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DOI: 10.1159/000350129
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Ba ca/Seoane/Seña ís/A ce: Fas-Induced As ocy e P oli e a ion
Cellula Physiology
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Cellula Physiology
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Depa amen o de Fisioloxía,Facul ade de Medicina, Uni e sidade de San iago de
Compos ela, 15705 San iago de Compos ela (Spain)
Tel.+34881812290, Fax +34981574145, E-Mail ic o [email p o ec ed]
D . Víc o M. A ce
Fas/CD95 Liga ion Induces
P oli e a ion o P ima y Fe al
As ocy es Th ough a Mechanism
In ol ing Caspase 8-Media ed ERK
Ac i a ion
Olga Ba caa Ma cos Seoane Rosa Mª Seña ís Víc o M. A ce
Depa amen o de Fisioloxía, Facul ade de Medicina. Uni e sidade de San iago de Compos ela,
15782 San iago de Compos ela, Spain; aP esen add ess: Neu oscience and B ain
Technologies Depa men , I alian Ins i u e o Technology, Genoa, I aly
Key Wo ds
As ocy es • Fas/CD95 • ERK • PI-3K • Caspase 8
Abs ac
Backg ound: Fas/CD95 is he bes -s udied membe o he dea h ecep o (DR) supe amily
in he cen al ne ous sys em whe e i can igge cellula esponses o he han apop osis,
including he p omo ion o neu ogenesis and neu i ogenesis, s imula ion o he p og ession
o gliomas, and egula ion o he immune esponse o as ocy es. Me hods: We ha e
in es iga ed he ole o Fas/CD95 in he egula ion o he p oli e a ion o e al as ocy es
in i o, as well as he signalling pa hways in ol ed. Resul s: Fas/CD95 liga ion s imula ed
he p oli e a ion o p ima y e al as ocy es, h ough a mechanism ha depends on he
ac i a ion o caspase 8 and subsequen phospho yla ion o ex acellula signal egula ed
kinase (ERK). In e es ingly his p oli e a i e e ec is only obse ed wi h a low dose o he
Fas/CD95 agonis . In con as , when p ima y as ocy es a e challenged wi h a high dose
o he Fas/CD95 agonis signi ican cell dea h occu s. Conclusions: Ou indings suppo
ha , besides i s e ec s on cell su i al, Fas/CD95 may play a complex and p ominen ole in
he egula ion o as ocy e p oli e a ion du ing de elopmen .
Copy igh © 2013 S. Ka ge AG, Basel
Cell Physiol Biochem 2013;32:111-120
DOI: 10.1159/000350129
Published online: July 12, 2013
© 2013 S. Ka ge AG, Basel
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Ba ca/Seoane/Seña ís/A ce: Fas-Induced As ocy e P oli e a ion
Cellula Physiology
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Cellula Physiology
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In oduc ion
The su ace-bound ecep o Fas (CD95) is a membe o he dea h ecep o (DR)
supe amily ha was ini ially iden i ied in he immune sys em as an apop osis-inducing
ecep o [1, 2]. Once ac i a ed, Fas/CD95 igge s apop osis h ough ec ui men o he
in acellula adap o Fas-associa ed dea h domain (FADD, also called MORT1). FADD in u n
ec ui s caspase 8 h ough in e ac ion wi h hei dea h e ec o domains (DED), leading o
he assembly o a speci ic dea h-inducing signalling complex (DISC) a he in acellula egion
o he Fas/CD95 ecep o . The ec ui men o caspase 8 o he DISC esul s in au op o eoly ic
ac i a ion o he enzyme, which subsequen ly clea es caspase 3. In some cells, caspase 8
is inadequa e o ac i a e p ocaspase 3 and Fas/CD95-induced apop osis signaling equi es
ampli ica ion h ough p o eoly ic ac i a ion o he p o-apop o ic Bcl-2 amily membe Bid
(Bcl-2 inhibi o y BH3-domain p o ein) [3-5].
As occu s wi h o he dea h domain (DD)-con aining ecep o s, Fas/CD95 has now
eme ged as an impo an egula o o se e al non-apop o ic unc ions in a wide ange o
cell ypes [6, 7]. Thus, Fas/CD95 has been shown o inc ease he p oli e a ion o human
T lymphocy es and human ib oblas s in i o [8, 9], o accele a e li e egene a ion in
mice subjec ed o pa ial hepa ec omy [10], o e en o p omo e umou g ow h [11-15].
O he non-apop o ic ac i i ies o Fas/CD95 liga ion include gene a ion o p oin lamma o y
cy okines and chemokines in a ious cell ypes, including as ocy es [16, 17], induc ion o
a hype ophic esponse in cul u ed ca diomyocy es [18], s imula ion o he ma u a ion o
dend i ic cells [19], induc ion o neu ogenesis [20, 21], o p omo ion o neu i e ou g ow h
[21, 22]. Al hough, he physiological impo ance o non-apop o ic unc ions o Fas/CD95
emains con o e sial, hey ha e led o he gene al assump ion o Fas/CD95 as a dea h
ecep o .
