ATP activates DNA synthesis by acting on P2X receptors in human osteoblast-like MG-63 cells

Ei'Ichiro Nakamura1, Yasuhito Uezono2, Ken'Ichiro Narusawa1, Izumi Shibuya3, Yosuke Oishi1, Masahiro Tanaka1, Nobuyuki Yanagihara2, Toshitaka Nakamura1, and Futoshi Izumi2

1 Departments of Orthopedic Surgery and 2 Pharmacology and 3 First Department of Physiology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu 807-8555, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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In human osteoblast-like MG-63 cells, extracellular ATP increased [3H]thymidine incorporation and cell proliferation and synergistically enhanced platelet-derived growth factor- or insulin-like growth factor I-induced [3H]thymidine incorporation. ATP-induced [3H]thymidine incorporation was mimicked by the nonhydrolyzable ATP analogs adenosine 5'-O-(3-thiotriphosphate) and adenosine 5'-adenylylimidodiphosphate and was inhibited by the P2 purinoceptor antagonist suramin, suggesting involvement of P2 purinoceptors. The P2Y receptor agonist UTP and UDP and a P2Y receptor antagonist reactive blue 2 did not affect [3H]thymidine incorporation, whereas the P2X receptor antagonist pyridoxal phosphate-6-azophenyl-2',4-disulfonic acid inhibited ATP-induced [3H]thymidine incorporation, suggesting that ATP-induced DNA synthesis was mediated by P2X receptors. RT-PCR analysis revealed that MG-63 cells expressed P2X4, P2X5, P2X6, and P2X7, but not P2X1, P2X2, and P2X3, receptors. In fura 2-loaded cells, not only ATP, but also UTP, increased intracellular Ca2+ concentration, and inhibitors for several Ca2+-activated protein kinases had no effect on ATP-induced DNA synthesis, suggesting that an increase in intracellular Ca2+ concentration is not indispensable for ATP-induced DNA synthesis. ATP increased mitogen-activated protein kinase activity in a Ca2+-independent manner and synergistically enhanced platelet-derived growth factor- or insulin-like growth factor I-induced kinase activity. Furthermore, the mitogen-activated protein kinase kinase inhibitor PD-98059 totally abolished ATP-induced DNA synthesis. We conclude that ATP increases DNA synthesis and enhances the proliferative effects of growth factors through P2X receptors by activating a mitogen-activated protein kinase pathway.

calcium imaging; cell proliferation; extracellular nucleotide; mitogen-activated protein kinase; osteoblast


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IT IS WELL KNOWN that intracellular ATP plays fundamental roles in nucleic acid synthesis, ion channel modulation, energy metabolism, and enzyme regulation. Accumulating evidence has shown that extracellular ATP also acts as an extracellular signaling molecule mediating cell-cell communication in nonneuronal cells (31, 45) and as a neurotransmitter or neuromodulator in the peripheral and central nervous system (10, 11). Extracellular ATP is known to exert its effects via a family of specific receptors termed P2 purinoceptors. The P2 purinoceptors are divided into the P2X receptors, which are ligand-gated ion channels, and the P2Y receptors, which are G protein-coupled receptors (29). Up to seven P2X subtypes (P2X1-P2X7) (5) and several types of P2Y receptors (P2Y1, P2Y2, P2Y4, and P2Y6) have been cloned, and a wide range of tissue expression of P2X and P2Y receptors has been reported (29).

Fracture healing of bone occurs through a complex sequence of cellular processes, and proliferation and differentiation of osteoblasts have pivotal roles in bone formation (2). Platelet-derived growth factor (PDGF) or insulin-like growth factor I (IGF-I) has been suggested to modulate the healing of bone fractures, in part by promoting proliferation and differentiation of osteoblasts, possibly in an autocrine/paracrine manner (1, 2).

It has been reported that at sites of tissue injury and inflammation, nucleotides, including ATP, are released from damaged cells (3) or from activated platelets or leukocytes (31), and they reach concentrations sufficient to activate purinoceptors (14). Thus ATP is considered to function as an autocrine/paracrine mediator to regulate osteoblast activity in fracture healing and inflammation. Recently, ATP has been shown to be released by mechanical stresses, such as stretch, compression, or shear stress, in a variety of cells, including osteoblasts (16, 19). In bone tissues, much attention has been paid to how mechanical stimuli transduce the signal to the osteoblasts or osteocytes (28). Although extracellular ATP is considered a possible candidate involved in the mechanical responses of the bone (19), it is not certain whether ATP mediates cellular responses and what are the signal transduction pathways of the ATP-induced cellular events in osteoblasts.

