Blocking Notch3 Signaling Abolishes MUC5AC Production in Airway Epithelial Cells From Asthmatics

Andrew T Reid, Kristy S Nichol, Punnam Chander Veerati, Fatemeh Moheimani, Anthony Kicic, Stephen M Stick , Nathan W Bartlett, Chris L Grainge, Peter A B Wark, Philip M Hansbro, Darryl A Knight

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Abstract

Abstract
Rationale: In asthma, goblet cell numbers are increased within the airway epithelium, perpetuating the production of mucus that is more difficult to clear and results in airway mucus plugging. Notch1, 2 or 3, or a combination of these have been shown to influence differentiation of airway epithelial cells. How the expression of specific Notch isoforms differs in fully differentiated adult asthmatic epithelium and whether Notch influences mucin production following differentiation is currently unknown.
Objectives: We aimed to quantify different Notch isoforms in the airway epithelium of severe asthmatics and to examine the impact of Notch signaling on mucin MUC5AC. Human lung sections and primary bronchial epithelial cells from asthmatics and non-asthmatics were used in this study.
Methods: Primary bronchial epithelial cells were differentiated at air-liquid interface for 28 days. Notch isoform expression was analyzed by Taqman qPCR. Immunohistochemistry was used to localize and quantify Notch isoforms in human airway sections. Notch signaling was inhibited in vitro using dibenzazepine or Notch3-specific small interfering RNA, followed by analysis of MUC5AC.
Measurements and main results: NOTCH3 was highly expressed in asthmatic airway epithelium compared to non-asthmatic. Dibenzazepine significantly reduced MUC5AC production in air-liquid interface cultures of primary bronchial epithelial cells, concomitant with a suppression of NOTCH3 intracellular domain protein. Specific knockdown using NOTCH3 siRNA recapitulated the dibenzazepine-induced reduction in MUC5AC.
Conclusions: We demonstrate that NOTCH3 is a regulator of MUC5AC production. Increased NOTCH3 signaling in the asthmatic airway epithelium may therefore be an underlying driver of excess MUC5AC production.
Original languageEnglish
JournalAmerican Journal of Respiratory Cell and Molecular Biology
Early online date10 Jan 2020
DOIs
Publication statusE-pub ahead of print - 10 Jan 2020

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Copyright © 2020 by the American Thoracic Society

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Reid, A. T., Nichol, K. S., Veerati, P. C., Moheimani, F., Kicic, A., Stick , S. M., Bartlett, N. W., Grainge, C. L., Wark, P. A. B., Hansbro, P. M., & Knight, D. A. (2020). Blocking Notch3 Signaling Abolishes MUC5AC Production in Airway Epithelial Cells From Asthmatics. American Journal of Respiratory Cell and Molecular Biology. https://doi.org/10.1165/rcmb.2019-0069OC