SIRT1 regulates differentiation of mesenchymal stem cells by deacetylating ?-catenin.

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by  Simic, Petra

OPEN TRANSPARENT ResearchArticle
ACCESS PROCESS SIRT1regulatesdifferentiationofMSCs
SIRT1 regulates differentiation
of mesenchymal stem cells by
deacetylating b-catenin
Petra Simic1, Kayvan Zainabadi1, Eric Bell1, David B. Sykes2, Borja Saez2, Sutada Lotinun3,
Roland Baron3, David Scadden2, Ernestina Schipani4, Leonard Guarente1*
Keywords: bone; cartilage;fat; Mesenchymalstemcells(MSCs)aremulti-potentcellsthatcandifferentiateinto
mesenchymal stem cells;sirtuin osteoblasts,adipocytes,chondrocytesandmyocytes.Thispotentialdeclineswith
aging. We investigated whether the sirtuin SIRT1 had a function in MSCs by
creating MSC specific SIRT1 knock-out (MSCKO) mice. Aged MSCKO mice (2.2
DOI10.1002/emmm.201201606
years old) showed defects in tissues derived from MSCs; i.e. a reduction in
subcutaneous fat, cortical bone thickness and trabecular volume. Young mice
ReceivedMay25,2012
RevisedNovember12,2012 showed related but less pronounced effects. MSCs isolated from MSCKO mice
AcceptedNovember14,2012 showed reduced differentiation towards osteoblasts and chondrocytes in vitro,
but no difference in proliferation or apoptosis. Expression of b-catenin targets
important for differentiation was reduced in MSCKO cells. Moreover, while b-
cateninitself(T41Amutantresistanttocytosolicturnover)accumulatedinthe
nuclei of wild-type MSCs, it was unable to do so in MSCKO cells. However,
mutatingK49RorK345Rinb-catenintomimic deacetylationrestored nuclear
localizationanddifferentiationpotentialinMSCKOcells.WeconcludethatSIRT1
deacetylates b-catenin to promote its accumulation in the nucleus leading to
transcription of genes forMSC differentiation.
INTRODUCTION However,MSCsarenotaswellcharacterizedasotheradultstem
cells,suchashaematopoieticstemcells(HSCs).
Mesenchymalstemcells(MSCs)aremultipotentcellsthatcan Osteogenic, adipogenic, chondrogenic andmyogenicpoten-
be isolated from bone marrow, can proliferate in vitro with tial is compromised in MSCs of aged rats (Asumda & Chase,
fibroblast-likemorphology,andcanbeinducedtodifferentiate 2011). In addition, the number of MSCs with osteogenic,
intoavarietyofmesenchymaltissues,includingbone,cartilage, chondrogenic and adipocyte potential has been reported to
tendon, fat, bone marrow stroma and muscle (Pittenger et al, decline in aging potentially contributing to age-related osteo-
1999;Prockop,1997;Uccellietal,2008).MSCscanmostreliably porosis,osteoarthritisandlossofsub-cutaneousfat(Cartwright
beidentifiedbythemonoclonalantibodySTRO1(Loeuilletetal, etal,2007;Changetal,2011;D’Ippolitoetal,1999).Agingalso
2001), which recognizes a cell surface protein on MSCs. compromises the efficacy of MSC differentiation to myocytes
and regeneration of damaged myocardial tissue (Asumda &
Chase,2011;Khanetal,2011),leadingtomusclewastingand
(1) Glenn Laboratory for the Science of Aging, Department of Biology, increased frailty (Goldspink, 2012). Indeed, transplantation of
MassachusettsInstituteofTechnology,Cambridge,MA,USA MSCsfromyoungdonorsdelaysaginginmiceandprolongsthe
(2) Center for Regenerative Medicine, Massachusetts General Hospital, lifespan(Shenetal,2011).Agingalsocausesaninappropriate
Boston,MA,USA shift of MSC commitment in bone from the osteoblast to the
(3) Department of Oral Medicine, Harvard School of Dental Medicine,
adipocyte lineage (Moerman et al, 2004), resulting in an
Infection,andImmunity,Boston,MA,USA
increaseinbonemarrowadipogenesis(Jiangetal,2008).
(4) MGH-HarvardMedicalSchool,EndocrinologyUnit,Boston,MA,USA
Wntsignallingviab-cateninplaysanimportantbutcomplex
*Correspondingauthor:Tel:þ16172587360;Fax:þ16174524130;
E-mail:[email protected] roleintheself-renewalanddifferentiationofMSCs(Lingetal,
(cid:1)2013TheAuthors.PublishedbyJohnWileyandSons,LtdonbehalfofEMBO.Thisisanopenaccessarticleunder
thetermsoftheCreativeCommonsAttributionLicense(CCBY3.0),whichpermitsuse,distributionandreproduction
430 inanymedium,providedtheoriginalworkisproperlycited. EMBOMolMed(2013)5,430–440
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2009).Wntsignalingcanexertapositiveeffectonself-renewal masscharacterizedbydecreasedboneformationandincreased
in some cases (Boland et al, 2004; Cho et al, 2006), and a marrow adipogenesis (Cohen-Kfir et al, 2011). SirT1 hetero-
negativeeffectinothers(Qiuetal,2007).Thesedifferencesmay zygotesalsoshowincreasedosteoarthritisandincreasedlevels
beduetodifferentresponsesofMSCstodifferentdosesofWnt of chondrocyte apoptosis in cartilage (Gabay et al, 2012). In
signaling (De Boer et al, 2004). Similarly, in osteogenesis SIRT1 muscle specific knock-out mice, endurance, electron
Wntsignallinghasbeenreportedtobepositive(Bennettetal, transport chain activity and voluntary wheel running-induced
2005; Gaur et al, 2005), negative (Boland et al, 2004; mitochondrial biogenesis are not impaired, but responses of
Choetal,2006)orstagedependent(Eijkenetal,2008;Kahler these same parameters to calorie restriction are severely
et al, 2006). In chondrogenesis, similar complexities are compromised(Philpetal,2011;Schenketal,2011).
also evident (Etheridge et al, 2004; Hartmann & Tabin, 2001; As SIRT1 levels affect bone, fat, cartilage and muscle, we
Tulietal,2003). investigated the role of SIRT1 in the common precursor cells,
Sir2-related proteins or sirtuins are highly conserved NAD- MSCs, by creating MSC specific SIRT1 knock-out (MSCKO)
dependentdeacetylasesthatwereshowntoregulatelifespanin mice. Our findings reveal important roles of SIRT1 in MSC
lowerorganisms(Tissenbaum&Guarente,2001;Viswanathan function.
