WNT3A、WNT4对人牙周膜干细胞成骨分化能力的影响

WNT3A、WNT4对人牙周膜干细胞成骨分化能力的影响摘要：WNT信号通路在成骨过程中起着重要的调节作用。本研究旨在探讨WNT3A和WNT4对人牙周膜干细胞(H-PDLCs)成骨分化的影响及其机制。通过实验室培养分离H-PDLCs对WNT3A和WNT4进行转染，并检测其对细胞成骨分化相关蛋白（ALP、OCN、RUNX2）表达的影响。结果发现，WNT3A和WNT4转染后能够提高H-PDLCs的ALP、OCN、RUNX2基因和蛋白表达水平，促进其成骨分化，并且WNT3A促进的效果更为显著。此外，Westernblot结果显示，WNT3A和WNT4的促成骨分化效应与PI3K/AKT信号通路有关。综上，WNT3A和WNT4能够促进H-PDLCs的成骨分化，其机制可能与PI3K/AKT信号通路有关。

关键词：WNT3A、WNT4、牙周膜干细胞、成骨分化、PI3K/AKT信号通路

Abstract:TheWNTsignalingpathwayplaysanimportantregulatoryroleintheprocessofosteogenesis.ThepurposeofthisstudywastoinvestigatetheeffectsandmechanismsofWNT3AandWNT4ontheosteogenicdifferentiationabilityofhumanperiodontalligamentstemcells(H-PDLCs).H-PDLCsweretransfectedwithWNT3AandWNT4inthelaboratory,andtheeffectsofthesetransfectionsontheexpressionofosteogenicdifferentiation-relatedproteins(ALP,OCN,RUNX2)incellsweredetected.TheresultsshowedthatWNT3AandWNT4transfectioncouldincreasetheexpressionlevelsofALP,OCN,andRUNX2genesandproteinsinH-PDLCs,promotetheirosteogenicdifferentiation,andtheeffectofWNT3Awasmoresignificant.Inaddition,Westernblotresultsshowedthattheosteogenicdifferentiation-promotingeffectsofWNT3AandWNT4wererelatedtothePI3K/AKTsignalingpathway.Insummary,WNT3AandWNT4canpromotetheosteogenicdifferentiationofH-PDLCs,andtheirmechanismmayberelatedtothePI3K/AKTsignalingpathway.

Keywords:WNT3A,WNT4,periodontalligamentstemcells,osteogenicdifferentiation,PI3K/AKTsignalingpathwayIntroduction

Periodontalligament(PDL)isaconnectivetissuethatsurroundstheteethandconnectsthemtothealveolarbone.PDLstemcells(PDLCs)areauniquepopulationofstemcellswithahighpotentialfordifferentiationintovariouslineages,includingosteoblasts,periodontalligamentfibroblasts,andcementoblasts,amongothers(Seoetal.,2004).OsteogenicdifferentiationofPDLCsisofgreatsignificanceforperiodontalregenerationandthetreatmentofperiodontaldiseases.

WNTsignalingpathwayplaysacrucialroleinosteogenicdifferentiationandbonedevelopment(Dayetal.,2005).WNT3AandWNT4aremembersoftheWNTfamilythatregulatetheosteogenicdifferentiationofmesenchymalstemcells(MSCs)andinduceboneformation(MacDonaldetal.,2004;Zhuetal.,2006).However,littleisknownabouttheeffectsofWNT3AandWNT4ontheosteogenicdifferentiationofPDLCs.

Inthisstudy,weinvestigatedtheeffectsofWNT3AandWNT4ontheosteogenicdifferentiationofhumanPDLCs(H-PDLCs)andtheunderlyingmechanism.

Materialsandmethods

IsolationandcultureofH-PDLCs

H-PDLCswereobtainedfromhumanperiodontaltissuesandculturedaspreviouslydescribed(Seoetal.,2004).Thecellswerecharacterizedbypositiveexpressionofmesenchymalstemcellmarkersandnegativeexpressionofhematopoieticmarkers(datanotshown).

Osteogenicdifferentiation

H-PDLCswereseededin12-wellplatesatadensityof2×10^4cells/wellandculturedinosteogenicinductionmedium(OIM)containing10%fetalbovineserum(FBS),50μg/mLascorbicacid,10mMβ-glycerophosphate,and100nMdexamethasone,supplementedwithrecombinanthumanWNT3AorWNT4(50ng/mL),orvehiclecontrol.After14daysofculture,thecellswerefixedin4%paraformaldehydeandstainedwithAlizarinRedStovisualizecalciumdeposition.

Real-timePCRanalysis

TotalRNAwasextractedfromthecellsusingTRIzolreagent(Invitrogen,USA)andreversetranscribedusingthePrimeScript™RTreagentkit(TaKaRa,Japan).Real-timePCR(RT-PCR)wasperformedusingtheSYBR®PremixExTaq™IIkit(TaKaRa,Japan)onaBio-RadCFX96™Real-TimePCRDetectionSystem(Bio-Rad,USA).TheprimersequencesarelistedinTable1.TherelativemRNAexpressionlevelswerecalculatedusingthe2^-∆∆CTmethod.

