From the Departments of Internal Medicine
and ** Pathology, Yale University School of Medicine, New
Haven, Connecticut 06520
Received for publication, July 31, 2002, and in revised form, January 21, 2003
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ABSTRACT |
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Hepatic stellate cells are the primary cell
type responsible for matrix deposition in liver fibrosis, undergoing a
process of transdifferentiation into fibrogenic myofibroblasts. These cells, which undergo a similar transdifferentiation process when cultured in vitro, are a major target of the profibrogenic
agent transforming growth factor- The transforming growth factors Hepatic stellate cells (HSC) are the primary target of fibrogenic
stimuli in the diseased liver. In the setting of chronic liver disease,
HSC transdifferentiate, adopting a myofibroblast-like phenotype
characterized in part by proliferation and the deposition of abnormal
matrix. This phenotypic change, termed "activation," has been
modeled in vitro by culturing HSCs on uncoated plastic. Freshly isolated HSC appear undifferentiated and are traditionally termed "quiescent," whereas cells grown on uncoated plastic for 5 to 7 days become activated. In response to TGF- TGF- Smad2 and Smad3 are clearly functionally distinct, although the details
of their differing functions are not well understood. They have
different expression patterns, produce different phenotypes in null
mice, and demonstrate different effects on at least one promoter
(16-20). Smad3 binds DNA directly, whereas Smad2 does not (21-24).
Expression of a dominant negative Smad3 but not Smad2 prevents
TGF- In this report we have examined TGF- Materials--
Media (minimum essential medium and medium 199 (M199)) were obtained from Invitrogen (Grand Island, NY) and
Cellgro (Herndon, VA), FBS was from Gemini Biosciences (Woodland, CA),
and antibiotics were from Invitrogen. TGF- HSC Isolation--
Primary rat hepatic stellate cells were
isolated from livers of Sprague-Dawley rats weighing 500-700 g by a
modification of a method described previously (27). Rats were
anesthetized with sodium pentobarbital. In situ liver
perfusion and digestion was performed with Pronase E (2.4 mg/ml, Roche
Molecular Biochemicals, Chicago, IL) and collagenase B (0.3-0.45
mg/ml, Roche Molecular Biochemicals), and the resulting liver cell
suspension was purified by density gradient centrifugation using 8.2%
Nycodenz. HSC were plated on uncoated plastic at a density of 5 × 106 per 10-cm diameter plate and maintained in M199/10%
FBS supplemented with glutamine and antibiotics (penicillin, 100 units/ml; streptomycin, 100 units/ml; gentamicin, 0.1 mg/ml; and
Fungizone (Invitrogen), 2.5 µg/ml). Cell viability was greater
than 90% as assessed by trypan blue exclusion. Purity was 90-95% as
assessed by desmin immunostaining and the typical light microscopic
appearance of the lipid droplets. Cells were considered to be quiescent
at day 1, 24 h after plating. Cells at day 7 were considered
activated, with greater than 95% staining positive for Proliferation Assays--
After 1, 4, or 7 days in culture,
primary HSC were washed twice with serum-free M199, then incubated in
M199/0.3% FBS with or without TGF- RNA Isolation and Northern Blotting--
A rat Smad2 cDNA
probe was generated by reverse transcription (RT)-PCR with
primers derived from GenBankTM accession number AB010147
(sense, CGATGCTCAAGCATGTCCTA; antisense, CGCTCTGGGTTTTGACTAGC). Results
from Northern blots with this probe were confirmed with a second probe
generated from primers encompassing a different region of the coding
sequence (sense, TACCACTCTCTCCCCTGTCAAT; antisense,
TCTCTCCAACCCTCTGGTTTAG). A cDNA encoding rat Smad3 (GenBankTM U66479) was a generous gift of Yun Chen (Indiana
University) (28); a KpnI restriction fragment representing
the entire coding region was purified with the QIAquick gel extraction
kit (Qiagen, Valencia, CA). The probe for
HSC at days 1, 4, and 7 were treated with 100 pM TGF- Western Blotting--
For immunoblotting of PAI-1, HSC at days
1, 4, or 7 after isolation were treated with 100 pM
TGF-
For immunoblotting of Smad proteins, HSC at days 1, 4, or 7 after
isolation were rinsed, serum-starved for 15 min, then treated with or
without 100 pM TGF-
The activated (phosphorylated) form of Smad2 was detected using a
polyclonal rabbit anti-phosphopeptide antibody as previously described
(30). The phosphorylated form of Smad3 was detected with a polyclonal
rabbit antibody raised against a synthetic peptide comprising the
C-terminal 13 amino acids of Smad3, in which two phosphoserine residues
were incorporated at the extreme C terminus (KMGSPSIRCSSPVSP), coupled to keyhole limpet
hemocyanin as a carrier protein (31-33). The antiserum was
affinity-purified by negative selection using a keyhole limpet
hemocyanin-agarose column, followed by chromatography using an
Affi-Gel-10 (Bio-Rad, Hercules, CA) matrix to which unphosphorylated Smad2 and Smad3 peptides had been coupled (34). The final purification step consisted of a positive selection using the phosphorylated Smad3
peptide coupled to Affi-Gel-10 matrix (34). The antibody was eluted
using 3 M sodium thiocyanate, immediately neutralized using
100 mM Tris, and dialyzed against PBS for 48 h. The
specificity and sensitivity of the anti-phospho-Smad3 antibody were
confirmed by enzyme-linked immunosorbent assay as previously described
(30). Blots were washed and incubated with horseradish
peroxidase-coupled secondary antibody, and signals were detected by ECL.
