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• W2014694687 abstract "The functional interaction (“cross-talk”) of integrins with growth factor receptors has become increasingly clear as a basic mechanism in cell biology, defining cell growth, adhesion, and motility. However, no studies have addressed the microdomains in which such interaction takes place nor the effect of gangliosides and tetraspanins (TSPs) on such interaction. Growth of human embryonal WI38 fibroblasts is highly dependent on fibroblast growth factor (FGF) and its receptor (FGFR), stably associated with ganglioside GM3 and TSPs CD9 and CD81 in the ganglioside-enriched microdomain. Adhesion and motility of these cells are mediated by laminin-5 ((LN5) and fibronectin (FN) through α3β1 and α5β1 integrin receptors, respectively. When WI38 cells or its transformant VA13 cells were adhered to LN5 or FN, α3β1 or α5β1 were stimulated, giving rise to signaling to activate FGFR through tyrosine phosphorylation and inducing cell proliferation under serum-free conditions without FGF addition. Types and intensity of signaling during the time course differed significantly depending on the type of integrin stimulated (α3β1 versus α5β1), and on cell type (WI38 versus VA13). Such effect of cross-talk between integrins and FGFR was influenced strongly by the change of GM3 and TSPs. (i) GM3 depletion by P4 caused enhanced tyrosine phosphorylation of FGFR and Akt followed by MAPK activation, without significant change of ceramide level. GM3 depletion also caused enhanced co-immunoprecipitation of FGFR with α3/α5/β1 and of these integrins with CD9/CD81. (ii) LN5- or FN-dependent proliferation of both WI38 and VA13 was strongly enhanced by GM3 depletion and by CD9/CD81 knockdown by siRNA. Thus, integrin-FGFR cross-talk is strongly influenced by GM3 and/or TSPs within the ganglioside-enriched microdomain. The functional interaction (“cross-talk”) of integrins with growth factor receptors has become increasingly clear as a basic mechanism in cell biology, defining cell growth, adhesion, and motility. However, no studies have addressed the microdomains in which such interaction takes place nor the effect of gangliosides and tetraspanins (TSPs) on such interaction. Growth of human embryonal WI38 fibroblasts is highly dependent on fibroblast growth factor (FGF) and its receptor (FGFR), stably associated with ganglioside GM3 and TSPs CD9 and CD81 in the ganglioside-enriched microdomain. Adhesion and motility of these cells are mediated by laminin-5 ((LN5) and fibronectin (FN) through α3β1 and α5β1 integrin receptors, respectively. When WI38 cells or its transformant VA13 cells were adhered to LN5 or FN, α3β1 or α5β1 were stimulated, giving rise to signaling to activate FGFR through tyrosine phosphorylation and inducing cell proliferation under serum-free conditions without FGF addition. Types and intensity of signaling during the time course differed significantly depending on the type of integrin stimulated (α3β1 versus α5β1), and on cell type (WI38 versus VA13). Such effect of cross-talk between integrins and FGFR was influenced strongly by the change of GM3 and TSPs. (i) GM3 depletion by P4 caused enhanced tyrosine phosphorylation of FGFR and Akt followed by MAPK activation, without significant change of ceramide level. GM3 depletion also caused enhanced co-immunoprecipitation of FGFR with α3/α5/β1 and of these integrins with CD9/CD81. (ii) LN5- or FN-dependent proliferation of both WI38 and VA13 was strongly enhanced by GM3 depletion and by CD9/CD81 knockdown by siRNA. Thus, integrin-FGFR cross-talk is strongly influenced by GM3 and/or TSPs within the ganglioside-enriched microdomain. Cell growth and associated differentiation are