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Autophagy6:8,1224-1226;November16,2010;2010LandesBioscienceAutophagicPunctum1224AutophagyVolume6Issue8Punctumto:RajawatYS,HiliotiZ,BossisI.
RetinoicAcidinducesautophagosomematurationthroughredistributionofthecation-independentmannose-6-phosphatereceptor.
AntioxidRedoxSignal2010;Inpress;PMID:20812861;DOI:10.
1089/ars.
2010.
3491.
Keywords:vitaminA,autophagosomematuration,amphisomes,autophagicefficiency,cation-independentmannose-6-phosphatereceptor,Beclin1,phospho-mTOR,phospho-Akt1Submitted:09/22/10Revised:09/28/10Accepted:09/29/10Previouslypublishedonline:www.
landesbioscience.
com/journals/autophagy/article/13793DOI:10.
4161/auto.
6.
8.
13793*Correspondenceto:IoannisBossis;Email:bossisi@umd.
eduAutophagyisanintracellularcata-bolicprocessthatrespondswithgreatsensitivitytonutrientavailability,implyingthatcertainmacro-ormicro-nutrientsareinvolved.
Wefoundthatretinoicacidpromotesautophagosomematurationthroughapathwayindepen-dentfromtheclassicnuclearretinoidreceptors.
Retinoicacidredistributesthecation-independentmannose-6-phop-shatereceptorfromthetrans-Golgiregiontomaturingautophagosomalstructuresinducingtheiracidification.
Manipulationoftheautophagicactivitybyretinoidscouldhaveenormoushealthimplications,sincetheyareessentialdietarycomponentsandfrequentlyusedpharmaceuticals.
VitaminAisanessentialdietarycom-ponentthatisinvolvedinseveralphysi-ologicalprocesses,suchasreproduction,embryonicdevelopment,tissueremodel-ing,visionandimmunefunction.
VitaminAdeficiencyhasbeenextensivelylinkedtoincreasedoxidativestress,inflammationandneurodegenerationandhistoricallyisconsideredasanutritionallyacquiredimmunodeficiencydisease.
Naturalandsyntheticretinoidsplayamajorroleinregulatingcellulargrowthanddifferentia-tion,andcaninduceapoptosisinawidevarietyofmalignantcells.
Themecha-nismhoweverbywhichretinoidsprotectagainstviralandbacterialinfectionsandsuppresstumorigenesisisstillunknown.
Regulationofautophagybymacro-nutrient(proteins,carbohydrates,lipids)availabilityiswellrecognized.
Ingen-eral,aminoaciddeprivationdirectlytrig-gerstheautophagicresponse,whereasAutophagyAtargetforretinoicacidsYogendraRajawat,ZoeHiliotiandIoannisBossis*DepartmentofVeterinaryMedicine;UniversityofMaryland;MDUSAcarbohydratesandlipidsaffectautophagyindirectlythroughtheinsulin/glucagonsignalingpathway.
WiththeexceptionofafewreportsonvitaminD3andsele-nium,theeffectofmicronutrientsandparticularlyvitaminAonautophagyhasnotbeenstudied.
Itisnoteworthy,how-ever,tomentionthateithervitaminAorautophagicdeficiencycanpotentiallyleadtosimilarpathophysiologicalconditions.
Toinvestigatetheroleofretinoicacidinautophagy,weinitiallysubjectedtran-sientlyandstablyCFP-LC3transfectedHeLacellstolowmicromolardosesofATRA(all-transretinoicacid)andnoticedadecreaseinthesteadystatelevelsofCFP-LC3-labeledautophagosomesbyconfocalmicroscopy.
However,wedidnotobserveanychangesintheratioofCFP-LC3-ItoCFP-LC3-IIbywesternblotting.
Bothprocedures,though,gavesimilarresultswhenthecellsweretreatedwithrapamycin,whichinducesautophagosomebiogenesis.
TodeterminewhetherATRAcouldaffectthesteadystatelevelsofautophagosomesbyinhibitingtheirfor-mation,weexaminedtheproteinlev-elsofBeclin1,phosphorylatedmTORandphosphorylatedAkt1underretinoidstimulation.
Ourstudiessuggestedthatretinoidsdonotaffecttheearlystagesofautophagosomeformation.
