Phosphoinositide 3 Kinase Mediates Toll-Like Receptor 4-Induced Activation of NF-κB 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-κB and transcription of many genes involved in the inflammatory response. In this study, we examined the signaling pathways involved in NF-κB 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-1β. 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-1β-induced Akt activity. Moreover, a dominant-negative mutant of p85 (p85-DN) inhibited the NF-κB luciferase activity, IL-6 production, and IκBα degradation elicited by LPS and IL-1β but not that stimulated by tumor necrosis factor alpha. The dominant-negative mutant of Akt partially inhibited the NF-κB luciferase activity evoked by LPS and IL-1β. However, expression of a constitutively activated Akt failed to induce NF-κB 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-1β signaling leading to NF-κB activation in endothelial cells and that Akt is necessary but not sufficient for NF-κB activation by TLR4.

Inhibition of Neutrophil Apoptosis by TLR Agonists in Whole Blood: Involvement of the Phosphoinositide 3-Kinase/Akt and NF-κB Signaling Pathways, Leading to Increased Levels of Mcl-1, A1, and Phosphorylated Bad

“…Indeed, it was recently reported that Akt modulates NF-B-dependent transcription in TLR2-stimulated PMN by modifying phosphorylation of the p65 subunit (7). Nevertheless, Akt is necessary but not sufficient for NF-B activation by TLR (51).…”

Lipopolysaccharide signaling in endothelial cells

“…88 Consistent with this, enforced expression of various dominant negative mutants has shown that PI3K activation is downstream of MyD88/IRAK/TRAF6, but not TIRAP. 89,90 Further, PI3K and its downstream target kinase, Akt, appear to be an important component of LPS-induced NF-kB activity following its translocation to the nucleus. 88,90 RIP is a serine/threonine kinase that plays a critical role in LPS-induced activation of NF-kB.…”

Inhibition of phosphoinositide 3-kinase enhances TRIF-dependent NF-κB activation and IFN-β synthesis downstream of Toll-like receptor 3 and 4

“…PI3K was shown to regulate MyD88-dependent events [23][24][25][26] but its role in the TRIF-dependent signaling cascade has not been demonstrated so far. To investigate this question, we first determined the effects of pharmacological inhibition of PI3K activity on IFN-b synthesis and activation of NF-jB and IRF3 transcription factors in human monocytederived DC stimulated by poly(I:C) or LPS used as TLR3 and TLR4 ligands, respectively.…”