Phorbol ester-stimulated NF-kappaB-dependent transcription: roles for isoforms of novel protein kinase C

Cell Signal. 2008 Jul;20(7):1338-48. doi: 10.1016/j.cellsig.2008.03.001. Epub 2008 Mar 18.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Calcium / metabolism
  • Carbazoles / pharmacology
  • Cell Line, Tumor
  • DNA / metabolism
  • Humans
  • Indoles / pharmacology
  • Intracellular Space / drug effects
  • Intracellular Space / metabolism
  • Isoenzymes / metabolism
  • Lamins / metabolism
  • Maleimides / pharmacology
  • Models, Biological
  • NF-kappa B / metabolism*
  • Phorbol Esters / pharmacology*
  • Protein Binding / drug effects
  • Protein Kinase C / metabolism
  • Protein Kinase C-delta / metabolism*
  • Protein Kinase C-epsilon / metabolism*
  • Protein Kinase Inhibitors
  • RNA, Small Interfering / metabolism
  • Transcription, Genetic / drug effects*
  • Transfection
  • Tumor Necrosis Factor-alpha / pharmacology


  • 2-(1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl)-3-(1H-indol-3-yl)maleimide
  • Carbazoles
  • Indoles
  • Isoenzymes
  • Lamins
  • Maleimides
  • NF-kappa B
  • Phorbol Esters
  • Protein Kinase Inhibitors
  • RNA, Small Interfering
  • Tumor Necrosis Factor-alpha
  • phorbol-12-myristate
  • DNA
  • protein kinase D
  • Protein Kinase C
  • Protein Kinase C-delta
  • Protein Kinase C-epsilon
  • bisindolylmaleimide I
  • Calcium