Directional secretomes reflect polarity-specific functions in an in vitro model of human bronchial epithelium

Abstract

The polarity of the conducting airway epithelium is responsible for its directional secretion. This is an essential characteristic of lung integrity and function that dictates interactions between the external environment (apical) and subepithelial structures (basolateral). Defining the directional secretomes in the in vitro human bronchial epithelial (HBE) differentiated model could bring valuable insights into lung biology and pulmonary diseases. Normal primary HBE cells (n = 3) were differentiated into respiratory tract epithelium. Apical and basolateral secretions (24 h) were processed for proteome profiling and pathway analysis. A total of 243 proteins were identified in secretions from all HBE cultures combined. Of these, 51% were classified as secreted proteins, including true secreted proteins (36%) and exosomal proteins (15%). Close examination revealed consistent secretion of 69 apical proteins and 13 basolateral proteins and differential secretion of 25 proteins across all donors. Expression of Annexin A4 in apical secretions and Desmoglein-2 in basolateral secretions was validated using Western blot or ELISA in triplicate independent experiments. To the best of our knowledge, this is the first study defining apical and basolateral secretomes in the in vitro differentiated HBE model. The data demonstrate that epithelial polarity directs protein secretion with different patterns of biological processes to the apical and basolateral surfaces that are consistent with normal bronchial epithelium homeostatic functions. Applying this in vitro directional secretome model to lung diseases may elucidate their molecular pathophysiology and help define potential therapeutic targets.

Figure 1.

Average transepithelial electrical resistance of human bronchial epithelial (HBE) cell cultures measured between Days 14 and 21 of air–liquid interface (ALI). Dashed line represents average TEER in normal HBE cultures at Days 14 and 21 of ALI as previously described (47).

Figure 2.

Characterization of proteins in apical and basal secretomes. Identified proteins (n = 243) in apical and basolateral secretions of all normal HBE cell cultures (n = 3) after incubating in protein-free media for 24 hours and excluding cytokeratins (n = 29). (A) Classification of protein location based on Uniprot and SecretomeP databases; exosome distinction based on previous study (27). (B) Adjusted protein characterization based on abundance determined by average normalized spectral count of detected peptides (from ProteoIQ).

Figure 3.

Biological processes of uniquely secreted apical and basolateral proteins. Using the PANTHER database, a total of 55 processes were associated with the 31 proteins found in all apical secretions and with fewer than two peptides in basolateral secretions for all cell cultures. A total of 56 processes were identified for the 13 proteins present in all basolateral secretions and with fewer than two peptides in apical secretions.

Figure 4.

Western blot analysis of Annexin A4. A total of 20 μg of protein from apical (Ap) and basal (Ba) samples for all three cultures (1–3) were loaded. Results confirm proteomic findings of Annexin A4 only in apical, and not basolateral, secretions. Positive control (+): cell lysate; negative control (−): loading buffer.

Figure 5.

Expression of Desmoglein-2 in HBE secretions. Average protein concentration (± SEM) for each HBE cell culture was measured using ELISA. Statistical differences between amount of Desmoglein-2 in apical and basolateral secretions for each cell culture were based on Wilcoxon signed-rank test. *P < 0.05.