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.     

A human homolog of the yeast Ssk2/Ssk22 MAP kinase kinase kinases, MTK1, mediates stress-induced activation of the p38 and JNK pathways.

A human homolog of the yeast Ssk2 and Ssk22 mitogen-activated protein kinase kinase kinases (MAPKKK) was cloned by functional complementation of the osmosensitivity of the yeast ssk2delta ssk22delta sho1delta triple mutant. This kinase, termed MTK1 (MAP Three Kinase 1), is 1607 amino acids long and is structurally highly similar to the yeast Ssk2 and Ssk22 MAPKKKs. In mammalian cells (COS-7 and HeLa), MTK1 overexpression stimulated both the p38 and JNK MAP kinase pathways, but not the ERK pathway. MTK1 overexpression also activated the MKK3, MKK6 and SEK1 MAPKKs, but not the MEK1 MAPKK. Furthermore, MTK1 phosphorylated and activated MKK6 and SEK1 in vitro. Overexpression of a dominant-negative MTK1 mutant [MTK1(K/R)] strongly inhibited the activation of the p38 pathway by environmental stresses (osmotic shock, UV and anisomycin), but not the p38 activation by the cytokine TNF-alpha. The dominant-negative MTK1(K/R) had no effect on the activation of the JNK pathway or the ERK pathway. These results indicate that MTK1 is a major mediator of environmental stresses that activate the p38 MAPK pathway, and is also a minor mediator of the JNK pathway.     

Apoptosis signalling by 4-hydroxynonenal: a role for JNK-c-Jun/AP-1 pathway

4-Hydroxynonenal (HNE) is one of the most abundant aldehyde components of ox-LDL and it exerts various effects on intracellular and extracellular signaling cascades. In this mini-review, a brief synopsis of HNE-modulated signaling pathways will be presented mainly focused on cell death, including recent studies from our laboratory. The results of a number of studies demonstrate the ability of HNE to induce apoptosis and ROS formation in a dose-dependent manner. Several signaling pathways have been shown to be modulated by HNE, including MAP kinases, PKC isoforms, cell-cycle regulators, receptor tyrosine kinases and caspases. In order to get insight into the mechanisms of apoptotic response by HNE, MAP kinase and caspase activation pathways have been studied in 3T3 fibroblasts; HNE induced early activation of JNK and p38 proteins but down-regulated the basal activity of ERK-1/2. We have shown that HNE-induced release of cytochrome c from mitochondria, caspase-9 and caspase-3 activation. Activation of AP-1 along with increased c-Jun and phospho-c-Jun levels could be inhibited by pretreatment of cells with certain molecules such as resveratrol. Additionally, overexpression of dominant negative c-Jun and JNK1 in 3T3 fibroblasts prevented HNE-induced apoptosis, which indicated a role for JNK-c-Jun/AP-1 pathway. JNK-dependent induction of c-Jun/AP-1 activation data in the literature indicates a critical potential role for JNK in the cellular response against toxic products of lipid peroxidation.     

Cocoa procyanidins attenuate 4-hydroxynonenal-induced apoptosis of PC12 cells by directly inhibiting mitogen-activated protein kinase kinase 4 activity

