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Transforming Growth Factor-

1 Induces an
Epithelial-to-Mesenchymal Transition State
in Mouse Hepatocytes in Vitro
*
Received for publication, February 2, 2007, and in revised form, May 16, 2007 Published, JBC Papers in Press, May 19, 2007, DOI 10.1074/jbc.M700998200
Aki Kaimori
‡
, James Potter
‡
, Jun-ya Kaimori
§
, Connie Wang
§1
, Esteban Mezey
‡
, and Ayman Koteish
‡2
From the
‡
Division of Gastroenterology and Hepatology and
§
Division of Nephrology, Department of Medicine,
The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
Liver fibrosis is a progressive pathologic process that involves
deposition of excess extracellular matrix leading to distorted
architecture and culminating in cirrhosis. The role of trans-
forming growth factor-

(TGF-

) as a key molecule in the devel-
opment and progression of hepatic fibrosis via the activation of
hepatic stellate cells, among other fibroblast populations, is
without controversy. We hereby show that TGF-

1 induces an
epithelial-to-mesenchymal transition (EMT) state in mature
hepatocytes in vitro. EMT state was marked by significant up-
regulation of
␣
1
(I) collagen mRNA expression and type I colla-
gen deposition. Similar changes were found in a “normal”
mouse hepatocyte cell line (AML12), thus confirming that
hepatocytes are capable of EMT changes and type I collagen
synthesis. We also show that in hepatocytes in the EMT state,
TGF-

1 induces the snail-1 transcription factor and activates
the Smad2/3 pathway. Evidence for a central role of the TGF-

1/Smad pathway is further supported by the inhibition of EMT
by Smad4 silencing using small interference RNA technology. In
conclusion, TGF-

1, a known pro-apoptotic cytokine in mature
hepatocytes, is capable of mediating phenotypic changes and
plasticity in the form of EMT, resulting in collagen deposition.
Our findings support a potentially crucial role for EMT in the
development and progression of hepatic fibrogenesis.
Liver fibrosis results from increased deposition of type I col-
lagen within the hepatic extracellular space and constitutes a
common cardinal signature to all forms of liver injury, regard-
less of etiology (1). End-stage liver fibrosis is recognized clini-
cally as cirrhosis. Since their initial description, hepatic stellate
cells (HSC)
3
have dominated the field of liver fibrogenesis
(2–4). Indeed, their role is central in hepatic fibrosis (5). Unfor-
tunately, despite several discoveries pertaining to HSC activa-
tion and mechanisms of collagen deposition, no substantial
anti-fibrotic therapies have been developed in order to halt the
progression to cirrhosis and or reverse established fibrosis.
Although resident tissue fibroblasts are traditionally consid-
ered as the principal source of fibrosis, there has been increas-
ing interest in the ability of epithelial cells to assume not only a
mesenchymal phenotype (known as epithelial-to-mesenchy-
mal transition (EMT)) but also to undertake mesenchymal
function(s), i.e. contribute to fibrosis formation. Indeed, EMT
has been established as a major mechanism for the deposition
of extracellular matrix in renal and pulmonary fibrosis injury
models (6 – 8).
Several lines of evidence support an important role for
TGF-

1 signaling in the initiation and progression of liver
fibrosis (9). In mature (i.e. adult) hepatocytes, TGF-

1is
responsible for inhibition of cell proliferation and induction of
apoptosis (10 –12). Interestingly, TGF-

1 is the most estab-
lished mediator and regulator of EMT (13). It has been shown
that TGF-

1 mediates EMT by inducing snail-1 transcription
factor and tyrosine phosphorylation of Smad2/3 with subse-
quent recruitment of Smad4 (14 –16).
EXPERIMENTAL PROCEDURES
Hepatocyte Isolation and Cell Culture—12-Week-old
C57BL-6 mice were purchased from The Jackson Laboratory
(Bar Harbor, ME). Mice were anesthetized with ketamine (50
mg/kg intraperitoneally) and xylazine (10 mg/kg intraperitone-
ally). Livers were perfused in situ through the portal vein, ini-
tially with calcium- and magnesium-free Hanks’ balanced salt
solution (Invitrogen) containing 0.5 m
M EDTA (Invitrogen) fol-
lowed by Dulbecco’s modified Eagle’s media (DMEM; Invitro-
gen) containing collagenase (Sigma) until the liver lost its firm
texture. The soft liver was removed and gently shaken in
DMEM containing collagenase at 37 °C for 10–15 min. The
homogenate was filtered and centrifuged for 2 min. The pellet
was washed three times with DMEM and then resuspended in
DMEM supplemented with 10% fetal bovine serum (Invitro-
gen), 100 units/ml penicillin G, 100
g/ml streptomycin, 250
ng/ml amphotericin B (Invitrogen), and 5
g/ml insulin
* This work was supported by National Institutes of Health Grant
P50DK57325 and United States Public Health Service Grant AA000626. The
costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked “advertise-
ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1
Present address: Division of Nephrology and Hypertension, University of
Kansas Medical Center, 3901 Rainbow Blvd., Sudler 4015, Mail Stop 3002,
Kansas City, KS 66160.
2
To whom correspondence should be addressed: The Johns Hopkins Univer-
sity School of Medicine, Division of Gastroenterology and Hepatology, 720
Rutland Ave., Ross Research Bldg. 933C, Baltimore, MD 21205. Tel.: 410-
614-0144; Fax: 410-955-9677; E-mail: akoteish@jhmi.edu.
3
The abbreviations used are: HSC, hepatic stellate cells; TGF-

, transforming
growth factor-

; EMT, epithelial-to-mesenchymal transition; siRNA, small
interference RNA; ANOVA, analysis of variance; GAPDH, glyceraldehyde-3-
phosphate dehydrogenase; DMEM, Dulbecco’s modified Eagle’s media;
PBS, phosphate-buffered saline; qRT, quantitative reverse transcription;
␣
-SMA,
␣
-smooth muscle actin; IP, immunoprecipitation; IF, immunofluo-
rescence; DAPI, 4⬘,6-diamidino-2-phenylindole; ERK, extracellular signal-
regulated kinase; PI3K, phosphatidylinositol 3-kinase; IB, immunoblotting.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 30, pp. 22089 –22101, July 27, 2007
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
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(Sigma) (Culture Medium). Cell number and viability were
assessed by counting an aliquot in the presence of 0.1% trypan
blue. Cells were plated on 60-mm culture plates previously
coated with collagen (Cohesion, Palo Alto, CA). Incubation was
carried out at 37 °C under 5% CO
2
. After 18 h, the culture
medium was replaced with serum-free medium (same compo-
sition as Culture Medium except that fetal bovine serum was
replaced with 9.6 ng/ml dexamethasone (Fujisawa, Deerfield,
IL), 10
g/liter epidermal growth factor, 0.5 mg/liter trans-
ferrin, 5
g/liter selenium, 0.5 mg/liter linoleic acid, and 0.5
mg/liter fetuin) (serum-free Culture Medium). Recombinant
human transforming growth factor-

1 (TGF-

1) (R & D Sys-
tems, Minneapolis, MN) was added once, i.e. at time of switch-
ing to serum-free medium, at a final concentration of 2 ng/ml.
The cells without TGF-

