Adenovirus-mediated gene transfer of dominant-negative Smad4 blocks TGF-beta signaling in pancreatic acinar cells

Lizhi Zhang2, Kathleen Graziano2, Trinh Pham2, Craig D. Logsdon1, and Diane M. Simeone2

Departments of 1 Physiology and 2 Surgery, University of Michigan Medical School, Ann Arbor, Michigan 48109


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Transforming growth factor-beta (TGF-beta ) is a potent inhibitor of pancreatic acinar cell growth. Smad4 is a central mediator in the TGF-beta signaling pathway. To study the effect of Smad4 on pancreatic growth, cell cycle protein expression, and the expression of a TGF-beta -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-beta 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-beta inhibition of [3H]thymidine incorporation, with maximal effects on day 3. AddnSmad4 also completely blocked TGF-beta -mediated growth inhibition in the presence of basic fibroblast growth factor. We next examined the effects of AddnSmad4 on TGF-beta -induced expression of the cell cycle regulatory proteins p21Cip1 and p27Kip1. TGF-beta induced upregulation of p21Cip1, which was completely blocked by AddnSmad4. AddnSmad4 also inhibited TGF-beta -induced expression of the TGF-beta -responsive luciferase reporter 3TP-Lux. These results show that Smad4 is essential in TGF-beta -mediated signaling in pancreatic acinar cells.

growth regulation; pancreas; cell cycle; Smad proteins


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

TRANSFORMING GROWTH FACTOR-beta (TGF-beta ) 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-beta has been demonstated to be an important inhibitor of acinar and ductal cell growth in vitro (2, 22). TGF-beta has also been shown to be important in maintaining differentiation of pancreatic exocrine and endocrine cells. In a whole organ culture system, TGF-beta had an inhibitory effect on acinar cell growth but also increased the number of islet cells differentiated from ductular-like structures, indicating that TGF-beta may have cell-specific effects on pancreatic differentiation (9, 31). Furthermore, transgenic mice expressing a dominant-negative TGF-beta 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-beta signaling pathway in the pancreas is underscored by the fact that perturbation of the TGF-beta signaling cascade in the pancreas has been associated with the development of pancreatic cancer (7, 8, 35, 36).

TGF-beta 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-beta 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-beta receptors has been described, the intracellular pathways by which the TGF-beta 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-beta family of serine/threonine receptors. Among them, Smad2 and Smad3 respond to TGF-beta . Smad2 and Smad3 are phosphorylated directly by TGF-beta -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-beta 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-beta signaling (5, 6, 10, 38), other recent reports have suggested that Smad4 is dispensible for TGF-beta signaling. For example, it has been found that in several cell lines, TGF-beta -mediated induction of fibronectin synthesis does not require Smad4 (13). In addition, TGF-beta 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-beta -mediated responses in normal pancreatic tissues remains to be determined.

In this study we sought to determine if dominant- negative Smad4 blocks TGF-beta -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-beta -mediated growth responses in pancreatic acinar cells. We demonstrated that Smad4 is required for TGF-beta -mediated growth inhibition and p21Cip1 induction in pancreatic acinar cells. Furthermore, we also showed that dominant-negative Smad4 inhibited TGF-beta -induced expression of the TGF-beta -responsive luciferase reporter 3TP-Lux. These data indicate that Smad4 is of central importance in mediating the effects of TGF-beta on acinar cells.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. The following reagents were purchased: TGF-beta 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-beta 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-beta -responsive 3TP-Lux luciferase reporter in pGL2 was described previously (33). A recombinant adenovirus expressing 3TP-Lux was generated by cutting the 3TP-Lux construct out of pGL2 with SalI and SmaI and ligating the 3TP-Lux construct into the shuttle vector pAdtrack cut with SalI and EcoRV. The resultant adenovirus was generated as described in the previous paragraph.

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-beta 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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Fig. 1.   Adenovirus-mediated gene delivery in primary pancreatic acinar cells. Isolated mouse pancreatic acini were infected with an adenoviral vector containing dominant-negative Smad4 driven by an elastase promoter and green fluorescent protein (GFP) driven by a separate cytomegalovirus (CMV) promoter [adenovirus containing dominant- negative COOH terminal truncated Smad4 (AddnSmad4)] at a titer of 106 plaque-forming units (pfu)/mg acinar protein for indicated times and examined. A: bright-field image 8 h after infection; B and C: fluorescent microscopic images of pancreatic acini infected for 8 h and 72 h, respectively. Representative acini are depicted.



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Fig. 2.   Time course of expression of dominant-negative Smad4 by adenoviral-mediated gene delivery (AddnSmad4). Acini were infected with 106 pfu/mg acinar protein of AddnSmad4 or adenovirus expressing GFP alone (AdGFP) for indicated times. Control acini were grown in enhanced media alone without addition of adenovirus. Acini were then lysed, and protein was harvested for Western blotting using an anti-Smad4 antibody. Results are of 2 separate experiments. d, Day.



