Hsp72-mediated suppression of c-Jun N-terminal kinase is implicated in development of tolerance to caspase-independent cell death

Pretreatment with mild heat shock is known to protect cells from severe stress (acquired thermotolerance). Here we addressed the mechanism of this phenomenon by using primary human fibroblasts. Severe heat shock (45 degrees C, 75 min) of the fibroblasts caused cell death displaying morphological characteristics of apoptosis; however, it was caspase independent. This cell death process was accompanied by strong activation of Akt, extracellular signal-regulated kinase 1 (ERK1) and ERK2, p38, and c-Jun N-terminal (JNK) kinases. Suppression of Akt or ERK1 and -2 kinases increased cell thermosensitivity. In contrast, suppression of stress kinase JNK rendered cells thermoresistant. Development of thermotolerance was not associated with Akt or ERK1 and -2 regulation, and inhibition of these kinases did not reduce acquired thermotolerance. On the other hand, acquired tolerance to severe heat shock was associated with downregulation of JNK. Using an antisense-RNA approach, we found that accumulation of the heat shock protein Hsp72 is necessary for JNK downregulation and is critical for thermotolerance. The capability of naive cells to withstand moderate heat treatment also appears to be dependent on the accumulation of Hsp72 induced by this stress. Indeed, exposure to 45 degrees C for 45 min caused only transient JNK activation and was nonlethal, while prevention of Hsp72 accumulation prolonged JNK activation and led to massive cell death. We also found that JNK activation by UV irradiation, interleukin-1, or tumor necrosis factor was suppressed in thermotolerant cells and that Hsp72 accumulation was responsible for this effect. Hsp72-mediated suppression of JNK is therefore critical for acquired thermotolerance and may play a role in tolerance to other stresses.     

Differential role of reactive oxygen species in the activation of mitogen-activated protein kinases and Akt by key receptors on B-lymphocytes: CD40, the B cell antigen receptor, and CXCR4

Background Antibodies produced by B-lymphocytes play a key role in the host defense against infection. The development, survival, and activation of B cell is regulated by multiple receptors including the B cell antigen receptor (BCR), which detects the presence of pathogens, CD40, which binds co-stimulatory molecules on activated T cells, and chemokines such as SDF-1 (CXCL12) that play key roles in B cell development and trafficking. Signaling by many receptors results in the generation of reactive oxygen species (ROS) that function as second messengers by regulating the activity of redox-sensitive kinases and phosphatases. We investigated the role of ROS in signaling by the BCR, CD40, and CXCR4, the receptor for SDF-1. We focused on activation of ERK, JNK, p38, and Akt, kinases that regulate multiple processes including cell survival, proliferation, and migration. Results Using the anti-oxidants N-acetyl L-cysteine (NAC) and ebselen to deplete intracellular ROS, we identified a differential requirement for ROS in the activation of ERK, JNK, p38, and Akt by these receptors. We found that CD40 activated JNK, p38, and Akt via redox-dependent pathways that were sensitive to ROS depletion by NAC and ebselen. In contrast, BCR-induced activation of ERK, JNK, p38, and Akt was not affected by ROS depletion. We also found that CXCR4-induced Akt activation was ROS-dependent even though activation of the ERK, JNK, and p38 MAP kinases by CXCR4 occurred via ROS-independent pathways. Conclusion The differential requirement for ROS in the activation of ERK, JNK, p38, and Akt by the BCR, CD40, and CXCR4 likely reflects the multiplicity of upstream activators for each of these kinases, only some of which may be regulated in a redox-dependent manner. These findings support the idea that ROS are important second messengers in B cells and suggest that oxidants or anti-oxidants could be used to modulate B cell activation.     

Signals for death and differentiation: a two-step mechanism for in vitro transformation of adult islets of Langerhans to duct epithelial structures

Phenotypic change of adult pancreatic islets has been implicated in the development of certain pancreatic cancers and in islet transplant failure. The aim of this study was to characterize intracellular events that mediate changes in adult islet phenotype. Using an in vitro islet-to-duct transformation model, canine islets were induced to undergo phenotypic transformation to duct-like epithelial structures through a two-stage process. Stage one was characterized by widespread islet cell apoptosis associated with the formation of cavitary spaces within the islets. During this stage, c-Jun N-terminal regulated kinase (JNK) and caspase-3 activities were elevated, while extracellular signal-regulated kinase (ERK) and Akt activities were decreased. The second stage of the process was characterized by an inversion in the balance in activity between these signal transduction pathways and by a concomitant decrease in apoptosis. The transformed islets were no longer immunoreactive for islet cell hormones, but expressed the duct epithelial cell marker CK-AE1/AE3. In contrast to islet cells, these duct epithelial cells were highly proliferative. To clarify the role of the identified changes in signal transduction events, we performed additional studies using pharmacological inhibitors of enzyme activity and demonstrated that inhibition of JNK and caspase-3 activity prevented cystic transformation. Our results indicate that the balance in signaling activity between ERK/Akt and JNK/caspase-3 appears to be an important regulator of islet cell death and differentiation.     