1 (control) and those with TGF-

1
were harvested at 0.5, 1, 6, 24, 48, and 72 h.
Mouse Hepatocyte Cell Line, AML12 Culture and Treatment—
A nontumorigenic mouse hepatocyte cell line, i.e. AML12
(CRL-2254), was purchased from ATCC (Manassas, VA). The
cell line was maintained in DMEM/F-12 medium (Invitrogen)
supplemented with 10% fetal bovine serum (Invitrogen), 5
g/ml insulin, 5
g/ml transferrin, 5 ng/ml selenium (BIO-
SOURCE), 40 ng/ml dexamethasone (Fujisawa), and 100 ng/ml
amphotericin B (Invitrogen) (Complete Growth Medium).
AML12 cells were seeded at ⬃60% confluence in Complete
Growth Medium. The cells were changed to serum-free
medium, 18 h later, after washing twice with calcium- and mag-
nesium-free phosphate-buffered saline (PBS). Similar to pri-
mary hepatocytes, TGF-

1 (R & D Systems) was added at a final
concentration of 2 ng/ml. The cells without TGF-

1 (control)
and those with TGF-

1 were harvested at 0.5, 1, 6, 24, 48, and
72 h after TGF-

1 treatment.
Real Time RT-PCR Analysis (qRT-PCR)— qRT-PCR was per-
formed by ABI Prism 7900HT (Applied Biosystem) using SYBR
Green PCR Master Mix (Applied Biosystems, Foster City, CA).
Specific forward and reverse primers were purchased from Qia-
gen. Data analysis was performed using ABI Prism 7900HT SDS
2.0 software (Applied Biosystem). 2
⌬Ctn
⫽ 2
CtGAPDH ⫺ Cttarget
calculation was used for relative expression value.
Preparation of Cell Lysate—AML12 cells and mouse primary
hepatocytes were lysed with cell lysis buffer (20 m
M Tris-HCl,
pH 7.5, 150 m
M NaCl, 1 mM Na
2
EDTA, 1 mM EGTA, 1% Triton,
2.5 m
M sodium pyrophosphate, 1 mM

-glycerophosphate, 1
m
M Na
3
VO
4
,1
g/ml leupeptin) (Cell Signaling, Beverly, MA)
and protease inhibitor mixture (Roche Applied Science) on ice.
Cells were scraped and transferred to 1.5-ml Eppendorf tubes
and rotated for1hat4°C,followed by centrifugation at
14,000 ⫻ g for 10 min at 4 °C. The resulting supernatants were
stored in aliquots at ⫺80 °C until required. Protein concentra-
tion in the cell lysate solution was determined using BCA pro-
tein assay kit (Pierce).
Western Blot Analysis—The cell lysate was mixed with 6⫻
SDS sample buffer (350 m
M Tris, pH 6.8, 30% glycerol, 10% SDS,
0.012% bromphenol blue) supplemented with 5%

-mercapto-
ethanol (Sigma). Samples were heated at 100 °C for 10 min
before loading and being separated on precasted 10% or 4 –15%
SDS-polyacrylamide gels (Bio-Rad). Proteins were electro-
transferred to a nitrocellulose membrane (Millipore, Bedford,
MA) in transfer buffer containing 25 m
M Tris, 192 mM glycine,
and 20% methanol at 4 °C for 1 h. Nonspecific binding to the
membrane was blocked for1hatroom temperature with 5%
nonfat milk in TTBS buffer (20 m
M Tris, 500 mM sodium NaCl,
and 0.1% Tween 20). Membranes were then incubated for 16 h
at 4 °C with various primary antibodies in blocking buffer con-
taining 5% nonfat milk at the dilution specified by the manufac-
turers. The following primary antibodies were used: mouse
anti-E cadherin (BD Biosciences), goat anti-vimentin (Abcam,
Cambridge, MA), rabbit anti-type I collagen (Calbiochem), rab-
bit anti-AKT (Cell Signaling), rabbit anti-phospho-AKT (Cell
Signaling), rabbit anti-ERK1/2 (Cell Signaling), rabbit anti-
phospho-ERK1/2 (Cell Signaling), rabbit anti-phospho-Smad2
(Cell Signaling), mouse anti-Smad4 (Santa Cruz Biotechnol-
ogy), mouse anti-
␣
-tubulin (Sigma) and mouse anti-GAPDH
(Abcam). After washing in TTBS buffer, the membranes were
incubated with horseradish peroxidase-conjugated secondary
antibody (Pierce) for1hatroom temperature in 5% nonfat milk
dissolved in TTBS. Membranes were then washed with TTBS
FIGURE 1. TGF-

1 induces a mesenchymal morphology in hepatocytes in
vitro. A, adult mouse hepatocytes were cultured in the absence (control) or
presence of 2 ng/ml TGF-

1 for 72 h in serum-free medium. Immunofluores-
cence staining for F-actin (green) and albumin (red) in primary hepatocytes
was evaluated by confocal laser microscopy. DAPI stained nuclei blue. Con-
trast the fibrillar polymerization of F-actin stress fibers after TGF-

1 treatment
to the localized membrane-bound F-actin distribution in controls. Scale bar,
20
m. B, whole protein lysates from TGF-

1-treated hepatocytes and con-
trols (i.e. non-TGF-

-treated) were probed for
␣
-SMA. None of the hepatocyte
preparations were positive for
␣
-SMA. The HSC lane represents a whole pro-
tein lysate obtained from an HSC preparation and used as a positive control
for
␣
-SMA. Each blot was reprobed with GAPDH to ensure equal loading of
each lane. C, AML12 hepatocytes under similar conditions (as described for
primary hepatocytes in A) show similar fibrillar F-actin polymerization 72 h
after TGF-

1 treatment. Scale bar, 20
m.
EMT in Hepatocytes
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buffer extensively, and the signals were visualized using the
enhanced chemiluminescence system (ECL, Amersham Bio-
sciences). Bands were quantified with Image-J software
(National Institutes of Health, Bethesda). Relative protein
abundance in each sample was normalized to that of GAPDH or
␣
-tubulin.
Immunoprecipitation (IP)—2
g of rabbit anti-Smad2/3
(Upstate Biotechnologies, Inc., Lake Placid, NY) was added to
510
g of whole protein cell lysate, and the mixture was incu-
bated with rotation for3hat4°C.After the addition of 30
lof
protein G-Sepharose beads (Amersham Biosciences), the mix-
ture was incubated with rotation overnight at 4 °C. Sample mix-
ture was centrifuged for 5 min at 3000 rpm at 4 °C, and the
beads were then washed three times with cell lysis buffer (20
m
M Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Na
2
EDTA, 1 mM
EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM

-glyc-
erophosphate, 1 m
M Na
3
VO
4
,1
g/ml leupeptin) which
included protease inhibitor mixture (Roche Applied Science).
The pellet was resuspended in 1⫻ SDS sample buffer (50 m
M
Tris, pH 6.8, 10% glycerol, 2% SDS, 0.1% bromphenol blue, 5%

-mercaptoethanol) and boiled for 5 min. Samples were loaded
on precasted 4 –15% SDS-polyacrylamide gels and analyzed by
Western blotting.
Immunofluorescence (IF) Staining—Mouse primary hepato-
cytes for immunofluorescence staining of E-cadherin and
vimentin were plated on collagen-coated coverslips in 6-well
culture dishes and treated in the same method as above
described.
Mouse primary hepatocytes for immunofluorescence
staining of procollagen/type I collagen were plated on posi-
tively charged, noncollagen-coated, coverslips seeded in
6-well culture dishes (to confirm de novo synthesis of type I
collagen). Incubation was carried out at 37 °C under 5% CO
2
.
After 18 h, culture medium was replaced with serum-free
medium. TGF-

