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56:3

s li and others AG and UAG induce β-casein 56:3 213–225 Research expression

AG and UAG induce β-casein expression via activation of ERK1/2 and AKT pathways

Sunan Li1, Juxiong Liu1, Qingkang Lv1, Chuan Zhang2, Shiyao Xu1, Dongxue Yang1, Bingxu Huang1, Yalong Zeng1, Yingjie Gao1 and Wei Wang1 Correspondence should be addressed 1College of Veterinary Medicine, Jilin University to W Wang 2Department of Endocrinology and Metabolism, The Second Hospital of Jilin University, Email Changchun, China [email protected]

Abstract

The ghrelin peptides were found to circulate in two major forms: acylated ghrelin (AG) Key Words and unacylated ghrelin (UAG). Previous studies showed that AG regulates β-casein (CSN2) ff acylated ghrelin expression in mammary epithelial cells. However, little is known about the mechanisms ff unacylated ghrelin by which AG regulates CSN2 gene and protein expression. Evidence suggests that UAG ff β-casein has biological activity through GHSR1a-independent mechanisms. Here, we investigated ff bovine mammary the possible GHSR1a-mediated effect of UAG on the expression of CSN2 in primary epithelial cells bovine mammary epithelial cells (pbMECs) isolated from lactating cow. We found that both AG and UAG increase the expression of CSN2 in a dose-dependent manner in pbMECs in comparison with the control group. Increased expression of CSN2 was blocked by [D-Lys3]-GHRP-6 (an antagonist of the GHSR1a) and NF449 (a Gs-α subunit inhibitor)

Journal of Molecular Endocrinology in pbMECs. In addition, both AG and UAG activated AKT/protein kinase B (AKT) and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways, whereas [D-Lys3]-GHRP-6 and NF449 inhibited the phosphorylation of AKT and ERK1/2 in pbMECs respectively. Blockade of ERK1/2 and AKT signaling pathways prevented the expression of CSN2 induced by AG or UAG. Finally, we found that both AG and UAG cause cell proliferation through identical signaling pathways. Taken together, these results demonstrate that both AG and UAG act on ERK1/2 and AKT signaling pathways to facilitate the expression of CSN2 Journal of Molecular in a GHSR1a-dependent manner. Endocrinology (2016) 56, 213–225

Introduction

Acylated (AG) and unacylated ghrelin (UAG) are secretagogue receptor 1a (GHSR1a) (Howard et al. 1996, circulating peptides encoded by the preproghrelin Barnett et al. 2010, Muller et al. 2015). The presence of gene peptides and are mainly secreted by the X/A-like an acyl side chain (mainly n-octanoic acid) attached to enteroendocrine cells of the stomach (Kojima et al. 1999, the AG peptide is required for full agonism of GHSR1a Muller et al. 2015). AG stimulates growth hormone (GH) (Barnett et al. 2010, Heppner et al. 2014, Kern et al. 2014). release and positive energy balance by binding in the In addition to the gut, AG and GHSR1a were hypothalamus and pituitary gland to the only functional expressed in mammary glands and cultured primary ghrelin receptor that has been characterized thus far, GH mammary epithelial cells (MECs) (Zhang et al. 2013,

http://jme.endocrinology-journals.org © 2016 Society for Endocrinology Published by Bioscientifica Ltd. DOI: 10.1530/JME-15-0287 Printed in Great Britain Downloaded from Bioscientifica.com at 09/24/2021 04:48:20PM via free access

10.1530/JME-15-0287 Journal of Molecular Endocrinology involving G-protein-dependent pathways,highlighting AG haveidentifedseveralsignalingmechanisms (G alpha subunits (G Gi alphasubunit(G classified intofourfamiliesaccordingtotheir subunits termedalpha,beta,andgamma.G-proteinsare associated withagroupofG-proteinsconsistingthree the majorclassesofcellsurface receptors andare CSN2 isnotclear. a similarpathwaytoAGregulatetheexpression of yet tobeidentifed.Moreover, whetherUAGcanactivate role ofUAGintheexpressionCSN2pbMECshas et al but fullagonismofbindingbyUAGtoGHSR1a( 2007 the effects of AG are mimicked by UAG ( of CSN2remainspoorlyunderstood.Inseveralstudies, to AG,theregulatingactionofUAGinexpression 2008 modifed anddes-acylAG( metabolism thatdidnotdistinguishbetweenacyl- regulation ofAGsecretioninthecontextenergy have notbeenexamined. epithelialcells(pbMECs) bovinemammary in primary effects andmechanismsofAGinmilkproteinsynthesis with control group ( in MECsoflactatinggoataftertreatedwithAGcompared colleagues showedthatlevelsofCSN2proteinincreased expression in rats ( gland AG significantly increased mammary Northern blotanalysisrevealedthatdailyinjectionof & Dovc2015 goat primary mammary cells( mammary goat primary cell lines ( mammary conditions indifferentspecies,suchasseveralbovine was previouslyshowntobeexpressedunder differentiation.CSN2 used asamarkerformammary the most abundant protein in cow milk and is often in waterbuffaloes( al et cows( and milkcomponentsinratsdairy Fukumori intake, and glucose concentrations ( lactation. Inadditiontoincreasingplasmainsulin, food peptides is of interest. AG has a specificactionduring Therefore, understandingtheregulatingactionsofthese Jedrzejczak &Szatkowska2014 DOI: 10.1530/JME-15-0287 http://jme.endocrinology-journals.org Research α q). Attempts to elucidate the peripheral effects of G-protein-coupled receptors (GPCRs)are one of A numberofreportshavebeenpublishedonthe . 2007 . 2003 , , Reano Delhanty , t al et ) andaffectsmilkfatproteinsynthesis Heppner e al et ), and mammary glandexplantsfromcows. ), andmammary . 2013 t al et .

