Overexpression of ╬▓-Catenin is Responsible for the Development of Portal Hypertension During Liver Cirrhosis.код для вставкиСкачать
THE ANATOMICAL RECORD 292:818–826 (2009) Overexpression of b-Catenin is Responsible for the Development of Portal Hypertension During Liver Cirrhosis JIAN-JUN HONG,1,2 FEI-YAN PAN,1 YAN QIAN,1 LI-CHENG CHENG,1 HONG-XIA ZHANG,1 BIN XUE,1 AND CHAO-JUN LI1,3* 1 The Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing, China 2 Medical Department, The Afﬁliated Hospital of Nanjing University of TCM, Nanjing, China 3 Model Animal Research Center (MARC), Nanjing University, Nanjing, China ABSTRACT b-catenin functions as both a structural protein and a transcriptional activator. In this study, we examined the expression of b-catenin in human cirrhotic livers, and administered adenoviruses carrying the b-catenin or DTCF4 genes to cirrhotic rats to investigate the role of b-catenin in the development of liver cirrhosis development. b-catenin expression was associated with liver cirrhosis development in cirrhotic human and rat liver. bcatenin adenovirus was capable of accelerating cirrhosis progress but this progression was unaffected by administration of DTCF4 adenovirus. b-catenin was mainly located in the intercellular regions between liver cells and was highly concentrated in the hepatic sinusoid wall, where a-smooth muscle actin (SMA) was also mainly distributed. The binding of b-catenin to aSMA was also increased in cirrhotic liver. Portal vein blood pressure was signiﬁcantly increased in the group administered b-catenin adenovirus, but not in that receiving DTCF4 adenovirus. These results suggest that high concentrations of b-catenin at the hepatic intercellular membrane and the hepatic sinusoid wall contribute to hepatic hyperpiesia in liver cirrhosis patients. b-catenin functions as a structural molecule, but not as a signaling molecule, during liver cirrhosis development. Anat Rec, 292:818–826, C 2009 Wiley-Liss, Inc. 2009. V Key words: b-catenin; liver cirrhosis; hepatic hyperpiesia; a-smooth muscle actin; Wnt signaling Liver cirrhosis occurs as a result of a wide variety of liver diseases including alcoholic hepatitis, nonalcoholic steatohepatitis, viral hepatitis, and cholestatic liver diseases (Wells, 2006). The development of liver cirrhosis is a progressive pathological process involving multiple cellular and molecular events that ultimately lead to distortion of the normal liver architecture (Friedman, 2000). When the liver is insulted by hepatotoxic factors, excess matrix proteins are deposited in the extracellular space, causing alterations in the microenvironment and resulting in liver ﬁbrosis and ﬁnally cirrhosis (Friedman, 2000, 2003; Bataller and Brenner, 2005; Wells, 2006). Hepatic stellate cells (HSCs) are the key ﬁbrogenic cells and play a pivotal role in liver ﬁbrosis (Eng and C 2009 WILEY-LISS, INC. V Grant sponsor: National Natural Science Foundation of China; Grant number: 30800574; Grant sponsor: Natural Science of Foundation of Jiangsu Province of China; Grant number: BK2008432; Grant sponsor: Natural Science of Foundation of the Jiangsu High Education Institutions of China; Grant number: 07KJD180103. *Correspondence to: Chao-Jun Li, Professor, The Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing 210046, China. Fax: þ086-25-85891870. E-mail: firstname.lastname@example.org Received 29 August 2008; Accepted 10 February 2009 DOI 10.1002/ar.20897 Published online in Wiley InterScience (www.interscience.wiley. com). b-CATENIN AND LIVER PORTAL HYPERTENSION Friedman, 2000; Friedman, 2004; Bataller and Brenner, 2005). HSCs are activated during wound healing in the liver, and they secrete an array of mediators of ﬁbrosis, such as transforming growth factor-b for ﬁbrogenesis, matrix metalloproteinase-2 for matrix degradation, monocyte chemotactic-1 and platelet-derived growth factor for leukocyte and HSC chemotaxis, and endothelin (ET)-1 for contractility (Eng and Friedman, 2000; Friedman, 2000, 2003; Bataller and Brenner, 2005; Wells, 2006). This process of advanced liver ﬁbrosis ﬁnally results in liver cirrhosis. The activated HSCs also express substantial