Proteomic Analysis of Primary Human Airway Epithelial Cells Exposed to the Respiratory Toxicant Diacetyl

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.