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Fig. 3.   Effect of AddnSmad4 on transforming growth factor-beta (TGF-beta )-mediated growth inhibition in pancreatic acinar cells. Acinar cells infected with either no virus (control), AdGFP, or AddnSmad4, each at a titer of 106 pfu/mg acinar protein, were cultured for 1, 2, and 3 days in the presence of 100 pM TGF-beta . [3H]thymidine incorporation assays were performed, and values are a percentage of control. Samples were run in triplicate, and results are means ± SE for 3 experiments (*P < 0.05 vs. control at day 1; Dagger P < 0.05 vs. control at day 3).



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Fig. 4.   Effect of AddnSmad4 on TGF-beta -mediated growth inhibition in pancreatic acinar cells stimulated with basic fibroblast growth factor (bFGF). Acinar cells infected with either AddnSmad4 or AdGFP at a titer of 106 pfu/mg acinar protein were cultured for 3 days in the presence of 1 nM bFGF; 100 pM TGF-beta were added in combination where indicated. [3H]thymidine incorporation assays were performed, and values are a percentage of control. Samples were run in triplicate, and results are means ± SE for 3 separate experiments.



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Fig. 5.   Effect of adenoviral-mediated gene transfer of dominant- negative Smad4 on TGF-beta -induced expression of p21Cip1 in mouse pancreatic acini. Short-term cultures of pancreatic acini were transfected for 8 h with no virus (lanes 1, 2), AddnSmad4 (lanes 3, 4), or AdGFP (lanes 5, 6), each at a titer of 106 pfu/mg acinar protein. Cells were then treated with TGF-beta (100 pM) for 24 h. Cell lysates were prepared and submitted to Western blotting using anti-p21Cip1 or anti-p27Kip1 antibodies (A, B). C: p21Cip1 data in graph form. Values are a percentage of control, and results are of 3 separate experiments (*P < 0.05 vs. control).



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Fig. 6.   Effect of dominant-negative Smad4 on TGF-beta -induced expression of the 3TP-Lux luciferase reporter. Acini were coinfected with 3TP-Lux and either AddnSmad4 or AdGFP, each at a concentration of 106 pfu/mg acinar protein. Eight hours after infection 100 pM TGF-beta was added, and after 12 h luciferase activity was measured and normalized with GFP activity (see MATERIALS AND METHODS). Data are normalized, with the level of expression in the absence of ligand set to 1. Values were determined in triplicate and are means ± SE for 3 independent experiments. *P < 0.05 vs. control (no TGF-beta ).

For investigation of the effects of dominant-negative Smad4 on TGF-beta responsiveness using the 3TP-Lux luciferase reporter, acini were isolated and plated onto 12-well culture plates coated with air-dried rat tail collagen. Acini were coinfected with adenovirus expressing 3TP-Lux (Ad3TP-Lux) and either AddnSmad4 or AdGFP, each added at a titer of 106 pfu/mg protein for various times (see description in Fig. 1-6 legends).

[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-beta were added to ligand-treated groups, and TGF-beta -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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-beta 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-beta (22). To determine the role of Smad4 in TGF-beta -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-beta (100 pM) and incubated for 1, 2, and 3 days in standard culture media, and [3H]thymidine incorporation was determined. TGF-beta 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-beta -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-beta -mediated growth inhibition; therefore, the effects of AddnSmad4 were not due to adenoviral infection.

The ability of AddnSmad4 to block TGF-beta -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-beta (62 ± 3% vs. control; n = 3; P < 0.05). The ability of TGF-beta 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-beta 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-beta 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-beta 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-beta (100 pM) for 16 h. Western blotting was performed to examine levels of p21Cip1 and p27Kip1 protein. As shown in Fig. 5, TGF-beta (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-beta to induce p21Cip1 expression, but control AdGFP had no effect, demonstrating that Smad4 is necessary for TGF-beta -induced p21Cip1 expression in pancreatic acinar cells.

The role of Smad4 in TGF-beta -mediated signaling in pancreatic acinar cells was further demonstrated by examining the effect of dominant-negative Smad4 on TGF-beta -induced expression of 3TP-Lux, a luciferase reporter under the control of a TGF-beta 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-beta . 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-beta to activate 3TP-Lux, whereas AdGFP had no effect (Fig. 6).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The results of our study demonstrated that Smad4 is necessary for TGF-beta -mediated growth inhibition, p21Cip1 expression, and TGF-beta -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-beta -induced inhibition of both basal and bFGF-stimulated [3H]thymidine incorporation. AddnSmad4 also inhibited TGF-beta -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-beta signaling. For example, targeted deletion of Smad4 through homologous recombination in colorectal cancer HCT 116 cells abrogated signaling from TGF-beta and from the TGF-beta 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-beta -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 (Arg100right-arrowThr) 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-beta -mediated responses in certain cell types. In two Smad4-null pancreatic cancer cell lines, TGF-beta 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-beta responses in a Smad4-null breast cancer cell line studied in parallel. The growth inhibitory response to TGF-beta in the two Smad4-null pancreatic cell lines was dependent on an intact ras effector pathway. Hocevar et al. (13) have demonstrated that TGF-beta -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-beta -induced responses may reflect differences based on cell type and cellular environment. In addition, many studies investigating the TGF-beta signaling pathway have been performed in transformed cell lines that contain other genetic mutations that may cloud the analysis of study results.

