key: cord-0254273-1zgvswnq
authors: Reynolds, Susan D.; Hill, Cynthia L.; Alsudayri, Alfahdah; Lallier, Scott W.; Wijeratne, Saranga; Tan, ZhengHong; Chiang, Tendy; Cormet-Boyaka, Estelle
title: MUCOSECRETORY LUNG DISEASE: DIFFERENT ASSEMBLIES OF JAG1 AND JAG2 DETERMINE TRACHEOBRONCHIAL CELL FATE
date: 2022-02-10
journal: bioRxiv
DOI: 10.1101/2022.01.29.478334
sha: 6284d12c93899767c2551de7d188dec2776ad037
doc_id: 254273
cord_uid: 1zgvswnq

Mucosecretory lung disease compromises airway epithelial function and is characterized by goblet cell hyperplasia and ciliated cell hypoplasia. These 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 Jagged2 (JAG2) is unclear. This study used primary human bronchial air-liquid- interface cultures, gamma secretase inhibition, and neutralizing antibodies to show: 1) JAG1 and JAG2 were necessary for secretory progenitor cell fate determination; 2) JAG2 suppressed squamous differentiation; and 3) pausing of the ciliated cell differentiation process after Notch inhibition. Histological, cell fractionation, cell surface biotinylation, and ubiquitination analyses demonstrated that all cells were JAG1 positive but that little JAG1 was present on the cell surface. In contrast, JAG2 was expressed in a positive-negative pattern and was abundant on the cell surface. Glycogen synthase kinase 3 (GSK3) and tankyrase inhibition studies showed that GSK3 regulated JAG2 trafficking, and that this mechanism was WNT-independent. Collectively, these data indicate that variation in JAG2 trafficking creates regions of high, medium, and low ligand expression. Thus, distinct assemblies of JAG1 and JAG2 may regulate Notch signal strength and determine the fate of tracheobronchial stem/progenitor cells. Graphical Abstract Different assemblies of JAG1 and JAG2 may determine Notch signal strength and cell fate within the tracheobronchial epithelium. A cell which interacts with JAG1+ cells (blue squares) receives a low Notch signal (light yellow square). A cell which interacts with a mixture of JAG1+ and JAG1+/JAG2+ cells (purple squares) receives a medium (med) Notch signal (medium yellow square). A cell which interacts with JAG1+/JAG2+ cells receives a high Notch signal (bright yellow square).

The conducting airway epithelium protects the lung from inspired environmental agents via 

Ciliated cells and differentiation intermediates were identified by ACT staining using mouse-anti-144 ACT (Sigma, 1/8000). JAG1 was evaluated using a mouse-anti-JAG1 C-terminal specific 145 antibody (BD Biosciences #612346, 1/50). JAG2 was evaluated using a rabbit-anti-JAG2 N-146 terminal specific antibody (Cell Signaling C23D2, 1/50) and a rabbit-anti-JAG2 C-terminal 147 specific antibody (Cell Signaling C83A8, 1/50). HES1 was evaluated using a goat-anti-HES1 148 (Santa Cruz #sc-13842, 1/40). Rbpjk was evaluated using a rabbit-anti-RBPJK (Sigma 149 #AB5790, 1/100). Primary antibodies were detected with ALEXA-488 or ALEXA-594 labeled 150 secondary antibodies (Jackson Immunological, 1/500). Nuclei were detected with DAPI. Cell 151 type frequency was quantified as previously described (39).

Cell fractionation: Cells were separated into cytoplasmic and nuclear fractions using a NE-PER 153 kit (Thermo Scientific, P178833). 4x Laemmli's Buffer/10% β-mercaptoethanol (BME) was 154 diluted 1/4 into the protein fraction and the mixture was incubated at 96 ˚C for 10 minutes.

