Phorbol ester-stimulated NF-kappa B-dependent transcription : Roles for isoforms of novel protein kinase C

Since protein kinase C (PKC) isoforms are variously implicated in the activation of NF-kappa B, we have investigated the role of PKC in the activation of NF-kappa B-dependent transcription by the diacyl glycerol (DAG) mimetic, phorbol 12-myristate 13-acetate (PMA), and by tumour necrosis factor (TNF) a in pulmonary A549 cells. The PKC selective inhibitors, Ro31-8220, Go6976, GF109203X and Go6983, revealed no effect on TNF alpha-induced NF kappa B DNA binding and a similar lack of effect on serine 32/36 phosphorylated I kappa B alpha and the loss of total I kappa B alpha, indicates that activation of the core IKK-I kappa B alpha-NF-kappa B cascade by TNF alpha does not involve PKC. In contrast, differential sensitivity of an NF-kappa B-dependent reporter to Ro31-8220, Go6976, GF109203X and Go6983 (EC50S 0.46 mu M, 0.34 mu M, > 10 mu M and > 10 mu M respectively) suggests a role for protein kinase D in transcriptional activation by TNF alpha. Compared with TNFa, PMA weakly induces NF-kappa B DNA binding and this effect was not associated with serine 32/36 phosphorylation of I kappa B alpha. However, PMA-stimulated NF-kappa B DNA binding was inhibited by Ro31-8220 (10 mu M), GF109203X (10 mu M) and Go6983 (10 mu M), but not by Go6976 (10 mu M), suggesting a role for novel PKCisoforms. Furthermore,a lack of positive effect of calcium mobilising agents on both NF-kappa B DNA binding and on transcriptional activation argues against major roles for classical PKCs. This, combined with the ability of both GF109203X and Go6983 to prevent enhancement of TNF alpha-induced NF-kappa B-dependent transcription by PMA, further indicates a role for novel PKCs in NF-kappa B transactivation. Finally, siRNA-mediated knockdown of PKC delta and epsilon, expression did not affect TNFa-induced NF-kappa B-dependent transcription. However, knockdown of PKC delta expression significantly inhibited PMA-stimulated luciferase activity, whereas knockdown of PKC epsilon was without effect. Furthermore, combined knockdown of PKC delta and E revealed an increased inhibitory effect on PMA-stimulated NF-kappa B-dependent transcription suggesting that PMA-induced NF-kappa B-dependent transcription is driven by novel PKC isoforms, particularly PKC delta and epsilon. (c) 2008 Elsevier Inc. All rights reserved