Sodium vanadate inhibits apoptosis in malignant glioma cells: A role for Akt/PKB

The dual signal hypothesis of apoptosis holds that a common signal can activate both apoptotic and proliferative pathways. The fate of a cell is dependent on which of these two pathways predominates. In the MAPK family of kinases, ERK and JNK have been proposed to mediate apoptosis whereas the PI3K-stimulated kinase, Akt/PKB, has been shown to inhibit apoptosis. The object of this study was to determine the role of these kinases in a glioma model of apoptosis. We have previously shown that K252a induces apoptosis and inhibits kinase activity. In this study we confirm these results and shown that the protein tyrosine phosphatase inhibitor sodium vanadate activates ERK, JNK and Akt/PKB, but does not stimulate proliferation. Vanadate did protect T98G cells from K252a-induced apoptosis, an effect that was abolished by addition of the PI3K inhibitor wortmannin. This suggests that PI3K and Akt/PKB may be responsible for mediating vanadate's protective effect on glioma cells. We conclude that the intracellular balance between protein phosphorylation pathways is a critical determinant of both cell proliferation and cell death.     

Interplay between PI3K/Akt and MAPK signaling pathways in DNA-damaging drug-induced apoptosis

In order to elucidate the role of the mitogen-activated protein kinases, including JNK, p38 MAPK and ERK, as well as the survival-associated PI3K/Akt signaling pathway, in the response to chemotherapy, we have conducted a comparative study regarding the effects of doxorubicin on these pathways. Doxorubicin was determined to elicit the apoptosis of NIH3T3 cells in a dose-dependent manner. Prior to cell death, both Akt and p38 MAPK were transiently activated, and subsequently inactivated almost wholly, whereas ERK and JNK evidenced sustained activations in response to the drug treatment. The inhibition of PI3K/Akt and p38 MAPK both accelerated and enhanced doxorubicin-induced apoptosis and ERK inhibition apparently exerted negative effect on apoptosis. The modulation of PI3K/Akt activation by treatment of LY294002 or expression of Akt mutants such as Akt-DN or Myr-Akt exerted a significant effect on the activation of ERK1/2. We also observed that PI3K/Akt and sustained ERK activation were associated intimately with the etoposide-induced apoptosis. Taken together, our results clearly suggest that the differential regulation of the PI3K/Akt, ERK1/2, and p38 MAPK signaling pathways are crucial in the context of DNA-damaging drug-induced apoptosis, and this has compelled us to propose that the sustained activation of ERK1/2 pathway may be generally involved in the apoptosis induced by anticancer DNA-damaging drugs, including doxorubicin and etoposide.     

The EGF receptor activates ERK but not JNK Ras-dependently in basal conditions but ERK and JNK activation pathways are predominantly Ras-independent during cardiomyocyte stretch

Myocardial stretch is a major determinant of ventricular hypertrophy, a physiological adaptational process that can be detrimental, leading to heart failure. Therapies aimed to limit the development of cardiac hypertrophy are thus currently evaluated. Among possible targets, the small G protein Ras and the epidermal growth factor receptor (EGFR) have been shown to be involved during stretch but their precise role in the activation of the major actors of hypertrophy, the mitogen activated protein kinases (MAPK) ERK and JNK is not well known. Our goal was thus was to evaluate precisely the activation pathways of ERK and JNK during stretch, with an emphasis on the role of the EGFR. For this purpose, neonatal rat cardiomyocytes in culture were stretched for different time durations. As measured by Western blot of their phosphorylated forms, ERK and JNK were activated by stretch. Ras inhibition decreased basal ERK phosphorylation but had no effect on stretch-induced ERK activation. Under basal conditions, EGFR activated ERK in a classical Ras-dependent manner. Upon stretch, EGFR transactivation activated ERK through both Ras-dependent and Ras-independent pathways. Interestingly, we also show that the Akt pathway participates in stretch-induced ERK activation with an involvement of EGFR. Unlike ERK, JNK activation is independent of either EGFR or PI3 kinase but dependent on other tyrosine kinases. In conclusion these data show different Ras-dependent and Ras-independent pathways in basal conditions and during stretch with a previously unrecognized role of Akt in the activation of ERK.     