2014 α α Gil Nakahara i), Gsalphasubunit(G et al . 2014 ), AGalsoaffectsmilkproduction 12/13), and Gq alpha subunit Zhang Jedrzejczak & Szatkowska 2014 ). Laterstudiesrevealedaweak t al et . 2014 , . 2013 ThidarMyint &Kuwayama Pinkney 2014 t al et Zhang , t al et ). However, thepotential s Ogorevc &Dovc2015

li . 2013 © 2016SocietyforEndocrinology andothers ). . 2003 et al β Itoh -casein (CSN2)is ). However, the . 2013 Printed inGreatBritain ). Zhang and Granata e al et ). Incontrast α CSN2 s), G12/G13 α , Nakahara subunit: Ogorevc . 2006 in vitro mRNA Gauna et al ), ), ). , .

pancreatic the ERK1/2andAKTpathwaysindifferentcells,suchas cell proliferation and prevent apoptosis via activation of physiological responses.BothAGandUAGstimulate diverse signalingmechanismandsubsequentlydistinct tissues andcells,activationofGHSR1amaytriggera Kern the complexityofGHSR1aactivation( lactating cows.Furthermore,thesignalpathwaysthatare AG and UAG on the expression of CSN2 in pbMECs of GHSR1a inpbMECsoflactatingcowsisnotknown. AKT pathwaysandinducetheexpressionofCSN2via However, whetherAGandUAGactivatetheERK1/2 activated inresponsetomilkyield( and ERK1/2pathways.Moreover, theAKTpathwayis can beaffectedbyIGF-IsignalingactivatingtheAKT 2011 Yu were isolatedusingaprocedure describedpreviously (d 150–200 after parturition) from healthy Holstein cows PbMECs from the alveolar tissue of four mid-lactation PbMECs cultures andtreatments MK2206 (AKT)wasfromSelleckchem(Houston,DE,USA). Pharma Inc.(Tokyo, Japan).Theselective inhibitorof inhibitor ofYM-254890(Gq11)wasprovidedbyAstellas obtained fromTOCRIS(Ellisville,MO,USA).Theselective pertussis toxin(PTX,G Biotechnology. Theselectiveinhibitorsof NF449 (G anti- IgG-HRP, rabbit anti-cytokeratin 18 (CK-18), and rabbit obtained fromSigma.Theantibodiesforgoatanti-rabbit tor cocktail2,proteaseinhibitorcocktail,andDAPIwere insulin, prolactin,hydrocortisone,phosphataseinhibi GHSR1a, andCSN2werepurchased fromAbcam.Bovine from CellSignalingTechnology Inc.AntibodiestoAG, Tyr204), AKT(Ser473),andP-AKT(Ser473)werepurchased bodies toP-ERK1/2(Thr202/Tyr204), ERK1/2 (Thr202/ Phoenix Pharmaceuticals,Inc.(Belmont,CA,USA).Anti Octanoyl), and[D-Lys3]-GHRP-6 wereobtainedfrom Human AG(Ser3-Decanoyl),UAG(Ser3-Des- Reagents Materials andmethods and UAGwerealsoinvestigated. involved intheexpressionofCSN2inducedbybothAG expression AG andUAGinduce Published byBioscientifica Ltd. et al The aimofthisstudywasto evaluate theeffectsof β ). Inaddition,bothpbMECnumberandactivity -tubulin (1–19)werepurchased fromSantaCruz t al et . 2014 . 2014 β -cells, skeletalmusclecells( ), andhumanendothelialcells( , β Yin -casein t al et α Downloaded fromBioscientifica.com at09/24/202104:48:20PM i), andU0126(ERK1/2)were . 2014 ). Dependingonthe Murney 56 Evron Reano : 3 et al et al Xiang et al . 2015 . 2014 . 2014 214 et al α s), via freeaccess ). - - , . ,

Journal of Molecular Endocrinology then treatedfordifferenttimeperiodswithAGorUAG. the cellswerepre-incubatedwithinhibitorsfor1 until mRNAorproteinextraction.Forsomeexperiments, or 24 of AGorUAG(0,0.01,0.1,1,10,and100 pbMECs frompassages1–4. detected byquantitativeRT-PCR andwesternblotin expression ofCSN2,ghrelin(GHRL),andGHSR1awere immunofluorescence andflowcytometry. Thegene CK-18 wasidentifiedasamarkerofpbMECsbyboth (1 (100 fetal bovineserum,penicillin(100 were maintainedinDMEM-F12,supplementedwith10% ( e al.2012 Sorg et http://jme.endocrinology-journals.org DOI: 10.1530/JME-15-0287 Research

μg/mL), andprolactin(1 The cellsweretreatedwithincreasingconcentrations

mg/mL), bovine insulin (5

h. Thecellswerethencollectedandfrozenat−80°C , Jedrzejczak &Szatkowska2014

μg/mL). Theexpressionof

s μg/mL), hydrocortisone

µ/mL), streptomycin Printed inGreatBritain

ng/mL) for12 ). Cells

h, and 4 from pbMECsandreversedtranscribedintocDNA Totalods, thepbMECswereharvested. RNAwasextracted After treatment with AG or UAG for different time peri- Quantitative RT-PCR SYBR GreenQuantiTect RT-PCR Kit.Thecomparative2 mRNA levelsofvariousgenes were evaluatedusingthe TTCCAGCTCGTC-3′; CSN2, 5′-GGCTATGGCTCCTAAGC 5′-ATGCCCGCCCCGTGGACCAT-3′ and5-AACCGGAT GACT-3′ and5-CAAGTTCCAGGGCCGGTAAT-3′; GHRL, primers wereused:GHSR1a,5′-CCGACAATGACTCGCT via 1.5%agarosegelelectrophoresis.Thefollowing the housekeeping gene. The PCR products were visualiz levels of CSN2, method wasusedtocalculatetherelativegeneexpression expression AG andUAGinduceβ

