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Enhanced Fibroblast Growth Factor 5 Expression in Stromal and Exocrine

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Enhanced Fibroblast Growth Factor 5 Expression in Stromal and Exocrine 134 Gut 1998;43:134–139 Enhanced fibroblast growth factor 5 expression in stromal and exocrine elements of the pancreas in chronic pancreatitis Gut: first published as 10.1136/gut.43.1.134 on 1 July 1998. Downloaded from T Ishiwata, M Kornmann, H G Beger, M Korc Abstract ies of NIH 3T3 fibroblasts with human tumour Background—Fibroblast growth factor 5 DNA.5 FGF-5 encodes a glycosylated 267 (FGF-5) belongs to a group of mitogenic amino acid protein that contains a hydrophobic and angiogenic heparin binding growth N-terminal sequence which allows its secretion factors but its potential role in chronic from its cells of origin.6 It is overexpressed in a inflammatory conditions is not known. number of human malignancies, including Aims—To compare FGF-5 expression in hepatomas and carcinomas of the bladder, the normal pancreas and in the pancreas of endometrium, and pancreas.6–8 patients with chronic pancreatitis (CP) and Although FGF-5 is known to be expressed in to characterise FGF-5 expression and se- inflammatory cells,8 the potential role of cretion in TAKA-1 cells, an immortalised FGF-5 in chronic inflammatory conditions is Syrian hamster pancreatic duct cell line. not known. Therefore, in the present study, we Methods and results—Northern blotting characterised FGF-5 expression in chronic revealed the presence of a 4.0 kb FGF-5 pancreatitis (CP), an inflammatory disease of mRNA transcript in both normal and CP the pancreas that may ultimately result in exo- tissue samples. Densitometric analysis crine and endocrine dysfunction.9 Along with indicated that the transcript levels were enlarged pancreatic ducts, pseudoductular increased by a factor of 1.44 in CP tissue hyperplasia, loss of parenchyma, and acinar cell samples compared with normal tissue degeneration there is significant replacement of samples (p=0.039). By immunohisto- the functional parenchyma by fibrosis in chemisty and in situ hybridisation, FGF-5 association with proliferation of fibroblasts and 10 was faintly expressed in ductal and islet infiltration by inflammatory cells. We now cells in the normal pancreas. In contrast, report that FGF-5 expression is increased in in CP tissue samples, there was abundant CP where it is localised in ductal, acinar, and http://gut.bmj.com/ expression of FGF-5 in ductal, acinar, and islet cells and in fibroblasts within the connec- islet cells, as well as in periductal fibrob- tive tissue. Furthermore, it is expressed and lasts. FGF-5 was also expressed in secreted by TAKA-1 cells, an immortalised TAKA-1 cells as determined by Northern hamster pancreatic duct cell line. blotting. By immunoblotting of heparin- sepharose precipitates, TAKA-1 cells were Methods shown to secrete FGF-5 into the medium. on September 29, 2021 by guest. Protected copyright. PATIENTS AND TISSUE SAMPLES Conclusion—Exocrine and stromal de- Normal human pancreatic tissue samples rived FGF-5 has the potential to partici- (from four men and two women; median age pate in autocrine and paracrine pathways 24.5 years, range 22–44) were obtained that may contribute to the pathobiology of through a donor programme. Chronic pan- Departments of chronic pancreatitis. creatitis tissue samples were obtained from one Medicine, Biological (Gut 1998;43:134–139) female and eight male patients (median age 50 Chemistry and Keywords: chronic pancreatitis; fibroblast growth years, range 33–60) undergoing pancreatic Pharmacology, 11 University of factor; in situ hybridisation surgery. Pancreatic adenocarcinoma tissue California, Irvine, samples were obtained from 10 female and six California, USA male patients (median age 64 years, range T Ishiwata Fibroblast growth factors (FGFs) are a group 44–77) undergoing surgery for pancreatic can- M Kornmann cer as recently described.8 On surgical removal M Korc of homologous heparin binding polypeptides that are involved in various biological processes samples were either immediately frozen in liq- Department of including stimulation of fibroblast proliferation uid nitrogen and stored at −80°C until RNA General Surgery, and angiogenesis.1 The FGF family currently extraction or fixed in Bouin’s solution for University of Ulm, consists of 14 members: acidic FGF (aFGF or 18–20 hours and embedded in paraYn wax for Ulm, Germany histological analysis. Studies involving human H G Beger FGF-1), basic FGF (bFGF or FGF-2), FGF-3 (int-2), FGF-4 (hst/kFGF), FGF-5, FGF-6, tissue were approved by the Ethics Committee Correspondence to: keratinocyte growth factor (KGF or FGF-7), of the University of Ulm, Germany and the Dr M Korc, Division of androgen induced growth factor (AIGF or Human Subjects Committee of the University Endocrinology, Diabetes, of California, Irvine, California, USA. and Metabolism, Med. Sci. I FGF-8), glia activating factor (GAF or FGF- 2 C240, University of 9), FGF-10, and FGF-11–14 