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Overexpression of ╬▓-Catenin is Responsible for the Development of Portal Hypertension During Liver Cirrhosis.

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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: licj@njnu.edu.cn
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.
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