u-0126 and benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone

CD95 is a major apoptosis receptor that induces caspase activation and programmed cell death in susceptible cells. CD95-induced apoptosis can be blocked by peptidic caspase inhibitors such as benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone or Ile-Glu-Thr-Asp-fluoromethyl ketone. Here we show that stimulation of CD95 in the presence of these inhibitors induces necrosis and expression of various proinflammatory cytokines in primary T lymphocytes, such as TNF-alpha, IFN-gamma and granulocyte/macrophage colony-stimulating factor. In the absence of caspase inhibition CD95 stimulation did not result in cytokine expression, indicating that this proinflammatory signaling pathway is suppressed by active caspases. Further analysis with A3.01 T cells revealed that the proinflammatory signaling activity of CD95 was mediated by MEK/ERK, p38 and NF-kappaB signaling pathways. These findings point to a pivotal role of caspases not only as mediators of apoptosis but also as enzymes that prevent proinflammatory signaling during CD95-induced apoptosis. Moreover, our findings may be useful for the development of novel pharmacological strategies.

Topics: Amino Acid Chloromethyl Ketones; Antigens, CD; Apoptosis; Butadienes; Cell Line; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; fas Receptor; Gene Expression Regulation; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Hydrogen-Ion Concentration; Imidazoles; Inflammation; Interferon-gamma; MAP Kinase Kinase 1; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Necrosis; NF-kappa B; Nitriles; Oligopeptides; p38 Mitogen-Activated Protein Kinases; Protein Serine-Threonine Kinases; Pyridines; Receptors, Tumor Necrosis Factor; Receptors, Tumor Necrosis Factor, Type I; Signal Transduction; T-Lymphocytes; Tumor Necrosis Factor-alpha

Coxsackievirus B3 (CVB3) is the most common human pathogen for viral myocarditis. We have previously shown that the signaling protein p21(ras) GTPase-activating protein (RasGAP) is cleaved and that mitogen-activated protein kinases (MAPKs) ERK1/2 are activated in the late phase of CVB3 infection. However, the role of intracellular signaling pathways in CVB3-mediated myocarditis and the relative advantages of such pathways to host or virus remain largely unclear. In this study we extended our prior studies by examining the interaction between CVB3 replication and intracellular signaling pathways in HeLa cells. We observed that CVB3 infection induced a biphasic activation of ERK1/2, early transient activation versus late sustained activation, which were regulated by different mechanisms. Infection by UV-irradiated, inactivated virus capable of receptor binding and endocytosis triggered early ERK1/2 activation, but was insufficient to trigger late ERK1/2 activation. By using a general caspase inhibitor (zVAD.fmk) we further demonstrated that late ERK1/2 activation was not a result of CVB3-mediated caspase cleavage. Treatment of cells with U0126, a selective inhibitor of MAPK kinase (MEK), significantly inhibited CVB3 progeny release and decreased virus protein production. Furthermore, inhibition of ERK1/2 activation circumvented CVB3-induced apoptosis and viral protease-mediated RasGAP cleavage. Taken together, these data suggest that ERK1/2 activation is important for CVB3 replication and contributes to virus-mediated changes in host cells. Our findings demonstrate coxsackievirus takeover of a particular host signaling mechanism and uncover a prospective approach to stymie virus spread and preserve myocardial integrity.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Butadienes; Caspase Inhibitors; Cysteine Proteinase Inhibitors; Enterovirus; Enzyme Inhibitors; HeLa Cells; Humans; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Nitriles; Proviruses; ras GTPase-Activating Proteins; Ultraviolet Rays; Viral Proteins; Virus Replication

Hemin is present in intracranial hematomas in high micromolar concentrations and is a potent, lipophilic oxidant. Growing evidence suggests that heme-mediated injury may contribute to the pathogenesis of CNS hemorrhage. Extracellular signal-regulated kinases (ERKs) are activated by oxidants in some cell types, and may alter cellular vulnerability to oxidative stress. In this study, the effect of hemin on ERK activation was investigated in cultured murine cortical astrocytes, and the consequence of this activation on cell viability was quantified. Hemin was rapidly taken up by astrocytes, and generated reactive oxygen species (ROS) within 30 min. Increased immunoreactivity of dually phosphorylated ERK1/2 was observed in hemin-treated cultures at 30-120 min, without change in total ERK. Surprisingly, ERK activation was not attenuated by concomitant treatment with antioxidants (U74500A or 1,10-phenanthroline) at concentrations that blocked ROS generation. Cell death commenced after 2 h of hemin exposure and was reduced by antioxidants and by the caspase inhibitor Z-VAD-FMK. Cytotoxicity was also attenuated by MEK inhibition with PD98059 or U0126 at concentrations that were sufficient to prevent ERK activation. Whereas the effect of Z-VAD-FMK on cell survival was transient, the effect of MEK inhibitors was long-lasting. MEK inhibitors had no effect on cellular hemin uptake or subsequent ROS generation. The present results suggest that hemin activates ERK in astrocytes via a mechanism that is independent of ROS generation. This activation sensitizes astrocytes to hemin-mediated oxidative injury.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antioxidants; Astrocytes; Blood Proteins; Butadienes; Cell Survival; Cells, Cultured; Cerebral Cortex; Cysteine Proteinase Inhibitors; Enzyme Activation; Flavonoids; Free Radicals; Hemin; MAP Kinase Kinase Kinase 1; Mice; Mitogen-Activated Protein Kinases; Nitriles; Oxidative Stress; Phenanthrolines; Pregnatrienes; Protease Inhibitors; Protein Serine-Threonine Kinases; Reactive Oxygen Species; Signal Transduction

Cisplatin activates multiple signal transduction pathways involved in coordinating cellular responses to stress. Here we demonstrate a requirement for extracellular signal-regulated protein kinase (ERK), a member of the mitogen-activated protein kinase family in mediating cisplatin-induced apoptosis of human cervical carcinoma HeLa cells. Cisplatin treatment resulted in dose- and time- dependent activation of ERK. That elevated ERK activity contributed to cell death by cisplatin was supported by several observations: 1) PD98059 and U0126, chemical inhibitors of the MEK/ERK signaling pathway, prevented apoptosis; 2) pretreatment of cells with TPA, an activator of the ERK pathway, enhanced their sensitivity to cisplatin; 3) suramin, a growth factor receptor antagonist that greatly suppressed ERK activation, likewise inhibited cisplatin-induced apoptosis; and, finally, 4) HeLa cell variants selected for cisplatin resistance showed reduced activation of ERK following cisplatin treatment. Cisplatin-induced apoptosis was associated with cytochrome c release and subsequent caspase-3 activation, both of which could be prevented by treatment with the MEK inhibitors. However, the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone protected HeLa cells against apoptosis without affecting ERK activation. Taken together, our findings suggest that ERK activation plays an active role in mediating cisplatin-induced apoptosis of HeLa cells and functions upstream of caspase activation to initiate the apoptotic signal.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Blotting, Western; Butadienes; Caspase 3; Caspases; Cell Line; Cell Survival; Cisplatin; Cysteine Proteinase Inhibitors; Cytochrome c Group; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Flavonoids; HeLa Cells; Humans; Indoles; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Models, Biological; Nitriles; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Signal Transduction; Suramin; Tetradecanoylphorbol Acetate; Time Factors