Abstract
Nicotine C-oxidation is primarily catalyzed by CYP2A6 in humans. This enzymatic activity exhibits a large interindividual variability, which to a great extent is caused by genetic polymorphisms in the CYP2A6 gene. There are large interindividual differences in CYP2A6 mRNA and protein levels, but little is known about the transcriptional regulation of CYP2A6, which can, e.g., explain such differences. Using transient transfections of 5′-deleted CYP2A6 promoter constructs in human hepatoma B16A2 cells, we show that maximal promoter activity was harbored in the sequence spanning from –112 to –61. Putative response elements for the transcription factors hepatocyte nuclear factor-4 (HNF-4)α, CCAAT-box/enhancer binding protein (C/EBP)α, C/EBPβ, and octamer transcription factor-1 (Oct-1) were identified in this region, and electrophoretic mobility shift assays showed that these transcription factors bind to the predicted elements. To determine the relevance of these sites, expression vectors for these transcription factors were cotransfected with CYP2A6 promoter constructs in HepG2 cells. HNF-4α, C/EBPα, and Oct-1 exerted an activating effect, whereas overexpression of C/EBPβ reduced CYP2A6 promoter activity. To confirm the importance of these sites in vivo, mutated CYP2A6 reporter constructs were injected into mouse liver. Mutation of either HNF-4 or C/EBP-Oct-1 motifs significantly decreased promoter activity, 52 and 26% of wildtype, respectively, whereas when both motifs were mutated the activity in mice decreased to 14% of wild type. In conclusion, the data indicate that the constitutive hepatic expression of CYP2A6 is governed by an interplay between the transcription factors HNF-4α, C/EBPα, C/EBPβ, and Oct-1. These results will be important for the identification of new polymorphisms affecting CYP2A6 gene expression.
Footnotes
-
This study was supported in part by National Institutes of Health, National Institute of General Medical Sciences Grant 1-R01 GM60548-01A2 and the Swedish Research Council and the Swedish Cancer Foundation. M.P. was granted a postdoctoral fellowship from the Institute of Environmental Medicine, Karolinska Institutet. C.R.-A. was a recipient of a Marie Curie fellowship from the European Union Community program Quality of Life and Management of Living Resources under contract number QLG5-CT-2002-51733.
-
doi:10.1124/jpet.104.081570.
-
ABBREVIATIONS: P450, cytochrome P450; SNP, single nucleotide polymorphism; HNF, hepatocyte nuclear factor; EMSA, electrophoretic mobility shift assay; C/EBP, CCAAT-box/enhancer binding protein; DR, direct repeat; Oct-1, octamer transcription factor-1; CAR, constitutive androstane receptor; DBP, D-box binding protein; RXR, retinoid-X-receptor; LAP, liver-enriched transcriptional activating protein; LIP, liver-enriched transcriptional inhibitory protein; NF-I, nuclear factor I; PCR, polymerase chain reaction.
-
↵1 These authors contributed equally to the experimental work and preparation of the manuscript.
-
↵2 Current address: Department of Molecular Medicine, Karolinska Institutet, Stockholm, Sweden.
- Received December 3, 2004.
- Accepted January 21, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
JPET articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|