cyclooxygenase 2 inhibits sapk activation in neuronal apoptosis
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Cyclooxygenase 2 inhibits SAPK activation in neuronal apoptosis
Bradley Miller, Yu-Wen E. Chang,1 and Andrey Sorokin*
Department of Medicine, Cardiovascular Research Center, Medical College of Wisconsin, Milwaukee, WI 53226-0509, USA
Received 9 December 2002
Abstract
Cyclooxygenase 2 (COX-2) expressed in cultured neuronal PC12 cells under inducible promoter protects cells from trophic
withdrawal apoptosis. Stimulation of SAPK is thought to play a significant role in initiation of PC12 cell death. We have therefore
examined whether COX-2 expression inhibits trophic withdrawal-mediated activation of SAPK. SAPK activity increased during the
first 6 h after NGF removal in mock-transfected PC12 cells. COX-2 expression attenuated the increase of SAPK, as detected by
Western blot analysis with phosphorylation state specific anti-SAPK antibodies and by SAPK activity assays. We propose that
COX-2 attenuated SAPK activation by preventing activation of nNOS, which occurs, as we have shown before, via COX-2-
mediated expression of dynein light chain (DLC). Activation of SAPK in neuronal cell death was attenuated by DLC expression.
These observations support a role for NO production and SAPK activation in the neuronal death mechanisms.
� 2002 Elsevier Science (USA). All rights reserved.
Keywords: Cyclooxygenase 2; Nitric oxide; Apoptosis; SAPK/JNK; PC-12 cells; Dynein light chain; NOS
Cyclooxygenase 2 (COX-2) has emerged as a key
player in the regulation of apoptosis in many types ofcells, including macrophages [1], renal medullary inter-
stitial cells [2], prostate cancer cell lines [3,4], esophageal
adenocarcinoma cells [5], pancreatic cancer cells [6], and
PC12 rat pheochromocytoma cells [7]. To date, several
distinct mechanisms have been proposed to explain the
anti-apoptotic effect of COX-2, namely: (a) arachidonic
acid depletion,which prevents the productionof ceramide
and activation of neutral sphyngomyelinase [8]; (b)modulation of expression of the anti-apoptotic protein
Bcl-2 [9,10]; (c) enhancement of Akt activation [11,12];
and (d) counteracting NO-mediated apoptotic cell death,
either via modulation of expression of prosurvival gene
dynein light chain (DLC) (also termed protein inhibitor of
nNOS), coupled to inhibition of NO production [13], or
via regulation of cellular susceptibility toward NO [14].
SAPK and p38 MAPK pathways are two majormammalian mitogen-activated protein kinase (MAPK)
signaling pathways activated by environmental stress
and inflammatory cytokines, which participate in reg-ulating apoptosis [15]. Trophic withdrawal apoptosis in
PC12 cells or primary cultures of neurons results in
activation of SAPK and p38 MAPK pathways [16,17].
It has been suggested that NGF withdrawal in PC12
cells recruits SAPK, resulting in AP-1-regulated ex-
pression of Fas ligand [10,16]. Inhibition of SAPK/JNK
by overexpression of the JNK binding domain of
scaffolding protein JIP-1 prevents apoptosis in sympa-thetic neurons and provides compelling evidence that
SAPK point of action in preventing apoptotic death
induced by NGF withdrawal lies upstream of mito-
chondrial dysfunction [18]. The molecular details of
initiation of mammalian stress-activated signal trans-
duction pathways have only begun to be dissected [19]
and the exact mechanisms of NGF withdrawal-depen-
dent SAPK and p38 MAPK activation remain to beelucidated.
Our studies suggest that generation of NO by
nNOS is partially responsible for SAPK activation
in PC12 cells following NGF withdrawal and that
COX-2-dependent overexpression of DLC is the
mechanism of cell protection from NO-mediated
SAPK activation.
Biochemical and Biophysical Research Communications 300 (2003) 884–888
www.elsevier.com/locate/ybbrc
BBRC
* Corresponding author. Fax: 1-414-456-6515.
E-mail address: sorokin@mcw.edu (A. Sorokin).1 Present Address: Pierce Milwaukee LLC, Milwaukee, WI 53202-
1009, USA.
0006-291X/02/$ - see front matter � 2002 Elsevier Science (USA). All rights reserved.doi:10.1016/S0006-291X(02)02947-9
Materials and methods
Cell culture. Stably transfected PC12 cell lines PC-MT (mock-
transfected) and PCXII (expressing COX-2 under isopropyl-1-thio-b-
DD-galactopyranoside (IPTG)-inducible promoter), constructed with
the lacSwitch gene expression system (Stratagene) [7] and stable cell
lines PC-Off (mock control) and PC-DLC (overexpressing DLC),
constructed with the retroviral gene delivery and expression system
RevTet-Off (Clontech) were cultivated as described previously [13].
