Filamin A-mediated Down-regulation of the Exchange Factor Ras-GRF1 Correlates with Decreased Matrix Metalloproteinase-9 Expression in Human Melanoma Cells
2007; Elsevier BV; Volume: 282; Issue: 20 Linguagem: Inglês
10.1074/jbc.m611430200
ISSN1083-351X
AutoresTie-Nian Zhu, Hua‐Jun He, Sutapa Kole, Theresa D’Souza, Rachana Agarwal, Patrice J. Morin, Michel Bernier,
Tópico(s)Signaling Pathways in Disease
ResumoThe actin-binding protein filamin A (FLNa) is associated with diverse cellular processes such as cell motility and signaling through its scaffolding properties. Here we examine the effect of FLNa on the regulation of signaling pathways that control the expression of matrix metalloproteinases (MMPs). The lack of FLNa in human M2 melanoma cells was associated with constitutive and phorbol ester-induced expression and secretion of active MMP-9 in the absence of MMP-2 up-regulation. M2 cells displayed stronger MMP-9 production and activity than their M2A7 counterparts where FLNa had been stably reintroduced. Using an MMP-9 promoter construct (pMMP-9-Luc), in vitro kinase assays, and genetic and pharmacological approaches, we demonstrate that FLNa mediated transcriptional down-regulation of pMMP-9-Luc by suppressing the constitutive hyperactivity of the Ras/MAPK extracellular signal-regulated kinase (ERK) cascade. Experimental evidence indicated that this phenomenon was associated with destabilization and ubiquitylation of Ras-GRF1, a guanine nucleotide exchange factor that activates H-Ras by facilitating the release of GDP. Ectopic expression of Ras-GRF1 was accompanied by ERK activation and elevated levels of MMP-9 in M2A7 cells, whereas a catalytically inactive dominant negative Ras-GRF1, which prevented ERK activation, reduced MMP-9 expression in M2 cells. Our results indicate that expression of FLNa regulates constitutive activation of the Ras/ERK pathway partly through a Ras-GRF1 mechanism to modulate the production of MMP-9. The actin-binding protein filamin A (FLNa) is associated with diverse cellular processes such as cell motility and signaling through its scaffolding properties. Here we examine the effect of FLNa on the regulation of signaling pathways that control the expression of matrix metalloproteinases (MMPs). The lack of FLNa in human M2 melanoma cells was associated with constitutive and phorbol ester-induced expression and secretion of active MMP-9 in the absence of MMP-2 up-regulation. M2 cells displayed stronger MMP-9 production and activity than their M2A7 counterparts where FLNa had been stably reintroduced. Using an MMP-9 promoter construct (pMMP-9-Luc), in vitro kinase assays, and genetic and pharmacological approaches, we demonstrate that FLNa mediated transcriptional down-regulation of pMMP-9-Luc by suppressing the constitutive hyperactivity of the Ras/MAPK extracellular signal-regulated kinase (ERK) cascade. Experimental evidence indicated that this phenomenon was associated with destabilization and ubiquitylation of Ras-GRF1, a guanine nucleotide exchange factor that activates H-Ras by facilitating the release of GDP. Ectopic expression of Ras-GRF1 was accompanied by ERK activation and elevated levels of MMP-9 in M2A7 cells, whereas a catalytically inactive dominant negative Ras-GRF1, which prevented ERK activation, reduced MMP-9 expression in M2 cells. Our results indicate that expression of FLNa regulates constitutive activation of the Ras/ERK pathway partly through a Ras-GRF1 mechanism to modulate the production of MMP-9. Matrix metalloproteinases (MMPs) 5The abbreviations used are: MMP, metalloproteinase; ECM, extracellular matrix; EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; NF-κB, nuclear factor κB; FLNa, filamin A; GEF, guanine-nucleotide exchange factor; ΔCDC25, mutant form of Ras-GRF1 lacking the C-terminal CDC25 domain; PMA, phorbol 12-myristate 13-acetate; IκBα, inhibitor of NF-κB; RasN17, dominant-negative mutant of