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

Foster MW, Gwinn WM, Kelly FL, Brass DM, Valente AM, Moseley MA, Thompson JW, Morgan DL, Palmer SM.
Journal of Proteome Research (2017) DOI: https://doi.org/10.1021/acs.jproteome.6b00672 PMID: 27966365

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Publication

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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.

Figures

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Figure 1. Hematoxylin and eosin staining of TBEC cross-sections shows evidence of DA vapor injury.

H&E staining was performed on cross sections of formalin-fixed and paraffin-imbedded PBS- and DA-exposed TBECs from (A) donor 1, (B) donor 2, (C) donor 3, and (D) donor 4. Images are representative of three sections per donor.

• Figure 1 (304 KB)

Figure 2. Hierarchical clustering of differentially expressed proteins and phosphoproteins.

(A) Proteome data were filtered to contain proteins quantified by two or more peptides, with a CV of <30% for triplicate QC injections and p < 0.1 for a paired t test w/FDR correction. The remaining 874 proteins were normalized using Z-scoring and were visualized by 2D hierarchical clustering (Ward method). Cluster 7 (green) is composed predominantly of proteins expressed in multiciliated airway cells, and clusters 10–12 (yellow) are composed predominantly of proteins expressed during squamous differentiation (see Results).
(B) Phosphopeptides were filtered by CV < 30% and p < 0.1 and visualized as in (C). Data were analyzed using JMP (SAS, Cary, NC).

• Figure 2 (510 KB)

Figure 3. Cilia-specific marker, RSPH4A, was markedly reduced after DA treatment.

(A) The levels of RSPH4A and GAPDH in cell lysates were visualized by Western blotting. Western blotting performed on a replicate across the entire MW range (Figure S3) showed that only a single antigen was recognized by this antibody. (B) Immunofluorescence was performed on cross sections of formalin-fixed and paraffin-imbedded TBECs to visualize RSPH4A (green, Alexa Fluor 488), β-catenin (red, Alexa Fluor 594), and nuclei (blue, DAPI) in donor 3. Localization of RSPH4A in ciliated cells is shown by blue arrow (upper image), and localization to basal cells is shown by yellow arrow (lower image). Images in panel B are representative of three different sections per condition (Figure S4A,B), and results were replicated in single sections from donors 2 and 4 (Figure S4C–F).

• Figure 3 (242 KB)

Figure 4. Markers of squamous differentiation are increased in DA-exposed cells.

(A) The levels of TGM1, repetin, and GAPDH in cell lysates were visualized by Western blotting.
(B,C) Immunofluorescence was performed on cross sections of formalin-fixed and paraffin-imbedded TBECs to visualize (B) TGM1 (green, Alexa Fluor 488), β-catenin (red, Alexa Fluor 594), and nuclei (blue, DAPI) in donor 3, and (C) repetin (green, Alexa Fluor 488), β-catenin (red, Alexa Fluor 594), and nuclei (blue, DAPI) in donor 3. Localization of repetin and TGM1 immunostaining is indicated by yellow arrows in panels B and C, respectively. Nonspecific staining of transwell membrane by secondary antibody is indicated by gray arrow. Images in panel B are representative of three different sections per condition (Figure S5A,B), and results were replicated in a single section from donor 2 (Figure S5C,D). Images in panel C are representative of three different sections per condition (Figure S6A,B), and results were replicated in single sections from donors 2 and 4 (Figure S6C−F).

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Figure 5. DA exposure induces cross-linking of basal cell cytokeratins.

(A) Fold changes were plotted for the basal cell keratins and clara cell marker CCSP, as determined by proteomic analysis. Note that keratin expression values were recalculated from isoform-specific peptides (Tables S7 and S8).
(B) The expression and “crosslinking” of cytokeratins was visualized by Western blotting of whole cell lysates. For K14, K6, and K17, panels labeled as (high MW) are longer exposures of the identical membranes from which the ∼50 kDa keratin monomers were visualized.

• Figure 5 (279 KB)

Figure 6. DA exposure results in hyperphosphorylation of keratin 6.

(A–D) Parallel reaction monitoring was used to analyze a mixture of monophosphorylated, stable isotope-labeled peptide standards with sequence 31SGFSSVSVSR40 (precursor m/z 551.7438). Isoform-specific product ions are shown for the peptides assigned to (A) 31(pSer)GFSSVSVSR40, (B) 31SGF(pSer)SVSVSR40, (C) 31SGFS(pSer)VSVSR40, and (D) 31SGFSSV(pSer)VSR40. The position of the properly assigned peak is indicated by an arrow. Note that the peptides containing pSer31 and pSer35 are virtually overlapping but can be distinguished based on unique product ions and retention times (as shown in Figure S8).
(E) K6 phosphopeptides were quantified by PRM. For each donor, phosphopeptide ratios were determined for DA versus PBS samples. Multiply phosphorylated peptides were quantified by label-free area-under-the-curve (AUC), and monophosphorylated peptides were quantified by AUC and normalized to SIL internal standards. Keratin 6 protein ratios are also shown (from Figure 5).
(F) Data in panel A were normalized to ratio of K6 protein.

• Figure 6 (412 KB)

Tables

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Table 1. Protein Signature of Diacetyl Injury to Multiciliated Cells^a.

aProteins ranked by average fold change of ∼−5-fold or greater in DA- versus PBS-treated HBECs (quantified by two or more peptides, CV < 30% for replicate injections of QC pool; p < 0.1, paired t test w/FDR correction) and localization to cytoplasm/membranes of ciliated cells of human bronchi by the Human Protein Atlas (http://www.proteinatlas.org).
bProteins with nuclear localization.

• Table 1 (160 KB)

Table 2. Phosphoprotein Signature of Diacetyl Injury to Multiciliated Cells^a.

aPhosphoproteins ranked by number of unique phosphopeptides with ∼−5-fold or greater in DA- versus PBS-treated HBECs (CV < 30% for replicate injections of QC pool; p < 0.1, paired t test w/FDR correction) and localization to cytoplasm/membranes of ciliated cells of human bronchi by the Human Protein Atlas (http://www.proteinatlas.org).
bProteins with nuclear localization.

• Table 2 (150 KB)

Table 3. Protein Signature of Squamous Differentiation^a.

aProteins ranked by average fold change in donors 2–4 in DA- versus PBS-treated HBECs (quantified by 2 or more peptides, CV < 30% for replicate injections of QC pool; required >2-fold change for donors 2–4).

• Table 3 (45 KB)

Supplemental Materials

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Supporting Information

• Table S1 (10 KB)
• Table S2 (4 MB)
• Table S3 (948 KB)
• Table S4 (10 KB)
• Table S5 (1 MB)
• Table S6 (2 MB)
• Table S7 (95 KB)
• Table S8 (14 KB)