Published byBioscientifica Ltd. μg totalRNA,asdescribedpreviously( GHRL, and JME-15-0287. is availableathttp://dx.doi.org/10.1530/ (passages 1–4).Afullcolourversion of thisfigure expression ofCSN2,AG,andGHSR1a inpbMECs cultured pbMECs(passages1–4).(E) Protein Gene expressionofCNS2,GHSR1aand GHRL in of CK-18inpbMECs(200×magnification).(D) flow cytometry. (C)Immunofluorescencelabeling expression ofCK-18inpbMECswasstudiedby epithelial cellsarevisible(bar Morphology ofculturedpbMECs.Islands Cultured anddetectionofpbMECs.(A) Figure 1 -casein Downloaded fromBioscientifica.com at09/24/202104:48:20PM GHSR1a, with 56 Li : -actin serving as β-actin serving 3 et al. 2013

1000 μm).(B)The ). The 215 −ΔΔct ed via freeaccess

Journal of Molecular Endocrinology for 2 10 Slides wereﬁxedwith4% paraformaldehyde in PBSfor cells/well wereseededinaslide in24-wellplatesfor24 h. To evaluate the expression of CK-18 in pbMECs, 1 Immunofluorescence assay with AGorUAG. incubated withinhibitorsfor1 of threetimes.Forsomeexperiments,thecellswerepre- treatment, and the experiments were repeated a minimum control. Eachexperimentincludedfvereplicatesper expressed asthepercentage relativetotheuntreated quantifed bymeasuringtheabsorbanceat570 for 10 4 h andthenincubatedwith150 μL ofdimethylsulfoxide cells wereincubatedwith1 mg/mL MTTforapproximately and 100 ng/mL) for24,48,and72 h. Aftertreatment,the cells/well andtreatedwithAGorUAG(0,0.01,0.1,1,10, Cells wereseededin96-wellplatesatadensityof5 Cell viabilityassay visualized usingtheECLdetectionsystem(Millipore). temperature (RT).Theimmunoreactiveproteinswere atroom incubated withgoatanti-rabbitIgG-HRPfor1 h membranes werewashedfourtimeswith0.1%TBS-Tand CSN2 (1:1000),AG(1:500),or (1:2000), AKTP-AKT(1:1000),GHSR1a(1:4000), with thespecifcantibodiestoP-ERK1/2(1:1000),ERK1/2 milk, themembraneswereincubatedovernightat4°C Millipore, Billerica,MA,USA).Afterblockingwith5% transferred toPVDFmembrane(ChemiconInternational, samples (40 cocktail 2,andproteaseinhibitorcocktail.Protein KCl; 0.1 lysis buffercontaining10 usingRIPAwere washedwithice-coldPBSandharvested with ourpublishedprotocol( periods, whole-cellproteinswereextractedinaccordance After treatmentwithAGorUAGfordifferenttime Western blotanalysis CAGGGACAGCACA-3 β ACA-3 -actin, 5 DOI: 10.1530/JME-15-0287 http://jme.endocrinology-journals.org Research min atRT. Afterblockingwith3%normal goatserum h atRT, theslideswereincubatedwith primary ′ and5 min. Theopticaldensityofthesampleswas mM EGTA; EDTA; 0.1 mM phosphataseinhibitor ′ -TCACCAACTGGGACGACA-3 μg) weresubjectedto12%SDS–PAGE and ′ -GAGAAAGGGACAGCACGGAC-3 ′ . mM HEPES,pH7.5;10 h andthentreatedfor24 h Li β s -tubulin (1:10,000).The

nm and -GCATA ′ ; and × × mM 10 10 4 4

1 ×10 the cellswereseededinto6-wellplatesatadensityof To evaluatethepopulationofCK-18-expressingpbMECs, Flow cytometryassay fluorescence microscope. 10 min. Afterstaining,slideswereviewedwithaninverted to stainthecellnucleiataconcentrationof1.43 goat anti-rabbitIgG(1:500)for1 washing, theslideswereincubatedwithPE-conjugated antibodies toCK-18(1:200)overnightat4°C.After experiments; * (C and D) Thedataarepresentedas themean UAG. ProteinexpressionofCSN2was examinedbywesternblot. (B) Cellswereincubatedfor24hwith increasingconcentrationsofAGor UAG. Geneexpressionof (A) Cellswereincubatedfor12hwith increasingconcentrationsofAGor AG andUAGinduceCSN2geneproteinexpressioninpbMECs. Figure 2 expression AG andUAGinduce Published byBioscientifica Ltd. 6 cells/well.After24 P .5 ** <0.05, β P CSN2 -casein

< 0.01 vsnon-treated(NT)controlgroup). wasdeterminedbyquantitativeRT-PCR. Downloaded fromBioscientifica.com at09/24/202104:48:20PM h, thecellswereaspiratedand h atRT. DAPIwasused ± s . 56 d . ( : n 3 independent >3 µM for 216 via freeaccess Journal of Molecular Endocrinology 99% CK-18expressionwereusedinthesubsequent the purityofpbMECs.Cellsfromapopulationwith expression ofCK-18beforeeachexperimenttoensure We AG andUAGpromote the expression ofCSN2inpbMECs Results statistically signifcant. as themean with similarresultsatleastthreetimes.Dataarepresented Dunnett’s test.Ineachpanel,experimentswererepeated among groups was carriedwithanANOVA followedby InStat Software,SanDiego,CA,USA).Thecomparison The datawereanalyzedusingGraphPadPrism5(GraphPad Statistical analysis used todeterminethepopulationofCK-18 (BDBiosciences,USA)was after washing.Flowcytometry incubated withdonkeyanti-rabbitIgG-FITCfor1 stained withCK-18(1:500)for16 washed withPBStwice.Afterblocking,thecellswere http://jme.endocrinology-journals.org DOI: 10.1530/JME-15-0287 Research frst detected the morphology ( ± s . d . A P- value of0.05or0.01wasconsidered s

