Toxicology in Vitro 27 (2013) 211–219

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Toxicology in Vitro

journal homepage: www.elsevier .com/locate / toxinvi t

Arsenic inhibits the adipogenic differentiation of mesenchymal stem cellsby down-regulating peroxisome proliferator-activated receptor gammaand CCAAT enhancer-binding proteins

Santosh Yadav a, Muralidharan Anbalagan b, Yongli Shi c, Feng Wang d, He Wang b,⇑a Tulane Cancer Center, Tulane University, New Orleans, LA 70112, USAb Department of Cellular and Structural Biology, Tulane University, New Orleans, LA 70112, USAc Department of Environmental Health Sciences, School of Public Health and Tropical Medicine, Tulane University, New Orleans, LA 70112, USAd Department of Laboratory Medicine, Southwest Hospital, Third Military Medical University, Chongqing, People’s Republic of China

a r t i c l e i n f o a b s t r a c t

Article history:Received 5 January 2012Accepted 16 October 2012Available online 26 October 2012

Keywords:Mesenchymal stem cellsArsenicAdipogenesisDifferentiationPPAR-cC/EBP family proteins

0887-2333/$ - see front matter Crown Copyright � 2http://dx.doi.org/10.1016/j.tiv.2012.10.012

Abbreviations: MSCs, mesenchymal stem cells; Ptor-activated receptor-gamma; C/EBPs, CCAAT/enhaarsenite; ORO, Oil Red O staining.⇑ Corresponding author. Address: Department of En

1430 Tulane Ave., Tulane University, Box TW-2100, NTel.: +1 504 9881081; fax: +1 504 988 1726.

E-mail address: hwang2@tulane.edu (H. Wang).

Arsenic remains a top environmental concern in the United States as well as worldwide because of its glo-bal existence and serious health impacts. Apoptotic effect of arsenic in human mesenchymal stem cells(hMSCs) has been identified in our previous study; the effects of arsenic on hMSCs remain largelyunknown. Here, we report that arsenic inhibits the adipogenic differentiation of human mesenchymalstem cells (hMSCs). Arsenic reduced the formation of lipid droplets and the expression of adipogenesis-related proteins, such as CCAAT enhancer binding protein-(C/EBPs), peroxisome proliferator-activatedreceptor-gamma (PPAR-c), and adipocyte fatty acid–binding protein aP2 (aP2). Arsenic mediates thisprocess by sustaining PPAR-c activity. In addition, inhibition of PPAR-c activity with T0070907 andup-regulation with its agonist troglitazone, showed the direct association of PPAR-c and arsenic-mediatedinhibition of differentiating hMSCs. Taken together, these results indicate that arsenic inhibits adipogenicdifferentiation through PPAR-c pathway and suggest a novel inhibitory effect of arsenic on adipogenicdifferentiation in hMSCs.

Crown Copyright � 2012 Published by Elsevier Ltd. All rights reserved.

1. Introduction

Arsenic is recognized as a group 1 carcinogen by the Interna-tional Agency for Research on Cancer (IARC, 1987). Its exposureis associated with lung, bladder, kidney, liver, and non-melanomaskin cancers (Pershagen, 1981; Smith et al., 1992; Kumar et al.,2010). Arsenic-induced effects at the cellular level involve impair-ment in cellular proliferation, differentiation, transformation(McNeely et al., 2008; Hester et al., 2009; Ruiz-Ramos et al.,2009) apoptotic cell death and cytotoxicity (Nasr et al., 2008;Yadav et al., 2010; Zhao et al., 2012).

