Regulation of cholangiocytes by InsP3 receptor isoforms

描述（由申请人提供）：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，而且这必须随后被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移植的20%，是儿科移植患者中最常见的肝病原因。胆管细胞中胆汁分泌部分受胞质Ca 2+（Ca 2+）调节，并且肌醇1，4，5-三磷酸受体（InsP 3R）的功能是如何调节这些Ca 2+信号的重要组成部分。在胆管细胞中发现了第二种细胞内蛋白多囊蛋白-2（PC 2），但对其在这些细胞中的Ca 2+信号传导中的作用知之甚少。PC 2中的突变导致多囊肾病（PKD），其与大的充满液体的囊肿相关。身体的大多数细胞表达PC 2以及InsP 3R的所有三种亚型，尽管是以细胞特异性比率表达。在三种InsP 3 R亚型中，III型是在肾脏和胆管（PKD囊肿形成的主要部位）中表达的主要亚型。在这个提议中，我们将探索InsP 3R和PC 2之间相互作用的功能效应。我们假设PC 2优先与InsP 3R的III型亚型相互作用，使具有这种亚型的细胞，如肾细胞和胆管细胞，在PC 2突变时比具有InsP 3R I型和II型的细胞更容易形成囊肿，例如肝细胞。我们还假设，由PC 2结合的InsP 3R将具有比未结合的受体更低的活性，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的反应。这一假设将通过以下具体目标进行调查：1。了解PC 2和InsP 3R之间的分子相互作用。具体来说，我们将监测InsP 3R的哪些亚型与PC 2相互作用，PC 2的哪个片段影响InsP 3R功能，以及这些相互作用如何通过PC 2中的突变进行修饰。我们将测试PC 2和特定致病突变对InsP 3R类型通道特性的影响，使用脂质双层技术在分子水平上研究这些相互作用。2.了解PC 2和InsP 3R之间相互作用的细胞后果。我们将监测这些受体中的每一个在胆管细胞中的Ca 2+信号传导中发挥的作用，以及这些相互作用如何导致下游信号传导。胆管细胞系MzCha 1细胞将被工程化以表达每种InsP 3R亚型和PC 2。我们将使用全细胞水平的真实的时间Ca 2+成像来测试Ca 2+信号的同种型特异性。3.了解PC 2-InsP 3R相互作用在天然胆管细胞Ca 2+信号组织和分泌功能中的作用，如在分离的微灌注胆管段中所确定的。我们将监测用InsP 3R亚型或PC 2的siRNA处理的导管中碳酸氢盐分泌的改变，以及表达与InsP 3R相互作用的PC 2片段的导管中碳酸氢盐分泌的改变。这项工作应确定负责Ca 2+信号在胆管细胞的分子机制，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PC 2在其他分泌性上皮细胞中的信号转导和刺激-分泌偶联中的作用，并可能有助于解释为什么PC 2突变差异地影响表达这种蛋白质的各种组织。公共卫生相关性：该提案将确定胆管细胞内的信号传导如何调节胆汁分泌。这一信息对于理解胆汁淤积的基础很重要，胆汁淤积是胆汁分泌受损的肝脏疾病的常见表现。此外，这项工作有可能建立一个新的模式，在其他类型的上皮组织分泌的调节以及。

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.