TLR2-ICAM1-Gadd45α Axis Mediates the Epigenetic Effectof Selenium on DNAMethylation and Gene Expression in KeshanDisease

Guang Yang & Yanhe Zhu & Xin Dong & Zongming Duan &

Xiaolin Niu & Jin Wei

Received: 17 January 2014 /Accepted: 15 April 2014 /Published online: 9 May 2014# Springer Science+Business Media New York 2014

Abstract Keshan disease (KD) is a fatal dilated cardio-myopathy with unknown etiology, and selenium deficiencyis considered the main cause of KD. Several observationsimplicate a role for altered DNA methylation in seleniumdeficiency-related diseases. The aim of the present studywas to investigate the epigenetic effects of selenium (Se)on DNA methylation and gene expression in Keshandisease. Using methylated DNA immunoprecipitation chip(MeDIP-Chip) and quantitative RT-PCR, we identified twoinflammatory-related genes (TLR2 and ICAM1) that weredifferentially methylated and expressed between normalindividuals and KD patients. Results from DNA methyla-tion profile between KD patients and normal individualsshowed that selenium deficiency decreased methylation ofCpG islands in promoter regions of TLR2 and ICAM1and upregulated messenger RNA (mRNA) and proteinlevels of TLR2 and ICAM1. In rat animal model of Keshandisease, selenite treatment could increase TLR2 and ICAM1promoter methylation, suppress these genes expression, andreduce infiltration of myocardial inflammatory cells. In cellculture model of Keshan disease, we found 5-Aza-dC (DNMT1inhibitor) treatment in the presence of selenium-reducedmRNA and protein levels of DNMT1 regardless of TLR2 andICAM1 promoter methylation status and expression levels ofthese genes. Selenite treatment suppressed the expression of theGadd45α, TLR2, and ICAM1 in a concentration-dependent

manner, while selenium deficiency increased the expressionof the Gadd45α, TLR2, and ICAM1 and decreased TLR2and ICAM1 promoter methylation level in a time-dependentmanner. Our results revealed that TLR2-ICAM1-Gadd45α axismight play an important role in gene-specific active DNAdemethylation during inflammatory response in myocardium.

Keywords Keshan disease . DNAmethylation . Gadd45α .

TLR2 . ICAM1

Introduction

Keshan disease (KD), a potentially fatal form of congestive(or dilated) cardiomyopathy, first found in Keshan county(northeast China), is characterized by pulmonary edema andheart failure [1–4]. As one of several types of nutritionaldiseases, Keshan disease is unique in that it primarily affectsyoung women and children [5]. Keshan disease resultsfrom a selenium deficiency which is a nutrient we receivein our diet from eating foods that were grown inselenium-enriched soils [6]. Because of that factor, Keshandisease can be found anywhere that the level of seleniumpresent in the soil is low [7].

The molecular and immune mechanisms in the pathogen-esis of Keshan disease are becoming better understood, andstudies in both experimental models and human clinical trialsdemonstrate the important relevance of dysregulated geneexpression [8, 9]. Furthermore, transcriptomic analyses ofanimal models of Keshan disease showed a consistentand distinct pattern of gene expression, and dysregulatedexpression of both coding and noncoding genes directlyaffects cardiomyopathy development and progression [10].Epigeneticmechanisms such as histone acetylation, microRNA,and DNA cytosine-methylation have been shown to play

Electronic supplementary material The online version of this article(doi:10.1007/s12011-014-9985-8) contains supplementary material,which is available to authorized users.

G. Yang :Y. Zhu :X. Dong : Z. Duan :X. Niu (*) : J. WeiDepartment of Cardiology, The Second Affiliated Hospital, KeyLaboratory of Environment and Genes Related to Diseases ofEducation Ministry, Xi’an Jiaotong University School of Medicine,Xi’an, Shaanxi 710004, People’s Republic of Chinae-mail: xlinniu1@126.com

Biol Trace Elem Res (2014) 159:69–80DOI 10.1007/s12011-014-9985-8

important roles in the development of cardiomyopathy, withoutinvolving any change in the underlying DNA sequence[11–14]. DNA cytosine-methylation alters accessibility for tran-scription factor complexes at a local level and, like histonemodifications, affects chromatin structure at regional andgenome-wide levels [15]. Awell-characterized functional effectof DNA methylation is thus control of gene expression [16]. Inthis respect, hypomethylation of the 5’ promoter end of a genecorrelates with increased expression of the gene, whereas hyper-methylation within the body of a gene is linked to increasedgene expression [14–16].

DNA hypermethylation is commonly associated with in-creased levels or altered function of DNA methyltransferases(DNMTs) [17]. Additionally, histone modifications, particu-larly methylation and acetylation, may also be involved intranscriptional silencing of a number of genes in humandiseases such as cancer [17–19]. Growth arrest and DNA-damage-inducible protein 45 alpha (Gadd 45α) plays a pivotalrole in cellular stress responses and is implicated in DNArepair, cell cycle arrest, and apoptosis [20, 21]. Recently, itwas proposed that Gadd 45α is a key regulator of active DNAdemethylation by way of its role in DNA repair [22]. It wasreported that Gadd 45α overexpression activated transcriptionfrom methylation-silenced reporter plasmids and promotedglobal DNA demethylation [23]. Gadd45α specifically inter-acts with the catalytic site of DNMT1 and inhibits methylationactivity in vitro [24].

