Department of Physiology, University of Michigan, Ann Arbor, Michigan 48109-0622
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ABSTRACT |
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CCK stimulates pleiotrophic responses in pancreatic acinar cells; however, the intracellular signaling pathways involved are not well understood. To evaluate the role of the ras gene product in CCK actions, a strategy involving in vitro adenoviral-mediated gene delivery of a dominant-negative mutant Ras (RasN17) was utilized. Isolated acini were infected with various titers of either a control adenovirus or an adenoviral construct expressing RasN17 for 24 h before being treated with CCK. Titer-dependent expression of RasN17 in the acini was confirmed by Western blotting. Infection with control adenovirus [106-109 plaque-forming units/mg acinar protein (multiplicity of infection of ~1-1,000)] had no effect on CCK stimulation of acinar cell amylase release, extracellular-regulated kinase (ERK) or c-Jun kinase (JNK) kinases, or DNA synthesis. In contrast, infection with adenovirus bearing rasN17 increased basal amylase release, inhibited CCK-mediated JNK activation, had no effect on CCK activation of ERK, and inhibited DNA synthesis. These data demonstrate important roles for Ras in specific actions of CCK on pancreatic acinar function.
cell proliferation; mitogen-activated protein kinase; c-Jun kinase; secretion
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INTRODUCTION |
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CHOLECYSTOKININ IS BOTH a hormone and a neuropeptide. CCK is broadly distributed in the central nervous system and may influence anxiety, neuroleptic activity, and arousal (9). Within the gastrointestinal tract, CCK has a wide variety of physiological actions, including the stimulation of gallbladder contraction and pancreatic secretion and the inhibition of gastric emptying (25). In the pancreas, CCK acts primarily on the acinar cells where it has a variety of physiological actions. CCK is a potent secretagogue (42); it stimulates acinar cell protein synthesis (42), the expression of protooncogenes (30), and the expression of a specific subset of digestive enzymes genes (24). CCK has a trophic influence on the pancreas, leading to both hypertrophy and hyperplasia (37). At supraphysiological concentrations, CCK can induce pancreatitis (38). However, the cellular signaling pathways and mechanisms involved in these diverse actions of CCK are not well understood.
At a mechanistic level, the best understood pancreatic acinar cell response to CCK is the stimulation of secretion. Rat pancreatic acinar cells possess CCK-A receptors, which are typical seven-transmembrane-spanning receptors coupled to heterotrimeric G proteins (41). Specifically, the CCK-A receptor appears to interact with the Gq class of G proteins. Effects of CCK-A receptor activation on secretion are due to Gq-mediated activation of phosphoinositide-specific phospholipase C, leading ultimately to increases in inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) (43). IP3 causes the release of intracellular Ca2+, and, in combination with DAG, these two second messengers lead to the activation of protein kinase C (7, 43). Increases in cellular Ca2+ and protein kinase C activity appear to account fully for the effects of CCK on acinar cell secretion (43).
In contrast to effects on secretion, the cellular mechanisms involved in the trophic actions of CCK on pancreatic acinar cells are not well understood. These actions likely involve a complex network of intracellular signaling pathways. Of particular interest in the regulation of the pancreatic acinar cell is the protooncogene ras. Ras is a GTP-binding molecule known to be upstream of several cell-signaling pathways, including two mitogen-activated protein (MAP) kinase pathways, the extracellular-regulated kinase (ERK) and c-Jun kinase (JNK) (12, 16, 32) pathways. ras is mutated to an oncogenic form in a very high proportion of pancreatic cancers (2). CCK has been shown to activate ERK (14) and JNK (10) in pancreatic acinar cells. These Ras-regulated pathways are known to be important in a variety of cellular functions, including growth control, in fibroblasts. However, whether CCK-mediated activation of these kinases involves Ras, or alternative pathways, remains controversial (11, 15).
