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• W2080647590 abstract "Reactive oxygen species have been shown to play an important role in the regulation of distinct signaling cascades, many of which act upon the production of matrix metalloproteinases (MMP). Using a series of redox-engineered cell lines we have previously demonstrated that MMP-1 expression is sensitive to the alterations in the steady state production of H2O2 (Ranganathan, A. C., Nelson, K. K., Rodriguez, A. M., Kim, K. H., Tower, G. B., Rutter, J. L., Brinckerhoff, C. E., Epstein, C. J., Huang, T. T., Jeffrey, J. J., and Melendez, J. A. (2001) J. Biol. Chem. 276, 14264–14270). In the present study, we investigate the molecular mechanisms involved in the H2O2-mediated induction of MMP-1. Mutational analysis of an MMP-1 promoter indicates that both the single nucleotide polymorphism creating an Ets binding site at –1607 and a proximal AP-1 site at –1602 are required for maximal H2O2-dependent transcription. The redox-sensitive MMP-1 protein expression requires activation of both ERK1/2 and JNK pathways. Importantly, JNK signaling is largely responsible for the H2O2 sensitivity of the MMP-1 promoter, whereas ERK1/2 contributes to both its basal and H2O2 dependence. H2O2 control of Ets-1 expression was ERK1/2-dependent whereas that of c-Jun requires both ERK1/2 and JNK signaling. Chromatin immunoprecipitation assays indicate that binding of the histone acetyltransferase, p300, and the transcription factors Ets-1 and c-Jun to the MMP-1 promoter is redox sensitive. The redox sensitivity of MMP-1 expression is also associated with an increase in the abundance of oxidatively inactivated protein-tyrosine phosphatases. Targeted cytosolic or mitochondrial scavenging of H2O2 prevented all of the aforementioned signals. These studies provide substantial insight into the mechanisms underlying the redox-dependent control of MMP-1 and may lead to the development of novel targeted antioxidant-based inhibitory therapies for controlling MMP-1 expression during degenerative disease processes. Reactive oxygen species have been shown to play an important role in the regulation of distinct signaling cascades, many of which act upon the production of matrix metalloproteinases (MMP). Using a series of redox-engineered cell lines we have previously demonstrated that MMP-1 expression is sensitive to the alterations in the steady state production of H2O2 (Ranganathan, A. C., Nelson, K. K., Rodriguez, A. M., Kim, K. H., Tower, G. B., Rutter, J. L., Brinckerhoff, C. E., Epstein, C. J., Huang, T. T., Jeffrey, J. J., and Melendez, J. A. (2001) J. Biol. Chem. 276, 14264–14270). In the present study, we investigate the molecular mechanisms involved in the H2O2-mediated induction of MMP-1. Mutational analysis of an MMP-1 promoter indicates that both the single nucleotide polymorphism creating an Ets binding site at –1607 and a proximal AP-1 site at –1602 are required for maximal H2O2-dependent transcription. The redox-sensitive MMP-1 protein expression requires activation of both ERK1/2 and JNK pathways. Importantly, JNK signaling is largely responsible for the H2O2 sensitivity of the MMP-1 promoter, whereas ERK1/2 contributes to both its basal and H2O2 dependence. H2O2 control of Ets-1 expression was ERK1/2-dependent whereas that of c-Jun requires both ERK1/2 and JNK signaling. Chromatin immunoprecipitation assays indicate that binding of the histone acetyltransferase, p300, and the transcription factors Ets-1 and c-Jun to the MMP-1 promoter is redox sensitive. The redox sensitivity of MMP-1 expression is also associated with an increase in the abundance of oxidatively inactivated protein-tyrosine phosphatases. Targeted cytosolic or mitochondrial scavenging of H2O2 prevented all