Article Text

Download PDFPDF

Fibrinogen induces cytokine and collagen production in pancreatic stellate cells
  1. A Masamune,
  2. K Kikuta,
  3. T Watanabe,
  4. K Satoh,
  5. M Hirota,
  6. S Hamada,
  7. T Shimosegawa
  1. Division of Gastroenterology, Tohoku University Graduate School of Medicine, Sendai, Japan
  1. Dr A Masamune, Division of Gastroenterology, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574 Japan; amasamune{at}int3.med.tohoku.ac.jp

Abstract

Objective: Fibroblasts in the area of fibrosis in chronic pancreatitis and of the desmoplastic reaction associated with pancreatic cancer are now recognised as activated pancreatic stellate cells (PSCs). Recent studies have shown strong expression of fibrinogen, the central protein in the haemostasis pathway, in the stromal tissues of pancreatic cancer and chronic pancreatitis, suggesting that PSCs are embedded in and exposed to abundant fibrinogen in these pathological settings. The effects of fibrinogen on cell functions in PSCs were examined here.

Methods: PSCs were isolated from human pancreas tissues of patients undergoing operations for pancreatic cancer, and from rat pancreatic tissues. The effects of fibrinogen on key cell functions and activation of signalling pathways in PSCs were examined.

Results: Fibrinogen induced the production of interleukin 6 (IL6), interleukin 8 (IL8), monocyte chemoattractant protein-1, vascular endothelial growth factor, angiopoietin-1 and type I collagen, but not proliferation or intercellular adhesion molecule-1 expression. Fibrinogen increased α-smooth muscle actin expression and induced the activation of nuclear factor-κB (NF-κB), Akt and three classes of mitogen-activated protein kinases (extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase and p38 mitogen-activated protein kinase (MAPK)). Fibrinogen-induced IL6 and IL8 production was inhibited by antibodies against αvβ3 and α5β1 integrins, suggesting that these integrins worked as counter receptors for fibrinogen in PSCs. In addition, fibrinogen-induced production of these cytokines was abolished by an inhibitor of NF-κB, and partially inhibited by inhibitors of ERK and p38 MAPK.

Conclusion: Fibrinogen directly stimulated profibrogenic and proinflammatory functions in PSCs.

View Full Text

Statistics from Altmetric.com

In 1998, star-shaped cells in the pancreas, namely pancreatic stellate cells (PSCs), were identified and characterised.1 2 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 myofibroblast-like cells, which express α-smooth muscle actin (α-SMA), actively proliferate and produce extracellular matrix (ECM) components such as type I collagen.15 In addition, PSCs have a variety of cell functions. For example, upon activation, PSCs acquire a proinflammatory phenotype; activated PSCs express intercellular adhesion molecule (ICAM)-16 and produce cytokines and chemokines such as interleukin 6 (IL6),7 IL8,8 and monocyte chemoattractant protein (MCP)-1.5 The ability of PSCs to induce angiogenesis has been shown recently.9 It has been established that activated PSCs play a pivotal role in the development of pancreatic fibrosis in chronic pancreatitis (CP).15

Fibrinogen (FBG) is a 340 kDa heterotrimer protein composed of pairs of three non-identical polypeptide chains designated as Aα, Bβ and γ.10 11 FBG is converted to fibrin by the proteolytic action of thrombin. FBG functions in primary haemostasis in support of platelet aggregation and in secondary haemostasis in the formation of an insoluble fibrin clot.10 11 In addition to these well-known roles in blood coagulation, recent studies have shown that FBG plays important roles in a variety of processes including cell–cell interaction, inflammation, wound healing and carcinogenesis.1014 Importantly, FBG has been recognised as a component of the ECM.13 14 Several reports have shown overexpression of FBG in pancreatic cancer and in CP.1518 Quantitative proteomic analysis showed that FBG β and γ were upregulated in pancreatic tissue, pancreatic juice and serum from patients with pancreatic cancer and with CP.15 16 Immunostaining revealed strong expression of FBG in stromal tissues of pancreatic cancer and CP.17 18 Compared with other epithelial neoplasms, pancreatic cancer has an extremely rich and dense fibrotic stroma surrounding the tumour cells, called the desmoplastic reaction.19 20 PSCs are now thought to be responsible for the dense stroma associated with pancreatic cancer.1921 Strong expression of FBG in the stroma tissues of pancreatic cancer and CP suggests that PSCs are embedded in and exposed to abundant FBG in these pathological settings. However, the possible regulation of cell functions by FBG in PSCs remains unknown. We report here that FBG induces the production of cytokines and type I collagen in an integrin-dependent manner in PSCs.

