Critical Role of Integrin α5β1 in Urokinase (uPA)/Urokinase Receptor (uPAR, CD87) Signaling
2003; Elsevier BV; Volume: 278; Issue: 32 Linguagem: Inglês
10.1074/jbc.m304694200
ISSN1083-351X
AutoresTakehiko Tarui, Nicholas M. Andronicos, Ralf-Peter Czekay, Andrew P. Mazar, Khalil Bdeir, Graham C. Parry, Alice A. Kuo, David J. Loskutoff, Douglas B. Cines, Yoshikazu Takada,
Tópico(s)Blood Coagulation and Thrombosis Mechanisms
ResumoUrokinase-type plasminogen activator (uPA) induces cell adhesion and chemotactic movement. uPA signaling requires its binding to uPA receptor (uPAR/CD87), but how glycosylphosphatidylinositol-anchored uPAR mediates signaling is unclear. uPAR is a ligand for several integrins (e.g. α5β1) and supports cell-cell interaction by binding to integrins on apposing cells (in trans). We studied whether binding of uPAR to α5β1 in cis is involved in adhesion and migration of Chinese hamster ovary cells in response to immobilized uPA. This process was temperature-sensitive and required mitogen-activated protein kinase activation. Anti-uPAR antibody or depletion of uPAR blocked, whereas overexpression of uPAR enhanced, cell adhesion to uPA. Adhesion to uPA was also blocked by deletion of the growth factor domain (GFD) of uPA and by anti-GFD antibody, whereas neither the isolated uPA kringle nor serine protease domain supported adhesion directly. Interestingly, anti-α5 antibody, RGD peptide, and function-blocking mutations in α5β1 blocked adhesion to uPA. uPA-induced cell migration also required GFD, uPAR, and α5β1, but α5β1 alone did not support uPA-induced adhesion and migration. Thus, binding of uPA causes uPAR to act as a ligand for α5β1 to induce cell adhesion, intracellular signaling, and cell migration. We demonstrated that uPA induced RGD-dependent binding of uPAR to α5β1 in solution. These results suggest that uPA-induced adhesion and migration of Chinese hamster ovary cells occurs as a consequence of (a) uPA binding to uPAR through GFD, (b) the subsequent binding of a uPA·uPAR complex to α5β1 via uPAR, and (c) signal transduction through α5β1. Urokinase-type plasminogen activator (uPA) induces cell adhesion and chemotactic movement. uPA signaling requires its binding to uPA receptor (uPAR/CD87), but how glycosylphosphatidylinositol-anchored uPAR mediates signaling is unclear. uPAR is a ligand for several integrins (e.g. α5β1) and supports cell-cell interaction by binding to integrins on apposing cells (in trans). We studied whether binding of uPAR to α5β1 in cis is involved in adhesion and migration of Chinese hamster ovary cells in response to immobilized uPA. This process was temperature-sensitive and required mitogen-activated protein kinase activation. Anti-uPAR antibody or depletion of uPAR blocked, whereas overexpression of uPAR enhanced, cell adhesion to uPA. Adhesion to uPA was also blocked by deletion of the growth factor domain (GFD) of uPA and by anti-GFD antibody, whereas neither the isolated uPA kringle nor serine protease domain supported adhesion directly. Interestingly, anti-α5 antibody, RGD peptide, and function-blocking mutations in α5β1 blocked adhesion to uPA. uPA-induced cell migration also required GFD, uPAR, and α5β1, but α5β1 alone did not support uPA-induced adhesion and migration. Thus, binding of uPA causes uPAR to act as a ligand for α5β1 to induce cell adhesion, intracellular signaling, and cell migration. We demonstrated that uPA induced RGD-dependent binding of uPAR to α5β1 in solution. These results suggest that uPA-induced adhesion and migration of Chinese hamster ovary cells occurs as a consequence of (a) uPA binding to uPAR through GFD, (b) the subsequent binding of a uPA·uPAR complex to α5β1 via uPAR, and (c) signal transduction through α5β1. Urokinase-type