Aryl Hydrocarbon Receptor Signaling Prevents Activation of Hepatic Stellate Cells and Liver Fibrogenesis in Mice

Gastroenterology 2019;-:1–14 Q1 Q2 Q3 Q20 Aryl Hydrocarbon Receptor Signaling Prevents Activation of Hepatic Stellate Cells and Liver Fibrogenesis ...

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Gastroenterology 2019;-:1–14

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Aryl Hydrocarbon Receptor Signaling Prevents Activation of Hepatic Stellate Cells and Liver Fibrogenesis in Mice Jiong Yan,1,* Hung-Chun Tung,1,* Sihan Li,1 Yongdong Niu,1 Wojciech G. Garbacz,1 Peipei Lu,1 Yuhan Bi,1 Yanping Li,2 Jinhan He,2 Meishu Xu,1 Songrong Ren,1 Satdarshan P. Monga,3 Robert F. Schwabe,4 Da Yang,1 and Wen Xie1,5 1

Center for Pharmacogenetics and Department of Pharmaceutical Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania; Department of Pharmacy, West China Hospital, Sichuan University, Chengdu, Sichuan, China; 3Department of Pathology and Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; 4Department of Medicine, Columbia University, New York, New York; 5Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, Pennsylvania 2

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BACKGROUND & AIMS: The role of aryl hydrocarbon receptor (AhR) in liver ﬁbrosis is controversial because loss and gain of AhR activity both lead to liver ﬁbrosis. The goal of this study was to investigate how the expression of AhR by different liver cell types, hepatic stellate cells (HSCs) in particular, affects liver ﬁbrosis in mice. METHODS: We studied the effects of AhR on primary mouse and human HSCs, measuring their activation and stimulation of ﬁbrogenesis using RNA-sequencing analysis. C57BL/6J mice were given the AhR agonists 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 2-(10 H-indole-30 -carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE); were given carbon tetrachloride (CCl4); or underwent bile duct ligation. We also performed studies in mice with disruption of Ahr speciﬁcally in HSCs, hepatocytes, or Kupffer cells. Liver tissues were collected from mice and analyzed by histology, immunohistochemistry, and immunoblotting. RESULTS: AhR was expressed at high levels in quiescent HSCs, but the expression decreased with HSC activation. Activation of HSCs from AhRknockout mice was accelerated compared with HSCs from wildtype mice. In contrast, TCDD or ITE inhibited spontaneous and transforming growth factor b–induced activation of HSCs. Mice with disruption of Ahr in HSCs, but not hepatocytes or Kupffer cells, developed more severe ﬁbrosis after administration of CCl4 or bile duct ligation. C57BL/6J mice given ITE did not develop CCl4induced liver ﬁbrosis, whereas mice without HSC AhR given ITE did develop CCl4-induced liver ﬁbrosis. In studies of mouse and human HSCs, we found that AhR prevents transforming growth

factor b–induced ﬁbrogenesis by disrupting the interaction of SMAD3 with b-catenin, which prevents the expression of genes that mediate ﬁbrogenesis. CONCLUSIONS: In studies of human and mouse HSCs, we found that AhR prevents HSC activation and expression of genes required for liver ﬁbrogenesis. Development of nontoxic AhR agonists or strategies to activate AhR signaling in HSCs might be developed to prevent or treat liver ﬁbrosis. Keywords: Cell-Speciﬁc Effect; Gene Regulation; Signal Transduction; Xenobiotic Receptor.

*Authors share co-ﬁrst authorship. Abbreviations used in this paper: a-SMA, a-smooth muscle actin; Ahr, aryl hydrocarbon receptor; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ARNT, aryl hydrocarbon receptor nuclear translocator; ChIP, chromatin immunoprecipitation; CUL4B, Cullin-4B; DRE, dioxinresponsive element; ECM, extracellular matrix; FICZ, 6-formylindolo[3,2-b] carbazole; HSC, hepatic stellate cell; ITE, 2-(10 H-indole-30 -carbonyl)thiazole-4-carboxylic acid methyl ester; KO, knockout; Lrat, lecithinretinol acyltransferase; mRNA, messenger RNA; PCR, polymerase chain reaction; RNA-seq, RNA sequencing; TCDD, 2,3,7,8-tetrachlorodibenzo-pdioxin; TGF, transforming growth factor; SRE, xenobiotic response element. © 2019 by the AGA Institute 0016-5085/$36.00 https://doi.org/10.1053/j.gastro.2019.05.066

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WHAT YOU NEED TO KNOW BACKGROUND AND CONTEXT The liver contains multiple cell types. We investigated how the expression of aryl hydrocarbon receptor (AHR) by different cell types affects liver ﬁbrosis and activation of hepatic stellate cells (HSCs) in mice. NEW FINDINGS AHR signaling prevents activation of HSCs expression of genes required for liver ﬁbrogenesis.

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LIMITATIONS Although human HSCs were used and bioinformatic analyses were performed on human liver samples, more human studies will further enhance the human relevance. IMPACT Non-toxic AHR agonists or strategies to activate AHR signaling in HSCs might be developed to prevent or treat liver ﬁbrosis.

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iver ﬁbrosis, deﬁned as the excessive intercellular accumulation of extracellular matrix (ECM) proteins in the liver, is strongly associated with chronic viral or nonviral liver injuries.1 Advanced liver ﬁbrosis causes liver cirrhosis, leading to portal hypertension and liver failure. Liver ﬁbrosis is also a major risk factor for the development of hepatocellular carcinoma. Activated hepatic stellate cells (HSCs) are the major cell population responsible for the production of ECM proteins and proﬁbrogenic cytokines.2 Residing in the space of Disse between the sinusoidal endothelial cells and hepatocytes, HSCs are characterized by their expression of desmin and glial ﬁbrillary acidic protein in the quiescent state and a-smooth muscle actin (a-SMA) in the activated state. The activation of HSCs, which is characterized by increased ﬁbrogenic gene expression and proliferation, is central to the pathogenesis of liver ﬁbrosis. As such, understanding the molecular basis of HSC activation will help develop strategies to prevent and treat liver ﬁbrosis. The transforming growth factor (TGF) b pathway that integrates a myriad of injury signals is pivotal for HSC activation.3 Emerging evidence also suggested the role of Wnt–b-catenin signaling in promoting HSC activation and its crosstalk with the TGFb pathway in the ﬁbrotic diseases.4 However, how Wnt signaling, particularly the b-catenin– mediated canonical pathway, promotes the ﬁbrogenic progression is not fully understood. The aryl hydrocarbon receptor (AhR), highly expressed in the liver, is a well-established xenobiotic receptor that senses environmental toxicants and regulates xenobiotic metabolism.5 Many industrial pollutants, such as polycyclic aromatic hydrocarbons, are AhR ligands.6 One compound that is widely used as a tool for studying the toxicologic effect of AhR is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a prototypical xenobiotic activator of AhR. As a liganddependent transcriptional factor, AhR signals through its DNA-binding motif and diverse protein partners.6 Upon binding by TCDD, AhR translocates into the nucleus, where

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it heterodimerizes with AhR nuclear translocator (ARNT) on the xenobiotic response elements (XREs) to regulate the transcription of a battery of TCDD-responsive genes involved in xenobiotic catabolism, inﬂammatory response, and metabolic reprogramming. Chromatin immunoprecipitation (ChIP)–sequencing analysis has shown non-XRE binding of AhR in the genome, suggesting that AhRresponsive genes are not limited to those harboring XREs.7 Subsequent studies, mainly through the characterization of AhR–/– mice, have implicated AhR and its endogenous ligands in tissue development and pathophysiology, including liver ﬁbrosis.8,9 The AhR signaling has also been implicated in the homeostasis of energy metabolism, gut microbiota, stem cell differentiation, circadian rhythm, and adaptive immunity.10 In addition to its function as a transcriptional factor, AhR also participates in proteasomedependent proteolysis by functioning as a substrate-speciﬁc adaptor that targets selected proteins for degradation in the Cullin-4B (CUL4B)–ubiquitin E3 ligase complex CUL4BAhR.11 The reported substrate proteins for CUL4BAhR include estrogen receptor a, androgen receptor, and b-catenin.11,12 The role of AhR in liver ﬁbrosis has been intriguing and controversial. On one hand, AhR–/– mice exhibited spontaneous liver ﬁbrosis.8 On the other hand, treatment of mice with TCDD or constitutive activation of AhR in the hepatocytes sensitized mice to methionine- and choline-deﬁcient or high-fat diet–induced liver ﬁbrosis.13–15 The liver is an organ of multiple cell types, including hepatocytes, HSCs, and Kupffer cells. It is unclear whether AhR has a cell-type speciﬁc role in liver ﬁbrosis. More speciﬁcally, it has not been reported whether and how AhR plays a role in HSC activation and liver ﬁbrosis. In this study, we uncovered an unexpected role of AhR in preventing HSC activation and attenuating liver ﬁbrosis. Knockout (KO) of AhR in HSCs was sufﬁcient to cause spontaneous liver ﬁbrosis and sensitize mice to experimental liver ﬁbrosis. In contrast, the nontoxic AhR agonist 2-(10 H-indole-30 -carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) exhibited antiﬁbrotic activity in vivo.

Materials and Methods Detailed information about chemical reagents, antibodies, and real-time polymerase chain reaction (PCR) primers is provided in Supplementary Tables 1–3.

Experimental Animals and Histology

Wild-type C57BL/6J (000664), AhRﬂ/ﬂ (Ahrtm3.1Bra/J, 006203), albumin-Cre (B6.Cg-Tg(Alb-Cre)21Mgn/J, 003574), and LysM-Cre (B6.129P2-Lyz2tm1(cre)Ifo/J, 004781) mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Lecithin-retinol acyltransferase (Lrat)–Cre mice have been previously described.2 AhRﬂ/ﬂ mice were crossbred with albumin-Cre, LysM-Cre, or Lrat-Cre mice to generate cell type– speciﬁc AhR-KO mice. For the toxicologic comparison of TCDD and ITE, 6-week-old male C57BL/6J mice were treated with vehicle (dimethyl sulfoxide), TCDD (25 mg/kg), or ITE (10 mg/ kg) once a week for 2 weeks by intraperitoneal injection. The

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animals were killed 6 hours after the third injection. For the carbon tetrachloride (CCl4) model of liver ﬁbrosis, 9- to 10week-old male mice were intraperitoneally injected with CCl4 (1 mL/g body weight, 1:3 diluted in corn oil, twice a week) for 4 weeks. The animals were killed 72 hours after the ﬁnal CCl4 injection. When ITE was used to treat CCl4-induced liver ﬁbrosis, ITE (10 mg/kg) was administrated every other day along with CCl4 (0.5 mL/g body weight) in corn oil by intraperitoneal injection for 4 weeks. The animals were terminated 6 hours after the ﬁnal dose of ITE. For the bile duct ligation model of liver ﬁbrosis, 8- to 9-week-old mice were subject to common bile duct ligation, and the animals were killed 14 days after the surgery. See the Supplementary Materials for histologic details. The use of mice was in accordance with the University of Pittsburgh Institutional Animal Care and Use Committee.

