22 29 —1 Universidad Complutense de Madrid (Dpto. de Bioquímica y Biología Molecular 1) REGULACIÓN DE LA SECRECIÓN DEL FACTOR DE CRECIMIENTO NERVIOSO EN CÉLULAS GLIALES. MECANISMOS DE TRANSDUCCIÓN A TRAVÉS DE MENSAJEROS LIPÍDICOS ¡1 ID DII II MUllID *5 ! 098396 UNIVERSIDAD COMPLUTENSE TESIS DOCTORAL por Ismael Galve Roperh Directores: Dra. Inés Díaz-Laviada Dr. Amador Haro Ramos
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22 29 —1
UniversidadComplutensedeMadrid
(Dpto. deBioquímica y Biología Molecular 1)
REGULACIÓN DE LA SECRECIÓNDEL FACTOR
DE CRECIMIENTO NERVIOSOEN CÉLULAS GLIALES.
MECANISMOS DE TRANSDUCCIÓNA TRAVÉS DE
MENSAJEROSLIPÍDICOS
¡1 ID DII II MUllID*5 ! 098396UNIVERSIDAD COMPLUTENSE
TESISDOCTORAL
por
IsmaelGalveRoperh
Directores:
Dra. InésDíaz-Laviada
Dr. AmadorHaroRamos
V0 B0 de los Directores:
Este trabajo de investigación ha sido realizado en el Departamento de
Bioquímicay Biología Molecular 1 de la Facultad de CienciasBiológicas de la
Universidad Complutensede Madrid, con la dirección de la Dra. Inés Díaz-
Laviaday el Dr. AmadorHaro Ramos.Parte de la investigaciónfue realizadaen
la unidad 298 del INSERM de Angers (Francia) en colaboración con el Dr.
Philippe Brachet y en la unidad 466 del INSERM de Toulouse (Francia) en
colaboracióncon el Dr. Thierry Levade,a los que quiero manifestarmt s¿ncero
Abstract Add¡t¡on ofphosphat¡dylcholine-hydrolyzingphospho-upase C (PC-PLC) to cultured glial celis increased tbe leveis ofnene growth factor (NGF) mRNA and the amount of ceil-se-creted NGF. The effect of PC-PLC wns 2.5 times higher (hanthat elicited by 4¡3-phorbol 12¡3-myristate 13a-acetate. In celísin which protein kinase C (PKC) was fully inhihited or don-regulated, (he effect of PC-PLC ivas reduced — though still evi-dent — and similar to that exerted by sphingosine. Results thusindicate that PC-PLC induces the synthesis of NGF by glial celisby a PKC-dependent and PKC-independent mechanisms.
Nerve growth factor (NGF) is a ncurotrophic protein re-qtiired for thc development and survival of several populationsof neurons [1]. In the central ncrvous system, NGF is synthe-sized by certain neurons, but during brain devclopnient andunder diffcrent paihological alterations of ihe ncrvous system,there isa local increase in the production of NGF by astrocytes[2—5].Thus, substantial present effort is directed toward anundcrstanding of the influences that control NGF synthesis,becauseof thc possibility that increasing the endogenous syn-diesisof NGF might provide clinical advantage in degenerativediseasesof central nervous system [6]. However, little is knownabout the elTectors that induce the synthesis of NGE by thosecelís and about how this synthetic process is up-regulated. Itseernstlíat several NGF-inducing agents act through differentpatlíways. One of them is the signalling network involving theactivation of protein kinase C (PKC>. Studies performed withcultured astrocytesand fibroblasts have shown that 4/3-phorbol12/3-myristate lh-acetate(PMA>, a welI-documented PKC ac-tivator, induces dic synthesis and secretion of NGF, and thiseffect is counteracted by PKC inhibitors like H-7 or H-9 [7,9].PMA induces the expression of c-fos, the product of whichenhancesNGF synthesisby the same route as PMA [5,9,10].
PKC is physiologically activated by diacylglycerol (DAG)resulting from agonist-induced hydrolysis of inositol phosphol-ipids [II]. However, this DAG rapidly disappears and seenis tobe responsible for a transient activation of PKC [12]. In con-trast,hydrolysisof othermembrane phospholipids, particularlyphosphatidylcholine(PC), produces DAG at a relatively laterphase.This DAG may be the responsible for the sustainedactivation of PKC that leads to long-term cellular responses
*Correspondingauthor.
[13]. Mechanisms of cellular responseto PC hydrolysis havebeen demonstrated to occur ja many cdl types (reviewed in[12,141).Despiteextensivestudies, the biocheniical mechanisnisunderlying signal-induced activation of PC breakdown are stillpoorly defined.PhospholipaseC (PLC)-catalyzedhydrolysisofPC has been shown to be activated in response to a number ofagonists [15],in transiormed celís [161and iii signalling cascadestriggeredby growth factors [17]. PC-PLC provides a positivesignal for PKC by inducing its translocation to membranethough not its down-regulation [161.It has also been shown thatPC hydrolysis by a PC-PLC mimics the ability of carbachol toinhibit adenylyl cyclase [18].Furthermore, a role of PC-specif’cphospholipase O (PLO) in signal transduction has been de-scribed [13,19].
In the present study we investigated thc possible relationshipbctwecn PC hydrolysis and PKC activation in the control ofNGF synthcsis by astrocytcs. Previous experimental evidenceshowed that activation of PKC with PMA stiniulated NGFsynthesis by glial celís [7]while exogenously added PC-PLC wasable to activate PKC in fibroblasts [18]. This provinded us atool to investigate whether NGF synthesis by glial celís dependson PC breakdown,
2. Experimental
2. 1. Culture con~li¡ionsPrimary cultures of rat brain glial celís werc prepared from cerebral
hemispheresof l—2-day-old rat pups. Cerebral hemisphcres wcre dis-sected in phosphate buffered satine (PBS> supplcmcnted with 0.33%glucose and treated with trypsin (5 mg/mI, 30 mm at 37’C). Trypsin wassubsequently inhibited by Ihe addition of [0%foetal calf serum (FCS),before treatment with DNase 1 (lOpglml, 5 mm at 370C). Brain celíswere dissociated by fluxation with a Pasteur pipcttc and ncxt sedi-mented by low speed centrifugation (1000 xg, 5 mm). The pellet wasgently resuspended, and celís were seeded at a density of 3 x lo4 cellslcml. AII plasdc supports were previously coated with 5 pghnl poly-L-ornithine in water. Celís were cultured for 3 weeks in basal mediumcontaining a mixture of Dulbeccos modilied Fagle medium and Ham’sFI? (1/1, vlv), supplemented withO.66% glucose, Spglml streptomycin,5 U/ml penicillin and 10% FCS. Three days before the experiment, FCSwas removed and celís were transferred to a chemically defined mediumconsisting of basal medium supplemented with 25 pg/ml insulin, lOOpg/ml human transferrin, 20 nM progesterone, 5OpM putrescine and30 nM sodiumselenite. Immunocytochemical studies showed that 90%of cultured celís were positive for glial fibrillary acidic protein, indicat-ing that cultures were largely enriched in astrocytes.
2.2. Assayof NGFaud NGF,,,RNACdl supernatants were collected 24 h after the different treatments
described iii the text and diluted la 1 volume of PBS containing 0.1%Tween-20 and 0.5% gelatin. NOF released by the celís was assayed intriplicate. with a double-site-ELISA, using a monoclonal anti-NGFantibody, coupled or not to t-galactosidase (Boehringer Manheim),according to [20].Northern blot analysis was performed by standardprocedures [~1~After 6 h of treatment, total RNA was extracted from
celísby the LiCI/urea method. Glyoxal-treated RNAs were fractionated‘o agarose gel, transíerred to a nylon membrane by capillary blotting,and hybridized with a “P-Iabelled NGF cDNA and a amyloid precur-sor protein (API’) cONA, used to verify the loading of the geis.
2.3. Idení/ficatio,, of PKC by innnunobloti~igGlial celís (treated as described in the legeod to Fig. 3) were scraped
oto 0.5 ml of ice-cold 50 mM Tris-bICI, pH 7.4,5mM EDTA, lO mM2-mercaptoethanolcontaining 1 /ig]ml leupeptin, ¡ pg/ml aprotinin, lOpg/ml benzamidin, 5 pg/ml soybean tripsin inhibitor and 0.1 mMPMSE, and rapidly centr¡fuged at 5.000xg br 5 mio to eliminatedebries. Following denaturation in SOS sample bulfer, proteins wereresolved in a 8% SOS-PAGE and transferred electrophoretically ontonitrocellulose membranes (Bio-Rad). Membranes were blocked with5% of fat-free dried milk and incubated with 2 pg/ml of specific anti-PKC antiserum (done MC5, Amersham International). Following jo-cubation with horseradish conjugated anti-mouse antibody, the blotswere developed with an enhanced chemiluminescence detection kit(Amersham International).
2.4. Ot/er c/,e,niealsTissueculture plastic wares were purchased from Nunc (Denmark),
culture media from Gibco (France) and FCS fron, Purobio (France).PC-PLC and bisindolylmaleimide (OF l09203X) were fron, Calbio-chem (USA). Sphingosine and most other reagents were obtained fromSigma Chemicals (USA).
3. 1{esults
3.]. PC-PLC enhancesMe synthesisof NGFCultured glial celís were treated for 24 E with different doses
of exogenously added PC-PLC from Bacillus cereusand NGFlevels were determined in the supernatant media 24 h later.Results presented in Fig. 1 show a dose-dependent and satura-ble increase in the extracellular levels of NGF, which becomemaximal at 0.5 U/mI of PC-PLC. Dosesover ¡ U/mI were toxicto celís. Stimulation of celís with lOO nM of PMA was quantí-tatively lower than that induced by PC-PLC(Fig. 1).
In order to determine whether PC-PLC Fiad a correspondingon the pool ofNGF transcripts, total RNAwas extracted fromcelís pretreated with or without the enzyme. Northern blotanalysis showed that PC-PLC produced a marked increase in
400 -
o,o-LA-oz 200 -
o No PMA
Ápc—PLC pc—puc Pc—PLC Pc—PLc
odd¡tion ~O0nM O.IU/rnl O.25U/rnl O.5U/mI Iu/mIFig. 1. Effect of increasing concentrations of PC-PLC on the produc-tion of NGF. Glial celis were incubated with no additions, PMA (lOOnM), or PC-PLC (0.1 U/mí, 0.25 U/mI, 0.5 U/mI and t U/mí). Themedium was collected after 24 h and NGF was quantitied by donhie-site ELISA. Values are means ±S.D. of 3 independent experimentsassayed in triplicate.
NG F
A PP
1 2 3 4 5
Fig. 2. Accumulation ofNGF mRNA in astrocytes. Constant aniountsof total RNAs were fractionated on ari agarose gel, blotted and hybrid-ized with a radiolabelled NGF cDNA probe. Lane 1: control; lane 2:EMA lOO nM; lane 3: PC-PLCO.5 U/mí; lane4: celis treated with EMA500 nM for 48 h and te last 24 h with EMA lOO nM; and lane 5: cellstreated with EMA 500 nM for 48 h and Pie Iast 24 h with PC-PLC0.5U/ml. Essentially identical results were obtained in two independentexperiments.
NGF niRNA, ascomparedto controls, indicating that PC-PLCis able to promote NGF synthesis (Fig. 2, lanes 1 and 3). Thisetfect is more potent than that elicited by PMA (Fig. 2, lane 2).
3.2. The induction of NGFsyníhesisby PC-PLC is pací/oíl>’dependeníon PKC
To investigate whether PKC activation is required for thePC-PLC-triggered induction of NGF synthesis, PKC wasdown-regulated in glial celis by pretreating the cdl cultures witha supramaximal dose ofPMA (500 nM) for 48 E. This treatmentcompletely removed PKC from astrocytes as determined byWestern blot analysis with a specific monoclonal anti-PKCantibody (Fig. 3). Interestingly, induction of NGF synthesis byPC-PLC emerged reduced but not abolished, in celis in whichPKC wasdown-regulated (Table I), whereas NGF induction
Table 1EffectofPKC inhibitionanddown-regulationandeffectofsphingosineon the levels of ceIl-secreted NGF measured by ELISA
Additions NGF pg./ml
None 34.68±lOEMA lOO nM 196.51 ±90PC-PLC 0.5 U/mI 490.54±119EMA lOO nM + SIM 2pM 3141±3PC-PLC 0.5 U/ml + BlM 2pM 130.30±23EMA 500 nM (48 h) + EMA lOO nM 32.87±2EMA 500 nM (48 h) + PC-PLC 0.5 U/ml 171.50±27Sphingosine lo pM 164.80±l2
The values represent the means ±S.E.M. of 2 independent experi-ments assayed in triplicate.
302
ID. Laviada ev al. IFEBSLeliers 364 (1995)301—304 303
1 2 3 4
97.4K >
69K h-
Fig. 3. Western blot analysis of PKC levels in cdl lysates. Lane 1:control; lime 2: PMA lOO nM; lane 3: celís treated with PMA 500 nMfor 48 h and the last 24 h with PMA 100 nM; and lane 4: cells treatedwith PMA 500 nM for 48 h and the last 24 h with PC-PLC 0.5 U/nl.
as elicited by PMA was completely abolished in down-regu-lated celís. This effect was also observed at the niRNA leveis,as determined by Northern blot analysis (Fig. 2, lanes 4 and 5).
Further experimental evidence to support these data wasobtained by thc use of bisindolylmaleimide (BlM), a potent andhiglyspecific inhibitorofPKC[21]. Inhibition ofPKCby BlMcomplctcly abolished the Increase ni NGF levels induced byPMA, whcrcas treatrncnt olcelís with BlM only reduced in partthe increase in NOF levels produced by PC-PLC (Table 1).Hence, PKC seems to be only partially involved in the NGFinducing response elicited by PC-PLC.
3.3. Splz/ugosine mercases l/w producílon of NGFIn order to study whether another mechanism of signal
transduction could be involved in the synthesis of NGF, wedetermined the effect of sphyngosine on NGF secretion by glialcelís. Results reported in Table 1 show that sphingosine in-creases the extracellular levels of NGF to ¡he same extent asPC-PLC does in PKC down-regulated celís.
4. Discuss¡on
Despite the recent efforts performed to unravel the mecha-nismsof NGF induction, the signal-transducing pathways lead-¡ng to the synthesis of this neurotrophic protein remain largelyunknown. Many ofIhe agents which are able to stimulatc NGFsynthesis are dependent on PKC activation [9]. Howcvcr, ster-oids and 1 ,25-dihydroxyvitamin D3 have been shown to induceNGF protein and mRNA by a mechanism difYerent to serun,and PMA, and independent of PKC and c-fos gene activation[22,24].Although this indicates ¡bat the the control of NGFsynthesis does not depend on a uniquc pathway, the diffcrentputative mechanisms up-regulating NGF synthesis operate byincreasing the steady-statc levels of intracellular NGF mRNA.
in this repor¡ wc demonstrate that a PC-PLC induces thesynthesis of NGFby glial celís by a mechanism ¡bat is partiallydependent on PKC activation. Thc fact that PC-PLC is able toproduce an accumulation of NGF transcripts indicates that atleast part of the stimulatory etTect takes place at ¡he pre-trans-lational level. One of the particular features of thc action ofPC-PLC, alone or in combination with phosphatidatephosphohydrolase, is that ¡he increase in intracellular DAGconcentration is prolonged, indicating that it may be responsi-ble for long-term cellular responses [13]. Our results are inagreement with this notion since NGF is generalíy considered
to becoded by a late expression gene. Results also indicate thatPC-PLC acts by at least two difl’erent pathways, one dependenton PKC and another independent of PKC. Wc and othcrs havepreviously found that PC-PLC triggers sorne cellular responses,namely induction of DNA synthesis or activation of nucleartranscription factor systems by a PKC-independcnt mechanism[17,24]. It has been described that PC-PLC is coupícd ¡o asphingomyelinasc that catalyzes the breakdown of sphingomy-clin into ceramide [24]. A second messenger function for thesphingomyelin cycle and ceramide in the control of ccli growth,differentiation and apoptosis has beenrecently proposed [25].Our data show that sphingosine induces NGF synthesis ¡o asimilar extent than the PKC-independent mechanism elicitedby PC-PLC. TEe effect exerted by sphyngosine may be medi-ated by its conversion to ceramide, as it has been suggested forother biological responses mediated by sphingosine [26].On theother hand, vitamin D3, which induces NGF by a PKC-inde-pendent mcchanism, has been shown to elicit an early andreversible hydrolisis of sphingomyelin with a concomitant gen-eration of ceramide [27]. Two mechanisnis may be thereforeinvolved in the generation of NGF by astrocytes, one repre-sented by serum and DAG acting through PKC, and anoiherone induced by vitamin D3 acting through the sphingomyelincycle. Our results suggest that thc breakdown of PC by a PLCmay trigger both mechanisms.
Áck,,oiíledg>,w~,is.The authors thank Drs. M. Guzmán and D. Wionbr Iruitful discussions and critical reading of the manuscript. This warkhas been supported by a coopcration program betwecn INSERM(France) and CSIC (Spain).
References
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[21Bakhit, C., Armanini, M., Bennett, G.L., Wong, W.L.T.. Hansen,SE. and Taylor, R. (1991) Brain Res. 560, 76—83.
[3] Lu, B., Yokoyama, M.. Dreyfus, CF. and Black, IB. (1991)J. Neurosci. II, 318—326.
[4] Alter, CA., Armanini, M., Dugich-Djordjevic. M., Bcnnett, OL.,Williams, R., Fcinglass, 5., Anicetti. y., Sinicropi, D. and Babbit,C. (1992)3. Neurochen,. 59, 2167—2177.
[5] Hengerer, B., Lindholm, D., Heurnann, R., Ruther, U,. Wagner,EF. and Thoenen, H. (1990) Proc. Natí. Acad. Sci. USA 87,3899—3903.
[6] Carswell 5. (1993) Exp. Neurol. 124, 36—42.[7] Neveu, 1., Jehan, F.. Houlgatte, R., Wion, D. and Brachet, P.
(1992) Brain Res. 570, 316—322.[8] Wion, D., Mac Grogan, D., Houlgatte, R. and Brachet, P. (1990)
FEBS Lett. 262, 42-44.[9] D’MeIlo, SR. and Heinrich, 0. (1990)3. Neurochem. 55, 718—721.
[¡O]Mocchetti, L., De Bernardi, MA., Szekely, AM., Albo, H.,Brooker, 0. and Costa, E. (1989) Proc. Natí. Acad. Sci. USA 86,3891—3895.
[II] Dennis, EA., Rhee, SG., Billah. M.M. and Hannum, YA. (1991)FASEB 3. 5, 2068—2077.
[19] Nakamura, S. and Nishizuka, Y. (1994) J. Biochem. 115,1029—1034.
[20] Houlgatte, R., Mallat, M., Brachet, P. and Prochianta, A. (1989)J. Neurosci. Res. 24, 143—152.
[21] Toullec, D., Pianetti, P., Coste, H. Bellevergue P., Grand-Perret,T., Ajakane. M., Baudet, V., Boissin, P., Boursier, E., Loriolle, F.,Duhamel, L., Charon, D. and Kirilovsky J. (1991) J. Biol. Chem.266. 15771—15781.
