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Role of Endoproteolytic Processing in the Adhesive and Signaling Functions of αvβ5 Integrin

2000; Elsevier BV; Volume: 275; Issue: 43 Linguagem: Inglês

10.1074/jbc.m004834200

1083-351X

Virginie Berthet, Véronique Rigot, Serge Champion, J. Secchi, Francis Fouchier, Jacques Marvaldi, José Luis,

Platelet Disorders and Treatments

Some integrin α subunits undergo a post-translational cleavage in their extracellular domain. However, the role of this cleavage in integrin function is unclear. Enzymes involved in this maturation belong to the subtilisin-like endoprotease family (convertases). To understand the role of the α subunit cleavage in integrin function, we have designed stable transfectants (PDX39P cells) expressing α1-PDX, a convertase inhibitor. Immunoprecipitation of cell surface proteins from PDX39P showed that α3, α6 and αvintegrins lack endoproteolytic cleavage. We have compared adhesion between PDX39P cells and mock-transfected cells on different extracellular matrix proteins. No difference in adhesion could be observed on laminin-1 and type I collagen, while attachment of PDX39P cells to vitronectin (ligand of the αvβ5integrin) was dramatically reduced. The reduced adhesion of PDX39P cells was not due to changes in integrin affinity as determined by solid-phase receptor assay in a cell-free environment. Intracellular signaling pathways activated by αv integrin ligation were also affected in PDX39P cells. It thus seems that the absence of endoproteolytic cleavage of αv integrins has important consequences on signal transduction pathways leading to alterations in integrin function such as cell adhesion. Some integrin α subunits undergo a post-translational cleavage in their extracellular domain. However, the role of this cleavage in integrin function is unclear. Enzymes involved in this maturation belong to the subtilisin-like endoprotease family (convertases). To understand the role of the α subunit cleavage in integrin function, we have designed stable transfectants (PDX39P cells) expressing α1-PDX, a convertase inhibitor. Immunoprecipitation of cell surface proteins from PDX39P showed that α3, α6 and αvintegrins lack endoproteolytic cleavage. We have compared adhesion between PDX39P cells and mock-transfected cells on different extracellular matrix proteins. No difference in adhesion could be observed on laminin-1 and type I collagen, while attachment of PDX39P cells to vitronectin (ligand of the αvβ5integrin) was dramatically reduced. The reduced adhesion of PDX39P cells was not due to changes in integrin affinity as determined by solid-phase receptor assay in a cell-free environment. Intracellular signaling pathways activated by αv integrin ligation were also affected in PDX39P cells. It thus seems that the absence of endoproteolytic cleavage of αv integrins has important consequences on signal transduction pathways leading to alterations in integrin function such as cell adhesion. extracellular matrix monoclonal antibody mitogen-activated protein kinase Dulbecco's phosphate-buffered saline bovine serum albumin Pseudomonas exotoxin A α1-antitrypsin polyacrylamide gel electrophoresis nonreducing reducing focal adhesion kinase extracellular signal-regulated kinase phorbol 12-myristate 13-acetate Integrins are transmembrane glycoproteins, composed of noncovalently associated α and β subunits, that are involved in cell-extracellular matrix (ECM)1 and cell-cell interactions (1Aplin A. Howe A. Alahari S. Juliano R. Pharmacol. Rev. 1998; 50: 197-263PubMed Google Scholar). Many integrin α chains undergo a post-translational endoproteolytic cleavage. The α3, α5, α6, α7, α8, α9, αv, and αIIb subunits are cleaved in the membrane-proximal extracellular region, resulting in a heavy chain that is disulfide-linked to a membrane spanning light chain (2Hemler M. Mecham R. McDonald J. Receptors for Extracellular Matrix Proteins. Academic Press, San Diego, CA1991: 255-299Google Scholar). The α4 and αE subunits can also be cleaved, but at unusual positions, near the middle and in the N-terminal region of the molecule, respectively (3Teixidó J. Parker C.M. Kassner P.D. Hemler M.E. J. Biol. Chem. 1992; 267: 1786-1791Abstract Full Text PDF PubMed Google Scholar, 4Shaw S.K. Cepek K.L. Murphy E.A. Russell G.J. Brenner M.B. Parker C.M. J. Biol. Chem. 1994; 269: 6016-6025Abstract Full Text PDF PubMed Google Scholar). Endoproteolytic cleavage of integrin α subunits occurs at specific sites comprising pairs of basic amino acids. Post-translational proteolysis is a common mechanism required for the synthesis of biologically active proteins in bacteria, fungi, yeast, invertebrates, and mammals (5Denault J.