The αvβ3 Integrin Regulates α5β1-mediated Cell Migration toward Fibronectin
1997; Elsevier BV; Volume: 272; Issue: 46 Linguagem: Inglês
10.1074/jbc.272.46.29380
ISSN1083-351X
AutoresKeiko O. Simon, Elka M. Nutt, Dicky G. Abraham, Gideon A. Rodan, Le T. Duong,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoThis study examines the interactions of αvβ3 and α5β1 in the regulation of cell migration. Human embryonic kidney (HEK) 293 cells that express α5β1 endogenously were transfected with αvβ3 and β3 mutants, and their attachment and migration to fibronectin (Fn) and vitronectin (Vn) were measured. An αvβ3 blocking antibody and the αvβ3 ligand cyclic G-Pen-GRGDSPC-A inhibited α5β1-mediated migration toward Fn, but not attachment to Fn. This function was αvβ3-specific since αvβ5 transfection and αvβ5 blocking antibody did not produce this effect. Mutations introduced into the β3 integrin subunit to dissect this phenomenon revealed the following. Disruption of the ligand binding domain by the Glanzmann thrombasthenia mutation β3-D119Y constitutively abolished migration toward both Vn and Fn, and attachment to Vn but not to Fn. Insertion of the Glanzmann mutation β3-S752P into the cytoplasmic domain or its truncation (β3-Δ717) abolished binding to Vn but not to Fn. Inhibition of migration toward Fn was inhibited in these cells by αvβ3 blocking antibody. αvβ3-mediated inhibition was, however, abolished by truncation of the transmembrane domain (β3-Δ693). These findings demonstrate αvβ3 regulation of α5β1-mediated cell migration and suggest that the β3 transmembrane domain is essential for this function. This study examines the interactions of αvβ3 and α5β1 in the regulation of cell migration. Human embryonic kidney (HEK) 293 cells that express α5β1 endogenously were transfected with αvβ3 and β3 mutants, and their attachment and migration to fibronectin (Fn) and vitronectin (Vn) were measured. An αvβ3 blocking antibody and the αvβ3 ligand cyclic G-Pen-GRGDSPC-A inhibited α5β1-mediated migration toward Fn, but not attachment to Fn. This function was αvβ3-specific since αvβ5 transfection and αvβ5 blocking antibody did not produce this effect. Mutations introduced into the β3 integrin subunit to dissect this phenomenon revealed the following. Disruption of the ligand binding domain by the Glanzmann thrombasthenia mutation β3-D119Y constitutively abolished migration toward both Vn and Fn, and attachment to Vn but not to Fn. Insertion of the Glanzmann mutation β3-S752P into the cytoplasmic domain or its truncation (β3-Δ717) abolished binding to Vn but not to Fn. Inhibition of migration toward Fn was inhibited in these cells by αvβ3 blocking antibody. αvβ3-mediated inhibition was, however, abolished by truncation of the transmembrane domain (β3-Δ693). These findings demonstrate αvβ3 regulation of α5β1-mediated cell migration and suggest that the β3 transmembrane domain is essential for this function. Cell migration is essential for many biological processes, including development, wound healing, and hemostasis. In addition, several pathologic processes such as cancer metastases, inflammation, thrombosis, and restenosis are dependent on cell migration. To generate the necessary traction forces required for movement, cells depend on adhesive interactions with the substratum, mediated at least in part by integrins. The α5β1 and α4β1 integrins mediate migration toward fibronectin (Fn) 1The abbreviations used are: Fn, fibronectin; Col I, collagen type I; Col IV, collagen type IV; FnR, fibronectin receptor; HUVEC, human umbilical vein endothelial cell; Ln, laminin; Vn, vitronectin; VnR, vitronectin receptor; IAP, integrin-associated protein; mAb, monoclonal antibody; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; uPAR, urokinase-type plasminogen activator receptor; MEM, minimal essential medium. (1Akiyama S.K. Yamada S.S. Chen W.