Ras; HA, hemagglutinin; WT, wild type. 5The abbreviations used are: MMP, metalloproteinase; ECM, extracellular matrix; EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; NF-κB, nuclear factor κB; FLNa, filamin A; GEF, guanine-nucleotide exchange factor; ΔCDC25, mutant form of Ras-GRF1 lacking the C-terminal CDC25 domain; PMA, phorbol 12-myristate 13-acetate; IκBα, inhibitor of NF-κB; RasN17, dominant-negative mutant of Ras; HA, hemagglutinin; WT, wild type. play a crucial role in degradation of extracellular matrix (ECM) associated not only with normal growth and development but also with various pathological conditions such as tumor invasion and angiogenesis (1Sternlicht M.D. Werb Z. Annu. Rev. Cell Dev. Biol. 2001; 17: 463-516Crossref PubMed Scopus (3226) Google Scholar). Melanoma progression, as in many other cancers, is associated with invasion into surrounding tissues, which depends on MMP-mediated proteolytic degradation of the basement membrane and ECM. Among its members, the 72-kDa gelatinase A (MMP-2) and 92-kDa gelatinase B (MMP-9) are thought to be key enzymes for degrading type IV collagen, a major component of the basement membrane. Contributions of these enzymes in both physiological and pathophysiological processes such as tumor cell invasion and metastasis have been well documented (2Himelstein B.P. Canete-Soler R. Bernhard E.J. Dilks D.W. Muschel R.J. Invasion Metastasis. 1994; 14: 246-258PubMed Google Scholar, 3Itoh T. Tanioka M. Matsuda H. Nishimoto H. Yoshioka T. Suzuki R. Uehira M. Clin. Exp. Metastasis. 1999; 17: 177-181Crossref PubMed Scopus (283) Google Scholar, 4Johansson N. Kahari V.M. Histol. 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Biol. 2004; 24: 5496-5509Crossref PubMed Scopus (113) Google Scholar, 13Sato H. Seiki M. Oncogene. 1993; 8: 395-405PubMed Google Scholar, 17Kato Y. Lambert C.A. Colige A.C. Mineur P. Noel A. Frankenne F. Foidart J.M. Baba M. Hata R. Miyazaki K. Tsukuda M. J. Biol. Chem. 2005; 280: 10938-10944Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Filamin A (FLNa), a member of the non-muscle actin-binding protein family, is a widely expressed molecular scaffold that regulates signaling events involved in cell shape change and motility by interacting with integrins, transmembrane receptor complexes, adaptor molecules, and second messengers (18Feng Y. Walsh C.A. Nat. Cell Biol. 2004; 6: 1034-1038Crossref PubMed Scopus (413) Google Scholar, 19Scott M.G. Pierotti V. Storez H. Lindberg E. Thuret A. Muntaner O. Labbe-Jullie C. Pitcher J.A. Marullo S. Mol. Cell. Biol. 2006; 26: 3432-3445Crossref PubMed Scopus (115) Google Scholar, 20Stossel T.P. Condeelis J. Cooley L. Hartwig J.H. Noegel A. Schleicher M. Shapiro S.S. Nat. Rev. Mol. Cell. Biol. 2001; 2: 138-145Crossref PubMed Scopus (820) Google Scholar). Mutations in FLNa gene underlie a spectrum of human disorders, such as skeletal dysplasia and the localized cerebral cortical neuronal migration disorder known as periventricular nodular heterotopia (21Parrini E. Ramazzotti A. Dobyns W.B. Mei D. Moro F. Veggiotti P. Marini C. Brilstra E.H. Dalla Bernardina B. Goodwin L. Bodell A. Jones M.C. Nangeroni M. Palmeri S. Said E. Sander J.W. Striano P. Takahashi Y. Van Maldergem L. Leonardi G. Wright M. Walsh C.A. Guerrini R. Brain. 2006; 129: 1892-1906Crossref PubMed Scopus (272) Google Scholar, 22Robertson S.P. Twigg S.R. Sutherland-Smith A.J. Biancalana V. Gorlin R.J. Horn D. Kenwrick S.J. Kim C.A. Morava E. Newbury-Ecob R. Orstavik K.H. Quarrell O.W. Schwartz C.E. Shears D.J. Suri M. Kendrick-Jones J. Wilkie A.O. Nat. Genet. OPD-spectrum Disorders Clinical Collaborative Group. 