Repression of Smad7 mediated by DNMT1 determines hepatic stellate cell activation and liver fibrosis in rats

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Repression of Smad7 mediated by DNMT1 determines hepatic stellate cell activation and liver fibrosis in rats夽

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Er-Bao Bian a,b , Cheng Huang a,b , Hua Wang a,b , Xiao-Xia Chen a,b , Lei Zhang b , Xiong-Wen Lv b , Jun Li a,b,∗ a b

Institute for Liver Diseases of Anhui Medical University (AMU), China School of Pharmacy, Anhui Medical University, Hefei 230032, China

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h i g h l i g h t s • Upregulation of DNMT1 expression in liver fibrosis. • Epigenetic repression of Smad7. • Hypermethylation of the Smad7contributes to the activation of Smad2 and Smad3 pathways.

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Article history: Received 8 August 2013 Received in revised form 23 October 2013 Accepted 28 October 2013 Available online xxx

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Keywords: Liver fibrosis Hepatic stellate cell (HSC) DNA methylation DNA methyltransferase 1 (DNMT1) Smad7

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1. Introduction

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Conversion of hepatic stellate cells (HSCs) into hepatic myofibroblasts is a necessary event during the development of liver fibrosis. DNA methyltransferase 1 (DNMT1), which catalyzes DNA methylation and subsequently leads to the transcriptional repression of profibrotic genes, is selectively induced in myofibroblasts from diseased livers. Treatment of HSC with the DNA methylation inhibitor, 5-aza-2 deoxycytidine (5-azadC), prevented TGF-␤1-induced proliferation and alpha-smooth muscle actin (␣SMA) and collagen expression. 5-AzadC also rescued TGF-␤1-induced suppression of Smad7 expression which occurs during HSC activation. Similarly, silencing the expression of the DNMT1 gene ameliorated the suppression of Smad7 expression by TGF-␤1. In addition, DNMT1 inhibition, by 5-azadC or DNMT1 silencing, prevented the phosphorylation of Smad2 and Smad3. These studies suggest that epigenetic repression of Smad7 promotes the phosphorylation of Smad2 and Smad3 that may be an important molecular mechanism for perpetuated HSC activation and liver fibrosis. © 2013 Published by Elsevier Ireland Ltd.

Myofibroblasts are essential cells involved in wound repair, wound contraction, recruitment of inflammatory cells and remodeling of the extracellular matrix (ECM) to promote the scar formation that subsequently protects against infection and further tissue damage. Persistence and proliferation of myofibroblasts

Abbreviations: ␣-SMA, alpha-smooth muscle actin; Col1a1, alpha1(1) collagen; ECM, extracellular matrix; TGF-␤1, transforming growth factor-␤1; DNMT1, DNA methyltransferase 1; HSC, hepatic stellate cell; 5-azadC, 5-aza-2 -deoxycytidine; RT, reverse transcription; siRNA, short interfering RNA; RNAi, RNA interference. 夽 This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited. ∗ Corresponding author at: Institute for Liver Diseases of Anhui Medical University, School of Pharmacy, Anhui Medical University, Mei Shan Road, Hefei, Anhui Province 230032, China. Tel.: +86 551 65161001; fax: +86 551 65161001. E-mail addresses: [email protected], [email protected] (J. Li).

in chronic injuries is associated with progressive deposition of collagen-rich ECM and formation of cross-linked scars also known as fibrotic tissue (Desmouliere et al., 2005). In normal livers, HSCs are quiescent, vitamin A-storing, adipogenic cells, however, upon liver injury HSCs undergo a major phenotypic change to become hepatic myofibroblasts leading to de novo expression of smooth muscle ␣-actin (␣-SMA), alpha1(1) collagen(Col1a1) and the secretion of the pro-fibrogenic growth factor; TGF-␤1 (Friedman, 2008a). TGF-␤1 is an important profibrogenic cytokine that accelerates fibrosis formation by triggering the proliferation and the differentiation of fibroblasts into myofibroblasts, a process known as HSC activation. The activation of HSC is a major contributor to the pathobiology of chronic fibrotic disease (Desmouliere et al., 1993; Roberts et al., 1986). A previous study demonstrated that immortalized rat liver stellate cell (HSC-T6) is a well-characterized model of activated hepatic stellate cells characterized by increased proliferation and collagen synthesis (Friedman et al., 1997). In vitro, TGF-␤1 accelerates HSC transformation into myofibroblasts by increasing the expression of cellular markers such as ␣-smooth muscle actin (␣-SMA), and up-regulates collagen protein expression (Gressner

0378-4274/$ – see front matter © 2013 Published by Elsevier Ireland Ltd. http://dx.doi.org/10.1016/j.toxlet.2013.10.038

Please cite this article in press as: Bian, E.-B., et al., Repression of Smad7 mediated by DNMT1 determines hepatic stellate cell activation and liver fibrosis in rats. Toxicol. Lett. (2013), http://dx.doi.org/10.1016/j.toxlet.2013.10.038

