Am J Physiol Cell Physiol AJP: Gastrointestinal and Liver Physiology
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Am J Physiol Cell Physiol 290: C1100-C1108, 2006. First published December 21, 2005; doi:10.1152/ajpcell.00465.2005
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RECEPTORS AND SIGNAL TRANSDUCTION

Autocrine loop between TGF-beta1 and IL-1beta through Smad3- and ERK-dependent pathways in rat pancreatic stellate cells

Hiroyoshi Aoki,1 Hirohide Ohnishi,1 Kouji Hama,1 Takako Ishijima,1 Yukihiro Satoh,1 Kazunobu Hanatsuka,1 Akira Ohashi,1 Shinichi Wada,1 Tomohiko Miyata,1 Hiroto Kita,1 Hironori Yamamoto,1 Hiroyuki Osawa,1 Kiichi Sato,1 Kiichi Tamada,1 Hiroshi Yasuda,2 Hirosato Mashima,3 and Kentaro Sugano1

1Department of Gastroenterology, Jichi Medical School, Tochigi; 2Division of Gastroenterology, Showa University Fujigaoka Hospital, Kanagawa; and 3Department of Gastroenterology, University of Tokyo School of Medicine, Tokyo, Japan

Submitted 19 September 2005 ; accepted in final form 16 November 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Pancreatic stellate cells (PSCs) are activated during pancreatitis and promote pancreatic fibrosis by producing and secreting ECMs such as collagen and fibronectin. IL-1beta has been assumed to participate in pancreatic fibrosis by activating PSCs. Activated PSCs secrete various cytokines that regulate PSC function. In this study, we have examined IL-1beta secretion from culture-activated PSCs as well as its regulatory mechanism. RT-PCR and ELISA have demonstrated that PSCs express IL-1beta mRNA and secrete IL-1beta peptide. Inhibition of TGF-beta1 activity secreted from PSCs by TGF-beta1-neutralizing antibody attenuated IL-1beta secretion from PSCs. Exogenous TGF-beta1 increased IL-1beta expression and secretion by PSCs in a dose-dependent manner. Adenovirus-mediated expression of dominant-negative (dn)Smad2/3 expression reduced both basal and TGF-beta1-stimulated IL-1beta expression and secretion by PSCs. Coexpression of Smad3 with dnSmad2/3 restored IL-1beta expression and secretion by PSCs, which were attenuated by dnSmad2/3 expression. In contrast, coexpression of Smad2 with dnSmad2/3 did not alter them. Furthermore, inhibition of IL-1beta activity secreted from PSCs by IL-1beta-neutralizing antibody attenuated TGF-beta1 secretion from PSCs. Exogenous IL-1beta enhanced TGF-beta1 expression and secretion by PSCs. IL-1beta activated ERK, and PD-98059, a MEK1 inhibitor, blocked IL-1beta enhancement of TGF-beta1 expression and secretion by PSCs. We propose that an autocrine loop exists between TGF-beta1 and IL-1beta in activated PSCs through Smad3- and ERK-dependent pathways.

fibrosis; cytokine; chronic pancreatitis

PANCREATIC STELLATE CELLS (PSCs) were recently identified, isolated, and characterized (4, 6). In the normal pancreas, PSCs possess fat droplets containing vitamin A, are quiescent, and can be defined by desmin-positive but {alpha}-smooth muscle actin ({alpha}-SMA)-negative staining (4). When cultured in vitro, PSCs are autoactivated (autotransformed) and change their morphological and functional features (6). PSCs commence losing vitamin A-containing lipid droplets, highly proliferating, increasing expression of {alpha}-SMA, and producing and secreting ECM components such as collagen and fibronectin. Namely, PSCs are autotransformed to myofibroblast-like cells. In vivo PSCs are also activated during both human and experimental pancreatic fibrosis (13). Therefore, PSCs are thought to play an important role in pancreatic fibrogenesis.

