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• W1986330147 abstract "Few molecules have been shown to confer cell motility. Although the motility-arresting properties of anti-CD9 monoclonal antibody (mAb) suggest the transmembrane 4 superfamily (TM4SF) member CD9 can induce a motorgenic signal, gene transfection studies have failed to confirm this hypothesis. We report here that ectopic expression of human CD9 (CD9h) and feline CD9 (CD9f) in the CD9-negative, poorly motile, human B cell line Raji dramatically enhances migration across fibronectin- and laminin-coated polycarbonate filters. Migration of Raji/CD9h and Raji/CD9f on either substrate was inhibited by the anti-CD9 mAb 50H.19 and by the anti-β1 integrin mAb AP-138. Migration of Raji/CD9h on laminin was potently inhibited by the anti-VLA-6 integrin mAb GoH3 and by the anti-VLA-4 integrin mAb 44H6, whereas migration of Raji/CD9h on fibronectin was inhibited only by mAb 44H6. Since CD9h-transfected Raji cells adhered to fibronectin as effectively as mock transfectants, expression of CD9 enhanced motility, but not adhesion. CD9-enhanced migration was inhibited by the protein tyrosine kinase inhibitor herbimycin A suggesting that tyrosine phosphorylation played a role in the generation of a motorgenic signal. Raji/CD9h transfectants adherent to fibronectin expressed 6-fold higher levels of phosphotyrosine than Raji. Raji/CD9f transfectants also phosphorylated proteins on tyrosine more effectively than Raji including a protein of 110 kDa which was phosphorylated on the motility-inducing substrates laminin and fibronectin, but not on bovine serum albumin. Our results support a role for CD9 in the amplification of a motorgenic signal in B cells involving β1 integrins and the activation of protein tyrosine kinases. Few molecules have been shown to confer cell motility. Although the motility-arresting properties of anti-CD9 monoclonal antibody (mAb) suggest the transmembrane 4 superfamily (TM4SF) member CD9 can induce a motorgenic signal, gene transfection studies have failed to confirm this hypothesis. We report here that ectopic expression of human CD9 (CD9h) and feline CD9 (CD9f) in the CD9-negative, poorly motile, human B cell line Raji dramatically enhances migration across fibronectin- and laminin-coated polycarbonate filters. Migration of Raji/CD9h and Raji/CD9f on either substrate was inhibited by the anti-CD9 mAb 50H.19 and by the anti-β1 integrin mAb AP-138. Migration of Raji/CD9h on laminin was potently inhibited by the anti-VLA-6 integrin mAb GoH3 and by the anti-VLA-4 integrin mAb 44H6, whereas migration of Raji/CD9h on fibronectin was inhibited only by mAb 44H6. Since CD9h-transfected Raji cells adhered to fibronectin as effectively as mock transfectants, expression of CD9 enhanced motility, but not adhesion. CD9-enhanced migration was inhibited by the protein tyrosine kinase inhibitor herbimycin A suggesting that tyrosine phosphorylation played a role in the generation of a motorgenic signal. Raji/CD9h transfectants adherent to fibronectin expressed 6-fold higher levels of phosphotyrosine than Raji. Raji/CD9f transfectants also phosphorylated proteins on tyrosine more effectively than Raji including a protein of 110 kDa which was phosphorylated on the motility-inducing substrates laminin and fibronectin, but not on bovine serum albumin. Our results support a role for CD9 in the amplification of a motorgenic signal in B cells involving β1 integrins and the activation of protein tyrosine kinases. INTRODUCTIONCD9, a 22-24-kDa cell surface glycoprotein, highly expressed in developing B cells, blood platelets, neuroblastoma cell lines, normal and transformed epithelia, peripheral glia, and neurones (1Kersey J. Le Bien T. Abramson C. Newman R. Sutherland R. Greaves M. J. Exp. Med. 1982; 153: 726-731Crossref Scopus (236) Google Scholar, 2Boucheix C. Soria C. Mirshahi M. Soria J. Perrot J.Y. Fournier N. Billard M. Rosenfeld C. FEBS Lett. 1983; 161: 289-295Crossref PubMed Scopus (92) Google Scholar, 3Kemshead J.T. Fritschy J. Asser U. Sutherland R. Greaves M.F. Hybridoma. 