Expression
of
suppressor
of
cytokine
signaling
1
and
3
in
ligature-induced
periodontitis
in
rats
Joa˜
o
Antonio
Chaves
de
Souza
a,
Andressa
Vilas
Boas
Nogueira
a,
Pedro
Paulo
Chaves
de
Souza
c,
Joni
Augusto
Cirelli
a,
Gustavo
Pompermaier
Garlet
b,
Carlos
Rossa
Jr.
a,*
aDepartmentofDiagnosisandSurgery,SchoolofDentistryatAraraquara,UnivEstadualPaulista–UNESP,RuaHumaita,1680-Centro,
Araraquara,SP,14801-903,Brazil
bDepartmentofBiologicalSciences,SchoolofDentistryatBauru,UniversityofSa˜o,Paulo–USP.Al.Octa´vioPinheiroBrisola,9-75,Bauru,SP,
17012-901,Brazil
cDepartmentofPhysiologyandPathology,SchoolofDentistryatAraraquara,UnivEstadualPaulista–UNESP,RuaHumaita,1680-Centro,
Araraquara,SP,14801-903,Brazil
1.
Introduction
Periodontitis is characterised by degradation of connective tissueandalveolar bone, whichoccurs asaconsequence of thehostresponsetothemicrobialchallengeoriginatingfromthe dental biofilm.1 According to the current paradigm, disease
progressionwithactivedegradationofperiodontaltissuesisa consequence of an unbalanced host–microbial interaction.2
Even though tissuedestruction may be induced directly by toxins and products of microbial metabolism, most of the damage is associated with the host immune/inflammatory response elicited by these microorganisms, usually charac-terisedbythepredominanceofpro-insteadof anti-inflammato-rycytokines.3,4Therefore,thecontrolofinflammatorymediators
by endogenous mechanisms and the balance between pro-inflammatorycytokines andtheirantagonistswill ultimately determinetheseverityandextentoftissuedestruction.5,6
a
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t
i
c
l
e
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n
f
o
Articlehistory:
Accepted25March2011
Keywords:
Signaltransduction STATtranscriptionfactors Januskinases
Suppressorofcytokinesignaling proteins
Periodontaldisease
a
b
s
t
r
a
c
t
Objective: :Evaluate expressionof inducible negative regulators of JAK/STATpathway andtheir targetproteinsduringthecourseofligature-inducedexperimentalperiodontaldiseaseinrats.
Design: :Ratsweresacrificed07,15and30days afterdiseaseinductionforhistological evaluationofperiodontalinflammationandmacroscopicanalysisofalveolarboneloss. SOCSexpressionandtheactivationstatusofSTAT1andSTAT3wereevaluatedingingival biopsiesbyrealtimePCRandWesternblot.
Results: :Ligature-induced modelpresented significantprogressivebone lossfrom 7to 30days.Inflammationwasevidentandsimilarfor07and15days;however,adecreaseon severityat the endofthe experimental period was observed.There was a significant (p<0.05)increaseonSOCS1andSOCS3geneexpressioninPDcomparedtocontrolgroup at15and30days.TheSOCS1andSOCS3proteinexpressionandactivationofSTAT1and STAT3wereincreasedinearlierperiodsintheligaturemodel.
Conclusion: : This study suggeststhat SOCS1 andSOCS3 were directlycorrelated with regulatorymechanismoftheinflammatoryprocessresponsiblefortheperiodontaldisease destruction.
# 2011ElsevierLtd.Allrightsreserved.
*Correspondingauthor.Tel.:+551633016509;fax:+551633016433. E-mailaddress:crossajr@foar.unesp.br(C.RossaJr.).