Matrix Deposition Assays--
Collagen secretion was measured by
differential collagenase sensitivity. Cells were labeled with 5 µCi/ml [2,3,4,5-3H]proline for 24 h in the
presence of 100 pM TGF-
To measure fibronectin production, HSC were treated with 100 pM TGF-
Glycosaminoglycan secretion was measured by treating HSC with 100 pM TGF- Neutralizing Antibody and Inhibitor Treatments--
For
treatments with neutralizing antibodies and inhibitors, cells were
rinsed twice with M199, then incubated with pan-TGF- Culturing rat HSC on uncoated plastic is a well-established
in vitro model of HSC activation. HSC at day 1 after
isolation demonstrate a quiescent phenotype, with a round shape,
abundant lipid droplets, and a lack of TGF- TGF- Smad 4 Activation--
To study the activation of Smad4 in HSC,
cytoplasmic and nuclear lysates from cells at different stages of
activation were immunoblotted with an antibody against the entire Smad4
coding sequence. There was no significant change in overall levels of Smad4 in cells 1, 4, or 7 days after isolation (Fig.
3A). Treatment with TGF-
A recent study reported the existence of widely expressed Smad4
isoforms that constitutively shuttle between the nucleus and cytoplasm
and are able to form complexes with Smad2 in the nucleus (41). To
determine whether this accounts for the Smad4 we observed constitutively in the nucleus, we treated day 7 cells with leptomycin B, an inhibitor of CRM1-mediated nuclear export. There was no significant accumulation of Smad4 in the nucleus after leptomycin B
treatment, indicating that the shuttling observed by Pierreux et
al. (41) does not explain our observations.
Differences in Smad2 and Smad3 Activation--
Smad2
phosphorylation in response to TGF-
Consistent with the stable total protein levels of Smads 2 and 3 (Fig.
4B and data not shown), no change in the message levels of
either Smad was seen with short (0-90 min) or long (24 h) term treatment of cells with TGF- The FYVE Protein SARA Decreases with Activation--
The linker
protein SARA recruits Smad2 to the TGF- Constitutively Phosphorylated and Nuclear Smads Do Not Result from
Autocrine TGF- Autocrine Activin Signaling Does Not Cause Constitutive Smad
Activation--
It has been reported that the TGF- Our data demonstrate in an in vitro model of HSC
activation that: 1) there is a dissociation between the growth
regulatory and matrix-inducing effects of TGF- We clearly demonstrate in this physiological system that there is a
dissociation between the cell cycle and matrix effects of TGF- In activated rat HSC in culture, TGF- This is the first description of differential Smad activation in HSC
during the process of transdifferentiation. The mechanism for the
switch from Smad2 to Smad3 is not obvious, because the total
populations of Smads 2 and 3 do not change at the protein or RNA level.
In the context of the finding of Goto et al. (46) that SARA
is required for Smad2- but not Smad3-mediated TGF- Dooley et al. (49) have shown TGF- The implications of the observed differential Smad activation for
TGF- The expression of the linker protein SARA in HSC has not previously
been described. This protein, which binds phosphatidylinositol 3-phosphate through its FYVE domain, is important for localization of
the Smads and their recruitment to the TGF- Our data demonstrate constitutive Smad 2 activation in day 7 HSC (Figs.
4 and 5). This could result from endogenous TGF- The TGF- The role of the antagonist Smad7 in moderating TGF- TGF- In summary, we demonstrate that Smad signaling pathways can be
differentially activated during cellular differentiation, specifically in primary HSC in culture. We are currently examining whether differential Smad activation is responsible for the differences in
TGF- (TGF-
). We have studied
activation of the TGF-
downstream signaling molecules Smads 2, 3, and 4 in hepatic stellate cells (HSC) cultured in vitro for
1, 4, and 7 days, with quiescent, intermediate, and fully
transdifferentiated phenotypes, respectively. Total levels of Smad4,
common to multiple TGF-
superfamily signaling pathways, do not
change as HSC transdifferentiate, and the protein is found in both
nucleus and cytoplasm, independent of treatment with TGF-
or the
nuclear export inhibitor leptomycin B. TGF-
mediates activation of
Smad2 primarily in early cultured cells and that of Smad3 primarily in
transdifferentiated cells. The linker protein SARA, which is required
for Smad2 signaling, disappears with transdifferentiation.
Additionally, day 7 cells demonstrate constitutive phosphorylation and
nuclear localization of Smad 2, which is not affected by pretreatment
with TGF-
-neutralizing antibodies, a type I TGF-
receptor kinase
inhibitor, or activin-neutralizing antibodies. These results
demonstrate essential differences between TGF-
-mediated signaling
pathways in quiescent and in vitro transdifferentiated hepatic stellate cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TGF-
s)1 are
multifunctional peptide growth factors with variable cellular effects,
including growth inhibition and matrix induction. They are potent
profibrogenic agents, with roles in multiple fibrotic diseases (1). In
particular, TGF-
is a key mediator of hepatic fibrosis, as
demonstrated by an increase in TGF-
production at sites of matrix
deposition, the development of fibrosis in laboratory animals
engineered to overproduce TGF-
, and the effectiveness of
anti-TGF-
therapies in mitigating experimentally-induced
fibrosis (2-9).
