A Critical Role for IκB Kinase β in Metallothionein-1 Expression and Protection against Arsenic Toxicity*
- ‡Department of Environmental Health, University of Cincinnati Medical Center, Cincinnati, Ohio 45267-0056 and §Department of Molecular Microbiology and Immunology, University of Southern California, Los Angeles, California 90033
- 1 To whom correspondence should be addressed: Dept. of Environmental Health, University of Cincinnati, 123 E. Shields St., Cincinnati, OH 45267-0056. Tel.: 513-558-0371; Fax: 513-558-0974; E-mail: xiay{at}email.uc.edu.
Abstract
Arsenic is a widespread environmental toxic agent that has been shown to cause diverse tissue and cell damage and at the same time to be an effective anti-cancer therapeutic agent. The objective of this study is to explore the signaling mechanisms involved in arsenic toxicity. We show that the IκB kinase β (IKKβ) plays a crucial role in protecting cells from arsenic toxicity. Ikkβ-/- mouse 3T3 fibroblasts have decreased expression of antioxidant genes, such as metallothionein 1 (Mt1). In contrast to wild type and IKKβ-reconstituted Ikkβ-/- cells, IKKβ-null cells display a marked increase in arsenic-induced reactive oxygen species (ROS) accumulation, which leads to activation of the MKK4-c-Jun NH2-terminal kinase (JNK) pathway, c-Jun phosphorylation, and apoptosis. Pretreatment with the antioxidant N-acetylcysteine (NAC) and expression of MT1 in the Ikkβ-/- cells prevented JNK activation; moreover, NAC pretreatment, MT1 expression, MKK4 ablation, and JNK inhibition all protected cells from death induced by arsenic. Our data show that two signaling pathways appear to be important for modulating arsenic toxicity. First, the IKK-NF-κB pathway is crucial for maintaining cellular metallothionein-1 levels to counteract ROS accumulation, and second, when this pathway fails, excessive ROS leads to activation of the MKK4-JNK pathway, resulting in apoptosis.
Footnotes
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↵2 The abbreviations used are: ROS, reactive oxygen species; IKK, inhibitor of nuclear factor-κB kinase; MKK4, mitogen-activated protein kinase kinase 4; JNK, c-Jun NH2-terminal kinase; NAC, N-acetyl-l-cysteine; MAP, mitogen-activated protein; NF-κB, nuclear factor-κB; IκBα, inhibitor of NF-κB; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling; DCFDA, 2′,7′-dichlorofluorescein-diacetate; CM-H2DCFDA, chloromethyl DCFDA; ES cells, embryonic stem cells; SOD, superoxide dismutase; TNF, tumor necrosis factor; MT, metallothionein; ERK, extracellular signal-regulated kinase; PARP, poly(ADP-ribose) polymerase; MEF, mouse embryonic fibroblast; DAPI, 4,6-diamidino-2-phenylindole; LDH, lactate dehydrogenase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; WT, wild type; As, arsenic.
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↵* This work was supported in part by National Institutes of Health Grants ES11798 (to Y. X.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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- Received March 23, 2007.
- Revision received May 23, 2007.
