Journal of Neuro-Oncology 42: 131–136, 1999. © 1999 Kluwer Academic Publishers. Printed in the Netherlands.
Laboratory Investigation
PAR 1-type thrombin receptors are involved in thrombin-induced calcium signaling in human meningioma cells Roland Kaufmann1 , Stephan Patt2 , Robert Kraft2 , Michael Zieger1 , Peter Henklein3 , Gunter Neupert2 and G¨otz Nowak1 1 Research Unit ‘Pharmacological Hemostaseology’, 2 Institute of Pathology (Neuropathology), Friedrich Schiller University Jena, Germany; 3 Institute of Biochemistry, Humboldt University, Berlin, Germany
Key words: proteinase-activated receptor 1, PAR 1, meningioma cells, thrombin, calcium signaling Summary Thrombin is known to play a role as regulator in tumor spreading and tumor growth. Proteinase-activated receptor 1 (PAR 1)-type thrombin receptors were identified in different cancer cells including human glioblastoma cells. Thus a function of PAR 1 in brain tumors may be suggested. In this study, the presence of PAR 1-type thrombin receptors was investigated in primary cell cultures established from operated human meningiomas from two 59- and 79-year-old women. Characterization of PAR 1 on binding level was performed using immunofluorescence studies with the monoclonal anti-PAR 1 antibody Mab 61-1 directed against a domain in the NH2 -terminus of PAR 1. These binding sites constitute functional thrombin receptors that are involved in thrombin-induced signaling in human meningioma cells as demonstrated by investigation of alpha-thrombin- and PAR 1-activating hexapeptide (TRAP-6)-induced [Ca2+ ]i mobilization. To our knowledge, this is the first report demonstrating thrombin-induced intracellular signaling in human meningioma cells mediated by the PAR 1-type thrombin receptor.
Introduction In recent years, a subfamily of G protein-coupled receptors that are capable to mediate intracellular signaling in response to cleavage by serine proteinases has been identified. There are 4 members of this receptor family designated proteinase-activated receptors (PARs): PAR 1 [1] and PAR 3 [2] which are cleaved and activated by thrombin, PAR 2, a receptor for trypsin-like enzymes [3], and PAR 4 recently cloned and characterized as a receptor for both thrombin and trypsin [4]. The best studied member of the proteinase-activated receptor family is the PAR 1-type thrombin receptor. Thrombin binds to this receptor and cleaves it in the NH2 terminal portion. The exposed new NH2 -terminus has been proposed to function as a ‘tethered peptide ligand’ binding to the thrombin receptor in specific regions to cause receptor activation [5]. Synthetic peptides of 5–14 amino acids (thrombin receptor activating peptides, TRAP’s), corresponding to the tethered ligand
sequence, have been found to be agonists for receptor activation [1]. Besides in a variety of non-neoplastic cells, functional PAR 1-type thrombin receptors have been identified in different tumor cell lines [6–9] suggesting a role of PAR 1 concerning thrombin-induced effects in tumors including promotion of tumor cell adhesion to endothelium and extracellular matrix as well as enhancement of metastatic capacity and growth of tumors [10–12]. Since the presence of functional PAR 1-type thrombin receptors has been demonstrated in primary cultures of human glioblastomas [13] the question raises whether thrombin receptors might also be involved in the development and progression of other brain tumor entities. Following this idea, we used primary cultures established from human meningiomas and characterized PAR 1-type thrombin receptors on binding level by immunofluorescence studies with a monoclonal antiPAR 1 antibody. To demonstrate that these binding sites constitute functional thrombin receptors the effect
132 of alpha-thrombin and PAR 1 activating hexapeptide (TRAP-6) on intracellular calcium mobilization was investigated in individual human meningioma cells.
Confocal immunofluorescence microscopy
Human alpha-thrombin (2975 NIH-Units/mg protein) was purchased from Kordia Laboratory Supplies, Leiden, the Netherlands, fluo-3-acetoxymethylester was from Sigma Chemie, Deisenhofen, Germany, and the human monoclonal anti-fibroblast Mab 5B5 from Dako Diagnostica (Cat-No. M 0877), Glostrup, Denmark. The monoclonal anti-thrombin receptor Mab 61-1 directed against the domain PESKATNATLDPRSF of the thrombin receptor N-terminus including the thrombin cleavage site was a gift from Dr. Vanitha Ramakrishnan, COR Therapeutics, Inc., South San Francisco, USA.
