The transcriptional regulation of human multidrug resistance associated protein 4 gene expression by xenobiotics

Abstract

Multidrug resistance-associated protein 4 (MRP4/ABCC4) is a member of the multidrug resistance-associated protein (MRP) family of transporters. Like other MRPs, MRP4 is an organic anion transporter, but it has the unique ability to transport cyclic nucleotides and acyclic nucleoside monophosphate analogs such as anti-viral agents (i.e. 9-(2-phosphonylmethoxyethyl) adenine [PMEA] and azidothymidine monophosphate [AZT]). Moreover, MRP4 has also been identified as a resistance factor for anti-cancer agents such as 6-mercaptopurine, 6-thioguanine, methotrexate and camptothecins (e.g. topotecan, irinotecan). Thus, due to its transport profile, the up-regulation of MRP4 may have a serious impact on the efficacy of nucleoside-based anti-viral treatments and anti-cancer chemotherapy. To date, the transport properties of human MRP4 (hMRP4) protein has been fairly well characterized. However, the same is not true for the regulation of the expression of the human MRP4 gene. Numerous in vivo and in vitro studies suggest that MRP1, MRP2 and MRP3 are regulated transcriptionally by nuclear receptors (NRs). In addition, several animal studies have provided evidences that MRP4 can be regulated by nuclear receptors.Hence, this study aims to examine the role of xenobiotic-activated NRs in the transcriptional regulation of hMRP4. Treatment of human hepatocarcinoma derived HepG2 and Huh7 cells indicates that hMRP4 can be transcriptionally induced by AhR activators (e.g. N2-Napthoflavone and 3-Methylcholanthrene), RAR activators (e.g. all-trans retinoic acid and 13-cis retinoic acid) as well as PXR activator (e.g. Pregnenolone-16N1-carbonitrile). All the xenobiotics used did not affect cell viability. Transient transfection of the hMRP4 5b -flanking region-luciferase reporter construct also produced similar responses to the same AhR, RAR, PXR activators. In addition, the induction of the hMRP4 promoter was dependent on the dose of the activators and the level of expression of the corresponding nuclear receptor. Analysis of the promoter region using the MatInspector program led to the prediction of the presence of putative AhR, RAR and PXR response elements on the promoter. Deletion or mutation of each of the predicted response elements was carried out and this led to the identification of two AhR response elements (AREs) at -2126/-2120 and -1452/-1446, a RAR response element (RRE) at -1764/-1759 and a PXR response element (PRE) at -1225/-1215 on the promoter of the hMRP4 gene. In conclusion, our present findings provide evidences to suggest that hMRP4 promoter can be activated by xenobiotics via several NR-mediated pathways.