Oncogene (2002) 21, 6317 – 6327 ª 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc Thioredoxin reductase regulates AP-1 activity as well as thioredoxin nuclear localization via active cysteines in response to ionizing radiation Shervin Karimpour1,7, Junyang Lou2,7, Lilie L Lin2, Luis M Rene2, Lucio Lagunas2, Xinrong Ma3, Sreenivasu Karra3, C Matthew Bradbury1, Stephanie Markovina2, Prabhat C Goswami4, Douglas R Spitz4, Kiichi Hirota5, Dhananjaya V Kalvakolanu3, Junji Yodoi6 and David Gius*,1 1Radiation Oncology Branch, Radiation Oncology Sciences Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA; 2Section of Cancer Biology, Mallinckrodt Institute of Radiology, Washington University School of Medicine, St. Louis, Missouri, USA; 3Greenebaum Cancer Center, Department of Microbiology and Immunology, Molecular and Cellular Biology Program, University of Maryland School of Medicine, Baltimore, Maryland, USA; 4Free Radical and Radiation Biology Program, Department of Radiology, University of Iowa, Iowa City, Iowa, USA; 5Department of Anesthesia, Kyoto University Hospital, Kyoto University, Kyoto, Japan; 6Department of Biological Responses, Institute for Virus Research, Kyoto University, Kyoto, Japan A recently identified class of signaling factors uses TR enzyme as a signaling factor in the regulation of AP- critical cysteine motif(s) that act as redox-sensitive 1 activity via a cysteine motif located in the protein. ‘sulfhydryl switches’ to reversibly modulate specific Oncogene (2002) 21, 6317 – 6327. doi:10.1038/sj.onc. signal transduction cascades regulating downstream 1205749 proteins with similar redox-sensitive sites. For example, signaling factors such as redox factor-1 (Ref-1) and Keywords: thioredoxin reductase; thioredoxin; AP-1; transcription factors such as the AP-1 complex both redox; ionizing radiation contain redox-sensitive cysteine motifs that regulate activity in response to oxidative stress. The mammalian thioredoxin reductase-1 (TR) is an oxidoreductase selenocysteine-containing flavoprotein that also appears Introduction to regulate multiple downstream intracellular redox- sensitive proteins. Since ionizing radiation (IR) induces The cytotoxicity of IR is primarily mediated via the oxidative stress as well as increases AP-1 DNA-binding production of reactive oxygen intermediates (ROI) from activity via the activation of Ref-1, the potential roles of intracellular H2O. ROI (in order of sequential reduction - TR and thioredoxin (TRX) in the regulation of AP-1 from O2, superoxide [O2 ], hydrogen peroxide [H2O2], activity in response to IR were investigated. Permanently and hydroxyl radical [.OH]) are normally produced by transfected cell lines that overexpress wild type TR cells as side products of electron transfer reactions demonstrated constitutive increases in AP-1 DNA- during cellular respiration (Halliwell and Gutterridge, binding activity as well as AP-1-dependent reporter gene 1988). Ideally, a metabolically active cell should strike a expression, relative to vector control cells. In contrast, balance between ROI production and the cellular permanently transfected cell lines expressing a TR gene antioxidant defense system, resulting in either a neutral with the active site cysteine motif deleted were unable to or slightly reduced cellular environment (Halliwell and induce AP-1 activity or reporter gene expression in Gutterridge, 1990). The accumulation of abnormally response to IR. Transient genetic overexpression of high levels of ROI, as a result of any source including either the TR wild type or dominant-negative genes IR, can create a condition referred to as ‘oxidative demonstrated similar results using a transient assay stress’ that damages cells by lipid peroxidation and system. One mechanism through which TR regulates amino acid disruption (Storz et al., 1990). AP-1 activity appears to involve TRX sub-cellular In response to a wide variety of environmental localization, with no change in the total TRX content stresses including IR, a class of proto-oncogenes of the cell. These results identify a novel function of the (including c-Fos and c-Jun) referred to as immediate early response genes are activated (Abate et al., 1991; Gius et al., 1990; Kerppola and Curran, 1995). These genes encode nuclear transcription factors (i.e., AP-1) *Correspondence: D Gius, Section Chief, Molecular Radiation Oncology, Radiation Oncology Branch, Radiation Oncology Sciences involved in the transmission of inter- and intracellular Program, Center for Cancer Research, National Cancer Institute, information through multiple cellular signaling path- National Institutes of Health, 9000 Rockville Pike, Bldg 10, Room ways (Holbrook and Fornace, 1991; Kerr et