2003; 33: 487-491Google Scholar). In addition, the ability of tissue factor to promote vascular remodeling and tumor cell metastasis have been shown to be mediated by interaction with FLNa (23Ott I. Fischer E.G. Miyagi Y. Mueller B.M. Ruf W. J. Cell Biol. 1998; 140: 1241-1253Crossref PubMed Scopus (275) Google Scholar), and remodeling of the cytoskeleton has been identified as having a role in cell migration and acquisition of invasive behavior (24de Curtis I. EMBO Rep. 2001; 2: 277-281Crossref PubMed Scopus (75) Google Scholar, 25Yamazaki D. Kurisu S. Takenawa T. Cancer Sci. 2005; 96: 379-386Crossref PubMed Scopus (510) Google Scholar). Thus, the ability of FLNa to act as integrator of cell mechanics (20Stossel T.P. Condeelis J. Cooley L. Hartwig J.H. Noegel A. Schleicher M. Shapiro S.S. Nat. Rev. Mol. Cell. Biol. 2001; 2: 138-145Crossref PubMed Scopus (820) Google Scholar) is of significant interest, and has led us to hypothesize that this scaffold protein provides a molecular pathway to support metastasis and motility through regulation of MMP expression in melanoma. The present study was designed to determine the importance of FLNa in the regulation of MMP-2 and MMP-9 levels in the human melanoma M2 cell line. Using FLNa-deficient M2 cells and M2 cells stably expressing normal concentration of human FLNa (M2A7 cells) we found a significant reduction in MMP-9 secretion and activity upon FLNa expression, and have assessed here the intracellular mechanisms responsible. Our data indicate a dampening in the constitutive activation of the Raf-1/MEK/ERK cascade in M2A7 cells through a decrease in steady-state levels of GTP-bound Ras. A number of studies have been reported on the important role of guanine-nucleotide exchange factors (GEFs) in Ras activation (26Arozarena I. Aaronson D.S. Matallanas D. Sanz V. Ajenjo N. Tenbaum S.P. Teramoto H. Ighishi T. Zabala J.C. Gutkind J.S. Crespo P. J. Biol. Chem. 2000; 275: 26441-26448Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 27Quilliam L.A. Rebhun J.F. Castro A.F. Prog. Nucleic Acids Res. Mol. Biol. 2002; 71: 391-444Crossref PubMed Google Scholar). FLNa interacts with Trio, a RhoGEF that controls RhoG/Rac1 GTPase function (28Bellanger J.M. Astier C. Sardet C. Ohta Y. Stossel T.P. Debant A. Nat. Cell Biol. 2000; 2: 888-892Crossref PubMed Scopus (181) Google Scholar); however, it remains unclear whether FLNa can limit the GTP-bound Ras levels through down-regulation of GEFs that target Ras. In this study, we present evidence that expression of FLNa was associated with reduction of Ras-GRF1 levels through ubiquitylation-mediated proteolysis. Ectopic expression of Ras-GRF1 but not the ΔCdc25 mutant form of Ras-GRF1 was correlated with an increase in the Raf-1/ERK pathway and the levels of MMP-9. These results suggest that FLNa may be a negative regulator of Ras-GRF1, thereby blocking the sustained activation of the Ras/ERK signal transduction pathway required for MMP-9 expression in melanoma. Materials—All cell culture reagents were purchased from Invitrogen (Carlsbad, CA) and Cellgro (Herndon, VA), except fetal bovine serum (FBS), which was from Hyclone (Logan, UT). Phorbol 12-myristate 13-acetate (PMA), BMS345541, U0126, and manumycin A were purchased from Calbiochem-EMD Biosciences, Inc. (La Jolla, CA). Cell Culture—Human M2 melanoma cells and FLNa-expressing M2A7 cell clone were originally described by Cunningham et al. (29Cunningham C.C. Gorlin J.B. Kwiatkowski D.J. Hartwig J.H. Janmey P.A. Byers H.R. Stossel T.P. Science. 1992; 255: 325-327Crossref PubMed Scopus (498) Google Scholar). These cells were maintained in α-MEM medium supplemented with 10 mm HEPES (pH 7.4), 0.25% sodium bicarbonate, 2 mm l-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, 8% newborn calf serum, and 2% fetal bovine serum. Experiments were performed on passage 6–16 cells. DNA Constructs and Transfections—The pREP4 expression vector containing the human wild-type FLNa construct (GenBank™/EBI Data Bank accession number NM_001456) was a gift from Y. Ohta (Harvard Medical School, Boston, MA). The pKH3 expression vector containing the mouse HA-tagged Ras-GRF1 and pCEFLAU5 vector expressing the rat ΔCdc25 mutant of Ras-GRF1 (truncation of the Cdc25 domain) were