Growth-dependent inhibition of CCAAT enhancer-binding protein (C/EBP alpha) gene expression during hepatocyte proliferation in the regenerating liver and in culture.

Abstract

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).

Regulation of expression of murine transferrin receptor 2

“…C/EBP-␣ is highly abundant in normal liver, and it is down-regulated during proliferation of hepatocytes. 38 This is consistent with our reporter assay, which indicated that C/EBP-␣ enhanced TfR2 promoter activity ( Figure 5). Another tissue in which high expression of TfR2 occurs is the erythroid compartment.…”

Adenovirus-mediated Transfer of CCAAT/Enhancer-binding Protein-α Identifies a Dominant Antiproliferative Role for This Isoform in Hepatocytes

“…However, since the differences among C/EBP isoform transcriptional activities appear more qualitative than quantitative (14), it has been difficult to attribute unique functions to C/EBP␣. Work in neoplastic hepatocyte lines and primary hepatocytes has not resolved this dilemma because these systems express relatively high levels of C/EBP␤, but very little C/EBP␣ endogenously (21,46). Furthermore, standard transfection techniques transfer exogenous C/EBP␣ to Ͻ20% of the cultured cells (18 -20), making it difficult to identify treatmentrelated differences because of low signal-to-noise ratios.…”

“…liver regeneration after 70% partial hepatectomy (21,30,46,47), although both the rate of hepatocyte proliferation and the transcription of some hepatocyte-specific genes increase significantly (48). Finally, in hepatoma cell lines and hepatocytederived hybridomas, loss of C/EBP␣ does not extinguish the hepatocyte phenotype (49,50).…”

“…Attempts to modulate the relative levels of these isoforms in vitro and to examine the resulting phenotype have been thwarted, in part, because the transfection efficiency of both primary adult and neoplastic hepatocytes is relatively poor, in the range of 20 -30% (18 -20). Furthermore, basal expression of C/EBP␣ is low in these cell lines (21), unlike in mature hepatocytes, where C/EBP␣ is strongly and constitutively expressed (10,11). To overcome these experimental difficulties and to clarify the role of C/EBP␣ in mature hepatocytes, replication-defective adenovirus vectors were used to efficiently overexpress the mouse C/EBP␣ gene in a conditionally transformed rat hepatocyte line that can be induced to express C/EBP␤ and C/EBP␦ but that has little endogenous C/EBP␣ expression.…”

Roles of CCAAT/Enhancer-binding Proteins in Regulation of Liver Regenerative Growth

“…Decreases in C/EBP␣ are preceded by the induction of other C/EBP isoforms. The level of C/EBP␤ and C/EBP␦ mRNAs, proteins, and DNA binding activities increases in the early pre-replicative period following PH and returns to base line as hepatocytes enter S phase (7)(8)(9)(10).…”