, culture-activated cells produce extracellular matrix that is similar in composition to
that seen in the fibrotic liver (7, 10-13).
signals through the sequential activation of two cell surface
serine/threonine kinase receptors, the type II and type I (T
RI). The
activated T
RI phosphorylates Smad2 or Smad3, members of the Smad
family of cytoplasmic and nuclear signaling molecules, which are
specific for the signaling pathways of TGF-
and the related growth
factor activin. Phosphorylated Smads 2 and 3 form heteromeric complexes
with Smad4, a Smad common to the signaling pathways of multiple TGF-
superfamily members, and move into the nucleus where they join other
transcription factors to form transcriptionally active complexes (14).
Recent work has also demonstrated a role for the protein
Smad anchor for receptor
activation (SARA), an FYVE domain linker protein that
recruits Smad2 to the TGF-
receptor complex and is required for
maximal Smad2-mediated TGF-
signaling (15).
-mediated inhibition of adipocyte differentiation (25).
Furthermore, studies with passaged fibroblasts derived from Smad2 or
Smad3 null mouse embryos suggest that, although both Smads contribute
to TGF-
-mediated growth inhibition and plasminogen activator
inhibitor (PAI)-1 up-regulation, Smad3 is primarily responsible for the
autocrine production of TGF-
and Smad2 for matrix
metalloproteinase-2 up-regulation (26).
signaling in primary HSC as
they undergo in vitro activation. We demonstrate that there is a dissociation between the cell cycle and matrix effects of TGF-
in HSC, with growth inhibition occurring only in phenotypically quiescent cells while TGF-
-mediated matrix deposition is observed in
HSC at all stages. Smad4 in these cells is constitutively expressed in
both nucleus and cytoplasm independent of TGF-
treatment. Smads 2 and 3, however, are phosphorylated and translocate into the nucleus in
response to TGF-
, although the pattern varies with the state of
activation: Smad2 phosphorylation and nuclear translocation occurs
primarily in quiescent and intermediate cells, whereas Smad3 activation
occurs primarily in activated cells. SARA is expressed in quiescent HSC
but is lost with in vitro activation in parallel with the
loss of TGF-
-mediated Smad2 phosphorylation. In day 7 HSC, in
addition to TGF-
-responsive Smad3 activation, we also
observed constitutive phosphorylation and nuclear localization of Smad2. Treatment with a T
RI kinase inhibitor or with TGF-
or
activin-neutralizing antibodies does not diminish this constitutive activation. These results demonstrate essential differences between TGF-
-mediated Smad signaling pathways in quiescent and in
vitro-transdifferentiated HSC, and suggest that both
TGF-
-dependent and -independent Smad activation occurs
in activated HSC.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 was from R&D Systems
(Minneapolis, MN).
-smooth
muscle actin. For all experiments in which cells at different stages
were compared, the cells were from the same isolation. All experiments
were repeated with cells from different animals, as noted in the figure legends.
1 for 24 h. 2 µCi/ml
[3H]thymidine was added for the last 18 h of
incubation. Labeled cells were washed twice with PBS and lysed in 1 M NaOH; lysates were counted in a liquid scintillation counter.
1(I) collagen was
generated by restriction digestion of the cDNA and included the
entire coding region (29). The probe for rat decorin was generated by
RT-PCR using primers from GenBankTM sequence Z12298 (sense,
CTCTGGCATAATCCCTTACGAC; antisense, GGTATGCAAGTCCTTCAGGTTC). Rat
-actin cDNA was from Clontech (Palo Alto,
CA). cDNAs were labeled with [32P]dCTP with the
Megaprime labeling kit (Amersham Biosciences, Piscataway, NJ) according
to the manufacturer's instructions.
1
for 24 h in 0.3% serum, or for shorter periods (0-90 min) in
serum-free media. This concentration of TGF-
1 (~2.5 ng/ml) is
predicted to be saturating. Total RNA was extracted with the RNeasy kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. Aliquots of 15-20 µg of total RNA were electrophoretically separated on 1% denaturing agarose gels, transferred to Hybond-N+ nylon membranes (Amersham Biosciences, Piscataway, NJ), prehybridized for 30 min at 42 °C in NorthernMax hybridization buffer (Ambion, Inc.,
Austin, TX), and hybridized with [32P]dCTP-labeled probes
at 42 °C overnight. After hybridization, membranes were washed with
0.2× SSC/0.2% SDS at 42 °C for 15 min, followed by two washes at
55 °C. Blots were exposed to x-ray film at
70 °C for
24-72 h.
1 for 24 h in the presence of 0.3% serum. Cells were lysed
in hypotonic buffer (20 mM NaCl, 10 mM KCl, 1.5 mM MgCl2, 10 mM Hepes, pH 7.4, 1%
Triton X-100, 1.5 mM phenylmethylsulfonyl fluoride), and
lysates were equalized for protein content and separated by SDS-PAGE,
transferred to nitrocellulose, and immunoblotted with antibody against
PAI-1 (5 µg/ml, American Diagnostica, Inc., Greenwich, CT).
