Extracellular ATP signalling mechanisms in mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) are considered to have promising applications in cell therapy and tissue regeneration. The chemical molecules in the local environments are known to be important in controlling the functions of MSCs. The intrinsic mechanisms activated by such extracellular molecules are not fully understood. ATP is a well-established extracellular signalling molecule and activates two structurally and functionally different subfamilies of purinergic receptors on the cell surface, ATP-gated ion channel P2X receptors and G-protein-coupled P2Y receptors. Activation of the P2Y receptors leads to depletion of intracellular Ca2+ store and subsequent activation of the store-operated Ca2+ (SOC) channels. ATP is released by MSCs constitutively or in response to stimulation. This study using MSCs isolated from human dental pulp (hDP-MSCs), investigated the effects of extracellular ATP on proliferation, migration, osteogenic and adipogenic differentiation and the underlying signalling mechanisms. Exposure of hDP-MSC to extracellular ATP up to 300 μM resulted in no effect on cell proliferation as shown in the MTT assays and cell counting. ATP at 30 μM promoted cell migration using the wound healing assays. ATP also inhibited the ALP expression and activity or osteogenesis of hDP-MSCs in basal medium (BM) and osteogenic differentiation medium (ODM), and by contrast increased the number of Oil red O-stained, fact droplet-containing cells or adipogenesis of hDP-MSCs cultured in adipogenic differentiation medium (ADM). RT-PCR revealed consistent expression of mRNA transcripts for P2X4, P2X6, P2X7, P2Y1, P2Y11 and also the SOC channel components, Orai1, Orai2, Orai3, Stim1 and Stim2. Single cell imaging and FLEXstation experiments in a combination of pharmacological intervention using selective inhibitors or genetic intervention using specific siRNA provide consistent evidence to show functional expression of the P2X7, P2Y1 and P2Y11 receptors and Stim1/Orai SOC channels. For example, ATP-induced Ca2+ responses were inhibited by AZ11645373 (a P2X7 antagonist), 2-APB (a SOC channel inhibitor), Synta66 (a SOC channel inhibitor) or siP2X7, siP2Y1, siP2Y11, siOrai1, siOrai2, siOrai3 and siStim1. Treatment with AZ11645373 or siP2X7 significantly inhibited ATP-induced stimulation of cell migration, but had no effect on ATP-induced inhibition of osteogenesis and stimulation of adipogenesis. Treatment with siP2Y1 or siP2Y11 strongly blocked ATP-induced stimulation of cell migration and adipogenesis, and inhibition of osteogenesis. Finally, treatment with siStim1/siOrai1 attenuated ATP-evoked stimulation of cell migration and inhibition of osteogenesis. ATP-induced increase in adipogenesis was reduced by treatment with 2-APB and siOrai3 but not with siStim1/siOrai1. In summary, the present study shows that extracellular ATP regulates cell migration via the P2X7, P2Y1 and P2Y11 receptors and Stim1/Orai1 SOC channels, osteogenic differentiation mainly via the P2Y1 and P2Y11 receptors and Stim1/Orai1 SOC channels, and adipogenic differentiation via the P2Y1 and P2Y11 receptor and Orai3 in hDP-MSCs. These findings provide a better understanding of the mechanisms underlying the actions of extracellular ATP on hDP-MSCs and useful information that facilitates developing better applications of hMSCs for regenerative medicines.