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P2Y receptors activated by diadenosine polyphosphates reestablish Ca2+ transients in achondroplasic chondrocytes
Bone 42 (2008) 516 – 523 www.elsevier.com/locate/bone
P2Y receptors activated by diadenosine polyphosphates reestablish Ca 2+ transients in achondroplasic chondrocytes Ana Guzmán-Aránguez a , Marta Irazu a , Avner Yayon b , Jesús Pintor a,⁎ a
Departamento de Bioquímica y Biología Molecular IV, E.U. Óptica, Universidad Complutense de Madrid, c/Arcos de Jalón s/n 28037 Madrid, Spain b ProChon Biotech Ltd. Kiryat Weizmann, Science Park, Rehovot 76114, Israel Received 26 July 2007; revised 27 September 2007; accepted 24 October 2007 Available online 13 November 2007
Abstract Achondroplasia is the most common type of dwarfism, characterised by a mutation in the gene that encodes the fibroblast growth factor receptor 3 (FGFR3). Achondroplasia mainly affects the chondrocytes and therefore bones do not grow properly since intracellular pathways are altered. In this sense, defective calcium signaling by mutant FGFR3 has been previously described. The purpose of this study was to investigate the presence of purinergic P2Y receptors and how the activation of these receptors can have influence on defective calcium signaling observed in achondroplasic chondrocytes. The presence of P2Y receptors was determined by immunocytochemical and western blot techniques. Calcium mobilization after stimulation with nucleotides, dinucleotides, or, FGF9 application, was measured using the ratiometric dye fura-2/AM and fluorescence imaging. Our results demonstrate the expression of P2Y1, P2Y2, P2Y6 and P2Y11 receptors in achondroplasic chondrocytes, as well as the activation of these receptors after nucleotides and dinucleotides exposure. The altered calcium signaling of achondroplasic chondrocytes was confirmed, since FGF9 treatment fails to induce calcium mobilization. However, achondroplasic chondrocytes pre-treated with Ap4A are able to respond with increases in intracellular calcium after FGF9 stimulation. These findings show the rescue effect of diadenosine tetraphosphate (Ap4A), acting by means of P2Y receptors, on defective calcium response triggered by achondroplasic FGFR3. © 2007 Elsevier Inc. All rights reserved. Keywords: P2Y receptors; Calcium; Achondroplasia; Chondrocytes; FGFR3
Introduction Chondrocytes are important cells in the endochondral bone growth in mammals. Several molecules, hormones, cytokines and growth factors drive the progress of epiphyseal growth plate chondrocytes through proliferative, prehypertrophic, hypertrophic and terminal hypertrophic stages during endochondral ossification. Among all of them, transforming growth factors betas (TGF-βs), bone morphogenetic proteins (BMP), Indian hedgehog (Ihh), insulin-like growth factors, (IGFs), fibroblast growth factors (FGFs), interleukins (ILs) and others, interplay an organised sequence of events that permit the normal development of bones [1,2]. Alteration in any of these substances, or more commonly in ⁎ Corresponding author. Dep. Bioquímica, E.U. Óptica, Universidad Complutense de Madrid, c/Arcos de Jalón s/n, 28037 Madrid, Spain. Fax: +34 91 3946885. E-mail address: [email protected] (J. Pintor). 8756-3282/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2007.10.023
any of their receptors, will produce changes in ossification that will lead to a group of pathologies termed as bone dysplasias. One of these bone syndromes is achondroplasia, the most common type of dwarfism. Achondroplasia is a genetic pathology due to mutation in the gene that encodes the fibroblast growth factor receptor 3 (FGFR3), being the mutation in 97% of the patients a Glycine to Arginine substitution at position 380. FGFR3 is activated after the binding of FGF. This activation induces receptor dimerization and a subsequent receptor autophosphorylation in the intracellular domain. Autophosphorylated tyrosine residues and adjacent amino-acids provide binding sites for different signaling proteins which initiate downstream cascades (STAT, mitogen-activated protein kinase cascade, phosphoinositide-3 kinase pathway), leading to a cellular response. The achondroplasic mutant induces an overstimulation of intracellular signal transduction pathways related to proliferation and hypertrophic differentiation stages of epiphyseal growth plate chondrocyte [3].
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Apart from the sustained signaling induced by the mutant receptor, the defective ability of achondroplasic FGFR3 to mobilize calcium has been also observed, thus suggesting the importance of this cation in the development of this pathology [4]. Achondroplasia does not have any pharmacological treatment. This orphan disease needs the development of strategies involving new molecules suitable for the treatment of this disorder. An interesting group of molecules, which has been scarcely investigated in relation with chondrocytes, are nucleotides. ATP, UTP and diadenosine polyphosphates are extracellular active naturally occurring substances involved in many biochemical and physiological processes. Thus, they take part in neurotransmission, secretion and vasodilation process, and they also mediate long-term events such as cell proliferation, differentiation and death involved in development and regeneration [5]. As a consequence of such a wide physiological involvement, these compounds can be used for the treatment of some pathologies [6]. All these compounds present the ability to activate membrane receptors termed P2 purinoceptors. Metabotropic P2 receptors are termed P2Y receptors and the existence of 8 different subtypes has been described. These receptors are coupled to phospholipase C (PLC) and inositol triphosphate and diacylglicerol generation, although some can be negatively coupled to the adenylate cyclase. On the other hand, P2X are ionotropic receptors involved in fast actions mediated by the nucleotides in different tissues [7,8]. The existence of P2 receptors in normal chondrocytes has already been described [9]. Nevertheless, the presence and the effect of P2 receptors have not been studied, mainly P2Y, in achondroplasic chondrocytes. Interestingly, P2 receptors can transactivate tyrosine kinase receptors, including growth factor receptors like epidermal growth factor receptor or platelet derived growth factor receptor [10]. The aim of this experimental work is to describe the presence of P2Y receptors in mouse achondroplasic chondrocytes and to study the intracellular Ca2+ changes triggered as a consequence of dinucleotide and nucleotide applications. The compounds were tested at various concentrations and some experiments were performed by reducing the extracellular calcium concentration by means of a mixture Ca2+/EGTA in order to know the contribution of an intracellular mechanism on the calcium transients. To clarify the P2Y receptor or receptors involved in the effect of dinucleotides, different P2 antagonists were used. In addition, we analyze whether or not the calcium responses induced by activated P2 receptors can have an influence on altered calcium signal observed in achondroplasic chondrocytes, as well as the intracellular mechanisms involved in this action. Materials and methods Achondroplasic chondrocytes Achondroplasic mouse chondrocytes and normal mouse chondrocytes were kindly provided by ProChon Biotech Ltd. (Israel). Chondrocytes were obtained from the articular cartilage of normal and hFGFR3G380R transgenic mice [11]. Cells were cultured in Dulbecco's Modified supplemented with 15% fetal bovine serum, penicillin and streptomycin. Chondrocytes were maintained at 37°C in atmosphere containing 5% CO2.
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Immunocytochemistry and western blotting For the immunohistochemical study we used achondroplasic chondrocytes glued to coverslips pre-treated with poly-L-Lysine. Covers were treated with 4% p-formaldehyde (w/v) for 15min and washed twice with phosphate-buffered saline (PBS) medium afterwards. Cells were incubated overnight at room temperature in PBS containing 1% bovine serum albumin (BSA) and anti-P2Y primary antibodies (Alomone Labs., Jerusalem, Israel). The dilutions of the primary antibodies were: anti-P2Y1, 1:200; anti-P2Y2, 1:500; anti-P2Y4, 1:500; anti-P2Y6, 1:200 and anti-P2Y11, 1:1000. The covers were washed three times in PBS in the presence of 3% BSA and were incubated for 1h with the secondary antibody which was also diluted (1:500) in PBS/BSA solution. As secondary antibody goat anti-rabbit IgG-TRITC (tetramethylrhodamine isothiocyanate) (40μg/ml) from Sigma (St. Louis, MO, USA) was used. The covers were washed three times with PBS and mounted following standard procedures. Controls were carried out following the same procedures but substituting the primary antibody by the same volume of PBS/BSA solution. Cells were analyzed by confocal microscopy using a Zeiss Axiovert 200M microscope equipped with a LSM 5 Pascal confocal module. Achondroplasic chondrocytes were observed with a Zeiss 63× oil immersion lens, numerical aperture 1.40. TRICT was monitored by exciting at 543nm wavelength. Differential interference contrast (Nomarski, DIC), was performed with the same 63× lens bypassed through the corresponding polarizers and analyzers. All the images were managed by the LSM 5 Pascal software. Western blot analysis was performed by homogenizing the chondrocytes with a buffer lysis that contained Hepes 50mM pH 7.5, Triton 2.5% (w/v), EDTA 10mM, phenylmethylsulphonylfluoride 0.2mM and leupeptin 5μg/ml. After homogenization, proteins were quantified by Bio-Rad protein assay (BioRad laboratories, Hercules, CA, USA). Protein samples (40μg) were separated by SDS-PAGE (10% acrylamide gel). Proteins were transferred to nitrocellulose membranes (Amersham-Pharmacia-Biotech, Buckinghamshire, UK). Following transfer, the membranes were washed with phosphate-buffered saline and blocked for 1h at room temperature with 5% (w/v) skimmed milk powder in PBS. Blots were then incubated overnight at 4°C with primary antibodies in 5% (w/v) skimmed milk powder dissolved in PBS-Tween 20 (0.05% by volume). The dilutions of primary antibodies were as follows: anti-P2Y1, 1:200, antiP2Y2, 1:500; anti-P2Y4, 1:200; anti-P2Y6, 1:200 and anti-P2Y11, 1:1000. The primary antibodies were removed and the blots extensively washed with PBSTween 20. Blots were then incubated for 1h at room temperature with the secondary antibody (anti-rabbit IgG coupled to horseradish peroxidase, from Sigma) at 1:1000 dilution in 5% (w/v) skimmed milk powder dissolved in PBS/ Tween 20. Following removal of the secondary antibody, blots were extensively washed as above and developed using the Enhanced Chemiluminescence detection system (Amersham-Pharmacia-Biotech, Buckinghamshire, UK). Films were scanned and a densitometric analysis was performed using Kodak GL 200 Imaging system and Kodak Molecular Imaging software (Kodak, Rochester, NY, USA).