1 for 15 min. For 24-h treatments, cells were incubated with or without 100 pM TGF-
1 for
24 h in the presence of 0.3% serum. Cells were chilled, washed
with PBS, and pelleted. Pellets were resuspended in low salt buffer (20 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM
NaVO4, 1 mM EGTA, 1 mM EDTA, 0.2%
Nonidet P-40, 10% glycerol, 1 mM phenylmethylsulfonyl
fluoride, 1 µg/ml Complete (Roche Molecular Biochemicals, Chicago,
IL)), incubated on ice for 10 min, and centrifuged (8000 × g, 2 min, 4 °C). Supernatants were collected and
considered to be the cytoplasmic fraction. Pellets were resuspended in
high salt buffer (low salt buffer with 420 mM NaCl, 20%
glycerol, and no Nonidet P-40), incubated on ice for 30 min, and
centrifuged as before. Supernatants were collected as the nuclear
fraction. Protein concentrations were equalized within the two groups
(cytoplasmic and nuclear), and equal aliquots within each group were
separated by reducing SDS-PAGE, transferred to nitrocellulose, blocked
with 5% nonfat milk in TBS with 0.1% Tween 20, and immunoblotted in
the same buffer with the appropriate antibodies at the following
concentrations: HSP-70 (1 µg/ml, Stressgen Biotechnologies Corp.,
Victoria, BC), proliferating cell nuclear antigen (PCNA, 1:300, Sigma,
St. Louis, MO),
-tubulin (0.4 µg/ml, Santa Cruz Biotechnology,
Inc., Santa Cruz, CA), Smad2 (1.25 µg/ml, Transduction Laboratories,
Lexington, KY), Smad3 (2.5 µg/ml, Zymed Laboratories
Inc., San Francisco, CA), Smad4 (1.0 µg/ml, clone B-8, Santa
Cruz Biotechnology, Inc.), and SARA (1.0 µg/ml, Santa Cruz
Biotechnology, Inc.). For immunoblotting of total protein (Fig. 4,
A and D), cells were lysed with total lysis
buffer (1% Triton X-100, 0.5% deoxycholic acid, 10 mM
EDTA, in PBS) with protease inhibitors, and treated as for the lysates described above. The specificity of the antibodies against Smads 2 and
3 and SARA were confirmed by immunoblotting control lysates from COS7
cells transfected with vector alone or a cDNA encoding the specific protein.
1. Media were collected,
equalized according to the DNA content of the associated cell layer,
digested with collagenase, trichloroacetic acid-precipitated, and counted in a scintillation counter, as described previously (35).
1 in 0.3% serum for 16 h. Cells were then
incubated with minimal essential medium lacking cysteine and methionine
for 2 h, followed by the addition of
[35S]cysteine/methionine (Express, PerkinElmer Life
Sciences, Boston, MA) at 100 µCi/ml for an additional 2 h.
TGF-
1 was present throughout. The supernatant was collected and
immunoprecipitated with antibody against rat fibronectin (1 µl/ml
supernatant, Invitrogen). Immunoprecipitants were separated by
SDS-PAGE, and gels were fluorographed with 2,5-diphenyloxazole, dried, and autoradiographed.
1 in 0.3% serum for 24 h, the last 16 in
the presence of 1 µCi/ml
D-[6-3H]glucosamine HCl (Amersham
Biosciences). Media were removed and spun three times in Amicon 10 microcentrifuge tubes to remove excess label, then counted.
-neutralizing antibodies (R&D systems, 1 µg/ml, overnight), activin-neutralizing antibodies (R&D Systems, 0.06 or 0.6 µg/ml, overnight), leptomycin B
(Sigma, 20 ng/ml, 0-60 min), or T
RI kinase inhibitor NPC-34016 (a
gift of David Liu, Scios, Inc., 0.1 µM, overnight). After
incubation, cells were rinsed, lysed, and prepared for immunoblotting
as above. The antibodies and inhibitors had been previously tested and
demonstrated to be both effective and non-toxic to HSC at the
concentrations used (data not shown).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-smooth muscle actin
expression. Cells at day 7 are spread out, have few lipid droplets, and
express
-smooth muscle actin, consistent with the activated
phenotype. Cells at day 4 have an intermediate phenotype. We used this
model system to study differences in TGF-
signaling pathways at
different stages of phenotypic differentiation. Data in the literature
regarding the effects of TGF-
on quiescent and activated cells are
contradictory, and the definition of quiescence and activation varies;
therefore, as a preface to studying TGF-
signaling in HSC, we
systematically defined their response to TGF-
.
-mediated Inhibition of DNA Synthesis--
We first
characterized the growth response to TGF-
. As assayed by
[3H]thymidine incorporation, quiescent HSC (day 1)
demonstrated marked inhibition of DNA synthesis in response to TGF-
(10-500 pM), with a decrease in incorporation of up to
83% and an IC50 of 10 pM (Fig.
1), similar to what has been reported for
phenotypically quiescent cells by other groups (36). In contrast, cells
at days 4 and 7 after isolation demonstrated no change in DNA synthesis even with 500 pM TGF-
, although these cells displayed a
3.4-fold higher baseline rate of thymidine incorporation in day 7 than in day 1 cells (Fig. 1).
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Fig. 1.
TGF-
inhibits DNA synthesis in primary HSC at day 1 after isolation
but not at days 4 and 7. Primary HSC at days 1, 4, and 7 after
isolation were treated with TGF-
1 for 24 h in 0.3% serum then
assayed for incorporation of [3H]thymidine, as described
under "Experimental Procedures." Values (mean ± S.D.;
n = 4) are normalized to incorporation in the absence
of TGF-
. The data shown are from one of three experiments with
similar results (*, p < 0.05; **, p < 0.01).
