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Ningning Wang, Ronald W. Raab, Robert L. McKown, Gordon W. Laurie; Lacritin Rapidly Suppresses Inflammatory Stress In Human Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3712.
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The rapid stress response of interferon-gamma-sensitized human corneal epithelial cells to TNF includes the self-catabolic process known as autophagy. Autophagy is both a regulator and effector of innate and adaptive immunity. The human prosecretory mitogen lacritin, normally present in human tears, counters autophagy in a biphasic dose-dependent manner within 1 - 5 min. Here we explore the mechanism.
Human corneal epithelial (HCE; Riken) cells were sensitized with INFG and then treated with TNF in the presence of 10 nM lacritin or inactive C-25 lacritin for 0, 1, 5 or 15 min or at 0, 0.1, 1, 10, 100 or 1000 nM lacritin or C-25. In some cases, PLD inhibitor FIPI or SRC inhibitor PP2 were included. HCE cell lines constitutively overexpressing the AKT kinase dead mutant K179M or AKT T308A/S473A were developed and similarly treated. Lysates were collected and blotted for autophagosome marker LC3, and LC3 immunoprecipitates were blotted for mediators of autophagosome formation and turnover including ATG4B, ATG3, ATG7, ATG12, ULK1 and p62.
A) Lacritin, but not C-25, reduced the quantity of lipidated autophagosome marker LC3-II within 1 - 5 min via a biphasic dose response that is optimal at 10 nM. B) Ubiquitin-binding and NFkB pathway activator p62 (SQSTM1) binds LC3 via a biphasic lacritin, but not C-25 dose response, that is optimal at 10 nM. C) LC3 binding to LC3 activating protease ATG4B, to LC3 activating E1-like protein ATG7, to ATG12 and to autophagic serine/threonine kinase ULK1 was absent at 1 min in lacritin, but not C-25, treated cells. D) In ATG3 immunoprecipitations, lacritin appears to block ATG3-FLIP (short form) ligation. E) FIPI and PP2 both blocked lacritin anti-autophagic activity, as did AKT T308A/S473A but not AKT K179M.
Lacritin signaling rapidly blocks the cellular machinery needed for autophagosome formation when human corneal epithelial cells are subjected to stress from inflammatory cytokines. PLD, SRC and S473 AKT1 appear to be involved.
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