Selenium (Se) is an essential trace element for humans,animals, and some species of microorganism [25, 26].Several microbiological Se-containing enzymes have beenalso identified, e.g., glycine reductase, formate dehydroge-nase, hydrogenase, nicotinic acid hydroxylase, xanthinedehydrogenase, and thiolase. Recent data suggest thatsome dietary supplements, including selenium (Se), mayprevent many diseases (cancer, Alzheimer's disease, heartdisease, cardiovascular disease, etc.) by modifications ofepigenetic processes in the cell [27, 28]. Studies haveshown that Se inhibited DNMT expression and activity[28, 29]. Thus, Se may regulate expression of some anti-cancer genes by epigenetic modifications of DNA andhistones. Several large population-based intervention trialsusing oral administration of sodium selenite tablets showedsignificant reduction of Keshan disease incidence [30, 31].

In the present study, the peripheral blood DNAmethylationprofile between KD patients and normal individualswere preliminarily determined to evaluate the relation-ship between the blood selenium levels and DNA meth-ylation levels. Then, both cell and animal models ofKeshan disease were established to determine whetherserum selenium levels and the chemical form of seleniumwould affect DNA methylation and gene expression patterns,and the epigenetic regulatory mechanisms of selenium inKeshan disease were explored.

Materials and Methods

Patients Collection and Test Procedures

This study conformed to the Basic Principles of the Declara-tion of Helsinki and was approved by the human researchcommittee of Xi'an Jiaotong University. All participantssigned informed consent statements prior to participation. Atotal of 71 randomly selected KD patients were recruited froma KD endemic area in Shaanxi Province, China. All patientswere asked to fill in a questionnaire about their family historyof KD, living standard, dietary customs, and selenium supple-ment status. KD was diagnosed according to “The DiagnosticStandard of the Keshan Disease” (GB 17021–1997 Diagnos-tic standard for Keshan disease, China) [32]. All patientsunderwent electrocardiography (ECG), radiography, andechocardiography, as well as blood sampling. All patientswere ECG abnormal with or without cardiac dilation. Weselected 78 age- and sex-matched healthy internal-controlsubjects with similar living standards and dietary customsfrom the same area, as well as 212 healthy external controlsnot living in the area. A case–control comparison of selectedsubject baseline characteristics is in Table 1.

Measurement of Whole Blood Selenium Concentration

Samples for Se analysis were acid-digested prior to the Seanalysis, and serum selenium concentration was analyzed byhydride generation atomic absorption spectrometry (AFS2201A, Beijing, China) as previously reported [33, 34].

Methylated-Cytosine DNA Immunoprecipitation–MicroarrayChip (MeDIP-Chip)

Genomic DNA was extracted from peripheral blood usinga commercially available DNA extraction kit (Invitrogen,Carlsbad, CA, USA). The genomic DNA sample wasrandomly sheared by sonication to generate fragmentsbetween 100 and 800 bp. Four micrograms of DNAsample was saved as input and the rest heated to 95 °C for10 min and immediately placed on ice. Immunoprecipitationwas performed using 2.5 μg of α-5′methyl-cytosine antibodyper microgram of sheared gDNA in IP buffer (20 mMNa-phosphate, pH 7.0, 1 M NaCl, 2 % Triton-X100).Samples were rotated overnight at 4 °C, and 10 μl of 50 %Protein-AAgarose slurry (pre-washed in 0.1 % BSA-PBS andequilibrated in IP buffer) was subsequently added permicrogram of DNA. Samples were rotated for further2.5 h and washed three times with IP buffer before elutionusing 250 μl lysis buffer (1 M Tris–HCl, pH 8.0, 0.5 MEDTA, 10 % SDS, 280 μg/ml Proteinase K) and incubationfor 2 h at 55 °C. MeDIP was purified and precipitated usingphenol and chloroform:isoamyl alcohol.

70 Yang et al.

Four micrograms of input and MeDIP for each samplewere labelled with Cy3 and Cy5, respectively, and co-hybridized to the Nimblegen “CpG island and promoter”microarray chip (Roche Nimblegen, Madison, WI , USA).This Nimblegen array chip comprises of 385,000 isothermalprobes of between 50 and 75 mer length with a median probespacing of 101 bp and is based on the HG18 human genomeassembly. These probes cover all reported human Refseq genepromoters (24,659) that ranged from −800 to +200 bp relativeto transcription start sites (TSS) and all reported CpG islands(28,226) annotated on the UCSC genome browser.

Bisulfite Treatment of gDNA, PCR, and Massive ParallelAmplicon Sequencing

Bisulfite (BS) conversion of gDNAwas performed using theEZ DNA Methylation-Gold kit (Zymo Research, Irvine, CA,USA) according to the manufacturer’s protocol. PCR wasperformed using BS-treated gDNA samples as template andBS-specific primers that were designed at a minimum lengthof 20 bp against selected differentially methylated region(DMRs) using the MethPrimer program. (http://www.urogene.org/methprimer/index1.html).

Primers were tested to confirm the amplification of BS-treated gDNA only, and individual PCR products were run on2 % agarose gels to verify product size. Amplicon sequencereads corresponding to the BS-PCR products were aligned tothe Human Reference Genome, and the extent of DNA meth-ylation was determined by comparing the total number of Cs(methylated) to Ts (unmethylated) for each CpG site in asingle DMR. Methylation fraction values with a detection pvalue greater than 0.01 were set to “missing.”