controlled by growth factors, and their receptors that have tyrosine kinases at the cytoplasmic domain (1Cohen S. Carpenter G. King L. J. Biol. Chem. 1980; 255: 4834-4842Abstract Full Text PDF PubMed Google Scholar, 2Schlessinger J. Trends Biochem. Sci. 1988; 13: 443-447Abstract Full Text PDF PubMed Scopus (295) Google Scholar, 3Ullrich A. Schlessinger J. Cell. 1990; 61: 203-212Abstract Full Text PDF PubMed Scopus (4569) Google Scholar). Cell adhesion to extracellular matrix and motility are mediated by integrin receptors consisting of various combinations of α and β subunits specific for extracellular matrix components, laminin (LN), 1The abbreviations used are: LN, laminin; BSA, bovine serum albumin; DLU, digital light unit; MJ, 1-deoxymannojirimycin; EGFR, epidermal growth factor receptor; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; FN, fibronectin; GEM, ganglioside-enriched microdomain; GFR, growth factor receptor; GlcCer, glucosyl-ceramide (Glcβ1Cer); GM3, NeuAcα3Galβ4Glcβ1Cer; GSL, glycosphin-golipid; HDEF, human dermal epithelial fibroblast; MAPK, mitogen-activated protein kinase; d-erythro-MAPP, (1S,2R)-d-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol; MEM, minimum essential medium; P4, d-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol; PBS, phosphate-buffered saline; PDMP, d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol; siRNA, small interfering RNA; TSP, tetraspanin. 1The abbreviations used are: LN, laminin; BSA, bovine serum albumin; DLU, digital light unit; MJ, 1-deoxymannojirimycin; EGFR, epidermal growth factor receptor; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; FN, fibronectin; GEM, ganglioside-enriched microdomain; GFR, growth factor receptor; GlcCer, glucosyl-ceramide (Glcβ1Cer); GM3, NeuAcα3Galβ4Glcβ1Cer; GSL, glycosphin-golipid; HDEF, human dermal epithelial fibroblast; MAPK, mitogen-activated protein kinase; d-erythro-MAPP, (1S,2R)-d-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol; MEM, minimum essential medium; P4, d-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol; PBS, phosphate-buffered saline; PDMP, d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol; siRNA, small interfering RNA; TSP, tetraspanin. fibronectin (FN), and collagen (4Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8941) Google Scholar, 5Ruoslahti E. J. Clin. Investig. 1991; 87: 1-5Crossref PubMed Scopus (1477) Google Scholar). In contrast, some integrins on hematopoietic cells mediate cell-to-cell interaction based on their binding capability to Ig-like receptors (ICAM-1, VCAM-1) (6Hemler M.E. Annu. Rev. Immunol. 1990; 8: 365-400Crossref PubMed Google Scholar). Growth factor receptors (GFRs) and integrin receptors clearly differ in their domain structure, mechanism of generating signaling, and localization in the membrane microdomain. Epidermal GFR (EGFR) and platelet-derived GFR are claimed to be associated with caveolar membrane (7Liu P. Ying Y. Ko Y.-G. Anderson R.G.W. J. Biol. Chem. 1996; 271: 10299-10303Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 8Mineo C. James G.L. Smart E.J. Anderson R.G.W. J. Biol. Chem. 1996; 271: 11930-11935Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar), whereas integrin receptors are soluble in 1% Triton X-100 and are considered to be located outside of the caveolar membrane or “raft” (9Anderson R.G.W. Annu. Rev. Biochem. 1998; 67: 199-225Crossref PubMed Scopus (1711) Google Scholar). However, in recent studies, integrin receptors have often been found associated with tetraspanins (TSPs) (10Mannion B.A. Berditchevski F. Kraeft S-K. Chen L.B. Hemler M.E. J. Immunol. 