amounts of a-smooth muscle actin (a-SMA). Increased expression of a-SMA results in an augmentation of contractility that impedes portal blood ﬂow, both by constricting individual sinusoids and by contracting the cirrhotic liver (Racine-Samson et al., 1997; Rockey, 2001). This causes hepatocellular dysfunction and increases intrahepatic resistance to blood ﬂow, all of which can result in hepatic insufﬁciency and portal hypertension, digestive tube hemorrhage, and abdominal dropsy (Albanis and Friedman, 2001; Gines et al., 2004). We recently showed that Wnt signaling was aberrantly activated in the hepatocellular carcinoma (HCC) cell line BEL-7402 (Zhao et al., 2004). As a pivotal component of Wnt signaling, b-catenin functions both as a structural protein that links adhesion receptors of the cadherin family to the actin cytoskeleton, enhancing intercellular adhesion, and as a transcriptional activator that mediates Wnt signal transduction (Adams and Nelson, 1998; Ben-Ze’ev and Geiger, 1998; Bullions and Levine, 1998; Behrens, 1999; Nelson and Nusse, 2004). It has been shown that increased cell proliferation owing to the aberrant activation of b-catenin might contribute to the progression of several kinds of cancers, including colorectal cancer, liver cancer, and ovarian carcinoma (Korinek et al., 1997; Palacios and Gamallo, 1998; Morin, 1999; Polakis, 1999; Thompson and Monga, 2007). Shackel et al. analyzed the gene expression in hepatitis C virus (HCV)-associated cirrhosis and identiﬁed many differentially expressed genes including those associated with inﬂammation, cirrhosis, proliferation, signaling, apoptosis, and oxidative stress. b-catenin and two secreted frizzled-related proteins (sFRP), SARP3 (sFRP5) and FRITZ (sFRP3), were found to be increased in HCV-associated cirrhosis (Shackel et al., 2002). b-catenin was increased in the ﬁbrous septa of proliferating bile duct structures in autoimmune hepatitis-, HCV- and HBV-associated cirrhosis, and primary biliary cirrhosis (Shackel et al., 2002). However, controversies remain concerning the pathophysiological roles of b-catenin during liver ﬁbrogenesis (Kondo et al., 1999; Vona et al., 2004). Kondo et al. failed to detect either accumulation or mutation of b-catenin in early HCC, suggesting that its accumulation and mutation might be associated with malignant progression of HCC (Kondo et al., 1999). Vona et al., however, argued against an impact of b-catenin in the initial step of tumor cell invasion (Vona et al., 2004). Thus, the role of b-catenin in liver disease still remains unclear. In this report, we examined the relationship between the expression and distribution of b-catenin and liver cirrhosis progression in human liver samples and also investigated the function of b-catenin using the CCl4-induced liver cirrhosis rat model. 819 TABLE I. Clinical patient data No. of patients Male/Female Age Normal liver Low-grade cirrhosis High-grade cirrhosis 4 4/2 53.7 14.2 8 4/4 52.1 16.4 19 11/8 50.3 13.7 Patients were grouped as follows: normal liver, low-grade cirrhosis, and high-grade cirrhosis. Informed consent was obtained from each patient and this study conformed to the ethical guidelines of China. The extent of liver cirrhosis was staged by pathologists according to the standards of The Chinese Society of Infectious Diseases and Parasitology and The Chinese Society of Hepatology of The Chinese Medical Association. There were no signiﬁcant differences in the ages between the three groups (P > 0.05). MATERIALS AND METHODS Patients Liver specimens were obtained from 27 patients with liver cirrhosis who underwent liver resection at the Department of Surgery of JiangSu Hospital of Traditional Chinese Medicine, and from six noncirrhotic controls undergoing liver transplantation surgery. Informed consent was obtained from each patient and this study conformed to the ethical guidelines of China. Clinical information is given in Table 1. The extent of hepatocellular disease was staged by pathologists according to the standards of the Chinese Society of Infectious Diseases and Parasitology and the Chinese Society of Hepatology of the Chinese Medical Association (2000). Antibodies