Phosphorylation of p65 is required for zinc oxide nanoparticle-induced interleukin 8 expression in human bronchial epithelial cells

Abstract

Background: Exposure to zinc oxide (ZnO) in environmental and occupational settings causes acute pulmonary responses through the induction of proinflammatory mediators such as interleukin-8 (IL-8).

Objective: We investigated the effect of ZnO nanoparticles on IL-8 expression and the underlying mechanisms in human bronchial epithelial cells.

Methods: We determined IL-8 mRNA and protein expression in primary human bronchial epithelial cells and the BEAS-2B human bronchial epithelial cell line using reverse-transcriptase polymerase chain reaction and the enzyme-linked immunosorbent assay, respectively. Transcriptional activity of IL-8 promoter and nuclear factor kappa B (NFkappaB) in ZnO-treated BEAS-2B cells was measured using transient gene transfection of the luciferase reporter construct with or without p65 constructs. Phosphorylation and degradation of IkappaBalpha, an inhibitor of NF-kappaB, and phosphorylation of p65 were detected using immunoblotting. Binding of p65 to the IL-8 promoter was examined using the chromatin immunoprecipitation assay.

Results: ZnO exposure (2-8 microg/mL) increased IL-8 mRNA and protein expression. Inhibition of transcription with actinomycin D blocked ZnO-induced IL-8 expression, which was consistent with the observation that ZnO exposure increased IL-8 promoter reporter activity. Further study demonstrated that the kappaB-binding site in the IL-8 promoter was required for ZnO-induced IL-8 transcriptional activation. ZnO stimulation modestly elevated IkappaBalpha phosphorylation and degradation. Moreover, ZnO exposure also increased the binding of p65 to the IL-8 promoter and p65 phosphorylation at serines 276 and 536. Overexpression of p65 constructs mutated at serines 276 or 536 significantly reduced ZnO-induced increase in IL-8 promoter reporter activity.

Conclusion: p65 phosphorylation and IkappaBalpha phosphorylation and degradation are the primary mechanisms involved in ZnO nanoparticle-induced IL-8 expression in human bronchial epithelial cells.

Figure 1

ZnO exposure increases IL-8 mRNA and protein expression in human bronchial epithelial cells. (A) IL-8/GAPDH mRNA level in ALI-cultured primary human bronchial epithelial cells treated with 2–8 μg/mL ZnO, (B) confluent BEAS-2B cells treated with 8 μg/mL ZnO for 2 hr or 4 hr, and (C) confluent BEAS-2B cells treated with 2, 4, or 8 μg/mL ZnO for 4 hr. IL-8 mRNA levels were determined by RT-PCR. (D) IL-8 protein in BEAS-2B cells treated with 4 or 8 μg/mL ZnO for 6 hr; assayed protein content was determined using ELISA. Data shown are representative of three separate experiments. *p < 0.05 compared with 0 μg/mL ZnO.

Figure 2

Transcriptional regulation is involved in ZnO-induced IL-8 expression. (A) IL-8/GAPDH mRNA level in confluent BEAS-2B cells pretreated with 10 μg/mL Act D for 30 min, then stimulated with 8 μg/mL ZnO for 2 hr. Dimethyl sulfoxide served as a vehicle control for Act D. IL-8 mRNA levels were determined by RT-PCR. (B) IL-8 promoter reporter activity in BEAS-2B cells grown to 40–50% confluence and transfected with p1.5IL-8-luc and pSV-β-galactosidase constructs using FuGENE 6 transfection reagent. Twenty-four hours after transfection, cultures were incubated with keratinocyte basal medium overnight; the cells were then treated with 8 μg/mL ZnO for 6 hr before being lysed. Luciferase activity was estimated as luciferase count/β-galactosidase count (luc/gal). Data shown are representative of three separate experiments. *p < 0.05 compared with DMSO plus ZnO. **p < 0.05 compared with 0 μg/mL ZnO.

Figure 3

ZnO exposure induces NFκB activation in BEAS-2B cells. (A) Phosphorylation of IκBα in cells pretreated with 20 μM MG132 for 30 min (to prevent IκBα degradation) before further stimulation with 8 μg/mL ZnO for 15, 30, 60, or 120 min, or 100 ng/mL TNFα for 30 min; cell lysates were separated by SDS-PAGE and immunoblotted with anti-phospho-IκBα (Ser32/36) and anti-IκBα antibodies. (B) IκBα degradation in cells treated with 8 μg/mL ZnO for 15, 30, 60, or 120 min, or 100 ng/mL TNFα for 30 min; cell lysates were separated by SDS-PAGE and immunoblotted with anti-IκBα and anti-actin antibodies. (C) NFκB reporter activity, estimated as luciferase count/β-galactosidase count, in cells grown to 40–50% confluence, transfected with pNFκB-luc and p-SV-β-galactosidase constructs, and treated with 8 μg/mL ZnO for 6 hr. Data shown are representative of three separate experiments. *p < 0.05 compared with 0 μg/mL ZnO.

Figure 4

NFκB is required for ZnO-induced IL-8 gene transcription, as shown by luciferase activity. BEAS-2B cells grown to 40–50% confluence were transfected with p1.5IL-8-luc (IL-8 wild-type) or p1.5IL-8-κB-luc (IL-8 mutant), as described in “Materials and Methods,” prior to treatment with 8 μg/mL ZnO for 6 hr. Data shown are representative of three separate experiments. *p < 0.05 compared with ZnO treatment in the IL-8 wild-type group.

Figure 5

Phosphorylation of p65 NFκB mediates ZnO-induced IL-8 gene transcription in BEAS-2B cells. (A) Binding of the IL-8 gene promoter in cells treated with 8 μg/mL ZnO for 2 hr, as detected by the ChIP assay using anti-p65 antibody. Precipitates from the antibody against IgG served as a negative control, and 2% of the diluted DNA was used as loading control. The target sequence for PCR was located around the IL-8 gene promoter. (B) Phosphorylation of p65 NFκB in cells treated with 8 μg/mL ZnO for 15–60 min and analyzed by SDS-PAGE and immunoblotting with anti-phospho-p65/RelA (Ser536), anti-phospho-p65/RelA (Ser276), or anti-p65/RelA antibodies. (C) IL-8 promoter reporter activity, estimated as luciferase count/β-galactosidase count, in cells grown to 40–50% confluence and co-transfected with wild-type p65 construct or a mutated version, as described in “Materials and Methods,” prior to treatment with 8 μg/mL ZnO for 6 hr. Data shown are representative of three separate experiments. *p < 0.05 compared with the p65 wild-type group.