Phosphorylation of p65 Is Required for Zinc Oxide Nanoparticle–Induced Interleukin 8 Expression in Human Bronchial Epithelial Cells

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 (NFκB) 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 IκBα, an inhibitor of NF-κB, 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 μg/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 κB-binding site in the IL-8 promoter was required for ZnO-induced IL-8 transcriptional activation. ZnO stimulation modestly elevated IκBα 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 IκBα phosphorylation and degradation are the primary mechanisms involved in ZnO nanoparticle-induced IL-8 expression in human bronchial epithelial cells

The 3′-untranslated region of human interleukin-8 mRNA suppresses IL-8 gene expression

Although adenosine/uridine (AU)-rich sequences in the 3′-untranslated region (UTR) of the interleukin-8 (IL-8) gene have been suggested to contribute to its post-transcriptional regulation, the molecular basis whereby this occurs still needs to be understood. To investigate the role of the 3′-UTR on human IL-8 gene regulation, chimeric reporter genes were generated by adding full length or differentially deleted 3′-UTR of the IL-8 gene to chloramphenicol acetyltransferase (CAT). Addition of the entire IL-8 3′-UTR markedly reduced CAT mRNA and protein expression in COS 7 cells. In a reporter gene study, IL-8 3′-UTR destabilized CAT mRNA, which was dependent on active transcription in COS 7 cells. A 357-base sequence (nucleotides (nt) 2387–2743 of genomic DNA) within 3′-UTR, designated e, suppressed CAT gene expression by accelerating CAT mRNA turnover. A 26-base AU-rich sequence (nt 2552–2577) within e, containing four AUUUA pentamers that form two UAUUUAUU and one UUAUUUAU octamers, did not suppress CAT gene expression. However, deletion of the AU-rich sequences attenuated the inhibitory effect of e on CAT gene expression. Elimination of the first 100 bases (nt 2386–2486) attenuated the potency of fragment e, but much weaker than elimination of the first 146 bases (nt 2387–2533). This study gives new insights in unravelling the molecular mechanisms involved in the post-transcriptional regulation of the IL-8 gene