OriginalArticle

LocalizationofHedgehogSignalingPathwayComponentsDuringExocrineRegeneration

inL-Arginine-InducedAcutePancreatitisinRats

NorihisaSuzuki＊

DepartmentofMolecularGastroenterologyandHepatology,

KyotoPrefecturalUniversityofMedicineGraduateSchoolofMedicalScience

Abstract:BackgroundandAim:Elucidatingthemolecularandcellularmechanismsunderlyingexocrinepancreaticregenerationrepresentsakeystepindevelopingtreatmentforexocrinepancreaticinsufficiency.TheaimofthisstudywastoinvestigatethelocalizationofHedgehog（Hh）signalingpathwaycomponentsduringexocrineregenerationinL-arginine-inducedacutepancreatitisinrats.AcutepancreatitiswasinducedbysingleintraperitonealinjectionofL-arginine（450mg/100g）.Beforesacrifice,5-bromo-2’-deoxyuridinewasintravenouslyinjectedtolabelproliferatingcellsinexocrineregeneration.LocalizationoftheHhreceptorsSmoothened（Smo）andPatched1（Ptch1）andoftheHhtranscriptionfactorglioblastoma（Gli）2andpancreaticprogenitormarkerpancreaticandduodenalhomeoboxfactor-1（Pdx1）wasassessedbyimmunostaining.ProliferationofPdx1-positivecellstoformtubularcomplexesandreplacenecrotictissueoccurredbyday3afterL-arginineinjection;acinarcellproliferationwaspredominantwhilethenumberoftubularcomplexesdecreasedonday5,andexocrineregenerationwasalmostcompleteonday14.Inthecontrolpancreata,Smo,Ptch1,andGli2werelocalizedtoisletandductalcells.Duringexocrineregeneration,theseproteinswerealsolocalizedtotubularcomplexes.TheseresultssuggestthattheHhpathwayplaysanimportantroleinregulatingPdx1-positiveprogenitorcellactivityduringexocrineregeneration.

KeyWords:Hedgehogsignalingpathway,Exocrineregeneration,Acutepancreati-tis,Progenitorcell,Pdx1.

Introduction

Themechanismsinvolvedinexocrineregenerationinresponsetoacutepancreatitisorotheracute

Hhsignalinginexocrineregeneration 171J.KyotoPref.Univ.Med.126（3），171～182，2017．

Received:January30,2017.Accepted:January30,2017＊CorrespondencetoNorihisaSuzuki465Kajii-cho,Kawaramachi-Hirokoji,Kamigyo-ku,Kyoto,602-8566,Japannorisuzu@koto.kpu-m.ac.jp

pancreaticinjuryinhumansarenotfullyunderstood.However,previousstudiesbasedonanimalmodelshaveprovidedincreasingevidencethatexocrineregenerationintheadultpancreascanbeinducedinresponsetoacutepancreaticinjury,althoughthenormaladultpancreashasalowrateofcellturnover１‐３）.Themajormechanisminvolvedinexocrineregenerationoftheadultpancreasisself-duplicationofacinarcells.Previousstudiesusingvariousmodelsofacutepancreaticinjuryhaveproposedthatexocrineregenerationmayalsoinvolvetheproliferationanddifferentiationofstem/progenitorcells４‐８）.Understandingthemechanismsthatdrivetheproliferationanddifferentiationoftheseprogenitorcellsrepresentsakeystepingeneratinganewtherapythatinvolvestheuseofstemcellsforexocrineinsufficiencycausedbyacutenecrotizingpancreatitisorchronicpancreatitis.Thereareseveralcandidatemarkersforprogenitorcellsthatparticipateinexocrineregeneration,

