Expression profiles of MUC mucins and trefoil factor family (TFF) peptides in the intrahepatic biliary system: Physiological distribution and pathological significance

ARTICLE IN PRESS PROGRESS IN HISTOCHEMISTRY AND CYTOCHEMISTRY Progress in Histochemistry and Cytochemistry 42 (2007) 61–110 www.elsevier.de/proghi

REVIEW

Expression profiles of MUC mucins and trefoil factor family (TFF) peptides in the intrahepatic biliary system: Physiological distribution and pathological significance Motoko Sasaki, Hiroko Ikeda, Yasuni Nakanuma Department of Human Pathology, Kanazawa University Graduate School of Medicine, Takaramachi 13-1, Kanazawa 920-8640, Japan

Abstract Mucin secreted by mucosal epithelial cells plays a role in the protection of the mucosal surface and also is involved in pathological processes. So far, MUC1–4, 5AC, 5B, 6–8, 11–13 and 15–17 genes coding the backbone mucin core protein have been identified in humans. Their diverse physiological distribution and pathological alterations have been reported. Trefoil factor family (TFF) peptides are mucin-associated molecules co-expressed with MUC mucins and involved in the maintenance of mucosal barrier and the biological behavior of epithelial and carcinoma cells. Intrahepatic biliary system is a route linking the bile canaliculi and the extrahepatic bile duct for the excretion of bile synthesized by hepatocytes. Biliary epithelial cells line in the intrahepatic biliary system, secreting mucin and other molecules Abbreviations: AAs, amino acids; ADPKD, autosomal dominant polycystic kidney disease; AMA, antimitochondrial antibodies; AMOP, adhesion-associated domain in MUC4 and other proteins; BECs, biliary epithelial cells; BMH, biliary microhamartoma; CK, cysteine knot motif; CKs, cytokeratins; CNSDC, chronic non-suppurative destructive cholangitis; CVH, chronic viral hepatitis; Cys, cysteine; DMBT1, deleted in malignant brain tumor-1; EBO, extrahepatic bile duct obstruction; EGF, epidermal growth factor; EGF-R, EGF receptor; EMA, epithelial membrane antigen; GI, gastrointestinal; HCC, hepatocellular carcinoma; HC-CC, combined hepatocellular cholangiocarcinoma; ICC, intrahepatic cholangiocarcinoma; IL, interleukin; ITF, intestinal trefoil factor; LPS, lipopolysaccharide; MAb, monoclonal antibody; NIDO, nidogen domain; PBC, primary biliary cirrhosis; PK, protein kinase; SEA, sea urchin sperm protein, enterokinase and agrin; TFF, trefoil factor family; TGF-a, transforming growth factor-a; TM, transmembrane domain; TNF-a, tumor necrosis factor-a; VWC, von Willebrand factor C-like domains; VWD, von Willebrand factor D-like domains; VWF, von Willebrand factor. Corresponding author. Tel.: +81 76 265 2197; fax: +81 76 234 4229. E-mail address: [email protected] (M. Sasaki). 0079-6336/$ - see front matter r 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.proghi.2007.02.001

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involved in the maintenance and regulation of the system. In this review, the latest information regarding properties, expression profiles and regulation of MUC mucins and TFF peptides in the intrahepatic biliary system is summarized. In particular, we focus on the expression profiles and their significance of MUC mucins in developmental and normal livers, various hepatobiliary diseases and intrahepatic cholangiocarcinoma. r 2007 Elsevier GmbH. All rights reserved. Keywords: MUC mucin core protein; Trefoil factor family (TFF); Intrahepatic biliary system; Biliary epithelial cells; Hepatolithiasis; Cholangiocarcinoma; Biliary epithelial dysplasia

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 Anatomy and embryology of the intrahepatic biliary system . . . . . . . . . . . . . . . . 64 2.1. The normal morphology of the intrahepatic bile ducts and peribiliary glands . . 65 2.1.1. The intrahepatic bile ducts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 2.1.2. The intrahepatic peribiliary glands . . . . . . . . . . . . . . . . . . . . . . . . . 67 2.2. Development of intrahepatic biliary system . . . . . . . . . . . . . . . . . . . . . . . . 68 Properties of MUC mucins and TFF peptides . . . . . . . . . . . . . . . . . . . . . . . . . . 70 3.1. Properties of MUC mucins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 3.1.1. Structures of mucins and carbohydrate side chains . . . . . . . . . . . . . 70 3.1.2. Classification of MUC mucin gene products . . . . . . . . . . . . . . . . . . 71 3.1.3. Membrane-bound mucins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 3.1.4. Secreted gel-forming mucins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 3.1.5. Small soluble mucins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 3.1.6. Regulation of MUC mucins expression. . . . . . . . . . . . . . . . . . . . . . 78 3.2. Properties of TFF peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78 Detection of mRNA and protein expression of MUC mucins and TFF peptides . . 80 4.1. Detection of mRNA expression of MUC mucins and TFF peptides . . . . . . 80 4.2. Detection of protein expression of MUC mucins and TFF peptides. . . . . . . 80 Expression of MUC mucins and TFF peptides in the fetal and normal adult intrahepatic biliary system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 5.1. Physiological expression of MUC mucins . . . . . . . . . . . . . . . . . . . . . . . . . 81 5.2. Physiological expression of TFF peptides . . . . . . . . . . . . . . . . . . . . . . . . . 84 Expression of MUC mucins and TFF peptides in the intrahepatic biliary system in non-tumorous hepatobiliary diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 6.1. Expression of MUC mucins and TFFs peptides in hepatolithiasis . . . . . . . . 84 6.1.1. Hepatolithiasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 6.1.2. Augmented expression of gel-forming MUC mucins in hepatolithiasis . 84 6.1.3. Coordinated expression of TFF peptides with MUC mucins in hepatolithiasis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 6.1.4. Regulation of the expression of MUC mucins in hepatolithiasis . . . . 88 6.2. Expression of MUC mucins and TFFs peptides in primary biliary cirrhosis (PBC) and other inflammatory hepatobiliary diseases . . . . . . . . . . . . . . . . . 88 6.2.1. PBC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 6.2.2. Altered expression of MUC mucins in PBC and other inflammatory hepatobiliary diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

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Altered expression of TFF peptides in PBC and other inflammatory hepatobiliary diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 6.3. Expression of MUC mucins in the hepatobiliary cystic diseases. . . . . . . . . . 90 6.3.1. The hepatobiliary cystic diseases . . . . . . . . . . . . . . . . . . . . . . . . . . 90 6.3.2. Altered expression of MUC mucins in the cystic liver diseases . . . . . 91 MUC and TFF expression in biliary epithelial dysplasia and cholangiocarcinoma . . 91 7.1. Intrahepatic cholangiocarcinoma (ICC). . . . . . . . . . . . . . . . . . . . . . . . . . . 91 7.1.1. ICC associated with hepatolithiasis . . . . . . . . . . . . . . . . . . . . . . . . 92 7.1.2. ICC associated with CVH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 7.2. Pathology of ICC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 7.3. Expression of MUC mucins in ICC and biliary epithelial dysplasia . . . . . . . 93 7.3.1. Increased expression of MUC5AC in biliary epithelial dysplasia . . . . 93 7.3.2. Expression profiles and its significance of MUC mucins in ICC . . . . 94 7.4. Expression of TFF peptides in ICC . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 7.5. Expression of MUC mucins and TFF peptides in gallbladder carcinoma, extrahepatic bile duct carcinoma and ampullary carcinoma . . . 100 Concluding remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

1. Introduction The intrahepatic biliary system, which links to the bile canaliculi at the proximal side and to the extrahepatic ducts at the distal site, is a route for the excretion of bile synthesized by hepatocytes. The intrahepatic biliary system, also called the intrahepatic bile ducts, comprises a complex three-dimensional network of conduits within the liver (the biliary tree) starting with Hering canals and ending at the hepatic hilum. The intrahepatic bile ducts are lined by specialized epithelial cells called biliary epithelial cells (BECs) or cholangiocytes (Lazaridis et al., 2004). Hepatocytes produce primary hepatic bile, which percolates through the intrahepatic bile ducts; during this journey, bile is modified by BECs via a series of secretory and absorptive processes that provide additional bile water (BECs secrete 40% of daily bile production in humans) or secrete HCO 3 to induce the alkaline state (Lazaridis et al., 2004). BECs interact with the immune system and microorganisms and are also involved in drug metabolism. To accomplish these functions, BECs display morphological and functional heterogeneity along the intrahepatic bile ducts. In normal livers, the smallest ductules are rimmed by no more than a few minimally differentiated cuboidal epithelial cells. The BECs lining large bile ducts gradually become columnar and more differentiated mucus-secreting cells. The intrahepatic biliary system is known to share many physiological and structural characteristics of the gastrointestinal (GI) tract, including the contact of the mucosal surface with toxic substances and the intraluminal secretion of mucin. Mucins are heavily glycosylated, generally high-molecular-weight proteins that are synthesized by the epithelial cells in many organs; for example, the GI, respiratory and genito-urinary tract, and play a role in the protection of mucosal surface. They

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consist of protein backbone structures (apomucins) and many carbohydrate side chains. There are two structurally and functionally distinct classes of mucins: secreted gel-forming mucins (MUC2, MUC5AC, MUC5B and MUC6) and transmembrane mucins (MUC1, MUC3, MUC4, MUC12 and MUC17), although the products of some MUC genes do not fit well into either class (MUC7, MUC8, MUC9, MUC13, MUC15 and MUC16) (details are described below). Each MUC mucin shows a characteristic distribution in organs and cell types (Kim and Gum, 1995; Gum et al., 1999; Horinouchi et al., 2003). In these MUC mucins, MUC1 mucin is most widely distributed in epithelial and carcinoma cells. The epitope of epithelial membrane antigen (EMA), which is a common immunohistochemical marker to detect epithelial cells and carcinomas, is on the MUC1 mucin of the glycosylated form. Altered mucin gene expression has been reported in inflammatory diseases and carcinomas of the GI tract and breast (Kim and Gum, 1995; Gum et al., 1999; Horinouchi et al., 2003). Accumulating data suggest that each MUC mucin has different properties; for example, the presence of an epidermal growth factor (EGF)like domain, a transmembrane region and a gel-forming capacity, and may have diverse functions. Therefore, the altered expression of MUC mucin appears to be somewhat involved in the pathogenesis of inflammatory diseases and in tumor biology. For example, MUC1 over-expression is most evident, and these rigid mucin glycoproteins on cancer cells play a role in metastasis by inhibiting tumor cell adhesion and in escaping from immune surveillance. MUC4 is a novel intramembrane ligand for receptor tyrosine kinase ErbB2 (HER-2) (Komatsu et al., 2001), and the increased expression of MUC4 is related to a poorer prognosis in patients with intrahepatic cholangiocarcinoma (ICC) of the mass-forming type (Shibahara et al., 2004a). In this review, we summarize the latest information regarding properties, expression profiles and regulation of mucin and trefoil factor family (TFF) peptides in the intrahepatic biliary system. In particular, we focus on the expression profiles and their significance of MUC mucins in developmental and normal livers, various hepatobiliary diseases, biliary epithelial dysplasia as a precancerous lesion and ICC.

2. Anatomy and embryology of the intrahepatic biliary system The liver is the largest organ in the body, it weighs 1200–1500 g and comprises one fiftieth of the total adult body weight. The liver and its companion extrahepatic biliary tree and gallbladder dominate the right upper quadrant of the abdomen. Residing at the crossroads between the digestive tract and the rest of body, the liver has the enormous task of maintaining the body’s metabolic homeostasis. This includes the processing of dietary amino acids (AAs), carbohydrates, lipids and vitamins; synthesis of serum proteins; and detoxification and excretion of endogenous waste products and pollutant xenobiotics into bile. Hepatocytes comprise about 60% of the liver, and the other components include sinusoidal endothelial cells, Kupffer cells and BECs of the intrahepatic biliary system.

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2.1. The normal morphology of the intrahepatic bile ducts and peribiliary glands 2.1.1. The intrahepatic bile ducts The intrahepatic biliary system is a route for the excretion of bile synthesized by hepatocytes and links to the bile canaliculi at the proximal side and to the extrahepatic ducts at the distal site. The intrahepatic biliary system comprises a complex three-dimensional network of conduits within the liver starting with Hering canals and ending at the hepatic hilum. The intrahepatic branching of the bile ducts is visualized in a biliary injection cast showing the ‘‘biliary tree’’ (Fig. 1) (Portmann and Nakanuma, 2002). There is no sharp delineation of the various segments, the intrahepatic biliary system is divided into the intrahepatic large bile ducts and small bile ducts (Nakanuma and Sasaki, 1989). Table 1 summarizes the classification and features of the intrahepatic bile ducts. Fig. 2 shows the representative features of BECs in large and small intrahepatic bile ducts. The large bile ducts correspond to the right and left hepatic ducts, segmental (large perihilar) ducts, and the first and second branches of the large area ducts. The large bile ducts are grossly visible, and are characterized by the presence of a fibrous ductal wall and surrounding intrahepatic peribiliary glands. The large bile ducts are lined with tall columnar cells (Fig. 2). The intrahepatic peribiliary glands are constantly located around the intrahepatic large bile ducts as described below (Terada et al., 1987; Nakanuma et al., 1994). The intrahepatic small bile ducts, which are recognizable by light microscopy correspond to the septal and interlobular bile ducts as well as to bile ductules (Nakanuma and Sasaki, 1989). Like the large intrahepatic bile ducts, the septal bile ducts (4100 mm in diameter) are lined by tall columnar cells with basal nuclei and are surrounded by a layer of hypocellular collagen (Fig. 2). In contrast, the interlobular bile ducts are connected to the bile canalicular network by ductules or cholangioles (o15 mm diameter) similarly lined by cuboidal cells and the canals of Hering that are lined partly by BECs and partly by hepatocytes. Neither ductules nor

Fig. 1. Gross appearance of the normal liver and the intrahepatic biliary system. (A) Crosssection of normal liver. (B) Biliary cast of the normal adult liver prepared at autopsy. CBD, common bile duct.