However,itiswellacceptedinthefieldthat,uponbio-genesis,autophagosomesprogressthroughaseriesofmaturationeventsbeforefusionwithlysosomesandformationofautoly-sosomes.
Therefore,changesinthepHoftheautophagosomallumenorfusionwiththeacidiclysosomescouldinducefluorescentquenchingofCFP/GFP/EYFPfusionproteins.
Indeed,treatmentofwww.
landesbioscience.
comAutophagy1225AutophagicPunctumAutophagicPunctum(acidified)autophagosomesdonotexist.
Inaddition,themechanismbywhichautophagosomesbecomeacidifiedisnotfullyunderstood;however,fusionwithlateacidicendosomeshasbeensuggested.
Inoureffortstodeterminethemech-anismofretinoicacid-inducedauto-phagosomematuration,weperformedseveralstudiesusingspecificantagonistsandagonistsoftheclassicretinoicacidreceptors(RARsandRXRs).
Ourobser-vationsstronglysuggestedthattheclas-sicRARsandRXRsreceptorsdonotmediatetheeffectsofretinoidsonauto-phagosomematuration.
Recentevidence,mCherry-LC3transfectedcells(mCherryisacidresistant)withATRAdidnotresultinsignificantchangeinthenumberofautophagosomes/auto-lysosomes.
Tofurtherverifythisobservation,wesub-jectedGFP-mCherry-LC3(pHsensitivereporter)transfectedcellstoATRAandobservedasignificantreductionintheratioofyellow/redpuncta.
Theseobser-vationssuggestedthatATRAeitherpro-motesautophagosome/lysosomefusionorinducesautophagosomeacidification(e.
g.
,bythegenerationofamphisomes).
ItisworthmentioningthatreliablemarkerstodistinguishbetweenearlyandmaturingFigure1.
Inductionofautophagosome(AP)maturationbyretinoidsthroughredistributionofthecation-independentmannose-6phosphaterecep-tor(CIMPR).
InitialformationofAPstakesplacebyengulfingofcargowithinthephagophore(PG).
ThePGelongatesandclosestoformavesiclethroughaprocessmediatedbytheLC3conjugationsystem.
APsthenundergomaturationbeforefusionwithlysosomesandformationofautolyso-somes.
Thismaturationprocesspotentiallyreliesonacquisitionofthevacuolar-typeprotonATPasepumpthatmediatesacidification.
Thisacidifica-tioncanbeaccomplishedbyeitherfusionofAPswithasubsetoflateendosomesandmultivesicularbodiesenrichedinprotonpumpstoformamphisomes,orbydirecttranslocationoftheprotonpumpfromtheTGNtoAPs.
TranslocationoftheprotonpumpandotherhydrolyticenzymesreliesonCIMPR.
BindingofretinoicacidtoCIMPR-enzymecomplexesintheTGNinducestheirtranslocationtolateendosomes(A)orAPs(B)andpromotesacidification.
thoughhassuggestedthatotherretinoidresponsepathwaysthatareindependentofthenuclearreceptorsmayexist.
Althoughthebiologicalsignificanceofretinoicacidbindingtoalternativeintracellularsitesisnotunderstood,itwasofinteresttousthatphotoaffinitylabelingstudieshaveshowndirectbindingofATRAtothecat-ion-independentmannose-6-phosphate/IGFIIreceptor(CIMPR)withhighaffin-ity.
CIMPRisaubiquitouslyandconstitu-tivelyexpressedlargeglycoprotein(~300kDa)thatplaysafundamentalroleinendocytosisanddegradationofextracel-lularligands(IGF-II,uPAR),andsorting1226AutophagyVolume6Issue8andtransportingmannose-6-phosphatebearingglycoproteins(suchashydrolases)fromthetrans-Golginetwork(TGN)toendosomes.
Toinvestigatepotentialeffectsofretinoidsontheintracellulartraffick-ingofCIMPR,wepreparedmGFP-andmRFP-taggedfull-lengthCIMPRcon-structs.
Underbasalconditions,thefluo-rescentCIMPRfusionproteinsaremostlylocalizedintheperinuclearTGN,somevesicularcompartmentsandtheplasmamembrane.
