Functional analysis of the polymorphism − 211C>T in the regulatory region of the human ABCC3 gene

Abstract

The multidrug resistance protein 3 (MRP3/gene symbol: ABCC3) is an ATP-dependent efflux pump mediating the transport of endogenous glucuronides and conjugated drug metabolites across cell membranes. In humans the hepatic expression of ABCC3 mRNA seems to be influenced by the polymorphism C>T at the position − 211 in the promoter of the ABCC3 gene. The aim of this study was to investigate the possible mechanisms of how this SNP influences the MRP3 expression. Promoter luciferase reporter gene constructs representing 0.5, 1.1, 4.4, and 8.1 kb upstream of the translational start site were cloned with cytosine or thymine at position − 211 and transfected into HepG2, Caco-2, and LS174T cells. Reporter gene activity was dependent on the length of the promoter sequence but interestingly not on the nucleotide at position − 211. Cotransfection with FTF cDNA (Fetoprotein Transcription Factor) binding to elements near the − 211 polymorphism increased promoter activity in all constructs except the 0.5 kb fragment also independently of the − 211 SNP. Taken together, we did not find any influence of the − 211C>T ABCC3 promoter polymorphism on either the basal or the FTF induced reporter gene activity. Whether other tissue specific mechanisms reveal an impact of this SNP on the in vivo regulation of MRP3 remains to be determined.

Introduction

The human MRP3 protein (gene symbol ABCC3) belongs to the ATP-binding cassette transporter family C (ABCC) which consists of 12 members. Nine of these comprise the subfamily of the multidrug resistance proteins (König et al., 1999b, Borst et al., 2000, Borst et al., 2007, Kruh and Belinsky, 2003). MRPs are integral membrane proteins mediating the ATP-dependent export of organic anions out of cells. MRP3 is a organic anion transporter with a preference for glucuronidated compounds (Zelcer et al., 2005, Zelcer et al., 2006, Zamek-Gliszczynski et al., 2006). Therefore, typical substrates include estradiol-17β-glucuronide (Hirohashi et al., 1999, Hirohashi et al., 2000) and bilirubin-glucuronides (Lee et al., 2004). Furthermore, cytotoxic agents like etoposide and methotrexate (Hirohashi et al., 1999, Zeng et al., 1999, Zelcer et al., 2001) are substrates for MRP3, demonstrating the involvement of this export pump in drug resistance. MRP3 protein was detected in liver, kidney, intestine, adrenals, pancreas, and gallbladder (Scheffer et al., 2002). In polarized cells MRP3 is located in the basolateral membrane as shown for example in hepatocytes (König et al., 1999a) and gallbladder epithelial cells (Rost et al., 2001). The hepatic MRP3 expression is highly variable in humans (Lang et al., 2004). The upregulation of MRP3/Mrp3 in humans (Scheffer et al., 2002) and rats (Ogawa et al., 2000) under cholestasis and in the absence of the apically localized family member MRP2 (ABCC2) as seen e.g. in patients with Dubin–Johnson syndrome (König et al., 1999a) suggests a compensatory efflux mechanism in these species under conditions where the apical efflux of substances is impaired. However, it is still unknown whether MRP3 has the ability to protect human liver from toxic bile salts during cholestasis especially since recent studies have demonstrated that there is no difference in serum bile salt levels and liver damage between Mrp3 knock out and wild type mice after bile duct ligation (Belinsky et al., 2005, Zelcer et al., 2006). Several polymorphisms in the ABCC3 gene have been identified and analyzed so far (Lang et al., 2004, Lee et al., 2004). While the polymorphism 3890A>G (Arg1297His) has no effect on the protein localization or the transport function (Lee et al., 2004), the polymorphism − 211C>T, located in the 5′-regulatory region of the ABCC3 gene, was associated with significantly lower ABCC3 transcript levels and a trend towards lower protein expression in human liver. In Caucasians, the frequencies of the CT and TT genotypes are 58.6% and 21.2%, respectively. Furthermore it was shown that this polymorphism could affect the binding of nuclear proteins to the ABCC3 promoter (Lang et al., 2004). The − 211C>T polymorphism is located in a region that contains putative bile salt-responsive elements including two α-1 fetoprotein transcription factor (FTF)-like elements (Galarneau et al., 1996). It has been shown that this transcription factor may play a role in the expression of MRP3 mRNA and protein in response to bile acids (Inokuchi et al., 2001, Bohan et al., 2003). While the − 211C>T polymorphism seems to affect the ABCC3 mRNA expression in human liver, in acute leukaemia the role of this polymorphism is not clear. Doerfel et al. (2006) showed that this polymorphism determines neither the expression of the ABCC3 gene nor the response to therapy. In addition the chance of survival in patients with acute myeloid leukaemia (AML) was unchanged. Interestingly, Risch and co-workers (2006) found that the T-allele of the − 211 polymorphism was associated with a better response of AML patients to chemotherapy and a better survival of AML patients.

The aim of our study was to clarify these discrepancies. To gain more insight into the functional consequences of this polymorphism we established several reporter gene constructs and analyzed the effect of the − 211C>T exchange in 3 different cell lines. In addition, the possible effect of the transcription factor FTF was analyzed.