Dysfunctional ErbB2, an EGF receptor family member, hinders repair of airway epithelial cells from asthmatic patients

Abstract

Background: Genetic and genomic data increasingly point to the airway epithelium as critical to asthma pathogenesis. Epithelial growth factor (EGF) family members play a fundamental role in epithelial differentiation, proliferation, and repair. Although expression of erythroblastosis oncogene B2 (ErbB2) mRNA, an EGF family receptor, was reported to be lower in asthmatic patients, little is understood about its functional role.

Objective: We sought to determine whether decreased ErbB2 activation in freshly isolated human airway epithelial cells (HAECs) from asthmatic patients associated with impaired wound closure in vitro.

Methods: An in vitro scratch-wound model of air-liquid interface cultured and freshly isolated HAECs were compared between HAECs from healthy control subjects (HCs) and asthmatic patients in relation to ErbB2.

Results: Freshly brushed HAECs from asthmatic patients had impaired ErbB2 activation compared with those from HCs. In an in vitro scratch-wound model, HAECs from asthmatic patients showed delayed wound closure compared with HAECs from HCs. Cell proliferation, as assessed based on [³H] thymidine incorporation after wounding, and expression or activation of ErbB2 and cyclin D1 at the leading edge of the wound were lower in HAECs from asthmatic patients and HCs. A selective ErbB2 tyrosine kinase inhibitor, mubritinib, impaired wound closure and decreased cyclin D1 expression in healthy HAECs, with less effect on cells from asthmatic patients, supporting diminished activity in asthmatic patients.

Conclusion: These results implicate a primary defect in the ErbB2 pathway as constraining epithelial repair processes in asthmatic patients. Restoration of homeostatic ErbB2 function should be considered a novel asthma therapeutic target.

Keywords: Asthma; air-liquid interface culture; airway inflammation; cell proliferation; cyclin D1; epidermal growth factor receptor; epithelial cell; erythroblastosis oncogene B2; wound repair.

Fig. 1.. in vivo phosphorylated ErbB2 protein expression in freshly isolated HAECs is lower in asthmatic than in healthy subjects.

Total and phosphorylated ErbB2 (tErbB2 and pErbB2) protein expression in freshly isolated HAECs was analyzed by western blotting (A-G) and immunofluorescent staining of cytospin samples (H-I). (A, D) There were no differences in tErbB2 protein expression across the groups. (B, E) pErbB2 protein and (C, F) the ratio of phosphorylated to total ErbB2 (p/t ErbB2) was lower in asthmatic HAECs, but without difference by severity. (G) Representative western blots for ErbB2 among freshly isolated HAECs. (H) Representative immunofluorescent pictures for pErbB2 among cytospin HAECs samples. Green: pErbB2, Blue: nucleus. The white bar represents 100 μm. (I) pErbB2 positive cells were less in asthmatic HAECs than in healthy HAECs. Each bar represents median with interquartile range. Statistical analysis was performed by Wilcoxon rank sum test. Symbols; ○: healthy control, ▲: mild-to-moderate asthma, ■: severe asthma. Abbreviations: HC, Healthy control; MMA, Mild-to-moderate asthma; SA, Severe asthma.

Fig. 2.. Wound closure and cell proliferation after wounding is impaired in asthmatic HAECs.

(A) Wound closure was decreased in asthmatic HAECs compared to healthy HAECs in air-liquid interface (ALI) culture. HAECs were cultured in ALI for 7 days. Wound area was measured at baseline and 7 hours after wounding, and percentage of wound closure at 7 hours after wounding was calculated. Each bar represents mean ± SEM. Statistical analysis was performed by Student’s t test. (B) Asthmatic patients with the worst HAEC wound closure (WC) (bottom quartile) had higher FeNO than those whose wound closure overlapped with normal wound repair (top quartile). Hi-%WC was defined as %WC >45%, and Lo-%WC was defined as %WC <30%. (C) Cell proliferation after wounding was impaired in asthmatic HAECs compared to healthy HAECs. [³H] thymidine incorporation at 16 hours after wounding was higher in healthy HAECs than in asthmatic HAECs. The fold-change of [³H] thymidine incorporation between 3 to 16 hours after wounding was significantly higher in healthy HAECs than in asthmatic HAECs. Statistical analyses were performed by Student’s t test and Wilcoxon rank sum test, respectively. Symbols; ○: healthy control, ▲: mild-to-moderate asthma, ■: severe asthma.

Fig. 3.. Wounding induced CCND1 expression is reduced in asthmatic HAECs.

(A) CCND1 mRNA expression. (B) CCND1protein expression. (C) Representative western blot for ErbB2 and CCND1 protein expression with or without wounding. (D) Representative immunofluorescent staining images for CCND1 at wound leading edge. Red: CCND1, Blue: nucleus. The white bar represents 300 μm. (E) CCND1 positive cells along wound leading edge were higher in healthy as compared to asthmatic HAECs. Each bar represents mean ± SEM. Statistical analysis was performed by Student’s t test. Symbols; ○: healthy control, ▲: mild-to-moderate asthma, ■: severe asthma.

Fig. 4.. Phosphorylation of ErbB2 after wounding is impaired in asthmatic HAECs.

(A) ERBB2 mRNA expression. (B) Total ErbB2 protein expression. (C) Phosphorylated ErbB2 protein expression. (D) Ratio of phosphorylated to total ErbB2 protein (P = 0.09 and 0.04 for difference in ratio between HC and asthma before and after wounding, respectively). (E) Representative immunofluorescent staining pictures and their intensity profile for phosphorylated ErbB2 and F-actin at wound leading edge. Red: phosphorylated ErbB2, Green: F-actin, Blue: nucleus. The white bar represents 100 μm. (F) Phosphorylated ErbB2 expression along leading edge of the wound was higher in healthy as compared to asthmatic HAECs. Each bar represents mean ± SEM. Statistical analysis was performed by Student’s t test. Symbols; ○: healthy control, ▲: mild-to-moderate asthma, ■: severe asthma.

Fig. 5.. Inhibition of ErbB2 phosphorylation impairs wound closure and the effect is greater in HC HAECs.

(A) Mubritinib (100nM) treatment decreased wound closure after 7 hours of wounding with greater impact in healthy HAECs as compared to asthmatic HAECs (P = 0.04 for difference in decrease in wound closure between HCs and asthma). (B) CCND1 protein expression was inhibited by mubritinib treatment in healthy HAECs, but not in asthmatic HAECs (P = 0.04 for difference between HC and asthma). (C) Representative western blot for ErbB2 and CCND1 protein expression with or without mubritinib treatment. (D) Phosphorylation of ErbB2 along leading edge of the wound was impaired by mubritinib primarily in healthy HAECs. Representative immunofluorescent staining for phosphorylated ErbB2 and F-actin at leading edge of the wound. Red: phosphorylated ErbB2, Green: F-actin, Blue: nucleus. The white bar represents 100 μm. (E) Intensity ratio of phosphorylated ErbB2 to F-actin at wound leading edge was decreased by mubritinib in healthy HAECs, not in asthmatic HAECs. Each bar represents mean ± SEM. Statistical analysis was performed by Student’s t test.