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Matrix Metalloproteinase-9 Silencing by RNA Interference Triggers the Migratory-adhesive Switch in Ewing's Sarcoma Cells

2003; Elsevier BV; Volume: 278; Issue: 38 Linguagem: Inglês

10.1074/jbc.m304300200

ISSN

1083-351X

Autores

Josiane Sancéau, Sandrine Truchet, Brigitte Bauvois,

Tópico(s)

Cell Adhesion Molecules Research

Resumo

Enhanced expression of (pro)matrix metalloproteinase-9 (MMP-9) is associated with human tumor invasion and/or metastasis. COH cells derived from a highly invasive and metastatic Ewing's sarcoma constitutively express proMMP-9. Transfection of a double stranded RNA that targets the MMP-9 mRNA into COH cells depleted the corresponding mRNA and protein as demonstrated by reverse transcriptase-PCR, enzyme-linked immunosorbent assay, and gelatin zymography. proMMP-9 extinction resulted in the following: (i) decreased spreading on extracellular matrix (fibronectin, laminin, collagen IV)-coated surfaces, (ii) inhibition of migration toward fibronectin, and (iii) induced aggregation, which was specifically disrupted by a function-blocking E-cadherin antibody. MMP-9 knockdown concomitantly resulted in increased levels of surface E-cadherin, redistribution at the plasma membrane of β-catenin, and its physical association with E-cadherin. Moreover, induction of E-cadherin-mediated adhesion was associated with RhoA activation and changes in paxillin cytoskeleton. Finally, an inhibitor of gelatinolytic activity of pro-MMP9 did not reduce COH cell migration confirming that the enzymatic property of COH MMP-9 was not required for migration toward fibronectin. Overall, our observations define a novel critical role for proMMP-9 in providing a cellular switch between stationary and migratory cell phases. Enhanced expression of (pro)matrix metalloproteinase-9 (MMP-9) is associated with human tumor invasion and/or metastasis. COH cells derived from a highly invasive and metastatic Ewing's sarcoma constitutively express proMMP-9. Transfection of a double stranded RNA that targets the MMP-9 mRNA into COH cells depleted the corresponding mRNA and protein as demonstrated by reverse transcriptase-PCR, enzyme-linked immunosorbent assay, and gelatin zymography. proMMP-9 extinction resulted in the following: (i) decreased spreading on extracellular matrix (fibronectin, laminin, collagen IV)-coated surfaces, (ii) inhibition of migration toward fibronectin, and (iii) induced aggregation, which was specifically disrupted by a function-blocking E-cadherin antibody. MMP-9 knockdown concomitantly resulted in increased levels of surface E-cadherin, redistribution at the plasma membrane of β-catenin, and its physical association with E-cadherin. Moreover, induction of E-cadherin-mediated adhesion was associated with RhoA activation and changes in paxillin cytoskeleton. Finally, an inhibitor of gelatinolytic activity of pro-MMP9 did not reduce COH cell migration confirming that the enzymatic property of COH MMP-9 was not required for migration toward fibronectin. Overall, our observations define a novel critical role for proMMP-9 in providing a cellular switch between stationary and migratory cell phases. Invasion and metastasis of tumor cells is a multiple process that depends on uncontrolled interactions between adjacent cells and/or cells and their extracellular environment (1Egeblad M. Werb Z. Nat. Rev. Cancer. 2002; 2: 161-174Crossref PubMed Scopus (5079) Google Scholar, 2Tomanek R.J.S. Chatteman G.C. Anat. Rec. 2000; 261: 126-135Crossref PubMed Scopus (136) Google Scholar). These interactions are mediated directly by specific adhesion receptors and indirectly by extracellular proteinases that mediate degradation of the extracellular matrix (ECM). 