To examine the roles of Ras in pancreatic acinar cell function, we expressed a dominant-negative mutant in the acinar cells and examined the effects on several different CCK-mediated actions. Dominant-negative molecules provide a very specific means of inhibiting the actions of their normal counterparts. To express the dominant-negative molecule in normal pancreatic acinar cells, we utilized adenoviral-mediated gene transfer in vitro. Previously, adenovirus has been shown to efficiently transfer genes into the pancreas in vivo (34). Initially, we determined the appropriate conditions for adenoviral infection. Then, we confirmed that adenoviral infection leads to the expression of the dominant-negative Ras molecule. Finally, we tested the effects of expression of the dominant-negative Ras on CCK-stimulated secretion, ERK and JNK activation, and DNA synthesis. The expression of the dominant-negative Ras increased basal cell secretion, blocked CCK-stimulated effects on acinar cell JNKs and DNA synthesis, but had no effect on CCK activation of ERKs. These data indicate that Ras is of central importance to some but not all of the effects of CCK on acinar cell functions.
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MATERIALS AND METHODS |
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Materials. DMEM, fetal bovine serum (FBS), penicillin, streptomycin, and amphotericin B were obtained from GIBCO (Grand Island, NY). Tissue culture plastic ware was obtained from Sarstedt (Newton, NC). CCK-8 sulfated was obtained from Bachem (Torrance, CA), [3H]thymidine was from Amersham (Arlington Heights, IL), TCA was from J. T. Baker (Phillipsburg, NJ), Bio-Rad protein reagent was from Bio-Rad Laboratories (Hercules, CA), and Phadebas reagent was from Pharmacia (Columbus, OH).
Construction of adenoviral vectors. A recombinant adenovirus expressing a dominant-negative Ras was generated by cloning the human H-ras cDNA with a serine-to-asparagine substitution at amino acid position 17 (gift of L. Feig, Tufts University, Boston, MA) into the multiple cloning site of the vector pAD.CMV-Link.1 (gift of Dr. Krishna J. Fisher, University of Pennsylvania). The p21-H-rasN17 cDNA was isolated from the plasmid pXVR (18) by digestion with BamH I and Bgl II; the resulting full-length cDNA was blunt ended using the Klenow fragment of E. coli DNA polymerase I and subcloned into the EcoR V site of the pAD.CMV-Link vector (pAD.CMV-Link.1.RasN17).
The pAD.CMV-Link.1.RasN17 and the pJM17 vector (Microbix Biosystems, Toronto, Ontario, Canada) were cotransfected into 80% confluent 293 cells using the CaPO4-DNA coprecipitation technique. After 8-10 days, the death of the 293 cells indicated that a new recombinant virus coding for the p21-H-RasN17 protein had been generated. The viral DNA was then extracted from the 293 cells and confirmed by Southern blot technique using the rasN17 cDNA as a probe. A single clone of recombinant adenovirus was isolated through serial dilution using a plaque assay. The expression of the recombinant adenovirus was performed as previously described with 293 cells (20), and the virus was subsequently concentrated on a cesium chloride gradient. The concentration of the recombinant adenovirus was assessed on the basis of the absorbency at 260 nm and on a limiting dilution plaque assay. For controls, we utilized an adenovirus bearing thePancreatic acinar isolation, infection, and culture. Pancreatic acini were isolated from male Wistar rats as previously described (21). After isolation, acini were suspended in 10 ml of Waymouth's medium and aliquots were sonicated in 0.1 N NaOH and assayed for protein using Bio-Rad protein reagent following the manufacturer's suggested protocol. For growth studies, cells were cultured by a modification of a previously described method (22). Cells were suspended in a basal media of Waymouth's medium containing 0.5% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 50 µg/ml amphotericin B, 0.5 mM IBMX, and 0.2 mg/ml soybean trypsin inhibitor and seeded at a density of ~0.1 mg/ml into 24-well culture plates coated with air-dried rat tail collagen. Adenovirus was added at various titers between 105 and 108 viral particles/ml as indicated. After 24 h, hormones and growth factors were added to the medium as indicated.
For investigation of the effects of adenoviral infection on amylase release, or activation of MAP kinases, pancreatic acini were cultured in suspension as described previously (28). For these experiments, acini were aliquoted into 12 × 75-mm polyethylene test tubes at ~1 mg protein/ml. Adenovirus was added at various titers between 106 and 109 pfu/ml as indicated. The acini were then allowed to sit for 10 min before being diluted to 10 ml with basal medium and transferred into 100-mm dishes. Acini were cultured for 24 h in suspension using medium identical to that described above but without the addition of FBS. All cultures were maintained in a humidified atmosphere of 5% CO2 in air at 37°C.Expression of adenoviral delivered -galactosidase.
