This proposal attempts to explore the cellular and molecular mechanisms of ethanol effects on hepatocyte growth since one of its major actions on the liver is a capacity to strikingly inhibit the regenerative response following cellular injury. We have found that human insulin receptor substrate (hIRS-1) overexpression as a transgene in the mammalian liver in the context of physiologic concentrations of hepatocyte growth factors leads to enhanced tyrosyl phosphorylation of the protein followed by increased binding to adapter molecules such as Grb2 and Syp and subsequent activation of both the P13K and the Ras/MAPK/MAPKK cascades. The biologic consequences of these protein- protein interactions and activation events are the stimulation of hepatocyte proliferation. Therefore, we have established a new animal model to study the effect of ethanol on liver growth in the context of this pathway. There is now direct evidence to suggest that ethanol substantially influences downstream events of the hIRS-1 mediated signal transduction cascade. To investigate ethanol actions on the liver, we plan to: 1. Study the effect of chronic ethanol feeding on the hIRS-1 mediated hepatocyte growth factor signal transduction cascade in a transgenic mouse model. In this regard, we will assess: a) the expression and tyrosyl phosphorylation of the insulin and IGF-1 receptors, b) the expression and tyrosyl phosphorylation of hIRS-1, c) binding of hIRS-1 to Grb2, Syp and P13K proteins, d) activation of Ras/Raf/MAPK/MAPKK and P13K cascades, e) phosphorylation of CREB on serine133 by CREB kinase, f) upregulation of c-fos and c-jun genes and g) effects on hepatocyte proliferation as well as 2. Explore the effects of ethanol on the expression, tyrosyl phosphorylation and function of specific hIRS-1 domains including: a) tyrosyl phosphorylation of the pleckstrin homology (PH) and phosphotyrosine binding (PTB) regions in the N-terminus of hIRS-1, b) biologic consequences of ethanol exposure on specific SH2 binding domains in the C-terminus of hIRS-1 with respect to effects on the generation of downstream events including the activation of the Ras/MAPK/MAPKK and P13K cascades, c) subsequent upregulation of the insulin responsive GAPDH gene and d) stimulation of cell growth. Our major hypothesis is that the hIRS-1 mediated pathway is central to activation of liver growth and chronic ethanol consumption leads to molecular alterations of protein phosphorylation and subsequent interactions of downstream effector molecules in this cascade.
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