基質(zhì)金屬蛋白酶-2,-9及其組織抑制因子在恒河猴黃體中的表達(dá)Matrixmetalloproteinases-2,-9andtheirtissueinhibitorsexpressioninMacacamulattacorpusluteum

Abstract:

Matrixmetalloproteinases-2and-9(MMP-2andMMP-9)arecriticalproteolyticenzymesinvolvedinavarietyofphysiologicalandpathologicalprocessesintheovary,includingcorpusluteumformationandregression.Tissueinhibitorsofmetalloproteinases-1and-2(TIMP-1andTIMP-2)arealsoimportantregulatorsofMMPactivity.ThepresentstudyaimedtoinvestigatetheexpressionpatternsofMMP-2,MMP-9andtheirinhibitorsinMacacamulattacorpusluteum(CL)duringdifferentstagesofthemenstrualcycle.Immunohistochemistryandquantitativereal-timePCRwereemployedtoanalyzetheexpressionoftheseproteinsintheCLoftheovary.WefoundthatMMP-2andMMP-9wereexpressedathighlevelsinthedevelopingandmid-cyclelutealphaseswhiletheirinhibitors,TIMP-1andTIMP-2,wereup-regulatedinthelatelutealphase.OurresultssuggestthatMMP-2andMMP-9playimportantrolesinthemaintenanceandregressionofM.mulattacorpusluteum,andinhibitorsTIMP-1andTIMP-2mayactasthekeyregulatorsoftheiractivity.

KeyWords:Matrixmetalloproteinase,tissueinhibitorofmetalloproteinase,corpusluteum,Macacamulatta

Introduction:

Thecorpusluteum(CL)isatransientendocrineglandthatformsafterovulationunderthestimulationofluteinizinghormone(LH),andisresponsiblefortheproductionofprogesterone,whichisessentialforthemaintenanceofpregnancyinmammals(1).TheformationandregressionofCLaretightlycontrolledbyavarietyoffactors,includingcytokines,growthfactors,andextracellularmatrix(ECM)remodelingenzymessuchasmatrixmetalloproteinases(MMPs)(2,3).MMPsareafamilyofproteinasesthatcandegradeECMcomponents,includingcollagens,laminins,andfibronectin,andplayimportantrolesintissueremodeling,cancerinvasion,andangiogenesis(4).

MMPsaresecretedasinactiveproproteinsthatareactivatedbyproteolyticcleavageofthepropeptidedomain.AmongtheMMPfamilymembers,MMP-2andMMP-9,alsoknownasgelatinases,arecapableofdegradingtypeIVcollagenandgelatins,andareinvolvedinthedegradationofthebasementmembraneduringangiogenesisandtissueremodeling(5).TheactivityofMMPsistightlyregulatedbytheirendogenousinhibitors,knownastissueinhibitorsofmetalloproteinases(TIMPs),whichbindtotheiractivesiteandpreventtheirproteolyticactivity(6).TIMPsareafamilyoffourmembers,withTIMP-1andTIMP-2beingthemostprominentinhibitorsofMMP-2andMMP-9(7).

AlthoughtherolesofMMP-2,MMP-9,andtheirinhibitorsTIMP-1andTIMP-2intheCLhavebeenwidelystudiedinotherspecies,theexpressionpatternsandtheirfunctionalrolesduringdifferentstagesofthemenstrualcycleremainunclearinMacacamulatta.Therefore,thepurposeofthisstudywastoinvestigatetheexpressionpatternsofMMP-2,MMP-9andtheirinhibitorsinM.mulattaCLduringdifferentstagesofthemenstrualcycle.

MaterialsandMethods:

Animals:

M.mulattafemaleswerehousedintheanimalfacilityofKunmingPrimateResearchCenter.Themenstrualcyclesoftheanimalsweremonitoredbymeasuringserumprogesteronelevels,andCLtissueswerecollectedfromtheovariesatdifferentstagesofthemenstrualcycle.ThisstudywasapprovedbytheAnimalCareandUseCommitteeofKunmingPrimateResearchCenter.

Immunohistochemistry:

FrozensectionsofM.mulattaCLwerefixedin4%paraformaldehydefor15min,permeabilizedwith0.1%TritonX-100,andblockedin5%bovineserumalbuminfor1h.Thesectionswereincubatedwithanti-MMP-2,anti-MMP-9,anti-TIMP-1oranti-TIMP-2antibodies(1:100dilution;Abcam,USA)overnightat4℃,followedbyincubationwithsecondaryantibodies(1:200dilution;JacksonImmunoResearch,USA)for1hatroomtemperature.AfterwashingwithPBS,thesectionswerestainedwithDAPIandvisualizedusingafluorescencemicroscope.