TGF-beta1 is one of the major profibrogenic cytokines in various tissues. Recently, TGF-beta1 has been implicated in the etiology of pancreatic fibrosis. It activates PSCs and promotes pancreatic fibrosis (5, 15). TGF-beta1 intracellular signaling is mediated and modulated primarily by the mothers against decapentaplegic homolog (Drosophila)-related proteins (Smads) (14, 18). Upon TGF-beta1 binding to the TGF-beta type II receptor, the type II receptor kinase phosphorylates the TGF-beta type I receptor, leading to activation of the type I receptor. The activated type I receptor kinase phosphorylates Smad homologs 2 and 3 (Smad2/3). Phosphorylated Smad2/3 forms oligomeric complexes with Smad4, and the complexes then translocate into the nucleus. These complexes subsequently activate the transcription of target genes. Thus dual Smad2/3-dependent pathways exist in TGF-beta1 intracellular signaling. Until recently, however, the distinction between Smad2- and Smad3-dependent pathways has been uncertain because of the lack of a methodology to assess their respective roles. Smad2 and Smad3 compete for both the TGF-beta receptor and Smad4 binding during their activation; thus their overexpression blocks endogenous Smad3 and Smad2 functions. Therefore, the possibility remains that the effects of Smad2 and Smad3 overexpression on cell functions result from competitive inhibition rather than from enhanced Smad2 and Smad3 activity due to their overexpression. To exclude this possibility, we developed a novel method to analyze the independent roles of Smad2 and Smad3 in TGF-beta1 signal transduction by coexpressing dominant-negative (dn)Smad2/3 with either Smad2 or Smad3 (25). The dnSmad2/3 mutant was generated by substituting Glu for Asp407 of smad3, which renders smad3 defective in TGF-beta receptor-dependent phosphorylation. Nevertheless, this mutant possesses a dominant-negative effect on both Smad2 and Smad3 (12). The expression of dnSmad2/3 blocks both endogenous Smad2 and Smad3 functions at the TGF-beta receptor-dependent phosphorylation step. Coexpression of either Smad2 or Smad3 with dnSmad2/3 rescues only the Smad2- or Smad3-dependent pathway, respectively, permitting the separation of the Smad2- and Smad3-specific signaling pathways (25).

IL-1beta is a potent proinflammatory cytokine and is known to play major roles in the progression of acute pancreatitis leading to chronic pancreatitis with fibrosis (8). Furthermore, IL-1beta was recently shown to enhance PSC activation and is thought to promote pancreatic fibrosis by activating PSCs (19).

Activated PSCs have been shown to secrete cytokines that modulate PSC function, such as activin A (24) and IL-6 (31). Our working hypothesis states that activated PSCs express and secrete IL-1beta. Because TGF-beta1 is central to the regulation of PSC function (5, 19), we assume that TGF-beta1 may regulate IL-1beta expression and secretion of activated PSCs. We thus conducted this study to assess the regulatory mechanism of IL-1beta production in culture-activated PSCs. We report herein that TGF-beta1 enhances IL-1beta mRNA expression and peptide secretion by activated PSCs in an autocrine manner. We have further shown, using the adenovirus-mediated double-expression method described above, that a Smad3-dependent, Smad2-independent signaling pathway mediates TGF-beta1-enhanced IL-1beta expression and secretion of PSCs. We finally have demonstrated that IL-1beta increases TGF-beta1 expression and secretion by PSCs via an ERK-dependent pathway, indicating the existence of an autocrine loop between IL-1beta and TGF-beta1 in activated PSCs.


   MATERIALS AND METHODS
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 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, anti-IL-1beta, and anti-TGF-beta1 antibodies were obtained from R&D Systems (Abingdon, UK). DNase I was purchased from Roche (Basel, Switzerland). Collagenase P was obtained from Boehringer Mannheim (Mannheim, Germany). Anti-ERK antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antiphosphorylated ERK antibody was obtained from Cell Signaling Technology (Beverly, MA). Horseradish peroxidase (HRP)-conjugated donkey anti-goat IgG, HRP-conjugated donkey anti-mouse IgG, and HRP-conjugated donkey anti-rabbit IgG antibodies were purchased from Jackson ImmunoResearch (West Grove, PA). PD-98059 was obtained from Calbiochem (San Diego, CA).

Isolation and culture of rat PSCs. Rat PSCs were prepared as described previously (4). Briefly, rat pancreas was digested in Gey's balanced salt solution supplemented with 0.05% collagenase P, 0.02% pronase, and 0.1% DNase I. 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 solution were collected, washed, and resuspended in Iscove's modified DMEM containing 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin. PSCs were cultured in a 5% CO2 atmosphere at 37°C. All experiments were performed using culture-activated PSCs between passages 2 and 3.

Western blot analysis. Western blot analysis was performed as described previously (23) using ECL reagent to visualize secondary antibodies.

Adenoviral infection. Recombinant adenoviruses of Smads were kindly provided by Dr. Kohei Miyazono (University of Tokyo, Tokyo, Japan). For a single adenoviral infection, cells were infected with a recombinant adenovirus at a dose of 10 plaque-forming units (PFU) per cell in the culture media described above. In those experiments using double adenovirus infection, cells were infected with dnSmad2/3 adenovirus (Ad-dnSmad2/3) at a dose of 10 PFU/cell concomitantly with Smad2 (Ad-Smad2) or Smad3 (Ad-Smad3) adenovirus at doses of 5 or 10 PFU/cell. Subsequent experiments were performed 48 h after infection. An adenovirus expressing beta-galactosidase (Ad-LacZ) was used as an infection control.

