22 29 1

Universidad Complutense de Madrid

(Dpto. de Bioqumica y Biologa Molecular 1)

REGULACIN DE LA SECRECIN DEL FACTOR

DE CRECIMIENTO NERVIOSO EN CLULAS GLIALES.
MECANISMOS DE TRANSDUCCIN A TRAVS DE
MENSAJEROS LIPDICOS

*5

! 1ID DII IIMUllID

098396

UNIVERSIDAD COMPLUTENSE

TESIS DOCTORAL
por

Ismael Galve Roperh

Directores:
Dra. Ins Daz-Laviada
Dr. Amador Haro Ramos

V0 B0 de los Directores:

Este trabajo de investigacin ha sido realizado en el Departamento de

Bioqumica y Biologa Molecular 1 de la Facultad de Ciencias Biolgicas de la
Universidad Complutense de Madrid, con la direccin de la Dra. Ins DazLaviada y el Dr. Amador Haro Ramos. Parte de la investigacin fue realizada en
la unidad 298 del INSERM de Angers (Francia) en colaboracin con el Dr.
Philippe Brachet y en la unidad 466 del INSERM de Toulouse (Francia) en
colaboracin con el Dr. Thierry Levade, a los que quiero manifestar mt sncero
agradecimiento.

1- INTRODUCCIN
1. Sistemas de sealizacin celular
1.1 Los lpidos como mensajeros intracelulares
Fosfolipasas de glicerolipidos
La ruta de la esfingomielinasa-ceramida
1.2 Sistemas de fosforilacin de protenas
Sistemas de fosforilacin en cascada regulados extracelularmente
MAP quinasas
SAPK/JNK y pSS
La isoforma ~ de protena quinasa C

1.3 Regulacin de la expresin gnica

2. El factor de crecimiento nervioso
2.1 El gen de NGF
2.2 La protena NGF
2.3 Localizacin de la sntesis de NGF
2.4 Regulacin de la sntesis de NGF
3. Los astrocitos y otras clulas gliales
4. Objetivos
II- RESULTADOS Y DISCUSIN
1. Regulacin de la sntesis y secrecin de NGF por mensajeros
lipdicos en cultivos primarios de astrocitos.
-

Introduccin

1.1 Phosphatidylcholine-phospholipase C mediates the induction of

nerve growth factor in cultured glial cels

FEES Letters 364:301-304 (1995)

1.2 Induction of nerve growth factor synthesis by sphingomyelinase
and ceramide in primary astrocyte cultures

Mol. Brain Res. 52:95.102 (1997)

-

Resultados y Discusin

2. Regulacin de la protena quinasa O ~ (PKC

~)por mensajeros ilpdicos en

clulas gliales
-

Introduccin

2.1 Addlition of phosphatidylcholirie-phospholipase

C induces ceDular

redistribution and phosphorylation of protein kinase C ~in C6 glial celis

Neurosci. Letters 219:68-70 (1996)

2.2 Ceramide-induced translocation of protein kinase C ~ in primary
cultures of astrocytes

FEBS Letters 415:271-274 (1997)

-

Resultados y Discusin

3. Regulacin de la sntesis y secrecin de NGF por el factor de

necrosis tumoral (TNFcz) y lipopolisacrido (LPS) en astrocitos en
cultivo primario
-

Introduccin

3.1 Evidence for the lack of involvement of sphingomyelin generation

in the tumor necrosis factor-induced secretion of NGF in primary
astrocytes

J. Neurochem., en prensa
3.2 Regulation of NGF secretion and mRNA expression by bacterial
lipopolysaccharide in primary cultures of rat astrocytes

1 Neurosci. Res. 49:569-575 (1997)

-

Resultados y Discusin

III- DISCUSIN GENERAL Y CONCLUSIONES

IV- BIBLIOGRAFA

ABREVIATURAS
ARF,

BDNF,

Factor de ADP.ribosilacin
Factor neurotrfico derivado de cerebro

BlM,

Bisindoleilmaleimida 0F109203X

cAMP,

AMP cclico
Protena quinasa activada por ceramida
Protena fosfatasa activada por ceramida
Protena homloga del gen de ciclo celular de levaduras 42

CAPK,
CAPP,
cdc42,

CNTF,
CRE,
DAG,
Elk-1,
ciAP,
GDNF,
GFs,
Grb2-sos,
hsp,
ICER,
IL,
PS

JARs,
JNR,
LPA,
LPS,

MAPK,
MEK,
mRNA,
NGF,
PA,
PAR,
PC,

PE,
PI
PMA,
PIlA,
PKC,
PLO,
PS,
Rho,
SAPK,
SER,

SF,
SM, SMasa,
SNC, SNP,
src,

SRE,
SRF,
STAT,

TNF,
Trk,
zIP

Factor ciliar neurotrfico

Elemento sensible a cAMP
Diacilglicerol
Factor de transcripcin similar a Ets

Protena activadora actividad GTPasa

Factor neurotrfico derivado de gua
Factores de crecimiento

Complejo proteico mediador de la activacin de MAPK

Protena de choque trmico
Represor temprano inducible por cAMP
Interleuquina
Inositol 1,4,5-trisfosfato
Quinasas del tipo Jano
Protena quinasa del extremo N terminal de c-Jun
Acido lisofosfatdico

Lipopolisacrido
Protena quinasa activada por mitgenos
Protena quinasa de MAPK
cido ribonuclico mensajero
Factor de crecimiento nervioso
Acido fosfatdico
Protena quinasa activada por p2i
Fosfatidileolina
Fosfatidiletanolamina
Fosfatidilinositol
4r3-forbol 123-miristato iSa-acetato

Protena quinasa dependiente de cAMP

Protena quinasa C
Fosfolipasa O
Fosfatidilserina

Protena homologa de Ras

Protena quinasa activada por estrs
Protena quinasa de SAPK
Esfingosina
Esfingomielina, Esfingomielinasa
Sistema nervioso central, Sistema nervioso perifrico
Protena con dominio de homologa SH
Elemento de respuesta al suero
Factor de respuesta al suero
Protenas contndn de tranalucrin de ~ales yknrde tranaripcn
Factor de necrosis tumoral
Receptor de neurotrofinas con actividad de tirosina qumasa

Protena que interacciona con PKC ~

Introduccin

1. SISTEMAS DE SEALIZACIN CELULAR

La regulacin de la funcin celular depende de la transmisin de informacin
del exterior al interior de la clula, lo que le permite responder de forma adecuada a
un entorno cambiante y dinmico. Este proceso de comunicacin intercelular se realiza a travs de mensajeros qumicos de diferente naturaleza. Los sistemas de transduccin de seales estn formados por un componente receptor y otro efector que induce la respuesta celular, pudiendo estos dos componentes funcionales formar o no
parte de la misma entidad proteica. La unin del agonista al receptor promueve una
respuesta celular mediada por la regulacin del componente efector. Generalmente,
los

mensajeros

intracelulares

elercen

su

efecto

travs

de

fosforilacin-

desfosforilacin de protenas. Por ltimo, los factores de transcripcin permiten la

regulacin de la expresin gnica.
A continuacin se presentarn algunos de los componentes de sistemas de
transduccin de seales ms directamente relacionados con el trabajo de investigacin desarrollado en la presente tesis doctoral, destacando por ello, su papel en sistema nervioso y en particular en astrocitos y otras clulas gliales

Primero se describi-

rn los distintos sistemas de generacin de mensajeros lipdicos intracelulares a partir de glicerolpidos. Posteriormente se revisarn los actuales conocimientos sobre el
papel de los esfingolipidos en la regulacin celular a travs de ceramidas. El estudio de
los sistemas de fosforilacin de protenas incluir una breve descripcin aclaratoria de los
sistemas de fosforilacin regulados extracelularmente, MAPK y SAPKJJNK/p38, que se
completar con el estudio de las caractersticas de la isoforma ~ de protena quinasa C.

1.1 LOS LPIDOS COMO MENSAJEROS INTRACELULARES

Adems de su funcin estructural, las membranas biolgicas desempean un
importante papel como fuente de generacin de mensajeros intracelulares y extracelulares, que permiten la comunicacin intercelular, la regulacin del crecimiento, diferenciacin, secrecin, migracin o apoptosis celular (Prescott, 1997). La investigacin de los mecanismos de generacin de mensajeros celulares de naturaleza lipdica
constituye uno de los campos de la bioqumica de mayor avance en los ltimos aos.

La accin de diferentes enzimas con actividad de fosfolipasa sobre glicerolpidos genera algunos de los mensajeros lipdicos mejor conocidos y caracterizados (Exton,
1997a;b; Leslie, 1997; Rhee y Bae, 1997). Recientemente se ha descrito la existencia
de esfingolipidos con importantes efectos reguladores de la actividad celular, en lo

Introduccin

que se ha venido en llamar la ruta de la esfingomielinasa o de las ceramidas (Testi,

1996; Hannun, 1996; Spiegel et al., 1997).
FOSFOLIPASAS DE GLICEROLPIDOS
Fosfolipasa C

La actividad de la fosfolipasa U (PLC) sobre el fosfolipido genera diadilglicerol

(DAG) y el grupo polar fosforilado, que pueden ser inositoles fosforilados, el ms importante el inositol 1,4,5-trisfosfato (IPs), para la fosfolipasa C especfica de fosfatidilinositol (PI-PLC) o fosforilcolina en el caso de la fosfolipasa C especfica de fosfatidil-

colina (PC-PLC) (revisado por Exton 1997a; Rhee y Bae, 1997).

Fosfolipasa C de fosfatidilinositol
Los productos resultantes de la actividad de la PI-PLC actan como mensaje-

ros intracelulares: el DAG como activador de protena quinasa C (PKC) y el IP3 regu24 intracelular. Adems, un descenso en los niveles de
lando la concentracin de Ca
Ps en la membrana plasmtica tiene otras consecuencias funcionales importantes en
la clula, ya que algunos de estos fosfolipidos son el punto de unin a la membrana de
protenas con dominios de homologa con la plecstrina, entre ellas la propia PI-PLC.
Los FIs son adems substrato de la enzima PI-3 quinasa, y as mismo cofactores de la
fosfolipasa D especfica de PC (Rhee y Bae, 1997). Existen diferentes formas de PIPLC, las formas j3 se caracterizan por ser activadas fundamentalmente por receptores
de siete segmentos transmembranares, a travs de las subunidades

aq

activas de pro-

tenas O heterotrimricas (Chen et al., 1996), o por subunidades j3y. La activacin de

las formas PJ-PLCy tiene lugar mayoritariamente a travs de receptores con actividad
de tirosina quinasa (Rhee y Bae, 1997). El Ca2~ es necesario para la actividad de las
distintas formas de PI-PLC, siendo las formas 6 especialmente sensibles. La actividad
de PI-PLC puede verse modulada tambin por cido fosfatdico, cido araquidnico e
IPa, lo que permite suponer la existencia de una activacin de PJ-PLC secundaria tras
la activacin de receptores acoplados a PLD, cPLA2, o PJ-3 quinasa (Rhee y Bae,
1997). La actividad de PI-PLC en astrocitos se activa por distintos agonistas como
insulina, glutamato, bradiquinina o angiotensina II (Chen et al., 1996; Ruiz-Albusac
et al., 1997; Tallant et al., 1997).

1n traduccin

Fosfolipasa C de fosfatidilcolina
La PC-PLC esta implicada en la regulacin del control de la proliferacin ce-

lular (Daz-Laviada et al., 1990; Bjorkoy et al., 1995; 1997; Cheng et al., 1997). El
aumento de DAG intracelular resultante de la hidrlisis de PC es de larga duracin y
se mantiene elevado ms all del aumento transitorio generado por la PI-PLC
(Asaoka et al., 1996; Nakamura, 1996). Este aumento de DAG, permite explicar la
activacin de PKC a largo plazo necesaria en la regulacin del crecimiento y prolife-

racin celular, siendo este proceso de especial importancia en las clulas gliales
(Klein et al., 1995). El aumento en los niveles de DAG est en estrecha relacin con la
transformacin celular (Laurentz et al., 1996). Como veremos ms adelante la activacin de PC-PLC se ha relacionado con la cascada de fosforilacin de MAPK y con la

PKC ~ (VanDijk et al., 1997a;b).

Fosfolipasa D
El substrato especficq de la PLD es la PC, aunque de forma secundaria puede
actuar sobre fosfatidiletanolamina (PE) o PI. La accin de la PLD sobre la PC genera
cido fosfatdico (PA) y colina libre (Fig. 1). El PA generado puede actuar directamente como mensajero intracelular, ser transformado en DAG por accin de una fosfatidato fosfohidrolasa, o ser metabolizado en cido lisofosfatdico (revisado por Exton,
1997a;b). Alternativamente la PLD puede catalizar una reaccin de transfosfatidilacin por la que se transfiere el grupo fosfato a un alcohol primario, lo que se utiliza
para la medida de su actividad.
La PLD, aunque mayoritaria en la membrana plasmtica, se encuentra tambin en el ncleo, retculo endoplsmico, Golgi y citoplasma. La PLD se estimula por
PI, Ca 2+ y las protenas O monomricas ARF y Rho A (Exton, 1997a;b). Existen varias
formas de PLD que se diferencian en su regulacin, siendo la actividad PLD del Golgi
ms sensible a activacin por ARF, mientras que la forma de membrana plasmtica
se activa fundamentalmente por Rho A y la forma citoslica por Ca2~. La PKC activa
la PLD, aunque no existen evidencias directas de una fosforilacin directa sobre la
propia PLD (Exton, 1997a;b).
La PLD cumple una importante funcin reguladora en la fisiologa del sistema
nervioso (Klein et al., 1995) y su actividad parece ms importante, en trminos generales, que la actividad de la PI-PLC en la regulacin de la funcionalidad de los astrocitos (Jehan et al., 1995; Mangoura et al., 1995). En los astrocitos en cultivo primario
existen importantes diferencias de regulacin de PLD y en general en los sistemas de

Introduccin

sealizacin celular segn el origen anatmico del sistema nervioso central (SNC) de
los cultivos (Mangoura et al., 1995; Tallant y Higson, 1997).
El PA generado posee un efecto mitognico que puede explicarse por su transformacin en DAG, activador de PKC (Asoka et al., 1996; Nakamura, 1996), por su
efecto modulador de Raf- 1, componente de la cascada de MAPK (Ghosh y Bell, 1997) o
por su transformacin en LPA (Keller et al., 1997). En astrocitos algunos factores mitgenicos como trombina o endotelinas activan la PLD (Desagher, et al., 1997). Adems, los niveles de PLD se regulan durante la diferenciacin inducida por cAMP de

las clulas de glioma C6 y en la apoptosis inducida por C2-ceramida en estas mismas

clulas (Yoshimura et al., 1997).

Ceramidas y PLD
Las ceramidas generadas por accin de esfingomielinasas inhiben la actividad
de PLD (Venable et al., 1996; Gomez-Muoz et al., 1997) lo que potencia el efecto
inhibidor del crecimiento de estos esfingolpidos (Hannun, 1996). La inhibicin de
PLD por ceramidas se debe al bloqueo de la translocacin a membrana plasmtica de
Rho A, ARF, cdc42 y determinadas isoformas de PKQ (Abousalham et al., 1997). Por
otro lado, la esfingosina-1-fosfato, que se caracteriza por ejercer un efecto fundamentalmente mitognico en las clulas (Spiegel et al., 1997), activa PLD (Desai et al.,
1992). Existe por tanto una regulacin cruzada entre las vas de sealizacin a travs
de esfingolipidos y glicerolpidos que aumentan la complejidad de los mecanismos de
regulacin celular (Gomez-Muoz et al., 1997).

Fosfolipasa A2
La accin de la PLA2 libera el cido graso situado en la posicin sn-2 del fosfo-

lpido, generando el lisofosfolpido correspondiente (revisado por Leslie, 1997) (Fig. 1).
Existen distintas formas de PLA2, una forma secretada, una forma independiente de
2~ y la forma citoslica, con caractersticas reguladoras especficas cada una de
Ca
ellas. La actividad de la PLA2 citoslica, cPLA
2, es especialmente importante en los

astrocitos, interviniendo en la regulacin del proceso de gliosis (Clemens et al., 1996).

La liberacin de cido araquidnico por cPLA2 regula la formacin de diversos mensajeros lpidos. La completa activacin de cPLA2 implica primero su fosforilacin por
2~, su unin a la membrana. Aunque la PKC
MAPK y posteriormente, gracias al Ca
incrementa la actividad de cPLA
2, esta activacin parece ser debida a un efecto indirecto mediado por la activacin previa de MAPK. TNFa, IL-1j3, ATP y glutamato in-

Introduccin

crementan los niveles de cPLA2 y aumentan su actividad en diferentes modelos celu-

lares, entre ellos astrocitos y clulas de glioma C6 (Stella et al., 1997).

PA
I

LisoPC

LiscSM

CatiRa
Aa

PLD

PLA2

PLA2

PC

SM
PLO

CDPCot

SMaso

rcouna

P-Colina

DAG

Ceramida

Fig. 1 Mensajeros lipdicos generados por accin de diferentes fosfolipasas sobre

fosfatidileolina (PC> y esfingomielina (SM).

LA RUTA DE LA ESFINGOMIELINASA-CERAMIDA
La identificacin de los esfingolpidos como reguladores de los procesos de
transduccin de seales comenz con la caracterizacin del efecto inhibidor de la es2. Actualmente se conoce
fingosina (SP) sobre la actividad de PKC dependiente de Ca
la existencia de la denominada ruta de sealizacin de la esfingomielinasa (SMasa),
tambin llamada de las ceramidas por ser este uno de sus productos con actividad
biolgica (revisado Hannun et al., 1996; Testi, 1996). La degradacin de la esfingomielina (SM) conleva la generacin de ceramida (Figs. 1 y 2), que posteriormente
puede ser transformada en SF, SF-1-P, o ceramida-1-fosfato que tambin pueden actuar como mensajeros celulares (Merrill et al., 1996; Spiegel et al., 1997). El aumento
de ceramida es transitorio y la SM se resintetiza incorporando el grupo fosforilcolina
procedente de PC a travs de la sintasa de SM (Fig. 1).
TNFx, IL-1[3, Fas, o la vitamina DS fueron los primeros activadores identificados de este nuevo sistema de transduccin de seales (mm et al., 1991; Hannun et
al., 1996). Posteriormente se ha descrito que el efecto de la vitamina DS es debido a

Introduccin

un mecanismo autocrino de regulacin mediado por la induccin de TNFa (Geilen et

al., 1997). La lista de inductores de esta va de sealizacin incluye, entre otros, agentes quimioteraputicos, radiaciones ionizantes, NGF, interfern y (Casaccia-Bonnefil
et al., 1996a; Hannun et al., 1996). La SM est localizada de forma mayoritaria en la
cara externa de la membrana plasmtica, existiendo distintas fracciones (pools) de
SM implicadas en la sealizacin celular. As la SM sensible a hidrlisis estimulada
por TNFx est localizada en la cara interna de la membrana plasmtica (Andrieu et
al., 1996) y la SM sensible a NGF se localiza en determinados microdominios de
membrana enriquecidos en caveolina (Bilderback et al., 1997).
Existen distintos tipos de SMasas con caractersticas de regulacin diferentes.
Se ha descrito la existencia de una actividad SMasa de membrana dependiente de
una SMasa citoslica independiente de Mg2~, una SMasa denominada cida, por
poseer un pH ptimo de actuacin cido, que se localiza en los endosomas y una SMasa nuclear (Andrieu et al., 1994; Albi y Magni, 1997). Las distintas formas de SMasa
parecen intervenir en diferentes mecanismos de sealizacin celular, lo que puede
explicar la existencia de respuestas celulares opuestas activadas por la ruta de la
SMasa. En el caso del receptor de 55 kDa del TNFct, existen dos dominios citoslicos
que activan de forma independiente la SMasa cida y la SMasa neutra. La activacin
de la SMasa cida est precedida por la activacin de PC-PLC. El aumento en los niveles intracelulares de ceramidas desencadenado por este mecanismo sera responsable de la activacin del factor nuclear de transcripcin NF-KB (Schtze et al., 1992;
Wiegmann et al., 1994). El otro dominio citoslico interacciona con una protena
adaptadora FAN y activa SMasa neutra (Adam-Klages et al., 1996). La generacin de
ceramidas por esta rama de sealizacin puede activar la cascada de MAPK y de este
modo inducir la activacin de cPLA2 (Adam-Klages et al., 1996). Sin embargo, en
otros modelos, la activacin de cPLA2 es un requisito previo necesario para la generacin de ceramidas estimulada por TNFcz (Jayadev et al., 1997).
Las ceramidas pueden activar una protena fosfatasa de tipo 2A, CAPP, que
fue identificada en clulas de glioblastoma T9 y que puede regular el estado de fosforilacin de c-Jun (Gonzalez et al., 1996). Adems, existe una protena quinasa activada por ceramida (CAPK) que recientemente se ha identificado como la quinasa supresora de Ras (Zhang et al., 1997a), y que puede fosforilar y activar Raf-1 (Yao et al.,
1995).

1ntraduccin

R~Fa

nvip
MW

0$,
SF-1-P~

SF

deino

CAPK
\~af4

,,gPLA2

+
+

.PRaIFERAUN ~ELUL4R
DIFERENcJAaNAFOPItEI
RFLSL4INMUNE

Hg. 2 Fsqirnn de la ruta de seializacin celularde las ceramidas

La estimulacin por TNFa y el aumento intracelular de ceramidas incrementan la actividad MAPK (Sasaki et al., 1995; Modur et al., 1996), sin embargo, parece
que es ms importante la activacin de esta quinasa por SF-1-P y factores de crecimiento (Coroneos et al., 1996; Pyne et al., 1996). Las ceramidas son potentes activa-

dores de SAIPK/JNK (Westwick et al., 1995), lo que puede explicar su efecto apopttico (Verheij et aL, 1996). El aumento en los niveles de DAG intracelular inhibe la
apoptosis, lo que se explica por su efecto inhibidor en la generacin de ceramida
(Laurenz et al., 1996). Existe por tanto un complejo mecanismo de regulacin de la
proliferacin y muerte celular en funcin del contexto fisiolgico en el que se generan
los distintos tipos de mediadores lipdicos.

Introduccin

Efectos biolgicos de las ceramidas

Las ceramidas se han relacionado con la inhibicin del crecimiento, la diferenciacin celular y con la apoptosis estimulada por citoquinas inflamatorias y Fas
(Hannun, 1996). Adems de ejercer un importante control de los procesos del crecimiento celular y axonal, las ceramidas regulan la expresin gnica a travs de factores de transcripcin como NF-KB o AP-1, procesos de endocitosis y canales de K~
(Hannun et al., 1996). Los efectos antiproliferativos de las ceramidas pueden estar
mediados por la disminucin de los niveles de c-myc a travs de CAPP. La elevacin
de los niveles de ceramida puede detener las clulas en las etapas Go/Ol del ciclo celular, manteniendo la protena de retinoblastoma pRb en un estado hipofosforilado.
En sistema nervioso, las ceramidas pueden inducir la muerte celular en oligodendrocitos, mientras que los astrocitos son resistentes a este fenmeno (CasacciaBonnefil et al., 1996a;b). En cultivos neuronales se ha observado que las ceramidas
pueden inducir o impedir la apoptosis, segn el origen o momento de desarrollo
(Wiesner y Dawson, 1996; Scharwz y Futerman, 1997). Sin embargo, parece que la
apoptosis inducida por ciertos activadores de la va de las ceramidas como Fas, puede
ser independiente de la generacin de ceramidas (Watts et al., 1997). En cualquier
caso, existen distintas fracciones de SM implicados en la sealizacin celular, y no
todas son capaces de inducir apoptosis en la clula (Zhang et al., 1997b).
La mayor parte del conocimiento del papel de los esfingolpidos en la sealizacin celular se fundamenta en la generacin de ceramida concomitante a la degradacin de SM. Sin embargo, la degradacin de los niveles de ceramida por la ceramidasa, o

la regulacin de su sntesis son tambin cruciales en el control de los niveles

intracelulares de ceramida (Bose et al., 1995; Biewlaska et al., 1996; NikolovaHarakashian et al., 1997). La diferente regulacin que ejercen citoquinas y factores

1+

citoquin,
Factores de
crecimiento
Ceramida.1-P
CAPP

Factores de
crecimiento

Ceramidasa

SAPKJJNK

APOPTOSLS

MAPK **

cPLA24

SP

4~

cAMP

SP quinasa

PRC C

REGIJLAcIO
Efl>RESIN
CNIO?.

\4

LPA

<

PA

PC

Ca
cAMP

MAFE

PROLIFERACIN
CELULAR

Fig. 3 Esquema de la regulaci6n que sobre la ruta de las ceramidas ejercen citoquinas por.inflamatoriaa y factores de crecimiento

Introduccin

de crecimiento sobre SMasa, ceramidasa y quinasa de SE puede determinar el balance entre los niveles de ceramidas y SF/SF-1-P lo que permite controlar el balance
muerte celular/diferenciacin o proliferacin a travs de la activacin de distintas
cascadas de fosforilacin (Fig.S) (Coroneos et al., 1995; 1996; Cuvilhier et al., 1996; Pyne et
al., 1997).

1.2 SISTEMAS DE FOSFORILACIN DE PROTENAS

La fosforilacin y desfosforilacin de protenas por protena quinasas y fosfatasas constituye uno de los procesos reguladores ms comunes en la clula. Su organizacin en cascadas de fosforilacin permite una amplificacin de la seal, una gran
sensibilidad en la respuesta, la integracin de la informacin procedente de diferentes
estmulos as como la aceleracin de la respuesta celular. En eucariotas, la fosforilacin de protenas con fines reguladores se lleva a cabo fundamentalmente en senna/treonina o tirosina, resultando excepcional la posibilidad de fosforilacin de ambos
tipos de aminocidos (actividad dual). A continuacin se hace una breve descripcin
de diferentes

cascadas

de

fosforilacin

reguladas

extracelularmente:

MAPK,

SAPK/JNK y p88. Adems se tratarn de forma especfica las caractersticas reguladoras de la isoforma ~ de la protena quinasa C.

SISTEMAS

DE

FOSFORILACIN

EN

CASCADA

REGULADOS

EXTRACELULARMENTE
Los sistemas de fosforilacin de protenas regulados por estmulos extracelulares incluyen al menos tres grandes grupos de sealizacin: MAPKs

SAPK/JNKs y

pSS (Fig. 4). Los primeros se caracterizan por ser activados fundamentalmente por
agentes mitognicos que actan a travs de receptores con actividad de tirosina quinasa (Denhart, 1996). Los segundos se activan por estrs celular y citoquinas pro.
inflamatorias a travs de receptores que carecen de actividad de tirosina quinasa
(Kyriakis y Avruch, 1996). Algunas de las serina/treonina quinasas que forman parte
de estos sistemas de fosforilacin se caracterizan por precisar de una doble fosforilacin en tirosina y treonina, para alcanzar su mximo grado de activacin, lo que permite un control muy preciso en su regulacin (Denhardt, 1996; Kyriakis y Avruch,

1996).

lo

Introduccin

MAP quinasas
El sistema de fosforilacin por la cascada de MAPK incluye la activacin sucesiva de Ras/Raf-1/MEK y MAPK (Fig. 4). Esta va de transduccin de seales constituye uno de los principales sistemas de control de proliferacin celular tanto en clulas gliales como de diferente origen (Cazaubon et al., 1997; Van Brocklyn et al., 1997).
El NGF se encuentra entre la gran variedad de mensajeros con receptores de actividad de tirosina quinasa que utilizan este sistema de transduccin de seales (Segal y
Greenberg, 1996). En general, la activacin de receptores con actividad de tirosina
quinasa por sus agonistas induce su autofosforilacin y dimerizacin. Los residuos de
tirosina fosforilados son reconocidos por una serie de protenas que contienen una
estructura de homologa en dominios denominados SH2 (src homology 2), estando el
reconocimiento de tirosinas fosforiladas condicionado por el contexto de aminocidos
en el que se encuentran. El receptor fosforilado une directamente o a travs de protenas adaptadoras,

el complejo citoslico preexistente Grb2-Sos (revisado por

Denhart, 1996). Sos es una protena activadora del intercambio de GDP por GTP, lo
que permite la activacin de Ras localizada en la membrana plasmtica y que pertenece a la superfamilia de las protenas G monomricas. En la regulacin de Ras adems de protenas que facilitan el intercambio GTP-GDP, participan protenas activadoras de su actividad GTPasa, GAP, importantes para la integracin de la informacin de seales procedentes de Ras y Rho (Denhart, 1996). Los receptores con siete
segmentos transmembranares tambin pueden inducir respuestas mitognicas a travs de la cascada de MAPKs (revisado por Post y Brown, 1996). As, la activacin de
PI-PLC va Gq/11 genera DAG que junto con el aumento de Ca2~ intracelular promovido por IPs permiten la plena activacin de determinadas PKCs, lo que puede desembocar en la activacin de la cascada de IVIAPK. La activacin de Gi, adems de
inhibir la adenilato ciclasa, libera subunidades J3y que pueden activar Ras a travs de
tirosinas quinasa citoslicas como src.

