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Enhancer of Zeste Homologue 2 Inhibition Attenuates TGF-? Dependent Hepatic Stellate Cell Activation and Liver Fibrosis.

ABSTRACT: BACKGROUND & AIMS:Transdifferentiation of hepatic stellate cells (HSCs) into myofibroblasts is a key event in the pathogenesis of liver fibrosis. Transforming growth factor ? (TGF-?) and platelet-derived growth factor (PDGF) are canonical HSC activators after liver injury. The aim of this study was to analyze the epigenetic modulators that differentially control TGF-? and PDGF signaling pathways. METHODS:We performed a transcriptomic comparison of HSCs treated with TGF-? or PDGF-BB using RNA sequencing. Among the targets that distinguish these 2 pathways, we focused on the histone methyltransferase class of epigenetic modulators. RESULTS:Enhancer of zeste homolog 2 (EZH2) was expressed differentially, showing significant up-regulation in HSCs activated with TGF-? but not with PDGF-BB. Indeed, EZH2 inhibition using either a pharmacologic (GSK-503) or a genetic (small interfering RNA) approach caused a significant attenuation of TGF-?-induced fibronectin, collagen 1?1, and ?-smooth muscle actin, both at messenger RNA and protein levels. Conversely, adenoviral overexpression of EZH2 in HSCs resulted in a significant stimulation of fibronectin protein and messenger RNA levels in TGF-?-treated cells. Finally, we conducted in vivo experiments with mice chronically treated with carbon tetrachloride or bile duct ligation. Administration of GSK-503 to mice receiving either carbon tetrachloride or bile duct ligation led to attenuated fibrosis as assessed by Trichrome and Sirius red stains, hydroxyproline, and ?-smooth muscle actin/collagen protein assays. CONCLUSIONS:TGF-? and PDGF share redundant and distinct transcriptomic targets, with the former predominating in HSC activation. The EZH2 histone methyltransferase is preferentially involved in the TGF-? as opposed to the PDGF signaling pathway. Inhibition of EZH2 attenuates fibrogenic gene transcription in TGF-?-treated HSCs and reduces liver fibrosis in vivo. The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE119606 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE119606).

SUBMITTER: Martin-Mateos R

PROVIDER: S-EPMC6282644 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Enhancer of Zeste Homologue 2 Inhibition Attenuates TGF-β Dependent Hepatic Stellate Cell Activation and Liver Fibrosis.

Martin-Mateos Rosa R De Assuncao Thiago M TM Arab Juan Pablo JP Jalan-Sakrikar Nidhi N Yaqoob Usman U Greuter Thomas T Verma Vikas K VK Mathison Angela J AJ Cao Sheng S Lomberk Gwen G Mathurin Philippe P Urrutia Raul R Huebert Robert C RC Shah Vijay H VH

Cellular and molecular gastroenterology and hepatology 20180915 1

<h4>Background & aims</h4>Transdifferentiation of hepatic stellate cells (HSCs) into myofibroblasts is a key event in the pathogenesis of liver fibrosis. Transforming growth factor β (TGF-β) and platelet-derived growth factor (PDGF) are canonical HSC activators after liver injury. The aim of this study was to analyze the epigenetic modulators that differentially control TGF-β and PDGF signaling pathways.<h4>Methods</h4>We performed a transcriptomic comparison of HSCs treated with TGF-β or PDGF-B ...[more]

