The long range goal of the proposed studies is to identify therapeutic strategies to prevent hepatocyte apoptosis in cholestatic liver disease. Hepatocyte apoptosis accompanies congenital and acquired cholestatic hepatobiliary disorders and is due, in part, to the retention of endogenous compounds normally excreted in bile. Bile acids are among these retained toxins. Bile acids induce apoptosis in primary cultures of hepatocytes and in hepatoma cells. Our results show that the hormonal intracellular messenger cAMP protects against bile acid induced apoptosis. The anti-apoptotic effect of cAMP is largely independent of classical cAMP signaling through protein kinase A, but requires cAMP mediated activation of phosphoinositide-3 kinase (PI3K). In our studies cAMP mediated cytoprotection and PI3K activation occurs through a novel cAMP stimulated pathway involving activation of cAMP dependent guanine exchange: factor (cAMP-GEF) which signals through the small GTP binding protein Rap. Analogue specific activation, of cAMP-GEF in hepatocytes activates Rap 1 and confers PI3K dependent protection from bile acid induced apoptosis. An initial aim of these studies is to characterize the mechanism (s) whereby cAMP binding to cAMP-GEF activates PI3K and to demonstrate that Rap 1 activation is a downstream effector of cAMP's anti-apoptotic effect.
A second aim of these studies is to identify downstream PI3K dependent effectors which mediate cAMP cytoprotection. We will determine if cAMP/PI3K dependent survival involves activation of a PI3K/Akt/glycogen synthase kinase 3 beta pathway.
Our third aim i s to identify the molecular targets of cAMP in the apoptotic machinery. Initial investigations will examine the effect of cAMP on death receptors dependent signaling mechanisms. Dissection of the signaling pathways and molecular mediators of cAMP mediated cytoprotection might lead to the discovery of signaling molecules that can be targeted for antiapoptotic drug therapy.
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