P2 purinoceptors are expressed in several species of osteoblasts, and extracellular ATP causes an increase in intracellular Ca2+ concentration ([Ca2+]i) in osteoblasts and osteoblast-like cells (19, 22, 34). In osteoblasts, a recent study has shown that extracellular ATP increased [Ca2+]i as well as DNA synthesis and cell proliferation in MC/3T3-E1 mouse osteoblast-like cells (36). In addition, ATP synergistically potentiated the PDGF-induced DNA synthesis in the cells (36). However, no direct proof was presented regarding the involvement of [Ca2+]i in the effect of ATP. It is not clear how extracellular ATP mediates cell proliferation and which types of ATP receptors are involved in the ATP-induced cellular responses in osteoblasts.

In the present study we focused on the characterization of ATP receptor subtypes and intracellular signal transduction pathways responsible for the ATP-induced proliferative effects in osteoblasts. We also investigated how ATP synergistically activates the growth factor-induced cell proliferation. To this end, we employed human osteoblast-like MG-63 cells, which have been widely used to investigate the signaling pathway of osteoblasts (8, 27). We showed that extracellular ATP causes DNA synthesis via activation of P2X, but not P2Y, receptors in a mitogen-activated protein (MAP) kinase-dependent manner.


    MATERIALS AND METHODS
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Materials. MG-63 human osteosarcoma cells were obtained from the American Type Culture Collection (Rockville, MD). Culture plates or dishes were obtained from Falcon (Meylan Cedex, France). ATP was obtained from Kohjin (Tokyo, Japan); UTP, UDP, adenosine 5'-O-(3-thiotriphosphate) (ATPgamma S), fura 2-AM, and herbimycin A from Calbiochem (La Jolla, CA); 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM from Dojindo (Kumamoto, Japan); alpha beta -mATP and suramin from Research Biochemicals International (Natick, MA); pyridoxal phosphate-6-azophenyl-2',4-disulfonic acid (PPADS) and reactive blue 2 from Tocris Cookson (Bristol, UK); human PDGF-BB from Pepro Tech (Rocky Hill, NJ); human IGF-I from Sumitomo Pharmacy (Tokyo, Japan); H-7 and KN-93 from Seikagaku Kogyo (Tokyo, Japan); staurosporine from Kyowa Hakko (Tokyo, Japan); dibutyryl-cAMP (db-cAMP), myelin basic protein (MBP), and IBMX from Sigma Chemical (St. Louis, MO); MEM from Nissui (Tokyo, Japan); fetal bovine serum (FBS) from Biocell (Rancho Dominguez, CA); [3H]thymidine (5.0 Ci/mmol) and p42/44 MAP kinase assay kit from Amersham Japan (Tokyo, Japan); and activated form of p42/44 MAP kinases from Stratagene (La Jolla, CA). The Abacus cell proliferation kit was obtained from Clontech (Palo Alto, CA). Other chemicals were analytic grade and were obtained from Nacalai Tesque (Kyoto, Japan).

Cell culture. MG-63 cells were cultured at 37°C in MEM (pH 7.2-7.4) containing 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin under a humidified atmosphere of 95% air-5% CO2. The cells were detached by exposure to 0.05% trypsin, washed twice with PBS, and seeded in 24- or 96-well 35- or 60-mm dishes at a density of 1.75 × 104 cells/cm2. Quiescence was induced by incubating cells for 24 h in MEM containing 10% FBS and then for 48 h in MEM containing 0.1% FBS. The medium was changed to serum-free MEM just before experiments (36).

DNA synthesis. Cultured cells were seeded in 24-well plates (3.5 × 104 cells/well, 1.75 × 104 cells/cm2), rendered quiescent, and treated with various test compounds in serum-free MEM for 24 h. [3H]thymidine (37 kBq) was added to the serum-free MEM during the last 2 h of the 24-h incubation. Antagonists were added 1 h before ATP stimulation and maintained throughout the experiment. Cells were washed twice with ice-cold PBS, once with 5% (wt/vol) TCA, and once with ethyl alcohol-diethyl ether (3:1, vol/vol) and then harvested with 0.3 M NaOH. After neutralization with 0.6 M HCl, the suspension was passed through a cellulose acetate filter, and the retained radioactivity was determined with a liquid scintillation spectrometer (model LS7000, Beckman, Fullerton, CA).

Cell proliferation. Cells were seeded at a density of 4.5 × 103/well (1.75 × 104 cells/cm2) in 96-well plates, rendered quiescent, and treated with ATP or ATPgamma S. The cells were incubated in a CO2 incubator for 48 h, and then the number of cells was counted with the Abacus cell proliferation kit. Briefly, cells were assayed for acid phosphatase activity as described by the manufacturer; incubation with the substrate solution was performed for 1 h at 37°C. The nonspecific (basal) acid phosphatase activity was measured as that in serum-free MEM without cells. Absorbance was measured at 405 nm with an automated plate reader (model MAP 9300, Shimadzu, Kyoto, Japan).

