Toxicology 323 (2014) 42–50

DNMT3A silencing RASSF1A promotes cardiac fibrosis throughupregulation of ERK1/2

Hui Tao a,b, Jing-Jing Yang c,**, Ze-Wen Chen a,b, Sheng-Song Xu a,b, Xiao Zhou a,b,Hong-Ying Zhan a,b, Kai-Hu Shi a,b,*aDepartment of Cardiothoracic Surgery, The Second Hospital of Anhui Medical University, Fu Rong Road, Hefei, Anhui 230601, ChinabCardiovascular Research Center, Anhui Medical University, Hefei 230601, ChinacDepartment of Pharmacology, The Second Hospital of Anhui Medical University, Hefei 230601, China

A R T I C L E I N F O

Article history:Received 28 April 2014Received in revised form 12 June 2014Accepted 13 June 2014Available online 16 June 2014

Keywords:Ras association domain family 1 isoform ACardiac fibroblastsCardiac fibrosisSmoothmuscle a-actinDNA methyltransferase 3A

A B S T R A C T

Cardiac fibrosis contributes to the pathogenesis of atrial fibrillation (AF). The molecular mechanismsunderlying the cardiac fibrosis remain unclear. However, Ras association domain family 1 isoform A(RASSF1A) is a regulatory tumor suppressor, which is important for pathogenesis of cardiac fibrosis andfibroblasts activation. Moreover, DNA methylation plays a central role in the maintenance of cardiacfibrosis. DNA methyltransferases 3A (DNMT3A) is a critical participant in the epigenetic silencing ofregulatory genes. Here, we report that the downregulation of RASSF1A in cardiac fibrosis is associatedwith DNMT3A. Treatment of cardiac fibroblasts with DNMT3A inhibitor 5-AzadC blocked proliferation. 5-AzadC also prevented the loss of RASSF1A expression that occurs during activated cardiac fibroblasts. Todetermine the underlying molecular mechanisms, we hypothesized that cardiac fibrosis is controlled byDNMT3A. We demonstrated that downregulation of RASSF1A is associated with cardiac fibrosis andfibroblasts activation. Knockdown of DNMT3A elevated RASSF1A expression in activated cardiacfibroblasts. Moreover, we investigated the effect of RASSF1A on the Ras/ERK pathway. Upregulation of p-ERK1/2 was detected in activated cardiac fibroblasts with decreased RASSF1A expression. Our resultshave shown that DNMT3A likely plays an essential role in RASSF1A mediated upregulation of ERK1/2 inrat cardiac fibrosis. DNMT3A and RASSF1A may serve as a new mechanism for cardiac fibrosis.

ã 2014 Elsevier Ireland Ltd. All rights reserved.

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Toxicology

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1. Introduction

Cardiac fibrosis results in an increased deposition of theextracellular matrix (ECM) proteins fibronectin and collagen (deHaas et al., 2014). Cardiac fibrosis contributes to pathogenesis ofatrial fibrillation (AF), which is the most common arrhythmiaencountered in clinical practice and remains a major cause ofmorbidity and mortality (Li et al., 2013; Velmurugan et al., 2013).However, cardiac fibroblasts, the most numerous cell type in theheart, are a major factor in the pathogenesis of cardiac fibrosis(Williams et al., 2014). Moreover, cardiac fibroblasts activation isalso associated with an increase in type I collagen (Col1A1) andsmooth muscle a-action (a-SMA) expression, and an increase incellular proliferation (Driesen et al., 2014). Numerous evidence

* Corresponding author. Tel.: +86 551 63869531; fax: +86 551 63869531.** Corresponding author.

E-mail addresses: yjjncs01@126.com (J.-J. Yang), ayskh3@hotmail.com(K.-H. Shi).

http://dx.doi.org/10.1016/j.tox.2014.06.0060300-483X/ã 2014 Elsevier Ireland Ltd. All rights reserved.

pointed to transforming growth factor-b1 (TGF-b1) and platelet-derived growth factor-BB (PDGF-BB) having a key role in themyocardial remodeling process, particularly in cardiac fibrosis(Leask, 2010; Tao et al., 2014; Zhang et al., 2014). In addition, PDGF-BB, as a multifunctional peptide, plays an important role inregulation of numerous physiologic and pathophysiologic process-es, including cell proliferation, differentiation and so on (Son et al.,2014).