1996; 157: 2039-2047PubMed Google Scholar, 11Maecker H.T. Todd S.C. Levy S. FASEB J. 1997; 11: 428-442Crossref PubMed Scopus (801) Google Scholar), and integrin·TSP complexes are insoluble in 0.5% Triton X-100 (12Kazui A. Ono M. Handa K. Hakomori S. Biochem. Biophys. Res. Commun. 2000; 273: 159-163Crossref PubMed Scopus (45) Google Scholar) or in 1% Brij 98 (10Mannion B.A. Berditchevski F. Kraeft S-K. Chen L.B. Hemler M.E. J. Immunol. 1996; 157: 2039-2047PubMed Google Scholar, 13Ono M. Handa K. Withers D.A. Hakomori S. Biochem. Biophys. Res. Commun. 2000; 279: 744-750Crossref PubMed Scopus (87) Google Scholar, 14Ono M. Handa K. Sonnino S. Withers D.A. Nagai H. Hakomori S. Biochemistry. 2001; 40: 6414-6421Crossref PubMed Scopus (133) Google Scholar, 15Kawakami Y. Kawakami K. Steelant W.F.A. Ono M. Baek R.C. Handa K. Withers D.A. Hakomori S. J. Biol. Chem. 2002; 277: 34349-34358Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 16Hemler M.E. Annu. Rev. Cell Dev. Biol. 2003; 19: 397-422Crossref PubMed Scopus (629) Google Scholar). Gangliosides are capable of interacting with and modulating the function of various GFRs (17Zheng M. Fang H. Tsuruoka T. Tsuji T. Sasaki T. Hakomori S. J. Biol. Chem. 1993; 268: 2217-2222Abstract Full Text PDF PubMed Google Scholar, 18Mutoh T. Tokuda A. Miyada T. Hamaguchi M. Fujiki N. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5087-5091Crossref PubMed Scopus (391) Google Scholar, 19Toledo M.S. Suzuki E. Handa K. Hakomori S. J. Biol. Chem. 2004; 279: 34655-34664Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), integrins (20Cheresh D.A. Pytela R. Pierschbacher M.D. Klier F.G. Ruoslahti E. Reisfeld R.A. J. Cell Biol. 1987; 105: 1163-1173Crossref PubMed Scopus (194) Google Scholar, 21Zheng M. Fang H. Hakomori S. J. Biol. Chem. 1994; 269: 12325-12331Abstract Full Text PDF PubMed Google Scholar), Src family kinases, small G-proteins, and various other signal transducers (for review see Refs. 22Yates A.J. Rampersaud A. Ledeen R.W. Hakomori S. Yates A.J. Schneider J.S. Yu R.K. Sphingolipids as Signaling Modulators in the Nervous System. 845. New York Academy of Sciences, New York1998: 57-71Google Scholar and 23Hakomori S. Handa K. Iwabuchi K. Yamamura S. Prinetti A. Glycobiology. 1998; 8: xi-xviiiCrossref PubMed Scopus (185) Google Scholar). Glycosphingolipids (GSLs) in general have been implicated as inhibiting signal transduction, because various signaling molecules (e.g. c-Src, phospholipase Cγ) are activated when GSLs are depleted (24Shu L. Lee L. Shayman J.A. J. Biol. Chem. 2002; 277: 18447-18453Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 25Shu L. Shayman J.A. J. Biol. Chem. 2003; 278: 31419-31425Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar) by GlcCer synthase inhibitors (26Lee L. Abe A. Shayman J.A. J. Biol. Chem. 1999; 274: 14662-14669Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar) (see “Discussion”). Since the discovery of integrin-induced EGFR activation (27Wang F. Weaver V.M. Petersen O.W. Larabell C.A. Dedhar S. Briand P. Lupu R. Bissell M.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14821-14826Crossref PubMed Scopus (542) Google Scholar), “cross-talk” between integrins and GFRs has been a hot topic in various aspects of cell biology (28Moro L. Dolce L. Cabodi S. Bergatto E. Erba E.B. Smeriglio M. Turco E. Retta S.F. Giuffrida M.G. Venturino M. Godovac-Zimmermann J. Conti A. Schaefer E. Beguinot L. Tacchetti C. Gaggini P. Silengo L. Tarone G. Defilippi P. J. Biol. Chem. 2002; 277: 9405-9414Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar) (for review see Ref. 29Eliceiri B.P. Circ. Res. 2001; 89: 1104-1110Crossref PubMed Scopus (311) Google Scholar). However, the possible effect on such cross-talk of gangliosides, TSPs, or other membrane components in microdomains has not yet been studied. We previously observed that