2024年4月10日发(作者：袭识)

BiochimicaetBiophysicaActa1821(2012)1453–1461

ContentslistsavailableatSciVerseScienceDirect

BiochimicaetBiophysicaActa

journalhomepage:/locate/bbalip

Theroleofacidsphingomyelinaseandcaspase5inhypoxia-inducedHuRcleavage

andsubsequentapoptosisinhepatocytes

QunZhu

a,c,1

,LiankuLin

a,b,1

,QiCheng

d

,QingXu

b

,JingmeiZhang

e

,StephenTomlinson

f

,JunfeiJin

b,

⁎

,

XiaopingChen

d,

⁎

,SongqingHe

a,b,

⁎⁎

a

DepartmentofHepatobiliarySurgery,AffiliatedHospitalofGuilinMedicalUniversity,Guilin,541001,Guangxi,People'sRepublicofChina

LaboratoryofLiverInjuryandRepairMolecularMedicine,GuilinMedicalUniversity,Guilin,541001,Guangxi,People'sRepublicofChina

c

DepartmentofEndocrinology,TheSecondAffiliatedHospitalofNanjingMedicalUniversity,Nanjing,210011,Jiangsu,People'sRepublicofChina

d

HepaticSurgeryCenter,TongjiHospital,TongjiMedicalCollege,HuazhongUniversityofScienceandTechnology,Wuhan,430030,Hubei,People'sRepublicofChina

e

DepartmentofBiochemistryandMolecularBiology,MedicalUniversityofSouthCarolina,Charleston,SC29425,USA

f

DepartmentofMicrobiologyandImmunology,DarbyChildren'sResearchInstitute,MedicalUniversityofSouthCarolina,Charleston,SC29425,USA

b

articleinfoabstract

ApreviousdatashowedthatthehypoxiamimeticcompoundCoCl

2

inducedcleavageofHuRandsubse-

previouslydemonstratedthatexposureofNT-2

humanneuronalprecursor

itisknownthatCoCl

2

inducescleavageofHuR,weinvestigatedwhetherthereisalinkbetweenHuRcleavage

andtheobservedsphingolipidchangesincellsexposedtohypoxia,andwhetherthislinkisassociatedwith

reofhepatocytestodirecthypoxiabymeansofahypoxicchamber

vationinceramidelevelswas

associatedwithactivat

dataraisethepossibilitythatacidsphingomyelinaseandcaspase5areeachpotentialtargetsfortreating

hypoxia(ischemia)-inducedliverinjury.

©htsreserved.

Articlehistory:

Received8March2012

Receivedinrevisedform14July2012

Accepted2August2012

Availableonline11August2012

Keywords:

Acidsphingomyelinase

Ceramide

Caspase5

HuRcleavage

Apoptosis

uction

Hypoxic(ischemic)liverinjuryoccursduringlivertransplantation,

electiveliverresection,trauma,cellulardamageis

involvedinthepathophysiologyofischemicliverinjuryresulting

fromhypoxicinsult[1].Althoughitisknownthathypoxia-induced

cellapoptosisplaysanimportantroleinhepatocytedamageduring

theseprocesses,themechanismofhypoxia-inducedcellinjuryisstill

notwellestablished.

ThereisincreasingevidencesuggestingthattheRNA-binding

protein,HuR,belongingtotheembryoniclethalabnormalvision

phenotype/Hufamily,canregulatestress-inducedapoptosis[2–4].

zi'sgrouphaselegantlydemonstratedthatHuRcleavage

products,includingHuR-CP1andHuR-CP2,participateinstress-

inducedapoptosis[5].Inadditiontostressorssuchasheatshock

proteins,staurosporineandtheproteasomeinhibitorMG132,CoCl

2

alsoinducedHuRcleavageandledtoapoptosisinoralcancercells

⁎.:+867732809503;fax:+867732822703.

⁎⁎Correspondenceto:,DepartmentofHepatobiliarySurgery,AffiliatedHospitalof

GuilinMedicalUniversity,Guilin,541001,Guangxi,People'.:+86

7732809503;fax:+867732822703.

E-mailaddresses:Jinj100@(),chenxp@(),

hesong@().

1

Bothauthorscontributedequallytothiswork.

1388-1981/$–seefrontmatter©htsreserved.

