Role of CREB on Heme Oxygenase-1 Induction in Adrenal Cells: Involvement of the PI3K Pathway

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f astort, e m repetto and CREB mediates adrenal HO-1 57: 2 113–124 Research others induction by cAMP

Role of CREB on heme oxygenase-1 induction in adrenal cells: involvement of the PI3K pathway

F Astort1,*, E M Repetto1,*, L Rocha-Viegas2, M E Mercau1, S Sanchez Puch1, C V Finkielstein3, A Pecci2 and C B Cymeryng1

1Departamento de Bioquímica Humana, Facultad de Medicina, Universidad de Buenos Aires, CEFYBO-CONICET, Buenos Aires, Argentina 2Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Correspondence Aires, IFIBYNE-CONICET, Buenos Aires, Argentina should be addressed 3Integrated Cellular Responses Laboratory, Department of Biological Sciences, Polytechnic Institute and to C B Cymeryng State University, Blacksburg, Virginia, USA Email *(F Astort and E M Repetto contributed equally to this work) [email protected]

Abstract

In addition to the well-known function of ACTH as the main regulator of adrenal Key Words steroidogenesis, we have previously demonstrated its effect on the transcriptional ff heme oxygenase stimulation of HO-1 expression, a component of the cellular antioxidant defense system. ff cAMP/PKA In agreement, we hereby demonstrate that, in adrenocortical Y1 cells, HO-1 induction ff adrenocortical cells correlates with a significant prevention of the generation of reactive oxygen species ff CREB 2+ induced by H2O2/Fe . ACTH/cAMP-dependent activation of redox-imbalanced related ff PI3K factors such as NRF2 or NFκB and the participation of MAPKs in this mechanism was, however, discarded based on results with specific inhibitors and reporter plasmids. We Journal of Molecular Endocrinology suggest the involvement of CREB in HO-1 induction by ACTH/cAMP, as transfection of cells with a dominant-negative isoform of CREB (DN-CREB-M1) decreased, while overexpression of CREB increased HO-1 protein levels. Sequence screening of the murine HO-1 promoter revealed CRE-like sites located at −146 and −37 of the transcription start site and ChIP studies indicated that this region recruits phosphorylated CREB (pCREB) upon cAMP stimulation in Y1 cells. In agreement, H89 (PKA inhibitor) or cotransfection with DN-CREB-M1 prevented the 8Br-cAMP-dependent increase in luciferase activity in cells transfected with pHO-1[−295/+74].LUC. ACTH and cAMP treatment induced the activation of the PI3K/Akt signaling pathway in a PKA-independent mechanism. Inhibition of this pathway prevented the cAMP-dependent increase in HO-1 protein levels and luciferase activity in cells transfected with pHO-1[−295/+74].LUC. Finally, here we show a crosstalk between the cAMP/PKA and PI3K pathways that affects the binding of Journal of Molecular Endocrinology p-CREB to its cognate element in the murine promoter of the Hmox1 gene. (2016) 57, 113–124

Introduction

ACTH is the main regulator of glucocorticoid synthesis apoptosis (Gallo-Payet & Payet 2003). Many of these in the adrenal cortex where it also affects other biological effects are mediated by ACTH binding to the MC2R functions such as differentiation, mitogenesis and receptor and downstream activation of the cAMP/PKA

http://jme.endocrinolovgy-journals.org © 2016 Society for Endocrinology Published by Bioscientifica Ltd. DOI: 10.1530/JME-16-0005 Printed in Great Britain Downloaded from Bioscientifica.com at 09/24/2021 11:53:44PM via free access

10.1530/JME-16-0005 Journal of Molecular Endocrinology and membersofthe AP-1(activating protein-1) 2-related factor 2), heat-shock factor proteins, NF-κ factors, includingNRF2 (nuclearfactor-erythroid regulation thatinvolves multipletranscription and humanHO-1promoters aresubjecttoacomplex 2002, Ryter & Choi controlled atthetranscriptionallevel(Maines1988 , Astort et al.2009). steroidogenesis (Pomeraniec involved inthefine-tuningregulationofadrenal systems been recentlyincludedamongthemodulatory glucocorticoid response,hemeoxygenaseactivityhas steroid production.Byattenuatingthehormone-induced activity hasbeenalsoinvolvedinthemodulation of Tyrrell 2000, & Ryter Bonkovsky1999, & activated bystressfulsignals(Elbirt has beenrecognizedasacellulardefensemechanism catabolism, theactivityofinducibleisoformHO and CO.Inadditiontothiswell-knownroleinheme (and thenbilirubin)withtheconcomitantreleaseofiron heme catabolism,theconversionoftobiliverdin treatment (Pomeraniecet al.2004). lipoperoxides andproteincarbonylstriggeredbyH cells correlatedwiththepreventionofgeneration induction ofhemeoxygenase1(HO-1)inadrenocortical we havepreviouslyshownthattheACTH-dependent Hanukoglu 2006, al.2002, several antioxidantmechanisms(Chinnet associated withoxidativestress,adrenalcellsactivate 2014). To copewiththepotentiallydamagingeffects other oxygenatedspecies(Hanukoglu2006, of reactive oxygen species (ROS), lipoperoxides and O catalyze thehydroxylationofsteroidsandreduction cytochrome P450enzymes,mixedfunctionoxidases, multistep processwheremitochondrialandmicrosomal 2003a . binding protein(C/EBP)couldbeincluded(Mannaet al the steroidogenicfactor1(SF1)andCAAT enhancer binding protein(CREB),theGATA-4 bindingprotein, genes. Amongthem,thecAMPresponseelement the transcriptionalstimulationofsteroidogenic-related activation ofseveraltranscriptionfactorsengagedin and PKA activityisinvolvedinthephosphorylation pathway (Gallo-Payet2016).Inturn,stimulationof as incomplete reduction in O stimulated steroidogenesisgeneratesoxidativestress, DOI: 10.1530/JME-16-0005 http://jme.endocrinology-journals.org 2 Research Research towater. Evenunderphysiologicalconditions,ACTH- The inductionofHO-1expressionisprimarily Heme oxygenasescatalyzetherate-limitingstepin Glucocorticoid synthesis in the adrenal cortex isa , b , Stocco et al.2005). e al.2009 ). Amongthem, Battista et Alam & Cook 2003).Bothmouse Alam & Cook Bach 2002).Hemeoxygenase et al. 2004 , 2 leads to the production others f