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et al., 2002). After binding to its receptors, TGF-␤1 activates downstream signal transduction pathways, including Smad2 and Smad3, to mediate fibrosis, which is negatively modulated by Smad7, an inhibitor of TGF-beta signaling (Derynck and Zhang, 2003; Kavsak et al., 2000). Smad7 silencing can result from epigenetic modifications including histone modifications, microRNA (Liu et al., 2010; Simonsson et al., 2005). In addition, gene silencing can be achieved by DNA methylation which is one of three epigenetic mechanisms (Bian et al., 2012). We hypothesized that epigenetic control of Smad7 via DNA methylation in HSC would lead to the discovery of novel and critical epigenetic regulators of fibrogenesis. The present study determines that DNA methylation regulates the repression of Smad7 transcription in hepatic myofibroblasts. In addition, experimental evidence indicates that DNA methylation has a wide range of functions involved in controlling the myofibroblast phenotype and liver fibrosis in rats.

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2. Materials and methods

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2.1. CCl4 liver injury model

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Animals were provided by the Experimental Animal Center of Anhui Medical University. Liver fibrosis was generated by 12-week treatments in adult male Sprague-Dawley (200–220 g) rats with CCl4 (CCl4 /olive oil, 1:1 (vol./vol.) per kg body weight by intraperitoneal injection twice weekly) as previously described (Oakley et al., 2005). Vehicle control animals were injected intraperitoneally with 1 ml of olive oil per kg body weight at the same time intervals. 24 h after the last CCl4 injection, rats were sacrificed and liver tissues were harvested for further analysis. Rat livers were then inflated with 10% formalin solution and embedded in paraffin. 4-␮m thick sections were prepared, deparaffinized with xylene, and then rehydrated in water through a graded ethanol series. The liver tissues were stained with hematoxylin and eosin (H&E) and Masson staining after fixation with 10% formalin.

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2.2. Cell culture and cell treatment with TGF-ˇ1

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The HSC-T6 cell line was obtained from Shanghai FuMeng Gene Biological Corporation (Shanghai, China). HSC-T6 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA), supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM l-glutamine, and 10% fetal calf serum. Cell cultures were maintained at 37 ◦ C at an atmosphere of 5% CO2 . HSC-T6 cells were incubated for 48 h after passaging and serum-starved with 0.5% FCS for 24 h before adding 5 ng/ml TGF-␤1 (Peprotech, USA).

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2.3. 5-Aza-2 -deoxycytidine treatment

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HSC-T6 cells were seeded overnight in tissue culture dishes. 1 ␮M 5-azadC (Sigma-Aldrich, St. Louis, MO) was added and refreshed every 24 h for 48 h.

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2.4. Immunofluorescence staining

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Five-␮m-thick sectioned tissues from rats livers or HSC-T6 cells were fixed with 4% paraformaldehyde. The tissues and HSC-T6 cells were permeabilized with 0.2% Triton X-100 in 1% bovine serum albumin (BSA) for 10 min, blocked with 5% BSA for one hour at room temperature. Incubation with Mouse DNMT1 monoclonal antibodies (1:50 dilution; SantaCruz Biotechnology, Santa Cruz, CA) and rabbit ␣-SMA monoclonal antibodies (1:50 dilution; Boster, China) at 4 ◦ C overnight was followed by goat anti-mouse and mouse anti-rabbit IgG, FITC conjugated (1:100 dilution; Kwbio, Peiking, China). The cells were mounted with SlowFade Gold antifade reagent with DAPI (Sigma, MO, USA) and images were taken using fluorescence microscopy. Analysis was performed by counting the number of lipid droplets containing a specific amount of pixels using ImageJ software.

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2.5. MTT cell viability assay

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Cells (5 × 103 ml–1 ) were cultured with 5-azadC and TGF-␤1 for various durations in 96-well plates. After exposure, 5 mg/ml MTT was added to each well and incubated with cells at 37 ◦ C for 4 h. After removal of supernatant, 150 ␮l of DMSO was added to each well. The optical density (OD) was measured at 550 nm. All experiments were performed in triplicate and repeated at least three times. Viability was calculated according to the following formula: viability % = T/C × 100%, where T and C refer to the absorbance of transfection group and cell control, respectively.

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2.6. Quantitative real-time PCR

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Total RNA was extracted from rat livers or from HSC-T6 cells using TRIzol reagent (Invitrogen). The first-strand cDNA was synthesized from total RNA

