Ethanol-induced HO-1 and NQO1 are differentially regulated by HIF-1alpha and Nrf2 to attenuate inflammatory cytokine expression

Abstract

Oxidative stress plays an important role in alcohol-induced inflammation and liver injury. Relatively less is known about how Kupffer cells respond to oxidative stress-induced expression of heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase (NQO1) to blunt inflammation and liver injury. We showed that Kupffer cells from ethanol-fed rats and ethanol-treated rat Kupffer cells and THP-1 cells displayed increased mRNA expression of HO-1, NQO1, and hypoxia-inducible factor-1α (HIF-1α). Our studies showed that silencing with HIF-1α and JNK-1 siRNAs attenuated ethanol-mediated mRNA expression of HO-1, but not NQO1, whereas Nrf2 siRNA attenuated the mRNA expression of both HO-1 and NQO1. Additionally, JunD but not JunB formed an activator protein-1 (AP-1) oligomeric complex to augment HO-1 promoter activity. Ethanol-induced HO-1 transcription involved antioxidant response elements, hypoxia-response elements, and an AP-1 binding motif in its promoter, as demonstrated by mutation analysis of the promoter, EMSA, and ChIP. Furthermore, livers of ethanol-fed c-Jun(fl/fl) mice showed reduced levels of mRNA for HO-1 but not of NQO1 compared with ethanol-fed control rats, supporting the role of c-Jun or the AP-1 transcriptional complex in ethanol-induced HO-1 expression. Additionally, attenuation of HO-1 levels in ethanol-fed c-Jun(fl/fl) mice led to increased proinflammatory cytokine expression in the liver. These results for the first time show that ethanol regulates HO-1 and NQO1 transcription by different signaling pathways. Additionally, up-regulation of HO-1 protects the liver from excessive formation of inflammatory cytokines. These studies provide novel therapeutic targets to ameliorate alcohol induced inflammation and liver injury.

FIGURE 1.

Ethanol augments HO-1 mRNA expression in rKCs. A, shown is ethanol-induced mRNA expression of HO-1 and HIF-1α in KCs isolated from ethanol-fed rats (E-rKCs) as determined by qRT-PCR. The data represent the -fold increase after ethanol feeding compared with isocaloric fed control in KCs. B, shown is ethanol-induced mRNA expression of HO-1 and HIF-1α in ethanol-treated rKCs. The data of ethanol-treated rKCs represent a -fold increase in mRNA expression after treatment with ethanol (100 mm) for the indicated time period compared with no ethanol treatment. All mRNA expression levels were normalized to GAPDH mRNA levels, and the data shown represent three independent experiments (mean ± S.D.). p values are denoted as: ***, p < 0.001; and **,. p < 0.01.

FIGURE 2.

Ethanol augments HO-1 expression in THP-1 via Src kinase, PI 3-kinase, p38 MAP kinase, NADPH oxidase, and JNK-1 but not JNK-2. A, shown is then time course of ethanol (100 mm)-induced HO-1 mRNA expression in THP-1 cells. B, shown is ethanol-induced HO-1 mRNA expression in THP-1 that were transiently transfected with p47 siRNA, p38 MAPK siRNA, Src-1 siRNA, dominant negative PI 3-kinase, JNK-1 siRNA, JNK-2 siRNA, and mock siRNA as a control followed by ethanol treatment (100 mm) for 8 h. C, shown is the time course of ethanol (100 mm)-induced HIF-1α mRNA expression in THP-1 cells. D, shown is ethanol-induced HIF-1α mRNA expression in THP-1 cells that were transiently transfected with p47 siRNA and dominant negative PI 3-kinase plasmid and mock siRNA as a control followed by ethanol treatment. qRT-PCR data of ethanol-treated THP-1 represent the -fold increase in mRNA expression after treatment with ethanol (100 mm) for 8 h compared with no ethanol treatment; the latter mRNA levels were normalized to a value of 1.0. All mRNA expression levels for genes were normalized to GAPDH mRNA levels, and the data shown represent three independent experiments (mean ± S.D.). *** p < 0.001; ns, not significant. E, shown is the time course of ethanol (100 mm)-induced HIF-1α protein expression in nuclear extracts of THP-1. The data were expressed as -fold change, normalized to β-actin and relative to untreated control. F, shown is ethanol-mediated HIF-1α protein expression in nuclear extracts of THP-1, in response to ethanol treatment (100 mm) for 2 h. Where indicated, cells were preincubated with the inhibitors DPI and LY294002 for 30 min before ethanol treatment. The data are representative of three independent experiments. Densitometric analysis of protein bands was determined, and the values are expressed relative to untreated control, normalized to β-actin. Vertical lines indicate repositioned gel lanes in E and F.

