Bacterial infection, a complication following burn and trauma, initiates responses of the innate immune system that include rapid gene induction in myeloid (MO) and non-myeloid lineage cells. Induced genes encode cytokines, adhesive proteins and enzymes that produce proinfIammatory mediators such as oxidants or lipid metabolites. While these changes form an important part of the host defense system they unfortunately also contribute to the pathogenesis of septic shock. When Gram-negative bacteria are present it is trace amounts of endotoxin (LPS) that begins this cascade of responses. LPS-induced cell activation occurs via receptor-dependent mechanisms which involve transfer of LPS from the extracellular milieu to the cell surface via LPS binding proteins. Here our focus is on understanding how the signal from LPS is transduced in MO. In MO, a consequence of LPS binding to its receptor is triggering of kinase cascades culminating in activation of protein kinases known as MAP kinases (MAPKs). Studies during the previous grant period led to the discovery of a new member of the MAPK superfamily; we termed this enzyme p38. Subsequent work resulted in our discovery of three related but structurally distinct enzymes [MAP kinase kinases, (MKKs)] capable of activating p38. Recent evidence suggests that activation of phosphoprotein phosphatases may also be important in the LPS signal transduction cascade. It is our hypothesis that p38 activation is causally related to LPS-induced gene expression. To test this hypothesis we will perform experiments using biochemical and molecular biologic approaches to identity the predominant MKK involved in LPS-induced p38 activation, to identity substrates of p38 in order to better understand how p38 might regulate LPS-induced gene expression and to evaluate the role of phosphoprotein phosphatases in LPS-induced signalling. These studies will provide basic information that is essential to bridge the gap in our knowledge about events occurring after LPS binds to its receptor on the surface of myeloid lineage cells.
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