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Am J Physiol Gastrointest Liver Physiol 287: G1175-G1181, 2004. First published August 19, 2004; doi:10.1152/ajpgi.00339.2004
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HORMONES AND SIGNALING

Pressure activates rat pancreatic stellate cells

Shiro Watanabe, Yoshikuni Nagashio, Hiroshi Asaumi, Yoko Nomiyama, Masashi Taguchi, Mitsuo Tashiro, Yasuyuki Kihara, Hayato Nakamura, and Makoto Otsuki

Third Department of Internal Medicine, University of Occupational and Environmental Health, Japan, School of Medicine, Kitakyushu 807-8555, Japan

Submitted 29 July 2004 ; accepted in final form 17 August 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pancreatic stellate cells (PSCs) play a central role in development of pancreatic fibrosis. In chronic pancreatitis, pancreatic tissue pressure is higher than that of the normal pancreas. We here evaluate the effects of pressure on the activation of rat PSCs. PSCs were isolated from the pancreas of Wistar rat using collagenase digestion and centrifugation with Nycodenz gradient. Pressure was applied to cultured rat PSCs by adding compressed helium gas into the pressure-loading apparatus to raise the internal pressure. Cell proliferation rate was assessed by 5-bromo-2'-deoxyuridine (BrdU) incorporation. MAPK protein levels and {alpha}-smooth muscle actin ({alpha}-SMA) expression were evaluated by Western blot analysis. Concentration of activated transforming growth factor-{beta}1 (TGF-{beta}1) secreted from PSCs into culture medium was determined by ELISA. Collagen type I mRNA expression and collagen secretion were assessed by quantitative PCR and Sirius red dye binding assay, respectively. Application of pressure significantly increased BrdU incorporation and {alpha}-SMA expression. In addition, pressure rapidly increased the phosphorylation of p44/42 and p38 MAPK. Treatment of PSCs with an MEK inhibitor and p38 MAPK inhibitor suppressed pressure-induced cell proliferation and {alpha}-SMA expression, respectively. Moreover, pressure significantly promoted activated TGF-{beta}1 secretion, collagen type I mRNA expression, and collagen secretion. Our results demonstrate that pressure itself activates rat PSCs and suggest that increased pancreatic tissue pressure may accelerate the development of pancreatic fibrosis in chronic pancreatitis.

activation; fibrosis; chronic pancreatitis; mitogen-activated protein kinase; transforming growth factor-{beta}

CHRONIC PANCREATITIS IS CHARACTERIZED by loss of functional tissue and its replacement by extracellular matrix (ECM)-rich connective tissue (11, 29). It is well known that pancreatic tissue pressure in chronic pancreatitis is higher than that in normal pancreas (20, 32). In the animal model of chronic pancreatitis, the parenchymal fibrosis and restrictive fibrotic capsule enveloping the gland prevents dissipation of the main pancreatic duct pressure (23). This could account for the increase of tissue pressure in chronic pancreatitis. Recent studies (3–5) have demonstrated that activated pancreatic stellate cells (PSCs) are considered key cells in the pathogenesis of pancreatic fibrosis. PSCs can be identified by the presence of vitamin A-containing lipid droplets in the cytoplasm and positive staining for stellate cell markers, such as desmin and glial fibrillary acidic protein (2). PSCs become activated as indicated by increased cell proliferation, expression of the cytoskeletal protein {alpha}-smooth muscle actin ({alpha}-SMA), and collagen synthesis when exposed to factors, such as proinflammatory cytokines, growth factors, oxidant stress, and ethanol and its oxidative metabolite acetaldehyde (3, 4). Because PSCs are situated at the base of pancreatic acini with their extended cytoplasmic processes encircling the basal aspect of acinar cells, PSCs might be subjected to mechanical forces, such as tissue pressure in chronic pancreatitis. Nevertheless, the effects of pressure on PSCs have not been elucidated.