the GEM fraction of WI38 cells prepared in 1% Brij 98 contains stably associated FGFR and high levels of TSPs CD9 and CD81, in addition to c-Src, Csk (C-terminal Src kinase), and Lyn (19Toledo M.S. Suzuki E. Handa K. Hakomori S. J. Biol. Chem. 2004; 279: 34655-34664Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Similar GEM components were found in VA13, although their proportions differed greatly; i.e. the VA13 GEM fraction contained much higher (4–5-fold) levels of FGFR and c-Src, a much lower level of TSP (only 15–20%) than WI38 GEM, and an undetectable level of Csk. In the present study we found integrins α3β1 and α5β1 in WI38 and VA13 GEM prepared in 1% Brij 98, in addition to the above components. We also found a striking functional interaction between integrins and FGFR and studied the effects of ganglioside GM3, TSPs CD9/CD81, and receptor N-glycosylation on such interaction. Applying P4 (to deplete GM3), RNA interference (to knock down CD9/CD81), and N-glycosylation processing inhibitor 1-deoxymannojirimycin (DMJ), we found that GM3 and/or CD9/CD81 strongly affect the functional interaction between integrins and FGFR and, consequently, affect FGF-independent proliferation in both WI38 and VA13 cells. GM3, associated with CD9/CD81 or with FGFR, may inhibit integrin-induced signaling through Akt, c-Src, and MAPK to activate FGFR. The exact mechanism of this signaling pathway remains to be studied, but ceramide may not be involved, since GM3 depletion by P4 does not significantly change ceramide level. Cells—Human embryonal lung diploid fibroblast WI38 and its SV40-transformed cell line VA13 were from American Type Culture Collection (ATCC, Manassas, VA). WI38 passage numbers 18–26 were used for experiments. WI38 and VA13 were grown in minimum essential medium (MEM, Invitrogen) supplemented with 10% fetal bovine serum (Hyclone Laboratories, Logan, UT), 1 mm sodium pyruvate, and penicillin/streptomycin at 37 °C, 5% CO2. LN5-producing HDEF cells, kindly donated by Dr. William G. Carter (Fred Hutchinson Cancer Research Center, Seattle), were grown in serum-free keratinocyte growth medium (KGM, Cambrex Bioscience, Walkersville, MD) supplemented with SingleQuots® KBM (Cambrex). Antibodies—Anti-P-MAPK (Thr202/Tyr204) rabbit IgG, anti-MAPK (p42/44) rabbit IgG, anti-P-Src (Tyr416) rabbit IgG, anti-P-FGFR (Tyr653–Tyr654) rabbit Ig, anti-P-Akt (Ser473) rabbit IgG, and anti-Akt rabbit IgG were from Cell Signaling Technologies, Beverly, MA; anti-c-Src (SRC2) rabbit IgG, anti-caveolin-1 rabbit Ig, and anti-FGFR-3 rabbit IgG were from Santa Cruz Biotechnology, Santa Cruz, CA; anti-FGFR (Ab-1) mouse IgG1 was from Oncogene Science, Cambridge, MA; anti-α3 rabbit Ig, anti-α5 rabbit Ig, and anti-β1 rabbit Ig were from Chemicon, Temecula, CA, anti-CD81 mouse IgG2a from Beckman Coulter, Brea, CA, and anti-CD9 mouse IgG1 from BD Biosciences; anti-β-actin mouse IgG was from Sigma; goat anti-rabbit IgG-horseradish peroxidase was from Transduction Laboratories, Lexington, KY, and goat anti-mouse Ig-horseradish peroxidase from Santa Cruz Biotechnology. Reagents—FN from human plasma, poly-l-lysine, DMJ, and swain-sonine were from Sigma; siRNA-CD9 and siRNA-CD81 were from Ambion, Austin, TX; d-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (P4) was kindly donated by Dr. James A. Shayman, University of Michigan; and d-erythro-MAPP was from Matreya, Pleasant Gap, PA. Other reagents were from Sigma unless indicated otherwise. WI38 or VA13 (at least 5 × 107) cells cultured as described above were used for preparation of postnuclear fractions in 1% Brij 98 followed by sucrose density gradient centrifugation to isolate 12 fractions, including GEM (fractions 4–6), as described previously (15Kawakami Y. Kawakami