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

Abstract

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/EBPα in mature hepatocytes, replication-defective adenovirus vectors were used to efficiently and homogeneously overexpress the mouse C/EBPα gene in a SV40 virus- conditionally transformed rat hepatocyte line that can be induced to express C/EBPβ and C/EBPδ but that has little endogenous C/EBPα expression. Hepatocytes were infected with a recombinant adenovirus vector carrying the cDNA for C/EBPα driven by Rous sarcoma virus promoter elements (AdCEBPα) or a similar vector carrying the Escherichia coli lacZ gene (Adβgal). Staining for β-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 AdCEBPα had 50-fold higher levels of C/EBPα mRNA and protein than those infected with Adβgal, but similar expression of C/EBPβ. Infection with AdCEBPα inhibited proliferation in cells expressing little C/EBPβ, 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/EBPβ. Although overexpression of C/EBPα consistently increased C/EBPα DNA binding activity, it was not sufficient for albumin expression. Infection with AdCEBPα only increased albumin mRNA levels in nontransformed cells that also expressed relatively high levels of C/EBPβ. Thus, in hepatocytes, C/EBPα has a dominant antiproliferative function, but must interact with other factors to regulate hepatocyte-specific gene expression.