1982; 1: 109-123Crossref PubMed Scopus (49) Google Scholar, 4Kaprielian Z. Cho K.-O. Hajiargyrou M. Patterson P.H. J. Neurosci. 1995; 15: 562-573Crossref PubMed Google Scholar) is a member of the transmembrane 4 protein superfamily (TM4SF). 1The abbreviations used are: TM4SFtransmembrane 4 superfamilyVLAvery late antigen or β1 integrinsRHAMMreceptor for hyaluronic acid-mediated motilitymAbmonoclonal antibodyPAGEpolyacrylamide gel electrophoresisBSAbovine serum albuminFACSfluorescence-activated cell sorter. TM4SF proteins possess two external loops and four hydrophobic domains of membrane-spanning length (5Boucheix C. Benoit P. Frachet P. Billard M. Worthington R.E. Gagnon J. Uzan G. J. Biol. Chem. 1991; 266: 117-122Abstract Full Text PDF PubMed Google Scholar, 6Horejsi V. Vlcek C. FEBS Lett. 1991; 288: 1-4Crossref PubMed Scopus (149) Google Scholar, 7Wright M.D. Tomlinson M.G. Immunol. Today. 1994; 15: 588-594Abstract Full Text PDF PubMed Scopus (332) Google Scholar, 8Lanza F. Wolf D. Fox C.F. Kieffer N. Seyer J.M. Fried V.A. Coughlin S.R. Phillips D.R. Jennings L.K. J. Biol. Chem. 1991; 266: 10638-10645Abstract Full Text PDF PubMed Google Scholar). The putative transmembrane regions and certain residues within the external loops are highly conserved suggesting the proteins perform closely related functions. However, the nature of those functions is not well understood(6Horejsi V. Vlcek C. FEBS Lett. 1991; 288: 1-4Crossref PubMed Scopus (149) Google Scholar). There is evidence that TM4SF proteins play a role in the initiation of signals controlling cell proliferation. For example, CD53 is found exclusively on subsets of proliferating thymocytes(9Paterson D.J. Green J.R. Jefferies W.A. Puklavec M. Williams A.F. J. Exp. Med. 1987; 165: 1-13Crossref PubMed Scopus (57) Google Scholar), CD81 exerts both positive and negative effects on the proliferation of T and B lymphoid cell lines(10Tedder T.F. Zhou L.-J. Engel P. Immunol. Today. 1994; 15: 437-442Abstract Full Text PDF PubMed Scopus (239) Google Scholar), and an anti-CD9 mAb was recently shown to induce the proliferation of Schwann cells(11Hajiargyrou M. Patterson P.H. J. Neurosci. 1995; 15: 574-583Crossref PubMed Google Scholar). Some family members may also regulate adhesive and morphogenetic functions. CD9 is expressed at high density on peripheral blood platelets(12Jennings L.K. Fox C.F. Kouns W.C. McKay C.P. Ballou L.R. Schultz H.E. J. Biol. Chem. 1990; 265: 3815-3822Abstract Full Text PDF PubMed Google Scholar), and anti-CD9 mAb are exceptional platelet agonists (2Boucheix C. Soria C. Mirshahi M. Soria J. Perrot J.Y. Fournier N. Billard M. Rosenfeld C. FEBS Lett. 1983; 161: 289-295Crossref PubMed Scopus (92) Google Scholar, 13Gorman D.J. Castaldi P.A. Zola H. Berndt M.C. Nouv. Rev. Fr. Hematol. 1985; 27: 255-259PubMed Google Scholar) when Fc-receptor interactions are not impeded(14Worthington R.E. Carroll R.C. Boucheix C. Br. J. Haematol. 1990; 74: 216-222Crossref PubMed Scopus (110) Google Scholar). Immobilized, but not soluble, F(ab′)2 fragments of anti-CD9 mAb activate platelets(15Griffith L. Slupsky J. Seehafer J. Boshkov L. Shaw A.R.E. Blood. 1991; 78: 1753-1759Crossref PubMed Google Scholar), and immobilized, but not soluble, antibody induces proliferation in nerve cells(11Hajiargyrou M. Patterson P.H. J. Neurosci. 