S100B Protein Stimulates Microglia Migration via RAGE-dependent Up-regulation of Chemokine ...

14 Pages·2011·2.66 MB·English

THEJOURNALOFBIOLOGICALCHEMISTRYVOL.286,NO.9,pp.7214–7226,March4,2011
©2011byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PrintedintheU.S.A.
S100B Protein Stimulates Microglia Migration via
RAGE-dependent Up-regulation of Chemokine Expression
and Release*□S
Receivedforpublication,July28,2010,andinrevisedform,December29,2010Published,JBCPapersinPress,January5,2011,DOI10.1074/jbc.M110.169342
RobertaBianchi,EiriniKastrisianaki1,IleanaGiambanco,andRosarioDonato2
FromtheDepartmentofExperimentalMedicineandBiochemicalSciences,SectionofAnatomy,UniversityofPerugia,
06122Perugia,Italy
The Ca2(cid:1)-binding protein of the EF-hand type, S100B, is differentiationviaactivationoftheSrc/PI3KmoduleandPI3K-
abundantlyexpressedinandsecretedbyastrocytes,andrelease dependentstimulationofAktandRhoAactivitiesandhenceof
ofS100Bfromdamagedastrocytesoccursduringthecourseof thesupramolecularorganizationofF-actin(2).S100Balsoreg-
acute and chronic brain disorders. Thus, the concept has ulatesthestateofassemblyofmicrotubulesandtypeIIIinter-
emergedthatS100Bmightactanunconventionalcytokineora mediatefilaments(3)andthecytosolicCa2(cid:1)concentration(4).
damage-associated molecular pattern protein playing a role Inadditiontohavingintracellularregulatoryactivities,S100B
in the pathophysiology of neurodegenerative disorders and alsoexertsextracellulareffects.Indeed,astrocytessecretethe
inflammatory brain diseases. S100B proinflammatory effects proteinconstitutivelyandtoalargerextentundertheactionof
requirerelativelyhighconcentrationsoftheprotein,whereasat severalstimuliincludingtheproinflammatorycytokine,TNF-(cid:1)
physiological concentrations S100B exerts trophic effects on (seeforreviewRef.1).Moreover,levelsofbrainS100Bareele-
neurons.Mostifnotalloftheextracellular(trophicandtoxic) vatedintheagingbrainandinseveralpathologicalconditions
effectsofS100Binthebrainaremediatedbytheengagementof such as Alzheimer disease, brain infarct, epilepsy, and infec-
RAGE(receptorforadvancedglycationendproducts).Weshow tious diseases, as well as in Down syndrome (5, 6) in conse-
herethathighS100Bstimulatesmurinemicrogliamigrationin quence of S100B human gene mapping to chromosome
BoydenchambersviaRAGE-dependentactivationofSrckinase, 21q22.3 (7). For example, hypertrophic astrocytes in peri-in-
Ras,PI3K,MEK/ERK1/2,RhoA/ROCK,Rac1/JNK/AP-1,Rac1/ farct areas and in neuritic plaques in Alzheimer disease and
NF-(cid:2)B,and,toalesserextent,p38MAPK.Recruitmentofthe DownsyndromeshowelevatedexpressionlevelsofS100B,and
adaptorprotein,diaphanous-1,amemberoftheforminprotein S100B can be detected outside hypertrophic astrocytes (8, 9),
family,isalsorequiredforS100B/RAGE-inducedmigrationof pointing to secretion/release of high amounts of the protein
microglia. The S100B/RAGE-dependent activation of diapha-
undertheseconditions.Also,oldbutnotyoungS100Btrans-
nous-1/Rac1/JNK/AP-1, Ras/Rac1/NF-(cid:2)B and Src/Ras/PI3K/
genic mice show hypertrophic astrocytes and an enhanced
RhoA/diaphanous-1resultsintheup-regulationofexpression expressionof(cid:2)-amyloidprecursorproteininnearbyneurons
of the chemokines, CCL3, CCL5, and CXCL12, whose release
(10). Lastly, S100B TG mice show increased susceptibility to
and activity are required for S100B to stimulate microglia
perinatalhypoxia-ischemia(11),andoverexpressionofS100B
migration. Lastly, RAGE engagement by S100B in microglia
hasbeenshowntoaccelerateAlzheimerdisease-likepathology
resultsinup-regulationofthechemokinereceptors,CCR1and
with enhanced astrogliosis and microgliosis (12). Because of
CCR5. These results suggests that S100B might participate in
theseobservations,theconcepthasemergedthatS100B,when
thepathophysiologyofbraininflammatorydisordersviaRAGE-
present in the brain extracellular milieu in relatively high
dependent regulation of several inflammation-related events
amounts, might act as an unconventional cytokine and/or a
includingactivationandmigrationofmicroglia.
damage-associated molecular pattern molecule playing a role
in the pathophysiology of neurodegenerative disorders and
inflammatory brain diseases (1, 5–13). Indeed, treatment of
S100BisaCa2(cid:1)-bindingproteinoftheEF-handtypeabun- astrocyteswithS100Bresultsinup-regulationofexpressionof
dantlyexpressedinastrocytes(1).S100Bhasbeenimplicatedin inducible NOS, stimulation of inducible NOS activity, NO
theregulationofastrocyteshape,migration,proliferationand
release, and NO-dependent killing of astrocytes and co-cul-
tured neurons (14, 15) and in stimulation of release of the
inflammatorycytokineIL-1(cid:2)(16).Neuronsandmicrogliaalso
*ThisworkwassupportedbyAssociazioneItalianaperlaRicercasulCancro
were shown to be targets of extracellular S100B. Specifically,
Project 6021 and Fondazione Cassa di Risparmio di Perugia Grants
2007.0218.020and2009.020.0021(toR.D.). S100Bwasshowntocauseneuronaldeathbyincreasingreac-
□S Theon-lineversionofthisarticle(availableathttp://www.jbc.org)contains tiveoxygenspeciesproductioninneurons(17,18),toup-regu-
supplementalFigs.S1–S3.
1Present address: Institute of Molecular Oncology, Biomedical Sciences lateinducibleNOSexpressioninandstimulateNOreleaseby
ResearchCenter“AlexanderFleming,”Vari,Greece. microgliainthepresenceofbacterialendotoxinorinterferon-(cid:3)
2To whom correspondence should be addressed: Dept. of Experimental (19, 20), to up-regulate the expression in and the release of
Medicine and Biochemical Sciences, University of Perugia, Via del IL-1(cid:2)andTNF-(cid:1)bymicroglia(14,21–23),toup-regulatethe
Giochetto, 06122 Perugia, Italy. Tel.: [phone]; Fax: 39-075-
5857451;E-mail:[email protected]. expressionoftheproinflammatoryenzyme,COX-2,inmicro-
7214 JOURNALOFBIOLOGICALCHEMISTRY VOLUME286•NUMBER9•MARCH4,2011
This is an Open Access article under the CC BY license.
