Anatoliy I. Masyuk

Characterization of PKD Protein-Positive Exosome-Like Vesicles

Proteins associated with autosomal dominant and autosomal recessive polycystic kidney disease (polycystin-1, polycystin-2, and fibrocystin) localize to various subcellular compartments, but their functional site is thought to be on primary cilia. PC1+ vesicles surround cilia in Pkhd1(del2/del2) mice, which led us to analyze these structures in detail. We subfractionated urinary exosome-like vesicles (ELVs) and isolated a subpopulation abundant in polycystin-1, fibrocystin (in their cleaved forms), and polycystin-2. This removed Tamm-Horsfall protein, the major contaminant, and subfractionated ELVs into at least three different populations, demarcated by the presence of aquaporin-2, polycystin-1, and podocin. Proteomic analysis of PKD ELVs identified 552 proteins (232 not yet in urinary proteomic databases), many of which have been implicated in signaling, including the molecule Smoothened. We also detected two other protein products of genes involved in cystic disease: Cystin, the product of the mouse cpk locus, and ADP-ribosylation factor-like 6, the product of the human Bardet-Biedl syndrome gene (BBS3). Our proteomic analysis confirmed that cleavage of polycystin-1 and fibrocystin occurs in vivo, in manners consistent with cleavage at the GPS site in polycystin-1 and the proprotein convertase site in fibrocystin. In vitro, these PKD ELVs preferentially interacted with primary cilia of kidney and biliary epithelial cells in a rapid and highly specific manner. These data suggest that PKD proteins are shed in membrane particles in the urine, and these particles interact with primary cilia.

Cholangiocyte cilia express TRPV4 and detect changes in luminal tonicity inducing bicarbonate secretion

Cholangiocytes, epithelial cells lining the biliary tree, have primary cilia extending from their apical membrane into the ductal lumen. Although important in disease, cilia also play a vital role in normal cellular functions. We reported that cholangiocyte cilia are sensory organelles responding to mechanical stimuli (i.e., luminal fluid flow) by alterations in intracellular Ca2+ and cAMP. Because cholangiocyte cilia are also ideally positioned to detect changes in composition and tonicity of bile, we hypothesized that cilia also function as osmosensors. TRPV4, a Ca2+-permeable ion channel, has been implicated in signal transduction of osmotic stimuli. Using purified rat cholangiocytes and perfused intrahepatic bile duct units (IBDUs), we found that TRPV4 is expressed on cholangiocyte cilia, and that hypotonicity induces an increase in intracellular Ca2+ in a TRPV4-, ciliary-, and extracellular calcium-dependent manner. The osmosensation of luminal tonicity by ciliary TRPV4 induces bicarbonate secretion, the main determinant of ductal bile formation, by a mechanism involving apical ATP release. Furthermore, the activation of TRPV4 in vivo, by its specific agonist, 4αPDD, induces an increase in bile flow as well as ATP release and bicarbonate secretion. Our results suggest that cholangiocyte primary cilia play an important role in ductal bile formation by acting as osmosensors.

MicroRNA15a modulates expression of the cell-cycle regulator Cdc25A and affects hepatic cystogenesis in a rat model of polycystic kidney disease

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.

Cholangiocyte primary cilia are chemosensory organelles that detect biliary nucleotides via P2Y12 purinergic receptors

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-beta and PKA RII-alpha 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 forskolin-induced cAMP levels, respectively. In NRCs, the forskolin-induced cAMP increase was also lowered by 1.3-fold in response to ATP-gammaS, 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 P2Y(12) 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.

CHOLANGIOCYTE PRIMARY CILIA IN LIVER HEALTH AND DISEASE

The epithelial cells lining intrahepatic bile ducts (i.e., cholangiocytes), like many cell types in the body, have primary cilia extending from the apical plasma membrane into the bile ductal lumen. Cholangiocyte cilia express proteins such as polycystin‐1, polycystin‐2, fibrocystin, TRPV4, P2Y12, AC6, that account for ciliary mechano‐, osmo‐, and chemo‐sensory functions; when these processes are disturbed by mutations in genes encoding ciliary‐associated proteins, liver diseases (i.e., cholangiociliopathies) result. The cholangiociliopathies include but are not limited to cystic and fibrotic liver diseases associated with mutations in genes encoding polycystin‐1, polycystin‐2, and fibrocystin. In this review, we discuss the functions of cholangiocyte primary cilia, their role in the cholangiociliopathies, and potential therapeutic approaches. Developmental Dynamics 237:2007–2012, 2008.

Exosomes in the pathogenesis, diagnostics and therapeutics of liver diseases

Exosomes are small (30-100 nm in diameter) extracellular membrane-enclosed vesicles released by different cell types into the extracellular space or into biological fluids by exocytosis as a result of fusion of intracellular multivesicular bodies with the plasma membrane. The primary function of exosomes is intercellular communication with both beneficial (physiological) and harmful (pathological) potential outcomes. Liver cells are exosome-releasing cells as well as targets for endogenous exosomes and exosomes derived from cells of other organs. Despite limited studies on liver exosomes, initial observations suggest that these vesicles are important in liver physiology and pathophysiology. In this review, we briefly summarize the recent findings on liver exosomes, their functions and significance for novel diagnostic and therapeutic approaches.