1995; 15: 574-583Crossref PubMed Google Scholar). These findings suggest that CD9 may transduce signals involving immobilized ligands such as extracellular matrix proteins. Integrins are heterodimeric adhesion molecules linking extracellular matrix proteins to an active cytoskeleton(16Hynes R. Cell. 1987; 48: 549-554Abstract Full Text PDF PubMed Scopus (3082) Google Scholar). We have reported that anti-CD9 mAb promote physical association between CD9 and the β3 integrin GPIIb-IIIa(17Slupsky J.R. Seehafer J.G. Tang S.C. Masellis-Smith A. Shaw A.R.E. J. Biol. Chem. 1989; 264: 12289-12293Abstract Full Text PDF PubMed Google Scholar), and that anti-CD9 mAb induce homotypic and heterotypic adhesive interactions in pre-B cells through pathways which may involve β1 integrins(18Masellis-Smith A. Jensen G.S. Seehafer J.G. Slupsky J.R. Shaw A.R.E. J. Immunol. 1990; 144: 1607-1613PubMed Google Scholar, 19Masellis-Smith A. Shaw A.R.E. J. Immunol. 1994; 152: 2768-2777PubMed Google Scholar). Very recently, these observations have been extended by a report that CD9 physically associates with β1 integrins(20Rubinstein E. Le Naour F. Billard M. Prenant M. Boucheix C. Eur. J. Immunol. 1994; 24: 3005-3013Crossref PubMed Scopus (142) Google Scholar). CD9 may therefore interact with integrins to participate in the transduction of integrin-dependent signals across the plasma membrane (17Slupsky J.R. Seehafer J.G. Tang S.C. Masellis-Smith A. Shaw A.R.E. J. Biol. Chem. 1989; 264: 12289-12293Abstract Full Text PDF PubMed Google Scholar, 20Rubinstein E. Le Naour F. Billard M. Prenant M. Boucheix C. Eur. J. Immunol. 1994; 24: 3005-3013Crossref PubMed Scopus (142) Google Scholar).Antibodies which uniquely prevent cell motility in a variety of tumor cell lines were found to recognize a protein subsequently identified as CD9 suggesting that CD9 is a positive regulator of cell motility(14Worthington R.E. Carroll R.C. Boucheix C. Br. J. Haematol. 1990; 74: 216-222Crossref PubMed Scopus (110) Google Scholar). However, ectopic expression of CD9 in heterologous tumor cell lines either failed to confer motility (20Rubinstein E. Le Naour F. Billard M. Prenant M. Boucheix C. Eur. J. Immunol. 1994; 24: 3005-3013Crossref PubMed Scopus (142) Google Scholar, 21Ikeyama S. Koyama M. Yamaoka M. Sasada R. Miyake M. J. Exp. Med. 1993; 177: 1231-1237Crossref PubMed Scopus (274) Google Scholar) or in some cases suppressed migratory and metastatic activity(21Ikeyama S. Koyama M. Yamaoka M. Sasada R. Miyake M. J. Exp. Med. 1993; 177: 1231-1237Crossref PubMed Scopus (274) Google Scholar). We chose to study CD9 function in cells of the B lymphocyte lineage in which CD9 expression is modulated in a stage-specific manner. Pre-B lymphocytes express high levels of CD9, and we have observed that transformed pre-B cell lines readily penetrate fibronectin-coated polycarbonate filters. B cell lines on the other hand lack CD9 and are poorly migratory in Transwell assays(21Ikeyama S. Koyama M. Yamaoka M. Sasada R. Miyake M. J. Exp. Med. 1993; 177: 1231-1237Crossref PubMed Scopus (274) Google Scholar). To investigate the possibility that CD9 plays a role in integrin-dependent motility, we transfected cDNA encoding human and feline CD9 into the immature B cell Raji which lacks CD9. Raji expresses the β1 integrin VLA-4, a fibronectin receptor found in highly motile cells (22Albelda S.M. Mette S.A. Elder D. Stewart R.M. Damjanovich R.M. Herlyn M. Buck C.A. Cancer Res. 1990; 50: 6757-6764PubMed Google Scholar, 23Bao L. Pigott R. Matsumura Y. Baban D. Tarin D. Differentiation. 1993; 52: 239-246Crossref PubMed Scopus (35) Google Scholar, 24Willett B.J. Neil J.C. Mol. Immunol. 