S100BStimulatesMicrogliaMigrationRAGE-dependently
glia(22),andtosynergizewithIL-1(cid:2)andTNF-(cid:1)toup-regulate S100Bconcentrationwascalculatedusingthemolecularmass
COX-2expressioninmicroglia(23).Mostoftheseeffectswere oftheS100Bdimer,i.e.21kDa.
observedatrelativelyhighdosesofS100B,pointingtotheneed Cell Culture—The murine BV-2 microglial cell line was
ofaccumulationoftheproteininthebrainextracellularspace obtainedandcharacterizedasdescribed(20,42,43).Thecells
for it to act as an inflammatory cytokine/damage-associated werecultivatedinRPMI1640containing10%heat-inactivated
molecular pattern factor and/or a neurotoxic factor. Also, FBS (Hyclone Laboratories, Lagan, UK) supplemented with
S100B was shown to bring about those effects by engaging L-glutamine(4mM),penicillin(100units/ml),andstreptomycin
RAGE(receptorforadvancedglycationendproducts)forthe (0.1 mg/ml) in H O-saturated 5% CO atmosphere at 37°C.
2 2
mostpart(24).RAGEisamultiligandreceptorbelongingtothe BV-2microgliaweretestedperiodicallyandresultednegative
immunoglobulin superfamily playing an important role in for mycoplasma contamination. Primary microglia were iso-
innateimmuneresponseincludingmacrophagemigrationand lated from 6-day-old CD rat (Charles River), WT C57BL/6
activation(25–28).However,atthenanomolarconcentrations (CharlesRiver)mouse,andRage(cid:2)/(cid:2)mousebrainandcharac-
atwhichS100Bisfoundinthebrainextracellularspaceunder terizedandcultivatedasdescribed(44).BV-2microgliastably
normal physiological conditions and at the very beginning of transfectedwithhumanRAGEcDNA(BV-2/RAGEmicroglia),
brain insult (1), S100B was reported to exert trophic effects, human RAGE(cid:3)cyto cDNA (BV-2/RAGE(cid:3)cyto microglia), or
protecting neurons against pro-apoptotic stimuli, promoting empty vector (BV-2/mock microglia) were obtained as de-
neurite outgrowth again via RAGE engagement (17, 29–32), scribed(45).RAGE(cid:3)cytoisaRAGEmutantlackingthecyto-
andreducingtheactivationofmicrogliabyneurotoxicagents plasmic and transducing domain (46, 47). BV-2 microglia
(33). express RAGE (45, 46), and BV-2/RAGE microglia express
Microglia, the brain-resident macrophages, become acti- larger amounts of the receptor compared with WT BV-2
vated in case of brain injury, thus contributing to the patho- microglia, whereas BV-2/RAGE(cid:3)cyto microglia express
physiologyofinflammatorybraindiseasesandbraindamageby endogenousRAGEplusthesignaling-deficientRAGEmutant,
releasing NO and inflammatory products such as prostaglan- RAGE(cid:3)cyto (45). BV-2/mock, BV-2/RAGE, and BV-2/
dins,cytokines,andchemokines(34–36).However,according RAGE(cid:3)cyto microglia were cultivated as above except that
to recent views, microglia are active players in brain tissue gentamicin (5 (cid:5)g/ml) was used instead of penicillin and
homeostasis under normal physiological conditions, continu- streptomycin.
ously patrolling the territory, exerting a protective action by Migration Assay—Migration assays were performed using
virtueoftheirabilitytokeeptheneuronalandastrocyticextra- Boydenchambers(poresize,8(cid:5)m)(Falcon).CellsinDMEM
cellular milieu clean, and likely resolving mild degree brain wereseededintheupperchamber,andtheinsertwasplacedin
insults(37,38).Microgliaalsoreleasechemokinesthatstimu- thelowerchamberofa24-welldishcontainingDMEMplusor
latethemigrationandproliferationofcerebellargranulecells minusS100Bandincubatedat37°Cfor2–6haccordingtothe
andcorticalneuronprogenitors,thuspromotingneurogenesis manufacturer’sinstructions.Thecellsontheuppersideofthe
(38–40). filterswereremovedwithcotton-tippedswabs,andthefilters
Weshowherethat:1)atproinflammatorybutnotlowdoses, werefixedinmethanolfor2minandstainedwith0.05%crystal
S100BstimulatesmicrogliamigrationviaRAGEsignaling-de- violetinPBSfor15minfollowedbyrepresentativecountsof10
pendentup-regulationoftheexpressionofCCL3,CCL5,and randomlyselectedmicroscopefields.
CXCL12 chemokines and their release, and 2) at low doses TransfectionsandOtherTreatmentsofMicroglia—Transient
S100B, however, can stimulate RAGE-overexpressing micro- transfectionswerecarriedoutusingLipofectamine2000asrec-
gliamigration.TheseS100BeffectsrequireRAGE-dependent ommendedbythemanufacturer.Briefly,microgliaculturedin
activation of Src kinase, Ras, MEK/ERK1/2, PI3K/Akt, Rac1- 10%FBSwithoutantibioticsweretransfectedwithexpression
Cdc42, JNK/activating protein-1 (AP-1),3 nuclear factor (cid:4)B plasmidN17Rac1,N17Cdc42,N17Ras,orN19RhoA(constitu-
(NF-(cid:4)B),theRhoA-associatedkinase(ROCK),and,toalesser tively inactive forms of Rac1, Cdc42, Ras, and RhoA, respec-
extent,p38MAPKandthepresence/activityofdiaphanous-1. tively), I(cid:4)B(cid:1)SR (a nonphosphorylatable form of the NF-(cid:4)B
Thepresentobservationssupporttheconceptthatextracellu- inhibitor,I(cid:4)B(cid:1))(48),oremptyvector.After24h,themedium