of the aforementioned signals. These studies provide substantial insight into the mechanisms underlying the redox-dependent control of MMP-1 and may lead to the development of novel targeted antioxidant-based inhibitory therapies for controlling MMP-1 expression during degenerative disease processes. The matrix metalloproteinase (MMP) 5The abbreviations used are: MMP, matrix metalloproteinase; JNK, c-Jun N-terminal kinase; ERK1/2, extracellular signal-regulated kinase 1/2; SNP, single nucleotide polymorphism; NAC, N-acetyl-l-cysteine; PBS, phosphate-buffered saline; ChIP, chromatin immunoprecipitation assay; MAPK, mitogen-activated protein kinase; PTP, proteintyrosine phosphatase; ROS, reactive oxygen species; CMV, cytomegalovirus. family consists of at least 25 zinc-dependent proteases that degrade multiple components of the extracellular matrix (ECM) (1Wenk J. Brenneisen P. Wlaschek M. Poswig A. Briviba K. Oberley T.D. Scharffetter-Kochanek K. J. Biol. Chem. 1999; 274: 25869-25876Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Under normal physiological conditions MMPs are important for development and wound healing; however, their augmented expression is associated with numerous disease pathologies (2Stamenkovic I. J. Pathol. 2003; 200: 448-464Crossref PubMed Scopus (896) Google Scholar). For example, MMPs are important at many stages of metastasis and the high level expression of the MMP family member, MMP-1, has been linked to poor prognosis in many types of cancers (3Gao C.Q. Sawicki G. Suarez-Pinzon W.L. Csont T. Wozniak M. Ferdinandy P. Schulz R. Cardiovasc. Res. 2003; 57: 426-433Crossref PubMed Scopus (121) Google Scholar, 4Hart C.A. Scott L.J. Bagley S. Bryden A.A. Clarke N.W. Lang S.H. Br. J. Cancer. 2002; 86: 1136-1142Crossref PubMed Scopus (43) Google Scholar, 5Kuruganti P.A. Wurster R.D. Lucchesi P.A. J. Neurooncol. 2002; 56: 109-117Crossref PubMed Scopus (23) Google Scholar, 6Nagata M. Fujita H. Ida H. Hoshina H. Inoue T. Seki Y. Ohnishi M. Ohyama T. Shingaki S. Kaji M. Saku T. Takagi R. Int. J. Cancer. 2003; 106: 683-689Crossref PubMed Scopus (139) Google Scholar, 7Baker E.A. Bergin F.G. Leaper D.J. Br. J. Surg. 2000; 87: 1215-1221Crossref PubMed Scopus (112) Google Scholar, 8Nikkola J. Vihinen P. Vlaykova T. Hahka-Kemppinen M. Kahari V.M. Pyrhonen S. Melanoma Res. 2001; 11: 157-166Crossref PubMed Scopus (23) Google Scholar, 9Sheu B.C. Lien H.C. Ho H.N. Lin H.H. Chow S.N. Huang S.C. Hsu S.M. Cancer Res. 2003; 63: 6537-6542PubMed Google Scholar). Within the MMP-1 promoter, Brinckerhoff and co-workers (10Rutter J.L. Mitchell T.I. Buttice G. Meyers J. Gusella J.F. Ozelius L.J. Brinckerhoff C.E. Cancer Res. 1998; 58: 5321-5325PubMed Google Scholar) identified a single nucleotide polymorphism (SNP) guanine insertion at –1607 (1G → 2G) base pairs that enhances the basal rate of transcription by creation of an Ets binding domain. Ets transcription factors include a large family of helix-turn-helix proteins (11Sharrocks A.D. Nat. Rev. Mol. Cell. Biol. 2001; 2: 827-837Crossref PubMed Scopus (827) Google Scholar) that normally do not bind DNA alone, but preferentially form coactivator complexes with transcription factors, like activator protein-1 (AP-1) (12Westermarck J. Kahari V.M. FASEB J. 1999; 13: 781-792Crossref PubMed Scopus (1400) Google Scholar). The proto-oncoproteins Fos and Jun, which comprise the AP-1 complex, can homo- or heterodimerize and bind its cognate consensus sequence (TGACTCA) in the regulatory domains of many genes including various MMP family members (13Whitmarsh A.J. Davis R.J. J. Mol. Med. 1996; 74: 589-607Crossref PubMed Scopus (1397) Google Scholar). Both Ets and AP-1 play a critical role in regulating the expression of various MMP family members, particularly that of MMP-1 (14Westermarck J. Seth A. Kahari V.M. Oncogene. 