TreatmentwithretinoidsinparallelexperimentsinducessignificantredistributionofCIMPRfusionproteinstoperipheralvesicularstructuresthatarenotlabeledwithCFP-LC3orLAMP-1-RFP(lysosomalmarker)butarepositiveformCherry-LC3.
ThesedatasuggestedthatretinoicacidinducesredistributionofCIMPRintoacidifiedautophagosomes(oramphisomes).
TofurtherunderstandtheroleofCIMPRinautophagosomematuration,weutilizedsiRNA-mediatedsilencingofendogenousCIMPRlevels.
KnockdownofCIMPRleadstoremarkableaccu-mulationofnonacidifiedimmatureautophagosomesandRab9-labeledlateendosomes,buthasnoeffectontheabundanceoflysosomes.
Inadditionthiseffectcannotbereversedbyretinoicacidtreatment,furtherdemonstratingthattheeffectsofthesecompoundsonautophagosomematurationaremedi-atedthroughCIMPR.
AccumulationofearlynonacidifiedautophagosomesintheabsenceofCIMPRindicatestheimportanceofthisglycoproteininauto-phagosomematuration,butalsosuggeststhatautophagosomeacidificationmightberequiredbeforefusionwithlyso-somes.
ItcanbespeculatedthatCIMPRisrequiredforacidificationofasubsetoflateendosomes,whichinturnmedi-ateautophagosomematurationthroughfusion.
Alternatively,directtranslocationofCIMPRtoautophagosomesmaybemediatingtheiracidification(Fig.
1).
Pharmacologicalmodulationofdis-ruptedautophagicactivityhasbeensug-gestedasastrategyfortherapieswithinawidespectrumofpathologicalsituationsincludingcancer,neurologicaldiseases,prematureagingandinfectiousdiseases.
Althoughretinoidsareamultitargetingclassofcompoundsthatcanmodulateseveralphysiologicalandcellularpro-cesses,weidentifiedanovelmechanisminvolvingtheCIMPRbywhichretinoidsaffectautophagy.
Thisfindingcanlaythefoundationforthedevelopmentofnewandspecificretinoidanaloguesthatcouldenhanceorreduceautophagicactivity.
RetinoicAcidinducesautophagosomematurationthroughredistributionofthecation-independentmannose-6-phosphatereceptor.
AntioxidRedoxSignal2010;Inpress;PMID:20812861;DOI:10.
1089/ars.
2010.
3491.
Keywords:vitaminA,autophagosomematuration,amphisomes,autophagicefficiency,cation-independentmannose-6-phosphatereceptor,Beclin1,phospho-mTOR,phospho-Akt1Submitted:09/22/10Revised:09/28/10Accepted:09/29/10Previouslypublishedonline:www.
landesbioscience.
com/journals/autophagy/article/13793DOI:10.
4161/auto.
6.
8.
13793*Correspondenceto:IoannisBossis;Email:bossisi@umd.
eduAutophagyisanintracellularcata-bolicprocessthatrespondswithgreatsensitivitytonutrientavailability,implyingthatcertainmacro-ormicro-nutrientsareinvolved.
Wefoundthatretinoicacidpromotesautophagosomematurationthroughapathwayindepen-dentfromtheclassicnuclearretinoidreceptors.
Retinoicacidredistributesthecation-independentmannose-6-phop-shatereceptorfromthetrans-Golgiregiontomaturingautophagosomalstructuresinducingtheiracidification.
Manipulationoftheautophagicactivitybyretinoidscouldhaveenormoushealthimplications,sincetheyareessentialdietarycomponentsandfrequentlyusedpharmaceuticals.
VitaminAisanessentialdietarycom-ponentthatisinvolvedinseveralphysi-ologicalprocesses,suchasreproduction,embryonicdevelopment,tissueremodel-ing,visionandimmunefunction.
VitaminAdeficiencyhasbeenextensivelylinkedtoincreasedoxidativestress,inflammationandneurodegenerationandhistoricallyisconsideredasanutritionallyacquiredimmunodeficiencydisease.
Naturalandsyntheticretinoidsplayamajorroleinregulatingcellulargrowthanddifferentia-tion,andcaninduceapoptosisinawidevarietyofmalignantcells.
