title: P2Y6 nucleotide receptors activate NF-kappaB and increase survival of osteoclasts.
abstract: 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-kappaB, a key transcription factor regulating osteoclastogenesis. Immunofluorescence was used to detect the p65 subunit of NF-kappaB, which upon activation translocates from the cytosol to nuclei. Low levels of NF-kappaB 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-kappaB 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-kappaB localization or osteoclast survival, consistent with a direct action. Moreover, SN50 (cell-permeable peptide inhibitor of NF-kappaB) 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-kappaB signaling and enhance osteoclast survival.
authors.affiliation: Canadian Institutes of Health Research Group in Skeletal Development and Remodeling, Department of Physiology and Pharmacology and Division of Oral Biology, Faculty of Medicine & Dentistry, The University of Western Ontario, London, Canada.
authors.forename: Jasminka
authors.initials: J
authors.lastname: Korcok
authors.forename: Lin N
authors.initials: LN
authors.lastname: Raimundo
authors.forename: Xiaobing
authors.initials: X
authors.lastname: Du
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Sims
authors.forename: S Jeffrey
authors.initials: SJ
authors.lastname: Dixon
mesh_terms: D021581:Active Transport, Cell Nucleus; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001842:Bone and Bones / Q000378:metabolism / Q000473:pathology; D002118:Calcium / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D017931:DNA Primers / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006023:Glycoproteins / Q000378:metabolism; D007249:Inflammation; D008024:Ligands; D008856:Microscopy, Fluorescence; D016328:NF-kappa B / Q000378:metabolism*; D010010:Osteoclasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D053244:Osteoprotegerin; D061988:Proteasome Inhibitors; D058910:Purinergic P2 Receptor Agonists; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058472:Receptors, Purinergic P2Y1; D058473:Receptors, Purinergic P2Y2; D018124:Receptors, Tumor Necrosis Factor; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013997:Time Factors; D051996:Transcription Factor RelA; D014530:Uridine Diphosphate / Q000378:metabolism; D014544:Uridine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology
chemical_list: D017931:DNA Primers; D004791:Enzyme Inhibitors; D006023:Glycoproteins; C501301:INS 48823; D008024:Ligands; D016328:NF-kappa B; D053244:Osteoprotegerin; C546384:P2ry1 protein, rat; C546387:P2ry2 protein, rat; D061988:Proteasome Inhibitors; D058910:Purinergic P2 Receptor Agonists; D018160:Receptors, Cytoplasmic and Nuclear; D018048:Receptors, Purinergic P2; D058472:Receptors, Purinergic P2Y1; D058473:Receptors, Purinergic P2Y2; D018124:Receptors, Tumor Necrosis Factor; C506032:Tnfrsf11b protein, rat; D051996:Transcription Factor RelA; C099845:purinoceptor P2Y6; D014530:Uridine Diphosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000469:Alkaline Phosphatase; D002118:Calcium; D014544:Uridine Triphosphate