Hakata antigen, a new member of the ficolin/opsonin p35 family, is a novel human lectin secreted into bronchus/alveolus and bile.

Hakata antigen was first reported as a serum protein that reacted with an autoantibody from patients with systemic lupus erythematosus. Recently, it has been found that Hakata antigen is a new member of the ficolin/opsonin p35 family, which is a distinct lectin family, on the basis of homology of structures and the common characteristic of possessing lectin activity. In this study we analyzed the tissue distribution of Hakata antigen. Hakata antigen mRNA and protein were generated in the lung and liver. In the lung, Hakata antigen was produced by both ciliated bronchial epithelial cells and Type II alveolar epithelial cells and was secreted into the bronchus and alveolus. In the liver, Hakata antigen was produced by bile duct epithelial cells and hepatocytes and was also secreted into the bile duct. These results demonstrate that Hakata antigen is a unique lectin protein that exists not only in serum but also in bronchus/alveolus and bile, and indicate that Hakata antigen plays a role in bronchus/alveolus and bile under physiological conditions.     

Cell of origin of distinct cultured rat liver epithelial cells, as typed by cytokeratin and surface component selective expression

The cell of origin of the nonparenchymal epithelioid cells that emerge in liver cell cultures is unknown. Cultures of rat hepatocytes and several types of nonparenchymal cells obtained by selective tissue dispersion procedures were typed with monoclonal antibodies to rat liver cytokeratin and vimentin, polyvalent antibodies to cow hoof cytokeratins and porcine lens vimentin, and monoclonal antibodies to surface membrane components of ductular oval cells and hepatocytes. Immunoblot analysis revealed that, in cultured rat liver nonparenchymal epithelial cells, the anti-rat hepatocyte cytokeratin antibody recognized a cytokeratin of relative mass (Mr) 55,000 and the anti-cow hoof cytokeratin antibody reacted with a cytokeratin of Mr 52,000, while the anti-vimentin antibodies detected vimentin in both cultured rat fibroblasts and nonparenchymal epithelial cells. Analyses on the specificity of anti-cytokeratin and anti-vimentin antibodies toward the various cellular structures of liver by double immunofluorescence staining of frozen tissue sections revealed unique reactivity patterns. For example, hepatocytes were only stained with anti-Mr 55,000 cytokeratin antibody, while the sinusoidal cells reacted only with the anti-vimentin antibodies. In contrast, epithelial cells of the bile ductular structures and mesothelial cells of the Glisson capsula reacted with all the anti-cytokeratin and anti-vimentin antibodies. It should be stressed, however, that the reaction of the anti-vimentin antibodies on bile ductular cells was weak. The same analysis on tissue sections using the anti-ductular oval cell antibody revealed that it reacted with bile duct structures but not with the Glisson capsula. The anti-hepatocyte antibody reacted only with the parenchymal cells. The differential reactivity of the anti-cytokeratin and anti-vimentin antibodies with the various liver cell compartments was confirmed in primary cultures of hepatocytes, sinusoidal cells, and bile ductular cells, indicating that the present panel of antibodies to intermediate filament constituants allowed a clear-cut distinction between cultured nonparenchymal epithelial cells, hepatocytes, and sinusoidal cells. Indirect immunofluorescence microscopy on nonfixed and paraformaldehyde-fixed cultured hepatocytes and bile ductular cells further confirmed that both anti-hepatocyte and anti-ductular oval cell antibodies recognized surface-exposed components on the respective cell types.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structure and function of xanthine oxidoreductase: where are we now?

Xanthine oxidoreductase (XOR) is a complex molybdoflavoenzyme, present in milk and many other tissues, which has been studied for over 100 years. While it is generally recognized as a key enzyme in purine catabolism, its structural complexity and specialized tissue distribution suggest other functions that have never been fully identified. The publication, just over 20 years ago, of a hypothesis implicating XOR in ischemia-reperfusion injury focused research attention on the enzyme and its ability to generate reactive oxygen species (ROS). Since that time a great deal more information has been obtained concerning the tissue distribution, structure, and enzymology of XOR, particularly the human enzyme. XOR is subject to both pre- and post-translational control by a range of mechanisms in response to hormones, cytokines, and oxygen tension. Of special interest has been the finding that XOR can catalyze the reduction of nitrates and nitrites to nitric oxide (NO), acting as a source of both NO and peroxynitrite. The concept of a widely distributed and highly regulated enzyme capable of generating both ROS and NO is intriguing in both physiological and pathological contexts. The details of these recent findings, their pathophysiological implications, and the requirements for future research are addressed in this review.     