1995; 32: 417-423Crossref PubMed Scopus (13) Google Scholar, 25MacLean G.D. Seehafer J.G. Shaw A.R.E. Kieran M.W. Longenecker B.M. J. Natl. Cancer Inst. 1982; 69: 357-364PubMed Google Scholar, 26Mould A.P. Askari J.A. Craig S.E. Clements J. Garratt A.N. Humphries M.J. J. Biol. Chem. 1994; 269: 27224-27230Abstract Full Text PDF PubMed Google Scholar) implicated in invasiveness, and metastasis(22Albelda S.M. Mette S.A. Elder D. Stewart R.M. Damjanovich R.M. Herlyn M. Buck C.A. Cancer Res. 1990; 50: 6757-6764PubMed Google Scholar, 23Bao L. Pigott R. Matsumura Y. Baban D. Tarin D. Differentiation. 1993; 52: 239-246Crossref PubMed Scopus (35) Google Scholar), as well as the laminin receptor VLA-6. In our experiments, Raji cells penetrated laminin or fibronectin-coated filters poorly, suggesting they might lack an accessory molecule required for migration on integrin-dependent substrates. We report here that ectopic expression of CD9 in Raji cells exposed to laminin and fibronectin dramatically enhanced both their ability to penetrate polycarbonate filters and their ability to phosphorylate protein targets on tyrosine.EXPERIMENTAL PROCEDURESMolecular Cloning of CD9A full-length cDNA encoding CD9 was selected using the anti-CD9 mAb 50H.19 from an endothelial cDNA library assembled in the expression vector lgt11 (Clontech) and the 1.2-kilobase insert subcloned into PTZ-19, transformed into XL1-Blue, and single-stranded template DNA isolated for sequencing. The clone contains the entire coding region for CD9 flanked by 51-base pair 5′- and 450 base pair 3′-untranslated regions and is identical with the published coding sequence(8Lanza F. Wolf D. Fox C.F. Kieffer N. Seyer J.M. Fried V.A. Coughlin S.R. Phillips D.R. Jennings L.K. J. Biol. Chem. 1991; 266: 10638-10645Abstract Full Text PDF PubMed Google Scholar). The insert was cloned into the eukaryotic Epstein Barr virus episomal plasmid expression vector pREP4 (Invitrogen), transfected into Raji by electroporation, and selected by hygromycin resistance, followed by immunoselection on immobilized mAb 50H.19. Mock-transfected controls containing the vector alone were selected on the basis of hygromycin-resistance. A full length cDNA encoding feline CD9 was obtained using the rapid amplification cDNA ends technique as described(24Willett B.J. Neil J.C. Mol. Immunol. 1995; 32: 417-423Crossref PubMed Scopus (13) Google Scholar), cloned into the cDNA3 expression vector, and transfected into the same strain of Raji by electroporation, followed by immunoselection with anti-CD9 mAb.Cell Lines, mAb, and FACS AnalysisHOON and Raji are pre-B cell and B cell lines, respectively. They were obtained from Dr. Michelle Letarte (University of Toronto) and Dr. B. M. Longenecker (University of Alberta) and maintained in RPMI 1640, 10% fetal calf serum. For FACS analysis cells were stained with mAb TS2/7 against VLA-1 (T Cell Diagnostics, Cambridge, MA), mAb P1E6 against VLA-2, (Life Technologies, Inc.), mAb P1B5 against VLA-3 (Life Technologies, Inc.), mAb 44H6 against VLA-4 (kindly provided by Dr. Michelle Letarte), mAb P1D6 against VLA-5 (Life Technologies, Inc.), and mAb GoH3 against VLA-6 (AMAC, BioCan, Mississauga, Ontario) using a FACScan cell sorter (Becton Dickinson) as described previously(18Masellis-Smith A. Jensen G.S. Seehafer J.G. Slupsky J.R. Shaw A.R.E. J. Immunol. 1990; 144: 1607-1613PubMed Google Scholar). mAb 50H.19 is an anti-CD9 mAb first raised against a melanoma cell line(25MacLean G.D. Seehafer J.G. Shaw A.R.E. Kieran M.W. Longenecker B.M. J. Natl. Cancer Inst. 1982; 69: 357-364PubMed Google Scholar). mAb 4B4 (Coulter Corp., Miami, FL) and mAb AP-138 recognize the β1 integrin subunit. Isotype-matched controls were included, and >10,000 events accumulated per sample. Data were analyzed using the Lysis II program.Immunoprecipitation and Immunoblotting106 HOON cells were lysed in 0.5 ml of RIPA buffer, precleared with Protein A-Sepharose, and immunoprecipitated by addition of 5 μg of the anti-CD9 mAb 50H.19 together with 30 μl of preswollen Protein A-Sepharose or Protein A-Sepharose