Loss of cilia causes embryonic lung hypoplasia,liver fibrosis,and cholestasis in the talpid3 ciliopathy mutant

Sonic hedgehog plays an essential role in maintaining hepatoblasts in a proliferative non-differentiating state during embryogenesis. Transduction of the Hedgehog signaling pathway is dependent on the presence of functional primary cilia and hepatoblasts, therefore, must require primary cilia for normal function. In congenital syndromes in which cilia are absent or non-functional (ciliopathies) hepatorenal fibrocystic disease is common and primarily characterized by ductal plate malformations which underlie the formation of liver cysts, as well as less commonly, by hepatic fibrosis, although a role for abnormal Hedgehog signal transduction has not been implicated in these phenotypes. We have examined liver, lung and rib development in the talpid³ chicken mutant, a ciliopathy model in which abnormal Hedgehog signaling is well characterized. We find that the talpid³ phenotype closely models that of human short-rib polydactyly syndromes which are caused by the loss of cilia, and exhibit hypoplastic lungs and liver failure. Through an analysis of liver and lung development in the talpid³ chicken, we propose that cilia in the liver are essential for the transduction of Hedgehog signaling during hepatic development. The talpid³ chicken represents a useful resource in furthering our understanding of the pathology of ciliopathies beyond the treatment of thoracic insufficiency as well as generating insights into the role Hedgehog signaling in hepatic development.     

Intermedilysin induces EGR-1 expression through calcineurin/NFAT pathway in human cholangiocellular carcinoma cells

Intermedilysin (ILY) is a cholesterol-dependent cytolysin produced by Streptococcus intermedius, which is associated with human brain and liver abscesses. Although intrahepatic bile duct cells play a valuable role in the pathogenesis of liver abscess, the molecular mechanism of ILY-treated intrahepatic bile duct cells remains unknown. In this study, we report that ILY induced a nuclear accumulation of intracellular calcium ([Ca²⁺]i) in human cholangiocellular cells HuCCT1. We also demonstrate that 10 ng/ml ILY induced NFAT1 dephosphorylation and its nuclear translocation in HuCCT1 cells. In contrast to the result that ILY induced NF-κB translocation in human hepatic HepG2 cells, ILY did not affect NF-κB localization in HuCCT1 cells. Dephosphorylation and nuclear translocation of NFAT1 caused by ILY were prevented by [Ca²⁺]i calcium chelator, BAPTA/AM, and calcineurin inhibitors, cyclosporine A and tacrolimus. ILY induced early growth response-1 (EGR-1) expression and it was inhibited by the pre-treatment with cyclosporine A, indicating that the calcineurin/NFAT pathway was involved in EGR-1 expression in response to ILY. ILY-induced calcineurin/NFAT1 activation and sequential EGR-1 expression might be related to the pathogenesis of S. intermedius in human bile duct cells.     

A model of in vitro UDP-glucuronosyltransferase inhibition by bile acids predicts possible metabolic disorders

Increased levels of bile acids (BAs) due to the various hepatic diseases could interfere with the metabolism of xenobiotics, such as drugs, and endobiotics including steroid hormones. UDP-glucuronosyltransferases (UGTs) are involved in the conjugation and elimination of many xenobiotics and endogenous compounds. The present study sought to investigate the potential for inhibition of UGT enzymes by BAs. The results showed that taurolithocholic acid (TLCA) exhibited the strongest inhibition toward UGTs, followed by lithocholic acid. Structure-UGT inhibition relationships of BAs were examined and in vitro-in vivo extrapolation performed by using in vitro inhibition kinetic parameters (K_i) in combination with calculated in vivo levels of TLCA. Substitution of a hydrogen with a hydroxyl group in the R1, R3, R4, R5 sites of BAs significantly weakens their inhibition ability toward most UGTs. The in vivo inhibition by TLCA toward UGT forms was determined with following orders of potency: UGT1A4 > UGT2B7 > UGT1A3 > UGT1A1 ∼ UGT1A7 ∼ UGT1A10 ∼ UGT2B15. In conclusion, these studies suggest that disrupted homeostasis of BAs, notably taurolithocholic acid, found in various diseases such as cholestasis, could lead to altered metabolism of xenobiotics and endobiotics through inhibition of UGT enzymes.     

The role of ERK-RSK signaling in the proliferation of intrahepatic biliary epithelial cells exposed to microcystin-leucine arginine

Microcystin-leucine arginine (MC-LR) is a potent specific hepatotoxin produced by cyanobacteria in diverse water systems, and it has been documented to induce liver injury and hepatocarcinogenesis. However, its toxic effects on intrahepatic biliary epithelial cells have not been invested in detail. In this study, we aimed to investigate the effects of MC-LR exposure on the intrahepatic biliary epithelial cells in the liver. MC-LR was orally administered to mice at 1 μg/L, 7.5 μg/L, 15 μg/L, or 30 μg/L for 180 consecutive days for histopathological and immunoblot analysis. We observed that MC-LR can enter intrahepatic bile duct tissue and induce hyperplasia of mice. Human primary intrahepatic biliary epithelial cells (HiBECs) were cultured with various concentrations of MC-LR for 24 h, meanwhile the cell viability and proteins level were detected. Western blotting analysis revealed that MC-LR increased RSK phosphorylation via ERK signaling. RSK participated in cell proliferation and cell cycle progression. Taken together, after chronic exposure, MC-LR-treated mice exhibited abnormal bile duct hyperplasia and thickened bile duct morphology through activating the ERK-RSK signaling. These data support the potential toxic effects of MC-LR on bile duct tissue of the liver.     

