Acrolein causes transcriptional induction of phase II genes by activation of Nrf2 in human lung type II epithelial (A549) cells

Abstract

Acrolein, an α,β-unsaturated aldehyde, is by far the strongest electrophile present in cigarette smoke which is involved in several lung pathophysiological conditions. Acrolein depletes glutathione and creates thiol imbalance. Acrolein due to thiol imbalance as well as covalent modification of cysteine is known to inhibit the activity of redox sensitive transcription factors such as NF-κB and AP-1. Exposure of human type II lung epithelial (A549) cells to non-lethal dose of acrolein (150 fmol/cell for 1 h) depletes 80% of intracellular glutathione and increases the transcription of γ-glutamylcysteine synthetase (γ-GCS) at 6–12 h post-treatment, which helps in replenishing the glutathione to normal level. Acrolein treatment activates transcription of phase II genes in general, as indicated by an increase in mRNA for NAD (P) H:quinone oxidoreductase (NQO1). Western blot analysis revealed the increased level of the transcription factor, Nrf2 in the nuclear extract from acrolein treated cells. Electrophoretic mobility shift assay shows increased binding of nuclear proteins to human antioxidant response element (ARE) consensus sequence after treatment with acrolein. The involvement of Nrf2 in ARE mediated transcriptional activation in response to acrolein exposure has been confirmed by human NQO1-ARE reporter assay. The ability of acrolein to transcriptionaly activate genes responsible for phase II enzymes may form the basis of resistance against cell death and can have implications in cigarette smoke related lung carcinogenesis.

Introduction

Acrolein is the most reactive amongst aldehydes present in cigarette smoke and vehicle exhaust (Eiserich et al., 1995, Kehrer and Biswal, 2000). In cigarette smoke, it is emitted at up to 230 μg per cigarette (Ghilarducci and Tjeerdema, 1995). Acrolein is highly toxic and is associated with various lung diseases including chronic obstructive pulmonary disease (COPD) (Borchers et al., 1999a, Borchers et al., 1998, Borchers et al., 1999b). Acrolein is also a byproduct generated during lipid peroxidation (Uchida et al., 1998), and acrolein–protein adducts have been associated with increased oxidative stress in brain lesions of Alzheimer's disease (Calingasan et al., 1999), diabetic glomerular lesions (Suzuki et al., 1999), and the pathogenesis of artherosclerosis (Uchida et al., 1998). 1,N2-propanodeoxyguanosine adducts derived from acrolein in oral tissue of cigarette smoker is 4.4-fold more than that of non-smokers (Nath et al., 1998). More recently, acrolein has been shown to help in transformation of mouse embryo fibroblast cells suggesting its role as an important mediator of cell transformation under oxidative stress (Takabe et al., 2001). Acrolein induces a pre-neoplastic marker enzyme glutathione S-transferase P-form (GST-P) in rat liver (Satoh et al., 2001). U.S. Environmental Protection Agency has placed acrolein in the category of possible human carcinogen (U.S. EPA, 1993). Although its acute toxicity has been well described, the mechanism(s) underlying effects observed at non-lethal concentrations remains to be deciphered.

Acrolein creates redox imbalance by conjugation with thiols including GSH and covalently modifying cysteine, lysine and histidine residues of proteins by Michael addition, leading to inhibition of NF-κB and AP-1 (Biswal et al., 2002, Horton et al., 1999). The carcinogenic property of acrolein is attributed to its ability to bind to GSH and form adducts with DNA (Kehrer and Biswal, 2000). GSH is critical for cellular antioxidant defenses, especially in protecting lung epithelial cells from oxidant injury and inflammation (Rahman and MacNee, 1999, Rahman and MacNee, 2000, Rahman et al., 1999). Severe depletion of GSH by acrolein has been implicated in bronchoalveolar lavage (BAL) of smokers. Patients with COPD showed a significant thiol deficiency compared to a non-smoker/non-COPD group. The intracellular thiol deficiency significantly correlated with reduced lung function (Tager et al., 2000). Cigarette smoke exposed human bronchial epithelial cells (BEAS-2B) have also been shown to have severe depletion of GSH (Arora et al., 2001). However, the molecular effects of acrolein have not been extensively studied (Kehrer and Biswal, 2000).

Cellular GSH level is modulated by transcriptional activation of the rate limiting enzyme, γ-GCS for glutathione biosynthesis. Increase in GSH protects against cell death in general, either by annihilation of free radicals or by conjugation with toxicants or carcinogens. Increase in GSH and phase II enzymes such as GST, Quinone reductase (NQO1) have been long known to mediate chemoresistance in cancer cells (Goto et al., 2001, Tew et al., 1998). The induction by xenobiotics as well as the basal activity of many of the phase II enzymes such as GST family, NQO1, heme oxygenase-1 (HO-1) and γ-GCS are transcriptionaly mediated in a coordinate manner by an electrophile response element (EpRE) or antioxidant response element (ARE) present in their promoter (Hayes and McMahon, 2001). The EpRE or ARE core sequence 5′ (G/C)TGA(C/G)NNNGC(A/G)-3′ may contain an embedded AP-1 binding site which is often flanked by AP-1 or AP-1 like sequences (Wild et al., 1999). Recent studies have shown that Nrf2, a cap ‘n’ collar basic leucine zipper transcription factor, plays a central role in the transcriptional activation of phase II enzymes via EpRE (Hayes, 2001, Kwak et al., 2001, McMahon et al., 2001).

The protein assembly at EpRE remains an area of intense investigation with the involvement of several transcriptional regulators such as small Maf proteins, CBP/p300 and p160 family co-activators and ARE binding protein-1 (Zhu and Fahl, 2001). Nrf2 is sequestered in the cytosol by interaction with the cytosolic inhibitor protein, keap1. The mechanism of activation of Nrf2 is far from being clear but it has been hypothesized that xenobiotics either directly or through activation of kinases perturb the association with keap1 leading to nuclear translocation of Nrf2 (Hayes and McMahon, 2001).

Since acrolein is known to be an important mediator of cell transformation under oxidative stress (Takabe et al., 2001) and activates pre-neoplastic marker GST-P (Satoh et al., 2001), we have investigated its ability to activate Nrf2 leading to upregulation of phase II gene(s), which can have implications in resistance against cell death and carcinogenesis in lung epithelial cells.