β-Arrestin prevents cell apoptosis through pro-apoptotic ERK1/2 and p38 MAPKs and anti-apoptotic Akt pathways

Our previous studies have shown that β-arrestin 2 plays an anti-apoptotic effect. However, the mechanisms by which β-arrestin contribute to anti-apoptotic role remain unclear. In this study, we show that a deficiency of either β-arrestin 1 or β-arrestin 2 significantly increases serum deprivation (SD)-induced percentage of apoptotic cells. β-arrestin 2 deficient-induced apoptosis was inhibited by transfection with β-arrestin 2 full-length plasmid, revealing that SD-induced apoptosis is dependent on β-arrestin 2. Furthermore, in the absence of either β-arrestin 1 or β-arrestin 2 significantly enhances SD-induced the level of pro-apoptotic proteins, including cleaved caspase-3, extracellular-signal regulated kinase 1/2 (ERK1/2) and p38, members of mitogen-activated protein kinases (MAPKs). In addition, a deficiency of either β-arrestin 1 or β-arrestin 2 inhibits phosphorylation of Akt. The SD-induced changes in cleaved caspase-3, ERK1/2 and p38 MAPKs, Akt, and apoptotic cell numbers could be blocked by double knockout of β-arrestin 1/2. Our study thus demonstrates that β-arrestin inhibits cell apoptosis through pro-apoptotic ERK1/2 and p38 MAPKs and anti-apoptotic Akt signaling pathways.     

Signaling events in amyloid beta-peptide-induced neuronal death and insulin-like growth factor I protection

Amyloid beta-peptide (Abeta) is implicated as the toxic agent in Alzheimer's disease and is the major component of brain amyloid plaques. In vitro, Abeta causes cell death, but the molecular mechanisms are unclear. We analyzed the early signaling mechanisms involved in Abeta toxicity using the SH-SY5Y neuroblastoma cell line. Abeta caused cell death and induced a 2- to 3-fold activation of JNK. JNK activation and cell death were inhibited by overexpression of a dominant-negative SEK1 (SEK1-AL) construct. Butyrolactone I, a cdk5 inhibitor, had an additional protective effect against Abeta toxicity in these SEK1-AL-expressing cells suggesting that cdk5 and JNK activation independently contributed to this toxicity. Abeta also weakly activated ERK and Akt but had no effect on p38 kinase. Inhibitors of ERK and phosphoinositide 3-kinase (PI3K) pathways did not affect Abeta-induced cell death, suggesting that these pathways were not important in Abeta toxicity. Insulin-like growth factor I protected against Abeta toxicity by strongly activating ERK and Akt and blocking JNK activation in a PI3K-dependent manner. Pertussis toxin also blocked Abeta-induced cell death and JNK activation suggesting that G(i/o) proteins were upstream activators of JNK. The results suggest that activation of the JNK pathway and cdk5 may be initial signaling cascades in Abeta-induced cell death.     

Differential signaling mechanisms of HNP-induced IL-8 production in human lung epithelial cells and monocytes

Human neutrophil peptides (HNP) kill microorganisms but also modulate immune responses through upregulation of the chemokine IL-8 by activation of the nucleotide P2Y(6) receptor. However, the intracellular signaling mechanisms remain yet to be determined. Human lung epithelial cells (A549) and monocytes (U937) were stimulated with HNP in the absence and presence of the specific kinase inhibitors for Src, extracellular signal-regulated kinase-1 and -2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK), c-Jun-N-terminal kinases (JNK), and Akt. HNP induced a rapid phosphorylation of the kinases in both cell types associated with a dose-dependent, selective production of IL-8 among 10 cytokines assayed. The HNP-induced IL-8 production was blocked by the Src tyrosine kinase inhibitor PP2, MEK1/2 inhibitor U0126, and the phosphatidylinositol 3 kinase (PI3K) inhibitor LY294002, but not by the JNK inhibitor SP600125 in both cell types. Treatment with the p38 inhibitor SB203580 attenuated the HNP-induced IL-8 production only in monocytes. Blockade of Src kinase blunted HNP-induced phosphorylation of the ERK1/2 and Akt but not p38 in monocytes. In contrast, Src inhibition had no effect on phosphorylation of the other kinases in the lung epithelial cells. We conclude that the activation of ERK1/2 and PI3K/Akt pathways is required for HNP-induced IL-8 release which occurs in a Src-independent manner in lung epithelial cells, while is Src-dependent in monocytes.     

