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A3802 - Inhibition of NOTCH3 Signaling Abolishes MUC5AC Production in Human Airway Epithelial Cells
Author Block: A. T. Reid1, K. S. Nichol2, L. Wei1, F. Moheimani1, N. W. Bartlett1, P. M. Hansbro3, P. A. B. Wark4, C. Grainge4, D. A. Knight1; 1School of Biomedical Sciences and Pharmacy, The University of Newcastle, Callaghan, Australia, 2School of Medicine and Public Health, The University of Newcastle, Callaghan, Australia, 3Immunology/Microbiology, The University of Newcastle, Callaghan, Australia, 4Respiratory and Sleep Medicine, John Hunter Hospital, New Lambton Heights, Australia.
Rationale: Mucus overproduction and accumulation is a key feature in severe asthmatics. These pathologies are directly linked to increased hospitalizations and morbidity. Accumulation of mucus in the airway is known to be directly influenced by levels of MUC5AC, the predominant mucin glycoprotein secreted by goblet cells. Notch, a key family of signaling receptor proteins, regulates airway mucus production by directing epithelial cell differentiation to produce goblet cells or ciliated cells. However little is known regarding Notch’s role during mucus production following differentiation, particularly in asthma. Aim/Hypothesis: To examine the impact of inhibiting Notch signaling on MUC5AC production in fully differentiated primary bronchial epithelial cells (pBECs) from control subjects as well as asthmatics. We hypothesize that Notch inhibition will downregulate MUC5AC production and secretion from airway epithelial cells. Methods: pBECs from 5-6 donors of each cohort were cultured at air-liquid interface (ALI) for 25 days to promote multicellular differentiation. At this time, cells were treated with the gamma-secretase inhibitor dibenzazepine (DBZ), a potent inhibitor of Notch signaling. Cells were treated for 96h during which, apical lining fluid was collected every 24h for assessment of MUC5AC release. At the completion of the experiment, samples were also collected for protein, mRNA and histological analysis. To assess the direct role of Notch3 on MUC5AC, siRNA knockdown of Notch3 was also performed in monolayer cultures of human epithelial cells. Results: DBZ treatment significantly reduced MUC5AC expression and release in cells from both cohorts as assessed by qPCR, ELISA and IHC. Western blotting/qPCR revealed significant reduction of NOTCH3 intracellular domain (NICD3) in DBZ treated pBECs. Notch3 siRNA knockdown significantly reduced MUC5AC protein which was not associated with any reduction in the goblet cell marker protein CLCA1. Conclusion: Notch3 inhibition reduced MUC5AC expression and secretion from differentiated pBECs, independent of goblet cell number. This reduction is NOTCH3 dependent and occurs in pBECs from asthmatics and non-asthmatics. This suggests that Notch3 regulates MUC5AC production outside of Notch’s well characterized role during secretory cell differentiation.