RESUMEN
Epithelial-mesenchymal transdifferentiation (EMT) is a critical morphogenic event that occurs during embryonic development and during the progression of various epithelial tumors. EMT can be induced by transforming growth factor (TGF)-beta in mouse NMuMG mammary epithelial cells. Here, we demonstrate a central role of helix-loop-helix factors, E2A and inhibitor of differentiation (Id) proteins, in TGF-beta-induced EMT. Epithelial cells ectopically expressing E2A adopt a fibroblastic phenotype and acquire migratory/invasive properties, concomitant with the suppression of E-cadherin expression. Id proteins interacted with E2A proteins and antagonized E2A-dependent suppression of the E-cadherin promoter. Levels of Id proteins were dramatically decreased by TGF-beta. Moreover, NMuMG cells overexpressed Id2 showed partial resistance to TGF-beta-induced EMT. Id proteins thus inhibit the action of E2A proteins on the expression of E-cadherin, but after TGF-beta stimulation, E2A proteins are present in molar excess of the Id proteins, thus over-riding their inhibitory function and leading to EMT.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Mesodermo/citología , Mesodermo/efectos de los fármacos , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Animales , Benzamidas/farmacología , Cadherinas/genética , Cadherinas/metabolismo , Proteínas de Unión al ADN/genética , Dioxoles/farmacología , Células Epiteliales/metabolismo , Expresión Génica , Regulación de la Expresión Génica , Proteína 2 Inhibidora de la Diferenciación , Mesodermo/metabolismo , Ratones , Regiones Promotoras Genéticas/genética , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Proteínas Represoras/genética , Factores de Transcripción/genéticaRESUMEN
Apoptosis signal-regulating kinase 1 (ASK1) is a MAP kinase kinase kinase (MAPKKK) that activates the JNK and p38 MAP kinase cascades and is activated in response to oxidative stress such as hydrogen peroxide (H(2)O(2)). A yeast two-hybrid screening identified a serine/threonine protein phosphatase 5 (PP5) as a binding partner of ASK1. PP5 directly dephosphorylated an essential phospho-threonine residue within the kinase domain of ASK1 and thereby inactivated ASK1 activity in vitro and in vivo. The interaction between PP5 and ASK1 was induced by H(2)O(2) treatment and was followed by the decrease in ASK1 activity. PP5 inhibited not only H(2)O(2)-induced sustained activation of ASK1 but also ASK1-dependent apoptosis. Thus, PP5 appears to act as a physiological inhibitor of ASK1-JNK/p38 pathways by negative feedback.
Asunto(s)
Retroalimentación Fisiológica/fisiología , Quinasas Quinasa Quinasa PAM/metabolismo , Proteínas Nucleares/metabolismo , Estrés Oxidativo/fisiología , Fosfoproteínas Fosfatasas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Células COS , Línea Celular , Células HeLa/enzimología , Humanos , Peróxido de Hidrógeno/farmacología , Riñón/citología , Riñón/metabolismo , MAP Quinasa Quinasa Quinasa 5 , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Quinasas Quinasa Quinasa PAM/genética , Proteínas Nucleares/genética , Proteínas Nucleares/farmacología , Oxidantes/farmacología , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/farmacología , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Transducción de Señal/fisiología , Transfección , Factor de Necrosis Tumoral alfa/farmacología , Técnicas del Sistema de Dos HíbridosRESUMEN
Apoptosis signal-regulating kinase (ASK) 1 is activated in response to various cytotoxic stresses including TNF, Fas and reactive oxygen species (ROS) such as H(2)O(2), and activates c-Jun NH(2)-terminal kinase (JNK) and p38. However, the roles of JNK and p38 signaling pathways during apoptosis have been controversial. Here we show that by deleting ASK1 in mice, TNF- and H(2)O(2)-induced sustained activations of JNK and p38 are lost in ASK1(-/-) embryonic fibroblasts, and that ASK1(-/-) cells are resistant to TNF- and H(2)O(2)-induced apoptosis. TNF- but not Fas-induced apoptosis requires ROS-dependent activation of ASK1-JNK/p38 pathways. Thus, ASK1 is selectively required for TNF- and oxidative stress-induced sustained activations of JNK/p38 and apoptosis.
Asunto(s)
Apoptosis/fisiología , Quinasas Quinasa Quinasa PAM/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Animales , Apoptosis/efectos de los fármacos , Células Cultivadas , Activación Enzimática , Peróxido de Hidrógeno/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos , MAP Quinasa Quinasa Quinasa 5 , Quinasas Quinasa Quinasa PAM/genética , Ratones , Ratones Noqueados , Transducción de Señal , Factor de Necrosis Tumoral alfa/farmacología , Receptor fas/farmacología , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Mechanical force or mechanical stress modulates intracellular signal pathways, including the mitogen-activated protein kinase (MAP kinase) cascades. In our system, cell stretching activated and cell contraction inactivated all three MAP kinase pathways (MKK1/2-extracellular signal-regulated kinase (ERK), MKK4 (SEK1)-cJun N-terminal kinase (JNK) and MKK3/6-p38 pathways). However, little is known about the molecular mechanisms that link the mechanical force to the MAP kinase cascades. To test whether Ras and Rap1 are possible components in the stretch-activated MAP kinase pathways, we examined if Ras and Rap1 were activated by cell stretching and if inhibition of their activity decreased the stretch-enhanced MAP kinase activity. Rap1 was activated by cell stretching and inactivated by cell contraction, whereas Ras was inactivated by cell stretching and activated by cell contraction. Rap1GapII and SPA-1, downregulators of Rap1 activity, decreased the stretch-enhanced p38 activity, whereas a dominant-negative mutant of Ras (RasN17) did not inhibit the stretch-initiated activation of MAP kinases. Furthermore, overexpression of Rap1 enhanced p38 activity but not ERK or JNK activity. These results indicate that Rap1 is involved in transducing the stretch-initiated signal to the MKK3/6-p38 pathway, but not to the MEK1/2-ERK or the MKK4 (SEK1)/MKK7-JNK pathway. Thus, Rap1 plays a unique role in force-initiated signal transduction.
