P2Y6 Nucleotide Receptors Activate NF-κB and Increase Survival of Osteoclasts
2005; Elsevier BV; Volume: 280; Issue: 17 Linguagem: Inglês
10.1074/jbc.m410764200
ISSN1083-351X
AutoresJasminka Korčok, Lin Raimundo, Xiaobing Du, Stephen M. Sims, S. Jeffrey Dixon,
Tópico(s)Macrophage Migration Inhibitory Factor
ResumoNucleotides, released from cells during inflammation and by mechanical stimulation, act through the P2 family of nucleotide receptors. Previous studies have demonstrated the expression of P2Y1 and P2Y2 receptors in osteoclasts. The aim of this study was to determine whether osteoclast P2Y receptors signal through NF-κB, a key transcription factor regulating osteoclastogenesis. Immunofluorescence was used to detect the p65 subunit of NF-κB, which upon activation translocates from the cytosol to nuclei. Low levels of NF-κB activation were observed in untreated rabbit osteoclasts and in those exposed to 2-methylthio ADP (P2Y1 agonist) or ATP or UTP (P2Y2 agonists). In contrast, UDP or INS48823 (P2Y6 agonists) induced a significant increase in the number of cells exhibiting NF-κB activation, a process sensitive to the proteasome inhibitor lactacystin. In osteoclasts purified by micromanipulation, reverse transcription-PCR revealed the presence of P2Y1, P2Y2, and P2Y6 receptor transcripts, and application of agonists for these receptors induced the transient rise of cytosolic calcium. Treatment of rat osteoclasts with UDP or INS48823, but not 2-methylthio ADP or UTP, increased osteoclast survival. Osteoprotegerin (a decoy receptor for RANK ligand) did not significantly alter the effects of UDP on NF-κB localization or osteoclast survival, consistent with a direct action. Moreover, SN50 (cell-permeable peptide inhibitor of NF-κB) suppressed the enhancement of cell survival induced by UDP and INS48823. Our findings demonstrate the presence of functional P2Y6 receptors in osteoclasts. Thus, nucleotides, following their release at sites of inflammation and mechanical stimulation, can act through P2Y6 receptors to initiate NF-κB signaling and enhance osteoclast survival. Nucleotides, released from cells during inflammation and by mechanical stimulation, act through the P2 family of nucleotide receptors. Previous studies have demonstrated the expression of P2Y1 and P2Y2 receptors in osteoclasts. The aim of this study was to determine whether osteoclast P2Y receptors signal through NF-κB, a key transcription factor regulating osteoclastogenesis. Immunofluorescence was used to detect the p65 subunit of NF-κB, which upon activation translocates from the cytosol to nuclei. Low levels of NF-κB activation were observed in untreated rabbit osteoclasts and in those exposed to 2-methylthio ADP (P2Y1 agonist) or ATP or UTP (P2Y2 agonists). In contrast, UDP or INS48823 (P2Y6 agonists) induced a significant increase in the number of cells exhibiting NF-κB activation, a process sensitive to the proteasome inhibitor lactacystin. In osteoclasts purified by micromanipulation, reverse transcription-PCR revealed the presence of P2Y1, P2Y2, and P2Y6 receptor transcripts, and application of agonists for these receptors induced the transient rise of cytosolic calcium. Treatment of rat osteoclasts with UDP or INS48823, but not 2-methylthio ADP or UTP, increased osteoclast survival. Osteoprotegerin (a decoy receptor for RANK ligand) did not significantly alter the effects of UDP on NF-κB localization or osteoclast survival, consistent with a direct action. Moreover, SN50 (cell-permeable peptide inhibitor of NF-κB) suppressed the enhancement of cell survival induced by UDP and INS48823. Our findings demonstrate the presence of functional P2Y6 receptors in osteoclasts. Thus, nucleotides, following their release at sites of inflammation and mechanical stimulation, can act through P2Y6 receptors to initiate NF-κB signaling and enhance osteoclast survival. Nucleotides, such as ATP and UTP, are signaling molecules that mediate diverse biological effects following their release into the extracellular fluid. In trauma, nucleotides are released from damaged cells and activated platelets (1Born G.V. Kratzer M.A. J. Physiol. 