Redox regulates COX-2 upregulation and cell death in the neuronal response to cadmium

www.elsevier.com/locate/cellsig

Cellular Signalling 16 (2004) 343–353

Redox regulates COX-2 upregulation and cell death in the neuronal

response to cadmium

Patricia Rockwell*, Jennifer Martinez, Luena Papa, Evan Gomes

Department of Biological Sciences, Hunter College of The City University of New York, 695 Park Ave., New York, NY 10021, USA

Received 9 August 2003; received in revised form 9 August 2003; accepted 19 August 2003

Abstract

We reported previously that cadmium, an oxidative stressor, induced cyclooxygenase-2 (COX-2) upregulation in mouse neuronal cells

that culminated in cell death. Herein, we show that cadmium induces reactive oxygen species (ROS) that activate c-Jun N-terminal kinase

(JNK) and p38 mitogen-activated protein kinase (MAPK) and their substrates, activating transcription factor 2 (ATF-2), CRE-binding protein

(CREB) and c-Jun. This response is accompanied by induction of heme-oxygenase-1 (HO-1), poly(ADP-ribose) polymerase cleavage and a

caspase-independent cell death. Inhibition of p38 MAPK, but not JNK, suppressed COX-2 protein expression and the cytotoxic response

induced by cadmium. Selective inhibitors of phosphatidylinositol-3-kinase (PI3-K), LY294002, and flavoproteins, dipheneylene iodonium

chloride (DPI), attenuated cadmium-induced ROS and stress kinase activation, suggesting that ROS can signal the COX-2 upregulation and

neuronal cell death mediated by p38 MAPK. Collectively, these findings implicate PI3-K, a flavoprotein, p38 MAPK and COX-2 in a

neuronal redox-regulated pathway that mediates cadmium-induced oxidative stress.

D 2003 Elsevier Inc. All rights reserved.

Keywords: Cadmium; Oxidative stress; Reactive oxygen species; Cyclooxygenase-2; Caspase; Stress-activated kinases

1. Introduction

Increasing evidence attributes the cellular damage in

neurodegenerative disorders such as Alzheimer’s disease

(AD) to oxidative stress [1]. Under pathological condi-

tions, excessive amounts of ROS can modify proteins,

lipids and DNA and alter their function. Alternatively,

ROS can serve as second messengers of redox-sensitive

signaling pathways [2]. Thus, oxidative stress may disrupt

neuronal cell homeostasis through aberrant gene expres-

sion from ROS-activated signaling pathways. However, the

mechanisms that contribute to these events are not well

characterized.

0898-6568/$ - see front matter D 2003 Elsevier Inc. All rights reserved.

doi:10.1016/j.cellsig.2003.08.006

Abbreviations: AD, Alzheimer’s disease; ATF-2, activating transcrip-

tion factor 2; Cd2+, cadmium; COX-1 and COX-2, cyclooxygenase-1 and

cyclooxygenase-2; CREB, CRE-binding protein; DPI, dipheneylene

iodonium chloride; ERK1/2, extracellular signal-regulated kinases; HO-1,

Heme oxygenase 1; JNK, c-Jun NH2-terminal kinase; NAC, N-acetyl-

cysteine; PI3-K, phosphatidylinositol-3-kinase; PGE2, prostaglandin E2.

* Corresponding author. Tel.: +1-212-650-3234; fax: +1-212-772-

5227.

E-mail address: [email protected] (P. Rockwell).

There is growing evidence that members of the mitogen-

activated protein kinase (MAPK) family may play a central

role in neurodegeneration (reviewed in Ref. [3]). MAPK

signaling cascades comprise a highly conserved cascade of

proline-directed serine/threonine kinases connecting cell

surface receptors to regulatory targets in response to various

stimuli [3]. Mammals express at least three distinct groups of

MAPKs: extracellular signal-regulated kinases (ERK)-1/2,

c-Jun NH2-terminal kinases (JNK) and p38 MAPK that are

activated by specific upstream MAPK kinases. In neuronal

cells, the activation of ERK1/2 is mainly associated with

cellular proliferation, differentiation and development in

response to growth factors. In contrast, the JNK and p38

MAPK signaling cascades are activated by environmental

stress and inflammatory cytokines and have been shown to

promote neuronal cell death [4]. The JNK and p38 MAPK

signaling pathways can also be strongly activated by stress-

induced ROS production or a mild oxidative shift of the

intracellular thiol/disulfide redox state [5]. Upon phosphor-

ylation, JNK can mediate activation of transcription factors

such as, c-Jun, ATF-2 and ELK-1 whereas activated p38

MAPK can target substrates that include ATF-2, and CREB.

Consequently, the magnitude and duration of JNK and p38

P. Rockwell et al. / Cellular Signalling 16 (2004) 343–353344

MAPK signaling cascades induced by harmful stimuli may

play an important role in the physiological outcome of the

neuronal stress response. Both JNK and p38 MAPK were

implicated as contributors to neurodegeneration by their

ability to mediate intracellular stress events in transgenic

mouse models of AD [6,7]. There is also substantial evi-

dence that the onset of neurodegeneration results from an

inflammatory response involving cyclooxygenase-2 (COX-

2) and its proinflammatory product, prostaglandin E2

(PGE2), which can be induced by different regulatory path-

ways including p38 MAPK [8,9]. Furthermore, p38 MAPK

activation and COX-2 induction are implicated as contrib-

utors to neuronal damage in AD in response to oxidative

stress [10,11]. More recently, the proinflammatory cytokine,

interleukin (IL)-1alpha was shown to induce COX-2 in a

ROS-dependent manner in nonneuronal cells [12].