Raf- 1
La protena Raf- 1 es una serina/treonina quinasa activada por la interaccin
con Ras, para la que posee dos posibles sitios de unin (revisado por Morrison y Cuter, 1997). La activacin de Raf-1 depende de su translocacin a la membrana que es
dirigida por la protena Ras activa (Stokoe et al., 1994), de modo que su translocacin,
independientemente del grado de fosforilacin, determina su activacin (Stokoe y
McCormick, 1997). La protena Raf-1 esta asociada de forma constitutiva con las pro-

11

Introduccin

tenas hsp 90, hsp50 y 14-3-3. La interaccin con 14-3-3 favorece el mantenimiento de

Raf-1 en su forma inactiva dado que interfiere la unin de Ras a travs del segundo
sitio (Clark et al., 1997). La protena 14-3-3 es una protena que une residuos de senna fosforilados posible componente de una familia de protenas an desconocida,
anlogas a las que poseen dominios 5112 que unen tirosinas fosforiladas, y que permitiran la formacin de complejos multiproteicos (Muslin et al., 1996).
Raf-1 se puede activar por interaccin especfica con ceramidas, lo.que permite
establecer una relacin directa entre la ruta de sealizacin de las ceramidas y la
activacin de MAPK (Huwiler et al., 1996). Adems, el PA, producto de la accin de
PLD, tambin puede activar Raf-1 (Ghosh y Bel, 1997). Del estudio de la secuencia
de Raf- 1 y del anlisis funcional de sus distintos dominios, se ha postulado la existencia de un elevado grado de analoga con la familia de PKC (Ghosh y Bel, 1997).
La regulacin de Raf-1 por los sistemas de fosforilacin es compleja y dista de
ser bien comprendida. Por un lado la fosfonilacin en tirosina por src parece ser necesaria para su plena activacin junto con la interaccin con Ras-GTP (Marais et al.,
1995). Sin embargo, existen sistemas en los que la activacin de Raf-1 no va acompaada de su fosforilacin en tirosina (Wartmann et al., 1997). La fosforilacin de Raf-1
en treonina o serma por CAPK o por PKC promueve tambin su activacin (Yao et al.,
1995). La isoforma

de PKC atpica puede activarse por Ras (Diaz-Meco et al., 1994)

lo que podra explicar la activacin de MEK/MAPK por PKC

vez a travs de la activacin directa de PKC

(Berna et al., 1995), tal

sobre Raf.1 (VanDijck et al., 1997a).

Por otro lado, la fosforilacin de una serma especfica por PKA inhibe Raf-1, lo que
indica la existencia de regulacin cruzada entre diferentes sistemas de transduccin
de seales (Hafner et al., 1994). En cualquier caso, el grado de fosforilacin es fundamental en la regulacin de la actividad de Raf-1, y su hiperfosforilacin conduce a
una menor afinidad por la membrana plasmtica y constituye uno de los mecanismos
de inactivacin (Wartmann et al., 1997).
La activacin de Raf-1 por fosforilacin permite activar las protena MEKs de
especificidad dual (MEK1 y MEK2). La fosforilacin de MEKs por Raf-1 est dirigida
por la existencia de dominios 5H3 ricos en prolina. Posteriormente MEK activa las
protenas quinasas MAPK1 y MAPK2. Estas enzimas pueden translocarse al ncleo y
por fosforilacin activar substratos como protenas asociadas a microtubulos, protenas quinasas nibosomales, cPLA2 y factores de transcripcin como Elik-1 y c-myc, lo

que permite el control de importantes procesos celulares como la proliferacin, la organizacin del citoesqueleto y la regulacin de la expresin gnica.

Introduccin

12

Regulacin de la actividad MAPK en astrocitos

La activacin de la cascada de MAPKs es esencial en la regulacin de la proliferacin de los astrocitos. Factores mitognicos como endotelinas, FGF, PDGF, los
ganglisidos o el pptido P estimulan la actividad de MAPK de forma sostenida
(Kurino et aL, 1996; Lazarini et al., 1996; Luo et al., 1996; Cazaubon et al., 1997;
VanBrocklyn et al., 1997). El aumento en los niveles intracelulares de cAMP inhibe la

proliferacin de astrocitos y activa la diferenciacin hacia un fenotipo astrocitario en

las clulas de glioma C6 (Chen et al., 1996; Anciaux et al., 1997), estando la inhibicin de la proliferacin directamente relacionada con la inhibicin de la actividad
MAPK (Kurino et al., 1996; Prins et al., 1996). La inhibicin de MAPK por el aumento intracelular de cAMP se observa tambin en la activacin de lVIAPK inducida por el
lipopolisacrido (LPS) bacteriano (Willis y Nisen, 1996).
La activacin de MAPK por endotelinas en astrocitos ocurre por un doble mecanismo: el clsico de fosforilacin en tirosina del adaptador Shc, asociacin a Grb2 y
activacin de Raf-1 (Lazarini et al., 1996) y por un mecanismo dependiente de Rho y
protenas de matriz extracelular (Cazaubon et al., 1997).
La actividad MAPK es importante tambin en la regulacin del espacio sinptico, condicionado por la regulacin del volumen celular de los astrocitos (Porter y
McCarthy, 1997). La reduccin del volumen celular en los astrocitos en respuesta a
un estmulo hipoosmtico tiene lugar a travs de un aumento intracelular de Ca2~, va
IPa y la activacin de MAPK dependiente de PI-3 quinasa (Schliess et al., 1996).

SAPK/JNK Y P38
De modo semejante a la cascada de fosforilacin Ras/RafMAPK activada por
factores mitognicos, se ha descrito la existencia de sistemas de transduccin activados por estrs celular (rayos UV, rayos X) y citoquinas inflamatorias (revisado por
Kyriakis y Avruch, 1996). Estos sistemas de transduccin de seales generan en la
clula fundamentalmente respuestas inhibitorias del crecimiento, diferenciacin o
muerte celular (Coroneos et al., 1996; Hannun, 1996; Brenner et al., 1997). Los receptores de TNFct o IL-1[3, carecen de actividad de tirosina quinasa y su activacin va
asociada a la activacin de protenas tirosina quinasas citoslicas como JAKs
(quinasas del tipo Jano) que se autofosforilan y fosforilan el receptor permitiendo la
unin de protenas adaptadoras (Baker y Reddy, 1996). La activacin de SAPK/JNK
est precedida por la hidrlisis de SM y generacin de ceramida, y las ceramidas son
capaces de activar SAPK/JNK de forma especfica (Westwick et al., 1995), lo que

Introduccin

13

permite atribuir un papel a este mensajero en su regulacin. En efecto, la apoptosis

inducida por citoquinas inflamatorias y estrs celular puede estar mediada por la
activacin de SAPK/JNK a travs de ceramida (Verheij, et al., 1996). En concreto, en
cultivos primarios de astrocitos, la activacin de SAPK por TNFct coincide con su activacin por la adicin de SMasa exgena o ceramidas permeables, sin embargo, la
cintica de activacin de SAPK por SMasa y ceramidas no coincide con la inducida

por el TNFct (Zhang et al., 1996).

La activacin de SAPKs por los receptores de membrana se lleva a cabo fundamentalmente por las protenas G monomricas Rac y Cdc42 de la familia Rho,
siendo la activacin de SAPK independiente de la accin directa de Ras (Kyriakis et
al., 1994). Estas protenas activan MEKI{1 a travs de las protenas PAR, que inhiben la actividad GTPsica de las protenas Rho. La protena MEKK1, situada a nivel
de Raf-1 en la cascada de fosforilacin mitognica (Fig. 4), es el activador de
SEK1/MKK4 y tambin de MEK (Kyriakis y Avruch, 1996). La protena SEK1MKK4
es

la protena homloga de MEK responsable de la activacin especfica de SAPK e

incapaz de activar MAPK. Las SAPKs, tambin se denominan quinasas del extremo
N terminal de c-Jun (JNKs) debido a que este factor de transcripcin es uno de sus
substratos, junto a otros como ATF-2 y Elk-1 (Kyriakis et al., 1994). Recientemente,
se ha demostrado que este sistema de transduccin de seales es fundamental en la
regulacin del proceso de gliognesis y diferenciacin de los astrocitos a partir de clulas progenitoras corticales (Bonni et al., 1997).

1~nA
ffiPAK

____

IMaua~a

1~

4,

c~z~
M

c-flz c-jw~ EIk-L c~nr,

~FEE

oJu,~ Hk-1

M4HCAP2,~ Hk-1

BE~ZIJLAB~

Hg. 4 Fs~.nnr de las pdr4el~ mandas de fEftri1ai6nr~u1adas exlnnlulanmt

Introduccin

14

Adems del sistema SAPK/JNK existe en clulas de mamferos una serie de

protenas homlogas al sistema de sealizacin de estrs celular osmtico en levaduras (HOG1)(Takekawa et al., 1997), denominado p38 (Fig. 4). Esta cascada de fosforilacin activada por estrs celular y citoquinas inflamatorias es paralela a SAPK e
implica a las quinasas MKK3/MKK6 (Kyriakis y Avruch, 1996).
La activacin de Ras por agentes mitognicos puede activar de forma secundaria SAPK a travs de la activacin de MEKK1 o directamente de Rac-cdc42 (Kyriakis
y Avruch, 1996). La existencia de puntos de regulacin cruzada entre las distintas
cascadas de fosforilacin descritas, permite su coordinacin en diferentes sistemas
celulares como los astrocitos (Korzus et al., 1997).

LA ISOFORMA ~ DE PROTENA QUINASA C

El trmino PKC engloba una familia de protenas quinasas caracterizadas inicialmente por su actividad dependiente de Ca2~ y DAG (Inoue et al., 1977). Actualmente se han descrito al menos 12 isoformas de PKC que se agrupan en funcin de su
estructura y caractersticas funcionales en tres categoras. Las PKCs convencionales

a, [31,[311y y, cPKCs, que precisan para su actividad de Ca2~, DAG (o sus anlogos los
steres de forbol) y fosfatidilserina (PS). Las PKCs denominadas nuevas 6, s, e, ~/L
(ratn/humano), nPKCs, tienen los mismos requerimientos de cofactores excepto que
no precisan de Ca2~. Por ltimo la categora de las PKCs atpicas Ah. (ratn/humano) y
~,

aPKCs, nicamente requieren para su actividad de PS, y son independientes del

Ca2~ o del DAG (revisado por Jaken, 1996; Hoffman, 1997). El Ca2~ se une al denominado dominio C2 y el DAG y sus anlogos al dominio Cl. Las distintas isoformas de
PKC se expresan en astrocitos y en clulas de glioma C6 (Ballestas y Benveniste,
1995; Chen et al., 1995; 1996), con la excepcin de la forma cPKC y (Roisin y Deschepper, 1995>. Las caractersticas intrnsecas reguladoras de cada grupo de PKC deter-

minan que cada tipo sea activado preferentemente por determinados sistemas de
transduccin de seales. As, las nPKCs parecen ser activadas fundamentalmente por
la generacin de DAG va PLD, mientras que las cPKCs precisan para su completa
activacin del aumento de Ca2~ intracelular que acompaa a la hidrlisis de fosfoinostidos va PI-PLC (Ha y Exton, 1993).
La activacin de PKC va acompaada de su translocacin desde la fraccin
citoslica a la bicapa lipdica, donde encontrar la PS necesaria para su actividad enzmtica. La PKC despus de una activacin persistente sufre una proteolisis parcial,

Introduccin

15

seguido de un proceso de degradacin posterior que conduce a una disminucin de los

niveles de enzima, denominado downregulation (Jaken, 1996; Hoffman, 1997).

PKC atpica
El grupo de las PKCs atpicas incluye dos isoformas X/t y

4.

La ausencia del

dominio C2, y el poseer nicamente una estructura en dedo de zinc en Cl, explica
2~ y DAG (Wooten et al., 1997). La PKC 4 se puede activar por
su independencia de Ca
el producto de la PI-3 quinasa (Nakanishi et al., 1993), y tambin por PA (Limatola et
al., 1994). La PKC

es por sus caractersticas estructurales resistente a la activacin

y downregulation por steres de forbol, como ocurre en los astrocitos (Ballestas y

Benveniste, 1995; Chen et al., 1995), y esta enzima podra estar implicada en el control de la diferenciacin astrocitaria (Yoshimura et al., 1997). Resulta interesante la
observacin de que la activacin de MAPK por endotelinas sea independiente de la
actividad de PKCs sensibles a downregulation por steres de forbol (Tournier et al.,
1994). Adems, la PKC

se encuentra

implicada en la diferenciacin de clulas PC12

por NGF (Coleman y Wooten, 1994), el proceso de potenciacin a largo plazo, longterm potentiation (Hrabetova y Sacktor, 1996), y el control de la proliferacin celular

(Berra et al., 1993; Lehrich y Forrest, 1994). La funcionalidad de la PKC

est nti-

mamente asociada a su localizacin subcelular (Wooten et al., 1996), y el proceso de

diferenciacin celular precisa de la translocacin de PKC
(Wooten et al., 1997). La localizacin subcelular de PKC
su interaccin con protenas especificas

a la matriz nuclear

puede estar regulada por

como ZIP (zeta interacting protein) o PICK

(Puls et al., 1997). La interaccin con este tipo de protenas puede ser importante para aumentar la especificidad de la interaccin de aPKCs con los lpidos que actan
como cofactores (Diaz-Meco et al., 1996). La translocacin nuclear de PKC

puede

venir condicionada tambin por la presencia de mensajeros lipdicos, como el PA generado va PLD nuclear, o por la existencia de secuencias especficas de sealizacin
nuclear (Wooten et al., 1997).

Sistemas de transduccin de seales y PKC

La PKC

acta de forma cooperativa con Ras y podra coordinar los efectos de

PC-PLC y PI-3 quinasa en el control de la proliferacin celular (Nakanishi et al.,

1993; Diaz-Meco et al., 1994), actuando en un nivel anterior a IVIAPK (Berra et al.,
1995). La PKC

activada por la PC-PLC es un activador de Raf-1, lo que explicara la

16

Introduccin

existencia de un proceso de activacin de MAPK independiente de Ras (van Dijk et

al., 1997a).
El modelo de Moscat y colaboradores propone que la PKC

es un importante

regulador de la mitognesis e inhibidor de la apoptosis (Berra et al., 1997). Sin embargo, la PKC

revierte el fenotipo transformado inducido por la expresin de v-Raf

en clulas 3T3 (Kieser et al., 1996), y su sobrexpresin no tiene consecuencias mitognicas en estas mismas clulas (Crespo et al., 1995; Montaner et al., 1995).
La PKC

parece estar implicada en el mecanismo de transduccin de seales

del TNFa e IL-1[3, a travs de la activacin de la SMasa cida previa activacin de la

PC-PLC (Lozano et al., 1994; Carson y Hart, 1996). El producto de accin de la SMasa, la ceramida, es en efecto un activador de PKC
mida activan PKC

4,

4.

Concentraciones bajas de cera-

pero niveles mayores revierten esta activacin. Por ltimo el

cido araquidnico compite con la ceramida, inhibiendo as la activacin que esta

ejerce sobre PKC

4 (Mller

et al., 1995). La PKC

4 puede

servir por tanto como meca-

nismo de integracin de seales mitognicas o de inhibicin del crecimiento.

1.3 REGULACIN DE LA EXPRESIN GNICA

La regulacin de la expresin gnica esta basada en su activacin o inhibicin
por los denominados factores de transcripcin y puede considerarse la ltima etapa
del proceso de transmisin de informacin que comienza en la membrana plasmtica.
La propia estructura del DNA en asociacin con histonas y las modificaciones qumicas de stas permiten mecanismos de control adicionales. Para ejercer su accin, los
factores de transcripcin deben localizarse en el ncleo e interaccionar con el DNA.
La regulacin a travs de factores de transcripcin puede ejercerse a travs de la regulacin de su translocacin al ncleo o por la induccin de su sntesis por seales
extracelulares. El estado de fosforilacin es esencial en la regulacin de factores de
transcripcin, favoreciendo cambios conformacionales que permiten exponer secuencias de sealizacin nuclear, modificando el estado de oligomerizacin de la protena,
regulando la interaccin protena-DNA o regulando su interaccin con el sistema celular de transcripcin (Hill y Treisman, 1995). A continuacin se describe el sistema
de regulacin de tres tipos fundamentales de factores de transcripcin.
El factor de transcripcin NF-KB est localizado en el citosol en asociacin con
la subunidad IKB que ejerce un efecto inhibidor que impide su transiocacin al ncleo.
Citoquinas inflamatorias, estrs celular, LPS o infeccin viral, entre otros, son activadores de NF-KB (Kohler et al., 1997). La fosforilacin de IicB permite su degrada-

Introduccin

cin, y la consiguiente translocacin de NF-KB al ncleo donde ejerce su efecto regulador de la transcripcin (ONeill y Kaltschmidt, 1997). La transcripcin de numerosos genes est regulada por el factor NF-KB, y es esencial en la funcionalidad glial y
neuronal (ONeill y Kaltschmidt, 1997). La identificacin de la protena quinasa responsable de la fosforilacin y degradacin de IKB, es actualmente uno de los objetivos
para el conocimiento de los mecanismos implicados en la activacin de este factor de
transcripcin (Isral, 1997).
Otro tipo de factores de transcripcin esta representado por c-Fos. Esta protena pertenece al grupo de los genes de expresin temprana. La activacin celular induce la expresin del gen c-fos. La protena c-Fos sintetizada constituye homodmeros, o
heterodmeros con c-Jun, complejos AP-1, que regulan la expresin gnica. El promotor de c-fos posee un elemento denominado SRE (serum response element) que es
un sitio de unin de factores de transcripcin activados por el suero. La activacin de
SRE requiere de la unin del factor SRF y de Elk-1. Elk-1 es activado por fosforilacin
a travs de la cascada de las MAPK, de modo que la translocacin nuclear de MAPK
permite su fosforilacin y activacin de Elk-1 (Denhart, 1996).
Las protenas STATs, son factores de transcripcin activados por su fosforilacin en la membrana plasmtica a travs de tirosina quinasas JAKs asociadas a receptores de la familia de las citoquinas. La fosforilacin permite la dimerizacin de
STATs y su translocacin al ncleo (Darnel, 1997). Al igual que ocurre con el factor
AP-1, la distinta composicin de los oligmeros puede condicionar la especificidad en
la interaccin complejo proteico-secuencia de DNA diana.
Tan importante como la activacin de los factores de transcripcin es su vuelta
al estado basal. Las protenas fosfatasas revierten la activacin de los factores de
transcripcin activados por fosforilacin. La induccin de la sntesis de factores inhibitorios, como IKB o de ICER, que compite en la unin de factores a CRE (cAMP responsive element), es otro modo de inactivacin de la expresin gnica. La comunicacin entre los sistemas de transduccin de seales y los factores de transcripcin no
es unidireccional, y estos ltimos tambin pueden regular la actividad de los primeros. As, la activacin de NF-KB puede activar la PICA de forma independiente de los
niveles de cAMP a travs de la disociacin de complejos NF-KB-IKB-PKA que mantienen la PICA en estado inactivo (Zhong et al., 1997).
Un mismo estmulo extracelular puede regular diferentes factores de trans-

cripcin, y las clulas reciben multitud de estmulos de forma simultnea. Esto sumado a los efectos cruzados de regulacin entre distintas rutas de sealizacin celu-

Introduccin

18

lar, permite imaginar una situacin en la que la regulacin de la expresin gnica

est modulada por un delicado balance entre numerosos sistemas de control.

2. EL FACTOR DE CRECIMIENTO NERVIOSO
El factor de crecimiento nervioso (NGF) fue el primer factor de crecimiento,

supervivencia y diferenciacin neuronal caracterizado y con el que se constituy la

familia de las neurotrofinas (Levi-Montalcini, 1987). El NGF fue descubierto como el
factor difusible responsable de la produccin de un halo de fibras nerviosas en un explante de ganglios simpticos y sensoriales de embrin de poo (Levi-Montacini y
Hamburger, 1953). La teora neurotrfica sostiene que las neuronas compiten entre s
por una cantidad limitada de neurotrofinas proporcionadas por el tejido inervado. Las
neurotrofinas juegan un papel esencial en la regulacin del desarrollo del sistema

nervioso, la plasticidad sinptica del sistema nervioso adulto y en el mantenimiento

de su integridad estructural (Hefti, 1994). La ausencia de NGF, o una disminucin
local en sus niveles, impide el desarrollo neuronal normal y explica la apoptosis fisiolgica durante el periodo neonatal. Adems el propio NGF participa en la activacin

de procesos apoptticos mediados por el receptor p75~R en neuronas deficientes en

trkA (Dechant y Barde, 1997). La familia de las neurotrofinas incluye adems el factor neurotrfico derivado del cerebro (BDNF) y las neurotrofinas NT-3, NT4/5 y NT-6
(Varon et al., 1995; Lewin y Barde, 1996). Cada tipo de neurotrofina ejerce su efecto

de forma preferente en determinadas poblaciones neuronales y en momentos especficos del desarrollo. Existen otras protenas con efectos neurotrficos como el factor
neurotrfico derivado de la gla (GDNF) o el factor ciliar neurotrfico (CNTF) con caractersticas diferenciales que las separan de la familia de las neurotrofinas.
Adems de los efectos convencionales del NGF en el sistema nervioso, este
factor, desempea un papel crucial en la regulacin de las relaciones entre el sistema
nervioso, inmune y neuroendocrino, existiendo una comunicacin bidireccional entre
sistema nervioso y sistema inmune (Levi-Montalcini et al., 1996; Merril y Benveniste, 1996). El NGF ejerce un efecto proliferativo sobre linfocitos T y B, constituye un
factor autocrino de supervivencia de los linfocitos B memoria y estimula la produccin
de inmunoglobulinas (Torcia et al., 1996>. Adems, acta como factor quimiotctico
regulando la funcionalidad de microglia y macrfagos y es un mediador de la respuesta de fase aguda (Gilad y Gilad, 1995; Elliabes et al., 1996). La existencia de elevados
niveles de NGF en algunas situaciones patolgicas como esclerosis mltiple o encefa-

Introduccin

19

litis alrgica experimental (Bonini et al., 1996; DeSimone et al., 1996), evidencian la
importancia de la interconexin entre el sistema inmune y el sistema nervioso.

21. EL GEN DEL NGF

El gen del NGF se ha donado en diferentes organismos, estando situado en el
hombre en la regin lpl3 del cromosoma 1 (Scott et al., 1983; Carrier et al., 1996). El
gen de NGF est formado por cuatro exones (Fig. 5) y su transcripcin da lugar a la
sntesis de cuatro transcritos (A, B, C y D), debidos a la existencia de un doble sitio de
iniciacin de la transcripcin y por procesamiento diferencial (splicing) del premRNA, siendo el principal transcrito de 1.35 kb (Selby et al., 1987).
La regin de 5 kb situada delante del primer exn posee los elementos necesanos responsables de la regulacin especfica de tejido del gen (Alexander et al., 1989).
El promotor principal del gen de NGF posee una caja TATA y CCAAT, elementos
fundamentales para la unin de la RNA polimerasa y sus cofactores. En rata, las cajas TATA estn situadas en la posicin -43 y -23, separadas por 15 nucletidos. Las
cajas CCAAT, asimilables a cajas CAAT, estn situadas en las posiciones -379 y -546.
Existen dos elementos represores en las posiciones -500 y -120 y tambin dos secuencias activadoras entre -120 y -39 (DMello y Heinrich, 1991a; Cartwright et al., 1992).
En la regin +33 a +50, en el lmite del exn lb con el intrn siguiente existe un elemento regulador AP-1 (Fig. 5) (Hengerer et al., 1990), y en la posicin -669 se encuentra un posible sitio de regulacin por NF-KB (Jehan et al., 1993). La existencia de una
secuencia AP- 1 permite explicar la induccin de NGF por estmulos activadores de cfos y c-jun como el PMA, suero, cAIVIP y dao cerebral (Jehan et al., 1993; Onteniente
et al., 1994; Colangelo et al., 1996). Sin embargo, existen otros elementos reguladores

H?fSAViti?

APi

7&~ F~

ATO

Hg 5

~
-S2kb
ATO

ntain~prnica del ~nde NYen mta lis ~m~uks rqx~ntanks e>n~

enblann sr ~gior~ u tmdtridas, mllacks crdiflmnnrt~ del pmzmsr de N3Fyen ~is
~ m.~tm la ~nnlifica,t
de la piemadxa

Introduccin

20

an no caracterizados que explicaran la induccin de NGF por otros inductores como

la vitamina D3 (Rush et al., 1995). En el exn 3 existe adems un segundo promotor
capaz de inducir la transcripcin (Racke et al., 1996).
El mRNA del NGF posee en el extremo 3 una secuencia no codificante rica en
AU, denominada 3IJTR, que acta en cis determinando la elevada inestabilidad del
mRNA de NGF, posiblemente a travs de la interaccin en trans con protenas de
unin a RNA (Tang et al., 1997). La inestabilidad del mRNA de NGF condiciona una
vida media de los transcritos muy corta (Onteniente et al., 1994), lo que explicara el
hecho de que numerosos agentes inductores de la sntesis del NGF, ejerzan su accin
a travs de la activacin de la transcripcin del gen de NGF (Carswell, 1993; Racke et
al., 1996).
%2 LA PROTENA NGF
El NGF se sintetiza como un precursor, preproNGF, de 34 kDa a partir del
transcrito A. Tras la hidrlisis de los 25 aminocidos del pptido seal, se genera el
proNGF, y a partir de este el [3-NGFmaduro, responsable de la actividad biolgica.
La forma madura del [3-NGF est formada por dos cadenas idnticas asociadas de
forma no covalente y con tres puentes disulfuro intracatenarios. El [3-NGFse encuentra formando parte de un complejo multiprotico de dos subunidades a, un homodmero [3y dos subunidades y. La subunidad y es una arginina peptidasa responsable
del procesamiento del 13-NGF (Edwards et al., 1988). La funcin exacta de la subunidad a se desconoce pero se cree que protege el [3-NGFde la degradacin enzimtica.
Todos los determinantes necesarios para el adecuado proceso de maduracin y secrecin del NGF se conservan en la secuencia del NGF maduro (Heymach et al., 1996>.
El NGF sintetizado es inmediatamente secretado, siendo los niveles intracelulares de
NGF muy pequeos (Furukawa et al., 1987). En clulas de naturaleza neuroendocrina, la secrecin puede ocurrir por grnulos de secrecin y estar bajo control de neurotransmisores (Missale et al., 1996). Se han descrito recientemente anticuerpos naturales antiNGF que podran tener una funcin transportadora del NGF en suero
(Dicou y Nerriere, 1997).
Estudios cristalogrficos han permitido determinar la estructura tridimensional del NGF, que incluye dos lminas

[3antiparalelas

unidas por tres cortos segmen-

tos de gran variabilidad conformacional (McDonald y Chao, 1995). Los tres puentes
disulfuro se agrupan en un motivo estructural denominado nudo de cisteinas. Estu-

Introduccin

21

dios de funcionalidad por mutagnesis dirigida han permitido identificar los aminocidos necesarios para la unin especfica y activacin del receptor (Woo y Neet, 1996).
2.3 LOCALIZACIN DE LA SNTESIS DEL NGF
Tejidos perifricos
La sntesis de NGF en los tejidos perifricos se correlaciona de forma positiva
con el grado de inervacin simptica o sensorial (Korsching y Thoenen, 1983; Davies
et al., 1987). Tejidos como la piel, el iris, o el corazn tienen un contenido relativamente alto de NGF y NGF mRNA (Heumann y Thoenen, 1986). La sntesis de NGF es
tambin significativa en prstata, testculos, tiroides y tejido adiposo pardo (Nisoli et
al., 1996; Chen et al., 1997).
El NGF sintetizado en los tejidos diana alcanza los ganglios simpticos y sensoriales por transporte retrgrado (Korsching y Thoenen, 1988; von Bartheld et al.,
1996). En estos ganglios, al igual que en el nervio citico, los niveles de mRNA son
muy bajos, pero el contenido en NGF es elevado. La axotomia del nervio citico produce un aumento bifsico en los niveles de NGF mRNA (Heuman et al., 1987), primero por la liberacin de factores sricos (Spranger et al., 1990; Neveu et al., 1992), y
posteriormente por la secrecin de citoquinas, fundamentalmente IL-1[3 por la invasin del nervio por macrfagos activados (Hattori et al., 1993; Pshenichkin y Wise,
1995; Dekosky et al., 1996).
Sistema nervioso central
De manera similar a lo que ocurre en el sistema nervioso perifrico (SNP), el
NGF es sintetizado por los tejidos diana de las neuronas colinrgicas del cerebro basal anterior, que constituyen las principales clulas sensibles al NGF (Robner et al.,
1997). As, la sntesis est directamente relacionada con el grado de inervacin colinrgica (Korsching et al., 1985; Robner et al., 1997). Mientras que en el SNP la sntesis se realiza por clulas no neuronales, en SNC la sntesis tiene lugar en las neuronas, encontrndose elevados niveles de NGF en el hipocampo y en menor medida en
la corteza y bulbo olfativo (Korsching et al., 1985; Ayer-Lelievre et al., 1988; Rocamora et al., 1996). Las neuronas constituyen la fuente mayoritaria de NGF en el SNC
adulto y ejercen un efecto represor de su expresin en astrocitos (Vig et al., 1992),
pero durante el desarrollo o en caso de lesin, los astrocitos pueden expresar el NGF
de forma significativa (Chakrabarti et al., 1990; Lu et al., 1991; Arendt et al., 1995).

