Increased sensitivity of Nrf2−/− mice to CS-induced oxidative stress. (A) Immunohistochemical staining for 4HNE in lung sections from Nrf2+/+ and Nrf2−/− mice exposed to air or CS for 1 mo. Lung sections from the CS-exposed Nrf2−/− mice show greater staining for 4HNE than the CS-exposed Nrf2+/+ mice and the respective air-exposed control mice. The image is representative of results from three animals in each group. Magnification: ×40. (B) Immunohistochemical staining clearly shows the presence of an increased number of 8-Oxo-dG–positive alveolar epithelial cells in the airways (indicated by arrow in the inset and middle bottom panel) of CS-exposed Nrf2-deficient mice compared with wild-type mice (middle upper panel). Airways of room air–exposed mice shows few or no 8-oxo-dG–positive cells (left panels). Magnification: ×20 (inset magnification: ×40) (n = 3).

Down-regulation of Nrf2 gene expression by Nrf2 siRNA decreased the expression of GPX2 in A549 cells. (A) Screening of Nrf2 siRNA duplexes. Cells transfected with nontargeting SS siRNA– and vehicle-treated cells were used as controls. (B) Nrf2 siRNA duplex-2 (showing maximum knockdown) was transfected into A549 cells. Total RNA was isolated 72 h after transfection and transcript level of Nrf2 its target gene GCLc were quantified by real-time RT-PCR. (C) Transfection of A549 cells with Nrf2 siRNA inhibited NQO1-ARE luciferase activity. (D) Transient transfection of A549 cells with Nrf2 siRNA inhibited the expression of Nrf2 in a time-dependent manner. (E) Down-regulation of GPX2 expression in cells transfected with Nrf2 siRNA. The inhibition of Nrf2 and GPX2 mRNA expression was progressive from 24 to 72 h after Nrf2 siRNA transfection (n = 3 independent experiments). * Significant when compared with the respective SS siRNAs at P ⩽ 0.05.

ARE mediates the transcriptional regulation of the GPX2 gene. (A) Comparison of the first exon and the 5′ upstream regulatory regions of the human, mouse, and rat GPX2 genes. The short ARE core, long ARE consensus, GPX2-ARE, ARE from the human HO-1 gene (StREb), human NQO1, and human GCLm (EpRE) are aligned. The vertical bar indicates nucleotides that are identical between the human, mouse, and rat ARE as well as long ARE, HO-1 ARE. The abbreviations follow the standard IUPAC nomenclature (M = A or C, R = A or G, Y = C or T, W = A or T, S = G or C). (B) Deletion analysis of GPX2 promoter by transient transfection in A549 cells. A 2.569-kb portion of the GPX2 promoter was cloned into pGL3 basic luciferase reporter vector (−2,030/+539) to monitor the activity of this promoter. Two deletion constructs (−1,029/+539 and −140/+539) were prepared by PCR. All three constructs were transfected into A549 cells transiently transfected with SS, Nrf2, or Keap1 siRNA, and luciferase activities were measured after 48 h. The three constructs had similar reporter activity. The −140/+539 deletion construct displayed the maximum reporter activity. * Significant when compared with vector control transfected with respective siRNA; ^† significant when compared with SS siRNA; ^‡ significant when compared with Nrf2 siRNA. (C) Reporter constructs (ARE-Mu1 and ARE-Mu2) bearing mutations in the ARE (−140/+539) were generated by using the −140/+539 deletion construct. These constructs were transiently transfected into A549 cells already transfected with siRNA, and luciferase reporter activity was monitored. Mutation of the ARE core sequence completely abolished the reporter activity of the deletion construct. * Significant when compared with ARE-WT. (D) 201 bp (−140/+61) ARE enhancer sequence was amplified from the full-length promoter and cloned in pTAL vector. Mutations were introduced in the ARE core sequence (ARE-Mu1 and ARE-Mu2) by site-directed mutagenesis. These constructs were transfected into A549 cell lines transiently transfected with siRNA and luciferase activity was measured. Luciferase activities were normalized by measuring the Renilla luciferase activity from a cotransfected reporter vector. * Significant when compared with ARE-WT (n = 3 independent experiments). Values are mean ± SE from three different experiments (P < 0.05).