Measurement of [Ca2+]i. The method of [Ca2+]i measurements has been described elsewhere (35). MG-63 cells were rendered quiescent and then incubated in HEPES-buffered solution (HBS) containing (in mM) 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose (pH adjusted to 7.4 with NaOH), with the addition of 5 µM fura 2-AM for 1 h. The perfusion fluid was HBS warmed to 37°C, and cells were continuously perfused at a constant flow rate of 1.5 ml/min. Ca2+-free solution was a modified HBS containing 2 mM EGTA without CaCl2 and MgCl2. Fluorescence was measured from fura 2-loaded cells in the perfusion chamber, which had a glass coverslip bottom and was positioned on the stage of an inverted microscope (model TMD-300, Nikon, Tokyo, Japan), with use of a Ca2+-imaging system equipped with an intensified charge-coupled device camera (Quanticell700, JEOL, Tokyo, Japan). Fluorescence intensities at 510 nm with excitation at 340 and 380 nm were recorded at an interval of 5 s. [Ca2+]i in individual cells was calculated from the ratio of fluorescence images measured with excitation at 340 nm to those with excitation at 380 nm by use of the equation of Grynkiewicz et al. (15). Autofluorescence in MG-63 cells was negligible compared with the fluorescence in the fura 2-loaded cells.

RT-PCR of P2X receptor mRNA. Total RNA was extracted from quiescent MG-63 cells (60-mm dish) with a Midi kit (Qiagen, Hilden, Germany), and RT-PCR was performed with a thermal cycler (Perkin-Elmer, Norwalk, CT) with use of an RT-PCR kit (Toyobo, Osaka, Japan). Seven independent forward and reverse primers specific for P2X1-P2X7 receptors were designed on the basis of the cloned human P2X receptors of GenBank (Table 1). The sizes of PCR products expected on the basis of each primer pair are also shown in Table 1. Reverse transcription was performed in a final volume of 20 µl by use of random primers and an RT supplied with the RT-PCR kit. PCR was performed in a final volume of 50 µl containing 1 µM primers, 1 mM each deoxynucleoside triphosphate, 2.5 U of recombinant Taq DNA polymerase, 10 U of RNase inhibitor, and the RT-PCR buffer supplied with the kit. The PCR was performed under the following conditions: 30 cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 90 s. At the end of the PCR, samples were kept at 72°C for 10 min for final extension and stored at 4°C. The amplification products were separated by electrophoresis (2% agarose gel) and visualized by GelStar staining (Takara, Osaka, Japan).

                              
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Table 1.   Individual P2X receptor primers for RT-PCR

cAMP assay. Cellular cAMP in MG-63 cells was measured with a conventional RIA. Briefly, cultured cells (35-mm dish) were rendered quiescent and then washed three times with Krebs-Ringer HEPES (KR-HEPES) buffer containing (in mM) 154 NaCl, 5.6 KCl, 1.1 MgSO4, 2.2 CaCl2, and 25 HEPES (pH adjusted to 7.4 with NaOH) and incubated at 37°C for 10 min with or without ATP, UTP, IGF-I, or PDGF in 1 ml of KR-HEPES buffer containing 0.5 mM IBMX. After reaction, cells were rapidly scraped with 1 ml of 0.1 M HCl, boiled at 95°C for 5 min, and then centrifuged at 10,000 g for 5 min. The supernatants were assayed for cAMP with a 125I-cAMP RIA kit (Yamasa, Chiba, Japan). Protein concentrations were determined according to the method of Bradford (4).

Measurement of MAP kinase assay in cell lysates. Cells seeded at a density of 1.75 × 104 cells/cm2 in 60-mm dishes were rendered quiescent and then incubated with KR-HEPES buffer in the absence or presence of ATP or growth factors. In some experiments, the cells were preincubated with Ca2+-free KR-HEPES buffer supplemented with 2 mM EGTA and 25 µM BAPTA-AM (pH 7.4) without CaCl2 for 15 min and incubated with ATP in the same medium. Cells were then washed, scraped, and lysed with 150 µl of lysis buffer containing (in mM) 50 HEPES (pH 7.4), 5 EGTA, 60 beta -glycerophosphate, 1 sodium orthovanadate, 6 dithiothreitol, 2 CaCl2, 1 phenylmethylsulfonyl fluoride and 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 0.5% Triton X-100, and the cell lysates were centrifuged at 15,000 g for 30 min. For measuring the MAP kinase activity, we employed two kinase assay procedures. First, the kinase activity was determined with a p42/44 MAP kinase assay kit according to the manufacturer's instructions. Second, the MAP kinase activity was measured using an in-gel protein kinase assay with MBP as a substrate for MAP kinase, as described previously (41). Briefly, the lysates (40 µg/lane) were dissolved in Laemmli buffer and separated on SDS-polyacrylamide (10%) gels containing 0.5 mg/ml MBP. Proteins were fixed by the gels with 20% 2-propanol in buffer A [50 mM HEPES (pH 7.4) and 5 mM 2-mercaptoethanol]. SDS was then removed from the gel by washing with buffer A, and the enzyme was denatured with 6 M guanidine and renatured. After renaturation, the gel was incubated in a kinase reaction buffer containing (in mM) 25 HEPES (pH 7.4), 2 2-mercaptoethanol, 0.1 EGTA, and 5 MgCl2 and 50 µCi of [gamma -32P]ATP for 1 h. The gel was subsequently washed several times with the stop solution (5% TCA and 1% sodium pyrophosphate), dried, and then subjected to autoradiography. The MAP kinase activity as determined by the phosphorylation of 42- and 44-kDa bands (p42MAPK and p44MAPK, respectively) was confirmed by eluting an activated purified MAP kinase. The band of p44MAPK was optically measured with the MACBAS system (Fujifilm, Tokyo, Japan).

