Departments of 1 Surgery and 2 Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48109
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ABSTRACT |
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Transforming growth
factor- (TGF-
) inhibits pancreatic acinar cell growth. In many
cell types, TGF-
mediates its growth inhibitory effects by
activation of Smad proteins. Recently, it has been reported that Smad
proteins may interact with the mitogen-activated protein (MAP) kinase
signaling pathways. In this study, we report on the interactions
between the TGF-
and MAP kinase signaling pathways in isolated rat
pancreatic acinar cells. TGF-
activated the MAP kinases
extracellular signal-related kinases (ERKs) and p38 in pancreatic
acinar cells, but had no effect on c-jun
NH2-terminal kinase activity. Activation of MAP kinase by
TGF-
was maximal 4 h after treatment. The ability of TGF-
to
activate ERKs was concentration dependent and dependent on protein
synthesis. TGF-
's stimulation of ERK activation was blocked by
PD-98059, an inhibitor of MAP kinase kinase 1, and by
adenoviral transfer of dominant negative RasN17.
Furthermore, adenoviral-mediated expression of dominant negative Smad4
blocked the ability of TGF-
to activate acinar cell MAP kinase,
demonstrating that this activation is downstream of Smads. The
biological relevance of ERK activation by TGF-
was indicated by
demonstrating that inhibition of ERK signaling by PD-98059 blocked the
ability of TGF-
to activate the transcription factor activator
protein-1. These studies provide new insight into the signaling
mechanisms by which TGF-
mediates biological actions in pancreatic
acinar cells.
transforming growth factor-
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INTRODUCTION |
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TRANSFORMING GROWTH
FACTOR- (TGF-
) is a ubiquitous growth factor
that plays a key role in regulation of growth and differentiation in a
wide variety of cell types. TGF-
has been demonstrated to be an
important inhibitor of pancreatic growth in vitro and in vivo. In
cultured pancreatic acinar cells, TGF-
inhibited basal cell growth
and growth stimulated by a variety of trophic factors, including
cholecystokinin (CCK) and epidermal growth factor (EGF) (28). TGF-
has also been shown to inhibit pancreatic
duct cell proliferation (4). Studies performed using a
pancreatic whole organ culture system demonstrated that TGF-
is
important in maintaining early differentiation of both pancreatic
exocrine and endocrine cells (36). The role of TGF-
in
pancreatic differentiation and growth has also been examined using
transgenic animals. In transgenic mice expressing a dominant negative
TGF-
type II receptor targeted to acinar cells, which results in
resistance to TGF-
-mediated signals, the pancreas showed increased
proliferation and dedifferentiation of abnormally proliferating acinar
cells (5). Interestingly, disruption of the TGF-
pathway in many organs, including the pancreas, appears to be important
in cancer development (30). Deleted in pancreatic
carcinoma 4 (DPC4), a gene frequently mutated or deleted in pancreatic
cancer (17), was found to be identical to Smad4, which is
part of the TGF-
signaling pathway. DPC4 was renamed Smad4 as a
result of an agreed change in the nomenclature to avoid confusion in
the field rather than due to independent discovery of the gene.
TGF- is a member of a large family of structurally related growth
factors, including activin, inhibin, and bone morphogenic proteins
(30). TGF-
exerts its biological effects by interacting with two types of transmembrane receptors (types I and II) with protein
serine/threonine kinase activity (31). The type II
receptor is involved in initial ligand binding; once the ligand is
bound, type II receptors bind to type I receptors, forming a complex. Type I receptors are then phosphorylated by the kinase domain of type
II receptors, resulting in the propagation of downstream signals.
Although these initial steps in receptor activation have been defined,
until recently, little has been known of the downstream effectors of
TGF-
.
Smad proteins have been identified as key signaling molecules in
TGF--related signaling pathways. Smad2 and Smad3 are specific to the
TGF-
signaling pathway and are phosphorylated by activated TGF-
receptors on serine residues within a conserved SSXS motif at the COOH
terminus of the protein (29). Phosphorylation of Smad2 and
Smad3 allows complex formation with Smad4, a common effector shared by
different TGF-
family pathways. Once formed, this Smad4-containing
complex moves into the nucleus. In the nucleus, Smad complexes activate
transcription of defined genes. The ability of Smad complexes to
activate transcription is a result of their ability to interact
directly with promoter sequences as well as their ability to interact
and cooperate functionally with various other transcription factors
(40).
