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• W1975178229 abstract "Integrin-ligand binding regulates tumor cell motility and invasion. Cell migration also involves the Rho GTPases that control the interplay between adhesion receptors and the cytoskeleton. We evaluated how specific extracellular matrix ligands modulate Rho GTPases and control motility of human squamous cell carcinoma cells. On laminin-5 substrates, the epithelial cells rapidly spread and migrated, but on type I collagen the cells spread slowly and showed reduced motility. We found that RhoA activity was suppressed in cells attached to laminin-5 through the α3 integrin receptor. In contrast, RhoA was strongly activated in cells bound to type I collagen and this was mediated by the α2 integrin. Inhibiting the RhoA pathway by expression of a dominant-negative RhoA mutant or by directly inhibiting ROCK, reduced focal adhesion formation and enhanced cell migration on type I collagen. Cdc42 and Rac and their downstream target PAK1 were activated following adhesion to laminin-5. PAK1 activation induced by laminin-5 was suppressed by expression of a dominant-negative Cdc42. Moreover, constitutively active PAK1 stimulated migration on collagen I substrates. Our results indicate that in squamous epithelial cells, collagen-α2β1 integrin binding activates RhoA, slowing cell locomotion, whereas laminin-5-α3β1 integrin interaction inhibits RhoA and activates PAK1, stimulating cell migration. The data demonstrate that specific ligand-integrin pairs regulate cell motility differentially by selectively modulating activities of Rho GTPases and their effectors. Integrin-ligand binding regulates tumor cell motility and invasion. Cell migration also involves the Rho GTPases that control the interplay between adhesion receptors and the cytoskeleton. We evaluated how specific extracellular matrix ligands modulate Rho GTPases and control motility of human squamous cell carcinoma cells. On laminin-5 substrates, the epithelial cells rapidly spread and migrated, but on type I collagen the cells spread slowly and showed reduced motility. We found that RhoA activity was suppressed in cells attached to laminin-5 through the α3 integrin receptor. In contrast, RhoA was strongly activated in cells bound to type I collagen and this was mediated by the α2 integrin. Inhibiting the RhoA pathway by expression of a dominant-negative RhoA mutant or by directly inhibiting ROCK, reduced focal adhesion formation and enhanced cell migration on type I collagen. Cdc42 and Rac and their downstream target PAK1 were activated following adhesion to laminin-5. PAK1 activation induced by laminin-5 was suppressed by expression of a dominant-negative Cdc42. Moreover, constitutively active PAK1 stimulated migration on collagen I substrates. Our results indicate that in squamous epithelial cells, collagen-α2β1 integrin binding activates RhoA, slowing cell locomotion, whereas laminin-5-α3β1 integrin interaction inhibits RhoA and activates PAK1, stimulating cell migration. The data demonstrate that specific ligand-integrin pairs regulate cell motility differentially by selectively modulating activities of Rho GTPases and their effectors. Cell migration is essential for a number of biological and pathological processes, including normal development, angiogenesis, wound repair, and tumor invasion and metastasis. The process of cell spreading and migration on extracellular matrix (ECM) 1The abbreviations used are: ECM, extracellular matrix; DMEM, Dulbecco's modified Eagle's medium; GAPs, GTPase activating proteins; GFP, green fluorescent protein; GST, glutathione S-transferase; MBP, myelin basic protein; PAK, p21-activated kinase; PLL, poly-l-lysine; ROCK, Rho-associated coiled-coil kinase; SCC, squamous cell