/10.1016/.2012.08.005

[6].CoCl

2

isahypoxiamimickingagent[7].HuRRNAbindingprotein

hasthreeRNArecognitionmotifs(RRM1,RRM2andRRM3)anda

HuRnucleocytoplasmicshuttling(HNS)domainbetweenRRM2and

RRM3[8].HuRcleavageproducts,includingHuR-CP1(RRM1–RRM2)

andHuR-CP2(RRM3),areproducedbycaspases(cysteinyland

aspartate-specificproteases)whichcleaveHuRRNAbindingprotein

ataspartate226[5].Currently,12caspases(caspases1–10,13and14)

es,includinginitiatorcaspases

andexecutionercaspases,aretheprincipleeffectormoleculesofapopto-

sis[9].Somestudieshaveshownthatcaspase3andcaspase7[5]or

caspase8/caspase3[10]areinvolvedinHuRcleavageunderconditions

eviouslyshownthatcaspase3and

caspase7participateinHuRcleavageunderhypoxicconditions[6].

Caspase3isactivatedbycaspase5-mediatedcleavageofpro-caspase3

[11,12],andactivatedcaspase3isinvolvedinHuRcleavagethatis

inducedbydifferentstimuli,isesthepossibility

thatcaspase5maybeinvolvedinhypoxia-inducedHuRcleavage.

Ceramideisabioactivesecondmessengermoleculewhichcanbe

generatedviasphingomyelinhydrolysisthroughtheactionofsphin-

gomyelinase[13],andceramideisawell-knowninducerofapoptosis

[14].Currently,fivedistinctsphingomyelinaseshavebeenidentified

basedontheirsubcellularlocalization,dependenceoncations,and

preferredoptimalpHforactivity[15].Acidsphingomyelinase(ASMase),

oneofthefivemembers,isinvolvedinceramidegenerationinresponse

l./BiochimicaetBiophysicaActa1821(2012)1453–1461

toapoptoticstimuli[16–18].Acidsphingomyelinaseisactivatedinre-

sponsetostimulationwithLPS,TNF-α,γ-irradiation,CD95,andIL-β

[19–23].HypoxicconditionscanalsotriggeractivationofASMase,

leadingtoceramideelevationandcaspase3activationinNT-2cells[24].

Basedonthesedata,itisreasonabletospeculatethatunderhypoxic

conditionsthereisalinkbetweenceramideelevationandcaspase-

eforeexaminedthecross-talkbetween

hepatocytesexposedtohypoxicconditions,weexaminedthemecha-

nismofHuRcleavage,anddeterminedtherolesofASMaseandcaspase

5inthecleavageofHuRfollowinghypoxia.

alsandmethods

ndmaterials

Cryopreservedplateablehumanhepatocytes(M/F00995-P),

InVitroGroCPMedium(Z990003),andInVitroGroHIMedium

(Z990012)wereobtainedfromInVitroTechnologies(Baltimore,MD).

C

2

-ceramide,C

2

-dihydroceramideandC

6

-ceramidewerefromAvanti

PolarLipids(Alabaster,AL).Anti-HuRantibodywasfromSantaCruz

Biotechnology(SantaCruz,CA),andβ-actinantibodywaspurchased

fromAbcam(Cambridge,MA).Horseradishperoxidase(HRP)-

conjugatedanti-rabbitandmouseIgGwerefromBio-Rad(Hercules,

CA).TheECLWesternblotdetectionkitwasfromGEHealthcare(Salt

LakeCity,UT).FuGENE6transfectionreagentwasfromRoche

(Indianapolis,IN)andMaxiprepkitsfromQiagen(SantaClarita,CA).

Pan-caspaseinhibitorzVAD-fmkandcaspaseinhibitorsetwere

purchasedfromAxxora(SanDiego,CA)andCalbiochem(LaJolla,CA),

e5assaykitwasfromCalbiochem,andcaspase5

cDNAfromOriGene(Rockville,MD).

entofhepatocyteswithhypoxia

Cryopreservedplateablehumanhepatocyteswerethawedand

eredilutedinCPmedium

toaconcentrationof0.7×10

6

cells/cyteswereplatedon

collagen-coatedplates,andthenplacedina37°C,5%CO

2

incubator.

