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Trypsin is produced by and activates protease-activated receptor-2 in human cancer colon cells
Life Sciences 70 (2002) 1359–1367
Trypsin is produced by and activates protease-activated receptor-2 in human cancer colon cells Evidence for new autocrine loop Robert Ducroc*, Claire Bontemps, Katia Marazova, Hélène Devaud, Dalila Darmoul, Marc Laburthe Neuroendocrinologie et Biologie Cellulaire Digestives, INSERM U410, Faculté de Médecine X. Bichat, 16 rue Henri Huchard, 75018 Paris, France Received 3 July 2001; accepted 19 September 2001
Abstract In this work, we showed that human colon cancer cell lines produce trypsin which can activate a receptor for trypsin, the protease-activated receptor-2 (PAR-2), in these cells. RT-PCR experiments showed that trypsinogen transcripts were present in four colon cancer cell lines : T84, Caco-2, HT-29 and Cl.19A. By Western blot analysis we found a 25 kDa immunoreactive band identified as trypsinogen I in cell lysates and in the corresponding culture media. Concentrations of trypsin in cell media were found in nanomolar range, thus compatible with activation of protease-activated receptor 2 (PAR-2). This was further demonstrated in a colon cancer cell line (H-29) Ca21i assay since increases in Ca21i were observed in response to media from T84, Caco-2 or Cl.19A cells that were similar to that observed with 2–5 nM trypsin and were abolished by trypsin inhibitor. Altogether, these data show that colon cancer cell lines produce and secrete trypsin at concentrations compatible with activation of PAR-2. They support possible autocrine/paracrine regulation of PAR-2 activity by trypsin in colon cancer cells. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Proteinase; Colon; Cancer
Introduction Protease-activated receptor-2 [PAR2) is a member of the new subfamily of proteolytically activated receptors that belong to the superfamily of seven transmembrane-spanning G-protein coupled receptors [1,2]. Like other PARs, PAR-2 is activated through proteolytic cleavage of extracellular N-terminal domain that reveals a new amino-terminus which acts as a ‘thetered * Corresponding author. INSERM Unité 410, Faculté de Médecine Xavier Bichat, 16 rue Henri Huchard, BP 416, 75870 Paris Cedex 18, France. Tel.: 33-1-44-85-61-33; fax: 33-1-42-28-87-65. E-mail address: [email protected] (R. Ducroc) 0024-3205/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 5 1 9 -3
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ligand’. PAR-2 is expressed in different tissues, including the gastrointestinal and respiratory tracts, pancreas, liver, kidney, ovary, skin and bone, and also in epithelial and endothelial cell lines, in muscle cells and neurons, neutrophils, and certain tumour cell lines [3]. In the gastrointestinal tract, PAR-2 is highly expressed at membrane of epithelial cells, smooth muscle cells and neurons [4]. Although the physiological role for PAR-2 in gastrointestinal tract is not completely understood, it has been shown that PAR-2 stimulate mucus secretion in sublingual glands [5], control gastrointestinal motility [6] and modulate ion transport in small intestine and colon [7,8]. PAR-2 is activated by trypsin or tryptase [1, 2]. Pancreatic trypsin is the most powerfull known activator of PAR-2 [1]. There is however a discrepancy between the wide tissular distribution of PAR-2 as in epithelia of lung or skin, endothelial and smooth muscle cells of the blood vessel wall on the one hand and the availability of pancreatic trypsin that is restricted to bile duct and small intestine lumen, on the other. Moreover, the concentrations of pancreatic trypsin liberated into intestine lumen are in micromolar range, in far excess to the concentration established to activate PAR-2 receptor in small intestinal cells. These contradictions stimulated recent search for biologically-relevant proteases that can activate PAR-2 in non-pancreatic tissues that express the receptor. It was indeed found that PAR-2activating trypsin-like proteases are expressed in cells or tissues such as lung epithelium [9], skin [10] or spermatozoa [11]. It was also discovered that trypsin is expressed at low level in various non-pancreatic tissues [10] and in some tumours [12–14]. Interestingly, we recently demonstrated that human colon cancer cells express PAR-2 and that subnanomolar concentrations of trypsin acting at PAR-2 promote dramatic increase of colon cancer cell proliferation [15]. It was thus tempting to speculate that trypsin can be produced by colon cancer cells and can activate PAR-2 at membrane of same or adjacent cells in an autocrine or paracrine regulation. Here we demonstrated that trypsin is indeed synthetized by and released from human colon cancer cells at concentrations that are sufficient to activate PAR-2 on colon cancer cells. Methods Materials The PAR-2 activating peptide AP2 (SLIGKV) and its reverse sequence RP (VKGILS) were obtained from Neosystem (Strasbourg, France). Dulbecco’s modified eagle’s medium (DMEM), Ham F-12 medium and fetal calf serum (FCS) were purchased from Gibco-BRL (Cergy Pontoise, France). Steptavidin peroxidase was purchased from Amersham (Amersham Pharmacia Biotech, Saclay, France). Trypsin (pancreatic porcine trypsin, 12,800 U/mg), type III-O trypsin inhibitor and all other reagents were from Sigma. (Sigma, St Louis Mo). Cells and culture conditions The human colon cancer lines T84, Caco-2 and HT29 were obtained from the ATCC (Rockville, MD). HT29 clone 19 A cell line referred to as Cl.19A [16] was a gift from Dr. C. Laboisse (Nantes, France). T84, Caco-2 and Cl.19A are fully differenciated cells wheras HT29 cells are undifferentiated cells [17]. HT29 (passage 164–175) and Cl.19A cells (passage
