Cigarette Smoke Regulates the Competitive Interactions between NRF2 and BACH1 for Heme Oxygenase-1 Induction

Abstract

Cigarette smoke has been shown to trigger aberrant signaling pathways and pathophysiological processes; however, the regulatory mechanisms underlying smoke-induced gene expression remain to be established. Herein, we observed that two smoke-responsive genes, HO-1 and CYP1A1, are robustly induced upon smoke by different mechanisms in human bronchial epithelia. CYP1A1 is mediated by aryl hydrocarbon receptor signaling, while induction of HO-1 is regulated by oxidative stress, and suppressed by N-acetylcysteine treatment. In light of a pivotal role of NRF2 and BACH1 in response to oxidative stress and regulation of HO-1, we examined if smoke-induced HO-1 expression is modulated through the NRF2/BACH1 axis. We demonstrated that smoke causes significant nuclear translocation of NRF2, but only a slight decrease in nuclear BACH1. Knockdown of NRF2 attenuated smoke-induced HO-1 expression while down-regulation of BACH1 had stimulatory effects on both basal and smoke-induced HO-1 with trivial influence on NRF2 nuclear translocation. Chromatin immunoprecipitation assays showed that smoke augments promoter-specific DNA binding of NRF2 but suppresses BACH1 binding to the HO-1 promoter ARE sites, two of which at -1.0 kb and -2.6 kb are newly identified. These results suggest that the regulation of NRF2 activator and BACH1 repressor binding to the ARE sites are critical for smoke-mediated HO-1 induction.

Keywords: HO-1; NRF2; airway epithelium; gene regulation; smoke.

Figure 1

Smoke induction of HO-1 in human airway epithelial cells. (A) HO-1 protein levels in NHBE cells were analyzed by Western blot assays at 3, 6 and 24 h after the start of smoke treatment (10% cigarette smoke extract) as shown in the top of the figure. β-tubulin was used to normalize the protein loading in the SDS-PAGE. Bottom, the mean results for densitometric scans of three blots from three separate experiments were expressed as fold relative to that of untreated NHBE cells at 3 h. * p < 0.05 (n = 3; mean ± S.D.); (B) HBE1 cells were treated with cigarette smoke and collected at indicated time points. The cell lysates were subjected to immunoblotting for HO-1 protein levels (top). Nucleolin was used to normalize the protein loading in the SDS-PAGE. The mean results for densitometric scans of three blots from three separate experiments are shown in the bottom panel as fold relative to that of untreated HBE1 cells at 30 min. * p < 0.05 (n = 3; mean ± S.D.); (C) HBE1 cells were exposed to smoke extract for 24 h and cell morphology was photographed.

Figure 2

Differential mechanisms of cigarette smoke-induced HO-1 and CYP1A1 gene expression. (A,B) HBE1 cells were pre-treated with 1 or 5 μM AhR antagonist followed by cigarette smoke at 180 mL for 20 min and analyzed for HO-1 (A) and CYP1A1 (B) mRNA level by real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) at 24 h. * p <0.05 (n = 3; mean ± S.E.M.); (C,D) HBE1 cells were pre-treated with 2.5 mM N-acetylcysteine (NAC) and then exposed to smoke treatment for 24 h. The mRNA levels of HO-1 (C) and CYP1A1 (D) were assessed by qRT-PCR. * p < 0.05 (n = 3; mean ± S.E.M.).

Figure 3

Cigarette smoke alters the nuclear levels of NRF2 and BACH1 in NHBE and HBE1 cells. (A,B) NHBE (A) and HBE1 (B) cells were collected at indicated time points after smoke exposure and extracted for nuclear proteins. The NRF2 and BACH1 protein levels in the nucleus were examined by Western blot assays (top). Nucleolin was used to normalize the protein loading in the SDS-PAGE gel. The mean results for densitometric scans of three blots from three separate experiments are shown in the bottom panel as fold relative to that of untreated NHBE cells at 3 h (A) or untreated HBE1 cells at 30 min (B). * p < 0.05 (n = 3; mean ± S.D.).