K. Steelant W.F.A. Ono M. Baek R.C. Handa K. Withers D.A. Hakomori S. J. Biol. Chem. 2002; 277: 34349-34358Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 19Toledo M.S. Suzuki E. Handa K. Hakomori S. J. Biol. Chem. 2004; 279: 34655-34664Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). GSLs, TSPs CD9/CD81, FGFR and its activated form P-FGFR, integrins α3, α5, and β1, and various signal transducers (c-Src, Csk, Akt, MAPK) and their activated forms were determined by Western blot analysis as described previously (15Kawakami Y. Kawakami K. Steelant W.F.A. Ono M. Baek R.C. Handa K. Withers D.A. Hakomori S. J. Biol. Chem. 2002; 277: 34349-34358Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 19Toledo M.S. Suzuki E. Handa K. Hakomori S. J. Biol. Chem. 2004; 279: 34655-34664Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). LN5—HDEF cells grown as described above were harvested with 0.05% trypsin, 0.53 mm EDTA. This step was repeated twice to ensure that all HDEF cells were detached. Trypsin activity in plates and in cell suspension was inhibited by adding soybean trypsin inhibitor (Invitrogen) at 100 μg/ml in PBS. Plates were washed twice with PBS, blocked with serum-free MEM containing 1% heat-inactivated (10 min at 60 °C) BSA (MEM-BSA), stored at 4 °C, and used within 2 days. FN—5 μg/ml FN in PBS was coated in bacterial plates (Fisher) overnight at 4 °C. Plates were washed with PBS, blocked with MEM-BSA, stored at 4 °C, and used within 2 days. Poly-l-lysine—0.1% poly-l-lysine in PBS was coated in bacterial plates (Fisher) for 5 min at room temperature. Plates were washed with PBS, blocked with MEM-BSA, stored at 4 °C, and used within 2 days. Cells were grown in culture medium to ∼95% confluence, washed with and starved overnight in serum-free MEM, and harvested with trypsin/EDTA. Trypsin activity was inhibited with soybean trypsin inhibitor (100 μg/ml). Cells were centrifuged and suspended in serum-free MEM at 5 × 105 cells/ml. To decrease basal phosphorylation of signaling molecules, cells were rotated for 1 h at 37 °C.A portion of this cell suspension was used as control cells without adhesion. The rest of the cells were added (2 ml suspension; 4 × 104 cells/cm2) on 6-cm plates precoated with LN5, FN, or poly-l-lysine as described above and incubated for various durations at 37 °C, to study the changes induced in FGFR activation and other signal transduction. Cells were harvested by rubber scraper in ice-cold PBS containing sodium vanadate, centrifuged, and lysed in radioimmunoprecipitation assay buffer (30 mm HEPES, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 5 mm EDTA, 1 mm NaVO4,50mm NaF, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml pepstatin A, 10 μg/ml leupeptin, 75 units/ml aprotinin). Lysate was collected after centrifugation at 2000 × g for 10 min. After protein quantification, ∼30 μg of protein was loaded per well, and the degree of phosphorylation of standard signaling molecules was analyzed by Western blot using anti-P-FGFR, anti-P-Src, anti-P-Akt, and anti-P-MAPK antibodies. LN5-, FN-, and poly-l-lysine-coated 96-well plates were prepared as described above. WI38 and VA13 cells were treated for 72 h by incubation with 1000 nm P4 (19Toledo M.S. Suzuki E. Handa K. Hakomori S. J. Biol. Chem. 2004; 279: 34655-34664Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 30Li R. Manela J. Kong Y. Ladisch S. J. Biol. Chem. 2000; 275: 34213-34223Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), incubation with 7500 nm DMJ, or co-transfection with 20 nm CD9 and CD81 siRNA. Nontreated cells adhered to LN5 or FN were used as control. For negative control of CD9/CD81 siRNA transfectant, Silencer™ Negative Control #1 siRNA (Ambion; proprietary