Arsenic can also induce bone-marrow toxicity (Feussner et al.,1979); mesenchymal stem cells (MSCs) are non-hematopoieticcomponents of bone marrow, which show a stromal nature by pro-viding extracellular matrix components, cytokines, and growth fac-

012 Published by Elsevier Ltd. All r

PARc, peroxisome prolifera-ncer-binding proteins; iAsIII,

vironmental Health Sciences,ew Orleans, LA 70112, USA.

tors to the maintenance of stem cell niches (Raaijmakers et al.,2010; Van Den Berg et al., 1998; Walenda et al., 2011; Striogaet al., 2012). MSCs are much of importance due to their therapeuticpotential including immunosuppressive (Sundin et al., 2011),immunomodulatory (Marigo and Dazzi, 2011; Engela et al., 2010)and cytoprotective response (Mougiakakos et al., 2011). Apart fromits direct effects on cells of the immune system, MSCs alsomodulate the microenvironment by secreting anti-fibrotic andpro-angiogenic factors, allowing the transition of the inflammatoryresponse to injury into one of repair, as reviewed by Reinders et al.(2010) and Li et al. (2012).

MSCs are thought to be the precursor cells for adipocytes andosteocytes (Sekiya et al., 2002, 2004; Friedenstein et al., 1974). InMSC niches, signaling by the canonical wingless (Wnt) pathwayleads to the differentiation of MSCs into osteoblasts. In contrast, acti-vation of the peroxisome proliferator-activated receptor-gamma(PPAR-c) mediated pathway result in adipocyte differentiation. Animbalance in the linage regulators may change the commitment ofthe MSCs (Krause et al., 2010). Changes in the cellular microenviron-ment, such as increased oxidative stress or reactive oxygen speciesor in the presence of carcinogens, MSCs found to show increasedsenescence, poor differentiation, and cell death (Yadav et al., 2010;

ights reserved.

212 S. Yadav et al. / Toxicology in Vitro 27 (2013) 211–219

Song et al., 2010). The presence of arsenic in the cellular environ-ment of C3H 10T1/2 cells suppresses adipogenic differentiation bydown-regulating the expression of PPAR-c (Wauson et al., 2002).These observations confirm the hypothesis that arsenic might playa role in the adipogenic differentiation potential of MSCs. However,there is no evidence for its effect on the differentiation potential ofMSCs. Here we use this cell model to examine the effects of arsenite(chemical compound of arsenic or iAsIII) on the differentiation po-tential of human mesenchymal stem cells (hMSCs).

It has been shown that exposure of arsenic has significant im-pact on the differentiation of keratinocytes (Kachinskas et al.,1994) and adipocytes (Trouba et al., 2000). However, the moleculardetails for arsenic to inhibit are not known. In fact the induction ofdifferentiation requires a comprehensive reprogramming of geneexpression, the most notable among these factors are membersof the C/EBP and PPAR families of transcription factors. In fact, itis now well accepted that both PPAR-c and C/EBPa function as crit-ical regulators of adipogenesis because deficiency of either of theseproteins prevents the development of white adipose tissue in themouse (Linhart et al., 2001; Rosen et al., 1999).

This study describes inhibition of adpogenic differentiation byarsenite by down-regulating transcription factors PPAR-c and C/EBPs (a and b). This study also explore the role of iAsIII on Wnt sig-naling, which has been previously reported to block apoptosis andregulate differentiation of mesenchymal progenitors cells throughinhibition of glycogen synthase kinase 3 and stabilization of b-catenin. Also component of Wnt signaling pathway (Wnt3a) foundto block adipogenesis by b-catenin independent pathway (Kennelland MacDougald, 2005).

In the previous studies, most experiments focused on themolecular regulation of adipogenesis by arsenite were performedon mouse cell line 3T3-L1 (Xue et al., 2011; Paul et al., 2007). How-ever, these cells are preadipocytes, already committed to the adi-pogenic lineage, data about the determination of mesenchymalstem cells into preadipocytes in the presence of arsenic is still lack-ing. In the present study we aimed to gain insights into the molec-ular interruption by arsenic in the developmental process usinghuman mesenchymal stem cells at cellular and molecular level.Altogether, this study evaluated the adipogenic differentiation ofhMSCs in the presence of arsenic, a potent carcinogen, and its im-pact on signaling systems, which may impair MSC lineagecommitments.