Neurodegenerative disorders such as Alzheimer's disease (AD) are associated with oxidative stress, and it has been suggested that apoptosis is a crucial pathway in neuronal cell death in AD patients. 4-Hydroxynonenal (HNE), one of the aldehydic products of membrane lipid peroxidation, is reported to be elevated in the brains of AD patients and mediates the induction of neuronal apoptosis in the presence of oxidative stress. In this study, we investigated the HNE-induced apoptosis mechanism and the protective effects of the cocoa procyanidin fraction (CPF) and its major antioxidant procyanidin B2 against the apoptosis induced by HNE in rat pheochromocytoma (PC12) cells. HNE-induced nuclear condensation and increased sub-G1 fraction, both of which are markers of apoptotic cell death, were inhibited by CPF and procyanidin B2. Intracellular reactive oxygen species (ROS) accumulation was attenuated by pretreatment with CPF and procyanidin B2. CPF and procyanidin B2 also prevented HNE-induced poly(ADP-ribose) polymerase cleavage, antiapoptotic protein (Bcl-2 and Bcl-X_L) down-regulation, and caspase-3 activation. Activation of c-Jun N-terminal protein kinase (JNK) and mitogen-activated protein kinase kinase 4 (MKK4) was attenuated by CPF and procyanidin B2. Moreover, CPF and procyanidin B2 bound directly to MKK4 and inhibited its activity. Data obtained with SP600125, a selective inhibitor of JNK, revealed that JNK is involved in HNE-induced apoptosis through the inhibition of PARP cleavage and caspase-3 activation in PC12 cells. Collectively, these results indicate that CPF and procyanidin B2 protect PC12 cells against HNE-induced apoptosis by blocking MKK4 activity as well as ROS accumulation.     

Eicosapentaenoic acid inhibits UV-induced MMP-1 expression in human dermal fibroblasts

Ultraviolet (UV) irradiation regulates UV-responsive genes, including matrix metalloproteinases (MMPs). Moreover, UV-induced MMPs cause connective tissue damage and the skin to become wrinkled and aged. Here, we investigated the effect of eicosapentaenoic acid (EPA), a dietary omega-3 fatty acid, on UV-induced MMP-1 expression in human dermal fibroblasts (HDFs). We found that UV radiation increases MMP-1 expression and that this is mediated by p44 and p42 MAP kinase (ERK) and Jun-N-terminal kinase (JNK) activation but not by p38 activation. Pretreatment of HDFs with EPA inhibited UV-induced MMP-1 expression in a dose-dependent manner and also inhibited the UV-induced activation of ERK and JNK by inhibiting ERK kinase (MEK1) and SAPK/ERK kinase 1 (SEK1) activation, respectively. Moreover, inhibition of ERK and JNK by EPA resulted in the decrease of c-Fos expression and c-Jun phosphorylation/expression induced by UV, respectively, which led to the inhibition of UV-induced activator protein-1 DNA binding activity. This inhibitory effect of EPA on MMP-1 was not mediated by an antioxidant effect. We also found that EPA inhibited 12-O-tetradecanoylphorbol-13-acetate- or tumor necrosis factor-alpha-induced MMP-1 expression in HDFs and UV-induced MMP-1 expression in HaCaT cells. In conclusion, our results demonstrate that EPA can inhibit UV-induced MMP-1 expression by inhibiting the MEK1/ERK/c-Fos and SEK1/JNK/c-Jun pathways. Therefore, EPA is a potential agent for the prevention and treatment of skin aging.     

ASK1 regulates influenza virus infection-induced apoptotic cell death

Apoptosis occurs in influenza virus (IV)-infected cells. There are a number of mechanisms for the regulation of apoptosis. However, the molecular mechanism of IV infection-induced apoptosis is still controversial. Apoptosis signal-regulating kinase1 (ASK1) is a ubiquitously expressed mitogen-activated protein kinase kinase kinase (MAPKKK) that activates the SEK1-c-Jun N-terminal kinase (JNK) and MKK3/MKK6-p38 MAPK signaling cascades. ASK1 has been implicated in cytokine- and stress-induced apoptosis. Here, we show the following: (1) IV infection activated ASK1 and concomitantly phosphorylated JNK and p38 MAPK in human bronchial epithelial cells; (2) the activation of JNK and p38 MAPK but not extracellular-regulated kinase (ERK) in embryonic fibroblasts (MEFs) derived from ASK1 knockout mice (ASK1(-/-) MEFs) was depressed compared to MEFs derived from wild type mice (ASK1(+/+) MEFs); and (3) ASK1(-/-) MEFs were defective in IV infection-induced caspase-3 activation and cell death. These results indicate that apoptosis in IV-infected BEC is mediated through ASK1-dependent cascades.     