Distinctive regulation and function of PI 3K/Akt and MAPKs in doxorubicin-induced apoptosis of human lung adenocarcinoma cells

Regulation and function of PI 3K/Akt and mitogen-activated protein kinases (MAPKs) in doxorubicin-induced cell death were investigated in human lung adenocarcinoma cells. Doxorubicin induced dose-dependent apoptosis of human lung adenocarcinoma NCI-H522 cells. Prior to cell death, both Akt and the MAPK family members (MAPKs: ERK1/2, JNK, and p38) were activated in response to the drug treatment. The kinetics of the inductions for Akt and MAPKs are, however, distinct. The activation of Akt was rapid and transient, activated within 30 min of drug addition, then declined after 3 h, whereas the activations of three MAPKs occurred later, 4 h after addition of the drug and sustained until cell death occurred. Inhibition of PI 3K/Akt activation had no effect on MAPKs' activation, suggesting that the two pathways are independently activated in response to the drug treatment. Inhibition of PI 3K/Akt and p38 accelerated and enhanced doxorubicin-induced cell death. On the contrary, inhibition of ERK1/2 or JNK had no apparent effect on the cell death. Taken together, these results suggest that PI 3K/Akt and MAPKs signaling pathways are all activated, but with distinct mechanisms, in response to doxorubicin treatment. Activation of PI 3K/Akt and p38 modulates apoptotic signal pathways and inhibits doxorubicin-induced cell death. These responses of tumor cells to cancer drug treatment may contribute to their drug resistance. Understanding of the mechanism and function of the responses will be beneficial for the development of novel therapeutic approaches for improvement of drug efficacy and circumvention of drug resistance.     

肠道病毒A71型致人扁桃体上皮细胞系UT-SCC-60B凋亡和S期阻滞的研究

肠道病毒A71型(Enterovirus A71,EV-A71)是手足口病的重要病原体,为研究EV-A71感染人扁桃体上皮细胞后对细胞凋亡和细胞周期的影响,确定ERK1/2、JNK1/2、PI3K/Akt和含半胱氨酸的天冬氨酸蛋白水解酶(Cysteinyl aspartate specific proteinase,Caspase)的作用,本文以人扁桃体上皮细胞系UT-SCC-60B为细胞模型,CCK-8试剂盒检测EV-A71对UT-SCC-60B的抑制率、流式细胞仪检测EV-A71感染组和抑制剂处理组的凋亡和细胞周期、Caspase活力检测试剂盒测定Caspase-3,Caspase-8,Caspase-9活力。EV-A71以感染剂量和感染时间依赖方式抑制UT-SCC-60B增殖;EV-A71感染致UT-SCC-60B发生细胞凋亡,抑制ERK1/2、JNK1/2和PI3K/Akt能够降低UT-SCC-60B细胞凋亡比例;EV-A71感染UT-SCC-60B后发生S期阻滞,抑制ERK1/2、JNK1/2、PI3K/Akt和Caspase阻止UT-SCC-60B发生S期阻滞;EV-A71感染UT-SCC-60B能够活化Caspase-3,Caspase-8,Caspase-9且ERK1/2、JNK1/2和PI3K/Akt调控Caspase-3,Caspase-8,Caspase-9活力。因此,EV-A71能够导致人扁桃体上皮细胞UT-SCC-60B发生凋亡和S期阻滞,并且ERK1/2、JNK1/2、PI3K/Akt和Caspase参与凋亡和S期阻滞的调控。     

P2X7 nucleotide receptors mediate caspase-8/9/3-dependent apoptosis in rat primary cortical neurons