1The abbreviations used are: ECM, extracellular matrix; MMP, matrix metalloproteinase; CAM, cellular adhesion molecule; ICAM-1, intercellular adhesion molecule 1; VCAM, vascular cell adhesion molecule; NCAM, neural cell adhesion molecule; ES, Ewing's sarcoma; ds, double stranded; m, mouse; Ab, antibody; mAb, monoclonal antibody; pTyr, phosphotyrosine; FCS, fetal calf serum; TRITC, tetramethylrhodamine isothiocyanate; RT, reverse transcriptase; PBS, phosphate-buffered saline; VEGF, vascular epidermal growth factor; RNAi, RNA-mediated interference; si-RNA, small interfering RNA; FACS, fluorescence-activated cell sorter; Fn, fibronectin; Lm, Laminin; Col, Collagen IV; BSA, bovine serum albumin; snc, negative control si-RNA.1The abbreviations used are: ECM, extracellular matrix; MMP, matrix metalloproteinase; CAM, cellular adhesion molecule; ICAM-1, intercellular adhesion molecule 1; VCAM, vascular cell adhesion molecule; NCAM, neural cell adhesion molecule; ES, Ewing's sarcoma; ds, double stranded; m, mouse; Ab, antibody; mAb, monoclonal antibody; pTyr, phosphotyrosine; FCS, fetal calf serum; TRITC, tetramethylrhodamine isothiocyanate; RT, reverse transcriptase; PBS, phosphate-buffered saline; VEGF, vascular epidermal growth factor; RNAi, RNA-mediated interference; si-RNA, small interfering RNA; FACS, fluorescence-activated cell sorter; Fn, fibronectin; Lm, Laminin; Col, Collagen IV; BSA, bovine serum albumin; snc, negative control si-RNA.Many of the relevant proteinases belong to the matrix metalloproteinases (MMPs), which are a family of related zinc-containing proteinases that have the ability to degrade ECM (3Brinckerhoff C.E. Matrisian L. Nat. Rev. Mol. Cell. Biol. 2002; 3: 207-214Crossref PubMed Scopus (959) Google Scholar). One member of the MMP family, MMP-9 (gelatinase B, 92 kDa) is capable of degrading type I, IV, V, VII, and XI collagens and laminin (1Egeblad M. Werb Z. Nat. Rev. Cancer. 2002; 2: 161-174Crossref PubMed Scopus (5079) Google Scholar, 4Opdenakker G. Van Damme J. Verh. K. Acad. Geneeskd. Belg. 2002; 64: 105-136PubMed Google Scholar). Such proteolytic ability suggests that MMP-9 ultimately regulates cell migration, tumor growth, and angiogenesis (1Egeblad M. Werb Z. Nat. Rev. Cancer. 2002; 2: 161-174Crossref PubMed Scopus (5079) Google Scholar, 2Tomanek R.J.S. Chatteman G.C. Anat. Rec. 2000; 261: 126-135Crossref PubMed Scopus (136) Google Scholar, 4Opdenakker G. Van Damme J. Verh. K. Acad. Geneeskd. Belg. 2002; 64: 105-136PubMed Google Scholar). MMP-9 is overexpressed in many human solid and hematological malignancies (2Tomanek R.J.S. Chatteman G.C. Anat. Rec. 2000; 261: 126-135Crossref PubMed Scopus (136) Google Scholar, 5Janowska-Wieczorek A. Marquez L. Matsuzaki A. Hashmi H.R. Larratt L.M. Boshkov L.M. Turner A.R. Zhang M.C. Edwards D.R. Kossakowska A.E. Br. J. Haematol. 1999; 105: 402-411Crossref PubMed Scopus (97) Google Scholar, 6Kossakowska A. Urbanski S. Janowska-Wieczorek A. Leuk. Lymphoma. 2000; 39: 485-493Crossref PubMed Scopus (32) Google Scholar, 7Bauvois B. Dumont J. Mathiot C. Kolb J.P. Leukemia. 2002; 16: 791-798Crossref PubMed Scopus (87) Google Scholar). MMP-9 promoter activity is induced coincidentally with invasion during tumor progression (8Kupferman M.E. Fini M. Muller W.J. Weber R. Cheng Y. Muschel R.J. Am. J. Pathol. 2000; 157: 1777-1783Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 9Lynch C. Matrisian L. Differentiation. 2002; 70: 561-573Crossref PubMed Scopus (311) Google Scholar). Further, in vitro overexpression of MMP-9 confers a metastatic phenotype (10Bernhard E.J. Muschel R. 