RESUMEN
The p21-activated kinase (PAK1) is a serine-threonine protein kinase that is activated by binding to the Rho family small G proteins Rac and Cdc42hs. Both Rac and Cdc42hs have been shown to regulate the activity of the transcription factor NFkappaB. Here we show that expression of active Ras, Raf-1, or Rac1 in fibroblasts stimulates NFkappaB in a PAK1-dependent manner and that expression of active PAK1 can stimulate NFkappaB on its own. Similarly, in macrophages activation of NFkappaB as well as transcription from the tumor necrosis factor alpha promoter depends on PAK1. In these cells lipopolysaccharide is a potent activator of PAK1 kinase activity. We also demonstrate that expression of active PAK1 stimulates the nuclear translocation of the p65 subunit of NFkappaB but does not activate the inhibitor of kappaB kinases alpha or beta. These data demonstrate that PAK1 is a crucial signaling molecule involved in NFkappaB activation by multiple stimuli.
Asunto(s)
FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Células 3T3 , Animales , Línea Celular , Núcleo Celular/enzimología , Activación Enzimática , Fibroblastos/enzimología , Humanos , Quinasa I-kappa B , Lipopolisacáridos/metabolismo , Macrófagos/enzimología , Ratones , FN-kappa B/genética , Plásmidos , Regiones Promotoras Genéticas , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-raf/metabolismo , Proteínas Recombinantes/metabolismo , Transducción de Señal , Factor de Transcripción ReIA , Transcripción Genética , Transfección , Factor de Necrosis Tumoral alfa/metabolismo , Quinasas p21 Activadas , Proteína de Unión al GTP rac1/metabolismo , Proteínas ras/metabolismoRESUMEN
Endotoxin triggers many of the inflammatory, hemodynamic, and hematological derangements of Gram-negative septic shock. Recent genetic studies in mice have identified the Toll-like receptor 4 as the transmembrane endotoxin signal transducer. The IL-1 intracellular signaling pathway has been implicated in Toll-like receptor signal transduction. LPS-induced activation of the IL-1 receptor-associated kinase (IRAK), and the influence of IRAK on intracellular signaling and cellular responses to endotoxin has not been explored in relevant innate immune cells. We demonstrate that LPS activates IRAK in murine macrophages. IRAK-deficient macrophages, in contrast, are resistant to LPS. Deletion of IRAK disrupts several endotoxin-triggered signaling cascades. Furthermore, macrophages lacking IRAK exhibit impaired LPS-stimulated TNF-alpha production, and IRAK-deficient mice withstand the lethal effects of LPS. These findings, coupled with the critical role for IRAK in IL-1 and IL-18 signal transduction, demonstrate the importance of this kinase and the IL-1/Toll signaling cassette in sensing and responding to Gram-negative infection.
Asunto(s)
Lipopolisacáridos/toxicidad , Macrófagos/inmunología , Proteínas Quinasas/fisiología , Receptores de Interleucina-1/fisiología , Animales , Activación Enzimática/inmunología , Quinasa I-kappa B , Inmunidad Innata , Quinasas Asociadas a Receptores de Interleucina-1 , Dosificación Letal Mediana , Sistema de Señalización de MAP Quinasas/inmunología , Macrófagos/enzimología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/metabolismo , FN-kappa B/fisiología , Proteínas Quinasas/deficiencia , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Choque Séptico/genética , Choque Séptico/inmunología , Choque Séptico/mortalidad , Transducción de Señal/inmunología , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
We have investigated the activation of ERK2, a serine/threonine kinase necessary for transmission of mitogenic signals, in cells derived from mouse embryos homozygous for a null mutation of the insulin-like growth factor (IGF)-1R gene (R- cells) and from wild-type littermates (W cells), respectively. Stimulation of quiescent W cells with IGF-1, epidermal growth factor (EGF), or with a combination growth factors induced both a maximal transient and a prolonged activation of ERK2, whereas platelet-derived growth factor or a combination of platelet-derived growth factor and EGF resulted only in transient activation of ERK2. In contrast, stimulation of R cells with IGF-1, EGF, or combinations of growth factors resulted in a transient and submaximal activation of ERK2. Reintroduction of a wild-type human IGF-1R or of a C-terminus IGF-1R mutant, but not of a juxtamembrane mutant IGF-1R, into R- cells was able to restore ERK2 activation to wild-type levels. Thus, prolonged ERK2 activation in mouse embryo fibroblasts stimulated with purified growth factors is largely dependent on a signal generated by the IGF-1R.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Sustancias de Crecimiento/farmacología , Factor I del Crecimiento Similar a la Insulina/metabolismo , Receptores de Somatomedina/fisiología , Animales , Línea Celular , Activación Enzimática/fisiología , Humanos , Factor I del Crecimiento Similar a la Insulina/farmacología , Ratones , Proteína Quinasa 1 Activada por Mitógenos , Fragmentos de Péptidos/metabolismo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Receptores de Somatomedina/química , Receptores de Somatomedina/metabolismoRESUMEN
The adverse effects of lipopolysaccharide (LPS) are primarily mediated by tumor necrosis factor-alpha (TNF-alpha). TNF-alpha production by LPS-stimulated macrophages is regulated both transcriptionally and post-transcriptionally. Transcriptional regulation of the TNF-alpha gene is dependent on nuclear factor-kappaB (NF-kappaB). We examined the signaling pathways involved in the regulation of NF-kappaB that lead to TNF-alpha promoter activity. We determined a role for one or both of the recently identified inhibitor of NF-kappaB kinases, IkappaB kinase-1 and IkappaB kinase-2, in LPS induction of an NF-kappaB reporter and of TNF-alpha promoter activity. IkappaB kinase activation is one of the earliest signaling events known to be induced by LPS. Furthermore, our results suggest roles for the IkappaB kinases NF-kappaB-inducing kinase and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1 in the regulation of IkappaB kinase-2, as well as in LPS-induced TNF-alpha transcription.
