title: 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.
  
  
  CONCLUSIONS
  p65 phosphorylation and IkappaBalpha phosphorylation and degradation are the primary mechanisms involved in ZnO nanoparticle-induced IL-8 expression in human bronchial epithelial cells.
authors.affiliation: Center for Environmental Medicine, Asthma, and Lung Biology, University of North Carolina, Chapel Hill, 27599, USA. Weidong_Wu@med.unc.edu
authors.forename: Weidong
authors.initials: W
authors.lastname: Wu
authors.forename: James M
authors.initials: JM
authors.lastname: Samet
authors.forename: David B
authors.initials: DB
authors.lastname: Peden
authors.forename: Philip A
authors.initials: PA
authors.lastname: Bromberg
mesh_terms: D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism*; D016209:Interleukin-8 / Q000235:genetics / Q000378:metabolism*; D008168:Lung / Q000166:cytology; D000072000:NF-KappaB Inhibitor alpha; D053758:Nanoparticles / Q000633:toxicity*; D010766:Phosphorylation / Q000187:drug effects; D011401:Promoter Regions, Genetic / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051996:Transcription Factor RelA / Q000378:metabolism*; D015034:Zinc Oxide / Q000633:toxicity*
chemical_list: D020672:I-kappa B Proteins; D016209:Interleukin-8; C000606204:NFKBIA protein, human; D051996:Transcription Factor RelA; D000072000:NF-KappaB Inhibitor alpha; D015034:Zinc Oxide