Apoptosis is a major cause of cell death in the nervous system. It plays a role in embryonic and early postnatal brain development and contributes to the pathology of neurodegenerative diseases. Here, we report that activation of the P2X₇ nucleotide receptor (P2X₇R) in rat primary cortical neurons (rPCNs) causes biochemical (i.e., caspase activation) and morphological (i.e., nuclear condensation and DNA fragmentation) changes characteristic of apoptotic cell death. Caspase-3 activation and DNA fragmentation in rPCNs induced by the P2X₇R agonist BzATP were inhibited by the P2X₇R antagonist oxidized ATP (oATP) or by pre-treatment of cells with P2X₇R antisense oligonucleotide indicating a direct involvement of the P2X₇R in nucleotide-induced neuronal cell death. Moreover, Z-DEVD-FMK, a specific and irreversible cell permeable inhibitor of caspase-3, prevented BzATP-induced apoptosis in rPCNs. In addition, a specific caspase-8 inhibitor, Ac-IETD-CHO, significantly attenuated BzATP-induced caspase-9 and caspase-3 activation, suggesting that P2X₇R-mediated apoptosis in rPCNs occurs primarily through an intrinsic caspase-8/9/3 activation pathway. BzATP also induced the activation of C-jun N-terminal kinase 1 (JNK1) and extracellular signal-regulated kinases (ERK1/2) in rPCNs, and pharmacological inhibition of either JNK1 or ERK1/2 significantly reduced caspase activation by BzATP. Taken together, these data indicate that extracellular nucleotides mediate neuronal apoptosis through activation of P2X₇Rs and their downstream signaling pathways involving JNK1, ERK and caspases 8/9/3.     

Analysis of Ras-dependent signals that prevent caspase-3 activation and apoptosis induced by cytokine deprivation in hematopoietic cells

In hematopoietic cells, Ras has been implicated in signaling pathways that prevent apoptosis triggered by deprivation of cytokines, such as interleukin-3 (IL-3). However, the mechanism whereby Ras suppresses cell death remains incompletely understood. We have investigated the role of Ras in IL-3 signal transduction by using the cytokine-dependent BaF3 cell line. Herein, we show that the activation of the pro-apoptotic protease caspase-3 upon IL-3 removal is suppressed by expression of activated Ras, which eventually prevents cell death. For caspase-3 suppression, the Raf/extracellular signal-regulated kinase (ERK)- or phosphatidylinositol 3-kinase (PI3-K)/Akt-mediated signaling pathway downstream of Ras was required. However, inhibition of both pathways did not block activated Ras-dependent suppression of cell death-associated phenotypes, such as nuclear DNA fragmentation. Thus, a pathway that is independent of both Raf/ERK and PI3-K/Akt pathways may function downstream of Ras, preventing activated caspase-3-initiated apoptotic processes. Conditional activation of c-Raf-1 also suppressed caspase-3 activation and subsequent cell death without affecting Akt activity, providing further evidence for a PI3-K/Akt-independent mechanism.     

Inhibiting Src family tyrosine kinase activity blocks glutamate signalling to ERK1/2 and Akt/PKB but not JNK in cultured striatal neurones

Glutamate receptor activation of mitogen-activated protein (MAP) kinase signalling cascades has been implicated in diverse neuronal functions such as synaptic plasticity, development and excitotoxicity. We have previously shown that Ca2+-influx through NMDA receptors in cultured striatal neurones mediates the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt/protein kinase B (PKB) through a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent pathway. Exposing neurones to the Src family tyrosine kinase inhibitor PP2, but not the inactive analogue PP3, inhibited NMDA receptor-induced phosphorylation of ERK1/2 and Akt/PKB in a concentration-dependent manner, and reduced cAMP response element-binding protein (CREB) phosphorylation. To establish a link between Src family tyrosine kinase-mediated phosphorylation and PI 3-kinase signalling, affinity precipitation experiments were performed with the SH2 domains of the PI 3-kinase regulatory subunit p85. This revealed a Src-dependent phosphorylation of a focal adhesion kinase (FAK)-p85 complex on glutamate stimulation. Demonstrating that PI3-kinase is not ubiquitously involved in NMDA receptor signal transduction, the PI 3-kinase inhibitors wortmannin and LY294002 did not prevent NMDA receptor Ca2+-dependent phosphorylation of c-Jun N-terminal kinase 1/2 (JNK1/2). Further, inhibiting Src family kinases increased NMDA receptor-dependent JNK1/2 phosphorylation, suggesting that Src family kinase-dependent cascades may physiologically limit signalling to JNK. These results demonstrate that Src family tyrosine kinases and PI3-kinase are pivotal regulators of NMDA receptor signalling to ERK/Akt and JNK in striatal neurones.     