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Table 1. Classification and terminology of the intrahepatic biliary system Terminology

Diameter (mm)

Remarks

Large bile ducts Left and right hepatic ducts

4800

Segmental ducts

400–800

Area ducts

300–400

Columnar (cylindrical) epithelium surrounded by dense fibrous tissue and elastic fibers Peribiliary glands Columnar (cylindrical) epithelium surrounded by dense fibrous tissue and elastic fibers Peribiliary glands Lower columnar (cylindrical) epithelium Peribiliary glands

Small intrahepatic bile ducts Septal bile ducts

4100

Interlobular bile ducts

15–100

Medium-sized Small Bile ductures (cholangioles)

40–100 15–40 o15

Canals of Hering

Lower columnar (cylindrical) – cuboidal epithelium Cuboidal epithelium accompanied by artery

Located in periphery of portal tracts (near limiting plate) Lined by hepatocytes, partly by biliary epithelial cells

Fig. 2. Histology of the intrahepatic bile ducts in the normal liver. (A) Intrahepatic large bile duct. Columnar biliary epithelial cells are lining. (B) Intrahepatic small bile duct (interlobular bile duct). Cuboidal epithelial cells form a tubular structure (arrow) in a portal tract. A, artery; PV, portal vein. HE staining.

canals of Hering are clearly identifiable in hematoxylin and eosin stained sections of normal liver, but they become obvious in various pathological conditions (Portmann and Nakanuma, 2002).

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The intrahepatic bile ducts are lined by specialized epithelial cells called BECs or cholangiocytes (Lazaridis et al., 2004). Immunostaining for cytokeratins (CKs) 7 and 19 is widely used to identify BECs. CKs are the intermediate filaments of epithelial cells and 19 different types have been identified (Moll et al., 1982). Hepatocytes produce primary hepatic bile, which percolates through the intrahepatic bile ducts; during this journey, bile is modified by BECs via a series of secretory and absorptive processes that provide additional bile water (BECs secrete 40% of daily bile production in humans) or secrete HCO 3 to induce the alkaline state) (Lazaridis et al., 2004). BECs interact with the immune system and microorganisms and are also involved in drug metabolism. To accomplish these functions, BECs display morphological and functional heterogeneity along the biliary tree. In normal livers, the smallest ductules are rimmed by no more than a few minimally differentiated cuboidal epithelial cells. The BECs lining progressively large bile ducts gradually become more differentiated, mucus-secreting cells (Table 1). The intrahepatic biliary system is known to share many physiological and structural characteristics of the GI tract, including the contact of the mucosal surface with toxic substances and the intraluminal secretion of mucin. 2.1.2. The intrahepatic peribiliary glands The intrahepatic peribiliary glands are present in the large intrahepatic bile ducts (Terada et al., 1987; Nakanuma et al., 1994). In approximate 60% of normal livers, the glands are observed to be present in hepatic and segmental bile ducts, and in about 40% they are seen in the hepatic, segmental and area ducts (Terada et al., 1987). The intrahepatic peribiliary glands are composed of the following two elements: intramural and extramural glands (Terada et al., 1987; Nakanuma et al., 1994). The lobules of individual extramural glands, which are branched tubuloalveolar seromucous glands do not communicate with hepatocytes, but do communicate with bile ducts via conduits (Terada et al., 1987). The intramural glands are non-branching or sparsely branching tubular mucous glands located within the bile duct wall. The intramural glands are composed of tall columnar cells with clear cytoplasm and basally situated nuclei. Hyperplasia of the intramural glands is frequently seen in hepatolithiasis (Fig. 3). In contrast, the extramural glands are composed of many lobules that are located in the connective tissue outside the bile duct wall (Fig. 3) (Terada et al., 1987; Nakanuma et al., 1994). The lobules of the extramural glands are surrounded by concentrically arranged dense connective tissues and are composed of many serous and mucous acini; some lobules are composed largely of serous acini, others of mucous acini, and still others of a mixture of serous and mucous acini. Serous acini outnumber mucous acini (Terada et al., 1987). The mucous cells of the mucous acini consist of tall columnar cells with a clear cytoplasm and basally located nuclei, while cells of serous acini are composed of low columnar or cuboidal cells with basophilic cytoplasm. Endocrine cells including argentaffin and argyrophil cells and cells containing several hormones (somatostatin, serotonin and pancreatic polypeptide) have been detected in the extramural glands of the peribiliary glands (Kurumaya et al., 1989, 1990).

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Fig. 3. Histology of the peribiliary glands in hepatolithiasis. (A) Hyperplasia of the intramural peribiliary gland in hepatolithiasis. (B) Hyperplasia of mucous acini of the extramural peribiliary gland. Extramural glands are arranged as lobules surrounded by fibrous tissue. HE staining.

2.2. Development of intrahepatic biliary system In human embryos, the liver first appears at the end of the 3rd week of development (MacSween et al., 2002). Its parenchyma is of endodermal origin and arises from the liver bud or hepatic diverticulum, which develops as a hollow midline outgrowth from the ventral wall of the future duodenum. During the 4th week budlike clusters of epithelial cells extend forwards and outwards from the hepatic diverticulum into the mesenchymal stroma, in which has appeared a hepatic sinusoidal plexus, fed by a vitelline venous plexus draining blood from the wall of the yolk sac. As the epithelial buds grow into the septum transversum, they break up into thick anastomosing epithelial sheets which meet and enmesh vessels of the hepatic sinusoidal plexus, forming the primitive hepatic sinusoids. The caudal part of the hepatic diverticulum does not contribute to the invading sheets of primitive hepatocytes but instead forms the epithelial primordium of the cystic duct and gallbladder. The hepatic bud gives rise not only to the epithelial parenchyma – the future hepatocytes – but also to the epithelial lining of the branching duct system, from its main stem, the common bile duct, to its terminal twigs, the smallest ductules, the canals of Hering (MacSween et al., 2002).The intrahepatic ducts, which link the bile canaliculi and the extrahepatic ducts, develop from the limiting plate of hepatoblasts which surround the branches of the portal vein (Bloom, 1925–26). The early structures of ductal elements were well demonstrated using immunohistochemical detection for CKs and cell surface markers (Burt et al., 1987; Van Eyken et al., 1988; Gall and Bhathal, 1989; Shah and Gerber, 1989; Desmet, 1992). Normal adult hepatocytes express CKs 8 and 18, whereas intrahepatic bile ducts, in addition, express CKs 7 and 19. During the first 7–8 weeks of embryonic development, no intrahepatic bile ducts are evident and the epithelial cells express CKs 8, 18 and 19. About 9–10 weeks (27–30 mm embryos) primitive hepatocytes (hepatoblasts) surrounding large portal vein branches near the liver hilum express these CKs more intensely and form a layer of cells which ensheaths the mesenchyme of the primitive

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portal tracts to form the so-called ductal plate (the ductal plate stage) (Fig. 4) (Bloom, 1925–26; Van Eyken et al., 1988). This is followed by a second but discontinuous layer of epithelial cells showing a similar phenotype change and so a segmentally double-layered plate is formed. From 12 weeks onwards a lumen develops in segments of the ductal plates forming double-layered cylindrical or tubular structures. Further remodeling of the plate occurs; invading connective tissue separates it from the liver parenchyma and the tubular structures become incorporated into the mesenchyme surrounding the portal vein branches (the remodeling ductal plate stage). An anastomosing network of bile ducts is formed, excess ductal epithelium undergoes resorption and bile ducts appear within the definitive portal tracts (the remodeled bile duct stage). The entire process of duct development progresses centrifugally from the porta hepatis and also from the larger to smaller portal tracts. However, this process may not be complete at 40 weeks of gestation, and full expression of CK7 is not found until about 1 month postpartum. Thus, the intrahepatic bile-duct system is still immature at birth (Van Eyken et al.,

Fig. 4. Development of the ductal plate and of the intrahepatic bile ducts. (A and B) Human fetus of 8 weeks’ gestation. Ductal structure is formed at the edge of portal area (arrows). (B) Ductal structure at the edge of portal area expresses CK19 (arrows). Immunohistochemical staining for CK19. (C) Human fetus of 13 weeks gestation. Double-layered ductal plate forms tubular structure focally (arrows). (D) Human fetus of 20 weeks’ gestation. Note the remodeled bile duct (arrow). Ductal plate structure still exists at the border (arrowheads).

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1988). Failure of remodeling and resorption produces the ductal plate malformation and may also be significant in the production of various congenital malformations of the intrahepatic biliary tree (Summerfields et al., 1986; Desmet, 1992). It is not known what factors determine whether hepatoblasts differentiate in one direction to hepatocytes, or in the other to duct epithelium. However, differentiation to ductal epithelium is associated, as first noted by Bloom (Bloom, 1925–26), by contact of primitive hepatic epithelium with young connective tissue developing in the portal tracts. There is now experimental evidence to indicate that the association is indeed one of cause and effect: in ectopic grafts of embryonic mouse liver, immature hepatocytes which are in contact with vascular endothelium tend to differentiate into mature hepatocytes, while those in contact with connective tissue cells differentiate into ductal epithelium (Shiojiri, 1984; Shiojiri and Sugiyama, 2004).

3. Properties of MUC mucins and TFF peptides Mucins are high-molecular weight epithelial glycoproteins with a high content of clustered oligosaccharides, O-glycosidically linked to tandem repeat peptides within threonine, serine and proline. Mucins are synthesized and secreted together with TFF peptides by the epithelial cells in many organs; for example in the GI, respiratory and genito-urinary tract, and play a role in the protection of mucosal surface. The high molecular secreted mucins and TFF peptides are responsible for the rheological properties of the mucus layer covering mucosa. TFF peptides have many other physiological functions in addition to their rheological properties, such as promotion of epithelial cell migration and anti-apoptotic properties, as described below (Hoffmann and Joba, 1995; Hoffmann et al., 2001). The collective physical properties of mucins (i.e., length, charge density, macromolecular architecture, interaction and mutual influences) have been related to a specific set of rheological parameters for the mucus gel (Ellingham et al., 1999). Interactions between mucosal epithelia and cells of the immune system are also, at least partly, achieved by mucin molecules or mucin domains (Leiper et al., 2001; Mitoma et al., 2003; Wahrenbrock et al., 2003; Aknin et al., 2004). Thus, the permeability of epithelia and both secretion and synthesis of mucins are complex responses to environmental and physiological factors. 3.1. Properties of MUC mucins 3.1.1. Structures of mucins and carbohydrate side chains Mucin consists of protein backbone structures (apomucins or mucin core protein) and many carbohydrate side chains. The carbohydrate content of mature mucins can account for 50–90% of the weight of the glycoprotein, and usually occurs in clusters. The presence of clustered O-linked oligosaccharides arises from the underlying presence of threonine- and serine-rich tandem repeat peptides and from the

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specificity of the polypeptidyl GalNAc transferases responsible for their synthesis. These clustered O-linked oligosaccharides result in extended regions of the molecule that has highly multiple binding sites for carbohydrate-specific antibodies and lectins. Oligosaccharides are not evenly distributed along the linear molecule, but concentrated in discrete regions, encoded by repeat sequences in the mucin gene (tandem repeats). These features, preserved in all the wet epithelia mucins protect, import on large polymeric mucins an appearance approaching a feathered wiggly worm: a central core with tufts of sugar chains (Paulsen and Berry, 2006). The peptide core, which is the stiffest part of these long polymers, is most likely coiled in solution, while the oligosaccharide chains that richly and unevenly decorate it occupy most of and determine the size of the spheroid molecular volume. The O-glycosidically linked olidosaccharhides of mucins can be described in terms of core type, backbone type and peripheral structures. Mucins produced by carcinoma cells have differences in both core carbohydrates and in peripheral carbohydrate structures that have been investigated as diagnostic and prognostic markers (Byrd and Bresalier, 2004). Cancer mucins typically have increases in three core structures: Tn antigen (GalNAcaThr/Ser), TF antigen (Galb3GalNAc) and sialyl Tn (NeuAca6GalNac). The type 3 cores (GlcNAcb3GalNAc) predominate in normal tissue, mucin is lacking in colon cancer mucins. There are cancer-associated alterations in the peripheral carbohydrates of mucins including a decrease in O-acetyl-sialic acid and a decrease in sulfation. There are cancer-associated increases in sialyl LeX and related structures on mucins and other glycoproteins that can serve as ligands for selectins, increasing the metastatic capacity of carcinoma cells (Byrd and Bresalier, 2004). 3.1.2. Classification of MUC mucin gene products The similarity in general architectures of mucin genes leads to the hypothesis that they arose by duplication of a common ancestral gene (Desseyn et al., 2000), with perhaps further local doubling and mobilization to a protective and interactive function. This is, however, accompanied by a large degree of variability, both genetic and epigenetic. The mucin molecules are regularly referred to by the same names, MUC1, MUC2, etc., for humans, Muc1, Muc2, etc. for their animal orthologues. Genes encoding mucin proteins have been identified and named chronologically as MUC1–MUC17 (Table 2). Nomenclature of the MUC genes has not been straightforward. MUC3A and MUC3B, both located at 7q22 correspond to two genes originally designated MUC3. Simultaneous discovery, for example, revealed MUC5A, MUC5B and MUC5C, and two of the proposed genes turned out to be one, hence MUC5AC. Conversely, cDNAs designated to MUC11 and MUC12 may be derived from two closely related genes or may be parts of the same gene. Murine MUC10 and MUC14 have no human counterparts, so far. MUC9, encoding the mucin-like structure glycoprotein oviductin, has been renamed OVGP1. MUC18, now named MCAM, was the original designation of a cell adhesion molecule in the immunoglobulin gene superfamily, not a mucin. The entire glycoprotein is often referred to by MUC gene name, for example, MUC2 mucin containing apomucins protein encoded by MUC2.

