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Matrix metalloproteinase-9 silencing by RNA interference triggers the migratory-adhesive switch in
Ewing’s sarcoma cells
Josiane Sancéau, Sandrine Truchet, Brigitte Beauvois
To cite this version:
Josiane Sancéau, Sandrine Truchet, Brigitte Beauvois. Matrix metalloproteinase-9 silencing by RNA
interference triggers the migratory-adhesive switch in Ewing’s sarcoma cells: MMP-9 silencing alters
Ewing’s sarcoma motility. Journal of Biological Chemistry, American Society for Biochemistry and
Molecular Biology, 2003, 278 (38), pp.36537-36546. �10.1074/jbc.M304300200�. �hal-02678906�
Matrix Metalloproteinase-9 Silencing by RNA Interference Triggers the Migratory-adhesive Switch in Ewing’s Sarcoma Cells*
Received for publication, April 24, 2003, and in revised form, June 12, 2003 Published, JBC Papers in Press, July 7, 2003, DOI 10.1074/jbc.M304300200
Josiane Sance´au‡, Sandrine Truchet§, and Brigitte Bauvois‡¶
From the‡Unite´ 365 INSERM, Institut Curie, 75248 Paris cedex 05, France and§USM503 MNHN, UMR 8646 CNRS-MNHN, U565 INSERM, Muse´um National d’Histoire Naturelle, 75005 Paris, France
Enhanced expression of (pro)matrix metalloprotein- ase-9 (MMP-9) is associated with human tumor invasion and/or metastasis. COH cells derived from a highly in- vasive 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 de- monstrated by reverse transcriptase-PCR, enzyme- linked immunosorbent assay, and gelatin zymogra- phy. proMMP-9 extinction resulted in the following: (i) decreased spreading on extracellular matrix (fibronec- tin, laminin, collagen IV)-coated surfaces, (ii) inhibition of migration toward fibronectin, and (iii) induced aggre- gation, which was specifically disrupted by a function- blocking E-cadherin antibody. MMP-9 knockdown con- comitantly resulted in increased levels of surface E-cadherin, redistribution at the plasma membrane of
-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 gelatino- lytic activity of pro-MMP9 did not reduce COH cell mi- gration confirming that the enzymatic property of COH MMP-9 was not required for migration toward fibronec- tin. 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 (1, 2).
These interactions are mediated directly by specific adhesion receptors and indirectly by extracellular proteinases that me- diate degradation of the extracellular matrix (ECM).1
Many of the relevant proteinases belong to the matrix met- alloproteinases (MMPs), which are a family of related zinc-
containing proteinases that have the ability to degrade ECM (3). 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 (1, 4). Such proteolytic ability suggests that MMP-9 ultimately regulates cell migration, tumor growth, and angiogenesis (1, 2, 4). MMP-9 is overexpressed in many human solid and hematological malignancies (2, 5–7). MMP-9 pro- moter activity is induced coincidentally with invasion during tumor progression (8, 9). Further,in vitro overexpression of MMP-9 confers a metastatic phenotype (10 –12). Conversely, selective suppression of MMP-9 by antisense gene transfer impairs in vitro cell migration of glioma cells (13, 14) and osteoclast-like cells (15). Different families of adhesion recep- tors are likely to play a role in directing cell motility and include integrins, CAMs, and cadherins (16, 17). Cadherins function by connecting cells to each other by homophilic inter- actions, in which they bind selectively to identical cadherin types. A cytoplasmic protein termed -catenin interacts di- rectly with the cadherin cytoplasmic domain and indirectly with the cytoskeleton via␣-catenin, which interacts with actin and ␣-actinin (18). Signal transduction pathways from cad- herin to RhoA, Rac1, and Cdc42 have been identified recently (19, 20).
Compiled studies indicate a clear relationship between loss of cadherin expression and increased invasiveness in tumor cells (21–23). Moreover, various carcinoma are shown to exhibit an inverse relationship between MMP-9 and E-cadherin ex- pression (24 –31). 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 (32).
ES is characterized by a specific recurrent balanced chromo- somal translocation t(11;22) (q24;q12) (33). Previous studies from our laboratory (34 –36) 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 (37–39) 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
-catenin/RhoA/paxillin signaling pathways in a manner inde- pendent of its enzyme activity.
MATERIALS AND METHODS
Reagents—Goat F(ab⬘)2fragment anti-mouse and anti-rat fluoresce- in-conjugated Ig, irrelevant rat isotype IgG2a, mouse (m) IgG1, IgG2a, mIgG2b, and monoclonal antibodies (mAbs) specific for 1 (4B4, mIgG1),␣1(HP-2B6, mIgG1),␣2(Gi9, mIgG1),␣4(HP2/1, mIgG1),␣5
(SAM1, mIgG2a), ␣6 (GoH3, rIgG2a), ␣v (AMF7, mIgG1), ICAM-1 (84H10, mIgG1), E-cadherin (67A4, mIgG1), and CD44 (LAZ221ALL,
* This work was supported by grants from INSERM. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶To whom correspondence should be addressed: Unite´ 365 INSERM, Institut Curie, 26, rue d’Ulm, 75248 Paris cedex 05, France. Tel.:
33-1-42-34-67-20; Fax: 33-1-44-07-07-85; E-mail: bbauvois@curie.fr.