Primary Hepatic Stellate Cell Isolation and Sorting, Adenovirus, Lentivirus, Plasmids, and Cell Transfection Primary mouse HSCs were isolated as previously reported.16 For the RNA-sequencing (RNA-seq) analysis, HSCs were further puriﬁed by vitamin A–based ﬂuorescenceactivated cell sorting, as described.16 See the Supplementary Materials for details about primary HSC isolation and sorting, adenovirus, lentivirus, plasmids, and cell transfection.

RNA-Sequencing Analysis RNA-seq was performed at the Health Sciences Sequencing Core at the Children’s Hospital of Pittsburgh. Gene expression was analyzed by gene set enrichment analysis.17 See the Supplementary Materials for details of bioinformatic analysis of the RNA-seq results and the Gene Expression Omnibus data sets.

Immunoﬂuorescence, Immunoprecipitation and Western Blot, Chromatin Immunoprecipitation, and Quantitative Real-Time Polymerase Chain Reaction These were performed as described18; Supplementary Materials for details.

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Statistics Statistical analysis was performed using Prism GraphPad 7.0 (GraphPad, San Diego, CA). All results are presented as means ± standard error of the mean of at least 3 replicates. Statistical differences between groups were determined using unpaired 2-tailed Student t test or 1-way analysis of variance with post hoc Tukey test. P values less than .05 were considered statistically signiﬁcant.

Results Treatment of Mice with the Nontoxic Aryl Hydrocarbon Receoptor Ligand ITE Ameliorates CCl4-Induced Liver Fibrosis

The classical AhR ligand TCDD induces liver ﬁbrosis in mice.14 TCDD is known to be toxic to the liver, so we

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speculated that liver ﬁbrosis might have been secondary to TCDD-induced hepatotoxicity and inﬂammation. We decided to test the effect of ITE, a nontoxic tryptophan metabolite and endogenous AhR agonist,19 on liver ﬁbrosis. Treatment of mice with TCDD for 2 weeks caused typical hepatotoxicity as indicated by neutrophil inﬁltration and collagen deposition (Figure 1A), consistent with a previous report.20 In contrast, the liver of ITE-treated mice showed no signs of hepatotoxicity and ﬁbrosis (Figure 1A). The lack of ﬁbrogenic activity of ITE was conﬁrmed by measuring the expression of ﬁbrogenic marker genes (Figure 1B). We then tested the effect of ITE on CCl4induced liver ﬁbrosis, as outlined in Figure 1C. Treatment with ITE ameliorated CCl4-induced liver ﬁbrosis, which was supported by decreased collagen deposition and ﬁbrogenesis as shown by Masson’s trichrome staining, Sirius Red staining, and immunostaining of a-SMA (Figure 1D). The suppression of a-SMA in ITE- and CCl4treated mice was further veriﬁed by Western blotting (Figure 1E) and real-time PCR (Figure 1F). Consistent with the relief of liver ﬁbrosis, the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were decreased in ITE-treated mice compared with their vehicle-treated counterparts (Figure 1G). The hepatic expression of Cyp1a1 and Cyp1a2, 2 typical AhR target genes, was induced by ITE, suggesting that AhR was efﬁciently activated (Figure 1H). We also determined whether ITE can mitigate existing liver ﬁbrosis by treating mice with ITE after the initiation of the CCl4 model, as outlined in Supplementary Figure 1A. The posttreatment with ITE also attenuated CCl4-induced liver ﬁbrosis, as shown by histology (Supplementary Figure 1B) and measurement of a-SMA protein expression (Supplementary Figure 1C). ITE posttreatment induced the expression of Cyp1a1 and Cyp1a2 (Supplementary Figure 1D) but had little effect on serum ALT and AST levels (Supplementary Figure 1E).

Aryl Hydrocarbon Receptor Is Highly Expressed in Hepatic Stellate Cells, and the Expression of Aryl Hydrocarbon Receptor Inversely Correlates With Hepatic Stellate Cell Activation In Vitro and In Vivo To investigate whether AhR in the HSCs mediates the antiﬁbrotic effect of ITE, we compared the expression of AhR in primary hepatocytes and HSCs isolated from the same mice. There was an approximately 4-fold increase in the messenger (mRNA) expression of both Ahr and its DNA-binding partner Arnt in HSCs compared with hepatocytes (Figure 2A). The basal expression of Cyp1a1 and Cyp1b1 was also markedly higher in HSCs (Figure 2A). These results were consistent with a recently reported proteomic analysis in which HSCs were shown to express a higher level of AhR than hepatocytes.21 Treatment of primary HSCs isolated from wild-type mice with TCDD induced the expression of Cyp1a1, Cyp1a2, and Cyp1b1, but the induction was abolished in HSCs isolated from AhR–/– mice (Figure 2B), suggesting that AhR in HSCs is transcriptionally functional. Moreover, we found that the

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Figure 1. Treatment of mice with the nontoxic AhR ligand ITE ameliorates CCl4-induced liver ﬁbrosis. (A, B) Six-week-old male C57BL/6J mice were treated with vehicle (dimethyl sulfoxide [DMSO]), TCDD (25 mg/kg), or ITE (10 mg/kg) once a week for 2 weeks (n ¼ 4 per group) before being analyzed for histology by (A) H&E and Sirius Red staining original magniﬁcation 20) and (B) the expression of ﬁbrogenic genes by real-time PCR. Arrows indicate neutrophil inﬁltration in H&E and collagen deposition in the Sirius Red staining. (C–H) Eight-week-old male C57BL/6J mice were treated with (C) vehicle or ITE (10 mg/kg) together with CCl4 (0.5 mL/g body weight) 3 times a week for 4 weeks (n ¼ 6 per group) (D) Histology was analyzed by Masson’s trichrome staining (MTS), Sirius Red staining, and a-SMA immunostaining (original magniﬁcation, 5), with the quantiﬁcation of Sirius Red and a-SMA staining shown on the right. The (E) hepatic protein and (F) mRNA expression levels of a-SMA were measured by Western blotting and real-time PCR, respectively. The (G) serum levels of ALT and AST and (H) hepatic expression of Cyp1a1 and Cyp1a2 were also measured. All data were presented as mean ± standard error of the mean. *P < .05, **P < .01. N.S., not statistically signiﬁcant; Veh, vehicle.

expression of AhR was inversely correlated with the activation of HSCs. When primary HSCs were subject to spontaneous activation in culture, the expression of AhR and its target gene Cyp1a1 in primary mouse (Figure 2C) and human (Figure 2D) HSCs decreased with the onset of HSC activation in a time-dependent manner. The expression of Acta2 was measured to validate HSC activation. Interestingly, we observed a major induction of Cyp1b1, another AhR target gene, with the onset of HSC activation (Supplementary Figure 2), suggesting that it was not a general decline in drug metabolism enzymes that was responsible for the phenotype. In contrast, deactivation of HSCs by inducing adipogenic differentiation in fully

activated HSCs was correlated with an increased expression of AhR (Supplementary Figure 3). The ﬁbrosis-responsive down-regulation of AhR in HSCs was also conﬁrmed in vivo because the primary HSCs isolated from CCl4-treated mice showed a decreased expression of Ahr compared with HSCs isolated from vehicle-treated mice (Figure 2E). Moreover, analysis of 2 Gene Expression Omnibus data sets, alcoholic hepatitis (GSE28619) and cirrhosis (GSE89377), showed that both ﬁbrosis-prone liver diseases are associated with a decreased expression of AHR and its target gene CYP1A2 (Figure 2F), suggesting that the ﬁbrosis-responsive down-regulation of AHR is conserved in humans.

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Figure 2. AhR is highly expressed in HSCs, and the expression of AhR inversely correlates with HSC activation. (A) Primary hepatocytes (HEPs) and HSCs were isolated from the same mouse liver. Gene expression was determined immediately after cell isolation by real-time PCR (n ¼ 4). (B) Primary HSCs were isolated from WT or AhR–/– (KO) mice and treated with TCDD (20 nmol/L) for 6 days. Gene expression was determined by real-time PCR (n ¼ 4). (C) Primary mouse or (D) human HSCs were culture-activated for the indicated duration. Gene expression was determined by real-time PCR (n ¼ 4). (E) Primary mouse HSCs were isolated from male mice treated with 4 doses of CCl4 (0.5 mL/g body weight, every 3 days, n ¼ 3) or vehicle (n ¼ 4). (F) Gene expression was determined immediately after cell isolation by real-time PCR. Two data sets (GSE28619 and GSE89377) from the Gene Expression Omnibus database were analyzed for the expression levels of AHR and CYP1A2. All data are presented as mean ± standard error of the mean. *P < .05, **P < .01. N.D., not detectable; Veh, vehicle; WT, wild type.

Pharmacologic Activation or Forced Expression of Aryl Hydrocarbon Receptor Inhibits Hepatic Stellate Cell Activation To determine whether the dynamic expression of AhR during HSC activation is functionally relevant, we treated primary mouse HSCs with TCDD and found that it inhibited the expression of ﬁbrogenic genes at both the protein (Figure 3A) and mRNA (Figure 3B) levels. The activation of AhR by TCDD was veriﬁed by the induction of Cyp1b1

(Supplementary Figure 4A). Treatment of primary mouse HSCs with 6-formylindolo[3,2-b]carbazole (FICZ) and ITE, 2 endogenous AhR agonists, also inhibited ﬁbrogenic gene expression (Supplementary Figure 4B). TCDD or ITE had little effect on HSC proliferation and apoptosis, as shown by bromodeoxyuridine labeling (Supplementary Figure 4C) and terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling staining (Supplementary Figure 4D), respectively. The inhibitory effect of TCDD on

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Figure 3. Pharmacologic activation or forced expression of AhR inhibits HSC activation. (A) Primary mouse HSCs were treated with TCDD (50 nmol/L) for 2, 4, and 6 days. Protein levels from the culture medium or whole-cell lysates were detected by Western blotting. (B) Primary mouse HSCs were treated with TCDD (20 nmol/L) for 4 days. Gene expression was determined by real-time PCR (n ¼ 3). (C, D) Primary mouse HSCs were isolated from WT and AhR–/– mice and treated with TCDD (20 nmol/L) for 6 days. (C) Cell morphology was analyzed by light-ﬁeld microscopy and (D) gene expression was determined by real-time PCR (n ¼ 4). (E) Primary human HSCs were treated with 3-methylcholanthrene (2 mmol/L) for 6 days. Gene expression was determined by real-time PCR (n ¼ 4). (F, G) Primary mouse HSCs were infected with adenovirus overexpressing AhR (multiplicity of infection ¼ 10) and treated with or without TCDD (50 nmol/L). (F) The expression levels of a-SMA and Ki67 were detected by immunoﬂuorescence, and (G) gene expression was determined by real-time PCR (n ¼ 3). (H, I) The experiments were the same as in F and G, except that primary human HSCs were used. Scale bar ¼100 mm. All data were presented as mean ± standard error of the mean. *P < .05, **P < .01.

