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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
Division of Gastroenterology, Tohoku University Graduate School of Medicine, Sendai, Japan
Received February 19, 2004; accepted March 31, 2004.
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Abstract |
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-smooth muscle actin and produce extracellular matrix components. Activated PSCs are implicated in the pathogenesis of pancreatic fibrosis and inflammation. We here evaluated the effects of SP600125 (anthra[1,9-cd]pyrazole-6 (2H)-one), an inhibitor of c-Jun NH2-terminal kinase (JNK), on the activation of PSCs. PSCs were isolated from rat pancreas tissue and used in their culture-activated, myofibroblast-like phenotype unless otherwise stated. Activation of JNK was determined by Western blotting using anti-phosphospecific JNK and c-Jun antibodies. Activation of transcription factors was determined by electrophoretic mobility shift assay. The effects of SP600125 on the key parameters of activation (chemokine production, collagen production, and proliferation) were examined. The effect of SP600125 on the activation of freshly isolated PSCs in culture also was examined. Interleukin-1
activated both 46- and 54-kDa JNK, whereas platelet-derived growth factor-BB activated only 46-kDa JNK. SP600125 inhibited interleukin-1-induced JNK activity and activator protein-1 activation, but it did not affect the activation of extracellular-regulated kinase, p38 mitogen-activated protein kinase, and nuclear factor-
B. SP600125 inhibited platelet-derived growth factor-induced proliferation, inducible monocyte chemoattractant protein-1 production, and serum-induced type I collagen production. Although SP600125 did not inhibit the transformation, it attenuated the proliferation of freshly isolated PSCs in culture. Collectively, our results suggest a role of JNK in the activation of PSCs, and a potential application of JNK inhibitors for the treatment of pancreatic fibrosis and inflammation.
; Bachem et al., 1998). In normal pancreas, stellate cells are quiescent and can be identified by the presence of vitamin A-containing lipid droplets in the cytoplasm. In response to pancreatic injury or inflammation, they are transformed ("activated") from their quiescent phenotype into highly proliferative myofibroblast-like cells that express the cytoskeletal protein -smooth muscle actin (-SMA) and produce type I collagen and other extracellular matrix components. Many of the morphological and metabolic changes associated with the activation of PSCs in animal models of fibrosis also occur when these cells are grown in serumcontaining medium in culture on plastic. Therefore, culture of primary PSCs on plastic has been accepted as an established model that mimics the phenotypic changes that occur during the process of PSC activation after pancreatic injury (Apte et al., 1999). There is accumulating evidence that PSCs, like hepatic stellate cells, are responsible for the development of pancreatic fibrosis (Apte et al., 1998; Bachem et al., 1998; Haber et al., 1999). In addition, PSCs may participate in the pathogenesis of acute pancreatitis (Haber et al., 1999; Masamune et al., 2002c). The activation of signaling pathways such as p38 mitogen-activated protein (MAP) kinase (Masamune et al., 2003c) and Rho-Rho kinase (Masamune et al., 2003b) is likely to play a role in PSC activation. However, the precise intracellular signaling pathways in PSCs are largely unknown. Obviously, the signaling mechanisms regulating the activation of PSCs are potential targets for the development of new treatments for pancreatic fibrosis and inflammation.
Members of the MAP kinase family, extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38 MAP kinase, are central elements that transduce the signal generated by growth factors, cytokines, and stress (Chang and Karin, 2001). Each member of this kinase family is activated by phosphorylation and subsequently translocates into the cell nucleus. Once in the nucleus, it phosphorylates and activates transcription factors such as activator protein-1 (AP-1) and nuclear factor B (NF-B), ultimately resulting in the transcription of specific genes. JNK, also called stress-activated protein kinase, is activated by external stimuli through a kinase cascade (Davis, 2000; Chang and Karin, 2001). Once activated, JNK phosphorylates serine-63 and -73 residues of c-Jun and increases the transcription activity of the AP-1 complex (Davis, 2000; Chang and Karin, 2001). JNK is encoded by three genes: JNK-1, JNK-2, and JNK-3 (Davis, 2000). Each JNK may be expressed as either a full-length (54-kDa) protein or as a C-terminally truncated (46-kDa) form that arises from differential mRNA splicing (Davis, 2000). The JNK-1 and JNK-2 genes are expressed ubiquitously. In contrast, the JNK-3 gene has a more limited pattern of expression and is largely restricted to brain, heart, and testis (Davis, 2000). MAP kinases play a role in a variety of cellular processes, including cell proliferation, cell survival, apoptosis, and cytokine production (Davis, 2000; Chang and Karin, 2001). We have previously reported that activation of ERK plays a central role in platelet-derived growth factor (PDGF)-induced proliferation of PSCs (Masamune et al., 2003a) and that p38 MAP kinase plays a role in proliferation, collagen production, and chemokine production (Masamune et al., 2003c). In addition, p38 MAP kinase plays a role in the transformation of quiescent PSCs to activated, myofibroblast-like phenotype (Masamune et al., 2003c). On the other hand, little is known about the activation of JNK or about the signaling outcomes of this pathway in PSCs, at least in part due to the lack of specific inhibitors. We here evaluated the effects of SP600125 (anthra[1,9-cd]pyrazole-6 (2H)-one), a newly developed inhibitor of JNK (Bennett et al., 2001), on the activation parameters of PSCs.