1The abbreviations used are: ECM, extracellular matrix; MMP, ma- trix metalloproteinase; CAM, cellular adhesion molecule; ICAM-1, in- tercellular adhesion molecule 1; VCAM, vascular cell adhesion mole- cule; 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, tetramethylrho- damine isothiocyanate; RT, reverse transcriptase; PBS, phosphate-buff- ered 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.
THEJOURNAL OFBIOLOGICALCHEMISTRY Vol. 278, No. 38, Issue of September 19, pp. 36537–36546, 2003
© 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
This paper is available on line at http://www.jbc.org
36537
mIgG1) were obtained from Coulter Immunotech (Coultronics, France).
The mAb specific for␣3(P1B5, mIgG1) was from Dako Corporation (Carpinteria, CA). mAbs used here were chosen for their ability to block cell adhesion to extracellular matrix (40). Human plasma fibronectin, laminin purified from Englebreth-Holm Swarm mouse tumor, bovine serum albumin, and mAbs against ␣-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),-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),-cate- nin (clone 2H4A7, mIgG1), and integrin1(clone DE9, mIgG1) anti- bodies were from Upstate Biotechnology. Phosphopaxillin (pTyr118, rab- bit 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 recombi- nant 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-SepharoseTM4 fast flow was from Amersham Biosciences. Human collagen type IV was from BD Bio- sciences. Renaissance Enhanced Luminol Reagent Plus was from PerkinElmer Life Sciences. M-PERTM-mammalian protein extraction reagent and NE-PERTMnuclear 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 (41), were maintained in RPMI 1640 (Invitrogen) containing 10% heat-inactivated FCS (Myoclone Plus;
Invitrogen) and 10g/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 as- sessed 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 (7). Samples were preincubated for 60 min with 0.5 mMamino-phenyl mercuric acid (Sigma), which activates the pro- form 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 incu- bated under the same conditions.
RNAi Experiments—The si-RNA sequence targeting human MMP-9 chosen in this study (from mRNA sequence; GenBankTM 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⬘over- hangs 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 se- quence was 5⬘-CAUCACCUAUUGGAUCCAAdTdT-3⬘. Antisense si- RNA was 5⬘-UUGGAUCCAAUAGGUGAUGdTdT-3⬘. For annealing of si-RNAs, mixture of complementary single stranded RNAs (at equimo- lar concentration) was incubated in annealing buffer (20 mMTris-HCl pH 7.5, 50 mMNaCl, and 10 mMMgCl2) 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 MirusTransIT-TKO transfection rea- gent (Mirus Corp., Interchim, France) according to the manufacturer’s instructions. Unless otherwise described, transfection used 20 nMRNA duplex in 0.5 ml of transfection medium OPTIMEM 1 without FCS per 5⫻105cells for 6 h and then the medium volume was adjusted to 1.5 ml per well with RPMI 2% FCS. SilencerTMnegative 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 (34). 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 (42).1-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 (43). VEGF cDNA was am- plified 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 (44). 18 S ribosomal RNA was used as an internal control (QuantumRNATM18 S Internal Standard;
Ambion Inc). The PCR products were visualized by electrophoresis in 1.6% agarose gel containing 0.2 g/ml ethidium bromide. The NIH Image 1.4411 software was used for the analysis.
Flow Cytometry—Intact cells were immunostained as described pre- viously (40). Clones used were 1 (4B4), ␣1 (HP-2B6), ␣2 (Gi9), ␣4
(HP2/1),␣5(SAM1),␣6(GoH3), ICAM-1 (84H10), VCAM-1 (BBIG-V1), NCAM (ERIC-1), CD44 (LAZ221ALL), ␣3 (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 (40).
Cells (4⫻105cells 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 14m). Results from triplicates (mean⫾S.D.) were expressed as relative cell adhesion (number of attached cells/total num- ber 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 us- ing the method described previously (45). After adjusting the cell den- sity to 1⫻106cells/ml in 0.1% BSA-RPMI, 100,000 cells in 100l were added to the top chamber of a 24-transwell apparatus (6.5-mm diame- ter, 8-m pore size; Costar 3422, Corning Inc., Corning, NY) in the absence or presence of fibronectin (25g) 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 m) 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⫻107cells/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 (36). Equivalent amounts of protein were sepa- rated on an 8 or 10% ProSieve® 50 gel (BioWhittaker Molecular Ap- plications), transferred to nitrocellulose (Schleicher & Schuell), and blotted as described previously (34). For co-immunoprecipitation exper- iments, cells were lysed in 400l 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 (40). For E-cadherin immunocomplexes, the-catenin was first revealed followed by the total E-cadherin hybridization, and con- versely for-catenin complexes, E-cadherin was first revealed before total-catenin hybridization.