1 was added at the onset of the experiment
(i.e. time of change to serum-free medium) at a final concen-
tration of 2 ng/ml.
FIGURE 2. TGF-

1 down-regulates E-cadherin and up-regulates vimentin
in hepatocytes. A, hepatocytes isolated from 12-week-old C57BL6 mice and
cultured for 24, 48, and 72 h in serum-free medium in the absence (control)or
presence of 2 ng/ml TGF-

1. E-cadherin (panel i) and vimentin (panel ii) mRNA
expression were examined by qRT-PCR. Error bars represent means ⫾ S.E. of
triplicate wells from three independent experiments. **, p ⬍ 0.05 versus con-
trol at same time point (Student’s t test). B, panel i, whole protein cell lysates
were immunoblotted with specific antibody against E-cadherin. Each blot
was reprobed with GAPDH to ensure equal loading of each lane. Results are
representative data of at least three separate experiments. Panel ii, relative
amounts of E-cadherin protein were normalized to GAPDH and expressed
relative to time 0 (i.e. cells harvested at time of change to serum-free
medium). Time 0 was arbitrarily set to 1. The error bars represent mean ⫾ S.E.
of at least three independent experiments. *, p ⬍ 0.05 versus time 0 cells
(ANOVA); **, p ⬍ 0.05 versus control cells at same time point (Student’s t test).
R.E.V., relative expression value.
FIGURE 3. Panels i–iii, TGF-

1 induces EMT-type changes in primary hepato-
cytes. Immunofluorescence staining for E-cadherin (green) and vimentin (red)
in primary hepatocytes were evaluated by confocal laser microscopy. DAPI
stained nuclei blue. There was apparent near loss of cell membrane-bound
E-cadherin in TGF-

1-treated cells in a time-dependent manner. Diffuse
vimentin staining was observed in control cells, and TGF-

1-treated hepato-
cytes exhibited a fibrillar vimentin pattern most noted at 48 and 72 h. Also
note that TGF-

1 caused hepatocytes to assume a spindle-shaped morphol-
ogy, which was not observed in controls. Scale bar, 50
m.
EMT in Hepatocytes
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AML12 cells for immunofluorescence staining were plated
on noncollagen-coated (as recommended by ATCC) positively
charged coverslips seeded in 6-well culture dishes and treated
using the same methods as described above. At 0.5, 1, 24, 48,
and 72 h after changing to serum-free medium, cells without
TGF-

1 (control) and those with
TGF-

1 were washed with PBS
three times and fixed with cold
methanol:acetone (1:1) for 10 min
on ice. Cells were fixed with 3.8%
paraformaldehyde in PBS for 10 min
at room temperature and permeabi-
lized in 0.5% Triton X-100/PBS for 5
min at room temperature. After
washing with PBS for 5 min (three
times), cells were blocked with Pro-
Block (Dako Cytomation, Carpinte-
nia, CA) for 30 min at room temper-
ature, then with 5% serum (from
same species as secondary antibody)
in PBS for 60 min, and then incu-
bated with the specific primary anti-
bodies. The following primary anti-
bodies were used for
immunostaining: goat anti-mouse
albumin (Immunology Consultants
Laboratory), rat anti-E cadherin
(clone ECCD-2) (Zymed Laborato-
ries Inc.), goat anti-vimentin
(Abcam), and rabbit anti-mouse
anti-procollagen/type I collagen
(Chemicon, Temecula, CA). Alexa
fluor-488, Alexa fluor-594 (Molecu-
lar probes, Eugene, OR), or Texas
Red (Abcam)-conjugated secondary
antibodies were used. For F-actin
staining, we used Alexa fluor-488
phalloidin (Molecular Probes). Cells
were co-stained with 4⬘,6-dia-
midino-2-phenylindole (DAPI), HCl
(Molecular Probes), to visualize
the nuclei. Stained cells were
mounted with fluorescent mount-
ing medium (Dako Cytomation)
and viewed by confocal laser
microscopy (PerkinElmer Life Sci-
ences). All exposure gains and rates
are consistent among samples.
Small Interference RNA (siRNA)
Experiment—Culture medium was
changed 4 h after the isolation of
primary hepatocytes, and the cells
were cultured in the presence of
negative control siRNA (Qiagen) or
Smad4 siRNA (Qiagen) for 48 h.
Transfection of siRNA was per-
formed according to the manufac-
turer’s instructions (Qiagen). 10
M
was determined to be the most effective siRNA concentration
for Smad4 silencing. Hence, 10
M siRNA with HiPerFect
transfection reagent (Qiagen) in serum-free culture medium
was incubated for 10 min at room temperature (transfection
mixtures) and added directly to the cultured cells. The AML12
FIGURE 4. TGF-

1 suppresses E-cadherin and up-regulates vimentin in a normal mouse hepatocyte cell
line (AML12). AML12 cells were cultured in the absence (control) or presence of 2 ng/ml TGF-

1 for 72 h in
serum-free medium. A, levels of E-cadherin (panel i) and vimentin (panel ii) mRNA in AML12 cells were quanti-
fied by qRT-PCR. Error bars represent mean ⫾ S.E. of triplicate wells from at least three separate experiments. **,
p ⬍ 0.05 versus control at same time point (Student’s t test). B, panel i, whole cell protein lysates were immu-
noblotted with specific anti-E-cadherin antibodies. The same blot was reprobed with
␣
-tubulin to ensure equal
loading of each lane. Results are representative of at least three independently performed experiments. Panel
ii, Western blots were quantified and normalized to
␣
-tubulin loading control. E-cadherin/
␣
-tubulin showed
the relative amounts of E-cadherin. The immunoreactivity at time 0 was arbitrarily set to 1. The error bars
represent means ⫾ S.E. from at least three independent experiments. *, p ⬍ 0.05 versus time 0 (ANOVA); **, p ⬍
0.05 versus control cells at same time point (Student’s t test). C, immunofluorescence for E-cadherin (green) and
vimentin (red) in AML12 cells in the absence (control) or presence of 2 ng/ml TGF-

1 for 72 h in serum-free
medium were examined by confocal laser microscopy. DAPI stained nuclei blue. The expression of cell mem-
brane-bound E-cadherin was reduced in TGF-

1-treated cells. Vimentin filament assumed a fibrillar rearrange-
ment in TGF-

1-treated cells, as opposed to a homogeneous cytoplasmic distribution in controls. Note the
spindle-shaped morphology assumed by hepatocytes 72 h after TGF-

1. Scale bar, 50
m. R.E.V., relative
expression value.
EMT in Hepatocytes
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hepatocyte cells were plated with transfection mixtures and
cultured for 48 h.
The primary hepatocytes and the AML12 cells with the
transfection mixture were changed to serum-free medium after
washing twice with PBS. TGF-

1 (R & D Systems) was added at
a final concentration of 2 ng/ml. The cells without TGF-

1
(control) and those with TGF-

1 were harvested 1 h and 72 h
later.
Statistical Analysis—Values are expressed as means ⫾ S.E.
All values were derived from measurements of at least three
independently performed experiments. Statistical analyses for
comparisons over time course were performed with repeated
measure analysis of variance (ANOVA) and post hoc Bonfer-
roni/Dunn’s correction. Student’s t test was used for the com-
parison of control and TGF-

