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Redox regulates COX-2 upregulation and cell death in the neuronal response to cadmium
Cellular Signalling 16 (2004) 343 – 353 www.elsevier.com/locate/cellsig
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 conditions, 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 expression from ROS-activated signaling pathways. However, the mechanisms that contribute to these events are not well characterized.
Abbreviations: AD, Alzheimer’s disease; ATF-2, activating transcription 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-acetylcysteine; PI3-K, phosphatidylinositol-3-kinase; PGE2, prostaglandin E2. * Corresponding author. Tel.: +1-212-650-3234; fax: +1-212-7725227. E-mail address: [email protected] (P. Rockwell). 0898-6568/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2003.08.006
There is growing evidence that members of the mitogenactivated 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 stressinduced ROS production or a mild oxidative shift of the intracellular thiol/disulfide redox state [5]. Upon phosphorylation, 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
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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 evidence that the onset of neurodegeneration results from an inflammatory response involving cyclooxygenase-2 (COX2) and its proinflammatory product, prostaglandin E2 (PGE2), which can be induced by different regulatory pathways including p38 MAPK [8,9]. Furthermore, p38 MAPK activation and COX-2 induction are implicated as contributors 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 contributed 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 phosphorylation 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
polyclonal COX-2 and HO-1 were from Santa Cruz Biotechnology, (Santa Cruz, CA). Rabbit antibodies to phosphorylated 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 (Beverly, MA) as well as the p38 MAPK in vitro kinase assay and secondary antibodies conjugated to horse radish peroxidase. 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 immortalized 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 concentration 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 measures the cleavage of the tetrazolium salt MTS by mitochondrial dehydrogenases in viable cells.
2.1. Materials 2.5. Protein determination N-acetyl-cysteine (NAC) and CdSO4 (Cd2 +) were purchased from Sigma. Fetal bovine serum, Dulbecco’s modified Eagle’s medium, hygromycin and geneticin were from Invitrogen Life Technologies (Carlsbad, CA). SP-600125 was from Biomol Research Laboratories (Plymouth Meeting, PA). SB202190, z-VAD-fmk and NS398 were from Calbiochem. 2V,7V-dichlorofluorescein-diacetate (DCF-DA) was purchase from Molecular Probes (Eugene, OR). Enzyme immunoassay reagents for PGE2 assays were from Cayman Chemical (Ann Arbor, MI). Anti-human Goat
Protein determinations were performed with a bicinchoninic 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
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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 fluoride and supplemented with a Complete Protease Inhibitor Cocktail tablet (Roche Diagnostics) according to manufacturer’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 peroxidase. Antigens were detected by chemiluminescence (Pierce). Where indicated, band intensities of scanned blots were quantified using a Molecular Dynamics densitometer.
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(Beckman Coulter) according to the manufacturer’s directions. Substrate utilization was measured by fluorescence and quantified using the Molecular Dynamics Typhoonk 9410 Imaging system with ImageQuant software (Amersham Pharmacia Biotech). Activity was determined as described under ROS measurements. 2.11. Statistical analyses Data are expressed as the mean F 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 (Duncan’s t-test). Overall statistical significance required p < 0.05 and the required level for multiple contrasts was adjusted lower using a Bonferroni approach.
2.7. PGE2 assays PGE2 levels in culture medium were determined using an enzyme-linked immunosorbant assay (ELISA) kit (Cayman Chemical) according to the manufacturer’s protocol. 2.8. Measurement of phospho-p38 MAPK activity In vitro kinase assays of p38 MAPK activity were analyzed in treated cells according to the manufacturer’s protocol. Briefly, cell extracts were incubated overnight with an immobilized anti-phospho-p38 MAPK (Thr 180 /Try 182 ) 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. Phosphorylation 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-dichlorofluoresceindiacetate (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 Dynamics 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
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 were subjected 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.
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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 phosphorylation of ERK1/2 at Thr202/Tyr204, JNK at Thr183/ Tyr185 and p38 MAPK at Thr180/Tyr182 using phosphospecific 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 phosphorylation 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.