For identification of infected acinar cells, acini were stained with
5-bromo-4-chloro-3-indolyl
-D-galactopyranoside (X-gal) after 24 h of infection. Acini were washed twice in PBS and then fixed
for 1 min with 2% formaldehyde-3% glutaraldehyde. They were then
washed twice in PBS and incubated at 37°C in a color solution consisting of 5 mM K3Fe(CN)6, 5 mM
K4Fe(CN)6, and 2 mM MgCl2 in PBS
with 0.1% X-gal.
Western blotting of Ras.
To determine the overexpression of
RasN17 in infected acini, Western
blots were performed using a polyclonal anti-H-Ras antibody (C-20)
(Santa Cruz Biotechnology, Santa Cruz, CA). Acini that had been
cultured for 24 h after infection with the indicated titers of
rasN17-bearing virus were pelleted by
centrifugation, washed once with ice-cold PBS, and sonicated for 7 s in
lysis buffer (20 mM Tris · HCl, pH 7.8, 150 mM NaCl,
2 mM EDTA, pH 8.0, 50 mM -glycerolphosphate, 0.5% NP-40, 0.1 M DTT,
40 µg/ml phenylmethylsulfonyl fluoride, 4 µg/ml leupeptin, and 1 mM
Na3VO4).
The amount of protein in the cell extracts was assessed by the Bio-Rad
protein assay reagent. Cell extracts (50 µg protein) were
electrophoretically separated on a 12% SDS polyacrylamide gel and
transferred to a nitrocellulose membrane. Briefly, blots were blocked
with 5% milk in Tris-buffered saline-0.05% Tween 20 (TBST) for 1 h at
room temperature followed by the incubation (1 h) of the blots with Ras
antibody (100 ng/ml in 3% BSA in TBST). After the blots were washed
with TBST, they were incubated with horseradish peroxidase-conjugated
secondary antibody. The bands were visualized using enhanced
chemiluminescence (ECL) reagent (Amersham).
Analysis of CCK-stimulated secretion. Amylase secreted in response to CCK-8 by acini cultured for 24 h was assayed as described previously (28). Briefly, infected and control acini were washed and resuspended in HEPES-buffered Ringer (HR) (in mM: 142 NaCl, 5.6 KCl, 2.2 CaCl2, 3.6 NaHCO3, 1 MgCl2, 5.6 D-glucose, and 30 HEPES buffer, pH 7.4). Each treatment group of acini was divided into two conditions, unstimulated and stimulated with 1 nM CCK-8. The medium was collected after 15 min at 37°C, and the concentration of amylase in the medium was measured using the Phadebas reagent. Results were expressed as a percentage of initial acinar amylase content.
Analysis of CCK-induced increases in intracellular Ca2+ concentration. Analysis of intracellular Ca2+ concentration was conducted on acini cultured overnight in suspension. Acini were incubated with 5 µM fura 2-AM at 37°C for 30 min and then washed and resuspended in HR buffer. Fura-loaded acini were transferred to a closed chamber, mounted on the stage of a Zeiss Axiovert inverted microscope, and continuously superfused at 1 ml/min with HR buffer at 37°C. Measurement of emitted fluorescence and calibration of these signals to yield a measurement of intracellular Ca2+ were performed using an Attofluor digital imaging system (Rockville, MD), exactly as described previously (45).
Analysis of lactate dehydrogenase release.
Lactate dehydrogenase (LDH) released by acini into the overnight
culture medium was determined by measuring LDH activity using a kit
from Sigma (procedure no. DG 1340-UV). Pancreatic acini were isolated,
an aliquot was taken to determine total LDH activity, and other
aliquots (1 mg protein) were either uninfected or infected with
rasN17- or -galactosidase-bearing virus
(titer of 109 pfu/mg protein).
Acini were cultured for 24 h and pelleted by centrifugation. LDH
activity was assayed using a 100-µl sample of the supernatant.
Results were expressed as enzyme activity that was released into
overnight culture medium as a percentage of total enzyme activity in
the initial sample.