2. Materials and methods

2.1. Cell culture and differentiation

hMSCs from adult human bone marrow were obtained from thecenter for the preparation and distribution of adult stem cells ofTulane University (New Orleans, LA, USA), as frozen vials of pas-sage one (P-1) (http://www.som.tulane.edu/gene_therapy/distrib-ute.shtml) which were well characterized for multipotentdifferentiation, and these cells were negative for hematopoieticmarkers (CD34, CD36, CD117, and CD45), and positive for CD29(95%), CD44 (>93%), CD49c (99%), CD49 (>70%), CD59(>99%),CD90 (>99%), CD105 (>99%), and CD166 (>99%). To culture hMSCs,alpha-MEM supplemented with FBS (16.5%) (Atlanta Biologicals,Lawrenceville, GA, USA) and 2 mM L-glutamine and penicillin/streptomycin (1%) (GIBCO, Invitrogen, Carlsbad, CA, USA) was used.Cells were maintained with culture conditions such as 37 �C tem-perature with 5% CO2. hMSCs (1 � 106/per vial) were plated in a15-cm diameter dish and allowed to reach up to 70–75% conflu-ence level (3–4 days). After appropriate confluency level, MSCswere harvested with Trypsin/1 mM EDTA (0.25%) (GIBCO, Invitro-gen), and re-plated in various culture plates according to the

experimental design. In the entire study, hMSCs from passage 2–5 were used from the same donor.

200,000 cells/well were plated in a 6-well plates, at 75–80%confluency, cells were induced with differentiation media (MEM-alpha, 16.5% FBS, 0.5 lM dexamethasone, 0.5 lM isobutyl-methyl-xanthine and 50 lM indomethacin) followed by treatment with ar-senic in relevant media. Treatment was in continuation till hMSCsbecame differentiated into mature adipocytes. Arsenic treatmentused in this study was non-cytotoxic to the hMSCs, as previouslydetermined in our laboratory (Yadav et al., 2010). No cytologicaldamage was noted except the inhibition of fat droplet formation.

The compounds troglitazone and T0070907 obtained from Sig-ma Aldrich and were dissolved in 100% DMSO. All stock solutionswere stored at �20 �C. An inorganic arsenite (iAsIII) compoundwas also purchased from Sigma–Aldrich (Sigma Aldrich, St. Louis,MO, USA) with the purity of 99%, as stated by Sigma. For eachexperiment, the various concentrations of arsenic (i.e., 0.2–4 lM)was freshly prepared using aMEM media without FBS Troglitazoneand T0070907 (a selective ligand for peroxisome proliferator-acti-vated receptor gamma).

2.2. Analysis of cell viability by MTT assay

Cell viability was measured by MTT cell proliferation assay asdescribed previously (Yadav et al., 2010). The MTT assay kit waspurchased from Sigma (St. Louis, MO, USA). For this assay200,000 cells were plated in 6-well plates, left for differentiationand treated with iAsIII (0.2–4 lM). Each experimental set was donein triplicates. At the end of differentiation period (i.e., 21st day),MTT dye was added to each well and incubated for additional 2 hand the reaction was stopped by the addition of formazan-solubi-lization reagent. Optical density in each well was determined at570 nm using the FLUOstar Optima multi detection plate reader(BMG Labtech, Cary, NC). Background absorbance of the mediumin the absence of cells was subtracted from all measured values,untreated hMSCs were considered as 100% viable cells.

2.3. Oil Red O staining and quantification

hMSCs were plated and then stimulated for adipogenic differen-tiation media alone or with iAsIII as described in cell culture anddifferentiation section. At day 21, the differentiated hMSCs werestained with Oil Red O and quantified (Sen et al., 2001). Briefly,hMSCs were fixed in formalin for 1 h, washed with PBS stainedwith 0.5% Oil Red O stain prepared in isopropyl alcohol for20 min (Sigma Aldrich, St. Louis Missouri, USA), and then washedthree times with PBS. After morphological examination cells werede-stained with absolute isopropanol for 15 min. The optical den-sity in each well was determined at 570 nm using the FLUOstar Op-tima multi-detection plate reader (BMG Labtech, Cary, NC).

