Am J Physiol Cell Physiol Watch the video to learn how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Cell Physiol 292: C259-C268, 2007. First published July 12, 2006; doi:10.1152/ajpcell.00030.2006
0363-6143/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/1/C259    most recent
00030.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via ISI Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Aoki, H.
Right arrow Articles by Sugano, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Aoki, H.
Right arrow Articles by Sugano, K.

RECEPTORS AND SIGNAL TRANSDUCTION

Cyclooxygenase-2 is required for activated pancreatic stellate cells to respond to proinflammatory cytokines

Hiroyoshi Aoki, Hirohide Ohnishi, Kouji Hama, Satoshi Shinozaki, Hiroto Kita, Hiroyuki Osawa, Hironori Yamamoto, Kiichi Sato, Kiichi Tamada, and Kentaro Sugano

Department of Gastroenterology, Jichi Medical School, Tochigi, Japan

Submitted 25 January 2006 ; accepted in final form 10 July 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cyclooxygenase-2 (COX-2) mediates various inflammatory responses and is expressed in pancreatic tissue from patients with chronic pancreatitis. To examine the role of COX-2 in chronic pancreatitis, we investigated its participation in regulating functions of pancreatic stellate cells (PSCs), using isolated rat PSCs. COX-2 was expressed in culture-activated PSCs but not in freshly isolated quiescent PSCs. TGF-beta1, IL-1beta, and IL-6 enhanced COX-2 expression in activated PSCs, concomitantly increasing the expression of {alpha}-smooth muscle actin ({alpha}-SMA), a parameter of PSC activation. The COX-2 inhibitor NS-398 blocked culture activation of freshly isolated quiescent PSCs. NS-398 also inhibited the enhancement of {alpha}-SMA expression by TGF-beta1, IL-1beta, and IL-6 in activated PSCs. These data indicate that COX-2 is required for the initiation and promotion of PSC activation. We further investigated the mechanism by which cytokines enhance COX-2 expression in PSCs. Adenovirus-mediated expression of dominant negative Smad2/3 inhibited the increase in expression of COX-2, {alpha}-SMA, and collagen-1 mediated by TGF-beta1 in activated PSCs. Moreover, dominant negative Smad2/3 expression attenuated the expression of COX-2 and {alpha}-SMA enhanced by IL-1beta and IL-6. Anti-TGF-beta neutralizing antibody also attenuated the increase in COX-2 and {alpha}-SMA expression caused by IL-1beta and IL-6. IL-6 as well as IL-1beta enhanced TGF-beta1 secretion from PSCs. These data indicate that Smad2/3-dependent pathway plays a central role in COX-2 induction by TGF-beta1, IL-1beta, and IL-6. Furthermore, IL-1beta and IL-6 promote PSC activation by enhancing COX-2 expression indirectly through Smad2/3-dependent pathway by increasing TGF-beta1 secretion from PSCs.

transforming growth factor-beta; interleukin; Smad; autocrine; pancreatic fibrosis

CYCLOOXYGENASE (COX) is the rate-limiting enzyme that catalyzes arachidonic acid conversion to prostaglandins. COX exists as two isoforms, COX-1 and COX-2. COX-1 is constitutively expressed in most types of cells and exerts such diverse physiological functions as gastric mucosal protection and platelet aggregation (9). In contrast, COX-2 is an inducible isoform in response to various proinflammatory and mitogenic stimuli. COX-2 induction leads to the increase in prostaglandin production that is responsible for the progression of inflammation. Recently, it has been shown that COX-2 is expressed in pancreatic tissue from patients with chronic pancreatitis (16, 26). The inflammation in the pancreatic tissue of such patients is caused by repeated acute pancreatic injury, mainly due to excessive alcohol intake (6). Although COX-2 expression in pancreatic tissue from patients with chronic pancreatitis suggests the participation of COX-2 in chronic pancreatic inflammation and fibrosis, the precise mechanism is unknown.

Pancreatic stellate cells (PSCs) have recently been identified, isolated, and characterized (2, 5). In the normal pancreas, PSCs possess fat droplets containing vitamin A and are quiescent (2). When cultured in vitro, PSCs are autoactivated (autotransformed), changing their morphological and functional features (5). PSCs commence losing vitamin A-containing lipid droplets, proliferate, express {alpha}-smooth muscle actin ({alpha}-SMA), and produce and secrete extracellular matrix components such as collagen and fibronectin. Thus PSCs are autotransformed to myofibroblast-like cells. In vivo, PSCs also are activated during pancreatic fibrosis in humans and animal models (12). Therefore, PSCs are thought to play an important role in pancreatic fibrogenesis in patients with chronic pancreatitis.

PSCs respond to various proinflammatory and profibrogenic cytokines including TGF-beta1, IL-1beta, and IL-6 (3, 22). Of these, a major profibrogenic cytokine, TGF-beta1, regulates multiple functions of PSCs. For example, TGF-beta1 1) stimulates extracellular matrix synthesis, 2) promotes PSC activation and increases {alpha}-SMA expression, 3) attenuates proliferation in an autocrine manner, and 4) reduces matrix metalloproteinase-3 and -9 expression (28). Furthermore, TGF-beta1 expression is upregulated in chronic pancreatitis tissues (12). Thus TGF-beta1 has been implicated in the etiology of pancreatic fibrosis.

TGF-beta1 intracellular signaling is mediated and modulated primarily by Sma- and Mad-related proteins (Smads) (13, 21). Upon TGF-beta1 binding to its receptor, Smad2 and Smad3 are phosphorylated by the receptor and form oligomeric complexes with Smad4; the complexes then translocate into the nucleus. These complexes subsequently activate the transcription of target genes. In addition to the Smad-dependent pathway, there exist Smad-independent TGF-beta signaling pathways, for example, mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase (4). Thus TGF-beta1 intracellular signaling occurs via Smad-dependent and Smad-independent pathways.

