FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2043945964> ?p ?o ?g. }

• W2043945964 endingPage "35553" @default.
• W2043945964 startingPage "35545" @default.
• W2043945964 abstract "Cell motility is highly dependent on the organization and function of microdomains composed of integrin, proteolipid/tetraspanin CD9, and ganglioside (Ono, M., Handa, K., Sonnino, S., Withers, D. A., Nagai, H., and Hakomori, S. (2001) Biochemistry 40, 6414–6421; Kawakami, Y., Kawakami, K., Steelant, W. F. A., Ono, M., Baek, R. C., Handa, K., Withers, D. A., and Hakomori, S. (2002) J. Biol. Chem. 277, 34349–34358), later termed “glycosynapse 3” (Hakomori, S., and Handa, K. (2002) FEBS Lett. 531, 88–92, 2002). Human bladder cancer cell lines KK47 (noninvasive and nonmetastatic) and YTS1 (highly invasive and metastatic), both derived from transitional bladder epithelia, are very similar in terms of integrin composition and levels of tetraspanin CD9. Tetraspanin CD82 is absent in both. The major difference is in the level of ganglioside GM3, which is several times higher in KK47 than in YTS1. We now report that the GM3 level reflects glycosynapse function as follows: (i) a stronger interaction of integrin α3 with CD9 in KK47 than in YTS1; (ii) conversion of benign, low motility KK47 to invasive, high motility cells by depletion of GM3 by P4 (d-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol) treatment or by knockdown of CD9 by the RNA interference method; (iii) reversion of high motility YTS1 to low motility phenotype like that of KK47 by exogenous GM3 addition, whereby the α3-to-CD9 interaction was enhanced; (iv) low GM3 level activated c-Src in YTS1 or in P4-treated KK47, and high GM3 level by exogenous addition caused Csk translocation into glycosynapse, with subsequent inhibition of c-Src activation; (v) inhibition of c-Src by “PP2” in YTS1 greatly reduced cell motility. Thus, GM3 in glycosynapse 3 plays a dual role in defining glycosynapse 3 function. One is by modulating the interaction of α3 with CD9; the other is by activating or inhibiting the c-Src activity, possibly through Csk translocation. High GM3 level decreases tumor cell motility/invasiveness, whereas low GM3 level enhances tumor cell motility/invasiveness. Oncogenic transformation and its reversion can be explained through the difference in glycosynapse organization. Cell motility is highly dependent on the organization and function of microdomains composed of integrin, proteolipid/tetraspanin CD9, and ganglioside (Ono, M., Handa, K., Sonnino, S., Withers, D. A., Nagai, H., and Hakomori, S. (2001) Biochemistry 40, 6414–6421; Kawakami, Y., Kawakami, K., Steelant, W. F. A., Ono, M., Baek, R. C., Handa, K., Withers, D. A., and Hakomori, S. (2002) J. Biol. Chem. 277, 34349–34358), later termed “glycosynapse 3” (Hakomori, S., and Handa, K. (2002) FEBS Lett. 531, 88–92, 2002). Human bladder cancer cell lines KK47 (noninvasive and nonmetastatic) and YTS1 (highly invasive and metastatic), both derived from transitional bladder epithelia, are very similar in terms of integrin composition and levels of tetraspanin CD9. Tetraspanin CD82 is absent in both. The major difference is in the level of ganglioside GM3, which is several times higher in KK47 than in YTS1. We now report that the GM3 level reflects glycosynapse function as follows: (i) a stronger interaction of integrin α3 with CD9 in KK47 than in YTS1; (ii) conversion of benign, low motility KK47 to invasive, high motility cells by depletion of GM3 by P4 (d-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol) treatment or by knockdown of CD9 by the RNA interference method; (iii) reversion of high motility YTS1 to low motility phenotype like that of KK47 by exogenous GM3 addition, whereby the α3-to-CD9 interaction was enhanced; (iv) low GM3 level activated c-Src in YTS1 or in P4-treated KK47, and high GM3 level by exogenous addition caused Csk translocation into glycosynapse, with subsequent inhibition of c-Src activation; (v) inhibition of c-Src by “PP2” in YTS1 greatly reduced cell motility. Thus, GM3 in glycosynapse 3 plays a dual role in defining glycosynapse 3 function. One is by modulating the interaction of α3 with CD9; the other is by activating or inhibiting the c-Src activity, possibly through Csk translocation. High GM3 level decreases tumor cell motility/invasiveness, whereas low GM3 level enhances tumor cell motility/invasiveness. Oncogenic transformation and its reversion can be explained through the difference in glycosynapse organization. The interaction of tumor cells with their microenvironment may define the direction of tumor development and the degree of malignancy (1Liotta L.A. Kohn E.C. Nature. 