Using a mouse neuronal model system, we showed

previously that cadmium (Cd2 +), a potent mediator of

oxidative stress, induced COX-2 upregulation that contrib-

uted to cell death [13]. Cadmium also induced glutathione

depletion and lipid peroxidation, suggesting that cellular

redox changes and ROS mediated the cytotoxic effect of the

heavy metal. However, the regulatory intermediates linking

these events are unknown. Herein, we show that the

neuronal response to Cd2 + is accompanied by increased

ROS production, HO-1 induction and sustained phosphor-

ylation of the stress-activated kinases, JNK and p38 MAPK,

and their downstream targets, c-Jun, ATF-2 and CREB. A

blockade of p38 MAPK function reduced neuronal COX-2

protein expression mediated by Cd2 + and promoted cell

survival whereas JNK activity was dispensable for cell

death. The loss in cell viability induced by p38 MAPK

involved caspase-dependent apoptosis and cell death by a

caspase-independent mechanism. Inhibitors of PI3-K

(LY294002) and NADPH oxidase-like flavoproteins, DPI,

suppressed ROS production and the stress response induced

by Cd2 +. Together, our data suggest that a signaling cascade

comprising PI3-K, a flavoprotein and p38 MAPK mediate

COX-2 upregulation and cell death mechanisms induced by

Cd2 + in a redox-dependent manner.

2. Materials and methods

2.1. Materials

N-acetyl-cysteine (NAC) and CdSO4 (Cd2 +) were pur-

chased from Sigma. Fetal bovine serum, Dulbecco’s mod-

ified Eagle’s medium, hygromycin and geneticin were from

Invitrogen Life Technologies (Carlsbad, CA). SP-600125

was from Biomol Research Laboratories (Plymouth Meet-

ing, PA). SB202190, z-VAD-fmk and NS398 were from

Calbiochem. 2V,7V-dichlorofluorescein-diacetate (DCF-DA)was purchase from Molecular Probes (Eugene, OR). En-

zyme immunoassay reagents for PGE2 assays were from

Cayman Chemical (Ann Arbor, MI). Anti-human Goat

polyclonal COX-2 and HO-1 were from Santa Cruz Bio-

technology, (Santa Cruz, CA). Rabbit antibodies to phos-

phorylated and nonphosphorylated forms of ERK1/2, JNK,

p38 MAPK, c-Jun, ATF-2 and CREB, the cleaved form of

mouse PARP, were from Cell Signaling Technology (Bev-

erly, MA) as well as the p38 MAPK in vitro kinase assay

and secondary antibodies conjugated to horse radish perox-

idase. Western blotting detection reagents and nitrocellulose

membranes were from Pierce Endogen (Rockford, IL). The

Cell Titer Assay System for cell viability was from Promega

(Madison, WI) and the Caspase 3/7 Whole Cell Assay Kit

was from Beckman Coulter (Fullerton, CA).

2.2. Cell cultures

HT4 cells are a mouse hippocampal cell line immortal-

ized with a recombinant temperature sensitive mutant of

SV40 large T antigen [13]. The cells are maintained at 33

jC in Dulbecco’s modified Eagle’s medium containing 5%

normal fetal bovine serum, and 100 units/ml penicillin, 100

Ag/ml streptomycin in 5% CO2 and cultured as previously

described [13]. To induce differentiation, the cells are

transferred to 39 jC for 3 days followed by a transfer to

37 jC for experimental treatments.

2.3. Cell treatments

HT4 cells were plated in 10-cm plates at a concentration

of 5� 105 cells/ml and cultured as described above. The

culture medium was replaced and cells were pretreated with

the selective inhibitor, antioxidant or vehicle (0.5% DMSO)

as indicated for 1 h at 37 jC followed by the addition of

CdSO4 at the concentrations indicated for 24 or 40 h at 37

jC as described.

2.4. Cell viability assay

Cells were plated in 96-well microtiter plates at a con-

centration of 1�104 cells/well and cultured and pretreated

as described above. Following cell treatments for 40 h at 37

jC, the culture medium was replaced and cell survival was

determined using a colorimetric assay (Promega) that meas-

ures the cleavage of the tetrazolium salt MTS by mitochon-

drial dehydrogenases in viable cells.

2.5. Protein determination

Protein determinations were performed with a bicincho-

ninic acid assay according to manufacture’s instructions

(Pierce).

2.6. Preparation of cell extracts for Western blotting

Cells were treated for 24 h at 37 jC, washed twice with

phosphate-buffered saline, and then harvested in a lysis

buffer containing 20 mM Tris–HCl at pH 7.4, 150 mM

Fig. 1. Cd2 + treatments activate stress kinase pathways in HT4 neuronal

cells. Cells were incubated in the absence and presence of 3–30 AM Cd2 +

(A and B) as indicated. Following incubation for 24-h at 37 jC, cells weresubjected to immunoblot analysis as described in Materials and methods

using antibodies that specifically recognize phosphorylated (arrows) and

nonphosphorylated ERK1/2, JNK, p38 MAPK, CREB, ATF-2 and c-Jun.

P. Rockwell et al. / Cellular Signalling 16 (2004) 343–353 345

NaCl, 0.5% Nonidet P-40, 10% glycerol, 1 mM EDTA, 100

mM sodium fluoride, 10 mM sodium pyrophosphate, 4 mM

EDTA, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluo-

ride and supplemented with a Complete Protease Inhibitor

Cocktail tablet (Roche Diagnostics) according to manufac-

turer’s directions. Lysates were then centrifuged at

15,000� g for 10 min to sediment the particulate matter.

Equivalent amounts of protein (20 Ag) from each lysate

were resolved by SDS-polyacrylamide gel electrophoresis

under reducing conditions on 10% polyacrylamide gels and

then transferred to nitrocellulose membranes. Blots were

blocked and probed with the appropriate primary antibody

overnight at 4 jC. Blots were washed and incubated with a

secondary antibody to IgG conjugated to horseradish per-

oxidase. Antigens were detected by chemiluminescence

(Pierce). Where indicated, band intensities of scanned blots

were quantified using a Molecular Dynamics densitometer.

