross-linking (Fig. 3D, 4), we next examined the effect of integrin effects of C8-LacCer and cholesterol on the actin cytoskeleton. On clustering on cell retraction and detachment. Cells were transfected internalization of BODIPY-LacCer via caveolae, we observed with GFP-actin and incubated with h -integrin IgG, and plasma BODIPY-LacCer fluorescence in intracellular punctae that 1 membrane–associated actin was visualized using TIRF microscopy. appeared to be associated with actin filaments under both basal As seen in Supplementary Fig. S2 (and Supplemental Video), selec- conditions and in cells stimulated with h -integrin IgG and C8- 1 ted regions of the cell in close proximity to the coverslip were LacCer (Supplementary Fig. S1). Previous studies have shown that retracted on warming to 37jC. Similar results to those in Fig. S2 Cav1 can be phosphorylated at Tyr14 by src family kinases in were also seen using C8-LacCer or cholesterol treatments of cells response to integrin activation (40). Further, on phosphorylation, (data not shown). Finally, we monitored cell detachment by counting Cav1 has been shown to localize at or near focal adhesions (40, 41). Thus, we investigated the possibility that C8-LacCer and cholesterol treatment stimulated Cav1 phosphorylation. Treatment of cells 2 with C8-LacCer, mhCD/cholesterol, or h1-integrin antibody for Unpublished data. www.aacrjournals.org 8237 Cancer Res 2005; 65: (18). September 15, 2005

Figure 3. C8-LacCer treatment promotes integrin activation and signaling. A, integrin activation by C8-LacCer. Fibroblasts were treated with C8-LacCer and h1-integrin IgG at low temperature as in Fig. 1. The cells were then fixed and immunostained using the HUTS-4 antibody that only recognizes h1-integrin in its active conformation followed by fluorescent secondary antibody. In some experiments, cells were pretreated with mhCD. Cellular fluorescence was quantified by image analysis and is expressed in arbitrary units. Data are mean F SD using measurements of 30 cells for each condition. Means of all treatments were significantly greater (P < 0.001) than that of the untreated control. B, src activity and C8-LacCer. Cells were untreated or treated with C8-LacCer or h1-integrin IgG as in Fig. 1, warmed to 37jC and src activation subsequently assayed in vitro. In some experiments, cells were infected with an adenovirus expressing Dyn1K44A or pretreated with mhCD to deplete cholesterol as indicated. Data are mean F SD. Src activity is expressed as fold stimulation relative to untreated cells. Means for C8-LacCer, h1-integrin, DynDN, and h1-integrin IgG were significantly (P < 0.005) different than that of the untreated control. The mean for the mhCD, h1-integrin IgG-treated sample was significantly different from that of the h1-integrin IgG-treated sample (P < 0.005) and the control (P < 0.05). C, integrin clustering induces phosphorylation of Cav1. Cells were incubated with C8-LacCer, mhCD/cholesterol, or anti-h1-integrin IgG for 30 minutes at 10jC to induce integrin clustering (see Fig. 1). Cells were then lysed and samples (20 Ag/lane) were run on 12% SDS-PAGE gels. Samples were then transferred to polyvinylpyrrolidone membranes and immunoblotted using an antibody against Tyr14-phosphorylated Cav1. In some cases, cells were pretreated with adenoviral KI-src. D, P-Cav1 relocalizes to focal adhesions on integrin cross-linking. Cells were incubated F anti-h1-integrin IgG for 30 minutes at 10jC, washed, and further warmed for 0 or 15 minutes at 37jC. Samples were then fixed and stained with rhodamine phalloidin and anti-Tyr14 P-Cav1 antibody. Note the localization of P-Cav1 at the tips of actin filaments and focal adhesions and the fragmentation of the actin cytoskeleton in the sample, which was treated with anti-h1-integrin IgG and warmed 15 minutes at 37jC. Bar, 10 Am.

the number of attached cells at various time points after warming Discussion j h to 37 C following treatment with 1-integrin IgG or C8-LacCer. At In this study, we investigated the mechanisms by which 40 minutes, there was a significant f50% loss of attached cells (P < plasma membrane lipid composition may affect h1-integrin 0.001, compared with untreated controls at 40 minutes) using either function. At low temperatures that prevented endocytosis, we treatment (Fig. 4C). This decrease in attached cells was not due to observed that addition of C8-LacCer or cholesterol to fibroblasts loss of cell viability as evidenced by trypan blue dye exclusion (data induced the formation or coalescence of glycosphingolipid- not shown). In addition, when the detached cells were washed and enriched microdomains on the plasma membrane in living cells replated, they reattached and grew normally (data not shown). as visualized using a fluorescent glycosphingolipid analogue,

