Departments of 1 Physiology and 2 Surgery, University of Michigan Medical School, Ann Arbor, Michigan 48109
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ABSTRACT |
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Transforming growth factor- (TGF-
) is a potent inhibitor
of pancreatic acinar cell growth. Smad4 is a central mediator in the
TGF-
signaling pathway. To study the effect of Smad4 on pancreatic growth, cell cycle protein expression, and the expression of a TGF-
-responsive promoter in vitro, we constructed an adenovirus containing dominant-negative COOH terminal truncated Smad4 (AddnSmad4) downstream of the rat elastase promoter. Acinar cells expressed dominant-negative Smad4 within 8 h after infection, and expression persisted for 72 h. Mouse pancreatic acini were infected with either AddnSmad4 or control adenovirus expressing green fluorescent protein, and TGF-
was added 8 h after infection. Acinar cells were then incubated for 1, 2, or 3 days, and
[3H]thymidine incorporation was determined. AddnSmad4
significantly reduced TGF-
inhibition of [3H]thymidine
incorporation, with maximal effects on day 3. AddnSmad4 also
completely blocked TGF-
-mediated growth inhibition in the presence
of basic fibroblast growth factor. We next examined the effects of
AddnSmad4 on TGF-
-induced expression of the cell cycle regulatory
proteins p21Cip1 and p27Kip1. TGF-
induced
upregulation of p21Cip1, which was completely blocked by
AddnSmad4. AddnSmad4 also inhibited TGF-
-induced expression of the
TGF-
-responsive luciferase reporter 3TP-Lux. These results show that
Smad4 is essential in TGF-
-mediated signaling in pancreatic acinar cells.
growth regulation; pancreas; cell cycle; Smad proteins
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INTRODUCTION |
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TRANSFORMING GROWTH
FACTOR- (TGF-
) is a member of a large family of
structurally related peptides that play a key role in regulating cell
fate by controlling a variety of biological processes including
cellular proliferation, differentiation, apoptosis, and
extracellular matrix production. In the pancreas, TGF-
has been
demonstated to be an important inhibitor of acinar and ductal cell
growth in vitro (2, 22). TGF-
has also been shown to be
important in maintaining differentiation of pancreatic exocrine and
endocrine cells. In a whole organ culture system, TGF-
had an
inhibitory effect on acinar cell growth but also increased the number
of islet cells differentiated from ductular-like structures, indicating
that TGF-
may have cell-specific effects on pancreatic differentiation (9, 31). Furthermore, transgenic mice
expressing a dominant-negative TGF-
type II receptor directed to
acinar cells exhibited increased proliferation of acinar cells with
altered differentiation to a more ductular-type phenotype
(3). The relevance of understanding the TGF-
signaling
pathway in the pancreas is underscored by the fact that perturbation of
the TGF-
signaling cascade in the pancreas has been associated with
the development of pancreatic cancer (7, 8, 35, 36).
TGF- signaling is initiated by interaction with two types of
transmembrane receptors (type I and type II) with serine/threonine kinase activity. Upon binding of TGF-
to type II receptors, type I
receptors are recruited and activated by phosphorylation, resulting in
propagation of downstream signaling. Although the molecular mechanism
of activation of these TGF-
receptors has been described, the
intracellular pathways by which the TGF-
signal is transduced from
the membrane to the nucleus have only recently begun to be defined.
Smad proteins have been identified as a family of highly conserved
intracellular proteins that function as signaling molecules downstream
from the TGF- family of serine/threonine receptors. Among them,
Smad2 and Smad3 respond to TGF-
. Smad2 and Smad3 are phosphorylated
directly by TGF-
-receptor complexes at the SSXS motif at the COOH
terminus of the proteins (23, 38). Phosphorylation of
Smad2 and Smad3 is followed by heteromerization with Smad4 and
subsequent accumulation of Smad complexes in the nucleus
(24). Smad4, which is not phosphorylated, acts as a common
mediator and is central to signaling pathways involving multiple
TGF-
family ligands. In the nucleus, Smad complexes activate
transcription of defined genes by cooperative interactions with various
other transcription factors and also by direct interactions with
promoter sequences. Of interest, Smad4 was originally identified as
deleted in pancreatic carcinoma locus 4 (DPC4), a tumor suppressor gene
that is inactivated either by deletion or by mutation in ~50%
pancreatic cancers (11). Interestingly, Smad4 is mutated in only a small proportion of other cancers (1, 18, 26, 32).
