Autoantibodies specific for alpha-fodrin fragments are found in the tissues of persons afflicted with Sjögren’s syndrome (SS). However, the mechanism for alpha-fodrin degradation remains elusive. The following experiments utilized Par C5 cells to examine the role of P2X7 receptor (P2X7R) in apoptosis, particularly in the cleavage and release of alpha-fodrin, an apparent SS autoantigen. Five mM ATP stimulation induced apoptotic cell death with a sustained Ca2+ influx, which was mimicked in HEK cells transfected with P2X7R. ATP also induced cleavage of alpha-fodrin mediated by caspase-3 and calpain, releasing alpha-fodrin fragments through membrane blebs. However, both apoptotic cell death and alpha-fodrin cleavage were inhibited in the presence of 300 μM oxidized-ATP (ox-ATP), an irreversible blocker of P2X7R, or in Ca2+-free solution. We concluded that P2X7R plays an important role in apoptosis and alpha-fodrin degradation in salivary epithelial cells, providing an important clue elucidating the presence of alpha-fodrin fragments in SS tissues.
Get full access to this article
View all access options for this article.
References
1.
Bellone M -2000-. Apoptosis, cross-presentation, and the fate of the antigen specific immune response. Apoptosis5:307–314.
2.
Jung EM, Lee TJ, Park JW, Bae YS, Kim SH, Choi YH, et al. -2008-. The novel phospholipase C activator, m-3M3FBS, induces apoptosis in tumor cells through caspase activation, down-regulation of XIAP and intracellular calcium signaling. Apoptosis13:133–145.
3.
Kahaly GJ, Bang H, Berg W, Dittmar M -2005-. Alpha-fodrin as a putative autoantigen in Graves’ ophthalmopathy. Clin Exp Immunol140:166–172.
4.
Li J, Ha YM, Ku NY, Choi SY, Lee SJ, Oh SB, et al. -2004-. Inhibitory effects of autoantibodies on the muscarinic receptors in Sjögren’s syndrome. Lab Invest84:1430–1438.
5.
Limesand KH, Barzen KA, Quissell DO, Anderson SM -2003-. Synergistic suppression of apoptosis in salivary acinar cells by IGF1 and EGF. Cell Death Differ10:345–355.
6.
Maniati E, Potter P, Rogers N, Morley B -2008-. Control of apoptosis in autoimmunity. J Pathol214:190–198.
7.
Maruyama T, Saito I, Hayashi Y, Kompfner E, Fox RI, Burton DR, et al. -2004-. Molecular analysis of the human autoantibody response to alpha-fodrin in Sjögren’s syndrome reveals novel apoptosis-induced specificity. Am J Pathol165:53–61.
Miyazaki K, Takeda N, Ishimaru N, Omotehara F, Arakaki R, Hayashi Y -2005-. Analysis of in vivo role of alpha-fodrin autoantigen in primary Sjögren’s syndrome. Am J Pathol167:1051–1059.
10.
Nagaraju K, Cox A, Casciola-Rosen L, Rosen A -2001-. Novel fragments of the Sjögren’s syndrome autoantigens alpha-fodrin and type 3 muscarinic acetylcholine receptor generated during cyto-toxic lymphocyte granule-induced cell death. Arthritis Rheum44:2376–2386.
11.
Nobile M, Monaldi I, Alloisio S, Cugnoli C, Ferroni S -2003-. ATP-induced, sustained calcium signalling in cultured rat cortical astrocytes: evidence for a non-capacitative, P2X7-like-mediated calcium entry. FEBS Lett538:71–76.
12.
North RA -2002-. Molecular physiology of P2X receptors. Physiol Rev82:1013–1067.
13.
Novak I -2003-. ATP as a signaling molecule: the exocrine focus. News Physiol Sci18:12–17.
14.
Orrenius S, Zhivotovsky B, Nicotera P -2003-. Regulation of cell death: the calcium-apoptosis link. Nat Rev Mol Cell Biol4:552–565.
15.
Routsias JG, Tzioufas AG -2007-. Sjögren’s syndrome—study of autoantigens and autoantibodies. Clin Rev Allergy Immunol32: 238–251.
16.
Sergeev IN, Li S, Colby J, Ho CT, Dushenkov S -2006-. Polymethoxylated flavones induce Ca-2+--mediated apoptosis in breast cancer cells. Life Sci80:245–253.
17.
Sisto M, Lisi S, Castellana D, Scagliusi P, D’Amore M, Caprio S, et al. -2006-. Autoantibodies from Sjögren’s syndrome induce activation of both the intrinsic and extrinsic apoptotic pathways in human salivary gland cell line A-253. J Autoimmun27:38–49.
18.
Surprenant A, Rassendren F, Kawashima E, North RA, Buell G -1996-. The cytolytic P2Z receptor for extracellular ATP identified as a P2X receptor -P2X7-. Science272:735–738.
19.
Tombal B, Denmeade SR, Gillis JM, Isaacs JT -2002-. A supramicromolar elevation of intracellular free calcium -[Ca-2+-]-i-- is consistently required to induce the execution phase of apoptosis. Cell Death Differ9:561–573.
20.
Wang KK -2000-. Calpain and caspase: can you tell the difference? Trends Neurosci23:20–26; erratum in Trends Neurosci 23:59, 2000.
21.
Wang Q, Wang L, Feng YH, Li X, Zeng R, Gorodeski GI -2004-. P2X7 receptor-mediated apoptosis of human cervical epithelial cells. Am J Physiol Cell Physiol287:1349–1358.
22.
Wang Y, Virji AS, Howard P, Sayani Y, Zhang J, Achu P, et al. -2006-. Detection of cleaved alpha-fodrin autoantigen in Sjögren’s syndrome: apoptosis and co-localisation of cleaved alpha-fodrin with activated caspase-3 and cleaved poly-ADP-ribose- polymerase -PARP- in labial salivary glands. Arch Oral Biol51:558–566.
23.
Willeke P, Gaubitz M, Schotte H, Becker H, Mickholz E, Domschke W, et al. -2007-. Clinical and immunological characteristics of patients with Sjögren‘s syndrome in relation to alpha-fodrin antibodies. Rheumatology -Oxford-46:479–483.
24.
Young FM, Phungtamdet W, Sanderson BJ -2005-. Modification of MTT assay conditions to examine the cytotoxic effects of amitraz on the human lymphoblastoid cell line, WIL2NS. Toxicol In Vitro19:1051–1059.
25.
Zhang X, Zhang M, Laties AM, Mitchell CH -2005-. Stimulation of P2X7 receptors elevates Ca2+ and kills retinal ganglion cells. Invest Ophthalmol Vis Sci46:2183–2191.
