Abstract
Basal cells (BCs) are stem/progenitor cells of the mucociliary airway epithelium, and their differentiation is orchestrated by the NOTCH signaling pathway. NOTCH3 receptor signaling regulates BC to club cell differentiation; however, the downstream responses that regulate this process are unknown. Overexpression of the active NOTCH3 intracellular domain (NICD3) in primary human bronchial epithelial cells (HBECs) on in vitro air–liquid interface culture promoted club cell differentiation. Bulk RNA-seq analysis identified 692 NICD3-responsive genes, including the classical NOTCH target HEYL, which increased in response to NICD3 and positively correlated with SCGB1A1 (club cell marker) expression. siRNA knockdown of HEYL decreased tight junction formation and cell proliferation. Further, HEYL knockdown reduced club, goblet and ciliated cell differentiation. In addition, we observed decreased expression of HEYL in HBECs from donors with chronic obstructive pulmonary disease (COPD) vs. normal donors which correlates with the impaired differentiation capacity of COPD cells. Finally, overexpression of HEYL in COPD HBECs promoted differentiation into club, goblet and ciliated cells, suggesting the impaired capacity of COPD cells to generate a normal airway epithelium is a reversible phenotype that can be regulated by HEYL. Overall, our data identify the NOTCH3 downstream target HEYL as a key regulator of airway epithelial differentiation.
Highlights
Introduction iationsThe mucociliary epithelium is a multicellular tissue that lines the conducting airways and functions as a barrier to protect the lung from environmental insults [1,2,3,4]
NOTCH3 receptor signaling plays a key role in regulating differentiation of the mucociliary airway epithelium during health and in chronic lung diseases, including asthma, IPF and chronic obstructive pulmonary disease (COPD) [5,14,19,23,27,33]
Studies have shown that NOTCH3 signaling impacts airway epithelial differentiation in a context-dependent manner with both suppression and activation of NOTCH3 signaling leading to pathological changes in airway epithelial structure [5,14,19,23,27,33]
Summary
Primary HBECs from normal donors (nonsmokers and smokers) and COPD smokers were purchased commercially (catalog number CC-2540 and 00195275, Lonza, Morristown, NJ, USA) and cultured as described [14]. HBECs from a single normal nonsmoker donor were cultured on ALI for 9 days and harvested for scRNA-seq analysis. Amplified cDNA was cleaned with 0.6X SPRISelect reagent (catalog number B23318, Beckman Coulter, Pasadena, CA, USA). Cleaned cDNA was quality checked on an Agilent Tapestation 4150 (catalog number G2992AA, Agilent, Santa Clara, CA, USA) using a High Sensitivity D5000 ScreenTape (catalog number 5067-5592, Agilent) and quantified using Qubit dsDNA High Sensitivity Assay Kit Tapestation 4150 using a High Sensitivity D1000 ScreenTape (catalog number 5067-5584, Agilent) and quantified using Qubit dsDNA High Sensitivity Assay Kit. Libraries were diluted to 4 nM prior to sequencing on a NovaSeq 6000 S1 flow cell with 28 cycles for read 1 and 90 cycles for read 2. Summary metrics revealed 11,567 cells with an average of 8452 reads/cell and a median of 1382 genes/cell. scRNA-seq data were visualized using Loupe Cell Browser (10X Genomics)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