Although a number of studies have supported the critical role of Smad4
in TGF- signaling (5, 6, 10, 38), other recent reports
have suggested that Smad4 is dispensible for TGF-
signaling. For
example, it has been found that in several cell lines, TGF-
-mediated
induction of fibronectin synthesis does not require Smad4
(13). In addition, TGF-
responsiveness has been
identified in several pancreatic cancer cell lines that lack Smad4
(21, 33). On the basis of these studies, the role of Smad4
in TGF-
-mediated responses in normal pancreatic tissues remains to
be determined.
In this study we sought to determine if dominant- negative Smad4 blocks
TGF--mediated growth inhibitory effects on pancreatic acinar cells.
To examine the role of Smad4, we expressed a dominant-negative mutant
of Smad4 utilizing adenoviral-mediated gene transfer in vitro. We
hypothesized that Smad4 was necessary for TGF-
-mediated growth
responses in pancreatic acinar cells. We demonstrated that Smad4 is
required for TGF-
-mediated growth inhibition and p21Cip1
induction in pancreatic acinar cells. Furthermore, we also showed that
dominant-negative Smad4 inhibited TGF-
-induced expression of the
TGF-
-responsive luciferase reporter 3TP-Lux. These data indicate
that Smad4 is of central importance in mediating the effects of TGF-
on acinar cells.
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MATERIALS AND METHODS |
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Materials.
The following reagents were purchased: TGF-1 and basic fibroblast
growth factor (bFGF) from R & D Systems (Minneapolis, MN); chromatographically purified collagenase from Worthington Biochemical (Freehold, NJ); soybean trypsin inhibitor (SBTI) and
3-isobutyl-1-methylxanthine (IBMX) from Sigma (St. Louis, MO);
[3H]thymidine from Amersham (Arlington Heights, IL); all
other reagents were purchased from GIBCO (Grand Island, NY).
Preparation of acini.
The preparation of pancreatic acini was performed as previously
described (14). Briefly, pancreatic tissue was obtained from male Swiss Webster mice and digested with collagenase (100 U/ml)
and incubated at 37°C for 45 min with shaking (120 cycles/min). Acini
were then mechanically dispersed by trituration of tissue through
polypropylene pipettes of decreasing orifice size (3.0, 2.4, and 1.2 mm) and filtration through a 150-µm mesh nylon cloth. Acini were
purified by centrifugation at 50 g for 3 min through a
solution containing 4% bovine serum albumin and were resuspended in
enhanced media that consisted of DMEM containing 0.5% FBS, 100 U/ml
penicillin, 100 µg/ml streptomycin, 0.5 mM IBMX, and 0.1 mg/ml SBTI.
The dispersed acini were aliquoted in tissue culture dishes and treated
with TGF- and various treatments for specified times. Cells were
maintained in a humidified atmosphere of 5% CO2 in air at
37°C during incubation times.
Construction of adenoviral vectors. A recombinant adenovirus expressing a dominant-negative Smad4 was generated by subcloning an EcoRI-HindIII fragment containing the rat elastase I enhancer/promoter (gift of Ray MacDonald, UT Southwestern) into pCMV5/dnSmad4(1-514), which contains a COOH terminal truncated dominant-negative Smad4 gene (gift of J. Massague, Sloan-Kettering). The elastase promoter/Smad4(1-514) construct was then cut out of pCMV5 with EcoRI and SmaI and subcloned into a replication-defective adenoviral vector as described (12). Briefly, the dominant-negative Smad4 construct was bluntly ligated into the EcoRV site of the shuttle vector (pAdTrack), which has an independent cytomegalovirus-driven green fluorescent protein (GFP) expression construct as a marker. The resultant plasmid was linearized with PmeI and cotransfected into Escherichia coli BJ5183 cells with an adenoviral backbone plasmid pAdEasy-1. Recombinants were selected for kanamycin resistance and confirmed using multiple restriction enzyme digest analyses. The linearized recombinant was transfected into HEK-293 cells, where the recombinant adenovirus was generated and packaged. Recombinant adenovirus was harvested after 7-10 days, followed by subsequent viral purification on a cesium chloride gradient. GFP expression was used to assist in viral titering.
The TGF-Pancreatic acinar cell infection and culture.
Isolated acini were suspended in DMEM containing 0.5% FBS, 100 U/ml
penicillin, 100 µg/ml streptomycin, and 0.1 mg/ml SBTI. For growth
studies, acini were plated onto 24-well culture plates coated with
air-dried rat tail collagen. Adenovirus expressing dominant-negative
Smad4 (AddnSmad4) or control adenovirus expressing GFP alone (AdGFP)
was added at a titer of 106 plaque-forming units (pfu)/mg
acinar protein for various times (see description in Fig. 1-6
legends). TGF- and other treatments were added to the medium
as indicated. Infection efficency was evaluated by assessing GFP
expression using fluorescent microscopy.