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highest qualityavailable. (Buenos Aires, Argentina). All other chemicals were of the from Sigma.ACTHwasobtained fromELEALaboratories (Life Technologies), while8Br-cAMPwaspurchased DNAse I and TAQ polymerase were from Invitrogen penicillin, streptomycin, MMVL reverse transcriptase, Biotechnology and Bio-Rad, respectively. Fetal calf serum, antirabbit-IgG antibodieswereobtainedfromSantaCruz respectively. Actinandperoxidase-conjugated goat Cruz BiotechnologyandCellSignalingTechnology Phospho-CREB (Ser133)antibodieswerefromSanta New EnglandBiolabs(Ipswich,MA,USA).CREBand and thoseforpAkttotalAktwereboughtfrom StressGen BiotechnologiesCorp.(Victoria, Canada, The antibodyraisedagainstHO-1wasacquiredfrom Chemicals Materials andmethods of thePI3K/AKTpathway. CREB bindingtothisregionisalsoaffectedbyactivation region in the murine HO-1 byACTH,andidentifiedaCREB-responsivebinding CREB activationinthetranscriptionalstimulationof present report,weanalyzedtheinvolvementofPKAand molecular mechanismhasremainedelusive.Inthe PKA pathway(Pomeraniec of HO-1 by ACTH involves the activation of the cAMP/ et al. 2003, modulator ofantioxidantgeneexpression(Bedogni a roleinregulatingROSdetoxificationasdirect al.2009).CREBactivationhasbeenshowntoplay et 2002, CREB (Lonze & Ginty ofbeen linkedtothePKA-dependentphosphorylation . 2007).ActivationoftheHO-1promoterhasalso et al promotes the induction of antioxidant genes (Kensler the cytosolandtranslocatestonucleuswhereit Upon stimulation,NRF2dissociatesfromKEAP1in proteosomal degradationbyubiquitinmodification. like ECH-associatedprotein1(KEAP1)andtargetedto maintained inthecytoplasmbyinteractingwithKlech- cytoprotective genes.Inbasalconditions,NRF2is has beenlinkedtotheinductionofHO-1andother factor, asaconsequenceofcellularredoximbalance, al.2008).ActivationoftheNRF2transcription et Choi2005, & response elements(AREs)(Ryter response elementsoftenembeddedinantioxidant bZIP(basic region/leucinezipper)families,with induction bycAMP CREB mediatesadrenalHO-1 Published byBioscientifica Ltd. Although wehavepreviouslyshownthatinduction Kronke et al. 2003). Hmox1 gene proximal promoter. Downloaded fromBioscientifica.com at09/24/202111:53:44PM t al.2004),theunderlying et . 2003, Kronke et al 57 57 : : 2 2 Wright 114 114 Surh via freeaccess

Journal of Molecular Endocrinology 2 H2DCF israpidlyoxidizedbyROStothefluorescent uptake bythecellsandhydrolysiscellularesterases, for 1 incubated inPBScontaining10 treatments, cells werewashedtwicewithPBSand then al.2014, previously (Astortet diacetate, MolecularProbes,Invitrogen)asdescribed probe 2′,7-dichlorodihydrofluoresceindiacetate(H2DCF ROS generationwasmeasuredusingthenonfluorescent Determination ofintracellularROSgeneration bluedyeexclusiontest. viability wasdeterminedbytrypan Ham’s F10 2 twice with PBS and medium was replaced with serum-free described previously (Schimmer 1979). Cells were washed 485 was confirmed by DNA sequencing (MACROGEN, Korea). was confirmedbyDNAsequencing (MACROGEN,Korea). 1(E1).LUC] (Promega).The identity ofreporterplasmids template andclonedintoapGL3 promoterplasmid[pHO- 523 murine HO-1promoterwasamplified byPCR(− [pHO-1(− only the(− ( Brigham and Women’s Hospital, Boston, MA, USA) andCritical CareDivision, Perrella(Pulmonary M Dr [pHO-1(− promoter clonedintothebasicpGL2reportervector the (− The Plasmids unitsrelativetototalproteincontent. in arbitrary Ortemberg, Germany).ROSgenerationlevelsareexpressed microplate reader, FLUOstarOmega(BMGLabtech, a humidifiedatmosphereof5%CO penicillin Gand270 bovine (2.5%)andhorse(12.5%)serum,200 in Ham’s F10mediumcontainingheat-inactivatedfetal and coworkers(1966). Cells were grown inmonolayers adrenocortical tumorcelllineestablishedbyYasumura Collection) isanACTH-andcAMP-responsivemouse The adrenalY1cellline(CCL-79,AmericanType Culture Cell culture andtreatments the pHO-1(− promoter wasgeneratedbyPCRamplificationusing ). A pGL2 reporter plasmid containing . 2005).ApGL2reporterplasmidcontaining Chung et al ʹ http://jme.endocrinology-journals.org DOI: 10.1530/JME-16-0005 7 Research ʹ -dichlorofluorescein. Fluorescence intensity (excitation -dichlorofluorescein. Fluorescenceintensity(excitation bp) usingthepHO-1(− nm, emission535 h at37°C.CellswerewashedtwicewithPBS.Upon luciferase reporter plasmid containing HO-1 luciferasereporterplasmidcontaining 4045/+ 295;+ 4045;+ h before the initiation of the experiments. Cell h before the initiation of the experiments. Cell 295/+ 74) fragmentfromthemurineHO-1 4045/+ 74).LUC]. The distal enhancer E1 from the 74).LUC]. ThedistalenhancerE1fromthe 74).LUC] was generously provided by 74).LUC] wasgenerouslyprovidedby 74) fragmentfromthemurineHO-1 nm) was determined in a multimode nm) wasdeterminedinamultimode 74).LUC construct as template 74).LUC construct as template μ g/mL streptomycin sulfate, in g/mL streptomycinsulfate,in 4045/+ e al.2014).After Mercau et others f