Afteranovernightincubation,hepatocyteswereswitchedintoInVitroGro

ereincubatedovernightinHImedium[25]andthen

theplateswereplacedinahypoxicchamber(Billups–Rothenberg),

flushedwith95%N

2

and5%CO

2

,andincubatedfor12–72hat37°C

[26,27].OxygenconcentrationwasmeasuredwithanYSImodel55CE

dissolvedoxygenprobe(YSI,Marion,MA).Underhypoxicconditions

usedinthisstudy,O

2

concentrationwas0.09±0.001mg/l.

-polyacrylamidegelelectrophoresisandWesternblotting

Hepatocyteswerecollectedandlysedinlysisbuffer(PBS,1%Triton

X-100,0.1%SDS,and25μg/mlphenylmethylsulfonylfluoride).Cell

lysateswerenormalizedforproteinconcentrationusingtheBio-Radpro-

-polyacrylamidegelelectrophoresis(12%)andWestern

blottinganalyseswereperformedasdescribed[24].Forimmunoblotting

analysis,theblotswereprobedwithanti-HuRandanti-β-actin

detectionsystemwasusedtovisualizelabeled

proteinbands.

deandsphingomyelinanalyses

Hypoxiatreatedhepatocyteswerecollectedatindicatedtime

points,fortifiedwithinternalstandards,extractedwithethylacetate/

isopropylalcohol/water(60:30:10,v/v/v),evaporatedtodryness,and

reconstitutedin100μlofmethanol[28].Simultaneousanalysesof

ceramidesandsphingomyelinswereperformedonaThermoFinnigan

TSQ7000triplequadrupolemassspectrometeroperatinginamultiple

reactionmonitoringpositiveionizationmode[28].Thephosphate

contentsofthelipidextractswereusedtonormalizetheMSmeasure-

mentsofsphingolipids.

omyelinaseassays

Hepatocytesweregrownto70–80%confluency,andsubjectedtohyp-

lswerethencollectedandsubjectedtoinvitroacid

sphingomyelinaseenzymaticassaysusing[

14

C]sphingomyelinaccording

tothepreviouslydescribedprotocol[29,30].

-timepolymerasechainreaction

TotalRNAwasisolatedfromhepatocytesusingtheRNeasyminikit

(Qiagen).First-strandcDNAwassynthesizedwiththeiScriptcDNAsyn-

thesiskit(Bio-RadLaboratories,Hercules,CA).PrimersusedforASMase

wereasfollows:forward:5′-TGGCTCTATGAAGCGATGGC-3′andre-

verse5′-TTGAGAGAGATGAGGCGGAGA-3′.ASMasemRNAexpres-

sionwasnormalizedtothatofβ-β-actinprimers

usedare:forward:5′-ATTGGCAATGAGCGGTTCC-3′andreverse:

5′-GGTAGTTTCGTGGATGCCACA-3′.Real-timePCRwasperformed

induplicateusinga25μlreactionmixturecontaining1.0μlreverse

transcriptionmixture,0.2μMofbothprimers,and12.5μliQSYBR

GreenSupermix(Bio-Rad).Real-timePCRwasrunintheiCycler

real-timedetectionsystem(Bio-Rad)-

eragestartingquantity(SQ)offluorescenceunitswasusedforanalysis.

QuantificationwascalculatedusingtheSQoftargetedcDNArelativeto

thatofβ-actincDNAinthesamesample.

deelevationisinvolvedinhypoxia-inducedHuRcleavageinhepatocytes.A.

WesternblotanalysisofHuRcleavageinhepatocytelysatesafterexposuretodirecthypoxia

usingahypoxicchamber(Billups–Rothenberg).BlotswereprobedforbothHuRandβ-actin

(usedasloadingcontrol).HuRindicatesthe36kDafull-lengthHuR,andHuRcleavage

isofceramide(Cer)andsphingomyelin(SM)content

wereextractedfromthehepatocytesand

expressedasapercentageofcontrolcells.C.

cytesweretreated

with40μMC

2

-ceramide(C

2

-Cer),C

2

-dihydroceramide(C

2

-dhCer),aninactiveceramide

compound,andC

6

-ceramide(C

6

-Cer)for6h,andcelllysatesanalyzedforHuRcleavage

andβ-actin.

