Effect of PACAP on MAP kinases, Akt and cytokine expressions in rat retinal hypoperfusion

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ContentslistsavailableatSciVerseScienceDirect

Neuroscience Letters

j o ur na l ho me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / n e u l e t

Effect of PACAP on MAP kinases, Akt and cytokine expressions in rat retinal hypoperfusion

Aliz Szabo

^a

, Bese Danyadi

^b

, Eszter Bognar

^a

, Krisztina Szabadﬁ

^c

, Eszter Fabian

^b

, Peter Kiss

^b

, Laszlo Mester

^a

, Sridharan Manavalan

^b,d

, Tamas Atlasz

^e

, Robert Gabriel

^c

, Gabor Toth

^f

, Andrea Tamas

^b

, Dora Reglodi

^b,∗,1

, Krisztina Kovacs

^a,1

aDepartmentofBiochemistryandMedicalChemistry,UniversityofPecs,Pecs,Hungary

bDepartmentofAnatomy,PTE-MTA“Lendulet”PACAPResearchTeam,UniversityofPecs,Pecs,Hungary

cDepartmentofExperimentalZoologyandNeurobiology,UniversityofPecs,Pecs,Hungary

dDepartmentofBasicSciences,CollegeofProfessionalStudies,NationalUniversityofHealthSciences,St.Petersburg,FL,USA

eDepartmentofSportbiology,UniversityofPecs,Pecs,Hungary

fDepartmentofMedicalChemistry,UniversityofSzeged,Szeged,Hungary

h i g h l i g h t s

EffectsofintravitrealPACAPwastestedinhypoperfusion-inducedretinalinjury.

PACAPattenuatesinﬂammatorycytokineresponseuponischemiaintheretina.

PACAPinducesERK1/2andAktactivationinischemicretina.

PACAPinhibitsJNKandp38MAPKexpression.

a r t i c l e i n f o

Articlehistory:

Received5May2012

Receivedinrevisedform15June2012 Accepted17June2012

Keywords:

Retinalischemia PACAP Cytokinearray ERK JNK p38MAPK Akt

a b s t r a c t

Pituitaryadenylatecyclaseactivatingpolypeptide(PACAP)isknownforitspotentneuroprotectiveeffects, includingtheretinoprotectiveactionsinseveraltypesofretinalinjuries.Wehaveshownearlierthat PACAPtreatmentcausesactivationofprotectivepathwaysandinhibitionofpro-apoptoticsignalingin excitotoxicretinallesions.Theaimofthepresentstudywastogaininsightintotheinvivoprotec- tivemechanismofPACAPinretinalhypoperfusioninjuryinducedbybilateralcommoncarotidartery occlusion(BCCAO).RatsunderwentBCCAOandreceivedintravitrealPACAP(PACAP38)treatment.We investigatedtheactivationleveloftheprotectiveAktpathwayaswellasthedifferentmitogenactivated proteinkinases(MAPKs)byWesternblotanalysisandtheexpressionofcytokinesusingacytokinearray kit.WefoundthatPACAPtreatmentalonedidnotinfluencethephosphorylationofAktortheMAPKs, butdecreasedthehypoperfusion-inducedactivationofbothp38MAPKandJNKandincreasedtheacti- vationoftheprotectiveAktandERK1/2inhypoperfusedretinas.Thecytokineprofilewasdramatically changedafterBCCAO,withmostcytokinesandchemokinesshowinganincrease,whichwasattenuated byPACAP(suchasCINC,CNTF,fractalkine,sICAM,IL-1,LIX,Selectin,MIP-1,RANTESandTIMP-1).Inaddi- tion,PACAPincreasedtheexpressionofVEGFandthymuschemokine.Thepresentresultsprovidefurther insightintotheneuroprotectivemechanisminducedbyPACAPinischemicretinalinjuries,showingthat PACAPameliorateshypoperfusioninjuryinvolvingAkt,MAPKpathwaysandanti-inflammatoryactions.

∗Correspondingauthorat:DepartmentofAnatomy,UniversityofPecs,7624Pecs, Szigetiu12,Hungary.Tel.:+367253600135398;fax:+367236393.

E-mailaddress:dora.reglodi@aok.pte.hu(D.Reglodi).