As details regarding the molecular mechanisms of Smad protein signaling
have been determined, interactions between these proteins and other
signaling pathways have been reported. Recently, mitogen-activated protein (MAP) kinases have been implicated in TGF- signal
transduction. MAP kinases are serine/threonine protein kinases that are
activated by a variety of cell surface receptors and function in signal cascade pathways that control expression of genes involved in many
cellular processes. To date, three MAP kinase subgroups have been
identified: extracellular signal-regulated kinases (ERKs), which are
stimulated by growth factors and induce cellular proliferation and
differentiation, and c-jun NH2-terminal kinases
(JNKs) and p38, which represent separate pathways primarily activated
by cellular stresses, ultraviolet irradiation, and cytokines. ERKs, p38, and JNKs have been reported to be activated by TGF-
in a number
of different cell types (2, 15, 18-20, 34, 35). In
addition, it has been recently reported that Smads 1, 2, and 3 contain
consensus sites for MAP kinases within their linker regions and that
Smad1 is phosphorylated at a MAP kinase consensus site in response to
EGF in mink lung epithelial cells, resulting in its inactivation
(26). This suggests that activation of MAP kinase
signaling pathways may modulate Smad activity more generally. While
most studies investigating the TGF-
signaling pathway have been
conducted in transformed tissue culture cells, it is known that TGF-
affects a wide variety of cellular responses and that significant
differences exist in this complex signaling pathway in different cells.
The intracellular signaling pathways through which TGF-
acts to
generate cellular responses in normal pancreatic cells remain largely
undefined. The pancreas, with its high incidence of mutations in this
pathway and the likely importance of Smads in pancreatic cancer, is
arguably one of the most important tissues in which to define this pathway.
The purpose of this study was to investigate the interactions between
TGF- signaling and MAP kinases in pancreatic acinar cells. In this
study, we found that TGF-
activated the MAP kinases ERKs and p38 but
not JNKs. The effect of TGF-
on MAP kinases was concentration
dependent and occurred with a time course different from CCK and
different from that reported for TGF-
-induced MAP kinase activation
in other cell types. TGF-
-induced activation of ERK required protein
synthesis and was dependent on both Ras and MAP kinase kinase 1 (MEK1).
The effects of TGF-
on MAP kinase were also found to depend on Smad4
functioning, because expression of a dominant negative Smad4 blocked
this effect. The biological significance of ERK activation by TGF-
was proven by demonstrating that blockade of ERK signaling by PD-98059
inhibited the ability of TGF-
to induce DNA binding of the
transcription factor activator protein-1 (AP-1).
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MATERIALS AND METHODS |
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Materials.
The following reagents were purchased: TGF-1 from R&D Systems
(Minneapolis, MN); CCK-8 from Research Plus (Bayonne, NJ); EGF from
Collaborative Biomedical Products (Bedford, MA); DMEM, fetal bovine
serum, penicillin, and streptomycin from GIBCO (Grand Island, NY); the
MEK1 inhibitor PD-98059 from New England Biolabs (Beverly, MA); and
Bio-Rad protein reagent from Bio-Rad Laboratories (Hercules, CA). All
other reagents were obtained from Sigma Chemical (St. Louis, MO).
Preparation of acini.
Pancreatic acini were isolated from Wistar rats as previously described
(24). In brief, pancreatic tissue was obtained from male
Wistar rats and digested with collagenase (100 U/ml) and incubated at
37°C for 45 min with shaking (120 cycles/min). Acini were
mechanically dispersed by trituration of tissue through polypropylene pipettes of decreasing orifice size and passed through a 150-µm mesh
nylon cloth. Acini were then purified by centrifugation at 50 g for 3 min in a solution that contained 4% BSA and were
resuspended in enhanced media that consisted of DMEM containing 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.1 mg/ml soybean
trypsin inhibitor. The dispersed acini were aliquoted onto six-well
plates. After a 2-h preincubation period to allow cells to become
quiescent, TGF- and pharmacological agents were added for indicated
times. Cells were maintained in a humidified atmosphere of 5%
CO2 in air at 37°C during incubation times.