2.4. Immuno-fluorescence staining

hMSCs were plated onto the six well plates which were pre-loaded with sterile coverslip followed by induction for adipogenicdifferentiation in the presence and absence of iAsIII as described incell culture and differentiation section. At day 21, cells werewashed briefly with PBS (0.5% BSA), fixed in formalin (Sigma–Al-drich, St. Louis Missouri, USA) for 30 min at room temperature,permeabilized using 0.5% Triton-X 100 in PBS and then blockedwith 0.5% bovine serum albumin (BSA) in PBS for 15 min at roomtemperature. The cells were incubated with a rabbit monoclonalantibody against PPAR-c (Cell Signaling technology Inc., MA,USA) overnight at 4 �C, followed by probing with a secondary anti-body conjugated to Rhodamine (Alexa-fluro 488, Invitrogen Rock-ford, IL). Nuclei were stained with 4,6-diamidino-2-phenylindole

S. Yadav et al. / Toxicology in Vitro 27 (2013) 211–219 213

(DAPI; Molecular Probes, Eugene, OR). Fat droplets were visualizedusing the BODIPY lipid probe (4,4-difluoro-3a, 4a diaza-s-indacene) fluorophore (Molecular Probes, Eugene, OR). Afterrinsing in PBS, the cells were mounted with anti-fade (MolecularProbes, Eugene, OR) and analyzed using a fluorescent microscope(Leica, Germany). Images were captured using fluorescence excita-tion maxima from 500 to 650 nm. To determine the percentage ofPPAR-c expressing cells, the number of positive cells and total cellswere counted manually. For each condition, at least 2 slides wereexamined and total of 100 cells were analyzed per slide.

2.5. Western blot analysis

hMSCs were treated as described in cell culture and differentia-tion section in the presence and absence of arsenic. For protein iso-lation, the cells were washed with ice cold PBS and collected with arubber scraper. After centrifugation at 10,000g for 30 min, at 4 �C,cell pellets were re-suspended in RIPA buffer (25 mM Tris (pH7.5), 50 mM NaCl, 0.5% sodium deoxycholate, 2% Nonidet P-40,0.2% sodium dodecyl sulfate, 1 mM phenylmethylsulphonyl fluo-ride (Sigma St. Louis, MO, USA). Laemmli sample buffer was usedfor loading, samples were heated at 95 �C for 5 min and then sepa-rated onto the 10% SDS–PAGE gel and transferred to nitrocellulosemembranes (Bio-Rad, Hercules, CA, USA). The membranes wereblocked with TBS-T (v/v tris buffered saline and 0.1% Tween 20)containing 5% non-fat milk. After blocking the non-specific bindingsites, the membranes were incubated with the following primaryantibodies: anti-C/EBP-a, anti-C/EBP-b, anti-PPAR-c, anti-b-actin,(Cell signaling technology Inc., MA, USA). For visualization, infraredIRDye-labeled secondary antibodies with a channel of 680 or800CW (Li-COR Biosciences, Nebraska, USA) were used. Blots werevisualized and analyzed for densitometry using the Odyssey Infra-red Imaging System (Li-COR Biosciences, Nebraska, USA).

2.6. Immunoprecipitation

hMSCs were plated and stimulated for adipogenic differentia-tion alone or treated with iAsIII as described previously. After incu-bation with iAsIII, cells were lysed using RIPA buffer, and proteinconcentrations were determined using the Bio-Rad Protein Assaykit (Bio-Rad, Hercules, CA, USA) as per manufacturer’s instructions.For Immunoprecipitation (IP), 500 lg of protein was used for IPusing PPAR-c antibody (IP-grade antibody; Cell Signaling technol-ogy Inc., MA, USA) and ChIP-grade protein G magnetic beads whilerotating overnight at 4 �C. Next day, beads were separated using amagnetic separation rack (Cell Signaling technology Inc., MA, USA)and washed three times with ice-cold cell lysis buffer. After the fi-nal wash, freshly prepared 1X sample buffer was added to eachtube and heated at 95 �C for 5 min. The beads were separated fromthe protein antibody complex. Samples were electrophoresedusing a 10% SDS gel, transferred to nitrocellulose membrane andprobed with C/EBPa or C/EBPb antibody.

