Regulation of cholangiocytes by InsP3 receptor isoforms

描述（申请人提供）：胆汁分泌是肝脏的主要功能之一。为了维持胆汁流动，不仅肝细胞必须分泌胆汁，而且还必须由胆管上皮细胞或胆管细胞进一步修饰和调节。胆管细胞功能异常导致胆汁淤积，这是肝脏疾病的主要表现。胆汁淤积性肝病占美国肝脏移植的20%，是儿童移植患者中最常见的肝病原因。胆管细胞的胆汁分泌部分受胞质Ca2+ (Ca2+)调节，而肌醇1,4,5-三磷酸受体（InsP3R）的功能是这些Ca2+信号调节的重要组成部分。在胆管细胞中发现了第二种细胞内蛋白polycytin -2 (PC2)，但对其在这些细胞中Ca2+信号传导中的作用知之甚少。PC2突变导致多囊肾病（PKD），多囊肾病与大的充满液体的囊肿有关。身体的大多数细胞都表达PC2以及InsP3R的所有三种同工异构体，尽管是在细胞特异性比例上。在三种InsP3R亚型中，III型是主要在肾脏和胆管中表达的亚型，而胆管是PKD中囊肿形成的主要部位。在本提案中，我们将探讨InsP3R和PC2之间相互作用的功能效应。我们假设PC2优先与InsP3R的III型异构体相互作用，使得具有这种异构体的细胞，如肾细胞和胆管细胞，在PC2突变时比具有InsP3R I型和II型的细胞（如肝细胞）更容易形成囊肿。我们还假设，与PC2结合的InsP3R比未结合的受体具有更低的活性，这种相互作用可以被调节，并且这种相互作用将转化为完整细胞中的改变反应。这一假设将通过以下具体目的进行调查：1。以了解PC2和InsP3R之间的分子相互作用。具体来说，我们将监测InsP3R的哪些同工异构体与PC2相互作用，PC2的哪个片段影响InsP3R的功能，以及这些相互作用如何被PC2突变修饰。我们将测试PC2和特定致病突变对InsP3R型通道特性的影响，使用脂质双分子层技术在分子水平上研究这些相互作用。2. 了解PC2和InsP3R相互作用对细胞的影响。我们将监测这些受体在胆管细胞Ca2+信号传导中的作用，以及这些相互作用如何导致下游信号传导。一种胆管细胞细胞系MzCha1细胞将被改造以表达每种InsP3R亚型和PC2。我们将在全细胞水平上使用实时Ca2+成像来测试Ca2+信号的异构体特异性。3. 了解PC2-InsP3R相互作用对天然胆管细胞Ca2+信号组织和分泌功能的影响，在分离的微灌注胆管段中确定。我们将监测用siRNA处理过的InsP3R异构体或PC2的导管以及表达与InsP3R相互作用的PC2片段的导管中碳酸氢盐分泌的变化。这项工作应该确定负责胆管细胞Ca2+信号的分子机制，这将有助于我们了解胆汁淤积的分子基础。更一般地说，这些研究将概述PC2在其他分泌上皮的信号转导和刺激-分泌偶联中的作用，并可能有助于解释为什么PC2突变对表达该蛋白的各种组织有不同的影响。公共卫生相关性：该提案将确定胆管细胞内的信号如何调节胆汁分泌。这一信息对于理解胆汁淤积的基础非常重要，胆汁淤积是肝脏疾病中胆汁分泌受损的常见表现。此外，这项工作也有可能为其他类型上皮组织的分泌调节建立一个新的范例。

DESCRIPTION (provided by applicant): Bile secretion is one of the principal functions of the liver. In order to maintain bile flow, not only must hepatocytes secrete bile, but this must then be modified and conditioned further by bile duct epithelial cells, or cholangiocytes. Abnormal cholangiocyte function results in cholestasis, which is a cardinal manifestation of liver disease. Cholestatic liver diseases are responsible for 20% of liver transplants in the US, and are the most common cause of liver disease among pediatric transplant patients. Bile secretion in cholangiocytes is regulated in part by cytosolic Ca2+ (Ca2+) and the function of inositol 1,4,5- trisphosphate receptors (InsP3R) is an important component of how these Ca2+ signals are regulated. A second intracellular protein polycystin-2 (PC2) is found in cholangiocytes but little is known about its role in Ca2+ signaling in these cells. Mutations in PC2 leads to polycystic kidney disease (PKD) which is associated with large fluid filled cysts. Most cells of the body express PC2 as well as all three isoforms of the InsP3R, albeit in cell-specific ratios. Of the three InsP3R isoforms, the type III is the predominant isoform expressed in kidney and in bile ducts, the primary sites for cyst formation in PKD. In this proposal we will explore the functional effects of the interaction between the InsP3R and PC2. We hypothesize that PC2 preferentially interacts with the type III isoform of the InsP3R, making cells with this isoform, such as kidney cells and cholangiocytes, more likely to form cysts when PC2 is mutated than are cells with InsP3R types I and II, exemplified by hepatocytes. We also hypothesize that the InsP3R bound by PC2 will have lower activity than the unbound receptor, that this interaction can be regulated, and that the interactions will translate to altered responses in intact cells. This hypothesis will be investigated through the following specific aims: 1. to understand the molecular interactions between PC2 and the InsP3R. Specifically, we will monitor which isoforms of the InsP3R interact with PC2, which segment of PC2 affects InsP3R function, and how these interactions are modified by mutations in PC2. We will test the effect of PC2 and specific pathogenic mutations on the channel properties of the InsP3R type using lipid bilayer techniques to study these interactions at a molecular level. 2. To understand the cellular consequences of interactions between PC2 and the InsP3R. We will monitor the contribution that each of these receptors plays in Ca2+ signaling in cholangiocytes and how these interactions lead to downstream signaling. A cholangiocyte cell line, MzCha1 cells, will be engineered to express each of the InsP3R isoforms and PC2. We will test the isoform specificity of the Ca2+ signals using real time Ca2+ imaging at the whole cell level. 3. To understand the effect of PC2-InsP3R interactions in the organization of Ca2+ signals and secretory function in native cholangiocytes, as determined in isolated microperfused bile duct segments. We will monitor alterations in bicarbonate secretion in ducts treated with siRNA for InsP3R isoforms or PC2, and in ducts expressing fragments of PC2 that interact with the InsP3R. This work should identify the molecular mechanisms responsible for Ca2+ signaling in cholangiocytes, which will help us to understand the molecular basis for cholestasis. More generally these studies will outline the role of PC2 in signal transduction and stimulus-secretion coupling in other secretory epithelia as well and may help explain why PC2 mutations differentially affect the various tissues that express this protein. PUBLIC HEALTH RELEVANCE: This proposal would determine how signaling inside bile duct cells regulates bile secretion. This information is important for understanding the basis for cholestasis, a common manifestation of liver disease in which bile secretion is impaired. Moreover, this work has the potential to establish a novel paradigm for regulation of secretion in other types of epithelial tissue as well.