title: Phosphoinositide 3 kinase mediates Toll-like receptor 4-induced activation of NF-kappa B in endothelial cells.
abstract: Many of the proinflammatory effects of gram-negative bacteria are elicited by the interaction of bacterial lipopolysaccharide (LPS) with Toll-like receptor 4 (TLR4) expressed on host cells. TLR4 signaling leads to activation of NF-kappa B and transcription of many genes involved in the inflammatory response. In this study, we examined the signaling pathways involved in NF-kappa B activation by TLR4 signaling in human microvascular endothelial cells. Akt is a major downstream target of phosphoinositide 3 kinase (PI3-kinase), and PI3-kinase activation is necessary and sufficient for Akt phosphorylation. Consequently, Akt kinase activation was used as a measure of PI3-kinase activity. In a stable transfection system, dominant-negative mutants of myeloid differentiation factor 88 (MyD88) and interleukin-1 (IL-1) receptor-associated kinase 1 (IRAK-1) (MyD88-TIR and IRAK-DD, respectively) blocked Akt kinase activity in response to LPS and IL-1 beta. A dominant-negative mutant (Mal-P/H) of MyD88 adapter-like protein (Mal), a protein with homology to MyD88, failed to inhibit LPS- or IL-1 beta-induced Akt activity. Moreover, a dominant-negative mutant of p85 (p85-DN) inhibited the NF-kappa B luciferase activity, IL-6 production, and I kappa B alpha degradation elicited by LPS and IL-1 beta but not that stimulated by tumor necrosis factor alpha. The dominant-negative mutant of Akt partially inhibited the NF-kappa B luciferase activity evoked by LPS and IL-1 beta. However, expression of a constitutively activated Akt failed to induce NF-kappa B luciferase activity. These findings indicate that TLR4- and IL-1R-induced PI3-kinase activity is mediated by the adapter proteins MyD88 and IRAK-1 but not Mal. Further, these studies suggest that PI3-kinase is an important mediator of LPS and IL-1 beta signaling leading to NF-kappa B activation in endothelial cells and that Akt is necessary but not sufficient for NF-kappa B activation by TLR4.
authors.affiliation: Department of Medicine, University of Washington, Seattle, Washington 98195, USA.
authors.forename: Xianwu
authors.initials: X
authors.lastname: Li
authors.forename: Joan C
authors.initials: JC
authors.lastname: Tupper
authors.forename: Douglas D
authors.initials: DD
authors.lastname: Bannerman
authors.forename: Robert K
authors.initials: RK
authors.lastname: Winn
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Rhodes
authors.forename: John M
authors.initials: JM
authors.lastname: Harlan
mesh_terms: D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D006090:Gram-Negative Bacteria / Q000472:pathogenicity; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology; D053592:Interleukin-1 Receptor-Associated Kinases; D015850:Interleukin-6 / Q000096:biosynthesis; D008070:Lipopolysaccharides / Q000633:toxicity; D008562:Membrane Glycoproteins / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D011956:Receptors, Cell Surface / Q000378:metabolism*; D015398:Signal Transduction; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D007375:Interleukin-1; D053592:Interleukin-1 Receptor-Associated Kinases; D015850:Interleukin-6; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D019869:Phosphatidylinositol 3-Kinases; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D011956:Receptors, Cell Surface; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha; C495345:TLR4 protein, human; C494918:AKT1 protein, human