larS100Bactasadamage-associatedmolecularpatternfactor was changed to RPMI 1640, and the cells were detached by
participatinginthepathophysiologyofbraininflammatorydis- agitationandlayeredontheupperwellofBoydenchambersfor
orders via RAGE-dependent regulation of several inflamma- transmigrationassay.Transfectionefficiencywasestimatedby
tion-related events such as activation and migration of transfectingparallelcellswithGFPcDNA.Thepercentageof
microglia. GFP-positivecells(20–25%)wasdeterminedbyfluorescence-
activated cell sorter analysis. Parallel cells were analyzed for
EXPERIMENTALPROCEDURES viability by trypan blue exclusion assay and by
S100B—RecombinantbovineS100B,whichis97%identical a tetrazolium-based (3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
tomouseS100B,wasexpressedandpurifiedasreported(17,41) nyltetrazolium bromide) colorimetric assay. No significant
and made free of bacterial endotoxin as described (23). The changescouldberegisteredinthenumbersofcellstransfected
withexpressionplasmidsoremptyvector(datanotshown).For
RNA interference, microglia were transfected with Stealth
3Theabbreviationsusedare:AP-1,activatingprotein-1;ROCK,RhoA-associ- RNAi Negative Universal Control or diaphanous-1 siRNA
atedkinase;HMGB1,highmobilitygroupprotein1. (Invitrogen)usingLipofectamine2000accordingtothemanu-
MARCH4,2011•VOLUME286•NUMBER9 JOURNALOFBIOLOGICALCHEMISTRY 7215
S100BStimulatesMicrogliaMigrationRAGE-dependently
facturer’s instructions. After 72 h the cells were cultivated in GACTTGGT-3(cid:5)forCCL3,5(cid:5)-TTCCCTGTCATCGCTTGCT-
RPMI 1640 and processed as described in Fig. 3. Where CT-3(cid:5) and 5(cid:5)-CGGATGGAGATGCCGATTTT-3(cid:5) for CCL5,
required,thecellsweretreatedfor1hwithPP2(Calbiochem) 5(cid:5)-ATTGTATGGTCAACACGCACG-3(cid:5) and 5(cid:5)-TTTGAAC-
(20(cid:5)M),SP600125(Calbiochem)(20(cid:5)M),LY294002(10(cid:5)M) GTCTCTGTCCCGAG-3(cid:5) for CXCL1, 5(cid:5)-GCCCACTTCAT-
(Alexis), NSC23766 (Calbiochem) (50 (cid:5)M), PD98059 (Calbi- AACCTCCA-3(cid:5) and 5(cid:5)-ATCACTTCCACATCAGCACA-3(cid:5)
ochem)(30(cid:5)M),Bay11-7082(Calbiochem)(5(cid:5)M),SB203580 for CXCL7, 5(cid:5)-CAAGGTCGTCGCCGTGCTG-3(cid:5) and 5(cid:5)-
(Calbiochem) (5 (cid:5)M), Y27632 (Calbiochem) (10 (cid:5)M), BoxA GCTCAGGCTGACTGGTTTACCG-3(cid:5) for CXCL12, 5(cid:5)-
(HMG Biotech) (200 ng/ml), pertussis toxin (Sigma) (100 AGGTGACTGAGGTGATTGCC-3(cid:5) and 5(cid:5)-CTGTGGATG-
ng/ml), RAGE-neutralizing antibody (N16; Santa Cruz Bio- GAGATATAGAACTGG-3(cid:5)forCCR1,5(cid:5)-CGAGCCCGAAC-
technology, 10 (cid:5)g/ml) (49), or 10 (cid:5)g/ml nonimmune IgG in TGTGACTTTTG-3(cid:5) and 5(cid:5)-GTCTTCTTCACCCTCTGGA-
RPMI1640beforemigrationassay. TAGCG-3(cid:5) for CCR3, 5(cid:5)-GGTGGAGGAGCAGGGAGAAC-
Western Blot Analyses—BV-2 microglia (3 (cid:4) 105) were GAG-3(cid:5) and 5(cid:5)-CTTTCAGGAACCCAGCGGTGAGAC-3(cid:5)
seededin24-multiwellplatesandcultivatedinthepresenceor forCCR5,and5(cid:5)-CCTCTGCCTGGTGACTCTGG-3(cid:5)and5(cid:5)-
absence of S100B. The cells were then solubilized with 2.5% AGGAGGAGGTGGAGGGATGG-3(cid:5) for diaphanous-1 in a
SDS,10mMTris-HCl,pH7.4,0.1Mdithiothreitol,0.1mMtosyl- reactionvolumeof20(cid:5)lcontainingRealMasterMixandSYBR
sulfonyl phenylalanyl chloromethyl ketone protease inhibitor solution(Eppendorf).Thereactionmixtureswereincubatedin
(RocheAppliedScience)forWesternblotanalyses.Phosphor- athermocycler(Stratagene)andanalyzedbyMultiplexQuan-
ylatedSrc,JNK,ERK1/2,p38MAPK,Akt,andp65NF-(cid:4)Bwere titativePCRSystem.Housekeeping(cid:2)-actinmRNAwasusedas
detectedusingapolyclonalanti-phosphorylatedSrc(Ser-416) acontrol(primers5(cid:5)-AGCCATGTACGTAGCCATCC-3(cid:5)and
(1:1,000; Cell Signaling Technology), JNK (Thr-183/Tyr-185) 5(cid:5)-CTCTCAGCTGTGGTGGTGAA-3(cid:5)).
antibody (1:1,000; Cell Signaling Technology), a polyclonal Determination of Chemokines by ELISA—BV-2 microglia
anti-phosphorylated (Thr-202/Tyr-204) ERK1/2 antibody (5 (cid:4) 105/ml) were cultivated in the presence or absence of
(1:2,000; Cell Signaling Technology), a polyclonal anti-phos- S100B.After6h,theculturemediaweretakenup,centrifuged,
phorylated (Thr-180/Tyr-182) p38 MAPK antibody (1:1,000; andsubjectedtoELISAusingcommercialkits(R&DSystem)
Cell Signaling Technology), polyclonal anti-phosphorylated to measure CCL3 and CCL5. Each sample was tested in
(Ser-473)Akt(1:1000;CellSignalingTechnology),andapoly- triplicate.
clonalanti-phosphorylated(Thr-534)p65NF-(cid:4)B(1:1,000;Cell Immunofluorescence—BV-2microglia(5(cid:4)105/ml)werecul-
Signaling Technology) antibody, respectively. Total Src, tivated for 3 h in thepresence or absence of S100B on glass
ERK1/2,p38MAPK,Akt,andp65NF-(cid:4)Bweredetectedusinga coverslipsandprocessedfordetectionofF-actinandmicrotu-
polyclonalanti-Src(1:1,000;CellSignalingTechnology),anti- bulesasdescribed(2).