h at4°C.Thecellswere li © 2016SocietyforEndocrinology andothers Fig. 1 Printed inGreatBritain + cells. A) and the h atRT higher in these pbMECs treated with AG (1–100 that theexpressionofCSN2proteinwassignifcantly of eitherAGorUAGinpbMECs.Theresultsshowedthat treatment for12or24 gene andproteinexpressionofCSN2weredetectedafter AG ontheexpressionofCSN2.To testthishypothesis,the with UAG has similar but maybe less potent effects than al et a weakeragonistofGHSR1acomparedwithAG( of CSN2inpbMECsisnotknown.Inaddition,UAG 2013 goats( mRNA expressioninMECsfromdairy pbMECs atpassages1–4( of CSN2,AG,andGHSR1awerealsostablyexpressedin passage 1 and passage 2( at passages1–4,althoughthegenelevelwaslower that genesof pbMECs frompassage1to4.Theresultsshowed expression ofCSN2,AG,andGHSR1aincultured experiments ( expression ( 10 compared withthenon-treated(NT)controlgroup,only expression AG andUAGinduce Published byBioscientifica Ltd. ng/mL AGorUAGsignifcantlyincreased Previous studiesshowedthatAGincreases . 2007 ). However, whetherAGinfluencestheexpression ). Therefore,wehypothesizedthattreatment Fig. 2 CSN2 Fig. 1 A, BandC).Western blotresultsshowed β treatment). # mean ± (E, F, G,andH)Thedataarepresentedasthe quantitative RT-PCR and(CD)westernblot. expression ofCSN2wasexaminedby (AandB) or 24hwithAGUAG.Thegeneandprotein GHRP-6 (100 cells werepre-treatedfor1hwith[D-Lys3]- a GHSR1a-dependentmannerinpbMECs.The AG andUAGincreasedtheexpressionofCSN2in Figure 3 , P -casein

B andC).We nextdetectedthe < GHRL 0.01 vsNT; * h withincreasingconcentrations Downloaded fromBioscientifica.com at09/24/202104:48:20PM s Fig. 1 . Fig. 1 d . ( , and n μg/mL) andthenincubatedfor12 idpnet experiments; independent >3 D). Moreover, the proteins E). P .5 ** <0.05, GHSR1a 56 P : 3

wereexpressed < 0.01 vsAGorUAG Zhang CSN2 ng/mL) Gauna gene 217 et al CSN2 via freeaccess .

Journal of Molecular Endocrinology GHSR1a-dependent mechanism( regulate peripheralglucosemetabolismthrougha Previous research reported thatbothAGandUAG expression ofCSN2inpbMECs GHSR1a andG for AGandUAGinsubsequentexperiments. results, 10 data givenattheendofthisarticle).Consideringabove ( signi Our resultsshowedthatbothAGandUAGhaveno UAG influencestheexpressionofGHSR1ainpbMECs. and D).Inaddition,wealsoinvestigatedwhetherAG ( expression ofCSN2wereobserved mL and100 ( and peakedat10 Supplementary Figure1 Supplementary P DOI: 10.1530/JME-15-0287 http://jme.endocrinology-journals.org

Research <

0.01; f cant effectonGHSR1ageneandproteinexpression Fig. 2

ng/mL was selectedas the best concentration

ng/mL, signi α B, C and D). For UAG treatment at 10 s are required forAG-andUAG-induced

ng/mL andthendecreasedat100 f , seesectiononsupplementary cantly highervaluesofprotein s

0.01; Printed inGreatBritain t al et i. 2 Fig. . 2014

ng/mL B, C

ng/ ). intracellular secondmessengerscoupledtoG expression ofCSN2comparedwiththeNTcontrolgroup. [D-Lys3]-GHRP-6 alonehadnosigni ( level ofCSN2wasalsosuppressedby[D-Lys3]-GHRP-6 ( gene levelof ( of CSN2inpbMECscomparedwiththeNTcontrolgroup UAG signi or UAGfor24 [D-Lys3]-GHRP-6 (100 GHSR1a activation, pbMECs were pre-incubated with AG and UAG on the expression of CSN2 depends on Zhang dependency ofGHSR1asignaling( antagonist andhasbeenwidelyusedtoinvestigatethe [D-Lys3]-GHRP-6 is regarded as a highly selective GHSR1a expression AG andUAGinduce P P Fig. 3 Published byBioscientifica Ltd.

< <

0.01; 0.01; AG bindingtoGHSR1ahasbeenshownactivate A, B, C, D, E, F, G, and H). However, the increased t al et Fig. 3 Fig. 3 f cantly increasedgeneandproteinexpression . 2013 C, D, G, and H). However, stimulation with A, B,E,andF).Moreover, increasedprotein CSN2

h. TheresultsshowedthatbothAGand ). To determinewhethertheeffectof β treatment). # mean (E, F, G,andH)Thedataarepresentedasthe quantitative RT-PCR and(C and D)westernblot. expression ofCSN2wasexaminedby (AandB) with AGorUAG.Thegeneandprotein NF449 (10μM)andthenincubatedfor12or24h pbMECs. Thecellswerepre-treatedfor1hwith AG- andUAG-inducedexpressionofCSN2in G Figure 4 P -casein wasinhibitedby[D-Lys3]-GHRP-6 α