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(pκ al.2008, et pcDNA3.1/V5HisB-mNRF2 (NRF2FL)(Innamorato Both p3xARE.LUC(pNRF2.LUC)reporterplasmid, For overexpression assays,10 Transfection ofY1cellsandluciferaseassay (FCEyN, UBA,CABA,Argentina)(Gionoet al.2001). (pCRE.LUC), werekindlysuppliedbyDrECánepa Montminy 1989) andaluciferasereporterforCREBp4xCRE.LUC & obtained fromRSV-CREB (Gonzalez siteS133A amutationinthephosphorylation carrying (RSV-CREB), thedominant-negativeisoform(DN-CREB-M1) containing theentirewild-typecodingregionofCREB al.2006).Anexpressionvector Argentina) (Squarizeet were kindlyprovidedbyDrOCoso(FCEyN,UBA,CABA, Tris–HCl pH7.4,250 Y1 cellswerewashedtwice inPBSandlysed20 Immunoblot analysis luminometer (BMGLABTECH,Germany). Luminescence wasmeasuredinaFLUOstarOmegaplate and valueswerenormalizedtoβ-galactosidaseactivity. using theSteady-GloLuciferaseAssaySystem(Promega) pCMV-βGAL (ratio8:1:1). plasmid (RSV-CREB orDN-CREB-M1 orpGEX-4T-3) and in acombinationofreporterplasmid,anexpression plates andtransfectedwith0.2 In anothersetofexperiments,cellswereseededin96-well medium, andthefollowingdayweretreatedaccordingly. to manufacturer’s instructions in serum-free OptiMEM LUC), andpCMV-βGAL inLipofectamine2000according LUC orpHO-1(E1).LUCpNRF2(ARE).LUCp4xCRE. plasmids: pHO-1(−4045/+74).LUCor295;74). transfected withluciferase0.2 on 96-wellplates.Onedayafter, cellsweretransiently incubated foradditional24 Ham’s F10mediumwithsera,andthecellswerefurther transfection mediumwasaspiratedandreplacedwithfresh was includedascontroloftransfection.Threehourslater, medium; pCMV-β manufacturer’s instructionsinserum-freeOptiMEM 2000 Transfection Reagent(Invitrogen)accordingto control) weretransientlytransfectedwithLipofectamine plasmids (RSV-CREB orDN-CREB-M1pGEX-4T-3 as 24-well plates.Twenty fourhourslater, 0.5 induction bycAMP CREB mediatesadrenalHO-1 Published byBioscientifica Ltd. B-LUC), containingfiveNFκ Luciferase activitywasdeterminedincellhomogenates For luciferasereporterassays,10 e al.2004)andPGL3-NFκ McMahon et GAL (β mM NaCl,1%Triton X-100,1 -galactosidase expression plasmid) -galactosidase expressionplasmid) Downloaded fromBioscientifica.com at09/24/202111:53:44PM h beforeanytreatment. 5 cells per well were seeded in cellsperwellwereseededin µg ofthefollowingreporter µg oftotalDNAperwell B response elements, B responseelements, 4 57 cellswereseeded : 2 µg of expression µg ofexpression B-LUC B-LUC 115 mM mM mM via freeaccess

Journal of Molecular Endocrinology Na nescence Reagent, Amersham Pharmacia Biotech, nescence Reagent,AmershamPharmaciaBiotech, visualized by chemiluminescence (Enhanced Chemilumi CREB orβ with the following antisera: HO-1, p-AKT, AKT, p-CREB, 60 0.15 were blockedinTBS-Tween (50 9.2, 192 Bio-Rad Trans-Blot SDsystemwith25 polyvinylidinefluoride membranesfor1 After electrophoresis,proteinsweretransferredto and phosphataseinhibitorscocktails(Sigma-Aldrich). DOI: 10.1530/JME-16-0005 http://jme.endocrinology-journals.org Research Research min at room temperature and then incubated overnight min atroomtemperatureand then incubatedovernight 3 VO M NaCl,0.05%Tween 20)with5%skimmedmilkfor 4 , 100 mM glycine and 20% methanol. Membranes mM glycineand20%methanol.Membranes -ACTIN at 4°C. Specific protein bands were -ACTIN at 4°C. Specific protein bands were mM NaF, 1 mM EGTA, with1Xproteases others f

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repetto and V in a V ina Diagenode (Denville,NJ,USA)asanegativecontrol. Millipore (EMDMillipore)andIgG(kch-504-250)from Y1 cells,usingChIPAb+Phospho-CREB (Ser133)from . 2014)withchromatinobtainedfromadrenocortical et al as described previously (Bertucci et al. 2013, Chromatin immunoprecipitation assays were performed Chromatin immunoprecipitation (ChIP)assay San Jose,CA,USA). Fluorchem software(V. 4.1.0,AlphaInnotechCorporation, andquantified with theAlphaEase System (GE Healthcare) Chalfont,Bucks, UK)inanImageQuant Imaging Little induction bycAMP CREB mediatesadrenalHO-1 Published byBioscientifica Ltd. ***P Tukey’s testorbyStudent’s t further incubatedwith100 probe 2′ Materials andMethodswiththeoxidant-sensing generation wasdeterminedasdescribedin ACTH fortheindicatedperiodsoftimeandROS (A) Y1cellswereincubatedwith12.5 Figure 1 12.5 (B) Y1 cellswerepreincubatedwithorwithout mean ± determined inlysedcells.Resultsare indicatedas the ratioofluciferasetoβ (JNKi) or5 absence of50 without 1 1(− 24 stimulated withorwithout1 construct [pHO-1(− obtained byPCRperformedfromtheoriginal promoter fragmentofthemurineHmox1gene, with areporterplasmidcontainingproximal were transfectedwithpHO-1(− LUC andNRF2FLareshownascontrol;(G)Y1cells from cotransfectionexperimentswithpHO-1(E1). 8Br-cAMP or12.5 further stimulatedwithorwithout1 the murineHO-1promoter[pHO-1(E1).LUC]and reporter plasmidcontainingtheE1enhancerof for 24 or without1 region pHO-1(− containing 4045 cells weretransfectedwithareporterplasmid 12.5 plasmid pκ (D) Y1 cellsweretransfectedwiththereporter pNRF2.LUC andNRF2FLareshownascontrol; 24 without 1 plasmid pNRF2.LUCandstimulatedwithor (C) Y1cellsweretransfectedwiththereporter preincubation inthepresenceorabsenceofACTH; HO-1 andβ Below, arepresentativewesternblotshowing for 1 h. (H)Y1cellsweretransfectedwithpHO- h. Resultsfromcotransfectionexperimentswith 295/+ mIU/mL ACTHfor5 mIU/mL ACTHor10