which are also 34 California, Irvine, California known as FGF homologous factors 1–4. The CELL CULTURE 92697, USA. FGF-5 gene was originally discovered as a TAKA-1 immortalised Syrian hamster pancre- Accepted for publication human oncogene which had acquired trans- atic duct cells were a gift from Dr Parviz Pour, 19 January 1998 forming potential, based on transfection stud- University of Nebraska, Omaha, Nebraska, FGF-5 expression in chronic pancreatitis 135 USA, and were grown in Dulbecco’s modified stead of primary antibodies did not yield any Eagle’s/F12 medium at 37°C in humidified air immunoreactivity. with 5% CO2. Medium contained 100 U/ml penicillin, 100 µg/ml streptomycin, and 5% IN SITU HYBRIDISATION Gut: first published as 10.1136/gut.43.1.134 on 1 July 1998. Downloaded from foetal bovine serum. Tissue sections were placed on 3-amino- propylmethoxysilane coated slides, deparaYn- ised, and incubated sequentially at 23°C for 20 NORTHERN BLOT ANALYSIS minutes with 0.2 N HCl and for 15 minutes Northern blotting was carried out as previously ° 8 12 with 20 µg/ml proteinase K at 37 C. The sec- described. Total RNA was extracted, size tions were then postfixed for five minutes in fractionated, electrotransferred to nylon mem- PBS containing 4% paraformaldehyde, and branes, and hybridised under high stringency quenched twice with glycine (2 mg/ml) in PBS. conditions with a 306 bp human FGF-5 cDNA 8 They were then incubated in 50% (vol/vol) fragment. A 190 bp mouse 7S cDNA was used formamide/2× saline sodium citrate (SSC) for as loading control.12 cDNAs were labelled with 32 one hour. The hybridisation buVer contained [á- P]dCTP (3000 Ci/mmol) by random 0.6 M NaCl, 1 mM EDTA, 10 mM Tris-HCl priming prior to hybridisation. The resulting (pH 7.5), 0.25% sodium dodecyl sulphate autoradiographs were scanned and the ratio of (SDS), 200 µg/ml yeast tRNA, 1× Denhart’s the optical densities of the RNA levels solution, 10% dextran sulphate, 40% forma- 8 (FGF-5:7S) was calculated for each sample. mide, and 50 ng/ml digoxigenin labelled FGF-5 riboprobe. Hybridisation was initiated IMMUNOHISTOCHEMISTRY by applying 100 µl of the hybridisation buVer to Immunohistochemistry was carried out as pre- each section, followed by incubation in a moist ° viously described.8 Briefly, 4 µm paraYnwax chamber for 16 hours at 42 C. For immuno- embedded tissue sections were immunostained logical detection, the Genius 3 non-radioactive using the streptavidin-peroxidase technique nucleic acid detection kit was used (Boe- (Kirkegaard & Perry Laboratories Inc., Gaith- hringer, Mannheim, Germany). The sections ersburg, Maryland, USA). After deparaYnisa- were then washed sequentially with 50% × ° × tion and blocking of endogenous peroxidase formamide/2 SSC for 30 minutes at 50 C, 2 ° × activity, sections were incubated for 15 minutes SSC for 20 minutes at 50 C, 0.2 SSC for 20 ° minutes at 50°C, and 0.1× SSC for 30 minutes at 23 C with 10% normal rabbit serum and ° then incubated with highly specific goat at 50 C. The sections were washed briefly with polyclonal antihuman FGF-5 antibodies (1/ buVer 1 solution (100 mM Tris-HCl and 150 100 in phosphate buVered saline (PBS) mM NaCl, pH 7.5) and incubated with 1% containing 1% BSA; Santa Cruz Biotechnol- (wt/vol) blocking reagents in buVer 1 solution ° http://gut.bmj.com/ ogy, Santa Cruz, California, USA) at 4°C for for 60 minutes at 23 C. After washing briefly 8 with buVer 1, the sections were incubated for 16 hours. Bound antibody was detected with a ° biotinylated rabbit antigoat IgG secondary 30 minutes at 23 C with a 1/2000 dilution of antibody and streptavidin-peroxidase complex alkaline phosphatase conjugated polyclonal (Kirkegaard & Perry), using diaminobenzidine sheep antidigoxigenin Fab fragment containing 0.2% Tween 20. The sections were then tetrahydrochloride (DAB) as the substrate. For ° insulin staining guinea pig polyclonal antipor- washed twice for 15 minutes at 23 C with buVer 1 solution containing 0.2% Tween 20 cine insulin antibodies (1/1000 in PBS con- on September 29, 2021 by guest. Protected copyright. taining 1% BSA; Dako Corp., Carpinteria, and equilibrated with buVer 3 (100 mM Tris-HCl, 100 mM NaCl, 50 mM MgCl ,pH California, USA), cross reactive with human 2 insulin, and biotinylated goat antiguinea pig 9.5) for two minutes. The sections were then IgG secondary antibodies (Kirkegaard & incubated with colour solution containing Perry) were used after incubation with 10% nitroblue tetrazolium and X phosphate in a normal goat serum. Sections were counter- dark box for two to three hours. After the reac- stained with Mayer’s haematoxylin. Omission tion was stopped with Tris-EDTA (TE) buffer, of primary antibodies or incubation in the the sections were mounted in aqueous mount- presence of non-immunised goat serum in- ing medium. PURIFICATION AND IMMUNOBLOTTING OF FGF-5 PROTEIN FGF-5 protein purification was carried out as previously described.78 TAKA-1 cells were grown in complete medium containing 5% FBS to 90% confluency in T75 flasks.
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