Cells were fixed with 3% paraformaldehyde and permeabilized with ice-cold 80% methanol. After incubation with DMEM supplemented with 10% FCS for 1 h, the preparation was incubated for 1 h at room temperature with either anti-PAR 1 Mab 61-1 or human anti-fibroblast Mab AS 02, respectively in PBS/0.1% BSA (1 : 100). The preparation was then washed three times with PBS and was next exposed to the secondary antibody (fluorescein isothiocyanate-conjugated antimouse-lgG at 1 : 100 and 0.1% BSA/PBS, Santa Cruz, USA, or Cy3-conjugated anti mouse-lgG at 1 : 100 and 0.1% BSA/PBS, Dianova). For confocal microscopy, the preparation was mounted with Aqua-Poly/Mount (Polysciences, Inc., Warrington, PA, USA) under a glass coverslip. An LSM 410 invert (Carl Zeiss, G¨ottingen, Germany) with an argon ion and helium– neon laser was used. Results were reproduced by at least six independent experiments.
Peptide synthesis
Calcium measurements
The PAR-1-activating peptide TRAP-6 (SFLLRN) and the peptide PESKATNATLDPRSF were synthesized by fmoc strategy on an ABI-Peptide-Synthesizer 433A. Protection of side chains were carried out for Ser as tertBut, for Asn as Trt, for Asp and Glu as O-But, for Lys as Boc and for Arg as Pbf. Cleavage of peptide from resin was performed with trifluoro acetic acid, 5% H2 O und 3% triisopropylsilane. Purification of the crude peptides was carried out by using preparative HPLC on a 40 × 300 mm Vydac C18-column with a flow rate of 100 ml per minute under standard conditions.
[Ca2+ ]i was measured in single cells with fluo-3, a fluorescence indicator of free Ca2+ , as described elsewhere [14]. Cells were grown on Lab Tek chambered borosilicate coverglass (Nunc GmbH & Co. KG, Wiesbaden, Germany) precoated with poly-L-lysine and washed twice with HEPES buffer (pH 7.4) containing 10 mM HEPES, 145 mM NaCl, 0.5 mM Na2 HPO4 , 6 mM glucose, 1 mM MgSO4 , 1.5 mM CaCl2 . Cells were incubated for 15 min at 37◦ C in the same buffer containing 2.0 µM fluo-3 acetoxymethylester. After fluo-3 loading the cells were washed twice and reincubated in HEPES buffer. For calcium measurement in single cells, an inverted confocal laser scanning microscope (LSM 410, Carl Zeiss, G¨ottingen, Germany) was used. Fluorescence images were collected by using the 488 nm argon ion laser line and the intracellular calcium concentration was calculated using the equation [Ca2+ ]i = Kd × (F − Fmin )/(Fmax − F ). The Ca2+ affinity of fluo-3 (Kd ) is 400 nM [14]. Fmax was obtained by addition of 10 mM ionomycin (+6 mM CaCl2 ), Fmin by addition of 20 mM EGTA.
Materials and methods
Preparation of tumor cell culture Tumor tissue for cell culture was taken from operated meningiomas from a 59-year-old and a 79-year-old woman, respectively. After removal of blood and blood vessels, the tumor tissue was mechanically dissected using scalpels. Subsequent trypsination (0.2% trypsin in calcium- and magnesium-free phosphate buffered saline) was performed for 25 min at 37◦ C. Trypsin was removed by centrifugation and resuspension of cells was carried out in DMEM supplemented with 10% fetal calf serum. Cells were routinely cultured at 37◦ C in a humidified atmosphere of 5% CO2 . The culture medium was changed every 2–3 days.