Analysis of CCK-stimulated DNA synthesis. The rate of DNA synthesis was measured using a modification of a previously described [3H]thymidine incorporation assay (22). Cells were exposed to hormones and growth factors for 24 h after which 0.1 µCi/ml [3H]thymidine was added for an additional 24 h (total time in culture: 3 days). 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 using 0.3 ml of 0.1 N NaOH. Radioactivity in the dissolved cell pellet was measured by liquid scintillation counting. Incorporation of [3H]thymidine was expressed as a percentage of total counts per minute observed in control cells.
Analysis of MAP kinase phosphorylation. To analyze effects of CCK-8 stimulation and dominant-negative Ras expression on levels of active MAP kinases, we conducted Western blotting experiments using anti-phospho-MAP kinase antibodies that recognize the phosphorylated forms of ERKs or JNKs (Anti-ACTIVE ERK polyclonal antibody and Anti-ACTIVE JNK polyclonal; Promega, Madison, WI). To evaluate protein levels of JNK, we used anti-JNK1 polyclonal antibody (C-17) (Santa Cruz Biotechnology). For these assays, 25 µg of protein from cell free extracts were subjected to electrophoresis on a 10% SDS-polyacrylamide gel and then electrophoretically transferred to nitrocellulose membrane. The membrane was blocked with 5% milk in TBST (pH 7.6) for 1 h and then incubated with either the anti-phospho-ERK antibody (25 ng/ml in 3% BSA in TBST) or the anti-phospho-JNK antibody (1:500 in 5% milk in TBST). After washing with TBST, membranes were incubated with anti-rabbit IgG antibody conjugated with horseradish peroxidase for 1 h. The phospho-ERK and -JNK bands, respectively, were then detected using ECL reagent, and the emitted light was recorded on X-ray film and quantified by densitometry.
Statistical analysis. Statistical analysis was carried out by using a commercial statistical program (InStat; Graphpad Software, San Diego, CA). Differences between individual conditions and controls were tested with ANOVA. In the analyses, differences were considered significant when P < 0.05.
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RESULTS |
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Adenoviral-mediated gene delivery to acinar cells in vitro.
To deliver a dominant-negative Ras mutant to rat pancreatic acinar
cells, we tested a variety of cell transfection techniques, including
calcium phosphate, DEAE dextran, and commercial liposomal preparations,
without success (data not shown). Next, we tested the ability of
recombinant adenoviral vectors to infect acinar cells in vitro.
Isolated pancreatic acini were treated with adenovirus (108 pfu/mg acinar protein)
bearing the -galactosidase gene, cultured for 24 h, and then fixed
and stained using X-gal as a substrate. A large majority of acinar
cells stained blue, indicating successful expression of the exogenous
gene (Fig. 1). Nearly all acini showed some
staining, and in most the percentage of cells staining was >80%. It
was observed that the number of staining cells increased over time in
the staining solution, suggesting that there was a wide degree of
variability in the levels of gene expression.
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Effects of dominant-negative ras gene expression on secretion.
Infection of isolated pancreatic acini in vitro with an adenovirus
bearing rasN17 led to a concentration-dependent
increase in the expression of Ras (Fig. 4).
Obvious increases in RasN17 were
observed at titers of 107, and
very high levels were noted at 109
pfu/mg acinar protein.
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Effects of dominant-negative ras gene expression on acinar cell
ERKs.
Next, we determined whether the adenoviral-mediated delivery of
dominant-negative Ras interfered with the ability of CCK-8 to stimulate
ERK or JNK in acinar cells. Acini were infected with various titers of
adenovirus bearing -galactosidase or
rasN17, cultured for 24 h to allow
expression of the exogenous gene, and then stimulated with CCK-8 to
activate the kinases. Infection with adenovirus bearing
rasN17 up to
109 pfu/mg acinar protein had no
effect on the ability of CCK-8 to stimulate activation of p42 and p44
MAP kinases (Fig. 7). However, RasN17 expression did inhibit ERK
phosphorylation stimulated by epidermal growth factor (EGF) (Fig. 7).
The inhibition of EGF-stimulated ERK phosphorylation was apparent with
108 and nearly complete with
109 pfu/mg acinar protein.
Infection with the control virus had no effect on activation of ERK
stimulated by either CCK-8 or EGF (Fig. 7).
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Effects of dominant-negative ras gene expression on acinar cell DNA
synthesis.