Measurement of IL-1beta and TGF-beta1 peptide secretion. Secretion of IL-1beta and TGF-beta1 peptides was measured by determining their concentration in the culture medium using commercially available ELISA kits (Biosource International, Camarillo, CA, and DRG International, Mountainside, NJ) according to the manufacturers' instructions.

RT-PCR. Total RNA was isolated from PSCs using TRIzol reagent (Life Technologies/GIBCO-BRL, Grand Island, NY). First-strand cDNA was made from total RNA using the ReverTra Ace system (Toyobo, Tokyo, Japan) according to the manufacturer's instructions. PCR for TGF-beta1 was performed using a PCR kit for rat TGF-beta1 (Maximbio, San Francisco, CA) according to the manufacturer's instructions. PCR for rat IL-1beta and GAPDH was performed using the following primers: rat IL-1beta sense, 5'-TCCTAGGAAACAGCAATGGTCG-3', rat IL-1beta antisense, 5'-TTCAT-CCCATACCACGGACAAC-3'; and rat GAPDH sense, 5'-CATGACCAC-AGTCCATGCCATC-3', rat GAPDH antisense, 5'-CACCCTGTTGCTGTAGCCATATTC-3'. The reactions were conducted using the following cycle conditions: denaturation at 94°C for 0.5 min, annealing at 45°C for 1 min, and extension at 72°C for 1 min for 30 cycles.

Statistical analysis. The data were analyzed using ANOVA to determine statistical significance, and P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
IL-1beta is secreted from PSCs. We first examined IL-1beta secretion from PSCs. As shown in Fig. 1A, IL-1beta peptide was detected in PSC culture medium. In particular, IL-1beta concentration in PSC culture medium was markedly increased after 48-h incubation. No IL-1beta activity was detected in fresh culture medium. These data indicate that IL-1beta is secreted from PSCs.


Figure 1
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  		Fig. 1. Anti-TGF-beta1-neutralizing antibody inhibited IL-1beta secretion from activated pancreatic stellate cells (PSCs). A: IL-1beta secretion from PSCs. IL-1beta concentration in culture medium was determined by ELISA 1–3 days after the culture medium was changed. Values are means ± SE of 3 independent experiments. *P < 0.01 vs. control (time 0). B: effect of anti-TGF-beta1-neutralizing antibody and nonimmune IgG on IL-1beta secretion from PSCs. Concentration of IL-1beta secreted from PSCs into culture medium was determined by ELISA after 48-h incubation with indicated amounts of anti-TGF-beta1 antibody ({circ}) or nonimmune IgG (bullet). Values are means ± SE of 3 independent experiments. *P < 0.05 vs. control.

 
Autocrine TGF-beta1 stimulates IL-1beta secretion from PSCs. Knowing that IL-1beta is secreted from PSCs, we next attempted to elucidate the mechanism that regulates IL-1beta secretion from PSCs. Because TGF-beta1 regulates various functions of PSCs in an autocrine manner (5, 15, 25), we examined the effect of anti-TGF-beta1 antibody, which neutralizes TGF-beta1 bioactivity, on IL-1beta secretion from PSCs. As shown in Fig. 1B, anti-TGF-beta1 antibody decreased IL-1beta secretion from PSCs in a dose-dependent manner, indicating that autocrine TGF-beta1 enhances IL-1beta secretion from PSCs.

Exogenous TGF-beta1 increased IL-1 expression and secretion by PSCs. As an independent experiment to examine the effect of TGF-beta1 on IL-1beta secretion from PSCs, we added exogenous TGF-beta1 to PSC culture medium and determined secreted IL-1beta from PSCs. Because maximal IL-1beta secretion from PSCs was observed in 48-h incubation, we first examined the effect of TGF-beta1 on IL-1beta expression and secretion at this time point. As shown in Fig. 2, exogenous TGF-beta1 enhanced IL-1beta mRNA expression in PSCs (Fig. 2A). In addition, exogenous TGF-beta1 augmented IL-1beta secretion from PSCs in a dose-dependent manner (Fig. 2B). We next examined exogenous TGF-beta1 effect on IL-1beta expression and secretion at earlier time points. As shown in Fig. 2, C and D, 2 ng/ml TGF-beta1 started enhancing IL-1beta mRNA expression and secretion within 3 and 6 h of incubation, respectively. These data imply that TGF-beta1 enhances IL-1beta expression and secretion by PSCs.