1 Theseauthorscontributedequallytothepresentwork.

1. Introduction

Pituitaryadenylatecyclaseactivatingpolypeptide(PACAP)isa neuropeptidewithmiscellaneousactionsinthenervoussystem andperipheralorgans[38,41,49].TheeffectsofPACAPintheeye arealsodiverse.Actionsonthepupillaryreﬂex,tearsecretionand retinohypothalamiccircadiansystem,aswellastrophiceffectsdur- ingretinaldevelopmenthavebeendescribed[7,17,20,24].Oneof thewell-studiedeffectsofPACAPintheeyeisitsretinoprotection 0304-3940/$–seefrontmatter© 2012 Elsevier Ireland Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.neulet.2012.06.044

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94 A.Szaboetal./NeuroscienceLetters523 (2012) 93–98

action [4]. PACAP protects against various harmful stimuli in theretina,suchasmonosodiumglutamate-,NMDA-orkainate- inducedexcitotoxicinjury,UVlight-inducedretinaldegeneration anddiabeticretinopathy[3,5,6,16,28,40,43,44].

Retinalhypoperfusionorischemiaactivatesinﬂammatoryreac- tions and induces apoptotic pathways that eventually lead to degenerationandvisualimpairment[33].Ischemiaisassociated withseveralretinalvasculardiseasesincludingdiabeticretinopa- thy, retinal artery occlusion and carotid artery stenosis [14].

Bilateralcommoncarotidarteryocclusion (BCCAO)causesmild cerebralhypoperfusion[18]and,dependingonratstrain,itleadsto retinaldegeneration.Wehavepreviouslystudieddifferentneuro- protectivestrategiesusingthisratmodel[42].Wehavealsoshown thatboth PACAPandVIP areretinoprotectiveinchronicretinal hypoperfusion[2,45].

The neuroprotective effects of PACAP seem to be mediated byinfluencing signaling pathwaysatdifferent levels.The main effectsinvolvedaretheanti-apoptotic,anti-inflammatoryandanti- oxidanteffects.AsafirststepininvestigatingthePACAP-induced retinoprotectivemechanism,wehavestudiedsignalingpathways in glutamate-induced excitotoxic degeneration.We have found thatPACAPinhibitspro-apoptoticpathways, whileitstimulates anti-apoptoticintracellular processes[35,36].However,itis not known,whichmechanismsareinvolvedintheprotectiveeffects ofPACAPinhypoperfusion-inducedinjury.Mitogenactivatedpro- teinkinases(MAPKs)andAktsignalingplayanimportantrolein theneuroprotectiveeffectsofPACAP.Also,PACAPisknowntoinflu- encelevelsofcytokinesandchemokinesafterinjuries,whichare importantmediatorsinischemicprocesses[8,11,13].Therefore,we investigatedtheactivationlevelofMAPKs,Aktandcytokinesinrat retinaexposedtoBCCAO.

2. Materialsandmethods

MaleWistarratsweighing250–300gweresubjectedtoperma- nentBCCAO.Experimentalprocedureswereperformedfollowing institutionalethicalguidelines(BA02/2000-24/2011).Underisoﬂu- raneanesthesia,bilateralcommoncarotidarterieswereexposed and ligated with a 3-0 ﬁlament. Immediately following the BCCAOoperation,PACAP38(100nmol/3␮lsaline)wasintravitre- allyinjectedintotherighteyewithaHamiltonsyringe.Theleft

eyereceivedthesame volumeofvehicletreatment.Agroupof animalsunderwentanesthesiaandallstepsofthesurgicalproce- dure,exceptligationofthecarotidarteries.Theseanimalsserved assham-operatedsaline-orPACAP-treatedanimals.

ForWesternblotexperiments,retinasfrom33 animalswere removedafter5,30 and 60minin ordertoinvestigatethesig- naling pathways activated within the first few minutes after an ischemic insult (n=6 animal/group in the ischemic groups and 5 animals/group in the sham-operated groups). Samples were processed for Western blot analysis as described earlier [35,36].Membraneswereprobedovernightat4^◦Cwiththepri- maryantibodies:phospho-specificanti-Akt-1Ser⁴⁷³(1:1000;R&D Systems, Hungary), phospho-specific anti-ERK1/2 Thr²⁰²/Tyr²⁰⁴, phospho-specific anti-SAPK/JNK Thr¹⁸³/Tyr¹⁸⁵, phospho-specific anti-p38MAPK(1:1000;CellSignalingTechnology,USA),andanti- glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:2000;