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using the Thermoscript RT-PCR System (Takara) according to the manufacturer’s instructions. Quantitative real-time PCR analyses of DNMT1, ␣-SMA, Col1a1, Smad7 and ␤-actin were performed by using an ABI Prism 7500 machine. The mRNA levels of ␤-actin were used as an internal control. Real time PCR was carried out under a standard protocol using the following primers: TGF-␤1 (forward: reverse: 5 -AGCGCACGATCATGTTGGAC5 -CGGCAGCTGTACATTGACTT-3 ; DNMT1 (forward: 5 -GGAAAGGAGGAGACTACTAC-3 ; reverse: 3 ), 5 -TCTCACTTGCCACCCACACA-3 ), ␣-SMA (forward: 5 -CGAAGCGCAGAGCAAGAGAreverse: 5 -CATGTCGTCCCAGTTGGTGAT-3 ), Col1a1 (forward: 3 ; reverse: 5 -TGTAGGCTACGCTGTTCTTGCA5 -GATCCTGCCGATGTCGCTAT-3 , Smad7 (forward: 5 -ACTGGTGCGTGGTGGCATACTGG-3 ; reverse: 3 ), ␤-actin (forward: 5 5 -GCCGATCTTGCTCCTCACTTTCTG-3 ),   ACCACAGCTGAGAGGGAAATCG-3 ; reverse: 5 -AGAGGTCTTTACGGATGTCAACG-3 ). Real-time PCR conditions were as followed; 42 ◦ C for 30 min; 95 ◦ C for 10 min, 40 cycles of amplification at 95 ◦ C for 20 s, 62 ◦ C for 30 s, 72 ◦ C for 30 s. Melt curve analysis was performed at 95 ◦ C for 30 s, 60 ◦ C for 30 min, 95 ◦ C for 30 s. The relative mRNA expression was calculated from three different experiments. The fold-change for mRNA relative to ␤-actin was determined by the formula: 2−  Ct .

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2.7. RNA interference (RNAi) analysis

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HSC-T6 cells were cultured in serum-free DMEM for 12 h. On the day of transfection, the cells were plated in DMEM supplemented with 10% FBS at a density of 2 × 105 cells/ml and were transfected with siRNA-DNMT1 or a negative control siRNA (GenaPharma, China) using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s protocol. The culture medium was changed 6 h after transfection, and TGF-␤1 (Peprotech, USA) was added at a concentration of 5 ng/ml. The sequences of oligonucleotides used are as follows: siRNA-DNMT1: 5 -CCCAGAGUAUGCACCAAUATT-3 ; 5 -UAUUGGUGCAUACUCUGGGTT-3 , negative control siRNA: 5 -UUCUCCGAACGUGUCACGUTT-3 ; 5 -ACGUGACACGUUCGGA GAATT-3 .

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2.8. Western blotting

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Rat liver tissue and cells were lysed with RIPA lysis buffer (Beyotime, China). Whole extracts were prepared, and protein concentrations were determined using the BCA protein assay kit (Boster, China). Whole-cell extracts (30 or 50 ␮g) were then fractionated by electrophoresis through an 8% or 12% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). Gels were run at a 120 V for 2 h before transfer onto a PVDF membrane (Millipore Corp., Billerica, MA, USA). After blocking against non-specific protein binding, nitrocellulose blots were incubated for 1 h with primary antibodies diluted in TBS/Tween20 (0.075% Tween 20) containing 3% Marvel. Anti-phospho-Smad2, anti-Smad2, anti-phospho-Smad3 and anti-Smad3 antibodies (Cell Signaling, Beverly, MA, USA) were diluted 1:1000. Rabbit polyclonal anti-␣-SMA (Proteintech, USA) was diluted 1:600. Smad7, DNMT1 and ␤-actin (Santa Cruz, CA, USA) were diluted 1:400. Mouse monoclonal anti-Col1a1 (Abcam ab90395) was diluted 1:1000. Following incubation with the primary antibody, blots were washed three times in TBS/Tween-20 before incubation for 1 h with goat anti-mouse or mouse anti-rabbit horseradish peroxidase conjugated antibody at a 1:10 000 dilution in TBS/Tween-20 containing 5% milk. After extensive washing in TBS/Tween-20, the blots were rinsed with distilled water and proteins were detected using the enhanced chemiluminescence system. Proteins were visualized with ECL-chemiluminescent kit (ECL-plus, Thermo Scientific).

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2.9. Methylation-specific polymerase chain reaction

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The methylation of Smad7 promoter region was determined by methylationspecific PCR (MSP) using bisulfite-modified DNA. Genomic DNA was extracted using the QIAamp DNA mini kit (Axygen). Two primer sets were used to amplify the promoter region of the Smad7 gene that incorporated a number of CpG sites, one specific for the methylated sequence (Smad7-M, forward: 5 -CGGGGTTAGTAGTTTATGCG3 ; reverse: 5 -CAAATCCTCGACGAAAACTT-3 ) and the other for the unmethylated sequence (Smad7-U, forward: 5 -GGTTGGGGTTAGTAGTTTATGTG-3 ; reverse: 5 CCAAATCCTCAACAAAAACTTCC-3 ). M and U, PCR products of methylated and unmethylated alleles, respectively, the polymerase chain reactions for Smad7-M and Smad7-U were carried out in a 50 ␮L volume containing 1× polymerase chain reaction buffer (15 mmol/L MgCl2 ), 2.5 mmol/L mixture of dNTPs, 10 pM of each primer, 4 U HotStart Taq DNA polymerase (Qiagen, Frankfurt, Germany), and 25–50 ng of bisulfite-modified DNA. Amplification was performed in a thermocycler with the following conditions: 95 ◦ C for 3 min, cycled at 94 ◦ C for 30 s, 57 ◦ C for 30 s, and 72 ◦ C for 45 s (35 cycles), followed by an extension at 72 ◦ C for 7 min. Methylation-specific PCR experiments were performed at least in duplicate.

