Thioredoxin reductase regulates AP-1 activity as well as thioredoxin nuclear localization via active cysteines in response to ionizing radiation Shervin Karimpour 1,7 , Junyang Lou 2,7 , Lilie L Lin 2 , Luis M Rene 2 , Lucio Lagunas 2 , Xinrong Ma 3 , Sreenivasu Karra 3 , C Matthew Bradbury 1 , Stephanie Markovina 2 , Prabhat C Goswami 4 , Douglas R Spitz 4 , Kiichi Hirota 5 , Dhananjaya V Kalvakolanu 3 , Junji Yodoi 6 and David Gius* ,1 1 Radiation Oncology Branch, Radiation Oncology Sciences Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA; 2 Section of Cancer Biology, Mallinckrodt Institute of Radiology, Washington University School of Medicine, St. Louis, Missouri, USA; 3 Greenebaum Cancer Center, Department of Microbiology and Immunology, Molecular and Cellular Biology Program, University of Maryland School of Medicine, Baltimore, Maryland, USA; 4 Free Radical and Radiation Biology Program, Department of Radiology, University of Iowa, Iowa City, Iowa, USA; 5 Department of Anesthesia, Kyoto University Hospital, Kyoto University, Kyoto, Japan; 6 Department of Biological Responses, Institute for Virus Research, Kyoto University, Kyoto, Japan A recently identified class of signaling factors uses critical cysteine motif(s) that act as redox-sensitive ‘sulfhydryl switches’ to reversibly modulate specific signal transduction cascades regulating downstream proteins with similar redox-sensitive sites. For example, signaling factors such as redox factor-1 (Ref-1) and transcription factors such as the AP-1 complex both 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 to regulate multiple downstream intracellular redox- sensitive proteins. Since ionizing radiation (IR) induces oxidative stress as well as increases AP-1 DNA-binding activity via the activation of Ref-1, the potential roles of TR and thioredoxin (TRX) in the regulation of AP-1 activity in response to IR were investigated. Permanently transfected cell lines that overexpress wild type TR demonstrated constitutive increases in AP-1 DNA- binding activity as well as AP-1-dependent reporter gene expression, relative to vector control cells. In contrast, permanently transfected cell lines expressing a TR gene with the active site cysteine motif deleted were unable to induce AP-1 activity or reporter gene expression in response to IR. Transient genetic overexpression of either the TR wild type or dominant-negative genes demonstrated similar results using a transient assay system. One mechanism through which TR regulates AP-1 activity appears to involve TRX sub-cellular localization, with no change in the total TRX content of the cell. These results identify a novel function of the TR enzyme as a signaling factor in the regulation of AP- 1 activity via a cysteine motif located in the protein. Oncogene (2002) 21, 6317 – 6327. doi:10.1038/sj.onc. 1205749 Keywords: thioredoxin reductase; thioredoxin; AP-1; redox; ionizing radiation Introduction The cytotoxicity of IR is primarily mediated via the production of reactive oxygen intermediates (ROI) from intracellular H 2 O. ROI (in order of sequential reduction from O 2 , superoxide [O 2 - ], hydrogen peroxide [H 2 O 2 ], and hydroxyl radical [ . OH]) are normally produced by cells as side products of electron transfer reactions during cellular respiration (Halliwell and Gutterridge, 1988). Ideally, a metabolically active cell should strike a balance between ROI production and the cellular antioxidant defense system, resulting in either a neutral or slightly reduced cellular environment (Halliwell and Gutterridge, 1990). The accumulation of abnormally high levels of ROI, as a result of any source including IR, can create a condition referred to as ‘oxidative stress’ that damages cells by lipid peroxidation and amino acid disruption (Storz et al., 1990). In response to a wide variety of environmental stresses including IR, a class of proto-oncogenes (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) involved in the transmission of inter- and intracellular information through multiple cellular signaling path- ways (Holbrook and Fornace, 1991; Kerr et al., 1992). One possible role for the induction of these transcrip- tion factors is to modulate the expression of specific target genes involved in a protective or reparative cellular response to the damaging effects of oxidative Received 8 January 2002; revised 28 May 2002; accepted 7 June 2002 *Correspondence: D Gius, Section Chief, Molecular Radiation Oncology, Radiation Oncology Branch, Radiation Oncology Sciences Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 9000 Rockville Pike, Bldg 10, Room B3B69, Bethesda, Maryland, 20892-1002301, USA; E-mail: giusd@mail.nih.gov 7 These two authors contributed equally to this work Oncogene (2002) 21, 6317 – 6327 ª 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc