LATS1/2 suppress NFκB and aberrant EMT initiation to permit pancreatic progenitor differentiation

Abstract

The Hippo pathway directs cell differentiation during organogenesis, in part by restricting proliferation. How Hippo signaling maintains a proliferation-differentiation balance in developing tissues via distinct molecular targets is only beginning to be understood. Our study makes the unexpected finding that Hippo suppresses nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) signaling in pancreatic progenitors to permit cell differentiation and epithelial morphogenesis. We find that pancreas-specific deletion of the large tumor suppressor kinases 1 and 2 (Lats1/2PanKO) from mouse progenitor epithelia results in failure to differentiate key pancreatic lineages: acinar, ductal, and endocrine. We carried out an unbiased transcriptome analysis to query differentiation defects in Lats1/2PanKO. This analysis revealed increased expression of NFκB activators, including the pantetheinase vanin1 (Vnn1). Using in vivo and ex vivo studies, we show that VNN1 activates a detrimental cascade of processes in Lats1/2PanKO epithelium, including (1) NFκB activation and (2) aberrant initiation of epithelial-mesenchymal transition (EMT), which together disrupt normal differentiation. We show that exogenous stimulation of VNN1 or NFκB can trigger this cascade in wild-type (WT) pancreatic progenitors. These findings reveal an unexpected requirement for active suppression of NFκB by LATS1/2 during pancreas development, which restrains a cell-autonomous deleterious transcriptional program and thereby allows epithelial differentiation.

Fig 1. LATS1/2 kinases are required for pancreatic lineage differentiation.

(A) Representative confocal images of pLATS1/2, MUC1, and CDH1 immunostaining of sections of WT pancreas at E9.75 are shown. Arrowhead indicates pLATS1/2 expression adjacent to MUC1⁺ lumens. Scale = 50 μm (left panel), 10 um (right panel). (B) WT and (F) Lats1/2^PanKO pancreata (outlined in black) at P1. Black arrowhead indicates Lats1/2^PanKO pancreatic rudiment. (C–E, G–I) Confocal images of INS, GCG, and CDH1; AMY and MUC1; or DBA and MUC1 immunostaining of WT and Lats1/2^PanKO pancreata at P1. “*” denotes absence of endocrine, acinar, or ductal proteins. Scale = 25 μm. (J) Normalized mRNA expression of endocrine genes Pdx1, Neurog3, Gcg, and Syn; ductal gene Sox9; acinar genes Cpa1 and Nr5a2; Notch ligand Jag1; and YAP1/TAZ/TEAD target [26]. Cyr61 were compared in WT and Lats1/2^PanKO pancreata at E13.5 (n = 3 embryos per genotype). Data are shown as mean ± SEM. Statistical significance was determined by Student t test (*p < 0.05; **p < 0.01; ***p < 0.001). Underlying numerical values can be found in S1 Data. (K, L) Confocal images of GCG and CDH1 immunostaining of WT and Lats1/2^PanKO pancreata at E15.5. Scale = 25 μm. (M–O) Confocal images of CDH1, GCG, and pYAP1 immunostaining of WT, Lats1/2^PanKO, and Pdx1Cre^early;Lats1/2^f/f;Yap1^f/f;Taz^f/wt (Lats1/2/Yap1^PanKOTaz^HET) pancreata at E12.5 (n = 3 embryos per genotype). Arrows indicate loss of pYAP1 expression in Lats1/2-deficient pancreata. Nuclei were counterstained with DAPI (blue). Scale = 25 μm. AMY, amylase; AU, arbitrary units; CDH1, E-cadherin; Cpa1, carboxypeptidase 1; Cyr61, cysteine rich angiogenic inducer 61; DAPI, 4’,6-diamidino-2-phenylindole; DBA, Dolichos biflorus agglutinin; E, embryonic day; GCG, glucagon; INS, insulin; Jag1, jagged1; LATS1/2, large tumor suppressor kinases 1 and 2; MUC1, mucin-1; Neurog3, neurogenin3; Nr5a2, nuclear receptor subfamily 5 group A member 2; P, postnatal day; Pdx1, pancreatic and duodenal homeobox 1; pLATS1/2, phospho-LATS1/2; pYAP1, phospho-yes-associated protein 1; Sox9, sex determining region Y-box 9 protein; Syn, synaptophysin; TAZ, transcriptional coactivator with PDZ-binding motif; TEAD, TEA domain transcription factor; WT, wild type.

Fig 2. Lats1/2 deletion initiates EMT transition of pancreatic progenitors.