-mediated Matrix Deposition--
We also characterized
TGF-
-mediated changes in the expression of various matrix
components. Collagen mRNA synthesis and secretion are modestly but
significantly increased in response to TGF-
at days 4 and 7 (Fig.
2, A and B).
Similar findings were seen for deposited collagen (data not shown).
PAI-1 protein expression was increased (1.4- to 2.3-fold) with TGF-
treatment at all three time points (Fig. 2B). Similar
findings were seen for fibronectin (Fig. 2D) and
glycosaminoglycans, including decorin (Fig. 2, A (middle panel) and E). TGF-
-mediated increases
in expression were modest (generally less than 2-fold), and, although
baseline synthesis of matrix components increased in activated cells,
the degree of up-regulation in response to TGF-
was similar in
quiescent and activated cells. Although it is often written that
TGF-
is strongly profibrogenic in HSC, most studies of rat HSC in
the literature report less than 3-fold induction of matrix components by TGF-
(13, 36-40).
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Fig. 2.
TGF- treatment of
quiescent and activated cells up-regulates production of matrix
proteins. Primary HSC at days 1, 4, and 7 after isolation were
treated with 100 pM TGF-
1 as described. A,
primary HSC at days 1, 4, and 7 were treated with 100 pM
TGF-
1 for 0, 15, 30, or 90 min (left panel) or 0 (
) or
24 h (+; right panel) then analyzed by Northern
blotting for collagen
1(I) (top panel) and decorin
(middle panel). To analyze loading, the same blot was
stripped and re-analyzed with a probe for
-actin (lower
panels). B, collagen secretion was measured by
differential collagenase sensitivity of
[3H]proline-labeled secreted proteins and was increased
in response to TGF-
at days 4 and 7 (*, p < 0.05 for both days; six samples for each condition). C, HSC
treated (+) or not (
) with TGF-
were lysed, and lysates were
immunoblotted with an antibody raised against PAI-1 (upper
panel). As a control, aliquots of the same lysate were
immunoblotted with an antibody against
-tubulin. D, HSC
were treated with TGF-
for 16 h and metabolically labeled, as
described under "Experimental Procedures." Media were collected and
immunoprecipitated with an antibody against fibronectin. For
comparison, experiments with fibronectin and PAI-1 performed in
parallel with Mv1Lu cells, widely used for TGF-
studies, demonstrate
a 3- to 4-fold enhancement of synthesis with TGF-
treatment (data
not shown). E, HSC were treated with TGF-
1 for 24 h
and labeled with [3H]glucosamine to determine secreted
glycosaminoglycans, as described. Data are shown as percent increase
for plus TGF-
compared with minus TGF-
. **, p < 0.05; *, p < 0.06 for
/+ TGF-
comparison. Six
replicates were counted for each condition. Results were similar for
matrix deposition of glycosaminoglycans (data not shown). Results for
each panel are representative of three experiments.
for 15 min resulted in minimal increases in nuclear Smad4 at days 1 and
4, although Smad4 was present in the nucleus even in the absence of
TGF-
. Immunodetection of blots with cytoplasmic (HSP-70) and nuclear
(PCNA) marker proteins confirmed the purity of the different
fractions.
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Fig. 3.
Smad4 is constitutively present in the
nucleus and cytoplasm, independent of
TGF- . A, primary HSC at days
1, 4, and 7 were treated plus or minus 100 pM TGF-
1 for
15 min and then lysed. Lysates were separated into nuclear and
cytoplasmic fractions and were immunoblotted with antibodies against
Smad4 (top panel) and, to confirm the purity of the
fractions, HSP70 (middle panel) and PCNA (lower
panel). Sizes are indicated in kilodaltons. All three blots were
prepared from the same lysate. The results are representative of five
independent experiments. We consistently see two faster bands in the
cytoplasm at day 4, with a corresponding reduction in the intensity of
the major signal. The significance of proteolysis of Smad4, especially
at day 4, is not known, although it has been observed by others (68).
B, primary HSC at day 7 after isolation were placed in
serum-free media, then treated with leptomycin B (20 ng/ml) for 0, 15, 30, or 60 min, or with TGF-
1 (100 pM;
) or TGF-
and leptomycin B (L/
) for 15 min, then lysed and
immunoblotted with antibodies against Smad4. There was no significant
nuclear accumulation of Smad4 with leptomycin B treatment. Serially
stripping the membrane and re-immunoblotting with antibodies against
Smad2 and Smad3 gives similar negative findings (data not shown). This
blot is representative of results obtained in three separate
experiments.
changed dramatically as cells
underwent in vitro activation (Fig.
4). This is shown for both total lysates
and nuclear and cytoplasmic fractions. Treatment of cells at days 1 and
4 with TGF-
for 15 min resulted in a dramatic increase in
phosphorylation of Smad2 (Fig. 4, A and B) as
well as a shift from cytoplasm to nucleus (Fig. 4B). In
contrast, Smad3 was phosphorylated and translocated to the nucleus in
response to TGF-
primarily at days 4 and 7 (Fig. 4, A and
B). Interestingly, there was a significant portion of
constitutively phosphorylated and constitutively nuclear Smad2 and, to
a lesser extent, Smad3, present at days 4 and 7.
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Fig. 4.
