人肝细胞膜蛋白MRP3（ABCC3）在阻塞性胆汁淤积肝组织中表达变化及其调节机制
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摘要
背景与目的:肝脏是胆酸合成、分泌、胆汁形成及其再循环(肠肝循环)的重要器官。胆汁淤积是指胆汁的生成和/或胆汁内溶质排出障碍,一些正常从胆汁中排泄的物质如胆酸、胆红素和胆固醇在血液内异常堆积[1,2]。而胆酸、胆红素等物质的排泄则主要靠肝细胞膜上的胆酸转运蛋白系统,它们包括MRPs﹑OATPs﹑NTCP﹑Bsep等等[3]。其中,MRPs(multidrug resistance-assocated proteins)家族,是一类非常重要的有机阴离子转运蛋白,属于ABC转运体超家族,目前已知至少有9种(MRP1-9)。主要分布于肝脏﹑肾脏﹑肠道等器官组织。MRPs的分子结构基本都包含跨膜区(TMD)与核苷酸结合域(NBD)。它们的主要功能是转运葡萄糖醛酸结合物﹑GSH﹑硫酸盐﹑或某些抗癌药物等等[4]。
多耐药相关蛋白3(multidrug resistance-assocated protein 3,MRP3)是近年新克隆的一种转运蛋白基因,主要分布于肠道、肾脏及肾上腺,而在正常肝细胞基底侧膜上表达很低或不表达[5]。但是在胆道结扎所致的急性胆汁淤积大鼠模型的肝脏中MRP3的表达量却非常高[6]。同时进一步研究发现在人类胆汁淤积性疾病(如:胆道梗阻、原发性毛细胆管性肝硬化、Dubin-Johnson综合征)时,MRP3在肝细胞基底侧膜表达量也大为增加,而同时肝细胞小管面的MRP2的表达量却急剧下降[7]。随着对MRP3转运功能研究进一步的深入,发现其转运底物与MRP2具有广泛的同源性,都能转运大部分胆酸,尤其是结合型胆酸,因此,MRP3的高表达被认为是代偿性的,以补偿小管面膜上MRP2的下调。所以,人们普遍认为MRP3的表达增高是在胆汁淤积状态下肝细胞的一种代偿性自我保护反应[6,8]。因此,随着对MRP3调节的分子机制的进一步研究深入,有可能在不久的将来开发出治疗胆汁淤积疾病的特效药物,并且对临床寻找肝胆汁淤积治疗对策也具重要的临床价值。
当胆汁淤积发生时,肝脏MRP3基因mRNA转录表达与核受体CPF﹑RXRα﹑RARα﹑Sp1﹑PXR等基因相关。已经有文献报道,在体外细胞培养与胆道结扎的动物模型实验研究表明,核受体CPF/Lrh-1能够上调MRP3/Mrp3 mRNA的表达[6];RXRα:RARα异二聚体则能够抑制MRP3的表达[9]。Sp1,PXR等也对MRP3的基因表达起一定的调节作用[10],但是其具体机制尚不清楚。而且目前的实验研究主要集中在体外细胞培养和动物模型实验,直接针对以“人”为研究对象的实验鲜有报道,人体组织的研究结果与体外细胞培养或动物实验常存在差异,如:用内毒素处理的大鼠Mrp2 mRNA的表达水平急剧下降,而当以“人”为研究对象时MRP2 mRNA的表达水平却维持不变,但是二者Mrp2/MRP2蛋白水平的变化却均急剧降低[11]。本实验通过收集40例人体肝组织标本,对阻塞性胆汁淤积时MRP3上调相关的基因进行研究,从而进一步探讨与研究阻塞性胆汁淤积时人肝细胞膜转运蛋白MRP3的转录调控机制。
方法
通过对收集的40例标本分组(对照组:正常肝组织;淤胆组:淤胆肝组织)后,分别在mRNA水平(RT-PCR)与蛋白水平(Western blot)检测分析膜转运蛋白MRP3表达变化,以及与其调节相关核受体基因CPF﹑RXRα与RARα表达变化。通过免疫荧光检测膜转运蛋白MRP3与核受体CPF在正常肝与淤胆肝组织的表达以及定位情况。通过EMSA来检测MRP3基因启动子区反应元件与核受体CPF结合活性在正常肝与淤胆肝组织的变化。