acommonlyusedmarkerbeingpancreaticandduodenalhomeoboxfactor-1（Pdx1）.Pdx1,amemberofthelargefamilyofhomeodomain-containingproteins,isatranscriptionfactorexpressedinembryonicpancreaticprogenitorcellsthatsubsequentlygiverisetodifferentiatedacinar,islet,andductalcells.Pdx1isinvolvedinthedifferentiationofembryonicpancreaticprogenitorcellsandisessentialforpancreaticdevelopment９）.Intheadultpancreas,Pdx1expressionisrestrictedtoβcellsandisrequiredformaintainingmatureβ-cellfunction.PreviousstudieshaveshownstrikingproliferationofPdx1-positivecellsandnewlyformedduct-likestructurescalledtubularcomplexesduringexocrineregenerationandhavesuggestedthatPdx1-positivecellswithinthetubularcomplexesaretheprogenitorsofacinarcells.However,theoriginofpancreaticacinarcellsremainscontroversial,withputativepancreaticstemcells,ductalprogenitors,acinartransdifferentiation,orcirculatingprogenitorsallhavingbeensuggestedassources１０‐１３）.TheHedgehog（Hh）signalingpathwayplaysanimportantrolebyregulatingcriticalcellfate

decisions,including proliferation,apoptosis,migration,and differentiation during embryonicdevelopment,adulttissuehomeostasis,andregenerationofmanyorgans;activationofthispathwayiswidelyobservedinmanycancers１４‐２０）.Inmammals,thereare3typesofHhligands:Sonichedgehog（Shh）,Indianhedgehog（Ihh）,andDeserthedgehog（Dhh）.HhsignalingisinducedbyligandbindingtothePatched1（Ptch1）Hhreceptorontargetcells,withthesubsequentreleaseofSmoothened（Smo）leadingtoactivationoftheglioblastoma（Gli）1andGli2transcriptionfactorsandupregulationofGlitargetgenes２１）２２）.Thus,cellsthatexpressSmo,Ptch1,andGli2areHh-responsive.Hhsignalingplaysanimportantroleinembryonicpancreaticdevelopmentandpancreaticcancer.

AfewstudieshavedescribedtheinvolvementofHhsignalinginexocrinepancreaticregeneration.Fendlichetal.reportedthatHhsignalingwasupregulatedaftertheinductionofcerulein-inducedacutepancreatitisandshowedthatinhibitionofHhsignalingbypharmacologicalandgenetictechniquesresultedinimpairedexocrineregeneration,suggestingthatHhsignalingisrequiredforexocrineregeneration２３）.However,thesestudiesdidnotevaluatethelocalizationofHh-responsivecellsindetail,andtheinvolvementofHhsignalinginexocrineregenerationhasnotbeeninvestigatedinotherexperimentalmodelsofacutepancreaticinjury.Mostpreviousstudiesonthemechanismofexocrineregenerationhaveusedexperimentalmodels

ofacutepancreaticinjury,includingcerulein-inducedacutepancreatitis,partialpancreatectomy,andtheduct-ligationmodel１）２４）.TheL-arginine-inducedacutepancreatitismodelisanexperimentalacutenecrotizingpancreatitismodelthatisinducedbyintraperitonealadministrationofL-argininetoratsandmice.Inthismodel,selectivedamagetopancreaticacinarcellleadstotheiralmostcompletedepletion,

172 NorihisaSuzuki

followedbyacinarcellregeneration.Thisisthereforeausefulmodelforevaluatingthemolecularmechanismthatunderliesexocrineregeneration２５‐２９）.Theaimsofthepresentstudywere（1）tocharacterizetheinvolvementofPdx1-positive

progenitorcellsinexocrineregenerationbymonitoringthecellproliferationoftubularcomplexesandPdx1immunolocalizationand（2）tocharacterizeHh-responsivecellsbyimmunolocalizationoftheHhsignalingcomponentsduringexocrineregenerationfollowingL-arginine-inducedacutepancreatitisinrats.TheseapproacheswereusedtoinvestigateapossiblelinkbetweenHhsignalingandPdx1-positiveprogenitorcellsinexocrineregeneration.

