Effects of fibrogenic mediators on the development of pancreatic fibrosis in a TGF-beta 1 transgenic mouse model

R. Vogelmann, D. Ruf, M. Wagner, G. Adler, and A. Menke

Department of Internal Medicine I, University of Ulm, 89081 Ulm, Germany


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The pancreas morphology of transgenic mice that overexpress transforming growth factor-beta 1 (TGF-beta 1) in the pancreas resembles partially morphological features of chronic pancreatitis, such as progressive accumulation of extracellular matrix (ECM). Using this transgenic mouse model, we characterized the composition of pancreatic fibrosis and involved fibrogenic mediators. On day 14 after birth, fibrotic tissue was mainly composed of collagen type I and III. At this time, mRNA levels of TGF-beta 1 were increased. On day 70, the ECM composition was expanded by increased deposition of fibronectin, whereas connective tissue growth factor, fibroblast growth factor (FGF)-1, and FGF-2 mRNA expression levels were elevated in addition to TGF-beta 1. In parallel, the number of pancreatic stellate cells (PSC) increased over time. In vitro, TGF-beta 1 stimulated collagen type I expression but not fibronectin expression in PSC, in contrast to FGF-2, which stimulated both. This confirms that TGF-beta 1 mediates pancreatic fibrosis through activation of PSC and deposition of collagen type I and III at early time points. Furthermore, this points to an indirect mechanism in which TGF-beta regulates pancreatic ECM assembly by induction of additional growth factors.

transforming growth factor-beta ; connective tissue growth factor; fibroblast growth factor-2; chronic pancreatitis; pancreatic stellate cells


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IN CHRONIC PANCREATITIS, exogenous and endogenous function is disturbed due to increased deposition of extracellular matrix (ECM) proteins. The development of pancreatic fibrosis in chronic pancreatitis is poorly understood (11, 16, 17). The comprehension of diseases dominated by ECM was expanded by identifying ECM-producing cells. It is well known that fibroblasts produce and maintain ECM architecture (8). However, recent evidence demonstrates that fat-storing cells significantly contribute to ECM production (13, 19). These cells, named stellate cells, are characterized by their ability to store vitamin A. Following activation, they change into myofibroblast-like cells with positive staining for alpha -smooth muscle actin and desmin. Recently, cells with similar characteristics were identified in the pancreas and named pancreatic stellate cells (PSC) (1, 3). They are shown to produce ECM proteins such as collagen type I and III and fibronectin. Among other growth factors, they are mainly activated by transforming growth factor (TGF)-beta 1 (2, 3).

Members of the TGF-beta superfamily play a role in cell proliferation and differentiation, tissue repair, and recycling (25). TGF-beta 1 was found to be overexpressed in inflammatory pancreatic diseases (6, 34). Several groups showed an upregulation of TGF-beta 1 during regeneration of acute pancreatitis in humans and in animal models (12, 27, 34, 35). ECM deposition and fibroblast proliferation were paralleled by an increase of TGF-beta 1 in cerulein-induced pancreatitis in the rat (12). This effect could be partially reversed by antibodies against TGF-beta 1 (27). Moreover, repeated injections of recombinant TGF-beta 1 into mice after recurrent episodes of acute pancreatitis led to pancreatic fibrosis (35). This suggests that TGF-beta 1 is involved in the development of pancreatic fibrosis.

Other growth factors, such as connective tissue growth factor (CTGF), were also found to be overexpressed in inflammatory pancreatic diseases (6, 36). CTGF is a cysteine-rich mitogenic peptide leading to cell proliferation and ECM production in fibroblasts. Connective tissue cells secrete CTGF after activation by TGF-beta 1, and therefore CTGF acts downstream of TGF-beta 1 (14). However, it is not known how CTGF and TGF-beta 1 interact with each other during the development of pancreatic fibrosis.

Transgenic mice overexpressing active TGF-beta 1 selectively in the pancreas show enhanced proliferation of fibroblasts, deposition of ECM, and inhibition of acinar and centroacinar cell proliferation (23, 30). In the present study, we investigated the development of pancreatic fibrosis in the TGF-beta 1 transgenic mouse model generated by Sanvito and colleagues (30). We demonstrated an early increase in the number of PSC in the pancreas of TGF-beta 1 transgenic mice. This was paralleled by deposition of collagen type I and III. At this time, TGF-beta 1 was the only growth factor found to be overexpressed. Later, on day 70, when fibronectin was added to the collagen deposition, other growth factors, such as CTGF, fibroblast growth factor (FGF)-1, and FGF-2, were also increased. In vitro, FGF-2 stimulated fibronectin and collagen type I mRNA expression in PSC, whereas TGF-beta 1 was only capable of enhancing collagen type I expression. This suggests a cooperation of TGF-beta 1, CTGF, FGF-1, and FGF-2 in the development of pancreatic fibrosis.


