Heregulinβ activates storeoperated Ca2+ channels through cerbB2 receptor leveldependent pathwayWord格式.docx

Heregulinβ activates storeoperated Ca2+ channels through cerbB2 receptor leveldependent pathwayWord格式.docx

《Heregulinβ activates storeoperated Ca2+ channels through cerbB2 receptor leveldependent pathwayWord格式.docx》由会员分享，可在线阅读，更多相关《Heregulinβ activates storeoperated Ca2+ channels through cerbB2 receptor leveldependent pathwayWord格式.docx（12页珍藏版）》请在冰豆网上搜索。

revised23November2006. 

Availableonline12December2006.

Abstract

Theheregulinβ（HRGβ）isaligandtoactivatec-erbB2/c-erbB3interactionandcansubsequentlyincreasescytosolic[Ca2+]i.Inthetwohumanbreastcancercelllines,MCF-7showsalowc-erbB2expressionlevel,whereasSK-BR-3overexpressc-erbB2receptor.Inthisarticle,wehavefoundthatinMCF-7,HRGβinducedCa2+releasefromtheendoplasmicreticulums（ER）andsubsequentlyactivatedCa2+entryviastore-operatedCa2+channel（SOC）.However,inSK-BR-3,HRGβfailedtoinduceCa2+releaseandCa2+entry.RNAinterferencetodecreasec-erbB2levelinSK-BR-3resultedinreactivationofHRGβ-evokedCa2+releaseandCa2+entryviaSOC,whichwassimilartothatofMCF-7.Inaddition,intheabsenceofHRGβ,aconstitutiveactivationofSOCwasobservedinSK-BR-3ratherthaninMCF-7andc-erbB2-siRNAtreatedSK-BR-3.Comparedtothecellswithlowc-erbB2level,c-erbB2mighttendtointeractwithc-erbB3intherestingstateinthecellswithhighc-erbB2level,whichresultedindifferent[Ca2+]iresponsestoHRGβ.InSK-BR-3,theCa2+mobilizationinthepresenceorintheabsenceofHRGβwascompletelyblockedbyPLCinhibitorU73122.Insummary,ourresultsindicatethatHRGβ-inducedSOCwasregulatedbyc-erbB2levelanddependentonactivationofPLCinhumanbreastcancercells.

Keywords:

HRGβ;

Store-operatedCa2+channel（SOC）;

C-erbB2;

PLC;

Human

breastcancer

cells

ArticleOutline

Materialsandmethods

Materials

Cellculture

[Ca2+]imeasurements

Mn2+quenchingmeasurements

RNAinterference（RNAi）

Westernblotting

Statisticalanalysis

Results

EffectofHRGβon[Ca2+]iinhumanbreastcancerwithdifferentexpressionlevelsofc-erbB2

SOCwasresponsibleforCa2+entryinthepresenceorintheabsenceofHRGβ

HRGβ-inducedCa2+mobilizationwasdependentonPLC

Discussion

Acknowledgements

References

CytosolicCa2+signalsrepresentaconvergentpointofmanysignaltransductionpathwaysandmodulateadiversearrayofcellularactivitiesrangingfromshort-termresponsessuchascontractionandsecretion,tolonger-termcontroloftranscription,celldivision,andcelldeath.Innon-excitablecells,intracellularcalciummobilizationgenerallyinvolvesinternalCa2+releasefromendoplasmicreticulums（ER）andfollowingCa2+entryacrosstheplasmamembrane[1]and[2].Thestimulationoftyrosinekinasereceptorbymanyhormonesleadstostimulationofreceptortyrosinekinase-PLC-γpathwaystoproduceinositol1,4,5-trisphosphate（IP3）.IP3couplestotheIP3receptor（IP3R）3locatedonERandinducesCa2+release[3]and[4].ThesubsequentCa2+entryinthisprocessisgenerallyknownasstore-operatedorcapacitativeCa2+entrythroughaspecifictypeofCa2+channeltermedstore-operatedCa2+channel（SOC）[1],[2]and[5].Previousstudieshavedemonstratedthatepidermalgrowthfactor（EGF）inducesCa2+signalsviadifferentmechanismsindifferentcelllines.Zhangetal.foundthatinhumansalivarycells,EGFstimulatedCa2+influxbyalternativemechanismsdistinctfromCa2+entryviaSOC[6]andanotherlaboratoryprovidedevidencethatEGF-activatedSOCviaaPLC-dependentbutIP3Rindependentpathwayinhumanglomerularmesangialcells[7].

