Constitutive androstane receptor–vitamin D receptor crosstalk: Consequence on CYP24 gene expression

doi:10.1016/j.bbrc.2007.06.003

Biochemical and Biophysical Research Communications

Volume 360, Issue 1, 17 August 2007, Pages 76-82

https://doi.org/10.1016/j.bbrc.2007.06.003 Get rights and content

Abstract

We previously reported that the pregnane X receptor (PXR) interferes with vitamin D receptor (VDR) target genes, notably CYP24, by targeting the same responsive elements. Since PXR and constitutive androstane receptor (CAR) share responsive elements in the promoter of their target genes, we wondered whether CAR also interferes with CYP24 expression. The current study shows that: (i) CAR-RXR heterodimer binds to and transactivates the proximal promoter of CYP24 (−1200/+22) and both VDRE-1 and VDRE-2 which control its expression in response to 1,25-dihydroxyvitamin D₃, (ii) androstanol an inverse agonist of hCAR inhibits transactivation of VDREs by hCAR, (iii) mutations of either VDRE-1 or -2 half sites inhibit hCAR-mediated transactivation, and (iv) in primary human hepatocytes (n = 11) CITCO, a specific hCAR agonist, is an inducer of CYP24 as well as of CYP2B6 and CYP3A4 mRNAs. In conclusion, CAR/PXR and VDR bind to and transactivate the same response elements in CYP24 promoter.

Section snippets

Materials and methods

Chemicals. Culture media, dimethylsulfoxide, rifampicin, 1α,25-dihydroxyvitamin D₃ (1,25-(OH)₂D), and culture medium additives were from Sigma (Saint Louis, MO). γ-[³²P]dATP was from Amersham International (Amersham, England). SR12813 were purchased from Tebu-bio (Le Perray en Yvelines, France).

Plasmids. The following plasmids have been described previously: pSG5-Δ^ATG-hPXR, pcDNA3.1-his-tagged PXR, pSG5-mPXR, pSG5-mRXRα, pSG5-hVDR, pGL3-hCYP24 (−1200/22)-LUC, p(NR1)3-tkLUC, p(VDRE-1)3-tkLUC,

CAR binds to and transactivates VDRE-1 and VDRE-2 of CYP24 promoter

Analysis of VDR- and CAR-binding to specific response elements has been assessed by electromobility shift assay. Radiolabeled CYP2B6 NR1 (a typical target of CAR/RXR) and CYP24 VDRE-1 and VDRE-2 oligonucleotides were incubated in the absence or presence of CAR, VDR, and RXRα proteins prepared by a transcription–translation coupled system, either alone or in association, before being loaded onto the gel. PXR and his-tagged PXR proteins were also included in these series of experiments as control

Discussion

The results reported here show that: (i) CAR/RXR heterodimer binds to and transactivates the proximal promoter of CYP24 (−1200/+20) and both VDRE-1 and VDRE-2 which control the expression of CYP24 in response to 1,25(OH)₂-D, (ii) androstanol a well-characterised inverse agonist of hCAR inhibits the transactivation of VDREs by hCAR, (iii) mutations of either VDRE-1 or -2 half sites inhibit hCAR-mediated transactivation, and (iv) in primary human hepatocytes CITCO, a specific CAR agonist, is an

References (36)

B. Endres et al.
26-Hydroxylation of 1alpha,25-dihydroxyvitamin D3 does not occur under physiological conditions
Arch. Biochem. Biophys.
(2001)
K.S. Chen et al.
Cloning of the human 1 alpha,25-dihydroxyvitamin D-3 24-hydroxylase gene promoter and identification of two vitamin D-responsive elements
Biochim. Biophys. Acta
(1995)
D.M. Kerry et al.
Transcriptional synergism between vitamin D-responsive elements in the rat 25-hydroxyvitamin D3 24-hydroxylase (CYP24) promoter
J. Biol. Chem.
(1996)
Y.E. Timsit et al.
CAR and PXR: the xenobiotic-sensing receptors
Steroids
(2007)
J.J. Eloranta et al.
Coordinate transcriptional regulation of transport and metabolism
Methods Enzymol.
(2005)
L. Drocourt et al.
Expression of CYP3A4, CYP2B6, and CYP2C9 is regulated by the vitamin D receptor pathway in primary human hepatocytes
J. Biol. Chem.
(2002)
S. Gerbal-Chaloin et al.
Transcriptional regulation of CYP2C9 gene. Role of glucocorticoid receptor and constitutive androstane receptor
J. Biol. Chem.
(2002)
J.M. Maglich et al.
Identification of a novel human constitutive androstane receptor (CAR) agonist and its use in the identification of CAR target genes
J. Biol. Chem.
(2003)
T. Sueyoshi et al.
The repressed nuclear receptor CAR responds to phenobarbital in activating the human CYP2B6 gene
J. Biol. Chem.
(1999)
L.B. Moore et al.
Orphan nuclear receptors constitutive androstane receptor and pregnane X receptor share xenobiotic and steroid ligands
J. Biol. Chem.
(2000)

D.J. Mangelsdorf et al.

The RXR heterodimers and orphan receptors

Cell

(1995)

J.P. Aumais et al.

Function of directly repeated half-sites as response elements for steroid hormone receptors

J. Biol. Chem.

(1996)

Z. Zhou et al.

Identification and characterization of a novel androgen response element composed of a direct repeat

J. Biol. Chem.

(1997)

C. Frank et al.

Identification of pregnane X receptor binding sites in the regulatory regions of genes involved in bile acid homeostasis

J. Mol. Biol.

(2005)

C.A. Vyhlidal et al.

Development and refinement of pregnane X receptor (PXR) DNA binding site model using information theory: insights into PXR-mediated gene regulation

J. Biol. Chem.

(2004)

M. Ellfolk et al.

Isolation and properties of the CYP2D25 promoter: transcriptional regulation by vitamin D3 metabolites

Biochem. Biophys. Res. Commun.

(2006)

F. Hosseinpour et al.

Phenobarbital suppresses vitamin D(3) 25-hydroxylase expression: A potential new mechanism for drug-induced osteomalacia

Biochem. Biophys. Res. Commun.

(2007)

C. Carlberg

Current understanding of the function of the nuclear vitamin D receptor in response to its natural and synthetic ligands

Recent Results Cancer Res.

(2003)

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Constitutive androstane receptor–vitamin D receptor crosstalk: Consequence on CYP24 gene expression

Abstract

Section snippets

Materials and methods

CAR binds to and transactivates VDRE-1 and VDRE-2 of CYP24 promoter

Discussion

Arch. Biochem. Biophys.

Biochim. Biophys. Acta

J. Biol. Chem.

Steroids

Methods Enzymol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Cell

J. Biol. Chem.

J. Biol. Chem.

J. Mol. Biol.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Current understanding of the function of the nuclear vitamin D receptor in response to its natural and synthetic ligands

Recent Results Cancer Res.