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[3H]thymidine incorporation assay. The rate of DNA synthesis in cultured pancreatic acini was measured using a [3H]thymidine incorporation assay as previously described (27). Acini were exposed to growth factors for indicated times after which 0.1 µCi/ml of [3H]thymidine was added for an additional 24 h. Subsequently, the medium was removed and cells were precipitated with 10% TCA for 15 min on ice. The cells were then rinsed twice in ice-cold 10% TCA and dissolved in 0.1 N NaOH. Radioactivity in the dissolved cell pellet was measured by liquid scintillation counting. [3H]thymidine incorporation was expressed as a percentage of total counts per minute observed in control cells.
Immunoblot analysis. Dispersed acini were treated (for description see Fig. 1-6 legends). Whole cell lysates of pancreatic acini 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 PMSF). 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-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Dominant-negative Smad4 immunoblot analysis was performed using a monoclonal anti-Smad4 antibody, which recognizes both mutant and wild-type Smad4 (B-8) (Santa Cruz Biotechnology, Santa Cruz, CA). To evaluate levels of the cell cycle inhibitory proteins p21Cip1 and p27Kip1, we used anti-p21Cip1 and anti-p27Kip1 polyclonal antibodies (Santa Cruz Biotechnology). Images were visualized by using the enhanced chemiluminensce detection system (Amersham Biotech, Piscataway, NJ). Film images were scanned with an Agfa Arcus II scanner (Bayer Corp, Ridgefield Park, NJ) to create a digital image.
Luciferase assays.
Luciferase assays were performed as previously described
(33). Eight hours after adenoviral infection 100 pM of
TGF- were added to ligand-treated groups, and TGF-
-induced
luciferase activity was assayed after 12 h. Luciferase activities
were determined in triplicate transfections and normalized to GFP
expression levels using fluorescent microscopy. GFP expression was
determined by counting the number of green fluorescent cells in three
separate high-powered fields and dividing this number by the total
number of cells in those three fields. Normalized luciferase activity was calculated as measured luciferase activity/GFP expression.
Data analysis. Data are expressed as means ± SE. Where appropriate, significance of difference between means was analyzed by ANOVA. P < 0.05 was considered significant.
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RESULTS |
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To examine the role of Smad4 in mouse pancreatic acini, we
expressed a dominant-negative mutant of Smad4 utilizing
adenoviral-mediated gene transfer in vitro. When truncated at its COOH
terminus, Smad4 has been shown to act as a dominant-negative inhibitor
of the normal TGF- response (38). We created a
construct by using a pancreatic tissue-specific elastase I
promotor/enhancer to direct dominant-negative Smad4 expression to
pancreatic acini. To determine the efficiency of adenoviral-mediated
gene delivery to pancreatic acinar cells in vitro, isolated mouse
pancreatic acini were treated with AddnSmad4, which contains GFP driven
by a separate cytomegalovirus promotor as a marker. Acini were cultured
for time periods ranging from 8 to 72 h and observed using
fluorescent microscopy. A bright-field image obtained 8 h after
adenoviral infection revealed healthy-appearing pancreatic acini (Fig.
1A). Adenoviral infection lead
to a titer-dependent increase in acinar cells expressing GFP. At a
titer of 106 pfu/mg acinar protein (approximate
multiplicity of infection of 10 viral particles/cell), ~90% of the
acini were found to be infected, similar to what we have previously
reported (27). Acini demonstrate green fluorescence
as early as 8 h after adenoviral infection, indicating GFP
expression (Fig. 1B). GFP expression remained elevated
72 h after adenoviral infection, at which point the acini begin to
grow and divide (Fig. 1C).
We next determined the level of dominant-negative Smad4 expression in infected acinar cells by Western blotting by using an anti-Smad4 antibody that recognizes both wild-type Smad4 and the COOH terminal truncated Smad4 mutant. Significant expression of dominant-negative Smad4 was evident after 1 day, was maximal at 2 days, and persisted at a high level for 3 days (Fig. 2). Specificity was confirmed by infecting acini with AdGFP. Faint bands were present in control and AdGFP-treated cells, indicating that the level of endogenous Smad4 in pancreatic acini was low.
It has been shown previously that growth of mouse pancreatic acini in
vitro can be inhibited by TGF- (22). To determine the
role of Smad4 in TGF-
-mediated growth inhibition in pancreatic acinar cells, isolated mouse pancreatic acini were given no adenovirus (control) or were infected with either AddnSmad4 or AdGFP (both at
106 pfu/mg acinar protein) for 8 h. Acini were then
treated with TGF-
(100 pM) and incubated for 1, 2, and 3 days in
standard culture media, and [3H]thymidine incorporation
was determined. TGF-
inhibited basal [3H]thymidine
incorporation, with a maximal effect on day 3 (56 ± 7% of control on day 1; n = 3;
P < 0.05; Fig. 3).
