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，肝细胞不仅必须分泌胆汁，而且还必须由胆管上皮细胞或胆管细胞进一步修改和调节胆汁。胆管细胞功能异常导致胆汁淤积，是肝病的主要表现。胆汁淤积性肝病是美国20%的肝移植手术的原因，也是儿童肝移植患者中最常见的肝病原因。胆管细胞的胆汁分泌在一定程度上受细胞内钙离子(Ca~(2+))的调节，而1，4，5-三磷酸肌醇受体(InsP3R)的功能是这些钙信号调节的重要组成部分。另一种细胞内蛋白多囊蛋白-2(PC2)在胆管细胞中被发现，但对其在这些细胞中的钙信号转导中的作用知之甚少。PC2基因的突变会导致多囊肾病(PKD)，这种疾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的所有三种亚型，尽管是以细胞特有的比例表达。在三种InsP3R亚型中，III型是在肾脏和胆管中主要表达的亚型，而肾和胆管是PKD中形成囊变的主要部位。在这个提案中，我们将探索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型亚型相互作用，使具有这种异构体的细胞，如肾细胞和胆管细胞，在PC2突变时更有可能形成包囊，而不是带有InsP3RI型和II型的细胞，以肝细胞为例。我们还假设，与PC2结合的InsP3R的活性将低于未结合的受体，这种相互作用是可以调节的，并且这种相互作用将转化为完整细胞中的改变反应。这一假说将通过以下具体目的进行研究：1.了解PC2和InsP3R之间的分子相互作用。具体地说，我们将监测InsP3R的哪些亚型与PC2相互作用，PC2的哪个片段影响InsP3R的功能，以及这些相互作用是如何被PC2的突变所改变的。我们将使用脂质双层技术来测试PC2和特定的致病突变对InsP3R型通道特性的影响，以在分子水平上研究这些相互作用。2.了解PC2和InsP3R相互作用的细胞后果。我们将监测这些受体在胆管细胞内钙信号传导中的作用，以及这些相互作用如何导致下行信号传导。一种胆管细胞系MzCha1细胞将被改造成表达每一种InsP3R亚型和PC2。我们将在整个细胞水平上使用实时钙成像来测试钙信号的异构体特异性。3.了解PC2-InsP3R相互作用对离体胆管微灌流段胆管细胞钙信号组织和分泌功能的影响。我们将监测用InsP3R亚型或PC2的siRNA处理的管道中碳酸氢盐分泌的变化，以及表达与InsP3R相互作用的PC2片段的管道中的变化。这项工作将有助于确定胆管细胞内钙信号转导的分子机制，有助于我们理解胆汁淤积的分子基础。更广泛地说，这些研究将概述PC2在其他分泌上皮细胞的信号转导和刺激-分泌耦合中的作用，并可能有助于解释为什么PC2突变对表达该蛋白的不同组织产生不同的影响。与公共健康相关：这项提议将决定胆管细胞内的信号如何调节胆汁分泌。这些信息对于了解胆汁淤积的基础很重要，胆汁淤积是肝病的一种常见表现，胆汁分泌受损。此外，这项工作也有可能为调节其他类型的上皮组织的分泌建立一个新的范例。

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.