Adenovirus-mediated transfer of CCAAT/enhancer-binding protein-alpha identifies a dominant antiproliferative role for this isoform in hepatocytes

CCAAT/enhancer-binding protein (C/EBP) isoforms are thought to be important regulators of the hepatocyte phenotype, However, the specific physiological roles of different isoforms are poorly understood because hepatocytes express multiple C/EBPs, and various isoforms have overlapping functions, To identify the functions of C/EBP alpha in mature hepatocytes, replication-defective adenovirus vectors were used to efficiently and homogeneously overexpress the mouse C/EBP alpha gene in a SV40 virus conditionally transformed rat hepatocyte line that can be induced to express C/EBP beta and C/EBP delta but that has little endogenous C/EBP alpha expression, Hepatocytes were infected with a recombinant adenovirus vector carrying the cDNA for C/EBP alpha driven by Rous sarcoma virus promoter elements (AdCEBP alpha) or a similar vector carrying the Escherichia coli lacZ gene (Ad beta gal). Staining for beta-galactosidase demonstrated an infection efficiency of 100% at a multiplicity of infection of 25 plaque-forming units/cell and persistence of foreign gene expression for at least 9 days. Cultures infected with AdCEBP alpha had 50-fold higher levels of C/EBP alpha mRNA and protein than those infected with Ad beta gal, but similar expression of C/EBP beta, Infection with AdCEBP alpha inhibited proliferation in cells expressing little C/EBP beta, even when proliferation was driven by the SV40 transforming antigen, and also blunted mitogenic induction of the c-myc proto-oncogene in nontransformed cells with high levels of C/EBP beta, Although overexpression of C/EBP alpha consistently increased C/EBP alpha DNA binding activity, it was not sufficient for albumin expression, Infection with AdCEBP alpha only increased albumin mRNA levels in nontransformed cells that also expressed relatively high levels of C/EBP beta. Thus, in hepatocytes, C/EBP alpha has a dominant antiproliferative function, but must interact with other factors to regulate hepatocyte-specific gene expression.