Ras association domain family 1 isoform A (RASSF1A) is a tumorsuppressor and the methylation thereof is known to be involved inmany human cancers (Duan et al., 2013; Yang et al., 2014).However, alterations in the regulation of the Ras/MAPK pathwayare frequent in cardiac fibrosis (Arumugam et al., 2013; Li et al.,2013). RASSF1A is a tumor suppressor gene that can regulate Ras/MAPK pathway negatively (Thaler et al., 2012). The Ras/ERK1/2 isactivated in cardiac fibroblasts by PDGF-BB and promotes cellularproliferation. Inhibiting ERK1/2 activity represses cell proliferationand collagen gene expression in activated cardiac fibroblasts(Pramod and Shivakumar, 2014). RASSF1A has been found to beinactivated by promoter methylation in some human tumors

Abbreviations

AF atrial fibrillation5-AzadC 5-aza-20-deoxycytidinea-SMA smooth muscle a-actionCol1A1 type I collagenDNMT3A DNA methyltransferase 3ADNMTs DNA methyltransferasesECM extracellular matrixPDGF-BB platelet-derived growth factor-BBRASSF1A Ras association domain family 1 isoform ATGF-b1 transforming growth factor-b1

H. Tao et al. / Toxicology 323 (2014) 42–50 43

(Wang et al., 2014). However, little is known about how RASSF1Adownregulated induces fibrotic change via ERK1/2 in cardiacfibrosis.

Gene silencing can be achieved by several epigenetic mecha-nisms: DNA methylation, microRNAs, histone modification, and soon (Hewitt et al., 2014). DNA methylation of CpG dinucleotides is akey epigenetic modification that influences gene expression (Kellerand Yi 2014; Tao et al., 2013). CpG methylation is catalyzed by afamily of DNA methyltransferase enzymes comprising of threemembers – DNMT1, DNMT3A and DNMT3B (Valente et al., 2014).In neural progenitors, DNMT3A has been shown to enable theexpression of neurogenic genes through gene body methylation(Wu et al., 2010). DNMT3A acts as a critical participant in theepigenetic silencing of the regulatory genes (Challen et al., 2012).Treatment of cardiac fibroblasts with the DNMT3A inhibitor 5-aza-20-deoxycytidine (5-AzadC) blocks cardiac fibroblasts activationand proliferation (Watson et al., 2014).

However, DNMT3A controls RASSF1A expression in cardiacfibroblasts activation has not been investigated. Given the role ofRASSF1A in mediating ERK1/2 activation, we hypothesized thatRASSF1A plays an important role in regulating cellular functionsassociated with cardiac fibroblasts and fibrosis. In this article, weinvestigated that DNMT3A induces rat cardiac fibrosis throughRASSF1A mediated upregulation of ERK1/2.

2. Materials and methods

2.1. Reagents

Isoprenaline was purchased from Shanghai Hefeng ChemistryPlant (Shanghai, China). Mouse monoclonal antibodies for a-SMAwere purchased from Boster (Wuhan, China), goat anti-RASSF1Apolyclonal antibody was purchased from Santa Cruz (California,USA), DNMT3A polyclonal antibody were purchased from Abcam(Cambridge, UK). ERK1/2, p-ERK1/2 antibodies were purchasedfrom Cell Signaling (Beverly, MA, USA). PDGF-BB was purchasedfrom Peprotech Company (Peprotech, USA). 5-aza-20-deoxycyti-dine was obtained from Sigma Company (Sigma–Aldrich, St. Louis,MO). DNMT3A, RASSF1A, a-SMA, Col1A1 primers were producedby the Shanghai Sangon Biological and Technological Company(Shanghai, China). Streptavidin peroxidase (SP) immunohisto-chemical kit was obtained from the Zhongshan BiotechnologyCorporation (Beijing, China). Reverse transcription reaction systemand SYBR Green Real Master Mix were purchased from MBIFermentas Corporation (Ontario, Canada). Secondary antibodiesfor goat anti-rabbit immunoglobulin (Ig), G horse radish peroxi-dase (HRP), rabbit anti-goat IgG HRP, goat anti-mouse IgG HRPwere obtained from Santa Cruz Biotechnology (Santa Cruz,California, USA).