MATERIALS AND METHODS

Materials

Collagenase P and recombinant human IL1β were purchased from Roche Applied Science (Mannheim, Germany). Human recombinant platelet-derived growth factor (PDGF)-BB was from R&D Systems (Minneapolis, Minnesota, USA). Rabbit antibody against FBG was from Dako (Glostrup, Denmark). Goat anti-type I collagen antibody was from Southern Biotechnology (Birmingham, Alabama, USA). Rabbit antibodies against mitogen-activated protein kinases (MAPKs), Akt and inhibitor of nuclear factor-κB (NF-κB) (IκB-α) were from Cell Signaling Technology (Beverly, Massachusetts, USA). Mouse antibodies against α5β1 and αvβ3 integrins were from Chemicon (Temecula, California, USA). Fluorescein isothiocyanate (FITC)-labelled mouse antibody against ICAM-1 was from Immunotech (Marseille, France). Rabbit antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was from Trevigen (Gaithersburg, Maryland, USA). Bay11-7082, U0126, SB203580, SP600125 and wortmannin were from Calbiochem-Novabiochem (San Diego, California, USA). All other reagents including plasminogen-free human FBG type I were from Sigma-Aldrich (St Louis, Missouri, USA) unless specifically described.

Cell culture

Human PSCs were isolated from the resected pancreas tissue of patients undergoing operation for pancreatic cancer as previously described,20 with approval from the Ethics Committee of Tohoku University School of Medicine. Experiments were performed using human PSCs from at least three independent preparations and those between passages three and nine after isolation. Rat PSCs were prepared as previously described22 from the pancreas tissues of male Wistar rats (Japan SLC, Hamamatsu, Japan) using Nycodenz solution (Nycomed Pharma, Oslo, Norway) after perfusion with 0.03% collagenase P. All animal procedures were performed in accordance with the National Institutes of Health Animal Care and Use Guidelines. Cell purity was >90% as assessed by a typical star-like configuration and by detecting vitamin A autofluorescence. Cells between passages two and four were used, except for those using freshly isolated PSCs. Cells were maintained in Ham’s F-12/Dulbecco’s modified essential medium supplemented with 10% heat-inactivated fetal bovine serum, penicillin sodium and streptomycin sulfate. Unless specifically described, we incubated PSCs in serum-free medium for 24 h before the addition of experimental reagents. For some experiments, inhibitors of signal transduction pathways or antibodies against integrins were added at 30 min before the treatment with FBG. We examined the effects of bovine serum albumin (BSA) as a control to clarify whether the effects of FBG were due to non-specific protein binding to PSCs.

Human pancreatic cancer AsPC-1 cells (American Type Culture Collection, Manassas, Virginia, USA) were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum, penicillin sodium and streptomycin sulfate.

Expression of FBG

Immunohistochemical staining

Pancreas tissues were removed from 10 patients undergoing operation (5 patients with pancreatic cancer and 5 patients with CP), and fixed by immersing in 4% paraformaldehyde overnight at 4°C. The specimens were embedded in regular paraffin wax and cut into 4 μm sections. Immunostaining for FBG was performed as previously described23 using a streptavidin–biotin–peroxidase complex detection kit (Histofine Kit; Nichirei, Tokyo, Japan). Briefly, tissue sections were deparaffinised and rehydrated in phosphate-buffered saline. Following antigen retrieval with the target retrieval solution (Dako), endogenous peroxidase activity was blocked by incubation with 0.3% hydrogen peroxide. After immersion in 10% normal goat serum, the sections were incubated with rabbit anti-FBG antibody (at 1:100 dilution) overnight at 4°C. The slides were incubated with biotinylated goat anti-rabbit immunoglobulin G (IgG) antibody, followed by peroxidase-conjugated streptavidin. Finally, colour was developed by incubating the slides for several minutes with diaminobenzidine (Dojindo, Kumamoto, Japan). H&E staining was performed on serial sections.

Reverse transcription-PCR

Total RNA was prepared from PSCs using the RNeasy total RNA preparation kit (Qiagen, Germantown, Maryland, USA). Total RNA (∼200 ng) was reverse transcribed, and the resultant cDNA was subjected to PCR. Specific primer sets are shown in table 1. FBG was amplified using 35 cycles at 94°C (for 1 min), at 60°C (for 1 min) and at 72°C (for 1 min). A 5 μl aliquot of the 30 μl of PCR products was separated by 2% agarose gel electrophoresis and visualised under ultraviolet light after gels had been stained with ethidium bromide.

