RESUMEN
A hallmark of inflammation is the burst-like formation of certain proteins, initiated by cellular stress and proinflammatory cytokines like interleukin 1 (IL-1) and tumor necrosis factor, stimuli which simultaneously activate different mitogen-activated protein (MAP) kinases and NF-kappaB. Cooperation of these signaling pathways to induce formation of IL-8, a prototype chemokine which causes leukocyte migration and activation, was investigated by expressing active and inactive forms of protein kinases. Constitutively active MAP kinase kinase 7 (MKK7), an activator of the stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) pathway, induced IL-8 synthesis and transcription from a minimal IL-8 promoter. Furthermore, MKK7 synergized in both effects with NF-kappaB-inducing kinase (NIK). Activation of the IL-8 promoter by either of the kinases required functional NF-kappaB and AP-1 sites. While NIK and MKK7 did not affect degradation of IL-8 mRNA, an active form of MKK6, which selectively activates p38 MAP kinase, induced marked stabilization of the transcript and further increased IL-8 protein formation induced by NIK plus MKK7. Consistently, the MAP kinase kinase kinase MEKK1, which can activate NF-kappaB, SAPK/JNK, and p38 MAP kinases, most potently induced IL-8 formation. These results provide evidence that maximal IL-8 gene expression requires the coordinate action of at least three different signal transduction pathways which cooperate to induce mRNA synthesis and suppress mRNA degradation.
Asunto(s)
Citocinas/farmacología , Interleucina-8/biosíntesis , Sistema de Señalización de MAP Quinasas , FN-kappa B/metabolismo , Estabilidad del ARN , ARN Mensajero/metabolismo , Sitios de Unión , Quimiotaxis de Leucocito , Proteínas Quinasas JNK Activadas por Mitógenos , MAP Quinasa Quinasa 7 , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Factor de Transcripción AP-1/metabolismo , Transcripción Genética , Quinasa de Factor Nuclear kappa BRESUMEN
The cytokine interleukin-1 (IL-1) is a major inflammatory hormone which activates a broad range of genes during inflammation. The signaling mechanisms triggered by IL-1 include activation of several distinct protein kinase systems. The stress-activated protein kinase (SAPK), also termed Jun N-terminal kinase (JNK), is activated particularly strongly by the cytokine. In an attempt to delineate its role in activation of gene expression by IL-1, we inhibited the IL-1-induced SAPK/JNK activity by stable overexpression of either a catalytically inactive mutant of SAPKbeta (SAPKbeta(K-R)) or antisense RNA to SAPKbeta in human epidermal carcinoma cells. A detailed analysis of signal transduction in those cells showed that activation of neither NFkappaB nor p38 mitogen-activated protein kinase was affected, suggesting that we achieved specific blockade of the SAPK/JNK. In untransfected and vector-transfected KB cells, IL-1 induced a strong increase in expression of IL-6 and IL-8 mRNA, along with the synthesis of high amounts of the proteins. In two KB cell clones stably overexpressing the mutant SAPKbeta(K-R), and three clones stably overexpressing antisense RNA to SAPKbeta, expression of IL-6 and IL-8 in response to IL-1 was strongly reduced at both the mRNA and protein level. These data indicate that the SAPK/JNK pathway provides an indispensable signal for IL-1-induced expression of IL-6 and IL-8.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Regulación Neoplásica de la Expresión Génica/inmunología , Interleucina-1/farmacología , Interleucina-6/genética , Interleucina-8/genética , Células KB/inmunología , Proteínas Quinasas Activadas por Mitógenos , Proteínas Quinasas Dependientes de Calcio-Calmodulina/biosíntesis , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Activación Enzimática , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Interleucina-1/fisiología , Interleucina-6/biosíntesis , Interleucina-8/biosíntesis , Proteínas Quinasas JNK Activadas por Mitógenos , Células KB/efectos de los fármacos , Células KB/enzimología , Cinética , Sistemas de Lectura Abierta , Reacción en Cadena de la Polimerasa , ARN sin Sentido/farmacología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/farmacología , Transfección , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Activation of NF kappa B is considered one way IL-1 exerts its costimulatory effect on T cell activation and IL-2 production. However, T helper 2 cells do not synthesize IL-2 upon IL-1 stimulation, although they depend on IL-1 for proliferation. The involvement of NF kappa B in IL-2 production was therefore addressed in two different murine cell lines: D10N, a T helper 2 cell line that does not synthesize IL-2, and EL4 6.1., a thymoma cell line producing IL-2 when stimulated with IL-1. In both cell types IL-1 activated the DNA binding activity of the NF kappa B heterodimers p50/65 and p50/c-rel via the IL-1 type I receptor. In D10N cells, however, the p50/p50 homodimer is present in large amounts in unstimulated cells, whereas the heterodimers were only activated by IL-1-treatment. In contrast, only marginal amounts of the p50/p50 homodimer were found in EL4 6.1. cells, but a strong activation of heterodimers was induced by IL-1. The findings are compatible with the concept of p50 homodimer being inhibitory by replacing the p50/p65 or p50/c-rel heterodimer from nuclear kappa B binding sites. The inability of D10N cells to respond to IL-1 exposure with IL-2 formation might therefore be due to their high constitutive levels of p50 homodimers.