    MATERIALS AND METHODS
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MATERIALS AND METHODS
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Materials. Polyclonal antiserum against collagen type I was obtained from Chemicon International (Temecula, CA). Antisera against collagen type III and IV were acquired from Rockland (Gilbertsville, PA). Antisera against fibronectin was supplied by Biomol (Hamburg, Germany). Monoclonal antibodies against alpha -smooth muscle actin, beta -actin, and desmin were purchased from Sigma Chemical (St. Louis, MO), and those against FGF-2 were received from Transduction Laboratories (Lexington, KY). The polyclonal antibody against CTGF, fisp12, and its cDNA clone were kindly provided by C. Wenger (University of Ulm). TGF-beta 1 was obtained from Pepro Tech (Rocky Hill, NY), and FGF-2 was purchased from Boehringer Mannheim (Mannheim, Germany). cDNA clones for alpha 1(I), alpha 1(III), and alpha 1(IV) collagen and for fibronectin were purchased from the American Type Culture Collection (ATCC nos. 61322, 61234, 65036, and 61038; Rockville, MD). The cDNA clone for 18S mRNA was made available by T. Gress (University of Ulm).

Animals. TGF-beta 1 transgenic mice, expressing TGF-beta 1 under control of a rat insulin II gene promoter, were a generous gift from Sanvito et al. (30). They were crossbred to C57BL/6 mouse strain (Charles River, Sulzfeld, Germany) and kept as heterozygotes for experiments. All experiments were performed according to the guidelines of local Animal Use and Care Committees. Transgenesis was determined on tail DNA (22) by PCR and Southern blot analysis. Primers for PCR analysis and the TGF-beta /human growth hormone cDNA probes for the transgene for Southern blot analysis were used as described by Sanvito et al. (30).

Protein studies. For protein analysis, samples were homogenized in protein lysis buffer (0.5 g tissue/ml) according to Laemmli (62.5 mM Tris · HCl, pH 6.8, 2% SDS, 10% glycerol, 5% mercaptoethanol, and 0.2% bromphenol blue) containing 5 µM aprotinin (Bayer-Leverkusen, Leverkusen, Germany), 1 mM pefabloc, 10 mM leupeptin, 10 µm pepstatin, and 5 mM soybean trypsin inhibitor (all Boehringer-Mannheim). Thereafter, probes were incubated for 10 min at 95°C, and 15 µl of each probe were analyzed by SDS-PAGE according to standard procedures (21). Quantity of protein was equalized by Coomassie blue staining of the gel. The fractionated proteins were blotted by semidry technique onto nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). For immunodetection, blots were incubated for 1 h at room temperature with first antibody as indicated and then incubated for 1 h with secondary peroxidase-coupled antiserum (Pierce, Rochester, NY). Antibody detection was carried out using an enhanced chemiluminescence reaction system (Pierce).

RNA studies. RNA was extracted according to Chomczynski and Sacchi (4). For Northern blots, 30 µg of total RNA were transferred on Hybond-N membranes by capillary blotting. Blots were hybridized with [32P]dCTP-labeled purified cDNA probes as described earlier (27). Membranes were exposed to X-ray films (Kodak XAR, Rochester, NY) at -70°C for 7-10 days. The mRNA expression was evaluated using quantitative RT-PCR analysis (TaqMan; PE Applied Biosystems, Norwalk, CT) (10). RT of 2 µg total RNA (20 mM Tris · HCl, 50 mM KCl, 2.5 mM MgCl2, 10 mM dithiothreitol, 0.5 µM random hexamer primer, 0.5 mM each dNTP, and 200 units Superscript II RT; GIBCO BRL, Rockville, MD) was carried out in duplicate and further processed independently. PCR was performed in triplicate using the primer combinations listed below (Sybr Green PCR core reagents; PE Applied Biosystems) and normalized to the endogenous 18S mRNA level for each reaction (ribosomal control reagent; PE Applied Biosystems). Relative quantification of the target cDNA in transgenic mice compared with littermate controls was established using the Delta -Delta -Cycle Threshold (CT) method. Results were expressed as means ± SD. Specific amplification was confirmed by electrophoresis on a 4% low-melting agarose gel, resulting in bands of the predicted size.