< s iscoupledtoGHSRandrequiredfor

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μg/mL) for1

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s . d . ( n

idpnet experiments; independent 3 P

.5 ** 0.05,

h, followedwithAG Granata f 56 cant effectsonthe P : 3

0.01 vsAGorUAG t al et α s, G . 2007 218 α i/o, via freeaccess , Journal of Molecular Endocrinology or UAGinpbMECsremainsunknown.To investigate subunits coupletoGHSR1amediatetheeffectofAG depending onthecelltype.However, whichG-protein signaling pathways and mediates different effects al et proliferation ( and G http://jme.endocrinology-journals.org DOI: 10.1530/JME-15-0287 Research . 2014 α q/11 andenhancedGHrelease,appetite, ). GHSR1acouplestodifferentintracellular Granata et al . 2007 , s

Evron li © 2016SocietyforEndocrinology andothers et al Printed inGreatBritain . 2014 , Reano AG- andUAG-inducedgeneproteinexpressionof with NF449resultedinacompleteblockadeofboth incubated for12or24 receptor antagonistsrespectively. Next,thecellswere which areselectiveG NF449 (10 this mechanism,thecellswerepre-incubated for 1 expression AG andUAGinduce Published byBioscientifica Ltd. μM), YM254890(10 * ( showing meanofP-ERK1/2andP-AKT levels. G-protein antagonist.(C,D,E,andF) Bargraph inhibited byNF449,aG of ERK1/2andAKTactivatedbyAG or UAGwas and P-AKTantibodies.(AB)Phosphorylation to westernblottingusingERK1/2,P-ERK1/2,AKT, points, celllysateswerepreparedandsubjected for 5,30,and60min.Attheindicatedtime UAG inthepresenceorabsenceofNF449(10μM) pbMECs. CellswereincubatedwitheitherAGor phosphorylation inducedbyAGorUAGin G Figure 6 n β P experiments; P-ERK1/2 andP-AKTlevels.( (C, D,E,andF)Bargraphshowingmeanof abolished upontreatmentwith[D-Lys3]-GHRP-6. phosphorylation ofERK1/2andAKTwere antibodies. (AandB)AG-UAG-induced blotting usingERK1/2,P-ERK1/2,AKT, andP-AKT were preparedandsubjectedtowestern min. Attheindicatedtimepoints,celllysates [D-Lys3]-GHRP-6 (100μg/mL)for5,30,and60 either AGorUAGinthepresenceabsenceof pbMECs. CulturedpbMECswereincubatedwith phosphorylation inGHSR-dependentmanner AG andUAGinduceERK1/2AKT Figure 5 vs AGorUAGtreatment). α idpnet experiments; independent >3 -casein .5 ** <0.05, s isrequiredforERK1/2andAKT α s, Gq/11,andGi/oprotein-coupled h withAGorUAG.Pre-incubation Downloaded fromBioscientifica.com at09/24/202104:48:20PM P

< # 0.01 vsAGorUAGtreatment). P

< μM), andPTX(10 0.01 vsNT; * α s subunit-selective 56 : 3 n independent >3 P .5 ** <0.05, # P .1 s NT; vs <0.01 P h with <0.01 219 μM), via freeaccess Journal of Molecular Endocrinology AKT wassignifcantlyincreasedcomparedwiththeNT ofERK1/2and treatment withAG,thephosphorylation and followedwithAGorUAGfor5,30,60 cells werepre-incubatedwith[D-Lys3]-GHRP-6 for1 activation of ERK1/2 and AKT signaling in pbMECs. The assessed thepotentialroleofGHSR1ainregulating the 2007 of phospho-ERK1/2andphospho-AKT( conditions, GHSR1acouplestoG Recent studieshavedemonstratedthatunderdeﬁned pbMECs viaactivationofGHSR1aandG AG andUAGinduceERK1/2AKTphosphorylationin the expressionofCSN2inpbMECs. likely coupledtoG implied thatactivationofGHSR1abyAGandUAGis CSN2 inpbMECs( AG- andUAG-inducedmRNAproteinexpressionof treatment withPTXandYM-254890hadnoeffectonboth CSN2 ( DOI: 10.1530/JME-15-0287 http://jme.endocrinology-journals.org Research , Evron P <0.01; et al Fig. 4 . 2014 Supplementary Figure2 Supplementary α s, notGq/11andGi/o,promotes A, B, C, D, E, F, G, and H). However, , Kern et al α s

s, resultinginactivation li . 2014 © 2016SocietyforEndocrinology andothers α ). Thus, we next s Printed inGreatBritain ). Theseresults Granata min. After t al et h . In addition, stimulation of pbMECs with UAG leads In addition,stimulationofpbMECswithUAGleads ( by NF449at5,30,and60 min and C),increasedphospho-AKTwasalsosuppressed at30and60 minERK1/2 phosphorylation ( or UAG.Inagreement,NF449alsoinhibitedAG-induced mediate theincreasedexpressionofCSN2inducedbyAG 2014 ERK1/2 andAKTsignaling( receptor, which, in turn, has been shown to activate induced expressionofCSN2involvesaG respectively ( phospho-AKT comparedtowiththeNTcontrolgroup, minforphospho-ERK1/2 and by [D-Lys3]-GHRP-6 at5 of ERK1/2 and AKT induced by UAG was also blocked C, andD).Inaddition, the increasedphosphorylation 60 [D-Lys3]-GHRP-6 at 60 control groupbutsuppressedafterpre-incubationwith which is reduced by NF449 at 5, 30, and 60 min ( which isreducedbyNF449at5,30,and60 min of ERK1/2, to signifcantly increased phosphorylation expression AG andUAGinduce Published byBioscientifica Ltd. min forphospho-AKT, respectively( Our previousresultsshowedthatAG-andUAG- ). Therefore, we investigated whether these pathways ). Therefore,weinvestigatedwhetherthesepathways P < 0.01; β * ( H) Thedataarepresentedasthemean RT-PCR and(CD)westernblot.(E,F, Gand CSN2 wasexaminedby(AandB)quantitative or 24h.Thegeneandproteinexpression of U0126 (10μM),followedbyAGorUAGfor12 pbMECs. Thecellswerepre-treatedfor1hwith through theERK1/2signalingpathwayin AG andUAGinducetheexpressionofCSN2 Figure 7 n -casein P idpnet experiments; independent >3 .5 ** <0.05, Fig. 5 min forphospho-ERK1/2, or 5and Downloaded fromBioscientifica.com at09/24/202104:48:20PM B, E,andF). Granata P

< 0.01 vsAGorUAGtreatment). P < 0.01; < 0.01; t al et 56 α P : s protein-coupled s protein-coupled 3 < 0.01; < 0.01; . 2007 P Fig. 6 < 0.01; # P .1 s NT; vs <0.01 A and D). A and D). , P Yu Yu Fig. 5 < 0.01; < 0.01; ± Fig. 6 s 220 . t al et d . A, via freeaccess A .