& Guarente, 2011) and affect diseases of aging in mammals,
suchasdiabetes,inflammationandneurodegenerativediseases
(Donmez&Guarente,2010).TheSir2orthologSIRT1isknown RESULTS
to deacetylate numerous mammalian transcription factors
important for aging and disease (Guarente, 2011; Imai et al, PhenotypesofMSCKOmice
2000). Transgenic mice with roughly twofold higher levels of To test the role of SIRT1 in mesenchymal progenitor cells we
SIRT1 expression globally are protected against metabolic generated MSC specific SIRT1 knock-out (MSCKO) mice by
decline due to aging (Banks et al, 2008; Bordone et al, 2007; crossing mice with cre recombinase expression driven by the
Herranzetal,2010).Importantly,thesemicearealsoprotected Prx-1 promoter (Logan et al, 2002) with SIRT1 floxed exon 4
againstaging-inducedboneloss(Herranzetal,2010).Lowto mice (Cheng et al, 2003; Fig 1A). Prx-1 is a homeobox gene
moderate SirT1 overexpression also protects mice from expressedinmesenchymalprogenitorcellsandisimportantin
paraquat-inducedcardiacstressandapoptosis, anddelaysthe limbgrowth(Caloetal,2010;Loganetal,2002).Allmicewere
onset of age-dependent heart muscle dysfunctions (Alcendor in a pure C57BL/6 background. We analysed both young
etal,2007). (2-month-old) andaged(2.2yearsold)MSCKOandwild-type
Conversely, mice with reduced SIRT1 levels show defects. (fl/fl) mice. Aged MSCKO mice had a 64% reduction in the
Sirt1þ/(cid:1)heterozygousfemalemiceexhibitareductioninbone subcutaneous fat weight (Fig 1B) with smaller adipocytes
Figure1.TheroleofSIRT1inmesenchymalprogenitorcellsinaged(2.2yearsold)miceinvivo.
A. WesternblotanalysisofSIRT-1inmesenchymalprogenitorcellsfromthebonemarrow.
B. Subcutaneousfatweightoffl/flandMSCKOmice.Bars,SD;(cid:2)p¼0.01,t-test,n¼8pergroup.
C. Haemalaun–eosinstainingofsubcutaneousadiposetissue,20(cid:3)magnification.
D. Bodyweightoffl/flandMSCKOmice.Bars,SD;(cid:2)p¼0.006versusfl/fl,t-test,n¼8pergroup.
E. Bloodtryglyceridelevelinfl/flandMSCKOmice.Bars,SD;(cid:2)p¼0.04,t-test,n¼8pergroup.
F. Bonemarrowhistologyoffl/flandMSCKOmice(haemalaun–eosinstaining,5(cid:3)magnification).
G–I. LongitudinalmicroCTimage(G)throughfemurdepictingareasofmidshaftanddistalfemuranalysedinexperiments.MicroCTimagesofthe(H)midshaft
femur(left)and(I)distalfemur,Ct.Th,corticalthickness;TV/BVtrabecularvolume/bonevolume;resultsaremean(cid:4)SD;(cid:2)p¼0.01,t-test,n¼6pergroup.
J,K. Histomorphometryoffemurs.(J)BFR/BS,boneformationrate/bonesurface,Bars,SD;(cid:2)p¼0.02,t-test,n¼6pergroup;(K)ObS/BS,osteoblastsurface/bone
surface,Bars,SD;(cid:2)p¼0.04,t-test,n¼6pergroup.
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SIRT1regulatesdifferentiationofMSCs
(Fig 1C), and an overall reduction of 29% in body weight Information Fig S5B). Analysis of lymphocytes in haemato-
(Fig1D).Therewasnochangeinvisceralfat,consistentwiththe poieticorgansrevealedanincreaseinCD4þlymphocytesanda
fact that there was no excision of SIRT1 in the visceral fat decreaseinTregulatory(Treg)lymphocytesinMSCKOthymus,
compartment (Supporting Information Fig S1A–C). However, andadecreaseinTregsinMSCKOspleen.Theincreaseinblood
total adipose tissue volume was decreased (Supporting T cells included both CD4þ and CD8þ lymphocytes, but there
Information Fig S1C). Blood tryglycerides were increased was no effect on these cells in the bone marrow (Supporting
(Fig1E)andcholesterollevelwasunchangedinMSCKOmice InformationFigS6).