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Cell Biol. 2002; 158: 1133-1144Crossref PubMed Scopus (73) Google Scholar). Different families of adhesion receptors are likely to play a role in directing cell motility and include integrins, CAMs, and cadherins (16Alahari S.K. Redding P. Juliano R.L. Int. Rev. Cytol. 2002; 220: 145-184Crossref PubMed Scopus (64) Google Scholar, 17Juliano R. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 283-323Crossref PubMed Scopus (493) Google Scholar). Cadherins function by connecting cells to each other by homophilic interactions, in which they bind selectively to identical cadherin types. A cytoplasmic protein termed β-catenin interacts directly with the cadherin cytoplasmic domain and indirectly with the cytoskeleton via α-catenin, which interacts with actin and α-actinin (18Wong N. Pignalli M. Am. J. Pathol. 2002; 160: 389-401Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). Signal transduction pathways from cadherin to RhoA, Rac1, and Cdc42 have been identified recently (19Fukata M. Kaibuchi K. Nat. Rev. Mol. Cell. Biol. 2001; 2: 887-897Crossref PubMed Scopus (352) Google Scholar, 20Braga V. Curr. Opin. Cell Biol. 2002; 14: 546-556Crossref PubMed Scopus (275) Google Scholar).Compiled studies indicate a clear relationship between loss of cadherin expression and increased invasiveness in tumor cells (21Kaihara T. Kusala T. Kawamata H. Oda Y. Fujii S. Morita K. Imura J. Fujimori T. Pathobiology. 2001; 69: 172-178Crossref PubMed Scopus (18) Google Scholar, 22Conacci-Sorrell M. Zhurinsky J. Ben-Ze'ev A. J. Clin. Invest. 2002; 109: 987-991Crossref PubMed Scopus (517) Google Scholar, 23Vizirianakis I. Chen Y. Kantak S. Tsiftsoglou A. Kramer R. Int. J. Oncol. 2002; 21: 135-144PubMed Google Scholar). Moreover, various carcinoma are shown to exhibit an inverse relationship between MMP-9 and E-cadherin expression (24Anzai H. Kidatai Y. Bucana C.D. Sanchez R. Omoto R. Fidler I.J. Differentiation. 1996; 60: 119-127Crossref PubMed Google Scholar, 25Llorens A Rodrigo I. Lopez-Barcons L. Gonzalez-Garrigues M. Lozano E. Vinyals A. Quintanilla M. Cano A. Fabra A. Lab. Invest. 1998; 78: 1131-1142PubMed Google Scholar, 26Kuniyasu H. Ellis L.M. Evans D. Abbruzzese J. Fenoglio C. Bucana C. Cleary K. Tahara E. Fidler I.J. Clin. Cancer Res. 1999; 5: 25-33PubMed Google Scholar, 27Sato F. Shimada Y. Watanabe G. Uchida S. Makino T. Imamura M. Br. J. Cancer. 1999; 80: 1366-1372Crossref PubMed Scopus (73) Google Scholar, 28Kuniyasu H. Troncoso P. Johnston D. Bucana C. Tahara E. Fidler I.J. Pettaway C.A. Clin Cancer Res. 2000; 6: 2295-2308PubMed Google Scholar, 29Roy S. Herbst R. Yano S. Kuniyasu H. Khuri F. Bucana C. Guo F. Liu D. Kemp B. Lee J. Hong W. Fidler I.J. Clin. Cancer Res. 2000; 6: 790-797PubMed Google Scholar, 30Slaton J.W. Karashima T. Perrotte P. Inoue K. Kim S.J. Izawa J. Kedar D. McConkey D.J. Millikan R. Sweeney P. Yoshikawa C. Shuin T. Dinney C.P. Clin. Cancer Res. 2001; 7: 2840-2853PubMed Google Scholar, 31Inoue K. Kamada M. Slaton J.W. Fukata S. Yoshikawa C. Tamboli P. Dinney C.P. Shuin T. Clin. Cancer Res. 2002; 8: 1863-1870PubMed Google Scholar). However, the molecular links between these divergent profiles of expression for MMP-9 and E-cadherin remain unknown.Ewing's sarcoma (ES) is a malignant childhood bone and soft tissue tumor known to be highly aggressive and invasive (32Paulussen M. Ahrens S. Lehnert M. Taeger D. Hense H. Wagner A. Dunst J. Harms D. Reiter A. Henze G. Niemeyer C. Gobel U. Kremens B. Folsch U. Aulitzky W. Voute P. Zoubek A. Jurgens H. Ann. Oncol. 