Elsevier

Experimental Cell Research

Volume 290, Issue 2, 1 November 2003, Pages 234-245
Experimental Cell Research

Regular article
Transcription factor Nrf2 activation by inorganic arsenic in cultured keratinocytes: involvement of hydrogen peroxide

https://doi.org/10.1016/S0014-4827(03)00341-0Get rights and content

Abstract

Inorganic arsenic is a well-documented human carcinogen that targets the skin. The induction of oxidative stress, as shown with arsenic, may have a bearing on the carcinogenic mechanism of this metalloid. The transcription factor Nrf2 is a key player in the regulation of genes encoding for many antioxidative response enzymes. Thus, the effect of inorganic arsenic (as sodium arsenite) on Nrf2 expression and localization was studied in HaCaT cells, an immortalized human keratinocyte cell line. We found, for the first time, that arsenic enhanced cellular expression of Nrf2 at the transcriptional and protein levels and activated expression of Nrf2-related genes in these cells. In addition, arsenic exposure caused nuclear accumulation of Nrf2 in association with downstream activation of Nrf2-mediated oxidative response genes. Arsenic simultaneously increased the expression of Keap1, a regulator of Nrf2 activity. The coordinated induction of Keap1 expression and nuclear Nrf2 accumulation induced by arsenic suggests that Keap1 is important to arsenic-induced Nrf2 activation. Furthermore, when cells were pretreated with scavengers of hydrogen peroxide (H2O2) such as catalase–polyethylene glycol (PEG-CAT) or Tiron, arsenic-induced nuclear Nrf2 accumulation was suppressed, whereas CuDIPSH, a cell-permeable superoxide dismutase (SOD) mimic compound that produces H2O2 from superoxide (·O2), enhanced Nrf2 nuclear accumulation. These results indicate that H2O2, rather than ·O2, is the mediator of nuclear Nrf2 accumulation. Additional study showed that arsenic causes increased cellular H2O2 production and that H2O2 itself has the ability to increase Nrf2 expression at both the transcription and protein levels in HaCaT cells. Taken together, these data clearly show that arsenic increases Nrf2 expression and activity at multiple levels and that H2O2 is one of the mediators of this process.

Introduction

Inorganic arsenic is a well-recognized human carcinogen and exposure is associated with an increased risk for tumors of the skin, bladder, liver, kidney, lung, prostate, and other tissues [1], [2], [3], [4]. Inorganic arsenic is also active in rodent models, producing liver tumors after transplacental exposure [5] and skin tumors in combination with UV irradiation or phorbol esters [6], [7]. However, the molecular mechanisms of inorganic arsenic carcinogenesis remain poorly understood. Arsenic can stimulate production of reactive oxygen species (ROS) [8] and alters cellular sulfhydryl levels [9], whereas glutathione depletion enhances arsenic cytotoxicity [10]. ROS resulting from an imbalance between antioxidants and oxidants during arsenic metabolism has been implicated as a possible etiologic factor in arsenic toxicity and carcinogenesis [11] and as a general factor in carcinogenic potential of inorganics [12]. The precise cause of arsenic-induced ROS is unclear and its molecular impact has not been well defined.

The transcription factor NF-E2-related factor 2 (Nrf2), a member of the Cap'n'Collar family of bZIP proteins, is a central mediator in the activation of a variety of genes encoding for antioxidative and phase 2 drug-metabolizing enzymes through antioxidant response elements (AREs) [13], [14]. The human ARE is a cis-element that contains one perfect and one imperfect AP1 binding element [15]. AREs have been found in the 5′-flanking region of many genes involved with cytoprotection from oxidative stress, such as glutathione S-transferase (GST) [16], NAD(P)H:quinone reductases (NQO1) [17], γ-glutamylcysteine synthetase heavy (γ-GCSh) and light subunits (γ-GCSL) [18], and heme oxygenase 1 (HO-1) [19] among many others [20], [21], [22]. The induction of these enzymes can be viewed as a strategy for cellular protection against the adverse effects of excess ROS production, including, potentially, carcinogenesis. Prestera et al. [23] previously identified arsenic as one of 28 compounds that can activate an incompletely defined GST enhancer element from the 5′ upstream region inserted into a plasmid containing a human growth hormone reporter. Furthermore, overexpression of HO-1, an Nrf2-activated gene [19], is common with arsenic exposure [24]. In this regard, peritoneal macrophages obtained from Nrf2-deficient mice show a greatly diminished induction of HO-1 when compared to wild-type cells after exposure to sodium arsenite [25]. Although these data infer a potential role for Nrf2 in the cellular response to arsenic [23], [25], little is specifically known about the effects of this metalloid on the Nrf2-mediated response to oxidative stress.

