The GOALS of this long-standing program remain to understand the function and dysfunction of hepatic epithelia. We focus on cholangiocytes because of their pathophysiologic importance, the novel techniques we have established for their study, and the progress we have made in understanding their role in the pathogenesis of and therapies for the cholangiopathies. In this renewal, we continue to concentrate on the polycystic liver diseases (PLD), a group of incurable genetic syndromes characterized by hepatic cysts arising from cholangiocytes. Our OVERALL OBJECTIVES are to explore the importance in PLD of TGR5, a G- protein-coupled receptor linked to intracellular cAMP (a signaling pathway central to hepatic cystogenesis), and to clarify the role of cholangiocyte autophagy in modulating expression of discrete microRNAs (miRNAs) involved in cyst progression. Our preliminary data show that: (a) cholangiocytes of animal models of and humans with autosomal dominant or recessive polycystic kidney disease (ADPKD and ARPKD) are characterized by: i) increased expression of TGR5;ii) decreased levels of two miRNAs (miR-204 and miR- 345) that target the mRNAs of cell cycle proteins involved in hepatic cystogenesis;and iii) increased autophagy, with localization of miR-204 and miR-345 in autophagosomes;(b) in vitro activation of TGR5 in cystic cholangiocytes results in: i) decreased miR-345 and miR-204;and ii) increased cell proliferation and cyst expansion;(c) conversely, in vitro inhibition of autophagy: i) increases miR-204 and miR-345;and ii) reduces cell proliferation and cyst expansion;and (d) cross-breeding of Gpbar1-/- (TGR5-deficient) mice with Pkhd1del2/del2 mice (animal model of ARPKD) diminishes hepatic cyst formation in double mutant mice. Thus, our results support the CENTRAL HYPOTHESIS that overexpression of TGR5 in cystic cholangiocytes induces autophagy of discrete miRNAs (i.e., miRNAutophagy) via the cAMP signaling pathway, thereby increasing expression of cell cycle proteins and promoting hepatic cystogenesis. We will test this novel hypothesis by using sophisticated biochemical and molecular techniques, animal models and human tissues to dissect the mechanisms and pathologic consequences of TGR5-regulated miRNAutophagy and to explore novel therapeutic interventions for PLD. Our proposal has three integrated SPECIFIC AIMS. First, we will test the hypothesis that overexpression of TGR5 and subsequent cAMP elevation in cystic cholangiocytes accelerates autophagy of discrete miRNAs (miR-204 and miR-345). Second, we will test the hypothesis that TGR5-regulated autophagy of miR-204 and miR-345 in cystic cholangiocytes results in increased expression of target cell cycle proteins, cell cycle abnormalities, hyperproliferation and hepatic cystogenesis. Finally, we will test the hypothesis that genetic and/or pharmacologic manipulations of TGR5 and autophagy in animal models of PLD influence disease progression. Results from these innovative experiments will: i) clarify the mechanisms of TGR5-regulated autophagy of discrete miRNAs, an unexplored area of cell biology;ii) identify the cellular consequences of TGR5 overexpression and increased miRNAutophagy in cystic cholangiocytes;and iii) assess in rodent models of PLD whether genetic or pharmacologic interventions affecting TGR5 and autophagy influence hepatic cystogenesis.
This proposal examines the mechanisms and consequences of cholangiocyte overexpression of TGR5 (a G-protein-coupled receptor linked to cAMP signaling) and the roles of microRNAs and autophagy in TGR5 regulation in the polycystic liver diseases. The results of these experiments could lead to new and effective therapies for these incurable conditions.
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