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2.10. Statistical analysis

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Data are represented as mean ± standard error of the mean (SE). Statistical analysis was performed using a one-way ANOVA followed by students t-test. Changes in mRNA or protein expression levels were determined by creating a ratio of mRNA (relative expression) or protein (densitometric values) levels compared to respective house-keeping controls. Significance was defined as P < 0.05.

Please cite this article in press as: Bian, E.-B., et al., Repression of Smad7 mediated by DNMT1 determines hepatic stellate cell activation and liver fibrosis in rats. Toxicol. Lett. (2013), http://dx.doi.org/10.1016/j.toxlet.2013.10.038

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3. Results

3.4. Epigenetic regulation of Smad7 gene transcription

3.1. DNMT1 expression is induced by wound healing process in myofibroblasts

Expression of Smad7 mRNA and protein was downregulated in livers of CCL4 -induced rats compared to vehicle control livers (Fig. 5A and B). To investigate the mechanism underlying the decreased Smad7 expression, we analyzed whether Smad7 promoter region hypermethylation is responsible for the downregulation of Smad7 expression. Methylation-specific PCR analysis indicated that the promoter region of Smad7 gene from the liver tissues of CCl4-treated rats in which Smad7 expression was downregulated were strongly methylated, whereas the liver tissues from vehicle-treated rats in which Smad7 expression was present had unmethylated Smad7 promoter region (Fig. 5C). To confirm that changes in Smad7 gene expression during HSC activation occurred in vitro, the levels of Smad7 mRNA and protein expression were also quantified in HSC-T6 cells. Smad7 expression was indeed downregulated in HSC-T6 cells exposed to TGF-␤1 treatment compared to basal levels (Fig. 5D and E). To further confirm that Smad7 expression changes were due to methylation in vitro, HSC-T6 cells were stimulated with TGF-␤1 then treated with 1 ␮mol/L 5-azadC for 48 h. 5-AzadC treatment reversed TGF␤-induced aberrant hypermethylation of the Smad7 gene, the decrease of Smad7 mRNA and protein expression (Fig. 5F, G and H). Moreover, DNMT1 knockdown with RNAi in HSC-T6 cells ameliorated Smad7 methylation, and restored the expression of Smad7 mRNA and protein (Fig. 5I, J and K). These results suggest that decreased Smad7 expression during HSC activation is associated with reversible epigenetic mechanisms, such as DNA methylation.

To test our hypothesis that an epigenetic modification, in particular hypermethylation, is involved in liver fibrogenesis, we treated rats with CCl4 to induce liver fibrosis. The degree of liver fibrosis was dramatic increased in CCL4 -treated rat as determined by H&E staining. These data were confirmed through Masson’s trichrome staining, which identifies collagen deposition (Fig. 1A). Immunofluorescence revealed more nuclear DNMT1 labeling in livers of rat that had received CCL4 as compared to vehicle control livers (Fig. 1B). Similarly, real time-PCR analysis determined that the mRNA expression level of DNMT1 was significantly increased in the livers from CCl4 -treated rats compared with those from vehicletreated rats along with the induction of mRNA level of Col1a1 and ␣-SMA, markers of HSC activation (Fig. 1C). Moreover, ␣-SMA and Col1a1 protein expression was also markedly increased in the livers from rats treated with CCl4 compared with that in vehicle-treated groups (Fig. 1D). To demonstrate that TGF-␤1 might contribute to the enhanced expression of DNMT1 in the fibrotic livers, we examined TGF-␤1 level in liver and found that, similar to DNMT1, TGF-␤1 mRNA expression was induced in fibrotic livers (Fig. 1C). These results suggest that DNMT1 plays an important role in the pathobiology of liver fibrosis. 3.2. Upregulation of DNMT1 expression in HSCs by the treatment of TGF-ˇ1 To confirm that changes in DNMT1 gene expression induced by TGF-␤1 during HSC activation also occur in vitro, we examined the expression levels of DNMT1 mRNA in cultured HSCs exposed to TGF-␤1. The steady state mRNA levels of DNMT1, ␣-SMA and Col1a1 was induced in HSC-T6 cells stimulated with TGF-␤1 from 24 h to 48 h compared to basal level expression (0 h) (Fig. 2A). Similarly, TGF-␤1 treatment markedly up-regulated expression of DNMT1, ␣-SMA and Col1a1 protein in HSC-T6 cells (Fig. 2B). These results provide additional evidence that epigenetic mechanisms may involve in HSC activation through the induction of TGF-␤1. 3.3. DNMT1 inhibition by 5-azadC or DNMT1 silencing prevents TGF-ˇ1-mediated HSC activation To examine the potential role of DNMT1 in the process of TGF␤1-induced HSC activation, HSC-T6 cells were exposed to TGF-␤1 and/or 5-azadC, a DNA methyltransferase (DNMT) inhibitor. As illustrated in Fig. 3A, 5-azadC treatment had a profound inhibitory effect on TGF-␤1-induced HSC-T6 cells at different time points. Expression of DNMT1 and a-SMA protein by immunofluorescent staining were increased in TGF-␤1-treated HSC, however, 5-azadC significantly reduced TGF-␤-induced expression of DNMT1 and a-SMA protein (Fig. 3B). In addition, treatment with 5-azadC abolished TGF-␤1-mediated up-regulation of DNMT1, ␣-SMA and Col1a1 mRNA expression (Fig. 3C). Similarly, Western blotting analysis showed that 5-azadC treatment also significantly reduced DNMT1, ␣-SMA and Col1a1 protein expression (Fig. 3D). We next determined whether DNMT1 plays a role in TGF-␤1induced HSC activation. As illustrated in Fig. 4A and B, TGF-␤1 treatment markedly up-regulated mRNA and protein expression of ␣-SMA and Col1a1 in HSC-T6 cells. However, decreasing DNMT1 expression through a DNMT1-specific siRNA, TGF-␤1 did not increase the expression of ␣-SMA and Col1a1 mRNA and/or protein (Fig. 4A and 4B). These results suggest that TGF-␤1-induced HSC activation expression is dependent on DNMT1 expression.

