The involvement of MAPK signaling pathways in determining the cellular response to p53 activation: cell cycle arrest or apoptosis

The effect of ERK, p38, and JNK signaling on p53-dependent apoptosis and cell cycle arrest was investigated using a Friend murine erythroleukemia virus (FVP)-transformed cell line that expresses a temperature-sensitive p53 allele, DP16.1/p53ts. In response to p53 activation at 32 degrees C, DP16.1/p53ts cells undergo p53-dependent G(1) cell cycle arrest and apoptosis. As a result of viral transformation, these cells express the spleen focus forming env-related glycoprotein gp55, which can bind to the erythropoietin receptor (EPO-R) and mimics many aspects of EPO-induced EPO-R signaling. We demonstrate that ERK, p38 and JNK mitogen-activated protein kinases (MAPKs) are constitutively active in DP16.1/p53ts cells. Constitutive MEK activity contributes to p53-dependent apoptosis and phosphorylation of p53 on serine residue 15. The pro-apoptotic effect of this MAPK kinase signal likely reflects an aberrant Ras proliferative signal arising from FVP-induced viral transformation. Inhibition of MEK alters the p53-dependent cellular response of DP16.1/p53ts from apoptosis to G(1) cell cycle arrest, with a concomitant increase in p21(WAF1), suggesting that the Ras/MEK pathway may influence the cellular response to p53 activation. p38 and JNK activity in DP16.1/p53ts cells is anti-apoptotic and capable of limiting p53-dependent apoptosis at 32 degrees C. Moreover, JNK facilitates p53 protein turnover, which could account for the enhanced apoptotic effects of inhibiting this MAPK pathway in DP16.1/p53ts cells. Overall, these data show that intrinsic MAPK signaling pathways, active in transformed cells, can both positively and negatively influence p53-dependent apoptosis, and illustrate their potential to affect cancer therapies aimed at reconstituting or activating p53 function.     

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.     

Synthesis,antiproliferative and apoptosis induction potential activities of novel bis(indolyl)hydrazide-hydrazone derivatives

In recent years, indole-indazolyl hydrazide-hydrazone derivatives with strong cell growth inhibition and apoptosis induction characteristics are being strongly screened for their cancer chemo-preventive potential. In the present study, N-methyl and N,N-dimethyl bis(indolyl)hydrazide-hydrazone analog derivatives were designed, synthesized and allowed to evaluate for their anti-proliferative and apoptosis induction potential against cervical (HeLa), breast (MCF-7 and MDA-MB-231) and lung (A549) cancer cell lines relative to normal HEK293 cells. The MTT assay in conjunction with mitochondrial potential assays and the trypan blue dye exclusion were employed to ascertain the effects of the derivatives on the cancer cells. Further, mechanistic studies were conducted on compound 14a to understand the biochemical mechanisms and functional interactions with various signaling pathways triggered in HeLa and MCF-7 cells. Compound 14a induced apoptosis via caspase independent pathway through the participation of mitogen-activated protein kinases (MAPK) such as extracellular signal related kinase (ERK) and p38 as well as p53 pathways. It originates the activation of pro-apoptotic proteins such as Bak and Mcl-1s and also strongly induced the generation of reactive oxygen species. In downstream signaling pathway, activated p53 protein interacted with MAPK pathways, including SAPK/c-Jun N-terminal protein kinase (JNK), p38 and ERK kinases resulting in apoptotic cell death. The involvement of MAPK cascades such as p38, ERK and p38 on compound 14a induced apoptotic cell death was evidenced by the fact that the inclusion of specific inhibitors of p38, ERK1/2 and JNK MAPK (SB2035809, PD98059 and SP600125) prevented the compound 14a towards induced apoptosis. The results clearly showed that MAP kinase cascades were crucial for apoptotic response in compound 14a induced cellular killing and were dependent on p53 activity. Based on the results, compound 14a was identified as a promising candidate for cancer therapeutics and these findings furnish a basis for further in vivo experiments on anti-proliferative activity.     

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.     