GPX2 confers protection against oxidative damage in lung epithelial cells. (A) A549 cells were transfected with GPX2 siRNA and non targeting SS siRNA. Cells were harvested after 72 h and GPX2 message was quantified by real-time RT-PCR. (B) Inhibition of GPX2 enhances the production of lipid hydroperoxides in lung epithelial A549 cells. Knockdown of GPX2 by siRNA significantly increased the production of thiobarbutyric acid substances in tBHP-treated A549 cells. Data are mean ± SEM of three independent experiments. * Significant when compared with vehicle-treated cells; ^† significant when compared with SS siRNA–transfected cells. ^†P ⩽ 0.05. (C) Enhanced sensitivity of Nrf2 siRNA– and GPX2 siRNA–transfected A549 cells to CS condensate. Cells were exposed to CS for 48 h and viable cells were determined by MTT assay. Data are represented as percentage of viable cells relative to the vehicle-treated control. Data are means of six independent replicates, combined to generate the mean ± SD for each concentration. *P < 0.01 relative to SS siRNA.

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Nrf2 induces GPX2 expression in A549 cells. (A) Screening of Keap1 siRNA duplexes. Cells transfected with nontargeting SS siRNA– and vehicle-treated cells were used as controls. (B) Transient transfection of A549 cells with duplex-3 of keap1 siRNA inhibited the expression of keap1 and upregulated the expression of NQO1 mRNA. * Significant when compared with SS siRNA transfected cells. (C) Transfection of A549 cells with keap1 siRNA significantly increased NQO1-ARE luciferase activity. ^† Significant when compared with vector control transfected with the respective siRNA; * significant when compared with SS siRNA. (D) Transient transfection of A549 cells with keap1 siRNA significantly inhibited the expression of Keap1. * Significant when compared with SS siRNA. (E) Up-regulation of GPX2 expression in cells transfected with Keap1 siRNA. * Significant when compared with SS siRNA. (F) Western blot analysis of A549 cells transfected with Nrf2, keap1, or SS siRNA. Total protein was isolated 72 h after transfection, and GPX2 protein levels were determined. β-Actin was used as the loading control. (G) Quantification of GPX2 protein in the Western blots of A549 cells transfected with Nrf2, Keap1, and SS siRNA (mean ± SE of the GPX2/actin protein ratio in three independent samples). * Significant when compared with SS siRNA; ^† significant when compared with Nrf2 siRNA-transfected A549 cells. (H) Induction of GPX2 expression in response to multiple oxidative stress inducing agents. Cells transfected with Nrf2 siRNA or SS siRNA were exposed to tBHP (400 μM), tBHQ (400 μM), and CS condensate (100 μg/ml), and expression of GPX2 was measured by real-time RT-PCR. *Significant when compared with vehicle-treated sample. Data are mean ± SEM of three independent experiments (P ⩽ 0.05).

EMSA was used to determine the DNA-binding activity of Nrf2 to GPX2 ARE. (A) Nuclear proteins isolated from the lungs of CS-exposed Nrf2+/+ mice showed increased binding to GPX2 ARE than the nuclear proteins from the lungs of CS-exposed Nrf2−/− mice or air-exposed control mice. A 100-fold molar excess of cold WT-ARE was used as a competitor oligo. Octamer transcription factor 1 consensus sequence was used as the endogenous control. (B) EMSA analysis with the nuclear proteins isolated from the A549 cells transfected with Nrf2, SS, or Keap1 siRNA. Incubation with 50- and 100-fold molar excess unlabeled cold GPX2 WT-ARE significantly abolished the binding suggesting the specificity of the binding of nuclear proteins to GPX2 ARE. However, incubation with mutant AREs did not abolish the binding of nuclear proteins to GPX2 ARE. (C) Supershift assay with anti-Nrf2 antibody revealed the binding of Nrf2 to GPX2 ARE. Normal rabbit IgG1 was used as the control. (D) CHIP assay was performed with A549 cells transfected with Nrf2 siRNA, Keap1 siRNA, or SS siRNA. No DNA, PCR without DNA. (E) Quantification of CHIP assay results (GPX2 promoter binding-antibody/input ratio). Filled bars, Keap siRNA; open bars, SS siRNA; shaded bars, Nrf2 siRNA.