The dysfunctional regulation of signal transduction pathways is central to the pathogenesis of a number of clinically significant inflammatory conditions including systemic inflammation (SIRS), atherosclerosis and autoimmune diseases. The mitogen- activated protein kinases (MAPKs) comprise a major mechanism by which cells respond to proinflammatory stimuli. A clear picture of how proinflammatory stimuli activate mammalian MAPK pathways and reprogram gene expression would be extremely beneficial in the development of novel and anti inflammatory treatment strategies. We find that the mammalian STE20 homologue germinal center kinase (GCK), through the MAPK-kinase-kinases mixed-lineage kinases (MLKs)-2 and -3), is required for activation of the Jun-N-terminal kinase (JNK) and p38 MAPK groups by a clinically relevant subset of pathogen-associated molecular patterns (PAMPs-molecular moieties, produced by microbial pathogens, which trigger innate immune responses): bacterial lipopolysaccharide (LPS), peptidoglycan (PGN) and flagellin (FliC). GCK is also required for these PAMPs to stimulate, in macrophages, stabilization of the mRNA for tumor necrosis factor (TNF), the release of TNF, and the induction of a large number of proinflammatory cytokines/chemokines, key initiating events in innate immunity. Consistent with this, gck-/- mice are strikingly resistant to LPS-induced lethal endotoxic shock. Our central hypothesis is that GCK is essential to LPS, PGN and FliC signaling. GCK is recruited by these stimuli and, in turn, activates MLKs-2 and -3, and, downstream of these, JNK and p38. JNK and p38 function at the transcriptional level to foster the expression of proinflammatory and immune genes. p38 also acts post- transcriptionally to affect the production of important proinflammatory mediators. We posit that in vivo, GCK-mediated signaling is pivotal to the responses to LPS, PGN and FliC and to the production of a competent innate immune response. We will use biochemical methods and cells isolated from gck-/- mice to determine the role of GCK and its effectors in PAMP-stimulated cellular inflammatory responses (cytokine induction, phagocytosis and monocyte-endothelial cell interaction). Mutant GCK constructs will be used to """"""""rescue"""""""" the GCK knockout and for structure-function analysis. Finally, we will use our gck-/- mice to determine the in vivo functions of the GCK pathway in promoting lethal pathogen-induced inflammation and in host-defense. These studies will provide important details of a previously unknown signaling pathway required for inflammatory responses.
ATTACHMENT) The dysfunctional regulation of signal transduction pathways is central to the pathogenesis of a number of clinically significant inflammatory conditions including atherosclerosis, sepsis, and autoimmune diseases. The mitogen-activated protein kinases (MAPKs) are activated by a wide variety of agents including inflammatory stimuli, and comprise a major mechanism by which cells respond to adaptive and pathologic changes in environmental or physiologic circumstances. The studies described in this application will dissect an important mechanism of MAPK pathway activation by inflammatory stimuli, and link this pathway to innate immune regulation;the results will pave the way towards the development of novel anti inflammatory treatment strategies.