Upregulation of Beclin-1 expression and phosphorylation of Bcl-2 and p53 are involved in the JNK-mediated autophagic cell death

Though the activation of c-Jun NH2-terminal kinase (JNK) has been reported to be essential for autophagic cell death in response to various stressors, the molecular links between JNK activation and autophagic cell death signaling remain elusive. Here we report that, in the JNK-dependent autophagic cell death of HCT116 cells induced by an agonistic single chain variable fragment antibody, HW1, against human death receptor 5 (DR5), JNK activation upregulated Beclin-1 expression and induced Bcl-2 and p53 phosphorylation. Further, the p53-deficient HCT116 cells showed less susceptibility to the HW1-mediated autophagic cell death than the wild type cells, suggesting that JNK-mediated p53 phosphorylation promotes the autophagic cell death. Our results suggest that DR5-stimulated JNK activation and its consequent fluxes into the pro-autophagic signaling pathways contribute to the autophagic cell death in cancer cells.     

Ceramide pathways modulate ethanol-induced cell death in astrocytes

We showed previously that alcohol exposure during in vivo brain development induced astroglial damage and caused cell death. Because ceramide modulates a number of biochemical and cellular responses to stress, including apoptosis, we now investigate whether ethanol-induced cell death in astrocytes is mediated by ceramide signalling pathways triggering apoptosis. Here we show that both ethanol and ceramide are able to induce apoptotic death in cultured astrocytes, in a dose-dependent manner, and that C2-ceramide addition potentiates the apoptotic effects of ethanol. Cell death induced by ethanol is associated with stimulation of neutral and acidic sphingomyelinase (SMase) and ceramide generation, as well as with activation of stress-related kinases, c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38) and extracellular signal-regulated kinase (ERK) pathways. We also provide evidence for the participation of JNK and p38 in ethanol-induced cell death, because pharmacological inhibitors of these kinases largely prevent the apoptosis induced by ethanol or by ethanol and C2-ceramide. Furthermore, we show that ethanol-induced ERK activation triggers the stimulation of cyclo-oxygenase-2 (COX-2) and the release of prostaglandin E2, and that blockade of the mitogen-activated protein kinase kinase (MEK)/ERK pathway by PD98059 abolishes the up-regulation of COX-2 induced by ethanol plus ceramide, and decreases the ethanol-induced apoptosis. These results strongly suggest that ethanol is able to stimulate the SMase-ceramide pathway, leading to the activation of signalling pathways implicated in cell death. These findings provide an insight into the mechanisms involved in ethanol-induced astroglial cell death during brain development.     

Modulation of nitric oxide-induced apoptotic death of HL-60 cells by protein kinase C and protein kinase A through mitogen-activated protein kinases and CPP32-like protease pathways.

To define the signaling pathways during NO-induced apoptotic events and their possible modulation by two protein kinase systems, we explored the involvement of three structurally related mitogen-activated protein kinase subfamilies. Exposure of HL-60 cells to sodium nitroprusside (SNP) strongly activated p38 kinase, but did not activate c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK). In addition, SNP-induced apoptosis was markedly blocked by the selective p38 kinase inhibitor (SB203580) but not by MEK1 kinase inhibitor (PD098059), indicating that p38 kinase serves as a mediator of NO-induced apoptosis. In contrast, treatment of cells with phorbol 12-myristate 13-acetate (PMA) strongly activated not only JNK but also ERK, while not affecting p38 kinase. However, although SNP by itself weakly activated CPP32-like protease, SNP in combination with PMA markedly increased the extent of CPP32-like protease activation. Interestingly, N6,O2-dibutylyl cAMP (DB-cAMP) significantly blocked SNP- or SNP plus PMA-induced activation of CPP32-like protease and the resulting induction of apoptosis. DB-cAMP also blocked PMA-induced JNK activation. Collectively, these findings demonstrate the presence of specific up- or down-modulatory mechanisms of cell death pathway by NO in which (1) p38 kinase serves as a mediator of NO-induced apoptosis, (2) PKC acts at the point and/or upstream of JNK and provides signals to potentiate NO-induced CPP32-like protease activation, and (3) PKA lies upstream of either JNK or CPP32-like protease to protect NO- or NO plus PMA-induced apoptotic cell death in HL-60 cells.     