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HSC activation was AhR dependent, because the inhibitory effect was abolished in HSCs isolated from AhR–/– mice, as evaluated by cell morphology (Figure 3C) or the expression of ﬁbrogenic marker genes (Figure 3D). A similar pattern of inhibition was observed in primary human HSCs treated with the human AHR activator 3-methylcholanthrene (Figure 3E). The activation of AHR by 3-methylcholanthrene was veriﬁed by the induction of CYP1A2 (Supplementary Figure 4E). Inhibition of HSC activation was also observed in primary mouse HSCs infected with adenovirus expressing the mouse AhR (Ad-AhR), as shown by the immunoﬂuorescence of aSMA and Ki67 (Figure 3F) and the expression of ﬁbrogenic genes (Figure 3G). Adenoviral overexpression of AhR achieved a similar inhibition of human HSC activation (Figures 3H and I). The adenoviral overexpression of AhR and induction of AhR target genes in the mouse (Supplementary Figure 4F) and human (Supplementary Figure 4G) HSCs was validated by real-time PCR.

Knockout of Aryl Hydrocarbon Receptor Promotes Hepatic Stellate Cell Activation In Vitro Consistent with our hypothesis that the down-regulation of AhR contributes to the activation of HSCs, HSCs isolated from AhR–/– mice showed enhanced culture-induced activation as shown by cell morphology (Figure 4A) and induction of ﬁbrogenic marker genes (Figure 4B). Increased protein expression of a-SMA and Col1A1 in AhR–/– HSCs was veriﬁed by Western blotting (Figure 4C), and induction of a-SMA and Ki67 was veriﬁed by immunoﬂuorescence (Figure 4D). To better assess the effect of AhR KO on HSC activation, we performed RNA-seq analysis on primary HSCs isolated from wild-type and AhR–/– mice after vitamin A autoﬂuorescence-based ﬂuorescence-activated cell sorting.16 The RNA-seq results are shown in Supplementary Table 4, with the major pathways affected presented in Supplementary Figure 5A. AhR–/– HSCs exhibited an altered transcriptome, including an elevated expression of ﬁbrogenic markers, such as Col1a1, Col1a2, Col3a1, and Lox (Figure 4E). Several pathways related to HSC activation were selected for gene set enrichment analysis.17 We found a signiﬁcant up-regulation of Extracellular Matrix, Cell Proliferation, Cytokine Receptor Binding, and Wnt Signaling Pathway in AhR–/– HSCs (Figure 4F). The Cell Cycle Progression Pathways were also signiﬁcantly up-regulated, whereas TGFb Pathway and Epithelial Mesenchymal Transition showed a trend of increase (Supplementary Figure 5B). Overall, the RNA-seq results supported the notion that AhR–/– HSCs were activated with an elevated Wnt signaling. When the RNA-seq results were integrated with a previously reported AHR-binding element (dioxin-responsive element [DRE]) database,22 we found that 4901 out of 17,128 genes have at least 1 potential DRE (Supplementary Table 5) but that the DRE-harboring genes are not enriched in either up-regulated or down-regulated gene sets (Supplementary Table 6). It was also noted that many of the ﬁbrogenic genes, such as Col1a2, do not contain DREs. The

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activation of AhR–/– HSCs was attenuated when the expression of AhR was reconstituted by adenoviral infection, as shown by the protein expression of a-SMA and Col1A1 (Figure 4G), the mRNA expression of ﬁbrogenic genes (Figure 4H), and immunoﬂuorescence of a-SMA and Ki67 (Figure 4I).

Knockout of Aryl Hydrocarbon Receptor in Hepatic Stellate Cells Sensitizes Mice to Liver Fibrosis and Abolishes the Antiﬁbrotic Activity of ITE To determine whether AhR suppresses HSC activation and ﬁbrosis in vivo, we generated HSC-speciﬁc AhR-KO mice (HSC-KO) by crossbreeding the AhR ﬂoxed (AhRﬂ/ﬂ, ﬂox) mice20 with the Lrat-Cre transgenic mice expressing Cre in HSCs under the control of the Lrat gene promoter2 (Supplementary Figure 6A). The AhR expression in HSCs isolated from HSC-KO mice was decreased by 75% compared with the ﬂox mice (Supplementary Figure 6B). Compared with the ﬂox mice, HSC-KO mice exhibited more pronounced liver ﬁbrosis when challenged with CCl4, as evidenced by increased collagen deposition and expression of a-SMA and desmin (Figure 5A). The increased protein expression of a-SMA (Figure 5B) and mRNA expression of collagen genes (Figure 5C) in HSC-KO mice were veriﬁed by Western blotting and real-time PCR, respectively. The HSCKO mice also exhibited increased sensitivity to bile duct ligation–induced liver ﬁbrosis, as evidenced by increased expression of a-SMA (Supplementary Figure 6C) and histologic analysis (Supplementary Figure 6D), but without affecting the serum ALT and AST levels (Supplementary Figure 6E). Furthermore, the antiﬁbrotic effects of ITE were abolished in HSC-KO mice, as shown by histology (Figure 5D), ﬁbrogenic gene expression (Figure 5E), and serum ALT and AST levels (Figure 5F), suggesting the HSC AhR is required for the antiﬁbrotic activity of ITE. To determine whether the hepatocyte AhR plays a role in the development of liver ﬁbrosis, we also generated the hepatocyte-speciﬁc AhR-KO mice (HEP-KO) by crossbreeding the AhR ﬂoxed mice with the albumin-Cre transgenic mice (Supplementary Figure 6F). Compared with ﬂox mice, HEP-KO mice showed a similar sensitivity to CCl4induced liver ﬁbrosis (Supplementary Figure 6F). It has been reported that the whole-body AhR–/– mice exhibited spontaneous periportal liver ﬁbrosis,8,9 which was veriﬁed by our own analysis (Supplementary Figure 7). To determine the loss of AhR in which cell type is responsible, we used the HEP-KO and HSC-KO mice, as well as the Kupffer cell–speciﬁc AhR-KO mice (MAC-KO). The MAC-KO mice were generated by crossbreeding the AhR ﬂoxed mice with the LysM-Cre transgenic mice. Compared with the whole-body AhR–/– mice, only HSC-KO mice, but not HEP-KO or MAC-KO mice, showed spontaneous liver ﬁbrosis, as shown by Sirius Red staining and immunostaining of a-SMA (Supplementary Figure 7).

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Figure 4. KO of AhR promotes HSC activation in vitro. (A, B) Primary mouse HSCs were isolated from WT and AhR–/– (KO) mice and culture-activated for the indicated duration. (A) Cell morphology was analyzed by light-ﬁeld microscopy, and (B) gene expression was determined by real-time PCR (n ¼ 4). (C) Primary mouse HSCs were isolated and culture-activated for 24 or 48 hours. The protein expression was determined by Western blotting. (D) Primary mouse HSCs were isolated and cultureactivated for 48 hours. The expression levels of a-SMA and Ki67 were detected by immunoﬂuorescence. (E) Primary mouse HSCs were isolated and ﬂuorescence-activated cell sorting–puriﬁed and directly subject to RNA-seq analysis. Differentially expressed genes are shown by a volcano plot with several ﬁbrogenic marker genes annotated. The red dots and blue dots indicate genes that are signiﬁcantly up-regulated (663 genes) and down-regulated (66 genes), respectively. The cutoff was set as 0.05 for the P value and 1.5 for log2(FC), where FC indicates the fold change of KO vs WT. (F) Gene set enrichment analysis of the RNA-seq results. (G–I) Primary mouse HSCs isolated from KO mice were infected with adenovirus overexpressing AhR and treated with or without TCDD (50 nmol/L). The gene expression was determined by (G) Western blotting, (H) real-time PCR (n ¼ 4), or (I) immunoﬂuorescence. Scale bar ¼ 100 mm. All data are presented as mean ± standard error of the mean. *P < .05, **P < .01. Ctrl, control; WT, wild type.

Aryl Hydrocarbon Receptor Attenuates Transforming Growth Factor b–Stimulated Fibrogenesis by Inhibiting Smad3-Mediated Transcriptional Activation of Fibrogenic Genes The TGFb-Smad3 signaling stimulates liver ﬁbrogenesis and is central to HSC activation. Indeed, we showed that the recruitment of phosphorylated Smad3 onto the Acta2

and Col1a1 gene promoters was increased in CCl4-treated liver (Supplementary Figure 8A, left panel). Moreover, the CCl4-responsive recruitment of phosphorylated Smad3 onto the Acta2 gene promoter was inhibited by the cotreatment of ITE (Supplementary Figure 8A, right panel). Having shown the inhibitory effect of AhR on the spontaneous activation of HSCs, we went on to determine

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Figure 5. KO of AhR in HSCs sensitizes mice to liver ﬁbrosis and abolishes the antiﬁbrotic activity of ITE. (A–C) 9- to 10-weekold male AhRﬂox/ﬂox (ﬂox) and HSC-KO mice were treated with CCl4 (1 mL/g body weight) twice a week for 4 weeks (n ¼ 5 per group). (A) The liver histology was analyzed by Sirius Red staining and immunostaining of a-SMA and desmin (original magniﬁcation, 10, with the quantiﬁcations of staining shown on the bottom). (B) The a-SMA protein level was determined by Western blotting, and (C) the mRNA expression levels of Col1a1 and Col1a2 were determined by real-time PCR. (D–F) The 8- to 10-week-old male HSC-KO mice were treated with vehicle or ITE (10 mg/kg) together with CCl4 (0.5 mL/g body weight) 3 times a week for 4 weeks (n ¼ 3 per group). (D) Liver tissues were analyzed for histology by Masson’s trichrome (MTS) and Sirius Red staining (original magniﬁcation, 5), (E) the expression of ﬁbrogenic gene by real-time PCR (top) or Western blotting (bottom), and (F) the serum ALT and AST levels. All data are presented as mean ± standard error of the mean. *P < .05, **P < .01; N.S., not statistically signiﬁcant; Veh, vehicle.

whether AhR can also inhibit TGFb-stimulated HSC activation. Treatment of primary mouse HSCs with TGFb induced the expression of ﬁbrogenic genes as expected, but the inductions were largely attenuated by the cotreatment of TCDD (Figure 6A). The attenuation of TGFbinduced expression of a-SMA by TCDD was conﬁrmed by immunoﬂuorescence (Figure 6B). Next, we overexpressed AhR in the human stellate cell line LX2, in which the endogenous AhR had little expression and little response to AhR ligands, whereas overexpression of AhR restored the responsiveness to AhR ligands (Supplementary Figure 8B). Overexpression of AhR in LX2 cells signiﬁcantly attenuated the response to the TGFb stimulation, and ligand treatment modestly enhanced the attenuation, as shown by gene expression analysis by real-time PCR (Figure 6C) or Western blotting (Figure 6D).