Results In this study, PAR 1-type thrombin receptors were investigated in primary cultures established from
133 surgically removed meningiomas diagnosed histologically as benign transitional meningioma and meningiotheliomatous meningioma (WHO malignancy grade I), respectively. Histological and immunohistological analysis on paraffin-embedded operation material showed positive reactions for endothelial membrane antigen, vimentin and desmoplakin. The primary meningioma cell cultures were observed to grow in whorls as it is typically for meningeal tumor cells. Immunofluorescence studies First, presence of PAR 1 was investigated in primary cultures of human meningioma cells by using a monoclonal antibody specific for the amino-terminus of the PAR 1-type thrombin receptor. Figure 1 shows immunofluorescence staining of PAR 1 by Mab 61-1 binding in a primary culture of human meningioma cells. Enhancement of immunoreactivity with no
Figure 1. Confocal microscopy of PAR 1 in a primary cultured human meningioma. Cells were treated with Mab 61-1 (1 : 100) and a secondary FITC-conjugated anti-mouse lgG. Fluorescence images were obtained with a confocal laser scanning microscope (LSM 410 invert). The picture is a representative of six independent experiments.
definite detection of the exact receptor localization could be demonstrated. When Mab 61-1 was preincubated with the antigenic peptide PESKATNATLDPRSF at concentrations ≥50 µM, inhibition of the antibody binding was observed in the cells (data not shown). In addition, by using the monoclonal human anti-fibroblast Mab 5B5 that is known to react with high selectivity with human fibroblasts and myoepithelial cells in normal and inflammatory tissue [15,16] no immunoreactivity could be observed in cells of similar morphology as those seen in Figure 1 (data not shown). Intracellular calcium concentration The effect of alpha-thrombin and the PAR 1-activating hexapeptide SFLLRN on intracellular Ca2+ mobilization in meningioma cells was investigated by using confocal laser fluorescence microscopy. The alphathrombin- and TRAP-6-induced rise in [Ca2+ ]i was transient and reached a peak within 10–20 sec for both alpha-thrombin (Figure 2) and TRAP-6 (data not shown). Preincubation of human meningioma cells
Figure 2. Time course of alpha-thrombin (final conc. 1.0 nM) induced [Ca2+ ]i mobilization in a single human meningioma cell. The cell was loaded according to Materials and methods. Each data point represents the mean value of 10 measurements with SE < 10%.
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Figure 3. Effect of anti-PAR 1 Mab 61-1 on thrombin-induced calcium mobilization in single human meningioma cells. The cells were preincubated with Mab 61-1 (final concentration 50 nM) for 30 min and subsequently stimulated with alpha-thrombin (final concentration 1.0 nM). Calcium mobilization was measured as described in Materials and methods. Intracellular calcium concentration in the presence or absence of alpha-thrombin was presented as 100% or 0% on the vertical axis, respectively. The data are the mean ± SE from measurements of 20 single cells.
with Mab 61-1 for 30 min resulted in approximately 70% reduction of alpha-thrombin-induced calcium signal (Figure 3). Furthermore, when Mab 61-1 was preincubated with the peptide PESKATNATLDPRSF using as antigen (50 µM), it could no longer block [Ca2+ ]i stimulated by alpha-thrombin (data not shown). Next, we investigated the effect of repeated alphathrombin and TRAP-6 application on Ca2+ mobilization in human meningioma cells. Under the conditions used in our experiments, a further calcium mobilization could be noted only when human meningioma cells were stimulated with alpha-thrombin after TRAP-6 application (Figure 4). In further investigations, cells used for calcium experiments were characterized subsequently by immunofluorescence staining using the monoclonal anti-fibroblast antibody Mab 5B5. No fibroblast immunoreactivity could be observed for the
Figure 4. [Ca2+ ]i mobilization in human meningioma cells after repeated application of alpha-thrombin (A), TRAP-6 application after thrombin stimulation (B) repeated TRAP-6 application (C) and alpha-thrombin application after TRAP-6 stimulation (D) (final concentration of alpha-thrombin: 1.0 nM, TRAP-6: 10 µM). Second application of test agents was performed after a short washout.