Immunohistological analysis of glutathione transferase A4 distribution in several human tissues using a specific polyclonal antibody.

We examined the cellular distribution of glutathione transferase A4 (GSTA4) in various human tissues by indirect immunoperoxidase using a specific polyclonal antibody raised in rabbit. This enzyme was localized in hepatocytes, bile duct cells, and vascular endothelial cells in liver, upper layers of keratinocytes and sebaceous and sweat glands in skin, proximal convoluted tubules in kidney, epithelial cells of mucosa and muscle cells in colon, muscle cells in heart, and neurons in brain. Staining was increased in pathological situations such as cirrhosis, UV-irradiated skin, and myocardial infarction and was strongly decreased in hepatocellular carcinoma. These results strongly support the view of a close correlation between cellular GSTA4 localization and the formation of reactive oxygen species in the tissues investigated.     

Bile system morphogenesis defects and liver dysfunction upon targeted deletion of HNF1beta

The inactivation of the Hnf1beta gene identified an essential role in epithelial differentiation of the visceral endoderm and resulted in early embryonic death. In the present study, we have specifically inactivated this gene in hepatocytes and bile duct cells using the Cre/loxP system. Mutant animals exhibited severe jaundice caused by abnormalities of the gallbladder and intrahepatic bile ducts (IHBD). The paucity of small IHBD was linked to a failure in the organization of duct structures during liver organogenesis, suggesting an essential function of Hnf1b in bile duct morphogenesis. Mutant mice also lacked interlobular arteries. As HNF1beta is not expressed in these cells, it further emphasizes the link between arterial and biliary formation. Hepatocyte metabolism was also affected and we identified hepatocyte-specific HNF1beta target genes involved in bile acids sensing and in fatty acid oxidation.     

Xanthine oxidoreductase: a journey from purine metabolism to cardiovascular excitation-contraction coupling

Xanthine oxidoreductase (XOR) is a ubiquitous complex cytosolic molybdoflavoprotein which controls the rate limiting step of purine catabolism by converting xanthine to uric acid. It is known that optimum concentrations of uric acid (UA) and reactive oxygen species (ROS) are necessary for normal functioning of the body. The ability of XOR to perform detoxification reactions, and to synthesize UA and reactive oxygen species (ROS) makes it a versatile intra- and extra-cellular protective "housekeeping enzyme". It is also an important component of the innate immune system. The enzyme is a target of drugs against gout and hyperuricemia and the protein is of major interest as it is associated with ischemia reperfusion (I/R) injury, vascular disorders in diabetes, cardiovascular disorders, adipogenesis, metabolic syndrome, cancer, and many other disease conditions. Xanthine oxidoreductase in conjugation with antibodies has been shown to have an anti-tumor effect due to its ability to produce ROS, which in turn reduces the growth of cancer tissues. Apart from this, XOR in association with nitric oxide synthase also participates in myocardial excitation-contraction coupling. Although XOR was discovered over 100 years ago, its physiological and pathophysiological roles are still not clearly elucidated. In this review, various physiological and pathophysiological functional aspects of XOR and its association with various forms of cancer are discussed in detail.     

Xanthine oxidoreductase: A journey from purine metabolism to cardiovascular excitation-contraction coupling

Xanthine oxidoreductase (XOR) is a ubiquitous complex cytosolic molybdoflavoprotein which controls the rate limiting step of purine catabolism by converting xanthine to uric acid. It is known that optimum concentrations of uric acid (UA) and reactive oxygen species (ROS) are necessary for normal functioning of the body. The ability of XOR to perform detoxification reactions, and to synthesize UA and reactive oxygen species (ROS) makes it a versatile intra- and extra-cellular protective “housekeeping enzyme”. It is also an important component of the innate immune system. The enzyme is a target of drugs against gout and hyperuricemia and the protein is of major interest as it is associated with ischemia reperfusion (I/R) injury, vascular disorders in diabetes, cardiovascular disorders, adipogenesis, metabolic syndrome, cancer, and many other disease conditions. Xanthine oxidoreductase in conjugation with antibodies has been shown to have an anti-tumor effect due to its ability to produce ROS, which in turn reduces the growth of cancer tissues. Apart from this, XOR in association with nitric oxide synthase also participates in myocardial excitation-contraction coupling. Although XOR was discovered over 100 years ago, its physiological and pathophysiological roles are still not clearly elucidated. In this review, various physiological and pathophysiological functional aspects of XOR and its association with various forms of cancer are discussed in detail.     