[22] Wion, D., Mac Grogan, D.. Neveu, 1., Jehan, F., Houlgatte, R.and Brachet, P. (1991) J. Neurosci. Res. 28, 110—114.
Astrocytessynthesizenenegrowthfactor (NGF) iri responseto pro-inflammatory cytokines.To further study
the signalingmechanisminvolvedin this induction of NEW production,the sphingomyelin(SM) pathwaywasstuclied.Addition of exogenousneutralSMase(Staphylococcusaureus)or C2-ceramideto primary cultures ofnewbornrat corticalastrocyteselicited adose-responseincreaseof NGF synthesis,with maximal effect at 1U/ml anil 25 ~tM,respectively. Induction of NGF synthesis by SMase and ceramidewas shown to beindependentof classicalPKC activity. Intracellular cAMP-raisingagents,such asforskolin and 3-isobutyl-1-methylxanthine,partially preventedthe SMase- and C2-ceramide-inducedsecretionof NEW to the cclisupernatant.PD098059andapigenin, inbibitors of the mitogen-activatedprotein (MAP) kinasepathway,produceda dose-responseinhibition of the SMase-andC2-cer-inducedreleaseof NGF. Thisobservationpointste the possibility that regulation of NGF synthesisandsecretionby the SMasepathwaymay be mecliateddownstreamby the MAP kinasecascade.As a matterof fact, pretreatmentof astrocyteswith SMaseor Ca-ceramide led to an increasedphosphorylationof raf-1. Moreover, MAP kinase activity was enhanceclinastrocytestreatedwith SMaseor both ceramiclesIn conclusion,resultasuggestthat the SMasepathwaymaycontrol NGF synthesisin the centralnervoussystem,andraisethe possibilityof aninvolvementof the MAPkinasecascadein thisprocesa.Copyright1997 ElsevierScienceB.V.
1. IntroductionWithin the last decadesphingolipidshave emergedas activeparticipantsin the regulationof a wide
rangeof cellular responses.In particular, sphingomyelin(SM) breakdownhasbeenimplicated in theregulation of celí growth, diflerentiation, transformation and apoptosis. SM is hydrolyzed bysphingomyelinase(SMase), releasing diffusible ceramidesthat function as second messengersinsignaling pathways [reviewed in 12, 13, 33, 34]. Ceramidesmay modulate the activity of differentserine/threonineproteinkinasesasceramide-activatedprotein kinase (CAPK) andprotein kinaseC ~,
aswell asceramide-activatedprotein phosphatase(CAPP) [13]. Downstreamsignalingoccuraat least inpart through the mitogen-activatedprotein (MAP) kinasecascadeand NFKB translocation[2, 13, 29, 33,38]. An increasing number of ceil-surface receptorsare currently being show to generatesignals thattrigger SM breakdown.Accumulationof ceramidesderived from SM hydrolysishasbeendescribedtooccurin responseto different extracellular agentsincluding la,25-dihydroxyvitamin D3, tumor necrosisfactora (TNFa), interleukin-1[3 (IL-1[3), IL-2, andinterferon-y [13, 33, 34].
TNFa and ILs are pivotal mediatorsof inflammation and immuneresponsesin the central nervoussystem(CNS). Such responsesare followed by the rapid activationof brain residentmacrophagesandsubsequentactivationof astrocytes,that constitutethe mostabundantnon-neuralcelísin the CNS [23].Astrocytes play an important role in a wide range of functions which are essentialfor adequatedevelopment,functionality, and pathologicalprocessof the CNS [3]. It is now well establishedthatastrocytesareinvolved in theinflammatoryresponsein the brainlike that occuringin multiple sclerosisand septicshock [22]. Oneof the responsesof reactiveastrocytesduring inflammatoryprocessand afterinjury is the synthesisand releaseof neurotrophicfactorssuch asnerve growth factor (NGF) [4, 6, 40].NGF production by glial celís during such circunstancesmay limit the extent of neuronalloss andpromoteregenerativeprocessesto restoreneuronalfunction [28, 39]. NGF synthesisby astrocytesis ahighly regulatedprocesswhich includescomplexinteractionsamongdifferentsignalingpathways.Pro-inflammatorycytokines,IL-113 andTNFa arewell known stimulatorsof NGF synthesis[14, 27, 28]. Onthe otherhand, lipopolysaccharide,the molecule responsiblefor the iniflammatory responseduring theseptic shock, is also a potent inducer of NGF synthesis and secretion by astrocytesand brainmacrophages[11, 21]. It has also been describedthat la,25-dihydroxyvitamin Da stimulates NQFsynthesis and release by cultured glial celis [25]. As stated aboye, both cytokines and la,25-dihydroxyvitamin D3 havebeenreportedto induce ceramideaccumulation[13, 26]. We havepreviouslydescribedthe involvementof aphosphatidylcholine-phospholipaseC (PC-PLC) in the regulationof NGFsynthesisby astrocytes[19], which in turn has been shown to mediateTNFa activation of NF-KBthrough SM breakdown [30]. Therefore, it seemsinteresting to study the possible function of SMbreakdownandceramidereleasein theregulationof NGF synthesisby astrocytes.
We reportherethe stimulatoryeffect of exogenousadditionof both, neutral SMaseandcelí-permeableceramideson NGF synthesis and secretion in primary cultures of newborn rat astrocytes.Thecharacteristicsof this NGF regulatory pathway were studiedwith specialemphasison the possibleeffectson theMAP kinasecascadeand its crosstalkwith othersignaltransductionpathways.
2. Materials andmethods2.1. MaterjaisTissue culture plastic wares and fetal calf serum(FCS) were from Nunc (Denmark). Culture media
wasfrom Biowhitakker (Belgium). NeutralSMase(5. aureus),3-isobutyl-l-methylxanthine(IBMX) and4[3-phorbol 12j3-myristate(PMA) were from Sigma Chemicals(USA). BisindolylmaleimideOF 109203X(BlM), forskolin (FK) and celí permeableceramide analogswere from Calbiochem (USA). 32Pi andDMEM Pi free were from ICN PharmaceuticalsInc. (USA). Anti-NGF monoclonalantibodieswere fromBoehringerManheim (Germany).Anti-raf-1 polyclonal antibody was from Santa Cruz Biotechnology(USA), and anti-rabbit agarose-linkedIgO from TransductionLaboratories(UK). p42/p44MAP kinasepeptidesubstrateandreagentsfor kinaseassaywerefrom Amersham(U.K.).
2.2. Pri¡nary culturesofctstroglial cellsCortical astrocyteswere derivedfrom 1-2 day oíd rats and culturedas previously described[19]. Celis
wereseededat a densityof 3x104cells/cm2on plastieplatespreviouslycoatedwith 5 gg/ml dl-polyornithine
in water. Celís were culturedfox 3 weeksin basalmediumconsistingof a mixture of DulbeccosmodifiedEagle medium (DMEM) and Ham’s F12 (1:1, y/y), with 0.66% glucose, 5 pg/ml streptomycin,5 U/mlpenicilhin, andsupplementedwith 10% (FCS).The primary culturesconsistedof 95% astrocytesasjudgedby immunocytochemicalstainingof glial fibrilary acidicprotein.
Three daysbeforethe experiment,the FCScontainingmediumwasremovedandcelísweretransferredto a chemically-definedmedium consisting of serum-freebasal medium supplemented.with 25 pg/mlinsulin, 50 gg/ml humantransferrin,20 nM progesterone,50 pM putrescine,and30 nM sodiumselenite.
2.3. RNAanalysisami enzjane-linked¿inmunosorbeníassay(ELISA)Afler the indicatedtimes of treatmentRNA extractionwas carriedout accordingte the LiC]/urea method.
Northernblot analysis was algo performedby standardprocedures.Glyoxal-treatedRNA was fractionatedinagarosegela, transferredte Hybond-Nmembranesby capillaryblotting,andhybridizedseriallywith a~P-labelledprobe of mouse NGF cDNA. The NOF probe was te 917 bp NGF DNA cloned by Scott et al. [31].
2
1 n duction of nerve growth factor synthesis by sphingomyelinase and ceramide in primary astrocyte cultures
Standardizationof RNA loading was routinely controlled by hybridization of the blots with amyloidprecursorprotein cDNA probe [32]. Densitometricanalyseswere performedwith Phosphorímager445 SI(MolecularDynamics).
Fox extracellular NGF determination, ceil supernatantswere collected 24 hours after treatmentasdescribedin the text, diluted in onevolume of phosphatebuffer salme(PBS)containing0.1%Tween20 and0.5% gelatin, andconservedfrozenuntil quantitation.NGF releasedby thecelis wasassayedin triplicate,by a double-siteELISA, usinga monoclonalanti-NGFantibody,coupledor not to ~-galactosidase accordingto anexperimentalprotocoldescribedbefore[19].
2.4. Raf-1irnrnunoprecipitationImmunoprecipitationwas performedas described previously [10]. Briefly, celís cultured in 57 cm2
dishes,were transferredto chemically definedmediumthreedays before the experiment.The mediumwas replacedby Pi-free DMEM and incubatedfor 4 hours with 100 j.tCi 32Pi. Celís were stimulatedasdescribedin the text. Lysatesweresubsequentobtainedby treatingcelíswith a buffer containing50mMTris pH 7.5, 2 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1 mg/ml bovine serumalbumin, and 150 mMNaCí. Immunoprecipitation was carried out by incubation with 7.5 pg/ml of anti-raf-1 polyclonalantibody andprecipitationwith anti-rabbitagarose-linkedIgO. Phosphorylationwasdeterminedin theimmunoprecipitatesby SDS-PAGEandautoradiographyof the gels. Gelswere previously stainedwithCoomasieblue in order to verify the loading of the geis.AutorradiographicFuji-films were subjectedtodensitometricanalysisusingtheSigma-Gelprogram.
2.5. MAPkñtaseassayAstrocyteswereincubatedfor 15 mm at 370C with agentsshownin Table 2, andthereactionterminatedby washingwith ice-cold PBSand adding500 pl of lysis buffer containing10 mM Tris (pH 7.4), 150 mMNaCí, 2mM EGTA, 2 mM DTT, 40 pg/ml digitonin, 1 mM orthovanadate,1 mM PMSF, 10 pg/mlleupeptin, and 10 pg/ml aprotinin, at 40C. Cellular debriswereprecipitatedat 25000x g fox 20 mm andMAP kinaseactivity wasmeasuredin thesupernatant.Extracts(15 pl) were thenassayedby adding10pl. of the substratebuffer (containing 6 mM substratepeptide, 75 mM Hepes, 300 pM sodiumorthovanadate,and0.05% sodium azide,pH 7.4), and 5 pl of ATP buffer (containing0.3 mM [y-32P]ATP(300 pCi/mi) and90 mM MgCl2). ASter 30 mm incubationat 30~ C., reactionwasterminatedby adding10pl of 300 mM orthophosphoricacid. Thirty pl of each samplewas spottedonto phosphocellulosediscs,washedtwo times fox 2 mm in 1% acetieacid andthen twicefor 2 mm in distilled water. Radioactivityofeachdiscwasdeterminedby scintillation counting.
3. Results3.1. Enhaucedsynthesisan.d secretion.of NGF by exogeuousadd¿tionofneutral SMaseand C2-cerarnideIn orderto study the possibleinvolvement of the SMasepathwayin the regulationof NGF synthesis,
primary culturesof astrocyteswereexposedto increasingconcentrationsof neutralSMase.An increasedsecretionof NGF to thecelí supernatantwasobservedat 250 mU/ml with maximal activationat 1 U/ml(15-foldincrease)(Fig. la). Time-courseexperimentsshowedthat maximal concentrationof NGF on thesupernatantpeakedat 72 h of treatment(Fig. 2). In order to know whether the effect observedwithSMasewas dueto the releaseof the secondmessengerceramide,the syntheticcelí-permeableanalog N-acetylsphingosine(Ce-cer)was used.Treatment of celís with C2-cer also enhancedNGF synthesisandsecretionfrom celís, with maximal stimulation observedat 25 pM C2-cer (Fig. ib). The specificity of C
2-cer effect was supportedby the fact that Ce-dihydroceramide,an inactive analog, had no effect onsecretedNGF at the sameconcentrations(Table 1).
Northernblot analyseswereusedto testwhetherthe contentof NGF mRNAin treatedcells waselicitedin concert.Fig. 3 showsthat after 24 h of treatment,NGF mRNA levelswerehigher in bothSMaseandceramide-treatedcelís.Therefore,it seemsthat ceramide-inducedNGF secretionis alsoaccompaniedby an increasein the mRNA levelsof theneurotrophicfactor.In fact, treatmentof celís inthe presenceof actinomycinO andcycloheximide,inhibitora of DNA transcriptionandproteinsynthesis,respectively,abrogatedincreasedlevelsof NGF in thecelí supernatantelicited by SMaseandceramide(datanot show).
3
1. Oalve-Roperhetat.
fico
700
000
~ 500
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200
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‘1
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100
:100
250
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o
Fig. 1 Dose-responseinduction of secretedNGF by exogenousneutral sphingomyelinaseaud C2-ceramide.Primary cultures ofastrocytes were stimulated with increasing concentrations of SMase (Fig. la) and N-acetylsphingosine (Fig. ib) for 24 h.Extracellular NGF was assayed with a double-siteELISA. Dataare meana±S.D. from threeindependent experiments. Asterisksindicate significant differences from the corresponding controis (* P<0.05, ** P<O.Ol, by Studentst-test>.
To further investigatethe mechanismmediatingceramideregulationof NGF synthesis,we investigatedif Ca2~ and diacylglycerol-dependentPKC was required for ceramide action. We have previouslydescribed that the use of bisindolylmaleimide (BlM), a potent and highly specific PKC inhibitor,completelyabolished4~-phorbol12~3-myristate(PMA) inducedincreaseof secretedNGF [19]. Treatmentof culturedastrocyteswith 2 MM BlM slightly inhibited SMase-inductionof NGF secretion,whereasC
2-cer-induction of NOF secretionwas unaffected (Table 1). This observationpoints to a mainly PKC-independentmechanismfor inductionof NEW by ceramide.
The study of a possible involvement of the cAMP signaling pathway was undertakenusing thediterpene forskolin (FR), a well known activator of adenylyl cyclase, and 3-isobutyl-l-methylxantine(IBMX), a phosphodiesteraseinhibitor. FR and IBMX alone failed to increaseNGF secretion(data notshown),whereasin the presenceof SMaseor CQ-certhey partially antagonizedthe stimulatory effect ofthe latter (40% and 60%inhibition respectively)(Table 1). Thusa regulatorycrosstalkamongthe SMaseand cAIVIP pathwaysseemsto exist. The factthat intracellular cAMP raising agentshavebeendescribedas inhibitors of the MAP kinasecascade[18, 35, 37], and the possibleinvolvementof tIte MAP kinasecascadeon the ceramide-activatedsignal transductionprocess[29], promptedus to investigatetIte roleof the MAP kinasecascadeon the ceramide-inductedstimulationof NOF production.
Fig. 2 Time-courseof NGF secretionafter treatment with exogenousneutral sphingomyelinaseand C2-ceraniide. Primarycultures of astrocyteswere treated with vehicle alone(e), maximal dose 1 U/ml of SMase(U)(Ieft axis) and25gM C2-cer(V)(rightaxis). At the indicate times ceil-secreted NGF was assayed.Data are the means± S.E.M of two independentexperinientswithdeterminations in triplicate.
3.3. Involvernen,toftire MA.P kinasecascadeiii tire induct¿on.of NGFsecretion,by cerarnideTo investigate tIte poasible role of the MAP kinase cascadein ceramide action, experimentswith
PD098059or apigenin were conducted.PD098059has beendescribedas a selectiveinhibitor of MAPkinasekinase(MEK) [8]. It binds to unactivatedMEK preventingita activationby raf-1 or MEK kinase[1]. Apigenin is aninhibitor of tIte MAP kinasecascade,probablyby acting at tIte raf-1 kinaselevel [17].Treatmentof primary astrocyteswith 100 ±MPD098059abolishedtIte induction of NGF secretionelicitedby SMaseand C2-cer (Table 1). TIte dose-responsecurve of inhibition showedan ICsoaround20pM (Fig. 4A). Treatmentof celíswith 25 pM apigeninpreventedtheSMaseandC2-cerinductedsecretionof NOF (65% and 85% inhibition respectively)(Table 1), the ICsovalue is approximately10 ¡.tM (Fig. 4B).Therefore, data indicate that the IVIAP kinase cascademay be implicated in the regulationof NGFsynthesisby SM hydrolysis.
3.4. Cera,nideincreasestirepirospirorylation extentofraf 1 anáMAPk¿naseactiuityTo further study tIte intracellular effects of ceramide, experimentswere undertakento study the
phosphorylationstateof raf-1. Immunoprecipitationof raf-1 from 32P-labelledcefls is shown in Fig. 5.Short treatmentof astrocyteswith SMaselead to an increasedphosphorylationof raf-1, whereasC2-cerfailed to induce raf-1 phosphorylation.In order to asaesthe role of endogenousceramidesin raf-lregulation we employed the more physiologicalceramide analog N-octanoylceramide(Ca-cer).Ca-certreatmentof celís resultedin an enhancedphosphorylationof raf-1, similar to that elicited by SMase(Fig. 5). SMaseand Cs-cer-inducedphosphorylationwasstill detectableafter 30 minutesof atimulation(datanotshown).Changesin thephosphorylationstateof raf-1 will result changesin ita kinaseactivity.Thisobservationthusconfirma previousreportsabouttIte role of ceramidein raf-1 phosphorylation[2,38]
MAP kinaseactivity was meassuredasphosphorylationlevelsof a highly selectivepeptidesubstratefox p42/p44MAP kinase [5]. Astrocyte treatmentwith C2-cer, Cs-cer and SMase evokedMAP kinaseactivation (Table 2). Activation elicited by SMase was luigher tItan that elicited by both ceramideanalogs.Specificity of MAP kinase activationwas corroboratedby the lack of effect of the inactive C
2-dihydroceramide(Table2). Moreoverpretreatmentwith the IVIAP kinasecascadeinhibitor
Table 1. Effect of different agentajo sphingomyelinaseand C2-cerinduction of NGFsecretion.Primaryculturesof astrocyteswereincubatedwith the indicatedagentsfor 24h. 2 kM BlM, 10 gM FK, 100 gM IBMX, 25 pM API and 100 pM PD 098059wereadded 1hourprior to 1 U/mI SMaseor 25 ~íM C2-cerstimulation. Data are the means±5.D. ofthe numberof independentexperimentaindicated in eachcase.Statistical analysiswasperformed by Studentst-test, significant differences are indicated: a, P.c0.01 versusincubationswith no addition; b, P<o.01,versusincubationswith SMasealone;c, P.c 0.01,versusincubationswith Ctcer alone).
1 2 3
NQP e e
APP~~S fl
Fig. 3 Northernblot anatysisof NGF mRNA accumulation.Lane1, untreatedcelís;lane 2,1 U/ml SMase;lane 3, 25 ~tMC2-cer,After 24 h incubationof Northern blot wasperformedas describedin Matherial and Methodawith NGF and API’ cDNA probes.Essentially identical results were obtainedlo threeindependentexperiments.
