Liver injury, mediated in part by TNF, is often an important component of multiple organ failure associated with sepsis-induced acute lung injury. Furthermore, the liver is thought to be a significant site (in addition to the lung) of cytokine production during sepsis. This project will explore an important signal transduction pathway in liver that leads to production of TNF during sepsis (ie. redox regulation of NFkappaB). The redox mediated events that later the activity of kinases and/or phosphatases involved in the activation of NFkappaB/TNF pathways are poorly defined. The focus of this proposal is to evaluate redox sensitive regulatory pathways for IkappaBalpha and IkappaBbeta in the liver which control the availability and composition of transcriptional NFkappaB complexes in the nucleus following LPS exposure. We hypothesize that both the hype and the subcellular localization of ROS may be involved in regulating these pathways. TO this end, we will utilize a genetic approach to study the effects of reducing ROS formation in liver cell lines (modeling hepatocytes and Kupffer cells) and mouse liver on activation of NFkappaB/TNF pathways following LPS exposure. Recombinant adenoviruses expressing the free radical scavengers, MnSOD, CU/ZnSOD, ecSOD, catalase, iNOS, and eNOS will be used to modulate the redox state of cells. Such an approach will provide an experimental paradigm for linking ROS formation in the liver to subsequent redox regulated phosphorylation events involved in the activation of NFkappaB pathways. Preliminary data has demonstrated that ectopic expression of the mitochondrial form of SOD (MnSOD), prior to endotoxin challenge, decreases both the levels of NFkappaB in the nucleus and serum TNF levels 10-fold as compared to vector controls. Furthermore, the fact that beneficial effects were not observed with the cytoplasmic form of SOD (Cu/ZnSOD), suggested that mitochondrial mediated redox events may preferentially effect the signal transduction cascades involved in TNF production. In a second model of endotoxin NFkappaB activation, we have evidence that the phosphorylation of IkappaBalpha and IkappaBbeta are differentially regulated by mitochondrial and cytoplasmic redox states. These model systems together with subcellular fractionation techniques of cytoplasmic and mitochondrial compartments will allow us to dissect the mechanisms of redox regulated phosphorylation events which control NFkappaB activation following endotoxin exposure. Therapeutic intervention for endotoxin mediated injury has traditionally centered on ameliorating the detrimental effects produced by cytokines such as TNFalpha. Findings from these studies may lead to alternative therapeutic approaches by which redox modulation of the liver can be used to abrogate the detrimental of systemic cytokine production during sepsis.
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