Contribution of Mature Hepatocytes to Biliary Regeneration in Rats with Acute and Chronic Biliary Injury

Whether hepatocytes can convert into biliary epithelial cells (BECs) during biliary injury is much debated. To test this concept, we traced the fate of genetically labeled [dipeptidyl peptidase IV (DPPIV)-positive] hepatocytes in hepatocyte transplantation model following acute hepato-biliary injury induced by 4,4’-methylene-dianiline (DAPM) and D-galactosamine (DAPM+D-gal) and in DPPIV-chimeric liver model subjected to acute (DAPM+D-gal) or chronic biliary injury caused by DAPM and bile duct ligation (DAPM+BDL). In both models before biliary injury, BECs are uniformly DPPIV-deficient and proliferation of DPPIV-deficient hepatocytes is restricted by retrorsine. We found that mature hepatocytes underwent a stepwise conversion into BECs after biliary injury. In the hepatocyte transplantation model, DPPIV-positive hepatocytes entrapped periportally proliferated, and formed two-layered plates along portal veins. Within the two-layered plates, the hepatocytes gradually lost their hepatocytic identity, proceeded through an intermediate state, acquired a biliary phenotype, and subsequently formed bile ducts along the hilum-to-periphery axis. In DPPIV-chimeric liver model, periportal hepatocytes expressing hepatocyte nuclear factor-1β (HNF-1β) were exclusively DPPIV-positive and were in continuity to DPPIV-positives bile ducts. Inhibition of hepatocyte proliferation by additional doses of retrorsine in DPPIV-chimeric livers prevented the appearance of DPPIV-positive BECs after biliary injury. Moreover, enriched DPPIV-positive BEC/hepatic oval cell transplantation produced DPPIV-positive BECs or bile ducts in unexpectedly low frequency and in mid-lobular regions. These results together suggest that mature hepatocytes but not contaminating BECs/hepatic oval cells are the sources of periportal DPPIV-positive BECs. We conclude that mature hepatocytes contribute to biliary regeneration in the environment of acute and chronic biliary injury through a ductal plate configuration without the need of exogenously genetic or epigenetic manipulation.     

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.     

Identification, characterization, and immunolocalization of a nucleoside triphosphate diphosphohydrolase in pig liver

Different isoforms of nucleoside triphosphate diphosphohydrolases (NTPDases; EC 3.6.1.5), also identified as ATP diphosphohydrolases, have been previously described in mammalian tissues. We report here the biochemical characterization of NTPDases in the pig liver. Optimum pH of catalysis is more acidic for this enzyme than for NTPDases (neutral or alkaline pH) found in other mammalian tissues. It is less sensitive to bile salts than the bovine spleen NTPDase. Calculated Km values for ATP and ADP (31 and 21 microM, respectively) are slightly higher than those reported for the latter enzyme. Electrophoretograms of these enzymes also show different migration patterns. Western blots with Ringo, an antibody that recognizes the different isoforms of mammalian NTPDases, show a small but reproducible difference in estimated molecular masses (75 kDa for liver vs 78 kDa for spleen NTPDase). A second antibody, generated against a different sequence of NTPDase I, does not recognize the liver enzyme, thereby indicating some differences in primary structure. Immunolocalization produced a strong signal on hepatocytes, epithelial cells of the bile duct system, and vascular cells. Immunoreactivity was variable among hepatocytes of different lobules and among hepatocytes within a given lobule. In general, those located in the perilobular zone were more reactive than those located in the central zone and in the periphery of the centrolobular vein.     

