Assemblies of JAG1 and JAG2 determine tracheobronchial cell fate in mucosecretory lung disease
Susan D. Reynolds, … , Tendy Chiang, Estelle Cormet-Boyaka
Susan D. Reynolds, … , Tendy Chiang, Estelle Cormet-Boyaka
Published July 12, 2022
Citation Information: JCI Insight. 2022;7(15):e157380. https://doi.org/10.1172/jci.insight.157380.
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Research Article Cell biology Stem cells

Assemblies of JAG1 and JAG2 determine tracheobronchial cell fate in mucosecretory lung disease

Abstract

Mucosecretory lung disease compromises airway epithelial function and is characterized by goblet cell hyperplasia and ciliated cell hypoplasia. Goblet and ciliated cell types are derived from tracheobronchial stem/progenitor cells via a Notch-dependent mechanism. Although specific arrays of Notch receptors regulate cell fate determination, the function of the ligands Jagged1 (JAG1) and JAG2 is unclear. This study examined JAG1 and JAG2 function using human air-liquid-interface cultures that were treated with γ-secretase complex (GSC) inhibitors, neutralizing peptides/antibodies, or WNT/β-catenin pathway antagonists/agonists. These experiments revealed that JAG1 and JAG2 regulated cell fate determination in the tracheobronchial epithelium; however, their roles did not adhere to simple necessity and sufficiency rules. Biochemical studies indicated that JAG1 and JAG2 underwent posttranslational modifications that resulted in generation of a JAG1 C-terminal peptide and regulated the abundance of full-length JAG2 on the cell surface. GSC and glycogen synthase kinase 3 were implicated in these posttranslational events, but WNT agonist/antagonist studies and RNA-Seq indicated a WNT-independent mechanism. Collectively, these data suggest that posttranslational modifications create distinct assemblies of JAG1 and JAG2, which regulate Notch signal strength and determine the fate of tracheobronchial stem/progenitor cells.

Authors

Susan D. Reynolds, Cynthia L. Hill, Alfahdah Alsudayri, Scott W. Lallier, Saranga Wijeratne, Zheng Hong Tan, Tendy Chiang, Estelle Cormet-Boyaka

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Figure 5

JAG1 and JAG2 trafficking to the cell surface.

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JAG1 and JAG2 trafficking to the cell surface. Human bronchial basal cel...
Human bronchial basal cells were differentiated in ALI cultures using H&H medium. (A and B) On day 8, cultures were fixed under nonpermeabilization conditions, and JAG1 (green) and JAG2 (red) were detected by dual immunofluorescence staining. Nuclei were stained with DAPI (blue). (B) A higher-magnification image of the boxed region in A. Yellow arrows, JAG2-positive cells. Scale bars: 5 μm (A); 20 μm (B). (C–F) After imaging, the cultures were permeabilized and restained for JAG1 (green), JAG2 (red), and DAPI (blue). Arrows, antigen-positive cells. Scale bars: 20 μm. (G) On day 8, cell surface proteins were labeled with biotin, biotinylated proteins were recovered, and the bound (BIO) and unbound (UnB) fractions were evaluated by Western blot. Two samples were evaluated for full-length JAG1, JAG2, or ATP1A1. (H) On day 8, cell lysates were reacted with TUBE beads that bind to proteins decorated with 4 or more ubiquitin moieties. The bound fraction was treated with vehicle (-DUB) or a broad spectrum debubiquitinase (+DUB). Western blots were generated from the input (In), unbound (Un), -DUB, and +DUB samples and were evaluated for full-length JAG1 and JAG2. *, Ubiquitinated JAG2; **, deubiquitinated JAG2. (I and J) On day 7, cultures were treated with vehicle (DMSO), 1.77 μg/mL cycloheximide (CHX), 10 μM FLI-06 (FLI), or 10 μM MITmAB (MIT) and lysed for Western blot analysis of (I) full-length JAG1 and (J) full-length JAG2. All quantitative data are presented as the mean ± SD, n = 3. Normally distributed data were analyzed by t test. Nonnormally distributed data were analyzed by Mann-Whitney test.