AddnSmad4 blocked TGF-
-mediated growth inhibition, achieving
statistical significance on the third day (124 ± 17 vs. 56 ± 7% of control at time 0; n = 3;
P < 0.05). AdGFP had no effect on TGF-
-mediated growth inhibition; therefore, the effects of AddnSmad4 were not due to
adenoviral infection.
The ability of AddnSmad4 to block TGF--mediated growth inhibition in
pancreatic acinar cells in the presence of a growth stimulus was also
examined. Treatment of acinar cells with bFGF (1 nM) for a 3-day period
resulted in stimulation of [3H]thymidine incorporation at
142 ± 30% of control values (P < 0.05;
n = 3; Fig. 4).
[3H]thymidine incorporation was blocked below control
levels when cells were coincubated with 100 pM TGF-
(62 ± 3%
vs. control; n = 3; P < 0.05). The
ability of TGF-
to block the growth stimulatory effects of bFGF was
abrogated by infection with AddnSmad4 (151 ± 30% of control;
n = 3; P < 0.05), whereas control
AdGFP had no effect.
In many cell types, TGF- has been shown to inhibit cell growth by
increasing expression of the cyclin-dependent kinase (CDK) inhibitor
p21Cip1, which in turn inhibits the enzymatic activities of
cyclin D-CDK4/6 and cyclin E-CDK2 complexes, leading to
cell cycle arrest at the late phase of G1
(19). TGF-
has also been demonstrated to inhibit proliferation in some cell types by inducing expression of the CDK
inhibitor p27Kip1, which binds to and inhibits the
activity of cyclin E-CDK2 (15). To evaluate the
ability of TGF-
to induce expression of the cell cycle inhibitory
proteins p21Cip1 and p27Kip1 and examine the
role of Smad4 in this process, isolated acinar cells were infected with
no virus (control), AddnSmad4, or AdGFP for 8 h and then treated
with TGF-
(100 pM) for 16 h. Western blotting was performed to
examine levels of p21Cip1 and p27Kip1 protein.
As shown in Fig. 5, TGF-
(100 pM)
stimulated the expression of p21Cip1 1.7 ± 0.05-fold
compared with control (n = 3; P < 0.05) but did not induce expression of p27Kip1. AddnSmad4
completely inhibited the ability of TGF-
to induce p21Cip1 expression, but control AdGFP had no effect,
demonstrating that Smad4 is necessary for TGF-
-induced
p21Cip1 expression in pancreatic acinar cells.
The role of Smad4 in TGF--mediated signaling in pancreatic acinar
cells was further demonstrated by examining the effect of
dominant-negative Smad4 on TGF-
-induced expression of 3TP-Lux, a
luciferase reporter under the control of a TGF-
signal-responsive promoter. The 3TP-Lux construct contains three repeats of a
12-O-tetradecanoylphorbol 13-acetate promoter linked to a
luciferase reporter gene (37). Infection with Ad3TP-Lux
resulted in a clear activation of luciferase reporter activity in
response to TGF-
. To determine if this response was Smad4 dependent,
we evaluated reporter gene activation after cotransfection with
either AddnSmad4 or control AdGFP. AddnSmad4 inhibited
the ability of TGF-
to activate 3TP-Lux, whereas AdGFP had no effect
(Fig. 6).
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DISCUSSION |
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The results of our study demonstrated that Smad4 is
necessary for TGF--mediated growth inhibition,
p21Cip1 expression, and TGF-
-responsive 3TP-Lux activity
in normal pancreatic acinar cells. Infection of pancreatic acinar cells
with an adenoviral vector containing a dominant-negative COOH terminal
truncated Smad4 resulted in high levels of dominant-negative Smad4
expression. AddnSmad4 blocked TGF-
-induced inhibition of both basal
and bFGF-stimulated [3H]thymidine incorporation.
AddnSmad4 also inhibited TGF-
-induced p21 expression and 3TP-Lux
luciferase activity in pancreatic acini.
