Cholestasis is a common manifestation of liver disease. Cholestasis is typically due to disorders specifically affecting cholangiocytes, which play a major role in bile secretion. They are responsible for secretion of bicarbonate into bile and modulating the biliary contents of other constituents as well. The type III inositol trisphosphate receptor (InsP3R-3) is the primary intracellular calcium release channel in cholangiocytes and previous studies from our laboratory have shown that apically-localized InsP3R-3 controls bicarbonate secretion. We also have found that in most ductular forms of human cholestasis, as well as in multiple animal models, there is loss of InsP3R-3 expression, underlying the importance of calcium homeostasis in normal cholangiocyte function and its dysregulation in cholestasis. We hypothesize that restoration of InsP3R- 3 expression could ameliorate cholestasis and improve biliary bicarbonate secretion. Thus the long term objective of this proposal is to understand the molecular mechanisms of InsP3R-3 regulation in normal and cholestatic liver and study the therapeutic effect of restoration of InsP3R-3 expression on liver function in cholestatic liver disease. In order to achieve this objective, the proposal will e implemented with the following specific aims: (1) The molecular factors governing the regulation of InsP3R-3 gene expression will be defined by studying the transcription factors (TFs) and microRNAs (miRs) that regulate the InsP3R-3 promoter and mRNA respectively;(2) The cellular mechanisms that direct the InsP3-R3 to the subapical region of cholangiocytes including targeting sequences and interacting proteins that lead to such localization will be established; (3) Molecular mechanisms that lead to loss of InsP3R-3 expression and function in multiple animal models of cholestasis will be determined and will also be verified in human cholestatic liver samples. Further, we will investigate whether restoration of expression of these factors using adenoviral overexpression of specific TFs/miRs-antimiRs in cholestasis results in improvement of the disease. Together, the data derived from these studies will improve our understanding of the regulation of secretion in cholangiocytes and other polarized epithelia and has the potential to lead to design of novel therapeutic approaches in a variety of cholestatic liver diseases that afflict humans.