Statistical analysis. Values are means ± SE. For comparison of two groups, the Mann-Whitney U test was performed, and for comparison between multiple groups, one-way ANOVA followed by Fisher's test was performed. P < 0.05 was considered significant.


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Extracellular ATP increases [3H]thymidine incorporation and cell proliferation in MG-63 cells. Quiescent MG-63 cells were stimulated with ATP. Treatment of the cells with ATP at 100 µM for 24 h increased [3H]thymidine incorporation to 183.2 ± 6.8% of the control value (P < 0.05), and the effect was concentration dependent (EC50 = 4.2 µM; Fig. 1A). We also measured cell number with a cell proliferation assay kit. Treatment of the cells with 100 µM ATP or the hydrolysis-resistant analog of ATP ATPgamma S (100 µM) for 48 h significantly increased the cell number to 113.0 ± 4.4 and 136.0 ± 2.2% of the control value, respectively (Fig. 1B).


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Fig. 1.   Effect of ATP on [3H]thymidine incorporation or cell proliferation. MG-63 cells were rendered quiescent by incubation for 24 h in MEM containing 10% fetal bovine serum and then for 48 h in MEM containing 0.1% fetal bovine serum. The medium was changed to serum-free MEM just before experiments. A: cells were stimulated with ATP for 24 h, and [3H]thymidine (10 µCi/ml) was added for the last 2 h of the 24-h incubation. After reaction, [3H]thymidine incorporation into the cells was measured (n = 12). Values (means ± SE) are expressed as a percentage of values obtained in the absence of ATP (control). * P < 0.05 vs. control. B: cells were stimulated with 100 µM ATP or 100 µM adenosine 5'-O-(3-thiotriphosphate) (ATPgamma S) for 48 h, and the number of cells was counted using the Abacus cell proliferation kit (n = 12). Values (means ± SE) are expressed as a percentage of values obtained in the absence of ATP or ATPgamma S (control = 10,212 ± 298 cells/well). * P < 0.05 vs. control.

ATP synergistically activates polypeptide growth factor-induced [3H]thymidine incorporation. Quiescent cells were stimulated with ATP and/or several polypeptide growth factors. IGF-I or PDGF, each given at 10 ng/ml for 24 h, also increased [3H]thymidine incorporation to 575.9 ± 57.7 or 550.7 ± 22.4% of the control values, respectively (Fig. 2A). When ATP was added with IGF-I or PDGF, the nucleotide caused synergistic increases in [3H]thymidine incorporation to 986.8 ± 197.2 or 763.4 ± 53.4% of the control values, respectively (Fig. 2A). This synergistic action was observed in other growth factors, such as basic fibroblast growth factor, but not hepatocyte growth factor (data not shown). ATP augmented the IGF-I (10 ng/ml)-induced [3H]thymidine incorporation in a concentration-dependent manner (Fig. 2B). The EC50 of ATP with IGF-I (EC50 = 5.0 µM) was essentially similar to the EC50 of ATP.


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Fig. 2.   Synergistic effects of ATP and polypeptide growth factors on [3H]thymidine incorporation. A: quiescent cells were stimulated with 100 µM ATP with or without insulin-like growth factor I (IGF-I, 10 ng/ml) or platelet-derived growth factor (PDGF, 10 ng/ml) for 24 h, and [3H]thymidine incorporation was measured. Values (means ± SE, n = 6) are expressed as a percentage of values obtained in the absence of ATP and growth factors (control, None). * P < 0.05 vs. ATP(-). # P < 0.05 vs. None. B: concentration-response curves for ATP-induced [3H]thymidine incorporation. Cells were stimulated with ATP with or without IGF-I (10 ng/ml) for 24 h. Values (means ± SE, n = 6) are expressed as a percentage of values obtained in the absence of ATP and growth factors (control).

Effects of various purinergic agonists or antagonists on [3H]thymidine incorporation. To determine whether the mitogenic effects of extracellular ATP are mediated by activation of P2 purinoceptors, selective agonists or antagonists for P2 purinoceptors were tested. The hydrolysis-resistant analogs of ATP, ATPgamma S, and AMP-PNP were more effective than ATP when tested at the same concentration (100 µM), suggesting that the effects of ATP were not due to P1 purinoceptor activation by adenosine or other hydrolyzed metabolites of ATP (Fig. 3A). alpha beta -mATP (100 µM), a selective agonist for P2X1 and P2X3 receptors, failed to mimic the ATP response. UTP or its hydrolyzed form UDP (100 µM each), which stimulates a family of P2Y receptors, except P2Y1 receptors, inhibited the [3H]thymidine incorporation by ~30% (Fig. 3A).