Up o now, he molecula mechanisms leading o he ac i a ion o he non-apop o ic
ac ions o Fas/CD95 ha e no been comple ely elucida ed. Howe e , i is widely accep ed
ha he essen ial componen s o he apop o ic signaling machine y may also exe a
p ominen ole in he egula ion o he non-apop o ic unc ions o Fas/CD95 [6, 23]. In his
ega d, caspase 8 has been epo ed o play an essen ial ole in he hepa ic egene a ion
ollowing pa ial hepa ec omy in mice [24] o in he s imula ion o he ma u a ion o
cul u ed monocy es [25]. Simila ly, blockade o he unc ion o FADD esul s in de ec i e li e
egene a ion [26] and impai ed p oli e a ion o B and T lymphocy es [27, 28].
Fas/CD95 is he bes -s udied DR in he cen al ne ous sys em (CNS), and p o ides a
good example o how di e gen he DR-media ed biological e ec s may be. Wi hin he CNS
Fas/CD95 exp ession has been epo ed in bo h neu ons and glial cells, including as ocy es
and oligodend ocy es [ e ised in 16]. Howe e , while bo h neu ons and oligodend ocy es
a e suscep ible o Fas/CD95-media ed apop osis, as ocy es a e qui e esis an o Fas/CD95
dea h [17, 29-34]. In addi ion, Fas/CD95 can igge cellula esponses o he han apop osis
in CNS cells, including he p omo ion o neu ogenesis and neu i ogenesis [21, 22, 35, 36], he
s imula ion o he p og ession o gliomas [12-14] and he egula ion o he immune esponse
o as ocy es [37-39].
In an a emp o gain u he insigh in he biological unc ions o Fas/CD95 signalling
in as ocy es, we in es iga ed i s ole in he egula ion o he p oli e a ion o e al as ocy es
in i o. Ou indings demons a e ha Fas/CD95 liga ion induces a p oli e a i e esponse
in p ima y e al as ocy es, ia ac i a ion o caspase 8 and subsequen phospho yla ion
o ex acellula signal egula ed kinase (ERK). In e es ingly, and in keeping wi h p e ious
epo s in bo h B and T cells [7, 40], his p oli e a i e esponse seems o be dose-dependen :
while low doses o he Fas/CD95 agonis s imula e cell p oli e a ion, highe doses p omo e
cell dea h. Al oge he , ou indings suppo a p ominen ole o Fas/CD95 in he egula ion
o he cellula a e o as ocy es du ing de elopmen .
Cell Physiol Biochem 2013;32:111-120
DOI: 10.1159/000350129
Published online: July 12, 2013
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Cellula Physiology
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Cellula Physiology
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Ma e ials and Me hods
Cell cul u es and ea men s
P ima y monolaye cul u es o co ical as ocy es we e es ablished om e al (emb yonic day 15,
E15) o neona al (pos na al day 4.5, P4.5) Sp ague-Dawley a ce eb al co ices as p e iously desc ibed
[41, 42]. The ca e and use o all expe imen al animals we e in acco dance wi h ins i u ional guidelines. Cells
we e pla ed in Falcon polys y ene cul u e dishes (BD Biosciences, San Jose, CA) and g own in Dulbecco’s
modi ied Eagle’s medium (Sigma-Ald ich, S . Louis, MO) supplemen ed wi h 5% e al bo ine se um (FBS)
(In i ogen Co po a ion, Ca lsbad, CA), 2 mM glu amine, 2.5 U/mL penicillin, and 2.5 µg/mL s ep omycin
(all om Sigma-Ald ich). The cul u es we e main ained a 37°C in a humidi ied a mosphe e o 5% CO2 o
one week and he ea e cells we e ypsinized and subcul u ed o he di e en expe imen s. As ocy es
we e cha ac e ized in cul u e by immuno luo escence using a monoclonal an ibody agains glial ib illa y
acidic p o ein (GFAP) (Dako Diagnos icos, Ba celona, Spain) [43]. Hams e an i-Fas/CD95 an ibody (Jo2)
and i s iso ype con ol (IgG2λ2) we e pu chased om BD Biosciences. The speci ic caspase 8 inhibi o
Z-IETD- mk was pu chased om Calbiochem (San Diego, CA). The ERK inhibi o PD98059 and he selec i e
phospha idylinosi ol 3-kinase (PI-3K) inhibi o LY294002 we e pu chased om Sigma-Ald ich.