-B. Leduc R. FEBS Lett. 1996; 379: 113-116Crossref PubMed Scopus (71) Google Scholar). However, the role of endoproteolytic cleavage of integrin α subunits is not clear. The cleavage is conserved, not only in different α chains but also across species, suggesting that it might be of functional importance. It has been established, by site-directed mutagenesis of cleavage sites, that uncleaved αIIbβ3 and α4β1 are able to mediate cell adhesion to their ligands (3Teixidó J. Parker C.M. Kassner P.D. Hemler M.E. J. Biol. Chem. 1992; 267: 1786-1791Abstract Full Text PDF PubMed Google Scholar, 6Kolodziej M. Vilaire G. Gonder D. Poncz M. Bennett J. J. Biol. Chem. 1991; 266: 23499-23504Abstract Full Text PDF PubMed Google Scholar). However, it has been reported that a defect in α6 cleavage impairs the inside/out signaling of uncleaved α6β1 integrins induced by phorbol ester, indicating that cleavage is necessary for proper integrin function (7Delwel G.O. Hogervorst F. Sonnenberg A. J. Biol. Chem. 1996; 271: 7293-7296Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). The search for mammalian analogues of kexin, a yeast endoprotease, has led to the discovery of the subtilisin/kexin-like family of proprotein convertases. These calcium-dependent serine proteases can be subdivided into four groups according to their cellular localization and tissue distribution (for reviews and updates, see Refs. 8Steiner D. Rouillé Y. Gong Q. Martin S. Carroll R. Chan S. Diabetes Metab. 1996; 22: 94-104PubMed Google Scholar, 9Seidah N.G. Chrétien M. Curr. Opin. Biotechnol. 1997; 8: 602-607Crossref PubMed Scopus (241) Google Scholar, 10Nakayama K. Biochem. J. 1997; 327: 625-635Crossref PubMed Scopus (704) Google Scholar, 11Zhou A. Webb G. Zhu X. Steiner D.F. J. Biol. Chem. 1999; 274: 20745-20748Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar). Furin and PC7, belonging to the first group, are widely expressed in tissues and mainly localized to the trans-Golgi network. PC1 and PC2 are primarily expressed in neural and endocrine cells and are found essentially within secretory granules. The third group includes PC5A and PACE4, widely distributed in tissues and localized within the trans-Golgi network and secretory granules, and PC5B (also called PC6), mostly found within thetrans-Golgi network. PC4, exclusively expressed in germ cells, constitutes the fourth group. The general consensus sequence cleaved by these enzymes contains the motif (K/R)-X n-(K/R)↓, where n = 0, 2, 4, or 6. In vitro and phage display data revealed that theX residue and those following the cleavage site define the fine specificity of each enzyme (12Matthews D. Goodman L. Gorman C. Wells J. Protein Sci. 1994; 3: 1197-1205Crossref PubMed Scopus (104) Google Scholar, 13Jean F. Boudreault A. Basak A. Seidah N. Lazure C. J. Biol. Chem. 1995; 270: 19225-19231Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Due to their potential pharmacological interest, several inhibitors of proprotein convertases have been designed (14Jean F. Stella K. Thomas L. Liu G. Xiang Y. Reason A. Thomas G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7293-7298Crossref PubMed Scopus (242) Google Scholar). In humans, a naturally occurring mutation of α1-antitrypsin (α1-AT), known as α1-AT Pittsburgh, changes the specificity of this serpin from an inhibitor of elastase into a potent inhibitor of thrombin (15Owen M. Brennan S. Lewis J. Carrel R. N. Engl. J. Med. 1983; 309: 694-698Crossref PubMed Scopus (323) Google Scholar). Another variant of α1-AT, called α1-AT Portland (α1-PDX), has been engineered by Anderson et al. (16Anderson E. Thomas L. Hayflick J. Thomas G. J. Biol. Chem. 1993; 268: 24887-24891Abstract Full Text PDF PubMed Google Scholar) and described as a potent inhibitor of convertases. Recent findings shows that α1-PDX is a selective inhibitor for furin and, to a lesser extent, for PC5B (14Jean F. Stella K. Thomas L. Liu G. Xiang Y. Reason A. Thomas G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7293-7298Crossref PubMed Scopus (242) Google Scholar). This inhibitor blocks the convertases dependent processing of various precursors (17Denault J.-B. D'Orléans-Juste P. Masaki T. Leduc R. J. Cardiovasc. Pharmacol. 1995; 26: S47-S50Crossref PubMed Scopus (23) Google Scholar, 18Decroly E. Wouters S. Di Bello C. Lazure C. Ruysschaert J.