Mechanisms of Bile Secretion and Cholestasis

说明(申请人提供)：胆汁分泌是肝脏的一项主要功能，在肝脏疾病中经常受到损害，导致胆汁淤积综合征。这项自1973年以来一直资助的赠款申请的长期目标一直是确定决定胆汁分泌的肝细胞基本运输机制的特征，并在细胞和分子水平上确定导致胆汁淤积性肝病的这些机制的变化。在这项竞争性更新提案中，具体目标是#1.了解人肝细胞基底膜转运体(特别是MRP4和OST1-OST2)适应性调节的分子机制，这些是淤胆性肝损伤适应性反应的重要决定因素，并基于这些信息设计新的治疗方法。特别是，我们将试图表征人类MRP4启动子的转录调控因子，并继续评估OST2在人类异构体促进胆盐转运体OST1-OST2的适应性调节中的作用，并确定Ost1基因敲除小鼠胆管结扎的效果。为了探索新的治疗方法，我们将评估全反式维甲酸+熊去氧胆酸对人转运蛋白基因在HepG2细胞和胆汁淤积动物模型中表达的协同治疗效果。在目标2中，我们将通过关注泛素/蛋白酶体降解途径作为调节胆盐输出泵(BSEP)表面表达的决定因素，来研究在正常和胆汁淤积性肝损伤中调节BSEP表达的决定因素。最后，在目标3中，我们将研究与维持小管顶膜结构极性有关的蛋白质的功能作用。为此，我们将确定支架蛋白在ABC转运蛋白MRP2的靶向和调控中的作用。我们还将通过使用siRNA敲除Fic1来评估FIC1在人类细胞系和大鼠肝细胞中调节人FXR表达和活性的作用，以及确定Fic1在维持大鼠肝细胞胶原蛋白夹心培养中的小管结构和BSEP和MRP2功能中的作用。公共卫生相关叙述：这项赠款申请支持的研究重点是了解肝脏如何适应胆汁产生障碍(称为胆汁淤积症)造成的损伤。胆汁形成是一种重要的功能，在各种胆汁淤积性肝病中，胆汁形成的损害通常会导致进行性胆汁淤积和肝功能衰竭，从而导致死亡或肝移植。通过在细胞和分子水平上了解胆汁转运蛋白在胆汁淤积性肝损伤中的适应性机制，我们希望设计新的治疗策略来增强这些适应性反应，并延缓或逆转这些潜在的致命肝病的进展。

DESCRIPTION (provided by applicant): Bile secretion is a major function of the liver which is frequently impaired in diseases of the liver resulting in the syndrome of cholestasis. The long term objectives of this grant application, funded continuously since 1973, have been to characterize the basic transport mechanisms in hepatocytes that determine the secretion of bile and to define at the cellular and molecular level, alterations in these mechanisms that result in cholestatic liver disease. In this competitive renewal proposal the specific aims are #1. To understand the molecular mechanisms for adaptive regulation of basolateral hepatocyte membrane transporters (specifically MRP4 and OST1-OST2) in human liver that are important determinants of the adaptive response in cholestatic liver injury and to devise new therapies based on this information. In particular we will attempt to characterize the transcriptional regulators of the human MRP4 promoter as well as to continue to assess the role of OST2 in the adaptive regulation of the human heteromeric, facilitated bile salt transporter, OST1-OST2, and to determine the effect of bile duct ligation in the Ost1 knock out mice. In order to examine new therapeutic approaches we will assess the synergistic therapeutic effects of the combination of all-trans retinoic acid + ursodeoxycholic acid on human transporter gene expression in HepG2 cells and in animal models of cholestasis. In Aim # 2. we will examine the determinants by which the expression of the bile salt export pump (Bsep) is regulated in normal and cholestatic liver injury by focusing on the role of the ubiquitin/proteasome degradation pathway as a determinant of the regulation of surface expression of Bsep. Finally in Aim # 3. we will investigate the functional roles of proteins involved in maintaining canalicular apical membrane structural polarity. To do this we will determine the role of scaffolding proteins in the targeting and regulation of the ABC transporter, Mrp2. We will also assess the role of FIC1 in the regulation of human FXR expression and activity in human cell lines and rat hepatocytes by knocking down Fic1 using siRNA, as well as determining the role of Fic1 in maintaining canalicular structure and Bsep and Mrp2 function in rat collagen sandwich hepatocyte cultures. Public Health Relevance Narrative: This grant application supports research that focuses on understanding how the liver adapts to injury resulting from impairment of bile production (known as cholestasis). Bile formation is a vital function and its impairment in a variety of cholestatic liver diseases often results in progressive cholestasis and liver failure that can result in death or liver transplantation. By understanding the mechanism of adaptations of bile transporter proteins at the cellular and molecular level in cholestatic liver injury, we hope to design new therapeutic strategies that will augment these adaptive responses and retard or reverse the progression of these potentially fatal liver disorders.