These results most likely reflect the autoregulatory mechanism 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 performed 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 pretreated 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 determine whether pretreatments with SB202190 mediated a corresponding loss in COX-2 activity, culture medium from
Fig. 2. Selective inhibition of p38 MAPK by SB202190 suppresses COX-2 protein expression, and PGE2 levels and promotes cell survival in Cd2 +-treated cells. (A) Cells were treated in the absence (Control) and presence of 15 AM of Cd2 + alone (Cd) and following pretreatments for 1 h with 10 AM SP-600125 (Cd/SP) or 10 AM SB202190 (Cd/SB). Following 24-h incubation at 37 jC, cells were subjected to immunoblot analyses using an antibody that recognizes COX-2. (B) PGE2 levels were measured for the cell treatments indicated using an ELISA as described in Materials and methods. Data represent the fold increase in PGE2 (pg/mg) relative to untreated controls. (C) Activated p38 MAPK was immunoprecipitated from Cd2 +-treated cells and subjected to an in vitro kinase assay in the absence and presence of SB202190 using an ATF-2 fusion protein as substrate. Kinase activity was detected by immunoblot analysis using an antibody that detects phosphorylated ATF-2. Data are representative of three independent experiments. (D) Cells were incubated with 15 AM Cd2 + following 1-h pretreatments with the selective inhibitors as indicated. Following a 40-h incubation at 37 jC, cell viability was measured by the MTS assay as described in Materials and methods. Data represent the F S.E.M. of the percent cell viability relative to their respective controls in the absence of Cd2 + (100%) from at least three independent experiments. The asterisk (*) indicates values that are significantly different ( p < 0.05) from cells treated with Cd2 +.
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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. 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 inhibition 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 MAPK mediates 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 AM S20B2190 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 +.
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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 caspase-3 and -7 activation (Fig. 4C). Pretreatments with the pan caspase inhibitor, z-VAD-fmc, abrogated PARP cleavage 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).
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 AM Cd2 + 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 were harvested for immunoblot analyses with antibodies specific for cleaved PARP, COX 2 and the phosphorylated forms of CREB and c Jun (arrows).
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 phosphorylation correlated with the SB202190-mediated decrease in the COX-2 upregulation and cytotoxicity induced by Cd2 +. 3.4. Cd2+ induces caspase activation and caspase-independent cell death in HT4 neuronal cell Caspase activation is implicated as an important mechanism that triggers stress-induced neuronal cell death [19].
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 phosphorylated (arrows) and nonphosphorylated forms of JNK and p38 MAPK as well as antibodies that detect COX-2, COX-1, HO-1 and cleaved PARP.
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3.5. The antioxidant enzyme HO-1 is a marker of the Cd2+induced stress response and is abrogated by N-acetylcysteine (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 nonneuronal 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 upregulation (compare Fig. 6A with Figs. 1 and 4A). Since redox can influence stress kinase activation we speculated that NAC pretreatments would alleviate the Cd2 +-induced phosphorylation 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 concentrations 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
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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 intracellular 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 Cd 2 + (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,
Fig. 7. PI3-K mediates the Cd2 +-induced stress response including induction of the antioxidant enzyme, HO-1. (A) Cells were incubated in the absence and presence of 15 AM Cd2 + alone and following 1-h pretreatments with AM concentrations of the selective inhibitors as indicated. Following a 40-h incubation at 37 jC, cell viability assays were performed using the MTS assay described in the legend to Fig. 2D. (B) HT4 cells were incubated in the absence (lane 1) and presence of 15 mM cadmium alone (lane 2) or cadmium following pretreatments with the 10 AM LY294002 (LY; lane 3). After 24-h at 37 jC, cell lysates were prepared for immunoblot analyses using antibodies that detect phosphorylated p38 and c-Jun, COX-2, COX-1 and cleaved PARP. (C) Cells were incubated in the absence (lane 1) and presence of 15 AM Cd2 + (Cd) alone (lane 2) and following 1-h pretreatments with SP-600125 (SP; lane 3) or SB202190 (SB; lane 4). Lysates were subjected to immunoblot analysis using an antibody that specifically recognizes HO-1 (arrow).
