Tumor necrosis factor (TNF)-alpha-induced IL-8 expression in gastric epithelial cells: role of reactive oxygen species and AP endonuclease-1/redox factor (Ref)-1

Abstract

TNF-alpha contributes to oxidative stress via induction of reactive oxygen species (ROS) and pro-inflammatory cytokines. The molecular basis of this is not well understood but it is partly mediated through the inducible expression of IL-8. As redox factor-1 (Ref-1), is an important mediator of redox-regulated gene expression we investigated whether ROS and Ref-1 modulate TNF-alpha-induced IL-8 expression in human gastric epithelial cells. We found that TNF-alpha treatment of AGS cells enhanced nuclear expression of Ref-1 and potently induced IL-8 expression. Overexpression of Ref-1 enhanced IL-8 gene transcription at baseline and after TNF-alpha treatment whereas Ref-1 suppression and antioxidant treatment inhibited TNF-alpha-stimulated IL-8 expression. TNF-alpha-mediated enhancement of other pro-inflammatory chemokines like MIP-3 alpha and Gro-alpha was also regulated by Ref-1. Although TNF-alpha increased DNA binding activity of Ref-1-regulated transcription factors, AP-1 and NF-kappaB, to the IL-8 promoter, promoter activity was mainly mediated by NF-kappaB binding. Silencing of Ref-1 in AGS cells inhibited basal and TNF-alpha-induced AP-1 and NF-kappaB DNA binding activity, but not their nuclear accumulation. Collectively, we provide the first mechanistic evidence of Ref-1 involvement in TNF-alpha-mediated, redox-sensitive induction of IL-8 and other chemokines in human gastric mucosa. This has implications for understanding the pathogenesis of gastrointestinal inflammatory disorders.

Fig. 1

NAC inhibits TNF-α-induced IL-8 protein (A) and mRNA (B) production in AGS cells. AGS cells were pre-treated with 0 mM, 10 mM, 20 mM, or 40 mM NAC for 30 min prior to treatment with 20 ng/ml TNF-α or left untreated (controls). After 3 h the supernatants were assayed for IL-8 protein by ELISA (A). TNF-α stimulated the secretion of IL-8 protein by AGS cells, and pre-treatment with 20 mM and 40 mM NAC significantly suppressed TNF-α-induced IL-8 protein (*P < 0.05 versus cells that were treated with TNF-α only). (B) IL-8 mRNA expression was assayed by real-time RT-PCR 1 h post-treatment, and the graph depicts the levels of IL-8 mRNA (normalized to 18S rRNA) detected. IL-8 mRNA expression was increased by TNF-α treatment when compared to untreated cells, and pre-treatment with NAC at all concentrations tested (10, 20, or 40 mM NAC) significantly decreased TNF-α-induced IL-8 mRNA (* P < 0.05 versus cells that were treated with TNF-α only). Results represent the mean ± SEM (n = 3).

Fig. 2

(A) Effect of Ref-1 suppression on TNF-α-induced IL-8 expression in AGS cells. AGS cells transfected with 50 nM Ref-1 siRNA or mock-transfected cells were treated 48 h post-transfection with 20 ng/ml TNF-α or left untreated. Our previous studies demonstrated that IL-8 expression was no different in AGS cells that were transfected with scrambled siRNA versus mock-transfected cells [32]. IL-8 mRNA expression levels were assayed by real-time RT-PCR 1 h post-treatment. The graph depicts the percent IL-8 mRNA expression (normalized to 18S rRNA) of TNF-α-stimulated cells transfected with Ref-1 siRNA compared to TNF-α-stimulated cells transfected with Lipofectamine 2000 alone. TNF-α-induced IL-8 mRNA expression was significantly inhibited (*P < 0.05) in AGS cells transfected with Ref-1 siRNA compared to cells transfected with Lipofectamine 2000 alone. (B) Forty-eight hours post-transfection, cells were stimulated with TNF-α for 3 h and the supernatants were assayed for IL-8 by ELISA. Results are expressed as pg IL-8/mg total protein concentration. In cells expressing normal levels of Ref-1, TNF-α stimulated a significant increase in IL-8 protein. In cells transfected with Ref-1 siRNA, TNF-α-induced IL-8 protein was significantly inhibited (*P < 0.05 when compared to TNF-α-stimulated mock-transfected cells). All data represent the mean ± SEM of three separate experiments.

Fig. 3

siRNA-mediated silencing of Ref-1 inhibits TNF-α-induced AP-1 and NF-κB DNA binding activity but does not block nuclear accumulation of these transcription factors. AGS cells transfected with 50 nM Ref-1 siRNA (+) or non-transfected (−) cells were treated with 20 ng/ml TNF-α 48 h post-transfection. Untreated cells served as controls. Nuclear extracts were used to bind to radiolabeled AP-1 or NF-κB probes. (A) A representative EMSA autoradiogram showing AP-1 activation in non-transfected or Ref-1 siRNA-transfected AGS cells treated for 1 h with TNF-α. The arrow indicates the activated AP-1 complex. (B) A representative EMSA autoradiogram showing NF-κB activation in non-transfected or Ref-1 siRNA-transfected AGS cells treated with TNF-α for 2 h. The arrow indicates the activated NF-κB complex. The specificity of the shifted AP-1 and NF-κB complexes were analyzed using a 50-fold excess of unlabeled specific competitor or unlabeled non-specific competitor as indicated. (C) Non-transfected control cells (lanes 1 and 4), and cells transfected with 50 nM control siRNA (lanes 2 and 5) or Ref-1 siRNA (lanes 3 and 6) were treated with 20 ng/ml TNF-α or left untreated 48 h post-transfection for 1 or 2 h. After harvesting, nuclear extracts were analyzed by SDS–PAGE immunoblotting for NF-κB p50, p65, c-Jun, c-Fos. Ref-1 levels were assessed in nuclear and total cellular extracts. The data shown are representative of four separate experiments.