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Materials and Methods |
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-32P]ATP was obtained from Amersham Biosciences UK, Ltd. (Buckinghamshire, Little Chalfont, UK). Rat recombinant PDGF-BB was purchased from R&D Systems (Minneapolis, MN). Recombinant human interleukin (IL)-1 and tumor necrosis factor (TNF)- were obtained from Roche Applied Science (Mannheim, Germany). Rabbit antibodies against phosphorylated JNK, phosphorylated ERK, phosphorylated p38 MAP kinase, phosphorylated c-Jun, phosphorylated p70 S6 kinase, total JNK, total ERK, total p38 MAP kinase, and inhibitor of NF-B(IB)- were purchased from Cell Signaling Technology, Inc. (Beverly, MA). Rabbit antibody against glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was obtained from Trevigen (Gaithersburg, MD). SP600125 and SB203580 [4-(4-flurophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)imidazole] were purchased from Calbiochem (La Jolla, CA). All other reagents were obtained from Sigma-Aldrich (St. Louis, MO) unless specifically described.
Cell Culture. All animal procedures were performed in accordance with the National Institutes of Health Animal Care and Use Guidelines. Rat PSCs were prepared from the pancreas tissues of male Wistar rats (Japan SLC Inc., Hamamatsu, Japan) weighing 200 to 250 g as described previously using the Nycodenz solution (Nycomed Pharma, Oslo, Norway) after perfusion with 0.03% collagenase P (Masamune et al., 2003a). The cells were resuspended in Ham's F-12 containing 10% heat-inactivated fetal bovine serum (ICN Biomedicals, Aurora, OH), penicillin sodium, and streptomycin sulfate. Cell purity was always more than 90% as assessed by a typical star-like configuration and by detecting vitamin A autofluorescence. All experiments were performed using cells between passages 2 and 5 except for those using freshly isolated PSCs. Unless specifically described, we incubated PSCs in serum-free medium for 24 h before the addition of experimental reagents.
Western Blotting. The level of activated, phosphorylated JNK was determined by Western blotting as described previously (Masamune et al., 2002d). Briefly, cells were lysed in sodium dodecyl sulfate buffer (62.5 mM Tris-HCl at pH 6.8, 2% sodium dodecyl sulfate, 10% glycerol, 50 mM dithiothreitol, and 0.1% bromphenol blue) for 15 min on ice. The samples were then sonicated for 2 s, heated for 5 min, and centrifuged at 12,000g for 5 min to remove insoluble cell debris. Cellular proteins (approximately 100 µg) were fractionated on a 10% sodium dodecyl sulfate-polyacrylamide gel. They were transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA), and the membrane was incubated overnight at 4°C with rabbit anti-phosphospecific JNK antibody. After incubation with peroxidase-conjugated goat anti-rabbit IgG antibody for 1 h, proteins were visualized using an ECL kit (Amersham Biosciences UK, Ltd.). Levels of total JNK, ERK (phosphorylated and total), p38 MAP kinase (phosphorylated and total), phosphorylated c-Jun, phosphorylated p70 S6 kinase, IB-, and G3PDH were determined in a similar manner.
Cell Viability Assay. Cell viability was assessed by the MTT assay. After treatment with SP600125 at the indicated concentrations for 72 h, MTT solution was added to the cells at a final concentration of 500 µg/ml and the incubation continued at 37°C for 4 h. After the incubation period, the medium was aspirated and the formazan product was solubilized with dimethyl sulfoxide. Cell viability was determined by the differences in absorbance at wavelength 570 minus 690 nm.
Electrophoretic Mobility Shift Assay. After 1-h incubation with IL-1, approximately 5 x 106 cells were harvested, nuclear extracts were prepared, and electrophoretic mobility shift assay was performed as described previously (Masamune et al., 1996). Double-stranded oligonucleotide probes for NF-B (5'-AGTTGAGGGGACTTTCCCAGGC-3') and AP-1 (5'-CGCTTGATGAGTCAGCCGGAA-3') were end-labeled with [-32P]ATP. Nuclear extracts (approximately 5 µg) were incubated with the labeled oligonucleotide probe for 20 min at 22°C and electrophoresed through a 4% polyacrylamide gel. Gels were dried and autoradiographed at –80°C overnight. A 100-fold excess of unlabeled oligonucleotide was incubated with nuclear extracts for 10 min before the addition of the radiolabeled probe in the competition assays.