2001; 411: 375-379Crossref PubMed Scopus (2051) Google Scholar). Such an interaction is likely based on the structure and function of the microdomain at the tumor cell surface interfacing with the normal cell microdomain and with extracellular matrix components, particularly at the basement membrane. A crucial event for the progression of many types of tumor cells of epithelial origin is their adhesion/motility/invasion on the basement membrane underlying epithelial cells. In this process, specific microdomains of tumor cells are considered to interact with laminin-5 (“epiligrin”) (2Carter W.G. Ryan M.C. Gahr P.J. Cell. 1991; 65: 599-610Abstract Full Text PDF PubMed Scopus (670) Google Scholar) or laminin-10/11 (3Gu J. Fujibayashi A. Yamada K.M. Sekiguchi K. J. Biol. Chem. 2002; 277: 19922-19928Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), which are major components of epithelial basement membrane and ligands of integrin α3 or α3β1 (4Tsuji T. Kawada Y. Kai-Murozono M. Komatsu S. Han S.A. Takeuchi K. Mizushima H. Miyazaki K. Irimura T. Clin. Exp. Metastasis. 2002; 19: 127-134Crossref PubMed Scopus (103) Google Scholar). Such microdomains, having proteolipid/tetraspanin (PLP/TSP) 2The abbreviations used are: PLP/TSPproteolipid/tetraspaninC/Mchloroform/methanolCskC-terminal Src kinaseco-IPco-immunoprecipitationFITCfluorescein isothiocyanateGEMGSL-enriched microdomainGSLglycosphingolipidHPTLChigh-performance thin-layer chromatographyPLPproteolipidPNFpostnuclear fractionPP24-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidineP4d-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanolRNAiRNA interferenceTSPtetraspaninmAbmonoclonal antibodyRTreverse transcription. CD9, integrin α3β1, and ganglioside GM3 (5Ono M. Handa K. Withers D.A. Hakomori S. Cancer Res. 1999; 59: 2335-2339PubMed Google Scholar, 6Kawakami 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 (105) Google Scholar, 7Satoh M. Ito A. Nojiri H. Handa K. Numahata K. Ohyama C. Saito S. Hoshi S. Hakomori S. Int. J. Oncol. 2001; 19: 723-731PubMed Google Scholar), are capable of controlling cell adhesion and motility (6Kawakami 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 (105) Google Scholar, 8Hakomori S. Handa K. FEBS Lett. 2002; 531: 88-92Crossref PubMed Scopus (118) Google Scholar, 9Hemler M.E. Annu. Rev. Cell Dev. Biol. 2003; 19: 397-422Crossref PubMed Scopus (658) Google Scholar, 10Toledo M.S. Suzuki E. Handa K. Hakomori S. J. Biol. Chem. 2004; 279: 34655-34664Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), in contrast to “caveolar membrane” or “raft,” which is cholesterol-dependent (11Simons K. Toomre D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 31-39Crossref PubMed Scopus (5187) Google Scholar), has no integrins (12Anderson R.G.W. Annu. Rev. Biochem. 1998; 67: 199-225Crossref PubMed Scopus (1727) Google Scholar), and is not involved in cell adhesion and motility. GM3/TSP/integrin microdomains have therefore been termed “glycosynapse” (8Hakomori S. Handa K. FEBS Lett. 2002; 531: 88-92Crossref PubMed Scopus (118) Google Scholar, 13Hakomori S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 225-232Crossref PubMed Scopus (472) Google Scholar), in analogy to the microdomain involved in immunocyte adhesion/antigen presentation with concurrent signaling, termed immunosynapse (14Krummel M.F. Davis M.M. Curr. Opin. Immunol. 