Regulation of Multidrug Resistance-associated Protein 2 (ABCC2) by the Nuclear Receptors Pregnane X Receptor, Farnesoid X-activated Receptor, and Constitutive Androstane Receptor

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 16␣-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 ␣ (RXR␣, 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.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. Recently, two orphan nuclear receptors, the farnesoid X-activated receptor (FXR, 1 NRIH4) and the pregnane X receptor (PXR, NR1I2) were shown to be activated by an overlapping spectrum of bile acids (1-5). These results indicate that bile acids function as hormonal ligands, in addition to their well established roles in the solubilization and absorption of lipids and fat-soluble vitamins from the intestinal lumen.

The apical conjugate efflux pump ABCC2 (MRP2)

“…The region between nucleotides −517 and −197 in front of the translation initiation codon is important for human ABCC2 expression [160], with the transcriptional start-site being at nucleotide −247. In addition, a hormone response element in the rat Abcc2 promoter (ER-8) was identified, which is bound by heterodimers of the retinoid X receptor α (RXRα, NR2B1 [129]) with the ligand-activated transcription factors farnesoid X receptor (FXR, NR1H4), pregnane X receptor (PXR, NR1I2), or constitutive androstane receptor (CAR, NR1I3) [73]. These nuclear receptors are, for example, activated by bile acids via FXR [132] by various xenobiotics such as the antibiotic rifampicin, the synthetic glucocorticoid dexamethasone, and pregnenolone 16α-carbonitrile via PXR [8, 41,85], or by phenobarbital via CAR [161].…”

“…These nuclear receptors are, for example, activated by bile acids via FXR [132] by various xenobiotics such as the antibiotic rifampicin, the synthetic glucocorticoid dexamethasone, and pregnenolone 16α-carbonitrile via PXR [8, 41,85], or by phenobarbital via CAR [161]. Knowledge of the presence of the hormone response element ER-8 in the rat Abcc2 promoter [73] may now explain studies which describe the induction of Abcc2 mRNA and Abcc2 protein in primary cultures of rat hepatocytes by a number of xenobiotics, including the carcinogen 2-acetylaminofluorene, the anticancer drug cisplatin, the antifungal agent clotrimazole, the antibiotic cycloheximide, dexamethasone, the chemopreventive agents oltipraz and sulforaphane, phenobarbital, and pregnenolone 16α-carbonitrile [23, 73,75,98,137]. Additional factors are probably involved in ABCC2/ Abcc2 regulation in vivo because phenobarbital or oltipraz treatment of rats did not increase Abcc2 mRNA expression [23,40,42,68].…”

Regulatory network of lipid-sensing nuclear receptors: roles for CAR, PXR, LXR, and FXR

“…They partly do so by inducing the same target genes including the canalicular bile acid transporter multidrug-resistance-associated protein 2 and 3 (MRP2/ABCC2 and MRP3/ABCC3) [80][81][82]. However, the xenosensors also increase alternate, compensatory pathways for lowering hepatic bile acid levels by inducing their hydroxylation, conjugation and subsequent excretion via blood and urine [50,51,54,55,83].…”

“…The ability of LXR to induce Cyp7a1 in mice and rats makes these animals extremely resistant to a high cholesterol diet whereas other species, including man, rapidly develop hypercholesterolemia under comparable conditions. Accordingly, high cholesterol-fed mice that transgenically express human CYP7A1 in a mouse Cyp7a1 knockout background lack induction of CYP7A1 and become hypercholesterolemic [66,67] Similar to FXR, CAR and PXR promote metabolism and excretion of bile acids.They partly do so by inducing the same target genes including the canalicular bile acid transporter multidrug-resistance-associated protein 2 and 3 (MRP2/ABCC2 and MRP3/ABCC3) [80][81][82]. However, the xenosensors also increase alternate, compensatory pathways for lowering hepatic bile acid levels by inducing their hydroxylation, conjugation and subsequent excretion via blood and urine [50,51,54,55,83].…”

Nuclear receptors CAR and PXR in the regulation of hepatic metabolism

“…As mentioned earlier, CAR and PXR are inexorably linked with regard to regulation of xenobiotic metabolizing enzymes. PXR and CAR coordinately regulate a multitude of genes in bile acid metabolism including the conjugating enzyme UGT1A1 and the transporters MRP2 and OATP2 (Guo et al 2002;Kast et al 2002;Stedman et al 2005). Double knockout mice that lack both CAR and PXR have provided further insight into CAR/PXR crosstalk with regard to bile acid metabolism.…”