Several models have been proposed for the activation of the Nrf2-ARE-mediated pathway. The most commonly invoked model for regulation of Nrf2 function is dissociation of Nrf2 from a cytoskeleton-associated protein Keap1 (human/rat homolog also known as KIAA0132/INrf2), which appears to act as a cytoplasmic repressor of Nrf2 [26]. When Nrf2 dissociates from Keap1, it can translocate to the nucleus and there interact with other transcription factors, bind to the ARE, and subsequently stimulate expression of target genes [26], [27]. Modification of sulfhydryl groups in Keap1 [28] and phosphorylation of Nrf2 through a protein kinase C-(PKC) based mechanism [29] appear to mediate the dissociation of Nrf2 from Keap1. Recent results demonstrated that the phosphatidylinositol 3-kinase (PI3-kinase) pathway regulates the rearrangement of actin microfilaments and depolymerization of actin facilitates the nuclear translocation of Nrf2 [30], [31]. Furthermore, mitogen-activated protein (MAP) kinase pathways activated by extracellular signal-regulated kinase kinase kinase 1 (MEKK1), transforming growth factor-β-activated kinase (TAK1), and apoptosis signal-regulating kinase (ASK1) are reported to be signaling pathways for Nrf2 activation [32]. Interestingly, Nrf2 has been shown to autoregulate its own expression through an ARE-like element located in the proximal region of the Nrf2 promoter, leading to persistent nuclear accumulation of Nrf2 and protracted induction of target genes [33]. An increase of Nrf2 stabilization represents another important posttranscriptional regulation mechanism that can enhance Nrf2 activity [34], [35]. Thus, it is likely that multiple mechanisms play a role in Nrf2-induced gene expression in response to oxidative stress.

It is well established that arsenic can induce oxidative stress [8], [9] and that Nrf2 is a key player in the cellular oxidative stress response [13], [14]. However, a clear interrelationship between Nrf2 and arsenic-mediated oxidative stress has not been established. In the present study, the effects of inorganic arsenic exposure on Nrf2 expression and nuclear translocation, as well as the downstream expression of Nrf2 related genes, were studied. We selected HaCaT cells, a human keratinocyte cell line that models the skin as a target of arsenic carcinogenesis. In this study, we provide direct evidence that arsenic activates the Nrf2 system probably through production of H2O2. This indicates the Nrf2 gene as a novel target of inorganic arsenic and provides evidence of a primary molecular response in a potential target cell of this important inorganic carcinogen.

Section snippets

Chemicals

Sodium m-arsenite (NaAsO2), copper (II) 3,5-diisopropyl salicylate hydrate (CuDIPSH), 4,5-dihydroxy-1,3-benzenedisulfolic acid (Tiron), and catalase–polyethylene glycol (PEG-CAT) were obtained from Sigma-Aldrich (St. Louis, MO).