结果
1.阻塞性胆汁淤积时,通过RT-PCR实验研究对膜转运蛋白基因MRP3与核受体基因CPF﹑RXRα﹑RARα等在mRNA水平表达进行分析,结果表明:MRP3以及上述基因在mRNA水平表达均显著增高,增高约3~4倍左右。
2.免疫荧光检测结果表明,阻塞性胆汁淤积时,膜转运蛋白MRP3在肝细胞基底侧膜的表达明显增加。而CPF的表达,在核内与胞浆内均有增加。
3.人肝脏阻塞性胆汁淤积时,通过Western blot实验对膜转运蛋白基因MRP3与核受体基因CPF﹑RXRα﹑RARα等在蛋白水平表达进行分析,结果表明: MRP3蛋白水平表达增高约4倍。CPF﹑RXRα﹑RARα在核内表达增高约6~8倍;而在胞浆内,RARα显著增高约13倍;RXRα增高约2.5倍,CPF增高约4倍。
4.人肝脏阻塞性胆汁淤积时,通过EMSA检测结果表明:MRP3基因启动子区反应元件与核受体CPF结合活性显著增加,约3.5倍。
讨论
MRP3(ABCC3)在正常肝细胞膜低表达或不表达。而当胆汁淤积发生时,肝细胞基底侧膜的MRP3表达却显著增高。同时,又由于MRP3转运底物与MRP2有着广泛的同源性,所以人们普遍认为MRP3的高表达是对MRP2的一种代偿,以减轻胆汁淤积时肝细胞受胆酸毒性损伤的程度,具有护肝的作用。因此,对MRP3表达调节的分子机制的研究,对寻找胆汁淤积治疗对策有着非常重要的临床意义。
本实验通过对人体肝组织标本研究分析得知,阻塞性胆汁淤积时人肝细胞膜转运蛋白MRP3在肝细胞基底侧膜的表达量显著增高。MRP3基因启动子区反应元件与核受体CPF结合活性显著增高,这些充分证明核受体CPF能够上调人肝细胞膜转运蛋白MRP3基因的转录表达功能。这与先前相关文献报道的体外细胞培养以及胆道结扎的胆汁淤积动物模型的实验结果相一致[5,6,7,8,9]。而核受体RXRα﹑RARα不论是在mRNA水平还是蛋白水平均显著增高,这与文献报道的体外细胞培养以及胆道结扎的胆汁淤积动物模型的实验结果结果相反[9]。尤其值得注意的是,核受体蛋白RARα在胞浆内的表达增高约13倍,而在核内增高约6倍,这与RXRα﹑CPF在核内以及胞浆的表达增高的情况相反。这可能提示RARα进入细胞核后,在某些因素的作用下,引起它由细胞核内向胞浆转移的核外转移现象,这需要进一步的研究证实。以上实验结果充分说明了人肝脏MRP3调节的分子机制与胆道结扎动物模型或体外细胞培养实验所建立的调节机制存在某些差异。另外,核受体蛋白RXRα﹑RARα﹑CPF在胞浆与核内的分布可能还存在差异,这可能提示其他的调节机制也参与了MRP3基因转录表达。
结论
通过本实验研究,我们证实了人肝脏阻塞性胆汁淤积时核受体CPF对MRP3基因转录表达具有上调的功能。核受体RXRα、RARα在胆汁淤积时表达显著增高,这与相关文献报道的胆道结扎动物模型或体外细胞培养的实验结果不同,这可能提示其他的调节机制也参与了MRP3基因的转录表达,有待进一步深入研究。
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.
Result
1. 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. Disscussion
MRP3 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.
Conclusion
Our 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.