The cAMP Effectors Epac and Protein Kinase A (PKA) Are Involved in the Hepatic Cystogenesis of an Animal Model of Autosomal Recessive Polycystic Kidney Disease (ARPKD)

PCK rats, an animal model of autosomal recessive polycystic kidney disease (ARPKD), develop cholangiocyte‐derived liver cysts associated with increased intracellular adenosine 3′,5′‐cyclic adenosine monophosphate (cAMP), the inhibition of which suppresses cyst growth. We hypothesized that elevated cAMP stimulates cholangiocyte proliferation via two downstream effectors, exchange proteins activated by cAMP (Epac1 and Epac2 isoforms) and protein kinase A (PKA), and that intracellular calcium is also involved in this process. Assessment of Epac isoforms and PKA regulatory subunits at the messenger RNA and protein level showed that cultured normal rat cholangiocytes express Epac1, Epac2, and all regulatory PKA subunits. Epac isoforms and the PKA RIβ subunit were overexpressed in cultured PCK cholangiocytes. Proliferation analysis in response to Epac and PKA activation indicated that both normal and PCK cholangiocytes increase their growth upon Epac‐specific stimulation, while PKA‐specific stimulation results in differential effects, suppressing proliferation in normal cholangiocytes but accelerating this process in PCK cholangiocytes. On the other hand, both PKA and Epac activation of cystic structures generated by normal and PCK cholangiocytes when cultured under three‐dimensional conditions resulted in increased cyst growth, particularly in PCK‐cholangiocyte derived cysts. Pharmacological inhibitors and small interfering RNA–mediated gene silencing demonstrated the specificity of each effector activation, as well as the involvement of MEK‐ERK1/2 signaling in all the observed effector‐associated proliferation changes. Hyperproliferation of PCK cholangiocytes in response to PKA stimulation, but not to Epac stimulation, was found to be associated with decreased intracellular calcium, and restoration of calcium levels blocked the PKA‐dependent proliferation via the PI3K/AKT pathway. Conclusion: Our data provide strong evidence that both cAMP effectors, Epac and PKA, and the levels of intracellular calcium are involved in the hepatic cystogenesis of ARPKD. (HEPATOLOGY 2009;49:160‐174.)

Biliary dysgenesis in the PCK rat, an orthologous model of autosomal recessive polycystic kidney disease.

Hepatic polycystic disease occurs alone or in combination with polycystic kidney disease (PKD). In autosomal recessive PKD (ARPKD), liver lesions are the major cause of morbidity and mortality in older patients. ARPKD is caused by a mutation to PKHD1 and the PCK rat is an orthologous model of disease. Recently, we showed that fibrocystin, Pkhd1 protein, is localized to primary cilia in rat cholangiocytes and that disruption of its ciliary expression results in biliary cystogenesis. This study describes biliary phenotype in the PCK rat using micro-computed tomography scanning and three-dimensional reconstruction, and light, scanning, and transmission microscopy. Our results show that the biliary tree undergoes extensive remodeling resulting in bile duct dilatation, focal budding, and formation of cysts that are initially connected to bile ducts, but throughout time separate from them. Progressive liver enlargement results from massive cyst formation while liver parenchymal volume remains unchanged. Cilia in cystic cells are abnormal consistent with the notion that the primary defect in ARPKD resulting in cystogenesis may be linked to ciliary dysfunction. Our results suggest that the PCK rat is a useful model for studies of biliary cystogenesis and treatment options of inherited cystic liver disease.

Physiology of cholangiocytes.

Cholangiocytes are epithelial cells that line the intra- and extrahepatic ducts of the biliary tree. The main physiologic function of cholangiocytes is modification of hepatocyte-derived bile, an intricate process regulated by hormones, peptides, nucleotides, neurotransmitters, and other molecules through intracellular signaling pathways and cascades. The mechanisms and regulation of bile modification are reviewed herein.

Cholangiociliopathies: genetics, molecular mechanisms and potential therapies.

Purpose of review The present review summarizes recent knowledge on polycystic liver diseases (PCLDs), mechanisms of hepatic cystogenesis and potential therapies for these conditions. Recent findings PCLD may be classified as cholangiociliopathies. In PCLD associated with polycystic kidney disease, cell proliferation is one of the major mechanisms of cystogenesis, whereas in isolated PCLD (autosomal dominant polycystic liver disease), disrupted cell adhesion may be more important in cyst progression. In cystic cholangiocytes, overexpression of ion transporters and water channels facilitates fluid secretion into the cystic lumen, and growth factors, estrogens and cytokines promote cholangiocyte proliferation. With age, cholangiocytes lining liver cysts acquire features of mesenchymal cells contributing to hepatic fibrocystogenesis. A novel mechanism of liver cyst expansion in PCLD involves microRNA regulatory pathways. Hyperproliferation of cystic cholangiocytes is linked to abnormalities in cell cycle progression and microRNA expression. Decreased levels of miR-15a are coupled to upregulation of its target – the cell cycle regulator, Cdc25A. Cholangiocyte cilia in liver cysts are structurally abnormal. Somatostatin analogues and sirolimus reduce liver cyst volume in PCLD patients. Summary Clarification of molecular mechanisms of hepatic cystogenesis provides an opportunity for the development of targeted therapeutic options in PCLD.