Hedgehog Signaling Overcomes an EZH2-Dependent Epigenetic Barrier to Promote Cholangiocyte Expansion

<div><p>Background & Aims</p><p>Developmental morphogens play an important role in coordinating the ductular reaction and portal fibrosis occurring in the setting of cholangiopathies. However, little is known about how membrane signaling events in ductular reactive cells (DRCs) are transduced into nuclear transcriptional changes to drive cholangiocyte maturation and matrix deposition. Therefore, the aim of this study was to investigate potential mechanistic links between cell signaling events and epigenetic regulators in DRCs.</p><p>Methods</p><p>Using directed differentiation of induced pluripotent stem cells (iPSC), isolated DRCs, and <i>in vivo</i> models, we examine the mechanisms whereby sonic hedgehog (Shh) overcomes an epigenetic barrier in biliary precursors and promotes both cholangiocyte maturation and deposition of fibronectin (FN).</p><p>Results</p><p>We demonstrate, for the first time, that Gli1 influences the differentiation state and fibrogenic capacity of iPSC-derived hepatic progenitors and isolated DRCs. We outline a novel pathway wherein Shh-mediated Gli1 binding in key cholangiocyte gene promoters overcomes an epigenetic barrier conferred by the polycomb protein, enhancer of zeste homolog 2 (EZH2) and initiates the transcriptional program of cholangiocyte maturation. We also define previously unknown functional Gli1 binding sites in the promoters of cytokeratin (CK)7, CK19, and FN. Our <i>in vivo</i> results show that EZH2 KO mice fed the choline-deficient, ethanolamine supplemented (CDE) diet have an exaggerated cholangiocyte expansion associated with more robust ductular reaction and increased peri-portal fibrosis.</p><p>Conclusion</p><p>We conclude that Shh/Gli1 signaling plays an integral role in cholangiocyte maturation <i>in vitro</i> by overcoming an EZH2-dependent epigenetic barrier and this mechanism also promotes biliary expansion <i>in vivo</i>.</p></div

Working Model.

<p>An epigenetic program, initiated by Shh, and mediated by EZH2 loss, links cholangiocyte maturation to the deposition of a provisional FN matrix, shedding light on the simultaneous processes of biliary repair and peri-portal scar formation. FN may serve as a provisional scaffold for subsequent recruitment and activation of hepatic stellate cells or portal myofibroblasts.</p

Shh activation and FN production accompany cholangiocyte differentiation.

<p>A. Stepwise differentiation toward cholangiocytes in five phases: induced pluripotent stem cells (iPSC), definitive endoderm (DE), hepatic specification (HS), hepatic progenitor (HP), and iPSC-derived cholangiocytes (iDC). B. RNA sequencing data showing upregulation of Shh, FN, and the biliary cytokeratins, CK7 and CK19. C. RT-PCR showing increases in Shh, FN, CK7, and CK19 mRNA levels. The data are shown as means ± standard error. *<i>p</i> < 0.05.</p

Shh critically mediates cholangiocyte differentiation.

<p>A. Reduced Shh dosing reduces mRNA levels of CK7, CK19, and FN (++Shh = 100 ng/ml Shh administered for 8 days, +Shh = 100 ng/ml Shh administered for 4 days, -Shh = no Shh administration). B. Inhibition of Shh signaling using CPN at HS, HP and iDC phases impairs cholangiocyte maturation and FN deposition as assessed by protein levels of CK7, CK19, and released FN. Bar graphs on the right depict the densitometry results showing quantitative reductions in FN, CK7 and CK19. C. SMO shRNA at the HS, HP and iDC phases of differentiation reduces acquisition of CK7 and CK19, and release of FN in media. Bar graphs on the right depict the densitometry results showing quantitative reductions in SMO, FN, CK7 and CK19. The data are shown as means ± standard error. *p < 0.05, ** p <0.01, ***p<0.001.</p

Gli1 binding activates transcription of FN, CK7, and CK19.

<p>A. RT-PCR for Gli1 shows increased mRNA during cholangiocyte differentiation. B. Western blotting demonstrates increased Gli1 protein during cholangiocyte differentiation. Bar graphs on the right depict the densitometry results. C. ChIP assay revealing increased Gli1 binding to the FN, CK7, and CK19 promoters with increasing Shh dosing FN (++Shh = 100 ng/ml Shh administered for 8 days, +Shh = 100 ng/ml Shh administered for 4 days, -Shh = no Shh administration). D. Luciferase assays demonstrating basal and Shh-stimulated luciferase activity with the FN promoter before and after truncation. E. Luciferase assays demonstrating basal and Shh-stimulated luciferase activity with the CK7 promoter before and after truncation. F. Luciferase assays demonstrating basal and Shh-stimulated luciferase activity with the CK19 promoter before and after truncation. The data are shown as means ± standard error. *p < 0.05.</p

RNA seq identifies EZH2 as a regulator of cholangiocyte differentiation.

<p>A. Heatmap of differential expression analysis of RNA sequencing data from HS, HP, iDC, NHC, IHC, and hepatocyte sequencing data. B. Ingenuity pathway analysis of the hepatocyte-specific and cholangiocyte-specific clusters. C. Heatmap of differential expression analysis of epigenetic regulators from RNA sequencing of HS, HP, iDC, NHC, IHC, and hepatocyte sequencing data. D. Analysis of the known EZH2 interactome showing an increasing number of EZH2 target genes upregulated as cholangiocyte differentiation proceeds.</p

EZH2 behaves as an epigenetic barrier for cholangiocyte differentiation.

<p>A. Western blotting on cell lysates demonstrates the acquisition of CK19 and CK7 along with FN production during biliary maturation. B. Western blotting on nuclear extracts demonstrates loss of the EZH2 and the associated H3K27me3 repressive mark as iPSC differentiation proceeds. C. Western blot demonstrates that Shh stimulation of isolated DRCs results in loss of EZH2 and H3K27me3 with concomitant acquisition of CK7. D. Western blotting on cell lysates of iPSC differentiation samples demonstrates that adenoviral overexpression of EZH2 inhibits the acquisition of CK7 and CK19 as well as FN secretion. E. Western blotting of isolated ductular reactive cells shows that EZH2 overexpression correlates with H3K27me3 and loss of CK7 and CK19. Bar graphs on the right side represent densitometry of CK7, CK19, and H3K27me3 protein levels. F. Quantitative RT-PCR shows that EZH2 overexpression correlates with loss of CK7, CK19, and FN mRNA expression. The data are shown as means ± standard error. *p < 0.05.</p

EZH2 KO exacerbates CDE-induced biliary expansion.

<p>A. Immunohistochemistry for EZH2 demonstrates robust nuclear staining for EZH2 in the liver of wild type animals that is absent in the knockout animals. B. H&E staining, trichrome staining, and immunohistochemistry demonstrated enhanced ductular reaction following CDE feeding in EZH2 KO animals including peri-portal fibrosis, ductular proliferation, fibronectin deposition, and expansion of CK7 positive DRCs. C. Quantification of the CK7+ area shows increased CDE-induced ductular reaction following EZH2 KO. D. Quantification of the Pico Sirius Red+ area shows increased CDE-induced peri-portal fibrosis following EZH2 KO. The data are shown as means ± standard error. ** <i>p</i> < 0.01, *** <i>p</i> < 0.001.</p