Cell p oli e a ion assays
In o de o e alua e he e ec o Fas/CD95 liga ion on as ocy e p oli e a ion, 4x104 cells we e seeded
in Falcon 24-well pla es, and ea ed wi h wi h di e en concen a ions o he agonis ic an ibody (5 o 10 µg/
mL) o wi h he iso ype an ibody (IgG2λ2) o 24 h. Cell numbe was de e mined wi h a Neubaue coun ing
chambe . To assess he le el o cell p oli e a ion, as ocy es we e labelled wi h b omodeoxyu idine (B dU)
using he FITC-B dU Flow Ki (BD Biosciences) as desc ibed in he manu ac u e ’s p o ocol. B ie ly, B dU
was used a a inal concen a ion o 10µM, o he las 6 h. A e washing wi h he BD pe m/wash bu e ,
cells we e ixed and pe meabilized wi h he BD cy o ix/cy ope m bu e . As ocy es we e hen ea ed wi h
DNase o 1 h a 37°C and incuba ed wi h a luo esceina ed an i-B dU an ibody and wi h 4 µg/mL o Hoechs
33258, o 20 min a oom empe a u e. Cells we e examined unde an Olympus luo escence mic oscope
(IX70, Olympus Op ical Co, Tokyo, Japan) wi h he app op ia e il e combina ion and pho og aphed a a
40x magni ica ion wi h a DP10 mic oscope digi al came a (Olympus Op ical Co). Fou andom ields we e
pho og aphed o each eplica e.
Assessmen o apop osis
Apop o ic cell dea h was assessed by luo escence mic oscopic analysis o cell DNA s aining pa e ns
wi h Hoechs 33258, as p e iously desc ibed [41]. Cells we e incuba ed wi h 4 µg/mL o Hoechs 33258
(Sigma-Ald ich) o 40 min a 37°C and, a e washing, cell mo phology was examined unde an Olympus
luo escence mic oscope (IX70, Olympus Op ical Co.) wi h he app op ia e il e combina ion. Fou andom
ields we e pho og aphed o each eplica e, and cells we e sco ed o apop osis by hei nuclea mo phology
(sh inkage, condensa ion, and agmen a ion) and he highe in ensi y o blue luo escence.
Wes e n blo ing
Fo phospho-ERK and phospho-Ak de e mina ion, cells we e main ained in 1% FBS o 48 h ollowed
by se um s a a ion o 2 h. Cells we e collec ed by cen i uga ion, and he pelle was hen lysa ed by hea ing
a 95°C o 5 min in 1% SDS, and immedia ely cooled a 4°C o 15 min wi h ice-cold lysis bu e (50 mM Hepes,
pH 7.5; 150 mM NaCl; 10% Glyce ol; 1% T i on X-100; 5mM EGTA; 1.5 mM MgCl2; 20 mM Na4P2O7; 20 mM
Na3VO4; 50 µg/mL ap o inin and 4 mM phenylme hylsul onyl luo ide). A e cen i uga ion (15000 g, 15
min, 4°C) o sepa a e cellula deb is, he lysa es we e esol ed in a 12% SDS-PAGE, and elec o ans e ed
on o a ni ocellulose pape (P o an; Schleiche and Schuell, Dassel, Ge many). ERK phospho yla ion was
de e mined wi h a speci ic an ibody ha ecognizes phospho-p44/42 (E k1/2) (Cell Signalling Technology
Inc, Dan e , MA). To al ERK was de e mined o loading con ol (Cell Signalling Technology). Fo phospho-
Ak de e mina ion, memb anes we e p obed wi h a comme cial ki (Phosphoplus Ak Se 473 An ibody
Ki , New England Biolabs, USA) ha allows speci ic ecogni ion o bo h non-phospho yla ed and se in-
phospho yla ed Ak . Immuno eac i e bands we e de ec ed wi h a wes e n-ligh chemiluminiscence
de ec ion sys em (ECL, GE Heal hca e Bio-Sciences AB, Uppsala, Sweden), pho og aphed (Hype ilmECL, GE
Heal hca e Bio-Sciences) and scanned wi h a GelDoc sys em (Bio-Rad, He cules, CA).
Cell Physiol Biochem 2013;32:111-120
DOI: 10.1159/000350129
Published online: July 12, 2013
© 2013 S. Ka ge AG, Basel
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Ba ca/Seoane/Seña ís/A ce: Fas-Induced As ocy e P oli e a ion
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Cellula Physiology
and Biochemis y
S a is ical analysis
Resul s a e p esen ed as he mean+s anda d e o (SE). The e ec o he di e en ea men s was
compa ed wi h hei espec i e con ols by using he non-pa ame ic Mann-Whi ney es . S a is ical
signi icance was es ablished a p<0.05.
Resul s
Fas/CD95 liga ion p omo es p oli e a ion in p ima y e al as ocy es
Fas/CD95 engagemen wi h he lowe dose o he agonis ic an ibody (5 µg/mL)
esul ed in a signi ican inc ease in he cell numbe as compa ed wi h con ol cells (Fig. 1A).