Fractionation Studies—Following cell transfection for the indicated times, cells were washed twice with cold PBS before resuspension in HEM buffer (2 mMMgCl2, 10 mMHepes, pH 7.5, 2 mMEDTA, 0.2 mM
EGTA) (46) 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⫻gto 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
l). Protein concentrations were determined using DCTMprotein 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. Kin- dler 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
g/ml), a monoclonal-catenin Ab (clone 2A4H7; 1g/ml), or an isotype mIgG1 (1g/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-con- jugated 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 (47). Labeled samples were further analyzed by confocal microscopy on an Nikon microscope equipped with the Bio-Rad Laser-Sharp MRC-1024 confocal laser scan- ning software, using a Nikon Fluor⫻100 oil-immersion objective and the 488- and 568-nm excitation wavelengths of the laser (47). The fluorescence of-catenin and E-cadherin was analyzed concurrently in the same cell samples.
RESULTS
Efficient 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 lev- els 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 SilencerTMnegative 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 nMds-RNA-transfected COH cells showed that induced MMP-9 silencing could be main- tained for at least 3 days (corresponding to seven generation times) (Fig. 1C). Importantly, snc-RNA and ds-RNA transfec- tion had no effect on the mRNA levels of two unrelated genes i.e.VEGF and integrin1 (Fig. 1B).
The RT-PCR results were confirmed by enzyme-linked im- munoadsorbent assay. COH snc-RNA-transfected cells cul- tured 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,com- pare lanes 4and6) 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, comparelane 8withlanes 4and6) corroborating the undetect- FIG. 1.Efficient inhibition of MMP-9
in COH cells using RNAi.A, the si-RNA sequence used for targeted silencing of MMP-9. The sense and antisense strand si-RNA oligonucleotides chosen are shown.B, the cDNAs from snc-RNA (20 nM) and ds-RNA (1–20 nM) cells cultured for up 3 days were used as templates for PCR reactions using specific primers for MMP-9, VEGF (A), integrin1, or 18 S ribosomal RNA as described under “Ma- terials and Methods.”C, the cDNAs from snc-RNA (20 nM) and ds-RNA (20 nM) cells cultured for up 4 days were used as templates for PCR reactions using spe- cific primers for MMP-9 or 18 S ribosomal RNA as described under “Materials and Methods.”D, MMP-9 and VEGF produc- tion (ng/ml) in the culture supernatants of COH snc-RNA (20 nM) or ds-RNA (1–20 nM) cells were determined by enzyme- linked immunosorbent assays.E, analysis of gelatinolytic activity in the culture me- dia of COH parental, snc-RNA (20 nM), and ds-RNA (20 nM) cells cultured for 2 days. The enzymatic activity of MMP-9 was analyzed using zymography per- formed with equal amounts of protein loaded. Gelatinolytic activities are de- tected asclear bands in the gel. Activa- tion of pro-MMP-9 by amino-phenyl mer- curic acid (0.5 mM) results in an 82-kDa active form.
MMP-9 Silencing Alters Ewing’s Sarcoma Motility 36539
able MMP-9 transcript levels (Fig. 1C). Preincubation of COH supernatants with amino-phenyl mercuric acid, which acti- vates 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 effi- ciently and specifically inhibits endogenous MMP-9 gene ex- pression in COH cells.
MMP-9 Silencing Induces E-cadherin-mediated Cell-Cell Ad- hesion—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 (48), 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 cells (Fig. 2A). Although their prolif- eration rates were almost comparable (Fig. 2B), the morpholo- gies of cultured COH ds-RNA cells appeared strikingly differ- ent from parental and snc-RNA cells (Fig. 2C). Parental COH cells cultured as adherent monolayers showed a fibroblastic- like phenotype (Fig. 2C,a) as was seen with snc-RNA cells (Fig.
2C,b). However, upon ds-RNA treatment for 48 h, typical cell aggregates were formed (Fig. 2C,c) in a dose-dependent man- ner (Fig. 2C,d-f). Kinetic studies indicated that aggregation was already observed within the first 24 h and was optimal at days 2–3 of ds-RNA treatment (data not shown).
Studies have linked cell-cell adhesion to CAMs and cad- herins (17, 22). As seen in Table I, parental COH cells ex- pressed low to intermediate levels of ICAM-1, VCAM, E-cad-
herin, and NCAM on the cell surface as assessed by flow cytometry. The levels of ICAM-1, VCAM, and NCAM remained unchanged upon snc-RNA and ds-RNA treatments for up 3 days (see Table I for day 2; data not shown for day 3). In contrast, after 2 to 3 days of transfection, aggregated ds-RNA cells consistently expressed E-cadherin more strongly (see Ta- ble I and Fig. 3A) than parental and snc-RNA cells, suggesting the importance of this adhesion molecule for cell-cell adhesion.
Indeed, aggregation of ds-RNA cells was inhibited by a neu- tralizing antibody against E-cadherin (Fig. 3B). Together, these data indicate that MMP-9 silencing promotes cell-cell contacts through the contribution of E-cadherin. We also as- sessed whether MMP-9 silencing prevented E-cadherin inacti- vation involving the proteolytic cleavage of its extracellular domain by MMP-9. However, we demonstrated that MMP-9 inhibitor did not affect the profiles of surface expression of ICAM-1, VCAM, NCAM, and E-cadherin of MMP-9-silenced cells as compared with control and snc-RNA cells (Table II).
