In adult hepatocytes, differentiation and proliferation are mutually exclusive events. For example, in the adult liver, differentiated hepatocytes are quiescent in regard to proliferation, and when proliferation occurs, differentiated functions, such as albumin gene expression are dramatically reduced. Studies with primary cultures of adult rat hepatocytes have provided evidence that cell-type specific growth regulatory mechanisms exist, but the liver-specific factors regulating hepatic cell proliferation have not yet been identified. We have reported the isolation of a clone encoding for a liver-activator protein, LAP, a major albumin promoter binding protein that confers liver-specific gene expression (14). Because LAP is a major liver nuclear protein binding to the albumin promoter D-site, and D-binding and -transcriptional activities are down-regulated in rapidly dividing hepatocytes upon the induction of liver regeneration, we studied whether LAP could modulate hepatic cell proliferation. We found that LAP inhibits progression of the cell cycle before the G1/S boundary, and that a liver-inhibitory protein (LIP) translated from LAP mRNA antagonizes this effect of LAP. To gain insight into the mechanisms underlying the quiescent state of differentiated hepatocytes, we will study whether hepatocyte expression of LAP is dissociated from cell proliferation during both development and hepatic regeneration in rats. In addition, we will determine the structural/functional relationship between LAP and cell cycle arrest, including the role of specific phosphorylations, and the mechanisms by which LAP down-regulates c-JUN and AP-1 activity. We will investigate the role of LAP in the maintenance of the quiescent state of differentiated hepatocytes. Hepatocytes will be treated with LAP antisense oligonucleotides, or transfected with either LAP antisense or LIP expression vectors. The pathophysiological significance of the effects of LAP on hepatocyte proliferation will be validated in mice bearing liver-specific and inducible LAP antisense or LIP (""""""""trans- dominant negative"""""""") transgenes.
The specific aims of this proposal are to assess: 1. The dissociation of LAP expression and hepatocyte proliferation in the liver. 2. The modulation of cell cycle progress by LAP in hepatoma cells. 3. The role of LAP in the maintenance of the quiescent state of differentiated hepatocytes. 4. The role of LAP phosphorylation on cell cycle progress in hepatic cells. 5. The regulation of c-JUN and AP-1 activity by LAP. 6. The modulation of hepatocyte proliferation in transgenic mice.
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