| Abstract: |
Antisense RNA technology was employed to specifically inhibit the expression of the protein kinase Cβ (PKCβ) 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)-α and β, and to characterise its controversial involvement in the expression of IL-2 and its receptor (IL-2R). Transfection of cells with an antisense PKCβ construct (as-PKCα-pREP3) significantly increased IL-2R/CD25 expression in phorbol 12-myristate 13-acetate (PMA)-stimulated as-PKCβ-pREP3 transfectants, in contrast to Jurkat cells transfected with a control as-PKCα-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-PKCβ-pREP3 transfectants, showed decreased PKCβ protein levels (40%, 50% and 60%, respectively, as determined by western blotting) and mRNA levels. The specific inhibition was confirmed in immunoblots for other PKC (α, δ, ε, ζ, θ, and λ/ι) isoforms and in immunoprecipitates from representative (c2/asb2) clones. Stimulation of PKCβ-depleted clones significantly increased CD25 expression but decreased IL-2 production (similarly to as-PKCβ-pREP3 transfectants) and IL-2 message levels. CD69 expression and IL-8 secretion were significantly decreased, but TNFβ message levels were highly increased in asb2/asb3 clones, without affecting TNFα 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 PKCβ functions, affecting CD69 expression and IL-8 production, and support the requirement for PKCβ in IL-2 secretion/transcription and IL-2R regulation. Copyright © by Biolife, s.a.s. |
| Keywords: |
signal transduction; mitogen activated protein kinase; protein expression; protein phosphorylation; human cell; interleukin 2; reverse transcription polymerase chain reaction; gene expression; map kinase signaling system; stress activated protein kinase; interleukin 8; transfection; phosphorylation; blotting, western; lymphoma, t-cell; lymphocyte activation; cytokines; tumor necrosis factor alpha; genetic transfection; interleukin-8; messenger rna; lymphoma cell; western blotting; immunoprecipitation; mitogen activated protein kinase 1; mitogen activated protein kinase 3; protein kinase c; gene induction; antigens, cd; t lymphocyte activation; interleukin-2; jurkat cells; interleukin-2 receptor alpha subunit; complementary rna; synaptophysin; phorbol 13 acetate 12 myristate; protein kinase c delta; antigens, differentiation, t-lymphocyte; map kinases; pkc isoforms; surface antigens; activating transcription factor 2; cd69 antigen; interleukin 2 receptor; ionomycin; lymphotoxin; protein kinase c beta; protein kinase c epsilon; protein kinase c lambda; protein kinase c theta; protein kinase c zeta; lectins, c-type; mitogen-activated protein kinase 8; receptors, interleukin-2; rna, antisense
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