plasminogen activator (uPA) 1The abbreviations used are: uPA, urokinase-type plasminogen activator; uPAR, uPA receptor; scuPA, single-chain uPA; CHO, Chinese hamster ovary; GFD, the growth factor domain; LMW, low molecular weight; mAb, monoclonal antibody: MAPK, mitogen-activated protein kinase; scuPA; ATF, amino-terminal fragment; BSA, bovine serum albumin. is a highly restricted serine protease that converts the zymogen plasminogen to the active plasmin. Plasmin, in turn, mediates pericellular proteolysis of extracellular matrix proteins in the path of cellular invasion (1Preissner K.T. Kanse S.M. May A.E. Curr. Opin. Cell Biol. 2000; 12: 621-628Crossref PubMed Scopus (207) Google Scholar, 2Ossowski L. Aguirre-Ghiso J.A. Curr. Opin. Cell Biol. 2000; 12: 613-620Crossref PubMed Scopus (359) Google Scholar). uPA has also been shown to induce adhesion and chemotactic movement of myeloid cells (3Waltz D.A. Sailor L.Z. Chapman H.A. J. Clin. Invest. 1993; 91: 1541-1552Crossref PubMed Scopus (137) Google Scholar, 4Gyetko M.R. Todd III, R.F. Wilkinson C.C. Sitrin R.G. J. Clin. Invest. 1994; 93: 1380-1387Crossref PubMed Scopus (291) Google Scholar), to induce cell migration in human epithelial cells (5Busso N. Masur S.K. Lazega D. Waxman S. Ossowski L. J. Cell Biol. 1994; 126: 259-270Crossref PubMed Scopus (260) Google Scholar) and bovine endothelial cells (6Odekon L.E. Sato Y. Rifkin D.B. J. Cell. Physiol. 1992; 150: 258-263Crossref PubMed Scopus (171) Google Scholar), and to promote cell growth (7Rabbani S.A. Mazar A.P. Bernier S.M. Haq M. Bolivar I. Henkin J. Goltzman D. J. Biol. Chem. 1992; 267: 14151-14156Abstract Full Text PDF PubMed Google Scholar, 8Aguirre Ghiso J.A. Kovalski K. Ossowski L. J. Cell Biol. 1999; 147: 89-104Crossref PubMed Scopus (467) Google Scholar, 9Fischer K. Lutz V. Wilhelm O. Schmitt M. Graeff H. Heiss P. Nishiguchi T. Harbeck N. Kessler H. Luther T. Magdolen V. Reuning U. FEBS Lett. 1998; 438: 101-105Crossref PubMed Scopus (61) Google Scholar). These signaling functions of uPA do not require its proteolytic activity. uPA is composed of three independently folded domain structures, growth factor domain (GFD) (residue 1–43), kringle domain (residue 50–131), and serine protease domain (residue 159–411). Enzymatic digestion of uPA by plasmin generates an amino-terminal fragment (ATF) that consists of the GFD and kringle domains and the low molecular weight fragment (LMW-uPA), possessing serine protease activity. uPA binds with high affinity through GFD (10Appella E. Blasi F. Ann. N. Y. Acad. Sci. 1987; 511: 192-195Crossref PubMed Scopus (19) Google Scholar) to a cell-surface receptor (uPAR/CD87) that has been identified in many cell types (1Preissner K.T. Kanse S.M. May A.E. Curr. Opin. Cell Biol. 2000; 12: 621-628Crossref PubMed Scopus (207) Google Scholar). uPAR is a glycosylphosphatidylinositol-anchored 35–55-kDa glycoprotein. It is generally accepted that uPA-mediated signaling requires prior binding to uPAR. However, the mechanism by which uPAR mediates signaling events is still to be fully elucidated. A major problem in understanding how uPA signals derives from the fact that uPAR has no transmembrane structure, leading to the proposal that hypothetical transmembrane adapters may be involved in this process (11Resnati M. Guttinger M. Valcamonica S. Sidenius N. Blasi F. Fazioli F. EMBO J. 1996; 15: 1572-1582Crossref PubMed Scopus (303) Google Scholar). Among the candidate transmembrane adapters are the integrins, a family of cell adhesion receptor heterodimers that interact with many extracellular matrix and cell-surface ligands (12Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9026) Google Scholar). At least 18 α and 8 β subunits have been identified. Integrin-ligand interaction is involved