a v a i l a b l e a t ww w . sci e n ce di r e ct . com
journalhomepage:http://www.elsevier.com/locate/aob
Manycytokinesthatparticipateonperiodontaldestruction such as interleukins and interferons signal through Janus Kinase/SignalTransducerandActivatorofTranscription(JAK/ STAT)pathway.Theactivationofthispathwayisessentialfor the signaling ofcytokines and other stimuli that regulates inflammatorygeneexpression.Thebindingofthecytokineto its specific receptor activates the associated JAK, which phosphorylates the cytoplasmic domain of the receptor to allowtherecruitmentandtyrosinephosphorylationofSTAT. ActivatedSTATsdimeriseandtranslocatetothenucleus,where theyworkastranscriptionfactorstoregulategeneexpression.7
Inflammatorycytokinegeneexpressionisaprocessstrictly regulated by various mechanisms, including the negative regulationofintracellularsignaling.Endogenousproteinsare involvedinthisprocess,butthemechanismsbywhichthese proteinsregulategeneexpressionarestillelusive,especially inperiodontaldisease.TheSuppressorofCytokineSignaling (SOCS)familyofproteinsmodulatesinafairlyspecificmanner theJAK/STATpathway,whichiscriticalinsignaltransduction ininflammation.8,9
TheSOCSfamilyconsistsofeightproteins(SOCS1to7,and cytokine-inducibleSH2-domain-containingprotein)thatcanbe inducedinresponsetoawiderangeofcytokineswithpro-and anti-inflammatoryactivities.Theyinterferewithsignalingfrom theinducingcytokineinaclassicnegativefeedbackloopandalso regulatesignalingdownstreamofothercytokinesinacross-talk manner.10Whilethemechanismsofcytokinesignalingcontrol
inperiodontaldiseaseremainelusive,SOCS1and3areexpressed in established periodontal lesions.11 SOCS1 and SOCS3 are
inducedbycytokinesthatsignalthroughJAK/STATpathway, including TNF-a, IFN-g, IL-6, and IL-10 and function and endogenousinhibitorsoftheactivationofJAK/STAT,reducing thecellulareffectsofthesecytokinesandalsoinhibitingtheir expression.8,12,13 Therefore, SOCS1and 3aresupposed to be
involvedinthenegativeregulationofinflammatorynetworks relevantintheperiodontaldiseasespathogenesis.11
However, while in vivo studies showed increased SOCS (particularly of SOCS1 and SOCS3) mRNA expression in periodontallesions the kineticsof SOCS expression during the course of experimental periodontal disease remains unknown.Thus,abetterunderstandingofnegativeregulatory mechanisms of JAK/STAT pathway during inflammatory responsemayleadtoimportantinformationonperiodontal diseasepathogenesisandalsoprovideatherapeutic perspec-tive based on the modulation of pro-inflammatory gene expression.ThisstudyevaluatedthekineticsofSOCS1and SOCS3expressioninligature-induced modelofperiodontal diseaseinrats.WealsoevaluatedthemRNAexpressionof TNF-a,IL-6andIL-10thataredirecttargetsofSOCSproteins and the mRNA expression of RANKL, OPG and that were showntoberelevantforpathogenesisofperiodontaldisease andmaybeindirecttargetsofSOCSproteins.
2.
Methods
and
materials
2.1. Experimentaldesign
Male adult Wistar (Norvegicus albinus) rats (N=36) were obtained from the Multidisciplinary Center for Biological
Investigation (CEMIB-UNICAMP).Theanimals,weighing ap-proximately250geach,weremaintainedwithfoodandwater ad libitum.Theexperimentalprotocolwas approvedbythe Ethical Committee on Animal Experimentation (protocol number 23/2007)oftheSchoolofDentistryat Araraquara– UNESPandperformedinaccordancewiththeguidelinesfrom the BrazilianCollege for Animal Experimentation (COBEA). General anesthesia was induced with intramuscular injec-tions ofketamineand xylazinechloridrate at 0.08mL/100g bodyweightand0.04mL/100gbodyweight,respectively.The animalsweredividedintotwoexperimentalgroups:A– sham-operatedgroup(n=9) –animalswereanaesthetisedbut no ligatureswereplacedonthelowermolarsB–experimental group(n=27)–acottonthreadligaturewasplacedaroundthe cervical area ofthe lower firstmolars bilaterallytoinduce experimentalperiodontaldisease.