Introduccin

22

Sntesis de NGF in vitro

La sntesis de NGF

iii

vitro se produce en una gran diversidad de tipos celula-

res. Las neuronas procedentes de distintas localizaciones del SNC como hipocampo,
septo, estriado y corteza sintetizan NGF pero con diferente intensidad (Houlgatte et
al., 1989). Los cultivos primarios de clulas gliales son tambin capaces de sintetizar
NGF (Neveu et al., 1992; Jehan et al., 1995; Brodie, 1996; Boutros et al., 1997), sin
que la procedencia de los astrocitos condicione los niveles de sntesis (Houlgatte et al.,
1989). Cultivos primarios de clulas cardicas, clulas musculares lisas, clulas de
Schwann, clulas mesangiales (Plss et al., 1995; Creedon y Tuttle, 1997) adems de
macrofagos y linfocitos T CD 4~, entre otras clulas del sistema inmune, son capaces
de producir NGF (Lambiase et al., 1997). Para el estudio de la regulacin del gen de
NGF se han utilizado lneas derivadas de fibroblastos, L929 o 3T3 (Wion et al., 1990;
DMello y Heinrich, 1991b). El NGF es producido adems, por clulas de glioma C6
(Fukumoto et al., 1994; Colangelo et al., 1996), clulas neuroendocrinas, neuroblastos
y lneas osteoblsticas (Charrasse et al., 1992; Jehan et al., 1996; Heymach et al.,
1996).
2.4 REGULACIN DE LA SNTESIS DE NGF
La regulacin de la sntesis de NGF es el principal mecanismo de regulacin
de los niveles de NGF extracelulares (Carswell et al., 1993; Racke et al., 1996) y por
ello su conocimiento resulta de suma importancia teniendo en cuenta el potencial teraputico de las neurotrofinas en general y del NGF en particular (Carswell, 1993;
Rush et al., 1995). Dado que el NGF no es capaz de atravesar la barrera hematoenceflica, no es posible su inyeccin sistmica para tratar de paliar determinadas situaciones neuropatolgicas (Han et al., 1997). La investigacin aplicada en esta rea va
encaminada a identificar posibles moduladores de la expresin del NGF o de otras
neurotrofinas y la utilizacin de clulas genticamente modificadas que podran ser
encapsuladas y transplantadas.
La sntesis de NGF por clulas en cultivo depende del estado de crecimiento
celular. As, en cultivos no confluentes, los niveles de sntesis de NGF son elevados,
reducindose la sntesis conforme se alcanza la confluencia debido a una inhibicin
por contacto celular (Furukawa et al 1987; Houlgatte et al., 1989; Lu et al., 1991). En
el proceso de gliosis, que sigue a un proceso de lesin, se liberan factores mitognicos
y citoquinas pro-inflamatorias que explican una induccin de la sntesis y secrecin

Introduccin

23

de NGF tanto in vivo como in vitro (Arendt et al., 1995; Psenichkin y Wise, 1995;
Dekosky et al., 1996).
Factores proliferativos
La utilizacin de medios de cultivo celular qumicamente definidos permiti
poner en evidencia el efecto estimulador del suero en la sntesis de NGF. El efecto del
suero es parcialmente dependiente de protenas U sensibles a toxina pertusis y ha
sido observado tanto en fibroblastos como en astrocitos (Spranger et al., 1990; Neveu
et al., 1992). Como en SNC los astrocitos no estn en contacto con el plasma, el efecto
inductor del suero podra reflejar la situacin tras una lesin vascular, que puede ser
imitada

iii

vitro por el modelo experimental de lesin del nervio citico (Heumann et

al., 1987). La expresin de c-fos precede al aumento de la sntesis de NGF por suero y
por lesin del nervio citico (Hengerer et al., 1990). La induccin de la sntesis de
NGF por el suero es dependiente de la activacin de cPKCs. En este sentido el PMA
es tambin un potente inductor de la sntesis de NGF, y la regulacin por los dos inductores tiene caractersticas comunes (Neveu et al., 1992). Itt vivo, parece haber un
equilibrio entre la regulacin positiva que ejerce la PKC en la expresin del NGF y el
efecto inhibidor que ejercen los glucocorticoides (Neveu et al., 1992). La induccin de
NGF dependiente de PKC se inhibe por un aumento en la concentracin intracelular
de Ca2~, lo que se puede explicar por los distintos requerimientos de Ca24 de las diferentes isoformas de PKC, o por efecto del Ca2~ en la downregulation de cPKCs
(Jehan et al., 1995). Los factores de crecimiento fueron algunos de los primeros estimuladores conocidos de la sntesis del NGF y el EGF, FGFa/b y el TGF[3 estimulan la
produccin de NGF por astrocitos (Yoshida y Gage, 1992).
Factores proliferativos de astrocitos como la trombina y los nucletidos de
guanina, tambin estimulan la sntesis y secrecin de NGF (Middlemiss et al., 1995;
Debeir et al., 1996). La induccin por trombina es dependiente de PKC y fisiolgicamente puede estar relacionada con la exposicin de las clulas gliales a proteasas tras
un proceso traumtico.
Mediadores de la inflamacin
La IL-1[3 y el TNFa son importantes citoquinas reguladoras de la respuesta
inflamatoria. A nivel perifrico son sintetizadas por los macrfagos activados, y en el
SNC por la microglia y por los astrocitos reactivos (Merril y Benveniste, 1996). Estas
dos citoquinas pro-inflamatorias son potentes estimuladores de la sntesis y secrecin

24

Introduccin

de NGF. As, la IL-l[3 es la responsable de la induccin de NGF que ocurre

itt

vivo

tras la lesin del nervio citico o un trauma cortical (Heumann et al., 1987; Dekosky
et al., 1996). Adems, la induccin de NGF por IL-1[3 y TNFct se observa tambin in
varo en astrocitos (Carman-Krzan y Wise, 1993; Psenichkin y Wise, 1995). La induccin de NGF no est restringida a clulas gliales, habindose observado tambin en
fibroblastos y cultivos primarios de hipocampo formados por neuronas y clulas gliales (Hattori et al., 1993; 1996).
La regulacin de la produccin de NGF ejercida por la IL-1[3 ocurre a diferentes niveles: aumento de la transcripcin y estabilizacin de los transcritos formados.
El efecto de la IL-1[3 es independiente de la actividad de cPKCs (Psenichkin et al.,
1994) y no se ve afectado por el aumento en los niveles intracelulares de cAMP,
(Carman-Krzan y Wise, 1993; Han et al., 1994), ni tampoco por el aumento en la concentracin intracelular de Ca2~ (Friedman et al., 1992). Parece que en general, la hidrlisis de fosfoinositidos tiene poca importancia en la regulacin de la sntesis del
NGF (Jehan et al., 1995), y el efecto de la IL-1[3 es tambin independiente de esta
ruta de sealizacin celular (Friedman et al., 1992).
El efecto de la IL-1[3 se atribuye a la generacin de cido araquidnico va activacin de PLA2, que posteriormente es metabolizado a leucotrienos (Carman-Krzan y
Wise, 1993). En lo que se refiere al TNFa, la induccin de NGF parece estar mediada
por el receptor de 55 kDa, que es el receptor mayoritario en astrocitos (Hattori et al.,
1996; Dopp et al., 1997).

Las catecolamnas
La activacin de receptores [3-adrenrgicos, o el aumento de los niveles intracelulares de cAMP, puede ejercer en determinadas circunstancias un efecto activador
de la sntesis y secrecin de NGF tanto in vivo como en clulas gliales (Condorelli et
al., 1994; Fukumoto et al., 1994). Sin embargo, en otras circunstancias carece de efecto (Friedman et al., 1992; Neveu et al., 1992), o ejerce un efecto supresor de la induccin por suero, PMA o TGF-f31 (Han et al., 1994; Jehan et al., 1995). Las diferencias
en la respuesta a agonistas adrenrgicos parecen ser debidas a diferencias en el estado de crecimiento del cultivo celular (Furukawa et al., 1987; Han et al., 1994).

Regulacin de la sntesis de NGF por diversos factores

La sntesis y secrecin de NGF in vitro se puede modular por multitud de factores, a continuacin se citan nicamente algunos de ellos: Citoquinas como IL-4, IL-

Introduccin

25

5, IL-lO y el factor activador de plaquetas poseen un efecto estimulador de la sntesis

de NGF (Awatsuji et al., 1993; Brodie, 1996), sin embargo el interfern y ejerce un
efecto inhibidor de la sntesis de NGF (Awatsuji et al., 1995). El interfern

[3y

PDGFBB pueden estimular o inhibir la produccin de NGF en funcin de las condiciones y tipo de cultivo celular utilizado (Plss et al., 1995; Boutros et al., 1997; Creedon
y Tuttle, 1997).
La 1,25-dihidroxivitamina D3 ejerce un efecto estimulador de la sntesis de
NGF en distintos modelos celulares, entre ellos astrocitos y clulas de glioma C6
(Neveu et al., 1994; Jehan et al., 1996; Han et al., 1997). La vitamina D3 posee un
efecto aditivo en la induccin de NGF por IL-1[3, sinrgico con el TGF-[31 y contrarresta el efecto supresor que ejerce la corticosterona (Pshenichkin et al., 1994; Han et al.,
1997). La activacin de la microglia en un proceso traumtico del SNC podra explicar
un aumento local en los niveles de vitamina D3 que estimulara la sntesis de NGF
por los astrocitos (Neveu et al., 1994). Los glucocorticoides regulan la sntesis de NGF
de modo complejo y especfico del tipo celular. En astrocitos, los glucocorticoides inhiben la secrecin basal e inducida por PMA, suero, vitamina D3, o citoquinas proinflamatorias (Neveu et al., 1992; Psenichkin et al., 1994; Han et al., 1997>.
La expresin del gen de NGF est controlada adems por el neurotransmisor
glutamato a travs de la induccin de NO, la organizacin del citoesqueleto y el estrs
oxidativo (Naveilhan et al., 1994; Persichini et al., 1994; Baudet et al., 1995).
3. LOS ASTROCITOS Y OTRAS CELULAS GLIALES
Los astrocitos son las clulas no neuronales mayoritarias del SNC, y se agrupan junto con los oligodendrocitos y la microgla en el trmino general de clulas
gliales (revisado por Porter y McCarthy, 1997>. Existen dos tipos morfologicamente
diferentes de astrocitos, protoplsmicos situados en la materia gris y astrocitos fibrosos fundamentalmente en las fibras mielinicas.
En 1983 Raff y colaboradores demostraron la existencia de dos tipos de astrocitos en cultivos de nervio ptico perinatal procedentes de dos tipos celulares diferentes. Estos estudios pusieron d manifiesto la existencia de un progenitor celular O-2A
que puede diferenciarse en oligodendrocitos y astrocitos de tipo 2. Las clulas progenitoras O-2A se caracterizan por ser reconocidas por un anticuerpo monoclonal A2B5,
y se diferencian en presencia de suero mayoritariamente en astrocitos de tipo 2,
mientras en un medio qumicamente definido se diferencian en oligodendrocitos
(Temple y Raff, 1985). La protena glial fibrilar cida (GFAP), constituyente de los

Introduccin

27

1995). De este modo en situaciones de traumatismo, lesin, infeccin bacteriana o

viral los astrocitos pueden proteger, favorecer la regeneracin del tejido daado o regular el proceso inflamatorio. Los astrocitos reactivos pueden expresar el complejo
mayor de histocompatibilidad y actuar como clulas presentadoras de antgeno en la
respuesta inmune mediada por linfocitos T (Hurwitz et al., 1995; Niikcevich et al.,
1997>.
Las clulas de glioma CO
Las clulas C6 de glioma proceden de un glioma de rata inducido qumicamente y se caracterizan por su naturaleza indiferenciada con potencial astroctico y olidodendrocitario. Las clulas C6 pueden expresar GFAP y se utilizan como modelo en el
estudio de la regulacin de la sntesis de NGF (Fukumoto et al., 1994; Colangelo et
al., 1996). Adems se utilizan como modelo celular en el estudio de los mecanismos de
transduccin de seales en clulas gliales (Chen et al., 1995; Anciaux et al., 1997;
Yoshimura et al., 1997a;b).

Introduccin

28

4. OBJETIVOS
Del NGF, la primera neurotrofina descubierta, existe un conocimiento pormenorizado sobre su funcionalidad y estructura. El papel del NGF en el normal funcionamiento del sistema nervioso, y su posible utilidad teraputica han impulsado el
estudio de los mecanismos de sntesis de este factor. Esta rea de investigacin ha
permitido identificar agentes moduladores de la sntesis de NGF en distintos modelos
experimentales. La presente tesis doctoral pretende profundizar en el estudio de los
mecanismos de sealizacin celular conducentes a la activacin de la sntesis y secrecin de este factor neurotrfico en clulas gliales, establecindose los siguientes objetivos:
1. El estudio de mecanismos de transduccin de seales implicados en
la regulacin de la sntesis de NGF, centrado en la generacin de mensajeros lipdicos intracelulares clsicos y en la recientemente caracterizada ruta de las ceramidas.
2. Caracterizacin de los posibles sistemas de fosforilacin con los que
est coordinada la generacin de mensajeros lipdicos y de los factores
de transcripcin implicados.
3. Establecimiento del posible papel de cada uno de los niveles anteriormente mencionados en el mecanismo de accin de estimuladores de
la expresin de NGF.

II- RESULTADOS Y DISCUSIN

1. REGULACIN DE LA SNTESIS Y
SECRECIN DE NGF POR
MENSAJEROS LIPDICOS EN
CULTIVOS PRIMARIOS DE
ASTROCITOS

Resultados y Discusin

29

Con este captulo se introduce el tema principal objeto de esta

tesis doctoral, el papel de mensajeros de naturaleza lipdica en la
regulacin de la sntesis de NGF por clulas gliales. El estudio de los
mensajeros intracelulares de origen lipdico y su mecanismo de
accin ha sido una de las reas de la bioqumica que ms ha
avanzado

durante

los

ltimos

aos,

proporcionando nuevos

conocimientos sobre el funcionamiento celular (Prescott, 1997). As,

se ha pasado de considerar a los lpidos de membrana como
componentes poco ms que estructurales a importantes reguladores
de las funciones celulares. El trabajo se centra primeramente en el
estudio del efecto de la adicin exgena de PC-PLC sobre la sntesis y
secrecin de NGF en cultivos primarios de clulas gliales. La accin
estimuladora de la PC-PLC en la sntesis de NGF sugiere un posible
papel de la ruta de la SMasa-ceramida, puesto que ambos sistemas
han sido directamente relacionados (Schtze et al., 1992; Miller et
al., 1994; Wiegman et al., 1994). Se estudia a continuacin la
importancia de la ruta de sealizacin por ceramidas en la
regulacin de la sntesis de NGF, as como los posibles sistemas de
transduccin de seales implicados. La utilizacin de anlogos
permeables de ceramida, o la adicin de SMasa exgena desencadena
una cascada de procesos intracelulares que conducen a un aumento
en la produccin de NGF. Los resultados que se exponen evidencian
la existencia de una regulacin cruzada de la sntesis de NGF entre
vas de sealizacin que involucran tanto a glicerolpidos como a
esfingolpidos.

FF55 Letters 364 (1995) 301304

FEHS 15486

Phosphatidylcholine-phospholipase C mediates the induction of nerve

growth factor in cultured glial celis
Ins D. Laviadaa*, Cristel Baudetb, Ismael Galvc-Roperh5, Philippe Naveilhanb, PhiIippe Brachetb
~

dc Bioqumica y Biologa Molecular, Facultad dc Biologa. Universidad Complutcnse. 28040 Madrid, Spain
bCe,,Ire Hospitalier Rgional Universitaire. INSERM U.298, 49100 Angers, France

Received 30 March 1995

Abstract Addton ofphosphatdylcholine-hydrolyzingphosphoupase C (PC-PLC) to cultured glial celis increased tbe leveis of
nene growth factor (NGF) mRNA and the amount of ceil-secreted NGF. The effect of PC-PLC wns 2.5 times higher (han
that elicited by 43-phorbol 123-myristate 13a-acetate. In cels
in which protein kinase C (PKC) was fully inhihited or donregulated, (he effect of PC-PLC ivas reduced though still evident and similar to that exerted by sphingosine. Results thus
indicate that PC-PLC induces the synthesis of NGF by glial celis
by a PKC-dependent and PKC-independent mechanisms.
Kev ,orcls: Nerve growth factor; phosphatidylcholine; Protein
kinase C: Sphingosne; Chal ccli; Astrocyte

1. Introduction
Nerve growth factor (NGF) is a ncurotrophic protein reqtiired for thc development and survival of several populations
of neurons [1]. In the central ncrvous system, NGF is synthesized by certain neurons, but during brain devclopnient and
under diffcrent paihological alterations of ihe ncrvous system,
there isa local increase in the production of NGF by astrocytes
[25].Thus, substantial present effort is directed toward an
undcrstanding of the influences that control NGF synthesis,
because of thc possibility that increasing the endogenous syndiesis of NGF might provide clinical advantage in degenerative
diseases of central nervous system [6]. However, little is known
about the elTectors that induce the synthesis of NGE by those
cels and about how this synthetic process is up-regulated. It
seerns tlat several NGF-inducing agents act through different
patlways. One of them is the signalling network involving the
activation of protein kinase C (PKC>. Studies performed with
cultured astrocytes and fibroblasts have shown that 4/3-phorbol
12/3-myristate lh-acetate (PMA>, a welI-documented PKC activator, induces dic synthesis and secretion of NGF, and this
effect is counteracted by PKC inhibitors like H-7 or H-9 [7,9].
PMA induces the expression of c-fos, the product of which
enhances NGF synthesis by the same route as PMA [5,9,10].
PKC is physiologically activated by diacylglycerol (DAG)
resulting from agonist-induced hydrolysis of inositol phospholipids [II]. However, this DAG rapidly disappears and seenis to
be responsible for a transient activation of PKC [12]. In contrast, hydrolysis of other membrane phospholipids, particularly
phosphatidylcholine (PC), produces DAG at a relatively later
phase. This DAG may be the responsible for the sustained
activation of PKC that leads to long-term cellular responses
*Corresponding author.

[13]. Mechanisms of cellular response to PC hydrolysis have

been demonstrated to occur ja many cdl types (reviewed in
[12,141).Despite extensive studies, the biocheniical mechanisnis
underlying signal-induced activation of PC breakdown are still
poorly defined. Phospholipase C (PLC)-catalyzed hydrolysis of
PC has been shown to be activated in response to a number of
agonists [15],in transiormed cels [161
and iii signalling cascades
triggered by growth factors [17]. PC-PLC provides a positive
signal for PKC by inducing its translocation to membrane
though not its down-regulation [161.
It has also been shown that
PC hydrolysis by a PC-PLC mimics the ability of carbachol to
inhibit adenylyl cyclase [18].Furthermore, a role of PC-specifc
phospholipase O (PLO) in signal transduction has been described [13,19].
In the present study we investigated thc possible relationship
bctwecn PC hydrolysis and PKC activation in the control of
NGF synthcsis by astrocytcs. Previous experimental evidence
showed that activation of PKC with PMA stiniulated NGF
synthesis by glial cels [7] while exogenously added PC-PLC was
able to activate PKC in fibroblasts [18]. This provinded us a
tool to investigate whether NGF synthesis by glial cels depends
on PC breakdown,
2. Experimental
2. 1. Culture con~liions

Primary cultures of rat brain glial cels werc prepared from cerebral

hemispheres of l2-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 37C). Trypsin was
subsequently inhibited by Ihe addition of [0%foetal calf serum (FCS),
before treatment with DNase 1 (lO pglml, 5 mm at 370C). Brain cels
were dissociated by fluxation with a Pasteur pipcttc and ncxt sedimented by low speed centrifugation (1000 xg, 5 mm). The pellet was
gently resuspended, and cels were seeded at a density of 3 x lo4 cellsl
cml. AII plasdc supports were previously coated with 5 pghnl poly-Lornithine in water. Cels were cultured for 3 weeks in basal medium
containing a mixture of Dulbeccos modilied Fagle medium and Hams
FI? (1/1, vlv), supplemented withO.66% glucose, Spglml streptomycin,
5 U/ml penicillin and 10% FCS. Three days before the experiment, FCS
was removed and cels were transferred to a chemically defined medium
consisting of basal medium supplemented with 25 pg/ml insulin, lOO
pg/ml human transferrin, 20 nM progesterone, 5OpM putrescine and
30 nM sodium selenite. Immunocytochemical studies showed that 90%
of cultured cels were positive for glial fibrillary acidic protein, indicating that cultures were largely enriched in astrocytes.
2.2. Assay of NGF aud NGF ,,,RNA

Cdl 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 cels was assayed in
triplicate. with a double-site-ELISA, using a monoclonal anti-NGF
antibody, coupled or not to t-galactosidase (Boehringer Manheim),
according to [20].Northern blot analysis was performed by standard
procedures [~1~
After 6 h of treatment, total RNA was extracted from

0014-5793195/S9.50 1995 Federation of European Biochemical Societies. AII rights reserved.

SSDI 0014-5793(95)00414-9

D. Laviada el at/FERS Leuers 364 (/995) 301304

302
cels by the LiCI/urea method. Glyoxal-treated RNAs were fractionated
o agarose gel, transerred 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. Iden/ficatio,, of PKC by innnunobloti~ig
Glial cels (treated as described in the legeod to Fig. 3) were scraped
oto 0.5 ml of ice-cold 50 mM Tris-bICI, pH 7.4,5 mM EDTA, lO mM
2-mercaptoethanol containing 1 /ig]ml leupeptin, pg/ml aprotinin, lO
pg/ml benzamidin, 5 pg/ml soybean tripsin inhibitor and 0.1 mM
PMSE, and rapidly centrfuged at 5.000 xg br 5 mio to eliminate
debries. Following denaturation in SOS sample bulfer, proteins were
resolved in a 8% SOS-PAGE and transferred electrophoretically onto
nitrocellulose membranes (Bio-Rad). Membranes were blocked with
5% of fat-free dried milk and incubated with 2 pg/ml of specific antiPKC antiserum (done MC5, Amersham International). Following jocubation with horseradish conjugated anti-mouse antibody, the blots
were developed with an enhanced chemiluminescence detection kit
(Amersham International).

NG F

A PP

2.4. Ot/er c/,e,nieals

Tissue culture 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, Calbiochem (USA). Sphingosine and most other reagents were obtained from
Sigma Chemicals (USA).

Fig. 2. Accumulation ofNGF mRNA in astrocytes. Constant aniounts

of total RNAs were fractionated on ari agarose gel, blotted and hybrid-

3. 1{esults
3.]. PC-PLC enhances Me synthesis of NGF

Cultured glial cels were treated for 24 E with different doses

of exogenously added PC-PLC from Bacillus cereus and NGF
levels were determined in the supernatant media 24 h later.
Results presented in Fig. 1 show a dose-dependent and saturable increase in the extracellular levels of NGF, which become
maximal at 0.5 U/mI of PC-PLC. Doses over U/mI were toxic
to cels. Stimulation of cels with lOO nM of PMA was quanttatively lower than that induced by PC-PLC (Fig. 1).
In order to determine whether PC-PLC Fiad a corresponding
on the pool ofNGF transcripts, total RNA was extracted from
cels pretreated with or without the enzyme. Northern blot
analysis showed that PC-PLC produced a marked increase in

400

200

ized with a radiolabelled NGF cDNA probe. Lane 1: control; lane 2:

EMA lOO nM; lane 3: PC-PLCO.5 U/m; lane4: celis treated with EMA
500 nM for 48 h and te last 24 h with EMA lOO nM; and lane 5: cells
treated with EMA 500 nM for 48 h and Pie Iast 24 h with PC-PLC 0.5
U/ml. Essentially identical results were obtained in two independent
experiments.

NGF niRNA, as compared to controls, indicating that PC-PLC

is able to promote NGF synthesis (Fig. 2, lanes 1 and 3). This
etfect is more potent than that elicited by PMA (Fig. 2, lane 2).
3.2.

The induction of NGF synhesis by PC-PLC is pac/ol>

dependen on PKC
To investigate whether PKC activation is required for the
PC-PLC-triggered induction of NGF synthesis, PKC was

down-regulated in glial celis by pretreating the cdl cultures with

a supramaximal dose ofPMA (500 nM) for 48 E. This treatment
completely removed PKC from astrocytes as determined by
Western blot analysis with a specific monoclonal anti-PKC
antibody (Fig. 3). Interestingly, induction of NGF synthesis by
PC-PLC emerged reduced but not abolished, in celis in which
PKC was down-regulated (Table I), whereas NGF induction

o,
oLA-

o
z

Table 1

EffectofPKC inhibitionanddown-regulationandeffectofsphingosine
on the levels of ceIl-secreted NGF measured by ELISA

No

PMA

pcPLC pcpuc PcPLC PcPLc

Iu/mI
Fig. 1. Effect of increasing concentrations of PC-PLC on the production of NGF. Glial celis were incubated with no additions, PMA (lOO
nM), or PC-PLC (0.1 U/m, 0.25 U/mI, 0.5 U/mI and t U/m). The
medium was collected after 24 h and NGF was quantitied by donhiesite ELISA. Values are means S.D. of 3 independent experiments
assayed in triplicate.
oddtion ~O0nM

O.IU/rnl

O.25U/rnl O.5U/mI

Additions

NGF pg./ml

None
EMA lOO nM

34.68lO
196.51 90

PC-PLC 0.5 U/mI

490.54119
31413
130.3023
32.87 2
171.5027
164.80 l2

EMA lOO nM + SIM 2pM

PC-PLC 0.5 U/ml + BlM 2pM

EMA 500 nM (48 h) + EMA lOO nM
EMA 500 nM (48 h) + PC-PLC 0.5 U/ml
Sphingosine lo pM

The values represent the means S.E.M. of 2 independent experiments assayed in triplicate.

303

ID. Laviada ev al. IFEBS Leliers 364 (1995) 301304

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: cels treated with PMA 500 nM
for 48 h and the last 24 h with PMA 100 nM; and lane 4: cells treated
with 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-regulated cels. 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 was
obtained by thc use of bisindolylmaleimide (BlM), a potent and
higlyspecific inhibitorofPKC[21]. Inhibition ofPKCby BlM
complctcly abolished the Increase ni NGF levels induced by
PMA, whcrcas treatrncnt olcels with BlM only reduced in part

the increase in NOF levels produced by PC-PLC (Table 1).

Hence, PKC seems to be only partially involved in the NGF
inducing response elicited by PC-PLC.

to be coded by a late expression gene. Results also indicate that

PC-PLC acts by at least two diflerent pathways, one dependent

on PKC and another independent of PKC. Wc and othcrs have
previously found that PC-PLC triggers sorne cellular responses,
namely induction of DNA synthesis or activation of nuclear
transcription factor systems by a PKC-independcnt mechanism
[17,24]. It has been described that PC-PLC is coupcd o a
sphingomyelinasc that catalyzes the breakdown of sphingomyclin into ceramide [24]. A second messenger function for the
sphingomyelin 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 a
similar extent than the PKC-independent mechanism elicited
by PC-PLC. TEe effect exerted by sphyngosine may be mediated by its conversion to ceramide, as it has been suggested for
other biological responses mediated by sphingosine [26].On the
other hand, vitamin D3, which induces NGF by a PKC-independent mcchanism, has been shown to elicit an early and
reversible hydrolisis of sphingomyelin with a concomitant generation of ceramide [27]. Two mechanisnis may be therefore
involved in the generation of NGF by astrocytes, one represented by serum and DAG acting through PKC, and anoiher
one induced by vitamin D3 acting through the sphingomyelin
cycle. Our results suggest that thc breakdown of PC by a PLC
may trigger both mechanisms.
ck,,oiledg>,w~,is. The authors thank Drs. M. Guzmn and D. Wion
br Iruitful discussions and critical reading of the manuscript. This wark

3.3. Splz/ugosine mercases l/w produclon of NGF

has been supported by a coopcration program betwecn INSERM

In order to study whether another mechanism of signal

transduction could be involved in the synthesis of NGF, we
determined the effect of sphyngosine on NGF secretion by glial
cels. Results reported in Table 1 show that sphingosine increases the extracellular levels of NGF to he same extent as
PC-PLC does in PKC down-regulated cels.

(France) and CSIC (Spain).

4. Discusson
Despite the recent efforts performed to unravel the mechanisms of NGF induction, the signal-transducing pathways leadng to the synthesis of this neurotrophic protein remain largely
unknown. Many ofIhe agents which are able to stimulatc NGF
synthesis are dependent on PKC activation [9]. Howcvcr, steroids and 1 ,25-dihydroxyvitamin D

3 have been shown to induce

NGF 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 NGF
synthesis does not depend on a uniquc pathway, the diffcrent
putative mechanisms up-regulating NGF synthesis operate by
increasing the steady-statc levels of intracellular NGF mRNA.
in this repor wc demonstrate that a PC-PLC induces the
synthesis of NGF by glial cels by a mechanism bat is partially
dependent on PKC activation. Thc fact that PC-PLC is able to
produce an accumulation of NGF transcripts indicates that at
least part of the stimulatory etTect takes place at he pre-translational level. One of the particular features of thc action of
PC-PLC, alone or in combination with phosphatidate
phosphohydrolase, is that he increase in intracellular DAG
concentration is prolonged, indicating that it may be responsible for long-term cellular responses [13]. Our results are in
agreement with this notion since NGF is generaly considered

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Research report
Induction of nerve growth factor synthesis by sphingomyelinase and
ceramide in primary astrocyte cultures
Ismael Galve~Roperh~,
Amador Haro~, and Ins Daz.Laviadab
Opto. Bioqumica y Biologa Molecular L Facultad de Biologa, Universidad Complutense, 28040 Madrid, Spain
Opto Bioqubuica y Biologa Molecular, Facultad de Medicina, Universidad de Alcal de Henares, 28871 Alcal de Henares,
Madrid, Spain

nene growth factor (NGF) iri response to pro-inflammatory cytokines. To further study
the signaling mechanism involved in this induction of NEW production, the sphingomyelin (SM) pathway was
stuclied. Addition of exogenous neutral SMase (Staphylococcus aureus) or C2-ceramide to primary cultures of
newborn rat cortical astrocytes elicited a dose-response increase of NGF synthesis, with maximal effect at 1
U/ml anil 25 ~tM,respectively. Induction of NGF synthesis by SMase and ceramide was shown to be
independent of classical PKC activity. Intracellular cAMP-raising agents, such as forskolin and 3-isobutyl- 1methylxanthine, partially prevented the SMase- and C2-ceramide-induced secretion of NEW to the ccli
supernatant. PD098059 and apigenin, inbibitors of the mitogen-activated protein (MAP) kinase pathway,
produced a dose-response inhibition of the SMase- and C2-cer-induced release of NGF. This observation points
te the possibility that regulation of NGF synthesis and secretion by the SMase pathway may be mecliated
downstream by the MAP kinase cascade. As a matter of fact, pretreatment of astrocytes with SMase or Caceramide led to an increased phosphorylation of raf-1. Moreover, MAP kinase activity was enhancecl in
Astrocytes synthesize

astrocytes treated with SMase or both ceramicles In conclusion, resulta suggest that the SMase pathway may

control NGF synthesis in the central nervous system, and raise the possibility of an involvement of the MAP
kinase cascade in this procesa. Copyright 1997 Elsevier Science B.V.
Key words: Astrocyte, Cerarnide, Mitogen-activated protein kinase, Nene grnwth factor, Rol- 1, Sphingomyelinase.
Abbreviations: BlM, bisindolylmaleum , cAMP, cyclic AMP; C2-cer, N-acetylsphingosine; FK forskolin; IBMX, 3-isobutyl-1methylxantliine; IL, interleuln; MAP kinase, mitogen-activatedprotein ldnase; NGF, nene growth factor; PMA, 4~-phorhol 123myristate; SM, sphingomyelin; TNF, tumor necrosis factor.

