1 Division of Gastroenterology
and Hepatology, Cytokines, growth factors, and alterations in
the extracellular matrix composition may play a role in maintaining
hepatic stellate cells (HSC) in the activated state that is responsible for hepatic fibrogenesis. However, the signal transduction pathways that are stimulated by these factors in HSC remain to be fully elucidated. Recent evidence indicates that the mitogen-activated protein kinase (MAPK) family, including c-Jun
NH2-terminal kinase (JNK) and
extracellular signal-regulated kinase (ERK), plays an important role in
the cellular response to stress. The aims of this study were to
investigate whether fibronectin (FN) or the inflammatory cytokines
interleukin-1
interleukin-1; liver; mitogen-activated protein kinase; metalloproteinase; tumor necrosis factor- PATHOLOGICAL FIBROSIS IN THE liver is mediated by
activated hepatic stellate cells (HSC), also known as lipocytes or Ito
cells (13, 17). These cells have a myofibroblastic phenotype with the
ability to proliferate and synthesize large quantities of extracellular
matrix components. Several factors have been proposed to perpetuate the
fibrogenic process in activated HSC, including inflammatory cytokines
and alterations in the extracellular matrix (13, 17). However, the
intracellular signaling pathways that are activated by these factors in
HSC are poorly understood. Recent evidence in other cell types
indicates that binding to some cytokine receptors or to integrins may
activate certain serine-threonine protein kinases, including
extracellular signal-regulated kinase (ERK) (10, 11, 29) and c-Jun
NH2-terminal kinase (JNK) (5, 26,
43). Therefore, activation of JNK and ERK might play a role in the
response of HSC to hepatic injury and inflammation. JNK activation
leads to the phosphorylation of c-Jun, which can then associate as
c-Jun homodimers or c-Jun/c-Fos heterodimers comprising the
transcription factor AP-1 (3). The activation of ERK can induce the
cellular levels of c-Fos, thus also upregulating AP-1 activity (3).
AP-1 influences the expression of several genes, including those for
collagenase (2, 41), gelatinase (12), and transin/stromelysin (33, 38).
Alterations in hepatic matrix composition as a result of changes in the
activity of metalloproteinases may play an important role in hepatic
fibrogenesis (13). Activated HSC synthesize a variety of products,
including collagen (14), fibronectin (FN) (34), proteoglycans (16),
transin (the rat homologue of stromelysin) (42), type IV collagenase
(4), and tissue inhibitors of metalloproteinases (21). Collagenases,
gelatinases, and stromelysins comprise classes of matrix
metalloproteinases that are important mediators of tissue remodeling in
response to growth factors, cytokines, or perturbations in the
extracellular matrix (24). Transin appears to be important during
inflammation due to its activation of interstitial collagenases in
association with plasmin (18) and its ability to degrade components of
the basement membrane (24). Transin production is stimulated by a wide
variety of growth factors and cytokines, and its hepatic gene
expression is upregulated after partial hepatectomy (1) and carbon
tetrachloride-induced injury (19). In this study, we demonstrate that
FN and the two inflammatory cytokines [interleukin-1 HSC isolation and culture.
HSC were isolated from male Sprague-Dawley rats (retired breeders;
Harlan Sprague Dawley, Indianapolis, IN), utilizing a sequential pronase and collagenase perfusion technique, as we described previously (35). HSC were purified by density-gradient centrifugation using arabinogalactan, and viability and purity were assessed as previously described (35). Cells were plated on six-well uncoated plastic tissue
culture dishes (Corning Glass Works, Corning, NY) for kinase assays or
on T-75 flasks for isolation of RNA. Cells were grown to confluency at
37°C in an atmosphere of 5%
CO2 in Dulbecco's modified
Eagle's medium (DMEM) (Sigma Chemical, St. Louis, MO) supplemented
with 12 mM NaHCO3, 2 × 105 U/l penicillin, 0.2 g/l
streptomycin, and 10% Cosmic calf serum (HyClone, Logan, UT). HSC were
utilized for experiments between passages
5 and
7.
JNK assay.
After HSC reached confluency in 6-well culture dishes, the cultures
were maintained for 48 h in serum-free medium. The medium was removed,
replaced with DMEM alone or DMEM containing either soluble FN
(0.1-100 µg/ml) (Sigma Chemical), TNF- ERK assay.
After HSC reached confluency in 6-well culture dishes, the cultures
were maintained for 48 h in serum-free medium. The medium was removed,
replaced with DMEM alone or DMEM containing either soluble FN
(0.1-100 µg/ml), TNF- Electrophoretic mobility shift assay for AP-1.
Confluent cultures of HSC were serum starved for 48 h and then
incubated for 18 h at 37°C in DMEM with or without either FN (100 µg/ml), TNF- Western blotting for nuclear c-Jun and c-Fos.
Confluent cultures of HSC were serum starved for 48 h and then
incubated for 18 h at 37°C in DMEM with or without either FN (100 µg/ml), TNF- Transfection and measurement of chloramphenicol
O-acetyltransferase activity.