MaterialsandMethods

AnimalsMaleWistarrats（OrientalYeast,Chiba,Japan）weighing200-230gwereprovidedwithwaterand

laboratorychowadlibitum.ExperimentalprotocolswereapprovedbytheAnimalCareCommitteeofKyotoPrefecturalUniversityofMedicine.InductionofExperimentalAcutePancreatitisToinduceacutepancreatitis,ratsweregivenasingleintraperitonealinjectionofL-arginine

monohydrochloride（450mg/100gbodyweight;NacalaiTesque,Kyoto,Japan）asa22.5％solutionin0.1MNaCl.Controlratswereinjectedwithanequalvolumeof0.15MNaCl.Theratsweresacrificedunderanesthesiabyintraperitonealinjectionofsodiumpentobarbital（40mg/kgbodyweight;Sigma-Aldrich,St.Louis,MO）.L-arginine-treatedratsweresacrificedon3,5,or14daysafterinjection.Thecontrolratsweresacrificedimmediatelyafterinjection（day0）.Onehourbeforesacrifice,5-bromo-2’-deoxyuridine（BrdU）（20mg/kgbodyweight;Sigma-Aldrich）wasinjectedintravenouslyintocontrolandarginine-treatedrats.Theirpancreatawereexcisedshortlyafterdeathandwereimmediatelyfixedin4％neutral-bufferedparaformaldehydeforimmunohistochemicalanalysis.ImmunohistochemistryParaffin-embeddedsections（4-μm thick）werepreparedusingstandardmethodology. After

deparaffinization,antigenretrievalwasperformedbyheatingthesectionsto95°Cfor10mininREALtargetretrievalsolution（Dako,Glostrup,Denmark）.ForBrdUstaining,thesectionswerethenincubatedin2NHCLatroomtemperaturefor90min.Forimmunostaining,endogenousperoxidaseactivitywasblockedbyincubationfor30minin0.3％ hydrogenperoxideinmethanol.Thesectionswerethenincubatedwiththeprimaryantibodiesinphosphate-bufferedsalineovernightat4°C.Theprimaryantibodieswereanti-BrdU（347580;BectonDickinson,LincolnPark,NJ;1：100）,anti-Pdx1

（NKR059;Transgenic,Kobe,Japan;1：250）,anti-Gli2（GTX27195,Genetex,Irvine,CA;1：250）,anti-Smo（sc-6366;SantaCruzBiotechnology,SantaCruz,CA;1：250）,andanti-Ptch1（sc-6149;SantaCruzBiotechnology;1：250）.Thesectionswerethenincubatedwiththesecondaryantibodiesfor30minatroomtemperatureandvisualizedusingdiaminobenzidinetetrahydrochloride.ThesecondaryantibodieswereHistofineSimpleStainRatMAXPeroxidase（mouse）forBrdU,RatMAXPeroxidase

（rat）forPdx1andGli2staining,andRatMAXPeroxidase（goat）forSmoandPtch1（Nichirei,Tokyo,Japan）.Thesectionswerecounterstainedwithhematoxylinusingstandardmethodology.Theprimaryantibodieswereomittedinthenegativecontrols.Thenegativecontrolsshowednoimmunoreactivity.BrdULabelingIndexThepercentageofBrdU-positivenucleiwasquantifiedintubularcomplexesandacinarcells.For

Hhsignalinginexocrineregeneration 173

eachtimepoint,sectionstakenrandomlyfrom 5L-arginine-injectedand5controlanimalswereexaminedat200×magnification.Atleast150nucleiwerecountedforeachexperimentalcondition.StatisticalAnalysisDatawereexpressedasthemeans±SEM.Statisticalanalyseswereperformedusingone-way

analysisofvariancefollowedbyDunnett’spost-hoctestusingJMP11software（SASInstitute,Cary,NC）.P-valuesof＜0.05wereconsideredstatisticallysignificant.

Results

HistologicalChangesinthePancreasinL-Arginine-InducedAcutePancreatitisOnday3,necrotictissueswerereplacedbythecharacteristicfeaturesofexocrineregeneration,

includingtheformationofduct-liketubularcomplexes,whichappearedascylindricaltubescontainingflattenedcuboidalcellssurroundingalargelumen（Fig.1Band1C）.Onday14,acinarcellregenerationwasnearlycompleteandonlyafewtubularcomplexeswereobserved（Fig.1D）.ProliferatingCellsinL-Arginine-InducedAcutePancreatitisBrdUlabelingwasperformedtoevaluatethecellproliferationoftubularcomplexesandacinarcells