2.7. RT-PCR analysis for gene expression

Total RNA from differentiated hMSCs (treated and untreated)was extracted using TRIZOL reagent (Invitrogen Carlsbad, CA,USA) according to the manufacturer’s instructions. cDNA wasamplified using the following primers: PPAR-c 50-aaggccattttct-caaacga-30 (Sense) and 50-aggagtgggagtggtcttcc-30(antisense); CEB-Pa 50-attgcctaggaacacgaagcacga-30 (sense) and 50-tttagcagagacgcgcacattcac-30 (antisense); CEBPb-50-agtaatgggtcaggcattcaggct-30

(Sense) and 50-atctcttctcaagcagggttccca-30 (antisense); Adiponectin5-accactatgatggctccact-3 (sense) and 5-ggtgaagagcatagccttgt-3(antisense). RT-PCR was performed using one-step RT-PCR kit(iQ-5 Bio-Rad laboratories, Inc., CA, USA) according to the manufac-

turer’s instructions. In brief, total RNA, 10 ng, was added to each re-verse transcriptase reaction, containing the kit master mix and theappropriate PCR primers (20 pmol) in 50 lL total volume. RT-PCRwas carried out for 30 min at 50 �C followed by a 15 min step at95 �C. Amplifications were performed for 30 cycles. Each cycle in-cluded denaturation at 94 �C for 1 min, annealing at an appropriatetemperature for 1 min, and extension at 72 �C for 1 min, followedby a final extension at 72 �C for 10 min. The number of cycleswas determined in preliminary experiments to be within the expo-nential range of RT-PCR amplification. Ct value was calculated withthe mRNA levels specific vs. the corresponding levels of GAPDH. Allthe experiments were repeated at least twice.

2.8. Statistical analyses

The data were the average of three independent experiments(n = 3). The values are expressed as the mean ± standard deviation(SD). For the determination of statistical significance, the resultswere analyzed by a one-way ANOVA (Graph Pad Prism version4), followed by the Tukey’s test for multiple comparison test, p va-lue less than 0.05 was considered statistically significant.

3. Results

3.1. iAsIII did not induce cell death but inhibits the adipogenesis inhMSCs

To investigate effects of iAsIII on triglyceride accumulation dur-ing human preadipocyte/adipocyte differentiation in vitro, cellswere cultured in ‘differentiation medium’ (n = 21 days) in the ab-sence or presence of 0.2–4 lM of iAsIII concentration. The totalamount of lipid accumulation was determined (Fig. 1A and B). iAsIII

significantly decreased triglyceride levels in a dose-dependentfashion in hMSCs (p < 0.0001). iAsIII-treated hMSCs failed to under-go adipocyte differentiation, as indicated by the presence of fewerlipid-accumulating cells (Fig. 1). To investigate the viability of dif-ferentiated hMSCs upon iAsIII treatment, we studied cell viabilityby MTT assay; iAsIII at the levels of 0.2–4 lM did not change viabil-ity of hMSCs (Fig. 1C).

3.2. iAsIII inhibits PPAR-c expression in hMSCs

It is well known that PPAR-c (a member of the nuclear hormonereceptor superfamily) plays a critical role in adipocyte differentia-tion most importantly to maintain adipocyte phenotype(Desvergne and Wahli, 1999; Liu et al., 2011). To address the ques-tion whether iAsIII mediated inhibition of adipogenesis is associatedwith PPAR-c, we further determined expression level of PPAR-c iniAsIII treated hMSCs. For this study, differentiated hMSCs wereexposed to various concentrations of iAsIII (0.2–4 lM). Immunoflu-orescence analysis showed that iAsIII gradually diminishes expres-sion level of PPAR-c as shown in Fig. 2A and B which wasaccompanied by low lipid droplets formation, resultant reducedBODIPY positive cells. This effect was further confirmed by proteinand mRNA expression level of PPAR-c (>2 fold reduction) as shownin Fig. 2C and D. However, at concentrations less than 0.2 lM, iAsIII

did not change the expression level of PPAR-c (data not shown).

