Transcriptional regulation of human CYP3A4 basal expression by CCAAT enhancer-binding protein alpha and hepatocyte nuclear factor-3 gamma.
Cytochrome P450 3A4 (CYP3A4) is involved in the metabolism of more than 50% of currently used therapeutic drugs, yet the mechanisms that control CYP3A4 basal expression in liver are poorly understood. Several putative binding sites for CCAAT/enhancer-binding protein (C/EBP) and hepatic nuclear factor 3 (HNF-3) were found by computer analysis in CYP3A4 promoter. The use of reporter gene assays, electrophoretic mobility shift assays, and site-directed mutagenesis revealed that one proximal and two distal C/EBP alpha binding sites are essential sites for the trans-activation of CYP3A4 promoter. No trans-activation was found in similar reporter gene experiments with a HNF-3 gamma expression vector. The relevance of these findings was further explored in the more complex DNA/chromatin structure within endogenous CYP3A4 gene. Using appropriate adenoviral expression vectors, we found that both hepatic and nonhepatic cells overexpressing C/EBP alpha had increased CYP3A4 mRNA levels, but no effect was observed when HNF-3 gamma was overexpressed. In contrast, overexpression of HNF-3 gamma simultaneously with C/EBP alpha resulted in a greater activation of the CYP3A4 gene. This cooperative effect was hepatic-specific and also occurred in CYP3A5 and CYP3A7 genes. To investigate the mechanism for HNF-3 gamma action, we studied its binding to CYP3A4 promoter and the effect of the deacetylase inhibitor trichostatin A. HNF-3 gamma was able to bind CYP3A4 promoter at a distal position, near the most distal C/EBP alpha binding site. Trichostatin A increased C/EBP alpha effect but abolished HNF-3 gamma cooperative action. These findings revealed that C/EBP alpha and HNF-3 gamma cooperatively regulate CYP3A4 expression in hepatic cells by a mechanism that probably involves chromatin remodeling.
Rodríguez-Antona
C
C
Departamento de Bioquímica, Facultad de Medicina, Universidad de Valencia, Valencia, Spain.
Bort
R
R
Jover
R
R
Tindberg
N
N
Ingelman-Sundberg
M
M
Gómez-Lechón
M J
MJ
Castell
J V
JV
D000256:Adenoviridae / Q000235:genetics; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000502:physiology*; D002478:Cells, Cultured; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D005822:Genetic Vectors; D051543:Hepatocyte Nuclear Factor 3-gamma; D022781:Hepatocytes / Q000187:drug effects / Q000201:enzymology / Q000502:physiology; D056572:Histone Deacetylase Inhibitors; D006801:Humans; D009687:Nuclear Proteins / Q000502:physiology*; D011401:Promoter Regions, Genetic / Q000502:physiology; D014157:Transcription Factors; D014158:Transcription, Genetic; D015533:Transcriptional Activation / Q000187:drug effects; D014162:Transfection
D022763:CCAAT-Enhancer-Binding Protein-alpha; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; C496505:FOXA3 protein, human; D056572:Histone Deacetylase Inhibitors; D009687:Nuclear Proteins; D014157:Transcription Factors; D051543:Hepatocyte Nuclear Factor 3-gamma; D003577:Cytochrome P-450 Enzyme System; C104464:CYP3A protein, human; C510164:CYP3A5 protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human