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Table 2. Characteristics of human MUC genes MUC gene

Locus

Domainsa TM VWD Other

Tandem Expression sites repeats No. of AAs

Membranebound MUC1

1q21

+

SEA,

20

MUC3A &3B

7q22

+

SEA, EGF

17

MUC4

3q29

+ 1

MUC11 &12 MUC17

7q22 7q22

+ +

NIDO, AMOP, 16 EGF SEA, EGF 28 SEA, EGF 59

Secreted gelforming MUC2

11p15.5

4

MUC5AC

11p15.5

4

MUC5B

11p15.5

4

MUC6

11p15.5

3

Other, unclassified MUC7

VWC, CK, Cys- 23 rich VWC, CK, Cys- 8 rich VWC, CK, Cys- 29 rich CK 169

MUC8

4q1321 12q24

MUC13

3q13.3 +

MUC15

11p14.3 +

No TR

MUC16 (CA125)

19p14.3 +

165

SEA, EGF

Mammary glands, pancreas, etc. Small intestine, gallbladder Trachea, stomach, salivary gland Colon Duodenum, transverse colon

Colon, trachea Stomach (foveolar), trachea Stomach, trachea, gallbladder Stomach (pyloric gland), trachea

23

Salivary glands

13 and 41

Trachea (submucosal gland) Colon, trachea, hematopoietic Colon, breast, small intestine Ovarian cancer, ocular surface

15

a TM, transmembrane domain; VWD, von Willebrand factor D-like domain; VWC, von Willebrand factor C-like domain; CK, cysteine knot; SEA, sea urchin sperm protein, enterokinase, and agrin module; EGF, epidermal growth factor-like domain; AMOP, Adhesion-associated domain in MUC4 and other proteins; NIDO, nidogen domain; AAs, amino acids.

There are two structurally and functionally distinct classes of mucins: transmembrane mucins (MUC1, MUC3, MUC4, MUC12 and MUC17) and secreted gel-forming mucins (MUC2, MUC5AC, MUC5B and MUC6), although

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the products of some MUC genes do not fit well into either class (MUC7, MUC8, MUC9, MUC13, MUC15 and MUC16) (Table 2). Each MUC mucin shows a characteristic distribution in organs and cell types (Kim and Gum, 1995; Gum et al., 1999; Horinouchi et al., 2003) (Table 2). Most organs seem to synthesize more than one type of mucin, although a particular mucin type may predominate in a particular organ. Altered mucin gene expression has been reported in inflammatory diseases and carcinomas of the GI tract and breast (Kim and Gum, 1995; Gum et al., 1999; Horinouchi et al., 2003). Accumulating data suggest that each MUC mucin has different properties; for example, the presence of an EGF-like domain, a transmembrane region and a gel-forming capacity; and may have diverse functions. Therefore, the altered expression of MUC mucin appears to be somehow involved in the pathogenesis of inflammatory diseases and in the biological behavior of carcinoma. 3.1.3. Membrane-bound mucins Membrane-bound mucins include the products of the MUC1, MUC3A, MUC3B, MUC4, MUC12 and MUC17 genes. Fig. 5 shows the structure of representative membrane-bound mucins, MUC1 and MUC4. These mucins all have a transmembrane hydrophobic domain with a large N-terminal extracellular mucin-like domain and a C-terminal cytoplasmic domain. The cytoplasmic domains in the membranebound mucins have motifs suggestive of a role in signal transduction. Except for MUC4, they have a sea urchin sperm protein, enterokinase and agrin (SEA) module in the extracellular domain that may function in regulating or binding carbohydrates side chains or may serve as a proteolytic site for the generation of a receptor-ligand pair from a membrane glycoprotein precursor (Wreschner et al., 2002). MUC1 is unlike the other membrane-bound mucins in that it lacks EGF-like domains. The function of EGF-like motifs in MUC3A, MUC3B, MUC4, MUC12 and MUC17 is

Fig. 5. Molecular structure of membrane-bound mucins MUC1 and MUC4. Both mucins have transmembrane (TM) domain. MUC1 and MUC4 have SEA domain and 2 EGF domains, respectively. SEA, sea urchin sperm protein, enterokinase and agrin; EGF, epidermal growth factor.

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unclear, but these domains could be involved in epithelial growth modulations. There is evidence that not all membrane-bound mucins are controlled by the same mechanism: a selective augmentation of MUC4 and MUC16, but MUC1 was not observed after retinoic acid or serum was added to a conjunctival cell line (Hori et al., 2004), while the eicosanoid 15-(S)-hydroxy-5,8,11,13-eicosatetraenoic acid is a selective secretogogue for MUC1 (Jumblatt et al., 2002). MUC1. MUC1 mucin has been well characterized (Gendler et al., 1991; Kim and Gum, 1995; Irimura et al., 1999). The proteins encoded by the MUC1 gene have a large extracellular domain with a variable number (20–120 in different individuals) of tandem repeats of the 20-AA peptide PAPGSTAPPAHGVTSAPDTR (Fig. 5). Most murine monoclonal antibodies recognize the DTR portion of the MUC1 tandem repeat peptide. MUC1 can serve as a target for tumor-specific cytotoxic T cells in breast cancer patients (Jerome et al., 1991). The MUC1 gene can generate isoforms; MUC1/TM, MUC1/Y and MUC1/SEC by alternative splicing. MUC1/ TM, the full-length form of MUC1 is cleaved to form a heterodimer with an extracellular, O-glycosylated subunit bound to a transmembrane subunit (Wreschner et al., 2002). MUC1/SEC, a soluble glycoprotein, can interact with MUC1/Y, a truncated transmembrane protein, to induce signal transduction. MUC1/Y is preferentially expressed on breast and ovarian cancer cells (Hartman et al., 1999). The expression of the isoforms of MUC1 has not been fully clarified in the intrahepatic biliary system, so far. MUC1 mucin is widely distributed in epithelial cells and carcinoma cells and the epitope of EMA, a useful immunohistochemical marker for epithelial cells and carcinomas, is on the MUC1 mucin of the glycosylated form. MUC1 overexpression is most evident in carcinoma cells and these rigid mucin glycoproteins on cancer cells play a role in metastasis by inhibiting tumor cell adhesion and in escaping from immune surveillance (van de Wiel-van Kemenade et al., 1993). It is also known that MUC1 acts as docking protein for signaling molecules (Li et al., 2001; Huang et al., 2005). For example, the MUC1 oncoprotein is aberrantly overexpressed by most human carcinomas and associates with b-catenin (Huang et al., 2005). Dysregulation of b-catenin is of importance for the development of diverse human malignancies. The EGF receptor (EGF-R) regulates interaction of the human DF3/MUC1 carcinoma antigen with c-Src and b-catenin (Li et al., 2001). Furthermore, a recent report revealed that RNA interference suppression of MUC1 reduces the growth rate and metastatic phenotype of human pancreatic cancer cells (Tsutsumida et al., 2006). MUC4. MUC4 is the human homologue of rat ASGP sialomucin complex. MUC4 was originally identified in trachea, but is also expressed in normal stomach, ovary, salivary gland and so on. It has 208 tandem 48 bp repetitive units, a transmembrane domain (TM), and three EGF-like domains (Fig. 5). There are five variant forms resulting from alternative splicing (Choudhury et al., 2000). The EGFlike domains are essential for secretion and interact with the receptor tyrosine kinase ErbB2 (HER-2), which can trans-activate the gene promoter (Komatsu et al., 2001; Perez et al., 2003). In cells transiently transfected with SMC/Muc4, ErbB2 is translocated to the apical membrane – where the mucin is expressed in polarized

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epithelia – suggesting a mucin role in ErbB signaling (Ramsauer et al., 2003). MUC4 mucin is also involved in the regulation of p27. MUC4 is considered to be a tumorassociated molecule from the potential role of MUC4 as a marker for adenocarcinoma of the pancreas. The expression of MUC4 in ICC of the massforming type is a new independent factor for poor prognosis (Shibahara et al., 2004a). MUC3A, 3B, MUC12 and MUC17. MUC3A, 3B, MUC12 and MUC17 genes on chromosome 7q22 are evolutionally related to each other (Gum et al., 2002). MUC3A, originally designated MUC3, was identified in a small intestinal cDNA library by screening with antibody against deglycosylated human small intestinal mucin (Gum et al., 1990). MUC3 is a very large cell surface glycoprotein present in columnar epithelial cells of small intestine and colon, rather than in goblet cells. A second MUC3 gene has been detected on 7q22 (Pratt et al., 2000). MUC3A and MUC3B are over 94% identical, suggesting that they arose from a relatively recent gene duplication. Though little information is available about factors that regulate expression of MUC3, there have been recent reports that probiotic bacteria can induce MUC3 expression (Mack et al., 1999). MUC11 and MUC12 are down-regulated in colonic carcinoma and are mapped to 7q22, near the location of MUC3 (Williams et al., 1999). MUC11 and MUC12 cDNA could be derived from the same gene, but differences in their expression patterns suggest that MUC11 and MUC12 are two distinct genes, as for MUC3A and MUC3B. MUC17 is also on chromosome 7q22 and the sequence is close to MUC12 (Gum et al., 2002). MUC17 expression occurs almost exclusively in the intestine, with highest levels in the duodenum and transverse colon. Functions of these mucins remain largely to be clarified, so far. Interestingly, a recent report revealed that The MUC3 mucin cysteine (Cys)-rich domain plays an active role in epithelial restitution, and represents a potential novel therapeutic agent for intestinal wound healing (Ho et al., 2006). 3.1.4. Secreted gel-forming mucins The four genes encoding secreted gel-forming mucins are contiguous on chromosome 11p15.5 in the order MUC6/MUC2/MUC5AC/MUC5B and appear to be evolutionally related (Desseyn et al., 1998, 2000; Van Seuningen et al., 2001). These gel-forming mucins (MUC2, MUC5AC, MUC5B and MUC6) are characteristically distributed in the distinct organs and cells. For example, MUC2, MUC5AC and MUC6 are expressed in colonic epithelia, gastric foveolar epithelium and gastric pyloric glands. These mucin genes are very large, and yield very large mRNAs (Table 2, Fig. 6). Each has a central region with a variable number of tandem repeats (VNTR), but there is little similarity among the different mucins, in the sequence of the VNTR-encoded threonine-, serine- and proline-rich repeat peptides. Two structural features that are conserved are the presence of sequences homologous to von Willebrand factor D-domains (VWD, thought to be involved in mucin oligomerization to for gels) and a C-terminal cysteine-knot motif (CK, thought to be involved in the initial dimerization of apomucins monomers) (Fig. 6). MUC2, MUC5AC and MUC5B also have sequenced homologous to von Willebrand factor

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Fig. 6. Molecular structure of secreted gel-forming mucins. The D domains are similar to domains in the von Willbrand factor (VWD), and are involved in mucin assembly. The cysteine-rich domains play a role in intra- and intermolecular bonds. CK, cysteine knots.

