Enhancement of cytokine-mediated NF-κB activation by phosphatidylinositol 3-kinase inhibitors in monocytic cells

Abstract

Nuclear factor-κB (NF-κB) is a transcription factor that plays crucial roles in inflammation and immunity. Understanding the positive and negative regulation of NF-κB activity is therefore of fundamental importance. A few previous studies reported that inhibition of the phosphatidylinositol 3-kinase (PI3K)–Akt pathway enhances lipopolysaccharide (LPS)-induced activation of NF-κB. However, many aspects of the PI3K negative regulation of NF-κB activation remain to be clarified. The present study was conducted to shed light on cell-type specificity, stimulus specificity, and upstream mechanisms of the enhanced NF-κB activation by PI3K inhibitors. Gel shift assays showed that LY294002 (LY29) potently increased interleukin (IL)-1-induced NF-κB DNA binding in human monocytic THP-1 cells. Moreover, another PI3K inhibitor 3-methyladenine also strongly enhanced IL-1-induced NF-κB DNA binding, while LY303511, an inactive analogue of LY29, did not increase the NF-κB DNA binding. Compared with LY29, wortmannin (WM) effected only a marginal enhancement of NF-κB DNA binding. LY29 treatment also augmented tumor necrosis factor (TNF)-mediated NF-κB DNA binding. Furthermore, LY29, but not WM, increased cyclooxygenase (COX)-2 mRNA expression by IL-1 or TNF in THP-1 cells. Likewise, prostaglandin E 2 production by IL-1 was increased by LY29, but not by WM. Western blot analysis demonstrated that IκB kinase (IKK) activation as well as IκB-α degradation and NF-κB nuclear translocation was elevated by LY29 and WM. Among the tested cell lines (HL-60, ECV304, Hep-2, and Molt-4), only HL-60, a promyelocytic cell line, showed enhanced NF-κB DNA binding by LY29. These results suggest that pharmacological inhibition of PI3K enhances the NF-κB-activating pathways by IL-1 through augmentation of IKK activation in myeloid/monocytic cells and the NF-κB enhancement is more robustly achieved by LY29 than by WM.