Cholangiocytes - Nicholas F. LaRusso

Overview

The objectives of my laboratory are to apply the fundamental concepts and broad technologies of cell/molecular biology to understand hepatic epithelial cell function and dysfunction. Our focus is principally on cholangiocytes (epithelial cells that line intrahepatic bile ducts) because of their biologic and clinical importance, the new hypotheses and techniques we have developed for their study, and the role we have played in advancing cholangiocyte pathobiology — an underserved area of research. Currently, my lab has two major parallel programs focused on cholangiocyte pathobiology, each supported by an R01 grant from NIH.

First, 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:

1. cholangiocyte cilia are mechano-, chemo- and osmo-sensors;
2. exosomes (biliary vesicles derived from cholangiocyte multivesicular bodies [MVBs]) interact with cilia triggering changes in cAMP that affect expression of selected microRNAs (miR-15a);
3. 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
4. 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 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 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 (Pkd2^ws25/- KO mouse).

Second, we focus on the interactions between cholangiocytes and Cryptosporidium parvum (C. parvum), an emerging pathogen causing intestinal and biliary cryptosporidiosis. Recent evidence from our lab indicates that:

1. the cellular expression and regulation of key receptors (toll-like receptors [TLRs]) and specific intracellular signaling pathways (NF-kB activation) are involved in cholangiocyte C. parvum recognition and strategic defense; and
2. endogenous microRNA (miRNA)-mediated post-transcriptional gene regulation is involved in cholangiocyte defense responses to C. parvum infection.

Thus, we test the central hypothesis that C. parvum-cholangiocyte interactions activate host-cell TLR/NF-kΒ signaling cascades initiating cholangiocyte defense responses including upregulation of both TLRs and antimicrobial peptides (defensins) through both transcriptional and post-transcriptional (endogenous miRNA-mediated) gene regulation.

In our three integrated specific aims, we test the hypotheses that:

1. cholangiocyte TLRs recognize C. parvum and modulate NF-kΒ activation resulting in upregulation and antimicrobial peptides (human beta-defensins [HBDs]) and TLRs through NF-kB mediated transcriptional regulation;
2. miRNA-mediated post-transcriptional repression of TLR expression normally exists in cholangiocytes, is regulated by TLR/NF-kΒ signals and is involved in C. parvum-induced upregulation of TLRs; and
3. expression of TLRs and HBDs in cholangiocyte are required for eradication of C. parvum infection in the biliary tract in in vitro and in vivo experimental models.