2002; 14: 66-74Crossref PubMed Scopus (175) Google Scholar). Among the glycosynapses, GM3-CD9-integrin complex termed “glycosynapse 3” (8Hakomori S. Handa K. FEBS Lett. 2002; 531: 88-92Crossref PubMed Scopus (118) Google Scholar) was suggested previously to play a role in the regulation of cell motility (see “Discussion”). proteolipid/tetraspanin chloroform/methanol C-terminal Src kinase co-immunoprecipitation fluorescein isothiocyanate GSL-enriched microdomain glycosphingolipid high-performance thin-layer chromatography proteolipid postnuclear fraction 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine d-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol RNA interference tetraspanin monoclonal antibody reverse transcription. GSLs, including GM3, have been implicated as inhibitors of signal transduction, because various signaling molecules such as c-Src and phospholipase C-γ (15Shu L. Lee L. Shayman J.A. J. Biol. Chem. 2002; 277: 18447-18453Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 16Shu L. Shayman J.A. J. Biol. Chem. 2003; 278: 31419-31425Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar), and growth factor receptor tyrosine kinases (17Toledo M.S. Suzuki E. Handa K. Hakomori S. J. Biol. Chem. 2005; 280: 16227-16234Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), are activated when GSLs are depleted by P4, a GlcCer transferase inhibitor (18Lee L. Abe A. Shayman J.A. J. Biol. Chem. 1999; 274: 14662-14669Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). The goal of the present study was to clarify contrasting composition of GM3, α3, and CD9 in glycosynapse 3 and their interaction and to define motility/invasive properties of two closely related human bladder cancer cell lines: KK47 with noninvasive, low motility phenotype, and YTS1 with invasive, high motility phenotype. Our results make clear the dual functional role of GM3 in glycosynapse 3 as follows: (i) high or low GM3 level promotes or inhibits the interaction of α3 with CD9 in order to stabilize or de-stabilize the α3-CD9-GM3 complex within the microdomain; (ii) high or low GM3 level activates or inhibits c-Src through association or dissociation with Csk within the same microdomain. Through either process, the GM3 level regulates tumor cell motility/invasiveness. Oncogenic conversion or phenotypic reversion from invasive to noninvasive cells is associated with change of the GM3 level, which mediates the functional alteration of the α3 interaction with CD9 and of the degree of c-Src activation with Csk interaction. The present study may help explain the mechanism by which glycosynapse organization and composition define oncogenic conversion or reversion to normal cell phenotypes, as suggested in previous studies of bladder cancer (7Satoh M. Ito A. Nojiri H. Handa K. Numahata K. Ohyama C. Saito S. Hoshi S. Hakomori S. Int. J. Oncol. 2001; 19: 723-731PubMed Google Scholar, 19Kakizaki H. Yamaguchi T. Suzuki H. Kubota Y. Ishii N. Numasawa K. Suzuki K. Jpn. J. Urol. 1990; 81: 204-209Crossref PubMed Scopus (4) Google Scholar, 20Masters J.R. Hepburn P.J. Walker L. Highman W.J. Trejdosiewicz L.K. Povey S. Parkar M. Hill B.T. Riddle P.R. Franks L.M. Cancer Res. 1986; 46: 3630-3636PubMed Google Scholar), colorectal cancer (5Ono M. Handa K. Withers D.A. Hakomori S. Cancer Res. 1999; 59: 2335-2339PubMed Google Scholar, 6Kawakami 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 (105) Google Scholar, 21Ono M. Handa K. Sonnino S. Withers D.A. Nagai H. Hakomori S. Biochemistry. 2001; 40: 6414-6421Crossref PubMed Scopus (137) Google Scholar), and Jun-transformed cells (22Miura Y. Kainuma M. Jiang H. Velasco H. Vogt P.K. Hakomori S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 16204-16209Crossref PubMed Scopus (65) Google Scholar). Cells—The YTS1 cell line was established from invasive human urinary bladder cancer (23Kakizaki H. Numasawa K. Suzuki K. Jpn. J. Urol. 1986; 77: 1790-1795Crossref PubMed Scopus (9) Google Scholar) and was donated by H. Kakizaki (Department of Urology, Yamagata University, Yamagata, Japan). The KK47 cell line was established from noninvasive, superficial bladder cancer (24Hisazumi H. Kanokogi M. Nakajima K. Kobayashi T. Tsukahara K. Naito K. Kuroda K. Matsubara F. Jpn. J. Urol. 1979; 70: 485-494Crossref PubMed Scopus (9) Google Scholar) and was donated by T. Masuko (Department of Hygienic Chemistry, Faculty of Pharmaceutical Science, Tohoku University, Sendai, Japan). Both cell lines were grown in RPMI 1640 containing 10% fetal bovine serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin at 37 °C, 5% CO2. Antibodies—Mouse anti-GM3 IgG3 mAb