h. ROS generation was determined as in (A). h. ROSgenerationwasdeterminedasin(A). < h; (F) Y1 cells were transfected with a h; (F)Y1cellsweretransfectedwitha 0.001 vscontrol,byANOVA followed by s Downloaded fromBioscientifica.com at09/24/202111:53:44PM . 7 e 74).LUC and further incubated with or 74).LUC andfurtherincubatedwithor ′ . mM 8Br-cAMP inthepresenceor mM 8Br-cAMP or12.5 dichlorodihydrofluorescein diacetate; dichlorodihydrofluorescein diacetate; m µM SB203580 (p38i). Data are shown as µM SB203580(p38i).Dataareshown as B.LUC and stimulated with or without B.LUC andstimulatedwithorwithout -ACTIN levelsafterthe5 . ( mM 8Br-cAMP or12.5 n µM PD98059(MEKi),1 ) *P =3), 4045/+ bp of the murine HO-1 promoter bp ofthemurineHO-1promoter mIU/mL ACTHfor24 295/+ .5 **P <0.05, 74).LUC and stimulated with 74).LUC andstimulatedwith h, washed with PBS and h, washedwithPBSand µg/mL LPSfor24 74).LUC], and further 74).LUC], andfurther 57 57 -galactosidase activity -galactosidase activity µM H -test. : : mM 8Br-cAMP for 2 2 4045/+ mIU/mL ACTH for mIU/mL ACTHfor 0.01, <0.01, 2 O Rocha-Viegas mIU/mL ACTH mIU/mL ACTH 2 h of h of M FeSO /80 µM mM mM µM SP600125 µM SP600125 mIU/mL mIU/mL 74).LUC or 74).LUC or h. Results h. Results h; (E) Y1 h; (E)Y1 116 116 4 via freeaccess

Journal of Molecular Endocrinology of input(mean was normalizedtoinputandexpressedaspercentage GAACAGAG-3ʹ. Chromatin immunoprecipitation signal ATGC TGAGTTGTGAT-3ʹ; reverse:5-CTGAGGCTGAGG distal enhancerE1(−3979; reverse: 5ʹ-AAACTCTGGAGGCGACTGG-3andforthe 5ʹ-ACGTGACCCGCGTACTTAAA-3(−42;+30), forward: ʹ; following primersspecificfortheHO-1promoterregion OpticonTM System cycler (MJResearch) withthe was performedbyreal-timePCRusingDNAEngine Quantification ofchromatinimmunoprecipitation different whenP (GraphPad Software).Datawereconsideredsignificantly using GraphPadInStatversion3.06forWindows post hoct-testorStudent’s t was performedbyone-wayANOVA followedbyTukey’s Data areexpressedasmean Statistical analysis http://jme.endocrinology-journals.org DOI: 10.1530/JME-16-0005 Research ± s <0.05. . e . m . , n =3). 3843), forward: 5ʹ-GCTGGA −3843), forward: -test asconsiderednecessary ± s . others f e

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HO-1 expression,andthentreatedwithH preincubated withACTHduring5 of ROSgeneration.Moreover, whencellswere least 2 addition andthisparameterremainedelevatedforat significantly increasedalready30 presence of ACTH, showed that ROS generation was in murineadrenocorticalY1cellsincubatedthe Results fromatimecourseexperiment,performed of reactiveoxygenspecies(ROS)uponACTHtreatment. adrenocortical cells,wefirstevaluatedthegeneration demonstrating theinductionofHO-1byACTHin a cellularredoximbalanceandourpreviousresults of antioxidantenzymesinducedbythegeneration Taking intoconsiderationthatHO-1belongstoafamily Results involvement oftheNrf2pathwayininduction ( that werepreincubatedintheabsenceofhormone generation wassignificantlylowercomparedwithcells induction bycAMP CREB mediatesadrenalHO-1 i. 1B).Theseresultspromptedustoanalyzethe Fig. Published byBioscientifica Ltd. i. 1A),confirmingtheACTH-dependence h (Fig. ANOVA followedbyTukey’s test. ) ***P (n =3), Results areshownasmean(%ofinput) with normalrabbitIgGwasusedas control. the murineHmox1gene.Immunoprecipitation corresponding todistalandproximal regionsof by qPCRtotesttheenrichmentinsequences The precipitatedDNAfragmentswereanalyzed subjected toChIPassayswithp-CREBantibody. with orwithout1 shown inboldletters.(F)Cellswereincubated and relativepositionofCRE-likebindingsitesare MatInspector software.Textbox detailsequences mouse HO-1promoterwasperformedwiththe (E) Analysisofthe−295/+74regionfrom ***P **P <0.01, corresponding controland induction) ± analysis ofdataisshownasmean(fold HO-1 andβ-ACTINantibodies.Densitometry prepared andanalyzedbywesternblottingwith 5 incubated withorwithout1 amounts ofDN-CREB-M1(A)orRSV-CREB (B)and cAMP. Y1cellsweretransfectedwithincreasing Involvement ofCREBintheinductionHO-1by Figure 2 and D,resultsareshownasmean incubated with1 pCRE.LUC andDN-CREB-M1further lower panels.(C)Y1cellswerecotransfectedwith experiments. Representativeimagesareshownin or RSV-CREB, obtainedfromthreeindependent 8Br-cAMP-stimulated cells withoutDN-CREB-M1 cotransfected withpCRE.LUC Luciferase activitywasdeterminedincells h. Aftertreatments,proteinextractswere Downloaded fromBioscientifica.com at09/24/202111:53:44PM < s . e 0.001 vsrespectivecontrol,by . m < . .0 vs ***P <0.001 **P < 0.01, , n =3, mM 8Br-cAMP for24 0.001 vscorrespondingcontrol. mM 8Br-cAMP for2 h, inordertoinduce min afterhormone 57 ### : mM 8Br-cAMP for 2 ± P RSV-CREB. InC .0 vs < 0.001 2 O ± 2 s . /Fe e . h andthen m h. (D) . ± , n =3, 2 + s . , ROS e 117 . m . via freeaccess Journal of Molecular Endocrinology promoter (− reporter plasmidcontaining4KbpofthemurineHO-1 (TSS), wealsoperformedtransienttransfectionswitha motif located4Kbpupstreamthetranscriptionstartsite indicated thepresenceofanNRF2consensusbinding Since sequenceanalysisofthemurineHO-1promoter Y1 cellsdidnotactivatetheNFκ experiments, wedemonstratedthatACTHtreatmentof activated bythecAMPpathway. Inanothersetof suggesting thatthistranscriptionfactorwasnot cAMP, 1C), 8Br-cAMP, treatmentswereeffective(Fig. activity, neitherACTHnorapermeableanalogof of NRF2(NRF2FL)significantlyincreasedluciferase cells. Ourresultsshowedthatwhileoverexpression an NRF2reporter, pNRF2.LUC,wereperformedinY1 of HO-1byACTH.Thus,transienttransfectionswith ACTH-dependent inductionofHO-1isnotmediatedby ( murine distalenhancerE1andtheNrf2consensussite pHO-1(E1).LUC, areporterplasmidcontainingthe 8Br-cAMP norACTHtreatmentswereabletostimulate E). However,increased luciferaseactivity(Fig. 1 neither of thesecellswith8Br-cAMPorACTHsignificantly − DOI: 10.1530/JME-16-0005 http://jme.endocrinology-journals.org Research Research 4045/− 3524, 4045/+ Fig. 1F) furthersupportingtheideathat 74). Resultsshowedthatstimulation others f

