P2X7 purinoceptor promotes survival or cytotoxicity depending on extracellular adenosine triphosphate (ATP) stimulus intensity controlling its ion channel or P2X7-dependent large pore (LP) functions. Mechanisms governing this operational divergence and functional idiosyncrasy are ill-understood. We have discovered a feedback loop where sustained activation of P2X7 triggers release of active matrix metalloproteinase 2 (MMP-2), which halts ion channel and LP responses via the MMP-2-dependent receptor cleavage. This mechanism operates in cells as diverse as macrophages, dystrophic myoblasts, P2X7-transfected HEK293, and human tumour cells. Given that serum-born MMP-2 activity also blocked receptor functions, P2X7 responses in vivo may decrease in organs with permeable capillaries. Therefore, this mechanism represents an important fine-tuning of P2X7 functions, reliant on both cell-autonomous and extraneous factors. Indeed, it allowed evasion from the ATP-induced cytotoxicity in macrophages and human cancer cells with high P2X7 expression levels. Finally, we demonstrate that P2X7 ablation eliminated gelatinase activity in inflamed dystrophic muscles in vivo. Thus, P2X7 antagonists could be used as an alternative to highly toxic MMP inhibitors in treatments of inflammatory diseases and cancers.

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Running title: mdx mutation affects calcium signalling in myoblastsHighlights:1. NGS reveals changes in Ca 2+ signalling-related RNAs in mdx myoblasts 2. Mdx myoblasts exerts increased susceptibility to P2RY2-mediated stimulation 3. Levels of several Ca 2+ signalling-related proteins are changed in mdx myoblasts 4. P2RY2 agonist slows-down mdx myoblasts motility SummaryPathophysiology of Duchenne Muscular Dystrophy is still elusive. Although progressive damage to muscle fibres is a cause of muscle deterioration leading to premature death, there is a growing body of evidence indicating that the triggering effects of DMD mutation are present at the very early stage of muscle development. Previously, elevated activity of P2X7 receptors and increased store-operated calcium entry were shown in myoblasts derived from mdx mice. Here, the metabotropic extracellular ATP/UTP-evoked response has been investigated. Sensitivity to antagonist, effect of gene silencing and cellular localization studies showed that elevated purinergic responses in mdx myoblasts have been caused by increased expression of P2Y2 but not P2Y4 receptors. These alterations have physiological implications as shown by reduced motility of mdx myoblasts upon treatment with P2Y2 agonist. The ultimate increase in intracellular calcium in dystrophic cells reflected complex alterations of calcium homeostasis identified in the RNA seq data and with significant modulation at the protein level including a decrease of Gq11 subunit , PMCA, IP3-receptor and elevation of PLC, SERCA and NCX. In conclusion, whereas specificity of dystrophic myoblast excitation by extracellular ATP is determined by specific receptor overexpression, the intensity of this altered response depends on relative activities of downstream calcium regulators that also accompany the Dmd gene mutation. These results confirm that phenotypic effects of DMD emerge in undifferentiated muscle cells and not only due to the absence of dystrophin protein in myofibres. Therefore, the pathogenesis of DMD and the relevance of current therapeutic approaches may need re-evaluation.