Specific inhibition of protein kinase Cbeta expression by antisense RNA affects the activation of Jurkat T lymphoma cells.
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.
Cervino
M C
MC
Departamento de Bioquimica y Biologia Molecular, Universidad de Santiago de Compostela, Santiago de Compostela, Spain.
Lopez-Lago
M A
MA
Vinuela
J E
JE
Barja
P
P
D051698:Activating Transcription Factor 2 / Q000378:metabolism; D015703:Antigens, CD / Q000032:analysis; D000945:Antigens, Differentiation, T-Lymphocyte / Q000032:analysis; D015153:Blotting, Western; D006801:Humans; D007376:Interleukin-2 / Q000235:genetics; D053645:Interleukin-2 Receptor alpha Subunit / Q000032:analysis; D016209:Interleukin-8 / Q000096:biosynthesis; D019169:Jurkat Cells; D037181:Lectins, C-Type / Q000032:analysis; D008213:Lymphocyte Activation; D016399:Lymphoma, T-Cell / Q000276:immunology*; D020935:MAP Kinase Signaling System; D048055:Mitogen-Activated Protein Kinase 8 / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000032:analysis / Q000037:antagonists & inhibitors / Q000502:physiology*; D064546:Protein Kinase C beta; D016372:RNA, Antisense / Q000235:genetics*; D015375:Receptors, Interleukin-2 / Q000235:genetics; D014162:Transfection; D000099192:CD69 Antigens
D051698:Activating Transcription Factor 2; D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D007376:Interleukin-2; D053645:Interleukin-2 Receptor alpha Subunit; D016209:Interleukin-8; D037181:Lectins, C-Type; D048055:Mitogen-Activated Protein Kinase 8; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D016372:RNA, Antisense; D015375:Receptors, Interleukin-2; D000099192:CD69 Antigens; C497105:ATF2 protein, human; C508017:IL2RA protein, human