Cell lysis: RIPA buffer (42) was used to lyse cells for immunoprecipitation, poly-ubiquitination, 156 and western blot analysis. The apical and basal surfaces of ALI cultures were washed with 8 The cleared lysate was incubated with a titration-determined amount of antibody (usually 1:100).

This mixture was rocked for 1 hr at 4 ˚C. The remaining 100 µl of washed beads were added to 169 the sample and rocked for 2 hr at 4 ˚C. The beads were collected using a neodymium magnet, 170 washed three times with RIPA Buffer, resuspended in 1x Laemmli's Buffer plus 2.5% BME, and 171 incubated at 96 ˚C for 10 minutes. The supernatant was kept as Unbound Fraction. denoted as the negative of its inverse (note that there will be no fold change values between -1 212 and 1, and that the fold changes of "1" and "-1" represent the same value). Transcripts were 213 considered significantly differentially expressed using a 10% false discovery rate (DESeq2 214 adjusted p value <= 0.1). GSEA was used to identify treatment dependent changes in signaling. 

Cells that contained many basal bodies and occasional short cilia (bristle cells, SFig 2C-D) were 269 identified on days 8-12 ( Fig 1B) . Finally, during Stage IV, each docked centriole (now termed a 270 basal body) nucleates a motile 9+2 ciliary axoneme and the ciliary appendage is formed. Cells 271 defined many long motile cilia (ciliated cells, SFig 2E-F) were detected on days 8-12 (Fig 1C) .

Goblet cells also exhibited three morphological phenotypes that were defined by low, medium, 

Pausing of the Notch signal: Accumulation of ciliated cell differentiation intermediates suggested 293 that GSI treatment paused the ciliation process. To investigate this mechanism, cultures were 294 treated with LY for 4 days and recovered (recovery) or treated with LY for 8 days (continuous, 295 Fig 2A) . On differentiation day 12, ciliated and goblet cell intermediates were quantified. Cells 296 with a primary cilium were not detected in these cultures (Fig 2B) . Both bristle and ciliated cells 297 were detected, and their frequency did not vary between the recovery and continuous treatment 298 protocols (Fig 2B) . Goblet cells defined by low, medium, and high expression of MUC5B were 299 identified but their frequency did not vary with treatment. Since continuous Ly treatment did not 300 cause an increase in bristle or ciliated cell frequency, these data indicated that GSI treatment 301 paused ciliated cell differentiation.

Since Notch signaling in the tracheobronchial epithelium has been attributed to JAG1 and 303 JAG2, the impact of GSI treatment on JAG1 or JAG2 protein abundance was determined.

Cultures were treated with LY or DAPT on days 4 and 6 and protein expression was analyzed 305 by western blot on day 8. GSI treatment did not alter JAG1 abundance (Fig 2C) . In contrast, GSI 306 treatment significantly increased the abundance of full-length JAG2 (Fig 2D) . These data 307 suggested that GSI treatment paused the differentiation process by increasing JAG2. (Fig 3A, D, E) and differentiation day 2 (Fig 3B) , JAG1 was expressed in a 320 subset of cells and was in the perinuclear/nuclear domain. JAG1 did not colocalize with β-321 catenin, a component of the adherens junction (Fig 3F-G) . On differentiation day 4 (Fig 3C) , 322 JAG1 was detected in most cells and some variation in protein abundance was noted. 

Since the previous studies indicated that most if not all JAG1 was in a perinuclear compartment 332 and that JAG1 was not acting as transcription factor, the relationship between transcription and 333 translation was evaluated. Jag1 mRNA was quantified by RNAseq and JAG1 protein levels 334 were quantified by Western blot. Jag1 abundance decreased as a function of time ( Fig 3J) . In 335 contrast, JAG1 abundance was constant over time ( Fig 3K) . These data suggested that a post-336 translational mechanism regulated JAG1 abundance.