多耐药相关蛋白3(multidrug resistance-assocated protein 3,MRP3)是近年新克隆的一种转运蛋白基因,主要分布于肠道、肾脏及肾上腺,而在正常肝细胞基底侧膜上表达很低或不表达[5]。但是在胆道结扎所致的急性胆汁淤积大鼠模型的肝脏中MRP3的表达量却非常高[6]。同时进一步研究发现在人类胆汁淤积性疾病(如:胆道梗阻、原发性毛细胆管性肝硬化、Dubin-Johnson综合征)时,MRP3在肝细胞基底侧膜表达量也大为增加,而同时肝细胞小管面的MRP2的表达量却急剧下降[7]。随着对MRP3转运功能研究进一步的深入,发现其转运底物与MRP2具有广泛的同源性,都能转运大部分胆酸,尤其是结合型胆酸,因此,MRP3的高表达被认为是代偿性的,以补偿小管面膜上MRP2的下调。所以,人们普遍认为MRP3的表达增高是在胆汁淤积状态下肝细胞的一种代偿性自我保护反应[6,8]。因此,随着对MRP3调节的分子机制的进一步研究深入,有可能在不久的将来开发出治疗胆汁淤积疾病的特效药物,并且对临床寻找肝胆汁淤积治疗对策也具重要的临床价值。
当胆汁淤积发生时,肝脏MRP3基因mRNA转录表达与核受体CPF﹑RXRα﹑RARα﹑Sp1﹑PXR等基因相关。已经有文献报道,在体外细胞培养与胆道结扎的动物模型实验研究表明,核受体CPF/Lrh-1能够上调MRP3/Mrp3 mRNA的表达[6];RXRα:RARα异二聚体则能够抑制MRP3的表达[9]。Sp1,PXR等也对MRP3的基因表达起一定的调节作用[10],但是其具体机制尚不清楚。而且目前的实验研究主要集中在体外细胞培养和动物模型实验,直接针对以“人”为研究对象的实验鲜有报道,人体组织的研究结果与体外细胞培养或动物实验常存在差异,如:用内毒素处理的大鼠Mrp2 mRNA的表达水平急剧下降,而当以“人”为研究对象时MRP2 mRNA的表达水平却维持不变,但是二者Mrp2/MRP2蛋白水平的变化却均急剧降低[11]。本实验通过收集40例人体肝组织标本,对阻塞性胆汁淤积时MRP3上调相关的基因进行研究,从而进一步探讨与研究阻塞性胆汁淤积时人肝细胞膜转运蛋白MRP3的转录调控机制。
方法
通过对收集的40例标本分组(对照组:正常肝组织;淤胆组:淤胆肝组织)后,分别在mRNA水平(RT-PCR)与蛋白水平(Western blot)检测分析膜转运蛋白MRP3表达变化,以及与其调节相关核受体基因CPF﹑RXRα与RARα表达变化。通过免疫荧光检测膜转运蛋白MRP3与核受体CPF在正常肝与淤胆肝组织的表达以及定位情况。通过EMSA来检测MRP3基因启动子区反应元件与核受体CPF结合活性在正常肝与淤胆肝组织的变化。
结果
1.阻塞性胆汁淤积时,通过RT-PCR实验研究对膜转运蛋白基因MRP3与核受体基因CPF﹑RXRα﹑RARα等在mRNA水平表达进行分析,结果表明:MRP3以及上述基因在mRNA水平表达均显著增高,增高约3~4倍左右。
2.免疫荧光检测结果表明,阻塞性胆汁淤积时,膜转运蛋白MRP3在肝细胞基底侧膜的表达明显增加。而CPF的表达,在核内与胞浆内均有增加。
3.人肝脏阻塞性胆汁淤积时,通过Western blot实验对膜转运蛋白基因MRP3与核受体基因CPF﹑RXRα﹑RARα等在蛋白水平表达进行分析,结果表明: MRP3蛋白水平表达增高约4倍。CPF﹑RXRα﹑RARα在核内表达增高约6~8倍;而在胞浆内,RARα显著增高约13倍;RXRα增高约2.5倍,CPF增高约4倍。
4.人肝脏阻塞性胆汁淤积时,通过EMSA检测结果表明:MRP3基因启动子区反应元件与核受体CPF结合活性显著增加,约3.5倍。
讨论
MRP3(ABCC3)在正常肝细胞膜低表达或不表达。而当胆汁淤积发生时,肝细胞基底侧膜的MRP3表达却显著增高。同时,又由于MRP3转运底物与MRP2有着广泛的同源性,所以人们普遍认为MRP3的高表达是对MRP2的一种代偿,以减轻胆汁淤积时肝细胞受胆酸毒性损伤的程度,具有护肝的作用。因此,对MRP3表达调节的分子机制的研究,对寻找胆汁淤积治疗对策有着非常重要的临床意义。
本实验通过对人体肝组织标本研究分析得知,阻塞性胆汁淤积时人肝细胞膜转运蛋白MRP3在肝细胞基底侧膜的表达量显著增高。MRP3基因启动子区反应元件与核受体CPF结合活性显著增高,这些充分证明核受体CPF能够上调人肝细胞膜转运蛋白MRP3基因的转录表达功能。这与先前相关文献报道的体外细胞培养以及胆道结扎的胆汁淤积动物模型的实验结果相一致[5,6,7,8,9]。而核受体RXRα﹑RARα不论是在mRNA水平还是蛋白水平均显著增高,这与文献报道的体外细胞培养以及胆道结扎的胆汁淤积动物模型的实验结果结果相反[9]。尤其值得注意的是,核受体蛋白RARα在胞浆内的表达增高约13倍,而在核内增高约6倍,这与RXRα﹑CPF在核内以及胞浆的表达增高的情况相反。这可能提示RARα进入细胞核后,在某些因素的作用下,引起它由细胞核内向胞浆转移的核外转移现象,这需要进一步的研究证实。以上实验结果充分说明了人肝脏MRP3调节的分子机制与胆道结扎动物模型或体外细胞培养实验所建立的调节机制存在某些差异。另外,核受体蛋白RXRα﹑RARα﹑CPF在胞浆与核内的分布可能还存在差异,这可能提示其他的调节机制也参与了MRP3基因转录表达。