Primer design. If possible, primers were designed spanning exon-intron borders according to the published murine GenBank sequences. Regions of high similarity between the individual genes were excluded. Furthermore, each primer was checked in a similarity search for possible cross-recognition of related or unrelated genes. Optimal primer concentrations were established in previous experiments. The following forward (FP) and reverse (RP) primers and primer concentrations were used: TGF-beta 1-FP (300 nM), 5'-GTACAGCAAGGTCCTTGCCCT-3'; TGF-beta 1-RP (300 nM), 5'-TAGTAGACGATGGGCAGTGGC-3'; TGF-beta 2-FP (300 nM), 5'-GCAGAGTTCAGGGTCTTCCG-3'; TGF-beta 2-RP (300 nM), 5'-CAGCGTCTGTCACGTCGAA-3'; TGF-beta 3-FP (50 nM), 5'-TGACCCACGTCCCCTATCA-3'; TGF-beta 3-RP (900 nM), 5'-TCTCCTGAGTGCAGCCTTCC-3'; fisp12-FP (300 nM), 5'-GTGTGCACTGCCAAAGATGGT-3'; fisp12-RP (300 nM), 5'-ACACCCACTCCTTGCAGCATT-3'; FGF-1-FP (300 nM), 5'-TATACGGCTCGCAGACACCAA-3'; FGF-1-RP (50 nM), 5'-AACCAGTTCTTCTCCGCATGC-3'; FGF-2-FP (300 nM), 5'-AGCGACCACACGTCAAACTAC-3'; FGF-2-RP (300 nM), 5'-CAGCCGTCCATCTTCCTTCATA-3'; platelet-derived growth factor (PDGF)-A-chain-FP (300 nM), 5'-CCCATTCGCAGGAAGAGAAGTA-3'; PDGF-A-chain-RP (900 nM), 5'-TTGACGCTGCTGGTGTTACAA-3'; PDGF-B-chain-FP (300 nM), 5'-GCAAGAGTGTGGGCAGGGTTAT-3'; PDGF-B-chain-RP (300 nM), 5'-GAATCAGGCATCGAGACAGACG-3'; insulin-like growth factor (IGF)-I-FP (900 nM), 5'-AGATGTACTGTGCCCCACTGAA-3'; IGF-I-RP (900 nM), 5'-CTTCCTTCTGAGTCTTGGGCAT-3'; TGF-alpha -FP (300 nM), 5'-GGCTGCCAGCCAGAAGAA-3'; TGF-alpha -RP (300 nM), 5'-ACAGGTGATAATGAGGACAGCCA-3'.

Morphological studies. Specimens of pancreatic tissue were fixed in 4% paraformaldehyde and embedded in paraffin. Sections (5 µm) were deparaffinized and stained with hematoxylin and eosin for light microscopic evaluation. Other sections were deparaffinized, pretreated in the microwave in citrate buffer (10 mM, pH 6.0), and blocked with calcium- and magnesium-free (CMF)-PBS containing 3% BSA for 1 h at room temperature for immunofluorescence. The first antibody was diluted in CMF-PBS plus 0.3% BSA and incubated for 1 h at room temperature. The staining was visualized by a second Cy3-conjugated antibody (1 h at room temperature), and examination was performed by use of an Axiophot microscope (Zeiss, Oberkochen, Germany) or Leica confocal microscope TCS-4 (Leica, Wetzlar, Germany).

To estimate collagen and total protein content in histological sections, we stained 10-µm sections with fast green and sirius red in accordance with the method of López-de León and Rojkind (24). Dye was eluted by washing with NaOH-methanol, and concentrations were determined in a spectrophotometer at 605 and 540 nm. Collagen and total protein content were mathematically determined from absorbance at 605 and 540 nm (33).

Cell isolation. PSC were isolated by outgrowth techniques from mouse pancreas as described previously (3). Briefly, pancreatic tissue of TGF-beta transgenic as well as wild-type mice was mechanically dissociated and seeded in uncoated culture wells in the presence of DMEM containing 10% FCS. After incubation at 37°C in a 10% CO2-air humidified atmosphere, tissue blocks were removed and culture medium was changed. Outgrown cells were trypsinized and transferred to new culture plates. For immunocytochemical characterization, nonspecific binding was blocked with CMF-PBS containing 3% BSA for 1 h at room temperature. The first antibody was diluted in CMF-PBS plus 0.3% BSA incubated for 1 h at room temperature. The staining was visualized by a second Cy3-conjugated antibody (1 h at room temperature), and examination was performed by use of an Axiophot microscope.

For protein and RNA studies, cells were stimulated by incubation with DMEM containing 10% FCS and TGF-beta 1 or FGF-2 (20 ng/ml each) for 18 h. Protein and RNA were extracted and processed as described in Protein studies and RNA studies.


    RESULTS
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Compared with the pancreas of wild-type mice (Fig. 1A), we found a progressive accumulation of ECM in TGF-beta 1 transgenic mice. On day 14 after birth, ECM deposition started around endocrine cells (Fig. 1B). The amount of ECM was increasing and continuously replaced exocrine tissue (Fig. 1C). In 330-day-old transgenic mice, most of the acini were replaced by fibrotic tissue (Fig. 1D).


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Fig. 1.   Pancreatic tissue cross-sections. d, Day. Hematoxylin and eosin staining of wild-type tissue (wt) on day 70 after birth (A; magnification, ×180) and transforming growth factor (TGF)-beta 1 transgenic mouse pancreas on day 14 (B), day 70 (C), and day 330 (D) after birth (magnification, ×150). B: arrows indicate endocrine cells. C: black arrowheads indicate extracellular matrix (ECM) deposition and open arrowheads indicate exocrine tissue.