After7,15,and30daysoftheligatureplacement(baseline), 3 animals fromthe controlgroup and 9 animals fromthe experimentalgroupweresacrificedperperiodbyanesthetic overdose.Themandibularjawswerehemisected,andhalfof the blocksamplesincludingmolarswiththeir surrounding tissuesweresubmittedtoroutinehistologicalprocessingtobe used in the stereometric evaluation. Theother halfof the blockshadthegingivaltissuearoundthefirstmolarscarefully dissectedforextractionoftotalRNAandproteinforRT-qPCR and western blot analysis. Afterdissection of the gingival tissues,thesampleswereimmersedin3%hydrogenperoxide for24htoremoveremainingsofttissues.Subsequently,these samples were stored in 70% ethanol and used for the macroscopicassessmentofboneresorption.
2.2. Macroscopicanalysisofboneresorption
Theareaofboneresorptioninthelingualsurfaceofthefirst molars was measured macroscopically. Briefly, the pieces wereremovedfromalcohol,dried,immersed for5minina solutioncontaining0.7g/Lofmethyleneblueandwashedwith tapwatertoremovetheexcessdye.
Digitalimagesofthelingualsurfacesofstainedfirstmolars wereobtainedatastandardised908anglewiththemid-lingual aspectofthefirstmolarswithastereomicroscope(LeicaMZ6– 20magnification).Thetotalareaofexposedroot(CEJ-bone crest) surfaces stained in blue (the crown enamel is not stained)ontheimageswasmeasuredbyanexaminerblindto experimental groups using software Image tool 3.0. An increase on the area of exposed roots in comparison to control,non-ligated,teethindicatesalveolarboneresorption.
2.3. Stereometry
32400mm2gridwith9
4squaresof30mmwasconstructed usinganimagemanaging/editorsoftware(AdobePhotoshop CS5)andoverlayedonthedigitalimagesobtainedfromthe histologicalsections.Theregionofinterestfortheanalysis wasrepresentedbythewholegrid,whichwaspositionedina submarginalarea ofthebuccalandlingual surfaces, repre-sentingtheconnectivetissuesubjacenttothegingivalsulcus (the apical border of the junctional epithelium and tooth structurewereusedasupper andlateral limitsofthegrid, respectively).Asingleexaminer,whowaspreviouslytrained andcalibrated(datanotshown)andblindtothepurposeofthe experiment, performed the stereometric analysis using a point-countingtechnique.Thefollowingstructuresobserved on each intersection point of the grid were recorded: fibroblastic cells, extracellular matrix, vascular structures andinflammatorycells.Thisprocedureallowsthe quantita-tiveassessmentofinflammatoryreactioninthevicinityofthe aggression.Foreachspecimen,thevaluesobtainedfromthe measurements from each surfaces were combined and averages and standard deviations were calculated. The presenceofeachstructurewasexpressedasapercentageof thetotalareaanalyzedinaccordancewithOdzeetal.14
2.4. Reversetranscription-real-timePCR
TotalRNAwasextractedfromtissuesamplesusingRNAqueous 4PCRkit,accordingtothemanufacturer’sprotocol(Ambion). Thequantityandpurityoftotal RNAweredeterminedona Biomate3(ThermoElectronCorporation)spectrophotometer byevaluatingtheabsorbanceat260nmandthe260/280nm ratio,respectively.TheintegrityoftotalRNAwasconfirmedby electrophoresis of0.5mg oftotalRNA in1% formaldehyde– agarosegels,followedbyvisualisationofthebands correspond-ingto18Sand28SribosomalRNAintheappropriateratio(1:2) underUVtransillumination.ComplementaryDNAwas synthe-sised byreverse transcription of400ngoftotal RNAin the presenceofOligo(dT), reversetranscriptase enzime,MgCl2, dNTPs, RNAse inhibitor, according to the manufacturer’s protocol (Applied Biosystems). Real-time polymerase chain reaction(qPCR)wasperformedusingaStepOnethermocycler (Applied Biosystems). The reactionincluded 1mLof the RT reactionproductina20mLtotalvolumePCRreactionmixthat included:8mLofnuclease-freewater,10mLofTaqManqPCR master mix and 1mL of TaqMan gene expression assays, includingforward,reverseprimersandfluorophore-conjugated probe (Applied Biosystems)for rat genes (seeTable 1). The cyclingconditions usedfor all primers werepre-optimised:
508Cfor2minand40cyclesof:958Cfor15sand608Cfor1min. Thedeterminationoftherelativelevelsofgeneexpressionwas performedusingthecyclethresholdmethodandnormalisedto thehousekeepinggeneGAPDH.Resultsarerepresentedasthe mean mRNAexpressionfromduplicatemeasurements nor-malised by internal control GAPDH and expressed as fold changeoverthelevelsdeterminedincDNAsamplesprepared fromhealthy(non-ligated)controlgingivaltissues.
