Cigarette Smoke Activates NFκB and Induces Muc5b Expression in Mouse Middle Ear Cells

Authors

  • Diego Preciado MD, PhD,

    Corresponding author
    1. From the Center for Genetic Medicine Research, Children's National Medical Center, Washington, D.C., U.S.A
    2. Division of Pediatric Otolaryngology–Head and Neck Surgery, Children's National Medical Center, Washington, D.C., U.S.A
    • Diego Preciado, MD, PhD, Division of Pediatric Otolaryngology/Head and Neck Surgery, Children's National Medical Center, 111 Michigan Ave NW, Washington, D.C. 20010, U.S.A.
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  • Jezhin Lin MD,

    1. Department of Otolaryngology–Head and Neck Surgery, University of Minnesota, Minneapolis, Minnesota, U.S.A.
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  • Beverly Wuertz BS,

    1. Department of Otolaryngology–Head and Neck Surgery, University of Minnesota, Minneapolis, Minnesota, U.S.A.
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  • Mary Rose PhD

    1. From the Center for Genetic Medicine Research, Children's National Medical Center, Washington, D.C., U.S.A
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  • This work was supported by a Triological Society Career Development Award and by an Avery Scholar Research Award from Children's National Medical Center to d.p.

Abstract

Objectives: Cigarette smoke exposure is a significant risk factor in the development of otitis media (OM). Nuclear factor kappa B (NF-κB) 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-κB 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-κB 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-κB 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-κB 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-κB 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-κB.

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