Calcium measurements Mouse achondroplasic chondrocytes were plated at a density of 2 × 104 cells/cm2 on 15mm diameter glass coverslips 1day before using. Subconfluent cultures were loaded with 5μM fura-2/AM (Molecular Probes, Breda, The Netherlands) for 30min at 37°C in incubation buffer (121mM NaCl, 4.7mM KCl, 5mM NaHCO3, 1.2mM KH2PO4, 1.2mM MgSO4, 2mM CaCl2, 10mM glucose, 10mM HEPES and 0.01% BSA, at pH 7.4 with NaOH). Coverslips with fura-2 loaded cells were transferred into an open flow chamber (1ml incubation buffer) mounted on the heated stage of a TE 200 inverted epifluorescence microscope (Nikon, Düsseldorf, Germany). The wavelength of the incoming light was selected with the aid of a monochromator (12nm bandwidth; Kinetic Imaging Ltd., Liverpool, United Kingdom) to 405nm. This wavelength corresponded to the peak of fluorescence of Ca2+-free fura-2 solutions in our system (peak shifted to a longer wavelength owing to lower transmission of the objective at shorter wavelengths). Twelve-bit images were acquired by a Hamamatsu C-4880-80 multiformat CCD camera controlled by Kalcium PC software (Kinetic Imaging Ltd.). The exposure time was 822ms and the changing time 5ms. The images were acquired continuously and buffered on a fast SCSI disk. After a stabilization period of 10min, image pairs were obtained alternately every 2s, and for a total of 8min, at excitation wavelengths of 340 and
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Fig. 1. Immunolocalization of P2Y receptors in achondroplasic chondrocytes. Cells were grown in cover slips, fixed, subjected to immunostaining with different P2Y receptor antibodies and analyzed by confocal microscopy. Images were taken with a magnification: ×63. Scale bars = 50 μm.
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bisindolymaleimide I, PDbu, pyridoxalphosphate-6-azophenyl-2,4-disulfonic acid (PPADS), suramin, N6-methyl-2′-deoxyadenosine-3′,5′-bisphosphate (MRS2179) and reactive blue-2 (RB-2) were purchased from Sigma Chemical (St. Louis, MO, USA). Other reagents were analytical grade from Merk (Darmstadt, Germany).
Data analysis Five different chondrocyte cultures were used along the present experimental work. Usually, a number of about 50 chondrocytes per field were individually analyzed in their ability to respond to the different compounds. Results are given as mean ± S.E.M. Two-way analysis of variance (ANOVA) test was used to evaluate statistical differences between control and nucleotide effects. Agonist potencies were calculated using a four-parameter logistic equation and the Origin software package (OriginLab Corporation, Northampton, MA, USA). pD2 values were transformed into EC50 values, which represent the concentration of agonist at which 50% of the maximal effect is achieved. When it is appropriated, single experimental traces are represented in the figures; these represent at least six determinations performed with equivalent results.
Results Presence of P2Y receptors in achondroplasic chondrocytes
Fig. 2. P2Y receptors expression in achondroplasic chondrocytes. (A) Cell lysates (40 μg total protein) were analyzed by western blot. A representative blot is shown for each P2Y receptor. (B) The histograms represent the intensity of the band corresponding each P2Y receptor expressed as arbitrary units (sum of the background subtracted pixels within a rectangle encompassing the entire band). 380nm (10nm bandwidth filters), to excite the Ca2+ bound and Ca2+ free forms of this ratiometric dye, respectively. The emission wavelength was 510nm (120-nm bandwidth filter). [Ca2+]i values were calculated on a single cell basis from the 340to 380-nm fluorescence ratios at each time point. In both control and experimental groups, Ca2+ was recorded for 1min before drug application and during 7min. Calcium single dose experiments were performed by applying the agonists at a final concentration of 100μM. For the concentration–response studies, the nucleotides were applied at variable doses ranging from 10− 9M to 10− 3M. For those experiments involving antagonists, they were applied at a final concentration of 100μM 30min before the treatment with the dinucleotide (also at 100μM). For some antagonists such as suramin, 1-[amino-5-(2,7-dichloro-6acetomethoxy-3-oxo-3H-xanthen-9-yl)phenoxy]-2-(2′-amino-5′-methylphenoxy) ethane-N,N,N′,N′-tetraacetic acid pentaacetoxy-methyl ester (FLUO-3AM) (Molecular Probes, Breda, The Netherlands), was used. The measurement conditions were almost identical but the excitation and detection wavelengths were 485nm and 538nm respectively. Free calcium measurements were carried out with a mixture formed of 50μM EGTA and 38μM CaCl2 as described [12,13]. For studies of calcium mobilization induced with fibroblast growth factor 9 (FGF9), normal and achondroplasic chondrocytes were stimulated with 25ng/ml of FGF9 alone or after pre-incubation with 100μM diadenosine tetraphosphate (Ap4A). PLC inhibitors U73122, U73143 were applied at 3μM, 10min before the addition of FGF9 (25ng/ml), since longer pre-incubations clearly produced cell damage. Protein Kinase C (PKC) inhibitor bisindolymaleimide I was preincubated at a concentration of 10μM, 30min before the application of FGF9 (25ng/ml). The PKC activator phorbol 12, 13 dibutyrate (PDbu) were applied at 1μM concentrations 30min before the addition of FGF9 (25ng/ml).
Drugs UTP and ATP were purchased from Amersham Biosciences, Inc. (Piscataway, NJ, USA); UDP, diadenosine triphosphate (Ap3A), diadenosine tetraphosphate (Ap4A), diadenosine pentaphosphate (Ap5A), FGF9, U73122, U73143,
Achondroplasic chondrocytes displayed an extended aspect when observed under the Nomarsky interferential microscopy (Fig. 1). When these cells were incubated with commercial antibodies against P2Y receptors, a moderate immunoreactivity for P2Y1, P2Y2, P2Y6 and P2Y11 was detected, while they showed no labeling for the P2Y4 antibody (Fig. 1). Western blots confirmed the results obtained by means of the immunocytochemical technique. P2Y1, P2Y2, P2Y6 and P2Y11 bands appeared with molecular weights close to those already presented in the literature (Fig. 2A). P2Y11 and P2Y2 receptors
Fig. 3. Intracellular Ca2+ increases in achondroplasic chondrocytes after their challenge with dinucleotides and mononucleotides. Cells grown in cover slips were loaded with fura-2 AM in a buffer containing CaCl2 (2 mM) for 30 min. Cover slips were then mounted in a perfusion chamber, and intracellular Ca2+ levels were analyzed after stimulation with the different compounds using a multiple excitation microfluorescence system. (A) Representative Ca2+ transients elicited by Ap3A, Ap4A and Ap5A, all at 100 μM. (B) Intracellular Ca2+ increases produced by the superfusion of either ATP, UTP, ADP and UDP, all at 100 μM.
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Fig. 5. Effect of the removal of extracellular Ca2+ on the responses elicited by diadenosine polyphosphates and mononucleotides. The removal of the extracellular calcium by means of a mixture of Ca2+/EGTA demonstrates that only a small part of the reordered ion increments are due to the presence of P2X receptors. Values represent the mean± S.E.M. of six independent experiments. ⁎p b 0.1 versus the effect of nucleotide without the removal of the extracellular calcium.