in many biological and pathological situations, including cell anchorage and migration, cell-cell interaction during immune response, development, wound healing, vascular remodeling, and cancer metastasis and invasion (13Hynes R.O. Lander A.D. Cell. 1992; 68: 303-322Abstract Full Text PDF PubMed Scopus (762) Google Scholar). Integrins transduce signals from outside cells through their interaction with specific ligands. uPAR has been shown to associate with integrins by co-immunocoprecipitation, immunocolocalization, and resonance energy transfer approaches (14Xue W. Kindzelskii A.L. Todd III, R.F. Petty H.R. J. Immunol. 1994; 152: 4630-4640PubMed Google Scholar, 15Xue W. Mizukami I. Todd III, R.F. Petty H.R. Cancer Res. 1997; 57: 1682-1689PubMed Google Scholar, 16Wei Y. Lukashev M. Simon D.I. Bodary S.C. Rosenberg S. Doyle M.V. Chapman H.A. Science. 1996; 273: 1551-1555Crossref PubMed Scopus (698) Google Scholar). However, it has not been established whether the association of uPAR with integrins is responsible for uPA-mediated signaling. We have recently reported that recombinant soluble uPAR is a ligand for several β1 and β3 integrins (17Tarui T. Mazar A.P. Cines D.B. Takada Y. J. Biol. Chem. 2001; 276: 3983-3990Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar), and we postulated that uPAR can transduce signals through the integrin signaling pathway upon binding to integrin in trans. However, it is still unclear whether uPAR binds to integrins as a ligand when both are present on the same membrane (in cis). It has been proposed that integrins "laterally associate" with uPAR (for review, see Ref. 2Ossowski L. Aguirre-Ghiso J.A. Curr. Opin. Cell Biol. 2000; 12: 613-620Crossref PubMed Scopus (359) Google Scholar) and play a role in uPA·uPAR-initiated signaling events. However, the role of the integrin itself in the uPA·uPAR signaling is unclear, since in the current models other integrin ligands (e.g. fibronectin) appear to be essential for uPA·uPAR signaling (8Aguirre Ghiso J.A. Kovalski K. Ossowski L. J. Cell Biol. 1999; 147: 89-104Crossref PubMed Scopus (467) Google Scholar). In this study we designed experiments to identify the role of integrin α5β1 in uPA·uPAR signaling using recombinant uPA fragments and Chinese hamster ovary (CHO) cells overexpressing uPAR or mutant α5β1. We found that cells adhered to immobilized uPA in a signaling-dependent manner. Anti-uPAR antibody, depletion of uPAR, and deletion of the GFD of uPA effectively blocked cell adhesion to uPA, suggesting that binding of uPA to uPAR through GFD is critical for cell adhesion to uPA. Interestingly, anti-α5 antibody, RGD peptide, and function-blocking α5β1 mutations blocked cell adhesion to uPA, suggesting that α5β1 is critical to this process as well. uPA-induced migration of CHO cells also required GFD of uPA, uPAR, and α5β1. We demonstrated that uPA induced RGD-dependent binding of uPAR to α5β1 in solution. These results suggest that uPA-induced signaling in CHO cells involves a process in which (a) uPA binds to uPAR, (b) the uPA·uPAR complex binds to α5β1 as a ligand in cis, and c) signal transduction is initiated through α5β1. Monoclonal antibody (mAb) KH72 (anti-α5) was a kind gift of K. Miyake (University of Tokyo, Tokyo, Japan). mAb 135-13C (anti-α6) (18Falcioni R. Sacchi A. Resau J. Kennel S. Cancer Res. 1988; 48: 816-821PubMed Google Scholar) was a kind gift of S. J. Kennel (Oak ridge National Laboratory, Oak Ridge, TN). mAbs P1F6 (anti-αvβ5), HA5 (anti-α5), and VC5 (anti-α5) were purchased from Chemicon (Temecula, CA). Anti-uPAR monoclonal antibody (3B10) (19Min H.Y. Semnani R. Mizukami I.F. Watt K. Todd III, R.F.D. Liu D.Y. J. Immunol. 