1 treatment groups at the same
time point. A p value ⬍0.05 was considered to indicate statisti-
cal significance. ANOVA and Student’s t test were performed
using StatView software package (Abacus Concepts Inc., Berke-
ley, CA).
RESULTS
TGF-

1 Induces Cytoskeletal Rearrangement in Hepatocytes
in Vitro—Primary adult (12-week-old) mouse hepatocytes were
cultured in serum-free medium with or without TGF-

1(2
ng/ml). After 72 h in culture, hepatocytes were co-immuno-
stained for F-actin and albumin and examined by confocal
microscopy. In control hepatocytes, F-actin was detected in the
cell-cell junction with a peri-cell membrane distribution, and
TGF-

1-treated cells acquired a spindle-shaped morphology
with polarization of the F-actin stress fibers throughout the cell
(Fig. 1A). This phenotypic appearance suggested that mature
hepatocytes can acquire a mesenchymal phenotype. Co-ex-
pression of albumin, in both controls and TGF-

1-treated cells,
indicates that the cells are hepatocytes (Fig. 1A). Moreover, we
confirmed the lack of expression of
␣
-smooth muscle actin
(
␣
-SMA), a marker of activated hepatic stellate cells (HSC), in
both control and TGF-

1-treated hepatocytes using Western
blot analysis (Fig. 1B). Furthermore, to confirm that our results
are not because of HSC or fibroblast contamination of our pri-
mary culture, the experiments were reproduced using a “nor-
mal” mouse hepatocyte cell line (AML12); TGF-

1-treated
cells, but not controls, exhibited a similar pattern of F-actin
rearrangement to primary hepatocytes after TGF-

1 treatment
(Fig. 1C).
TGF-

1 Induces an EMT State in Hepatocytes—E-cadherin is
a universal epithelial marker that plays a key role in the main-
tenance of cellular integrity, and down-regulation of its expres-
sion marks early EMT (16, 17). We evaluated the pattern of
E-cadherin expression in response to TGF-

1 treatment;
E-cadherin mRNA levels were decreased in the TGF-

1-
treated hepatocytes as compared with controls, p ⬍ 0.05 (Fig.
2A, panel i). Similarly, Western blot analysis showed that
E-cadherin protein levels were reduced in the TGF-

1-treated
hepatocytes, p ⬍ 0.05 (Fig. 2B).
To demonstrate whether the TGF-

1-induced phenotypic
changes and down-regulation of epithelial markers were
accompanied by acquisition/up-regulation of mesenchymal
makers, we evaluated the expression of vimentin, a filament
used to identify mesenchymal cells, as well as cells in EMT (10).
Both groups of hepatocytes (i.e. controls and TGF-

1-treated)
expressed vimentin mRNA, which increased over time. How-
ever, the expression of vimentin mRNA was significantly
increased in the TGF-

1-treated hepatocytes as compared with
controls, p ⬍ 0.05 (Fig. 2A, panel ii). Co-immunofluorescence
staining for E-cadherin and vimentin, evaluated by confocal
microscopy, revealed near loss of cell membrane-bound E-cad-
herin by 72 h after TGF-

1. Down-regulation of the mem-
brane-bound E-cadherin occurs in parallel with fibrillar rear-
rangement of vimentin filament in the cytoplasm hepatocytes
48 and 72 h after TGF-

1 exposure. These changes were absent
in the controls (Fig. 3).
TGF

1 Induces an EMT State in the AML12 Hepatocytes—
To further test our hypothesis, we examined whether the EMT
changes observed in primary hepatocytes could be confirmed in
the AML12 mouse hepatocyte cell line. E-cadherin mRNA lev-
els were repressed by TGF-

1 treatment, p ⬍ 0.05 (Fig. 4A,
panel i). Western blot analysis showed that E-cadherin protein
levels were also reduced in the AML12 cells after TGF-

1 treat-
FIGURE 5. TGF-

1 induces de novo type I collagen synthesis in primary
hepatocytes in vitro. A, panel i, qRT-PCR for
␣
1
(I) collagen mRNA in primary
hepatocytes reveals increased expression in response to TGF-

1. Panel ii, qRT-
PCR, showing that TGF-

1 does not up-regulate FSP-1 mRNA in primary hepa-
tocytes, provides further proof that collagen synthesis was not because of
fibroblast contamination. Error bars represent mean ⫾ S.E. of triplicate wells
from three separately performed experiments. **, p ⬍ 0.05 versus control at
same time point (Student’s t test). B, panel i, whole protein cell lysates were
immunoblotted with specific anti-type I collagen antibody. The same blot
was reprobed with GAPDH to ensure equal loading of each lane. Panel ii,
relative amounts of type I collagen proteins were normalized to GAPDH and
expressed relative to time 0. Time 0 was arbitrarily set to 1. The error bars
represent mean ⫾ S.E. of at least three independent experiments. **, p ⬍ 0.05
versus control cells at same time point (Student’s t test). R.E.V., relative expres-
sion value.
EMT in Hepatocytes
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ment, p ⬍ 0.05 (Fig. 4B). Similar to primary hepatocytes, vimen-
tin mRNA levels were up-regulated in TGF-

1-treated AML12
cells at 48 and 72 h, p ⬍ 0.05 (Fig. 4A, panel ii).
Phenotypic examination by immunohistochemistry using
confocal microscopy was also performed on AML12 cells in
order to further delineate the distribution pattern of these epi-
thelial and mesenchymal markers (Fig. 4C). The findings are
consistent with those observed in primary hepatocytes, and
provide further proof that our results are due to EMT and not to
potential fibroblast contamination and overgrowth.
TGF-

1 Induces Type I Collagen Expression in Primary
Hepatocytes—To evaluate whether the acquisition of a mesen-
chymal phenotype by hepatocytes (as suggested by changes in
morphology and cytoskeleton rearrangement) is accompanied
by mesenchymal defining function(s), we determined whether
primary hepatocytes could contribute to the production of
extracellular matrix (18). For this purpose, we examined the
expression of
␣
1
(I) collagen mRNA by hepatocytes. TGF-

1
induced
␣
1
(I) collagen mRNA 72 h after treatment (Fig. 5
A,
panel i). Next, we evaluated the expression of fibroblast-
specific protein (FSP1), a member of the S100 family of cal-
cium-binding proteins (S1004A) that is specific to fibro-
blasts (16). Lack of FSP1 up-regulation after TGF-

1 further
supports that increased
␣
1
(I) collagen mRNA expression by
TGF-

1 is not because of fibroblast contamination/over-
growth (Fig. 5A, panel ii).
Subsequently, we examined the type I collagen protein
expression in whole cell lysates, and as shown in Fig. 5B, type I
collagen was produced by hepatocytes. Levels of type I collagen
were higher in the TGF-

1-treated hepatocytes than in their
FIGURE 6. Panels i–iii, immunofluorescence staining for type I collagen in pri-
mary hepatocytes. Co-immunostaining for albumin (red) and procollagen/
type I collagen (green) in adult primary mouse hepatocytes examined by con-
focal microscopy. Cells were cultured in the absence (control) or presence of
2 ng/ml TGF-

1 for 24, 48, and 72 h in serum-free medium. DAPI stained
nuclei blue. TGF-

1-treated hepatocytes increased de novo type I collagen
synthesis 48 and 72 h after treatment. Note the cytoplasmic staining of pro-
collagen. Albumin co-staining confirms the hepatocyte identity of the colla-
gen-producing cells. Scale bar, 20
m.
FIGURE 7. TGF-