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BODIPY-LacCer. This microdomain formation resulted in Glycosphingolipids and cholesterol induce microdomain h 1-integrin clustering within these domains, activation of these formation and B1-integrin clustering. We showed that addition of integrins, followed by activation of src kinase, and an increase in either C8-LacCer or cholesterol to fibroblasts stimulated the P-Cav1 levels. Addition of C8-LacCer followed by a brief incu- formation of plasma membrane lipid microdomains enriched in j bation at 37 C resulted in a stimulation of the caveolar endo- BODIPY-LacCer and caused h1-integrins to cluster within these h cytosis of 1-integrins, a rearrangement of the actin cytoskeleton, microdomains. h1-Integrin cross-linking with integrin IgG had translocation of RhoA away from the plasma membrane, actin similar effects. These glycosphingolipid-enriched microdomains depolymerization, and cell detachment. These data indicate that formed at low temperature, where endocytosis is prevented. Micro- glycosphingolipids and cholesterol in plasma membrane microdomain formation was disrupted by mhCD, indicating a requirement domains can affect integrin function both by modulating integrin for cholesterol in this process. However, glycosphingolipid-enriched clustering and by regulating internalization from the plasma microdomain formation occurred even when src activity was membrane. inhibited, suggesting that signaling via src was not needed for the

Figure 4. C8-LacCer treatment results in translocation of RhoA from the plasma membrane and promotes cell detachment. A, RhoA is translocated away from the plasma membrane by integrin clustering. Cells were transfected with GFP-RhoA and either untreated or treated with h1-integrin IgG or C8-LacCer for 30 minutes at 10jC. Samples were washed and further incubated for 0 (1-3)or15(4-6) minutes at 37jC. Plasma membrane–associated fluorescence was visualized by TIRF microscopy. Bar, 10 Am. B, quantitation of plasma membrane–associated GFP-RhoA under various conditions. Cells were treated as in (A) and GFP images were acquired using TIRF and conventional epifluorescence microscopy. TIRF intensity values (mean F SD) are expressed as a percentage of total GFP fluorescence measured in the same cells using images acquired by epifluorescence. Means of all treated samples were significantly different (P < 0.002) than the corresponding untreated control sample. C, integrin clustering by C8-Lacer or h1-integrin antibody induced cell detachment. Samples were incubated for 30 minutes at 10jC with buffer alone (control) or with C8-LacCer or anti-h1-integrin IgG, washed, and warmed to 37jC for the indicated times. Cell detachment was quantified by counting the number of cells remaining on the culture dish at the indicated times. Data are means F SD from three experiments and are expressed as percent of untreated controls. Means for C8-LacCer-treated samples at 20 and 40 minutes and h1-integrin IgG-treated samples at 40 minutes were significantly (P < 0.05) lower than their corresponding untreated controls. Detached cells were viable as indicated by trypan blue staining and by their ability to replate after washing (see text). www.aacrjournals.org 8239 Cancer Res 2005; 65: (18). September 15, 2005