2.2. Animal models

Forty adult male Sprague-Dawley (SD) rats weighing 200–220 gwere purchased from the Experimental Animal Center of AnhuiMedical University. The SD rats were randomly divided into twogroups (twenty rats per group). All animal experiments wereapproved by the Anhui Medical University Institutional AnimalCare and Use Committee. The male SD rats were injectedsubcutaneously with ISO (2.4 mg/kg/day, 7 days, n = 20) to induceexperimental cardiac fibrosis. Cardiac fibrosis was produced by acommon ISO as previously described (Miyoshi et al., 2014). Oneweek later, the animals were anesthetized, killed and their heartswere harvested. Cardiac tissue specimens were fixed in 4%phosphate-buffered paraformaldehyde. Other specimens weresnap-frozen in liquid nitrogen and stored at �80 �C for RNA andprotein analysis.

2.3. Cell culture and treatment

Cardiac fibroblasts were harvested from SD neonate rats andcultured. Cardiac fibroblasts cells can be cultured with PDGF-BB(10 ng/ml) on plastic in serum-containing media. The mediumcontaining PBS only was regarded as a control. Cardiac fibroblastscells were cultured on plastic in 90% DMEM medium (GIBCO,Invitrogen Corporation, NY) supplemented with 10% fetal bovineserum(GIBCO,InvitrogenCorporation,NY),100 U/mlpenicillinsulfateand 100 U/ml streptomycin, pH 7.4, in a CO2 incubator (ThermoElectronic, USA) with a humidified atmosphere of 5% CO2 at 37 �C.

2.4. Treatment with 5-aza-20-deoxycytidine

Cardiac fibroblasts cells were seeded overnight in 24-wellplates, 5-AzadC (Sigma–Aldrich, St. Louis, MO) was added and wasrefreshed every 24 h until the 48 h treatment finished. The mediumcontaining PBS only was regarded as a control.

2.5. MTT assay

A cell proliferation assay was performed with MTT kit (Sigma,St. Louis, MO) according to the manufacturer's instruction. Viablecells were counted by trypan blue staining. Cardiac fibroblasts cells(5 �103/ml) were cultured with various concentrations of 5-AzadCand PDGF-BB for 24, 48 h in 96-well plates. After culture, 5 mg/mlMTT (Sigma) reagent was added and incubated for 1 h at 37 �Cbefore adding DMSO to dissolve formazan crystals and measuringin triplicate at 490 nm wavelength using a Thermomax microplatereader (bio-tekEL, USA). All experiments were performed intriplicate and repeated at least three times.

2.6. Enzyme-linked immunosorbent assay (ELISA)

Blood samples were taken from the rat. Total protein wasextracted from plasma was centrifuged at 3000 rpm for 10 min.Plasma concentrations of PICP (R&D Systems, USA), PIIINP (UscnLife Science Inc., Wuhan, China), and PDGF-BB (R&D Systems, USA)were determined by enzyme immunoassay (EIA) using commercialassay systems as per the manufacturer's instructions. Finally, theOD (optical density) of each well was immediately assayed at450 nm using a micro-plate reader (Thermo Fisher, USA).

2.7. Histopathology

Cardiac specimens were fixed at 10% neutral buffered formalinand then embedded in paraffin for light microscopic examinationand sectioned at a thickness of 5 mm. Hematoxylin and eosin (H&E)

Table 1Comparison of collagen I and PCIII in various groups (x � s, n = 20).

Group PICP/mg l�1 PIIINP/mg l�1 PDGF-BB/mg l�1

Normal 101.45 � 26.11 78.69 � 23.21 20.69 � 6.13Model 175.78 � 51.49* 129.58 � 41.26* 41.16 � 11.34*

* p < 0.01 vs normal.

44 H. Tao et al. / Toxicology 323 (2014) 42–50

staining was used to examine the changes in liver pathology. Thecollagen deposition in liver tissue was evaluated by Masson'strichrome staining. Each section was assessed under light micro-scopic fields.