Journal of Molecular Endocrinology blockaded the phosphorylation ofAKTinpbMECs blockaded thephosphorylation addition, stimulatedwithMK2206for1 also inhibitedbyU0126inpbMECs( and proteinlevelsofCSN2inducedbyAGUAGwere (datanotshown).Thegene of ERK1/2phosphorylation treatment withU0126resultedinasignifcantblockade of ERK1/2andAKTrespectively. Ourresultsshowedthat with U0126andMK2206,whicharespecifcinhibitors CSN2 inpbMECs,thecellswerepre-incubatedfor1 AKT pathwaysinAG-andUAG-inducedexpressionof To further determine the potential role of ERK1/2 and activating ERK1/2andAKTsignaling AG andUAGstimulatetheexpression ofCSN2by ofERK1/2andAKTinpbMECs. phosphorylation that GHSR1aandG was foundusinginhibitorsalone.Theseresultssuggested decreased at 30 Fig. 6 http://jme.endocrinology-journals.org DOI: 10.1530/JME-15-0287 Research B and E). Moreover, inducedphospho-AKTwasalso E). and B min ( α P s are required for AG- and UAG-induced s arerequiredforAG-andUAG-induced <0.01; Fig. 6 B andF);however, no effect s

but also suppressed apoptosis in pancreatic Both AGandUAGnotonlystimulatedcellproliferation AG andUAGstimulatecellproliferation inpbMECs of ERK1/2andAKTinpbMECs. expression of CSN2 might depend on the phosphorylation these resultsdemonstratedthatAG-andUAG-induced of CSN2 compared with the NT control group. Together, MK2206 alone had no signifcant effects on the expression ( of CSN2wasalsosuppressedbyMK2206inpbMECs (data notshown).Increasedgeneandproteinexpression proliferation wasenhancedfollowingincreasedAG Fig. 9 UAG treatmentonthegrowthofpbMECs.Asshownin the effects of increasing the concentration of AG and et al muscle, andmacrophages( stimulation for24,48,and72 expression AG andUAGinduce P Published byBioscientifica Ltd. <0.01; . 2014 , comparedwiththeNTcontrolgroup,pbMECs i. 8 Fig. , Li et al. ). However, stimulationwithU0126and β * independent experiments; The dataarepresentedasthemean (A andB)westernblot(CD).(E, F, G,andH) examined by(EandF)quantitativeRT-PCR and The geneandproteinexpressionof CSN2 were (10 μM),followedbyAGorUAGfor1224h. The cellswerepre-treatedfor1hwithMK2206 through theAKTsignalingpathwayinpbMECs. AG andUAGinducetheexpressionofCSN2 Figure 8 2015 -casein P .5 ** <0.05, Downloaded fromBioscientifica.com at09/24/202104:48:20PM ). Thus, fnally, weinvestigated P Gauna

< 0.01 vsAGorUAGtreatment). h, withthemaximum t al et 56 : # 3 P .1 s NT; vs <0.01 . 2007 β -cells, skeletal ± s . , d . ( Evron 221 n >3 via freeaccess Journal of Molecular Endocrinology Figure 9 experiments; MK2206. Values are presentedasthemean UAG-induced cellproliferationwereinhibitedbyD-Lys, NF449,U0126,or MK2206, followedbyAGorUAGfor24and48h.(CD)AG- 72 (A andB)AGUAGmarkedlyenhancedcellproliferationat24,48,or without AGorUAG.Then,cellproliferationwasmeasuredinaMTTassay. increasing concentrationsofAGorUAGfor24,48,and72hwith AG andUAGinducepbMECproliferation.PbMECswereculturedwith and UAG-inducedproliferation inpbMECs,thecells there werenocytotoxiceffects onthegrowthofpbMECs. enhanced astheconcentration ofAGandUAG.However, results suggestedthatcell proliferationwasmarkedly above basallevelafter48and72 a dose-dependent manner at 24 addition, UAGsignifcantlyinducedcellproliferationin proliferative responseat100 DOI: 10.1530/JME-15-0287 http://jme.endocrinology-journals.org h. Thecellswerepre-treatedfor1hwithD-Lys, NF449,U0126,or Research To examine whether GHSR1aandG # P

