Withdrawal of Survival Factors Results in Activation of the JNK Pathway in Neuronal Cells Leading to Fas Ligand Induction and Cell Death

The JNK pathway modulates AP-1 activity. While in some cells it may have proliferative and protective roles, in neuronal cells it is involved in apoptosis in response to stress or withdrawal of survival signals. To understand how JNK activation leads to apoptosis, we used PC12 cells and primary neuronal cultures. In PC12 cells, deliberate JNK activation is followed by induction of Fas ligand (FasL) expression and apoptosis. JNK activation detected by c-Jun phosphorylation and FasL induction are also observed after removal of either nerve growth factor from differentiated PC12 cells or KCl from primary cerebellar granule neurons (CGCs). Sequestation of FasL by incubation with a Fas-Fc decoy inhibits apoptosis in all three cases. CGCs derived from gld mice (defective in FasL) are less sensitive to apoptosis caused by KCl removal than wild-type neurons. In PC12 cells, protection is also conferred by a c-Jun mutant lacking JNK phosphoacceptor sites and a small molecule inhibitor of p38 mitogen-activated protein kinase and JNK, which inhibits FasL induction. Hence, the JNK-to-c-Jun-to-FasL pathway is an important mediator of stress-induced neuronal apoptosis.     

ASK1-p38 MAPK/JNK signaling cascade mediates anandamide-induced PC12 cell death

Anandamide is a neuroimmunoregulatory molecule that triggers apoptosis in a number of cell types including PC12 cells. Here, we investigated the molecular mechanisms underlying anandamide-induced cell death in PC12 cells. Anandamide treatment resulted in the activation of p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), and p44/42 MAPK in apoptosing cells. A selective p38 MAPK inhibitor, SB203580, or dn-JNK, JNK1(A-F) or SAPKbeta(K-R), blocked anandamide-induced cell death, whereas a specific inhibitor of MEK-1/2, U0126, had no effect, indicating that activation of p38 MAPK and JNK is critical in anandamide-induced cell death. An important role for apoptosis signal-regulating kinase 1 (ASK1) in this event was also demonstrated by the inhibition of p38 MAPK/JNK activation and death in cells overexpressing dn-ASK1, ASK1 (K709M). Conversely, the constitutively active ASK1, ASK1DeltaN, caused prolonged p38 MAPK/JNK activation and increased cell death. These indicate that ASK1 mediates anandamide-induced cell death via p38 MAPK and JNK activation. Here, we also found that activation of p38 MAPK/JNK is accompanied by cytochrome c release from the mitochondria and caspase activation (which can be inhibited by SB203580), suggesting that anandamide triggers a mitochondrial dependent apoptotic pathway. The caspase inhibitor, zVAD, and the mitochondrial pore opening inhibitor, cyclosporine A, blocked anandamide-induced cell death but not p38 MAPK/JNK activation, suggesting that activation of these kinases may occur upstream of mitochondrial associated events.     

Apoptosis of PC12 cells by 4-hydroxy-2-nonenal is mediated through selective activation of the c-Jun N-terminal protein kinase pathway

Cytotoxic lipid peroxides such as 4-hydroxy-2-nonenal (HNE) are produced when cells are exposed to toxic chemicals. However, the mechanism by which HNE induces cell death has been poorly understood. In this study, we investigated the molecular mechanism of HNE-induced apoptosis in PC12 cells by measuring the activities of the mitogen-activated protein (MAP) kinases involved in early signal transduction pathways. Within 15-30 min after HNE treatment, c-Jun N-terminal protein kinase (JNK) was maximally activated, before returning to control level after 1 h post-treatment. In contrast, activities of extracellular signal regulated kinase (ERK) and p38 MAP kinase remained unchanged from their basal levels. SEK1, an upstream kinase of JNK, was also activated (phosphorylated) within 5 min after HNE treatment and remained activated for up to 60 min. Marked activation of the JNK pathway through SEK1 was demonstrated by the transient transfection of cDNA for wild type SEK1 and JNK into COS-7 cells. Furthermore, significant reductions in JNK activation and HNE-induced cell death were observed when the dominant negative mutant of SEK1 was co-transfected with JNK. Pretreatment of PC12 cells with a survival promoting agent, 8-(4-chlorophenylthio)-cAMP, prevented both the HNE-induced JNK activation and apoptosis. Nonaldehyde, a nontoxic aldehyde, caused neither apoptosis nor JNK activation. Pretreatment of PC12 cells with SB203580, a specific inhibitor of p38 MAP kinase, had no effect on HNE-induced apoptosis. All these data suggest that the HNE-mediated apoptosis of PC12 cells is likely to be mediated through the selective activation of the SEK1-JNK pathway without activation of ERK or p38 MAP kinase.     