HSC were transfected with an AP-1 responsive
chloramphenicol O-acetyltransferase
(CAT) reporter construct
(12-O-tetradecanoylphorbol-13-acetate response element-CAT) (gift of Dr. Gary Fisher). HSC (5 × 105 cells) were plated
overnight on 35-mm plates to reach 50% confluency. After a change of
medium, the cells were transfected with 3 µg of purified construct
DNA utilizing lipofectamine (Life Technologies, Gaithersburg, MD). The
cultures were incubated for 24 h at 37°C, and the medium was
replaced with fresh DMEM containing 10% Cosmic calf serum for 24 h to
allow the cells to recover. The medium was then replaced with DMEM, and
the cells were serum starved for 48 h before exposure to FN (100 µg/ml), IL-1 RNA isolation and ribonuclease protection assay.
Untreated (control) HSC and HSC treated for 18 h with FN (100 µg/ml),
IL-1 Statistical analysis.
The results are expressed as means ± SE. Statistical difference
between groups was assessed using two-tailed Student's
t-test or analysis of variance with
P <0.05 considered as significant.
FN, IL-1
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
(IL-1
) and tumor necrosis factor-
(TNF-
)
activate JNK, ERK, and AP-1 activity in HSC and induce the gene
expression of the matrix metalloproteinase transin. Treatment of HSC
with FN resulted in an up to 4.5-fold increase in ERK activity and a
2.1-fold increase in JNK activity. IL-1
and TNF-
produced up to a
fourfold increase in JNK activity and a twofold increase in ERK
activity. We then compared the effects of FN, IL-1
, and TNF-
on
AP-1 activity and metalloproteinase mRNA induction. All three compounds
increased AP-1 binding and promoter activity, and transin mRNA levels
were increased 1.8-fold by FN, 2.2-fold by IL-1
, and 2.8-fold by
TNF-
. Therefore, FN and inflammatory cytokines increase MAPK
activity, stimulate AP-1 activity, and increase transin gene expression
in HSC. Signal transduction pathways involving the MAPK family may play
an important role in the regulation of matrix metalloproteinase
expression by cytokines and FN in HSC.
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
(IL-1
) and
tumor necrosis factor-
(TNF-
)] stimulate JNK and ERK activity,
increase AP-1 activity, and induce the gene expression of transin in
HSC.
METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
(0.1-100 ng/ml) (Sigma Chemical), or IL-1
(1.5-150 ng/ml) (Genzyme, Cambridge, MA) and incubated for 15 min at 37°C. The cells were then placed on
ice, rinsed twice with ice-cold phosphate-buffered saline, and lysed by
incubating at 4°C for 30 min in 200 µl of solubilization buffer
containing 25 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 300 mM NaCl, 1.5 mM
MgCl2, 0.1% Triton X-100, 20 mM
-glycerophosphate, 0.1 mM sodium orthovanadate, 0.01 mg/ml aprotonin, 0.01 mg/ml leupeptin, and 0.5 mM phenylmethylsulfphonyl fluoride (PMSF). The lysates were centrifuged at 2,000 g for 10 min at 4°C, and the
supernatants were removed and diluted with 3 vol of the above-mentioned
buffer containing 20 mM HEPES, 50 mM NaCl, 0.1 mM EDTA, and 0.05%
Triton X-100. Cell extracts (40 µg of protein) were then
mixed with 10 µg of a glutathione
S-transferase-c-Jun fusion protein
bound to glutathione-agarose beads (26) for 18 h at 4°C. Under
these incubation conditions, JNK in the cell extracts binds tightly to
the c-Jun portion of the fusion protein. At the end of the incubation,
the beads and associated proteins were pelleted and washed three times
in a buffer containing 20 mM HEPES, 50 mM NaCl, 0.1 mM EDTA, 2.5 mM
MgCl2, and 0.05% Triton
X-100. JNK-mediated phosphorylation of the c-Jun portion
of the fusion protein was performed in 30 µl of kinase buffer (pH
7.6) containing 20 mM HEPES, 20 mM
MgCl2, 5 µCi of
[32P]ATP (New England
Nuclear, Boston, MA), 20 mM
-glycerophosphate, 0.1 mM sodium
orthovanadate, 20 mM p-nitrophenyl
phosphate, 2 mM dithiothreitol, and 20 mM ATP at 30°C for 20 min.
The reactions were terminated by the addition of 2× Laemmli
buffer, and the 32P-labeled c-Jun
fusion protein was resolved by sodium dodecyl sulfate
(SDS)-polyacrylamide gel electrophoresis. After autoradiography, quantitation of the band corresponding to the
32P-labeled c-Jun fusion protein
was performed with a scanning laser densitometer and ImageQuant
software (Molecular Dynamics, Sunnyvale, CA).