Fig. 4. Concentration–response curves for dinucleotides and mononucleotides. (A) Representative Ca2+ traces of single achondroplasic chondrocytes challenged by graded concentrations of Ap4A. (B) Concentration–response curves for diadenosine polyphosphates and mononucleotides. Graded concentrations starting from 10− 9 M to 10− 3 M were tested. Curves represent the increase in the intracellular Ca2+ concentration after subtracting the basal cytosolic calcium (82 ± 7 nM). Values are the mean ± S.E.M. of four independent experiments.
showed the highest levels of expression, whereas a lower amount of P2Y1 and P2Y6 receptors was found by densitometric analysis (Fig. 2B). Effects of dinucleoside polyphosphates in intracellular calcium in chondrocytes When achondroplasic chondrocytes were challenged by the dinucleoside polyphosphates: diadenosine triphosphate, Ap3A, diadenosine tetraphosphate, Ap4A and diadenosine pentaphosphate, Ap5A (all at 100μM), changes in the cytosolic calcium were observed (Fig. 3A). The increases in calcium varied from 175 ± 12nM in the case of Ap3A to 198 ± 8nM in the case of Ap4A and 188 ± 7nM in the case of Ap5A (n = 6). Mononucleotides such as ATP, UTP, ADP and UDP also induced intracellular calcium increases (Fig. 3B). ATP and UTP provided calcium increases of 300 ± 10nM and 276 ± 9nM respectively (both at 100μM). Lower intracellular calcium changes were produced by the nucleosides diphosphate ADP and UDP,
the values being 105 ± 5nM and 95 ± 8nM for ADP and UDP respectively (n = 6, Fig. 3B). Concentration–response curves for dinucleotides and mononucleotides The behavior of the system at various agonist concentrations was assessed (Fig. 4). Diadenosine tetraphosphate depicted a gradual increase in the calcium concentration values (Fig. 4A). Ap4A and other mono and dinucleotides records were transformed to generate concentration–response curves. At a wide range of concentrations (10− 9M to 10− 3M), ATP provided the highest increment of intracellular calcium, followed by UTP, Ap4A, Ap5A and Ap3A (Fig. 4B, Table 1). The analysis of pD2 values obtained from the concentration–response curves (Table 1), allowed to determine the following potency order: Ap3A ≥ Ap5A = ATP = UTP ≥ Ap4A.
Table 1 Concentration–response values for diadenosine polyphosphates and other mononucleotides in achondroplasic chondrocytes Compound
pD2 (EC50)
Maximal Ca2+ increase (nM)
Ap3A Ap4A Ap5A ATP UTP
− 5.7 ± 0.1 (1.99 μM) − 5.9 ± 0.1 (1.25 μM) − 5.8 ± 0.2 (1.58 μM) − 5.8 ± 0.2 (1.25 μM) − 5.8 ± 0.2 (1.58 μM)
147 ± 17 198 ± 10 181 ± 13 289 ± 21 263 ± 12
Values represent the mean ± S.E.M. of four independent experiments.
Fig. 6. Effect of P2 antagonists on the Ca2+ increases induced by Ap4A. (A) Effect of MRS 2179, suramin, PPADS, and RB-2 (all at 100 μM) on the effect elicited by Ap4A (100 μM). Among all the tested antagonists, PPADS was the most effective antagonizing the effect of Ap4A while RB-2 was almost unable to reverse the effect of the dinucleotide. Values represent the mean ± S.E.M. of six independent experiments. ⁎⁎p b 0.05 vs control; ⁎⁎⁎p b 0.001 vs control.
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The extracellular calcium concentration was reduced by means of a mixture Ca2+/EGTA and experiments were performed under these conditions. As shown in Fig. 5, extracellular Ca2+ removal partially reduced the effect of some of the tested di-and mononucleotides, indicating the presence of ionotropic P2X receptors in achondroplasic chondrocytes. Studies with P2 antagonists The selective P2Y1 antagonist N6-methyl-2′-deoxyadenosine-3′,5′-bisphosphate (MRS2179) and the non-selective P2 antagonists suramin, pyridoxal phosphate 6-azophenyl-2′,4′disulfonate (PPADS) and reactive blue 2 (RB-2) were tested at a concentration of 100μM. The antagonists presented different effects on the Ap4A induced calcium increase in achondroplasic chondrocytes. In particular, PPADS was the most effective in reversing the effect triggered by Ap4A, reducing the calcium signal to 15% of control (Fig. 6). The P2Y1 selective antagonist MRS2179 and suramin reduced Ap4A signal to 38% and 40% of control values respectively (Fig. 6). RB-2 was unable to produce significant change in the cellular response elicited by diadenosine tetraphosphate (all the experiments n = 6).
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Recovery of FGFR3 calcium transients in achondroplasic chondrocytes after treatment with Ap4A The mobilization of calcium in response to stimulation with FGF9, the preferred ligand for FGFR3, was measured in both normal and achondroplasic cells. In accordance with other papers [4], the native FGFR3 induced a calcium increase after FGF9 treatment (Fig. 7A), whereas the achondroplasic FGFR3 was not able to trigger calcium mobilization (Fig. 7C). Interestingly, in achondroplasic chondrocytes, pre-treatment with Ap4A restored the ability of the FGFR3 to mobilize intracellular Ca2+, the achondroplasic cells behaving apparently as normal chondrocytes (Fig. 7D and B). In the absence of extracellular calcium, the FGF9-induced Ca2+ signal after previous Ap4A exposure was detected (Fig. 7E) although the area under the curve was slightly lower than the Ca2+ response observed when extracellular calcium was present (Fig. 7D), suggesting a transmembrane calcium influx contribution to calcium transients. Additional experiments, in which Ap4A was pre-applied for 2 min, removed, and cells were challenged alone with FGF9 30 min after the dinucleotide addition, were carried out.
Fig. 7. Intracellular Ca2+ increases in achondroplasic chondrocytes after their challenge with FGF9 and after previous Ap4A pre-incubation. Cells grown in cover slips were loaded with fura-2 AM in a buffer containing CaCl2 (2 mM) for 30 min. Cover slips were then mounted in a perfusion chamber, and intracellular Ca2+ levels were analyzed after stimulation with the different compounds using a multiple excitation microfluorescence system. Representative Ca2+ transients shown correspond to: Normal chondrocytes (A) or achondroplasic chondrocytes (C) stimulated with 25 ng/ml FGF9. Normal chondrocytes (B) and achondroplasic chondrocytes (D) pretreated with 100 μM Ap4A 2 min before treatment with 25 ng/ml FGF9. Extracellular calcium was removed by means of a mixture of Ca2+/EGTA and achondroplasic chondrocytes were pre-treated with Ap4A 2 min before treatment with 25 ng/ml FGF9 (E). Achondroplasic chondrocytes were pre-treated with Ap4A and after 30 min 25 ng/ml FGF9 was added (F).
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As observed in Fig. 7F, the pre-treatment of Ap4A was enough to allow FGF9 alone to produce a Ca2+ increase similar to that observed when Ap4A and FGF9 are applied consecutively. This result support that the Ap4A action is mediated by a P2Y receptor, rather than by a direct action on the FGFR3 (n = 9). Intracellular mechanisms involved in the Ap4A effect on FGF9-induced calcium mobilization in achondroplasic chondrocytes The P2Yantagonist PPADS blocked the Ap4A action (Fig. 8), confirming that this diadenosine polyphosphate acts by means of P2Y stimulation. On the other hand, treatment with PLC inhibitor U73122 prevented the Ap4A effect on calcium increase whereas its inactive analog U73143 failed to inhibit this effect (Fig. 8). These results suggest that PLC acts as a mediator in the Ap4A effect, this being a common intracellular mechanism in P2Y receptors. Pre-treatment of achondroplasic chondrocytes with the PKC inhibitor Bisindolymaleimide I abolished Ap4A-induced calcium signaling triggered by FGF9 (Fig 8), indicating the involvement of PKC in Ap4A-induced calcium mobilization. The use of phorbol 12, 13 dibutyrate (PDbu) allowed to confirm this involvement. Thus, treatment of achondroplasic chondrocytes with 1 μM of PDbu caused a marked calcium increase when FGF9 was applied, even higher than that induced by Ap4A pre-treatment.
Fig. 8. Intracellular mechanism used by Ap4A to mediate its action on FGF9induced Ca2+ response. Achondroplasic chondrocytes were treated with 25 ng/ml FGF9 alone or after 100 μM Ap4A pre-treatment. The participation of P2Y receptor was verified by means of the use of the antagonist PPADS. This compound was added for 30 min before incubation with Ap4A and subsequent FGF9 stimulation. To elucidate the role of PLC in the Ap4A effect, achondroplasic chondrocytes were pre-treated with PLC inhibitor U73122 (3 μM), or its inactive analog U73143 (3 μM) for 10 min before incubation with Ap4A and subsequent FGF9 stimulation. In order to test the influence of PKC, cells were pre-incubated with the PKC inhibitor Bisindolymaleimide I (10 μM) for 30 min before stimulation with Ap4A and subsequent FGF9 stimulation. Alternatively, to confirm PKC involvement, achondroplasic chondrocytes were treated with PDbu before stimulation with FGF9. The recordered Ca2+ increments are summarized and shown as a bars diagram. Values represent the mean ± S.E.M. of a total of nine experiments performed with three different batches of cells.