1992; 148: 3636-3642PubMed Google Scholar) was kindly provided by R. F. Todd III (University of Michigan Medical Center, Ann Arbor, MI). The polyclonal anti-uPAR has been described previously (20Gum R. Juarez J. Allgayer H. Mazar A. Wang Y. Boyd D. Oncogene. 1998; 17: 213-225Crossref PubMed Scopus (47) Google Scholar). The anti-uPA kringle antibody (Ab963) was a kind gift from J. Henkin (Abbott Laboratories, Abbott Park, IL). Anti-uPA kringle and anti-LMW uPA mAbs linked to Sepharose 4B were from IKTEK Ltd. (Moscow, Russia). A mAb against soluble uPAR (clone D2D3–813, IgG1κ) was raised against the soluble uPAR D2D3 fragment. Strategic Biosolutions (Newark, DE) generated the ascites and purified the antibody using a 50-ml Amersham Biosciences protein A-Sepharose fast flow column. GRGDS and GRGES peptide were purchased from Advanced ChemTech (Louisville, KY). Phosphatidylinositol-specific phospholipase C was obtained from Glyko, Inc. (Novato, CA). PD98059 was purchased from Calbiochem. Protein G-agarose was from Amersham Biosciences. Na-125I was purchased from PerkinElmer Life Sciences, and Iodo beads were from Pierce. CHO cells were obtained from the American Type Culture Collection (Manassas, VA). CHO cells expressing the three domain forms of human uPAR (designated uPAR-CHO) have been described (17Tarui T. Mazar A.P. Cines D.B. Takada Y. J. Biol. Chem. 2001; 276: 3983-3990Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). The α5-deficient CHO cells (B2 variant) expressing human α5 (wild type or mutant) have been described (21Irie A. Kamata T. Puzon-McLaughlin W. Takada Y. EMBO J. 1995; 14: 5542-5549Crossref PubMed Scopus (91) Google Scholar). Generation of Wild-type uPA and uPA Fragments—cDNA encoding wild-type single-chain uPA (scuPA) was generated and subcloned into pMT/BiP/V5 (Invitrogen) as described previously (22Bdeir K. Kuo A. Mazar A. Sachais B.S. Xiao W. Gawlak S. Harris S. Higazi A.A. Cines D.B. J. Biol. Chem. 2000; 275: 28532-28538Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). cDNA encoding the amino-terminal fragment (ATF, amino acids 1–143), kringle (amino acids 47–143), ΔGFD-scuPA (amino acids 47–411), and FLAG-LMW-uPA (amino acids 136–411) were generated by PCR with full-length UK/pUN121 (23Axelrod J.H. Reich R. Miskin R. Mol. Cell. Biol. 1989; 9: 2133-2141Crossref PubMed Scopus (126) Google Scholar) as a template. The PCR products were digested with BamH1 and XhoI and subcloned into pMT/BiP/V5 at the BglII and XhoI sites. Recombinant proteins were expressed using the Drosophila expression system (Invitrogen) in Schneider S2 cells according to the manufacturer's recommendations. Wild-type scuPA, ΔGFD-scuPA, and FLAG-LMW-uPA were purified from S2 medium by affinity chromatography using anti-LMW uPA mAb immobilized onto Sepharose (IKTEK Ltd.). ATF 1–143 and kringle were purified from S2 medium by affinity chromatography using an anti-kringle uPA mAb immobilized onto Sepharose (IKTEK Ltd.). Synthesis of soluble uPAR (D2D3 form) has been described (17Tarui T. Mazar A.P. Cines D.B. Takada Y. J. Biol. Chem. 2001; 276: 3983-3990Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Adhesion Assays—Adhesion assays were performed as previously described (24Zhang X.P. Kamata T. Yokoyama K. Puzon-McLaughlin W. Takada Y. J. Biol. Chem. 1998; 273: 7345-7350Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). Briefly, wells in 96-well Immulon-2 microtiter plates (Dynatech Laboratories, Chantilly, VA) were coated with 100 μl of phosphate-buffered saline (10 mm phosphate buffer, 0.15 m NaCl, pH 7.4) containing substrates at a concentration of 50–1000 nm and were incubated1hat37 °C. Remaining protein binding sites were blocked by incubating with 0.2% BSA (Calbiochem) for 1 h at room temperature. Cells (105 cells/well) in 100 μl of Hepes-Tyrode buffer (10 mm HEPES, 150 mm NaCl, 12 mm NaHCO3, 0.4 