Identification of the functional vitamin D response elements in the human MDR1 gene
Introduction
P-glycoprotein (P-gp), which is encoded by the multidrug resistance 1 (MDR1) gene, transports a wide range of compounds, such as drugs and xenobiotics, from intracellular to extracellular compartments [1]. P-gp is expressed on the apical surface of epithelial cells of tissues including intestine, kidney, liver, and brain, and plays an important role in drug absorption, renal secretion, biliary excretion, and brain distribution [1]. CYP3A4 is the most abundantly expressed human cytochrome P450 contributing to drug metabolism, and P-gp and CYP3A4 share many substrates, inhibitors, inducers, and tissue distribution patterns [2]. Therefore, it has been hypothesized that the expressions of MDR1 and CYP3A4 have similar regulatory mechanisms. In fact, both genes are directly regulated by nuclear receptors, pregnane X receptor (PXR) and constitutive androstane receptor (CAR) [3], [4], [5], [6].
Previous reports revealed that 1α,25-dihydroxyvitamin D3 (1,25-(OH)2D3), the most active metabolite of vitamin D3, regulates the expression of MDR1 mRNA and P-gp protein. The treatment of LS180 cells, a human colon carcinoma cell line, with 1,25-(OH)2D3 led to a significant increase in MDR1 mRNA and P-gp protein levels [7], [8], [9]. In human airway epithelium-derived Calu-3 cells, treatment with 1,25-(OH)2D3 caused elevated P-gp expression [10]. Furthermore, Olaizola et al. reported that the uptake of [99mTc]-sestamibi (which is known to be a substrate of P-gp and is excreted by P-gp [11], [12]) by the parathyroid glands of uremic patients was suppressed by pulse administration of 1,25-(OH)2D3 for 2 weeks, suggesting that P-gp induction by 1,25-(OH)2D3 leads to increased [99mTc]-sestamibi efflux [13].
The biological activity of 1,25-(OH)2D3 is mainly mediated via the vitamin D receptor (VDR), a member of the nuclear receptor superfamily. VDR forms a heterodimer with retinoid X receptor (RXR) and binds to the vitamin D response element (VDRE) in the regulatory region of genes. The VDRE by which the gene is regulated positively is generally composed of a direct repeat (DR) of the consensus hexamer (half-site) sequence of 5′-RGKTCA-3′ (R = A or G, and K = G or T) spaced by three or four nucleotides (DR3 or DR4), or an everted repeat spaced by 6, 7, 8, or 9 nucleotides (ER6, ER7, ER8, or ER9) [14], [15], [16], [17]. However, negative response elements for 1,25-(OH)2D3 have been identified in several genes down-regulated by 1,25-(OH)2D3, such as human pituitary transcription factor-1 gene, in which an imperfect DR2 motif acts as a negative VDRE [18].
Previously, Geick et al. reported that the induction of MDR1 by rifampin is mediated by PXR which binds to a DR4 located between −7.9 and −7.8 kbp upstream from the transcription start site [3]. Burk et al. reported that CAR also induces MDR1 expression by binding to several DR4s located in the same region [4]. Recently, we reported that thyroid hormone receptor (TR) regulates the expression of MDR1 by binding to several DRs located in the same region [19]. It was reported that 1,25-(OH)2D3 also regulates CYP3A4 induction through the binding of VDR/RXRα to some PXR response elements [14]. These results suggest that VDR also binds to several DR motifs in the same region of the MDR1 gene and regulates the expression of MDR1. However, this theory requires substantiation.
There is variation amongst individuals in intestinal MDR1 expression [20]. Since 1,25-(OH)2D3 induces the expression of MDR1, the 1,25-(OH)2D3-mediated induction process might be involved in this inter-individual variation. Furthermore, vitamin D is widely prescribed and influences the induction of P-gp, which potentially affects pharmacokinetics. Therefore, the role of vitamin D in the mechanism of MDR1 expression is worthy of investigation. In this study, we investigated that how 1,25-(OH)2D3 regulates the expression of MDR1 using the intestinal epithelial cell line Caco-2. We demonstrate that the induction of MDR1 by 1,25-(OH)2D3 is mediated by VDR/RXRα binding to several VDREs located between −7880 and −7810 bp upstream of the MDR1 gene, in which every VDRE additively contributes to the 1,25-(OH)2D3 response.
