Regulation of multidrug resistance-associated protein 2 (ABCC2) by the nuclear receptors pregnane X receptor, farnesoid X-activated receptor, and constitutive androstane receptor

Heidi R. Kast, Bryan Goodwin, Paul T. Tarr, Stacey A. Jones, Andrew M. Anisfeld, Catherine M. Stoltz, Peter Tontonoz, Steve Kliewer, Timothy M. Willson, Peter A. Edwards

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Abstract

The multidrug resistance-associated protein 2 (MRP2, ABCC2), mediates the efflux of several conjugated compounds across the apical membrane of the hepatocyte into the bile canaliculi. We identified MRP2 in a screen designed to isolate genes that are regulated by the farnesoid X-activated receptor (FXR, NR1H4). MRP2 mRNA levels were induced following treatment of human or rat hepatocytes with either naturally occurring (chenodeoxycholic acid) or synthetic (GW4064) FXR ligands. In addition, we have shown that MRP2 expression is regulated by the pregnane X receptor (PXR, NR1I2) and constitutive androstane receptor (CAR, NR1I3). Thus, treatment of rodent hepatocytes with PXR or CAR agonists results in a robust induction of MRP2 mRNA levels. The dexamethasone- and pregnenolone 16α-carbonitrile-dependent induction of MRP2 expression was not evident in hepatocytes derived from PXR null mice. In contrast, induction of MRP2 by phenobarbital, an activator of CAR, was comparable in wild-type and PXR null mice. An unusual 26-bp sequence was identified 440 bp upstream of the MRP2 transcription initiation site that contains an everted repeat of the AGTTCA hexad separated by 8 nucleotides (ER-8). PXR, CAR, and FXR bound with high affinity to this element as heterodimers with the retinoid X receptor α (RXRα, NR2B1). Luciferase reporter gene constructs containing 1 kb of the rat MRP2 promoter were prepared and transiently transfected into HepG2 cells. Luciferase activity was induced in a PXR-, CAR-, or FXR-dependent manner. Furthermore, the isolated ER-8 element was capable of conferring PXR, CAR, and FXR responsiveness on a heterologous thymidine kinase promoter. Mutation of the ER-8 element abolished the nuclear receptor response. These studies demonstrate that MRP2 is regulated by three distinct nuclear receptor signaling pathways that converge on a common response element in the 5′-flanking region of this gene.

Original languageEnglish (US)
Pages (from-to)2908-2915
Number of pages8
JournalJournal of Biological Chemistry
Volume277
Issue number4
DOIs
StatePublished - Jan 25 2002

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Cytoplasmic and Nuclear Receptors
Hepatocytes
Genes
Luciferases
Rats
Pregnenolone Carbonitrile
Bile Canaliculi
Retinoid X Receptors
Chenodeoxycholic Acid
Messenger RNA
Thymidine Kinase
5' Flanking Region
Transcription Initiation Site
Hep G2 Cells
Response Elements
Phenobarbital
Reporter Genes
Dexamethasone
Rodentia
Nucleotides

ASJC Scopus subject areas

  • Biochemistry

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Regulation of multidrug resistance-associated protein 2 (ABCC2) by the nuclear receptors pregnane X receptor, farnesoid X-activated receptor, and constitutive androstane receptor. / Kast, Heidi R.; Goodwin, Bryan; Tarr, Paul T.; Jones, Stacey A.; Anisfeld, Andrew M.; Stoltz, Catherine M.; Tontonoz, Peter; Kliewer, Steve; Willson, Timothy M.; Edwards, Peter A.

In: Journal of Biological Chemistry, Vol. 277, No. 4, 25.01.2002, p. 2908-2915.

Research output: Contribution to journalArticle

Kast, Heidi R. ; Goodwin, Bryan ; Tarr, Paul T. ; Jones, Stacey A. ; Anisfeld, Andrew M. ; Stoltz, Catherine M. ; Tontonoz, Peter ; Kliewer, Steve ; Willson, Timothy M. ; Edwards, Peter A. / Regulation of multidrug resistance-associated protein 2 (ABCC2) by the nuclear receptors pregnane X receptor, farnesoid X-activated receptor, and constitutive androstane receptor. In: Journal of Biological Chemistry. 2002 ; Vol. 277, No. 4. pp. 2908-2915.
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abstract = "The multidrug resistance-associated protein 2 (MRP2, ABCC2), mediates the efflux of several conjugated compounds across the apical membrane of the hepatocyte into the bile canaliculi. We identified MRP2 in a screen designed to isolate genes that are regulated by the farnesoid X-activated receptor (FXR, NR1H4). MRP2 mRNA levels were induced following treatment of human or rat hepatocytes with either naturally occurring (chenodeoxycholic acid) or synthetic (GW4064) FXR ligands. In addition, we have shown that MRP2 expression is regulated by the pregnane X receptor (PXR, NR1I2) and constitutive androstane receptor (CAR, NR1I3). Thus, treatment of rodent hepatocytes with PXR or CAR agonists results in a robust induction of MRP2 mRNA levels. The dexamethasone- and pregnenolone 16α-carbonitrile-dependent induction of MRP2 expression was not evident in hepatocytes derived from PXR null mice. In contrast, induction of MRP2 by phenobarbital, an activator of CAR, was comparable in wild-type and PXR null mice. An unusual 26-bp sequence was identified 440 bp upstream of the MRP2 transcription initiation site that contains an everted repeat of the AGTTCA hexad separated by 8 nucleotides (ER-8). PXR, CAR, and FXR bound with high affinity to this element as heterodimers with the retinoid X receptor α (RXRα, NR2B1). Luciferase reporter gene constructs containing 1 kb of the rat MRP2 promoter were prepared and transiently transfected into HepG2 cells. Luciferase activity was induced in a PXR-, CAR-, or FXR-dependent manner. Furthermore, the isolated ER-8 element was capable of conferring PXR, CAR, and FXR responsiveness on a heterologous thymidine kinase promoter. Mutation of the ER-8 element abolished the nuclear receptor response. These studies demonstrate that MRP2 is regulated by three distinct nuclear receptor signaling pathways that converge on a common response element in the 5′-flanking region of this gene.",
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AU - Goodwin, Bryan

AU - Tarr, Paul T.

AU - Jones, Stacey A.

AU - Anisfeld, Andrew M.

AU - Stoltz, Catherine M.

AU - Tontonoz, Peter

AU - Kliewer, Steve

AU - Willson, Timothy M.

AU - Edwards, Peter A.

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