These results are consistent with a number of reports in the literature
demonstrating that Smad4 is necessary for TGF- signaling. For
example, targeted deletion of Smad4 through homologous recombination in
colorectal cancer HCT 116 cells abrogated signaling from TGF-
and
from the TGF-
family member activin (39). Two studies
examining both transient and stable transfection of Smad4 into a
Smad4-null cell line (MDA-MB468 breast carcinoma cells) revealed that
transfection of wild-type Smad4 restored both growth inhibition and
induction of a TGF-
-sensitive reporter construct (5,
6). In addition, Le Dai et al. (20) recently
reported the development of an inducible system in which wild-type
Smad4 or a Smad4 containing a tumor-derived mutation was activated when
cell lines stably expressing wild-type or mutant Smad4 proteins fused
to a murine estrogen receptor domain were treated with
4-hydroxytamoxifen. Nuclear translocation of wild-type Smad4 resulted
in a decrease in growth rate, attributable to cell cycle arrest at the
G1 phase and induction of apoptosis, whereas
nuclear translocation of Smad4 containing a tumor-derived mutation
critical for DNA binding (Arg100
Thr) demonstrated an
"oncogenic" phenotype, with an increased number of cells in S phase
and decreased apoptosis.
In contrast, it has recently been reported that Smad4 is not necessary
for some TGF--mediated responses in certain cell types. In two
Smad4-null pancreatic cancer cell lines, TGF-
growth inhibitory and
transcriptional responses were found to be Smad4 independent (21,
33). In one of these studies, this observation contrasted with
absolute Smad4 dependence of TGF-
responses in a Smad4-null breast
cancer cell line studied in parallel. The growth inhibitory response to
TGF-
in the two Smad4-null pancreatic cell lines was dependent on an
intact ras effector pathway. Hocevar et al. (13) have demonstrated that TGF-
-induced fibronectin
synthesis in human fibrosarcoma cells occurs through a c-Jun
NH2 terminal kinase-dependent, Smad4-independent pathway.
These variations in Smad4 dependence of TGF-
-induced responses may
reflect differences based on cell type and cellular environment. In
addition, many studies investigating the TGF-
signaling pathway have
been performed in transformed cell lines that contain other genetic
mutations that may cloud the analysis of study results.
TGF- has been shown to inhibit cell proliferation by causing growth
arrest in the G1 phase of the cell cycle. Progression through G1 is dependent on formation, activation, and
subsequent deactivation of G1 cyclin-CDK complexes,
primarily cyclin D-CDK4 and cyclin E-CDK2 complexes (15,
28). TGF-
-induced G1 cell cycle arrest has been
attributed, at least in part, to its regulatory effects on the
activities of cyclin-dependent kinase inhibitors, including
p21Cip1 and p27Kip1, and on the levels
and activities of the G1 cyclins and CDKs (15,
28). TGF-
has been shown to induce p21Cip1
expression in a number of different cell types, including human prostate epithelial cells, primary cultured rat hepatocytes, and HaCaT
cells (30, 34, 4). In the current study, treatment of
pancreatic acinar cells with TGF-
resulted in induction of p21Cip1 expression. In contrast, TGF-
had no effect on
levels of p27Kip1 in pancreatic acinar cells. TGF-
has
been reported to induce p27Kip1 expression in some cell
types, including human prostate epithelial cells and murine B cell
lines (30, 17) but not in others (34, 29). In
the latter, TGF-
does not increase expression of p27Kip1
but rather regulates p27Kip1 by affecting its ability to
bind to and inactivate cyclin E-CDK2 complexes in G1. The
ability of TGF-
to regulate p27Kip1 binding and
inactivation of cyclin E-CDK2 complexes in pancreatic acinar cells was
not addressed in the current study.
To further define the signaling pathways through which TGF-
induces p21Cip1 in pancreatic acinar cells, we treated
cells with a dominant-negative mutant of Smad4. We demonstrated that
the ability of TGF-
to induce p21Cip1 expression is
dependent on Smad4. This is consistent with recent work that has shown
that Smad4 can activate p21Cip1 gene transcription in human
hepatoma Hep G2 cells via functional interactions with the
transcription factor Sp1 (25). It is of note, however,
that TGF-
has also been shown to induce p21Cip1
expression by signaling pathways other than the Smads. Hu et al.
(16) demonstrated that in human keratinocyte HaCaT cells, constitutively active components in the MAPK pathway activate p21Cip1 expression and inhibitors or dominant-negative
constructs for the MAPK pathway significantly decrease
p21Cip induction by TGF-
while having no effect on Smad
activity. This suggests that although the ability of TGF-
to induce
p21Cip1 is dependent on Smad4 in some cells, in other
cells, TGF-
-induced p21Cip1 expression may occur through
activation of the MAPK pathway.
To further strengthen the evidence that Smad4 is involved in
TGF--mediated responses in pancreatic acinar cells, we examined the
effect of dominant-negative Smad4 on TGF-
-induced activity of the
TGF-
-responsive 3TP-Lux luciferase reporter. The 3TP-Lux reporter is
a sensitive indicator of TGF-
signaling and has been used as a
standard for assessing TGF-
-dependent transcriptional responses.