结论
通过本实验研究,我们证实了人肝脏阻塞性胆汁淤积时核受体CPF对MRP3基因转录表达具有上调的功能。核受体RXRα、RARα在胆汁淤积时表达显著增高,这与相关文献报道的胆道结扎动物模型或体外细胞培养的实验结果不同,这可能提示其他的调节机制也参与了MRP3基因的转录表达,有待进一步深入研究。
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.
Result
1. 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. Disscussion
MRP3 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.
Conclusion
Our 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.
引文
1. Kullak-Ublick GA, Stieger B, Meier PJ.Enterohepatic bile salt transporters in normal physiology and liver disease. Gastroenterology. 2004 Jan;126(1):322-42. Review.
2. Pauli-Magnus C, Meier PJ. Hepatocellular transporters and cholestasis. J Clin Gastroenterol. 2005 Apr;39(4):S103-10.
3. Meier PJ, Stieger B.Bile salt transporters.Annu Rev Physiol. 64:635-61. Review(2002).
4. Piet Borst, Raymond Evers, Marcel Kool, Jan Wijinholds. A family of drug transporter:the multidrug resistance-associate proteins. Journal of the National Cancer Insitute.2000 Aug;92(16):1295-1302.Review.
5. Kiuchi Y, Suzuki H, Hirohashi T, et al. Enhanced expression of the human multidrug resisitance protein 3 by bile salt in human enterocytes.A transcriptional control of a plausible bile acid transporter.J Biol Chem276:46822-46829,2001.
6. Bohan A, Chen WS, Denson LA, Held MA, Boyer JL. Tumor necrosis factor alpha-dependent up-regulation of Lrh-1 and Mrp3(Abcc3) reducees liver injury in obstructive cholestasis. J Biol Chem. 2003 Sep 19;278(38):36688-98.
7. Kullak-ublick G A, Stieger B, Meier P J. Enterohepatic bile salt transporters in normal physiology and liver disease [J]. Gastro enterology, 2004, 126 (1): 322-342.
8. Alan Bohan, Wen-Sheng Chen, Lee A. Denson, et al. MRP3/Mrp3 (ABCC3/Abcc3) is up-regulated by TNF via cholesterol-7-hydroxylase promoter factor (CPF) and is hepatoprotective in obstructive cholestasis. Hepatology 2003; 38(4) Suppl. 1:168A
9. Wensheng Chen,Shi-Ying Cai,Shuhua Xu,et al.Nuclear receptors RXRα: RARαare repressors for human MRP3 expression.Am J physiol Gastrointest Liver Physiol ,2007, 292:G1221-G1227.