carcinoma; WT, wild type; mAb, monoclonal antibody.1The abbreviations used are: ECM, extracellular matrix; DMEM, Dulbecco's modified Eagle's medium; GAPs, GTPase activating proteins; GFP, green fluorescent protein; GST, glutathione S-transferase; MBP, myelin basic protein; PAK, p21-activated kinase; PLL, poly-l-lysine; ROCK, Rho-associated coiled-coil kinase; SCC, squamous cell carcinoma; WT, wild type; mAb, monoclonal antibody. involves integrin receptors and dynamic changes in the cytoskeleton. Migration represents a multi-step process including formation of adhesive protrusions at the leading edge, release of adhesions, and cell rear retraction (1Lauffenburger D.A. Horwitz A.F. Cell. 1996; 84: 359-369Abstract Full Text Full Text PDF PubMed Scopus (3254) Google Scholar). The complex interplay between integrins and the cytoskeleton is regulated by specific signaling pathways that are not completely understood.The Rho family GTPases, particularly Rho, Rac, and Cdc42, modulate many aspects of cytoskeletal function that occur during migration (2Clark E.A. King W.G. Brugge J.S. Symons M. Hynes R.O. J. Cell Biol. 1998; 142: 573-586Crossref PubMed Scopus (528) Google Scholar, 3Nobes C.D. Hall A. J. Cell Biol. 1999; 144: 1235-1244Crossref PubMed Scopus (1204) Google Scholar, 4Ridley A.J. Schwartz M.A. Burridge K. Firtel R.A. Ginsberg M.H. Borisy G. Parsons J.T. Horwitz A.R. Science. 2003; 302: 1704-1709Crossref PubMed Scopus (3773) Google Scholar, 5Horwitz R. Webb D. Curr. Biol. 2003; 13: R756-R759Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 6Raftopoulou M. Hall A. Dev. Biol. 2004; 265: 23-32Crossref PubMed Scopus (1132) Google Scholar). Rac1 seems to be essential in most cells for the protrusion of lamellipodia at the leading edge and for forward cell movement. In contrast, RhoA is required to maintain substrate adhesion during cell movement and to produce contractile force in the forwarding migrating cell. The main function of Cdc42 is to maintain cell polarity and initiate the formation of filopodium.The role of individual Rho GTPases in migration may depend on cell type. The contribution of RhoA in motility has been established in specific cell types such as colon carcinoma cells, hepatoma cells, and lymphoma cells (7Stam J.C. Michiels F. van der Kammen R.A. Moolenaar W.H. Collard J.G. EMBO J. 1998; 17: 4066-4074Crossref PubMed Scopus (202) Google Scholar, 8O'Connor K.L. Nguyen B.K. Mercurio A.M. J. Cell Biol. 2000; 148: 253-258Crossref PubMed Scopus (180) Google Scholar, 9Itoh K. Yoshioka K. Akedo H. Uehata M. Ishizaki T. Narumiya S. Nat. Med. 1999; 5: 221-225Crossref PubMed Scopus (559) Google Scholar). However, many studies on the role of Rho proteins in migration have used fibroblasts (10Schmitz A.A. Govek E.E. Bottner B. Van Aelst L. Exp. Cell Res. 2000; 261: 1-12Crossref PubMed Scopus (506) Google Scholar, 11Etienne-Manneville S. Hall A. Nature. 2002; 420: 629-635Crossref PubMed Scopus (3795) Google Scholar). Other studies show that increased Rac activity promotes migration and invasion of lymphocytes (7Stam J.C. Michiels F. van der Kammen R.A. Moolenaar W.H. Collard J.G. EMBO J. 1998; 17: 4066-4074Crossref PubMed Scopus (202) Google Scholar, 12Michiels F. Habets G.G. Stam J.C. van der Kammen R.A. Collard J.G. Nature. 1995; 375: 338-340Crossref PubMed Scopus (506) Google Scholar). For lung adenocarcinoma cells (13Gu J. Sumida Y. Sanzen N. Sekiguchi K. J. Biol. Chem. 2001; 276: 27090-27097Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar), Rac1 can inhibit the motility of epithelial cells because of an increase in the formation of cadherin junctional adhesions (14Hordijk P.L. ten Klooster J.P. van der Kammen R.A. Michiels F. Oomen L.C. Collard J.G. Science. 