MMP-9 Silencing Impairs COH Cell Migration—In parallel, to analyze the impact of MMP-9 silencing on migration of COH cells, we used transmigration assays with Transwells (Costar) where the lower chamber was uncoated or coated with ECM proteins Fn, Lm, and Col. As shown in Fig. 4A, a significant percentage of parental COH cells spontaneously transmi- grated, and Fn significantly increased their migration. The two other ECM proteins Col and Lm did not promote as efficient migration as Fn (data not shown). Importantly, COH cell mi- gration was not inhibited by the MMP-9 inhibitor 2(R)-2-[(4- biphenylsulfonyl)amino]-3-phenylpropionic acid (Fig. 4A). As expected, gelatinolytic activity of recombinant proMMP-9, as well as that present in the conditioned medium of COH cells, was down-regulated by this inhibitor (Fig. 4B) ascertaining its potency.
Migration toward Fn was significantly reduced for day 2 ds-RNA COH cells (50% inhibition) compared with day 2 snc- RNA cells or parental cells (Fig. 4A). Visualization of migrated cells indicated cell integrity for both snc-RNA and ds-RNA cells (Fig. 4C). A kinetic course similar to that of cell-cell contact formation was observed for inhibition of migration (maximal inhibition at day 2–3; data not shown for day 3). Altogether, these experiments support a positive role for proMMP-9 in reduced COH cell migration secondary to increased cell-cell adhesion.
MMP-9 Silencing Inhibits COH Cell Spreading to Fibronec- tin-, Laminin-, and Collagen IV-coated Surfaces—We further examined the impact of MMP-9 silencing on adhesion of COH cells to extracellular matrix components. Parental COH cells were shown to express intermediate to high levels of (␣1⫺␣6)1
integrins as determined by FACS analysis (Table I), and ad- here to Fn, Lm, and Col but not to BSA (Fig. 4D). No detectable level of2and3subunits was observed (data not shown). We employed function-blocking Abs to identify the cell adhesion receptors involved in cell attachment. These experiments indi- cated that cell adhesion was mediatedvia␣51for fibronectin,
␣21, and CD44 for laminin and␣11and␣21for Col, respec- tively (Fig. 4D). Day 2 transfection with snc-RNA or ds-RNA did not modify the rates of binding of COH cells to Fn, Lm, and Col (Fig. 4E). However, a significant difference in morphology of attached cells was observed. Adherence of snc-RNA cells, like parental cells (data not shown), was accompanied by an appar- ent spreading of many cells on each substrate-coated well (Fig.
4F, a, c, and e) whereas adherent ds-RNA transfected COH cells retained a rounded morphology (Fig. 4F,b,d, andf). These morphological changes were, however, independent of the lev- els of cell surface expression of snc-RNA and ds-RNA cell ad- hesion molecules involved in COH cell adhesion (Table I).
FIG. 2.Cellular characteristics of COH snc-RNA and ds-RNA cells.A, COH snc-RNA (20 nM) and ds-RNA (20 nM) cells cultured for 3 days were assayed for expression of Apo-2.7 antigen by immunofluores- cence in a BD Biosciences flow cytometer as described under “Materials and Methods.” Typical FACS profiles are shown. Theopen histograms represent background fluorescence obtained with the isotype and, the filled histogramsrepresent specific fluorescence obtained with Apo-2.7.
B, COH snc-RNA (20 nM) and ds-RNA (20 nM) cells were cultured for up 3 days, harvested, and counted. One experiment representative of four separate experiments is shown.C, following 2 days of culture, COH parental (a), snc-RNA (20 nM;b), and ds-RNA (1–20 nM;c-f) cells were examined under the microscope and photographed. Magnification,
⫻160 (a-c) and⫻40 (d-f).
Effects of MMP-9 Silencing on Paxillin Phosphorylation and RhoA Activity—Because adhesive interactions (between adja- cent cells or cells and ECM) are critically influenced by the organization of the cytoskeleton, we further explored the effects of MMP-9 silencing on various cytoskeletal related proteins.
COH snc-RNA cells, like parental cells, express different pro- teins of the cytoskeleton including actin, talin, vinculin, and paxillin (Fig. 5A). The total steady state protein levels of actin, actinin, vinculin, and talin were not modified upon 3 days of ds-RNA treatment (Fig. 5A). With regard to␣-actinin, a time- dependent increased signal in the Western blot was unexpect- edly observed under both snc- and ds-RNA conditions (Fig. 5A).
Paxillin migrates in SDS-PAGE as two close bands between 66 and 70 kDa (49) and in lower molecular mass forms (44 – 46 kDa) (50, 51). COH snc-RNA cells exhibited high molecular mass forms of paxillin around 66 kDa (Fig. 5A). Total steady state protein levels of 66-kDa paxillin increased with time in COH cells transfected with ds-RNA compared with the snc- RNA cells, and this was associated with the clear appearance of 44 – 46-kDa paxillin forms on day 2 (Fig. 5A). The 66-kDa form of paxillin contains two critical tyrosine phosphorylation sites at Tyr31 and Tyr118 (52). Immunoblot analysis with an Ab against phosphotyrosine residue 118 (pTyr118-paxillin) showed a significant decrease (3-fold) in phosphopaxillin levels in ds- RNA COH cells compared with snc-RNA cells (Fig. 5A). Thus, MMP-9 silencing in COH cells appears associated with a de- crease in 66-kDa phosphopaxillin reactivity. We further exam- ined two tyrosine kinases, FAK and PYK2, implicated previ- ously (52) in the phosphorylation of paxillin. Activation of the kinase activities of FAK and PYK2 leads first to autophospho-
TABLE II
Effect of an MMP-9 inhibitor on cell-cell adhesion molecules on COH cells in absence or presence of an MMP-9 inhibitor
COH parental cells were cultured in absence or in presence of the MMP-9 inhibitor 2(R)-2-[(4-biphenylsulfonyl)amino]-3-phenyl propionic acid (25M) or its Me2SO control for 1 day. Then, cells were assayed for expression of I-CAM-1, V-CAM, E-cadherin, and N-CAM by immuno- fluorescence.
