Activation of P2X7 receptors causes isoform-specific translocation of protein kinase C in osteoclasts.
Nucleotides, released in response to mechanical or inflammatory stimuli, signal through P2 nucleotide receptors in many cell types. Osteoclasts express P2X7 receptors (encoded by P2rx7) - Ca(2+)-permeable channels that are activated by high concentrations of extracellular ATP. Genetic disruption of P2rx7 leads to increased resorption and reduced skeletal response to mechanical stimuli. To investigate whether P2X7 receptors couple to activation of protein kinase C (PKC), RAW 264.7 cells were differentiated into multinucleated osteoclast-like cells and live-cell confocal imaging was used to localize enhanced green fluorescent protein (EGFP)-tagged PKC. Benzoylbenzoyl-ATP (BzATP; a P2X7 agonist) induced transient translocation of PKCalpha to the basolateral membrane. UTP or ATP (10 microM), which activate P2 receptors other than P2X7, failed to induce translocation. Moreover, BzATP failed to induce PKC translocation in osteoclasts derived from the bone marrow of P2rx7(-/-) mice, demonstrating specificity for P2X7. BzATP induced a transient rise of cytosolic Ca(2+), and removal of extracellular Ca(2+) abolished the translocation of PKCalpha that was induced by BzATP (but not by phorbol ester). We examined the isoform specificity of this response, and observed translocation of the Ca(2+)-dependent isoforms PKCalpha and PKCbetaI, but not the Ca(2+)-independent isoform PKCdelta. Thus, activation of P2X7 receptors specifically induces Ca(2+)-dependent translocation of PKC to the basolateral membrane domain of osteoclasts, an aspect of spatiotemporal signaling not previously recognized.
Armstrong
Souzan
S
Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada N6A 5C1.
Pereverzev
Alexey
A
Dixon
S Jeffrey
SJ
Sims
Stephen M
SM
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004789:Enzyme Activation; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D010010:Osteoclasts / Q000166:cytology / Q000378:metabolism*; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000378:metabolism*; D058486:Receptors, Purinergic P2X7; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D007527:Isoenzymes; C546469:P2rx7 protein, mouse; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; D011993:Recombinant Fusion Proteins; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D002118:Calcium