(SupportingInformationFigS2A).MSCKOmicehadnosignsof
fattyliverdisease,buthadincreasedfastingglucose,suggestive TheeffectofSIRT1ondifferentiationofMSCsinvitro
ofmetabolicsyndrome(SupportingInformationFigS2BandC). Bonemarrowcellsofwild-typeandMSCKOmicewereplatedto
Cortical bone thickness and trabecular volume/bone volume selectforadherent,growingcellsthroughreiterativecycles.This
werereduced25and23%(Fig1F–I),respectively,asaresultof procedure has been shown to select for MSCs (Soleimani &
reducednumbersofosteoblastsandareducedboneformation Nadri,2009).Todemonstratethatthisprocedureyieldedmulti-
rate (Fig 1J and K). However, there was no change in bone potent MSCs in our hands, we performed limit dilution and
marrow adipocytes (Fig 1F). Cartilage of the long bones in placed single cells into separate wells of a 96-well plate and
MSCKO mice showed no morphological change (Supporting allowedthemtoproliferate.Thenumberoftotalcellsperwellor
Information Fig S3). Muscles in MSCKO mice showed no apoptoticcellsperwellwasnotdifferent,comparingwild-type
difference by the grip strength functional test (Supporting (fl/fl) and MSCKO cells (Fig 3A and B). Cells from each well
Information Fig S4A and B). Young MSCKO mice had a less were then treated with reagents to differentiate them into
pronounced reduction of subcutaneous fat weight (35%, osteoblastsoradipocytes.Singlewild-type(fl/fl)cellswereable
Fig 2A), without any change in the overall body weight togiverisetoosteoblastsoradipocytesatafrequencybetween
(Fig 2B). However, adipocytes in the subcutaneous fat were 60and70%,indicatingthattheputativeMSCsisolatedbythis
smaller (Fig 2C), and there was an increase in blood methodaremulti-potent(Fig3C).ColoniesfromsingleMSCKO
tryglycerides (Fig 2D). The effect of SIRT1 loss on bone was cells gave rise to fewer osteoblasts, and more of these cells
also less pronounced in young mice, affecting only trabecular continued to express the STRO1 MSC marker, indicating that
thickness,withoutasignificantchangeofoverallbonevolume they remained in the undifferentiated state (Fig 3D). These
(Fig2E–G). findingssuggestedthatourMSCsareindeedmulti-potent,but
LossofSIRT1fromMSCsalsohadanimpactonimmunecells, indicate a reduced ability of MSCKO cells to differentiate into
which are derived from HSCs, consistent with earlier sugges- osteoblasts.
tionsofinteractionsbetweenHSCsandMSCs(Ghannametal, To further test whether the cells we isolated were multi-
2010).WeobservedanincreaseinT-lymphocytesinblood,but potent,weinsertedaGFP-expressingretrovirusintowild-type
not B-lymphocytes, monocytes, granulocytes or erythrocytes (fl/fl) and MSCKO derived cells, selected 15–20 cells micro-
(SupportingInformationFigS5A).Therewasalsoanincrease scopicallyforanMSCmorphology,andinjectedthemintoearly
in the spleen size and weight in MSCKO mice (Supporting blastocysts to follow their potential to differentiate during
Figure2.TheroleofSIRT1inmesenchymalprogenitorcellsinyoung(2-month-old)miceinvivo.
A,B. Subcutaneousfat(A)andoverallbodyweight(B)offl/flandMSCKOmice.Bars,SD;p¼0.05,t-test,n¼6pergroup.
C. Haemalaun–eosinstainingofsubcutaneousadiposetissue,20(cid:3)magnification.
D. Bloodtryglyceridelevelinfl/flandMSCKOmice.Bars,SD;p¼0.04,t-test,n¼6pergroup.
E. Bonemarrowhistologyoffl/flandMSCKOmice(haemalaun–eosinstaining,5(cid:3)magnification).
F. MicroCTimagesofthedistalfemur,transversalsectionontheleft,3Dlongitudinalreconstructionontheright.
G. MicroCTanalysisofdistalfemur.TV/BV,trabecularvolume/bonevolume,Tb.N,trabecularnumber,Tb.Th.trabecularthickness,Bars,SD;(cid:2)p¼0.05,t-test,
n¼6pergroup.
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Figure3.TheeffectofSIRT1ondifferentiationofMSCsinvitro.
A–D. SinglecellMSCproliferationanddifferentiation.Differentiationtoosteoblastsontheleftandtoadipocytesontheright.(A)Cellnumberperwell(n¼12
wellspergroup),(B)FACSanalysisofapoptoticannexinVpositivecellsperwell(n¼6wellspergroup),(C)FACSanalysisofALPþosteoblastsperwell
((cid:2)p¼0.04,t-test,n¼12wellspergroup)ontheleftandFACSanalysisofFABP4þadipocytesperwell(n¼12wellspergroup)ontheright,(D)FACSanalysis
ofSTRO-1þmesenchymalprogenitorcellsperwell((cid:2)p¼0.03ontheleftandp¼0.05ontheright,t-test,n¼6wellspergroup);bars,SD.
E. FACSanalysisofpercentageofMSCsinthebonemarrowofold(left,(cid:2)p¼0.01,t-test,n¼6pergroup)andyoung(right)mice.
F. Alkalinephosphatase(ALP)stainingofMSCs(isolatedbytheplatingmethod)differentiatedtowardsosteoblasts.BFUboneformingunits,numbersare
mean(cid:4)SD;(cid:2)p¼0.001,t-test,n¼12wellspergroup.
G. AlcianbluestainingofMSCsdifferentiatedtowardsadipocytes.CFU/alcianblueþ,colonyformingunits/Alcianbluepositive,numbersaremean(cid:4)SD;
(cid:2)p¼0.03,t-test,n¼6wellspergroup.
H. LipidTOXstainingofMSCsdifferentiatedtowardsadipocytes,(cid:2)p¼0.05,t-test,n¼6wellspergroup.
I. ALPstainingofMSCs(isolatedbytheplatingmethod)afterfl/flandMSCKObonemarrowtransplantation(BMT)anddifferentiatedtowardsosteoblasts.