Differential activation of c-Jun NH2-terminal kinase and p38 pathways during FTY720-induced apoptosis of T lymphocytes that is suppressed by the extracellular signal-regulated kinase pathway

FTY720 is a novel immunosuppressive drug derived from a metabolite from Isaria sinclairii that is known to induce apoptosis of rat splenic T cells. In this study, we examined the intracellular signaling pathway triggered by FTY720. Treatment of human Jurkat T lymphocytes with FTY720-induced apoptosis characterized by DNA fragmentation. The same treatment induced activation of protein kinases such as c-Jun NH2-terminal kinase (JNK), p38/CSBP (CSAID-binding protein), and a novel 36-kDa myelin basic protein (MBP) kinase, but not extracellular signal-regulated kinase (ERK). Pretreatment of Jurkat cells with DEVD-CHO blocked FTY720-induced DNA fragmentation as well as the activation of p38/CSBP. However, DEVD-CHO treatment failed to inhibit FTY720-induced activation of JNK and the 36-kDa MBP kinase. We have also demonstrated that activation of the ERK signaling pathway completely suppressed the FTY720-induced apoptotic process including activation of caspase 3 and activation of JNK and the 36-kDa MBP kinase. Furthermore, transient expression of constitutively active mitogen-activated protein kinase/ERK kinase (MEK) protected the cells from FTY720-induced cell death. The effect of MEK was canceled by coexpression of a mitogen-activated protein kinase phosphatase, CL100. These results indicate that JNK and p38 pathways are differentially regulated during FTY720-induced apoptosis and that activation of ERK pathway alone is sufficient to cancel the FTY720-induced death signal.     

JNK and p38 MAPK are independently involved in tributyltin-mediated cell death in rainbow trout (Oncorhynchus mykiss) RTG-2 cells

Mitogen-activated protein kinases (MAPKs) are a family of Ser/Thr protein kinases that transmit various extracellular signals to the nucleus inducing gene expression, cell proliferation, and apoptosis. Recent studies have revealed that organotin compounds induce apoptosis and MAPK phosphorylation/activation in mammal cells. In this study, we elucidated the cytotoxic mechanism of tributyltin (TBT), a representative organotin compound, in rainbow trout (Oncorhynchus mykiss) RTG-2 cells. TBT treatment resulted in significant caspase activation, characteristic morphological changes, DNA fragmentation, and consequent apoptotic cell death in RTG-2 cells. TBT exposure induced the rapid and sustained accumulation of phosphorylated MAPKs, including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAP kinase (p38 MAPK). Further analysis using pharmacological inhibitors against caspases and MAPKs showed that TBT also induced cell death in a caspase-independent manner and that p38 MAPK is involved in TBT-induced caspase-independent cell death, whereas JNK is involved in the caspase-dependent apoptotic pathway. Thus, TBT employs at least two independent signaling cascades to mediate cell death in RTG-2 cells. To our knowledge, this is the first study revealing the relationship between MAPK activation and TBT cytotoxicity in RTG-2 cells.     

Requirement of the JNK-associated Bcl-2 pathway for human lactoferrin-induced apoptosis in the Jurkat leukemia T cell line

The cell proliferation of p53-deficient Jurkat T cells is controlled after prolonged exposure to human lactoferrin (Lf). However, the molecular mechanism by which Lf influences these cellular responses remains unclear. In this study, we demonstrate that Lf-induced apoptosis in Jurkat T cells occurs in a dose- and time-dependent manner via the regulation of c-Jun N-terminal kinase (JNK) activity. Jurkat cells exposed to Lf for 1 day, especially at concentrations in excess of 500 microg/ml, showed typical apoptosis, as indicated by decreased cell viability and increased Annexin V binding. Our results also showed that Lf induced the activation of caspase 9 and caspase 3 activation, as demonstrated by our detection of cleaved caspases and PARP. Lf-induced apoptosis did not influence Bcl-2 expression via an ERK1/2 phosphorylation pathway, but was rather associated with the level of Bcl-2 phosphorylation. The treatment of cells with the specific JNK inhibitor SP600125, but not the p38 MAPK inhibitor SB203580, revealed that the JNK-Bcl-2 signaling cascade is required for Lf-induced apoptosis. When JNK activation was abolished by SP600125, no Bcl-2 phosphorylation was detected, and the Lf-treated Jurkat cells did not undergo cell death. These findings indicate that Lf functions as a biological mediator of apoptosis in the human leukemia Jurkat T-cell line, via the JNK-associated Bcl-2 signaling pathway.     

