Phorbol ester-stimulated NF-kappaB-dependent transcription: roles for isoforms of novel protein kinase C.
Since protein kinase C (PKC) isoforms are variously implicated in the activation of NF-kappaB, we have investigated the role of PKC in the activation of NF-kappaB-dependent transcription by the diacyl glycerol (DAG) mimetic, phorbol 12-myristate 13-acetate (PMA), and by tumour necrosis factor (TNF) alpha in pulmonary A549 cells. The PKC selective inhibitors, Ro31-8220, Gö6976, GF109203X and Gö6983, revealed no effect on TNFalpha-induced NF-kappaB DNA binding and a similar lack of effect on serine 32/36 phosphorylated IkappaBalpha and the loss of total IkappaBalpha indicates that activation of the core IKK-IkappaBalpha-NF-kappaB cascade by TNFalpha does not involve PKC. In contrast, differential sensitivity of an NF-kappaB-dependent reporter to Ro31-8220, Gö6976, GF109203X and Gö6983 (EC(50)s 0.46 microM, 0.34 microM, >10 microM and >10 microM respectively) suggests a role for protein kinase D in transcriptional activation by TNFalpha. Compared with TNFalpha, PMA weakly induces NF-kappaB DNA binding and this effect was not associated with serine 32/36 phosphorylation of IkappaBalpha. However, PMA-stimulated NF-kappaB DNA binding was inhibited by Ro31-8220 (10 microM), GF109203X (10 microM) and Gö6983 (10 microM), but not by Gö6976 (10 microM), suggesting a role for novel PKC isoforms. Furthermore, a lack of positive effect of calcium mobilising agents on both NF-kappaB 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 TNFalpha-induced NF-kappaB-dependent transcription by PMA, further indicates a role for novel PKCs in NF-kappaB transactivation. Finally, siRNA-mediated knockdown of PKCdelta and epsilon expression did not affect TNFalpha-induced NF-kappaB-dependent transcription. However, knockdown of PKCdelta expression significantly inhibited PMA-stimulated luciferase activity, whereas knockdown of PKCepsilon was without effect. Furthermore, combined knockdown of PKCdelta and epsilon revealed an increased inhibitory effect on PMA-stimulated NF-kappaB-dependent transcription suggesting that PMA-induced NF-kappaB-dependent transcription is driven by novel PKC isoforms, particularly PKCdelta and epsilon.
Holden
Neil S
NS
Airways Inflammation Group, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada.
Squires
Paul E
PE
Kaur
Manminder
M
Bland
Rosemary
R
Jones
Carol E
CE
Newton
Robert
R
D002118:Calcium / Q000378:metabolism; D002227:Carbazoles / Q000494:pharmacology; D045744:Cell Line, Tumor; D004247:DNA / Q000378:metabolism; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D034882:Lamins / Q000378:metabolism; D008301:Maleimides / Q000494:pharmacology; D008954:Models, Biological; D016328:NF-kappa B / Q000378:metabolism*; D010703:Phorbol Esters / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism; D051745:Protein Kinase C-delta / Q000378:metabolism*; D051744:Protein Kinase C-epsilon / Q000378:metabolism*; D047428:Protein Kinase Inhibitors; D034741:RNA, Small Interfering / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects*; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
C465664:2-(1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl)-3-(1H-indol-3-yl)maleimide; D002227:Carbazoles; D007211:Indoles; D007527:Isoenzymes; D034882:Lamins; D008301:Maleimides; D016328:NF-kappa B; D010703:Phorbol Esters; D047428:Protein Kinase Inhibitors; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha; C119222:phorbol-12-myristate; D004247:DNA; C089212:protein kinase D; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D051744:Protein Kinase C-epsilon; C070515:bisindolylmaleimide I; D002118:Calcium