DH2 (25Dohi T. Nores G. Hakomori S. Cancer Res. 1988; 48: 5680-5685PubMed Google Scholar) was established in this laboratory. Mouse anti-CD9 IgG1 mAb was from Pharmingen. Rabbit anti-α3 and -α5 and mouse anti-β1 were from Chemicon International (Temecula, CA). Rabbit anti-Src, -P-Src (Tyr-416), -P-Src (Tyr-527), and -Csk were from Cell Signaling Technologies (Beverly, MA). Anti-β-actin and anti-γ-tubulin mouse IgG were from Sigma. Goat anti-mouse IgG labeled with horseradish peroxidase and goat anti-mouse IgG1 labeled with Texas Red were from Southern Biotech (Birmingham, AL). Goat anti-rabbit IgG labeled with horseradish peroxidase and goat anti-rabbit IgG labeled with FITC were from Santa Cruz Biotechnology (Santa Cruz, CA). Goat anti-mouse IgM + IgG labeled with FITC was from BIOSOURCE. Reagents—Gangliosides GM3 and GM1 were from Matreya (Pleasant Gap, PA). d-Threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (P4) (18Lee L. Abe A. Shayman J.A. J. Biol. Chem. 1999; 274: 14662-14669Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar) was originally established and kindly donated by J. A. Shayman (University of Michigan). FITC-labeled cholera toxin subunit B was from Sigma. 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), an Src family kinase inhibitor (26Hanke J.H. Gardner J.P. Dow R.L. Changelian P.S. Brissette W.H. Weringer E.J. Pollok B.A. Connelly P.A. J. Biol. Chem. 1996; 271: 695-701Abstract Full Text Full Text PDF PubMed Scopus (1790) Google Scholar), was from Biomol (Plymouth Meeting, PA). Micro-BCA protein assay reagent kit was from Pierce. Immunostain horseradish peroxidase-1000 kit was from Konica (Tokyo, Japan). Vecstain ABC kit was from Vector Laboratories (Burlingame, CA), and protein A/G-agarose was from Santa Cruz Biotechnology. Other reagents were from Sigma unless described otherwise. Total cell lysate was prepared as described previously (6Kawakami 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 (105) Google Scholar). Briefly, ∼1 × 107 cells were collected, and the cell pellet was suspended in 1 ml of RIPA buffer (1% Triton X-100, 150 mm NaCl, 25 mm Tris, pH 7.5, 0.5% sodium deoxycholate, 0.1% SDS, 5 mm pyrophosphate, 50 mm NaF) containing 75 units of aprotinin and 2 mm phenylmethanesulfonyl fluoride. The suspension was kept on ice for 30 min and Dounce homogenized (10–15 strokes). The lysate was centrifuged at 15,000 rpm for 15 min at 4 °C, and the supernatant was subjected to SDS-PAGE and Western blot after determination of protein concentration. The membranes were reblotted with anti-β-actin antibody after stripping with Re-Blot Plus solution (Chemicon). The intensity of Western blot was determined by densitometry using Scion image program. GSLs were extracted and analyzed as described previously (27Nakamura K. Suzuki M. Taya C. Inagaki F. Yamakawa T. Suzuki A. J. Biochem. (Tokyo). 1991; 110: 832-841Crossref PubMed Scopus (38) Google Scholar, 28Aoki H. Satoh M. Mitsuzuka K. Ito A. Saito S. Funato T. Endoh M. Takahashi T. Arai Y. FEBS Lett. 2004; 567: 203-208Crossref PubMed Scopus (28) Google Scholar). Briefly, YTS1 and KK47 cells were grown until ∼90% confluence in 15-cm dishes and washed three times with PBS (137 mm NaCl, 8.1 mm Na2HPO4, 2.68 mm KCl, 1.47 mm KH2PO4, pH 7.4). ∼2×107 cells were collected and extracted twice with C/M (2:1). The extracts were dried under a N2 stream. To remove phospholipids, the dried residue was dissolved in 2 ml of 0.1 m NaOH in methanol and incubated at 40 °C for 2 h. After neutralization with 1 n HCl, fatty acids were extracted twice with 2 ml of hexane. GSLs in the lower phase were evaporated, dissolved in water, and applied to 3 ml of BondElut C18 columns (Varian, Harbor City, CA) to remove salt. Columns were washed with water, and GSLs were eluted with C/M (2:1). Solvents were evaporated, and equal aliquots of GSLs dissolved in C/M (1:1) were subjected to HPTLC plate (Merck), developed with C/M, 0.2% CaCl2 in H2O (50:40:10), and stained with 0.5% orcinol in 2 n sulfuric acid to visualize GSLs, or immunostained with DH2 using Vecstain ABC kit and Immunostain horseradish peroxidase-1000 kit according to the manufacturer's instructions. For analysis of GSLs in GEM, after fractionation of