MAP kinase immunoblot analysis. Dispersed acini were treated as described in the figure legends. Whole cell lysates were prepared by incubating cells in ice-cold lysis buffer (20 mM Tris, pH 7.8, 2 mM EDTA, 50 mM NaF, 1% Triton X-100, 5 µg/ml leupeptin, 5 µg/ml pepstatin, and 0.5 mM phenylmethylsulfonyl fluoride). Cells were sonicated for 8 s and then placed on ice for 15 min. The lysates were then centrifuged at 14,000 g for 15 min at 4°C and assayed for protein by the Bio-Rad protein assay reagent. Equal amounts of protein (30 µg) were resolved by SDS-7.5% PAGE and transferred to nitrocellulose membranes. MAP kinase immunoblot analysis was performed using anti-phospho-MAP kinase antibodies that recognize the phosphorylated forms of ERKs (cat. no. V8031), JNKs (cat. no. V7931; Promega, Madison, WI), and p38 (cat. no. 9211; New England Biolabs). Equal loading of protein in all blots was validated by measuring total protein levels of ERKs with a K-23 anti-ERKs polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Images were visualized with an enhanced chemiluminescence detection system (Amersham). Film images were scanned with an Agfa Arcus II (Bayer, Ridgefield Park, NJ) to create a digital image.
In-gel ERK assay.
In-gel ERK assays on SDS-polyacrylamide gels were carried out as
described by Dabrowski et al. (9). In brief, 10%
polyacrylamide gels were cast containing 0.5 mg/ml of myelin basic
protein. Protein from pancreatic acini lysates (20 µg) were subjected
to electrophoresis. After electrophoresis, gels were washed four times
with 125 ml of 20% isopropyl alcohol in 50 mM Tris, pH 8.0, to remove
SDS and then with two washes of 125 ml of denaturing buffer containing 6 M guanidine hydrochloride and 5 mM 2-mercaptoethanol in 50 mM Tris,
pH 8.0. The enzymes on the gel were then renatured by washing four
times with buffer containing 50 mM Tris, 0.04% Tween 40, and 5 mM
2-mercaptoethanol at 4°C for 20 h. The renatured gel was
incubated in assay buffer that contained 40 mM HEPES, pH 8.0, 10 mM
MgCl2, 2 mM dithiothreitol (DTT), and 0.1 mM EGTA at 30°C for 30 min. Kinase activity was determined by incubating the gel in 60 ml of the same buffer plus 20 µM ATP and 90 µCi of
[-32P]ATP at room temperature for 1 h. The
reaction was terminated by removal of the gel into 250 ml of 5%
trichloroacetic acid and 10 mM sodium pyrophosphate solution, followed
by multiple washes over a 2-h period to eliminate nonspecific
radioactivity in the gel. The activities of protein kinases were then
measured in dried gels with a Bio-Rad GS-250 molecular imager.
p38 in vitro kinase assay.
p38 activity was determined by measuring its ability to phosphorylate
glutathione S-transferase (GST)-activating transcription factor-2 (ATF-2) in vitro. p38 immunoprecipitates were incubated in a
kinase buffer with GST-ATF-2 and [-32P]ATP for 30 min
at 30°C. The reaction was terminated by adding 4× SDS sample buffer,
and the products were separated by 10% SDS-PAGE. Quantitation was
performed with a Bio-Rad GS-250 molecular imager.
