Ca2+ waves in hepatocytes: Mechanisms and effects

项目摘要 胆汁分泌是肝脏的主要功能之一。胆汁淤积，或胆汁流动受损，是一个主要的 肝脏疾病的表现。胆汁淤积性肝病是一组重要的疾病， 代表儿科肝移植的最常见适应症，占所有肝脏移植的十分之一。 移植本项目研究上皮生物学中的一个基本生物学机制， 用于调节健康和疾病中肝细胞的分泌。肝细胞中的钙信号形成 主要是通过从肌醇1，4，5-三磷酸（InsP 3）受体（InsP 3R）释放钙，InsP 3R是InsP 3- 内质网（ER）门控钙通道。II型InsP 3R（InsP 3R-2）是主要的 InsP 3R是肝细胞中的一种同种型，占InsP 3R总库的80%，并且最集中在一个专门的 泪小管膜下的内质网区域。肝细胞中的钙信号通常开始为钙 起源于该区域的波，肝细胞中InsP 3R-2的顶端池对于介导 钙波和小管分泌。相比之下，I型InsP 3R（InsP 3R-1），其构成了 肝细胞中剩余20%的InsP 3R池，优先定位于不同的亚细胞区域， 不影响分泌。该项目的假设是，调节表达的因素和 InsP 3R-2亚细胞分布也调节肝细胞分泌，因此胆汁淤积由这些介导 对InsP 3R-2的影响。特别是，我们将测试胆汁淤积性肝病是否部分是由于受损的 InsP 3R-2的表达和/或小管周围靶向，这反过来又损害了细胞中的极化钙波。 细胞溶质和下游事件，包括胆汁酸转运蛋白Bsep和有机阴离子的调节 转运蛋白Mrp 2。这一假设将通过以下具体目标进行检验：（1）我们将确定 调节肝细胞中InsP 3R-2表达的遗传和表观遗传因子。(2)康贝特人将以 InsP 3R-2靶向ER-小管膜界面的分子基础和细胞效应。(3)我们 将确定InsP 3R-2表达和/或小管周围靶向是否以及如何在肝细胞中受损 胆管胆汁淤积症我们还将确定正在开发的新疗法的疗效 用于治疗胆汁淤积性疾病部分地通过调节InsP 3R-2来介导。这些研究将 在我们对信号微区建立的方式的理解上开辟了新的天地， 肝细胞，并有可能建立新的范式，将这些观察转化为 了解一系列临床显著人类肝脏疾病的机制基础。

PROJECT SUMMARY Bile secretion is one of the principal functions of the liver. Cholestasis, or impaired bile flow, is a cardinal manifestation of liver disease. Cholestatic liver diseases are an important group of disorders, which collectively represent the most common indication for pediatric liver transplant and account for one in ten of all liver transplants. This project investigates a basic biological mechanism in epithelial biology that is directly relevant for the regulation of secretion in hepatocytes in health and disease. Calcium signals in hepatocytes are formed largely by calcium release from the inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R), which is an InsP3- gated calcium channel in the endoplasmic reticulum (ER). The type II InsP3R (InsP3R-2) is the predominant isoform in hepatocytes, constituting 80% of the total pool of InsP3Rs, and is most concentrated in a specialized region of the ER beneath the canalicular membrane. Calcium signals in hepatocytes generally begin as calcium waves that originate in this region, and the apical pool of InsP3R-2 in hepatocytes is important for mediating calcium waves and canalicular secretion. In contrast, the type I InsP3R (InsP3R-1), which constitutes the remaining 20% of the InsP3R pool in hepatocytes, preferentially localizes to a different subcellular region and does not affect secretion. The hypothesis of this project is that the factors that regulate the expression and subcellular distribution of InsP3R-2 also regulate hepatocyte secretion, so that cholestasis is mediated by these effects on InsP3R-2. In particular, we will test whether cholestatic liver diseases are due in part to impaired expression and/or peri-canalicular targeting of InsP3R-2, which in turn impairs polarized calcium waves in the cytosol and downstream events including regulation of the bile acid transporter Bsep and the organic anion transporter Mrp2. This hypothesis will be tested through the following specific aims: (1) We will determine the genetic and epigenetic factors that regulate expression of InsP3R-2 in hepatocytes. (2) We will determine the molecular basis for, and cellular effects of, targeting InsP3R-2 to the ER-canalicular membrane interface. (3) We will determine whether and how InsP3R-2 expression and/or pericanalicular targeting is impaired in hepatocytes during canalicular cholestasis. We also will determine whether the efficacy of new therapies being developed for treatment of cholestatis disorders is mediated in part by modulating InsP3R-2. Collectively, these studies will break new ground in our understanding of the ways in which signaling microdomains are established in hepatocytes, and have the potential to establish new paradigms for translating these observations into understanding the mechanistic basis for a range of clinically significant human liver diseases.