-T. Yamada K.M. J. Cell Biol. 1989; 109: 863-875Crossref PubMed Scopus (511) Google Scholar, 2Wu C. Fields A.J. Kapteijn B.A.E. McDonald J.A. J. Cell Sci. 1995; 108: 821-829PubMed Google Scholar). The vitronectin receptors (VnR) αvβ3 and αvβ5 have been implicated in the migration of a variety of cell types including smooth muscle (3Clyman R.I. Mauray F. Kramer R.H. Exp. Cell Res. 1992; 200: 272-284Crossref PubMed Scopus (170) Google Scholar), keratinocytes (4Kim J.P. Zhang K. Chen J.D. Kramer R.H. Woodley D.T. J. Biol. Chem. 1994; 269: 26926-26932Abstract Full Text PDF PubMed Google Scholar), leukocytes (5Hendley B. Lawson M. Marcantonio E.E. Maxfield F.R. Blood. 1996; 87: 2038-2048Crossref PubMed Google Scholar, 6Lawson M.A. Maxfield F.A. Nature. 1995; 377: 75-79Crossref PubMed Scopus (478) Google Scholar), endothelial cells (7Leavesley D.I. Schwartz M.A. Rosenfeld M. Cheresh D.A. J. Cell. Biol. 1993; 121: 163-170Crossref PubMed Scopus (349) Google Scholar), and neural crest cells (8Delannet M. Martin F. Bossy B. Cheresh D.A. Reichardt L.F. Duband J.-L. Development. 1994; 120: 2687-2702Crossref PubMed Google Scholar), and were shown to play a role in melanoma metastases (9Nip J. Brodt P. Cancer Metastasis Rev. 1995; 14: 241-252Crossref PubMed Scopus (53) Google Scholar, 10Seftor R.E.B. Seftor E.A. Gehlsen K.R. Stetler-Steveson W.G. Brown P.D. Ruoslahti E. Hendrix M.J.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1557-1561Crossref PubMed Scopus (433) Google Scholar) and angiogenesis (11Brooks P.C. Clark R.A.F. Cheresh D.A. Science. 1994; 264: 569-571Crossref PubMed Scopus (2742) Google Scholar). Migration requires the fine control of integrin association with and release from the extracellular matrix. These interactions generate signals that are subsequently transmitted to the cytoskeleton. It has been suggested that integrin activity is itself regulated by interaction with substrate or by inside-out signaling (12Hynes R. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9014) Google Scholar,13Schwartz M.A. Schaller M.D. Ginsberg M.H. Annu. Rev. Cell Dev. Biol. 1995; 11: 549-599Crossref PubMed Scopus (1470) Google Scholar) and that cell migration is regulated, in part, by the cycling of integrins between cytoplasmic compartments and the cell surface (14Bretscher M.S. EMBO J. 1992; 8: 1341-1348Crossref Scopus (173) Google Scholar,15Bretscher M.S. Cell. 1996; 85: 465-467Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Modulation of integrin affinity or avidity has been clearly observed in the platelet integrin αIIbβ3, and the β1 and β2 integrins of lymphocytes and leukocytes (12Hynes R. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9014) Google Scholar). In resting cells, these receptors exist in a low affinity ligand binding state and change to a high affinity binding state upon cellular activation. Platelets are activated by various agonists such as thrombin, collagen, and ADP (16Smythe S.S. Joneckis C. Parise L.V. Blood. 1993; 81: 2827-2843Crossref PubMed Google Scholar) that increase intracellular pH and Ca2+. Cellular activation of β2 integrins varies with cell type. Neutrophils and monocytes are activated by phorbol esters and by inflammatory mediators, such as tumor necrosis factor, platelet-activating factor, fMet-Leu-Phe, and lipids (12Hynes R. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9014) Google Scholar). The T-lymphocyte αLβ2 can be activated by phorbol esters or by cross-linking of the cell surface molecules CD2 or CD3 (17Dustin M.L. Springer T.A. Nature. 