1 induces expression of type I collagen in AML12 hepa-
tocyte cell line. A,
␣
1
(I) collagen mRNA was examined by qRT-PCR. Error bars
represent mean ⫾ S.E. of triplicate wells from three independent experi-
ments. **, p ⬍ 0.05 versus control at same time point (Student’s t test). B, panel
i, whole protein cell lysates were immunoblotted with anti-type I collagen
antibody. Blots were reprobed with
␣
-tubulin to ensure equal loading of each
lane. Panel ii, collagen (I)/
␣
-tubulin was quantitated in each lane. The immu-
noreactivity at time 0 cells is set to 1. The error bars represent means ⫾ S.E. of
three independent experiments. *, p ⬍ 0.05 versus baseline collagen (I) in time
0 cells (ANOVA); **, p ⬍ 0.05 versus collagen (I) in same time point control cells
(Student’s t test). R.E.V., relative expression value.
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control counterparts. Furthermore, the primary hepatocytes
were immunostained with anti-procollagen/type I collagen
antibody. As shown in Fig. 6, collagen fibrils were clearly
detected in the extracellular space 48 and 72 h after TGF-

1
treatment. Moreover, the intracellular detection of procollagen
in albumin-positive cells supports collagen production by
hepatocytes.
TGF-

1 Treatment Stimulates Type I Collagen Production by
AML12 Hepatocytes—To further confirm that the source of
type I collagen was the primary hepatocytes, rather than fibro-
blasts or other unaccounted cell types that may have potentially
contaminated our primary hepatocyte preparation, the AML12
cell line was evaluated for type I collagen production.
␣
1
(I) col
-
lagen mRNA and type I collagen protein were up-regulated
after TGF-

1 treatment (Fig. 7). Immunohistochemistry stain-
ing confirmed that AML12 hepatocytes synthesize procolla-
gen/type I collagen in response to TGF-

1 treatment (Fig. 8).
TGF-

1 Induces snail-1 and Smad4 Activation in
Hepatocytes—snail-1, a repressor of E-cadherin transcription,
is induced by TGF-

1 (19). The snail gene functions as a key
regulator of EMT in vitro and in vivo (14, 20). Therefore, we
examined whether the expression of snail in TGF-

1-treated
and control hepatocytes is consistent with EMT. In fact, snail
mRNA was up-regulated at 0.5 and 1 h after TGF-

1 exposure
in AML12 and primary hepatocytes, respectively (Fig. 9A, pan-
els i and ii). This is consistent with other reports and with our
observation of E-cadherin down-regulation (21).
Next, we evaluated the TGF-

1 pathways that are associated
with EMT change. Both Smad-dependent and Smad-independ-
ent (i.e. Akt- and ERK-dependent) TGF-

pathways have been
implicated in EMT in various epithelial cell types (8, 18, 22, 23).
First, we evaluated the Smad-independent pathway by exam-
ining Akt and ERK1/2 activation. The level and differential
expression of phospho-Akt (P-Akt) and phospho-ERK1/2
(P-ERK1/2), the active forms of Akt and ERK1/2, were similar,
i.e. not significantly different between control and TGF-

1-
treated hepatocytes (Fig. 9B, panel i). Similarly, P-AKT and
FIGURE 8. Immunofluorescence for type I collagen in AML12 hepatocytes
after TGF-

1 treatment. Co-immunostaining for albumin (red) and procol-
lagen/type I collagen (green) in AML12 cells in the absence (control) or pres-
ence of 2 ng/ml TGF-

1 at 24, 48, and 72 h (shown in panels i, ii, and iii, respec-
tively) in serum-free medium, examined by confocal laser microscopy. Cell
nuclei are demonstrated by DAPI (blue). Assembly of type I collagen is dem-
onstrated in TGF-

1-treated AML12 cells at 72 h. Note the intracellular pro-
collagen staining in TGF-

1-treated cells. Albumin staining is consistent with
the hepatocyte identity of the cells. Scale bar, 50
m.
FIGURE 9. TGF-

1 induces snail-1 in hepatocytes in EMT state and lack of
evidence for Akt/Erk1/2 activation. A, qRT-PCR reveals increased snail-1
mRNA levels in primary (panel i) and AML12 hepatocytes (panel ii) after
TGF-

1 treatment. B, whole cell lysates were obtained and analyzed by West-
ern blotting for total and phosphorylated forms of Akt and ERK1/2 proteins
using appropriate specific antibodies in primary hepatocytes (panel I) and
AML12 cells (panel ii). GAPDH and
␣
-tubulin were used as loading controls.
There was no notable TGF-

1-mediated up-regulation of Akt and/or Erk1/2 in
either primary or AML12 cells, thus suggesting that the non-Smad pathway
was not the likely pathway for TGF-

1 induced EMT changes. R.E.V., relative
expression value.
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P-ERK1/2 had similar patterns and levels of expression in both
control and TGF-

1-treated AML12 cells (Fig. 9B, panel ii).
These observations suggested that Smad-independent TGF-

signaling is not significantly involved in mediating EMT.
Next, we examined the Smad-dependent signaling pathway,
i.e. Smad2/3 and Smad4 activation. IP assays confirmed binding
of Smad2/3 to Smad4, hence their activation (Fig. 10A, panel i).
P-Smad2 levels increased after TGF-

1 treatment (Fig. 10A,
panel ii), and immunohistochemistry staining confirmed the
translocation (i.e. activation) of the Smad2/3 complex to the
nucleus of hepatocytes 0.5 h after TGF-

1 treatment (Fig. 10B).
AML12 cells were similarly examined for evidence of
Smad2/3 activation after TGF-

treatment. IP (Fig. 11A) and
immunohistochemistry (Fig. 11B) studies were consistent with
Smad2/3 and Smad4 binding and activation.
Taken together, the Smad-dependent signaling pathway was
clearly activated in TGF-

1-treated primary and AML12 hepa-
tocytes and not in their control counterparts. This suggests that
TGF-

1-induced EMT changes in hepatocytes were predomi-
nantly mediated via Smad signaling and not via the non-Smad,
Akt, and/or ERK1/2 pathway.
Smad4-siRNA Reverses EMT in Hepatocytes in Vitro—To
demonstrate the central role of the TGF-

1/Smad signal on
EMT induction, siRNA technology was used for smad4 gene
silencing in both primary and AML12 hepatocytes (24, 25). The
efficiency of siRNA transfection and resultant smad4 gene
silencing in primary and AML12 hepatocytes was evaluated by
IP, qRT-PCR, and IF. An additional arm using control-siRNA
(i.e. nonsilencing, negative control-siRNA) was added to all
experiments to rule out nonspecific off-target effects that may
be unrelated to smad4 gene silencing.
In primary hepatocytes, the activation of the Smad signal was
abrogated after transfection with Smad4-siRNA; the TGF-

1-
induced binding of Smad4 to Smad2/3, a sine-qua-non of the
TGF-

1/Smad activation, was repressed, as confirmed by IP
(Fig. 12A). Gene silencing of Smad4 was confirmed by qRT-
PCR. The Smad4 mRNA levels were significantly decreased up
to 72 h after Smad4-siRNA transfection in control hepatocytes;
consistent with Smad4 gene silencing. TGF-