alone, and eluted with SDS-PAGE loading buffer. Following separation on a 5-20% gradient gel, the proteins were transferred to nitrocellulose and immunoblotted with horseradish peroxidase-conjugated mAb 50H.19. Positive bands were detected by enhanced chemiluminescence (Amersham International, Buckinghamshire, United Kingdom).Adhesion AssayCells were grown in RPMI 1640 supplemented with 10% fetal calf serum and washed in RPMI containing 0.5% BSA. 2 × 106 cells in a volume of 250 μl of RPMI 1640, 0.5% BSA were added to a 24-well plate in which wells were precoated for 2 h with varying concentrations of human plasma fibronectin (Life Technologies, Inc.) in phosphate-buffered saline at 37° C and blocked for 1 h with 1% BSA(26Mould A.P. Askari J.A. Craig S.E. Clements J. Garratt A.N. Humphries M.J. J. Biol. Chem. 1994; 269: 27224-27230Abstract Full Text PDF PubMed Google Scholar). Control wells were incubated with BSA alone. The effect of inhibitors was determined by preincubation with the agent for 20 min at 23° C and addition to the assay without further dilution. Cells were preincubated at 37° C for 90 min before dislodging the loosely adherent cells by agitation on a rotary shaker at 110 rpm for 5 min. Detached cells were quantitated in an automated cell counter (Coulter Electronics, Hialeah, FL).Motility AssayCells were washed and resuspended in RPMI 1640, 0.5% BSA. 1.5 × 105 cells in a volume of 100 μl were applied to the upper chamber of 6.5-mm diameter Transwells (Costar, Sin-Can Inc., Calgary, Alberta), and 600 μl of RPMI 1640, 0.5% BSA were added to the lower chamber. Polycarbonate filters (8-μ diameter pores) were precoated with 100 μl of protein solution for 2 h and blocked with 1% BSA(26Mould A.P. Askari J.A. Craig S.E. Clements J. Garratt A.N. Humphries M.J. J. Biol. Chem. 1994; 269: 27224-27230Abstract Full Text PDF PubMed Google Scholar). Cells were incubated for 18 h at 37° C, and cells migrating to the lower chamber were quantitated in an automated cell counter (Coulter Electronics). Examination of the lower surface of the filter confirmed that transmigrating cells did not adhere to the filter, but accumulated in the lower chamber.Analysis of Phosphotyrosine250-μl aliquots of Raji or Raji/CD9 transfectants at concentrations of 5 × 105 to 8 × 106 cells/ml were introduced into the wells of a 24-well tissue culture plate (Costar) precoated with fibronectin and blocked with BSA. Cells were incubated for 90 min at 37° C and then lysed in RIPA buffer (pH 8.0) containing 0.02 M Tris-HCl, 0.001 M Na2HPO4, 137 mM NaCl, 0.5% Nonidet P-40, 0.25% sodium deoxycholate; 1 μg/ml leupeptin, pepstatin A, antipain, and Trasylol; 1 mM EGTA; 1 mM iodoacetamide; 1 mM sodium orthovanadate. Proteins were separated by 5-20% SDS-PAGE, transferred to nitrocellulose, immunoblotted with the horseradish peroxidase-conjugated anti-phosphotyrosine mAb PY20 (Transduction Laboratories, Lexington, KY), and developed using enhanced chemiluminescence.RESULTSEctopic Expression of CD9 in RajiCD9 was strongly expressed in lysates of Raji cells transfected with the human CD9 cDNA insert (Raji/CD9h), but not in Raji transfected with pREP4 alone (Fig. 1, lanes E and C). The level of expression was very similar to that of the highly motile pre-B cell line HOON (Fig. 1, lane A). FACS analysis demonstrated that our strain of Raji cells expressed predominantly VLA-4 and VLA-6 among β1 integrins, and that ectopic expression of CD9 did not qualitatively affect their VLA profile (Fig. 2). However, ectopic expression of both human and feline CD9 markedly increased the expression of VLA-6 (Fig. 2) suggesting that CD9 may preferentially affect the transport or assembly of this integrin.Figure 2:FACS analysis of VLA antigen and CD9 expression on Raji, Raji transfected with human