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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