Phenotype Percentages of positive cells
Parental ⫹Me2SO ⫹Inhibitor
relative intensity of fluorescence
E-cadherin 36 (138) 20 (138) 30 (135)
N-CAM 50 (20) 54 (20) 52 (20)
I-CAM-1 ⬍5 ⬍5 ⬍5
V-CAM ⬍10 ⬍10 ⬍10
FIG. 3.E-cadherin expression on COH cells before and after MMP-9 silencing and efficient inhibition of aggregation by anti- E-cadherin.A, COH snc-RNA (20 nM) and ds-RNA (20 nM) cells cul- tured for 2 and 3 days were assayed for expression of E-cadherin by immunofluorescence in a BD Biosciences flow cytometer as described under “Materials and Methods.” Typical FACS profiles are shown. The open histogramsrepresent background fluorescence obtained with the isotype, and thefilled histogramsrepresent specific fluorescence ob- tained with E-cadherin (67A4) Ab.B, snc-RNA-treated COH cells re- main as adherent single cells (a) whereas ds-RNA transfected cells form cell aggregates (dandg). Isotype mIgG1 (20g/ml) (b, e, andh) or E-cadherin antibody (c,f, andi) (clone HECD-1; 20g/ml) treatment of snc-RNA and ds-RNA transfected COH cells. Aggregation of ds-RNA cells is exclusively blocked by the addition of E-cadherin antibody (fand i). Magnification,⫻160.
TABLE I
Expression of cell-cell and cell-ECM adhesion molecules on COH cells before and after MMP-9 silencing
Parental COH cells (control) and snc-RNA and ds-RNA COH cells cultured under similar conditions for 2 days were then assayed for expression of I-CAM-1, V-CAM, E-cadherin, N-CAM, CD44,␣1–␣6and1integrins by immunofluorescence in a BD Biosciences flow cytometer, as described under “Materials and Methods.” Percentages of positive cells are means⫾S.D. of three to five separate experiments. The relative intensity of fluorescence (number in parentheses) is an indication of the level of expression.
Percentages of positive cells⫾S.D.
Parental snc-RNA ds-RNA
relative intensity of fluorescence
I-CAM-1 ⬍5 ⬍5 ⬍5
V-CAM 22⫾14 (4) 15⫾8 (4) 13⫾6 (4)
E-cadherin 23⫾10 (135) 31⫾19 (138) 59⫾26 (135)
N-CAM 37⫾10 (18) 40⫾7 (18) 44⫾12 (18)
␣1 59 (16) 64⫾24 (19) 69⫾29 (17)
␣2 77 (35) 61⫾17 (13) 58⫾18 (11)
␣3 64 (22) 79⫾3 (10) 60⫾3 (10)
␣4 33 (6) 27⫾12 (5) 36⫾15 (5)
␣5 91⫾3 (49) 91⫾6 (47) 93⫾5 (47)
␣6 20 (5) 18⫾15 (5) 16⫾5 (5)
1 ⬎95 (116) ⬎95 (107) ⬎95 (110)
CD44 66⫾12 (24) 54⫾27 (19) 60⫾23 (21)
MMP-9 Silencing Alters Ewing’s Sarcoma Motility 36541
FIG. 4.Impact of MMP-9 silencing on COH cell adhesion to Fn-, Lm-, and Col-coated surfaces and COH migration toward Fn.A, COH parental cells were cultured in the absence or presence of the MMP-9 inhibitor 2(R)-2-[(4-biphenylsulfonyl)amino]-3-phenylpropionic acid (25
M) or its Me2SO (DMSO) control for 1 day prior to being plated (1⫻105/chamber) in the Transwells in which the lower side was uncoated (⫺) or coated with Fn (⫹) overnight at 37 °C in serum-free RPMI medium containing 0.1% BSA. snc-RNA (20 nM) and ds-RNA (20 nM) COH cells were cultured for 2 days prior to being plated (1⫻105/chamber) in the Transwells. The data represent the mean number of cells that migrated to the lower chamber in triplicate samples for one experiment, representative of three separate experiments for parental, snc-RNA, and ds-RNA cells.B, the gelatinolytic activities of recombinant pro-MMP-9 (0.5, 1, and 10 ng) and in the culture media of COH parental cells were analyzed using zymography performed in the absence (minus) and in the presence of MMP-9 inhibitor (10⫺4M).C, light microscopy of adherent snc-RNA and ds-RNA cells that migrated in the lower chamber. Magnification,⫻160.D, COH parental cells were added to Fn-, Lm-, Col-, and BSA-coated wells, and the plates were incubated at 37 °C for 60 min. Attached living cells were quantitated as described under “Materials and Methods.” 100%
relative adherence represented adherence of untreated COH cells. Data represent the mean⫾S.D. of four separate experiments performed in
rylation (52, 53). Our data, however, indicated that FAK/PYK2 were not phosphorylated upon MMP-9 silencing thus excluding their involvement in decreased paxillin phosphorylation (data not shown).