(0.1-100 ng/ml), or IL-1
(1.5-150 ng/ml) and incubated for 15 min at 37°C. The cells
were then lysed as described for the JNK assay. Anti-ERK1 antibody (2 µg) (Santa Cruz Biotechnology, Santa Cruz, CA) was added to the
lysate and incubated with gentle rotation at 4°C. After 5 h,
protein A-Sepharose was added and the incubation was continued
overnight at 4°C. ERK1 immune complexes were washed three times
with fresh solubilization buffer, then three times with LiCl buffer
[0.5 M LiCl, 100 mM tris(hydroxymethyl)aminomethane (Tris) pH
7.6], and finally with assay buffer [20 mM
Tris · HCl, pH 7.6, 1.5 mM ethylene
glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic
acid (EGTA), 50 mM
-glycerophosphate, 1 mM dithiothreitol, and
0.03% Brij 35]. The ERK1 immune complexes were pelleted by
centrifugation for 5 min at 10,000 g,
and the pellets were resuspended in kinase buffer (20 mM
Tris · HCl, pH 7.3, 10 mM
MgCl2, 50 mM ATP). For measurement
of ERK activity, the immune complexes were incubated for 45 min at
30°C with myelin basic protein (10 µg per sample) and 5 µCi
[32P]ATP (Amersham).
The reaction was terminated by addition of 2× Laemmli buffer, and
the samples were subjected to SDS-polyacrylamide electrophoresis. After
autoradiography, quantitation of the band corresponding to the
32P-labeled myelin basic protein
was performed with a scanning laser densitometer and ImageQuant
software (Molecular Dynamics).
(10 ng/ml), or IL-1
(15 ng/ml). Nuclear extracts were prepared using the procedure described by Schreiber et al. (37).
Cells were harvested in a solution containing 20 mM
Tris · HCl, pH 7.8, 5 mM
MgCl2, 0.5 mM dithiothreitol, 0.3 M sucrose, 1 mM PMSF, 0.2 mM EGTA, 5 mM
-glycerophosphate, and
protease inhibitors. Nuclear extracts were obtained after
centrifugation at 12,000 g, and
binding assays were established in 25 µl of binding buffer, as
described previously (8), using 3 µg of nuclear extract per reaction.
In the binding reaction, nuclear extracts were incubated for 15 min at
20°C with 0.1 ng (10,000 counts/min) of
32P-end-labeled AP-1 consensus
oligonucleotide (Santa Cruz Biotechnology). For competition studies,
50-fold excess of either unlabeled AP-1 oligonucleotide or an unrelated
oligonucleotide (OCT-1) was incubated with nuclear extracts for 5 min
before the addition of the labeled AP-1 oligonucleotide. To resolve the
oligonucleotide-protein complex, aliquots of the binding reaction were
electrophoresed on 4% nondenaturing polyacrylamide gels and after
drying, radioactivity on the gels was visualized by autoradiography.
(10 ng/ml), or IL-1
(15 ng/ml). Nuclear extracts were prepared as described above, and 5 µg aliquots were
electrophoresed on 10% polyacrylamide gels. Separated proteins were
then transferred to polyvinylidene difluoride membranes (Millipore,
Boston, MA), and the membranes were probed with primary antibodies to
c-Jun or c-Fos (Santa Cruz Biotechnology). Goat anti-rabbit
immunoglobulin G horseradish peroxidase conjugate (Biorad) was added as
the secondary antibody, and specific protein bands were visualized
using enhanced chemiluminescence (ECL; Amersham, Arlington Heights, IL)
following the manufacturer's suggested protocol.
(15 ng/ml), or TNF-
(10 ng/ml) for 18 h. Cell
extracts were prepared, normalized for protein, and assayed for CAT
activity, as previously described (15). Quantitation of CAT activity
was done by direct scanning of autoradiographs from thin-layer
chromatography plates, using a scanning laser densitometer and
ImageQuant software (Molecular Dynamics). The CAT assay was performed
in triplicate on four independent cell preparations.
(15 ng/ml), or TNF-
(10 ng/ml) were lysed by the addition of
RNA Stat-60 (1 ml/5 × 106
cells) (Teltest, Friendswood, TX). RNA was extracted with 0.2 vol of
chloroform and precipitated with 0.5 vol of isopropanol. The RNA
pellets were washed with 75% ethanol and resuspended in 1 mM EDTA. The
ribonuclease (RNase) protection assay was performed as previously
described (32) with the following modifications. Briefly, 40 µg of
RNA was hybridized with an antisense
[32P]UTP-labeled RNA
probe prepared by in vitro transcription of a fragment isolated from
the cDNA for rat transin (25). An EcoR I/BamH I fragment was isolated and
subcloned into Bluescript KS plasmid (Promega, Madison, WI) with the
orientation of the T7 promoter
giving antisense RNA and the T3 promoter yielding sense RNA. The
antisense RNA probe protected 400 bp of the transin mRNA. Sty I digest of the cDNA for
glyceraldehyde-3-phosphate dehydrogenase (Ambion, Austin, TX) (280 bp
fragment) was included in the hybridization reaction as a control.