cells used in measurement of free intracellular calcium mobilization (data not shown). Discussion First results on cellular functions of the ‘tethered ligand’ thrombin receptor PAR 1 in primary cultures of human glioblastomas suggest its role in glial or neuroepithelial tumors [13]. Here, we used primary cultures established from human meningiomas. To evaluate the origin of the cells immunofluorescence studies with a monoclonal anti-fibroblast antibody were performed. In the primary meningioma cultures,
135 no anti-fibroblast immunoreactivity could be observed. Since this antibody was demonstrated to detect fibroblasts on a primary glioblastoma culture in which cocultivated fibroblasts were present (Kaufmann and Patt, unpublished observations) a fibroblast origin of the cultured meningiomas may be excluded with high probability. The finding that the meningioma cultures were grown in whorls, which is typical for this kind of cells [19], is a further indication for their meningothelial nature. Using primary meningioma cultures, we investigated PAR 1-type thrombin receptors by receptor binding with a well-characterized monoclonal antihuman PAR 1 antibody [17,18]. The thrombin receptor PAR 1 is known to be a cell surface receptor. But additionally, an intracellular PAR 1 pool could be demonstrated in Rat 1 cells transfected with the human PAR 1 [20]. In human meningioma cells, the exact localization of thrombin receptors remains unknown and has to be elucidated in further investigations especially using electron microscopy. The finding that the Mab 61-1 antigenic peptide PESKATNATLDPRSF inhibited binding of Mab 61-1 is a further evidence for the presence of PAR 1 binding sites in human meningioma cells. To demonstrate that these sites constitute functional thrombin receptors their connection with the calcium system was investigated. Stimulation of human meningioma cells both with alpha-thrombin or TRAP-6 resulted in a transient mobilization of free intracellular calcium. TRAP-6 mimicked the effect of alpha-thrombin on [Ca2+ ]i and Mab 61-1 inhibited alpha-thrombin-induced calcium mobilization partly. Thus it may be concluded that PAR 1 is involved in thrombin-induced calcium signaling in human meningioma cells. Since a further calcium signal could be observed in human meningioma cells stimulated with alpha-thrombin after TRAP-6 PAR 1-independent mechanisms might contribute additionally to alphathrombin-induced calcium signaling in human meningioma cells. This was supported by the finding that Mab 61-1 known to block PAR 1-mediated signaling completely (V. Ramakrishnan, personal communication) was unable to act in this way in the meningioma cells. The most likely explanation might be an interaction of alpha-thrombin with other types of thrombin receptors, PAR 3 and PAR 4, recently cloned and characterized [2,4]. Taken together, the data imply that primary human meningioma cultures express functional PAR 1-type thrombin receptors. Thrombin has been demonstrated to play a role in both tumor spreading and growth
[10,11]. Since meningiomas are usually benign tumors thrombin may be suggested to function as a growth regulator by interaction with PAR 1-type thrombin receptors in neuroepithelial tumors.
Acknowledgements We are grateful to Prof. Kalff, Clinic of Neurosurgery, Friedrich Schiller University Jena, Germany, for providing tumor material and to Dr. Vanitha Ramakrishnan, COR Therapeutics, Inc., South San Francisco, for the gift of Mab 61-1. We thank also Mrs. Raabe and Mrs. Faeseke for excellent technical assistance. This work was supported by grants from the Deutsche Krebshilfe (10-1254 Ka 2) and from the Max-Planck Society.
References 1. Vu TKH, Hung DT, Wheaton DT, Coughlin SR: Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 64: 1057–1068, 1991 2. Ishihara H, Conolly AW, Zeng D, Kahn ML, Zheng YW, Timmons C, Tram T, Coughlin SR: Protease-activated receptor 3 is a second thrombin receptor in humans. Nature 386: 502–506, 1997 3. Nystedt S, Emilsson K, Wahlestedt C, Sundelin J: Molecular cloning of a potential proteinase activated receptor: Proc Natl Acad Sci USA 91: 9208–9212, 1994 4. Xu W-F, Andersen H, Whitmore TE, Presnell SR, Yee DP, Ching A, Gilbert T, Davie EA, Foster CA: Cloning and characterization of human protease-activated receptor 4. Proc Natl Acad Sci USA 95: 6642–6646, 1998 5. Nanevicz T, Ishii M, Wang L, Chen M, Chen J, Turck CW, Cohen F, Coughlin SR: Mechanism of thrombin receptor agonist specificity. Chimeric receptors and complementary mutations identify an agonist recognition site. J Biol Chem 270: 21619–21625, 1995 6. Wojtukiewicz MZ, Tang DG, Ben-Josef E, Nenaud C, Walz DA, Honn KV: Solid tumor cells express functional ‘tethered ligand’ thrombin receptor. Cancer Res 55: 698–704, 1995 7. Nierodzik ML, Bain RM, Liu LX, Shivji M, Takeshita K, Karpatkin S: Presence of seven transmembrane thrombin receptor on human tumour cells: effect of activation on tumour adhesion to platelets and tumor metastasis in vivo. Br J Haematol 92: 452–457, 1996 8. Kaufmann R, Schafberg H, Rudroff C, Nowak G: Thrombin receptor activation results in calcium signalling and
136
9.