2.7. PGE2 assays

PGE2 levels in culture medium were determined using

an enzyme-linked immunosorbant assay (ELISA) kit (Cay-

man Chemical) according to the manufacturer’s protocol.

2.8. Measurement of phospho-p38 MAPK activity

In vitro kinase assays of p38 MAPK activity were ana-

lyzed in treated cells according to the manufacturer’s proto-

col. Briefly, cell extracts were incubated overnight with an

immobilized anti-phospho-p38 MAPK (Thr180/Try182)

bound to agarose beads. Immunoprecipitated phospho-p38

MAPK was assayed in vitro in the presence of 100 AM cold

ATP and 2 Ag ATF-2 fusion protein as a substrate. Phosphor-ylation of ATF-2 was measured by Western blotting using an

antibody that detects phosphorylation of ATF-2 at Thr71.

2.9. Measurements of ROS generation

Cells were plated as described for the cell viability assays.

To detect accumulation of OH� radicals, H2O2 or their

downstream free radical products, medium was removed

and cells were washed twice with PBS followed by the

addition of the fluorescent dye 2V,7V-dichlorofluorescein-diacetate (H2DCF-DA, Molecular Probes) at 10 Ag/ml. After

incubation at 37 jC for 10 min, cells are analyzed and

quantified for green fluorescence using the Molecular Dy-

namics Typhoonk 9410 Imaging System with ImageQuant

software (Amersham Pharmacia Biotech). Mean fluorescent

data are determined as units/Ag protein and expressed as the

percent increase over untreated control samples (treated/

untreated� 100) from at least three independent experiments.

2.10. Measurements of caspase 3/7 activity

Cells were plated as described for cell viability assays.

Caspase-3/7 activity was by measured using a cell-based kit

(Beckman Coulter) according to the manufacturer’s direc-

tions. Substrate utilization was measured by fluorescence

and quantified using the Molecular Dynamics Typhoonk9410 Imaging system with ImageQuant software (Amer-

sham Pharmacia Biotech). Activity was determined as

described under ROS measurements.

2.11. Statistical analyses

Data are expressed as the meanF S.E.M. of experiments

that were performed in triplicate and replicated at least three

times. Effects were evaluated with one-way analysis of

variance (ANOVA) followed by pairwise contrasts (Dun-

can’s t-test). Overall statistical significance required p < 0.05

and the required level for multiple contrasts was adjusted

lower using a Bonferroni approach.


3. Results

3.1. JNK and p38 MAPK are activated in response to Cd2+

To explore whether MAPK signaling pathways are

activated by Cd2 +, cell lysates were prepared from HT4

cells treated with increasing concentrations of CdSO4 (3–

30 AM) and analyzed by Western blotting for the phos-

phorylation of ERK1/2 at Thr202/Tyr204, JNK at Thr183/

Tyr185 and p38 MAPK at Thr180/Tyr182 using phospho-

specific antibodies. At concentrations of 15 and 30 AM,

Cd2 + induced increased phosphorylation of p38 MAPK and

the p46 and p54 JNK isoforms, but not ERK1/2 (Fig. 1A).

The antibody recognizing phosphorylated JNK also

detected an unknown immunoreactive protein (f p50) that

was consistently observed in untreated and treated HT4

cells. Cd2 + also induced a concentration-dependent phos-

phorylation of the stress kinase substrates, c-Jun at Ser63

and ATF-2 at Thr71 and CREB phosphorylation at Ser133

(Fig. 1B). A reprobing of blots indicated equal amounts of

total protein per lane except for c-Jun where the total and

activated protein levels increased in a coordinated manner.

Fig. 2. Selective inhibition of p38 MAPK by SB202190 suppresses COX-2 prote

cells. (A) Cells were treated in the absence (Control) and presence of 15 AM of C

(Cd/SP) or 10 AM SB202190 (Cd/SB). Following 24-h incubation at 37 jC, cellsCOX-2. (B) PGE2 levels were measured for the cell treatments indicated using

increase in PGE2 (pg/mg) relative to untreated controls. (C) Activated p38 MAPK

kinase assay in the absence and presence of SB202190 using an ATF-2 fusion prot

an antibody that detects phosphorylated ATF-2. Data are representative of three ind

1-h pretreatments with the selective inhibitors as indicated. Following a 40-h incub

Materials and methods. Data represent the F S.E.M. of the percent cell viability rel

three independent experiments. The asterisk (*) indicates values that are significan

These results most likely reflect the autoregulatory mecha-

nism characteristic of activated c-Jun [14]. Interestingly, the

apparent induction of stress kinase-related proteins at 15

AM Cd2 + (Fig. 1A and B, lane 4) is coincident with the

high induction levels of COX-2 observed previously [13].

Given these results, subsequent cell treatments were per-

formed with 15 AM Cd2 +.

3.2. p38 MAPK mediates COX-2 protein expression, PGE2

production and cell survival in Cd2+-treated cells

To investigate the relationship between stress kinase

activation and COX-2 upregulation, HT4 cells were pre-

treated with pharmacological antagonists of p38 MAPK

(SB202190) and JNK (SP-600125) prior to exposure to 15

AM Cd2 +. The results showed that selective inhibition of

p38 MAPK, but not JNK, reduced the COX-2 protein levels

induced by Cd2 + (Fig. 2A). These findings are consistent

with reports that activated p38 MAPK can modulate COX-2

protein expression in various cell types [15–17]. To deter-

mine whether pretreatments with SB202190 mediated a

corresponding loss in COX-2 activity, culture medium from

in expression, and PGE2 levels and promotes cell survival in Cd2 +-treated

d2 + alone (Cd) and following pretreatments for 1 h with 10 AM SP-600125

were subjected to immunoblot analyses using an antibody that recognizes

an ELISA as described in Materials and methods. Data represent the fold

was immunoprecipitated from Cd2 +-treated cells and subjected to an in vitro

ein as substrate. Kinase activity was detected by immunoblot analysis using

ependent experiments. (D) Cells were incubated with 15 AMCd2 + following

ation at 37 jC, cell viability was measured by the MTS assay as described in

ative to their respective controls in the absence of Cd2 + (100%) from at least

tly different ( p< 0.05) from cells treated with Cd2 +.