sequence) was transfected. All cells were starved overnight in serum-free MEM. Cells were harvested with trypsin/EDTA, and trypsin was neutralized by soybean trypsin inhibitor. Cells were centrifuged, suspended in MEM-BSA (2 × 104 cells/50 μl), rotated at 37 °C for 1 h, and transferred (50 μl suspension) to the coated plates. After 30 min, 1.5 μCi of [3H]thymidine was added per well. After a 3-h incubation at 37 °C, cells were washed five times with PBS, harvested with trypsin/EDTA, collected in Eppendorf tubes, centrifuged at 3000 rpm, and suspended in scintillation liquid. Incorporated [3H]thymidine was measured by scintillation counter. Four independent experiments were conducted. WI38 and VA13 (2 × 106 cells) were grown in culture medium in 150-mm plates. After 16 h at 37 °C, the medium was replaced by fresh complete medium with or without 1000 nm P4, 7500 nm DMJ, or 7500 nm swainsonine for 72 h. Cells were starved in serum-free MEM overnight and processed further as described under “Activation of FGFR and Other Signal Transducers (c-Src, Akt, MAPK) following Stimulation of Integrin Receptors.” FGFR-Integrin Receptor Interaction Studied by Co-immunoprecipitation—1% Brij lysate (postnuclear fraction) (15Kawakami Y. Kawakami K. Steelant W.F.A. Ono M. Baek R.C. Handa K. Withers D.A. Hakomori S. J. Biol. Chem. 2002; 277: 34349-34358Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) was subjected to co-immunoprecipitation, using a ProFound™ Mammalian Co-immunoprecipitation kit (Pierce) according to the manufacturer's instructions. Briefly, 100 μg of antibody (anti-FGFR, anti-α3, or anti-α5) was immobilized with 100 μl of antibody-coupling gel. Lysate containing ∼1.5 mg of protein was incubated at 4 °C overnight with antibody-affixed gel, washed three times with co-immunoprecipitation buffer, and eluted four times with elution buffer (50 μl). Eluates were analyzed by SDS-PAGE and Western blot. Normal mouse IgG was used as control. WI38 and VA13 siRNA Transfection—WI38 and VA13 cells were cultured at a density of 9 × 105 (for 100-mm dish) or 2 × 106 (for 150-mm dish). One day later, cells were co-transfected with 20 nm siRNA each for CD9 (sense, 5′-GGAGUCUAUAUUCUGAUCG-3′; antisense, 5′-CGAUCAGAAUAUAGACUCC-3′) and CD81 (sense, 5′-GGACCAGAUCGCCAAGGAU-3′; antisense, 5′-AUCCUUGGCGAUCUGGUCC-3′), using Oligofectamine (Invitrogen) according to the manufacturer's protocol. The gene-silencing effect was analyzed 3 days after transfection by Western blot. To decide the best sequence for CD9 knockdown, we also tested the sequence (sense, 5′-GGAUGAGGUGAUUAAGGAA-3′; antisense, 5′-UUCCUUAAUCACCUAAUCC-3′), which was less effective. The sequence for CD81 knockdown was taken from Zhang et al. (31Zhang J. Randall G. Higginbottom A. Monk P. Rice C.M. McKeating J.A. J. Virol. 2004; 78: 1448-1455Crossref PubMed Scopus (309) Google Scholar). The degree of expression of CD9 and CD81 in original WI38 and VA13 and in those co-transfected with siRNA was determined by Western blot with 10 μg of protein. Silencer™ Negative Control #1 siRNA (Ambion) was used as negative control, under the same conditions as for CD9 and CD81 siRNA. Cells were grown as described under “Cells, Antibodies, and Reagents.” Different groups were treated with P4 for 24, 48, or 72 h. For the 24-h treatment, subconfluent cells were cultured in serum-free MEM containing P4. For the 48-h treatment, cells were cultured in medium with serum and then in medium without serum, both containing P4 and both for 24 h. For the 72-h treatment, cells were cultured in medium with serum for 48 h and then without serum for 24 h, with both media containing P4. Within each group, subgroups were treated with 0, 100, or 1000 nm P4. All subgroups were subjected