mm NaH2PO4, 2.5 mm KCl, 0.1% glucose, 0.02% BSA) supplemented with 2 mm MgCl2 were added to the wells and incubated at 37 °C for 1 h unless stated otherwise. After non-bound cells were removed by rinsing the wells with the same buffer, bound cells were quantified by measuring endogenous phosphatase activity (25Prater C.A. Plotkin J. Jaye D. Frazier W.A. J. Cell Biol. 1991; 112: 1031-1040Crossref PubMed Scopus (188) Google Scholar). Antibodies were used at a 250-fold dilution for ascites (KH72 and 135-13C) and at 10 μg/ml for purified antibodies or IgG. Data are shown as means ± S.D. of three independent experiments. We confirmed that equivalent amounts of the fragments and mutants of uPA were coated on the plate by enzyme-linked immunosorbent assay (data not shown). Mitogen-activated Protein Kinases (MAPK) Activation Assay—uPAR-CHO cells were plated into 6-ell tissue culture plates at 2 × 106 cells/ml in Dulbecco's modified Eagle's medium supplemented with 0.5% fetal calf serum, 1× penicillin-streptomycin-glutamine solution and incubated for 2 days at 37 °C in a 95% air, 5% CO2 humidified atmosphere. The cell culture media was removed, and the cells were washed once with prewarmed serum-free Dulbecco's modified Eagle's medium. The cells were incubated for 3 h with serum-free Dulbecco's modified Eagle's medium with or without the MEK inhibitor PD98059 (500 μm) at 37 °C in a 95% air, 5% CO2 humidified atmosphere. The cells were stimulated with different concentrations of soluble scuPA for 5 min at 37 °C. The reaction was terminated by removing the stimulation media and washing the cells with 1 ml of ice-cold phosphate-buffered saline containing 1 mm Na3PO4 followed by incubation with 100 μl of ice-cold radioimmune precipitation assay buffer for 20 min on ice. The whole cell lysate was collected, and the nuclear material was pelleted by centrifugation at 14,000 × g for 10 min. The supernatant from each treatment was retained and stored at –20 °C until required. Whole cell lysates (40 μg of protein) were fractionated using 4–20% SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose for Western blotting. The membranes were blocked for 1 h at room temperature with blocking buffer (10 mm Tris-HCl, 150 mm NaCl, pH 7.5, supplemented with 0.1% Tween 20 and 5% BLOTTO; Biorad, Hercules, CA). To determine the phosphorylation changes in MAPK the membranes were washed 3 times for 5 min with wash buffer (10 mm Tris-HCl, 150 mm NaCl, pH 7.5, supplemented with 0.1% Tween 20) and incubated overnight at 4 °C with a 1:1000 dilution of rabbit anti-phospho-p44/42 MAPK (Thr-202/Tyr-204) antibody (Cell Signaling Technology, Beverly, MA) in blocking buffer. The blots were washed 3 times for 5 min with wash buffer and probed with a 1:2000 dilution of anti-rabbit horseradish peroxide-conjugated secondary antibody (Cell Signaling Technology) in blocking buffer at room temperature for 1 h. The blots were washed 3 times for 5 min and developed using the Immun-Star horseradish peroxide chemiluminescence substrate kit (Bio-Rad). The blots were stripped by incubating with stripping buffer (0.1 m glycine, pH 2.6, and 2% SDS) for 30 min at 50 °C. The blots were washed 3 times with 10 mm Tris-HCl, 150 mm NaCl, pH 7.5, re-blocked for 1 h at room temperature with blocking buffer, washed 3 times for 5 min, incubated with a 1:1000 dilution of rabbit anti-p44/42 MAPK (Cell Signaling Technology) overnight at 4 °C in blocking buffer, and processed as above to determine the total MAPK levels of each lane. Migration Assays—Cell migration was analyzed using tissue culture-treated 24-well Transwell plates (Costar, Cambridge, MA) with polycarbonate membranes of pore size 8 μm. The lower side of the filter was coated with various concentrations (20–200 nm) of