Omeprazole transactivates human CYP1A1 and CYP1A2 expression through the common regulatory region containing multiple xenobiotic-responsive elements
Introduction
CYP1A1 and CYP1A2, members of the cytochrome P450 (CYP) gene superfamily, are involved in the detoxification of xenobiotics such as pharmaceutical drugs and environmental pollutants as well as metabolic activation of these compounds. These enzymes are highly inducible. The exposure to certain types of compounds including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3-methylcholanthrene (3MC), or β-naphthoflavone (BNF) enhances the transcription of CYP1A1 and CYP1A2 to elevate these protein levels. This transactivation is mediated by a receptor-type transcription factor, aryl hydrocarbon receptor (AHR) [1], [2]. AHR is normally retained in the cytoplasm and translocates into nucleus upon ligand binding. It then heterodimerizes with AHR nuclear translocator and the heterodimer binds to the cis-element called xenobiotic-responsive element (XRE) located in the promoter region of target genes to activate their transcription. The abovementioned chemicals (i.e. TCDD, 3MC, and BNF) causing CYP1A1 and CYP1A2 induction are shown as strong AHR ligands [1], [2].
The molecular mechanism of the AHR-mediated activation of CYP1A genes have been extensively studied on CYP1A1 and several XREs are identified in the 5′-flanking region of human as well as rodent CYP1A1 genes. In contrast, the molecular mechanism of CYP1A2 induction has been unclear. Because Cyp1a2 induction by TCDD or 3MC is deficient in Ahr-null mice, AHR is believed to be involved in the CYP1A2 induction. However, no XRE sequence has been identified in the CYP1A2 promoter sequence in previous reports.
Human CYP1A1 and CYP1A2 genes are located on chromosome 15 in a head-to-head orientation and share about 23 kb 5′-flanking region. This has raised the possibility that these genes share a common regulatory element(s). To test this hypothesis, we have recently prepared dual reporter gene constructs containing the 23 kb sequence, with which we are able to measure CYP1A1 and CYP1A2 transcriptional activities simultaneously as different reporters, luciferase (Luc) and secreted alkaline phosphatase (SEAP), respectively [3]. The transient transfection assays with the various dual reporter constructs have demonstrated that the region from −464 to −1829 of human CYP1A1, which contains five XREs, is necessary for 3MC- and BNF-induced expression of not only CYP1A1 but CYP1A2 as well [3].
Omeprazole, a proton pump inhibitor with a benzimidazole structure, has been shown to induce CYP1A1 and CYP1A2 in human hepatoma cell lines and human liver in vitro and in vivo [4], [5], [6], [7], [8]. The molecular mechanism of this induction, however, remains obscure. In contrast to TCDD, 3MC, and BNF, omeprazole is shown to activate AHR without its direct binding [9], [10], [11], [12], [13] and this activation seems to require an additional cellular factor(s) [12], [14], [15], [16]. To understand the mechanism for the omeprazole induction of human CYP1A1 and CYP1A2, here we have sought to identify an omeprazole-responsive promoter region by transient transfection assays using the dual reporter constructs.
Section snippets
Materials
Omeprazole, lansoprazole, albendazole, BNF, 3MC, and primaquine were purchased from Sigma–Aldrich (St. Louis, MO) and their chemical structures are shown in Fig. 1. Dimethyl sulfoxide (DMSO) was from Wako Pure Chemicals (Osaka, Japan). HepG2 cells were obtained from Riken Bioresource Center (Tsukuba, Japan).
Reporter gene assays
The single and dual reporter plasmids for human CYP1A1 and/or CYP1A2 were reported previously [3]. HepG2 cells were cultured in Dulbecco's modified Eagles medium (Sigma–Aldrich) supplemented
Selectivity of omeprazole for the CYP1A1 and CYP1A2 transactivation
In this study, we have employed human hepatoma HepG2 cells to investigate the omeprazole-dependent transactivation of human CYP1A1 and CYP1A2 genes because various CYP1A-inducing compounds including omeprazole increased both CYP1A1 and CYP1A2 mRNA levels in the cells [3], [8], [17], [18], [19] and the expression of AHR and AHR nuclear translocator was reported in the cell line [17], [19]. The full-length dual reporter construct, pd-1A1/1A2, containing the nucleotides from −23,411 to +1039 of
Discussion
Omeprazole increases CYP1A1 and CYP1A2 levels in human hepatocytes in vitro and in vivo. Although AHR is involved in this induction, its molecular mechanism is believed to be different from that for TCDD, 3MC, and BNF. Recently, we have prepared the dual reporter gene constructs, which enable us to determine transcriptional activities of human CYP1A1 and CYP1A2 simultaneously as Luc and SEAP activities, respectively. In the present study, we have employed these constructs to investigate
Acknowledgments
This work was supported by Grant-in-Aid from Ministry of Education, Culture, Sports, Sciences and Technology, the Ministry of Health, Labor, and Welfare of Japan.
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Current address: Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai, Miyagi 981-8558, Japan.