Role Of P2X7 And P2Y2 Purinoceptors In Osteogenic And Adipogenic Differentiation Of Bone Marraw Derived Mesenchymal Stem Cells

ObjectiveTo study the roles of P2X7 and P2Y2 purinoceptors in osteogenic and adipogenic differentiation of rat BMSCs.Methods1. In vitro experiment(1) Expression of P2Y2 and P2X7 purinoceptors were assessed by immunofluorescence, Western-blot and RT-qPCR.(2) The proliferation and cell activity of BMSCs were assessed by CCK-8 proliferation assay.(3) Differentiation of BMSCs were determined by measuring the mRNA and protein expression levels of osteogenic-(RUNX2, ALP and OPN) and adipogenic-related (PPARy, FABP4 and Adipsin) markers, alkaline phosphatase (ALP) staining, alizarin red staining and Oil Red O staining.(4) The siRNAs were used for silence genes of P2X7 and P2Y2 purinoceptors to assess the roles of the two receptors in BMSCs differentiation.(5) Signaling pathways were assessed by Western-blot.2. In vivo experiment(1) To investigate the effect of P2X7 receptors activation on osteogenic and adipogenic differentiation of BMSCs in vivo, we established an ovariectomized (OVX) osteoporosis mice model to test P2X7 receptors activation on adipocytes formation, trabecular and cortical bone parameters in vivo by microCT and H&E staining.(2) The detection indexes included BV/TV (bone volume/tissue volume), Tb. Th (trabecular thickness), Tb. Sp (trabecular separation), Th. N (trabecular number), Ct. Th (comparable cortical thickness), Ct. Ar/Tt. Ar (cortical area/total area) and N. Adi/Ma. Ar (numbers of adipocytes normalized by the area of bone marrow).Results1. Expression of P2X7 and P2Y2 receptors in BMSCs were confirmed by immunofluorescence. Expression of the P2X7 receptor was increased in BMSCs cultured in an osteogenic medium and decreased in BMSCs cultured in adipogenic medium in a time-dependent manner, whereas the P2Y2 receptor was just the opposite.2. Activation of P2X7 receptor by BzATP resulted in increase in the gene expression of osteoblastic markers, the expression of alkaline phosphatase and bone mineralization, and decrease in the gene expression of adipogenic markers and the number of adipocytes generated by BMSCs. microCT analysis showed that BzATP treatment ameliorated the micro-architecture of trabecular bones in OVX mice, while cortical bone parameters were unaffected. H&E staining analysis showed that was increase in the volume of trabecular bone and number of trabecular bone, and decrease in bone marrow adipocytes in BzATP-treated OVX mice compared with OVX mice.3. Activation of P2X7 receptor by BzATP transduced to ERK1/2 and JNK signaling pathways. This transduction was prevented by BBG, U0126 and SP600125. U0126 and SP600125 prevented BzATP-induced up-regulation of osteogenic-related genes expression and down-regulation of adipogenic-related genes expression by BMSCs.4. UTP decreased the gene expression of osteoblastic markers, the expression of alkaline phosphatase and bone mineralization, and increased the gene expression of adipogenic markers and the number of lipid droplet generated by activating the P2Y2 receptor.5. Activation of P2Y2 receptor by UTP transduced to ERK1/2 signaling pathway. This transduction was prevented by U0126 and siRNA targeting the P2Y2 receptor. U0126 prevented the effects of UTP on osteogenic- and adipogenic-related gene expression after 24 h of culture.Conclusions1. BzATP activates the differentiation of BMSCs into osteoblasts but not adipocytes by stimulating ERK1/2 and JNK signaling pathways in a P2X7R-dependent way.2. UTP suppresses the osteogenic and enhances the adipogenic differentiation of BMSCs by activating the P2Y2 receptor. The ERK1/2 signaling pathway mediates the early stages of this process.