Delayed and persistent ERK1/2 activation is required for 4-hydroxytamoxifen-induced cell death

Tamoxifen (Tam), and its active metabolite, 4-hydroxytamoxifen (OHT), compete with estrogens for binding to the estrogen receptor (ER). Tam and OHT can also induce ER-dependent apoptosis of cancer cells. 10-100nM OHT induces ER-dependent apoptosis in approximately 3 days. Using HeLaER6 cells, we examined the role of OHT activation of signal transduction pathways in OHT-ER-mediated apoptosis. OHT-ER activated the p38, JNK and ERK1/2 pathways. Inhibition of p38 activation with SB203580, or RNAi-knockdown of p38alpha, moderately reduced OHT-ER mediated cell death. A JNK inhibitor partly reduced cell death. Surprisingly, the MEK1/2 inhibitor, PD98059, completely blocked OHT-ER induced apoptosis. EGF, an ERK1/2 activator, enhanced OHT-induced apoptosis. OHT induced a delayed and persistent phosphorylation of ERK1/2 that persisted for >80h. Addition of PD98059 as late as 24h after OHT largely blocked OHT-ER mediated apoptosis. The antagonist, ICI 182,780, blocked both the long-term OHT-mediated phosphorylation of ERK1/2 and OHT-induced apoptosis. Our data suggests that the p38 and JNK pathways, which often play a central role in apoptosis, have only a limited role in OHT-ER-mediated cell death. Although rapid activation of the ERK1/2 pathway is often associated with cell growth, persistent activation of the ERK1/2 pathway is essential for OHT-ER induced cell death.     

The kinase domain of MEKK1 induces apoptosis by dysregulation of MAP kinase pathways

MAP kinase pathways comprise a group of parallel protein phosphorylation cascades, which are involved in signaling triggered by a variety of stimuli. Previous findings suggested that the ERK and the JNK pathways have opposing roles in regulating proliferation and survival or apoptosis and that apoptosis can be promoted by inhibiting the ERK pathway or by activation of the JNK pathway. In order to test this hypothesis and explore whether it can be exploited as a strategy for killing human cancer cells, we used gene transfer experiments with a range of cancer cell lines. We expressed the catalytic fragment of human MEKK1 to activate JNK and the Ras-binding domain (RBD) of Raf-1 to inhibit the Ras-ERK pathway. In addition, we designed several RBD-MEKK1 fusion proteins aiming to simultaneously activate the JNK and block the ERK pathway. We found that the MEKK1 proteins as well as the RBD alone could reduce colony formation in all cell lines. The survival time of MEKK1-expressing cells depended on the cell line. In HeLa cells, survival could be prolonged by inhibition of caspases but not by coexpression of the anti-apoptotic protein Bcl-2. Due to a lower kinase activity the RBD-MEKK1 fusion proteins were less effective in apoptosis induction than the MEKK1 kinase domain alone. Using mutant forms of Ras and Raf-1 we could show that the reduced kinase activity of RBD-MEKK1 fusion proteins was caused by binding to the Ras protein. The expression of lethal doses of MEKK1 resulted in a strong activation of all three major MAP kinase families JNK, ERK, and p38. Blocking these pathways either by coexpressing a dominant negative form of MKK4 or with inhibitors of MEK or p38 failed to inhibit apoptosis. This suggests that MEKK1 induces apoptosis by causing a general deregulation of MAP kinase signaling rather than by the activation of a single pathway.     

Role of mitogen-activated protein kinases in S-nitrosoglutathione-induced macrophage apoptosis

The inflammatory mediator nitric oxide (NO*) promotes apoptotic cell death based on morphological evidence, accumulation of the tumor suppressor p53, caspase-3 activation, and DNA fragmentation in RAW 264.7 macrophages. Since nitrosothiols may actually be the predominant form of biologically active NO* in vivo, we used S-nitrosoglutathione (GSNO) to study activation of extracellular signal-regulated protein kinases1/2 (ERK1/2), c-Jun N-terminal kinases/stress-activated protein kinases (JNK1/2), and p38 kinases. Moreover, we determined the role of mitogen-activated protein kinase signaling in the apoptotic transducing ability of GSNO. ERK1/2 became activated in response to GSNO after 4 h and remained active for the next 20 h. Blocking the ERK1/2 pathway by the mitogen-activated protein kinase kinase inhibitor PD 98059 enhanced GSNO-elicited apoptosis. p38 was activated as well, but inhibition of p38 with SB 203580 left apoptosis unaltered. Activation of JNK1/2 by GSNO showed maximal kinase activities between 2 and 8 h. Attenuating JNK1/2 by antisense-depletion eliminated the pro-apoptotic action of low GSNO concentrations (250 microM), whereas apoptosis proceeded independently of JNK1/2 at higher doses of the NO donor (500 microM). Decreased apoptosis by JNK1/2 depletion prevented p53 accumulation after the addition of GSNO, which positions JNK1/2 upstream of the p53 response at low agonist concentrations. In line, JNK1/2 activation proceeded unaltered in p53-antisense transfected macrophages. However, with higher GSNO concentrations apoptotic transducing pathways, including p53 accumulation, were JNK1/2 unrelated. The regulation of mitogen-activated protein kinases by GSNO may help to define cell protective and destructive actions of reactive nitrogen species.     