Alternatively, because paxillin can be activated viaa Rho- regulated pathway (51, 54), we further studied the effect of MMP-9 silencing on Rho-A protein in COH cells. RhoA activity is regulated by both translocation and GTP binding (19). No changes in total RhoA protein levels were observed in COH cellular extracts taken from snc-RNA or ds-RNA cells (Fig. 5A).
However, immunoprecipitation with Rhotekin-RBD beads re- sulted in a marked increase in the amount of GTP-Rho complex (activated Rho) in ds-RNA cells (Fig. 5A). Together, these re- sults indicate that MMP-9 silencing concomitantly alters two proteins,i.e. paxillin and RhoA, known to play a role in the rearrangement of the cytoskeleton.
MMP-9 Silencing Favors Complex Formation between E-cad- herin and-Catenin—We determined whether MMP-9 silenc- ing affects the steady state levels of NCAM and E-cadherin.
Western blotting showed that total protein levels of NCAM and E-cadherin were unchanged in COH ds-RNA cells compared
with the parental and snc-RNA cells (Fig. 5B). Because the association of the E-cadherin/-catenin complex is necessary for cadherin-mediated cell-cell adhesion (22, 55, 56), we next examined the ability of E-cadherin to bind-catenin. When the total cellular extracts from parental cells, snc-RNA, and ds- RNA transfected cells were subjected to immunoprecipitation with anti-E-cadherin and then analyzed for-catenin and E- cadherin levels, a higher level of-catenin was complexed with E-cadherin in ds-RNA cells than in parental and snc-RNA cells (Fig. 5C,left panel). This result was further confirmed by the reverse approach, that is, by assessing the level of E-cadherin associated with immunoprecipitated-catenin (Fig. 5C,right panel). These data demonstrate that upon MMP-9 silencing, E-cadherin and-catenin physically associate. We further an- alyzed the localization of -catenin before and after MMP-9 silencing. Both parental and snc-RNA-treated COH cells showed similar expression patterns of cytoplasmic and nuclear
-catenin whereas ds-RNA treatment resulted in a significant accumulation of-catenin in the cytoplasm (Fig. 5D). We then evaluated in which subcellular compartment-catenin accu- mulated.-Catenin was detectable in the membranous pool of
triplicate. Specificity of adhesion to Fn, Lm, and Col was assayed in the presence of CD44 Ab or␣1Abs (20g/ml). When used at the same concentration, isotype-matched antibodies (controls) were not inhibitory.E, adhesion of day 2 snc-RNA (20 nM) and ds-RNA (20 nM) cells to Fn, Lm, Col, and BSA. Data represent the means of experiments performed in triplicate.F, light microscopy of adherent snc-RNA (a,c, ande) and ds-RNA (b,d, andf) COH cells onto Fn (aandb), Lm (candd), and Col (eandf). May-Gru¨ nwald staining was used. Magnification,⫻160.Bar, 10M.
FIG. 5.Expression of cytoskeletal-related proteins in COH cells before and after MMP-9 silencing.A, equivalent amounts of cell extracts (40g) obtained from COH cells treated with snc-RNA or ds-RNA (20 nM) for 1–3 days were loaded on 8% ProSieve® gels in reducing conditions. Immunoblotting and protein detection with appropriate Abs to paxillin (PXC-10), pTyr118-paxillin and RhoA (mIgG1, cytoskeleton), vinculin (hVIN-1), talin (8D4),␣-actinin (EA53), and actin (AC-15) were performed as described under “Materials and Methods.” The level of GTP-bound RhoA was measured in the total extracts (500g of protein) by immunoprecipitation with beads coupled to the Rho-binding domain of the RhoA effector Rhotekin. Exposure times were⬍15 s for total RhoA and actin, 5 min for talin, vinculin, and␣-actinin, and⬎30 min for GTP-RhoA.B, equivalent amounts of total extracts (40g) obtained from COH parental cells or treated with snc-RNA or ds-RNA (20 nM) for 2 days were loaded on 8% ProSieve® and immunoblotted using E-cadherin (clone 34) and NCAM Abs.C, total extracts prepared were subjected to immunoprecipitation (IP) with anti-E-cadherin (clone 34) or-catenin (clone 14) Abs, followed by immunoblotting with anti-E-cadherin (H-108) and-catenin (clone 14) Abs. Nuclear (EN) and cytoplasmic (EC) extracts (D) or cytosolic and membranous pools obtained from COH parental cells or treated with snc-RNA or ds-RNA (20 nM) for 2 days (E) were fractioned as described under “Materials and Methods.” After solubilization, equivalent amounts of proteins were loaded on 8% ProSieve® gels and immunoblotted using-catenin (clone 14) and integrin1 (clone DE9) Abs.