Apoptosis. Cells were washed twice in serum-free DMEM and in-
cubated in either NGF differentiation medium for control or serum
free DMEM with rabbit neutralizing antibody to 2.5s NGF (diluted
1:500; Sigma) for the times indicated. The nNOS inhibitor N 5-(1-im-ino-3-buteny)-LL-orthine (LL-VNIO) (a gift from Owen Griffith, Medical
College of Wisconsin) [20] was added to cells as indicated. Cells were
harvested in lysis buffer and subjected to Western blot analysis.
SAPK/JNK activation assays. Lysates containing 150lg proteinwere immunoprecipitated with 10ll JNK-1 antibody (goat; SantaCruz) for 1.5 h at 4 �C prior to the addition of GammaBind Sepharose(Amersham Pharmacia) and incubation for an additional 1 h. Immu-
noprecipitates were washed twice in lysis buffer and twice in kinase
assay buffer containing 20mM Hepes, pH 7.4, 10mM MgCl2, and
0.2mM sodium orthovanadate and then incubated for 15min at 30 �Cin assay buffer containing 50lMATP, 5lCi [c-32P]ATP, and 0.035mg/ml ATF-2. The reaction was terminated by the addition of Laemmli
buffer and the proteins were subjected to SDS–PAGE electrophoresis
and visualized by autoradiography. Total SAPK/JNK activation as-
says were carried out using SAPK/JNK Assay Kit (Cell Signaling)
according to manufacturer�s instructions. This assay selectively mea-sures the total activity of all 10 isoforms of SAPK/JNK. Briefly 250lglysates were incubated overnight at 4 �C with 2lg c-Jun fusion proteinbeads and beads were precipitated, washed, and incubated in kinase
buffer supplemented with ATP for 30min at 30 �C. The reaction wasterminated by addition of Laemmli buffer and analyzed with phospho-
c-Jun (Ser63) immunoblot.
Western blot analysis. Cells were washed and harvested in lysis
buffer as described previously [21]. Cleared total cell lysates (20–40lg)were separated by Criterion SDS–PAGE (Bio-Rad) and transferred to
nitrocellulose membranes. Primary antibody incubations were carried
out at 4 �C overnight. Antibodies against COX-2 (N20), antibodiesagainst caspase-3 (H227), and against phospho-SAPK [p-JNK (G-7)
mouse monoclonal antibody (sc-6254)] were from Santa Cruz. Anti-
bodies against phospho-p38 were from New England Biolabs. Anti-
bodies against DLC (PIN) were either provided by Samie R. Jeffrey of
John Hopkins University or purchased from Santa Cruz. Appropriate
secondary antibody (HRP-conjugated, Bio-Rad) incubation was fol-
lowed by development with enhanced chemiluminescence (Amersham
Pharmacia).
Results and discussion
Overexpression of COX-2 inhibits activation of stress-
activated protein kinases in PC12 cells subjected to
trophic withdrawal apoptosis
Opposing effects of ERK and SAPK/p38 MAPK on
NGF-withdrawal apoptosis in PC12 cells have been es-tablished [17]. We have investigated whether COX-2
expression affects activation of the three major mam-
malian MAPK. SAPK and p38 MAPK activities
increased during the first 6 h after NGF removal in
Fig. 1. COX-2 inhibits activation of SAPK/JNK and p38 pathways upon NGF-withdrawal. (A) Differentiated PC-MT and PCXII cells were treated
in serum-free medium with anti-NGF at dilution 1:500 for the indicated periods of time (hours). Lysates from PC-MT and PCXII cells were resolved
by SDS–PAGE and immunoblotted with phosphorylation state specific polyclonal antibodies against either SAPK/JNK, or p38 MAPK, as well as
with antibodies against Cox-2, followed by Western blotting with antibodies recognizing p54 SAPK and p38 MAPK independent of phosphorylation
status. Positions of Cox-2, phosphorylated SAPK isoforms p54 and p46 (p-p54 and p-p46) as well as phosphorylated p38 MAPK (p-p38) are in-
dicated on the right. Experiment was repeated three times. (B) Lysates from PC-MT and PCXII cells treated in serum-free medium with anti-NGF at
dilution 1:500 for 6 h were subjected to SAPK activation assays. Immunoprecipitates obtained with JNK-1 antibodies were exploited for SAPK
immunocomplex kinase assay, using a downstream target for SAPK/JNK ATF-2, as a substrate (upper panel). Total SAPK/JNK activity was
assayed using c-Jun fusion protein beads and phospho-c-Jun antibodies (lower panel). Positions of c-Jun and ATF-2 are indicated on the right.