1984; 354: 419-429Crossref PubMed Scopus (215) Google Scholar). ATP is also released from nerve terminals in the peripheral and central nervous systems (2Burnstock G. Br. J. Anaesth. 2000; 84: 476-488Abstract Full Text PDF PubMed Scopus (324) Google Scholar, 3Edwards F.A. Gibb A.J. Colquhoun D. Nature. 1992; 359: 144-147Crossref PubMed Scopus (728) Google Scholar). Mechanical stimulation or fluid shear stress induces the release of nucleotides including ATP and UTP in a variety of non-neuronal cells (4You J. Jacobs C.R. Steinberg T.H. Donahue H.J. J. Biol. Chem. 2002; 277: 48724-48729Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 5Grygorczyk R. Hanrahan J.W. Am. J. Physiol. 1997; 272: C1058-C1066Crossref PubMed Google Scholar, 6Lazarowski E.R. Harden T.K. Br. J. Pharmacol. 1999; 127: 1272-1278Crossref PubMed Scopus (130) Google Scholar). In the extracellular environment, ATP and UTP are degraded by a family of enzymes collectively called ecto-nucleotidases (7Zimmermann H. Braun N. Kegel B. Heine P. Neurochem. Int. 1998; 32: 421-425Crossref PubMed Scopus (129) Google Scholar). ATP is degraded to ADP, AMP, and adenosine, whereas UTP is degraded to UDP, UMP, and uridine. ATP, UTP, as well as some of their degradation products are biologically active. Nucleotides act through the P2 family of cell surface receptors. P2Y receptors are G protein-coupled receptors that, in many cases, signal through the release of Ca2+ from intracellular stores, whereas P2X receptors are ligand-gated cation channels (8Ralevic V. Burnstock G. Pharmacol. Rev. 1998; 50: 413-492PubMed Google Scholar). To date, seven mammalian P2X receptors, P2X1–7, and at least eight P2Y receptors, P2Y1, 2, 4, 6, and 11–14, have been identified. P2X receptors are activated by adenine nucleotides, whereas P2Y receptors are activated by adenine and/or uridine nucleotides. P2Y1 receptors are activated selectively by ADP (9Jacobson K.A. Jarvis M.F. Williams M. J. Med. Chem. 2002; 45: 4057-4093Crossref PubMed Scopus (320) Google Scholar). The P2Y2 receptor is activated by ATP and UTP with equal potency, and the human P2Y4 receptor is activated selectively by UTP, whereas at the rat P2Y4 receptor ATP and UTP are equipotent. The P2Y6 receptor is activated selectively by UDP (8Ralevic V. Burnstock G. Pharmacol. Rev. 1998; 50: 413-492PubMed Google Scholar). Osteoclasts are multinucleated cells with the unique ability to resorb bone. Together with bone forming osteoblasts, they regulate bone mass. A variety of endocrine and paracrine factors control osteoclast formation and life span. Until recently, it was believed that the major determinants of bone resorption were osteoclast proliferation and activation. However, growing evidence has led to the proposal that the control of the osteoclast life span is critical for the regulation of bone resorption (10Parfitt A.M. Mundy G.R. Roodman G.D. Hughes D.E. Boyce B.F. J. Bone Miner. Res. 1996; 11: 150-159Crossref PubMed Scopus (292) Google Scholar, 11Boyce B.F. Hughes D.E. Wright K.R. Xing L. Dai A. Lab. Investig. 