To determine the effects of adenoviral-mediated transfer of
RasN17 into pancreatic acinar
cells on CCK-8-stimulated DNA synthesis, cells were put into a
thymidine incorporation assay. Cells were isolated, infected with or
without adenovirus bearing
rasN17 or a control virus,
and cultured for 24 h. Cultured cells were then treated with or without
CCK-8 (10 nM) for an additional 48 h, the last 24 h of which
[3H]thymidine was
included in the medium. Uninfected cells and cells infected with
control adenovirus responded to CCK-8 stimulation with an ~2-fold
increase in DNA synthesis (Fig. 9). In
contrast, acinar cells infected with adenovirus bearing
rasN17 showed a
titer-dependent reduction in response to CCK-8 stimulation of DNA
synthesis. At a titer of 109
pfu/mg acinar protein, RasN17
completely blocked the stimulatory effect of CCK-8 on acinar cell
growth. Similar results were observed when EGF was used as a growth
stimulus (data not shown).
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DISCUSSION |
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The complexities of the in vivo environment make it difficult to
ascertain intracellular signaling events involved in CCK-mediated growth effects. To circumvent this problem, researchers have turned to
studies in cell lines. However, acinar-derived cell lines are transformed and exhibit many nonacinar cell characteristics such that
results obtained with these cells do not fully reflect normal acinar
cell function. These problems are avoided with primary cultures. Acinar
cells from rats and mice cultured in vitro respond to various growth
regulatory factors. In vitro acinar cell DNA synthesis is stimulated by
CCK (29), EGF (26), fibroblast growth factor (22), and insulin (26) and
is inhibited by transforming growth factor- (27). Previously, a
limitation of this model was that genetic manipulation was not possible
in primary acinar cells. In this study, we show that adenoviral
technology allows genetic approaches to normal acinar cell biology
without the difficulties of transgenic or gene knockout strategies.
The functional importance of Ras activation in acinar cell biology has not been clear. An important role for Ras in acinar cell function was suggested by the observation that nearly 90% of pancreatic cancers exhibit somatic mutations in ras genes (2). Also, it is well recognized that the ras protooncogene is a central component of mitogenic signal transduction pathways (13). Expression of a dominant-negative Ras molecule has previously been shown to inhibit growth induced by a variety of growth factors in fibroblastic cells (17). It is also becoming increasingly clear that Ras functions at multiple levels in cell regulation. Ras has been shown to be upstream of a number of pathways important to cell function, including the ERK (8), the phosphoinositol-3-OH kinase (36), and the Rac/Rho (35) pathways. However, the functional importance of Ras in pancreatic acinar cells has not been previously investigated. In the current study, adenoviral-mediated expression of dominant-negative Ras had several effects on pancreatic acinar cell function.
As a sensitive measure of acinar cell function, we initially examined the effects of adenoviral infection on the ability of the cells to secrete amylase. Control adenovirus had no effect on either basal or CCK-8-stimulated amylase release at any titer examined. This indicates that at the titers utilized in the current study adenoviral infection per se was not overtly harmful to the acinar cells. In contrast to the control adenovirus, infection with dominant-negative Ras increased basal amylase release. The explanation for this stimulation of amylase release is not readily apparent. One proposal would be that Ras is required for normal cell maintenance and that inhibition of Ras has a deleterious effect on overall cell function, leading to a leaking of digestive enzymes from unhealthy cells. However, expression of dominant-negative Ras did not increase the release of LDH from cultured acini. In addition, expression of dominant-negative Ras did not inhibit the ability of CCK-8 to increase the intracellular Ca2+ concentration or activate ERK phosphorylation. Another proposal would be that Ras, or a Ras-regulated protein, normally plays an inhibitory role in cell secretion. Small Ras-like GTP-binding proteins are known to be important for vesicle trafficking (5). Therefore, it is possible that expression of dominant-negative Ras interfered with the secretory pathway. To our knowledge, the effects of expression of dominant-negative Ras on secretion have not previously been examined.
