Our GOALS are to continue to clarify the pathogenesis of polycystic liver disease (PLD) and develop effective therapies. PLD is a genetic ciliopathy for which there are no regulatory approved drugs. We reported that PLD cholangiocytes (PLDC) have increased cAMP, accelerated autophagy, and structurally and functionally malformed cilia. However, it remains unclear what mechanisms are involved, how they contribute to hepatic cystogenesis, or if they represent new therapeutic targets. Based on recent published observations and our preliminary data, our OVERALL OBJECTIVES are to explore the mechanistic relations between autophagy of ciliogenic proteins (i.e., proteins involved in cilia maintenance), pro-cystogenic signaling and hepatic cystogenesis. Our preliminary data show: i) autophagy, cilia, and cAMP signaling are the most altered biological processes in human PLDC vs. normal cholangiocytes (NHC) as assessed by RNAseq and gene enrichment analysis; ii) ciliogenic proteins are diminished in PLDC cilia but increased in autophagosomes isolated from PLDC; iii) in PLDC, HDAC6 is increased and ubiquitinates ciliogenic proteins; iv) in PLDC, the G- protein coupled bile acid receptor, TGR5, is depleted in cilia due to autophagic degradation of ciliogenic proteins that normally target TGR5 to cilia, is overexpressed at the apical membrane and its activation further increases cAMP; and v) genetic elimination of the ciliogenic protein, Kif3A, from cholangiocytes in mice leads to malformed TGR5-depleted cilia, TGR5 overexpression at the apical membrane, and hepatic cystogenesis. Thus, our data support the CENTRAL HYPOTHESIS that in PLD, HDAC6-dependent autophagy of ciliogenic proteins disrupts ciliogenesis resulting in malformed cholangiocyte cilia, ciliary depletion of TGR5, enhanced pro-cystogenic signaling and hepatic cyst growth. We will test this hypothesis by using biochemical, molecular and microscopic techniques, cultured and freshly isolated human and rodent normal and PLD cholangiocytes, unique animal models, and human tissue. Our proposal has three integrated SPECIFIC AIMS. First, we test the hypothesis that autophagy-mediated, HDAC6-dependent depletion of ciliogenic proteins disrupts ciliogenesis in PLDC resulting in malformed cilia. Second, we test the hypothesis that autophagy of ciliogenic proteins depletes cholangiocyte cilia of TGR5 and enhances pro-cystogenic signaling and cyst growth. Third, we test the hypothesis that pharmacologic interventions that target cholangiocyte autophagy and ciliogenesis (ciliotherapy) inhibit cystogenesis in vitro and in animal models of PLD. These innovative experiments will: i) clarify how autophagy of ciliogenic proteins leads to ciliary malformations; ii) address how HDAC6-mediated autophagy of ciliogenic proteins disrupts ciliary targeting of TGR5, thereby increasing pro-cystogenic cAMP signaling and cyst growth; and iii) explore how pharmacologic manipulations of autophagy and ciliogenesis affect cystogenesis (ciliotherapy). Our results will be informative for mechanisms of PLD progression and potentially transformative for new therapeutic approaches.
Polycystic liver disease (PLD) is an incurable genetic disorder characterized by progressive growth of hepatic cysts derived from bile duct cells (cholangiocytes). PLD causes considerable patient morbidity and there are no regulatory approved drugs for its treatment. Thus, with the goals of better understanding how cysts develop in PLD in order to identify effective therapies, this project explores the intersection between two cellular processes (defective autophagy and ciliogenesis) that underlie growth of liver cysts and are potential therapeutic targets.
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