Cell culture

The human keratinocyte cell line HaCaT is a spontaneously transformed human epithelial cell line developed by Boukamp et al. [36]. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U of penicillin/ml, and

Arsenic increases nuclear Nrf2 accumulation and induces NQO1 and γ-GCSh expression in HaCaT cells

As shown in Fig. 1, exposure to inorganic arsenic resulted in Nrf2 protein accumulation in the nuclear fraction of HaCaT cells in a time- and dose-dependent fashion that reached a peak at between 6 and 12 h. To evaluate the effects of the nuclear Nrf2 accumulation induced by arsenic on transcriptional activation through the ARE, the expression of the ARE-controlled genes NQO1 and γ-GCSh was assessed. Both NQO1 and γ-GCSh expression were induced concomitantly with arsenic induction of Nrf2 (

Discussion

Nrf2 is a ubiquitously expressed transcription factor that occurs in a wide range of tissues and cell types, including keratinocytes [13], [39]. Skin is a major target organ for the chronic toxic and carcinogenic effects of inorganic arsenic [1]. Nrf2 has been reported as an important regulator in the skin wound healing process accompanied with excess ROS production [39]. The present work clearly demonstrates the relationship between arsenic exposure and increased cellular and nuclear

Acknowledgements

We thank Drs. Jie Liu, William Achanzar, and Larry Keefer for their critical review of this article. We also thank Dr. David Miller for his assistance with the immunostaining.

References (51)

  • A.C Wild et al.

    Regulation of gamma-glutamylcysteine synthetase subunit gene expression by the transcription factor Nrf2

    J. Biol. Chem.

    (1999)
  • J Alam et al.

    Nrf2, a Cap'n'Collar transcription factor, regulates induction of the heme oxygenase-1 gene

    J. Biol. Chem.

    (1999)
  • M.K Kwak et al.

    Modulation of gene expression by cancer chemopreventive dithiolethiones through the Keap1-Nrf2 pathwayidentification of novel gene clusters for cell survival

    J. Biol. Chem.

    (2003)
  • T Ishii et al.

    Transcription factor Nrf2 coordinately regulates a group of oxidative stress-inducible genes in macrophages

    J. Biol. Chem.

    (2000)
  • H.C Huang et al.

    Phosphorylation of Nrf2 at Ser-40 by protein kinase C regulates antioxidant response element-mediated transcription

    J. Biol. Chem.

    (2002)
  • R Yu et al.

    Activation of mitogen-activated protein kinase pathways induces antioxidant response element-mediated gene expression via a Nrf2-dependent mechanism

    J. Biol. Chem.

    (2000)
  • D Stewart et al.

    Degradation of transcription factor Nrf2 via the ubiquitin-proteasome pathway and stabilization by cadmium

    J. Biol. Chem.

    (2003)
  • T Nguyen et al.

    Increased protein stability as a mechanism that enhances Nrf2-mediated transcriptional activation of the antioxidant response elementdegradation of Nrf2 by the 26S proteasome

    J. Biol. Chem.

    (2003)
  • C.H He et al.

    Identification of activating transcription factor 4 (ATF4) as an Nrf2-interacting protein. Implication for heme oxygenase-1 gene regulation

    J. Biol. Chem.

    (2001)
  • S Dhakshinamoorthy et al.

    Small maf (MafG and MafK) proteins negatively regulate antioxidant response element-mediated expression and antioxidant induction of the NAD(P)H:Quinone oxidoreductase1 gene

    J. Biol. Chem.

    (2000)
  • W Shi et al.

    The role of arsenic-thiol interactions in metalloregulation of the ars operon

    J. Biol. Chem.

    (1996)
  • A.M Bode et al.

    The paradox of arsenicmolecular mechanisms of cell transformation and chemotherapeutic effects

    Crit. Rev. Oncol. Hematol.

    (2002)
  • T.J Preston et al.

    Scavenging of extracellular H2O2 by catalase inhibits the proliferation of HER-2/Neu-transformed rat-1 fibroblasts through the induction of a stress response

    J. Biol. Chem.

    (2001)
  • C.O Abernathy et al.

    Arsenichealth effects, mechanisms of actions, and research issues

    Environ. Health Perspect.

    (1999)
  • T.W Gebel

    Arsenic and drinking water contamination

    Science

    (1999)
  • Cited by (0)

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