Extracellular nucleotides act through P2X7 receptors to activate NF-kappaB in osteoclasts.
Nucleotides, released in response to mechanical and other stimuli, act on P2 receptors in osteoclasts and other cell types. In vitro studies of osteoclasts from rabbits and P2X7 receptor-deficient mice revealed that P2X7 receptors couple to activation of the key transcription factor NF-kappaB.


BACKGROUND
Osteoclasts express functional P2X4 and P2X7 receptors, which are ATP-gated cation channels. Knockout (KO) of the P2X7 receptor has revealed its role in regulating bone formation and resorption, but the underlying signals are not known. The transcription factor NF-kappaB plays a key role in the response of osteoclasts to RANKL and other cytokines. The aim of this study was to examine whether P2X receptors on osteoclasts signal through NF-kappaB.


METHODS
Osteoclasts were isolated from neonatal rabbits or wildtype (WT) and P2X7 receptor KO mice. Immunofluorescence was used to detect the p65 subunit of NF-kappaB, which, on activation, translocates from the cytosol to the nuclei. The concentration of cytosolic free Ca2+ ([Ca2+]i) was monitored in single osteoclasts loaded with fura-2.


RESULTS
In control samples, few rabbit osteoclasts demonstrated nuclear localization of NF-kappaB. Benzoyl-benzoyl-ATP (BzATP, a P2X7 agonist, 300 microM) induced nuclear translocation of NF-kappaB after 3 h in approximately 45% of rabbit osteoclasts. In contrast, a low concentration of ATP (10 microM, sufficient to activate P2X4 and P2Y2, but not P2X7 receptors) did not induce nuclear translocation of NF-kappaB. Because BzATP activates multiple P2 receptors, we examined responses of osteoclasts derived from WT and P2X7 receptor KO mice. Treatment with BzATP for 30 minutes increased nuclear localization of NF-kappaB in osteoclasts from WT but not KO mice, showing involvement of P2X7 receptors. Both ATP (10 microM) and BzATP (300 microM) caused transient elevation of [Ca2+]i, indicating that rise of calcium alone is not sufficient to activate NF-kappaB. Pretreatment of rabbit osteoclasts with osteoprotegerin inhibited translocation of NF-kappaB induced by RANKL but not by BzATP, establishing that the effects of BzATP are independent of RANKL signaling.


CONCLUSIONS
These findings show that P2X7 nucleotide receptors couple to activation of NF-kappaB in osteoclasts. Thus, nucleotides, released at sites of inflammation or in response to mechanical stimuli, may act through NF-kappaB to regulate osteoclast formation and activity.
Korcok
Jasminka
J
CIHR Group in Skeletal Development and Remodeling, Department of Physiology and Pharmacology and Division of Oral Biology, Faculty of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada.
Raimundo
Lin N
LN
Ke
Hua Z
HZ
Sims
Stephen M
SM
Dixon
S Jeffrey
SJ
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000494:pharmacology; D051379:Mice; D018345:Mice, Knockout; D016328:NF-kappa B / Q000378:metabolism*; D010010:Osteoclasts / Q000378:metabolism*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D058910:Purinergic P2 Receptor Agonists; D053245:RANK Ligand; D011817:Rabbits; D053246:Receptor Activator of Nuclear Factor-kappa B; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058848:Receptors, Purinergic P2X4; D058486:Receptors, Purinergic P2X7; D015398:Signal Transduction / Q000187:drug effects*
D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D016328:NF-kappa B; C548227:P2rx4 protein, mouse; C546469:P2rx7 protein, mouse; D010975:Platelet Aggregation Inhibitors; D058910:Purinergic P2 Receptor Agonists; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D018048:Receptors, Purinergic P2; D058848:Receptors, Purinergic P2X4; D058486:Receptors, Purinergic P2X7; C505687:Tnfrsf11a protein, mouse; C505689:Tnfsf11 protein, mouse; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D002118:Calcium