The involvement of calcium and MAP kinase signaling pathways in the production of radiation-induced bystander effects

Much evidence now exists regarding radiation-induced bystander effects, but the mechanisms involved in the transduction of the signal are still unclear. The mitogen-activated protein kinase (MAPK) pathways have been linked to growth factor-mediated regulation of cellular events such as proliferation, senescence, differentiation and apoptosis. Activation of multiple MAPK pathways such as the ERK, JNK and p38 pathways have been shown to occur after exposure of cells to radiation and a variety of other toxic stresses. Previous studies have shown oxidative stress and calcium signaling to be important in radiation-induced bystander effects. The aim of the present study was to investigate MAPK signaling pathways in bystander cells exposed to irradiated cell conditioned medium (ICCM) and the role of oxidative metabolism and calcium signaling in the induction of bystander responses. Human keratinocytes (HPV-G cell line) were irradiated (0.005-5 Gy) using a cobalt-60 teletherapy unit. The medium was harvested 1 h postirradiation and transferred to recipient HPV-G cells. Phosphorylated forms of p38, JNK and ERK were studied by immunofluorescence 30 min-24 h after exposure to ICCM. Inhibitors of the ERK pathway (PD98059 and U0126), the JNK pathway (SP600125), and the p38 pathway (SB203580) were used to investigate whether bystander-induced cell death could be blocked. Cells were also incubated with ICCM in the presence of superoxide dismutase, catalase, EGTA, verapamil, nifedipine and thapsigargin to investigate whether bystander effects could be inhibited because of the known effects on calcium homeostasis. Activated forms of JNK and ERK proteins were observed after exposure to ICCM. Inhibition of the ERK pathway appeared to increase bystander-induced apoptosis, while inhibition of the JNK pathway appeared to decrease apoptosis. In addition, reactive oxygen species, such as superoxide and hydrogen peroxide, and calcium signaling were found to be important modulators of bystander responses. Further investigations of these signaling pathways may aid in the identification of novel therapeutic targets.     

Surfactin induces apoptosis in human breast cancer MCF-7 cells through a ROS/JNK-mediated mitochondrial/caspase pathway

Surfactin has been known to inhibit proliferation and induce apoptosis in cancer cells. However, the molecular mechanisms involved in surfactin-induced apoptosis remain poorly understood. The present study was undertaken to elucidate the underlying network of signaling events in surfactin-induced apoptosis of human breast cancer MCF-7 cells. In this study, surfactin caused reactive oxygen species (ROS) generation and the surfactin-induced cell death was prevented by antioxidants N-acetylcysteine (NAC) and catalase, suggesting involvement of ROS generation in surfactin-induced cell death. Surfactin induced a sustained activation of the phosphorylation of ERK1/2 and JNK, but not p38. Moreover, surfactin-induced cell death was reversed by PD98059 (an inhibitor of ERK1/2) and SP600125 (an inhibitor of JNK), but not by SB203580 (an inhibitor of p38). However, the phosphorylation of JNK rather than ERK1/2 activation by surfactin was blocked by NAC/catalase. These results suggest that the action of surfactin on MCF-7 cells was via ERK1/2 and JNK, but not via p38, and the ERK1/2 and JNK activation induce apoptosis through two independent signaling mechanisms. Surfactin triggered the mitochondrial/caspase apoptotic pathway indicated by enhanced Bax-to-Bcl-2 expression ratio, loss of mitochondrial membrane potential, cytochrome c release, and caspase cascade reaction. The NAC and SP600125 blocked these events induced by surfactin. Moreover, the general caspase inhibitor z-VAD-FMK inhibited the caspase-6 activity and exerted the protective effect against the surfactin-induced cell death. Taken together, these findings suggest that the surfactin induces apoptosis through a ROS/JNK-mediated mitochondrial/caspase pathway.     