Fig. 3. p38 MAPK and JNK induced transcription factors are activated in

response to Cd2 +. Cells were incubated in the absence (lane 1) and

presence of 15 AM Cd2 + alone (lane 2) and following 1-h pretreatments

with SB202190 (SB; lane 3) or SP-600125 (SP; lane 4). After 24 h at

37 jC, cells were harvested for immunoblot analyses using antibodies

that specifically recognize the phosphorylated (arrows) forms of CREB,

ATF-2 and c-Jun (arrows). Data are representative of at least three

independent experiments.


treated cells was analyzed for its product, PGE2. A blockade

of p38 MAPK function abrogated PGE2 levels in Cd2 +-

treated cells to levels that were equivalent to those obtained

from pretreatments with NS398, a selective inhibitor of

COX-2 function (Fig. 2B). In vitro kinase assays confirmed

that phosphorylated p38 MAPK was a functional enzyme in

the presence of Cd2 + that was sensitive to inhibition by

SB202190 (Fig. 2C). For these experiments, activated p38

MAPK was selectively immunoprecipitated from treated

cells and shown to phosphorylate its substrate, an ATF-2

fusion protein, in the absence but not the presence of

SB202190. Since we showed previously that COX-2 inhi-

bition promoted cell survival, we investigated whether stress

kinase activation played a regulatory role in mediating

Cd2 +-induced cytotoxicity. Pretreatments with SB202190

but not the JNK inhibitor SP-600125 exerted a significant

reduction in neuronal cell death following prolonged (40 h)

Fig. 4. p38 MAPKmediates PARP cleavage and caspase activation in Cd2 +-

treated cells. (A) Cells were incubated in the absence and presence of 3–30

AM Cd2 + using the protocol described in the legend to Fig. 1. Lysates were

subjected to immunoblot analysis using an antibody that specifically

recognizes the cleaved form (89 kDa) of mouse PARP (arrow). (B) Cells

were incubated in the absence (lane 1) and presence of 15 AM Cd2 + (Cd)

alone (lane 2) and following 1-h pretreatments with SB202190 (SB; lane 3) or

SP-600125 (SP; lane 4). After 24 h at 37 jC, cells were harvested for

immunoblot analysis for detection of the cleaved form (89 kDa) of mouse

PARP (arrow). Densitometer measurements of PARP cleavage (units as

pixels) are shown below the blot. (C) Cells were treated with 15 AM Cd2 +

(Cd) alone or following pretreatments with 10 AMS20B2190 as described in

B and lysates were analyzed for caspase 3/7 activation as described in

Materials and methods. Fluorescence was measured and quantified using a

Molecular Dynamics Typhoon Phosphorimager. Normalized caspase

activities represent percent caspase activity (units/Ag protein) relative to

respective controls in the absence of Cd2 + (100%) from at least there

independent experiments. The asterisk (*) indicates values that are

significantly different ( p< 0.05) from cells treated with Cd2 +.

exposure to 15 AM Cd2 + (Fig. 2D). A further assessment of

putative signaling pathways associated with Cd2 +-induced

cytotoxicity showed that pretreatments with either H89, an

inhibitor of cAMP-dependent protein kinase A (PKA), or 8-

(4-chlorophenylthio)-cAMP, an analogue of cAMP that

inhibits JNK-mediated neuronal cell death, failed to reverse

the stress response induced by the heavy metal (data not

shown) [18]. Together, these data demonstrated a direct

regulatory link between p38 activity and the upregulation of

COX-2 and its proinflammatory product, PGE2, in neuronal

cells exposed to Cd2 +.

Fig. 5. Caspase inhibition blocks PARP cleavage but fails to promote

survival in Cd2 +-treated cells. (A) Cells were incubated in the absence and

presence of 15 AMCd2 + alone (lane 2) and following 1-h pretreatments with

50 AM z-VAD-fmk and incubated for 40-h at 37 jC. Cell viability assays

were performed using the MTS assay described in the legend to Fig. 2D.

(B) Cells were pretreated with the concentrations of z-VAD-fmk indicated

followed by the addition of 15 AM Cd2 +. After 24-h at 37 jC, cells wereharvested for immunoblot analyses with antibodies specific for cleaved

PARP, COX 2 and the phosphorylated forms of CREB and c Jun (arrows).

Fig. 6. NAC abrogates the stress response in Cd2 +-treated cells. (A) Cells

were incubated in the absence and presence of 3–30 AM Cd2 + using the

protocol described in the legend to Fig. 1. Lysates were subjected to

immunoblot analysis using antibodies that specifically recognize HO-1. (B)

Cells were incubated with 15 AM Cd2 + alone and following 1-h

pretreatments with increasing (1–20 mM) concentrations of NAC as

indicated. Following 24-h at 37 jC, cells were harvested and analyzed by

immunoblotting using antibodies that specifically recognize the phosphory-

lated (arrows) and nonphosphorylated forms of JNK and p38 MAPK as

well as antibodies that detect COX-2, COX-1, HO-1 and cleaved PARP.


3.3. p38 MAPK inhibition reduces the activation levels of

ATF-2, CREB and p-c-Jun in response to Cd2+

To further delineate stress kinase activation induced by

Cd2 +, we investigated which substrates were targeted by

JNK and p38 MAPK. Inhibition of p38 MAPK by

SB202190 decreased the Cd2 +-induced phosphorylation

of ATF-2 and CREB while JNK inhibition suppressed

phosphorylation of c-Jun, but not ATF-2 (Fig. 3). These

findings revealed that a loss in ATF-2 and CREB phos-

phorylation correlated with the SB202190-mediated de-

crease in the COX-2 upregulation and cytotoxicity induced

by Cd2 +.