Mechanical forces, such as pressure, stretch, and share stress, play important roles in various pathophysiological processes including the development of cardiovascular and renal diseases. Myocardium responds to chronic pressure or volume overload resulting in cardiac hypertrophy. This process is characterized by enlargement of cardiac myocytes, activation and proliferation of cardiac fibroblasts, differentiation of fibroblasts into myofibroblasts, and excessive accumulation of ECM protein (31). Mechanical overloading to vascular walls initiates a series of biochemical events that have been postulated to form the basis for arteriosclerosis and vascular remodeling (15, 38). In the kidney, increases in intraglomerular pressure are known to predispose to the development of glomerular sclerosis, which is characterized by accumulation of glomerular ECM within the glomerulus (1, 13). In in vitro studies, mechanical force has been shown to stimulate the synthesis of ECM proteins, such as collagen in cultured cardiac fibroblasts (9), vascular smooth muscle cells (6, 28), and mesangial cells (41).

From these observations, to determine whether the increase of pancreatic tissue pressure accelerates the development of pancreatic fibrosis, we investigated the effects of pressure on the activation of rat PSCs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials. Chemicals were obtained from the following sources: DMEM, penicillin, and streptomycin were from GIBCO-Life Technologies (Grand Island, NY). Anti-phosporylated (phospho)-p44/42 MAPK antibody, anti-p44/42 MAPK antibody, anti-phospho-p38 MAPK antibody, anti-p38 MAPK antibody, and MEK inhibitor PD-98059 were purchased from Cell Signaling Technology (Beverly, MA). p38 Kinase inhibitor SB-203580 was purchased from Calbiochem (La Jolla, CA). Antibodies to {alpha}-SMA for Western blot analysis were provided by Sigma-Aldrich (St. Louis, MO). Multiwell plates and cell culture flasks were purchased from Iwaki Glass (Funabashi, Japan).

Isolation and culture of PSCs. All animal procedures were performed in accordance with the guidelines of animal experiments of University of Occupational and Environmental Health, Japan, School of Medicine. Rat PSCs were isolated as detailed previously (2). Briefly, the pancreas was digested with a mixture of collagenase, pronase, and DNase in Gey's balanced salt solution. The resultant suspension of cells was centrifuged in a 13.2% Nycodenz gradient at 1400 g for 20 min. Stellate cells separated into a fuzzy band just above the interface of the Nycodenz solution and the aqueous buffer. This band was harvested, and the cells were washed and then resuspended in DMEM containing 10% FBS, 4 mM glutamine, and antibiotics (penicillin 100 U/ml; streptomycin 100 µg/ml). PSCs cultured in uncoated plastic wells (passages 2–5) were used for all experiments.

Pressure-loading apparatus. We used the pressure-loading apparatus, described previously (24). The apparatus consisted of a resealable steel chamber with inlet and outlet ports (Miwa, Osaka, Japan). The inlet port was connected through a tube to a reservoir of compressed helium, whereas the exit port was connected through a tube to a sphygmomanometer and an air-release valve. Compressed helium gas was pumped in the chamber to raise the internal pressure. During the delivery of helium gas into apparatus, no prepacked room air was released, so that the partial pressure of the gases originally contained in the chamber, such as oxygen, nitrogen, and carbon dioxide were kept constant, consistent with Boyle-Charles's law, as described previously (37). The plates and the flasks used for experiments were placed on a warm plate (37°C) inside the chamber. The partial pressure of oxygen, temperature, and pH of the incubation medium in the plates remained constant throughout the experiments.

Application of pressure to PSCs. PSCs were plated at equal seeding densities into uncoated plastic wells in DMEM with 10% FBS and incubated for 24 h. The cells were then serum starved for 24 h by incubation in DMEM containing 0.1% FBS, and then exposed to pressure for various periods of time.

Cell proliferation rate. To assess the effect of pressure application on cell proliferation rate, the BrdU incorporation assay was carried out by using a cell proliferation ELISA BrdU kit (Roche Applied Science, Tokyo, Japan) according to the manufacturer's instruction. PSCs were seeded and exposed to pressure from 0 to 120 mmHg for 0–120 min. After 24 h of incubation, the cells were labeled with 10 µM of BrdU solution and incubated for an additional 24 h at 37°C. After centrifugation, the labeling medium was removed from the microplates, the cells were dried and fixed, and the cellular DNA was denatured with FixDenat solution (Roche Applied Science, Tokyo, Japan) for 30 min at room temperature. A mouse anti-BrdU monoclonal antibody conjugated with peroxidase was added to each well and the plates were incubated again at room temperature for 2 h. After the plates were washed, tetramethylbenzidine was added and the cells were incubated for 30 min at room temperature. Finally, the absorbance of the samples was measured by a microplate reader at 370 nm (reference wavelength 492 nm).