Figure 2
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  		Fig. 2. Effect of exogenous TGF-beta1 on IL-1beta expression and secretion by PSCs. A and C: IL-1beta mRNA expression was determined by RT-PCR after 48-h incubation with indicated amounts of TGF-beta1 (A) or after incubation for indicated times with 2 ng/ml TGF-beta1 (C). B and D: concentration of IL-1beta secreted into culture medium from PSCs was quantified by ELISA after 48-h incubation with indicated amounts of TGF-beta1 (B) or after incubation for indicated times with (open bars) and without (solid bars) 2 ng/ml TGF-beta1 (D). Values are means ± SE of 3 independent experiments. *P < 0.05, **P < 0.01 vs. control (B). #P < 0.05, ##P < 0.01 vs. control without TGF-beta1 (D).

 
Autocrine TGF-beta1 enhances IL-1beta expression and secretion by PSCs through Smad-dependent pathways. We next examined the intracellular signaling pathway through which TGF-beta1 stimulates IL-1beta expression and secretion by PSCs. Because Smad proteins are the major mediators of TGF-beta1 signaling, we investigated Smad-dependent-pathways using adenoviral vectors of Smad2 (Ad-Smad2), Smad3 (Ad-Smad3), and dnSmad2/3 (Ad-dnSmad2/3). We used an adenovirus expressing beta-galactosidase (Ad-LacZ) as an infection control. We previously reported that >98% of PSCs are infected with these adenoviral vectors and expressed each Smad protein sufficiently (25). In addition, the infections of these adenoviruses do not affect TGF-beta1 mRNA expression or peptide secretion by PSCs (25). Therefore, we can observe the effect of these adenoviral infections on IL-1beta expression and secretion by PSCs modulated by autocrine TGF-beta1, regardless of the effect of the infection on the amount of autocrine TGF-beta1. When Smad-dependent pathways were blocked by Ad-dnSmad2/3, both basal IL-1beta mRNA expression (Fig. 3A; compare first and third lanes) and basal peptide secretion (Fig. 3B; compare first and third columns) of PSCs were attenuated. Because infection with Ad-dnSmad2/3 does not alter autocrine TGF-beta1 secretion from PSCs as reported previously (25), these data indicate that TGF-beta1 stimulates IL-1beta mRNA expression and peptide secretion in an autocrine manner via a Smad-dependent pathway. Moreover, Ad-dnSmad2/3 attenuated exogenous TGF-beta1-enhanced IL-1beta expression and secretion (Fig. 3, A and B), thus reinforcing our suggestion that TGF-beta1 stimulates IL-1beta expression and secretion via a Smad-dependent pathway.


Figure 3
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  		Fig. 3. Effect of dominant-negative (dn)Smad2/3 expression on IL-1beta expression and secretion by PSCs. PSCs were infected with 10 plaque-forming units (PFU)/cell of adenovirus expressing beta-galactosidase (Ad-LacZ) or dnSmad2/3 adenovirus (Ad-dnSmad2/3) and then incubated for 48 h with or without 2 ng/ml TGF-beta1. A: after incubation, IL-1beta mRNA expression was determined by RT-PCR using GAPDH mRNA expression as a control. B: concentration of IL-1beta secreted from PSCs into culture medium during 48-h incubation was determined by ELISA. Values are means ± SE of 3 independent experiments. *P < 0.05 vs. first bar. **P < 0.01 vs. second bar.

 
Smad3-dependent pathway mediates TGF-beta1 stimulation to IL-1beta expression and secretion by PSCs. We subsequently tried to elucidate whether the Smad2- or Smad3-dependent pathway mediates TGF-beta1 stimulation of IL-1beta expression and secretion by PSCs. Ad-Smad3, but not Ad-Smad2, increased 1) basal IL-1beta mRNA expression (Fig. 4A) and 2) basal and TGF-beta1 stimulated IL-1beta secretion by PSCs (Fig. 4B). Because both Smad2 and Smad3 competitively inhibit one another at the receptor and smad4 binding sites, the effect of Smad3 overexpression on IL-1beta expression and secretion by PSCs may result from the inhibition of endogenous Smad2 and may not imply the involvement of Smad3-dependent pathway in TGF-beta1-enhanced IL-1beta expression and secretion. To exclude this possibility, we investigated the specific roles of Smad2 and Smad3 in eliciting autocrine TGF-beta1-stimulated IL-1beta expression and secretion after coinfection of PSCs with Ad-dnSmad2/3 and either Ad-Smad2 or Ad-Smad3. On the one hand, coinfection with Ad-Smad3 precluded Ad-dnSmad2/3 inhibition of IL-1beta expression (Fig. 5A) and secretion (Fig. 5B). On the other hand, coinfection with Ad-Smad2 did not alter Ad-dnSmad2/3 inhibition of IL-1beta expression or secretion (Fig. 5). These data suggest that TGF-beta1 increases the expression and secretion of IL-1beta by PSCs via a Smad3-dependent, Smad2-independent pathway.