ERK1/2 antibody (1:20,000; Sigma), a polyclonal anti-p38 StatisticalAnalysis—Eachexperimentwasrepeatedatleast
MAPK antibody (1:2,000; Cell Signaling Technology), poly- threetimes.Representativeexperimentsaredepictedinthefig-
clonalanti-Akt(1:1000;CellSignalingTechnology),andapoly- uresunlessstatedotherwise.Thedataweresubjectedtoanaly-
clonalanti-p65NF-(cid:4)Bantibody(1:1,000;SantaCruzBiotech- sisofvariancewithStudent-Newman-Keulsposthocanalysis
nology),respectively.AnalysisofculturemediumHMGB1was using a statistical software package (GraphPad Prism version
performedasdescribed(50).Briefly,culturemediawereclari- 4.00; GraphPad Software, San Diego, CA). Statistical signifi-
fiedbycentrifugation,addedwith1/100volumeof2%sodium cancewasassumedwhenp(cid:6)0.05.
deoxycholate,andsubjectedtoprecipitationwith1/10volume
RESULTS
of100%trichloroaceticacid.Theresultantpelletswereresus-
pendedinLaemmlibufferandtitratedwith1NNaOHtoobtain S100BStimulatesMicrogliaTransmigrationinaRAGE-de-
thenormalbluecolorofthesamplebuffer,boiledfor5min,and pendentManner—Whenassayedat2h,S100BstimulatedBV-2
subjectedtoWesternblottingusingananti-HMGB1antibody microgliatransmigrationinBoydenchambersat5and10(cid:5)M
(BD PharMingen). A monoclonal anti-(cid:1)-tubulin (1:10,000; butnotatlowerconcentrations(supplementalFig.S1A).How-
Sigma)wasusedtomonitorproteinloadingonSDSgels.Per- ever,whenassayedat6h,S100BstimulatedBV-2/WTmicro-
oxidase-conjugated secondary antibodies were from Sigma. gliatransmigrationdose-dependentlywithmaximumstimula-
Antibodies were diluted in blocking buffer (10 mM Tris-HCl, tion at 1 (cid:5)M and decreasing effects at higher concentrations
pH7.4,0.1MNaCl,5%nonfatdriedmilkpowder,0.1%Tween (Fig.1A).RatprimarymicrogliarespondedtoS100Bagainwith
20).TheimmunereactionwasdevelopedbyECL(SuperSignal noeffectsatnanomolardosesandstimulationoftransmigra-
WestPico;Pierce). tionat1(cid:5)M(Fig.1B).However,addingS100B(0.1–1.0(cid:5)M)to
RealTimePCR—BV-2microgliawereincubatedwithS100B thecellsintheupperwellofBoydenchambersresultedinno
as shown in Figs. 4 and 5. Where appropriate, the cells were changesinS100B-inducedmicrogliatransmigrationrelativeto
pretreated for 1 h with 20 (cid:5)M PP2, 20 (cid:5)M SP600125, 10 (cid:5)M theinternalcontrol(i.e.noS100Bpresentinupperwells)(Fig.
LY294002, 50 (cid:5)M NSC23766, 30 (cid:5)M PD98059, 5 (cid:5)M Bay 1C), suggesting that S100B might not be a chemoattractant
11-7082,or5(cid:5)MSB203580.TotalcytoplasmicRNAwasiso- towardmicrogliaperse,butratheritmightactinanindirect
latedfromBV-2microgliausingtheTRIzolreagentmethod.To manner.
detect CCL3, CCL5, CXCL1, CXCL7, and CXCL12 mRNAs, In the absence of S100B, the extent of migration of BV-2
cDNA(0.1(cid:5)g/sample)wasincubatedwithprimers5(cid:5)-TTCT- microgliaoverexpressingRAGE(cid:3)cyto,aRAGEmutantlacking
GCTGACAAGCTCACCCT-3(cid:5)and5(cid:5)-ATGGCGCTGAGAA- the cytoplasmic and transducing domain (BV-2/RAGE(cid:3)cyto
7216 JOURNALOFBIOLOGICALCHEMISTRY VOLUME286•NUMBER9•MARCH4,2011
S100BStimulatesMicrogliaMigrationRAGE-dependently
FIGURE1.S100BstimulatesBV-2microgliamigrationinaRAGE-dependentmanner.A,migrationassayswereperformedusingBoydenchambers.The
cells(1(cid:4)105)wereseededontothetopofTranswell(cid:2)migrationchambersandallowedtomigratefor6htowardS100Bormediumalonenegativecontrol
placedinthelowerchamber.B,conditionswereasinAexceptthatBV-2orratprimarymicrogliawerepretreatedwithnonimmuneIgGoraRAGEneutralizing
antibodyfor60minandthentransferredtotheupperchamber.C,conditionswereasinAexceptthatS100B(0–1.0(cid:5)M)wasaddedtothecellsplacedonthe
upperwellofBoydenchambers,whereindicated.D,conditionswereasinAexceptthatWTBV-2microglia(BV-2/WT),mock-transfectedBV-2microglia
(BV-2/mock),BV-2microgliastablyexpressingaRAGEmutantlackingthecytoplasmicandtransducingRAGEdomain(BV-2/RAGE(cid:3)cyto),orBV-2microglia
stablyoverexpressingfull-lengthRAGE(see“ExperimentalProcedures”)wereused.E,BV-2microgliawerecultivatedinDMEMfor6hinthepresenceof
increasingS100Bconcentrations.Theculturemediawerecollected,trichloroaceticacid-precipitatedasdescribedunder“ExperimentalProcedures,”and
subjectedtoWesternblottingusingananti-HMGB1antibody.Residualcellswerelysed,andcelllysateswereprobedwithanti-HMGB1antibody.Alsoshown
isaWesternblotoftubulin.F,BV-2microgliawerepretreatedwithBoxAandallowedtomigratefor6htowardmediumaloneplacedinthelowerchamber.The
resultsareexpressedasthemeans(cid:7)S.D.(n(cid:8)3)(A–D,F,andG).G,mouseWTandRage(cid:2)/(cid:2)microgliaweresubjectedtomigrationassayasdescribedinAin
thepresenceofincreasingS100Bconcentrations.*,significantlydifferentfromcontrol(firstcolumnsinA,C,F,andG)orfrominternalcontrol(firstcolumnsfrom
leftineachgroupinBandD).#,significantlydifferentfromthecorrespondingcolumnintheBV-2/WT(cid:1)IgGgrouportheprimarymicrogliagroupinBandin
theBV-2/mockgroupinD.