Role of apoptosis signal-regulating kinase in regulation of the c-Jun N-terminal kinase pathway and apoptosis in sympathetic neurons

We have previously shown that nerve growth factor (NGF) withdrawal-induced death requires the activity of the small GTP-binding protein Cdc42 and that overexpression of an active form of Cdc42 is sufficient to mediate neuronal apoptosis via activation of the c-Jun pathway. Recently, a new mitogen-activated protein (MAP) kinase kinase kinase, apoptosis signal-regulating kinase 1 (ASK1) which activates both the c-Jun N-terminal kinase (JNK) and p38 MAP kinase pathways and plays pivotal roles in tumor necrosis factor- and Fas-induced apoptosis, has been identified. Therefore, we investigated the role of ASK1 in neuronal apoptosis by using rat pheochromocytoma (PC12) neuronal cells and primary rat sympathetic neurons (SCGs). Overexpression of ASK1-DeltaN, a constitutively active mutant of ASK1, activated JNK and induced apoptosis in differentiated PC12 cells and SCG neurons. Moreover, in differentiated PC12 cells, NGF withdrawal induced a four- to fivefold increase in the activity of endogenous ASK1. Finally, expression of a kinase-inactive ASK1 significantly blocked both NGF withdrawal- and Cdc42-induced death and activation of c-jun. Taken together, these results demonstrate that ASK1 is a crucial element of NGF withdrawal-induced activation of the Cdc42-c-Jun pathway and neuronal apoptosis.     

Ebselen inhibits tumor necrosis factor-alpha-induced c-Jun N-terminal kinase activation and adhesion molecule expression in endothelial cells

Tumor necrosis factor-alpha (TNF-alpha) stimulates expression of endothelial cell (EC) genes that may promote atherosclerosis in part by an activation of mitogen-activated protein (MAP) kinases. Ebselen (2-phenyl-1,2-benzisoselenazol-3[2H]-one), a selenoorganic compound, is effective for acute ischemic stroke; however, its effect on EC has not yet been elucidated. We examined the effect of ebselen on TNF-alpha-induced MAP kinase activation and adhesion molecule expression in cultured human umbilical vein endothelial cells (HUVEC). Extracellular signal-regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK) and p38 were rapidly and significantly activated by TNF-alpha in HUVEC. TNF-alpha-induced JNK activation was inhibited by ebselen, whereas ERK1/2 and p38 were not affected. Apoptosis signal-regulated kinase 1 (ASK1) was suggested to be involved in TNF-alpha-induced JNK activation because transfection of kinase-inactive ASK1 inhibited TNF-alpha-induced JNK activation. Ebselen inhibited TNF-alpha-induced TNF receptor-associated factor 2 (TRAF2)-ASK1 complex formation and phosphorylation of stress-activated protein kinase ERK kinase 1 (SEK1), which is an upstream signaling molecule of JNK. Finally, TNF-alpha-induced activator protein-1 (AP-1) and nuclear factor-kappaB (NF-kappaB) activation and resultant intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expressions were inhibited by ebselen. Specific inhibitors for JNK and NF-kappaB also inhibited TNF-alpha-induced ICAM-1 and VCAM-1 expressions in HUVEC. These findings suggest that ebselen prevents TNF-alpha-induced EC activation through the inhibition of TRAF2-ASK1-SEK1 signaling pathway, which leads to JNK activation. Inhibition of JNK by ebselen may imply its usefulness for the prevention of atherosclerosis relevant to EC activation.     

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.     

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.     

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.     

