The angiotensinogen gene encodes the only known glycoprotein precursor of angiotensin II, a potent octapeptide vasopressor, and is activated during the hepatic acute phase reaction. Hormonal mediators of the acute phase response include macrophage derived cytokines (IL-1alpha, IL-6, TNFalpha) and adrenally secreted glucocorticoids. The angiotensinogen gene is transcriptionally activated through distinct cis-acting regulatory elements, the cytokine responsive element APRE, and the glucocorticoid responsive element GRE, both of which are targets for inducible DNA binding factors. The cytokine responsive element in the angiotensinogen 5' flanking sequences is active only in the presence of glucocorticoids, thus suggesting a functional interaction between the two adjacently located cis regulatory elements. The objective of this study is to define the interaction of DNA binding proteins to result in the transcriptional activation of the angiotensinogen gene during the acute phase response. By gene transfer experiments using wild-type and mutated angiotensinogen/luciferase reporter genes introduced into cultured hepatic cells, functional activity of specific mutations within the two cooperating cis elements will be measured. DNA binding studies, electrophoretic gel shift assay and DNAse I footprinting, using purified liver nuclear protein and cDNA-encoded proteins expressed in E. Coli and the APRE binding factors. Conventional protein purification and screening of protein expression cDNA libraries will be used to obtain structural information of the APRE DNA binding factors. Characterization of the interactions of proteins involved in binding these two cis elements will provide insight into how transcriptional coupling occurs to produce this acute phase homeostatic response. Understanding the molecular events involved in transactivation by the inducible APRE binding factor BPi, a factor indistinguishable from nuclear factor kappaB previously identified to be important in the activation of the HIV promoter in lymphoid cells and activation of the amyloid A protein subunit of amyloid fibrils, will be relevant to disease states such as AIDS and AIDS related complex, secondary amyloidosis in chronic inflammatory diseases and cachexia of malignancy.
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