The P2X7 receptor may be a novel therapeutic target to promote osteogenic differentiation and mineralization of postmenopausal bone marrow stromal cells

JB Noronha-Matos1, A Sá-e-Sousa1, J Coimbra1, S Gomes1, R Rocha3, J Marinhas3, R Freitas3, J Morais-Neves3, MA Costa1,2, P Correia-de-Sá1. 1Laborat ó rio Farmacologia e Neurobiologia, UMIB, Instituto Ci ê ncias Biom é dicas Abel Salazar, University of Porto, Departamento de Imuno-Fisiologia e Farmacologia, Largo Prof. Abel Salazar, 2, 4099-003 Porto, Portugal, 2Dept. Qu í mica, UMIB, Instituto Ci ê ncias Biom é dicas Abel Salazar, University of Porto, Departamento de Qu í mica, Largo Prof. Abel Salazar, 2, 4099-003 Porto, Portugal, 3Serv. Ortopedia e Traumatologia, Centro Hospitalar de Gaia, Espinho, Rua de Santa Catarina, 1288, 4000-447 Porto, Portugal

Adult mesenchymal stromal progenitor/stem cells (BMSCs) have extensive proliferative ability while retaining multilineage differentiation potential. BMSCs are able to differentiate into osteoblast cell lineage and extracellular nucleotides may command osteogenic differentiation of these cells (see e.g. Noronha-Matos et al., 2012, J. Cell. Physiol. 227, 2694-709) through the activation of P2Y and P2X purinoceptors. Single nucleotide polymorphisms of the P2X7 receptor gene are associated with increased risk of fractures in postmenopausal women. P2X7 receptors are expressed in both osteoblasts and osteoclasts, but their role in bone formation remains controversial. This study was designed to investigate the role of P2X7 receptors on osteogenic differentiation and mineralization of BMSCs (positive for CD105, CD29 and CD117 mesenchymal cell markers and negative for hematopoietic surface markers, like CD14 and CD45) isolated from postmenopausal female patients (71±3 years old, n=18) undergoing total hip replacement. Ethical approval and informed consent for using the biological material was obtained. Cultures were characterized for cell proliferation (MTT assay), total protein content (method of Lowry) and alkaline phosphatase (ALP) activity during 30 days. Mineralization was investigated at day 43 (Alizarin red test). Single-cell [Ca2+]i oscillations was evaluated after loading the cells with Fluo-4NW (2.5 µM, 45 min at 37ºC). ATP (100 µM) and the P2X7 agonist BzATP (100µM) transiently increased [Ca2+]i in parallel to the formation of non-selective cell-membrane pores visualized by TO-PRO-3 (1 µM) dye uptake. Formation of reversible plasma membrane blebs (zeiosis) was observed in 38±1% (n=3) and in 70±1% (n=6) of the cells exposed to 100µM ATP and BzATP, respectively. Zeiosis was observed independently of extracellular Ca2+; depletion of intracellular Ca2+ stores by thapsigargin (2 µM) did not prevent blebbing. BzATP-induced zeiosis was almost completely abolished by inhibiting phospholipase C (PLC), protein kinase C (PKC) and Rho-kinase, respectively with U73122 (3 µM), chelerythrine (5 µM) and H1152 (3 µM). Incubation with BzATP (100µM) anticipated osteogenic differentiation and promoted mineralization of BMSC cultures; BzATP (100µM) increased alkaline phosphatase activity by 329±131% (at culture day 4, n=6) and favoured mineralization of bone-nodules by 388±88% (at culture day 43, n=3). Effects of BzATP (100µM) on differentiation and mineralization of BMSC cultures were significantly (P<0.05) attenuated upon blocking P2X7 receptors with A438079 (3µM) or by inhibiting Rho-kinase activity with H1152 (3µM). The expression of P2X7 receptors on BMSCs exhibiting osteoblast cell-markers (e.g. type I collagen, osteocalcin) was confirmed by confocal microscopy. Data suggest that activation of P2X7 receptors favors early osteogenic differentiation and mineralization of postmenopausal BMSCs through a mechanism that depends on cytoskeleton rearrangements (pore formation and blebbing) operated by the PLC / PKC / Rho-kinase intracellular pathway. These findings indicate that P2X7 receptor may be a novel therapeutic target to promote osteogenic differentiation and mineralization of postmenopausal bone marrow stromal cells, which might be relevant in bone disorders, such as osteoporosis and rheumatoid arthritis.

Work supported by FCT (FEDER funding, PTDC/SAU-OSM/73576/2006 and UMIB-215/94); JBNM is in receipt of a PhD Studentship (SRFH/BD/68584/2010).