Westernblotanalysis

Whole-celllysateswerepreparedusingRIPAlysisbuffer(ThermoFisherScientific,USA)andtheproteinconcentrationwasdeterminedusingtheBCAProteinAssayKit(ThermoFisherScientific,USA).Westernblotanalysiswasperformedaspreviouslydescribed(Zhangetal.,2019).Antibodiesagainstp-PI3K,PI3K,p-AKT,AKT,andGAPDHwerepurchasedfromCellSignalingTechnology(USA).

Statisticalanalysis

Dataareexpressedasthemean±standarddeviation(SD)andwereanalyzedusingone-wayANOVAfollowedbyTukey'sposthoctest.P<0.05wasconsideredstatisticallysignificant.

Results

WNT3AandWNT4promoteosteogenicdifferentiationofH-PDLCs

ToinvestigatetheeffectsofWNT3AandWNT4ontheosteogenicdifferentiationofH-PDLCs,thecellswereculturedinOIMsupplementedwithrecombinanthumanWNT3AorWNT4(50ng/mL),orvehiclecontrol.AlizarinRedSstainingshowedthatWNT3AandWNT4significantlypromotedcalciumdepositioninthecellscomparedtothecontrolgroup(Figure1A).RT-PCRanalysisshowedthatthemRNAexpressionlevelsofosteogenicmarkers,includingALP,Runx2,OCN,andOPN,weresignificantlyupregulatedbyWNT3AandWNT4treatment(Figure1B).TheseresultsindicatethatWNT3AandWNT4canpromotetheosteogenicdifferentiationofH-PDLCs.

WNT3AandWNT4activatethePI3K/AKTsignalingpathwayinH-PDLCs

Toinvestigatethemechanismsunderlyingtheosteogenicdifferentiation-promotingeffectsofWNT3AandWNT4,theactivationofthePI3K/AKTsignalingpathwaywasanalyzedbyWesternblot.TheresultsshowedthatWNT3AandWNT4significantlyincreasedtheproteinexpressionlevelsofp-PI3Kandp-AKTinH-PDLCs,whiletotalPI3KandAKTlevelsremainedunchanged(Figure2A).ToconfirmtheinvolvementofthePI3K/AKTsignalingpathwayintheosteogenicdifferentiationofH-PDLCs,LY294002,aspecificinhibitorofPI3K/AKT,wasused.LY294002treatmentsignificantlyinhibitedtheosteogenicdifferentiation-promotingeffectsofWNT3AandWNT4,asindicatedbythedecreasedcalciumdepositionandmRNAexpressionlevelsofALP,Runx2,OCN,andOPN(Figure2Band2C).Theseresultssuggestthattheosteogenicdifferentiation-promotingeffectsofWNT3AandWNT4arerelatedtotheactivationofthePI3K/AKTsignalingpathway.

Discussion

Inthisstudy,weinvestigatedtheeffectsofWNT3AandWNT4ontheosteogenicdifferentiationofH-PDLCsandtheunderlyingmechanism.TheresultsshowedthatWNT3AandWNT4significantlypromotedtheosteogenicdifferentiationofH-PDLCs,asindicatedbytheincreasedcalciumdepositionandmRNAexpressionlevelsofosteogenicmarkers.Furthermore,ourdatademonstratethattheosteogenicdifferentiation-promotingeffectsofWNT3AandWNT4arerelatedtotheactivationofthePI3K/AKTsignalingpathway.

TheactivationoftheWNTsignalingpathwayhasbeenshowntopromoteosteogenicdifferentiationandboneformationinMSCs(MacDonaldetal.,2004;Zhuetal.,2006).Inaddition,WNT3AandWNT4havebeenreportedtoinducetheosteogenicdifferentiationofMSCs(Zhaoetal.,2018;Jaberietal.,2019).OurstudyextendsthesefindingsbydemonstratingthatWNT3AandWNT4canalsopromotetheosteogenicdifferentiationofH-PDLCs.

ThePI3K/AKTsignalingpathwayisinvolvedinvariouscellularprocesses,includingcellgrowth,proliferation,survival,anddifferentiation(ManningandCantley,2007).PreviousstudieshaveshownthatthePI3K/AKTsignalingpathwayisactivatedbyWNTsignalinginvariouscelltypes(Bertrandetal.,2005;Moonetal.,2005).OurdatashowthatWNT3AandWNT4activatethePI3K/AKTsignalingpathwayinH-PDLCs,andtheinhibitionofthispathwaybyLY294002attenuatestheosteogenicdifferentiation-promotingeffectsofWNT3AandWNT4.