and Reagents Mouse monoclonal antibody against a-SMA was purchased from DAKO (Carpinteria, CA). Rabbit polyclonal antibody against b-catenin was purchased from Santa Cruz Bio-technology (Santa Cruz, CA). Rhodamine phalloidin was purchased from Molecular Probes. Other chemicals including CCl4 and olive oil were obtained from Sigma unless otherwise indicated. Induction of Cirrhosis by CCl4 and Adenovirus Administration Male Sprague Dawley rats (200–300 g) were obtained from the Nanjing Medicine University Animal Center. Liver damage was induced by hypodermic injection of CCl4 twice weekly for 2 months (40% sterile CCl4 in olive oil at a dosage of 0.02 mL/kg). All rats (N ¼ 70) were given standard chow. Water containing 10% alcohol was supplied. At the 9th week, all surviving male rats (30) were randomly divided into four groups (phosphatebuffered saline (PBS) mock, control adenovirus, Ad-bcatenin, Ad-DTCF4). Adenovirus was injected via the tail vein at 1 109 particles three times a week for 2 weeks. At Week 11, the portal vein pressure and the portal vein blood ﬂow of all animals were measured, the animals were sacriﬁced, and the livers subjected to histological analysis using hematoxylin/eosin or immunhistochemical staining. All experiments were performed according to the criteria of the Committee for the Care and Use of Laboratory Animals of Nanjing Medicine University. 820 HONG ET AL. Immunohistochemistry Frozen liver tissues were sectioned at 5 lm. The sections were incubated with primary antibody (1:100 dilution), followed by horseradish peroxidase-conjugated secondary antibody. The signal was developed using a streptavidin peroxidase kit (Beijing Zhongshan Company, Beijing, China). Immunoprecipitation and Western Blotting Tissue and whole-cell lysates were prepared according to standard protocols. The protein concentration was measure using the Brandford method. For immunoprecipitation experiments, a total of 200 lg protein was incubated at 4 C with l lg of anti-b-catenin or a-SMA antibodies. After precipitation with 30 lL protein A/G beads (Roche, Mannhein, Germany) on a rotation machine at 4 C overnight, samples were washed three times with 800 lL lysis buffer and once with PBS. Pellets were then boiled for 2 min in 2 sample loading buffer and analyzed by Western blotting. For Western blotting, equal amounts of protein were loaded, resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred onto polyvinylidene diﬂuoride membranes (Bio-Rad, Hercules, CA). The membranes were then incubated with the appropriate primary antibody, as indicated. Bound antibody was visualized using alkaline phosphatase-conjugated secondary antibodies. Adenovirus Generation and Infection The generation, ampliﬁcation, and titering of adenovirus carrying wild-type b-catenin and dominant-negative TCF4 genes were performed according to the simpliﬁed system described by He et al. (1998). The genes tagged with myc were inserted between the BglII and HindIII sites of pShuttle-CMV and recombined with pAdEasy-1 in BJ5183 bacteria. The virus was generated in 293A cells. Viral particles were puriﬁed by cesium chloride density gradient centrifugation. Measurement of Portal Vein Blood Pressure and Blood Flow Volume All rats were anaesthetized by intraperitoneal injection of 40 mg/kg pentobarbital sodium. The abdominal area was exposed and the portal vein of the liver was carefully dissected from the surrounding tissues. The portal vein blood ﬂow was measured using an electromagnetic blood ﬂowmeter (MFV-3200, Nihon Kohden, Japan). The longest segment of the portal vein blood vessel was carefully lifted and the probe head (lumen diameter 1.5 or 2 mm) was inserted, without twisting or bending. For portal vein pressure measurement, an 18G pinhead with catheter was inserted into the portal vein. The catheter was connected to a highly sensitive pressure transducer (TP-400T, Nihon Kohden) and the data transcribed on a multichannel recorder (Polygraph system, Nihon Kohden). Statistical Analyses All data are expressed as mean SD. Statistical signiﬁcance was determined by Student t test or by ANOVA TABLE 2. Comparison of b-catenin expression