3.3. iAsIII induced inhibition is mediated through the down-regulatedexpression of adipogenic transcription factor PPAR-c

To determine whether PPAR-c is directly associated in arsenicmediated inhibition of adipogenesis, specific antagonist of PPAR-c, T0070907 was used. Differentiation of hMSCs was induced inthe presence and absence of iAsIII (2 lM) and T0070907 (1 lM).Co-treatment of iAsIII with T0070907 has supplementary negative

Fig. 1. Effect of iAsIII on the adipogenic differentiation of hMSCs. (A) Images of Oil Red O staining in iAsIII treated (0.2, 2 and 4 lM) hMSCs during the process of differentiationinto adipocytes. (B) Quantification of Oil red O staining (absorbance at 540 nm). The bars represent the mean value ± SD from two samples per treatment regimen, ⁄P < 0.05and ⁄⁄⁄P < 0.001 vs. untreated hMSCs. (C) Cells viability of hMSCs in the presence of iAsIII (0.2, 2 and 4 lM), was determined by MTT assay. The data were the average of threeindependent experiments (n = 3). The values are expressed as the mean ± standard deviation (SD). A p value less than 0.05 were considered statistically significant.

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effect on iAsIII-mediated inhibition of PPAR-c and adiponectin pro-tein expression level as shown in Fig. 3A and B; is indicating thatiAsIII inhibits adipogenic differentiation of hMSCs via PPAR-cdependent pathway. To further confirm the essential roles ofPPAR-c in iAsIII-mediated suppression of adipogenic differentia-tion, we quantify the mRNA levels of PPAR-c and adiponectin(aP2). Quantitative real-time PCR analysis showed that co-treat-ment with T0070907 reduced expression level of PPAR-c and aP2illustrating a significant negative association between T0070907treatment and iAsIII as shown in Fig. 3C.

From above study it was evident that adipogenesis is sup-pressed by inhibition of PPAR-c, to further confirm this mechanismwe used the specific agonist of PPAR-c troglitazone. Co-treatmentwith troglitazone reversed iAsIII mediated inhibition of PPAR-c

protein expression as shown in Fig. 3D and E. To further confirmthis rescue, we assessed the mRNA levels of PPAR-c quantitativereal-time PCR analysis showed that treatment with 10 lM of trog-litazone rescues iAsIII (2 and 4 lM)-induced inhibition of PPAR-cand aP2 (Fig. 3F). The expression level of PPAR-c protein was alsorescued to 72% of the normal protein levels in the differentiationcontrol.

3.4. iAsIII activates Wnt signaling in hMSCs during adipogenesis

We next studied the effect of iAsIII treatment on Wnt signaling,which is a negative regulator of adipogenesis. Wnt3a, adverselyaffects the decision of hMSCs to adopt the adipogenic lineage,interestingly a significant up-regulation in the expression level of

Fig. 2. iAsIII inhibits expression of PPAR-c in hMSCs during adipogenic differentiation. (A) hMSCs were grown on coverslips followed by differentiation with adipogeneicmedia, in the presence or absence of iAsIII for the indicated times. Cell were fixed, and counter-stained for PPAR-c (FITC, Green) with the combination of BODIPY+ cells (Texas-Red) and DAPI staining for nuclear localization (Blue). (B) Fluorescent intensity of PPAR-c and BODIPY+ cells as analyzed by Image j software. The bars represent the meanvalue ± SD from 2 samples per treatment regimen. (C) mRNA expressions level of PPAR-c in iAsIII treated (0.2, 2 and 4 lM) and untreated differentiated hMSCs. (D) Expressionpatterns of PPAR-c protein as determined by western blotting. (For interpretation of the references to color in this figure legend, the reader is referred to the web version ofthis article).

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Wnt3a protein was noticed (Fig. 4A and B) this result was furtherconfirm by mRNA expression level of Wnt3a (Fig. 4C and D). Theseresults indicate that iAsIII also activates Wnt signaling however, nosignificant changes were seen in low-density lipoprotein receptor-related protein 6 (LRP) or phospho-LRP-6 protein expression and inmRNA expression level (Fig. 4A, C and E) which is a large, highlyconserved receptor that binds numerous types of ligands, invokesseveral different signal transduction pathways.

