Role of the P2X7 receptor in the osteogenic differentiation of mesenchymal cells and in osteoclast fusion

摘要

This manuscript by Sun and colleagues implicate the importance of P2X7 receptors in the differentiation of mesenchymal stem cells towards bone forming osteoblasts [ class="CitationRef">href="#CR2">2]. In a series of well-designed and performed experiments, they tested and verified that 1) shockwaves induce ATP release from human mesenchymal stem cells (hMSCs), 2) shockwave-induced ATP release will activate P2X7 receptors and p38 MAP kinase (MAPK) signalling pathways, and 3) the downstream consequence of this enhanced osteogenic differentiation of hMSCs. class="Para">The first part of this study verified that shockwave treatment could cause ATP release from hMSCs based on the previous observation of shockwave-induced ATP release from T cells [ class="CitationRef">href="#CR3">3]. Isolated hMSCs (validated as CD73 and CD105 positive and clean of contamination from CD34 and CD45 positive hematopoietic lineage cells) were treated with shockwaves and cell viability and ATP release assessed. When cells were subjected to 100 shockwave impulses significant release of ATP in the μM range was observed. However, 200 shockwave impulses induced significant extracellular ATP release but this was found to be due to shockwave induce cell damage. class="Para">In the following part of the manuscript, the authors performed a set of experiments to test if shockwave treatment activates P2X7 receptors, p38 MAPK, and the downstream transcription factors c-fos and c-jun. Using western blotting, the phosphorylation of p38 MAPK of hMSCs were determined and found to be significantly enhanced either after shockwave treatment (200 impulses) or exogenous ATP treatment (100?μM). In addition, using real-time RT-PCR and immunocytochemical staining the expression of P2X7 receptors by the hMSCs was confirmed and found to be up-regulated following shockwaves treatment (100 impulses) and 1?μM exogenous ATP at both the mRNA and protein levels. Treating hMSCs with apyrase (an enzyme to hydrolyse extracellular ATP), P2X7-siRNA, PPADS (non-selective P2 antagonist), and KN-62 (P2X7 receptor antagonist) completely abolished the p38 MAPK activation induced by either shockwave or exogenous ATP treatment. These results confirmed that P2X7 receptors are involved in the shockwave and ATP induced p38 MAPK activation. Furthermore, using real-time RT-PCR, the mRNA levels of c-fos and c-jun were significantly increased and reach peak levels within 45?min after cell stimulation of either shockwave or ATP treatment. Silencing or blocking of P2X7 receptors or inhibiting the p38 MAPK activation led to significantly decreased shockwave- and ATP- induced c-fos and c-jun transcription, supporting the notion that shockwave or ATP treatment induced c-fos and c-jun transcription via P2X7 receptors and p38 MAPK. class="Para">Finally, the physiological consequence of shockwave-induced ATP release and P2X7 receptor activation was determined by examining the osteogenic differentiation of hMSCs. The ability of shockwave or extracellular ATP to induce hMSCs towards bone forming osteoblasts was confirmed with the observation of a dose-dependent increase in osteoblastic differentiation markers alkaline phosphatase (ALP), osteocalcin (OC) and bone-nodule formation (measured by calcium deposit binding Alizarin Red). class="Para">The authors suggest the possible molecular mechanism of shockwave-induced osteogenic response. Briefly, shockwave induces acute ATP release from hMSCs which, via P2X7 receptors, activates p38 MAPK and this subsequently enhances the transcription of c-fos and c-jun driving the differentiation of hMSCS towards osteoblasts via the up regulation of the activator protein 1 (AP-1) [ class="CitationRef">href="#CR4">4].