The angiontensinogen 9AGT) gene encodes the only known precursor of angiotensin II (AII), a potent vasopressor octapeptide. Under normal conditions, plasma AGT circulates a concentrations that are limiting for maximal activity of the renin angiotensin system. Not surprisingly, hyper-expressing AGT alleles have been linked to human kindreds with essential hypertension. Our long term goal is to characterize the mechanisms regulating AT expression in the hepatocyte, as this cell type determines circulating plasma AGT levels. Transcriptional regulation of AGT expression is the primary mechanism for inducible changes in AGT secretion in response to physiological (hormonal) regulators. The vasopressor hormone, AII, and the cytokine, tumor necrosis factor-alpha (TNFalpha), control AGT transcription by influencing activity of the nuclear factor -kappaB (NF-kappaB) transcription factor by two different mechanisms. TNFalpha induces cytoplasmic to nuclear translocation of NF- kappaB following proteolysis of the inhibitory IkappaBalpha protein by calcium activated neutral endoproteases (calpains). By contrast, AII uses an independent mechanisms for NF-kappaB activation, by recruiting large pre-existing nuclear NF-kappaB1 isoforms to bind to the APRE (without changes in their steady-state abundance). Following NF-kappaB activation through the cytokine pathway, NF-kappaB activity is attenuated by the synthesis of cytoplasmic ankyrin-repeat proteins IkappaBalpha and a novel ankyrin-repeat protein not previously known to be expressed in hepatocytes, the proto-oncogene Bcl-3. The hypothesis pursued in this project will be that the vasopressor peptide AII and the cytokine, TNFalpha control NF-kappaB activity via fundamentally different mechanisms. During the next project period, we propose three specific aims: 1) to define mechanisms for NF-kappaB1 transcriptional activation by transcript analysis of alternatively spliced NF-kappaB1 isoforms and over-expression assays. We will assay for the presence of co-regulators in NF-kappaB binding by DNA affinity assays and the role of PKC in NF-kappaB by selective down-regulation; 2) to characterize the effect of TNFalpha on the calpain-calpastatin pathway and its contribution at hormone inducible IkappaBalpha proteolysis; and 3) to define mechanisms for attenuation NF-kappaB activity through resynthesis of cytoplasmic ankyrin-repeat containing protein (Bcl-3) by Western immunoblot and non-denaturing immunoprecipitation. These studies will define pathways for NF-kappaB activation using a gene relevant to the cardiovascular system, and elucidate new therapeutic targets that may be useful in modulating blood pressure in secondary (renovascular) and hereditary forms of essential hypertension.'
