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.
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.
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