Antisense riboprobes were labeled and transcribed from their respective
cDNAs utilizing a transcription system (Gemini; Promega) with the
addition of [32P]UTP
(300 Ci/mmol). Hybridization and digestion to yield the protected
fragments were performed using an RPA II kit (Ambion). Samples were
electrophoresed on a 6% polyacrylamide gel, and the radioactive bands
were detected by autoradiography and quantitated by scanning laser
densitometry and ImageQuant software (Molecular Dynamics).
RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References
, and TNF-
increase JNK and
ERK activity.
Treatment of HSC with FN, IL-1
, or TNF-
increased JNK activity
above basal levels, although unstimulated HSC showed considerable basal
JNK activity (Figs.
1-3).
The addition of soluble FN to HSC cultures at concentrations of 10 and
100 µg/ml produced a 1.8- and 2.1-fold increase in JNK activity and a
4.5- and 4.3-fold increase in ERK activity, respectively (Fig. 1).
Treatment of HSC with FN at concentrations of 0.1 and 1 µg/ml
increased JNK activity by 1.2- and 1.4-fold and ERK activity by 2.1- and 3.0-fold, respectively. IL-1
at concentrations of 1.5, 15, and
150 ng/ml produced significant dose-dependent increases (1.7-, 2.3-, and 4.3-fold, respectively) in the phosphorylation of the c-Jun fusion protein (Fig. 2) and a twofold increase in ERK activity (data not
shown). Similar to IL-1
, TNF-
at concentrations of 1.0, 10, and
100 ng/ml elicited 2.4-, 4.7-, and 4.2-fold increases in JNK activity
and a 2.2-fold increase in ERK activation (Fig. 3).
View larger version (26K):
[in a new window]
Fig. 1.
Fibronectin (FN) stimulates c-Jun
NH2-terminal kinase (JNK) and
extracellular signal-regulated kinase (ERK) in hepatic stellate cells
(HSC). HSC were incubated in absence or presence of FN (0.1-100
µg/ml) for 15 min. JNK and ERK activity were measured in cell
extracts by determining phosphorylation of a c-Jun fusion protein
(top) and myelin basic protein,
respectively (see METHODS). Results
are expressed as fold increase over control values (means ± SE;
n = 5).
View larger version (20K):
[in a new window]
Fig. 2.
Interleukin-1 (IL-1
) stimulates JNK in HSC. HSC were treated with
IL-1
(1.5, 15, or 150 ng/ml) for 15 min. IL-1
caused progressive
increase in JNK activity, indicated by increased phosphorylation of
c-Jun fusion protein. Results are expressed as fold increase over
control values (means ± SE; n = 5).
View larger version (20K):
[in a new window]
Fig. 3.
Tumor necrosis factor- (TNF-
) increases JNK and ERK activity in
HSC. HSC were incubated with TNF-
(0.1-100 ng/ml) for 15 min.
JNK and ERK activity were measured by determining phosphorylation of
c-Jun fusion protein (top) or myelin
basic protein, respectively. Results are expressed as fold increase
over control values (means ± SE; n = 3).
FN, IL-1, and TNF-
increase AP-1
binding activity.
To determine if FN, IL-1
, and TNF-
stimulate AP-1 in HSC, an
electrophoretic mobility shift assay was performed to measure AP-1
binding activity in nuclear extracts. Figure
4 shows that FN, IL-1
, and TNF-
increased nuclear AP-1 binding in HSC. The shifted band was
competitively inhibited by the addition of a 50-fold excess of
unlabeled AP-1 nucleotide but not by an excess of an unrelated
oligonucleotide (OCT-1).
|
Western blotting for nuclear c-Jun and c-Fos.
To determine the nuclear levels of c-Jun and c-Fos, HSC were treated
with FN (100 µg/ml), IL-1 (15 ng/ml), or TNF-
(10 ng/ml) for 18 h and nuclear extracts were prepared and examined by Western analysis.
As shown in Fig. 5, IL-1
, TNF-
, and
FN caused an approximately twofold increase in nuclear c-Jun levels.
IL-1
and TNF-
also increased the nuclear levels of c-Fos by about
twofold. Unstimulated HSC contained detectable basal levels of c-Jun
and c-Fos. The increases in the nuclear levels of the AP-1 family
members, c-Jun and c-Fos, coincided with elevations in AP-1 binding and
AP-1 dependent gene expression (Figs. 4 and
6).
|
|
FN, IL-1, and TNF-
increase
expression of an AP-1 responsive reporter construct.
Transient transfections of HSC with an AP-1 responsive reporter
construct were performed to determine if the observed elevations in
AP-1 binding activity were associated with increased transcription of
an AP-1 responsive CAT construct. FN, IL-1
, and TNF-
elicited a
1.5-, 2.8-, and 2-fold increase, respectively, in CAT activity relative
to the activity in unstimulated cells (Fig. 6).
FN, IL-1, and TNF-
increase transin
mRNA levels.