In con as , cell numbe was no modi ied when p ima y as ocy es we e challenged wi h
a highe dose (10 µg/mL) o he an i-Fas/CD95 an ibody (Fig. 1A). Cell numbe was also
una ec ed by challenging p ima y as ocy es wi h he iso ype an ibody. The lack o e ec
o he highe dose o he Fas/CD95 agonis on cell numbe may depend, a leas in pa , on
he apop o ic esponse o p ima y as ocy es (Fig. 1B). A signi ican inc ease in he numbe
o apop o ic cells was obse ed when as ocy es we e ea ed wi h he highe dose o he
agonis . In con as , apop o ic cell dea h was no obse ed when p ima y as ocy es we e
challenged wi h he lowe dose o he Fas/CD95 agonis (Fig. 1B). To gain u he insigh
in o he mechanisms ha may accoun o he obse ed inc ease in cell numbe , p ima y
as ocy es we e ea ed wi h 5 µg/mL o an i-Fas/CD95 an ibodies in he p esence o B dU.
As expec ed, he aise in as ocy e numbe appea s o be seconda y o a s imula ion o cell
p oli e a ion as demons a ed by he inc eased B dU up ake (Fig. 1B and 1C).
Fig. 1. Fas/CD95 liga ion p omo es p oli e a ion in p ima y e al as ocy es. A. Fas/CD95 engagemen wi h
he lowe dose o he agonis ic an ibody (5 µg/mL) esul s in a signi ican inc ease in he cell numbe as
compa ed wi h con ol cells. The dashed line indica es he cell coun s a ime 0. Each ba ep esen s he
mean + S.E.M. o h ee independen expe imen s in iplica e. *=p<0.05 s. con ol. B. A signi ican inc ease
in he numbe o apop o ic cells is obse ed when as ocy es a e ea ed wi h he highe dose o he agonis
(10 µg/mL). Each ba ep esen s he mean + S.E.M. o h ee independen expe imen s in iplica e. *=p<0.05
s. con ol. C. T ea men o p ima y as ocy es wi h 5 µg/mL o an i-Fas/CD95 an ibodies inc eases B dU
up ake. Each ba ep esen s he mean + S.E.M. o h ee independen expe imen s in iplica e. * = p<0.05 s.
con ol. D. Rep esen a i e images o he esul s shown in panel C.
Cell Physiol Biochem 2013;32:111-120
DOI: 10.1159/000350129
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ERK ac i a ion is necessa y o Fas/CD95-induced p oli e a ion in p ima y e al as ocy es
To gain u he insigh in he molecula mechanisms esponsible o he Fas/CD95-
induced as ocy e p oli e a ion, we in es iga ed he in ol emen o he ERK and PI-3K/Ak
pa hways. Blockade o ERK phospho yla ion wi h he speci ic MEK-1 inhibi o PD98059
esul ed in a signi ican educ ion in Fas/CD95-induced p oli e a ion, wi hou a ec ing
cell su i al (Fig. 2A and 2B). Fu he mo e, Fas/CD95 engagemen clea ly s imula ed he
phospho yla ion o ERK in p ima y as ocy es, an e ec ha was comple ely abolished by
PD98059 (Fig. 2C). Simila ly, blockade o he PI-3K pa hway also esul ed in a dec ease in
Fas/CD95-induced p oli e a ion, wi hou a ec ing cell su i al (Fig. 2A and 2B). Howe e ,
Fig. 2. ERK ac i a ion is necessa y o Fas/CD95-induced as ocy e p oli e a ion in p ima y e al as ocy es.
A. Blockade o ei he ERK o Ak phospho yla ion wi h speci ic inhibi o s esul s in a signi ican educ ion
in Fas/CD95-induced p oli e a ion. The dashed line indica es he cell coun s a ime 0. Each ba ep esen s
he mean + S.E.M. o h ee independen expe imen s in iplica e. *=p<0.05 s. con ol; ◊=p<0.05 s. an i-
Fas/CD95. B. Blockade o ei he ERK o Ak phospho yla ion wi h speci ic inhibi o s does no a ec cell
su i al. Each ba ep esen s he mean + S.E.M. o h ee independen expe imen s in iplica e. C. Fas/CD95
engagemen s imula es he phospho yla ion o ERK in p ima y e al as oci es, an e ec ha is comple ely
abolished by PD98059. D. Ak phospho yla ion is unchanged a e Fas/CD95 ea men in p ima y e al
as oci es.
Fig. 3. ERK phospho yla ion depends on caspase
8 ac i a ion in p ima y e al as ocy es. A. Cell
p oli e a ion induced by Fas/CD95 liga ion is
comple ely ab oga ed when cells a e ea ed in he
p esence o an i e e sible inhibi o o caspase 8.
The dashed line indica es he cell coun s a ime
0. Each ba ep esen s he mean + S.E.M. o h ee
independen expe imen s in iplica e. *=p<0.05
s. con ol; ◊=p<0.05 s. an i-Fas/CD95. B and C.
Inhibi ion o caspase 8 dec eases he amoun o
phospho yla ed ERK de ec ed a e Fas/CD95
ac i a ion wi hou a ec ing phospho-Ak le els.
