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• W2014278192 abstract "The regulation of tissue turnover requires the coordinated activity of both local and systemic factors. Nucleotides exist transiently in the extracellular environment, where they serve as ligands to P2 receptors. Here we report that the localized release of these nucleotides can sensitize osteoblasts to the activity of systemic factors. We have investigated the ability of parathyroid hormone (PTH), a principal regulator of bone resorption and formation, to potentiate signals arising from nucleotide stimulation of UMR-106 clonal rat osteoblasts. PTH receptor activation alone did not lead to [Ca2+]i elevation in these cells, indicating no Gq coupling, however, activation of Gq-coupled P2Y1 receptors resulted in characteristic [Ca2+]i release. PTH potentiated this nucleotide-induced Ca2+ release, independently of Ca2+ influx. PTH-(1–31), which activates only Gs, mimicked the actions of PTH-(1–34), whereas PTH-(3–34), which only activates Gq, was unable to potentiate nucleotide-induced [Ca2+]i release. Despite this coupling of the PTHR to Gs, cAMP accumulation or protein kinase A activation did not contribute to the potentiation. 3-Isobutyl-1-methylxanthine, but not forskolin effectively potentiated nucleotide-induced [Ca2+]i release, however, further experiments proved that cyclic monophosphates were not involved in the potentiation mechanism. Costimulation of UMR-106 cells with P2Y1agonists and PTH led to increased levels of cAMP response element-binding protein phosphorylation and a synergistic effect was observed on endogenous c-fos gene expression following costimulation. In fact the calcium responsive Ca/cAMP response element of the c-fos promoter alone was effective at driving this synergistic gene expression. These findings demonstrate that nucleotides can provide a targeted response to systemic factors, such as PTH, and have important implications for PTH-induced signaling in bone. The regulation of tissue turnover requires the coordinated activity of both local and systemic factors. Nucleotides exist transiently in the extracellular environment, where they serve as ligands to P2 receptors. Here we report that the localized release of these nucleotides can sensitize osteoblasts to the activity of systemic factors. We have investigated the ability of parathyroid hormone (PTH), a principal regulator of bone resorption and formation, to potentiate signals arising from nucleotide stimulation of UMR-106 clonal rat osteoblasts. PTH receptor activation alone did not lead to [Ca2+]i elevation in these cells, indicating no Gq coupling, however, activation of Gq-coupled P2Y1 receptors resulted in characteristic [Ca2+]i release. PTH potentiated this nucleotide-induced Ca2+ release, independently of Ca2+ influx. PTH-(1–31), which activates only Gs, mimicked the actions of PTH-(1–34), whereas PTH-(3–34), which only activates Gq, was unable to potentiate nucleotide-induced [Ca2+]i release. Despite this coupling of the PTHR to Gs, cAMP accumulation or protein kinase A activation did not contribute to the potentiation. 3-Isobutyl-1-methylxanthine, but not forskolin effectively potentiated nucleotide-induced [Ca2+]i release, however, further experiments proved that cyclic monophosphates were not involved in the potentiation mechanism. Costimulation of UMR-106 cells with P2Y1agonists and PTH led to increased levels of cAMP response element-binding protein phosphorylation and a synergistic effect was observed on endogenous c-fos gene expression following costimulation. In fact the calcium responsive Ca/cAMP response element of the c-fos promoter alone was effective at driving this synergistic gene expression. These