3.4. Cd2+ induces caspase activation and caspase-inde-

pendent cell death in HT4 neuronal cell

Caspase activation is implicated as an important mech-

anism that triggers stress-induced neuronal cell death [19].

To investigate whether Cd2 + induced caspase activation in

our neuronal model system, HT4 cells were treated with

increasing concentrations (3–30 AM) and analyzed by

Western blotting for the cleaved form of poly(ADP-ribose)

polymerase (PARP), a substrate for caspase-3. PARP

cleavage was apparent at the same concentrations of

Cd2 + (15 and 30 AM) showing high induction levels of

stress-related proteins (compare Fig. 4A with Fig. 1).

Inhibitor studies revealed that a blockade of p38 MAPK

but not JNK activity in Cd2 +-treated cells decreased

PARP cleavage by 50% (Fig. 4B) and attenuated cas-

pase-3 and -7 activation (Fig. 4C). Pretreatments with the

pan caspase inhibitor, z-VAD-fmc, abrogated PARP cleav-

age but failed to promote cell survival, suggesting that

p38 MAPK mediated Cd2 +-induced neuronal cell death

through a caspase-independent pathway (Fig. 5A and B).

These results also indicated that the COX-2 upregulation

and c-Jun activation induced by Cd2 + were independent

events that are not a consequence of caspase activation

(Fig. 5B).

r Signalling 16 (2004) 343–353 349

3.5. The antioxidant enzyme HO-1 is a marker of the Cd2+-

induced stress response and is abrogated by N-acetylcys-

teine (NAC)

Our previous demonstration that pretreatments with 1

mM NAC alleviated COX-2 upregulation in HT4 neuronal

cells implicated ROS as a contributor to Cd2 +-induced

oxidative stress [13]. To monitor Cd2 +-induced changes in

intracellular redox, we addressed the possibility that HO-1, a

hallmark of oxidative stress and inflammation in nonneuro-

nal cells, was upregulated in HT4 neuronal cells [20].

Treatments with increasing concentrations of Cd2 + revealed

that HO-1 was induced in a pattern that correlated with

stress kinase activation, PARP cleavage and COX-2 upre-

gulation (compare Fig. 6Awith Figs. 1 and 4A). Since redox

can influence stress kinase activation we speculated that

NAC pretreatments would alleviate the Cd2 +-induced phos-

phorylation of the JNK and p38 MAPK signaling cascades.

Accordingly, pretreatments with increasing concentrations

(1–20 mM) of NAC completely suppressed the JNK and

p38 MAPK activation induced by Cd2 + at NAC concen-

trations of 5 mM and higher (Fig. 6B). These events were

accompanied by a concomitant loss in PARP cleavage and

HO-1 induction in a pattern that corresponded with the loss

in COX-2 but not COX-1 expression. Cell viability assays

P. Rockwell et al. / Cellula

Fig. 7. PI3-K mediates the Cd2 +-induced stress response including induction of th

presence of 15 AM Cd2 + alone and following 1-h pretreatments with AM concentra

37 jC, cell viability assays were performed using the MTS assay described in the

presence of 15 mM cadmium alone (lane 2) or cadmium following pretreatments w

prepared for immunoblot analyses using antibodies that detect phosphorylated p38

the absence (lane 1) and presence of 15 AM Cd2 + (Cd) alone (lane 2) and followin

Lysates were subjected to immunoblot analysis using an antibody that specificall

showed that each concentration of NAC promoted cell

survival in Cd2 +-treated cells (data not shown). These

results suggested that redox regulates the stress response

induced by Cd2 +.

3.6. PI3-K and flavoprotein inhibition attenuates the stress

response induced by Cd2+ in HT4 neuronal cells

To further explore the mechanism associated with Cd2 +-

induced oxidative stress, we investigated whether intracel-

lular redox changes in HT4 neuronal cells involved the lipid

kinase PI3-K. This approach was undertaken due to recent

evidence showing that PI3-K regulates oxidative stress in

neuronal cells by modulating HO-1 expression levels [21].

Cell viability experiments revealed that pretreatments with

selective inhibitors of PI3-K, LY294002 or wortmannin,

promoted cell survival in Cd2 +-treated cells to 75–100% of

the untreated controls (Fig. 7A). Western blot analyses of

LY294002-pretreated cells revealed that PI3-K inhibition

diminished the induction levels of HO-1, COX-2 but not

COX-1 protein expression, and suppressed the activation

levels of p38 MAPK, c-Jun and PARP cleavage in response

to Cd2 + (Fig. 7B). Furthermore, pretreaments with

SB202190 also elicited a partial reduction in HO-1 protein

levels, supporting the notion that p38 MAPK serves, in part,

e antioxidant enzyme, HO-1. (A) Cells were incubated in the absence and

tions of the selective inhibitors as indicated. Following a 40-h incubation at

legend to Fig. 2D. (B) HT4 cells were incubated in the absence (lane 1) and

ith the 10 AM LY294002 (LY; lane 3). After 24-h at 37 jC, cell lysates wereand c-Jun, COX-2, COX-1 and cleaved PARP. (C) Cells were incubated in

g 1-h pretreatments with SP-600125 (SP; lane 3) or SB202190 (SB; lane 4).

y recognizes HO-1 (arrow).


as a downstream mediator of PI3-K-mediated regulation of

the antioxidant enzyme (Fig. 7C). In contrast, JNK inhibi-

tion had no effect on HO-1 induction in response to Cd2 +.

Our findings are in accordance with the demonstration that

PI3-K upregulates HO-1 when ROS levels are increased

[21]. Moreover, both PI3-K and COX-2 inhibition by

LY294002 and NS398, respectively, suppressed caspase-3

and -7 activity in Cd2 +-treated cells to levels that resembled

those obtained with the p38 MAPK inhibitor SB202190

(Fig. 8A), implicating their participation in the same stress-

activated pathway.

