? Redox dependent upregulation of hepatocyte inducible nitric oxide synthase (iNOS) expression serves anti-oxidant and anti-apoptotic functions in sepsis and shock. However, in the setting of oxidative stress and pro-inflammatory cytokines which characterize sepsis and shock, the redox-sensitive mechanisms which enhance hepatocyte iNOS expression are unknown. In interleukin-1 beta (IL-1) stimulated rat hepatocytes exposed to superoxide, we demonstrated that iNOS protein expression and gene transcription is increased by oxidative stress. Subsequently, we characterized a redox-sensitive DR1 cis-acting enhancer element in the iNOS promoter. We then identified hepatocyte nuclear factor-4 alpha (HNF-4) as the corresponding transcription factor, confirmed its redox-sensitive enhancement of IL-1 induced iNOS gene transcription, and demonstrated that redox-sensitive HNF-4 DNA binding and transactivation are dependent upon its phosphorylation state. We hypothesize that a redox-dependent kinase pathway phosphorylates HNF-4 to enhance its DNA binding/ transactivation potential and increase hepatocyte iNOS gene transcription. Previous studies have not addressed a redox dependent signaling pathway which targets HNF-4 to enhance iNOS expression. We will focus on the following specific aims which are critical to defining the mechanisms underlying redox-induced phosphorylation of HNF-4 and its role as a redox-dependent enhancer of iNOS promoter activity in IL-1 stimulated rat hepatocytes. We will: 1) identify the amino acid residues of HNF-4 specifically phosphorylated in the presence of IL-1 and superoxide, 2) mutate the candidate amino acid residues to ablate phosphorylation and characterize mutations which inhibit HNF-4 DNA binding and/or transactivation in the presence of IL-1 and superoxide, 3) identify the kinase(s) which phosphorylate the amino acid residues that mediate redox-dependent HNF-4 DNA binding/ transactivation and confirm in vitro hepatocellular kinase functionality, and 4) confirm in vivo function of HNF-4 and its corresponding kinase in a murine model of endotoxemia. Our studies will define a novel and, as yet, poorly described redox-dependent signal transduction pathway which regulates iNOS as an antioxidant mechanism in hepatocytes. ? ?
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