Smad4 mediates activation of mitogen-activated protein kinases by TGF-beta in pancreatic acinar cells

Diane M. Simeone1, Lizhi Zhang1, Kathleen Graziano1, Barbara Nicke2, Trinh Pham1, Claus Schaefer2, and Craig D. Logsdon2

Departments of 1 Surgery and 2 Physiology, 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 ) inhibits pancreatic acinar cell growth. In many cell types, TGF-beta 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-beta and MAP kinase signaling pathways in isolated rat pancreatic acinar cells. TGF-beta 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-beta was maximal 4 h after treatment. The ability of TGF-beta to activate ERKs was concentration dependent and dependent on protein synthesis. TGF-beta '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-beta to activate acinar cell MAP kinase, demonstrating that this activation is downstream of Smads. The biological relevance of ERK activation by TGF-beta was indicated by demonstrating that inhibition of ERK signaling by PD-98059 blocked the ability of TGF-beta to activate the transcription factor activator protein-1. These studies provide new insight into the signaling mechanisms by which TGF-beta mediates biological actions in pancreatic acinar cells.

transforming growth factor-beta


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

TRANSFORMING GROWTH FACTOR-beta (TGF-beta ) is a ubiquitous growth factor that plays a key role in regulation of growth and differentiation in a wide variety of cell types. TGF-beta has been demonstrated to be an important inhibitor of pancreatic growth in vitro and in vivo. In cultured pancreatic acinar cells, TGF-beta inhibited basal cell growth and growth stimulated by a variety of trophic factors, including cholecystokinin (CCK) and epidermal growth factor (EGF) (28). TGF-beta has also been shown to inhibit pancreatic duct cell proliferation (4). Studies performed using a pancreatic whole organ culture system demonstrated that TGF-beta is important in maintaining early differentiation of both pancreatic exocrine and endocrine cells (36). The role of TGF-beta in pancreatic differentiation and growth has also been examined using transgenic animals. In transgenic mice expressing a dominant negative TGF-beta type II receptor targeted to acinar cells, which results in resistance to TGF-beta -mediated signals, the pancreas showed increased proliferation and dedifferentiation of abnormally proliferating acinar cells (5). Interestingly, disruption of the TGF-beta 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-beta 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-beta is a member of a large family of structurally related growth factors, including activin, inhibin, and bone morphogenic proteins (30). TGF-beta 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-beta .

Smad proteins have been identified as key signaling molecules in TGF-beta -related signaling pathways. Smad2 and Smad3 are specific to the TGF-beta signaling pathway and are phosphorylated by activated TGF-beta 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-beta 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-beta 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-beta 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-beta signaling pathway have been conducted in transformed tissue culture cells, it is known that TGF-beta 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-beta 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-beta signaling and MAP kinases in pancreatic acinar cells. In this study, we found that TGF-beta activated the MAP kinases ERKs and p38 but not JNKs. The effect of TGF-beta on MAP kinases was concentration dependent and occurred with a time course different from CCK and different from that reported for TGF-beta -induced MAP kinase activation in other cell types. TGF-beta -induced activation of ERK required protein synthesis and was dependent on both Ras and MAP kinase kinase 1 (MEK1). The effects of TGF-beta 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-beta was proven by demonstrating that blockade of ERK signaling by PD-98059 inhibited the ability of TGF-beta to induce DNA binding of the transcription factor activator protein-1 (AP-1).


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. The following reagents were purchased: TGF-beta 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-beta 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 [gamma -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 [gamma -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-beta , 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 [gamma -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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To determine the effects of TGF-beta on the activity of MAP kinases in pancreatic acinar cells, isolated acini were treated with TGF-beta for various times. TGF-beta (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-beta 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-beta (100 pM) to activate ERKs and p38, with significant effects at 4 h (Fig. 2, A and B).