< 0.01 vsNT; * P .5 ** <0.05, ng/mL ( P

< h, and was lower but still h ( 0.01 vsAGorUAGtreatment). ± s

s li . P d © 2016SocietyforEndocrinology andothers . ( <0.05; n P 3 independent independent >3 <0.05; α Printed inGreatBritain s mediateAG- Fig. 9 Fig. 9 B). These A). In Discussion G by AGorUAGwaslikelymediatedthroughaGHSR1aand group. These results showed that cell proliferation induced effect oncellproliferationcomparedwiththeNTcontrol NF449, U0126,andMK2206alonehadnosignifcant and D).Inaddition,stimulationwith[D-Lys3]-GHRP-6, acyl-modifed anddes-acylAGhavebeen published( food intakeincowsthatdidnotdistinguishbetween AG secretion in the context of energy metabolism and n interact withthereceptorinamannersimilartothat of one oftheactiveformsAGpeptideandthatitcould et al whose Ser3residueismodifedby and otheracylatedAGspecies,suchas exist, includingUAG,whichlacksanacylmodifcation, addition to modifcation isessentialforAGbiologicalactivity. In of AG,serine(Ser3),ismodifedbyanacylgroup;this as AG( other hormoneshavebeendiscoveredinbreastmilksuch prolactin, insulin, and hydrocortisone. More recently, hormones, predominantlythelactogenichormones: controlled bythecomplexinterplayofpeptideandsteroid protein synthesis( formilk can besuppliedwiththenutrientsnecessary glands metabolism ofmanyorganssothatthemammary activated byGHSR1aandG lactating cows.TheERK1/2andAKTsignalpathwaythatis gene andproteinexpressionofCSN2inpbMECs Our fndingsshowthatbothAGandUAGincreasethe in pbMECscomparedwithAGgroup( cell proliferationswereblockedbyU0126orMK2206 effect ofU0126andMK2206.BothAG-UAG-induced to AG-andUAG-inducedproliferation,weassessedthe activation oftheERK1/2andAKTpathwaysarerequired group ( proliferation inpbMECscomparedwithAGorUAG or NF449remarkablyreducedAG-UAG-induced results showedthatpre-incubationwith[D-Lys3]-GHRP-6 for 1 were pre-incubatedwith[D-Lys3]-GHRP-6 andNF449 Fukumori 2008 al et expression AG andUAGinduce -octanoyl AG.Anumberofreportsontheregulation Published byBioscientifica Ltd. α s signalcascade. The initiationoflactationdramaticallyaltersthe . 2011 . 2006 , h followedbyincubationwithAGorUAG.The Roche Savino P 0.05; < et al ). Ithasbeenshownthat , n ThidarMyint et al -octanoyl AG,otherformsofAGpeptide . 2013 et al . 2008 Fig. 9 Yu β . 2012 ). Inaddition,theeffectsof -casein e al et , C andD).To elucidatewhether ThidarMyint &Kuwayama2008 Downloaded fromBioscientifica.com at09/24/202104:48:20PM ). Thethirdaminoacidresidue t al et . 2015 α s likelymediatestheseeffects. . 2006 ) . Proteinproductionis n -decanoic acid( , n 56 -decanoyl AGwas Bradford &Allen P : 3 0.05; < n -decanoyl AG, n -octanoyl Fig. 9 222 Itoh Yoh via freeaccess C ,

Journal of Molecular Endocrinology of theGHSR1a,withactivityinhighnanomolarrange The causeofthisresultsmaybethatUAGisafullagonist achieved in10 Furthermore, highest protein expression of CSN2 was there isnosignifcantlyincreasedwhentreatedwithUAG. 1 ng/mL ofAGcomparedwithNTcontrolgroup; however, increased proteinexpressionofCSN2whentreatedwith effects betweenAGandUAG.We signifcantly observed tendency withAG-treatedgroup,therearesomedifferent CSN2 the geneexpressionof time thatUAG,similartoAG,signifcantlystimulated been elucidated.However, hereweshowforthefrst 2014 AG, UAGisnotacylatedatSer3( control group.Previousstudiessuggestedthatunlike concentration rangeof1–100 signifcantly higher in these pbMECs treated with a results showedthattheexpressionofCSN2proteinwas increased thegeneexpressionof pbMECs. We alsofndthatonly10 demonstrated thatAGandGHSR1awereexpressedin goats ( lactation in dairy of theepithelialcellsalveoliandductsthroughout of CSN2inpbMECslactationcows.With theexception our experiment. examined. Thus, and UAGinmilkproteinsynthesis However, theeffectsandmechanismsof detail ( and qualityinlactationcowshavebeendescribed AG onplasmaconcentrationsofglucoseandmilkyield a fullagonistofGHSR1aand inducessimilarmaximal GHSR1a agonist,otherdata suggestthatUAGisactually range. Although some data indicate that UAG is a weak concentrations withinthehigh-nanomolar ormicromolar recent studyreportedthatUAGinteractswithGHSR1a at are independentofGHSR1aactivation.However, amore led totheassumptionthatbiologicaleffectsofUAG low-nanomolar range(Heppner residue toactivateGHSR1aatconcentrations within the have demonstratedthatAGrequiresacylationattheSer3 to AGorUAGinpbMECsisunknown.Cell-basedassays ﬁndings ingoat(Zhang CSN2 intheculturedpbMECs,thisisagreementwith AG andUAGcoulddirectlymodulatetheexpressionof (Gauna http://jme.endocrinology-journals.org DOI: 10.1530/JME-15-0287 Research The speciﬁcreceptormediatingthesignalingresponses We frstevaluatedtheeffectsofAGonexpression ), and its effect on theexpression of CSN2has not mRNAaftertreatmentwithUAGexhibitedasimilar Itoh et al et al . 2007).Inaword,ourresultssuggestedthat ng/mL ofAG,whereas100 . 2006 n -octanoyl AGandUAGwereusedin , CSN2 Roche et al Zhang . 2013). . Althoughtheexpressionof et al ng/mL comparedwithNT et al s t al et