C-Domains (VWC), involved in binding of trefoil factors (Brodt et al., 1997)) and two–seven conserved 108-AA Cys-rich domains. Secreted gel-forming mucins protect the mucosal surface from infection and physical trauma. It is likely that secreted mucins form small gels or aggregates around foreign bodies or dead cells and eliminate them from the mucosal surface. Neutrophil enzymes are able to cleave mucins from apical cell membranes (Kim et al., 1987; Leiper et al., 2001). Mucin synthesis is not a fast process: pulse-chase studies indicated that it takes 2–4 h (Sheehan et al., 2004) to detect the first-labeled mucins in the cells. Mucin polypeptide is synthesized and undergoes dimerization, then becomes substituted with GalNAc residues, and then glycosylation proceeds to produce a mucin dimer. The last step in the process is multimerization. These oligomeric mucins follow a similar assembly to the von Willebrand factor (VWF) glycoprotein to yield long linear disulfide-linked chains (Sheehan et al., 2004). Secreted mucins are stored in secretion granules from which water is excluded, often in specialized cells such as goblet cells (Paulsen et al., 2004). MUC2. The human intestinal mucin encoded on the MUC2 gene is a major product of goblet cells of the colon and small intestine. Complete characterization of MUC2 has been complicated by the large size (40 kb) and repetitive nature of the gene (50%), and the large size (600–5000 kDa) and potential for variable posttranscriptional processing of its protein products that contain large repetitive subdomains (Gum et al., 1989; Griffiths et al., 1990; Rousseau et al., 2004). The difference of the amount of MUC2 mucin synthesis in colon cancers and cell lines correlate with altered biochemical and biological properties, including those with relevance to the metastatic progression of colon cancer (Bresalier et al., 1991; Sternberg et al., 1999). Both at the level of sequence homology and in molecular mechanisms responsible for the control of mucin transcription and expression it appears that the MUC2 gene and the MUC5AC gene have much in common. Common regulatory mechanisms (protein kinase (PK)-A, PKC, PKG and Ca2+ signaling, Sp1/Sp3) may account for the capability of mucous-secreting cells, during carcinogenesis, to express MUC2 and MUC5AC genes simultaneously (Van Seuningen et al., 2001). MUC5AC. MUC5AC is a gel-forming mucin containing the Cys-rich motifs in their C- and N-terminal domains necessary for oligomerization. MUC5AC is

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expressed in gastric foveolar cells and in tracheobronchial epithelial cells (Nguyen et al., 1990; Guyonnet Duperat et al., 1995). Increased expression of MUC5AC is well known in inflammatory airway diseases such as asthma and cystic fibrosis and is suggested to participate in the pathogenesis of these diseases (Thornton and Sheehan, 2004). Aberrant expression of MUC5AC is also reported in preneoplastic lesions and carcinoma arising in pancreatic ducts and intrahepatic and extrahepatic bile ducts as described later (Sasaki et al., 1996; Kim et al., 2002). MUC5B. MUC5B is a large gel-forming mucin encoded on chromosome 11p15.5 as well as other gel-forming mucins (MUC2, MUC5AC and MUC6) (Pigny et al., 1996). The large central exon of MUC5B is composed of 18 exons and spans 10,690 bp (Desseyn et al., 1997a, b). This region encodes a predicted 808-AA peptide containing 6 subdomains; the last 5 are Cys-rich and are similar to MUC2, MUC5AC and VWF. MUC5B is expressed mainly in bronchus glands and also in submaxillary glands, endocervix, gallbladder and pancreas (Keates et al., 1997). MUC6. MUC6 is a large gel-forming mucin thought to play a major role in protecting the GI tract from acid, proteases, pathogenic microorganisms and mechanical trauma (Toribara et al., 1993). The MUC6 gene is mapped to chromosome 11p15.5–p15.4 suggesting a clustering of secretory mucin genes (Toribara et al., 1993). MUC6 contains an N-terminal signal peptide, followed by several VWF-like domains, a large tandem repeat domain and a C-terminal CK domain (Rousseau et al., 2004). MUC6 is expressed in a wide variety of epithelial tissues including gastric pyloric glands, duodenal Brunner’s glands, gallbladder and seminal vesicle (Bartman et al., 1998). 3.1.5. Small soluble mucins MUC7 and MUC8 also encode mucin-like glycoproteins lacking a TM, but they are structurally dissimilar from the 11p15.5 mucins and cannot be classified as secreted gel-forming mucins. The molecular weights of mucins are characteristically very high, reflecting a large carbohydrate content, extensive oligomerization and very large apomucin proteins. In contrast, these mucins are secreted, but monomeric and do not form linear multimers. MUC7 is a salivary low-molecular-weight mucin that lacks the capacity to oligomerize (Bobek et al., 1993, 1996). MUC7. MUC7 encodes a protein of only 377 AA residues, with no sequence homology with other mucins, and is expressed only in the sublingual and submandibular glands. In saliva, MUC7 mucin is a low-specificity but avid bacterial binder, and thus fulfils an anti-bacterial role. This mucin is also an exception in that it polymerizes not as an N–N and C–C linear polymer but around a central structure, somewhat like spokes of a wheel (Mehrotra et al., 1998). MUC8. MUC8 is a tracheobronchial mucin and encoded on chromosome 12q24.3. Only small portions of MUC8 have been sequenced, revealing both 13-AA repeats and 41-AA repeats. Expression of MUC8 was detected in the submucosal glands in human trachea (Shankar et al., 1997) and nasolacrimal ducts. MUC8 has been also demonstrated as a ciliated cell marker in human nasal epithelium and the epithelium of paranasal sinuses (Jung et al., 2000; Kim et al., 2005) and is

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up-regulated by proinflammatory cytokines and under chronic inflammatory conditions (Jung et al., 2000; Kim et al., 2005). 3.1.6. Regulation of MUC mucins expression Common regulatory mechanisms might account for the capability of mucoussecreting cells to express several mucin genes simultaneously (Van Seuningen et al., 2001). 11p15 mucin genes are regulated at the transcriptional level by proinflammatory cytokines (interleukin (IL)-1b, IL-6, tumor necrosis factor (TNF)-a), pleiotropic cytokines (IL-4, IL-13, IL-9), bacterial lipopolysaccharide (LPS), growth factors (EGF, transforming growth factor (TGF-a)), lipid mediators (platelet aggregating factor), retinoids and hormones (Van Seuningen et al., 2001), as well as by extracellular Ca2+ (Verdugo et al., 1987), injury-induced pathways (Basbaum et al., 1999), and cells of the immune system. Although these pathways have not yet been fully clarified so far, the data regarding this important point are accumulating also in the intrahepatic biliary system as described later. 3.2. Properties of TFF peptides TFF-1,2,3 is a mucin-associated protein involved in the maintenance of mucosal barrier and restitution of lining epithelial cells (Poulsom et al.,1996; Podolsky, 1999, 1996; Tino and Wright, 1999). TFFs are synthesized and secreted by mucus-secreting cells (Poulsom et al., 1996; Podolsky, 1999; Tino and Wright, 1999). TFFs are a family of short peptides, rich in disulfide bonds that form intramolecular loops (Thim and May, 2005). TFFs are intimately associated with mucins: in amphibians TFFs are encoded by mucin genes (Hoffmann and Joba, 1995). In humans the separate genes encoding members of the TFFs are clustered on chromosome 21q22.3 (Beck et al., 1996; Chinery et al., 1996; Gott et al., 1996; Seib et al., 1997). In humans, 3 TFFs have been identified: TFF1 (originally called pS2); TFF2 (formerly called spasmolytic peptide, SP); and TFF3 (formerly called intestinal trefoil factor, ITF) (Tino and Wright, 1999) (Table 3). Their expression patterns are known to be characteristic for the anatomical portions of the GI tract. TFFs and 11p5.5 mucins (MUC2, MUC5AC and MUC6) are secreted coordinately in a site-specific fashion in the human GI tract. TFF1, 2 and 3 are up-regulated in the surface epithelial cells at the margin of gastric ulcer and also in inflammatory bowel disease (Hauser et al., 1993; Poulsom et al., 1996; Podolsky, 1999; Tino and Wright, 1999). The peptides contain trefoil domains, conserved sequences of 42 or 43 AAs in which 6 conserved residues are found in specific disulfide bridges: 1–5, 2–4, 3–6. These residues form a stable core with protruding, clover-like loops. The structure, functions and interactions of TFF peptides have recently been the subject of authoritative reviews (Baus-Loncar and Giraud, 2005; Hoffmann, 2005; Thim and May, 2005). TFF1. TFF1, also known as pS2, is a 60 amino-acids long mature peptide and contains a single trefoil domain. The Cys, three residues from the acid C-terminus, permits homo- and hetero-dimerization. The TFF1 dimer is the predominant form that co-elutes with MUC5AC on gradient centrifugation and is precipitated by an anti-MUC5AC antibody (Ruchaud-Sparagano et al., 2004). TFF1 is a product of

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Table 3. Characteristics of TFF peptides Name

Locus

No. of AAs

No. of TFF domains

Co-expressed MUC mucins

Expression sites

TFF1

21q22.3

60

1a

MUC5AC

TFF2

21q22.3

106

2

MUC6

TFF3

21q22.3

59

1a

MUC2, MUC5B, MUC8

Stomach (foveolar), conjunctiva, larynx Stomach (pyloric gland), duodenum (Brunner’s gland) Colon, salivary glands, respiratory tract, endocervix, conjunctiva, larynx

AAs, amino acids. a Dimerization.

gastric surface mucous cells together with the MUC5AC mucin (Poulsom et al., 1996; Podolsky, 1999; Tino and Wright, 1999; Ruchaud-Sparagano et al., 2004). TFF1 is suggested to have tumor-suppressor properties. In fact, TFF1 knockout mice developed multiple gastric adenomas and carcinomas (Lefebvre et al., 1996). Loss of TFF1 expression, somatic mutation of TFF1 gene and the methylation of TFF1 promoter have also been reported in human gastric cancer (Park et al., 2000) and ICC (Sasaki et al., 2003c). A recent study reported the loss of tumor-suppressor activity and a gain of invasiveness from single point mutations, constituting evidence for a functional role of TFF1 mutations in gastric cancer (Yio et al., 2006). TFF2. Mature TFF2 contains 106 AAs, with two trefoil domains and two Cys residues outside the trefoil domains (Tomasetto et al., 1990). Human TFF2 is an only member of the family to have one N-linked glycosylation site: TFF2 is glycosylated in gastric tissue, whereas in normal gastric juice both forms are present. The two loops of TFF2 are not only linked by disulfide bonds, but also by a peptide sequence, resulting in a very compact molecule. TFF2 is expressed in gastric mucous neck cells and cells at the pyloric glands together with MUC6 (Poulsom et al., 1996; Podolsky, 1999; Tino and Wright, 1999). TFF3. TFF3 is a mature peptide composed of 59 AAs and is secreted as a monomer or dimer. TFF3 has one TFF domain and a free Cys residue in the C terminal. TFF3 promotes migration of epithelial cells in vitro and enhances mucosal healing and epithelial restitution in vivo in the GI mucosa, where it co-localizes with MUC2. TFF3 is generated in intestinal goblet cells in combination with MUC2 (Poulsom et al., 1996; Podolsky, 1999; Tino and Wright, 1999). TFFs have distinct intracellular and mucosa actions. TFFs are known to modulate cell migratory processes (Dignass et al., 1994; Playford et al., 1995), regulate apoptosis (Lalani et al., 1999; Kinoshita et al., 2000) and enhance healing of the mucosa in vivo (Poulsom et al., 1996; Podolsky, 1999; Tino and Wright, 1999).

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For example, TFF3 knockout mice are sensitive to chemically induced mucosal injury because of impairment of mucosal healing and decrease of epithelial regeneration (Mashimo et al., 1996). TFFs are also known to stabilize the secreted mucins. Increase in gel viscosity is spectacular on addition TFF2 (10-fold), and with TFF3 dimers (Thim et al., 2002). TFF dimers are resistant to trypsin, chymotrypsin and carboxypeptidases, which supports a protective function in the GI tract, but not for cocktails of bacterial enzymes (Thim and May, 2005), which might suggest that interactions with mucins enhance this role. It is postulated that TFF peptides function as link peptides interacting non-covalently with mucins and influencing the rheological properties of mucous gels (Hauser et al., 1993). This hypothesis has been confirmed by in vitro studies demonstrating that TFF peptides increase the viscosity of mucin preparations (Thim et al., 2002) and that trefoils interact with the VWFlike domains of mucins (Tomasetto et al., 2000). The dimeric structure of TFF1 and TFF3 is ideally suited to form an entangled network (Hoffmann et al., 2001) with MUC5AC, also the structure of the two trefoil factors may give rise to different ways of interacting with their ligands (Muskett et al., 2003). Synthesis and release of TFFs are regulated by a number of environmental and local agents, estrogens, proinflammatory and anti-inflammatory cytokines (Baus-Loncar and Giraud, 2005).