Assays were repeated three times and representative experiments are shown. Results were evaluated using densitometry analysis and are presented in
arbitrary units below the corresponding panels. (C) PCXII cells were preincubated with indomethacin (10lM, 24 h) before they were treated inserum-free medium with anti-NGF at dilution 1:500 for the indicated periods of time (hours). Cell lysates were resolved by SDS–PAGE and im-
munoblotted with phosphorylation state specific polyclonal antibodies against SAPK/JNK. Positions of phosphorylated SAPK isoforms p54 and p46
(p-p54 and p-p46) are indicated on the right. Experiment was repeated two times.
B. Miller et al. / Biochemical and Biophysical Research Communications 300 (2003) 884–888 885
PC-MT cells (Fig. 1). COX-2 expression attenuated theincrease of SAPK and p38 MAPK signaling, as detected
by Western blot analysis with phosphorylation state
specific anti-SAPK and anti-p38 MAPK antibodies
(Fig. 1A) and by SAPK activity assays using either
ATF2 or c-Jun fusion protein as substrates (Fig. 1B).
This inhibitory effect of COX-2 expression was nullified
by pretreatment with cyclooxygenase inhibitor indo-
methacin (Fig. 1C). NGF withdrawal was reported tolead to inhibition of ERK in PC12 cells [17]. In PC-MT
cells we also observed some decrease of ERK activity,
detected by kinase assay using myelin basic protein as a
substrate for immunoprecipitated ERK1. In PCXII cells
expressing COX-2 there was no significant change in
ERK activity (data not shown). Our data are in full
accord with published observations that agents, which
inhibit the activation of JNK/SAPK and p38 MAPKsignaling pathways, while ERK is not suppressed, pro-
tect neurons against apoptosis [22].
The COX-2 effect was shown to be specific for certain
SAPK-activating stimuli such as NGF-withdrawal. The
ability of TNFa or such stress agents as anisomycin,sorbitol, and wortmannin to stimulate SAPK and p38
MAPK kinases was not affected by COX-2 overexpres-sion in PC12 cells (Fig. 2). These observations support
the role of COX-2 in regulation of upstream events in-
volved in activation of SAPK in neuronal cells under-
going apoptosis and argue against the possibility of
direct attenuation of SAPK function/activity by COX-2.
COX-2 effect upon SAPK is mimicked by overexpression
of dynein light chain
Although, like many other critical regulators of cell
metabolism, COX-2 is likely to exert its effects through
several effectors, we have previously identified inhibition
of nNOS activity achieved by COX-2-mediated regula-
tion of DLC expression as one of the major factors in
COX-2 ability to inhibit trophic withdrawal apoptosis in
PC-12 cells [13]. As we have shown previously, COX-2
expression controls association of the nNOS monomersinto dimers via regulation of expression of DLC [13]
(only dimers of all NOS isoforms represent active forms
of enzymes, whereas monomers are inactive [23]). In
PC12 cells DLC expression can be stimulated by addi-
tion of prostaglandins, products of COX-2 enzymatic
activity. In order to explore whether COX-2 effect upon
SAPK activation is mediated by induced DLC expres-
sion, we compared SAPK activation after NGF with-drawal in PC-Off (parental) cells and in PC-DLC cells,
which are characterized by overexpression of DLC,
obtained with the help of the retroviral gene delivery
and expression system RevTet-Off (Clontech) [13]. Ac-
tivation of SAPK was attenuated by DLC overexpres-
sion (Fig. 3), arguing that with all probability COX-2
effect is indeed mediated via inhibition of nNOS activity.