1997; 14: 2651-2660Crossref PubMed Scopus (134) Google Scholar). The MMP-1 SNP is associated with a higher risk of metastasis in patients with a variety of distinct cancers (15Wood G.A. Archer M.C. Exp. Biol. Med. (Maywood). 2001; 226: 799-803Crossref PubMed Scopus (5) Google Scholar, 16Ye S. Dhillon S. Turner S.J. Bateman A.C. Theaker J.M. Pickering R.M. Day I. Howell W.M. Cancer Res. 2001; 61: 1296-1298PubMed Google Scholar, 17Noll W.W. Belloni D.R. Rutter J.L. Storm C.A. Schned A.R. Titus-Ernstoff L. Ernstoff M.S. Brinckerhoff C.E. Am. J. Pathol. 2001; 158: 691-697Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 18Zhu Y. Spitz M.R. Lei L. Mills G.B. Wu X. Cancer Res. 2001; 61: 7825-7829PubMed Google Scholar, 19Hinoda Y. Okayama N. Takano N. Fujimura K. Suehiro Y. Hamanaka Y. Hazama S. Kitamura Y. Kamatani N. Oka M. Int. J. Cancer. 2002; 102: 526-529Crossref PubMed Scopus (120) Google Scholar). More striking is the finding that both Ets-1 and MMP-1 immunoreactivity is high in stromal tissue adjacent to the leading edge of several tumor types (12Westermarck J. Kahari V.M. FASEB J. 1999; 13: 781-792Crossref PubMed Scopus (1400) Google Scholar). The two-electron reduction product of oxygen, H2O2 has emerged as a potent signaling molecule and can modulate MMP expression and activity (20Argiles J.M. Lopez-Soriano J. Busquets S. Lopez-Soriano F.J. FASEB J. 1997; 11: 743-751Crossref PubMed Scopus (128) Google Scholar). H2O2-sensitive signaling molecules ERK1/2 and JNK are important in regulating MMP-1 expression (12Westermarck J. Kahari V.M. FASEB J. 1999; 13: 781-792Crossref PubMed Scopus (1400) Google Scholar, 21Frost J.A. Geppert T.D. Cobb M.H. Feramisco J.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3844-3848Crossref PubMed Scopus (203) Google Scholar, 22Han Z. Boyle D.L. Chang L. Bennett B. Karin M. Yang L. Manning A.M. Firestein G.S. J. Clin. Investig. 2001; 108: 73-81Crossref PubMed Scopus (722) Google Scholar). Furthermore, numerous reports indicate that transcription factors important for MMP-1 expression are also redox-sensitive (23Clerk A. Michael A. Sugden P.H. Biochem. J. 1998; 333: 581-589Crossref PubMed Scopus (162) Google Scholar, 24Sanij E. Hatzistavrou T. Hertzog P. Kola I. Wolvetang E.J. Biochem. Biophys. Res. Commun. 2001; 287: 1003-1008Crossref PubMed Scopus (42) Google Scholar). Jun-N-terminal kinase (JNK) phosphorylates and activates AP-1 members including c-Jun and JunB (25Reddy S.P. Mossman B.T. Am. J. Physiol. Lung Cell Mol. Physiol. 2002; 283: L1161-L1178Crossref PubMed Scopus (137) Google Scholar) whereas c-Jun, c-Fos, FosB, Fra-1, and Ets family member, Ets-1, are sensitive to ERK activation (25Reddy S.P. Mossman B.T. Am. J. Physiol. Lung Cell Mol. Physiol. 2002; 283: L1161-L1178Crossref PubMed Scopus (137) Google Scholar). Because both AP-1 and Ets transcription factors are subject to regulation by JNK and/or ERK and are redox-regulated, we sought to determine the role these factors play in the H2O2 -dependent regulation of MMP-1. Using well characterized redox-engineered HT-1080 fibrosarcoma cell lines (26Rodriguez A.M. Carrico P.M. Mazurkiewicz J.E. Melendez J.A. Free Radic. Biol. Med. 2000; 29: 801-813Crossref PubMed Scopus (115) Google Scholar) this study demonstrates that the proximal Ets and AP-1 binding sites in the MMP-1 promoter are required for maximal H2O2-dependent expression. We also established that JNK confers redox sensitivity to the MMP-1 promoter whereas both ERK and/or JNK are required for maximal basal promoter activity and the expression of AP-1 and Ets-1. Lastly, both c-Jun and Ets-1 and the histone acetyltransferase, p300 are recruited to the region of the MMP-1 SNP in response to alterations in the steady state production