An RNase protection assay was used to measure the levels of transin
mRNA in HSC (Fig. 7). Treatment with FN
resulted in a 1.8-fold increase (P < 0.01) in transin mRNA levels, whereas treatment with IL-1
and
TNF-
caused a 2.2- and 2.8-fold increase, respectively (P < 0.001). These data demonstrate
that FN, IL-1
, and TNF-
increase the steady-state levels of
transin mRNA in HSC.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Activated HSC are the major source of extracellular matrix components
during hepatic fibrosis (13, 17). Activated HSC have been observed in
human liver disease and in a variety of animal models of hepatic
fibrosis, including iron overload, chronic cholestasis, ethanol
administration, and chronic carbon tetrachloride (7, 13, 17). The
perpetuation of the activated phenotype of HSC may depend on a complex
interplay between inflammatory cytokines, altered extracellular matrix,
and continued hepatocellular injury (13, 17). In the present study, we
demonstrate that FN and two inflammatory cytokines (IL-1 and
TNF-
) activate JNK, ERK, and AP-1 in HSC and upregulate transin gene
expression in these cells. The activation of these mitogen-activated
protein kinase (MAPK) signaling pathways by FN, IL-1
, and TNF-
may contribute to their effects on the fibrogenic state of HSC.
The deposition of FN is increased early in inflammation (30), and FN is
a constituent of the abnormal matrix laid down in hepatic fibrosis
(22). FN is found in two main forms: a circulating form and a cellular
form (30). Circulating levels of FN have been reported to be ~300
µg/ml (28), and the concentration of cellular FN present within the
extracellular matrix of the liver may vary according to the degree of
injury and inflammation. Both forms of FN contain an RGD
cell-binding sequence that can bind to the
5
1-integrin
in cell plasma membranes, and this integrin has recently been detected
in cultured human HSC (9). In other cells, activation of
MAPK by FN appears dependent on integrin binding, receptor aggregation,
and tyrosine kinase activity (27). Recent evidence indicates that,
unlike circulating FN, the cellular FN produced during hepatic injury
contains a segment (EIIIA) that is particularly efficacious in
activating HSC (22). Because the current experiments used the
circulating form of FN to activate ERK and JNK, it is not known what
effect hepatic cellular FN would have on these signaling pathways.
Previous studies in rabbit fibroblasts indicate that the effects of FN
on the expression of transin/stromelysin may be complex. When these
fibroblasts were plated on dishes coated with FN, the expression of
stromelysin was not changed, but when a mixed substrate of FN and
tenascin was used, it caused induction (39). In addition, plating these
cells on a fragment of FN containing the RGD region caused induction of
stromelysin, whereas another region of the molecule inhibited
stromelysin expression through binding to the 4
1-integrin
(20). It is not known whether HSC express the
4
1-integrin.
Because our experiments used HSC attached to plastic and endogenously
produced matrix, the effects of soluble FN that we observed may have
been influenced by cooperative effects of attachment to other
extracellular matrix molecules. Characterization of the integrin
composition of rat HSC and the effects of attachment to different
matrices will be useful to more fully evaluate possible integrin-mediated signaling in HSC.
Hepatic inflammation elicits the release of a wide variety of
cytokines, including IL-1 and TNF-
, from activated macrophages and other inflammatory cells. The ability of IL-1
and TNF-
to stimulate JNK in HSC is consistent with previous reports in fibroblasts (5) and monocytes (43). The four- to fivefold increase in JNK activity
induced by IL-1
and TNF-
in HSC is lower than the increases
reported in some other cell types (5, 43). Besides the possibility of
an inherent difference in responsiveness of HSC to these agonists, the
high basal JNK activity in unstimulated HSC may have decreased the fold
stimulation seen with IL-1
and TNF-
.
The current results indicate that FN, IL-1, and TNF-
stimulate
both JNK and ERK in HSC, with IL-1
and TNF-
having a more pronounced effect on JNK activity, whereas FN is a more potent stimulant of ERK than JNK. Evidence is accumulating from a variety of
cell types that several mediators can activate multiple MAPK pathways,
albeit with different efficacies (11). One potential site of
integration of JNK and ERK signaling is at the level of the
transcription factor AP-1 (3). A major target of JNK is the
transcriptional activation domain of c-Jun: c-Jun forms the AP-1
transcription factor either as a homodimer or as a heterodimer with
c-Fos (3, 23). The activation of ERK can induce the cellular levels of
c-Fos thus also stimulating AP-1 activity (3, 23). AP-1 regulates
transcription at promoters containing the 12-O-tetradecanoylphorbol-13-acetate-response element, and
previous work in several cell types has indicated a role for AP-1 in
regulating transin/stromelysin gene expression (33, 38). The present study demonstrates that FN, IL-1
, and TNF-
activate both AP-1 activity and transin mRNA levels in HSC. Alcorn et al. (1) have
observed that the hepatic mRNA levels of c-Jun and transin are
increased after partial hepatectomy, and they suggested that c-Jun-dependent AP-1 activity may be responsible for the induction of
transin gene expression.
