Extracellular ATP Induces Cytokine Expression and Apoptosis through P2X7 Receptor in Murine Mast Cells

Abstract

Extracellular ATP and other nucleotides act through specific cell surface receptors and regulate a wide variety of cellular responses in many cell types and tissues. In this study, we demonstrate that murine mast cells express several P2Y and P2X receptor subtypes including P2X7, and describe functional responses of these cells to extracellular ATP. Stimulation of bone marrow-derived mast cells (BMMC), as well as MC/9 and P815 mast cell lines with millimolar concentrations of ATP, resulted in Ca2+ influx across the cellular membrane and cell permeabilization. Moreover, brief exposures to ATP were sufficient to induce apoptosis in BMMCs, MC/9, and P815 cells which involved activation of caspase-3 and -8. However, in the time period between commitment to apoptosis and actual cell death, ATP triggered rapid but transient phosphorylation of multiple signaling molecules in BMMCs and MC/9 cells, including ERK, Jak2, and STAT6. In addition, ATP stimulation enhanced the expression of several proinflammatory cytokines, such as IL-4, IL-6, IL-13, and TNF-α. The effects of ATP were mimicked by submillimolar concentrations of 3-O-(4′-benzoyl)-benzoyl-benzoyl-ATP, and were inhibited by pretreatment of mast cells with a selective blocker of human and mouse P2X7 receptor, 1[N,O-bis(5-isoquinolinesulphonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine, as well as oxidized ATP. The nucleotide selectivity and pharmacological profile data support the role for P2X7 receptor as the mediator of the ATP-induced responses. Given the importance of mast cells in diverse pathological conditions, the ability of extracellular ATP to induce the P2X7-mediated apoptosis in these cells may facilitate the development of new strategies to modulate mast cell activities.

P2 receptor-mediated signaling in mast cell biology

“…Expression of P2X 1 receptor was confirmed by FACS while expression of P2X 3 receptor by immunoblotting [54]. Interestingly, BMMCs do not express the P2X 4 receptor on the surface, as was shown by FACS analysis and confirmed by immunoblotting, despite the presence of mRNA transcript [9,54]. This could be explained by the recent finding that P2X 4 receptor contains a non-canonical tyrosine-based sorting motif which leads to constitutive endocytosis and recycling of the P2X 4 receptor [55].…”

“…We have shown that P815 mastocytoma cells (derived from DBA/2 mice) express mRNA for all P2X and P2Y receptors, while BMMCs derived from C57BL/6 mice express mRNA for P2X [1][2][3][4] , P2X [6][7] , and all P2Y receptors with the exception of P2Y 2 [9]. MC/9 cells (derived from fetal liver of a (B6 X A/J)F 1 mouse) express P2X 1-2 , P2X 4 , P2X 6-7 , P2Y 6 , P2Y 13 , and P2Y 14 receptors [9]. The expression of P2X 7 receptor protein on BMMCs, P815, and MC/9 cells was shown by immunoblotting analysis [9] and fluorescenceactivated cell sorter (FACS) [54].…”

“…MC/9 cells (derived from fetal liver of a (B6 X A/J)F 1 mouse) express P2X 1-2 , P2X 4 , P2X 6-7 , P2Y 6 , P2Y 13 , and P2Y 14 receptors [9]. The expression of P2X 7 receptor protein on BMMCs, P815, and MC/9 cells was shown by immunoblotting analysis [9] and fluorescenceactivated cell sorter (FACS) [54]. Expression of P2X 1 receptor was confirmed by FACS while expression of P2X 3 receptor by immunoblotting [54].…”

“…, or oxidized ATP (oATP) [9]. In the P2X 7 receptor knockout (P2X 7 −/− ) BMMCs, ATP triggered P2X 1 and P2X 3 receptor activation.…”

“…Years later, families of P2Y and P2X purinergic receptors were cloned and characterized. Mast cells are not only a great source of ATP which is stored in their secretory granules and released upon stimulation [7], but they also express a variety of purinoceptors which trigger mast cell degranulation, cytokine secretion, chemotaxis, and apoptotic cell death [3,8,9]. In this review, we discuss the role of the P2 receptors and nucleotide signaling in mast cell biology.…”