Construction of adenoviral vectors. An EcoRI-HindIII fragment containing the rat elastase 1 promoter (gift of Ray MacDonald, University of Texas, Southwestern) was subcloned into pCMV5/Smad4(1-514) containing a COOH-terminal truncated dominant negative Smad4 gene (gift of J. Massague, Sloan-Kettering). An EcoRI-SmaI fragment containing the elastase promoter/Smad4(1-514) construct from pCMV5 was then subcloned into a replication-defective adenoviral vector as described (22). Briefly, the construct was bluntly ligated into the EcoRV site of the shuttle vector (pAd.Track). The resultant plasmid was linearized and cotransformed into Escherichia coli BJ5183 cells with an adenoviral backbone plasmid pAdEasy-1. Recombinants were selected for kanamycin resistance, and recombination was confirmed using multiple restriction enzyme digest analyses. The linearized recombinant plasmid was transfected into HEK-293 cells, and recombinant adenovirus was harvested after 7-10 days, followed by subsequent viral purification on a cesium chloride gradient. Green fluorescent protein, encoded by a gene in the viral backbone, was used to assist in viral titering. A recombinant adenovirus expressing dominant negative RasN17 (human H-ras cDNA with a serine-to-asparagine substitution at amino acid 17) has been previously described by our laboratory (33).
Pancreatic acinar cell infection and culture.
Isolated acini were suspended in DMEM that contained 100 U/ml
penicillin, 100 µg/ml streptomycin, and 0.1 mg/ml soybean trypsin inhibitor and plated onto 12-well culture plates coated with air-dried rat tail collagen. Adenovirus was added at varying titers as indicated. After 8 h, TGF-, EGF, or CCK was added to the medium as
indicated. MAP kinase immunoblot analysis was carried out as described earlier.
Electrophoretic mobility shift assay.
Acini were prepared and collected by centrifugation and washed with PBS
plus 1 mM EDTA. The pellet was homogenized with a motor-driven pestle
in a buffer that contained 2 M sucrose, 10% glycerol, 10 mM HEPES, 25 mM KCl, 2 mM EDTA, 150 mM spermine, 500 mM spermidine, 1 mM
DTT, and protease inhibitors. Nuclei were collected at 30,000 rpm and
resuspended in lysis buffer. Nuclear protein (5 µg) was incubated
with gel shift binding buffer [10 mM HEPES, 10% glycerol, 1 mM DTT, 1 mg/10 ml poly(dI-dC), and 5 mg/10 ml BSA] and an AP-1 oligonucleotide
probe labeled with [-32P]ATP using T4 polynucleotide
kinase. The oligonucleotide probes were provided by the Promega gel
shift assay system (cat. no. E3300). In competition experiments, the
extract was preincubated for 30 min with 50-fold molar excess of cold
competitor. Reactions were analyzed on a 10 × 12-cm,
nondenaturing, 4% acrylamide gel, 0.75 mm thick. Gels were transferred
to Whatman paper on a gel dryer, exposed to a Bio-Rad GS-250 screen
overnight, and then analyzed on a Bio-Rad molecular imager.
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RESULTS |
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To determine the effects of TGF- on the activity of MAP kinases
in pancreatic acinar cells, isolated acini were treated with TGF-
for various times. TGF-
(100 pM) stimulated the activity of the MAP
kinases ERK (2.8-fold) and p38 (2.4-fold), but had no effect on JNK
activity (Fig. 1). This result was
different from what is observed for the gastrointestinal
hormone CCK. It has been previously shown that CCK rapidly activates
all three MAP kinases in rat pancreatic acini (12, 37). In
this study, CCK (1 nM) stimulated the activities of the three MAP
kinases, ERK, p38, and JNK (2.3-, 2.5-, and 2.7-fold, respectively).
Moreover, compared with the effects of CCK, which were significant at
10 min, the effects of TGF-
were significant only after 4 h.
ERK and p38 kinase assays, using myelin basic protein and recombinant GST-ATF2, respectively, as substrates, confirmed the ability of TGF-
(100 pM) to activate ERKs and p38, with significant effects at 4 h
(Fig. 2, A and B).
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Subsequently, studies were concentrated on specifically examining the
mechanisms involved in the effect of TGF- on ERKs. An extended time
course indicated that TGF-
induced maximal activation of ERKs at
4 h, which persisted at 8 h, and returned to basal levels by
24 h (Fig. 3A). There was
no evidence of ERK activation by TGF-
at time points earlier than 10 min (data not shown). This was a different time course than that
observed with CCK, which produced maximal activation of ERKs at 10 min,
with a return toward basal levels by 4 h (Fig. 3B). The
concentration dependence of TGF-
's effect was then analyzed.
