Cyclosporin A Up-Regulates and Activates Protein Kinase C-ζ in EBV-Infected and EBV-Transformed Human B-Cells

BackgroundProtein Kinase C (PKC) is a family of enzymes that plays a key role in cell signaling pathways leading to cellular activation and proliferation. Conventional PKC (cPKC) is dependent on calcium for activation. We have proposed that cyclosporin A (CsA), despite being a calcineurin inhibitor, will activate PKC in B cells, thus promoting Epstein-Barr virus (EBV)-induced transformation. Here we show that CsA promoted atypical PKC isoform PKC-ζ in B cells.Materials and methodsWestern-blot was used to assay PKC-ζ protein level in EBV-B cells. Confocal microscopy was used to assay PKC-ζ translocation from cytosol to cell membrane, a known process of PKC activation.ResultsCsA (500 ng/mL) time dependently increased PKC-ζ from control of 7055 units to 7145, 10,805, 10,914, and 12,705 units, respectively, after 15 min, 1 h, 12 h, and 24 h of incubation in EBV-transformed human B-cell line (LCL). CsA increased PKC-ζ expression was inhibited 50% by Vit.E (40 μM) indicating that this effect may be due to oxidative stress induced by CsA. Indeed, after oxidant H2O2 (0.1 mM) treatment, PKC-ζ protein level in LCL cells increased 124%, 257%, 349%, and 359% after 15 min, 1 h, 12 h, and 24 h of culture compared with control. Addition of Vit.E (40 μM) in H2O2 (0.1 mM) treatment and then with Vit.E in the culture decreased PKC-ζ level in LCL cells 26%, 20%, 41%, and 60% after 15 min, 1 h, 12 h, and 24 h of culture. In confocal microscopy in Jurkat T cell line, phorbol 12-myristate 13-acetate (PMA) activated cPKC isoform PKCα after 30 min treatment and activated PKC-ζ after 60 min treatment. CsA inhibited PMA activation of PKC-α, but not PKC-ζ. CsA alone did not activate PKC-α or PKC-ζ in Jurkat T cells. In LCL and in EBV-infected human B-cells, PMA stimulated PKC-α activation after 30 min treatment and stimulated PKC-ζ activation after 60 min treatment. CsA inhibited PMA activation of PKC-α, but not PKC-ζ. In addition, CsA activated PKC-ζ in the EBV-transformed and EBV-infected human B cells.ConclusionThese experiments show that CsA-induced oxidative stress caused PKC-ζ up-regulation in LCL cells, and show the differential effect of CsA in the PKC signaling pathways in T cells versus B cells. CsA-induced PKC-ζ activation may be an important signaling step in EBV-induced post-transplant lymphoproliferative disorders.