Western blot analysis for MAPK and {alpha}-SMA. Western blot analysis was performed by using an equal amount of protein extracted from PSCs with antibody MAPK and {alpha}-SMA. PSCs were harvested by trypsinization after the application of pressure, and cell lysates were obtained by using a commercially available kit (Cellytic-M; Sigma). Samples were then centrifuged at 2,200 g for 10 min, and supernatant was harvested for measurement of MAPK and {alpha}-SMA levels. Protein concentration was determined by the Bradford method (7). Supernatant was prepared for one-dimensional SDS-PAGE. Proteins (5–20 µg/lane) were then separated by 8% SDS-PAGE and transferred onto polyvinylidene difluoride membrane (Hybond-P; Amersham Pharmacia Biotech, UK). Membranes were blocked with 10% fat-free dry milk for 60 min in PBS (pH 7.4) and then incubated with antibody in PBS containing 0.05% Triton X-100 (pH 7.4) for 60 min at room temperature. After being washed, membranes were incubated with appropriate IgG antibody conjugated with horseradish peroxidase in PBS for 60 min at room temperature. Antibody binding was detected by an enhanced chemiluminescence detection system (ECL Plus; Amersham Pharmacia Biotech, UK) and exposed to X-ray films (scientific bioimaging film; Kodak, Rochester, NY).

Treatment of PSCs with the MEK inhibitor PD-98059. To assess the effect of MEK inhibitor PD-98059 (an inhibitor of the MAPK cascade leading to p44/42 activation) on pressure-induced cell proliferation in PSCs, cells were plated at equal seeding densities and pretreated with 50 µM of PD-98059 for 60 min in DMEM. This was followed by a further incubation for 24 h after the application of pressure at 80 mmHg for 60 min. PSCs were labeled with BrdU and incubated for an additional 24 h. BrdU incorporation assay was then carried out as detailed in Cell proliferation rate. Cell viability was assessed by trypan blue staining.

Treatment of PSCs with the p38 MAPK inhibitor SB-203580. To investigate the effect of p38 MAPK inhibitor SB-203580 on pressure-induced {alpha}-SMA expression in PSCs, cells were plated at equal seeding densities into an uncoated plastic flask and then treated with 10 µM of SB-203580 for 60 min in DMEM. Preliminary studies using a range of concentrations (2–20 µM) of SB-203580 had identified 10 µM as the optimum concentration for use in our experiments. This was followed by a further incubation for 12 h after the application of pressure at 80 mmHg for 60 min. Cell viability was assessed by trypan blue staining. {alpha}-SMA expression in the cells was then assessed by Western blot analysis as described in Western blot analysis for MAPK and {alpha}-SMA.

Assay of transforming growth factor-{beta}1 by ELISA. PSCs grown to subconfluence were serum-starved for 24 h and exposed to pressure at 80 mmHg for 60 min. This was followed by a further incubation for 6 and 12 h after cells wered exposed to pressure. Activated transforming growth factor-{beta}1 (TGF-{beta}1) was measured in conditioned medium using commercial kits from BioSource International (Camarillo, CA) according to the manufacturer's instructions.