1 treatment failed
to induce Smad4, as evidenced by comparable Smad4 mRNA
levels to the untreated (control) Smad4-siRNA-transfected
hepatocytes. This effect was not observed in the control siRNA
group (Fig. 12B).
FIGURE 10. TGF-

1 activates Smad2/3-dependent pathway in primary
hepatocytes. A, whole protein cell lysates from TGF-

1-treated and control
primary hepatocytes at 0.5-, 1-, and 6-h time points were immunoprecipi-
tated (IP) for Smad2/3 and protein G-Sepharose. Panel i, immunoblotting (IB)
for Smad4 reveals increased binding to Smad2/3 in TGF-

1-treated hepato-
cytes. Panel ii, IB with P-Smad2 antibody reveals increased levels (hence acti-
vation) of P-Smad2 after TGF-

1 treatment. Panel iii, IB with anti-Smad2/3
antibody is shown as a control; increased Smad4 binding to Smad2/3 and
increased P-Smad2 levels were not because of increased Smad2/3 levels in
TGF-

1-treated hepatocytes. B, co-immunostaining for albumin (red) and
Smad2/3 (green) in hepatocytes were examined by confocal microscopy. Cell
nuclei are demonstrated by DAPI ( blue). At 0.5 h, Smad2/3 translocates into
the nuclei of TGF-

1-treated primary hepatocytes: a sine qua non of its acti-
vation. No appreciable translocation of Smad2/3 was noted in control cells.
Co-staining for albumin confirms the hepatocyte identity of the cells. Scale
bar, 50
m.
FIGURE 11. TGF-

1 induces activation of Smad2/3 pathway in AML12 cell
line. A, whole protein cell lysates from control and TGF-

1-treated AML12
hepatocytes at 0.5-, 1-, and 6-h time points were immunoprecipitated (IP)
with anti-Smad2/3 and protein G-Sepharose. Panel i, IB with Smad4 reveals
increased binding of Smad2/3 to Smad4. Panel ii, similarly, IB for P-Smad2
reveals increased P-Smad2 levels in AML12 hepatocytes after TGF-

1 treat-
ment. Panel iii, IB for Smad2/3 was performed to control for equal loading.
B, co-immunostaining of AML12 cells for albumin (red) and Smad2/3 (green)
was examined by confocal microscopy. Cell nuclei were stained with DAPI
(blue). Smad2/3 translocates to the nucleus 0.5 h after TGF-

1 treatment but
not in the controls. Scale bar, 50
m.
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Whether Smad4-siRNA could reverse the TGF-

1-induced
EMT was then examined. In fact, the suppression of E-cadherin
mRNA expression by TGF-

1 treatment was partly, but signif-
icantly, preserved after Smad4-siRNA treatment (Fig. 12B).
snail-1, a repressor of E-cadherin transcription, was shown to
be induced 1 h after TGF-

1 treat-
ment (Fig. 9A). As shown in Fig.
12B, panel ii, snail-1 mRNA was sig-
nificantly repressed by Smad4-
siRNA in TGF-

1-treated hepato-
cytes. Vimentin mRNA expression
was decreased slightly, but not sig-
nificantly, by Smad4-siRNA in
TGF-

1-treated primary hepato-
cytes (Fig. 12B). On the other hand,
the reduction of albumin mRNA
expression by TGF-

1 was pre-
vented after Smad4-siRNA trans-
fection. Most importantly, the up-
regulation of
␣
1
(I) collagen mRNA
by TGF-

1 was significantly sup-
pressed by Smad4-siRNA (Fig. 12B).
The effect of Smad4-siRNA on
the expression of E-cadherin and
vimentin was then confirmed by
IF. Consistent with qRT-PCR
results, E-cadherin was partly
recovered after Smad4 RNA
knockdown in TGF-

1-treated
primary hepatocytes. Also, vimen-
tin staining was equally intense
after siRNA transfection, hence
consistent with the nonsignificant
decrease in vimentin mRNA after
Smad4-siRNA treatment. Nota-
bly, however, vimentin assumed a
less fibrillar pattern and exhibited
a more diffuse, granular cytoplas-
mic distribution (Fig. 13). Next, to
confirm the reduction of de novo
type I collagen production by
Smad4-siRNA in primary hepato-
cytes, slides were immunostained
with anti-procollagen/type I colla-
gen antibody. As shown in Fig. 14,
TGF-

1-treated hepatocytes that
were transfected with Smad4-
siRNA exhibited a significant
decrease in type I collagen. This
effect was not seen in the control-
siRNA-treated hepatocytes. Fur-
thermore, cellular morphology of
TGF-

1-treated hepatocytes re-
verted from a spindle shape to a
more cuboidal/hexagonal shape,
after smad4 silencing. This effect
was not observed in control-siRNA-
transfected and TGF-

1-treated
hepatocytes that preserved a spindle-shaped morphology.
Consistent with the results obtained with primary hepato-
cytes, Smad4-siRNA repressed the binding of Smad4 to
Smad2/3 in TGF-

1-treated AML12 cells, as confirmed by IP
assay (Fig. 15A). This lack of binding was because of Smad4
FIGURE 12. Smad4 silencing suppresses EMT in TGF-

1-treated primary hepatocytes. A, whole protein cell
lysates (extracted at 1 h) from serum-starved, TGF-

1-treated, and control (i.e. non-TGF-

1-treated) primary
hepatocytes that were transfected with control-siRNA or Smad4-siRNA were immunoprecipitated (IP) for
Smad2/3 and protein G-Sepharose. Panel i, consistent with Smad4 silencing, IB for Smad4 reveals decreased
Smad4 binding to Smad2/3 in Smad4-siRNA-transfected hepatocytes. Panel ii, IB with anti-Smad2/3 antibody is
shown as a loading control. B, primary hepatocytes transfected with control-siRNA or Smad4-siRNA were then
cultured with TGF-

1(black bars) or in the absence thereof (i.e. control in white bars): qRT-PCR results are shown
for 72 h after TGF-

1 treatment (panel i) and 1 h after treatment (panel ii). Panel i, E-cadherin, vimentin, albumin,
and
␣
1
(I) collagen; and panel ii, snail-1 mRNA expression levels were evaluated. Results are consistent with
inhibition of EMT after Smad4 silencing; Smad4-siRNA prevented E-cadherin loss and abrogated
␣
1
(I) collagen
and snail-1 expression. The decrease in vimentin expression with Smad4-siRNA was not statistically significant.
qRT-PCR for Smad4 was performed to confirm that Smad4 mRNA expression was indeed repressed by Smad4-
siRNA transfection. Error bars represent mean ⫾ S.E. of triplicate wells from three representative experiments.
**, p ⬍ 0.05; Smad4-siRNA versus control-siRNA (Student’s t test). R.E.V., relative expression value.
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silencing, as reflected by a significant decrease in Smad4 mRNA
expression in the non-TGF-

1-treated (control) AML12
hepatocytes. This effect was not seen after control-siRNA
transfection. Similarly, TGF-

1-treated AML12 cells that
were transfected with Smad4-siRNA failed to induce the
Smad4 gene (Fig. 15B).
The TGF-

1-induced suppression of E-cadherin mRNA was
inhibited after Smad4-siRNA treatment (Fig. 15B). Consis-
tently, snail-1 mRNA expression was repressed by Smad4-
siRNA in TGF-

1-treated AML12 cells (Fig. 15B). Increased
expression of vimentin mRNA after TGF-