Quantitativereal-timePCR:

TotalRNAwasisolatedfromM.mulattaCLtissuesusingTRIzol(Invitrogen,USA)accordingtothemanufacturer’sinstructions.TheRNAwasreverse-transcribedtocDNAusingaRevertAidFirstStrandcDNASynthesiskit(ThermoFisherScientific,USA)andquantitativereal-timePCRwasperformedusinga7500FastReal-TimePCRsystem(AppliedBiosystems,USA)withaPowerSYBRGreenPCRMasterMix(AppliedBiosystems,USA).TheprimersequencesofMMP-2,MMP-9,TIMP-1,TIMP-2,andGAPDHareshowninTable1.GAPDHwasusedasaninternalcontrolfornormalization.

Statisticalanalysis:

DatawereanalyzedusingGraphPadPrismsoftware(version7.0)andexpressedasmean±SEM.One-wayANOVAfollowedbyTukey’smultiplecomparisontestorStudent’st-testwasusedforstatisticalanalysis.P<0.05wasconsideredstatisticallysignificant.

Results:

ExpressionofMMP-2,MMP-9,TIMP-1,andTIMP-2inM.mulattaCLatdifferentstagesofthemenstrualcycle:

ToinvestigatetheexpressionpatternsofMMP-2,MMP-9,TIMP-1,andTIMP-2inM.mulattaCLatdifferentstagesofthemenstrualcycle,immunohistochemistryandquantitativereal-timePCRwereemployed.TheresultsshowedthatMMP-2andMMP-9werehighlyexpressedinthedevelopingandmid-cyclelutealphases,butdecreasedinthelatelutealphase(Fig.1A,B).Incontrast,TIMP-1andTIMP-2wereup-regulatedinthelatelutealphase(Fig.1C,D).Quantitativereal-timePCRconfirmedtheresultsofimmunohistochemistry,showingthatMMP-2andMMP-9mRNAlevelsweresignificantlyhigherinthedevelopingandmid-cyclelutealphasesthaninthelatelutealphase(P<0.05),whileTIMP-1andTIMP-2mRNAlevelsweresignificantlyhigherinthelatelutealphase(P<0.01)(Fig.2).

Discussion:

OurstudyinvestigatedtheexpressionpatternsofMMP-2,MMP-9,TIMP-1,andTIMP-2inM.mulattaCLatdifferentstagesofthemenstrualcycle.OurdatashowedthatMMP-2andMMP-9werehighlyexpressedinthedevelopingandmid-cyclelutealphases,butdecreasedinthelatelutealphase,consistentwiththepreviousstudiesonotherspeciessuchasratsandcows(8,9).ThisindicatesthatMMP-2andMMP-9mayplayimportantrolesintheformationandmaintenanceofM.mulattaCLduringtheearlylutealphase,andthencontributetotheregressionofCLduringthelatelutealphase.

Incontrast,theexpressionofTIMP-1andTIMP-2wasup-regulatedinthelatelutealphase,indicatingthatTIMP-1andTIMP-2mayactasthekeyregulatorsofMMP-2andMMP-9activityduringtheregressionofM.mulattaCL.ThisisconsistentwithpreviousstudiesonhumanCL(10).IthasbeenreportedthattheexpressionofTIMP-1andTIMP-2iscontrolledbytheprogesteronereceptor(11).Therefore,theup-regulationofTIMP-1andTIMP-2expressioninthelatelutealphasemayberelatedtothedecreasedlevelsofprogesterone.

Conclusion:

Inconclusion,ourstudyinvestigatedtheexpressionpatternsofMMP-2,MMP-9,TIMP-1,andTIMP-2inM.mulattaCLatdifferentstagesofthemenstrualcycle,providinginsightsintotheirfunctionalrolesduringtheformationandregressionofCL.OurdatasuggestthatMMP-2andMMP-9playimportantrolesinthemaintenanceandregressionofM.mulattaCL,andtheirinhibitorsTIMP-1andTIMP-2mayactasthekeyregulatorsoftheiractivity.FurtherstudiesareneededtoinvestigatethemechanismsunderlyingtheregulationofMMPactivitybyTIMPsinM.mulattaCL.

Acknowledgments:

ThisworkwassupportedbytheNationalNaturalScienceFoundationofChina(#81860282),theYunnanAppliedBasicResearchProject(#2017FB149),andtheKunmingPrimateResearchCenter(#KPRC-2020-06).

DeclarationofInterest:

Theauthorsdeclarenoconflictsofinterest.Thefundingagencieshadnoroleinthedesign,analysis,orinterpretationofthedataorthewritingofthemanuscript.

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Table1.Primersequencesusedforreal-timePCR

GenePrimersequence

MMP-2Forward:5’-GACACCTGGTTCTTAAAGGTCA-3’Reverse:5’-TGGTCTCGCGGCGTCCAG-3’

MMP-9Forward:5’-AGGTGACCGAGATGACATCCC-3’Reverse:5’-CCACCCGAGTGTAACCATAGC-3’

TIMP-1Forward:5’-CACAGGGGACAGCGCGCG-3’Reverse:5’-AGCCCAGGACATCGGCTCATT-3’

TIMP-2Forward:5’-AGTTCTGTCTGCGCGCGCTAT-3’Reverse:5’-GCTCGGCTGAGCGAACTCAT-3’

GAPDHForward:5’-GGATTTGGTCGTATTGGG-3’Reverse:5’-GGAAGATGGTGATGGGATT-3’

Figure1.ImmunohistochemicalanalysisoftheexpressionofMMP-2,MMP-9,TIMP-1,andTIMP-2inM.mulattaCLatdifferentstagesofthemenstrualcycle.(A)MMP-2,(B)MMP-9,(C)TIMP-1,(D)TIMP-2.Scalebar:100μm.

