Background & Aims-Each cholangiocte in the biliary tree has a primary cilium extending from the apical plasma membrane into the ductal lumen. While the physiological significance of cholangiocyte cilia is unknown, studies in renal epithelia suggest that primary cilia possess sensory functions. Here, we tested the hypothesis that cholangiocyte cilia are sensory organelles that detect and transmit luminal bile flow stimuli into intracellular Ca 2+ ([Ca 2+ ] i ) and cAMP signaling.

Hyperproliferation of bile duct epithelial cells due to cell-cycle dysregulation is a key feature of cystogenesis in polycystic liver diseases (PCLDs). Recent evidence suggests a regulatory role for microRNAs (miRNAs) in a variety of biological processes, including cell proliferation. We therefore hypothesized that miRNAs may be involved in the regulation of selected components of the cell cycle and might contribute to hepatic cystogenesis. We found that the cholangiocyte cell line PCK-CCL, which is derived from the PCK rat, a model of autosomal recessive polycystic kidney disease (ARPKD), displayed global changes in miRNA expression compared with normal rat cholangiocytes (NRCs). More specific analysis revealed decreased levels of 1 miRNA, miR15a, both in PCK-CCL cells and in liver tissue from PCK rats and patients with a PCLD. The decrease in miR15a expression was associated with upregulation of its target, the cell-cycle regulator cell division cycle 25A (Cdc25A). Overexpression of miR15a in PCK-CCL cells decreased Cdc25A levels, inhibited cell proliferation, and reduced cyst growth. In contrast, suppression of miR15a in NRCs accelerated cell proliferation, increased Cdc25A expression, and promoted cyst growth. Taken together, these results suggest that suppression of miR15a contributes to hepatic cystogenesis through dysregulation of Cdc25A.

Masyuk AI, Gradilone SA, Banales JM, Huang BQ, Masyuk TV, Lee S-O, Splinter PL, Stroope AJ, LaRusso NF. Cholangiocyte primary cilia are chemosensory organelles that detect biliary nucleotides via P2Y 12 purinergic receptors. Am J Physiol Gastrointest Liver Physiol 295: G725-G734, 2008. First published August 7, 2008 doi:10.1152/ajpgi.90265.2008.-Cholangiocytes, the epithelial cells lining intrahepatic bile ducts, contain primary cilia, which are mechano-and osmosensory organelles detecting changes in bile flow and osmolality and transducing them into intracellular signals. Here, we asked whether cholangiocyte cilia are chemosensory organelles by testing the expression of P2Y purinergic receptors and components of the cAMP signaling cascade in cilia and their involvement in nucleotide-induced cAMP signaling in the cells. We found that P2Y 12 purinergic receptor, adenylyl cyclases (i.e., AC4, AC6, and AC8), and protein kinase A (i.e., PKA RI-␤ and PKA RII-␣ regulatory subunits), exchange protein directly activated by cAMP (EPAC) isoform 2, and A-kinase anchoring proteins (i.e., AKAP150) are expressed in cholangiocyte cilia. ADP, an endogenous agonist of P2Y 12 receptors, perfused through the lumen of isolated rat intrahepatic bile ducts or applied to the ciliated apical surface of normal rat cholangiocytes (NRCs) in culture induced a 1.9-and 1.5-fold decrease of forskolininduced cAMP levels, respectively. In NRCs, the forskolin-induced cAMP increase was also lowered by 1.3-fold in response to ATP-␥S, a nonhydrolyzed analog of ATP but was not affected by UTP. The ADP-induced changes in cAMP levels in cholangiocytes were abolished by chloral hydrate (a reagent that removes cilia) and by P2Y 12 siRNAs, suggesting that cilia and ciliary P2Y12 are involved in nucleotide-induced cAMP signaling. In conclusion, cholangiocyte cilia are chemosensory organelles that detect biliary nucleotides through ciliary P2Y 12 receptors and transduce corresponding signals into a cAMP response. liver; ADP; adenylyl cyclases; cAMP; protein kinase A; exchange protein directly activated by cAMP; A-kinase anchoring protein 150 CHOLANGIOCYTES, the epithelial cells lining intrahepatic bile ducts (IBDs), contain primary cilia, nonmotile, solitary organelles extending from the apical plasma membrane into the ductal lumen (21,28,29). In many cell types, including cholangiocytes, primary cilia function as sensory organelles detecting multiple (i.e., mechano-, osmo-, chemo-) stimuli and transducing them into intracellular signaling (12,16,27,40,46). However, although increasing evidence suggests the ability of primary cilia to act as mechano-and osmosensors (16, 24, 28, 34 -37, 40 -42, 49, 50, 55), less data support their chemosensory functions. To function as chemosensory organelles, primary cilia should possess receptors and associated signaling cascades through which signals induced by specific ligands are transmitted into the cell. Such mechanism exists in Caenorhabditis elegans neuronal primary cilia, which express specific G protein-coupled...

In polycystic liver (PLD) and kidney (PKD) diseases, increased cAMP levels trigger hepato-renal cystogenesis. We previously reported that reduction of elevated cAMP by targeting somatostatin receptors (SSTRs) with Octreotide (OCT; a somatostatin analogue that preferentially binds to SSTR2), inhibits cyst growth. We now compare effects of OCT to Pasireotide (PAS; a more potent somatostatin analogue with broader receptor specificity) on: (i) cAMP levels, cell cycle, proliferation and cyst expansion in vitro using cholangiocytes derived from control and PCK rats (a model of autosomal recessive PKD [ARPKD]), healthy human beings and patients with autosomal dominant PKD (ADPKD); and (ii) hepato-renal cystogenesis in vivo in PCK rats and Pkd2WS25/− mice (a model of ADPKD). Expression of SSTRs was assessed in control and cystic cholangiocytes of rodents and human beings. Concentrations of IGF and VEGF (both involved in indirect action of somatostatin analogs), and expression and localization of SSTRs after treatment were evaluated. We showed that PAS was more potent (by 30–45%) than OCT in reducing cAMP and cell proliferation, affecting cell cycle distribution, decreasing growth of cultured cysts in vitro and inhibiting hepato-renal cystogenesis in vivo in PCK rats and Pkd2WS25/− mice. The levels of IGF1 (but not VEGF) were reduced only in response to PAS. Expression of SSTR1 and SSTR2 (but not SSTR3 and SSTR5) was decreased in cystic cholangiocytes compared to control. While both OCT and PAS increased immunoreactivity of SSTR2, only PAS up-regulated SSTR1; neither drug affected cellular localization of SSTRs. Conclusion PAS is more effective than OCT in reducing hepato-renal cystogenesis in rodent models; therefore it might be more beneficial for the treatment of PKD and PLD.

Background & Aims-In polycystic liver diseases, cyst formation involves cholangiocyte hyperproliferation. In PCK rats, an animal model of autosomal recessive polycystic kidney disease (ARPKD), decreased [Ca 2+ ] i in cholangiocytes is associated with hyperproliferation. We recently showed Trpv4, a calcium-entry channel, is expressed in normal cholangiocytes and its activation leads to [Ca 2+ ] i increase. Thus, we hypothesized that pharmacological activation of Trpv4 might reverse the hyperproliferative phenotype of PCK cholangiocytes.