NF-κB is a characteristic transcriptional factor which has been shown to regulate production of acute inflammatory mediators and to be involved in the pathogenesis of many inflammatory lung diseases. There has been some evidence that PI3K/Akt pathway could activate NF-κB in human cell lines. However, the effect of PI3K/Akt pathway on the activation of NF-κB varied depending on the cell lines used in the experiments. In this study we evaluated the effect of PI3K/Akt pathway on the activation of NF-κB in human respiratory epithelial cell lines.

BEAS-2B, A549 and NCI-H157 cell lines were used in this experiment. To evaluate the activation of Akt activation and IκB degradation, cells were analysed by western blot assay using phospho-specific Akt Ab and IκB Ab. To block PI3K/Akt pathway, cells were pretreated with wortmannin or LY294002 and transfected with dominant negative Akt (DN-Akt). For IKK activity, immune complex kinase assay was performed. To evaluate the DNA binding affinity and transcriptional activity of NF-κB, electrophoretic mobility shift assay (EMSA) and luciferase assay were performed, respectively.

In BEAS-2B, A549 and NCI-H157 cell lines, Akt was activated by TNF-α and insulin. Activation of Akt by insulin did not induce IκBα degradation. Blocking of PI3K/Akt pathway via wortmannin/LY294002 or DN-Akt did not inhibit TNF-α- induced IκBα degradation or IKK activation. Inhibition of PI3K/Akt did not affect TNF-α-induced NF-κB activation. Overexpression of DN-Akt did not block TNF-α-induced transcriptional activation of NF-κB, but wortmannin enhanced TNF-α-induced in NF-κB transcriptional activity.

PI3K/Akt was not involved in TNF-α-induced IκBα degradation or transcriptional activity of NF-κB in human respiratory epithelial cell lines.