between early- and late-stage liver cirrhosis Normal liver Early liver cirrhosis Advanced liver cirrhosis Number of specimens Grade 1 Grade 2 Grade 3 6 8 19 0 7 1 0 1 6 0 0 12 For analysis of results, ﬁve ﬁelds were randomly chosen in each tissue section and the proportion of b-catenin-positive hepatocytes was determined. Tissue with 0%–25% of b-catenin-positive hepatocytes was deﬁned as Grade 1, 25%–50% of positive staining was termed Grade 2, and 50%–100% of positive staining was termed Grade 3. The level of b-catenin expression was positively correlated with the stages of liver cirrhosis (P < 0.0001). in the case of comparison of multiple groups. P < 0.05 was considered signiﬁcant. RESULTS The Expression and Distribution of b-Catenin is Related to Liver Cirrhosis Progression The level of b-catenin expression was positively correlated with the stages of liver cirrhosis (P < 0.0001, Table 2). In normal hepatic tissues, only weak immunostaining of b-catenin was observed at the hepatocyte membrane, except at the interlobular bile duct epithelia (arrow), where b-catenin was strongly stained (Fig. 1A). In the cirrhotic liver tissues, b-catenin expression was increased in progressive stages. The most intensely stained sample was from the advanced stage of liver cirrhosis (Fig. 1B). Moreover, numerous hyperplastic interlobular bile ducts were identiﬁed in the advanced cirrhotic liver sample, and b-catenin was strongly stained in the duct epithelial cells (arrow, Fig. 1C). In the cirrhotic tissue, b-catenin was not only strongly concentrated at the hepatocyte membrane but was also positively stained in the cytosol (Fig. 1D). The Expression of b-Catenin in Rat Cirrhotic Liver We established an animal model of liver cirrhosis and examined the expression of b-catenin during cirrhosis progression. Although invasive lymphocytes were found in the liver after 5 weeks of hepatic injury, no pseudolobule formed (data not shown). A high level of micronodular cirrhosis was detected after 12 weeks of CCl4 treatment, with obvious nodular cirrhosis, which was continuous and extended throughout the entire tissue section (Fig. 2A, arrow). The hepatocytes contained large numbers of fatty granules (star) and balloon degeneration of hepatocytes was also observed (triangle) after 12 weeks of treatment (Fig. 2A). b-catenin expression increased with the development of liver cirrhosis in these animal models. At the early stage of hepatic injury, little b-catenin was detected in the cytosol and the cell membrane (data not shown), whereas b-catenin was highly expressed in the membranes of the hepatocytes, comparable to the situation in human liver at advanced stages of hepatic cirrhosis (Fig. 2B). These b-CATENIN AND LIVER PORTAL HYPERTENSION 821 Fig. 1. The expression and distribution of b-catenin is related to the development of human liver cirrhosis. A: Normal, control hepatic tissues; only weak immunostaining of b-catenin was observed at the hepatocyte membrane, except at the interlobular bile duct epithelia. B: High-grade liver cirrhosis tissues; b-catenin showed the most intense staining in the cell membrane and cytosol. C: High-grade liver cirrhosis tissues; b-catenin was also strongly stained in the duct epithelial cells. D: High-grade liver cirrhosis tissues; b-catenin was not only strongly concentrated at the membrane of the hepatocytes but was also positively stained in the cytosol. Scale: 10 lm. data further conﬁrm the hypothesis that b-catenin expression is related to liver cirrhosis progression. was also distorted with collagen ﬁber hyperplasia in bcatenin adenovirus-infected rats (Fig. 3C). Obvious cirrhotic lobules separated by well-delineated ﬁbrous septae with collagen deposition (arrow) were found in this group. Overexpression of b-Catenin Accelerates the Progress of Liver Cirrhosis To examine the function of b-catenin in liver cirrhosis, we administered recombinant b-catenin adenovirus to rats. Western blotting conﬁrmed the overexpression of bcatenin (Fig. 3A). Pathological analysis showed that liver cirrhosis development was signiﬁcantly increased in the b-catenin group (P ¼ 0.023, 7/10). Administration of DTCF4 adenovirus, however, produced