Millipore,Hungary).Membraneswerewashedsixtimesfor5min inTrisbufferedsaline(pH=7.5)containing0.2%Tweenpriorto additionofgoatanti-rabbitoranti-mousehorseradishperoxidase- conjugatedsecondary antibody (1:3000; BioRad, Hungary).The antibody–antigen complexes were visualized by means of enhancedchemiluminescence.Afterscanning,resultswerequan- tiﬁedbymeansofScionImageBeta4.2program.Allexperiments were performedat least fourtimes. Data are expressed asthe mean±SEM.Statisticalanalysiswasperformedbyanalysisofvari- anceandunpairedStudent’st-test.Differenceswithpvaluesbelow 0.05wereconsideredtobesigniﬁcant.

Forsemiquantitativecytokinearray,retinas(n=18from9ani- mals/group) in the ischemic and sham-operated groups) were removedafter24hofischemiaandhomogenizedinPBSwithpro- teaseinhibitors. Sampleswere pooled in three replicates(n=3 per replicate). Triton X-100 was added to a ﬁnal concentra- tion of 1%. The samples were stored at −80^◦C prior to use.

CytokinearrayfromtissuehomogenateswasperformedusingRat Cytokine ArrayPanel A Array kit from R&D Systems (Biomed- ica,Budapest,Hungary).Afterblockingthearraymembranesfor 1handadding thereconstitutedDetectionAntibodyCoctailfor another 1h at room temperature, the membranes were incu- batedwith1.5mloftissue homogenatesat2–8^◦Covernighton arockingplatform.Afterwashingwithbuffer3timesandaddi- tion of horseradishperoxidase-conjugated Streptavidin to each

Fig.1. (A)RepresentativeWesternblotsshowingactivation(phosphorylation)ofAktandMAPkinasesinsham-operated,sham+PACAP-treated,ischemic(inducedbyBCCAO) andBCCAO+PACAP-treatedretinas.Samplesweretaken5,30and60minaftertreatments.GAPDHwasusedasinternalcontrol.(B)StatisticalanalysisofWesternblotresults.

Dataaregivenasmean±SEM.**p<0.01,***p<0.001vscontrol,^##p<0.01;^###p<0.001vsischemicretinas.

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Fig.1. (A)(Continued).

membrane we exposed them to a chemiluminescent detection reagent(AmershamBiosciences,Hungary)thensideuptoanX-ray ﬁlmcassette[46].

3. Results

3.1. EffectofPACAPtreatmentonischemiainducedsignaling pathways

Insham-operatedgroups,nomarkeddifferencesinthephos- phorylationleveloftheexaminedfactorswerefounduponPACAP

treatment, only phosphorylation of Akt was slightly elevated byPACAPinsham-operatedretinas. Ischemiaitselfalsoslightly increasedthephosphorylationstateofAkt,butPACAPtreatment ledtoamarkedsigniﬁcantfurtherincreaseatalltime-pointsfol- lowingischemia(Fig.1AandB).ERK1/2phosphorylationwasclose tothedetectionlimitinshamoperatedsaline-treatedretinaswith littleeffectafterPACAPtreatment.Ischemiainducedastrongphos- phorylationofERK1/2comparedtosham-operation,andthiswas furtherincreasedbyPACAP.Wefoundtheactivationslightlymore intensiveafter30 than after60min,suggesting a fastresponse afteranischemicinsult.InPACAP-treatedretinas,thesigniﬁcant

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Fig.2.Representativecytokinearraytodetecttheprotectiveeffectsof100nM PACAPinretinaexposedtoischemicinjurybybilateralcommoncarotidarteryocclu- sion(BCCAO).Thepanelsshowarraysfromsham-operatedcontrol(upperpanel), BCCAOischemic(middlepanel)andBCCAO+PACAP-treated(lowerpanel)retinas.

Thetableindicatestheexaminedfactorsineachbox,highlightingwitharrowsthe changesobservedafterPACAPtreatment.

activationofERK1/2appearedalready5minaftertheinductionof ischemia,furthersupportingthisrapidreaction.

Inthesham-operatedeyes,wecouldnotdetectmarkedphos- phorylationofSAPK/JNK.Ischemiainducedtheactivationofthis kinaseasitwasrevealedby itsslightlyincreasedphosphoryla- tionafter5minandstrongelevationafter30and60min.However, administrationofPACAPcausedsigniﬁcantdecreaseinthephos- phorylationstateofSAPK/JNKatalltime-pointsafterischemia.A phosphorylationpatternquitesimilartothatofJNKwasdetectedin caseofp38MAPK.Nophosphorylationofthiskinasewasobserved insham-operatedanimalsandwe foundnodifferencebetween salineandPACAP-treatedretinas.Ischemiainducedastrongphos- phorylationofp38MAPKthatwasattenuatedbyPACAPtreatment 30and60minafterBCCAO(Fig.1AandB).