generously provided by P. Crespo (Instituto de Investigaciones Biomedicas, Universidad de Cantabria-CSIC, Santander, Spain) (26Arozarena I. Aaronson D.S. Matallanas D. Sanz V. Ajenjo N. Tenbaum S.P. Teramoto H. Ighishi T. Zabala J.C. Gutkind J.S. Crespo P. J. Biol. Chem. 2000; 275: 26441-26448Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The pUSEamp expression vector containing the dominant negative RasN17 mutant was purchased from Upstate Biotechnology Inc. (Lake Place, NY; 21-104). The pGL3-basic vector expressing wild type human MMP-9 promoter (–670/+54 fragment, GenBank™/EBI Data Bank accession number D10051) luciferase construct and the MMP-9 promoter constructs harboring point mutation in either the AP1 or NF-κB binding site fused to luciferase were generously provided by H. Sato (Cancer Research Institute, Kanazawa University, Kanazawa, Japan) (13Sato H. Seiki M. Oncogene. 1993; 8: 395-405PubMed Google Scholar). M2 and M2A7 cells were plated in duplicates, and transfected with either pcDNA3.1 or FLNa construct at a ratio of 4 μg of plasmid/60-mm dish, using Lipofectamine2000 (Invitrogen). In other experiments, RasN17 (2 μg), HA-Ras-GRF1 (1–2 μg), or AU5-ΔCdc25 (2–4 μg) were transfected into M2 and M2A7 cells (35-mm2 dishes) with MMP-9 luciferase (1 μg) and pSV-β-galactosidase (0.2 μg) constructs. At 30 h post-transfection, the cells were serum-starved for different times as described, rinsed in phosphate-buffered saline, and lysed in reporter lysis buffer (Promega, Inc., Madison, WI) for luciferase and β-galactosidase assays (kits from Promega) and Western blot analysis. Gelatin Zymography—Growth-arrested M2 and M2A7 cells were incubated in the serum-free MEM for 24 h at 37 °C. Gelatinase activity in conditioned medium was measured by zymography. An equal amount of proteins from the concentrated conditioned medium (25 μl) was separated on a 10% Zymogram gel containing 0.1% gelatin, and the gel was incubated in the zymogram developing buffer (Invitrogen) for 16 h at 37 °C to evaluate activities of MMP-9 and MMP-2. The gel was stained with Coomassie Blue G-250 and then destained to reveal areas of gelatinolytic activity. Semi-quantitative RT-PCR—Cells were serum-starved for 24 h and then cellular RNA was extracted with TRIzol, followed by RT-PCR reaction. MMP-9 forward (5′-GATGCGTGGAGAGTCGAAAT-3′) and reverse (5′-CACCAAACTGGATGACGATG-3′) primers, MMP-2 forward (5′-ACAAAGAGTTGGCAGTGCAA-3′) and reverse (5′-CACGAGCAAAGGCATCATCC-3′) primers, and GAPDH forward (5′-GAAGGTGAAGGTCGGAGTC-3′) and reverse (5′-GAAGATGGTGATGGGATTTC-3′) primers were synthetized by Integrated DNA Technologies, Inc. (Coralville, IA). For all reactions, PCR conditions comprised 25 cycles of 94 °C (45 s), 50 °C (45 s) and 72 °C (45 s). PCR products were resolved by 2% agarose gel electrophoresis, and the relative intensity of MMP-2 (303 bp), MMP-9 (329 bp) and GADPH (220 bp) bands was determined following ethidium bromide staining and quantitated using ImageQuant software (Amersham Biosciences Life Science). Samples were standardized for equal expression of GADPH. Quantitative Real Time PCR—cDNA were prepared from treated M2 and M2A7 cells, and the quantitative PCR analysis was performed by using TaqMan® Gene Expression Assay (Part 4324018) from Applied Biosystems (Foster City, CA). Primers for MMP-9 (catalog number Hs00234579), MMP-2 (catalog number Hs00234422), and reference gene GAPDH (catalog number Hs99999905) were predesigned by Applied Biosystems according to the sequences available from NCBI with the accession numbers NM_004994.2, NM_004530.2, and NM_002046.3, respectively. Each reaction was carried out in tetraplicate in the ABI 7300 Real Time PCR System. To determine the relative quantitation of gene expression, the comparative CT (threshold cycle) method was used (30Livak K.J. Schmittgen T.D. Methods. 