Wnt signaling regulates hepatobiliary repair following cholestatic liver injury in mice

Abstract

Hepatic repair is directed chiefly by the proliferation of resident mature epithelial cells. Furthermore, if predominant injury is to cholangiocytes, the hepatocytes can transdifferentiate to cholangiocytes to assist in the repair and vice versa, as shown by various fate-tracing studies. However, the molecular bases of reprogramming remain elusive. Using two models of biliary injury where repair occurs through cholangiocyte proliferation and hepatocyte transdifferentiation to cholangiocytes, we identify an important role of Wnt signaling. First we identify up-regulation of specific Wnt proteins in the cholangiocytes. Next, using conditional knockouts of Wntless and Wnt coreceptors low-density lipoprotein-related protein 5/6, transgenic mice expressing stable β-catenin, and in vitro studies, we show a role of Wnt signaling through β-catenin in hepatocyte to biliary transdifferentiation. Last, we show that specific Wnts regulate cholangiocyte proliferation, but in a β-catenin-independent manner.

Conclusion: Wnt signaling regulates hepatobiliary repair after cholestatic injury in both β-catenin-dependent and -independent manners. (Hepatology 2016;64:1652-1666).

Figure 1. Spatial analysis of Wls and Wnts after DDC diet

(A) Periportal (PP), pericentral (PC), and portal triad (PT) areas were dissected from H&E-stained frozen liver sections. (B) Expression of periportal and pericentral markers is shown by qRT-PCR. (C) Wls expression was detected mainly in the PT region, as shown by qRT-PCR. (D) Wnts 7A, 7B, and 10A were dramatically upregulated in the livers of DDC-fed mice compared to controls. (E) Expression of Wnt7A and Wnt7B was detected in the PT after DDC diet.

Figure 2. Cellular analysis of Wls and Wnts after DDC diet

(A) Parenchymal cells (PC), EpCAM+ cells, and CD45+ cells were separated from non-parenchymal cells after liver perfusion. (B) Expression of EpCAM, albumin (Alb), and Wls in PC, CD45+ cells, and EpCAM+ cells is shown by qRT-PCR. (C) Wls, Wnt7A, Wnt7B, and Wnt10A expression increases in EpCAM+ cells after DDC treatment, while expression of Wnt2 decreases after DDC, as shown by qRT-PCR.

Figure 3. Wnt7B and Wnt10A induce proliferation of cholangiocytes, while Wnt7A increases Sox9 expression in hepatocytes

(A) Expression of Wnt7A, Wnt7B, and Wnt10A expression in small cholangiocyte (sm-cc) cell cultures after transfection with Wnt-expressing plasmids. (B) A significant increase in proliferation as determined by Wst-8 assay was evident in sm-cc cells expressing Wnt7B or Wnt10A plasmid, but not Wnt7A. (C) Human 293T cells expressing either Wnt7A, Wnt7B, or Wnt10A were co-cultured with either mouse AML12 cells or primary mouse hepatocytes. (D) Wnt mRNA expression in 293T cells after transfection. (E) Mouse Sox9 and GAPDH mRNA expression in total cell lysates demonstrates the specificity of these primers in identifying gene expression from mouse hepatocytes only. (F) 293T cells expressing Wnt7A induce Sox9 expression in co-cultured AML12 cells and primary hepatocytes, as measured by qRT-PCR. * p < 0.05, ** p < 0.01.

Figure 4. Recombinant Wnt7A protein induces expression of biliary markers in vitro through activation of β-catenin

(A) Expression of EpCAM and Sox9 is increased in AML12 cells treated with recombinant human Wnt7A. (B) Sox9 expression is significantly increased in Hep3B cells treated with recombinant human Wnt7A. (C) Treatment of Hep3B cells with recombinant human Wnt7A increases TOPflash reporter activity. *-p<.05; ***-p<0.001

Figure 5. Expression of biliary markers increases in S45D mice after DDC diet

(A) qRT-PCR shows that expression of EpCAM and CK19 is increased in S45D mice after 1 month of DDC as compared to WT controls. (B) IHC shows the presence of Sox9 in the nuclei of S45D periportal hepatocytes after DDC, which is absent in WT. *-p<.05 vs. WT T0; #-p<.05 vs. S45D T0; %-p<.05 vs. WT DDC

Figure 6. Wls KO have more hepatic damage, less ductular proliferation, and decreased survival after DDC diet

(A) Serum total bilirubin and ALT levels are higher in Wls KO than WT after DDC diet. * p < 0.05. (B) Kaplan-Meier survival analysis shows a significant decrease in survival of Wls KOs on DDC diet. (C) Quantification of immunohistochemistry for A6 (200×), CK19 (100×), Sirius red (100×), and αSMA (100×) shows a significant decrease in ductular proliferation in Wls KO after DDC. * p < 0.05.

Figure 7. Cholangiocyte proliferation and hepatocyte-to-cholangiocyte transdifferentiation are disrupted in Wls KO after DDC diet

(A) Representative images of WT and KO livers stained for biliary marker A6 (green) and hepatocyte marker HNF4α (red) are shown. Many large hepatocytes expressing A6 are seen in WT after DDC treatment, whereas the number of A6+HNF4α+ cells was significantly decreased in KO. (B) Representative images of WT and KO livers stained for biliary marker CK19 (green) and Sox9 (red) are shown. The presence of CK19 negative cells expressing Sox9, which is indicative of incompletely differentiated cholangiocytes derived from transdifferentiating hepatocytes, is significantly higher in WT than in KO after DDC treatment. (C) Representative images of WT and KO livers stained for CK19 (green) and proliferation marker Ki67 (red) are shown. The number of proliferating cholangiocytes is higher in WT after DDC treatment than in KO. * p < 0.05. Images taken at 100×; magnifications are 400×.

Figure 8. Like DDC, BDL-induced Wnts regulate β-catenin dependent hepatobiliary transdifferentiation and β-catenin independent cholangiocyte proliferation

(A) Expression of Wnt7A, Wnt7B, and Wnt10A increases in WT mouse livers after BDL. (B) Wnt7A, Wnt7B, and Wnt10A are also among the top Wnt genes expressed in fibrotic septa of bile duct ligated rats compared to age-matched controls. (C) Wnt7A, Wnt7B, and Wnt10A are primarily expressed in the fibrotic septa (corresponding to the periportal compartment in DDC) after rat BDL. (D) LRP5/6 KO have fewer hepatocytes (arrows) expressing A6 after BDL compared to WT controls. Images taken at 200× magnification. (E) β-catenin siRNA significantly decreases β-catenin protein in sm-cc cells; however, transcriptional activity of β-catenin, which is minimal under normal growth conditions, is not further decreased by β-catenin siRNA. (F) Inhibiting β-catenin does not decrease survival or proliferation of sm-cc cells.