(A–J) Confocal images of VIM, CDH1, TAGLN, KRT19, and ACTA2 immunostains in WT and Lats1/2^PanKO pancreata at (A–C, G) E11.5, (D, H) E12.5, or (E, F, I, J) E14.5 (n = 3 embryos per stage per genotype). “*” denotes absence of protein expression within pancreas (outlined in white). (G) Arrowhead indicates mosaic ACTA2 expression in pancreas progenitors at E11.5. Nuclei were counterstained with DAPI (blue). Scale = 25 μm. (K) Schematic illustrating partial EMT following Lats1/2 deletion, with acquisition of mesenchymal proteins including VIM, ACTA2, and TAGLN, normally absent from WT pancreas epithelium, and persistent CDH1. ACTA2, alpha smooth muscle actin; CDH1, E-cadherin; E, embryonic day; EMT, epithelial-mesenchymal transition; KRT19, cytokeratin 19; TAGLN, transgelin; VIM, vimentin; WT, wild type.

Fig 3. LATS1/2 kinases are required to suppress Vnn1 in early pancreatic progenitors.

(A) RNA isolation from pancreas buds at E11.0 (39–40 somites) for RNA-seq of total RNA from individual WT (n = 5) and Lats1/2^PanKO (n = 3) embryos. Pancreatic bud is outlined (black). The heat map was generated using genes that were differentially expressed by at least 2-fold in Lats1/2^PanKO (mutant) compared to WT (control). Gene down-regulation and up-regulation are indicated in green and red, respectively. (B) Selected pathways from KEGG pathway analysis are shown. The x-axis indicates (-log₁₀) fold change, and the vertical red line designates significance, with a threshold of 1.5-fold change. Underlying data analysis can be found in S1 Data. (C, D) RNA ISH of Vnn1 antisense probe on WT and Lats1/2^PanKO pancreata (outlined in black) at E11.5 (n = 3 embryos per genotype). “*” denotes undetectable Vnn1 mRNA in WT pancreas epithelium. Scale = 50 μm. (E) Normalized Vnn1 mRNA expression was compared in Lats1/2^PanKO and WT pancreata at E13.5 (n = 3 embryos per genotype). Data are shown as mean ± SEM. Statistical significance was determined by Student t test (**p < 0.01). Underlying numerical values can be found in S1 Data. (F, G) Confocal images of VNN1 and CDH1 immunostains of WT and Lats1/2^PanKO pancreata at E11.5 (n = 3 embryos per genotype). Scale = 10 μm. (H) TEAD motif analysis in the Vnn1 promoter. (I, J) Confocal images of GFP, VNN1, and CDH1 immunostains of WT and Ptf1a-rtTA;Yap1-GFP (Yap1^GFP) pancreata at E12.5 (n = 3 embryos per genotype). Arrowheads denote immunopositivity for both GFP and VNN1. Arrow points to cell with low GFP and no VNN1 expression. Scale = 20 μm. AU, arbitrary units; C, control; CDH1, E-cadherin; CTNNB1, catenin beta 1; E, embryonic day; GFP, Green Fluorescent Protein; ISH, in situ hybridization; KEGG, Kyoto Encyclopedia of Genes and Genomes; M, mutant; RNA-seq, RNA sequencing; TEAD, TEA domain transcription factor; TSS, transcription start site; Vnn1, vanin1; WT, wild type.

Fig 4. Lats1/2 deletion, or the VNN1 product cysteamine, induces ROS.

(A, B) Compressed Z stack confocal images of CellROX, a green fluorogenic probe that detects ROS, on WT and Pdx1Cre^ERT2;Lats1^f/f;Lats2^f/f (Lats1/2^i-PanKO) pancreas explants (n = 5 explants per genotype). Daily tmx induction was performed on WT and Lats1/2^i-PanKO embryos from E8.5 to E11.5. Lower panels depict overlay of CDH1 immunostaining (outlined in white). Scale = 100 μm. (C) The percent area covered by CellROX⁺ pixels within the pancreatic CDH1⁺ progenitor epithelium was quantified in WT and Lats1/2^i-PanKO explants. (D) Normalized mRNA expression of oxidative stress-responsive Hmox1 was compared in WT and Lats1/2^PanKO pancreas at E13.5 (n = 3 embryos per genotype). (E–H) Compressed Z stack confocal images of CellROX plus CDH1 immunostains of WT pancreas explants treated with 4 mM cysteamine and/or 10 μM NAC (n = 3–7 explants per treatment group). (F) Cysteamine provoked ROS in WT epithelium and mesenchyme (arrowheads). Scale = 150 μm. (I) The percent area covered by CellROX⁺ pixels (above a set threshold) within the pancreatic epithelium was quantified using ImageJ. (J–M) Compressed Z stack confocal images of CellROX plus CDH1 and TAGLN immunostaining on WT and Lats1/2^i-PanKO pancreas explants, treated O/N with antioxidant NAC or vehicle control (n = 3 explants per genotype per treatment). Daily tmx induction was performed on WT and Lats1/2^i-PanKO embryos from E8.5 to E11.5. Scale = 100 μm. (N) The percent area covered by TAGLN⁺ pixels within the pancreatic CDH1⁺ progenitor epithelium was quantified in WT and Lats1/2^i-PanKO explants. Data are shown as mean ± SEM. Statistical significance was determined by Student t test (*p < 0.05; **p < 0.01; ***p < 0.001). Underlying numerical values can be found in S1 Data. AU, arbitrary units; CDH1, E-cadherin; E, embryonic day; Hmox1, heme oxygenase 1; NAC, N-acetyl-cysteine; O/N, overnight; ROS, reactive oxygen species; TAGLN, transgelin; tmx, tamoxifen; VNN1, vanin1; WT, wild type.

Fig 5. Lats1/2 deletion or ROS stimulates NFκB1 and RELA.

(A, B) Confocal images of pNFκB1 and CDH1 immunostains of WT and Lats1/2^PanKO pancreata at E12.0 (n = 3 embryos per genotype). Scale = 20 μm. (C) The average percentage of pNFκB1⁺ epithelial cells/total epithelial cells per pancreas section at E12.0 was calculated. (D, E) Confocal images of pNFκB1 and pHH3 immunostaining of WT and Lats1/2^PanKO pancreata at E14.5 (n = 3 embryos per genotype). Scale = 20 μm. (F) Western blotting from E12.5 WT and Lats1/2^PanKO pancreata using pRELA and GAPDH antibodies (n = 3 embryos per genotype). (G, H) Confocal images of CDH1 and pRELA immunostained WT pancreas explants treated with 200 μM H₂O₂ for 2 hours (n = 4 explants per treatment group). Arrowheads in (H) indicate pRELA⁺ nuclei. (I) The average number of pRELA⁺ nuclei per field of view were quantified. Data are shown as mean ± SEM. Statistical significance was determined by Student t test (*p < 0.05). Underlying numerical values can be found in S1 Data. (J) Lats1/2 deficiency increases expression of the pantetheinase VNN1, which produces cysteamine, followed by stimulation of ROS and subsequent NFκB activation. CDH1, E-cadherin; E, embryonic day; NFκB, nuclear factor kappa-light-chain-enhancer of activated B cells; pHH3, phospho-histone H3; pNFκB1, phospho-nuclear factor kappa B subunit 1; pRELA, phospho-RELA proto-oncogene, NF-KB subunit; ROS, reactive oxygen species; VNN1, vanin1; WT, wild type.

Fig 6. NFκB is necessary and sufficient to initiate pancreatic EMT.

(A–D) Compressed Z stack images of VIM⁺ immunostains of vehicle- or anatabine-treated WT and Lats1/2^PanKO pancreas explants (n = 3–6 explants per genotype per treatment group). Scale = 50 μm. (B) Arrowhead indicates ectopic VIM expression in Lats1/2^PanKO epithelium. (C, D) Anatabine also decreased mesenchymal VIM expression (asterisks). (E) The percent area covered by VIM⁺ pixels within the pancreatic epithelium (above a set threshold) was quantified in anatabine- and vehicle-treated Lats1/2^PanKO and WT explants. Data are shown as mean ± SEM. Statistical significance was determined by Student t test (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). (F, G) Compressed Z stack confocal images of CDH1 and VIM immunostains of WT pancreas explants treated with 10 μg/mL PMA (n = 3 explants per treatment). Masked VIM (VIM within CDH1⁺ progenitor surface volume) was used to exclude mesenchymal VIM. Scale = 50 μm. (F’,G’) Colocalization (white) was calculated above common thresholds and visualized using Imaris. (H) The percent area of VIM+CDH1 colocalization pixels was quantified using ImageJ. (I, J) Mean VIM⁺ pixel intensity and the percent area of VIM⁺ pixels (above a common threshold) within WT pancreatic epithelium were quantified using ImageJ. Underlying numerical values can be found in S1 Data. (K, L) Slice view through Z stack shows VIM (green) + CDH1 (red). (L) Arrowheads indicate VIM within the CDH1⁺ epithelial layer. (M) PMA hyperactivates NFκB, which initiates EMT in pancreatic progenitors. CDH1, E-cadherin; EMT, epithelial-mesenchymal transition; NFκB, nuclear factor kappa light chain enhancer of activated B cells; PMA, Phorbol 12-myristate 13-acetate; VIM, vimentin; WT, wild type.

Fig 7. LATS1/2 restrict NFκB to maintain epithelial identity and coordinate proliferation and differentiation of pancreatic progenitors.

(A) During embryonic development, LATS1/2 kinases exquisitely control subcellular localization of YAP1/TAZ, thereby maintaining pancreas progenitors and coordinating morphogenesis, cell proliferation. NFκB TFs also shuttle between the nucleus and cytoplasm, depending on activation, and coordinate cell proliferation. (B) Lats1/2 deficiency triggers NFκB activators like VNN1, a pantetheinase enzyme that converts pantetheine to pantothenic acid and cysteamine. VNN1 products increase ROS and activate NFκB, leading to initiation of EMT and mesenchymal gene expression. EMT, epithelial-mesenchymal transition; LATS1/2, large tumor suppressor kinases 1 and 2; NFκB, nuclear factor kappa light chain enhancer of activated B cells; ROS, reactive oxygen species; TAZ, transcriptional coactivator with PDZ-binding motif; TF, transcription factor; VNN1, vanin1; YAP1, yes-associated protein 1.