Table 1 Sequences of PCR primer sets used to amplify fibrinogen

Immunofluorescent staining

Immunofluorescent staining was performed as previously described.24 PSCs were directly plated on μ-slides (ibidi, Munich, Germany), incubated overnight at 37°C and fixed in 4% paraformaldehyde. After blocking with 10% normal goat serum, cells were incubated with rabbit anti-FBG antibody (at 1:100 dilution) overnight at 4°C. After washes, cells were incubated with FITC-conjugated goat anti-rabbit IgG antibody (Jackson Immunoresearch, West Grove, Pennsylvania, USA) for 1 h. After washes, cells were analysed for fluorescence using a confocal laser scanning microscope (ECLIPSE TE2000-U, Nikon, Tokyo, Japan). Nuclear counterstaining was performed using propidium iodide.

Induction of subcutaneous tumours in nude mice

To determine whether PSCs produce FBG in vivo, 2×106 AsPC-1 cells were injected alone on the left side of the back, or together with PSCs (2×106 cells) on the right side, subcutaneously into nude mice (Charles River Laboratories Japan, Yokohama, Japan).20 The animals were sacrificed after 4 weeks, and tumours were removed. Expression of FBG was determined by immunohistochemical staining. In addition, double immunofluorescent staining of FBG and α-SMA was performed. Briefly, tissue sections were deparaffinised and rehydrated. Following antigen retrieval, the slides were blocked with 10% normal goat serum, and incubated with the mouse Ig-blocking reagent (Vector Labs, Burlingame, California, USA) for 1 h. The slides were incubated with rabbit anti-FBG antibody (at 1:100 dilution) and mouse anti-α-SMA antibody (at 1:200 dilution) overnight at 4°C. After washes, the slides were incubated with FITC-conjugated goat anti-rabbit IgG antibody and Alexa Fluor 546-labelled goat anti-mouse IgG antibody (Molecular Probes, Eugene, Oregon, USA). After washes, the slides were analysed for fluorescence using a confocal laser scanning microscope. Nuclear counterstaining was performed using 4′,6-diamidino-2-phenylindole (DAPI).

Cell proliferation assay

Cell proliferation was assessed using a commercial kit (Cell proliferation ELISA, BrdU; Roche Applied Science). This is a colourimetric immunoassay based on the measurement of 5-bromo-2′-deoxyuridine (BrdU) incorporation during DNA synthesis. After 24 h incubation with FBG, cells were labelled with BrdU for 3 h. 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 optical density (OD)370–OD492.

Cell migration assay

Cell migration was examined by the modified Boyden’s chamber assay using a commercial kit (QCM chemotaxis colorimetric cell migration assays; Millipore, Bedford, Massachusetts, USA). Briefly, the cell suspension (2×105 cells/well) was added to the culture inserts (8 μm pore) coated with type I collagen in a 24-well companion plate. The lower chamber included FBG at the indicated concentrations. After 24 h incubation, the cell suspension in the upper chamber was aspirated, and the upper surface of the filter was carefully cleaned with cotton plugs. Cells that migrated through the polycarbonate membrane were stained, lysed, and quantified on a standard microplate at OD560.

ELISA

After 24 h incubation, cell culture supernatants were harvested and stored at −80°C until use. The levels of IL6, IL8, MCP-1, vascular endothelial growth factor (VEGF) and angiopoietin-1 in the culture supernatants were determined by ELISA using commercial kits (VEGF and rat IL6 assays from Pierce Chemical, Rockford, Illinois, USA; the other assays from R&D Systems).

Collagen assay

Collagen expression was determined by procollagen type I C-peptide (PICP) assay and by western blot analysis.

PICP assay

PSCs were plated into 24-well plates and grown to confluence. After 48 h incubation with FBG, culture supernatants were harvested and stored at −80°C until use. The level of PICP was determined using an ELISA kit (Takara Bio, Otsu, Japan).

Western blot analysis

Western blot analysis was performed as previously described.25 Cells were lysed in sodium dodecyl sulfate (SDS) buffer, and cellular proteins (∼100 μg) were fractionated on a 10% SDS–polyacrylamide gel (Bio-Rad, Hercules, California, USA). They were transferred to a nitrocellulose membrane (Bio-Rad), and the membrane was incubated overnight at 4°C with goat antibody against type I collagen. After incubation with peroxidase-conjugated anti-goat IgG antibody, proteins were visualised using an ECL kit (Amersham Biosciences, Buckinghamshire, UK). Levels of α-SMA and GAPDH were determined in a similar manner.