Numbersaremean(cid:4)SD;(cid:2)p¼0.01,t-test,n¼12wellspergroup.
embryogenesis.Both,wild-type(fl/fl)andMSCKOMSCsgave marrowshowedalargedecreaseintheMSCnumberinagedKO
risetomesenchymaltissuesconstitutingmanyorgans(Support- mice, but no significant change in MSC number in wild-type
ing Information Fig S7A–C). The distribution of cells in the versusMSCKOyoungmice(Fig3E).Thesefindingssuggestthat
organs was roughly similar between the groups, except for deletionofSIRT1promotesthelossofMSCswithaging.Wenext
3.5-foldmoreMSCKOMSCsdetectedinthymus,2.4-foldmore isolated MSCs from young mice by the reiterative plating
incraniumandbrainand0.5-foldlessinthelungs.However,it method and differentiated them. Consistent with our data on
is difficult to interpret this quantitation solely in light of singly cloned cells, we observed a reduced differentiation
differentiationpotential,becauseitsumstheabilitiesofinjected towards osteoblasts, and chondrocytes (48% and 33%,
cellstohometotissues,colonizethereanddifferentiate. respectively; Fig 3F and G), in MSCKO cells compared to
Inordertodeterminewhetherthenumberanddifferentiation wild-type, but no significant difference in differentiation
potential of MSCs were affected by aging, we characterized towards adipocytes or myocytes (Fig 3H). However, the
MSCsfrombothyoungandagedmice.FACSsortingforMSCs adipocytesweresmallerwithlesslipidasrevealedbyLipidTox
(STRO-1þ, glycophorin A(cid:1), CD 45(cid:1), CD 31(cid:1)) from the bone staining(Fig3H).Similartothesinglyclonedcellsabove,there
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was no difference between wild-type and MSCKO cell effectofMSCKOonreducedMSCdifferentiation(Fig4A).The
proliferation(SupportingInformationFigS8). activityofEX527wasconfirmedbyincreasedacetylationofp53,
TodistinguishwhetherthephenotypeofMSCKOMSCsisdue aknownSIRT1target(Fig4B).
toacellulardefectandnotasystemiceffect,weperformedbone
marrow transplantation from old fl/fl and MSCKO mice into SIRT1deacetylatesb-cateninandpromotesitsnuclear
irradiatedrecipientmice.Sixmonthsfollowingtheprocedure, localizationandactivityinmesenchymalprogenitorcells
weanalysedthedifferentiationpotentialoftransplantedMSCs. WenextinvestigatedthemechanismbywhichSIRT1promotes
Mice that received bone marrow from MSCKO animals had osteoblastdifferentiationinMSCs.Wefirsttestedwhetherthe
reduced differentiation of MSCs towards bone (Fig 3I), deacetylaseactivitywasrequired.Lentiviralexpressionofwild-
suggesting that observedphenotype ofMSCKO miceis dueto type (WT) Sirt1, but not a deacetylase-inactive (H363Y) point
a differentiation defect of MSCKO MSCs. Recipient mice mutantofSIRT1(SupportingInformationFigS10),wasableto
exhibited no difference in total body and subcutaneous fat restore the differentiation potential of MSCKO MSCs (Fig 4C).
weight nor bone and subcutaneous adipose tissue structure MicroarrayanalysisofMSCsshowedadifferenceintheoutput
(SupportingInformationFigS9). of the Wnt signalling pathway (Supporting Information
To test whether reduced differentiation of MSCs is due to Fig S11A) and qPCR analysis of the known target genes
SIRT1 activity, we treated fl/fl MSCs with the SIRT1 inhibitor revealed reduction of alp, C/ebp, Sox9 and runx2 in MSCs
EX527.InvitrotreatmentofMSCswithEX527recapitulatedthe (Fig 4D), bone, adipose tissue and cartilage of MSCKO mice
Figure4. SIRT1promotesnuclearlocalizationofb-cateninanditsactivityinmesenchymalprogenitorcells.
A. Alkalinephosphatasestainingoffl/flMSCstreatedwithSIRT1inhibitorEX527.BFUboneformingunits,mean(cid:4)SD,(cid:2)p¼0.004,t-test,n¼6wellspergroup.
B. Westernblotanalysisofp53acetylationasamarkerofSIRT1activityandbargraphshowingacetyl53/p53densitometryfoldchange.
C. AlkalinephosphatasestainingofMSCKOmesenchymalprogenitorcellsdifferentiatedtowardsboneaftertransfectionwithcontrolvector,SIRT1over-
expressingvirus,andSIRT1deacetylaseinactiveHYmutant.BFUboneformingunits,mean(cid:4)SD,(cid:2)p¼0.001,ANOVADunnetttest,n¼6wellspergroup.
D. qPCRanalysisofb-catenintargets,n¼4mesenchymalprogenitorsamplespergroup,(cid:2)p¼0.01–0.05,t-test.
E. Luciferasereporterassaysoffl/flandMSCKOMSCs.Transcriptionalactivityofb-catenininMSCswasdetectedusingpTOP-flash(containingtripleTcf/Lef1
bindingsites,thebasicthymidinekinasepromoterandfireflyluciferasereportergene)orpFOP-flash(containingmutatedTcf/Lef1bindingsites)plasmidsand
wasnormalizedtoRenillaluciferase.(cid:2)p¼0.003,t-test,n¼12wellspergroup,threeindependentexperiments.
F. Immunofluorescentstainingofb-cateninandDAPIinfl/flandMSCKOMSCstransfectedwithpCMV-b-cateninT41A(b-catenin(cid:2)).Thirty-fourplusorminus
eightpercentofcellsshowednuclearlocalizationinfl/flMSCs,while0%showednuclearstaininginMSCKOMSCs.