Apoptosis of PC12 cells by 4-hydroxy-2-nonenal is mediated through selective activation of the c-Jun N-Terminal protein kinase pathway

Cytotoxic lipid peroxides such as 4-hydroxy-2-nonenal (HNE) are produced when cells are exposed to toxic chemicals. However, the mechanism by which HNE induces cell death has been poorly understood. In this study, we investigated the molecular mechanism of HNE-induced apoptosis in PC12 cells by measuring the activities of the mitogen-activated protein (MAP) kinases involved in early signal transduction pathways. Within 15–30 min after HNE treatment, c-Jun N-terminal protein kinase (JNK) was maximally activated, before returning to control level after 1 h post-treatment. In contrast, activities of extracellular signal regulated kinase (ERK) and p38 MAP kinase remained unchanged from their basal levels. SEK1, an upstream kinase of JNK, was also activated (phosphorylated) within 5 min after HNE treatment and remained activated for up to 60 min. Marked activation of the JNK pathway through SEK1 was demonstrated by the transient transfection of cDNA for wild type SEK1 and JNK into COS-7 cells. Furthermore, significant reductions in JNK activation and HNE-induced cell death were observed when the dominant negative mutant of SEK1 was co-transfected with JNK. Pretreatment of PC12 cells with a survival promoting agent, 8-(4-chlorophenylthio)-cAMP, prevented both the HNE-induced JNK activation and apoptosis. Nonaldehyde, a nontoxic aldehyde, caused neither apoptosis nor JNK activation. Pretreatment of PC12 cells with SB203580, a specific inhibitor of p38 MAP kinase, had no effect on HNE-induced apoptosis. All these data suggest that the HNE-mediated apoptosis of PC12 cells is likely to be mediated through the selective activation of the SEK1-JNK pathway without activation of ERK or p38 MAP kinase.     

v-Crk, an effector of the nerve growth factor signaling pathway, delays apoptotic cell death in neurotrophin-deprived PC12 cells

v-Crk is a member of a class of SH2 and SH3-containing adaptor proteins that have been implicated in regulating the TrkA receptor tyrosine kinase and potentiating Nerve Growth Factor (NGF)-mediated neurite outgrowth in pheochromocytoma (PC12) cells (Hempstead et al, Mol. Cell Biol. 14: 1964 - 1971). Given the fact that NGF induces both differentiation and survival by binding to TrkA, we examined the rate of apoptotic cell death elicited by NGF-withdrawal in native, v-Crk, and TrkA-expressing PC12 cells. While more than 50% of native PC12 cells underwent apoptosis within 48 h of NGF withdrawal, the v-Crk and TrkA-expressing cells were much more resistant to apoptosis under these conditions, whereby approximately 70 and 95%, respectively, of the cells were alive. The ability of v-Crk to delay apoptosis required prior NGF-dependent differentiation, since naive undifferentiated v-Crk expressing PC12 cells or cells that express v-Crk mutants that are defective in NGF signaling were not protected from apoptosis during growth factor withdrawal. Moreover, addition of 50 ng/ml EGF to serum and NGF deprived v-Crk expressing cells, which also causes neurite outgrowth, promoted complete and long-term survival, although such EGF replacement had no neurotrophic effect on wild-type PC12 cells or PC12 cells overexpressing Human Bcl-2. These experiments suggest that v-Crk potentiation of a receptor tyrosine kinase under conditions of growth factor deprivation is essential for preventing apoptosis. However, unlike native PC12 cells, neither v-Crk or TrkA-expressing PC12 cells exhibited a G1 arrest when incubated for 2 weeks in NGF. Thus, v-Crk and TrkA may protect NGF deprived PC12 by preventing cell cycle arrest and hence an aborted entry into a defective cell cycle. Moreover, during NGF-withdrawal, v-CrkPC12 cells exhibited down regulation in MAP kinase and JNK activities while in native cells, these activities increased within 6 - 8 h after NGF deprivation. Thus, unlike v-Crk-mediated augmentation of differentiation, sustained activation of MAP kinase may not be required for v-Crk-induced cell survival.     

