The Phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 modulates cytokine expression in macrophages via p50 nuclear factor kappa B inhibition, in a PI3K-independent mechanism

The Phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002 (LY2), has been previously reported to inhibit nuclear factor kappa B (NF kappa B) activity, in a PI3K-independent mechanism. The goals of the current research were to determine the specificity of LY2 regarding NF kappa B subunits, and to identify relevant modulation of cytokine expression in LPS-stimulated macrophages. We found that LY2 specifically diminished the level of p50, but not p65, NF kappa B in the nucleus of LPS-stimulated mouse RAW264.7 macrophages and human THP-1 monocytes. This activity of LY2 was mimicked by its PI3K-inert analog LY303511 (LY3), but not by another PI3K inhibitor - wortmannin. We further show that LY2 inhibited LPS-induced IL-10 expression by RAW264.7 macrophages, in a PI3K-independent mechanism. Moreover, using a deletion mutant of an IL-10 promoter reporter gene we demonstrate that the activity of the NF kappa B enhancer site at the IL-10 promoter is regulated by LY2 in a PI3K-independent manner. Finally, both LY2 and LY3 elevated TNF alpha production in the LPS tolerant state which is regulated by p50 NF kappa B homodimers, but not before tolerance development. The effects of LY2 and LY3 on p50 translocation and on cytokine production in LPS-stimulated macrophages are thus consistent with specific PI3K-independent inhibition of p50 NF kappa B homodimer activity by LY2. (C) 2011 Elsevier Inc. All rights reserved.