1997; 278: 1464-1466Crossref PubMed Scopus (392) Google Scholar). High RhoA activity inhibits movement in fibroblasts and lung adenocarcinoma cells (13Gu J. Sumida Y. Sanzen N. Sekiguchi K. J. Biol. Chem. 2001; 276: 27090-27097Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 15Arthur W.T. Burridge K. Mol. Biol. Cell. 2001; 12: 2711-2720Crossref PubMed Scopus (380) Google Scholar). Thus, the balance among RhoA, Rac, and Cdc42 activities will determine whether a given cell remains stationary or is migratory.It is well known that ECM proteins can trigger cell spreading and motility through integrin-dependent regulation of Rho family members. The type of ECM also appears to have dramatic effects on the migratory response of the cell. For example, in Madin-Darby canine kidney epithelial cells, Rac activation enhanced migration on collagen but suppressed migration on laminin-1 or fibronectin substrates (16Sander E.E. van Delft S. ten Klooster J.P. Reid T. van der Kammen R.A. Michiels F. Collard J.G. J. Cell Biol. 1998; 143: 1385-1398Crossref PubMed Scopus (585) Google Scholar). During the initial phase of spreading on fibronectin, RhoA activity is reduced through activation of p190 RhoGAP as a result of Src and FAK signaling in fibroblasts (15Arthur W.T. Burridge K. Mol. Biol. Cell. 2001; 12: 2711-2720Crossref PubMed Scopus (380) Google Scholar, 17Ren X.D. Kiosses W.B. Schwartz M.A. EMBO J. 1999; 18: 578-585Crossref PubMed Scopus (1355) Google Scholar, 18Ren X.D. Kiosses W.B. Sieg D.J. Otey C.A. Schlaepfer D.D. Schwartz M.A. J. Cell Sci. 2000; 113: 3673-3678Crossref PubMed Google Scholar); subsequently, RhoA activity increases markedly (17Ren X.D. Kiosses W.B. Schwartz M.A. EMBO J. 1999; 18: 578-585Crossref PubMed Scopus (1355) Google Scholar). Rac1 and Cdc42 activities are known to be high during spreading and membrane protrusion (19Price L.S. Leng J. Schwartz M.A. Bokoch G.M. Mol. Biol. Cell. 1998; 9: 1863-1871Crossref PubMed Scopus (524) Google Scholar, 20Cox E.A. Sastry S.K. Huttenlocher A. Mol. Biol. Cell. 2001; 12: 265-277Crossref PubMed Scopus (247) Google Scholar). Other ECM proteins, such as laminin-10/11 (13Gu J. Sumida Y. Sanzen N. Sekiguchi K. J. Biol. Chem. 2001; 276: 27090-27097Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar) and laminin-8 (21Fujiwara H. Gu J. Sekiguchi K. Exp. Cell Res. 2004; 292: 67-77Crossref PubMed Scopus (57) Google Scholar), activate Rac1 to promote cell migration. However, α4β1 integrin interaction with fibronectin down-regulated RhoA activity and induced melanoma cell migration (22Moyano J.V. Maqueda A. Casanova B. Garcia-Pardo A. Mol. Biol. Cell. 2003; 14: 3699-3715Crossref PubMed Google Scholar). Another important issue concerns differences between the behavior of cells studied on two-dimensional surfaces versus three-dimensional matrices (23DeMali K.A. Wennerberg K. Burridge K. Curr. Opin. Cell Biol. 2003; 15: 572-582Crossref PubMed Scopus (430) Google Scholar).Previously, we showed that for squamous cell carcinoma (SCC) cells, laminin-5 ligand promotes rapid cell scattering, whereas fibronectin and collagen I do not (24Kawano K. Kantak S.S. Murai M. Yao C.C. Kramer R.H. Exp. Cell Res. 2001; 262: 180-196Crossref PubMed Scopus (57) Google Scholar). In the present study, we analyzed the integrin-mediated regulation of Rho GTPases and their downstream effectors resulting from adhesion to laminin-5 and type I collagen substrates in SCC cells. On laminin-5 substrate, α3β1 integrin preferentially inactivated RhoA and induced activation of Cdc42 and PAK1, thereby promoting migration of oral SCC cells. In contrast, on type I collagen, α2β1 integrin strongly activated RhoA, leading to enhanced focal contact formation, thereby hindering cell migration. These results suggest that Rho signaling in SCC may be important in defining cell