In this study, we examined the role of COX-2 in the regulation of PSC functions and found that COX-2 plays a pivotal role in the functional regulation of PSCs by multiple cytokines. We further showed that Smad2/3-dependent TGF-beta1 signaling is critical for the cytokine-mediated induction of COX-2 expression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. TGF-beta1, Nycodenz, pronase, and anti-{alpha}-SMA antibody were purchased from Sigma (St. Louis, MO). IL-1beta, IL-6, and anti-TGF-beta1 neutralizing antibody were obtained from R&D (Abrington, UK). DNase I was obtained from Roche (Basel, Switzerland). Collagenase P was obtained from Boehringer Mannheim (Mannheim, Germany). SB 203580, SP600125, and PD-98059 were obtained from Calbiochem (La Jolla, CA). NS-398, anti-COX-1, and anti-COX-2 antibodies were obtained from Cayman Chemical (Ann Arbor, MI). Anti-collagen 1 antibody was obtained from Rockland Immunochemicals (Gilbertsville, PA). Horseradish peroxidase (HRP)-conjugated donkey anti-goat IgG, HRP-conjugated donkey anti-mouse IgG, HRP-conjugated donkey anti-rabbit IgG, FITC-conjugated donkey anti-mouse IgG, and Cy3-conjugated donkey anti-rabbit IgG antibodies were obtained from Jackson ImmunoResearch (West Grove, PA).

Isolation and culture of rat pancreatic stellate cells. Rat PSCs were prepared as described previously (2). Briefly, rat pancreas was digested in Grey’s balanced salt solution supplemented with 0.05% collagenase P, 0.02% pronase, and 0.1% DNase. After filtration through nylon mesh, cells were centrifuged in a 13.2% Nycodenz gradient at 1,400 g for 20 min. PSCs in the band just above the interface of the Nycodenz solution and the aqueous layer were collected, washed, and resuspended in Iscove’s modified Dulbecco’s medium containing 10% fetal calf serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. PSCs were cultured in a 5% CO2 atmosphere at 37°C. The purity of isolated cells was confirmed to be >90% by observing their cytoplasmic droplets with vitamin A autofluorescence. We used PSCs between passages 2 and 3 as culture-activated PSCs in experiments, unless otherwise indicated.

Immunofluorescence microscopy. Cells were fixed with 2% formaldehyde in PBS, treated with Triton-X in PBS for 5 min, and incubated sequentially with blocking ACE (Snow Brand Milk Products, Tokyo, Japan), primary antibodies, and secondary antibodies. For double staining using anti-COX-2 and anti-{alpha}-SMA antibodies, Cy3-conjugated donkey anti-rabbit IgG and FITC-conjugated donkey anti-mouse IgG antibodies were used as secondary antibodies, respectively. Samples were examined under an Olympus BX51 microscope (Tokyo, Japan). Images were digitized and then processed using Photoshop 5.0 software (Adobe System, Mountain View, CA).

Western blotting. Western blotting was performed as described previously (23). Briefly, 10 µg of protein from whole cell lysates were separated on 10% SDS minigels and transferred to nitrocellulose membranes. Membranes were then probed with primary antibodies, followed by detection with peroxidase-conjugated secondary antibodies and visualization by enhanced chemiluminescence. Western blotting using anti-{alpha}-tubulin antibody was carried out as an internal control.

Densitometry. Western blot images were captured and digitized with a Fluor-S Multi-Imager (Bio-Rad, Hercules, CA). Densitometric evaluation of Western blot data was then carried out using Quantity One software (Bio-Rad).

Evaluation of prostaglandin E2 production. Production of prostaglandin E2 by PSCs was examined by determining the culture medium concentration of prostaglandin E2 released from PSCs, using a commercial enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturer’s instructions.

Adenovirus infection. Recombinant adenoviral vector for dominant negative Smad2/3 (AdDNSmad2/3) was kindly provided by Dr. K. Miyazono (University of Tokyo, Japan). Cells were infected with a recombinant adenoviral vector at a dose of 10 plaque-forming units per cell in the culture medium described above. An adenoviral vector expressing beta-galactosidase (AdLacZ) was used as an infection control.

Measurement of TGF-beta1 peptide secretion. TGF-beta1 peptide secretion was measured by determining its concentration in the culture medium with a commercial ELISA kit (DRG International, Mountainside, NJ) according to the manufacturer’s instructions.

Statistical analysis. The data were analyzed by one-way ANOVA with post hoc analysis using Dunnett’s test (see GoGoGoGoGoGoGoGoFig. 9) or Tukey’s test (see Figs. 5A and 6A) to determine statistical significance, and P < 0.05 was considered significant.


Figure 1
View larger version (59K):
[in this window]
[in a new window]

  		Fig. 1. Expression of cyclooxygenase (COX)-2 in activated pancreatic stellate cells (PSCs). A, a and c: Nomarski images of freshly isolated (a) and culture-activated PSCs (c); b, immunostaining of the same field as in a using anti-COX-2 antibody; d and e, double immunostaining of the same field as in c using anti-COX-2 antibody (d) and anti-{alpha}-smooth muscle actin ({alpha}-SMA) antibody (e). Bars, 10 µm. B: Western blotting of freshly isolated and culture-activated PSCs using primary antibodies against {alpha}-SMA, COX-2, and {alpha}-tubulin.