We then examined the effects of the flavoprotein inhib-

itor, DPI on the ROS production induced by Cd2 + in

comparative studies with LY294002 and NS398. The results

in Fig. 8B show that Cd2 + induced a twofold increase in

ROS that was reduced significantly by pretreatments with

LY294002, DPI, SB202190 and NS398. Comparative West-

ern blot analyses between cells pretreated with 0.1 AM DPI

and 10 AM SB202190 showed that DPI suppressed COX-2

upregulation ATF-2 activation and PARP cleavage to levels

Fig. 8. Caspase 3/7 activation and ROS production are induced by Cd2 + throug

absence and presence of 15 AM Cd2 + alone and following 1-h pretreatments wi

Normalized casapse activities were determined as described in the legend to Fig

described in A. To measure ROS, cells were washed twice with PBS followed

(H2DCF-DA, Molecular Probes) at 10 Ag/ml and incubated at 37 jC for an addition

fluorescence using the Molecular Dynamics Typhoonk 9410 Imaging System wit

percent mean fluorescence (units/Ag protein) relative to respective controls in the

asterisk (*) indicates values that are significantly different ( p< 0.05) from cells tr

presence of 15 mM Cd2 + alone (lane 2) or following pretreatments with 10 mM S

subjected to Western blot analyses as described under Materials and methods using

ATF-2, as well as COX-2 and cleaved PARP (arrows). Data are representative of

comparable to SB202190 and also diminished the phos-

phorylation of p38 MAPK and c-Jun induced by Cd2 + (Fig.

8C, compare lanes 2, 3 and 4). Since DPI can inhibit the

flavoproteins, NADPH oxidase and nitric oxide synthase,

further studies were performed to distinguish which enzyme

mediated the stress response induced by Cd2 +. Pretreat-

ments with a selective inhibitor of the enzyme, N-mono-

methyl-L-arginine methyl (L-NAME), had no effect on the

loss in cell viability, stress kinase activation and inflamma-

tion induced by Cd2 +, ruling out nitric oxide as the source

of ROS in our neuronal cell death paradigm (data not

shown). Collectively, these findings implicate PI3-K, and

a NADPH oxidase-like flavoprotein in a ROS generating

system that mediates Cd2 +-induced oxidative stress through

activation of the p38 MAPK signaling pathway.

Given these results, we proposed a model of the neuronal

response to oxidative stress in which p38 MAPK serves as

regulatory link between ROS and the inflammation and cell

death induced by Cd2 (Fig. 9). In this regard, ROS produc-

tion via PI3-K and a flavoprotein signal activation of the

h activation of PI3-K and a flavoprotein. (A) Cells were incubated in the

th 0.1 AM DPI, 10 AM LY294002, 10 AM SB202190 and 10 AM NS398.

. 4C. (B) Cells were treated for 24-h at 37 jC using the same protocol as

by the addition of the fluorescent dye 2V,7V-dichlorofluorescein-diacetateal 10 min. ROS levels were determined by quantifying the intensity of green

h ImageQuant software (Amersham Pharmacia Biotech). Data represent the

absence of Cd2 + (100%) from at least three independent experiments. The

eated with Cd2 +. (C) HT4 cells were incubated in the absence (lane 1) and

B202190 (SB; lane 3) or 0.1 mM DPI (lane 4). After 24 h, cell lysates were

antibodies that specifically recognize phosphorylated p38 MAPK, JNK and

at least three independent experiments.

Fig. 9. A model for the signaling pathway associated with Cd2 +-induced

oxidative stress in HT4 neuronal cells. ROS production generated through

PI3-K and a NADPH oxidase-like flavoprotein serves as effectors of stress

kinase activation and their downstream transcription factors c-Jun, ATF-2

and CREB. In this model, p38 MAPK, but not JNK, is a molecular

intermediate that signals upregulation of COX-2 and HO-1, the

phosphorylation of the transcription factors ATF-2 and CREB and caspase

activation that ultimately leads to cell death by a caspase-independent

mechanism. It is not known whether PI3-K stimulates NADPH oxidase

activity (?) as found in phagocytic cells [5].


JNK and p38 MAPK signaling pathways. Activated p38

MAPK, in turn, mediates upregulation of COX-2 and HO-1

protein expression, transcriptional activation of ATF-2 and

CREB together with the induction of caspase-dependent

(PARP cleavage) and -independent mechanisms that culmi-

nate in neuronal cell death.

4. Discussion

Although oxidative stress is implicated as a causative

factor in neurodegenerative disorders the signaling path-

ways linking free radical production with neuronal cell

death are not well characterized [1]. We provide evidence

that Cd2 +-induced oxidative stress in neuronal cells are

associated with sustained activation of the stress activated

kinases, JNK and p38 MAPK, and their downstream tran-

scription factors, c-Jun, ATF-2 and CREB. A blockade of

p38 MAPK downregulates neuronal COX-2 and PGE2

levels induced by Cd2 + and promotes cell survival, indicat-

ing its role as an upstream regulator of the COX-2-mediated

cell death in HT4 neuronal cells. Furthermore, p38 MAPK

and COX-2 inhibition reduce caspase activity in Cd2 +-

treated cells, suggesting that inflammation may trigger cell

death mechanisms as a neuronal response to oxidative

stress. Support for our findings are the demonstrations that

Cd2+ induces the JNK and p38 MAPK signaling pathways

and caspase-3 dependent cell death in several different cell

types and that both kinases associate with amyloid deposi-

tion and inflammation in degenerating neurons in AD

[6,7,11,22–26]. Apoptosis resulting from caspase activation

appears to play a critical role in the pathogenesis of several

neurodegenerative disorders by mechanisms that remain

unclear [19]. Nevertheless, cell death proceeds in Cd2 +-

treated cells when caspase activity is inhibited, indicating

that p38 MAPK and COX-2 can mediate an alternative

caspase-independent mechanism to promote neuronal cell

death. Recently, Cd2 + was shown to induce caspase-inde-

pendent cell death in human lung cells but, unlike our

results, this event occurred in the absence of PARP cleavage

[27]. Our results suggest that the cell death induced by Cd2 +

in neuronal cells involves recruitment of several cell death

processes that are mediated through the p38 MAPK signal-

ing pathway. Also, caspase inhibition had no effect on the

Cd2 +-induced levels of COX-2 and phosphorylated c-Jun

and CREB, indicating that they are independent stress-

activated events residing upstream from caspase activation.

