Nonsteroidal Anti-inflammatory Drugs Inhibit Matrix Metalloproteinase-2 via Suppression of the ERK/Sp1-mediated Transcription

Our previous data showed that nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit matrix metalloproteinase-2 (MMP-2) expression via repression of gene transcription in lung cancer cells. In this study, we investigate the molecular mechanism by which NSAIDs inhibit MMP-2. Promoter deletion and mutation analysis indicate that NSAIDs act via the Sp1 transcription factor binding site located between −91 and −84 in the MMP-2 promoter to suppress gene expression. Electrophoretic mobility shift assays show that Sp1 and Sp3 proteins constitutively bind to this consensus sequence and overexpression of Sp1 may enhance MMP-2 expression. NSAID treatment reduces Sp1 DNA binding activity and phosphorylation and attenuates MMP-2 expression. We also investigate the signaling pathway that mediates the effect of NSAIDs. Our results suggest that ERKs are involved in this process. First, NSAIDs suppress basal and serum-stimulated ERK activity. Second, a MEK inhibitor PD98059 inhibits MMP-2 promoter activity and Sp1 phosphorylation. Third, overexpression of constitutively active MEK1 stimulates Sp1 phosphorylation and MMP-2 promoter activity and antagonizes the inhibition of NSAIDs. Collectively, our data suggest that NSAIDs inhibit MMP-2 by blocking ERK/Sp1-mediated transcription. Our previous data showed that nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit matrix metalloproteinase-2 (MMP-2) expression via repression of gene transcription in lung cancer cells. In this study, we investigate the molecular mechanism by which NSAIDs inhibit MMP-2. Promoter deletion and mutation analysis indicate that NSAIDs act via the Sp1 transcription factor binding site located between −91 and −84 in the MMP-2 promoter to suppress gene expression. Electrophoretic mobility shift assays show that Sp1 and Sp3 proteins constitutively bind to this consensus sequence and overexpression of Sp1 may enhance MMP-2 expression. NSAID treatment reduces Sp1 DNA binding activity and phosphorylation and attenuates MMP-2 expression. We also investigate the signaling pathway that mediates the effect of NSAIDs. Our results suggest that ERKs are involved in this process. First, NSAIDs suppress basal and serum-stimulated ERK activity. Second, a MEK inhibitor PD98059 inhibits MMP-2 promoter activity and Sp1 phosphorylation. Third, overexpression of constitutively active MEK1 stimulates Sp1 phosphorylation and MMP-2 promoter activity and antagonizes the inhibition of NSAIDs. Collectively, our data suggest that NSAIDs inhibit MMP-2 by blocking ERK/Sp1-mediated transcription. matrix metalloproteinase nonsteroidal anti-inflammatory drug fetal calf serum electrophoretic mobility shift assay extracellular signal-regulated kinase mitogen-activated protein kinase/extracellular signal-regulated kinase kinase The matrix metalloproteinases (MMPs)1 are a family of zinc-dependent endopeptidases that can selectively degrade components of the extra cellular matrix (1Sterler-Stevenson W.G. Hewitt R. Corcoran M. Cancer Biol. 1996; 7: 147-154Crossref PubMed Scopus (339) Google Scholar). The MMP family consists of at least 20 enzymes and may be subgrouped into different types including collagenases, stromelysins, gelatinases, matrilysin, membrane-type MMPs, and metalloelastase, based on sequence characteristics and substrate specificity (2Chambers A.F. Matrisian L.M. J. Natl. Cancer Inst. 1997; 89: 1260-1270Crossref PubMed Scopus (1423) Google Scholar). MMPs are synthesized as inactive precursors and are activated by proteolytic cleavage (3Nagase H. Woessner J.F, Jr. J. Biol. Chem. 1999; 274: 21491-21494Abstract Full Text Full Text PDF PubMed Scopus (3826) Google Scholar, 4Kleiner D.E.J. Sterler-Stevenson W.G. Curr. Opin. Cell Biol. 1993; 5: 891-897Crossref PubMed Scopus (327) Google Scholar, 5Brew K. Dinakarpandian D. Nagase H. Biochim. Biophys. Acta. 2000; 1477: 267-283Crossref PubMed Scopus (1575) Google