findings demonstrate that nucleotides can provide a targeted response to systemic factors, such as PTH, and have important implications for PTH-induced signaling in bone. intracellular [Ca2+] parathyroid hormone, where not specified PTH = PTH-(1–34) fluorescent imaging plate reader basal salt solution 2-methylthioadenosine 5′-triphosphate 2-methylthioadenosine 5′-diphosphate 3-isobutyl-1-methylxanthine N 2,2′-O-dibutyrylguanosine 3′,5′-cyclic monophosphate cAMP response element-binding protein calcium and cAMP response element Dulbecco's modified Eagle's medium Nucleotides exist in the extracellular microenvironment of bone due to release from damaged cells at sites of injury, or via more controlled nonlytic release (1Bowler W.B. Tattersall J.A. Hussein R. Dixon C.J. Cobbold P.H. Gallagher J.A. Bone. 1998; 22: 3SCrossref Scopus (51) Google Scholar), where they differentially activate cell surface P2 receptors. Activation of Gq-coupled P2Y receptors leads to [Ca2+]i 1mobilization which has ultimately been found to induce processes including proliferation (2Dixon J.C. Bowler W.B. Littlewood-Evans A. Dillon J.P. Bilbe G. Sharpe G.R. Gallagher J.A. Br. J. Pharmacol. 1999; 127: 1680-1686Crossref PubMed Scopus (100) Google Scholar) and differentiation (3Koshiba M. Apasov S. Sverdlov V. Chen P. Erb L. Turner J.T. Weisman G.A. Sitkovsky M.V. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 831-836Crossref PubMed Scopus (77) Google Scholar) and to modulate osteoblast-induced bone formation (4Jones S.J. Gray C. Boyde A. Burnstock G. Bone. 1997; 21: 393-399Crossref PubMed Scopus (52) Google Scholar). The bone matrix synthesizing osteoblasts are thought to express multiple P2Y receptor subtypes (5Reimer W.J. Dixon S.J. Am. J. Physiol. 1992; 263: C1040-C1048Crossref PubMed Google Scholar), with expression profile changing as osteoblasts differentiate (6Dixon J.C. Bowler W.B. Walsh C.A. Gallagher J.A. Br. J. Pharmacol. 1997; 120: 777-780Crossref PubMed Scopus (45) Google Scholar). The mechanisms that determine focal responsiveness to systemic factors in bone to allow a local tissue turnover, characteristic of the remodeling process, remain unclear. However, our previous observations that coapplication of nucleotides and parathyroid hormone (PTH) can result in synergistic responses in osteoblasts (7Bowler W.B. Dixon C.J. Halleux C. Maier R. Bilbe G. Fraser W.D. Gallagher J.A. Hipskind R.A. J. Biol. Chem. 1999; 274: 14315-14324Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar) led us to hypothesize that locally released extracellular nucleotides could sensitize surrounding cells to the action of circulating hormones. Parathyroid hormone is one of the most important systemic regulators of bone and mineral homeostasis. The action of PTH on skeletal cells is complex and can result in the stimulation of both resorption and new bone formation (8Raisz L.G. Nature. 1965; 197: 1015-1017Crossref Scopus (68) Google Scholar, 9Dempster D.W. Cosman F. Parisien M. Shen V. Endocrine Rev. 1993; 14: 690-709Crossref PubMed Scopus (671) Google Scholar). This ability to stimulate coupled, but opposing processes, has been attributed to the nature of the receptor that transduces signals arising from PTH stimulation. The PTH1 receptor belongs to a subgroup of seven transmembrane receptors that include those responsive to calcitonin, secretin, and VIP (10Abou-Samra A.B. Juppner H. Force T. Freeman M.W. Kong X-F. Schipani E. Urena P. Richards J. Bonventre J.V. Potts Jr., J.T. Kronenberg H.M. Segre G.V. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2732-2736Crossref PubMed Scopus (1007) Google Scholar). These receptors are distinct in their ability to couple to both Gs and Gq and hence activate dual signal transduction pathways leading to both cyclic AMP formation and release of Ca2+from intracellular stores ([Ca2+]i). The coupling of the PTH receptor to Gs in osteoblasts is well characterized. Activation of the cAMP signaling pathway is responsible for a number of PTH-induced downstream responses, including expression of the c-fos proto-oncogene (11Tyson D.R. Swarthout J.T. Partridge N.C. Endocrinology. 1999; 140: 1255-1261Crossref PubMed Google Scholar). In addition, the use of truncated PTH fragments in vivo has confirmed that complete N-terminal sequence (vital for Gs activation) is essential to drive the anabolic skeletal effects of PTH (12ArmamentoVillareal R. Ziambaras K. AbbasiJarhomi S.H. Dimarogonas A. Halstead L. Fausto A. Avioli L.V. Civitelli R. J. Bone Min. Res. 1997; 12: 384-392Crossref PubMed Scopus (46) Google Scholar) and cAMP accumulation is often used as a measure of PTH receptor activation (13Du P.F. Seitz P.K. Cooper C.W. Endocrine. 2000; 12: 25-33Crossref PubMed Google Scholar). However, the nature of PTH receptor/Gq-coupling and subsequent downstream responses in osteoblasts following activation remains controversial. Both inositol 1,4,5-trisphosphate-dependent and independent mechanisms for PTH-induced [Ca2+]i release have been reported (14Dunlay R. Hruska K. Am. J. Physiol. 1990; 258: F223-F231PubMed Google Scholar, 15Tong Y. Zull J. Yu L. J. Biol. Chem. 1996; 271: 8183-8191Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). To further complicate the issue of PTH-induced [Ca2+]i release, recent studies in cell types other than osteoblasts have demonstrated that while PTH alone is unable to induce [Ca2+]i release, it can strongly potentiate the [Ca2+]i elevations induced by agonists acting at other Gq-coupled G-protein-coupled receptor. The mechanism behind this potentiation remains unclear but has been attributed to G protein subunit interaction or shuttling of calcium between intracellular stores (16Short A.D. Taylor C.T. J. Biol. Chem. 2000; 275: 1807-1813Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). In a study by Kaplan et al. (17Kaplan A.D. Reimer W.J. Feldman R.D. Dixon S.J. Endocrinology. 1995; 136: 1674-1684Crossref PubMed Google Scholar) using rat UMR-106 cells, PTH alone was shown to produce small [Ca2+]i elevations and also potentiated nucleotide-induced [Ca2+]i elevations. However, in contrast to these findings, we previously demonstrated that PTH alone did not elevate [Ca2+]i and was ineffective at potentiating nucleotide-induced responses in the human osteosarcoma cell line SaOS-2 (7Bowler W.B. Dixon C.J. Halleux C. Maier R. Bilbe G. Fraser W.D. Gallagher J.A. Hipskind R.A. J. Biol. Chem. 1999; 274: 14315-14324Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). In the same study, however, PTH and nucleotide costimulation of SaOS-2 cells did result in synergistic induction of gene expression. Considering the observations described above, the current study has addressed: (i) the controversy concerning the ability of PTH to effect [Ca2+]i release in the rat osteosarcoma cell line UMR-106, (ii) the mechanisms by which PTH can potentiate [Ca2+]i release induced by locally released nucleotides, and (iii) whether this potentiated [Ca2+]i response can ultimately drive downstream cellular responses in osteoblasts. Dulbecco's modified Eagle's medium (DMEM), α-modified Eagle's medium (α-MEM), Ham's F-12, and fetal calf/bovine serum were purchased from Life Technologies (United Kingdom or France). Human parathyroid hormones (PTH) 1–34 and 1–31, and bovine PTH-(3–34) were purchased from Peninsula Laboratories (UK). Nucleotides, bovine serum albumin, fluo-3 AM, potato apyrase, H-89, IBMX, dibutyryl-cGMP, SKF96365, nitrocellulose membranes, and