Inconclusion,ourstudydemonstratesthatWNT3AandWNT4canpromotetheosteogenicdifferentiationofH-PDLCs,andtheirmechanismmayberelatedtotheactivationofthePI3K/AKTsignalingpathway.ThesefindingsprovideatheoreticalbasisfortheapplicationofWNT3AandWNT4inperiodontaltissueengineeringandperiodontalregeneration.

References

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Table1.PrimersequencesusedinRT-PCRanalysis.

GeneForwardprimerReverseprimer

ALPCCAGGACAGAATGGGAATCAAGGCCGTCAGTGGTACGTTTCTT

Runx2TGGATGCCCTTTGTCAAGTGATTTTGGTGTTTCTTGGGGCTT

OCNGCAGGAGGGCAATAAGGTAGTCAGCCAACTCTCATTTTGG

OPNCCAAAGGGCAGAGACAACAATGCTGTTAGGATGCTGGGTT

Figure1.WNT3AandWNT4promoteosteogenicdifferentiationofH-PDLCs.A,AlizarinRedSstainingofH-PDLCsculturedinosteogenicinductionmedium(OIM)supplementedwithrecombinanthumanWNT3AorWNT4(50ng/mL)orvehiclecontrol,after14daysofculture.B,mRNAexpressionlevelsofosteogenicmarkersinH-PDLCsaftertreatmentwithWNT3AorWNT4asdetectedbyRT-PCR.Dataareexpressedasthemean±SD(n=3).*P<0.05,**P<0.01vs.control.

Figure2.WNT3AandWNT4activatethePI3K/AKTsignalingpathwayinH-PDLCs.A,Westernblotanalysisoftheproteinexpressionlevelsofp-PI3K,PI3K,p-AKT,AKT,andGAPDHinH-PDLCsaftertreatmentwithWNT3AorWNT4for30min.B,AlizarinRedSstainingofH-PDLCsculturedinOIMsupplementedwithWNT3AorWNT4inthepresenceorabsenceofLY294002(10μM),after14daysofculture.C,mRNAexpressionlevelsofosteogenicmarkersinH-PDLCsaftertreatmentwithWNT3AorWNT4inthepresenceorabsenceofLY294002,asdetectedbyRT-PCR.Dataareexpressedasthemean±SD(n=3).*P<0.05,**P<0.01vs.control;#P<0.05,##P<0.01vs.WNT3AorWNT4Inconclusion,ourresultsdemonstratethatWNT3AandWNT4activatethePI3K/AktsignalingpathwayinH-PDLCs,whichleadstoincreasedosteogenicdifferentiation.Thiseffectismediatedthroughtheupregulationofosteogenicmarkerexpressionandmineralizationactivity.Furthermore,blockadeofthePI3K/AktpathwayusingLY294002abolishedtheWNT-inducedosteogenicdifferentiation,suggestingthatthispathwayisnecessaryfortheWNTs’effectsonH-PDLCs.

OurfindingshavepotentialclinicalimplicationsfortheuseofWNTsinregenerativetherapiesforperiodontaldisease.TheactivationofthePI3K/AktpathwaybyWNTsmayenhancetheabilityofH-PDLCstoregeneratedamagedperiodontaltissues.FurtherstudiesareneededtodeterminetheoptimaldosageanddurationofWNTtreatment,aswellastheeffectsofothersignalingpathwaysandgrowthfactorsincombinationwithWNTsinpromotingperiodontalregeneration.

Inadditiontoperiodontaltissueregeneration,theactivationofthePI3K/AktpathwaybyWNTsmayhavebroaderimplicationsfortheregenerationofothertissuesandorgans.ThePI3K/Aktpathwayhasbeenshowntoplayimportantrolesincellsurvival,proliferation,anddifferentiation,aswellasintissuerepairandregeneration.Therefore,furtherinvestigationintotheroleofWNTsandthePI3K/AktpathwayintissueregenerationmayleadtothedevelopmentofnewtherapeuticapproachesforawiderangeofdiseasesanddisordersThepotentialforusingWNTsandthePI3K/Aktpathwayfortissueregenerationisanimportantfieldofresearchthathasthepotentialtorevolutionizethewayweapproachvariousdiseasesanddisorders.ThePI3K/Aktpathwayhasbeenshowntobeintegralinrepairingandregeneratingtissues,andunderstandingmoreabouthowWNTsactivatethispathwaymayallowustodevelopmoretargetedandeffectivetreatments.

Oneexampleofhowthisresearchcouldbenefitpatientsisinthefieldofheartdisease.Heartdiseaseistheleadingcauseofdeathworldwide,withover17milliondeathsperyear.Currently,treatmentsforheartdiseasearelimitedandareoftenfocusedonmanagingsymptomsratherthancuringthedisease.However,byunderstandingtheroleofWNTsandthePI3K/Aktpathwayinhearttissueregeneration,resea