2.8. Immunohistochemistry

Immunohistochemical staining was performed on paraffinembedded heart tissue sections of 5 mm thickness, which weredeparaffinized, treated with 0.3% endogenous peroxidase blockingsolution for 20 min. Sections were treated sequentially with 3%hydrogen peroxidase in methanol for 10 min at room temperatureand washed with PBS for 5 min three times to block endogenousperoxidase activity. After high pressure cooking retrieval and 5%bovine serum albumin (BSA) blocking, sections were incubatedovernight at 4 �C with one of the following primary antibodies: ratpolyclonal anti-RASSF1A (1:100), polyclonal anti-DNMT3A(1:200), anti-a-SMA (1:100). Primary antibodies were detectedby rabbit anti-mouse and goat anti-rabbit non-biotinylated regents(Zhongshan, Beijing, China), according to the instructions of themanufacturer. At least five random fields of each section wereexamined, and semiquantitative evaluations were analyzed with aPhoto and Image Auto Analysis System (Image-Pro-Plus, China).

2.9. Immunofluorescence

Cells were fixed with 4% (w/v) paraformaldehyde in PBS for10 min, permeabilized in 0.1% Triton X-100 in PBS for 10 min andwashed several times with PBS. The cells were then incubated in 5%(v/v) FBS in PBS for 30 min for blocking and stained with primaryantibody at room temperature (25 �C) for 1 h. Cardiac fibroblastswere stained with anti-DNMT3A, RASSF1A antibody, and sectionswere stained with the antibodies anti-DNMT3A and anti-a-SMA(Abcam, UK) overnight at 4 �C and then secondary antibodies for2 h at 37 �C. The nuclei were stained by DAPI (40,6-diamidino-2-phenylindole; Sigma). Coverslips were mounted on to microscopeslides using fluorescence mounting medium (Dako) and observedunder an inverted fluorescence microscope (Olympus).

2.10. Real-time fluorescent quantitation PCR assay

Total RNA was extracted from rat cardiac tissues and cardiacfibroblasts using TRIzol reagents (Invitrogen). 1 ml of reversetranscription (RT) product was subjected to real-time PCR analysis.The primers for rat DNMT3A, RASSF1A, a-SMA, Col1A1 werepurchased from the Shanghai Sangong Corporation and the SYBRGreen Real Master Mix was purchased from MBI Formentas (Ontario,Canada). The PCR was performed at 95 �C for 10 min followed by 40cycles at 95 �C for 15 s and at 60 �C for 1 min. The cycle threshold (CTvalue) of the target genes was normalized to that of b-actin to obtainthe delta CT (DCT). The ratio of the relative expression of target genesto b-actin was calculated by using the 2DCT formula. The sequencesof primers are listed as follows: b-actin (forward: 50-TGAG-CTGCGTGTGGCCCC TGAG-30; reverse: 50-GGGGCA TCGGAACCGCT-CATTG-30), a-SMA: (forward: 50-TGGCCACTGCTGCTTCCTCTTCTT-30;reverse: 50-GGGGCCAGCTTCGTCA TACTCCT-30), Col1A1: (forward:50-TACAGCACGCTTGTGGATG-30; reverse: 50-TTGAGTTTGGGTT-GTTGGTC- 30), DNMT3A: (forward: 50-GGCCCAT TCGATCTGGTGA;reverse: 50- CTTGGCTATTCTGCCGTGTTC-30), RASSF1A: (forward: 50-GAGACACCTGATCTTTCCCA -30; reverse: 50- CTGGAAGGCACTGAAACA -30).

2.11. RNA interference (RNAi) analysis

RNAi experiments in cardiac fibroblasts cells were performedby forward transfection in cultured cardiac fibroblasts using

Lipofectamine RNAi Max (Invitrogen) according to the manufac-turer's protocol. Small interfering RNA (siRNA) oligonucleotidesagainst DNMT3A genes or scrambled sequences were synthesizedby the Shanghai GenaPharma Corporation. The following siRNAsequences were used: si- DNMT3A (rat), 50-GCGUCACACAGAAG-CAUAUTT' (sense) and 50-AUAUGCUUCUGUGUGACGCTT-30 (anti-sense); si-control with scrambled sequence (negative controlsiRNA having no perfect matches to known rat genes), 50-UUCUCCGAACGUGUCACGUTT-30 (sense) and 50-ACGUGACAC-GUUCG GAGAATT-30 (antisense). The cells were transfected with2 mg siRNA targeting DNMT3A, or a negative control using a squarewave electroporator and allowed to grow for 48 h prior topreparation of RNA and whole cell extracts.