To elucidate the molecular mechanism underlying the inhibitory effect of AhR on TGFb-Smad stimulated ﬁbrosis, we initially considered the transcriptional activity of AhR as a potential mechanism. However, the reported ChIP-sequencing studies showed that AhR does not bind to the consensus TGFbresponsive element.7,23 Our own ChIP analysis conﬁrmed the lack of AhR binding to the TGFb-responsive elements in the promoter regions of COL1A1 and COL1A2 genes (Supplementary Figure 8C). We also examined the expression of negative regulators of the TGFb signaling pathway. There were no signiﬁcant changes in the expression levels of genes that are known to be involved in protein degradation (Smurf1 and Smurf2), receptor antagonism (Bambi), nuclear transport (Xpo1, Xpo4, and Ranbp3), or transcriptional repressors (Tgif, Ski, and Skil) (Supplementary Figure 8D). We then determined whether AhR has a direct interaction with the Smad proteins.

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Figure 6. AhR attenuates TGFb-stimulated ﬁbrogenesis by inhibiting Smad3-mediated transcriptional activation of ﬁbrogenic genes. (A) Primary mouse HSCs were treated with TGFb1 (2 ng/mL) in the absence or presence of TCDD (50 nmol/L) for 24 hours. Gene expression was determined by real-time PCR (n ¼ 3). (B) Primary mouse HSCs were treated with TGFb1 (2 ng/mL) in the absence or presence of TCDD (50 nmol/L) for 48 hours. The expression of a-SMA was determined by immunoﬂuorescence. Scale bars ¼ 200 mm. (C, D) LX2 cells were infected with adenovirus overexpressing AhR and treated with TCDD (50 nmol/L) and/or TGFb1 (5 ng/mL) for 24 hours. The gene expressions at the (C) mRNA and (D) protein levels were measured by real-time PCR (n ¼ 4) and Western blotting, respectively. The relative values of a-SMA protein expression in the Western blotting are labeled. (E) LX2 cells stably expressing human AhR were transfected with HA-Smad2, HA-Smad3, or HA-Smad4 expression vector. Protein interaction was determined by coimmunoprecipitation with an HA antibody followed by Western blotting. (F) LX2 cells infected with adenovirus overexpressing AhR were pretreated with or without TCDD (50 nmol/L) for 24 hours and then TGFb1 (5 ng/mL) with or without TCDD (50 nmol/L) for 4 hours. The recruitment of phosphorylated Smad3 onto the promoter regions of the COL1A1, COL1A2, and PAI1 genes were determined by ChIP coupled with real-time PCR on the recovered DNA (n ¼ 4). All data are presented as mean ± standard error of the mean. *P < .05; **P < .01.

Coimmunoprecipitation analysis showed that AhR can speciﬁcally interact with Smad3, but not Smad2 or Smad4 (Figure 6E). The interaction did not seem to inhibit the nuclear entry of Smad2/3 (Supplementary Figure 8E), but at the functional level, overexpression of AhR attenuated TGFb responsive recruitment of phosphorylated Smad3 onto the promoter regions of ﬁbrogenic genes (Figure 6F).

Aryl Hydrocarbon Receptor Disrupts the Interaction Between Smad3 and b-Catenin We reason that the AhR-Smad3 interaction may interfere with the accessibility of Smad3 to the Smad3-interacting

transcriptional factors that are involved in ECM gene expression. Among the tested proteins, b-catenin, myocyte enhancer factor 2C (MEF2C), serum response factor (SRF), and speciﬁcity protein 1 (SP1), only b-catenin exhibited substantial interaction with Smad3 in the LX2 cells (Figure 7A). The interaction between b-catenin and Smad3 was decreased by the overexpression of AhR and was further reduced by the TCDD treatment, which was correlated with an increased binding of Smad3 to AhR (Figure 7B). In contrast, the Smad3-Smad4 interaction was not affected by AhR (Figure 7B). We also showed that AhR decreased b-catenin’s binding to Smad3 in a dosedependent manner (Figure 7C), further suggesting that

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AhR competes with b-catenin for the binding of Smad3. Both AhR and b-catenin interact with Smad3 via the Mad homolog domain 2 (MH2) (Figure 7D). These results collectively suggested that AhR may attenuate TGFb-stimulated ﬁbrosis by sequestering Smad3 from b-catenin. b-Catenin is the effector of the canonical Wnt signaling pathway that is also responsive to TGFb stimulation.24 It has been reported that the coupling of the Smad pathway and b-catenin is required for optimal induction of ECM gene expression and ﬁbrogenesis.4 However, how b-catenin potentiates TGFb-Smad mediated ﬁbrogenesis and whether b-catenin is the target of the antiﬁbrogenic activity of AhR are unknown. We examined the kinetics of Smad2/3 phosphorylation. In the absence of AhR, overexpression of b-catenin was efﬁcient to prolong TGFb-stimulated Smad2/ 3 phosphorylation, but this effect was largely diminished by the overexpression of AhR (Figure 7E). At the functional level, the ﬁbrogenic gene expression induced by b-catenin was down-regulated in primary HSCs regardless of the TGFb treatment (Figure 7F).

Mechanism by Which Aryl Hydrocarbon Receptor Facilitates the Degradation of Phosphorylated Smad2/3 In our effort to determine the mechanism by which AhR facilitates the degradation of phosphorylated Smad2/3, we found that treatment of LXR2 cells with MLN4924, a panCullin inhibitor, abolished the inhibitory effect of AhR on the expression of ﬁbrogenic marker genes. as shown by Western blotting (Supplementary Figure 9A) and real-time PCR (Supplementary Figure 9B). This effect of MLN4924 was also conﬁrmed in primary human HSCs (Supplementary Figures 9C and D). These results suggested that Cullin E3 ligases may play a role in AhR-promoted degradation of phosphorylated Smad2/3. Indeed, treatment of MLN4924 or the proteasome inhibitor MG132 extended the levels of phosphorylated Smad2/3 in the presence of AhR overexpression (Supplementary Figure 9E). Because AhR has been reported as a component of the CUL4BAhR E3 ubiquitin ligase that targets the intestinal b-catenin for degradation,12 we examined whether the E3 ligase activity of AhR is required for its inhibitory effect on HSC activation. The E3 ligase-deﬁcient AhR mutant with the deletion of the acidic domain12 remained effective in decreasing the expression of ﬁbrogenic genes at the protein (Supplementary Figure 9F) and mRNA (Supplementary Figure 9G) levels, indicating that the E3 ligase activity of CUL4BAhR is dispensable.

Discussion In this study, we uncovered a novel function of AhR in HSCs and liver ﬁbrosis. The antiﬁbrogenic role of AhR in HSC activation and liver ﬁbrosis was a surprise, because AhR has been better known for its proﬁbrogenic activity. Treatment of mice with the xenobiotic AhR activator TCDD sensitizes mice to ﬁbrosis.13–15 TCDD and other polycyclic aromatic hydrocarbons are known to be toxic to the hepatocytes and to induce neutrophil inﬁltration, although the

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mechanism for the hepatotoxicity remains to be clearly deﬁned. For example, Cyp1a1–/– mice showed paradoxical protection from the toxicity of BaP25 and TCDD,26 whereas the expression of hepatic carboxylesterase 3 (CES3) was suggested to be important for TCDD-induced liver toxicity through the production of azelaic acid monoesters.27 Nevertheless, we speculate that the ﬁbrogenic activity of TCDD might be secondary to its hepatotoxicity and associated inﬂammation, a notion that has been supported by previous reports.28,29 The hepatotoxicity and inﬂammation associated with TCDD exposure may have been exacerbated when animals were challenged with the MCD or high-fat Q11 diet.13–15 In contrast, the nontoxic endogenous AhR ligand ITE protected mice from liver ﬁbrosis in an AhR-dependent manner. TCDD and ITE share an electron-rich fused aromatic ring system with a lipophilic property, suggesting that both compounds can efﬁciently penetrate the cell membrane. However, the metabolic stabilities of TCDD and ITE are different, which may have contributed to their differential effect on HSC activation and liver ﬁbrosis. TCDD is metabolically stable, with an estimated half-life of more than 7 years in human serum,30 which renders the persistent activation of AhR. The metabolic turnover rate of endogenous AhR ligands, on the other hand, is high because of the rapid clearance by the CYP1A and 1B enzymes, especially in the liver.31 Indeed, we found that the AhR-activating effect of ITE, but not TCDD, was inhibited by the cotransfection of CYP1B1 in a reporter gene assay (data not shown), suggesting that ITE can be metabolized by CYP1B1, whereas TCDD is resistant. Future studies with the use of Cyp1a- and Cyp1b-KO mice are necessary to clarify the relevance of their regulation in the ﬁbrogenic phenotype of AhR–/– mice. Nevertheless, it is tempting to speculate that the sustained activation of AhR by xenobiotic AhR ligands such as TCDD and transient activation of AhR by endogenous AhR ligands such as ITE may have accounted for the differential effects between the xenobiotic and endobiotic AhR ligands. Another interesting ﬁnding is the cell type–speciﬁc role of AhR in liver ﬁbrosis, which may have explained the counterintuitive observation that both AhR KO and xenobiotic activation of AhR sensitized mice to liver ﬁbrosis. The liver is an organ of multiple cell types. Most of our understanding of the hepatic function of AhR has been centered on the hepatocytes. In this study, we have presented compelling evidence that AhR is highly expressed and functional in HSCs. The expression of AhR in HSCs decreased rapidly with the onset of HSC activation, and HSCs isolated from AhR–/– mice exhibited increased spontaneous activation. More importantly, we showed that HSC-speciﬁc KO of AhR was sufﬁcient to cause spontaneous liver ﬁbrosis and sensitized mice to injury-induced liver ﬁbrosis. In contrast, mice bearing hepatocyte- or Kupffer cell–speciﬁc AhR KO did not show spontaneous liver ﬁbrosis. The mechanism by which AhR inhibits HSC activation was also interesting. We showed that AhR impairs bcatenin–dependent stabilization of phosphorylated Smad2/ 3. Treatment with the ubiquitination inhibitor MLN4924 or proteasome inhibitor MG132 attenuated the inhibitory effect of AhR on Smad2/3 phosphorylation and the