3.5. Epigenetic repression of Smad7 contributes to the activations of Smad2 and Smad3 pathway in HSCs To explore the potential signaling pathways effected by Smad7 downregulation during HSC activation, the phosphorylation levels of Smad2 and Smad3 were quantified. As shown in Fig. 6A, phosphorylation levels of Smad2 and Smad3 were significantly induced in the livers from CCl4 -treated rats compared to those from vehicletreated rats. To confirm that the phosphorylation of Smad2 and Smad3 occurred during HSC activation through TGF-␤1, HSC-T6 cells were treated with TGF-␤1 and the time-course expression and phosphorylation of Smad2 and Smad3 were determined. Sustained phosphorylation of Smad2 and Smad3 was induced by TGF-␤1 (Fig. 6B). To determine the effects of 5-azadC on the signal pathways involved in TGF-␤1-induced HSC activation, phosphorylation levels of Smad2 and Smad3 in 5-azadC-treated HSC were analyzed. The phosphorylation levels of Smad2 and Smad3 were decreased by 5-azadC treatment compared to untreated HSC-T6 cells (Fig. 6C). Furthermore, DNMT1 silencing also significantly prevented the phosphorylation of Smad2 and Smad3 by TGF-␤1 compared to control or scrambled RNAi-treated cells (Fig. 6D). These results suggest that epigenetic repression of Smad7 by DNA methylation contributes to the phosphorylation of Smad2 and Smad3 in HSC-T6 cells. 4. Discussion The essential role of myofibroblasts in wound-healing responses of solid organs is well established, as is the concept of persistence of myofibroblasts in chronic injury leading to fibrosis (Wynn, 2008). Hepatic myofibroblasts can be generated locally at the site of liver injury by transdifferentiation of resident hepatic stellate cells (HSCs) (Friedman, 2008b). Regulated transdifferentiation ensures the optimization of wound healing by controlling the transition of resident differentiated cells to myofibroblasts. In the present study,

Please cite this article in press as: Bian, E.-B., et al., Repression of Smad7 mediated by DNMT1 determines hepatic stellate cell activation and liver fibrosis in rats. Toxicol. Lett. (2013), http://dx.doi.org/10.1016/j.toxlet.2013.10.038

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Fig. 1. Expression of DNMT1, TGF-␤1, ␣-SMA and Col1a1 in vehicle control liver and fibrotic liver tissues. (A) Pathology observation of the experimental rat liver sections stained with hematoxylin and eosin (H&E) staining (×200) and Masson staining (×200) in vehicle control and CCl4 -treated liver tissue. (B) The levels of DNMT1 expression are analyzed via immunofluorescence staining in control liver tissue and liver fibrotic tissue. (C) Real-time PCR were performed to examine the mRNA level of DNMT1, TGF-␤1, ␣-SMA and Col1a1 in the liver tissues of CCl4 -treated rats or vehicle-treated groups. (D) Western blotting was performed to assess the protein level of ␣-SMA and Col1a1 in the liver tissues of CCl4 -treated rats or vehicle-treated groups. Each bar represents the mean ± SD of four independent experiments performed in duplicate. **p < 0.01 vs. vehicle control.

Please cite this article in press as: Bian, E.-B., et al., Repression of Smad7 mediated by DNMT1 determines hepatic stellate cell activation and liver fibrosis in rats. Toxicol. Lett. (2013), http://dx.doi.org/10.1016/j.toxlet.2013.10.038

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Fig. 2. Upregulation of DNMT1 expression in HSC-T6 cells by the treatment of TGF-␤1. (A) Real-time PCR were performed to examine the mRNA level of DNMT1, ␣-SMA and Col1a1 in TGF-␤1-treated HSC-T6 cells at the indicated time points. (B) Western blotting was performed to assess the protein level of DNMT1, ␣-SMA and Col1a1 in TGF-␤1-treated HSC-T6 cells at different time points. Each bar represents the mean ± SD of three independent experiments performed in duplicate. *p < 0.05, **p < 0.01 vs. 0 h.