phenotype.EXPERIMENTAL PROCEDURESReagents and Antibodies—Y-27632 and C3 transferase were purchased from Calbiochem; myelin basic protein (MBP) and poly-l-lysine (PLL) were from Sigma; type I collagen was from Cohesion Technologies (Palo Alto, CA). MAbs against Rac1, Cdc42, and paxillin (clone 165) were purchased from BD Transduction Laboratories (Lexington, KY). Anti-RhoA mAb and anti-PAK (N-20) polyclonal antibody were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-hemagglutinin mAb was purchased from Covance (Richmond, CA). Anti-laminin-5 blocking antibody (P3H9–2) and anti-vinculin mAb were obtained from Chemicon International, Inc. (Temecula, CA); anti-phospho-PAK1 (Thr423) polyclonal antibody and anti-Myc mAb were purchased from Cell Signaling Technology Inc. (Beverly, MA); VC5 (mouse anti-α5 integrin) and GoH3 (rat anti-α6 integrin) were purchased from BD Pharmingen (San Diego, CA). J143 (anti-human α3 integrin) was obtained from ATCC. VM1 and VM2 mAb (anti-α2 and α3 subunits, respectively) were described previously (25Zhang K. Kramer R.H. Exp. Cell Res. 1996; 227: 309-322Crossref PubMed Scopus (152) Google Scholar). AIIB2 (rat anti-human β1 integrin) was provided by Caroline Damsky (University of California, San Francisco). Anti-mouse and anti-rabbit secondary antibodies, conjugated to horseradish peroxidase for immunoblotting, or conjugated to fluorescein isothiocyanate for confocal and immunofluorescence microscopy, were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). Rhodamine-conjugated phalloidin was obtained from Molecular Probes (Eugene, OR).Cell Culture—Cells from the human SCC cell lines HSC-3 and UM-SCC-10A were maintained as described previously (24Kawano K. Kantak S.S. Murai M. Yao C.C. Kramer R.H. Exp. Cell Res. 2001; 262: 180-196Crossref PubMed Scopus (57) Google Scholar) in DMEM (Mediatech, Inc., Herndon, VA) supplemented with 10% fetal bovine serum (Gemini Bio-Products, Woodland, CA) and cultured at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. Cells used for adhesion and motility assays were serum-starved overnight.Purified laminin-5 was used as described previously for adhesion and motility assays (24Kawano K. Kantak S.S. Murai M. Yao C.C. Kramer R.H. Exp. Cell Res. 2001; 262: 180-196Crossref PubMed Scopus (57) Google Scholar, 25Zhang K. Kramer R.H. Exp. Cell Res. 1996; 227: 309-322Crossref PubMed Scopus (152) Google Scholar). Laminin-5 matrix substrate was derived from human SCC cells as described previously (24Kawano K. Kantak S.S. Murai M. Yao C.C. Kramer R.H. Exp. Cell Res. 2001; 262: 180-196Crossref PubMed Scopus (57) Google Scholar). In brief, cells were grown to confluence on tissue culture plates for 48 h. After washing with phosphate-buffered saline, cells were removed by treatment with 20 mm NH4OH for 5 min according to the method described previously (26Gospodarowicz D.J. Prog. Clin. Biol. Res. 1984; 145: 103-128PubMed Google Scholar, 27Langhofer M. Hopkinson S.B. Jones J.C. J. Cell Sci. 1993; 105: 753-764Crossref PubMed Google Scholar). The matrix was then extensively washed with phosphate-buffered saline before use.Adenoviral Infection—Adenovirus encoding constitutively active RhoA (V14RhoA) or dominant-negative RhoA (N19RhoA) was kindly provided by A. Hassid (University of Tennessee, Memphis, TN). Adenovirus encoding GFP or dominant-negative Cdc42 (N17Cdc42) was a gift of G. E. Davis (Texas A&M University System, College Station, TX). Adenovirus encoding wild-type (WT) PAK1 or constitutively active PAK1 (E423 PAK1) was from W. T. Gerthoffer (University of Nevada, Reno, NV). HSC-3 cells were infected with adenovirus at a multiplicity of infection of 500 in 1 ml of culture medium in 6-well plates or in 4 ml of culture medium in 10-cm plates. After 2 h incubation, 2 or 6 ml