Discussion The present experimental work describes the existence of P2Y receptors in achondroplasic chondrocytes as well as their activation and subsequent Ca2+ mobilization after being challenged with diadenosine polyphosphates and other mononucleotides. The influence of activated P2Y receptors on altered calcium signaling showed by achondroplasic FGFR3 in chondrocytes has been also examined. Some of the P2Y receptors identified in achondroplasic chondrocytes are present in normal chondrocytes. For instance, the existence of P2Y1 and P2Y2 receptors has been previously studied by other authors in normal chondrocytes [9,14,15]. Our studies have also contributed to define the presence of P2Y6 and P2Y11 receptors in achondroplasic chondrocytes. In addition, the existence of P2X receptors in achondroplasic chondrocytes has been detected, since the experiments performed in the absence of extracellular Ca2+ showed differences compared to those carried out in the presence of this ion. In this sense, other authors have identified by immunohistochemistry the existence of P2X2 and P2X5 receptors in rat chondrocytes [14]. Additionally, a putative involvement of a calcium-induced calcium release mechanism with capacitative calcium entry, such as a store-operated channel or transient receptor potential channel could be suggested in order to explain the difference of calcium signal induced by nucleotides in absence or in presence of calcium [16]. Diadenosine polyphosphates have not been tested before in their ability to increase intracellular Ca2+ in normal chondrocytes. Our results demonstrate that diadenosine polyphosphates seem to have the same efficacy and pharmacological potency to that exhibited by naturally occurring mononucleotides such as ATP and UTP. The activity of diadenosine polyphosphates in achondroplasic chondrocytes can be carried out by any of the P2Y receptors present in these cells. Since Ap3A, Ap4A and Ap5A can activate different P2Y receptors [17] is difficult to assign the possible receptor selectivity of these dinucleotides. Nevertheless, the use of P2 antagonists may contribute to consign what receptor is activated by the dinucleotides. Thus, the experiments with antagonists revealed that a part of the Ap4A action is due to the activation of a P2Y1 receptor, since its selective inhibitor, MRS2179, was able to reduce about 62% the dinucleotide response. The response was not completely abolished, suggesting the involvement of other P2Y receptors. P2Y2 would be a good candidate to be taken into consideration, since previous experiments with heterologous expressed receptors have determined that Ap4A acts preferentially on P2Y1 and P2Y2 receptors [18,19]. After determining the existence and functionality of P2Y receptors in achondroplasic chondrocytes, the other main objective of this work has been to analyze the possible effect of activated P2Y receptors on the lack of Ca2+ responses in FGF9stimulated achondroplasic chondrocytes. Some reports have described the increase of intracellular calcium levels induced by wild-type FGFR3 in response to FGF exposure [4,20]. Binding of FGF to FGFR3 leads to autophosphorylation of the receptor on several tyrosine residues, being the phosphorylation of Tyr760 essential for interaction and subsequent activation of PLCγ. Then, active PLCγ induces
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release of calcium from intracellular stores. This calcium increase was observed in the normal chondrocytes analyzed after FGF9 treatment, whereas the mutant FGFR3 of achondroplasic chondrocytes was not able to trigger such a calcium mobilization. This result is consistent with previous findings in which altered FGF-induced calcium signals were detected in cell lines with achondroplasia and thanatophoric dysplasia mutations [4]. Since phosphorylated Tyr760 and its flanking sequences constitute the major site of PLCγ association with FGFR3, a possible impediment for this interaction could be responsible for the different behavior shown by mutated FGFR3 in achondroplasic chondrocytes. In fact, a mutant FGFR1 in which Tyr766 (corresponding to Tyr760 in FGFR3) was exchanged for the totally lacked calcium response [21]. Taking into account that phosphorylated Tyr760 is also a binding site for other proteins involved in signal transduction pathways (STAT, MAPK cascade) [22,23], which are stimulated in a prolonged way by mutant FGFR3, the constant binding of these proteins could interfere with PLCγ binding and/or activation. Interestingly, in achondroplasic chondrocytes pre-treatment with Ap4A restored the achondroplasic FGFR3 ability to elicit intracellular Ca2+ increase. Ap4A seems to mediate its action by stimulating presumably a P2Y1 or P2Y2 receptor. Confirming this notion the analysis of downstream effectors that link Ap4A stimulation to FGF9-induced calcium mobilization in achondroplasic chondrocytes revealed that PLC and PKC, the classical second transduction systems coupled to P2Y receptors, act as key mediators of this process. Regarding the role of these effectors, PLC activation could be simply necessary for subsequent PKC stimulation. The activated PKC may phosphorylate the achondroplasic FGFR3 receptor regulating its activity. Alternatively, PKC could modulate the assembly of adaptor signaling complexes involved in prolonged signal transduction pathways, shifting the equilibrium towards the binding of PLCγ to phosphorylated Tyr760. In summary, activation of P2Y receptors, coupled to PLC and PKC, by Ap4A allow the achondroplasic chondrocytes to recover the intracellular calcium signaling, which is critical for different cellular events [20,24]. This finding suggests that activation of P2Y receptors may be a useful pharmacological tool to modify the altered biochemical and physiological patterns that occur in achondroplasia. Acknowledgments We thank PROCHON BIOTECH laboratories for providing us normal and achondroplasic chondrocytes. We wish to thank Dr. Antonio Rodríguez Artalejo for his valuable discussions and suggestions. This work has been supported by research grants from Comunidad de Madrid CAM GR/SAL/057372004, Fundación Magar, Fundación López Hidalgo and SantanderComplutense PR41/06-14962. References [1] Kobayashi T, Kronenberg H. Minireview: transcriptional regulation in development of bone. Endocrinology 2005;146:1012–7.
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[2] Provot S, Schipani E. Molecular mechanisms of endochondral bone development. Biochem Biophys Res Commun 2005;328:658–65. [3] L'Hote CG, Knowles MA. Cell responses to FGFR3 signalling: growth, differentiation and apoptosis. Exp Cell Res 2005;304:417–31. [4] Nguyen HB, Estacion M, Gargus JJ. Mutations causing achondroplasia and thanatophoric dysplasia alter bFGF-induced calcium signals in human diploid fibroblasts. Hum Mol Genet 1997;6:681–8. [5] Burnstock G. Pathophysiology and therapeutic potential of purinergic signaling. Pharmacol Rev 2006;58:58–86. [6] Fischer B., Therapeutic applications of ATP-(P2)-receptors agonists and antagonists. Exp. Opin. Ther. Patents 1999;9:385–399. [7] Boeynaems JM, Communi D, Gonzalez NS, Robaye B. Overview of the P2 receptors. Semin Thromb Hemost 2005;31:139–49. [8] North RA. Molecular physiology of P2X receptors. Physiol Rev 2002;82: 1013–67. [9] Caswell AM, Leong WS, Russell RG. Evidence for the presence of P2-purinoceptors at the surface of human articular chondrocytes in monolayer culture. Biochim Biophys Acta 1991;1074:151–8. [10] Daub H, Weiss FU, Wallasch C, Ullrich A. Role of transactivation of the EGF receptor in signalling by G-protein-coupled receptors. Nature 1996;379:557–60. [11] Segev O, Chumakov I, Nevo Z, Givol D, Madar-Shapiro L, Sheinin Y, et al. Restrained chondrocyte proliferation and maturation with abnormal growth plate vascularization and ossification in human FGFR-3(G380R) transgenic mice. Hum Mol Genet 2000;9:249–58. [12] Carbone E, Swandulla D. Neuronal calcium channels: kinetics, blockade and modulation. Prog Biophys Mol Biol 1989;54:31–58. [13] Verhage M, Besselsen E, Lopes da Silva FH, Ghijsen WE. Ca2+-dependent regulation of presynaptic stimulus-secretion coupling. J Neurochem 1989;53:1188–94. [14] Hoebertz A, Townsend-Nicholson A, Glass R, Burnstock G, Arnett TR. Expression of P2 receptors in bone and cultured bone cells. Bone 2000;27: 503–10. [15] Koolpe M, Pearson D, Benton HP. Expression of both P1 and P2 purine receptor genes by human articular chondrocytes and profile of ligandmediated prostaglandin E2 release. Arthritis Rheum 1999;42:258–67. [16] Ekstein J, Nasatzky E, Boyan BD, Ornoy A, Schwartz Z. Growth-plate chondrocytes respond to 17beta-estradiol with sex-specific increases in IP3 and intracellular calcium ion signalling via a capacitative entry mechanism. Steroids 2005;70:775–86. [17] Pintor J, Miras-Portugal MT. Receptors for diadenosine polyphosphates P2D, P2YApnA, P4 and dinucleotide receptors: are there too many? Trends Pharmacol Sci 2000;21:135. [18] Lazarowski ER, Watt WC, Stutts MJ, Boucher RC, Harden TK. Pharmacological selectivity of the cloned human P2U-purinoceptor: potent activation by diadenosine tetraphosphate. Br J Pharmacol 1995;116: 1619–27. [19] Patel K, Barnes A, Camacho J, Paterson C, Boughtflower R, Cousens D, et al. Activity of diadenosine polyphosphates at P2Y receptors stably expressed in 1321N1 cells. Eur J Pharmacol 2001;430:203–10. [20] Abe M, Naski MC. Regulation of sprouty expression by PLCgamma and calcium-dependent signals. Biochem Biophys Res Commun 2004;323: 1040–7. [21] Peters KG, Marie J, Wilson E, Ives HE, Escobedo J, Del Rosario M, et al. Point mutation of an FGF receptor abolishes phosphatidylinositol turnover and Ca2+ flux but not mitogenesis. Nature 1992;358:678–81. [22] Cross MJ, Lu L, Magnusson P, Nyqvist D, Holmqvist K, Welsh M, et al. The Shb adaptor protein binds to tyrosine 766 in the FGFR-1 and regulates the Ras/MEK/MAPK pathway via FRS2 phosphorylation in endothelial cells. Mol Biol Cell 2002;13:2881–93. [23] Hart KC, Robertson SC, Donoghue DJ. Identification of tyrosine residues in constitutively activated fibroblast growth factor receptor 3 involved in mitogenesis, Stat activation, and phosphatidylinositol 3-kinase activation. Mol Biol Cell 2001;12:931–42. [24] Zuscik MJ, D'Souza M, Ionescu AM, Gunter KK, Gunter TE, O'Keefe RJ, et al. Growth plate chondrocyte maturation is regulated by basal intracellular calcium. Exp Cell Res 2002;276:310–9.