Asunto(s)
Lipopolisacáridos/farmacología , Regiones Promotoras Genéticas/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Factor de Necrosis Tumoral alfa/genética , Animales , Línea Celular , Ratones , Mutación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Transducción de Señal , Quinasa de Factor Nuclear kappa BRESUMEN
CD40 engagement induces a variety of functional outcomes following association with adaptor molecules of the TNF receptor-associated factor (TRAF) family. Whereas TRAF2, -5, and -6 initiate NF-kappaB activation, the outcomes of TRAF3-initiated signaling are less characterized. To delineate CD40-induced TRAF3-dependent events, Ramos B cells stably transfected with a dominant negative TRAF3 were stimulated with membranes expressing recombinant CD154/CD40 ligand. In the absence of TRAF3 signaling, activation of p38 and control of Ig production were abrogated, whereas Jun N-terminal kinase activation and secretion of IL-10, lymphotoxin-alpha, and TNF-alpha were partially blocked. By contrast, induction of apoptosis, activation of NF-kappaB, generation of granulocyte-macrophage CSF, and up-regulation of CD54, MHC class II, and CD95 were unaffected by the TRAF3 dominant negative. Together, these results indicate that TRAF3 initiates independent signaling pathways via p38 and JNK that are associated with specific functional outcomes.
Asunto(s)
Linfocitos B/inmunología , Antígenos CD40/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Citocinas/metabolismo , Inmunoglobulinas/biosíntesis , Proteínas Quinasas Activadas por Mitógenos , Proteínas/fisiología , Receptores del Factor de Necrosis Tumoral/metabolismo , Animales , Apoptosis/inmunología , Linfocitos B/enzimología , Linfocitos B/metabolismo , Linfoma de Burkitt , Antígenos CD40/biosíntesis , Antígenos CD40/inmunología , Ligando de CD40 , Activación Enzimática/genética , Activación Enzimática/inmunología , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , Ligandos , Glicoproteínas de Membrana/metabolismo , Ratones , Transducción de Señal/inmunología , Factor 3 Asociado a Receptor de TNF , Transfección/inmunología , Células Tumorales Cultivadas , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
The adverse effects of lipopolysaccharide (LPS) are mediated primarily by tumor necrosis factor alpha (TNF-alpha). TNF-alpha production by LPS-stimulated macrophages is regulated at the levels of both transcription and translation. It has previously been shown that several mitogen-activated protein kinases (MAPKs) are activated in response to LPS. We set out to determine which MAPK signaling pathways are activated in our system and which MAPK pathways are required for TNF-alpha gene transcription or TNF-alpha mRNA translation. We confirm activation of the MAPK family members extracellular-signal-regulated kinases 1 and 2 (ERK1 and ERK2), p38, and Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), as well as activation of the immediate upstream MAPK activators MAPK/ERK kinases 1 and 4 (MEK1 and MEK4). We demonstrate that LPS also activates MEK2, MEK3, and MEK6. Furthermore, we demonstrate that dexamethasone, which inhibits the production of cytokines, including TNF-alpha, significantly inhibits LPS induction of JNK/SAPK activity but not that of p38, ERK1 and ERK2, or MEK3, MEK4, or MEK6. Dexamethasone also blocks the sorbitol but not anisomycin stimulation of JNK/SAPK activity. A kinase-defective mutant of SAPKbeta, SAPKbeta K-A, blocked translation of TNF-alpha, as determined by using a TNF-alpha translational reporting system. Finally, overexpression of wild-type SAPKbeta was able to overcome the dexamethasone-induced block of TNF-alpha translation. These data confirm that three MAPK family members and their upstream activators are stimulated by LPS and demonstrate that JNK/SAPK is required for LPS-induced translation of TNF-alpha mRNA. A novel mechanism by which dexamethasone inhibits translation of TNF-alpha is also revealed.