10. Micheline Piquette-Miller.Induction of ABCC3(MRP3) by pregnane X receptor active- tors.Drug metabolism and disposition,2003,31(11):1296-1299.
11. M. Trauner,M. Arrese, C.J. Soroka,et al.The rat canalicular conjugate export pump (Mrp2) is down-regulated in intrahepatic and obstructive cholestasis. Gastroenterology 1997,113:255-264.
12. Satyanarayana R. Pondugula, Cynthia Brimer-Cline,et al. A Phosphomimetic Mutation at Threonine-57 Abolishes Transactivation Activity and Alters Nuclear LocalizationPattern of Human Pregnane X Receptor.Drug metabolism and disposition 2009,37:719-730
13. Zhengliang Wu, John Y.L. Chiang.Transcriptional regulation of human oxysterol 7a-hydroxylase gene(CYP7B1) by Sp1.Gene,2001,272:191-197.
14. Trauner M, Arrese M, Lee H, Boyer JL, Karpen SJ. Endotoxin downregulates rat hepatic Ntcp gene expression via decreased activity of critical transcription factors. J Clin Invest 1998;101:2092–2100.
15. Gerald U.Denk,Carol J.Soroka,Yasuaki Takeyama,et al.Multidrug resistance-associated protein 4 is up-regulated in liver but down-regulated in kidney in obstructive cholestasis in the rat. Journal of Hepatology,2004,40:585-591.
16. Lee J, Azzaroli F, Wang L, Soroka CJ, Gigliozzi A, Setchell KD, et al.Adaptive regulation of bile salt transporters in kidney and liver in obstructive cholestasis in the rat. Gastroenterology 2001;121: 1473–1484.
1. Borst P. Multidrug resistant proteins. Semin Cancer Biol 1997;8:131–4.
2. Kuchler K, Sarkadi B, Szakacs G, editors. Structure and function of ABC transporters.Bio chim Biophys Acta 1999;1461:special issue.
3. Piet Borst, Raymond Evers, Marcel Kool, Jan Wijinholds. A family of drug transporter:the multidrug resistance-associate proteins. Journal of the National Cancer Insitute.2000 Aug;92(16):1295-1302.Review.
4. Kiuchi Y, Suzuki H, Hirohashi T, et al. Enhanced expression of the human multidrug resisitance protein 3 by bile salt in human enterocytes.A transcriptional control of a plausible bile acid transporter.J Biol Chem276:46822-46829,2001.
5. Bohan A, Chen WS, Denson LA, Held MA, Boyer JL. Tumor necrosis factor alpha-dependent up-regulation of Lrh-1 and Mrp3(Abcc3) reducees liver injury in obstructive cholestasis. J Biol Chem. 2003 Sep 19;278(38):36688-98.
6. Kullak-ublick G A, Stieger B, Meier P J. Enterohepatic bile salt transporters in normal physiology and liver disease [J]. Gastro enterology, 2004, 126 (1): 322-342.
7. Takeshi Uchiumi,Eiji Hinoshita,Sei Haga,et al. Isolation of a Novel Human Canalicular Multispecific Organic Anion Transporter, cMOAT2/MRP3, and Its Expression in Cisplatin-Resistant Cancer Cells with Decreased ATP-Dependent Drug Transport. Bioch-emical and Biophysical Research communications,1998,252:103-110.
8. Jorg Konig, Daniel Rost, YunHai Cui,et al. Characterization of the Human Multidrug Resistance Protein Isoform MRP3 Localized to the Basolateral HepatocyteMembrane.Hepatology,1999,29(4):1156-1163
9. Yu-Jui Yvonne Wan,Dahsing An,Yan Cai,et.al. Hepatocyte-Specific Mutation Establishes Retinoid X Receptorαas a Heterodimeric Integrator of Multiple Physiological Processes in the Liver. Molecular and Cellular Biology,2000, 20(12):4436-4444
10. Carmen Stanca, Diana Jung, Peter J. Meier et al. Hepatocellular transport proteins and their role in liver disease. World J Gastroenterol 2001, 7(2):157-169.