Reverse transcription and quantitative PCR. Gene expression of collagen type I was assessed by reverse transcription followed by real-time PCR (TaqMan; PE Applied Biosystems, Foster City, CA). In brief, PSCs were cultured in a 75-cm2 flask, serum starved for 24 h, and then pressure was applied at 80 mmHg for 60 min. Total RNA was isolated with Isogen (Nippon Gene, Tokyo, Japan), and reverse transcription was performed with the TaqMan Reverse Transcription Reagents (Applied Biosystems). An oligonucleotide probe was designed to anneal to the gene of interest between two PCR primers (Assay-on-Demand products purchased from Applied Biosystems). The probe was labeled with fluorescence with FAM (reporter dye) on the 5' end and TAMRA (quencher dye) on the 3' end. A similar probe and PCR primers were designed for GAPDH. The probe for this gene incorporated VIC as the reporter dye. PCR reactions including the primers and probes for these two genes and complementary DNA from cells were incubated. As the polymerase moves across the gene during the reaction, it cleaves the reporter dye from one end of each probe, which causes a fluorescent emission, which we measured with the ABI Prizm 7000 Sequence Detector (Applied Biosystems). The emissions recorded for each complementary DNA can then be converted into the level of expression for the gene normalized to the expression of GAPDH. TaqMan thermal cycler conditions were as follows: step 1, 50°C for 2 min; step 2, 95°C for 10 min; step 3, 95°C for 15 s; step 4, 62°C for 1 min; and steps 3 and 4 repeated for 40 cycles.

Assay of collagen secretion. Collagen secreted into culture medium by PSCs was determined by using Sircol Sirius red dye (Biocolor, Newtownabbey, UK) as described previously (50). Collagen was measured by spectrophotometry at 540 nm.

Statistical analysis. All values were expressed as the means ± SD of at least three independent experiments. The significance of changes was evaluated by using the two-tailed unpaired Student's t-test when data consisted of only two groups or by ANOVA when three or more groups were compared. A P value of <0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of pressure on cell proliferation. Cell proliferation in response to pressure was assessed by using the BrdU incorporation. We applied 0 to 120 mmHg pressure on PSCs based on a previous study (20), which revealed that the mean pancreatic tissue pressure in normal pancreas and in chronic pancreatitis is 10 to 20 mmHg and 80 to 120 mmHg, respectively. Application of pressure for 60 min resulted in a significant increase in BrdU incorporation in proportion to the level of applied pressure (Fig. 1A). Application of pressure at 80 mmHg to PSCs significantly promoted the incorporation of BrdU in a time-dependent manner (Fig. 1B). Cell viability assessed by a trypan blue exclusion test and light microscopy was always >90% throughout experiments. The cells appeared morphologically intact, and the number of detached cells was negligible during experiments. On the basis of these results, application of pressure at 80 mmHg for 60 min, which caused the maximum value of BrdU incorporation, was selected in the following experiments.



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  		Fig. 1. Effect of intensity (A) and duration (B) of pressure on 5-bromo-2'-deoxyuridine (BrdU) incorporation into pancreatic stellate cells (PSCs). A: PSCs in DMEM containing 0.1% FBS were subjected to a high pressure for 60 min at indicated pressure levels, and BrdU incorporation was determined. B: PSCs in DMEM containing 0.1% FBS were incubated in the absence or presence of pressure at 80 mmHg for the indicated periods. Twenty-four hours after pressurization, cells were further incubated for 24 h with BrdU. Data are means ± SD (n = 6 PSCs). *P < 0.0001 compared with control (absence of pressure).

 
Effects of pressure on phosphorylation of p44/42 MAPK. We then investigated the phosphorylation of MAPK in PSCs after exposure to pressure, because p44/42 MAPK pathway plays a major role in the regulation of growth and differentiation of PSCs (21, 33). Because it has been shown that phosphorylation of MAPK is observed within minutes in PSCs (33), we measured them after the application of pressure at 80 mmHg for 0 to 60 min. Phosphorylation of p44/42 MAPK after application of pressure was rapid and transient with peak phosphorylation at 2 min in p44 MAPK and 5 min in p42 MAPK (Fig. 2). Western blot analysis of cell extracts with anti-p44/42 antibody revealed no change in total levels of p44/42 protein.



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  		Fig. 2. Effect of pressure on p44/42 MAPK activation in cultured PSCs. PSCs were subjected to a pressure at 80 mmHg for indicated time intervals. Relative p44/p42 MAPK phosphorylation levels were determined from Western blot analysis by densitometric analysis. Data are means ± SD (n = 4 PSCs). *P < 0.05 vs. control (absence of pressure). Phospho, phosphorylated.