1989; 341: 619-624Crossref PubMed Scopus (1288) Google Scholar, 18van Kooyk Y. van de Wiel-van Kamenade P. Weder P. Kuijpers T.W. Figdor C.G. Nature. 1989; 342: 811-813Crossref PubMed Scopus (396) Google Scholar). These are some examples of integrin activation by inside-out signaling in response to extracellular agonists. An additional mechanism for modulating integrin activity is by another integrin heterodimer within the same cell. Ligation of the fibronectin receptor (FnR) by attachment of monocytes to Fn-coated surfaces promoted αMβ2-mediated phagocytosis of complement fragment C3b (19Wright S.D. Licht M.R. Craigmyle L.S. Silverstein S.C. J. Cell Biol. 1984; 99: 336-339Crossref PubMed Scopus (74) Google Scholar). Furthermore, ligation of the FnR expressed on the basal plasma membrane of these cells activated αMβ2 on the apical cell surface (20Wright S.D. Craigmyle L.S. Silverstein S.C. J. Exp. Med. 1983; 158: 1338-1343Crossref PubMed Scopus (162) Google Scholar), suggesting a signal transduction pathway. Ligation of the leukocyte response integrin, a β3 and unique α subunit-containing receptor (21Brown E.J. Goodwin J.L. J. Exp. Med. 1988; 167: 777-793Crossref PubMed Scopus (105) Google Scholar), and subsequent formation of the leukocyte response integrin/integrin-associated protein (IAP) complex was reported to enhance αMβ2 binding activity (22Van Strijp J.A.G. Russell D.G. Tuomanen E. Brown E.J. Wright S.D. J. Immunol. 1993; 151: 3324-3336PubMed Google Scholar, 23Ishibashi Y. Claus S. Relman D.A. J. Exp. Med. 1994; 180: 1225-1233Crossref PubMed Scopus (127) Google Scholar). In addition, binding of α5β1 to its ligand stimulates, in a protein kinase C-dependent manner, α2β1-mediated adherence to collagen type I (Col I), which subsequently results in secretion of interleukin-1 (24Pasqualini R. Bodorova J. Ye S. Hemler M.E. J. Cell Sci. 1993; 105: 101-111PubMed Google Scholar). An inflammatory response is thus induced by cell adhesion to extracellular matrix proteins. Furthermore, it was recently reported that αIIbβ3 regulates α5β1- and α2β1-mediated cell attachment to Fn and Col I by a conformation change induced by receptor occupancy (25Dı́az-González Forsyth F.J. Steiner B. Ginsberg M.H. Mol. Biol. Cell. 1996; 7: 1939-1951Crossref PubMed Scopus (155) Google Scholar). The αvβ3 integrin has previously been shown to negatively regulate α5β1 integrin function. Blystone and colleagues (26Blystone S.D. Graham I.L. Lindberg F.P. Brown E.J. J. Cell Biol. 1994; 127: 1129-1137Crossref PubMed Scopus (221) Google Scholar) have demonstrated that ligation of αvβ3 by antibodies or by Vn-coated surfaces inhibits α5β1-mediated phagocytosis of Fn-coated beads. It was suggested that this phenomenon is mediated by a phosphoserine/phosphothreonine signaling cascade, since it is blocked by the inhibitor H7 (26Blystone S.D. Graham I.L. Lindberg F.P. Brown E.J. J. Cell Biol. 1994; 127: 1129-1137Crossref PubMed Scopus (221) Google Scholar). Furthermore, it was shown that αvβ3 inhibition of α5β1 phagocytosis occurs as a result of αvβ3 interaction with IAP, and that this integrin cross-talk requires the cytoplasmic domain of the β3 integrin subunit (27Blystone S.D. Lindberg F.P. LaFlamme S.E. Brown E.J. J. Cell Biol. 1995; 130: 745-754Crossref PubMed Scopus (95) Google Scholar). The present study examines the role of αvβ3in the regulation of cell migration. We show that antibody ligation of αvβ3, addition of an αvβ3 peptide inhibitor, or a Glanzmann mutation in the ligand binding site of β3 not only inhibit migration toward Vn but also toward Fn. The αvβ3 modulation of α5β1-mediated migration toward Fn appears to be specific and unidirectional. In contrast to the cross-talk between integrins observed in phagocytosis (27Blystone