postnuclear fractions (PNF) through sucrose density gradient ultracentrifugation, low density fractions and high density fractions were applied to C18 column and processed as described above. PNF was prepared as described previously (6Kawakami 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 (105) Google Scholar). Briefly, ∼4 × 107 cells were collected, and the pellet was suspended in 1 ml of Brij 98 lysis buffer (1% Brij 98, 25 mm HEPES buffer, pH 7.5, 150 mm NaCl, 5 mm EDTA) containing 75 units of aprotinin and 2 mm phenylmethanesulfonyl fluoride. The suspension was kept on ice for 30 min and Dounce homogenized (10∼15 strokes). The lysate was centrifuged at 2,500 rpm for 7 min at 4 °C to remove nuclei and debris. PNF was subjected to sucrose density gradient ultracentrifugation to separate low density membrane fractions as described previously (6Kawakami 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 (105) Google Scholar). Fractions were separated and numbered 1–12 from top to bottom. Aliquots of each fraction, containing equal protein content (∼2.5 μg), were analyzed by SDS-PAGE and Western blot. Cell motility was studied by an improved method (29Scott W.N. McCool K. Nelson J. Anal. Biochem. 2000; 287: 343-344Crossref PubMed Scopus (21) Google Scholar) based on Ref. 30Albrecht-Buehler G. Cell. 1977; 11: 395-404Abstract Full Text PDF PubMed Scopus (384) Google Scholar. Briefly, 24-well plates were incubated with 1% bovine serum albumin for 24 h at 37 °C, washed with 100% ethanol, and dried. Gold sol suspension (gold colloidal particles) was prepared by adding 11 ml of H2O and 6 ml of 36.5 mm Na2CO3 to 1.8 ml of 14.5 mm AuHCl4. The mixture was boiled, and 1.8 ml of freshly prepared 0.1% formaldehyde solution was added. Gold sol suspension was put in each well and incubated for 40 min at room temperature, and the plate was washed with culture medium. Cells in culture medium (1 × 104/well) were plated onto gold sol-coated wells and incubated for 16 h at 37 °C. Migratory cells were observed and photographed under light microscope (Nikon). Migratory areas of 20 cells of each well were measured by Scion image program and expressed as square pixels. Interaction between CD9 and integrins was analyzed by co-IP as described previously (6Kawakami 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 (105) Google Scholar). Briefly, PNF (400 μl, containing 400 μg protein) was prepared as described above and mixed with protein A/G-agarose beads (∼30-μl bed volume). The mixture was placed on rotator at 4 °C for 3 h and centrifuged at 1,000 rpm to collect supernatant. ∼3 μg of antibody were added to the supernatant and rotated at 4 °C overnight; the antibodies used were rabbit anti-α3 and -α5 and mouse anti-β1. Protein A/G-agarose beads was added, rotated at 4 °C for 3 h, and centrifuged to collect the beads. After washing twice with lysis buffer, the immunoprecipitates were resolved in SDS-PAGE sample buffer and subjected to SDS-PAGE and Western blot with mouse anti-CD9 IgG1 antibody. The interaction between CD9 and α3 was also analyzed by confocal microscopy as described previously (6Kawakami 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 (105) Google Scholar). Briefly, YTS1 and KK47 cells were grown for 24 h on cover glass (12 mm diameter) placed in 24-well plates. Cells on cover glass were washed three times with PBS and fixed with 3.7% paraformaldehyde/PBS for 15 min. Fixed cells were washed three times with PBS, incubated with 1% bovine serum albumin, 0.1% NaN3, PBS for 30 min and incubated with mouse anti-CD9 IgG1 mAb for 1 h at room temperature. After washing and permeabilization with 0.05% Triton X-100 in PBS for 5 min, rabbit anti-α3 was added and incubated for 1 h at room temperature. Cells were incubated with a mixture of goat anti-mouse IgG1-labeled with Texas Red and goat anti-rabbit IgG labeled with FITC for 1 h at room temperature, washed, mounted with a drop of Glycergel mounting medium (Dako, Carpinteria, CA), and observed by laser-scanning confocal microscopy (Fluo-View™, Olympus, Tokyo, Japan) using an appropriate filter set. YTS1 cells were grown in a 10-cm dish for 24 h, and growth medium was changed to serum-free medium. GM3 was dissolved in serum-free