RESUMEN
The stress-activated pathways leading to activation of p38 MAP kinase (p38 MAPK) and c-jun N-terminal kinases (JNK) have been shown to be activated by pro-inflammatory cytokines, physical and chemical stresses as well as a variety of hematopoietic growth factors. One exception is interleukin (IL)-4, which does not activate this pathway in hematopoietic cell. We report here that in A431, a keratinocytic cell line, IL-4 activates Rac and Cdc42 and their downstream effector p21-activated kinase (PAK). Rac and Cdc42 appear to regulate a protein kinase cascade initiated at the level of PAK and leading to activation of p38 MAPK, since IL-4 stimulates tyrosine phosphorylation of p38 MAPK and increases its catalytic activity. As A431 cells are able to produce IL-6 in response to IL-4 stimulation, we assessed the involvement of p38 MAPK in IL-6 gene expression. A pyrimidazole compound, SB203580, a specific inhibitor of p38 MAPK, inhibits production and gene expression of IL-6. SB203580 reduced significantly the stability of IL-6 mRNA. Here we provide evidence that p38 MAPK is activated in response to IL-4 and is involved in IL-6 synthesis by stabilizing IL-6 mRNA.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Interleucina-4/metabolismo , Interleucina-6/metabolismo , Queratinocitos/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Humanos , Imidazoles/farmacología , Interleucina-4/farmacología , Interleucina-6/genética , Proteínas Quinasas JNK Activadas por Mitógenos , Queratinocitos/efectos de los fármacos , Sistema de Señalización de MAP Quinasas , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Piridinas/farmacología , Receptores de Interleucina-4/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Factor de Transcripción STAT6 , Transactivadores/genética , Transactivadores/metabolismo , Transcripción Genética , Quinasas p21 Activadas , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Interleukin-4 (IL-4) is a pleiotropic cytokine, which acts on both hematopoietic and non-hematopoietic cells, through different types of receptor complexes. In this study, we report that in human B cells, IL-4 caused rapid phosphorylation of Janus kinase (JAK) 1 and JAK3 tyrosine kinases. In keratinocytes, the hematopoietic-specific receptor common gamma(c) chain is not expressed and the IL-13 receptor alpha(1) (IL-13Ralpha(1)) participates in IL-4 signal transduction. In keratinocytes, IL-4 induced JAK1 and JAK2 phosphorylation but, unlike in immune cells, IL-4 did not involve JAK3 activation for its signaling. In both cell types, IL-4 induced phosphorylation and DNA binding activation of the signal transducer and activator of transcription (STAT) 6 protein. Furthermore, IL-4 stimulation of keratinocytes also induced tyrosine phosphorylation of STAT3 which was found to bind to the phosphorylated IL-13Ralpha(1). STAT3 however did not significantly translocate to the nucleus, nor did it bind with high affinity to target DNA sequences.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Interleucina-4/metabolismo , Receptores de Interleucina-4/metabolismo , Receptores de Interleucina/metabolismo , Transactivadores/metabolismo , Línea Celular , ADN/metabolismo , Activación Enzimática , Técnica del Anticuerpo Fluorescente , Humanos , Subunidad alfa1 del Receptor de Interleucina-13 , Queratinocitos/metabolismo , Linfocitos/metabolismo , Fosforilación , Pruebas de Precipitina , Unión Proteica , Proteínas Tirosina Quinasas/metabolismo , Receptores de Interleucina-13 , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT3 , Transducción de SeñalRESUMEN
Two genistein analogues (MD831 and MD833) have been synthesized and analyzed for their biological properties and their mechanism of action im comparison to genistein either in vitro or in intact cells. We showed that, in vitro, one of these compounds (MD831) inhibits the tyrosine kinase activity associated with the epidermal growth factor receptor (EGFR) as efficiently as genistein. However, treatment of A431 cells with these compounds did not result in any significant modification of EGFR tyrosine phosphorylation. Extracellular-signal regulated kinase (ERK) phosphorylation in cells stimulated by EGF was enhanced in the presence of MD831, whereas the other compounds, genistein and MD833, were able to activate the c-jun N-terminal kinase (JNK). This study showed that two structurally related compounds could elicit markedly different pharmacological effects on two signalling pathways, one involved in the mitogenic response and the other in the stress response. Such compounds may be useful to characterize signalling events involved in cell response to physiological stimuli.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Inhibidores Enzimáticos/farmacología , Receptores ErbB/efectos de los fármacos , Isoflavonas/farmacología , Proteínas Quinasas Activadas por Mitógenos , Transducción de Señal/efectos de los fármacos , Proteínas Quinasas Dependientes de Calcio-Calmodulina/efectos de los fármacos , Receptores ErbB/metabolismo , Genisteína , Técnicas In Vitro , Isoflavonas/síntesis química , Isoflavonas/química , Proteínas Quinasas JNK Activadas por Mitógenos , Fosforilación/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Most cytokines stimulate the p21ras pathway, leading to MAP kinase activation. One exception is interleukin-4 (IL-4), which has been shown not to activate this pathway in hematopoietic cells. However, IL-4 acts on a broad range of cells, including keratinocytes, in which it induces IL-6 production. We report here that IL-4 stimulation of human keratinocytic cell lines or primary cultures activates MAP kinase. In these cells, IL-4 stimulation induces the tyrosine phosphorylation of p42/44 MAP kinase as well as its catalytic activity. We also observed an increased phosphorylation of p46shc, an SH2-containing protein involved in the Ras pathway, as a result of IL-4 stimulation in human keratinocytic cell lines but not in T lymphocytes.
