RESUMEN
The present experiments were aimed to characterize in immortalized human HaCat keratinocytes the gene expression induced by paraquat and capsaicin, two agents known to induce cell death or to affect inflammatory and pain pathways, respectively. In particular, the following set of genes were analysed by qRealtime PCR: CXCL10,CXCL11, IL-10 (inflammatory and immune responses), TP73, BCL2, (apoptotic and anti-apoptotic genes), MMP9 (proteolysis), SOD-1, BAK-1 and CAT (peroxysomal and microsomal oxidation pathways). In this way, we were able to differentiate the two toxins since they had a different profile of gene expression. In fact, paraquata was found to activate set of genes involved in inflammatory (CXL10,CXL11 and IL-10), and cell death (BCL2, BAK-1, MMP9) pathways. Another specific site of action of paraquat was represented by an activation of the gene involved in SOD-1 transcription. On the contrary, capsaicin was found to produce only an up-regulation of BCL2, an anti-apoptotic gene and MMP9, whereas no significant changes were reported in genes involved in inflammatory and immune responses. Finally, in comparison to previous experiments carried out with TNF-alpha and IL-1beta, we have shown that paraquat produced a similar pattern of activation of set of genes involved both in inflammation and apoptosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Capsaicina/farmacología , Inflamación/genética , Queratinocitos/efectos de los fármacos , Paraquat/farmacología , Apoptosis/genética , Catalasa/genética , Línea Celular , Quimiocina CXCL10/genética , Quimiocina CXCL11/genética , Proteínas de Unión al ADN/genética , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inflamación/enzimología , Inflamación/inmunología , Interleucina-10/genética , Queratinocitos/enzimología , Queratinocitos/inmunología , Queratinocitos/patología , Metaloproteinasa 9 de la Matriz/genética , Proteínas Nucleares/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Superóxido Dismutasa/genética , Superóxido Dismutasa-1 , Proteína Tumoral p73 , Proteínas Supresoras de Tumor/genética , Proteína Destructora del Antagonista Homólogo bcl-2/genéticaRESUMEN
The present experiments were designed to characterize by microarray analysis the transcriptional responses of human keratinocytes (HaCat) to TNF-α and IL-1 ß, given alone or in combination, in order to better understand the mechanisms underlying inflammatory, immune responses and cell death in which both cytokines play a pathophysiological role. Significant differences in the percentage and quality of genes dysregulated by TNF-α and IL-1 ß were shown. Both cytokines activated a series of genes involved in inflammatory, immune response as well as in cell death. In our experimental conditions, TNF-α, in contrast to IL-1 ß, did not induce a significant level of apoptosis in keratinocytes. However, given together both cytokines produced a significant decrease in apoptotic cells and synergistic transcriptional response which was due to the activation of several specific genes occurring after application of each cytokine. TNF-α and IL-1 ß evoked apoptotic effect and transcriptional responses were linked to the stimulation of their specific receptors since a pre-treatment with monoclonal antibodies vs TNF-α and/or IL-1 ß receptors was able to significantly reduce them.