1999; 79: 83-94PubMed Google Scholar). Receptor activator of nuclear factor κB (RANK) 1The abbreviations used are: RANK, receptor activator of NF-κB; IκB, inhibitor of NF-κB; 2MeSADP, 2-methylthio ADP; NF-κB, nuclear factor κB; OPG, osteoprotegerin; PLC, phospholipase C; INS48823, P1-((2-benzyl-1,3-dioxolo-4-yl)uridine 5′) P3-(uridine 5′-) triphosphate; PBS, phosphate-buffered saline; ALP, alkaline phosphatase. ligand, a protein expressed by osteoblasts and activated T cells, acts directly on osteoclasts to stimulate their formation and activity (12Lacey D.L. Timms E. Tan H.L. Kelley M.J. Dunstan C.R. Burgess T. Elliott R. Colombero A. Elliott G. Scully S. Hsu H. Sullivan J. Hawkins N. Davy E. Capparelli C. Eli A. Qian Y.X. Kaufman S. Sarosi I. Shalhoub V. Senaldi G. Guo J. Delaney J. Boyle W.J. Cell. 1998; 93: 165-176Abstract Full Text Full Text PDF PubMed Scopus (4656) Google Scholar). RANK ligand signals by binding to its receptor RANK, a member of the tumor necrosis factor receptor superfamily. The soluble, decoy receptor osteoprotegerin (OPG) also binds RANK ligand, preventing signaling. Multiple downstream transcription factors are activated during RANK signaling, including activator protein 1 and NF-κB (13Boyle W.J. Simonet W.S. Lacey D.L. Nature. 2003; 423: 337-342Crossref PubMed Scopus (4988) Google Scholar). Inactive NF-κB resides in the cytoplasm bound to the inhibitory protein IκB. Phosphorylation of IκB leads to its ubiquitination and degradation by the proteasome. IκB degradation permits NF-κB to translocate to the nucleus where it regulates the expression of a variety of genes involved in inflammation and immunity, cell proliferation, responses to stress, and apoptosis (14Karin M. Lin A. Nat. Immunol. 2002; 3: 221-227Crossref PubMed Scopus (2470) Google Scholar). The essential role of NF-κB in osteoclast formation was demonstrated in mice deficient for both the p50 and p52 subunits of NF-κB (15Iotsova V. Caamano J. Loy J. Yang Y. Lewin A. Bravo R. Nat. Med. 1997; 3: 1285-1289Crossref PubMed Scopus (890) Google Scholar). These mice showed a pronounced osteopetrotic phenotype as a result of the absence of osteoclasts. Previous studies have shown that low concentrations of ATP stimulate the resorptive activity and formation of rodent osteoclasts (16Morrison M.S. Turin L. King B.F. Burnstock G. Arnett T.R. J. Physiol. 1998; 511: 495-500Crossref PubMed Scopus (124) Google Scholar). This effect was attributed to the P2Y1 receptor, because ADP and a selective P2Y1 agonist, 2MeSADP, stimulated osteoclast formation and resorption (17Hoebertz A. Meghji S. Burnstock G. Arnett T.R. FASEB J. 2001; 15: 1139-1148Crossref PubMed Scopus (88) Google Scholar). Signaling through many P2Y receptors involves activation of phospholipase C (PLC) leading to the formation of inositol 1,4,5-trisphosphate and release of Ca2+ from intracellular stores (8Ralevic V. Burnstock G. Pharmacol. Rev. 1998; 50: 413-492PubMed Google Scholar). In osteoclasts, several P2Y receptor agonists were shown to induce elevation of cytosolic free calcium concentration ([Ca2+]i) through this pathway (18Weidema A.F. Dixon S.J. Sims S.M. Am. J. Physiol. 2001; 280: C1531-C1539Crossref PubMed Google Scholar). In addition, immunocytochemistry and electrophysiological studies have shown the presence of P2Y2, P2X4, and P2X7 receptors in osteoclasts (19Hoebertz A. Townsend-Nicholson A. Glass R. Burnstock G. Arnett T.R. Bone. 2000; 27: 503-510Crossref PubMed Scopus (130) Google Scholar, 20Naemsch L.N. Weidema A.F. Sims S.M. Underhill T.M. Dixon S.J. J. Cell Sci. 1999; 112: 4425-4435Crossref PubMed Google Scholar, 21Naemsch L.N. Dixon S.J. Sims S.M. J. Biol. Chem. 2001; 276: 39107-39114Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Our previous work demonstrated that P2X7 receptors couple to NF-κB signaling in osteoclasts (22Korcok J. Raimundo L.N. Ke H.Z. Sims S.M. Dixon S.J. J. Bone Miner. Res. 2004; 19: 642-651Crossref PubMed Scopus (124) Google Scholar); however, it was unknown whether P2Y receptors activate NF-κB as well. It is also unknown whether other P2Y receptors