Zn2+-induced ERK activation mediated by reactive oxygen species causes cell death in differentiated PC12 cells

Recent studies have provided evidence that Zn2+ plays a crucial role in ischemia- and seizure-induced neuronal death. However, the intracellular signaling pathways involved in Zn2+-induced cell death are largely unknown. In the present study, we investigated the roles of mitogen-activated protein kinases (MAPKs), such as c-Jun N-terminal kinase (JNK), p38 MAPK and extracellular signal-regulated kinase (ERK), and of reactive oxygen species (ROS) in Zn2+-induced cell death using differentiated PC12 cells. Intracellular accumulation of Zn2+ induced by the combined application of pyrithione (5 microM), a Zn2+ ionophore, and Zn2+ (10 microM) caused cell death and activated JNK and ERK, but not p38 MAPK. Preventing JNK activation by the expression of dominant negative SEK1 (SEKAL) did not attenuate Zn2+-induced cell death, whereas the inhibition of ERK with PD98059 and the expression of dominant negative Ras mutant (RasN17) significantly prevented cell death. Inhibition of protein kinase C (PKC) and phosphatidylinositol-3 kinase had little effect on Zn2+-induced ERK activation. Intracellular Zn2+ accumulation resulted in the generation of ROS, and antioxidants prevented both the ERK activation and the cell death induced by Zn2+. Therefore, we conclude that although Zn2+ activates JNK and ERK, only ERK contributes to Zn2+-induced cell death, and that ERK activation is mediated by ROS via the Ras/Raf/MEK/ERK signaling pathway.     

Activation of the c-Jun N-terminal kinase (JNK) signaling pathway is essential during PBOX-6-induced apoptosis in chronic myelogenous leukemia (CML) cells

The mitogen-activated protein (MAP) kinase family is activated in response to a wide variety of external stress signals such as UV irradiation, heat shock, and many chemotherapeutic drugs and leads to the induction of apoptosis. A novel series of pyrrolo-1,5-benzoxazepines have been shown to potently induce apoptosis in chronic myelogenous leukemia (CML) cells, which are resistant to many chemotherapeutic agents. In this study we have delineated part of the mechanism by which a representative compound known as PBOX-6 induces apoptosis. We have investigated whether PBOX-6 induces activation of MAP kinase signaling pathways in CML cells. Treatment of K562 cells with PBOX-6 resulted in the transient activation of two JNK isoforms, JNK1 and JNK2. In contrast, PBOX-6 did not activate the extracellular signal-regulated kinase (ERK) or p38. Apoptosis was found to occur independently of the small GTPases Ras, Rac, and Cdc42 but involved phosphorylation of the JNK substrates, c-Jun and ATF-2. Pretreatment of K562 cells with the JNK inhibitor, dicoumarol, abolished PBOX-6-induced phosphorylation of c-Jun and ATF-2 and inhibited the induced apoptosis, suggesting that JNK activation is an essential component of the apoptotic pathway induced by PBOX-6. Consistent with this finding, transfection of K562 cells with the JNK scaffold protein, JIP-1, inhibited JNK activity and apoptosis induced by PBOX-6. JIP-1 specifically scaffolds JNK, MKK7, and members of the mixed-lineage kinase (MLK) family, implicating these kinases upstream of JNK in the apoptotic pathway induced by PBOX-6 in K562 cells.     

ZBP-89-induced apoptosis is p53-independent and requires JNK

ZBP-89 induces apoptosis in human gastrointestinal cancer cells through a p53-independent mechanism. To understand the apoptotic pathway regulated by ZBP-89, we identified downstream signal transduction targets. Ectopic expression of ZBP-89 induced apoptosis through the mitochondrial pathway and was accompanied by activation of all three MAP kinase subfamilies: JNK1/2, ERK1/2 and p38 MAP kinase. ZBP-89-induced apoptosis was markedly enhanced by ERK inhibition with U0126. In contrast, inhibiting JNK with a JNK1-specific peptide inhibitor or dominant-negative JNK2 expression abrogated ZBP-89-mediated apoptosis. The p38 inhibitor SB202190 had no effect on ZBP-89-induced cell death. Protein dephosphorylation assays revealed that ZBP-89 activates JNK via repression of JNK dephosphorylation. Oligonucleotide microarray analyses revealed that ectopic expression of ZBP-89 downregulated expression of the dual-specificity phosphatase MKP6. Overexpression of MKP6 blocked ZBP-89-induced JNK phosphorylation and PARP cleavage. In addition, ectopic expression of ZBP-89 repressed Bcl-xL and Mcl-1 expression, but had no effect on Bcl-2. Silencing ZBP-89 with small interfering RNA enhanced both Bcl-xL and Mcl-1 expression. Taken together, ZBP-89-mediated apoptosis occurs via a p53-independent mechanism that requires JNK activation.