PARP-1 induced cytoplasmic signaling pathways
PD 105589
Poly(ADP-ribose) polymerase (PARP-1) regulates different kinase pathways but the mechanistic
model for the regulation of a nuclear enzyme of the predominantly cytoplasmaticly regulated
kinase pathways has yet to be identified in many cases. We raised the possibility that PARP-1
inhibition activates MAPK phosphatases-1 (MKP-1) and inactivates JNK and p38 MAP kinases
in oxidative stress. These data indicated the significance of PARP-1 regulated MKP-1 expression,
but the precise mechanism has not been determined.
Because regulatory region of MKP-1 has heat shock responsive element and a cAMP response
element (CRE), we hypothesized that these transcription factors may play role in MKP-1
expression under oxidative stress induced PARP-1 activation. Our aim was to investigate the
effects of HSFs and CREBs under oxidative stress induced PARP-1 activation. Our results show
that suppression of HSF and CREB1 do not have any effect but the suppression of ATF4/CREB2
significantly decreased the MKP-1 expression.
PARP-1 induced poly(ADP-ribosyl)ation plays an important role in regulation of transcription
factors through ADP-ribosylation of its partner proteins or by physical association with
transcription factors. Our aim was also to investigate the precise mechanism of PARP-1 on MKP1 expression. Our results show that PARP-1 activation in oxidative stress induced poly-ADPribosylation of ATF4 and inactivated its binding to CRE giving a mechanistic way to the
inactivation of ATF4 dependent MKP-1 expression. PARP inhibitor dependent protection against
ROS induced mitochondrial depolarization and cell death can be regulated by PARP-1-ATF4MKP-1-JNK-p38 MAPK dependent retrograde pathway. These data indicate the identification of
1 a new P
PARP mediaated retrogrrade pathwaay by whichh PARP inhhibition throough ATF4 activation
activatess MKP-1 exxpression which
w
inactiivates JNK and p38MA
APK, and protects
p
mitoochondrial
integrityy and prevennts cell deatth.
The effect of hydroggen peroxidde on the exppression of M
MKP-1 in H
HSF-1,2,4 siilencing WR
RL-68 cells
NA were treated with
WRL-688 liver cellss transfectedd with a plassmid expresssing of HSF-1,2,4 siRN
0.3 mM
M H2O2 foor 3 hrs. Cellular eexpression of MKP-1 was expression asssessed by
immunooblotting usiing a specifi
fic primary aantibody. Our result shoows that levvel of MKP--1 was not
decreaseed in HSF-11,2,4 suppressed cells (F
Fig.1 A-C).
Fig.1.A
Fig.1.B Fig.1.C
2 The effect of hydroggen peroxidde on the exppression of M
MKP-1 in A
ATF4 silenciing WRL-688 cells
TF4 siRNA were treated with 0.3
WRL-688 liver cellss transfectedd with a plaasmid expresssing of AT
mM H2O2 for 3 hrss. The H2O2-treatment significantlly increasedd the phosphhorylation oof JNK1/2
and p38 MAP kinasse; howeverr, the PJ-34 with the H2O2-co-treattment only slightly deccreased the
phosphoorylation off these kinases. Phosphhorylation oof JNK1/2 and
a p38 MA
AP kinases show the
same result in the A
ATF4 supprressed cells, but the levvel of the sttress induceed MAP kinnases were
Cellular expression of
significaantly increaased compaared with thhe wild typpe cells (Figg.2.A,B). C
MKP-1 was expresssion assesseed by immunoblotting uusing a speccific primaryy antibody. Our result
was significcantly decreeased in A
ATF4 suppreessed cells (Fig.2.C).
shows tthat level oof MKP-1 w
These reesults suggeest that ATF
F4 is able to regulate MK
KP-1 activaation.