li ng/mL AGsignifcantly . 2008 © 2016SocietyforEndocrinology andothers Delhanty in vitro . 2014).Thisevidence CSN2 . 2013 , ng/mL forUAG. Printed inGreatBritain . Western blot Gil havenotbeen n -octanoyl AG ), our results et al t al et . 2013 . 2013, ). proliferation andglucosemetabolism( receptor stimulationasAG,suchtheregulationofcell et al prolactin-induced synthesis ofmilkproducts( G-protein signalingcascades playanimportantrolein GHRP-6. Moreover, previous research demonstrated that pre-incubated withtheinhibitorofGHSR1a,[D-Lys3]- or AKTinducedbyAGUAGwassuppressedafter 2007 and AKT comparedwith NT controlgroup ( by AGorUAGinducessignifcantactivationofERK1/2 and AKT. Inagreementwithpreviousfndings,stimulation ofERK1/2 signaling, weinvestigatedthephosphorylation tissue-specifc responseinGHSR1a-mediatedintracellular To uncover the molecular mechanism underlying the 2014 of G pbMECs. We fndthatonlyNF449,thespecicinhibitor GHSR1a inAG-andUAG-inducedexpressionofCSN2 have investigatedwhichG-proteinsubunitcouplesto as G which allowsittoactivateanassociatedG-proteinsuch GPCR, aconformationalchangeoccursinthe receptor (GPCR)family. WhenGHSR1abindstothe the expressionofCSN2inpbMECs. suggest thatUAGisanagonistofGHSR1aandregulates our by [D-Lys3]-GHRP-6, aninhibitor ofGHSR1a.Altogether, induced expressionofCSN2weresignifcantlyblocked on GHSR1a.OurresultsshowedthatbothAG-andUAG- of AGorUAGontheexpressionCSN2weredependent Pinkney 2014 types, suchaspancreatic inmanycell activation ofERK1/2andAKTisobserved ( of cellsurvival ERK1/2 andAKTplayimportantrolesintheregulation Evron ERK1/2 andAKT( by severalsignalingpathwaysincludingthereceptors undefned. DownstreamsignalingofGHSR1aismediated 2013 species ( with theexpressionofCSN2 activated GHSR1atoG-proteinsubtypes. not shown).Thisresultrevealedtheselectivecouplingof pbMECs, whereasPTXandYM-254890hadnoeffect(data expression ofCSN2atboththegeneandproteinlevelsin expression AG andUAGinduce Published byBioscientifica Ltd. . 1996 GHSR1a belongs to the class A G-protein-coupled Previous studiesdemonstratedthatAGiscorrelated in vitro α α ). However, ofERK1/2 increasedphosphorylation ), pancreatic ). However, themechanismsofthiscorrelationare s, signifcantlyreduced both AG and UAG-induced s, G et al Gil α , . 2014 resultsareconsistentwiththeliteratureand i/o, or G Larrea et al ). Thus,weinvestigatedwhethertheeffects . 2013 Xiang , β Reano et al -cells, andmacrophages( Gauna β -casein α q/11 ( . 1999 , et al Nakahara Downloaded fromBioscientifica.com at09/24/202104:48:20PM et al e al et β -cell lineHIT-T15 ( . 2011 Evron ). AGbindingtoGHSR1ahas invivo . 2014) . 2007 ). AG-orUAG-induced et al t al et . Itiswellknownthat or , 56 . 2003 in vitro Chung Xiang . 2014 : 3 Li Granata , indifferent Zhang t al et Evron ). Here, we et al et al Howard . 2015 . 2011 . 2013 et al 223 et al et al via freeaccess ). . . , . , Journal of Molecular Endocrinology AKT signalingpathways. ofERK1/2and likely mediatedbythephosphorylation the expressionofCSN2induced byeitherAGorUAGare induced expressionofCSN2 in pbMECsandthatincreased a G activity ( ERK1/2 and AKTpathways regulate pbMEC number and are consistentwithpreviousreportsthatactivationofthe cell proliferation induced by AG or UAG. These results inhibitors U0126andMK2206,respectively, alsosuppress proliferation inpbMECs.Inaddition,theERK1/2andAKT GHRP-6 andNF449abolishesAG-orUAG-inducedcell at 24 pbMECs proliferationinadose-dependentmanner our results also proved that both AG and UAG stimulate be regulatedthroughcellproliferation.Inagreement, that themilkprotein synthesis withintheMECsmay and excretedbyMECsduringlactation.We presume muscle cell( ( proliferation andinhibitapoptosisinpancreatic expression inpbMECs. to G that activation of GHSR1a by AG or UAG may couple ( role in milk protein synthesis in the lactating cows mTOR andSTAT5 signalingpathwaysplayanimportant pathways. Althoughpreviousstudiesindicatethat CSN2 viaactivationoftheERK1/2andAKTsignaling that bothAGandUAGstimulatedtheexpressionof UAG-induced expressionofCSN2inpbMECs,indicating and AKT resulted in a signifcant suppression of AG- and AKT respectively. ThechemicalinhibitionofERK1/2 MK2206, whicharespecifcinhibitorsofERK1/2and by AGorUAG,weexaminedtheeffectofU0126and pathway arerequiredfortheexpressionofCSN2induced AKT ( ofERK1/2and for AG-orUAG-inducedphosphorylation consistent withthereportofGranatathatG is induced byAGorUAGinpbMECs.Thisobservation 1 our resultsshowedthatonlytreatmentwithNF449for signaling pathwaysdependingonthecelltype.Here, al et of AKT, ERK1/2,AMPK,ormTORsignalpathways( coupled toG been showntoactivateintracellularsecondmessengers Granata Kojima DOI: 10.1530/JME-15-0287 http://jme.endocrinology-journals.org h inhibited the phosphorylation ofERK1/2andAKT h inhibitedthephosphorylation Research α Collectively, ourresultsdemonstratethatGHSR1a is It has shown that both AG and UAG promote cell To furtherdeterminewhethertheERK1/2andAKT α . 2011 s-coupled receptorinvolved intheAG-andUAG- Granata s andsignalstoERK1/2AKTincreaseCSN2 h, evenfor48 et al e al et Murney ). GHSR1acouplestodifferentintracellular Yu Yu . 1999 α et al s, G . 2007 et al et al α . 2007 ), here we are the i/o, andG . 2014 . 2015 ), endothelialcells,andskeletal h. Pre-treatmentwith[D-Lys3]- ). ). Milkproteinsaresynthesized ). α q/11, leadingtoactivation s

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