The biological effects of extracellular ATP are mediated by P2Y metabotropic and P2X ionotrophic receptors. Among P2X receptors, the P2X7R subtype exhibits unique molecular, functional and physiological features. P2X7R activation by low ATP concentrations reversibly opens a cationpermeable membrane channel. On the other hand, stimulation with high ATP concentrations induces formation of a large conductance pore permeable to high molecular weight molecules. The role of P2X7R in bone biology is only in part investigated and it is still not clear whether the net effects on bone are positive or negative. It has been demonstrated that osteoblasts (OBs) and osteoclasts (OCs) express P2X7R, but its participation in the differentiation of mesenchymal stem cells (MSCs) and in the regulation of bone formation and bone resorption is not still completely understood. The aim of this study was to investigate the role of P2X7R in human primary cells including MSCs (from Wharton’s jelly, WJ-MSCs, and bone marrow, BM-MSCs) and OBs in terms of ability of the cells to deposit mineral matrix, according to the scheme below: -Use of culture systems that are close to the physiological bone micro-environment. We cultured the cells in X3 Hypoxia Hood and Culture Combo – Xvivo System to mimic the hypoxic conditions in which the cells grow in vivo (1-3% oxygen pressure); -Treatment with P2X7R agonist (BzATP), P2X7R antagonists (A740003 or AZ11645373) or apyrase (scavenger of ATP and ADP) to elucidate the role of extracellular ATP in the deposition of mineral matrix; -Analysis of ectonucleotidases expression and activity to investigate the participation of these enzymes to produce inorganic phosphates for mineralization. Preliminary data indicate that: 1. After 48h of hypoxia preconditioning, there was no difference in the mRNA expression of P2X7R in WJ-MSCs, but there was a decreased of mRNA expression in BM-MSCs and OBs. No difference in the P2X7R protein (analyzed by Western Blotting) in the three different cell types; 2. The treatment with P2X7R agonist, P2X7R antagonists or apyrase was not toxic to the different cells; 3. Matrix mineralization analyzed by Alizarin Red staining decreased after treatment with P2X7R antagonist only in OB cells. We also demonstrated that the P2X7R protein increased after the exposure of BM-MSCs and OBs, but not WJ-MSCs, to osteogenic medium. With this planning we aim to elucidate the P2X7R role in the bone context and the potential of P2X7R as a therapeutic target for treatment of bone diseases.
The role of purinergic system in mature and precursor bone cells
Penolazzi L.;Lambertini E.;Falzoni S.;Sarti AC.;Di Virgilio F.;Piva R.
2018
Abstract
The biological effects of extracellular ATP are mediated by P2Y metabotropic and P2X ionotrophic receptors. Among P2X receptors, the P2X7R subtype exhibits unique molecular, functional and physiological features. P2X7R activation by low ATP concentrations reversibly opens a cationpermeable membrane channel. On the other hand, stimulation with high ATP concentrations induces formation of a large conductance pore permeable to high molecular weight molecules. The role of P2X7R in bone biology is only in part investigated and it is still not clear whether the net effects on bone are positive or negative. It has been demonstrated that osteoblasts (OBs) and osteoclasts (OCs) express P2X7R, but its participation in the differentiation of mesenchymal stem cells (MSCs) and in the regulation of bone formation and bone resorption is not still completely understood. The aim of this study was to investigate the role of P2X7R in human primary cells including MSCs (from Wharton’s jelly, WJ-MSCs, and bone marrow, BM-MSCs) and OBs in terms of ability of the cells to deposit mineral matrix, according to the scheme below: -Use of culture systems that are close to the physiological bone micro-environment. We cultured the cells in X3 Hypoxia Hood and Culture Combo – Xvivo System to mimic the hypoxic conditions in which the cells grow in vivo (1-3% oxygen pressure); -Treatment with P2X7R agonist (BzATP), P2X7R antagonists (A740003 or AZ11645373) or apyrase (scavenger of ATP and ADP) to elucidate the role of extracellular ATP in the deposition of mineral matrix; -Analysis of ectonucleotidases expression and activity to investigate the participation of these enzymes to produce inorganic phosphates for mineralization. Preliminary data indicate that: 1. After 48h of hypoxia preconditioning, there was no difference in the mRNA expression of P2X7R in WJ-MSCs, but there was a decreased of mRNA expression in BM-MSCs and OBs. No difference in the P2X7R protein (analyzed by Western Blotting) in the three different cell types; 2. The treatment with P2X7R agonist, P2X7R antagonists or apyrase was not toxic to the different cells; 3. Matrix mineralization analyzed by Alizarin Red staining decreased after treatment with P2X7R antagonist only in OB cells. We also demonstrated that the P2X7R protein increased after the exposure of BM-MSCs and OBs, but not WJ-MSCs, to osteogenic medium. With this planning we aim to elucidate the P2X7R role in the bone context and the potential of P2X7R as a therapeutic target for treatment of bone diseases.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