2001; 25: 402-428Crossref PubMed Scopus (123392) Google Scholar) and normalized to the amount of GADPH RNA present in each sample. Assays—To evaluate the amount of MMP-9 secretion, the conditioned medium from M2 and M2A7 cells was concentrated and assayed for MMP-9 protein by ELISA, according to the manufacturer’s protocol (Calbiochem). Ras activation assay was performed as followed: Briefly, M2 and M2A7 cells were rinsed in PBS and then serum-starved for 4 h before cell lysis. Ras activation was then determined using a RasGTPase Chemi ELISA kit, according to the manufacturer’s protocol (Active Motif, Carlsbad, CA). B-Raf and c-Raf kinase cascade assay kits were used to measure cellular Raf activities via phosphorylation of recombinant inactive MEK1 and its inactive substrate ERK2. Activation of ERK2 leads to phosphorylation of myelin basic protein as ERK substrate. In brief, after PBS washes, cells were lysed in ice-cold Tris lysis buffer (TLB: 20 mm Tris-HCl (pH 7.5), 137 mm NaCl, 25 mm β-glycerophosphate, 2 mm EDTA, 1 mm sodium orthovanadate, 2 mm Na2HPO4, 1% Triton X-100, 10% glycerol, 0.5 mm dithiothreitol, and protease inhibitor mixture set I (Calbiochem)]. The cell lysates were clarified by centrifugation (14,000 × g for 20 min at 4 °C), and protein content was measured using BCA protein assay kit (Pierce). 500 μg of soluble cellular extracts were immunoprecipitated with 2 μg of polyclonal anti-B-Raf (07-453) or c-Raf (07-396) antibody (Upstate Biotechnologies, Inc.) in the presence of protein A/G PLUS agarose beads (Calbiochem) for 2 h at 4°C. Immunoprecipitates were extensively washed and resuspended in kinase assay buffer (20 mm HEPES (pH 7.4), 25 mm β-glycerophosphate, 5 mm EGTA, 1 mm sodium orthovanadate, 1 mm dithiothreitol). In vitro kinase assays were then performed in two stages, according to the manufacturer’s protocol (Upstate Biotechnologies, Inc.). Subcellular Fractionation—Serum-starved cells were left untreated or treated either with 20 nm EGF or 100 nm PMA for 10 min. The cells were rinsed twice in ice-cold phosphate-buffered saline and scraped in lysis buffer (50 mm HEPES, pH 7.5, 50 mm NaCl, 1 mm MgCl2, 10 mm NaF, 10 mm sodium pyrophosphate, 0.5 mm sodium orthovanadate, 2 mm EDTA, 1 mm dithiothreitol, and protease inhibitor mixture set I). The cells were homogenized by passing them five times through a 23-gauge needle on ice and centrifuged at 1300 × g for 5 min to sediment nuclei and unbroken cells. The clarified homogenate was then centrifuged at 100,000 × g for 60 min at 4 °C. The supernatant was removed and saved as cytosol, and the pelleted membranes were washed twice in lysis buffer and then resuspended in TLB buffer. Protein concentration for the cytosol, and membrane fractions was determined by the BCA method. Immunoprecipitation and Western Blot Analysis—After cell lysis in immune precipitation buffer (25 mm HEPES, pH 7.4, 135 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 mm NaF, 1 mm sodium orthovanadate, 0.01% sodium azide, and protease inhibitor mixture set I), the clarified lysates (500 μg) were incubated with 2 μg of rabbit anti-Ras-GRF1, anti-SOS1 or anti-c-Myc antibodies for 16 h at 4 °C followed by the addition of 20 μl of protein A/G-agarose (Upstate Biotechnologies, Inc.) for 90 min at 4 °C. The immunocomplexes were washed two times with immune precipitation buffer, two times with 50 mm HEPES, pH 7.6, 0.1% Triton X-100 supplemented with 0.5 m NaCl, and twice with 50 mm HEPES, pH 7.6, 0.1% Triton X-100. Total cell extracts and immunoprecipitated complexes were separated by 4–12% gradient SDS-PAGE, and then subjected to immunoblotting with specific primary antibodies. The signals were quantified using a chemiluminescence detection system (Amersham Biosciences). The antibodies used in these studies included polyclonal antibodies that were raised against phospho-MEK1/2 (9121), phospho-ERK1/2 (7071), phospho-AKT (Ser473; 9271), phospho-IκBα (9241), total STAT3 (9132), MMP-9 (2270), and c-Fos (4384), and were purchased from Cell