in addition to P2Y1 and P2Y2 are expressed in authentic osteoclasts. The aim of this study was to examine which P2Y receptors are expressed in osteoclasts and to determine whether they signal through NF-κB. Our results show that in addition to previously demonstrated P2Y1 and P2Y2 receptors, osteoclasts express functional P2Y6 receptors. We also establish that P2Y6 receptors couple to activation of NF-κB and promote osteoclast survival. Materials—Medium 199 (containing 25 mm HEPES and 26 mm HCO3−), HCO3−-free Medium 199 (25 mm HEPES), heat-inactivated fetal bovine serum, and antibiotic solution (penicillin 10,000 units/ml; streptomycin 10,000 μg/ml; amphotericin B 25 μg/ml) were from Invitrogen. Nucleotides were purchased from Sigma. Mouse monoclonal antibody against the p65 subunit of NF-κB was from Santa Cruz Biotechnology (Santa Cruz, CA; catalog number sc-8008). Mounting medium (Vecta-Shield) and biotinylated goat anti-mouse IgG were from Vector Laboratories (Burlingame, CA). Fluorescein-conjugated streptavidin and fluorescent probes TOTO-3 and fura-2-AM were from Molecular Probes (Eugene, OR). The P2Y6-selective agonist INS48823 was a generous gift from Inspire Pharmaceuticals (Durham, NC). The cell-permeable peptide inhibitor of NF-κB, SN50, the inactive control peptide, SN50M, and the irreversible proteasome inhibitor, lactacystin, were from Calbiochem (La Jolla, CA). RANK ligand (recombinant protein consisting of human residues 151–316 fused at the N terminus to a linker peptide and a FLAG tag) was purchased from Alexis Corp. (San Diego, CA), and OPG (recombinant protein consisting of human residues 21–194) was from Research Diagnostics Inc. (Flanders, NJ). Osteoclast Isolation and Culture—Osteoclasts were isolated from the long bones of neonatal New Zealand White rabbits and Wistar rats according to previously described procedures (21Naemsch L.N. Dixon S.J. Sims S.M. J. Biol. Chem. 2001; 276: 39107-39114Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Rabbit and rat long bones were dissected free of soft tissues and cut with a scalpel to release bone fragments into 2–3 ml of osteoclast culture medium, which consisted of Medium 199 buffered with HEPES and HCO–3 supplemented with 15% fetal bovine serum and 1% antibiotic solution. The cells were suspended by repeated passage through a pipette and plated on glass coverslips. The rabbit bone cell preparations were maintained at 37 °C, 5% CO2 for 2 h after isolation, and then fresh culture medium was added. The cells were incubated at 37 °C, 5% CO2 for 2–7 days before use. The majority of nonosteoclastic cells were removed using Pronase E (0.001% in PBS with 0.5 mm EDTA) at room temperature (22–25 °C) with intermittent agitation. When the majority of nonosteoclastic cells had been removed (1–5 min), the Pronase E solution was replaced with fresh medium. Samples partially purified with Pronase E were used for immunofluorescence studies and calcium measurements. To obtain homogeneous populations of osteoclasts for RNA isolation, some samples were further purified as follows. The bottom and edge of coverslips were cleaned with cotton swabs to remove adherent cells and were then placed in medium. Osteoclasts were identified by phase contrast microscopy as having three or more nuclei. Cells with fewer than three nuclei were removed using a micromanipulator and janitor pipette (20Naemsch L.N. Weidema A.F. Sims S.M. Underhill T.M. Dixon S.J. J. Cell Sci. 1999; 112: 4425-4435Crossref PubMed Google Scholar), leaving pure preparations of 20–80 adherent multinucleated osteoclasts/coverslip. Following isolation, rat osteoclasts were incubated at 37 °C in 5% CO2 for 1 h, then gently washed with PBS to remove nonadherent cells, and incubated in fresh culture medium for 