of DMEM was added to the 6-well or 10-cm plates, respectively. After 24 h in culture, cells were processed for experiments as described below.Inhibitor Treatments—For treatment with the Rho inhibitor C3 transferase, cells were preincubated with 5 μg/ml C3 transferase in serum-free medium overnight. For treatment with the Rho-associated coiled-coil kinase (ROCK) inhibitor Y-27632, cells were preincubated with 25 μm Y-27632 in DMEM for 30 min before and included during the experiments.Cell Spreading Measurements—Suspended cells were seeded onto plates previously coated with collagen I or laminin-5 substrate for the indicated times at 37 °C in serum-free medium. After fixation with 4% paraformaldehyde, cells were stained with 2% Coomassie Brilliant Blue (Sigma) in 45% methanol and 10% acetic acid for 10 min (15Arthur W.T. Burridge K. Mol. Biol. Cell. 2001; 12: 2711-2720Crossref PubMed Scopus (380) Google Scholar). The relative areas in pixels of more than 20 individual cells were generated using Metamorph and NIH Image software. The average of the relative areas of cells plated on laminin-5 substrate for 30 min was chosen as the maximal cell area. The ratio of the cell area at the indicated time point to the maximal cell area was then determined.Immunofluorescent Staining—Cells were seeded onto chamber slides (Nalge Nunc International, Naperville, IL) coated with collagen I or laminin-5 and incubated at 37 °C for 1 h. After fixation with 4% paraformaldehyde for 10 min and permeabilization with 0.5% Nonidet P-40 in phosphate-buffered saline for 5 min, cells were incubated with primary antibodies (anti-paxillin mAb or anti-vinculin mAb) for 1 h, followed by incubation with goat anti-mouse fluorescein isothiocyanate-conjugated secondary antibodies. Rhodamine-conjugated phalloidin was used to co-stain polymerized actin filaments. Slides were mounted with Vectashield (Vector, Burlingame, CA) and viewed using a Nikon fluorescence microscope or a Bio-Rad Laboratories laser scanning confocal microscope (model MRC-1024).Immunoblotting—Cells were extracted with lysis buffer (50 mm Tris, pH 7.2, 500 mm NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 10 mm MgCl2, and complete protease inhibitor mixture (Roche Molecular Biochemicals) and processed for SDS-PAGE after adjusting for equal protein loading, estimated by using the BCA protein assay kit (Pierce). After transfer to nitrocellulose membranes (Millipore Corp., Bedford, MA), proteins were probed with primary antibodies and secondary horseradish peroxidase-coupled antibody. Blots were developed by chemiluminescence using the ECL system (Amersham Biosciences). The band intensities were measured by densitometry using NIH Image software (Scion Corp., Frederick, MD).Adhesion Assay—Cell adhesion was measured as described previously (28Yao C.C. Ziober B.L. Squillace R.M. Kramer R.H. J. Biol. Chem. 1996; 271: 25598-25603Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Briefly, 96-well plates were coated with type I collagen (10 μg/ml) and laminin-5 (2.5 μg/ml) at 37 °C for 1 h, followed by blocking with 0.1% bovine serum albumin. HSC-3 cells were incubated with or without blocking mAbs to integrin subunits for 30 min at 4 °C, and 2 × 104 cells were added to each well and allowed to attach for 20 min at 37 °C. Adherent cells were then quantified by a microcolorimetric assay (28Yao C.C. Ziober B.L. Squillace R.M. Kramer R.H. J. Biol. Chem. 1996; 271: 25598-25603Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar).Migration Assay—Time-lapse video microscopy was performed as described with a modification (29Denker S.P. Barber D.L. J. Cell Biol. 2002; 159: 1087-1096Crossref PubMed Scopus (342) Google Scholar). Briefly, cells were seeded onto 6-well plates (Falcon, Becton Dickinson