This study shows that FN, IL-1, and TNF-
increase nuclear AP-1
binding activity and the expression of an AP-1 reporter construct in
HSC. The induction of AP-1 binding activity (as determined by
electrophoretic mobility shift assay) was greater than the increase in
AP-1 dependent gene expression (as determined in transfected HSC). This
is not unexpected, since changes in AP-1 DNA binding do not always
reflect the transcriptional activity of this complex (23). The ability
of AP-1 to activate transcription is best assessed using an AP-1
reporter gene in transfection experiments (23), and the current results
indicate that IL-1
, TNF-
, and FN elicit a 2.8-, 2.0-, and
1.8-fold increase, respectively, in the expression of an AP-1 CAT
reporter construct in HSC. This correlates well with the increases in
steady-state transin mRNA levels produced by these three substances.
The effects of IL-1 and TNF-
on mRNA levels of AP-1 family
members and the constituents of the AP-1 complex have been examined in
other cell types. Treatment with these agonists elicits early increases
in c-Fos and c-Jun mRNA levels (6, 31, 41). The cell attachment region
of FN has also been shown to elicit elevations in c-Fos and c-Jun mRNA
that are associated with the subsequent expression of collagenase (40).
Thus the current consensus is that early increases in c-Jun and c-Fos
gene transcription lead to increases in the levels of these proteins,
with the formation of heterodimers of c-Fos and c-Jun or homodimers of
c-Jun, which can bind to AP-1 sites in DNA. In addition, the activation
of JNK results in the phosphorylation of c-Jun, which increases its transcriptional activity, and this mechanism may be responsible for
part of the increase in AP-1 activity (23). We have determined by
Western blotting that TNF-
, IL-1
, and FN elicit increases in
nuclear c-Jun levels, whereas TNF-
and IL-1
also increase nuclear
c-Fos, in association with elevations in AP-1 activity in HSC. In
accordance with what has been observed in other cell types, it seems
most likely that c-Jun homodimers and c-Jun/c-Fos heterodimers are the
major constituents of the AP-1 complex that is induced by TNF-
,
IL-1
, and FN in HSC. However, these results do not exclude the
possibility of a role for other AP-1 family members in FN- and
cytokine-mediated transcriptional activation within HSC.
In conclusion, the results of the current experiments suggest that activation of the JNK and ERK pathways by FN and inflammatory cytokines leads to an increase in AP-1 activity and transin gene expression in HSC. The upregulation of the gene expression for transin might result in increased production of this metalloproteinase, which could have several possible consequences, including activation of extracellular proteases in their proenzyme form and degradation or alteration of ECM components, which may further perpetuate HSC activation. Further understanding of terminal events elicited through the MAPK signaling pathways in HSC may provide insight into the mechanisms by which some inflammatory cytokines and FN may influence the fibrogenic activity of these cells.
![]() |
ACKNOWLEDGEMENTS |
---|
This study was supported by a National Research Service Award F-32 (to J. E. Poulos), National Institutes of Health Grants RO1 DK-41816 (to B. R. Bacon) and RO1 HL-40901 (to J. J. Baldassare), a Howard Hughes Medical Institute Medical Student Research Training Fellowship (to J. M. Bellezzo), and by a donation from the David Lichtenstein Foundation (to B. R. Bacon).
![]() |
FOOTNOTES |
---|
Portions of this study were presented at the annual meetings of the American Association for the Study of Liver Diseases (Chicago, IL, November 1995, 1996) and have been published in abstract form (Hepatology 22: 293A, 1995; Hepatology 24: 459A, 1996).
Address for reprint requests to: B. R. Bacon, Division of Gastroenterology and Hepatology, St. Louis Univ. Health Sciences Center, 3635 Vista Ave., St. Louis, MO 63110-0250.
Received 13 August 1996; accepted in final form 4 June 1997.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Alcorn, J. A.,
S. P. Feitelberg,
and
D. A. Brenner.
Transient induction of c-Jun during hepatic regeneration.
Hepatology
11:
909-915,
1990[Medline].
2.
Angel, P.,
M. Imagawa,
R. Chiu,
B. Stein,
R. J. Imbra,
H. J. Rahmsdorf,
C. Jonat,
P. Herrlich,
and
M. Karin.
Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor.
Cell
49:
729-739,
1987[Medline].
3.
Angel, P.,
and
M. Karin.
The role of Jun, Fos, and the AP-1 complex in cell-proliferation and transformation.
Biochim. Biophys. Acta
1072:
129-157,
1991[Medline].
4.
Arthur, M. J. P.,
S. L. Friedman,
F. J. Roll,
and
D. M. Bissell.
Lipocytes from normal rat liver release a matrix metalloproteinase that degrades basement membrane type IV collagen.
J. Clin. Invest.
84:
1076-1085,
1989[Medline].
5.
Bird, T. A.,
J. M. Kyriakis,
L. Tyshler,
M. Gayle,
A. Milne,
and
G. D. Virca.
Interleukin-1 activates p54 mitogen-activated protein (MAP) kinase/stress-activated protein kinase by a pathway that is independent of p21ras, Raf-1, and MAP kinase kinase.
J. Biol. Chem.
269:
31836-31844,
1994
6.