Methylation-Specific Polymerase Chain Reaction (MS-PCR)Analysis

DNAwas extracted from the cells using the DNeasy Tissue Kitand modified with sodium bisulfite using the CpGenome DNAModification Kit (Chemicon, Temecula, CA, USA) accordingto the manufacturer’s instructions. Bisulfite-treated DNA wasanalyzed by methylation-specific polymerase chain reaction

(MS-PCR) with primer pairs for the unmethylated or methyl-ated promoters of TLR2 and ICAM1 as described previously[35–38]. Polymerase chain reactions (PCRs) were run withHotStar Taq polymerase Plus (Qiagen, Hilden, Germany). Allreactions were visualized on 2 % agarose gels stained withethidium bromide.

Human ICAM1(methylated primers)

5′-TTGGGAGTTGTAAAGACGTTTTC-3′ (sense)

5′-GTCCACACCTAACTAACACGAA-3′ (antisense)

Human ICAM1(unmethylated primers)

5′-TTTGGGAGTTGTAAAGATGTTTTT-3′ (sense)

5′-CATCCACACCTAACTAACACAAA-3′ (antisense).

Human TLR2(methylated primers)

5′-TTCGGGAGAAGTAGGGGTTATAC-3′ (sense)

5′-GAAACCTACAAAATTAAAAAAACG-3′ (antisense)

Human TLR2(unmethylated primers)

5′-TTTTTGGGAGAAGTAGGGGTTATAT-3′ (sense)

5′-AAAACCTACAAAATTAAAAAAACACT-3′ (antisense)

Rat ICAM1 (methylatedprimers)

5′-TGTCGAGTTTTAGTGTATTTTTTCG-3′ (sense)

5′-CAACTTTTATAATCTCGAACCACG-3′ (antisense)

Rat ICAM1(unmethylated primers)

5′-GTTGAGTTTTAGTGTATTTTTTTGA-3′ (sense)

5′-AACTTTTATAATCTCAAACCACAAA-3′ (antisense).

Rat TLR2 (methylatedprimers)

5′-CTACCGAAAATCAAAAAAACGAA-3′ (sense)

5′-GGAGTCGTAGTTCGGTTACG-3′(antisense)

Rat TLR2 (unmethylatedprimers)

5′-AGGGGAGTTGTAGTTTGGTTATG-3′ (sense)

5′-CCCTACCAAAAATCAAAAAAACA-3′ (antisense)

RNA Extraction, RT-PCR and Quantitative PCR

Total RNA from samples (peripheral blood mononuclearcells, cells or heart tissues) was isolated using TRIzol

Table 1 Characteristics of KDpatients and normal individuals KD (n=71) Internal control (n=78) p

Age 46.6±10.2 44.1±13.8 0.125

Gender, male (%) 27 (38.0 %) 27 (34.6 %) 0.607

NYHA stage, n (%)

I 24 (33.8 %) 77 (98.7 %) <0.001

II 25 (35.2 %) 1 (1.3 %)

III 20 (28.2 %) 0 (0 %)

IV 2 (2.8 %) 0 (0 %)

Family history of KD, n (%) 29 (40.9 %) 1 (1.3 %) <0.001

TLR2-ICAM1-Gadd45α Axis Mediates the Epigenetic Effect of Selenium 71

reagent® (Sigma-Aldrich, St Louis, Missouri, USA) accord-ing to the manufacturer's protocol. RNA concentrations weredetermined by spectrophotometric measurement at wave-lengths of 260/280 nm. A 20 μl of complementary DNA(cDNA) was synthesized from 1 μg of Total RNA, using amixture of both oligo-dT and random hexamers and the“Superscript-III first strand cDNA synthesis kit” (Invitrogen,Paisley, UK). Integrity of RNA for all samples was checkedusing the 2100 Bioanalyzer (Agilent Technologies, SantaClara, California, USA). Conventional RT-PCRwas performedto analyze the messenger RNA (mRNA) expression. Quantita-tive real-time PCR for housekeeping genes was initially per-formed using 4 μl of 1:20 pre-diluted cDNA in a 20 μl reactionand Taqman Gene Expression Assays specific for GAPDHusing geNorm (http://medgen.ugent.be/,jvdesomp/genorm/#PrimerDesign) we determined that of these housekeepinggenes, GAPDH was most stable for both control and diseasedsamples. Quantitative PCR performed for target genes (TLR2and ICAM1) using validated Taqman Gene Expression Assayprimers (Applied Biosystems, Foster City, California, USA)was therefore normalized against GAPDH. Quantitative PCRfor both target genes and GAPDH were performed at leasttwice in triplicate on the same diluted cDNA samples. Theresults were analyzed by relative quantitation using theΔcyclethreshold method (ΔCt). Primers of DNMT have been de-scribed previously [39]. For beta-actin or GAPDH, the condi-tions were 20 cycles at 95 °C for 30 s, 55 °C for 30 s, and 72 °Cfor 30 s, followed by extension at 72 °C for 5 min. For TLR2,ICAM1 and Gadd45α, the conditions were 35–40 cycles at95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s, followed byextension at 72 °C for 10 min.

