GROWTH-DEPENDENT INHIBITION OF CCAAT ENHANCER-BINDING PROTEIN (C/EBP-ALPHA) GENE-EXPRESSION DURING HEPATOCYTE PROLIFERATION IN THE REGENERATING LIVER AND IN CULTURE

As an approach to understanding physiological mechanisms that control the proliferation of highly differentiated cells, we are addressing whether certain hepatic transcription factors participate in mechanisms that control the growth of hepatocytes. We have focused on CCAAT enhancer-binding protein (C/EBP-alpha), a transcription factor which is highly abundant in normal liver and is considered to regulate expression of many genes, including some involved in energy metabolism (S. L. McKnight, M. D. Lane, and S. Gluecksohn-Walsh. Genes Dev. 3:2021-2024, 1989). Using Northern (RNA) blot analysis, we have examined the expression of C/EBP-alpha mRNA during liver regeneration and in primary cultures of hepatocytes. C/EBP-alpha mRNA levels decrease 60 to 80% within 1 to 3 h after partial hepatectomy as the cells move from G0 to G1 and decrease further when cells progress into S phase. Run-on transcription analysis is in agreement with the Northern blot data, thus suggesting that C/EBP-alpha is transcriptionally regulated in regenerating liver. C/EBP-alpha mRNA expression also decreases dramatically during the growth of freshly isolated normal hepatocytes cultured under conventional conditions (on dried rat tail collagen; stimulated to proliferate by epidermal growth factor [EGF] and insulin). Cultures of hepatocytes on rat tail collagen in the presence or absence of EGF clearly show that within 3 h, EGF depresses C/EBP-alpha mRNA expression and that this effect is substantially greater by 4 h. Inhibition of protein synthesis in the liver by cycloheximide or in cultured hepatocytes by puromycin or cycloheximide effectively blocks the down-regulation of C/EBP-alpha gene expression, apparently by stabilizing the normal rapid turnover of the C/EBP-alpha mRNA (half-life of < 2 h). This drop in C/EBP-alpha gene expression in response to activation of hepatocyte growth is consistent with the proposal that C/EBP-alpha has an antiproliferative role to play in highly differentiated cells (R. M. Umek, A. D. Friedman, and S. L. McKnight, Science 251: 288-292, 1991).