Cell Proliferation Assay. Serum-starved PSCs (approximately 80% density) were treated with PDGF-BB (at 25 ng/ml) in the presence of SP600125 at the indicated concentrations. Cell proliferation was assessed using a commercial kit [cell proliferation enzyme-linked immunosorbent assay, 5-bromo-2'-deoxyuridine (BrdU); Roche Applied Science] according to the manufacturer's instruction. This is a colorimetric immunoassay based on the measurement of BrdU incorporation during DNA synthesis (Porstmann et al., 1985). After 24-h incubation with experimental reagents, cells were labeled with BrdU for 3 h at 37°C. Cells were fixed and incubated with peroxidase-conjugated anti-BrdU antibody. Then, the peroxidase substrate 3,3',5,5'-tetramethylbenzidine was added, and BrdU incorporation was quantitated by differences in absorbance at wave-length 370 minus 492 nm.
Monocyte Chemoattractant Protein (MCP)-1 Assay. After a 24-h incubation, cell culture supernatants were harvested and stored at –80°C until the measurement. MCP-1 level in the culture supernatants was measured by enzyme-linked immunosorbent assay (Pierce Chemical, Rockford, IL) according to the manufacturer's instruction.
Collagen Assay. PSCs were incubated in serum-free medium or in medium containing 5% fetal bovine serum in the presence or absence of SP600125 for 48 h. Type I collagen released into the culture supernatant was quantified by enzyme-linked immunosorbent assay as described previously (Moshage et al., 1990). Briefly, immunoassay plates (BD Biosciences, Franklin Lakes, NJ) were coated with diluted samples overnight at 4°C. After blocking with 5% dry milk in phosphate-buffered saline, plates were incubated with goat anti-rat type I collagen antibody (Southern Biotechnology Associates, Birmingham, AL). After washes, rabbit anti-goat IgG antibody conjugated with alkaline phosphatase was added, and incubated. Finally, P-nitrophenylphosphate was added as a substrate, and the collagen levels were determined by differences in absorbance at wavelength 405 minus 690 nm. Rat tail collagen type I was used as a standard. The collagen levels in each sample were normalized to the cellular DNA content, which was determined by a fluorometric assay, according to the method of Brunk et al. (1979). The results are expressed as a percentage of the untreated control.
Effect of SP600125 on Spontaneous Activation of PSCs in Culture. Freshly isolated PSCs were incubated with or without SP600125 (at 5 µM) in serum-free medium for 1 h and then fetal bovine serum was added at the final concentration of 5%. After 7-day incubation, morphological changes characteristic of PSC activation were assessed after staining with glial fibrillary acidic protein (GFAP) as described previously (Masamune et al., 2003d) using a streptavidin-biotin-peroxidase complex detection kit (Histofine kit; Nichirei, Tokyo, Japan). Briefly, cells were fixed with ice-cold methanol and then endogenous peroxidase activity was blocked by incubation in methanol with hydrogen peroxide for 5 min. After immersion in normal rabbit serum, the slides were incubated with mouse anti-GFAP antibody overnight at 4°C. The slides were incubated with biotinylated goat anti-mouse Ig antibody, followed by peroxidase-conjugated streptavidin. Finally, color was developed by incubating the slides for several minutes with diaminobenzidine (Dojindo). As a control, the primary antibody was replaced with phosphate-buffered saline. In addition, total cellular proteins (approximately 25 µg) were prepared after 7-day incubation, and the levels of -SMA and G3PDH were determined by Western blotting.
Statistical Analysis. The results were expressed as mean ± S.D. Luminograms and autoradiograms are representative of at least three experiments. Differences between experimental groups were evaluated by the two-tailed unpaired Student's t test. A p value less than 0.05 was considered statistically significant.
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and PDGF-BB Activated JNK. We first determined whether IL-1 and PDGF-BB, which play important roles in proinflammatory responses (Masamune et al., 2002c) and proliferation (Apte et al., 1999; Masamune et al., 2003a), respectively, activated JNK in PSCs. We assessed the activation of JNK by Western blotting using anti-phosphospecific JNK antibody. The antibody recognizes only phosphorylated forms of JNK, thus allowing the assessment of activation of this kinase. IL-1 induced phosphorylation of both 46- and 54-kDa isoforms of JNK in a time-dependent manner (Fig. 1A). PDGF-BB induced phosphorylation of 46-, but not 54-kDa isoform of JNK (Fig. 1B).