Cellular characteristics of epithelial cell lines from juvenile rat liver: selective induction of glutamine synthetase by dexamethasone

Four epithelial cell lines established from juvenile rat liver and selected on the basis of their capacity to prolong the lifespan of cocultured hepatocytes were compared with respect to several immunocytochemical markers (vimentin, cytokeratin 19, MAB 19C6), enzyme activities, and amino acid uptake systems. Their phenotypes were found to be quite different from that of hepatocytes and bile duct epithelial cells (BEC), but very similar among each other. In particular, a variety of functions affected by dexamethasone (DEX) or changing spontaneously in cultured hepatocytes and/or BEC, showed neither inducibility nor spontaneous changes in the four cell lines. Instead, the lines were inducible for glutamine synthetase (GS) by DEX, in contrast to hepatocytes and BEC but also to other juvenile or adult epithelial lines that did not support cocultured hepatocytes. In addition, they showed relatively high basal levels of GS activity, exceeding those found in adult epithelial cell lines and approaching the average values found for liver tissue. Basal as well as DEX-induced GS activity was reduced in the presence of newborn calf serum, while only DEX-induced but not basal activity was suppressed by glutamine.These results suggest an origin of these four juvenile epithelial cell lines different from that of hepatocytes as well as of BEC. Furthermore, they suggest the coherent acquisition of new functional properties during early phases of cultivation of these cell lines; the selective inducibility of GS by DEX and its suppression by glutamine are the most intriguing of these, because neither is found in any normal cell type present in rat liver.     

Liver stem/progenitor cells: their characteristics and regulatory mechanisms

Liver stem cells give rise to both hepatocytes and bile duct epithelial cells also known as cholangiocytes. During liver development hepatoblasts emerge from the foregut endoderm and give rise to both cell types. Colony-forming cells are present in the liver primordium and clonally expanded cells differentiate into either hepatocytes or cholangiocytes depending on culture conditions, showing stem cell characteristics. The growth and differentiation of hepatoblasts are regulated by various extrinsic signals. For example, periportal mesenchymal cells provide a cue for bipotential hepatoblasts to become cholangiocytes, and mesothelial cells covering the parenchyma support the expansion of foetal hepatocytes by producing growth factors. The adult liver has an extraordinary capacity to regenerate, and after 70% hepatectomy the liver recovers its original mass by replication of the remaining hepatocytes without the activation of liver stem cells. However, in certain types of liver injury models, liver stem/progenitor-like cells, known as oval cells in rodents, proliferate around the portal vein, while the roles of such cells in liver regeneration remain a matter of debate. Clonogenic and bipotential cells are also present in the normal adult liver. In this minireview we describe recent studies on liver stem/progenitor cells by focusing on extracellular signals.     

Increased serum level of xanthine oxidoreductase in liver transplanted patients

Xanthine oxidoreductase (XOR) leakage into serum has been observed in various types of liver pathology as well as after liver transplantation (LT). We determined the amount of XOR associated with LT to investigate the changes in serum enzyme level during the LT procedure and the post-operative period. Additionally, we examined whether there was any correlation between XOR levels and the surgical technique. XOR levels were measured by a competitive ELISA. In a first group of patients, the portal vein was flushed before the liver and systemic reperfusions, which occurred simultaneously. In the second group, the graft was flushed with blood from the portal vein before the systemic reperfusion. XOR showed a marked elevation in the caval effluent collected during LT and was higher compared to control serum levels at all time points that were examined after LT. The XOR levels during LT were also higher than samples taken pre-LT or from the portal blood flush before reperfusion. The XOR level was higher in Group 2 than in Group 1. Enhancement of the XOR serum level during LT was not derived from enterocytes, and it should be attributed to enzyme leakage from graft liver cells. We report the elevation of serum XOR during the three weeks following LT for the first time, as well as the influence of the graft reperfusion technique on XOR serum levels.     

Pathophysiologic implications of innate immunity and autoinflammation in the biliary epithelium

The most studied physiological function of biliary epithelial cells (cholangiocytes) is to regulate bile flow and composition, in particular the hydration and alkalinity of the primary bile secreted by hepatocytes. After almost three decades of studies it is now become clear that cholangiocytes are also involved in epithelial innate immunity, in inflammation, and in the reparative processes in response to liver damage. An increasing number of evidence highlights the ability of cholangiocyte to undergo changes in phenotype and function in response to liver damage. By participating actively to the immune and inflammatory responses, cholangiocytes represent a first defense line against liver injury from different causes. Indeed, cholangiocytes express a number of receptors able to recognize pathogen- or damage-associated molecular patterns (PAMPs/DAMPs), such as Toll-like receptors (TLR), which modulate their pro-inflammatory behavior. Cholangiocytes can be both the targets and the initiators of the inflammatory process. Derangements of the signals controlling these mechanisms are at the basis of the pathogenesis of different cholangiopathies, both hereditary and acquired, such as cystic fibrosis-related liver disease and sclerosing cholangitis. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.     