Thus, p38 MAPK plays a dualistic role in mediating Cd2 +-

induced cytotoxicity by regulating the ability of COX-2 to

produce the proinflammatory prostaglandin PGE2 and to

serve as an effector of caspase-independent cell death.

There is growing evidence that redox-sensitive signaling

pathways like JNK and p38 MAPK are strongly activated

by ROS or a mild oxidative shift of the intracellular thiol/

disulfide redox state [2,5]. Our finding that the antioxidants

NAC or DPI effectively inhibited the activation of the JNK

and p38 signaling cascades is consistent with reports impli-

cating ROS as an effector of Cd2 +-induced oxidative stress

[2,13,24,28]. Moreover, the p38 MAPK signaling pathway

can potentiate Cd2 +-induced glutathione depletion, suggest-

ing that stress kinase activation may exacerbate the neuronal

response to oxidative stress [24]. The fact that p38 MAPK

inhibition reduced caspase activation and promoted cell

survival after prolonged exposure (40 h) to Cd2 + also

supports its role as a critical modulator of redox-regulated

mechanisms. The finding that COX-2 inhibition reduced

caspase activation and ROS production in Cd2 +-treated cells

lends support to the notion that activated p38 MAPK

together with COX-2 upregulation and its proinflammatory

product PGE2 can contribute to the cellular damage in

neurodegeneration [10,22]. Furthermore, elevated levels of

COX-2 protein are associated with increased ROS produc-

tion and apoptosis in cultured cortical cells and in the brains

of patients afflicted with AD [22,29]. Conversely, the

activation of JNK and its downstream substrate c-Jun was

dispensable for Cd2 +-induced cytotoxicity in our cell death

paradigm, although JNK has been shown to contribute to

stress-mediated neuronal cell death [4,7,14]. Although high-

ly activated by Cd2 +, the role of JNK/c-Jun is unclear in our

neuronal model.

Superoxide anion (O2�) production by NADPH oxidase

and its subsequent conversion to H2O2 has been well

characterized as a source of oxidative stress that causes

apoptosis and cell death in phagocytic cells in a mechanism

that can involve PI3-K and p38 [2,30]. The possibility that a


similar mechanism functions during neurodegeneration is

supported by evidence that cytotoxic effects induced by

ROS in neuronal and astroglial cells occur through activa-

tion of NADPH oxidase [31–33]. Accordingly, LY294002

and DPI mitigate stress-induced ROS production and the

signaling pathways induced by cadmium, suggesting that

ROS generated by PI3-K and a NADPH oxidase-like

flavoprotein in Cd2 +-treated cells signals activation of p38

MAPK and COX-2. In agreement with this finding, PI3-K

was shown to regulate COX-2 expression in nonneuronal

cells [34]. This notion is further strengthened by the

observation that the gene expression of the p67phox cyto-

solic subunit of NADPH oxidase is upregulated in HT4 cells

in response to Cd2 + (unpublished results). Our results are in

contrast to reports showing that PI3-kinase serves an anti-

apoptotic role as a critical mediator of neuronal cell survival

[35]. It is conceivable that Cd2 + elicits a deregulation in

PI3K signaling, causing an imbalance that leads to excessive

ROS production, inflammation and ultimately cell death.

Together, our results suggest that ROS and the redox-

sensitive p38 MAPK signaling pathway play cooperative

roles in mediating Cd2 +-induced oxidative stress through

COX-2. The p38 MAPK-mediated induction of HO-1

correlates with COX-2 upregulation, stress kinase activation

and PARP cleavage, supporting its role as a biomarker of

cellular damage in neuronal cells [20,21]. This response

requires de novo synthesis since actinomycin D treatments

abolished HO-1 induction in Cd2 +-treated HT4 neuronal

cells (data not shown). However, the role of HO-1 in our

studies requires further attention since the enzyme can

confer cytoprotective or cytotoxic effects on neuronal cells

depending on the stress conditions [36]. In this regard, HO-1

and COX-2 were found to co-localize with phosphorylated

c-Jun in neurons following stress induced ischemia [37].

The finding that neuronal ATF-2, c-Jun and CREB were

phosphorylated in response to Cd2 + is consistent with

reports that these transcription factors are activated by

stressful stimuli such as UV, ionizing irradiation, ischemia

or inflammatory signals [5]. The parallel reductions in the

levels of COX-2 protein and activated ATF-2 and CREB by

p38 MAPK inhibition in Cd2 +-treated cells are consistent

with increasing evidence that these transcription factors play

regulatory roles in the p38 MAPK-mediated regulation of

COX-2 expression [15,38]. Indeed we observed that phos-

phorylated forms of ATF-2, c-Jun and CREB localize in the

nucleus in Cd2 +-treated HT4 neuronal cells and this re-

sponse is lost by p38 MAPK inhibition (unpublished

results). Our previous findings ruled out NFnB as the

transcription factor responsible for the elevated levels of

COX-2 induced by Cd2 + [13]. It is also conceivable that the

concomitant loss in ATF-2 activation and PARP cleavage

elicited by p38 MAPK inhibition in Cd2 +-treated cells

reflects its reported role as a transcription factor that

mediates neuronal apoptosis [39].