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repetto and ( only theproximalfragmentofHO-1promoter inareporterplasmidcontainingwas conserved NRF2 activation.Inaddition,stimulationby8Br-cAMP assayed (Fig. 2CandD). the activityofapCRE.LUCreporterinY1cellswasalso 2B).TheeffectofDN-CREB-M1orRSV-CREB on (Fig. RSV-CREB significantlyupregulatedHO-1proteinlevels of CREB in cells transfected with increasing amounts of 2A).Conversely, overexpression expression vector(Fig. cells transfectedwithincreasinglevelsofDN-CREB-M1 8Br-cAMP-induced increase inHO-1proteinlevels (DN-CREB-M1). Ourresultsshowedaninhibitionofthe a plasmidexpressingdominant-negativeformofCREB induction ofHO-1bycAMPinY1cellstransfectedwith in adrenalcells.To thatend,wefirstevaluatedthe the involvementofCREBoninductionHO-1 and activationofCREB.Therefore,wesoughttoanalyze pathway hasbeenassociatedwiththephosphorylation MAPK signalingpathways(Fig. 1H). the additionofspecificinhibitorsforERK,JNKorp38 activity ofpHO-1(− induction bycAMP CREB mediatesadrenalHO-1 − Published byBioscientifica Ltd. 295/+ It iswellknownthatstimulationofthecAMP/PKA i. 1G).8Br-cAMP-stimulatedluciferase 74) (Fig. control, experiments. **P are shownasmean normalized againstβ activity wasdeterminedincellextractsand 8Br-cAMP (rightpanel).(AandB)Luciferase of 12.5 and furtherincubatedinthepresenceorabsence incubated withorwithout10 transfected withpHO-1(− 1 with either12.5 expression plasmidDN-CREB-M1andstimulated cotransfected withpHO-1(− ACTH and8Br-cAMP. (A)Y1cellswere luciferase activityofpHO-1(− Involvement ofCREBinthestimulation Figure 3 conserved bases. Enclosed areascorrespondtoCREsites; *indicates norvergicus) originusingNCBIblastanalysistool. taurus), mouse(Musmusculus)andrat Rattus sapiens), monkey(Macacamulattabovine Bos promoter regionofHO-1fromhuman (Homo Comparison ofthesequencessurrounding+ ACTH byANOVA followedbyTukey’s test.(C) mM 8Br-cAMP (rightpanel)for24 295;+ mIU/mL ACTHfor24 Downloaded fromBioscientifica.com at09/24/202111:53:44PM ## P 74).LUC wasnotaffectedby <0.01, mIU/mL ACTH (left panel) or mIU/mL ACTH(leftpanel)or .1 ***P <0.01, ### ± P -galactosidase activity. Values s .

< e . 0.001 vs8Br-cAMP or m 57 57 . 295/+ , for three independent , forthreeindependent h (leftpanel)or1 295/+ : : 2 2 0.001 vs respective respective vs <0.001 295/+ μ M H89for30 74).LUC were 74).LUC were 74).LUC and the 74).LUC andthe 74).LUC by 74).LUC by h; (B) Y1 cells h; (B)Y1cells 118 118 min min mM mM via freeaccess 1 Journal of Molecular Endocrinology notion thattheproximalpromoterregionisinvolved 2F).Theseresultssupportthe upstream theTSS)(Fig. atthedistalenhancerE1(around4Kbpobserved the phospho-CREB to the proximal promoter region of 8Br-cAMP significantlyincreasedtherecruitmentof Results showedthatstimulationofY1cellswith to assessCREBrecruitmenttheendogenousgene. at −146and37. indicate twodifferentputativeCREbindingsiteslocated Suite, V3.1,Munich,Germany).ResultsshowedinFig. 2E MatInspector Release 8.2 software (Genomatix Software analysis oftranscriptionfactorbindingmotifswiththe 4Kbp ofthemurineHO-1promoter, weperformedan http://jme.endocrinology-journals.org DOI: 10.1530/JME-16-0005 Research Therefore, weperformedaChIPanalysisinorder In thesearch ofcAMP-responseelements(CRE)along Hmox1 gene,whilenosignificantbindingwas others f

