RESUMO
The enterobacterial pathogen Salmonella induces phagocyte apoptosis in vitro and in vivo. These bacteria use a specialized type III secretion system to export a virulence factor, SipB, which directly activates the host's apoptotic machinery by targeting caspase-1. Caspase-1 is not involved in most apoptotic processes but plays a major role in cytokine maturation. We show that caspase-1-deficient macrophages undergo apoptosis within 4-6 h of infection with invasive bacteria. This process requires SipB, implying that this protein can initiate the apoptotic machinery by regulating components distinct from caspase-1. Invasive Salmonella typhimurium targets caspase-2 simultaneously with, but independently of, caspase-1. Besides caspase-2, the caspase-1-independent pathway involves the activation of caspase-3, -6, and -8 and the release of cytochrome c from mitochondria, none of which occurs during caspase-1-dependent apoptosis. By using caspase-2 knockout macrophages and chemical inhibition, we establish a role for caspase-2 in both caspase-1-dependent and -independent apoptosis. Particularly, activation of caspase-1 during fast Salmonella-induced apoptosis partially relies on caspase-2. The ability of Salmonella to induce caspase-1-independent macrophage apoptosis may play a role in situations in which activation of this protease is either prevented or uncoupled from the induction of apoptosis.
Assuntos
Apoptose , Caspases/metabolismo , Macrófagos/citologia , Salmonella typhimurium/fisiologia , Animais , Proteínas de Bactérias/metabolismo , Caspase 1/genética , Caspase 1/metabolismo , Caspase 2 , Células Cultivadas , Grupo dos Citocromos c/metabolismo , Ativação Enzimática , Macrófagos/microbiologia , Proteínas de Membrana/metabolismo , CamundongosRESUMO
Invasive Salmonella induces macrophage apoptosis via the activation of caspase-1 by the bacterial protein SipB. Here we show that infection of macrophages with Salmonella causes the activation and degradation of Raf-1, an important intermediate in macrophage proliferation and activation. Raf-1 degradation is SipB- and caspase-1-dependent, and is prevented by proteasome inhibitors. To study the functional significance of Raf-1 in this process, the c-raf-1 gene was inactivated by Cre-loxP-mediated recombination in vivo. Macrophages lacking c-raf-1 are hypersensitive towards pathogen-induced apoptosis. Surprisingly, activation of the antiapoptotic mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) and nuclear factor kappaB pathways is normal in Raf-1-deficient macrophages, and mitochondrial fragility is not increased. Instead, pathogen-mediated activation of caspase-1 is enhanced selectively, implying that Raf-1 antagonizes stimulus-induced caspase-1 activation and apoptosis.
Assuntos
Apoptose , Macrófagos/citologia , Proteínas Proto-Oncogênicas c-raf/fisiologia , Salmonella typhimurium/fisiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/enzimologia , Caspase 1/metabolismo , Células Cultivadas , Ativação Enzimática , Leupeptinas/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Inibidores de Proteases/farmacologia , Proteínas Proto-Oncogênicas c-raf/metabolismoRESUMO
Activation of several different kinases characterizes the induction of apoptosis. Abelson virus transformed pre-B lymphocytes undergo apoptosis within 24 h of serum deprivation, PKA activation or gamma-irradiation, and the activity of two kinases of ca. 40 and 44 kDa is specifically induced during this apoptotic process. Bcl-2 expression prevents both apoptosis and the induction of these kinases. Immunologic and substrate similarities indicate that these kinases are related to the p38 family of MAP kinases. More mature cells of the B lymphocytic lineage, plasmacytomas, also exhibit induction of these kinases when apoptosis is induced by withdrawal of serum or IL-6. Treatment of the pre-B cells with ICE protease inhibitors when apoptotic stimuli are delivered prevents induction of the kinase activity, and partially inhibits apoptosis. These findings indicate that the induction of these 40 and 44 kDa p38 related kinases is a common feature of apoptosis in mouse B lymphocytic cells and may represent a step downstream of ICE proteases in the signal cascade that leads to programmed cell death.