Flow cytometry

Cell surface expression of αvβ3 and α5β1 integrins was examined by flow cytometry. PSCs were detached using 2 mM EDTA solution, and washed. Cells were incubated with mouse antibodies against αvβ3 and α5β1 integrin (at 1:200 dilution each) or non-specific mouse IgG (diluted to the same protein concentration as the anti-integrin antibodies) as a negative control for 1 h on ice. Cells were then washed, and incubated with FITC-conjugated goat anti-mouse IgG (at 1:100 dilution) for 30 min on ice. Cells were washed thoroughly and integrin expression was analysed in a FACScan flow cytometer (Becton Dickinson, Tokyo, Japan). Cell surface expression of ICAM-1 was examined in a similar manner using FITC-conjugated anti-ICAM-1 antibody.

Assessment of MAPK activation

Activation of MAPKs was examined by western blot analysis using antiphosphospecific MAPK antibodies as previously described.25 These antibodies recognise only phosphorylated forms of MAPKs, thus allowing the assessment of activation of the kinases. Levels of total MAPKs, Akt (phosphorylated at Ser473 and total) and IκB-α were determined by western blot analysis in a similar manner.

Luciferase assay

The reporter construct containing two IgG κ chain NF-κB-binding sites (GGGACTTTCC), inserted upstream of the firefly luciferase gene in the pXP2 vector,26 was generously provided by Dr Naofumi Mukaida (Kanazawa University, Japan). Approximately 1×106 PSCs were transfected with 2 μg of the reporter construct, along with 40 ng of pRL-TK vector (Promega, Madison, Wisconsin, USA) as an internal control, using the FuGENE6 reagent (Roche Diagnostics). After 24 h, transfected cells were treated with FBG for an additional 24 h. At the end of the incubation, cell lysates were prepared using a Pica Gene kit (Toyo Ink, Tokyo, Japan), and the light intensities were measured using a model AB-2200 luminescencer (Atto, Tokyo, Japan).

Statistical analysis

The results were expressed as mean (SD). Experiments were performed at least three times, and similar results were obtained. Representative luminograms and autoradiograms are shown. Differences between the groups were evaluated by analysis of variance (ANOVA), followed by Fisher’s test for post hoc analysis. A p value <0.05 was considered statistically significant.

RESULTS

FBG was expressed in the fibrotic areas of the pancreas

We first examined the expression of FBG in the resected pancreas of the patients undergoing operation. In agreement with previous reports,17 18 strong FBG expression in the stromal tissues was observed in all of the 10 specimens obtained from patients with pancreatic cancer and CP (fig 1). We examined whether PSCs by themselves expressed FBG. PSCs expressed mRNA for FBG (fig 2A). Immunofluorescent staining of cultured PSCs showed cytoplasmic expression of FBG (Fig. 2B), similar to the FBG staining previously reported in A549 human lung epithelial cells,13 and for the ECM constituents such as collagen and fibronectin in PSCs.1 2 27 We further examined the expression of FBG in subcutaneous tumours which developed in nude mice. Strong FBG expression was observed in the tumours arising from the combined injection of AsPC-1 cells and PSCs, whereas FBG expression was less evident in tumours growing after injection of AsPC-1 cells alone (fig 2C). Of note, positive FBG immunostaining was observed on α-SMA-positive PSCs surrounding cancer cells (Fig. 2D).

Figure 1

Fibrinogen (FBG) expression in the stromal tissues of patients with chronic pancreatitis (CP) and pancreatic cancer. Immunohistochemical staining for FBG was performed in the resected pancreas tissues of patients with pancreatic cancer or CP. H&E staining was performed on serial sections. Original magnification: ×100.

Figure 2

Pancreatic stellate cells (PSCs) express fibrinogen (FBG). (A) Total RNA was prepared from human PSCs, and the mRNA levels of FBG Aα, Bβ and γ were examined by reverse transcription-PCR. sm, size marker (100 bp ladder). (B) Expression of FBG in cultured PSCs was examined by immunofluorescent staining. Nuclear counterstaining was performed using propidium iodide. Original magnification: ×200. (C, D) 2×106 AsPC-1 cells were injected alone on the left side of the back, or together with PSCs (2×106 cells) on the right side, subcutaneously into nude mice. The animals were sacrificed after 4 weeks, and tumours were removed. Expression of FBG was determined by immunohistochemical staining (C). H&E staining was performed on serial sections. In addition, double immunofluorescent staining of FBG and α-smooth muscle actin (α-SMA) was performed (D). Positive FBG immunostaining was observed on α-SMA-positive PSCs surrounding cancer cells. Nuclear counterstaining was performed using methyl green (C) or 4,6′-diamidino-2-phenylindole (DAPI) (D). Original magnification: ×40 (C) or ×400 (D).