Labware) coated with different substrates (collagen I (10 μg/ml), laminin-5 (5 μg/ml), or PLL (10 μg/ml)) for 30 min. Plates were then examined in a Zeiss Axiovert inverted microscope with an X-Y scanning motorized stage (Carl Zeiss MicroImaging, Inc., Thornwood, NY) and maintained at 37 °C and 5% CO2. Images were collected at the indicated time intervals using a SPOT-RT CCD camera (Molecular Dynamics) and analyzed with the Openlab software system (Improvision Inc., Lexington, MA). The positions of individual nuclei were tracked to determine the relative migration rates.In the transwell migration assay, the undersides of the transwell (8-μm pore size; Corning Costar Corp., Cambridge, MA) were precoated with collagen (10 μg/ml) and laminin-5 (0.5 or 1.25 μg/ml). Next, 2 × 105 cells were loaded onto the upper chamber of the transwell, and the lower chamber was filled with serum-free medium. Cells were incubated for 3 h at 37 °C, fixed with 4% paraformaldehyde, and stained with crystal violet. Non-migrating cells retained on the upper side were removed by wiping with a cotton swab. Cells that had migrated through the filter were counted and averaged from 10 randomly chosen microscopic fields using a ×20 objective. Migration was taken as 100% for cells infected with control virus.Invasion Assay—Polymerized type I collagen gels were prepared by overlaying 30 μl of DMEM containing 2.4 mg/ml type I collagen to the upper chamber of each transwell and allowing gelation at 37 °C for 1 h. Next, HSC-3 cells (2 × 105) in 200 μl of serum-free DMEM were added on top of the collagen gel. Serum-free medium was then added to the lower chamber and incubated for 24 h at 37 °C. Cells were fixed and stained with crystal violet. Collagen gel and associated cells were removed with a cotton swab. Cells that had penetrated the collagen I gel and reached the underside of the filter membrane were then counted in 10 randomly chosen microscopic fields using a ×20 objective. For each experimental condition, three invasion chambers were used. The mean ± S.E. were determined. Data were expressed as the percentage of treated migratory cells compared with that of control cells.RhoA, Rac1, and Cdc42 Activity Assays—The construct expressing the Rho binding domain of ROCK fused to glutathione S-transferase (GST) was provided by M. A. Woodrow (University of California, San Francisco). The construct expressing PAK 75–132 fused to GST was a gift from P. N. Lowe (Medicines Research Center, GlaxoSmithKline, United Kingdom). Proteins were expressed in Escherichia coli BL21 and purified as described for pull-down assays (17Ren X.D. Kiosses W.B. Schwartz M.A. EMBO J. 1999; 18: 578-585Crossref PubMed Scopus (1355) Google Scholar).Prior to the assay, cells were cultured under serum-free conditions overnight. Cells were plated on different substrates for the indicated times and were lysed in 500 μl of 50 mm Tris, pH 7.5, 150 mm NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 10 mm MgCl2,1mm sodium vanadate, 1 mm NaF, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, and 2.5 μg/ml leupeptin. The lysates were then clarified by centrifugation. For estimating RhoA activity, 40 μg of cell lysates was used to measure total RhoA, and 1.5 mg of cell lysates was mixed with 30 μg of GST-ROCK and 90 μl of glutathione-agarose beads (Sigma) for 60 min at 4 °C. For estimating activity of Rac1 or Cdc42, 40 μg of cell lysates was used to measure total Rac1 or Cdc42, and 1.5 mg of cell lysates was mixed with 30 μg of GST-PAK and 90 μl of glutathione-agarose beads for 60 min at 4 °C. Beads were washed, and bound protein was eluted by boiling in Laemmli buffer. Samples were separated on 12% SDS-polyacrylamide gels, transferred to nitrocellulose, and then immunoblotted with anti-RhoA