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 6

Regulation of JAG1 and JAG2 abundance.

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Regulation of JAG1 and JAG2 abundance. Human bronchial basal cells were ...
Human bronchial basal cells were differentiated in ALI cultures using H&H medium. (A and B) On day 4 and day 6, the cultures were treated with vehicle (Veh; DMSO), CHIR99021 (CHIR), XAV939 (XAV), or LGK974 (LGK), and protein lysates were collected on day 8. CHIR and XAV concentrations are in μM. LGK concentrations are in pM. Western blots were used to (A) quantify full-length JAG1 and (B) full-length JAG2 abundance. Mean ± SD, n = 3. (C and D) Immunofluorescence was used to detect JAG2 in (C) Veh- and (D) CHIR-treated (5 μM) cultures on day 8. Red arrows, JAG2-high cell; white arrow, JAG2-negative cell; yellow arrow, redistributed JAG2; asterisks, JAG2-low cells. Scale bars: 10 μm. (E) JAG1 and JAG2 primary structure is represented by the vertical line with the amino-terminus (N-term) at the top and the carboxy-terminus (C-term) at the bottom. The red boxes represent the cysteine-rich domain (CRD), and green boxes represent the transmembrane domain (TM). Drawings are not to scale. Putative GSK3/CSNK phosphorylation sites are represented by asterisks, ADAM is represented by red arrows, and GSC cleavage sites are represented by green arrows. (F and G) Cells were cultured to day 8, lysed, and immunoprecipitated with a phospho-serine/phospho-threonine (pSer/pThr) antibody. Precipitates were analyzed for (F) JAG1 and (G) JAG2 using C-terminus specific antibodies. Two samples were analyzed. Black asterisk, high-molecular-weight protein; green asterisks, full-length protein; red asterisks, C-terminal fragments. (H and I) Cultures were treated with Veh or DKK1 on day 0 and day 2 and lysed on day 4 or treated on day 4 and day 6 and lysed on day 8. Drug concentrations are in ng/mL. Western blots were used to quantify (H) full-length JAG1 and (I) full-length JAG2 abundance. 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.