FBG stimulated the cell functions in human PSCs

We examined whether FBG affected the key cell functions of human PSCs. FBG did not induce proliferation, whereas PDGF-BB, one of the most potent mitogens for PSCs in vitro,22 did (fig 3A). FBG induced the production of IL6, IL8 and MCP-1 (fig 3B,C). FBG induced the production of these cytokines in a dose-dependent manner, with a peak at concentrations of ∼1 mg/ml. BSA up to 1 mg/ml did not induce the production of these cytokines (data not shown), suggesting that the effects of FBG were not due to non-specific protein binding to PSCs. FBG did not induce PSC migration (fig 3D). As previously reported in rat PSCs,6 human PSCs expressed ICAM-1 (fig 3E, left panel). ICAM-1 expression was enhanced by IL1β, but not by FBG (fig 3E, right panel). FBG increased type I collagen production, as assessed by PICP production (fig. 4A) and western blot analysis (fig 4B). BSA up to 1 mg/ml did not induce PICP production (data not shown). FBG increased the expression of α-SMA, a marker of PSC activation1 2 (fig 4B). In addition, FBG induced the production of proangiogenic factors VEGF and angiopoietin-1 (fig 4C,D).

Figure 3

Fibrinogen (FBG) induces the production of cytokines in pancreatic stellate cells (PSCs). (A–D) Serum-starved human PSCs were treated with FBG at the indicated concentrations for 24 h. (A) Cell proliferation was assessed by 5-bromo-2′-deoxyuridine (BrdU) incorporation ELISA, and the optical density (OD)370–OD492 of the sample is shown. Platelet-derived growth factor (PDGF)-BB (at 25 ng/ml) served as a positive control. (B, C) The levels of interleukin 6 (IL6), IL8 and monocyte chemoattractant protein-1 (MCP-1) in the culture supernatants were determined by ELISA. (D) Cell migration was examined by the modified Boyden’s chamber assay. Cells that migrated through the polycarbonate membrane were stained, lysed and quantified at OD560. **p<0.01 vs FBG at 0 mg/ml. (E) PSCs were left untreated (“Control”), or treated with FBG (at 1 mg/ml) or IL1β (at 2 ng/ml) for 24 h. Intercellular adhesion molecule-1 (ICAM-1) expression was examined by flow cytometry. For control, non-specific mouse IgG was used instead of mouse anti-ICAM-1 antibody in untreated cells (left panel). FL1-H, FL1 pulse height.

Figure 4

Fibrinogen (FBG) induces expression of type I collagen, α-smooth muscle actin (α-SMA) and proangiogenic factors. (A) Serum-starved human pancreatic stellate cells (PSCs) were treated with FBG at the indicated concentrations for 48 h, and the level of procollagen type I C-peptide (PICP) in the culture supernatants was determined by ELISA. *p<0.05, **p<0.01 vs FBG at 0 mg/ml. (B) PSCs were left untreated (“Cont”) or treated with FBG at 1 mg/ml for 48 h. Total cell lysates (∼50 μg) were prepared, and the levels of type I collagen, α-SMA and glyceraldehyde phosphate dehydrogenase (GAPDH) were determined by western blot analysis. (C, D) PSCs were treated with FBG at the indicated concentrations for 24 h, and the levels of vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) in the culture supernatants was determined by ELISA. Data shown are expressed as means (SD) (n = 6). **p<0.01 vs FBG at 0 mg/ml.

FBG induced IL6 and IL8 production via α5β1 and αvβ3 integrins

It has been shown previously that FBG interacts with cell surface integrins including α5β1 and αvβ3, as well as non-integrin receptors.10 28 FBG-induced production of cytokines and PICP was enhanced in the presence of Mn2+ (fig 5A), suggesting involvement of integrins.28 FBG-induced IL6 and IL8 production was inhibited by antibodies against α5β1 and αvβ3 integrins, but not by non-specific mouse IgG (fig 5B, data not shown), suggesting that α5β1 and αvβ3 integrins were involved in FBG-induced cytokine production. Indeed, activated PSCs expressed α5β1 and αvβ3 integrins as assessed by flow cytometry (fig 5C).