1 treatment was
slightly, but significantly, reduced after siRNA-induced smad4
silencing (Fig. 15B). Also, the reduction in albumin mRNA by
TGF-

1 was partly, but significantly, prevented by Smad4-
siRNA. Moreover, the induction of
␣
1
(I) collagen mRNA by
TGF-

1 was clearly suppressed by Smad4 silencing (Fig. 15B).
Immunofluorescence confirmed the above qRT-PCR results;
membrane-bound E-cadherin was preserved by Smad4 silenc-
ing in TGF-

1-treated AML12 cells (Fig. 16). Similar to primary
hepatocytes, the pattern of vimentin staining reverted from
fibrillar to granular in TGF-

1-treated, Smad4-siRNA-trans-
fected AML12 cells (Fig. 16). Also, as shown in Fig. 17, Smad4
silencing in AML12 hepatocytes suppressed the TGF

1-in-
duced type I collagen production. Overall, our results support a
central role of the TGF

1/Smad pathway in EMT induction.
DISCUSSION
In this study, we demonstrate that hepatocytes synthesize
type I collagen in response to low dose TGF-

1 (2 ng/ml), one
of the most ubiquitous and powerful mediators of fibrosis
across organ systems. Our results revive the seminal work by
Chojkier (26) that hepatocytes are able to produce collagen.
Those early findings were eclipsed by multiple criticisms that
undermined the ability of hepatocytes to contribute to hepatic
fibrosis (27). Currently, it is postulated that only HSC, portal
myofibroblasts, and mesenchymal cells of bone marrow origin
have a fibrotic potential in the liver (28, 29). Although other
studies have also shown that hepatocytes may synthesize colla-
gen, the notion persists that hepatocytes have an insignificant
role in the perpetuation of liver fibrosis, and hence progression
to cirrhosis. We suspect that hepatocytes play a potentially
important role in the genesis of liver fibrosis and progression to
cirrhosis.
Yet the contribution of epithelial cells to “fibrogenesis” is not
a novel concept. In fact, EMT refers to that very ability, which in
its “strict” definition implies that epithelial cells are at the origin
of local formation of interstitial fibroblasts. EMT implies that
FIGURE 13. Membrane-bound E-cadherin is preserved by Smad4 RNA
knockdown. Primary hepatocytes were transfected with control-siRNA or
Smad4-siRNA. Cells were cultured in the absence (control) or presence of 2
ng/ml TGF-

1 for 72 h. IF staining for E-cadherin (green) and vimentin (red)in
hepatocytes transfected with either Smad4 or control-siRNA was evaluated
by confocal laser microscopy. DAPI stained nuclei blue. Smad4-siRNA trans-
fection preserved membrane-bound E-cadherin in TGF-

1-treated hepato-
cytes, a finding consistent with EMT inhibition. Note the change in vimentin
pattern; Smad4-siRNA-treated hepatocytes demonstrated a granular, nonpo-
lymerized, distribution of vimentin, whereas control-siRNA-treated hepato-
cytes exhibited a polarized fibrillar vimentin pattern after TGF-

1 treatment.
Scale bar, 20
m.
FIGURE 14. Smad4-siRNA suppresses type I collagen synthesis in primary
hepatocytes. Primary hepatocytes, under similar conditions (as described in
Fig. 13), were immunostained for albumin (red) and procollagen/type I colla-
gen (green) and evaluated by confocal laser microscopy. DAPI stained nuclei
blue. Smad4-siRNA transfection reduced type I collagen synthesis and depo-
sition in TGF-

1 treated hepatocytes. Scale bar, 20
m.
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epithelial cells lose their “characteristic” epithelial phenotype
and markers in favor of acquiring mesenchymal markers. This
latter phenomenon has been studied and proven to occur in
many epithelial cells as follows: kidney tubular epithelia, mam-
mary glands, alveoli, and prostate (8, 17, 30, 31). Controversy in
EMT nomenclature remains, however, and in part stems from
the fact that mesenchymal markers lack specificity. To avoid
such confusion, we prefer the term “EMT state” and better
describe the phenotypic changes that hepatocytes undergo in
our in vitro system, namely that TGF-

1-treated hepatocytes
down-regulate epithelial markers in
favor of up-regulating mesenchy-
mal markers and inducing type I
collagen synthesis, a known fibro-
blast function. Hence, we do not
imply that hepatocytes transform
into fibroblasts but that they assume
a fibroblast-like phenotype and
function (collagen synthesis), while
down-regulating but preserving to a
certain extent the epithelial marker
E-cadherin. In the liver, EMT was
first noted to involve the hemato-
poietic lineage, i.e. stromal cells, in
the fetal liver and was later recog-
nized in fetal and neonatal hepato-
cytes when treated with TGF-

1or
upon serum starvation (20, 32).
Although it has been concluded that
EMT is not a major phenomenon in
mature hepatocytes because of ter-
minal differentiation, no studies
have been conducted to elucidate
the potential role of EMT in hepatic
fibrogenesis. Since its demonstra-
tion in other organs, namely lung,
kidney, prostate, and mammary
glands, little attention has been
given to EMT in mature hepato-
cytes and its potential implication in
liver fibrogenesis (8, 18, 23, 30, 31,
33). Lately, however, a few studies
have alluded to EMT potential in
liver cellular subpopulation (34, 35).
Several concerns arise when try-
ing to study EMT in a primary hep-
atocyte culture, the most critical
being contamination with resident
fibroblasts and/or stellate cells/
myofibroblasts. Moreover, TGF-

1,
a known inducer of apoptosis in
hepatocytes, may in fact favor the
overgrowth of such contaminating
cells. To address these major con-
cerns, we resorted to multiple strat-
egies as follows: (a) co-labeling with
hepatocellular/epithelial specific and
mesenchymal-type markers. Thus,
immunofluorescence has shown co-localization of E-cadherin
and vimentin, as well as collagen and albumin. Type I collagen
was detected intracellularly and extracellularly in albumin-pos-
itive cells, hence providing further proof that hepatocytes are
the source of synthesized collagen. This is expected because
collagen synthesis starts in the form of procollagen, which then
matures in the form of interlacing fibrils once secreted to extra-
cellular space. The next strategy is as follows: (b) assay for fibro-
blast-specific marker and a stellate cell-specific marker. To rule
out contamination with HSC and interstitial fibroblasts, we
FIGURE 15. Smad4 silencing inhibits TGF-

1-induced EMT in AML12 cells. A, AML12 cells were transfected
with control-siRNA or Smad4-siRNA. Whole protein cell lysates from TGF-

1-treated cells for 1 or 6 h and their
corresponding controls (i.e. non-TGF-

1-treated) were immunoprecipitated (IP) for Smad2/3 and protein
G-Sepharose. Panel i, IB for Smad4 reveals decreased Smad4 binding to Smad2/3 in Smad4-siRNA-transfected
hepatocytes. Panel ii, IB with anti-Smad2/3 antibody is shown as loading control. B, AML12 cells were trans-
fected with control-siRNA or Smad4-siRNA. mRNA was extracted at 72 h (panel i)and1h(panel ii) from TGF-