PD098059,.counteractedceramideinduction of MAP kinaseactivity (Table 2). Theseresults supportthe notion that signaltransductiontriggeredby SM hydrolysismay activateMAP kinaseandpoint toarole of tIte MAP kinasecascadein tIte regulationof NGF secretionby the SM pathway.
4. Discussion
The presentreportshowsthat exogenousadditionof neutralSMaseor C2-cer,a celípermeableanalogof ceramide,inducesa dose-responseandtime-dependentstimulation of NGF synthesisand secretion.The effect of addedSMaseindicatesthat endogenouslyproducedceramidesmay also be able to induceNGF synthesisandcorroboratesthe specificityof the ceramideanalogaction. Thefact that increased
6
Induction of nervegrowth factorsynthesisby sphingomyelinaseandceramidejo primary astrocytecultures
700
con
500
1 soóir;az— 300
200
100
o
‘ca
600-
500-
~ 4000-a— 200•
200
‘Co
o
Fig. 4 Dose-response inhibition by PD 095059 and apigenin of sphingomyelinaseand C2-ceramide induction of NGF secretion.Primar>’ cultures of astrocytes were incubatedwith 1 U/ml SMase (@) and 25 gM C2-cer (O) andthe indicateddosesof PD098059 (A) or apigenin(13) for 24 h., nadsecretedNGF subsequentassayed.Data are the meana±S.E.M. of two independentexperimenta, with determinations in triplicate.
NGF protein secretionto the celí supernatantwasaccompaniedby an increasein NGF mRNA levels,togetherwith the dataobtainedwith transcriptionand transíationinhibitors, supportsthe notion thatincreasedNGF outputis tIte result of an augmentedsynthesisof NGF.
Regulationof NGF production by a numberof agentsin primary astrocyteshasbeen shown to bedependenton PKC activation [7, 24]. However, IL-143 and vitamin Da induce NGF production bymechanismsindependentof PKC activity [25, 28]. Theseagentsare also well known to exert someoftheir effectsthroughtIte SM pathwayand tIte generationof ceramide[13, 26, 33]. It is wortIt noting thatlipopolysaccharide,which is able to mimic sorne of the effects of ceramide [16, 36] is also a PKC-independentinductor of NGF secretion [11]. We Itave previously reported that PC-PLC is able toenhanceNGF synthesisby primary astrocytesby a signaling mechanismwhich is only partiallydependentof PKC [19]. PC-PLCaction, may in turn be coupledto ceramidegeneration[30]. In fact,recentresults show that short treatmentof CG glioma celís with PC-PLClead to tIte translocationandphosphorylationof PKC ~ [10], which is one of the molecular targets of ceramide [20]. Orn- resultsindicatethat ceramideinductionof NGF in primary astrocyteculturesis independentof PKC. HencetheSM pathway may representone of tIte main PKC-independentsignaling pathwaysleading to theregulation of NGF production by glial celís. TItis is in line with previous reports showing tIternvolvementof the SM pathwayin the inductionof IL-6 synthesis,inhuman astrocytomacelís [9]. Raf-1is a memberof tIte MAP kinasecascadethat is activatedupon cellular stimulation with a numberofeffectorsaspro-inflammatorycytokines[2]. The precisernechanismby which raf-1 is
0 20 .40 00 00 tOD
PO 090059 I~JM]
H
0 5 lO 15 20 25
[API) iM
7
1 Galve-Roperhet al.
3
a
o
½
1%Y
Fig. 5 Immunoprecipitation of raf- 1.32Plabelledcelia were atimulatedfor 15 minuteswith the indicatedagentsand raf-l was,
subsequently immunoprecipitatedwith a polyclonal anti-raf-1 antibod>’. Immunoprecipitates were subjectedto SDS-PAGE andautoradiograph>’. Densitometrie dataare means±S.E.M. of two independent experiments.
Table 2. Effect of ceramideanalogaand sphingomyelinaseon MAP kinaseactivity. Primar>’ cultures of astrocyteswere incubatod with 1 U/mI SMase,25 vM C2-cer,or 25 ~tM C5-cerfor 15 mm. 100 ~M PD 098059was added 1 hourprior to stirnulation. Data, expressed as pmol phosphatetransferred/min/mg prutein, are the means±S.D. of threeindependent experimenta performed in triplicate. Statistical analysiswas performed by Studentst-tost, significantdifferences are indicated: a, P.cO.O1 versus incubationawith no addition; b, P.co.O1,versus incubationawith C2-ceralone; c, P.c 0.01, versus incubatinas with Cs-cer alone).
activated is as yet uncertain, but it is generally agreedthat changesin its stateof phosphorylationproducechangesin its kinaseactivity. Recentreportsindicatethat raf-1maybe phosphorylatedby TNF-activated CAPK, increasingita activity towards MEK [38]. Thereforeit seemathat the SM pathwaycould be linked with the MAP kinasecascade,probablyat tIte levelof raf-1,through tIte activationof theCAPK [2, 29, 38]. Our resultaindicatethat tIte regulationof NGF syntItesisby SMaseandceramidemaydependon the activationof theMAP kinasecascade.It shouldbe notedthat SMase-
8
v~ u:
Jaduction of nerve growth factor synthesis by sphingomyelinase and ceramide fa primar>’astrocytecultures
inductionof NGF secretionwasless sensitiveto MAP lcinaseinhibition tItan C2-cer-inductionof NGFsecretion,in concordancewith its greatereffect on MAP kinaseactivity.
It hasbeendescribedthat hyperphosphorylationof raf-1 inducedby intracellular cAMP-raisingagentsleads to the blockade of tIte MAP kinase cascade[37]. A similar inhibitory effect of tIte MAP kinasecascadeby cAMP-increasingagentshas also beendescribedin astroglial celís stimulatedwith growthfactorsandLPS [18, 35]. Resultsin tIte presentreportshowthat treatmentwith FK andIBMX abolisheshalf of the ceramideaction on NGF production by astrocytes.Thus, tIte existenceof an inhibitorycrosstalkbetweenthe SM and the cA1VIP pathwaysappearsto exist also in the regulation of NGFsynthesisby ceramide.
The possibleinvolvementof tIte IvIAP kinasecascadeasthe signalingmechanismunderlyingceramidegenerationis enforcedby the observedincreasein raf-1 phosphorylationandMAP kinaseactivity astheresult of short-term treatmentwith SMase or ceramide analogs. Short-chain ceramideanalogs aresupposedto be biologically more active than their long chain equivalentslikely due to the increasedsolubility of the former. However, long chain ceramideshavebeen describedto be more potent thanshort chain ceramidesin activating raf-1 [15].This is in concordancewith our results showing anrncreasedphosphorylationstateof raf-1 from Cs-cer-stimulatedtreatmentof cells,whereasC2-cerfailedto induce such effect. Phosphorylationof raf-1 by the ceramideanalogCa-cerhas beendescribedalsopreviouslyby otherauthors[38]. Thus,it seemsplausible that ceramideactionon NGF expressionmaybe mediatedby activationof theMAP kinasecascadeat the levelof raf-1 andp42/p44MAP kinase.
In conclusion, we report here tIte role of the SM pathway in tIte regulationof NGF synthesisandsecretionby cultured rat astrocytes.The action of ceramideon NGF production seemsto be partiallymediatedby activationof the MAP kinasecascade.The regulatoryaction of ceramidemay be relatedtopreviouslydescribedeffectsof pro-inflammatory cytokinessuch as IL-ljB andTNFa, aud is thereforeofmaximal importancein our understandingof the processesoccurring during inflammatorybrain lesionsandcertainneuropathologicaldisorders.
AcknowledgmentsThe authors wisIt to thank Dr. P. Brachet, and members of his laboratory for sharing their
methodologiesfor Northern blot, Dr. M. Guzmánand TAS. Branton for their advice and valuableassistance.Measurement of ELISA were performed at the Plan Nacional de Prevención de laMinusvalía, Hospital Gregorio Marañón, under the supervision of Dr. E. Dulín. This work wasfinancially supported by Comisión Interministerial Ciencia y Tecnología (SAF 96-0113), Fondo deInvestigacionesSanitarias(FIS 97/0039-01),andAcción IntegradaHispano-Francesa(N 96/154).
References[1] Alessi DR., CuendaA., CohenP., DudleyD.T., andSaltielAR. Fn 098059is a specificinhibitor of theactivationof niitogen-activatedprotein kinaselii vitro anáin vivo. J. Rio?. Client. 270 <1995)27489-27494.[2] Belka C., Wiegmann1<., Adam U., HollandR., NeulohM., HerrmannF., KronkeM. audBrachMA. Tumornecrosisfactor(TNlD-a activatesc-raf-1 kinasevia the p55 TN?F receptorengagingneutralsphingomyelinaseEMBOJ. 14(1995)1156-1165.[3] BhatNR. Signal transductionmechanismsin glial celis. fleo. Neurosci.17 (1995) 267-284.[4] Brodie C. Differentialeffectsof TItí andTh2 derivedcytokineson NGF synthesisby nxouseastrocytes.FIJESLetters394 <1996)117-120.[5] Clark-Lewis1., SangheraJ.S.andPelechS.L.Definition of a consensussequencebr peptidesubstraterecognitionby p44mpk,tIte meiosisactivatedmyelinbasicproteinkinase.J.Rio?. Chem.266 (1991)15180-15184.[6] Dekosky ST., GossJ.R., Miller P.D., StyrenSO.,KochanekF.M., andMarion O. Upregulationof nervegrowthfactorfollowing corticaltrauma.ExperimentalNeurology130 (1994) 173-177.[7] D’Mello SR.audHeinrichO. Inductionof nervegrowthfactorgeneexpressionby 12-O-tetradecanoylphorbol13-acetate.J. Neurochem.55 (1990)718-721.[8] DudleyD.T., PangL., DeckerS.J.,BridgesAS. andSaltiel AR. A syntheticinhibitor of the mitogen-activatedproteincascadeProc. Natí.Acad. Sci.USA 92 (1995)7686-7689.[9] FiebichHL., Lieh It, BergerM. andBauer5. Stimulationof the sphingomyelinpathwayinducesinterleukin-6geneexpressioninhumanastrocytomacelís.J. Neuroi,n,nuno?. 63(1995)207-211.
9
1. Galve-Roperhet al.
[10] Galve-Roperh1., MalpartidaJ.M.,HaroA. and Diaz-Laviada1. Addition of phosphatidylcholine-phospholipaseC inducescellularredistributionandphosphorylationof proteinkinaseC 4 in CG glialcelís.Neurosci.Letters219(1996)68-70.[11] Galve-Roperh1., MalpartidaJ.M., HaroA. BrachetP.and Ojaz-Laviada1. Regulationof nervegrowth factorsecretionandmRNA expressionby bacteriallipopolysaccharidein primaryculturesof ratastrocytes.J.Neurosa.Res.(in press).[12]HannunY. A., ObeidL. andObaibo0.5. Ceramide.A Novel secondmessengerandlipid mediator.In Belí R.B.et al. (Eds)Handbookoflipid research,uo?.8:Lipid second ~nessengers.PlenummPress,NewYork, 1996,Pp 177-204.[13]HannunY. A. Functionsof ceramidein coordinatingcellularresponsesto stress.Science274 (1996) 1855-1859.[14]Hattori A., HayashiE andKohnoM. Tumor necrosisfactor (TNF) stimulatestheproductionof nenegrowthfactor in fibroblastsvia the 5S-kOatype 1 TNF receptor.FIJESLetters379 (1996)157-160.[15]Huwiler A., BrunnerJ.,Hummel R., VervoordeldonkM., Stabel5., Van derBoshH. andPfeilschifterJ.Ceramide-bindingandactivationdefinesproteinkinasec-rafas a ceramide-activatedproteinkinase.Proc.Nat?.Acad. Sci. USA93 (1996)6959-6963.[16]JosephC.K., Wright SO.,BornmannW.G., RandolphJ.T. KumarER.,BittmanR., Liu it andKolesnickR.Bacteriallipopolysaccharidehasstructuralsimilarity to ceramideaudstimulatesceramide-activatedproteinkinasein myeloidcelís.J. Rio?. Client. 269 (1994)17606-17610.[17]Kuo M. andYang N. Reversionof v-H-ras-transformedMII 3T3 celís by apigeninthroughinhibiting mitogenactivatedprotein kinaseandits downstreamoncogenes.Biochirn. Biophys.Res.Comrnun..212 (1995)767-775.[18]KurinoM., FukunagaK., UshioY audMiyamoto E. Cyclic AiMP inhibits activationof mitogen-activatedproteinkinaseandcelí proliferationin responseto growth factorsin culturedrat cortical astrocytes.J.Neurochem.67(1996)2246-2255.[19]Laviada1.0., BaudetC., Galve-Roperh1., NaveilhanP. andBrachetP. Phosphatidylcholine-phospholipaseCmetliatesthe inductionof nervegrowthfactorin culturedglial celís.PEESLetters364 (1995)301-304.[20]Lozanoit, BerraE., MunicioMM., Diaz-MecoMT., Oominguez1., SanzL., andMoscatJ.ProteinkinaseC 4isoform is critical fox KB-dependentpromoteractivationby sphingomyelinase.J. Biol. Chein.269 <1994)19200-19202.[21]Mallat M., HoulgatteR., BrachetP. andProchiantzA. Lipopolysaccharide-stimulatedratbrainmacrophagesreleaseNGF in Vitro. Deueloprnenta?Bio?ogy133 (1989)309-311.[22]Merrilí SE. andBenvenisteEN. Cytokinesin infiammatorybrain lesions:helpful andharniful TrendsinNeuroscience19<1996)331-338.[23]Montero-Menei C.N., Sindji L., GarcionE., MegoM., CouezO., GamelinE. andDarcyF., Earlyeventaof theinfiammatoryreactioninducedin ratbrainby lipopolysaccharideintracerebralinjection:relativecontributionofperipheralmonocytesandactivatedmicroglia.Brain Res.724 (1996)55-66.[24]Neveu1., JehanP., HoulgatteR., Wion O. andBrachetP. Activation of nerve growthfactorsynthesisbyphorbol 12-myristate13-acetate:role of proteinkinaseC BrainRes.570 (1992)316-322.[25]Neven 1., NavejihanP., JehanF., Baudet C., Wion O., De LucaH.P. andBrachet1’. 1,25-Dihydroxyvitamin03regulatesthe synthesisof nervegrowth factorin primar>’ culturesof glial celís.Mo?. Brain Res.24(1994)70-76.[26] OkazakiT., BielawskaA., OomaeN., Belí R.M. andHannunYA. Characteristicsandpartialpurificationof anovel cytosolic,magnesiumindependent,neutralsphingomyelinaseactivatedin the earlysignaltransductionofla,25-dihydroxyvitaminDa-inducedHL-Go celí differentiation.J. Rio?. Client. 269 <1994)4070-4077.[27]PlússKM., Pfeilschifter5., Múhí H., Huwiler A., BoeckhC. andOtten U. Modulatoryroleof platelet-derivedgrowthfactor on cytokine-inducednenegrowth factorsynthesisin rat glomerularmesangialcelís.Rioche,n.eJ. 312(1996)707-711.[28]Pshenichkin5.?.,SzekelyAM., andWiseB.C. Transcriptionalandposttranscriptionalmechanismsinvolvedin theinterleukin-1,steroid,andproteinkinaseC regulationof nervegrowthfactorin corticalastrocytesel.Neurochern.63 (1994)419-428.[29] RainesMA., Kolesnick R.N. andGoldeD.W. Sphingomyelinaseandceramideactivatemitogen-activatedproteinkinasein mnyeloidHL-GO cells. el. Rio?. Client. 268 (1993)14572-14575.[30]Schutze5., PotthoffK., MachleidtT., BerkovicO., WiegmannK. andKronke M. TNF activatesNF-KB byphosphatidylcholine-specfficphospholipaseC-induced“acidic’ sphingomyelinbreakdownCeIl 11<1992)765-776.[31]ScottJ.,SelbyM., Urdea M., Belí 0., QuirogaM., andRutterW.J.Isolation andnucleotidesequenceof acDNA encodingtheprecursorof mousenenegrowthfactor.Nature 302 (1983)538-540.[32]Shivers BO. Hilbich C., Multhaup0., SalbaumM., BeyreutherK., anáSeebergPH. Alzheimersdiseaseamyloidogenicglycoprotein:expressionpatternin ratbrainsuggestsa role in cdl contact.EMROJ. 7 (1988)1365-1370.[33]Spiegel5., FosterO. andKolesnickR. Signaltransductionthroughlipid secondmessengers.Current Opinionin Cel? Bio?ogy8(1996)159-167.
Regulaciónde Raf-1 por ceramidas.1/ Translocaciónde Raf-1 a la fracción particuladaencultivosprimarios de astrocitostratadosdurante15 mm con SMasa(Saureus)1 U/mí, C8- yC2-ceramida25 pM. 2/ Cinéticade la transiocaciónde Raf-1 inducidapor C8-ceramida25 pM.3/ Inmunoprecipitaciónde la proteína Raf-1 revelada con un anticuerpopoliclonal anti-fosfotirosina.Lascélulasse estimularoncon CS-ceramida25 pM durante15 mm y dondeseindica preincubadasdurante 1 It. con H7 o tirfostina AG126 100 ~tM.Los resultadosde lafigura correspondenaun experimentorepresentativode otros 3 o 5 realizadosy se obtuvieronmediantelosprocedimientosdescritosenlas publicacionesincluidasen la presentememoria.
2. REGULACIÓN DE LA PKC ~ POR
MENSAJEROS LIPÍDICOS EN
CÉLULAS GLIALES
Resultadosy Discusión 50
En este capítulo se estudia el posible papel modulador de
diferentesmensajeroslipídicos sobre la isoforma ~ de PKC atípica.
La PKC ~ estárelacionadacon la activación de la cascadade MAPK
en diferentesmodelosexperimentales(Berraet al., 1995; Liao et al.,
1997). Por otro lado, las ceramidasejercenun efectoreguladorde la
actividad de PKC ~, queesresponsablede algunasde las respuestas
celulares de la vía de la SMasa-ceramidacomo la activación del
factor de transcripción NF-KB (Lozano et al., 1994; Múller et al.,
1995). Estasobservacionessugierenla posibilidadde que la proteína
PKC ~ este implicada en el sistema de transducción de señales
iniciado por la PC-PLC,mediandola activaciónde Raf-1 y cascadade
MAiPK por ceramidas,y regulandoen ultima instanciala síntesisde
NGF.
Primeramentese exponeel efecto de la actividad de la PC-
PLC exógenasobrela PKC ~ en la línea celular de glioma C6 y,
posteriormente,se estudia el efecto que en esta misma proteína,
PKC ~, tienen los análogos permeablesde ceramida y la SMasa
exógena.
