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S. Parameswaran, C. Spee, S. J. Ryan, R. Kannan, D. R. Hinton; A Novel Mechanism for Photoreceptor Protection: Selective Apical Secretion of B Crystallin From Polarized Human RRE Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3703.
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© ARVO (1962-2015); The Authors (2016-present)
Our laboratory showed that the small heat shock protein αB crystallin, has antiapoptotic properties in addition to its well known chaperone function. Our aim herein was to identify αB crystallin as a secretory protein in human RPE cells and characterize its asymmetry of secretion in human polarized RPE cells.
Early passage, confluent human fetal RPE cells grown in T75 flasks in DMEM were switched to serum free medium and the extracellular medium was collected and concentrated using filters. Exosomes were isolated using well established procedures. Release of αB crystallin to the extracellular medium through exosomes was confirmed by Western blot and immunogold transmission electron microscopy (TEM), using CD63 as an exosome marker. Secretion was also examined in the presence of lipid raft inhibitor (25µg/ml methyl-β-cyclodextrin) and classical protein transport inhibitor brefeldin (BF 7µg/ml), tunicamycin (TM 5µg/ml) and exosome inhibitor dimethyl amiloride (25µg/ml). Secretion of αB crystallin from the apical and basolateral domains was determined in polarized RPE monolayers with TER >340 Ω.cm2.
Western blot analysis and immunogold TEM revealed that RPE cells secrete αB crystallin into the extracellular medium. The secretion was by a nonclassical exosomal pathway. Inhibiting lipid rafts reduced the αB crystallin secretion significantly (p<0.01 vs controls). However, transport inhibitors BF and TM did not affect secretion. αB crystallin was predominantly secreted to the apical side toward the photoreceptors and the neural retina with undetectable secretion to the basolateral, choroidal side. In support, αB crystallin was found in the interphotoreceptor matrix (IPM) in retinal sections by immunofluorescence and TEM.
Our data show that αB crystallin is secreted by RPE through a lipid raft dependent exosomal pathway and the secretion is selective to the apical surface. Secretion of αB crystallin into the IPM could serve to protect the photoreceptors from injury.
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