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28–36) were routinely cultured in 25 cm2 plastic flasks (Costar, Cambridge, MA) as recommanded by ATCC. They were maintained at 378C in humidified atmosphere of 5% CO2/air in DMEM containing 4.5 g/L glucose, supplemented with 10% fetal calf serum (FCS). Caco-2 cells (passage 19–28) were cultured in same medium supplemented with 20% FCS and 1% nonessential amino acids. T84 cells (passage 62–80) were cultured in Ham F-12 supplemented with 10% FCS and 1% nonessential amino acids. All cell lines were analysed after confluence was reached. RT-PCR experiments Cells were rinced twice in PBS and total RNA was extracted using a commercial kit (RNAxel, Eurobio, Les Ulis, France). Total RNA was precipitated and resuspended in sterile H2O to produce working stock solutions with a final concentration of 1.0 mg/ml as verified by spectrophotometry (A260/ A280 ratio . 1.8). Two-microgram aliquots of each RNA stock were reverse-transcribed by the random hexanucleotide method using Moloney murine leukemia virus reverse transcriptase (Promega, France). Five microliters of the cDNA product were used in amplification reactions with oligonucleotide primers specific for human trypsinogen gene message. Oligonucleotide primers from separate exons were designed from published sequence of trypsinogen I [18] and purchased from Gibco BRL (Life Technologies, UK). The sequence of trypsinogen upstream primer and downstream primers are 59-CTCATCAGCGAACAGTGG-39 and 59-TTGGTGTAGACTCCAGGC -39, respectively. Nucleotide sequences for the two primers were identical in trypsinogen I and trypsinogen II [18]. Reaction was run in 10 mM tris-HCl, pH 8.3, containing 2.5 mM MgCl2, 50 mM KCl, 250 mM dNTP, and 0.4 units of Taq DNA polymerase (Promega, France) in the presence of 50 mM of each sequence-specific primers. Each of the 30 cycles of amplification was performed as follow : 94 8C for 40 seconds, 56 8 C for 45 seconds, 72 8C for 40 seconds. Control PCRs were performed by substituting water for cDNA and omitting RT during the DNA synthesis. Preparation of cell homogenate and cell culture media Except for determination of trypsin activity, the cell culture media were harvested with 1 mM phenylmethylsulfonyl fluoride (PMSF) and stored at 2808C. After the cells were washed twice in PBS, cell homogenate was obtained by freezing the cells at 2808C for 20 min and then adding 2 ml of a solution composed of 10 mM Tris, pH 7.5, 1 mM EDTA, pH 7, 100 mM NaCl and 1 mM PMSF at 378C. After scraping, the cells were sonicated and stored at 2808C. Cellular extract and media for determination of trypsin activity were collected without PMSF. Preparation of protein sample from serum-free conditioned media Cultured cells were washed twice with Ca21, Mg21-free Hank’s balanced salt solution and then medium was replaced with fresh serum-free DMEM medium (or serum-free Ham F-12 for T84 cells) for a further 2-day incubation. The resultant serum-free media were clarified by two-step centrifugation at 200g for 10 min and 15,000g for 10 min according to Hirahara et al. [19]. Clarified media were added with ammonium sulfate to a final concentration of 80% saturation and allowed to stand at 48C for 4 hr. The obtained protein precipitates were
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collected by centrifugation at 18,000g for 30 min, dissolved in and dialysed at 48C overnight against 20 mM Tris-HCl buffer (pH 7.5) using a dialyse membrane with a cut-off of 3,500 (Spectra/Por, Spectrum Laboratories Inc. USA). The dialysates were used as protein samples (100-fold concentrated conditioned media). Determination of trypsin-like activity Cellular extracts and media were collected and treated for trypsinogen activation as described [20] and trypsin-like activity was then determined using a commercial kit based on the liberation of a highly fluorescent BODIPY dye upon proteolysis of a quenched dyelabeled casein substrate (Molecular probes Inc., Eugene, OR). This method has been shown to be simple and reliable and to have a great sensitivity [21]. Assay was proceeded at 378C with BODIPY TR-X dye-labeled caseins (10 mg/ml) in PBS according to the recommendation of the manufacturer. Aliquots were then assayed in duplicate and trypsin-like concentration calculated from standard curves of trypsin (pancreatic porcine trypsin, E.C. 3.4.21.4, 12,800 U/mg). Results were expressed in mg or mg per mg of proteins. Western blotting Electrophoresis was performed through a 12% SDS-polyacrylamide slab gel according to Laemmli [22]. Gels were electroblotted to nitrocellulose sheets with a semi-dry blotting apparatus (Dual Caster gel, Hofster, Pharmacia Biotech, France). Detection of trypsinogen was done with a rabbit polyclonal antibody raised against human trypsinogen generously provided by Dr C. Figarella (Marseille, France). Horseradish peroxidase-labeled antirabbit antibody (Amersham Pharmacia Biotech, Saclay, France) was used as the second antibody. Non-immune serum activity was controlled in parallel. Positive control was human pancreatic duct lavage kindly given by Dr C. Figarella (Marseille, France). Molecular weight (Mr) markers (Rainbow molecular weight marker, Amersham Pharmacia Biotech, Saclay, France) used are recombinant proteins of Mr 15,000 to Mr 50,000. Blots were developed with chemiluminescence detection system (ECL, Western blotting, Amersham). Protein were assayed in duplicate by a microassay procedure (Bio-rad Laboratories, Münschen, Germany) with bovine serum albumin as standard. Measurement of Ca21i increase in HT29 cells Intracellular calcium concentrations of monolayers of HT29 cells were measured using Fura-2/AM as described [15]. The cells (1 3 103/well) were seeded onto glass coverslips and cultured for 3 days. The cells attached to coverslips were then loaded with 5 mM Fura-2/AM in Hepes-buffered saline (135 mM NaCl, 4.6 mM KCl, 1.2 mM MgCl2, 11 mM Hepes, 11 mM glucose, and 1.5 mM CaCl2 at pH 7.4) containing 0.01% pleuronic acid in the dark for 60 min at 378C. Cells were rinsed three times with the same buffer and coverslips were placed in a disposable cuvette in a volume of 1.5 ml buffer that was gently agitated and maintained at 378C. Cell preparations were excited at 340 and 380 nm alternately (every 2 seconds) and the fluorescence intensity emitted at 510 nm was measured using a photoscan micro-fluorimeter (Photon Technology International, Kontron, France). The ratio of fluorescence that was measured at the two wavelengths is proportional to [Ca21]i.