 
Effects of MEK inhibitor PD-98059 on pressure-induced cell proliferation. We then examined whether MEK inhibitor (PD-98059) can block the pressure-induced PSC proliferation. As shown in Fig. 3, PD-98059 significantly inhibited the pressure-induced increase in BrdU incorporation. These results suggest that pressure induces cell proliferation through p44/42 MAPK pathway.



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  		Fig. 3. Effect of MEK inhibitor (PD-98059) on pressure-induced PSC proliferation. PSCs were treated with (+) or without (–) PD-98059 (50 µM) for 60 min, and then pressure at 80 mmHg was applied for 60 min. Twenty-four hours after pressurization, cells were further incubated for 24 h with BrdU. Data are means ± SD (n = 6 PSCs). *P < 0.0001 vs. control (absence of pressure). N.S., not significant.

 
Effects of pressure on {alpha}-SMA expression. {alpha}-SMA is an important marker of PSC activation, and activated PSCs are the principal source of collagen in the fibrotic pancreas (3, 4). We evaluated whether pressure application activates PSCs. {alpha}-SMA expression is increased at 24 h after exposure to various stimuli in PSCs (35). For that reason, {alpha}-SMA expression was studied at 0 to 24 h after the application of pressure at 80 mmHg for 60 min. Western blot analysis for {alpha}-SMA revealed a single band in each lane corresponding to the known molecular weight of {alpha}-SMA (42 kDa) (Fig. 4). Densitometry of autoradiographs of Western blot analysis revealed that pressure (80 mmHg for 60 min) significantly increases {alpha}-SMA expression compared with controls at 12 h after the application of pressure. The increase of the levels of {alpha}-SMA expression remained at least 24 h after the application of pressure (data not shown).



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  		Fig. 4. Effect of pressure on {alpha}-smooth muscle actin ({alpha}-SMA) production by cultured PSCs. After the application of pressure at 80 mmHg for 60 min, the level of {alpha}-SMA protein was analyzed by Western blot. Cell extracts (5 µg protein) were subjected to Western blot analysis with anti-{alpha}-SMA antibody. Data are means ± SD (n = 4 PSCs). *P < 0.0001 vs. control (absence of pressure).

 
Effects of pressure on phosphorylation of p38 MAPK. We examined the effects of pressure on phosphorylation of p38 MAPK, which plays a role in mediating the ethanol- and acetaldehyde-induced {alpha}-SMA expression in PSCs (33). Application of pressure significantly increased p38 MAPK activation in PSCs (Fig. 5). The responses were rapid and transient with peak phosphorylation at 2 min after exposure, returning to basal values by 30 to 60 min. Western blot analysis of cell extracts with anti-p38 antibody revealed no change in total levels of p38 protein (Fig. 5A). SB-203580, a p38 kinase inhibitor, abolished the pressure-induced increase in {alpha}-SMA expression (Fig. 6). Trypan blue exclusion studies confirmed cell viability in the presence of p38 kinase inhibition (data not shown). These results suggest that pressure increases {alpha}-SMA expression via p38 MAPK pathway.



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  		Fig. 5. Activation of p38 kinase by pressure in rat PSCs. A: a representative Western blot analysis for p38 kinase activation showing an increase in expression of activated p38 kinase in PSCs exposed to pressure. B: densitometry analysis of Western blot showed a significant increase in p38 kinase activation in PSCs treated with pressure at 2 min compared with control (absence of pressure). n = 4 separate cell preparations; *P < 0.01.

 


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  		Fig. 6. Effect of SB-203580, a p38 MAPK inhibitor, on pressure-induced {alpha}-SMA expression. A: a representative Western blot showing increased {alpha}-SMA expression in PSCs treated with pressure alone, and a reduction in the pressure-induced increase in {alpha}-SMA expression to control levels in the presence of SB-203580. B: densitometry analyses of Western blot showed a significant increase in {alpha}-SMA expression in PSCs treated with pressure compared with control (absence of pressure). Treatment with SB-203580 abolished the pressure-induced increase in {alpha}-SMA expression in PSCs (n = 4 separate cell preparations; *P < 0.05).