Time course of ECM deposition. We analyzed the time course of different members of the ECM on protein and mRNA level in TGF-beta 1 transgenic mice compared with the wild type. As shown in Fig. 2A, collagen type I and III were increased on the protein and mRNA levels on day 14, whereas fibronectin expression levels remained low. From day 70, there was a prominent fibronectin expression documented on both protein and mRNA levels. The distribution pattern of fibronectin was diffuse throughout fibrotic areas around endocrine and remaining acinar cells on day 70 (Fig. 2C). Only little fibronectin was present in normal mouse pancreas. The distribution of collagen type I and III was similar in specific immunofluorescence staining (data not shown). Although a further increase of ECM was morphologically documented on day 330 (Fig. 1D), the collagen type I and III expression remained stable. However, fibronectin expression levels continued to increase from day 70 to day 330 (Fig. 2, A and B). Collagen type IV was also enhancing from day 14 to day 330 on the mRNA expression level. However, on the protein level the expression remained similar throughout the time course (Fig. 2, A and B). Laminin expression, known to be associated with basal membrane, was not increased at the examined time points (data not shown).


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Fig. 2.   ECM analysis of TGF-beta 1 transgenic mouse pancreas at different time points vs. wt on day 70. Expression of collagen type I, III, and IV and fibronectin on the protein (A) and RNA (B) levels on day 14, day 70, and day 330 is shown. Same Northern blot for collagen type I was reprobed with fibronectin cDNA probe and for collagen type III with collagen type IV cDNA probe, respectively. Equal RNA loading is confirmed by ethidium bromide staining of blotted membrane (B, control). C: immunohistochemistry of pancreas paraffin sections. Right: fibronectin expression in fibrotic areas of 70-day-old TGF-beta transgenic mouse pancreas. Left: control pancreas of wt on day 70 (magnification ×100).

To confirm this observation, we quantified collagen content in pancreatic tissue sections by colorimetric analysis. Figure 3 shows the pancreatic collagen content in micrograms per milligram of total protein at different time points. Wild-type mice had low collagen content, which was similar throughout the examined time points on day 14, day 30, day 70, and day 330. In 70-day-old wild-type mice, the collagen content was 14.3 ± 0.8 µg/mg total protein. In transgenic mice, the collagen content was constantly increasing from day 14 to day 70. On day 70, the pancreatic collagen content was 44.0 ± 6.7 µg/mg total protein. In older transgenic mice (day 330), there was no further increase in pancreatic collagen content in relation to total protein mass (46.5 ± 4.2 µg/mg total protein).


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Fig. 3.   Influence of TGF-beta 1 on pancreatic collagen content in transgenic mouse pancreas at different time points. Collagen content is measured by colorimetric analysis and is expressed in µg/mg total protein. The wt were 70 days old. Data represent means ± SE of 3 different mice using 3 sections for each animal at time points outlined.

Growth factors in TGF-beta 1 transgenic mouse pancreas. The relative TGF-beta 1 transcript level was upregulated 9.8 ± 1.8-fold on day 14 and 15.8 ± 1.9-fold on day 70 compared with the wild-type level at the related time points as determined by quantitative RT-PCR TaqMan analysis (Table 1). The RT-PCR TaqMan analysis revealed no significant enhancement of TGF-beta 2, TGF-beta 3, CTGF, FGF-1, FGF-2, IGF-I, PDGF A, and PDGF B on mRNA expression level on day 14. 

                              
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Table 1.   Relative expression of cytokines/growth factors

However, CTGF, a growth factor recently described to be involved in the regulation of TGF-beta effects, was significantly upregulated on day 70 by 2.6 ± 0.4-fold. This was confirmed by Northern blot analysis, in which mRNA expression level continued to be similar on day 330 compared with day 70 (Fig. 4). Furthermore, mRNA expression levels of FGF-1 (4.7-fold), FGF-2 (5.4-fold), and TGF-beta 2 (2.5-fold) were significantly increased 70 days after birth. The upregulation of FGF-2 was even more prominent on day 330, as demonstrated by Northern blot analysis (Fig. 4). The expression levels of PDGF A and B, IGF-I, and TGF-beta 3 remained similar in the pancreas of transgenic and wild-type mice, whereas TGF-alpha was significantly downregulated (Table 1).


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Fig. 4.   Northern blot analysis of connective tissue growth factor (CTGF) and fibroblast growth factor (FGF)-2 mRNA using radioactive labeled CTGF and FGF-2 cDNA probes. Expression in TGF-beta 1 transgenic mouse pancreas on day 14, day 70, and day 330 compared with wt on day 70 is shown. Bottom: equal RNA loading is demonstrated by ethidium bromide staining of blotted membrane.

To localize the origin of CTGF (Fig. 5A) and FGF-2 (Fig. 5B) production, we stained pancreatic tissue sections of 70-day-old transgenic mice by using specific antibodies for CTGF and FGF-2. This revealed oval-shaped cells with cytoplasmic staining sparing the nucleus. The cells were localized in or adjacent to fibrotic areas. In contrast, no cells were specifically stained in wild-type or 14-day-old transgenic pancreatic mouse tissue (Fig. 5).