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Results In a first set of experiments, we investigated the expression of trypsinogen in several colon cancer cell lines using RT-PCR. The primers used were designed to amplify for two forms of trypsinogen, including trypsinogen I and trypsinogen II mRNA. As shown in Figure 1, amplicon of the expected size (538 bp) was observed with RNA extracted from all cell lines tested including T84, Caco-2, HT29 and Cl.19A. PCR products were sequenced (not shown) showing complete identity with human trypsinogen I. We further analysed the different cell lines for trypsinogen production by Western blot using a human trypsinogen antibody that recognizes trypsinogen I and II. Non-activated human pancreatic juice samples which express both trypsinogen I and II [23] were run in parallel as positive controls. A 25-KDa immunoreactive band was found in cellular extracts from all cancer cell lines studied. This band was observed at the same size as the pancreatic trypsinogen I in control lane (Fig 2). Since, trypsinogen is a secreted protein, we examined if the immunoreactive band found in the different cell extracts was also present in the culture medium of these cells. The different cell lines were grown without serum for 48 h and media were harvested and concentrated. Fig 2 shows that immunoreactive bands were found in all the media samples at the same size as the trypsinogen I—like band found in the corresponding cells. These data indicate that trypsinogen I is not only synthetized but is also secreted in significant amount by T84, Caco-2, HT29 and Cl.19 A colon cancer cell lines. The trypsin activity was measured in the samples using a sensitive fluorimetric-based assay [21] and compared to standard curves established with porcine pancreatic trypsin. Trypsin activity could be measured in cell homogenates and cell culture media from all cell lines tested (Table 1). Assuming a molecular weight of 23,000 Da for active trypsin [23], the trypsin-like activity measured in the medium can be calculated as ranging from 3.1 to 5.0 nM (Table 1). To examine if the trypsin secreted by colon cancer cells was able to activate PAR-2 receptor in colon cancer cells, we tested the ability of culture media to promote an increase in [Ca21]i transient following the stimulation of PAR-2 receptor that is expressed in HT-29 cells [15]. HT29 cells exhibited significant (Ca21)i increase in response to 2 or 10 nM trypsin
Fig. 1. PCR-based detection of trypsinogen mRNAs in four different human colon cancer cell lines. Total RNA extracted from the colon cancer cells was reverse transcribed and PCR amplified with trypsinogen primers. Single amplicons of expected size were visualised in a 1.6% agarose gel with a 100-bp DNA ladder (M).
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Fig. 2. Western blot of trypsinogen I in different human colon cancer cell lines. 50 mg of cellular homogenate of T84, Caco-2, HT29 or Cl.19A cells and media from the same cell lines were run on 12% SDS-PAGE and challenged with human antitrypsin antibody. This antibody recognizes trypsinogen I (25 kDa) in human pancreatic duct lavage (Control).
(Fig 3A), or to the PAR-2 activating peptide SLIGKV (AP2, 100 mM) but not the reverse peptide (RP, 100 mM) (Fig 3B). Response to 2 or 10 nM trypsin was totally blocked by trypsin inhibitor (80mg/ml). Addition of 15 ml of T84 medium on HT29 cells resulted in a significant increase in intracellular calcium transient (Fig. 3C). This increase in (Ca21)i was prevented by incubation of the medium with trypsin inhibitor (80 mg/ml, Fig. 3C). As shown in Figure 3D and 3E, similar increases in (Ca21)i were observed in response to Caco-2 or Cl.19A media that were consistent in intensity with the response observed with 2–10 nM trypsin. Finally, the lack of effect of the media when first incubated with trypsin inhibitor was consistent with an activation of PAR-2 by trypsin present and active in the conditioned medium. Discussion Altogether, these results showed that human colon cancer cell lines secrete a trypsinogen/ trypsin that can activate PAR-2 at membrane of colon cancer cells. Efforts have been made to Table 1 Concentration of trypsin in cell homogenates and culture media from four different human colon cancer cell lines Cell homogenates Cell lines T84 Caco-2 HT29 HT29 clone 19A
Culture media
mg/mg protein
m/ml
nM
5.2 6 0.8 8.5 6 2.0 8.3 6 1.1 8.3 6 1.5
0.08 6 0.03 0.12 6 0.04 0.10 6 0.01 0.09 6 0.01
3.1 6 1.0 5.0 6 1.5 4.2 6 0.4 3.5 6 0.3
Trypsin was measured in cellular homogenates and in FCS-free conditioned media using a commercial kit based on liberation of fluorescent dye upon proteolysis of quenched–dye-labeled casein substrat (see Material & Method section). Standard curves were obtained with porcine type IX pancreatic trypsin (12,800 u/mg). Expression of trypsin activity in media in nM was calculated assuming a molecular mass of 23,000 for trypsin.