2.5. Westernblot
Activation of STAT1 and STAT3 as well as the global expressionofSOCS1andSOCS3wasassessedusingsamples oftotalproteinextractedfromgingivaltissuescollectedfrom ratssacrificedinthedifferentexperimentalperiods(7,15and 30daysafterligatureplacement).Adetergent-based extrac-tionbuffer(T-PER,TissueProteinExtractionReagent–Pierce) containingaproteaseinhibitorcocktail(ProteinStabilizing Cocktail–SantaCruz Biotechnology)wasused forprotein extraction.Thetissuesamplesweremaceratedin30mLof ice-coldbuffer,centrifugedfor5minat13,000RPMat48Candthe supernatant wascollected.Concentrationoftotal proteins wasdeterminedwithaBradford-basedassay(Bio-RadLab.) and 30mg oftotal protein were added to a sample buffer containing 2% SDS, 10mM of DTT as a reducing agent, glycerolandbromophenolbluedye(CellSignaling), heated-denaturated at978C for5minandchilled oniceof 5min beforeloadingon10%SDS–polyacrylamidegels. Electropho-resis on discontinuousacrylamidegels was carriedout at constant100Vfor90minandsubsequentlyelectrotransfered to0.4mmnitrocellulosemembranesusinga300mAconstant currentfor1h.Themembraneswereblockedfor1hin Tris-buffered saline containing 5% non-fat dry milk and 0.1% Tween-20andsubsequentlywashedfor10min(threetimes) withTBS–0.1%Tween-20.Themembraneswerethen incu-batedwithpre-optimiseddilutionsoftheprimaryantibodies overnight at 48C with mild agitation. Membranes were washed in TBS-T buffer three times for 10min each and incubated with secondaryantibodies conjugated to horse-radishperoxidase(1:5000dilutionintheblockingbuffer)for 1hatroomtemperatureandwashedagainthreetimesfor 10minwith TBS-Tbuffer. Thepresence oftarget proteins (GAPDH,SOCS1,SOCS3andtotalandphosphorylatedSTAT1 andSTAT3)onthenitrocellulosemembraneswasdetectedby using a chemiluminescence system (Pierce ECL Western Blotting Substrate)andexposing the membranesto radio-graphicfilms.
Table1–InventoriedTaqManPrimersandprobe(TaqManGeneExpressionAssays,AppliedBiosystems).
Targetgene AssayID Acession# Ampliconlength(bp)
GAPDH Rn99999916_s1 NM_017008.3 87
SOCS1 Rn00595838_s1 NM_145879.1 76
SOCS3 Rn00585674_s1 NM_053565.1 73
RANKL Rn00589289_m1 NM_057149.1 69
OPG Rn00563499_m1 NM_012870.2 75
TNF-a Rn01525859_g1 NM_012675.2 92
IL-6 Rn99999011_m1 NM_012589.1 90
2.6. Statisticalanalysis
Thepurposeofthestatisticalanalyseswas todetermineif thereweresignificantvariationsonthehistologicalstructures observed and on the levels of expression of target genes accordingtopresenceofexperimentalperiodontaldiseasein eachperiod.Comparisonoftheresultsineachexperimental periodaccordingtothe controlgroupwas performedusing unpairedStudent’st-test.
Moreover,wealsowantedtodetermineiftheareaofbone resorptioninthelingualsurfacevariedwithinthe experimen-tal periods. One-way Analysis of Variance test (ANOVA) followedby the Tukey post hoc test was usedto evaluate significantdifferencesamongexperimentalperiods.