TNF-α Induces Apoptosis Through JNK/Bax-Dependent Pathway in Differentiated,but not Naïve PC12 Cells

Differentiated PC12 cells have been used widely as a model for the analysis of neuronal degeneration. Some evidences showed that differentiated PC12 cells were more sensitive than naïve PC12 against apoptosis stimuli. However, the apoptosis mechanism of both types of PC12 cells was not fully known. In this study, the signaling pathways involved in tumor necrosis factor-α (TNF-α)-induced apoptosis in living differentiated and naïve PC12 cells were investigated using confocal microscope for the first time. Our results showed that during TNF-α-induced apoptosis, Bax translocation to mitochondria and cytochrome C (Cyt c) release from mitochondria were observed in differentiated PC12 cells, but not in naïve PC12 cells. Furthermore, the mRNA levels of bim, c-Jun N-terminal protein kinase 1 and 2 (JNK1 and JNK2) increased noticeably in differentiated PC12 cells. The apoptosis induced by TNF-α was inhibited by Z-IETD-fmk (specific inhibitor of caspase-8) but not SP600125 (specific inhibitor of JNK) in naïve PC12 cells. While in differentiated PC12 cells, the process of apoptosis could only be inhibited effectively by Z-IETD-fmk and SP600125 co-treatment, and SP600125 inhibited the Bax translocation to mitochondria implying that JNK mediated activation of Bax. The experimental data strongly demonstrated that TNF-α induced apoptosis through JNK/Bax-dependent pathway in differentiated, but not naïve PC12 cells.     

Regulation of gene expression by the amyloid precursor protein: inhibition of the JNK/c-Jun pathway

The amyloid precursor protein (APP) has been suggested to regulate gene expression. GeneChip analysis and in vitro kinase assays revealed potent APP-dependent repression of c-Jun, its target gene SPARC and reduced basal c-Jun N-terminal kinase (JNK) activity in PC12 cells overexpressing APP. UV-induced activation of the JNK signalling pathway and subsequent apoptosis were likewise reduced by APP and this effect could be mimicked by the indirect JNK inhibitor CEP-11004. Treatment with a gamma-secretase inhibitor did not affect APP-mediated downmodulation of the JNK signalling pathway, suggesting that the effects might be mediated via alpha-secretase processing of APP. In support of these data, overexpression of the Swedish mutant of APP did not inhibit SPARC expression, UV-induced JNK activation and cell death. Our data suggest an important physiological role of APP and alpha-secretase activity in the control of JNK/c-Jun signalling, target gene expression and cell death activation in response to cytotoxic stress.     

LIM kinase and Diaphanous cooperate to regulate serum response factor and actin dynamics

The small GTPase RhoA controls activity of serum response factor (SRF) by inducing changes in actin dynamics. We show that in PC12 cells, activation of SRF after serum stimulation is RhoA dependent, requiring both actin polymerization and the Rho kinase (ROCK)-LIM kinase (LIMK)-cofilin signaling pathway, previously shown to control F-actin turnover. Activation of SRF by overexpression of wild-type LIMK or ROCK-insensitive LIMK mutants also requires functional RhoA, indicating that a second RhoA-dependent signal is involved. This is provided by the RhoA effector mDia: dominant interfering mDia1 derivatives inhibit both serum- and LIMK-induced SRF activation and reduce the ability of LIMK to induce F-actin accumulation. These results demonstrate a role for LIMK in SRF activation, and functional cooperation between RhoA-controlled LIMK and mDia effector pathways.     

Piceatannol attenuates hydrogen-peroxide- and peroxynitrite-induced apoptosis of PC12 cells by blocking down-regulation of Bcl-XL and activation of JNK