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SP600125 Inhibited JNK Activity. To elucidate the roles of JNK for cell functions in PSCs, we used SP600125, a specific inhibitor of JNK (Bennett et al., 2001). We first examined the effect of SP600125 on the cell viability of PSCs. PSCs were incubated with increasing concentrations of SP600125 in serum-free medium for 72 h, and the cell viability was assessed by MTT assay. SP600125 up to 10 µM did not alter the cell viability, but above 10 µM, SP600125 was cytotoxic (Fig. 2). The results were confirmed by trypan blue dye exclusion test (data not shown). Based on these results, we used SP600125 up to 10 µM in the subsequent experiments.
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To show the inhibitory effect of SP600125 on JNK activity, phosphorylation of c-Jun, a downstream target of JNK, at serine-63 was assessed by Western blotting. In agreement with the result of JNK phosphorylation, IL-1 induced phosphorylation of c-Jun in a time-dependent manner (Fig. 3A). IL-1-induced c-Jun phosphorylation was inhibited by SP600125 in a dose-dependent manner (Fig. 3B). SP600125 at 10 µM almost completely abolished phosphorylation of c-Jun. Phosphorylation of c-Jun by PDGF was inhibited in a similar manner (data not shown).
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Previous studies have shown that cross talk may exist between JNK and other signaling pathways such as ERK and p38 MAP kinase (Davis, 2000; Chang and Karin, 2001). In addition, SP600125 might inhibit protein kinases other than JNK (Bain et al., 2003). We examined the effect of SP600125 on the activation of ERK, p38 MAP kinase, and p70 S6 kinase. IL-1-induced phosphorylation of ERK and p38 MAP kinase was not affected by SP600125 (Fig. 3C). PDGF-induced phosphorylation of p70 S6 kinase was not affected by SP600125 (Fig. 3D), suggesting that SP600125 specifically inhibited JNK activity and JNK was not located upstream of ERK, p38 MAP kinase, or p70 S6 kinase.
SP600125 Decreased AP-1, but not NF-B, Binding Activity. It is known that JNK pathway plays a role in the activation of transcription factors such as AP-1 and NF-B (Davis, 2000; Chang and Karin, 2001). We examined the role of JNK in the activation of these transcription factors in PSCs. IL-1 increased the AP-1- and NF-B-binding activities (Fig. 4, A and B). The specificity of these binding activities was confirmed by competition assays using 100-fold excess of unlabeled oligonucleotides (data not shown). SP600125 decreased the AP-1 binding activity, whereas NF-B binding activity was not altered. Phosphorylation and degradation of IB- are necessary for the activation of NF-B (Grilli et al., 1993). We examined the effect of SP600125 on the IL-1-induced degradation of IB- by Western blotting. IL-1 induced degradation of IB-, which was not inhibited by SP600125 (Fig. 4C), further supporting that SP600125 did not affect NF-B activation.
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SP600125 Did Not Alter the Expression of -SMA. It has been shown that culture-activated PSCs express -SMA, and -SMA expression has been accepted as a marker of PSC activation (Apte et al., 1998; Bachem et al., 1998). -SMA expression was confirmed in culture-activated PSCs by Western blotting (Fig. 5). As reported previously (Masamune et al., 2003c), a specific p38 MAP kinase inhibitor SB203580 decreased -SMA expression, whereas SP600125 did not.
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SP600125 Inhibited PDGF-Induced Proliferation. In agreement with the previous studies showing that PDGF-BB is a potent mitogen of PSCs in vitro (Apte et al., 1999; Masamune et al., 2003a), PDGF-BB significantly increased proliferation of PSCs (Fig. 6). PDGF-induced PSC proliferation was inhibited by SP600125 in a dose-dependent manner. The inhibitory effect was significant at as low as 1 µM.
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SP600125 Inhibited MCP-1 Expression. Activated PSCs may acquire the ability to modulate the recruitment and activation of inflammatory cells at least in part through the expression of MCP-1 (Masamune et al., 2002a). To clarify the role of JNK in MCP-1 expression, PSCs were treated with IL-1 or TNF- in the presence of SP600125. IL-1 and TNF- induced MCP-1 production, and SP600125 significantly, but not completely, decreased the inducible MCP-1 production (Fig. 7).
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SP600125 Inhibited Type I Collagen Production. It has been accepted that PSCs are the principal source of collagen in the fibrotic pancreas (Haber et al., 1999). We examined the effect of SP600125 on the serum-induced type I collagen release from PSCs into the culture supernatant by enzyme-linked immunosorbent assay. SP600125 decreased the type I collagen production in a dose-dependent manner (Fig. 8).