The primers were as follows:

Rat GAPDH(primers)

5′-ACCACAGTCCATGCCATCAC-3′ (sense)5′-TCCACCACCCTGTTGCTGTA-3′ (antisense)

Rat β-actin(primers)

5′-GACCGAGCGTGGCTACAGC-3′ (sense)5′-TGTCAGCGATGCCTGGGTAC-3′ (antisense).

Rat ICAM1(primers)

5′-GTGAGCGTCCATATTTAGGCATGG-3′ (sense)5′-CAGACACTAGAGGAGTGAGCAGG-3′ (antisense)

Rat TLR2(primers)

5′-TGCCTTTGTTTCCTACAGCG-3′ (sense)5′-ATCTTGCGCAGTTTGCAGAA-3′ (antisense)

Rat Gadd45α(primers)

5′-TCGAAAGGATGGACACGGTG-3′ (sense)5′-TAGCAACAGCTCTGCCAGCC-3′ (antisense)

Animals and Diets

Sprague–Dawley male rats (n=60) were purchased from VitalRiver Laboratories (VRL, Beijing, China). All rats werehoused individually in stainless steel wire-bottomed cages ina room with controlled temperature and light. The rats wererandomly divided into three groups (20 per group). The ratswere provided free access to demineralized water and purified

diet. The basal diet was a selenium-deficient, torula yeast–based diet. The basal diet contained 300 g/kg torula yeast,3 g/kg DL-methionine, 590 g/kg sucrose, 50 g/kg corn oil,35 g/kg selenium-deficient AIN-76A mineral mix, 12 g/kgcalcium carbonate, 10 g/kg AIN-76A vitamin mix, 0.1 g/kgcholine bitartrate, and 0.01 g/kg menadione sodium bisulfitecomplex. The basal diet was supplemented with 0, 0.1, or2 mg selenium/kg diet as sodium selenite (by analysis, thediets contained 0.004, 0.11, and 2.03 mg selenium/kg diet,respectively). The rats were fed with 0, 0.1, or 2 mg selenium/kg diet daily for 4 weeks.

This study was approved by the Animal Care Committee ofthe Grand Forks Human Nutrition Research Center, and therats were maintained in accordance with the guidelines for thecare and use of laboratory animals.

Animal Blood Samples Collection

Food was withheld overnight before the rats were anesthetizedwith xylazine (Mobay Corp., Shawnee, KS, USA) and keta-mine (Aveco Co., Fort Dodge, IA, USA), hemodynamicchanges were measured by carotid intubation method, struc-ture and morphology changes were observed under an elec-tron microscope, then anesthetized and killed by exsanguina-tion. Blood was collected by cardiac puncture into syringescontaining EDTA such that the final concentration was ~1 gEDTA/L blood. The heart was removed, flushed with saline,and then stored in 4 % formaldehyde at 4 °C. Then, myocar-dial tissue slices are dehydrated and embedded in paraffinwax; HE staining was performed to observe infiltration ofmyocardial inflammatory cells.

Cell Model Studies

Isolation, Culture, and Treatment of Primary Neonatal RatCardiomyocytes

Primary cultures of neonatal rat ventricular cardiomyocyteswere derived according to previously published procedures[53]. In brief, cardiomyocytes were obtained from 1- to 2-day-old neonatal Sprague–Dawley rats by means of a protocolapproved by the committee on animal care at Xi’an JiaotongUniversity (Xi’an, China). The ventricles were quartered,dissociated overnight at 4 °C in a 0.06 % (w/v) solution oftrypsin in Hanks’ balanced salt solution (HBSS, Gibco BRL,Gaithersburg, MD, USA), washed in culture medium(Dulbecco’s modified Eagle’s medium (DMEM) supplementedwith 10–20 % fetal bovine serum (FBS) and 5 % horse serum,10mMHEPES, 2mM l-glutamine, and penicillin–streptomycin[100 units/ml]; Gibco), and then subjected to a series of diges-tions (8 min at 37 °C; 100 g) in a 0.1 % (w/v) solution ofcollagenase type II in HBSS. The harvested cells were thenpooled and resuspended in culturemedium. Cells were preplated

72 Yang et al.

for one 60-min period to enrich for cardiomyocytes. The cellswere seeded at a density of 2×104 cells/cm2 in six-well plates(plates were coated with 5 μg/ml fibronectin+1 % gelatin;Sigma-Aldrich). Bromodeoxyuridine (BrdU, 100 μM; Sigma-Aldrich) was added to the growth medium for 3–4 days toeliminate proliferating fibroblast cells and progenitor cells [40]to ensure >95 % of myocytes remaining are rod-shaped andviable. Cells were seeded at a density of 2×104 cells per well andallowed to attach for 24 h, then the media was changed to 5 %FBS+5 % horse serum-containing DMEM before treatmentwith selenite, 5-Aza-dC for 7 days with media and treatmentagents refreshed every 2 days.