MARCH4,2011•VOLUME286•NUMBER9 JOURNALOFBIOLOGICALCHEMISTRY 7217
S100BStimulatesMicrogliaMigrationRAGE-dependently
microglia), was significantly lower than that detected using GTPases,Ras,Rac1,andCdc42viaRAGEligationinmicroglia
BV-2/mockmicroglia,whereasthenumberofmigratedRAGE- withensuingactivationofthetranscriptionfactorsNF-(cid:4)Band
overexpressing BV-2 microglia (BV-2/RAGE microglia) was AP-1, leading to up-regulation of COX-2, IL-1(cid:2), and TNF-(cid:1)
morethanfourtimeslargerthanthatofBV-2/mockmicroglia expression (22, 23), and others have reported that RAGE
underbasalconditions(Fig.1D).Theseresultssuggestedthat engagement results in activation of Src kinase in monocytes/
RAGEsignalingplaysanimportantroleinmicrogliamigration macrophagesandvascularsmoothmusclecells(63,64).Atpro-
andthatfactorsintheculturemediummightstimulatebasal inflammatory doses S100B enhanced the phosphorylation
microgliamigrationinaRAGE-mediatedmanner.Onecandi- (activation) levels of ERK1/2, p38 MAPK, JNK, Akt, and Src
dateRAGEagonistwiththeabilitytostimulatebasalmicroglia (Ser-416)kinasesandofNF-(cid:4)B(p65)inmicroglia(Fig.2Aand
transmigrationishighmobilitygroupprotein1(HMGB1),an supplemental Fig. S2). Inhibition of Src using PP2 and of the
establishedRAGEligand(51–53)releasedbyactivatedmacro- Akt upstream activator, PI3K, using LY294002, resulted in
phages (54) and implicated in RAGE-dependent migration of reducedabilityofS100BtoactivateAktwhileleavingthestim-
several cell types (55–58). Indeed, the microglia culture ulatoryeffectofS100BonNF-(cid:4)Bunaffected(Fig.2A).Bycon-
medium contained HMGB1 (Fig. 1E), which might explain trast, inhibition of Rac1 by NSC23766 significantly reduced
basalBV-2/mockmicrogliamigrationinthepresentassayand S100B-dependentactivationofNF-(cid:4)B(p65)(Fig.2A)(alsosee
the enhanced basal migration of BV-2/RAGE microglia com- Refs.22and23).Thissuggestedthatinadditiontosignalingto
pared with BV-2/mock microglia. Consistently, treating pri- Ras, Rac1, and Cdc42 to activate NF-(cid:4)B and AP-1 (22, 23),
mary and BV-2 microglia with BoxA, an HMGB1 antagonist S100B/RAGE activated a Src/PI3K/Akt module in microglia
(59),resultedinasignificantreductionofbasalmigration(Fig. that did not impinge on NF-(cid:4)B. However, S100B-dependent
1F).However,asmentionedearlier,addingS100Btothelower transmigrationofmicrogliawasabrogatedwhenthecellswere
compartment of Boyden chambers resulted in stimulation of pretreated with inhibitors of either Src kinase (PP2), JNK
migrationofratprimary,BV-2/mock,andBV-2/RAGEbutnot (SP600125), PI3K (LY294002), Rac1 (NSC23766), MEK/
BV-2/RAGE(cid:3)cytomicroglia(Fig.1,BandD),andpretreatment ERK1/2(PD98059),orNF-(cid:4)B(Bay11-7082)andsignificantly
ofprimaryandBV-2microgliawithaRAGEneutralizinganti- reducedbutnotabrogatedwhenthecellsweretreatedwithan
bodyresultedintheabrogationofS100B-stimulatedmigration inhibitorofp38MAPK(SB203580)(Fig.2B).Also,treatmentof
alongwithasignificantreductionofbasalmigration(Fig.1B). BV-2microgliawithY27632(Fig.2B),aspecificinhibitorofthe
These results suggested that the ability of S100B to stimulate RhoA-associated kinase, ROCK, or transfection with a domi-
microgliamigrationwasdependentonRAGEsignaling,andit nant negative mutant of either Ras, Rac1, Cdc42, or RhoA or
addedtobasal,HMGB1-inducedmigration.Consistently,at6h transfection with I(cid:4)B(cid:1)SR, a nonphosphorylatable mutant of
S100B did not stimulate the migration of Rage(cid:2)/(cid:2) microglia, theendogenousNF-(cid:4)Binhibitor,I(cid:4)B(cid:1),resultedinreductionof
whereastheproteinefficientlystimulatedmigrationofmouse basal and S100B-dependent migration (Fig. 2C). However,
WTmicroglia(Fig.1G).Also,similartoBV-2microglia,at2h S100B still stimulated JNK activity in the presence of the Src
S100B((cid:6)5(cid:5)M)stimulatedmouseWTbutnotRage(cid:2)/(cid:2)micro- inhibitor,PP2(Fig.2A),suggestingthatthestimulatoryeffectof
gliamigration(supplementalFig.S1,BandC).Notably,S100B S100BonmicrogliamigrationreliedonRAGE-dependentacti-
stimulatedmicrogliamigrationwiththesameefficacyirrespec- vation of a Ras/Rac1-Cdc42/NF-(cid:4)B, a Ras/MEK/ERK1/2/NF-
tiveoftheabsenceorpresenceofBoxA(Fig.1D),inaccordance (cid:4)B,aRas/Rac1-Cdc42/JNK/AP-1,andaSrc/Ras/PI3K/RhoA/
with the specificity of BoxA blocking effect toward HMGB1 ROCK pathway, and individual pathways were necessary for
(60).Thepresentresultsalsosuggestedthatalackofeffectsof S100B/RAGE-stimulated microglia migration. Interestingly, a
lowdosesofS100Bonmicrogliamigrationmightbedependent dramatic reduction of basal and S100B-stimulated microglia
on the inability of S100B to displace RAGE-bound HMGB1, migration was observed with 20–25% transfection efficiency
becauseHMGB1andS100BbothbindtoRAGEVdomain(51, using either dominant negative Ras, RhoA, Rac1, or cdc42 or
62). I(cid:4)B(cid:1)SR.Thissuggestedthatabolitionoftheactivityofanyone
Whereas at 1 (cid:5)M S100B caused an (cid:9)4-fold increase in the oftheseG-proteinsorI(cid:4)B(cid:1)SRin20–25%ofcellsmightresult
numberofBV-2/mockmigratedcells,theproteincausedonly in profound alteration of, for example, secretion of factors
an(cid:9)2-foldincreaseinthemigrationofBV-2/RAGEcells,com- requiredformicrogliamigration(ananalysisofwhichisbeyond
paredwithcontrols(Fig.1A).Thismightbeaconsequenceof the scope of the present work). Of note, no toxic effects of
the presence of HMGB1 in the microglia culture medium, transfectionthatmightresultinreductionofcellnumberswere
which might enhance basal migration of BV-2/RAGE cells. observed.Wereportedthattransfection(20–25%transfection
Thus, the stimulatory effect of S100B on microglia migration efficiency)ofmicrogliawiththeabovemutantsunderthesame
wasdependentonfunctionalRAGEand,toacertainextent,the conditionsusedinthepresentworkresultedinabrogationof
amountofexpressedRAGE. S100B ability to up-regulate COX-2 and to activate JNK and
Inaddition,lowS100BstimulatedthemigrationofRAGE- NF-(cid:4)B,againwithnoevidencefortoxiceffects(22).
