title: Proteomic Analysis of Primary Human Airway Epithelial Cells Exposed to the Respiratory Toxicant Diacetyl.
abstract: Occupational exposures to the diketone flavoring agent, diacetyl, have been associated with bronchiolitis obliterans, a rare condition of airway fibrosis. Model studies in rodents have suggested that the airway epithelium is a major site of diacetyl toxicity, but the effects of diacetyl exposure upon the human airway epithelium are poorly characterized. Here we performed quantitative LC-MS/MS-based proteomics to study the effects of repeated diacetyl vapor exposures on 3D organotypic cultures of human primary tracheobronchial epithelial cells. Using a label-free approach, we quantified approximately 3400 proteins and 5700 phosphopeptides in cell lysates across four independent donors. Altered expression of proteins and phosphopeptides were suggestive of loss of cilia and increased squamous differentiation in diacetyl-exposed cells. These phenomena were confirmed by immunofluorescence staining of culture cross sections. Hyperphosphorylation and cross-linking of basal cell keratins were also observed in diacetyl-treated cells, and we used parallel reaction monitoring to confidently localize and quantify previously uncharacterized sites of phosphorylation in keratin 6. Collectively, these data identify numerous molecular changes in the epithelium that may be important to the pathogenesis of flavoring-induced bronchiolitis obliterans. More generally, this study highlights the utility of quantitative proteomics for the study of in vitro models of airway injury and disease.
authors.forename: Matthew W
authors.identifier: 0000-0003-0212-2346
authors.initials: MW
authors.lastname: Foster
authors.affiliation: National Institute of Environmental Health Sciences , Research Triangle Park, North Carolina 27709, United States.
authors.forename: William M
authors.initials: WM
authors.lastname: Gwinn
authors.forename: Francine L
authors.initials: FL
authors.lastname: Kelly
authors.forename: David M
authors.initials: DM
authors.lastname: Brass
authors.forename: Ashlee M
authors.initials: AM
authors.lastname: Valente
authors.forename: M Arthur
authors.initials: MA
authors.lastname: Moseley
authors.forename: J Will
authors.initials: JW
authors.lastname: Thompson
authors.affiliation: National Institute of Environmental Health Sciences , Research Triangle Park, North Carolina 27709, United States.
authors.forename: Daniel L
authors.initials: DL
authors.lastname: Morgan
authors.forename: Scott M
authors.initials: SM
authors.lastname: Palmer
mesh_terms: D000293:Adolescent; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002923:Cilia / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D003931:Diacetyl / Q000633:toxicity*; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D005421:Flavoring Agents / Q000633:toxicity*; D005786:Gene Expression Regulation / Q000187:drug effects*; D063990:Gene Ontology; D006801:Humans; D053553:Keratin-6 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D058977:Molecular Sequence Annotation; D010766:Phosphorylation / Q000187:drug effects; D061251:Primary Cell Culture; D020543:Proteome / Q000235:genetics* / Q000378:metabolism; D020545:Respiratory Mucosa / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D014835:Volatilization; D055815:Young Adult
chemical_list: D005421:Flavoring Agents; D053553:Keratin-6; D020543:Proteome; D003931:Diacetyl
keywords: BO; PRM; RSPH4A; TGM1; butanedione; butter flavoring; cornified envelope; pentanedione; repetin; squamous metaplasia