peroxidase-coupled goat anti-rabbit antibodies were obtained from Sigma (UK). Phospho-CREB and CREB specific antisera were obtained from New England Biolabs (UK). Nitrocellulose membranes and enhanced chemiluminescence reagents were acquired from Amersham Pharmacia Biotech (UK) or PerkinElmer Life Sciences (Belgium). Zetabind hybridization membrane was purchased from Cuno (Meriden, CT). Luciferase lysis reagent and luciferase reagent were purchased fromPromega (UK). UMR-106 cells were kindly provided by T. J. Martin, Melbourne, Australia. UMR-106 rat osteoblast-like cells were cultured in α-MEM supplemented with 10% fetal calf serum, 100 μg/ml streptomycin, 100 units/ml penicillin, and 2 mml-glutamine. The cells were maintained at 37 °C in a humidified atmosphere of 95% air and 5% CO2. Confluent cells were either induced for subsequent preparation of whole cell extracts or RNA isolation, or harvested upon confluence for Ca2+ release measurements in the FLIPR system (18Kuntzweiler T.A. Arneric S.P. Donnelly-Roberts D.L. Drug Dev. Res. 1998; 44: 14-20Crossref Scopus (39) Google Scholar). The UMR-106 multi CRE c-fos reporter cell line was cultured in Ham's F-12 and DMEM (1:1) under similar conditions. Cells were grown in 225-cm2 vented cap flasks, harvested upon confluence, and resuspended in basal salt solution (BSS) composition (mm): NaCl (125), KCl (5Reimer W.J. Dixon S.J. Am. J. Physiol. 1992; 263: C1040-C1048Crossref PubMed Google Scholar), MgCl2 (1Bowler W.B. Tattersall J.A. Hussein R. Dixon C.J. Cobbold P.H. Gallagher J.A. Bone. 1998; 22: 3SCrossref Scopus (51) Google Scholar), CaCl2 (1.5), HEPES (25Donahue H.J. Fryer M.J. Erikson E.F. Heath H. J. Biol. Chem. 1988; 263: 13522-13527Abstract Full Text PDF PubMed Google Scholar), glucose (5Reimer W.J. Dixon S.J. Am. J. Physiol. 1992; 263: C1040-C1048Crossref PubMed Google Scholar), and 1 mg/ml bovine serum albumin, pH 7.3. Cells were loaded with 17 μmfluo-3-AM for 1 h at 37 °C with agitation and in the presence of apyrase (2 units/ml), after which cells were washed 3 times in BSS and aliquoted into 96-well black wall, clear base plates at a density of 2.5 × 105 cells/well. These were then centrifuged to obtain a confluent layer of cells on the base of the plate and cells were subsequently washed 4 times in BSS. Ca2+ flux was measured in all 96 wells simultaneously and in real time using a Fluorescent Imaging Plate Reader (FLIPR). When effects of PTH and nucleotides together were studied the cells were incubated for 60 s with PTH prior to exposure to nucleotide. The effects of IBMX and forskolin were assessed by their introduction to the cells 6 min prior to nucleotide addition, H-89 was introduced 10 min before the addition of nucleotides and dibutyryl-cGMP 30 min before nucleotide addition. In all cases [Ca2+]i release was measured for 1 min after nucleotide addition, a measurement of fluorescence being taken every second. Whole cell extracts were prepared as previously described (19Hipskind R.A. Baccarini M. Nordheim A. Mol. Cell. Biol. 1994; 14: 6219-6231Crossref PubMed Scopus (137) Google Scholar). At the appropriate time point, plates were placed on ice and the cell layer quickly washed twice with ice-cold phosphate-buffered saline (140 mm NaCl, 10 mm NaPO4, pH 7.3) containing 10 mmNaF and 100 μm Na3VO4. Cells were solubilized in 10 mm Tris-HCl, pH 7.05, 50 mmNaCl, 30 mm sodium pyrophosphate, 50 mm NaF, 5 mm ZnCl2, 1% (v/v) Triton X-100, 100 μm Na3VO4, 20 mmβ-glycerophosphate, 10 mm 4-nitrophenyl phosphate, 1 mm dithiothreitol, 0.5 mm benzamidine, 2.5 μg/ml aprotinin, leupeptin, pepstatin, 0.2 mmphenylmethylsulfonyl fluoride, and 200 nm okadaic acid. The cell layer was collected by scraping and lysis completed by vortexing