Western Blot Analysis

Cardiomyocyte lysates were resolved by 10 % SDS–PAGEand transferred to a nitrocellulose membrane. TLR2, ICAM1,and β-actin proteins were detected by use of antibodiesagainst TLR2, ICAM1, and β-actin (Santa Cruz Biotechnol-ogy, Santa Cruz, CA, USA) followed by horseradishperoxidase-conjugated secondary antibody. The protein bandswere visualized by the ECL detection system (Amersham,Arlington Heights, IL, USA). Anti-Gadd45α and DNMT1antibodies (Santa Cruz Biotechnology, Santa Cruz, CA,USA) were diluted at 1:2,000 and 1:500, respectively. Proteinbands were visualized on X-ray film using an enhancedchemiluminescence system (Rockford, IL, USA).

Statistical Analysis

Data are presented as mean±SEM and differences betweengroups were analyzed using one-way analysis of variance(ANOVA) with the SPSS 17.0 (SPSS Inc., Chicago, IL,USA). Analysis for MeDIP-chip was performed using theBATMAN algorithm as previously described [41]. Analysisfor quantitative PCR was performed using the nonparametricMann–Whitney t test, and two-tailed p values were used todetermine the statistical significance. Analysis for the associ-ation between DNA methylation and gene expression wasperformed using Spearman’s rank order correlation coeffi-cient. Student’s t test was used to determine the statisticaldifference between data at the level of p<0.05.

Results

Differentially Methylated Genes in KD (KD-DMRs)

To investigate whether DNA methylation in a subset ofgenomic loci might connect with Keshan disease (KD), weinitially set out to profile a series of blood samples of KDpatients and normal controls (Table 1) using MeDIP-chip(NimbleGen MeDIP-chip Protocol). We used the Nimblegen

“CpG island and promoter”microarray chip (RocheNimblegen,HG18_CpG_Promoter array) which covers all annotatedhuman Refseq gene promoters (24,659) and CpG islands(28,226) as annotated on the UCSC genome browser.Based on a differentially methylated region (DMR) T-statistic>+3.0 and <3.0, candidate regions (KD-DMRs)were identified using the validated algorithm-Bayesiantool that had been developed specifically for handlingMeDIP data (BATMAN) [41]. Of the target KD-DMRsmeeting these criteria, two candidates (TLR2 and ICAM1)were identified by gene ontology analysis and GeneCards torelate to inflammation (Fig. 1).

Next, we undertook methylation-specific PCR (MS-PCR)and real-time PCR for these KD-DMRs using other KD sam-ples. Bisulfite treatment of gDNA converts unmethylated-cytosine nucleotides to uracil but leaves methylated-cytosineresidues unaffected. This difference is then detected as a C/Tnucleotide polymorphism at each CpG site by subsequent MS-PCR, providing the gold-standard high-resolution informationabout the methylation status of a DNA region. Q-PCR analysisrevealed that the identified two genes were associated with thethree KD-DMRs, and statistically significant mRNA levelswere observed between control and KD patients (**p<0.01)(Fig. 2a, b). We further analyzed the correlation between geneexpression and differential methylation in each individualpatient directly and independently of disease and found, aspredicted, that 5’region methylation and gene expressionwere inversely correlated. Hypomethylation in the 5’ regionof TLR2 and ICAM1 in KD correlated with increased ex-pression of TLR2 and ICAM1 (Fig. 2c, d).

There was a significant difference of methylation in the 5’region of TLR2 and ICAM1 among the three groups, andmethylation in the 5’region of TLR2 and ICAM1 was lowestin blood DNA samples taken from KD group. Moreover,individual KD samples with the highest or lowest expressionof each of these genes mapped to the predicted methylationstate in their correlation between methylation and expressionindependently of disease state but are correlated with serumconcentration of selenium (Fig. 2e–h).

Hemodynamics Studies

The concentration of selenium significantly influenced thebody weight gain of the rats (p<0.01) (Fig. 3a). Rats fedselenium-supplemented diets (0.1 and 2 mg Se/kg diet)weighed significantly more than rats fed the selenium-deficient (0 mg Se/kg diet) diets (Fig. 3a). The results ofhemodynamic showed that LVP, +dp/dt max and −dp/dt maxin selenium-deficient group were significantly lower thanselenium-supplemented groups (p<0.05) (Table 2). The serumselenium concentrations of rats fed the selenium-deficient dietwere significantly lower than rats fed selenium-supplementeddiets (0.1 and 2 mg Se/kg diet) (p<0.01) (Fig. 3a).

TLR2-ICAM1-Gadd45α Axis Mediates the Epigenetic Effect of Selenium 73

Fig. 1 Differential DNAmethylation profile for twocandidate KD-DMRs. aMethodology work flow forMeDIP-chip. b Venn diagramsummarizing the number of DNAmethylation peaks and theiroverlap. Group 1#: externalcontrol (n=80); group 2#: internalcontrol (n=78); group 3#: Keshangroup (n=71). c Differentiallymethylated regions (DMR)analyzed by bisulfite mutagenesisand sequencing assay. 95 CpGslocated in the promoter and firstintron of the gene TLR2; 21CpGslocated in the promoter of thegene ICAM1

Fig. 2 Differential expression ofgenes related to KD-DMR.a Relative quantification of targetmRNAs (TLR2 and ICAM1) byreal-timeQ-PCR. bRepresentativeMS-PCR analysis of CpG islandmethylation in the TLR2 andICAM1 promoter region. Mmethylated, U unmethylated.c Comparison of blood seleniumconcentration andhypomethylation ratios of targetmRNAs in KD patients andcontrols. d) Blood seleniumconcentration and correspondingfrequency of variants for allsubjects. External control (n=80);internal control (n=78); Keshangroup (n=71). *, #p<0.01,significantly different fromexternal controls