Scholar). Additionally, naturally occurring tissue inhibitor of metalloproteinases can bind and suppress MMP function (6Wang Z. Juttermann R. Soloway P.D. J. Biol. Chem. 2000; 275: 26411-26415Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 7Caterina J.J. Yamada S. Caterina N.C. Longenecker G. Holmback K. Shi J. Yermovsky A.E. Engler J.A. Birkedal-Hansen H. J. Biol. Chem. 2000; 275: 26416-26422Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Therefore, MMP activity can be regulated at three steps: (a) gene expression, (b) proenzyme processing, and (c) inhibition of enzymatic activity. Among the human MMPs reported previously, MMP-2 (gelatinase-A) and MMP-9 (gelatinase-B) show substrate specificity toward type IV collagen, the major component of basement membrane, and their expressions are strongly linked with tumor metastasis in various types of human cancer (8Uhm J.H. Dooley N.P. Villemure J.G. Yong V.W. Clin. Exp. Metastasis. 1996; 14: 421-433Crossref PubMed Scopus (127) Google Scholar, 9Qin H. Moellinger J.D. Wels A. Windsor L.J. Sun Y. Meltzer S.J. J. Immunol. 1998; 161: 6664-6673PubMed Google Scholar, 10Callejas N.A. Casado M. Diaz-Guerra M.J.M. Bosca L. Martin-Sanz P. Hepatology. 2001; 33: 860-867Crossref PubMed Scopus (105) Google Scholar). Recent work showed a positive correlation between MMP-2 expression and tumor metastasis in human lung cancer (11Brown P.D. Bloxidge R.E. Stuart N.S.A. Gatter K.C. Carmichael J. J. Natl. Cancer Inst. 1993; 85: 574-578Crossref PubMed Scopus (281) Google Scholar). In addition, overexpression of MMP-2 is associated with unfavorable outcome and tumor recurrence in early stage lung cancer (12Cai M. Onoda K. Takao M. Kyoko I.Y. Shimpo H. Yoshida T. Yada I. Clin. Cancer Res. 2002; 8: 1152-1156PubMed Google Scholar, 13Passlick B. Sienel W. Seen-Hibler R. Wockel W. Thetter O. Mutschler W. Pantel K. Clin. Cancer Res. 2000; 6: 3944-3948PubMed Google Scholar). The mechanisms of MMP-2 gene activation in human cancer cells are not well defined. Originally, the MMP-2 gene has been thought to be refractory to modulation due to a lack of potential regulatory elements in the MMP-2 promoter (14Manviel A. J. Cell. Biochem. 1993; 53: 288-295Crossref PubMed Scopus (396) Google Scholar). However, a recent study demonstrated that the human MMP-2 promoter contains a number of cis-acting regulatory elements and that several transcription factors including Sp1, Sp3, and Ap-2 participate in the control of constitutive MMP-2 gene expression (15Qin H. Sun Y. Benveniste E.N. J. Biol. Chem. 1999; 274: 29130-29137Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). In addition, expression of MMP-2 can be modulated by different signal transduction pathways. For example, activation of the AKT kinase signaling pathway may stimulate MMP-2 activity and tumor invasion (16Park B.K. Zeng X. Glazer R.I. Cancer Res. 2001; 61: 7647-7653PubMed Google Scholar), whereas activation of the phosphatase and tensin homologue PTEN, a dual specificity phosphatase that effectively attenuates AKT activity, may suppress MMP-2 expression and metastasis (17Koul D. Parthasarathy R. Shen R. Davies M.A. Chintala S.K. Rao J.S. Sun Y. Benvenisite E.N. Liu T.J. Yung W.K. Oncogene. 1999; 20: 6669-6678Crossref Scopus (115) Google Scholar). Recent studies showed that nonsteroidal anti-inflammatory drugs (NSAIDs) exert potent anti-angiogenesis and anti-metastasis activity both in vitro and in vivo (18Masferrer J.L. Leahy K.M. Koki A.T. Zweifel B.S. Settle S.L. MarkWoerner S.B. Edwards D.A. Flickinger A.G. Moore R.J. Seilbert K. Cancer Res. 2000; 60: 1306-1311PubMed Google Scholar). Our previous study demonstrated that A549 human lung cancer cells expressed a high level of MMP-2, and two NSAIDs, NS398 and indomethacin, inhibited MMP-2 expression and enzymatic activity in these cells (19Pan M.R. Chuang L.Y. Hung W.C. FEBS Lett. 2001; 508: 365-368Crossref PubMed Scopus (70) Google Scholar). We also showed that these two NSAIDs suppressed MMP-2 via inhibition of gene transcription. In this study, we address the