Significance level was set to 5%. All calculations were performedusingGraphPadPrism5software(GraphPad,Inc., SanDiego,CA,USA).
3.
Results
3.1. Severityofinflammationassociatedwith
ligature-inducedperiodontitispeakedat7days
Therewasasignificantincreaseonthenumberofinflammatory cellsand vascularstructuresalready at 7days post-ligature placement.The overall changes on the composition of the connective tissue, including a decrease on the number of fibroblastsandonthedensityofcollagenisacommonfindingin
periodontaldisease.Theseverityofinflammationwas signifi-cantlyhigherincomparisontothecontrolgroupthroughoutthe 30-dayexperimentalperiod;butadecreaseininflammatorycell densityisobservedafter15days,aswellasatrendofincreasing numberoffibroblastsandextracellularmatrixat15and30days (Figs.1and2).
Cytokinegeneexpressioninthegingivaltissues corrobo-rate these findings, with a maximum increase of mRNA expression forbone-related cytokines RANKLand OPGand pro-inflammatorycytokinesTNF-aandIL-6at7days,followed byadecreaseat15and30days.Theseresultsalsoagreewith the findingthatanti-inflammatorycytokineIL-10tendedto increaseoverthe30day-experimentalperiod(Fig.4).
3.2. SOCS1andSOCS3proteinexpressionin
ligature-inducedperiodontaldiseaseiscorrelatedwithboneresorption
andinflammatorystatus
Expression of SOCS1 and 3 proteins were significantly increased already at 7days in the disease-induced group, followedbyasignificantdecreaseonremainingexperimental periods,althoughtheirexpression remainedhigherthanin the controlgroup(Fig.5).Theseresultsmirrorthoseofthe macroscopicanalysis ofboneresorptionand ofthe stereo-metry indicating a strong correlation of the inflammatory status and the expression of SOCS (Figs. 1–3). It is well documentedthatSOCSisexpressedatlowlevelsinhealthy periodontaltissues.11Ourresultsareinaccordancewiththese
findings.
Fig.1–Photomicrographsillustratinghistologicalsectionsofeachexperimentalperiod(control,7,15and30days)are shown.Inflammationassociatedwithligaturesdecreasesitsseverityinthesubmarginalareatowardstheendofthe experimentalperiod.Thegeneralcharacteristicsoftheinflammatoryreactionincludeincreaseincelldensitiesand vascularstructures,anddecreaseinthenumberoffibroblastsandextracellularmatrix.Theimageofacontrolsitedepicts theplacementofthe32,400mm2gridsonthesubmarginalarealimitedcoronallybytheapicalborderofthejunctional
Interestingly,activationofSTAT1andSTAT3inbothtotal andphosphorylatedformsfollowedtheexpressionofSOCS1 andSOCS3proteins,respectively.Asignificantactivationof STAT1andSTAT3wasobservedalreadyat7daysinanimals withligature-inducedperiodontaldisease.Another interest-ing finding is the decrease on the activation of these transcription factors on ligature sites after 15 and 30days (Fig.5).Thestereometricanalysissupportstheseresults,as thedensityofinflammatorycellsdecreasedatdays15and30 (Figs.1and2).
3.3. LackofcorrespondencebetweenSOCS1mRNA
expressionandproteinlevelsinligature-inducedperiodontitis
TheresultsshowthattherewasanincreaseonSOCS gene expression in ligature-induced periodontitis compared to controlgroupat 7,15and 30days(Fig. 4).Interestingly,the kineticsofSOCS3expressionatthemRNAlevelwasdirectly correlatedtotheexpressionattheproteinlevel.Surprisingly, for SOCS1 there was a lack of transcription–translation coupling, as mRNA levels did not correlate to protein expression. Considering the fact that RNA and protein sampleswereharvestedsimultaneouslyfromthesamewells, thismaysuggesttheinfluenceofpost-transcriptional regula-tion, which has been shown to play a role for SOCS1. Alternatively, the lack of correspondence between mRNA
andproteinlevelsmaymerelyreflectanincreasedefficiency of translation or a longer half-life of the protein. The mechanismofregulationofSOCSexpressionbyperiodontal diseasewillbeexploredinfuturestudies.