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(Supporting Information Fig S11B). The above findings etal,2011).Sincethesestudieswereallperformedinvitro,we
suggested that the Wnt signalling pathway, previously impli- chosetoinvestigatetheroleofSIRT1inthebiologyofMSCsby
cated in MSC differentiation (see Introduction section), was generatingMSC-specificknockoutmice(MSCKO)usingafloxed
defective in MSCKO cells and tissues derived from them. SIRT1alleleandacregenedrivenbythePrx-1promoter.Our
AnalysisofadditionalcomponentsoftheWntpathwayrevealed in vivo studies showed that loss of SIRT1 negatively impacts
the increase of receptor and co-receptor, Lrp5 and Fzd1, tissuesofmesenchymalorigin,especiallyinoldmice.Defectsin
expression,possiblybycompensatorymechanisms(Supporting bone and subcutaneous fat were strikingly evident in older
InformationFigS12A)andnoeffectontheexpressionofsome MSCKO mice, while milder phenotypes were found in young
other important bone related transcription factors like Smads mice.ThedefectsinboneintheMSCKOmicecouldbeattributed
(SupportingInformationFigS12B). to two effects. First, there was a significant aging-induced
b-Catenin is turned over by the proteasome, when it has depletionofSTRO1þbonemarrowcellsinthesemice,indicative
been phosphorylated by GSK3b Wnt functions as an extra- of aging-induced MSC loss. Second, as described below, the
cellularligandtotriggerinhibitionofGSK3b.Wntthusleadsto potentialofMSCstodifferentiateintobonewasalsoreducedby
the stabilization and nuclear accumulation of b-catenin and knockingoutSIRT1.Wealsoobservedaberrationsinnumbers
theactivationoftargetgeneslikerunx.Tofurthervalidatethe ofhelperandregulatoryTcellsinMSCKOmice,consistentwith
defect in the Wnt pathway in MSCKO cells, we transfected a theknownimmunomodulatorypropertiesofMSCs(Ghannam
b-catenin-responsiveluciferasereporterintocells,andobserved etal,2010;Shietal,2011).
a large defect in MSCKO cells compared to wild-type MSCs InordertostudytheeffectsofSIRT1 depletiononMSCsin
(Fig4E). more detail, we used an established protocol to isolate these
TolocatethedefectintheWntpathwayinMSCKOcells,we cellsfrombonemarrow(Soleimani&Nadri,2009).Importantly,
transfected wild-type MSCs and MSCKO cells with a T41A singlecellsisolatedinthiswayhadthepotentialtodifferentiate
mutant form of b-catenin, which resists GSK3b-mediated into bone and fat cells, indicating that they display multi-
turnover. We thus observed b-catenin in the nucleus of WT, potency,onehallmarkofstemcells.InjectingtheputativeMSCs
butnotofMSCKOcells(Fig4F).SIRT1inMSCsdeacetylatedb- intoblastocystsalsodemonstratedthattheyweremulti-potent.
catenin, since reduction of its activity by EX527, SIRT1 TheseMSCcellsalsoreplicatedinvitro,indicatingthattheyare
deacetylase inhibitor, led to acetylation of b-catenin in both capableofself-renewal,andnosignificantdifferencewasnoted
oldandyoungMSCs(Fig5AandB).Inaddition,whenMSCs between wild-type and MSCKO cells in replication. However,
differentiate,levelsofSIRT1decrease(Fig5A),andacetylation SIRT1 was important for the differentiation of these MSCs
of b-catenin increases (Fig 5A). We further tested whether b- towards the bone and cartilage. SIRT1 was dispensable for
cateninmutationsatK49andK345(residuespreviouslyshown differentiation to muscle or adipocytes, although the latter
tobeacetylated,Le´vyetal,2004;Wolfetal,2002)couldrescue differentiatedcellscontainedlessfatandweresmallerinsize.
the nuclear localization defect in MSCKO cells. Indeed, To test whether the differentiation defects in MSCKO cells
transfectionwithb-cateninmutantsK49R,K345Rorthedouble was due to a reduction SIRT1-mediated deacetylation of b-
mutant (Supporting Information Fig S13), led to nuclear catenin,wefirstshowedthatwild-typeSIRT1butnotacatalytic
localizationofb-catenininMSCKOcells(Fig5C),andrestored mutantcouldrescuethedefectinosteoblastdifferentiation.We
theirpotentialfordifferentiationtoosteoblasts(Fig5D). furtherfoundthatab-cateninreporterwasactivatedtoamuch
Finally, since b-catenin is an oncogene, we tested the higherlevelinwild-typeMSCsthaninMSCKOcells.Strikingly,
tumorigenic potential of MSCs with repressed or activated b- b-catenin was immuno-localized to nuclei in the wild-type
catenin.WeinjectedGFPmarkedwild-type(fl/fl),MSCKOand MSCs, but was extranuclear in MSCKO cells. b-Catenin is
MSCKOexpressingb-cateninK49RK345Rsubcutaneouslyinto acetylated by p300 at K345 (Le´vy et al, 2004) and by CREB-
nudemiceandscoredfortumours.WealsoinjectedMSCsover- bindingprotein(CBP)acetyltransferaseatK49(Wolfetal,2002)
expressingSIRT1(MSCTg).Noneoftheinjectedmicedisplayed in epithelial cells, and is deacetylated by SIRT1 in these cells
tumours. However, to our surprise MSCTg and MSCKO cells (Firestein et al, 2008). Indeed, b-catenin was hyperacetylated
expressingK49RK345Rb-catenininducedhairgrowthinnude uponSIRT1inhibition,andmutatingK49andK345toarginine
mice (Supporting Information Fig S14A). All injected MSCs singly or together rescued the nuclear localization defect and
migrated to the hair follicle dermal papilla that has mesen- bonedifferentiationdefectinMSCKOcells.Expressionofsome
chymalorigin(SupportingInformationFigS14BandC).Since b-catenin targets, like alp, runx2 and bmp4 is crucial for
b-cateninsignallingindermalpapillaiscrucialforhairgrowth differentiation of MSCs towards bone. Expression of all these
(Enshell-Seijffers et al,2010), these findings further support a targets was reduced in MSCKO MSC-derived tissues, and this
linkbetweenSIRT1andb-catenininMSCs. mightserveasamolecularexplanationfortheeffectsonbone
differentiation. However, in the case of adipose and cartilage
only a subset of important factors was affected, which might
DISCUSSION explainmilderphenotypeinthosetissues.