results that were not signiﬁcantly different from those of the control group (2/8, Table 3). Immunostaining results demonstrated that the overexpressed b-catenin was mainly distributed at the cell membrane (Fig. 3B). The liver architecture Overexpression of b-Catenin Increases Liver Portal Vein Blood Pressure Although we found that overexpression of b-catenin could accelerate liver cirrhosis progression, blocking the b-catenin signal by overexpression of DTCF4 had no obvious effect on this process. This suggests that b-catenin signaling might not be involved in liver cirrhosis development. We found that overexpression of b-catenin could result in collagen ﬁber hyperplasia, indicating that the status of the hepatic microcirculation was also 822 HONG ET AL. Fig. 2. The expression of b-catenin in cirrhotic rat liver. A: In cirrhotic rat liver, obvious nodular cirrhosis with deposition of well-delineated ﬁbrous septae (arrow) was found. The hepatocytes contained large numbers of fatty granules (star) and balloon degeneration of hepatocytes was also observed (triangle). B: b-catenin was highly expressed in the membranes of the hepatocytes. Scale: 10 lm. altered. We therefore measured the portal vein pressure and blood ﬂow in the rats. Portal vein pressure was signiﬁcantly increased only by b-catenin adenovirus administration (Fig. 4A). The pressure increased from 8.202 1.548 (control adenovirus, N ¼ 8) to 11.091 2.13 mmHg (b-catenin adenovirus, N ¼ 10) (P ¼ 0.004). However, the administration of adenoviruses with b-catenin or DTCF4 had no signiﬁcant effect on blood ﬂow (Fig. 4B). The results suggest that the b-catenin protein level may result in liver cirrhosis-associated portal vein pressure elevation. b-catenin/a-SMA complexes were found in cirrhotic liver tissue than in normal liver tissue (Fig. 5C). b-catenin and F-actin were also colocalized at the cell membrane in the cell–cell adhesion area in aggregated LO2 cells (Fig. 5D,E). These data suggest that colocalized b-catenin and actin might be involved in the constriction of individual sinusoids and contraction of the cirrhotic liver, thus modulating the increased portal vein pressure in cirrhotic liver tissue. DISCUSSION The Interaction Between b-Catenin and a-SMA in Cirrhotic Liver Might Be Associated With Hyperpiesia of the Portal Vein The mechanism whereby b-catenin expression could result in increased portal vein pressure remains unclear, but it is well known that b-catenin can work as a structural molecule by connecting actins and cadherins at the cell membrane where the cell is integrated with the extracelluler matrix. The hepatic sinusoid wall showed strong b-catenin staining in cirrhotic liver samples (Fig. 5A). a-SMA was also mainly distributed on the hepatic sinusoid wall (Fig. 5B), particularly in the branch of spindle-shaped cells (activated HSCs). Because activated HSCs impede portal blood ﬂow both by constricting individual sinusoids and by contracting the cirrhotic liver, the colocalization of b-catenin and a-SMA might be related to the development of portal hypertension in cirrhotic liver. We examined the binding of a-SMA and b-catenin in cirrhotic and normal liver tissues by immunoprecipitation. a-SMA and b-catenin interacted directly, and more The later stages of liver cirrhosis are often accompanied by complications such as portal vein hypertension, digestive tube hemorrhage, and abdominal dropsy (Albanis and Friedman, 2001; Gines et al., 2004). At the early stage of liver cirrhosis, HSCs are activated by liver injury resulting from hepatotoxic insults, including alcohol stimulation and virus infection. HSCs undergo a process of activation and exert increased contractility during the wound healing response (Racine-Samson et al., 1997; Rockey, 2001), leading to increased portal resistance. Meanwhile, activated HSCs can secrete ET-1, the key contractile stimulus of stellate cells (RacineSamson et al., 1997). Nitric oxide production is meanwhile decreased, reducing the physiological antagonism to ET-1 (Rockey and Chung, 1995; Gupta et al., 1998a,b). Thus, as liver disease progresses, the imbalance shifts in favor of ET-1, therefore