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Fig. 5.   Growth factors in TGF-beta 1 transgenic mice. Immunohistochemistry of pancreas paraffin sections using CTGF (A) and FGF-2 (B) antibodies. No staining of cells in wt (I) and TGF-beta 1 transgenic mouse pancreas on day 14 (II). Groups of CTGF- (A, III) and FGF-2- (B, III) positive cells sparing the nucleus on day 70 in fibrotic areas are shown (magnification, ×250).

ECM-producing cells. Immunofluorescence staining of pancreatic tissue of TGF-beta 1 transgenic mice showed an increased number of alpha -smooth muscle actin-positive cells in fibrotic areas (Fig. 6A). They were oval shaped and grouped in areas where fibrosis was close to normal-appearing acinar cells on day 14 (Fig. 6A, II) and in fibrotic areas on day 70 (Fig. 6A, III). They were not found in wild-type mice (Fig. 6A, I). The morphology and localization of these cells corresponded to the cells identified in Fig. 5, which were stained with CTGF or FGF-2, respectively.


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Fig. 6.   Pancreatic stellate cells (PSC) in TGF-beta 1 transgenic mice. A: immunohistochemistry of pancreas paraffin sections using alpha -smooth muscle actin antibody. There was no staining of cells in wt mouse pancreas. Blood vessels (V) served as internal positive control (I; magnification, ×250). alpha -Smooth muscle actin-positive cells in fibrotic areas close to acinar cells (A) in TGF-beta 1 transgenic mouse pancreas on day 14 (II; magnification, ×250). A large group of alpha -smooth muscle actin-positive cells sparing the nucleus are shown on day 70 (III; magnification, ×100). B: PSC growing out of TGF-beta 1 transgenic mouse pancreas after placing in cell culture medium reveal an intense staining with specific antibodies for alpha -smooth muscle actin, desmin, fibronectin, and collagen type III (magnification, ×600).

In in vitro analysis, cells were densely growing out of pancreatic tissue derived from TGF-beta 1 transgenic mice within days 1-3, in contrast to tissue from control mice. These cells were characterized as PSC by positive staining for alpha -smooth muscle actin and desmin (Fig. 6B).

Effect of growth factors on PSC. Primary cultures of PSC were immunohistochemically positive for fibronectin, collagen type I, and collagen type III (Fig. 6B; similar staining for collagen type I). RNA analyses of these cells revealed a low expression of fibronectin and collagen type I when cultured in DMEM only (Fig. 7). However, incubation of PSC with 20 ng/ml TGF-beta 1 in DMEM for 18 h exhibited a strong increase of collagen type I but not of fibronectin mRNA and protein expression levels. In contrast, incubation with 20 ng/ml FGF-2 in DMEM stimulated both fibronectin and collagen type I mRNA and protein expression levels in PSC in vitro (Fig. 7).


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Fig. 7.   Analysis of collagen type I and fibronectin protein and mRNA levels. Incubation of PSC in vitro for 18 h in DMEM (control) and DMEM containing 10% FCS and TGF-beta 1 or FGF-2 as indicated is shown. Left: protein expression. Right: mRNA expression. Bottom: equal loading is demonstrated by staining of blotted membrane using actin antibody for protein loading and radioactive labeled 18S rRNA cDNA probe for RNA loading, respectively.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we analyzed the development of pancreatic fibrosis in a TGF-beta 1 transgenic mouse model. Investigation of the time course of ECM deposition in this model revealed additional insights into the regulation of ECM by TGF-beta 1 in vivo. Soon after birth on day 14, ECM was mainly composed of collagen type I and type III in the transgenic mouse pancreas, whereas at the mRNA level, TGF-beta 1 was found to be overexpressed. However, in older mice collagen and fibronectin were the principal components of ECM.

As a possible source of ECM, we could identify PSC in younger and older mice. TGF-beta 1 was shown to be a major factor in stimulating the proliferation of PSC and their collagen synthesis capacity (2). Saotome et al. (31) also described fibroblast-like cells in periacinar regions possessing characteristics of myofibroblasts. In these cells, alpha -smooth muscle actin, the marker of myofibroblast-like phenotype, as well as collagen synthesis could be increased by TGF-beta 1 treatment.

In parallel to the increased fibronectin concentration in older animals, we could demonstrate CTGF and FGF-2 expression in the TGF-beta 1 transgenic mouse pancreas on day 70 by immunofluorescence staining. Both could be localized to oval-shaped cells in fibrotic areas. Staining of corresponding sections suggested myofibroblast-like cells as a source. TGF-beta 1 was shown to induce CTGF gene expression in fibroblasts (14) and caused FGF-2 and FGF receptor mRNA expression in human myofibroblastic liver cells, known as activated hepatic stellate cells (29). Furthermore, analysis of mRNA expression in the TGF-beta 1 transgenic mouse pancreas on day 70 revealed a significant increase of additional growth factors such as CTGF, TGF-beta 2, FGF-1, and FGF-2.

