title: Oxidative stress induced interleukin-32 mRNA expression in human bronchial epithelial cells.
abstract: BACKGROUND
  Chronic obstructive pulmonary disease (COPD) is characterized by airflow obstruction and persistent inflammation in the airways and lung parenchyma. Oxidative stress contributes to the pathogenesis of COPD. Interleukin (IL)-32 expression has been reported to increase in the lung tissue of patients with COPD. Here, we show that IFNγ upregulated IL-32 expression and that oxidative stress augmented IFNγ-induced-IL-32 expression in airway epithelial cells. We further investigated transcriptional regulation responsible for IFNγ induced IL-32 expression in human airway epithelial cells.
  
  
  METHODS
  Human bronchial epithelial (HBE) cells were stimulated with H2O2 and IFNγ, and IL-32 expression was evaluated. The cell viability was confirmed by MTT assay. The intracellular signaling pathways regulating IL-32 expression were investigated by examining the regulatory effects of MAPK inhibitors and JAK inhibitor after treatment with H2O2 and IFNγ, and by using a ChIP assay to identify transcription factors (i.e. c-Jun, CREB) binding to the IL-32 promoter. Promoter activity assays were conducted after mutations were introduced into binding sites of c-Jun and CREB in the IL-32 promoter. IL-32 expression was also examined in HBE cells in which the expression of either c-Jun or CREB was knocked out by siRNA of indicated transcription factors.
  
  
  RESULTS
  There were no significant differences of cell viability among groups. After stimulation with H2O2 or IFNγ for 48 hours, IL-32 expression in HBE cells was increased by IFNγ and synergistically upregulated by the addition of H2O2. The H2O2 augmented IFNγ induced IL-32 mRNA expression was suppressed by a JNK inhibitor, but not by MEK inhibitor, p38 inhibitor, and JAK inhibitor I. Significant binding of c-Jun and CREB to the IL-32 promoter was observed in the IFNγ + H2O2 stimulated HBE cells. Introducing mutations into the c-Jun/CREB binding sites in the IL-32 promoter prominently suppressed its transcriptional activity. Further, knocking down CREB expression by siRNA resulted in significant suppression of IL-32 induction by IFNγ and H2O2 in HBE cells.
  
  
  CONCLUSIONS
  IL-32 expression in airway epithelium may be augmented by inflammation and oxidative stress, which may occur in COPD acute exacerbation. c-Jun and CREB are key transcriptional factors in IFNγ and H2O2 induced IL-32 expression.
authors.affiliation: Department of Respiratory Medicine, Kyoto University Graduate School of Medicine, Kyoto, Japan.
authors.forename: Megumi
authors.initials: M
authors.lastname: Kudo
authors.forename: Emiko
authors.initials: E
authors.lastname: Ogawa
authors.forename: Daisuke
authors.initials: D
authors.lastname: Kinose
authors.forename: Akane
authors.initials: A
authors.lastname: Haruna
authors.forename: Tamaki
authors.initials: T
authors.lastname: Takahashi
authors.forename: Naoya
authors.initials: N
authors.lastname: Tanabe
authors.forename: Satoshi
authors.initials: S
authors.lastname: Marumo
authors.forename: Yuma
authors.initials: Y
authors.lastname: Hoshino
authors.forename: Toyohiro
authors.initials: T
authors.lastname: Hirai
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Sakai
authors.forename: Shigeo
authors.initials: S
authors.lastname: Muro
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Date
authors.forename: Michiaki
authors.initials: M
authors.lastname: Mishima
mesh_terms: D001980:Bronchi / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007371:Interferon-gamma / Q000494:pharmacology; D007378:Interleukins / Q000378:metabolism*; D020935:MAP Kinase Signaling System / Q000502:physiology; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology*; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology
chemical_list: D017362:Cyclic AMP Response Element-Binding Protein; C496971:IL32 protein, human; D007378:Interleukins; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D007371:Interferon-gamma; D006861:Hydrogen Peroxide