JAG2 subcellular location and molecular weight: JAG2 localization was further evaluated on 338 proliferation day 5 and differentiation days 4 and 8 (Fig 4A-C) . The cortical pattern was 339 observed on proliferation day 5 and JAG2-high cells were apparent on differentiation day 4. By 

The molecular weight of JAG2 was determined using two antibodies which were specific to the 344 N-terminal or C-terminal domains. This study detected full-length (~145 KDa) JAG2 on days 4 345 and 8 (Fig 4E) . To further examine JAG2 subcellular localization, co-immunoprecipitation was 346 used to determine if JAG2 was associated with the adherens junction. An N-terminal specific β-347 catenin antibody was used for immunoprecipitation and the precipitate was analyzed with N-348 terminal and C-terminal specific β-catenin antibodies. β-catenin was successfully isolated (Fig   349   4F ) and JAG2 was identified in the β-catenin complex.

Since the previous studies suggested that much of JAG2 was trafficking to or from the plasma 351 membrane, the relationship between transcription and translation was evaluated using RNAseq 352 and western blots. Jag2 abundance decreased as a function of time (Fig 4G) . In contrast, JAG2 353 abundance was constant over time ( Fig 4H) . Like JAG1, these data suggested a post-354 translational mechanism regulated JAG2 abundance. (Fig 5A) . In contrast, both treatments caused a significant decrease in 362 JAG2 abundance (Fig 5B) . Interestingly, the CHIR effect was limited to lower doses (5 and 10 363 µM); whereas the XAV effect was observed at all three doses. contrast, JAG2 was detected on the surface of many cells (Fig 6A-B) . Subsequent 424 permeabilization and staining detected both JAG1 and JAG2 and illustrated their typical 425 intracellular distributions (Fig 6C-F ).

Cell surface biotinylation was used to evaluate JAG1 and JAG2 trafficking to the plasma 427 membrane. These studies detected sodium/potassium ATPase (NKA) a known cell surface 428 protein as well as JAG1 and JAG2 (Fig 6G) . However, cell surface JAG2 was 5-10 times more 429 abundant than JAG1. Since interaction with a Notch receptor results in ligand ubiquitination and 430 internalization, polyubiquitination of JAG1 and JAG2 was analyzed. Little or no JAG1 was 431 detected in the bound (ubiquitin-positive) fraction (Fig 6H, upper panel) . In contrast, JAG2 was 432 detected in the bound fraction and the molecular weight of the captured JAG2 decreased after 433 treatment with a deubiquitinase (Fig 6H) . These biochemical studies indicated that JAG2 434 participated in Notch-signaling and raised the possibility that JAG1 was not involved.

Phosphorylation of JAG2: Immunofluorescence analysis demonstrated that CHIR treatment 436 caused a striking redistribution of JAG2 to a perinuclear location (Fig 6I-J) and was suggestive 437 of JAG2 degradation. JAG2 was not detected by immunofluorescence staining in XAV treated 438 cells (data not shown). CHIR is a potent and highly selective inhibitor of GSK3 (54). In contrast, 439 XAV inhibits tankyrase and regulates GSK3 by altering the kinase's localization within the cell 440 (55). Since both drugs decrease GSK3 activity and decrease JAG2 abundance, it was possible 441 19 that the shared mechanism was a GSK3-dependent decrease in JAG2 phosphorylation. To 442 evaluate this mechanism, differentiation day 8 cell lysates were immunoprecipitated with an 443 anti-pSerine/pThreonine (pS/pT) antibody and western blots were probed for JAG2 and positive 444 controls (KRT5 and YWHA). This study detected JAG2, KRT5 and YWHA in the bound fraction 445 ( Fig 6K) and indicated that JAG2 was phosphorylated on Serine and/or Threonine. Collectively, 446 these data suggested that normal JAG2 trafficking was dependent on phosphorylation by GSK3. 