molecular mechanism by which NSAIDs inhibit MMP-2 expression. A549 human lung cancer cells were cultured in Dulbecco’s modified Eagle’s medium and F-12 nutrition mixture supplemented with 10% heat-inactivated fetal calf serum (FCS) and antibiotics. NS398 and indomethacin were purchased from Biomol Co. (Plymouth Meeting, PA). Antibodies against human Sp1 and Sp3 were from obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-ERK and anti-phospho-ERK antibody were obtained from New England Biolabs (Beverly, MA). Anti-phosphoserine antibody was obtained from Sigma. LipofectAMINE reagent was from Invitrogen, and luciferase assay and β-galactosidase assay systems were from Promega (Madison, WI). Full-length human MMP-2 promoter-luciferase construct and a series of deletion or mutant constructs were created as described previously (15Qin H. Sun Y. Benveniste E.N. J. Biol. Chem. 1999; 274: 29130-29137Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar) and were kindly provided by Dr. E. N. Benveniste. CMV-Sp1, a expression vector in which Sp1 expression is under the control of the CMV promoter, was kindly provided by Dr. S. T. Smale (20Emami K.H. Navarre W.W. Smale S.T. Mol. Cell. Biol. 1995; 15: 5906-5916Crossref PubMed Scopus (105) Google Scholar). The constitutively active MEK1 vector CMV-MEK1 (21Mansour S.J. Matten W.T. Hermann A.S. Candia J.M. Rong S. Fukasawa K. Vande Woude G.F. Ahn N.G. Science. 1994; 265: 966-970Crossref PubMed Scopus (1253) Google Scholar) was a gift from Dr. M. Z. Lai. Activity of full-length or mutant MMP-2 promoter constructs was analyzed as described previously (22Lee T.H. Chuang L.Y. Hung W.C. Oncogene. 1999; 18: 4269-4274Crossref PubMed Scopus (33) Google Scholar). In brief, cells were plated onto six-well plates at a density of 100,000 cells/well and grown overnight. Cells were co-transfected with 2 μg of MMP-2 promoter constructs and 1 μg of pCMV-β-galactosidase plasmid for 5 h by the LipofectAMINE method. After transfection, cells were cultured in 10% FCS medium with vehicle (Me2SO) or drugs for 24 h. Luciferase and β-galactosidase activities were assayed by using the assay systems according to the procedures of the manufacturer (Promega). Luciferase activity was normalized for β-galactosidase activity in cell lysate and expressed as an average of three independent experiments. In some experiments, CMV-Sp1 or CMV-MEK1 was co-transfected with MMP-2 promoter constructs by using a similar transfection procedure. Preparation of nuclear extracts from control or drug-treated cells were carried out as described previously (23Lee T.H. Chuang L.Y. Hung W.C. Oncogene. 2000; 19: 3766-3773Crossref PubMed Scopus (62) Google Scholar). The oligonucleotide 5′-CAGAGAGGGGCGGCCCGAGTG-3′, corresponding to the human MMP-2 promoter sequence −97 to −76 was used as probe. The mutant Sp1 oligonucleotide has the sequence 5′-CAGATATCTAGATGATATCGTG-3′. The reaction mixture for EMSA contained 20 mm Tris-HCl, pH 7.6, 1 mmdithiothreitol, 2 mm MgCl2, 1 mmEDTA, 10% glycerol, 1% Nonidet P-40, 1 μg of poly(dI-dC), and 8–10 μg of nuclear proteins. Unlabeled wild type or mutant oligonucleotides or preimmune, Sp1, or Sp3 antibody was added into reaction mixture and incubated for 10 min at room temperature.32P-Labeled probe DNA (300,000 cpm) was added, and the binding reaction was allowed to proceed for another 20 min. After reaction, mixtures were resolved on 6% polyacrylamide gels at 200 V for 2 h. Gels were dried and subjected to autoradiography. Cells cultured in 10% FCS medium were treated with vehicle or drugs for 24 h, washed with phosphate- buffered saline, and harvested in a lysis buffer as described previously (24Hung W.C. Chang H.C. Pan M.R. Lee T.H. Chuang L.Y. Mol. Pharmacol. 