Fig.2.A
Fig.2.B
3 Fig.2.C
Effects oof MKP-1, A
ATF4 suppreession and oxidative
o
strress inducedd mitochonddrial depolaarization
Since m
mitochondriaal depolarizaation can bee the conseqquence as well as the caause of bothh apoptotic
and necrrotic cell deeath, we stuudied mitochondrial meembrane pootential of control, 0.3 mM H2O2
with or without 100μM PJ-34 for 30 minn in wild tyype and MK
KP-1, ATF44 suppressed cells by
PARP-1 prootected the cells
c
againsst oxidative stress, as
fluorescence microoscopy. Inhiibition of P
demonsttrated by ann increasedd fluorescennce value oof JC-1 red and a deccreased mitoochondrial
depolariization induuced JC-1 green fluorrescence vaalue. Althoough, these protective effect of
PARP-11 inhibition w
was abolishhed in MKP--1 and ATF
F4 suppresseed cells (Figg.3.A).
Statistical analysiss of mitochondrial depolarizatiion was vverified wiith flow ccytometric
measureement. Resuult shows thaat oxidativee stress induuced significcant mitochoondrial depoolarization
in wild--type, MKP
P-1 and AT
TF-4 suppressed cells. PARP-1 innhibition waas able to protect
p
the
mitochoondria againnst depolarrization in wild-type cells, althoough this pprotective effect
e
was
abolisheed in MKP-11 and ATF44 suppressedd cells (Fig.33.B-D).
4 Fig.3.A
Fig..3.C
Fig.3.B
Fig.3.D
5 ADP-ribbosylation of ATF4
Since thhe increasedd ADP-ribossylation by PARP-1 cann trigger thee cell deathh pathways aand it able
to modify the regulation of traanscription factors, wee studied pooly(ADP-ribbosy)lation oof control,
R level wass increased
0.3 mM H2O2 with or without 10μM PJ-344 for 30 minn in WRL-668 cells. PAR
RP-1 inhibittion was efffective by P
PJ-34, it deccreased the
in oxidaative stress ttreated cellss, while PAR
PAR levvels.
To test if ATF4 is a substrate by ADP-riibosylation under oxidaative stress condition, cells were
mM H2O2 with or w
without 10μ
μM PJ-34 for 30 miin. Then ATF4
A
was
treated with 0.3 m
immunooprecipitatedd and subbsequently ADP-ribosyylation of the proteiin was deetected by
immunooblot analysis using an poly(ADP-rribose) antibbody. Analyyzing the AD
DP-ribosylaated ATF4
verified that oxidaative stress induced P
PARP-1 actiivation triggger ADP-ribosylation of ATF4
(Fig.4).
Fig.4.
6 DNA binnding capaccity of ATF44 under oxiddative stresss induced AD
DP-ribosylaation
DAPA eexperimentss were perfformed to aanalyze thee ATF4 trannscription ffactor bindiing to the
respectivve CRE in 0.3 mM H2O2 treated cells with or without 10μM PJ-34 for 30 m
min. Probes
containiing consensuus sequencees of CRE and
a CRE m
mutation werre used to cclarify the ccomponent
binding to the CRE
E sites. Increeases in the binding of ATF4 to thhe CRE site after PJ-344 treatment
d
unnder oxidattive stress
and PJ--34, H2O2 co-treatmennt were ideentified, buut it was decreased
conditioon (Fig.5.).
Fig. 55.
7 Possiblee model for the cytoplassmic regulaation of cell survival by PARP inhibbition underr oxidative
stress viia ATF4 andd MKP-1.
Cytoplasmic and nuuclear evennts under PA
ARP inhibittion (Fig.6.)). Arrows: aactive proceesses; flatended arrrows: inhibbition; P: phhosphorylateed.
Fig.6.
This graant is given for 3 years, however, ddue to personal reasons I am unablee to continuue working
on the pproject from
m the end off the first yeear. Therefoore, the grannt has beenn ended in aaccordance
with thee rules.
Majorityy of the expperiments ddetailed in the
t researchh proposal have
h
been carried
c
out dduring the
first yeaar. Due to prremature ennding, the yeearly scheduuling of reseearch proposal has beenn modified
and fundds allocatedd for confereence attendaance were innstead used for purchasing chemicaals.
Results of the proj
oject will be submittedd for publication at a later time; details off accepted
publicattions will bee sent to the OTKA com
mmittee therreafter. 8