The Study Of Expression Of Hepatic Membrane Transporter Multidrug Resistance-Associated Protein3 (ABCC3) And The Mechanism Of Its Transcriptional Regulation In Obstructive Cholestatic Human Liver

Background and Purpose:The liver is vital to the health of the human organism, including the synthesis and secretion of cholic acid, the formation of bile salts and the enterohepatic circulation. Cholestasis refers to the generation of bile salts and the discharge of bile solute obstacles, which is the abnormal accumulation of the bile of substances such as acid, bilirubin and cholesterol in the blood[1,2]. The excretion of cholic acid, bilirubin and other substances is mainly on the organic acid transport system in human liver cell membranes, which include the MRPs, OATPs, NTCP, Bsep and so on[3]. The MRP family (multidrug resistance- assocated proteins, MRPs), an important organic anion transporter, belongs to the ATP-binding cassette superfamily. At present, at least nine of these transporters are known, and they are mainly distributed in the liver, intestinal, kidney and other tissues or organs. The MRP family contain at least one nucleotide-binding domain (NBD) and one transmembrane domain(TMD). And they can function as efflux pumps to the excretion of glucuronide-conjugates, GSH conjugates, sulfate conjugated organic anions, anticancer drug conjugates, and so on[4].MRP3, a new transporter gene, which is mainly distributed in the intestinal, kidney and adrenal, is cloned in recent years. MRP3 expression levels are very low in normal liver, but its expression was induced highly by bile duct ligation in rat. Recent studies have shown that the expression of MRP3 levels is also significantly increased in the basolateral membrane of liver cells, while the canalicular surface expression of MRP2 is showed a dramatic decrease in human cholestatic diseases such as biliary obstruction, primary biliary cirrhosis, and Dubin-Johnson syndrome. With the physiologic function of the MRP3 in depth study, it was found that both MRP2 and MRP3 share a similar substrate spectrum. They can transport the majority of bile acid, in particular, the combination of bile acid. Therefore, the high expression level of MRP3 is considered compensatory for the sharp decline in MRP2 protein expression and protects the liver against injury caused by cholestatic diseases. As the molecular mechanism of regulation of MRP3 further studies, it is not surprising that it is an effective drug for the treatment of cholestatic liver diseases that could be developed in the near future. And it also has important clinical value for the goal of treatment for cholestasis.Recent studies have shown that the expression of MRP3 in obstructive cholestasis may be transcriptionally regulated by CPF, RXRα, RARα, Sp1, PXR and others. A previous study reported that fetoprotein transcription factor/liver receptor homolog-1 is an activator of MRP3/Mrp3 expression, while the functional role of RXRα:RARαacts as a repressor[6,9]. And activator Sp1 and PXR also act in certain to regulate MRP3 expression [10]. However, the underlying mechanism in this regulation is not clear. The experimental research of cholestasis in present focused on in vitro cell and experimental animal models. Unfortunately, the result is usually different in humans. For example, in contrast to the dramatic reduction of Mrp2 mRNA levels in endotoxin-treated rats, MRP2 mRNA levels remained unchanged in humans, but the protein levels was dramatically reduced in both[11]. To further explore the transcriptional regulation mechanism of MRP3 gene expression in obstructive cholestasis in human livers, we investigated the related genes which might regulate the expression of MRP3 by collecting 40 cases of human liver tissue specimens,.Method:For statistical analysis, cases were divided into two groups (control group: normal liver tissues; cholestatic groups: cholestatic liver tissues). The mRNA and protein expression level of MRP3 and the related nuclear receptor genes including CPF, RXRαand RARαwhich may regulate the expression of MRP3, were determined by RT- PCR and western blot. Immunofluorescence and electrophoretic mobility shift assays were also used to study MRP3 protein subcelluar localization and MRP3 gene promoter activation, respectively. Result1. RT-PCR results: The mRNA expression of MRP3 and CPF, RXRα, RARαwere significantly increased 3 to 4-fold in cholestatic liver tissue.2. Immunofluorescence results: MRP3 was localized to the basolateral membrane of hepatocyte, nuclear receptor CPF was in certein increased and distributed in the nucleus and cytoplasm, both in cholestatic or normal liver tissue.3. West blot results: Expression of MRP3 protein was increased 4-fold, and the expression of CPF, RXRαand RARαproteins were increased 6 to 8-fold in the nucleus. In addition, the expression of CPF, RXRαand RARαwas increased about 4-, 2.5- and 13-fold in the cytoplasm.4. EMSA results: The binding activity of CPF to its responsive elements in MRP3 promoter region was markedly increased about 3.5-fold in cholestatic liver tissue. DisscussionMRP3 expression levels are very low in normal liver. In contrast, the expression of MRP3 localized to the basolateral membrane was markedly increased in cholestatic liver tissue. The high expression of MRP3 is considered compensatory for the sharp decline in MRP2 protein expression and protects the liver against injury caused by accumulation of toxic compunds in cholestatic diseases, because MRP2 and MRP3 have a similar substrate specificity, expumping compounds to bile or blood. Therefore, it has important clinical value for the goal of specific treatment for obstruct cholestasis.Our study has confirmed that the expression of MRP3 was markedly increased in cholestatic liver tissue. Then we have further demonstrated that MRP3 promoter activity may be increased by CPF in human cholestatic livers since the enhanced binding of CPF to the responsive elements in MRP3 promoter. These results are consistent with previous studies[5,6,7,8,9]. In contrast with the results reported in the other literature[9], the expression of RXRαand RARαeither at the mRNA levels or at the protein levels was significantly increased in cholestatic hepatocyte. In particular, the expression of RARαwas dramaticly increased 13-fold in the cytoplasm, but only 6-fold in the nucleus. This result implies that some other mechanisms might modulate RARαtranscriptional activity by transferring this nuclear receptor from the nuclei to the cytoplasm, which need to be further studied. Our results showed that there may be some differences existed in the mechanism of regulation of MRP3 gene expression between in human cholestasis and in vitro cell culture experiments, and also cholestatic animal model of bile duct ligation. Interestingly, the redistribution of nuclear receptor protein CPF, RXRαand RARαbetween the nuclei and cytoplasm might imply that other regulatory mechanisms also participating in MRP3 gene expression.ConclusionOur study has confirmed that the expression of MRP3 in humans was up-regulated by nuclear receptor protein CPF in obstructive cholestasis. The expression of RXRαand RARαwere increased in human cholestatic liver, which were different with previous studies even in in vitro human liver cell culture or cholestatic animal model, that might imply some other regulatory mechanism involved in MRP3 gene transcription expression and it need to be further studied.