2.12. Western blotting

Cardiac tissues and cardiac fibroblasts cells were lysed withlysis buffer (Beyotime, China). Total protein (30 mg) from samplesof interest were then fractionated by electrophoresis through a 12%SDS-PAGE. Gels were run at a 120 V for 1.5 h before transferringonto a PVDF membrane. After blockade of nonspecific proteinbinding, nitrocellulose blots were incubated for 1 h with primaryantibodies diluted in TBS/Tween20. Antibody to DNMT3A,RASSF1A, a-SMA, ERK1/2, p-ERK1/2 and b-actin was diluted in1:200–1:1000. Following incubation with primary antibodies,blots were washed three times in TBS/Tween-20 before incubationfor 1 h in goat anti-mouse or mouse anti-rabbit horseradishperoxidase conjugate antibody at 1:5000 dilution in TBS/Tween-20containing 5% skimmed milk. After extensive washing in TBS/Tween-20, the blots were processed with distilled water fordetection of antigen using the enhanced chemiluminescencesystem, Proteins were visualized with the ECL-chemiluminescentkit (ECL-plus, Thermo Scientific).

2.13. Statistical analysis

Quantitative data are expressed as mean � SD. Statisticalsignificance was determined by either the Student's t-test forcomparison between means or one-way analysis of variance with apost hoc Dunnett's test. If p < 0.05, the result was considered to bestatistically significant.

3. Results

3.1. Biochemical determinations

In the three weeks experiments, ISO treatment significantlyincreased the plasma PDGF-BB, PICP and PIIINP. Compared with thenormal rats, the model rats had obvious cardiac inflammation andinjury. These results showed that cardiac fibrosis was successfullytreated by ISO in rats (Table 1).

3.2. Pathological changes

Using H&E and Masson's trichrome staining, H&E stainingrevealed that the increase in cardiomyocyte width in the ISO group

Fig. 1. Pathological changes in isoprenaline (ISO)-caused rat cardiac fibrosis model. A piece of the heart tissue from each rat in the ISO rat model was fixed with formalin, andthen it was embedded in paraffin. Thin sections were cut and stained with hematoxylin and eosin (H&E), Masson's trichrome stain. Rats were grouped as vehicle (control) rathearts or ISO (fibrotic) rat hearts. Representative views from each group (n = 20/group) are presented. The numbers in the views represent the groups of rats in theexperiments.

H. Tao et al. / Toxicology 323 (2014) 42–50 45

compared with normal group (Fig. 1) and Masson's trichromestaining shown that cardiac collagen deposition compared withthe control. In total, one week after ISO-treatment, increasedfibrosis was observed in rat cardiac tissue.

3.3. DNMT3A overexpressed in cardiac fibroblasts activation andfibrosis

Immunohistochemistry demonstrated that a-SMA proteinexpression was significantly increased in the model groupscompared with the control. However, cardiac subjected to ISOinjury to induce fibrosis displayed strong DNMT3A staining infibrosis tissue. To help identify the DNMT3A positive cells weperformed dual staining with anti-DNMT3A and anti-a-SMA(Fig. 2A). Double immunofluroescence assay indicate thatDNMT3A and a-SMA were expressed in cardiac wound healingtissues. Therefore, these results have shown that DNMT3A anda-SMA overexpression in cardiac fibrosis tissue (Fig. 2B).

In vitro, we showed that DNMT3A and a-SMA mRNA expressionwas increased in activated cardiac fibroblasts (Fig. 2C). Moreover,western blot revealed that DNMT3A and a-SMA protein were alsoincreased in activated cardiac fibroblasts (Fig. 2D). These dataindicated that DNMT3A plays a key role in the pathobiology ofcardiac fibrosis and suggest that epigenetic mechanisms mayregulate the phenotype and function of the activated cardiacfibroblasts.