1. Introduction
Within

the last decade sphingolipids have emerged as active participants in the regulation of a wide

range of cellular responses. In particular, sphingomyelin (SM) breakdown has been implicated in the
regulation of cel growth, diflerentiation, transformation and apoptosis. SM is hydrolyzed by
sphingomyelinase (SMase), releasing diffusible ceramides that function as second messengers in
signaling pathways [reviewed in 12, 13, 33, 34]. Ceramides may modulate the activity of different

serine/threonine protein kinases as ceramide-activated protein kinase (CAPK) and protein kinase C
as well as ceramide-activated protein phosphatase
part through the mitogen-activated protein (MAP)

~,

(CAPP) [13]. Downstream signaling occura at least in

kinase cascade and NFKB translocation [2, 13, 29, 33,

38]. An increasing number of ceil-surface receptors are currently being show to generate signals that

trigger SM breakdown. Accumulation of ceramides derived from SM hydrolysis has been described to
occur in response to different extracellular agents including la,25-dihydroxyvitamin D

3, tumor necrosis

factor a (TNFa), interleukin-1[3 (IL-1[3), IL-2, and interferon-y [13, 33, 34].

Corresponding author. Fax: +34 1 885 45 85 E-mail: bqidm~bioqui.alcala.es

1. Galve Roperh et al.

TNFa and ILs are pivotal mediators of inflammation and immune responses in the central nervous
system (CNS). Such responses are followed by the rapid activation of brain resident macrophages and
subsequent activation of astrocytes, that constitute the most abundant non-neural cels in the CNS [23].
Astrocytes play an important role in a wide range of functions which are essential for adequate
development, functionality, and pathological process of the CNS [3]. It is now well established that
astrocytes are involved in the inflammatory response in the brain like that occuring in multiple sclerosis
and septic shock [22]. One of the responses of reactive astrocytes during inflammatory process and after
injury is the synthesis and release of neurotrophic factors such as nerve growth factor (NGF) [4, 6, 40].
NGF production by glial cels during such circunstances may limit the extent of neuronal loss and
promote regenerative processes to restore neuronal function [28, 39]. NGF synthesis by astrocytes is a
highly regulated process which includes complex interactions among different signaling pathways. Proinflammatory cytokines, IL-113 and TNFa are well known stimulators of NGF synthesis [14, 27, 28]. On
the other hand, lipopolysaccharide, the molecule responsible for the iniflammatory response during the
septic shock, is also a potent inducer of NGF synthesis and secretion by astrocytes and brain
macrophages [11, 21]. It has also been described that la,25-dihydroxyvitamin Da stimulates NQF
synthesis and release by cultured glial celis [25]. As stated aboye, both cytokines and la,25dihydroxyvitamin D3 have been reported to induce ceramide accumulation [13, 26]. We have previously
described the involvement of a phosphatidylcholine-phospholipase C (PC-PLC) in the regulation of NGF
synthesis by astrocytes [19], which in turn has been shown to mediate TNFa activation of NF-KB
through SM breakdown [30]. Therefore, it seems interesting to study the possible function of SM
breakdown and ceramide release in the regulation of NGF synthesis by astrocytes.
We report here the stimulatory effect of exogenous addition of both, neutral SMase and cel-permeable
ceramides on NGF synthesis and secretion in primary cultures of newborn rat astrocytes. The
characteristics of this NGF regulatory pathway were studied with special emphasis on the possible
effects on the MAP kinase cascade and its crosstalk with other signal transduction pathways.
2. Materials and methods

2.1. Materjais
Tissue culture plastic wares and fetal calf serum (FCS) were from Nunc (Denmark). Culture media
was from Biowhitakker (Belgium). Neutral SMase (5. aureus), 3-isobutyl-l-methylxanthine (IBMX) and
4[3-phorbol 12j3-myristate (PMA) were from Sigma Chemicals (USA). Bisindolylmaleimide OF 109203X
(BlM), forskolin (FK) and cel permeable ceramide analogs were from Calbiochem (USA). 32Pi and
DMEM Pi free were from ICN Pharmaceuticals Inc. (USA). Anti-NGF monoclonal antibodies were from
Boehringer Manheim (Germany). Anti-raf-1 polyclonal antibody was from Santa Cruz Biotechnology
(USA), and anti-rabbit agarose-linked IgO from Transduction Laboratories (UK). p42/p44 MAP kinase
peptide substrate and reagents for kinase assay were from Amersham (U.K.).
2.2. Prinary cultures ofctstroglial cells
Cortical astrocytes were derived
from 1-2onday
od plates
rats and
culturedcoated
as previously
described
[19]. Celis
4 cells/cm2
plastie
previously
with 5 gg/ml
dl-polyornithine
were
seeded
at awere
density
of 3x10
in
water.
Cels
cultured
fox 3 weeks in basal medium consisting of a mixture of Dulbeccos modified
Eagle medium (DMEM) and Hams F12 (1:1, y/y), with 0.66% glucose, 5 pg/ml streptomycin, 5 U/ml
penicilhin, and supplemented with 10% (FCS). The primary cultures consisted of 95% astrocytes as judged
by immunocytochemical staining of glial fibrilary acidic protein.
Three days before the experiment, the FCS containing medium was removed and cels were transferred
to a chemically-defined medium consisting of serum-free basal medium supplemented. with 25 pg/ml
insulin, 50 gg/ml human transferrin, 20 nM progesterone, 50 pM putrescine, and 30 nM sodium selenite.

RNA analysis ami enzjane-linked inmunosorben assay (ELISA)

Afler the indicated times of treatment RNA extraction was carried out according te the LiC]/urea method.
2.3.

Northern blot analysis was algo performed by standard procedures. Glyoxal-treated RNA was fractionated in
agarose gela, transferred te Hybond-N membranes by capillary blotting, and hybridized serially with a ~P-labelled
probe of mouse NGF cDNA. The NOF probe was te 917 bp NGF DNA cloned by Scott et al. [31].

1 n duction of nerve growth factor synthesis by sphingomyelinase and ceramide in primary astrocyte cultures

Standardization of RNA loading was routinely controlled by hybridization of the blots with amyloid
precursor protein cDNA probe [32]. Densitometric analyses were performed with Phosphormager 445 SI
(Molecular Dynamics).
Fox extracellular NGF determination, ceil supernatants were collected 24 hours after treatment as
described in the text, diluted in one volume of phosphate buffer salme (PBS) containing 0.1% Tween 20 and
0.5% gelatin, and conserved frozen until quantitation. NGF released by the celis was assayed in triplicate,
by a double-site ELISA, using a monoclonal anti-NGF antibody, coupled or not to ~-galactosidase according
to an experimental protocol described before [19].
2.4.

Raf-1 irnrnunoprecipitation

Immunoprecipitation was performed as described previously [10]. Briefly, cels cultured in 57 cm2
dishes, were transferred to chemically defined medium three days before the experiment. The medium
was replaced by Pi-free DMEM and incubated for 4 hours with 100 j.tCi 32Pi. Cels were stimulated as
described in the text. Lysates were subsequent obtained by treating cels with a buffer containing 50mM
Tris pH 7.5, 2 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1 mg/ml bovine serum albumin, and 150 mM
NaC. Immunoprecipitation was carried out by incubation with 7.5 pg/ml of anti-raf-1 polyclonal
antibody and precipitation with anti-rabbit agarose-linked IgO. Phosphorylation was determined in the
immunoprecipitates by SDS-PAGE and autoradiography of the gels. Gels were previously stained with
Coomasie blue in order to verify the loading of the geis. Autorradiographic Fuji-films were subjected to
densitometric analysis using the Sigma-Gel program.
2.5. MAP ktase assay
Astrocytes were incubated for 15 mm at 370C with agents shown in Table 2, and the reaction terminated
by washing with ice-cold PBS and adding 500 pl of lysis buffer containing 10 mM Tris (pH 7.4), 150 mM
NaC, 2mM EGTA, 2 mM DTT, 40 pg/ml digitonin, 1 mM orthovanadate, 1 mM PMSF, 10 pg/ml
leupeptin, and 10 pg/ml aprotinin, at 40C. Cellular debris were precipitated at 25000 x g fox 20 mm and
MAP kinase activity was measured in the supernatant. Extracts (15 pl) were then assayed by adding 10
pl. of the substrate buffer (containing 6 mM substrate peptide, 75 mM Hepes, 300 pM sodium
orthovanadate, and 0.05% sodium azide, pH 7.4), and 5 pl of ATP buffer (containing 0.3 mM [y-32P]ATP
(300 pCi/mi) and 90 mM MgCl2). ASter 30 mm incubation at 30~ C., reaction was terminated by adding 10
pl of 300 mM orthophosphoric acid. Thirty pl of each sample was spotted onto phosphocellulose discs,
washed two times fox 2 mm in 1% acetie acid and then twice for 2 mm in distilled water. Radioactivity of
each disc was determined by scintillation counting.
3. Results
3.1. Enhauced synthesis an.d secretion. of NGF by exogeuous addtion of neutral SMase and C2-cerarnide
In order to study the possible involvement of the SMase pathway in the regulation of NGF synthesis,
primary cultures of astrocytes were exposed to increasing concentrations of neutral SMase. An increased
secretion of NGF to the cel supernatant was observed at 250 mU/ml with maximal activation at 1 U/ml
(15-fold increase) (Fig. la). Time-course experiments showed that maximal concentration of NGF on the
supernatant peaked at 72 h of treatment (Fig. 2). In order to know whether the effect observed with
SMase was due to the release of the second messenger ceramide, the synthetic cel-permeable analog Nacetylsphingosine (Ce-cer) was used. Treatment of cels with C2-cer also enhanced NGF synthesis and
secretion from cels, with maximal stimulation observed at 25 pM C2-cer (Fig. ib). The specificity of C
2cer effect was supported by the fact that Ce-dihydroceramide, an inactive analog, had no effect on
secreted NGF at the same concentrations (Table 1).
Northern blot analyses were used to test whether the content of NGF mRNA in treated cells was
elicited in concert. Fig. 3 shows that after 24 h of treatment, NGF mRNA levels were higher in both
SMase and ceramide-treated cels. Therefore, it seems that ceramide-induced NGF secretion is also
accompanied by an increase in the mRNA levels of the neurotrophic factor. In fact, treatment of cels in
the presence of actinomycin O and cycloheximide, inhibitora of DNA transcription and protein synthesis,
respectively, abrogated increased levels of NGF in the cel supernatant elicited by SMase and ceramide
(data not show).

1. Oalve-Roperh et at.
fico
700
000

500

O0

,oo
200

loo
1
000

005

020

025

0.50

1.00

SMcse ti/ini

~oo
t,o
100

:100
250

0,

e
z

0.

~5o

10<3

Jo

o
0

26

50

C2.eer hM

Dose-response induction of secreted NGF by exogenous neutral sphingomyelinase aud C2-ceramide. Primary cultures of
astrocytes were stimulated with increasing concentrations of SMase (Fig. la) and N-acetylsphingosine (Fig. ib) for 24 h.
Extracellular NGF was assayed with a double-site ELISA. Data are meana S.D. from three independent experiments. Asterisks
indicate significant differences from the corresponding controis (* P< 0.05, ** P<O.Ol, by Students t-test>.
Fig. 1

3.2.

Signalingpathways responsible for tire induction. of NGFsecretion by cerarnide

To further investigate the mechanism mediating ceramide regulation of NGF synthesis, we investigated
if Ca2~ and diacylglycerol-dependent PKC was required for ceramide action. We have previously
described that the use of bisindolylmaleimide (BlM), a potent and highly specific PKC inhibitor,
completely abolished 4~-phorbol 12~3-myristate (PMA) induced increase of secreted NGF [19]. Treatment
of cultured astrocytes with 2 MM BlM slightly inhibited SMase-induction of NGF secretion, whereas C
2cer-induction of NOF secretion was unaffected (Table 1). This observation points to a mainly PKCindependent mechanism for induction of NEW by ceramide.

The study of a possible involvement of the cAMP signaling pathway was undertaken using the
diterpene forskolin (FR), a well known activator of adenylyl cyclase, and 3-isobutyl-l-methylxantine
(IBMX), a phosphodiesterase inhibitor. FR and IBMX alone failed to increase NGF secretion (data not
shown), whereas in the presence of SMase or CQ-cer they partially antagonized the stimulatory effect of
the latter (40% and 60% inhibition respectively) (Table 1). Thus a regulatory crosstalk among the SMase
and cAIVIP pathways seems to exist. The fact that intracellular cAMP raising agents have been described
as inhibitors of the MAP kinase cascade [18, 35, 37], and the possible involvement of tIte MAP kinase
cascade on the ceramide-activated signal transduction process [29], prompted us to investigate tIte role
of the MAP kinase cascade on the ceramide-inducted stimulation of NOF production.

1 nduction of nene growth factor synthesis by sphingomyelinase and ceramide in primary astrocyte cultures

600

3000

2500
400

2000

300
ej

e isco
2
ha
a

200

1000
100

500

o
0

20

40

60

50

100

120

140

150

time Cicure)
Fig. 2 Time-course of NGF secretion after treatment with exogenous neutral sphingomyelinase and C2-ceraniide. Primary
cultures of astrocytes were treated with vehicle alone (e), maximal dose 1 U/ml of SMase (U)(Ieft axis) and 25 gM C2-cer (V)(right
axis). At the indicate times ceil-secreted NGF was assayed. Data are the means S.E.M of two independent experinients with
determinations in triplicate.

3.3. Involvernen,t of tire MA.P kinase cascade iii tire inducton. of NGFsecretion, by cerarnide
To investigate tIte poasible role of the MAP kinase cascade in ceramide action, experiments with
PD098059 or apigenin were conducted. PD098059 has been described as a selective inhibitor of MAP
kinase kinase (MEK) [8]. It binds to unactivated MEK preventing ita activation by raf-1 or MEK kinase
[1]. Apigenin is an inhibitor of tIte MAP kinase cascade, probably by acting at tIte raf-1 kinase level [17].
Treatment of primary astrocytes with 100 MPD098059 abolished tIte induction of NGF secretion
elicited by SMase and C2-cer (Table 1). TIte dose-response curve of inhibition showed an ICso around 20
pM (Fig. 4A). Treatment of cels with 25 pM apigenin prevented the SMase and C2-cer inducted secretion
of NOF (65% and 85% inhibition respectively) (Table 1), the ICso value is approximately 10 .tM (Fig. 4B).
Therefore, data indicate that the IVIAP kinase cascade may be implicated in the regulation of NGF
synthesis by SM hydrolysis.
3.4. Cera,nide increases tire pirospiro rylation extent of raf 1 an MAP knase actiuity
To further study tIte intracellular effects of ceramide, experiments were undertaken to study the
phosphorylation state of raf-1. Immunoprecipitation of raf-1 from 32P-labelled cefls is shown in Fig. 5.
Short treatment of astrocytes with SMase lead to an increased phosphorylation of raf-1, whereas C2-cer
failed to induce raf-1 phosphorylation. In order to asaes the role of endogenous ceramides in raf-l
regulation we employed the more physiological ceramide analog N-octanoylceramide (Ca-cer). Ca-cer
treatment of cels resulted in an enhanced phosphorylation of raf-1, similar to that elicited by SMase
(Fig. 5). SMase and Cs-cer-induced phosphorylation was still detectable after 30 minutes of atimulation
(data not shown). Changes in the phosphorylation state of raf-1 will result changes in ita kinase activity.
This observation thus confirma previous reports about tIte role of ceramide in raf-1 phosphorylation [2,
38]
MAP kinase activity was meassured as phosphorylation levels of a highly selective peptide substrate
fox p42/p44 MAP kinase [5]. Astrocyte treatment with C2-cer, Cs-cer and SMase evoked MAP kinase
activation (Table 2). Activation elicited by SMase was luigher tItan that elicited by both ceramide
analogs. Specificity of MAP kinase activation was corroborated by the lack of effect of the inactive C
2dihydroceramide (Table 2). Moreover pretreatment with the IVIAP kinase cascade inhibitor

1.

Gatve-Roperh et al

Table 1
Addition
None
SMase
BlM + SMase
FR + SMase
IBMX + Smase
API + SMase
PD 098059 + Smase
02-cer
BlM + C2-cer
FK + C2-Cer
IBMX + C2-Cer
API + C2-Cer
PD 098059 + C2-Cer
C2-dihydrocer

pg NGF/ml
31.7
588.0
509.4
369.3
355.7
205.5
60.7
383.0
363.2
154.7
157.4
62.8
43.8
35.0

8.9
66.4
43.5
87.0
34.7
32.7
17.0
46.8
40.3
67.8
44.4
19.9
5.6
17.0

(n5>
(n3)
(n3)
(n3)

a
a
a, b
a, 1,

(n3) a, b
(n3) b
(n4) a
(n=3) a
(n4) a, c
(n4) a, c
(n4) c
(n3) c
(n3)

Table 1. Effect of different agenta jo sphingomyelinase and C2-cer induction of NGF

secretion. Primary cultures of astrocytes were incubated with the indicated agents for 24
h. 2 kM BlM, 10 gM FK, 100 gM IBMX, 25 pM API and 100 pM PD 098059 were added 1
hour prior to 1 U/mI SMase or 25 ~M C2-cer stimulation. Data are the means 5.D. of
the number of independent experimenta indicated in each case. Statistical analysis was
performed by Students t-test, significant differences are indicated: a, P.c0.01 versus
incubations with no addition; b, P<o.01, versus incubations with SMase alone; c, P.c 0.01,
versus incubations with Ctcer alone).

NQP

e e

APP~~S

fl

Fig. 3 Northern blot anatysis of NGF mRNA accumulation. Lane 1, untreated cels; lane 2,1 U/ml SMase; lane 3, 25 ~tMC2-cer,
After 24 h incubation of Northern blot was performed as described in Matherial and Methoda with NGF and API cDNA probes.
Essentially identical results were obtained lo three independent experiments.

PD098059,.counteracted ceramide induction of MAP kinase activity (Table 2). These results support
the notion that signal transduction triggered by SM hydrolysis may activate MAP kinase and point to
a role of tIte MAP kinase cascade in tIte regulation of NGF secretion by the SM pathway.
4. Discussion
The present report shows that exogenous addition of neutral SMase or C2-cer, a cel permeable analog
of ceramide, induces a dose-response and time-dependent stimulation of NGF synthesis and secretion.
The effect of added SMase indicates that endogenously produced ceramides may also be able to induce
NGF synthesis and corroborates the specificity of the ceramide analog action. The fact that increased

Induction of nerve growth factor synthesis by sphingomyelinase and ceramide jo primary astrocyte cultures

700

con
500

1 so
ir;
a
z

300

200
100

o
0

20

.40

PO

00

090059

00

tOD

I~JM]

ca
600-

500~

400

0-

200
200

Co
o
0

lO

15

20

25

[API) iM

Fig. 4 Dose-response inhibition by PD 095059 and apigenin of sphingomyelinase and C2-ceramide induction of NGF secretion.
Primar> cultures of astrocytes were incubated with 1 U/ml SMase (@) and 25 gM C2-cer (O) and the indicated doses of PD
098059 (A) or apigenin (13) for 24 h., nad secreted NGF subsequent assayed. Data are the meana S.E.M. of two independent
experimenta, with determinations in triplicate.

NGF protein secretion to the cel supernatant was accompanied by an increase in NGF mRNA levels,
together with the data obtained with transcription and transation inhibitors, supports the notion that
increased NGF output is tIte result of an augmented synthesis of NGF.
Regulation of NGF production by a number of agents in primary astrocytes has been shown to be
dependent on PKC activation [7, 24]. However, IL-143 and vitamin Da induce NGF production by
mechanisms independent of PKC activity [25, 28]. These agents are also well known to exert some of
their effects through tIte SM pathway and tIte generation of ceramide [13, 26, 33]. It is wortIt noting that
lipopolysaccharide, which is able to mimic sorne of the effects of ceramide [16, 36] is also a PKCindependent inductor of NGF secretion [11]. We Itave previously reported that PC-PLC is able to
enhance NGF synthesis by primary astrocytes by a signaling mechanism which is only partially
dependent of PKC [19]. PC-PLC action, may in turn be coupled to ceramide generation [30]. In fact,
recent results show that short treatment of CG glioma cels with PC-PLC lead to tIte translocation and
phosphorylation of PKC ~ [10], which is one of the molecular targets of ceramide [20]. Orn- results
indicate that ceramide induction of NGF in primary astrocyte cultures is independent of PKC. Hence the
SM pathway may represent one of tIte main PKC-independent signaling pathways leading to the

regulation of NGF production by glial cels. TItis is in line with previous reports showing tIte
rnvolvement of the SM pathway in the induction of IL-6 synthesis, inhuman astrocytoma cels [9]. Raf-1
is a member of tIte MAP kinase cascade that is activated upon cellular stimulation with a number of
effectors as pro-inflammatory cytokines [2]. The precise rnechanism by which raf-1 is

1 Galve-Roperh et al.

v~

u:

1%
a

32Plabelled celia were atimulated for 15 minutes with the indicated agents and raf-l was,
Fig. 5 Immunoprecipitation of raf- 1.
subsequently immunoprecipitated with a polyclonal anti-raf-1 antibod>. Immunoprecipitates were subjected to SDS-PAGE and
autoradiograph>. Densitometrie data are means S.E.M. of two independent experiments.

Table 2
Addition

pmol/min/mg
98.6 22.6
555.5 17.7 a
282.9 11.5 a

None
SMase
C2-cer
Ca-cer

290.1

2.0 a

C
2-dihydrocer

103.6 5.9

PD 098059 + C2-cer

125.3
155.9

PD 098059 + Cs-cer

9.6 b
8.4 c

Table 2. Effect of ceramide analoga and sphingomyelinase on MAP kinase activity. Primar> cultures of astrocytes
were incubatod with 1 U/mI SMase, 25 vM C2-cer, or 25 ~tM C5-cer for 15 mm. 100 ~M PD 098059 was added 1 hour
prior to stirnulation. Data, expressed as pmol phosphate transferred /min/mg prutein, are the means S.D. of three
independent experimenta performed in triplicate. Statistical analysis was performed by Students t-tost, significant
differences are indicated: a, P.cO.O1 versus incubationa with no addition; b, P.co.O1, versus incubationa with C2-cer
alone; c, P.c 0.01, versus incubatinas with Cs-cer alone).

activated is as yet uncertain, but it is generally agreed that changes in its state of phosphorylation
produce changes in its kinase activity. Recent reports indicate that raf-1 may be phosphorylated by TNF-

activated CAPK, increasing ita activity towards MEK [38]. Therefore it seema that the SM pathway
could be linked with the MAP kinase cascade, probably at tIte level of raf-1, through tIte activation of the
CAPK [2, 29, 38]. Our resulta indicate that tIte regulation of NGF syntItesis by SMase and ceramide may
depend on the activation of the MAP kinase cascade. It should be noted that SMase-

Jaduction of nerve growth factor synthesis by sphingomyelinase and ceramide fa primar> astrocyte cultures

induction of NGF secretion was less sensitive to MAP lcinase inhibition tItan C2-cer-induction of NGF

secretion, in concordance with its greater effect on MAP kinase activity.

It has been described that hyperphosphorylation of raf-1 induced by intracellular cAMP-raising agents
leads to the blockade of tIte MAP kinase cascade [37]. A similar inhibitory effect of tIte MAP kinase
cascade by cAMP-increasing agents has also been described in astroglial cels stimulated with growth
factors and LPS [18, 35]. Results in tIte present report show that treatment with FK and IBMX abolishes
half of the ceramide action on NGF production by astrocytes. Thus, tIte existence of an inhibitory
crosstalk between the SM and the cA1VIP pathways appears to exist also in the regulation of NGF
synthesis by ceramide.
The possible involvement of tIte IvIAP kinase cascade as the signaling mechanism underlying ceramide
generation is enforced by the observed increase in raf-1 phosphorylation and MAP kinase activity as the
result of short-term treatment with SMase or ceramide analogs. Short-chain ceramide analogs are
supposed to be biologically more active than their long chain equivalents likely due to the increased
solubility of the former. However, long chain ceramides have been described to be more potent than
short chain ceramides in activating raf-1 [15].This is in concordance with our results showing an
rncreased phosphorylation state of raf-1 from Cs-cer-stimulated treatment of cells, whereas C2-cer failed
to induce such effect. Phosphorylation of raf-1 by the ceramide analog Ca-cer has been described also
previously by other authors [38]. Thus, it seems plausible that ceramide action on NGF expression may
be mediated by activation of the MAP kinase cascade at the level of raf-1 and p42/p44 MAP kinase.
In conclusion, we report here tIte role of the SM pathway in tIte regulation of NGF synthesis and
secretion by cultured rat astrocytes. The action of ceramide on NGF production seems to be partially
mediated by activation of the MAP kinase cascade. The regulatory action of ceramide may be related to
previously described effects of pro-inflammatory cytokines such as IL-ljB and TNFa, aud is therefore of
maximal importance in our understanding of the processes occurring during inflammatory brain lesions
and certain neuropathological disorders.
Acknowledgments
The authors wisIt to thank Dr. P. Brachet, and members of his laboratory for sharing their
methodologies for Northern blot, Dr. M. Guzmn and TAS. Branton for their advice and valuable
assistance. Measurement of ELISA were performed at the Plan Nacional de Prevencin de la
Minusvala, Hospital Gregorio Maran, under the supervision of Dr. E. Duln. This work was
financially supported by Comisin Interministerial Ciencia y Tecnologa (SAF 96-0113), Fondo de
Investigaciones Sanitarias (FIS 97/0039-01), and Accin Integrada Hispano-Francesa (N 96/154).

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[3] Bhat NR. Signal transduction mechanisms in glial celis. fleo. Neurosci. 17 (1995) 267-284.
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[8] Dudley D.T., Pang L., Decker S.J., Bridges AS. and Saltiel AR. A synthetic inhibitor of the mitogen-activated
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[10] Galve-Roperh 1., Malpartida J.M., Haro A. and Diaz-Laviada 1. Addition of phosphatidylcholine-phospholipase
C induces cellular redistribution and phosphorylation of protein kinase C 4 in CG glial cels. Neurosci. Letters 219
(1996) 68-70.
[11] Galve-Roperh 1., Malpartida J.M., Haro A. BrachetP. and Ojaz-Laviada 1. Regulation of nerve growth factor
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[12] Hannun Y. A., Obeid L. and Obaibo 0.5. Ceramide. A Novel second messenger and lipid mediator. In Bel R.B.
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[14] Hattori A., Hayashi E and Kohno M. Tumor necrosis factor (TNF) stimulates the production of nene growth
factor in fibroblasts via the 5S-kOa type 1 TNF receptor. FIJES Letters 379 (1996) 157-160.
[15] Huwiler A., Brunner J., Hummel R., Vervoordeldonk M., Stabel 5., Van der Bosh H. and Pfeilschifter J.
Ceramide-binding and activation defines protein kinase c-raf as a ceramide-activated protein kinase. Proc. Nat?.
Acad. Sci. USA 93 (1996) 6959-6963.
[16] Joseph C.K., Wright SO., Bornmann W.G., Randolph J.T. Kumar ER., Bittman R., Liu it and Kolesnick R.
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[18] Kurino M., Fukunaga K., Ushio Y aud Miyamoto E. Cyclic AiMP inhibits activation of mitogen-activated protein
kinase and cel proliferation in response to growth factors in cultured rat cortical astrocytes. J. Neurochem. 67
(1996) 2246-2255.
[19] Laviada 1.0., Baudet C., Galve-Roperh 1., Naveilhan P. and Brachet P. Phosphatidylcholine-phospholipase C
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[20]Lozano it, Berra E., Municio MM., Diaz-Meco MT., Oominguez 1., Sanz L., and Moscat J. Protein kinase C 4
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[21]Mallat M., Houlgatte R., Brachet P. and Prochiantz A. Lipopolysaccharide-stimulated rat brain macrophages
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[22]Merril SE. and Benveniste EN. Cytokines in infiammatory brain lesions: helpful and harniful Trends in
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[23]Montero- Menei C.N., Sindji L., Garcion E., Mego M., Couez O., Gamelin E. and Darcy F., Early eventa of the
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10

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11

45

Resultados y Discusin

RESULTADOS Y DISCUSIN
Regulacin de la secrecin de NGF por la PC.PLC
El estudio de la accin de la PC-PLC en la regulacin de la sntesis de NGF se
justifica

por

dos importantes

razones.

Resultados

preliminares pusieron

manifiesto la importancia de la accin de esta fosfolipasa

de

C en relacin a otras de

diferente especificidad por el substrato. As, aunque la PI-PLC y PLD tienen un efecto

inductor en la secrecin de NGF, este es cinco veces menor que el mostrado por la PCPLC. En este sentido, el sistema de transduccin de seales acoplado a la hidrlisis
de fosfoinostidos, presente en astrocitos en cultivo primario, no parece ser uno de los
mecanismos de regulacin

de expresin del NGF (Jehan et al., 1995). A estas

observaciones se suma el hecho de que la hidrlisis de PC podra ser importante en la

generacin de un aumento de larga duracin en los niveles intracelulares de DAG

(Asaoka et al., 1996; Nakamura et al., 1996). Este efecto
permitira explicar ciertos mecanismos de regulacin

de la hidrlisis de PC

celular a largo plazo como el

crecimiento, la diferenciacin celular, o la expresin del gen de NGF, considerado de

expresin

tarda.