1-treated (black bars) and non-TGF-

1-treated cells (control, white bars). qRT-PCR results are shown as follows.
Panel i, E-cadherin, vimentin, albumin,
␣
1
(I)collagen; panel ii, snail-1. Smad4-siRNA prevented E-cadherin loss,
decreased vimentin expression, and abrogated
␣
1
(I) collagen and snail-1 expression and hence consistent with
EMT inhibition with Smad4 silencing. qRT-PCR for Smad4 was performed to confirm suppression of Smad4
mRNA expression by Smad4 siRNA. Error bars represent mean ⫾ S.E. of triplicate wells from three representa-
tive experiments. *, p ⬍ 0.05; Smad4-siRNA versus control-siRNA (student’s t test). R.E.V., relative expression
value.
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assayed for
␣
-SMA and FSP-1, two specific markers with high
specificity to both types of cells, respectively.
␣
-SMA was not
detected, therefore ruling out HSC contamination. Further-
more, qRT-PCR for FSP-1 did not show up-regulation after
TGF-

1 treatment, thus indicating that our results were
because of EMT in hepatocytes and not to contamination with,
or overgrowth of, fibroblasts. (c) We then resorted to a “nor-
mal” nontransformed hepatocyte cell line, i.e. AML12. EMT
changes were also confirmed in the AML12 mouse hepatocyte
cell line, thus confirming that the observed changes in primary
hepatocytes were indeed because of EMT changes rather than
to potential HSC, fibroblast, or other mesenchymal cell con-
tamination. Yet another concern arises when trying to address
EMT of hepatocytes in vitro, which is that hepatocytes dedif-
ferentiate in culture. Hence, hepatocytes take a more elongated
and flattened morphology. Although our cells may have dedif-
ferentiated to a certain degree (as expected when grown in cul-
ture), this is controlled for in the nontreated group. Changes
reported after TGF-

1 treatment are beyond what we may
expect from dedifferentiation alone, which by itself does not
account for collagen production.
Our results further support the general “EMT hypothesis”
and have two important potential implications. First, EMT may
be a continuum rather than an all or none phenomenon,
namely that further testing in vitro with a different experimen-
tal design is warranted to study the spectrum. Second, hepato-
cytes are to be viewed as perpetuators of hepatic fibrogenesis,
rather than as victims.
As another mean of evaluating our results, we examined known
mediators of the EMT mechanism reported in other cellular types
(kidney tubular epithelial and fetal hepatocytes). To date, it is
thought that the sole effect of TGF-

1 on “mature” hepatocytes is
to induce apoptosis (10, 12, 36). It is evident, however, that some
hepatocytes escape this fate by undergoing EMT (32). Mechanis-
tically, snail transcription factor is central to EMT (14, 20, 21).
snail-1 is activated by TGF-

and is implicated in the direct sup-
pression of E-cadherin transcription. Indeed, snail is known to
trigger EMT during embryonic development and tumor progres-
sion; hence, snail mutant mice die at gastrulation because of a
defective EMT (21). Moreover, snail is upstream of mesenchymal
markers such as vimentin (37). TGF-

1 exerts its effects by bind-
ing to the TGF-

type II receptor (T

RII) and subsequently
recruiting the TGF-

type I receptor (T

RI) (38). Smad2/3 and
Smad4 are known intracellular mediators of TGF-

1. Once phos-
phorylated by the activated TGF-

1 receptor, Smad2 and/or
Smad3 complex with Smad4 and translocate to the nucleus where
they regulate TGF-

1 target genes (15, 38, 39). It has been shown
that Smad2/3 activation is induced during EMT. Numerous
reports indicate a central role for Smads as mediators of TGF-

-
induced EMT and demonstrate their important role in fibrosis (18,
40–43). Smad-independent pathways have also been implicated
in the induction of EMT in certain cell types (23, 44). In Madin-
FIGURE 16. E-cadherin protein is preserved in AML12 cells after Smad4
knockdown. Immunofluorescence staining for E-cadherin (green) and
vimentin (red) in AML12 cells transfected with control-siRNA or Smad4-siRNA.
Cells were cultured in the absence (control) or presence of TGF-

1 for 72 h
and then were examined by confocal microscopy. DAPI stained nuclei blue.
Smad4-siRNA, not control-siRNA, transfection preserved membrane-bound
E-cadherin in TGF-

1-treated AML12 cells. Vimentin staining intensity was
decreased in the Smad4-siRNA-treated cells, not in controls. Notably, how-
ever, Smad4-siRNA-treated cells demonstrated a granular, nonpolymerized,
distribution of vimentin, whereas control-siRNA-treated cells preserved a
polarized fibrillar vimentin pattern after TGF-

1 treatment. Scale bar, 20
m.
FIGURE 17. Smad4 siRNA transfect suppressed type I collagen synthesis
in mouse hepatocyte cell line, AML12. Immunofluorescence staining for
albumin (red) and procollagen/type I collagen (green) in AML12 cells (same
conditions as described in Fig. 16) was examined by confocal laser micros-
copy. DAPI stained nuclei blue. Smad4-siRNA transfection inhibited type I
collagen deposition by TGF-

1-treated AML12 cells. Scale bar, 20
m.
EMT in Hepatocytes
22100 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282• NUMBER 30• JULY 27, 2007
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Darby canine kidney cells, TGF-

1 promotes EMT via mitogen-
activated protein kinase/extracellular signal-regulated kinase
kinase (MEK) and PI3K pathways that up-regulate snail expres-
sion (19, 45, 46). In NMuMG mammary cells, PI3K pathway was
found to be essential for TGF-

1-mediated EMT (47). Moreover,
Valdes et al. (48) reported activation of the PI3K pathway in fetal
rat hepatocytes that escape TGF-

1-induced apoptosis.
We have also shown that TGF-

1 mediates EMT in hepato-
cytes by the induction of snail-1 and activation of Smad4. This
is further supported by the abrogation of EMT after Smad4
RNA interference-mediated silencing (49). In fact, transfection
of primary and AML12 hepatocytes with Smad4-siRNA inhib-
its TGF-

1-induced EMT as follows: preserving the epithelial
phenotype (E-cadherin) and function (albumin), and most
importantly, inhibiting TGF-

1-induced type I collagen
expression. Even though vimentin was not significantly down-
regulated in primary hepatocytes, there was a notable reversal
of its intracellular polymerization. Smad4-siRNA transfection
caused depolymerization of vimentin fibrils to a more diffuse
granular pattern, consistent with EMT inhibition. The finding
that Smad4 silencing counteracts TGF-

1-induced EMT in
hepatocytes supports that Smad4 is central to EMT in hepato-
cytes and hence hepatic fibrogenesis.
Although specific therapies to inhibit the progression of liver
fibrosis are still not available, we propose that our findings may
open new avenues to the understanding of the development
and progression of hepatic fibrogenesis. This may lead to new
therapeutic options for hepatic fibrosis.
Acknowledgments—We thank Dr. Gregory Germino and Dr. Terry
Watnick of the Division of Nephrology, The Johns Hopkins University,
for their assistance and feedback.
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EMT in Hepatocytes
JULY 27, 2007 •VOLUME 282 •NUMBER 30 JOURNAL OF BIOLOGICAL CHEMISTRY 22101
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Koteish
Connie Wang, Esteban Mezey and Ayman
Aki Kaimori, James Potter, Jun-ya Kaimori,
in VitroState in Mouse Hepatocytes
an Epithelial-to-Mesenchymal Transition
1 InducesβTransforming Growth Factor-
Mechanisms of Signal Transduction:
doi: 10.1074/jbc.M700998200 originally published online May 19, 2007
2007, 282:22089-22101.J. Biol. Chem.
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