The Study Of Expressive Of Hepatic Membrane Transporter Multidrug Resistance-Associated Protein 3, 2(MRP3, MRP2) And Its Transcriptional Regulation Mechanism In Obstructive Cholestatic Human Liver

MRPs (multidrug resistance-associated proteins) family is a class of membrane glycoproteins which is located in a variety of cell surface polarity and mediates its substrate to transport from intracellular to the outside. The energy necessary for the substrate translocation across biomembranes is generated from the hydrolysis of ATP and intermediate phosphorylation of the transporter, enabling active transport of substrates against steep concentration gradients. It is a very important class of organic anion transporter protein, belongs to the ATP-binding domain cassette transmembrane transporter superfamily (ATP binding cassette, ABC-transporter proteins superfamily) [1]. There are now at least nine members of the multidrug resistance-associated protein family (ABCC1-6/MRP1-6, ABCC10-12/MRP7-9) and MRP1-8 are known as organic anion transporters. They can transport a variety of organic anions, including glucuronide-conjugates, GSH conjugated, sulfate, and some other anti-cancer drugs, etc [2]. MRP2 (cMOAT, canalicular multispecific organic anion transporter) mainly located on the apical membranes of hepatocytes, renal proximal tubules and intestinal, may be expressed in trophoblast cells[3,4]. Under normal physiological conditions, Mrp2/MRP2 expression is high in liver, whereas the expression is reduced in the BDL rats and Dubin-Johnson syndrome patients. Multidrug resistance-associated protein 3 (MRP3) is a new transporter gene which is recent cloned, shares the degree of amino acid homology with MRP1(58%) and MRP2 (48%)[5,6]respectively, and mainly distributed on basolateral membrane of liver cells, bile duct epithelium, intestinal epithelium and renal tubular epithelial cells. MRP3 can transport organic anions (including bile salts,17β-estradiol and some anti-cancer drugs) into the blood[7]. The previous studies have showed that MRP3 expression levels are very low or undetectable on basolateral membrane of normal hepatocyte, but its expression was induced highly by the bile duct ligation cholestatic rat model [8]. Also in human cholestatic diseases, such as Dubin-Johnson syndrome and familial hereditary jaundice, MRP3 expression is dramatically increased, while the liver cell canalicular surface expression level of MRP2 was decreased. This negative correlation between these two transporters suggests that the high expression of MRP3 may be a kind of compensatory response to impairement of MRP2 function in hepatic cholestasis [9,10] .Recent studies have reported that in vitro cell culture and / or animal models of experimental bile duct ligation, the hepatic MRP3 mRNA transcription is regulated by nuclear receptor CPF, RXRα:RARα, Sp1, PXR edc. Nuclear receptor CPF/Lrh-1 is able to increase MRP3/Mrp3 mRNA expression [11]. While SP1 is also a transcription activator of MRP3, but RXRα:RARαheterodimers may suppress the expression of MRP3 via inhibition of SP1 activity [12]. In addition, the regultiaon of CAR and PXR on MRP3 gene expression is so far uncertain[13,14]. It has been reported the activation of FXR could up-regulate 7α-hydroxylase (cholesterol-7-αhydroxylase, CYP7A1) gene and MRP2 transcription, induce the expression up-regulated in cultured mouse liver cells, promote the glucuronic esters of bilirubin excretion and feedback inhibit bile acid biosynthesis through repression the encoding of cholesterol[15,16], but the exact mechanism is unclear. So far, most studies were form in vitro cell culture or animal model experiments, there is rare data directly from human liver tissues. In this study, we have investigated MRP2 and MRP3 gene expression and the possible regulatory transcription mechanism of MRP3 in 40 cases of human liver tissue samples from obstructive cholestatic patients.MethodsBy collecting samples of 40 cases of group (control group, normal liver tissue; cholestasis Group, cholestatic liver tissue), mRNA and protein expression levels of membrane transporter MRP2, MRP3 and nuclear receptors FXR, SHP and SP1 were detected by reverse transcription polymerase chain reaction (RT-PCR) and western blot. Immunofluorescence was utilized to detect MRP3, MRP2 and SP1 expression and subcelluar localization in normal and cholestatic liver tissue. The binding abundance and specificity of nuclear receptor SP1, RXRα:RARαto MRP3 promoter DNA was identified by electrophoretic mobility shift assays(EMSA).Results1. RT-PCR results showed that in patients with obstructive cholestasis, the level of expression of MRP2 mRNA is not