overexpressingBV-2microglia(BV-2/RAGEmicroglia)(Fig.1, S100B/RAGE Recruits Diaphanous-1 in Microglia—Recent
AandC).ThissuggestedthatincreasingtheRAGEabundance workindicatedthatRAGEsignalstoRac1viadiaphanous-1to
mightmakelowS100Badditsstimulatoryeffectonmicroglia inducegliomacellmigration(65),anddiaphanous-1waspro-
migrationtothatofHMGB1. posedtoactivateSrc(66).Thus,weinvestigatedthepossibility
S100BStimulatesMicrogliaMigrationviaMultiplePathways— thatS100B-dependentRAGEengagementinmicrogliamight
We have previously shown that S100B signals to the small resultinrecruitmentofdiaphanous-1withconsequentactiva-
7218 JOURNALOFBIOLOGICALCHEMISTRY VOLUME286•NUMBER9•MARCH4,2011
S100BStimulatesMicrogliaMigrationRAGE-dependently
tionofmultiplepathwaysleadingtoNF-(cid:4)Band/orJNK/AP-1
activationandtomicrogliachemoattraction.Diaphanous-1is
an adaptor protein of the formin family that mediates the
effectsofRhoAoncellmotilityandthecytoskeleton(67–69)as
wellasofCdc42andRac1signaling(65),andCdc42mightplay
an important role in the activation of diaphanous-1 (70). We
foundthatknockdownofdiaphanous-1byRNAinterferencein
microglia (Fig. 3A) resulted in negation of S100B/RAGE-in-
ducedSrcandJNKactivation(Fig.3B)andmicrogliatransmi-
gration (Fig. 3C), but not of p38 MAPK, ERK1/2, or NF-(cid:4)B
activation (Fig. 3B). These results established that S100B/
RAGE-dependentactivationofSrcandJNKwasdependenton
diaphanous-1. However, the S100B/RAGE ability to activate
NF-(cid:4)BinmicrogliaprovednotcruciallydependentonSrcor
diaphanous-1activityandwaslikelydependentonactivationof
aRas/Rac1moduleaswellasaRas/MEK/ERK1/2and/orp38
MAPKpathway(22,23).Alternatively,thereducedactivityof
JNK that occurred in diaphanous-1 siRNA-treated microglia
(Fig.3B)mightresultinactivationofNF-(cid:4)B;itisknownthat
NF-(cid:4)BandJNKreciprocallymodulatetheiractivationstate(71,
72).
S100B Up-regulates CCL3, CCL5, and CXCL12 Chemokine
Expression via RAGE Engagement—Although the bell-shaped
patternofS100B-dependentmicrogliatransmigration(Fig.1A)
wassuggestiveofachemoattractanteffectoftheproteintoward
microglia,theresultsinFig.1CpointedtoanS100B-dependent
chemokinetic effect and/or S100B-induced secretion of che-
moattractantfactors.Thus,weinvestigatedthepossibilitythat
S100B might stimulate the expression and release of chemo-
kines.AsinvestigatedbyrealtimePCR,wefoundthatS100B
up-regulatedtheexpressionofthechemokinesCCL3(macro-
phage inflammatory protein-1a), CCL5 (RANTES), and
CXCL12(Fig.4A)butnotCXCL1orCXCL7(datanotshown).
Specifically, when used at 1 (cid:5)M, S100B up-regulated CCL3
expression with maximum stimulation at 3 h, significantly
reducedeffectat7and10h,andup-regulatedCCL5expression
withasmalleffectat1handahigheffectat3,7,and10h.S100B
also up-regulated the expression of CXCL12 at 30 min with
decliningeffectthereafter(Fig.4A).Athigherconcentrations
also S100B stimulated CCL3, CCL5, and CXCL12 expression
(supplemental Fig. S3). Thus, secretion of CCL3, CCL5, and
CXCL12 might contribute to the S100B-dependent transmi-
gration of microglia to a large extent. We concentrated on
CCL3andCCL5insubsequentanalyses.
microgliaweretreatedwith1(cid:5)MS100Bfor30min.WhererequiredBV-2
microgliawerepretreatedwith20(cid:5)MPP2(inhibitorofSrc),10(cid:5)MLY294002
(inhibitorofPI3K),or50(cid:5)MNSC23766(inhibitorofRac1)beforeexposureto1
(cid:5)MS100B.ThecellswereharvestedandprocessedforWesternblottingto
detectphosphorylatedSrc(Ser-416),ERK1/2,Akt,p38MAPK,JNK,andNF-(cid:4)B
(p65),asindicated.Shownisonerepresentativeexperimentofthree.B,con-
ditionswereasdescribedinthelegendtoFig.1DexceptthatBV-2microglia
werepretreatedfor30minwiththeindicatedinhibitorsandthentransferred
totheupperchambersformigrationassay.Theresultsareexpressedasthe
means(cid:7)S.D.(n(cid:8)3).C,S100BstimulatesmicrogliamigrationviaRAGE-de-
pendent activation of Ras, Rac1, Cdc42, and RhoA. Conditions were as
describedinthelegendtoFig.1BexceptthatBV-2microgliaweretransiently
transfectedwithdominantnegativemutant(DN)ofRas,Rac1,Cdc42orRhoA,
FIGURE2.S100BactivatesERK1/2,p38MAPK,JNK,Src(Ser-416),Akt,and I(cid:4)B(cid:1)-SR,oremptyvectorandthentransferredtotheupperchambersfor
NF-(cid:2)Binmicroglia,andS100B-inducedmigrationofmicrogliaisdiffer- migrationassay.Theresultsareexpressedasthemeans(cid:7)S.D.(n(cid:8)3).*,
entiallyregulatedbysignalingmoleculesdownstreamofRAGE.A,BV-2 significantlydifferentfromcontrol(firstcolumnfromleftinAandB).
MARCH4,2011•VOLUME286•NUMBER9 JOURNALOFBIOLOGICALCHEMISTRY 7219
S100BStimulatesMicrogliaMigrationRAGE-dependently
S100Beffectsonchemokineup-regulationweredependent
onRAGEsignalingbecausenoeffectsofS100BonCCL3and
CCL5 expression could be detected in BV-2/RAGE(cid:3)cyto
microglia (Fig. 4B), in BV-2/WT microglia pretreated with a
RAGEneutralizingantibody,orinRage(cid:2)/(cid:2)microglia(datanot
shown). Pharmacological inhibition of Src, Rac1, PI3K, JNK,
MEK/ERK1/2, p38 MAPK, or NF-(cid:4)B resulted in a significant
decrease in S100B-induced expression of CCL3 mRNA (Fig.
4C),althoughthestimulatoryeffectofS100Bonexpressionof
CCL5mRNAwasdecreasedbyinhibitionofRac1,PI3K,JNK,
orNF-(cid:4)B,butnotSrc,MEK/ERK1/2,orp38MAPK(Fig.4C).
However,knockdownofdiaphanous-1resultedinnegationof
the ability of S100B to up-regulate CCL3 and CCL5 mRNAs
(Fig. 4D). Thus, S100B-dependent engagement of RAGE
appearedtoimpactontheexpressionofthetwochemokines
viadifferentmolecularmechanisms,i.e.activationofadiapha-
nous-1/Rac1/JNK/AP-1, a Src/Ras/MEK/ERK1/2/NF-(cid:4)B, a
Src/Ras/p38MAPK/NF-(cid:4)B,andaRas/Rac1/NF-(cid:4)Bpathwayin
thecaseofCCL3andactivationofaRas/Rac1/NF-(cid:4)Band/or
JNK/AP-1 and a diaphanous-1/Rac1/NF-(cid:4)B pathway in the
caseofCCL5.