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as a downstream mediator of PI3-K-mediated regulation of the antioxidant enzyme (Fig. 7C). In contrast, JNK inhibition 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 stressactivated pathway. We then examined the effects of the flavoprotein inhibitor, 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 Western 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
comparable to SB202190 and also diminished the phosphorylation 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 +. Pretreatments with a selective inhibitor of the enzyme, N-monomethyl-L-arginine methyl (L-NAME), had no effect on the loss in cell viability, stress kinase activation and inflammation 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 production via PI3-K and a flavoprotein signal activation of the
Fig. 8. Caspase 3/7 activation and ROS production are induced by Cd2 + through activation of PI3-K and a flavoprotein. (A) Cells were incubated in the absence and presence of 15 AM Cd2 + alone and following 1-h pretreatments with 0.1 AM DPI, 10 AM LY294002, 10 AM SB202190 and 10 AM NS398. Normalized casapse activities were determined as described in the legend to Fig. 4C. (B) Cells were treated for 24-h at 37 jC using the same protocol as described in A. To measure ROS, 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 and incubated at 37 jC for an additional 10 min. ROS levels were determined by quantifying the intensity of green fluorescence using the Molecular Dynamics Typhoonk 9410 Imaging System with ImageQuant software (Amersham Pharmacia Biotech). Data represent the percent mean fluorescence (units/Ag protein) relative to respective controls in the absence of Cd2 + (100%) from at least three independent experiments. The asterisk (*) indicates values that are significantly different ( p < 0.05) from cells treated with Cd2 +. (C) HT4 cells were incubated in the absence (lane 1) and presence of 15 mM Cd2 + alone (lane 2) or following pretreatments with 10 mM SB202190 (SB; lane 3) or 0.1 mM DPI (lane 4). After 24 h, cell lysates were subjected to Western blot analyses as described under Materials and methods using antibodies that specifically recognize phosphorylated p38 MAPK, JNK and ATF-2, as well as COX-2 and cleaved PARP (arrows). Data are representative of at least three independent experiments.
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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 culminate in neuronal cell death.
4. Discussion Although oxidative stress is implicated as a causative factor in neurodegenerative disorders the signaling pathways 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 transcription 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, indicating 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-
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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-independent 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 signaling 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 stressactivated 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 implicating 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, suggesting 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 production 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 highly 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
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similar mechanism functions during neurodegeneration is supported by evidence that cytotoxic effects induced by ROS in neuronal and astroglial cells occur through activation 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 cytosolic 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 antiapoptotic 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 redoxsensitive 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 phosphorylated forms of ATF-2, c-Jun and CREB localize in the nucleus in Cd2 +-treated HT4 neuronal cells and this response 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. Although 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] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27]
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P. Rockwell et al. / Cellular Signalling 16 (2004) 343–353 [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, Kuwabara 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.
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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 conditions, 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 expression from ROS-activated signaling pathways. However, the mechanisms that contribute to these events are not well characterized.
Abbreviations: AD, Alzheimer’s disease; ATF-2, activating transcription 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-acetylcysteine; PI3-K, phosphatidylinositol-3-kinase; PGE2, prostaglandin E2. * Corresponding author. Tel.: +1-212-650-3234; fax: +1-212-7725227. E-mail address: [email protected] (P. Rockwell). 0898-6568/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2003.08.006
There is growing evidence that members of the mitogenactivated 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 stressinduced ROS production or a mild oxidative shift of the intracellular thiol/disulfide redox state [5]. Upon phosphorylation, 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
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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 evidence that the onset of neurodegeneration results from an inflammatory response involving cyclooxygenase-2 (COX2) and its proinflammatory product, prostaglandin E2 (PGE2), which can be induced by different regulatory pathways including p38 MAPK [8,9]. Furthermore, p38 MAPK activation and COX-2 induction are implicated as contributors 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 contributed 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 phosphorylation 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
polyclonal COX-2 and HO-1 were from Santa Cruz Biotechnology, (Santa Cruz, CA). Rabbit antibodies to phosphorylated 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 (Beverly, MA) as well as the p38 MAPK in vitro kinase assay and secondary antibodies conjugated to horse radish peroxidase. 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 immortalized 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 concentration 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 measures the cleavage of the tetrazolium salt MTS by mitochondrial dehydrogenases in viable cells.
2.1. Materials 2.5. Protein determination N-acetyl-cysteine (NAC) and CdSO4 (Cd2 +) were purchased from Sigma. Fetal bovine serum, Dulbecco’s modified Eagle’s medium, hygromycin and geneticin were from Invitrogen Life Technologies (Carlsbad, CA). SP-600125 was from Biomol Research Laboratories (Plymouth Meeting, PA). SB202190, z-VAD-fmk and NS398 were from Calbiochem. 2V,7V-dichlorofluorescein-diacetate (DCF-DA) was purchase from Molecular Probes (Eugene, OR). Enzyme immunoassay reagents for PGE2 assays were from Cayman Chemical (Ann Arbor, MI). Anti-human Goat
Protein determinations were performed with a bicinchoninic 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
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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 fluoride and supplemented with a Complete Protease Inhibitor Cocktail tablet (Roche Diagnostics) according to manufacturer’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 peroxidase. Antigens were detected by chemiluminescence (Pierce). Where indicated, band intensities of scanned blots were quantified using a Molecular Dynamics densitometer.