In summary, our results implicate a novel pathway in

neuronal cells whereby ROS generated by a flavoprotein

and PI3-K serve as upstream effectors of stress kinase

cascades to mediate Cd2 +-induced oxidative stress. Al-

though ROS elicits activation of both p38 MAPK and

JNK pathways, p38 MAPK functions as a regulatory link

between the induction of an inflammatory response, caspase

activation and neuronal cell death. These results implicate

p38 MAPK as a pivotal determinant of neuronal cell fate in

the neurodegenerative process and underscore the impact of

stress-induced redox changes on neuronal homeostasis.

Acknowledgements

This work was funded by grant IIRG-00-2396 from the

Alzheimer’s Association.

References

[1] Halliwell B. Drugs Aging 2001;18:685–716.

[2] Droge W. Physiol Rev 2002;82:47–95.

[3] Kyriakis JM, Avruch J. Physiol Rev 2001;81:807–69.

[4] Harper SJ, LoGrasso P. Cell Signal 2001;13:299–310.

[5] Rhee SG, Bae YS, Lee SR, Kwon J. Sci STKE 2000:PE1.

[6] Koistinaho M, Kettunen MI, Goldsteins G, Keinanen R, Salminen A,

Ort M, et al. Proc Natl Acad Sci U S A 2002;99:1610–5.

[7] Savage MJ, Lin YG, Ciallella JR, Flood DG, Scott RW. J Neurosci

2002;22:3376–85.

[8] Mirjany M, Ho L, Pasinetti GM. J Pharmacol Exp Ther 2002;301:

494–500.

[9] Pasinetti GM. Arch Gerontol Geriatr 2001;33:13–28.

[10] Hull M, Lieb K, Fiebich BL. Curr Med Chem 2002;9:83–8.

[11] Hensley K, Floyd RA, Zheng NY, Nael R, Robinson KA, Nguyen X,

et al. J Neurochem 1999;72:2053–8.

[12] Di Mari JF, Mifflin RC, Adegboyega PA, Saada JI, Powell DW.

Gastroenterology 2003;124:1855–65.

[13] Figueiredo-Pereira ME, Li Z, Jansen M, Rockwell P. J Biol Chem

2002;277:25283–9.

[14] Mielke K, Herdegen T. Prog Neurobiol 2000;61:45–60.

[15] Fiebich BL, Mueksch B, Boehringer M, Hull M. J Neurochem 2000;

75:2020–8.

[16] Guan Z, Buckman SY, Springer LD, Morrison AR. Adv Exp Med

Biol 1999;469:9–15.

[17] Lasa M, Mahtan KR, Finch A, Brewer G, Saklatvala J, Clark AR.

Mol Cell Biol 2000;20:4265–74.

[18] Soh Y, Jeong KS, Lee IJ, Bae MA, Kim YC, Song BJ. Mol Pharmacol

2000;58:535–41.

[19] Nicotera P. Toxicol Lett 2002;127:189–95.

[20] Takahashi S, Takahashi Y, Yoshimi T, Miura T. Cell Biochem Funct

1998;16:183–93.

[21] Salinas M, Diaz R, Abraham NG, Ruiz de Galarreta CM, Cuadrado

A. J Biol Chem 2003;278:13898–904.

[22] Bazan NG, Colangelo V, Lukiw WJ. Prostaglandins Other Lipid Me-

diat 2002; 68–69, 197–210.

[23] Zhu X, Raina AK, Rottkamp CA, Aliev G, Perry G, Boux H, et al.

J Neurochem 2001;76:435–41.

[24] Son MH, Kang KW, Lee CH, Kim SG. Biochem Pharmacol 2001;

15:1379–90.

[25] Lopez E, Figueroa S, Oset-Gasque MJ, Gonzalez MP. Br J Pharmacol

2003;138:901–11.

[26] Watjen W, Haase H, Biagioli M, Beyersmann D. Environ Health

Perspect 2002;110:865–7.

[27] Shih CM, Wu JS, Ko WC, Wang LF, Wei YH, Liang HF, et al. J Cell

Biochem 2003;89:335–47.


[28] Ercal N, Guer-Orhan H, Aykin-Burns N. Curr Top Med Chem

2001;6:529–39.

[29] Li L, Prabhakaran K, Shou Y, Borowitz JL, Isom GE. Toxicol Appl

Pharmacol 2002;185:55–63.

[30] Yamamori T, Inanami O, Sumimoto H, Akasaki T, Nagahata H, Ku-

wabara M. Biochem Biophys Res Commun 2002;293:1571–8.

[31] Tammariello SP, Quinn MT, Estus S. J Neurosci 2000;20:RC53.

[32] Kim YH, Koh JY. Exp Neurol 2002;177:407–18.

[33] Lee M, You HJ, Cho SH, Woo CH, Yoo MH, Joe EH, et al. Biochem J

2002;366:937–43.

[34] Tang Q, Gonzales M, Inoue H, Bowden GT. Cancer Res 2001;61:

4329–32.

[35] Rodgers EE, Theibert AB. Int J Dev Neurosci 2002;20(53):187–97.

[36] Schipper HM. Exp Gerontol 2000;35:821–30.

[37] Matsuoka Y, Okazaki M, Zhao H, Asai S, Ishikawa K, Kitamura YJ.

Cereb Blood Flow Metab 1999;19:1247–55.

[38] Tan Y, Rouse J, Zhang A, Cariati S, Cohen P, Comb MJ. EMBO J

1996;15:4629–42.

[39] Walton M, Woodgate AM, Sirimanne E, Gluckman P, Dragunow M.

Brain Res Mol Brain Res 1998;63:198–204.

Redox regulates COX-2 upregulation and cell death in the neuronal response to cadmium

Documents

Transcript of Redox regulates COX-2 upregulation and cell death in the neuronal response to cadmium