 
Effect of pressure on collagen type I mRNA expression and collagen secretion. Collagen type I mRNA expression was studied at 6, 12, and 24 h after the application of pressure at 80 mmHg for 60 min, because collagen type I mRNA expression significantly increases 12 h after exposure to serum (5). Pressure increased mean collagen type I mRNA expression (Fig. 7A). At 24 h after the application of pressure, collagen type I mRNA expression significantly increased compared with controls (absence of pressure). Pressure also enhanced collagen secretion from PSCs (Fig. 7B).



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  		Fig. 7. Effect of pressure on collagen type I mRNA expression and collagen secretion by PSCs. A: after the application of pressure at 80 mmHg for 60 min, the level of collagen type I mRNA expression was examined by real-time PCR as described in MATERIALS AND METHODS. B: collagen secretion from PSCs into culture medium during 48 h of incubation after pressure was quantified with Sirius red dye. Results are expressed as %collagen concentration of controls (absence of pressure). Data are means ± SD (n = 4 PSCs). *P < 0.05 vs. control (absence of pressure).

 
Effect of pressure on activated TGF-{beta}1 secretion. We examined whether pressure application stimulates TGF-{beta}1 secretion from PSCs. TGF-{beta}1 concentration in the culture medium after pressure application for 6 and 12 h was significantly higher compared with controls (absence of pressure) (Table 1).


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  		Table 1. Activated TGF- {beta}1 secretion from PSCs

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we found that pressure promotes the activation of PSCs. It is possible that the increase of pancreatic tissue pressure in chronic pancreatitis may be one of the predominant factors promoting a fibrotic response in PSCs.

Mechanical forces are important regulators of cell proliferation and differentiation in a variety of mammalian cells (18), and responses of cells to mechanical forces may contribute to pathological states, such as cardiac hypertrophy (42) and atherosclerosis (48). Our results suggest that mechanical force, such as pressure, is also an important regulator of PSC activation. There are at least two mechanisms responsible for the pressure-induced PSC activation. The first mechanism is the direct effect of pressure on PSCs via p44/42 and p38 MAPK. It is well known that the Ras-Raf-p44/42 signal transduction pathway plays a central role in the regulation of cell growth and differentiation in many types of cells (10). Moreover, mechanical force increases cell number through the activation of p44/42 MAPK in cultured vascular smooth muscle (51), mesangial (25), epithelial (8), and endothelial cells (47). Our data are in agreement with these findings. In recent years, there has been increasing interest in the evaluation of the signaling pathways mediating PSC activation in response to specific stimuli. In fact, PDGF, ethanol, and acetaldehyde activate the p44/42 MAPK pathway in PSCs (21, 33). In the present study, we have demonstrated that pressure also stimulates PSC proliferation through the p44/42 MAPK pathway, suggesting that increase of pancreatic tissue pressure in chronic pancreatitis may be implicated in the development of pancreatic fibrosis.

We have shown that pressure activates not only p44/42 MAPK but also p38 MAPK in PSCs, and that pressure transforms PSCs into a myofibroblastic phenotype, which exhibits positive staining for the cytoskeletal protein {alpha}-SMA through p38 MAPK pathway. This study provides strong evidence that p38 MAPK plays a role in mediating pressure-induced {alpha}-SMA expression in PSCs. Our results are in agreement with other studies (40, 44) showing that p38 MAPK mediates {alpha}-SMA expression in activated hepatic stellate cells (HSCs). In vascular smooth muscle cells, {alpha}-SMA expression is regulated through the activation of {alpha}-SMA promoter, and inhibition of p38 MAPK decreased arginine vasopressin-stimulated {alpha}-SMA promoter activity (16). In PSCs, the p38 MAPK pathway is also shown to mediate ethanol- and acetaldehyde-induced increases in {alpha}-SMA expression (33). Along this line, it appears that pressure-induced {alpha}-SMA expression is regulated, in part, through the activation of p38 MAPK in PSCs.