Pancreatic acini were incubated for 4 h with different
concentrations of TGF-
, and ERK activity was assessed. TGF-
stimulated ERKs in a concentration-dependent manner (Fig.
3C). The minimal response of MAP kinase to TGF-
stimulation was observed at a dose of 10 pM, while maximal responses were observed at 100 pM.
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To further understand the mechanisms involved in the effects of TGF-
on MAP kinase activity, the role of MEK1, an upstream ERK activator,
was investigated using PD-98059, a specific inhibitor of MEK1 activity.
Acini were pretreated with PD-98059 (50 µM) for 30 min, then left
untreated or stimulated with TGF-
(100 pM) in the continued presence
of PD-98059 for 4 h, and the level of ERK phosphorylation was
determined. PD-98059 lowered basal ERK phosphorylation and
significantly blocked increases in ERK phosphorylation induced by
TGF-
(Fig. 4A). To
determine when the responsiveness to PD-98059 occurs, experiments were
conducted examining the effect of PD-98059 added before, at the same
time, and 1, 2, and 3 h after addition of TGF-
. As shown in
Fig. 4B, PD-98059 blocked the ability of TGF-
to activate
ERKs when added as late as 3 h after addition of TGF-
. These
data demonstrated that the ability of TGF-
to activate ERKs was
dependent on MEK1 and indicated that the activity of MEK1 is required
at a late time after TGF-
stimulation. Pretreatment of acini with
PD-98059 had no effect on the ability of TGF-
to induce p38 activity
(data not shown), supporting the use of PD-98059 as a specific MEK1/ERK pathway inhibitor.
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Ras is a GTP-binding molecule known to be upstream of several cellular
signaling pathways, including the ERK pathway (11). To
determine whether TGF--mediated activation of ERKs involves Ras, we
expressed a dominant negative Ras mutant in pancreatic acinar cells
utilizing adenoviral-mediated gene transfer in vitro. Previous work in
our laboratory has found that primary pancreatic acinar cells cannot be
transfected using a variety of standard techniques, such as
Lipofectamine, calcium phosphate, and DEAE dextran, but can be
transfected with high efficiency using adenoviral vectors
(33). We utilized an adenoviral construct containing dominant negative RasN17 (AddnRas) to test the effects of
Ras on TGF-
-induced ERK activation. Acini were infected with no
virus (control), adenovirus bearing dominant negative
RasN17 (AddnRas), or adenovirus bearing green fluorescent
protein (AdGFP), each at a titer of 108 pfu/mg of
pancreatic protein for 8 h. AdGFP was used as a control to account
for any effects that might be due to adenoviral infection. Cells were
then treated with either TGF-
(100 pM) for 4 h or EGF (10 nM)
for 10 min. EGF has been shown previously to induce Ras-dependent ERK
activity in pancreatic acinar cells (10, 33) and was
included to serve as a positive control. AddnRas lowered basal levels
of ERK activity and blocked increases in ERK activity induced by both
TGF-
and EGF (Fig. 5). Infection with
the control AdGFP had no effect on activation of ERK stimulated by
either TGF-
or EGF.
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Because TGF--induced ERK activation occurred at 4 h,
experiments were performed to determine whether this activation was direct or required de novo protein synthesis. Acini were prepared and
pretreated with cyclohexamide (10 µg/ml) for 30 min before addition
of TGF-
for 4 h, a point at which maximal activation of MAP
kinases by TGF-
is observed. Cyclohexamide had no effect on basal
ERK activity and completely blocked the ability of TGF-
to activate
ERKs (Fig. 6). These data suggest that
TGF-
-induced activation of ERKs in pancreatic acinar cells requires
protein synthesis.
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It is known that TGF- mediates many of its cellular responses
through Smad-signaling molecules. Therefore, we investigated whether
Smad4, a common effector in the TGF-
signaling pathway, was required
for activation of ERKs. The role of Smad4 was determined by expressing
in pancreatic acinar cells a COOH-terminal, truncated Smad4 that has
been shown to possess dominant negative activity (41). An
adenoviral vector expressing dominant negative Smad4 (AddnSmad4)
was constructed under the control of an elastase promoter. This
adenovirus also expressed GFP driven by a separate cytomegalovirus promoter. Infection of acinar cells with AddnSmad4 (106
viral pfu/mg acinar protein), but not control AdGFP virus at the same
titer, completely blocked the ability of TGF-
to activate ERKs (Fig.