2001; 12: 1619-1630Abstract Full Text PDF PubMed Scopus (71) Google Scholar). ES is characterized by a specific recurrent balanced chromosomal translocation t(11;22) (q24;q12) (33Delattre O. Zucman J. Plougastel B. Desmaze C. Melot T. Peter M. Kovar H. Joubert I. de Jong P. Rouleau G. Nature. 1992; 359: 162-165Crossref PubMed Scopus (1506) Google Scholar). Previous studies from our laboratory (34Sanceau J. Hiscott J. Delattre O. Wietzerbin J. Oncogene. 2000; 19: 3372-3383Crossref PubMed Scopus (106) Google Scholar, 35Bouman L. Sanceau J. Rouillard D. Bauvois B. Biochem. J. 2002; 364: 719-724Crossref PubMed Google Scholar, 36Sanceau J Boyd D.D. Seiki M. Bauvois B. J. Biol. Chem. 2002; 277: 35766-35775Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) have demonstrated the inhibitory action of interferons on ES cell growth and MMP-9 expression. In the present investigation, we sought to determine whether MMP-9 extinction could affect ES cell behavior. Because recent reports (37Brantl S. Biochim. Biophys. Acta. 2002; 1575: 15-25Crossref PubMed Scopus (190) Google Scholar, 38McManus M. Sharp P.A. Nat. Rev. Genet. 2002; 3: 737-747Crossref PubMed Scopus (1207) Google Scholar, 39Paddison P. Caudy A. Hannon G.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1443-1448Crossref PubMed Scopus (496) Google Scholar) have demonstrated the utility of gene silencing by si-RNA in mammalian cells, we used a ds-RNA to interfere with the expression of MMP-9 gene. Our findings support the conclusion that MMP-9 constitutes a trigger for the switch between adhesive and migratory states of ES cells through β-catenin/RhoA/paxillin signaling pathways in a manner independent of its enzyme activity.MATERIALS AND METHODSReagents—Goat F(ab′)2 fragment anti-mouse and anti-rat fluoresce-in-conjugated Ig, irrelevant rat isotype IgG2a, mouse (m) IgG1, IgG2a, mIgG2b, and monoclonal antibodies (mAbs) specific for β1 (4B4, mIgG1), α1 (HP-2B6, mIgG1), α2 (Gi9, mIgG1), α4 (HP2/1, mIgG1), α5 (SAM1, mIgG2a), α6 (GoH3, rIgG2a), αv (AMF7, mIgG1), ICAM-1 (84H10, mIgG1), E-cadherin (67A4, mIgG1), and CD44 (LAZ221ALL, mIgG1) were obtained from Coulter Immunotech (Coultronics, France). The mAb specific for α3 (P1B5, mIgG1) was from Dako Corporation (Carpinteria, CA). mAbs used here were chosen for their ability to block cell adhesion to extracellular matrix (40Bauvois B. Rouillard D. Sanceau J. Wietzerbin J. J. Immunol. 1992; 148: 3912-3919PubMed Google Scholar). Human plasma fibronectin, laminin purified from Englebreth-Holm Swarm mouse tumor, bovine serum albumin, and mAbs against α-actinin (EA-53, mIgG1), talin (8D4, mIgG1), vinculin (hVIN-1, mIgG1), paxillin (PXC-10, mIgG1), phosphoserine (PSR45, mIgG1), phospho-PYK2 (pTyr579–580, rabbit polyclonal), and phospho-FAK (pTyr397, rabbit polyclonal) were from Sigma. E-cadherin (clone 34, mIgG2b), β-catenin (clone 14, mIgG1), PYK2 (clone 11, mIgG1), and FAK (clone 77, mIgG1) Abs were from BD Biosciences. E-cadherin (H-108, rabbit polyclonal) Ab was from Santa Cruz Biotechnology Inc. (Tebu, France). ILK (65.1.9, mIgG2b), β-catenin (clone 2H4A7, mIgG1), and integrin β1 (clone DE9, mIgG1) antibodies were from Upstate Biotechnology. Phosphopaxillin (pTyr118, rabbit polyclonal) Ab was from Cell Signaling Technology. Anti-actin (clone C4, mIgG1) was from ICN Biomedicals, Inc. Rho activation assay was from Cytoskeleton Inc. (Denver, CO). Fluorescein-conjugated affinity pure goat anti-mouse IgG and TRITC-conjugated affinity pure goat anti-rabbit IgG were from Jackson ImmunoResearch. Human recombinant MMP-9 and MMP-9 (4H3, mIgG1), E-cadherin (HECD-1, mIgG1), VCAM-1 (BBIG-V1, mIgG1), and NCAM (ERIC-1, mIgG1) Abs were from R & D Systems. Protein G-Sepharose™4 fast flow was from Amersham Biosciences. Human collagen type IV was from BD Biosciences. Renaissance Enhanced Luminol Reagent Plus was from PerkinElmer Life Sciences. M-PER™-mammalian protein extraction reagent and NE-PER™ nuclear and cytoplasmic extraction reagents were from Pierce. MMP-9 inhibitor 2(R)-2-[(4-biphenylsulfonyl)amino]-3-phenylpropionic acid was from Calbiochem.Cells—Human Ewing's sarcoma COH cells, wild-type p53 cells from a metastatic tumor localized on femur (41Hamelin R. Zucman J. Melot T. Delattre O. Thomas G. Int. J. Cancer. 