CD9 (Raji/CD9h), and Raji transfected with feline CD9 (Raji/CD9f). Cells were stained with mAb against VLA antigens and CD9 as described under “Experimental Procedures” followed by a fluorescein isothiocyanate-conjugated second antibody and analyzed on a FACScan. The fluorescence profiles of stained cells (black) are compared to isotype controls (white).View Large Image Figure ViewerDownload Hi-res image Download (PPT)CD9 Enhances Motility of Raji on FibronectinWhen Raji and Raji/CD9 transfectants were compared for their ability to transmigrate across fibronectin-coated polycarbonate filters, the transfectants exhibited a dramatically enhanced migratory capacity (Fig. 3A). Migration of the transfectants increased in proportion to the coating concentration of fibronectin, reaching a maximum at 3 μg/ml. In contrast, mock-transfected Raji cells barely migrated except at the highest coating concentration of 10 μg/ml. Since Raji has the capacity to migrate on fibronectin, but requires a considerably higher concentration to become motile, it suggests that CD9 expression may amplify a motorgenic signal induced by fibronectin. Tyrosine kinases have recently been implicated in cell motility(27Chen P. Gupta K. Wells A. J. Cell Biol. 1994; 124: 547-555Crossref PubMed Scopus (201) Google Scholar, 28Klemke R.L. Yebra M. Bayna E.M. Cheresh D.A. J. Cell Biol. 1994; 127: 859-866Crossref PubMed Scopus (248) Google Scholar). We therefore asked whether preincubation of the cells with the protein tyrosine kinase inhibitor herbimycin A would affect cell migration. Incubation of cells for 3.5 h with herbimycin A reduced tyrosine phosphorylation by >70% without affecting cell viability. Preincubation of Raji/CD9 transfectants with herbimycin A for this period inhibited migration by almost 80% (Table 1), indicating that tyrosine kinase activity was required for CD9-enhanced cell migration.Figure 3:A, motility of Raji and of Raji/CD9 transfectants on fibronectin-coated filters. Motility was assessed by counting the number of cells penetrating the lower chamber of a Transwell apparatus through a perforated polycarbonate filter (8-μ diameter pore). The abscissa shows the coating concentration of plasma fibronectin. Error bars = 1 S.D. The result is representative of six experiments. B, adhesion of Raji and Raji/CD9 to fibronectin-coated plates. Cells were plated on wells coated with varying amounts of plasma fibronectin, and adhesion-quantitated after 90 min by mechanically agitating the plate and counting the nonadherent cells. The abscissa shows the coating concentration of fibronectin. The result is representative of five experiments. Error bars = 1 S.D.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table 1 Open table in a new tab Since CD9 is reported to associate with β1 integrins(20Rubinstein E. Le Naour F. Billard M. Prenant M. Boucheix C. Eur. J. Immunol. 1994; 24: 3005-3013Crossref PubMed Scopus (142) Google Scholar), we investigated whether β1 integrins were involved in cell migration on fibronectin by preincubating the cells with mAb AP-138, an antibody which recognizes the β1 integrin subunit. mAb AP-138 inhibited cell motility by 59% indicating that β1 integrins play a regulatory role in the migratory behavior (Table 1). In keeping with reports that anti-CD9 mAb block cell movement in a variety of transformed cells(20Rubinstein E. Le Naour F. Billard M. Prenant M. Boucheix C. Eur. J. Immunol. 