aftertheinductionofpancreatitis.Inthecontrolpancreata,veryfewBrdU-labeledacinarcellsweredetected（Fig.2A）.Inall,0.18％±0.01％ofacinarcellswereBrdU-positiveinthecontrolgroup（Fig.3B）.Incontrast,onday3afterL-arginineinjection,manycellswithinthetubularcomplexeswereBrdU-positive（17.81％±3.18％;Fig.2Band3A）andthepercentageofBrdU-labeledacinarcellshadslightlyincreasedto6.16％±1.05％（Fig.2Band3B）.Onday5,BrdU-labeledacinarcellsweresignificantlyincreasedcomparedwiththecontrolgroup（23.00％±3.79％vs.0.18％±0.01％,P＜0.05;Fig.2Cand3B）,whereasfewertubularcomplexescontainedBrdU-labeledcells（3.81％±0.53％;Fig.2Cand3A）.Onday14,farfeweracinarcellswerelabeledwithBrdU（2.00％±0.80％;Fig.2Dand3B）.ImmunohistochemicalAnalysisofPdx1inL-Arginine-InducedAcutePancreatitisInthecontrolpancreata,Pdx1wasdetectedinthenucleusofisletandductalcellsbutnotinacinar

cells（Fig.4A）.Onday3afterL-arginineinjection,Pdx1wasdetectedinthenucleusoftubularcomplexesandinthenucleusofisletandductalcells（Fig.4B）;onday14,itwasdetectedinthenucleusoftheremainingtubularcomplexesandinthenucleusofisletandductalcellsbutnotinacinarcells（Fig.4C）.ImmunohistochemicalLocalizationofHhSignalingComponentsinL-Arginine-InducedAcutePancreatitisInthecontrolpancreata,Gli2wasdetectedmainlyinthenucleusofisletandductalcells,withweak

staininginsomeacinarcells（Fig.5A）.Onday3afterL-arginineinjection,Gli2wasdetectedinthenucleusoftubularcomplexesandinthenucleusofisletandductalcells,whereasonday14,itwasdetectedinthenucleusoftheremainingtubularcomplexes,isletandductalcells,andsomeacinarcells（Fig.5Band5C）.Inthecontrolpancreata,Smowasdetectedinthecytoplasmofisletcellsbutnotacinarcellsandductcells（Fig.6A）.Onday3afterL-arginineinjection,Smowasdetectedinthecytoplasmoftubularcomplexesandisletcells,withweakcytoplamicstaininginductalcells（Fig.6B）.Onday14afterL-arginineinjection,Smowasdetectedinthecytoplasmofisletcells,withweakcytoplasmicstainingintubularcomplexesandductalcells（Fig.6C）.Inthecontrolpancreata,Ptch1wasdetectedinthecytoplasmofisletandductalcellsbutnotinacinarcells（Fig.7A）.Onday3andday

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Hhsignalinginexocrineregeneration 175

Fig.1.HistologicalprogressionofexocrineregenerationinL-arginine-inducedacutepancreatitis.（A）Pancreatictissueinjectedwithsalineasacontrol.（B）Onday3,necroticcellswerereplacedbytubularcomplexes.（C）Tubularcomplexesappearedascylindricaltubeswithflattenedcuboidalcellssurroundingalargelumen.（D）Onday14,acinarcellregenerationwasnearlycompleteandtherewerefewertubularcomplexes.Hematoxylinandeosinstaining.Scalebar,100μm（A,B,andD）;200μm（C）.

Fig.2.ChangesinpancreaticBrdUimmunostaininginL-arginine-inducedacutepancreatitis（scalebar＝100μm）.（A）Incontrolpancreata,veryfewBrdU-labeledacinarcellsweredetected.（B）Onday3afterL-arginineinjection,manytubularcomplexcellswerepositiveforBrdUstaining.（C）Onday5,manyacinarcellswerepositiveforBrdU,whereasfewertubularcomplexcellswerepositiveforBrdU.（D）Onday14,afewBrdU-labeledacinarcellsweredetected.

14afterL-arginineinjection,Ptch1wasdetectedinthecytoplasmoftubularcomplexesandinthecytoplasmofisletandductalcells（Fig.7Band7C）.