P2Y receptors activated by diadenosine polyphosphates reestablish Ca 2+ transients in achondroplasic chondrocytes Ana Guzmán-Aránguez a , Marta Irazu a , Avner Yayon b , Jesús Pintor a,⁎ a
Departamento de Bioquímica y Biología Molecular IV, E.U. Óptica, Universidad Complutense de Madrid, c/Arcos de Jalón s/n 28037 Madrid, Spain b ProChon Biotech Ltd. Kiryat Weizmann, Science Park, Rehovot 76114, Israel Received 26 July 2007; revised 27 September 2007; accepted 24 October 2007 Available online 13 November 2007
Abstract Achondroplasia is the most common type of dwarfism, characterised by a mutation in the gene that encodes the fibroblast growth factor receptor 3 (FGFR3). Achondroplasia mainly affects the chondrocytes and therefore bones do not grow properly since intracellular pathways are altered. In this sense, defective calcium signaling by mutant FGFR3 has been previously described. The purpose of this study was to investigate the presence of purinergic P2Y receptors and how the activation of these receptors can have influence on defective calcium signaling observed in achondroplasic chondrocytes. The presence of P2Y receptors was determined by immunocytochemical and western blot techniques. Calcium mobilization after stimulation with nucleotides, dinucleotides, or, FGF9 application, was measured using the ratiometric dye fura-2/AM and fluorescence imaging. Our results demonstrate the expression of P2Y1, P2Y2, P2Y6 and P2Y11 receptors in achondroplasic chondrocytes, as well as the activation of these receptors after nucleotides and dinucleotides exposure. The altered calcium signaling of achondroplasic chondrocytes was confirmed, since FGF9 treatment fails to induce calcium mobilization. However, achondroplasic chondrocytes pre-treated with Ap4A are able to respond with increases in intracellular calcium after FGF9 stimulation. These findings show the rescue effect of diadenosine tetraphosphate (Ap4A), acting by means of P2Y receptors, on defective calcium response triggered by achondroplasic FGFR3. © 2007 Elsevier Inc. All rights reserved. Keywords: P2Y receptors; Calcium; Achondroplasia; Chondrocytes; FGFR3
Introduction Chondrocytes are important cells in the endochondral bone growth in mammals. Several molecules, hormones, cytokines and growth factors drive the progress of epiphyseal growth plate chondrocytes through proliferative, prehypertrophic, hypertrophic and terminal hypertrophic stages during endochondral ossification. Among all of them, transforming growth factors betas (TGF-βs), bone morphogenetic proteins (BMP), Indian hedgehog (Ihh), insulin-like growth factors, (IGFs), fibroblast growth factors (FGFs), interleukins (ILs) and others, interplay an organised sequence of events that permit the normal development of bones [1,2]. Alteration in any of these substances, or more commonly in ⁎ Corresponding author. Dep. Bioquímica, E.U. Óptica, Universidad Complutense de Madrid, c/Arcos de Jalón s/n, 28037 Madrid, Spain. Fax: +34 91 3946885. E-mail address: [email protected] (J. Pintor). 8756-3282/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2007.10.023
any of their receptors, will produce changes in ossification that will lead to a group of pathologies termed as bone dysplasias. One of these bone syndromes is achondroplasia, the most common type of dwarfism. Achondroplasia is a genetic pathology due to mutation in the gene that encodes the fibroblast growth factor receptor 3 (FGFR3), being the mutation in 97% of the patients a Glycine to Arginine substitution at position 380. FGFR3 is activated after the binding of FGF. This activation induces receptor dimerization and a subsequent receptor autophosphorylation in the intracellular domain. Autophosphorylated tyrosine residues and adjacent amino-acids provide binding sites for different signaling proteins which initiate downstream cascades (STAT, mitogen-activated protein kinase cascade, phosphoinositide-3 kinase pathway), leading to a cellular response. The achondroplasic mutant induces an overstimulation of intracellular signal transduction pathways related to proliferation and hypertrophic differentiation stages of epiphyseal growth plate chondrocyte [3].
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Apart from the sustained signaling induced by the mutant receptor, the defective ability of achondroplasic FGFR3 to mobilize calcium has been also observed, thus suggesting the importance of this cation in the development of this pathology [4]. Achondroplasia does not have any pharmacological treatment. This orphan disease needs the development of strategies involving new molecules suitable for the treatment of this disorder. An interesting group of molecules, which has been scarcely investigated in relation with chondrocytes, are nucleotides. ATP, UTP and diadenosine polyphosphates are extracellular active naturally occurring substances involved in many biochemical and physiological processes. Thus, they take part in neurotransmission, secretion and vasodilation process, and they also mediate long-term events such as cell proliferation, differentiation and death involved in development and regeneration [5]. As a consequence of such a wide physiological involvement, these compounds can be used for the treatment of some pathologies [6]. All these compounds present the ability to activate membrane receptors termed P2 purinoceptors. Metabotropic P2 receptors are termed P2Y receptors and the existence of 8 different subtypes has been described. These receptors are coupled to phospholipase C (PLC) and inositol triphosphate and diacylglicerol generation, although some can be negatively coupled to the adenylate cyclase. On the other hand, P2X are ionotropic receptors involved in fast actions mediated by the nucleotides in different tissues [7,8]. The existence of P2 receptors in normal chondrocytes has already been described [9]. Nevertheless, the presence and the effect of P2 receptors have not been studied, mainly P2Y, in achondroplasic chondrocytes. Interestingly, P2 receptors can transactivate tyrosine kinase receptors, including growth factor receptors like epidermal growth factor receptor or platelet derived growth factor receptor [10]. The aim of this experimental work is to describe the presence of P2Y receptors in mouse achondroplasic chondrocytes and to study the intracellular Ca2+ changes triggered as a consequence of dinucleotide and nucleotide applications. The compounds were tested at various concentrations and some experiments were performed by reducing the extracellular calcium concentration by means of a mixture Ca2+/EGTA in order to know the contribution of an intracellular mechanism on the calcium transients. To clarify the P2Y receptor or receptors involved in the effect of dinucleotides, different P2 antagonists were used. In addition, we analyze whether or not the calcium responses induced by activated P2 receptors can have an influence on altered calcium signal observed in achondroplasic chondrocytes, as well as the intracellular mechanisms involved in this action. Materials and methods Achondroplasic chondrocytes Achondroplasic mouse chondrocytes and normal mouse chondrocytes were kindly provided by ProChon Biotech Ltd. (Israel). Chondrocytes were obtained from the articular cartilage of normal and hFGFR3G380R transgenic mice [11]. Cells were cultured in Dulbecco's Modified supplemented with 15% fetal bovine serum, penicillin and streptomycin. Chondrocytes were maintained at 37°C in atmosphere containing 5% CO2.
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Immunocytochemistry and western blotting For the immunohistochemical study we used achondroplasic chondrocytes glued to coverslips pre-treated with poly-L-Lysine. Covers were treated with 4% p-formaldehyde (w/v) for 15min and washed twice with phosphate-buffered saline (PBS) medium afterwards. Cells were incubated overnight at room temperature in PBS containing 1% bovine serum albumin (BSA) and anti-P2Y primary antibodies (Alomone Labs., Jerusalem, Israel). The dilutions of the primary antibodies were: anti-P2Y1, 1:200; anti-P2Y2, 1:500; anti-P2Y4, 1:500; anti-P2Y6, 1:200 and anti-P2Y11, 1:1000. The covers were washed three times in PBS in the presence of 3% BSA and were incubated for 1h with the secondary antibody which was also diluted (1:500) in PBS/BSA solution. As secondary antibody goat anti-rabbit IgG-TRITC (tetramethylrhodamine isothiocyanate) (40μg/ml) from Sigma (St. Louis, MO, USA) was used. The covers were washed three times with PBS and mounted following standard procedures. Controls were carried out following the same procedures but substituting the primary antibody by the same volume of PBS/BSA solution. Cells were analyzed by confocal microscopy using a Zeiss Axiovert 200M microscope equipped with a LSM 5 Pascal confocal module. Achondroplasic chondrocytes were observed with a Zeiss 63× oil immersion lens, numerical aperture 1.40. TRICT was monitored by exciting at 543nm wavelength. Differential interference contrast (Nomarski, DIC), was performed with the same 63× lens bypassed through the corresponding polarizers and analyzers. All the images were managed by the LSM 5 Pascal software. Western blot analysis was performed by homogenizing the chondrocytes with a buffer lysis that contained Hepes 50mM pH 7.5, Triton 2.5% (w/v), EDTA 10mM, phenylmethylsulphonylfluoride 0.2mM and leupeptin 5μg/ml. After homogenization, proteins were quantified by Bio-Rad protein assay (BioRad laboratories, Hercules, CA, USA). Protein samples (40μg) were separated by SDS-PAGE (10% acrylamide gel). Proteins were transferred to nitrocellulose membranes (Amersham-Pharmacia-Biotech, Buckinghamshire, UK). Following transfer, the membranes were washed with phosphate-buffered saline and blocked for 1h at room temperature with 5% (w/v) skimmed milk powder in PBS. Blots were then incubated overnight at 4°C with primary antibodies in 5% (w/v) skimmed milk powder dissolved in PBS-Tween 20 (0.05% by volume). The dilutions of primary antibodies were as follows: anti-P2Y1, 1:200, antiP2Y2, 1:500; anti-P2Y4, 1:200; anti-P2Y6, 1:200 and anti-P2Y11, 1:1000. The primary antibodies were removed and the blots extensively washed with PBSTween 20. Blots were then incubated for 1h at room temperature with the secondary antibody (anti-rabbit IgG coupled to horseradish peroxidase, from Sigma) at 1:1000 dilution in 5% (w/v) skimmed milk powder dissolved in PBS/ Tween 20. Following removal of the secondary antibody, blots were extensively washed as above and developed using the Enhanced Chemiluminescence detection system (Amersham-Pharmacia-Biotech, Buckinghamshire, UK). Films were scanned and a densitometric analysis was performed using Kodak GL 200 Imaging system and Kodak Molecular Imaging software (Kodak, Rochester, NY, USA).