Asunto(s)
MAP Quinasa Quinasa 4 , Sistema de Señalización de MAP Quinasas/fisiología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas de Unión al GTP rap1/metabolismo , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Línea Celular , Activación Enzimática , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , MAP Quinasa Quinasa 1 , MAP Quinasa Quinasa 3 , MAP Quinasa Quinasa 6 , MAP Quinasa Quinasa 7 , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Transfección , Proteínas Quinasas p38 Activadas por Mitógenos , Proteínas ras/metabolismoRESUMEN
Apoptosis signal-regulating kinase (ASK) 1 was recently identified as a mitogen-activated protein (MAP) kinase kinase kinase which activates the c-Jun N-terminal kinase (JNK) and p38 MAP kinase pathways and is required for tumor necrosis factor (TNF)-alpha-induced apoptosis; however, the mechanism regulating ASK1 activity is unknown. Through genetic screening for ASK1-binding proteins, thioredoxin (Trx), a reduction/oxidation (redox)-regulatory protein thought to have anti-apoptotic effects, was identified as an interacting partner of ASK1. Trx associated with the N-terminal portion of ASK1 in vitro and in vivo. Expression of Trx inhibited ASK1 kinase activity and the subsequent ASK1-dependent apoptosis. Treatment of cells with N-acetyl-L-cysteine also inhibited serum withdrawal-, TNF-alpha- and hydrogen peroxide-induced activation of ASK1 as well as apoptosis. The interaction between Trx and ASK1 was found to be highly dependent on the redox status of Trx. Moreover, inhibition of Trx resulted in activation of endogenous ASK1 activity, suggesting that Trx is a physiological inhibitor of ASK1. The evidence that Trx is a negative regulator of ASK1 suggests possible mechanisms for redox regulation of the apoptosis signal transduction pathway as well as the effects of antioxidants against cytokine- and stress-induced apoptosis.
Asunto(s)
Apoptosis , Inhibidores Enzimáticos/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Tiorredoxinas/metabolismo , Animales , Encéfalo/metabolismo , Línea Celular , Cloruros/farmacología , Medio de Cultivo Libre de Suero , Activación Enzimática , Humanos , Peróxido de Hidrógeno/farmacología , Células L , Quinasas Quinasa Quinasa PAM , Mamíferos , Ratones , Oxidación-Reducción , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Transducción de Señal , Tiorredoxinas/biosíntesis , Tiorredoxinas/farmacología , Transfección , Factor de Necrosis Tumoral alfa/farmacología , Compuestos de Zinc/farmacologíaRESUMEN
Apoptosis Signal-regulating Kinase (ASK) 1 was identified that activated two different subgroup of MAP kinase kinase (MAPKK), SEK1 (or MKK4), and MKK3/MAPKK6 (or MKK6), which in turn activated stress-activated protein kinase (SAPK, also known as JNK: c-Jun amino-terminal kinase) and p38 subgroup of MAP kinases, respectively. It was suggested that ASK1 contributed to cytokine-induced apoptosis in some cell lines. In this report, for further investigation about roles of ASK1 in mammal, initial characterization of mouse ASK1 was done. The mouse cDNA encoding ASK1 was isolated from the mouse kidney cDNA library and the overall amino acid sequence similarity between the mouse and the human ASK1 was 91.9%. A database search revealed that the kinase domain of ASK1 is evolutionally well-concervedover species among nematode, fly, mouse, and human. Northern blot analysis identified a 6-kb transcript of ASK1 which is expressed in the various mouse adult tissues. Immunohistochemical analysis of mouse embryos (17 days post coitum) revealed a localized expression of ASK1 in developing skin, cartilage, and bone, suggesting a possible role of ASK1 in tissue development during embryogenesis as well as cytokine-induced apoptosis.
Asunto(s)
Proteínas Serina-Treonina Quinasas/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Histocitoquímica , Quinasas Quinasa Quinasa PAM , Ratones , Datos de Secuencia Molecular , Proteínas Serina-Treonina Quinasas/aislamiento & purificaciónRESUMEN
The mouse cDNA for apoptosis signal-regulating kinase 1 (ASK)1 was isolated. The overall amino acid sequence identity between the mouse and the human ASK1 was 91.9%. A database search revealed that the kinase domain of ASK1 is evolutionally well-conserved over species among nematode, fly, mouse and human. Northern blot analysis identified a 6-kb transcript of ASK1 which is expressed in the various mouse adult tissues including heart, brain, lung, liver and kidney. Immunohistochemical analysis of mouse embryos (17 days post coitum) revealed a localized expression of ASK1 in developing skin, cartilage and bone, suggesting a possible role for ASK1 in tissue development during embryogenesis as well as cytokine-induced apoptosis.