Discussion

Inthepresentstudy,wedemonstratedthatexocrineregenerationafterL-arginine-inducedacutepancreatitiswasinitiatedbyPdx1-positiveprogenitorcellproliferationfortubularcomplexformation,acinarcellproliferation,andacinarcell-mediatedregenerationcoincidentwiththedisappearanceoftubularcomplexes.WealsodemonstratedthatexpressionoftheseHhsignalingcomponentswasalmostcompletelyrestrictedtoisletandductalcellsinthecontrolpancreata;however,expressionoftheseproteinswasalsoobservedintubularcomplexesduringexocrineregeneration,indicatingthatthePdx1-positiveprogenitorcellswithintubularcomplexesareHh-responsive.Previousstudiesinvariousrodentexperimentalmodelsofacutepancreaticinjuryhavesuggested

thatPdx1-positiveprogenitorcellsformingtubularcomplexeshavestemcell-likepropertiesthatallowthemtodifferentiateintoacinarcellsbasedonthefollowing:1）thecoincidenceoftheproliferationofacinarcellsandthedisappearanceoftubularcomplexes;2）thecontinuumofdifferentiationfromtubularcomplextoacinarcells;and 3）expressionofmarkersofotherembryonicpancreaticprogenitorcells,suchasTcf2andSox9,Hnf1b,andFoxa2４）１２）１３）.WefirstexaminedtheproliferationofPdx1-positiveprogenitorcellsthatoccurredduringexocrineregeneration.OurresultsindicatedthatexocrineregenerationafterL-arginine-inducedacutepancreatitiswasinitiatedbytheappearanceandproliferationofPdx1-positiveprogenitorcellstoformtubularcomplexesandacinarcellsandthecoincidenceoftheprogressionofacinarcellproliferationandthedisappearanceoftubularcomplexes.TheseresultsindicatethatexocrineregenerationinL-arginine-inducedacutepancreatitisinvolvesbothtransientlyproliferatingPdx1-positiveprogenitorcellproliferationtoformtubularcomplexesandacinarcellproliferation,supportingpreviousnotionsthatPdx1-positiveprogenitorcellsmayhavethepotentialtodifferentiateintoacinarcells.ElucidatingthemechanismthatregulatesPdx1-positivecellsinexocrineregenerationmayprovide

animportantbasisforunderstandinghowPdx1-positiveprogenitorcellsproliferateanddifferentiateintonewacinarcells.Hhsignalingregulatestheregenerationofmanyorgansandtissuesthroughits

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Fig.3.BrdUlabelingindicesfortubularcomplexes（A）andacinarcells（B）inL-arginine-inducedacutepancreatitis.Datarepresentthemean±SEM（n＝5ineachgroup）.＊P＜0.05vs.controls（day0）.

Hhsignalinginexocrineregeneration 177

Fig.4.ImmunohistochemicalanalysisofPdx1expressioninL-arginine-inducedacutepancreatitis（scalebar＝100μm）.Incontrolpancreata（A）,Pdx1wasexpressedinisletandductalcells.Duringexocrineregeneration,Pdx1wasalsoexpressedintubularcomplexesonday3（B）andday14（C）afterL-arginineinjection.

Fig.5.ImmunohistochemicalanalysisofGli2expressioninL-arginine-inducedacutepancreatitis（scalebar＝100μm）.Incontrolpancreata（A）,Gli2wasexpressedinisletandductalcellsandweaklyexpressedinafewacinarcells.Duringexocrineregeneration,Gli2wasalsoexpressedintubularcomplexesonday3（B）andday14（C）afterL-arginineinjection.

178 NorihisaSuzuki

Fig.6.ImmunohistochemicalanalysisofSmoexpressioninL-arginine-inducedacutepancreatitis（scalebar＝100μm）.Incontrolpancreata（A）,Smowasexpressedinisletcells.Duringexocrineregeneration,Smowasalsoexpressedintubularcomplexesonday3（B）andday14（C）afterL-arginineinjection.