Calcium measurements Mouse achondroplasic chondrocytes were plated at a density of 2 × 104 cells/cm2 on 15mm diameter glass coverslips 1day before using. Subconfluent cultures were loaded with 5μM fura-2/AM (Molecular Probes, Breda, The Netherlands) for 30min at 37°C in incubation buffer (121mM NaCl, 4.7mM KCl, 5mM NaHCO3, 1.2mM KH2PO4, 1.2mM MgSO4, 2mM CaCl2, 10mM glucose, 10mM HEPES and 0.01% BSA, at pH 7.4 with NaOH). Coverslips with fura-2 loaded cells were transferred into an open flow chamber (1ml incubation buffer) mounted on the heated stage of a TE 200 inverted epifluorescence microscope (Nikon, Düsseldorf, Germany). The wavelength of the incoming light was selected with the aid of a monochromator (12nm bandwidth; Kinetic Imaging Ltd., Liverpool, United Kingdom) to 405nm. This wavelength corresponded to the peak of fluorescence of Ca2+-free fura-2 solutions in our system (peak shifted to a longer wavelength owing to lower transmission of the objective at shorter wavelengths). Twelve-bit images were acquired by a Hamamatsu C-4880-80 multiformat CCD camera controlled by Kalcium PC software (Kinetic Imaging Ltd.). The exposure time was 822ms and the changing time 5ms. The images were acquired continuously and buffered on a fast SCSI disk. After a stabilization period of 10min, image pairs were obtained alternately every 2s, and for a total of 8min, at excitation wavelengths of 340 and
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Fig. 1. Immunolocalization of P2Y receptors in achondroplasic chondrocytes. Cells were grown in cover slips, fixed, subjected to immunostaining with different P2Y receptor antibodies and analyzed by confocal microscopy. Images were taken with a magnification: ×63. Scale bars = 50 μm.
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bisindolymaleimide I, PDbu, pyridoxalphosphate-6-azophenyl-2,4-disulfonic acid (PPADS), suramin, N6-methyl-2′-deoxyadenosine-3′,5′-bisphosphate (MRS2179) and reactive blue-2 (RB-2) were purchased from Sigma Chemical (St. Louis, MO, USA). Other reagents were analytical grade from Merk (Darmstadt, Germany).
Data analysis Five different chondrocyte cultures were used along the present experimental work. Usually, a number of about 50 chondrocytes per field were individually analyzed in their ability to respond to the different compounds. Results are given as mean ± S.E.M. Two-way analysis of variance (ANOVA) test was used to evaluate statistical differences between control and nucleotide effects. Agonist potencies were calculated using a four-parameter logistic equation and the Origin software package (OriginLab Corporation, Northampton, MA, USA). pD2 values were transformed into EC50 values, which represent the concentration of agonist at which 50% of the maximal effect is achieved. When it is appropriated, single experimental traces are represented in the figures; these represent at least six determinations performed with equivalent results.
Results Presence of P2Y receptors in achondroplasic chondrocytes
Fig. 2. P2Y receptors expression in achondroplasic chondrocytes. (A) Cell lysates (40 μg total protein) were analyzed by western blot. A representative blot is shown for each P2Y receptor. (B) The histograms represent the intensity of the band corresponding each P2Y receptor expressed as arbitrary units (sum of the background subtracted pixels within a rectangle encompassing the entire band). 380nm (10nm bandwidth filters), to excite the Ca2+ bound and Ca2+ free forms of this ratiometric dye, respectively. The emission wavelength was 510nm (120-nm bandwidth filter). [Ca2+]i values were calculated on a single cell basis from the 340to 380-nm fluorescence ratios at each time point. In both control and experimental groups, Ca2+ was recorded for 1min before drug application and during 7min. Calcium single dose experiments were performed by applying the agonists at a final concentration of 100μM. For the concentration–response studies, the nucleotides were applied at variable doses ranging from 10− 9M to 10− 3M. For those experiments involving antagonists, they were applied at a final concentration of 100μM 30min before the treatment with the dinucleotide (also at 100μM). For some antagonists such as suramin, 1-[amino-5-(2,7-dichloro-6acetomethoxy-3-oxo-3H-xanthen-9-yl)phenoxy]-2-(2′-amino-5′-methylphenoxy) ethane-N,N,N′,N′-tetraacetic acid pentaacetoxy-methyl ester (FLUO-3AM) (Molecular Probes, Breda, The Netherlands), was used. The measurement conditions were almost identical but the excitation and detection wavelengths were 485nm and 538nm respectively. Free calcium measurements were carried out with a mixture formed of 50μM EGTA and 38μM CaCl2 as described [12,13]. For studies of calcium mobilization induced with fibroblast growth factor 9 (FGF9), normal and achondroplasic chondrocytes were stimulated with 25ng/ml of FGF9 alone or after pre-incubation with 100μM diadenosine tetraphosphate (Ap4A). PLC inhibitors U73122, U73143 were applied at 3μM, 10min before the addition of FGF9 (25ng/ml), since longer pre-incubations clearly produced cell damage. Protein Kinase C (PKC) inhibitor bisindolymaleimide I was preincubated at a concentration of 10μM, 30min before the application of FGF9 (25ng/ml). The PKC activator phorbol 12, 13 dibutyrate (PDbu) were applied at 1μM concentrations 30min before the addition of FGF9 (25ng/ml).
Drugs UTP and ATP were purchased from Amersham Biosciences, Inc. (Piscataway, NJ, USA); UDP, diadenosine triphosphate (Ap3A), diadenosine tetraphosphate (Ap4A), diadenosine pentaphosphate (Ap5A), FGF9, U73122, U73143,
Achondroplasic chondrocytes displayed an extended aspect when observed under the Nomarsky interferential microscopy (Fig. 1). When these cells were incubated with commercial antibodies against P2Y receptors, a moderate immunoreactivity for P2Y1, P2Y2, P2Y6 and P2Y11 was detected, while they showed no labeling for the P2Y4 antibody (Fig. 1). Western blots confirmed the results obtained by means of the immunocytochemical technique. P2Y1, P2Y2, P2Y6 and P2Y11 bands appeared with molecular weights close to those already presented in the literature (Fig. 2A). P2Y11 and P2Y2 receptors
Fig. 3. Intracellular Ca2+ increases in achondroplasic chondrocytes after their challenge with dinucleotides and mononucleotides. Cells grown in cover slips were loaded with fura-2 AM in a buffer containing CaCl2 (2 mM) for 30 min. Cover slips were then mounted in a perfusion chamber, and intracellular Ca2+ levels were analyzed after stimulation with the different compounds using a multiple excitation microfluorescence system. (A) Representative Ca2+ transients elicited by Ap3A, Ap4A and Ap5A, all at 100 μM. (B) Intracellular Ca2+ increases produced by the superfusion of either ATP, UTP, ADP and UDP, all at 100 μM.
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Fig. 5. Effect of the removal of extracellular Ca2+ on the responses elicited by diadenosine polyphosphates and mononucleotides. The removal of the extracellular calcium by means of a mixture of Ca2+/EGTA demonstrates that only a small part of the reordered ion increments are due to the presence of P2X receptors. Values represent the mean± S.E.M. of six independent experiments. ⁎p b 0.1 versus the effect of nucleotide without the removal of the extracellular calcium.
Fig. 4. Concentration–response curves for dinucleotides and mononucleotides. (A) Representative Ca2+ traces of single achondroplasic chondrocytes challenged by graded concentrations of Ap4A. (B) Concentration–response curves for diadenosine polyphosphates and mononucleotides. Graded concentrations starting from 10− 9 M to 10− 3 M were tested. Curves represent the increase in the intracellular Ca2+ concentration after subtracting the basal cytosolic calcium (82 ± 7 nM). Values are the mean ± S.E.M. of four independent experiments.
showed the highest levels of expression, whereas a lower amount of P2Y1 and P2Y6 receptors was found by densitometric analysis (Fig. 2B). Effects of dinucleoside polyphosphates in intracellular calcium in chondrocytes When achondroplasic chondrocytes were challenged by the dinucleoside polyphosphates: diadenosine triphosphate, Ap3A, diadenosine tetraphosphate, Ap4A and diadenosine pentaphosphate, Ap5A (all at 100μM), changes in the cytosolic calcium were observed (Fig. 3A). The increases in calcium varied from 175 ± 12nM in the case of Ap3A to 198 ± 8nM in the case of Ap4A and 188 ± 7nM in the case of Ap5A (n = 6). Mononucleotides such as ATP, UTP, ADP and UDP also induced intracellular calcium increases (Fig. 3B). ATP and UTP provided calcium increases of 300 ± 10nM and 276 ± 9nM respectively (both at 100μM). Lower intracellular calcium changes were produced by the nucleosides diphosphate ADP and UDP,
the values being 105 ± 5nM and 95 ± 8nM for ADP and UDP respectively (n = 6, Fig. 3B). Concentration–response curves for dinucleotides and mononucleotides The behavior of the system at various agonist concentrations was assessed (Fig. 4). Diadenosine tetraphosphate depicted a gradual increase in the calcium concentration values (Fig. 4A). Ap4A and other mono and dinucleotides records were transformed to generate concentration–response curves. At a wide range of concentrations (10− 9M to 10− 3M), ATP provided the highest increment of intracellular calcium, followed by UTP, Ap4A, Ap5A and Ap3A (Fig. 4B, Table 1). The analysis of pD2 values obtained from the concentration–response curves (Table 1), allowed to determine the following potency order: Ap3A ≥ Ap5A = ATP = UTP ≥ Ap4A.