2000; 58: 1398-1403Crossref PubMed Scopus (75) Google Scholar). Protein concentration was measured by using a BCA protein assay kit (Pierce), and an equal amount of proteins was subjected to SDS-PAGE. Proteins were then transferred to nitrocellulose membranes, and Sp1 or Sp3 protein levels were examined by probing the membranes with different primary antibodies. Enhanced chemiluminescence reagents were used to depict the protein bands on the membranes. For immunoprecipitation, equal amounts of cell proteins prepared as described above were incubated with Sp1 or Sp3 antibody at 4 °C for 4 h, and immunocomplexes were collected by adding protein A/G-agarose beads for another 1 h. The immunoprecipitates were resolved by SDS-PAGE, and the membranes were probed with anti-phosphoserine antibody to investigate the phosphorylation status of Sp1 or Sp3 protein. To test the effect of NSAIDs on serum- stimulated ERK activity, cells were first serum-starved for 24 h and treated with vehicle or drugs under serum-free conditions for another 24 h. Cells were then restimulated with 10% FCS for different times. After treatment, cellular proteins were extracted and were subjected to SDS-PAGE. ERK activation was investigated by probing the membranes with anti-phospho-ERK antibody. Equal loading of proteins in each lane was confirmed by probing the membranes with anti-ERK antibody. In some experiments, cells cultured under 10% FCS were incubated with vehicle or drugs for 24 h and harvested for analysis of the effect of NSAIDs on basal ERK activity in cells. To determinate the critical regions in the MMP-2 promoter responsible for the transcriptional inhibition by NSAIDs, a series of 5′-deletion mutants based on the 1659-bp MMP-2 promoter were transfected into A549 cells, and the effect of NS938 and indomethacin on the promoter activity was examined. Our previous data have shown that NS398 and indomethacin inhibited MMP-2 promoter activity in a concentration-dependent manner. In this study, we routinely used 100 μm of NS398 and indomethacin to treat cells. As shown in Fig. 1, NS398 and indomethacin at this concentration inhibited about 80% of MMP-2 promoter activity when compared with that of vehicle-treated cells. MMP-2 deletion constructs consisting of 1050 bp or bp to the transcription site were to NS398 and the effect of these two NSAIDs was in the and deletion Our data that the is located between and in the MMP-2 We that a potential Sp1 consensus is located this −91 to Therefore, we mutation of this Sp1 binding site the to NSAIDs. NS398 and indomethacin not inhibit promoter activity of the construct results suggest that NSAIDs inhibit MMP-2 expression via the Sp1 site located at −91 to −84 in the of the Sp1 site the effect of NSAIDs on MMP-2 promoter activity. Cells were transfected with deletion construct of MMP-2 promoter or deletion construct in which the Sp1 site located between −91 and −84 in the promoter was After transfection, cells were treated with vehicle or 100 μm of NS398 or indomethacin for 24 h. Cells were harvested for the of luciferase and β-galactosidase activity. The activity of vehicle-treated cells is as and the activity of or cells is expressed as a of the activity of vehicle-treated cells. are the from three independent To inhibition of MMP-2 promoter activity by NSAIDs is by in the between nuclear proteins with the promoter, EMSA assays were with oligonucleotides corresponding to the to of the wild type Our results that nuclear proteins constitutively to the oligonucleotides and two and were in the NSAID treatment the binding of nuclear proteins to the Our data also showed that of nuclear proteins with the is the binding be by an of oligonucleotides not by mutant We the nuclear factors that bind to this are proteins. As shown in Fig. the of Sp1 antibody the signal of not the signal of the Sp3 antibody inhibited the of not I. The of antibody lane effect on the of both data suggest that Sp1 and Sp3 proteins constitutively bind to the Sp1 site located between bp −91 and −84 in the MMP-2 promoter, and this binding is suppressed by NSAIDs. We also investigated the of Sp1 and Sp3 binding by NSAID is due to of Sp1 or Sp3 proteins. or drug-treated cells were and cellular proteins were extracted for Our data showed that the protein level of Sp1 or Sp3 was not treatment of NS398 