4.
Discussion
HumaninvivostudiessuggesttheinvolvementofSOCS1and SOCS3 inthe negativeregulationofimmune inflammatory networks in diseased periodontal tissues.13 However, such
datafromcross-sectionalstudiesdoesnotallowtheanalysis ofthekineticsofSOCSexpressionthroughoutdiseaseonset, neither its possible association with inflammatory cell migrationandwiththepathologicalchangesofthegingival tissues. In this scenario, experimental animal models of periodontitis arewidelyusedforabetterunderstanding of periodontaldiseasepathogenesisandtheinformationderived fromthesemodelsmaybeusefultootherchronic inflamma-toryconditions.
Theligaturemodelofexperimentalperiodontitishasbeen commonlyusedandconsideredbysomeauthorstobemore representativeofperiodontitisinhumansthanothermodels. Thejustificationforthispreferenceistheparticipationoflive microorganisms naturallypresent intheanimalspecies(in contrast to monoinfection models with microorganisms Fig.2–Stereometricanalysisindicatesthatligatureisassociatedwithasustainedinflammatoryreaction.Inflammation wasassessedin4sectionsperperiodbytherelativepresenceofinflammatorycells,vascularstructures,fibroblastsand extracellularmatrixin32,400mm2area(schematicallyshowninFig.1).Theseverityofinflammationwassignificantly
presentinhumansbutnotinrodents)withdiversevirulence factors, known as pathogen-associated molecular patterns (PAMPs), including toxins, microbial metabolism products, CpGDNAandpeptidoglycan.Thisgreaterdiversityofantigens mayresultinamorecomplexhostresponse;whichmayhave an effect on the profile of cytokine and inflammatory mediatorsinthegingivaltissues.However,theligaturemodel has limitedusefulness instudying natural mechanisms of infectivity since periodontal disease is facilitated by the ligature.
InthisstudyweshowincreasedexpressionofSOCS1and SOCS3,at themRNAandproteinlevel,indiseasedgingival tissueswhencomparedwithlevelsinhealthygingivaltissues fromcontrolanimals. SOCS areproduced inresponseto a varietyofstimuli,includingPAMPssuchasbacterial lipopoly-saccharide,characteristicallypresentinthebiofilm accumu-lated around ligatures, and pro-inflammatory cytokines, produced in response to the bacterial challenge. Afterthe inducing-stimuli and its production, SOCS proteins act as endogenousnegativeregulatorsofinflammatoryattenuating cytokine-inducedsignaltransductionaffectingprimarilythe JAK-STATpathway, as part ofa negativefeedback loop to suppressthedownstreameffectsofcytokines.Therefore,in accordance with our findings, SOCS is usually absent or minimallyexpressedinhealthytissues,andtheir up-regula-tion and differential expression in inflamed tissues is an important regulatory mechanism that may influence the
outcomeofinflammatoryreaction.12,15Theincreasedlevelsof
SOCSproteinsintheexperimentalgroupareconsistentwith data fromliterature showingthat SOCS expression canbe induced by inflammatory cytokines present in diseased periodontal tissues such as IL-6, INF-g and TNF-a.2,16,17
Furthermore, biopsiesofinflamed/diseased gingival tissues showhigherSOCS1and-3mRNAexpressionwhencompared withcontrolgroupwithoutdisease.11Inadditiontothe
host-derivedcytokines,theincreasedmicrobialburdenassociated withthetransitionfromperiodontalhealthtodiseasecanalso induceexpressionofSOCSproteins.18,19
SinceseveralinflammatorymediatorsmayregulateSOCS expression,20thenatureofinflammatoryprocessin
periodon-tal tissues can influence SOCS production by different cell types.OurresultsshowthattheexpressionofSOCSprotein mirror inflammation degree/intensityand boneloss during periodontaldiseaseprogression.Indiseasedtissues,already at7days,SOCSproteinexpressionhadasignificantincrease, followedbyasignificantdecreaseonremainingexperimental periods.TheseresultsindicateastrongassociationofSOCS expression and the inflammatory status and density of inflammatory cells, suggesting the kinetic involvement of thesecells,oritsproducts/cytokines,andSOCSexpression.