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in different systems, we analyzed the involvement of this associated withthe stimulation of the PI3K/AKTpathway between mouseandratpromoterregions(Fig. 3C). inthe−146CREsite a significanthomologywasobserved the human, monkey, bovineandratsequencesindicatesthat promoter regionofthemurineHmox1genewith 3B).Asequencecomparisonoftheproximal H89 (Fig. were incubatedinthepresenceofPKAinhibitor, 3A).Similarresultswereobtainedwhenthecells (Fig. of pHO-1(−295/+74).LUCinducedbyACTHor8Br-cAMP DN-CREB-M1 preventedtheincreaseinluciferaseactivity dependent pathway. in theinductionofHO-1byacAMP/PKA/CREB- induction bycAMP CREB mediatesadrenalHO-1 Published byBioscientifica Ltd. 37 CRE site is highly conserved amongspecies,while −37 CREsiteishighlyconserved As several antioxidant and protective effects have been As severalantioxidantandprotectiveeffectshavebeen In agreement,transfectionofY1cellswith ***P for threeindependentexperiments, **P <0.01, Values areshownasfoldinduction(mean normalized againstβ-galactosidaseactivity. activity wasdeterminedincellextracts and without 1 LY294002 for30 preincubated with0.25 cells transfectedwithpHO-1(−295/+74).LUCwere three independentexperimentsareshown.(F)Y1 antibodies (6 and AKTantibodies(2 were analyzedbywesternblottingwithpAKT 1 30 0.25 lower panels.(E)Y1cellswerepreincubatedwith antibodies. Representativeimagesareshownin western blottingwithHO-1andβ-ACTIN Protein extractswerepreparedandanalyzedby treated withorwithout1 with 0.25 an insetinFig. 4B.(D)Y1cellswereincubated after theinitiationoftreatmentisshownas control and8Br-cAMP-stimulated Y1cells2 western blotshowingpAKTandAKTlevelsin are showninlowerpanels.Arepresentative independent experiments.Representativeimages nuto (mean ± induction Densitometry analysisofdataisshownasfold western blottingwithp-AKTandAKTantibodies. prepared aftertreatmentsandanalyzedby 8Br-cAMP for2 further incubatedwithorwithout1 wortmannin and/or10 preincubated inthepresenceof0.25 different periodsoftime; (C)Y1cellswere 12.5 cells. Y1cellswereincubatedinthepresenceof Activation ofthePI3Kpathwayinadrenocortical Figure 4 8Br-cAMP, byANOVA followedbyTukey’s test. mM 8Br-cAMP for2 min andfurtherincubatedwithorwithout µM wortmanninor10 mIU/mL ACTH(A)or1 < 0.001 vscontroland Downloaded fromBioscientifica.com at09/24/202111:53:44PM μM wortmanninfor30 mM 8Br-cAMP for24 h). Representativeimagesfrom h. ACproteinextractswere min andfurthertreatedwithor s . e . m h or6 h) andHO-1β-ACTIN . µM H89for30 ), obtainedfromthree μM wortmanninor10 57 mM 8Br-cAMP for6 mM 8Br-cAMP (B)for µM LY 294002for # h. Proteinextracts : P <0.05, 2 h. Luciferase min andfurther mM ### µM min and .0 vs P <0.001 ± 119 h s . e µM . h. m via freeaccess . ) Journal of Molecular Endocrinology (A) Proteinextractswerepreparedand analyzedbywesternblotwith for 30 PI3K/AKT pathway. (A)Y1cellsweretreated with0.25 Modulation ofCREBactivationand binding totheHO-1promoterby Figure 5 DOI: 10.1530/JME-16-0005 http://jme.endocrinology-journals.org Research Research min andfurtherincubatedwithorwithout 1 others f