substrates. Coated filters were placed into a serum-free migration buffer (Dulbecco's modified Eagle's medium supplemented with 10 mm Hepes, 0.5% bovine serum albumin, and 1× penicillin-streptomycin), and cells (100 μl) suspended in the same buffer (8 × 105 cells/ml) were added to the upper chamber. The cells were incubated at 37 °C in 5% CO2 for 20 h. Cells in the upper chamber were removed by wiping, and those that migrated to the lower surface of the filters were fixed and stained with 0.5% crystal violet in 20% ethanol and counted. The result in each well is the mean cell number of 4 randomly selected high magnification microscopic fields from triplicate experiments. In some experiments, anti-integrin antibodies (10 μg/ml) were incubated with cells for 15 min before to the assay. Co-precipitation of uPAR and Integrin α5β1 1—Soluble uPAR was radioiodinated with Na-125I using Iodo beads (specific activity 14,500 cpm/ng). Purified human α5β1 integrin was obtained from Chemicon International. Purified α5β1 (6 μg/ml), mAb HA5 (9 μg/ml), 125I-labeled soluble uPAR (6 μg/ml), and uPA (12 μg/ml) were incubated with protein G-agarose beads either in the presence or absence of RGD peptide (150 μg/ml) in serum-free RPMI 1640 medium supplemented with 10 mm HEPES, pH 7.4, 0.02% bovine serum albumin at 4 °C for 4 h. As a control, experiments were performed in the absence of uPA. Beads were washed 3 times in RPMI supplemented with 10 mm HEPES, 0.02% bovine serum albumin. Bound materials were extracted into reducing SDS-PAGE sample buffer and analyzed by SDS-PAGE and autoradiography. Other Methods—Flow cytometric analysis and stress-fiber staining were performed as described before (17Tarui T. Mazar A.P. Cines D.B. Takada Y. J. Biol. Chem. 2001; 276: 3983-3990Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 26Takada Y. Puzon W. J. Biol. Chem. 1993; 268: 17597-17601Abstract Full Text PDF PubMed Google Scholar). uPAR-dependent Cell Adhesion to uPA—It has been reported that CHO cells express endogenous hamster uPAR (17Tarui T. Mazar A.P. Cines D.B. Takada Y. J. Biol. Chem. 2001; 276: 3983-3990Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). We detected low level endogenous hamster uPAR on the mock-transfected CHO cells with polyclonal anti-human uPAR antibodies (Fig 1a). Consistent with this finding, mock-transfected CHO cells adhered to immobilized uPA in a dose-dependent manner (Fig. 1c). We transfected CHO cells with cDNA encoding human uPAR and cloned stable cell lines expressing high levels of receptor (designated uPAR-CHO). uPAR-CHO cells express uPAR at a much higher level (Fig. 1b) and showed greater adhesion to uPA than mock-CHO cells (Fig. 1c). Anti-uPAR polyclonal rabbit IgG completely blocked adhesion of uPAR- and mock-transfected CHO cells to uPA (Fig. 1d). To further test whether uPAR is involved in this process, we treated uPAR-CHO cells with phosphatidylinositol-specific phospholipase C to remove the glycosylphosphatidylinositol-anchored uPAR. The phosphatidylinositol-specific phospholipase C treatment removed more than 90% of uPAR on the cell surface, as determined by flow cytometry with anti-uPAR (Fig. 1e), and markedly reduced the adhesion of uPAR-CHO cells to uPA (Fig. 1f). These results indicate that adhesion of these CHO cells to immobilized uPA is uPAR-dependent and that the level of adhesion to uPA correlates with the amount of uPAR. We also found that adhesion of uPAR-CHO cells to immobilized uPA occurred at 37 °C, but not at 4 °C, suggesting that this process is temperature-dependent and requires signal transduction (Fig. 2a). It has been reported that uPA induces activation of MAPK (27Nguyen D.H. Hussaini I.M. Gonias S.L. J. Biol. Chem. 1998; 273: 8502-8507Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). An