TheabovefindingssuggestthatinMSCs,SIRT1deacetylates
Several studies suggest an effect of SIRT1 on promoting b-catenin to trigger its concentration in the nucleus and
osteogenesis and decreasing adipogenesis of MSCs (Ba¨ckesjo¨ activation of genes for differentiation. Intriguingly, an earlier
etal,2006;Lietal,2011;Peltzetal,2012;Purietal,2012;Tseng studyinepithelialcells(Firesteinetal,2008)showedthatSIRT1
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SIRT1regulatesdifferentiationofMSCs
Figure5. SIRT1deacetylatesb-cateninleadingtonuclearlocalizationanddifferentiationofMSCs.
A. WesternblotanalysisofoldMSCstreatedwithSIRT1deacetylaseinhibitorEX527beforeandafterdifferentiationtoosteoblasts.SIRT1wasblottedbefore
immunoprecipitationofb-cateninandacetyl-lysinefollowingimmunoprecipitationofb-catenin.
B. Acetylationofb-catenininyoungMSCsfollowingthetreatmentwithSIRT1inhibitor,EX527.MSCsampleswereimmunoprecipitatedwithb-cateninantibody
andmembraneswereprobedwithacetyl-lysineandb-cateninantibody.
C. Immunofluorescentstainingofb-cateninandDAPIinfl/flandMSCKO.MSCsweretransfectedwiththestableformofb-catenin(T41A)andab-cateninmutant
K49RK345R.Nuclearstainingwas0%ofcellsforT41Aand29(cid:4)9%ofcellsforT41AK49RK345R.
D. AlkalinephosphatasestainingofMSCKOmesenchymalprogenitorcellsdifferentiatedtowardsboneaftertransfectionwiththecontrolvector,stableformof
b-catenin(T41A)andb-cateninmutantwhereK49R,K345R.%þarea,percentageofalkalinephosphatasepositiveareaofthewell;numbersaremean(cid:4)SD,
(cid:2)p¼0.005versuslacZ,ANOVADunnetttest.
436 (cid:1)2013TheAuthors.PublishedbyJohnWileyandSons,LtdonbehalfofEMBO. EMBOMolMed(2013)5,430–440
www.embomolmed.org ResearchArticle
PetraSimicetal.
deacetylationofb-cateninexertstheoppositeresult—eviction SIRT1 inhibitor EX527 (Sirtris Pharmaceuticals), was dissolved in
of b-catenin from the nucleus. More generally, epithelial and waterandwasusedin10mMconcentrationeveryotherdayduring
mesenchymalcellsshowdifferentlocalizationofb-catenin,for differentiationofMSCs.
example during the epithelial to mesenchymal transition
(Kalluri & Weinberg, 2009). How might deacetylation of Plasmidsandcelltransfections
b-cateninbySIRT1exertoppositeeffectsindifferentcelltypes? Stablecellsweregeneratedusinglentivirusorretrovirus.Theplasmids
Wespeculatethatthenuclearimportandexportmachinerymay expressingmSIRT1,deacetylaseinactiveHYSIRT1mutant,pBABEand
differinMSCsversusepithelialcells,suchthatthedeacetylated PRRL-GFPvectorswerepurchasedfromAddgene.siRNA-SIRT1plasmid
lysines in b-catenin are triggers for nuclear import in MSCs has been previously described (Picard et al, 2004). The b-catenin
butexportinepithelialcells.Thiswouldbetrue,forexample, expression vector pCMV-T41Ab-catenin, carrying a Myc-tagged
if both cell types had an export pathway recognizing the dominant stable b-catenin mutated at residue 41 (threonine to
deacetylated lysines, but MSCs uniquely possessed an import alanine), has been described previously (Wei et al, 2000). Viral
pathwaythatrecognizestheseresiduesandoverridestheexport productionwasperformedbyligationofplasmidintopENTR-D-TOPO
pathway. (Invitrogen) and then recombined into pLenti4-TO-V5-DEST (Invitro-
In summary, we show that SIRT1 affects MSCs by gen). Mutations of lysine residue K345 and/or K49 to R or A were
deacetylating b-catenin, which increases their potential for generated in this construct with the QuickChange XL site-directed
differentiationtobone,andtoalesserextentfat.Anothereffect mutagenesis kit (Stratagene) and verified by sequencing. Cells were
of SIRT1 is to counter the depletion of MSC number in aging virally infected as previously described (Bell et al, 2011). Transient
mice. Our findings may represent a fraction of the biology transfections were performed by using Fugene transfection reagent
influenced by the SIRT1/b-catenin interaction in MSCs. For (Roche).
example,weobservedthatMSCswithoverexpressedSIRT1or
expressingaconstitutivelydeacetylatedb-cateninmimichadan Mesenchymalprogenitorcellinjectionsintoblastocystand
increasedpotentialtogiverisetohairgrowthafterinjectioninto embryotransfers
nudemice.ItwillbeinterestingtostudytheeffectsofSIRT1in MSCsweremarkedwiththeviraltransfectionofGFP.Blastocystswere
stillotherbiologicalsystemsinwhichMSCsplayadefiningrole. injected with 15–20 MCSc each using micropipette (Longenecker &
Kulkarni, 2009). Blastocysts were transferred to the oviducts of
pseudopregnant F1 females mated to vasectomized F1 males of
MATERIALS AND METHODS proven sterility. Twelve embryos were transferred to oviduct of
recipientmouse.Therewerefourrecipientmiceintotal,tworeceiving
Mousestrains blastocystswithfl/flMSCsandtwoblastocystswithMSCKOMSCs.All
Severe combined immunodeficiency mice were purchased from experimentswereperformedinaccordancetoMassachusettsInstitute
Charles River Laboratories. All other mice were in congenic C57Bl/6 of Technology guidelines and regulations and were approved by
background. MSCKO mice were generated by crossing SIRT1 allele CommitteeonAnimalCare.
