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

Since protein kinase C (PKC) isoforms are variously implicated in the activation of NF-κB, we have investigated the role of PKC in the activation of NF-κB-dependent transcription by the diacyl glycerol (DAG) mimetic, phorbol 12-myristate 13-acetate (PMA), and by tumour necrosis factor (TNF) α in pulmonary A549 cells. The PKC selective inhibitors, Ro31-8220, Gö6976, GF109203X and Gö6983, revealed no effect on TNFα-induced NF-κB DNA binding and a similar lack of effect on serine 32/36 phosphorylated IκBα and the loss of total IκBα indicates that activation of the core IKK-IκBα-NF-κB cascade by TNFα does not involve PKC. In contrast, differential sensitivity of an NF-κB-dependent reporter to Ro31-8220, Gö6976, GF109203X and Gö6983 (EC50s 0.46 μM, 0.34 μM, > 10 μM and > 10 μM respectively) suggests a role for protein kinase D in transcriptional activation by TNFα. Compared with TNFα, PMA weakly induces NF-κB DNA binding and this effect was not associated with serine 32/36 phosphorylation of IκBα. However, PMA-stimulated NF-κB DNA binding was inhibited by Ro31-8220 (10 μM), GF109203X (10 μM) and Gö6983 (10 μM), but not by Gö6976 (10 μM), suggesting a role for novel PKC isoforms. Furthermore, a lack of positive effect of calcium mobilising agents on both NF-κB DNA binding and on transcriptional activation argues against major roles for classical PKCs. This, combined with the ability of both GF109203X and Gö6983 to prevent enhancement of TNFα-induced NF-κB-dependent transcription by PMA, further indicates a role for novel PKCs in NF-κB transactivation. Finally, siRNA-mediated knockdown of PKCδ and ε expression did not affect TNFα-induced NF-κB-dependent transcription. However, knockdown of PKCδ expression significantly inhibited PMA-stimulated luciferase activity, whereas knockdown of PKCε was without effect. Furthermore, combined knockdown of PKCδ and ε revealed an increased inhibitory effect on PMA-stimulated NF-κB-dependent transcription suggesting that PMA-induced NF-κB-dependent transcription is driven by novel PKC isoforms, particularly PKCδ and ε.