74 Yang et al.

HE staining showed that infiltration of myocardial inflam-matory cells in rats fed with the selenium-deficient diet wasmore abundant than rats fed selenium-supplemented diets(p<0.01). In contrast, when rats were fed selenium-supplemented diets (0.1 and 2 mg Se/kg diet), there was asignificant reduction in infiltration of myocardial inflammato-ry cells (p<0.01) (Fig. 3b). Further electronmicroscopic (EM)analysis showed that most of the myocardial mitochondria in

rats fed with the selenium-deficient diet were enlarged and hadirregular cristae arranged in a disorderly pattern, while myo-cardial mitochondria in rats fed 0.1 or 2.0 mg selenium/kg dietshowed no abnormalities (Fig. 3c).

Then, methylation status and expression level of TLR2 inrat myocardium were verified using MS-PCR and westernblot analyses. Rats fed with the selenium-deficient diet hadsignificantly higher promoter (TLR2 and ICAM1) DNA

Fig. 3 Effects of selenitedeficiency diet on DNAmethylation and gene expressionin rat model of Keshan disease.a Comparison of heart weight,body weight, heart/body weightindex, serum seleniteconcentration in three groups.b Infiltration of myocardialinflammatory cells in threetreatment group was analyzedwith HE staining. c Electronicmicroscopic analysis ofmyocardium ultrastructure.d Left: MS-PCR analysis ofmethylation status of ICAM1and TLR2 in rat myocardiumfrom three treatment group(M methylated, U unmethylated);Right: densitometricquantification analysis. e Left:Western blot analysis ofinflammatory related genes in ratmyocardium from three treatmentgroup. (2: 2 mg/kg selenite diet;0.1: 0.1 mg/kg selenite diet; 0:0 mg/kg selenite diet); Right:densitometric quantificationanalysis. f Q-PCR analysis ofinflammatory related genes in ratmyocardium from three treatmentgroups. (2: 2 mg/kg selenite diet;0.1: 0.1 mg/kg selenite diet; 0:0 mg/kg selenite diet). Data arerepresentative of 3–6 individualmice analyzed in at least twoindependent experiments.*p<0.05;**p<0.01

Table 2 Carotid artery hemody-namics of SD rat animal model ofKeshan disease (X ±S)

*p<0.05; comparedwith selenium-supplemented diets (0.1, 2 mg/kg)

n=14 Selenium-supplemented diets

(2 mg/kg)

Selenium-supplemented diets

(0.1 mg/kg)

Selenium-deficient

(0 mg/kg)

Sp 91.96±3.81 90.26±4.32 85.71±3.56

Dp 41.58±2.108 42.76±3.70 46.04±4.00

Lvp 110.49±1.29 111.00±3.72 98.69±3.24*

Lvedp −0.92±1.59 −1.21±1.44 −2.88±1.18+dp/dt max 6926.11±1609.33 6893.12±1757.37 5291.31±757.64*

−dp/dt max 4523.60±808.93 4366.00±1007.60 3863.32±348.00*

TLR2-ICAM1-Gadd45α Axis Mediates the Epigenetic Effect of Selenium 75

unmethylation but significantly higher TLR2 and ICAM1protein levels than rats fed with supplemental dietary selenium(0.1 and 2 mg Se/kg diet). Selenium treatment increased thelevel of myocardial DNAmethylation and inhibits the expres-sion of TLR2 and ICAM1 (Fig. 3d, e, f). Moreover, seleniumtreatment also decreased DNMT1 protein level (Fig. 3e).

Cell Culture Studies

Reduction of DNA Methylation and Reactivation of TLR2Expression by Selenium Deficiency in Rat NeonatalCardiomyocytes

We first verified methylation status and expression of TLR2and ICAM1 in rat cardiomyocytes using MS-PCR, RT-PCR,and western blot analyses. Rat cardiomyocytes cultured in theabsence of selenium (0 μM) had significantly lower promoter(TLR2 and ICAM1) methylation levels but significantlyhigher TLR2 and ICAM1 mRNA and protein levels than cellscultured in the presence of 0.5 or 1.5 μM selenium, treatmentwith selenite could partially remethylate the promoter ofTLR2 and ICAM1 and inhibit the expression of TLR2 andICAM1 (Fig. 4a, b). RT-PCR and western blot analyses dem-onstrated that regulation of TLR2 and ICAM1 expression byselenite was concentration-dependent after treatment with 0.5and 1.5 μM selenite for 4 days.