ELSEVIER
NiDROSCIINCELITTLE
NeuroscienceLetters 219 (1996) 68—70
Addition of phosphatidylcholine-phospholipaseC inducescellularredistribution and phosphorylation of protein kinase C ~ in C 6 glial celis
Departamentode Bioquímica y Biología Molecular 1, Facultad de Químicas, Universidad Complutense> 28040 Madrid, Spain
Received 7 August 1996; revised version received 5 October 1996: accepted8 October1996
Abstract
Phosphatidylcholine breakdown has beeri shown ro play a critical role in signal rransduction involving generation of a number ofsecond messengers [Exton,3.13., Biochim. Hiophys. Acta, 1212 (1994) 26-421. lo the present repofl we demonstrate by immunofluor-escence Ihat short-treatment of C 6 glial celis with phosphatidylcholine-hydrolyzing phospholipase C (PC-PLC), changes the intracel-lular localization of protein kinase C (PKC) ~ from the cytoplasm to a perinuclear region. Western blol analysis also showed aredistributiori of PKC ~ aher incubation of celís with PC-PLC. To tesL whether these changes were accompaniedby an activation ofihe enzyme.we measured the extent of phosphorylationof PKC ~by immunoprecipitationfrom >2P-labelled celís.Short-treatmentwithPC-PLCresulted in enhanced phosphorylation of the higher Mr PKC r in C 6 glial celís.
Keywords: Protein kinase C fl Phosphatidylcholine-phospholipase C; Glial celís
Proteinkinase C (PKC) is oneof themajor mediatorsofsignals upon externa! stimulation of celís by hormones,neurotransmitters,and growth factors. PKC has beenfound in mosímammaliantissues,but is particular!>’abun-dant in the brain [17]. ‘PEe manmalian PKC po]ypeptidesidentified to date by cONA cloning havebeenclassifiedinto three groups basedon two criteria, namel>’ structuralfeaturesand regulationby cofactor.TEe atypical PKCs <rand tIX isoforms)are dependent un phosphatidylserine,butdo not require Ca2~ urdiacylglycerolfor activation [4,13].
lo astrocytesand glial celís the presenceof PKC a,13,7,5, e and r isoformshas been reponed[5,7,11,14].Prufilesof PKC isoformsin rat astrocytesandC 6 gliomacelisaresimilar, although a higher expressionof &, e, and riso-forms in glioma celis hasbeendetected[14]. Recently,ithas beendemonstratedthat the PKC ~ isofonn plays animportantrole in mitogenicsignal transduction[1], neuro-na! differentiation [19], and maintenanceof long-termpotentiation [15]. Huwever, the precise mechanismbywhich PKC r activity is regulatedis not fully understood.PKC ? is not activatedby diacylglycerolor phorbol esters[13,18]andin manycelí typesincluding glial celis,PKC r
is neither transiocatedfrom cytusul to membranenurdown-regulatedin responsetu acuteur chrunictreatmentwith phorbol esters[2]. Activation of PKC r may beachievedby ceramide[10]. The secundmessengercera-mide is generatedby the action of sphingumyelinase,which is la tum functionally coupled tu phosphatidyl-hydrolyzing phospholipaseC <PC-PLC) [16].
We haverecentí>’ shown that PC-PLCenhancesnervegrowth factor synthesisin culturedgua!celisby amechan-ism which is partially independentof the classical iso-forms of PKC [8]. In the presentreponwe show thataddition of PC-PLC inducescellular redistributionandphosphurylationof PKC ~in C 6 glial celis.
Tu testwhetherPC-PLCcouldhaveaneffect un PKC rweanalyzedihe intracellularlucalizationandtheexteníofphosphorylationof this NCC isofurm. Short-term-treat-ment of C 6 cells with exogenousPC-PLCpruduced achangein the lucalizatiunuf PKC r fromn cytuplasmtu aperinuclearmembraneregion (Fig. lA,B). Non-specificlabeling was shownafter incubationwith anti-PKCtanti-bud>’ in thepresenceuf a competingpeptideusedtu rinsethe antibody (Fig. lC).
As a further proufof the effectuf PC-PLCun the loca-lization uf PKC ~, particulate,andsolublefractiunsuf celíswere separatedby centrifugation,andPKC ~wasdetected
Fig. 3. Phusphorylatiou lcvcl of I’KC ~. C 6 celís wcrc iucubatcd with‘i’—t,r¡ hopluospl¡inc (A u,crslMLm) :¡nd ihen citl~cr un rcmucd ( lanc ji or1 rcatctl ¡br 15 miii 1 U/u,1 PC— PLC ( lauc 2). A lcr imm uuoprccipilal ionwi(h un uí,ci-PKC ~> amihody. pcllcts wcrc rcsolvcd by clcctrophorcsisaud subjcc¡cd lo :iuloiadiography. Dcnsiíomciric aualysis was pcrlormcdjis dcscrihcd ¡br Wcslcrn blol. Thc markcrs on thc cli corrcspoud lo ihcuhigration position ol 97.4 :índ 66 kDa molecular wcighí u,,arkcrs. ¡den-
cal rcsults wcrc obíained u Il,rcc indcpeudcut cxpcrimcuts.
order to know whether transiocation of PKC ~ correlateswiíh an activation of the enzyme, we derermined the extentof phosphorylatiou of PKC tby imrnunoprecipilarion from32P-labeled C 6 cells. Thus, incubation of >2P-labeled celiswith PC-PLC lcd lo un enhanced phosphorylation of PKC~ (Fig. 3). lucreased phosphorylation of PKC ~ aflectedone form. probably corresponding lo 83 kDa, which agreeswith its observed tran.slocation. A similar effect has beendernonstrated in epidermal celís, where phorbol myristateacetate-treated celis showed an increased phosphorylationaffecting to higher-Mr PKC ~ isoform [12].
‘Paken togeiher these results indicate that treatment of C6 cells wilh PC-PLC causes a change in the intracellularlocalization of PKC ~which becomes associated to a peri-nuclear region. Redistribution of this kinase was accom-panied by an increase in the extent of phosphorylation of(he enzyme, confirming the hypothesis that PC-PLC treat-mcm of C 6 glioma celis leads to ihe translocation andconsequent activation of PKC ~ isoform.
‘Phe aurhors gratefully acknowlcdged Dr. M. Guzman forcrilical reading of (he manuscripts and Dr. P. Brachet forproviding C, glial celís. This work was suppor(ed by agrant from FIS (94/0300). 1. Galve has a fellowship fromUniversity Complutense.
II] Berra, E.. Díaz-Meco,MT., Dom<nguez.1., Municio, MM., Sanz,L., Lozano, i.. Chapkin. RS. and Moscar, J.. Protein Kinase C ~ísoforni is cricical ter miíogenic sigua! cransduction, CeIl, 74(1993)555—563.
[2] Chen, C., Protein kinase C a, 5, and ~in C 6 glioma celís. TPAinduces Iransiocation and down-regulaíion of convencional andnew PKC isoforms bu not atypical NCC ~ FEBS Lea., 332(1993) 169—173.
l~] Gott. A.L., Mellon, B.S.. Paton, A., Groome, N. aud Rumsby.MG., Ral brain glial celís in primary culture and subculturc con-tain the 8, e and ~subspccies of protein kinase C as weil as 11wconvenjional subspecies. Neurosci. Lctt., [7! (1994) 117—l’t)
[6] Kcranen, L.M., Dulil, EM. and Newton, A., Prolein kinasc C isrcgulated in vivo by thrce funclionally distinct phosphoryíalions.Curr. Biol., 5(1995)1394—1403.
171 Kumar, 1<., Kim, B.. Rupp, 1-1. aud Madl,ukar, 13., Exprcssiou ofprolein kin:íse C isozymcs in rut glial ccli Une, NeuroRcpor¡. 4(1993) 431—434.
181 Laviatla, ID., Baudct. C.. Galve-Ropcrh. 1.. Navcilhan. P. audBrachct, P., Phosphatidylcholinc-phospholipase C mediates thcintiuction of ncrvc growth factor in culturcd glial ccli,, FEHSLett., 364 (¡995) 301—304.
19] Lchrich, R.W. -md Forrest. iN., Proicin kinase C ~ is associaícdwith Ihe n,iíotic apparalus u primary ccli cultures of thc sharkrcclal glaud, J. Biol. Chcm., 269 (1994) 32446—32450.
líO] Lozano, J., Bcrra, E., Municio, MM., Diaz-Meco, MT.,Domiuguez. 1., Sanz, L. aud Moscat, 1., Prolein km-ase C ~ isoforinis critical for xB-dcpcndcut promoter activation by sphingo-myclinase, 1. Biol. Chem., 269 (1994) 19200—19202.
III] Masliah, E.. Yoshida, K.. Shimohama, 5., Gage, F.H. and Saiioh,T., D¡ffcrcuíial expression of protein kinase C isozymcs in rat gua!eclI cultures, Brain Res., 549 (1991) 106—111.
1121 Nishikawa, K.. Yamamoto, 5.. Nagumo, H.. Maruyama. K. audKaío. R., The presence of phorbol ester responsive and non-respon-<ve forms ofihe ~isozymeof prolein kinase C in mouse epidemialcdl,. Cdl. Signal.. 7(1995)491—504.
1131 Ono, Y.. Fujii, T., Ogica. K., Kikkawa, U., lgarashi, K. andNishizuka. Y., Proicin kinase C t subspecies from rat brain: utssírucíurc, cxprcssion, and propcrtics, Proc. Nalí. Acad. Sci. USA.86(1989)3099—3103.
[¡4] Roisin, M. aud Dcscheppcr, CF.. Idenlificadon and cellular loca-lizalion of prolcin kinase C isofoniis u cultures of ral type-lastrocytes, Brain Res., 701 (1995) 297—300.
lIS] Sacktor, T.C.. Osten, P., Valsamis, H., Jiang. X., Naik, MU. andSublecte, E., Persistent accivarion of (he ~isofoma, of procein kinaseC u the maintenance of long-leras potentiation, Proc. Nací. Acad.Sci. USA. 90(1993)8342—8346.
[¡6] Schtitze, 5., Polthoff, 1<., Machieidt. T.. Berkovic, D., Wiegmann,K. aud Krdnke, M., TNF activales NF-seR by phosphatidylcholine-spccilic phospholipase C-induced acidic sphingomyelinbreakdown, Cdl, 71(1992)765—776.
[171 Tanaka, C. and Nishizuka, Y., The protein kinase C family forneuronal signaling. Annu. Rey. Neurosci., 17 (1994) 551—567.
[18] Ways. D.K.. Cook, PP.. Webster, C. aud Parker. P., Effect ofphorbol esters on protein kinase C-~, J. Biol. Chem., 267 (1992)4799—4805.
[19] Wooren, MW., Zhou. O., Seibenhener, ML. aud Coleman, ES., Arole for zeta protein kinase C in nene growth factor-induced dii-ferentiation of PCI2 celís, Ccli Growth fliff., 5 (1994) 395—403.
FEBS 19133 FEBS Letters415 (¡997) 271—274
Ceramide-inducedtransiocation of protein kinase C ~ in primary culturesof astrocytes
Dcpartníent of Biochemistry and Molecular hiology. Facully of Medicine, Alcalá de Henares University, 28871 Alcalá de Henares. Madrid, Spain
Received21 Jul>’ 1997
Abstract The present research was undertaken to study thepossible involvement of> the atypical protein kinase C (PKC) ~ laceramide signal transduction la priniary cultures of ral astro-cytes. As shown by Western blot analysis, transiocation ofimmunoreactive PKC4 to the particulate fraction occurred uponexposere of astrocytes to ceil-permeable ceramide analogs or toexogenous spliingomyelinase. The particulate fraction ma>’correspoad to a perinuclear area, as indicated by immunocyto-chemical techoiques. Furthermore, treatment of celís with N-octanoylsphingosine lcd fo aB increased phosphorylation ofPKC4. Results thus show that stimulation of PKC4 may beone of the intracellular events triggered by activation of thesphingomyelin pathway.
Xc>’ ítords: Protein kinase C 4; Ceramide; Sphingomyelin;Astrocyte
1. Introduction
Sphingolipids are currentí>’ recognized as active participantsin the regulation of a wide range of cellular responses such asregulation of ccli growth, differentiation, transformation anddeath [1].Sphingomyelin (SM) breakdown occurs in multiplecdl typcs in response to a variety of extracellular mediatorsíncluding cytokines cg. tumor necrosis factor a (TNFa), in-terleukiii-l~ (lL-l~). nterferon-y and other agents such asnerve growth factor (NGF), la,25-dihydroxyvitamin O>,Fas ligand and ionizing radiation (reviewed iii [¡—3]).Recep-tor-mediatedhydrolysisof SM generatesceramide,which ma>’funetion as a second messenger activating sever-al serine/threo-fine protein kinascs and phosphatases [4].Among the former,one of the atypical isoforms of protein kinase C (PKC),PKC4, has been proposed as an intracellular target of ceram-ide action [5,61.These atypical PKC members (4 and ?Jt) aredependent on phosphatidylserine but are not affected by diac-ylglycerol, phorbol esters or Ca2~ [7,8]. Although PKC¿ hasbeen shown to be involved in the control of a number ofcellular functions including rnitogenic signal transduction [9],neurona] differentiation [¡01 and long-terni potentiation [II],the signal mechanisms downstream PKC4 activation are notweIl known. PKC has been suggested to play a role in theactivation of the mitogen-activated protein kinase (MAPK)
pathway [12]. PKC has also beenshown to be involved inthe activation of nuclear factor KB <NF-KB), which is a land-mark of the TNFa mechanism of action. Little is knownabout the specific signal transductionpathways linking TNFreceptorsto NF-KB. A connectiorthasbeenproposedbetweenTNFa receptor and phosphatidylcholinephospholipaseC(PC-PLC), which is coupled to sphingomyelinase(SMase),resulting in the generation of ceramide[6,13,14].We haverecentí>’ demonstratedthat PC-PLCinducescellular redistrib-ution and phosphorylationof PKC4 in glial cells [15]. Thepresentwork was thus undertakento test whether PKC4may also be a targetfor ceramide action in primaryculturesof astrocytes.
2. Materinls and n,ethuds
2.1. MaterialsAnti-PKC~ antibod>’, raised against amino acids 577—592 of rat
PKC~. was from Life Technologies Inc. (Gaithesburg. MD, USA).Fluorescein-conjugated donkey anti-rabbit IgO was from Amersham(Little Chaltfont, UK). o-erythro-N-acetylsphingosine (C2-ceramide),o-erythro-N-octanoylsphingosine (C8-ceramide) and o-erythro-dihy-dro-N-acetylsphingosine (DHC) were froro Calbiochero (La Jolla,CA, USA). Protein A-agarose was from Transduction Laboratories(Lexington, KV, USA). Neutral SMase (5. aureus) and alí other re-agenis were from Sigma Chemicals (St Louis, MO. USA).
22. .Prin,ary cultures of asírocytesCortical astrocytes were derived from ¡—2 day oíd rats and cultured
as previously described [16]. CelIs were seeded at a density of 3X ¡0’cells/cm> on plastic plates previously coated with 5 1kg/ml dl-polyor-nithine in water. The primary cultures consisted of 95% astrocytes asjudged by immunocytochemical staining of glial fibrillary acidic pro-tein.
Three days before the experiment, the serum-containing mediumwas removed and celís were transferred to a chemically defined me-dium consisting of serum-free DMEM:Hams FIZ (1:1, v:v) mediumsupplemented with 25 gg/ml insulin, 50 ¡sg/ml human transferrin, 20nM progesterone, 50 ¡iM putrescine, and 30 nM sodium selenite.
2.3. Western blol analysis of PKCCAfter stimulation, celís were washed with ice-cold PBS and subse-
quently homogenized in 50 mM Tris-UCI, pH 7.5, containing 5 mMEDTA, 1 mM EGTA, ¡OmM p-mercaptoethanol, 1 mM PMSF, 5 ¡sglml leupeptin, 2 ¡sg/ml aprotinin, ¡O ¡sg/ml soybean trypsin inhibitorand 10 ¡sg/ml benzamidine. Soluble and particulate fractions wereobtained after centrifugation for 60 mm at 40000Xg. Proteins werethen resolved by SDS-PAGE and transferred onto nitrocellulose. lm-inunodetection of PKCI was carried out by incubating membraneswith anti-PKC4 polyclonal antibody and developing with an enhancedchemiluminescence reaction kit (Amersham).
2.4. Inununocytochemical proceduresAstrocytes were grown in glass coverslips in serum-containing me-
dium and at three weeks made quiescent by serum starvation. Celíswere stimulated with different agents, washed with phosphate bufl7ersalme (PBS) asidcoverslipsimmediately immersed in 3.7% formalde-hyde/PBS for 5 mm. After washing in PBS, celís were treated with
272 L Galve-Roperh el a!¡FEAS Letters 415 (1997) 271—2 74
0.05% Triton x-íoo and subsequently incubated with 40 ¡sg/mI poíy-clonal anti-PKC~ antibod>’ for 60 mm and fluorescein-conjugated~inti-rabbitIgO for 60 additional mm. Coverslips were then mountedwith glycerol containing 0.1% p-phenylenediamine and subjected tofluorescence microscopy.
2.5. Plíosplíorylation of PKC<Phosphorylation of PKCC was performed after loading of celís with32Pi and immunoprecipitation as previously described [¡7]. Imniuno-
precipitation ws carried out by incubation with 7.5 ¡sg/ml of ant¡-PKC polyclonal antibody asid precipitation with agarose-¡inked pro-tein A. Phosphorylation was determined in the immunoprecipitates bySDS-PAGE and autoradiography. GeIs were previously stained w¡thCoomasie blue in order to verify the appropriate loading of the geis.Autoradiography Fuji films were subjected to dens¡tometric analysisusing Ihe Sigma-Gel program.
3. Results atad discussion
3.1. Transloca:ion of PKC4 and idenílficauion byinununoblo t ting
To study whether SM-ase and cerarnides may have an effectun the subcellular distribution of PKCC, primary cultures ofastroc>’tes were treated with neutral SMase or celí-permeableceramide analogs, C
2-ceram¡de and C8-ceramide. As shown inFig. 1, PKC! appcars to be expressed in primar>’ cultures ofastrocytes as a single 75 kDa isoform in both the particulateand the soluble fraction, with approximately equal amounts oftotal enz>’me in each fraction in non-stimu]ated cells (55% and45% respective!>’) [¡8]. C8- and Q-ceramide, as well as exog-enous SMase, induced a signiflcant increase in immunoreac-tive PKC ita the particulate fraction with a concomitant de-crease in the soluble fraction (Fig. 1). The time course of C8-ceramide-induced PKCC translocation to the particulate frac-
2.00
<3
ce
cd
0>
8o
e0>
~0>
1.75
1.50
1.25
1.00
0.75
particulate fracuon soluble fracecon
FKCC — — — — — — —
0.00
Fig. 1. Localintion of PKCC by immunoblotting. Astrocytes werestimukted with vehicle, ¡ U/ml SMase (S. aureus), 25 ¡4M C8-cer-anide or 25 ¡sM C>-ceramide for ¡5 mi The upper panel shows arepresentative luminogram of the immunoblots. The lower panelshows the mcans ±S.D. of densitometric analyses from three mdc-pendent experiments expressed in arbitrar>’ units referred tu densito-metric units from control celís. Statistical analysis was performed byStudent’s .t-test. Significantí>’ different vs. the corresponding subcel-lular fraction of control celís: t .P-c0.025; **: P<O.O¡.