Assuntos
Apoptose/fisiologia , Linfócitos B/enzimologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Genes bcl-2/genética , Proteínas Quinases Ativadas por Mitógeno , Animais , Apoptose/genética , Linfócitos B/fisiologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/química , Linhagem Celular Transformada , Ativação Enzimática , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
In vitro addition of 16,16'-dimethyl prostaglandin E2 to Golgi-rich membrane fraction in final concentration of 0.1 microgram/1 mg of protein increased generally the activity of galactosyltransferase in comparison with control. The percentage of phospholipids in the whole fraction was similar in both investigated groups, only the sum of phosphatidylethanolamine+phosphatidic acid was significantly lower after addition of dmPGE2 than in the control (0.001 < P < 0.01).
Assuntos
16,16-Dimetilprostaglandina E2/farmacologia , Galactosiltransferases/metabolismo , Fígado/efeitos dos fármacos , Fígado/enzimologia , Animais , Feminino , Complexo de Golgi/enzimologia , Complexo de Golgi/metabolismo , Técnicas In Vitro , Fígado/metabolismo , N-Acetil-Lactosamina Sintase/metabolismo , Fosfolipídeos/metabolismo , Ratos , Ratos WistarRESUMO
Activation of the extracellularly regulated kinase (ERK) pathway is part of the early biochemical events that follow lipopolysaccharide (LPS) treatment of macrophages or their infection by virulent and attenuated Salmonella strains. Phagocytosis as well as the secretion of invasion-associated proteins is dispensable for ERK activation by the pathogen. Furthermore, the pathways used by Salmonella and LPS to stimulate ERK are identical, suggesting that kinase activation might be solely mediated by LPS. Both stimuli activate ERK by a mechanism involving herbimycin-dependent tyrosine kinase(s) and phosphatidylinositol 3-kinase. Phospholipase D activation and stimulation of protein kinase C appear to be intermediates in this novel pathway of MEK/ERK activation.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Lipopolissacarídeos/farmacologia , Macrófagos/imunologia , Fosfatidilinositol 3-Quinases/fisiologia , Fosfolipase D/fisiologia , Salmonella typhimurium/fisiologia , Androstadienos/farmacologia , Benzoquinonas , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Flavonoides/farmacologia , Lactamas Macrocíclicas , Macrófagos/enzimologia , Fagocitose/efeitos dos fármacos , Proteína Quinase C/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Quinonas/farmacologia , Rifabutina/análogos & derivados , Transdução de Sinais , WortmaninaRESUMO
The activation of kinases of the mitogen-activated protein kinase superfamily initiated by lipopolysaccharide (LPS) plays an important role in transducing inflammatory signals. The pathway leading to the induction of stress-activated protein kinases in macrophages stimulated with LPS was investigated. The activation of Jun N-terminal kinases (JNK) by LPS is herbimycin sensitive. Using specific inhibitors, it was shown that the pathway involves the activation of phosphoinositide 3-kinase (PI 3-K). However, in contrast to previous reports, the small GTPases Cdc42 and Rac are not required downstream of PI 3-K for JNK activation. Instead, the phosphoinositides produced by PI 3-K stimulate protein kinase C (PKC) zeta activation through PDK1. In turn, activation of this atypical PKC leads to the stimulation of phosphatidylcholine phospholipase C (PC-PLC) and acidic sphingomyelinase (ASMase). It is therefore proposed that PKCzeta regulates the PC-PLC/ASMase pathway, and it is hypothesized that the resultant ceramide accumulation mediates the activation of the SEK/JNK module by LPS.