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Fig. 1.   Effect of transforming growth factor-beta (TGF-beta ) and cholecystokinin (CCK) on the phosphorylation of mitogen-activated protein kinases (MAP kinases or MAPK) in rat pancreatic acini. Acini were treated with 100 pM TGF-beta or 1 nM CCK octapeptide (CCK-8) for the indicated periods of time. Cells were then lysed, and cell lysates (30 µg protein) were resolved on SDS-PAGE, blotted, and probed with anti-phospho MAP kinase antibodies, followed by enhanced chemiluminescence detection. Arrows indicate phosphorylated (activated) forms of p38 (A); extracellular signal-related kinases (ERKs; B; p42MAPK and p44MAPK); and c-jun NH2-terminal kinase (JNK; C; p46JNK and p55JNK). Data shown are representative of 5 separate experiments.



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Fig. 2.   Effect of TGF-beta on ERK and p38 activity by kinase assays. Pancreatic acini were treated with 100 pM TGF-beta or 1 nM CCK-8 for the indicated periods of time. A: in-gel ERK assays were performed as described in MATERIALS AND METHODS using myelin basic protein as a substrate. B: p38 in vitro kinase assays were performed as described using glutathione S-transferase-activating transcription factor-2 as a substrate. The data presented are representative of 2 experiments, each performed in duplicate.

Subsequently, studies were concentrated on specifically examining the mechanisms involved in the effect of TGF-beta on ERKs. An extended time course indicated that TGF-beta 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-beta 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-beta 's effect was then analyzed. Pancreatic acini were incubated for 4 h with different concentrations of TGF-beta , and ERK activity was assessed. TGF-beta stimulated ERKs in a concentration-dependent manner (Fig. 3C). The minimal response of MAP kinase to TGF-beta stimulation was observed at a dose of 10 pM, while maximal responses were observed at 100 pM.


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Fig. 3.   Time- and concentration-dependent effects of TGF-beta on ERK activity in rat pancreatic acini. A: acini were treated with 100 pM TGF-beta for indicated periods of time. B: acini were treated with CCK (1 nM) for indicated periods of time. C: acini were incubated with TGF-beta at increasing concentrations (1-100 pM) for 4 h. Cell lysates were prepared and submitted to Western blotting with anti-phospho (P) ERK antibody. The membrane was stripped and reprobed with an antibody to the nonphosphorylated (nonactive) form of ERK to serve as a control for protein loading in C.

To further understand the mechanisms involved in the effects of TGF-beta 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-beta (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-beta (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-beta . As shown in Fig. 4B, PD-98059 blocked the ability of TGF-beta to activate ERKs when added as late as 3 h after addition of TGF-beta . These data demonstrated that the ability of TGF-beta to activate ERKs was dependent on MEK1 and indicated that the activity of MEK1 is required at a late time after TGF-beta stimulation. Pretreatment of acini with PD-98059 had no effect on the ability of TGF-beta to induce p38 activity (data not shown), supporting the use of PD-98059 as a specific MEK1/ERK pathway inhibitor.


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Fig. 4.   Effect of the MAP kinase kinase 1 (MEK1) inhibitor, PD-98059, on TGF-beta -mediated activation of ERKs in rat pancreatic acini. Acini were prepared and preincubated with or without PD-98059 (50 µM) for 30 min. A: acini were then treated with TGF-beta (100 pM) for 4 h in the absence and presence of PD-98059 (lanes 3 and 4). B: acini were prepared and treated with TGF-beta for 4 h after either pretreatment with PD-98059, treatment with PD-98059 at the same time as addition of TGF-beta , or treatment with PD-98059 either 1, 2, or 3 h after adding TGF-beta . Western blotting was performed using an anti-phospho ERK antibody. The results are representative of 3 separate experiments.

Ras is a GTP-binding molecule known to be upstream of several cellular signaling pathways, including the ERK pathway (11). To determine whether TGF-beta -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-beta -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-beta (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-beta and EGF (Fig. 5). Infection with the control AdGFP had no effect on activation of ERK stimulated by either TGF-beta or EGF.