FIGURE 3.

Cellular signaling pathway of ethanol-induced Nrf2 and NQO1 expression in THP-1 cells. A and B, THP-1 were transiently transfected with indicated siRNA or mock siRNA constructs or PI 3-kinase expression plasmid followed by ethanol treatment (100 mm) for 8 h. qRT-PCR data of Nrf2 and NQO1 mRNA expression were normalized to GAPDH mRNA levels. The data of ethanol-treated THP-1 represent the -fold increase in mRNA expression compared with no ethanol treatment. The data shown represent three independent experiments (mean ± S.D.). p values are denoted as: *** p < 0.001; * p < 0.05; ns, not significant, p > 0.05.

FIGURE 4.

Ethanol-induced expression of HO-1 and NQO1 mRNA utilize divergent set of transcription factors. A, shown is ethanol-induced HO-1 mRNA expression in THP-1 cells that were transiently transfected with indicated siRNA or mock siRNA as a control followed by ethanol treatment (100 mm) for 8 h. B, shown is ethanol-induced mRNA expression of HIF-1a, Nrf2, HO-1, and NQO1 in C-rKCs that were transiently transfected with indicated siRNA or mock siRNA constructs followed by ethanol treatment for 8 h. qRT-PCR data represent the -fold increase in mRNA expression after ethanol treatment compared with untreated cells. All mRNA expression levels were normalized to GAPDH mRNA levels, and the data shown represent three independent experiments (mean ± S.D.). p values are denoted as: ***, p < 0.001 and ns (not significant), p > 0.05. C, shown is ethanol-mediated HO-1 protein expression in nuclear extracts from ethanol-treated rat Kupffer cells. Where indicated cells were preincubated with the inhibitors DPI, LY294002, and SB203580 for 30 min before ethanol treatment (100 mm) for 12 h. The data are expressed as the -fold change, normalized to β-actin and relative to untreated cells. The data are representative of three independent experiments.

FIGURE 5.

Identification of functional cis-acting elements in ethanol-induced HO-1 promoter activation. A, schematics of human HO-1 promoter (−3936/−1 bp, relative to the transcription start site), indicating the location of HRE-1 to −3, proximal and distal AP-1 binding sites, and AREs. B, shown is ethanol-induced −9.2kb-HO-1-luc and −4.5kb-HO-1-luc human promoter activity in THP-1 cells. Where indicated, the ARE enhancer region (E1) of the −4.5-kb HO-1 promoter was deleted (rE1). C, shown is the effect of pharmacological inhibitors on HO-1 promoter activity in response to ethanol treatment. Where indicated, cells were preincubated with the indicated inhibitors for 30 min before ethanol treatment for 8 h. RLU, relative luciferase unit. D, THP-1 cells were transiently transfected with indicated siRNAs or Dn PI 3-kinase expression plasmid followed by ethanol treatment for 8h. E, shown is the effect of mutations of AP-1 (AP-1_1, AP-1_2, and AP-1_3) and HRE (HRE-1) binding sites on −4.5-kb HO-1 promoter activity in response to ethanol treatment. Luciferase assay data are expressed as -fold change and have been normalized relative to the change in luciferase activity of the untreated controls and to transfection efficiency with β-galactosidase. The data shown represent three independent experiments in duplicate (means ± S.D.). p values are denoted as ***, p < 0.001; **, p < 0.01, and ns (not significant).

FIGURE 6.