The second mechanism responsible for pressure-induced PSC activation is the indirect effect of pressure on PSCs mediated by TGF-{beta}1. We have shown that pressure stimulated secretion of the activated TGF-{beta}1 into the medium. This is in agreement with the previous study (43) in rat HSCs that mechanical force induces TGF-{beta} synthesis. TGF-{beta}1 plays a profibrotic role in a variety of organs including the liver, kidneys, and pancreas (14, 22, 45, 49). Overexpression of TGF-{beta}1 has been revealed in human chronic pancreatitis (17, 45, 49). Transgenic mice overexpressing TGF-{beta} in islet cells develop fibrosis of the exocrine pancreas. Moreover, inhibition of TGF-{beta} by anti-TGF-{beta} antibody reduced ECM production in rat cerulein pancreatitis (34). In vitro studies, TGF-{beta}1 promotes PSC activation and ECM production (3, 5, 26).

An excessive production of ECM proteins in PSCs is considered to be responsible for the development of chronic pancreatitis. Our results indicate that mechanical forces due to an increase in pancreatic tissue pressure has been proposed as one of the factors leading to an increase in the production of ECM proteins in PSCs. Similarly, application of mechanical force to vascular smooth muscle cells also increases collagen synthesis that was shown to be due to the actions of TGF-{beta} (46). In the present study, TGF-{beta}1 secretion from PSCs was elevated at 6 and 12 h after exposure, and {alpha}-SMA expression was increased at 12 h but not at 6 h after pressurization (Data not shown). Increases in collagen type I mRNA expression and collagen secretion were observed at 24 and 48 h after exposure, respectively. Thus the time course of these events suggest that the increase in pressure-induced TGF-{beta}1 secretion may initiate pancreatic fibrosis, in part by activating PSCs, which express collagen type I mRNA and secrete collagen. Mechanical force is shown to regulate autocrine growth factors, such as TGF-{beta}, IGF, and PDGF in vascular smooth muscle cells (12, 36, 46). Because it is well known that several growth factors, particularly TGF-{beta}1, have an activating effect on PSCs (21), we suggest that the mechanism of mechanical force-induced PSC activation may appear to involve the intermediary action of autocrine production of growth factors including TGF-{beta}1.

Pancreatic ductal and tissue pressure are increased in patients with chronic pancreatitis (20, 32). These events can be also observed in the animal model of chronic pancreatitis (23, 39). In this model, the restrictive fibrotic capsule enveloping the gland prevents dissipation of the main pancreatic duct pressure. This could account for the increase of tissue pressure in chronic pancreatitis. There is no trigger of the increase in tissue pressure, unless the fibrosis exists in the pancreas. However, if once the pancreatic fibrosis is revealed, pancreatic tissue pressure will be increased. Thereafter, increase of pancreatic tissue pressure might accelerate the development of pancreatic fibrosis via PSC activation. From our results, the increase of pancreatic tissue pressure is one of the important factors that may accelerate the development of pancreatic fibrosis in chronic pancreatitis.

The actual mechanisms underlying the pressure-induced enhancement of PSC activation have not been elucidated yet. Recently, investigators have identified how mechanical forces are sensed and transduced into biochemical signals by multiple pathways within the cardiac, vascular, and mesangial cells (19, 27, 42). The proliferative effect of mechanical force is dependent, at least in part, on the secretion of PDGF in response to the mechanical signal (51). The second pathway of importance uses integrin-mediated signaling to transmit mechanical force. This can be inhibited by blocking integrins with Arg-Gly-Asp (RGD) peptides (52). Integrins are likely to be key mechanosensors. Indeed, mechanical force resulted in activation of intracellular MAPK signaling pathways in cardiac fibroblasts, an effect clearly demonstrated to be integrin dependent (30). In parallel, ion channels and other unknown mechanical receptors presumably transduce the mechanical signal. Additional studies are required to determine how mechanical forces are sensed and transduced into biochemical signals in PSCs.

In summary, we reported the impact of mechanical force on PSC activation. We suggest that pressure has a profibrotic effect on PSCs, and these observations provide new insights for understanding the development of pancreatic fibrosis. Furthermore, these data indicate that the increased pancreatic tissue pressure that occurs in chronic pancreatitis might accelerate the development of pancreatic fibrosis.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Otsuki, Third Dept. of Internal Medicine, Univ. of Occupational and Environmental Health, Japan, School of Medicine, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan (E-mail: mac-otsk{at}med.uoeh-u.ac.jp)

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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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