Fig.7.ImmunohistochemicalanalysisofPtch1expressioninL-arginine-inducedacutepancreatitis（scalebar＝100μm）.Incontrolpancreata（A）,Ptch1wasexpressedinisletandductalcells.Duringexocrineregeneration,Ptch1wasalsoexpressedintubularcomplexesonday3（B）andday14（C）afterL-arginineinjection.

effectsonstem/progenitorcells１８）２８）.Previousstudiesusingacerulein-inducedacutepancreatitismousemodelshowedthattheblockingofHhsignalinginhibitedacinarcellregenerationbutdidnotinhibitPdx-1-positiveprogenitorcellproliferationintubularcomplexes,suggestingthatHhsignalingisinvolvedinPdx1-positiveprogenitorcelldifferentiationwithintubularcomplexesduringexocrineregeneration２３）.TogainsomeinsightintoapossiblelinkbetweenHhsignalingandPdx1-positiveprogenitorcellsinexocrineregeneration,weaimedtoidentifyandlocalizeHh-responsivecellsusingadifferentrodentmodelofacutepancreaticinjury.OurresultsindicatedthatHhsignalingisactivatedintubularcomplexesduringexocrineregeneration,underscoringtheimportanceofHhsignalingincontrollingPdx1-positiveprogenitorcellstoformtubularcomplexesduringexocrineregeneration,althoughwedidnotinvestigatethefunctionofHhsignalingintheregenerationofPdx1-positiveprogenitorcellsindetail.FurtherresearchisneededtoelucidatetherolesofHhsignalinginregulatingcellfatedecisionsinacinarcellregeneration,suchasHhtargetgeneresponses,whichmayplayimportantrolesinregulatingtheproliferationordifferentiationofPdx1-positiveprogenitorcells.Previousstudieshaveproposedthatthemechanismsunderlyingexocrineregeneration

recapitulatesomeaspectsofembryonicpancreaticdevelopment.ManystudieshavereportedthatPdx1-positiveprogenitorcellswithintubularcomplexeshavecharacteristicssimilartoembryonicpancreaticprogenitorcellsduringearlypancreaticdevelopment,e.g.,thelossofmatureacinarcellmarkerssuchasamylase,co-expressionofembryonicpancreaticprogenitorcellmarkers,andactivationofotherembryonicsignalingpathwayssuchastheNotchandWntsignalingpathways１０）３０）３１）.Hhsignalingisalsoessentialforembryonicpancreaticdevelopment. Duringtheearlystages,Hhsignalingmustspecificallybedownregulatedinembryonicpancreaticprogenitorcellsfornormaldevelopmenttooccur３２）３３）.Incontrast,atlaterstagesofpancreaticdevelopmentandinadults,Hhsignalingisactivatedinisletandductalcells３４）.Previousstudiesoncerulein-inducedacutepancreatitishaveindicatedthatablockadeofHhsignalingresultsintheinhibitionofexocrineregeneration,suggestingthepositiveroleofHhsignalinginexocrineregeneration,incontrasttoitsnegativeroleinpancreaticdevelopment２３）.OurstudyindicatedthatPdx1-positiveprogenitorcellswithintubularcomplexesduringexocrineregenerationareHh-responsive,incontrasttotheexclusionofHhsignalinginembryonicpancreaticprogenitorcells.TheseresultsprovideadditionalinsightsintothedifferencesbetweenthemolecularmechanismsbywhichHhsignalingregulatesexocrineregenerationandembryonicpancreaticdevelopment.ThereisaccumulatingevidencethattightregulationofHhsignalingisessentialforadulttissueregenerationafteracuteinjury.ThisprocessinvolvesHhsignalingbeingtransientlyandreversiblyactivatedafteracuteinjury,withtheexpansionofHh-responsivecells,aswellasthelossofHh-responsivecellswhenrecoveryiscompleted１８）.Incontrast,chronicorpersistentinjuryresultsinaprolongedincreaseinHhsignaling,leadingtoHh-responsivecellproliferationforaslongastheinjurypersistsandfinallyresultinginirreversibleactivationofthesignalingpathway,whichleadstocancerformation２８）.Inthisstudy,wedemonstratedthattransientactivationofHhsignalinginexocrineregenerationischaracterizedbyatransientexpansionofHh-responsivecellstoformtubularcomplexesandreductionofHh-responsivecellsasregenerationprogresses,suggestingthattransientactivationandtightcontrolofHhsignalingisimportantinexocrineregeneration.Ontheotherhand,constitutiveanduncontrolledHhpathwayactivationhasbeenimplicatedinthedevelopmentofchronicpancreatitisandpancreaticcancer３５‐３８）.Inchronicpancreatitis,thepancreaticparenchymaisdestroyedandreplacedbypersistenttubularcomplexes. Thesetubularcomplexesinchronicpancreatitisalsoexpress