containing a floxed exon 4 (Chen et al, 2008) with Cre-expressing
mice driven by the mesenchymal progenitor cell-specific Prx1-cre Mesenchymalprogenitorcellsubcutaneousinjectionsin
promoter (Jackson Laboratory). MSCTG mice were generated by SCIDmice
crossingaSIRT1loxP flankedmice(Firesteinetal, 2008)withmice MSCcellsweresuspendedinPBS.SCIDmicewereanesthetizedwith
havingPrx1crepromoter.Allmicewerehousedat258Cand12:12h isofluorane.Weinjected106cellssubcutaneouslyintothedorsalflank.
light/dark cycle. All experiments were performed in accordance to SCID mice were sacrificed 14 days following the subcutaneous
MassachusettsInstituteofTechnologyguidelinesandregulationsand injections. All experiments were performed in accordance to
wereapprovedbyCommitteeonAnimalCare. Massachusetts Institute of Technology guidelines and regulations
andwereapprovedbyCommitteeonAnimalCare.
Cellsandtreatment
Primary mesenchymal progenitor cells were isolated from the bone Bonemarrowtransplantation
marrow of tibias and femurs and then cultured with trypsinization Five-week-old CD45.2 recipient mice were irradiated with a single
after the cells became confluent according to previously published doseof9.5Gy.Thefollowingday,bonemarrowsfromfl/flandMSCKO
protocolSoleimani&Nadri,2009).ApurifiedpopulationofMSCswas donor C57Bl/6 mice were injected via tail vein into recipient mice
obtained3weeksaftertheinitiationofculture.ForasinglecellMSC (Sharabi and Sachs, 1989). Mice were left for 6 months and bone
experiment one MSC for transferred into one well of 96-well plate marrowMSCswereanalysed.
usingmicroscopeandmicropipette.
Primary MSCs were differentiated towards adipocytes using 0.5mM MicroCTanalysisofbonesandadiposetissue
isobutyl-methylxanthine (IBMX), 1mM dexamethasone, 10mM in- The microcomputerized tomography apparatus (mCT 40) and the
sulin; towards osteoblasts using 0.1mM dexamethasone, 50mM analyzing software used in these experiments were obtained from
ascorbate-2-phosphate, 10mM b-glycerophosphate; and towards SCANCOMedicalAG(Bassersdorf,Switzerland).Thedistalfemurwas
chondrocytes using 6.25mg/ml insulin, 10ng/ml TGF-b1, 50nM scannedin250slices,each5-mmthickinthedorsoventraldirection
ascorbate-2-phosphate in a-MEM media with 10% foetal bovine (Simic et al, 2006). Three-dimensional reconstruction of bone was
serumand1%antibioticfor2–3weeks(Zuketal,2001). performed using the triangulation algorithm. The trabecular bone
EMBOMolMed(2013)5,430–440 (cid:1)2013TheAuthors.PublishedbyJohnWileyandSons,LtdonbehalfofEMBO. 437
ResearchArticle www.embomolmed.org
SIRT1regulatesdifferentiationofMSCs
The paper explained
PROBLEM:
ThisstudyaddressesthepotentialroleofSIRT1inadultMSCs, MSCs,andwasrescuedbymutatinglysines49and345in
whichcandifferentiateintoosteoblasts,adipocytes,chondro- b-catentintoarginine.WeconcludethatSIRT1deacetylates
cytesandmyocytes. b-catenininMSCstopromotegeneexpressionimportantin
differentiationtoosteoblasts.
RESULTS:
IMPACT:
OldSIRT1MSC-specificknockoutmiceshowedareductionin
MSCnumberanddefectsintissuesderivedfromMSCs.In OurfindingssuggestthatSIRT1maybeatherapeutictargetto
addition,MSCsisolatedfromknockoutmicehadalower fosterMSCdifferentiationtopreserveintegrityofMSC-derived
potentialfordifferentiationintoosteoblastsinvitro.Thisdefect tissuesinaginganimals.
wasassociatedwithadefectinb-cateninfunctioninknockout
volume,trabecularandcorticalthicknessweredirectlymeasuredon gend)andannexinV(BD)primaryantibodiesandAlexaFluor488and
three-dimensionalimagesusingthemethoddescribedbyHildebrand AlexaFluor647secondaryantibodiesandcountedbyaFACSCalibur
et al (Hildebrand et al, 1999). Quantification of adipose tissue was FlowCytometer(BDBiosciences).Haematopoieticcellswereanalysed
preformed in the abdominal region from L1 to L7 using previously usingB220,Mac1,Gr1,Ter119,CD3,Lin,Kit,Sca,CD48,CD150,CD34,
describedmethod(Judexetal,2010). CD16/32, CD45 and CD31 primary antibodies and Pac Orange, Pac
Blues, APC, PE-Cy7,FITC, PE andPE-Cy5 secondaryantibodies. Flow
Histology,histomorphometryandimmunohistochemistry cytometricanalysisformarkerexpressionwasperformedusingFlowJo
Bone,adipose,cartilageandmuscletissuehistologywasexaminedon software(Ashland,OR,USA).
formalin fixed, paraffin embedded sections 3mm thick stained by
haemalaun–eosin(6sections/organ). RNAisolationandanalysis
For histomorphometry of bones MSC fl/fl and MSCKO animals TotalRNAfromMSCs,bonemarrow,adiposetissueandmusclewas
were given a subcutaneous injection of the fluorochrome calcein at isolatedbyusingTrizol(Qiagen).cDNAwassynthesizedfromtotalRNA
10mg/kg (Sigma) at 4 and 14 days before death.The femurs were by SuperScript III reverse transcriptase (Invitrogen) with random
removed at death, prepared for histomorphometric analysis, and primers.The cDNA was then subjected to real time q-PCR analysis
quantifiedusingacomputer-aidedimageanalysissystem(BioquantII, with gene specific primers in the presence of CYBR green (Bio-Rad).