Prolactin receptors in rat cholangiocytes: Regulation of level and isoform ratio is sex independent

The presence of prolactin receptor and peculiarities of its isoform expression in bile duct cells (cholangiocytes) differentially isolated from rat liver under different conditions were investigated in the present study. Normal cholangiocytes express prolactin receptor at relatively low level comparable to those of some prolactin-dependent tissues. Long receptor isoform is predominant in cholangiocytes but not in hepatocytes. The prolactin receptor level increases significantly under obstructive cholestasis due to evaluation of long and appearance of short isoforms. In rat cholangiocytes, unlike other tissues, the main positive regulators of prolactin receptor expression are cholestasis-induced factors instead of sex hormone and prolactin levels. Long isoform is predominant and induced primarily by cholestasis-induced factors. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 2, pp. 229–236.     

Cellular localization and biochemical analysis of mammalian CDC50A, a glycosylated β-subunit for P4 ATPases

CDC50 proteins are β-subunits for P4 ATPases, which upon heterodimerization form a functional phospholipid translocation complex. Emerging evidence in mouse models and men links mutations in P4 ATPase genes with human disease. This study analyzed the tissue distribution and cellular localization of CDC50A, the most abundant and ubiquitously expressed CDC50 homologue in the mouse. The authors have raised antibodies that detect mouse and human CDC50A and studied CDC50A localization and glycosylation status in mouse liver cells. CDC50A is a terminal-glycosylated glycoprotein and is expressed in hepatocytes and liver sinusoidal endothelial cells, where it resides in detergent-resistant membranes. In pancreas and stomach, CDC50A localized to secretory vesicles, whereas in the kidney, CDC50A localized to the apical region of proximal convoluted tubules of the cortex. In WIF-B9 cells, CDC50A partially costains with the trans-Golgi network. Data suggest that CDC50A is present as a fully glycosylated protein in vivo, which presumes interaction with distinct P4 ATPases.     

Bile acid transporters

In liver and intestine, transporters play a critical role in maintaining the enterohepatic circulation and bile acid homeostasis. Over the past two decades, there has been significant progress toward identifying the individual membrane transporters and unraveling their complex regulation. In the liver, bile acids are efficiently transported across the sinusoidal membrane by the Na⁺ taurocholate cotransporting polypeptide with assistance by members of the organic anion transporting polypeptide family. The bile acids are then secreted in an ATP-dependent fashion across the canalicular membrane by the bile salt export pump. Following their movement with bile into the lumen of the small intestine, bile acids are almost quantitatively reclaimed in the ileum by the apical sodium-dependent bile acid transporter. The bile acids are shuttled across the enterocyte to the basolateral membrane and effluxed into the portal circulation by the recently indentified heteromeric organic solute transporter, OSTα-OSTβ. In addition to the hepatocyte and enterocyte, subgroups of these bile acid transporters are expressed by the biliary, renal, and colonic epithelium where they contribute to maintaining bile acid homeostasis and play important cytoprotective roles. This article will review our current understanding of the physiological role and regulation of these important carriers.     

Influence of Obstructive Cholestasis and Sex Hormones on the Ratio of mRNA of Two Alternative Prolactin Receptor Isoforms in Rat Hepatocytes

The effects of obstructive cholestasis and sex hormones on the total content of prolactin receptor mRNA and ratio of mRNAs of its short and long isoforms have been studied in rat hepatocytes. Obstructive cholestasis caused insignificant changes in total content of prolactin receptor mRNA, but the proportion of mRNA of the long isoform increased. Comparison of prolactin receptor mRNA levels in gonadectomized and intact animals revealed opposite effects of male and female sex hormones on total mRNA, but both groups of these hormones increased the proportion of prolactin receptor short form mRNA. Changes in ratio of mRNA of receptor isoforms found in rat hepatocytes under obstructive cholestasis did not depend on levels of sex hormones. Obstructive cholestasis and sex hormones are suggested to regulate the content of long and short prolactin receptor isoforms in hepatocytes independently.     

Genetic determinants of cholangiopathies: Molecular and systems genetics

Familial cholangiopathies are rare but potentially severe diseases. Their spectrum ranges from fairly benign conditions as, for example, benign recurrent intrahepatic cholestasis to low-phospholipid associated cholelithiasis and progressive familial intrahepatic cholestasis (PFIC). Many cholangiopathies such as primary biliary cholangitis (PBC) or primary sclerosing cholangitis (PSC) affect first the bile ducts (“ascending pathophysiology”) but others, such as PFIC, start upstream in hepatocytes and cause progressive damage “descending” down the biliary tree and leading to end-stage liver disease. In recent years our understanding of cholestatic diseases has improved, since we have been able to pinpoint numerous disease-causing mutations that cause familial cholangiopathies. Accordingly, six PFIC subtypes (PFIC type 1–6) have now been defined. Given the availability of genotyping resources, these findings can be introduced in the diagnostic work-up of patients with peculiar cholestasis. In addition, functional studies have defined the pathophysiological consequences of some of the detected variants. Furthermore, ABCB4 variants do not only cause PFIC type 3 but confer an increased risk for chronic liver disease in general. In the near future these findings will serve to develop new therapeutic strategies for patients with liver diseases. Here we present the latest data on the genetic background of familial cholangiopathies and discuss their application in clinical practice for the differential diagnosis of cholestasis of unknown aetiology. As look in the future we present “system genetics” as a novel experimental tool for the study of cholangiopathies and disease-modifying genes. This article is part of a Special Issue entitled: Cholangiocytes in Health and Disease edited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.