Transcriptional Regulation of the Human CYP2A6 Gene

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 5Ј-deleted CYP2A6 promoter constructs in human hepatoma B16A2 cells, we show that maximal promoter activity was harbored in the sequence spanning from Ϫ112 to Ϫ61. Putative response elements for the transcription factors hepatocyte nuclear factor-4 (HNF-4)␣, CCAAT-box/enhancer binding protein (C/EBP)␣, C/EBP␤, 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-4␣, C/EBP␣, and Oct-1 exerted an activating effect, whereas overexpression of C/EBP␤ 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-4␣, C/EBP␣, C/EBP␤, and Oct-1. These results will be important for the identification of new polymorphisms affecting CYP2A6 gene expression.

Dexamethasone-Mediated Up-Regulation of Human CYP2A6 Involves the Glucocorticoid Receptor and Increased Binding of Hepatic Nuclear Factor 4α to the Proximal Promoter

“…CYP2A6, thereby confirming this suspicion. It has been previously demonstrated, by adenovirus-mediated antisense targeting in cultured human primary hepatocytes, that HNF4␣ is a general activator of CYP2A6 (Pitarque et al, 2005). HNF4-REs have also been identified in the 5Ј flanking region of the hCYP2C8 (Ferguson et al, 2005) and hCYP2C9 genes.…”

“…However, an HNF4-RE present in this region has previously been shown to be pivotal in the transcriptional regulation of CYP2A6 (Pitarque et al, 2005). To determine whether the HNF4-RE plays a role in DEX-responsiveness, the HNF4-RE within p2A6-2469-LUC was mutated by site-directed mutagenesis and used in luciferase reporter assays as described above.…”

“…Moreover, response elements for HNF-1␣, CCAAT/enhancer-binding protein ␣, CCAAT/ enhancer-binding protein ␤, and Oct-1 transcription factors in the proximal promoter are involved in CYP2A6 transcriptional regulation (Pitarque et al, 2005). Characterization of the CYP2A6 promoter conducted in this study examines the mechanisms of glucocorticoid-mediated induction.…”

Transcription Factor and Drug-Metabolizing Enzyme Gene Expression in Lymphocytes from Healthy Human Subjects

“…CYP2C8 expression is regulated by CAR, PXR, GR, and hepatic nuclear factor 4␣ in the liver (Ferguson et al, 2005). Finally, constitutive hepatic expression of CYP2A6 is governed by interplay between the TF hepatic nuclear factor-␣, CCAAT/enhancer-binding protein-␣, CCAAT/enhancer-binding protein-␤, and octamer TF 1 (Pitarque et al, 2005). It is also possible to describe activation through the antioxidant response element, which could cross-talk with the xenobiotic response elements (Miao et al, 2004).…”

Transcription Factors Potentially Involved in Regulation of Cytochrome P450 Gene Expression

“…Just as Nrf2, they have a characteristic C-terminal domain responsible for DNA binding, characterized by the structure of basic-leucine zipper. C/EBP may participate in the transcriptional regulation of some cytochrome P450 genes, such as CYP2A6, CYP2B6, CYP2C9, CYP2D6, CYP3A4, CYP3A5 and CYP3A7 (Pitarque et al, 2005). In hepatocytes, this regulation takes place in cooperation with HNFs and other transcription factors.…”