Signaling Technology (Beverly, MA). Monoclonal anti-ubiquitin (sc-8017) and anti-phospho-Jun (sc-822), and polyclonal antibodies directed against SOS1 (sc-256), Ras-GRF1 (sc-863), clathrin (sc-9069), ERK1/2 (sc-094), c-Src (sc-18), IKKα (sc-7607), and p89TFIIH (sc-293) were from Santa Cruz Biotechnology (Santa Cruz, CA). The monoclonal antibodies against FLNa, panRas (OP40), and phospho-Raf-1 (Ser338; 05-538) were purchased from Research Diagnostics, Inc. (Flanders, NJ), Calbiochem and Upstate Biotechnology Inc., respectively. FLNa Attenuates Constitutive and Phorbol Ester-mediated MMP-9 Secretion and Activity—In vivo, MMP-2 and -9 are two major gelatinases produced by human melanomas, and their proteolytic activity is thought to be necessary in both physiological and pathophysiological processes. Gelatin zymography was carried out to resolve the two closely related activities. The level of active MMP-9 was markedly higher in M2 cells when compared with M2A7 cells, both in the absence and the presence of PMA (Fig. 1A). Under these conditions, MMP-2 activity levels were found to be very low and unresponsive to PMA stimulation (Fig. 1A). To verify whether the presence of FLNa differentially affected MMP-9 and MMP-2 at the mRNA level, cells were serum-starved, lysed, and mRNA was analyzed by RT-PCR. The results showed that the MMP-9 PCR product was present in M2 cells, but not in M2A7 cells (Fig. 1B), consistent with the gelatin zymography data. In contrast, MMP-2 mRNA level was comparable in both cell lines following normalization with GADPH mRNA. Real time PCR analysis was also carried out and demonstrated a ∼30-fold increase in MMP-9 mRNA levels in PMA-stimulated M2 cells as compared with unstimulated cells (Fig. 1C, filled bars). Pretreatment with the MEK inhibitor U0126 sharply reduced the PMA response. In contrast, no significant increase in the amount of MMP-9 mRNA was observed in M2A7 cells post-PMA treatment (Fig. 1C, open bars). MMP-2 mRNA levels were unresponsive to PMA stimulation in both M2 and M2A7 cells (data not shown). In keeping with these observations, all subsequent experiments were performed studying the regulation of MMP-9. The differential ability of M2 and M2A7 cells at promoting MMP-9 expression and activity was associated with differences in MMP-9 secretion (Fig. 1D). Using an ELISA assay to measure the amount of MMP-9 released in the medium, we showed that control M2 cells secreted MMP-9 at a rate of 0.12 ± 0.05 ng/ml/24 h, while no response was observed with M2A7 cells. A significant increase in MMP-9 secretion of ∼8-fold above the control was found upon a 24-h exposure of M2 cells to the phorbol ester PMA (100 nm) (Fig. 1D). In contrast, MMP-9 secretion was found to be below the detection limit following stimulation of M2A7 cells with PMA. These results were corroborated by gelatinase activity (Fig. 1A, lanes 3–6). As anticipated, pharmacological inhibition of the ERK/MAPK pathway with U0126 blocked the basal and PMA-induced release of MMP-9 in M2 cells (Fig. 1D). Taken together, our data suggest that FLNa is a negative regulator of MMP-9 expression. FLNa Attenuates the MMP-9 Promoter—The human MMP-9 promoter (–670/+54) has been previously found to contain binding sites for AP-1, NF-κB and SP-1 proteins (13Sato H. Seiki M. Oncogene. 1993; 8: 395-405PubMed Google Scholar). In a first series of experiments, wild type and mutant forms of the promoter were used to delineate the gene elements needed for constitutive and PMA-induced MMP-9 gene expression in M2 cells (Fig. 2A). More than 3.8-fold induction of the wild-type MMP-9 promoter construct was observed in response to PMA. It is interesting to note that mutation of the AP-1-binding site markedly reduced promoter activity in control and PMA-stimulated cells, whereas inactivation of the NF-κB binding site elicited a ∼40% decrease in the PMA response (Fig. 2A). We then addressed whether pharmacological