Rac1 mediates intestinal epithelial cell apoptosis via JNK

Apoptosis plays a key role in the maintenance of a constant cell number and a low incidence of cancer in the mucosa of the intestine. Although the small GTPase Rac1 has been established as an important regulator of migration of intestinal epithelial cells, whether Rac1 is also involved in apoptosis is unclear. The present study tested the hypothesis that Rac1 mediates TNF-alpha-induced apoptosis in IEC-6 cells. Rac1 is activated during TNF-alpha-induced apoptosis as judged by the level of GTP-Rac1, the level of microsomal membrane-associated Rac1, and lamellipodia formation. Although expression of constitutively active Rac1 does not increase apoptosis in the basal condition, inhibition of Rac1 either by NSC-23766 (Rac1 inhibitor) or expression of dominant negative Rac1 protects cells from TNF-alpha-induced apoptosis by inhibiting caspase-3, -8, and -9 activities. Inhibition of Rac1 before the administration of apoptotic stimuli significantly prevents TNF-alpha-induced activation of JNK1/2, the key proapoptotic regulator in IEC-6 cells. Inhibition of Rac1 does not modulate TNF-alpha-induced ERK1/2 and Akt activation. Inhibition of ERK1/2 and Akt activity by U-0126 and LY-294002, respectively, increased TNF-alpha-induced apoptosis. However, inhibition of Rac1 significantly decreased apoptosis in the presence of ERK1/2 and Akt inhibitors, similar to the effect observed with NSC-23766 alone in response to TNF-alpha. Thus, Rac1 inhibition protects cells independently of ERK1/2 and Akt activation during TNF-alpha-induced apoptosis. Although p38 MAPK is activated in response to TNF-alpha, inhibition of p38 MAPK did not decrease apoptosis. Rac1 inhibition did not alter p38 MAPK activity. Thus, these results indicate that Rac1 mediates apoptosis via JNK and plays a key role in proapoptotic pathways in intestinal epithelial cells.     

A new platinum(II) compound anticancer drug candidate with selective cytotoxicity for breast cancer cells

[Pt(O,O′-acac)(γ-acac)(DMS)] (PtAcD) is able to induce apoptosis in various human cancer cells, including the cisplatin-resistant human breast carcinoma MCF-7 cells. Here, to confirm that PtAcD has the potentiality for therapeutic intervention, we studied its effects in primary cultured epithelial breast cells obtained from cancers and also from the corresponding histologically proven non-malignant tissue adjacent to the tumor. We demonstrated that PtAcD (1) is more cytotoxic in cancer than in normal breast cells; (2) activated NAD(P)H oxidase, leading to PKC-ζ and PKC-α tanslocations; (3) activated antiapoptotic pathways based on the PKC-α, ERK1/2 and Akt kinases; (4) activated PKC-ζ and, only in cancer cell PKC-δ, responsible for the sustained phosphorylation of p38 and JNK1/2, kinases both of which are involved in the mitochondrial apoptotic process. Moreover, crosstalk between ERK/Akt and JNK/p38 pathways affected cell death and survival in PtAcD-treated breast cell. In conclusion, this study adds and extends data that highlight the pharmacological potential of PtAcD as an anti breast cancer drug.     

Amylin-induced cytotoxicity is associated with activation of caspase-3 and MAP kinases

Nanomolar concentrations of human amylin promote death of RINm5F cells in a time- and concentrationdependent manner. Morphological changes of chromatin integrity suggest that cells are predominantly undergoing apoptosis. Human amylin induces significant activation of caspase-3 and strong and sustained phosphorylation of stress-activated protein kinases, c-Jun N-terminal kinase (JNK) and p38, that precedes cell death. Extracellular signal-regulated kinase (ERK) activation was not concomitant with JNK and/or p38 activation. Activation of caspase-3 and mitogen-activated protein kinases (MAPKs) was detected by Western blot analysis. Addition of the MEK1 inhibitor PD 98059 had no effect on amylin-induced apoptosis, suggesting that ERK activation does not play a role in this apoptotic scenario. A correlative inhibition of JNK activation by the immunosuppressive drug FK506, as well as a selective inhibition of p38 MAPK activation by SB 203580, significantly suppressed procaspase-3 processing and the extent of amylin-induced cell death. Moreover, simultaneous pretreatment with both FK506 and SB 203580, or with the caspase-3 inhibitor Ac-DEVD-CHO alone, almost completely abolished procaspase-3 processing and cell death. Thus, our results suggest that amylin-induced apoptosis proceeds through sustained activation of JNK and p38 MAPK followed by caspase-3 activation.