-M. Seidah N. J. Biol. Chem. 1996; 271: 3042-3050Abstract Full Text Full Text PDF Scopus (102) Google Scholar, 19Benjannet S. Savaria D. Laslop A. Munzer J.S. Chrétien M. Marcinkiewicz M. Seidah N.G. J. Biol. Chem. 1997; 272: 26210-26218Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 20Logeat F. Bessia C. Brou C. LeBail O. Jarriault S. Seidah N. Israël A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8108-8112Crossref PubMed Scopus (578) Google Scholar, 21Cui Y. Jean F. Thomas G. Christian J. EMBO J. 1998; 17: 4535-4543Crossref PubMed Scopus (200) Google Scholar). In a previous report (22Lehmann M. Rigot V. Seidah N.G. Marvaldi J. Lissitzky J.C. Biochem. J. 1996; 317: 803-809Crossref PubMed Scopus (54) Google Scholar), we showed that α3, α6, and αv integrin subunits are not processed in the furin-deficient LoVo-C5 cell. Moreover, pro-forms of α integrin subunits are cleaved both by recombinant convertasesin vitro and ex vivo after overexpression in LoVo cells (22Lehmann M. Rigot V. Seidah N.G. Marvaldi J. Lissitzky J.C. Biochem. J. 1996; 317: 803-809Crossref PubMed Scopus (54) Google Scholar, 23Lissitzky J. Luis J. Munzer J. Benjannet S. Parat F. Chrétien M. Marvaldi J. Seidah N. Biochem. J. 2000; 346: 133-138Crossref PubMed Scopus (98) Google Scholar). In the present study, we investigated the functional importance of the α subunits cleavage during the interaction of integrins with ECM proteins. We have designed stable transfectants of α1-PDX in HT29-D4 adenocarcinoma cells. These transfected cells, bearing uncleaved integrin α subunits, displayed a reduced attachment to vitronectin. This alteration was correlated with defects in the intracellular signaling pathways activated by αvintegrin ligation. Dulbecco's modified Eagle's medium was purchased from Life Technologies, Inc. (Cergy-Pontoise, France) and fetal bovine serum from BioWhittaker (Fontenay-sous-Bois, France). PBS was from Oxoid (Basingstoke, United Kingdom). The synthetic peptides RGDS and RGES, Pseudomonas exotoxin A, BSA, type I collagen, poly-l-lysine, and crystal violet were obtained from Sigma (St-Quentin Fallavier, France). Sulfosuccinimidyl-6-(biotinamido) hexanoate (NHS-LC-biotin) was from Pierce. TMB peroxidase EIA substrate kit was obtained from Bio-Rad (Ivry-sur-Seine, France) and protein G-agarose beads from Roche Molecular Biochemicals (Meylan, France). The light-based ECL detection kit was from Amersham Pharmacia Biotech (Les Ulis, France). Laminin-1 and vitronectin were prepared according to Timpl et al. (24Timpl R. Rohde H. Gehron-Robey P. Rennard S.I. Foidart J.M. Martin G.R. J. Biol. Chem. 1979; 254: 9933-9937Abstract Full Text PDF PubMed Google Scholar) and Yatogho et al. (25Yatogho T. Izumi M. Kashiwagi H. Hayashi M. Cell Struct. Funct. 1988; 13: 281-292Crossref PubMed Scopus (439) Google Scholar), respectively. Rat mAb 69.6.5 against αv integrin was produced as described previously (26Lehmann M. Rabenandrasana C. Tamura R. Lissitzky J.-C. Quaranta V. Pichon J. Marvaldi J. Cancer Res. 1994; 54: 2102-2107PubMed Google Scholar). Mouse mAb M-Kid2 (anti-α3), mouse mAb AMF-7 (anti-αv), and rat mAb GoH3 (anti-α6) were from Immunotech (Marseille, France). Anti-active MAPK polyclonal antibody was obtained from Promega (Madison, WI). Mouse mAbs anti-phosphotyrosine (PY20) and anti-paxillin (clone 349) were from Transduction Laboratories (Lexington, KY). Rabbit polyclonal anti-FAK antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Sheep anti-mouse and donkey anti-rabbit immunoglobulins conjugated to horseradish peroxidase were purchased from Amersham Pharmacia Biotech. HT29-D4 cells, derived from a human colon adenocarcinoma, were transfected with a pBK-CMV expression vector containing the full-length α1-PDX cDNA (gift of G. Thomas, Portland, OR). Clones of stable transfected cells (controlled by Northern blot) were isolated and tested for their sensitivity toPseudomonas exotoxin A (PEA), a toxin activated after cleavage by convertases, which has proved to be an indicative assay for furin activity (22Lehmann M. Rigot V. Seidah N.G. Marvaldi J. Lissitzky J.C. Biochem. J. 1996; 317: 803-809Crossref PubMed Scopus (54) Google Scholar, 27Moehring J. Inocencio N. Robertson B. Moehring T. J. Biol. Chem. 1993; 268: 2590-2594Abstract Full Text PDF PubMed Google Scholar). Among the clones tested, the clone PDX39 presented a pronounced increase in PEA resistance compared with HT29-D4 cells or to the clone PDX0 transfected with the empty vector. To generate cells expressing higher levels of α1-PDX inhibitor, the clone PDX39 was cultured in the presence of 1 μg/ml PEA. This cell subpopulation, resistant to PEA and expressing high levels of α1-PDX (controlled by reverse transcription polymerase chain reaction), was referred as PDX39P. The cell surface expression of integrin subunits was determined by flow cytometry and by immunoprecipitation of biotinylated cell surface proteins as described elsewhere (28Rigot V. Lehmann M. André F. Daemi N. Marvaldi J. Luis J. J. Cell Sci. 1998; 111: 3119-3127Crossref PubMed Google Scholar). Adhesion assays were performed as described previously (28Rigot V. Lehmann M. André F. Daemi N. Marvaldi J. Luis J. J. Cell Sci. 1998; 111: 3119-3127Crossref PubMed Google Scholar, 29Kadi A. Pichard V. Lehmann M. Briand C. Braguer D. Marvaldi J. Rognoni J.