Cell Physiol Biochem 2013;32:111-120
DOI: 10.1159/000350129
Published online: July 12, 2013
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and in con as wi h he e ec o Fas/CD95 engagemen on ERK phospho yla ion, we we e
unable o ind any consis en e ec o Fas/CD95 liga ion on Ak phospho yla ion (Fig. 2D).
ERK phospho yla ion depends on caspase 8 ac i a ion in p ima y e al as ocy es
In o de o in es iga e he in ol emen o caspase-8 ac i a ion in Fas/CD95-induced
p oli e a ion, we ea ed he cells in he p esence o an i e e sible inhibi o o caspase 8. As
Fig. 3A shows, he cell p oli e a ion induced by Fas/CD95 liga ion was comple ely ab oga ed
unde hese expe imen al condi ions, hus con i ming ha ac i a ion o caspase 8 is in ol ed
in Fas/CD95-induced p oli e a ion. In e es ingly, inhibi ion o caspase 8 also esul ed in a
dec ease in he amoun o phospho yla ed ERK de ec ed a e Fas/CD95 ac i a ion (Fig. 3B),
hus sugges ing ha ERK phospho yla ion also depends on caspase 8 ac i i y. In con as ,
phospho-Ak le els we e no modi ied by z-IETD-FMK ea men (Fig. 3C).
Fas/CD95 liga ion ails o p omo e cell p oli e a ion in p ima y neona al as ocy es
The in ol emen o caspase 8 in Fas/CD95-induced p oli e a ion was u he in es iga ed
by ea ing a neona al as ocy es wi h Fas/CD95 agonis . As p e iously epo ed by ou
g oup [34], caspase 8 gene is inac i a ed in neona al as ocy es and, he e o e, we ook
ad an age o his condi ion o con i m he in ol emen o caspase 8 in Fas/CD95-induced
p oli e a ion. In keeping wi h ou hypo hesis, ea men o neona al as ocy es wi h ac i a ing
an i-Fas/CD95 an ibodies was unable o p omo e cell p oli e a ion. Only a sligh dec ease in
cell numbe was obse ed, hough i did no each s a is ical signi icance (Fig. 4B). Also in
keeping wi h he esul s ob ained when caspase 8 ac i i y was inhibi ed in e al as ocy es,
Fas/CD95 liga ion was unable o induce ERK phospho yla ion in neona al as ocy es (Fig.
4C).
Fig. 4. Fas/CD95 liga ion ails o p omo e cell p oli e a ion in p ima y neona al as ocy es. A. Neona al
as ocy es a e esis an o Fas/CD95-d i en apop osis. Each ba ep esen s he mean + S.E.M. o h ee
independen expe imen s in iplica e. B. T ea men o neona al as ocy es wi h ac i a ing an i-Fas/CD95
an ibodies is unable o p omo e cell p oli e a ion. The dashed line indica es he cell coun s a ime 0. Each
ba ep esen s he mean + S.E.M. o h ee independen expe imen s in iplica e. C. Fas/CD95 liga ion is
unable o induce ERK phospho yla ion in neona al as ocy es.
Cell Physiol Biochem 2013;32:111-120
DOI: 10.1159/000350129
Published online: July 12, 2013
© 2013 S. Ka ge AG, Basel
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Discussion
Fas/CD95 liga ion p omo es p oli e a ion in p ima y e al as ocy es
Despi e he exp ession o Fas/CD95 in as ocy es is well documen ed, i s physiological
ole s ill emains o be elucida ed. As a membe o he DR amily, Fas/CD95 has been
epo ed o p omo e apop osis in p ima y as ocy es [17, 34]. Howe e , p ima y as ocy es
a e gene ally esis an o Fas/CD95 killing, a leas unde basal condi ions [34, 37, 44, 45];
and esis ance o Fas/CD95 killing has been e en epo ed in glioma cells [14, 44]. Al hough
he easons o his esis ance a e no comple ely unde s ood, we ha e ecen ly p oposed
ha silencing o caspase-8 gene may be a key ac o con olling he ou come o as ocy es
upon Fas/CD95 engagemen [34]. Howe e , o he mechanisms such as he exp ession
le els o Fas/CD95, he p esence o he Fas-associa ed p o ein phospha ase-1 (FAP-1), o
he ac i a ion o cellula caspase 8 (FLICE)-like inhibi o y p o ein (c-FLIP), may also con e
as ocy e esis ance o Fas/CD95-media ed cell dea h [45].