Figure 5

Fibrinogen (FBG) induces the production of interleukin 6 (IL6) and IL8 through α5β1 and αvβ3 integrins. (A) Human pancreatic stellate cells (PSCs) were treated with FBG (at 1 mg/ml) in the absence (0 μM) or presence (20 μM) of Mn2+. After 24 h (for IL6, IL8 and monocyte chemoattractant protein-1 (MCP-1)) or 48 h (for procollagen type I C-peptide (PICP)), cell culture supernatants were harvested, and the levels of IL6, IL8, MCP1 and PICP were determined by ELISA. The vertical line is in ng/ml for cytokines and μg/ml for PICP. **p<0.01 vs Mn2+ at 0 μM. (B) PSCs were left untreated (“Cont”), or treated with FBG (at 1 mg/ml) in the absence or presence of antibodies against α5β1 or αvβ3 integrins (at 10 μg/ml). After 24 h, cell culture supernatants were harvested, and the levels of IL6 and IL8 were determined by ELISA. **p<0.01 vs FBG only. (C) Expression of α5β1 and αvβ3 integrins in human PSCs was examined by flow cytometry. For control, non-specific mouse IgG was used instead of mouse anti-integrin antibody. FL1-H, FL1 pulse height.

FBG activated NF-κB, MAPKs and Akt

We then examined the effects of FBG on the activation of signal transduction pathways. We used FBG at 1 mg/ml because maximal induction of cytokines and type I collagen was observed at this concentration. FBG activated NF-κB, as assessed by the luciferase assay (fig 6A). FBG induced transient degradation of IκB-α, further supporting its ability to activate NF-κB (fig 6B). In addition, FBG activated three classes of MAPKs (extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPK) and Akt in a time-dependent manner (Fig. 6C).

Figure 6

Fibrinogen (FBG) induces activation of nuclear factor-κB (NF-κB), mitogen-activated protein kinases (MAPKs) and Akt. (A) Human pancreatic stellate cells (PSCs) were transfected with the luciferase vector (2× NF-κB) along with the pRL-TK vector as an internal control. After 24 h, the transfected cells were left untreated (control; “Cont”), or treated with FBG at the indicated concentrations or interleukin 1β (IL1β) (at 2ng/ml). After another 24 h incubation, intracellular luciferase activities (“RLU”) were determined. The data represent mean values (SD), calculated from three independent experiments as fold induction compared with the activity observed in control. **p<0.01 vs control. IL1, interleukin 1. (B, C) Human PSCs were treated with FBG (at 1 mg/ml) for the indicated times. Total cell lysates (∼100 μg) were prepared, and the levels of inhibitor of NF-κB (IκB-α), glyceraldehyde phosphate dehydrogenase (GAPDH), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 MAPK and Akt were determined by western blot analysis. P, phosphorylated.

Roles of intracellular signalling pathways in migration and PICP production

We examined the roles of activated NF-κB, MAPKs and Akt pathways in FBG-induced cytokine production. For this purpose, we employed specific inhibitors of these signalling pathways. We employed Bay11-7082 (at 5 μM, an inhibitor of NF-κB), U0126 (at 5 μM, an inhibitor of the ERK pathway), SB203580 (at 25 μM, an inhibitor of p38 MAPK), SP600125 (at 10 μM, an inhibitor of the JNK pathway) and wortmannin (at 100 nM, an inhibitor of the phosphatidylinositol 3-kinase–Akt pathway). We have previously shown that these inhibitors effectively inhibited the respective target pathways in PSCs at the concentrations used in this study.2225 Bay11-7082 abolished FBG-induced production of IL6 and IL8, suggesting a central role for NF-κB activation (Fig. 7). FBG-induced IL6 and IL8 production was in part inhibited by U0126 and SB203580, whereas SP600125 and wortmannin were ineffective. Thus, FBG induced IL6 and IL8 production mainly through the activation of NF-κB and in part that of the ERK and p38 MAPK pathways.

Figure 7

Roles of signalling pathways in fibrinogen (FBG)-induced interleukin 6 (IL6) and IL8 production. Serum-starved human pancreatic stellate cells (PSCs) were left untreated (“Cont”), or treated with FBG (at 1 mg/ml) in the absence or presence of inhibitors of signalling pathways. We employed Bay11-7082 (“Bay”, at 5 μM, a specific inhibitor of nuclear factor-κB (NF-κB), U0126 (“U”, at 5 μM, an inhibitor of the extracellular signal-regulated kinase (ERK) pathway), SB203580 (“SB”, at 25 μM, an inhibitor of p38 mitogen-activated protein kinase (MAPK), SP600125 (“SP”, at 10 μM, an inhibitor of the c-Jun N-terminal kinase (JNK) pathway) and wortmannin (“W”, at 100 nM, an inhibitor of the phosphatidylinositol 3-kinase–Akt pathway). After 24 h incubation, culture supernatants were harvested, and the levels of IL6 and IL8 were determined by ELISA. **p<0.01 vs FBG only.