Figure 4

Effects of NRF2 and BACH1 on the induction of HO-1 by cigarette smoke. HBE1 cells were transfected with control non-specific or gene-specific siRNA. After 48 h of transfection, these cells were exposed to smoke extract for additional 24 h and then subjected to qRT-PCR. (A,B) Cells were transfected with siRNA targeting NRF2 and BACH1. The knockdown efficiency of NRF2 siRNA assessed by qRT-PCR was 81% in air exposed cells and 92% in smoke exposed cells (A); in BACH1 siRNA treatment, the knockdown efficiency was 81% in air exposed cells and 79% in smoke exposed cells (B); smoke had no effect on siRNA knockdown. (C,D) Cells with siRNA knockdown of NRF2 and BACH1 followed by smoke exposure were subjected to qRT-PCR for HO-1 (C) and CYP1A1 (D) expression. Results are expressed as the mean across three independent experiments with error bars representing standard errors of measurements (S.E.M.). * p < 0.05.

Figure 5

Contributions of NRF2 and BACH1 to HO-1 protein level and cell viability in response to smoke exposure. (A) HBE1 cells transfected with siRNA targeting NRF2 and BACH1 were subsequently exposed to cigarette smoke. After 24 h of exposure, cells were collected for protein analysis by immunoblotting. (B–D) The relative expression of HO-1/β-tubulin (B); NRF2/Nucleolin (C); and BACH1/Nucleolin (D) were quantified from three separate experiments as fold relative to that of untreated HBE1 cells with control siRNA. * p < 0.05 (n = 3; mean ± S.D.); (E) HBE1 cells with NRF2 or BACH1 siRNA transfection were exposed to cigarette smoke for 72 h and cell viability was determined by MTS assay (n = 3; mean ± S.D.). * p < 0.05.

Figure 5

Contributions of NRF2 and BACH1 to HO-1 protein level and cell viability in response to smoke exposure. (A) HBE1 cells transfected with siRNA targeting NRF2 and BACH1 were subsequently exposed to cigarette smoke. After 24 h of exposure, cells were collected for protein analysis by immunoblotting. (B–D) The relative expression of HO-1/β-tubulin (B); NRF2/Nucleolin (C); and BACH1/Nucleolin (D) were quantified from three separate experiments as fold relative to that of untreated HBE1 cells with control siRNA. * p < 0.05 (n = 3; mean ± S.D.); (E) HBE1 cells with NRF2 or BACH1 siRNA transfection were exposed to cigarette smoke for 72 h and cell viability was determined by MTS assay (n = 3; mean ± S.D.). * p < 0.05.

Figure 6

The binding sites and abilities of NRF2 and BACH1 to the HO-1 promoter. (A) Diagram of putative ARE enhancer sites in the HO-1 promoter regions for NRF2 and BACH1 binding and primers used for the ChIP assays; (B) ChIP assay analysis for binding of NRF2 and BACH1 to the HO-1 gene ARE. HBE1 cells after smoke treatment were collected at 6 h, fixed in formaldehyde, and ChIP analysis was performed using IgG, NRF2 and BACH1 antibodies. The immunoprecipitated chromatin was PCR-amplified, run on agarose gel and photographed. The mean enrichment intensities are shown in the bottom panel as fold relative to that of untreated HBE1 cells (n = 3; mean ± S.D.).

Figure 7

The schematic diagram of smoke-mediated HO-1 induction. Under stress-free conditions, NRF2 is associated with KEAP1 and undergoes proteolysis. Upon exposure to cigarette smoke, NRF2 is dissociated from KEAP1 and translocated into nucleus. Nuclear NRF2 competes with BACH1 for binding to the HO-1 promoter ARE sites, leading to HO-1 transcription and antioxidant activity.