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N. Wang, V. Talla, R. L. McKown, G. W. Laurie; Lacritin-Dependent Anti-Autophagic Survival Signaling in Human Corneal Epithelial Cells Involves Phospho-Dependent and Non-Canonical Phospho-Independent AKT Pathways. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4176.
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Human tear protein lacritin is protective against INFG/TNF-dependent death of human corneal epithelial cells. Cell death is surprisingly non-apoptotic and is blocked by inhibiting autophagy. Inhibitor studies and dominant negative FOXO3a, suggest that lacritin/AKT/FOXO3a and IKK/FOXO3a signaling is involved. Here we examine signaling mechanisms using phosphoblotting, inhibitor and cell survival studies.
Human corneal epithelial (HCE; Riken) cells were sensitized with INFG then treated with TNF in the absence or presence of lacritin or C-25, and/or different doses of inhibitors AKTIV, AKTVIII or PI103. Phosphorylation of AKT on threonine 308 and serine 473, and AKT-dependent activation of pGSK3 were monitored by Western blotting from anti-AKT immunoprecipitates. Parallel pFOXO3a; caspase 9, 3, 7; and PARP cleavage blotting were performed.
A) Inhibitors of phospho-independent (AKTIV) and -dependent (AKTVIII) AKT signaling block lacritin survival signaling including phosphorylation of FOXO3a, but subsequent cell death appears to differ with lacritin-dependent autophagy unaffected by AKTIV, but inhibited by AKTVIII. B) Lacritin promotes phosphorylation of AKT on serine 473, but not threonine 308 in keeping with lack of survival effect of PI3K inhibitor PI103. C) Phosphorylation slightly precedes phosphorylation of FOXO3a, yet AKTVIII has little effect on AKT kinase activity. AKTIV, however, does inhibit AKT kinase activity. D) INFG/TNF promotes cleavage of PARP, and caspases 9, 3 and 7 without DNA laddering - suggesting the possible involvement of IAP’s. Lacritin has no apparent effect on cleavage.
Taken together, these data suggest both phospho-dependent and-independent lacritin AKT/FOXO3a survival signaling. The phospho-independent pathway is non-canonical, with some similarity at the outset to the recently described incretin-triggered survival pathway in pancreatic beta cells.
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