Figure 4
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  		Fig. 4. Effect of Smad2 and Smad3 overexpression on IL-1beta expression and secretion of PSCs. PSCs were infected with 10 PFU/cell of Ad-LacZ, Ad-Smad2, or Ad-Smad3. A: IL-1beta mRNA expression was determined by RT-PCR after 48-h incubation using GAPDH mRNA expression as a control. B: concentration of IL-1beta secreted from PSCs into culture medium during 48-h incubation with or without 2 ng/ml TGF-beta1 was determined by ELISA. Values are means ± SE of 3 independent experiments. *P < 0.05 vs. first bar. **P < 0.01 vs. second bar.

 

Figure 5
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  		Fig. 5. Effect of coexpression of Smad2 or Smad3 with dnSmad2/3 on IL-1beta expression and secretion by PSCs. For double-adenovirus infection experiments, PSCs were infected with the combination of Ad-Smad2 and Ad-dnSmad2/3 or Ad-Smad3 and Ad-dnSmad2/3. Single infection of Ad-LacZ or Ad-dnSmad2/3 was used as a control. A: after 48-h incubation, IL-1beta mRNA expression was determined by RT-PCR using GAPDH mRNA expression as an internal control. B: concentration of IL-1beta secreted from PSCs into culture medium during 48-h incubation was determined by ELISA. Values are means ± SE of 3 independent experiments. *P < 0.05, **P < 0.01 vs. control.

 
Autocrine IL-1beta promotes TGF-beta1 secretion from PSCs. Because IL-1beta has been reported to modulate PSC function (17, 19), we hypothesized that IL-1beta may increase the autocrine secretion of TGF-beta1 from PSCs. To test this hypothesis, we first examined the effect of anti-IL-1beta antibody, which neutralizes IL-1beta bioactivity, on TGF-beta1 secretion from PSCs. As shown in Fig. 6, anti-IL-1beta antibody added into culture medium attenuated TGF-beta1 secretion from PSCs in a dose-dependent manner. In contrast, nonimmune IgG did not affect TGF-beta1 secretion from PSCs. These data indicate that autocrine IL-1beta stimulated TGF-beta1 secretion from PSCs.


Figure 6
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  		Fig. 6. Effect of anti-IL-1beta-neutralizing antibody and nonimmune IgG on TGF-beta1 secretion from PSCs. Concentration of TGF-beta1 secreted from PSCs into culture medium was determined by ELISA after 48-h incubation with indicated amounts of anti-IL-1beta antibody ({circ}) or nonimmune IgG (bullet). Values are means ± SE of 3 independent experiments. *P < 0.05, **P < 0.01 vs. control.

 
IL-1beta enhances TGF-beta1 mRNA expression and peptide secretion by PSCs through an ERK-dependent pathway. We next examined the effect of exogenous IL-1beta on TGF-beta1 expression and secretion by PSCs. Because secreted IL-1beta concentration in culture medium reaches a maximum after 48 h of incubation, we first examined the effect of IL-1beta on TGF-beta1 mRNA expression and peptide secretion by PSCs at this time point. As shown in Fig. 7, A and B, exogenous IL-1beta added to culture medium enhanced TGF-beta1 mRNA expression and peptide secretion in a dose-dependent manner. We next examined IL-1beta effect on TGF-beta1 expression and secretion at earlier time points. As shown in Fig. 7, C and D, 10 ng/ml IL-1beta started augmenting TGF-beta1 mRNA and peptide secretion in 3 and 6 h of incubation, respectively. These data reinforce that IL-1beta enhances TGF-beta1 secretion from PSCs. Because ERK is one of IL-1beta signaling mediators (16), we subsequently examined the participation of the ERK-dependent pathway in IL-1beta stimulation of TGF-beta1 expression and secretion by PSCs. To this end, we blocked ERK activation using the MEK1 inhibitor PD-98059. We previously reported that pretreatment of PSCs with 10 nM PD-98059 for 48 h successfully blocks TGF-beta1-induced ERK activation (25). Pretreatment of the cultures with PD-98059 attenuated exogenous IL-1beta enhancement of TGF-beta1 mRNA expression in PSCs (Fig. 8A; compare second and fourth lanes). Moreover, PD-98059 pretreatment decreased both basal and IL-1beta-stimulated TGF-beta1 peptide secretion from PSCs (Fig. 8B). Finally, we have confirmed that IL-1beta activates ERK in PSCs (Fig. 8C). These data indicate that IL-1beta stimulates TGF-beta1 expression and secretion by PSCs via an ERK-dependent pathway.