1994; 57: 336-340Crossref PubMed Scopus (46) Google Scholar), were maintained in RPMI 1640 (Invitrogen) containing 10% heat-inactivated FCS (Myoclone Plus; Invitrogen) and 10 μg/ml gentamycin in a humidified 37 °C incubator (5% CO2). COH cells were tested free of mycoplasma as assessed by RT-PCR (VenorGeM; Biovalley S. A.). In preliminary experiments, we controlled that trypsin treatment did not alter the levels of COH cell surface adhesion molecules (integrins, CAMs, and E-cadherin) as assessed by flow cytometry. Thus, cells were rapidly trypsinized and washed twice in PBS before adhesion and migration assays.Gelatinolytic Zymography—Analysis of MMP-9 activity was carried out in 7.5% (w/v) SDS-polyacrylamide gels containing 0.1% gelatin (w/v) as described elsewhere (7Bauvois B. Dumont J. Mathiot C. Kolb J.P. Leukemia. 2002; 16: 791-798Crossref PubMed Scopus (87) Google Scholar). Samples were preincubated for 60 min with 0.5 mm amino-phenyl mercuric acid (Sigma), which activates the proform to the activated form.Enzyme-linked Immunoadsorbent Assays—The culture supernatants from COH cells were harvested under sterile conditions and frozen before MMP-9 and VEGF contents were determined using commercial enzyme-linked immunosorbent assay kits provided by R & D Systems. Controls included FCS-supplemented RPMI 1640 medium alone incubated under the same conditions.RNAi Experiments—The si-RNA sequence targeting human MMP-9 chosen in this study (from mRNA sequence; GenBank™ accession number NM-004994) corresponds to the coding region 377–403 relative to the first nucleotide of the start codon (target = 5′-AAC ATC ACC TAT TGG ATC CAA ACT AC-3′). Computer analysis using the software developed by Ambion Inc. confirmed this sequence to be a good target. si-RNAs were 21 nucleotides long with symmetric 2-nucleotide 3′ overhangs composed of 2′-deoxythymidine to enhance nuclease resistance. The si-RNAs were synthesized chemically and high pressure liquid chromatography purified (Genset, Paris, France). Sense si-RNA sequence was 5′-CAUCACCUAUUGGAUCCAAdTdT-3′. Antisense si-RNA was 5′-UUGGAUCCAAUAGGUGAUGdTdT-3′. For annealing of si-RNAs, mixture of complementary single stranded RNAs (at equimolar concentration) was incubated in annealing buffer (20 mm Tris-HCl pH 7.5, 50 mm NaCl, and 10 mm MgCl2) for 2 min at 95 °C followed by a slow cooling to room temperature (at least 25 °C) and then proceeded to storage temperature of 4 °C. Before transfection, cells cultured at 50% confluence in 6-well plates (10 cm2) were washed two times with OPTIMEM 1 (Invitrogen) without FCS and incubated in 1.5 ml of this medium without FCS for 1 h. Then, cells were transfected with MMP-9-RNA duplex formulated into Mirus TransIT-TKO transfection reagent (Mirus Corp., Interchim, France) according to the manufacturer's instructions. Unless otherwise described, transfection used 20 nm RNA duplex in 0.5 ml of transfection medium OPTIMEM 1 without FCS per 5 × 105 cells for 6 h and then the medium volume was adjusted to 1.5 ml per well with RPMI 2% FCS. Silencer™ negative control 1 si-RNA (Ambion Inc.) was used as negative control under similar conditions (20 nm).RT-PCR—RNA extraction from Ewing cells and subsequent cDNA synthesis were conducted as described previously (34Sanceau J. Hiscott J. Delattre O. Wietzerbin J. Oncogene. 