for 60 s. The lysate was clarified by centrifugation at 14,000 × g at 4 °C for 15 min and the protein concentration determined with Bradford's reagent using bovine serum albumin as standard. An aliquot for Western analysis was denatured immediately in 2% SDS, 2% glycerol, 50 mm Tris-HCl, pH 6.8, 1% 2-mercaptoethanol and the remaining supernatant stored at −70 °C. 15 μg of whole cell extracts were separated on an 8.5% SDS-polyacrylamide electrophoresis minigel and transferred electrophoretically to a nitrocellulose membrane. The filter was blocked for 1 h at room temperature in 5% (w/v) low fat milk powder-TBST (10 mm Tris-HCl, pH 7.5, 100 mm NaCl, 0.1% Tween 20), followed by incubation overnight at 4 °C with the specific antiserum indicated below in blocking buffer. After washing in TBST, the blots were incubated for 1 h at room temperature with peroxidase-coupled goat anti-rabbit antibody, diluted 1/1500 in 5% (w/v) low fat milk powder-TBST. After washing, the immune complexes were visualized using enhanced chemiluminescence. The antisera used were: anti-CREB and anti-phospho Ser133CREB, diluted 1:1000. Total RNA was extracted from control and stimulated cells according to the protocol of Chomczynski and Sacchi (20Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-161Crossref PubMed Scopus (63232) Google Scholar). Briefly, cells were lysed in 4 m guanidine thiocyanate, 0.5% Sarkosyl, 0.1 m mercaptoethanol, 25 mm sodium citrate, pH 7.0, followed by acid phenol/chloroform extraction. RNA was stored as a precipitate at −70 °C. 10 μg of total RNA was denatured and electrophoresed through a 0.8% (w/v) agarose gel containing 3.7% formaldehyde (v:v), followed by transfer to Zetabind hybridization membrane. Blots were prehybridized at 65 °C in 50% formamide, 5 × SSC, 5 × Denhardt's reagent, 1% SDS, 50 mm sodium phosphate buffer, pH 6.8, 5 mg/ml total calf liver RNA, 200 μg of tRNA, and probed with a c-fosriboprobe spanning exons 3 and 4 of the human c-fos gene mixed with a riboprobe derived from the rat gapdh cDNA as previously described (19Hipskind R.A. Baccarini M. Nordheim A. Mol. Cell. Biol. 1994; 14: 6219-6231Crossref PubMed Scopus (137) Google Scholar). Membranes were washed for 30 min at 65 °C with 0.2 × SSC, 1% SDS solution and mRNAs visualized using phosphorstorage technology and autoradiography with Kodak X-AR film and intensifying screens at −70 °C. RNA loading and integrity were measured by UV shadowing of the filter prior to hybridization. Reporter cells were seeded into white 96-well plates (Dynex) at a density of 96,000 cells/well in 50% DMEM, 50% Ham's F-12 containing 10% fetal calf serum. Cells were incubated for 24 h, after which time medium was replaced by 50% DMEM, 50% Ham's F-12 medium containing 0.1% bovine serum albumin and incubated for a further 24 h. Cells were induced for 4 h with ×10 concentration of agonist stock solution prepared in serum-free medium to the final concentrations indicated in Fig. 9. After induction, cells were washed twice in cold phosphate-buffered saline and lysed in luciferase cell culture lysis reagent for 15 min at room temperature (10 μl/well). A microtiter plate luminometer (ML 3000, Dynex Technologies) was set up in the enhanced flash mode (maximum sensitivity, delay time = 2 s, integration time = 10 s). Luciferase reagent (100 μl) was automatically added and light emission was measured every 10 ms during a period of 10 s. Data was recorded as the peak value of relative light units. Treatments were performed in triplicate and the mean fold increase in luciferase activity was calculated relative to mock-induced cells, which was taken as 1-fold. Since osteoblasts have been described to express numerous subtypes of the P2Y family, and