 

Figure 2
View larger version (29K):
[in this window]
[in a new window]

  		Fig. 2. Effect of TGF-beta1 on COX-2 expression and activity in PSCs. Activated PSCs were incubated with the indicated amounts of TGF-beta1 for 48 h. A: Western blotting was performed using anti-collagen-1, anti-{alpha}-SMA, anti-COX-1, anti-COX-2, and anti-{alpha}-tubulin antibodies. B: densitometry of Western blotting results shown in A. Values are means for 3 independent experiments. C: COX-2 activity was examined by determining the prostaglandin E2 (PGE2) concentration secreted into culture medium from activated PSCs during the incubation. Values are means ± SE for 3 independent experiments.

 

Figure 3
View larger version (42K):
[in this window]
[in a new window]

  		Fig. 3. Effects of IL-1beta and IL-6 on COX-2 expression and activity in PSCs. Activated PSCs were stimulated with the indicated amounts of IL-1beta or IL-6 for 48 h. A: Western blotting was performed using anti-COX-2, anti-{alpha}-SMA, and anti-{alpha}-tubulin antibodies. B: densitometry of Western blotting results shown in A. Values are means for 3 independent experiments. C: COX-2 activity was examined by determining the PGE2 concentration secreted into culture medium from activated PSCs during the incubation. Values are means ± SE for 3 independent experiments.

 

Figure 4
View larger version (36K):
[in this window]
[in a new window]

  		Fig. 4. Effect of COX-2 inhibitor NS-398 on activation of freshly isolated PSCs. Twelve hours after the isolation, 100 µM NS-398 was added into the culture medium or cells were left untreated. Western blotting was carried out using anti-{alpha}-SMA and anti-{alpha}-tubulin antibodies at indicated times after the isolation.

 

Figure 5
View larger version (23K):
[in this window]
[in a new window]

  		Fig. 5. Effect of COX-2 inhibitor NS-398 on COX-2 activity and {alpha}-SMA and collagen-1 expression in activated PSCs. PSCs were preincubated in the presence or absence of 100 µM NS-398 for 30 min, followed by stimulation with 30 pM TGF-beta1 for 48 h. A: COX-2 activity was examined by determining the PGE2 concentration secreted into culture medium from activated PSCs. Values are means ± SE for 3 independent experiments. *P < 0.05. N.S., not significant. B: Western blotting was performed using anti-{alpha}-SMA, anti-collagen-1, and anti-{alpha}-tubulin antibodies. C: densitometry of Western blotting results shown in B. Values are means for 3 independent experiments.

 

Figure 6
View larger version (36K):
[in this window]
[in a new window]

  		Fig. 6. Effect of COX-2 inhibitor NS-398 on COX-2 activity and {alpha}-SMA expression enhanced by IL-1beta or IL-6 in activated PSCs. PSCs were incubated in the presence or absence of 100 µM NS-398 for 30 min, followed by stimulation with 10 ng/ml IL-1beta or 100 ng/ml IL-6 for 48 h. A: COX-2 activity was determined by measuring secreted PGE2 concentration using the enzyme immunoassay kit. Values are means ± SE for 3 independent experiments. *P < 0.05. B: {alpha}-SMA expression was examined with Western blotting (top). Values for the densitometry of Western blotting (bottom) are means for 3 independent experiments.

 

Figure 7
View larger version (27K):
[in this window]
[in a new window]

  		Fig. 7. Effect of dominant negative Smad2/3 expression on the TGF-beta1-stimulated induction of COX-2, {alpha}-SMA, and collagen-1 in activated PSCs. A: PSCs were infected with adenovirus vector expressing dominant negative Smad2/3 (AdDNSmad2/3) or beta-galactosidase (AdLacZ). Twenty-four hours after infection, cells were incubated in the presence or absence of 30 pM TGF-beta1 for 48 h. Western blotting was carried out using anti-COX-2, anti-{alpha}-SMA, anti-collagen-1, and anti-{alpha}-tubulin antibodies. B: densitometry of Western blotting results shown in A. Values are means for 3 independent experiments.

 

Figure 8
View larger version (32K):
[in this window]
[in a new window]

  		Fig. 8. Effect of dominant negative Smad2/3 expression on IL-1beta- or IL-6-stimulated induction of COX-2 and {alpha}-SMA in activated PSCs. A: PSCs were infected with AdDNSmad2/3 or AdLacZ. Twenty-four hours after infection, cells were incubated in the presence or absence of 10 ng/ml IL-1beta or 100 ng/ml IL-6 for 48 h. Western blotting was carried out using anti-COX-2, anti-{alpha}-SMA, and anti-{alpha}-tubulin antibodies. B: densitometry of Western blotting results shown in A. Values are means for 3 independent experiments.

 

Figure 9
View larger version (11K):
[in this window]
[in a new window]

  		Fig. 9. Effect of exogenous IL-6 on TGF-beta1 secretion from activated PSCs. Cells were stimulated with the indicated amounts of IL-6 for 48 h. TGF-beta1 secretion was then examined using ELISA. Values are means ± SE for 3 independent experiments. *P < 0.05 vs. control.

 

   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
COX-2 expression in PSCs. In our initial attempt to examine the role of COX-2 in the regulation of PSC functions, we examined the expression of COX-2 in both quiescent and culture-activated PSCs. Freshly isolated PSCs were used for experiments 12 h after isolation. Immunocytochemistry using anti-COX-2 antibody showed that COX-2 was not expressed in freshly isolated quiescent PSCs as determined by the presence of lipid droplets (Fig. 1A, a and b). In contrast, COX-2 was expressed in culture-activated PSCs as determined by their absence of droplets, fibroblast-like shape, and {alpha}-SMA expression, a parameter of PSC activation (5) (Fig. 1A, c–e). In addition, Western blotting revealed that freshly isolated PSCs did not express either COX-2 or {alpha}-SMA (Fig. 1B). However, culture-activated PSCs expressed both COX-2 and {alpha}-SMA (Fig. 1B). These data suggest that COX-2 expression is induced in PSCs during their activation.

