Cigarette smoke activates NFκB-mediated Tnf-α release from mouse middle ear cells.
OBJECTIVE
: Cigarette smoke exposure is a significant risk factor in the development of otitis media. NF-κB is a transcription factor known to mediate cigarette smoke effects in multiple cell types. We hypothesized that stimulation of murine middle ear epithelial cells (MEEC) with cigarette smoke condensate (CSC) activates NF-κB resulting in upregulation of proinflammatory cytokines.


METHODS
: In vitro model of cultured murine middle ear epithelial cells.


METHODS
: Time course CSC stimulation of MEEC was performed. Antibody microarrays were then utilized to simultaneously measure 40 inflammatory cytokines. Enzyme-linked immunosorbent assay (ELISA) and quantitative reverse transcriptase-polymerase chain reaction were performed to validate and further evaluate array results. Luciferase reporter assays were performed to evaluate NF-κB activation with CSC in MEEC. Chromatin immunoprecipitation (ChIP) assays were performed to determine whether CSC induces NF-κB interaction with the Tnf-α promoter.


RESULTS
: Multiple cytokines showed significant increases with CSC exposure. ELISA studies demonstrated that Tnf-α secretion increased the most. CSC stimulation likewise increased Tnf-α mRNA abundance and induced promoter activity 4.8-fold in a Tnf-α reporter plasmid. Reporter assays demonstrated 4.84-fold activation of NF-κB with CSC. ChIP assays demonstrated NF-κB binding to canonical κB sites in the Tnf-α promoter with CSC stimulation.


CONCLUSIONS
: CSC activates NF-κB in MEEC. Furthermore, this activation results in CSC induced Tnf-α promoter activation, gene expression, and levels in cell secretions. Laryngoscope, 120:2508-2515, 2010.
Center for Genetic Medicine Research, Georgetown University, Washington, DC 20010, USA. dpreciad@cnmc.org
Diego
D
Preciado
Elaine
E
Kuo
Samaneh
S
Ashktorab
Peter
P
Manes
Mary
M
Rose
D000818:Animals; D002460:Cell Line; D004195:Disease Models, Animal; D004432:Ear, Middle / Q000378:metabolism* / Q000473:pathology; D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000378:metabolism / Q000473:pathology; D005786:Gene Expression Regulation; D047468:Immunoprecipitation; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism*; D010033:Otitis Media / Q000209:etiology / Q000378:metabolism / Q000473:pathology; D012313:RNA / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012907:Smoking / Q000009:adverse effects* / Q000378:metabolism / Q000473:pathology; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000235:genetics
D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D012313:RNA