Table 1 Concentration–response values for diadenosine polyphosphates and other mononucleotides in achondroplasic chondrocytes Compound
pD2 (EC50)
Maximal Ca2+ increase (nM)
Ap3A Ap4A Ap5A ATP UTP
− 5.7 ± 0.1 (1.99 μM) − 5.9 ± 0.1 (1.25 μM) − 5.8 ± 0.2 (1.58 μM) − 5.8 ± 0.2 (1.25 μM) − 5.8 ± 0.2 (1.58 μM)
147 ± 17 198 ± 10 181 ± 13 289 ± 21 263 ± 12
Values represent the mean ± S.E.M. of four independent experiments.
Fig. 6. Effect of P2 antagonists on the Ca2+ increases induced by Ap4A. (A) Effect of MRS 2179, suramin, PPADS, and RB-2 (all at 100 μM) on the effect elicited by Ap4A (100 μM). Among all the tested antagonists, PPADS was the most effective antagonizing the effect of Ap4A while RB-2 was almost unable to reverse the effect of the dinucleotide. Values represent the mean ± S.E.M. of six independent experiments. ⁎⁎p b 0.05 vs control; ⁎⁎⁎p b 0.001 vs control.
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The extracellular calcium concentration was reduced by means of a mixture Ca2+/EGTA and experiments were performed under these conditions. As shown in Fig. 5, extracellular Ca2+ removal partially reduced the effect of some of the tested di-and mononucleotides, indicating the presence of ionotropic P2X receptors in achondroplasic chondrocytes. Studies with P2 antagonists The selective P2Y1 antagonist N6-methyl-2′-deoxyadenosine-3′,5′-bisphosphate (MRS2179) and the non-selective P2 antagonists suramin, pyridoxal phosphate 6-azophenyl-2′,4′disulfonate (PPADS) and reactive blue 2 (RB-2) were tested at a concentration of 100μM. The antagonists presented different effects on the Ap4A induced calcium increase in achondroplasic chondrocytes. In particular, PPADS was the most effective in reversing the effect triggered by Ap4A, reducing the calcium signal to 15% of control (Fig. 6). The P2Y1 selective antagonist MRS2179 and suramin reduced Ap4A signal to 38% and 40% of control values respectively (Fig. 6). RB-2 was unable to produce significant change in the cellular response elicited by diadenosine tetraphosphate (all the experiments n = 6).
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Recovery of FGFR3 calcium transients in achondroplasic chondrocytes after treatment with Ap4A The mobilization of calcium in response to stimulation with FGF9, the preferred ligand for FGFR3, was measured in both normal and achondroplasic cells. In accordance with other papers [4], the native FGFR3 induced a calcium increase after FGF9 treatment (Fig. 7A), whereas the achondroplasic FGFR3 was not able to trigger calcium mobilization (Fig. 7C). Interestingly, in achondroplasic chondrocytes, pre-treatment with Ap4A restored the ability of the FGFR3 to mobilize intracellular Ca2+, the achondroplasic cells behaving apparently as normal chondrocytes (Fig. 7D and B). In the absence of extracellular calcium, the FGF9-induced Ca2+ signal after previous Ap4A exposure was detected (Fig. 7E) although the area under the curve was slightly lower than the Ca2+ response observed when extracellular calcium was present (Fig. 7D), suggesting a transmembrane calcium influx contribution to calcium transients. Additional experiments, in which Ap4A was pre-applied for 2 min, removed, and cells were challenged alone with FGF9 30 min after the dinucleotide addition, were carried out.
Fig. 7. Intracellular Ca2+ increases in achondroplasic chondrocytes after their challenge with FGF9 and after previous Ap4A pre-incubation. Cells grown in cover slips were loaded with fura-2 AM in a buffer containing CaCl2 (2 mM) for 30 min. Cover slips were then mounted in a perfusion chamber, and intracellular Ca2+ levels were analyzed after stimulation with the different compounds using a multiple excitation microfluorescence system. Representative Ca2+ transients shown correspond to: Normal chondrocytes (A) or achondroplasic chondrocytes (C) stimulated with 25 ng/ml FGF9. Normal chondrocytes (B) and achondroplasic chondrocytes (D) pretreated with 100 μM Ap4A 2 min before treatment with 25 ng/ml FGF9. Extracellular calcium was removed by means of a mixture of Ca2+/EGTA and achondroplasic chondrocytes were pre-treated with Ap4A 2 min before treatment with 25 ng/ml FGF9 (E). Achondroplasic chondrocytes were pre-treated with Ap4A and after 30 min 25 ng/ml FGF9 was added (F).
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As observed in Fig. 7F, the pre-treatment of Ap4A was enough to allow FGF9 alone to produce a Ca2+ increase similar to that observed when Ap4A and FGF9 are applied consecutively. This result support that the Ap4A action is mediated by a P2Y receptor, rather than by a direct action on the FGFR3 (n = 9). Intracellular mechanisms involved in the Ap4A effect on FGF9-induced calcium mobilization in achondroplasic chondrocytes The P2Yantagonist PPADS blocked the Ap4A action (Fig. 8), confirming that this diadenosine polyphosphate acts by means of P2Y stimulation. On the other hand, treatment with PLC inhibitor U73122 prevented the Ap4A effect on calcium increase whereas its inactive analog U73143 failed to inhibit this effect (Fig. 8). These results suggest that PLC acts as a mediator in the Ap4A effect, this being a common intracellular mechanism in P2Y receptors. Pre-treatment of achondroplasic chondrocytes with the PKC inhibitor Bisindolymaleimide I abolished Ap4A-induced calcium signaling triggered by FGF9 (Fig 8), indicating the involvement of PKC in Ap4A-induced calcium mobilization. The use of phorbol 12, 13 dibutyrate (PDbu) allowed to confirm this involvement. Thus, treatment of achondroplasic chondrocytes with 1 μM of PDbu caused a marked calcium increase when FGF9 was applied, even higher than that induced by Ap4A pre-treatment.
Fig. 8. Intracellular mechanism used by Ap4A to mediate its action on FGF9induced Ca2+ response. Achondroplasic chondrocytes were treated with 25 ng/ml FGF9 alone or after 100 μM Ap4A pre-treatment. The participation of P2Y receptor was verified by means of the use of the antagonist PPADS. This compound was added for 30 min before incubation with Ap4A and subsequent FGF9 stimulation. To elucidate the role of PLC in the Ap4A effect, achondroplasic chondrocytes were pre-treated with PLC inhibitor U73122 (3 μM), or its inactive analog U73143 (3 μM) for 10 min before incubation with Ap4A and subsequent FGF9 stimulation. In order to test the influence of PKC, cells were pre-incubated with the PKC inhibitor Bisindolymaleimide I (10 μM) for 30 min before stimulation with Ap4A and subsequent FGF9 stimulation. Alternatively, to confirm PKC involvement, achondroplasic chondrocytes were treated with PDbu before stimulation with FGF9. The recordered Ca2+ increments are summarized and shown as a bars diagram. Values represent the mean ± S.E.M. of a total of nine experiments performed with three different batches of cells.