There is mounting evidence implicating the accumulation of intracellular reactive oxygen species (ROS) and reactive nitrogen species (RNS) in the pathogenesis of neurodegenerative disorders, including Alzheimer's disease. Recently, considerable attention has been focused on identifying naturally occurring antioxidants that are able to reduce excess ROS and RNS, thereby protecting against oxidative stress and neuron death. The present study investigated the possible protective effects of piceatannol (trans-3,4,3',5'-tetrahydroxystilbene), which is present in grapes and other foods, on hydrogen-peroxide- and peroxynitrite-induced oxidative cell death. PC12 rat pheochromocytoma (PC12) cells treated with hydrogen peroxide or SIN-1 (a peroxynitrite-generating compound) exhibited apoptotic death, as determined by nucleus condensation and cleavage of poly(ADP-ribose)polymerase (PARP). Piceatannol treatment attenuated hydrogen-peroxide- and peroxynitrite-induced cytotoxicity, apoptotic features, PARP cleavage and intracellular ROS and RNS accumulation. Treatment of PC12 cells with hydrogen peroxide or SIN-1 led to down-regulation of Bcl-X(L) and activation of caspase-3 and -8, which were also inhibited by piceatannol treatment. Hydrogen peroxide or SIN-1 treatment induced phosphorylation of the c-Jun-N-terminal kinase (JNK), which was inhibited by piceatannol treatment. Moreover, SP600125 (a JNK inhibitor) significantly inhibited hydrogen-peroxide- and peroxynitrite-induced PC12 cell death, revealing inactivation of the JNK pathway as a possible molecular mechanism for the protective effects of piceatannol against hydrogen-peroxide- and peroxynitrite-induced apoptosis of PC12 cells. Collectively, these findings suggest that the protective effect of piceatannol against hydrogen-peroxide- and peroxynitrite-induced apoptosis of PC12 cells is associated with blocking the activation of JNK and the down-regulation of Bcl-XL.     

Neurotrophic Factors Prevent Ceramide-Induced Apoptosis Downstream of c-Jun N-Terminal Kinase Activation in PC12 Cells

Abstract: Neurotrophic factors prevent apoptosis of PC12 cells in serum-free medium. The present study determines whether neurotrophic factors can prevent ceramide-induced apoptosis in PC12 cells and investigates the role that c-Jun N-terminal kinase (JNK) activation may play in this system. Ceramide-induced apoptosis was inhibited by nerve growth factor, basic fibroblast growth factor, pituitary adenylyl cyclase-activating peptide, 4-(8-chlorophenylthio)cyclic AMP, and the caspase inhibitor benzyloxycarbonyl-Val-Ala- dl -Asp fluoromethyl ketone (zVAD-FMK). It was surprising that inhibition of extracellular signal-regulated kinase and/or phosphatidylinositol 3-kinase did not markedly block the protective effects exerted by neurotrophic factors against ceramide-induced apoptosis, suggesting that neurotrophic factors can promote survival independently of these signaling pathways. Treatment of PC12 cells with ceramide resulted in a time-dependent increase in JNK activity. However, neither neurotrophic factors nor zVAD-FMK attenuated ceramide-stimulated JNK activation. Further experiments indicated that ceramide-induced apoptosis in PC12 cells requires new protein synthesis, and that nerve growth factor and zVAD-FMK can prevent apoptosis after JNK activity has been detected. These results indicate that ceramide-induced JNK activation is an early event and may be required for the expression of essential components of the apoptotic machinery. It is anticipated that neurotrophic factors inhibit ceramide-induced apoptosis by affecting signaling events downstream of JNK activation.     

Signaling and function of caspase and c-jun N-terminal kinase in cisplatin-induced apoptosis

Caspases and c-Jun N-terminal kinase (JNK) are activated in tumor cells during induction of apoptosis. We investigated the signaling cascade and function of these enzymes in cisplatin-induced apoptosis. Treatment of Jurkat T-cells with cisplatin induced cell death with DNA fragmentation and activation of caspase and JNK. Bcl-2 overexpression suppressed activation of both enzymes, whereas p35 and CrmA inhibited only the DEVDase (caspase-3-like) activity, indicating that the activation of these enzymes may be differentially regulated. Cisplatin induced apoptosis with the cytochrome c release and caspase-3 activation in both wild-type and caspase-8-deficient JB-6 cells, while the Fas antibody induced these apoptotic events only in wild-type cells. This indicates that caspase-8 activation is required for Fas-mediated apoptosis, but not cisplatin-induced cell death. On the other hand, cisplatin induced the JNK activation in both the wild-type and JB-6 cells, and the caspase-3 inhibitor Z-DEVD-fmk did not inhibit this activation. The JNK overexpression resulted in a higher JNK activity, AP-1 DNA binding activity, and metallothionein expression than the empty vector-transfected cells following cisplatin treatment. It also partially protected the cells from cisplatin-induced apoptosis by decreasing DEVDase activity. These data suggest that the cisplatin-induced apoptotic signal is initiated by the caspase-8-independent cytochrome c release, and the JNK activation protects cells from cisplatin-induced apoptosis via the metallothionein expression.     