1–3 h before use. To quantify survival, the numbers of rat osteoclasts on coverslips were determined using a phase contrast microscope at time 0 (time of addition of test substances) and at 18 h. Osteoclasts were identified as having three or more nuclei. The number of surviving osteoclasts was expressed as a percentage of the number of osteoclasts at time 0 (100% = 98 ± 3 osteoclasts/coverslip, mean ± S.E., n = 278). These procedures were approved by the Council on Animal Care of the University of Western Ontario. Determination of NF-κB Localization by Immunofluorescence—Following 5–6 days in culture, rabbit osteoclasts on glass coverslips were incubated with test agents in osteoclast culture medium at 37 °C and, at the indicated times, washed in PBS, fixed with 4% paraformaldehyde (10 min), washed in PBS (2 × 10 min), permeabilized with 0.1% Triton X-100 in PBS (10 min), washed in PBS (2 × 5 min), and blocked with 1% normal goat serum in PBS (blocking solution) for 1–2 h at room temperature. Osteoclasts were incubated overnight at 4 °C with primary antibody to the p65 subunit of NF-κB (diluted 1:100 in blocking solution). The coverslips were then washed and incubated for 2 h at room temperature with biotinylated secondary antibody diluted 1:100 in blocking solution, followed by washing and incubation with fluorescein-conjugated streptavidin (1:100 in PBS) for 30 min at room temperature. The nuclei were stained with TOTO-3 (2 μm), the coverslips were washed and mounted on slides, and the cells were observed using a Zeiss LSM 510 laser-scanning confocal microscope. All of the osteoclasts on each coverslip were examined (usually 150–200 osteoclasts/coverslip). The osteoclasts were rated positive for nuclear localization of NF-κB if the fluorescence intensity of three or more nuclei exceeded that of the cytoplasm. Reverse Transcription-PCR—Partial nucleotide sequences for rabbit P2Y receptors and ALP were obtained by PCR amplification of genomic DNA from rabbit brain using primers based on sequences previously published for rats and humans. These primer sequences were: P2Y1, sense 5′-GGCTTCCAGTTCTACTACCT-3′ and antisense 5′-GAGACTTGCTAGACCTCTTG-3′ (NM012800); P2Y2, sense 5′-ACACTAACCTCTACTGCAGCATCC-3′ and antisense 5′-TGACACCTGACTGAGCTGCTAT-3′ (NM017255); P2Y6, sense 5′-CATCCTCTTCCTCACCTGCATC-3′ and antisense 5′-CAGGAAGCTGATGGCAAAGGCA-3′ (NM176798); P2Y11, sense 5′-CACCTGCATCAGCCTCAACC-3′ and antisense 5′-CAGTGCTCTTGGCGTCCTCT-3′ (AF030335); and alkaline phosphatase (ALP), sense 5′-ATGTCTGGAACCGCACTGAA-3′ and antisense 5′-TCACCGTCCACCACCTTGTA-3′ (NM007431). For P2Y1, P2Y6 receptors, and ALP, the outer primers were the same as those used for PCR amplification of genomic DNA. For P2Y2 and P2Y11 receptors, rabbit sequences were used to design outer primers. The nested primers were also designed based on rabbit sequences (Table I). ALP was used as a control for osteoclast purity.Table IOligonucleotide primers used to detect P2Y receptors and alkaline phosphatase (ALP) by reverse transcription-PCRPrimerSenseAnti-senseSizebpP2Y1 outerGGCTTCCAGTTCTACTACCTGAGACTTGCTAGACCTCTTG804P2Y1 nestedCCATCTGGATGTTCGTCTTCTCTGAGGTGGTGTCATAGCA409P2Y2 outerTGTCTGAACGTCGTGGCGCTCAGACCAGGATGACGGCGAA506P2Y2 nestedTTCCTGTGCCGCCTCAAGACCATGACGGAGCTGTAGGCCA435P2Y6 outerCATCCTCTTCCTCACCTGCATCCAGGAAGCTGATGGCAAAGGCA421P2Y6 nestedCACCTGCATCAGCTTCCAGCGTCAGCAGGAAGCCAATGACCGTG259P2Y11 outerACTCGCCTTCTCCCGCCTCATTCTCTGTGCGTGACATGCTGG237P2Y11 nestedTGCAACAGGACCCAGCCCGAGTAGGCCAGCAGGGTGAGCA141ALP outerATGTCTGGAACCGCACTGAATCACCGTCCACCACCTTGTA478ALP nestedCCTTCGCTCTCCGAGATGGTTTCTTGTCCGTGTCGCT305 Open table in a new tab Total RNA was isolated from coverslips containing purified rabbit osteoclasts or bone cells (containing stromal cells and cells of both