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Infection and Immunity, August 2003, p. 4414-4420, Vol. 71, No. 8
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.8.4414-4420.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Phosphoinositide 3 Kinase Mediates Toll-Like Receptor 4-Induced Activation of NF-{kappa}B in Endothelial Cells

Xianwu Li,1 Joan C. Tupper,1 Douglas D. Bannerman,2 Robert K. Winn,3 Christopher J. Rhodes,4,5 and John M. Harlan1*

Departments of Medicine,1 Surgery,3 Pharmacology, University of Washington, Seattle, Washington 98195,4 Immunology and Disease Resistance Laboratory, USDA-Agricultural Research Service, Beltsville, Maryland 20705,2 Pacific Northwest Research Institute, Seattle, Washington 981225

Received 30 December 2002/ Returned for modification 7 February 2003/ Accepted 16 May 2003

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-1ß. 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-1ß-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-1ß 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-1ß. 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-1ß 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.

* Corresponding author. Mailing address: Division of Hematology, University of Washington, Harborview Medical Center, Box 359756, Seattle, WA 98104-2499. Phone: (206) 341-5314. Fax: (206) 341-5312. E-mail: jharlan{at}u.washington.edu.

Editor: J. T. Barbieri

Infection and Immunity, August 2003, p. 4414-4420, Vol. 71, No. 8
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.8.4414-4420.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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