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SP600125 Did Not Block the Transformation, but Inhibited the Proliferation of Freshly Isolated PSCs. We finally examined whether SP600125 blocked the transformation of PSCs from quiescent to myofibroblast-like phenotype in culture. Freshly isolated PSCs were incubated with SP600125 (at 5 µM) in 5% serum-containing medium for 7 days. Morphological changes characteristic of PSC activation were assessed after staining with GFAP. After 7 days, PSCs treated with serum-free medium only were small and circular, with lipid droplets present in many cells and with slender dendritic processes (Fig. 9A). PSCs treated with 5% serum-containing medium showed transformation into cells with a myofibroblast-like phenotype (Fig. 9B). Regardless of the presence of SP600125, PSCs showed transformation into cells with a myofibroblast-like phenotype (Fig. 9, C and D). Indeed, -SMA expression was induced regardless of the SP600125 treatment, further indicating the transformation to a myofibroblast-like phenotype (Fig. 9E). It should be noted that serum-induced PSC proliferation was inhibited by SP600125 (Fig. 9, C and F). Thus, SP601025 did not inhibit the transformation, but it did inhibit the proliferation of freshly isolated PSCs.
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and PDGF-BB in PSCs. JNK has apparently molecular masses of 46 and 54 kDa that are largely, but not exclusively, composed of the JNK1 and JNK2 isoforms, respectively (Ip and Davis, 1998). Interestingly, the pattern of JNK activation was different between IL-1 and PDGF-BB; IL-1 activated both 46- and 54-kDa JNK, whereas PDGF-BB activated only 46-kDa JNK. The individual JNK proteins are highly homologous and the functional differences between each isoform remain unclear. To elucidate the role of JNK in PSCs, we used SP600125, a novel inhibitor of JNK (Bennett et al., 2001). We here showed that SP600125 inhibited key parameters of PSC activation, including proliferation, MCP-1 production, and type I collagen production. These effects seem not to be due to nonspecific cytotoxicity, because the concentrations of the reagents used in this study did not cause cell death. In hepatic stellate cells, Reeves et al. (2000) suggested that JNK might be involved for the activation of hepatic stellate cells. They reported that constitutive activity of JNK was higher in transformed cells compared with quiescent cells. Due to the lack of the specific JNK inhibitor at that time, role of JNK in cell functions was not further pursued.
Activated PSCs may acquire the ability to modulate the recruitment and activation of inflammatory cells. MCP-1 expression by myofibroblasts has been shown to be increased in fibrous tissue sections from patients with chronic pancreatitis (Saurer et al., 2000). Furthermore, MCP-1 may act as a fibrosis-promoting chemokine; MCP-1 stimulated collagen gene expression via endogenous up-regulation of transforming growth factor- in rat lung fibroblasts (Gharaee-Kermani et al., 1996). Therefore, control of MCP-1 expression is an important therapeutic target for pancreatic fibrosis as well as inflammation. We have previously shown that IL-1 and TNF- induced expression of MCP-1 in PSCs (Masamune et al., 2002a). SP600125 here inhibited IL-1- or TNF--induced MCP-1 expression, suggesting that JNK is required for optimal MCP-1 expression in PSCs. This is in agreement with the previous report showing that JNK was necessary for IL-1-induced MCP-1 expression in human mesangial cells (Lee et al., 2003). We have previously reported that ethanol and acetaldehyde at clinically relevant concentrations activated JNK, but they failed to induce MCP-1 expression in PSCs (Masamune et al., 2002b). Thus, activation of JNK is required but not sufficient for optimal MCP-1 expression in PSCs.
JNK plays a key role is the regulation of AP-1 transcription activity (Chang and Karin, 2001). The JNK substrate c-Jun forms homodimers or heterodimers with c-Jun or c-Fos to form the AP-1 complex (Chang and Karin, 2001). SP600125 here decreased the AP-1 binding activity, suggesting a role of JNK for IL-1-induced AP-1 activation. On the other hand, cross talk between the MAP kinase and NF-B pathways has been demonstrated in recent studies (Lee et al., 1998). We examined whether JNK activation by IL-1 is an upstream signaling event in the pathway leading to NF-B activation and consequent MCP-1 production in PSCs. Although SP600125 was effective in blocking MCP-1 production, it did not have any apparent effect on IL-1-induced NF-B binding activity or IB- degradation. Thus, activation of JNK is not required for IL-1-induced NF-B activation in PSCs. Because inhibition of NF-B by pyrrolidine dithiocarbamate abolished MCP-1 production (Masamune et al., 2002a), JNK and NF-B pathways may exert independent regulatory effects on IL-1-induced MCP-1 production.
Although SP600125 has been widely used as a JNK inhibitor, a recent study showed that SP600125 inhibited several other kinases in vitro, including p70 S6 kinase, AMP-dependent protein kinase, and cyclin-dependent protein kinase 2/cyclin A (Bain et al., 2003). In this study, SP600125 did not inhibit the activation of ERK, p38 MAP kinase, p70 S6 kinase, and NF-B. But, it is still possible that SP600125 exhibited the observed effects through the inhibition of other kinases. The development of more selective JNK inhibitors will clarify this issue.