al., 1992). B3B69, Bethesda, Maryland, 20892-1002301, USA; One possible role for the induction of these transcrip- E-mail: [email protected] 7These two authors contributed equally to this work tion factors is to modulate the expression of specific Received 8 January 2002; revised 28 May 2002; accepted 7 June target genes involved in a protective or reparative 2002 cellular response to the damaging effects of oxidative Radiation-induced regulation of AP-1 by thioredoxin reductase and thioredoxin S Karimpour et al 6318 stress induced by exogenous cytotoxic agents such as One intracellular function of TRX is to activate IR (Karin and Smeal, 1992; Kerppola and Curran, protein-nucleic acid interactions of nuclear transcrip- 1995; Xanthoudakis et al., 1992). tion factors via redox regulation (Hirota et al., 1999, c-Fos and c-Jun are members of a multigene family 2000a; Wei et al., 2000). It has been previously shown implicated in a number of signal transduction cascades that following exposure to IR (Wei et al., 2000) or associated with growth, differentiation, neuronal exci- other oxidants (Hirota et al., 1999, 2000a), TRX tation, and cellular stress, and thus provide a useful translocates into the nucleus, where it interacts with model for the investigation of stimulus-evoked altera- another redox-sensitive signaling protein, redox factor- tions in gene expression (Abate et al., 1990, 1991; Kerr 1, and appears to regulate the nuclear transcription et al., 1992). c-Fos and c-Jun comprise part of the factor complex, AP-1. The current study demonstrates mammalian transcription factor AP-1 and heterodi- the essential role of TR as a redox-sensitive signaling merize to bind to a specific DNA sequence referred to factor functioning upstream of TRX in the regulation as the AP-1 site; this binding activates the expression of AP-1 activity and demonstrates that the active site of downstream target genes (Franza et al., 1988). In cysteine residues are essential in mediating IR-induced this regard, the induction of c-Fos and c-Jun is an ideal activation as well as the regulation of stress-induced paradigm for studying the role of early response genes TRX nuclear translocation. in response to oxidative cellular stress induced by IR. One post-translational mechanism that modulates c- Results Fos/c-Jun DNA-binding activity in vitro involves changes in oxidation-reduction (redox) state of the Characterization of TRX and TR overexpressing cell lines protein complex (Abate et al., 1991; Xanthoudakis et al., 1992). In this process, redox regulation is mediated We have previously demonstrated that AP-1 DNA- by a conserved cysteine residue flanked by basic amino binding activity is induced following exposure to IR via acids lysine and arginine (KCR) located in the basic a pathway that appears to involve redox regulation of DNA-binding domain of both c-Fos and c-Jun (Abate et TRX and Ref-1 (Wei et al., 2000). However, the IR- al., 1990). This specific motif appears to be conserved in induction of AP-1 dependent gene expression and the all of the c-Fos- and c-Jun-related proteins and is also upstream factors regulating TRX as well as the role of present, in a slightly modified form, in several other the active site cysteine residues in the process had not redox sensitive transcription factors (Abate et al., 1990; been resolved. To address these issues, a series of Xanthoudakis et al., 1992; Kerppola and Curran, 1995). permanent cell lines derived from MCF-7 or HeLa Since IR induces AP-1 DNA-binding activity as well as were constructed that stably overexpress: (1) a control alters the intracellular oxidation/reduction state of the expression vector (pcDNA-X); (2) a wild-type TRX- cell via the generation of ROIs, it seems logical to containing vector (pcDNA-TRX); (3) a TRX mutant- investigate if other signaling proteins upstream of AP-1 containing vector (pcDNA-TRX-dm); (4) a wild type containing conserved cysteines regulate AP-1 activity in TR-containing vector (pCXN2-myc-TR-wt); or (5) a response to IR. We have recently shown that thioredox- mutant TR containing vector (pCXN2-myc-mTR). in and Ref-1 undergo changes in redox state that These plasmids have been previously described (Hu et contribute to the activation of AP-1 DNA-binding al., 2001; Ma et al., 2001) activity (Wei et al., 2000). These results suggest a role To confirm the expression of these exogenous genes for additional redox-sensitive signaling pathways in in these permanently transfected cell lines, Western blot regulating cellular responses to IR. analyses were performed. pcDNA-TRX-wt and TR, TRX, and NADPH comprise a highly conserved, pcDNA-TRX-dm cell lines demonstrated a sevenfold ubiquitous system (Mustacich and Powis, 2000)