As occu s in o he cell ypes, Fas/CD95 can also ansduce non-apop o ic signals in
p ima y as ocy es [37, 39, 46]. In keeping wi h his, ou indings demons a e ha Fas/
CD95 liga ion can induce a p oli e a i e esponse in p ima y e al as ocy es, bu no in
p ima y neona al as ocy es, hus suppo ing a possible ole o Fas/CD95 in he egula ion
o as ocy e p oli e a ion du ing de elopmen . Rema kably, his p oli e a i e esponse
appea s o be concen a ion-dependen , since i could be only obse ed when e al
as ocy es we e challenged wi h he lowe dose o he agonis . In con as , s imula ion wi h
high concen a ions o an i-Fas/CD95 an ibodies esul ed in a di e en scena io in which
p ima y as ocy es unde wen apop osis. In e es ingly, a simila dual e ec has been also
epo ed in bo h T and B lymphocy es [7, 40]. In hose cells, low doses o Fas/CD95 agonis s
d ama ically inc ease cell p oli e a ion and ac i a ion, while high doses o he agonis may
induce cell dea h [7, 40]. Fu he mo e, a bimodal e ec on as ocy e p oli e a ion/dea h has
been also ecen ly epo ed o in e e on β [47] hus u he illus a ing he complexi y o
he mechanisms in ol ed in he egula ion o as ocy e a e du ing de elopmen .
Caspase 8 ac i a ion is necessa y o Fas/CD95-induced as ocy e p oli e a ion
To he p esen , li le is known abou he molecula mechanisms in ol ed in he con ol
o Fas/CD95-induced p oli e a ion. Howe e , i is now widely accep ed ha he essen ial
componen s o he p o-dea h ac i i y a e also c i ically in ol ed in i s non-apop o ic
consequences. This is he case o caspase 8, he p ima y caspase ha is ec ui ed o he DISC
and, consequen ly, he mos apical componen o he Fas/CD95 dea h signaling pa hway [3,
7]. I has been epo ed ha ec ui men and ac i a ion o caspase 8 may be also necessa y
o he non-apop o ic unc ions o Fas/CD95 and, in his ega d, caspase 8 is equi ed o
cy okine-induced p oli e a ion o hemopoie ic p ogeni o cells and T cells, and i is also
necessa y o cy okine-induced monocy e di e en ia ion [25, 48, 49]. In addi ion, capase
8 posi i ely con ols p oli e a ion and cell-cycle p og ession in p ima y hepa ocy es [50].
The esul s p esen ed he e demons a e he impo ance o caspase 8 in he con ol o he
p oli e a ion o e al as ocy s since (i) Fas/CD95-induced p oli e a ion o e al as ocy es is
coun e ac ed by inhibi ion o caspase 8 ac i i y, and (ii) Fas/CD95-induced p oli e a ion is
absen in neona al as ocy es, in which, allegedly caspase 8 gene is inac i a ed [34].
I is p esen ly unclea whe he hese p oli e a i e e ec s o caspase 8 equi e he
p ese a ion o i s enzyma ic ac i i y. Howe e , i is impo an o indica e ha enzyma ic
inhibi ion o caspase 8 impai s he Fas/CD95-induced p oli e a ion in T cells [48], myeloid
p ogeni o s [49] o p ima y e al as ocy es ( he p esen s udy). Howe e , we canno ule
ou ha some o he unc ions o caspase 8, o e en caspase 8 independen mechanisms [40]
may be also in ol ed in he p oli e a i e e ec s o Fas/CD95.
Caspase 8-media ed ERK ac i a ion is in ol ed in Fas/CD95-induced as ocy e p oli e a ion
To u he es ablish he unc ional ele ance o Fas/CD95-d i en p oli e a ion, we
analyzed some o he signaling pa hways ha may be in ol ed in his esponse. The
Cell Physiol Biochem 2013;32:111-120
DOI: 10.1159/000350129
Published online: July 12, 2013
© 2013 S. Ka ge AG, Basel
www.ka ge .com/cpb 118
Ba ca/Seoane/Seña ís/A ce: Fas-Induced As ocy e P oli e a ion
Cellula Physiology
and Biochemis y
Cellula Physiology
and Biochemis y
impo ance o ERK phospho yla ion in ansducing he non-apop o ic unc ions o Fas/
CD95 has been p e iously demons a ed in di e en cell ypes [13, 21, 22, 40]. Rema kably,
he consequences o Fas/CD95-induced ERK ac i a ion include he p omo ion o umo
p og ession o Fas/CD95 apop osis- esis an umo cells [12-14] o in p ima y e al
as ocy es ( he p esen s udy). Al hough he molecula link be ween Fas/CD95 engagemen
and ERK phospho yla ion has no been unco e ed ye , based in ou indings, i appea s ha
he p ocess equi es he enzyma ic ac i i y o caspase 8. In con as wi h ou indings, i
has been epo ed ha ERK ac i a ion is no blocked in a B lymphoblas oid cell line by he
addi ion o he pan-caspase inhibi o Z-VAD- mk [40]. Al hough we do no p esen ly know
he easons o hese disc epancies, we canno ule ou he exis ence o di e ences in he
caspase 8 ac i a ion wi hin he DISC, o he pa icipa ion o ye uncha ac e ized p o eins.