h formation of these microdomains. The clustering of 1-integrin in inhibited by DN-Dyn1 expression, suggesting that integrin inter- these domains seems to be the initial step in integrin activation and nalization (or stimulated caveolar endocytosis) is required for the signaling. This premise is supported by our demonstration that observed changes in RhoA distribution. This result is similar to a h C8-LacCer treatment caused a change in 1-integrin to its active report by del Pozo et al. (47), which showed that the GTPase, rac1, conformation (Fig. 3A). Further, both src activity and P-Cav1 levels is prevented from translocating away from the plasma membrane are increased on C8-LacCer addition. Our results are consistent with when lipid microdomain endocytosis is inhibited in serum- previous studies showing that integrin activation by ligands or cross- stimulated 3T3 cells. linking antibodies can trigger the movement of integrins into lipid Regulation of integrin functions by glycosphingolipids and microdomains (18, 46). Several studies have shown that the cross- cholesterol. The results of these studies have elucidated two linking of glycosphingolipid-enriched microdomain constituents important mechanisms by which plasma membrane glycosphingo- h (e.g., glycophosphoinositol-linked proteins and GM1 ganglioside) lipids can modulate 1-integrin function: enhancement of integrin can also stimulate integrin clustering (15, 16). However, to our clustering and stimulation of integrin removal from the plasma knowledge, our study is the first demonstration that increases of membrane by endocytosis. Although these two mechanisms seem plasma membrane glycosphingolipid or cholesterol composition can to modulate integrin function in opposite directions, both are rapidly promote integrin clustering within microdomains. consistent with previous observations. First, because integrin Induction of glycosphingolipid-enriched microdomains clustering is known to increase the avidity of integrins for ECM results in stimulated endocytosis via caveolae. The clustering and other ligands (48, 49), the observation that C8-LacCer and of glycosphingolipids into microdomains seems to be a prerequisite cholesterol promote the clustering of integrin at the plasma for the stimulation of caveolar endocytosis observed in this study. membrane may be directly related to stimulatory effects of glyco- Addition of C8-LacCer, cholesterol, and cross-linking of h1-integrin sphingolipids on integrin binding shown in some other studies. at low temperature each resulted in the clustering of BODIPY- For example, the stimulation of integrin-mediated adhesion of h LacCer and 1-integrins together in microdomains. Each of these platelets to collagen by gangliosides from neuroblastoma cells treatments also elicited a significant stimulation of endocytosis via (mainly GD2 ganglioside) and atherosclerotic plaques (mainly GD3 caveolae when cells were warmed to 37jC. Stimulated uptake was and GM3 gangliosides; refs. 9–11) could be due to effects on consistent with endocytosis via caveolae based on its selective integrin clustering. Similarly, studies in various cell types, which inhibition by several pharmacologic inhibitors and dominant- have shown a direct relationship between integrin-dependent cell h negative proteins (Fig. 2; ref. 25). C8-LacCer and 1-integrin cross- adhesion and glycosphingolipid levels (9, 13, 14), could reflect the linking each stimulated caveolar endocytosis and src activity to a modulation of integrin clustering by changes in glycosphingolipid similar degree and were not additive in their effects, suggesting composition. that both treatments stimulated endocytosis via similar mecha- Although C8-LacCer addition initially causes a stimulation of h j nisms. The observations that C8-LacCer and cholesterol cause 1-integrin clustering and activation over time at 37 C, C8-LacCer clustering of microdomains provides a partial mechanistic expla- promotes the internalization of h1-integrin (Fig. 2), presumably nation for the selective stimulation of caveolar endocytosis that we leading to reduced levels of integrin at the plasma membrane. Loss of reported previously (ref. 25; i.e., these treatments induce the integrin at the cell surface may then lead to cell detachment selective clustering of glycosphingolipids and integrins into regions (Fig. 4C). Inhibitory effects of gangliosides (GT1b and GD3) on of the plasma membrane, which then become sites for endocytosis integrin-based cell adhesion and migration have been shown for via caveolae). Importantly, our study suggests that glycosphingo- keratinocytes binding to fibronectin (50, 51). GD3 has also been lipids and cholesterol at the plasma membrane may play important reported to inhibit the binding of neurepithelial cells to fibronectin roles in regulating the endocytic rate of integrins from the cell (52). It remains to be determined if the observed inhibitory effects of surface. some gangliosides on integrin function are due to stimulated C8-lactosylceramide and cholesterol induce signaling via integrin endocytosis and loss from the cell surface or other mecha- B1-integrin and reorganization of the actin cytoskeleton. nisms (e.g., reducing integrin clustering by supplanting integrin Treatment of fibroblasts with C8-LacCer or cholesterol initiated a association with other more favorable lipids). The net overall effects series of signaling events consistent with signaling via integrins. On of exogenous glycosphingolipids on integrin function may ultimately incubation of cells with C8-LacCer or cholesterol at 10jC, depend on the particular glycosphingolipids and integrin hetero- h1-integrins clustered in microdomains as stated above and were dimers present, the duration and temperature of treatment, and the found to be in an activated conformation as shown by binding with presence or absence of integrin ligands. Our studies thus far have the HUTS-4 antibody. Src was activated by treatment with only shown that C8-LacCer could affect integrin clustering and C8-LacCer. This stimulation of src activity was not inhibited by endocytosis. However, our previous study showing that, in addition h DN-Dyn1, suggesting that src elevation precedes 1-integrin to C8-LacCer, GM1 ganglioside could also stimulate caveolar endocytosis. At 10jC, P-Cav1 levels were increased by C8-LacCer endocytosis (25) suggests that C8-LacCer may not be unique among or cholesterol, similar to results seen with stimulation of cells with glycosphingolipids in its ability to regulate h1-integrin function. h1-integrin IgG. This increase in P-Cav1 levels was blocked by Further study will be required to determine the specific effects of KI-src expression, consistent with the idea that stimulation of src individual glycosphingolipid species on the clustering and endo- activity precedes P-Cav1 elevation. On a brief (5-15 minutes) warm- cytosis of other members of the integrin family. up to 37jC, C8-LacCer caused a rearrangement of the actin cytoskeleton, concomitant with translocation of RhoA away from Acknowledgments the plasma membrane. By 40 minutes after treatment, f50% of treated cells became detached from glass coverslips presumably as Received 3/9/2005; revised 6/28/2005; accepted 7/8/2005. The costs of publication of this article were defrayed in part by the payment of page a result of integrin internalization and cytoskeletal changes. charges. These articles must therefore be hereby marked advertisement in accordance Interestingly, RhoA movement from the plasma membrane was with 18 U.S.C. Section 1734 solely to indicate this fact.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2005 American Association for Cancer Research. The Glycosphingolipid, Lactosylceramide, Regulates β1 -Integrin Clustering and Endocytosis

Deepak K. Sharma, Jennifer C. Brown, Zhijie Cheng, et al.

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