Discussion The present experimental work describes the existence of P2Y receptors in achondroplasic chondrocytes as well as their activation and subsequent Ca2+ mobilization after being challenged with diadenosine polyphosphates and other mononucleotides. The influence of activated P2Y receptors on altered calcium signaling showed by achondroplasic FGFR3 in chondrocytes has been also examined. Some of the P2Y receptors identified in achondroplasic chondrocytes are present in normal chondrocytes. For instance, the existence of P2Y1 and P2Y2 receptors has been previously studied by other authors in normal chondrocytes [9,14,15]. Our studies have also contributed to define the presence of P2Y6 and P2Y11 receptors in achondroplasic chondrocytes. In addition, the existence of P2X receptors in achondroplasic chondrocytes has been detected, since the experiments performed in the absence of extracellular Ca2+ showed differences compared to those carried out in the presence of this ion. In this sense, other authors have identified by immunohistochemistry the existence of P2X2 and P2X5 receptors in rat chondrocytes [14]. Additionally, a putative involvement of a calcium-induced calcium release mechanism with capacitative calcium entry, such as a store-operated channel or transient receptor potential channel could be suggested in order to explain the difference of calcium signal induced by nucleotides in absence or in presence of calcium [16]. Diadenosine polyphosphates have not been tested before in their ability to increase intracellular Ca2+ in normal chondrocytes. Our results demonstrate that diadenosine polyphosphates seem to have the same efficacy and pharmacological potency to that exhibited by naturally occurring mononucleotides such as ATP and UTP. The activity of diadenosine polyphosphates in achondroplasic chondrocytes can be carried out by any of the P2Y receptors present in these cells. Since Ap3A, Ap4A and Ap5A can activate different P2Y receptors [17] is difficult to assign the possible receptor selectivity of these dinucleotides. Nevertheless, the use of P2 antagonists may contribute to consign what receptor is activated by the dinucleotides. Thus, the experiments with antagonists revealed that a part of the Ap4A action is due to the activation of a P2Y1 receptor, since its selective inhibitor, MRS2179, was able to reduce about 62% the dinucleotide response. The response was not completely abolished, suggesting the involvement of other P2Y receptors. P2Y2 would be a good candidate to be taken into consideration, since previous experiments with heterologous expressed receptors have determined that Ap4A acts preferentially on P2Y1 and P2Y2 receptors [18,19]. After determining the existence and functionality of P2Y receptors in achondroplasic chondrocytes, the other main objective of this work has been to analyze the possible effect of activated P2Y receptors on the lack of Ca2+ responses in FGF9stimulated achondroplasic chondrocytes. Some reports have described the increase of intracellular calcium levels induced by wild-type FGFR3 in response to FGF exposure [4,20]. Binding of FGF to FGFR3 leads to autophosphorylation of the receptor on several tyrosine residues, being the phosphorylation of Tyr760 essential for interaction and subsequent activation of PLCγ. Then, active PLCγ induces
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release of calcium from intracellular stores. This calcium increase was observed in the normal chondrocytes analyzed after FGF9 treatment, whereas the mutant FGFR3 of achondroplasic chondrocytes was not able to trigger such a calcium mobilization. This result is consistent with previous findings in which altered FGF-induced calcium signals were detected in cell lines with achondroplasia and thanatophoric dysplasia mutations [4]. Since phosphorylated Tyr760 and its flanking sequences constitute the major site of PLCγ association with FGFR3, a possible impediment for this interaction could be responsible for the different behavior shown by mutated FGFR3 in achondroplasic chondrocytes. In fact, a mutant FGFR1 in which Tyr766 (corresponding to Tyr760 in FGFR3) was exchanged for the totally lacked calcium response [21]. Taking into account that phosphorylated Tyr760 is also a binding site for other proteins involved in signal transduction pathways (STAT, MAPK cascade) [22,23], which are stimulated in a prolonged way by mutant FGFR3, the constant binding of these proteins could interfere with PLCγ binding and/or activation. Interestingly, in achondroplasic chondrocytes pre-treatment with Ap4A restored the achondroplasic FGFR3 ability to elicit intracellular Ca2+ increase. Ap4A seems to mediate its action by stimulating presumably a P2Y1 or P2Y2 receptor. Confirming this notion the analysis of downstream effectors that link Ap4A stimulation to FGF9-induced calcium mobilization in achondroplasic chondrocytes revealed that PLC and PKC, the classical second transduction systems coupled to P2Y receptors, act as key mediators of this process. Regarding the role of these effectors, PLC activation could be simply necessary for subsequent PKC stimulation. The activated PKC may phosphorylate the achondroplasic FGFR3 receptor regulating its activity. Alternatively, PKC could modulate the assembly of adaptor signaling complexes involved in prolonged signal transduction pathways, shifting the equilibrium towards the binding of PLCγ to phosphorylated Tyr760. In summary, activation of P2Y receptors, coupled to PLC and PKC, by Ap4A allow the achondroplasic chondrocytes to recover the intracellular calcium signaling, which is critical for different cellular events [20,24]. This finding suggests that activation of P2Y receptors may be a useful pharmacological tool to modify the altered biochemical and physiological patterns that occur in achondroplasia. Acknowledgments We thank PROCHON BIOTECH laboratories for providing us normal and achondroplasic chondrocytes. We wish to thank Dr. Antonio Rodríguez Artalejo for his valuable discussions and suggestions. This work has been supported by research grants from Comunidad de Madrid CAM GR/SAL/057372004, Fundación Magar, Fundación López Hidalgo and SantanderComplutense PR41/06-14962. References [1] Kobayashi T, Kronenberg H. Minireview: transcriptional regulation in development of bone. Endocrinology 2005;146:1012–7.
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[2] Provot S, Schipani E. Molecular mechanisms of endochondral bone development. Biochem Biophys Res Commun 2005;328:658–65. [3] L'Hote CG, Knowles MA. Cell responses to FGFR3 signalling: growth, differentiation and apoptosis. Exp Cell Res 2005;304:417–31. [4] Nguyen HB, Estacion M, Gargus JJ. Mutations causing achondroplasia and thanatophoric dysplasia alter bFGF-induced calcium signals in human diploid fibroblasts. Hum Mol Genet 1997;6:681–8. [5] Burnstock G. Pathophysiology and therapeutic potential of purinergic signaling. Pharmacol Rev 2006;58:58–86. [6] Fischer B., Therapeutic applications of ATP-(P2)-receptors agonists and antagonists. Exp. Opin. Ther. Patents 1999;9:385–399. [7] Boeynaems JM, Communi D, Gonzalez NS, Robaye B. Overview of the P2 receptors. Semin Thromb Hemost 2005;31:139–49. [8] North RA. Molecular physiology of P2X receptors. Physiol Rev 2002;82: 1013–67. [9] Caswell AM, Leong WS, Russell RG. Evidence for the presence of P2-purinoceptors at the surface of human articular chondrocytes in monolayer culture. Biochim Biophys Acta 1991;1074:151–8. [10] Daub H, Weiss FU, Wallasch C, Ullrich A. Role of transactivation of the EGF receptor in signalling by G-protein-coupled receptors. Nature 1996;379:557–60. [11] Segev O, Chumakov I, Nevo Z, Givol D, Madar-Shapiro L, Sheinin Y, et al. Restrained chondrocyte proliferation and maturation with abnormal growth plate vascularization and ossification in human FGFR-3(G380R) transgenic mice. Hum Mol Genet 2000;9:249–58. [12] Carbone E, Swandulla D. Neuronal calcium channels: kinetics, blockade and modulation. Prog Biophys Mol Biol 1989;54:31–58. [13] Verhage M, Besselsen E, Lopes da Silva FH, Ghijsen WE. Ca2+-dependent regulation of presynaptic stimulus-secretion coupling. J Neurochem 1989;53:1188–94. [14] Hoebertz A, Townsend-Nicholson A, Glass R, Burnstock G, Arnett TR. Expression of P2 receptors in bone and cultured bone cells. Bone 2000;27: 503–10. [15] Koolpe M, Pearson D, Benton HP. Expression of both P1 and P2 purine receptor genes by human articular chondrocytes and profile of ligandmediated prostaglandin E2 release. Arthritis Rheum 1999;42:258–67. [16] Ekstein J, Nasatzky E, Boyan BD, Ornoy A, Schwartz Z. Growth-plate chondrocytes respond to 17beta-estradiol with sex-specific increases in IP3 and intracellular calcium ion signalling via a capacitative entry mechanism. Steroids 2005;70:775–86. [17] Pintor J, Miras-Portugal MT. Receptors for diadenosine polyphosphates P2D, P2YApnA, P4 and dinucleotide receptors: are there too many? Trends Pharmacol Sci 2000;21:135. [18] Lazarowski ER, Watt WC, Stutts MJ, Boucher RC, Harden TK. Pharmacological selectivity of the cloned human P2U-purinoceptor: potent activation by diadenosine tetraphosphate. Br J Pharmacol 1995;116: 1619–27. [19] Patel K, Barnes A, Camacho J, Paterson C, Boughtflower R, Cousens D, et al. Activity of diadenosine polyphosphates at P2Y receptors stably expressed in 1321N1 cells. Eur J Pharmacol 2001;430:203–10. [20] Abe M, Naski MC. Regulation of sprouty expression by PLCgamma and calcium-dependent signals. Biochem Biophys Res Commun 2004;323: 1040–7. [21] Peters KG, Marie J, Wilson E, Ives HE, Escobedo J, Del Rosario M, et al. Point mutation of an FGF receptor abolishes phosphatidylinositol turnover and Ca2+ flux but not mitogenesis. Nature 1992;358:678–81. [22] Cross MJ, Lu L, Magnusson P, Nyqvist D, Holmqvist K, Welsh M, et al. The Shb adaptor protein binds to tyrosine 766 in the FGFR-1 and regulates the Ras/MEK/MAPK pathway via FRS2 phosphorylation in endothelial cells. Mol Biol Cell 2002;13:2881–93. [23] Hart KC, Robertson SC, Donoghue DJ. Identification of tyrosine residues in constitutively activated fibroblast growth factor receptor 3 involved in mitogenesis, Stat activation, and phosphatidylinositol 3-kinase activation. Mol Biol Cell 2001;12:931–42. [24] Zuscik MJ, D'Souza M, Ionescu AM, Gunter KK, Gunter TE, O'Keefe RJ, et al. Growth plate chondrocyte maturation is regulated by basal intracellular calcium. Exp Cell Res 2002;276:310–9.