Fig 1.
Acinar cell-specific Lats1/2 deletions caused pancreatic inflammation and fibrosis in adult mice.
P, PL, L1/2, PL1KO, and PL2KO mice were administrated with 180 mg/kg/d of TAM for 5 consecutive days via i.p., respectively (n = 6). Pancreata were collected at Day 5 after the final injection. (A) Histological morphology of P, L1/2, PL, PL1KO, and PL2KO mice. L1/2, PL1KO, and PL2KO mice were littermate controls. (B) Western blot showing the levels of LATS1, LATS2, phospho-YAP1, YAP1, and TAZ in P and PL mice. Tubulin was used as the internal control. (C) YAP1 and TAZ IHC staining in P and PL mice. (D) Immunofluorescence of YFP (Green) and CK19 (Red) in P and PL mice. Nuclei stained with DAPI (Blue). (E) P and PL pancreata stained with anti-CD45 antibody. (F) P and PL pancreata stained with anti-αSMA antibody, and Sirius Red, respectively. αSMA, α-smooth muscle actin; CD45, ; CK19, cytokeratin 19; IHC, immunohistochemistry; i.p., intraperitoneal; L1/2, ; LATS1, large tumor suppressor 1; P, control; PL, double knockout; PL1KO, Lats1 knockout; PL2KO, Lats2 knockout; TAM, tamoxifen; TAZ, transcriptional coactivator with PDZ binding motif; YAP1, yes-associated protein 1.
Fig 2.
Loss of YAP1/TAZ in acinar cells rescues Lats1/2-deletion–induced pancreatic inflammation and fibrosis.
P, PL, and PLTY mice were subjected to 180 mg/kg/d of TAM for 5 consecutive days via i.p. (n = 6). Pancreata were collected 5 days later. (A) Western blot of Lats1, Lats2, YAP1, and TAZ in P, PL, and PLTY mice. Tubulin was used as the internal control. (B) Histological examination by HE staining among P, PL, and PLTY groups. (C) Immunofluorescence staining of αSMA (Green), CD45 (Red), and ADM (Green + Red) among P, PL, and PLTY groups. Nuclei stained with DAPI (blue). Quantification of αSMA and CD45 immunostaining in P, PL, and PLTY mice showed 90.1% reduction of inflammation and 95.2% reduction of stromal reaction in PLTY mice compared with PL mice. ADM was quantified by counting YFP and CK19 double positive cell numbers. There was an 84.5% reduction of ADM in PLTY mice compared with PL mice (n = 6). **P < 0.01. Underlying numerical values can be found in S1 Data. αSMA, α-small muscle actin; ADM, acinar-to-ductal metaplasia; CD45, cluster of differentiation antigen 45; CK19, cytokeratin 19; HE, hemtoxylin–eosin; i.p., intraperitoneal; LATS1, large tumor suppressor 1; P, control; PL, double knockout; PLTY, quadruple deletions of Lats1/2 and YAP1/TAZ; TAM, tamoxifen; TAZ, transcriptional coactivator with PDZ binding motif; YAP1, yes-associated protein 1.
Fig 3.
Loss of Lats1/2 does not affect acinar cell proliferation, apoptosis, or ADM directly.
(A) Schematic diagram of experimental design (n = 3). (B) PL mice were injected once with 45 mg/kg of TAM. Cell proliferation, apoptosis, and ADM were analyzed on Day 4, Day 8, and Day 12 by co-staining anti-YFP with anti-Ki67, anti-cleaved-caspase-3, anti-amylase, and anti-CK19 antibodies. Nuclei stained with DAPI (Blue). Percentages of Ki67, cleaved caspase-3 (n = 3), and YFP (n = 4) cells were quantified by IHC profiler score. Underlying numerical values can be found in S1 Data. (C) YAP1 and TAZ were detected by IHC in PL mice after injection with 45 mg/kg of TAM. ADM, acinar-to-ductal metaplasia; CK19, cytokeratin 19; IHC, immunohistochemistry; Ki67, antigen identified by monoclonal antibody Ki 67; LATS1, large tumor suppressor 1; P, control; PL, double knockout; TAM, tamoxifen; TAZ, transcriptional coactivator with PDZ binding motif; YAP1, yes-associated protein 1; YFP, yellow fluorescent protein.
Fig 4.
Lats1/2 deletion in acinar cells results in PSC activation.
PL mice were injected once with 180 mg/kg of TAM; pancreata were collected and embedded at Day 0, Day 5, Day 10, Day 15, and Day 20 after injection, respectively (n = 4–6). (A) Morphological changes over time (Day 0, Day 5, Day 10, Day 15, Day 20) were examined by HE staining. (B) Anti- αSMA, anti-CD45, and anti-CK19 antibodies were used to stain activated PSCs, immune cells, and ductal-like cells, respectively. (C) αSMA, YAP1, and TAZ IHC staining in consecutive sections at Day 10. (D) αSMA and CD45 IHC staining in consecutive sections at Day 10. (E) αSMA and CK19 IHC staining in consecutive sections at Day 10. αSMA, α-smooth muscle actin; CD45, cluster of differentiation antigen 45; CK19, cytokeratin 19; HE, hematoxylin–eosin; IHC, immunohistochemistry; LATS1, large tumor suppressor 1; PL, double knockout; PSC, pancreatic stellate cell; TAM, tamoxifen; TAZ, transcriptional coactivator with PDZ binding motif; YAP1, yes-associated protein 1.