2024年4月10日发(作者：袭识)

BiochimicaetBiophysicaActa1821(2012)1453–1461

ContentslistsavailableatSciVerseScienceDirect

BiochimicaetBiophysicaActa

journalhomepage:/locate/bbalip

Theroleofacidsphingomyelinaseandcaspase5inhypoxia-inducedHuRcleavage

andsubsequentapoptosisinhepatocytes

QunZhu

a,c,1

,LiankuLin

a,b,1

,QiCheng

d

,QingXu

b

,JingmeiZhang

e

,StephenTomlinson

f

,JunfeiJin

b,

⁎

,

XiaopingChen

d,

⁎

,SongqingHe

a,b,

⁎⁎

a

DepartmentofHepatobiliarySurgery,AffiliatedHospitalofGuilinMedicalUniversity,Guilin,541001,Guangxi,People'sRepublicofChina

LaboratoryofLiverInjuryandRepairMolecularMedicine,GuilinMedicalUniversity,Guilin,541001,Guangxi,People'sRepublicofChina

c

DepartmentofEndocrinology,TheSecondAffiliatedHospitalofNanjingMedicalUniversity,Nanjing,210011,Jiangsu,People'sRepublicofChina

d

HepaticSurgeryCenter,TongjiHospital,TongjiMedicalCollege,HuazhongUniversityofScienceandTechnology,Wuhan,430030,Hubei,People'sRepublicofChina

e

DepartmentofBiochemistryandMolecularBiology,MedicalUniversityofSouthCarolina,Charleston,SC29425,USA

f

DepartmentofMicrobiologyandImmunology,DarbyChildren'sResearchInstitute,MedicalUniversityofSouthCarolina,Charleston,SC29425,USA

b

articleinfoabstract

ApreviousdatashowedthatthehypoxiamimeticcompoundCoCl

2

inducedcleavageofHuRandsubse-

previouslydemonstratedthatexposureofNT-2

humanneuronalprecursor

itisknownthatCoCl

2

inducescleavageofHuR,weinvestigatedwhetherthereisalinkbetweenHuRcleavage

andtheobservedsphingolipidchangesincellsexposedtohypoxia,andwhetherthislinkisassociatedwith

reofhepatocytestodirecthypoxiabymeansofahypoxicchamber

vationinceramidelevelswas

associatedwithactivat

dataraisethepossibilitythatacidsphingomyelinaseandcaspase5areeachpotentialtargetsfortreating

hypoxia(ischemia)-inducedliverinjury.

©htsreserved.

Articlehistory:

Received8March2012

Receivedinrevisedform14July2012

Accepted2August2012

Availableonline11August2012

Keywords:

Acidsphingomyelinase

Ceramide

Caspase5

HuRcleavage

Apoptosis

uction

Hypoxic(ischemic)liverinjuryoccursduringlivertransplantation,

electiveliverresection,trauma,cellulardamageis

involvedinthepathophysiologyofischemicliverinjuryresulting

fromhypoxicinsult[1].Althoughitisknownthathypoxia-induced

cellapoptosisplaysanimportantroleinhepatocytedamageduring

theseprocesses,themechanismofhypoxia-inducedcellinjuryisstill

notwellestablished.

ThereisincreasingevidencesuggestingthattheRNA-binding

protein,HuR,belongingtotheembryoniclethalabnormalvision

phenotype/Hufamily,canregulatestress-inducedapoptosis[2–4].

zi'sgrouphaselegantlydemonstratedthatHuRcleavage

products,includingHuR-CP1andHuR-CP2,participateinstress-

inducedapoptosis[5].Inadditiontostressorssuchasheatshock

proteins,staurosporineandtheproteasomeinhibitorMG132,CoCl

2

alsoinducedHuRcleavageandledtoapoptosisinoralcancercells

⁎.:+867732809503;fax:+867732822703.

⁎⁎Correspondenceto:,DepartmentofHepatobiliarySurgery,AffiliatedHospitalof

GuilinMedicalUniversity,Guilin,541001,Guangxi,People'.:+86

7732809503;fax:+867732822703.

E-mailaddresses:Jinj100@(),chenxp@(),

hesong@().

1

Bothauthorscontributedequallytothiswork.

1388-1981/$–seefrontmatter©htsreserved.