Figure2.RelativemRNAexpressionofMMP-2,MMP-9,TIMP-1,andTIMP-2inM.mulattaCLatdifferentstagesofthemenstrualcycle.Dataareexpressedasmean±SEM.*P<0.05,**P<0.01comparedwiththelatelutealphase.TheexpressionandactivityofMMPsandtheirinhibitorsareessentialfornormalovarianfunction,includingfolliculardevelopment,ovulation,andcorpusluteumformationandregression.ThepresentstudydemonstratedthatMMP-2andMMP-9werehighlyexpressedinthedevelopingandmid-cyclelutealphasesofM.mulattaCL,indicatingtheirimportantrolesinthemaintenanceandregressionofCL.Incontrast,theup-regulationofTIMP-1andTIMP-2expressioninthelatelutealphasesuggestedtheirkeyrolesasregulatorsofMMP-2andMMP-9activityduringtheregressionofM.mulattaCL.TheexpressionofTIMP-1andTIMP-2iscontrolledbytheprogesteronereceptor,andtheirup-regulationmayberelatedtothedecreasedlevelsofprogesteroneinthelatelutealphase.ThesefindingsprovidenewinsightsintothemolecularmechanismsunderlyingtheregulationofMMP-2,MMP-9,TIMP-1,andTIMP-2expressionduringdifferentstagesofthemenstrualcycleinM.mulatta,andmayhaveimportantimplicationsforunderstandingthepathogenesisofhumanreproductivedisorders.FurtherstudiesareneededtoexplorethefunctionalrolesoftheseproteinsinM.mulattaCLandtheirpotentialastherapeutictargetsforreproductivedisorders.Inadditiontotheirrolesinnormalovarianfunction,MMPsandtheirinhibitorsalsoplayimportantrolesinovarianpathologies,suchasovariancancer,endometriosis,andpolycysticovarysyndrome(PCOS).DysregulatedMMP-2andMMP-9expressionhasbeenimplicatedinovariancancerinvasionandmetastasis,whiledecreasedTIMP-1andTIMP-2expressionhasbeenassociatedwithincreasedovariancancercellinvasionandpoorprognosis.Similarly,MMP-2andMMP-9havebeenfoundtobeup-regulatedinendometrioticlesions,contributingtotheirinvasionandproliferation,whileTIMP-1andTIMP-2havebeenshowntoinhibitendometrioticcellinvasion.InPCOS,increasedMMP-2andMMP-9expressionhasbeenimplicatedinexcessiveovarianangiogenesisandfolliculararrest,whiledecreasedTIMP-1andTIMP-2expressionhasbeenassociatedwithabnormalfolliculargrowthandsteroidogenesis.

TargetingMMPsandtheirinhibitorshasthereforeemergedasapotentialtherapeuticstrategyforovarianpathologies.SeveralMMPinhibitorshavebeendevelopedandtestedinclinicaltrialsforovariancancer,withpromisingresultsintermsofreducedtumorinvasionandimprovedsurvival.InhibitorsofTIMP-1andTIMP-2havealsobeeninvestigatedaspotentialtherapeuticagentsinendometriosisandPCOS,withsomepreclinicalevidencesuggestingtheirefficacyinreducingovariancellproliferationandangiogenesis.

Overall,theexpressionandactivityofMMPsandtheirinhibitorsarecrucialfornormalovarianfunctionandareimplicatedinvariousovarianpathologies.Furtherresearchisneededtofullyelucidatethefunctionalrolesoftheseproteinsinovarianhealthanddisease,andtoadvancethedevelopmentoftargetedtherapiesforreproductivedisorders.Inadditiontotheirpotentialastherapeutictargets,MMPsandtheirinhibitorsalsohavediagnosticandprognosticvalueinovarianpathologies.StudieshavefoundthatMMP-2andMMP-9expressionlevelscanbeusedtopredicttheaggressivenessofovariancancerandthelikelihoodoftumorrecurrence,whileTIMP-1andTIMP-2levelsareassociatedwithabetterprognosis.

Furthermore,MMPsandtheirinhibitorsmayalsoserveasbiomarkersforthediagnosisandmonitoringofotherovariandisorders.Forexample,MMP-9hasbeenproposedasapotentialbiomarkerforendometriosis,asitsexpressionisincreasedintheserumofpatientswiththecondition.Likewise,decreasedlevelsofTIMP-1andTIMP-2havebeenassociatedwithPCOSandmayserveasdi