3.4. RASSF1A decreased in cardiac fibroblasts activation and fibrosis

RASSF1A mRNA expressions in rat cardiac fibrosis tissue weredecreased significantly. However, Col1A1 mRNA expressions in ratcardiac fibrosis tissue was increased significantly (Fig. 3A).Furthermore, western blot shown that an obvious decrease for

RASSF1A protein expression in model group compared with thevehicle group (Fig. 3B).

In vitro, western blot shown that RASSF1A protein expressiondecreased during activated cardiac fibroblasts, which was treatedwith PDGF-BB (Fig. 3C). The QRT-PCR suggested expression levelsof RASSF1A mRNA were decreased in activated cardiac fibroblasts(Fig. 3D). What's more, we showed that RASSF1A protein wasexpressed at detectable levels in cardiac fibroblasts, which wasuntreated with PDGF-BB. However, the protein, mRNA expressionlevels of Col1A1 in activated cardiac fibroblasts increasedsignificantly higher than those from the control group (Fig. 3Cand D).

3.5. DNMT3A inhibits RASSF1A expression in cardiac fibroblastsactivation

As shown in Fig. 4A and B, treatment of cardiac fibroblasts withDNMT3A inhibitor 5-AzadC, it elevated expressions level ofRASSF1A mRNA and protein suggesting that an epigeneticmechanism was operated to inhibit RASSF1A expression inactivated cardiac fibroblasts. However, the protein, mRNA expres-sion levels of Col1A1 and a-SMA decreased significantly than thegroup from 5-AzadC untreated cells. Moreover, treatment ofcardiac fibroblasts with 5-AzadC had a profound inhibitory effecton PDGF-BB-induced fibroblasts proliferation (Fig. 4C).

To determine whether DNMT3A inhibits RASSF1A mRNAexpression, cardiac fibroblasts were transfected at high efficiencywith an siRNA vector designed to inhibit DNMT3A expression(Fig. 5A). Cardiac fibroblasts transfected with DNMT3A siRNAexpressed higher levels of RASSF1A protein and mRNA relative tocells transfected with a control siRNA (Fig. 5A and B). These dataindicated that DNMT3A repressions RASSF1A expression inactivated cardiac fibroblasts.

Fig. 2. DNMT3A, a-SMA overexpression in rat cardiac fibrosis and fibroblasts. (A) DNMT3A immunostaining on sections of vehicle rat heart or ISO (fibrotic) rat hearts. Imagesshow absence of DNMT3A in vehicle rat heart and selective DNMT3A expression located in fibrotic lesions of diseased heart. Images show a-SMA staining localized selectivelyto smooth muscle cells lining vessels of vehicle rat heart and to myofibroblasts in association with fibrotic lesions in diseased heart. (B) Double immunofluroescence assayindicate that DNMT3A and a-SMA were overexpressed in cardiac fibrosis tissues. (C) Rat heart tissues RNA was isolated, and DNMT3A, a-SMA expression were evaluated byqRT-PCR. Total RNA isolated from 0, 24 and 48 h cultures with PDGF-BB of rat CFs, DNMT3A, a-SMA expression were evaluated by qRT-PCR. (D) Total protein isolated from 0, 24and 48 h cultures with PDGF-BB of rat CFs, DNMT3A, a-SMA expression were analyzed by western blotting. Results are mean � SD of triplicate experiments. *p < 0.05,**p < 0.01 vs control (vehicle).

46 H. Tao et al. / Toxicology 323 (2014) 42–50

3.6. RASSF1A suppressed ERK1/2 signal pathway

In our study, RASSF1A mRNA expression was decreased, withconcomitantRas/ERK1/2 pathwayactivationin rat cardiac fibrosis. Inorder to further explore the underlying mechanism for the RASSF1Ain cardiac fibroblasts activation, wedetectedthe effectofRASSF1Aonthe Ras/ERK1/2 pathway. ERK1/2 is a downstream factor in Rassignaling. Western blotting showed that RASSF1A suppressed ERK1/2 phosphorylation, which promoted the cellular proliferation.Upregulation of p-ERK1/2 was discovered in activated cardiacfibroblasts with decreased RASSF1A expression (Fig. 6A).