osteoblast and osteoclast lineages) using the RNeasy mini kit (Qiagen). Genomic DNA was removed by incubation with 0.05–0.1 unit/ml DNase I (amplification grade; Invitrogen) at 25 °C for 15 min followed by heat inactivation at 65 °C for 10 min. Typically, the RNA from a single coverslip was divided into two equal fractions. One fraction was not subjected to reverse transcription to detect genomic DNA contamination, and the second fraction was reverse transcribed into single-stranded cDNA using Superscript II (Invitrogen) according to the manufacturer's instructions. A portion (20–25%) of the resulting cDNA (or control sample that did not undergo reverse transcription) was used as template in each PCR performed with AdvanTaq Plus polymerase (Clontech Laboratories, Palo Alto, CA). Each 25-μl reaction contained 1 × AdvanTaq Plus PCR buffer, 0.2 mm of each dNTP, 0.2 mm primer, and 10% dimethyl sulfoxide (no dimethyl sulfoxide was used for PCR of P2Y1). PCR was performed with a hot start at 95 °C for 2 min, followed by 35–36 cycles at 94 °C for 30 s, 64 °C for 30 s, and 68 °C for 1 min, with a final extension at 68 °C for 3 min. PCR using nested primers was carried out on first round PCR products using the protocol described above. PCR products were separated on a 1% agarose gel containing 0.5 μg/ml ethidium bromide and detected under UV illumination. Identities of amplified products were verified by sequencing of selected samples. Fluorescence Measurement of Cytosolic Free Ca2+Concentration— [Ca2+]i of single osteoclasts loaded with fura-2 was monitored using microfluorimetric techniques. Cells on glass coverslips were incubated for 40 min at room temperature in HCO–3-free osteoclast culture medium containing 1.5 μm fura-2-AM. The coverslips were then placed in a chamber mounted on the stage of a Nikon Diaphot inverted phase contrast microscope and bathed in HCO–3-free osteoclast culture medium supplemented with 15% fetal bovine serum and 1% antibiotic solution. The ratio of fluorescence emission at 510 nm with alternate excitation wavelengths of 345 and 380 nm was measured using a Deltascan illumination system (Photon Technology International, London, Canada) as described previously (18Weidema A.F. Dixon S.J. Sims S.M. Am. J. Physiol. 2001; 280: C1531-C1539Crossref PubMed Google Scholar). Test substances were dissolved in the HCO–3-free osteoclast culture medium and applied locally to cells by pressure ejection from a micropipette. Statistical Analyses—Data are expressed as means ± S.E. The sample size (n) indicates the number of osteoclasts for Ca2+ fluorescence determinations or the number of separate cell preparations for immunofluorescence and survival studies. Unless otherwise indicated, the data were analyzed by one-way analysis of variance followed by a Bonferroni's post test. The differences were accepted as statistically significant at p < 0.05. UDP Induces NF-κB Translocation in Rabbit Osteoclasts— Activation of NF-κB results in its translocation from the cytoplasm to the nuclei. Our previous work demonstrated that immunofluorescence is an effective tool for examining NF-κB activation in rabbit osteoclasts (22Korcok J. Raimundo L.N. Ke H.Z. Sims S.M. Dixon S.J. J. Bone Miner. Res. 2004; 19: 642-651Crossref PubMed Scopus (124) Google Scholar). To determine whether P2Y receptors activate NF-κB, rabbit osteoclasts were exposed to various P2Y receptor agonists. UDP (10 μm), an agonist at P2Y6 receptors, increased the proportion of osteoclasts displaying nuclear localization of NF-κB with a maximal effect at 3 h of exposure (Fig. 1, A and D). Control osteoclasts showed low levels of nuclear NF-κB for up to 4 h (Fig. 1B). The extent of NF-κB translocation was dependent on UDP concentration, with maximal effects