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Fig. 3.   Effects of various purinergic agonists (A) or antagonists (B) on [3H]thymidine incorporation. A: quiescent cells were stimulated with various purinergic agonists at 100 µM for 24 h, and [3H]thymidine incorporation was determined. Values (means ± SE, n = 6-12) are expressed as a percentage of values obtained in the absence of each agonist (control). AMP-PNP, adenosine 5'-adenylylimidodiphosphate. * P < 0.05 vs. control. B: quiescent cells preincubated with or without 10 µM suramin, 30 µM pyridoxal phosphate-6-azophenyl-2',4-disulfonic acid (PPADS), or 100 µM reactive blue 2 for 1 h were stimulated with 100 µM ATP for 24 h, and [3H]thymidine incorporation was determined. Antagonists were present throughout ATP stimulation. Values (means ± SE, n = 6-12) are expressed as a percentage of values obtained in the absence of ATP and each antagonist (control, None). * P < 0.05 vs. ATP(-).

Suramin, a nonselective antagonist for P2X and P2Y receptors (18), caused a complete inhibition of the ATP-induced [3H]thymidine incorporation at 10 µM (Fig. 3B). PPADS, an antagonist for the P2X receptor (12), also completely inhibited the ATP-induced [3H]thymidine incorporation. On the other hand, reactive blue 2, an antagonist for P2Y1 receptors (7), failed to inhibit the ATP-induced [3H]thymidine incorporation even at 100 µM, which is reported to inhibit the P2Y1 receptor-mediated cellular responses in other types of cells (44) (Fig. 3B).

RT-PCR analysis of the P2X receptor subtypes in MG-63 cells. RT-PCR analysis showed that MG-63 cells expressed mRNAs for the P2X4, P2X5, P2X6, and P2X7, but not the P2X1, P2X2, and P2X3 receptors (Fig. 4). The bands for P2X4, P2X5, P2X6, and P2X7 receptors were sequenced and found to be identical to the reported human P2X receptors. When we used mRNAs as templates prepared from rat vas deferens, which expresses the P2X1 receptor (45), or rat pheochromocytoma PC-12 cells, which express P2X2 and P2X3 receptors (25), a fragment for P2X1, P2X2, and P2X3 receptors was amplified with the corresponding primer pairs for each rat P2 purinoceptor (data not shown).


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Fig. 4.   RT-PCR analysis of P2X receptor mRNAs in MG-63 cells. To identify the P2X receptor mRNAs, total RNA from MG-63 cells was reverse transcribed (+) or not (-) and amplified by PCR with each primer pair described in Table 1. Primers for glyceraldehyde-3-phosphate dehydrogenase (G3PDH) were used as internal standard and generated a 450-bp fragment. Amplification products were electrophoresed on 2% agarose gel and visualized by GelStar staining. Lane M, DNA marker.

Ca2+ imaging of MG-63 cells stimulated with nucleotides or growth factors. [Ca2+]i responses to extracellular nucleotides and/or growth factors were analyzed in fura 2-loaded MG-63 cells. ATP (100 µM) increased [Ca2+]i in MG-63 cells (Fig. 5A), and the peak increase in [Ca2+]i from the baseline (Delta [Ca2+]i) was 866.0 ± 55.6 nM (n = 65; Fig. 5B). UTP (100 µM) also increased [Ca2+]i (n = 43), with Delta [Ca2+]i of 781.2 ± 73.4 nM. PDGF (10 ng/ml) increased [Ca2+]i, with Delta [Ca2+]i of 561.7 ± 81.8 nM (n = 12). In contrast, IGF-I did not increase [Ca2+]i (n = 45; Fig. 5, A and B), even when IGF-I was applied for a longer period (up to 5 min; data not shown). The selective agonist alpha beta -mATP did not elicit any increase in [Ca2+]i (data not shown). In addition, ATP and UTP increased [Ca2+]i, even when cells were perfused with Ca2+-free buffer, and the amplitude of the [Ca2+]i increase was similar to that obtained with normal, Ca2+-containing buffer (data not shown). ATP did not further increase [Ca2+]i when added with PDGF or IGF-I compared with the increase in [Ca2+]i caused by ATP alone (Fig. 5B).


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Fig. 5.   Effects of various compounds on intracellular Ca2+ concentration ([Ca2+]i) in MG-63 cells loaded with fura 2. A: pseudocolor [Ca2+]i images of MG-63 cells. Cells loaded with 5 µM fura 2 were challenged with ATP (100 µM), UTP (100 µM), IGF-I (10 ng/ml), or PDGF (10 ng/ml) for 2 min. Each image was taken ~30 s after drug application, when the [Ca2+]i response reached a peak. Each panel is 250 × 250 µm. Each pair of top (control) and bottom (stimulated) panels represents the same field. B: summary data for the amplitude of the [Ca2+]i increase from baseline. Values are means ± SE.