3.5. iAsIII inhibits interaction between PPAR-c and C/EBPs familyproteins (a and b)

We next examined whether the block in differentiation has anyeffect on PPAR-c and C/EBPs interactions, thus next we examinedexpression level of C/EBPs (a and b) family proteins and their inter-action with PPAR-c in the presence of iAsIII (4 lM). Our resultshowed that the treatment of iAsIII inhibits protein and mRNAexpression level of C/EBPa and b as shown in Fig. 5A and B. Impor-tantly, we found that the interaction between PPAR-c and C/EBPs(a and b) was reduced in the presence of iAsIII as examined byimmunoprecipitation analysis (Fig. 5C and D).

4. Discussion

The formation of new adipocytes from precursor cells in adipo-genesis is important for normal adipose tissue function. Impairedadipogenesis may contribute to the pathogenesis of obesity-associated conditions, such as insulin resistance, hyperlipidemiaand type II diabetes. Adipogenesis involves the activation of cas-cade of transcription factors, which coordinate the expression ofgenes responsible for adipocyte function (Farmer, 2006).Adipogenic stimuli induce the rapid expression of transcriptionfactors, such as members of C/EBP family and PPAR-c. These factorsthen promote the expression of the master adipogenic transcrip-tion factor such as PPAR-c. However, the presence of external fac-tors in the cellular microenvironment may regulate or interferewith the normal process of adipogenesis by modulating the activityof these transcriptional events. The presence of arsenic in the cel-lular environment of C3H 10T1/2 cells suppresses adipogenic dif-ferentiation by down-regulating the expression of PPAR-c(Wauson et al., 2002). These observations confirm the hypothesisthat arsenic might play a role in the adipogenic differentiationpotential of MSCs. However, there is no evidence for this role in

Fig. 3. Expression of PPAR-c in the presence of its anatagonist-/agonist and iAsIII. (A, B and C). Protein and mRNA expression level of PPAR-c and adiponectin in the presenceof PPAR-c antagonist T0070907 and iAsIII. (D–F) combined effect of iAsIII and troglitazone on PPAR-c protein and mRNA expression level in differentiating hMSCs. Error barsrepresent SD (n = 3) for a representative single experiment. ⁄p < 0.05; ⁄⁄p < 0.01; ⁄⁄⁄p < 0.001.

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human MSCs. In the present study we examine the effects of ar-senic on the differentiation potential of MSCs. This study also dem-onstrated the molecular details of the inhibitory effects of arsenicon cell differentiation of MSCs. We show that arsenite could inhibitthe expression of the PPAR-c and C/EBP (a and b) and disrupt theinteraction between them. We also found that arsenite treatmentinduced PPAR-c phosphorylation but decreased the total PPAR-cexpression.

PPAR-c, which is a member of the nuclear receptor superfamily,is the master regulator of adipogenesis and, therefore, is essentialfor adipogenesis (Linhart et al., 2001; Tontonoz et al., 1994; Leeet al., 2010). Differentiation of hMSCs into adipocytes results fromsequential induction of transcription factors C/EBPb, C/EBPd, PPAR-c, and C/EBPa (Rangwala and Lazar, 2000) and (Rosen and Spiegel-man, 2000). Any variation among these factors can influence thesequential process of adipogenesis because expression of one

Fig. 4. Role of iAsIII on Wnt3a signaling during adipogenic differentiation of hMSCs. (A, B and C) protein expression level of Wnt3a, LRP-6 and phospho-LRP-6 in the presenceof iAsIII. (D and E) mRNA expression level of Wnt3a and LRP-6 respectively, data are given as mean ± standard deviation (n = 3) of a representative experiment.

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factor regulates others. During adipogenesis C/EBPb and C/EBPd areinduced immediately and transiently mediates the expression ofPPAR-c and C/EBPa (Christy et al., 1991; Wu et al., 1995; Yehet al., 1995). Arsenic treatment to hMSCs found to inhibit PPAR-c, that plays a critical role in the expression of most of the adipo-cyte-specific genes (Tontonoz et al., 1995) and is able to convertnonadipogenic cells, such as fibroblasts and myoblasts, to adipo-cytes (Hu et al., 1995) and (Tontonoz et al., 1994). These data sug-gest the PPARc and C/EBPb may be potential targets for adipogenicintervention.