of H2O2. Thus, H2O2 plays an important role in regulating chromatin remodeling events that lead to optimal MMP-1 transcriptional activity. These findings indicate that diseases which are associated with augmented MMP-1 production may be amenable to targeted antioxidant based therapeutic intervention. Cell Culture and Reagents—All indicated cell lines were maintained in 25-cm2 flasks in MEM containing 10% fetal calf serum, 1000 units/ml penicillin, 500 μg/ml streptomycin, and 1 mg/ml neomycin in a 37 °C incubator containing 5% CO2. Constructions of the recombinant Sod2 and catalase plasmids and transfections were previously described in detail (26Rodriguez A.M. Carrico P.M. Mazurkiewicz J.E. Melendez J.A. Free Radic. Biol. Med. 2000; 29: 801-813Crossref PubMed Scopus (115) Google Scholar, 27Bai J. Rodriguez A.M. Melendez J.A. Cederbaum A.I. J. Biol. Chem. 1999; 274: 26217-26224Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar) and their ability to alter the steady state production of H2O2 has been demonstrated in detail. Cells were treated with recombinant human tumor necrosis factor (TNF) (R & D Systems, Minneapolis, MN), U0126, U0124, PD98059, SB203580 (Calbiochem, San Diego, CA), or SP600125 (Amersham Biosciences, Piscataway, NJ). Ebselen, N-acetyl-l-cysteine (NAC), Trolox (Oxis Int.), and butylated hydroxytoluene (BHT) were used at concentrations that have been reported to inhibit UVA-dependent MMP-1 expression (28Polte T. Tyrrell R.M. Free Radic. Biol. Med. 2004; 36: 1566-1574Crossref PubMed Scopus (74) Google Scholar). The antioxidant porphyrin, was a kind gift of Dr. B. Kalayanaraman of the Medical College of Wisconsin. All reagents were obtained from Sigma unless otherwise indicated. Construction and Transient Transfection of MMP-1 Deletion Constructs—The full-length human MMP-1 promoter/luciferase reporter plasmids (1G and 2G) contained the firefly luciferase gene under the transcriptional control of the human MMP-1 promoter in a pGL3 basic reporter vector (Promega, Madison, WI) and were kindly provided by Dr. Constance Brinckerhoff (Dartmouth University). Transfection and analysis of MMP-1 promoter luciferase activity have been described in detail by Nelson et al. (29Nelson K.K. Ranganathan A.C. Mansouri J. Rodriguez A.M. Providence K.M. Rutter J.L. Pumiglia K. Bennett J.A. Melendez J.A. Clin. Cancer Res. 2003; 9: 424-432PubMed Google Scholar). Treatments with MAPK inhibitors, PD98059 (50 μm), SB203580 (20 μm), U0126 (50 μm), U0124 (50 μm), and SP600125 (10 μm) were performed 4-h post-transfection for 18 h following removal and addition of complete medium. Construction of Mutant AP-1, Sod2, and Scrambled 1G MMP-1 Promoter Constructs—An active site mutant form of human MnSOD (H26L) (abbreviated mutSod2) was generated by the Dr. Larry W. Oberley laboratory at the University of Iowa (Iowa City, IA), and shown to have ∼70% less activity compared with wild-type human MnSOD (30Kim A. Oberley L.W. Oberley T.D. Free Radic. Biol. Med. 2005; 39: 1128-1141Crossref PubMed Scopus (15) Google Scholar). The mutant 1G and AP-1 promoter constructs were prepared by changing the wild-type 1G from 5′-AAAGATATGACTTA-3′ to the form 5′-AAACTTATGACTTA-3′ (m1G) or to the mutant AP-1 and m1G form 5′-AAACTTAGTCATTA-3′ (m1G mAP). An AatII and EcoRV site located 5′ and 3′ to the mutation, respectively, allowed for digestion of flanking sequences and subsequent ligation into a previously digested pGL3-MMP-1 construct with ends compatible with the digested fragment. The respective sense and antisense primer pairs used for mutant construction are described below: AatII: sense, 5′-CAGTGTATGAGACTCTTCC-3′; EcoRV: antisense, 5′-CAGTGGAGAAACACTGGC-3′; 1G: scrambled, 5′-AATAATTAGAAACTTATGACTTAT-3′, 5′-ATAAGTCATAAGTTTCTAATTATT-3′; 1G and AP-1 double mutants, 5′-AATAATTAGAAACTTAGTCATTAT-3′, 5′-ATAATGACTAAGTTTCTAATTATT-3′. The corresponding products of these reactions were combined and used as the template for the AatII and the EcoRV primers to create the final fragment containing the appropriate mutant Ets or mutant Ets/mutant AP-1 sites. The native sites and mutations (bold) are as follows: 1GmAP-1, 5′-.... GATATGACTTATCT... -3′ 5′-... GATAGTCATTATCT... -3′, m1G 5′-.... TAGAAAGATATGA... -3′ 5′-... TAGAAACTTATGA... -3′, m1GmAP, 5′-.... TAGAAAGATATGACTTATCT... -3′ 5′-... TAGAAACTTAGTCATTATCT...