2.00
aa
1-
‘o
uSt
0>Etee0>
1.75
1.50
1.25
1.00
0.75
0.50
Fig. 2. Time course of C8-ceramide-induced translocation of PKC~.Astrocytes were stimulated with 25 tM C1-ceramide forO, 5, ¡5, 60and ¡80 mm, The upper panel shows a representative luminogramof the inmunoblots. The lower panel shows the means±S.E.M.ofdensitometric analyses from two independent experiments expressedIn írbitrary units referred to densitometrie units from control celís.
tion is shown in Fig. 2. PKCC redistribution was airead>’evident after 5 mm treatment and was ¡siaximal after 15 mmof ceramide stimulation. These flndings indicate that activa-tion of the SM pathway induces PKC~ translocation to theparticulate fraction of rat astrocytes.
3.2. Redistribuí ion of PXC0 itt primary astrocytesTo further stud>’ the intracellular localization of PKC.
ummunocytochemical analyses were perfornied. As shown inFig. 3, exposure of celís to eithcr SMase or ceramide analogsinduced PKC¿ translocation to a perinuclear area. The elTectof added SMase indicates that endogesiously produced ceram-¡des are also able to induce PKC! cellular redistribution,therefore corroborating the specificity of ceramide analog ac-tion. Perinuclear signal was stronger in SMase-treated and C5-ceramide-treated celís (Fig. 3C and D) thasi in C2-ceramide(Fig. 3E). Ihis may be probabí>’ due to the longer alkyl chainof the former, which makes it more similar to natural occur-ring ceramides. Like ¡si Western blot asial>’sis (see aboye), C8-ceramide asid SMase-generated ceramides produced a greateretlect thasi the short chain Crceramide. Therefore alkyl chainlength may pía>’ an important role in ceramide action. Thiseffect has also been observed in raf-l activation by ceramides[19]. Ihe emerging h>’pothesis from this observatiosis is thatsuch structural requirements may explain divergesit effects ofdifferesit ceramides as a consequence of its subcellular origin,and the specific SM pool involved [20]. ¡si this context, adifferent action of intracellularí>’ generated ceramides asidexogenously added ceramides in the induction of apoptosishas been recentí>’ reported [21].
The speciflcity of tbe effectof ceramides is supported by thefact that inactive ceranuide analog DHC did not induce an>’chasige in the subcellular localization of PKC~ (Fig. 3B).
0 5 15 60 180 0 5 15 60 180mm
274 1. Galve-Roperh et al/FEBS Letrer, 415 (1997) 2 71—274
assistance in cdl culturing. This work was supported by grants froniComisión Interministerial de Ciencia y Tecnología (SAF 96/0113) andFondo de ¡nevestigaciosies Sanitarias (97/0039-01).
References
[II Spicgcl, 5.. Foster, D. asid Kolesnick, R. (1997) Curr. Opin. CdlBiol. 8,159—167.
[2] Hannun, Y. (1996) Scicnce 274, 1855—1859.[3] Testi, R. (1996) Trends Biocbem. Sci. 21, 468—471.[4] BalIou, L.R., Lauderkind, S.J.!’., Roslosiiec, E. and Raghow, R.
[19]Huwiler, A., Brusisier, 1., Hummel, R., vervoordeldosik, M., Sta-bel, 5., Van Den Bosch, H. atad Pfeilschifter, J. (1996) Proc. Natí.Acad. Sci. USA 93, 6959—6963.
[20] Asidrieu, N., Salvayre, R. and Levade, T. (1996) Eur. 1. Biochem.236, 738—745.
[22]Lehrich, R.W. and Forrest, J.N. (¡994) 3. Biol. Ches,. 269,32446-32450.
[23]G~,rcia-Rocha, M., Avila, J. and Lozasio, 3. (1997) Exp. Cdl.Res. 230, 1—8.
[24]Westermann, P., Ksioblich, M., Maier, O., Lindschau, C. asidHaller, H. (1996) Biochero. 3. 320. 651—658.
[25]Kerancsi, L.M., Dutil, EM. and Newton, A.C. (¡995) Curr. Riol.5, ¡394—1403.
[261Gomez, J.. Martinez dc Aragon, A., Rosiay, P., Pitton, C., Gar-cia, A., Silva, A., Fressio, M., Alvarez, F. and Rebollo, A. (1995)Eur. 1. Imniunol. 25. 2673—2678.
[27]Yavin. E., Inserte-Igual, 3. iind Gil, 5. (1995) J. Neurochen,. 65,2594—2602.
uno de los componentesdel grupo de las PKCs atípicas,la PKC 4 (Hoffman, 1997).
Los nivelesde PKC 4, no disminuyenpor exposiciónprolongadaaésteresde forbol, es
decir no experimentan“downregulation’, tanto en astrocitos, como en la línea de
glioma C6 (Ballestasy Benveniste, 1995; Chen et al., 1995). Los datos obtenidos,
muestrancomo la PC-PLC induce un cambioen la localización subcelularde PKC 4,acompañadode un aumentodel estadode fosforilación de la enzimaen la línea de
glioma C6. Lasvariacionesenel estadode fosforilaciónpuedenreflejar cambiosen el
estado de activación de la isoforma de PKC estudiada (Jaken, 1996). Estas
observacionesson compatiblescon quela PKC 4 puedadesempeñarun papel en la
regulación de la producción de NGF por células gliales inducida por PC-PLC
resistentea BlM y “downregulation” de PKC.
La estimulación de la producción de NGF promovida por ceramidas en
astrocitospresentacoincidenciascon la regulación por la PC-PLC que sugierenque
Departa~nento de Bioquímica y Biología Molecular L Facultad deBiología, Universidad Complutense, 28040 Madrid,
Spain. b Laboratoire de Biochintie “Maladies Métaboliques’-INSEBM U. 466. Instituí Louis Rugnard, 31408 Toulouse,
France r Departamento deRioquintica y Biología Moleeula,-, Facultad deMedicina,,Universidad de Alcalá de Henares,
28871 Alcalá de Henares, Spain
The signal mechanismunderlying tumor necrosisfactor a <TNFa) upregulationof nenegrowth factor (NGF) productionwas studied in primary rat astrocytes.Since ceramide is
also able to induce NGF secretion and becauseTNFa is a known agonist of the
sphingomyelin-ceramidepathway, we investigated whether the TN?Fcz-induced NGFsecretion b>’ primary astrocytesis mediatedby ceramide. TNFa stimulation of NGFsecretionwas shown to be independentof protein kinase C, abrogatedby the tyrosinephosphoproteinphosphataseinhibitor phenylarsineoxide (PAO), and independentof the
activationof the mitogenactivatedprotein kinasecascade.In markedcontrast,inhibition of
MAP kinasecounteractedthe NGF secretioninducedby ceramide.TNFa stimulation of the
nucleartranseriptionfactorNF-KB waspreventedby celí pretreatmentwith PAO, whereasceramideand sphingomyelinasehad a marginal effect on NF-KB activation. Moreover,TNFa failed to activate the sphingomyelinpathwas’, as indicated by the lack of 5PMdegradationandthe absenceof cerarnidegeneration.To further clarifr the roleof NF-KB inNGF synthesis,EMSA were performedwith a NF-KB site from the NGF proanoter.The
absenceof signfficantbindingof NF-KB to theNGF genepromoterindicatestheexistenceofan indirect mechanisanfor NF-KB regulation of NGF synthesis. Altogether, our datastrongly suggest that TNFcz-mediatedupregulation of NGF occurs independentlyofceramidegeneration.
(IL-1j3) areimportantmediatorsof inflammationandimmuneresponsesin the CNS.Such responsesare followed by the rapid activation of brain resident macrophagesandsubsequentactivationof astrocytes(Merrilí andBenveniste,1996).Oneof theresponsesofreactiveastrocytesis the synthesis and releaseof neurotrophicfactors such as nenegrowthfactor (NGF) (Brodie, 1996; Yu et al., 1996). NGF production by glial celis during suchcircumstancesmay limit the extent of neuronalloss and promote regenerativeprocessestorestoreneuronalfunction (Pshenichkinet al., 1994).
~ outhor. Ismael Galve-Roperh, Departamento de Bioquímica y Biología Molecular 1, Facultad de
NGF synthesis by astrocytesis a finely regulated processwhich includes complexinteractionsamongdifferent signalingpathways.Pro-inflammatorycytokines such as TNFaare well known stimulatorsof NGF synthesis(Hattori et al., 1993;Pshenichkinet al., 1994).TNFa is a pleiotropic cytokine which exerts most of its biological effects, including NGFinduction, throughthe 55 kDa TNFct receptor(Hattori et al., 1996),which is the TNFareceptorpredominantlyexpressedin astrocytes(Dopp et al., 1997). Someof the early eventstriggeredby TNFa include the activation of phospholipaseAz (Jayadevet al., 1994), neutral and acidicsphingomyelinases(SPMases) as well as phosphatidylcholine-phospholipaseC (PC-PLC)(Schtttze et al., 1992; Andrieu et al., 1995; Wiegman et al., 1994). The signaling cascadeanvolving sphingomyelin(5PM) hydrolysis andsubsequentceramidegenerationconstitutestheso-calledsphingomyelin-ceramidepathway(Hannun,1996;Spiegelet al., 1996).
We have recently demonstratedthat N-aeetylsphingosine (C2-ceramide), a celí-permeantceramide analog, and exogenousbacterial SPMase,which elevatesintracellularceramide, are potent inducersof NGF synthesisby primary astrocytes(Galve-Roperhet al.,1997a). Ceramideaction as secondmessengermay involve stimulation of different moleculartargets as ceramide-activatedprotein kinase, ceramide-activatedprotein phosphatase,andatypicalPKC 4 (Hannun, 1996;Spiegelet al., 1996).As a matterof fact, ceramidesareabletotrigger PKC 4 transiocationandphosphorylationin primary astrocytecultures(Galve-Roperhet al., 1997c). Activation of the MAPK cascadeby the stimulation of SPM-ceramidepathwayhas also beenreported,and may be related to Raf-1 activation (Huwiler et al., 1996), whichmay in turn be activatedby direct interactionwith PKC 4 (Van Dijk et al., 1997).
Another characteristieintracellular action induced by TNFx is the early activationofthe nuclear transcription factor NF-KB (Schútzeet al., 1992). The exact role of ceramidegeneration in TNFct-activationof NF-KB 15 still controversial (Hannun, 1996). Thus, somereportsindicatethat ceramidegenerationthroughacidicSEMasemay be responsiblefor NF-KBactivation (Machleidt et al., 1994; Wiegman et al., 1994), whereasothers have not foundevidencefor the role of ceramidein NF-KB stimulation (Betts et al., 1993;Andrieu et al., 1995;Gamardet al., 1997;ZumbansenandStoffel, 1997).
Thiswork wasthereforeundertakenin order to further studythe role of ceramideandother signal transductionpathwaysinvolved in TNFa-mediatedNGF synthesisand secretionby primary astrocytes.The role of nuclear transcription factor NF-KB in NOF generegulationwasalso assessed.
2. Materlalsandmethods
2.1 MateríaisTissue cultureplastic wareswerefrom Nune(Denmark). Culture media andfetal calf
serumwerefrom Biowhitakker (Belgium). The bisindolylmaleimideOF 109203X, forskolin, PD098059 andcelí permeableceramide analogswere from Calbiochem(USA). TNFcz was fromPreproTech-Tebu(France).T4 polynucleotide kinase, NF-KB protein and NF-KB consensusoligonucleotidewere from Promega(France). Bacterial SPMase(5. aureus), dibutyryl cyclicAMP andthe restof reagentswere from SigmaChemicals(USA).
2.2 Primary culturesofastrocytecelis
Cortical astrocyteswere derived from 1-2 day oíd rats and cultured as previouslydescribed(Galve-Roperhet al., 1997a).Cellswereseededat a densityof 3x104 cells/cm2on plasticplatespreviouslycoatedwith 5 pg/ml dl-polyornithine in water. Celíswereculturedfor 3 weeksinbasalmediumconsistingof a mixture of Dulbeccosmodified Eaglemedium (DMEM) and HamsF12 (1:1,y/y), with 0.66%glucose,5 pg/ml streptomycin,SU/ml penicilhin, andsupplementedwith10% fetal calf serum. The primary cultures consisted of 95% astrocytesas judged byimmunocytochemicalstaining of glial fibrillary acidic protein. Primary cultures of astrocyteswereincubatedfor threedaysbefore the experiments,in chemically-definedmedium(consisting
For extracellular NGF determination, ceil supernatantswere collected 48 h aftertreatmentas describedin the text, diluted in one volume of phosphatebuffer salme (PBS)containing0.1% Tween 20 and 0.5% gelatin, and conservedfrozen at -20 0C until quantitation.NQF releasedby the cells wasassayedin triplicate, by a double-siteELISA, usinga monoclonalanti-NGF antibody, coupled or not to 13-galactosidaseaccording to an experimental protocoldescribedbefore(Laviadaet al., 1995).
2.4 Preparationofnuclearaudcytosolicextracta
Nuclear and cytosolic extracts were obtained according to a previously describedmethod(Andrieu et al., 1995).Briefly, ceil pelletswerehomogenizedat 4 0C in 400 pl of bufferA (10 mM Hepes,pH 7.8, 10 mM KCl, 1mM MgCl2, 0.5 mM EDTA, 0.1 mM EGTA, 1 mM
dithiothreitol, lmM phenylmethylsulfonyl fluoride and 10 pg/ml leupeptin). The celís wereallowedto swell for 15 mm on ice, IgepalCA-630 (0.6% final concentration)wasadded,andthetubeswerevortexed.Nucleiwere pelletedby centrifugationat 1200 x g for 20 mm at 4 0C. Thesupernatantcontainingthe cytosolic fraction was centrifugedat 14 000 x g and the resultingsupernatantwas usedfor Westernblot analysis.The nuclei were suspendedin buffer A andafter sedimentationat 2000 x g for 20 mm, they were suspendedin 25 pl of buffer C (20 mMHepes. pH 7.8, 400 mM NaCí, lmM MgCl2, 0.5 mM EDTA, 0.1 mM EGTA, 1 mMphenylmethylsulfonyl fluoride and 10 pg/ml leupeptin)and incubatedfor 30 mm on ice. Aftercentrifugation at 15 000 x g for 20 mm, the supernatantwas collectedand storedat -80 0C.Proteinconcentrationsweredeterminedby the bicinchoninicacidmethod.
2.5Electrophoreticmobility shift assays
Oligonucleotideprobesused were end-labeledusing T4 polynucleotidekinase and [y-32P]ATP accordingto the supplier’s instructions.Binding reactionswere performedin a totalvolume of 20 pl with the radiolabeledoligonucleotide(250000cpm), bindingbuffer (containing2 mM Hepes,pH 7.8, 50 mM NaCí, 1 mM MgCl2, 05 mM EDTA, 0.5 mM dithiotreitol, 4%glycerol, 1.2 jtg poly (dIdC), and 0.4 pg of bovine serumalbumin) and 5 pg of nuclearextract.After incubationfor 20 mm at room temperature,the samplesweresubiectedto electrophoresison 4% acrylamide gel for 3-4 h at 100 y. Dried gel was exposedto an X-ray film (Hyperfilm,Amersham)at .800 C or quantifiedby Phosphorímager445 SI (Molecular Dynamics).
The following oligonucleotideswere usedin EMSA: NF-KB consensus,AGT TOA 000GAC TTT CCC AGO C; mutatedNF-KB, AOT TGA TTC TCG AAA CCC AGO C; NF-KBNGF,OCA TTG CAO 000 CCT CCC AGO Mil and mutatedNF-KBNGF, OCA TTG CAT AGC CCTCCC AGO MR.
Primary cultures of astrocytes were mcubated before the experiment in thechemically-definedmedium with 1 pCi/ml of [methyl-3H]choline. After 72 h incubation, tlieradioactivemediumwasremovedandcells werechasedfor 2 h in the chemically-defmedmedium.Then, cells were stimulatedwith TNFa for the indicated times, scrapedand inmediatelysubjectedto low speedcentrifugationandfi-ozen at -20 0C.
Lipid extraction and analysis were performed according tu previously describedprocedures(Andrieu et al., 1994). Briefly, ceil pelletsweresuspendedin distilled water andceliswere disruptedat 40C by briefsonication(10 s). An aliquotwas takenfor protein determination.The remaiingwasextractedwith 2.5 ml of chloroform/methanol(2:1, vf~’). The lowerpliase waswashedwith a syntheticupperphaseconsistingof chloroform/methanol/water(3:48:47,y/y/y) andevaporatedunder nitrogen.Then the lipid extractwas subiectedto mild alkaline hydrolysis asfollows: the residuewasdissolvedin 0.25ml cbloroform and0.25ml methanolic0.5 M NaOH, andincubatedovernightat 37 0C. ASter additionof 0.85ml of chloroform,0.25ml of methanolic0.5 MHCl, 0.43ml of water and0.5 ml of chloroform/methanol(2:1, y/y) the upperphase(contaiingthe[3H]-choline label releasedfrom phosphatidylcholine)was countedfor radioactivity. The lowerphasewas washedtwice with the synthetic upperphase,and the washingswere countedforradioactivity. The organic phase (containing the SPM) was evaporated and counted forradioactivity. In sorneexperiments,the massof total phospholipidsandSPM wasquantitatedbymeasuringtheir phosphatecontent(Ames, 1966).
Ceramidequantitationwas carriedout using E. coli diacylglycerol kinase (Amersham,UR)and [y-32P]ATPaccordingto previouslypublishedprocedures(VanVeldhovenet al., 1992).
3. Results
3.1 TNFa-iutduced NG)? secretion iii primar-y astrocytes is independen!of PKC and MI4PKactivíties
In order to investigatethe regulationof NOF synthesisby TNFa, primary astrocyteswere exposed to the cytokine and the NOF secretedinto the extracellularmedium wasquantifiedby ELISA. Incubationof astrocyteswith TNFa inducedadose-dependentincreaseinthe NGF presentin the ceil supernatant,with a maximal effect at 50 ng/ml (datanot included).The enhancedsynthesis and secretion reached a maximal level after 48 h of cytokinestimulation.
In anattemptto clarify the possiblesignalingpathwaysinvolved in TNFaupregulationof NOF, celís werepretreatedwith the bisindolylmaleimide0F109203X(BlM), a highly specificinhibitor of PKC. We havepreviouslydemonstratedthat BlM treatmentof astrocytesis abletomimic the effect of PKC down-regulationon the regulationof NGF secretionin glial celís(Laviada et al., 1995).BlM did not affect TNFa-stimulatedNOF secretion,thereforeindicatinga PKC-independentmechanismof cytokine effect (Fig. lA). On the other hand, the cAMPanalogdibutyryl-cyclic AMP (db-cAMP) and the cAMP raising agentforskolin (FK) partiallyinhibited cytokinestimulatedNOF secretion(30% and25% inhibition, respectively).
The MAP kinase dependencyof TNFa-induced NGF secretion was studied bypretreatmentof the cells with the MEK inhibitor PD098059(Alessi et al., 1995), which is aMeto preventMAP kinase activation in primary astrocytes(Oalve-Roperhet aL, 1997a). SpecificMEK inhibition did not result in a decrease,but rather led to a slight increase,of TNFa-inducedNGF secretion,indicating a MAP kinaseindependentmechanism.Thiswasin markedcontrastwith NGF inductionby cell-permeantceramideand exogenouslyaddedSPMase(Fig.iB), where incubation with PD098059resulted in a 80 and 90% inhibition respectively,stronglysuggestingthat TNFa andceramidesactthroughdifferentpathways.