previous studies to receptor profile UMR-106 cells have been incomplete, we used the FLIPR system to fully investigate P2Y receptor expression by these cells. The effects of a series of known P2 agonists on [Ca2+]i elevation were measured in fluo-3-loaded cells. The agonist potency order (p[A]50) was as follows: 2-MeSADP (5.27 ± 0.09) > 2-MeSATP (4.89 ± 0.15) > ADP (4.60 ± 0.15) > ATP (4.57 ± 0.11), suggesting that P2Y1is the predominant P2 receptor expressed by UMR-106 cells (Fig.1). While AMP, αβMeATP, and UDP were inactive at concentrations between 0.1 and 100 μm (data not shown), UTP evoked a small [Ca2+]i elevation (Fig. 1). This profile differs from that suggested by Kaplan et al. (17Kaplan A.D. Reimer W.J. Feldman R.D. Dixon S.J. Endocrinology. 1995; 136: 1674-1684Crossref PubMed Google Scholar) in an earlier study, where predominant functional effects were elicited by P2Y2/Y4 receptor agonists. We have previously reported heterogeneity of P2 receptor expression in populations of primary and clonal osteoblasts (7Bowler W.B. Dixon C.J. Halleux C. Maier R. Bilbe G. Fraser W.D. Gallagher J.A. Hipskind R.A. J. Biol. Chem. 1999; 274: 14315-14324Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), a process that appears to be differentiation-dependent (6Dixon J.C. Bowler W.B. Walsh C.A. Gallagher J.A. Br. J. Pharmacol. 1997; 120: 777-780Crossref PubMed Scopus (45) Google Scholar). Diverse experimental culture methods or passage number may therefore account for the apparent differences in receptor profile between cells in this study and those used in the study of Kaplan et al. (17Kaplan A.D. Reimer W.J. Feldman R.D. Dixon S.J. Endocrinology. 1995; 136: 1674-1684Crossref PubMed Google Scholar). In a previous study using UMR-106 cells PTH potentiated [Ca2+]i elevations induced by certain nucleotide agonists (17Kaplan A.D. Reimer W.J. Feldman R.D. Dixon S.J. Endocrinology. 1995; 136: 1674-1684Crossref PubMed Google Scholar). In one of our earlier studies, however, using the human osteosarcoma cell line SaOS-2, we observed no potentiation of nucleotide-induced [Ca2+]i release following costimulation with PTH (7Bowler W.B. Dixon C.J. Halleux C. Maier R. Bilbe G. Fraser W.D. Gallagher J.A. Hipskind R.A. J. Biol. Chem. 1999; 274: 14315-14324Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). To clarify these differences, the effects of human PTH-(1–34) (100 ng/ml), in combination with known nucleotide agonists, on [Ca2+]i were investigated in UMR-106 cells using the FLIPR. In contrast to the earlier study of Kaplan et al. (17Kaplan A.D. Reimer W.J. Feldman R.D. Dixon S.J. Endocrinology. 1995; 136: 1674-1684Crossref PubMed Google Scholar), we repeatedly saw no elevation of [Ca2+]i in response to PTH (0.1–500 ng/ml, data not shown). We did, however, observe a striking potentiation to the [Ca2+]i response with all nucleotides tested (Fig.2 shows just ADP for clarity). A potentiated calcium response was observed when cells were stimulated with UTP and PTH, therefore confirming that P2Y2 receptors are expressed by these cells, albeit at low levels. To begin to investigate the mechanism behind this observed potentiation we wondered whether calcium influx contributed to this response. Again using the FLIPR we found that in the absence of extracellular Ca2+, ADP and PTH costimulation induced a [Ca2+]i response approximately six times greater than that induced by ADP alone (Fig.3 A). However, this response was depressed in comparison to that resulting from the same experiment performed in the presence of extracellular Ca2+ (Fig.3 A). Interestingly, introduction of the receptor-activated calcium channel blocker SKF96365 resulted in an increase in the observed potentiation between ADP