and indomethacin Therefore, NSAIDs Sp1 binding activity via Sp1 and Sp3 are we investigated NSAIDs the phosphorylation status of Sp1 and or drug-treated cells were and Sp1 or Sp3 was by antibody and protein A/G-agarose The immunoprecipitates were subjected to and probed with anti-phosphoserine antibody. As shown in Fig. phosphorylation of Sp1 and Sp3 was NSAID inhibit not protein level of Sp1 and cells were cultured in the vehicle or NS398 or indomethacin for 24 h, and cellular proteins were or were used to probe the nitrocellulose membranes and to investigate the level of these two proteins. cells were treated as described and Sp1 or Sp3 proteins were by of antibody and protein The immunoprecipitates were subjected to and the membranes were probed with anti-phosphoserine antibody to investigate the phosphorylation status of Sp1 and The membranes were also probed with or antibody to that similar amounts of Sp1 or Sp3 proteins were in the To Sp1 MMP-2 promoter activity, different amounts of CMV-Sp1 expression vector were co-transfected with or MMP-2 promoter As shown in CMV-Sp1 stimulated MMP-2 promoter activity in a manner. CMV-Sp1 also activated the promoter construct CMV-Sp1 not promoter activity Our results the that Sp1 is a for the MMP-2 We the signaling pathway by which NSAIDs inhibit MMP-2 expression. Recent studies indicate that DNA binding activity of Sp1 can be regulated by phosphorylation. of the protein that may Sp1 and enhance DNA binding activity is M. A. Biochem. Biophys. Res. 1999; PubMed Scopus Google Scholar). also demonstrated that various NSAIDs the mitogen-activated protein kinase signaling R.J. Cancer Res. 2001; 61: Google Scholar, M. Cancer Res. 2002; Scholar). Therefore, we test NS398 can ERK activity in A549 cells. Our results showed that NS398 and indomethacin inhibited basal ERK activity Additionally, of NS398 inhibited serum-stimulated ERK activation results were in cells not NSAIDs suppress MMP-2 expression via inhibition of ERK is that of ERK activity be to the effect of NSAIDs. To test this MMP-2 promoter construct was co-transfected with the CMV-MEK1 which expressed the constitutively active MEK1 under control of the CMV promoter, and the effect of NSAIDs on the MMP-2 promoter activity was As shown in ERK activity was in cells transfected with CMV-MEK1 vector not control We that expression of active MEK1 the inhibition of MMP-2 by NSAIDs results suggest that NSAIDs inhibit MMP-2 via of ERK activity. Our data showed that ERK and Sp1 are involved in the of MMP-2. Therefore, we two First, modulation of ERK activity may MMP-2 expression in A549 cancer cells. We investigated the effect of a MEK inhibitor PD98059 on MMP-2 promoter activity, and we that PD98059 inhibited MMP-2 promoter activity in a Additionally, treatment of PD98059 the phosphorylation of Sp1 in A549 cells We an of ERK activity by expression of CMV-MEK1 enhance basal MMP-2 promoter activity. The full-length MMP-2 promoter construct was co-transfected with CMV-MEK1 or control vector into A549 cells. We that a of MMP-2 promoter activity was in cells transfected with CMV-MEK1 transfection of CMV-MEK1 Sp1 phosphorylation Second, we investigated Sp1 is involved in MMP-2 expression. Our results that CMV-MEK1 activated the deletion construct not the deletion construct CMV-Sp1 also not the promoter activity not Collectively, our results suggest that the ERK signaling pathway MMP-2 expression via the Sp1 site in the promoter of ERK activation on MMP-2 promoter activity and Sp1 cells were co-transfected with 2 μg of full-length MMP-2 promoter or the or deletion construct and with 2 μg of control or CMV-MEK1 After transfection, cells were cultured in 10% FCS medium for 24 h and MMP-2 promoter activity was are the from three independent experiments. cells were transfected with control or CMV-MEK1 vector and were cultured in 10% FCS medium for 24 h. proteins were harvested for with antibody. The immunoprecipitates were subjected to and probed with anti-phosphoserine antibody to investigate Sp1 