PMID: 30539787

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Project description:During biliary disease, cholangiocytes become activated by various pathological stimuli, including transforming growth factor β (TGF-β). The result is an epigenetically regulated transcriptional program leading to a pro-fibrogenic microenvironment, activation of hepatic stellate cells (HSCs), and progression of biliary fibrosis. This study evaluated how TGF-β signaling intersects with epigenetic machinery in cholangiocytes to support fibrogenic gene transcription. We performed RNA sequencing in cholangiocytes with or without TGF-β. Ingenuity pathway analysis identified "HSC Activation" as the highly up-regulated pathway, including overexpression of fibronectin 1 (FN), connective tissue growth factor, and other genes. Bioinformatics identified enhancer of zeste homologue 2 (EZH2) as an epigenetic regulator of the cholangiocyte TGF-β response. EZH2 overexpression suppressed TGF-β-induced FN protein in vitro, suggesting FN as a direct target of EZH2-based repression. Chromatin immunoprecipitation assays identified an FN promoter element in which EZH2-mediated tri-methylation of lysine 27 on histone 3 is diminished by TGF-β. TGF-β also caused a 50% reduction in EZH2 protein levels. Proteasome inhibition rescued EZH2 protein and led to reduced FN production. Immunoprecipitation followed by mass spectrometry identified ubiquitin protein ligase E3 component N-recognin 4 in complex with EZH2, which was validated by western blotting in vitro. Ubiquitin mutation studies suggested K63-based ubiquitin linkage and chain elongation on EZH2 in response to TGF-β. A deletion mutant of EZH2, lacking its N-terminal domain, abrogates both TGF-β-stimulated EZH2 degradation and FN release. In vivo, cholangiocyte-selective knockout of EZH2 exacerbates bile duct ligation-induced fibrosis whereas MDR2-/- mice are protected from fibrosis by the proteasome inhibitor bortezomib. Conclusion: TGF-β regulates proteasomal degradation of EZH2 through N-terminal, K63-linked ubiquitination in cholangiocytes and activates transcription of a fibrogenic gene program that supports biliary fibrosis.

Project description:BACKGROUND:Hepatic fibrosis is caused by chronic liver injury and may progress toward liver cirrhosis, and even hepatocellular carcinoma. However, current treatment is not satisfactory. Therefore, there is a mandate to find novel therapeutic targets to improve therapy, and biomarkers to monitor therapeutic response. METHODS:Liver fibrosis was induced by carbon tetrachloride (CCl4) or thioacetamide (TAA) in wild type (WT) or CTHRC1-/- mice, followed by immunofluorescence and immunohistochemical analyses. CTHRC1 monoclonal antibody (mAb) was used to abrogate the effect of CTHRC1 in vitro and in vivo. RESULTS:Here, we reported that collagen triple helix repeat containing 1 (CTHRC1), a secreted protein derived from hepatic stellate cells (HSCs), was significantly up-regulated in fibrotic liver tissues. CTHRC1 promoted HSCs transformation from a quiescent to an activated state, and enhanced migratory or contractile capacities of HSCs by activating TGF-? signaling. Meanwhile, CTHRC1 competitively bound to Wnt noncononical receptor and promoted the contractility but not activation of HSCs. CCl4 or TAA-induced liver fibrosis was attenuated in CTHRC-/- mice compared with littermate control, while a monoclonal antibody of CTHRC1 suppressed liver fibrosis in WT mice treated with CCl4 or TAA. INTERPRETATION:We demonstrated that CTHRC1 is a new regulator of liver fibrosis by modulating TGF-? signaling. Targeting CTHRC1 could be a promising therapeutic approach, which can suppress TGF-? signaling and avoid the side effects caused by directly targeting TGF-?. CTHRC1 could also be a potential biomarker for monitoring response to anti-fibrotic therapy. FUND: This study was supported by the National Natural Science Foundation of China (ID 81672358, 81871923, 81872242, 81802890), the Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant Support (ID 20181708), the Natural Science Foundation of Shanghai (ID 17ZR1428300, 18ZR1436900), and Shanghai Municipal Health Bureau (ID 2018BR32). The funders did not play a role in manuscript design, data collection, data analysis, interpretation nor writing of the manuscript.

Dataset Information

Enhancer of Zeste Homologue 2 Inhibition Attenuates TGF-? Dependent Hepatic Stellate Cell Activation and Liver Fibrosis.

Publications

Enhancer of Zeste Homologue 2 Inhibition Attenuates TGF-β Dependent Hepatic Stellate Cell Activation and Liver Fibrosis.

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