1994; 24: 3005-3013Crossref PubMed Scopus (142) Google Scholar, 21Ikeyama S. Koyama M. Yamaoka M. Sasada R. Miyake M. J. Exp. Med. 1993; 177: 1231-1237Crossref PubMed Scopus (274) Google Scholar, 29Miyake M. Koyama M. Seno M. Ikeyama S. J. Exp. Med. 1991; 174: 1347-1354Crossref PubMed Scopus (190) Google Scholar), we observed that the anti-CD9 mAb 50H.19 inhibited migration of the transfectants by 70.4% (Table 1). mAb 50H.19 and mAb AP-138 also blocked motility of the highly motile pre-B cell line HOON by 71.2 and 72.4%, respectively. Transfection of CD9 therefore confers motility upon a poorly motile B cell line which is inhibitable by anti-CD9 mAb in the manner of pre-B cells which constitutively express the protein.CD9 Does Not Confer Enhanced Adhesion to FibronectinB cells exposed to surfaces coated with fibronectin adhere weakly in comparison to pre-B cells(30Bernardi P. Patel V.P. Lodish H.F. J. Cell Biol. 1987; 105: 489-498Crossref PubMed Scopus (70) Google Scholar). To investigate whether CD9 influenced the avidity of cellular adhesion of B lymphocytes, we quantitated binding of Raji and Raji/CD9 to surfaces coated with fibronectin. Because B lymphoid cells possess relatively low avidity for most extracellular matrix proteins, we developed a low stringency adhesion assay in which the usual method of detaching loosely adherent cells by washing was replaced by a 10-min period of mechanical agitation. Using this assay, we found that both Raji and Raji/CD9 transfectants adhered to fibronectin in a dose-dependent manner, and that the CD9 transfectants adhered similarly to mock-transfected controls (Fig. 3B). Since ectopic expression of CD9 did not significantly affect adhesion of the B cell line CD9 does not appear to influence motility by increasing the level of cellular avidity for fibronectin or by inducing a generalized state of cellular activation. Preincubation of the cells with herbimycin A did not affect adhesion, indicating that adhesion unlike CD9-enhanced motility does not require tyrosine phosphorylation (results not shown).CD9-transfected Raji Exhibit Enhanced Migration on LamininAlthough Raji possesses VLA-6, a laminin receptor, it is poorly motile on laminin. We asked whether cells transfected with CD9 would show enhanced motility on laminin. Raji/CD9h transfectants demonstrated a dramatic increase in motility over Raji controls on polycarbonate filters coated with laminin which increased with the coating concentration between 1 and 10 μg/ml (Fig. 4A). Raji transfected with feline CD9 also exhibited a laminin-dependent increase in migration over mock-transfected controls (Fig. 4B). The ability of CD9 from two different species to confer large increases in cell motility on laminin substrates strongly suggests that motility enhancement is a fundamental property of CD9. In a second experiment, Raji/CD9f was found to show enhanced migration on both fibronectin and laminin, but not BSA (Fig. 5), confirming that CD9 confers motility on substrates recognized by the two major Raji VLA-antigens.Figure 4:A, migration of Raji and Raji/CD9h through laminin-coated polycarbonate filters. Polycarbonate filters were coated with between 1 and 10 μg/ml of laminin, and cells were applied to the upper chamber of a Transwell apparatus. Cells penetrating to the lower chamber over 18 h were quantitated by electronic cell counting. B, migration of Raji and Raji/CD9f through laminin-coated polycarbonate filters. Polycarbonate filters were coated with between 1 and 10 μg/ml of laminin, and the number of cells penetrating to the lower chamber of a Transwell apparatus was determined over 18 h by electronic cell counting.