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Proteasomal Degradation of Enhancer of Zeste Homologue 2 in Cholangiocytes Promotes Biliary Fibrosis.

ABSTRACT: During biliary disease, cholangiocytes become activated by various pathological stimuli, including transforming growth factor ? (TGF-?). 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-? signaling intersects with epigenetic machinery in cholangiocytes to support fibrogenic gene transcription. We performed RNA sequencing in cholangiocytes with or without TGF-?. 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-? response. EZH2 overexpression suppressed TGF-?-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-?. TGF-? 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-?. A deletion mutant of EZH2, lacking its N-terminal domain, abrogates both TGF-?-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-? 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.

SUBMITTER: Jalan-Sakrikar N

PROVIDER: S-EPMC6819212 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Proteasomal Degradation of Enhancer of Zeste Homologue 2 in Cholangiocytes Promotes Biliary Fibrosis.

Jalan-Sakrikar Nidhi N De Assuncao Thiago M TM Shi Guang G Aseem Sayed Obaidullah SO Chi Cheng C Shah Vijay H VH Huebert Robert C RC

Hepatology (Baltimore, Md.) 20190622 5

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 ...[more]

PMID: 31070797

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Project description: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).

Dataset Information

Proteasomal Degradation of Enhancer of Zeste Homologue 2 in Cholangiocytes Promotes Biliary Fibrosis.

Publications

Proteasomal Degradation of Enhancer of Zeste Homologue 2 in Cholangiocytes Promotes Biliary Fibrosis.

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