Role of hsp105 in protection against stress-induced apoptosis in neuronal PC12 cells.

hsp105alpha is a stress protein characteristically highly expressed in the brain compared with other tissues in mammals. Here, to examine whether hsp105alpha plays a pivotal role in the nervous system, we tested the capability of hsp105alpha to protect against apoptosis in rat neuronal PC12 cells. Various stress treatments such as serum deprivation, heat shock, hydrogen peroxide, etoposide, and actinomycin D induced apoptosis in PC12 cells with characteristic shrinking of nuclei and chromatin. However, PC12 cells that constitutively overexpressed mouse hsp105alpha exhibited a strong protective effect against apoptosis induced by these stress treatments. Cleavage of poly(ADP-ribose) polymerase induced in PC12 cells by these treatments was inhibited in the constitutively overexpressed hsp105alpha cells. Furthermore, c-Jun N-terminal kinase (JNK) was activated in the cells treated with heat shock but not other treatments, and the heat-induced JNK activation was inhibited by the constitutive expression of hsp105alpha.Thus, hsp105alpha prevents not only heat-induced apoptosis by inhibiting JNK activation, but also prevents the apoptosis induced by other stressors through different pathways, and may play important roles in neuronal protection.     

Overexpression of atypical PKC in PC12 cells enhances NGF-responsiveness and survival through an NF-kappaB dependent pathway.

Removal of atypical PKC blocks NGF-induced differentiation of PC12 cells.1 We now examine the consequences that overexpression of atypical PKCs had upon NGF responses. PC12 cells were stably transfected with either PKC-iota or PKC-zeta. Overexpression of atypical PKCs markedly enhanced NGF- induced neurite outgrowth as well as enhanced NGF-stimulated JNK kinase. Cotransfection of HA-JNK1 along with increasing concentrations of PKC-iota, resulted in dose-dependent phosphorylation of GST c-Jun (1 - 79). NGF treatment of PC12 cells resulted in activation of NF-kappaB. In comparison, overexpression of atypical PKC-iota was by itself sufficient to activate NF-kappaB and shift the kinetics of NGF-induced kappaB activity. Furthermore, transfection of full-length antisense PKC-iota blocked basal and NGF-stimulated NF-kappaB. Differentiated and undifferentiated PC12 cells overexpressing atypical PKC-iota were protected from serum deprivation-induced cell death. Collectively, these findings demonstrate that atypical PKC-iota lies in a pathway that regulates NF-kappaB and contributes to both neurotrophin-mediated differentiation and survival signaling.     

Cadmium activates the mitogen-activated protein kinase (MAPK) pathway via induction of reactive oxygen species and inhibition of protein phosphatases 2A and 5

Cadmium (Cd), a highly toxic environmental pollutant, induces neurodegenerative diseases. Recently we have demonstrated that Cd may induce neuronal apoptosis in part through activation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase 1/2 (Erk1/2) pathways. However, the underlying mechanism remains enigmatic. Here we show that Cd induced generation of reactive oxygen species (ROS), leading to apoptosis of PC12 and SH-SY5Y cells. Pretreatment with N-acetyl-L-cysteine (NAC) scavenged Cd-induced ROS, and prevented cell death, suggesting that Cd-induced apoptosis is attributed to its induction of ROS. Furthermore, we found that Cd-induced ROS inhibited serine/threonine protein phosphatases 2A (PP2A) and 5 (PP5), leading to activation of Erk1/2 and JNK, which was abrogated by NAC. Overexpression of PP2A or PP5 partially prevented Cd-induced activation of Erk1/2 and JNK, as well as cell death. Cd-induced ROS was also linked to the activation of caspase-3. Pretreatment with inhibitors of JNK (SP600125) and Erk1/2 (U0126) partially blocked Cd-induced cleavage of caspase-3 and prevented cell death. However, zVAD-fmk, a pan caspase inhibitor, only partially prevented Cd-induced apoptosis. The results indicate that Cd induction of ROS inhibits PP2A and PP5, leading to activation of JNK and Erk1/2 pathways, and consequently resulting in caspase-dependent and -independent apoptosis of neuronal cells. The findings strongly suggest that the inhibitors of JNK, Erk1/2, or antioxidants may be exploited for prevention of Cd-induced neurodegenerative diseases.     

