P2X7 receptor mediated alpha-fodrin degradation and release in Par C5

Objectives: The cleaved forms of alpha-fodrin act as autoantigens producing autoantibodies in patients with Sjögren's Syndrome. However, the mechanisms regulating alpha-fodrin degradation and its release have not been elucidated. In this study, we investigated P2X7 receptor (P2X7R) -mediated alpha-fodrin degradation in Par C5 cells. Methods: ATP-induced cell death was assessed by MTT assay. Intracellular calcium was measured using microspectrofluorimetry. Enzyme activities were assessed by immunoblotting. Results: 5 mM ATP or 500 µM Bz-ATP, a specific P2X7R agonist, induced apoptosis and cell membrane blebbing, but 300 µM Ox-ATP, a P2X7R antagonist, inhibited all ATP-induced apoptosis. 5 mM ATP evoked a biphasic increase in cytosolic Ca2+ concentration which could only be elicited in ATP-stimulated HEK cells following transfection with P2X7R. ATP also induced cleavage of alpha-fodrin, which was mediated by caspase-3, caspase-9, release of cytochrome C and calpain activation. We found that degraded alpha-fodrins are released through membrane blebs. Conclusion: We conclude that cleavage and release of alpha-fodrin are mediated by P2X7R. Our results strongly suggest that P2X7R activation is a strong candidate inducing the autoimmune response via cleavage of alpha-fodrin in epithelial cells.