Loss of NF-κB Control and Repression of Prdx6 Gene Transcription by Reactive Oxygen Species-driven SMAD3-mediated Transforming Growth Factor β Signaling (2009)

Activation of transforming growth factor β (TGFβ) in response to increased reactive oxygen species (ROS) leads to pathophysiology of cells/tissues by overmodulation of gene transcription. PRDX6 plays a rheostat role in regulating gene transcription by controlling cellular ROS in maintaining homeostasis; thus, fine tuning of Prdx6 expression is required to optimize ROS levels. Using Prdx6- and Smad3-depleted cells, we show that Prdx6−/− cells bear active NF-κB and Smad3, and repression of Prdx6 transcription in redox-active cells (Prdx6−/−) is due to ROS-induced dominant Smad3-mediated TGFβ signaling. The Prdx6 promoter (−1139 bp) containing repressive Smad3-binding elements and NF-κB sites showed reduced promoter activity in Prdx6−/− cells, and the activity was restored in Smad3−/− cells. Mutation of repressive Smad3-binding elements eliminated the repression of the Prdx6 promoter. Revival of promoter activity by application of TGFβ1 antibody to Prdx6−/− and Smad3−/− cells with increased Prdx6 mRNA and protein conferred resistance to TGFβ- and H2O2-induced insult, demonstrating that repression of Prdx6 transcription is Smad3-dependent. Promoter activity in Smad3−/− or Prdx6−/− cells was moderately increased by disruption of NF-κB sites, suggesting the role of NF-κB in tuning of Prdx6 expression. Findings revealed a mechanism of repression and regulation of PRDX6 expression in cells facing stress or aging and provided clues for antioxidant(s)-based new approaches in preventing ROS-driven deleterious signaling. PRDX6 (peroxiredoxin 6) is a member of an emerging peroxiredoxin family that has GSH peroxidase as well as acidic Ca2+-independent phospholipase A2 activities (1Fatma N. Kubo E. Sharma P. Beier D.R. Singh D.P. Cell Death Differ. 2005; 12: 734-750Crossref PubMed Scopus (75) Google Scholar, 2Fatma N. Singh D.P. Shinohara T. Chylack Jr., L.T. J. Biol. 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The peroxiredoxins function in concert to detoxify reactive oxygen species (ROS) 2The abbreviations used are: ROSreactive oxygen speciesLEClens epithelial cellSBESmad-binding elementRSBErepressive Smad3-binding elementTGFβtransforming growth factor βTIETGFβ-inhibitory elementCATchloramphenicol acetyltransferaseELISAenzyme-linked immunoassayNACN-acetylcysteineMTS3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2 to 4-sulfophenyl)-2H-tetrazolium saltAbantibodyH2-DCF-DA2′,7′-dichlorodihydrofluorescein diacetate. and play a cytoprotective role by removing ROS and by limiting ROS levels regulating cell survival signaling (1Fatma N. Kubo E. Sharma P. Beier D.R. Singh D.P. Cell Death Differ. 2005; 12: 734-750Crossref PubMed Scopus (75) Google Scholar, 3Fisher A.B. Dodia C. Manevich Y. Chen J.W. Feinstein S.I. J. Biol. Chem. 1999; 274: 21326-21334Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 4Kubo E. Fatma N. Akagi Y. Beier D.R. Singh S.P. Singh D.P. Am. J. 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However, given the role of ROS as a mediator of normal or redox signaling, we believe that optimal regulation of Prdx6 expression may require fine tuning to avoid overshooting the desired beneficial effects to the point of perturbing the delicate redox balance essential to maintenance of normal cellular function. Recently, using targeted inactivation of the Prdx6 gene, we reported activation and expression of TGFβ and TGFβ-mediated suppression of Prdx6 mRNA and protein (1Fatma N. Kubo E. Sharma P. Beier D.R. Singh D.P. Cell Death Differ. 2005; 12: 734-750Crossref PubMed Scopus (75) Google Scholar, 4Kubo E. Fatma N. Akagi Y. Beier D.R. Singh S.P. Singh D.P. Am. J. Physiol. Cell Physiol. 2008; 294: C842-C855Crossref PubMed Scopus (101) Google Scholar, 5Kubo E. Miyazawa T. Fatma N. Akagi Y. Singh D.P. Mech. Ageing Dev. 2006; 127: 249-256Crossref PubMed Scopus (50) Google Scholar, 31Gallagher B.M. Phelan S.A. Free Radic. Biol. Med. 2007; 42: 1270-1277Crossref PubMed Scopus (51) Google Scholar), suggesting that this molecule may have a role in repression of Prdx6 transcription. Quantification by staining with H2DCF-DA established a higher prevalence of ROS in these cells. Also, the activation of TGFβ in Prdx6-deficient cells was associated with ROS and inhibited by PRDX6 overexpression or MnTBAP, a superoxide dismutase mimetic and a known ROS inhibitor, in view of our earlier finding(s) that Prdx6-depleted cells bear higher levels of ROS and are thereby more susceptible to oxidative stress and apoptotic cell death (1Fatma N. Kubo E. Sharma P. Beier D.R. Singh D.P. Cell Death Differ. 2005; 12: 734-750Crossref PubMed Scopus (75) Google Scholar, 2Fatma N. Singh D.P. Shinohara T. Chylack Jr., L.T. J. Biol. Chem. 2001; 276: 48899-48907Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 3Fisher A.B. Dodia C. Manevich Y. Chen J.W. Feinstein S.I. J. Biol. 