Extracellular ATP signalling mechanisms in mesenchymal stem cells

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Human mesenchymal stem cells (hMSCs) are considered to have promising \napplications in cell therapy and tissue regeneration. The chemical molecules in the \nlocal environments are known to be important in controlling the functions of MSCs. \nThe intrinsic mechanisms activated by such extracellular molecules are not fully \nunderstood. ATP is a well-established extracellular signalling molecule and \nactivates two structurally and functionally different subfamilies of purinergic \nreceptors on the cell surface, ATP-gated ion channel P2X receptors and \nG-protein-coupled P2Y receptors. Activation of the P2Y receptors leads to depletion \nof intracellular Ca2+ store and subsequent activation of the store-operated Ca2+ (SOC) \nchannels. ATP is released by MSCs constitutively or in response to stimulation. This \nstudy using MSCs isolated from human dental pulp (hDP-MSCs), investigated the \neffects of extracellular ATP on proliferation, migration, osteogenic and adipogenic \ndifferentiation and the underlying signalling mechanisms. \nExposure of hDP-MSC to extracellular ATP up to 300 μM resulted in no effect on \ncell proliferation as shown in the MTT assays and cell counting. ATP at 30 μM \npromoted cell migration using the wound healing assays. ATP also inhibited the \nALP expression and activity or osteogenesis of hDP-MSCs in basal medium (BM) \nand osteogenic differentiation medium (ODM), and by contrast increased the \nnumber of Oil red O-stained, fact droplet-containing cells or adipogenesis of \nhDP-MSCs cultured in adipogenic differentiation medium (ADM). \nRT-PCR revealed consistent expression of mRNA transcripts for P2X4, P2X6, \nP2X7, P2Y1, P2Y11 and also the SOC channel components, Orai1, Orai2, Orai3, \nStim1 and Stim2. Single cell imaging and FLEXstation experiments in a \ncombination of pharmacological intervention using selective inhibitors or genetic \nintervention using specific siRNA provide consistent evidence to show functional \nexpression of the P2X7, P2Y1 and P2Y11 receptors and Stim1/Orai SOC channels. \nFor example, ATP-induced Ca2+ responses were inhibited by AZ11645373 (a P2X7 \nantagonist), 2-APB (a SOC channel inhibitor), Synta66 (a SOC channel inhibitor) or \nsiP2X7, siP2Y1, siP2Y11, siOrai1, siOrai2, siOrai3 and siStim1. \nTreatment with AZ11645373 or siP2X7 significantly inhibited ATP-induced \nstimulation of cell migration, but had no effect on ATP-induced inhibition of \nosteogenesis and stimulation of adipogenesis. Treatment with siP2Y1 or siP2Y11 \nstrongly blocked ATP-induced stimulation of cell migration and adipogenesis, and \ninhibition of osteogenesis. Finally, treatment with siStim1/siOrai1 attenuated \nATP-evoked stimulation of cell migration and inhibition of osteogenesis. \nATP-induced increase in adipogenesis was reduced by treatment with 2-APB and \nsiOrai3 but not with siStim1/siOrai1. \nIn summary, the present study shows that extracellular ATP regulates cell migration \nvia the P2X7, P2Y1 and P2Y11 receptors and Stim1/Orai1 SOC channels, \nosteogenic differentiation mainly via the P2Y1 and P2Y11 receptors and \nStim1/Orai1 SOC channels, and adipogenic differentiation via the P2Y1 and P2Y11 \nreceptor and Orai3 in hDP-MSCs. These findings provide a better understanding of \nthe mechanisms underlying the actions of extracellular ATP on hDP-MSCs and \nuseful information that facilitates developing better applications of hMSCs for \nregenerative medicines. \n