The rat angiotensinogen gene will be used to study the mechanisms by which changes in gene expression in liver and adipose tissue are induced during the acute-phase. Tumor necrosis factor and interleukin-1 are elevated during the acute-phase, these inflammatory cytokines activate rat angiotensinogen gene transcription through a previously identified cis-acting promoter element that binds a family of CCAAT/Enhancer Binding Protein (C/EBP)-like proteins. The composition of this family of proteins is altered during the acute-phase, suggesting a role for C/EBP- like proteins in mediating effects of cytokines on gene expression. The levels of one member, LAP, increase whereas C/EBP and a newly identified inhibitor, CHOP-10, fall. Experiments to characterize the mechanisms and consequences of cytokine-induced changes in C/EBP-family members are proposed. Because C/EBP-binding sites are important to the expression of genes that play a role in metabolic regulation, these studies will advance our understanding of the molecular pathogenesis of some of the symptoms associated with the acute-phase (such as weight loss) and provide insights into interactions between the immune system and the renin-angiotensin system. Members of the family of C/EBP-like proteins readily dimerize through a leucine zipper domain. Novel probes, consisting of the dimerization domain of LAP and C/EBP fused to a high affinity protein kinase-A phosphorylation site, will be labeled in vitro and used to detect acute- phase induced changes in C/EBP-like proteins immobilized on nitrocellulose blots. Bacterially expressed C/EBP and LAP coupled to a solid support will be used as an affinity matrix for microscale purification of dimerizing proteins from nuclear extracts. These experiments will identify proteins that are candidates for playing a role in regulating gene transcription during the acute-phase. The probe will be used to isolate cDNA clones encoding new C/EBP-like proteins of relevance to the acute-phase. The newly identified proteins will be analyzed for their ability to alter the behavior of the C/EBP-like complex in vitro and in transfected cells, in vivo. Characterization of the proteins will be guided by several hypotheses: a) Some dimerization partners will exhibit specificity for C/EBP and LAP. b) Dimerization can be altered by hormonally-induced post-translational modification (predominantly changes in phosphorylation state). c) Dimerization can affect the DNA binding properties of the heteromeric complex, its transactivation potency, as well as the stability and intracellular localization of the dimerizing partners. It is predicted that these mechanisms are utilized for transduction of hormonal signals into changes in gene expression. The biochemical models will be complemented by the studies on albino-lethal mice (an animal model for tyrosinemia type I) which overexpress CHOP-10 and exhibit severe defects in activating genes important in intermediary metabolism.
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