TGF-beta 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-beta -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-beta 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-beta resulted in induction of p21Cip1 expression. In contrast, TGF-beta had no effect on levels of p27Kip1 in pancreatic acinar cells. TGF-beta 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-beta 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-beta 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-beta induces p21Cip1 in pancreatic acinar cells, we treated cells with a dominant-negative mutant of Smad4. We demonstrated that the ability of TGF-beta 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-beta 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-beta while having no effect on Smad activity. This suggests that although the ability of TGF-beta to induce p21Cip1 is dependent on Smad4 in some cells, in other cells, TGF-beta -induced p21Cip1 expression may occur through activation of the MAPK pathway.

To further strengthen the evidence that Smad4 is involved in TGF-beta -mediated responses in pancreatic acinar cells, we examined the effect of dominant-negative Smad4 on TGF-beta -induced activity of the TGF-beta -responsive 3TP-Lux luciferase reporter. The 3TP-Lux reporter is a sensitive indicator of TGF-beta signaling and has been used as a standard for assessing TGF-beta -dependent transcriptional responses. TGF-beta 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-beta by using 3TP-Lux. Several groups have shown that 3TP-Lux is not activated by TGF-beta 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-beta -induced 3TP-Lux response in Mv1Lu cells, whereas wild-type Smad4 restored TGF-beta -induced 3TP-Lux activity.

Understanding the molecular mechanisms of TGF-beta signaling in normal pancreatic cells may have important clinical implications. TGF-beta resistance occurs in many cancer types, including pancreatic cancer. Loss of the growth inhibitory response to TGF-beta 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-beta signaling pathway and Smad4 in the pancreas and suggest which TGF-beta -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-beta 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-beta signaling in normal pancreatic acinar cells. TGF-beta -induced growth inhibition and p21Cip1 induction were found to be dependent on Smad4. Smad4 was also required for activation of the TGF-beta -responsive 3TP-Lux reporter. These studies demonstrate that Smad4 is a critical and necessary component of the TGF-beta signaling pathway in acinar cells. Further understanding of the TGF-beta signaling pathway may provide insight into the pathological alterations that occur in pancreatic cancer.


    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. We also thank Dr. Vogelstein for use of the AdEasy system.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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[Abstract/Free Full Text].

4.   Datto, MB, Yong Y, and Wang XF. Functional analysis of the transforming growth factor beta  responsive elements in the WAF1/Cip1/p21 promoter. J Biol Chem 270: 28623-28628, 1995[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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-beta 1 (TGF-beta 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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

13.   Hocevar, BA, Brown TL, and Howe PH. TGF-beta induces fibronectin synthesis through a c-Jun N-terminal kinase-dependent, Smad4-independent pathway. EMBO J 18: 1345-1356, 1999[Abstract/Free Full Text].

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[Abstract/Free Full Text].

15.   Hu, PP, Datto MB, and Wang XF. Molecular mechanisms of transforming growth factor-beta signaling. Endocr Rev 19: 349-363, 1998[Abstract/Free Full Text].

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-beta . J Biol Chem 274: 35381-35387, 1999[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

21.   Le Dai, JL, Schutte M, Bansal RK, Wilentz RE, Sugar AY, and Kern SE. Transforming growth factor-beta 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-beta (TGF-beta 1) inhibits pancreatic acinar cell growth. Am J Physiol Gastrointest Liver Physiol 262: G364-G368, 1992[Abstract/Free Full Text].

23.   Macias-Silva, M, Abdollah S, Hoodless PA, Pirone R, Attisano L, and Wrana JL. MADR2 is a substrate of the TGFbeta receptor and its phosphorylation is required for nuclear accumulation and signaling. Cell 87: 1215-1224, 1996[ISI][Medline].

24.   Massague, J. TGF-beta 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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

28.   Ravitz, MJ, and Wenner CE. Cyclin-dependent kinase regulation during G1 phase and cell cycle regulation by TGF-beta . 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-beta . 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[Abstract/Free Full Text].

31.   Sanvito, F, Herrera PL, Montesano R, Orci L, and Vassalli JD. TGF-beta 1 influences the relative development of the exocrine and endocrine pancreas in vitro. Development 120: 3451-3462, 1994[Abstract/Free Full Text].

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 TGFbeta 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-beta 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 TGFbeta 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 beta  and activin signaling in colorectal cancer cells. Proc Natl Acad Sci USA 95: 2412-2416, 1998[Abstract/Free Full Text].


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