It is of note that NO induces SAPK activation inparallel with induction of apoptosis in cardiomyocytes
[24]. We have established that LL-VNIO, a potent and
selective inhibitor for nNOS, is able to prevent upreg-
Fig. 2. COX-2 does not inhibit SAPK/JNK and p38 MAPK activation
in response to TNFa and stress agents. Undifferentiated PC-MT andPCXII cells were incubated with IPTG for 2 days to induce COX-2
expression. Thereafter, cells were either incubated in growth medium
and left untreated or were treated with 1% horse serum DMEM for 2 h
prior to addition of the following stress agents: wortmannin (100 nM,
30min) (Wort); TNFa (10 ng/ml, 15min); anisomycin (100 nM, 30min)(Aniso); and sorbitol (300mM, 15min) (Sorb). Lysates from PC-MT
(upper panel) and PCXII (lower panel) cells were resolved by SDS–
PAGE and immunoblotted with phosphorylation state specific poly-
clonal antibodies against either SAPK/JNK, or p38 MAPK, as well as
with antibodies recognizing SAPK and p38 MAPK independent of
phosphorylation status. Positions of phosphorylated SAPK isoforms
p54 and p46 (p-p54 and p-p46) as well as phosphorylated p38 MAPK
(p-p38) are indicated on the right. Experiment was repeated two times.
Fig. 3. DLC overexpression suppresses SAPK/JNK phosphorylation
upon NGF-withdrawal. Lysates from differentiated PC-Off and PC-
DLC cells treated in serum-free medium by anti-NGF (1:500 dilution)
for 3 and 6 h were subjected to SDS–PAGE and immunoblotted with
phosphorylation state specific SAPK antibodies followed by Western
blotting with antibodies recognizing p54 SAPK independent of phos-
phorylation status. Expression of DLC was also determined by Wes-
tern blotting with corresponding antibodies. The positions of DLC and
SAPK isoforms are indicated on the right. Experiment was repeated
six times.
886 B. Miller et al. / Biochemical and Biophysical Research Communications 300 (2003) 884–888
ulation of SAPK in NGF-deprived PC-MT cells (Fig. 4).
Moreover, treatment of PC-MT cells with LL-VNIO in-
hibited activation of caspase-3 after NGF withdrawal
(Fig. 4). Taken together our data suggest that nNOS
activity plays an important role in activation of SAPK
in differentiated PC12 cells induced by NGF withdrawal
to apoptosis.As mentioned earlier, COX-2 is anti-apoptotic in
many types of cells, but the case of nervous system is
more complex. COX-2 was shown to contribute to the
pathophysiology of age-related diseases by promoting
neuronal apoptosis in an age-dependent manner in
transgenic mice generated to express COX-2 in neurons
[25]. On the contrary in cultured PC12 cells expressing
COX-2 under inducible promoter the pro-survival effectof COX-2 is revealed by multiple methods [7,13]. This
contradiction between effects of COX-2 in PC12 cells
and transgenic mouse model in which COX-2 is over-
expressed constitutively in neurons can be probably at-
tributed to the difference in the experimental systems in
which COX-2 regulatory effects were studied. It must be
borne in mind that pro-apoptotic action of COX-2
overexpressed constitutively in mice neurons was re-vealed only at 14 months of age. At 8 months of age
there was no difference between transgenic and non-
transgenic littermates in the number of apoptotic cells
[25].
We suggest that activation of SAPK is mediated by
generation of NO and COX-2 prevents activation of
nNOS and, as a result, activation of SAPK. We have
previously demonstrated that COX-2-regulated expres-sion of DLC is responsible for blunting the activation
of nNOS enzymatic activity in PC12 cells undergoing
apoptosis [13]. It must be mentioned that although ourdata and data from other laboratories support the idea
that NO upregulates SAPK activity [24,26,27], the role
of NO-mediated activation of SAPK in regulation of
apoptosis is contradictory. While NO-donor (SNAP)
induced apoptosis in cardiomyocyte via SAPK-medi-
ated AP-1 activation [24,26], SAPK activation induced
by another NO donor protected cardiocytes cells from
NO-mediated cell death [27]. Furthermore, NO wasreported to negatively regulate SAPK by means of
S-nitrosylation [28]. These contradictions notwith-
standing, in PC12 cells and in sympathetic neurons the
induction of SAPK activity is clearly a pro-apoptotic
factor, contributing to the cell death induced by NGF
withdrawal [17,18,29].
In summary, our data show that COX-2 expression in
PC12 cells, while inhibiting trophic withdrawal apop-tosis, blunts SAPK and p38 MAPK activation. We
propose that in PC12 cells undergoing apoptosis, SAPK
activation is partially triggered by NO generation by
nNOS. Further experiments will clarify whether COX-2
will have similar effects in primary sympathetic neurons.
Acknowledgments
This work was supported by National Institutes of Health Grants
HL22563 and DK41684 (to A.S.). Authors thank Dr. Michael J. Dunn
(Medical College of Wisconsin, Milwaukee, USA) for his continuous
support and advice. Current address of Yu-Wen E. Chang is Pierce
Milwaukee LLC, Milwaukee, WI 53202-1009.
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