Dominant-negative Ras, but not control virus, inhibited CCK-8-stimulated DNA synthesis. Further investigations will be required to determine the specific effectors downstream of Ras whose activation is required for acinar cell DNA synthesis. One possible mechanism for this effect could involve inhibition of the activation of ERK. The role of Ras in the activation of this MAP kinase pathway has been well characterized (8). Activated Ras stimulates the activation of Raf, a MAP ERK kinase kinase. Raf in turn phosphorylates and activates MAP ERK kinase (MEK1 or MEK2). The MEKs phosphorylate and activate ERK1 and ERK2. This pathway appears to be particularly important for growth factor stimulation of mitogenesis. Thus it is not surprising that in the current study dominant-negative Ras expression inhibited ERK activation by EGF in a titer-dependent manner. This may also explain the inhibitory effects of dominant-negative Ras on EGF-stimulated acinar cell DNA synthesis. However, expression of dominant-negative Ras, even at the highest titer examined, did not inhibit CCK-8-mediated ERK activation.
Activation of the ERK pathway in a Ras-independent manner has previously been shown for a variety of other receptors (1, 6, 39). One important Ras-independent mechanism for activation of the ERK pathway involves protein kinase C, which directly activates Raf (40). It was previously shown that direct activation of pancreatic acinar cell protein kinase C using phorbol esters activated ERK to a level similar to that achieved by CCK-8 (11, 14). Also, inhibition of protein kinase C using either staurosporine (14) or GF-109203X (11) blocked the ability of CCK-8 to activate ERK in acinar cells. Together with the current studies, these data suggest that the major pathway for CCK stimulation of the ERK pathway involves protein kinase C and does not require activation of Ras. However, it has recently been reported that RasN17 expression blocks some, but not all, Ras-mediated signals (31). Therefore, the current results should not be interpreted to necessarily indicate that Ras plays no role in CCK-mediated activation of ERKs. These results indicate that the mechanisms involved in CCK stimulation of ERKs are different from those utilized by typical tyrosine kinase receptor-mediated growth factors. A full understanding of these results will require a better understanding of Ras signaling.
In contrast to its lack of effects on the ERK pathway, expression of dominant-negative Ras inhibited the ability of CCK-8 to activate the JNK pathway. Unlike the ERK pathway in which Raf is the primary upstream kinase, there are many upstream kinases that appear to regulate the JNK pathway (16). Ras is able to activate the JNK pathway in some but not all cell types. The mechanisms involved in CCK stimulation of the JNK pathway have not been elucidated in pancreatic acinar cells. However, it was previously noted that activation of protein kinase C does not stimulate the JNK pathway in these cells (10). The current data indicate that Ras activity may play an important role in the pathway from CCK receptor occupation to JNK activation. The biological role of activated JNK in the acinar cell is currently not understood. The secretagogue specificity and CCK concentration dependence of JNK activation correlate with secretagogue-induced pancreatitis, leading to the speculation that this pathway may play a role in this disease (19). Activation of the JNK pathway is often associated with cell stasis or in some cases apoptosis (23, 44). However, activation of JNK has been associated with hypertrophy of cardiac myocytes (33), and strong evidence supports a primary role for this kinase in regulating DNA synthesis in hepatocytes (3). Therefore, further investigation will be necessary to determine the role of JNK in acinar cell growth regulation.
In summary, the current study utilized adenovirus-mediated gene transfer to investigate the role of Ras protein in CCK-8-mediated actions in pancreatic acinar cells. Adenovirus-mediated expression of foreign genes was time and titer dependent. Dominant-negative Ras induced an increase in basal acinar cell amylase release. Expression of dominant-negative Ras also inhibited CCK-8-stimulated activation of the JNK pathway and DNA synthesis. In contrast, expression of dominant-negative Ras had no effect on CCK-8-stimulated activation of the ERK pathway. Further studies will be needed to identify the critical downstream effectors involved in these effects of Ras inhibition on acinar cell function. The current data support a critical role for Ras in specific effects of CCK-8 on pancreatic acinar cells.
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ACKNOWLEDGEMENTS |
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We acknowledge the efforts of Terrence Grady and Katharina Detjen who were involved at the initiation of this project.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-35912 and Michigan Gastrointestinal Peptide Center Grant DK-34933.
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. §1734 solely to indicate this fact.
Address for reprint requests: C. D. Logsdon, Box 0622, Dept. of Physiology, Univ. of Michigan, Ann Arbor, MI 48109.
Received 16 March 1998; accepted in final form 25 October 1998.
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