4. Detection of mRNA and protein expression of MUC mucins and TFF peptides 4.1. Detection of mRNA expression of MUC mucins and TFF peptides In situ hybridization is widely used to detect the expression and distribution of MUC mRNA in tissue sections. Both radioisotope-labeled and digoxigenin (nonisotope)-labeled oligonucleotides are usually used as probes (Sasaki et al., 1998c; Buisine et al., 2000). Oligonucleotides encompassing the tandem repeat sequence of each MUC gene are synthesized (Sasaki et al., 1998c; Buisine et al., 2000). The specificity of each oligonucleotide probe is confirmed by sequence database search (NCBI). MUC mucin is abundantly produced, when expressed, and multiple tandem repeat regions are present in each MUC mRNA. The mRNA expression of TFFs is also detectable by in situ hybridization (Sasaki et al., 2003c). For the detection of TFFs mRNA, digoxigenin-labeled RNA probes are synthesized using reverse transcriptase polymerase chain reaction products (Cone and Schlaepfer, 1997). Positive signals are clearly detected in the cytoplasm of MUC mucins and TFFs expressing cells on tissue sections. Both frozen tissue sections and formalin-fixed, paraffin-embedded tissue sections are available to detect the mRNA expression of MUC mucins and TFFs. 4.2. Detection of protein expression of MUC mucins and TFF peptides The protein expression of MUC mucins and TFFs is detected in tissue section by immunohistochemical methods. For MUC1 mucin expression, several mouse

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monoclonal antibodies detecting different glycosylated forms are available (Sasaki and Nakanuma, 1996a; Higashi et al., 1999). DF3 (CA15-3; Toray-Fuji bionichs, Tokyo, Japan) (Kufe et al., 1984), SM3 (Cymbus Bioscience, Southampton, UK) (Burchell et al., 1987) and NCL-MUC1-CORE (Novocastra, New Castle, UK) recognize MUC1 core peptide. DF3 binding to MUC1 mucin was reportedly enhanced by the presence of carbohydrate. EMA (Dako, Santa Barbara, CA), HMFG1 (Cosmo Bio, Tokyo, Japan) (Taylor-Papadimitriou et al., 1981) and NCL-MUC-1-GP (Novocastra) recognize the glycosylated form of MUC1. For other types of MUC mucin expression, rabbit or chicken polyclonal antibodies raised against synthetic tandem repeat peptide of each MUC mucin have been used (Sasaki et al., 1995, 1996, 1998c). Monoclonal antibodies are also commercially available for the detection of MUC2 (Ccp58), MUC5AC (CLH2 and 45M1) and MUC6 (CLH5) (Novocastra). For MUC3 and MUC5B mucin expression, monoclonal antibody (MAb) M3.1 and goat polyclonal antibody are also commercially available, respectively (Santa Cruz biotech., Santa Cruz, CA) (Sasaki et al., 2005b). Mouse monoclonal antibodies are commercially available for the immunohistochemical detection of TFF1 (anti-pS2 protein, clone BC04, Dako, Santa Barbara, CA) and TFF2 (anti-human SP, clone GE16C, YLEM, Via Gramsci, Italy) (Sasaki et al., 2003c, 2004a, b). We have used a rabbit polyclonal antibody (anti-ITF; kindly provided by Dr. Kataoka in Miyazaki medical school, Japan (Uchino et al., 2000)) for the detection of TFF3 (Sasaki et al., 2004a, b). Other polyclonal antibodies against TFF3 have been raised and used in previous studies (Podolsky, 1999; Wiede et al., 1999; Kimura et al., 2002; Srivatsa et al., 2002).

5. Expression of MUC mucins and TFF peptides in the fetal and normal adult intrahepatic biliary system 5.1. Physiological expression of MUC mucins The expression profile of MUC mucin in fetal and normal postnatal (adult) livers is summarized in Fig. 7. As described above, the process of intrahepatic bile duct development is staged as the ductal plate stage, the remodeling ductal plate stage, and the remodeled bile duct stage in fetal livers (Van Eyken et al., 1988). The ductal plate is an excess structure consisting of a double-layered cylinder at the peripheral hepatic parenchyma. The ductal plate stage is characterized by the formation of these ductal plates. The remodeling ductal plate stage is characterized by the incorporation of ductal plate cells into the mesenchyma; as well as by the gradual disappearance of the ductal plates (Van Eyken et al., 1988). The remodeled bile duct stage is characterized by new bile ducts in the portal tracts as well as by the disappearance of the ductal plate. The development of intrahepatic bile ducts proceed from the hilar to the peripheral portions (Van Eyken et al., 1988). In the fetal liver, BECs in remodeled new bile ducts in the portal tracts, either at the hilar level or at the peripheral level, frequently expressed MUC1 mucin, both the

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Fig. 7. Expression profile of MUC mucins in fetal and normal adult livers and hepatolithiasis BD, bile duct. ( ), weak and focal expression; *U, G, unglycosylated and glycosylated form; *G, glycosylated form.

unglycosylated (detected by MAb DF3) and glycosylated form (detected by antiEMA) on their luminal surface (Sasaki et al., 1995) (Fig. 8). Ductal plates also focally expressed MUC1 mucin. In addition, the remodeled bile ducts in the fetal liver focally express MUC6 mucin. By contrast, in the adult liver, the BECs of intrahepatic large bile ducts constantly express MUC3 mucin, whereas those of small bile ducts do not (Fig. 8) (Sasaki et al., 1995, 1998d; Vandenhaute et al., 1997). Although MUC3 is a membrane-binding type mucin, the immunohistochemical MUC3 expression is usually observed in the supranuclear cytoplasm in BECs and carcinomas. This may reflect the soluble form of MUC3 produced by alternative splicing (Crawley et al., 1999). MUC1 mucin of unglycosylated form (detected by DF3) is not expressed in adult intrahepatic bile ducts, whereas MUC1 mucin of the glycosylated form (detected by EMA) is expressed in intrahepatic large bile ducts. These findings suggest that the glycosylation status of MUC1 mucin is altered before and after birth. BECs of intrahepatic large and small bile ducts express MUC4 mucin focally and weakly (Sasaki et al., unpublished data). BECs of intrahepatic large bile ducts express MUC5B constantly, while MUC5B expression is focal and weak in BECs of intrahepatic small bile ducts (Sasaki et al., unpublished data). MUC2 and MUC5AC mucin are absent in the intrahepatic biliary elements of the fetal and adult livers. These data suggest that BECs switch MUC1 mucin expression before birth to that of MUC3 after birth (Sasaki et al., 1995). The characteristic transition may be similar to the changes in the hepatocellular expression of alpha-fetoprotein and albumin during the perinatal period. The molecular mechanism involved in the induction of MUC3 mucin and the altered glycosylation of MUC1 mucin after birth remain to be addressed. Interestingly, MUC1 is highly expressed in oval cell fraction and regarded as a hepatic progenitor cell surface marker in the adult rat liver in a recent study (Yovchev et al., 2007).

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Fig. 8. MUC1 and MUC3 expression in fetal and normal adult livers. (Top) Fetal intrahepatic bile ducts (arrow) and ductal plates frequently express MUC1 mucin (unglycosylated form) (arrow), but not MUC3. (Bottom) In contrast, intrahepatic large bile ducts after birth mainly express MUC3 mucin, whereas MUC1 mucin of the unglycosylated form is not expressed. Immunohistochemical staining for MUC1 and MUC3 was detected using monoclonal antibody, DF3, and rabbit polyclonal antibody, M3P.

There are several studies reporting MUC expression in the gallbladder, extrahepatic bile ducts, and the ampulla of Vater (van Klinken et al., 1998; Sasaki et al., 1999; Yamato et al., 1999a, b; Zhou et al., 2004; Paulsen et al., 2006). In the fetal gallbladder, MUC3, MUC6, MUC5B and MUC1 are detected by in situ hybridization (Buisine et al., 2000). In the adult gallbladder, the strong expression of MUC3, the moderate expression of MUC5B and MUC6, and the weak expression of MUC1, MUC2 and MUC5AC are detected by in situ hybridization (van Klinken et al., 1998; Sasaki et al., 1999; Yamato et al. 1999a, b; Buisine et al., 2000). MUC4 mucin is not detected in fetal and adult gallbladder (Buisine et al., 2000). In the adult extrahepatic bile duct and the ampulla of Vater, the expression of MUC1, MUC3, MUC4, MUC5AC, MUC5B, MUC6, MUC7 and MUC8 are noted, but MUC2 expression is absent (Zhou et al., 2004; Paulsen et al., 2006). The constant expression of MUC3 and MUC5B expression in adult intrahepatic large bile ducts is similar to that in the adult gallbladder and the ampulla of Vater (Table 2, Fig. 8).

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5.2. Physiological expression of TFF peptides In normal adult intrahepatic biliary system, TFF1 and TFF3 are focally and faintly expressed in BECs in large bile ducts (Sasaki et al., 2004a, b). TFF1 and TFF3 are expressed in the supranuclear cytoplasm. TFF2 is not expressed in large bile ducts in normal livers at all. A small number of extramural peribiliary glands observed around large bile ducts in normal liver express TFF2 and TFF3 to a mild degree (Sasaki et al., 2004a). No apparent expression of TFF peptides is detected in BECs in small bile ducts (Sasaki et al., 2004a, b).

6. Expression of MUC mucins and TFF peptides in the intrahepatic biliary system in non-tumorous hepatobiliary diseases Altered mucin gene expression has been reported in various non-tumorous inflammatory or cystic hepatobiliary diseases (Yamashita et al., 1993; Sasaki et al., 1998b, c, 2005b; Sasaki and Nakanuma, 1996a) as well as those in the GI tract and breast (Kim and Gum, 1995; Gum et al., 1999; Horinouchi et al., 2003). Most studies focus on hepatolithiasis in which mucin is an important factor in the pathogenesis (Yamashita et al., 1993; Sasaki et al., 1998c, 2005b). Inflammatory conditions, for example, inflammatory cytokines, reactive oxygen species and infection-related components are suggested to play a role in the altered expression of MUC mucins. Since MUC mucins have diverse properties and functions, the altered expression of MUC mucin may be involved in the pathogenesis of inflammatory diseases. 6.1. Expression of MUC mucins and TFFs peptides in hepatolithiasis 6.1.1. Hepatolithiasis Hepatolithiasis is not rare in the Far East (Nakanuma et al., 1985; Nakayama et al., 1986), and chronic proliferative cholangitis of the intrahepatic large bile duct with mucus hypersecretion is a key lesion in the process of stone formation (Nakanuma et al., 1988) (Fig. 9). Histologically, biliary mucosa in the liver with hepatolithiasis shows a marked proliferation of intramural and extramural peribiliary glands accompanied by chronic inflammation and fibrosis (chronic proliferative cholangitis) (Nakanuma et al., 1988) (Figs. 3 and 9). The majority of these stones are of the calcium-bilirubinate type (Nakayama et al., 1986), which differs from pure cholesterol and black pigment stones in their composition and etiology (Trotman et al., 1974). The hepatoliths of calcium-bilirubinate type usually include a high content of cholesterol (Shoda et al., 2001, 2003). Bacterial infection, bile stasis and an alteration of the bile composition are thought to be responsible for the nucleation, formation and maturation of intrahepatic stones (Nakayama et al., 1986; Shoda et al., 2003) (Table 4). 6.1.2. Augmented expression of gel-forming MUC mucins in hepatolithiasis In the intrahepatic biliary system with hepatolithiasis, surface epithelial cells express MUC5AC mucin (gastric foveolar type) and proliferative peribiliary glands

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Fig. 9. Gross appearance, mucin histochemistry and MUC mucin expression in hepatolithiasis. (A) The gross appearance of hepatolithiasis. Asterisk indicates calcium-bilirubinate stones compacted in the intrahepatic large bile ducts. (B) The intrahepatic large bile duct containing hepatoliths includes proliferated peribiliary glands (arrows) and abundant secreted mucin in the lumen of large bile ducts (asterisks). Alcian blue (pH2.5)/PAS staining. (C) MUC2 is expressed in the cytoplasm of goblet cells at the intestinal metaplasia. CDX2 is expressed in the nuclei of goblet cells at intestinal metaplasia. Immunohistochemical staining for MUC2 and CDX2. (D) In hepatolithiasis, surface epithelial cells and a part of proliferated peribiliary glands express MUC5AC mucin (left). MUC6 mucin (right) is mainly expressed in proliferated peribiliary glands. Immunohistochemical staining for MUC5AC (rabbit polyclonal antibody, M5P-b1) and MUC6 (chicken polyclonal antibody, M6P) and mRNA expression detected with in situ hybridization using digoxigenin-labeled oligonucleotide probe for MUC5AC and MUC6.

express MUC6 mucin (gastric pyloric gland type) (Sasaki et al., 1998d, 2003c, 2005b) (Figs. 7 and 9). The expression profile of MUC mucin in the biliary mucosa with hepatolithiasis resembles that of the gastric mucosa. The altered expression of MUC mucins may play a role in the initiation and progression of hepatolithiasis, since newly expressed MUC5AC and MUC6 mucin have the capacity for viscous gel formation (Sasaki et al., 1998d). MUC2, colon-type mucin, is also newly expressed in the biliary tract with hepatolithiasis at the site of intestinal metaplasia and hyperplasic epithelia. As shown in Fig. 9, nuclear expression of the CDX2 homeobox gene, which induces intestinal differentiation, is co-localized with MUC2 expression at the site of intestinal metaplasia. This finding suggests that CDX2 induces the