Previous studies assessing the role of the JNK pathway in type I collagen production have reported conflicting results. In human mesangial cells, transient transfection of a dominant negative mutant of JNK had no effect on transforming growth factor-1-induced 1(I) collagen gene transcription (Hayashida et al., 1999). Ultraviolet irradiation-induced c-Jun interfered with procollagen gene transcription and reduced type I procollagen mRNA levels in human skin (Fisher et al., 2000). In hepatic stellate cells, acetaldehyde induced 1(I) collagen gene expression through the activation of JNK in rat hepatic stellate cells (Chen and Davis, 2000). Nevertheless, in quiescent hepatic stellate cells, inhibition of JNK did not decrease, but increased steady-state level of 1(I) collagen mRNA, whereas the effect was less pronounced in activated hepatic stellate cells (Schnabl et al., 2001). Thus, the role of JNK in collagen production depends on the type of cells and of stimuli. Chen and Davis (2000) reported that JNK used the basic transcription element binding protein binds to a distal GC box in the 5' upstream region and mediated acetaldehyde-induced, JNK-dependent 1(I) collagen gene expression in rat hepatic stellate cells. Further studies are necessary to clarify the detailed mechanisms by which SP600125 decreased type I collagen production in PSCs.
c-Jun and JNK have been implicated as a positive regulator of cell proliferation and of G1-to-S phase progression (Bost et al., 1997; Behrens et al., 2001). Fibroblasts with only mutated alleles of c-Jun containing serine-to-alanine substitutions at 63 and 73 have a proliferation defect, but they grow faster than c-Jun null cells (Behrens et al., 2001). Inhibition of JNK blocked epidermal growth factor-stimulated growth but not basal growth in human A549 lung carcinoma cells (Bost et al., 1997). On the other hand, it also has been reported that the requirement for c-Jun during the progression through G1 phase in fibroblasts is independent of phosphorylation of serine-63 and -73 (Wisdom et al., 1999). Activation of JNK has been shown to play a role in the apoptosis induction (Chang and Karin, 2001). Thus, again, it seems that the role of JNK pathway and of c-Jun in proliferation is cell type- and stimuli-specific. Our study shows that SP600125 inhibited PSC proliferation independently of the activation stage, as in the case of hepatic stellate cells (Schnabl et al., 2001).
The potential of JNK inhibitors as therapeutics in inflammatory, vascular, neurodegenerative, diabetic, and oncological diseases has attracted considerable interest (Manning and Davis, 2003). SP600125 has been shown to reduce paw swelling in a rat model of inflammatory arthritis (Han et al., 2001). Another JNK inhibitor curcumin has been shown to decrease the severity of amiodarone-induced pulmonary fibrosis in rats (Punithavathi et al., 2003). Because SP600125 blocked profibrogenic and proinflammatory actions in activated PSCs, it would be interesting to examine whether inhibition of JNK might provide new therapeutic strategies for pancreatic fibrosis and inflammation. On the other hand, we previously reported that troglitazone, a ligand of the peroxisome proliferator-activated receptor-, modulated profibrogenic and proinflammatory actions in a similar manner to SP600125 (Masamune et al., 2002a). Of note, inhibition of JNK can activate the peroxisome proliferator-activated receptor- (Camp et al., 1999). Recently, Shimizu et al. (2002) have reported that troglitazone prevented the progression of pancreatic inflammatory process and fibrosis in an animal model of chronic pancreatitis, suggesting that PSCs are potential targets of antifibrogenic and anti-inflammatory strategies in vivo. Experiments designed to test this hypothesis are under way in our laboratory.
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Footnotes |
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ABBREVIATIONS: PSC, pancreatic stellate cell; -SMA, -smooth muscle actin; MAP kinase, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal kinase; AP-1, activator protein-1; NF-B, nuclear factor-B; PDGF, platelet-derived growth factor; SP600125, anthra[1,9-cd]pyrazole-6 (2H)-one; MTT, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide; IL, interleukin; TNF, tumor necrosis factor; IB, inhibitor of nuclear factor-B; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; SB203580, 4-(4-flurophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)imidazole; BrdU, bromo-2'-deoxyuridine; GFAP, glial fibrillary acidic protein; MCP-1, monocyte chemoattractant protein-1.
Address correspondence to: Dr. Atsushi Masamune, Division of Gastroenterology, Tohoku University Graduate School of Medicine, 1-1 Seiryo-cho, Aoba-ku, Sendai 980-8574 Japan. E-mail: amasamune{at}int3.med.tohoku.ac.jp
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References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Apte MV, Haber PS, Applegate TL, Norton ID, McCaughan GW, Korsten MA, Pirola RC, and Wilson JS (1998) Periacinar stellate-shaped cells in rat pancreas: identification, isolation and culture. Gut 43: 128–133.