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Fig. 5.   Effect of adenoviral-mediated transfer of dominant negative RasN17 on TGF-beta -induced activation of ERKs. Short-term cultures of pancreatic acini were transfected for 8 h with no virus (lanes 1-3), dominant negative Ras adenovirus (AddnRas, lanes 4-6), or a control green fluorescent protein adenovirus (AdGFP, lanes 7-9), each at a titer of 108 pfu/mg acinar protein. Cells were then treated with 100 pM TGF-beta (lanes 2, 5, and 8) for 4 h or epidermal growth factor (EGF; lanes 3, 6, and 9) for 10 min. Cell lysates were prepared and submitted to Western blotting with anti-phospho ERK antibody. The membrane was stripped and reprobed with an antibody to the nonphosphorylated (nonactive) form of ERK to serve as a control for protein loading. The results are representative of 3 separate experiments.

Because TGF-beta -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-beta for 4 h, a point at which maximal activation of MAP kinases by TGF-beta is observed. Cyclohexamide had no effect on basal ERK activity and completely blocked the ability of TGF-beta to activate ERKs (Fig. 6). These data suggest that TGF-beta -induced activation of ERKs in pancreatic acinar cells requires protein synthesis.


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Fig. 6.   Effect of the protein synthesis inhibitor cyclohexamide (CH) on TGF-beta -induced ERK activity in rat pancreatic acini. Acini were prepared and preincubated with or without cyclohexamide (10 µg/ml) for 30 min. Acini were then treated with TGF-beta (100 pM) for 4 h in the absence and presence of cyclohexamide. Cell lysates were prepared and Western blotting performed using an anti-phospho ERK antibody. The results are representative of 3 separate experiments.

It is known that TGF-beta mediates many of its cellular responses through Smad-signaling molecules. Therefore, we investigated whether Smad4, a common effector in the TGF-beta 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-beta 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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Fig. 7.   Effect of adenoviral-mediated transfer of dominant negative Smad4 on induction of ERK activity by TGF-beta and CCK. Short-term cultures of pancreatic acini were transfected for 8 h with no virus (lanes 1 and 2), dominant negative Smad4 adenovirus (AddnSmad4, lanes 3 and 4), or a control GFP adenovirus (AdGFP, lanes 5 and 6), each at a titer of 106 pfu/mg pancreatic protein. Cells were then treated with TGF-beta (100 pM) for 4 h (A) or CCK (1 nM) for 30 min (B). Cell lysates were prepared and submitted to Western blotting with anti-phospho ERK antibody (A). The membrane was stripped and reprobed with an antibody to the nonphosphorylated (nonactive) form of ERK to serve as a control for protein loading in A. The results are representative of 3 separate experiments.

To determine the biological relevance of ERK activation by TGF-beta , we examined the role of ERKs in TGF-beta 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-beta altered AP-1 activity in pancreatic acinar cells, cells were treated for 4 h with TGF-beta (100 pM), chosen because it is the time point at which there is maximal activation of ERKs by TGF-beta . Nuclear proteins were then isolated and examined in an electrophoretic mobility shift assay (EMSA). EMSAs showed that TGF-beta 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-beta to induce AP-1 binding, suggesting that the ability of TGF-beta to activate AP-1 is dependent on ERK activity.