Apoptosis signal-regulating kinase 1 (ASK1) induces neuronal differentiation and survival of PC12 cells

Apoptosis signal-regulating kinase 1 (ASK1) is a ubiquitously expressed mitogen-activated protein kinase kinase kinase that activates the c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase signaling cascades. We report here that expression of constitutively active ASK1 (ASK1DeltaN) induces neurite outgrowth in the rat pheochromocytoma cell line PC12. We found that p38 and to a lesser extent JNK, but not ERK, were activated by the expression of ASK1DeltaN in PC12 cells. ASK1DeltaN-induced neurite outgrowth was strongly inhibited by treatment with the p38 inhibitor SB203580 but not with the MEK inhibitors, suggesting that activation of p38, rather than of ERK, is required for the neurite-inducing activity of ASK1 in PC12 cells. We also observed that ASK1DeltaN induced expression of several neuron-specific proteins and phosphorylation of neurofilament proteins, confirming that PC12 cells differentiated into mature neuronal cells by ASK1. Moreover, ASK1DeltaN-expressing PC12 cells survived in serum-starved condition. ASK1 thus appears to mediate signals leading to both differentiation and survival of PC12 cells. Together with previous reports indicating that ASK1 functions as a pro-apoptotic signaling intermediate, these results suggest that ASK1 has a broad range of biological activities depending on cell types and/or cellular context.     

Overexpression of protein kinase C isoforms protects RAW 264.7 macrophages from nitric oxide-induced apoptosis: involvement of c-Jun N-terminal kinase/stress-activated protein kinase, p38 kinase, and CPP-32 protease pathways

Nitric oxide (NO) induces apoptotic cell death in murine RAW 264.7 macrophages. To elucidate the inhibitory effects of protein kinase C (PKC) on NO-induced apoptosis, we generated clones of RAW 264.7 cells that overexpress one of the PKC isoforms and explored the possible interactions between PKC and three structurally related mitogen-activated protein (MAP) kinases in NO actions. Treatment of RAW 264.7 cells with sodium nitroprusside (SNP), a NO-generating agent, activated both c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38 kinase, but did not activate extracellular signal-regulated kinase (ERK)-1 and ERK-2. In addition, SNP-induced apoptosis was slightly blocked by the selective p38 kinase inhibitor (SB203580) but not by the MAP/ERK1 kinase inhibitor (PD098059). PKC transfectants (PKC-beta II, -delta, and -eta) showed substantial protection from cell death induced by the exposure to NO donors such as SNP and S-nitrosoglutathione (GSNO). In contrast, in RAW 264.7 parent or in empty vector-transformed cells, these NO donors induced internucleosomal DNA cleavage. Moreover, overexpression of PKC isoforms significantly suppressed SNP-induced JNK/SAPK and p38 kinase activation, but did not affect ERK-1 and -2. We also explored the involvement of CPP32-like protease in the NO-induced apoptosis. Inhibition of CPP32-like protease prevented apoptosis in RAW 264.7 parent cells. In addition, SNP dramatically activated CPP32 in the parent or in empty vector-transformed cells, while slightly activated CPP32 in PKC transfectants. Therefore, we conclude that PKC protects NO-induced apoptotic cell death, presumably nullifying the NO-mediated activation of JNK/SAPK, p38 kinase, and CPP32-like protease in RAW 264.7 macrophages.     

Differential activation of mitogen-activated protein kinases in AGS gastric epithelial cells by cag+ and cag- Helicobacter pylori.

The aim of this study was to determine whether Helicobacter pylori activates mitogen-activated protein (MAP) kinases in gastric epithelial cells. Infection of AGS cells with an H. pylori cag+ strain rapidly (5 min) induced a dose-dependent activation of extracellular signal-regulated kinases (ERK), p38, and c-Jun N-terminal kinase (JNK) MAP kinases, as determined by Western blot analysis and in vitro kinase assay. Compared with cag+ strains, cag- clinical isolates were less potent in inducing MAP kinase, particularly JNK and p38, activation. Isogenic inactivation of the picB region of the cag pathogenicity island resulted in a similar loss of JNK and p38 MAP kinase activation. The specific MAP kinase inhibitors, PD98059 (25 microM; MAP kinase kinase (MEK-1) inhibitor) and SB203580 (10 microM; p38 inhibitor), reduced H. pylori-induced IL-8 production in AGS cells by 78 and 82%, respectively (p < 0.01 for each). Both inhibitors together completely blocked IL-8 production (p < 0.001). However, the MAP kinase inhibitors did not prevent H. pylori-induced IkappaBalpha degradation or NF-kappaB activation. Thus, H. pylori rapidly activates ERK, p38, and JNK MAP kinases in gastric epithelial cells; cag+ isolates are more potent than cag- strains in inducing MAP kinase phosphorylation and gene products of the cag pathogenicity island are required for maximal MAP kinase activation. p38 and MEK-1 activity are required for H. pylori-induced IL-8 production, but do not appear to be essential for H. pylori-induced NF-kappaB activation. Since MAP kinases regulate cell proliferation, differentiation, programmed death, stress, and inflammatory responses, activation of gastric epithelial cell MAP kinases by H. pylori cag+ strains may be instrumental in inducing gastroduodenal inflammation, ulceration, and neoplasia.     

