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Specific inhibition of protein kinase Cbeta expression by antisense RNA affects the activation of Jurkat T lymphoma cells.
Cervino, M C; Lopez-Lago, M A; Vinuela, J E; Barja, P.
Afiliação
  • Cervino MC; Departamento de Bioquimica y Biologia Molecular, Universidad de Santiago de Compostela, Santiago de Compostela, Spain.
J Biol Regul Homeost Agents ; 24(3): 273-85, 2010.
Article em En | MEDLINE | ID: mdl-20846475
Antisense RNA technology was employed to specifically inhibit the expression of the protein kinase Cbeta (PKCbeta) isoform in Jurkat cells, to explore its influence on the expression of surface antigens (CD69) and the cytokines interleukin-8 (IL-8), tumour necrosis factor (TNF)-alpha and beta, and to characterise its controversial involvement in the expression of IL-2 and its receptor (IL-2R). Transfection of cells with an antisense PKCbeta construct (as-PKCbeta-pREP3) significantly increased IL-2R/CD25 expression in phorbol 12-myristate 13-acetate (PMA)-stimulated as-PKCbeta-pREP3 transfectants, in contrast to Jurkat cells transfected with a control as-PKCalpha-pREP3 plasmid. IL-2 production, in contrast, was strongly inhibited in both transfectant populations stimulated by PMA plus the calcium ionophore ionomycin. Three clones (asb1/asb2/asb3), selected from as-PKCbeta-pREP3 transfectants, showed decreased PKCbeta protein levels (40 percent, 50 percent and 60 percent, respectively, as determined by western blotting) and mRNA levels. The specific inhibition was confirmed in immunoblots for other PKC (alpha, delta, epsilon, gamma, theta, and lambda lambda/tau) isoforms and in immunoprecipitates from representative (c2/asb2) clones. Stimulation of PKCbeta-depleted clones significantly increased CD25 expression but decreased IL-2 production (similarly to as-PKCbeta-pREP3 transfectants) and IL-2 message levels. CD69 expression and IL-8 secretion were significantly decreased, but TNFbeta message levels were highly increased in asb2/asb3 clones, without affecting TNFalpha secretion. Analysis of the mitogen-activated protein kinase (MAP Kinase) signalling pathway showed unaltered extracellular signal regulated kinase 1/2 (ERK1/2) and p38 phosphorylation but increased activation of c-Jun N-terminal kinase (JNK1) and its substrate, the transcription factor ATF-2 (activated transcription factor-2), which are involved in IL-2 gene expression. Our results revealed new PKCbeta functions, affecting CD69 expression and IL-8 production, and support the requirement for PKCbeta in IL-2 secretion/transcription and IL-2R regulation.
Assuntos
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Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Proteína Quinase C / Linfoma de Células T / RNA Antissenso Limite: Humans Idioma: En Revista: J Biol Regul Homeost Agents Ano de publicação: 2010 Tipo de documento: Article
Buscar no Google
Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Proteína Quinase C / Linfoma de Células T / RNA Antissenso Limite: Humans Idioma: En Revista: J Biol Regul Homeost Agents Ano de publicação: 2010 Tipo de documento: Article