-3′. All mutants were confirmed by DNA sequencing. Immunoblotting—Media from confluent cells were analyzed for MMP-1 by Western blotting. Complete media from the cells were normalized to cell count and incubated overnight at 4 °C with 50 μl of heparin-Sepharose beads (Amersham Biosciences). The beads were centrifuged at 1,000 rpm for 5 min and boiled for 5 min in Hanks Buffer Salt Solution, followed by the addition of 5× loading dye containing 5% 2-mercaptoethanol. Eluates were then analyzed on a 10% SDS-PAGE followed by Western immunoblotting using monoclonal MMP-1 antibody (R&D Systems) at 1:400 in Tris-buffered saline containing 0.1% Tween 20 and 5% milk followed by incubation with a horseradish peroxidase-conjugated anti-mouse secondary antibody at a 1:4000 dilution for 1 h at 25°C (Amersham Biosciences). Detection of the proteins was performed by the addition of Pierce SuperSignal Chemiluminescent Substrate for 5 min and exposure to Kodak MS radiographic film (Kodak, Rochester, NY). JNK immunoblotting was performed on cell lines grown to 90% confluence and washed three times with ice-cold 1× PBS. Nuclear preparation was performed for phosphospecific JNK, whereas whole cell lysates were used for total JNK Western blots. For total cell lysates, cells were resuspended in 1× PBS/EDTA and sonicated. Protein concentrations were determined using the BCA protein assay according to the manufacturer's instructions (Pierce). 30 μg of protein was resolved on 10% SDS-PAGE and immunoblotted as described above. Blots were incubated with a rabbit polyclonal phosphospecific JNK antibody (BIOSOURCE, Camarillo, CA) at 1:1000 in TTBS containing 5% milk followed by incubation with horseradish peroxidase-conjugated anti-rabbit IgG (Amersham Biosciences) at 1:100,000 for 1 h at room temperature. Immunoreactive JNK was visualized as described above. The immunoblots were stripped and reprobed using rabbit polyclonal antibody (BIOSOURCE) that recognizes JNK regardless of its phosphorylation state at a 1:1000 dilution overnight. The blot was washed and subsequently incubated with horseradish peroxidase-conjugated secondary anti-rabbit antibody (Amersham Biosciences) at 1:100,000, and immunoreactive protein was detected as described above. Fra-1, c-Jun, c-Fos, and Ets-1 were analyzed by Western blotting with either rabbit anti-human Fra-1, Ser63 c-Jun, Ser73 c-Jun, c-Jun, c-Fos monoclonal (broad or specific), or Ets-1 polyclonal antibodies, (Santa Cruz Biotechnology, Santa Cruz, CA) at 1:1000 dilution. Blots were then incubated with the appropriate secondary horseradish peroxidase-conjugated antibodies (1:5000) and developed as described above. Phospho-c-Jun blots were performed using nuclear lysates. In-gel Phosphatase Assay—In-gel phosphatase assays and phosphotyrosine immunoprecipitations were performed essentially as described by Meng et al. (31Meng T.C. Fukada T. Tonks N.K. Mol. Cell. 2002; 9: 387-399Abstract Full Text Full Text PDF PubMed Scopus (895) Google Scholar). Chromatin Immunoprecipitation (ChIP)—Confluent cells lines were treated with 37% formaldehyde to cross-link proteins to DNA for 10 min at 37 °C. 0.33 ml of 1.25 m glycine was added to the flasks to stop the reaction. The