Interaction of hepatocyte nuclear factors in transcriptional regulation of tissue specific hormonal expression of human multidrug resistance-associated protein 2 (abcc2)

Abstract

Multidrug resistance-associated protein 2 (MRP2) (ABCC2) is an ATP-binding cassette membrane protein located primarily on apical surface of hepatocytes that mediates transport of conjugated xenobiotics and endogenous compounds into bile. MRP2 is highly expressed in hepatocytes, and at lower levels in small intestines, stomach and kidney. Previous reports have characterized mammalian MRP2 promoters, but none have established the molecular mechanism(s) involved in liver enriched expression. This study aims to investigate the mechanism of hepatic MRP2 regulation.

A 2130 bp of MRP2 promoter was cloned from PAC-1 clone P108G1–7, to identify putative liver specific/hormone responsive functional DNA binding sites. Using deletion analysis, site specific mutagenesis and co-transfection studies, liver specific expression was determined.

MRP2 promoter-LUC constructs were highly expressed in liver cell lines compared to non-liver cells. The region extending from − 3 to+ 458 bp of MRP2 promoter starting from AUG contained the potential binding sites for CAAATT box enhancer binding protein (C/EBP), hepatocytes nuclear factor 1, 3 and 4 (HNF1, HNF3, and HNF4. Only HNF1 and HNF4 co-transfection with MRP2 luciferase increased expression. Site specific mutational analysis of HNF1 binding site indicated an important role for HNF1α. HNF4α induction of MRP2 was independent of HNF1 binding site. C/EBP, HNF3, and HNF6 inhibited HNF1α while HNF4α induced MRP2 luciferase expression and glucocorticoids stimulated MRP2 expression.

This study emphasizes the complex regulation of MRP2 with HNF1α and HNF4α playing a central role. The coordinated regulation of xenobiotic transporters and oxidative conjugation may determine the adaptive responses to cellular detoxification processes.

Introduction

Major functions of hepatocytes involve sinusoidal extraction, intracellular metabolism and biliary excretion of endogenous and exogenous lipophilic compounds (Kim, 2002). Xenobiotics, unconjugated bilirubin and hormones are efficiently cleared from sinusoidal blood circulation into liver by basolateral transporters, including OATP-C (SLCO1B1) and also conjugated with phase II enzymes such as UDP-glucuronyltransferase (UGT1A1), sulfotransferase or glutathione transferase (Cui et al., 2001, Hagenbuch and Meier, 2004, Kullak-Ublick et al., 2004). After conjugation, the water soluble metabolites are excreted primarily into bile by the multidrug resistance-associated protein 2 (MRP2) or ABCC2 (Paulusma and Oude Elferink, 1997). Since MRP2 mediates the rate-limiting step in biliary excretion of many drugs and conjugated bilirubin, it is not surprising that recent studies indicate that biliary excretion plays an important role in the overall bioavailability of their metabolism (Bodo et al., 2003).

The transcriptional regulation of multi drug resistance is a complex process involving several key transcriptional cascades (Eloranta et al., 2005, Geier et al., 2003). Nuclear transcriptional factors such as Y-box protein1, FXR, SHP, PXR, CAR, and HNF4α have been reported to modulate the regulation of transport and drug metabolizing genes (Eloranta et al., 2005, Geier et al., 2003, Kast et al., 2002).

MRP2 is an ATP-binding cassette membrane protein that belongs to the MRP transporter subfamily. MRP2 mediates transport of xenobiotics that are generally conjugated with glucuronide, sulphate or glutathione (Borst et al., 2000, Paulusma and Oude Elferink, 1997). MRP2 is found at the apical pole primarily of hepatocytes, but also at lower content in the intestines and kidneys (Gerk and Vore, 2002). Mutant rat strains lacking Mrp2 expression such as transport deficient (TR⁻) Wistar rat and the Eisai hyperbilirubinemic rat (EHBR) display low biliary secretion of xenobiotic conjugates and endogenous compounds such as conjugated bilirubin (Paulusma and Oude Elferink, 1997). Similarly, hereditary deficiency of MRP2 expression in humans, known as the Dubin–Johnson syndrome, causes reduced biliary elimination of bilirubin glucuronides (Kartenbeck et al., 1996, Paulusma et al., 1997) and other organic anions except bile acids.

Regulation of hepatic expression of MRP2 is still poorly understood. In rats, down regulation of Mrp2 has been shown to be associated with administration of lipopolysacharide, ethinyl estradiol and cholestasis (Gerk and Vore, 2002). On the other hand, cellular hydration, reactive oxygen species, drugs and hormones have been demonstrated to increase Mrp2 (Fardel et al., 2001, Qadri et al., 2006, Schrenk et al., 2001). Potential molecular processes for the regulation of rat and human MRP2 have been described, but their specific sites of action and tissue specific regulation is not evident. Both rat and human MRP2 promoters have been cloned and partially characterized (Geier et al., 2003, Hisaeda et al., 2004, Kauffmann and Schrenk, 1998, Kauffmann et al., 2001, Stockel et al., 2000, Tanaka et al., 1999). Potentially important sites in the human promoter which may be involved in the regulation of MRP2 were identified at − 678 to + 197 and include putative sites such as C/EBPβ, HNF1, USF, CBF/NF-Y and Sp1 (Hisaeda et al., 2004, Kauffmann et al., 2001, Stockel et al., 2000; Tanaka et al., 1999a). However, sites for basal liver-specific expression were not demonstrated in either rat or human promoters (Kauffmann et al., 2001).

Hepatocyte nuclear factor 1α (HNF1α) was one possible candidate which may be important in regulation of liver-specific expression since putative DNA binding sites have been identified in human, rat and mouse genes (Vollrath et al., 2006). In addition, HNF1α is important in modifying hepatic expression of xenobiotic transporters such as OATP-C (SLC21A6) and in both phase I and II conjugation steps including glucuronyl transferases and cytochrome P450s (Bernard et al., 1999, Bock and Kohle, 2004, Paulson et al., 1990, Song et al., 1998). Therefore, it is speculated that HNF1α, a liver enriched transcription factor, might also be responsible for basal hepatic expression of MRP2. Since many conjugating enzymes as well as Mrp2 in rats are induced by administration of glucocorticoids (Jemnitz et al., 2002), we examined whether Dexamethasone also regulated the human MRP2 promoter.

The results in this report demonstrate that HNF1α is a key transcription factor responsible for basal liver specific MRP2 expression. In addition, we also identified a putative HNF4α DNA binding site is also involved in regulation of human MRP2 expression. In contrast, HNF3, HNF6 and C/EBP decrease both basal and HNF1 and HNF4 induced expression of MRP2 promoter. Finally, our results indicate that glucocorticoids induce the human as well as the rat MRP2 promoter in vitro through a proximal promoter site that is independent of HNF1α.