and PTH (Fig. 3 B). Thus calcium influx is not the major component in the potentiation we observe. To further elucidate the mechanisms behind the observed potentiated [Ca2+]i release we used truncated forms of the PTH peptide, which have previously been reported to have selective stimulatory effects upon the G-proteins that couple second messenger signaling pathways to the PTH receptor. It has been reported that stimulation of the PTH receptor by PTH-(1–34) results in activation of both Gq and Gs and therefore utilizes both PKC and adenylyl cyclase to initiate signaling cascades (10Abou-Samra A.B. Juppner H. Force T. Freeman M.W. Kong X-F. Schipani E. Urena P. Richards J. Bonventre J.V. Potts Jr., J.T. Kronenberg H.M. Segre G.V. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2732-2736Crossref PubMed Scopus (1007) Google Scholar). We report here that PTH-(1–31) (100 ng/ml), which stimulates adenylyl cyclase but not PKC (21Jouishomme H. Whitfield J.F. Gagnon L. Maclean S. Isaacs R. Chakravarthy B. Durkin J. Neugebauer W. Willick G. Rixon R.H. J. Bone Min. Res. 1994; 9: 943-949Crossref PubMed Scopus (124) Google Scholar), is able to potentiate ADP-induced Ca2+responses as effectively as the full-length peptide (PTH-(1–34)) (Fig.4 A). In contrast, PTH-(3–34) (100 ng/ml) which does not activate adenylyl cyclase (22Goltzman D. Peytremann A. Callahan E. Tregear G.W. Potts Jr., J.T. J. Biol. Chem. 1975; 250: 3199-3203Abstract Full Text PDF PubMed Google Scholar) was unable to potentiate the [Ca2+]i release induced by ADP (Fig. 4 A). To confirm that this peptide was functionally interacting with the PTH receptor it was introduced in combination with ADP and the active peptides, PTH-(1–34) or -(1–31). PTH-(3–34) was shown to act as a functional antagonist at the PTH receptor as it depressed the [Ca2+]i release induced by ADP in combination with either PTH-(1–34) or PTH-(1–31) (Fig.4 B). As a main downstream target for activated adenylyl cyclase pathways, we next investigated the potential role of PKA in driving potentiated [Ca2+]i release in UMR-106 cells. Preincubation of UMR-106 cells with the PKA inhibitor H-89 (1–100 μm) did not affect potentiated [Ca2+]i release induced by ADP and PTH, indicating that PKA activity is not necessary to initiate the potentiation (Table I). H-89 was inhibitory in these cells, since it blocked PTH-induced phosphorylation of CREB on Ser133, the residue phosphorylated by active PKA (Fig.5).Table IH-89 does not depress ADP/PTH-induced potentiated Ca2+ elevationsAgonist(s)Response of ADP (10−4m) + PTH (100 ng/ml)ADP22%ADP + PTH100%ADP + PTH + H89104%H-89 (1–100 μm) was introduced to fluo-3-loaded UMR-106 cells 10 min before ADP (100 μm) and PTH (100 ng/ml) addition. Ca2+ release was measured according to fluorescence every 30 s for 10 min after H-89 addition, then every second for a further 1 min after ADP/PTH addition. Fluorescence is expressed as a percentage of the response to 100 μm ADP + 100 ng/ml PTH. H-89 had no effect on the potentiation at any concentration tested, figures show effects of 10 μm H-89 (n = 3). Open table in a new tab H-89 (1–100 μm) was introduced to fluo-3-loaded UMR-106 cells 10 min before ADP (100 μm) and PTH (100 ng/ml) addition. Ca2+ release was measured according to fluorescence every 30 s for 10 min after H-89 addition, then every second for a further 1 min after ADP/PTH addition. Fluorescence is expressed as a percentage of the response to 100 μm ADP + 100 ng/ml PTH. H-89 had no effect on the potentiation at any concentration tested, figures show effects of 10 μm H-89 (n = 3). The results above showed that potentiation occurs with a PTH peptide that activates a signaling pathway involving adenylyl cyclase but not PKA. To