CTGF is known as a downstream mediator of TGF-beta action on fibroblasts. Wenger et al. (36) could identify slightly to moderately elevated transcript levels for CTGF in 15 studied tissue samples of chronic pancreatitis. Also, di Mola et al. (6) recently showed an upregulation of CTGF mRNA expression levels in human chronic pancreatitis tissues. They revealed a positive correlation of CTGF expression and degree of fibrosis in these samples.

Growth factors such as FGF-1 and FGF-2 were also found to be overexpressed in human chronic pancreatitis, and a role in pancreatic fibrosis was implicated (9). Here we could demonstrate the upregulation of FGF-1 and FGF-2 in the TGF-beta 1 transgenic mouse pancreas on day 70. Little is known about the potential of FGF-1 in fibrosis development. A role of FGF-1 in the proliferation of interstitial fibroblasts in human kidney diseases was suggested (20). FGF-2 is predominantly recognized for its mitogenic effect on fibroblasts (29). The effects of FGF-2 on synthesis of ECM components by various cell types were contradictory (7, 15, 32). However, in fibroblasts derived from the pancreas, FGF-2 stimulated mRNA concentrations of collagen type I and III and fibronectin (26).

Because CTGF and FGF-2 were not increased at early stages, this suggests that TGF-beta 1 alone is sufficient to induce pancreatic fibrosis by the induction of PSC and the associated collagen expression in this transgenic mouse model. However, CTGF and FGF-2 appear to be cofactors at later stages. The concomitant effect of TGF-beta 1, FGF-2, and CTGF in older animals was combined with a change in ECM composition when fibronectin expression was getting prominent and collagen deposition remained constant.

This was confirmed by our in vitro analysis. Thereby, TGF-beta 1 stimulated collagen type I but not fibronectin expression in cells that grew out of the TGF-beta 1 transgenic mouse pancreas in cell culture medium. In vitro, these cells were alpha -smooth muscle actin- and desmin-positive, identifying them as mouse PSC. However, TGF-beta 1 was described to stimulate collagen type I and III and fibronectin expression in fibroblasts and human PSC in vitro (3, 18). Furthermore, TGF-beta 1 was shown to regulate fibronectin gene transcription in different cell lines, including fibroblasts. The regulation occurred rapidly and did not require protein synthesis, indicating a direct mechanism (5). However, it is well known that TGF-beta exhibits differential effects on the same cell type under different experimental conditions. There is evidence that early cultured PSC show different ECM expression and responsiveness to growth factor stimulation compared with fibroblast cell lines (18, 19). In contrast, it was also shown that protein synthesis was required for TGF-beta to induce ECM protein gene expression, indicating that some other intermediate factor or factors may be involved in the signaling pathways (5, 28). One of these factors was shown to be CTGF, stimulating fibronectin and collagen type I synthesis in cultured fibroblasts after being induced by TGF-beta (14). In the present study, the difference that TGF-beta did not directly influence fibronectin expression in mouse PSC underscores the idea that TGF-beta effects were partially mediated by induction of additional peptide growth factors and emphasizes the complexity in the control of ECM composition in vivo. Our in vitro data were in agreement with the in vivo situation, in which TGF-beta 1 stimulated the activation and proliferation of PSC on day 14, which produced and deposited predominantly collagen matrix.

The complexity of ECM control by TGF-beta is also accentuated by the findings of collagen type IV regulation in this in vivo model. In vitro data in hepatic stellate cells showed the induction of collagen type IV by TGF-beta 1 (18). However, despite the induction of collagen type IV on the mRNA level, it remained constant on the protein expression level throughout the examined stages of life of TGF-beta transgenic mice compared with the wild type.

Furthermore, we could demonstrate that FGF-2 stimulated collagen type I and fibronectin expression and synthesis in mouse PSC in vitro. As outlined above, FGF-2 was shown to induce collagen and fibronectin expression in human PSC and fibroblasts derived from pancreatic tissue (3, 26). The mouse PSC could also be demonstrated in vivo in the TGF-beta 1 transgenic mouse pancreas on day 70 after birth, when fibronectin and FGF-2 expression was increased. The in vitro effect of FGF-2 on fibronectin expression in mouse PSC suggests a concomitant effect of TGF-beta 1 and FGF-2 on fibronectin and collagen production in this transgenic mouse model at a later time point.