MMP-9 Silencing Alters Ewing’s Sarcoma Motility 36543
parental and snc-RNA cells (Fig. 5E). A marked accumulation of membranous-catenin was seen with ds-RNA transfected cells (Fig. 5E). The integrin1was used as control to normalize the levels of-catenin (Fig. 5E). We next examined the cellular distribution of-catenin and E-cadherin in snc-RNA and ds- RNA cells by indirect immunofluorescence analysis using spe- cific Abs (Fig. 6). Diffuse intracellular-catenin staining was observed in control snc-RNA cells that is consistent with ab- sence of E-cadherin-mediated cell adhesion (Fig. 6,a).-Cate- nin and E-cadherin were absent from cell junctions in control snc-RNA cells (Fig. 6,a andc). In contrast, MMP-9-silenced cells presented a typical pattern of-catenin and E-cadherin staining at the level of cell-cell contacts (Fig. 6, b and d).
Together, these findings indicate that MMP-9 silencing favored localization of -catenin/E-cadherin complex at the plasma membrane.
DISCUSSION
Like all MMPs, MMP-9 is synthesized as a preproenzyme, secreted as an inactive zymogen form (proMMP-9), and re- quires proteolytic removal of a portion of its N-terminal domain to become activated (1, 4). Compiled studies have implicated MMP-9 in cell invasion in physiological and pathological pro- cesses by providing the basis for the mechanism of ECM deg- radation (1, 4). Indeed, tissue inhibitors of MMPs in general inhibit tumor cell invasion, angiogenesis, and metastasis (57).
However, discrepant data showed that the active form of MMP-9 (13, 14), as well as its proenzyme form (58 – 64), are associated with tumor invasionin vitro. Therefore, the actual mechanism of (pro)MMP-9-mediated invasion remains unclear.
In this study, we used as a model of Ewing’s sarcoma, meta- static COH cells that constitutively express high levels of 92- kDa proMMP-9. Using an RNA silencing approach, we have found that MMP-9 in its proenzyme state provides a critical link between cell motility and cell-cell adhesion.
We first showed that MMP-9 silencing affects motile proper- ties of COH cells. ProMMP-9 silencing by RNAi significantly decreases spreading of COH cells on Fn, Lm, and Col and strongly reduces their migration toward Fn. Although other authors showed recently (65) that proMMP-9 binding to gelatin or collagen type IV resulted in an enzymatic activation of proMMP-9 without loss of its NH2-terminal propeptide, the scenario is unlikely in our system, because cell migration is not inhibited by an MMP-9 inhibitor. Therefore, our results strongly suggest that MMP-9-silenced transfectants are less motile than their parental cells because of the loss of proMMP-9 protein devoid of enzymatic activity.
COH cells cultured as monolayers express a fibroblastic mor- phology characteristic of metastatic cells. Our data demon- strate that, concomitantly to the loss of migration, proMMP-9 silencing in COH cells favored a morphology different from that of the parental cells and more specifically cell aggregates. This was because of up-regulation of E-cadherin levels at the cell surface, because an Ab against E-cadherin efficiently blocked cell-cell adhesion. Malignant transformation is often character- ized by disruption of cell-cell adhesion, which can be achieved by down-regulating the expression of cadherin and/or-catenin (22). Moreover, clustering of cell surface cadherins is associated with the recruitment of-catenin to the cadherin-associated complex (17). Although the levels of total E-cadherin and
-catenin remained unchanged upon ds-RNA treatment, we demonstrated an enhanced physical association between E-cadherin/-catenin proteins by coimmunoprecipitation. As expected,-catenin was detected at the plasma membrane at cell-cell junctions of MMP-9-silenced cells. Together, our data indicate that surface stabilization of E-cadherin/-catenin com- plex in MMP-9-silenced COH cells leads to the induction of E-cadherin-mediated cell-cell adhesion, which, in turn, dimin- ishes the motility. Our data are in agreement with the litera- ture documenting the involvement of the cadherin/-catenin pathway in tumorigenesis (23, 66). Disruption of adhesion sys- tems can contribute to tumor development (22, 23) whereas increased levels of cadherins reduce tumorigenic properties (67).
In addition, biological and synthetic inhibitors of MMP ac- tivities have been suggested to promote cell-cell adhesion by at least preventing the cadherin ectodomain cleavage, thus stabi- lizing cadherin-mediated cell-cell contacts (68, 69). MMP-3 can cleave E-cadherin (67) whereas MMP-9 cleaves ICAM-1 (70).
Similarly, an MMP activity seems to be responsible for the shedding of VCAM-1 (71). Here, we demonstrate that an inhib- itor of MMP-9 affected neither COH cell adhesion and migra- tion nor the profiles of surface expression of ICAM-1, VCAM, NCAM, and E-cadherin. These results therefore provide evi- dence of the following. (i) The integrities of CAMs and E- cadherin on COH cells are independent of proMMP-9. (ii) The impact of proMMP-9 silencing on E-cadherin-mediated cell-cell adhesion is not related to decreased proteolysis of surface E-cadherin.