Figure 7
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  		Fig. 7. Effect of IL-1beta on TGF-beta1 expression and secretion by PSCs. A and C: TGF-beta1 mRNA expression was determined by RT-PCR after 48-h incubation with indicated amounts of IL-1beta (A) or after incubation for indicated times with 10 ng/ml IL-1beta (C). GAPDH mRNA expression was determined as an internal control. B and D: concentration of TGF-beta1 secreted into culture medium from PSCs was quantified by ELISA after 48-h incubation with indicated amounts of IL-1beta (B) or after incubation for indicated times with (open bars) or without (solid bars) 10 ng/ml IL-1beta (D). Values are means ± SE of 3 independent experiments. *P < 0.05 vs. control (B). #P < 0.05, ##P < 0.01 vs. controls without IL-1beta (D).

 

Figure 8
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  		Fig. 8. Effect of MEK1 inhibitor PD-98059 on TGF-beta1 expression and secretion by PSCs. A and B: after 2-h pretreatment with or without 10 nM PD-98059, cultured PSCs were incubated for 48 h in the presence or absence of 10 ng/ml IL-1beta. A: after incubation, TGF-beta1 mRNA expression was determined by RT-PCR using GAPDH mRNA expression as an internal control. B: TGF-beta1 peptide secreted into culture medium from PSCs during 48-h incubation quantified by ELISA. Values are means ± SE (n = 3). *P < 0.05 vs. first bar. **P < 0.001 vs. second bar. C: effect of IL-1beta on ERK activation in PSCs. Cells were incubated with 10 ng/ml IL-1beta for indicated times. ERK activation was then determined using Western blot analysis with antiphosphorylated ERK antibody (top). Western blot analysis using anti-ERK antibody was performed as an internal control (bottom).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we have demonstrated that anti-TGF-beta1 and anti-IL-1beta neutralizing antibodies attenuate IL-1beta and TGF-beta1 secretion from activated PSCs, respectively. Furthermore, IL-1beta expression and secretion by PSCs are enhanced by TGF-beta1 via a Smad3-dependent pathway. We also found that IL-1beta stimulates TGF-beta1 expression and secretion by PSCs via an ERK-dependent pathway. Accordingly, TGF-beta and IL-1beta stimulate each other's expression and secretion via Smad3- and ERK-dependent pathways, respectively, indicating the existence of an autocrine loop between IL-1beta and TGF-beta1 in activated PSCs. Because both TGF-beta1 and IL-1beta are fibrogenic factors, our observations suggest an interaction between TGF-beta1 and IL-1beta during pancreatic fibrosis.

IL-1beta is a major proinflammatory cytokine and plays a critical role in various inflammatory diseases of a variety organs (10). For example, at the onset of acute pancreatitis, IL-1beta is produced in and secreted from pancreatic acinar cells and transmigrated leukocytes and subsequently mediates systemic inflammatory responses, such as systemic circulatory failure and respiratory distress (8, 21). Repeated acute pancreatitis results in chronic pancreatitis with irreversible parenchymal destruction and glandular fibrosis. Pancreatic fibrosis is characterized by disorganized collagen deposition and acinar cell atrophy (8, 29). Although systemic inflammatory responses are readily resolved in chronic pancreatitis, the serum level of IL-1beta remains significantly elevated in these patients (7), and IL-1beta is known to promote pancreatic fibrosis. Currently, it is assumed that local cytokine sources are more important than distant sources because cytokines function exclusively in their immediate pericellular milieu (11). Therefore, in studies of the mechanism of inflammatory diseases, considerable attention has been focused on both autocrine and paracrine sources of cytokines (11). In acute pancreatitis, numerous inflammatory cells infiltrate pancreas tissues and secrete various cytokines, which induce both local and systemic inflammatory responses (22). Although not as severe as in acute pancreatitis, the infiltration of inflammatory cells into pancreatic tissues also is frequently observed in chronic pancreatitis (9a). Thus inflammatory cells, including macrophages, are assumed to be important sources of proinflammatory cytokines in chronic pancreatitis. In addition to inflammatory cells, activated PSCs also secrete various cytokines. However, the mechanism that regulates cytokines production and secretion by activated PSCs has not been resolved. We designed the present study to examine IL-1beta expression and secretion by culture-activated PSCs and to elucidate the molecular mechanism whereby TGF-beta1 regulates this expression.