Assuntos
Isoenzimas/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno , Lipopolissacarídeos/farmacologia , Macrófagos/enzimologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteína Quinase C/metabolismo , Fosfolipases Tipo C/metabolismo , Androstadienos/farmacologia , Benzoquinonas , Linhagem Celular , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Lactamas Macrocíclicas , MAP Quinase Quinase 4 , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Quinonas/farmacologia , Rifabutina/análogos & derivados , Transdução de Sinais , Esfingomielina Fosfodiesterase/metabolismo , Wortmanina , Proteína cdc42 de Ligação ao GTP/fisiologiaRESUMO
The interaction between bacteria and macrophages is central to the outcome of Salmonella infections. Salmonella can escape killing by these phagocytes and survive and multiply within them, giving rise to chronic infections. Cytokines produced by infected macrophages are involved in the early gastrointestinal pathology of the infection as well as in the induction and maintenance of the immune response against the invaders. Jun N-terminal kinases (JNK) are activated by inflammatory stimuli and play a role in cytokine production. We have investigated the signaling routes leading to JNK activation in Salmonella-infected macrophages and have discovered that they differ radically from the mechanisms operating in epithelial cells. In particular, activation of the JNK kinase stress and extracellular-activated kinase 1 (SEK1) and of JNK in macrophages occurs independently of actin rearrangements and of the GTPases Cdc42 and Rac, essential mediators in other cells. Activation of JNK is effected by a novel pathway comprising tyrosine kinase(s), phosphoinositide 3-kinase and, likely, atypical protein kinase C zeta. SEK1 is stimulated by a distinct mechanism involving phosphatidylcholine-phospholipase C and acidic sphingomyelinase. Dominant-negative SEK1 can block JNK activation by LPS, but not by Salmonella. These data demonstrate that SEK1 and JNK are activated independently in Salmonella-infected macrophages and offer experimental support for the concept that incoming signals can direct the selective coupling of downstream pathways to elicit highly specific responses. Inhibitors of stress kinase pathways are receiving increasing attention as potential anti-inflammatory drugs. The precise reconstruction of stimulus-specific pathways will be instrumental in predicting/evaluating the effects of the inhibitors on a given pathological condition.
Assuntos
MAP Quinase Quinase 4 , Macrófagos/enzimologia , Macrófagos/imunologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Salmonella typhimurium/imunologia , Animais , Benzoquinonas , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Ativação Enzimática/imunologia , Inibidores Enzimáticos/farmacologia , GTP Fosfo-Hidrolases/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Proteínas Quinases JNK Ativadas por Mitógeno , Lactamas Macrocíclicas , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Fagocitose/imunologia , Proteína Quinase C/fisiologia , Quinonas/farmacologia , Rifabutina/análogos & derivados , Salmonella typhimurium/efeitos dos fármacos , Transdução de Sinais/imunologia , Esfingomielina Fosfodiesterase/fisiologia , Transfecção , Fosfolipases Tipo C/fisiologiaRESUMO
Phosphorylation of protein kinase C (PKC) provides an amplitude control that operates in conjunction with allosteric effectors. Under many conditions, PKC isotypes appear to be highly phosphorylated; however, the cellular inputs that maintain these phosphorylations are not characterized. In the present work, it is shown that there is a differential phosphorylation of PKCdelta in adherent versus suspension cultures of transfected HEK-293 cells. It is established that integrin activation is sufficient to trigger PKCdelta phosphorylation and that this signals through phosphoinositide 3-kinase (PI3-kinase) to stimulate the phosphorylation of two sites, T505 and S662. The loss of signal input to PKCdelta in suspension culture is dependent on the tumour suppressor gene PTEN, which encodes a bi-functional phosphotyrosine/phosphoinositide 3-phosphate phosphatase. In the PTEN(-/-) UM-UC-3 bladder carcinoma cell line grown in suspension, transfected PKCdelta no longer accumulates in a dephospho-form on serum removal. By contrast, in a UM-UC-3-derivative cell line stably expressing PTEN, PKCdelta does become dephosphorylated under these conditions. Employing the PTEN Gly(129)-->Glu mutant, which is selectively defective in lipid phosphatase activity, it was established that it is the lipid phosphatase activity that controls PKCdelta phosphorylation. The evidence indicates that PKCdelta phosphorylation and its latent activity are maintained in serum-deprived adherent cultures through integrin-matrix interactions. This control acts through a pathway involving a lipid product of PI3-kinase in a manner that can be suppressed by PTEN.