observed at 10–100 μm UDP (Fig. 1E). In contrast to its action on osteoclasts, UDP did not induce NF-κB translocation in rabbit bone marrow stromal cells. After 3 h of exposure, 15 ± 7% of vehicle-treated and 16 ± 8% of UDP-treated stromal cells (Fig. 1C) showed nuclear localization of NF-κB (n = 3, p > 0.05). Thus, UDP induces activation of NF-κB in rabbit osteoclasts but not bone marrow stromal cells. To examine whether NF-κB activation occurs through P2Y receptors other than P2Y6, osteoclasts were exposed to 2MeSADP (P2Y1 receptor agonist) or UTP (P2Y2 receptor agonist) for 3 h, and the effect was compared with that of UDP (Fig. 2). In these experiments, osteoclasts exposed to 2MeSADP (10 μm) or UTP (10 μm) showed low levels of nuclear localization of NF-κB. Similarly, our previous results showed predominantly cytoplasmic localization of NF-κB in osteoclasts exposed to a low concentration of ATP (10 μm, a concentration sufficient to activate multiple P2 receptors including P2Y2 and P2X4 but not P2X7) (22Korcok J. Raimundo L.N. Ke H.Z. Sims S.M. Dixon S.J. J. Bone Miner. Res. 2004; 19: 642-651Crossref PubMed Scopus (124) Google Scholar). In contrast, treatment with UDP (10 μm) significantly increased the percentage of osteoclasts with nuclear localization of NF-κB (Fig. 2, p < 0.05). Therefore, the P2Y6 receptor agonist UDP, but not agonists at other P2Y receptors, activates NF-κB in rabbit osteoclasts. The canonical pathway for NF-κB activation involves proteasome-mediated IκB degradation. To investigate the mechanism of UDP-induced NF-κB activation, we examined the effects of the specific proteasome inhibitor, lactacystin (23Lee D.H. Goldberg A.L. Trends Cell Biol. 1998; 8: 397-403Abstract Full Text Full Text PDF PubMed Scopus (1252) Google Scholar). Lactacystin (10 μm) abolished UDP-induced NF-κB activation (Fig. 2B), consistent with involvement of the classical pathway for NF-κB activation. Expression of P2Y Receptors in Rabbit Osteoclasts—The presence of P2Y1 and P2Y2 receptors as well as P2X4 and P2X7 receptors has been previously shown in osteoclasts (17Hoebertz A. Meghji S. Burnstock G. Arnett T.R. FASEB J. 2001; 15: 1139-1148Crossref PubMed Scopus (88) Google Scholar, 18Weidema A.F. Dixon S.J. Sims S.M. Am. J. Physiol. 2001; 280: C1531-C1539Crossref PubMed Google Scholar, 19Hoebertz A. Townsend-Nicholson A. Glass R. Burnstock G. Arnett T.R. Bone. 2000; 27: 503-510Crossref PubMed Scopus (130) Google Scholar, 20Naemsch L.N. Weidema A.F. Sims S.M. Underhill T.M. Dixon S.J. J. Cell Sci. 1999; 112: 4425-4435Crossref PubMed Google Scholar, 21Naemsch L.N. Dixon S.J. Sims S.M. J. Biol. Chem. 2001; 276: 39107-39114Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). To examine whether additional P2Y receptors are expressed on osteoclasts, reverse transcription-PCR was performed on heterogeneous populations of bone marrow cells (bone cells) as well as on osteoclasts purified by micromanipulation (osteoclasts). Bone marrow cells, containing cells of both osteoblast and osteoclast lineages, possessed transcripts for P2Y1, P2Y2, P2Y6, and P2Y11 receptors and ALP (used as a marker for cells of the osteoblast lineage) (Fig. 3Ai). In purified osteoclasts, expression of P2Y1 and P2Y2 receptors was confirmed. Moreover, the presence of P2Y6 receptor transcripts and the absence of P2Y11 transcripts were shown for the first time (Fig. 3Aii). P2Y1 and P2Y2 receptor agonists cause elevation of [Ca2+]i in osteoclasts (18Weidema A.F. Dixon S.J. Sims S.M. Am. J. Physiol. 2001; 280: C1531-C1539Crossref PubMed Google Scholar). We next examined whether P2Y6 receptors in osteoclasts also couple to Ca2+ signaling. Application of 2MeSADP (10 μm), UTP (10 μm), and UDP (10 μm) to single fura-2-loaded rabbit osteoclasts induced a transient rise in [Ca2+]i (Fig. 3B), indicating that P2Y1, P2Y2, and P2Y6 receptors induce Ca2+ signaling. In Jurkat T cells, elevations of [Ca2+]i enhance the activation of NF-κB (24Dolmetsch R.E. Xu K. Lewis R.S. Nature. 