Activation of P2X7 receptors causes isoform-specific translocation of protein kinase C in osteoclasts

Abstract

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) – Ca2+-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 PKCα to the basolateral membrane. UTP or ATP (10 μM), 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 Ca2+, and removal of extracellular Ca2+ abolished the translocation of PKCα that was induced by BzATP (but not by phorbol ester). We examined the isoform specificity of this response, and observed translocation of the Ca2+-dependent isoforms PKCα and PKCβI, but not the Ca2+-independent isoform PKCδ. Thus, activation of P2X7 receptors specifically induces Ca2+-dependent translocation of PKC to the basolateral membrane domain of osteoclasts, an aspect of spatiotemporal signaling not previously recognized.

P2X7 nucleotide receptor signaling potentiates the Wnt/β-catenin pathway in cells of the osteoblast lineage

“…These findings are consistent with observations in cultures of cerebellar granule neurons, in which activation of P2X7 inhibits GSK3β in a manner dependent upon PKC signaling [13]. Moreover, in osteoclasts, P2X7 couples to activation of various Ca 2+ -sensitive PKC isoforms including PKCα and PKCβ [39]. We have shown previously that activation of P2X7 in osteoblast-like cells couples to PI3K/AKT signaling [17], a pathway associated with GSK3β inhibition [38].…”

Expression, signaling, and function of P2X7 receptors in bone

“…To determine whether P2X7 receptors activate PKC in osteoclasts, Armstrong et al employed murine macrophage RAW 264.7 cells, which differentiate into multinucleated osteoclast-like cells in the presence of RANK ligand. Stimulation of differentiated RAW 264.7 cells with BzATP-induced transient translocation of PKCα to the plasma membrane [60]. In contrast, UTP and low concentrations of ATP, which activate P2 receptors other than P2X7, failed to elicit this response.…”

“…Moreover, BzATP failed to induce PKC translocation in osteoclasts derived from the bone marrow of P2rx7 −/− mice, demonstrating specificity for P2X7. Translocation was temporally correlated with BzATP-induced elevations in [Ca 2+ ] i and dependent on the presence of extracellular Ca 2+ , thereby implicating P2X7-mediated Ca 2+ influx in PKC activation [60]. A recent study identified signaling through PKC as being essential for enhanced osteoclast survival in response to extracellular acidification [70].…”

The role of purinergic signalling in the musculoskeletal system

“…Other work has demonstrated that P2X7 receptor activation induces the translocation of NFĸB (Korcok et al, 2004) and an isoform specific PKC in osteoclasts and their precursors (Armstrong et al, 2009). The P2X7 receptor may additionally play a role in the intercellular communication between osteoblasts and osteoclasts, cytoskeletal reorganisation at the sealing zone and the delivery and secretion of lytic granules into the resorption lacunae (Hazama et al, 2009;Jorgensen et al, 2002).…”