TGF-beta1, IL-1beta, and IL-6 enhanced the expression and the activity of COX-2 in activated PSCs. Since TGF-beta1 plays a central role in regulating PSC functions such as increasing collagen and {alpha}-SMA expression (3), we next examined the effect of TGF-beta1 on the expression and activity of COX-2 in activated PSCs. As shown in Fig. 2, A and B, TGF-beta1 enhanced COX-2 expression in activated PSCs concurrently with an increase in {alpha}-SMA and collagen-1 expression in a dose-dependent manner. In contrast, the expression of COX-1, which occurs constitutively in various types of cells, and {alpha}-tubulin was not altered by TGF-beta1. Furthermore, TGF-beta1 augmented COX-2 activity in PSCs (Fig. 2C). In addition to responding to TGF-beta1, PSCs also respond to various proinflammatory cytokines, including IL-1beta and IL-6 (22). We therefore examined the effect of these cytokines on COX-2 expression and activity in PSCs. As shown in Fig. 3, both IL-1beta and IL-6 increased COX-2 expression (A and B) and COX-2 activity (C) while concomitantly enhancing {alpha}-SMA expression (A and B) in activated PSCs. These data suggest the possibility that COX-2 may mediate the regulation of activated PSC functions by TGF-beta1, IL-1beta, and IL-6.

COX-2 inhibitor NS-398 repressed PSC culture activation. To examine the role of COX-2 in the regulation of PSC functions, we investigated the effect of NS-398, which specifically inhibits COX-2 activity, on PSC culture activation using freshly isolated PSCs. Since the maximal inhibition of basal COX-2 activity in activated PSCs was observed with 100 µM NS-398 treatment (data not shown), which is consistent with a previous report (8), we utilized 100 µM NS-398 for the current study. As shown in Fig. 4, at 48 h after isolation, a very low-level {alpha}-SMA signal was observed in both NS-398-treated and control cells. In control cells, however, {alpha}-SMA expression was increased at 72 and 96 h after the isolation. In contrast, {alpha}-SMA expression did not increase at 72 h, and its signal in NS-398-treated cells was much smaller than that in control cells even at 96 h. These data indicate that blockade of COX-2 activity by NS-398 inhibited PSC activation, suggesting that COX-2 is necessary for PSC activation.

NS-398 inhibited the responses of activated PSCs to TGF-beta1, IL-1beta, and IL-6. We next examined the role of COX-2 in the regulation of PSC functions by cytokines. For this purpose, we first investigated the effect of NS-398 on TGF-beta1-mediated enhancement of {alpha}-SMA and collagen-1 expression in activated PSCs. As shown in Fig. 5A, TGF-beta1 augmented COX-2 activity in PSCs. When PSCs were pretreated with 100 µM NS-394, basal COX-2 activity was decreased and TGF-beta1 failed to increase it. Under this condition, moreover, TGF-beta1 also failed to increase the expression of {alpha}-SMA and collagen-1 (Fig. 5, B and C). These data indicate that COX-2 mediates the stimulatory effect of TGF-beta1 on {alpha}-SMA and collagen-1 expression in activated PSCs, suggesting that TGF-beta1 increases {alpha}-SMA and collagen-1 expression by inducing COX-2 expression. We then examined the effect of NS-398 on the enhancement of {alpha}-SMA expression by IL-1beta and IL-6. As shown in Fig. 6, both IL-1beta and IL-6 enhanced COX-2 activity and {alpha}-SMA expression in activated PSCs but not when PSCs were pretreated with 100 µM NS-398 (Fig. 6). These data indicate that COX-2 also mediates the stimulatory effect of IL-1beta and IL-6 on {alpha}-SMA expression in activated PSCs.

Dominant negative Smad2/3 expression blocked the enhancement of COX-2 and {alpha}-SMA expression by TGF-beta1, IL-1beta, and IL-6 in activated PSCs. Since COX-2 mediates the effects of multiple cytokines on activated PSC functions, we tried to elucidate the mechanism of COX-2 induction by cytokines in activated PSCs. Since TGF-beta1 is critical for the regulation of PSC functions, we first examined the signaling pathway through which TGF-beta1 increases COX-2 expression. TGF-beta signaling occurs by Smad2/3-dependent and Smad2/3-independent pathways (4, 21). To identify the pathway through which TGF-beta1 enhances COX-2 expression in PSCs, we examined the effect of dominant negative Smad2/3 expression with an adenovirus vector (AdDNSmad2/3) on COX-2 expression in PSCs. The dominant negative Smad2/3 mutant was generated by substituting Glu for Asp-407 of Smad3, which renders it defective in TGF-beta receptor-dependent phosphorylation (11). Nevertheless, this mutant possesses a dominant negative effect on both Smad2 and Smad3 (11). We utilized an adenovirus vector expressing beta-galactosidase (AdLacZ) as an infection control. We previously reported that >98% of PSCs are infected with these adenovirus vectors and express the corresponding proteins (24). As shown in Fig. 7, TGF-beta1 increased COX-2 expression and consequently enhanced {alpha}-SMA and collagen-1 expression in activated PSCs infected with AdLacZ. In contrast, TGF-beta1 did not increase COX-2 expression and failed to enhance {alpha}-SMA and collagen-1 expression in cells infected with AdDNSmad2/3. These data indicate that TGF-beta1 increases COX-2 expression through a Smad2/3-dependent pathway and that the Smad2/3-dependent pathway is important for TGF-beta1 regulation of PSC functions. We further examined the effect of dominant negative Smad2/3 expression on the enhancement of COX-2 expression by IL-1beta and IL-6. Both IL-1beta and IL-6 increased COX-2 and {alpha}-SMA expression in activated PSCs infected with AdLacZ (Fig. 8). Surprisingly, the promoting effect of IL-1beta and IL-6 on COX-2 expression was inhibited by infection with AdDNSmad2/3. Moreover, IL-1beta and IL-6 failed to increase {alpha}-SMA expression in cells infected with AdDNSmad2/3. These data suggest that IL-1beta and IL-6 increase COX-2 expression, at least in part, through a Smad2/3-dependent pathway and that COX-2 is critical for the enhancement of {alpha}-SMA expression in PSCs by IL-1beta and IL-6.