assess whether elevated cAMP could drive potentiated [Ca2+]i release, cells were costimulated with ADP and the cell permeable adenylyl cyclase activator, forskolin (50 μm), in the absence of extracellular calcium. Forskolin was unable to potentiate ADP-induced calcium responses (TableII). Forskolin alone was shown to elevate intracellular cAMP levels in these cells and to induce CREB phoshorylation via PKA phosphorylation (data not shown). We also tested the ability of the nonspecific phosphodiesterase inhibitor, IBMX (1 mm) to potentiate ADP-induced [Ca2+]i release. In contrast to forskolin, IBMX effectively potentiated ADP-induced [Ca2+]i release (Table II).Table IIIBMX, but not forskolin can potentiate nucleotide-induced [Ca2+]i releaseAgonist(s)Response of ADP (10−4m) + PTH (100 ng/ml)ADP65%ADP + PTH100%ADP + forskolin64%ADP + IBMX96%Forskolin (50 μm), IBMX (1 mm), and PTH (100 ng/ml) were introduced to fluo-3-loaded UMR-106 cells 6 min before ADP addition (ADP concentration range, 0.3 μm to 1 mm). Ca2+ release was measured according to fluorescence every second for 1 min after ADP addition. Fluorescence is expressed as a percentage of the response to 100 μmADP + 100 ng/ml PTH (n = 3). Open table in a new tab Forskolin (50 μm), IBMX (1 mm), and PTH (100 ng/ml) were introduced to fluo-3-loaded UMR-106 cells 6 min before ADP addition (ADP concentration range, 0.3 μm to 1 mm). Ca2+ release was measured according to fluorescence every second for 1 min after ADP addition. Fluorescence is expressed as a percentage of the response to 100 μmADP + 100 ng/ml PTH (n = 3). Since the results obtained with forskolin and IBMX suggested that accumulation of cGMP, downstream of the activated PTH receptor, may be causing the potentiation of ADP-induced [Ca2+]i levels, we costimulated cells with ADP and the cell permeable cGMP analogue, dibutyryl-cGMP (300 μm), in the absence of extracellular calcium. Dibutyryl-cGMP was unable to potentiate ADP-induced [Ca2+]i elevations (Fig.6), eliminating accumulation of this cyclic monophosphate as a possible mechanism of potentiation. Dibutyryl-cGMP alone produced no [Ca2+]i elevation (data not shown). Phosphorylation of CREB on Ser133 is strongly linked to signaling-induced transcriptional activation. Ser133 is a substrate for PKA but also for kinases activated by elevated intracellular calcium. We investigated whether ADP and PTH alone or in combination induced CREB phosphorylation in UMR-106 cells. We performed Western analysis of whole cell extracts using antisera directed against CREB phosphorylated on Ser133. The P2Y1 agonist ADP (100 μm) induced low CREB activation following 15 min stimulation, while PTH-(1–34) (100 ng/ml) gave rise to a robust transcription factor phosphorylation. In combination, however, agonists induced levels of CREB activation greater than those seen with either agonist alone. Quantitative values for CREB and phospho-CREB were obtained by densitometric analysis (Fig.7). To determine the impact of the observed potentiated calcium response on gene expression we measured the effects of P2Y1 agonists and PTH on the levels of endogenous c-fos transcription in UMR-106 cells. This proto-oncogene is of particular relevance when studying osteoblasts as it has been implicated in many of the processes that govern skeletal tissue remodeling (23Grigoriadis A.E. Wang Z-Q. Cecchini M.G. Hofstetter W. Felix R. Fleish H.A. Wagner E.F. Science. 1994; 266: 443-448Crossref PubMed Scopus (1082) Google Scholar). Stimulation of quiescent UMR-106 cells with the P2Y1 agonists ADP and 2-MeSATP (10 μm) for 45 min had a very weak effect on c-fos mRNA expression, as assessed by Northern analysi" @default.
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