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Table 4. Expression profiles of TFF1, 2, 3 and MUC2, 5AC, MUC6 in hepatolithiasis and control livers TFF1

MUC5AC

TFF2

MUC6

TFF3

MUC2

Hepatolithiasis Large bile ducts At erosion Peribiliary glands

++ +++ +

++ ++ /+

/+  +++

+ + +++

++ ++ ++

+ /+ +

EBO livers Large bile ducts Peribiliary glands

/+ 

/+ /+

 +

+ +

+ +

 

Normal livers Large bile ducts Peribiliary glands

/+ 

 /+

 +

+ +

/+ +

 

EBO, extrahepatic bile duct obstruction; , negative; /+, weak and very focal expression, if present; +, focal expression; ++, moderate expression; +++, extensive expression.

expression of MUC2 mucin in intrahepatic large bile duct in hepatolithiasis (Sasaki et al., 1998d; Ishikawa et al., 2004). Furthermore, the focal expression of MUC1 and increased expression of MUC3 and MUC5B are also noted in large bile ducts and peribiliary glands in hepatolithiasis. Although the significance of the increased expression of MUC3 mucin remains unclear, the increased expression of MUC5B, which is one of the gel-forming mucins, may contribute to the increased viscosity of the bile and lithogenesis. The gel-forming mucins are also known to play an important role in the early stage of cholesterol gallstone formation (MacPherson and Pemsingh, 1997; Wang et al., 1997). Interestingly, recent studies revealed that increased gallbladder epithelial MUC1 mucin also enhances lithogenesis by promoting gallbladder cholesterol absorption and impairing gallbladder motility in transgenic for the human MUC1 gene mice (Wang et al., 2006). 6.1.3. Coordinated expression of TFF peptides with MUC mucins in hepatolithiasis The TFF are mucin-associated proteins that are important for mucosal defense and the repair of GI epithelia (Poulsom et al., 1996; Podolsky, 1999; Longman et al., 2000). In humans, three types of TFF (TFF1–3) and their characteristic and coordinated distribution together with MUC mucin have been reported (Table 3). That is a combination of TFF1 with MUC5AC and that of TFF2 with MUC6 which are generated in gastric surface mucous cells and gastric pyloric glands, respectively. TFF3 is co-expressed with MUC2 in intestinal goblet cells, and also co-expressed with MUC5B and MUC8 (Wiede et al., 1999). The expression of TFF1, TFF2 and TFF3 are augmented markedly in the biliary mucosa in hepatolithiasis in coordination with gel-forming mucin (Fig. 10) (Sasaki et al., 2003c, 2004a).Thus, the expression of TFF1 and TFF2 is almost parallel to the expression of MUC5AC and MUC6, respectively. TFF3 is co-expressed with MUC2

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Fig. 10. Expression of TFF1, 2 and 3 in hepatolithiasis. (A) Biliary epithelial cells (BECs) in the large bile duct (arrowheads) as well as intramural peribiliary glands (arrows) express TFF1 intensely at the supranuclear cytoplasm. (B) BECs in intramural peribiliary glands express TFF2 in the cytoplasm (arrows). (C) BECs express TFF3 at the supranuclear cytoplasm widely in large bile ducts and intramural peribiliary glands. Immunohistochemical staining for TFF1 (A), TFF2 (B) and TFF3 (C).

at the site of intestinal metaplasia (Sasaki et al., 2004a). Furthermore, TFF3 is more widely distributed in BECs in large bile ducts and peribiliary glands (Sasaki et al., 2004a, b). It is conceivable that widely distributed TFF3 may be co-expressed with MUC5B. TFF3 is detected in hepatic bile samples of hepatolithiasis by immunoblotting (Sasaki et al., 2005b). Since TFFs are known to interact with MUC2 and MUC5AC and possibly raise the viscosity of mucin (Tomasetto et al., 2000), it is likely that over-secreted TFFs in the biliary tract with hepatolithiasis may couple with MUC5AC and MUC2 mucin, increase the viscosity of secreted biliary mucin and contribute to the formation of hepatoliths (Sasaki et al., 2003c, 2004b). In addition, an extensive expression of TFF1 and TFF3 is noted in BECs at the margin of erosion of biliary mucosa in hepatolithiasis, whereas TFF2 expression is not augmented in BECs at the margin of erosion (Sasaki et al., 2003c, 2004a). This finding suggests that TFF1 and TFF3 may play a role in the repair of the biliary mucosa in the intrahepatic large bile ducts. Furthermore, we have reported the augmented expression of mucin-associated molecule that is, deleted in malignant brain tumor-1 (DMBT1) (Mollenhauer et al., 1997, 2001), which is a candidate of the TFF receptor in BECs in the biliary tract with hepatolithiasis and suggested the possible participation of this molecule in lithogenesis (Sasaki et al., 2003a). Bovine gallbladder mucin that has scavenger receptor cysteine-rich domains accelerating cholesterol crystallization was identified as an alternative splicing form of DMBT1. In our previous report, DMBT1 protein is frequently detected in hepatic bile samples of hepatolithiasis (50%), but not in the other bile samples (Sasaki et al., 2003a). The percentage of cholesterol in the intrahepatic canaliculi is significantly higher in the patients with DMBT-1 positive bile samples (Sasaki et al., 2003a). Taken together, it

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is conceivable that the increased expression of gel-forming MUC mucins, TFF peptides and DMBT1 may play a role cooperatively in lithogenesis in addition to mucosal defense and mucosal repair in hepatolithiasis. 6.1.4. Regulation of the expression of MUC mucins in hepatolithiasis The regulatory factors of increased and altered MUC expression in BECs have not as yet been fully clarified. LPS, a component of the cell wall of gram-negative bacteria up-regulates MUC2 and MUC5AC immediately in cultured mouse BECs via TNF-a and COX-2 related pathways (Zen et al., 2002). In our recent study, the chronic exposure of mouse cultured BECs to bacterial components such as LPS and peptideglycan induced the expression of the CDX2 homeobox gene and the following MUC2 expression via Toll-like receptor signaling pathway (Ikeda et al., in press). Various inflammatory cytokines including TNF-a also up-regulated the expression of CDX2 and MUC2 in cultured BECs (Ikeda et al., press). Furthermore, it is reported recently that EGF-R legends (either EGF or TGF-a) cause significant increases in MUC5AC mRNA and protein expression in the presence of TNF-a, whereas expression of the other gallbladder mucins MUC1, MUC3 and MUC5B was unchanged (Finzi et al., 2006). These findings suggest that chronic inflammatory conditions may be important for the alteration and over-expression of MUC mucins, especially a gel-forming MUC5AC and MUC2 mucins in BECs and may play a role in lithogenesis. 6.2. Expression of MUC mucins and TFFs peptides in primary biliary cirrhosis (PBC) and other inflammatory hepatobiliary diseases 6.2.1. PBC PBC is an organ specific autoimmune disease and presents with chronic progressive cholestasis and liver failure. It usually affects middle-aged women (Gershwin et al., 1987; Sasaki et al., 2000), and often leads to liver failure and liver transplantation (Kaplan, 1996; Portmann and Nakanuma, 2001). PBC is characterized histologically as a cholangitis of small bile ducts (chronic non-suppurative destructive cholangitis; CNSDC) eventually followed by extensive loss of small bile ducts (Nakanuma and Ohta, 1979; Kaplan, 1996; Sasaki et al., 2000; Portmann and Nakanuma, 2001). PBC is serologically characterized by the presence of antimitochondrial antibodies (AMA) (Kaplan, 1996) and B-cell and T-cell-mediated immune reaction targeting an inner lipoyl domain of E2-component of pyruvate dehydrogenase (Fussey et al., 1988; Shimoda et al., 1998; Kita et al., 2002; Kamihira et al., 2003). There have been several studies reporting the altered characteristics of BECs in PBC. Most of these changes are related to epithelial damage and suggested to be reactive changes irrespective to diseases (Sasaki et al., 1994, 1998b, 2000, 2004b, 2006a; Yasoshima et al., 1998). There have been few changes of BECs that are truly specific for PBC. Recently, we have reported that BECs in the damaged small bile ducts show features of cellular senescence in PBC and we speculated that the cellular

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senescence may be involved in the pathogenesis of progressive bile duct loss in PBC (Sasaki et al., 2005a, 2006b). 6.2.2. Altered expression of MUC mucins in PBC and other inflammatory hepatobiliary diseases BECs express physiologically a glycosylated form of MUC1 detected by antiEMA antibody in the intrahepatic small bile ducts. MUC4 and MUC5B mucins are also expressed focally and weakly in BECs in normal small bile ducts (Fig. 7). There have been few studies to focus on MUC expression in the hepatobiliary diseases affecting intrahepatic small bile ducts such as PBC (Sasaki and Nakanuma, 1996b; Sasaki et al., 1998b), whereas there are several studies reporting the altered expression of TFFs in hepatobiliary diseases (Kimura et al., 2002; Srivatsa et al., 2002; Nozaki et al., 2004; Sasaki et al., 2004b; Idilman et al., 2005), as described below. MUC1. In normal liver, MUC1 mucin of unglycosylated form detected by DF3 is infrequently and focally expressed in BECs in small bile ducts (Fig. 11). In contrast, MUC1 mucin of unglycosylated form is frequently and strongly positive in the luminal surface of BECs in small bile ducts in PBC (Fig. 11) and chronic viral hepatitis (CVH) due to hepatitis B or C viral infection. In particular, a high level of MUC1 mucin of unglycosylated type is expressed in BECs in small bile ducts involved in CNSDC in PBC (Fig. 11) and hepatitic bile duct injuries in CVH, C. It is well known that hepatitic bile duct injury is frequently seen in CVH, C (Kaji et al., 1994; Scheuer, 1994). However, the hepatitic bile duct injury does not lead to bile duct loss (Kaji et al., 1994; Scheuer, 1994). Therefore, frequent expression of MUC1 mucin of unglycosylated type may be a reactive change to injuries in BECs (Sasaki and Nakanuma, 1996b). It is of interest that the increased expression of MUC1 mucin (unglycosylated type) was rather infrequently detected in bile ductules in PBC and CVH, C (Sasaki and Nakanuma, 1996b), while the increased expression of MUC6 mucin is frequent in bile ductules, as described below (Sasaki et al., 1998b). MUC6. MUC6 mucin is focally and weakly expressed in small bile ducts in normal livers (Sasaki et al., 1998b). The expression of MUC6 mucin is increased in small bile ducts and bile ductules in CVH and to a lesser degree in other hepatobiliary diseases including PBC (Fig. 11). The extent of MUC6 expression in bile ductules is in parallel to the degree of active inflammation in CVH (Sasaki et al., 1998b). Therefore, it is conceivable that MUC6 mucin may be up-regulated in inflammatory conditions and may play a role as a cytoprotective agent (Sasaki et al., 1998b). 6.2.3. Altered expression of TFF peptides in PBC and other inflammatory hepatobiliary diseases The intrahepatic biliary tree shows a site-characteristic expression and induction of TFF1, 2, 3 and DMBT1. In large bile ducts, TFF1 and 3 are constitutively expressed and increased in pathologic bile ducts (Sasaki et al., 2004b). Similarly, the increased expression of TFF1 and 3 has been reported in the large bile ducts in several diseased livers (Kimura et al., 2002; Srivatsa et al., 2002). In small bile ducts, TFF2/DMBT1 is induced in damaged ducts, irrespective of etiologies. For example,

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Fig. 11. Expression of MUC1, MUC6 and TFF2 in PBC and control livers (A) MUC1 is not expressed in biliary epithelial cells (BECs) in the small bile duct (arrow) in normal liver. (B) MUC1 is expressed on the luminal surface of BECs in the small bile duct (arrow) in PBC liver. (C) MUC6 is expressed on the luminal surface and the cytoplasm of the small bile duct (arrow) in PBC liver. (D) TFF2 is expressed on the luminal surface of BECs in the small bile duct (arrow) in PBC liver. Immunohistochemical staining for MUC1 (A and B), MUC6 (C) and TFF2 (D).

the augmented expression of TFF2/DMBT1 is observed in BECs involved in CNSDC in PBC (Fig. 11) and hepatitic bile duct lesions in CVH. The expression of TFF1 and 3 are also increased in BECs involved in CNSDC in PBC (Sasaki et al., 2004b). TFF3 expression is regulated by IL-6 via signal transducer and activator of transcription signaling pathway and that TFF3 contributes to BEC migration (Nozaki et al., 2004). 6.3. Expression of MUC mucins in the hepatobiliary cystic diseases 6.3.1. The hepatobiliary cystic diseases Hepatic fibropolycystic disease consists of autosomal dominant polycystic kidney disease (ADPKD), autosomal recessive polycystic kidney disease, choledochal cyst, Meckel syndrome, solitary or simple hepatic cysts, and biliary microhamartoma (BMH) (Summerfields et al., 1986). Among this category, ADPKD is known to

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occur in approximately 1 in 500 to 1 in 1000 live births (Gabow, 1993). Two major genes responsible for ADPKD have been identified and characterized in detail: PKD1 and PKD2, mapping on chromosomes 16p13.3 and 4q21–23, respectively (The European polycystic kidney disease consortium, 1994; Mochizuki et al., 1996). Multiple hepatic cysts are a major manifestation in ADPKD (Carone et al., 1994), and BMHs are also frequently observed in polycystic liver, being considered to be a precursor of hepatic cysts (Gabow et al., 1990; Ramos et al., 1990; Carone et al., 1994). In addition, some of these hepatic cysts are proposed to arise from the cystic dilatation of peribiliary glands (Kida et al., 1992), because they are concentrated along the intrahepatic biliary tree and are intermingled with peribiliary glands showing various dilatation. These data suggest that hepatic cysts in ADPKD arise from BMHs (cysts in parenchyma) and also from intrahepatic peribiliary glands (peribiliary cysts) (Gupta et al., 1999; Kudo, 2001). In ADPKD, hepatic cysts generally develop during adult stage, later than the formation of polycystic kidney, and the hepatic cysts themselves are rarely symptomatic. 6.3.2. Altered expression of MUC mucins in the cystic liver diseases We have reported that MUC1 mucin of unglycosylated form, detected by MAbs DF3 and SM3, is frequently expressed in BECs lining cysts and BMHs, independent of the type of cystic diseases (Sasaki and Nakanuma, 1996a). Interestingly, MUC1 mucin of unglycosylated type is frequently expressed in BECs in histologically normal bile ducts in polycystic livers including ADPKD. In contrast, mature MUC1 mucin (glycosylated form), detected by MAb HMFG-1, is observed in BECs lining cysts, while it is rarely seen in BECs in BMH and intrahepatic bile ducts. These findings suggest that the expression of MUC1 mucin of unglycosylated form may represent the immature feature of BECs and that it may be related to the cystogenesis in ADPKD and other cystic liver diseases (Sasaki and Nakanuma, 1996a). In contrast, the expression of MUC1 mucin of glycosylated form may be related to the late cystogenic process in these cystic diseases.