In conclusion, transgenic mice selectively overexpressing TGF-beta 1 in the pancreas revealed an increased production and deposition of ECM in pancreatic tissue. PSC were identified as a source of this ECM assembly. At early time points, when TGF-beta 1 was the predominant fibrotic factor, the matrix was mainly composed of collagen type I and III. Later, when fibronectin was also a prominent part of ECM, growth factors such as CTGF and FGF-2 were also found to be increased. In vitro, FGF-2 is able to stimulate fibronectin and collagen expression, whereas TGF-beta 1 mainly increased collagen expression in mouse PSC. This points to an indirect mechanism by which TGF-beta regulates pancreatic ECM assembly by induction of additional growth factors. Future studies will be necessary to look further into the cross-talk of the different growth factors to understand the complexity of fibrosis development in inflammatory pancreatic diseases.


    ACKNOWLEDGEMENTS

We would like to thank L. Orci (Dept. of Morphology, University of Geneva, Switzerland) for kindly providing the TGF-beta transgenic mouse model. Furthermore, we thank S. Braeg for excellent technical assistance and C. Wenger for kindly providing the CTGF cDNA.


    FOOTNOTES

This work was supported by grant KN 200 DFG Forschergruppe obtained from the Deutsche Forschungsgemeinschaft.

Address for reprint requests and other correspondence: R. Vogelmann, Dept. of Internal Medicine I, Univ. of Ulm, Robert-Koch-Str. 8, 89081 Ulm, Germany (E-mail: roger.vogelmann{at}medizin.uni-ulm.de).

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.

Received 7 January 2000; accepted in final form 20 July 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   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].

2.   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].

3.   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[ISI][Medline].

4.   Chomczynski, P, and Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156-159, 1987[ISI][Medline].

5.   Dean, DC, Newby RF, and Bourgeois S. Regulation of fibronectin biosynthesis by dexamethasone, transforming growth factor beta, and cAMP in human cell lines. J Cell Biol 106: 2159-2170, 1988[Abstract].

6.   Di Mola, FF, Friess H, Martignoni ME, Di Sebastiano P, Zimmermann A, Innocenti P, Graber H, Gold LI, Korc M, and Buchler MW. Connective tissue growth factor is a regulator for fibrosis in human chronic pancreatitis. Ann Surg 230: 63-71, 1999[ISI][Medline].

7.   Drago, J, Nurcombe V, Pearse MJ, Murphy M, and Bartlett PF. Basic fibroblast growth factor upregulates steady-state levels of laminin B1 and B2 chain mRNA in cultured neuroepithelial cells. Exp Cell Res 196: 246-254, 1991[ISI][Medline].

8.   Elsässer, HP, Adler G, and Kern HF. Fibroblast structure and function during regeneration from hormone-induced acute pancreatitis in the rat. Pancreas 4: 169-178, 1989[ISI][Medline].

9.   Friess, H, Yamanaka Y, Buchler M, Beger HG, Do DA, Kobrin MS, and Korc M. Increased expression of acidic and basic fibroblast growth factors in chronic pancreatitis. Am J Pathol 144: 117-128, 1994[Abstract].

10.   Gibson, UE, Heid CA, and Williams PM. A novel method for real time quantitative RT-PCR. Genome Res 6: 995-1001, 1996[Abstract].

11.   Gress, TM, Menke A, Bachem M, Müller-Pillasch F, Ellenrieder V, Weidenbach H, Wagner M, and Adler G. Role of extracellular matrix in pancreatic diseases. Digestion 59: 625-637, 1998[ISI][Medline].

12.   Gress, TM, Müller-Pillasch F, Elsässer HP, Bachem M, Ferrara C, Weidenbach H, Lerch M, and Adler G. Enhancement of transforming growth factor beta 1 expression in the rat pancreas during regeneration from caerulein-induced pancreatitis. Eur J Clin Invest 24: 679-685, 1994[ISI][Medline].

13.   Gressner, AM, and Bachem MG. Molecular mechanisms of liver fibrogenesis---a homage to the role of activated fat-storing cells. Digestion 56: 335-346, 1995[ISI][Medline].

14.   Grotendorst, GR. Connective tissue growth factor: a mediator of TGF-beta action on fibroblasts. Cytokine Growth Factor Rev 8: 171-179, 1997[Medline].

15.   Hurley, MM, Abreu C, Harrison JR, Lichtler AC, Raisz LG, and Kream BE. Basic fibroblast growth factor inhibits type I collagen gene expression in osteoblastic MC3T3-E1 cells. J Biol Chem 268: 5588-5593, 1993[Abstract/Free Full Text].

16.   Kennedy, RH, Bockman DE, Uscanga L, Choux R, Grimaud JA, and Sarles H. Pancreatic extracellular matrix alterations in chronic pancreatitis. Pancreas 2: 61-72, 1987[Medline].

17.   Klöppel, G, and Maillet B. The morphological basis for the evolution of acute pancreatitis into chronic pancreatitis. Virchows Arch 420: 1-4, 1992.

18.   Knittel, T, Janneck T, Muller L, Fellmer P, and Ramadori G. Transforming growth factor beta 1-regulated gene expression of Ito cells. Hepatology 24: 352-360, 1996[ISI][Medline].