La induccin de NGF por la accin de la PC-PLC exgena es parcialmente

dependiente de la actividad de cPKCs, dependientes de Ca2~ y DAG, como pone de
manifiesto el efecto inhibidor de la bisindoleilmaleimida OF 109203X (BlM). Este
efecto coincide con el observado al disminuir los niveles de PKC por exposicin

prolongada de los astrocitos a steres de forbol (downregulation). Se observa

adems que la SF, producto del metabolismo de las ceramidas por una ceramidasa
(Nikolova-Karakashian et al., 1997), es capaz de inducir la secrecin de NOF en igual
extensin que la PC-PLC en clulas preincubadas con BlM o con los niveles de PKC
disminuidos por el efecto de downregulation causado por steres de forbol. Estas

observaciones coinciden con evidencias experimentales en otros sistemas celulares,

que implican a la PC-PLC como posible activador de la va de sealizacin de la

SMasa-ceramida, responsable de la generacin de ceramidas (Schtze et al., 1992;

Mller et al., 1994; Wiegman et al., 1994). Se plantea por tanto la hiptesis de que la
accin de la PC-PLC podra activar la sntesis de NGF por un doble mecanismo:
generacin de DAG y activacin de cPKCs, y por otro activacin de la hidrlisis de
esfingolipidos.

Para estudiar el efecto de la va de la SMasa-ceramida en la sntesis de NGF

utilizamos el anlogo permeable de ceramida, C2-ceramida y el tratamiento con
SMasa exgena de

Staphyococcus aureus. La estructura de las ceramidas permeables

Resultados y Discusin

46

con una cadena adilada ms corta facilita el paso de estos compuestos al interior
celular (Biewlaska et al., 1992). Por otra parte el tratamiento con SMasa exgena
genera ceramida endgena por la hidrlisis de la SM situada en la cara externa de la
membrana plasmtica (Olivera et al., 1992). Aunque se ha descrito la existencia de
ciertas diferencias en las respuestas inducidas por estos anlogos y las ceramidas de
origen endgeno (Andrieu et al., 1996; Zhang et al., 1997), la utilizacin de este tipo
de aproximacin experimental se considera vlida para el estudio de la regulacin de
numerosos procesos celulares por la va de sealizacin de la SMasa-ceramida
(Fiebich et al., 1995; Yao et al., 1995; Casaccia-Bonnefil et al., 1996; Cuvillier et al.,
1996; Zhang et al., 1997).
Regulacin de la secrecin de NGFpor diferentes componentes de la va de la SMasa
Uno de los primeros interrogantes que se plantean al estudiar el papel deJa
va de la SMasa-ceramida en la regulacin de la sntesis del NGF es el de determinar
cual o cuales de sus intermediarios pueden ser responsables del efecto regulador de la
sntesis de NGE. La SF induce un aumento de aproximadamente cinco veces en los
niveles extracelulares de NGF respecto de las clulas control. Sin embargo, el
derivado fosforilado, SF-1-P, potencial mediador activo de la SE (Spiegel et al., 1997)
carece de efecto en la secrecin de NGF (resultados no incluidos). Por otro lado, las
ceramidas son inductores significativamente ms potentes de la secrecin de NGF

que la SF. Estas observaciones hacen suponer que el efecto de la SF podra estar
mediado por su paso a ceramida (Goldkorn et al., 1991), sin descartar un efecto
directo de la propia SE.
La diferencia observada entre ceramida y SE en la regulacin de la sntesis de
NGF, supone una aportacin al debate sobre la significacin biolgica que tiene la
induccin de NGE por los mensajeros derivados de esfingolipidos. As, se ha postulado
que mientras que la generacin de ceramida es responsable de respuestas biolgicas
como la diferenciacin celular, supresin del crecimiento o apoptosis (revisado por
Hannun, 1996; Testi, 1996), la SE y SE-1-P parecen ser inductores de respuestas
mitognicas (Olivera y Spiegel, 1993; Spiegel et al., 1997). En este sentido, se ha
propuesto recientemente que citoquinas pro-inflamatorias y factores de crecimiento
favorecen la acumulacin de uno u otro tipo de mensajeros en funcin de la diferente
regulacin que ejercen sobre la actividad SMasa, ceramidasa, y SE quinasa (Coroneos
et al., 1995; Nikolova-Karakashian et al., 1997), de manera que el balance entre los
dos tipos de esfingolpidos podra regular el equilibrio entre diferenciacin/muerte

Resultados y Discusin

47

celular y divisin/proliferacin celular. Se ha demostrado recientemente que, en

efecto, la SF-1P es capaz de revertir la muerte celular programada inducida por
ceramida (Cuvillier et al., 1996). Si este esquema es vlido en clulas gliales, la
sntesis de NGF estara asociada a los procesos de diferenciacin celular e inhibicin
del crecimiento, y por tanto estimulada y coordinada con respuestas de estrs celular.
La estimulacin por ceramidas no resulta aparentemente citotxica
La generacin de ceramidas adems de inducir apoptosis en numerosos
sistemas celulares, parece ser uno de los mecanismos responsables de la citotoxicidad
de citoquinas pro-inflamatorias como el TNFcx (llannun, 1996; Testi, 1996). Por todo
ello, es importante comprobar que la incubacin de las clulas con los diferentes
moduladores utilizados no es citotxica. Esto se comprob cualitativamente dada la

ausencia de clulas despegadas del soporte de cultivo despus de los tratamientos de

24 horas. Adems, se analiz la viabilidad celular tras las incubaciones mediante el
ensayo de MTT, basado en la reduccin del anillo de tetrazolio por la actividad de
las deshidrogenasas mitocondriales. En el intervalo de concentraciones utilizado de
C2-ceramida slo las clulas tratadas con C2-ceramida 50 ~.iMpresentan una menor

viabilidad celular, que coincide con una disminucin en los niveles de NGF
extracelulares. La ausencia de efecto citotxico de las ceramidas sobre cultivos
primarios de astrocitos, concuerdan con resultados de otros autores que evidencian
que los astrocitos son resistentes a la muerte celular inducida a travs de la ruta de
la SMasa-ceramida (Casaccia-Bonnefil et al., 1996a;b).
Regulacin de la sntesis y secrecin de NGF por el aumento en los niveles
intracelulares de ceramida
Los resultados expuestos ponen de manifiesto un significativo aumento en la
produccin de NGF inducido por el anlogo permeable de ceramida, C2-ceramida, as
como por accin de SMasa exgena. La induccin de NGF por la generacin de
ceramidas es independiente de la activacin de cPKCs dependientes de Ca2~ y DAG,
como pone de manifiesto la ausencia de inhibicin por BlM. La induccin de la
sntesis de NGF estimulada por ceramidas se inhibe parcialmente por el aumento de
los niveles de cAMP. El efecto inhibidor del cAMP sobre la induccin de NGF Coincide
con el observado para otros inductores de NGF como el suero, PMA o TGF-fBl (Jehan
et al., 1993;1995; Han et al., 1994).

48

Resultados y Discusin

Implicacin de la cascada de MAPK en la sntesis de NGF inducida por ceramidas

Los resultados obtenidos muestran como la secrecin de NGF inducida por
ceramidas se inhibe por apigenina y el inhibidor sinttico PD 098059, lo que sugiere
que la induccin depende de la activacin de la cascada de MAPK (Alessi et al., 1995).
En efecto, tanto los anlogos permeables de ceramida como la SMasa exgena
incrementan la actividad p42/p44 MAPK en cultivos primarios de astrocitos,

confirmndose la especificidad de esta activacin por el inhibidor PD 098059. El

estudio detallado de la cintica de activacin de MAPK por C2- y C8-ceramida mostr
que en todos los tiempos ensayados la activacin de MAPK es mayor en clulas
tratadas con C8-ceramida que con C2-ceramida (datos no incluidos). Estas
observaciones confirman que los anlogos de ceramida de cadena acilada ms larga
son mejores activadores de Raf-1 (Huwiler et al., 1996). La activacin de MAPK por

ceramidas se mantiene al menos durante 60 minutos, un efecto sostenido que apoya

un posible papel de este sistema de fosforilacin en la regulacin celular a medio y
largo plazo, como la sntesis de NGF.
El papel de la activacin de MAPK por ceramidas para explicar algunos de sus
efectos biolgicos como la apoptosis, es objeto de controversia (Sasaki et al., 1995;
Coroneos et al., 1996; Pyne et al., 1996; Brenner et al., 1997). Sin embargo, es de
destacar que los anlogos permeables de ceramida inducen un aumento de la
actividad MAPK que es del mismo orden de magnitud que la activacin de MAPK
producida por otros inductores de la sntesis de NGF como TNFct o LPS (Mallat et al.,
1989; Hattori et al., 1996).
Estudio detallado del posible mecanismo de activacin de MAPKpor ceramidas
La activacin de MAPK por ceramidas puede explicarse a travs del efecto
activador de estos lpidos sobre Raf-1 (Yao et al., 1995; Huwiler et al., 1996). La
estimulacin de los astrocitos con C8-ceramida o SMasa, produce un aumento en los

niveles de fosforilacin de la protena Raf-1 inmunoprecipitada, lo que explicara la

activacin observada de MIAPK. Puesto que la activacin de Raf-1 se asocia con una
translocacin a membrana plasmtica (Stokoe et al., 1994), se estudi la posible
translocacin de Raf-1 tras la estimulacin por ceramidas. Como se muestra en la

figura de la siguiente pgina, el tratamiento con ceramidas (SMasa exgena 1 U/ml o

anlogos permeables 25 M) durante 15 mm

aumenta los niveles de protena en

fraccin particulada. El efecto de la OS-ceramida es de nuevo de mayor intensidad

que el obtenido con la C2-ceramida, lo que coincide con los resultados previamente

49

Resultados y Discusin

descritos. La transiocacin de Raf-1 a membrana inducida por C8-ceramida ocurre

muy rpidamente, desde los primeros 5 minutos de estimulacin, y se mantiene al
menos durante los primeros 60 mm. La translocacin y activacin de Raf- 1 va
acompaada de la fosforilacin en tirosina y serina/treonina (Morrison y Cuter,
1997). La fosforilacin en tirosina es uno de los marcadores caractersticos de
activacin de la protena dependiente de Ras (Marais et al., 1995) y la fosforilacin en
serina/treonina puede estar mediada por PKC o por la propia protena quinasa
activada por ceramida CAPK (Yao et al., 1995; Morrison y Cuter, 1997). En la figura
inferior se muestra como el aumento de los niveles de fosforilacin de Raf-1 en clulas
marcadas con 32p, tambin se pone de manifiesto con un anticuerpo especfico para
tirosina fosforilada. La especificidad del marcaje se demuestra por la ausencia de
efecto del inhibidor H7 de serina/treonina quinasas, mientras que el inhibidor de
tirosina quinasa, tirfostina AG126, revierte parcialmente la fosforilacion.

0>

el

O
+

<

~5

o
o

~4

<3)

~~

<3)

0)

O
.~

00~H

00000

Raf- 1

Regulacin de Raf-1 por ceramidas. 1/ Translocacin de Raf-1 a la fraccin particulada en

cultivos primarios de astrocitos tratados durante 15 mm con SMasa (Saureus) 1 U/m, C8- y
C2-ceramida 25 pM. 2/ Cintica de la transiocacin de Raf-1 inducida por C8-ceramida 25 pM.
3/ Inmunoprecipitacin de la protena Raf-1 revelada con un anticuerpo policlonal antifosfotirosina. Las clulas se estimularon con CS-ceramida 25 pM durante 15 mm y donde se
indica preincubadas durante 1 It. con H7 o tirfostina AG126 100 ~tM.Los resultados de la
figura corresponden a un experimento representativo de otros 3 o 5 realizados y se obtuvieron
mediante los procedimientos descritos en las publicaciones incluidas en la presente memoria.

2. REGULACIN DE LA PKC ~ POR

MENSAJEROS LIPDICOS EN
CLULAS GLIALES

Resultados y Discusin

50

En este captulo se estudia el posible papel modulador de

diferentes mensajeros lipdicos sobre la isoforma ~ de PKC atpica.
La PKC ~ est relacionada con la activacin de la cascada de MAPK
en diferentes modelos experimentales (Berra et al., 1995; Liao et al.,
1997). Por otro lado, las ceramidas ejercen un efecto regulador de la
actividad de PKC

~,

que es responsable de algunas de las respuestas

celulares de la va de la SMasa-ceramida como la activacin del

factor de transcripcin NF-KB (Lozano et al., 1994; Mller et al.,
1995). Estas observaciones sugieren la posibilidad de que la protena
PKC ~ este implicada en el sistema de transduccin de seales
iniciado por la PC-PLC, mediando la activacin de Raf-1 y cascada de
MAiPK por ceramidas, y regulando en ultima instancia la sntesis de
NGF.

Primeramente se expone el efecto de la actividad de la PCPLC exgena sobre la PKC ~ en la lnea celular de glioma C6 y,
posteriormente, se estudia el efecto que en esta misma protena,
PKC

~,

exgena.

tienen los anlogos permeables de ceramida y la SMasa

ELSEVIER

Neuroscience Letters 219 (1996) 6870

NiDROSCIINCE
LITTLE

Addition of phosphatidylcholine-phospholipase C induces cellular

redistribution and phosphorylation of protein kinase C ~ in C 6 glial celis
Ismael Galve-Roperh, Jose M. Malpartida, Amador Haro, Ins Diaz~Laviada*
Departamento de Bioqumica y Biologa Molecular 1, Facultad de Qumicas, Universidad Complutense> 28040 Madrid, Spain
Received 7 August 1996; revised version received 5 October 1996: accepted 8 October 1996

Abstract
Phosphatidylcholine breakdown has beeri shown ro play a critical role in signal rransduction involving generation of a number of
second messengers [Exton, 3.13., Biochim. Hiophys. Acta, 1212 (1994) 26-421. lo the present repofl we demonstrate by immunofluorescence Ihat short-treatment of C 6 glial celis with phosphatidylcholine-hydrolyzing phospholipase C (PC-PLC), changes the intracellular localization of protein kinase C (PKC) ~ from the cytoplasm to a perinuclear region. Western blol analysis also showed a
redistributiori of PKC ~ aher incubation of cels with PC-PLC. To tesL whether these changes were accompanied by an activation of
2P-labelled cels. Short-treatment with
ihe enzyme. we measured the extent of phosphorylation of PKC ~by immunoprecipitation from >
PC-PLC resulted in enhanced phosphorylation of the higher Mr PKC r in C 6 glial cels.
Keywords: Protein kinase C

fl

Phosphatidylcholine-phospholipase C; Glial cels

Protein kinase C (PKC) is one of the major mediators of

signals upon externa! stimulation of cels by hormones,

neurotransmitters, and growth factors. PKC has been
found in mos mammalian tissues, but is particular!> abundant in the brain [17]. PEe manmalian PKC po]ypeptides
identified to date by cONA cloning have been classified
into three groups based on two criteria, namel> structural
features and regulation by cofactor. TEe atypical PKCs
and tIX isoforms) are dependent un phosphatidylserine, but
do not require Ca2~ ur diacylglycerol for activation [4,13].
lo astrocytes and glial cels the presence of PKC a, 13,7,
5, e and isoforms has been reponed [5,7,11,14]. Prufiles
of PKC isoforms in rat astrocytes and C 6 glioma celis are

<r

similar, although a higher expression of &, e, and risoforms in glioma celis has been detected [14]. Recently, it
has been demonstrated that the PKC ~ isofonn plays an
important role in mitogenic signal transduction [1], neurona! differentiation [19], and maintenance of long-term
potentiation [15]. Huwever, the precise mechanism by
which PKC r activity is regulated is not fully understood.
PKC ? is not activated by diacylglycerol or phorbol esters
[13,18] and in many cel types including glial celis, PKC r
*

Corresponding author. Tel.: +34 1 3944668; fax: *34 1 3944159;

e-mail: idl@solea.quim.ucm.es

is neither transiocated from cytusul to membrane nur

down-regulated in response tu acute ur chrunic treatment
with phorbol esters [2]. Activation of PKC r may be
achieved by ceramide [10]. The secund messenger ceramide is generated by the action of sphingumyelinase,
which is la tum functionally coupled tu phosphatidylhydrolyzing phospholipase C <PC-PLC) [16].
We have recent> shown that PC-PLC enhances nerve
growth factor synthesis in cultured gua! celis by a mechanism which is partially independent of the classical isoforms of PKC [8]. In the present repon we show that
addition of PC-PLC induces cellular redistribution and
phosphurylation of PKC ~ in C 6 glial celis.
Tu test whether PC-PLC could have an effect un PKC r
we analyzed ihe intracellular lucalization and the exten of
phosphorylation of this NCC isofurm. Short-term-treatment of C 6 cells with exogenous PC-PLC pruduced a
change in the lucalizatiun uf PKC r fromn cytuplasm tu a
perinuclear membrane region (Fig. lA,B). Non-specific
labeling was shown after incubation with anti-PKC tantibud> in the presence uf a competing peptide used tu rinse
the antibody (Fig. lC).
As a further prouf of the effect uf PC-PLC un the localization uf PKC ~, particulate, and soluble fractiuns uf cels
were separated by centrifugation, and PKC ~ was detected

0304.3940C6/$12.O0 1996 Elsevier Science lreland Ltd. AII rights reserved

Pl? S0304-3940(96)13165-7
6

1. Oale-Roper/t e: al. / Neuroscience Letters 219 (996) 6870

70

II] Berra, E.. Daz-Meco, MT., Dom<nguez. 1., Municio, MM., Sanz,
L., Lozano, i.. Chapkin. RS. and Moscar, J.. Protein Kinase C ~
97.4 kfla-

soforni is cricical ter miogenic sigua! cransduction, CeIl, 74(1993)

555563.

[2] Chen, C., Protein kinase C a, 5, and ~in C 6 glioma cels. TPA
induces Iransiocation and down-regulaion of convencional and
66 kDa-

o
=
a

(o

.0
(<3

o
o
9(/

new PKC isoforms bu not atypical NCC ~ FEBS Lea., 332

(1993) 169173.
[31Ufaz-Laviada, t., Larrodera, P., Nieto, J.L., Cornet, ME.. MazMeco, MT., Snchez, Mi.. Guddal, PH.. Johansen, T., Haro, A.
and Moscal, 1., Mechanism of inhibition of adenylate cyclase by
phospholipase C-catalyzed hydrolysis of phosphaidylcholine. J.
Biol. Chem., 266 (1991) 11701176.
14] Excon, J.H., Phosphaidylcholine breakdown anil signal Iran,duction, Biochim. Biopl,ys. Ada. 1212 (1994> 2642.
l~] Gott. A.L., Mellon, B.S.. Paton, A., Groome, N. aud Rumsby.
MG., Ral brain glial cels in primary culture and subculturc contain the 8, e and ~subspccies of protein kinase C as weil as 11w
convenjional subspecies. Neurosci. Lctt., [7! (1994) 117lt)
[6] Kcranen, L.M., Dulil, EM. and Newton, A., Prolein kinasc C is
rcgulated in vivo by thrce funclionally distinct phosphoryalions.
Curr. Biol., 5(1995)13941403.
171 Kumar, 1<., Kim, B.. Rupp, 1-1. aud Madl,ukar, 13., Exprcssiou of

prolein kin:se C isozymcs in rut glial ccli Une, NeuroRcpor. 4

(1993) 431434.
181 Laviatla, ID., Baudct. C.. Galve-Ropcrh. 1.. Navcilhan. P. aud

Fig. 3. Phusphorylatiou lcvcl of IKC ~. C 6 cels wcrc iucubatcd with

it,r hopluosplinc (A u,crslMLm) :nd ihen citl~cr un rcmucd ( lanc ji or
1 rcatctl br 15 miii 1 U/u,1 PC PLC ( lauc 2). A lcr imm uuoprccipilal ion
wi(h un u,ci-PKC ~> amihody. pcllcts wcrc rcsolvcd by clcctrophorcsis
aud subjcccd lo :iuloiadiography. Dcnsiomciric aualysis was pcrlormcd
jis dcscrihcd br Wcslcrn blol. Thc markcrs on thc cli corrcspoud lo ihc
uhigration position ol 97.4 :nd 66 kDa molecular wcigh u,,arkcrs. dencal rcsults wcrc obained u Il,rcc indcpeudcut cxpcrimcuts.

order to know whether transiocation of PKC ~ correlates

wih an activation of the enzyme, we derermined the extent
of phosphorylatiou of PKC tby imrnunoprecipilarion from
32P-labeled C 6 cells. Thus, incubation of >2P-labeled celis
with PC-PLC lcd lo un enhanced phosphorylation of PKC
~ (Fig. 3). lucreased phosphorylation of PKC ~ aflected
one form. probably corresponding lo 83 kDa, which agrees
with its observed tran.slocation. A similar effect has been
dernonstrated in epidermal cels, where phorbol myristate
acetate-treated celis showed an increased phosphorylation
affecting to higher-Mr PKC ~ isoform [12].
Paken togeiher these results indicate that treatment of C
6 cells wilh PC-PLC causes a change in the intracellular
localization of PKC ~which becomes associated to a perinuclear region. Redistribution of this kinase was accompanied by an increase in the extent of phosphorylation of
(he enzyme, confirming the hypothesis that PC-PLC treatmcm of C 6 glioma celis leads to ihe translocation and
consequent activation of PKC ~ isoform.
Phe aurhors gratefully acknowlcdged Dr. M. Guzman for
crilical reading of (he manuscripts and Dr. P. Brachet for
providing C, glial cels. This work was suppor(ed by a
grant from FIS (94/0300). 1. Galve has a fellowship from
University Complutense.

Brachct, P., Phosphatidylcholinc-phospholipase C mediates thc

intiuction of ncrvc growth factor in culturcd glial ccli,, FEHS
Lett., 364 (995) 301304.
19] Lchrich, R.W. -md Forrest. iN., Proicin kinase C ~ is associacd
with Ihe n,iotic apparalus u primary ccli cultures of thc shark
rcclal glaud, J. Biol. Chcm., 269 (1994) 3244632450.
lO] Lozano, J., Bcrra, E., Municio, MM., Diaz-Meco, MT.,
Domiuguez. 1., Sanz, L. aud Moscat, 1., Prolein km-ase C ~ isoforin
is critical for xB-dcpcndcut promoter activation by sphingomyclinase, 1. Biol. Chem., 269 (1994) 1920019202.
III] Masliah, E.. Yoshida, K.. Shimohama, 5., Gage, F.H. and Saiioh,

T., Dffcrcuial expression of protein kinase C isozymcs in rat gua!

eclI cultures, Brain Res., 549 (1991) 106111.

1121 Nishikawa, K.. Yamamoto, 5.. Nagumo, H.. Maruyama. K. aud

Kao. R., The presence of phorbol ester responsive and non-respon<ve forms ofihe ~isozymeof prolein kinase C in mouse epidemial
cdl,. Cdl. Signal.. 7(1995)491504.
1131 Ono, Y.. Fujii, T., Ogica. K., Kikkawa, U., lgarashi, K. and
Nishizuka. Y., Proicin kinase C t subspecies from rat brain: uts
srucurc, cxprcssion, and propcrtics, Proc. Nal. Acad. Sci. USA.
86(1989)30993103.
[4] Roisin, M. aud Dcscheppcr, CF.. Idenlificadon and cellular localizalion of prolcin kinase C isofoniis u cultures of ral type-l
astrocytes, Brain Res., 701 (1995) 297300.
lIS] Sacktor, T.C.. Osten, P., Valsamis, H., Jiang. X., Naik, MU. and
Sublecte, E., Persistent accivarion of (he ~isofoma, of procein kinase
C u the maintenance of long-leras potentiation, Proc. Nac. Acad.
Sci. USA. 90(1993)83428346.
[6] Schtitze, 5., Polthoff, 1<., Machieidt. T.. Berkovic, D., Wiegmann,
K. aud Krdnke, M., TNF activales NF-seR by phosphatidylcholinespccilic phospholipase
C-induced acidic sphingomyelin
breakdown, Cdl, 71(1992)765776.
[171 Tanaka, C. and Nishizuka, Y., The protein kinase C family for
neuronal signaling. Annu. Rey. Neurosci., 17 (1994) 551567.
[18] Ways. D.K.. Cook, PP.. Webster, C. aud Parker. P., Effect of
phorbol esters on protein kinase C-~, J. Biol. Chem., 267 (1992)

47994805.
[19] Wooren, MW., Zhou. O., Seibenhener, ML. aud Coleman, ES., A
role for zeta protein kinase C in nene growth factor-induced diiferentiation of PCI2 cels, Ccli Growth fliff., 5 (1994) 395403.

FEBS Letters 415 (997) 271274

FEBS 19133

Ceramide-induced transiocation of protein kinase C ~ in primary cultures

of astrocytes
Ismael Galve-RoperM, Amador Haroa, Ins Daz~Laviadab,*
~Deparnentof Riochemis:ry aud Molecular Biology Facul:y of Biology. Complutense University, 28040 Madrid. Spain
Dcpartnent of Biochemistry and Molecular hiology. Facully of Medicine, Alcal de Henares University, 28871 Alcal de Henares. Madrid, Spain

Received 21 Jul> 1997

Abstract The present research was undertaken to study the
possible involvement of> the atypical protein kinase C (PKC) ~ la
ceramide signal transduction la priniary cultures of ral astrocytes. As shown by Western blot analysis, transiocation of
immunoreactive PKC4 to the particulate fraction occurred upon
exposere of astrocytes to ceil-permeable ceramide analogs or to
exogenous spliingomyelinase. The particulate fraction ma>
correspoad to a perinuclear area, as indicated by immunocytochemical techoiques. Furthermore, treatment of cels with Noctanoylsphingosine lcd fo aB increased phosphorylation of
PKC4. Results thus show that stimulation of PKC4 may be
one of the intracellular events triggered by activation of the
sphingomyelin pathway.
1997 Federation of Furopean Biochemical Societies.
Xc> tords: Protein kinase C

4;

pathway [12]. PKC has also been shown to be involved in

the activation of nuclear factor KB <NF-KB), which is a landmark of the TNFa mechanism of action. Little is known
about the specific signal transduction pathways linking TNF

receptors to NF-KB. A connectiort has been proposed between

TNFa receptor and phosphatidylcholine phospholipase C
(PC-PLC), which is coupled to sphingomyelinase (SMase),
resulting in the generation of ceramide [6,13,14]. We have
recent> demonstrated that PC-PLC induces cellular redistribution and phosphorylation of PKC4 in glial cells [15]. The
present work was thus undertaken to test whether PKC4
may also be a target for ceramide action in primary cultures
of astrocytes.

Ceramide; Sphingomyelin;

Astrocyte

2. Materinls and n,ethuds

2.1. Materials

Anti-PKC~ antibod>, raised against amino acids 577592 of rat

1. Introduction

PKC~. was from Life Technologies Inc. (Gaithesburg. MD, USA).

Fluorescein-conjugated donkey anti-rabbit IgO was from Amersham

Sphingolipids are current> recognized as active participants

in the regulation of a wide range of cellular responses such as
regulation of ccli growth, differentiation, transformation and
death [1]. Sphingomyelin (SM) breakdown occurs in multiple
cdl typcs in response to a variety of extracellular mediators
ncluding cytokines cg. tumor necrosis factor a (TNFa), interleukiii-l~ (lL-l~). nterferon-y and other agents such as
nerve growth factor (NGF), la,25-dihydroxyvitamin O>,
Fas ligand and ionizing radiation (reviewed iii [3]).Receptor-mediated hydrolysis of SM generates ceramide, which ma>
funetion as a second messenger activating sever-al serine/threofine 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 ceramide action [5,61.These atypical PKC members (4 and ?Jt) are
dependent on phosphatidylserine but are not affected by diac2~ [7,8]. Although PKC has
ylglycerol, phorbol esters or Ca
been shown to be involved in the control of a number of
cellular functions including rnitogenic signal transduction [9],
neurona] differentiation [01 and long-terni potentiation [II],
the signal mechanisms downstream PKC4 activation are not
weIl known. PKC has been suggested to play a role in the
activation of the mitogen-activated protein kinase (MAPK)

(Little Chaltfont, UK). o-erythro-N-acetylsphingosine (C2-ceramide),

o-erythro-N-octanoylsphingosine (C8-ceramide) and o-erythro-dihydro-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 al other reagenis were from Sigma Chemicals (St Louis, MO. USA).
22. .Prin,ary cultures of asrocytes
Cortical astrocytes were derived from 2 day od 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-polyornithine in water. The primary cultures consisted of 95% astrocytes as
judged by immunocytochemical staining of glial fibrillary acidic pro-

tein.
Three days before the experiment, the serum-containing medium
was removed and cels were transferred to a chemically defined medium consisting of serum-free DMEM:Hams FIZ (1:1, v:v) medium
supplemented with 25 gg/ml insulin, 50 sg/ml human transferrin, 20
nM progesterone, 50 iM putrescine, and 30 nM sodium selenite.
2.3. Western blol analysis of PKCC
After stimulation, cels were washed with ice-cold PBS and subse-

Corresponding author. Fax: +34 (1) 885 45 85.

E-mail: bqidm~bioqui.alcala.es

quently homogenized in 50 mM Tris-UCI, pH 7.5, containing 5 mM

EDTA, 1 mM EGTA, OmM p-mercaptoethanol, 1 mM PMSF, 5 sgl
ml leupeptin, 2 sg/ml aprotinin, O sg/ml soybean trypsin inhibitor
and 10 sg/ml benzamidine. Soluble and particulate fractions were
obtained after centrifugation for 60 mm at 40000Xg. Proteins were
then resolved by SDS-PAGE and transferred onto nitrocellulose. lminunodetection of PKCI was carried out by incubating membranes
with anti-PKC4 polyclonal antibody and developing with an enhanced
chemiluminescence reaction kit (Amersham).

