Orai1/Stim1 channel as a downstream mechanism also plays a significant role in ATP-induced Ca 21 signaling. ATP concentration-dependently stimulates hDP-MSC migration. Pharmacological and genetic interventions of the expression or function of the P2X7, P2Y 1 and P2Y 11 receptors, and Orai1/Stim1 channel support critical involvement of these Ca 21 signaling mechanisms in ATP-induced stimulation of hDP-MSC migration. Taken together, this study provide evidence to show that purinergic P2X7, P2Y 1 , and P2Y 11 receptors and store-operated Orai1/Stim1 channel represent important molecular mechanisms responsible for ATP-induced Ca 21 signaling in hDPMSCs and activation of these mechanisms stimulates hDP-MSC migration. Such information is useful in building a mechanistic understanding of MSC homing in tissue homeostasis and developing more efficient MSC-based therapeutic applications. STEM CELLS 2016;34:2102-2114 SIGNIFICANCE STATEMENTATP is an important signaling molecule that regulates diverse cell functions. Mesenchymal stem cells (MSCs) have promising potential of therapeutic application but the migration capacity of MSCs limits the effectiveness of MSC-based therapies. MSCs release ATP and here we provide evidence to show that ATP stimulates MSC migration through purinergic P2X7, P2Y 1 , and P2Y 11 receptors. Our study is the first to find that Orai1 and Sitm1 form Ca 21 -release-activated-Ca 21 channel as downstream Ca 21 signaling mechanism mediating ATP-induced stimulation of MSC migration. These results reveal novel mechanisms regulating MSC migration. Such information is desirable in optimization of MSC cultures for therapeutic use.

Extracellular ATP-induced Ca2+ signalling is critical in regulating diverse physiological and disease processes. Emerging evidence suggests high concentrations of extracellular ATP in tumour tissues. In this study, we examined the P2 receptor for ATP-induced Ca2+ signalling in human hepatocellular carcinoma (HCC) cells. Fura-2-based measurements of the intracellular Ca2+ concentration ([Ca2+]i) showed that extracellular ATP induced an increase in the [Ca2+]i in human HCC Huh-7 and HepG2 cells. NF546, a P2Y11 receptor agonist was equally effective in inducing an increase in the [Ca2+]i. In contrast, agonists for the P2X receptors (αβmeATP and BzATP), P2Y1 receptor (MRS2365) or P2Y2 receptor (MRS2768) were ineffective. In addition, ATP/NF546-induced increases in the [Ca2+]i were strongly inhibited by treatment with NF340, a P2Y11 receptor antagonist. Immunofluorescent confocal imaging and western blotting analysis consistently demonstrated the P2Y11 receptor expression in Huh-7 and HepG2 cells. Transfection with P2Y11-specific siRNA attenuated the P2Y11 receptor protein expression level and also reduced NF546-induced increase in the [Ca2+]i. Importantly, immunohistochemistry revealed that the P2Y11 receptor was expressed at very high level in human HCC tissues and, by contrast, it was barely detected in normal liver tissues. Trans-well cell migration assay demonstrated that ATP and NF546 induced concentration-dependent stimulation of Huh-7 cell migration. Treatment with NF340 prevented ATP-induced stimulation of cell migration. Taken together, our results show carcinoma-specific expression of the P2Y11 receptor and its critical role in mediating ATP-inducing Ca2+ signalling and regulating cell migration in human HCC cells.

Extracellular ATP and other nucleotides induce autocrine and/or paracrine purinergic signalling via activation of the P2 receptors on the cell surface, which represents one of the most common signalling mechanisms. Mesenchymal stem cells (MSC) are a type of multipotent adult stem cells that have many promising applications in regenerative medicine. There is increasing evidence to show that extracellular nucleotides regulate MSC functions and P2 receptor‐mediated purinergic signalling plays an important role in such functional regulation. P2 receptors comprise ligand‐gated ion channel P2X receptors and G‐protein‐coupled P2Y receptors. In this review, we provide an overview of the current understanding with respect to expression of the P2X and P2Y receptors in MSC and their roles in mediating extracellular nucleotide regulation of MSC proliferation, migration and differentiation. J. Cell. Physiol. 232: 287–297, 2017. © 2016 Wiley Periodicals, Inc.

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