4
150
125
100
a:0 75z
50
25
o
1000
800
-~ 800
a:0zo. 400
200
o
Mg. 1. Regulationof TNFa-, C2-ceramide-aná SPMase-stimulatedNGF secretion.Primary culturesofrat astrocyteswere incubatedin the presenceof 2 ¡sM BlM, 500 ¡sM db-cAMP, 10 ¡sM FK, 100 ¡sM PD098059 or 500 nM PAO 1 h prior to 50 ng/ml TN?Fct(A) or 25 ¡sM C2-ceramide,1 U/ml bacterialSPMase<B) treatmentfor 48 h. NGF in thesupernatantswasassayedwith a doublesiteELISA. Dataare means±S.E.M. of 3 to 4 independentexperimentswith determinationsin triplicate. Agentsalone,which hadnoeffect on NGF seeretionhave been omitted for clarification. Statistieal analysis was performed byStudent’st-test;significantdifferencesare indicated:* PczO.025,** P<0.001.Statisticalanalysisfor TNFaandceramidestimulation is relatedto controlcelis,andwheninhibitors are usedthestatisticsla referredtu thevalueswith thestimulant alone.
Preincubationof primary astrocyteswith the tyrosine phosphoproteinphosphataseinhibitor phenylarsineoxide (PAO) completely abolishedthe TNFa-stimulatedNOF secretion.PAO hasbeenshownto preventNF-KB activationby TNFain botEmyeloid andepithelial cells(Singh and Aggarwal, 1995). Therefore, tliese results indicate that TNFcz regulationof NOFsynthesisis independentof PKC and MAPK activities, is partially inhibited by cAMP raisingagents,and mayinvolve the nucleartranscriptionfactor NF-KB.
5
3.2Regulation.of thenucleartranscriptionfactorNF-KB by TNFa
To determine the effect of TNFct on the nuclear transcription factor NF-a,electrophoreticmobility shilt assays(EMSA) were undertaken.Treatment of astrocyteswithTNFa ledto the activationof NF-KB, asdemonstratedin the EMSA by theformation of aspecificbandwhich was competedoff by an excessof unlabeledprobe. Fig. 2 showsthe time course ofTNFct-inducedNF-KB activation.Nucleartranslocationof NF-wB wasalreadyevident5 mm afterceil stimulationwith a maximaleffect at 30 mm of treatment.Although the nucleartranscriptionfactor AP-1 was also activatedin primary astrocytesby TNFa, AP-1 activation was transient,with maximalactivationonly 5 mm after TNFct stimulationandreturningto basallevelawithin15 mm (datanotshown).
We further investigatedNF-KB regulationby TNFa to identily possibleanalogieswithTNFa-stimulatedNOF secretion.As shownin Fig. 3, PAO completelyblockedNF-a activation,in agreementwith previousreportsindicating the role of tyrosinephosphoproteinphosphataseinNF-KB activationby TNFa (SinghandAggarwal, 1995). NF-KB activationwasslightly inhibitedby PICC inhibition asshown by the effect of BlM on TNFa sitimulation. Increasedintracelularlevelsof cAMP evokedby FK partially blocked the ability of TNFa tu activate NF-KB. Similarresultswere obtainedwith db-cAMP.The specifityof NF-KB
-~ TNF‘-4
Lo ‘~oo — Cj CD -~
NF-KB complex
Fig. 2. Time-courseof TNFa activationof NF-KB. Primar>’ astrocyteswere stimulatedfor the indicatedtimes with 35 ng/ml TNFcz, andthen nuclearextractssubjectedto EMSA. The gel is representativeof twoindependentexperiments.
activation by TNFa was demonstratedby the inhibitory effect exertedby the sermeproteaseinhibitor dichloroisocoumarin(DCIC) (datanot shown).Indeed,pretreatmentof celiswith thisproteaseinhibitor abolishedTNFx-induced NF-KB activation, which is concordancewith apreviousreportthat demonstratedthe existenceof aserine-likeproteasecrucialfor tlie controlof NF-KB activationby TNFa (Machleidtet al., 1994).
Some of the intracellular effects of TNFa have been related to its abiity to induceceramidegenerationupon SPM hydrolysis (Hannunet al., 1996; Spiegelet al., 1996). In orderto test theposaiblerole of ceramidegenerationin TNFastimulationof NF-KB activation,we
6
-~
zZ
~zz o
-q ~ +
o Z -ct~--~ -D ~ 4o E~~-dQW E-~
NF-kB complex
Fig. 3. Regulationof NF-KB activation by TNFa andceramíde.Pnmaryastrocyteswere treatedfor 30mm with 35 ng/ml TNFa, 25 ¡iM CS-ceramideamI 1 U/ml SPMase.Where indicated, celís werepreincubatedfor 30 mm with 5 ¡sM PAO, 2 ¡sM BlM or 500 ¡sM db-cAIMP. Results shown arerepresentativeof 3 to 4 independentexperiments.
assessedthe ability of ceramidetreatmentto activate NF-KB in primary rat astrocytes.Asshown in Fig. 3, treatmentwith the cell-permeantN-octanoylceramide(Ca-cerarnide)or withbacterial SPMase,which elevatesthe cellular ceramideconcentration,also stimulated thenuclear tranalocationof NF-KB. However, in agreementwith previousobservationson othercell types(Andrieu et al., 1995), their stimulatoryeffect wasmuch weakerthan that exertedbyTNFa.
3.3¡kB-adegr-adationoccur-stít co¡rcer-t with NF-,cBactivation.¿u TNFa-stirnulatedastrocytes
Dissociationof the inhibitory moiety, IkB, of cytosolic NF-KB complexesallows NF-KBactivation and translocationto the nucleus (O’Neill and Kaltschmidt, 1997). Therefore, wemeasuredthe levels of IkB-a protein in the cytosol of TNFa- and ceramide-stimulatedcelísbyWesternblot. Fig. 4A showsthat thereis a dramaticdepletionof the inhibitory subunitlvelsin TNFa-stimulatedcelís, which occura in parallel to the observedactivation of NF-KB. Inagreementwith resultsobtainedby EMSA, exogenousSPMase,Ca-,and C2-ceramidesproduceda slight decreasein cytosolic leveis of IkB-a (Fig. 4A); however, this decreasewas not aspronouncedas in TNFcz-treatedcelís.Degradationof IkB-a waspreventedby celípretreatmentwith PAO prior to TNFaor Ca-ceramidestimulation (Fig. 4B).
To further investigatewhether the SPM-ceramidepathwaywas activatedby TNFa inprimary astrocytes,SPM and ceramidelevels were measuredas describedin Materials andMethods.Althoughslight variationscould be observed,TNFa stimulationsfor up to 120 mmdid not evokeany significant decreaseof 5PM levels nor any increaseof intracellular ceramidelevels (Fig. SA). SPM massmeasurementswere carriedout leading to identical resulta(n3;
7
datanot included). Furthermore,neitherSPM nor ceramidelevels were affectedby TNFa inthe C6 glioma celí line (n3, datanot included). Measurementof phosphatidylcholine(PC)anddiacylglycerol (DAO) levels also indicated the absenceof a pool of PC sensitive to TNFa inastrocytes(Fig 5B).
JkB
IkB
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a,
‘-4 ‘-4a)e e
Cl CCr) o U)
-4o
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~ oz ~H ~
‘-4a)e
CCo
z
Fig. 4. Westernblot analysisof IkB-a subunit on cytosolic extractsfrom stimulatedastrocytes.Al Celíswerestimulatedwith vehicle, 25 ¡sM C2-ceramide,25 ¡sM Ca-ceramide,1 U/ml SPMase,or 35 ng/ml TNFafor 30 mm. B/ Celís were ineubatedwith or without 5 ¡sM PAO for 30 mm prior to TNFa or Cs-ceramidestimulation.
The NOF gene promoter contains a NF-KB like binding site located 669 base pairsupstream of the TATA signal (Jehanet al., 1993). To examine whether this site is apotential target for activatedNF-KB to regulateNOF genetranscription,gel shift assayswereperformedwith purified nucleartranscription NF-KB and with the oligonucleotidesofthe proposedsite of regulation. Fig. 6 shows that, although this NF-KBNGF probe is able tobind speeifically the nuclear transeription factor (seeresult with mutatedoligonucleotide),the intensity of the binding is weak as compared to the binding to NF-KBconsensus
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120
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80
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20
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Fig. 5. Lipid quantifleationafter TNFx stimulation.Primary rat astrocyteswere labeledwith [H]cholinefor 72 h. Then, celís were incubatedwith 35 ng/ml TNFz and at the indicatedtime points incubationswerestopped.SPM (Fig SA, -@-> andPC (Fig. 5B, -@-) levelswere determinedasdescribedin ‘Materialsand Methods”.Phospholipidlevelsareexpressedas percentageof thevalue observedat time O. Valuesrepresentthe mean±S.E.M. of 4 different experiments.Ceramide(Fig. SA, -O-) andnAO (Fig. SB, -O-)levels weredeterminedby the DAG kinasemethod.Valuesareexpressedas percentageof radiactivity attime O of 3 independentexperiments.Statistical analysis using the Student’s t-test revealed nosigsiificant differences.
oligonucleotide.In fact, cellular NF-KB presentin the nuclearextractsfrom TNFa-stimulatedcelíswasnot ableto bindto the NF-KBNGF site (datanotshown).
4. Discussion
Resultsfrom the presentreportpoint tu a differential effectof TNFa versusexogenousceramideor SPMasein the regulationof NOF synthesisandsecretionby primary astrocytes.Inspite of the many efforts tu elucidate the mechanismsthat control NOF synthesis, theregulationof this complexprocessremainslargely unlknown. We havepreviouslydescribedtheinvolvementof exogenousPC-PLCin the regulationof NGF synthesisby astrocytes(Laviadaetal., 1995). PC-PLC in turn has also been reported tu mediate TNFa activation of NF-KBthroughacidic5PMbreakdown(Schútzeet al., 1992;Wiegmannet al., 1994).NGF inductionbyPC-PLCis partially dependenton PKC activity (Laviadaet al., 1995),whereasTNFa inductionof NOE synthesisin primary astrocytesseemstu be independentof PKC activity (the presentreport). This is also true for N01’ induction by cell-permeantceramidesand exogenousbacterialSPMase(Oalve-Roperhet al., 1997).
O lO 10 45 60 75 90
TIME (coja)
9 15 30 45 60 75 90
TIME (mio)
9
The existenceof crosstallcsamongdifferent signaling pathways,which allows a tightcontrol of NOF synthesis, is evidencedby the inhibitory effect of increasedintracellular cAMPlevels on TNFz-stimulatedNGF production.We havepreviously describeda similar inhibitoryeffect of thecAiMP cascadeon ceramide-andlipopolysaccharide-inducedNGF synthesis(Oalve-Roperhet al., 1997a; 1997b),althoughthe exactmechanismunderlyingthis processis not wellunderstood.IncreasedcAMP levels have been shown to prevent MAP kinase activation inastrocytes(Kurino et al., 1996; Willis and Nisen, 1996). However, although TNFa increasesp42/p44MAP kinaseactivity in primary astrocytes(unpublishedobservation),this activationwould not be responsiblefor theinduction of NOF synthesis,asshownby the lack of inhibitoryeffect of P0098059(Fig. 1). Our datarather indicatethat NOF induction by TNFct in primaryastrocytesis independentof MAP kinase activity, in sharp contrast with the effect ofceramides,which activate NGF synthesisby a p42/44 MAPK activity-dependentmechanism(Oalve-Roperhet al., 1997a,andpresentreport). The inhibitory effect of cAiMP may also beattributedto the observeddecreaseof TNFct-activationof NF-KB (the presentreport; Pahanetal., 1997).
uE
a> ceci ci a az 8 8
o,,~ -~ -~ ~ ci a
z8
z z+ + + + rL ~~ z z
NF-kB complex
Fig. 6. Analysis of NF-KB binding to different oligonucleotides.Gel retardationassayswere performedwith 400 ng of the purffied NF-KB protein (1’) andthe correspondingradiolabeledprobes.Lanes1, P +NF-KB NGF probe;2, P 4 NF-KB NGF mutatedprobe;3, P + NF-KB consensusprobe; 4, P + NF-KB consensusmutatedprobe;5, free NF-KB NGF probeand6; free NF-KB consensusprobe.
Our resultsshow that in primaryrat astrocytesthe nucleartranscription factor NF-KB18 rapidly activated by TNFx stimulation. The ability of TNFa to activate NF-KB is wellestablished,butmuchcontroversystill existsaboutthe signalingmechanismsinvolved insuch a stimulation. Sornereportsindicatethat ceramidegeneration,by acidic SPMase,rnayberesponsiblefor NF-KB activation (Schútzeet al., 1992;Machleidtet al., 1994;Wiegmannet al.,1994). In contrast,othershavenot found. evidencefor the involvementof ceramidein NF-KBstimulation (Bettset al., 1993; Andrieu et al., 1995;Gamardet al., 1997). Moreover,evidencehasbeenpresentedagainsta role of acidic SPMasein the effectsof TNFa (Andrieuet al., 1994;ICuno et al., 1994; Zumbansenand Stoffel, 1997). In our hands,exogenousSPMaseand Cs-
lo
ceramide,which stimulateNOF synthesisto a muchgreaterextentthan TNFct, activatedNF-KB in primary astrocytes,althoughto a lesserextentthan TNFa. In addition, IkB-a egradationwaslessaffectedby ceramidesthanby TNFa.Recently,Modur et al. (1996)havedescribedthatin endothelialcelís TNFa is aMe tu activate Raf-1 througli a ceramide-dependentpathway,whereasNF-KB activation 15 achieved by a ceramide-independentmechanism.Our datasuggeststhat primary astrocytesrnay behavein sucha way. As a matter of fact, ceramideisableto induce subeellularredistributionof tEn atypicalPKC 4 in glial cells (Oalve-Roperhetal., 1997c), andthis effect coincideswith increasedRaf-1 pliosphorylationandMAPK activity(Oalve-Roperhet al., 1997a).
The existenceof a ceramide-independentmechanismfor the TNFa-inducedstimulationof NF-KB was confirmed by the lack of significant 5PM hydrolysisupon celí stimulation. It iswurth notingthat the absenceof activation of the SPM-ceramidepathwayby TNFamight beacommon featureof glial celís, since murine C6 glioma celís and humanU2S1MOglioma celísare also resistantto TNFa-stimulatedSPM hydrolysis (datanot shown;Richard et al., 1996).Moreover, although ceramidesare able to stimulate stress-activatedprotein kinase in glialcelís, TNFcz activationof SAPK doesnot seemto involve the ceramidepathway (Zhanget al.,1996).Re TNFx-sensitiveSPM pool involved in signaltransductionhasbeensuggestedto belocated in the inner leaflet of the plasmamembrane(Andrieu et al., 1996). The absenceofsignificant variations of 5PM mass levels, togetherwith the absenceof ceramidegeneration,rule out the possibility that metabolic labeling with [3H]cholinemay not be able tu label theTNF~-sensitivesignalingpooíof SPMin confluentastrocytes.
The origin of the endogenousceramidesresponsiblefor the inductionof NOF synthesisin the celímaybe linkedtu otherpossiblegenerationsourcesof ceramidethan5PM hydrolysis,e.g. resulting frum stimulated “de novo’ synthesis or inhibited degradation.Theseprucesseshavealreadybeenshownto mediatesorneof the biological effectsuf sphingolipids(Boseet al.,1995; Bielawskaet al., 1996;Paumenet al., 1997). For instance,the generationof ceramidebya mechanisminvulving a reducedrate of catabolism would lead to a prugressiveand slowaccumulation of the lipid within the ceIl, and this may be of great importancein regulatinglong-term responses(Bielawskaet aL, 1996).
In summary,our resultsare in line with previousreportspointing to the existenceofceramide-dependentand ceramide-independenteffects of TNFa. Our data provide strongevidence that TNFa and ceramide act by different mechanismsin the regulation of NOFsecretionby astrocytes.This is evidencedby their differentdependencyun MAP kinaseactivity,the lack of SPM-ceramideactivation by TNFa, and their different ability tu activateNF-KB.Finally, our results indicatethat, althoughNF-KB transcriptionfactor doesnot appeartu play adirect role on NGF genetranscriptiun, TNFa-induction of NGF synthesismayrely un previousNF-kB activatiun.
AcknowledgmentsWe are indebtedto Dr. M. Ouzmánfor fruitful commentsun the manuscript,Dr. U.
Wiun for the NGF promoteruligonucleotidesequences,F. Valiño andDr. E. Dulín for technicalassistance.I.O.R. was recipient of a FEBS shurt-term fellowship. This work was financiallysupportedby SpanishCICYT (SAF 96/0113),FIS (97/0039),CAM (6648)andINSERM grantstuT.L.
II
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activatiunof mitogen-activatedproteinkinaseñ~ vitro andin vivo. ~J.Riol. Chan.270,27489-27494.AmesB.N. (1966)Assayofinorganiephosphate,totalphosphateandphosphatases.Meth.Enzyrnol.8, 115-118.Andrieu N., Salvayre R., and Levade T. (1994) Evidence againsí involvement of the acid lysosomal
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[3
Joarnal of NeuroscienceResearch49:569—575(1997)
Regulation of Nerve Growth Factor Secretionand mRNA Expressionby BacterialLipopolysaccharide in Primary Cultures of RatAstrocytesIsmaelGalve-Roperb,’JoséM. Malpartida,1 Amador Haro,1 Ph¡l¡ppeBrachet,2and InésDíaz~Laviada3*‘Dcpartrnent of Hiochcmistry asid Molecular Biology, Faculty Biology, Univcrsity Complutense, Madrid, Spain2Centre Hospitalier Régional Universitaire, INSERM U.298, Angers, France3Departínent of Biochemistry asid Molecular Biology, Faculty Medicine, University of Alcalá,Alcalá de Henares, Madrid, Spain
The present work was undertaken to study tbe effectof bacterial lipopolysacebaride(LPS), a potent activa-tor of the hostinfianimatory response,on dicsynthesisof nerve growtb factor (NGF) by newborn rat brainastrocytes. Treatment of pr¡mary rat astroglial celíscultured in chemieally defined medium with LPSresulted in a dose-dependentaccumulation of NGFmRNA, andan increasedreleaseof NGF protein lii theeclI ¡nedium. NGF mRNA levels were maximal after24 brof stimulation (8-fold mercase),whereasextraed-lular NGF peakedafter 72 bours of treatment (17-foldmercase).This dramatie mercaseof extraecílular NCFwasabrogated¡f celís wcrc treated witb actinomycinD or cycloheximidc, a fact whieh implies tbat titeaceumulat¡on of extracellular NGF by LPS-treatcdcelís requires DNA transcr¡ption and RNA transía-tion. Stimulation of NGE syntbcs¡sand seeretionwas:(i) unaifeeted hy treatment witb thc protein kinase Cinhibitor b¡sindolylmaleimide, asid (II) preventedbyforskolin and 3-isobutyl-1-metbylxanthine,two agentswhieb mercaseeAMP levels. Inhibition of LPS cfTeetivasalso obtained with apigenin, a proposed inhibitorof tite mitogen-netivatedprotein kinase pathway. Re-sults thus showthat LPS stimulates NGF synthcsisbyastroglial celís througb a meehanismtbat is indepen-dent of protein kinase C (PKC), antagonized bycAMP-elevatingaguils, and probably mediatedbytite mitogen-activatedprotein kinase cascade.Titedataraisetite possibility that LPS exertsstimulatoryeffeetson NGF synthesistbat are independentof tboscelicited by astroeytc-dcrived inflammatory lympho-kines sueb as IL-113, TNflx or TGF~1. J. Neurosci.Res.49:569—575,1997. o 1997 Wilcy-Liss, Inc.