Effects of inhibitors for various protein kinases on ATP-induced [3H]thymidine incorporation. The protein kinase C inhibitors H-7 (10 µM) and staurosporine (3 nM) or the Ca2+/calmodulin-dependent protein kinase II inhibitor KN-93 (0.3 µM) had little effect on ATP-induced [3H]thymidine incorporation at concentrations reported to sufficiently inhibit each kinase activity in several types of cells (30) (Fig. 6). On the other hand, herbimycin A (3 µM), an inhibitor for Src-related intracellular protein tyrosine kinases (37), completely inhibited the ATP-induced [3H]thymidine incorporation (Fig. 6).


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Fig. 6.   Effects of inhibitors for various protein kinases on ATP-induced [3H]thymidine incorporation. Quiescent cells were pretreated for 1 h in the presence or absence of H-7 (10 µM), staurosporine (3 nM), KN-93 (0.3 µM), or herbimycin A (3 µM) before stimulation with ATP (100 µM) for 24 h. The inhibitors were present throughout the incubation with ATP, and then [3H]thymidine incorporation was determined. Values (means ± SE, n = 6) are expressed as a percentage of values obtained in the absence of ATP and each inhibitor (control, None). * P < 0.05 vs. ATP(-).

Involvement of the cAMP pathway in the ATP- or growth factor-induced [3H]thymidine incorporation. Increase or decrease in intracellular cAMP levels is known to inhibit or stimulate DNA synthesis, respectively, in a variety of cell types (32). We determined the effects of elevating intracellular cAMP on [3H]thymidine incorporation and also measured cAMP levels in response to ATP, PDGF, or IGF-I. The cAMP-elevating agent forskolin and the membrane-permeable cAMP analog db-cAMP significantly inhibited the control level of [3H]thymidine incorporation by up to 40% (100 ± 7, 160 ± 10, 51 ± 8, and 61 ± 9% for control, 100 µM ATP, 50 µM forskolin, and 1 mM db-cAMP, respectively, n = 6 each). cAMP assay showed that although forskolin elevated cAMP (12.0 ± 1.2 and 280.0 ± 19.7 pmol/well for control and forskolin, respectively, n = 6, P < 0.05), neither ATP and UTP (100 µM each) nor the growth factors (10 ng/ml each) changed the cAMP levels (14.0 ± 2.2, 12.0 ± 1.1, 10.5 ± 1.1, and 12.0 ± 2.1 pmol/well for ATP, UTP, IGF-I, and PDGF, respectively, n = 6 each). ATP, when added with each growth factor, did not alter the cAMP level (11.5 ± 1.1 and 12.1 ± 2.1 pmol/well for IGF-I + ATP and PDGF + ATP, respectively, n = 6 each).

Involvement of a MAP kinase or a phosphatidylinositol-3 kinase pathway in ATP-induced [3H]thymidine incorporation. To examine whether the ATP-induced mitogenic effect is mediated through activation of a MAP kinase or a phosphatidylinositol-3 kinase (PI-3 kinase) pathway, a selective inhibitor for each kinase was employed. PD-98059, a highly selective inhibitor for MAP kinase kinase (9), inhibited ATP-induced [3H]thymidine incorporation in a concentration-dependent manner (Fig. 7A). By contrast, wortmannin, a selective inhibitor for PI-3 kinase (33), did not have significant inhibitory effects on ATP-induced [3H]thymidine incorporation (Fig. 7B).


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Fig. 7.   Effects of PD-98059 or wortmannin on ATP-induced [3H]thymidine incorporation. Quiescent cells were pretreated with or without PD-98059 (A) or wortmannin (B) for 1 h before stimulation with ATP for 24 h. Each inhibitor was present throughout the incubation with ATP, and then [3H]thymidine incorporation was determined. Values (means ± SE, n = 6) are expressed as a percentage of values obtained in the absence of ATP and each inhibitor (control). * P < 0.05 vs. ATP(-).

Effects of ATP and growth factors on MAP kinase activity. ATP (100 µM) increased MAP kinase activity, which reached a peak within 5 min and then slightly decreased at 10 min (Fig. 8A). In-gel protein kinase assay showed that ATP (100 µM), IGF-I (10 ng/ml), or PDGF (10 ng/ml) increased MAP kinase activity, as evidenced by the ability of cell lysates to phosphorylate MBP (Fig. 8B). When ATP was added with the growth factors, the nucleotide caused synergistic increases in MAP kinase activity (Fig. 8B). The ATP-induced phosphorylation of MAP kinase was completely inhibited by PD-98059 (data not shown). ATP also activated MAP kinase activity, even in the Ca2+-free, BAPTA-AM-containing KR-HEPES buffer (Fig. 8B). In fura 2-loaded MG-63 cells treated with the same Ca2+-free buffer, neither ATP nor UTP (each 100 µM) increased [Ca2+]i (n = 26; data not shown).


