Abstract
Hypoxia has diverse stimulatory effects on human adipose-derived stem cells (ASCs). In the present study, we investigated whether hypoxic culture conditions (2% O2) suppress spontaneous mineralization and osteogenic differentiation of ASCs. We also investigated signaling pathways and molecular mechanisms involved in this process. We found that hypoxia suppressed spontaneous mineralization and osteogenic differentiation of ASCs, and up-regulated mRNA and protein expression of Insulin-like growth factor binding proteins (IGFBPs) in ASCs. Although treatment with recombinant IGFBPs did not affect osteogenic differentiation of ASCs, siRNA-mediated inhibition of IGFBP3 attenuated hypoxia-suppressed osteogenic differentiation of ASCs. In contrast, overexpression of IGFBP3 via lentiviral vectors inhibited ASC osteogenic differentiation. These results indicate that hypoxia suppresses spontaneous mineralization and osteogenic differentiation of ASCs via intracellular IGFBP3 up-regulation. We determined that reactive oxygen species (ROS) generation followed by activation of the MAPK and PI3K/Akt pathways play pivotal roles in IGFBP3 expression under hypoxia. For example, ROS scavengers and inhibitors for MAPK and PI3K/Akt pathways attenuated the hypoxia-induced IGFBP3 expression. Inhibition of Elk1 and NF-κB through siRNA transfection also led to down-regulation of IGFBP3 mRNA expression. We next addressed the proliferative potential of ASCs with overexpressed IGFBP3, but IGFBP3 overexpression reduced the proliferation of ASCs. In addition, hypoxia reduced the osteogenic differentiation of bone marrow-derived clonal mesenchymal stem cells. Collectively, our results indicate that hypoxia suppresses the osteogenic differentiation of mesenchymal stem cells via IGFBP3 up-regulation.
Highlights
Hypoxia has diverse effects on human adipose-derived stem cells (ASCs) [1,2,3,4]
We examined whether siRNA-mediated inhibition of Insulin-like growth factor binding proteins (IGFBPs) proteins could attenuate hypoxiaiSnidnucceedexosugpepnroeusssiornecoofmobsitneaongetnIiGc FdBifPfesrednitdiatnioont. aWffeectptrheepaoresdteosgiReNniAcsdoifffeIrGenFBtiPa3ti–o5n, aonfdASCs, we further examined whether endogenous intracellular IGFBPs affected osteogenic differentiation
We found that reactive oxygen species (ROS) mediated IGFBP3 expression in ASCs under hypoxia (2% O2)
Summary
Hypoxia has diverse effects on human adipose-derived stem cells (ASCs) [1,2,3,4]. For example, hypoxia increased the expression of Oct and Rex, and inhibited the aging/senescence of ASCs during culture [5]. We first examined whether hypoxic culture conditions (2% O2) suppress the spontaneous mineralization and osteogenic differentiation of ASCs. we investigated signaling pathways and molecular mechanisms involved in this process. We examined whether siRNA-mediated inhibition of IGFBP proteins could attenuate hypoxia-induced suppression of osteogenic differentiation. Hs eyxppoexciatesdu,phpyrepsosexdiath(2e%os)tienodguenciecd the mRNdAiffeerxepnrtieastsioionnoof fclIoGnFaBl PB3Mi-nMcSlCosnaalt B7Man-Md S1C4 sda(Fyisg(uFriegu6rAe ).BH).yOpsotxeioagesnuipcpirnedsuscetdiotnhemoarskteeorsgenic diffesruecnhtiaastioRnUoNfXc2lo, noastleBocMal-cMinS, Cans dato7staenridx 1w4erdeaysisgn(Fifiigcaunrtely6Bre).dOucsetdeobgyenhiycpionxdiaucintioDnMmEaMrkaenrsdsuch as ROUDNMX2(,Fiogsutreeo6cDal)c.iInn, aadndditioosnt,esriiRxNwAe-rmeesdiiganteidficiannhitblyitiroendoufcIeGdFBbPy3hsyigpnoifixciaanitnlyDatMteEnuMateadndtheODM (FiguhOrysepte6ooDxgie)a.n-sIinuc pianpddrduesictstiieoodnno,msstiaeRrokNgeeArsn-ismcucdehdiffaieasrtReendUtNiiantXhio2i,nboiotsitfoeconlcooanlfcaIilnGB,FMaBn-dPM3oSssCtiegsrniaxitfwi7ceaarnnetdsliyg1n4aitfidtecanaynustal(yFteirgdeudtruhecee6hdEy,bFpy).oxiasupphryepsosexida oatst7edoagyesn(iFcigduifrfee6reGn)taiantdio1n4 doafycslo(FniagluBreM6-HM).SCs at 7 and 14 days (Figure 6E,F) Osteogenic induction markers such as RUNX2, osteocalcin, and osterix were significantly reduced by hypoxia at 7 days (Figure 6G) and 14 days (Figure 6H). Induucceedd the mRNA expreessssiioonn ooff IGFBPP33 in clonal BM-MSCs; (B) hypoxia suppresseed the osteogenic ddiiffffeerreennttiiaattiioonn ooff cclloonnaall BBMM--MMSSCCss aatt 77 aanndd 1144ddaayyss((4400××); (CC) qquuaannttiifificcaation ooff AARRSS ssttaaiinniinngg wwaass mmeeaassuurreedd aatt 77ddaayyss;; ((DD)) oosstteeooggeenniicc iinndduuccttiioonn mmaarrkkeerrss ssuucchh aass RRUUNNXX22,, oosstteeooccaallcciinn,, aanndd oosstteerriixxwweerree ssiiggnniifificcaannttllyy rreedduucceedd bbyy hhyyppooxxiiaa iinn DDMMEEMM aanndd OODDMM;; ((EE)) ssiiRRNNAA--mmeeddiiaatteedd iinnhhiibbiittiioonn ooff IIGGFFBBPP33 ssiiggnniifificcaannttllyyaatttetennuuaateteddththeehyhpyopxoixai-as-uspupprpersessesdedosotesotegoegneicndicifdfeirfefenrteianttiioantioonf colofncalol nBaMl -BMMS-CMs SaCt 7s aant d7 1a4ndda1y4s (d4a0y×s);(4(F0)×q);u(aFn)tiqfiucaantitoifnicoaftiAonRSosftaAinRiSngstwaiansinmgeawsausremd eaats7udreadysa; t(G7,Hda) yoss;te(oGg,eHn)icoisntdeougcteinoinc minadrukcetrisonsumcharakseRrsUsNucXh2,aossRteUoNcaXlc2i,no,satneodcoaslcteinri,xawnderoessteigrinxifiwcearnetlsyigrnedifuiccaendtlbyyrehdyupcoexdiabayt h7ydpaoyxsia(Ga)t a7ndda1y4s d(Gay) san(Hd)1. 4Ndoarymso(xHia).: Nbloarcmk obxairas:, bhlyapcokxbiaa:rsw, hhyitpeobxairas:.w* hpi
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