medium by sonication and standing for 24 h at room temperature, added to cells in serum-free medium at concentrations of 10, 20, and 50 μm, and incubated at 37 °C for 24 h. GM1 was exogenously added in the same way in a separate experiment. Cells were analyzed by co-IP and confocal microscopy for interaction between CD9 and α3 and by phagokinetic gold sol assay for cell motility. KK47 cells were grown in a 10-cm dish for 24 h at 37 °C. The medium was changed to fresh growth medium with or without P4 (1 μm) and further incubated for 72 h. The cells were analyzed by co-IP and confocal microscopy for interaction between CD9 and α3 and by phagokinetic gold sol assay for cell motility. Ceramide in P4-treated cells was analyzed by HPTLC with C/M/water (65:25:4) and charring in 3% cupric acetate and 10% phosphoric acid for 20 min at 130 °C, on 400 μg of cellular protein basis, as described previously (31Rani C.S. Abe A. Chang Y. Rosenzweig N. Saltiel A.R. Radin N.S. Shayman J.A. J. Biol. Chem. 1995; 270: 2859-2867Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 32Li R. Manela J. Kong Y. Ladisch S. J. Biol. Chem. 2000; 275: 34213-34223Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Plasmid Construct—A plasmid-based system for production of RNAi was made in pSUPER vector (Oligoengine, Seattle, WA) according to the manufacturer's instructions, as described previously (33Sui D. Wilson J.E. Biochem. Biophys. Res. Commun. 2004; 319: 768-773Crossref PubMed Scopus (8) Google Scholar, 34Brummelkamp T.R. Bernards R. Agami R. Science. 2002; 296: 550-553Crossref PubMed Scopus (3971) Google Scholar). Four oligonucleotides (68 nucleotides) encoding CD9-specific sequences and one oligonucleotide (68 nucleotides) encoding a control scrambled sequence were constructed (CD9-specific sequences or control scrambled sequences shown in italics): A, 5′-GATCCCCgctgttcggatttaacttcatTTCAAGAGAatgaagttaaatccgaacagcTTTTTGGAAA-3′ and 3′-GGGcgacaagcctaaattgaagtaAAGTTCTCTtacttcaatttaggcttgtcgAAAAACCTTTTCGA-5′; B, 5′-GATCCCCacaggagtctatattctgatcTTCAAGAGAgatcagaatatagactcctgtTTTTTGGAAA-3′ and 3′-GGGtgtcctcagatataagactagAAGTTCTCTctagtcttatatctgaggacaAAAAACCTTTTCGA-5′; C, 5′-GATCCCCccaagagcatcttcgagcaagTTCAAGAGActtgctcgaagatgctcttggTTTTTGGAAA-3′ and 3′-GGGggttctcgtagaagctcgttcAAGTTCTCTgaacgagcttctacgagaaccAAAAACCTTTTCGA-5′; D, 5′-GATCCCCggattgctgtccttgccattgTTCAAGAGAcaatggcaaggacagcaatccTTTTTGGAAA-3′ and 3′-GGGcctaacgacaggaacggtaacAAGTTCTCTgttaccgttcctgtcgttaggAAAAACCTTTTCGA-5′; Control, 5′-GATCCCCggtaagcattatcttcaagtcTTCAAGAGAgacttgaagataatgcttaccTTTTTGGAAA-3′ and 3′-GGGccattcgtaatagaagttcagAAGTTCTCTctgaacttctattacgaatggAAAAACCTTTTCGA-5′. Transfection—These plasmids were co-transfected with pPUR vector (Clontech) into KK47 cells by electroporation. Two days after transfection, puromycin (0.5 μg/ml) was added for selection. After cloning, CD9 mRNA level was measured by RT-PCR, and CD9 expression was measured by flow cytometry and Western blot. RT-PCR—Total RNA was isolated from transfectants by RNeasy minikit (Qiagen, Valencia, CA). cDNA was prepared from 1 μg of total RNA using Superscript II kit (Invitrogen). PCR was performed using TaqDNA polymerase (Promega, Madison, WI) according to the manufacturer's instruction, with CD9 primers (sense, 5′-TTGGACTATGGCTCCGATTC-3′; antisense, 5′-AGCATGCACTGGGACTCCT-3′), yielding a 175-bp product, and with β-actin primers (sense, 5′-AACCGCGAGAAGATGACCCAG-3′; antisense, 5′-CTCCTGCTTGCTGATCCACAT-3′), yielding a 721-bp product. Flow Cytometry—Cells were detached by trypsin/EDTA and washed with PBS. Aliquots of cells (1 × 105) were incubated with mouse anti-CD9 IgG1 for 1 h on ice, washed with PBS, incubated with goat anti-mouse IgM + G-labeled with FITC for 40 min on ice, fixed in 2% paraformaldehyde/PBS, and analyzed using a Coulter EPICS XL flow cytometer (Beckman Coulter, Fullerton, CA). c-Src and its phosphorylation at Tyr-416 (for activation) and at Tyr-527 (for inhibition) were determined by Western blot analysis in KK47 versus YTS1 cells, using phosphorylation site-specific antibodies. PNF, low density membrane fractions (fractions 4–6), and high density membrane fractions (fractions 10–12) separated through sucrose density gradient ultracentrifugation were