Section snippets
Plasmid constructs
Human VDR cDNA was amplified from human kidney Marathon-Ready cDNA (Clontech Laboratories Inc., Palo Alto, CA, USA) with the primers 5′-ATGGAGGCAATGGCGGC-3′ and 5′-TCAGGAGATCTCATTGCCAAACAC-3′ using a TaKaRa LA Taq (Takara Bio Inc., Shiga, Japan). The resulting DNA fragment was subcloned into the pEF6/V5-His-TOPO vector (Invitrogen, Carlsbad, CA, USA) and this expression plasmid (pEF6/V5-hVDR) was used for the transfection. The sequences were verified by DNA sequencing. The pEF6/V5-hVDR plasmid
Identification of the 1,25-(OH)2D3-responsive region in the MDR1 gene
To investigate the mechanism of MDR1 gene expression induced by 1,25-(OH)2D3, we performed a luciferase reporter gene assay using an intestinal epithelial cell line, Caco-2, which expresses VDR at relatively lower level [9]. The cells were transfected with a reporter plasmid containing the 5′-upstream region from −10082 to +117 bp of MDR1 (pMD10082L) in the presence or absence of an expression plasmid encoding VDR. Following the treatment with either vehicle (DMSO) or 1,25-(OH)2D3, luciferase
Discussion
Several studies have shown that 1,25-(OH)2D3 induces the expression of MDR1[7], [22]. However, it remains unclear how 1,25-(OH)2D3 regulates MDR1 expression. In this study, we demonstrated that the induction of MDR1 by 1,25-(OH)2D3 is mediated by VDR/RXRα binding to the region located between −7.9 and −7.8 kbp upstream from the transcriptional start site of the human MDR1 gene (Fig. 5).
As shown in Fig. 1B, the region located between −7880 and −7817 bp is essential for VDR-mediated induction. This
Acknowledgements
We thank Dr. Shuichi Koizumi (Yamanashi University) for providing the human RXRα cDNA. This work was supported in part by grants from the Ministry of Health, Labor and Welfare of Japan and the Japan Health Sciences Foundation (Research on Publicly Essential Drugs and Medical Devices).
References (40)
- et al.
Nuclear receptor response elements mediate induction of intestinal MDR1 by rifampin
J Biol Chem
(2001) - et al.
Orphan nuclear receptors constitutive androstane receptor and pregnane X receptor share xenobiotic and steroid ligands
J Biol Chem
(2000) - et al.
The effects of culture conditions on CYP3A4 and MDR1 mRNA induction by 1alpha,25-dihydroxyvitamin D(3) in human intestinal cell lines, Caco-2 and LS180
Drug Metab Pharmacokinet
(2005) - et al.
Expression of CYP3A4, CYP2B6, and CYP2C9 is regulated by the vitamin D receptor pathway in primary human hepatocytes
J Biol Chem
(2002) - et al.
Thyroid hormone receptor mediates human MDR1 gene expression—identification of the response region essential for gene expression
Arch Biochem Biophys
(2008) - et al.
The expression of CYP2B6, CYP2C9 and CYP3A4 genes: a tangle of networks of nuclear and steroid receptors
Biochim Biophys Acta
(2003) - et al.
Ligand-triggered stabilization of vitamin D receptor/retinoid X receptor heterodimer conformations on DR4-type response elements
J Mol Biol
(2000) - et al.
Salt concentration determines 1,25-dihydroxyvitamin D3 dependency of vitamin D receptor–retinoid X receptor–vitamin D-responsive element complex formation
Arch Biochem Biophys
(1997) - et al.
CYP3A4, CYP3A5, and MDR1 in human small and large intestinal cell lines suitable for drug transport studies
J Pharm Sci
(2001) - et al.