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Fig. 3. Effect of different conditioned media on calcium signalling in HT-29 cells. HT-29 cells cultivated on glass slides were loaded for 60 min at 378C using Fura-2/AM. A: effect of 2 nM trypsin or 10 nM trypsin. B: The addition of (100 mM) reverse peptide (RP, 100 mM) was followed by PAR-2 activating peptide SLIGKV (AP2, 100 mM). C. Effect of T84 conditioned medium. The effect was abolished by incubation with trypsin inhibitor D. Similar experiment with medium from Caco-2 cells. E. Similar experiment with medium from Cl.19A. These figures are representative of 3 experiments.
identify biologically-relevant proteases that can possibly activate PAR-2 in gastrointestinal tract as recently reviewed [2]. Pancreatic trypsin is know to activate PAR-2 with high potency. It is unlikely that trypsin activity originating from exocrine pancreas remain in the lumen of colon with efficient activity [24], but the importance of locally secreted trypsin at the vicinity of colon tumours was recently emphasized : i) normal epithelial cells surrounding colon tumour cells are likely a source of active trypsin [10]; ii) blood vessels surrounding
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tumours also express trypsin [25]; iii) some human colon cancer cell lines have been shown to produce and secrete trypsinogens [12–14]. Indeed COLO 205, a colon adenocarcinoma cell line express tumour-associated trypsinogen-2 (TAT-2), a serine-protease related to trypsinogen II [13], and HT29 and Caco-2 cells produce trypsinogen I [12]. Here we extend these observations to other cancer cell lines and we demonstrate that trypsin from colon cancer cell lines can also activate PAR-2 in colon cancer cells with a potency similar to that of pancreatic trypsin. This is in line with recent report indicating that trypsin-2, a trypsin-like protease expressed by COLO-205 cell line is equally effective to pancreatic trypsin to cleave PAR-2 [26]. It has been suggested from studies in pancreas that trypsin-like enzymes secreted by tumour cells could directly regulate growth of pancreatic cells in an autocrine manner by interacting with PAR-2 [2]. In gastric carcinoma cells, it was recently showed that trypsinogen secreted by tumour cells, when activated to trypsin, can stimulate the growth and adhesiveness of the producer cells in an autocrine manner [27]. Although there is increasing evidences for different roles of PAR-2 in normal intestine, the consequence of activation of PAR-2 in colon cancers had not been studied until recently. Studies from our laboratory indicate that functional PAR-2 is expressed in several colon cancer cell lines [15]. Moreover, activation of PAR-2 by trypsin in these colon cancer cell lines is associated with dramatic increase in proliferation. It is therefore likely that serine-proteases like trypsin-1 secreted by colonic tumours could control their growth through interaction with PAR-2. In this context, our results suggest an autocrine/ paracrine regulation of human colonic tumours by trypsin. Acknowledgments Dr. C. Figarella (GRGE Marseille, France) for generous gift of antitrypsin antibodies and human pancreatic juice. Dr. K. Marazova was supported by a fellowship from INSERM (Poste Jaune). Dr. K. Marazova present address is : Department of Pharmacology and Toxicology, Medical University of Sofia, 2 Zdrave st., 1431 Sofia, Bulgaria References 1. Déry O, Corvera CU, Steinhoff M, Bunnett NW. Proteinase-activated receptors: novel mechanisms of signaling by serine proteases. American Journal of Physiology 1998; 74 (6):C1429–52. 2. O’Brien PJ, Molino M, Kahn M, Brass LF. Protease activated receptors: theme and variations. Oncogene 2001 (13); 20:1570–81. 3. Bohm SK, Kong W, Bromme D, Smeekens SP, Anderson DC, Connolly A, Kahn M, Nelken NA, Coughlin SR, Payan DG, Bunnett NW. Molecular cloning, expression and potential functions of the human proteinaseactivated receptor-2. Biochemical Journal 1996; 314 (3):1009–16. 4. Vergnolle N, Wallace JL, Bunnet NW, Hollenberg MD. Protease-activated receptors in inflammation, neuronal signaling and pain. Trends in Pharmacological Sciences 2001; 22 (3):146–52. 5. Kawabata A, Morimoto N, Nishikawa H, Kuroda R, Oda Y, Kakehi KA. Activation of protease-activated receptor-2 (PAR-2) triggers mucin secretion in the rat sublingual gland. Biochemical Biophysical Research Communications 2000; 270 (1):298–302. 6. Corvera CU, Déry O, McConalogue K, Bohm SK, Khitin LM, Caughey GH, Payan DG, Bunnett NW. Mast cell tryptase regulates rat colonic myocytes through proteinase-activated receptor 2. Journal of Clinical Investigations 1997; 100 (6): 1383–93. 7. Kong W, McConalogue K, Khitin LM, Hollenberg MD, Payan DG, Bohm SK, Bunnett NW. Luminal trypsin
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Trypsin is produced by and activates protease-activated receptor-2 in human cancer colon cells Evidence for new autocrine loop Robert Ducroc*, Claire Bontemps, Katia Marazova, Hélène Devaud, Dalila Darmoul, Marc Laburthe Neuroendocrinologie et Biologie Cellulaire Digestives, INSERM U410, Faculté de Médecine X. Bichat, 16 rue Henri Huchard, 75018 Paris, France Received 3 July 2001; accepted 19 September 2001
