A Critical Role for IκB Kinase β in Metallothionein-1 Expression and Protection against Arsenic Toxicity

Zhimin Peng,Ebrahim Zandi,Howard G. Shertzer,Ying Xia,Li Peng,Yunxia Fan

doi:10.1074/jbc.m702510200

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 IkappaB kinase beta (IKKbeta) plays a crucial role in protecting cells from arsenic toxicity. Ikkbeta(-)(/)(-) mouse 3T3 fibroblasts have decreased expression of antioxidant genes, such as metallothionein 1 (Mt1). In contrast to wild type and IKKbeta-reconstituted Ikkbeta(-)(/)(-) cells, IKKbeta-null cells display a marked increase in arsenic-induced reactive oxygen species (ROS) accumulation, which leads to activation of the MKK4-c-Jun NH(2)-terminal kinase (JNK) pathway, c-Jun phosphorylation, and apoptosis. Pretreatment with the antioxidant N-acetylcysteine (NAC) and expression of MT1 in the Ikkbeta(-)(/)(-) 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-kappaB 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.

Highlights

As a redox-active metalloid, arsenic in a dose-dependent manner elicits an immediate burst of intracellular reactive oxygen species (ROS)
IKK␤ Protects Cells from Arsenic Toxicity—The identity of the cells used for these studies was confirmed by genotypic analyses, which detected the presences of the Ikk␤null alleles [40] in Ikk␤Ϫ/Ϫ and Ikk␤Ϫ/Ϫ-R but not in wild type cells (Fig. 1A)
GTCATGGATGACC; Sod2, 5Ј-GCACATTAACGCGCAG- showed that expression of IKK␤ proteins, apparent in wild type, ATCA and 5Ј-AGCCTCCAGCAACTCTCCTT; Sod1, 5Ј-TGG- Ikk␤Ϫ/Ϫ-R, and Ikk␤Ϫ/Ϫ-Ad, was completely absent in Ikk␤Ϫ/Ϫ

Summary

Introduction

As a redox-active metalloid, arsenic in a dose-dependent manner elicits an immediate burst of intracellular ROS Numerous studies have shown that chronic exposure to arsenic from drinking water in humans and experimental animals are often associated with increased oxidative stress, a state when cellular ROS production exceeds antioxidant capacity (19 –22) Depending on their level and persistence, excessive ROS can cause diverse pathophysiological conditions, such as oxidative damage to DNA, proteins, and lipids, that may contribute to cell transformation and tumorigenesis [23, 24]. Upon stimulation by various growth factors, cytokines and cellular stressors, the I␬B kinase (IKK) complexes consisting of ␣, ␤, and ␥ subunits are activated, which phosphorylate I␬B on serine/threonine residues This phosphorylation leads to I␬B ubiquitination and subsequent proteolytic degradation, thereby unmasking the nuclear localization signal on NF-␬B and allowing its nuclear translocation. Cells and mice defective in NF-␬B signaling are more susceptible to oxidative damage induced by TNF␣, H2O2, and arsenic [36]

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