Fig 5.
Loss of Lats1/2 induces M2 macrophage polarization.
P and PL mice were treated with 180 mg/kg/d of TAM for 5 consecutive days; cells were isolated from both groups 4 days later (n = 3). (A) F4/80 IHC staining in P and PL mice on Day 4 after TAM injection. (B) M2 macrophage-specific genes Ym-1, Ccl17, Tgf-β and IL10 were elevated in sorted macrophages of PL mice. (C) M1 macrophage-specific genes Tnf-α, Socs1, and Nos2 were not significantly changed. **P < 0.01, *P < 0.05. Underlying numerical values can be found in S1 Data. IHC, immunohistochemistry; LATS1, large tumor suppressor 1; P, control; PL, double knockout; TAM, tamoxifen.
Fig 6.
RNA-seq analysis of LATS1/2 regulated genes in mouse pancreas.
(A) Heat map analysis showed differential gene expression patterns at different days (n = 3–5). (B) Left: K-means clustering analysis showed the gene expression patterns of 2 classes (up-regulated and down-regulated genes) that were changed with time. Right: GO term analysis of the function of top up-regulated and down-regulated genes. (C) Validation of profibrotic gene expressions by qPCR. (D) The small lesions were stained with anti- αSMA, anti-SPP1, anti-YAP1, and anti-TAZ antibodies in consecutive sections. White arrow indicates region in which αSMA is already present, while red arrow indicates region in which SPP1 expression has occurred but αSMA is not yet present. (E) Co-stain of αSMA (Red) with SPP1 (Green) in P and PL (Day 2 and Day 3) mice by immunofluorescence. (F) Isolated PSCs were treated with CTGF (5 nM) or SPP1 (50 nM) for 3 days (n = 3). Untreated group serves as the control. Anti-αSMA antibody was used to stain activated PSCs (Green). Cell proliferation was evaluated by EdU incorporation (White). Nuclei stained with DAPI (Blue). (G) SPP1 IHC staining in control, AP, and CP mice. Quantitative analysis for αSMA and EdU incorporation in CTGF or SPP1-stimulated cells (n = 3). *P < 0.05. Underlying numerical values can be found in S1 Data. αSMA, α-smooth muscle actin; AP, acute pancreatitis; CP, chronic pancreatitis; CTGF, connective tissue growth factor; EdU, 5-ethynyl-2'-deoxyuridine; IHC, immunohistochemistry; LATS1, large tumor suppressor 1; P, control; PL, double knockout; PSC, pancreatic stellate cell; qPCR, quantitative PCR; RNA-seq, RNA sequencing; SPP1, secreted phosphoprotein 1; TAZ, transcriptional coactivator with PDZ binding motif; YAP1, yes-associated protein 1.
Fig 7.
Depletion of CTGF partially abolishes pancreatic inflammation and fibrosis in Lats1/2 double knockout mice.
(A) Ctgf mRNA expression in P and PL mice (n = 3). **P < 0.01. (B) Western blot of CTGF protein levels in P, PL, and PLTY mice. Tubulin served as the internal control. (C) Schematic diagram of experimental design (n = 6 per group). Mice without TAM injection served as control group. FG-3019: CTGF-neutralizing antibody. (D) Top: histological examination by HE staining; center: immunofluorescent staining of αSMA (Green); bottom: immunofluorescent staining of CD45 (Red). Nuclei stained with DAPI (blue). (E) Quantitative analysis of FG-3019 treatment effects on Lats1/2 deletion-induced ADM, PSC activation, and immune cell infiltration in the mouse pancreas (n = 6); *P < 0.05. Underlying numerical values can be found in S1 Data. αSMA, α-smooth muscle actin; ADM, acinar-to-ductal metaplasia; CD45, ; CTGF, connective tissue growth factor; HE, hematoxylin–eosin; IgG, human Immunoglobulin G control; LATS1, large tumor suppressor 1; P, control; PL, double knockout; PLTY, quadruple deletions of Lats1/2 and YAP1/TAZ; PSC, pancreatic stellate cell; TAM, tamoxifen; TAZ, transcriptional coactivator with PDZ binding motif; YAP1, yes-associated protein 1.
Fig 8.
The Hippo signaling pathway is involved in inflammation and fibrosis in caerulein-induced pancreatitis mouse model.
(A) Schematic diagram of induction of AP. n = 3. (B) The AP was analyzed by HE examination on Day 1, Day 2, Day 3, Day 4, and Day 6 after the induction. (C) Western blot of LATS1, LATS2, P-YAP1, YAP1, and TAZ in untreated and AP mice. Untreated mice served as the control group. Tubulin was used as the internal control. (D) Schematic diagram of induction of CP (n = 4). (E) HE examination of CP mice. Untreated mice served as the control group. Sirius Red was used to detect collagen deposition in CP mice. (F) Western blot of LATS1, LATS2, YAP1, and TAZ in untreated and CP mice. AP, acute pancreatitis; CP, chronic pancreatitis; HE, hematoxylin–eosin; LATS1, large tumor suppressor 1; P-YAP1, phospho-YAP1; TAZ, transcriptional coactivator with PDZ binding motif; YAP1, yes-associated protein 1.