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

Nuclear factor-kappa B (NF-kappa B) is a transcription factor that plays crucial roles in inflammation and immunity. Understanding the positive and negative regulation of NF-kappa B 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-kappa B. However, many aspects of the PI3K negative regulation of NF-kappa B 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-kappa B activation by PI3K inhibitors. Gel shift assays showed that LY294002 (LY29) potently increased interleukin (IL)-1-induced NF-kappa B DNA binding in human monocytic THP-1 cells. Moreover, another PI3K inhibitor 3-methyladenine also strongly enhanced IL-1-induced NF-kappa B DNA binding, while LY303511, an inactive analogue of LY29, did not increase the NF-kappa B DNA binding. Compared with LY29, wortmannin (WM) effected only a marginal enhancement of NF-kappa B DNA binding. LY29 treatment also augmented tumor necrosis factor (TNF)mediated NF-kappa B 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 I kappa B kinase (IKK) activation as well as I kappa B-alpha degradation and NF-kappa B 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-kappa B DNA binding by LY29. These results suggest that pharmacological inhibition of PI3K enhances the NF-kappa B-activating pathways by IL-1 through augmentation of IKK activation in myeloid/monocytic cells and the NF-kappa B enhancement is more robustly achieved by LY29 than by WM. (c) 2006 Elsevier B.V All rights reserved.