The current proposal represents a series of experiments designed to determine the major signal transduction pathways involved in interleukin-1beta stimulation of cyclooxygenase-2(COX-2) and the inducible nitric oxide synthase (iNOS) gene expression. Since IL-1beta can regulate the production of biologically active prostaglandins and NO through increases in the expression of COX-2 and iNOS genes, we would therefore like: (1) to determine whether or not the JNK pathway is involved in the activation of the gene; (2) we would also like to expand experiments to determine whether or not IL-1beta can activate PI 3- kinase which can then convert PIP2 to PIP3 which is a known activator of PKC/zeta. Based on the recent information that PKC/zeta can phosphorylate I/kappa/B, we have put forth the hypothesis that the activation of PKC/zeta then leads to the phosphorylation of I/kappa/B and its subsequent proteolysis and disassociation from NF/kappa/B leading to the activation of certain genes. The experiments are planned in a stepwise fashion to determine whether the signaling pathway functions with respect to expression of the COX-2 and iNOS genes. In a third set of experiments, we will then evaluate whether or not ras and the rho family of GTP-binding proteins functions upstream of both the activation of PI3-kinase, p38 or the JNK pathway. Experiments are then planned to evaluate through deletion constructs the important regulatory sequences present in the proximal promoter of the COX-2 gene which respond to IL-1 as the extracellular ligand and how these transcriptional factors interact with serum induced transcriptional factors for full expression of the cyclooxygenase gene. We feel that these pathways are important in the full understanding of the mechanisms by which inflammatory cytokines can induce PGE2 production in the renal mesangial cell which may be an important consequence of the inflammatory mediators on the biology of the renal mesangial cell.