-B. Luis J. Biochem. Biophys. Res. Commun. 1998; 246: 690-695Crossref PubMed Scopus (50) Google Scholar). Briefly, cells were harvested in single cell suspension by treatment with 0.53 mm EDTA in PBS, added to wells coated with purified ECM proteins, and allowed to adhere to the substrata for 2 h at 37 °C. After washing, attached cells were stained by 0.1% crystal violet and lysed with 1% SDS. Absorbance was then measured at 600 nm. Serum-starved cells in suspension were washed twice with 10 mm Tris, pH 7.4, and resuspended in the same buffer containing a mixture of protease inhibitors (1 mm phenylmethylsulfonyl fluoride, 500 units/ml aprotinin, 1 μg/ml leupeptin, 1 μm pepstatin, 1 mm iodoacetamide, and 1 mm o-phenanthroline). Cells were disrupted with a Potter homogenizer and homogenate was centrifuged at 600 × gfor 10 min at 4 °C. Plasma membranes were recovered by centrifuging the supernatant at 50,000 × g for 45 min and then solubilized for 45 min at 4 °C with 50 mm Tris, pH 7.4, 150 mm NaCl (TBS) containing 1%n-octyl-β-d-glucopyranoside, 1 mmMgCl2, 1 mm CaCl2, and the mixture of protease inhibitors. The extract was clarified by centrifugation at 15,000 × g for 10 min at 4 °C, and the protein concentration was determined by the Bio-Rad DC protein assay. MaxiSorbTM microtiter plate wells (Nunc) were coated for 2 h at 37 °C with 10 μg/ml vitronectin in 50 μl of TBS and blocked with 1% BSA for 1 h at room temperature. Vitronectin-coated wells were incubated with 100 μl of solubilized membranes (300 μg of protein) overnight at 4 °C. Unbound proteins were removed by four washes with TBS containing 0.2% Tween 20 and 1% BSA. αv binding was detected by sequential incubations for 1 h at 37 °C with 100 μl of 10 μg/ml mouse mAb AMF-7 and at room temperature with rabbit anti-mouse IgG (1/1000) and anti-rabbit IgG-horseradish peroxidase-conjugated antibodies. Finally, 100 μl of peroxidase substrate was added for 10 min at room temperature, the reaction was stopped by adding 100 μl of 0.5 mol/liter H2SO4, and absorbance was measured at 450 nm. Confluent cells were washed twice with serum-free medium and incubated in Dulbecco's modified Eagle's medium containing 0.1% BSA for 24 h at 37 °C. Serum-starved cells were harvested in single cell suspension, added (5 × 106 cells in 1 ml) to 9.6-cm2 wells coated with 10 μg/ml poly-l-lysine or vitronectin, and allowed to adhere to the substrata for the indicated periods of time at 37 °C. After three washes with PBS, attached cells were lysed with 20 mmTris-HCl, pH 8, 200 mm NaCl, 1 mm EDTA, and 1% Triton X-100 (RIPA buffer) containing 10 mm sodium orthovanadate, 10 mm sodium pyrophosphate, 10 mm NaF, and the mixture of protease inhibitors. Lysates were clarified by centrifugation and analyzed by immunoblot, after immunoprecipitation, or directly with an anti-active MAPK polyclonal antibody as described previously (30André F. Rigot V. Remacle-Bonnet M. Luis J. Pommier G. Marvaldi J. Gastroenterology. 1999; 116: 64-77Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). For immunoprecipitation, 600–900 μg of proteins were incubated with 1 μg of anti-FAK or 2 μg of anti-paxillin antibodies overnight at 4 °C and then with protein G-agarose for 45 min. Pellets were washed three times with RIPA buffer, three times with RIPA buffer, 500 mm NaCl, and once with PBS. Immunoprecipitated proteins were resolved by SDS-PAGE and blotted onto a nitrocellulose sheet. Membranes were blocked in PBS, 4% BSA, 0.2% Tween 20 and probed overnight at 4 °C with PY20 antibody in PBS, 0.8% BSA, 0.2% Tween 20. Blots were then visualized with horseradish peroxidase-conjugated secondary antibodies. When necessary, nitrocellulose membranes were stripped and reprobed with anti-MAPK (1/1000), anti-FAK (0.2 μg/ml), or anti-paxillin (0.1 μg/ml) antibodies. α1-PDX has been described as a potent inhibitor of convertases (16Anderson E. Thomas L. Hayflick J. Thomas G. J. Biol. Chem. 1993; 268: 24887-24891Abstract Full Text PDF PubMed Google Scholar), which are responsible for the cleavage of α integrin subunits (22Lehmann M. Rigot V. Seidah N.G. Marvaldi J. Lissitzky J.C. Biochem. J. 1996; 317: 803-809Crossref PubMed Scopus (54) Google Scholar, 23Lissitzky J. Luis J. Munzer J. Benjannet S. Parat F. Chrétien M. Marvaldi J. Seidah N. Biochem. J. 2000; 346: 133-138Crossref PubMed Scopus (98) Google Scholar). We therefore expressed the α1-PDX inhibitor in HT29-D4 cells, and we selected stable transfectants. The clone PDX39 was further selected on the basis of its resistance to PEA, a toxin activated by convertases (27Moehring J. Inocencio N. Robertson B. Moehring T. J. Biol. Chem. 1993; 268: 2590-2594Abstract Full Text PDF PubMed Google Scholar), to obtain a population (PDX39P cells) expressing high levels of the α1-PDX inhibitor (data not shown). Among the cleavable integrins, HT29-D4 cells express α3β1, α6β4, αvβ5, and αvβ6(26Lehmann M. Rabenandrasana C. Tamura R. Lissitzky J.