Brenner, D. A.,
M. A. O'Hara,
P. Angel,
M. Chojkier,
and
M. Karin.
Prolonged activation of Jun and collagenase genes by tumor necrosis factor-.
Nature
337:
661-663,
1989[Medline].
7.
Britton, R. S.,
and
B. R. Bacon.
Role of free radicals in liver diseases and hepatic fibrosis.
Hepatogastroenterology
4:
343-348,
1994.
8.
Bulla, G. A.,
V. DeSimone,
R. Cortese,
and
R. E. K. Fournier.
Extinction of 1-antitrypsin gene expression in somatic cell hybrids: evidence for multiple controls.
Genes Dev.
6:
316-327,
1992[Abstract].
9.
Carloni, V.,
R. G. Romanelli,
M. Pinzani,
G. Laffi,
and
P. Gentilini.
Expression and function of integrin receptors for collagen and laminin in cultured human hepatic stellate cells.
Gastroenterology
110:
1127-1136,
1996[Medline].
10.
Chen, C.,
M. S. Kinch,
T. H. Lin,
K. Burridge,
and
R. L. Juliano.
Integrin-mediated cell adhesion activates mitogen-activated protein kinases.
J. Biol. Chem.
269:
26602-26605,
1994
11.
Denhardt, D. T.
Signal-transducing protein phosphorylation cascades mediated by Ras/Rho proteins in the mammalian cell: the potential for multiplex signalling.
Biochem. J.
318:
729-747,
1996[Medline].
12.
Fini, M. E.,
J. D. Bartlett,
M. Matsubara,
W. B. Rinehart,
M. K. Mody,
M. T. Girard,
and
M. Rainville.
The rabbit gene for 92-kDa matrix metalloproteinase. Role of AP1 and AP2 in cell type-specific transcription.
J. Biol. Chem.
269:
28620-28628,
1994
13.
Friedman, S. L.
The cellular basis of hepatic fibrosis. Mechanisms and treatment strategies.
N. Engl. J. Med.
328:
1828-1835,
1993
14.
Friedman, S. L.,
F. J. Roll,
J. Boyles,
and
D. M. Bissell.
Hepatic lipocytes: the principal collagen producing cells of normal rat liver.
Proc. Natl. Acad. Sci. USA
82:
8681-8685,
1985[Abstract].
15.
Gorman, C. M.,
L. F. Moffat,
and
B. H. Howard.
Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells.
Mol. Cell. Biol.
2:
1044-1051,
1982[Medline].
16.
Gressner, A. M.
Time-related distribution profiles of sulfated glycosaminoglycans in cells, cell surfaces, and media of cultured rat liver fat storing cells.
Soc. Exp. Biol. Med.
196:
307-315,
1991[Abstract].
17.
Gressner, A. M.
Cytokines and cellular crosstalk involved in the activation of fat-storing cells.
J. Hepatol.
22:
28-36,
1995[Medline].
18.
He, C. S.,
S. M. Wilhelm,
A. P. Pentland,
B. L. Marmar,
G. A. Grant,
A. Z. Eise,
and
G. I. Golberg.
Tissue cooperation in a proteolytic cascade activating human interstitial collagenase.
Proc. Natl. Acad. Sci. USA
86:
2632-2636,
1989[Abstract].
19.
Herbst, H.,
O. Heinrichs,
D. Schuppan,
S. Milani,
and
H. Stein.
Temporal and spatial patterns of transin/stromelysin RNA expression following toxic injury in rat liver.
Virchows Arch. B Cell Pathol.
60:
295-300,
1991[Medline].
20.
Huhtala, P.,
M. J. Humphries,
J. B. McCarthy,
P. M. Tremble,
Z. Werb,
and
C. H. Damsky.
Cooperative signaling by 5
1 and
4
1 integrins regulates metalloproteinase gene expression in fibroblasts adhering to fibronectin.
J. Cell Biol.
129:
867-879,
1995[Abstract].
21.
Iredale, J. P.,
G. Murphy,
R. M. Hembrix,
S. L. Friedman,
and
M. J. P. Arthur.
Human hepatic lipocytes synthesize tissue inhibitor of metalloproteinases-1.
J. Clin. Invest.
90:
282-287,
1992[Medline].
22.
Jarnagin, W. R.,
D. C. Rockey,
V. E. Koteliansky,
S.-S. Wang,
and
D. M. Bissell.
Expression of variant fibronectins in wound healing: cellular sources and biological activity of the EIIIA segment in rat hepatic fibrogenesis.
J. Cell Biol.
127:
2037-2048,
1994[Abstract].
23.
Karin, M.
The regulation of AP-1 activity by mitogen-activated protein kinases.
J. Biol. Chem.
270:
16483-16484,
1995
24.
Matrisian, L. M.
The matrix-degrading metalloproteinases.
Bioessays
14:
455-463,
1992[Medline].
25.
Matrisian, L. M.,
N. Glaichenhaus,
M. C. Gesnel,
and
R. Breathnach.
Epidermal growth factor and oncogenes induce the transcription of the same cellular mRNA in rat fibroblasts.