FBG stimulated cell functions in rat PSCs

Finally, we examined whether FBG stimulated cell functions of rat PSCs isolated by density gradient centrifugation. FBG induced the production of IL6 and MCP-1 in a dose-dependent manner (fig 8A). FBG increased the expression of type I collagen and α-SMA (fig 8B). FBG did not induce the transformation of freshly isolated PSCs into myofibroblast-like cells (data not shown), suggesting that the FBG responsiveness might differ depending on the stages of activation.

Figure 8

Fibrinogen (FBG) induces the expression of cytokines, type I collagen and α-smooth muscle actin (α-SMA) in rat pancreatic stellate cells (PSCs). (A) Serum-starved rat PSCs were treated with FBG at the indicated concentrations for 24 h. The levels of interleukin 6 (IL6) and monocyte chemoattractant protein-1 (MCP-1) in the culture supernatants were determined by ELISA. **p<0.01 vs FBG at 0 mg/ml. (B) Rat PSCs were left untreated (“Cont”) or treated with FBG at 1 mg/ml for 48 h. Total cell lysates (∼50 μg) were prepared, and the levels of type I collagen, α-SMA and glyceraldehyde phosphate dehydrogenase (GAPDH) were determined by western blot analysis.

DISCUSSION

We showed here that FBG directly stimulated cell functions of PSCs. FBG induced IL6, IL8, MCP-1, type I collagen and proangiogenic factors, but not proliferation, migration or ICAM-1 expression. This pattern of induction is distinct from that of IL1β, tumour necrosis factor α, transforming growth factor β1 or PDGF-BB.3 4 68 In addition, FBG increased α-SMA expression, a marker of activated PSCs.1 2 FBG induced production of cytokines, type I collagen and proangiogenic factors in a dose-dependent manner, with a peak at concentrations of ∼1 mg/ml. The local FBG concentrations in the pancreas are unknown, but there a few studies measuring the plasma FBG concentrations in patients with pancreatic cancer.17 29 Preston et al reported that the plasma FBG concentration was 4.7 mg/ml in patients with pancreatic cancer, which was greater than that in healthy controls.29 Similarly, Bloomston et al reported that the plasma FBG concentration in patients with pancreatic cancer, measured by enzymatic analysis, was 0.514 (0.641) mg/ml, which was greater than that of healthy controls.17 It has been shown that FBG expression was upregulated in pancreatic juice in patients with pancreatic cancer and with CP,16 suggesting that the FBG concentration is high locally in the pancreas. Interestingly, we showed here that activated PSCs by themselves expressed FBG, suggesting an autocrine regulation of cell functions in PSCs. The abundant FBG present in the stromal tissues is thought to derive from exudation of plasma FBG due to increased vascular permeability and subsequent deposition.10 11 18 However, recent studies have shown the ability of human cancer cells and epithelial cells to synthesise and secrete FBG.14 The origin of FBG in the stromal tissues may, therefore, be due in part to endogenous synthesis and deposition.

FBG induced IL6, IL8 and MCP-1 production, but not ICAM-1 expression in PSCs. It has been shown that FBG induces proinflammatory responses in several cells, but the effects were cell type specific.28 30 31 For example, FBG induced IL8 and MCP-1 production in integrin-dependent manners in human umbilical vein endothelial cells.28 FBG induced IL8, but not MCP-1, production in human monocytes.30 FBG induced ICAM-1 and chemokine expression in human synovial fibroblasts.31 Interestingly, IL6 is known to be the major inducer of FBG,32 suggesting that FBG-induced IL6 production might regulate FBG expression in PSCs in an autocrine/paracrine manner. We showed here that FBG activated NF-κB, Akt and three classes of MAPKs in PSCs. Little is known as to the effect of FBG on the activation of signal transduction pathways. FBG activated NF-κB in human umbilical vein endothelial cells.28 FBG activated p38 MAPK and Akt in human vascular smooth muscle cells.33 FBG-induced IL6 and IL8 production was almost completely inhibited by an inhibitor of NF-κB, and partially by inhibitors of ERK and p38 MAPK, suggesting important roles for these signalling pathways. Activation of signalling pathways which play key roles in a variety of cellular processes suggests that FBG alters a variety of cell functions in PSCs.