S100B Stimulates CCL3 and CCL5 Release via RAGE
Engagement—At 1 (cid:5)M but not at 10 nM, S100B stimulated
CCL3 and CCL5 secretion by microglia at 6 h (Fig. 5A), and
pre-treatmentwithaRAGEneutralizingantibodybluntedthis
effect(Fig.5B).Also,noS100B-dependentstimulationofCCL3
andCCL5secretioncouldbeobservedusingBV-2/RAGE(cid:3)cyto
microglia (Fig. 5A) orRage (cid:2)/(cid:2) microglia (data not shown).
Moreover, treatment of microglia with pertussis toxin, an
inhibitor of chemokine receptor-associated G proteins, abol-
ishedS100B/RAGE-inducedmicrogliamigration(Fig.5B).By
contrast,S100BdidnotstimulateHMGB1releasefrommicro-
gliaat6h(Fig.1C).Theseresultssupportedthepossibilitythat
S100B/RAGE-inducedmicrogliamigrationmightbemediated
bysecretedchemokines.
We also found that the conditioned medium (20 h) from
control BV-2/mock microglia caused a (cid:9)100% increase in
migrationofBV-2/mockmicroglia,comparedwiththecontrol
(Fig.5C),likelybecauseofthepresenceofbasallevelsofchemo-
kines(Fig.4A),whereastheconditionedmediumfromS100B-
treated BV-2/mock microglia caused a (cid:9)700% increase in
migration,likelybecauseoftherelativelyhighlevelsofchemo-
kinesreleasedundertheactionofS100B(Fig.5C).However,the
two conditioned media caused the same, modest (i.e. (cid:9)20%)
FIGURE3.Roleofdiaphanous-1inS100B/RAGE-dependentstimulation
ofmicrogliamigration.A,treatmentofmicrogliawithdiaphanous-1siRNA increase in migration of BV-2/RAGE(cid:3)cyto microglia, that is,
reducesdiaphanous-1expressionasinvestigatedbyrealtimePCR.B,knock- themigratoryperformanceofBV-2/RAGE(cid:3)cytomicrogliawas
downofdiaphanous-1resultsinreductionofS100B-dependentactivationof
JNK,butnotNF-(cid:4)B,ERK1/2,orp38MAPK,andreductionofbasalandS100B- not enhanced by either conditioned medium. This suggested
dependentactivationofSrc.Theconditionswereasdescribedinthelegend that S100B-stimulated RAGE signaling played an important
toFig.2A,exceptthatBV-2microgliaweretransientlytransfectedwithdiaph-
rolenotonlyintheactivationofsignalingpathwaysleadingto
anous-1siRNAornonsilencingsiRNAbeforeprocessingforWesternblotting.
Shownisonerepresentativeexperimentofthree.C,S100B/RAGE-dependent the up-regulation of the expression of certain chemokines; it
chemoattractionofmicrogliaisdependentondiaphanous-1inpart.Condi-
might also impact on the extent of expression of chemokine
tionswereasdescribedinthelegendtoFig.2AexceptthatBV-2microglia
weretransientlytransfectedwithdiaphanous-1siRNAornonsilencingsiRNA receptors and/or the molecular machinery responsible for
andthentransferredtoBoydenchambersformigrationassay.Theresultsare microglialocomotion.Thislatterpossibilitywassupportedby
expressedasthemeans(cid:7)S.D.(n(cid:8)3).*,significantlydifferentfromcontrol
(firstcolumnsfromleftinAandC).#,significantlydifferent frominternal the observation that whereas inhibition of RAGE function
control. resultedinasignificantlyreducedbasalmigration(Fig.1,Aand
B),treatmentwithpertussistoxindidnot(Fig.5B).
7220 JOURNALOFBIOLOGICALCHEMISTRY VOLUME286•NUMBER9•MARCH4,2011
S100BStimulatesMicrogliaMigrationRAGE-dependently
FIGURE4.S100Bup-regulatestheexpressionofCCL3,CCL5,andCXCL12chemokinesinaRAGE-dependentmanner.A,BV-2/mockmicrogliawere
treatedwithincreasingconcentrationsofS100Bfortheindicatedtime,andtotalmRNAwasextractedandsubjectedtorealtimePCRforquantificationofCCL3,
CCL5,andCXCL12mRNAs.B,sameasinAexceptthatBV-2/RAGE(cid:3)cytomicrogliawereused.C,sameasinAexceptthatBV-2microgliawerepretreatedwith
theindicatedinhibitors,andanalyseswererestrictedtoCCL3andCCL5.D,diaphanous-1siRNA-treatedandcontrolBV-2microgliawereanalyzedforCCL3and
CCL5expressionbyrealtimePCR.Theresultsareexpressedasthemeans(cid:7)S.D.(n(cid:8)3).
S100BStimulatesCCR1andCCR5ExpressioninMicrogliavia wasrequiredforinductionofCCR1andCCR5inmicroglia,S100B
RAGEEngagement—Inadditiontoup-regulatingCCL3andCCL5 enhancingtheeffectofRAGE;and2)lackofstimulationofBV-2/
expression(Fig.5A),S100Balsoup-regulatedtheexpressionofthe RAGE(cid:3)cyto microglia migration upon treatment with BV-2/
CCL3andCCL5receptors,CCR1andCCR5,withnoeffectson mock microglia conditioned media (Fig. 5A) was dependent on
CCR3,asmeasuredat5h(Fig.5D).Notably,basalexpressionlev- defectiveexpressionofthetwochemokinereceptors,inpart.
elsofCCR1andCCR5inBV-2/RAGE(cid:3)cytomicrogliawere(cid:9)75% S100B Causes Cytoskeleton Rearrangements in Microglia
smallerthaninBV-2/mockmicroglia,andS100Bdidnotchange in a RAGE-dependent Manner—Whereas the few BV-2/
them(Fig.5D).Theseresultssuggestedthat:1)RAGEsignaling RAGE(cid:3)cyto microglial cells found on the inferior side of the
MARCH4,2011•VOLUME286•NUMBER9 JOURNALOFBIOLOGICALCHEMISTRY 7221
S100BStimulatesMicrogliaMigrationRAGE-dependently
FIGURE5.S100BstimulatesCCL3andCCL5secretionandCCR1andCCR5expressioninaRAGE-dependentmanner.A,BV-2/mockandBV-2/RAGE(cid:3)cyto
microgliaweretreatedfor6hwithincreasingdosesofS100B.CulturemediawereanalyzedforCCL3andCCL5contentbyELISA.B,conditionswereas
describedforFig.1AexceptthatBV-2microgliawerepretreatedwithpertussistoxin(PTX)beforemigrationassay.C,conditionedmediafromcontrol(CM)and
S100B-treated(CM/S100B)BV-2/mockmicrogliastimulateBV-2/mockbutnotBV-2/RAGE(cid:3)cytomicrogliamigration.BV-2/mockmicrogliaweretreatedfor20h
withvehicleor1(cid:5)MS100B.TheculturemediawerecollectedandaddedtothelowercompartmentofBoydenchambers,andBV-2/mockorBV-2/RAGE(cid:3)cyto
microgliawereaddedtotheuppercompartmentandallowedtomigratefor6h.D,BV-2/mockandBV-2/RAGE(cid:3)cytomicrogliaweretreatedfor5hwithvehicle
or1(cid:5)MS100B,andCCR1,CCR3,andCCR5expressionlevelsweremeasuredbyrealtimePCR.Theresultsareexpressedasthemeans(cid:7)S.D.(n(cid:8)3).*,
significantlydifferentfromcontrol(firstcolumnfromleftinD).