To determine the underlying mechanisms responsible forpromoter hypomethylation and upregulation of TLR2 andICAM1, rat cardiomyocytes were treated with 5-Aza-dC inthe presence of 1.5 μM selenium. Cardiomyocytes were treat-ed with 5 μM 5-Aza-dC in the presence of 1.5 μM seleniumfor 7 days; MS-PCR showed that 5-Aza-dC treatment did notchange the promoter methylation status of TLR2 and ICAM1(Fig. 4c, d). The dose of selenite used in the study was anontoxic dose, which caused slight cell growth inhibition with4.6 and 14 % decreases in cell viability at days 7 and 14,respectively (supplementary Figure 1). The doses of 5-Aza-dCused in the study had optimal effects on DNA methylationbut caused significant cell killing (supplementary Figure 1).Consistent with the results of MS-PCR, mRNA and pro-tein expression levels of TLR2 and ICAM1 were observedin cells treated with selenite, but not with 5-Aza-dC in thepresence of 1.5 μM selenium (Fig. 4e). Treatment with 5-Aza-dC, a potent DNMT1 (DNA methyltransferase) inhib-itor, is known to induce demethylation. In our experiment,selenium treatment also inhibited expression level ofDNMT1, which has also been reported by other investiga-tors. 5-Aza-dC showed no effect on TLR2 and ICAM1methylation levels in the presence of selenium, whileDNMT1 was significantly inhibited. All these results demon-strated that selenium-induced TLR2 and ICAM1 methylationlevels were DNMT1 independent.

Gadd45α Might Mediate the Effect of Selenite Deficiencyon DNA Demethylation in Rat Cardiomyocytes

The regulatory mechanism of selenite deficiency-inducedTLR2 and ICAM1 hypomethylation in rat cardiomyocytes isconsidered to be mediated by DNA demethylase activity butnot DNMT1. Recently, a role of the growth arrest and DNA-damage-inducible protein Gadd45α in DNA demethylationhas been reported, but whether Gadd45α participates in activeDNA demethylation at a gene-specific pol II promoter andpromotes mRNA transcription in rat cardiomyocytes is stillunknown. To determine whether selenite treatment wouldalter activity of Gadd45α in rat cardiomyocytes treated withselenite or 5-Aza-dC, protein level of Gadd45αwas measuredwith western blot analyses (Fig. 5). Protein level of Gadd45αwas significantly decreased in rat cardiomyocytes treated withselenite in a concentration-dependent manner (Fig. 5a).Whereas under selenium deficiency condition (cells treatedin the absence of selenium), Gadd45α expression showed anincreasing trend as selenium intervention (selenite deficiency)time increased, and TLR2 and ICAM1 promoter demethyla-tion level, mRNA and protein expression level of TLR2 andICAM1 gradually increased (Fig. 5b, c). These results sug-gested that Gadd45α might mediate the effect of selenitedeficiency on DNA demethylation of TLR2 and ICAM1.

Discussion

In this study, we used a rare but fatal form of dilated cardio-myopathy (disease of the heart muscle, DCM), namely KD, asa model to study the epigenetic regulation in the pathophysi-ology of the disease. Keshan disease is a cardiomyopathyendemic to areas in China where low soil selenium contentis prevalent [1]. Studies have shown a relationship of thisdisease to low dietary intake of selenium and low blood ortissue selenium content and a reduction in the incidence ofcardiomyopathy with selenium supplementation [7, 42, 43].Keshan disease can also lead to higher rates of cancer, cardio-vascular disease, hypertension, and strokes [1–7].

DNA methylation and post-translational histone modifica-tions are two important epigenetic events in regulation of geneexpression and maintenance of cellular function [44]. DNAmethylation is a potent mechanism for silencing gene expressionandmaintaining genome stability in the face of a vast quantity ofrepetitive DNA, which can otherwise mediate illegitimate re-combination events and cause transcriptional deregulation ofnearby genes [44, 45]. Given the critical role of DNA methyl-ation in gene expression and cell differentiation, it seems obvi-ous that errors in methylation could give rise to a number ofdevastating consequences, including various diseases [44–47].A number of DNA methylation and histone-modifying agentsare currently being evaluated in preventing and treating diseases

76 Yang et al.

[48]. Recent data suggest that some dietary factors may beinvolved in epigenetic modifications to regulate cellular functionand modify cardiovascular diseases [12–14]. Se is an essentialnutritional element and has been demonstrated to be a potentialchemopreventive agent for cardiovascular disease [49].

In the current study, the adult rat cardiomyocytes as a modelsystem and inorganic form of selenium (sodium selenite) wereemployed to elucidate the epigenetic regulatory mechanisms ofTLR2 and ICAM1. TLR2 and ICAM-1 play crucial roles inmodulating inflammatory responses and are thought to contrib-ute to cardiomyopathy development and progression. Our re-sults demonstrate that selenium-deficient diet increased proteinlevels of TLR2 and ICAM1, whereas selenite treatment de-creased protein levels of TLR2, ICAM1 and DNMT1. Thepromoters of TLR2 and ICAM1 were demethylated and thegenes were reexpressed at mRNA and/or protein levels incardiomyocytes cultured in the absence of selenium. Theseresults indicated that selenite could inhibit some inflammatorygene expression by DNA methylation-related chromatin mod-ification. Take TLR2 as an example, we further demonstrated

the role of selenium deficiency in Gadd45α mediated DNAdemethylation. The selenium-deficient diet increased expres-sion of Gadd45α and modulated TLR2 promoter hypomethy-lation. In contrast, in the presence of 1.5 μM selenium, 5-Aza-dC treatment changed neither methylation nor the expression ofTLR2 and ICAM1. These results suggested that selenite mightinhibit these genes, at least in part, by downregulatingGadd45α with resultant remethylation of their promoters. Ad-ditionally, Se has been demonstrated to inhibit demethylationactivity. These results reveal that Gadd45α plays an essentialrole in gene-specific active DNA demethylation duringcardiomyocytes exposed to low selenium levels for a long time.Thus, inhibition of Gadd45α expression is probably also in-volved in the selenite-mediated alterations of DNAmethylationobserved in our study. Our results suggest that Gadd45α ismost likely the target gene of Se. Additionally, our studydemonstrates that selenite decreased levels of Gadd45α, sug-gesting that Se may have a broad effect on DNA methylationand demethylation. This is supported by the results of demeth-ylation of multiple genes in our study.