-C. Quaranta V. Pichon J. Marvaldi J. Cancer Res. 1994; 54: 2102-2107PubMed Google Scholar, 28Rigot V. Lehmann M. André F. Daemi N. Marvaldi J. Luis J. J. Cell Sci. 1998; 111: 3119-3127Crossref PubMed Google Scholar). The α6 subunit is synthesized as a 140-kDa precursor and then converted to a 120-kDa species by endoproteolytic processing (31Rigot V. André F. Lehmann M. Lissitzky J.-C. Marvaldi J. Luis J. Eur. J. Biochem. 1999; 261: 659-666Crossref PubMed Scopus (20) Google Scholar). To determine whether α1-PDX expression impairs the endoproteolytic processing of α6, cells were surface-biotinylated, and integrins were immunoprecipitated with specific antibodies against α6 subunit. Immunopurified proteins were then analyzed by SDS-PAGE under nonreducing (NR) or reducing (R) conditions (Fig.1). As illustrated in Fig. 1 A, nonreduced α6 immunoprecipitates, composed of α6 chains (140 kDa) associated with β4subunits (190 kDa), displayed a similar pattern in the different cell populations. However, when the disulfide bridges were broken, the electrophoretic profiles of α6 immunoprecipitates were obviously different depending on the cell population. Indeed, in HT29-D4 cells and in cells transfected with the empty vector (PDX0 cells), the apparent molecular mass of α6 was reduced from 140 to 120 kDa upon reduction, while a major band of 150 kDa corresponding to noncleaved subunit (α6NC) was observed in PDX39P cells. Only a limited amount of cleaved α6 subunit (α6C) was seen on the gel for PDX39P. These data, similar to those obtained with the cleavage-deficient LoVo cells (22Lehmann M. Rigot V. Seidah N.G. Marvaldi J. Lissitzky J.C. Biochem. J. 1996; 317: 803-809Crossref PubMed Scopus (54) Google Scholar), indicate that in PDX39P the large majority of the α6 integrin subunit was not endoproteolytically processed. In the case of PDX39 cells, about half of the α6 chains were cleaved, confirming that the selection of PDX39 cells in the presence of a high concentration of PEA increased α1-PDX expression and led to a strong impairment of α6 cleavage. These observations were extended to the αv and α3 subunits using the same experimental approach. As seen on Fig. 1 B, most αv chain, that associated with β5 and β6 subunits, failed to be cleaved in PDX39P cells, as observed with α6 subunit. The amount of normally processed α3 is more difficult to evaluate because cleaved form (α3C) comigrated with reduced β1 (Fig. 1 C). However, the bands corresponding to noncleaved (α3NC) and nonreduced α3 chains from PDX39P have the same intensity, suggesting that virtually all α3 subunits are in an uncleaved form. Thus, the expression of high levels of α1-PDX inhibitor blocked almost totally the endoproteolytic maturation of all the cleavable α integrin subunits expressed in PDX39P cells. Moreover, the ratio cleaved/noncleaved form correlated with the expression level of α1-PDX (data not shown). As observed above after cell surface biotinylation and immunoprecipitation, the expression of α1-PDX does not seem to affect the labeling intensity of integrin subunits. To confirm these results by a more quantitative approach, cells were examined for cell surface expression of integrin subunits by indirect immunofluorescence using specific mAbs. Flow cytometry analysis indicated that the extent of integrin expression at the cell surface is quite similar whatever the cell population studied (Fig.2). This demonstrates that the defect in α chains processing did not impair the exportation of the heterodimers to the plasma membrane. To examine the importance of the α chain cleavage for integrin function, we first measured the adhesion of transfected cells to purified ECM proteins and to poly-l-lysine as an integrin-independent substrate. Adhesion of PDX39P cells to laminin-1 and collagen type I was quite similar to control cells, as was the attachment to poly-l-lysine (Fig.3 A). On the contrary, adhesion of PDX39P cells to vitronectin was significantly reduced when compared with HT29-D4 or PDX0 cells. The integrin αvβ5, the unique receptor for vitronectin in HT29-D4 (26Lehmann M. Rabenandrasana C. Tamura R. Lissitzky J.