ATP release due to Thy-1–integrin binding induces P2X7-mediated calcium entry required for focal adhesion formation

Abstract

SummaryThy-1, an abundant mammalian glycoprotein, interacts with avb3 integrin and syndecan-4 in astrocytes and thus triggers signaling events that involve RhoA and its effector p160ROCK, thereby increasing astrocyte adhesion to the extracellular matrix. The signaling cascade includes calcium-dependent activation of protein kinase Ca upstream of Rho; however, what causes the intracellular calcium transients required to promote adhesion remains unclear. Purinergic P2X7 receptors are important for astrocyte function and form large non-selective cation pores upon binding to their ligand, ATP. Thus, we evaluated whether the intracellular calcium required for Thy-1-induced cell adhesion stems from influx mediated by ATP-activated P2X7 receptors. Results show that adhesion induced by the fusion protein Thy-1-Fc was preceded by both ATP release and sustained intracellular calcium elevation. Elimination of extracellular ATP with Apyrase, chelation of extracellular calcium with EGTA, or inhibition of P2X7 with oxidized ATP, all individually blocked intracellular calcium increase and Thy-1-stimulated adhesion. Moreover, Thy-1 mutated in the integrin-binding site did not trigger ATP release, and silencing of P2X7 with specific siRNA blocked Thy-1-induced adhesion. This study is the first to demonstrate a functional link between avb3 integrin and P2X7 receptors, and to reveal an important, hitherto unanticipated, role for P2X7 in calcium-dependent signaling required for Thy-1-stimulated astrocyte adhesion.

Purinoreceptor P2X7 Regulation of Ca2+ Mobilization and Cytoskeletal Rearrangement Is Required for Corneal Reepithelialization after Injury

“…28 These studies are supported by others demonstrating that P2X7-dependent Ca 2þ signaling is necessary for focal adhesion formation and migration in astrocytes. 22 On the basis of this evidence, we hypothesized that P2X7 activation and the subsequent change in P2X7 localization after injury promote rapid cytoskeletal rearrangements at the leading edge. Immunofluorescence studies demonstrated that focal adhesion proteins in cells that were scratch wounded in the presence or absence of oxATP were altered.…”

“…P2X7 signaling has been shown in lung epithelial cells to be upstream of focal adhesion formation. 22 We therefore analyzed whether inhibition of P2X7 in corneal epithelial cells altered focal adhesions after injury. Cultures were incubated in the presence or absence of oxATP, and scratchwounded cells were stained for talin or vinculin.…”

Integrin-mediated transactivation of P2X7R via hemichannel-dependent ATP release stimulates astrocyte migration

“…We previously demonstrated that the P2X7R is required to increase Ca 2+ uptake and cell adhesion to the underlying matrix [20]. Here, we set out to study whether P2X7R is also required for Thy-1-stimulated migration of astrocytes.…”

“…Strong astrocyte adhesion induced by Thy-1 also requires increased extracellular adenosine 5'-triphosphate (ATP), activation of the purinergic P2X7 receptor (P2X7R), and Ca 2+ entry into the cell [20]. Whether this molecular mechanism is also required for cell migration and how ATP is released from the cells to the extracellular media are questions that remain to be explored.…”

“…We observed that incubation of DITNC1 astrocytes with the nucleotidase enzyme, Apyrase, precluded Thy-1-induced migration ( 1.6 ± 0.1, p < 0.001). The latter concentration was used in subsequent experiments (unless indicated otherwise) and coincides with that employed in our previous study [20].…”