TGF-
induced luciferase activity in pancreatic acini infected with
an adenovirus expressing 3TP-Lux, which was inhibited by coexpression
of dominant-negative Smad4 but not control GFP adenovirus. This is the
first reported use of a reporter assay to investigate signaling
pathways in primary pancreatic cells. These results are consistent with
those published in the literature examining the role of Smad4 in
TGF-
by using 3TP-Lux. Several groups have shown that 3TP-Lux is not
activated by TGF-
in Smad4-deficient cell lines, but this signaling
pathway is restored upon reintroduction of Smad4 (5, 6,
33). In a separate study, Zhang et al. (38)
demonstrated that overexpression of COOH terminal truncated dominant-negative Smad4 inhibited the normal TGF-
-induced 3TP-Lux response in Mv1Lu cells, whereas wild-type Smad4 restored
TGF-
-induced 3TP-Lux activity.
Understanding the molecular mechanisms of TGF- signaling in
normal pancreatic cells may have important clinical implications. TGF-
resistance occurs in many cancer types, including pancreatic cancer. Loss of the growth inhibitory response to TGF-
at the cellular level is likely an important step in malignant progression. Several lines of evidence indicate that Smad4 acts as a tumor suppressor gene. The data presented in this study demonstrate the
functional relationship between the TGF-
signaling pathway and Smad4
in the pancreas and suggest which TGF-
-mediated responses are likely
to be affected by mutations in Smad4. These studies implicate Smad4,
with its high frequency of deletion or mutation in pancreatic cancer,
as a potential therapeutic target to restore TGF-
responsiveness,
and hence growth control, to pancreatic tumor cells.
In summary, we have utilized adenoviral technology as a tool to
decipher the mechanisms of TGF- signaling in normal pancreatic acinar cells. TGF-
-induced growth inhibition and p21Cip1
induction were found to be dependent on Smad4. Smad4 was also required
for activation of the TGF-
-responsive 3TP-Lux reporter. These
studies demonstrate that Smad4 is a critical and necessary component of
the TGF-
signaling pathway in acinar cells. Further understanding of
the TGF-
signaling pathway may provide insight into the pathological
alterations that occur in pancreatic cancer.
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ACKNOWLEDGEMENTS |
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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. We also thank Dr. Vogelstein for use of the AdEasy system.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant K08-DK-02637-1 and funds from the University of Michigan Gastrointestinal Peptide Center (National Institute of Diabetes and Digestive and Kidney Diseases Grant 5-P30-DK-34933). D. Simeone is also a recipient of an American College of Surgeons Faculty Research Fellowship Award.
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 17 March 2000; accepted in final form 22 January 2001.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Barrett, MT,
Schutte M,
Kern SE,
and
Reid BJ.
Allelic loss and mutational analysis of the DPC4 gene in esophageal carcinoma.
Cancer Res
56:
4351-4353,
1996[Abstract].
2.
Bisgaard, HC,
and
Thorgeirsson SS.
Evidence for a common cell of origin for primitive epithelial cells isolated from rat liver and pancreas.
J Cell Physiol
147:
333-343,
1991[ISI][Medline].
3.
Bottinger, E,
Jakubczak JL,
Roberts IS,
Mumy M,
Hemmati P,
Bagnall K,
Merlino G,
and
Wakefield LM.
Expression of a dominant-negative mutant TGF-beta type II receptor in transgenic mice reveals essential roles for TGF-beta in regulation of growth and differentiation in the exocrine pancreas.
EMBO J
16:
2621-2633,
1997
4.
Datto, MB,
Yong Y,
and
Wang XF.
Functional analysis of the transforming growth factor responsive elements in the WAF1/Cip1/p21 promoter.
J Biol Chem
270:
28623-28628,
1995
5.
De Caestecker, MP,
Hemmati P,
Larisch-Bloch S,
Ajmera R,
Roberts AB,
and
Lechleider RJ.
Characterization of functional domains within smad4/DPC4.
J Biol Chem
272:
13690-13696,
1997
6.
DeWinter, JP,
Roelen BAJ,
ten Dijke P,
van der Burg B,
and
van den Eijnden-van Raaij AJM
DPC4 ( SMAD4) mediates transforming growth factor-1 (TGF-
1) induced growth inhibition and transcriptional response in breast tumour cells.
Oncogene
14:
1891-1899,
1997[ISI][Medline].
7.
Friess, H,
Yamanaka Y,
Buchler M,
Ebert M,
Beger HG,
Gold LI,
and
Korc M.