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we provide evidence that epigenetic repression of Smad7 by DNA methylation promotes HSC activation and liver fibrosis in rats. DNA methylation is catalyzed by several known biologically active DNA methyltransferases: Dnmt1, Dnmt3a and Dnmt3b and others (Okano et al., 1999; Quina et al., 2006). DNMT1 is responsible for the maintenance of pre-existing DNA methylation patterns after replication (Zhang et al., 2011). DNMT1 up-regulation has been reported in many types of cancers, such as gastric, hepatic, endometrial, prostate, pancreatic and bladder cancer, suggesting that over-expression of DNMT1 plays a significance role during carcinogenesis (Etoh et al., 2004; Liao et al., 2008; Morey Kinney

et al., 2008; Nakagawa et al., 2005; Peng et al., 2006; Saito et al., 2003). We demonstrate that the expression of DNMT1 is increased in the fibrotic livers from CCl4 -treated livers. DNMT1, but DNMT3a and DNMT3b, has previously been shown to be induced in TGF-␤treated mouse kidney fibroblasts and to regulate the expression of genes involved in fibroblast activation (Bechtel et al., 2010). TGF␤ is a multifunctional cytokine, with an essential role in wound healing and tissue repair after injury (Blobe et al., 2000). The induction of DNMT1 by TGF-␤1 during HSC activation suggests that DNMT1 may have a potential role in the pathogenesis of liver fibrosis.

Please cite this article in press as: Bian, E.-B., et al., Repression of Smad7 mediated by DNMT1 determines hepatic stellate cell activation and liver fibrosis in rats. Toxicol. Lett. (2013), http://dx.doi.org/10.1016/j.toxlet.2013.10.038

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Fig. 3. Effects of 5-azadC on TGF-␤1-induced HSC activation. HSC-T6 cells were divided into untreated (control), TGF-␤1-treated HSC-T6 cells (model) or 5-azadC plus TGF␤1-treated HSC-T6 cells at different time points. (A) MTT was performed to examine the effect of 5-azadC on TGF-␤1-induced HSC proliferation. (B) Immunofluorescence staining was performed to assess the protein level of DNMT1 and ␣-SMA at different groups. (C) Real-time PCR were performed to examine the mRNA level of DNMT1, ␣-SMA and Col1a1 at different groups. (D) Western blotting was performed to assess the protein level of DNMT1, ␣-SMA and Col1a1 at different groups. Each bar represents the mean ± SD of three independent experiments performed in duplicate. *p < 0.05, **p < 0.01 vs. control, # p < 0.05, ## p < 0.01 vs. model.

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Inhibition of DNMT1 activity may reduce hypermethylation of repressive genes and promote its re-expression, and reverse the phenotype of malignant tumors (Szyf, 2001). Recent evidence suggests that DNMT1 is rapidly degraded through the proteasomal pathway upon treatment of cells with 5-azadC, an DNMTs inhibitor (Ghoshal et al., 2005). 5-AzadC effectively delays the morphological features of HSC activation and inhibits proliferation of cultured

HSC, a behavioral phenotype characteristically associated with HSC activation (Mann et al., 2007). Our work demonstrates that 5-azadC reduces cell viabilities and prevents TGF-␤1-induced HSC activation. In addition, 5-azadC treatment in HSC identified Smad7 as a mRNA transcript that is repressed during HSC activation in a DNA methylation-dependent manner. Smad7 is a negative regulator of

Please cite this article in press as: Bian, E.-B., et al., Repression of Smad7 mediated by DNMT1 determines hepatic stellate cell activation and liver fibrosis in rats. Toxicol. Lett. (2013), http://dx.doi.org/10.1016/j.toxlet.2013.10.038

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Fig. 4. Effect of DNMT1 gene silencing on TGF-␤1-induced HSC activation. After DNMT1 siRNA or siControl transfection, the exposure HSC-T6 cells to TGF-␤1 for 2 day. (A) Real-time PCR were performed to examine the mRNA level of DNMT1, ␣-SMA and Col1a1 in RNAi-treated HSC-T6 cells with or without TGF-␤1. (B) Western blotting was performed to assess the protein level of DNMT1, ␣-SMA and Col1a1 in RNAi-treated HSC-T6 cells with or without TGF-␤1. Each bar represents the mean ± SD of two to four independent experiments performed in duplicate. *p < 0.05, **p < 0.01 vs. scrambled RNAi.