TGF- treatment
results in different patterns of phosphorylation and nuclear
translocation of Smads 2 and 3. A, primary HSC at days
1, 4, and 7 were treated with TGF-
for 15 min, then lysed and
immunoblotted with antibodies specific for phospho-Smad2 (upper
panel) and phospho-Smad3 (lower panel). The
images shown are from the same blot, stripped and re-probed.
B, the blot from Fig. 3A, of nuclear and
cytoplasmic isolates of HSC at days 1, 4, and 7 treated with (+) or
without (
) TGF-
1 for 15 min, was serially stripped and probed with
antibodies against phospho-Smad2 (top panel), total Smad2
(second panel), and total Smad3 (bottom panel). A
separate blot (third panel) was probed with antibodies
against phospho-Smad3. C, primary HSC at days 1, 4, and 7 were treated with 100 pM TGF-
1 for 0, 15, 30, or 90 min
(left panel) or 0 (
) or 24 h (+; right
panel) then analyzed by Northern blotting for Smad2 and Smad3
mRNA expression, as indicated. When the signal was corrected for
loading by
-actin expression, there was no change in mRNA levels
with either activation or short or long term TGF-
treatment. The
same pattern of three bands at days 4 and 7 for Smad2 was seen on
different blots with a non-overlapping probe (data not shown).
D, the blot from Fig. 3A was stripped and
re-probed with an antibody against SARA (upper panel),
demonstrating that SARA expression is lost as activated HSC in culture.
Similar results were obtained from blots using total lysates from cells
treated with plus or minus TGF-
for 15 min (lower panel).
These blots are representative of three independent experiments.
(Fig. 4C), although an
additional transcript for Smad2 was seen at days 4 and 7. This band was
specific: an identical pattern was also seen on different blots probed
with a different, non-overlapping probe (data not shown). Two or more bands specific for Smad2 by Northern blotting have been reported previously (25, 42, 43), although their significance is not known. A
splice variant of Smad2 that, unlike the wild type, is able to bind
DNA, has been reported (24) and may account for this additional
band, although one recent report demonstrated neither multiple bands
for Smad2 nor changes in the splice variant with HSC activation (45).
In contrast to other reports (45), we consistently observed a single
transcript for Smad3 in HSC.
receptor complex and is
necessary for maximal Smad2-mediated TGF-
signaling (15). The
relationship between SARA and Smad3 is less clear, and one report
suggests that SARA is required for Smad2- but not Smad3-mediated
TGF-
signaling (46). We therefore examined HSC undergoing in
vitro activation for changes in SARA expression, looking for
correlations between SARA expression and Smad activation. By Western
immunoblotting, we noted a dramatic decrease in SARA levels in HSC from
day 1 to day 4 after isolation; SARA was not detectable in HSC cultured
for 7 days (Fig. 4D).
Signaling--
We observed a significant amount of
constitutively phosphorylated Smads 2 and 3, particularly Smad2,
present in the nucleus at days 4 and 7 (Fig. 4B). This does
not represent contamination of the different fractions (see controls in
Fig. 3A); additionally, two different TGF-
-responsive
cell lines (L6 myoblasts and LLC-PK1 proximal tubule
epithelial cells) demonstrated phospho-Smad2 only in nuclear
fractions.2 One possibility
is that this represents a response to autocrine TGF-
production,
although increased production of active (as opposed to latent) TGF-
has not been observed in culture-activated cells (12, 47-49), and the
responsiveness of our cells to exogenous TGF-
in other assays argues
against saturation of the TGF-
signaling machinery. To answer this
question definitively, we treated HSC with both TGF-
-neutralizing
antibodies (Fig. 5, B and
C) and with a specific T
RI kinase inhibitor (Fig. 5,
A and B). Neither treatment altered the baseline
nuclear localization or constitutive phosphorylation of Smad2 or Smad3,
indicating that it is not the result of autocrine TGF-
production.
View larger version (40K):
[in a new window]
Fig. 5.
Constitutive activation of Smads 2 and 3 at
day 7 is not the result of autocrine TGF- or
activin signaling. A, HSC at days 1, 4, and 7 were
treated overnight with 0.1 µM NPC-34016 T
RI inhibitor,
then lysed and analyzed by immunoblotting with antibodies against
phospho-Smad2, total Smad2, or total Smad3 (the same blot was stripped
and reprobed sequentially). A separate blot was probed with antibody
against phospho-Smad3. B, HSC at day 7 were treated with
either the inhibitor (as in A) or with 1 µg/ml of a
pan-TGF-
-neutralizing antibody (T) and analyzed as in
A. C, HSC were treated overnight with antibodies
against TGF-
(as in B (T)) or against activin
(0.06 (A1) or 0.6 µg/ml (A10))
overnight, then lysed and immunoblotted as above with antibodies
against Smad2 or (using the same lysates) Smad 3. All blots shown are
representative of at least three independent experiments.
superfamily
member activin is produced in an autocrine fashion by activated HSC
(50). Because activin downstream signaling pathways share Smad2 and Smad3 with TGF-
signaling pathways, we treated day 7 HSC with neutralizing antibodies against activin A and immunoblotted the lysates
to determine whether activin is responsible for the activation state or
distribution of Smads 2 and 3 (Fig. 5C). We saw no change in
the constitutive nuclear localization of Smads 2 or 3, indicating that
it is not the result of autocrine activin signaling.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
as HSC become
activated; 2) activation is associated with a shift in TGF-
signaling pathways such that TGF-
predominantly activates Smad2 in
quiescent cells and Smad3 in activated cells; 3) Smad4 is present
constitutively in the nucleus and cytoplasm of HSC, and its
distribution does not change in response to TGF-
; 4) Smad 2 and, to
a lesser extent, Smad3, are constitutively activated in activated HSC
in vitro; and 5) this constitutive activation is not due to
autocrine TGF-
or activin signaling.