Enhanced expression of transforming growth factor beta isoforms in pancreatic cancer correlates with decreased survival.
Gastroenterology
105:
1846-1856,
1993[ISI][Medline].
8.
Freeman, JW,
Mattingly CA,
and
Strodel WE.
Increased tumorigenicity in the human pancreatic cell line MIA PaCa-2 is associated with aberrant regulation of an IGF-1 autocrine loop and lack of expression of the TGF-beta type RII receptor.
J Cell Physiol.
165:
155-163,
1995[ISI][Medline].
9.
Gittes, GK,
Galante PE,
Hanahan D,
Rutter WJ,
and
Debas HT.
Lineage-specific morphogenesis in the developing pancreas: role of mesenchymal factors.
Development
122:
439-447,
1996
10.
Grau, AM,
Zhang L,
Wang W,
Evans DB,
Abbruzzese L,
Zhang W,
and
Chiao PJ.
Induction of p21waf1 expression and growth inhibition by transforming growth factor beta involve the tumor suppressor gene DPC4 in human pancreatic adenocarcinoma cells.
Cancer Res
57:
3929-3934,
1997[Abstract].
11.
Hahn, SA,
Schutte M,
Hoque AT,
Moskaluk CA,
da Costa LT,
Rozenblum E,
Weinstein CL,
Fischer A,
Yeo CJ,
Hruban RH,
and
Kern SE.
DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1.
Science
271:
350-353,
1996[Abstract].
12.
He, TC,
Zhou S,
da Costa LT,
Yu J,
Kinzler KW,
and
Vogelstein B.
A simplified system for generating recombinant adenoviruses.
Proc Natl Acad Sci USA
95:
2509-2514,
1998
13.
Hocevar, BA,
Brown TL,
and
Howe PH.
TGF- induces fibronectin synthesis through a c-Jun N-terminal kinase-dependent, Smad4-independent pathway.
EMBO J
18:
1345-1356,
1999
14.
Hoshi, H,
and
Logsdon CD.
Both low and high affinity CCK receptor states mediate trophic effects on pancreatic acinar cells.
Am J Physiol Gastrointest Liver Physiol
265:
G1177-G1181,
1993
15.
Hu, PP,
Datto MB,
and
Wang XF.
Molecular mechanisms of transforming growth factor- signaling.
Endocr Rev
19:
349-363,
1998
16.
Hu, PP,
Shen X,
Huang D,
Liu Y,
Counter C,
and
Wang XF.
The MEK pathway is required for stimulation of p21WAF1/CIP1 by transforming growth factor-.
J Biol Chem
274:
35381-35387,
1999
17.
Kamesaki, H,
Nishizawa K,
Michaud GY,
Cossman J,
and
Kiyono T.
TGF-beta induces the cyclin-dependent kinase inhibitor p27Kip1 mRNA and protein in murine B cells.
J Immunol
160:
770-777,
1998
18.
Kim, SK,
Fan Y,
Papadimitrakopoulou V,
Clayman G,
Hittelman WN,
Hong WK,
Lotan R,
and
Mao L.
DPC4, a candidate tumor suppressor gene, is altered infrequently in head and neck squamous carcinoma.
Cancer Res
56:
2519-2521,
1996[Abstract].
19.
Laiho, M,
Decaprio JA,
Ludlow JW,
Livingston DM,
and
Massague J.
Growth inhibition by TGF-beta linked to suppression of retinoblastoma protein phosphorylation.
Cell
62:
175-185,
1990[ISI][Medline].
20.
Le Dai, J,
Bansal RK,
and
Kern SE.
G1 cell cycle arrest and apoptosis induction by nuclear Smad4/Dpc4: phenotypes reversed by a tumorigenic mutation.
Proc Natl Acad Sci USA
96:
1427-1432,
1999
21.
Le Dai, JL,
Schutte M,
Bansal RK,
Wilentz RE,
Sugar AY,
and
Kern SE.
Transforming growth factor- responsiveness in DPC4/SMAD4-null cancer cells.
Mol Carcinog
26:
37-43,
1999[ISI][Medline].
22.
Logsdon, CD,
Keyes L,
and
Beauchamp RD.
Transforming growth factor- (TGF-
1) inhibits pancreatic acinar cell growth.
Am J Physiol Gastrointest Liver Physiol
262:
G364-G368,
1992
23.
Macias-Silva, M,
Abdollah S,
Hoodless PA,
Pirone R,
Attisano L,
and
Wrana JL.
MADR2 is a substrate of the TGF receptor and its phosphorylation is required for nuclear accumulation and signaling.
Cell
87:
1215-1224,
1996[ISI][Medline].
24.