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HSC activation and hepatic fibrosis in vitro and in vivo since deletion of Smad7 promotes, whereas Smad7 over-expression protects, against this process (Dooley et al., 2003, 2008; Hamzavi et al., 2008). Smad7 is a potential master transcriptional repressor of HSC transdifferentiation that undergoes loss of expression during HSC activation. Consistent with the previous reports, we observed the loss of Smad7 in fibrotic liver and during HSC activation induced by TGF-␤1. Loss of Smad7 expression can result from epigenetic modifications including microRNA, histone modification and promoter hypermethylation (Xia et al., 2013; Glenisson et al., 2007;

Matsumura et al., 2011). DNA methylation patterns are critical for generating cellular diversity and maintaining distinct gene expression profiles (Ausio et al., 2003). In many disease processes such as cancer, gene promoter CpG islands acquire abnormal hypermethylation, which results in heritable transcriptional silencing (Hu et al., 2010). DNA methylation, therefore, exerts control over Smad7 transcriptional regulators of the myofibroblast phenotype. To clearly establish whether DNMT1 silencing affects Smad7 expression, we studied these changes in the HSC-T6 cell line in order to determine the effects of silencing Smad7 expression through siRNA

Please cite this article in press as: Bian, E.-B., et al., Repression of Smad7 mediated by DNMT1 determines hepatic stellate cell activation and liver fibrosis in rats. Toxicol. Lett. (2013), http://dx.doi.org/10.1016/j.toxlet.2013.10.038

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knockdown conditions. Knockdown of DNMT1 by RNAi inhibited HSC activation, and restored Smad7 gene expression. These results suggest that DNA methylation is involved in decreasing the expression of Smad7 during HSC activation. Phosphorylation of Smad2 and Smad3, which are pivotal signaling events in TGF-␤1-induced gene transcription, lead to activation of these proteins and translocation into the nucleus where they induce expression of ␣-SMA (Cho et al., 2012). Activation of TGF-␤/Smad signaling is a key mechanism of liver fibrosis in both experimental and chronic human liver diseases (Inagaki and

Okazaki, 2007; Liu et al., 2011; Novo and Parola, 2008). The functional importance of TGF-␤/Smad signaling in liver fibrosis has been demonstrated through studies using Smad3 knockout mice, which are protected against dimethylnitrosamine-induced hepatic fibrosis (Latella et al., 2009). Here, we demonstrate that the activity of Smad2 and Smad3 were enhanced during TGF-␤1-induced HSC activation. Smad7, an inhibitory molecule of TGF-␤1 signaling, is known to block the phosphorylation of Smad2 and Smad3 (Chung et al., 2009). Over-expression of Smad7 in liver attenuates TGF␤/Smad signaling, shown by inhibiting Smad2/3 phosphorylation,

Fig. 5. Effect of inhibition of DNMT1 on Smad7 in TGF-␤1-induced HSC activation. (A) Real-time PCR were performed to examine the mRNA level of Smad7 in the liver tissues of CCl4 -treated rats or vehicle-treated groups. (B) Western blotting was performed to assess the protein level of Smad7 in the liver tissues of CCl4 -treated rats or vehicle-treated groups. (C) Results of MSP analysis of Smad7 gene in the liver tissues from CCl4-treated rats and vehicle-treated groups. M and U, PCR products of methylated and unmethylated alleles, respectively; bottom, frequencies of CpG methylation of Smad7 gene promoter by MSP. (D) Total RNAs were isolated from TGF-␤1-treated HSC-T6 cells for 48 h. The expression of Smad7 was assessed by real-time PCR. (E) Western blotting was performed to assess the protein level of Smad7 in TGF-␤1-treated HSC-T6 cells at different time points. (F) HSC-T6 cells were treated with or without TGF-␤1 and 5-azadC for 2 days. Real-time PCR analyses of Smad7 were performed. (G) HSC-T6 cells were treated with or without TGF-␤1 and 5-azadC for 2 days. Western blotting analyses of Smad7 was performed. (H) MSP analysis of Smad7 promoter from untreated HSC-T6 cells and TGF-␤1-treated HSC-T6 cells challenged with or without 5-azadC for 48 h. M and U, PCR products of methylated and unmethylated alleles, respectively; bottom, frequencies of CpG methylation of Smad7 gene promoter by MSP. (I) Total RNAs were isolated from RNAi-treated cells with or without TGF-␤1, and subjected to real-time PCR analyses. (J) Whole cell extracts were isolated from RNAi-treated cells with or without TGF-␤1, and subjected to Western blot analyses. Each bar represents the mean ± SD of three to five independent experiments performed in duplicate. *p < 0.05, **p < 0.01 vs. vehicle control, control or scramble control # p < 0.05, ## p < 0.01 vs. model. (K) MSP analysis of Smad7 promoter in TGF-␤1-treated HSCs-T6 cells with RNAi transfection. M and U, PCR products of methylated and unmethylated alleles, respectively; bottom, frequencies of CpG methylation of Smad7 gene promoter by MSP.