10.
11.
12.
13.
14. 15.
PKC-dependent stimulation of DNA synthesis in Hep2 laryngeal carcinoma cells. Cancer 80: 2068–2074, 1997 Rudroff C, Schafberg H, Nowak G, Weinel R, Scheele J, Kaufmann R: Characterization of functional thrombin receptors in human pancreatic tumor cells (MIA PaCa-2). Pancreas 16: 189–194, 1998 Zacharski LR: Small cell carcinoma of the lung: interaction with the blood coagulation mechanism and treatment with anticoagulants. Onkologie 10: 264–270, 1987 Zacharski LR, Constantini V: Blood coagulation activation in cancer: Challenges for cancer treatment. Haemostaseologie 15: 14–20, 1995 Fenton II JW, Ofosu FA, Henrikson PK, Brezniak DV, Hassouna HI: Thrombin participation in cancers. In: Pifarr´e R (ed) New Anticoagulants for the Cardiovascular Patient. Hanley & Belfus, Inc., Philadelphia, 1997, pp 517–520 Kaufmann R, Patt S, Schafberg H, Kalff R, Neupert G, Nowak G: Functional thrombin receptor PAR1 in primary cultures of human glioblastoma cells. NeuroReport 9: 709–712, 1998 Tsien RY: Fluorescence probes of cell signaling. Annu Rev Neurosci 12: 227–253, 1989 Konttinen YT, Nyk¨anen P, Nordstr¨om D, Saari H, Sandelin J, Santavirta S, Kouri TI: DNA synthesis in prolyl 4hydroxylase positive fibroblasts in situ in synovial tissue. An autoradiography – immunoperoxidase double labelling study. J Rheumatol 16: 339–345, 1989
16. Janin A, Konttinen YT, Gr¨onblad M, Karhunen P, Gosset D, Malmstr¨om M: Fibroblast markers in labial salivary gland biopsies in progressive systemic sclerosis. Clin Exp Rheumatol 8: 237–242, 1990 17. Norton KJ, Scarborough RM, Kutok JL, Escobedo M-A, Nannizzi L, Coller BS: Immunologic analysis of the cloned thrombin receptor activation mechanism: evidence supporting receptor cleavage, release of the N-terminal peptide, and insertion of the tethered ligand into a protected environment. Blood 82: 2125–2136, 1993 18. Blackhart BD, Cuenco G, Toda T, Scarborough RM, Wolf DL, Ramakrishnan V: The anion-binding exosite is critical for the high affinity binding of the thrombin receptor. Growth factors 11: 17–28, 1994 19. Weisman AS, Villemure J-G, Kelly PA: Regulation of DNA synthesis and growth of cells derived from primary human meningiomas. Cancer Res 46: 2545–2559, 1986 20. Hein L, Ishii K, Coughlin SR, Kobilka BK: Intracellular targeting and trafficing of thrombin receptors. A novel mechanism for resensitization of a G protein-coupled receptor. J Biol Chem 269: 27719–27726, 1994 Address for offprints: Roland Kaufmann, Research Unit ‘Pharmacological Hemostaseology’, Medical Faculty at the Friedrich Schiller University Jena, Drackendorfer Str. 1, 07747 Jena, Germany; Tel.: +49-3641-30 4424; Fax: +49-3641-304412; E-mail:
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