19.   Knittel, T, Kobold D, Saile B, Grundmann A, Neubauer K, Piscaglia F, and Ramadori G. Rat liver myofibroblasts and hepatic stellate cells: different cell populations of the fibroblast lineage with fibrogenic potential. Gastroenterology 117: 1205-1221, 1999[ISI][Medline].

20.   Kuo, NT, Norman JT, and Wilson PD. Acidic FGF regulation of hyperproliferation of fibroblasts in human autosomal dominant polycystic kidney disease. Biochem Mol Med 61: 178-191, 1997[ISI][Medline].

21.   Laemmli, UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685, 1970[ISI][Medline].

22.   Laird, PW, Zijderveld A, Linders K, Rudnicki MA, Jaenisch R, and Berns A. Simplified mammalian DNA isolation procedure. Nucleic Acids Res 19: 4293, 1991[ISI][Medline].

23.   Lee, MS, Gu D, Feng L, Curriden S, Arnush M, Krahl T, Gurushanthaiah D, Wilson C, Loskutoff DL, Fox H, and Sarvetnick N. Accumulation of extracellular matrix and developmental dysregulation in the pancreas by transgenic production of transforming growth factor-beta 1. Am J Pathol 147: 42-52, 1995[Abstract].

24.   López-de León, A, and Rojkind M. A simple micromethod for collagen and total protein determination in formalin-fixed paraffin-embedded sections. J Histochem Cytochem 33: 737-743, 1985[Abstract].

25.   Massague, J, and Weis Garcia F. Serine/threonine kinase receptors: mediators of transforming growth factor beta family signals. Cancer Surv 27: 41-64, 1996[ISI][Medline].

26.   Menke, A, Lutz MP, Ludwig CU, Gress TM, and Adler G. Regeneration after acute pancreatitis: influences of peptide growth factors. In: Acute Pancreatitis---Novel Concepts in Biology and Therapy, edited by Büchler M, Uhl W, Friess H, and Malfertheiner P.. Berlin: Blackwell Wissenschafts-Verlag, 1999, p. 129-141.

27.   Menke, A, Yamaguchi H, Gress TM, and Adler G. Extracellular matrix is reduced by inhibition of transforming growth factor beta1 in pancreatitis in the rat. Gastroenterology 113: 295-303, 1997[ISI][Medline].

28.   Roberts, AB, and Sporn MB. The transforming growth factor betas. In: Peptide Growth Factors and Their Receptors-Handbook of Experimental Pathology, edited by Sporn MB, and Roberts AB.. Heidelberg: Springer-Verlag, 1990, p. 419-472.

29.   Rosenbaum, J, Blazejewski S, Preaux AM, Mallat A, Dhumeaux D, and Mavier P. Fibroblast growth factor 2 and transforming growth factor beta 1 interactions in human liver myofibroblasts. Gastroenterology 109: 1986-1996, 1995[ISI][Medline].

30.   Sanvito, F, Nichols A, Herrera PL, Huarte J, Wohlwend A, Vassalli JD, and Orci L. TGF-beta 1 overexpression in murine pancreas induces chronic pancreatitis and, together with TNF-alpha, triggers insulin-dependent diabetes. Biochem Biophys Res Commun 217: 1279-1286, 1995[ISI][Medline].

31.   Saotome, T, Inoue H, Fujimiya M, Fujiyama Y, and Bamba T. Morphological and immunocytochemical identification of periacinar fibroblast-like cells derived from human pancreatic acini. Pancreas 14: 373-382, 1997[ISI][Medline].

32.   Tan, EM, Rouda S, Greenbaum SS, Moore JH, Jr, Fox JW, IV, and Sollberg S. Acidic and basic fibroblast growth factors down-regulate collagen gene expression in keloid fibroblasts. Am J Pathol 142: 463-470, 1993[Abstract].

33.   Valderrama, R, Navarro S, Campo E, Camps J, Gimenez A, Parés A, and Caballeria J. Quantitative measurement of fibrosis in pancreatic tissue. Int J Pancreatol 10: 23-29, 1991[ISI][Medline].

34.   Van Laethem, JL, Deviere J, Resibois A, Rickaert F, Vertongen P, Ohtani H, Cremer M, Miyazono K, and Robberecht P. Localization of transforming growth factor beta 1 and its latent binding protein in human chronic pancreatitis. Gastroenterology 108: 1873-1881, 1995[ISI][Medline].

35.   Van Laethem, JL, Robberecht P, Résibois A, and Devière J. Transforming growth factor beta promotes development of fibrosis after repeated courses of acute pancreatitis in mice. Gastroenterology 110: 576-582, 1996[ISI][Medline].

36.   Wenger, C, Ellenrieder V, Alber B, Lacher U, Menke A, Hameister H, Wilda M, Iwamura T, Beger HG, Adler G, and Gress TM. Expression and differential regulation of connective tissue growth factor in pancreatic cancer cells. Oncogene 18: 1073-1080, 1999[ISI][Medline].


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