Apte MV, Haber PS, Darby SJ, Rodgers SC, McCaughan GW, Korsten MA, Pirola RC, and Wilson JS (1999) Pancreatic stellate cells are activated by proinflammatory cytokines: implications for pancreatic fibrogenesis. Gut 44: 534–541.
Bachem MG, Schneider E, Gross H, Weidenbach H, Schmidt RM, Menke A, Siech M, Beger H, Grunert A, and Adler G (1998) Identification, culture and characterization of pancreas stellate cells in rats and humans. Gastroenterology 115: 421–432.[CrossRef][Medline]
Bain J, McLauchlan H, Elliott M, and Cohen P (2003) The specificities of protein kinase inhibitors: an update. Biochem J 371: 199–204.[CrossRef][Medline]
Behrens A, Sabapathy K, Graef I, Cleary M, Crabtree GR, and Wagner EF (2001) Jun N-terminal kinase 2 modulates thymocyte apoptosis and T cell activation through c-Jun and nuclear factor of activated T cell (NF-AT). Proc Natl Acad Sci USA 98: 1769–1774.
Bennett BL, Sasaki DT, Murray BW, O'Leary EC, Sakata ST, Xu W, Leisten JC, Motiwala A, Pierce S, Satoh Y, et al. (2001) SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase. Proc Natl Acad Sci USA 98: 13681–13686.
Bost F, McKay R, Dean N, and Mercola D (1997) The JUN kinase/stress-activated protein kinase pathway is required for epidermal growth factor stimulation of growth of human A549 lung carcinoma cells. J Biol Chem 272: 33422–33429.
Brunk KC, Jones KC, and James TW (1979) Assay for nanogram quantities of DNA in cellular homogenates. Anal Biochem 2: 497–500.
Camp HS, Tafuri SR, and Leff T (1999) c-Jun N-terminal kinase phosphorylates peroxisome proliferator-activated receptor-gamma1 and negatively regulates its transcriptional activity. Endocrinology 140: 392–397.
Chang L and Karin M (2001) Mammalian MAP kinase signalling cascades. Nature (Lond) 410: 37–40.[CrossRef][Medline]
Chen A and Davis BH (2000) The DNA binding protein BTEB mediates acetaldehyde-induced, Jun N-terminal kinase-dependent alphaI(I) collagen gene expression in rat hepatic stellate cells. Mol Cell Biol 20: 2818–2826.
Davis R (2000) Signal transduction by the JNK group of MAP kinases. Cell 103: 239–252.[CrossRef][Medline]
Fisher GJ, Datta S, Wang Z, Li XY, Quan T, Chung JH, Kang S, and Voorhees JJ (2000) c-Jun-dependent inhibition of cutaneous procollagen transcription following ultraviolet irradiation is reversed by all-trans retinoic acid. J Clin Investig 106: 663–670.[Medline]
Gharaee-Kermani M, Denholm EM, and Phan SH (1996) Costimulation of fibroblast collagen and transforming growth factor beta1 gene expression by monocyte chemoattractant protein-1 via specific receptors. J Biol Chem 271: 17779–17784.
Grilli M, Chiu JJ-S, and Lenardo MJ (1993) NF-B and Rel: participants in a multiform transcriptional regulatory system. Int Rev Cytol 143: 1–62.[Medline]
Haber PS, Keogh GW, Apte MV, Moran CS, Stewart NL, Crawford DH, Pirola RC, McCaughan GW, Ramm GA, and Wilson JS (1999) Activation of pancreatic stellate cells in human and experimental pancreatic fibrosis. Am J Pathol 155: 1087–1095.
Han Z, Boyle DL, Chang L, Bennett B, Karin M, Yang L, Manning AM, and Firestein GS (2001) c-Jun N-terminal kinase is required for metalloproteinase expression and joint destruction in inflammatory arthritis. J Clin Investig 108: 73–81.[CrossRef][Medline]
Hayashida T, Poncelet AC, Hubchak SC, and Schnaper HW (1999) TGF-beta1 activates MAP kinase in human mesangial cells: a possible role in collagen expression. Kidney Int 56: 1710–1720.[CrossRef][Medline]
Ip YT and Davis RJ (1998) Signal transduction by the c-Jun N-terminal kinase (JNK) - from inflammation to development. Curr Opin Cell Biol 10: 205–219.[CrossRef][Medline]
Lee FS, Peters RT, Dang LC, and Maniatis T (1998) MEKK1 activates both IkappaB kinase alpha and IkappaB kinase beta. Proc Natl Acad Sci USA 95: 9319–9324.