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Figure 7. AhR disrupts the interaction between Smad3 and b-catenin. (A) LX2 cells were stimulated with TGFb1 (5 ng/mL) for 3 hours. Protein interaction was determined by coimmunoprecipitation with an anti-Smad3 antibody followed by Western blotting. Arrows indicate the speciﬁc protein bands. (B) LX2 cells were infected with adenovirus overexpressing AhR and treated with or without TCDD (50 nmol/L). Protein interaction was determined by coimmunoprecipitation with an anti-Smad3 antibody. (C) LX2 cells were infected with increasing doses of Ad-AhR. Protein interaction was determined by coimmunoprecipitation with an anti-Smad3 antibody. Arrows indicate the speciﬁc protein bands. SE and LE indicate short exposure and long exposure, respectively. (D) LX2 cells stably transfected with AhR were transiently transfected with HA-Smad3 plasmids (FL, full-length; MH1-L, MH1 domain plus linker; L-MH2, linker plus MH2 domain). Protein interaction was determined by coimmunoprecipitation with an anti-HA antibody. (E) LX2 cells were infected with Ad-AhR and/or Ad-b-catenin and then stimulated with TGFb1 (5 ng/mL) for 1 hour before harvesting at indicated time points. The levels of phosphorylated Smad2/3 were detected by Western blotting with the relative quantitative values labeled. (F) Primary human HSCs were infected with Ad-AhR with or without Ad-b-catenin and treated with TCDD (50 nmol/L) and TGFb1 (5 ng/mL) as indicated. Gene expression was determined by real-time PCR. All data are presented as mean ± standard error of the mean. *P < .05; **P < .01. IP, immunoprecipitation; Veh, vehicle.

expression of ﬁbrogenic genes. AhR has been reported to have E3 ligase activity.11,12 Interestingly, the E3-dead mutant AhR remained efﬁcient in inhibiting HSC activation. Instead, we found that AhR attenuated TGFb-stimulated ﬁbrogenesis by interacting with Smad3, which may have sequestered Smad3 from interacting with b-catenin. TGFb is known to be proﬁbrogenic in HSCs, and Smad3 is a key effector of TGFb signaling. The canonical Wnt signaling and its downstream effector b-catenin have been suggested to play a role in ﬁbrotic responses.4 Indeed, our RNA-seq

results showed an up-regulation of both the Wnt and TGFb pathways in AhR–/– HSCs. Our results suggest that bcatenin stabilizes the transcriptionally active phosphorylated Smad2/3. It has been reported that b-catenin interacts with the C-terminal MH2 domain of Smad3.32 This MH2 domain is also the binding site for E3 ligases.33 Therefore, it is reasonable to speculate that the stabilization of phosphorylated Smad2/3 protein by b-catenin could have been achieved through the prevention of ubiquitination. The E3 ligase responsible for AhR-dependent destabilization of

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phosphorylated Smad2/3 remains to be deﬁned. The AhRSmad3 interaction and the sequestration of Smad3 from bcatenin suggest a plausible mechanism by which AhR inhibits HSC activation and liver ﬁbrosis. Although our RNAseq results suggested that AhR may not directly inhibit HSC activation as a genomic repressor of the ﬁbrogenic genes, we cannot exclude the transcriptional role of AhR as a mechanism to prevent HSC activation. It remains possible that certain AhR-regulated genes play a role in mediating the antiﬁbrogenic effect of AhR. Among limitations, much of our mechanistic characterization was done through in vitro studies. Previous reports suggested that the TGFb-Smad pathway was activated in AhR–/– mice,34,35 which was consistent with our results. However, future studies are necessary to determine whether ﬁbrosis caused by AhR KO requires the TGFb-Smad signaling in vivo and in HSCs by using Ctnnb1 and Smad2/3/ 4 ﬂoxed mice. In addition, it is unclear whether AhRregulated Smad3–b-catenin interaction is also applicable in other biological events, such as embryonic stem cell differentiation (Supplementary Table 7). In summary, we have established AhR as an antiﬁbrogenic transcriptional factor in HSCs. Our results strongly suggest that development of nontoxic AHR agonists and strategies that preferentially activate AhR in HSCs may represent novel approaches to prevent and treat liver ﬁbrosis.

Supplementary Material Note: To access the supplementary material accompanying this article, visit the online version of Gastroenterology at www.gastrojournal.org, and at https://doi.org/10.1053/ j.gastro.2019.05.066.

References 1. Bataller R, Brenner DA. Liver ﬁbrosis. J Clin Invest 2005; 115:209–218. 2. Mederacke I, Hsu CC, Troeger JS, et al. Fate tracing reveals hepatic stellate cells as dominant contributors to liver ﬁbrosis independent of its aetiology. Nat Commun 2013;4:2823. 3. Tsuchida T, Friedman SL. Mechanisms of hepatic stellate cell activation. Nat Rev Gastroenterol Hepatol 2017; 14:397–411. 4. Akhmetshina A, Palumbo K, Dees C, et al. Activation of canonical Wnt signalling is required for TGF-b-mediated ﬁbrosis. Nat Commun 2012;3:735. 5. Beischlag TV, Morales JL, Hollingshead BD, et al. The aryl hydrocarbon receptor complex and the control of gene expression. Crit Rev Eukaryot Gene Expr 2008; 18:207–250. 6. Jackson D, Joshi A, Elferink C. Ah receptor pathway intricacies; signaling through diverse protein partners and DNA-motifs. Toxicol Res 2015;4:1143–1158. 7. Lo R, Matthews J. High-resolution genome-wide mapping of AHR and ARNT binding sites by ChIP-Seq. Toxicol Sci 2012;130:349–361.

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8. Fernandez-Salguero P, Pineau T, Hilbert DM, et al. Immune system impairment and hepatic ﬁbrosis in mice lacking the dioxin-binding Ah receptor. Science 1995; 268(5211):722–726. 9. Schmidt JV, Su G, Reddy JK, et al. Characterization of a murine Ahr null allele: involvement of the Ah receptor in hepatic growth and development. Proc Natl Acad Sci U S A 1996;93:6731–6736. 10. Nebert DW. Aryl hydrocarbon receptor (AHR): “pioneer member” of the basic-helix/loop/helix per-Arnt-sim (bHLH/PAS) family of “sensors” of foreign and endogenous signals. Prog Lipid Res 2017;67:38–57. 11. Ohtake F, Baba A, Takada I, et al. Dioxin receptor is a ligand-dependent E3 ubiquitin ligase. Nature 2007; 446(7135):562–566. 12. Kawajiri K, Kobayashi Y, Ohtake F, et al. Aryl hydrocarbon receptor suppresses intestinal carcinogenesis in ApcMin/þ mice with natural ligands. Proc Natl Acad Sci U S A 2009;106:13481–13486. 13. He J, Hu B, Shi X, et al. Activation of the aryl hydrocarbon receptor sensitizes mice to nonalcoholic steatohepatitis by deactivating mitochondrial sirtuin deacetylase Sirt3. Mol Cell Biol 2013;33:2047–2055. 14. Pierre S, Chevallier A, Teixeira-Clerc F, et al. Aryl hydrocarbon receptor–dependent induction of liver ﬁbrosis by dioxin. Toxicol Sci 2014;137:114–124. 15. Duval C, Teixeira-Clerc F, Leblanc AF, et al. Chronic exposure to low doses of dioxin promotes liver ﬁbrosis development in the C57BL/6J diet-induced obesity mouse model. Environ Health Perspect 2017; 125:428–436. 16. Mederacke I, Dapito DH, Affò S, et al. High-yield and high-purity isolation of hepatic stellate cells from normal and ﬁbrotic mouse livers. Nat Protoc 2015;10:305–315. 17. Subramanian A, Tamayo P, Mootha VK, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression proﬁles. Proc Natl Acad Sci U S A 2005;102:15545–15550. 18. Gao J, Yan J, Xu M, et al. CAR suppresses hepatic gluconeogenesis by facilitating the ubiquitination and degradation of PGC1a. Mol Endocrinol 2015; 29:1558–1570. 19. Song J, Clagett-Dame M, Peterson RE, et al. A ligand for the aryl hydrocarbon receptor isolated from lung. Proc Natl Acad Sci U S A 2002;99:14694–14699. 20. Walisser JA, Glover E, Pande K, et al. Aryl hydrocarbon receptor-dependent liver development and hepatotoxicity are mediated by different cell types. Proc Natl Acad Sci U S A 2005;102:17858–17863. 21. Azimifar SB, Nagaraj N, Cox J, et al. Cell-type-resolved quantitative proteomics of murine liver. Cell Metab 2014; 20:1076–1087. 22. Dere E, Forgacs AL, Zacharewski TR, et al. Genomewide computational analysis of dioxin response element location and distribution in the human, mouse, and rat genomes. Chem Res Toxicol 2011;24:494–504. 23. Dere E, Lo R, Celius T, et al. Integration of genome-wide computation DRE search, AhR ChIP-chip and gene expression analyses of TCDD-elicited responses in the mouse liver. BMC Genomics 2011;12:365.

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24. Jian H, Shen X, Liu I, et al. Smad3-dependent nuclear translocation of b-catenin is required for TGF-b1-induced proliferation of bone marrow-derived adult human mesenchymal stem cells. Genes Dev 2006;20:666–674. 25. Uno S, Dalton TP, Shertzer HG, et al. Benzo[a]pyreneinduced toxicity: paradoxical protection in Cyp1a1(-/-) knockout mice having increased hepatic BaP-DNA adduct levels. Biochem Biophys Res Commun 2001; 289:1049–1056. 26. Uno S, Dalton TP, Sinclair PR, et al. Cyp1a1(-/-) male mice: protection against high-dose TCDD-induced lethality and wasting syndrome, and resistance to intrahepatocyte lipid accumulation and uroporphyria. Toxicol Appl Pharmacol 2004;196:410–421. 27. Matsubara T, Tanaka N, Krausz KW, et al. Metabolomics identiﬁes an inﬂammatory cascade involved in dioxinand diet-induced steatohepatitis. Cell Metab 2012; 16:634–644. 28. Ozeki J, Uno S, Ogura M, et al. Aryl hydrocarbon receptor ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin enhances liver damage in bile duct-ligated mice. Toxicology 2011;280:10–17. 29. Lamb CL, Cholico GN, Pu X, et al. 2,3,7,8Tetrachlorodibenzo-p-dioxin (TCDD) increases necroinﬂammation and hepatic stellate cell activation but does not exacerbate experimental liver ﬁbrosis in mice. Toxicol Appl Pharmacol 2016;311:42–51. 30. Pirkle JL, Wolfe WH, Patterson DG, et al. Estimates of the half-life of 2,3,7,8-tetrachlorodibenzo-p-dioxin in Vietnam veterans of Operation Ranch Hand. J Toxicol Environ Health A 1989;27:165–171. 31. Wincent E, Bengtsson J, Bardbori AM, et al. Inhibition of cytochrome P4501-dependent clearance of the endogenous agonist FICZ as a mechanism for activation of the aryl hydrocarbon receptor. Proc Natl Acad Sci U S A 2012;109:4479–4484.