mAb, anti-Rac1 mAb, or anti-Cdc42 mAb.Protein Kinase Assay—Serum-starved HSC-3 cells or WT PAK1-transduced cells were plated on collagen I or laminin-5 substrate for 30 min. Cell lysates were collected using lysis buffer (10 mm HEPES, pH 7.5, 150 mm NaCl, 1 mm EDTA, 0.6% Triton X-100, 20 mm glycerophosphate, 10% glycerol, 5 mm sodium fluoride, 1 mm sodium vanadate, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin and aprotinin) as described by Royal et al. (30Royal I. Lamarche-Vane N. Lamorte L. Kaibuchi K. Park M. Mol. Biol. Cell. 2000; 11: 1709-1725Crossref PubMed Scopus (242) Google Scholar). Cell lysate (800 μg) was immunoprecipitated with anti-PAK1 (N-20) antibody for 1 h, followed by incubation with 40 μl of protein G-Sepharose (40% suspension) for a second 1-h incubation at 4 °C. The immune complex was washed twice with the lysis buffer and once with the kinase buffer (20 mm HEPES, pH 7.5, 20 mm MgCl2, 1 mm EDTA, 20 mm glycerophosphate, 1 mm dithiothreitol, 5 mm sodium fluoride, 0.1 mm sodium vanadate, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin and aprotinin). The immunoprecipitated PAK1 activity was assayed using MBP as a substrate. The kinase reaction was performed for 30 min at 30 °C in 50 μl of kinase buffer containing 5 μg of MBP, 100 μm cold ATP, and 6 μCi of [γ-32P]ATP. The reaction was terminated by adding Laemmli sample buffer, followed by boiling. Proteins were separated by electrophoresis on a 12% SDS-polyacrylamide gel. Following autoradiography, band intensities were measured by densitometry. PAK1 expression levels were evaluated by Western blotting with the anti-PAK1 (N-20) antibody.RESULTSEnhanced Cell Spreading and Motility on Laminin-5 Substrates—Previous studies using cell aggregates had revealed that, compared with collagen, fibronectin, and laminin-1, laminin-5 had the most potential to disrupt cell-cell adhesions and promote cell scattering (24Kawano K. Kantak S.S. Murai M. Yao C.C. Kramer R.H. Exp. Cell Res. 2001; 262: 180-196Crossref PubMed Scopus (57) Google Scholar). We analyzed the effects of type I collagen and laminin-5 on single cell spreading and migration. Cells were able to attach but spread slowly on type I collagen over the 30-min time period (Fig. 1A). On laminin-5 substrates, cells spread rapidly and were nearly fully spread by 20 min after seeding, whereas cells on collagen I showed only a partial spreading during this time period. In addition, cells displayed an array of lamellopodia and microspikes on the laminin-5 substrate but on type I collagen cells showed only small lamellopodia (Fig. 1B). The time course of adhesion of cells to type I collagen and laminin-5 was indistinguishable (data not shown).Next, the role of specific integrin receptors involved in mediating adhesion was determined using anti-integrin blocking antibodies. HSC-3 cells have significant levels of α2, α3, and α6 integrins and low levels of α5 and αv integrins (24Kawano K. Kantak S.S. Murai M. Yao C.C. Kramer R.H. Exp. Cell Res. 2001; 262: 180-196Crossref PubMed Scopus (57) Google Scholar, 31Matsumoto K. Nakamura T. Kramer R.H. J. Biol. Chem. 1994; 269: 31807-31813Abstract Full Text PDF PubMed Google Scholar). Treatment of cells with anti-α2 or anti-β1 integrin antibody effectively blocked cell adhesion to type I collagen, whereas antibodies to α3 or α5 integrin was without effect. On laminin-5, treatment of cells with anti-α3 or anti-β1 integrin antibody inhibited cell adhesion (Fig. 1C). Interestingly, anti-α6 antibody had little inhibitory activity, suggesting that α6β1 and α6β4 integrins are not the dominant adhesion receptors for laminin-5 in these cells.Formation of Focal Adhesions on Type I Collagen and Laminin-5—To establish the role of substrate adhesions in migration, we seeded cells