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(Beckman Coulter) according to the manufacturer’s directions. Substrate utilization was measured by fluorescence and quantified using the Molecular Dynamics Typhoonk 9410 Imaging system with ImageQuant software (Amersham Pharmacia Biotech). Activity was determined as described under ROS measurements. 2.11. Statistical analyses Data are expressed as the mean F 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 (Duncan’s t-test). Overall statistical significance required p < 0.05 and the required level for multiple contrasts was adjusted lower using a Bonferroni approach.
2.7. PGE2 assays PGE2 levels in culture medium were determined using an enzyme-linked immunosorbant assay (ELISA) kit (Cayman Chemical) according to the manufacturer’s protocol. 2.8. Measurement of phospho-p38 MAPK activity In vitro kinase assays of p38 MAPK activity were analyzed in treated cells according to the manufacturer’s protocol. Briefly, cell extracts were incubated overnight with an immobilized anti-phospho-p38 MAPK (Thr 180 /Try 182 ) 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. Phosphorylation 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-dichlorofluoresceindiacetate (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 Dynamics 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
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 were subjected 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.
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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 phosphorylation of ERK1/2 at Thr202/Tyr204, JNK at Thr183/ Tyr185 and p38 MAPK at Thr180/Tyr182 using phosphospecific 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 phosphorylation 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.
These results most likely reflect the autoregulatory mechanism 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 performed 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 pretreated 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 determine whether pretreatments with SB202190 mediated a corresponding loss in COX-2 activity, culture medium from
Fig. 2. Selective inhibition of p38 MAPK by SB202190 suppresses COX-2 protein expression, and PGE2 levels and promotes cell survival in Cd2 +-treated cells. (A) Cells were treated in the absence (Control) and presence of 15 AM of Cd2 + alone (Cd) and following pretreatments for 1 h with 10 AM SP-600125 (Cd/SP) or 10 AM SB202190 (Cd/SB). Following 24-h incubation at 37 jC, cells were subjected to immunoblot analyses using an antibody that recognizes COX-2. (B) PGE2 levels were measured for the cell treatments indicated using an ELISA as described in Materials and methods. Data represent the fold increase in PGE2 (pg/mg) relative to untreated controls. (C) Activated p38 MAPK was immunoprecipitated from Cd2 +-treated cells and subjected to an in vitro kinase assay in the absence and presence of SB202190 using an ATF-2 fusion protein as substrate. Kinase activity was detected by immunoblot analysis using an antibody that detects phosphorylated ATF-2. Data are representative of three independent experiments. (D) Cells were incubated with 15 AM Cd2 + following 1-h pretreatments with the selective inhibitors as indicated. Following a 40-h incubation at 37 jC, cell viability was measured by the MTS assay as described in Materials and methods. Data represent the F S.E.M. of the percent cell viability relative to their respective controls in the absence of Cd2 + (100%) from at least three independent experiments. The asterisk (*) indicates values that are significantly different ( p < 0.05) from cells treated with Cd2 +.
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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. 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 inhibition 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 MAPK mediates 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 AM S20B2190 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 +.
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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 caspase-3 and -7 activation (Fig. 4C). Pretreatments with the pan caspase inhibitor, z-VAD-fmc, abrogated PARP cleavage 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).
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 AM Cd2 + 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 were harvested for immunoblot analyses with antibodies specific for cleaved PARP, COX 2 and the phosphorylated forms of CREB and c Jun (arrows).
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 phosphorylation correlated with the SB202190-mediated decrease in the COX-2 upregulation and cytotoxicity induced by Cd2 +. 3.4. Cd2+ induces caspase activation and caspase-independent cell death in HT4 neuronal cell Caspase activation is implicated as an important mechanism that triggers stress-induced neuronal cell death [19].