7A). AddnSmad4 had no effect
on the ability of CCK to induce ERK activity (Fig. 7B).
These data demonstrate that the effect of AddnSmad4 was specific.
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To determine the biological relevance of ERK activation by TGF-, we
examined the role of ERKs in TGF-
induction of DNA binding of the
transcription factor AP-1. AP-1 is a known nuclear target of the MAP
kinase signaling pathways (38). To determine whether TGF-
altered AP-1 activity in pancreatic acinar cells, cells were
treated for 4 h with TGF-
(100 pM), chosen because it is the
time point at which there is maximal activation of ERKs by TGF-
.
Nuclear proteins were then isolated and examined in an electrophoretic
mobility shift assay (EMSA). EMSAs showed that TGF-
increased AP-1
binding to its respective DNA binding site at 4 h (Fig.
8). Pretreatment of acinar cells with
PD-98059 (50 µM) significantly blocked the ability of TGF-
to
induce AP-1 binding, suggesting that the ability of TGF-
to activate
AP-1 is dependent on ERK activity.
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DISCUSSION |
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In the present study, we have demonstrated for the first time that
TGF- activates the MAP kinases ERKs and p38 in isolated rat
pancreatic acini. Interestingly, activation of both ERKs and p38 by
TGF-
did not occur until 4 h, persisted at 8 h, and was no
longer evident at 24 h. This represents a time course
significantly different from that for TGF-
-mediated activation of
MAP kinases in many other cell types. Previous studies have shown that
TGF-
-induced ERK activation in fetal rat lung fibroblasts
(3), intestinal epithelial cells (20), breast
cancer cells (16), and NIH/3T3 cells (32) was
maximal within 5 min of stimulation. However, recently, Finlay and
colleagues (14) demonstrated a time delay in activation of
ERKs in response to TGF-
in primary human lung fibroblasts. In these
cells, TGF-
stimulated ERK activity beginning at 2 h, with
maximal activity at 16 h. Interestingly, in this same study,
TGF-
induced rapid activation of p38. TGF-
has also been shown to
rapidly activate p38 in human neutrophils and in HEK-293 cells
(19, 35). In our studies, no effects were noted at these
short time points. Thus TGF-
influences MAP kinases with a different
time course in different cells. TGF-
also has a differential ability
to activate distinct MAP kinase signaling cascades in different cell
types. For example, TGF-
did not activate JNKs in pancreatic acinar
cells; however, TGF-
induced JNK activity in other cells, including
human-derived fibrosarcoma cells (23), Madin-Darby canine
kidney cells (1), Hep G2 (1), and Chinese hamster ovary cells (1).
The ability of TGF- to activate ERKs in pancreatic acinar cells was
shown to be dependent on both MEK1 and Ras. This is consistent with
studies performed in cultured hepatic stellate cells in which TGF-
-induced activation of ERKs was dependent on Ras and MEK1 (34) as well as studies in an intestinal epithelial cell
line that demonstrated that expression of dominant negative
RasN17 completely abrogated the TGF-
-mediated activation
of ERK1 (21). Activation of ERKs in pancreatic acinar
cells, however, may occur through signaling pathways other than Ras. It
has been previously shown that while EGF-mediated activation of ERKs is
Ras-dependent in acinar cells, the major mechanism for the
activation of ERKs by CCK is independent of Ras and likely involves a
protein kinase C-mediated activation of Raf (10, 33).