11. Shinichiro Takayanagi,Toshihisa Ishikawa. Molecular identification and characterization of rat Abcc1 cDNA: existence of two splicing variants and species difference in drug resistance profile.Journal of Experimental Therapeutics and Oncology,2003,3(3):136-146
12. Scott S. Auerbach, Richard Ramsden,et al. Alternatively spliced isoforms of the humanconstitutive androstane receptor. Nucleic Acids Res. 2003 June 15; 31(12): 3194–3207.
13. Hirohashi T, Suzuki H, Ito K, Ogawa K,et al.Hepatic expression of multidrug resistance-associated protein-like proteins maintained in Eisai hyperbilirubinemic rats. Mol Pharm-acol,1998,53:1068-1075.
14. Liying Cui, Luba Aleksandrov, Xiu-Bao Chang,et al.Domain Interdependence in the Biosynthetic Assembly of CFTR. J. Mol. Biol. (2007) 365, 981–994
15. Smith, P. C., Karpowich, N., Millen, L., Moody, J. E.,Rosen, J., Thomas, P. J. & Hunt, J. F. (2002). ATP binding to the motor domain from an ABC transporter drives formation of a nucleotide sandwich dimer. Mol.Cell, 10, 139–149.
16. Moody, J. E., Millen, L., Binns, D., Hunt, J. F. & Thomas, P. J. (2002). Cooperative, ATP-dependent association of the nucleotide binding cassettes during the catalytic cycle of ATP-binding cassette transporters.J. Biol. Chem. 277, 21111–21114.
17. Persson B, Argos P. Prediction of transmembrane segments in proteins utilising multiple sequence alignments. J Mol Biol 1994;237:182-192.
18. Kamata S, Kishimoto T, Kobayashi S & Miyazaki M. Expression and localization of ATP binding cassette (ABC) family of drug transporters in gastric hepatoid adenocarcin -omas. Histopathology ,2008,52, 747–754.
19. Flens MJ, Zaman GJ, van der Valk P et al. Tissue distribution of the multidrug resistance protein. Am. J. Pathol. 1996; 148:1237–1247.
20. Dean M, Rzhetsky A, Allikmets R. The human ATP-binding cassette (ABC) transportersuperfamily. Genome Res, 2001, 11:1156–1166.
21. Kruh GD, Zeng H, Rea PA et al. MRP subfamily transporters and resistance to anticanc- er agents. J. Bioenerg. Biomembr,2001,33:493–501.
22. M. Trauner, J.L. Boyer, Bile salt transporters: molecular characterization,function, and regulation, Physiol. Rev. 83 (2003) 633–671.
23. M. Trauner, P.J. Meier, J.L. Boyer, Molecular pathogenesis of cholestasis,N. Engl. J. Med. 339 (1998) 1217–1227.
24. M. Arrese, M. Trauner, Molecular aspects of bile formation and cholestasis,Trends Mol. Med. 9 (2003) 558–564.
25. G.A. Kullak-Ublick, B. Stieger, P.J. Meier, Enterohepatic bile salt transporters in normal physiology and liver disease, Gastroenterology 126(2004) 322–342.
26. M.S. Anwer, Cellular regulation of hepatic bile acid transport in healthand cholestasis, Hepatology 39 (2004) 581–590.
2. Pauli-Magnus C, Meier PJ. Hepatocellular transporters and cholestasis. J Clin Gastroenterol. 2005 Apr;39(4):S103-10.
3. Meier PJ, Stieger B.Bile salt transporters.Annu Rev Physiol. 64:635-61. Review(2002).
4. Piet Borst, Raymond Evers, Marcel Kool, Jan Wijinholds. A family of drug transporter:the multidrug resistance-associate proteins. Journal of the National Cancer Insitute.2000 Aug;92(16):1295-1302.Review.
5. Kiuchi Y, Suzuki H, Hirohashi T, et al. Enhanced expression of the human multidrug resisitance protein 3 by bile salt in human enterocytes.A transcriptional control of a plausible bile acid transporter.J Biol Chem276:46822-46829,2001.