To address the limitations of the JAG1 peptide studies, ALI cultures were treated with 468 neutralizing antibodies on days 8 and 10 and ciliated and secretory cell differentiation was 469 assayed on day 12. Treatment with anti-JAG1 or anti-JAG2 significantly increased bristle cell 470 frequency and decreased ciliated cell frequency (Fig 7C-D) . Neutralizing antibody treatment did 471 not alter the frequency of MUC5B-medium cells but significantly decreased the frequency 472 MUC5B-high cells (Fig 7E-F) . These data suggested that loss of either JAG1 or JAG2 caused 473 MUC5B-medium cells to generate bristle cells. To address the finding that anti-JAG1 or anti-474 JAG2 treatment decreased ciliated cell frequency, ALI cultures were treated with a mixture of 475 anti-JAG1 and anti-JAG2. This treatment significantly decreased bristle cell frequency but did 476 not alter ciliated cell frequency (Fig 7G-H) . The combined neutralizing antibody treatment did 477 not alter the frequency of MUC5B medium cells and significantly decreased the frequency of 478 MUC5B-high cells (Fig 7I-J) . These data in combination with the single antibody study indicated 479 that either JAG1 or JAG2 inhibited bristle cell to ciliated cell differentiation. 

This study also indicated that JAG2 paused the ciliated cell differentiation process (SFig 7C).

Initial support for pausing came from the finding that GSI treatment increased the frequency of 502 ciliated cell differentiation intermediates. These data suggested that Notch signaling fluctuated 503 from "off" to "on" as the cell progressed from one morphological intermediate to the next. These 504 data challenged the idea that ciliated differentiation occurred in the absence of a Notch signal 505 but also raised the possibility that the GSI-sensitive target was the ligand rather than the 506 receptor. Support for the latter idea comes from previous demonstration that JAG1 and JAG2 507 were cleaved by γ-secretase resulting in production of an intracellular domain which functions 508 as a transcription factor (51-53). Failure to detect the ~25 kDa intracellular domain may reflect 509 low sensitivity in the ALI model relative to ligand over-expression systems. 

Proteins were immunoprecipitated with an N-terminus (N-term) specific β-catenin antibody.

Precipitates were analyzed for β-catenin using a C-terminus (C-term) specific β-catenin 618 antibody, N-term specific β-catenin antibody, or a JAG2 N-term specific antibody. Three 619 samples were analyzed. G. Analysis of Jag2 mRNA abundance on differentiation days 2, 4, and 620 8. Mean ± SD (n=3). H. Western blot analysis of JAG2 protein abundance. Mean ± SD, N=3. G. 

Frequency of MUC5B-medium cells (I)

Supplemental Figure 1: Ciliated and goblet cell differentiation as a function of medium type

Human bronchial basal cells were differentiated in air-liquid-interface cultures using three 666 media: Wu, H&H (H&H), and complete Pneumacult (cPC). Cultures were fixed on days

Cell density was determined by quantifying the number of DAPI-stained nuclei per unit 668 area. B. Ciliated cells were identified by acetylated tubulin (ACT) staining and their frequency 669 was reported as the number ACT+ cells/number nuclei. C. Goblet cells were identified by 670 MUC5B staining and their frequency was

Human bronchial basal 673 cells were differentiated in air-liquid-interface cultures using H&H medium. A-B. Cells defined by 674 a primary cilium were identified by ACT staining on differentiation day 4. Panel B is a high 675 magnification image of the region identified in panel A. Arrows: white, primary cilium; yellow, 676 mitotic figure. C-D. Cells with bristle morphology were identified by ACT staining on 677 differentiation day 8

Ciliated cells defined were identified by ACT staining on differentiation 679 day 12. Panel F is a high magnification image of the region identified in panel E

Goblet cells were identified by MUC5B staining on differentiation day 8

Panel H is a high magnification image of the region identified in panel G. Arrows identify 682 MUC5B-low, -medium, and -high goblet cell intermediates