Themecha-nismhoweverbywhichretinoidsprotectagainstviralandbacterialinfectionsandsuppresstumorigenesisisstillunknown.
Regulationofautophagybymacro-nutrient(proteins,carbohydrates,lipids)availabilityiswellrecognized.
Ingen-eral,aminoaciddeprivationdirectlytrig-gerstheautophagicresponse,whereasAutophagyAtargetforretinoicacidsYogendraRajawat,ZoeHiliotiandIoannisBossis*DepartmentofVeterinaryMedicine;UniversityofMaryland;MDUSAcarbohydratesandlipidsaffectautophagyindirectlythroughtheinsulin/glucagonsignalingpathway.
WiththeexceptionofafewreportsonvitaminD3andsele-nium,theeffectofmicronutrientsandparticularlyvitaminAonautophagyhasnotbeenstudied.
Itisnoteworthy,how-ever,tomentionthateithervitaminAorautophagicdeficiencycanpotentiallyleadtosimilarpathophysiologicalconditions.
Toinvestigatetheroleofretinoicacidinautophagy,weinitiallysubjectedtran-sientlyandstablyCFP-LC3transfectedHeLacellstolowmicromolardosesofATRA(all-transretinoicacid)andnoticedadecreaseinthesteadystatelevelsofCFP-LC3-labeledautophagosomesbyconfocalmicroscopy.
However,wedidnotobserveanychangesintheratioofCFP-LC3-ItoCFP-LC3-IIbywesternblotting.
Bothprocedures,though,gavesimilarresultswhenthecellsweretreatedwithrapamycin,whichinducesautophagosomebiogenesis.
TodeterminewhetherATRAcouldaffectthesteadystatelevelsofautophagosomesbyinhibitingtheirfor-mation,weexaminedtheproteinlev-elsofBeclin1,phosphorylatedmTORandphosphorylatedAkt1underretinoidstimulation.
Ourstudiessuggestedthatretinoidsdonotaffecttheearlystagesofautophagosomeformation.
However,itiswellacceptedinthefieldthat,uponbio-genesis,autophagosomesprogressthroughaseriesofmaturationeventsbeforefusionwithlysosomesandformationofautoly-sosomes.
Therefore,changesinthepHoftheautophagosomallumenorfusionwiththeacidiclysosomescouldinducefluorescentquenchingofCFP/GFP/EYFPfusionproteins.
Indeed,treatmentofwww.
landesbioscience.
comAutophagy1225AutophagicPunctumAutophagicPunctum(acidified)autophagosomesdonotexist.
Inaddition,themechanismbywhichautophagosomesbecomeacidifiedisnotfullyunderstood;however,fusionwithlateacidicendosomeshasbeensuggested.
Inoureffortstodeterminethemech-anismofretinoicacid-inducedauto-phagosomematuration,weperformedseveralstudiesusingspecificantagonistsandagonistsoftheclassicretinoicacidreceptors(RARsandRXRs).
Ourobser-vationsstronglysuggestedthattheclas-sicRARsandRXRsreceptorsdonotmediatetheeffectsofretinoidsonauto-phagosomematuration.
Recentevidence,mCherry-LC3transfectedcells(mCherryisacidresistant)withATRAdidnotresultinsignificantchangeinthenumberofautophagosomes/auto-lysosomes.
Tofurtherverifythisobservation,wesub-jectedGFP-mCherry-LC3(pHsensitivereporter)transfectedcellstoATRAandobservedasignificantreductionintheratioofyellow/redpuncta.
Theseobser-vationssuggestedthatATRAeitherpro-motesautophagosome/lysosomefusionorinducesautophagosomeacidification(e.
g.
,bythegenerationofamphisomes).
ItisworthmentioningthatreliablemarkerstodistinguishbetweenearlyandmaturingFigure1.
Inductionofautophagosome(AP)maturationbyretinoidsthroughredistributionofthecation-independentmannose-6phosphaterecep-tor(CIMPR).
InitialformationofAPstakesplacebyengulfingofcargowithinthephagophore(PG).
ThePGelongatesandclosestoformavesiclethroughaprocessmediatedbytheLC3conjugationsystem.