Role of JNK-1, individual components of AP-1 and HIF-1a in ethanol-induced HO-1 promoter activity. A, shown is a time-dependent increase in JNK-1 phosphorylation in nuclear extracts from ethanol-treated THP-1 cells. The data were normalized to unphosphorylated JNK as a loading control and are expressed as -fold change. B, shown is the effect of indicated inhibitors on JNK-1 phosphorylation in response to ethanol treatment. THP-1 cells were preincubated with pharmacological inhibitors for 30 min followed by ethanol treatment for 10 min and analysis of nuclear extracts by Western blotting. The data in A and B are representative of three independent experiments. C, shown is the effect of transient transfection of c-Fos, c-Jun, JunD, and JunB expression plasmid on HO-1-luciferase promoter activity. THP-1 was transiently transfected with total 1 μg of indicated expression plasmid or combination of indicated plasmids. RLU, relative luciferase unit. D, shown is the effect of silencing with siRNAs for c-Jun, JunD, and JunB on ethanol-induced HO-1-luciferase promoter activity. Luciferase assay data are expressed as -fold change and have been normalized relative to the change in luciferase activity of the untreated controls and to transfection efficiency with β-galactosidase. The data shown represent three independent experiments in duplicate (means ± S.D.). p values are denoted as: ***, p < 0.001 and ns, not significant. E and F, EMSA of nuclear extracts from THP-1 utilizing oligonucleotide probes corresponding to HRE, ARE, and AP-1 binding sites in HO-1 promoter (the primer sequence listed in Table 1). Where indicated, THP-1 was transfected with siRNAs for HIF-1α, Nrf2, or c-Jun siRNA followed by ethanol treatment for 8 h and isolation of nuclear extracts. As denoted, oligonucleotide probes with mutated HRE, ARE, or AP-1 sites were used (Table 1). Additionally, a 50-fold excess of cold probe was added to the nuclear extracts as indicated. The data are representative of three independent experiments. G, ethanol augments HIF-1α binding to HRE sites (the primer sequence listed in Table 1, corresponding to HRE-2 and HRE-3 sites in the HO-1 promoter) as assessed by ChIP analysis. THP-1 was preincubated for 30 min with indicated inhibitors before ethanol treatment for 8 h. Soluble chromatin was immunoprecipitated with either HIF-1α antibody (upper panel) or control rabbit IgG (lower panel). Primers used to amplify the products flanking the HRE site in the HO-1 promoter are indicated in Table 1. The middle panel represents the amplification of input DNA before immunoprecipitation. Densitometric analysis was performed and is shown as the -fold change. Data are representative of three independent experiments. H, shown is ethanol-induced HO-1 protein expression in nuclear extracts of THP-1 involves HIF-1α, Nrf2, and c-Jun. Where indicated, cells were transfected with siRNAs for HIF-1α, Nrf2, or c-Jun before ethanol treatment. The data are expressed as -fold change, normalized to TBP and β-actin relative to untreated cells, and are representative of three independent experiments.

FIGURE 7.

Role of HO-1 and NQO1 in attenuating expression of proinflammatory cytokines in vitro and in vivo. A, mRNA from liver tissue of control isocaloric fed wild-type, ethanol-fed wild-type, isocaloric fed c-Jun^fl/fl, and ethanol-fed c-Jun^fl/fl mice were analyzed for the expression of HO-1 and NQO1. The data represent livers from six rats in each category, and the assay was performed in duplicate (mean ± S.D.). B, TNF-α and IL-1β protein release from THP-1 treated with ethanol is shown. Where indicated, cells were transfected with indicated siRNA or control mock siRNA constructs. The data represent four independent experiments, and the ELISA assay was performed in duplicate (mean ± S.D.). C, mRNA levels of HO-1 show inverse correlation with TNF-α and IL-1β mRNA levels in liver tissue. mRNA isolated from control isocaloric-fed wild-type, ethanol-fed wild-type, isocaloric-fed c-Jun^fl/fl, and ethanol-fed c-Jun^fl/fl mice were analyzed by qRT-PCR. The data represent livers from six mice in each category, and the assay was performed in duplicate (mean ± S.D.). p values are denoted as: ***, p < 0.001 and ns, not significant. D, shown is an illustration of proposed ethanol-induced cell signaling pathways involved in regulating Nrf2 and HO-1 transcription.