Abbreviations: C>-ceramide, N-acetylsphingosine; C

2.4. Inununocytochemical procedures

8-ceramide, 7Voctanoylsphingosine; DHC, dihydro-N-acetylsphingosine; IL, interIeukin; MAPK, mitogen-activated protein kinase; NGF, nene growth
factor; PC-PLC, phosphatidylcholine phospholipase C; PKC, protein
kinase C; SM, sphingomyelin; TNF, tumor necrosis factor

hyde/PBS for 5 mm. After washing in PBS, cels were treated with

Astrocytes were grown in glass coverslips in serum-containing medium and at three weeks made quiescent by serum starvation. Cels
were stimulated with different agents, washed with phosphate bufl7er
salme (PBS) asid coverslips immediately immersed in 3.7% formalde-

0014-5793/97/$17.00 1997 Federation of European Biochemical Societies. AII rights reserved.

PH SOO 4- 5 79 3(97)0098 5- x

L Galve-Roperh el a!FEAS Letters 415 (1997) 2712 74

272

x-oo and subsequently incubated with 40 sg/mI poyclonal anti-PKC~ antibod> for 60 mm and fluorescein-conjugated
~inti-rabbitIgO for 60 additional mm. Coverslips were then mounted
with glycerol containing 0.1% p-phenylenediamine and subjected to
fluorescence microscopy.
0.05% Triton

2.00

2.5. Plosplorylation of PKC<

Phosphorylation
of PKCC was
loading
of cels
with
32Pi
and immunoprecipitation
as performed
previously after
described
[7].
Imniunoprecipitation ws carried out by incubation with 7.5 sg/ml of antPKC polyclonal antibody asid precipitation with agarose-inked protein A. Phosphorylation was determined in the immunoprecipitates by
SDS-PAGE and autoradiography. GeIs were previously stained wth
Coomasie blue in order to verify the appropriate loading of the geis.
Autoradiography Fuji films were subjected to denstometric analysis
using Ihe Sigma-Gel program.

1.75

aa
1.50
1-

o
u
0>

E
3.1. Transloca:ion of PKC4 and idenlficauion by
inununoblo t ting

To study whether SM-ase and cerarnides may have an effect

un the subcellular distribution of PKCC, primary cultures of
astroc>tes were treated with neutral SMase or cel-permeable
ceramide analogs, C
2-ceramde and C8-ceramide. As shown in
Fig. 1, PKC! appcars to be expressed in primar> cultures of
astrocytes as a single 75 kDa isoform in both the particulate
and the soluble fraction, with approximately equal amounts of
total enz>me in each fraction in non-stimu]ated cells (55% and
45% respective!>) [8]. C8- and Q-ceramide, as well as exogenous SMase, induced a signiflcant increase in immunoreactive PKC ita the particulate fraction with a concomitant decrease in the soluble fraction (Fig. 1). The time course of C8ceramide-induced PKCC translocation to the particulate frac-

particulate fracuon
FKCC

soluble fracecon

2.00

1.75
<3

1.50
ce
cd

1.25

St

3. Results atad discussion

1.25

1.00
8
o
0>

e
0.75
0>

~0>

0.00

Fig. 1. Localintion of PKCC by immunoblotting. Astrocytes were

stimukted with vehicle, U/ml SMase (S. aureus), 25 4M C8-cer-

anide or 25 sM C>-ceramide for 5 mi The upper panel shows a

representative luminogram of the immunoblots. The lower panel
shows the mcans
S.D. of densitometric analyses from three mdcpendent experiments expressed in arbitrar> units referred tu densitometric units from control cels. Statistical analysis was performed by
Students .t-test. Significant> different vs. the corresponding subcellular fraction of control cels: t .P-c0.025; **: P<O.O.

1.00

e0>
te

0.75
0.50
0

15

60 180

mm

15

60 180

Fig. 2. Time course of C8-ceramide-induced translocation of PKC~.

Astrocytes were stimulated with 25 tM C1-ceramide forO, 5, 5, 60
and 80 mm, The upper panel shows a representative luminogram
of the inmunoblots. The lower panel shows the meansS.E.M.of
densitometric analyses from two independent experiments expressed
In rbitrary units referred to densitometrie units from control cels.
tion is shown in Fig. 2. PKCC redistribution was airead>
evident after 5 mm treatment and was siaximal after 15 mm
of ceramide stimulation. These flndings indicate that activation of the SM pathway induces PKC~ translocation to the
particulate fraction of rat astrocytes.
3.2. Redistribu ion of PXC0 itt primary astrocytes

To further stud> the intracellular localization of PKC.

ummunocytochemical analyses were perfornied. As shown in
Fig. 3, exposure of cels to eithcr SMase or ceramide analogs
induced PKC translocation to a perinuclear area. The elTect
of added SMase indicates that endogesiously produced ceramdes are also able to induce PKC! cellular redistribution,
therefore corroborating the specificity of ceramide analog action. Perinuclear signal was stronger in SMase-treated and C5ceramide-treated cels (Fig. 3C and D) thasi in C2-ceramide
(Fig. 3E). Ihis may be probab> due to the longer alkyl chain
of the former, which makes it more similar to natural occurring ceramides. Like si Western blot asial>sis (see aboye), C8ceramide asid SMase-generated ceramides produced a greater
etlect thasi the short chain Crceramide. Therefore alkyl chain
length may pa> an important role in ceramide action. This
effect has also been observed in raf-l activation by ceramides
[19]. Ihe emerging h>pothesis from this observatiosis is that
such structural requirements may explain divergesit effects of
differesit ceramides as a consequence of its subcellular origin,
and the specific SM pool involved [20]. si this context, a
different action of intracellular> generated ceramides asid
exogenously added ceramides in the induction of apoptosis
has been recent> reported [21].
The speciflcity of tbe effect of ceramides is supported by the
fact that inactive ceranuide analog DHC did not induce an>
chasige in the subcellular localization of PKC~ (Fig. 3B).

1. Galve-Roperh et al/FEBS Letrer, 415 (1997) 2 71274

274

assistance in cdl culturing. This work was supported by grants froni

Comisin Interministerial de Ciencia y Tecnologa (SAF 96/0113) and
Fondo de nevestigaciosies Sanitarias (97/0039-01).
References
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Biol. 8,159167.

[2] Hannun, Y. (1996) Scicnce 274, 18551859.

[3] Testi, R. (1996) Trends Biocbem. Sci. 21, 468471.
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(1996) Biochim. Biophys. Acta 1301, 273287.

[5] Lozasio, J., Berra, E.. Municio, MM., Diaz-Meco, M.T., Dominguez. 1., Sasiz, L. and Mosca!, J. (994) J. RbI. Chem. 269,
1920019202.

[6] MjIler. O., Ayoub. M.. Storz, P., Rennecke. J., Fabbro. D. and
Pflzenmaier, K. (995) EMBO J. 14, 19611969.
[7] Jaken, 5. (996) Curr. Opin. Biol. 8, 168173.
[8] Newton, A.C. (996) Curr. Biol. 6, 806809.
[9] Rerra, E., Obsa-Meco, MT., Dominguez, 1., Musilcio, MM.,
Sanz, L., Lozano, J., Chapkin. RS. and Moscat, J. (993) Ccli
74, 555563.
[0] Colcm~,n, ES. atad Wooten, M.W. (1994) J. Mol. Neurosci. 5,

395 7.
[II] Hrabctova, 5. atad Sacktor, T. (1996) J. Neurosci. 6, 53245333.
[2] Berra, E.. Diaz-Meco, MT., Lozano, J.. Frutos, 5., Municio,
MM., Snchez. P., Sanz, L. and Moscat, i. (995) EMBO 1.
4, 61576163.

[3] Schuitzc, 5., Pothofl, 1<., Machlcidt, T.. Berkovic, D., Wiegmann. K. atad Krnke, M. (1992) CeIl 71, 765776.
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Wiegmann, K. and Krnke, M. (996) J. Exp. Mcd. 184, 725

733-

[5] Galve-Ropcrh,

1.,

Mlpartida, J.M., Haro, A. asid Diaz-Laviada,

1. (996) Neurosci. Lett. 219, 6870.

[16] Laviada, 1.0., Baudet, C., Galve-Roperh, 1., Naveilhan, P. and

Brachet, P. (995) FEBS Lett. 364, 301304.
[17] Galve-Roperh, 1., Haro, A. asid Diaz-Laviada, 1. (1997) Mol.
Brain Res. (in press).
[8] Chen, C.C., Chang, 3. asid Chen, wc. (1995) Mol. Plnrsiiacol.
48, 3947.
[19] Huwiler, A., Brusisier, 1., Hummel, R., vervoordeldosik, M., Stabel, 5., Van Den Bosch, H. atad Pfeilschifter, J. (1996) Proc. Nat.
Acad. Sci. USA 93, 69596963.
[20] Asidrieu, N., Salvayre, R. and Levade, T. (1996) Eur. 1. Biochem.

236, 738745.
[21] Zhang, P., Lio, B., Jenkins, G.M., Hannun, Y. asid Obeid, L.M.
(1997) 3. Biol. Chem. 272, 96099612.
[22] Lehrich, R.W. and Forrest, J.N. (994) 3. Biol. Ches,. 269,
32446-32450.
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Res. 230, 18.

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Haller, H. (1996) Biochero. 3. 320. 651658.

[25] Kerancsi, L.M., Dutil, EM. and Newton, A.C. (995) Curr. Riol.
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Resultados y Discusin

58

RESULTADOS Y DISCUSIN
El efecto de la PC-PLC en la sntesis y secrecin de NGF en cultivos primarios

de astrocitos tiene un componente dependiente y otro independiente de la actividad

de cPKCs, sensible a Ca2~ y DAG. Como aproximacin al estudio de uno de los
posibles sistemas de transduccin de seales independiente de cPKCs, estudiamos

uno de los componentes del grupo de las PKCs atpicas, la PKC

Los niveles de PKC

4,

(Hoffman, 1997).

no disminuyen por exposicin prolongada a steres de forbol, es

decir no experimentan downregulation, tanto en astrocitos, como en la lnea de

glioma C6 (Ballestas y Benveniste, 1995; Chen et al., 1995). Los datos obtenidos,
muestran como la PC-PLC induce un cambio en la localizacin subcelular de PKC

4,

acompaado de un aumento del estado de fosforilacin de la enzima en la lnea de

glioma C6. Las variaciones en el estado de fosforilacin pueden reflejar cambios en el

estado de activacin de la isoforma de PKC estudiada (Jaken, 1996). Estas

observaciones son compatibles con que la PKC

pueda desempear un papel en la

regulacin de la produccin de NGF por clulas gliales inducida por PC-PLC

resistente a BlM y downregulation de PKC.

La estimulacin de la produccin de NGF promovida por ceramidas en

astrocitos presenta coincidencias con la regulacin por la PC-PLC que sugieren que

podran actuar por rutas comunes de sealizacin celular. En efecto, la estimulacin

de astrocitos por anlogos permeables de ceramida y SMasa exgena afectan a la
PKC

de manera semejante a lo observado con la PC-PLC. El aumento en los niveles

intracelulares de ceramida induce una redistribucin de la PKC

4,

que pasa de una

localizacin homognea en la clula a estar concentrada en una regin perinuclear.

Paralelamente a la translocacin de PKC 4 inducida por las ceramidas, se produce un
aumento en los niveles de fosforilacin de la enzima. Recientemente, estudios ms
detallados utilizando microscopia electrnica han permitido observar que en la
activacin de clulas PC12 se induce la translocacin de PKC

al ncleo, donde es

capaz de unirse a la cromatina (Wooten et al., 1997). Estas observaciones, junto con el
papel de la PKC

en la activacin de NF-KB por la SMasa (Lozano et al., 1994),

apuntan un posible papel de la PKC

en los efectos de regulacin de la expresin

gnica inducidos por los sistemas de transduccin de seales que involucran a esta
qrnnasa.

3. REGULACIN DE LA SNTESIS Y
SECRECIN DE NGF POR TNFot Y
LPS EN ASTROCITOS EN
CULTIVO PRIMARIO

59

Resultados y Discusin

Este apartado se refiere al estudio de la regulacin de la

produccin de NGF por la citoquina pro-inflamatoria TNFcz y el

efecto del lipopolisacrido bacteriano (Eseherichia coli). Algunos de
los efectos biolgicos de las citoquinas proinflamatorias, como el
TNFct y la IL-113 estn mediados por la va de transduccin de la

SMasa y consiguiente generacin de ceramidas (Hannun, 1996). Por

otro lado TNFcx e IL-1~ son potentes inductores de la sntesis de
NGF (Carman-Krzan et al., 1991; Hattori et al., 1993; Pshenichkin
et al., 1994). Por ello resulta de gran inters estudiar la posible
implicacin de la ruta de las ceramidas en la induccin de la sntesis
de NGF por el TNFa, uno de los principales reguladores fisiolgicos
del proceso de gliosis (Hurwizt et al., 1995; Merril y Benveniste,
1996). La segunda parte del captulo aborda el efecto inductor en la

sntesis de NGF de la exposicin al LPS de los astrocitos en cultivo

primario.
Por ltimo, se estudia el papel regulador del factor nuclear de
transcripcin NF-KB en la expresin del gen de NGF. Este factor de
transcripcin est

relacionado

con algunas

de las

respuestas

celulares inducidas por TNFa y LPS, as como con la va de

generacin de ceramidas (Yang et al., 1993; Machleidt et al., 1994;
Wiegman et al., 1994; Kohler y Joly, 1997) y podra por tanto

explicar

su

efecto

activador

de

la

sntesis

de

NGF.

Evidence for the lack ofinvolvement of sphingomyein hydrolysis in the tumor

necrosis factor-induced secretion of NGF in primary astrocyte cultures
Ismael Galve.Roperha>, Cristina Snchez, Bruno Sguib, Amador Haro, Ins Daz.Laviadac, Thierry
5
Levade
Departa~nento de Bioqumica y Biologa Molecular L Facultad de Biologa, Universidad Complutense, 28040 Madrid,
Spain. b Laboratoire de Biochintie Maladies Mtaboliques-INSEBM U. 466. Institu Louis Rugnard, 31408 Toulouse,
France r Departamento de Rio quintica y Biologa Moleeula,-, Facultad de Medicina,,Universidad de Alcal de Henares,
28871 Alcal de Henares, Spain

The signal mechanism underlying tumor necrosis factor a <TNFa) upregulation of nene
growth factor (NGF) production was studied in primary rat astrocytes. Since ceramide is
also able to induce NGF secretion and because TNFa is a known agonist of the
sphingomyelin-ceramide pathway, we investigated whether the TN?Fcz-induced NGF
secretion b> primary astrocytes is mediated by ceramide. TNFa stimulation of NGF
secretion was shown to be independent of protein kinase C, abrogated by the tyrosine
phosphoprotein phosphatase inhibitor phenylarsine oxide (PAO), and independent of the
activation of the mitogen activated protein kinase cascade. In marked contrast, inhibition of
MAP kinase counteracted the NGF secretion induced by ceramide. TNFa stimulation of the
nuclear transeription factor NF-KB was prevented by cel pretreatment with PAO, whereas

ceramide and sphingomyelinase had a marginal effect on NF-KB activation. Moreover,

TNFa failed to activate the sphingomyelin pathwas, as indicated by the lack of 5PM
degradation and the absence of cerarnide generation. To further clarifr the role of NF-KB in
NGF synthesis, EMSA were performed with a NF-KB site from the NGF proanoter. The
absence of signfficant binding of NF-KB to the NGF gene promoter indicates the existence of
an indirect mechanisan for NF-KB regulation of NGF synthesis. Altogether, our data

strongly suggest that TNFcz-mediated upregulation of NGF occurs independently of

ceramide generation.

Key words: astrocytes, ceramide, Nerve growth factor, NF-cB, sphisigomyein, tumor necrosis factor.
Abbreviations used: BlM, bisindolylmaleimide 0F109203X; DAG, diacylgl>cerol; db-cAMP, dibut>ryl cyclic
AMP; EMSA, electrophoretic mobility shift assay; FK. forskolin: MAPK, mitogen-ctivated protein kinase;
NGF, nerve growth factor; PAO, phen>larsine oxide; PC, phosphatidyleholine; PKC, protein kinase C; 5PM,
sphingomyelin; TNF, tumor necrosis factor
1. Introduction

Pro-inflammatory cytokines such as tumor necrosis factor a (TNFa) and interleukin 13

(IL-1j3) are important mediators of inflammation and immune responses in the CNS.
Such responses are followed by the rapid activation of brain resident macrophages and
subsequent activation of astrocytes (Merril and Benveniste, 1996). One of the responses of
reactive astrocytes is the synthesis and release of neurotrophic factors such as nene growth
factor (NGF) (Brodie, 1996; Yu et al., 1996). NGF production by glial celis during such
circumstances may limit the extent of neuronal loss and promote regenerative processes to
restore neuronal function (Pshenichkin et al., 1994).
~

outhor. Ismael Galve-Roperh, Departamento de Bioqumica y Biologa Molecular 1, Facultad de

Biologa, Universidad Complutense, 28040 Madrid, Spain. ig-usolea.quim.ucm.es Tel: (1) 394 46 68 Fax> (1) 894 46 72

NGF synthesis by astrocytes is a finely regulated process which includes complex

interactions among different signaling pathways. Pro-inflammatory cytokines such as TNFa
are well known stimulators of NGF synthesis (Hattori et al., 1993; Pshenichkin et al., 1994).
TNFa is a pleiotropic cytokine which exerts most of its biological effects, including NGF
induction, through the 55 kDa TNFct receptor (Hattori et al., 1996), which is the TNFa receptor
predominantly expressed in astrocytes (Dopp et al., 1997). Some of the early events triggered
by TNFa include the activation of phospholipase Az (Jayadev et al., 1994), neutral and acidic
sphingomyelinases (SPMases) as well as phosphatidylcholine-phospholipase C (PC-PLC)
(Schtttze et al., 1992; Andrieu et al., 1995; Wiegman et al., 1994). The signaling cascade
anvolving sphingomyelin (5PM) hydrolysis and subsequent ceramide generation constitutes the
so-called sphingomyelin-ceramide pathway (Hannun, 1996; Spiegel et al., 1996).
We have recently demonstrated that N-aeetylsphingosine (C2-ceramide), a celpermeant ceramide analog, and exogenous bacterial SPMase, which elevates intracellular
ceramide, are potent inducers of NGF synthesis by primary astrocytes (Galve-Roperh et al.,
1997a). Ceramide action as second messenger may involve stimulation of different molecular
targets as ceramide-activated protein kinase, ceramide-activated protein phosphatase, and
atypical PKC 4 (Hannun, 1996; Spiegel et al., 1996). As a matter of fact, ceramides are able to
trigger PKC 4 transiocation and phosphorylation in primary astrocyte cultures (Galve-Roperh
et al., 1997c). Activation of the MAPK cascade by the stimulation of SPM-ceramide pathway
has also been reported, and may be related to Raf-1 activation (Huwiler et al., 1996), which
may in turn be activated by direct interaction with PKC 4 (Van Dijk et al., 1997).
Another characteristie intracellular action induced by TNFx is the early activation of
the nuclear transcription factor NF-KB (Schtze et al., 1992). The exact role of ceramide
generation in TNFct-activation of NF-KB 15 still controversial (Hannun, 1996). Thus, some
reports indicate that ceramide generation through acidic SEMase may be responsible for NF-KB
activation (Machleidt et al., 1994; Wiegman et al., 1994), whereas others have not found
evidence for the role of ceramide in NF-KB stimulation (Betts et al., 1993; Andrieu et al., 1995;
Gamard et al., 1997; Zumbansen and Stoffel, 1997).
This work was therefore undertaken in order to further study the role of ceramide and
other signal transduction pathways involved in TNFa-mediated NGF synthesis and secretion
by primary astrocytes. The role of nuclear transcription factor NF-KB in NOF gene regulation
was also assessed.

2. Materlals and methods

2.1 Materais
Tissue culture plastic wares were from Nune (Denmark). Culture media and fetal calf
serum were from Biowhitakker (Belgium). The bisindolylmaleimide OF 109203X, forskolin, PD
098059 and cel permeable ceramide analogs were from Calbiochem (USA). TNFcz was from
PreproTech-Tebu (France). T4 polynucleotide kinase, NF-KB protein and NF-KB consensus
oligonucleotide were from Promega (France). Bacterial SPMase (5. aureus), dibutyryl cyclic
AMP and the rest of reagents were from Sigma Chemicals (USA).

2.2 Primary cultures of astrocyte celis

Cortical astrocytes were derived from 1-2 day od rats and cultured as previously
described (Galve-Roperh et al., 1997a). Cells were seeded at a density of 3x104 cells/cm2 on plastic
plates previously coated with 5 pg/ml dl-polyornithine in water. Cels were cultured for 3 weeks in
basal medium consisting of a mixture of Dulbeccos modified Eagle medium (DMEM) and Hams
F12 (1:1, y/y), with 0.66% glucose, 5 pg/ml streptomycin, SU/ml penicilhin, and supplemented with
10% fetal calf serum. The primary cultures consisted of 95% astrocytes as judged by
immunocytochemical staining of glial fibrillary acidic protein. Primary cultures of astrocytes
were incubated for three days before the experiments, in chemically-defined medium (consisting

of serum-free basal medium supplemented with 25 pg/ml insulin, 50 pg/ml human transferrin, 20
nM progesterone, 50 pM putrescine, and 30 nM sodium selenite).
2.3 ELISA of secreted NG)?

For extracellular NGF determination, ceil supernatants were collected 48 h after

treatment as described in the text, diluted in one volume of phosphate buffer salme (PBS)
containing 0.1% Tween 20 and 0.5% gelatin, and conserved frozen at -20 0C until quantitation.
NQF released by the cells was assayed in triplicate, by a double-site ELISA, using a monoclonal
anti-NGF antibody, coupled or not to 13-galactosidase according to an experimental protocol
described before (Laviada et al., 1995).
2.4 Preparation of nuclear aud cytosolic extracta
Nuclear and cytosolic extracts were obtained according to a previously described
method (Andrieu et al., 1995). Briefly, ceil pellets were homogenized at 4 0C in 400 pl of buffer
A (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 cels were
allowed to swell for 15 mm on ice, Igepal CA-630 (0.6% final concentration) was added, and the
tubes were vortexed. Nuclei were pelleted by centrifugation at 1200 x g for 20 mm at 4 0C. The
supernatant containing the cytosolic fraction was centrifuged at 14 000 x g and the resulting
supernatant was used for Western blot analysis. The nuclei were suspended in buffer A and
after sedimentation at 2000

x g for 20 mm, they were suspended in 25 pl of buffer C (20 mM

Hepes. pH 7.8, 400 mM NaC, lmM MgCl2, 0.5 mM EDTA, 0.1 mM EGTA, 1 mM
phenylmethylsulfonyl fluoride and 10 pg/ml leupeptin) and incubated for 30 mm on ice. After
centrifugation at 15 000 x g for 20 mm, the supernatant was collected and stored at -80 0C.
Protein concentrations were determined by the bicinchoninic acid method.
2.5 Electrophoretic mo bility shift assays
Oligonucleotide probes used were end-labeled using T4 polynucleotide kinase and [y-

32P]ATP according to the suppliers instructions. Binding reactions were performed in a total
volume of 20 pl with the radiolabeled oligonucleotide (250000 cpm), binding buffer (containing
2 mM Hepes, pH 7.8, 50 mM NaC, 1 mM MgCl2, 05 mM EDTA, 0.5 mM dithiotreitol, 4%
glycerol, 1.2 jtg poly (dIdC), and 0.4 pg of bovine serum albumin) and 5 pg of nuclear extract.

After incubation for 20 mm at room temperature, the samples were subiected to electrophoresis
on 4% acrylamide gel for 3-4 h at 100 y. Dried gel was exposed to an X-ray film (Hyperfilm,
Amersham) at .800 C or quantified by Phosphormager 445 SI (Molecular Dynamics).
The following oligonucleotides were used in EMSA: NF-KB consensus, AGT TOA 000
GAC TTT CCC AGO C; mutated NF-KB, AOT TGA TTC TCG AAA CCC AGO C; NF-KBNGF,

OCA TTG CAO 000 CCT CCC AGO Mil and mutated NF-KBNGF, OCA TTG CAT AGC CCT
CCC AGO MR.
2.6

Western blot autalysis of kB-a

Cytosolic extracts were subjected to SDS-PAOE on 8% polyacrylamide geis (Galve-

Roperh et al., 1997c). Then proteins were transferred onto Hybond-ECL membrane
(Amersham, UY.). Blots were blocked with 5% fat-free dried millc, and subsequently incubated
with IkB-a polyclonal antibody (Santa Cruz, USA). Membranes were then incubated with
secondary anti-rabbit peroxidase-labeled antibody and finally subjected to luminography with

an ECL detection kit (Amersham, U.R3.

2.7 Metabolic labeling of cellular choline-phospholipids an.d lipid analysis

Primary cultures of astrocytes were mcubated before the experiment in the

chemically-defined medium with 1 pCi/ml of [methyl-3H]choline. After 72 h incubation, tlie
radioactive medium was removed and cells were chased for 2 h in the chemically-defmed medium.
Then, cells were stimulated with TNFa for the indicated times, scraped and inmediately
subjected to low speed centrifugation and fi-ozen at -20 0C.
Lipid extraction and analysis were performed according tu previously described
procedures (Andrieu et al., 1994). Briefly, ceil pellets were suspended in distilled water and celis
were disrupted at 40C by brief sonication (10 s). An aliquot was taken for protein determination.
The remaiing was extracted with 2.5 ml of chloroform/methanol (2:1, vf~). The lower pliase was
washed with a synthetic upper phase consisting of chloroform/methanol/water (3:48:47, y/y/y) and
evaporated under

nitrogen. Then the lipid extract was subiected to mild alkaline hydrolysis as

follows: the residue was dissolved in 0.25 ml cbloroform and 0.25 ml methanolic 0.5 M NaOH, and
incubated overnight at 37 0C. ASter addition of 0.85 ml of chloroform, 0.25 ml of methanolic 0.5 M
HCl, 0.43 ml of water and 0.5 ml of chloroform/methanol (2:1, y/y) the upper phase (contaiing the
[3H]-choline label released from phosphatidylcholine) was counted for radioactivity. The lower
phase was washed twice with the synthetic upper phase, and the washings were counted for
radioactivity. The organic phase (containing the SPM) was evaporated and counted for
radioactivity. In sorne experiments, the mass of total phospholipids and SPM was quantitated by
measuring their phosphate content (Ames, 1966).
Ceramide quantitation was carried out using E. coli diacylglycerol kinase (Amersham, UR)
and [y-32P]ATPaccording to previously published procedures (Van Veldhoven et al., 1992).
3. Results
3.1 TNFa-iutduced NG)? secretion iii primar-y astrocytes is independen! of PKC and MI4PK
activties
In order to investigate the regulation of NOF synthesis by TNFa, primary astrocytes
were exposed to the cytokine and the NOF secreted into the extracellular medium was
quantified by ELISA. Incubation of astrocytes with TNFa induced a dose-dependent increase in
the NGF present in the ceil supernatant, with a maximal effect at 50 ng/ml (data not included).
The enhanced synthesis and secretion reached a maximal level after 48 h of cytokine

stimulation.
In an attempt to clarify the possible signaling pathways involved in TNFa upregulation
of NOF, cels were pretreated with the bisindolylmaleimide 0F109203X (BlM), a highly specific
inhibitor of PKC. We have previously demonstrated that BlM treatment of astrocytes is able to

mimic the effect of PKC down-regulation on the regulation of NGF secretion in glial cels
(Laviada et al., 1995). BlM did not affect TNFa-stimulated NOF secretion, therefore indicating

a PKC-independent mechanism of cytokine effect (Fig. lA). On the other hand, the cAMP
analog dibutyryl-cyclic AMP (db-cAMP) and the cAMP raising agent forskolin (FK) partially
inhibited cytokine stimulated NOF secretion (30% and 25% inhibition, respectively).
The MAP kinase dependency of TNFa-induced NGF secretion was studied by
pretreatment of the cells with the MEK inhibitor PD098059 (Alessi et al., 1995), which is aMe
to prevent MAP kinase activation in primary astrocytes (Oalve-Roperh et aL, 1997a). Specific
MEK inhibition did not result in a decrease, but rather led to a slight increase, of TNFainduced NGF secretion, indicating a MAP kinase independent mechanism. This was in marked
contrast with NGF induction by cell-permeant ceramide and exogenously added SPMase (Fig.
iB), where incubation with PD098059 resulted in a 80 and 90% inhibition respectively,
strongly suggesting that TNFa and ceramides act through different pathways.

150

125
100

a:0
z

75

50

25

1000

800

-~

800

a:
0
z
o.

400

200

Mg. 1. Regulation of TNFa-, C2-ceramide- an SPMase-stimulated NGF secretion. Primary cultures of

rat astrocytes were incubated in the presence of 2 sM BlM, 500 sM db-cAMP, 10 sM FK, 100 sM PD
098059 or 500 nM PAO 1 h prior to 50 ng/ml TN?Fct (A) or 25 sM C2-ceramide, 1 U/ml bacterial SPMase
<B) treatment for 48 h. NGF in the supernatants was assayed with a double site ELISA. Data are means
S.E.M. of 3 to 4 independent experiments with determinations in triplicate. Agents alone, which had no
effect on NGF seeretion have been omitted for clarification. Statistieal analysis was performed by
Students t-test; significant differences are indicated: * PczO.025, ** P<0.001. Statistical analysis for TNFa
and ceramide stimulation is related to control celis, and when inhibitors are used the statistics la referred
tu the values with the stimulant alone.