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Fig. 8.   Effect of the MEK1 inhibitor, PD-98059, on TGF-beta mediated activated induction of activator protein-1 (AP-1) binding in rat pancreatic acini. Acini were prepared and preincubated with or without PD-98059 (50 µM) for 30 min. Acini were then treated with TGF-beta (100 pM) for 4 h in the absence and presence of PD-98059. Electrophoretic mobility shift assay was performed using 32P-labeled AP-1 binding site oligonucleotide and 5 µg of acinar cell nuclear extract. Specificity of the AP-1 band is demonstrated by using excess cold, competing specific (AP-1) and nonspecific (Sp1) oligonucleotide. Data are representative of 3 separate experiments.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we have demonstrated for the first time that TGF-beta activates the MAP kinases ERKs and p38 in isolated rat pancreatic acini. Interestingly, activation of both ERKs and p38 by TGF-beta 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-beta -mediated activation of MAP kinases in many other cell types. Previous studies have shown that TGF-beta -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-beta in primary human lung fibroblasts. In these cells, TGF-beta stimulated ERK activity beginning at 2 h, with maximal activity at 16 h. Interestingly, in this same study, TGF-beta induced rapid activation of p38. TGF-beta 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-beta influences MAP kinases with a different time course in different cells. TGF-beta also has a differential ability to activate distinct MAP kinase signaling cascades in different cell types. For example, TGF-beta did not activate JNKs in pancreatic acinar cells; however, TGF-beta 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-beta 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-beta -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-beta -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-beta 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-beta 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-beta to activate ERKs; thus the action of TGF-beta is indirect. In the study previously cited by Finlay et al. (14), they demonstrated that delayed activation of ERKs and AP-1 by TGF-beta 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-beta -mediated activation of ERKs in pancreatic acinar cells, we performed Western blotting to determine whether TGF-beta induced expression of bFGF. TGF-beta 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-beta -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-beta - and Smad4-induced gene expression and TGF-beta -stimulated Smad4 transcriptional activity. In endothelial cells, MEK1 can selectively stimulate Smad2 transcriptional activation in the absence of exogenous TGF-beta stimulation (7). Furthermore, transfection of dominant negative ERK1 into NIH/3T3 fibroblasts abolished stimulation of plasminogen activator inhibitor-1 promoter activity by TGF-beta (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-beta 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-beta , while having no effect on Smad activity (25). In this case, ERKs could represent a distinct, parallel pathway from Smad signaling in TGF-beta regulation of p21Cip1 or may function downstream of Smads.

The results of our study clearly demonstrate that ERK activation by TGF-beta 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-beta -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-beta 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-beta -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-beta -mediated physiological responses are just beginning to be understood. It does not appear that cell type-specific modulation of cell growth by TGF-beta correlates with MAP kinase activity, based on several observations. They include the fact that TGF-beta -stimulated growth of Swiss 3T3 cells occurs without activation of MAP kinase, and TGF-beta 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-beta , 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-beta -induced MAP kinase activation in other cell types. For example, TGF-beta -stimulated activation of p38 in human neutrophils is necessary for TGF-beta -induced actin polymerization and chemotaxis (19). In a human fibrosarcoma-derived cell line, TGF-beta 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-beta is required for TGF-beta production (39), and, in some cell types, the MAP kinase pathway is required for stimulation of p21 by TGF-beta (25).

MAP kinase activation may modulate TGF-beta signaling by selective activation of specific transcription factors. We chose to investigate the functional consequences of TGF-beta -induced ERK activation in pancreatic acinar cells by examining the role of ERKs in TGF-beta 's ability to activate the transcription factor AP-1. We demonstrated that TGF-beta activated AP-1 at 4 h, the same time point at which TGF-beta induced maximal activation of ERKs. We also showed that AP-1 activation by TGF-beta was dependent on ERK signaling.

TGF-beta 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-beta -dependent transcription of a number of genes, including TGF-beta 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-beta , the mechanisms regulating AP-1 induction by TGF-beta are incompletely understood. Our data indicates that ERK signaling is important for AP-1 activation by TGF-beta in pancreatic acinar cells. This is supported by two recent studies performed in human lung fibroblasts and intestinal epithelial cells that demonstrated that TGF-beta -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-beta activates the MAP kinases ERKs and p38 in pancreatic acinar cells. The time course of MAP kinase activation by TGF-beta is different than that described in other cell types, with maximal effects 4 h after treatment. The ability of TGF-beta to activate ERKs is dependent on Smad4, MEK1, and Ras. ERK activation by TGF-beta 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-beta 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.


    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.


    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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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