Our objectives remain to understand the function and dysfunction of hepatic epithelia. We focus on cholangiocytes because of their pathobiologic importance and the novel techniques we have developed for their study. Here we concentrate on the cholangiociliopathies, a group of incurable genetic diseases manifesting biliary cysts with or without fibrosis;Autosomal Recessive and Autosomal Dominant Polycystic Kidney Disease (ARPKD and ADPKD) are the most important. We have shown that: (i) cholangiocyte cilia are mechano-, chemo- and osmo-sensors;(ii) exosomes [biliary vesicles derived from cholangiocyte multivesicular bodies (MVBs)] interact with cilia triggering changes in cAMP that affect expression of selected microRNAs (miR-15a);(iii) cystic cholangiocytes from the PCK rat, a model of ARPKD, have increased cAMP, decreased miR-15a, overexpressed Cdc25A (a cell cycle protein targeted by miR-15a), and increased Hedgehog (Hh) components (Ptc, Smo and Gli-2);and (iv) pharmacologic reduction of cAMP and upregulation of miR-15a with accompanying decrease of Cdc25A inhibits cell proliferation and cyst growth in vitro;in PCK rats, octreotide reduces cholangiocyte cAMP and inhibits hepatic cystogenesis. Thus, we will test the CENTRAL HYPOTHESIS that normal sensory/transducing activities of cholangiocyte cilia are disrupted in cystic liver disease leading to altered intracellular signaling and modified miRNA expression resulting in cholangiocyte hyperproliferation, ductal dysmorphogenesis, and hepatic cyst formation.
Our SPECIFIC AIMS will test three hypotheses: (i) biliary exosomes bind to cholangiocyte cilia with involvement of polycystin 1 (mutated in ADPKD) and/or fibrocystin (mutated in ARPKD) affecting intracellular signaling (cAMP, Hh) and miRNA expression (miR-15a);(ii) in cystic cholangiocytes, elevated cAMP reduces expression of miR-15a via transcriptional activation of the CREB/ICER/CRE pathway causing miR-15a target proteins (Cdc25A, Ptc, Erk1) to increase leading to hyperproliferation;and (iii) pharmacologic targeting of MEK, Smo and Cdc25A, components of the cAMP, Hh, and cell cycle machinery, respectively, inhibits cholangiocyte hyperproliferation in vitro and reduces hepatic cystogenesis in vivo in rodent models of ARPKD and ADPKD. We utilize new models including isolated exosomes, perfused bile ducts, isolated cholangiocytes and cilia, and cysts grown in 3-D culture;cultured normal and PCK cholangiocytes;and rodent models of ARPKD (PCK rat and Pkhd1 KO mouse) and ADPKD (Pkd2ws25/- KO mouse). Innovative aspects include: new in vitro techniques and animal models;new hypotheses regarding function and dysfunction of cholangiocyte cilia;new concepts regarding biliary exosomes and signaling via interactions with cilia;gene regulation by miRNAs in cholangiocyte proliferation;and new therapies for hepatic cystogenesis. Our results will yield new mechanistic insights into normal cholangiocyte function, clarify the molecular pathogenesis of the cholangiociliopathies, and test new drugs for treatment of fibrocystic liver disease.

Public Health Relevance

This proposal will both examine the cellular mechanisms by which cysts form in the liver as well as test new pharmacologic therapies to inhibit cyst development and progression in rodent models of ADPKD and ARPKD, the two most important, incurable, genetic cystic liver diseases. These diseases are associated with mutations in known genes and the protein products of these genes are expressed in cilia, antenna-like organelles that extend into the bile ducts from cholangiocytes, the cells from which cysts form. We believe that abnormalities in the sensory and transducing activities of cholangiocyte cilia are central to hepatic cyst formation.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK024031-34
Application #
8247144
Study Section
Hepatobiliary Pathophysiology Study Section (HBPP)
Program Officer
Serrano, Jose
Project Start
1978-12-01
Project End
2014-03-31
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
34
Fiscal Year
2012
Total Cost
$570,959
Indirect Cost
$193,090
Name
Mayo Clinic, Rochester
Department
Type
DUNS #
006471700
City
Rochester
State
MN
Country
United States
Zip Code
55905
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Larusso, Nicholas F; Masyuk, Tatyana V; Hogan, Marie C (2016) Polycystic Liver Disease: The Benefits of Targeting cAMP. Clin Gastroenterol Hepatol 14:1031-4
Lazaridis, Konstantinos N; LaRusso, Nicholas F (2015) The Cholangiopathies. Mayo Clin Proc 90:791-800
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Masyuk, Tatyana V; Masyuk, Anatoliy I; LaRusso, Nicholas F (2015) TGR5 in the Cholangiociliopathies. Dig Dis 33:420-5
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Gradilone, Sergio A; Habringer, Stefan; Masyuk, Tatyana V et al. (2014) HDAC6 is overexpressed in cystic cholangiocytes and its inhibition reduces cystogenesis. Am J Pathol 184:600-8
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Urribarri, Aura D; Munoz-Garrido, Patricia; Perugorria, María J et al. (2014) Inhibition of metalloprotease hyperactivity in cystic cholangiocytes halts the development of polycystic liver diseases. Gut 63:1658-67

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