Key words: lipopolysaecbaride; nervegrowtb factor;astroeytcs; protein kinase C
INTRODUCTION
Bacterial lipopolysaccharide(LPS) is the sole lipidpresentisi the outer membrasieof Gram-negativebacteria.It acts as a potent activatorof the host isifiammatoryresponse. In the cesitral nervoussystem (CNS), localinjection of LPS is followed by arapid activation of brainresident macrophages, the microglial celís, and by arecruitment of peripheral monocytesor macrophages(Montero-Menei et al., 1996). However, astrocytesbe-come activated in a secosid phase of the inflammatoryprocess. It is presently well established that astrocytesconstitute an importasit auxillbry ccli controlling theimmunological status of the CNS. These celis, whcsiactivatedwith LPS, produce lymphokisies or cytokinessuch as nitric oxide, IL-113, IL-6, TOP-pl asid TNFa(Kimberlin et al., 1995; Schmidlin asid Wiesingcr, 1995;Willis andNisen, 1995;Slegersasidjoniau, 1996) whicliare actively driving tite immune reactiosis. However,astrocytesarealso a seurceof neurotrophicfactors,asid
Abbreviations: BlM, bisindolylmalcimidc; cAMP. cyclic AMP; FCS,fetalcali scrum; IBMX, 3-isobutyl-1-methylxanthine;IL- 113, intcrlcu-kin- 13; IL-6. interleukin-6; LPS, lipopolysaccharidc;MAP kinase,mitogen-activatcd protein kinase; NGF, nerve growth factor; PKC,protein kinmc C; FMA, phorbol 1213-myristate 13a-acctatc; TGFI3I,transforming growth factor 31; TNFa, tumor necrosis factor u; Tyr,tyrphostin.Contract gr-ant sponsor: Comisión Interministerial de Cicncia y Tecno-logia; Contract gr-ant number: SAF 96-0113. Contract gr-ant sponsor:Fondo de Investigación Sanitaria; Contract gr-ant number: FIS 97/0039-01. Conrract gr-ant sponsor: Accion Integrada (Picasso Progrm);Contract grant number: 95/138.*Co~espondencc tu: Dr. Inés D. Laviada, Dept. of Biochemistry,Faculty of Medicine, University of Alcalá. 28871 Alcalá de Henares(Madrid), Spain. E-mail:id¡@’solea.quim.ucm.es
Received l4Fcbruary ¡997; Revised ¡SApril 1997; Accepted l5April¡997
these different mediators, when applied exogcnouslyalune or in combination, can act as inducers of thesynthesis of the neurotruphic factor nerve gruwth factor(NGF), at least in vitro, and pussibly in vivo (Pshenichkinet al., 1994;Pltiss et al., 1995; Hattori et al., 1996). In amuregeneral way, arelationship between CNS inflamma-tion and NGF accumulation exists, since levels of thisfactor appear significantly increased in cerebrospinalfluids of multiple sclerosis patients (Bracci-Laudiero etal., 1992).
NGF is mainly known asa neurunalsurvival factor,active un sorne peripheral fetal neuruns and, in the CNS,un cholinergic neurons uf basal forebrain (Varun a al.,1995). However, high aftinity recepturs for NGF wereidentified un pheripheral monocytesand/or T lympho-cytes (Ehrhard et al., 1993), while the factor may beproduced not unly by astrocytes, but also by activated
brain microglial celís (Mallat et al., 1989).Therefore,theinvolvement of NGF in the neuruimmuneinteractionswhich takeplaceduring an inllammatoryreactionin theCNS appears probable, but remains elusive. In thisgeneral cuntext, we have investigated whether LPS caninduce NGF synthesis in newborn rat astrocytes, andanalyzed the signalling pathway activated by LPS inthese celis. Previousstudies have shuwn that pruteintyrusine-kinase activatiun andnucleartranscription factorNF-KB transduceLPSresponsesin monocytes or astrucy-toma celís (Geng et al., 1993; Carísunand Aschmies,1995). LPS strungly activatesp42míPk and Raf-l, twoessential componentsof tIte mitugen-activatedprutein(MAP) kinase signal transductiunpathway (Willis andNisen, 1996). Agents tliat elevate intracellular cyclicAMI’ (cAMP) have been slouwn tu inhibit the MAPkinasepathway(Cook andMcCurmick, 1993;Wu et al.,1993) and tu decreaseLPS-inducedIL- I~B mRNA accu-mulatiunin humanastrocytes(Willis andNisen,1995).
Our resultssbow that LPS is a powerful inducerofNGF synthesisin astrocytes.Thecumpuundactsthrougha signal transduction pathway independent of proteinkinase C, antagonizedby cAMP-elevatingagents, andinvolving presumablyMAP kinaseactivatiun.
MATERIALS AND METHODS
Primary Culturesof Astroglial Celís
Cortical astruglial celís were derivedfrom 1- tu2-day-uld rats and cultured as previously described(Laviada et al., 1995).Celís were seededat a densityof3 >< lO~ cells/cm2un plastic platespreviouslycoatedwith5 ~ig/mldl-pulyurnitbine in water.Celíswereculturedfor3 weeks in basal medium cunsisting uf a mixture ofDulbecco’smodified Faglemedium(DMEM) and Ham’sFI 2 (1:1,y/y), with0.66%glucose, 5 pg/ml streptomycin,5 U/ml penicillin, andsupplemented with 10% fetal calf
serum (FCS). Microglia-oligudendroglia-freeastroglialcultures were obtained after 2 hours uf orbital shaking.Ihe primary cultures cunsisted uf 95% astrucytes asjudged by immunocytuchemical staining uf glial fibril-lary acidic protein. Time course, duse response andNurtbern blut experiments were carried orn in 57-cm2plates. The rest uf the experiments were done in 6-welldishes.
Three days before the experiment, at day 21, theserum-containing medium was removed and celís weretransferred tu a chemically defined medium cunsisting ofserum-free basal medium supplemented with 25 ng/mlinsulin, 50 pg/ml humantransferrin, 20 nM prugesterone,50 jiM putrescine, and 30 nM sodium selenite.
After the indicated times uf treatment, RNA extrac-tiun wascarried out accordingtu the LiCI/ureamethod.Nurthern blot analysis was also performedby standardprocedures(Jehanet al., 1995).Glyuxal-treatedRNA wasfractionatedin agarosegels, transferred tu Hybond-Nmembranesby capillaryblotting, andhybridizedseriallywith a32P-labelledprubeof muuseNGF cONA.The NGFprobewas the 917-bpNGF DNA cloned by Scottet al.(1983). Standardizationof RNA loading was routinelycontrolled by hybridization of tite blots with amyluidprecursorprotein cDNA prube (Shivers et al., 1988).Densitumetricanalyseswereperfurmedwith Phosphorlm-ager445 SI (MolecularDynamics,Sunnyvale, CA).
FurextracellularNGF determinatiun,celíssuperna-tantswerecollected24 huursafter treatmentas describedin tite text, diluted in une volume of phosphate-hufferedsalme(PBS)containing0.1%Tween20 and0.5%gelatin,andcunservedfruzenuntil quantitatiun.NGE releasedbytite celís was assayed in triplicate, by a double-siteELISA, usinga munoclonalanti-NGFantibudy,coupledur not tu fB-galactusidase(BuehringerMannheim,India-napulis, IN), according tu an experimental protocoldescribedbefore(Laviadaet al., 1995).
Otiter Citemicals
Tissue culture plastic wares were purchasedfrumNunc (Roskilde,Denmark),culturemediafrom BioWhit-taker (Verviers, Belgium), and FCS frum JRH Biosci-ences (Sussex,England). E. coli 0127:B8 LPS W wasfrom DIFCO Laboratories(Detroit, MI). It wasdissulvedand sonicatedin PBS prior tu utilization. Hybond-Nmembraneswere from Amersham(Little Chaltfont, En-gland). Apigenin, forskolin andbisidulylmaleimidewerefrom Calbiuchem(La Julia,CA). 3-Isobutyl-1 -methylxan-titine (IBMX) and otherreagentswere from Sigma (St.Louis,MO).
LipopolysaecharideRegulation of NGF Synthesis 571
RESULTS
Induetion of NGF Expression and Seeretionby LPSHigitly enrichedastruglial cultureswereexpusedtu
increasingconcentrationsuf E. cali LPS during24 hr,andsecretedNGF was determinedni the supernatantofcultured celís. As shown in Figure 1, LPS treatment-increasedthe amount of extracellularNGF in a dose-dependent manner. Maximal stimulation (1 0-fold overcontrol) wasobserved at 2 pg/ml. In additiun, Nortiternblut analysis of total cellularRNA wasusedtu studytiteeffect of LPS un NGF mRNA levels. Asimilarconcentra-tiun-dependent effect of LPS was observed un NGFmRNAaccumulatiun(Fig. 2).
Primary culturesof astrocyteswere treated with amaximal duseuf LFS (2 pg/ml) and extracellularNGFanéNGF mRNA accumulationwereassessedafter van-uus time periods. Figure 3 situws titat titere was noincreasein extracellularNGEconcentratiunafter 8 hr uftreatment. In contrast, a striking enhancementwas no-ticed after 24 itr. Maximal NGF secretionoccurredincelís treatedfur 72 itr with LPS (17-fold increaseuverbasal value). Densitumetricanalysis uf Nurthern blutspresentedin Figure 4 indicatedthat a 4-fold increaseinNGF transcripts touk place after 8 br of treatment,witereas maximal levels uf NGF mRNA were reachedafter 24 itr of treatment(8-fold mercase).
Tu assesswitetherLPS inductiunwasdependentunDNA transcriptiunandRNA transíatiun,weempluyedaninhibitur of DNA transcription,actinomycinO (Act O),and an inhibitur of prutein syntitesis, cycluiteximide(CHX). Resultssummarizedin Table 1 sbow that treat-mentwith either2.5pM Act O urSpM CHX completelyblocked tite augmentationof NGF secretiuninduced byLPS-
incubatedwith increasingconcentration of LPS during 24 hr. Atthis moment, NGF releasedin the supernatants was assayedwith a double-siteELISA. Dat-a are meatns1 S.E.M. of fourindependentexperirnents.Each determination was in triplicate.Maximal NGF secretionwasobtainedwith 2 iig/m¡ LPS.
NGF
1 2 3.01 67
PKC and Tyrosinc Kinasc Inhibit¡on Do Not AfTcettite Induction of NGF Seeretionby LPS
In an attempt to study tite patitways involved in thecontrol of NGF synthesisby LPS in astrocytes,celísweretreatedwith bisidolylmaleimide(BlM), a highly specificinhibitor of PKC, prior tu tite incubation with LPS.ClassicalPKCs are strongly activatedby phorbol estersas phorbol 12-myristate 13-acetate(PMA), whicit is aputent inducer of NGF genein astrocytes(Neveu et al.,1992). We itave previouslydescribedthattreatmentwiththe PKC inhibitor BlM abolisited PMA-induced NGF(Laviadaet al., 1995). Treatmentof astrucyteswitit SIMdid not affect LPS-inducedNGF secretiun(Table 1),indicating that tite signaltransductionpathwaytriggeredby LPS wasPKC-independent.
Tyrusine kinase inhibitors have been shuwn tublock sumeof tite LPS effects.Tu studywhetheractiva-tiun of tyrusinekinaseswas onvolved in NGF induction
APP~ ~ .9,,,Fg2N hnbo na oNOFmRNAa m uninducedby increasingconcentration of LPS. Celís were treatedwith increasingconcentrations of LPS. Lane 1,untreated celís;lane 2,0.001 pg/ml; lane3,0.01 ¡ig/ml; ¡une4,0.1 ¡ig/ml; ¡une5, 0.5 gg/ml; lane6, 2 gg/ml; lane 7, 10 gg/m¡. After 24 hrincubation, total RNA was isolated, electrophoresed,blottedand serially hybridized with a radiolabelled NGF and AP?cONA probes.Essentiallyidentical results were obtained inthreeindependentexperiments.
by LPS in astroglial celís, we empluyed the inhibiturstyrphustin A0126 and genistein.As situwn in Table 1,neititer tyrpitustin AGI2G nor genistein affected LPS-stimulatedsecretionof NGF.
1
n 1
572 Galvc-Roperhet al.
80
70
60
o>o.u-<3z
50
40
30
20
10
0
hours
Fig. 3. Time courseof NGF secretionafter treatmentwith LPS.Primary astrocytecultureswere incubatedin the presenceof 2~ig/mILPS. At the indicated times, cell-secretedNGF wasassayedwi¡h a double:siteELISA assay. Data are mean ±S.E.M. of two independent experiments with determinations intriplicate.Maximal inductionoccurredat 72 hoursof treatment.
Tite effect of raising intracellularcAME levels unLES-inducedNGF releaseby astroglialcelís was deter-mined by treating celís with eititer furskolin (FK) orIBMX. FK or IBMX alune had no effect un NGFsecretion. Huwever, a 1 -hr preincubation with theseagents led tu a complete inhibitiun uf LPS-stimulatedsecretiunof NGF. Celí treatmentwith thecAMEanaloguedibutyryl-cAMP exertedtite sameinitibitory effect (datanot sitown). Tu cunfirm tite antagonisticaction of cAMEun theinduction uf tite NGF geneby LES, Nortitern blotanalyseswere carneé uut under the same treatmentconditions.Figure 5 shuwsthat botit FK and IBMX docompletely block the accumulationof NGF transcriptswhicit uccursin responsetu aLES treatment.
Stimulation of NGF Produetion by LPS Is Initibitedby Apigenin
Tu furtiter examine tite pathway by witich LESinducesNGF syntitesis and secretion by astroglial celís,the plant fiavonuid apigenin (API) was used. Celís werepreincubated with apigenin for 1 hr, and titen treated .wititLES. Under sucit conditiuns, LPS failed tu induce anyaugmentatiun of tite amount of cdl secretedNGF (Table
NGF
1 23 4
‘OsÍAPPfl•
Fig. 4. Time courseof NGF mRNA accumulationin astroglialcelís treated with LPS.Celíswere treated at the indicated timeswith 2 pg/ml LPS and NGF mRNA accumulationassessedbyNorthern blot. Lane 1, 0 hours; lane 2, 8 hours; lame 3, 24hours; lane4,72 hours; lameS,¡20 hours.Essentially identicalresults were obtainedin two independent experiments.
TABLE 1. Efl’ect of Treatnient With Dillerení Agcnts o» LPS-Induced NGFSecretionby Astroglial CelIs*
*Primary cultures of astrocytcs were incubated in the presence of the
indicated agents ¡ hour prior tu LPS Ireatmcnt fur 24 hours. NGF u ihesupernatants was assayed with a double-site ELISA. Data are means ±S.E.M. of three indcpendent experiments with dewrminations intriplicate. Agents alune, which had no etfect un NGF secretion, havebeen omitted tu cl-arify Ihelable.Abbreviatiuns: Act D, -actinumycin D; CHX, cyclohcxiniide; BlM.bisindolylnnleimidc; Tyr AC126. tyrphostin A0126; FK, forskolin:IBMX, 3-isubutyl-l-methylxanthine; API, apigenin.
1). Tite compuundalsobluckedtheNGF mRNA accumu-lationin thecelís (Fig. 6)-Titedose-responsecurveshowstitat tite 1C50 valuefor apigenininhibition was approxí-mately lO ~iM(Fig. 7).
DISCUSSIONTite present study shows that LES is a putent
activaturof NGF secretionin primaryculturesof corticalastrocytes.LES inducedNGF synthesisanésecretiunbyastruglial celís in a cuncentration-dependentmanner.Adoseuf 10 ng/ml wasalreadyeffective but thestrongestresponsewasobtainedwitit a doseof 2 jig/ml LES. At titisconcentration,LES induced an accumulationuf NGFmRNA and NGF proteins whicit reacitedtiteir maiximallevelsafter24 itr and72 itr uf treatment,respectively.Titeprotein secreted in tite extracellular medium was in-
0 8 24 72 120
Lipopolysaceharide Regulation of NGF Syuthesis 573
12 3 4
NGF Oo,o-
I.l~0z
APPS••U~
Fig. 5. cAMP-clevatingagentscounteract NOF mRNA accumu-lation inducedby LPS. Astrocyte cultures were treated with noadditions (lane 1) or with 2 ¡ig/ml LPS alune (Infle 2) or in thepresenceof eitherforskolin (FK) lO ~M (Iane3) or3-isubutyl-¡-meihylxantbine (IBMX) lOO ¡iM (Infle 4). FK and IBMXwereadded1 hotir prior tu LPS treatment. Essentially identicalresulís were obtained in two independent experiments.
1 234
NGF
100
75
50
25
o
[Apigenin] pM
Fig. 7. Dose responseinhibition of LPS-inductedNGF secre-¡ion by apigenin. Astrocyte cultureswere incubated with 2ag/ml LPS andthe indicated doses of apigeninfor 24 hoursandNGF secreted to the supernatantswas assayedwith a double-siteELISA assay.Dataaremeans±S.E.M.of two independentexperimentswith determinationsin triplic~¡te.
ARR
Fig. 6. Apigenin inhibits NGF mRNA accumulationin as-troglial celís induced by LPS. Primary cultures were treatedwith no additions (lame 1) or with 2 ¡dg/ml LPS (lame 2), 25 pMapigenin (lane 3) or 2 ¡ig/ml LPS plus 25 14/ml apigenin (lane4). The latter was added 1 hour prior tu LPS treatment.Essentially identical results were obtaincd in twu independentexperiinents.
creased by afactoruf 1 7-fold, witicit is considerable.Titeeffect uf LPS requiredbutit prutein syntitesisandDNAtranscriptiun,as it was abrogatedby initibition uf cititerbiusyntiteticsteps,usingCHX anéActO.