increasing the contractile activity of stellate cells and impeding portal blood ﬂow both by constricting individual sinusoids and by contracting the cirrhotic liver (Racine-Samson et al., 1997). The activated HSCs also express substantial amounts of a-SMA and augment contractility (Rockey, b-CATENIN AND LIVER PORTAL HYPERTENSION 823 Fig. 3. The effect of adenovirus administration on the progress of liver cirrhosis. A: b-catenin adenovirus administration greatly increased b-catenin gene expression. B: b-catenin was mainly distributed in the cell membrane after b-catenin adenovirus administration. C: The liver architecture was altered, with collagen ﬁber hyperplasia in b-catenin adenovirus-infected rats. Cirrhotic lobules separated by welldelineated ﬁbrous septae with collagen deposition were also obvious (arrow). Scale: 10 lm. TABLE 3. b-catenin adenovirus accelerates the development of liver cirrhosis 2001). However, the intrinsic responsible for portal vein hypertension still remains unclear. Numerous studies have shown that aberrations in the intracellular signaling molecule, b-catenin, are involved in the pathogenesis of various types of malignancies (Korinek et al., 1997; Palacios and Gamallo, 1998; Morin, 1999). Zeng et al. recently identiﬁed 11 Wnts and nine Frizzleds that were normally expressed in adult mouse liver, and were differentially expressed in various cell types within the liver (Zeng et al., 2007). Speciﬁc differences in expression were also observed in active and resting states of various cell types. This suggested that the Wnt/b-catenin signaling pathway might be signiﬁcantly involved in different liver pathologies (Zeng et al., 2007). However, b-catenin can also function as a structural protein, regulating intercellular adhesion through binding the intracellular terminal of E-cadherins with the actin cytoskeleton. Treatment PBS Mock Control adenovirus DTCF4 adenovirus b-catenin adenovirus Cirrhosis development 0/8 1/8 2/8 7/10 Liver cirrhosis began to develop after 5 weeks of treatment. Adenovirus was administered at week 8, when the liver was at an early stage of cirrhosis. After 2 weeks administration, the expression of b-catenin was signiﬁcantly increased, as indicated by comparison of the luminescence of b-catenin-immunostained cirrhotic liver sections without HE staining (data not shown). The extent of liver cirrhosis was staged by pathologists according to the standards of The Chinese Society of Infectious Diseases and Parasitology and The Chinese Society of Hepatology of The Chinese Medical Association. 824 HONG ET AL. Fig. 4. Overexpression of b-catenin can increase the portal vein pressure but has no effect on portal vein blood ﬂow. A: Administration of b-catenin adenovirus signiﬁcantly increased the portal vein pressure (P < 0.05), whereas administration of DTCF4 adenovirus had no effect. B: Administration of both b-catenin and DTCF4 adenoviruses had no effect on portal vein blood ﬂow. In this study, we initially investigated the function of Wnt/b-catenin signaling during liver cirrhosis development. We determined the expression and distribution of b-catenin in human and rat cirrhotic liver and found an association between b-catenin and the progression of liver cirrhosis. However, although b-catenin adenovirus was capable of accelerating cirrhosis progress, blocking the Wnt/b-catenin signaling pathway by administration of an adenovirus carrying DTCF4 had no apparent effect on cirrhosis progression. This suggested that b-catenin signaling pathway was not involved in liver pathogenesis. However, higher levels of b-catenin were found in the intercellular regions of liver cells in b-catenin adenovirus-infected animals, and it is well known that b-catenin is associated with E-cadherin at the hepatocyte membrane. This connection forms a link between the cytoplasmic domain of the cadherins and the actin portion of the cytoskeleton, with signiﬁcant implications for cell– cell adhesion. Overloading of b-catenin at the hepatocyte membrane would enhance intercellular adhesion, thus having a mechanical effect on the hepatocytes (Thompson and Monga, 2007). We also found that b-catenin was highly