-C. Quaranta V. Pichon J. Marvaldi J. Cancer Res. 1994; 54: 2102-2107PubMed Google Scholar) and in PDX0 and PDX39P cells (not shown), does not require activation to mediate ligand binding. However, the absence of cleavage of α subunits could lead to the suppression of the constitutive integrin activation. We, therefore, assessed whether PMA or the divalent cation Mn2+ could restore a normal attachment of α1-PDX-expressing cells on vitronectin. As illustrated in Fig. 3 B, stimulation of PDX39P cells by PMA failed to restore cell adhesion to vitronectin. Moreover, treatment of cells by 1 mm (Fig. 3 B) or higher concentrations (not shown) of Mn2+ does stimulate cell adhesion, but in the same proportion for all cell types. These data indicate that uncleaved αvβ5 integrin present on PDX39P cells seems to be constitutively active, but it can be further activated by divalent ions as does cleaved integrin. Integrin-mediated cell adhesion may be altered by changes either in affinity of individual receptors for ligand or in integrin avidity (32Stewart M. Hogg N. J. Cell. Biochem. 1996; 61: 554-561Crossref PubMed Scopus (223) Google Scholar). It has been proposed that integrin interaction with cytoskeletal proteins that serve to anchor and cluster integrins regulates receptor avidity. We therefore used a solid-phase binding assay to test the interaction of the αvβ5 integrins with vitronectin in a cell-free environment. In these conditions, we observed no significant difference of binding between the solubilized αvβ5 integrins from PDX0 and those from PDX39P cells (Fig. 4). The interaction integrin/vitronectin was RGD- and divalent cation-dependent and was enhanced to the same extent in the presence of Mn2+. These results suggest that the reduced adhesion of PDX39P cells to vitronectin was not due to changes in the interaction of the integrins with their ligand. Previous studies on the role of cleavage on integrin function have focused on ligand binding (3Teixidó J. Parker C.M. Kassner P.D. Hemler M.E. J. Biol. Chem. 1992; 267: 1786-1791Abstract Full Text PDF PubMed Google Scholar, 6Kolodziej M. Vilaire G. Gonder D. Poncz M. Bennett J. J. Biol. Chem. 1991; 266: 23499-23504Abstract Full Text PDF PubMed Google Scholar, 7Delwel G.O. Hogervorst F. Sonnenberg A. J. Biol. Chem. 1996; 271: 7293-7296Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). However, cell adhesion to ECM results in the integrin-dependent activation of a number of signaling pathways that can be affected by cleavage. We therefore examined whether α1-PDX-expressing cells were able to elicit intracellular signal transduction events upon αvβ5 integrin ligation. Protein tyrosine phosphorylation is one of the earliest events detected in response to cell attachment to an ECM protein-coated surface. Adhesion of PDX0 cells to vitronectin led to a time-dependent increase in tyrosine phosphorylation of proteins in the molecular mass range of 110–125 kDa and around 70 kDa, whereas no enhanced phosphorylation could be observed with PDX39P cells (data not shown). In most cell types, the FAK accounts for a large proportion of the tyrosine phosphorylation in the 110–125-kDa region. Therefore, PDX0 and PDX39P cells were plated on vitronectin-coated dishes, and the phosphorylation status of FAK was assessed after immunoprecipitation. We observed a time-dependent increase in tyrosine phosphorylation of FAK from PDX0 cells after adhesion on vitronectin (Fig. 5 A). In PDX39P cells in suspension, the FAK phosphorylation level was very low compared with control cells and did not enhance in vitronectin-adherent cells. We repeated the experiment with an antibody against paxillin, a FAK-associated phosphoprotein. As observed with FAK, paxillin from PDX0 cells was tyrosine-phosphorylated in response to integrin ligation, while only a very slight phosphorylation could be detected in PDX39P cells (Fig. 5 B). The absence of tyrosine phosphorylation of FAK in PDX39P cells could be due to a defect in the FAK signaling pathway. However, as shown in Fig. 5 C, FAK could still be tyrosine phosphorylated after stimulation of PDX39P cells by neuropeptides, such as neurotensin, or by treatment with the microtubule-disrupting agent nocodazole. Because MAPK cascade is an important signaling pathway activated by integrins, we analyzed MAPK activation in response to cell adhesion using an antibody raised against the dually phosphorylated region within the catalytic core of the active form of ERK1 and ERK2. As illustrated in Fig. 6 A, adhesion of PDX0 cells to vitronectin resulted in the activation of ERK2, while ERK1 was barely activated. In