Preincubation of primary astrocytes with the tyrosine phosphoprotein phosphatase

inhibitor phenylarsine oxide (PAO) completely abolished the TNFa-stimulated NOF secretion.
PAO has been shown to prevent NF-KB activation by TNFa in botE myeloid and epithelial cells
(Singh and Aggarwal, 1995). Therefore, tliese results indicate that TNFcz regulation of NOF
synthesis is independent of PKC and MAPK activities, is partially inhibited by cAMP raising
agents, and may involve the nuclear transcription factor NF-KB.

3.2 Regulation. of the nuclear transcription factor NF-KB by TNFa

To determine the effect of TNFct on the nuclear transcription factor NF-a,
electrophoretic mobility shilt assays (EMSA) were undertaken. Treatment of astrocytes with
TNFa led to the activation of NF-KB, as demonstrated in the EMSA by the formation of a specific
band which was competed off by an excess of unlabeled probe. Fig. 2 shows the time course of
TNFct-induced NF-KB activation. Nuclear translocation of NF-wB was already evident 5 mm after
ceil stimulation with a maximal effect at 30 mm of treatment. Although the nuclear transcription
factor AP-1 was also activated in primary astrocytes by TNFa, AP-1 activation was transient,
with maximal activation only 5 mm after TNFct stimulation and returning to basal levela within
15 mm (data not shown).
We further investigated NF-KB regulation by TNFa to identily possible analogies with
TNFa-stimulated NOF secretion. As shown in Fig. 3, PAO completely blocked NF-a activation,
in agreement with previous reports indicating the role of tyrosine phosphoprotein phosphatase in
NF-KB activation by TNFa (Singh and Aggarwal, 1995). NF-KB activation was slightly inhibited
by PICC inhibition as shown by the effect of BlM on TNFa sitimulation. Increased intracelular
levels of cAMP evoked by FK partially blocked the ability of TNFa tu activate NF-KB. Similar
results were obtained with db-cAMP. The specifity of NF-KB

-~

TNF

-4

o
o

Lo

Cj

CD

-~

NF-KB complex

Fig. 2. Time-course of TNFa activation of NF-KB. Primar> astrocytes were stimulated for the indicated
times with 35 ng/ml TNFcz, and then nuclear extracts subjected to EMSA. The gel is representative of two

independent experiments.
activation by TNFa was demonstrated by the inhibitory effect exerted by the serme protease
inhibitor dichloroisocoumarin (DCIC) (data not shown). Indeed, pretreatment of celis with this
protease inhibitor abolished TNFx-induced NF-KB activation, which is concordance with a

previous report that demonstrated the existence of a serine-like protease crucial for tlie control
of NF-KB activation by TNFa (Machleidt et al., 1994).
Some of the intracellular effects of TNFa have been related to its abiity to induce
ceramide generation upon SPM hydrolysis (Hannun et al., 1996; Spiegel et al., 1996). In order
to test the posaible role of ceramide generation in TNFa stimulation of NF-KB activation, we

-~

~zz

o
-q

o
NF-kB

Z -ct~--~

-D

~
~

E~~-dQW

4
E-~

complex

Fig. 3. Regulation of NF-KB activation by TNFa and ceramde. Pnmary astrocytes were treated for 30
mm with 35 ng/ml TNFa, 25 iM CS-ceramide amI 1 U/ml SPMase. Where indicated, cels were
preincubated for 30 mm with 5 sM PAO, 2 sM BlM or 500 sM db-cAIMP. Results shown are
representative of 3 to 4 independent experiments.
assessed the ability of ceramide treatment to activate NF-KB in primary rat astrocytes. As
shown in Fig. 3, treatment with the cell-permeant N-octanoylceramide (Ca-cerarnide) or with
bacterial SPMase, which elevates the cellular ceramide concentration, also stimulated the
nuclear tranalocation of NF-KB. However, in agreement with previous observations on other
cell types (Andrieu et al., 1995), their stimulatory effect was much weaker than that exerted by
TNFa.
3.3 kB-a degr-adation occur-s tt corcer-t with NF-,cB activation. u TNFa-stirnulated astrocytes

Dissociation of the inhibitory moiety, IkB, of cytosolic NF-KB complexes allows NF-KB
activation and translocation to the nucleus (ONeill and Kaltschmidt, 1997). Therefore, we
measured the levels of IkB-a protein in the cytosol of TNFa- and ceramide-stimulated cels by
Western blot. Fig. 4A shows that there is a dramatic depletion of the inhibitory subunit lvels
in TNFa-stimulated cels, which occura in parallel to the observed activation of NF-KB. In
agreement with results obtained by EMSA, exogenous SPMase, Ca-, and C2-ceramides produced
a slight decrease in cytosolic leveis of IkB-a (Fig. 4A); however, this decrease was not as
pronounced as in TNFcz-treated cels. Degradation of IkB-a was prevented by cel pretreatment
with PAO prior to TNFa or Ca-ceramide stimulation (Fig. 4B).
3.4 The sphingornyelin-cer-arnide pathway is mt activated by TNFa n. pr-linar-y astr-ocytes
To further investigate whether the SPM-ceramide pathway was activated by TNFa in
primary astrocytes, SPM and ceramide levels were measured as described in Materials and

Methods. Although slight variations could be observed, TNFa stimulations for up to 120 mm
did not evoke any significant decrease of 5PM levels nor any increase of intracellular ceramide
levels (Fig. SA). SPM mass measurements were carried out leading to identical resulta (n3;

data not included). Furthermore, neither SPM nor ceramide levels were affected by TNFa in
the C6 glioma cel line (n3, data not included). Measurement of phosphatidylcholine (PC) and
diacylglycerol (DAO) levels also indicated the absence of a pool of PC sensitive to TNFa in
astrocytes (Fig 5B).
a,

-4

-4

-4

-4

a)
e

Cl

CC

r)

U)

JkB
A
-.4

a,

e
CC

-4

e
o

o
IkB

a)

o
z
~
H
~
-~

-4

e
CC

o
~

Fig. 4. Western blot analysis of IkB-a subunit on cytosolic extracts from stimulated astrocytes. Al Cels
were stimulated with vehicle, 25 sM C2-ceramide, 25 sM Ca-ceramide, 1 U/ml SPMase, or 35 ng/ml TNFa
for 30 mm. B/ Cels were ineubated with or without 5 sM PAO for 30 mm prior to TNFa or Cs-ceramide
stimulation.

3.5 NF-KB does not directly r-egulate NG)?gene expresson.

The NOF gene promoter contains a NF-KB like binding site located 669 base pairs
upstream of the TATA signal (Jehan et al., 1993). To examine whether this site is a

potential target for activated NF-KB to regulate NOF gene transcription, gel shift assays
were performed with purified nuclear transcription NF-KB and with the oligonucleotides of
the proposed site of regulation. Fig. 6 shows that, although this NF-KBNGF probe is able to
bind speeifically the nuclear transeription factor (see result with mutated oligonucleotide),
the intensity
consensus

of the binding is weak as compared to the binding to NF-KB

1 Oc
120
->

lid

E<3
E

leo

90
80
0

so

lO

10

45

60

75

90

75

90

TIME (coja)

130
20
lo

so

90

a
79
el
9

15

30

45

60

TIME (mio)

Fig. 5. Lipid quantifleation after TNFx stimulation. Primary rat astrocytes were labeled with [H]choline

for 72 h. Then, cels were incubated with 35 ng/ml TNFz and at the indicated time points incubations
were stopped. SPM (Fig SA, -@-> and PC (Fig. 5B, -@-) levels were determined as described in Materials
and Methods. Phospholipid levels are expressed as percentage of the value observed at time O. Values
represent the mean S.E.M. of 4 different experiments. Ceramide (Fig. SA, -O-) and nAO (Fig. SB, -O-)

levels were determined by the DAG kinase method. Values are expressed as percentage of radiactivity at
time O of 3 independent experiments. Statistical analysis using the Students t-test revealed no
sigsiificant differences.

oligonucleotide. In fact, cellular NF-KB present in the nuclear extracts from TNFa-stimulated
cels was not able to bind to the NF-KBNGF site (data not shown).
4.

Discussion

Results from the present report point tu a differential effect of TNFa versus exogenous
ceramide or SPMase in the regulation of NOF synthesis and secretion by primary astrocytes. In
spite of the many efforts tu elucidate the mechanisms that control NOF synthesis, the
regulation of this complex process remains largely unlknown. We have previously described the
involvement of exogenous PC-PLC in the regulation of NGF synthesis by astrocytes (Laviada et
al., 1995). PC-PLC in turn has also been reported tu mediate TNFa activation of NF-KB
through acidic 5PM breakdown (Schtze et al., 1992; Wiegmann et al., 1994). NGF induction by
PC-PLC is partially dependent on PKC activity (Laviada et al., 1995), whereas TNFa induction
of NOE synthesis in primary astrocytes seems tu be independent of PKC activity (the present

report). This is also true for N01 induction by cell-permeant ceramides and exogenous
bacterial SPMase (Oalve-Roperh et al., 1997).

The existence of crosstallcs among different signaling pathways, which allows a tight
control of NOF synthesis, is evidenced by the inhibitory effect of increased intracellular cAMP
levels on TNFz-stimulated NGF production. We have previously described a similar inhibitory
effect of the cAiMP cascade on ceramide- and lipopolysaccharide-induced NGF synthesis (OalveRoperh et al., 1997a; 1997b), although the exact mechanism underlying this process is not well
understood. Increased cAMP levels have been shown to prevent MAP kinase activation in
astrocytes (Kurino et al., 1996; Willis and Nisen, 1996). However, although TNFa increases
p42/p44 MAP kinase activity in primary astrocytes (unpublished observation), this activation
would not be responsible for the induction of NOF synthesis, as shown by the lack of inhibitory
effect of P0098059 (Fig. 1). Our data rather indicate that NOF induction by TNFct in primary

astrocytes is independent of MAP kinase activity, in sharp contrast with the effect of
ceramides, which activate NGF synthesis by a p42/44 MAPK activity-dependent mechanism
(Oalve-Roperh et al., 1997a, and present report). The inhibitory effect of cAiMP may also be
attributed to the observed decrease of TNFct-activation of NF-KB (the present report; Pahan et
al., 1997).

ci

E
ce
a

a>

ci

ci

o,

,~

-~

-~

z+

z+

+
rL ~
~
z z

z8

NF-kB complex

Fig. 6. Analysis of NF-KB binding to different oligonucleotides. Gel retardation assays were performed
with 400 ng of the purffied NF-KB protein (1) and the corresponding radiolabeled probes. Lanes 1, P +
NF-KB NGF probe; 2, P 4 NF-KB NGF mutated probe; 3, P + NF-KB consensus probe; 4, P + NF-KB consensus
mutated probe; 5, free NF-KB NGF probe and 6; free NF-KB consensus probe.

Our results show that in primary rat astrocytes the nuclear transcription factor NF-KB
rapidly activated by TNFx stimulation. The ability of TNFa to activate NF-KB is well
established, but much controversy still exists about the signaling mechanisms involved in
18

such a stimulation. Sorne reports indicate that ceramide generation, by acidic SPMase, rnay be
responsible for NF-KB activation (Schtze et al., 1992; Machleidt et al., 1994; Wiegmann et al.,
1994). In contrast, others have not found. evidence for the involvement of ceramide in NF-KB
stimulation (Betts et al., 1993; Andrieu et al., 1995; Gamard et al., 1997). Moreover, evidence
has been presented against a role of acidic SPMase in the effects of TNFa (Andrieu et al., 1994;
ICuno et al., 1994; Zumbansen and Stoffel, 1997). In our hands, exogenous SPMase and Cs-

lo

ceramide, which stimulate NOF synthesis to a much greater extent than TNFct, activated NFKB in primary astrocytes, although to a lesser extent than TNFa. In addition, IkB-a egradation
was less affected by ceramides than by TNFa. Recently, Modur et al. (1996) have described that
in endothelial cels TNFa is aMe tu activate Raf-1 througli a ceramide-dependent pathway,

whereas NF-KB activation

achieved by a ceramide-independent mechanism. Our data

behave in such a way. As a matter of fact, ceramide is
able to induce subeellular redistribution of tEn atypical PKC 4 in glial cells (Oalve-Roperh et
al., 1997c), and this effect coincides with increased Raf-1 pliosphorylation and MAPK activity
15

suggests that primary astrocytes rnay

(Oalve-Roperh et al., 1997a).

The existence of a ceramide-independent mechanism for the TNFa-induced stimulation
of NF-KB was confirmed by the lack of significant 5PM hydrolysis upon cel stimulation. It is

wurth noting that the absence of activation of the SPM-ceramide pathway by TNFa might be a
common feature of glial cels, since murine C6 glioma cels and human U2S1MO glioma cels
are also resistant to TNFa-stimulated SPM hydrolysis (data not shown; Richard et al., 1996).
Moreover, although ceramides are able to stimulate stress-activated protein kinase in glial
cels, TNFcz activation of SAPK does not seem to involve the ceramide pathway (Zhang et al.,
1996). Re TNFx-sensitive SPM pool involved in signal transduction has been suggested to be
located in the inner leaflet of the plasma membrane (Andrieu et al., 1996). The absence of
significant variations of 5PM mass levels, together with the absence of ceramide generation,
rule

out the possibility that metabolic labeling with [3H]choline may not be able tu label the

TNF~-sensitive signaling poo of SPM in confluent astrocytes.

The origin of the endogenous ceramides responsible for the induction of NOF synthesis
in the cel may be linked tu other possible generation sources of ceramide than 5PM hydrolysis,
e.g. resulting frum stimulated de novo synthesis or inhibited degradation. These prucesses
have already been shown to mediate sorne of the biological effects uf sphingolipids (Bose et al.,
1995; Bielawska et al., 1996; Paumen et al., 1997). For instance, the generation of ceramide by
a mechanism invulving a reduced rate of catabolism would lead to a prugressive and slow
accumulation of the lipid within the ceIl, and this may be of great importance in regulating
long-term responses (Bielawska et aL, 1996).
In summary, our results are in line with previous reports pointing to the existence of
ceramide-dependent and ceramide-independent effects of TNFa. Our data provide strong
evidence that TNFa and ceramide act by different mechanisms in the regulation of NOF

secretion by astrocytes. This is evidenced by their different dependency un MAP kinase activity,
the lack of SPM-ceramide activation by TNFa, and their different ability tu activate NF-KB.
Finally, our results indicate that, although NF-KB transcription factor does not appear tu play a
direct role on NGF gene transcriptiun, TNFa-induction of NGF synthesis may rely un previous
NF-kB activatiun.

Acknowledgments

We are indebted to Dr. M. Ouzmn for fruitful comments un the manuscript, Dr. U.
Wiun for the NGF promoter uligonucleotide sequences, F. Valio and Dr. E. Duln for technical
assistance. I.O.R. was recipient of a FEBS shurt-term fellowship. This work was financially
supported by Spanish CICYT (SAF 96/0113), FIS (97/0039), CAM (6648) and INSERM grants tu

T.L.

II

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acidic sphingomyelisiases in tumor necrosis factor signabng Cel 78, 1005-1015.

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bacterial lipopolysaccharide in cultured monocytes and astrocytes. Biochern. el. 313, 519-524.
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Zhang 1>., Miller B.S., Rosesizweig SA. asid Bhat NR. (1996) Activation uf the c-Jun N-terminal kinase/stressactivated proteni kinase ni primary glial cultures. el. Neurosci. Res. 46, 114-121.
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[3

Joarnal of Neuroscience Research 49:569575 (1997)

Regulation of Nerve Growth Factor Secretion

and mRNA Expression by Bacterial
Lipopolysaccharide in Primary Cultures of Rat
Astrocytes
Ismael Galve-Roperb, Jos M. Malpartida,1 Amador Haro,1 Phlppe Brachet,2
and Ins Daz~Laviada3*
Dcpartrnent of Hiochcmistry asid Molecular Biology, Faculty Biology, Univcrsity Complutense, Madrid, Spain
2Centre Hospitalier Rgional Universitaire, INSERM U.298, Angers, France
3Departnent of Biochemistry asid Molecular Biology, Faculty Medicine, University of Alcal,
Alcal de Henares, Madrid, Spain
The present work was undertaken to study tbe effect
of bacterial lipopolysacebaride (LPS), a potent activator of the host infianimatory response, on dic synthesis
of nerve growtb factor (NGF) by newborn rat brain
astrocytes. Treatment of prmary rat astroglial cels
cultured in chemieally defined medium with LPS
resulted in a dose-dependent accumulation of NGF
mRNA, andan increased release of NGF protein lii the
eclI nedium. NGF mRNA levels were maximal after
24 br of stimulation (8-fold mercase), whereas extraedlular NGF peaked after 72 bours of treatment (17-fold
mercase). This dramatie mercaseof extraeclular NCF
was abrogated f cels wcrc treated witb actinomycin
D or cycloheximidc, a fact whieh implies tbat tite
aceumulaton of extracellular NGF by LPS-treatcd
cels requires DNA transcrption and RNA transation. Stimulation of NGE syntbcss and seeretion was:
(i) unaifeeted hy treatment witb thc protein kinase C
inhibitor bsindolylmaleimide, asid (II) prevented by
forskolin and 3-isobutyl-1-metbylxanthine, two agents
whieb mercase eAMP levels. Inhibition of LPS cfTeet
ivas also obtained with apigenin, a proposed inhibitor
of tite mitogen-netivated protein kinase pathway. Results thus show that LPS stimulates NGF synthcsis by
astroglial cels througb a meehanism tbat is independent of protein kinase C (PKC), antagonized by

cAMP-elevating aguils, and probably mediated by

tite mitogen-activated protein kinase cascade. Tite
data raise tite possibility that LPS exerts stimulatory

INTRODUCTION
Bacterial lipopolysaccharide (LPS) is the sole lipid
present isi the outer membrasie of Gram-negative bacteria.
It acts as a potent activator of the host isifiammatory
response. In the cesitral nervous system (CNS), local
injection of LPS is followed by a rapid activation of brain
resident macrophages, the microglial cels, and by a
recruitment of peripheral monocytes or macrophages

(Montero-Menei et al., 1996). However, astrocytes become activated in a secosid phase of the inflammatory
process. It is presently well established that astrocytes
constitute an importasit auxillbry ccli controlling the
immunological status of the CNS. These celis, whcsi
activated with LPS, produce lymphokisies or cytokines
such as nitric oxide, IL-113, IL-6, TOP-pl asid TNFa
(Kimberlin et al., 1995; Schmidlin asid Wiesingcr, 1995;
Willis and Nisen, 1995; Slegers asid joniau, 1996) whicli
are actively driving tite immune reactiosis. However,
astrocytes are also a seurce of neurotrophic factors, asid

Abbreviations: BlM, bisindolylmalcimidc; cAMP. cyclic AMP; FCS,

fetal cali scrum; IBMX, 3-isobutyl-1 -methylxanthine; IL- 113, intcrlcukin- 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: Comisin Interministerial de Cicncia y Tecnologia; Contract gr-ant number: SAF 96-0113. Contract gr-ant sponsor:
Fondo de Investigacin Sanitaria; Contract gr-ant number: FIS 97/0039-

effeets on NGF synthesis tbat are independent of tbosc 01. Conrract gr-ant sponsor: Accion Integrada (Picasso Progrm);
elicited by astroeytc-dcrived inflammatory lympho- Contract grant number: 95/138.
kines sueb as IL-113, TNflx or TGF~1. J. Neurosci. *Co~espondencc tu: Dr. Ins D. Laviada, Dept. of Biochemistry,
Faculty of Medicine, University of Alcal. 28871 Alcal de Henares
Res. 49:569575, 1997. o 1997 Wilcy-Liss, Inc.
(Madrid), Spain. E-mail:id@solea.quim.ucm.es

Key words: lipopolysaecbaride; nerve growtb factor;

astroeytcs; protein kinase C
1997 Wiley-Lss,

Inc.

Received l4Fcbruary 997; Revised SApril 1997; Accepted l5April

997

570

Galve-Roperh

et

al.

these different mediators, when applied exogcnously

alune or in combination, can act as inducers of the
synthesis of the neurotruphic factor nerve gruwth factor
(NGF), at least in vitro, and pussibly in vivo (Pshenichkin
et al., 1994; Pltiss et al., 1995; Hattori et al., 1996). In a
mure general way, a relationship between CNS inflammation and NGF accumulation exists, since levels of this
factor appear significantly increased in cerebrospinal
fluids of multiple sclerosis patients (Bracci-Laudiero et
al., 1992).
NGF is mainly known as a neurunal survival 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 were
identified un pheripheral monocytes and/or T lymphocytes (Ehrhard et al., 1993), while the factor may be
produced not unly by astrocytes, but also by activated
brain microglial cels (Mallat et al., 1989). Therefore, the
involvement of NGF in the neuruimmune interactions
which take place during an inllammatory reaction in the
CNS appears probable, but remains elusive. In this
general cuntext, we have investigated whether LPS can
induce NGF synthesis in newborn rat astrocytes, and
analyzed the signalling pathway activated by LPS in
these celis. Previous studies have shuwn that prutein
tyrusine-kinase activatiun and nuclear transcription factor
NF-KB transduce LPS responses in monocytes or astrucytoma cels (Geng et al., 1993; Carsun and Aschmies,
1995). LPS strungly activates p42mPk and Raf-l, two
essential components of tIte mitugen-activated prutein
(MAP) kinase signal transductiun pathway (Willis and
Nisen, 1996). Agents tliat elevate intracellular cyclic
AMI (cAMP) have been slouwn tu inhibit the MAP
kinase pathway (Cook and McCurmick, 1993; Wu et al.,
1993) and tu decrease LPS-induced IL- I~B mRNA accumulatiun in human astrocytes (Willis and Nisen, 1995).
Our results sbow that LPS is a powerful inducer of
NGF synthesis in astrocytes. The cumpuund acts through
a signal transduction pathway independent of protein
kinase C, antagonized by cAMP-elevating agents, and
involving presumably MAP kinase activatiun.
MATERIALS AND METHODS
Primary Cultures of Astroglial Cels
Cortical astruglial cels were derived from 1- tu
2-day-uld rats and cultured as previously described
(Laviada et al., 1995). Cels were seeded at a density of
3 >< lO~ cells/cm2 un plastic plates previously coated with
5 ~ig/mldl-pulyurnitbine in water. Cels were cultured for
3 weeks in basal medium cunsisting uf a mixture of
Dulbeccos modified Fagle medium (DMEM) and Hams
FI 2 (1:1, y/y), with 0.66% glucose, 5 pg/ml streptomycin,
5 U/ml penicillin, and supplemented with 10% fetal calf

serum (FCS). Microglia-oligudendroglia-free astroglial

cultures were obtained after 2 hours uf orbital shaking.
Ihe primary cultures cunsisted uf 95% astrucytes as
judged by immunocytuchemical staining uf glial fibrillary acidic protein. Time course, duse response and
Nurtbern blut experiments were carried orn in 57-cm2
plates. The rest uf the experiments were done in 6-well
dishes.
Three days before the experiment, at day 21, the
serum-containing medium was removed and cels were
transferred tu a chemically defined medium cunsisting of
serum-free basal medium supplemented with 25 ng/ml
insulin, 50 pg/ml human transferrin, 20 nM prugesterone,
50 jiM putrescine, and 30 nM sodium selenite.
RNA Analysis and Enzyme-Linked

lmmunosorbent

Assay (ELISA)
After the indicated times uf treatment, RNA extrac-

tiun was carried out according tu the LiCI/urea method.

Nurthern blot analysis was also performed by standard
procedures (Jehan et al., 1995). Glyuxal-treated RNA was
fractionated in agarose gels, transferred tu Hybond-N
membranes by capillary blotting, and hybridized serially
with

a 32P-labelled prube of muuse NGF cONA. The NGF

probe was the 917-bp NGF DNA cloned by Scott et al.

(1983). Standardization of RNA loading was routinely
controlled by hybridization of tite blots with amyluid
precursor protein cDNA prube (Shivers et al., 1988).
Densitumetric analyses were perfurmed with Phosphorlmager 445 SI (Molecular Dynamics, Sunnyvale, CA).
Fur extracellular NGF determinatiun, cels supernatants were collected 24 huurs after treatment as described
in tite text, diluted in une volume of phosphate-huffered
salme (PBS) containing 0.1% Tween 20 and 0.5% gelatin,
and cunserved fruzen until quantitatiun. NGE released by
tite cels was assayed in triplicate, by a double-site
ELISA, using a munoclonal anti-NGF antibudy, coupled
ur not tu fB-galactusidase (Buehringer Mannheim, Indianapulis, IN), according tu an experimental protocol
described before (Laviada et al., 1995).
Otiter Citemicals
Tissue culture plastic wares were purchased frum
Nunc (Roskilde, Denmark), culture media from BioWhittaker (Verviers, Belgium), and FCS frum JRH Biosciences (Sussex, England). E. coli 0127:B8 LPS W was
from DIFCO Laboratories (Detroit, MI). It was dissulved
and sonicated in PBS prior tu utilization. Hybond-N
membranes were from Amersham (Little Chaltfont, England). Apigenin, forskolin and bisidulylmaleimide were
from Calbiuchem (La Julia, CA). 3-Isobutyl- 1 -methylxantitine (IBMX) and other reagents were from Sigma (St.
Louis, MO).

Lipopolysaecharide Regulation of NGF Synthesis

RESULTS
Induetion of NGF Expression and Seeretion by LPS

Higitly enriched astruglial cultures were expused tu

increasing concentrations uf E. cali LPS during 24 hr, and
secreted NGF was determined ni the supernatant of
cultured cels. As shown in Figure 1, LPS treatment
-increased the amount of extracellular NGF in a dosedependent manner. Maximal stimulation (1 0-fold over
control) was observed at 2 pg/ml. In additiun, Nortitern
blut analysis of total cellular RNA was used tu study tite
effect of LPS un NGF mRNA levels. A similar concentratiun-dependent effect of LPS was observed un NGF
mRNAaccumulatiun (Fig. 2).
Primary cultures of astrocytes were treated with a
maximal duse uf LFS (2 pg/ml) and extracellular NGF
an NGF mRNA accumulation were assessed after vanuus time periods. Figure 3 situws titat titere was no
increase in extracellular NGE concentratiun after 8 hr uf
treatment. In contrast, a striking enhancement was noticed after 24 itr. Maximal NGF secretion occurred in
cels treated fur 72 itr with LPS (17-fold increase uver
basal value). Densitumetric analysis uf Nurthern bluts
presented in Figure 4 indicated that a 4-fold increase in
NGF transcripts touk place after 8 br of treatment,
witereas maximal levels uf NGF mRNA were reached
after 24 itr of treatment (8-fold mercase).
Tu assess witether LPS inductiun was dependent un
DNA transcriptiun and RNA transatiun, we empluyed an
inhibitur of DNA transcription, actinomycin O (Act O),
and an inhibitur of prutein syntitesis, cycluiteximide
(CHX). Results summarized in Table 1 sbow that treatment with either 2.5 pM Act O urS pM CHX completely
blocked tite augmentation of NGF secretiun induced by

LPS PKC and Tyrosinc Kinasc Inhibiton Do Not AfTcet

tite Induction of NGF Seeretion by LPS

In an attempt to study tite patitways involved in the

control of NGF synthesis by LPS in astrocytes, cels were
treated with bisidolylmaleimide (BlM), a highly specific
inhibitor of PKC, prior tu tite incubation with LPS.
Classical PKCs are strongly activated by phorbol esters
as phorbol 12-myristate 13-acetate (PMA), whicit is a
putent inducer of NGF gene in astrocytes (Neveu et al.,
1992). We itave previously described that treatment with

the PKC inhibitor BlM abolisited PMA-induced NGF

(Laviada et al., 1995). Treatment of astrucytes witit SIM

60

50

40

a,
o.
u.. 30
C.D

571

20

10

0 0001 0.01

0.1

0.5

10

LPS pg/ml
Fig. 1. Nerve growth factor (NGF) secretion by astroglial cels
treated with increasing concentratiuns of lipopolysaccharide
(LPS). Primary cultures uf strocytes, 3 weeks od, were
incubated with increasing concentration of LPS during 24 hr. At
this moment, NGF released in the supernatants was assayed
with a double-site ELISA. Dat-a are meatns 1 S.E.M. of four
independent experirnents. Each determination was in triplicate.
Maximal NGF secretion was obtained with 2 iig/m LPS.

APP~

.01

NGF

Fg2N

hnbo

67

.9,,,
na

oNOFmRNAa

un

induced by increasing concentration of LPS. Cels were treated

with increasing concentrations of LPS. Lane 1, untreated cels;
lane 2,0.001 pg/ml; lane 3,0.01 ig/ml; une 4,0.1 ig/ml; une

5, 0.5 gg/ml; lane 6, 2 gg/ml; lane 7, 10 gg/m. After 24 hr

incubation, total RNA was isolated, electrophoresed, blotted
and serially hybridized with a radiolabelled NGF and AP?
cONA probes. Essentially identical results were obtained in
three independent experiments.

did not affect LPS-induced NGF secretiun (Table 1),

indicating that tite signal transduction pathway triggered
by LPS in astroglial cels, we empluyed the inhibiturs
by LPS was PKC-independent.
Tyrusine kinase inhibitors have been shuwn tu tyrphustin A0126 and genistein. As situwn in Table 1,
block sume of tite LPS effects. Tu study whether activa- neititer tyrpitustin AGI2G nor genistein affected LPStiun of tyrusine kinases was onvolved in NGF induction stimulated secretion of NGF.

572

Galvc-Roperh et al.

80

NGF

70

23

Os

60

APPfl

50

o>
o.
u- 40
<3

Fig. 4. Time course of NGF mRNA accumulation in astroglial

cels treated with LPS. Cels were treated at the indicated times
with 2 pg/ml LPS and NGF mRNA accumulation assessed by
Northern blot. Lane 1, 0 hours; lane 2, 8 hours; lame 3, 24
hours; lane 4,72 hours; lameS, 20 hours. Essentially identical
results were obtained in two independent experiments.