inhibition of the MEK/ERK or IKK/NF-κB pathway might reduce MMP-9 reporter activity. At 36 h post-transfection, the M2 cells were either untreated or treated with U0126 or the IKK inhibitor BMS345541 for 1 h prior to the addition of 100 nm PMA for 7 h (Fig. 2B). While basal MMP-9 reporter activity was reduced by ∼50% in cells treated with the MEK inhibitor U0126, PMA-mediated increases in pMMP-9-Luc activity felt sharply upon inhibition of either pathway as compared with control cells (Fig. 2B). These results are consistent with earlier findings suggesting that activation of the MMP-9 promoter requires synergistic cooperation between various cis-acting elements (12Ma Z. Shah R.C. Chang M.J. Benveniste E.N. Mol. Cell. Biol. 2004; 24: 5496-5509Crossref PubMed Scopus (113) Google Scholar, 13Sato H. Seiki M. Oncogene. 1993; 8: 395-405PubMed Google Scholar). Finally, a marked reduction in MMP-9 promoter activation was observed in FLNa-expressing M2A7 cells when compared with M2 cells treated in the absence or the presence of PMA (Fig. 2C). These results recapitulate the MMP-9 activity determined by gelatin zymography (Fig. 1A). Importantly, transient expression of FLNa in M2 cells elicited an increase in FLNa protein levels (Fig. 2C, upper panel), while inhibiting both the basal and PMA-induced MMP-9 gene promoter construct activation (Fig. 2C, lower panel). There was markedly reduced expression of MMP-9 protein in response to PMA in M2 cells transfected with FLNa (Fig. 2D, lanes 2 and 4, top). Reprobing the blots demonstrated similar expression of ERK1/2 (Fig. 2D, bottom). Taken together, these results provide evidence that the differences in MMP-9 expression between M2 and M2A7 cells are not attributable to clonal variations, but instead suggest that FLNa inhibits the expression of MMP-9. FLNa Reduces the Constitutive Activation of the Raf-1/ERK Cascade—To investigate the signaling pathways that are affected by FLNa expression, we focused on the Raf-MEK-ERK cascade and the PI 3-kinase/AKT pathway. Total lysates from M2 and M2A7 cells were analyzed by SDS-PAGE and immunoblotted with antibodies against phosphorylated (e.g. activated) Raf-1 (pSer338), MEK1/2, ERK1/2, and AKT. M2 cells had relatively high levels of phosphorylated forms of Raf-1, MEK1/2, and ERK1/2 in basal conditions that were increased severalfold in the presence of PMA stimulation (Fig. 3A). The stable expression of FLNa in M2A7 cells was associated with a reduction in basal phosphorylation levels of these intermediates when compared with M2 cells (Fig. 3A, lane 3 versus 2). No significant difference in AKT phosphorylation was observed between M2 and M2A7 cells (data not shown). Interestingly, one of the AP-1 subunits, the immediate-early gene c-Fos, was detected in M2 cells under basal conditions, and a decrease in c-Fos migration occurred following 15 min of PMA treatment (Fig. 3A), which is consistent with its phosphorylation. There was little c-Fos expression, if any, in M2A7 cells exposed in the absence or presence of PMA, despite the presence of ERK activation upon PMA stimulation. Because of the important role for NF-κB in regulating melanoma cell migration, we measured also the levels of phosphorylated IκBα in lysates from M2 and M2A7 cells, and found measurable IκBα phosphorylation in unstimulated M2 cells (Fig. 3A, fifth panel). Moreover, short-term treatment with PMA led to significant increase in phospho-IκBα levels in M2A7 cells, although to levels that were lower to those observed in M2 cells. These results support earlier findings about enhanced NF-κB activation in melanoma by phorbol esters (31La Porta C.A. Comolli R. Anticancer Res. 1998; 18: 2591-2597PubMed Google Scholar). Western blot analysis utilizing antibodies against c-Src and IKKα confirmed equal protein loading in each lane. Stable expression of FLNa in M2A7 cells is associated with reduction i
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