2000; 19: 3372-3383Crossref PubMed Scopus (106) Google Scholar). MMP-9 cDNA (296 bp) was amplified using the sense primer 5′-GGA GAC CTG AGA ACC AAT CTC-3′ and the antisense primer 5′-TCC AAT AGG TGA TGT TGT CGT-3′ according to published sequences (42Trocme C. Gaudin P. Berthier S. Barro C. Zaoui P. Morel F. J. Biol. Chem. 1998; 273: 20677-20684Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). β1-Integrin cDNA was amplified using the sense primer 5′-GTG AAT GGG AAC AAC GAG GTC-3′ and the antisense 5′-ACA ATA AAA CGA TAA ACG GAC-3′ according to published sequences (43De Arcangelis A. Lefebvre O. Mechine-Neuville A. Arnold C. Klein A. Remy L. Kedinger M. Simon-Assmann P. Int. J. Cancer. 2001; 94: 44-53Crossref PubMed Scopus (40) Google Scholar). VEGF cDNA was amplified using the sense primer 5′-ACA TCT TCC AGG AG ACC CTG ATG AG-3′ and the antisense 5′-GCA TTC ACA TTT GTT GTG CTG T-3′ according to published sequences (44Westphal J.R. Van't Hullenaar R. Peek R. Willems R. Crickard K. Crickard U. Askaa J. Clemmensen I. Ruiter D. De Waal R. Int. J. Cancer. 2000; 86: 768-776Crossref PubMed Scopus (106) Google Scholar). 18 S ribosomal RNA was used as an internal control (QuantumRNA™ 18 S Internal Standard; Ambion Inc). The PCR products were visualized by electrophoresis in 1.6% agarose gel containing 0.2 μg/ml ethidium bromide. The NIH Image 1.44 β11 software was used for the analysis.Flow Cytometry—Intact cells were immunostained as described previously (40Bauvois B. Rouillard D. Sanceau J. Wietzerbin J. J. Immunol. 1992; 148: 3912-3919PubMed Google Scholar). Clones used were β1 (4B4), α1 (HP-2B6), α2 (Gi9), α4 (HP2/1), α5 (SAM1), α6 (GoH3), ICAM-1 (84H10), VCAM-1 (BBIG-V1), NCAM (ERIC-1), CD44 (LAZ221ALL), α3 (P1B5), and E-cadherin (67A4). Analysis was performed in a FACS flow cytometer analyzer (BD Biosciences). Values are given as percentages of positive cells and relative intensity of fluorescence, which is an indication of the level of expression.Adhesion Assays—Twenty-four-well flat bottomed microtiter plates (Nunc) were coated overnight with fibronectin (Fn) or Laminin (Lm) or Collagen IV (Col) and blocked with BSA as described previously (40Bauvois B. Rouillard D. Sanceau J. Wietzerbin J. J. Immunol. 1992; 148: 3912-3919PubMed Google Scholar). Cells (4 × 105 cells per well in 1 ml of RPMI without FCS) were allowed to adhere to each substrate-coated well at 37 °C for 60 min. After PBS washing, adherent cells were trypsinized and quantitated using a cell Coulter Counter channelizer 256 (the diameters of living migrated cells ranging from 7 to 14 μm). Results from triplicates (mean ± S.D.) were expressed as relative cell adhesion (number of attached cells/total number of cells × 100). Specific adhesion to substrates was determined by subtracting the nonspecific attachment of cells to BSA-coated surfaces from cell attachment to coated surfaces. Morphology of attached cells was assessed by staining with the Hemacolor kit from Merck and subsequent light microscope examination.Migration Assays—The migration of COH cells was determined using the method described previously (45Ashida N. Arai H. Yamasaki M. Kita T. J. Biol. Chem. 2001; 276: 16555-16560Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). After adjusting the cell density to 1 × 106 cells/ml in 0.1% BSA-RPMI, 100,000 cells in 100 μl were added to the top chamber of a 24-transwell apparatus (6.5-mm diameter, 8-μm pore size; Costar 3422, Corning Inc., Corning, NY) in the absence or presence of fibronectin (25 μg) in the lower