analyzed. Csk was determined simultaneously in these fractions. To test the GM3 effect on c-Src activity, KK47 cells were pretreated with P4 for 72 h (see “Effect of P4 on GM3 and Ceramide Levels, Interaction of CD9 with α3, and Cell Motility in KK47 Cells”), followed by Western blot analysis of c-Src, Tyr-416, Tyr-527, and Csk. The intensity of γ-tubulin in each fraction was used as loading control. YTS1 cells were incubated with 50 μm GM3 in serum-free medium for various durations (1, 3, and 16 h) (see “Effect of Exogenous GM3 on Interaction of CD9 with α3 and Cell Motility in YTS1 Cells”), followed by sucrose density gradient ultracentrifugation to separate low density and high density fractions, and Western blot analysis of each fraction for determination of c-Src phosphorylation status and Csk translocation into low density fractions (GEM). YTS1 cells were seeded on a 15-cm dish and grown in RPMI with 10% fetal bovine serum. PP2, an Src tyrosine kinase inhibitor, was added to the medium at 0, 5, 10, and 20 μm and incubated at 37 °C for 24 h. Cells were subjected to phagokinetic gold sol assay as described above. Composition of KK47 Versus YTS1 Microdomain—First, we compared the major components of glycosynapse 3 (integrins α3, α5, and β1; PLP/TSP CD9 and CD82; gangliosides) in total cell lysate prepared in RIPA buffer from KK47 and YTS1 as shown in Fig. 1, A–C. Levels of the integrins were essentially the same in the two cell lines (Fig. 1A). The CD9 level was similar, and CD82 was absent in both cell lines (Fig. 1B). The ganglioside expressed in YTS1 was mainly GM2, whereas that in KK47 was mainly GM3, as revealed by TLC with orcinol/sulfuric acid staining. GM3 level in KK47 was 4–5 times higher than in YTS1 (Fig. 1C). The amounts of α3 and CD9 in low density insoluble membrane fraction and high density soluble fraction, separated by sucrose density gradient ultracentrifugation from PNF of cell lysate prepared with lysis buffer containing 1% Brij 98, were analyzed by SDS-PAGE followed by Western blot (Fig. 1D, top). α3 was enriched in fractions 4–6, whereas CD9 was enriched in fractions 4 and 5. The degree of enrichment of both components was higher in YTS1 than in KK47. Neither α3 nor CD9 was detectable in fractions 1–3 or fractions 7–9 from YTS1 or KK47 (data not shown). Gangliosides were concentrated in fractions 4–6 from both cell lines (Fig. 1D, bottom). The GM3 concentration in combined low density fractions (fractions 4–6) from KK47 and YTS1 was ∼15 ng/30 μg of protein and <2 ng/30 μg of protein, respectively. GM2 concentration in the same fractions from YTS1 and KK47 was ∼12 ng/30 μg of protein and <2 ng/30 μg of protein, respectively. Neither GM3 nor GM2 was detectable in fractions 1–3 or fractions 7–9 from YTS1 or KK47 (data not shown). Interactions of Components in KK47 Versus YTS1 Microdomain—Interactions were determined by co-IP and by confocal microscopy. Co-IP of α3 with CD9 and of β1 with CD9 was performed by the addition of anti-α3 or anti-β1 antibody to PNF, followed by Western blot with anti-CD9, as described under “Materials and Methods.” Co-IP of α3 with CD9 was much higher in KK47 than in YTS1, whereas co-IP of β1 with CD9 was weak (and similar) in the two cell lines. α5 was not co-immunoprecipitated with CD9 from either cell line (Fig. 2A). Co-localization of α3 with CD9 was significantly higher in KK47 than in YTS1, as indicated by the enhanced merge image in confocal microscopy (Fig. 2B). Difference in Motility of KK47 Versus YTS1 by Phagokinetic Gold Sol Assay—Transwell membrane motility through thick Matrigel was much higher for YTS1 than for KK47 (7Satoh M. Ito A. Nojiri H. Handa K." @default.
• W2043945964 created "2016-06-24" @default.
• W2043945964 creator A5020054555 @default.
• W2043945964 creator A5065926823 @default.
• W2043945964 creator A5070398233 @default.
• W2043945964 creator A5080700568 @default.
• W2043945964 creator A5080725230 @default.
• W2043945964 date "2005-10-01" @default.
• W2043945964 modified "2023-10-17" @default.
• W2043945964 title "A Specific Microdomain (“Glycosynapse 3”) Controls Phenotypic Conversion and Reversion of Bladder Cancer Cells through GM3-mediated Interaction of α3β1 Integrin with CD9" @default.