cells were washed twice with cold 1× PBS containing 1 μg/ml aprotinin, 1 μg/ml pepstatin, and 1 mm phenylmethylsulfonyl fluoride. The cells were scraped off in this buffer and pelleted at 2000 rpm for 4 min at 4 °C. 200 μl of warm SDS lysis buffer containing protease inhibitors was added and incubated for 10 min on ice. Cells were sonicated to generate ∼500-bp DNA fragments. Before adding antibody, 25 μl of lysate was saved as the input sample. Either 2 μl of Ets-1 (Santa Cruz Biotechnology) or c-Jun (Cell Signaling, Beverly, MA) antibody was added to the lysate with cold 1× PBS containing protease inhibitors and rocked overnight at 4 °C. The following day, 40 μl of protein A-agarose beads per sample (Santa Cruz Biotechnology) were washed three times with 1× PBS and twice with SDS lysis buffer, and beads were resuspended in lysis buffer containing protease inhibitors. 40 μl of beads was added to samples and incubated at 4 °C for 2 h with gentle agitation. Beads were washed twice with lysis buffer containing protease inhibitors, once with lysis buffer and 500 mm NaCl, once with 10 mm Tris-HCl, pH 8.0, 0.5 m LiCl, 0.5% IPEGAL, 0.5% sodium deoxycholate, 1 mm EDTA without protease inhibitors, and once with TE, pH 8.0. The precipitate was eluted from beads with 100 μl of 50 mm Tris-HCl, pH 8.0, 10 mm EDTA, 1% SDS for 15 min at 65 °C. Beads were pelleted for a few seconds at 14,000 rpm, and supernatant was transferred to a new tube. The eluates and input samples were incubated at 65 °C overnight to reverse cross-links. The following day, 250 μl of TE, 5 μg of glycogen, and 100 μg of proteinase K were added and incubated for 2 h at 37 °C. 55 μl of 4 m LiCl were added to each sample, and DNA was extracted using a standard phenol-chloroform method. The extracted DNA was kept in TE, pH 8.0 and subjected to PCR using MMP-1-specific primers to amplify the region between –1978 and –1523 of the MMP-1 promoter. Real Time ChIP PCR—One microliter of ChIP-derived DNA was used as template in a 20-μl reaction containing 10 μlof2× SYBR Green Master Mix (Bio-Rad). Real time thermal cycling was performed using a MyiQ Cycler thermal cycler (Bio-Rad), with continuous SYBR Green monitoring according to the manufacturer's recommendations, using iCycler software. All PCR reactions were performed in duplicate and included negative controls (no DNA). Pre-IP and post-IP DNA samples were amplified using previously described parameters. Ct values were obtained after completion of reaction, which is the cycle at which fluorescence rises above a baseline threshold. The DNA units in the samples were calculated as 2–(Ct IP–Ct Input) × 1000. Native Sod PAGE—PAGE was performed as described in detail by Rodriguez et al. (26Rodriguez A.M. Carrico P.M. Mazurkiewicz J.E. Melendez J.A. Free Radic. Biol. Med. 2000; 29: 801-813Crossref PubMed Scopus (115) Google Scholar). Statistical Analysis—Analysis of variance with α = 0.05 was used for processing the data. Two sample Student's t test was used as the post test. Values are expressed as means ± S.E. of the respective test or control group. Data are representative of at least three independent experiments. Identification of Redox-responsive MMP-1 Promoter Elements—We have previously reported that H2O2 enhances the activity of MMP-1 promoter containing the 2G SNP (29Nelson K.K. Ranganathan A.C. Mansouri J. Rodriguez A.M. Providence K.M. Rutter J.L. Pumiglia K. Bennett J.A. Melendez J.A. Clin. Cancer Res. 2003; 9: 424-432PubMed Google Scholar, 32Ranganathan A.C. Nelson K.K. Rodriguez A.M. Kim K.H. Tower G.B. Rutter J.L. Brinckerhoff C.E. Epstein C.J. Huang T.T. Jeffrey J.J. Melendez J.A. J. Biol. Chem. 2001; 276: 14264-14270Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Analysis of the full-length promoter