Further this study showed that arsenite inhibits the interactionbetween C/EBP (a and b), PPAR-c. C/EBP family proteins, includingC/EBP-a, C/EBP-b and C/EBP-c, are transcription factor homologuesof the CCAAT-enhancer-binding protein. They are expressed in adi-pocytes and participate in adipogenesis (Urs et al., 2010; Lim et al.,2011; Siersbaek et al., 2010). C/EBP-a, C/EBP-b both are importantfactors for adipogenesis where induction of C/EBP-b and togetherwith C/EBP-d expression leads to the induction of C/EBP-a expres-sion, and these transcription factors bind to other adipocyte pro-moters during adipocyte differentiation. Studies have

demonstrated that the lack or inhibition of the C/EBP family’s pro-tein expression could lead to impaired adipogenesis (Bezy et al.,2007). In this study, arsenic found to inhibits the expression ofPPAR-c and the C/EBP family members, these experiments estab-lish a clear role for arsenic in the fate of MSC lineage commitmentin vitro.

Wnts are a family of secreted glycoproteins that act in autocrineand paracrine manners to regulate cell proliferation, survival anddifferentiation (Krishnan et al., 2006; Baron et al., 2006). Wnt li-gands work through two pathways: canonical and non-canonical.The canonical Wnt signaling cascade mediates through the tran-scriptional regulator b-catenin, which is a downstream effector inthis pathway (Wang et al., 2010). In the absence of Wnts, cytoplas-mic b-catenin undergoes many molecular processes that end withubiquitination and proteasomal degradation, which favors cell dif-ferentiation. However, binding of canonical Wnt ligands, such asWnt1, Wnt3a, Wnt5b, Wnt7a and Wnt10b, to frizzled (FZD) recep-tors and the low-density lipoprotein-receptor-related protein (LRP)5 or LRP6 co-receptors inactivates the degradation complex andinhibits differentiation (Takada et al., 2009, 2010). This study eval-

C D

A B

IgG 2um control

Input PPAR-gamma

C/EBP-alpha

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IgG 2um control

Input PPAR-gamma

C/EBP-beta

IP

Beta -actin

C/EBPα

C/EBPβ

untreated 0.2 2 4

iAs III (μM)

Fig. 5. iAsIII decreases the expression of C/EBPs (a and b) and inhibit the interaction between PPAR-c and C/EBPs (a and b) in differentiating hMSCs. (A and B) protein andmRNA expression level of C/EBPa and C/EBPb in differentiating hMSCs in the presence of iAsIII. (C and D) mRNA expression of C/EBPa and C/EBPb followed by treatment withiAsIII (0.2–4 lM). The interaction between PPAR-c and C/EBPs was determined by immune-precipitation. Error bars represent SD (n = 3 independent experiments).

218 S. Yadav et al. / Toxicology in Vitro 27 (2013) 211–219

uated the effects of arsenite on Wnt signaling, that induces theexpression of Wnt3a which is known to adversely affect the adipo-genesis. Previous studies have demonstrated the effect of arseniteon various cell types and its role in differentiation. In the past, mostexperiments on the molecular regulation of adipogenesis were per-formed using the mouse cell line 3T3-L1 (Wauson et al., 2002).However, since these cells are preadipocytes that were alreadycommitted to the adipogenic lineage, data about the determinationof hMSCs into preadipocytes in the presence of arsenite is lacking.Our findings suggest that the cellular environment generated inthe presence of arsenic mediates its downstream effects on adipo-genesis. This study shows the impact of arsenite on cell-fate deter-mination, which may be involved in mediating the impairedadipogenesis.

Acknowledgements

This project is supported by the Tulane Cancer Center & Depart-ment of Environmental Health Science at Tulane University. Wethank the adult stem-cell core facility for providing the humanmesenchymal stem cells.

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