Human bronchial 684 basal cells were differentiated in air-liquid-interface cultures using H&H medium. Cells were 685 treated with vehicle (DMSO) or 25 µM DAPT as follows: treatment on differentiation days 0 and 686 2 and fix on day 4, treatment on differentiation days 2 and 4 and fix on day 6, treatment on 687 differentiation days 4 and 6 and fix on day 8, or treatment on differentiation days 8 and 10 and 688 fix on day 12

Human 690 bronchial basal cells were differentiated in air-liquid-interface cultures using three media

Cultures were 692 fixed on day 7. JAG1 (green, A-C) and JAG2 (red, D-F) were detected by dual 693 immunofluorescence. Nuclei were detected with DAPI (blue, G-I). Merged images (J-L). Arrows: 694 yellow

Supplemental Figure 5: Gene expression and gene set enrichment analysis. ALI cultures were 696 treated with vehicle, 10 µM CHIR or 10 µM XAV on differentiation days 4 and 6 and RNA was 697 purified on day 8. Transcriptional changes were interrogated by RNA-sequencing and Gene Set 698

A. GSEA for WNT/β-catenin genes in vehicle and CHIR treated 699 cultures. B. Heat map representation of WNT/β-catenin genes in vehicle and CHIR treated 700 cultures. Each column is a sample: samples 1-3 vehicle treated (VEH), samples 4-6 CHIR 701 treated. Each row is a gene

GSEA for WNT/β-catenin genes in vehicle and XAV treated cultures. D. Heat map 704 representation of WNT/β-catenin genes in vehicle and XAV treated cultures. Each column is a 705 sample: samples 1-3 vehicle (VEH treated), samples 7-9 XAV treated. Each row is a gene. Red 706 indicates upregulation. Blue indicates downregulation. Leading-edge genes are indicated by the 707 green line. Jag1 and Jag2 are indicated by the asterisks. E. GSEA for Notch genes in vehicle 708 and CHIR treated cultures. F. Heat map representation of Notch genes in vehicle and CHIR 709 treated cultures

Each row is a gene. Red indicates upregulation. Blue indicates downregulation

Each column is a sample: 714 samples 1-3 vehicle (VEH treated), samples 7-9 XAV treated. Each row is a gene. Red 715 indicates upregulation. Blue indicates downregulation. Leading-edge genes are indicated by the 716 green line. Jag1 and Jag2 are indicated by the asterisks. I. Analysis of Jag1 gene expression in 717 vehicle, CHIR, and XAV treated cultures

Analysis of Jag2 gene expression in 719 vehicle, CHIR, and XAV treated cultures. Data are presented as the Log2(fold change). Mean ± 720 SD (n=3)

Supplemental Figure 6: Analysis of leading-edge genes. ALI cultures were treated with vehicle, 722 10 µM CHIR or 10 µM XAV on differentiation days 4 and 6 and RNA was purified on day 8

Transcriptional changes were interrogated by RNA-sequencing and Gene Set Expression 724

Leading-edge genes, which determine the gene set Enrichment Score, were 725 extracted and organized to indicate similarities/differences across the treatments and gene sets

Red indicates downregulation. Blue indicates upregulation. Transc: Transcriptional targets of 727

WNT/β-catenin were based on literature reports. (transc): WNT/β-catenin targets identified by 728 29 similarity. Protein: Targets of glycogen synthase kinase (GSK3) and tankyrase (TNKS) were 729 based on literature reports. (protein): GSK3 and TNKS targets identified by similarity

A. 731 JAG1 or JAG2 permit Basal Progenitor Cell (BPC) differentiation to a Secretory Progenitor Cell 732 (SPC) and SPC differentiation to a bristle cell (green light). Both JAG1 and JAG2 are required 733 for SPC differentiation to a goblet cell (green light). B. JAG2 inhibits the Squamous Cell fate 734 (red light). C. JAG2 pauses progression from one ciliated cell differentiation intermediate to the 735 next

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