containing the 2G polymorphism indicated that the region between –1702 and –1602 is required for optimal activation of the MMP-1 promoter by H2O2. Within this 100-bp region, there are many Ets and AP-1 binding sites including the Ets site created by the SNP at position –1607 and a very proximal AP-1 site at –1602 within the MMP-1 promoter. Given the importance of the 2G SNP in MMP-1 promoter activity and that of AP-1 in MMP-1 regulation, the impact of mutations of these sites was assessed. The activities of the full-length 1G and 2G MMP-1 promoter constructs were increased in response to the mitochondrial production of H2O2 in cells overexpressing Sod2 (Fig. 1A). Mutating the AP-1 site in the context of the 1G (1GmAP) or 2G (2GmAP) promoter decreased MMP-1 promoter activity. Mutation of the 1G region (m1G) or both the 1G and AP-1 sites (m1GmAP1) further decreased MMP-1 promoter activity (Fig. 1C). Thus, both the 1G region and the AP-1 binding sites are required for activation of the MMP-1 promoter both basally and in response to alterations in the steady state production of H2O2. H2O2 Regulates AP-1 and Ets-1 Expression—The levels of AP-1 factors play an important role in regulating the expression of AP-1 responsive genes. To define whether intracellular H2O2 affects the expression of transcription factors that control MMP-1 promoter activity we analyzed protein levels of both AP-1 and ETS-1 in the redox-engineered cell lines. Cultured media from the indicated cell lines were collected and examined for the level of secreted MMP-1 (Fig. 2, A and B). MMP-1 levels were increased in response to enforced expression of Sod2. In addition to the Sod2-overexpressing HT-1080 cells, catalase-overexpressing cell lines were developed to test the role of H2O2 on gene expression. We have directed catalase expression to both the cytosolic and mitochondrial compartments that are distinct from its normal peroxisomal location. In this fashion signaling components that are sensitive to either mitochondrial or cytosolic H2O2 can be defined. Coexpression of catalase in the Sod2-overexpressing cells did not completely attenuate the expression of immunoreactive MMP-1 (Fig. 2, A and B). Sod2 overexpression also increased the protein levels of many of the AP-1 family members including; c-Fos, phosphorylated Jun, and the Ets family member ets-1. The Sod2-dependent increase in the expression of both the AP-1 and Ets family members was reversed by coexpression of catalase in either the cytosolic or mitochondrial compartment (Fig. 2, A and C). However, the AP-1 family member fra-2 was insensitive to changes in the steady state production of H2O2 resulting from antioxidant enzyme overexpression. JNK historically has been studied as the primary kinase responsible for phosphorylating c-Jun (33Hsu T.C. Young M.R. Cmarik J. Colburn N.H. Free Radic. Biol. Med. 2000; 28: 1338-1348Crossref PubMed Scopus (251) Google Scholar). Analysis of phosphorylated and total forms of JNK showed that its phosphorylation state and protein expression are also redox-dependent, and these increases may account for the increase expression of the phosphorylated forms of c-Jun in response to Sod2 expression (Fig. 2, A and B). These data establish that many of the factors that are involved in regulating MMP-1 expression are also under redox control. However, only a partial reduction in Sod2-driven MMP-1 expression was observed in response to catalase overexpression even when both Ets-1 and c-Fos were significantly attenuated. The Antioxidants N-Acetyl Cysteine, Ebselen, and Antioxidant Porphyrin Treatments Restrict Sod2-mediated MMP-1 Expression—The failure of catalase overexpression to completely reverse the Sod2-mediated increase in MMP-1 expression, prompted us to evaluate the