chamber. After overnight incubation at 37 °C in an atmosphere containing 5% CO2, living cells (with diameters ranging from 7 to 14 μm) that passed through the membrane were collected from the lower well and counted in a cell Coulter Counter channelizer 256. Results from triplicate wells were expressed as mean ± S.D.Protein Analysis—Following cell (1 × 107 cells/ml) transfection for the indicated times, cells were washed twice with cold PBS. Total cellular extracts, nuclear and cytoplasmic extracts were prepared as described previously (36Sanceau J Boyd D.D. Seiki M. Bauvois B. J. Biol. Chem. 2002; 277: 35766-35775Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Equivalent amounts of protein were separated on an 8 or 10% ProSieve® 50 gel (BioWhittaker Molecular Applications), transferred to nitrocellulose (Schleicher & Schuell), and blotted as described previously (34Sanceau J. Hiscott J. Delattre O. Wietzerbin J. Oncogene. 2000; 19: 3372-3383Crossref PubMed Scopus (106) Google Scholar). For co-immunoprecipitation experiments, cells were lysed in 400 μl of lysis buffer containing 0.5% Brij35 (Sigma). Cell lysates were immunoprecipitated with mAbs prebound to protein G-Sepharose at 4 °C overnight. Immune complexes were washed four times and then resolved on 8% ProSieve® gels as described previously (40Bauvois B. Rouillard D. Sanceau J. Wietzerbin J. J. Immunol. 1992; 148: 3912-3919PubMed Google Scholar). For E-cadherin immunocomplexes, the β-catenin was first revealed followed by the total E-cadherin hybridization, and conversely for β-catenin complexes, E-cadherin was first revealed before total β-catenin hybridization.Fractionation Studies—Following cell transfection for the indicated times, cells were washed twice with cold PBS before resuspension in HEM buffer (2 mm MgCl2, 10 mm Hepes, pH 7.5, 2 mm EDTA, 0.2 mm EGTA) (46Gilmore A.P. Metcalfe A. Romer L.H. Streuli C.H. J. Cell Biol. 2000; 149: 431-446Crossref PubMed Scopus (253) Google Scholar) containing protease inhibitor mixture and phosphatase inhibitor mixture II. Following ice incubation for 10 min, cells were lysed in a Dounce homogenizer. After centrifugation at 4 °C for 5 min at 500 × g to discard nucleus and subcellular organelles, the homogenates were centrifuged at 4 °C for 30 min at 45,000 × g. The cytosolic pool was collected, and the pellet (membrane-enriched fraction) was rapidly washed in HEM buffer and solubilized in Laemmli sample buffer (100 μl). Protein concentrations were determined using DC™ protein assay (Bio-Rad). Proteins from cytosolic and membrane-enriched pools were separated on an 8% ProSieve® 50 gel and immunoblotted as described above.Immunofluorescence Staining and Confocal Microscopy—Cells were washed twice with cold PBS and once with PBS containing 1% BSA before low speed cytocentrifugation to polylysine-treated slides (O. Kindler GmBH & Co., Freiburg, Germany). Cells were fixed in ice-cold methanol for 7 min and then washed twice with PBS. After 60 min of blocking in PBS containing 3% BSA at room temperature, cells were incubated in the same buffer with a polyclonal E-cadherin Ab (H108; 5 μg/ml), a monoclonal β-catenin Ab (clone 2A4H7; 1 μg/ml), or an isotype mIgG1 (1 μg/ml) for overnight at 4 °C. The preparations were washed three times for 15 min with PBS/0.05% Tween 20 and one wash with PBS, followed by an incubation for 90 min at room temperature with a fluorescein isothiocyanate-conjugated anti-mouse Ab or a TRITC-conjugated anti-rabbit Abboth diluted 1/1000 in PBS/BSA. Preparations were then washed three times with PBS/0.05% Tween 20 and once in PBS. Slides were mounted in Vectashield (47Truchet S. Wietzerbin J. Debey P. Mol. Reprod. Dev. 