/10.1016/.2012.08.005

[6].CoCl

2

isahypoxiamimickingagent[7].HuRRNAbindingprotein

hasthreeRNArecognitionmotifs(RRM1,RRM2andRRM3)anda

HuRnucleocytoplasmicshuttling(HNS)domainbetweenRRM2and

RRM3[8].HuRcleavageproducts,includingHuR-CP1(RRM1–RRM2)

andHuR-CP2(RRM3),areproducedbycaspases(cysteinyland

aspartate-specificproteases)whichcleaveHuRRNAbindingprotein

ataspartate226[5].Currently,12caspases(caspases1–10,13and14)

es,includinginitiatorcaspases

andexecutionercaspases,aretheprincipleeffectormoleculesofapopto-

sis[9].Somestudieshaveshownthatcaspase3andcaspase7[5]or

caspase8/caspase3[10]areinvolvedinHuRcleavageunderconditions

eviouslyshownthatcaspase3and

caspase7participateinHuRcleavageunderhypoxicconditions[6].

Caspase3isactivatedbycaspase5-mediatedcleavageofpro-caspase3

[11,12],andactivatedcaspase3isinvolvedinHuRcleavagethatis

inducedbydifferentstimuli,isesthepossibility

thatcaspase5maybeinvolvedinhypoxia-inducedHuRcleavage.

Ceramideisabioactivesecondmessengermoleculewhichcanbe

generatedviasphingomyelinhydrolysisthroughtheactionofsphin-

gomyelinase[13],andceramideisawell-knowninducerofapoptosis

[14].Currently,fivedistinctsphingomyelinaseshavebeenidentified

basedontheirsubcellularlocalization,dependenceoncations,and

preferredoptimalpHforactivity[15].Acidsphingomyelinase(ASMase),

oneofthefivemembers,isinvolvedinceramidegenerationinresponse

l./BiochimicaetBiophysicaActa1821(2012)1453–1461

toapoptoticstimuli[16–18].Acidsphingomyelinaseisactivatedinre-

sponsetostimulationwithLPS,TNF-α,γ-irradiation,CD95,andIL-β

[19–23].HypoxicconditionscanalsotriggeractivationofASMase,

leadingtoceramideelevationandcaspase3activationinNT-2cells[24].

Basedonthesedata,itisreasonabletospeculatethatunderhypoxic

conditionsthereisalinkbetweenceramideelevationandcaspase-

eforeexaminedthecross-talkbetween

hepatocytesexposedtohypoxicconditions,weexaminedthemecha-

nismofHuRcleavage,anddeterminedtherolesofASMaseandcaspase

5inthecleavageofHuRfollowinghypoxia.

alsandmethods

ndmaterials

Cryopreservedplateablehumanhepatocytes(M/F00995-P),

InVitroGroCPMedium(Z990003),andInVitroGroHIMedium

(Z990012)wereobtainedfromInVitroTechnologies(Baltimore,MD).

C

2

-ceramide,C

2

-dihydroceramideandC

6

-ceramidewerefromAvanti

PolarLipids(Alabaster,AL).Anti-HuRantibodywasfromSantaCruz

Biotechnology(SantaCruz,CA),andβ-actinantibodywaspurchased

fromAbcam(Cambridge,MA).Horseradishperoxidase(HRP)-

conjugatedanti-rabbitandmouseIgGwerefromBio-Rad(Hercules,

CA).TheECLWesternblotdetectionkitwasfromGEHealthcare(Salt

LakeCity,UT).FuGENE6transfectionreagentwasfromRoche

(Indianapolis,IN)andMaxiprepkitsfromQiagen(SantaClarita,CA).

Pan-caspaseinhibitorzVAD-fmkandcaspaseinhibitorsetwere

purchasedfromAxxora(SanDiego,CA)andCalbiochem(LaJolla,CA),

e5assaykitwasfromCalbiochem,andcaspase5

cDNAfromOriGene(Rockville,MD).

entofhepatocyteswithhypoxia

Cryopreservedplateablehumanhepatocyteswerethawedand

eredilutedinCPmedium

toaconcentrationof0.7×10

6

cells/cyteswereplatedon

collagen-coatedplates,andthenplacedina37°C,5%CO

2

incubator.