7. MUC and TFF expression in biliary epithelial dysplasia and cholangiocarcinoma 7.1. Intrahepatic cholangiocarcinoma (ICC) ICC is an intrahepatic malignant tumor composed of cells resembling those of bile ducts. ICC arises from any portion of the intrahepatic bile ducts epithelium, from intrahepatic large bile ducts (the segmental and area bile ducts and their first branches) or small bile ducts. Cholangiocarcinoma arising from the right and left hepatic ducts at or near their junction is called hilar cholangiocarcinoma and is considered to be an extrahepatic lesion (Nakanuma et al., 2000). ICC is a relatively rare tumor in most populations in the world but second among primary malignant liver tumors; about 5–15% of liver cancer are estimated to be ICC (Kinami et al.,

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1978; Nakanuma et al., 1985, 2000). The incidence and mortality of ICC are increasing in the United States and worldwide (Patel, 2002; Shaib et al., 2004). Although several etiological factors such as liver fluke infection (Vatanasapt et al., 1995, 1999) and hepatolithiasis (Kinami et al., 1978; Nakanuma et al., 1985), CVH (Terada et al., 1994; Sorensen et al., 1998; Kobayashi et al., 2000; Shaib et al., 2005) have been proposed, the cause of ICC remains speculative in many cases (Nakanuma et al., 2000; Gores, 2003). So far, early detection of ICC is still difficult, and the overall prognosis after surgical resection is poor compared with that of hepatocellular carcinoma (HCC) (Nakanuma et al., 2000). 7.1.1. ICC associated with hepatolithiasis As described above, chronic proliferative cholangitis is a main histological finding in hepatolithiasis. Gastric mucosal metaplasia and intestinal metaplasia are common in the intrahepatic biliary tracts in hepatolithiasis, but these changes of the intrahepatic biliary tracts are rare in normal and control diseased livers. In patients with hepatolithiasis, ICC is known to develop in approximately 10% of more than 1000 patients with hepatolithiasis patients (Kinami et al., 1978; Nakanuma et al., 1985) and the relative risk is at least 30-fold, when compared with usual ICC without any background liver diseases. Hepatolithiasis is regarded as a model disease of cholangiocarcinogenesis arising in chronic inflammatory conditions. Biliary epithelial dysplasia is frequently associated with hepatolithiasis and being accepted as a precursor lesion of ICC in the biliary tract with hepatolithiasis (Nakanuma et al., 1985, 2000). The stepwise development and progression through biliary epithelial dysplasia, non-invasive ICC and invasive ICC has been proposed in hepatolithiasis (Nakanuma et al., 1985). Regarding the terminology and classification of biliary epithelial dysplasia, biliary intraepithelial neoplasia classification, corresponding to pancreatic intraepithelial neoplasm classification (Hruban et al., 2001) is being proposed. Oxidative and nitrosative stress are candidate causative factors involved in the carcinogenesis arising in chronic inflammatory condition (Gores, 2003; Sirica, 2005), although details remain to be elucidated, so far. 7.1.2. ICC associated with CVH Accumulating data suggest that chronic advanced liver disease, particularly cirrhosis, due to hepatitis B and C viral infection, is a major factor in hepatocarcinogenesis in humans (Ikeda et al., 1993; Sorensen et al., 1998; Kuper et al., 2001; Shaib et al., 2005). Although most primary hepatic tumors arising in CVH and cirrhosis are HCC, ICC and combined hepatocellular cholangiocarcinoma (HC-CC) occur occasionally in such instances (Terada et al., 1994; Sorensen et al., 1998; Kobayashi et al., 2000; Shaib et al., 2005). Patients with ICC or HC-CC arising in CVH and cirrhosis usually have a poor prognosis when compared with ordinary HCC arising in CVH and cirrhosis (Kobayashi et al., 2000). It is conceivable that such a minor population of hepatic carcinoma in CVH and cirrhosis may have a characteristic carcinogenesis and biologic behavior, which are different from ordinary HCC in CVH and cirrhosis and also from ICC arising in the apparently normal liver.

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7.2. Pathology of ICC In gross appearance, ICC are classified into three representative types of growth pattern: mass-forming type, periductal-infiltrating type and intraductal-growth type (Japan, 1997; Nakanuma et al., 2000). The mass-forming type is an expansive nodule and is the most common. The tumor borders between the cancerous and noncancerous portions are relatively clear. The periductal-infiltrating type, which is usually associated with biliary stricture, is relatively common. The tumor exhibits diffuse infiltration along the portal pedicle. This type resembles hilar or extrahepatic bile duct carcinoma. The intraductal-growth type is less common. These tumors are confined within the dilated part of an intrahepatic extension beyond the bile duct walls. Some tumors of this type of ICC may have arisen from biliary papillomatosis after malignant transformation (Japan, 1997; Nakanuma et al., 2000). Histologically, most ICCs are adenocarcinomas showing tubular and/or papillary structures with a various fibrous stroma (Colombari and Tsui, 1995; Nakanuma et al., 2000). Adenocarcinoma growing in the hepatic parenchyma and portal pedicle show usually a significant heterogeneity of histological features and degrees of differentiation. Adenocarcinoma shows tubular, cord-like or papillary patterns. The carcinoma cells are small or large, cuboidal or columnar, and can be pleomorphic. ICC arising from the large intrahepatic bile ducts shows intraductal micropapillary growth and in situ spreading along the biliary lumen. Once there is invasion through the periductal tissue, the lesion may be a well-, moderately or poorly differentiated adenocarcinoma, with abundant fibrous stroma (Nakanuma et al., 2000). Infrequently, ICC may be composed of adenosquamous, squamous, cholangiolocellular, mucinous, signet-ring cell, sarcomatous, lymphoepithelioma-like, clear cell variant, mucoepidermoid carcinomas. There is no dominant histological type of ICC in cases associated with liver flukes or hepatolithiasis when compared to those in non-endemic areas. ICC associated with CVH and cirrhosis shows frequently the histological features of so-called cholangiolocellular carcinoma (Sasaki et al., 2003b), in which the carcinoma cells are arranged as small, regular, narrow tubular structures resembling bile ductules or canals of Hering (Steiner and Higginson, 1959). 7.3. Expression of MUC mucins in ICC and biliary epithelial dysplasia There have been studies addressing the profile of MUC expression in ICC (Yamashita et al., 1993; Sasaki and Nakanuma, 1994; Sasaki et al., 1996, 1998a, 2003c, 2005b; Higashi et al., 1999; Amaya et al., 2001; Shibahara et al., 2004a, b; Goto et al., 2005; Aishima et al., 2006) and biliary epithelial dysplasia as preneoplastic lesion (Sasaki et al., 1996, 2003c; Lee and Liu, 2001; Ishikawa et al., 2004). These studies have addressed the relationship I between the profile of MUC expression and clinical and histopathological features in ICC and speculative cholangiocarcinogenesis. 7.3.1. Increased expression of MUC5AC in biliary epithelial dysplasia Biliary epithelial dysplasia is being accepted as a precursor lesion of ICC in the biliary tract with hepatolithiasis. The stepwise development and progression through

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biliary epithelial dysplasia, non-invasive ICC and invasive ICC has been proposed in hepatolithiasis (Nakanuma et al., 1985). Biliary epithelial dysplasia is defined as follows: structurally, the dysplastic epithelial cells show hyperplasia, such as piled-up nuclei and micropapillary projection into the ductal lumina. Cytologically, the dysplastic epithelial cells have an increased nucleo-cytoplasmic ratio, a partial loss of nuclear polarity, and nuclear hyper-chromasia. In biliary epithelial dysplasia, the up-regulation of MUC5AC mucin, a gastric foveolar-type mucin, has been reported (Sasaki et al., 1996, 2003c; Lee and Liu, 2001; Ishikawa et al., 2004) (Fig. 12). The increased expression of MUC5AC is coupled with the increased expression of TFF1 in biliary epithelial dysplasia. This indicates that gastric metaplasia is an early event in the stepwise carcinogenesis of ICC, especially of ICC associated with hepatolithiasis. Interestingly, the aberrant expression of MUC5AC has also been reported in the early step of pancreatic carcinoma (Kim et al., 2002). Therefore, increased expression of MUC5AC may be a common feature suggesting the early step of carcinogenesis in pancreatobiliary system. Focal expression of MUC1 and MUC2 mucin is also reported (Sasaki et al., 1996). MUC3 mucin is frequently expressed in biliary epithelial dysplasia as well as non-dysplastic BECs in the large bile ducts. However, it is of note that the expression of MUC3 mucin is focally decreased in biliary epithelial dysplasia (Sasaki et al., 1996). The increased expression of MUC4 is also seen in biliary epithelial dysplasia associated with hepatolithiasis (Lee and Liu, 2001). 7.3.2. Expression profiles and its significance of MUC mucins in ICC The increased expression of MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC6 and MUC7 has been reported in ICC, so far (Yamashita et al., 1993; Sasaki and Nakanuma, 1994; Sasaki et al., 1996, 1998a, 2003c, 2005b; Higashi et al., 1999; Amaya et al., 2001; Shibahara et al., 2004a, b; Goto et al., 2005; Aishima et al., 2006) (Fig. 13). The molecular mechanisms involved in the altered regulation of mucin genes and the significance of the altered expression of MUC mucin in carcinoma cells have not been fully clarified so far and further studies are needed.

Fig. 12. Expression of MUC5AC and TFF1 in biliary epithelial dysplasia associated with hepatolithiasis. MUC5AC (A) and TFF1 (B) are strongly expressed in the cytoplasm of dysplastic biliary cells. Immunohistochemical staining for MUC5AC (A) and TFF1 (B).

