title: Inhibition of phosphoinositide 3-kinase enhances TRIF-dependent NF-kappa B activation and IFN-beta synthesis downstream of Toll-like receptor 3 and 4.
abstract: Phosphoinositide 3-kinases (PI3K) are known to regulate Toll-like receptor (TLR)-mediated inflammatory responses, but their impact on the different pathways of TLR signaling remains to be clarified. Here, we investigated the consequences of pharmacological inhibition of PI3K on Toll-IL-1 receptor domain-containing adapter-inducing IFN-beta (TRIF)-dependent signaling, which induces IFN-beta gene expression downstream of TLR3 and TLR4. First, treatment of monocyte-derived dendritic cells (DC) with wortmannin or LY294002 was found to enhance IFN-beta expression upon TLR3 or TLR4 engagement. In the same models of DC activation, PI3K inhibition increased DNA-binding activity of NF-kappaB, but not interferon response factor (IRF)-3, the key transcription factors required for TLR-mediated IFN-beta synthesis. In parallel, wortmannin-treated DC exhibited enhanced levels of IkappaB kinase (IKK)-alpha/beta phosphorylation and IkappaB-alpha degradation with a concomitant increase in NF-kappaB nuclear translocation. Experiments carried out in HEK 293T cells stably expressing TLR3 or TLR4 confirmed that inhibition of PI3K activity enhances NF-kappaB-dependent promoters as well as IFN-beta promoter activities without interfering with transcription at the positive regulatory domain III-I. Furthermore, wortmannin enhanced NF-kappaB activity induced by TRIF overexpression in HEK 293T cells, while overexpression of catalytically active PI3K selectively attenuated TRIF-mediated NF-kappaB transcriptional activity. Finally, in co-immunoprecipitation experiments, we showed that PI3K physically interacted with TRIF. We conclude that inhibition of PI3K activity enhances TRIF-dependent NF-kappaB activity, and thereby increases IFN-beta synthesis elicited by TLR3 or TLR4 ligands.
authors.affiliation: Institute for Medical Immunology, Université Libre de Bruxelles, Charleroi, Belgium.
authors.forename: Ezra
authors.initials: E
authors.lastname: Aksoy
authors.forename: Wim
authors.initials: W
authors.lastname: Vanden Berghe
authors.forename: Sophie
authors.initials: S
authors.lastname: Detienne
authors.forename: Zoulikha
authors.initials: Z
authors.lastname: Amraoui
authors.forename: Kathrine A
authors.initials: KA
authors.lastname: Fitzgerald
authors.forename: Guy
authors.initials: G
authors.lastname: Haegeman
authors.forename: Michel
authors.initials: M
authors.lastname: Goldman
authors.forename: Fabienne
authors.initials: F
authors.lastname: Willems
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000502:physiology*; D000730:Androstadienes / Q000494:pharmacology; D002867:Chromones / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism; D003713:Dendritic Cells / Q000276:immunology / Q000378:metabolism; D015536:Down-Regulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D050838:Interferon Regulatory Factor-3; D016899:Interferon-beta / Q000096:biosynthesis*; D008070:Lipopolysaccharides / Q000276:immunology; D008562:Membrane Glycoproteins / Q000378:metabolism*; D009025:Morpholines / Q000494:pharmacology; D016328:NF-kappa B / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011956:Receptors, Cell Surface / Q000378:metabolism*; D051196:Toll-Like Receptor 3; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic / Q000502:physiology; D000077191:Wortmannin
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; D000730:Androstadienes; D002867:Chromones; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D050838:Interferon Regulatory Factor-3; D016899:Interferon-beta; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D009025:Morpholines; D016328:NF-kappa B; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011956:Receptors, Cell Surface; D051196:Toll-Like Receptor 3; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014157:Transcription Factors; D000077191:Wortmannin; C494232:IRF3 protein, human; C470409:TICAM1 protein, human; C495344:TLR3 protein, human; C495345:TLR4 protein, human; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one