TheerbB/epidermalgrowthfactorreceptor（EGFR）familyconsistsoffourproteins:

EGFR（c-erbB1）,c-erbB2,c-erbB3andc-erbB4,whichareinvolvedinsignaltransductionpathwaysthatregulatecellgrowthanddifferentiation.Overexpressionoramplificationofepidermalgrowthfactorreceptorisassociatedwithmalignancyandapoorprognosisinbreastcancer.Inthefourreceptors,c-erbB2isoverexpressedinabout30%ofhumanbreastcancerpatients[8],[9],[10]and[11].Heregulinβ（HRGβ）isamemberofEGF-likeligandstoleadtoerbB-receptorinteraction[11].Inthevariousheterodimersandhomodimers,thec-erbB2/c-erbB3dimerconstitutesahighestaffinityco-receptorforHRGβ[12],[13],[14],[15]and[16],whichresultsinactivationofPLCsignalpathwayandsubsequentmobilizationof[Ca2+]i[3],[4],[17]and[18].EGF-inducedrisein[Ca2+]iviaactivationofEGFRisderivedfromtheactivationofbothreleasefromER（triggeredbyIP3）andCa2+influxviaSOClocatedonplasmamembrane[19],[20]and[21].However,whetherCa2+influxstimulatedbyHRGβisthroughSOCandthecorrelationbetweenthec-erbB2receptorlevelandsignaltransductionpathwaymodulatedbycellular[Ca2+]iarestillunknown.

Here,weinvestigatedtheeffectsofc-erbB2receptorathighandlowlevelsonCa2+mobilization.OurstudyfocusedonMCF-7andSK-BR-3humanbreastcancercelllines,representingmodelswithdifferentc-erbB2expressionlevel:

MCF-7showsalowc-erbB2level,whereasSK-BR-3isobservedtooverexpressc-erbB2[22]and[23].Inthepresentstudy,weprovidedevidencesthatinthecellswithlowc-erbB2level（MCF-7andc-erbB2-siRNAtreatedSK-BR-3）,HRGβ-evokedintracellularCa2+mobilizationinvolvedtwophases（i.e.,aninitialrapidrisefollowedbyasustainedplateau）.InSK-BR-3cellswithhighlevelofc-erbB2receptor,HRGβfailedtoactivatefurtherincreaseofintracellular[Ca2+]ialthoughconstitutiveactivationofSOCwasmeasuredwithoutHRGβaddition.Inaddition,Ca2+mobilizationinthecellseitherwithlowc-erbB2levelorwithhighc-erbB2levelwasviaaPLC-dependentmechanism.Thus,c-erbB2receptorlevelhasanessentialroleinregulationofPLC-dependentCa2+mobilizationpathwayinhumanbreastcancercells.

Materialsandmethods

Materials

Heregulinβ（HRGβ）,1-[6-[（（17β）-3-methoxyestra-1,3,5[10]-trien-17-yl）amino]hexyl]-1H-pyrrole-2,5-dione（U73122）,Fura-2acetoxymethylester（Fura-2/AM）andthapsigargin（Tg）wereobtainedfromSigma.c-erbB2monoclonalantibodywaspurchasedfromNeomarkers.Allotherreagentswereofanalyticalgrade.2-Aminoethoxydiphenylborate（2-APB）wasagiftfromProf.JianwenChen（TheNationalLaboratoryofBiomacromolecules,InstituteofBiophysics,ChineseAcademyofSciences,China）.