In MMP-9-silenced COH cells, alterations of spreading, mi- gration, and E-cadherin-mediated cell-cell adhesion were indic- ative of changes in the functional association of cell adhesion molecules (integrins and E-cadherin) with the cytoskeletal net- work. The proteins of the Rho subfamily Cdc42, Rac, and Rho are signaling molecules primarily involved in remodeling the actin cytoskeleton (17, 20). In particular, Rho proteins are required for cadherin-mediated cell-cell adhesion and conse- quently influence many aspects of cell shape and movement (17, 19, 20). More specifically,-catenin, depending on its rel- ative amounts in the cytoplasm/nucleus or associated with cadherins at the plasma membrane, can influence cells toward migration or cell-cell adhesion by regulating the function of Rho proteins (20). Previous studies (20, 72) reported that RhoA activation increases the accumulation of cadherin/-catenin complexes at cell-cell contact in keratinocytes and epithelial cells. We similarly found a correlation among RhoA activation, preferential accumulation of cadherin/-catenin complexes at cell-cell junctions, and E-cadherin-mediated adhesion of MMP- 9-silenced COH cells.
Among cytoskeletal proteins that play a critical role in cell spreading and migration, paxillin localizes primarily to sites of cell adhesion to the ECM called focal adhesions (51, 52). Pax- illin is a 66-kDa cytoplasmic protein that binds to several FIG. 6.Immunofluorescence analysis of MMP-9-silenced cells
for E-cadherin and-catenin expression.Day 2 control snc-RNA cells (aandc) and ds-RNA cells (bandd) were stained with antibodies to-catenin (2A4H7) (aandb) or E-cadherin (H108) (candd). Pictures were obtained with a confocal microscope.
cytoskeletal, intracellular signaling, and adaptor molecules such as vinculin, pp125FAK, ILK, the cytoplasmic domain of integrin␣4, and modulators/effectors of Rho proteins (51, 73).
In MMP-9-silenced cells, reduction of cell spreading to ECM is accompanied by an increase in paxillin steady state protein levels but a decrease in paxillin tyrosine phosphorylation. Our data are consistent with prior studies (52), which demonstrate in various cell types that paxillin phosphorylation is an early and requisite step in cell spreading.
Cell shape changes result in modulation of various pheno- typic changes and alteration of MMP-9 expression (55, 74).
Inversely, our data indicate that proMMP-9 extinction induces cell shape changes (particularly those resulting from paxillin/
RhoA/-catenin pathways) that alter the adhesive and migra- tory properties of COH cells. Therefore, an unanswered ques- tion regarding the critical role of proMMP-9 for E-cadherin- mediated cell-cell adhesion and migration is how can proMMP-9 influence motile properties of COH cells? A number of recent papers have demonstrated that the hyaluronan recep- tor CD44 can serve as a docking molecule to retain secreted MMP-9 (under both pro and active forms) at the cell surface (75, 76). CD44 is involved in cell adhesion and trafficking, as well as in tumor growth, invasion, and metastasis (76 –78). The cytoplasmic domain of CD44 interacts with several cytoskeletal proteins including several guanine nucleotide exchange factors for Rho family GTPases. The interaction of CD44 with these guanine nucleotide exchange factors leads to activation of Rac1 and, under certain conditions, results in increased Rho activa- tion (76 –78). Parental COH cells constitutively express CD44, and MMP-9 silencing does not affect CD44 expression (Table I).
Thus, one can hypothesize that extracellular proMMP-9 influ- ences cytoskeleton rearrangements (involving RhoA/paxillin/- catenin signalings) through its association with surface CD44.
Alternatively, a yet unidentified signaling pathway in re- sponse to MMP-9 may lead to nuclear localization of-catenin, which,via members of the LEF/TCF family of DNA-binding
proteins, transactivates certain genes culminating in cell-cell adhesion. More specifically, association of nuclear -catenin with TCF-4 promotes the expression of several molecules that have important roles in the development and progression of tumors (18, 79, 80). Indeed, the transcriptional activity of MMP-7, MMP-26, and MT1-MMP is regulated by TCF-4 (79, 81, 82), and other MMPs including MMP-9 also possess a consensus TCF-4 regulatory element (79). Our results show that COH parental cells that express low levels of surface E-cadherin have high levels of nuclear -catenin while in- versely increase of surface E-cadherin in MMP-9-silenced cells is associated with decreased nuclear localization of-catenin.
Whether MMP-9 silencing results in the withdraw of nuclear
-catenin via the coordinating signals transduced through
-catenin/TCF-4 pathways has to be considered.
In conclusion, our observations highlight a novel aspect of the role of MMP-9, independently of its enzymatic activity, for influencing tumor cell fate. Loss of motility and concomitant induction of E-cadherin-mediated cell-cell adhesion of MMP-9- silenced COH cells are associated with stabilization of E-cad- herin/-catenin complex and reorganization of the cytoskeleton through paxillin/RhoA pathways. Our data suggest a model (Fig. 7) in which proMMP-9 can be critical for progression of malignancy by triggering the switch from a stationary state to a migratory state.
Acknowledgments—We thank Dr. Olivier Delattre and collaborators (Institut Curie, Unite´ INSERM 509) for kindly providing Ewing’s cell lines. We are very grateful to Dr. Douglas Boyd (M. D. Anderson Cancer Center, Houston, TX) for critical review of the manuscript. We thank Catherine Silvestri for valuable technical assistance and Dany Rouillard for dedicated helpfulness in FACS.
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