Abstract In this work, we showed that human colon cancer cell lines produce trypsin which can activate a receptor for trypsin, the protease-activated receptor-2 (PAR-2), in these cells. RT-PCR experiments showed that trypsinogen transcripts were present in four colon cancer cell lines : T84, Caco-2, HT-29 and Cl.19A. By Western blot analysis we found a 25 kDa immunoreactive band identified as trypsinogen I in cell lysates and in the corresponding culture media. Concentrations of trypsin in cell media were found in nanomolar range, thus compatible with activation of protease-activated receptor 2 (PAR-2). This was further demonstrated in a colon cancer cell line (H-29) Ca21i assay since increases in Ca21i were observed in response to media from T84, Caco-2 or Cl.19A cells that were similar to that observed with 2–5 nM trypsin and were abolished by trypsin inhibitor. Altogether, these data show that colon cancer cell lines produce and secrete trypsin at concentrations compatible with activation of PAR-2. They support possible autocrine/paracrine regulation of PAR-2 activity by trypsin in colon cancer cells. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Proteinase; Colon; Cancer
Introduction Protease-activated receptor-2 [PAR2) is a member of the new subfamily of proteolytically activated receptors that belong to the superfamily of seven transmembrane-spanning G-protein coupled receptors [1,2]. Like other PARs, PAR-2 is activated through proteolytic cleavage of extracellular N-terminal domain that reveals a new amino-terminus which acts as a ‘thetered * Corresponding author. INSERM Unité 410, Faculté de Médecine Xavier Bichat, 16 rue Henri Huchard, BP 416, 75870 Paris Cedex 18, France. Tel.: 33-1-44-85-61-33; fax: 33-1-42-28-87-65. E-mail address: [email protected] (R. Ducroc) 0024-3205/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 5 1 9 -3
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ligand’. PAR-2 is expressed in different tissues, including the gastrointestinal and respiratory tracts, pancreas, liver, kidney, ovary, skin and bone, and also in epithelial and endothelial cell lines, in muscle cells and neurons, neutrophils, and certain tumour cell lines [3]. In the gastrointestinal tract, PAR-2 is highly expressed at membrane of epithelial cells, smooth muscle cells and neurons [4]. Although the physiological role for PAR-2 in gastrointestinal tract is not completely understood, it has been shown that PAR-2 stimulate mucus secretion in sublingual glands [5], control gastrointestinal motility [6] and modulate ion transport in small intestine and colon [7,8]. PAR-2 is activated by trypsin or tryptase [1, 2]. Pancreatic trypsin is the most powerfull known activator of PAR-2 [1]. There is however a discrepancy between the wide tissular distribution of PAR-2 as in epithelia of lung or skin, endothelial and smooth muscle cells of the blood vessel wall on the one hand and the availability of pancreatic trypsin that is restricted to bile duct and small intestine lumen, on the other. Moreover, the concentrations of pancreatic trypsin liberated into intestine lumen are in micromolar range, in far excess to the concentration established to activate PAR-2 receptor in small intestinal cells. These contradictions stimulated recent search for biologically-relevant proteases that can activate PAR-2 in non-pancreatic tissues that express the receptor. It was indeed found that PAR-2activating trypsin-like proteases are expressed in cells or tissues such as lung epithelium [9], skin [10] or spermatozoa [11]. It was also discovered that trypsin is expressed at low level in various non-pancreatic tissues [10] and in some tumours [12–14]. Interestingly, we recently demonstrated that human colon cancer cells express PAR-2 and that subnanomolar concentrations of trypsin acting at PAR-2 promote dramatic increase of colon cancer cell proliferation [15]. It was thus tempting to speculate that trypsin can be produced by colon cancer cells and can activate PAR-2 at membrane of same or adjacent cells in an autocrine or paracrine regulation. Here we demonstrated that trypsin is indeed synthetized by and released from human colon cancer cells at concentrations that are sufficient to activate PAR-2 on colon cancer cells. Methods Materials The PAR-2 activating peptide AP2 (SLIGKV) and its reverse sequence RP (VKGILS) were obtained from Neosystem (Strasbourg, France). Dulbecco’s modified eagle’s medium (DMEM), Ham F-12 medium and fetal calf serum (FCS) were purchased from Gibco-BRL (Cergy Pontoise, France). Steptavidin peroxidase was purchased from Amersham (Amersham Pharmacia Biotech, Saclay, France). Trypsin (pancreatic porcine trypsin, 12,800 U/mg), type III-O trypsin inhibitor and all other reagents were from Sigma. (Sigma, St Louis Mo). Cells and culture conditions The human colon cancer lines T84, Caco-2 and HT29 were obtained from the ATCC (Rockville, MD). HT29 clone 19 A cell line referred to as Cl.19A [16] was a gift from Dr. C. Laboisse (Nantes, France). T84, Caco-2 and Cl.19A are fully differenciated cells wheras HT29 cells are undifferentiated cells [17]. HT29 (passage 164–175) and Cl.19A cells (passage
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28–36) were routinely cultured in 25 cm2 plastic flasks (Costar, Cambridge, MA) as recommanded by ATCC. They were maintained at 378C in humidified atmosphere of 5% CO2/air in DMEM containing 4.5 g/L glucose, supplemented with 10% fetal calf serum (FCS). Caco-2 cells (passage 19–28) were cultured in same medium supplemented with 20% FCS and 1% nonessential amino acids. T84 cells (passage 62–80) were cultured in Ham F-12 supplemented with 10% FCS and 1% nonessential amino acids. All cell lines were analysed after confluence was reached. RT-PCR experiments Cells were rinced twice in PBS and total RNA was extracted using a commercial kit (RNAxel, Eurobio, Les Ulis, France). Total RNA was precipitated and resuspended in sterile H2O to produce working stock solutions with a final concentration of 1.0 mg/ml as verified by spectrophotometry (A260/ A280 ratio . 