IL-6 increased TGF-beta1 expression and secretion by PSCs. Although the current results suggest the participation of a Smad2/3-dependent pathway in the expression of COX-2 and {alpha}-SMA induced by IL-1beta and IL-6, it is unlikely that the Smad2/3-dependent pathway directly mediates the stimulatory effect of IL-1beta and IL-6 in PSCs, since Smad proteins are intracellular signaling molecules specific to the TGF-beta family. In the regulation of activated PSC functions, TGF-beta1 acts in an autocrine manner (17, 28). We recently reported that IL-1beta enhances TGF-beta1 expression and secretion by activated PSCs (1). Therefore, we hypothesized that IL-1beta and IL-6 may increase COX-2 expression through an Smad2/3-dependent pathway by increasing TGF-beta1 secretion from PSCs. To test this hypothesis, we first investigated whether IL-6 enhances TGF-beta1 secretion from activated PSCs. As shown in Fig. 9, IL-6 increased TGF-beta1 peptide secretion by activated PSCs in a dose-dependent manner. These data reinforce the idea that IL-6, as well as IL-1beta, enhances COX-2 and {alpha}-SMA expression in PSCs by increasing autocrine TGF-beta1.

Anti-TGF-beta neutralizing antibody blocked the enhancement of COX-2 and {alpha}-SMA expression by IL-1beta and IL-6. To further examine the involvement of autocrine TGF-beta1 in the enhancement of COX-2 and {alpha}-SMA expression by IL-1beta and 1L-6, we investigated the effect of anti-TGF-beta neutralizing antibody on COX-2 and {alpha}-SMA expression in PSCs. When anti-TGF-beta neutralizing antibody was added in the culture medium, the stimulatory effects of IL-1beta and IL-6 on the COX-2 expression in activated PSCs were attenuated (Fig. 10). In addition, IL-1beta and IL-6 did not enhance {alpha}-SMA expression in PSCs in the presence of anti-TGF-beta neutralizing antibody. These data indicate that IL-1beta and IL-6 enhance COX-2 expression in PSCs, at least in part, through autocrine TGF-beta1, and that autocrine TGF-beta1 plays a pivotal role in the enhancement of {alpha}-SMA expression by IL-1beta and IL-6.


Figure 10
View larger version (30K):
[in this window]
[in a new window]

  		Fig. 10. Effect of anti-TGF-beta neutralizing antibody on IL-1beta- or IL-6-stimulated induction of COX-2 and {alpha}-SMA in activated PSCs. A: PSCs were pretreated with 50 µg/ml anti-TGF-beta neutralizing antibody or nonimmune IgG for 30 min, followed by stimulation with 10 ng/ml IL-1beta or 100 ng/ml IL-6 for 48 h. Western blotting was then carried out using anti-COX-2, anti-{alpha}-SMA, and anti-{alpha}-tubulin antibodies. B: densitometry of Western blotting results shown in A. Values are means for 3 independent experiments.

 
MAPK inhibitors did not block TGF-beta1 enhancement of COX-2 expression in activated PSCs. Our present data suggest that TGF-beta1 plays a central role in cytokine-induced COX-2 expression through Smad2/3-dependent pathway. However, TGF-beta1 intracellular signaling is mediated by MAPKs as well. In addition, in other types of cells, MAPKs function in COX-2 expression (7, 9). Therefore, we finally examined the participation of MAPKs in TGF-beta1-induced COX-2 expression in activated PSCs. For this purpose, we pretreated activated PSCs with SP600125 (10 µM, JNK inhibitor), PD-98059 (10 nM, ERK inhibitor), or SB 203580 (25 µM, p38MAPK inhibitor). It has been previously reported that these dosages of inhibitors successfully block each MAPK-dependent pathway in rat activated PSCs (19, 20, 25). As shown in Fig. 11, TGF-beta1 increased COX-2 expression in cells pretreated with SP600125, PD-98059, or SB 203580. These data reinforce the evidence that TGF-beta1 enhances COX-2 expression in activated PSCs mainly through Smad2/3-dependent pathway.