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Interestingly, MUC mucin profiles appear to be different between ICCs arising in the large bile duct and those in the small bile duct. ICC of hilar type arising from the large bile ducts, including those associated with hepatolithiasis frequently express MUC3 mucin, whereas, MUC3 expression is infrequent in ICC of peripheral type; ICC suggested to arise from small bile ducts (Sasaki et al., 1996). ICC associated with hepatolithiasis shows characteristics of ICC arising in large bile ducts (Fig. 13). Fig. 14 summarizes the expression profiles of MUC mucin during stepwise development and progression through biliary epithelial dysplasia, non-invasive ICC, and invasive ICC in the biliary tract with hepatolithiasis. In contrast, ICC associated with CVH and cirrhosis lacks MUC3 expression and shows characteristics of ICC arising in small bile ducts. The expression of MUC5AC is also rather low in ICC associated with CVH and cirrhosis (Sasaki et al., 1998a). ICC associated with CVH and cirrhosis frequently express MUC6 and MUC7 mucin (Sasaki et al., 1998a). The MUC mucin profile is similar to that of the cholangiocarcinoma elements of HC-CC, suggesting a common histogenesis of these types of carcinomas (Sasaki et al., 1998a). The increased expression of MUC1, MUC4 and MUC5AC is related to aggressive behavior of carcinoma cells and poorer prognosis, whereas the expression of MUC2 and MUC6 indicates better prognosis reportedly (Yamashita et al., 1993; Higashi et al., 1999; Amaya et al., 2001; Shibahara et al., 2004b; Goto et al., 2005; Aishima et al., 2006). In the standpoint of MUC mucin profiles, ICC could be categorized into intestinal type (MUC2-expressing), gastric (foveolar) type (MUC5AC-expressing) and pyloric gland type (MUC6-expressing), as proposed in gastric (Pinto-deSousa et al., 2002; Tsukamoto et al., 2005) and pancreatic carcinoma (Kim et al., 2002; Luttges et al., 2002; Terris et al., 2002; Adsay et al., 2004). A better clinical outcome has been reported in the carcinoma of ‘‘intestinal’’ type, when compared with those of ‘‘gastric’’ type in carcinomas in several organs including gastric and pancreatic carcinoma. MUC1. MUC1 is frequently expressed in the luminal surface of tubular structure, cell membrane and cytoplasm of the carcinoma cells in ICC (Fig. 13) and cholangiocarcinoma element of HC-CC (Yamashita et al., 1993; Sasaki and Nakanuma, 1994; Sasaki et al., 1996). The increased expression of MUC1 is evident especially in invasive ICC, when compared with non-invasive ICC. Increased MUC1 expression is associated with poor patient outcome in ICC (Higashi et al., 1999). Carcinoma cells expressing high levels of MUC1 have increased invasive and metastatic potential in many organs (Osako et al., 1993; Kim and Gum, 1995; Terris et al., 2002). MUC1 expression on carcinoma cells contributes to the inhibition of cell–cell and cell–substratum interaction and may play a role in the metastasis of carcinoma cells. Rigid MUC1 molecules may be involved in escaping from immune surveillance (van de Wiel-van Kemenade et al., 1993). It is also known that MUC1 acts as docking protein for signaling molecules (Li et al., 2001; Huang et al., 2005). For example, the MUC1 oncoprotein is aberrantly overexpressed in most human carcinomas and associates with b-catenin (Huang et al., 2005). Dysregulation of b-catenin is of importance for the development of diverse human malignancies. The EGF receptor regulates interaction of the human DF3/MUC1 carcinoma antigen

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Fig. 13. Gross appearance and expression of MUC mucins and TFF1 in intrahepatic cholangiocarcinoma associated with hepatolithiasis. (A) Gross appearance of intrahepatic cholangiocarcinoma associated with hepatolithiasis. Periductal infiltration of cholangiocarcinoma shows marked thickening of the intrahepatic bile ducts (arrows). Asterisks, hepatoliths. (B) TFF1 is expressed in the non-invasive carcinoma cells in the surface area (arrowheads), whereas TFF1 is not expressed in the invasive carcinoma cells (arrows). (C–E) MUC1 (C), MUC3 (D) and MUC5AC (E) mucins are expressed on the luminal surface (C) and in the cytoplasm (D and E) of carcinoma cells in intrahepatic cholangiocarcinoma associated with hepatolithiasis. Immunohistochemical staining for (C), MUC3 (D) and MUC5AC (E).

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Fig. 14. The expression profile of MUC mucin during stepwise development and progression through biliary epithelial dysplasia, non-invasive and invasive intrahepatic cholangiocarcinoma in hepatolithiasis. ICC, intrahepatic cholangiocarcinoma.

with c-Src and beta-catenin (Li et al., 2001). Furthermore, a recent report revealed that RNA interference suppression of MUC1 reduces the growth rate and metastatic phenotype of human pancreatic cancer cells (Tsutsumida et al., 2006). MUC2. MUC2 mucin is expressed in 21% of ICC arising in the large bile duct, whereas the expression is detected in 10% of ICC of peripheral type (Sasaki et al., 1996) (Fig. 15). The frequency of MUC2 expression is rather high in ICC associated with hepatolithiasis (29%). MUC2 itself has a property of a tumor suppressor gene (Velcich et al., 2002). In contrast to MUC1 expression indicating a poorer prognosis, MUC2 expression is higher in the mucinous type of tumors with a favorable outcome in biliary and pancreatic tumors (Yamashita et al., 1993; Higashi et al., 1999; Amaya et al., 2001; Terris et al., 2002). Recently, a specific group of intraductal papillary tumors and associated, mucinous carcinoma are categorized as counterparts of pancreatic intraductal papillary-mucinous neoplasm (Chen et al., 2001; Shibahara et al., 2004a). This type of tumor is occasionally associated with hepatolithiasis and the frequent and characteristic expression of MUC2 mucin has been noted (Ishikawa et al., 2004) (Fig. 15). In these cases, dilated intrahepatic large and extrahepatic bile ducts are filled with papillary dysplastic or carcinomatous biliary epithelia with delicate fibrovascular stalks and oversecretion of mucin (Chen et al., 2001). Mucinous ICC is characteristically associated with this type of tumor (Ishikawa et al., 2004). The MUC2 expression in the intrahepatic biliary system, including intestinal metaplasia, intraductal papillary tumors and mucinous carcinoma, is dependent on the CDX2 homeobox gene, which induces intestinal differentiation (Ishikawa et al., 2004) (Fig. 15). CDX2 is a key molecule in

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Fig. 15. CDX2-dependent MUC2 expression in biliary epithelial dysplasia and mucinous cholangiocarcinoma associated with hepatolithiasis. Left column, biliary epithelial dysplasia; Right column, mucinous ICC. MUC2 is expressed in the cytoplasm of goblet-type cells in biliary epithelial dysplasia and mucinous carcinoma. CDX2 is expressed in the nuclei of corresponding goblet-type cells in biliary epithelial dysplasia and mucinous carcinoma. Hematoxylin and eosin (top), and immunohistochemical staining for MUC2 (middle) and CDX2 (bottom).

intraductal papillary tumors associated with hepatolithiasis, gastric carcinoma and Barrett’s adenocarcinoma, in which transient or constant intestinal metaplasia is important in the carcinogenesis. Therefore, it is conceivable that a common factor,

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which promotes or regulates CDX2 expression, is closely related to the pathway of stepwise carcinogenesis in these carcinomas. Accumulating data suggest that CDX2 functions as a tumor suppressor, and CDX2-dependent regulation of cell proliferation may be an important factor in defining the prognosis of the patient. Forced expression of CDX2 in various intestinal epithelial cell lines inhibits cell proliferation and stimulates cell differentiation and apoptosis (Lorentz et al., 1999; Mallo et al., 1998). CDX2 restoration in CDX2-negative cells decreases the proliferative activity (Hinoi et al., 2003). CDX2 expression decreases with the tumor grade in human colon cancers and in chemically induced tumors in the rat (Ee et al., 1995). MUC3. ICC of hilar type arising from the large bile ducts, including those associated with hepatolithiasis frequently express MUC3 mucin (Fig. 13), whereas MUC3 expression is infrequent in ICC of peripheral type; ICC is suggested to arise from small bile ducts (Sasaki et al., 1996). Since MUC3 mucin is physiologically expressed in the intrahepatic large bile duct (Sasaki et al., 1995), the frequent expression of MUC3 mucin reflects the original phenotype of large bile ducts in ICC of hilar type. MUC3 expression is seen in 57% of ICC associated with hepatolithiasis (Sasaki et al., 1996), and the expression is rather frequent in noninvasive ICC. MUC5AC expression is frequently seen in ICC (Sasaki et al., 1996, 2003c). MUC4. MUC4 is a novel intramembrane ligand for receptor tyrosine kinase (ErbB2) (HER-2) (Komatsu et al., 2001) and is also involved in the regulation of p27. MUC4 is considered to be a tumor-associated molecule from the potential role of MUC4 as a marker for adenocarcinoma of the pancreas. MUC4 is frequently expressed in ICC associated with hepatolithiasis and biliary epithelial dysplasia (about 70%) in previous report (Lee and Liu, 2001) and according to our observation. The expression of MUC4 in ICC of the mass-forming type is a new independent factor for poor prognosis (Shibahara et al., 2004a). MUC5AC. MUC5AC, a gel-forming mucin of gastric foveolar type, is frequently over-expressed in ICC arising in the large bile ducts including ICC associated with hepatolithiasis (Fig. 13) (Sasaki et al., 1996, 2003c; Lee and Liu, 2001; Ishikawa et al., 2004; Aishima et al., 2006). Since MUC5AC is frequently expressed in biliary epithelial dysplasia (Fig. 12), ‘‘gastric metaplasia’’ is an early event in the stepwise carcinogenesis of ICC, especially of ICC associated with hepatolithiasis (Fig. 14). In contrast, MUC5AC expression is less frequent in ICC of peripheral type (Sasaki et al., 1996, 1998a; Aishima et al., 2006). MUC5AC expression in ICC is associated with a higher incidence of lymph node metastasis and is an independent prognostic factor by multivariate survival analysis (Aishima et al., 2006). MUC5B. The expression of MUC5B is focal and weak in the normal large bile ducts and the expression is increased in hepatolithiasis. MUC5B mucin is focally expressed in ICC, but the expression of MUC5B mRNA is reported to decrease in ICC in contrast to non-neoplastic biliary epithelium (Lee and Liu, 2001). MUC6 and MUC7. MUC6 mucin is frequently and widely expressed in ICC and cholangiocarcinoma components of HC-CC (Sasaki et al., 1998a). In mucinproducing bile duct carcinomas, aberrant expression of MUC6 (pyloric gland-type

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mucin) corresponds to the ‘‘cuboidal-type’’, not to ‘‘columnar-type’’ of mucinproducing bile duct tumor and may be related to the better prognosis (Goto et al., 2005; Aishima et al., 2006). MUC7 mucin was also frequently expressed in ICC and cholangiocarcinoma components of ICC (Sasaki et al., 1998a). 7.4. Expression of TFF peptides in ICC Up-regulation of TFF1 coupled with MUC5AC in biliary epithelium in hepatolithiasis, biliary epithelial dysplasia (Fig. 12), and non-invasive ICC (Fig. 13) may reflect the gastric metaplasia and early neoplastic lesion (Sasaki et al., 2003c). However, TFF1 expression is significantly decreased in invasive ICC (Fig. 13) despite preserved expression of MUC5AC (Sasaki et al., 2003c). Missense mutations are detected in a part of non-invasive and invasive ICC and loss of heterozygosity of the TFF1 gene is not detectable (Sasaki et al., 2003c). The decreased expression of TFF1 in invasive ICC may be explained by the methylation of TFF1 promoter region (Sasaki et al., 2003c). The decreased TFF1 expression may lead to an increased cell proliferation and then to an invasive character of ICC (Sasaki et al., 2003c). Recently, patients with cholangiocarcinoma who had amplification at chromosomal region 21q22-qter harboring candidate genes especially TFF and serine protease family showed poor prognosis, whereas patients who had deletion showed favorable prognosis (Muenphon et al., 2006). 7.5. Expression of MUC mucins and TFF peptides in gallbladder carcinoma, extrahepatic bile duct carcinoma and ampullary carcinoma There have been studies addressing the profile of MUC expression in gallbladder carcinoma (Sasaki et al., 1999; Yamato et al., 1999b; Ghosh et al., 2005; Takagawa et al., 2005; Wu et al., 2005), extrahepatic bile duct carcinoma (Tamada et al., 2002) and ampullary carcinoma (Kitamura et al., 1996; Gurbuz and Kloppel, 2004; Zhou et al., 2004) Khayyata et al., 2005). The relationship between the profiles of MUC expression and clinical outcomes and prognosis of the patients has been described (Zhou et al., 2004; Ghosh et al., 2005; Takagawa et al., 2005). Overall profiles are quite similar among ICC, extrahepatic bile duct carcinoma and ampullary carcinoma. Long with ICC, MUC1 is a marker of poorer prognosis, whereas MUC2 is a marker indicating better prognosis.

8. Concluding remarks BECs in the intrahepatic biliary system express a specific spectrum of MUC mucins and TFF peptides. The expression profile of MUC mucins and TFF peptides may be closely related to the physiological function of the intrahepatic biliary system. The altered expression profiles of MUC mucins and TFF peptides are seen in various hepatobiliary diseases. In particular, increased and aberrant expression of

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gel-forming mucins; MUC5AC, MUC2, MUC6 and MUC5B and TFF1 and TFF3 is evident in the intrahepatic biliary system in hepatolithiasis, suggesting that the MUC mucins and TFF peptides may be involved in its pathogenesis. Aberrant expression of MUC5AC and TFF1 is distinct in biliary epithelial dysplasia as a preneoplastic lesion, suggesting the alteration may be the early event in the multistep carcinogenesis in the intrahepatic biliary system. Diverse expression profiles of MUC mucin and TFF peptides in ICC may reflect two or more pathways of carcinogenesis, the diverse aggressiveness of carcinoma cells and also a prognosis of patients with ICC. There has been great progress in the cloning of MUC mucins and TFF peptides and characterization of their structures and physiological and pathological functions. However, a number of issues remain to be investigated regarding the regulation, the function and more detailed expression profiles of MUC mucins and TFF peptides in the intrahepatic biliary system. Acknowledgements This work is supported in part by a Grant-in Aid for Scientific Research (C) from the Ministry of Education, Culture, Sports and Science and Technology of Japan (15590297 and 18590325) and by a Research Grant from the Hokkoku Cancer Research Foundation.

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