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Genome-wide CRISPR Screening to Identify Drivers of TGF-β-Induced Liver Fibrosis in Human Hepatic Stellate Cells.

ABSTRACT: Liver fibrosis progression in chronic liver disease leads to cirrhosis, liver failure, or hepatocellular carcinoma and often ends in liver transplantation. Even with an increased understanding of liver fibrogenesis and many attempts to generate therapeutics specifically targeting fibrosis, there is no approved treatment for liver fibrosis. To further understand and characterize the driving mechanisms of liver fibrosis, we developed a high-throughput genome-wide CRISPR/Cas9 screening platform to identify hepatic stellate cell (HSC)-derived mediators of transforming growth factor (TGF)-β-induced liver fibrosis. The functional genomics phenotypic screening platform described here revealed the novel biology of TGF-β-induced fibrogenesis and potential drug targets for liver fibrosis.

SUBMITTER: Yu S

PROVIDER: S-EPMC9016707 | biostudies-literature | 2022 Apr

REPOSITORIES: biostudies-literature

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Genome-wide CRISPR Screening to Identify Drivers of TGF-β-Induced Liver Fibrosis in Human Hepatic Stellate Cells.

Yu Shan S Ericson Matthew M Fanjul Andrea A Erion Derek M DM Paraskevopoulou Maria M Smith Erin N EN Cole Banumathi B Feaver Ryan R Holub Corine C Gavva Narender N Horman Shane R SR Huang Jie J

ACS chemical biology 20220311 4

Liver fibrosis progression in chronic liver disease leads to cirrhosis, liver failure, or hepatocellular carcinoma and often ends in liver transplantation. Even with an increased understanding of liver fibrogenesis and many attempts to generate therapeutics specifically targeting fibrosis, there is no approved treatment for liver fibrosis. To further understand and characterize the driving mechanisms of liver fibrosis, we developed a high-throughput genome-wide CRISPR/Cas9 screening platform to ...[more]

PMID: 35274923

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Project description:BackgroundHepatic 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.MethodsLiver 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.ResultsHere, 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-β 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.InterpretationWe demonstrated that CTHRC1 is a new regulator of liver fibrosis by modulating TGF-β signaling. Targeting CTHRC1 could be a promising therapeutic approach, which can suppress TGF-β signaling and avoid the side effects caused by directly targeting TGF-β. 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.

Project description:Background & aimsTransdifferentiation 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.MethodsWe 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.ResultsEnhancer 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.ConclusionsTGF-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).

Project description:BackgroundLiver fibrosis and fibrosis-related hepatocarcinogenesis are a rising cause for morbidity and death worldwide. Although transforming growth factor-β (TGF-β) is a critical mediator of chronic liver fibrosis, targeting TGF-β isoforms and receptors lead to unacceptable side effect. This study was designed to explore the antifibrotic effect of Compound kushen injection (CKI), an approved traditional Chinese medicine formula, via a therapeutic strategy of rebalancing TGF-β/Smad7 signaling.MethodsA meta-analysis was performed to evaluate CKI intervention on viral hepatitis-induced fibrosis or cirrhosis in clinical randomized controlled trials (RCTs). Mice were given carbon tetrachloride (CCl4 ) injection or methionine-choline deficient (MCD) diet to induce liver fibrosis, followed by CKI treatment. We examined the expression of TGF-β/Smad signaling and typical fibrosis-related genes in hepatic stellate cells (HSCs) and fibrotic liver tissues by qRT-PCR, Western blotting, RNA-seq, immunofluorescence, and immunohistochemistry.ResultsBased on meta-analysis results, CKI improved the liver function and relieved liver fibrosis among patients. In our preclinical studies by using two mouse models, CKI treatment demonstrated promising antifibrotic effects and postponed hepatocarcinogenesis with improved liver function and histopathologic features. Mechanistically, we found that CKI inhibited HSCs activation by stabilizing the interaction of Smad7/TGF-βR1 to rebalance Smad2/Smad3 signaling, and subsequently decreased the extracellular matrix formation. Importantly, Smad7 depletion abolished the antifibrotic effect of CKI in vivo and in vitro. Moreover, matrine, oxymatrine, sophocarpine, and oxysophocarpine were identified as material basis responsible for the antifibrosis effect of CKI.ConclusionsOur results unveil the approach of CKI in rebalancing TGF-β/Smad7 signaling in HSCs to protect against hepatic fibrosis and hepatocarcinogenesis in both preclinical and clinical studies. Our study suggests that CKI can be a candidate for treatment of hepatic fibrosis and related oncogenesis.

Dataset Information

Genome-wide CRISPR Screening to Identify Drivers of TGF-β-Induced Liver Fibrosis in Human Hepatic Stellate Cells.

Publications

Genome-wide CRISPR Screening to Identify Drivers of TGF-β-Induced Liver Fibrosis in Human Hepatic Stellate Cells.

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