Moreover, 5-AzadC and DNMT3A siRNA treated cardiacfibroblasts were analyzed via western blotting. The upregulationof RASSF1A was induced by 5-AzadC treated, however, the proteinlevel of p-ERK1/2 was decreased by 5-AzadC treated compared tountreated cardiac fibroblasts cells (Fig. 6B).

4. Discussion

Cardiac fibrosis is a multi-factorial disease that occurs in severalpathological processes, including hypertension, diabetes, viralmyocarditis, genetic mutations and so on (Martinez-Martinez

Fig. 3. RASSF1A decreased in cardiac fibroblasts activation and fibrosis. (A) Rat heart tissues RNA was isolated, and RASSF1A, Col1A1, b-actin expression were evaluated byqRT-PCR. (B) Total protein isolated from rat heart tissues, RASSF1A, Col1A1, b-actin were analyzed by western blotting. (C) Total RNA isolated from 0, 24 and 48 h cultures withPDGF-BB of rat CFs, DNMT3A, Col1A1, b-actin expression were evaluated by qRT-PCR. (D) Total protein isolated from 0, 24 and 48 h cultures with PDGF-BB of rat CFs, DNMT3A,Col1A1, b-actin expression were analyzed by western blotting. Results are mean � SD of triplicate experiments. *p < 0.05, **p < 0.01 vs control (vehicle).

H. Tao et al. / Toxicology 323 (2014) 42–50 47

et al., 2014). Cardiac fibroblasts play a key role in the pathogenesisof cardiac fibrosis. To assess the possible mechanisms ofhypertension and inflammation-induced cardiac fibrosis, weevaluated the effects of biomarkers Col1A1 in the rat hearts.ELISA assay showed that the inflammation significantly increasedthe expression of PDGF-BB, PICP and PIIINP. These resultsdemonstrated that inflammatory stress markedly exacerbatedaccumulation in cardiac blood vessels and cardiac collagen

deposition, which contributed to the progression of cardiacfibrosis.

In the present study, DNMT3A has a specific role in permittinghematopoietic stem cell (HSC) differentiation, as in its absence,phenotypically normal stem cells accumulate but progressivelylose their differentiation capacity. DNMT3A-null HSCs show bothincreased and decreased methylation at distinct loci, includingsubstantial CpG island hypermethylation (Challen et al., 2012).Here we found that 5-AzadC normalizes the function of cardiac

Fig. 4. Treatment of rat CFs with 5-AzadC prevents their activation in vitro. (A) Activated CFs cells were cultured in the presence of 1 mM 5-AzadC for up to 48 h. Total RNA wasmade from the cells at time points 0, 24 and 48 h as well as the control untreated cells. RASSF1A, Col1A1, a-SMA and b-actin mRNA were measured by qRT-PCR. (B) ActivatedCFs cells were cultured in the presence of 1 mM 5-AzadC for up to 48 h. Whole-cell protein extracts were made from the cells at time points 0, 24 and 48 h as well as the controluntreated cells. RASSF1A, Col1A1, a-SMA and b-actin protein were analyzed by western blotting. (C) Rat CFs were plated out onto plastic in several separate dishes. 5 �103 CFscells were seeded in triplicate on day 0 and incubated in 10% DMEM or same media supplemented with 1 mM 5-AzadC for further 2 days. Proliferation was measured on day 3by adding 5 mg/ml MTT reagent per well for 4 h. Results are representative of at least three independent experiments. *p < 0.05,**p < 0.01 vs control, #p < 0.05,##p<0.01 vsmodel.

48 H. Tao et al. / Toxicology 323 (2014) 42–50

fibroblasts in vitro. This led us to hypothesize that DNA methylationaffect gene expression in fibroblasts is important in cardiacfibrosis.

Our work demonstrated that 5-AzadC reduced cardiac fibro-blasts cell viability by inhibition of cell proliferation. Generally, 5-AzadC is incorporated into DNA during replication, resulting insequestration of DNA methyltransferases (DNMTs), leading toreplication-dependent global demethylation and gene reactivation(Palii et al., 2008). DNMT3A may serve as a critical participant inthe epigenetic silencing of the regulatory genes (Vaid et al., 2012).

