Decreased histone deacetylase 2 impairs Nrf2 activation by oxidative stress - PubMed (original) (raw)

Decreased histone deacetylase 2 impairs Nrf2 activation by oxidative stress

Nicolas Mercado et al. Biochem Biophys Res Commun. 2011.

Abstract

Nuclear factor erythroid 2-related factor 2 (Nrf2) plays a crucial role in cellular defence against oxidative stress by inducing the expression of multiple anti-oxidant genes. However, where high levels of oxidative stress are observed, such as chronic obstructive pulmonary disease (COPD), Nrf2 activity is reduced, although the molecular mechanism for this defect is uncertain. Here, we show that down-regulation of histone deacetylase (HDAC) 2 causes Nrf2 instability, resulting in reduced anti-oxidant gene expression and increase sensitivity to oxidative stress. Although Nrf2 protein was clearly stabilized after hydrogen peroxide (H(2)O(2)) stimulation in a bronchial epithelial cell line (BEAS2B), Nrf2 stability was decreased and Nrf2 acetylation increased in the presence of an HDAC inhibitor, trichostatin A (TSA). TSA also reduced Nrf2-regulated heme-oxygenase-1 (HO-1) expression in these cells, and this was confirmed in acute cigarette-smoke exposed mice in vivo. HDAC2 knock-down by RNA interference resulted in reduced H(2)O(2)-induced Nrf2 protein stability and activity in BEAS2B cells, whereas HDAC1 knockdown had no effect. Furthermore, monocyte-derived macrophages obtained from healthy volunteers (non-smokers and smokers) and COPD patients showed a significant correlation between HDAC2 expression and Nrf2 expression (r=0.92, p<0.0001). Thus, reduced HDAC2 activity in COPD may account for increased Nrf2 acetylation, reduced Nrf2 stability and impaired anti oxidant defences.

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Figures

Fig. 1

Hydrogen peroxide (H2O2) increases Nrf2 protein and activity in BEAS2B cells. (A) Cells were treated H2O2 (75 μM) for 0.5–24 h and nuclear Nrf2 was measured by Western blotting and normalized to the oxidant insensitive protein lamin C (n = 3). (B) The same nuclear extracts were used for measurement of Nrf2 DNA binding (n = 3). (C) Western blot analysis of BEAS2B cell nuclear extracts stimulated with H2O2 (1–100 μM) for 30 min (n = 3). (D) BEAS2B cells were stimulated with H2O2 (75 μM) for 8 h and HO-1 mRNA expression was normalized to GNB2L1 and analysed using qRT-PCR; NT: non-treatment. Values represent means ± SEM, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005.

Fig. 2

HDAC2 inhibition reduces Nrf2 protein stability. (A) BEAS2B cells were treated with TSA (0.1–10 ng/ml) for 1.5 h prior 30 min exposure to H2O2 (75 μM) and nuclear Nrf2 was measured by Western blotting. (B) Nrf2 DNA binding activity was measured in nuclear extracts stimulated with trichostatin A (TSA 0.1–1000 ng/ml) for 1.5 h prior to 30 min exposure to H2O2 (75 μM; n = 3; ∗p < 0.05, ∗∗p < 0.01 vs. H2O2). (C) Cells were stimulated with TSA (10 ng/ml) for 180 min and nuclear extracts analyzed for Nrf2 and lamin A/C expression by Western blotting. NT: non-treatment; n = 3. (D) Cells were stimulated with cycloheximide (CXM 0.5 μg/ml) in the presence or absence of 30 min pre-treatment with TSA (50 ng/ml). Cells were also stimulated with CXM in the presence of H2O2 (50 μM) with or without pre-treatment with TSA for 30 min. Nuclear extracts were analyzed by Western blot for Nrf2 and TBP as nuclear control. NT: non-treatment; n = 3. NT vs. TSA and H2O2 vs. H2O2 + TSA for each time point (∗p < 0.05, ∗∗p < 0.01). (E) Cells were treated with TSA (10 ng/ml) for 1.5 h prior to 8 h with H2O2 (75 μM). HO-1 expression was measured by QRT-PCR with GNB2L1 used as control (n = 3; ∗p < 0.05 between NT and H2O2, #p < 0.05 between TSA and H2O2). (F) Mice were treated with intranasal TSA or vehicle (Veh) for 1 h prior to 5 h to cigarette smoke (CS) or air. HO-1 was measured against β-actin using QRT-PCR, (#p < 0.05 vs. Air/Veh, ∗p < 0.05 vs. CS/Veh, (n = 5/group)). (G) Cells were transfected with HDAC2 siRNA (H2 kd), HDAC1 siRNA (H1 kd) or a random oligonucleotide (RO) for 48 h and stimulated with H2O2 (50 μM) for 30 min. Nrf2, HDAC2, HDAC1 and Lamin A/C were measured by Western blot from whole cell extracts. Values represent means ± SEM, n = 4, ∗p < 0.05.

Fig. 3

HDAC2 and Nrf2 expression correlation in monocyte-derived macrophages. (A) MDMs from a healthy volunteer (HV), a smoking volunteer (SV) and two COPD patients were stimulated with H2O2 (50 μM) for 30 min prior to whole-cell extraction and Western blotting. The expression of Nrf2, DJ-1, HDAC2, Keap1 and β-actin are shown. The whole cell extracts from unstimulated BEAS2B cells was used as positive control (Ct). (B) MDMs from five healthy volunteers (HV), three smoking volunteers (SV) and five COPD patients were stimulated with H2O2 (50 μM) for 30 min and expression of Nrf2 was normalized against β-actin. (C) Nrf2 and HDAC2 expression were correlated in all samples (n = 13). (D) MDMs from four healthy volunteers were stimulated with TSA (50 ng/ml) for 1.5 h prior exposure to H2O2 (50 μM) for 30 min and the expression of Nrf2 was normalized against β-actin. Values represent means ± SEM. NT (Non-treatment). ∗p < 0.01 for NT vs. H2O2 and #p < 0.01 for H2O2 vs. TSA (H2O2).

Fig. 4

Nrf2 is acetylated with HDAC inhibition. (A) BEAS2B cells were treated with the proteasome inhibitor MG132 (5 μg/ml) for 1.5 h or with H2O2 (75 μM) for 30 min and Nrf2 and lamin C (control) were detected in nuclear extracts by Western blotting. (B) Nrf2 activity was determined in the same extracts using a TransAM kit, (n = 4), (∗∗p < 0.01 and ∗∗∗∗p < 0.0001 vs. NT [non-treatment]). (C) BEAS2B cells were stimulated with MG132 (5 μg/ml) for 2 h, with/without TSA (10 ng/ml for 1–2 h). Nrf2 was immunoprecipitated and acetylated-lysine (Ac-K) and HDAC2 were detected with the relevant-antibody. IP: immunoprecipitation, IB: immunoblot, WC: whole cell extracts. IgG: negative control for immunoprecipitation where no cellular lysates where used. The arrow (→) indicates the correct Nrf2 protein size in the whole cell extracts. (D) Band density of acetylated Nrf2 was determined and normalized to total Nrf2 expression (n = 3), ∗p < 0.05 vs. NT (non-treatment). Keap1 (E) and DJ-1 (F) were immunoprecipitaed from whole cell extracts under the same conditions used in (C).

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Decreased histone deacetylase 2 impairs Nrf2 activation by oxidative stress - PubMed (original) (raw)