Rosa Martin-Mateos 1 , 2 , Thiago M. De Assuncao 1 , Juan Pablo Arab 1 , 3 , Nidhi Jalan-Sakrikar 1 , Usman Yaqoob 1 , Thomas Greuter 1 , Vikas K. Verma 1 , Angela J. Mathison 4 , 5 , Sheng Cao 1 , Gwen Lomberk 5 , Philippe Mathurin 6 , Raul Urrutia 4 , 5 , Robert C. Huebert 1 , Vijay H. Shah 1 , ∗
15 September 2018
EZH2, Liver Fibrosis, Epigenetics, Histone Modifications, BDL, bile duct ligation, CCL4, carbon tetrachloride, ChIP, chromatin immunoprecipitation, CTGF, connective tissue growth factor, DKK1, Dickkopf-1, ECM, extracellular matrix, EZH2, enhancer of zeste homologue 2, FBS, fetal bovine serum, FDR, false discovery rate, H3K27me3, trimethylation of histone 3 at lysine 27, HCC, hepatocellular carcinoma, HMT, histone methyltransferase, HSC, hepatic stellate cell, IP, intraperitoneally, IPA, Ingenuity Pathway Analysis, logFC, logarithmic fold change, mRNA, messenger RNA, PCR, polymerase chain reaction, PDGF, platelet-derived growth factor, siRNA, small interfering RNA, α-SMA, α-smooth muscle actin, TGF-β, transforming growth factor β, VEGFA, vascular endothelial growth factor A, WT, wild type
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.
We performed a transcriptomic comparison of HSCs treated with TGF-β or PDGF-BB using RNA sequencing. Among the targets that distinguish these 2 pathways, we focused on the histone methyltransferase class of epigenetic modulators.
Enhancer of zeste homolog 2 (EZH2) was expressed differentially, showing significant up-regulation in HSCs activated with TGF-β 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-β–induced fibronectin, collagen 1α1, and α-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-β–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 α-smooth muscle actin/collagen protein assays.
TGF-β 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-β as opposed to the PDGF signaling pathway. Inhibition of EZH2 attenuates fibrogenic gene transcription in TGF-β–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)