S.D. Lindberg F.P. LaFlamme S.E. Brown E.J. J. Cell Biol. 1995; 130: 745-754Crossref PubMed Scopus (95) Google Scholar), deletion and mutation studies indicate that the transmembrane domain of β3 is important for generating the regulatory signal for α5β1-dependent migration. Human vitronectin (Vn) and mouse laminin (Ln) were purchased from Life Technologies, Inc. Human fibronectin (Fn) was purchased from NY Blood Center (New York, NY). Collagen type IV (Col IV) was purchased from Collaborative Biomedical Products (Bedford, MA). Antibodies against: αvβ3 (mAb LM609), α5 subunit (mAb CLB-705), αvβ5 (mAb P1F6), and α5β1 (mAb JB55). Anti-β1antibody (mAb 13) was purchased from Becton-Dickinson (San Jose, CA). Polyclonal rabbit anti-αv antiserum were purchased from Chemicon (Temecula, CA). Polyclonal rabbit anti-β3antibodies was a generous gift from Dr. Daniel Bollag and Patricia McQueney (Merck Research Laboratories, West Point, PA). These antibodies were raised against human αIIbβ3(28Abraham D.G. Nutt E.M. Bednar R.A. Bednar B. Gould R.J. Duong L.T. Mol. Pharmacol. 1997; 52: 227-236Crossref PubMed Scopus (9) Google Scholar). These antibodies were also recognize by immunoprecipitation the β3 subunit of the αvβ3integrin expressed in HEK 293 cells (Fig. 1 B); we therefore refer to these antibodies as anti-β3 antibodies. Full-length cDNA of the αv integrin subunit (selectable by hygromycin resistance) was generously provided by Dr. A. Schmidt (Merck Research Laboratories). The full-length cDNAs of the β3 and β5 integrin subunits were cloned from a human umbilical cord endothelial cell λgt11 5′ stretch cDNA library (CLONTECH, Palo Alto, CA). The β subunit constructs were cloned into pcDNA3 (InVitrogen, San Diego, CA) selectable by neomycin resistance. Deletion mutants of the β3 subunit, β3(Δ717) and β3(Δ693), were generated by introducing a stop codon after Lys-716 and Asp-692, respectively. These constructs were made by polymerase chain reaction using a common 5′ primer spanning theBamHI site and 3′ primer containing the appropriate mutation and a XhoI restriction site at its 3′ end. Amplified products were digested with the same restriction enzymes. The β3(D119Y) construct was made by polymerase chain reaction using a 5′ primer including the β3 5′-sequence with aHindIII site, and a 3′ primer spanning the KpnI site and a point mutation at the appropriate position. The amplified product was inserted directly into pcDNA3-β3 digested with HindIII-KpnI. The β3(D119A/Δ717) construct was made as a combination of both mutations. The β3(S752P) construct was made as described previously (29O'Toole T.E. Katagiri Y. Faull R.J. Peter K. Tamura R. Quaranta V. Loftus J.C. Shattil S.J. Ginsberg M.H. J. Cell Biol. 1994; 124: 1047-1059Crossref PubMed Scopus (580) Google Scholar). All constructs were characterized by sequence analysis and purified by CsCl centrifugation prior to transfection into cells. Restriction enzymes were purchased from Stratagene (La Jolla, CA) or New England Biolabs (Beverly, MA). Human embryonic kidney 293 cells (ATCC, Rockland, MD) were cultured in minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), 0.1 mg/ml kanamycin, and 2 mml-glutamine (Life Technologies, Inc.), and maintained at 37 °C and 5% CO2. Cells transfected with human αv and β3 integrin subunits were maintained in complete media with added 400 μg/ml G418 (Life Technologies, Inc.) and 50 μg/ml hygromycin (Calbiochem, San Diego, CA). Human umbilical vein endothelial cells (HUVECs; Cell Systems, Kirkland, WA) were maintained in MCDB (Sigma) supplemented with: 15% FBS (heat-inactivated), 0.2 mm KCl, 3 mmKH2PO4, 0.3 mm glycine, 90 μg/ml heparin (Sigma), 25 μg/ml endothelial mitogen (Sigma), 50 μg/ml kanamycin (Life Technologies, Inc.) on tissue culture plates coated with 50 μg/ml Col I (Celtrix Pharmaceuticals, Santa Clara, CA). Cells were used before passage 8. The constructs described above were transfected into HEK 293 cells by electroporation at 200 V, 960 microfarads using a GenePulser (Bio-Rad). Briefly, cells at 50% confluence were collected using trypsin-EDTA. After two washes in serum-free media, the cells (1 × 106 cells/ml) were incubated with 5 μg of plasmid DNA on ice for 30 min prior to electroporation. Cells were subjected to differential selection after 48 h in complete media containing 800 μg/ml G418 (Life Technologies, Inc.) and 100 μg/ml hygromycin (Calbiochem, San Diego, CA). In this study, all cell lines represent pools of at least six single clones. Surface expression of transfected integrins was characterized using flow cytometry analysis and immunoprecipitation, followed by Western blots. For flow cytometry analysis, cells were lifted by trypsin-EDTA and washed once with five volumes of MEM containing 10% FBS and twice in Dulbecco's phosphate-buffered saline. HEK 293 cells expressing αvβ3 and β3 mutants (2 × 105 cells/ml) were incubated with polyclonal anti-β3 antibodies (15 μg/ml), for 30 min at room temperature, followed by washing and incubation with FITC-conjugated donkey-anti-rabbit IgG antibodies (Jackson Laboratories, West Grove, PA) for 30 min at room temperature. Cells were then washed and resuspended in 250 μl of Flow buffer (100 mm HEPES buffer, pH 7.5, 150 mm NaCl, 3 mm KCl, and 1 mm CaCl2) and analyzed by flow cytometry using a FACScalibur instrument (Becton-Dickinson). Similarly, HEK 293 cells expressing αvβ5 were incubated with mAb P1F6 (20 μg/ml), followed by incubation with FITC-conjugated goat anti-mouse IgG antibodies (Jackson). Endogenous expression of α5β1 in all cell lines were detected using mAb JB55 (20 μg/ml) and followed by FITC-conjugated anti-mouse IgG antibodies as described above. Additionally, transfectants (1 × 106 cells) were surface-labeled with 2 mm Immunopure Sulfo-NHS-LC-Biotin (Pierce) and then solubilized in RIPA buffer (50 mm Tris, pH 7.5, 150 mm NaCl, 1 mm CaCl2, 1% Nonidet P-40, 0.5% deoxycholate) containing 1 mmphenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, and 100 μg/ml leupeptin. Cell extracts were immunoprecipitated using the polyclonal anti-β3 antibodies, followed by protein G-Sepharose (30Lantz L.M. Holmes K.L. Biotechniques. 1994; 18: 58-60Google Scholar). Precipitated proteins were separated on 8% SDS-polyacrylamide gel (Novex, San Diego, CA), followed by Western blotting and developed with horseradish peroxidase-conjugated streptavidin (Amersham). Similarly, expression of αvβ1 in these cells was detected by immunoprecipitation with mAb 13 (anti-β1) and subsequent blotting with anti-αv cytoplasmic domain polyclonal antibodies, then detected using horseradish peroxidase-conjugated anti-rabbit IgG antibodies (Amersham), followed by enhanced chemiluminescence (ECL) system (NEN Life Science Products). Cell migration was assayed using a Boyden chamber type apparatus (Neuroprobe, Cabin John, MD). Prior to assay, cells were loaded with a fluorescent marker, 5-chloromethyl fluorescein diacetate (Molecular Probes, Eugene, OR). Blocking antibodies or peptides were added to cells just prior to assay. Extracellular matrix proteins were diluted into serum-free media and placed in the bottom chamber. Labeled cells were washed with serum-free media and added to the upper chamber at a density of 20,000 cells/well. Normally, 3,000–10,000 cells (∼15–50% of total cells added) migrate in these assays. Cells were allowed to migrate through a polycarbonate filter (pore size 8 μm) for 15 h in a humidified incubator at 37 °C. The cells migrating to the bottom of the filter were detected using the Cytofluor fluorescence plate reader (Millipore, Bedford, MA). No migrated cells were detected when ligand was added to the upper well of the migration chamber. The number of migrated cells was calculated based upon standard curves for each cell line used in the experiment. Results are expressed as a mean value of triplicate or quadruplicate samples. Cells were lifted with trypsin-EDTA and washed four times with serum-free MEM. Cells (10,000 cells/well) were added to microtiter wells coated with Vn or Fn and allowed to attach at 37 °C in a humidified incubator for 30 min or 2 h. Non-attached cells were gently washed away, and attached cells were quantified by colorimetric detection of hexosaminidase enzymatic activity (31Landegren U. J. Immunol. Methods. 1984; 67: 379-388Crossref PubMed Scopus (601) Google Scholar) in a Vmax plate reader (Molecular Devices, Menlo Park, CA). The number of attached cells was quantitated using a standard curve for each cell line assayed and expressed as a mean value of triplicate samples. Stable transfectants of HEK 293 cells expressing αvβ3 (β3-WT) and αvβ5 (β5-WT) and β3 mutants were used in this study. The following mutants of β3 were constructed and co-expressed with wild type αv: truncations of the β3 subunit cytoplasmic domain (β3-Δ717) and of the transmembrane domain (β3-Δ693), insertion of the Glanzmann thrombasthenia mutations in the ligand binding site (β3-D119Y) or in the cytoplasmic domain (β3-S752P), and the combined β3-D119Y mutation with truncation of the cytoplasmic domain (β3-D119Y/Δ717). Surface expression of αvβ3 integrin and its mutants was determined by flow cytometry and immunoprecipitation followed by Western blots. In Fig.1 A, the surface expression of αvβ3 and its mutants was analyzed by flow cytometry using polyclonal anti-β3 antibodies. The level of integrin expression is compared with that in parental HEK 293 cells, which lack endogenous αvβ3 expression. Surface expression of the αvβ3 mutants is comparable to that present in cells expressing the wild-type αvβ3 integrin (β3-WT), with the exception of the β3-Δ693 cells, which express approximately 10-fold lower levels of mutant integrin. Therefore, we chose for comparison another HEK 293 cell line (β3-L) that expresses wild type αvβ3 at levels comparable to those in β3-Δ693 cells. The β3-S752P cells appear to be a mixed population as indicated by the broad histogram indicating varied levels of receptor expression (Fig. 1 A). In addition, heterodimer formation and surface expression of αvβ3 and all β3 mutants were also confirmed by surface biotinylation followed by immunoprecipitation with the anti-β3 antibodies (Fig. 1 B). Both αv (130 kDa) and β3 (110 kDa) subunits were immunoprecipitated from β3-WT and the β3mutants (β3-S752P and β3-D119Y). Deletion of the cytoplasmic domain (β3-Δ717, β3-D119Y/Δ717) and transmembrane domain (β3-Δ693) of the β3 subunit leads to a shift in the mobility of the β3 subunit bands (97 kDa) on the gels. Therefore, the β subunit mutations do not appear to disrupt normal subunit association or cell surface expression. The αvβ5 expression in transfected cells was also relatively high, and the α5β1 levels were similar to those in parental HEK 293 cells as shown in Fig.1 A. Therefore, the level of expression of the endogenous α5β1 integrin was not affected by overexpression of exogenous VnRs. HEK 293 cells express αvβ1 integrins, which function as Vn and Fn receptors in these cells (32Bodary S.C. McLean J.W. J. Biol. Chem. 1990; 265: 5938-5941Abstract Full Text PDF PubMed Google Scholar). We examined the relative levels of αvβ1 in β3-WT and β3 mutants, using immunoprecipitation