Regulation of multidrug resistance-associated protein 2 (ABCC2) by the nuclear receptors pregnane X receptor, farnesoid X-activated receptor, and constitutive androstane receptor.
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.
Kast
Heidi R
HR
Department of Biological Chemistry and Medicine, UCLA, Los Angeles, California 90095, USA.
Goodwin
Bryan
B
Tarr
Paul T
PT
Jones
Stacey A
SA
Anisfeld
Andrew M
AM
Stoltz
Catherine M
CM
Tontonoz
Peter
P
Kliewer
Steve
S
Willson
Timothy M
TM
Edwards
Peter A
PA
D000818:Animals; D001483:Base Sequence; D001647:Bile Acids and Salts / Q000378:metabolism; D015152:Blotting, Northern; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D000090702:Constitutive Androstane Receptor; D004268:DNA-Binding Proteins / Q000378:metabolism*; D018432:Drug Resistance, Multiple; D017930:Genes, Reporter; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D007555:Isoxazoles / Q000494:pharmacology; D008024:Ligands; D008099:Liver / Q000378:metabolism; D026901:Membrane Transport Proteins; D051379:Mice; D008954:Models, Biological; D008969:Molecular Sequence Data; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D010634:Phenobarbital / Q000494:pharmacology; D000077297:Pregnane X Receptor; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D021381:Protein Transport; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D011987:Receptors, Steroid / Q000378:metabolism*; D015398:Signal Transduction; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D000099150:Receptor, Farnesoid X-Activated
D001647:Bile Acids and Salts; D000090702:Constitutive Androstane Receptor; D004268:DNA-Binding Proteins; D007555:Isoxazoles; D008024:Ligands; D026901:Membrane Transport Proteins; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D010634:Phenobarbital; D000077297:Pregnane X Receptor; D012333:RNA, Messenger; D018160:Receptors, Cytoplasmic and Nuclear; D011987:Receptors, Steroid; D014157:Transcription Factors; D000099150:Receptor, Farnesoid X-Activated; C000715087:ABCC2 protein, human; C000625852:NR1I2 protein, human; C000716327:NR1I3 protein, human; C000625853:Nr1i2 protein, mouse; C000625854:Nr1i2 protein, rat; C000716328:Nr1i3 protein, mouse; C000716329:Nr1i3 protein, rat; C412815:GW 4064; C421109:multidrug resistance-associated protein 1