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32. Zhang M, Wang M, Tan X, et al. Smad3 prevents bcatenin degradation and facilitates b-catenin nuclear translocation in chondrocytes. J Biol Chem 2010; 285:8703–8710. 33. Gao S, Alarcón C, Sapkota G, et al. Ubiquitin ligase Nedd4L targets activated Smad2/3 to limit TGF-b signaling. Mol Cell 2009;36:457–468. 34. Corchero J, Martin-Partido G, Dallas SL, et al. Liver portal ﬁbrosis in dioxin receptor-null mice that overexpress the latent transforming growth factor-b-binding protein-1. Int J Exp Pathol 2004; 85:295–302. 35. Andreola F, Calvisi DF, Elizondo G, et al. Reversal of liver ﬁbrosis in aryl hydrocarbon receptor null mice by dietary vitamin A depletion. Hepatology 2004; 39:157–166.

1621 1622 1623 1624 1625 1626 1627 1628 1629 1630 1631 1632 1633 1634 1635 1636 1637 Received May 21, 2018. Accepted May 28, 2019. 1638 1639 Reprint requests 1640 Address requests for reprints to: Wen Xie, MD, PhD, 306 Salk Pavilion, Q5 Q6 University of Pittsburgh, Pittsburgh, PA 15261. e-mail: [email protected]. 1641 Q7 1642 Acknowledgments We thank Dr Jodi A. Flaws (University of Illinois Urbana-Champaign, Urbana, IL) 1643 for the Ad-AhR and control Ad-GFP adenovirus and Dr Tong-Chuan He 1644 (University of Chicago, Chicago, IL) for the Ad-b-catenin (S33Y). Author contributions: Wen Xie conceived and mentored this study. Jiong Yan Q12 1645 and Hung-Chun Tung designed and performed experiments, acquired and 1646 analyzed data, and wrote the draft of the manuscript. Hung-Chun Tung, 1647 Sihan Li, Yongdon Niu, Peipei Lu, Yuhan Bi, and Meishu Xu performed experiments. Yongdon Niu, Wojciech G. Garbacz, Peipei Lu, Jinhan He, 1648 Meishu Xu, Songrong Ren, Satdarshan P. Monga, Robert F. Schwabe, and 1649 Da Yang gave technical support and conceptual advice. Wen Xie, Jiong Yan, and Hung-Chun Tung wrote the manuscript. 1650 1651 Conﬂicts of interest Q8 The authors disclose no conﬂicts. 1652 1653 Funding This work was supported in part by National Institutes of Health grants Q9 Q18 1654 DK083952 and ES023438 to Wen Xie. 1655 1656 1657 1658 1659 1660 1661 1662 1663 1664 1665 1666 1667 1668 1669 1670 1671 1672 1673 1674 1675 1676 1677 1678 1679 1680

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Supplementary Methods Histology Liver tissues were ﬁxed overnight in 10% formalin phosphate followed by dehydration in alcohol and xylene. Dehydrated tissues were embedded in parafﬁn blocks, and 5-mm parafﬁn sections were prepared using microtome, dewaxed, and rehydrated before histologic staining. For H&E staining, hydrated tissue sections were stained in hematoxylin solution for 2 minutes, washed with tap water for 10 minutes, and differentiated in 1% acetic acid solution for 1 minute, followed by 10 quick dips in eosin solution. For Sirius Red staining, hydrated tissue sections were stained in 0.1% PicroSirius Red solution for 1 hour, followed by 2 washes in 1% acetic acid solution. For Masson’s Trichrome staining, hydrated tissue sections were reﬁxed in Bouin’s solution for 1 hour at 56 C, washed with running tap water for 10 minutes, stained in Weigert’s iron hematoxylin solution for 10 minutes, washed with running tap water for 10 minutes, stained in Biebrich scarlet-acid fuchsin solution (Sigma-Aldrich, St. Louis, MO) for 15 minutes, washed brieﬂy with distilled water, differentiated in phosphomolybdic-phosphotungstic acid solution (SigmaAldrich) for 15 minutes, stained in aniline blue solution (Sigma-Aldrich) for 10 minutes, and differentiated in 1% acetic acid solution for 5 minutes. After the major staining procedures, all the sections were dehydrated in alcohol and xylene and mounted in Permount mounting medium (Thermo Fisher Scientiﬁc, Pittsburgh, PA) for microscopic analysis. Immunohistochemistry was performed using the VECTASTAIN ABC HRP Kit (Vector Laboratories, Burlingame, CA). Parafﬁn-embedded liver sections were dewaxed and rehydrated before staining. Antigen retrieval was performed by incubating the sections in 10 mmol/L citric acid solution (pH 6.0) at 95 C for 15 minutes. Potential endogenous peroxidase activity was reduced using 0.3% H2O2 solution for 30 minutes. The specimens were then blocked in the serum from the species where the secondary antibody was produced for 1 hour before incubation with the primary antibodies (anti–aSMA and anti-desmin) overnight at 4 C. The next day, the tissue samples were incubated with the secondary antibody for 30 minutes followed by ABC reagents for 30 minutes. Positive staining was visualized using DAB Peroxidase (HRP) Substrate Kit (Vector Laboratories). Nuclei were stained with hematoxylin QS (Vector Laboratories). After the major staining procedures, all the sections were dehydrated in alcohol and xylene and mounted in Permount mounting medium (Thermo Fisher Scientiﬁc) for microscopic analysis. Quantiﬁcation of Sirius Red and a-SMA staining was performed by threshold analysis of randomly selected ﬁelds of view per slide (magniﬁcation, 10) and presented as a percentage of the positive staining vs the total area using Image J software (National Institutes of Health, Bethesda, MD).

Primary Hepatic Stellate Cell Isolation, Culture, and Fluorescence-Activated Cell Sorting Primary mouse HSCs were isolated according to the previously reported protocol, with minor modiﬁcations.1

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Brieﬂy, in situ 2-step perfusion was performed as previously described, followed by a single-step Nycodenz gradient separation (NycoPrep; Axis-Shield, Oslo, Norway).2 The purity of the isolated HSCs was >90%, as determined by the vitamin A auto- ﬂuorescence from the stellate cells. For RNA-seq analysis, HSCs were isolated and pooled from 3 8-week-old wild-type male mice and 8 8-week-old AhR–/– male mice and further puriﬁed by vitamin A–based ﬂuorescence-activated cell sorting (FACS) by using a FACSAria Instrument from Becton Dickinson (Franklin Lakes, NJ). 1.2 106 wild-type HSCs and 7.8 105 AhR–/– HSCs sorted by FACS were immediately lysed for RNA library preparation to achieve ultrapure HSC isolates without plating artifacts. The FACS procedures were not applied to the cultured HSCs because of the negative impact on HSC survival, attachment, and activation. For in vivo HSC activation,3 8- to 9-week-old male wild-type mice were treated with 4 intraperitoneal injections of CCl4 (0.5 mL/g body weight, 1:3 diluted in corn oil, every 3 days). HSCs were isolated 48 hours after the last CCl4 injection and subject to immediate RNA extraction and real-time PCR analysis. The primary human HSCs were isolated from the nonparenchymal fraction of in vitro digested human livers (Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA) following the same protocol as for primary mouse HSC isolation. The primary HSCs were cultured in Dulbecco’s modiﬁed Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and ampicillin/ streptomycin. For the adipogenic reversion of activated HSCs, cells were treated with MDI medium that contains 3- Q16 isobutyl-1-methylxanthine (0.5 mmol/L), dexamethasone (1 mmol/L), insulin (10 mg/mL), and rosiglitazone (10 mmol/L) for 72 hours. The immortalized human stellate cell line LX2 was obtained from Dr Scott Friedman (Mount Sinai School of Medicine, New York, NY). The LX2, 293A, and 293T cells were maintained in DMEM containing 10% FBS and penicillin/streptomycin.

Adenovirus, Lentivirus, Plasmids, and Cell Transfection Adenovirus-overexpressing mouse AhR and control green ﬂuorescent protein virus were a gift from Dr Jodi A. Flaws (University of Illinois Urbana-Champaign, Urbana, IL).4 Adenovirus-overexpressing b-catenin (S33Y) and control green ﬂuorescent protein virus were a gift from Dr Tong-Chuan He (The University of Chicago Medical Center, Chicago, IL).5 All the adenoviruses were ampliﬁed in 293A cells and puriﬁed using cesium chloride gradient ultracentrifugation based on the previously reported procedures, with some minor modiﬁcations.6 Lentiviral vector pCDHpuro (System Biosciences, Palo Alto, CA) carrying human full-length AhR (wild type) and AhR with acidic domain deletion (DAcidic, amino acids 500–600 deleted) were cloned using standard molecular cloning techniques. The lentiviral packaging vectors psPAX2 (no. 12260) and pMD2.G (no. 12259) were obtained from Addgene (Cambridge, MA). Lentivirus production was performed in 293T cells by transfection with the lentiviral vectors (pCDH-

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AhR_WT or pCDH-AhR_DAcidic), together with the packaging vectors psPAX2 and pMD2.G. Lentiviruses were collected from the culture supernatants 48 hours after transfection and ﬁltered through a 0.45-mm nitrocellulose membrane. To establish the stable cell lines, LX2 cells were infected with lentiviruses plus polybrene (8 mg/mL) and selected with puromycin (0.5 mg/mL). The HA-Smad2, HASmad3 (FL, MH1-L, L-MH2), and HA-Smad4 were cloned by using standard molecular cloning techniques. Cell transfection was performed using TransIT-LT1 or TransIT-X2 (Mirus Bio, Madison, WI).

Immunoﬂuorescence Primary HSCs were grown on slide chambers and treated as desired. Cells were ﬁxed in ice-cold methanol for 10 minutes, followed by blocking with phosphate-buffered saline (PBS)/0.25% Triton X-100 (PBS-T) containing 5% donkey serum for 30 minutes. Slides were incubated in the PBS-T with primary antibody against a-SMA or Ki67 overnight at 4 C followed by incubation with a ﬂuorochrome-conjugated secondary antibody for 1 hour at room temperature in the dark. Slides were mounted in 40 ,6diamidino-2-phenylindole–containing medium for microscopic analysis.