Changes in the expression or activity of DNMT3A may thereforeprovide mechanisms by which the cardiac fibroblasts proliferationis generated and maintained. However, immunohistochemicalassays show that DNMT3A is overexpressed in cardiac fibrosis, andimplicate DNMT3A as a regulator of cardiac fibrosis. Its expressioncorrelated strongly with the activation process as measured byinduction of Col1A1 and a-SMA, known markers of cardiacfibroblasts activation. To discover this idea, we used a candidate

gene approach focusing on mechanisms responsible for transcrip-tional suppression of RASSF1A. The rationale for this approach isbased on the fact that RASSF1A is a suppressor of fibrosis disease.We have shown that decreased RASSF1A expression is associatedwith cardiac fibrosis. Silencing of DNMT3A in cardiac fibroblastsinhibited activation as detected by the increase in DNMT3Aexpression. DNMT3A silencing might have prevented Ras/ERK1/2signaling in cardiac fibroblasts and hence inhibited activation andgrowth.

Furthermore, our studies emphasize Ras signal pathway as apotential therapeutic target for cardiac fibrosis, confirming previousstudies that suggested a prominent role of Ras signaling in cardiacfibrosis (Li et al., 2011). Phosphorylated ERK1/2 was examined in theratcardiacfibrosistissueswithreducedRASSF1Aexpression.Previousstudies have shown that increased Ras signaling within the fibroticmicroenvironment is due to autocrine and paracrine growth factorstimulation (Bechtel et al., 2010; Nagalingam et al., 2013). Our results

Fig. 5. DNMT3A siRNA directed against DNMT3A, which is able to alleviate RASSF1A expression. (A) 5 �104 CFs cells were seeded onto 10 cm dishes and transfected with 1 mgsiRNA-DNMT3A which includes an expression cassette for green fluorescent protein to enable visualization of transfected cells. CFs cells transfected with siRNA-DNMT3A orcontrol siRNA-Scrambled were harvested 48 h after transfection, total RNA made. DNMT3A, RASSF1A and b-actin were evaluated by qRT-PCR (B) CFs cells transfected withsiRNA-DNMT3A or control siRNA-scrambled were harvested 48 h after transfection, whole-cell extracts made. DNMT3A, RASSF1A and b-actin were analyzed by westernblotting. Results shown were representative of three independent experiments. *p < 0.05, **p < 0.01 vs control, #p < 0.05,##p < 0.01 vs model.

Fig. 6. RASSF1A suppressed ERK1/2 signal pathway. (A) CFs cells were treated with PDGF-BB (10 ng/ml) for 24, 48 h. Expression of RASSF1A, p-ERK1/2, ERK1/2 was estimatedin parallel by western blotting. The expression of b-actin was used as a control. (B) Activated HSC were cultured in the presence of 1 mM 5-AzadC for up to 24, 48 h as well as

H. Tao et al. / Toxicology 323 (2014) 42–50 49

50 H. Tao et al. / Toxicology 323 (2014) 42–50

show a new mechanism of epigenetic RASSF1A silencing causingincreased intrinsic Ras activity in cardiac fibroblasts.

In summary, this may be the first report of DNMT3A thatinhibits RASSF1A expression as a new mechanism of cardiacfibrosis. RASSF1A may affect cardiac fibroblasts activation byinfluencing ERK1/2 signal pathway. These findings demonstratedthat epigenetic and pharmacological disruptions of DNMT3Areduced the cardiac fibroblasts activation and attenuated fibrosis.Future studies are needed to explicate the mechanisms ofepigenetic therapeutics for cardiac fibrosis.

5. Conclusion

Cardiac fibroblasts play a key role in the pathological process ofcardiac fibrosis. The ERK1/2 signaling pathway has long beenrecognized as the intracellular signal transduction enzymes whichare critically involved in regulating fibroblasts proliferation.DNMT3A knockdown by siRNAs inhibited the RASSF1A expression,which influences ERK1/2 signal pathway activation. These resultsindicated that DNMT3A silencing RASSF1A promotes cardiacfibrosis through upregulation of ERK1/2.

Conflict of interest

The authors declare that there are no conflicts of interest.

Transparency document

The Transparency document associated with this article can befound in the online version.

Acknowledgement

This project was supported by Anhui Provincial Natural ScienceFoundation (1308085MH117, 1408085MH175).

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