concentrated in the hepatic sinusoid wall, where SMA was also mainly distributed. HSCs, as one type of sinusoidal lining cells, are central to the process of cirrhosis as the major source of ﬁbrillar and nonﬁbrillar matrix proteins. Recent DNA microarray analysis of quiescent and activated rat HSCs by Jiang et al. has shown that although genes involved in the noncanonical Wnt pathway were upregulated, b-catenin activation was absent (Jiang et al., 2006). Zeng et al. also found no difference in the overall expression of Wnt/b-catenin between active and resting stellate and Kupffer cells (Zeng et al., 2007). Therefore, the high levels of b-catenin found in the hepatic sinusoid wall might have other functions unrelated to its signaling characteristics. Several kinds of cells lining the hepatic sinusoid wall including Kupffer cells and HSCs. Kupffer cells can release thromboxane A2 and thus increase portal pressure when activated by bacterial infections (Steib et al., 2007). Activated HSCs express a-SMA (Friedman, 1993; Bataller and Brenner, 2001). The expression of a-SMA markedly strengthens the contractility of HSCs and so changes the microcirculation of the hepatic sinusoid (Racine-Samson et al., 1997; Rockey, 2001). We therefore suggest that b-catenin increased the contractile activity of stellate cells through its role as a structural protein linking the extracellular matrix and the intracellular cytoskeleton, rather than through its role as signaling molecule. This hypothesis was supported by the observation that b-catenin and a-SMA formed more complexes in cirrhotic liver than in normal liver tissue. The enhanced linkage of b-catenin and actin was also conﬁrmed by the fact that b-catenin and actin were colocalized at the intercellular membrane in aggregated LO2 cells. The portal vein pressure in b-catenin-overexpressing rats was signiﬁcantly increased only in the group administered b-catenin adenovirus, but not in that receiving DTCF4 adenovirus, whereas portal vein blood ﬂow was not affected by either b-catenin adenovirus or DTCF4 adenovirus. In this study, we found that b-catenin inﬂuenced liver cirrhosis development through its role as a structural molecule, but not as a signaling molecule. We also demonstrated for the ﬁrst time that accumulation of b-catenin at the hepatic intercellular membrane and hepatic sinusoid wall contributed to hepatic hyperpiesia in liver cirrhosis patients. Our results suggest a potential new b-CATENIN AND LIVER PORTAL HYPERTENSION Fig. 5. b-catenin and a-SMA were colocalized on the hepatic sinusoid wall in cirrhotic liver. A: b-catenin was found on the hepatic sinusoid wall (arrow) in the cirrhotic tissue sample. B: a-SMA was also located on the hepatic sinusoid wall in the cirrhotic sample. C: Immu- 825 noprecipitation and Western blotting showed that b-catenin and aSMA could form complexes in cirrhotic liver. b-catenin (D) and actin (E) were also colocalized in the cell–cell contact area in aggregated LO2 cells. Scale: 10 lm. 826 HONG ET AL. therapeutic target for the treatment of hyperpiesia and digestive tube hemorrhage in patients with liver cirrhosis. This has strong clinical implications because of the currently limited therapeutic opportunities available at the exacerbation stage of liver cirrhosis. LITERATURE CITED Adams CL, Nelson WJ. 1998. Cytomechanics of cadherin-mediated cell–cell adhesion. Curr Opin Cell Biol 10:572–577. Albanis E, Friedman SL. 2001. Hepatic ﬁbrosis pathogenesis and principles of therapy. Semin Liver Dis 5:315–334. Bataller R, Brenner DA. 2001. Hepatic stellate cells as a target for the treatment of liver ﬁbrosis. Semin Liver Dis 21:437–451. Bataller R, Brenner DA. 2005. Liver ﬁbrosis. J Clin Invest 115: 209–218. Behrens J. 1999. Cadherins and catenins: role in signal transduction and tumor progression. Cancer Metastasis Rev 18:15–30. Ben-Ze’ev A, Geiger B. 1998. Differential molecular interactions of b-catenin and plakoglobin in adhesion, signaling and cancer. Curr Opin Cell Biol 10:629–639. Bullions LC, Levine AJ. 1998. The role of b-catenin in cell adhesion, signal transduction, and cancer. Curr Opin Oncol 10:81–87. ChineseSociety of Infectious Diseases and Parasitology and Chinese Society of Hepatolgy of Chinese Medical Association. 