the case of PDX39P, ERKs are already activated in suspended cells and adhesion to vitronectin resulted in an additional increase in activation of ERK2. On the contrary, activation of ERK1 decreases with time of adhesion. A recent report demonstrated that activation of MAPK might suppress the activation of integrins and thus negatively regulate cell adhesion and motility (33Hughes P. Renshaw M. Pfaff M. Forsyth J. Keivens V. Schwartz M. Ginsberg M. Cell. 1997; 88: 521-530Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar). Thus, it is possible that the reduced attachment of PDX39P cells to vitronectin could be due to the higher activity of MAPK in these cells. We therefore measured cell adhesion when MAPK is activated by epidermal growth factor or, on the contrary, when its activation is impaired by PD98059. As illustrated in Fig.6 B, we observed no difference in cell adhesion (upper panel), whatever the activation status of MAPK (lower panels). Thus, neither impediment of MAPK activation in PDX39P cells, nor activation of MAPK in PDX0 cells had any effect on adhesion to vitronectin (Fig. 6 B). In the present work we have examined the importance of α subunit cleavage for integrin function. We have generated stable transfectants (PDX39P cells) expressing high levels of α1-PDX, a potent and selective inhibitor of convertases and especially of furin and PC5B (14Jean F. Stella K. Thomas L. Liu G. Xiang Y. Reason A. Thomas G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7293-7298Crossref PubMed Scopus (242) Google Scholar). The α1-PDX-expressing cells displayed plasma membrane integrins mainly under an uncleaved form, although a minor cleavage could be observed. This result confirms that the convertase family of serine proteases is involved in the post-translational processing of integrins α chains, as we reported previously (22Lehmann M. Rigot V. Seidah N.G. Marvaldi J. Lissitzky J.C. Biochem. J. 1996; 317: 803-809Crossref PubMed Scopus (54) Google Scholar,23Lissitzky J. Luis J. Munzer J. Benjannet S. Parat F. Chrétien M. Marvaldi J. Seidah N. Biochem. J. 2000; 346: 133-138Crossref PubMed Scopus (98) Google Scholar). Endoproteolytic cleavage is often required to generate active proteins from inactive precursors. Here we report that PDX39P cells, which display uncleaved integrins on cell surface, showed a reduced adhesion to vitronectin through the αvβ5 integrin. However, we found no evidence that cleavage of the α subunits has any major effect on cell adhesion to other ECM proteins, such as laminin-1 and collagen type I. This absence of effect is likely due to the involvement of noncleavable β1 integrins, such as α1β1 or α2β1, which support HT29-D4 cell adhesion to these ECM proteins (26Lehmann M. Rabenandrasana C. Tamura R. Lissitzky J.-C. Quaranta V. Pichon J. Marvaldi J. Cancer Res. 1994; 54: 2102-2107PubMed Google Scholar, 28Rigot V. Lehmann M. André F. Daemi N. Marvaldi J. Luis J. J. Cell Sci. 1998; 111: 3119-3127Crossref PubMed Google Scholar). We neither observed any difference between cells when using the integrin-independent substrate poly-l-lysine. The reduced adhesion of PDX39P cells to vitronectin was not due to changes in αvβ5 expression, as neither transfection nor PEA selection altered the amount of integrin subunits at the cell surface. For several reasons, abolishing α subunit cleavage neither seems to cause important alterations in integrin structure. (i) α/β association still occurred because heterodimers containing the uncleaved α3, α6, or αv subunits were expressed on the cell surface, as also reported for the furin-deficient cell line LoVo (22Lehmann M. Rigot V. Seidah N.G. Marvaldi J. Lissitzky J.C. Biochem. J. 1996; 317: 803-809Crossref PubMed Scopus (54) Google Scholar) and mutated αIIb and α4 subunits (3Teixidó J. Parker C.M. Kassner P.D. Hemler M.E. J. Biol. Chem. 1992; 267: 1786-1791Abstract Full Text PDF PubMed Google Scholar,6Kolodziej M. Vilaire G. Gonder D. Poncz M. Bennett J. J. Biol. Chem. 1991; 266: 23499-23504Abstract Full Text PDF PubMed Google Scholar). (ii) PDX39P cells attached with the same efficiency than parental cells or empty vector transfected cells to two mAbs (69.6.5 and AMF-7) recognizing distinct αv epitopes (not shown). (iii) The function-blocking mAb 69.6.5 inhibited adhesion to vitronectin with similar dose-effect profiles whatever the cell type (not shown). The integrin adhesive function can be regulated by intracellular signals, a phenomenon known as inside-out signaling (reviewed in Refs.34Gumbiner B.M. Cell. 1996; 84: 345-357Abstract Full Text Full Text PDF PubMed Scopus (2945) Google Scholar and 35Brown E. Hogg N. Immunol. Lett. 1996; 54: 189-193Crossref