Afteranovernightincubation,hepatocyteswereswitchedintoInVitroGro

ereincubatedovernightinHImedium[25]andthen

theplateswereplacedinahypoxicchamber(Billups–Rothenberg),

flushedwith95%N

2

and5%CO

2

,andincubatedfor12–72hat37°C

[26,27].OxygenconcentrationwasmeasuredwithanYSImodel55CE

dissolvedoxygenprobe(YSI,Marion,MA).Underhypoxicconditions

usedinthisstudy,O

2

concentrationwas0.09±0.001mg/l.

-polyacrylamidegelelectrophoresisandWesternblotting

Hepatocyteswerecollectedandlysedinlysisbuffer(PBS,1%Triton

X-100,0.1%SDS,and25μg/mlphenylmethylsulfonylfluoride).Cell

lysateswerenormalizedforproteinconcentrationusingtheBio-Radpro-

-polyacrylamidegelelectrophoresis(12%)andWestern

blottinganalyseswereperformedasdescribed[24].Forimmunoblotting

analysis,theblotswereprobedwithanti-HuRandanti-β-actin

detectionsystemwasusedtovisualizelabeled

proteinbands.

deandsphingomyelinanalyses

Hypoxiatreatedhepatocyteswerecollectedatindicatedtime

points,fortifiedwithinternalstandards,extractedwithethylacetate/

isopropylalcohol/water(60:30:10,v/v/v),evaporatedtodryness,and

reconstitutedin100μlofmethanol[28].Simultaneousanalysesof

ceramidesandsphingomyelinswereperformedonaThermoFinnigan

TSQ7000triplequadrupolemassspectrometeroperatinginamultiple

reactionmonitoringpositiveionizationmode[28].Thephosphate

contentsofthelipidextractswereusedtonormalizetheMSmeasure-

mentsofsphingolipids.

omyelinaseassays

Hepatocytesweregrownto70–80%confluency,andsubjectedtohyp-

lswerethencollectedandsubjectedtoinvitroacid

sphingomyelinaseenzymaticassaysusing[

14

C]sphingomyelinaccording

tothepreviouslydescribedprotocol[29,30].

-timepolymerasechainreaction

TotalRNAwasisolatedfromhepatocytesusingtheRNeasyminikit

(Qiagen).First-strandcDNAwassynthesizedwiththeiScriptcDNAsyn-

thesiskit(Bio-RadLaboratories,Hercules,CA).PrimersusedforASMase

wereasfollows:forward:5′-TGGCTCTATGAAGCGATGGC-3′andre-

verse5′-TTGAGAGAGATGAGGCGGAGA-3′.ASMasemRNAexpres-

sionwasnormalizedtothatofβ-β-actinprimers

usedare:forward:5′-ATTGGCAATGAGCGGTTCC-3′andreverse:

5′-GGTAGTTTCGTGGATGCCACA-3′.Real-timePCRwasperformed

induplicateusinga25μlreactionmixturecontaining1.0μlreverse

transcriptionmixture,0.2μMofbothprimers,and12.5μliQSYBR

GreenSupermix(Bio-Rad).Real-timePCRwasrunintheiCycler

real-timedetectionsystem(Bio-Rad)-

eragestartingquantity(SQ)offluorescenceunitswasusedforanalysis.

QuantificationwascalculatedusingtheSQoftargetedcDNArelativeto

thatofβ-actincDNAinthesamesample.

deelevationisinvolvedinhypoxia-inducedHuRcleavageinhepatocytes.A.

WesternblotanalysisofHuRcleavageinhepatocytelysatesafterexposuretodirecthypoxia

usingahypoxicchamber(Billups–Rothenberg).BlotswereprobedforbothHuRandβ-actin

(usedasloadingcontrol).HuRindicatesthe36kDafull-lengthHuR,andHuRcleavage

isofceramide(Cer)andsphingomyelin(SM)content

wereextractedfromthehepatocytesand

expressedasapercentageofcontrolcells.C.

cytesweretreated

with40μMC

2

-ceramide(C

2

-Cer),C

2

-dihydroceramide(C

2

-dhCer),aninactiveceramide

compound,andC

6

-ceramide(C

6

-Cer)for6h,andcelllysatesanalyzedforHuRcleavage

andβ-actin.