Figure 11
View larger version (41K):
[in this window]
[in a new window]

  		Fig. 11. Effect of MAPK inhibitors on TGF-beta1-stimulated COX-2 expression in activated PSCs. A: PSCs were pretreated with 10 µM SP600125, 10 nM PD-98059, or 25 µM SB 203580 for 2 h, followed by incubation with 30 pM TGF-beta1 for 48 h. Western blotting was carried out using anti-COX-2, anti-{alpha}-SMA, and anti-{alpha}-tubulin antibodies. B: densitometry of Western blotting results shown in A. Values are means for 3 independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we demonstrated that 1) COX-2 is important in both PSC activation and the cytokine regulation of activated PSC functions, 2) TGF-beta1 induces COX-2 expression through a Smad2/3-dependent pathway, and 3) IL-1beta and IL-6 indirectly increase COX-2 and {alpha}-SMA expression in activated PSCs, at least partly, through a Smad2/3-dependent pathway by enhancing autocrine TGF-beta1.

COX-2 plays a pivotal role in the progression of acute and chronic inflammatory responses in various tissues and organs. Although two groups have shown that COX-2 is expressed in pancreatic tissue from patients with chronic pancreatitis (16, 26), the function of COX-2 in chronic pancreatitis is uncertain. Since PSCs are activated during chronic pancreatitis and promote pancreatic fibrosis by producing and secreting multiple extracellular matrix components, our current observation that COX-2 is expressed in activated PSCs but not in quiescent ones strongly suggests COX-2 participation in the progression of chronic pancreatitis, especially in pancreatic fibrosis. Moreover, the fact that profibrogenic cytokine TGF-beta1 enhanced COX-2 expression and the COX-2 inhibitor NS-398 blocked TGF-beta1 enhancement of {alpha}-SMA and collagen-1 expression implies a role for COX-2 in promoting pancreatic fibrosis.

To date, studies on the regulation of COX-2 expression have shown the involvement of multiple intracellular signaling mediators. For instance, nuclear factor-{kappa}B, a transcription factor that plays a central role in inflammatory and immune responses, is involved in COX-2 induction by lipopolysaccharide in macrophages and colonic epithelial cells (9, 10, 15). In addition, MAPKs, including ERK, JNK, and p38MAPK also play a role in COX-2 expression (9). The signaling pathway through which TGF-beta1 induces COX-2 expression also has been elucidated; the COX-2 gene promoter region contains a TGF-beta response element (27, 29). Sheares et al. (27) reported that TGF-beta1 induces COX-2 expression through p38MAPK in human pulmonary artery muscle cells. Indeed, MAPK is one mediator of TGF-beta1 signaling (4). However, our present study showed that MAPK inhibitors did not inhibit TGF-beta1-induced COX-2 or {alpha}-SMA expression in activated PSCs (Fig. 11). Masamune et al.(19) previously reported that p38MAPK inhibitor SB 203580 inhibited {alpha}-SMA expression in PSCs during the culture activation. Consisting with their data, basal {alpha}-SMA expression in SB 203580-treated cells was relatively lower than in cells with other treatment (Fig. 11B). However, TGF-beta1 successfully increased both COX-2 and {alpha}-SMA expression in PSCs treated with SB 203580. Therefore, we suggest that TGF-beta1 enhances COX-2 and {alpha}-SMA expression in activated PSCs through a pathway independent of p38MAPK.

Intriguing are our present data indicating that both dominant negative Smad2/3 expression and anti-TGF-beta neutralizing antibody attenuated the enhancement of the COX-2 expression by IL-1beta and IL-6. These data imply that IL-1beta and IL-6 indirectly increase COX-2 expression in PSCs through autocrine TGF-beta1. We further reinforced this idea by showing that IL-6 as well as IL-1beta (1) augmented TGF-beta1 secretion by PSCs (Fig. 9). However, we do not rule out a direct effect of IL-1beta and IL-6 on COX-2 expression in PSCs. It is well known that IL-1beta directly modulates PSC functions through its specific intracellular signaling pathway (18). In the present study, dominant negative Smad2/3 expression did not completely inhibit IL-1beta- or IL-6-induced COX-2 expression (Fig. 8). In addition, although anti-TGF-beta neutralizing antibody abolished IL-6-mediated enhancement of COX-2 expression in PSCs, it partially attenuated the increase of COX-2 expression mediated by IL-1beta (Fig. 10). Thus IL-1beta and IL-6 could directly enhance COX-2 expression in PSCs. However, we propose that IL-1beta and IL-6 indirectly enhance COX-2 expression in PSCs, at least partly, through an Smad2/3-dependent pathway by increasing autocrine TGF-beta1.

COX-2 participation in gastrointestinal fibrosis has been intensively studied in the liver. Consistent with our current data, for example, studies by Cheng et al. (8) using an immortalized human hepatic stellate cell (HSC) line showed that COX-2 inhibitor NS-398 attenuated {alpha}-SMA expression in HSCs, indicating a role for COX-2 in promoting HSC activation. On the other hand, Hui et al. (14), using another immortalized human HSC line, showed that NS-398 increased basal and TGF-beta1-stimulated collagen-1 expression and that COX-2-derived prostaglandin E2 inhibited both basal and TGF-beta1-stimulated collagen-1 expression, suggesting an inhibitory role for COX-2 on activated HSC function (14). In the present study, we examined COX-2 participation concurrently in basal and TGF-beta1-stimulated expression of {alpha}-SMA and collagen-1. We demonstrated that COX-2 promotes both {alpha}-SMA and collagen-1 expression. However, there exists an apparent discrepancy between our data and that of Hui et al. (14). One possible explanation for the discrepancy is that the role of COX-2 in PSCs may be distinct from that in HSCs. Another possibility is that the COX-2 function might be variable in different immortalized HSC lines, since Hui et al. (14) and Cheng et al. (8) utilized different ones. We have extended the knowledge obtained from studies utilizing HSCs by elucidating the mechanisms of COX-2 induction in PSCs through the demonstration that TGF-beta1/Smad signaling is critical for COX-2 expression in PSCs.