Fig. 4 Effects of selenite and 5-Aza-dC on promoter methylation andexpression of TLR2 and ICAM1 in rat neonatal cardiomyocytes. a Left:MS-PCR analysis of CpG island methylation in the TLR2 and ICAM1promoter region (M methylated, U unmethylated); Right: densitometricquantification analysis. b Left: RT-PCR analysis of mRNA levels andWestern blot analysis of protein levels of TLR2 and ICAM1; Right:densitometric quantification analysis. Beta-actin was used as control forequal sample loading. c Left: MS-PCR analysis of dose-dependent effects

of 5-Aza-dC on methylation levels in the TLR2 and ICAM1 promoterregion (Mmethylated,U unmethylated); Right: relative methylation levelanalysis. dWestern blot analysis of effect of selenite on levels of DNMT1protein. e Left: Western blot and RT-PCR analysis of effect of 5-Aza-dCon levels of DNMT1, TLR2, and ICAM1; Right: relative methylationlevel analysis. Data are representative of three independent experiments.*p<0.05;**p<0.01

TLR2-ICAM1-Gadd45α Axis Mediates the Epigenetic Effect of Selenium 77

Studies from other investigators have suggested thatdietary selenium can inhibit Dnmt1 activity in vitro [50].Our studies differ from the previous studies in that weinvestigated Gadd45α protein expression in the cells atphysiological concentrations of selenium. In contrast, theprevious studies primarily investigated the effect of sele-nium on the activity of the purified enzyme in vitro atvery high concentrations of selenium [51]. However, theseresults all suggest that inhibition of Gadd45α activity bydietary selenium might be a mechanism for its chemopre-ventive effect.

In the current study, selenium deficiency treatment causedDNA hypomethylation in TLR2 and ICAM1 in both cellculture systems and animal models. However, seleniumdeficiency increased and selenium treatment decreased in-filtration of myocardial inflammatory cells in rats. Theseresults suggest that changes in DNA methylation might beresponsible for the chemopreventive effects of dietary sele-nium. Furthermore, our data may also indicate that selenitedeficiency and oxidative stress factors had a synergisticeffect on expression of inflammatory factors. Our resultsimplicated an association of plasma selenium levels with

gene methylation status, implicating that selenium treatmentmight protect selenium-deficient rats against myocardialinflammatory infiltration.

Conclusions

Gadd45a plays an essential role in active DNA demethylationduring rat cardiomyocytes grown in the absence of additionalselenium. Selenium treatment, which inhibits demethylationand overexpression of inflammatory factor, protects selenium-deficient rats against myocardial injury induced by inflamma-tory cytokine overexpression. Rat cardiomyocytes cultured inthe absence of selenium expressed significantly more Gadd45αand inflammatory proteins than cells cultured in the presence ofselenium. Evidence was presented that dietary seleniumcould inhibit activity of inflammatory factors [51, 52]. Takentogether, these results suggested that inhibition of Gadd45αactivity by dietary selenium may be responsible for itschemopreventive activity. However, further studies shouldbe performed to investigate the effect of dietary selenium

Fig. 5 Gadd45α might mediatethe effect of selenite deficiency onDNA demethylation in ratcardiomyocytes. a Left: Westernblot analysis of dose-dependenteffects of selenite on levels ofGadd45α, TLR2, and ICAM1protein; Right: Q-PCR analysis ofeffects of selenite on levels ofGadd45α, TLR2, and ICAM1mRNA. Cells were treated with0.5 or 1.5 μM selenite for 7 days.b. Left: Western blot analysis oftime-dependent effects of selenitedeficiency on levels of Gadd45α,TLR2, and ICAM1 protein;Right: Q-PCR analysis of effectsof selenite on levels of Gadd45α,TLR2, and ICAM1 mRNA. Cellswere treated with 0.5 or 1.5 μMselenite for 7 days. c Left: MS-PCR time-dependent effects ofselenite deficiency onmethylation levels in the TLR2and ICAM1 promoter region;Right: relative methylation levelanalysis. Data are representativeof three independent experiments.*p<0.05;**p<0.01

78 Yang et al.

on KD susceptibility, global and gene-specific DNA methyla-tion, Dnmt1 activity, and DNA demethylase activity, to deter-mine whether the chemopreventive effects of selenium may bedue to changes inDNAmethylation, DNAdemethylase activity,and/or Dnmt1 activity.

Acknowledgments This work was supported by the Chinese nationalnatural science fund project approval (NO-30972557), (NO-81170290).

Authorship Xiaolin Niu and JinWei conceived and designed the study.GuangYang performed the experiment, analyzed and interpreted the data,and wrote the initial draft of the manuscript. Yanhe Zhu, Xin Dong, andZongming Duan contributed to the experimental design, the statisticalanalysis, and the writing of the manuscript.

Conflict of interest The authors declare that they have no conflict ofinterest.

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