Cellculture

TheMCF-7humanbreastcancercelllinewaskindlyprovidedbyProf.JianwenChen.SK-BR-3wasobtainedfromAmericanTypeCultureCollection（Rockville,MD）.CellsweremaintainedinDulbecco’smodifiedEagle’smedium（Sigma）supplementedwith10%fetalbovineserum（PAA）at37 

°

Cinahumidified5%CO2incubator.

[Ca2+]imeasurements

Thecells（2 

×

105cells）wereplantedatlowdensityincompletemediumformorethan24 

honglassbottomdishesandwerestarvedfor4–6 

hinserum-freeDMEMbeforetheexperiments.[Ca2+]iwasmonitoredwithdualexcitationwavelengthfluorescencemicroscopyusingFura-2/AM.Inbrief,cellswereloadedwithFura-2/AM（1 

μM）byincubationfor40 

minin5%CO2incubatorinCa2+buffer（145 

mMNaCl,5 

mMKCl,1 

mMMgCl2,2 

mMCaCl2,10 

mMglycose,10 

mMHepes,pH7.4）andthenrinsedwithnominallyCa2+-freebuffer（145 

mMKCl,3 

mMMgCl2,10 

mMHepes,0.01 

mMEGTA,pH7.4）nolessthan20 

mintoremoveextracellularfreecalcium.Fura-2/AMwasexcitedwithlightfromamercurylampalternatelyfilteredto340or380 

nm.Ina37 

Cenvironmentalchamber,thefluorescenceimagesoftheattachedcellsonthebottomglass（40×

objective）werecapturedevery5 

satemissionof510 

nmonanNikonDiaphot300invertedmicroscopeequippedwithAquaCosmosMicroscopicImageAcquisition.AnalysissystemwasprovidedbyHamamatsuPhotonicsK.K.（Japan）.Thedigitizedfluorescenceratio（R340/R380）imagesofthecellsandthekineticchangeoftheratioineachcellwereprocessedonlineonaPCcomputer.Intheimagingsystemusedinthisinvestigation,theexcitationlightilluminatescellsonlyforveryshorttime（112 

ms）duringacquisitionofeachimage,butwasshutdownbeforenextimaging（5 

sapart）.Therefore,thephotobleachingoftheCa2+indicatorisnegligible.

Mn2+quenchingmeasurements

TherateofMn2+entry,asmeasuredbythequenchingofcellularFura-2/AMfluorescenceafteradditionofMn2+（0.2 

mMMnCl2）,hasbeenusedasameasureofstore-operatedCa2+influx.ThecellswereloadedwithFura-2/AMinCa2+bufferasdescribedaboveandthenrinsedwithnominallyCa2+-freebuffernolessthan20 

min.Thefluorescencewasexcitedat360 

nmandmeasuredat510 

nm.TheattachedcellsonthebottomglasswerestimulatedbyagonistsbeforeMn2+addition.TheslopeofMn2+entry-inducedreductionofFura-2/AMfluorescenceinitiatedbyadditionofTgorHRGβwasestimatedasdescribedbyCohenetal.[24].TheinitialslopesofthelineardeclineinFura-2fluorescencewereduringthe30 

simmediatelyafteradditionofMn2+.

RNAinterference（RNAi）

c-erbB2-siRNAwasasfollows:

sense:

5′-GGAGCUGGCGGCCUUCAUG,antisense:

5′-GCACAAGGCCGCCAGCUCC;

negativecontrolsiRNAwasasfollows:

5′-AAUAGUGUAUACGGCAUGC,antisense:

5′-CGAUGCCGUAUACACUAUU.Onedaybeforetransfection,cells（2 

105cells）wereplatedin1 

mlofgrowthmediumsothattheywillbe30–50%confluentatthetimeoftransfection.Transfectionof100 

nMsiRNAduplexeswascarriedoutusinglipofectamine2000transfectionreagent（Invitrogen）accordingtothemanufacturer’sinstructions.S