It is unclear why the effect of TGF- on MAP kinase activation occurs
in a delayed fashion in pancreatic acinar cells, while in most other
cell types, the response is much more rapid. Because of this delay, we
hypothesized that TGF-
may increase ERK activity indirectly by
inducing synthesis of protein(s) in pancreatic acinar cells, which
subsequently acts on the acinar cell to induce ERK activity. Our data
show that pretreatment of acinar cells with cyclohexamide, a protein
synthesis inhibitor, completely blocked the ability of TGF-
to
activate ERKs; thus the action of TGF-
is indirect. In the study
previously cited by Finlay et al. (14), they demonstrated
that delayed activation of ERKs and AP-1 by TGF-
in human lung
fibroblasts required the autocrine induction of basic fibroblast growth
factor (bFGF). To determine whether bFGF was serving a similar role in
TGF-
-mediated activation of ERKs in pancreatic acinar cells, we
performed Western blotting to determine whether TGF-
induced
expression of bFGF. TGF-
did not induce expression of bFGF in
pancreatic acinar cells (unpublished observations); therefore, it is
likely that another, as yet undetermined autocrine factor is required
for TGF-
-mediated ERK activation in acinar cells.
Smads and the MAP kinase signaling pathways have recently been
determined to have a number of functional interactions. In some cells,
the MAP kinase pathways have been implicated as positive regulators of
Smad-dependent effects. Atfi and colleagues (1, 2)
reported that dominant negative mutants of various components of the
stress-activated protein kinase/JNK pathway could inhibit TGF-- and
Smad4-induced gene expression and TGF-
-stimulated Smad4
transcriptional activity. In endothelial cells, MEK1 can selectively
stimulate Smad2 transcriptional activation in the absence of exogenous
TGF-
stimulation (7). Furthermore, transfection of
dominant negative ERK1 into NIH/3T3 fibroblasts abolished stimulation of plasminogen activator inhibitor-1 promoter activity by TGF-
(32).
In other cells, the classic MAP kinase/ERK pathway has been shown to act as a negative regulator of Smad signaling. For example, recently Kretzschmar and colleagues (26) demonstrated that ERKs can phosphorylate serines in four PXSP motifs in the linker region of Smad1. This phosphorylation inhibits nuclear translocation of Smad1 and Smad1-induced transcription activation, suggesting that ERKs exert antagonistic effects on Smads. In a similar manner, oncogenic Ras or activated forms of MEK1, both of which activate ERKs, can lead to the phosphorylation of Smad2 and Smad3 in the linker region, thereby inhibiting nuclear accumulation of Smad2 and Smad3 and Smad-dependent transcription (27). Together, these studies show that in many instances, MAP kinases function upstream from Smads.
A few recent studies suggest that in some cells, MAP kinases may have a
distinct pathway from Smads, and both may act synergistically. In
HEK-293 cells, TGF- activated p38, which subsequently activated ATF-2 (35). In these cells, both Smads 3 and 4 and p38
synergistically enhanced the activity of ATF-2, which acted as their
common nuclear target. In HaCaT cells, constitutively active components
of the ERK pathway activated p21Cip1 expression, and
inhibitors or dominant negative constructs of the ERK pathway blocked
p21Cip1 induction by TGF-
, while having no effect on
Smad activity (25). In this case, ERKs could represent a
distinct, parallel pathway from Smad signaling in TGF-
regulation of
p21Cip1 or may function downstream of Smads.
The results of our study clearly demonstrate that ERK activation by
TGF- in pancreatic acinar cells is downstream from Smad4. These
findings represent a fairly unique mechanism of interaction of MAP
kinases and Smads in mediating TGF-
-induced signals. Thus far, only
one report from the literature has demonstrated that MAP kinases play a
role as downstream effectors of Smads. In C2C12 cells, TGF-
activation of the transcription factor ATF-2 is
dependent on p38, and p38 activation can be blocked by a dominant
negative form of Smad4. These data indicate that Smad4 is upstream of
p38 in these cells (18). Together, all of these studies
highlight the complexity of interactions between TGF-
-stimulated MAP
kinase pathways and Smads that vary depending on cellular context. Thus MAP kinases may act upstream of Smads or may represent distinct pathways that function independently and synergistically with Smads,
or, in some cases, as in pancreatic acinar cells, MAP kinases may
function downstream from Smads.
The contributions of MAP kinase signaling pathways to TGF--mediated
physiological responses are just beginning to be understood. It does
not appear that cell type-specific modulation of cell growth by TGF-
correlates with MAP kinase activity, based on several observations.