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Fig. 8.   In vitro (A) and in-gel (B) MAP kinase assay. A: quiescent MG-63 cells were treated with ATP (100 µM). After reaction, the cell lysates were prepared, and MAP kinase activity was measured with a MAP kinase assay kit. Values (means ± SE, n = 6) are expressed as a percentage of the control value. * P < 0.05 vs. control. B: quiescent cells were treated with ATP and growth factors for 5 min, and then cell lysates were prepared. The cell lysates were then subjected to SDS-PAGE including myelin basic protein as a substrate. Autoradiography (bottom) shows ATP- and/or growth factor-induced MAP kinase activity (left) and Ca2+ dependency of the ATP-induced MAP kinase activity (right). Quantification of each band (top) represents the activity of p44MAPK (left, n = 3; right, n = 8). Values (means ± SE) are expressed as a percentage of values obtained in the absence of ATP and growth factors (control, None). * P < 0.05 vs. ATP(-). # P < 0.05 vs. None.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ATP receptors responsible for cell proliferation in MG-63 cells. We have shown that the nonhydrolyzable ATP analogs ATPgamma S and AMP-PNP, as well as ATP, activated DNA synthesis or cell proliferation, and the P2 purinoceptor antagonist suramin abolished the ATP-induced DNA synthesis, suggesting that ATP-induced DNA synthesis was mediated by P2-type, but not P1-type, purinoceptors. We also showed that UTP or UDP did not increase DNA synthesis and that PPADS, but not reactive blue 2, inhibited the ATP-induced DNA synthesis, indicating that P2X, but not P2Y, receptors are involved in the ATP-induced DNA synthesis in MG-63 cells. RT-PCR analysis revealed that mRNAs for receptors of P2X4, P2X5, P2X6, and P2X7, but not those of P2X1, P2X2, and P2X3, were expressed in MG-63 cells. Our results that the selective agonist for P2X1 and P2X3 receptor alpha beta -mATP neither activated the DNA synthesis nor increased [Ca2+]i also support the RT-PCR results. To our knowledge, this is the first evidence demonstrating that P2X receptors are expressed in osteoblasts.

P2X4, P2X6, and P2X7, among the P2X receptor subtypes, are reported to be insensitive to suramin and PPADS in expression studies with each of the cloned receptors (6, 29). Our results showed that suramin and PPADS almost completely inhibited the ATP-induced DNA synthesis, suggesting that P2X4, P2X6, and P2X7 receptors expressed in MG-63 cells may not be involved in the proliferative effects of ATP. From these results, P2X5 receptors could be responsible for the ATP-induced DNA synthesis. P2X5 receptors were expressed in rat heart, and an electrophysiological study with cells heterologously expressed in rat P2X5 receptors showed that the currents induced by ATP were completely inhibited by PPADS and suramin, and the effects of ATP were not mimicked by alpha beta -mATP (13). Furthermore, in rat pituitary gonadotrophs, ATP caused gonadotropin release through P2X2 and/or P2X5 receptors, where the ATP-induced events were inhibited by suramin but not mimicked by UTP or alpha beta -mATP (42). Our data, however, do not exclude the possibility of the involvement of recently reported heterooligomeric P2X receptors composed of different subtypes of P2X receptors [i.e., P2X2/3 (25) and P2X1/5 (23) or other combinations (43)] in ATP-induced cellular responses in MG-63 cells.

Intracellular signaling pathways involved in ATP-induced DNA synthesis. A previous study in osteoblasts showed that ATP increased [3H]thymidine incorporation in parallel with the increment of [Ca2+]i (36). Our results also showed that ATP increased [3H]thymidine incorporation and [Ca2+]i. However, we showed that not only ATP, but also UTP, which did not increase [3H]thymidine incorporation, increased [Ca2+]i with similar amplitudes. In addition, ATP and UTP increased [Ca2+]i, even in a Ca2+-free buffer, suggesting that the ATP-induced increase in [Ca2+]i is due to Ca2+ release from intracellular stores via P2Y receptors. Collectively, our results indicate that an increase in [Ca2+]i is not necessary for the ATP-induced DNA synthesis and that there is a [Ca2+]i increase-independent cell proliferation mechanism in MG-63 cells. The increment of [Ca2+]i via the P2Y receptors observed in our study could be associated with other cellular functions such as cell differentiation.

We further demonstrated that several Ca2+-dependent protein kinases, such as protein kinase C and Ca2+/calmodulin-dependent protein kinase II, are not involved in ATP-induced DNA synthesis. These results support the belief that [Ca2+]i-independent signaling pathways underlie the proliferative effects induced by ATP in MG-63 cells. On the other hand, herbimycin A completely inhibited ATP-induced DNA synthesis, suggesting that non-receptor-associated protein tyrosine kinase pathways are downstream of ATP receptors.