1.8). Two-microgram aliquots of each RNA stock were reverse-transcribed by the random hexanucleotide method using Moloney murine leukemia virus reverse transcriptase (Promega, France). Five microliters of the cDNA product were used in amplification reactions with oligonucleotide primers specific for human trypsinogen gene message. Oligonucleotide primers from separate exons were designed from published sequence of trypsinogen I [18] and purchased from Gibco BRL (Life Technologies, UK). The sequence of trypsinogen upstream primer and downstream primers are 59-CTCATCAGCGAACAGTGG-39 and 59-TTGGTGTAGACTCCAGGC -39, respectively. Nucleotide sequences for the two primers were identical in trypsinogen I and trypsinogen II [18]. Reaction was run in 10 mM tris-HCl, pH 8.3, containing 2.5 mM MgCl2, 50 mM KCl, 250 mM dNTP, and 0.4 units of Taq DNA polymerase (Promega, France) in the presence of 50 mM of each sequence-specific primers. Each of the 30 cycles of amplification was performed as follow : 94 8C for 40 seconds, 56 8 C for 45 seconds, 72 8C for 40 seconds. Control PCRs were performed by substituting water for cDNA and omitting RT during the DNA synthesis. Preparation of cell homogenate and cell culture media Except for determination of trypsin activity, the cell culture media were harvested with 1 mM phenylmethylsulfonyl fluoride (PMSF) and stored at 2808C. After the cells were washed twice in PBS, cell homogenate was obtained by freezing the cells at 2808C for 20 min and then adding 2 ml of a solution composed of 10 mM Tris, pH 7.5, 1 mM EDTA, pH 7, 100 mM NaCl and 1 mM PMSF at 378C. After scraping, the cells were sonicated and stored at 2808C. Cellular extract and media for determination of trypsin activity were collected without PMSF. Preparation of protein sample from serum-free conditioned media Cultured cells were washed twice with Ca21, Mg21-free Hank’s balanced salt solution and then medium was replaced with fresh serum-free DMEM medium (or serum-free Ham F-12 for T84 cells) for a further 2-day incubation. The resultant serum-free media were clarified by two-step centrifugation at 200g for 10 min and 15,000g for 10 min according to Hirahara et al. [19]. Clarified media were added with ammonium sulfate to a final concentration of 80% saturation and allowed to stand at 48C for 4 hr. The obtained protein precipitates were
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collected by centrifugation at 18,000g for 30 min, dissolved in and dialysed at 48C overnight against 20 mM Tris-HCl buffer (pH 7.5) using a dialyse membrane with a cut-off of 3,500 (Spectra/Por, Spectrum Laboratories Inc. USA). The dialysates were used as protein samples (100-fold concentrated conditioned media). Determination of trypsin-like activity Cellular extracts and media were collected and treated for trypsinogen activation as described [20] and trypsin-like activity was then determined using a commercial kit based on the liberation of a highly fluorescent BODIPY dye upon proteolysis of a quenched dyelabeled casein substrate (Molecular probes Inc., Eugene, OR). This method has been shown to be simple and reliable and to have a great sensitivity [21]. Assay was proceeded at 378C with BODIPY TR-X dye-labeled caseins (10 mg/ml) in PBS according to the recommendation of the manufacturer. Aliquots were then assayed in duplicate and trypsin-like concentration calculated from standard curves of trypsin (pancreatic porcine trypsin, E.C. 3.4.21.4, 12,800 U/mg). Results were expressed in mg or mg per mg of proteins. Western blotting Electrophoresis was performed through a 12% SDS-polyacrylamide slab gel according to Laemmli [22]. Gels were electroblotted to nitrocellulose sheets with a semi-dry blotting apparatus (Dual Caster gel, Hofster, Pharmacia Biotech, France). Detection of trypsinogen was done with a rabbit polyclonal antibody raised against human trypsinogen generously provided by Dr C. Figarella (Marseille, France). Horseradish peroxidase-labeled antirabbit antibody (Amersham Pharmacia Biotech, Saclay, France) was used as the second antibody. Non-immune serum activity was controlled in parallel. Positive control was human pancreatic duct lavage kindly given by Dr C. Figarella (Marseille, France). Molecular weight (Mr) markers (Rainbow molecular weight marker, Amersham Pharmacia Biotech, Saclay, France) used are recombinant proteins of Mr 15,000 to Mr 50,000. Blots were developed with chemiluminescence detection system (ECL, Western blotting, Amersham). Protein were assayed in duplicate by a microassay procedure (Bio-rad Laboratories, Münschen, Germany) with bovine serum albumin as standard. Measurement of Ca21i increase in HT29 cells Intracellular calcium concentrations of monolayers of HT29 cells were measured using Fura-2/AM as described [15]. The cells (1 3 103/well) were seeded onto glass coverslips and cultured for 3 days. The cells attached to coverslips were then loaded with 5 mM Fura-2/AM in Hepes-buffered saline (135 mM NaCl, 4.6 mM KCl, 1.2 mM MgCl2, 11 mM Hepes, 11 mM glucose, and 1.5 mM CaCl2 at pH 7.4) containing 0.01% pleuronic acid in the dark for 60 min at 378C. Cells were rinsed three times with the same buffer and coverslips were placed in a disposable cuvette in a volume of 1.5 ml buffer that was gently agitated and maintained at 378C. Cell preparations were excited at 340 and 380 nm alternately (every 2 seconds) and the fluorescence intensity emitted at 510 nm was measured using a photoscan micro-fluorimeter (Photon Technology International, Kontron, France). The ratio of fluorescence that was measured at the two wavelengths is proportional to [Ca21]i.