Hhsignalinginexocrineregeneration 179

embryonicpancreaticprogenitormarkers（includingPdx1）andexhibitactivationofHhandNotchsignaling,showingmorphologicalandmolecularcharacteristicssimilartothoseoftubularcomplexesinacutepancreaticinjury.However,aberrantactivationofHhsignalingintubularcomplexesduringchronicinflammation,whichcanbeinducedthroughNK-κBactivationduringinflammation,mayhavearoleinthepathogenesisandprogressionofchronicpancreatitis,furthercorrelatingwithpancreaticcancerdevelopment３９）.FurtherstudiesarerequiredtoelucidatethemechanismsunderlyingthetightregulationofHhsignalinginexocrineregenerationwithoutuncontrolledactivationthatpossiblycorrelateswiththedevelopmentofpancreaticcancer.

Conclusions

WedemonstratedthatexocrineregenerationafterL-arginine-inducedacutepancreatitisinvolvesPdx1-positiveprogenitorcellproliferationtoformtubularcomplexesandthattransientactivationofHhsignalingischaracterizedbytheexpressionofHhcomponentsintubularcomplexesduringexocrineregeneration.ThesefindingssuggestthatHhsignalingplaysanimportantroleincontrollingPdx1-positiveprogenitorcellfunctionduringexocrineregeneration,afindingthatneedstobeexploredfurther.

Acknowledgments

ThisworkwassupportedinpartbyJSPSKAKENHIGrantNumbers15590673and17790461,agrantfromthe

ResearchCommitteeofIntractablePancreaticDiseases（PrincipalInvestigator:TooruShimosegawa）providedbytheMinistryofHealth,LabourandWelfareofJapan,andagrantfromthePancreasResearchFoundationofJapan.

ConflictofInterests

Theauthorsdeclarethatthereisnoconflictofinterestregardingthepublicationofthispaper.

180 NorihisaSuzuki

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〈和文抄録〉

膵腺房細胞再生におけるヘッジホッグシグナル伝達系の局在の評価

鈴 木 教 久

京都府立医科大学大学院医学研究科消化器内科学

急性膵障害後の膵腺房細胞の再生過程で増殖するPdx1陽性細胞が，腺房細胞へと分化する前駆細胞の可能性が示唆され，このメカニズムを解明することが慢性膵炎などの膵外分泌腺機能不全に対する再生医療を開発する上で有用であると考えられる．本研究ではラットアルギニン急性膵炎モデルを用いて，腺房細胞再生過程におけるヘッジホッグシグナル伝達系（以下Hh系）とPdx1陽性細胞の関連を評価した．アルギニン投与後3日目に膵炎発症に伴う腺房細胞の壊死消失，Pdx1陽性細胞からなるTubularcomplexの出現増殖を認めた．投与後5日目に腺房細胞の増殖が優位になる一方，Tubularcomplexの減少を認め，投与後14日目には腺房細胞の再生はほぼ終了する．Hh系のレセプターであるSmoとPtch1，転写因子であるGli2は，正常膵ではラ氏島と導管に発現を認めたが，アルギニン投与後3日目及び14日目にはTubularcomplexにも発現を認めた．本研究において，Hh系がPdx1陽性細胞を制御することにより膵腺房細胞の再生に関与している可能性が示唆された．

キーワード：ヘッジホッグシグナル伝達系，膵腺房細胞の再生，急性膵炎，膵前駆細胞，Pdx1．

182 NorihisaSuzuki

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