Tumor necrosis factor (TNF) is a cytokine produced by macrophages and is implicated in the pathogenesis of septic shock and rheumatoid arthritis. An excellent system to study the signal transduction pathways involved in TNF biosynthesis is the lipopolysaccharide (LPS)- induced macrophage system. LPS activates macrophages and causes the release of toxic cytokines including TNF. The signaling pathways utilized by LPS to induce TNF biosynthesis are largely unknown but are believed to be dependent on the activation of tyrosine kinases and the subsequent activation of the three best described MAPK (mitogen activated protein kinase) pathways: MAPK/ERK (mitogen activated protein kinase/extracellular signal regulated kinase), p38, and the JNK/SAPK (Jun N-terminus kinase/stress activated protein kinase) pathways. Furthermore, these signaling pathways branch to regulate both transcription of the TNF gene and translation of TNF. The main objectives of this proposal are to determine the MAPK pathways involved in LPS signaling and to examine their involvement in the transcription and translation of TNF. This will be accomplished using kinase activation assays and transient transfection assays with various signaling molecule expression constructs and TNF transcriptional and translational reporter constructs.