6. Bohan A, Chen WS, Denson LA, Held MA, Boyer JL. Tumor necrosis factor alpha-dependent up-regulation of Lrh-1 and Mrp3(Abcc3) reducees liver injury in obstructive cholestasis. J Biol Chem. 2003 Sep 19;278(38):36688-98.
7. Kullak-ublick G A, Stieger B, Meier P J. Enterohepatic bile salt transporters in normal physiology and liver disease [J]. Gastro enterology, 2004, 126 (1): 322-342.
8. Alan Bohan, Wen-Sheng Chen, Lee A. Denson, et al. MRP3/Mrp3 (ABCC3/Abcc3) is up-regulated by TNF via cholesterol-7-hydroxylase promoter factor (CPF) and is hepatoprotective in obstructive cholestasis. Hepatology 2003; 38(4) Suppl. 1:168A
9. Wensheng Chen,Shi-Ying Cai,Shuhua Xu,et al.Nuclear receptors RXRα: RARαare repressors for human MRP3 expression.Am J physiol Gastrointest Liver Physiol ,2007, 292:G1221-G1227.
10. Micheline Piquette-Miller.Induction of ABCC3(MRP3) by pregnane X receptor active- tors.Drug metabolism and disposition,2003,31(11):1296-1299.
11. M. Trauner,M. Arrese, C.J. Soroka,et al.The rat canalicular conjugate export pump (Mrp2) is down-regulated in intrahepatic and obstructive cholestasis. Gastroenterology 1997,113:255-264.
12. Satyanarayana R. Pondugula, Cynthia Brimer-Cline,et al. A Phosphomimetic Mutation at Threonine-57 Abolishes Transactivation Activity and Alters Nuclear LocalizationPattern of Human Pregnane X Receptor.Drug metabolism and disposition 2009,37:719-730
13. Zhengliang Wu, John Y.L. Chiang.Transcriptional regulation of human oxysterol 7a-hydroxylase gene(CYP7B1) by Sp1.Gene,2001,272:191-197.
14. Trauner M, Arrese M, Lee H, Boyer JL, Karpen SJ. Endotoxin downregulates rat hepatic Ntcp gene expression via decreased activity of critical transcription factors. J Clin Invest 1998;101:2092–2100.
15. Gerald U.Denk,Carol J.Soroka,Yasuaki Takeyama,et al.Multidrug resistance-associated protein 4 is up-regulated in liver but down-regulated in kidney in obstructive cholestasis in the rat. Journal of Hepatology,2004,40:585-591.
16. Lee J, Azzaroli F, Wang L, Soroka CJ, Gigliozzi A, Setchell KD, et al.Adaptive regulation of bile salt transporters in kidney and liver in obstructive cholestasis in the rat. Gastroenterology 2001;121: 1473–1484.
1. Borst P. Multidrug resistant proteins. Semin Cancer Biol 1997;8:131–4.
2. Kuchler K, Sarkadi B, Szakacs G, editors. Structure and function of ABC transporters.Bio chim Biophys Acta 1999;1461:special issue.
3. Piet Borst, Raymond Evers, Marcel Kool, Jan Wijinholds. A family of drug transporter:the multidrug resistance-associate proteins. Journal of the National Cancer Insitute.2000 Aug;92(16):1295-1302.Review.
4. Kiuchi Y, Suzuki H, Hirohashi T, et al. Enhanced expression of the human multidrug resisitance protein 3 by bile salt in human enterocytes.A transcriptional control of a plausible bile acid transporter.J Biol Chem276:46822-46829,2001.
5. Bohan A, Chen WS, Denson LA, Held MA, Boyer JL. Tumor necrosis factor alpha-dependent up-regulation of Lrh-1 and Mrp3(Abcc3) reducees liver injury in obstructive cholestasis. J Biol Chem. 2003 Sep 19;278(38):36688-98.
6. Kullak-ublick G A, Stieger B, Meier P J. Enterohepatic bile salt transporters in normal physiology and liver disease [J]. Gastro enterology, 2004, 126 (1): 322-342.
7. Takeshi Uchiumi,Eiji Hinoshita,Sei Haga,et al. Isolation of a Novel Human Canalicular Multispecific Organic Anion Transporter, cMOAT2/MRP3, and Its Expression in Cisplatin-Resistant Cancer Cells with Decreased ATP-Dependent Drug Transport. Bioch-emical and Biophysical Research communications,1998,252:103-110.