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repetto and h. corresponding controland used ascontrol.Resultsareshown mean murine HO-1promoter. Immunoprecipitationwithnormalrabbit IgGwas sequences correspondingtotheproximal CREsites(−156;37)from DNA fragmentswereanalyzedbyqPCR totesttheenrichmentin then subjectedtoChIPassayswithp-CREBantibody. Theprecipitated *P experiments, ***P fold induction(mean p-CREB andCREBantibodies.Densitometryanalysisofdataisshownas by Tukey’s test. as foldinduction(mean extracts andnormalizedagainstβ-galactosidaseactivity. Values areshown with 1 preincubated with0.25 and AKTisshown.(B)Y1cellsweretransfectedwithpCRE.LUC, lower panel,Inset:Arepresentativewesternblotanalysiswithantip-AKT ANOVA followedbyTukey’s test.Representativeimagesareshowninthe region, suggesting that an active PI3K signaling pathway increase inpCREBbindingtotheproximalpromoter inhibitor completelyblockedthe8Br-cAMP-dependent that the PI3K proximal promoter of HO-1, we observed effect ofwortmanninonthebindingpCREBto further supportingthecrosstalkhypothesis(Fig. 5B). of luciferaseactivityincellstransfectedwithpCRE.LUC, increase inthepAKT/AKTratio(insert)andstimulation This treatment also prevented the 8BrcAMP-dependent increase inpCREB/CREBratiotriggeredby8Br-cAMP. that wortmanninslightlybutsignificantlypreventedthe 5Aindicated ResultspresentedinFig. phosphorylation. assessed CREB activation by determining Ser133-CREB cells with 8Br-cAMP in the presence of wortmannin and HO-1transcription. the cAMPandPI3Kpathwaysaffectingstimulationof results suggesttheestablishmentofacrosstalkbetween 4F).These inhibited bywortmanninandLY294002 (Fig. of theHO-1promoterpHO-1(− the reporterplasmidcontainingproximalfragment 4E).Luciferaseactivityof triggered by8Br-cAMP(Fig. the pAKT/AKTratioandinexpressionlevelsofHO-1 PI3K inhibitor, LY204002, also prevented the increase in determined bywesternblotting(Fig. 4D). Accordingly, the treatment alsoinhibitedHO-1inductionby8Br-cAMP, as Akt activation.AsfortheexpressionofHO-1,wortmannin but notbyH89,rulingouttheinvolvementofPKAon this effectwasinhibitedbythePI3Kinhibitor, wortmannin, activation ofthissignalingpathway. C, AsshowninFig. 4 4B)indicatingthe 4A)and 8Br-cAMP Fig. ( ACTH (Fig. by Figure 4showsatimecourseofAKTphosphorylation cells. induction ofHO-1bycAMPinY1 the in pathway induction bycAMP CREB mediatesadrenalHO-1 Published byBioscientifica Ltd. < 0.05 vscontrolbyStudent’s t-test.(C)Cellsweretreatedasin(A)and When ChIPanalysiswasperformedtoassessthe In ordertotestthishypothesis,wefirstincubatedY1 mM 8Br-cAMP for24 < 0.001 vscontroland ± s µM wortmanninfor30 . ± e . s m . e . ### h. Luciferaseactivitywasdeterminedincell ), obtainedfromthreeindependent . m P . ) forthreeindependentexperiments. < Downloaded fromBioscientifica.com at09/24/202111:53:44PM 0.001 vs8Br-cAMP, byANOVA followed ### P 295/+ ± < s . 0.001 vs8Br-cAMP, by e min andfurtherincubated . m . 57 57 .0 vs ***P <0.001 (n =3), 74).LUC was also 74).LUC wasalso : : 2 2 120 120 via freeaccess Journal of Molecular Endocrinology (including theE1enhancer containing anAREelement), is suggested,asROSgenerationinducedbyH with ACTH.TheroleofHO-1asanantioxidantenzyme in ROSgenerationadrenocorticalY1cellsincubated Accordingly, wehavepreviouslydemonstratedanincrease Pomeraniec al.1989, steroidogenesis (Bhaskeret ofautocrine/paracrinemodulators in thecategory In adrenal cells, HO activity has also been included antioxidant responseuponexposuretostressfulstimuli. in therecruitmentofpCREBtoHO-1promoter. HO-1. We alsosuggesttheinvolvementofPI3Kpathway CRE localizedwithintheproximalpromoterregionof ofCREBanditsbindingtoafunctional phosphorylation of theHmox1genebyACTH/cAMPrelieson In thisstudy, wereportthatthetranscriptionalstimulation Discussion region (Fig. 5C). fortheproperrecruitmentofpCREBtothis is necessary containing 4 activity inY1cellstransfected withareporterplasmid although 8Br-cAMP was able to stimulate luciferase luciferase activityofanNRF2 reporterplasmid,and First, neitherACTHnor8Br-cAMPtreatmentstimulated participation in the induction of HO-1 by ACTH/cAMP. ( with NO-donorshasbeenpreviouslydemonstrated NRF2 in HO-1 induction in adrenocortical cells stimulated pretreatment withACTH.Althoughtheinvolvement of significantly lowerincellswhereHO-1wasinduced by incompletely reduced products of O conditions, releasesreactiveoxygenspeciesbyleaking in adrenalcells,aprocessthat,evenunderphysiological 2004, Grionet al.2007). steroid levelsinbothY1cellsandrats(Pomeraniec HO-1 expression or activity results in significantly higher fact, wehavepreviouslydemonstratedthatinhibitionof cells toavoidanunregulatedsteroidogenicresponse.In generation, heme catabolism etc.), thus allowing the a mechanisminvolvinghemeoxygenaseactivity(CO generation of ROS and (b) inhibits steroidogenesis by the deleteriouseffectsassociatedwithanincreased twopurposes:(a)protectsthecellsfrom could serve that inductionofHO-1byACTHinadrenocorticalcells ACTH addition(Pomeraniec expression isinducedinadrenocorticalcells5–8 Astort http://jme.endocrinology-journals.org DOI: 10.1530/JME-16-0005 Research Induction of HO-1 has been widely recognized as an ACTH is the main regulator of glucocorticoid synthesis ACTH isthemainregulatorofglucocorticoidsynthesis ), several evidences led us to discard its . 2014),severalevidencesledustodiscardits et al Kbp of the HO-1 promoter of the murine gene t al.2004, et e al.2011).AsHO-1 Spiga et . 2004),wehypothesize et al others f

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repetto et al . 2009). and 2 O h after 2 was was . et al

( gene throughacAMP/PKA-dependentmechanism glucagon treatmentledtotheinductionofHmox1 Similar resultswereobtainedinhepaticcells,where stimulation ofHO-1byACTH(Pomeraniec of thecAMP/PKApathwayintranscriptional among themwehavedemonstratedtheparticipation al.2004, et 1986, by theactivationofcAMP/PKApathway(Kimura region. activity ofareporterplasmidcontainingonlytheE1 Finally, 8Br-cAMPtreatmentfailedtoinduceluciferase only 300 inareporterplasmidcarrying stimulation wasconserved the humanHmox1gene(Wright et al.2009). proximal E-boxlocatedwithin theproximalpromoterof This CREsitehasbeenalsoshowntoactinconcertwith a of HO-1byoxidizedphospholipids(Kronke CRE sitewasresponsibleforthePKA-mediatedinduction (between 3.8and4.9 human endothelial cells, a distal promoter fragment upstream theTSS(Immenschuh of HO-1involvesaCRE/AP-1sitelocated−665/654 In rathepatocytes,cGMP/PKG-dependentinduction al.1998a, (Immenschuh et to theoverexpressionofbiliverdinreductaseviaATF-2 the inhibition of protein phosphatases 1 and 2A and has been linked to the stimulation of cGMP/PKG, to cAMP-mediated inductionofHO-1. cells, suggesting the involvement of CREB in the ACTH/ increased HO-1proteinlevelsin8Br-cAMP-stimulatedY1 decreased, while overexpressing CREB (RSV-CREB) a dominant-negativeisoformofCREB(DN-CREB-M1) experimental system,targetingCREBexpressionwith al.2009, et of HO-1bydifferentstimuli(Kronke and bindingtoCREshasbeeninvolvedintheinduction transcriptional level.Moreover, CREBphosphorylation suitable candidateasamediatorfortheACTHeffectat the basaltranscriptionalmachinery. Itwasthereforea p300 linkingtheCRE/CREBcomplextocomponentsof of theco-activatorproteinCBP(CREB-bindingprotein)/ its transcriptional activity and promotes the recruitment binding siteslocatedat−146and −37 upstreamtheTSS. showed thepresenceof only twoputativeCREB induction bycAMP CREB mediatesadrenalHO-1 Immenschuh et al.1998b Published byBioscientifica Ltd. Several effects of ACTH in adrenal cells are mediated Analysis of4 Involvement ofCREsitesintheinductionHO-1 ofCREBonSer133byPKAregulates Phosphorylation Schimmer 1995, bp oftheproximalpromoterthatlacksARE. e al.2013, Park et Gallo-Payet 2016).Asstatedpreviously, kb fromthemurineHO-1 promoter kb upstreamtheTSS)containinga Gallo-Payet & Payet 2003, Downloaded fromBioscientifica.com at09/24/202111:53:44PM ). 2000, e al.2015).Inour Barnett et t al.1998a),whilein et e al.2004). Kravets et 57 . 2003, et al : 2 ). et al. 2004). . 2003). et al Sabban Wright 121 via freeaccess