to a determination of ceramide quantity, as described in the following section. Subgroups from the 48-h- and 72-h-treated cells were subjected to determination of integrin-induced FGFR tyrosine phosphorylation by Western blot analysis with antibodies to tyrosine-phosphorylated (Tyr653–Tyr654) FGFR as described above. The protein content applied in each Western blot was ∼10 μg, rather than 30 μg. This condition was required to distinguish the occurrence of FGFR activation under different P4 treatments. Cultured cells treated with P4 at various concentrations and durations (see above) were subjected to determination of relative ceramide quantity compared with nontreated cells. Cells (3 × 106) were extracted in chloroform/methanol/water (2:1:0.8, v/v/v) by vigorous agitation. Chloroform and water (1 ml each) were added, mixed, and centrifuged (3000 rpm, 5 min) to separate the upper from the lower phase (32Signorelli P. Hannun Y.A. Methods Enzymol. 2002; 345: 275-294Crossref PubMed Scopus (26) Google Scholar). Total phospholipids in the lower phase were determined by Scion densitometric analysis of blot from Dittmer-Lester reagent (33Dittmer J.C. Lester R.L. J. Lipid Res. 1964; 5: 126-127Abstract Full Text PDF PubMed Google Scholar) (Sigma) as described previously (34Suzuki E. Handa K. Toledo M.S. Hakomori S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 14788-14793Crossref PubMed Scopus (72) Google Scholar). Quantity of ceramide in the lower phase was determined as ceramide-1-[32P]phosphate following 32P-phosphorylation by diacylglycerol kinase (Calbiochem), precisely following the method of Bektas et al. (35Bektas M. Jolly P.S. Milstien S. Spiegel S. Anal. Biochem. 2003; 320: 259-265Crossref PubMed Scopus (20) Google Scholar). C16-ceramide (Matreya) was used as control. Ceramide-1-[32P]phosphate was separated on high performance TLC developed in chloroform/acetone/methanol/acetic acid/water (10:4: 3:2:1,v/v/v/v). The relative amount of ceramide-1-[32P]phosphate from P4-treated versus nontreated cells, using aliquots with the same quantity (30 nmol) of total phospholipid, was quantified as digital light units (DLU) using a phosphorimaging device (Cyclone Storage Phosphor Screen, PerkinElmer Life Sciences), and expressed as the ratio of DLU value (value in nontreated cells defined as 1.0). The relative ceramide level was also determined in cells treated with the ceramidase inhibitor d-erythro-MAPP (36Bielawska A. Greenberg M.S. Perry D. Jayadev S. Shayman J.A. McKay C. Hannun Y.A. J. Biol. Chem. 1996; 271: 12646-12654Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar) for 24 h and expressed as pmol/nmol phospholipid. WI38 or VA13 cells treated with 100 or 1000 nm P4 for 48 or 72 h were harvested. The postnuclear fraction was prepared and separated into 12 fractions by sucrose density gradient centrifugation as described above. Lipids present in each fraction were adsorbed on C18 silica gel column (Varian), separated from sucrose and other water-soluble components, eluted in chloroform/methanol 2:1, separated on TLC under conditions similar to those for ceramide, and detected by primulin spray. Fluorescence intensity corresponding to standard P4 was estimated as % of total P4 applied to cells. WI38 Cell Adhesion to LN5- or FN-coated Plates Induces Activation of FGFR and Other Signal Transducers—When WI38 cells were adhered to LN5- or FN-coated plates, FGFR was activated, as indicated by its tyrosine phosphorylation (at Tyr653–Tyr654) (Fig. 1A, columns a and b, row 1). FGFR activation occurred within 10 min to 1 h. FGFR activation was not observed in cell suspension without cell adhesion (Fig. 1A, column S, row 1) or in cells adhered on poly-l-lysine-coated plates (data not shown). In