Abstract
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/EBPα binding sites are essential sites for thetrans-activation of CYP3A4 promoter. Notrans-activation was found in similar reporter gene experiments with a HNF-3γ 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/EBPα had increasedCYP3A4 mRNA levels, but no effect was observed when HNF-3γ was overexpressed. In contrast, overexpression of HNF-3γ simultaneously with C/EBPα resulted in a greater activation of theCYP3A4 gene. This cooperative effect was hepatic-specific and also occurred in CYP3A5 andCYP3A7 genes. To investigate the mechanism for HNF-3γ action, we studied its binding to CYP3A4 promoter and the effect of the deacetylase inhibitor trichostatin A. HNF-3γ was able to bind CYP3A4 promoter at a distal position, near the most distal C/EBPα binding site. Trichostatin A increased C/EBPα effect but abolished HNF-3γ cooperative action. These findings revealed that C/EBPα and HNF-3γ cooperatively regulateCYP3A4 expression in hepatic cells by a mechanism that probably involves chromatin remodeling.
Footnotes
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This work was supported by the European Union, BIOTECH contract BIO4-CT96-0052 and BIOMED contract BMH4-CT86-0254 (Eurocyp). C. R.-A. was the recipient of a fellowship of Generalitat Valenciana.
- Abbreviations:
- P450
- cytochrome P450
- LETF
- liver-enriched transcription factor
- HNF
- hepatocyte nuclear factor
- C/EBP
- CCAAT enhancer-binding protein
- MOI
- multiplicity of infection
- EMSA
- electrophoretic mobility shift assay
- Ad-C/EBPα
- recombinant adenovirus encoding C/EBPα
- Ad-HNF-3γ
- recombinant adenovirus encoding HNF-3γ
- Ad-pAC
- recombinant adenovirus encoding pAC/CMVpLpA
- TSA
- trichostatin A
- PCR
- polymerase chain reaction
- bp
- base pair(s)
- CMV
- cytomegalovirus
- RT
- reverse transcription
- Received October 4, 2002.
- Accepted February 11, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
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