from cell lysates with anti-β1antibody (mAb 13), followed by immunoblotting with anti αv-cytoplasmic domain antibodies. A small reduction in αvβ1 was observed in β3-WT cells in the experiment presented in Fig. 1 C; however, we detected no significant difference in αvβ1between cells expressing αvβ3 or its mutants in repeated experiments. HEK 293 cells expressing αvβ3 attached (Fig.3 A), and spread on Vn (Fig.2 A). Parental cells attach loosely but fail to spread on Vn. In contrast, the β3-WT and β5-WT cells exhibit a well spread morphology. Cells with the Glanzmann mutations (β3-S752P and β3-D119Y) or the transmembrane truncation (β3-Δ693) show diminished spreading on and attachment to Vn by comparison to β3-WT cells (Figs. 2 Aand 3 A). Cells with the cytoplasmic domain truncation (β3-Δ717) plated on Vn have at the periphery projections of thin ruffled lamellipodia (Fig.2 A), which appear lucid and free of organelles. Cells overexpressing αvβ3 or its mutants spread (Fig. 2 B) and attach (Fig. 3 B) to Fn similarly to parental cells. Interestingly, the β5-WT cells are very well spread on Fn and contain many vacuoles (Fig. 2 B). Cells transfected with αvβ3 acquire, as expected, the ability to migrate toward Vn (Fig.4). Migration toward Fn via the endogenous α5β1 integrin is not altered by the presence of αvβ3 integrin (Fig. 4).Figure 2Morphology of cell lines expressing the indicated integrins: β3-WT, the β3 mutants, and β5-WT seeded on Vn or Fn. Cells were allowed to attach and spread on glass coverslips coated with Vn (5 μg/ml) (A) or Fn (25 μg/ml) (B) overnight in MEM containing 0.5% FBS. Photographs were taken using a 40× objective under phase contrast conditions.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Surface expression of αvβ3 enables migration toward vitronectin. Cells were fluorescently labeled and added to migration chambers containing either Vn (63 ng) or Fn (625 ng) in serum-free medium as described under "Materials and Methods." Cells were allowed to migrate overnight at 37 °C in a humidified incubator in serum-free medium. The number of migrated cells was quantified and expressed as means of quadruplicate samples ± S.E.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Treatment of β3-WT expressing cells with the anti-αvβ3blocking monoclonal antibody LM609 inhibits migration toward Vn by ∼98% (Fig. 5 A). Surprisingly, αvβ3 ligation also inhibits migration toward Fn by ∼81%. This cross-regulation phenomenon appears to be specific to αvβ3. Overexpression of the VnR αvβ5 also enables HEK 293 cells to migrate toward Vn. However, while antibodies against αvβ5 (mAb P1F6) block migration toward Vn by ∼89%, they do not affect migration toward Fn. The parental HEK 293 cells, which do not express αvβ3 or αvβ5, were used as controls. As expected, they do not migrate toward Vn and the addition of LM609 (Fig.5 A) or P1F6 (data not shown) does not alter migration toward Fn. To determine if αvβ3 cross-regulation of α5β1 integrin function is a consequence of the overexpression of the exogenous VnR, a cell line expressing lower levels of αvβ3 was also examined (β3-L) (Figs. 1 A and 5 B). Treatment of these cells with mAb LM609 resulted in similar inhibition of cell migration toward both Vn and Fn. As shown in Fig. 5 B, the level of inhibition of migration toward Fn is similar to that in highly expressing β3-WT. Therefore, in this range, the level of αvβ3 expression does not seem to alter the cross-regulation of α5β1 activity. The integrin cross-regulation produced by ligation of αvβ3 appears to be unidirectional, with signals from αvβ3 modulating α5β1 migratory function. Ligation of α5β1 with the anti-α5blocking antibody (mAb CLB-705) inhibits migration toward