phosphorylation The membranes were also probed with antibody to that similar amounts of Sp1 proteins were in the In the study, we investigate the molecular mechanism by which NSAIDs inhibit MMP-2 expression, and our results show that of MMP-2 by NSAIDs is via the signaling of this work are First, recent studies have that NSAIDs potent and activity both in in vivo (18Masferrer J.L. Leahy K.M. Koki A.T. Zweifel B.S. Settle S.L. MarkWoerner S.B. Edwards D.A. Flickinger A.G. Moore R.J. Seilbert K. Cancer Res. 2000; 60: 1306-1311PubMed Google Scholar, K.M. H. E. A.S. 1999; 5: PubMed Scopus Google Scholar). However, the molecular mechanisms of these are Our results indicate that NSAIDs may suppress MMP-2 gene transcription. Additionally, inhibition of another MMP involved in by NSAIDs has also been demonstrated A.T. Cancer Res. 2000; 60: Google Scholar). on these we suggest that of MMPs is of the mechanisms by which NSAIDs inhibit and Second, we have the mechanism by which NSAIDs inhibit MMP-2. Our results the first that NSAIDs suppress MMP-2 expression via inhibition of the signaling In with our a recent study demonstrated that Sp1 and Sp3 are for constitutive MMP-2 gene expression in human cells and that the Sp1 site located between bp −91 and −84 in the promoter is critical for constitutive activity of the MMP-2 gene (15Qin H. Sun Y. Benveniste E.N. J. Biol. Chem. 1999; 274: 29130-29137Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). Additionally, we that Sp1 is a for MMP-2 gene expression, overexpression of Sp1 stimulates MMP-2 promoter activity in A549 cells. we also the effect of Sp1 and Sp3 on MMP-2 promoter activity cells In with the results of (15Qin H. Sun Y. Benveniste E.N. J. Biol. Chem. 1999; 274: 29130-29137Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar), we that Sp1 and Sp3 are potent for MMP-2 not Recent studies indicate that DNA binding activity and activity of Sp1 can be regulated by as phosphorylation and J.D. J. J. Cell. 2001; Scopus Google Scholar, G. 1999; PubMed Scopus Google Scholar). our results showed that Sp1 DNA binding activity not the protein level was NSAID treatment, we that NSAID may a signaling pathway to Sp1 Our data this and suggest that the ERK signaling pathway is the for NSAIDs. We that of MMP-2 expression by NSAIDs was with inhibition of ERK activity and that overexpression of constitutively active MEK1 inhibition of ERK activity and MMP-2 expression. However, be that expression of active MEK1 the effect of is that NSAIDs may signaling to MMP-2 gene transcription. that has been in this study is the ERK signaling pathway MMP-2 expression. including have that overexpression of constitutively active MEK1 can ERK activity and stimulate MMP-2 expression in transfected cells R. G. H. Cell 1999; Google Scholar, H. S. T. K. N. M. Exp. Cell Res. 2000; PubMed Scopus Google Scholar). However, the mechanism by which ERK MMP-2 has not been Our demonstrated that CMV-MEK1 the deletion the deletion construct and mutation construct were not stimulated by In addition, we also demonstrated that modulation of ERK activity by PD98059 or CMV-MEK1 is with the of Sp1 phosphorylation in A549 lung cancer our results strongly suggest that of MMP-2 expression by the ERK signaling pathway is via modulation of Sp1 DNA binding activity. that are and 10 potential ERK phosphorylation in Sp1 and Sp3 We are the in which phosphorylation are by ERK activation and are suppressed by NSAID ERK is a of various signaling pathways. of different signaling as mutation of or overexpression of which are in human lung cancer cells Curr. Opin. 2000; PubMed Scopus Google Scholar, Hung J. K. J.A. Cancer Res. Google Scholar), may stimulate ERK activity and MMP-2 expression to and of cancer cells. Therefore, the ERK signaling pathway a for of tumor and Collectively, results from this work indicate that the signaling pathway the constitutive activity of the MMP-2 gene in lung cancer cells and NSAIDs suppress MMP-2 by the ERK/Sp1-mediated transcription. We Dr. E. N. Dr. S. T. and Dr. M. Z. for