30

20
10

TABLE 1. Eflect of Treatnient With Dilleren Agcnts o LPSInduced NGF Secretion by Astroglial CelIs*

0
0

24
hours

72

120

Addition

NGF pg/mI

Control
LPS 2 ig/ml
LPS 2 ag/ml + Act D 2.5 pM
LPS 2 ig/ml + CHX 5pM

6.23.0
09.3 6.6
9.9 2.5
2.0 .3
104.59

Fig. 3. Time course of NGF secretion after treatment with LPS.

Primary astrocyte cultures were incubated in the presence of 2
~ig/mILPS. At the indicated times, cell-secreted NGF was LPS 2 pg/ml + BlM 2pM
assayed wih a double:site ELISA assay. Data are mean LPS2pg/mI+TyrAGl26 100pM
S.E.M. of two independent experiments with determinations in LPS 2 pg/ml + genistein 00 pM
triplicate. Maximal induction occurred at 72 hours of treatment. LPS 2 4/ml 1- FK O pM
LPS 2 pg/ml + IBMX 00 pM

LPS 2 pg/ml + API 25 pM

Agents Elcvatng cAMP Levels Initibit LPS-Induced

Produetion of NGF
Tite effect of raising intracellular cAME levels un

LES-induced NGF release by astroglial cels was determined by treating cels with eititer furskolin (FK) or
IBMX. FK or IBMX alune had no effect un NGF
secretion. Huwever, a 1 -hr preincubation with these
agents led tu a complete inhibitiun uf LPS-stimulated
secretiun of NGF. Cel treatment with the cAME analogue
dibutyryl-cAMP exerted tite same initibitory effect (data

not sitown). Tu cunfirm tite antagonistic action of cAME

un the induction uf tite NGF gene by LES, Nortitern blot
analyses were carne uut under the same treatment
conditions. Figure 5 shuws that botit FK and IBMX do
completely block the accumulation of NGF transcripts
whicit uccurs in response tu a LES treatment.

05.026
03.6 5
25.60.7
21.77.5
22.9 2.2

*Primary cultures of astrocytcs were incubated in the presence of the

indicated agents hour prior tu LPS Ireatmcnt fur 24 hours. NGF u ihe
supernatants was assayed with a double-site ELISA. Data are means
S.E.M. of three indcpendent experiments with dewrminations in
triplicate. Agents alune, which had no etfect un NGF secretion, have
been 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

compuund also blucked the NGF mRNA accumulation in the cels (Fig. 6)-Tite dose-response curve shows
titat tite 1C50 value for apigenin inhibition was approxmately lO ~iM(Fig. 7).
DISCUSSION

Tite present study shows that LES is a putent

Stimulation of NGF Produetion by LPS Is Initibited
by Apigenin
Tu furtiter examine tite pathway by witich LES
induces NGF syntitesis and secretion by astroglial cels,
the plant fiavonuid apigenin (API) was used. Cels were
preincubated with apigenin for 1 hr, and titen treated .witit
LES. Under sucit conditiuns, LPS failed tu induce any
augmentatiun of tite amount of cdl secreted NGF (Table

activatur of NGF secretion in primary cultures of cortical

astrocytes. LES induced NGF synthesis an secretiun by
astruglial cels in a cuncentration-dependent manner. A
dose uf 10 ng/ml was already effective but the strongest
response was obtained witit a dose of 2 jig/ml LES. At titis
concentration, LES induced an accumulation uf NGF
mRNA and NGF proteins whicit reacited titeir maiximal
levels after 24 itr and 72 itr uf treatment, respectively. Tite
protein secreted in tite extracellular medium was in-

Lipopolysaceharide Regulation of NGF Syuthesis

12

NGF

573

100
75
o,
oI.l~

50

APPSU~
Fig. 5. cAMP-clevating agents counteract NOF mRNA accumulation induced by LPS. Astrocyte cultures were treated with no
additions (lane 1) or with 2 ig/ml LPS alune (Infle 2) or in the
presence of eitherforskolin (FK) lO ~M (Iane3) or3-isubutyl-meihylxantbine (IBMX) lOO iM (Infle 4). FK and IBMX
were added 1 hotir prior tu LPS treatment. Essentially identical
resuls were obtained in two independent experiments.

234

NGF

ARR
Apigenin inhibits NGF mRNA accumulation in astroglial cels induced by LPS. Primary cultures were treated
with no additions (lame 1) or with 2 dg/ml LPS (lame 2), 25 pM
apigenin (lane 3) or 2 ig/ml LPS plus 25 14/ml apigenin (lane
4). The latter was added 1 hour prior tu LPS treatment.
Essentially identical results were obtaincd in twu independent
experiinents.
Fig. 6.

creased by a factor uf 1 7-fold, witicit is considerable. Tite

effect uf LPS required butit prutein syntitesis and DNA
transcriptiun, as it was abrogated by initibition uf cititer
biusyntitetic steps, using CHX an ActO.

LES induction uf NGF by astrocytes may be tite

resuir uf a complex prucess cumprising tite variuus effects

uf LES un titose cels, as well as un otiter cel types titat

migitt be present in tite primary culture. However, tite
-experimental prucedure used in tite present wurk, whicit
provides itigitly enriched astrocytic cultures, points tu a
direct effect of LPS on NGF synthesis and secretion by
astrocytes. Nevertiteless, stimulation of NGF syntitesis
may not result from a direct action of LES un astrucytes,
but frum autocrine luups. Otiter products released by

25
o
0

10

15

20

25

[Apigenin]
pM
Fig. 7. Dose response inhibition of LPS-inducted NGF secreion by apigenin. Astrocyte cultures were incubated with 2
ag/ml LPS and the indicated doses of apigenin for 24 hours and
NGF secreted to the supernatants was assayed with a doublesite ELISA assay. Data are means S.E.M. of two independent
experiments with determinations in triplic~te.
astrocyte in response tu LES cuuld enitance tite expressiun of tite NGF gene. This is the case uf inflammatory
cytokines TNFx, TGFj3 and IL-lI3, witich have been
described as inducers of NGF synthesis (Spranger et al.,
1990; Pshenichkin et al., 1994; Pltiss et al., 1995; Hattori
et al., 1996). However, inductiun of cytokines by LES itas
been sitown tu uccur througit protein tyrosine kinase
activatiun (Geng et al., 1993). Tyrphostin AG126 itas
been sitown tu prevent LPS tuxicity in vivo an blucks
LPS-stimulated TNFct an nitric oxide (NO) production
by macropitages (Novogrodsky et al., 1994; Vanicitkin et
al., 1996). Genistein itas also been implicated in tite
initibition uf the syntitesis of IL-6 stimulated by IL-l3
(Carson an Ascitmies, 1995). Our results did not
evidence any effect of tite tyrosine kinase initibiturs
tyrpitostin AG 126 and genistein un the LES-induced NGF
productiun. Titerefure, our data suggest titat tite regulation of tite expression uf tite NGF gene elicited by LES is
nut mediated by TNEa, IL- 1, IL-6 or NO pruduction.
Tu furtiter study tite mechanism triggered by LPS
and leading tu a stimulatiun of NGF production by

astrucytes, we examined witether agents able tu increase

intracellular cAME can initibit tite effect of LES. Tite role
uf cAMP an prutein kinase A in NGF regulatiun by
astroglial cels is still nut clearly defined. Depending un
tite cel culture conditiuns, tite gruwth state of cels and
tite experimental mudel, different results itave been

574

Galve-Roperh ct al.

ubtained. Witile under certain cunditiuns, forskolin, cate- astrucytes by a mecitanism that seems tu be closely
chulamines or cAME analugues stimulated NGF synthesis, tite cumpuunds were fuund by otiters tu be ineffective
or able tu counteract tite promotory actiun of several
inducers of NGF syntitesis (Haitn et al., 1994; Jeitan eta!.,
1995). Our results fil witit this sciteme, since titey show
titat FK an IBMX block tite stimulatury effect uf LES,
butit at tite level uf NGF mRNA accumulation and
cel-secreted prutein. FK an IBMX have been involved
in the blockade of tite MAP kinase patitway in epidermal

growth factor (EGF)-stimulated cels titruugit a cAMEdependent increase in Raf-l pitospitorylatiun, witicit
prevents Ras-ependent activation of Raf-l (Cook an
McCormick, 1993; Wu et al., 1993). Our data inicate
that apigenin, a prupused initibitur of the MAP kinase
patitway, also inhibited LES-induced NGF synthesis.
Apigenin itas been demonstrated tu reverse the v-H-rastransformed phenutype uf NIH 3T3 cels titrough the
initibitiun uf tite MAE kinase-assuciated signal transduction patitway (Kuu an Yang, 1995). Our result suggests
that LES triggers in astroglial cels a MAP kinasedependent mecitanism whicit promutes tite activation of
tite NGF gene. It is notewortity titat activation uf tite MAP
kinase cascade by LPS, and its initibition by agents titat elevate cel levels of cAME, was recently reporte in a ituman astrocytic cel line, U-373 MG (WiIlis an Nisen, 1995).
Twu general mecitanisms appear tu promote tite
expression of tite NGF gene in astrucytes. Tite first une
uperates titrough tite activatiun of PKC, an is triggered
by scrum an iacylglycerol (DMello and l-Ieinricit,
1990; Neveu et al., 1992). Tite otiter une acts througit tite
sphingomyelin patitway, and is tituugitt tu be switcited un
by vitamin D3 (Neveu et al., 1994). We itave recently

related tu tite patitway activated by spitingumyelinase and

witich is independent of PKC. The data raise the pussibility titat stimulatiun uf tite synthesis of NGF is a irect
cunsequence uf LES actiun un astrocytes, rather than an
indirect response elicited by LES-induced cytukines such
as IL- 13, TNF~ or TGFj3.
NOTE ADDED IN PROOE

We itave recently observed that LES induces a

3-fuld stimulation of p42/p44 MAPK in primary cultures

uf astrocytes.
ACKNOWLEDGMENTS

We titank Dr. M. Guzmn for itelpful discussiun and

critical reaing of tite manuscript, and tu Dr. E. Duln br
iter assistance in the ELISA prucedure witit tite Fluoroskan Titis work was supported by grants frum Comisin
Interministerial de Ciencia y Tecnologa (SAE 96-0113),
from Fundo de Investigacin Sanitaria (FIS 97/0039-Dl)
an from Acciun Integrada (Eicassu Erugram) promoting tite cullaboration between France an Spain (95/138).
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Kronke M, Brach MA (995): Tumor necrosis factor (TNF)-u
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Bracci-Laudicro L, Abc L, Levi-Montalcini R, Buttinelli D, Schilter

D. Gillessen S, Ottcn U (992): Multiple sclcrosis patients

express increased levels of B-nerve growth factor in cerebrospi-

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Hattori A, Hayashi K, Kohno M (1996): Tumor necrosis factor (TNF)
stimulates Ihe production of nerve growh factor in flbroblasts
via the 55-kDa type 1 TNF receptor. FEBS Lett 379:157160.
Jehan F, Neveu 1, Naveilhan P, Wion O, Brachel P (995): lnteracions
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ioscph CK. Wright SO, Bornmann WC, Randolph JT, Kumar ER.
Bitman R, Liu J, Kolesnick RN (994): Bacierial lipopolysaccharide has structural similarity tu ceramide and stimulates
ccramide-activated protein kinase in myeloid cels. J Biol Chem
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Kimberlin 0W, Velasco 5, Paris MM, Hickey SM, McCracken GH,
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in he inflammatory response by lipopolisaccharide and temperature in cullured human astroglial cels. lmmunol lnvest 24:775
785.
Kolesnick R, CuIde DW (994): The spbingoniyelin pathway in tumor
nccrosis factor and intcrleukin-I signaling. Cdl 77:325328.
Kuo M, Yang N (995): Reversion of y-U-ras transformed NIH 3T3
cels by apigenin through inhibiling niitogcn activaled protein
kinase and its downstream oncogcncs. Biochem Biophys Res
Comniun 212:767775.
Laviada ID. Baudel C. Galve-Ropcrh 1, Naveilban P, Brachet P(1995):
Phosph-atidylcholine-phospholipase C mediates ihe induction of
nerve growth factor in cultured glial cels. FEBS Let 364:301
304.
Mallat M, Houlgate R, Brachet P, Prochianz A (989): Lipopolysaccharide-stimulatcd rat brain macrophagcs release NGF o vitro.
Dey Biul 33:309311.
Montero-Menei CN, Sindji L. Garcion E, Mege M, Conez O, Gamelin
E, Darcy F (996): Early evcnts of the intlammatory reaclion
induced in mt brain by lipopoysaccharide intracerebral injection: Relative coniribution of peripheral monocytes and acuivated microglia. Brain Res 724:5566.
Neveo 1, Jehan F, Houlgatte R, Wion O, Brachel P (992): Activalion of
nerve growth factor synthesis in prinlary glial cels by phorbol
12-niyristate 3-acetate: Role of prolein kinase C. Brain Res
570:316322.
Neveu 1. Naveilban P Jehan E. Haudet C. Wion DDe Loca HE, Bracher

P (994): l.25-Dihydroxyvitamin 0, rcgulates he synthesis of

nerve growh Lictor o primary culturcs of gilal edN. Mu Brain
Res 24:7076.
Novogrodsky A. Vanichkin A. Patya M. CuAL A. Osherov N. Levitzki
A (1994): Prevention of Iipopolysaccharide-induced leihal Luxcity by tyrusinc kinase inhibitors. Scicnce 264:13191322.
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575

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cels. Biochem 1 312:707711.
Pshenicbkin SP, Szekely AM, Wise 5 (1994): Transcriptional and
posttranscriptional mechanisms invulved in the intereukin-l,
stcroid, and protein kinase C regulation of nerve gruwth factor

In curical astrocytes. J Neurochem 63:419-428.

Schmidlin A, Wiesinger U (1995): Stimulation of arginine ransport
ana) nitric oxide production by ipopolysaccharide is mediated
by different signaling pathways in astrucytes. 1 Neurochem
65 :590594.
Scott J, Selby M. Urde-a M, BeIl 6, Quiroga M, Ruter WJ (1983):
solation and nucleotide sequence of cONA encoding he
precursor of mouse nerve growh factor. Nalure 302:538540.
Shivers BD. Hilbich C, Multhaup O, Salbaum M, Beyreuther K. Seeber

PH (988): Alzheimers disease amyloidogenic glycoprotein:

Expression pattern in rat brain suggess a role in cel contad.
EMBO Ji: 13651370.
Slegers H. Joniau M (996): Lipupolysaccharide-enhanced expression
uf interleukin-6 in dibutyryl cyclic AMP-differentiaed rat C6
glioma. 1 Neurochem 66:466473.
Spranger M, Lindholm O, Bandtlow C, Heumann R. Gnahn H.
Naher-Noe M, Thocncn U (990): Regulaion of nerve growth
factor (NGE) synhesis in the rar central nervuos sysem:
Coniparison between the elfects of interleukin-l and various
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2:6976.
Vanichkin A, Patya M, Gazi A. Leyitzki A, Novogrodsky A (1996):
Late administration of Iipophilic tyrusine kinasc inhibitor
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Neurusci 8:8594.
Willis SA, Nisen PO (995): lnhibiion of lipopolysaccharidc-induced
IL- 13 transcription by cyclic adenosine monophosphatc in
human astrocytic cels. J lmmunol 154:13991406.
Willis SA, Nisen PO (1996): Differential induction of the mitugenactivated protein kinase pathway by bacterial lipopolysaccharide in cultured munucyes and astrocytes. Biuchem 1 313:519
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Wright SO. Kolesnick R (1995): Dues endutoxin stimulale cels by
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Wu 1, Den P, Jelinek T. Wolfman A, Weber Mi, Sturgill TW (993):
Inhibition of he EGF-acivated MAP kinase signaling pathway
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Phosphorylation of Raf by ceramidc-activated protein kinase.
Nature 378:307310.

80

Resultados y Discusin

RESULTADOS Y DISCUSIN
Regulacin de la produccin de NGF por el TNFc Papel del NF-icB
Los resultados expuestos ponen de manifiesto el efecto inductor del TNFcz en

la sntesis de NGF. Del estudio de la regulacin del NGF por el TNFa se desprende
que este ejerce su efecto por un mecanismo diferente, y probablemente independiente,

de la generacin de ceramidas por accin de la SMasa. La sntesis de NGF inducida

por TNFa y LPS es independiente de cPKC, de forma similar al efecto promovido por
ceramidas. Sin embargo, la induccin de NGF por ceramidas parece estar mediada
por la activacin de la cascada de MAPK, mientras que el efecto del TNFct es
independiente

de este

sistema

de

fosforilacin. Esta

constituye la primera

observacin de que ambos mediadores, TNFa y ceramidas, ejercen su efecto de

manera independiente. La ausencia de hidrlisis de SM y de generacin de ceramidas
por TNFa en astrocitos, indica que esta citoquina no activa la sntesis de NGF a
travs

de la

SMasa.

Los resultados

descritos

indican

la existencia

de

otros

mecanismos endgenos distintos de la accin de la SMasa, como puede ser la

estimulacin de la sntesis

de novo o la inhibicin de su degradacin para incrementar

los niveles intracelulares

de ceramidas (Bose et al., 1995; Biewlaska et al., 1996;

Paumen et al., 1997). Es importante destacar que una protena viral de RIV-1
favorece la activacin de la SMasa por el TNFcz, lo que revela que este sistema de
transduccin de seales es funcional en clulas gliales en una situacin de infeccin

vrica (Richard et al., 1996). Experimentos realizados iii vivo muestran como la
inyeccin de otra protena viral de RJV-1 aumenta la expresin de NGF in vivo
(Bagetta et al., 1996). El conjunto de estas observaciones sugiere que el aumento de

los niveles intracelulares de ceramida puede ser importante en los astrocitos

reactivos, induciendo entre otras respuestas un aumento en la sntesis y secrecin de
NGF.

Por otra parte se observa que la induccin del NGF se inhibe por el xido de
fenilarsina, inhibidor de tirosina fosfatasas. Este modulador bloquea la activacin del
factor de transcripcin nuclear NF-KB, as como la degradacin de su subunidad
cituslica inhibidora

IkBa. Parece por tanto que NF-KB, est implicado en la

regulacin de la expresin de NGF por TNFct. El estudio del promotor del gen de NGF
indica la existencia de una posible secuencia reguladora que podra unir este factor de

transcripcin

(Jehan

et al.1993).

Sin

embargo,

los

experimentos

realizados

demuestran que, al menos in vitro, el oligonucletido correspondiente

a esta

secuencia no es capaz de unir el factor de transcripcin. Por tanto la expresin del gen

Resultados y Discusin

81

de NGF inducida por TNFa depende de NF-KB pero de forma indirecta y no por la

unin directa al promotor del gen de NGF. La inhibicin parcial que ejerce el cAMP
sobre la secrecin de NGF inducida por TNFct no puede ser atribuida en este caso a
una inhibicin de la cascada de MAPK, ya que este sistema de fosforilacin
aparentemente no esta implicado (Kurino et al., 1996, Willis y Nisen, 1996). Sin
embargo, la inhibicin parcial de la activacin de NF-KB por el aumento en los niveles
de cAMP intracelular (Pahan et al., 1997), constituye una alternativa para explicar el
efecto inhibidor del cAIVIP sobre la sntesis de NGF.

Regulacin de la secrecin de NGF por LPS

En lo que se refiere al LPS, los resultados obtenidos ponen de manifiesto el
potente efecto inductor del LPS

(E.coli) en la sntesis y secrecin del NGF por

astrocitos en cultivo primario.

Los inhibidores de actividad tirosina

quinasa

empleados no bloquean la secrecin de NEW inducida por LPS, lo que sugiere una
activacin directa del LPS y no un efecto indirecto mediado por la secrecin de
citoquinas pro-inflamatorias, inductores de la secrecin de NGF (Carman-Krzan et
al., 1991; Rattori et al., 1993; Pshenichkin et al., 1994). Para profundizar en este
aspecto, se ha estudiado tambin el posible papel del xido ntrico (NO) en la
induccin de NEW por LPS. El NO es un importante mensajero paracrino regulador
de diferentes situaciones patufisiolgicas en el SNC (Murphy et al., 1993; Schmidlin y
Wiesinger, 1995). La exposicin de astrocitos a LPS o a citoquinas pro-inflamatorias
activa la generacin de NO a travs de la NO sintasa inducible, por ello resulta
factible que la generacin de NO constituya un mecanismo mediador de la regulacin
de la sntesis de NGF. Los resultados obtenidos (no incluidos) con aminoguanidina
(inhibidor de la NO sintasa constitutiva e inducible), y L-NAME (inhibidor
competitivo de la NO sintasa constitutiva) ponen de manifiesto que este sistema de
transduccin de seales carece de efecto en la regulacin por LPS de los niveles de
NGF sintetizado. Por tanto, la secrecin de NGF inducida por LPS es independiente
de la produccin de NO y posiblemente tambin de citoquinas pro-inflamatorias. En
este contexto, es importante destacar que la estimulacin de la va de las ceramidas
puede simular algunas de las respuestas celulares inducidas por el LPS (Barber et

al., 1996).
La inhibicin a travs de la va del cAMP de la secrecin de NGF inducida por
LPS pone de manifiesto la existencia de mecanismos de regulacin que permiten un
control preciso de la sntesis de este factor neurotrfico. Los datos obtenidos con

Resultados y Discusin

82

apigenina, inhibidor de la cascada de MAPK, fueron confirmados posteriormente con

el uso de un inhibidor sinttico altamente especfico de MEK, denominado PD 098059
(Alessi et aL, 1995). El tratamiento simultneo de los astrocitos en cultivo primario
con PD 098059 y la dosis mxima de LPS disminuye en un 50% la estimulacin
mxima producida por LPS en ausencia del inhibidor. Puesto que el LPS es un
importante activador de NF-KB, el efecto inhibidor del cAMP en la sntesis de NGF
inducida por LPS puede, por tanto, ser explicado por una inhibicin de la cascada de
MAPK, como por la inhibicin de la activacin de NF-KB (Pahan et al., 1997).

III- DISCUSIN GENERAL Y

CONCLUSIONES

83

Discusin General y Conclusiones

III- DISCUSIN GENERAL Y CONCLUSIONES

El funcionamiento del SNC depende del equilibrio funcional entre sus distintos
componentes celulares y de una adecuada integracin y adaptacin del sistema a
unas condiciones externas variables. Los astrocitos constituyen un importante centro

de coordinacin funcional en el SNC, a travs de la expresin de neurotrofinas,

protenas

de adhesin, citoquinas y factores de crecimiento que regulan el

funcionamiento neuronal, su desarrollo y la plasticidad neuronal. Adems, los

astrocitos son activadores de respuestas de emergencia ante procesos de lesin o
infeccin mediante su contribucin al proceso de gliosis. En este contexto, los niveles
locales de neurotrofinas, como el NGF, favorecen el crecimiento de determinadas
poblaciones neuronales

en momentos especficos del desarrollo. Un descenso

programado en los niveles de NGF puede ser importante para restringir, eliminar o
modificar poblaciones neuronales locales. El propio NGF puede

desencadenar

procesos de apoptosis, necesarios durante el desarrollo del sistema nervioso. La

sntesis neurunal de NGF en el SNC puede ser sustituida por la sntesis glial, siendo
los astrocitos las clulas no neuronales ms abundantes del SNC. La induccin de la
sntesis de NGF por astrucitus activados puede proporcionar el suficiente suministro
de NGF en el SNC en determinadas situaciones.
La sntesis de NGF est muy regulada y controlada por las condiciones
extracelulares. In vivo, en una situacin fisiolgica normal y en el SNC adulto, la
expresin del NGF por astrocitos esta reprimida pero la activacin astrocitaria induce
su expresin.

Los resultados

mostrados

profundizan

en el conocimiento

de los

sistemas de transduccin de seales que activan la sntesis y secrecin de NGF por

astrocitos en cultivo primario. La ruta de la SMasa y la consiguiente generacin de
ceramidas presenta un alto grado de similitud con vas de transduccin de seales
clsicas y mejor conocidas como la generacin de DAG por activacin de diferentes
fosfolipasas. El DAG y la ceramida son estructuralmente

semejantes y una vez

generados por la fosfolipasa correspondiente permanecen embebidos en la membrana

plasmtica. De manera anloga al DAG, que acta dirigiendo a algunos de los tipos
de PKC a la membrana donde se activan, la ceramida podra actuar como punto de
unin dirigiendo su(s) protena(s) efectora(s) hacia la membrana, lo que implicara su
activacin. Las ceramidas pueden por tanto modular la actividad de sus efectores
induciendo cambios conformacionales de la protena, modificando su localizacin
subcelular por translocacin y favoreciendo el acceso a sus substratos. La existencia
de una modulacin diferencial de PKC ~ y Raf-1 por los anlogos de ceramida de

84

Discusin General y Conclusiones

diferente longitud del grupo acilo, puede explicarse desde el modelo del grupo alquilo
protuberante (Krnke, 1997). Segn este modelo, el grupo alquilo de la SF sera
reconocido por las protenas efectoras de los esfingolpidos, mientras el resto acilo
sera el responsable de la insercin en la membrana plasmtica. Si esto fuera as, la

C2-ceramida podra ser reconocida por las mismas protenas efectoras que ceramidas
de mayor longitud, pero carecera de la capacidad de dirigirlas a la membrana
plasmtica.

La induccin observada de NGF por accin de la PC-PLC, puede reflejar el

efecto regulador sobre la sntesis de NGF de los sistemas de control de proliferacin
celular, dado que la hidrlisis de PC constituye uno de sus mecanismos de regulacin.
El efecto promovido por la PC-PLC confirma, adems, la importancia de la generacin
de mensajeros lipdicos en la regulacin de la funcionalidad astrocitaria. La induccin
de la sntesis de NGF por la accin de la PC-PLC posee caractersticas comunes con el
efecto observado por la accin de la SMasa exgena y las ceramidas permeables, lo
que hace pensar que ambos efectos estn relacionados de forma sucesiva y
coordinada. El trabajo realizado en la presente tesis doctoral constituye un ejemplo de
la importancia de los mensajeros lipdicos en la regulacin de la funcionalidad celular.
Glicerolipidos y

esfingolpidos

son

importantes

reguladores

de

sistemas

de

fosforilacin de protenas y la regulacin cruzada que ejercen entre si aumenta la

complejidad de los mecanismos de control posibles.

El LPS, uno de los principales activadores de la respuesta inmune e

inflamatoria ante una situacin de infeccin bacteriana, induce un drstico aumento
en los niveles de NGF, sintetizado por astrocitos en cultivo primario. El efecto

regulador del LPS puede tener diferentes lecturas pero todas ellas relacionadas con
los resultados anteriormente descritos. La va de las ceramidas puede ser activada
por la exposicin celular a endotoxinas (Hannun, 1996), y se ha propuesto que el LPS
puede ejercer alguno de sus efectos por analoga 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

caractersticas comunes de la estimulacin de la sntesis de NGF por el LPS y

ceramidas sugieren que ambos efectos podran estar relacionados con la induccin de

NGF durante una situacin inflamatoria o de lesin.

El TNFcz, otro activador caracterstico pro-inflamatorio de la va de la SMasaceramidas (Hannun, 1996), promueve sin embargo un incremento en la sntesis de
NGF con algunas diferencias con la induccin observada por el aumento de los niveles

Discusin General y Conclusiones

intracelulares

85

de ceramida, lo que sugiere que ejerce su accin de manera

independiente de la hidrlisis de SM. En efecto, el TNFct no induce un aumento en los

niveles intracelulares de ceramida a travs de la hidrlisis de SM. La induccin de
NGF por un aumento en los niveles de ceramida se puede explicar por la activacin
de la ruta de la SMasa por otros activadores de este sistema de sealizacin celular, o

por un aumento en los niveles intracelulares debido a la regulacin de su sntesis o

degradacin.
Respecto a los sistemas de fosforilacin implicados, los resultados expuestos

constituyen la primera referencia, de la que tenemos constancia, que asigna un papel

a la cascada de las MAP quinasas en la regulacin de la expresin del NGF. La
importancia de este sistema de fosforilacin en la regulacin de la sntesis de NGF se
ve confirmada al observarse que distintos activadores de la sntesis de NGF dependen
en mayor o menor medida de la actividad MAPK. La protena quinasa PKC ~ es una
de las dianas de la accin de la PC-PLC y las ceramidas en los astrocitos, modificando
su localizacin subcelular y su grado de fosforilacin. La localizacin perinuclear de
PKC ~ inducida por la activacin de los astrocitos puede estar relacionada con efectos
de regulacin de la expresin gnica, como ocurre tambin en otros tipos celulares
gliales y neuronales (Carson y Hart, 1996; Wooten et al., 1997).
La activacin de PKC ~ por ceramidas puede ocurrir por interaccin directa,
adems las ceramidas son capaces de activar Raf- 1, lo que se relaciona con el efecto
activador de PKC ~ sobre MEK y IVIAPK. Recientemente se ha descrito que PKC ~ es
capaz de activar e interaccionar de forma directa con Raf-1, lo que explicara un
mecanismo de activacin de MAPK independiente de Ras desencadenado por la
activacin de la PC-PLC (Van Dijk et al., 1997a). Estas observaciones junto con los

resultados mostrados permiten proponer un modelo en el que en los cultivos

primarios de astrocitos puede existir una relacin entre la activacin de la PC-PLC y
la generacin de ceramidas por activacin de la SMasa. Las ceramidas generadas son
capaces de incrementar la actividad MAPK, pudiendo atribuirse esta activacin a su
efecto regulador sobre PKC ~ y Raf-1. Este sistema de transduccin de seales podra
constituir un importante sistema de regulacin de la sntesis de NGF en situaciones
de activacin astrocitaria como la que ocurre durante el proceso de gliosis.

86

Discusin General y Conclusiones

PGPIfla~na

penwabl~
S\4asa excgena

SF

UAW

sF-1-P

0
BaH

a
Fsqtnnn de ks prripaks asp&t~ ffitldiadm en lapi~ntn npnrina de tesisdcxtnl

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