Fn, but does not affect migration toward Vn in αvβ3expressing cells (Fig. 5 C). In addition, the αvβ3-mediated cross-regulation appears to be specific for Fn, since αvβ3 ligation did not affect cell migration on Ln or Col IV (data not shown). It has been reported that αvβ3 can mediate attachment to Fn (32Bodary S.C. McLean J.W. J. Biol. Chem. 1990; 265: 5938-5941Abstract Full Text PDF PubMed Google Scholar). Therefore, ligation of αvβ3 with mAb LM609 may directly inhibit its interaction with and, subsequently, migration toward Fn. To address this possibility, blocking antibodies to either the α5subunit (mAb CLB-705) or the β1 subunit (mAb 13) were used. Ligation of α5β1 with these antibodies in β3-WT cells resulted in 85–88% inhibition of migration toward Fn (Fig. 5 D), indicating that in the cell system used here, cell migration toward Fn requires accessible α5β1 integrins. Attachment of β3-WT cells to Fn was reduced by about 30% by the presence of the anti-αvβ3 blocking antibody LM609 (Fig.6 A), and separately by 60% by anti-β1 integrin antibodies. Combining both antibodies caused additive effects on attachment to Fn. The αvβ3 integrin thus participates in the attachment of β3-WT cells to Fn (33Charo I.F. Nannizzi L. Smith J.W. Cheresh D.A. J. Cell Biol. 1990; 111: 2795-2800Crossref PubMed Scopus (231) Google Scholar). Although the VnR αvβ5 was also shown to act as an FnR (24Pasqualini R. Bodorova J. Ye S. Hemler M.E. J. Cell Sci. 1993; 105: 101-111PubMed Google Scholar,34Cheresh D.A. Smith J.W. Cooper H.M. Quaranta V. Cell. 1989; 57: 59-69Abstract Full Text PDF PubMed Scopus (255) Google Scholar), in β5-WT cells only β1 blocking antibodies, not anti-αvβ5, inhibit attachment to Fn (Fig. 6 A). Attachment of β3-WT to Vn is reduced by 70% by LM609 (Fig. 6 B) and was not affected by β1antibodies. Therefore, αvβ3 functions as the predominant VnR in attachment of β3-WT cells to Vn. Similarly, in β5-WT cells, αvβ5 is the primary receptor for Vn, although anti-β1 antibodies seem to have an additive effect in the presence of anti-β5 antibodies. Antibodies to αvβ5 (mAb P1F6) inhibit attachment to Vn by 72%, whereas anti-β1 antibodies alone had little or no effect (Fig. 6 B). These data suggest that there may be a component of Vn attachment that is mediated by β1integrins and that this activity is enhanced by αvβ5 ligation, or conversely that α5β1 ligation modulates αvβ5 activity. The endogenous integrin αvβ1 in HEK 293 cells may be responsible for additional binding to Vn and Fn, and for the lack of complete inhibition of attachment to Vn by anti-αvβ3or anti-αvβ5 antibodies. The data presented above show that antibodies that ligate αvβ3 and block ligand binding to this receptor cross-regulate α5β1-mediated migration. We therefore examined whether αvβ3 ligands can inhibit α5β1 function. As shown in Fig.7, the preferential peptide inhibitor of αvβ3, the cyclic RGD peptide G-Pen-GRGDSPC-A (35Pierschbacher M.D. Ruoslahti E. J. Biol. Chem. 1987; 262: 17294-17298Abstract Full Text PDF PubMed Google Scholar), is a potent inhibitor of β3-WT cell migration toward Vn (IC50 ∼ 1 nm). In addition, it also strongly inhibits migration toward Fn (IC50 ∼ 2.5–5 nm). The cyclic peptide also weakly inhibits parental HEK 293 cell migration toward Fn (∼25% at 10 nm); however, this inhibition was much lower than for β3-WT cells migrating toward either Vn (∼92%) or Fn (∼80%). The effect on parental HEK 293 cells may be due to cross-reactivity of the peptide with endogenous integrins such as αvβ1. The effects of the αvβ3 ligand further support a role for αvβ3 in cross-regulation of migration toward Fn, and the low concentration (<5 nm) suggests that partial
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