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Results In a first set of experiments, we investigated the expression of trypsinogen in several colon cancer cell lines using RT-PCR. The primers used were designed to amplify for two forms of trypsinogen, including trypsinogen I and trypsinogen II mRNA. As shown in Figure 1, amplicon of the expected size (538 bp) was observed with RNA extracted from all cell lines tested including T84, Caco-2, HT29 and Cl.19A. PCR products were sequenced (not shown) showing complete identity with human trypsinogen I. We further analysed the different cell lines for trypsinogen production by Western blot using a human trypsinogen antibody that recognizes trypsinogen I and II. Non-activated human pancreatic juice samples which express both trypsinogen I and II [23] were run in parallel as positive controls. A 25-KDa immunoreactive band was found in cellular extracts from all cancer cell lines studied. This band was observed at the same size as the pancreatic trypsinogen I in control lane (Fig 2). Since, trypsinogen is a secreted protein, we examined if the immunoreactive band found in the different cell extracts was also present in the culture medium of these cells. The different cell lines were grown without serum for 48 h and media were harvested and concentrated. Fig 2 shows that immunoreactive bands were found in all the media samples at the same size as the trypsinogen I—like band found in the corresponding cells. These data indicate that trypsinogen I is not only synthetized but is also secreted in significant amount by T84, Caco-2, HT29 and Cl.19 A colon cancer cell lines. The trypsin activity was measured in the samples using a sensitive fluorimetric-based assay [21] and compared to standard curves established with porcine pancreatic trypsin. Trypsin activity could be measured in cell homogenates and cell culture media from all cell lines tested (Table 1). Assuming a molecular weight of 23,000 Da for active trypsin [23], the trypsin-like activity measured in the medium can be calculated as ranging from 3.1 to 5.0 nM (Table 1). To examine if the trypsin secreted by colon cancer cells was able to activate PAR-2 receptor in colon cancer cells, we tested the ability of culture media to promote an increase in [Ca21]i transient following the stimulation of PAR-2 receptor that is expressed in HT-29 cells [15]. HT29 cells exhibited significant (Ca21)i increase in response to 2 or 10 nM trypsin
Fig. 1. PCR-based detection of trypsinogen mRNAs in four different human colon cancer cell lines. Total RNA extracted from the colon cancer cells was reverse transcribed and PCR amplified with trypsinogen primers. Single amplicons of expected size were visualised in a 1.6% agarose gel with a 100-bp DNA ladder (M).
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Fig. 2. Western blot of trypsinogen I in different human colon cancer cell lines. 50 mg of cellular homogenate of T84, Caco-2, HT29 or Cl.19A cells and media from the same cell lines were run on 12% SDS-PAGE and challenged with human antitrypsin antibody. This antibody recognizes trypsinogen I (25 kDa) in human pancreatic duct lavage (Control).
(Fig 3A), or to the PAR-2 activating peptide SLIGKV (AP2, 100 mM) but not the reverse peptide (RP, 100 mM) (Fig 3B). Response to 2 or 10 nM trypsin was totally blocked by trypsin inhibitor (80mg/ml). Addition of 15 ml of T84 medium on HT29 cells resulted in a significant increase in intracellular calcium transient (Fig. 3C). This increase in (Ca21)i was prevented by incubation of the medium with trypsin inhibitor (80 mg/ml, Fig. 3C). As shown in Figure 3D and 3E, similar increases in (Ca21)i were observed in response to Caco-2 or Cl.19A media that were consistent in intensity with the response observed with 2–10 nM trypsin. Finally, the lack of effect of the media when first incubated with trypsin inhibitor was consistent with an activation of PAR-2 by trypsin present and active in the conditioned medium. Discussion Altogether, these results showed that human colon cancer cell lines secrete a trypsinogen/ trypsin that can activate PAR-2 at membrane of colon cancer cells. Efforts have been made to Table 1 Concentration of trypsin in cell homogenates and culture media from four different human colon cancer cell lines Cell homogenates Cell lines T84 Caco-2 HT29 HT29 clone 19A
Culture media
mg/mg protein
m/ml
nM
5.2 6 0.8 8.5 6 2.0 8.3 6 1.1 8.3 6 1.5
0.08 6 0.03 0.12 6 0.04 0.10 6 0.01 0.09 6 0.01
3.1 6 1.0 5.0 6 1.5 4.2 6 0.4 3.5 6 0.3
Trypsin was measured in cellular homogenates and in FCS-free conditioned media using a commercial kit based on liberation of fluorescent dye upon proteolysis of quenched–dye-labeled casein substrat (see Material & Method section). Standard curves were obtained with porcine type IX pancreatic trypsin (12,800 u/mg). Expression of trypsin activity in media in nM was calculated assuming a molecular mass of 23,000 for trypsin.