It is assumed that PSCs are activated by various cytokines in chronic pancreatitis (3, 22) and that activated PSCs promote pancreatic fibrosis by producing and secreting various extracellular matrix components. Thus a therapeutic strategy that inhibits PSC activation or increases PSC quiescence could be useful for the treatment of pancreatic fibrosis. We showed that COX-2 inhibitor NS-398 blocked the expression of {alpha}-SMA in freshly isolated quiescent PSCs during primary culture (Fig. 4). In addition, densitometry analyses of Western blotting results showed that the enhancement of COX-2 expression by TGF-beta1, IL-1beta, and IL-6 preceded or accompanied the increase in {alpha}-SMA expression (Figs. 2 and 3). Furthermore, NS-398 blocked the enhancement of {alpha}-SMA expression by TGF-beta1, IL-1beta, and IL-6 in activated PSCs (Figs. 5 and 6). These data suggest that COX-2 is essential for the increase of {alpha}-SMA expression mediated by these cytokines. Since {alpha}-SMA expression is a parameter of PSC transformation from quiescent to activated myofibrobast-like cells, these observations indicate that COX-2 inhibitor blocked the initiation of PSC transformation and attenuated its promotion by these cytokines. These data suggest that COX-2 inhibitors could be effective in the therapy of pancreatic fibrosis. Further in vivo studies on the effect of COX-2 inhibitors on pancreatic fibrosis are warranted.

In conclusion, we have shown that COX-2 plays a pivotal role in the response of PSCs to multiple cytokines and that TGF-beta1/Smad signaling is critical for COX-2 expression. These data provide insights for understanding the mechanism of pancreatic fibrosis and for developing novel therapeutic strategies for its treatment.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Kohei Miyazono (University of Tokyo) for dominant negative Smad2/3 adenovirus vector.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. Ohnishi, Dept. of Gastroenterology, Jichi Medical School, 3311-1 Yakushiji, Minamikawachi-cho, Kawachi-gun, Tochigi 329-0498, Japan (e-mail: hohnishi{at}jichi.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
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Aoki H, Ohnishi H, Hama K, Ishijima T, Satoh Y, Hanatsuka K, Ohashi A, Wada S, Miyata T, Kita H, Yamamoto H, Osawa H, Sato K, Tamada K, Yasuda H, Mashima H, Sugano K. Autocrine loop between transforming growth factor-beta1 and interleukin-1beta through Smad3- and ERK-dependent pathways in rat pancreatic stellate cells. Am J Physiol Cell Physiol 290: C1100–C1108, 2006.[Abstract/Free Full Text]

2. Apte MV, Haber PS, Applegate TL, Norton ID, McCaughan GW, Korsten MA, Pirola RC, Wilson JS. Periacinar stellate shaped cells in rat pancreas: identification, isolation, and culture. Gut 43: 128–133, 1998.[Abstract/Free Full Text]

3. Apte MV, Haber PS, Darby SJ, Rodgers SC, McCaughan GW, Korsten MA, Pirola RC, Wilson JS. Pancreatic stellate cells are activated by proinflammatory cytokines: implications for pancreatic fibrogenesis. Gut 44: 534–541, 1999.[Abstract/Free Full Text]

4. Attisano L, Wrana JL. Signal transduction by the TGF-beta superfamily. Science 296: 1646–1647, 2002.[Abstract/Free Full Text]

5. Bachem MG, Schneider E, Gross H, Weidenbach H, Schmid RM, Menke A, Siech M, Beger H, Grunert A, Adler G. Identification, culture, and characterization of pancreatic stellate cells in rats and humans. Gastroenterology 115: 421–432, 1998.[CrossRef][ISI][Medline]

6. Bentrem DJ, Joehl RJ. Pancreas: healing response in critical illness. Crit Care Med 31: S582–589, 2003.[CrossRef][ISI][Medline]

7. Chen W, Tang Q, Gonzales MS, Bowden GT. Role of p38 MAP kinases and ERK in mediating ultraviolet-B induced cyclooxygenase-2 gene expression in human keratinocytes. Oncogene 20: 3921–3926, 2001.[CrossRef][ISI][Medline]

8. Cheng J, Imanishi H, Liu W, Iwasaki A, Ueki N, Nakamura H, Hada T. Inhibition of the expression of {alpha}-smooth muscle actin in human hepatic stellate cell line, LI90, by a selective cyclooxygenase 2 inhibitor, NS-398. Biochem Biophys Res Commun 297: 1128–1134, 2002.[CrossRef][ISI][Medline]

9. Chun KS, Surh YJ. Signal transduction pathways regulating cyclooxygenase-2 expression: potential molecular targets for chemoprevention. Biochem Pharmacol 68: 1089–1100, 2004.[CrossRef][ISI][Medline]

10. D’Acquisto F, Iuvone T, Rombola L, Sautebin L, Di Rosa M, Carnuccio R. Involvement of NF-{kappa}B in the regulation of cyclooxygenase-2 protein expression in LPS-stimulated J774 macrophages. FEBS Lett 418: 175–178, 1997.[CrossRef][ISI][Medline]

11. Goto D, Yagi K, Inoue H, Iwamoto I, Kawabata M, Miyazono K, Kato M. A single missense mutant of Smad3 inhibits activation of both Smad2 and Smad3, and has a dominant negative effect on TGF-beta signals. FEBS Lett 430: 201–204, 1998.[CrossRef][ISI][Medline]