Lugea et al34 recently reported that persistent activation of PSCs and development of pancreatic fibrosis were observed in plasminogen-deficient mice. Because the deposition of FBG and fibrin is a critical feature of plasminogen-deficient mice,35 profibrogenic and proinflammatory actions of FBG might contribute to the persistent activation of PSCs and development of pancreatic fibrosis in that experimental model.

Wojtukiewicz et al18 showed different distributions of FBG and fibrin in the stromal tissues of pancreatic cancer. They showed that FBG existed abundantly throughout the tumour stroma, especially near blood vessels, whereas fibrin was readily detected in the stroma immediately surrounding nests of cancer cells.18 Therefore, FBG might be converted to fibrin through the action of cancer cell-derived thrombin, whereas PSCs distant from the cancer cells are unlikely to be exposed to fibrin. In this study, we focused on FBG rather than fibrin. Recent studies have shown unique biological effects of FBG without conversion to fibrin.13 33 Importantly, it has been shown that FBG assembles into a pre-established, mature ECM of fibroblasts without conversion to fibrin, and matrix–FBG fibrils co-localize with other fibrillar proteins such as fibronectin, laminin and collagen type IV.12 36 Thus, FBG demonstrates properties of matricellular proteins.37 Once secreted, matricellular proteins bind to ECM proteins including collagen type I and IV, fibronectin and laminin. Although not structurally related, matricellular proteins regulate similar biological functions during embryonic development, tissue injury and cancer development, mainly by promoting adhesion, migration and survival of cells.37 In a similar manner to FBG, significant upregulation of matricellular proteins including galectin-1,25 periostin,27 connective tissue growth factor38 and tenascin-C39 was demonstrated in the stromal tissues of pancreatic cancer and CP, as well as in activated PSCs.

We observed here that the presence of Mn2+ enhanced the production of cytokine and type I collagen, consistent with mediation of the responses through integrins.28 Inhibition of IL6 and IL8 production by antibodies to αvβ3 and α5β1 integrins provided additional evidence for integrin-mediated FBG effects. This is in agreement with a recent study showing that α5β1 integrin mediates connective tissue growth factor-induced adhesion, migration and type I collagen synthesis in PSCs.38 Expression of several matricellular proteins suggests that PSCs express other integrins to transduce extracellular signals. Indeed, we have found that PSCs express αvβ5 as well as αvβ3 and α5β1 integrins (A Masamune et al, unpublished observation).

Our study adds a new insight as to the role of FBG in the progression of pancreatic cancer from the viewpoint of tumour–stromal interaction, which has received increasing attention in recent years. Although a role for FBG has been suggested in stroma formation and subsequent tumour growth,40 41 the underlying molecular mechanisms remained largely unclear. Our study suggests that FBG contributes to tumour stroma formation and subsequent tumour growth in the pancreas through the direct stimulation of PSCs. Although it is still controversial, recent studies have supported the concept that the desmoplastic reaction created by the interaction between pancreatic cancer cells and PSCs favours the progression of pancreatic cancer.1921 Our results suggest that FBG contributes to creating a tumour-supportive microenvironment in the pancreas by sustaining fibrogenic stellate cell activity. It is likely that FBG exerts its influence on tumourigenesis by changing the microenvironment of tumour cells through the alternation of cell adhesion, the composition of the ECM and the activities of PSCs within and surrounding the tumour mass. In addition, it is possible that FBG and fibrin directly affect the cell behaviour of pancreatic cancer cells. Indeed, recent studies have shown that integrin-mediated cell attachment to the ECM components affects the cell behaviour of pancreatic cancer cells in integrin-dependent manners.42 Further elucidation of the roles of FBG in pancreatic fibrosis will facilitate better understanding and rational approaches for the treatment of pancreatic cancer and CP.

Acknowledgments

The authors are grateful to Kiiko Ogasiwa for technical assistance.

REFERENCES

View Abstract

Footnotes

  • Competing interests: None.

  • Funding: This work was supported in part by a Grant-in-Aid from the Japan Society for the Promotion of Science (to AM and KK), by the Pancreas Research Foundation of Japan (to AM and KK), by the Kanae Foundation for Life and Socio-Medical Science (to AM) and by the Uehara Memorial Foundation (to AM).

  • Ethics approval: This study was approved by the Ethics Committee of Tohoku University School of Medicine, Sendai, Japan.

Request Permissions

If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.

Linked Articles

  • Digest
    Robin Spiller Magnus Simren