Studies show that the function of SOCS is to prevent transduction of the cytokine signal by binding to specific receptor sites and ultimately preventing activation of STATs.12,21 Through anegativefeedbackregulatory
mecha-Fig.3–Boneresorptionareainthelingualsurfaceofthefirstmolarswasmeasuredmacroscopically.Digitalimageswere obtainedatastandardised908anglewiththemid-lingualaspectofmethylenebluestainedfirstmolarswitha
stereomicroscope.Thetotalareaofexposedrootsurfacesstainedinblue(thecrownenamelisnotstained)ontheimages wasmeasuredbyanexaminerblindtotheexperimentalgroupsusingsoftwareImagetool3.0.Anincreaseintheareaof exposedrootsincomparisontocontrol,non-ligatedteeth,indicatesalveolarboneresorption.*Significantdifferences (p<0.05)amongexperimentalperiods.(Forinterpretationofthereferencestocolorinthisfigurelegend,thereaderis
Fig.4–ExpressionofSOCS,bone-relatedcytokinesRANKLandOPG,pro-inflammatorycytokinesTNF-aandIL-6and
anti-inflammatorycytokineIL-10profileduringthecourseofligature-inducedperiodontaldisease.Reverse transcription-real-timePCRshowsthatSOCSexpressionincreasessignificantlyalreadyat7days,maintainingelevatedlevelsat15and 30days.mRNAexpressionofbone-relatedcytokinesRANKLandOPGandpro-inflammatorycytokinesTNF-aandIL-6
increaseat7days,followedbyadecreaseat15and30days,andanti-inflammatorycytokineIL-10tendedtoincreaseover the30day-experimentalperiod.mRNAexpressionwasnormalisedtotheexpressionofthehouse-keepinggeneGAPDH. Thebarsrepresentmeanfoldchangesandtheverticallinesthestandarddeviationofthemeanfoldchangeofsixanimals ineachexperimentalgroupincomparisontountreatedcontrol.*Significantdifferences(p<0.05)comparedwithcontrol
nism,increasingSTATactivityleadstoincreasedexpression ofSOCSinanattempttodecreasetheveryactivationstatusof theJAK/STAT pathway and,consequently,reduce the con-sequencesofprolongedactivationofSTAT,suchasincreased expressionofinflammatorycytokines(e.g.IL-1b,IL-6and TNF-a)associatedwithperiodontaltissuedestruction.8,22
Interest-inglyandinaccordancewiththeliterature,inthediseased periodontiumtheSOCS1andSOCS3proteinsexpressionlevels werecorrelatedwiththelevelsoftotalandphosphorylated (activated) STAT1 and STAT3, respectively. In fact, the increasedproductionofinflammatorymediatorsbyresident andinflammatorycellsintheresponsetostimulileadstoan increaseintheintracellularsignalingandintheactivationof these transcription factors.23 These data suggest that
in-creased expression of SOCS1 and SOCS3 may represent a mechanismofnegativeregulationinresponsetoactivityof STAT1andSTAT3,andmaybeanimportantmechanismin regulatingexpressionofgenesassociatedwithdegradationof connectivetissueandalveolarboneresorption.
EventhoughdeletionofSOCS1andSOCS3genesinmiceis lethal,24itistemptingtospeculatethatintheabsenceofthis
endogenousregulatorymechanismthehostresponsewould beexacerbatedintermsofseverityandduration,withamajor increase on the activation of STATs. In these conditions, inflammatory cytokine expression and tissue destruction associated with periodontal diseases and other conditions associated with chronic inflammation, would be severely aggravated.Experimentsintransgenicanimalswith tissue-specificdeletionofthesegeneswilldefinetheirrelevancefor theimmuneresponse.