LES induction uf NGF by astrocytesmay be titeresuiruf a complexprucesscumprisingtite variuuseffectsuf LES un titosecelís,as well as un otiter celí types titatmigitt be presentin tite primary culture. However, tite-experimentalprucedureusedin tite presentwurk, whicitprovidesitigitly enrichedastrocyticcultures,points tu adirect effect of LPS on NGF synthesisand secretionbyastrocytes.Nevertiteless,stimulation of NGF syntitesismay not result from a direct actionof LES un astrucytes,but frum autocrine luups. Otiter products releasedby
astrocyte in responsetu LES cuuld enitancetite expres-siun of tite NGF gene.This is thecaseuf inflammatorycytokinesTNFx, TGFj3 and IL-lI3, witich have beendescribedas inducersof NGF synthesis(Sprangeret al.,1990; Pshenichkinet al., 1994;Pltisset al., 1995;Hattoriet al., 1996).However,inductiunof cytokinesby LES itasbeen sitown tu uccur througit protein tyrosine kinaseactivatiun (Geng et al., 1993). Tyrphostin AG126 itasbeensitown tu preventLPS tuxicity in vivo anéblucksLPS-stimulatedTNFct anénitric oxide (NO) productionby macropitages(Novogrodskyet al., 1994;Vanicitkin etal., 1996). Genistein itas also been implicated in titeinitibition uf the syntitesis of IL-6 stimulatedby IL-l¡3(Caríson ané Ascitmies, 1995). Our results did notevidence any effect of tite tyrosine kinase initibiturstyrpitostinAG 126 andgenisteinuntheLES-inducedNGFproductiun. Titerefure,our datasuggesttitat tite regula-tion of tite expressionuf tite NGF geneelicitedby LES isnut mediatedby TNEa,IL- 1, IL-6 or NO pruduction.
Tu furtiter study tite mechanismtriggeredby LPSand leading tu a stimulatiun of NGF production byastrucytes,we examinedwitetheragentsabletu increaseintracellularcAME caninitibit tite effectof LES. Tite roleuf cAMP anéprutein kinase A in NGF regulatiun byastroglialcelís is still nut clearly defined.Dependinguntite celí cultureconditiuns,tite gruwth stateof celís andtite experimental mudel, different results itave been
0 5 10 15 20 25
574 Galve-Roperh ct al.
ubtained.Witile undercertaincunditiuns,forskolin, cate-chulaminesor cAME analuguesstimulatedNGF synthe-sis, tite cumpuundswere fuundby otiters tu be ineffectiveor able tu counteract tite promotory actiun of severalinducersof NGF syntitesis(Haitnetal., 1994;Jeitaneta!.,
1995). Our results fil witit this sciteme,sincetitey showtitat FK ané IBMX block tite stimulatury effect uf LES,butit at tite level uf NGF mRNA accumulation andcelí-secretedprutein.FK anéIBMX havebeeninvolvedin theblockadeof tite MAP kinasepatitwayin epidermalgrowth factor (EGF)-stimulatedcelís titruugit a cAME-dependent increase in Raf-l pitospitorylatiun, witicitpreventsRas-éependentactivation of Raf-l (Cook anéMcCormick, 1993; Wu et al., 1993). Our data inéicatethat apigenin,a prupusedinitibitur of the MAP kinasepatitway, also inhibited LES-induced NGF synthesis.Apigenin itas beendemonstratedtu reversethe v-H-ras-transformed phenutypeuf NIH 3T3 celís titrough theinitibitiun uf tite MAE kinase-assuciatedsignaltransduc-tion patitway (Kuu anéYang, 1995). Our result suggeststhat LES triggers in astroglial celís a MAP kinase-dependentmecitanismwhicit promutestite activation oftite NGF gene.It is notewortitytitat activationuf tite MAPkinasecascadeby LPS,and its initibition by agentstitat ele-vate celí levelsof cAME, wasrecentlyreporteéin a itu-man astrocyticcelí line, U-373MG (WiIlis anéNisen, 1995).
Twu general mecitanismsappeartu promote titeexpressionof tite NGF genein astrucytes.Tite first uneuperatestitrough tite activatiunof PKC, anéis triggeredby scrum ané éiacylglycerol (D’Mello and l-Ieinricit,1990;Neveuet al., 1992).Tite otiter uneactsthrougittitesphingomyelinpatitway,and is tituugitt tu beswitcited unby vitamin D3 (Neveu et al., 1994). We itave recentlydescribedtitat tite breakduwnof phosphatidylcholinebypitospitulipaseC may activatebotit mechanisms(Laviadaet al., 1995).Resultssituwn in tite presentreport inéicatetitat the regulatiunof NGF production by LPS in astro-cytes seemstu be independentof EKC activation, andtiterefore, LES could control NGF syntitesis by titesecuné mecitanism. Bacterial LPS has been sitown tutrigger similar cellular responsestu TNFa and IL-l~,witich areknuwn tu initiate a signalling pathwayinvolv-ing spitingomyelin itydrulisis (Kulesnick ané Golde,1994;Hannunet al., 1996) andRaf-l activation(Belkaetal., 1995). Recentstudiessuggesttitat LES is recugnizedby tite sameintracellularmoleculestitat biné tite seconémessengerceramide(Wrigitt anéKolesnick, 1995). Fur-titermore,LES canstimulatea ceramide-activatedpruteinkinasein HL-60 celís (Josepitet al., 1994) whicit appearscapableuf catalyzing tite pitosphorylatiunuf Raf (Yau etal., 1995). Titis suggeststitat the MA? kinasecascadeisinvolved in tite spitingomyelinpatitway. Therefore,ourresults indicate titat LPS stimulates NGF syntitesis in
astrucytes by a mecitanism that seemstu be closelyrelatedtu tite patitwayactivatedby spitingumyelinaseandwitich is independentof PKC.Thedataraisethe pussibil-ity titat stimulatiun uf tite synthesisof NGF is a éirectcunsequenceuf LES actiun un astrocytes,ratherthan anindirect responseelicited by LES-inducedcytukinessuchasIL- 13,TNF~ or TGFj3.
NOTE ADDED IN PROOE
We itave recently observedthat LES induces a3-fuld stimulationof p42/p44MAPK in primary culturesuf astrocytes.
ACKNOWLEDGMENTS
We titank Dr. M. Guzmánfor itelpful discussiunandcritical reaéingof tite manuscript,and tu Dr. E. Dulín ¡briter assistancein the ELISA prucedurewitit tite Fluoros-kan -
Titis work wassupportedby grantsfrum ComisiónInterministerialde Cienciay Tecnología(SAE 96-0113),from Fundode InvestigaciónSanitaria(FIS 97/0039-Dl)ané from “Acciun Integrada”(EicassuErugram)promot-ing tite cullaborationbetweenFranceanéSpain(95/138).
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Resultadosy Discusión 80
RESULTADOS Y DISCUSIÓN
Regulaciónde la producciónde NGFpor el TNFc¿ Papeldel NF-icB
Los resultadosexpuestosponen de manifiesto el efecto inductor del TNFcz en
la síntesisde NGF. Del estudiode la regulacióndel NGF por el TNFa se desprende
queesteejercesuefecto por un mecanismodiferente, y probablementeindependiente,
de la generaciónde ceramidaspor acciónde la SMasa.La síntesisde NGF inducida
por TNFa y LPS esindependientede cPKC, de forma similar al efectopromovidopor
ceramidas.Sin embargo, la inducción de NGF por ceramidaspareceestarmediada
por la activación de la cascadade MAPK, mientras que el efecto del TNFct es
independiente de este sistema de fosforilación. Esta constituye la primera
observaciónde que ambos mediadores,TNFa y ceramidas,ejercen su efecto de
maneraindependiente.La ausenciade hidrólisis de SM y de generaciónde ceramidas
por TNFa en astrocitos,indica que esta citoquina no activa la síntesis de NGF a
través de la SMasa. Los resultados descritos indican la existencia de otros
mecanismosendógenosdistintos de la acción de la SMasa, como puede ser la
estimulaciónde la síntesisde novoo la inhibición de su degradaciónparaincrementar
los niveles intracelulares de ceramidas (Bose et al., 1995; Biewlaska et al., 1996;
Paumen et al., 1997). Es importante destacar que una proteína viral de RIV-1
favorecela activación de la SMasapor el TNFcz, lo que revela queeste sistemade
transducciónde señaleses funcional en células gliales en una situación de infección
vírica (Richard et al., 1996). Experimentosrealizados iii vivo muestran como la
inyección de otra proteína viral de RJV-1 aumentala expresiónde NGF in vivo
(Bagettaet al., 1996). El conjunto de estasobservacionessugiereque el aumento de
los niveles intracelulares de ceramida puede ser importante en los astrocitos
reactivos,induciendoentre otras respuestasun aumentoen la síntesisy secreciónde
NGF.
Por otra parte se observaquela inducción del NGF se inhibe por el óxido de
fenilarsina, inhibidor de tirosina fosfatasas.Estemoduladorbloqueala activacióndel
factor de transcripciónnuclear NF-KB, así como la degradaciónde su subunidad
citusólica inhibidora IkBa. Parece por tanto que NF-KB, está implicado en la
regulaciónde la expresiónde NGF por TNFct. El estudiodel promotor del gende NGF
indica laexistenciade unaposiblesecuenciareguladoraquepodríaunir estefactor de
transcripción (Jehan et al.1993). Sin embargo, los experimentos realizados
demuestranque, al menos in vitro, el oligonucleótido correspondientea esta
secuenciano escapazde unir el factor de transcripción.Portanto laexpresióndel gen
Resultadosy Discusión 81
de NGF inducidapor TNFa dependede NF-KB pero de forma indirecta y no por la
unión directaal promotordel gen de NGF. La inhibición parcialque ejerceel cAMP
sobrela secreciónde NGF inducidapor TNFct no puedeser atribuida en estecasoa
una inhibición de la cascada de MAPK, ya que este sistema de fosforilación
aparentementeno esta implicado (Kurino et al., 1996, Willis y Nisen, 1996). Sin
embargo,la inhibición parcialde la activaciónde NF-KB por el aumentoen los niveles
de cAMP intracelular (Pahanet al., 1997), constituyeunaalternativaparaexplicarel
efectoinhibidor del cAIVIP sobrela síntesisde NGF.
Regulaciónde la secrecióndeNGFpor LPS
En lo que se refiere al LPS, los resultadosobtenidosponende manifiesto el
potente efecto inductor del LPS (E.coli) en la síntesis y secreción del NGF por
astrocitos en cultivo primario. Los inhibidores de actividad tirosina quinasa
empleadosno bloqueanla secreciónde NEW inducidapor LPS, lo quesugiere una
activación directa del LPS y no un efecto indirecto mediado por la secreción de
citoquinaspro-inflamatorias, inductores de la secreciónde NGF (Carman-Krzanet
al., 1991; Rattori et al., 1993; Pshenichkinet al., 1994). Para profundizar en este
aspecto, se ha estudiado también el posible papel del óxido nítrico (NO) en la
inducción de NEW por LPS. El NO es un importante mensajeroparacrino regulador
de diferentessituacionespatufisiológicasen el SNC (Murphy et al., 1993; Schmidliny
Wiesinger,1995). La exposiciónde astrocitosa LPS o a citoquinaspro-inflamatorias
activa la generaciónde NO a través de la NO sintasa inducible, por ello resulta
factible que la generaciónde NO constituyaun mecanismomediadorde la regulación
de la síntesis de NGF. Los resultadosobtenidos(no incluidos) con aminoguanidina
(inhibidor de la NO sintasa constitutiva e inducible), y L-NAME (inhibidor
competitivo de la NO sintasaconstitutiva)ponende manifiesto queestesistemade
transducciónde señalescarecede efecto en la regulaciónpor LPS de los niveles de
NGF sintetizado.Por tanto, la secreciónde NGF inducida por LPS es independiente
de la producciónde NO y posiblementetambiénde citoquinaspro-inflamatorias.En
estecontexto,esimportantedestacarque la estimulaciónde la vía de las ceramidas
puedesimular algunas de las respuestascelularesinducidas por el LPS (Barberet
al., 1996).
La inhibición a travésde la vía del cAMP de la secreciónde NGF inducidapor
LPS pone de manifiesto la existenciade mecanismosde regulaciónquepermiten un
control preciso de la síntesisde este factor neurotrófico. Los datos obtenidoscon
Resultadosy Discusión 82
apigenina,inhibidor de la cascadade MAPK, fueron confirmadosposteriormentecon
el usode un inhibidor sintético altamenteespecíficode MEK, denominadoPD 098059
(Alessi et aL, 1995). El tratamientosimultáneode los astrocitosen cultivo primario
con PD 098059 y la dosis máxima de LPS disminuye en un 50% la estimulación
máxima producida por LPS en ausenciadel inhibidor. Puesto que el LPS es un
importanteactivadorde NF-KB, el efecto inhibidor del cAMP en la síntesisde NGF
inducidapor LPS puede,por tanto, serexplicadopor una inhibición de la cascadade
MAPK, comopor la inhibición de la activaciónde NF-KB (Pahanet al., 1997).
III- DISCUSIÓN GENERAL Y
CONCLUSIONES
Discusión General y Conclusiones 83
III- DISCUSIÓN GENERAL Y CONCLUSIONES
El funcionamientodel SNC dependedel equilibrio funcional entresus distintos
componentescelularesy de una adecuadaintegración y adaptacióndel sistema a
unascondicionesexternasvariables. Los astrocitosconstituyen un importante centro
de coordinaciónfuncional en el SNC, a través de la expresiónde neurotrofinas,
proteínas de adhesión, citoquinas y factores de crecimiento que regulan el
funcionamiento neuronal, su desarrollo y la plasticidad neuronal. Además, los
astrocitosson activadoresde respuestasde emergenciaante procesosde lesión o
infecciónmediantesucontribuciónal procesode gliosis. En estecontexto, los niveles
locales de neurotrofinas,como el NGF, favorecen el crecimiento de determinadas
poblaciones neuronales en momentos específicos del desarrollo. Un descenso
“programado”en los niveles de NGF puedeserimportantepararestringir, eliminar o
modificar poblaciones neuronales locales. El propio NGF puede desencadenar
procesosde apoptosis, necesariosdurante el desarrollo del sistema nervioso. La
síntesisneurunalde NGF en el SNC puedeser sustituidapor la síntesisglial, siendo
los astrocitoslas células no neuronalesmásabundantesdel SNC. La inducción de la
síntesisde NGF por astrucitusactivadospuedeproporcionarel suficiente suministro
de NGF en el SNC endeterminadassituaciones.
La síntesis de NGF está muy regulada y controlada por las condiciones
extracelulares.In vivo, en una situación fisiológica normal y en el SNC adulto, la
expresióndel NGF por astrocitosestareprimidaperola activaciónastrocitariainduce
su expresión. Los resultados mostrados profundizan en el conocimiento de los
sistemasde transducciónde señalesque activanla síntesisy secreciónde NGF por
astrocitosen cultivo primario. La ruta de la SMasay la consiguientegeneraciónde
ceramidaspresentaun alto grado de similitud con vías de transducciónde señales
clásicasy mejor conocidascomo la generaciónde DAG por activación de diferentes
fosfolipasas. El DAG y la ceramida son estructuralmentesemejantesy una vez
generadospor la fosfolipasacorrespondientepermanecenembebidosen la membrana
plasmática.De maneraanálogaal DAG, que actúadirigiendo a algunosde los tipos
de PKC a la membranadondese activan,la ceramidapodría actuarcomo punto de
unión dirigiendo su(s)proteína(s)efectora(s)hacia la membrana,lo que implicaría su
activación. Las ceramidaspuedenpor tanto modular la actividad de sus efectores
induciendo cambios conformacionalesde la proteína, modificando su localización
subcelularpor translocacióny favoreciendoel accesoa sus substratos.La existencia
de una modulacióndiferencial de PKC ~ y Raf-1 por los análogosde ceramida de
Discusión General y Conclusiones 84
diferente longitud del grupo acilo, puedeexplicarsedesdeel modelo del grupo alquilo
protuberante(Krñnke, 1997). Según este modelo, el grupo alquilo de la SF sería
reconocidopor las proteínasefectorasde los esfingolípidos,mientras el resto acilo
seríael responsablede la inserción en la membranaplasmática. Si esto fuera así, la
C2-ceramidapodría ser reconocidapor las mismasproteínasefectorasqueceramidas
de mayor longitud, pero carecería de la capacidadde dirigirlas a la membrana
plasmática.
La inducción observadade NGF por acción de la PC-PLC, puede reflejar el
efectoregulador sobrela síntesisde NGF de los sistemasde control de proliferación
celular, dadoquela hidrólisis de PC constituyeuno de susmecanismosde regulación.
El efectopromovidoporla PC-PLCconfirma, además,la importanciade la generación
de mensajeroslipídicos en la regulaciónde la funcionalidadastrocitaria.La inducción
de la síntesisde NGF por la acciónde la PC-PLC poseecaracterísticascomunesconel
efecto observadopor la acción de la SMasaexógenay las ceramidaspermeables,lo
que hace pensar que ambos efectos están relacionados de forma sucesiva y
coordinada.El trabajo realizadoenla presentetesisdoctoralconstituyeun ejemplode
la importanciade los mensajeroslipídicosen la regulaciónde la funcionalidadcelular.
Glicerolipidos y esfingolípidos son importantes reguladores de sistemas de
fosforilación de proteínasy la regulación cruzadaque ejercen entre si aumentala
complejidadde los mecanismosde control posibles.
El LPS, uno de los principales activadores de la respuesta inmune e
inflamatoria anteuna situaciónde infecciónbacteriana,induce un drásticoaumento
en los niveles de NGF, sintetizado por astrocitos en cultivo primario. El efecto
regulador del LPS puedetener diferenteslecturaspero todas ellas relacionadascon
los resultadosanteriormentedescritos.La vía de las ceramidaspuede ser activada
por la exposicióncelulara endotoxinas(Hannun, 1996), y se hapropuestoqueel LPS
puede ejercer alguno de sus efectos por analogía estructural con las ceramidas
activando efectores similares a los activados por el aumento en los niveles
intracelulares de ceramidas como la CAPK (Wright y Kulesnick, 1995). Las
característicascomunesde la estimulación de la síntesis de NGF por el LPS y
ceramidassugierenque ambosefectospodrían estar relacionadoscon la inducción de
NGF duranteunasituacióninflamatoriao de lesión.
El TNFcz, otro activadorcaracterísticopro-inflamatoriode la vía de la SMasa-
ceramidas(Hannun, 1996), promuevesin embargoun incrementoen la síntesisde
NGF con algunasdiferenciasconla inducciónobservadapor el aumentode los niveles
Discusián General y Conclusiones 85
intracelulares de ceramida, lo que sugiere que ejerce su acción de manera
independientede la hidrólisis de SM. En efecto,elTNFct no induceun aumentoen los
niveles intracelularesde ceramidaa través de la hidrólisis de SM. La inducción de
NGF por un aumentoen los niveles de ceramida se puedeexplicar por la activación
de la ruta de la SMasapor otros activadoresde estesistemade señalizacióncelular, o
por un aumentoen los niveles intracelularesdebidoa la regulaciónde su síntesiso
degradación.
Respectoa los sistemasde fosforilación implicados, los resultadosexpuestos
constituyenla primerareferencia,de la quetenemosconstancia,queasignaun papel
a la cascadade las MAP quinasasen la regulación de la expresióndel NGF. La
importanciade estesistemade fosforilaciónen la regulaciónde la síntesisde NGF se
ve confirmadaal observarsequedistintos activadoresde la síntesisde NGF dependen
en mayoro menormedidade la actividadMAPK. La proteínaquinasaPKC ~ es una
de las dianasde la acciónde la PC-PLCy las ceramidasen los astrocitos,modificando
su localizaciónsubcelulary su grado de fosforilación. La localizaciónperinuclearde
PKC ~ inducidapor la activaciónde los astrocitospuedeestarrelacionadacon efectos
de regulaciónde la expresióngénica, como ocurre tambiénen otros tipos celulares
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