8. Jorg Konig, Daniel Rost, YunHai Cui,et al. Characterization of the Human Multidrug Resistance Protein Isoform MRP3 Localized to the Basolateral HepatocyteMembrane.Hepatology,1999,29(4):1156-1163
9. Yu-Jui Yvonne Wan,Dahsing An,Yan Cai,et.al. Hepatocyte-Specific Mutation Establishes Retinoid X Receptorαas a Heterodimeric Integrator of Multiple Physiological Processes in the Liver. Molecular and Cellular Biology,2000, 20(12):4436-4444
10. Carmen Stanca, Diana Jung, Peter J. Meier et al. Hepatocellular transport proteins and their role in liver disease. World J Gastroenterol 2001, 7(2):157-169.
11. Shinichiro Takayanagi,Toshihisa Ishikawa. Molecular identification and characterization of rat Abcc1 cDNA: existence of two splicing variants and species difference in drug resistance profile.Journal of Experimental Therapeutics and Oncology,2003,3(3):136-146
12. Scott S. Auerbach, Richard Ramsden,et al. Alternatively spliced isoforms of the humanconstitutive androstane receptor. Nucleic Acids Res. 2003 June 15; 31(12): 3194–3207.
13. Hirohashi T, Suzuki H, Ito K, Ogawa K,et al.Hepatic expression of multidrug resistance-associated protein-like proteins maintained in Eisai hyperbilirubinemic rats. Mol Pharm-acol,1998,53:1068-1075.
14. Liying Cui, Luba Aleksandrov, Xiu-Bao Chang,et al.Domain Interdependence in the Biosynthetic Assembly of CFTR. J. Mol. Biol. (2007) 365, 981–994
15. Smith, P. C., Karpowich, N., Millen, L., Moody, J. E.,Rosen, J., Thomas, P. J. & Hunt, J. F. (2002). ATP binding to the motor domain from an ABC transporter drives formation of a nucleotide sandwich dimer. Mol.Cell, 10, 139–149.
16. Moody, J. E., Millen, L., Binns, D., Hunt, J. F. & Thomas, P. J. (2002). Cooperative, ATP-dependent association of the nucleotide binding cassettes during the catalytic cycle of ATP-binding cassette transporters.J. Biol. Chem. 277, 21111–21114.
17. Persson B, Argos P. Prediction of transmembrane segments in proteins utilising multiple sequence alignments. J Mol Biol 1994;237:182-192.
18. Kamata S, Kishimoto T, Kobayashi S & Miyazaki M. Expression and localization of ATP binding cassette (ABC) family of drug transporters in gastric hepatoid adenocarcin -omas. Histopathology ,2008,52, 747–754.
19. Flens MJ, Zaman GJ, van der Valk P et al. Tissue distribution of the multidrug resistance protein. Am. J. Pathol. 1996; 148:1237–1247.
20. Dean M, Rzhetsky A, Allikmets R. The human ATP-binding cassette (ABC) transportersuperfamily. Genome Res, 2001, 11:1156–1166.
21. Kruh GD, Zeng H, Rea PA et al. MRP subfamily transporters and resistance to anticanc- er agents. J. Bioenerg. Biomembr,2001,33:493–501.
22. M. Trauner, J.L. Boyer, Bile salt transporters: molecular characterization,function, and regulation, Physiol. Rev. 83 (2003) 633–671.
23. M. Trauner, P.J. Meier, J.L. Boyer, Molecular pathogenesis of cholestasis,N. Engl. J. Med. 339 (1998) 1217–1227.
24. M. Arrese, M. Trauner, Molecular aspects of bile formation and cholestasis,Trends Mol. Med. 9 (2003) 558–564.
25. G.A. Kullak-Ublick, B. Stieger, P.J. Meier, Enterohepatic bile salt transporters in normal physiology and liver disease, Gastroenterology 126(2004) 322–342.
26. M.S. Anwer, Cellular regulation of hepatic bile acid transport in healthand cholestasis, Hepatology 39 (2004) 581–590.