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Fig. 3. Effect of different conditioned media on calcium signalling in HT-29 cells. HT-29 cells cultivated on glass slides were loaded for 60 min at 378C using Fura-2/AM. A: effect of 2 nM trypsin or 10 nM trypsin. B: The addition of (100 mM) reverse peptide (RP, 100 mM) was followed by PAR-2 activating peptide SLIGKV (AP2, 100 mM). C. Effect of T84 conditioned medium. The effect was abolished by incubation with trypsin inhibitor D. Similar experiment with medium from Caco-2 cells. E. Similar experiment with medium from Cl.19A. These figures are representative of 3 experiments.
identify biologically-relevant proteases that can possibly activate PAR-2 in gastrointestinal tract as recently reviewed [2]. Pancreatic trypsin is know to activate PAR-2 with high potency. It is unlikely that trypsin activity originating from exocrine pancreas remain in the lumen of colon with efficient activity [24], but the importance of locally secreted trypsin at the vicinity of colon tumours was recently emphasized : i) normal epithelial cells surrounding colon tumour cells are likely a source of active trypsin [10]; ii) blood vessels surrounding
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tumours also express trypsin [25]; iii) some human colon cancer cell lines have been shown to produce and secrete trypsinogens [12–14]. Indeed COLO 205, a colon adenocarcinoma cell line express tumour-associated trypsinogen-2 (TAT-2), a serine-protease related to trypsinogen II [13], and HT29 and Caco-2 cells produce trypsinogen I [12]. Here we extend these observations to other cancer cell lines and we demonstrate that trypsin from colon cancer cell lines can also activate PAR-2 in colon cancer cells with a potency similar to that of pancreatic trypsin. This is in line with recent report indicating that trypsin-2, a trypsin-like protease expressed by COLO-205 cell line is equally effective to pancreatic trypsin to cleave PAR-2 [26]. It has been suggested from studies in pancreas that trypsin-like enzymes secreted by tumour cells could directly regulate growth of pancreatic cells in an autocrine manner by interacting with PAR-2 [2]. In gastric carcinoma cells, it was recently showed that trypsinogen secreted by tumour cells, when activated to trypsin, can stimulate the growth and adhesiveness of the producer cells in an autocrine manner [27]. Although there is increasing evidences for different roles of PAR-2 in normal intestine, the consequence of activation of PAR-2 in colon cancers had not been studied until recently. Studies from our laboratory indicate that functional PAR-2 is expressed in several colon cancer cell lines [15]. Moreover, activation of PAR-2 by trypsin in these colon cancer cell lines is associated with dramatic increase in proliferation. It is therefore likely that serine-proteases like trypsin-1 secreted by colonic tumours could control their growth through interaction with PAR-2. In this context, our results suggest an autocrine/ paracrine regulation of human colonic tumours by trypsin. Acknowledgments Dr. C. Figarella (GRGE Marseille, France) for generous gift of antitrypsin antibodies and human pancreatic juice. Dr. K. Marazova was supported by a fellowship from INSERM (Poste Jaune). Dr. K. Marazova present address is : Department of Pharmacology and Toxicology, Medical University of Sofia, 2 Zdrave st., 1431 Sofia, Bulgaria References 1. Déry O, Corvera CU, Steinhoff M, Bunnett NW. Proteinase-activated receptors: novel mechanisms of signaling by serine proteases. American Journal of Physiology 1998; 74 (6):C1429–52. 2. O’Brien PJ, Molino M, Kahn M, Brass LF. Protease activated receptors: theme and variations. Oncogene 2001 (13); 20:1570–81. 3. Bohm SK, Kong W, Bromme D, Smeekens SP, Anderson DC, Connolly A, Kahn M, Nelken NA, Coughlin SR, Payan DG, Bunnett NW. Molecular cloning, expression and potential functions of the human proteinaseactivated receptor-2. Biochemical Journal 1996; 314 (3):1009–16. 4. Vergnolle N, Wallace JL, Bunnet NW, Hollenberg MD. Protease-activated receptors in inflammation, neuronal signaling and pain. Trends in Pharmacological Sciences 2001; 22 (3):146–52. 5. Kawabata A, Morimoto N, Nishikawa H, Kuroda R, Oda Y, Kakehi KA. Activation of protease-activated receptor-2 (PAR-2) triggers mucin secretion in the rat sublingual gland. Biochemical Biophysical Research Communications 2000; 270 (1):298–302. 6. Corvera CU, Déry O, McConalogue K, Bohm SK, Khitin LM, Caughey GH, Payan DG, Bunnett NW. Mast cell tryptase regulates rat colonic myocytes through proteinase-activated receptor 2. Journal of Clinical Investigations 1997; 100 (6): 1383–93. 7. Kong W, McConalogue K, Khitin LM, Hollenberg MD, Payan DG, Bohm SK, Bunnett NW. Luminal trypsin
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