.
TGF-
treatment resulted in growth inhibition in HSC only at day 1, but caused increased expression of matrix components in cells at all
stages of in vitro transdifferentiation; although the
background expression of matrix components was increased in culture-activated HSC, the -fold induction by TGF-
did not change significantly. Our results are consistent with descriptions of the
behavior of HSC in bile duct-ligated rats and smooth muscle cells in
human atherosclerotic lesions (3, 51). Interestingly, although there
are several reports of engineered or malignant cell lines that have
lost TGF-
-mediated growth inhibition while retaining matrix
responses, there are no reports of the reverse scenario (52-55), and
it has been suggested that there is a higher threshold of Smad
activation required for growth inhibition than for matrix induction (3,
52, 56). Whether this is correct has yet to be determined.
treatment results in the net
deposition of abnormal matrix by up-regulation of multiple matrix
components, including proteoglycans, collagen, and fibronectin, and
protease inhibitors (11, 13, 37, 40, 57, 58). The effect of TGF-
on
quiescent HSC in vitro has not been extensively studied. Our
results demonstrating that TGF-
up-regulates matrix production
modestly (less than 3-fold) are consistent with the published results
of other investigators (13, 36-40), although the various studies use
HSC at different time points after isolation. These data and our
finding that TGF-
up-regulates matrix production to similar degrees
in quiescent and in vitro activated cells (Fig. 2 and data
not shown) are consistent with the hypotheses of other investigators
that autocrine TGF-
signaling by activated cells is an incomplete
explanation for the action of TGF-
in liver fibrosis (49, 59).
Although HSC at day 1 after isolation have almost certainly started to
transdifferentiate by virtue of being in culture, the data nonetheless
show that TGF-
has significant matrix effects well before full
activation. The effects on quiescent cells are potentially important in
early liver disease and may explain in part why TGF-
is necessary
and sufficient for in vivo stellate cell activation and
liver fibrosis (2, 4, 5, 60-62).
signaling, the
loss of SARA expression as HSC activate could explain the
decrease in TGF-
-mediated Smad2 phosphorylation in cells from day 1 to day 7 after isolation, although this does not explain why Smad3 is
not phosphorylated in response to TGF-
treatment in day 1 cells. We
have noted significant changes in the population of all three TGF-
receptors in HSC at days 1, 4, and 7 of in vitro
culture.3 Although the
relationship between receptor expression and Smad activation is not
well understood, varying receptor expression is a potential cause of
differential Smad activation.
-responsive
phosphorylation and nuclear localization of Smads 2 and 3 in day 2 HSC
but not passaged cells. The same group demonstrated a peak in
TGF-
-mediated activation of a Smad3/4-specific luciferase reporter
construct in day 4 HSC, although Smad activation was not examined
directly in this second report (45). Other investigators, using a
spontaneously immortalized line of activated rat HSC, report that Smad2
is activated in response to TGF-
, whereas Smad3 is constitutively
activated (63). Some of the differences between these results and ours may relate to the use of passaged or immortalized versus
primary activated HSC.
signaling specificity are not clear. Although we demonstrate a
correlation between TGF-
-mediated growth inhibition and the activation of Smad2, the requirement for individual Smads in
TGF-
-mediated responses in HSC is not known, and knockouts of the
different Smads in HSC at different time points will be required to
determine definitively the signaling pathways required for each
response. Roberts et al. (64) hypothesize that Smad3 is a
critical element in fibrogenesis in general, although it is notable
that HSC from Smad3 null mice undergo normal activation on plastic and
show only a minor decrease in collagen
1(I) mRNA (65). Our data demonstrating that Smad3 activation occurs late in transdifferentiation in vitro are consistent with the findings from Smad3 null
mice, although both results raise questions about the actual function of Smad3 in activated HSC.
receptor complex (15).
Our data, showing loss of TGF-
-mediated Smad2 activation coincident
with the loss of SARA expression, are consistent with the finding that
SARA is required for Smad2- but not Smad3-mediated TGF-
signaling
(46). In addition, the recent report that localization of SARA to early
endosomes is required for Smad-mediated signaling has interesting
implications for understanding TGF-
signaling in HSC (66): basic
mechanisms of TGF-
signaling may be different in quiescent cells,
which have SARA, and activated cells, which have lost it.