PubMed Scopus (37) Google Scholar). This can be done either by altering the affinity of the individual integrins or by integrin clustering, which allows more efficient interaction and increased binding between cells and ECM. The reduced adhesion of PDX39P cells to vitronectin is most likely not due to changes in integrin affinity, as we noticed no significant difference of interaction between cleaved and uncleaved integrins in a solid-phase receptor assay. This result is in agreement with previous reports showing that the absence of cleavage of the α4and αIIb subunits did not affect ligand binding of the α4β1 and αIIbβ3integrins (3Teixidó J. Parker C.M. Kassner P.D. Hemler M.E. J. Biol. Chem. 1992; 267: 1786-1791Abstract Full Text PDF PubMed Google Scholar, 6Kolodziej M. Vilaire G. Gonder D. Poncz M. Bennett J. J. Biol. Chem. 1991; 266: 23499-23504Abstract Full Text PDF PubMed Google Scholar). However, these studies have no examined the role of integrin cleavage on intracellular signaling pathways. In the present study we show that the phosphorylation level of FAK and its associated phosphoprotein paxillin, in response to ligation of αvβ5 integrin, was very low in α1-PDX-expressing cells. Various signaling proteins have been involved in the inside-out modulation of integrin function. Thus, FAK phosphorylation induced by integrin ligation appears to contribute to stabilization of cell adhesion (36Miyamoto S. Teramoto H. Coso O. Gutkind J. Burbelo P. Akiyama S. Yamada K. J. Cell Biol. 1995; 131: 791-805Crossref PubMed Scopus (1106) Google Scholar). Such a positive feedback loop is also thought to occur in the context of protein kinase C activation during adhesion and spreading (37Chun J.-S. Jacobson B.S. Mol. Biol. Cell. 1993; 4: 271-281Crossref PubMed Scopus (66) Google Scholar). It thus can be hypothesized that the absence of cleavage of the αvβ5 integrin leads to the impairment of signal transduction by FAK (or another molecule) upon cell adhesion, that in turn might result in a reduced efficiency of PDX39P cells attachment to vitronectin. In support of this hypothesis, Delwel et al. (7Delwel G.O. Hogervorst F. Sonnenberg A. J. Biol. Chem. 1996; 271: 7293-7296Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) have shown that the cleavage of the α6A subunit is essential for activation of the integrin by the phorbol ester PMA, a protein kinase C activator. MAPKs (ERK1 and ERK2) are activated in response to a variety of extracellular signals and thus represent a convergence point for many signaling pathways. It is now clear that integrin-mediated cell adhesion can also lead to the activation of the MAPK cascade (for review, see Ref. 1Aplin A. Howe A. Alahari S. Juliano R. Pharmacol. Rev. 1998; 50: 197-263PubMed Google Scholar). In this work we show that ERK2 and, to a lesser extent, ERK1 are activated upon interaction of the αvβ5 integrin with vitronectin. We also reported that ERK2 was already activated in PDX39P cells kept in suspension and that the enzyme was further activated during adhesion. There is increasing evidence that suppression of integrin activation may be a physiological mechanism to control integrin-dependent cell adhesion and migration. The existence of signaling pathways acting as inhibitors of integrin activation has been proposed, and recently Ras/Raf-1-initiated activation of MAPK pathway has been shown to suppress the activation of β1 and β3 integrins (33Hughes P. Renshaw M. Pfaff M. Forsyth J. Keivens V. Schwartz M. Ginsberg M. Cell. 1997; 88: 521-530Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar). Thus, one possibility is that the reduced adhesion of PDX39P cells to vitronectin was the result of the high activity level of MAPK in these cells. However, this is likely not the case, because neither MAPK activation nor impediment of MAPK activation had any effect on adhesion to vitronectin. An alternative is the suppression of the constitutive integrin activation due to the absence of α subunits cleavage. Nevertheless, the integrin αvβ5 was already present as an active form in α1-PDX-expressing cells, as neither PMA nor the divalent cation Mn2+, two integrin activators, could restore a normal attachment of α1-PDX-expressing cells on vitronectin. It thus appears that the absence of endoproteolytic cleavage of the αv subunit affects integrin function by altering αvβ5-dependent signaling pathways. The molecular mechanism responsible of this dysfunction remains to be determined. We are grateful to M. Lehmann for the critical reading of the manuscript; to J.-C. Lissitzky for providing laminin-1; and to F. Parat, F. Giannellini, and P. Bremond for their expert technical assistance.