3.2. Cytokinearray

The expression of several cytokines was increased after ischemia,includingchemoattractantproteins,thechemokinesof theCINC(cytokine-inducedneutrophilchemoattractant)andMIP (macrophageinﬂammatoryprotein)families:CINC-1,CINC-2␣/␤, CINC-3andMIP-1␣,MIP-3␣.Severalinterleukinswerealsoacti- vated:IL-1␣,IL-1␤andIL-1ra,whileotherinterleukinsremained unchanged,suchasIL-2,IL-3,IL-4,IL-6andIL-10.Theactivationof theciliaryneurotrophicfactor(CNTF),fractalkine,sICAM-1(intercellularadhesionmolecule),LIX(lipopolysaccharideinducedCXC chemokine),L-selectin,RANTES(regulatedonactivation,normalt cellexpressedandsecreted),thymuschemokineandTIMP-1(tis- sueinhibitor ofmetalloproteinase)wasincreasedin theretinas thatunderwentBCCAOcomparedwiththecontrolgroups(Fig.2).

PACAPtreatmentattenuatedactivationofalltheabove-mentioned cytokines,asmeasuredbyacytokinearraysystem.Exceptionswere VEGFandthymuschemokine,theactivationofwhichwasfurther increaseduponPACAPtreatment.Theexpressionofothercytokines analyzedbythearraydidnotshowanymarkedchanges(Fig.2).

4. Discussion

Thepresentstudyshowedthat PACAPcounteractedchanges in the phosphorylation of MAPKs and levelsof cytokinesafter ischemicinjuryintheratretina.PACAPhasbeenshowntoinﬂu- encecellsurvivalandinﬂammatorypathwaysinseveralischemic injuries,includingischemiaofthebrainanddifferentperipheral organs[19,34,38,46].TheMAPKfamilyseemstoplayanimportant rolein thePACAP-inducedcellularprotectioninseveralmodels [31,47].Incerebellargranulecells,thebalancebetweenERKand JNKMAPKsiscriticalforsurvival,andPACAPinducesERKphospho- rylationwhileitinhibitsJNKphosphorylation,therebyattenuating ceramide-inducedcelldeath[48].Ourpresentresultsarealsoin accordancewithpreviousobservationsinglutamate-inducedretinalinjury, where PACAP had opposingeffects on ERKand JNK phosphorylation[35,36].p38MAPK,thethirdmainmemberofthe MAPKfamily,istypicallyconsideredtobeapro-apoptoticMAPK, thephosphorylationofwhichisinducedbymanycellularinjuries.

PACAPhasbeenshowntoinhibitthephosphorylationofp38MAPK inseveralstudies[35,36].Aktsignalingisgenerallyassociatedwith increasedcellularprotection,includingretinalischemia[14].Akt phosphorylationwasalsoinducedbyPACAPinthepresentstudy, similarlytoourearlierobservationsinexcitotoxicretinalinjury and otherdescriptionsonPACAP-mediated neuronal protection [31,37].

Theanti-inflammatoryeffects of PACAPofferanother mech- anismagainstischemicinjury intheretina.Theinvolvementof PACAPininflammatoryreactionshaslongbeenknown,butthe exacteffectsofPACAPininflammationdependoncelltype,tissue, age,pathological conditionsandseveralotherfactors.However, PACAPisusuallyconsideredtobeananti-inflammatorypeptide [12].Inthepresentstudy,wealsofoundthatadditionofPACAP counteracted changes in several cytokines and chemokines in ischemicretinalinjury.Theoverallpictureofcytokineexpression showsthatischemiadramaticallyinducedseveralcytokinesand lessactivationwasfoundinmostcytokinesafterPACAPtreatment, exceptforVEGFandthymuschemokine,whichwereelevatedafter PACAPtreatment.Thestrongactivationofseveralcytokinesfound inourstudyisinagreementwithotherreportsshowinginduced cytokineexpressionafterretinalischemia[23,50].Thealterations inducedbyPACAPstronglysuggestananti-inflammatoryroleof thepeptideinischemicretinalinjury,keepinginmindthatthis wascheckedatonetime-point,namely24haftertheinductionof ischemia.