Bromodeoxyuridine and Terminal Deoxynucleotidyl Transferase–Mediated Deoxyuridine Trisphosphate Nick-End Labeling Staining Mouse primary HSC were seeded in 96-well plates and incubated in DMEM containing 10% FBS overnight. Cells were then treated with various concentrations of TCDD (1 nmol/L, 10 nmol/L, 20 nmol/L, 50 nmol/L) or ITE (0.1 mmol/L, 1 mmol/L, 5 mmol/L, 10 mmol/L) for 48 hours. For bromodeoxyuridine (BrdU) staining, cells were incubated for an additional 24 hours in medium containing 10 mmol/L BrdU, followed by ﬁxation in ice-cold 4% paraformaldehyde in PBS for 15 minutes at room temperature. Next, the BrdUlabeled cells were incubated in 2N HCl for 20 minutes at 37 C to disrupt the DNA structure in the cells. After denaturation, 0.01% trypsin in PBS was added for neutralization. Nonspeciﬁc binding was blocked with rabbit serum in PBS for 20 minutes at room temperature, and the cells were then incubated with rat anti-BrdU antibody from Accurate (Westbury, NY) for 1 hour at 37 C, followed by incubation with goat anti-rat ﬂuorescein-labeled secondary antibody from Abcam (Cambridge, MA) for 1 hour at 37 C in the dark. For terminal deoxynucleotidyl transferase–mediated deoxyuridine trisphosphate nick-end labeling (TUNEL) staining, after paraformaldehyde ﬁxation, the cells were incubated with permeabilization solution (0.1% Triton X-100 in PBS) for 2 minutes on ice, followed by incubation with TUNEL reaction mixture from Roche (Pleasanton, CA) for 1 hour in 37 C in the dark. Cells pretreated with DNase I recombinant were used as a positive control. Hoechst from Invitrogen

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(Waltham, MA) was used to stain the nucleus. The cells were washed twice with PBS before microscopic analysis.

Immunoprecipitation and Western Blot For immunoprecipitation, cells were lysed in nondenaturing immunoprecipitation buffer (150 mmol/L NaCl, 50 mmol/L Tris-HCl at pH 7.5, 1% NP-40) supplemented with a protease-inhibitor cocktail (Sigma-Aldrich). The supernatants after sonication and centrifugation (14,000g, 15 minutes, 4 C) were incubated with the primary antibody against HA or Smad3 overnight at 4 C, followed by incubation with Protein A magnetic beads (New England Biolabs, Ipswich, MA) for 1 hour at 4 C. The beads were washed 6 times with ice-cold immunoprecipitation buffer and eluted with protein loading buffer. The eluents were analyzed by Western blot. For Western blot analysis, liver tissues were homogenized in ice-cold radioimmunoprecipitation assay buffer containing a protease inhibitor cocktail. Protein concentration was measured with the Pierce BCA Protein Assay Kit (Thermo Fisher Scientiﬁc). Cell lysates were prepared by adding protein loading buffer directly to the cells. Protein samples were heated for 10 minutes and loaded on the sodium dodecyl sulfate–polyacrylamide gel for electrophoresis. The proteins were transferred on the nitrocellulose membrane and blocked with 5% nonfat milk in 1 Tris-buffered saline solution with Tween-20. Primary antibodies were incubated overnight at 4 C, followed by incubation of horseradish peroxidase–conjugated secondary antibody on the next day before ﬁlm development using horseradish peroxidase–based Pierce ECL Western Blot Substrate or SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientiﬁc). The densitometric analysis of Western blot was performed using Image Lab software (Bio-Rad Laboratories, Hercules, CA).

Chromatin Immunoprecipitation Chromatin immunoprecipitation (ChIP) analysis was performed according to the previously reported protocol, with minor modiﬁcations.7 Brieﬂy, LX2 cells or liver tissues were ﬁxed in the formalin for 15 minutes, followed by quenching with glycine for 5 minutes at room temperature. Cells were scraped/collected and washed with ice-cold PBS twice. Cell pellets were resuspended in the ChIP buffer (150 mmol/L NaCl, 50 mmol/L Tris-HCl at pH 7.5, 5 mmol/L EDTA, 0.5% NP-40, 1% Triton X-100) supplemented with protease inhibitor cocktail, followed by sonication. The sheared chromatins were cleared by centrifugation (12,000g, 4 C, 10 minutes). Supernatants were incubated with normal rabbit immunoglobulin G or primary antibody against phosphorylated Smad3 overnight at 4 C, followed by incubation with Protein A magnetic beads for another 1 hour at 4 C. The immunoprecipitants were washed in the ChIP buffer 6 times and eluted in 10% Chelex-100 by heating for 10 minutes. The resultant DNA was analyzed with quantitative real-time PCR using the following speciﬁc primers, as previously reported8–10:

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AhR Inhibits Hepatic Stellate Cell Activation

Acta2_F: 50 -CAAGTCCTCAGCTAATGGCC-30 , Acta2_R: 50 GGGGATAAACATCCTAAGCC-30 , Col1a1_F: 50 CCTCTGCCTCTTCTTGAGAGC-30 , ol1a1_R: 50 -GGAGAGGAGCTAAGTGTGAAGC-30 , COL1A1_F: 50 CATTCCCAGCTCCCCTCTCT-30 , COL1A1_R: 50 -AGTCTACGTGGCAGGCAAGG-30 , COL1A2_F: 50 -CCTGAGCCAGTAACCACCTCC-30 , COL1A2_R: 50 CTTTCGAAGCTAACGTGGCAG-30 , PAI1_F: 50 -GCAGGACATCCGGGAGAGA-30 , PAI1_R: 50 -CCAATAGCCTTGGCCTGAGA-30 .

Supplementary References 1.

2.

3.

4.

Quantitative Real-Time Polymerase Chain Reaction Total RNA was extracted from cells or tissues using TriPure isolation reagent from Sigma- Aldrich. Reverse transcription was performed with a high-capacity complementary DNA reverse transcription kit from Applied Biosystems (Thermo Fisher Scientiﬁc). SYBR Green-based real-time PCR was performed with the QuantStudio 6 Flex Real-Time PCR System from Applied Biosystems. A standard curve method was used for data analysis with cyclophilin as the housekeeping gene.

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RNA Sequencing and Bioinformatic Analysis RNA-seq was performed by the Health Sciences Sequencing Core at Children’s Hospital of Pittsburgh, and gene expression proﬁles were generated. The RNA-seq data analysis was performed as we have previously described.11,12 In brief, to interpret the function of regulated genes in AhR–/– HSCs compared with those in wild-type HSC, the expression was ﬁrst log2 transformed, and then the gene set enrichment analysis was performed based on the gene list ranked by the gene expression fold changes between AhR–/– and the wild type, using hallmark gene sets, Kyoto Encyclopedia of Genes and Genomes gene sets, and gene ontology gene sets. To search for genes with a potential DRE, a previously reported database with DRE binding information (score ranging from 0.5 to 1) in the liver was used.13 The cutoff score for considering genes with functional DREs was set at 0.85. A chisquares test was performed to analyze the enrichment of the DRE in up-regulated or down-regulated genes. Differentially expressed genes were screened for Smad3 and b-catenin co-binding on the promoter regions based on 2 ChIPsequencing data sets of mouse embryonic stem cells previously published.14,15 The distance from ChIP-sequencing peak position to transcription start site for each gene was either provided (for Smad3) or calculated (for b-catenin). Genes with both Smad3 and b-catenin binding in the same promoter region were deﬁned as the distance between Smad3 and b-catenin binding peaks less than 200 base pairs. Two data sets from Gene Expression Omnibus database were analyzed with GEO2R to proﬁle gene expression in the liver samples from human volunteers with alcoholic hepatitis (GSE28619)16 and cirrhosis (GSE89377).

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Supplementary Figure 1. Therapeutic effect of ITE on existing liver ﬁbrosis. Related to Figure 1. Eight-week-old male C57BL/ 6J mice were treated with CCl4 (0.5 mL/g body weight) 3 times a week for 4 weeks before being treated with vehicle (Veh) or ITE (10 mg/kg) together with CCl4 (0.5 ml/g body weight) 3 times a week for another 2 weeks (n ¼ 6 per group). (A) Outline of animal treatment protocol. (B) Histologic analysis by Sirius Red staining and immunostaining of a-SMA (original magniﬁcation, 5) with the quantiﬁcation of Sirius Red–and á-SMA–positive areas shown on the right. (C) Hepatic protein expression of a-SMA was measured by Western blotting (quantiﬁcation and statistical analysis shown in parentheses). (D) Hepatic expression of Cyp1a1 and Cyp1a2 was measured by real-time PCR. (E) Serum AST and ALT activity. All the data are presented as mean ± standard error of the mean. *P < .05, **P < .01. ITE vs. vehicle.

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2101 2102 2103 2104 2105 2106 2107 2108 2109 2110 2111 2112 2113 2114 2115 2116 2117 2118 2119 2120 2121 2122 2123 2124 2125 2126 2127 2128 2129 2130 2131 2132 2133 2134 2135 2136 2137 2138 2139 2140 2141 2142 2143 2144 2145 2146 2147 2148 2149 2150 2151 2152 2153 2154 2155 2156 2157 2158 2159 2160

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Supplementary Figure 2. The expression of CYP1B1 is induced with the onset of HSC activation. Related to Figure 2. The same complementary DNA used in Figure 2C and D from primary mouse or human HSCs was analyzed by real-time PCR for the expression levels of Cyp1b1 and CYP1B1, respectively.

Supplementary Figure 3. Reversal of HSC activation by adipogenesis induces the expression of AhR. Related to Figure 2. Fully activated HSCs were treated with MDI medium containing 3-isobutyl-1-methylxanthine (0.5 mmol/L), dexamethasone (1 mmol/L), insulin (10 mg/mL), and rosiglitazone (10 mmol/L) for 72 hours. Gene expression was determined by real-time PCR. n ¼ 4, **P < .01.

FLA 5.6.0 DTD YGAST62695_proof 22 July 2019 9:10 pm ce

2221 2222 2223 2224 2225 2226 2227 2228 2229 2230 2231 2232 2233 2234 2235 2236 2237 2238 2239 2240 2241 2242 2243 2244 2245 2246 2247 2248 2249 2250 2251 2252 2253 2254 2255 2256 2257 2258 2259 2260 2261 2262 2263 2264 2265 2266 2267 2268 2269 2270 2271 2272 2273 2274 2275 2276 2277 2278 2279 2280

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=

Supplementary Figure 4. AhR is transcriptionally activated by its ligands, and AhR-mediated inhibition of HSC activation is independent of cell proliferation or apoptosis. Related to Figure 3. (A) The same complementary DNA used in Figure 3B from primary mouse HSCs was analyzed for the expression of Cyp1b1. (B) Primary mouse HSCs were treated with FICZ (400 nmol/L) or ITE (1 mmol/L) for 6 days. The protein expression of a-SMA from whole-cell lysate was detected by Western blotting. (C, D) Primary mouse HSCs were isolated and treated with TCDD or ITE of indicated concentrations. Cell proliferation and apoptosis were determined by BrdU labeling, followed by (C) immunoﬂuorescent staining and (D) TUNEL staining, respectively. Original magniﬁcation, 200. (E) The same complementary DNA used in Figure 3E from primary human HSCs was analyzed for the expression of CYP1A2. (F) The same complementary DNA used in Figure 3G from primary mouse HSCs was analyzed for the expression of Ahr and Cyp1b1. (G) The same complementary DNA used in Figure 3I from primary human HSCs was analyzed for the expression of Ahr and CYP1A1. DMSO, dimethyl sulfoxide; M, mol/L.