inhibitor to MEK-1 (PD98059) that prevents the activation of MAPK (extracellular signal-regulated kinases1/2) (Fig. 2b) consistently blocked adhesion of uPAR-CHO cells to uPA in a dose-dependent manner (Fig. 2c). These results suggest that this process requires extracellular signal-regulated kinase 1/2 activation (most likely induced by immobilized uPA). We found that immobilized uPA did not induce spreading or stress-fiber formation in uPAR-CHO cells, in contrast to fibronectin used as a positive control (Fig. 2d). Role of Integrins in uPAR-mediated Adhesion to uPA—It has been proposed that integrins may be critically involved in the uPA·uPAR signaling (2Ossowski L. Aguirre-Ghiso J.A. Curr. Opin. Cell Biol. 2000; 12: 613-620Crossref PubMed Scopus (359) Google Scholar). To examine this hypothesis we first determined whether integrins contribute to cell adhesion to uPA. To do so, we tested the effects of anti-integrin mAbs on adhesion of uPAR-CHO cells to uPA. CHO cells have endogenous hamster integrins α5β1, αvβ1, and αvβ5 (data not shown). We found that RGD peptide (100 μm) blocked adhesion of uPAR-CHO cells to uPA, but control RGE peptide did not (Fig. 3a). Consistent with the findings that RGD-dependent integrin(s) is involved in this process, anti-α5 mAb (KH72) completely blocked adhesion of uPAR-CHO to uPA, whereas anti-αvβ5 mAb (P1F6) or control ascites did not (Fig. 3a and data not shown). These results suggest that adhesion of uPAR-CHO cells to immobilized uPA is α5β1-dependent. Then we tested whether the level of α5β1 expression affects cell adhesion to uPA using the B2 variant of CHO cells, which expresses ∼2% of α5β1 compared with parental CHO cells (28Bauer J.S. Schreiner C.L. Giancotti F.G. Ruoslahti E. Juliano R.L. J. Cell Biol. 1992; 116: 477-487Crossref PubMed Scopus (116) Google Scholar). The B2 cells adhered to uPA at a level lower than CHO cells (Fig 3b). This adhesion was completely blocked by anti-uPAR polyclonal antibodies and anti-integrin α5 (data not shown). These results suggest that cell adhesion to uPA is dependent on the level of α5β1 but that a small amount of α5β1 on the B2 cells still supports the adhesion to uPA to some extent. Another possibility is that integrin αvβ1 may also be involved in this process, although we were not able to test this hypothesis since function-blocking anti-hamster αv mAb is not currently available. To test the specific contribution of α5β1 to uPA·uPAR-dependent cell adhesion, we used immobilized anti-uPAR mAb as a uPAR ligand. We found that uPAR-CHO cells adhered to anti-uPAR and that this adhesion was not inhibited by anti-integrin α5 mAb (Fig. 3c), suggesting that α5β1 was not required for this process. Taken together, adhesion of CHO cells to uPA requires uPAR and α5β1, and α5β1 is specifically involved in this process only when uPA is used as a ligand. The Domains of uPA Required for Cell Adhesion to uPA—To identify which uPA domains are involved in uPAR/α5β1-dependent cell adhesion to uPA, we used several uPA fragments including the ATF, the kringle domain, the LMW-uPA, ΔGFD-uPA, which lacks GFD, and ΔKringle-uPA, which lacks the kringle domain (Fig 4a). We found that ATF and ΔKringle-uPA supported cell adhesion at levels comparable with that of wild-type uPA. Kringle, LMW-uPA, or ΔGFD-uPA did not support the adhesion at all (Fig 4b). These results suggest that GFD is primarily involved in cell adhesion to uPA, but other domains are not. As a second approach, we tested whether mAbs to different domains of uPA block cell adhesion to uPA. We found that anti-GFD (AD3471) and anti-kringle mAb (Ab963) blocked adhesion of uPAR-CHO cells to uPA, whereas anti-LMW (UNG-5) did not (Fig 4c). Ab963 has been observed to inhibit the binding of