production,
although our Smad3 data clearly demonstrate that the TGF-
response
is not saturated (Fig. 4). We have shown in two different ways, with a
TGF-
signaling inhibitor and with neutralizing antibodies, that
autocrine TGF-
is not the cause (Fig. 5). This is consistent with
published data (12, 47-49) showing that, although there is a
significant amount of latent TGF-
produced by transdifferentiated HSC, there is little active ligand produced. Constitutive
phosphorylation of Smad2 in HSC from acutely injured rat livers and in
immortalized, highly passaged HSC has been reported by Tahashi et
al. (67), although this group reports inhibition of the
phosphorylation with TGF-
-neutralizing antibodies. Of note, there is
a significant amount of phospho-Smad2 in the cytoplasm of HSC, the
function of which is not known, although cytoplasmic phospho-Smad2 has been observed in other cells (41). Feldmann et al. (68) have reported sustained phosphorylation of Smads 2 and 3 in both the cytoplasm and nucleus of splenocytes and lymphocytes and have found
that this is correlated with resistance to TGF-
-mediated growth
inhibition, similar to our observations in HSC. The stability of the
cytoplasmic phospho-Smad2 we observed and its mechanism of degradation
are not known, because Smad2 is normally degraded by the proteasome
after nuclear translocation, possibly in the nucleus itself (69). It
has recently been demonstrated that Smads 2 and 3 are imported into the
nucleus by different mechanisms (70, 71) and that the import of Smad2
is phosphorylation-independent and blocked by the Smad2-SARA
interaction; TGF-
-mediated phosphorylation releases Smad2 from SARA
and unmasks the intrinsic nuclear import capabilities of Smad2. The
loss of SARA in activated HSC may therefore be an important factor in
the nuclear Smad2 we observed, although it does not explain the
constitutive phosphorylation.
superfamily member activin also signals through Smads 2 and
3, and it is secreted by in vitro activated HSC (50). We
considered this as a potential cause of the constitutive Smad activation we observed. Treatment of cells with an anti-activin antibody, however, did not alter the level of Smad activation. Similarly, there was no change in [3H]thymidine
incorporation or expression of
-smooth muscle actin or fibronectin
by day 7 HSC treated with activin-neutralizing antibodies (data not shown).
activity in
quiescent versus activated HSC is not known, although its mRNA is rapidly up-regulated in response to TGF-
treatment in early HSC but not passaged cells (45, 72). It has been suggested that
differential up-regulation of Smad7 in acute versus chronic liver injury determines whether injury resolution or fibrosis occurs
(67). We observed phosphorylation of either Smad2 or Smad3 in response
to TGF-
in quiescent and activated cells. This is consistent with
there being no difference in Smad7 activity between cells at day 1 and
day 7 after isolation or, alternatively, that Smad7 exerts a greater
inhibitory effect on Smad3 than on Smad2, or that the TGF-
response
is less sustained in activated compared with quiescent cells.
-induced Smad4 nuclear translocation in our system was minimal,
especially in activated HSC. A similar observation has been made in an
immortalized line of activated HSC (63). This finding may indicate that
TGF-
signaling in HSC is Smad4-independent, as has been shown for
some TGF-
responses in other cell systems (44, 73). We also observed
a significant fraction of Smad4 in the nucleus constitutively,
consistent with the data of Inagaki et al. (63) using an
immortalized line of HSC. It was recently reported that there are
widely expressed and functional Smad4 splice variants that shuttle
constitutively between nucleus and cytoplasm (41). We did not observe
accumulation of Smad4 in leptomycin B-treated cells, however, making
this an unlikely explanation. Smad2 and Smad4 can form active complexes
in the nucleus as well as the cytoplasm, suggesting that Smad4 could
play a role in TGF-
signaling in HSC even in the absence of
significant cytoplasmic to nuclear translocation (41).
-mediated growth inhibition and matrix induction in quiescent and activated HSC. The results from these studies will be important to
our understanding of TGF-
signaling as well as to the design of
rational anti-fibrotic therapies.
![]() |
ACKNOWLEDGEMENTS |
---|
The rat Smad3 construct was a generous gift
from Yun Chen (Indiana University), and the TRI kinase inhibitor
NPC-34016 was a generous gift from David Liu (Scios, Inc.). We are
grateful to Kathy Augustyn Harry, Tanya Arslanian, and the Yale Liver
Center for assistance with HSC isolation; to Lixia Guo for technical assistance; and to Michael Centrella and Oliver Eickelberg for helpful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Yale Liver Center (Grant DK34989 to R. G. W.), NIDDK, National Institutes of Health (NIH) (Grant DK58123 to R. G. W.), NCI, NIH (Grant CA41556 to M. R. and V. F. V.), and Chinese Scholarship Council (to C. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to this work.
¶ Present address: Institute of Liver Diseases, Shanghai University of Traditional Chinese Medicine, Shanghai 200032, People's Republic of China.
Present address: The Cancer Institute of New Jersey,
University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, New Brunswick, NJ 08903.
To whom correspondence should be addressed: Dept. of Medicine
(Gastroenterology), School of Medicine, University of Pennsylvania, 415 Curie Blvd., 670 CRB/6140, Philadelphia, PA 19104-6140. Tel.: 215-573-1860; Fax: 215-573-2024; E-mail: rgwells@
mail.med.upenn.edu.
Published, JBC Papers in Press, January 22, 2003, DOI 10.1074/jbc.M207728200
2 O. Eickelberg and R. G. Wells, unpublished observations.
3 M. D. A. Gaça and R. G. Wells, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
TGF-, transforming growth factor-
;
HSC, hepatic stellate cell(s);
T
RI, type I TGF-
receptor;
RT, reverse transcription;
PAI-1, plasminogen
activator inhibitor-1;
PCNA, proliferating cell nuclear antigen;
SARA, Smad anchor for receptor
activation;
FBS, fetal bovine serum;
PBS, phosphate-buffered saline;
M199, medium 199.
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