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Q17

2461 2462 2463 2464 2465 2466 2467 2468 2469 2470 2471 2472 2473 2474 2475 2476 2477 2478 2479 2480 2481 2482 2483 2484 2485 2486 2487 2488 2489 2490 2491 2492 2493 2494 2495 2496 2497 2498 2499 2500 2501 2502 2503 2504 2505 2506 2507 2508 2509 2510 2511 2512 2513 2514 2515 2516 2517 2518 2519 2520

2521 2522 2523 2524 2525 2526 2527 2528 2529 2530 2531 2532 2533 2534 2535 2536 2537 2538 2539 2540 2541 2542 2543 2544 2545 2546 2547 2548 2549 2550 2551 2552 2553 2554 2555 2556 2557 2558 2559 2560 2561 2562 2563 2564 2565 2566 2567 2568 2569 2570 2571 2572 2573 2574 2575 2576 2577 2578 2579 2580

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Supplementary Figure 5. Gene ontology and gene set enrichment analysis for the RNA-seq results. Related to Figure 4. (A) Gene ontology analysis based on selected pathways. (B) Gene set enrichment analysis plots of key pathways.

FLA 5.6.0 DTD YGAST62695_proof 22 July 2019 9:10 pm ce

Q19

2581 2582 2583 2584 2585 2586 2587 2588 2589 2590 2591 2592 2593 2594 2595 2596 2597 2598 2599 2600 2601 2602 2603 2604 2605 2606 2607 2608 2609 2610 2611 2612 2613 2614 2615 2616 2617 2618 2619 2620 2621 2622 2623 2624 2625 2626 2627 2628 2629 2630 2631 2632 2633 2634 2635 2636 2637 2638 2639 2640

2641 2642 2643 2644 2645 2646 2647 2648 2649 2650 2651 2652 2653 2654 2655 2656 2657 2658 2659 2660 2661 2662 2663 2664 2665 2666 2667 2668 2669 2670 2671 2672 2673 2674 2675 2676 2677 2678 2679 2680 2681 2682 2683 2684 2685 2686 2687 2688 2689 2690 2691 2692 2693 2694 2695 2696 2697 2698 2699 2700

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Supplementary Figure 6. Creation and characterization of the HSC-speciﬁc HSC-KO and hepatocyte-speciﬁc HEP-KO mice. Related to Figure 5. (A) Genotyping PCR for the HSC-KO mice. (B) HSCs were isolated from ﬂox and HSC-KO mice. The mRNA expression of Ahr was measured by real-time PCR. (C, D) 8- to 9-week-old ﬂox and HSC-KO mice were subject to bile duct ligation for 2 weeks (Female: n ¼ 8 for ﬂox and 4 for HSC-KO; male: n ¼ 7 for ﬂox and 6 for HSC-KO). (C) The a-SMA protein expression was determined by Western blotting, and (D) the histology was analyzed by Masson’s trichrome staining, Sirius Red staining, and immunostaining of a-SMA (original magniﬁcation, 10). (E) ALT and AST measurement in serum from ﬂox and HSC-KO mice shown in Figure 5A–C. (F) Genotyping PCR for the HEP- KO mice. (G) 9- to 10-week-old male ﬂox and HEP-KO mice were treated with CCl4 (1 mL/g body weight) twice a week for 4 weeks (n ¼ 5 per group). Representative Sirius Red staining is shown (original magniﬁcation, 10). WT, wild type.

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2701 2702 2703 2704 2705 2706 2707 2708 2709 2710 2711 2712 2713 2714 2715 2716 2717 2718 2719 2720 2721 2722 2723 2724 2725 2726 2727 2728 2729 2730 2731 2732 2733 2734 2735 2736 2737 2738 2739 2740 2741 2742 2743 2744 2745 2746 2747 2748 2749 2750 2751 2752 2753 2754 2755 2756 2757 2758 2759 2760

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Supplementary Figure 7. Ablation of AhR in HSCs, but not in the hepatocytes and Kupffer cells, causes spontaneous liver ﬁbrosis. Related to Figure 5. Histologic analysis of liver by Sirius Red staining and immunostaining of a-SMA in wholebody AhR–/– (Whole-KO), HSC-speciﬁc (HSC-KO), hepatocyte-speciﬁc (HEP-KO), and Kupffer cell–speciﬁc (MAC-KO) KO mice (original magniﬁcation, 10).

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2821 2822 2823 2824 2825 2826 2827 2828 2829 2830 2831 2832 2833 2834 2835 2836 2837 2838 2839 2840 2841 2842 2843 2844 2845 2846 2847 2848 2849 2850 2851 2852 2853 2854 2855 2856 2857 2858 2859 2860 2861 2862 2863 2864 2865 2866 2867 2868 2869 2870 2871 2872 2873 2874 2875 2876 2877 2878 2879 2880

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AhR Inhibits Hepatic Stellate Cell Activation

2881 2882 2883 2884 2885 2886 2887 2888 2889 2890 2891 2892 2893 2894 2895 2896 2897 2898 2899 2900 2901 2902 2903 2904 2905 2906 2907 2908 2909 2910 2911 2912 2913 2914 2915 2916 2917 2918 2919 2920 2921 2922 2923 2924 2925 2926 2927 2928 2929 2930 2931 2932 2933 2934 2935 2936 2937 2938 2939 2940

14.e11

2941 2942 2943 2944 2945 2946 2947 2948 2949 2950 2951 2952 2953 2954 2955 2956 2957 2958 2959 2960 2961 2962 2963 2964 2965 2966 2967 2968 2969 2970 2971 2972 2973 2974 2975 2976 2977 2978 2979 2980 2981 2982 2983 2984 2985 2986 2987 2988 2989 2990 2991 2992 2993 2994 2995 2996 2997 2998 2999 3000 FLA 5.6.0 DTD YGAST62695_proof 22 July 2019 9:10 pm ce

14.e12

3001 3002 3003 3004 3005 3006 3007 3008 3009 3010 3011 3012 3013 3014 3015 3016 3017 3018 3019 3020 3021 3022 3023 3024 3025 3026 3027 3028 3029 3030 3031 3032 3033 3034 3035 3036 3037 3038 3039 3040 3041 3042 3043 3044 3045 3046 3047 3048 3049 3050 3051 3052 3053 3054 3055 3056 3057 3058 3059 3060

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Supplementary Figure 8. Effect of AhR on the TGFb-Smad pathway. Related to Figure 6. (A) The recruitment of phosphorylated Smad3 onto the promoter regions of the mouse Acta2 and Col1a1 genes in vehicle- or CCl4-treated mouse liver tissues was determined by ChIP coupled with real-time PCR analysis (left). Shown on the right is the comparison of phosphorylated Smad3 recruitment onto the Acta2 gene promoter in livers treated with CCl4 alone or CCl4 þ ITE (n ¼ 3, *P < .05, **P < .01). (B) The same complementary DNA used in Figure 6C from LX2 cells was analyzed for the expression of Ahr, CYP1A1, and CYP1B1. (C) LX2 cells infected with adenoviral vector overexpressing AhR were treated with or without TCDD (50 nmol/L) for 6 hours. The recruitment of AhR onto the DNA regions of the human CYP1B1, COL1A1, and COL1A2 genes was determined by ChIP with an anti-AhR antibody coupled with real-time PCR analysis on the recovered DNA. (D) Primary mouse HSCs were infected with adenoviral vector overexpressing AhR and treated with or without TCDD (50 nmol/L). Gene expression was determined by real-time PCR (n ¼ 3). (E) LX2 cells were infected with adenoviral vector overexpressing AhR and treated with TCDD (50 nmol/L) before being stimulated by TGFb1 (5 ng/mL) with or without TCDD for 4 hours. The cytoplasmic and nuclear fractions were extracted and subject to Western blot analysis. Ctrl, control; DMSO, dimethyl sulfoxide; Ig, immunoglobulin; Veh, vehicle.

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3061 3062 3063 3064 3065 3066 3067 3068 3069 3070 3071 3072 3073 3074 3075 3076 3077 3078 3079 3080 3081 3082 3083 3084 3085 3086 3087 3088 3089 3090 3091 3092 3093 3094 3095 3096 3097 3098 3099 3100 3101 3102 3103 3104 3105 3106 3107 3108 3109 3110 3111 3112 3113 3114 3115 3116 3117 3118 3119 3120

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AhR Inhibits Hepatic Stellate Cell Activation

3121 3122 3123 3124 3125 3126 3127 3128 3129 3130 3131 3132 3133 3134 3135 3136 3137 3138 3139 3140 3141 3142 3143 3144 3145 3146 3147 3148 3149 3150 3151 3152 3153 3154 3155 3156 3157 3158 3159 3160 3161 3162 3163 3164 3165 3166 3167 3168 3169 3170 3171 3172 3173 3174 3175 3176 3177 3178 3179 3180

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14.e14

3241 3242 3243 3244 3245 3246 3247 3248 3249 3250 3251 3252 3253 3254 3255 3256 3257 3258 3259 3260 3261 3262 3263 3264 3265 3266 3267 3268 3269 3270 3271 3272 3273 3274 3275 3276 3277 3278 3279 3280 3281 3282 3283 3284 3285 3286 3287 3288 3289 3290 3291 3292 3293 3294 3295 3296 3297 3298 3299 3300

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Supplementary Figure 9. Mechanism by which AhR facilitates the degradation of phosphorylated Smad2/3. (A, B) LX2 cells were infected with adenoviral vector overexpressing AhR and treated with TCDD (50 nmol/L) with or without TGFb1 (5 ng/mL) or MLN4924 (0.5 mmol/L) for 24 hours as labeled. The (A) protein and (B) mRNA expressions of the genes were measured by Western blotting and real-time PCR, respectively. (C, D) The experiments were the same as described in A and B except that primary human HSCs were used. (E) LX2 cells infected with Ad-AhR were stimulated with TGFb1 (5 ng/mL) for 1 hour and were treated with MLN4924 (0.5 mmol/L) or MG132 (10 mmol/L) before being collected at indicated time points. The protein expression was detected by Western blotting with the relative quantitative values labeled. (F, G) LX2 cells stably expressing empty vector (EV), human AhR full length (WT), or AhR with acidic domain deleted (dA) were treated with TGFb1 (5 ng/mL) with or without 3MC (2 mmol/L) for 24 hours. The (F) protein and (G) mRNA expressions of the genes were measured by Western blotting and real-time PCR, respectively. All the data are presented by mean ± standard error of the mean. *P < .05, **P < .01. Ctrl, control; DMSO, dimethyl sulfoxide; h, hour; WT, wild type.

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Aryl Hydrocarbon Receptor Signaling Prevents Activation of Hepatic Stellate Cells and Liver Fibrogenesis in Mice

Aryl Hydrocarbon Receptor Signaling Prevents Activation of Hepatic Stellate Cells and Liver Fibrogenesis in Mice

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