ThefamilyofCINC(CINC1-3)isapro-inflammatorychemokine family,involvedinseveralinflammatoryprocesses,usuallyduring theacuteinflammatoryresponsephase[21].Compromiseinblood flowhasbeenshowntoinduceexpressionofthecytokineCINCalso intheretina[52]andsubstancesdecreasingCINClevelsareusually associatedwithdecreasedtissuedamage[1,27].Thisis thefirst reportshowingthatPACAPattenuateselevatedCINClevelsafter ischemiainduction in theretina.Fractalkine, anotherchemoat- tractant,wasalso reducedafterPACAP treatment.The effectof PACAPoninterleukinswasvariable.Severalinterleukinswerenot affected,whiletheincreasedexpressionofotherswasattenuated byPACAP. Taken together,PACAPdecreased levelsof IL-1 sub- classes,thereduction ofwhichhasbeenshowntobebeneficial inretinalischemicinjuries[15,25].

RANTESalsoparticipatesinretinalinflammatoryreactions.The chemokineRANTEShasthepotentialtoinfluencethemigration ofmemoryTcells and monocytesacrosstheblood-retinalbar- rierduringinflammatoryeyedisease[10].Hereweshowedthat PACAPwasabletodownregulatetheischemia-inducedupregu- lationofRANTESintheretina.EarlierstudiesfoundthatPACAP inducedsecretionofRANTES,forexampleinastrocytes[8]andin corticalneurons[39].Inthesestudies,releaseofRANTEShasbeen

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showntobeassociatedwithincreasedneuroprotection.Theroleof RANTESinretinalischemia,however,seemstobetheincreasein inflammatoryreactions,againstwhichPACAPmayprotectaccord- ingtoourfindings.Thisis,therefore,inaccordancewithfindings ofothersshowingthatPACAPandVIPinhibitchemokinereleasein microglialcells[11].

Severalother factors werefound tobe altered upon PACAP administration. Little is known about the effect of PACAP on these factors. Adhesion molecules have been demonstrated to be involved in several ophthalmic pathologies [26]. Similarly, theinvolvementofmatrixmetalloproteinasesinretinalischemic injuryhasbeenreportedbymanyauthors[30].MIP-1alphahas alsobeenfoundtobeelevatedafterretinalischemia[51].Inthe presentstudy,PACAPpreventedtheincreasedactivationofICAM, selectin,MIP-1alphaandTIMP-1.VEGF,ontheotherhand,was increasedafterPACAPtreatment.TheinducingeffectofPACAPon thelevelsofVEGFhasbeenreportedearlierinlungcancercells[32]

andinpituitaryfolliculostellatecells[22].VEGFisawell-studied angiogeneticfactorplayingaroleinseveralretinopathies,including retinopathyofprematurityanddiabeticretinopathy,butitalsohas aneuroprotectivefunction[53].Inretinalischemia,VEGFexpres- sionisaltereddependingonthetime,reperfusionandarterialor neuronallocalizationofVEGF[9,29].WhetherthisPACAP-induced increaseinVEGFhasaneuroprotectivefunction,aroleinreestab- lishingcirculationafterischemicretinainjury,oritplaysarolein inﬂammatoryprocesses,isnotknownatthemoment.Altogether, theexactquantiﬁcationofthechangesincytokineexpressionand activationisneededtobeperformedinfutureexperimentsfocus- ingonthecytokinesandchemokinesshowingthemostmarked changes.

Takentogether,thedramaticchangesinthecytokineproﬁleis counteractedbyPACAPtreatment.Thepresentresultsprovidefur- therinsightintotheneuroprotectivemechanisminducedbyPACAP inischemicinjuries,showingthatPACAPamelioratesischemicreti- nalinjuryinvolvingAkt,MAPKpathwaysandanti-inﬂammatory actions.

Acknowledgments

SupportbySROP4.1.2.B-10/2/KONV-20/0-0002,SROP-4.2.2/B- 10/1-2010-0029,OTKAK72592,104984,CNK78480,Momentum- ProgramoftheHungarianAcademyofSciences,BolyaiScholarship, Akira Arimura Foundation, National University of Health Sci- ences/Florida Campus Research Grant and Richter Gedeon CentenaryFoundationwasacknowledged.

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