Please cite this article in press as: Bian, E.-B., et al., Repression of Smad7 mediated by DNMT1 determines hepatic stellate cell activation and liver fibrosis in rats. Toxicol. Lett. (2013), http://dx.doi.org/10.1016/j.toxlet.2013.10.038

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Fig. 5. Effect of inhibition of DNMT1 on Smad7 in TGF-␤1-induced HSC activation. (A) Real-time PCR were performed to examine the mRNA level of Smad7 in the liver tissues of CCl4 -treated rats or vehicle-treated groups. (B) Western blotting was performed to assess the protein level of Smad7 in the liver tissues of CCl4 -treated rats or vehicle-treated groups. (C) Results of MSP analysis of Smad7 gene in the liver tissues from CCl4-treated rats and vehicle-treated groups. M and U, PCR products of methylated and unmethylated alleles, respectively; bottom, frequencies of CpG methylation of Smad7 gene promoter by MSP. (D) Total RNAs were isolated from TGF-␤1-treated HSC-T6 cells for 48 h. The expression of Smad7 was assessed by real-time PCR. (E) Western blotting was performed to assess the protein level of Smad7 in TGF-␤1-treated HSC-T6 cells at different time points. (F) HSC-T6 cells were treated with or without TGF-␤1 and 5-azadC for 2 days. Real-time PCR analyses of Smad7 were performed. (G) HSC-T6 cells were treated with or without TGF-␤1 and 5-azadC for 2 days. Western blotting analyses of Smad7 was performed. (H) MSP analysis of Smad7 promoter from untreated HSC-T6 cells and TGF-␤1-treated HSC-T6 cells challenged with or without 5-azadC for 48 h. M and U, PCR products of methylated and unmethylated alleles, respectively; bottom, frequencies of CpG methylation of Smad7 gene promoter by MSP. (I) Total RNAs were isolated from RNAi-treated cells with or without TGF-␤1, and subjected to real-time PCR analyses. (J) Whole cell extracts were isolated from RNAi-treated cells with or without TGF-␤1, and subjected to Western blot analyses. Each bar represents the mean ± SD of three to five independent experiments performed in duplicate. *p < 0.05, **p < 0.01 vs. vehicle control, control or scramble control # p < 0.05, ## p < 0.01 vs. model. (K) MSP analysis of Smad7 promoter in TGF-␤1-treated HSCs-T6 cells with RNAi transfection. M and U, PCR products of methylated and unmethylated alleles, respectively; bottom, frequencies of CpG methylation of Smad7 gene promoter by MSP.

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and protects against HSC activation and liver fibrosis (Dooley et al., 2003, 2008). We demonstrate that the inhibition of DNMT1 upregulated Smad7 expression. To further test the effect of 5-azadC on Smad2 and Smad3 pathways in the present study, we determined that 5-azadC treatment inhibited phosphorylation of Smad2 and Smad3 by TGF-␤1 and also lead to an increase of Smad7 expression. Similarly, DNMT1 knockdown also inhibited phosphorylation

of the Smad2 and Smad3 pathways in HSC-T6 cells by TGF-␤1. These results suggest that epigenetic silencing of Smad7 contributes to the phosphorylation of Smad2 and Smad3 in HSC-T6 cells. To the best of our knowledge, this is the first report of epigenetic silencing of Smad7 by DNA methylation as a mechanism of HSC activation and fibrosis. In summary, our report indicates that epigenetic repression of Smad7 contributes to the phosphorylation of Smad2

Please cite this article in press as: Bian, E.-B., et al., Repression of Smad7 mediated by DNMT1 determines hepatic stellate cell activation and liver fibrosis in rats. Toxicol. Lett. (2013), http://dx.doi.org/10.1016/j.toxlet.2013.10.038

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Fig. 6. Effects of epigenetic silencing of Smad7 on phosphorylation of Smad2 and Smad3 in TGF-␤1-induced HSC activation. (A) Western blotting was performed to assess the protein level of p-Smad2 and p-Smad3 in the liver tissues of CCl4 -treated rats or vehicle-treated groups. (B) Western blotting was performed to assess the protein level of p-Smad2 and p-Smad3 in TGF-␤1-treated HSC-T6 cells at different time points. (C) HSC-T6 cells were treated with or without TGF-␤1 and 5-azadC for 2 days. Western blotting analyses of p-Smad2 and p-Smad3 was performed. (D) Whole cell extracts were isolated from RNAi-treated cells with or without TGF-␤1, and subjected to Western blot analyses. Each bar represents the mean ± SD of three to four independent experiments performed in duplicate. *p < 0.05, **p < 0.01 vs. vehicle control, control or scramble control # p < 0.05, ## p < 0.01 vs. model.

Please cite this article in press as: Bian, E.-B., et al., Repression of Smad7 mediated by DNMT1 determines hepatic stellate cell activation and liver fibrosis in rats. Toxicol. Lett. (2013), http://dx.doi.org/10.1016/j.toxlet.2013.10.038

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and Smad3 pathways resulting in HSC activation. Future studies are needed to unravel how DNA methylation together with other repression mechanisms, such as microRNA and histone modification, generate distinct patterns of gene silencing in hepatic fibrosis. These studies clearly provide the incentive for additional investigations that may help gain a better understanding of the effects of Smad7 inactivation in hepatic fibrosis and could pave the way for the exploration of its potential targeting in the development of therapeutic modalities.

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Conflict of interest

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The authors declare no conflict of interest. Acknowledgement

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This project was supported by the National Science Foundation of China (Nos. 81072686, 81273526).

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