EMBO J.
4:
1435-1440,
1985[Abstract].
26.
Minden, A.,
A. Lin,
M. McMahon,
C. Lange-Carter,
B. Derijard,
R. J. Davis,
G. L. Johnson,
and
M. Karin.
Differential activation of ERK and JNK mitogen-activated protein kinases by Raf-1 and MEEK.
Science
266:
1719-1723,
1994[Medline].
27.
Miyamoto, S.,
H. Teramoto,
O. A. Coso,
J. S. Gutkind,
P. D. Burbelo,
S. K. Akiyama,
and
K. M. Yamada.
Integrin function: molecular hierarchies of cytoskeletal and signalling molecules.
J. Cell Biol.
131:
791-805,
1995[Abstract].
28.
Mohri, H.
Fibronectin and integrins interactions.
J. Investig. Med.
44:
429-441,
1996[Medline].
29.
Morino, N.,
T. Mimura,
K. Hamasaki,
K. Tobe,
K. Ueki,
K. Kikuchi,
K. Takehara,
T. Kadowaki,
Y. Yazaki,
and
Y. Nojima.
Matrix/integrin interaction activates the mitogen-activated protein kinase p44erk1 and p42erk2.
J. Biol. Chem.
270:
269-273,
1995
30.
Mosher, D. F.
Organization of the provisional fibronectin matrix: control by products of blood coagulation.
Thromb. Haemost.
74:
529-533,
1995[Medline].
31.
Muegge, K.,
T. M. Williams,
J. Kant,
M. Karin,
R. Chiu,
A. Schmidt,
U. Sigbenlist,
H. A. Young,
and
S. K. Durum.
Interleukin-1 costimulatory activity on interleukin-2 promoter via AP-1.
Science
32:
249-251,
1989.
32.
Poulos, J. E.,
W. R. Gower,
H. L. Fontanet,
G. W. Kalmus,
and
D. L. Vesely.
Cirrhosis with ascites: increased atrial natriuretic peptide messenger RNA expression in rat ventricle.
Gastroenterology
108:
1496-1503,
1995[Medline].
33.
Quinones, S.,
G. Buttice,
and
M. Kurkinen.
Promoter elements in the transcriptional activation of the human stromelysin-1 gene by the inflammatory cytokine, interleukin-1.
Biochem. J.
302:
471-477,
1994[Medline].
34.
Ramadori, G.,
H. Rieder,
T. H. Knittel,
H. P. Dienes,
and
K. Meyer-zum Buschenfelde.
Fat storing cells (FSC) of rat liver synthesize and secrete fibronectin. Comparison with hepatocytes.
J. Hepatol.
4:
190-197,
1987[Medline].
35.
Ramm, G. A.,
S. C. Y. Li,
L. Li,
R. S. Britton,
R. O'Neill,
Y. Kobayashi,
and
B. R. Bacon.
Chronic iron overload causes activation of rat lipocytes in vivo.
Am. J. Physiol.
268 (Gastrointest. Liver Physiol. 31):
G451-G458,
1995
37.
Schreiber, E.,
P. Matthias,
M. M. Müller,
and
W. Schaffner.
Rapid detection of octamer binding proteins with mini-extracts, prepared from a small number of cells.
Nucleic Acids Res.
17:
6419,
1989[Medline].
38.
Sirum-Connolly, K.,
and
C. E. Brinckerhoff.
Interleukin-1 or phorbol induction of the stromelysin promoter requires an element that cooperates with AP-1.
Nucleic Acids Res.
19:
335-341,
1991[Abstract].
39.
Tremble, P.,
R. Chiquet-Ehrismann,
and
Z. Werb.
The extracellular matrix ligands fibronectin and tenascin collaborate in regulating collagenase gene expression in fibroblasts.
Mol. Biol. Cell
5:
439-453,
1994[Abstract].
40.
Tremble, P.,
C. H. Damsky,
and
Z. Werb.
Components of the nuclear signaling cascade that regulate collagenase gene expression in response to integrin-derived signals.
J. Cell Biol.
129:
1710-1720,
1995.
41.
Vincenti, M. P.,
C. I. Coon,
O. Lee,
and
C. E. Brinckerhoff.
Regulation of collagenase gene expression by IL-1 requires transcriptional and post-transcriptional mechanisms.
Nucleic Acids Res.
22:
4818-4827,
1994[Abstract].
42.
Vyas, S. K.,
H. Leyland,
J. Gentry,
and
M. J. P. Arthur.
Rat hepatic lipocytes synthesize and secrete transin (stromelysin) in early primary culture.
Gastroenterology
109:
889-898,
1995[Medline].
43.
Yan, M.,
T. Dai,
J. C. Deak,
J. M. Kyriakis,
L. I. Zon,
J. R. Woodgett,
and
D. J. Templeton.
Activation of stress-activated protein kinase by MEKK1 phosphorylation of its activator SEK1.
Nature
372:
798-800,
1994[Medline].