They include the fact that TGF-
-stimulated growth of Swiss 3T3
cells occurs without activation of MAP kinase, and TGF-
inhibition of EGF-stimulated proliferation of mouse keratinocytes
(PAM212) occurs without inhibiting EGF-induced MAP kinase activation
(8). This lack of correlation between growth effects and
effects on ERKs is unique for TGF-
, because for most, if not all,
other growth factors, including platelet-derived growth factor and EGF,
their ability to modulate growth is closely associated with MAP kinase activation.
Several studies have demonstrated the functional consequences of
TGF--induced MAP kinase activation in other cell types. For example,
TGF-
-stimulated activation of p38 in human neutrophils is necessary
for TGF-
-induced actin polymerization and chemotaxis (19). In a human fibrosarcoma-derived cell line, TGF-
induction of fibronectin expression is dependent on JNK activity, and
interestingly, independent of Smad4 (23). In untransformed
lung and intestinal epithelial cells, activation of the MAPK pathway by
TGF-
is required for TGF-
production (39), and, in
some cell types, the MAP kinase pathway is required for stimulation of
p21 by TGF-
(25).
MAP kinase activation may modulate TGF- signaling by selective
activation of specific transcription factors. We chose to investigate the functional consequences of TGF-
-induced ERK
activation in pancreatic acinar cells by examining the role of ERKs in
TGF-
's ability to activate the transcription factor AP-1. We
demonstrated that TGF-
activated AP-1 at 4 h, the same time
point at which TGF-
induced maximal activation of ERKs. We also
showed that AP-1 activation by TGF-
was dependent on ERK signaling.
TGF- is a known activator of the transcription factor AP-1, which is
comprised of homo- or heterodimers of Fos and Jun proteins. AP-1 has been shown to be an effector molecule for TGF-
-dependent transcription of a number of genes, including TGF-
1
(39), Smad7 (6), interleukin-6
(13), and type I collagen (14). Although AP-1
is known to mediate many of the cellular responses to TGF-
, the
mechanisms regulating AP-1 induction by TGF-
are incompletely understood. Our data indicates that ERK signaling is important for AP-1
activation by TGF-
in pancreatic acinar cells. This is supported by
two recent studies performed in human lung fibroblasts and intestinal
epithelial cells that demonstrated that TGF-
-induced activation of
AP-1 is dependent on ERKs (14, 39). Although the direct
relationship between Smads and ERKs was not evaluated in these two
studies, we demonstrated that ERK activation in pancreatic acinar cells
was regulated by Smad4.
In summary, we have demonstrated that TGF- activates the MAP kinases
ERKs and p38 in pancreatic acinar cells. The time course of MAP kinase
activation by TGF-
is different than that described in other cell
types, with maximal effects 4 h after treatment. The ability of
TGF-
to activate ERKs is dependent on Smad4, MEK1, and Ras. ERK
activation by TGF-
is important in the regulation of DNA binding of
the transcription factor AP-1. These data, together with previous data
from other groups, suggest that multiple mechanisms exist for
interactions between the MAP kinase signaling pathways and Smads. In
addition, Smads may play a role in more than TGF-
family signaling.
Because many stimuli activate the ERK pathway, Smads may be involved in
modulating the cellular response from diverse signals, including other
growth factors.
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ACKNOWLEDGEMENTS |
---|
We thank R. MacDonald for the rat elastase 1 promoter/enhancer and J. Massague for the plasmid containing the COOH-terminal truncated dominant negative Smad4.
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FOOTNOTES |
---|
This work was supported by the American College of Surgeons Faculty Research Fellowship and National Institute of Diabetes and Digestive and Kidney Diseases Grants 5P30-DK-34933 (to Michigan Gastrointestinal Peptide Center) and K08-DK-02637-01 (to D. M. Simeone).
Address for reprint requests and other correspondence: D. M. Simeone, TC 2922D, Box 0331, Univ. of Michigan Medical Center, 1500 E. Medical Center Dr., Ann Arbor, MI 48109 (E-mail: simeone{at}umich.edu).
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.
Received 19 June 2000; accepted in final form 20 February 2001.
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