significant change, while MRP3 expression were significantly increased about 3-fold, and SP1 expression increased about 2-fold.2. Immunofluorescence assay results showed that, in the obstructive cholestasis, the liver cell membrane tubule surface transport protein MRP2 significantly reduced, while MRP3 expression was significantly increased at the basolateral membrane, and the expression of nuclear receptor SP1 was also significantly increased in the nuclei.3. Western blot quantitatively analyzed all the membrane transporters and nuclear receptor expression at protein level. MRP3 expression was increased at about 4-fold in total membrane fraction. SP1 was increased at about 3-fold and both FXR and SHP were reduced about 2～2.5-fold. Interestingly, MRP2 expression was significantly decreased at about 4-fold in total membrane fraction, meanwhile, there no changes was found in total cellular fraction.4. EMSA results showed that the binding abundance of SP1 to its responsive elements in MRP3 gene promoter region was significantly increased about 4-fold in human liver with obstructive cholestasis.DiscussionMRP3 (ABCC3) expression is at very low or undetectable level under physiological conditions. In contrast, its expression is significantly increased in cholestatic liver tissue. MRP3 and MRP2 transporter substrate has extensive homology, so people generally considered that the high expression of MRP3 is a kind of compensatory response for MRP2 function deficiency, which can expump the bile acids from the hepatocyte into the systemic blood to eliminate the accumulation of bile acids in cholestatic hepatocytes, and eventually exclude out of the body through the urine by kidney and reduce the extent of damage of toxial bile acids to the liver. This phenomenom is thought as an auto-protective response of hepatocyte.In current study, we have found that MRP2 expression was significantly reduced both in membrane fraction by western blot and subcellular distribution on the apical membrane of hepatocytes by immunofluorescence detection in obstructive cholestatic liver, while the expression of MRP3 on basolateral membrane was dramatically increased. These results are consistent with previous cell culture or animal model studies [7,8,9,10]. However, MRP3 mRNA was significantly increased, and MRP2 mRNA was not obviously changed. These results suggest that a post-transcriptional regulatory mechanism might be involved in expression of MRP2, and MRP2 was not changed in the total cellular protein level, it may be due to depression of MRP2 on the apical membrance which needs to be further studied. In the cholestasis state, the export pump MRP2 expression on the apical region of hepatocyte membrane was significantly reduced that impaired its function, so that bile acids, endogenous or exogenous compounds and toxic substances can not be sufficiently eliminated from hepatocytes by excretion into the bile. At the same time, the upregulation of MRP3 expression is a functional compensation, which could expump those toxic substances into the blood, and then excreted into the urine by kidney to diminish the liver injuries. At present, the regulatory mechanism of MRP2 and MRP3 expression in cholestasis is not completely clear. As previous studies from in vitro cell culture or in vivo animal models have found that the bile acid as ligands can regulate Mrp2 expression via nuclear receptors FXR/SHP [16,17,18]. There is rare study from human liver to directly demostrate previous findings, since some differences might be existed between human and rodent. In human cholestatic liver, we found that FXR and SHP expression were decreased, which may be associated with reduction of MRP2 expression that was consistent with previous studies. Nuclear receptors SP1 was significantly increased in cholestatic liver at both mRNA and protein levels. EMSA results also indicated that the binding abundance of SP1 to its two typical response elements GGGCGG, which was localized within -103～-97nts and -108～-114nts of MRP3 gene 5'-flanking promoter region, was significantly enhanced. Our data have first confirmed that, in the human obstructive cholestatic liver, nuclear receptor SP1 may participate in upregulation of MRP3 gene mRNA transcription.ConclusionThis study from clinical obstructive cholestatic liver tissues has found that down regulation of apical membrane transporter MRP2 in cholestatic hepatocyte is associated with the reduction of nuclear receptor FXR, SHP expression, which there may be a post-transcriptional regulation mechanism also involved. Nuclear receptor SP1 has played a important role in upregulation of basolateral membrane transporter MRP3 gene transcription.