Abstract
Pregnane X receptor (PXR) has been shown to form a heterodimer with retinoid X receptor α (RXRα) and to bind to the distal nuclear receptor-binding element 1 and an everted repeat separated by six nucleotides in the proximal promoter of the CYP3A4 gene. In the present study, a new rifampicin-responsive region, located at -7.6 kilobases upstream from the transcription initiation site, has been identified using reporter assays in HepG2 cells. This region contains a cluster of possible nuclear receptor-binding half-sites, AG(G/T)TCA-like sequence. Of these putative half-sites, we focused six half-sites and termed them α-η half-sites. Introduction of a mutation into either an α or β half-site of CYP3A4 reporter genes almost completely diminished the rifampicin-induced transcription. In electrophoretic mobility shift assays, PXR/RXRα heterodimer bound to the direct repeat separated by four nucleotides (DR4) formed with α and β half-sites. HepG2-based transactivation assays with the reporter gene constructs with or without mutations in the PXR binding element(s) demonstrated that this DR4 motif is essential for the transcriptional activation not only by rifampicin but also by various human PXR activators. In addition, reporter assays performed in human hepatocytes and mice with adenoviruses expressing luciferase derived from various CYP3A4 reporter genes and that expressing human PXR supported the results of experiments in HepG2 cells. These results suggest the obligatory role of the newly identified direct repeat separated by four nucleotides-type PXR binding element of the CYP3A4 gene for xenobiotic induction of CYP3A4.
Footnotes
-
This work was supported in part by a Grant-in Aid from the Ministry of Education, Culture, Sports, Sciences, and Technology of Japan and by the Ministry of Health, Labor, and Welfare of Japan.
-
This work was presented in poster form at the 8th International Meeting of the International Society for the Study of Xenobiotics, 2007 Oct 9-12, Sendai, Japan.
-
ABBREVIATIONS: P450, cytochrome P450; AdCont, control adenovirus; AdhPXR, adenovirus expressing human pregnane X receptor; ANOVA, analysis of variance; DMEM, Dulbecco's modified Eagle's medium; DMSO, dimethyl sulfoxide; dNR, distal nuclear receptor binding element; DR4, direct repeat separated by four nucleotides; EMSA, electrophoretic mobility shift assay; eNR3A4, essential distal nuclear receptor-binding element for CYP3A4 induction; ER6, everted repeat separated by six nucleotides; prER6, everted repeat separated by six nucleotides in the CYP3A4 proximal promoter; PXR, pregnane X receptor; kb, kilobase(s); MOI, multiplicity of infection; RXRα, retinoid X receptor α; TCID50, 50% titer culture infectious dose; PCR, polymerase chain reaction; RU486, 11β-(4-dimethylamino)phenyl-17β-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one; SR12813, tetraethyl 2-(3,5-di-tert-butyl-4-hydroxyphenyl)ethenyl-1,1-bisphosphonate; T0901317, N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]phenyl]-benzenesulfonamide.
-
↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
-
↵1 Current affiliation: Tohoku Pharmaceutical University, Sendai, Japan.
- Received July 18, 2008.
- Accepted December 15, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
MolPharm 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.
|