1998; 392: 933-936Crossref PubMed Scopus (1683) Google Scholar), so it is possible that Ca2+ is involved in nucleotide-induced activation of NF-κB in osteoclasts. Although all of the nucleotides tested caused a transient increase in [Ca2+]i, only the P2Y6 receptor agonist UDP activated NF-κB. Therefore, it appears that P2Y-mediated rise in [Ca2+]i alone is not sufficient to activate NF-κB in osteoclasts. The Role of P2Y6 Receptors in Osteoclast Survival—The physiological roles of P2Y6 receptors are poorly understood. In 1321N1 human astrocytes, activation of P2Y6 receptors prevents apoptosis induced by tumor necrosis factor α (25Kim S.G. Soltysiak K.A. Gao Z.G. Chang T.S. Chung E. Jacobson K.A. Biochem. Pharmacol. 2003; 65: 923-931Crossref PubMed Scopus (51) Google Scholar), suggesting that the P2Y6 receptor regulates cell survival. We assessed the effects of nucleotides on osteoclast survival using isolated rat osteoclasts, which spontaneously undergo apoptosis over a period of 24 h in culture. Osteoclasts treated with 2MeSADP (10 μm) or UTP (10 μm) had survival rates that were not significantly different from control (Fig. 4A). In contrast, RANK ligand (10 ng/ml) and UDP (10 μm) promoted osteoclast survival (Fig. 4A). Significant enhancement of osteoclast survival was observed at UDP concentrations of at least 3 μm (Fig. 4B). Possible Role of RANK Ligand in P2Y6 Receptor-mediated NF-κB Activation and Enhancement of Osteoclast Survival— Cells isolated from rabbit long bones include marrow stromal cells and cells of the osteoblast lineage. Osteoblasts express multiple P2Y receptors, including P2Y6 (26Maier R. Glatz A. Mosbacher J. Bilbe G. Biochem. Biophys. Res. Commun. 1997; 240: 298-302Crossref PubMed Scopus (33) Google Scholar). Thus, extracellular nucleotides may act on osteoblasts to up-regulate expression of RANK ligand, which could then bind to its receptor RANK on osteoclasts activating NF-κB and increasing survival. To examine this possibility, osteoclast preparations were treated with the decoy receptor for RANK ligand, OPG. OPG did not affect basal levels of NF-κB activation. Moreover, treatment with OPG (100 ng/ml) for 20 min prior to and during 3 h of incubation with UDP (10 μm) did not prevent NF-κB activation (Fig. 5Ai). On the other hand, pretreatment with OPG significantly inhibited NF-κB activation induced by exogenous RANK ligand (100 ng/ml, 30 min), demonstrating the effectiveness of OPG (Fig. 5Aii). In keeping with these findings, treatment with OPG (100 ng/ml) did not significantly alter osteoclast survival under basal conditions or in the presence of UDP (10 μm, 18 h) (Fig. 5B). In contrast, OPG abolished the increase in survival induced by exogenous RANK ligand (10 ng/ml) (Fig. 5B). Thus, UDP induces nuclear translocation of NF-κB and enhances osteoclast survival independently of RANK ligand. The Role of NF-κB Activation in Osteoclast Survival—NF-κB plays an important role in controlling survival of a number of cell types (14Karin M. Lin A. Nat. Immunol. 2002; 3: 221-227Crossref PubMed Scopus (2470) Google Scholar). To examine whether the effect of UDP on osteoclast survival is mediated through activation of NF-κB, we used the cell-permeable peptide inhibitor of NF-κB, SN50 (27Lin Y.Z. Yao S.Y. Veach R.A. Torgerson T.R. Hawiger J. J. Biol. Chem. 1995; 270: 14255-14258Abstract Full Text Full Text PDF PubMed Scopus (854) Google Scholar). Treatment of rabbit osteoclasts with SN50 (20 μm) 20 min prior to and during incubation
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