2000. The program of prevention and cure for viral hepatitis. Zhonghua Ganzangbing Zazhi 8:324–329. Eng FJ, Friedman SL. 2000. Fibrogenesis I. New insights into hepatic stellate cell activation: the simple becomes complex. Am J Physiol Gastrointest Liver Physiol 279:G7–G11. Friedman S. 2003. Liver ﬁbrosis—from bench to bedside. J Hepatol 38:38–53. Friedman SL. 1993. The cellular basis of hepatic ﬁbrosis—mechanisms and treatment strategies. N Engl J Med 328:1828–1835. Friedman SL. 2000. Molecular regulation of hepatic ﬁbrosis, an integrated cellular response to tissue injury. J Hepatol 275:2247– 2250. Friedman SL. 2004. Mechanisms of disease: mechanisms of hepatic ﬁbrosis and therapeutic implications. Nat Clin Pract Gastroenterol Hepatol 1:98–105. Gines P, Cardenas A, Arroyo V, Rodes J. 2004. Management of cirrhosis and ascites. N Engl J Med 350:1646–1654. Gupta TK, Toruner M, Groszmann RJ. 1998a. Role of nitric oxide. Digestion 59:413–415. Gupta TK, Toruner M, Chung MK, Groszmann RJ. 1998b. Endothelial dysfunction and decreased production of nitric oxide in the intrahepatic microcirculation of cirrhotic rats. Hepatology 28: 926–931. He T-C, Zhou S, da Costa LT, Yu J, Kinzler KW, Vogelstein B. 1998. A simpliﬁed system for generating recombinant adenoviruses. Proc Natl Acad Sci USA 95:2509–2514. Jiang F, Parsons CJ, Stefanovic B. 2006. Gene expression proﬁle of quiescent and activated rat hepatic stellate cells implicates Wnt signaling pathway in activation. J Hepatol 45:401–409. Kondo Y, Kanai Y, Sakamoto M, Genda T, Mizokami M, Ueda R, Hirohashi S. 1999. b-Catenin accumulation and mutation of exon 3 of the b-catenin gene in hepatocellular carcinoma. Jpn J Cancer Res 90:1301–1309. Korinek V, Barker N, Morin PJ, van Wichen D, de Weger R, Kinzler KW, Vogelstein B, Clevers H. 1997. Constitutive transcriptional activation by a b-catenin-Tcf complex in APC/ colon carcinoma. Science 275:1784–1787. Morin PJ. 1999. b-Catenin signaling and cancer. Bioessays 21:1021– 1030. Nelson WJ, Nusse R. 2004. Convergence of Wnt, b-catenin, and cadherin pathways. Science 303:1483–1487. Palacios J, Gamallo C. 1998. Mutations in the b-catenin gene (CTNNB1) in endometrioid ovarian carcinomas. Cancer Res 58:1344–1347. Polakis P. 1999. The oncogenic activation of b-catenin. Curr Opin Genet Dev 9:15–21. Racine-Samson L, Rockey DC, Bissell DM. 1997. The role of a1b1 integrin in wound contraction: a quantitative analysis of liver myoﬁbroblasts in vivo and in primary culture. J Biol Chem 272:30911–30917. Rockey DC. 2001. Hepatic blood ﬂow regulation by stellate cells in normal and injured liver. Semin Liver Dis 21:337–350. Rockey DC, Chung JJ. 1995. Inducible nitric oxide synthase in rat hepatic lipocytes and the effect of nitric oxide on lipocyte contractility. J Clin Invest 95:1199–1206. Shackel NA, McGuinness PH, Abbott CA, Gorrell MD, McCaughan GW. 2002. Insights into the pathobiology of Hepatitis C Virusassociated cirrhosis: analysis of intrahepatic differential gene expression. Am J Pathol 160:641–654. Steib CJ, Gerbes AL, Bystron M, op den Winkel M, Hartl J, Roggel F, Prufer T, Goke B, Bilzer M. 2007. Kupffer cell activation in normal and ﬁbrotic livers increases portal pressure via thromboxane A2. J Hepatol 47:228–238. Thompson MD, Monga SP. 2007. Wnt/b-catenin signaling in liver health and disease. Hepatology 45:1298–1305. Vona G, Estepa L, Béroud C, Damotte D, Capron F, Nalpas B, Mineur A, Franco D, Lacour B, Pol S, Bréchot C, PaterliniBréchot P. 2004. Impact of cytomorphological detection of circulating tumor cells in patients with liver cancer. Hepatology 39:792–797. Wells RG. 2006. Mechanisms of liver ﬁbrosis: new insights into an old problem. Drug Discov Today 3:489–495. Zhao DH, Hong JJ, Guo SY, Yang RL, Yuan J, Wen CJ, Zhou KY, Li CJ. 2004. Aberrant expression and function of TCF4 in the proliferation of hepatocellular carcinoma cell line BEL-7402. Cell Res 14:74–80. Zeng G, Awan F, Otruba W, Muller P, Apte U, Tan X, Gandhi C, Demetris AJ, Monga SP. 2007. Wnt’er in liver: expression of Wnt and frizzled genes in mouse. Hepatology 45:195–204.