Accumulating evidence has shown that stimulation of tyrosine kinase leads to the activation of several protein kinases such as MAP kinases, which are well known to be involved in cell proliferation (26). Our results showed that ATP increased MAP kinase activity, and the activation of MAP kinase and P2X receptor-mediated DNA synthesis were abolished by PD-98059, indicating that a MAP kinase pathway is involved in the P2X receptor-mediated DNA synthesis, which is inconsistent with the previous study in rat pheochromocytoma PC-12 cells (39). However, in contrast to the previous study in PC-12 cells (39), we demonstrated that ATP increased MAP kinase activity in a Ca2+-independent manner. The exact mechanism of the P2X receptors involved in the activation of the MAP kinase pathway remains to be determined; however, P2X receptors might transduce their signal to MAP kinases in a manner independent of ion influx. In fact, it has been recently reported that ionotropic glutamate receptors transduce the signal to the MAP kinase pathway in a tyrosine kinase Lyn-dependent, but a Ca2+ and Na+ influx-independent, manner (17).

Several growth factors, including PDGF, promote DNA synthesis by activating a PI-3 kinase pathway in MG-63 cells (40); however, we showed that the PI-3 kinase inhibitor wortmannin failed to inhibit the ATP-induced DNA synthesis. Taken together, the ATP-induced cell proliferation may be mediated through several protein kinase pathways, including the tyrosine kinases and the MAP kinases, but not the Ca2+-dependent protein kinases or the PI-3 kinase pathways.

Mechanisms by which ATP causes synergistic effects of DNA synthesis induced by the growth factors. The mechanism by which ATP accelerates the DNA synthesis induced by the growth factors in osteoblasts has not been well clarified. In the osteoblast cell line UMR-106, ATP was shown to augment the parathyroid hormone-induced [Ca2+]i increases, which may lead to modulation of the parathyroid hormone-induced cellular functions (20, 24). These results suggest that enhancement of [Ca2+]i increase results in the synergistic effects of cellular responses. Our results, however, showed that ATP administered with IGF-I or PDGF caused an increase in [Ca2+]i similar to that caused by ATP alone, suggesting that augmentation of the increase in [Ca2+]i does not account for the ATP-induced synergism of the growth factor-induced DNA synthesis in MG-63 cells.

The cAMP signaling pathways are involved in cell proliferation of a variety of the cells (32). Although we showed that an increase in cAMP levels inhibits the DNA synthesis in MG-63 cells, ATP did not change the levels regardless of the presence of the growth factors. These results suggest that cAMP-dependent pathways are not involved in the synergistic proliferative effects of ATP in MG-63 cells.

We showed that ATP augmented the MAP kinase activity induced by PDGF or IGF-I, and we hypothesized that ATP-mediated augmentation of growth factor-induced MAP kinase activity contributes to the synergistic proliferative effects of ATP. Previous reports have shown that, in human erythroid colony-forming cells, stem cell factor synergistically activated both DNA synthesis and MAP kinase activity induced by erythropoietin, and these effects were inhibited by PD-98059 (38). In Swiss 3T3 cells, the polypeptide bombesin synergistically activated the mitogenic effects of insulin by enhancing the p42 MAP kinase activity (21).

Physiological significance of the ATP-induced cellular responses in osteoblasts. ATP and related nucleotides are widely distributed in a variety of tissues, inasmuch as ATP is stored in cytosol or in secretory vesicles in most of the cells (14). A recent study showed that, in rat aortic smooth muscle cells, ATP released by mechanical stretch from the cells activates Jun NH2-terminal kinase/stress-activated protein kinase and MAP kinase pathways via activation of P2Y receptors in an autocrine/paracrine manner (16). On the basis of these results and our present results, it is possible to speculate that extracellular ATP released by mechanical stimulation or from damaged cells in bone fracture may contribute to the bone formation and remodeling.

In summary, we have demonstrated that extracellular ATP increased the DNA synthesis and also enhanced the proliferative effects induced by IGF-I or PDGF through P2X receptors, presumably of the P2X5 subclass, expressed in MG-63 cells and that ATP-induced DNA synthesis was mediated through a MAP kinase-dependent pathway.


    ACKNOWLEDGEMENTS

The authors are grateful to Drs. K. Inoue and I. Matsuoka for helpful comments on the study and to Dr. Tanimoto for help with the assay of MAP kinase.


    FOOTNOTES

This work was supported in part by grants from the Japan Orthopaedics and Traumatology Foundation (to Y. Oishi and Y. Uezono), the Ministry of Education, Science, Sports, and Culture of Japan (to K. Narusawa), and the University of Occupational and Environmental Health School of Medicine (to E. Nakamura).

Address for reprint requests and other correspondence: Y. Uezono, Dept. of Pharmacology, School of Medicine, Miyazaki Medical College, 5200 Kihara, Kiyotake, Miyazaki-gun 889-1692, Japan (E-mail: uezonoy{at}fc.miyazaki-med.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 9 August 1999; accepted in final form 24 February 2000.


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DISCUSSION
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