The PI-3K/Ak pa hway is ano he p ominen signalling mechanism in ol ed in some
o he non-apop o ic unc ions o Fas/CD95 [see 43 o e iew]; and PI-3K has been ecen ly
epo ed as an impo an media o o Fas/CD95 e ec s on glioma cells [36]. Repo edly,
Fas/CD95 s imula ion induces a s ong phospho yla ion o he ca aly ic uni o PI-3K in
bo h Ju ka and apop osis-sensi i e glioma cells [11]. Howe e , despi e we ha e ound ha
blockade o PI-3K signaling educes Fas/CD95-induced p oli e a ion, we ha e been unable o
demons a e any e ec o Fas/CD95 engagemen on he ac i a ion o he PI-3K/Ak pa hway.
In e es ingly, o he s udies ha e also ailed in de ec ing inc eased ac i i y o PI-3K in CD95
apop osis- esis an glioma cells [11, 14]. Fu he mo e, since ac i a ion o PI-3K by Fas/
CD95 does no equi es caspase ac i i y [14], i is emp ing o specula e ha he PI-3K/Ak
pa hway is no a key componen o he signaling mechanisms in ol ed in he non-apop o ic
e ec s o Fas/CD95 ac i a ion.
In summa y, we ha e ound ha Fas/CD95 engagemen may induce a dual e ec in e al
as ocy es: high doses o a Fas/CD95 agonis may induce cellula apop osis, while low doses
p omo e as ocy e p oli e a ion. The g ow h p omo ing unc ion o Fas/CD95 appea s o be
seconda y o caspase 8-media ed ac i a ion o he ERK signaling pa hway. Al oge he , ou
indings sugges he FasL-Fas/CD95 sys em may play an impo an ole in he egula ion o
as ocy e biology du ing de elopmen . In con as , he impo ance o he FasL-Fas/CD95
sys em in pos na al as ocy es may be limi ed by he ac ha hese cells do no exp ess
caspase-8.
Disclosu e
This wo k was suppo ed in pa by g an s om Xun a de Galicia (Axudas pa a
a consolidación das unidades de in es igación do Sis ema Galego de In es igación e
Inno ación).
Re e ences
1 I oh N, Yoneha a S, Ishii A, Yoneha a M, Mizushima S, Sameshima M, Hase A, Se o Y, Naga a S: The
polypep ide encoded by he cDNA o human cell su ace an igen Fas can media e apop osis. Cell
1991;66:233-243.
2 Oehm A, Beh mann I, Falk W, Pawli a M, Maie G, Klas C, Li-Webe M, Richa ds S, Dhein J, T au h BC,
Pons ingl H, K amme PH: Pu i ica ion and molecula cloning o he APO-1 cell su ace an igen, a membe
o he umo nec osis ac o /ne e g ow h ac o ecep o supe amily. Sequence iden i y wi h he an igen.
J Biol Chem 1992;267:10709-10715.
3 Wajan H: The Fas signaling pa hway: mo e han a pa adigm. Science 2002;296:1635-1636.
4 Cu in JF, Co e TG: Li e and le die: egula o y mechanisms in Fas-media ed apop osis. Cell Signal
2003;15:983-992.
5 Ka u ma MM, Khachigian LM: Signaling and ansc ip ional con ol o Fas ligand gene exp ession. Cell
Dea h Di e 2003;10:36-44.
6 S asse A, Jos PJ, Naga a S: The many oles o FAS ecep o signaling in he immune sys em. Immuni y
2009;30:180-192.
Cell Physiol Biochem 2013;32:111-120
DOI: 10.1159/000350129
Published online: July 12, 2013
© 2013 S. Ka ge AG, Basel
www.ka ge .com/cpb 119
Ba ca/Seoane/Seña ís/A ce: Fas-Induced As ocy e P oli e a ion
Cellula Physiology
and Biochemis y
Cellula Physiology
and Biochemis y
7 Paulsen M, Janssen O: P o- and an i-apop o ic CD95 signaling in T cells. Cell Commun Signal 2011;9:7.
8 Alde son M, A mi age RJ, Ma asko sky E, Tough TW, Roux E, Schooley K, Ramsdell F, Lynch DH: Fas
ansduces ac i a ion signals in no mal human T lymphocy es. J Exp Medcine 1993;178:2231-2235.
9 Agga wal BB, Singh S, LaPushin R, To pal K: Fas an igen signals p oli e a ion o no mal human diploid
ib oblas and i s mechanism is di e en om umo nec osis ac o ecep o . FEBS Le 1995;364:5-8.
10 Desba a s J, Newell MK: Fas engagemen accele a es li e egene a ion a e pa ial hepa ec omy. Na Med
2000; 6:920-923.
11 Gulbins E, He misson M, B enne B, G assme HU, Linde kamp O, Dichgans J, Welle M, Lang F: Cellula
s imula ion ia CD95 in ol es ac i a ion o phospho-inosi ide-3-kinase. P luge s A ch 1998;435:546-554.
12 Shinoha a H, Yagi a H, Ikawa Y, Oyaizu N: Fas d i es cell cycle p og ession in glioma cells ia ex acellula
signal- egula ed kinase ac i a ion. Cance Res 2000;60:1766-1772.
