Extracellular Nucleotides Act Through P2X7 Receptors to Activate NF-κB in Osteoclasts

UNLABELLED: 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. INTRODUCTION: 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. MATERIALS AND 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. CONCLUSION: 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.