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 phosphorylated (arrows) and nonphosphorylated forms of JNK and p38 MAPK as well as antibodies that detect COX-2, COX-1, HO-1 and cleaved PARP.
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3.5. The antioxidant enzyme HO-1 is a marker of the Cd2+induced stress response and is abrogated by N-acetylcysteine (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 nonneuronal 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 upregulation (compare Fig. 6A with Figs. 1 and 4A). Since redox can influence stress kinase activation we speculated that NAC pretreatments would alleviate the Cd2 +-induced phosphorylation 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 concentrations 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
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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 intracellular 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 Cd 2 + (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,
Fig. 7. PI3-K mediates the Cd2 +-induced stress response including induction of the antioxidant enzyme, HO-1. (A) Cells were incubated in the absence and presence of 15 AM Cd2 + alone and following 1-h pretreatments with AM concentrations of the selective inhibitors as indicated. Following a 40-h incubation at 37 jC, cell viability assays were performed using the MTS assay described in the legend to Fig. 2D. (B) HT4 cells were incubated in the absence (lane 1) and presence of 15 mM cadmium alone (lane 2) or cadmium following pretreatments with the 10 AM LY294002 (LY; lane 3). After 24-h at 37 jC, cell lysates were prepared for immunoblot analyses using antibodies that detect phosphorylated p38 and c-Jun, COX-2, COX-1 and cleaved PARP. (C) Cells were incubated in the absence (lane 1) and presence of 15 AM Cd2 + (Cd) alone (lane 2) and following 1-h pretreatments with SP-600125 (SP; lane 3) or SB202190 (SB; lane 4). Lysates were subjected to immunoblot analysis using an antibody that specifically recognizes HO-1 (arrow).
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as a downstream mediator of PI3-K-mediated regulation of the antioxidant enzyme (Fig. 7C). In contrast, JNK inhibition 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 stressactivated pathway. We then examined the effects of the flavoprotein inhibitor, 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 Western 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
comparable to SB202190 and also diminished the phosphorylation 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 +. Pretreatments with a selective inhibitor of the enzyme, N-monomethyl-L-arginine methyl (L-NAME), had no effect on the loss in cell viability, stress kinase activation and inflammation 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 production via PI3-K and a flavoprotein signal activation of the
Fig. 8. Caspase 3/7 activation and ROS production are induced by Cd2 + through activation of PI3-K and a flavoprotein. (A) Cells were incubated in the absence and presence of 15 AM Cd2 + alone and following 1-h pretreatments with 0.1 AM DPI, 10 AM LY294002, 10 AM SB202190 and 10 AM NS398. Normalized casapse activities were determined as described in the legend to Fig. 4C. (B) Cells were treated for 24-h at 37 jC using the same protocol as described in A. To measure ROS, 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 and incubated at 37 jC for an additional 10 min. ROS levels were determined by quantifying the intensity of green fluorescence using the Molecular Dynamics Typhoonk 9410 Imaging System with ImageQuant software (Amersham Pharmacia Biotech). Data represent the percent mean fluorescence (units/Ag protein) relative to respective controls in the absence of Cd2 + (100%) from at least three independent experiments. The asterisk (*) indicates values that are significantly different ( p < 0.05) from cells treated with Cd2 +. (C) HT4 cells were incubated in the absence (lane 1) and presence of 15 mM Cd2 + alone (lane 2) or following pretreatments with 10 mM SB202190 (SB; lane 3) or 0.1 mM DPI (lane 4). After 24 h, cell lysates were subjected to Western blot analyses as described under Materials and methods using antibodies that specifically recognize phosphorylated p38 MAPK, JNK and ATF-2, as well as COX-2 and cleaved PARP (arrows). Data are representative of at least three independent experiments.
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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 culminate in neuronal cell death.
4. Discussion Although oxidative stress is implicated as a causative factor in neurodegenerative disorders the signaling pathways 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 transcription 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, indicating 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-
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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-independent 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 signaling 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 stressactivated 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 implicating 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, suggesting 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 production 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 highly 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
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similar mechanism functions during neurodegeneration is supported by evidence that cytotoxic effects induced by ROS in neuronal and astroglial cells occur through activation 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 cytosolic 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 antiapoptotic 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 redoxsensitive 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 phosphorylated forms of ATF-2, c-Jun and CREB localize in the nucleus in Cd2 +-treated HT4 neuronal cells and this response 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. Although 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] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27]
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