Journal of Molecular Endocrinology pathway couldmodulatethe transcriptionalstimulation rat skeletalmuscle(Bavieraet al . 2010). described an Epac-dependent PI3Kactivationby cAMP in As analternativemechanism, Bavieraandcoworkers out theinvolvementofPKAonAKTphosphorylation. al. ), 2007 but not by H89, we ruled inhibitors (Bain et this effectwaspreventedbytheadditionofspecificPI3K levels.As leads toanincreaseinAktphosphorylation treatment ofY1cellswitheitherACTHor8Br-cAMPalso expression,that, inadditiontostimulatingHmox1gene Yang. 2013).Inthissense,ourresultsdemonstrated et al . 2012, against oxidativestress(Moet al signaling functionsasacytoprotectivemechanism the proximalpromoter region ofthemurineHmox1gene. induction ofHO-1mediatedbyCREBrecruitmentwithin gene. Thisis,toourknowledge,thefirstreporton promoter regioninthePKA-mediatedinductionofHmox1 results furthersupporttheinvolvementofthisproximal of thepHO-1(−295/+74).LUCreporterplasmid.These activation resultedinasignificantdecreasetheactivity H89) andmolecular(DN-CREB-M1)inhibitionofCREB inhibitor In agreement,bothpharmacological(PKA cAMP-stimulated Y1cells,asdeterminedbyChIPanalysis. of themurineHmox1generecruitedphospho-CREBin promoter (Wright et al.2009). reported by Wright and coworkers in the human HO-1 In particular, thislastsitecontainstheEboxsequence the in theproximalpromoterofratHmox1gene,while These CRE-likesitespresentahighhomologywiththose DOI: 10.1530/JME-16-0005 http://jme.endocrinology-journals.org Research Research Our resultsalsoindicated that thePI3Ksignaling It iswellknownthatactivationofPI3K/AKT Our resultsshowedthatthisproximalpromoterregion 37 site is conserved inallthespeciesanalyzed. −37 siteisconserved others f

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repetto and PI3K-dependent cascade(Fig. 6 ). crosstalk between the cAMP/PKA pathway and the show thatHO-1inductionismodulatedbyafunctional proximal regionoftheHO-1promoter. Our resultsalso and activationofCREBtop-CREBrecruitment a cAMP/PKA pathwaythatleadstothephosphorylation demonstrate thatHO-1inductioninvolvestheclassical stimulation of induces steroidogenesisandtriggersthetranscriptional (Tang et al.2007,Sakamoto2009). of thePI3Kpathwayhasbeenpreviouslydemonstrated in therecruitmentof CBP toNRF2or NFκB upon activation influencing HO-1transcription.Inthissense,anincrease pathway affectsthebindingofCREBco-activators,thus do notdiscardthepossibilitythatactivationofPI3K modulate thebindingofCREBtoHO-1promoter. We PI3K (such as AKT, PKCξ, RSK, SGK, p70S6K etc.) could suggesting thatproteinkinasesactivateddownstreamof role intherecruitmentofpCREBtoHO-1promoter, 8Br-cAMP, thispathwayappearstoplayasignificant by (but significantly)decreasedCREBphosphorylation HO-1 promoter. AlthoughPI3Kinhibition,onlyslightly status ofCREBand/orthebindingp-CREBto that PI3Kactivationcouldaffectthephosphorylation by 8Br-cAMP. Basedontheseresults,wehypothesized activation ofthepHO-1(−295/+74).LUCreporterinduced LY 294002treatmentsinhibitedHO-1inductionandthe of the perceived asprejudicingtheimpartiality oftheresearchreported. The authorsdeclarethatthereis no conflictofinterestthatcouldbe Declaration ofinterest induction bycAMP CREB mediatesadrenalHO-1 Published byBioscientifica Ltd. In summary, bindingof ACTHtotheMC2R Hmox1 gene triggered by cAMP, as wortmannin and Hmox1 gene. Inthepresentwork,we promoter region. to itscognatebindingsiteintheproximal results inthemodulationofbindingpCREB pathway bycAMP, byayetunknownmechanism, gene. Inaddition,stimulationofthePI3K proximal promoterregionofthemurineHmox1 the phosphorylationandbindingofCREBto and theactivationofPKA.This,inturn,triggers receptor), theconsequentgenerationofcAMP G-protein-coupled receptor(Melanocortintype2 ACTH ismediatedbyitsbindingtoamembrane Transcriptional stimulationofHO-1expressionby HO-1 expressioninmurineadrenocorticalcells. Signaling pathwaysinvolvedinACTH-induced Figure 6 Downloaded fromBioscientifica.com at09/24/202111:53:44PM 57 57 : : 2 2 122 122 via freeaccess

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