CELL BASED MODEL FOR STUDYING THE REGULATIONOF HUMAN ANGIOTENSINOGEN GENE.

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Objective. To address the underlying mechanism of increase in blood pressure and fibrotic transformation of liver by studying the regulation of AGT after ethanol exposure using cell based model. Background. Alcohol usage is linked to increased blood pressure and fibrotic transformation of the liver. Angiotensin II (Ang II), an octapeptide, is involved in blood pressure regulation and fibrotic transformation of the liver due to ethanol mediated death of hepatocytes. Ang II is produced from its precursor, AGT, by sequential action of renin followed by angiotensin converting enzyme. The blood AGT levels are less than the Michaelis-Menten constant (Km) of renin. Therefore, an increase in blood AGT levels would result in a corresponding increase in Ang II levels that might play crucial role in blood pressure regulation and fibrogenic activity. Increasing the AGT copy number in transgenics increases blood pressure by 8 mm Hg/copy and increased blood AGT levels are observed among hypertensives. Moreover, alcohol metabolism by the liver produces acetaldehyde by alcohol dehydrogenase and reactive oxygen species (ROS) by CYP2E1. Increased ROS mediated oxidative stress activates hypoxia inducible transcription factor-1alpha (HIF- 1alpha). In addition, alcohol-mediated hepatic injury results in immune responses, where the levels of interleukin(IL)-1beta and other cytokines become elevated. IL-1beta and acetaldehyde activate nuclear factor-kappa B (NF-kB) transcription factor. Due to lack of alcohol metabolism by hepatocytes, these transcription factors can be activated by treatment of hypoxia mimetics and relevant cytokines to study human AGT gene regulation. Methods. In addition to regular HepG2 which loses alcohol metabolism after several passages, a variant of HepG2 with stably expressing alcohol dehydrogenase (ALD) and Huh7 cell lines were used to study the alcohol mediated effects on AGT. To mimic HIF-1alpha and NF-kB activation, hypoxia mimetics and NF-kB activators were employed to investigate the effects on AGT secretion with normal hepatocytes. HepG2 and Huh7 hepatocytes were treated with deferoxamine (60nM and 120 nM), cobalt chloride (10nM and 20 nM), IL-1beta (10 ng/ml) and phorbol 12-myristate 13-acetate (PMA) (50 ng/ml) respectively for 4 and 12hrs. The HepG2 expressing ALD were exposed with 25, 50, and 100 mM ethanol for 4 hrs. After treatments, effects on AGT secretion were studied by western blotting. Results. It was observed that alcohol metabolism by HepG2 stably expressing alcohol dehydrogenase caused increased secretion of AGT. Moreover, deferoxamine, cobalt chloride, IL-1beta and PMA treatment also increased the secretion of AGT. Huh7 cells were less sensitive compared to HepG2 cells with HIF-1alpha and NFkappaB mediated activation of human AGT. Conclusion. It is concluded that cell based model can be employed to study the human AGT regulation by alcohol metabolism as well as HIF-1alpha and NF-kappaB activation. Grants. HPD Grant