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/EBPalpha in mature hepatocytes, replication-defective adenovirus vectors were used to efficiently and homogeneously overexpress the mouse C/EBPalpha gene in a SV40 virus-conditionally transformed rat hepatocyte line that can be induced to express C/EBPbeta and C/EBPdelta but that has little endogenous C/EBPalpha expression. Hepatocytes were infected with a recombinant adenovirus vector carrying the cDNA for C/EBPalpha driven by Rous sarcoma virus promoter elements (AdCEBPalpha) or a similar vector carrying the Escherichia coli lacZ gene (Adbetagal). 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 AdCEBPalpha had 50-fold higher levels of C/EBPalpha mRNA and protein than those infected with Ad-beta-gal, but similar expression of C/EBP-beta. Infection with AdCEBPalpha inhibited proliferation in cells expressing little C/EBPbeta, 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/EBPbeta. Although overexpression of C/EBPalpha consistently increased C/EBPalpha DNA binding activity, it was not sufficient for albumin expression. Infection with AdCEBPalpha only increased albumin mRNA levels in nontransformed cells that also expressed relatively high levels of C/EBPbeta. Thus, in hepatocytes, C/EBPalpha has a dominant antiproliferative function, but must interact with other factors to regulate hepatocyte-specific gene expression.
Diehl
A M
AM
Department of Medicine, Johns Hopkins University, Baltimore, Maryland 21205, USA.
Johns
D C
DC
Yang
S
S
Lin
H
H
Yin
M
M
Matelis
L A
LA
Lawrence
J H
JH
D000256:Adenoviridae; D000273:Adipose Tissue / Q000378:metabolism; D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics; D022762:CCAAT-Enhancer-Binding Proteins; D002455:Cell Division / Q000502:physiology*; D002460:Cell Line; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism*; D015870:Gene Expression; D016259:Genes, myc; D005822:Genetic Vectors; D006801:Humans; D007668:Kidney; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D051379:Mice; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D000090063:Proto-Oncogene Mas; D016271:Proto-Oncogene Proteins c-myc / Q000096:biosynthesis; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D013539:Simian virus 40; D013696:Temperature; D014162:Transfection
D022762:CCAAT-Enhancer-Binding Proteins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; C000715470:MAS1 protein, human; D009687:Nuclear Proteins; D000090063:Proto-Oncogene Mas; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D011994:Recombinant Proteins