Massague, J.
TGF- signal transduction.
Annu Rev Biochem
67:
753-791,
1998[ISI][Medline].
25.
Moustakas, A,
and
Kardassis D.
Regulation of the human p21/WAF1/Cip1 promoter in hepatic cells by functional interactions between Sp1 and Smad family members.
Proc Natl Acad Sci USA
95:
6733-6738,
1998
26.
Nagatake, M,
Takagi Y,
Osada H,
Uchida K,
Mitsudomi T,
Saji S,
Shimokata K,
Takahashi T,
and
Takahashi T.
Somatic in vivo alterations of the DPC4 gene at 18q21 in human lung cancers.
Cancer Res
56:
2718-2720,
1996[Abstract].
27.
Nicke, B,
Tseng MJ,
Fenrich M,
and
Logsdon CD.
Adenovirus-mediated gene transfer of RasN17 inhibits specific CCK actions on pancreatic acinar cells.
Am J Physiol Gastrointest Liver Physiol
276:
G499-G506,
1999
28.
Ravitz, MJ,
and
Wenner CE.
Cyclin-dependent kinase regulation during G1 phase and cell cycle regulation by TGF-.
Adv Cancer Res
71:
165-207,
1997[ISI][Medline].
29.
Reynisdottir, I,
Polyak K,
Iavarone A,
and
Massague J.
Kip/Cip and Ink4 Cdk inhibitors cooperate to induce cell cycle arrest in response to TGF-.
Genes Dev
9:
1831-1845,
1995[Abstract].
30.
Robson, CN,
Gnanapragasam V,
Byrne RL,
Collins AT,
and
Neal DE.
Transforming growth factor-beta 1 up-regulates p15, p21, p27 and blocks cell cycling in G1 in human prostate epithelium.
J Endocrinol
160:
257-266,
1999
31.
Sanvito, F,
Herrera PL,
Montesano R,
Orci L,
and
Vassalli JD.
TGF-1 influences the relative development of the exocrine and endocrine pancreas in vitro.
Development
120:
3451-3462,
1994
32.
Schutte, M,
Hruban RH,
Hedrick L,
Cho KR,
Nadasdy GM,
Weinstein CL,
Bova GS,
Isaacs WB,
Cairns P,
Nawroz H,
Sidransky D,
Casero RA,
Meltzer PS,
Hahn SA,
and
Kern SE.
DPC4 gene in various tumour types.
Cancer Res
56:
2527-2530,
1996[Abstract].
33.
Simeone, DM,
Pham T,
and
Logsdon CD.
Disruption of TGF signaling pathways in human pancreatic cancer cells.
Ann Surg
232:
73-80,
2000[ISI][Medline].
34.
Sugiyama, A,
Nagaki M,
Shidoji Y,
Moriwaki H,
and
Muto Y.
Regulation of cell cycle-related genes in rat hepatocytes by transforming growth factor beta 1.
Biochem Biophys Res Commun
238:
539-543,
1997[ISI][Medline].
35.
Venkatasubbarao, K,
Ahmed MM,
Swiderski C,
Harp C,
Lee EY,
McGrath P,
Mohiuddin M,
Strodel W,
and
Freeman JW.
Novel mutations in the polyadenine tract of the transforming growth factor type II receptor gene are found in a subpopulation of human pancreatic adenocarcinomas.
Genes Chromosomes Cancer
22:
138-144,
1998[ISI][Medline].
36.
Villanueva, A,
Garcia C,
Paules AB,
Vicente M,
Megias M,
Reyes G,
de Villalonga P,
Agell N,
Lluis F,
Bachs O,
and
Capella G.
Disruption of the antiproliferative TGF- signaling pathways in human pancreatic cancer cells.
Oncogene
17:
1969-1978,
1998[ISI][Medline].
37.
Wrana, JL,
Attisano L,
Carcamo J,
Zentella A,
Doody J,
Laiho M,
Wang XF,
and
Massague J.
TGF beta signals through a heteromeric protein kinase reporter complex.
Cell
71:
1003-1014,
1992[ISI][Medline].
38.
Zhang, Y,
Feng XH,
Wu RY,
and
Derynck R.
Receptor-associated Mad homologues synergize as effectors of the TGF response.
Nature
383:
168-172,
1996[ISI][Medline].
39.
Zhou, S,
Buckhaults P,
Zawel L,
Bunz F,
Riggins G,
Le Dai JL,
Kern SE,
Kinzler KW,
and
Vogelstein B.
Targeted deletion of Smad4 shows it is required for transforming growth factor and activin signaling in colorectal cancer cells.
Proc Natl Acad Sci USA
95:
2412-2416,
1998