Inadditiontodirectlymodulatingtissuedestruction,SOCS couldalsoimpactperiodontitisoutcomethroughthe modula-tionofhealingprocess.Indeed,invivostudiesdemonstratethe importanceofSOCS3innegativemodulationofgp130/STAT3 signalingpathwayinwoundhealing.TheabsenceofSOCS3 leads to an increased activity of STAT3 causing delay in healing.25,26Inourstudy,wefoundthatevenafter30daysof
ligatureplacement,mRNAandproteinlevelsofSOCS3remain elevatedinspiteofthedecreaseontheseverityof inflamma-tion.Infact,theapicalmigrationofthejunctionalepithelium increasingthedistancetothesiteofaggressionlocatedonthe gingival margin reduced the aggression and consequently decreasedtheseverityoftheinflammatoryinfiltrate.Thismay be followed by an attempt to repair the damaged tissues, whichischaracterisedbythetendencytoincreasethenumber offibroblastsandextracellularmatrixverifiedbystereometry. Thisinterpretationissupportedbythefactthatonceplaced, ligatures were kept throughout the 30-day experimental period;howevertheywerenotpushedfurtherapicallyeven ifthegingivalmarginreceded.ThissuggeststhatSOCS3may alsoparticipateinthehealingofperiodontaltissues.
Toourknowledge,thisisthefirst studytodescribethe kinetic profile of SOCS1 and SOCS3 expression during experimentalperiodontaldisease, anditsassociation with STATactivationprofile.Additionalstudieswillincludegain andloss offunctionexperimentstodeterminethe roleof theseproteinsinthemodulationofhostresponseassociated withchronicinflammationandalsotoverifypossiblenovel targetsofSOCSproteins fordirectprotein–protein interac-tions.
Insummary,ourstudyshowsthekineticsofSOCS1and SOCS3 mRNA and protein expression in the experimental modelofligature-inducedperiodontaldisease.Thekineticsof SOCS1andSOCS3expressionwasdirectlycorrelatedwiththe severityofinflammation,ofalveolarboneresorptionandthe levelofpro-inflammatorycytokineexpression,aswellaswith theactivationstatusofSTAT1andSTAT3transcriptionfactors duringthecourseofexperimentalperiodontaldisease.This suggests that theseproteins playan important rolein the modulationofhostresponse.
Understanding theroleofSOCSproteinsinthenegative regulationofcytokinesignaling,especiallyintheJAK/STAT pathway,mayprovidenovelinformationonthesusceptibility to periodontal diseases and also for therapeutic strategies basedonthemodulationoftheinflammatoryprocess.
Acknowledgements
Theauthorswanttoexpresstheirgratitudetotheresearch technicianAnaClaudiaGregolin daCostaMiranda (Depart-Fig.5–Totalproteinwasextractedfromgingivaltissue
samplesobtainedfromligatureandcontrolsitesat7,15 and30dayperiods.30mgoftotalproteinwasseparatedby
electrophoresisin10%acrylamidegelsunderdenaturing conditionsandtransferredtonitrocellulosemembranes. ActivationofSTAT1andSTAT3aswellastheglobal expressionofSOCS1andSOCS3wasdeterminedby incubatingthemembraneswiththespecificprimary antibodiesandrevealedwithHRP-conjugatedsecondary antibodiesandanECLsystem.ExpressionofSOCS1and3 proteinsandactivationofSTAT1andSTAT3inbothtotal andphosphorylatedformsweresignificantlyincreased alreadyatday7followedbyasignificantdecreaseinthe remainingexperimentalperiods.Theimagesare
mentofDiagnosisandSurgery,UNESPatAraraquara)forher technicalassistance.
Grantsupportwasprovidedbygrants#2007/06658-7and #2007/06332-4awardedbyFundac¸a˜odeAmparoaPesquisado EstadodeSa˜oPaulo(FAPESP)toC.R.J.andtransferredtoJ.A.C. Funding: Sao Paulo State Research SupportFoundation: FAPESP.AresearchsupportfoundationfromtheStateofSa˜o Paulo,Brazil.Grants#2007/06658-7and#2007/06332-4.
Conflictofinterest:None.
Ethical approval: By the Ethical Committee on Animal ExperimentationoftheSchoolofDentistryat Araraquara– UNESP(protocolnumber23/2007),wheretheinvivopartofthe studywasconducted.
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