• W2043945964 cites W109060764 @default.
• W2043945964 cites W1524346733 @default.
• W2043945964 cites W1525426202 @default.
• W2043945964 cites W1553517909 @default.
• W2043945964 cites W1564861448 @default.
• W2043945964 cites W1582740601 @default.
• W2043945964 cites W1602804242 @default.
• W2043945964 cites W1656339735 @default.
• W2043945964 cites W187359374 @default.
• W2043945964 cites W1923199073 @default.
• W2043945964 cites W1965046777 @default.
• W2043945964 cites W1967644185 @default.
• W2043945964 cites W1969202693 @default.
• W2043945964 cites W1971344702 @default.
• W2043945964 cites W1972049323 @default.
• W2043945964 cites W1988541237 @default.
• W2043945964 cites W1988683816 @default.
• W2043945964 cites W1991040915 @default.
• W2043945964 cites W1991593966 @default.
• W2043945964 cites W1997261294 @default.
• W2043945964 cites W2003096173 @default.
• W2043945964 cites W2005343690 @default.
• W2043945964 cites W2014694687 @default.
• W2043945964 cites W2017767253 @default.
• W2043945964 cites W2019193799 @default.
• W2043945964 cites W2028601165 @default.
• W2043945964 cites W2028641623 @default.
• W2043945964 cites W2029231027 @default.
• W2043945964 cites W2031174583 @default.
• W2043945964 cites W2036456072 @default.
• W2043945964 cites W2038783205 @default.
• W2043945964 cites W2041985227 @default.
• W2043945964 cites W2043762044 @default.
• W2043945964 cites W2045230676 @default.
• W2043945964 cites W2052963506 @default.
• W2043945964 cites W2054092526 @default.
• W2043945964 cites W2059510815 @default.
• W2043945964 cites W2062136146 @default.
• W2043945964 cites W2065766712 @default.
• W2043945964 cites W2075965099 @default.
• W2043945964 cites W2086996769 @default.
• W2043945964 cites W2090895449 @default.
• W2043945964 cites W2094465595 @default.
• W2043945964 cites W2101730120 @default.
• W2043945964 cites W2105225386 @default.
• W2043945964 cites W2105740478 @default.
• W2043945964 cites W2128963767 @default.
• W2043945964 cites W2129396527 @default.
• W2043945964 cites W2129968911 @default.
• W2043945964 cites W2157834399 @default.
• W2043945964 cites W2167933444 @default.
• W2043945964 cites W2186472020 @default.
• W2043945964 cites W2333590563 @default.
• W2043945964 cites W2404769540 @default.
• W2043945964 cites W4241599450 @default.
• W2043945964 cites W4250969231 @default.
• W2043945964 doi "https://doi.org/10.1074/jbc.m505630200" @default.
• W2043945964 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16103120" @default.
• W2043945964 hasPublicationYear "2005" @default.
• W2043945964 type Work @default.
• W2043945964 sameAs 2043945964 @default.
• W2043945964 citedByCount "110" @default.
• W2043945964 countsByYear W20439459642012 @default.
• W2043945964 countsByYear W20439459642013 @default.
• W2043945964 countsByYear W20439459642014 @default.
• W2043945964 countsByYear W20439459642015 @default.
• W2043945964 countsByYear W20439459642016 @default.
• W2043945964 countsByYear W20439459642017 @default.
• W2043945964 countsByYear W20439459642018 @default.
• W2043945964 countsByYear W20439459642019 @default.
• W2043945964 countsByYear W20439459642020 @default.
• W2043945964 countsByYear W20439459642021 @default.
• W2043945964 countsByYear W20439459642022 @default.
• W2043945964 countsByYear W20439459642023 @default.
• W2043945964 crossrefType "journal-article" @default.
• W2043945964 hasAuthorship W2043945964A5020054555 @default.
• W2043945964 hasAuthorship W2043945964A5065926823 @default.
• W2043945964 hasAuthorship W2043945964A5070398233 @default.
• W2043945964 hasAuthorship W2043945964A5080700568 @default.
• W2043945964 hasAuthorship W2043945964A5080725230 @default.
• W2043945964 hasBestOaLocation W20439459641 @default.
• W2043945964 hasConcept C104317684 @default.
• W2043945964 hasConcept C121608353 @default.
• W2043945964 hasConcept C127716648 @default.
• W2043945964 hasConcept C134088382 @default.
• W2043945964 hasConcept C170493617 @default.
• W2043945964 hasConcept C185592680 @default.
• W2043945964 hasConcept C195687474 @default.

• next