Liver cirrhosis has an increasing incidence and prevalence in the United States and is closely associated with the development of hepatocellular carcinoma, which has a dismal long-term prognosis. The mechanisms that lead to dysregulated cirrhotic hepatocyte growth are unknown. In our K08 and R03 awards we hypothesized that cirrhotic hepatocytes would be less sensitive to pro-apoptotic agents. We demonstrated that murine cirrhotic hepatocytes exhibited markedly decreased apoptosis after treatment with transforming growth factor beta (TGFbeta), tumor necrosis factor alpha (TNFalpha), and ultraviolet radiation. Gene expression profiles revealed decreased anti-oxidant gene expression. This is a curious finding in face of known increased reactive oxygen species (ROS) in CCI4-treated hepatocytes. In normal hepatocytes, we found that TGFbeta-induced apoptosis was dependent on increased ROS, caspase-8 activation, and the mitochondrial permeability transition (MPT). Cirrhotic hepatocytes exhibited no ROS burst, caspase activation, or MPT. Pre-treatment with the anti-oxidant, trolox, however, permitted TGFbeta-induced apoptosis in cirrhotic hepatocytes suggesting that markedly increased ROS in cirrhotic hepatocytes may protect against apoptosis. Furthermore in SmadS wild-type and knockout mice, we showed that SmadS was necessary for ROS generation and apoptosis. Also, changes in cirrhotic hepatocyte sensitivity to apoptosis may be due to altered extracellular matrix and integrin expression in the cirrhotic liver. In preliminary experiments, we have shown that CCI4-treated mouse hepatocytes have novel expression of the integrin alpha6beta1 compared to normal hepatocytes. These changes may mediate growth related signaling through focal adhesion kinase and related pathways. In this proposal we plan to investigate the following: (1) To determine differences between the TGFbeta-induced apoptotic and Smad signaling pathways that account for cirrhotic hepatocyte resistance to apoptosis. (2) To assess alterations in integrin expression and extracellular matrix related survival signaling pathways in normal and cirrhotic hepatocytes. (3) To determine if cirrhotic human hepatocytes are resistant to TGFbeta-induced apoptosis via mechanisms that are operative in a murine model.
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