12. Haber PS, Keogh GW, Apte MV, Moran CS, Stewart NL, Crawford DH, Pirola RC, McCaughan GW, Ramm GA, Wilson JS. Activation of pancreatic stellate cells in human and experimental pancreatic fibrosis. Am J Pathol 155: 1087–1095, 1999.[Abstract/Free Full Text]

13. Heldin CH, Miyazono K, ten Dijke P. TGF-beta signalling from cell membrane to nucleus through SMAD proteins. Nature 390: 465–471, 1997.[CrossRef][Medline]

14. Hui AY, Dannenberg AJ, Sung JJ, Subbaramaiah K, Du B, Olinga P, Friedman SL. Prostaglandin E2 inhibits transforming growth factor beta1-mediated induction of collagen {alpha}1(I) in hepatic stellate cells. J Hepatol 41: 251–258, 2004.[CrossRef][ISI][Medline]

15. Kojima M, Morisaki T, Izuhara K, Uchiyama A, Matsunari Y, Katano M, Tanaka M. Lipopolysaccharide increases cyclo-oxygenase-2 expression in a colon carcinoma cell line through nuclear factor- {kappa}B activation. Oncogene 19: 1225–1231, 2000.[CrossRef][ISI][Medline]

16. Koliopanos A, Friess H, Kleeff J, Roggo A, Zimmermann A, Buchler MW. Cyclooxygenase 2 expression in chronic pancreatitis: correlation with stage of the disease and diabetes mellitus. Digestion 64: 240–247, 2001.[CrossRef][ISI][Medline]

17. Kruse ML, Hildebrand PB, Timke C, Folsch UR, Schmidt WE. TGFbeta1 autocrine growth control in isolated pancreatic fibroblastoid cells/stellate cells in vitro. Regul Pept 90: 47–52, 2000.[CrossRef][ISI][Medline]

18. Masamune A, Sakai Y, Kikuta K, Satoh M, Satoh A, Shimosegawa T. Activated rat pancreatic stellate cells express intercellular adhesion molecule-1 (ICAM-1) in vitro. Pancreas 25: 78–85, 2002.[CrossRef][ISI][Medline]

19. Masamune A, Satoh M, Kikuta K, Sakai Y, Satoh A, Shimosegawa T. Inhibition of p38 mitogen-activated protein kinase blocks activation of rat pancreatic stellate cells. J Pharmacol Exp Ther 304: 8–14, 2003.[Abstract/Free Full Text]

20. Masamune A, Kikuta K, Suzuki N, Satoh M, Satoh K, Shimosegawa T. A c-Jun NH2-terminal kinase inhibitor SP600125 (anthra[1,9-cd]pyrazole-6 (2H)-one) blocks activation of pancreatic stellate cells. J Pharmacol Exp Ther 310: 520–527, 2004.[Abstract/Free Full Text]

21. Massague J. TGF-beta signal transduction. Annu Rev Biochem 67: 753–791, 1998.[CrossRef][ISI][Medline]

22. Mews P, Phillips P, Fahmy R, Korsten M, Pirola R, Wilson J, Apte M. Pancreatic stellate cells respond to inflammatory cytokines: potential role in chronic pancreatitis. Gut 50: 535–541, 2002.[Abstract/Free Full Text]

23. Ohnishi H, Mine T, Shibata H, Ueda N, Tsuchida T, Fujita T. Involvement of Rab4 in regulated exocytosis of rat pancreatic acini. Gastroenterology 116: 943–952, 1999.[CrossRef][ISI][Medline]

24. Ohnishi H, Miyata T, Yasuda H, Satoh Y, Hanatsuka K, Kita H, Ohashi A, Tamada K, Makita N, Iiri T, Ueda N, Mashima H, Sugano K. Distinct roles of Smad2-, Smad3-, and ERK-dependent pathways in transforming growth factor-beta1 regulation of pancreatic stellate cellular functions. J Biol Chem 279: 8873–8878, 2004.[Abstract/Free Full Text]

25. Ohnishi H, Miyata T, Yasuda H, Satoh Y, Hanatsuka K, Kita H, Ohashi A, Tamada K, Makita N, Iiri T, Ueda N, Mashima H, Sugano K. Distinct roles of Smad2-, Smad3-, and ERK-dependent pathways in transforming growth factor-beta1 regulation of pancreatic stellate cellular functions. J Biol Chem 279: 8873–8878, 2004.[Abstract/Free Full Text]

26. Schlosser W, Schlosser S, Ramadani M, Gansauge F, Gansauge S, Beger HG. Cyclooxygenase-2 is overexpressed in chronic pancreatitis. Pancreas 25: 26–30, 2002.[CrossRef][ISI][Medline]

27. Sheares KK, Jeffery TK, Long L, Yang X, Morrell NW. Differential effects of TGF-beta1 and BMP-4 on the hypoxic induction of cyclooxygenase-2 in human pulmonary artery smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 287: L919–L927, 2004.[Abstract/Free Full Text]

28. Shek FW, Benyon RC, Walker FM, McCrudden PR, Pender SL, Williams EJ, Johnson PA, Johnson CD, Bateman AC, Fine DR, Iredale JP. Expression of transforming growth factor-beta1 by pancreatic stellate cells and its implications for matrix secretion and turnover in chronic pancreatitis. Am J Pathol 160: 1787–1798, 2002.[Abstract/Free Full Text]

29. Yang X, Hou F, Taylor L, Polgar P. Characterization of human cyclooxygenase 2 gene promoter localization of a TGF-beta response element. Biochim Biophys Acta 1350: 287–292, 1997.[Medline]





This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/1/C259    most recent
00030.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via ISI Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Aoki, H.
Right arrow Articles by Sugano, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Aoki, H.
Right arrow Articles by Sugano, K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.