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Upon cell stimulation, IκBs become phosphorylated and subsequently ubiquitinylated. Released NF-κB translocates into the nucleus and binds to the target sequences and initiates transcription of "defense genes" involved in inflammatory responses and pathophysiology of cells and tissues. This molecule may sense ROS-induced stress, and its function may be altered in relation to the levels of ROS in cellular microenvironment. Earlier study has shown that proapoptotic and antiapoptotic roles of NF-κB depend on cell type and cell environment (57Uberti D. Carsana T. Francisconi S. Ferrari Toninelli G. Canonico P.L. Memo M. Biochem. Pharmacol. 2004; 67: 1743-1750Crossref PubMed Scopus (27) Google Scholar). Recently, two putative NF-κB sites have been predicted in the Prdx6 gene promoter (31Gallagher B.M. Phelan S.A. Free Radic. Biol. Med. 2007; 42: 1270-1277Crossref PubMed Scopus (51) Google Scholar) and have been thought to be involved in Prdx6 regulation. However, based on current study using transactivation and mutational analysis of the Prdx6 promoter coupled with Smad3- and Prdx6-deficient lens cells as models as well as biochemical assays, we think the control of NF-κB signaling in tuning of Prdx6 gene expression is attenuated in Prdx6-depleted cells (redox state) or aging cells, due to activation of Smad3-mediated TGFβ-induced repressive signaling. The process attenuates Nrf2 activation of Prdx6 gene transcription (58Chowdhury I. Mo Y. Gao L. Kazi A. Fisher A.B. Feinstein S.I. Free Radic. Biol. Med. 2009; 46: 146-153Crossref PubMed Scopus (121) Google Scholar) as well. Thus, autoregulation of Prdx6 disrupted in response to internal or external stressors. However, Web-based computer analysis (MatInspector; Genomatix) of the 5′-flanking region of the Prdx6 promoter revealed that it has various putative regulatory elements as follows: SP1, E2F-Myc, Nrf2, HIF1α, PBX1-MEIS1, EREs, GRE, GATA-1, HSF1, LEDGF, c-ETS-1, v-Myb, Oct1/Oct2, etc., including two sites of the NF-κB at −644 to −635 and −948 to −939 as well as two sites of RSBE (TIE) (45Chen C.R. Kang Y. Siegel P.M. Massagué J. Cell. 2002; 110: 19-32Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar, 47Frederick J.P. Liberati N.T. Waddell D.S. Shi Y. Wang X.F. Mol. Cell. Biol. 2004; 24: 2546-2559Crossref PubMed Scopus (190) Google Scholar) at −379 to −367 and −482 to −471 from the transcription start site. In the present investigation, using Prdx6−/−- and Smad3−/−-deficient lens epithelial cells (LECs) and a variety of biochemical assays, we examined the regulation of Prdx6 expression by ROS-driven Smad3-mediated TGFβ1-induced adverse signaling in cells facing oxidative stress, and we have shown this as a model to study the ROS-induced TGFβ1-mediated adverse signaling in LECs that may lead to degenerative disorders, including cataractogenesis. We identified two novel NF-κB-binding elements and two RSBE elements in the Prdx6 promoter and demonstrated how Smad3, a protein known to be involved in regulation of TGFβ target genes, may attenuate Prdx6 gene transcription and thereby negatively regulate survival signaling in cells in the redox state. Given the role of ROS as a mediator of normal signaling, we a rheostat role for NF-κB in regulation of Prdx6 expression to maintain physiological ROS levels. The studies reported how the gene is aging or oxidative stress and clues in the development of a or epithelial cell from from (Prdx6−/−) and and in with as earlier (1Fatma N. Kubo E. Sharma P. Beier D.R. Singh D.P. Cell Death Differ. 2005; 12: 734-750Crossref PubMed Scopus (75) Google Scholar). at the of R. Beier H. Beier D.R. 1997; 42: PubMed Scopus Google Scholar). Prdx6−/− of used the present of the and used as control specific in the at Smad3−/− from have been by Deng and X. Letterio J.J. Lechleider R.J. Chen L. Hayman R. Gu H. Roberts A.B. Deng C. EMBO J. 1999; 18: 1280-1291Crossref PubMed Google Scholar). The and of and the of have been in X. Letterio J.J. Lechleider R.J. Chen L. Hayman R. Gu H. Roberts A.B. Deng C. EMBO J. 1999; 18: 1280-1291Crossref PubMed Google Scholar). The by to Smad3−/− and lens epithelial cells from from the of and analysis was to the of a specific of in Smad3−/− and LECs LECs used for the Cell in as (1Fatma N. Kubo E. Sharma P. Beier D.R. Singh D.P. Cell Death Differ. 2005; 12: 734-750Crossref PubMed Scopus (75) Google Scholar, 4Kubo E. Fatma N. Akagi Y. Beier D.R. Singh S.P. Singh D.P. Am. J. Physiol. Cell Physiol. 2008; 294: C842-C855Crossref PubMed Scopus (101) Google Scholar). of protein a and with at the antibody from PRDX6 antibody Smad3, and antibody with protein by the with and to and with a expression and of the protein the earlier was and with antibody The 5′-flanking region (−1139 to bp) was from and of was by it to using the and of of different and of the Prdx6 promoter with sense and with and used in the present The was and used for the as follows: and repression of different of Prdx6 gene promoter to in and Prdx6−/− cells. The the putative elements in the Prdx6 promoter. of two NF-κB sites and and two repressive Smad3-binding elements and with NF-κB and two RSBE sites and with RSBE site Cells with or C. protein was and activity was The using a The the from was using the the or to or to and or to or to and to by point in the The used are in type and cells with and on The was and the was by The was using a Prdx6−/− or Smad3−/− LECs as well as LECs using with and of as reported earlier (1Fatma N. Kubo E. Sharma P. Beier D.R. Singh D.P. Cell Death Differ. 2005; 12: 734-750Crossref