TGF-beta1 participates in the regulation of a variety of PSC functions. For example, TGF-beta1 1) stimulates ECM synthesis, 2) promotes PSC activation, 3) attenuates proliferation in an autocrine manner, and 4) reduces matrix metalloproteinase (MMP)-3 and -9 expression (30). However, the intracellular signaling pathways through which TGF-beta1 exerts these diverse regulatory effects on PSC function have not been demonstrated. The facts that Smad proteins are major mediators of TGF-beta1 signaling and dual Smad2 and Smad3 pathways transduce TGF-beta1 stimuli raise questions concerning which pathway mediates the TGF-beta1 effects on cell function in various organs. However, because Smad2 and Smad3 are competitive with each other at TGF-beta1 receptor and Smad4 binding sites, it has been difficult to examine their specific roles in TGF-beta1 signal transduction. The targeted homozygous deletion of Smad2 and Smad3 genes in mice is a powerful tool for analyzing their functions in embryogenesis (9, 20 33, 34). However, because Smad2-knockout mice usually die in utero, elucidating the specific roles of Smad2 and Smad3 concurrently in differentiated cells derived from mature animals is difficult (20, 33). We developed a novel method to analyze Smad2- and Smad3-specific roles in TGF-beta1 signal transduction using adenovirus-mediated coexpression of Smad2 or Smad3 with dnSmad2/3 (25). We have demonstrated that TGF-beta1 enhances IL-1beta expression and secretion by activated PSCs via a Smad3-dependent pathway.

Our data regarding IL-1beta-enhanced TGF-beta1 expression and secretion by activated PSCs are important. IL-1beta has been shown to induce TGF-beta1 production in various cell types (1, 28). However, the intracellular signaling pathway through which IL-1beta stimulates TGF-beta1 expression is unclear. Because NF-{kappa}B is the proinflammatory transcription factor, the role of NF-{kappa}B in TGF-beta1 production during inflammatory responses has been studied. Rameshwar et al. (28) reported that IL-1beta-induced TGF-beta1 expression in monocytes obtained from patients with idiopathic myelofibrosis is dependent on NF-{kappa}B. However, NF-{kappa}B participation in TGF-beta1 induction, even that induced by IL-1beta, is thought to be indirect because of the absence of NF-{kappa}B binding site on the enhanced promoter region of the TGF-beta1 gene (26). To our knowledge, this report is the first to elucidate the intracellular signaling pathway for IL-1beta induction of TGF-beta1.

TGF-beta1 is secreted as a latent complex with TGF-beta propeptide and latent TGF-beta binding protein. After secretion, multiple TGF-beta activators, such as matrix MMP-2 and -9, convert latent TGF-beta1 to active TGF-beta1 by proteolytic cleavage of TGF-beta1 propeptide and latent TGF-beta binding protein (3). Recently, it was shown that PSCs express and secrete MMP-2 and -9 (27, 30). In addition, their expression and secretion by PSCs are regulated by various cytokines (27, 30). Thus it is an intriguing question whether IL-1beta controls autocrine TGF-beta1 activation by modulating the expression and secretion of MMP-2 and -9 by PSCs. A further study of the participation of MMP-2 and -9 in autocrine loop between IL-1beta and TGF-beta1 in activated PSCs is warranted.

In conclusion, we have shown the existence of autocrine loop between TGF-beta1 and IL-1beta in activated PSCs via Smad3- and ERK-dependent pathways, respectively. These observations provide new insights for understanding the mechanism of pancreatic fibrosis and developing a novel therapeutic strategy for its treatment.


   GRANTS
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 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, Tokyo, Japan) for Smad2, Smad3, and dnSmad2/3 adenoviral vectors.


   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
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 REFERENCES
 
1. Andriamanalijaona R, Felisaz N, Kim SJ, King-Jones K, Lehmann M, Pujol JP, and Boumediene K. Mediation of interleukin-1beta-induced transforming growth factor beta1 expression by activator protein 4 transcription factor in primary cultures of bovine articular chondrocytes: possible cooperation with activator protein 1. Arthritis Rheum 48: 1569–1581, 2003.[CrossRef][Web of Science][Medline]

3. Annes JP, Munger JS, and Rifkin DB. Making sense of latent TGFbeta activation. J Cell Sci 116: 217–224, 2003.[Abstract/Free Full Text]

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

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

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

7. Bamba T, Yoshioka U, Inoue H, Iwasaki Y, and Hosoda S. Serum levels of interleukin-1beta and interleukin-6 in patients with chronic pancreatitis. J Gastroenterol 29: 314–319, 1994.[CrossRef][Web of Science][Medline]

8. Bentrem DJ and Joehl RJ. Pancreas: healing response in critical illness. Crit Care Med 31, Suppl 8: S582–S589, 2003.[CrossRef][Web of Science][Medline]

9. Datto MB, Frederick JP, Pan L, Borton J, Zhuang Y, and Wang XF. Targeted disruption of Smad3 reveals an essential role in transforming growth factor beta-mediated signal transduction. Mol Cell Biol 19: 2495–2504, 1999.[Abstract/Free Full Text]

