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N. Wang, R. W. Raab, R. L. McKown, G. W. Laurie; Human Corneal Epithelial INFG/TNF-Dependent Cell Death (ITDCD) and Protection by Tear Prosecretory Mitogen Lacritin. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4265.
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Lacritin is a 12.3 kDa human tear protein secreted mainly by lacrimal acinar cells, with apparent contributions from corneal, conjunctival and meibomian epithelial cells. Mechanisms of lacritin cell targeting (Ma et al, J. Cell Biol. 174:1097-1106, 2006) and mitogenic signaling (Wang et al, J. Cell Biol. 174:689-700, 2006) have been partially worked out. However little is known of its suggested (Sharma et al, IOVS 2005; 46: E-Abstract 2119) cell protective activity against TNF. Inflammatory cytokines INFG and TNF are augmented in dry eye.
Recombinant human lacritin and negative control fragment C-25 (lacking 25 amino acids of lacritin’s C-terminus) were generated in E. coli and purified. Human corneal epithelial (HCE; Riken) cells were treated with TNF alone, or with INFG followed by TNF in the absence or presence of lacritin or C-25. Then HCE viability was assayed. Lacritin survival signaling was studied by phospho-blotting and with pharmacological inhibitors AKT-IV, STO-609, wedelolactone, EGCG, U0126, rapamycin and cyclosporine. Autophagic flux was examined using vinblastine and leupeptin. All reagents were optimized via dose response and time course analyses.
A) Sensitization with INFG, then treatment with TNF (and not TNF alone) is necessary to promote death. B) ITDCD appears to be non-apoptotic with no apparent DNA laddering or caspase-3 activation. Instead, ITDCD is coincident with LC3 lipidation, a marker of autophagy. C) Lacritin, but not C-25, is protective against ITDCD and rapidly blocks LC3 lipidation and promotes FOXO3a phosphorylation in a biphasic dose-dependent manner with a 10 nM optimum. C) Lacritin cell survival activity against ITDCD is inhibited by FOXO3a kinase inhibitors AKT-IV, STO-609 and wedelolactone, but not by EGCG or negative control U0126. Lacritin cell survival activity is also blocked by mTOR inhibitor rapamycin and calcineurin inhibitor cyclosporine. D) Cell flux studies in the absence or presence of leupeptin or baflomycin confirm that INFG/TNF treatment promotes autophagy.
Lacritin is protective against ITDCD in a biphasic dose dependent manner with a lacritin optimum of 10 nM. It appears to do so by rapidly promoting the phosphorylation of FOXO3a thereby retaining this pro-death transcription factor in the cytoplasm, and by blocking autophagy. However, whether ITDCD is by autophagy, or only coincident with autophagy, remains to be determined.
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