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5:Migration of Raji/CD9f through polycarbonate filters. Polycarbonate filters were coated with 10 μg/ml plasma fibronectin, or laminin, and the percentage of cells penetrating to the lower chamber of a Transwell apparatus was determined over 18 h by electronic cell counting. Error bars represent 1 S.D. about the mean.View Large Image Figure ViewerDownload Hi-res image Download (PPT)CD9 Enhanced Migration Is Inhibitable by Antibodies against VLA-4 and VLA-6The ability of anti-β1 integrin subunit mAb to inhibit the migration of Raji/CD9h on extracellular matrix implicates VLA in the generation of a motorgenic signal. To investigate which VLA is responsible for the inhibition, cells were preincubated with the anti-VLA-4 mAb 44H6 and with the anti-VLA-6 mAb GoH3. Both antibodies strongly inhibited migration of Raji/CD9h on laminin, and their combination completely prevented cells entering the lower chamber (Fig. 6A), whereas mAb 44H6, but not mAb GoH3, significantly inhibited migration on fibronectin-coated filters (Fig. 6B). Since VLA-6 selectively regulates migration on laminin, but cross-linking VLA-4 affects migration on either laminin or fibronectin, it suggests that VLA-4 plays an unexpected and essential role in B cell motility. Cells transfected with feline CD9 were similarly inhibited by both mAbs on laminin substrates, but the anti-VLA-6 mAb effectively inhibited migration on fibronectin implying a possible species difference (Fig. 6, C and D). The epitope recognized by the anti-VLA-4 mAb may be critical since the anti-VLA4 mAb P4G9 had no effect upon migration (results not shown). mAb against CD9 and the β1 integrin subunit also effectively inhibited migration of both CD9h and CD9f transfectants on either substrate (Fig. 6, A, B, C, and D). These results implicate CD9 and the β1 integrin subunit as major determinants of cell motility on laminin substrates through interactions involving VLA-4 and VLA-6.Figure 6:Effect of antibodies to VLA- antigens and CD9 on the migration of Raji/CD9h and Raji/CD9f through laminin- or fibronectin-coated polycarbonate filters. Cells were preincubated with the anti-CD9 mAb 50H.19, the anti-β1 subunit mAb AP-138, the anti-VLA-4 mAb 44H6, the anti-VLA-6 mAb GoH3, or a combination of mAb 44H6 and GoH3 at 10 μg/ml for 30 min at 37° C before introduction into the upper chamber of a Transwell apparatus. Cells penetrating to the lower chamber were quantitated over 18 h. A, Raji/CD9 on laminin-coated filters. B, Raji/CD9h on fibronectin-coated filters. C, Raji/CD9f on laminin-coated filters. D, Raji/CD9f on fibronectin-coated filters. Laminin and fibronectin were coated at a concentration of 10 μg/ml.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The Migratory Subset Is Enriched for CD9 ExpressionIf CD9 is directly responsible for migratory activity, we reasoned that CD9 might be preferentially expressed in cells migrating to the lower" @default.
• W1986330147 created "2016-06-24" @default.
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• W1986330147 date "1995-10-01" @default.
• W1986330147 modified "2023-09-28" @default.
• W1986330147 title "Ectopic Expression of Human and Feline CD9 in a Human B Cell Line Confers β1 Integrin-dependent Motility on Fibronectin and Laminin Substrates and Enhanced Tyrosine Phosphorylation" @default.
• W1986330147 cites W1302337 @default.
• W1986330147 cites W1493730509 @default.
• W1986330147 cites W1523413432 @default.
• W1986330147 cites W1532372627 @default.
• W1986330147 cites W1555648778 @default.
• W1986330147 cites W1567976120 @default.
• W1986330147 cites W1591179447 @default.
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• W1986330147 cites W2063170571 @default.
• W1986330147 cites W2077687259 @default.
• W1986330147 cites W2097619822 @default.
• W1986330147 cites W2101730120 @default.
• W1986330147 cites W2104373491 @default.
• W1986330147 cites W2113861564 @default.
• W1986330147 cites W2118375252 @default.
• W1986330147 cites W2125220487 @default.
• W1986330147 cites W2135215155 @default.
• W1986330147 cites W2156133105 @default.
• W1986330147 cites W2159800908 @default.
• W1986330147 cites W2161155632 @default.
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