13 Ba nha BC, Legemb e P, Pie as E, Bubici C, F anzoso G, Pe e ME: CD95 ligand induces mo ili y and
in asi eness o apop osis- esis an umo cells. EMBO J 2004;23:3175-3185.
14 Zuliani C, Klebe S, Klussmann S, Wenge T, Kenzelmann M, Sch eglmann N, Ma inez A, del Rio JA, So iano
E, Vod azka P, Kune R, G oene HJ, He I, K amme PH, Ma in-Villalba A: Con ol o neu onal b anching
by he dea h ecep o CD95 (Fas/Apo-1). Cell Dea h Di e 2006;13:31-40.
15 Chen L, Pa k SM, Tumano AV, Hau A, Sawada K, Feig C, Tu ne JR, Fu YX, Rome o IL, Lengyel E, Pe e ME:
CD95 p omo es umou g ow h. Na u e 2010;465:492-496.
16 Choi C, Ben enis e EN: Fas ligand/Fas sys em in he b ain: egula o o immune and apop o ic esponses.
B ain Res Re 2004;44:65-81.
17 Falsig J, La a M, Leis M: De ined in lamma o y s a es in as ocy e cul u es:co ela ion wi h suscep ibili y
owa ds CD95-d i en apop osis. J Neu ochem 2004;304:181-193.
18 Bado C, Rue en H, Muelle S, S ahme M, Geh ing D, Jung F, Ihling C, Zeihe AM, Dimmele S: Fas
ecep o signaling inhibi s glycogen syn hase kinase 3 be a and induces ca diac hype ophy ollowing
p essu e o e load. J Clin In es 2002;109:373-381.
19 Rescigno M, Pigue V, Valzasina B, Lens S, Zuble R, F ench L, Kindle V, Tschopp J, Riccia di-Cas agnoli P:
Fas engagemen induces he ma u a ion o dend i ic cells (DCs), he elease o in e leukin (IL)-1β, and he
p oduc ion o in e e on γ in he absence o IL-12 du ing DC-T cell cogna e in e ac ion: a new ole o Fas
ligand in in lamma o y esponses. J Exp Med 2000;192:1661-1668.
20 Ricci-Vi iani L,Pedini F, Mollina i C, Condo elli G, Bonci D, Bez A, Colombo A, Pa a i E, Peschle C, De Ma ia
R: Absence o caspase 8 and high exp ession o PED p o ec p imi i e neu al cells om cell dea h. J Exp
Med 2004;200:1257–1266.
21 Tamm C, Robe son JD, Sleepe E, Enoksson M, Emgå d M, O enius S, Cecca elli S: Di e en ial egula ion o
he mi ochond ial and dea h ecep o pa hways in neu al s em cells. Eu J Neu osci 2004;19:2613-2621.
22 Desba a s J, Bi ge RB, Mimouni-Rongy M, Weins ein DE, Pale me JS, Newell MK: Fas engagemen induces
neu i e g ow h h ough ERK ac i a ion and p35 up egula ion. Na Cell Biol 2003;5:118–125.
23 New on K, S asse A: Caspases signal no only apop osis bu also an igen-induced ac i a ion in cells o he
immune sys em. Gen De 2003;17:819-825.
24 Ben Moshe T, Ba ash H, Kang TB, Kim JC, Ko alenko A, G oss E, Schuchmann M, Ab amo i ch R, Galun
E, Wallach D: Role o caspase-8 in hepa ocy e esponse o in ec ion and inju y in mice. Hepa ology
2007;45:1014-1024.
25 Kang TB, Ben-Moshe T, Va olomee EE, Pewzne -Jung Y, Yoge N, Ju ewicz A, Waisman A, B enne O,
Ha ne R, Gus a sson E, Ramak ishnan P, Lapido T, Wallach D: Caspase-8 se es bo h apop o ic and
nonapop o ic oles. J Immunol 2004;173:976–2984.
26 Schuchmann M, Rucke F, Ga cia-Laza o JF, Ka g A, Bu g J, Kno N, Sieble J, Va olomee EE, Wallach D,
Sch eibe W, Lohse AW, Galle PR: MORT1/FADD is in ol ed in li e egene a ion. Wo ld J Gas oen e ol
2005;11:7248-7253.
27 New on K, Ha is AW, Ba h ML, Smi h KGC, S asse A: A dominan in e e ing mu an o FADD/Mo 1
enhances dele ion o au o eac i e hymocy es and inhibi s p oli e a ion o ma u e T lymphocy es. EMBO J
1998;17:706-718.
28 Im iyaz HZ, Rosenbe g S, Zhang Y, Rahman ZS, Hou YJ, Manse T, Zhang J: The Fas-associa ed dea h
domain p o ein is equi ed in apop osis and TLR-induced p oli e a i e esponses in B cells. J Immunol
2006;176:6852–6861.
