Elsevier

Biochemical Pharmacology

Volume 76, Issue 4, 15 August 2008, Pages 531-542
Biochemical Pharmacology

Identification of the functional vitamin D response elements in the human MDR1 gene

https://doi.org/10.1016/j.bcp.2008.05.030Get rights and content

Abstract

P-glycoprotein, encoded by the multidrug resistance 1 (MDR1) gene, is an efflux transporter and plays an important role in pharmacokinetics. The expression of MDR1 is induced by a variety of compounds, of which 1α,25-dihydroxyvitamin D3 is known to be an effective inducer. However, it remains unclear how 1α,25-dihydroxyvitamin D3 regulates the expression of MDR1. In this study, we demonstrated that the vitamin D receptor (VDR) induces MDR1 expression in a 1α,25-dihydroxyvitamin D3-dependent manner. Luciferase assays revealed that the region between −7.9 and −7.8 kbp upstream from the transcription start site of the MDR1 is responsible for the induction by 1α,25-dihydroxyvitamin D3. Electrophoretic mobility shift assays revealed that several binding sites for the VDR/retinoid X receptor α (RXRα) heterodimer are located between the −7880 and −7810 bp region, to which the three molecules of VDR/RXRα are able to simultaneously bind with different affinities. Luciferase assays using mutated constructs revealed that the VDR-binding sites of DR3, DR4(I), MdC3, and DR4(III) contribute to the induction, indicating that these binding sites act as vitamin D response elements (VDREs). The contribution of each VDRE to the inducibility was different for each response element. An additive effect of the individual VDREs on induced luciferase activity by 1α,25-dihydroxyvitamin D3 was also observed. These results indicate that the induction of MDR1 by 1α,25-dihydroxyvitamin D3 is mediated by VDR/RXRα binding to several VDREs located between −7880 and −7810 bp, in which every VDRE additively contributes to the 1α,25-dihydroxyvitamin D3 response.

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)

  • J.R. Walters et al.

    The effects of Vitamin D metabolites on expression of genes for calcium transporters in human duodenum

    J Steroid Biochem Mol Biol

    (2007)
  • M. Hewison et al.

    Extra-renal 25-hydroxyvitamin D3-1alpha-hydroxylase in human health and disease

    J Steroid Biochem Mol Biol

    (2007)
  • G.D. Kutuzova et al.

    1,25-Dihydroxyvitamin D3 regulates genes responsible for detoxification in intestine

    Toxicol Appl Pharmacol

    (2007)
  • S. Marchetti et al.

    Concise review: clinical relevance of drug drug and herb drug interactions mediated by the ABC transporter ABCB1 (MDR1, P-glycoprotein)

    Oncologist

    (2007)
  • V.J. Wacher et al.

    Overlapping substrate specificities and tissue distribution of cytochrome P450 3A and P-glycoprotein: implications for drug delivery and activity in cancer chemotherapy

    Mol Carcinog

    (1995)
  • O. Burk et al.

    A role for constitutive androstane receptor in the regulation of human intestinal MDR1 expression

    Biol Chem

    (2005)
  • G. Bertilsson et al.

    Identification of a human nuclear receptor defines a new signaling pathway for CYP3A induction

    Proc Natl Acad Sci USA

    (1998)
  • A. Pfrunder et al.

    Gene expression of CYP3A4, ABC-transporters (MDR1 and MRP1–MRP5) and hPXR in three different human colon carcinoma cell lines

    J Pharm Pharmacol

    (2003)
  • K.E. Thummel et al.

    Transcriptional control of intestinal cytochrome P-4503A by 1alpha,25-dihydroxy vitamin D3

    Mol Pharmacol

    (2001)
  • J. Patel et al.

    Transport of HIV-protease inhibitors across 1 alpha,25di-hydroxy vitamin D3-treated Calu-3 cell monolayers: modulation of P-glycoprotein activity

    Pharm Res

    (2002)
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