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Involvement of 3-phosphoinositide-dependent kinase 1 (PDK1) in the regulation of nitric oxide expression in LPS-stimulated macrophages Jian, Zhiqi


Macrophages are part of the innate immune system playing an important role in intestinal inflammation, and amplify the inflammatory response through the activation of Th-1 cytokines including nitric oxide (NO). Macrophages become activated as a result of exposure to microbial product lipopolysaccaride (LPS) as well as interferon-γ from T cells. One of the aspects that attract researchers' interests most is the induction of inducible nitric oxide (iNOS) and increased NO production in these cells. PDK1 is a key enzyme in linking extracellular signals to multiple effector pathways. The mechanism by which LPS induces NO synthesis in murine macrophages is incompletely understood, and a role for PDK1 had not been previously investigated. In this study we demonstrate the involvement of PDK1 in the regulation of nitric oxide expression in LPS-stimulated Raw 264.7 macrophages. N-α-tosyl-L-phenylalanyl chloromethyl ketone (TPCK) was used as an inhibitor of PDK1 signaling. The inhibition of PDK1 by TPCK led to the suppression of NF-kB activity, iNOS expression and NO production. What's more, Raw 264.7 macrophages transiently transfected with a kinase-dead PDK1 construct also showed decreased NF-kB reporter activity, iNOS protein expression and NO production. To further confirm our findings, we also created a stable cell line by transducing the lentiviral vectors expressing kinase-dead PDK1 into Raw 264.7 macrophages. The NF-kB pathway and the NO production were suppressed too in response to LPS compared to normal Raw 264.7 cells. Therefore, our results show for the first time that PDK1 is involved in NO expression in LPS-stimulated Raw264.7 macrophages and that this is associated with attenuation of LPS-mediated activation of the NF-kB pathway.

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