Fig. 4

Schematic representation of the luciferase-linked human IL-8 promoter constructs used in this study. The plasmids contain sequentially deleted 5′ flanking regions of the human IL-8 gene. Locations of transcription factor binding sites are indicated. The AP-1 binding site is located between –126 and –120 nucleotides, the NF-IL-6 binding site is between –94 and –81 nucleotides, and the NF-κB binding site is between –80 and –70 nucleotides. A consensus TATA box is located between –20 and –13 nucleotides.

Fig. 5

Effect of 5′ deletions and site-directed mutations in the human IL-8 promoter sequence on TNF-α-inducible IL-8 luciferase activity in cells with normal and over-expressed levels of Ref-1. AGS cells transfected with 5′ deletions of the hIL-8 Luc promoter were treated with 20 ng/ml TNF-α for 3 h. Untreated AGS cells served as controls. IL-8 luciferase activity normalized to protein, was expressed as fold induction for each construct relative to untreated cells (A). In cells bearing the −1498/+44 or the −99/+44 hIL-8/Luc constructs, TNF-α significantly stimulated IL-8 luciferase activity (*P < 0.05 when compared to untreated cells). In contrast, TNF-α did not induce IL-8 luciferase activity in cells transfected with the −54/+44 hIL-8/Luc construct. All data represent the mean relative luciferase activity ± SEM of triplicate samples from three separate experiments. AGS cells were transfected in triplicate with one of the site-mutated plasmids of the −162/+44 hIL-8/Luc promoter construct alone (B) or co-transfected with pFLAG-Ref-1 cDNA3.1 (C). Panel B depicts the percentage increase in normalized IL-8 luciferase activity of each plasmid in TNF-α treated compared tor untreated cells. In cells with basal levels of Ref-1 bearing the −162/+44 hIL-8/Luc construct, TNF-α significantly stimulated IL-8 transcription (*P < 0.05). Following TNF-α stimulation, mutation of the NF-κB binding site, but not the AP-1 binding site, significantly decreased TNF-α-induced promoter activity (^#P < 0.05 compared to TNF-α-treated cells transfected with −162/+44 hIL-8/Luc or −162/+44 hIL-8/Luc ΔAP-1). (C) The figure depicts the percentage increase in IL-8 luciferase activity of each plasmid in Ref-1 over-expressing cells compared to untreated cells with basal levels of Ref-1. In Ref-1 over-expressing cells, TNF-α significantly increased the IL-8 luciferase activity of the parent −162/+44 hIL-8/Luc plasmid, compared to untreated cells with constitutive levels of Ref-1 (*P < 0.05). Mutation of either the AP-1 or NF-κB binding sites significantly reduced IL-8 luciferase activity in stimulated cells that over-expressed Ref-1 (^#P < 0.05 compared to TNF-α treated cells co-transfected with −162/+44 hIL-8/Luc and pFLAG-Ref-1 cDNA3.1), but the difference in the IL-8 luciferase activity between the two mutated constructs was not significant. All data represent the mean ± SEM of three separate experiments.

Fig. 6

Confirmation of Ref-1 over-expression and the effects of over-expression on TNF-α-inducible IL-8 promoter activity in AGS cells transfected with the −1498/+44 hIL-8/Luc construct. (A) The 37 kDa bands demonstrate increased Ref-1 expression in cells transfected with 0.25 μg pFLAG-Ref-1 cDNA3.1 compared to non-transfected or mock-transfected cells by western blot. (B) The figure depicts the relative increase in normalized IL-8 luciferase activity of treated cells compared to untreated cells with basal levels of Ref-1. The over-expression of Ref-1 significantly (*P < 0.05) increased IL-8 luciferase activity in untreated control cells, and the IL-8 luciferase activity induced by TNF-α in cells with basal levels of Ref-1 was further enhanced in Ref-1 over-expressing cells following TNF-α stimulation (*P < 0.05). Data are expressed as mean relative luciferase activity of the −1498/+44 hIL-8/Luc construct compared to untreated cells with constitutive levels of Ref-1, and results represent the mean ± SEM of three separate experiments.

Fig. 7

Summary diagram illustrating a model for the mechanism by which Ref-1 regulates TNF-α-induced IL-8 in human gastric epithelial cells. TNF-α acts via its receptor, TNFR-1, on mitochondria to initiate caspase-mediated apoptosis that generates ROS, and/or on IκB kinase to activate NF-κB. ROS themselves can also activate NF-κB, and once activated, free NF-κB translocates into the nucleus. Similarly, either c-Jun/c-Jun homodimers or c-Jun/c-Fos heterodimers are activated by TNF-α or ROS through pathways involving mitogen-activated protein kinases leading to the activation of AP-1. Both ROS and TNF-α increase the expression of Ref-1 in gastric epithelial cells leading to nuclear translocation of Ref-1. The intranuclear presence of Ref-1 reductively modulates AP-1 and NF-κB activation, and in the reduced form, these transcription factors activate transcription by binding to DNA binding sites upstream of several redox-sensitive pro-inflammatory cytokine genes including IL-8.