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Title: PI3-KINASE MEDIATES TLR-4-INDUCED ACTIVATION OF NF-KAPPA B IN ENDOTHELIAL CELLS.

Author
item LI, XIANWU - UNIV WASHINGTON SEATTLE
item TUPPER, JOAN - UNIV WASHINGTON SEATTLE
item Bannerman, Douglas
item WINN, ROBERT - UNIV WASHINGTON SEATTLE
item RHODES, CHRISTOPHER - PACIFIC NW RES INST SEATT
item HARLAN, JOHN - UNIV WASHINGTON SEATTLE

Submitted to: Infection and Immunity
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/16/2003
Publication Date: 8/1/2003
Citation: LI, X., TUPPER, J.C., BANNERMAN, D.D., WINN, R.K., RHODES, C.J., HARLAN, J.M. PI3-KINASE MEDIATES TLR-4-INDUCED ACTIVATION OF NF-KAPPA B IN ENDOTHELIAL CELLS. INFECTION AND IMMUNITY. 2003. Vol. 71, pp. 4414-4420.

Interpretive Summary: In well-managed herds, Gram-negative bacteria cause approximately 50% of all cases of mastitis. Bacterial lipopolysaccharide (LPS), a highly pro-inflammatory component of the outer membrane of all Gram-negative bacteria, has been implicated in the pathogenesis of Gram-negative bacteria-induced mastitis. LPS elicits an array of host responses that are mediated by the activation of the transcription factor, NF-kappaB. These responses include the upregulation of adhesion molecules and the production of pro-inflammatory cytokines that contribute to host defense mechanisms. Although the receptor for LPS, Toll-like receptor-4 (Tlr-4) has been identified, the signaling pathways activated by LPS/Tlr-4 have yet to be fully elucidated. The present manuscript details the identification of key signaling molecules that are involved in LPS activation of PI3-kinase and the role of PI3-kinase in mediating NF-kappaB ctivation. Identification of these signaling pathways may lead to the development of novel therapeutics that can be used to enhance and/or downregulate signaling pathways involved in host response to Gram-negative mastitic infections.

Technical 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-kB and transcription of many genes involved in the inflammatory response. Endothelial cells (EC) are a major target of LPS as well as LPS-induced mediators such as interleukin-1beta (IL-1b) and tumor necrosis factor-alpha (TNFa). In this study, we examined the signaling pathways involved in NF-kB activation by TLR4 signaling in EC. We show that LPS, as well as the LPS-inducible cytokines IL-1b and TNFa, activate PI3-kinase in human dermal microvascular endothelial cells (HMEC). Using a stable transfection system, dominant-negative mutants of TLR adapter proteins, MyD88 (MyD88-TIR) and IRAK-1 (IRAK-DD), blocked Akt kinase activity in response to LPS and IL-1b. A dominant-negative mutant of Mal (Mal-P/H), a protein with homology to MyD88, failed to inhibit LPS- or IL-1b- induced Akt activity. Moreover, a dominant-negative mutant of p85 (p85-DN) abrogated the NF-kB luciferase activity, IL-6 production and IkBa degradation by LPS and IL-1b. In contrast, p85-DN failed to affect the NF-kB luciferase activity and IkBa egradation by TNFa. The dominant-negative mutant of Akt partially inhibited the NF-kB luciferase activity by LPS and IL-1b. However, expression of a constitutively-activated Akt failed to induce NF-kB luciferase activity. These findings indicate that TLR/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-1b signaling leading to NF-kB activation in EC and that Akt is necessary but not sufficient for NF-kB activation by TLR4.