Boydenchamberfilterexhibitedaroundand/orflatmorphol- nization of F-actin, a cytoskeleton component that, when
ogyregardlessoftheabsenceorpresenceofS100Binthebot- assembled into stress fibers, drives cell locomotion (73–75).
tomwell,BV-2/mockmicrogliaand,toalargerextent,BV-2/ Lamellipodiaformationatonecellside,whichdependsonRac1
RAGE microglia that had transmigrated in the presence of activationandisindicativeofmigration(73–75),wasdetected
S100B exhibited cell processes that are typical of highly acti- rarely in BV-2/RAGE(cid:3)cyto microglia, irrespective of the
vatedmicroglia(datanotshown).Toinvestigatethispointin absenceorpresenceofS100Bupto1(cid:5)M;onlyextremelyshort
moredetail,weexposedthethreeBV-2microglialclonesused cellprotrusionsweredetectedinasmallpercentage((cid:9)6%)of
inthepresentstudydirectlytoincreasingdosesofS100Band thecells(Fig.6).Bycontrastandinagreementwiththemigra-
analyzedthembyrhodamine-phalloidinstaining.Thisproce- tionresultsinBoydenchambers(Fig.1B),BV-2/mockmicro-
dureallowsforthevisualizationoflamellipodiaandtheorga- gliashowedlamellipodiain(cid:9)28,(cid:9)40,and(cid:9)95%ofthecellsin
7222 JOURNALOFBIOLOGICALCHEMISTRY VOLUME286•NUMBER9•MARCH4,2011
S100BStimulatesMicrogliaMigrationRAGE-dependently
theabsenceofS100Bandinthepresenceof10nMand1(cid:5)M
S100B, respectively (Fig. 6A). Lastly, BV-2/RAGE microglia
showedlamellipodiain(cid:9)35,(cid:9)70,and100%ofthecellsinthe
absenceofS100Bandinthepresenceof10nMand1(cid:5)MS100B,
respectively (Fig. 6A). Because these morphological changes
were detected as early as 3 h after exposure of microglia to
S100B, it is possible that they reflected the S100B-stimulated
abilityofRAGEtoenhanceRac1abilitytoinducelamellipodia
formation and RhoA/ROCK-dependent stimulation of acto-
myosincontraction(and,hence,cellmigration)inadditionto
theS100B/RAGEabilitytostimulateRac1signaling-dependent
chemokine expression. It is known that RAGE can signal to
Rac1inseveralcelltypes(22,47,49,65)andthatRAGEsignal-
ing to Rac1 is required for RAGE-mediated C6 glioma cell
migration(65).Thus,theS100B-stimulatedabilityofRAGEto
activateRac1mightserveadualfunctioninmicroglia,produc-
ing the cytoskeleton rearrangement required for cell shape
changes during locomotion and inducing chemokine expres-
sionandrelease.
DISCUSSION
WehaveshownthatS100Bstimulatesmicrogliatransmigra-
tioninBoydenchambersinaRAGE-anddose-dependentman-
ner.S100Benhancedthemigrationofprimarymicroglia,BV-2
microglia, and BV-2 microglia overexpressing RAGE but not
BV-2 microglia overexpressing a RAGE mutant lacking the
cytoplasmic and transducing domain, microglia pretreated
withaRAGEneutralizingantibody,orRage(cid:2)/(cid:2)microglia.The
stimulatory effect on microglia migration was detected using
S100Batproinflammatorydoses(e.g.1(cid:5)M)likethoseshownto
bepresentintheextracellularmilieuincaseofbraindamage(8,
9).Noeffectsoftheproteinatnanomolarconcentrationswere
detectedunlesstheamountofexpressedRAGEwasincreased,
inwhichcasenanomolardosesofS100Befficientlystimulated
microgliamigration.Theselatterfindingsmightbeexplained
bytheobservationthatRAGEundergoesligand-inducedoligo-
merization,whichappearstoberequiredforRAGEsignaling
(62, 76), or that RAGE ligands stabilize naturally occurring
RAGE oligomers, an event also proposed to be necessary for
RAGEsignaling(78);itispossiblethatincreasingtheamountof
expressed RAGE might either favor (low) S100B-dependent
RAGEoligomerizationandsignalingorincreasetheprobability
thatpreformedRAGEoligomersbecomestabilizedtherebysig-
naling. Overall, the fact that relatively high concentrations of
S100BarerequiredforRAGE-dependentstimulationofmicro-
glia migration suggests that the ability of RAGE to activate
downstream signaling pathways up-regulating chemokine
concentrationsofS100B,andfixed.Thecellsweresubjectedtoimmunofluo-
rescenceusingamonoclonalanti-tubulinantibody(green)andthentreated
withrhodamine-phalloidintostainF-actin(red).Thenucleiwerecounter-
stainedwithDAPI(blue).Shownisonerepresentativefieldforeachcondition.
A quantitative analysis is also shown. B, schematic representation of the
molecular mechanism whereby S100B stimulates microglia migration via
RAGEengagement.ThroughmultiplepathwaysS100B/RAGEstimulatesthe
expression (arrow) and release of chemokines that in turn chemoattract
microglia.Inaddition,S100BactivatesRAGE/diaphanous-1/Ras/PI3K/RhoA/
FIGURE6.A,S100BinducesshapechangesinBV-2microgliainaRAGE-de- ROCK and RAGE/diaphanous-1/Cdc42-Rac1 pathways that cause the
pendentmanner.BV-2/mock,BV-2/RAGE(cid:3)cyto,andBV-2/RAGEmicroglia cytoskeletonrearrangementandcellshapechangesrequiredformicroglia
were cultivated on glass coverslips, treated for 3 h with increasing motility.
MARCH4,2011•VOLUME286•NUMBER9 JOURNALOFBIOLOGICALCHEMISTRY 7223