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Gordon W Laurie, Ningning Wang, Jeffrey Romano, Robert L McKown; Anti-Stress and Basal Tear Stimulatory Tear Factor ‘Lacritin’ Promotes Acetylation of ATG101-Associated Proteins but has No Apparent Effect on Transepithelial Resistance. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2490.
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© ARVO (1962-2015); The Authors (2016-present)
Lacritin is a multifunctional tear protein whose active monomer is selectively downregulated in dry eye, and yet is required to restore health to inflamed epithelia and apparently also stimulates corneal sensory nerves to promote basal tearing. Our dual purpose was to further explore lacritin-dependent signaling mechanisms associated with lacritin mediator ATG101 that rapidly accelerates autophagy and promotes oxidative phosphorylation to restore health, and how lacritin might gain contact with corneal sensory nerves, possibly by transiently loosening tight junctions for lacritin access.
Cultured human corneal epithelial (HCE) cells were stressed with dry eye-associated inflammatory cytokines INFG and TNF in the presence of lacritin or inactive lacritin truncation mutant C-25. Cell lysates were subjected to affinity precipitation with anti-ATG101 antibodies. Affinity precipitates were then blotted using antibodies against acetyl-lysine. To study possible alteration of tight junctions, transepithelial resistance of HCE or human corneal-limbal epithelial (HCLE) monolayers or of HCLE stratified cultures in normal or low calcium media without or with lacritin or C-25 was measured.
Lacritin, but not C-25, promotes rapid acetylation of several ATG101 associated proteins in stressed HCE cells. Transepithelial resistance is greater in HCE cells than in HCLE cells, even of the latter in stratified culture. Addition of lacritin or C-25 equally decreased and then increased transepithelial resistance, whereas positive control low calcium media promoted the progressive loss of resistance that slowly returned up reintroduction of calcium.
Acetylation of ATG101 associated proteins upon lacritin, but not C-25 addition, in stressed HCE cells points to additional signaling mediators of lacritin’s health restorative activity. Lacritin appears to have no specific affect on transepithelial resistance, although non-specific loosening of the epithelium may be sufficient to gain neural access.
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