9a. De Angelis C, Valente G, Spaccapietra M, Angonese C, Del Favero G, Naccarato R, and Andriulli A. Histological study of alcoholic, nonalcoholic, and obstructive chronic pancreatitis. Pancreas 7: 193–196, 1992.[Web of Science][Medline]

10. Dinarello CA and Wolff SM. The role of interleukin-1 in disease. N Engl J Med 328: 106–113, 1993.[Free Full Text]

11. Friedman SL. Cytokines and fibrogenesis. Semin Liver Dis 19: 129–140, 1999.[Web of Science][Medline]

12. Goto D, Yagi K, Inoue H, Iwamoto I, Kawabata M, Miyazono K, and 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][Web of Science][Medline]

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

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

15. Kruse ML, Hildebrand PB, Timke C, Fölsch UR, and Schmidt WE. TGFbeta1 autocrine growth control in isolated pancreatic fibroblastoid cells/stellate cells in vitro. Regul Pept 90: 47–52, 2000.[CrossRef][Web of Science][Medline]

16. Kyriakis JM and Avruch J. Protein kinase cascades activated by stress and inflammatory cytokines. Bioessays 18: 567–577, 1996.[CrossRef][Web of Science][Medline]

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

18. Massagué J. TGF-beta signal transduction. Annu Rev Biochem 67: 753–791, 1998.[CrossRef][Web of Science][Medline]

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

20. Nomura M and Li E. Smad2 role in mesoderm formation, left-right patterning and craniofacial development. Nature 393: 786–790, 1998.[CrossRef][Medline]

21. Norman J. The role of cytokines in the pathogenesis of acute pancreatitis. Am J Surg 175: 76–83, 1998.[CrossRef][Web of Science][Medline]

22. Norman J. New approaches to acute pancreatitis: role of inflammatory mediators. Digestion 60, Suppl 1: 57–60, 1999.

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

24. Ohnishi N, Miyata T, Ohnishi H, Yasuda H, Tamada K, Ueda N, Mashima H, and Sugano K. Activin A is an autocrine activator of rat pancreatic stellate cells: potential therapeutic role of follistatin for pancreatic fibrosis. Gut 52: 1487–1493, 2003.[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, and Sugano K. 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. Perez JR, Higgins-Sochaski KA, Maltese JY, and Narayanan R. Regulation of adhesion and growth of fibrosarcoma cells by NF-{kappa}B RelA involves transforming growth factor beta. Mol Cell Biol 14: 5326–5332, 1994.[Abstract/Free Full Text]

27. Phillips PA, McCarroll JA, Park S, Wu MJ, Pirola R, Korsten M, and Wilson JS, and Apte MV. Rat pancreatic stellate cells secrete matrix metalloproteinases: implications for extracellular matrix turnover. Gut 52: 275–282, 2005.

28. Rameshwar P, Narayanan R, Qian J, Denny TN, Colon C, and Gascon P. NF-{kappa}B as a central mediator in the induction of TGF-beta in monocytes from patients with idiopathic myelofibrosis: an inflammatory response beyond the realm of homeostasis. J Immunol 165: 2271–2277, 2000.[Abstract/Free Full Text]

29. Satake K, Yamamoto T, and Umeyama K. A serial histologic study of the healing process after relapsing edematous acute pancreatitis in the rat. Surg Gynecol Obstet 165: 148–152, 1987.[Web of Science][Medline]

30. Shek FW, Benyon RC, Walker FM, McCrudden PR, Pender SL, Williams EJ, Johnson PA, Johnson CD, Bateman AC, Fine DR, and 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]

31. Shimada M, Andoh A, Hata K, Tasaki K, Araki Y, Fujiyama Y, and Bamba T. IL-6 secretion by human pancreatic periacinar myofibroblasts in response to inflammatory mediators. J Immunol 168: 861–868, 2002.[Abstract/Free Full Text]

32. Vesey DA, Cheung CWY, Cuttle L, Endre ZA, Gobé G, and Johnson DW. Interleukin-1beta induces human proximal tubule cell injury, {alpha}-smooth muscle actin expression and fibronectin production. Kidney Int 62: 31–40, 2002.[CrossRef][Web of Science][Medline]

33. Weinstein M, Yang X, Li C, Xu X, Gotay J, and Deng CX. Failure of egg cylinder elongation and mesoderm induction in mouse embryos lacking the tumor suppressor smad2. Proc Natl Acad Sci USA 95: 9378–9383, 1998.[Abstract/Free Full Text]

34. Yang X, Letterio JJ, Lechleider RJ, Chen L, Hayman R, Gu H, Roberts AB, and Deng C. Targeted disruption of SMAD3 results in impaired mucosal immunity and diminished T cell responsiveness to TGF-beta. EMBO J 18: 1280–1291, 1999.[CrossRef][Web of Science][Medline]




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