impact of various antioxidants on the redox-dependent induction of MMP-1. Treatment of cells with the glutathione precursor, N-acetyl cysteine, or the glutathione peroxidase mimetic ebselen completely reversed the Sod2-mediated increase in MMP-1 expression, whereas the lipid hydroperoxide scavengers trolox and butylated hydroxy toluene had a limited effect on MMP-1 expression. Treatment of Sod2-overexpressing cell lines with the redox-active porphyrin, an efficient H2O2 scavenger (34Konorev E.A. Kotamraju S. Zhao H. Kalivendi S. Joseph J. Kalyanaraman B. Free Radic. Biol Med. 2002; 33: 988-997Crossref PubMed Scopus (60) Google Scholar), also reversed the increase in MMP-1 expression in the Sod2-overexpressing cell lines. These findings indicate that efficient synthetic H2O2 scavengers can reverse the Sod2-dependent increases in MMP-1 expression. ERK1/2- and JNK-dependent Redox Regulation of MMP-1 and Transcription Factors—We have previously reported that ERK1/2 is critical for redox control of the MMP-1 promoter (32Ranganathan A.C. Nelson K.K. Rodriguez A.M. Kim K.H. Tower G.B. Rutter J.L. Brinckerhoff C.E. Epstein C.J. Huang T.T. Jeffrey J.J. Melendez J.A. J. Biol. Chem. 2001; 276: 14264-14270Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). ERK1/2 can enhance the expression or the phosphorylation state of Ets-1, c-Fos (35Seidel J.J. Graves B.J. Genes Dev. 2002; 16: 127-137Crossref PubMed Scopus (147) Google Scholar, 36McCarthy S.A. Chen D. Yang B.S. Ramirez J.J. Garcia Cherwinski H. Chen X.R. Klagsbrun M. Hauser C.A. Ostrowski M.C. McMahon M. Mol. Cell Biol. 1997; 17: 2401-2412Crossref PubMed Scopus (151) Google Scholar), and c-Jun (12Westermarck J. Kahari V.M. FASEB J. 1999; 13: 781-792Crossref PubMed Scopus (1400) Google Scholar), and impact gene expression. Whether the redox state of the cell modulates the expression of these factors via ERK1/2 has not been established. Analysis of MMP-1 immunoreactive protein in control and Sod2-overexpressing cell lines indicates that the redox-dependent increase in its production is sensitive to inhibition by the pharmacologic inhibitors of ERK1/2, PD98059, and U0126 (Fig. 3, A and B). Furthermore, redox-dependent MMP production was not significantly altered by the inactive U0126 analog, U0124, or the P38 MAP kinase inhibitor, SB203580 (Fig. 3B). The H2O2-dependent induction of Ets-1 and c-Fos was also attenuated by inhibition of ERK1/2 but not the inactive analog U0124 (Fig. 3A). Overexpression of Sod2 also increased c-Jun levels and was reversed by the ERK1/2 inhibition (data not shown). These findings suggest that H2O2 regulates the levels of key determinants involved in regulating MMP-1 transcription through ERK1/2. Thus, the redox-dependent increase in the levels of c-Fos, c-Jun, and Ets-1 are paralleled by an increase the extracellular production of MMP-1 that is ERK1/2-sensitive. To evaluate the influence of JNK on the redox-dependent control of Jun the various redox-engineered cell lines were treated with the pharmacological JNK inhibitor, SP600125 and phosphospecific c-Jun analyzed. Phosphorylation of Jun at Ser63 and Ser73 was increased in response to Sod2 overexpression relative to control cells, which was attenuated by overexpression of mitochondrial catalase but not cytosolic catalase(Fig. 4A). The JNK inhibitor decreased the phosphorylation of both serine residues suggesting that alterations in the steady state production of H2O2 regulate Jun phosphorylation th" @default.
• W2080647590 created "2016-06-24" @default.
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• W2080647590 title "Redox-dependent Matrix Metalloproteinase-1 Expression Is Regulated by JNK through Ets and AP-1 Promoter Motifs" @default.
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