R and M Biometrics, Nashville, TN) as previously described (Simic RelativeabundanceofmRNAwasnormalizedtorpl19.
etal,2006).
Westernblotting
Alkalinephosphatase,lipotoxandalcianbluestainingof OrgansandMSCswerehomogenizedinRIPAbufferincludingComplete
MSCs ProteaseInhibitor(Roche).Protein(100mg)wasloadedontoSDS–PAGE
MSCsafterdifferentiationwerestainedusingalkalinephosphtasekit gels and immunoblotted with anti-SIRT1 (Upstate), b-catenin (BD
(Sigma)andLipidTOXGreenNeutralLipidStain(Invitrogen)according Bioscience)andacetyl-lysine(CellSignalling)antibodies.
to manufacturers instructions. Toluidine staining of MSCs was
performed according to previously described protocol (Zuk et al, Luciferaseassay
2001).CellsizewasdeterminedusinganAxioImagerM1epifluores- Transcriptional activity of b-catenin in MSCs was detected using
cencemicroscope(Zeiss)andImageJsoftware(version1.37forMac pTOP-flash (containing triple Tcf/Lef1 binding sites, the basic
OS X; National Institutes of Health, Bethesda, MD). In each thymidine kinase promoter and firefly luciferase reporter gene) or
preparation, 400 cells from various parts of the tissue slice were pFOP-flash (containing mutated Tcf/Lef1 binding sites) plasmids
sizedforcalculationofmeancellsize. (Addgene). Luciferase activity was measured by Dual-Luciferase
Reporter Assay System (Promega).The firefly luciferase activity was
Immunofluorescentstaining normalizedtoRenilla.Theexperimentswereperformedintriplicates
MSCswerelabelledwithb-cateninantibody.Theslideswereexposed andwererepeatedthreetimes.
to Alexa Fluor 488, 568 and 594 anti-rabbit and anti-mouse
secondary antibodies (Invitrogen). The staining was examined with DetailsonmicroarrayanalysisofRNAcanbefoundintheSupporting
fluorescencemicroscope(ZeissAxioImager.M1). InformationMaterialsandMethods.
Flowcytometricanalysisandsorting Statisticalanalysis
MSCsweretrypsinized,resuspendedinthe FACSbufferandlabelled TheanalyseswereperformedusingANOVADunnetttest. Differences
with alkaline phosphatase (Abcam), FABP4 (Abcam), STRO-1 (Biole- wereconsideredsignificantifp<0.05.
438 (cid:1)2013TheAuthors.PublishedbyJohnWileyandSons,LtdonbehalfofEMBO. EMBOMolMed(2013)5,430–440
www.embomolmed.org ResearchArticle
PetraSimicetal.
Author contributions Sir2homolog(SIRT1)-deficientmice.ProcNatlAcadSciUSA100:10794-
PS designed and performed experiments, analysed data and 10799
wrote the paper; KZ started the colony of MSCKO mice; EB ChoHH,KimYJ,KimSJ,KimJH,BaeYC,BaB,JungJS(2006)EndogenousWnt
signalingpromotesproliferationandsuppressesosteogenicdifferentiation
performedexperiments;DBS,BSandDSperformedexperiments
inhumanadiposederivedstromalcells.TissueEng12:111-121
thatincludedanalysesoflymhocytes;SLdidhistomorphometric
Cohen-KfirE,ArtsiH,LevinA,AbramowitzE,BajayoA,GurtI,ZhongL,D’UrsoA,
analysis,ESpreparedbonehistology,RBperformedhistomor- ToiberD,MostoslavskyR,Dresner-PollakR(2011)Sirt1isaregulatorof
phometricanalysisandLGdesignedexperiments,analyseddata bonemassandarepressorofSostencodingforsclerostin,aboneformation
andwrotethepaper. inhibitor.Endocrinology152:4514-4524
D’IppolitoG,SchillerPC,RicordiC,RoosBA,HowardGA(1999)Age-related
osteogenicpotentialofmesenchymalstromalstemcellsfromhuman
vertebralbonemarrow.JBoneMinerRes14:1115-1122
Acknowledgements
DeBoerJ,WangHJ,VanBlitterswijkC(2004)EffectsofWntsignalingon
We wish to thank Eric O. Williams for help during the
proliferationanddifferentiationofhumanmesenchymalstemcells.Tissue
preparation of the manuscript. This work is supported by Eng10:393-401
Fulbright Association post-doctoral fellowship to P.S., F32CA DonmezG,GuarenteL(2010)Aginganddisease:connectionstosirtuins.
132358 to E.L.B. and grants from the NIH and a gift from the AgingCell9:285-290
GlennMedicalFoundationtoL.G. EijkenM,MeijerIM,WestbroekI,KoedamM,ChibaH,UitterlindenAG,PolsHA,
vanLeeuwenJP(2008)Wntsignalingactsandisregulatedinahuman
osteoblastdifferentiationdependentmanner.JCellBiochem104:568-579
Supporting Information is available at EMBO Molecular
Enshell-SeijffersD,LindonC,KashiwagiM,MorganBA(2010)Beta-catenin
MedicineOnline.
activityinthedermalpapillaregulatesmorphogenesisandregenerationof
hair.DevCell18:633-642
Conflict of interest statement: LG is co-chair of the SAB of EtheridgeSL,SpencerGJ,HeathDJ,GeneverPG(2004)Expressionprofilingand
Sirtris, a GSK company. The other authors declare that they functionalanalysisofwntsignalingmechanismsinmesenchymalstem
havenoconflictofinteres. cells.StemCells22:849-860
FiresteinR,BlanderG,MichanS,OberdoerfferP,OginoS,CampbellJ,
BhimavarapuA,LuikenhuisS,deCaboR,FuchsC,etal(2008)TheSIRT1
deacetylasesuppressesintestinaltumorigenesisandcoloncancergrowth.
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