onto the different ECM ligands and then evaluated focal adhesion formation by immunofluorescent staining. On type I collagen, we found that cells formed a high density of large, mature focal adhesions strongly stained by paxillin or vinculin antibodies (Fig. 1D). However, on laminin-5 substrate, paxillin- or vinculin-positive focal adhesions were limited in number and poorly stained. When cells were costained with rhodamine-phalloidin, an extensive array of polymerized actin and stress fibers was visible on collagen I but was minimal on laminin-5 (data not shown).The ability of type I collagen and laminin-5 to promote cell migration was assessed using time-lapse video microscopy. Analysis of cell tracts revealed that although HSC-3 cells attached to type I collagen and the poly-l-lysine control, they migrated poorly on these substrates (Fig. 2A). In contrast, cells plated on laminin-5 showed a mostly random course of migration but moved at a rate severalfold greater than cells plated on type I collagen (Fig. 2, A and B). Another SCC cell line, UM-SCC-10A, responded similarly as HSC-3 cells to the two substrates (Fig. 2C). To evaluate the role of integrin receptors in HSC-3 cell migration, we performed motility assays in the presence of inhibitory antibodies. Blocking antibodies against integrin α3 or β1 most effectively inhibited cell movement on laminin-5, whereas blocking antibodies against integrin α2 and α6 had negligible effect on motility (Fig. 2D). Although α6β1 and α6β4 can act as receptors for laminin-5 (32Nguyen B.P. Ryan M.C. Gil S.G. Carter W.G. Curr. Opin. Cell Biol. 2000; 12: 554-562Crossref PubMed Scopus (224) Google Scholar) and are both expressed on HSC-3 cells (31Matsumoto K. Nakamura T. Kramer R.H. J. Biol. Chem. 1994; 269: 31807-31813Abstract Full Text PDF PubMed Google Scholar), these integrins do not appear to be crucial for cell adhesion or migration. This indicates that α3β1 integrin is the primary receptor for adhesion to, and migration on, laminin-5. Importantly, these cells were mostly immobile on type I collagen but showed high rates of locomotion on laminin-5.Fig. 2Cell motility on different ECM substrates.A, HSC-3 cells were plated on type I collagen (Col I), laminin-5 (Ln-5) substrate, and PLL. Time-lapse images on different substrates were taken at 20-min intervals for 8 h. Cells were cultured in DMEM supplemented with 10% serum. Individual cell tracks are shown. B, the rate of cell migration on different substrates was estimated as detailed under “Experimental Procedures.” C, HSC-3 cells and SCC-10A cells were plated on type I collagen and laminin-5 substrates in serum-free medium. Time-lapse images were taken at 20-min intervals for 3 h. Cell migration rates of eight tracked cells in each condition are shown. D, the effect of anti-integrin antibodies on cell migration on laminin-5 substrate. Cells were incubated without mAb (Control) or with control antibody (IgG) or with function perturbing mAbs to α2 (VM1), α3 (J143), α6 (GoH3), or β1 (AIIB2) and then plated on laminin-5 substrate, and migration was followed by time-lapse microscopy over a 3-h period. The average migration speed was determined. Data represent the mean ± S.E. of 10 tracked cells. E, cell morphology on collagen I and laminin 5 followed by time-lapse phase microscopy. On collagen I substrate (a), cells display little locomoti" @default.
• W1975178229 created "2016-06-24" @default.
• W1975178229 creator A5027631631 @default.
• W1975178229 creator A5037789032 @default.
• W1975178229 date "2005-03-01" @default.
• W1975178229 modified "2023-10-17" @default.
• W1975178229 title "Integrin Engagement Differentially Modulates Epithelial Cell Motility by RhoA/ROCK and PAK1" @default.
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