2001; 60: 319-330Crossref PubMed Scopus (26) Google Scholar). Labeled samples were further analyzed by confocal microscopy on an Nikon microscope equipped with the Bio-Rad Laser-Sharp MRC-1024 confocal laser scanning software, using a Nikon Fluor ×100 oil-immersion objective and the 488- and 568-nm excitation wavelengths of the laser (47Truchet S. Wietzerbin J. Debey P. Mol. Reprod. Dev. 2001; 60: 319-330Crossref PubMed Scopus (26) Google Scholar). The fluorescence of β-catenin and E-cadherin was analyzed concurrently in the same cell samples.RESULTSEfficient Extinction of MMP-9 Expression in COH Cells by RNAi Strategy—We used an RNAi method to target MMP-9 in the ES COH cell line, which constitutively expresses high levels of MMP-9. The constructs we designed encoded an RNA that targets the MMP-9 mRNA (Fig. 1A). The 21-nucleotide-long target sequence had no homology with other members of the MMP family. The ds-RNA and Silencer™ negative control si-RNA (snc) were each tested for their ability to suppress MMP-9 specifically. We first assessed whether RNAi was dose- and time-dependent. COH cells were transfected with 1–20 nm of the ds-RNA for up 3 days. By RT-PCR analysis, a MMP-9-dependent ds-RNA-mediated inhibition was observed in a dose- and time-dependent manner (Fig. 1B). The snc-RNA (20 nm) was incapable of inhibiting MMP-9 gene expression (Fig. 1B) even when transfected with a 10-fold-excess of the saturating ds-RNA concentration (200 nm) (data not shown). The time-course assay performed with 20 nm ds-RNA-transfected COH cells showed that induced MMP-9 silencing could be maintained for at least 3 days (corresponding to seven generation times) (Fig. 1C). Importantly, snc-RNA and ds-RNA transfection had no effect on the mRNA levels of two unrelated genes i.e. VEGF and integrin β1 (Fig. 1B).The RT-PCR results were confirmed by enzyme-linked immunoadsorbent assay. COH snc-RNA-transfected cells cultured up to 3 days spontaneously released high amounts of MMP-9 into the culture conditioned medium whereas ds-RNA-transfected cells showed a marked time- and dose-dependent inhibition in MMP-9 protein levels (Fig. 1D, left panel). In accordance with RT-PCR data, levels of released VEGF by COH cells were not affected by ds-RNA transfection (Fig. 1D, right panel). Zymography analysis of the conditioned media from COH cells before and after snc-RNA treatment indicated the presence of a gelatinase activity at 92 kDa (Fig. 1E, compare lanes 4 and 6) consistent with the pattern of recombinant proMMP-9 (Fig. 1E, lane 1). In contrast, MMP-9 was barely evident in conditioned medium from ds-RNA cells (Fig. 1E, compare lane 8 with lanes 4 and 6) corroborating the undetectable MMP-9 transcript levels (Fig. 1C). Preincubation of COH supernatants with amino-phenyl mercuric acid, which activates the proform to the activated form, resulted in conversion of proMMP-9 to an active form of 82 kDa size (Fig. 1E, lanes 2, 5, and 7). Together, these findings indicate that RNAi efficiently and specifically inhibits endogenous MMP-9 gene expression in COH cells.MMP-9 Silencing Induces E-cadherin-mediated Cell-Cell Adhesion—RNAi led neither to cell necrosis (data not shown) nor to cell apoptosis as measured by means of fluorescence flow cytometry (Fig. 2A). Indeed, Apo 2.7 antigen, a mitochondrial membrane protein exposed on the surface of cells undergoing programmed cell death (48Zhang C. Ao Z. Seth A. Schlossman S. J. Immunol. 1996; 157: 3980-3987PubMed Google Scholar), was expressed on less than 10% of snc-RNA cells. The levels of Apo 2.7 on ds-RNA cells remained close to that of snc-RNA c

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