Lactogenic hormones regulate xanthine oxidoreductase and beta-casein levels in mammary epithelial cells by distinct mechanisms

Xanthine oxidoreductase (XOR) is a prominent component of the milk lipid globule, whose concentration is selectively increased in mammary epithelial cells during the transition from pregnancy to lactation. To understand how XOR expression is controlled in the mammary gland, we investigated its properties and regulation by lactogenic hormones in cultured HC11 mammary epithelial cells. XOR was purified as the NAD(+)-dependent dehydrogenase by benzamidine-Sepharose chromatography and was shown to be intact and to have biochemical properties similar to those of enzyme from other sources. Treating confluent HC11 cells with prolactin and cortisol produced a progressive, four- to fivefold, increase in XOR activity, while XOR activity in control cells remained constant. Elevated cellular XOR activity was correlated with increased XOR protein and was due to both increased synthesis and decreased degradation of XOR. Prolactin and cortisol increased XOR protein and mRNA in the presence of epidermal growth factor, which blocked the stimulation of beta-casein synthesis by these hormones. Further, hormonal stimulation of XOR was inhibited by genistein (a protein tyrosine kinase inhibitor) and by PD 98059 (a specific inhibitor of the MAP kinase cascade). These findings indicate that lactogenic hormones stimulate XOR and beta-casein expression via distinct pathways and suggest that a MAP kinase pathway mediates their effects on XOR. Our results provide evidence that lactogenic hormones regulate milk protein synthesis by multiple signaling pathways.     

Regulation of intrahepatic biliary duct morphogenesis by Claudin 15-like b

The intrahepatic biliary ducts transport bile produced by the hepatocytes out of the liver. Defects in biliary cell differentiation and biliary duct remodeling cause a variety of congenital diseases including Alagille Syndrome and polycystic liver disease. While the molecular pathways regulating biliary cell differentiation have received increasing attention (Lemaigre, 2010), less is known about the cellular behavior underlying biliary duct remodeling. Here, we have identified a novel gene, claudin 15-like b (cldn15lb), which exhibits a unique and dynamic expression pattern in the hepatocytes and biliary epithelial cells in zebrafish. Claudins are tight junction proteins that have been implicated in maintaining epithelial polarity, regulating paracellular transport, and providing barrier function. In zebrafish cldn15lb mutant livers, tight junctions are observed between hepatocytes, but these cells show polarization defects as well as canalicular malformations. Furthermore, cldn15lb mutants show abnormalities in biliary duct morphogenesis whereby biliary epithelial cells remain clustered together and form a disorganized network. Our data suggest that Cldn15lb plays an important role in the remodeling process during biliary duct morphogenesis. Thus, cldn15lb mutants provide a novel in vivo model to study the role of tight junction proteins in the remodeling of the biliary network and hereditary cholestasis.     

Biliary secretion of endotoxin and pathogenesis of primary biliary cirrhosis.

Previous studies suggested endotoxin, derived from the intestine through the portal blood to the liver, was predominantly metabolized by Kupffer cells. In the present study, fluorescent-labeled endotoxin injected into the rat portal vein was demonstrated not only in Kupffer cells but also in hepatocytes. Furthermore a great amount of labeled endotoxin was recovered in bile. In the livers of patients with primary biliary cirrhosis (PBC), immunohistochemistry demonstrated significant retention of endotoxin in the biliary epithelial cells, and treatment with ursodeoxycholic acid significantly reduced the retention in those cells. The study for detection of apoptosis demonstrated increased rates of apoptosis in hepatocytes and biliary epithelial cells in PBC liver, and the rate of apoptosis in biliary epithelial cells was significantly reduced after treatment with ursodeoxycholic acid. Immunohistochemistry in PBC liver demonstrated significant reduction of fluorescence intensity for a 7H6 antigen in biliary epithelial cells, indicating the increased paracellular permeability of bile ducts, because cellular immunolocalization of that antigen has been shown to be inversely correlated with the paracellular permeability of the tight junction. These results suggest that, in biliary epithelial cells, retention of endotoxin, increased apoptosis, and increased permeability of tight junctions may be involved in the pathogenesis of PBC.