uPA to whole cells despite the fact that its epitope has been mapped to the kringle domain. These results are consistent with the results with uPA fragments (Fig. 4b) with the exception of the effect of the anti-kringle antibody. GFD is required for uPA to bind to uPAR (10Appella E. Blasi F. Ann. N. Y. Acad. Sci. 1987; 511: 192-195Crossref PubMed Scopus (19) Google Scholar). Taken together these studies suggest that uPAR/α5β1-mediated cell adhesion to uPA is also critically dependent on the interaction with GFD. Mutations in Integrin α5 Affect Cell Adhesion to uPA—We have recently reported that uPAR is a ligand for several integrins (17Tarui T. Mazar A.P. Cines D.B. Takada Y. J. Biol. Chem. 2001; 276: 3983-3990Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Specifically, soluble uPAR supports integrin-mediated cell adhesion, and glycosylphosphatidylinositol-linked uPAR binds to integrins in apposing cells in trans and supports cell-cell interaction. We have reported that mutations in the ligand binding region of integrin α4 subunit blocked adhesion of α4β1-transfected CHO cells to immobilized soluble uPAR (17Tarui T. Mazar A.P. Cines D.B. Takada Y. J. Biol. Chem. 2001; 276: 3983-3990Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar), suggesting that uPAR binds to α4β1 as a ligand. These critical residues are located within the ligand binding site in integrins based on the crystal structure of integrin αvβ3 (29Xiong J.-P. Stehle T. Diefenbach B. Zhang R. Dunker R. Scott D.L. Andrzej J. Goodman S.L. Arnaout M.A. Science. 2001; 294: 339-345Crossref PubMed Scopus (1118) Google Scholar, 30Xiong J.P. Stehle T. Zhang R. Joachimiak A. Frech M. Goodman S.L. Arnaout M.A. Science. 2002; 296: 151-155Crossref PubMed Scopus (1410) Google Scholar). We suspected that cell adhesion to uPA in the present study involves interaction between uPAR and α5β1 on the same cell surface (in cis). We have reported that similar mutations (the Tyr-186 to Ala (Y186A), F187A, and W188A) in the ligand binding site of α5 blocked fibronectin binding to α5β1 (21Irie A. Kamata T. Puzon-McLaughlin W. Takada Y. EMBO J. 1995; 14: 5542-5549Crossref PubMed Scopus (91) Google Scholar). We therefore studied whether uPAR binds to α5β1 as a ligand during adhesion to uPA using these α5 mutants. We first tested the effects of these function-blocking mutations of α5 (21Irie A. Kamata T. Puzon-McLaughlin W. Takada Y. EMBO J. 1995; 14: 5542-5549Crossref PubMed Scopus (91) Google Scholar) on adhesion to soluble uPAR. We used B2 cells expressing wild type and Y186A, F187A, and W188A mutants of integrin α5 (designated α5-B2, α5/Y186A-B2, α5/F187A-B2, and α5/W188A-B2, respectively). Expression levels of integrin α5β1 among those transfectants were comparable as measured by flow cytometry with non-function blocking anti-human α5 mAb (VC-5) (21Irie A. Kamata T. Puzon-McLaughlin W. Takada Y. EMBO J. 1995; 14: 5542-5549Crossref PubMed Scopus (91) Google Scholar). CHO cells, but not B2 cells, bind to coated soluble uPAR upon Mn2+ activation (17Tarui T. Mazar A.P. Cines D.B. Takada Y. J. Biol. Chem. 2001; 276: 3983-3990Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar), indicating that α5β1 requires activation to bind to uPAR. We found that α5-B2 cells adhered to soluble uPAR if activated with Mn2+ (Fig 5a) and that the Y186A and W188A mutations completely, and the F187A mutation partially, blocked the adhesion to soluble uPAR. We then tested the effect of these α5 mutations on cell adhesion to uPA. In this experiment we used ATF instead of wild-type uPA to exclude the possible contribution of the serine protease domain because we found that both B2 and α5-B2 cells bind weakly to LMW-uPA when α5β1 is activ
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