Role of MAPK phosphatase-1 in sustained activation of JNK during ethanol-induced apoptosis in hepatocyte-like VL-17A cells

Ethanol metabolism plays a central role in activating the mitogen-activated protein kinase (MAPK) cascade leading to inflammation and apoptosis. Sustained activation of c-Jun N-terminal kinase (JNK), one of the MAPKs, has been shown to induce apoptosis in hepatocytes. MAPK phosphatase-1 (MKP-1) has been shown to dephosphorylate MAPKs in several cells. The aim of the study is to evaluate the role of MKP-1 in sustained JNK activation as a mechanism to explain ethanol-induced hepatocyte apoptosis. VL-17A cells (HepG2 cells overexpressing alcohol dehydrogenase and cytochrome P450-2E1) were exposed to ethanol for different time periods. Western blots were performed for MKP-1, phospho-JNK, phosphotyrosine, and protein kinase Cdelta (PKCdelta). Electrophoretic mobility shift assays for AP-1 were performed. Apoptosis was measured by caspase-3 activity assay, TUNEL, and 4',6-diamidino-2-phenylindole staining. Reactive oxygen species were neutralized by overexpressing both superoxide dismutase-3 and catalase genes using lentiviral vectors in VL-17A cells. Ethanol incubation markedly decreased the MKP-1 protein levels to 15% of control levels and was associated with sustained phosphorylation of p46 JNK and p54 JNK, as well as increased apoptosis. VL-17A cells overexpressing superoxide dismutase-3 and catalase, treatment with a tyrosine kinase inhibitor, or incubation of the cells with PKCdelta small interference RNAs significantly inhibited the ethanol-induced MKP-1 degradation and apoptosis. Ethanol-induced oxidative stress enhanced the tyrosine phosphorylation of PKCdelta, which in turn caused the proteasomal degradation of MKP-1, leading to sustained JNK activation and increased apoptosis in VL-17A cells.     

Role of c-Jun in cellular sensitivity to the microtubule inhibitor vinblastine

The role of c-Jun in the apoptotic response of cells to the microtubule inhibitor vinblastine was investigated using fibroblasts lacking or overexpressing c-Jun. c-Jun null cells were found to be more sensitive than wild-type cells at low (1-3 nM) concentrations of vinblastine, but showed essentially identical apoptotic responses as wild-type cells at a higher concentration of 10nM. In contrast, c-Jun overexpressing cells were highly vinblastine-resistant, with an IC50 of 12-fold greater than wild-type cells. The fate of cells exposed to lethal concentrations of vinblastine was examined by propidium iodide staining and flow cytometry. All cell types appeared to undergo mitotic arrest prior to apoptosis. Apoptosis of wild-type cells was associated with significant DNA re-replication. In contrast, DNA re-replication was much less prominent in vinblastine-treated c-Jun null cells and absent during apoptosis of c-Jun overexpressing cells. These results suggest that c-Jun plays a key role in the cellular sensitivity to vinblastine. In addition, c-Jun appears to regulate the pathway to cell death following mitotic arrest.     

Prevention of anti-IgM-induced apoptosis accompanying G1 arrest in B lymphoma cells overexpressing dominant-negative mutant form of c-Jun N-terminal kinase 1

A family of mitogen-activated protein (MAP) kinases comprising the extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs), and p38 MAP kinases are involved in proliferation and apoptosis. However, there are some arguments concerning the role of these kinases in Ag-induced B cell apoptosis. Two of the B lymphoma cell lines (CH31 and WEHI-231) susceptible to anti-IgM-induced apoptosis were used as a model. To address these issues, we examined the kinetics of anti-IgM-induced activation of MAP kinases and established cell lines overexpressing a dominant-negative (dn) mutant form of JNK1 (dnJNK1). Anti-IgM induced a sustained JNK1 activation with a peak at 8 h, with a marginal activation of ERK1/ERK2 in CH31 cells. The sustained JNK1 activation was not a secondary event through a caspase activation. The peak point of the JNK1 activation was just before the onset of a decline in mitochondrial membrane potential, which preceded anti-IgM-induced cell death. Following anti-IgM stimulation, dnJNK1 prevented a decline in mitochondrial membrane potential at 24 h, with a prolonged inhibition up to 72 h in WEHI-231, although it did so only partially during a later time period in CH31. The dnJNK1 cells also demonstrated diminished procaspase-3 activation and a decreased rate of apoptosis upon anti-IgM stimulation, with a concomitant increased arrest in G(1) phase, which could be explained by enhanced levels of cyclin-dependent kinase inhibitor p27(Kip1) protein. Thus, anti-IgM-induced JNK activation might be implicated in cell cycle progression as well as in apoptosis regulation, probably involving p27(Kip1) protein.