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The Journal of Immunology, 2008, 180: 4218-4226.
Copyright © 2008 by The American Association of Immunologists, Inc.

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Genetic Analysis of the Role of the PI3K-Akt Pathway in Lipopolysaccharide-Induced Cytokine and Tissue Factor Gene Expression in Monocytes/Macrophages1

James P. Luyendyk2,3, Gernot A. Schabbauer2,4, Michael Tencati, Todd Holscher, Rafal Pawlinski and Nigel Mackman5,6

Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037

LPS stimulation of monocytes/macrophages induces the expression of genes encoding proinflammatory cytokines and the procoagulant protein, tissue factor. Induction of these genes is mediated by various signaling pathways, including mitogen-activated protein kinases, and several transcription factors, including Egr-1, AP-1, ATF-2, and NF-{kappa}B. We used a genetic approach to determine the role of the phosphatidylinositol-3-kinase (PI3K)-protein kinase B (Akt) pathway in the regulation of LPS signaling and gene expression in isolated macrophages and in mice. The PI3K-Akt pathway is negatively regulated by the phosphatase and tensin homologue (PTEN). We used peritoneal exudate cells from Pik3r1-deficient mice, which lack the p85{alpha} regulatory subunit of PI3K and have reduced PI3K activity, and peritoneal macrophages from PTENflox/flox/LysMCre mice (PTEN–/–), which have increased Akt activity. Analysis of LPS signaling in Pik3r1–/– and PTEN–/– cells indicated that the PI3K-Akt pathway inhibited activation of the ERK1/2, JNK1/2, and p38 mitogen-activated protein kinases and reduced the levels of nuclear Egr-1 protein and phosphorylated ATF-2. Modulating the PI3K-Akt pathway did not affect LPS-induced degradation of I{kappa}B{alpha} or NF-{kappa}B nuclear translocation. LPS induction of TNF-{alpha}, IL-6, and tissue factor gene expression was increased in Pik3r1–/– peritoneal exudate cells and decreased in PTEN–/– peritoneal macrophages compared with wild-type (WT) cells. Furthermore, LPS-induced inflammation and coagulation were enhanced in WT mice containing Pik3r1–/– bone marrow compared with WT mice containing WT bone marrow and in mice lacking the p85{alpha} subunit in all cells. Taken together, our results indicate that the PI3K-Akt pathway negatively regulates LPS signaling and gene expression in monocytes/macrophages.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grant HL48872 (to N.M.), National Institutes of Health National Research Service Award F32 HL085983 (to J.P.L), Austrian Science Fund FWF P19850 (to G.A.S.), COBRE Grant P20 RR021940, and a Kansas IDeA Network of Biomedical Research Excellence Recruitment Package 5 P20 RR016475 (to J.P.L).

2 J.P.L. and G.A.S. contributed equally to this work.

3 Current address: Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160.

4 Current address: Institute for Vascular Biology and Thrombosis Research, Center for Biomolecular Medicine and Pharmacology, Medical University, Vienna, Austria.

5 Current address: Department of Medicine, University of North Carolina, Chapel Hill, NC 27599.

6 Address correspondence and reprint requests to Dr. Nigel Mackman, Division of Hematology/Oncology, Department of Medicine, 917 Mary Ellen Jones Building, 98 Manning Drive, CB 7035, Chapel Hill NC 27599. E-mail address: nigel_mackman{at}med.unc.edu

7 Abbreviations used in this paper: IKK, I{kappa}Bβ kinase; MAPK, mitogen-activated protein kinase; TF, tissue factor; PI3K, the phosphatidylinositol-3-kinase; PTEN, phosphatase and tensin homologue; WT, wild type; TAT, thrombin-antithrombin; PEC, peritoneal exudate cell; PM, peritoneal macrophage.







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