close association with FGFR activation, strong c-Src activation also occurred during the early stage (10 min to 1 h) of cell adhesion to LN5 or FN (Fig. 1A, columns a and b, row 3); Akt activation after adhesion to FN also occurred at the early stage (10 min to 1 h), whereas Akt activation after adhesion to LN5 occurred at a later stage (1–24 h) (Fig. 1A, row 5). In contrast, MAPK activation (P-MAPK) occurred at the later stage (1–24 h) regardless of adhesion to FN or LN5 (Fig. 1A, columns a and b, row 7). Only minimal activation was observed upon adhesion to poly-l-lysine (data not shown). VA13 Cell Adhesion to LN5-or FN-coated Plates Induces Activation of FGFR and Other Signal Transducers—Adhesion of VA13 cells to LN5- or FN-coated plates induced activation of FGFR by tyrosine phosphorylation (Fig. 1B, columns a and b, row 1) similar to that in WI38 cells. However, the pattern of associated signal transducer changes differed from that in WI38. c-Src activation occurred strongly at a later stage, i.e. 24 h after adhesion to both LN5 and FN (Fig. 1B, columns a and b, row 3). Akt activation was stronger when cells were adhered to FN than to LN5 and was higher at the early stage (10 min to 1 h) (Fig. 1B, column b, row 5). In contrast, MAPK activation was stronger when cells were adhered to LN5 than to FN and in both cases was higher at the later stage (4–24 h) (Fig. 1B, columns a and b, row 7). Quantitative Time Course Change of FGFR Phosphorylation Induced by Integrin-dependent Adhesion by WI38 or VA13 Cells on LN5 of FN—Because FGFR activation in terms of Tyr653–Tyr654 phosphorylation induced by LN5- or FN-dependent adhesion is of primary importance, the time course process was determined quantitatively as described in the legend for Fig. 1, C and D. The results indicate that FGFR activation occurred consistently at the early stage (10 min to 1 h) after cell adhesion to LN5 or FN in both WI38 and VA13 cells. Change of GM3 Level Alters Functional Interaction of Integrin with FGFR in WI38 and VA13 Cells—Ganglioside GM3 is the sole GSL present in low-density microdomain fraction of WI38 cells. ∼95% of total GM3 was depleted upon preincubation of cells with P4 (1000 nm, 72 h) (19Toledo M.S. Suzuki E. Handa K. Hakomori S. J. Biol. Chem. 2004; 279: 34655-34664Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Depletion of gangliosides by P4, at similar concentration and duration, had been observed previously in other types of cells (30Li R. Manela J. Kong Y. Ladisch S. J. Biol. Chem. 2000; 275: 34213-34223Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). A lower concentration of P4 or a shorter incubation time resulted in a lesser depletion of GM3 (see below). GM3 suppressed the process of LN5-induced FGFR activation, because depletion of GM3 by P4 significantly enhanced FGFR tyrosine phosphorylation. Such differential effect on FGFR phosphorylation in the presence versus absence of GM3 was more clear in LN5-induced than in FN-induced adhesion of WI38 cells (Fig. 2A, row 1 versus 3). A similar differential effect in the presence versus absence of GM3 was observed for Akt phosphorylation (Fig. 2A, row 5 versus 7). In contrast, in transformed VA13 cells, the presence of GM3 versus its depletion by P4 made no clear difference in the effect of integrin-induced activation on FGFR tyrosine phosphorylation (Fig. 2B, row 1 versus 3) or on Akt phosphorylation (row 5 versus 7). However, VA13 showed much higher MAPK phosphoryl" @default.
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• W2014694687 title "Effect of Ganglioside and Tetraspanins in Microdomains on Interaction of Integrins with Fibroblast Growth Factor Receptor" @default.
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