Assemblies of JAG1 and JAG2 determine tracheobronchial cell fate in mucosecretory lung disease
Susan D. Reynolds, Cynthia L. Hill, Alfahdah Alsudayri, Scott W. Lallier, Saranga Wijeratne, Zheng Hong Tan, Tendy Chiang, Estelle Cormet-Boyaka
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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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 3

JAG1 subcellular location.

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JAG1 subcellular location. Human bronchial basal cells were differentiat...
Human bronchial basal cells were differentiated in ALI cultures using H&H medium. (A–C) Immunofluorescence was used to detect JAG1 (red) on proliferation (A) day 3, (B) day 2, or (C) day 4. Nuclei were detected with DAPI (blue). Original magnification, ×400. (D and E) High-magnification images of the boxed region in A. Arrows indicate JAG1-positive cells. Original magnification, ×800. (F and G) Dual immunofluorescence analysis of JAG1 (green) and CTNNB1 (red) on day 4. Nuclei were detected with DAPI (blue). (F) Three-color and (G) 2-color images of the same region. Original magnification, ×800. (H) Western blot analysis of JAG1 on day 4 and 8. The full-length band is labeled JAG1. C-terminal fragments are shown by the bracket. Three samples per time point. (I) Cells were cultured to day 8, separated into cytoplasmic (C), nuclear (N), and insoluble (I) fractions or were unfractionated (T), and proteins were analyzed by Western blot. Top: TOP2A, a nuclear protein; middle: TUBA1A, a cytoplasmic protein; bottom: analysis of full-length JAG1 in the C and N fractions. Two different samples were analyzed. (J) Analysis of Jag1 mRNA abundance on day 2, day 4, and day 8. Mean ± SD (n = 3). (K) Western blot analysis of full-length JAG1 protein abundance on day 2, day 4, and day 8. Mean ± SD (n = 3). (L and M) Western blot analysis of full-length JAG1 abundance in cultures that were (L) treated with DMSO, 25 μM DAPT, or 10 μM LY on day 4 and day 6 and harvested on day 8 or (M) treated on day 8 and day 10 and harvested on day 12. 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.