Cigarette smoke activates NF kappa B and induces Muc5b expression in mouse middle ear cells.
OBJECTIVE
Cigarette smoke exposure is a significant risk factor in the development of otitis media (OM). Nuclear factor kappa B (NF-kappa B) is a ubiquitous transcription factor known to mediate cigarette smoke effects on gene regulation in multiple cell types. The MUC5B mucin gene contains several putative NF-kappa B sites in its promoter and is the predominant mucin expressed in human OM. We hypothesized that in vitro stimulation of a recently developed model system, murine middle ear epithelial cells (MEEC), with cigarette smoke condensate (CSC) activates NF-kappa B and subsequently induces Muc5b gene expression.


METHODS
Luciferase reporter assays, electromobility shift assays (EMSA), and quantitative microplate transcription factor assays (TFA) were performed to evaluate NF-kappaB activation with CSC in immortalized murine MEEC (mMEEC). Reverse transcriptase polymerase chain reaction (RT-PCR) assays and quantitative real time RT-PCR were performed to determine whether time course CSC stimulation upregulates Muc5b mRNA levels in differentiated mMEEC. Luciferase reporter assays were performed to determine whether CSC activates the Muc5b promoter.


RESULTS
Reporter assays, EMSA, and TFA demonstrated three- to five-fold dose-dependent activation of NF-kappa B with CSC in mMEEC. CSC stimulation likewise increased Muc5b mRNA abundance and induced reporter activity 1.8- to 4.8-fold in plasmids containing -556 and -255 base pairs upstream of the Muc5b transcriptional start site in mMEEC.


CONCLUSIONS
CSC activates NF-kappaB in immortalized MEEC. Furthermore, this activation correlates with CSC-induced Muc5b promoter activation and gene expression. Taken together, these results hint that much as in lung cells, the activation of mucins by cigarette smoke is mediated in part by NF-kappa B.
Preciado
Diego
D
Center for Genetic Medicine Research, Children's National Medical Center, Washington, DC 20010, USA. dpreciad@cnmc.org
Lin
Jezhin
J
Wuertz
Beverly
B
Rose
Mary
M
D000818:Animals; D002478:Cells, Cultured; D004432:Ear, Middle / Q000166:cytology*; D051379:Mice; D055257:Mucin-5B; D009077:Mucins / Q000096:biosynthesis*; D016328:NF-kappa B / Q000502:physiology*; D012906:Smoke; D014026:Nicotiana
C524578:MUC5B protein, human; D055257:Mucin-5B; D009077:Mucins; D016328:NF-kappa B; D012906:Smoke