The goal of the proposed research is to understand the role of MAP kinases in mediating the response of macrophages to lipopolysaccharide (LPS). LPS induces dramatic polyclonal activation of B lymphocytes. In macrophages, LPS induces the production and release of cytokines, inflammatory proteins, and arachidonic acid (AA) metabolites. These events play a central role in initiating local immune responses to bacterial pathogens and neoplasia. Systemic effects of these responses, such as fever and vasodilatation, may result in the clinical manifestations of septic shock. The long-term objective of this proposal is to better understand the biochemical and molecular responses of macrophages to LPS. The biochemical events to be studied focus on the protein phosphorylation cascade initiated by LPS stimulation, in which MAP kinase activation is a pivotal event. This proposal will employ the use of mammalian expression vectors to achieve expression of an epitope-tagged isoform of MAP kinase (Erk-2) cDNA in a murine macrophage cell line, RAW264.7. Immunoprecipitation and blotting experiments with both anti-epitope and anti-MAP kinase antibodies will identify transfected clones that express the tagged molecule. At present, antibodies to various MAP kinase isoforms cross-react; therefore, tagging these proteins will allow the study of specific isoforms. LPS-induced tyrosine phosphorylation of the epitope-tagged protein will be studied by immunoprecipitation and blotting experiments using an antiphosphotyrosine antibody. Protein kinase activity of the tagged molecule will also be measured to see if it increases in response to LPS. Stimulation via PKC-dependent and -independent pathways will be assessed by phorbol ester stimulation and the use of PKC inhibitors. Downstream targets of activated MAP kinase will be identified by overexpressing wild-type Erk-2 in RAW264.7 and stimulating with LPS. Targets identified in other systems that will be studied in macrophages include the transcription factors c-jun/AP-1 and p62TCF, the translation regulator ribosomal S6 kinase, and c-Raf. Nf-kappaB will also be studied as it is an important molecule in macrophage activation, but it may not itself be activated by MAP kinase. The effect of MAP kinase overexpression on the biological responses of macrophages to LPS will also be examined. After identifying targets of MAP kinase activation in response to LPS stimulation, vectors containing either antisense sequences or a mutated, inactive form of the enzyme will be used to inhibit endogenous synthesis of MAP kinase or inhibit its activation, respectively. These experiments are designed to examine the direct role MAP kinase plays in the regulation of LPS-induced signaling events. The approach of overexpressing either an intact or inactive kinase may have general usefulness for the study of other systems that incorporate MAP kinase phosphorylation in the signalling cascade.
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