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

doi:10.1074/jbc.m109326200

Heidi R Kast, Bryan Goodwin + Show 8 more

Open Access

https://doi.org/10.1074/jbc.m109326200

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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 16alpha-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 alpha (RXRalpha, 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.

Highlights

Members of the nuclear receptor superfamily of ligand-activated transcription factors have critical roles in many aspects of development and adult physiology, including cholesterol homeostasis, bile acid biosynthesis and transport, and xenobiotic metabolism
Activation of PXR results in increased expression of a number of cytochrome P450 (CYP) genes including Cyp3a11, CYP3A4, and various CYP2B subfamily members that are involved in the metabolism of a wide array of xenobiotics and endogenous substrates prior to their excretion into the bile [19, 20, 22,23,24,25,26,27]
We demonstrate that treatment of human, rat, or murine hepatocytes with ligands for FXR, PXR, or CAR results in increased expression of MRP2 mRNA

Summary

Introduction

Members of the nuclear receptor superfamily of ligand-activated transcription factors have critical roles in many aspects of development and adult physiology, including cholesterol homeostasis, bile acid biosynthesis and transport, and xenobiotic metabolism. We demonstrate that treatment of human, rat, or murine hepatocytes with ligands for FXR, PXR, or CAR results in increased expression of MRP2 mRNA. We demonstrate that ligands for FXR, PXR, or CAR can activate expression of reporter genes controlled either by the rat MRP2 proximal promoter or by two copies of the novel hormone response element identified in the MRP2 promoter.

Results

Conclusion