Lee MJ, Yang CW, Jin DC, Chang YS, Bang BK, and Kim YS (2003) Bone morphogenetic protein-7 inhibits constitutive and interleukin-1 beta-induced monocyte chemoattractant protein-1 expression in human mesangial cells: role for JNK/AP-1 pathway. J Immunol 170: 2557–2563.
Manning AM and Davis RJ (2003) Targeting JNK for therapeutic benefit: from junk to gold? Nat Rev Drug Discov 2: 554–565.[CrossRef][Medline]
Masamune A, Igarashi Y, and Hakomori S (1996) Regulatory role of ceramide in interleukin (IL)-1 beta-induced E-selectin expression in human umbilical vein endothelial cells. J Biol Chem 271: 9368–9375.
Masamune A, Kikuta K, Satoh M, Kume K, and Shimosegawa T (2003a) Differential roles of signaling pathways for proliferation and migration of rat pancreatic stellate cells. Tohoku J Exp Med 199: 69–84.[CrossRef][Medline]
Masamune A, Kikuta K, Satoh M, Sakai Y, Satoh A, and Shimosegawa T (2002a) Ligands of peroxisome proliferator-activated receptor- block activation of pancreatic stellate cells. J Biol Chem 277: 141–147.
Masamune A, Kikuta K, Satoh M, Satoh A, and Shimosegawa T (2002b) Alcohol activates activator protein-1 and MAP kinases in rat pancreatic stellate cells. J Pharmacol Exp Ther 302: 36–42.
Masamune A, Kikuta K, Satoh M, Satoh K, and Shimosegawa T (2003b) Rho kinase inhibitors block activation of pancreatic stellate cells. Br J Pharmacol 140: 1292–1302.[CrossRef][Medline]
Masamune A, Sakai Y, Kikuta K, Satoh M, Satoh A, and Shimosegawa T (2002c) Activated rat pancreatic stellate cells express intercellular adhesion molecule-1 in vitro. Pancreas 25: 78–85.[CrossRef][Medline]
Masamune A, Satoh M, Kikuta K, Sakai Y, Satoh A, and Shimosegawa T (2003c) Inhibition of p38 mitogen-activated protein kinase blocks activation of rat pancreatic stellate cells. J Pharmacol Exp Ther 304: 8–14.
Masamune A, Satoh M, Kikuta K, Suzuki N, and Shimosegawa T (2003d) Establishment and characterization of a rat pancreatic stellate cell line by spontaneous immortalization. World J Gastroenterol 9: 2751–2758.[Medline]
Masamune A, Satoh K, Sakai Y, Yoshida M, Satoh A, and Shimosegawa T (2002d) Ligands of peroxisome proliferator-activated receptor- induce apoptosis in AR42J cells. Pancreas 24: 130–138.[CrossRef][Medline]
Moshage H, Casini A, and Lieber CS (1990) Acetaldehyde selectively increases collagen synthesis in cultured rat fat-storing cells but not in hepatocytes. Hepatology 12: 511–518.[Medline]
Punithavathi D, Venkatesan N, and Babu M (2003) Protective effects of curcumin against amiodarone-induced pulmonary fibrosis in rats. Br J Pharmacol 139: 1342–1350.[CrossRef][Medline]
Porstmann T, Ternynck T, and Avrameas S (1985) Quantitation of 5-bromo-2-deoxyuridine incorporation into DNA: an enzyme immunoassay for the assessment of the lymphoid cell proliferative response. J Immunol Methods 82: 169–179.[CrossRef][Medline]
Reeves HL, Dack CL, Peak M, Burt AD, and Day CP (2000) Stress-activated protein kinases in the activation of rat hepatic stellate cells in culture. J Hepatol 32: 465–472.[CrossRef][Medline]
Saurer L, Reber P, Schaffner T, Buchler MW, Buri C, Kappeler A, Walz A, Freiss H, and Mueller C (2000) Differential expression of chemokines in normal pancreas and in chronic pancreatitis. Gastroenterology 118: 356–367.[CrossRef][Medline]
Schnabl B, Bradham CA, Bennett BL, Manning AM, Stefanovic B, and Brenner DA (2001) TAK1/JNK and p38 have opposite effects on rat hepatic stellate cells. Hepatology 34: 953–963.[CrossRef][Medline]
Shimizu K, Shiratori K, Hayashi N, Kobayashi M, Fujiwara T, and Horikoshi H (2002) Thiazolidinedione derivatives as novel therapeutic agents to prevent the development of chronic pancreatitis. Pancreas 24: 184–190.[CrossRef][Medline]
Wisdom R, Johnson RS, and Moore C (1999) c-Jun regulates cell cycle progression and apoptosis by distinct mechanisms. EMBO (Eur Mol Biol Organ) J 18: 188–197.[CrossRef][Medline]
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