Sphingosine-1-phosphate promotes mouse melanocyte survival via ERK and Akt activation

In this study, we investigated the signalling pathways induced by ultraviolet B (UVB) and the effects of sphingosine-1-phosphate on UVB-induced apoptosis of mouse melanocytes, Mel-Ab, and observed the cytoprotective effects of sphingosine-1-phosphate on UVB-induced apoptosis. Since sphingosine-1-phosphate is a well-known mitogenic agent, we thought it possible that the mitogenic effect of sphingosine-1-phosphate might contribute to cell survival. However, we found that sphingosine-1-phosphate significantly inhibits DNA synthesis. We next examined the regulation of the three major subfamilies of mitogen-activated protein (MAP) kinases and of the Akt pathway by sphingosine-1-phosphate against UVB-induced apoptosis. UVB irradiation resulted in the remarkable and sustained activation of c-Jun N-terminal kinase (JNK), while p38 MAP kinase was only transiently activated. The basal level of extracellular signal-regulated protein kinase (ERK) phosphorylation decreased 30 min after UVB irradiation, whereas the basal level of Akt phosphorylation was unaffected by UVB. We also found that sphingosine-1-phosphate potently stimulates the phosphorylation of both ERK and Akt, which are involved in the cell survival-signalling cascade. Furthermore, the specific inhibition of the ERK and Akt pathways by PD98059 and LY294002, respectively, restored the cytoprotective effect induced by sphingosine-1-phosphate. On the other hand, the p38 inhibitor SB203580 additively enhanced the cytoprotective effect on sphingosine-1-phosphate. Based on these results, we conclude that the activation of p38 MAP kinase plays an important role in UVB-induced apoptosis, and that sphingosine-1-phosphate probably exert its cytoprotective effect in Mel-Ab cells through ERK and Akt activation.     

Hypochlorous acid stimulation of the mitogen-activated protein kinase pathway enhances cell survival

We investigated the activation of three subfamilies of mitogen-activated protein kinases (MAP kinase), the extracellular regulated kinase (ERK1/2), p38, and c-Jun N-terminal kinase (JNK), by the myeloperoxidase-derived oxidant HOCl, in human umbilical vein endothelial cells (HUVEC) and human skin fibroblasts. Treatment of fibroblasts with 10-30 microM HOCl induced a dose-dependent increase in the tyrosine phosphorylation of several proteins. ERK1/2 was activated by exposure to sublethal concentrations of reagent HOCl or by HOCl generated by myeloperoxidase as shown by immune complex kinase assays. Maximum activation was seen at 20 microM and peak activation occurred within 10 min. Western blot analysis demonstrated activation of p38 with 30 microM HOCl, occurring at 15-30 min. No activation of JNK was detected in the concentration range investigated. These results show that HOCl is able to activate MAP kinases. Effective doses were considerably lower than with H2O2 and the lack of JNK activation contrasts with the activation frequently seen with H2O2. Exposure to HOCl caused a loss of viability in HUVEC that was markedly enhanced when ERK1/2 activation was inhibited by U0126. This suggests that the activation of ERK promotes cell survival in response to the oxidative challenge.