25-Hydroxyvitamin D-1alpha-hydroxylase in normal and malignant colon tissue
Lancet
(2001)
The effects of Vitamin D metabolites on expression of genes for calcium transporters in human duodenum
J Steroid Biochem Mol Biol
Extra-renal 25-hydroxyvitamin D3-1alpha-hydroxylase in human health and disease
J Steroid Biochem Mol Biol
1,25-Dihydroxyvitamin D3 regulates genes responsible for detoxification in intestine
Toxicol Appl Pharmacol
Concise review: clinical relevance of drug drug and herb drug interactions mediated by the ABC transporter ABCB1 (MDR1, P-glycoprotein)
Oncologist
Overlapping substrate specificities and tissue distribution of cytochrome P450 3A and P-glycoprotein: implications for drug delivery and activity in cancer chemotherapy
Mol Carcinog
A role for constitutive androstane receptor in the regulation of human intestinal MDR1 expression
Biol Chem
Identification of a human nuclear receptor defines a new signaling pathway for CYP3A induction
Proc Natl Acad Sci USA
Gene expression of CYP3A4, ABC-transporters (MDR1 and MRP1–MRP5) and hPXR in three different human colon carcinoma cell lines
J Pharm Pharmacol
Transcriptional control of intestinal cytochrome P-4503A by 1alpha,25-dihydroxy vitamin D3
Mol Pharmacol
Transport of HIV-protease inhibitors across 1 alpha,25di-hydroxy vitamin D3-treated Calu-3 cell monolayers: modulation of P-glycoprotein activity
Pharm Res
Cited by (63)
Bile acid metabolism and signaling, the microbiota, and metabolic disease
2022, Pharmacology and TherapeuticsEffects of vitamin D on drugs: Response and disposal
2020, NutritionCitation Excerpt :VD could significantly induce the expression and activity of P-gp (also known as MDR1], ATP-binding cassette sub-family B member 1 [ABCB1]; Table 3). VD induced the mRNA and protein expression of multidrug resistance-related protein 1A[MDR1A] in rat brain, small intestine [144], liver [146], and kidneys [143] by binding the VDR/RXR alpha heterodimer to multiple VD response elements (VDREs) [151], increased the removal of P-gp substrates (fluorescent P-gp substrate and quinidine) in the brain and kidneys [152,153], and reduced the exposure of digoxin to the blood and brain [132,154]. In addition, VD induced the expression of MDR-1 and P-gp in various cell lines, increased the translocation of digoxin, and decreased the accumulation of 5 (and 6)-carboxy-20, 70-dichlorofluorescein [15,17,155].
Hormones and Hormone Precursors of the Skin
2020, Hormonal Signaling in Biology and Medicine: Comprehensive Modern EndocrinologyHormones and Hormone Precursors of the Skin
2019, Hormonal Signaling in Biology and Medicine: Comprehensive Modern EndocrinologyVitamin D pathway genetic variants are able to influence sofosbuvir and its main metabolite pharmacokinetics in HCV mono-infected patients
2018, Infection, Genetics and EvolutionCitation Excerpt :Cdx2 G allele is associated to a reduced affinity to bind Cdx2 and a consequent decreased transcriptional activity (Arai et al., 2001; Khan et al., 2013). Our data confirm results reported by Fatiguso et al., who observed decreased ethambutol plasma levels at week 4, supposing a reduced VDR activity and a consequent enhanced expression of uptake transporters and/or reduced efflux, such as P-glycoprotein, which is able to transport SOF (Saeki et al., 2008). The CYP27B1 gene on chromosome 12q13.1-q13.3 encodes the 1a-hydroxylase enzyme, which converts the calcifediol into calcitriol, the major biological effector of the immune response (Overbergh et al., 2006).
Influence of 25-hydroxyvitamin D<inf>3</inf> and 1,25-dihydroxyvitamin D<inf>3</inf> on expression of P-glycoprotein and cytochrome P450 3A in sheep
2016, Journal of Steroid Biochemistry and Molecular BiologyCitation Excerpt :Both, P-gp and CYP3A, are regulated by a family of nuclear receptors, including the vitamin D receptor (VDR) and the pregnane X receptor (PXR) [11]. Functional vitamin D responsive elements have been demonstrated to be involved in up-regulation of human P-gp as well as in the induction of CYP3A expression in adenocarcinoma cells [12,13]. Furthermore, it has been shown that hydroxylated vitamin D metabolites can bind to human PXR [14].