APsthenundergomaturationbeforefusionwithlysosomesandformationofautolyso-somes.
Thismaturationprocesspotentiallyreliesonacquisitionofthevacuolar-typeprotonATPasepumpthatmediatesacidification.
Thisacidifica-tioncanbeaccomplishedbyeitherfusionofAPswithasubsetoflateendosomesandmultivesicularbodiesenrichedinprotonpumpstoformamphisomes,orbydirecttranslocationoftheprotonpumpfromtheTGNtoAPs.
TranslocationoftheprotonpumpandotherhydrolyticenzymesreliesonCIMPR.
BindingofretinoicacidtoCIMPR-enzymecomplexesintheTGNinducestheirtranslocationtolateendosomes(A)orAPs(B)andpromotesacidification.
thoughhassuggestedthatotherretinoidresponsepathwaysthatareindependentofthenuclearreceptorsmayexist.
Althoughthebiologicalsignificanceofretinoicacidbindingtoalternativeintracellularsitesisnotunderstood,itwasofinteresttousthatphotoaffinitylabelingstudieshaveshowndirectbindingofATRAtothecat-ion-independentmannose-6-phosphate/IGFIIreceptor(CIMPR)withhighaffin-ity.
CIMPRisaubiquitouslyandconstitu-tivelyexpressedlargeglycoprotein(~300kDa)thatplaysafundamentalroleinendocytosisanddegradationofextracel-lularligands(IGF-II,uPAR),andsorting1226AutophagyVolume6Issue8andtransportingmannose-6-phosphatebearingglycoproteins(suchashydrolases)fromthetrans-Golginetwork(TGN)toendosomes.
Toinvestigatepotentialeffectsofretinoidsontheintracellulartraffick-ingofCIMPR,wepreparedmGFP-andmRFP-taggedfull-lengthCIMPRcon-structs.
Underbasalconditions,thefluo-rescentCIMPRfusionproteinsaremostlylocalizedintheperinuclearTGN,somevesicularcompartmentsandtheplasmamembrane.
TreatmentwithretinoidsinparallelexperimentsinducessignificantredistributionofCIMPRfusionproteinstoperipheralvesicularstructuresthatarenotlabeledwithCFP-LC3orLAMP-1-RFP(lysosomalmarker)butarepositiveformCherry-LC3.
ThesedatasuggestedthatretinoicacidinducesredistributionofCIMPRintoacidifiedautophagosomes(oramphisomes).
TofurtherunderstandtheroleofCIMPRinautophagosomematuration,weutilizedsiRNA-mediatedsilencingofendogenousCIMPRlevels.
KnockdownofCIMPRleadstoremarkableaccu-mulationofnonacidifiedimmatureautophagosomesandRab9-labeledlateendosomes,buthasnoeffectontheabundanceoflysosomes.
Inadditionthiseffectcannotbereversedbyretinoicacidtreatment,furtherdemonstratingthattheeffectsofthesecompoundsonautophagosomematurationaremedi-atedthroughCIMPR.
AccumulationofearlynonacidifiedautophagosomesintheabsenceofCIMPRindicatestheimportanceofthisglycoproteininauto-phagosomematuration,butalsosuggeststhatautophagosomeacidificationmightberequiredbeforefusionwithlyso-somes.
ItcanbespeculatedthatCIMPRisrequiredforacidificationofasubsetoflateendosomes,whichinturnmedi-ateautophagosomematurationthroughfusion.
Alternatively,directtranslocationofCIMPRtoautophagosomesmaybemediatingtheiracidification(Fig.
1).
Pharmacologicalmodulationofdis-ruptedautophagicactivityhasbeensug-gestedasastrategyfortherapieswithinawidespectrumofpathologicalsituationsincludingcancer,neurologicaldiseases,prematureagingandinfectiousdiseases.
Althoughretinoidsareamultitargetingclassofcompoundsthatcanmodulateseveralphysiologicalandcellularpro-cesses,weidentifiedanovelmechanisminvolvingtheCIMPRbywhichretinoidsaffectautophagy.
Thisfindingcanlaythefoundationforthedevelopmentofnewandspecificretinoidanaloguesthatcouldenhanceorreduceautophagicactivity.
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