Abstract
Purpose: :
Dry eye is characterized by elevated tear inflammatory cytokines, including interferon-gamma (IFNG) and tumor necrosis factor-alpha (TNF) that when added to human corneal epithelial (HCE) cells promotes stress and diminish viability. Cells are rescued by inclusion of the tear protein lacritin. Characterization of the mechanism reveals that lacritin has no effect on some detectable caspase 9 and 3 cleavage, however it does rapidly diminish biochemical levels of LC3-II - a marker of autophagy that is elevated with INFG and TNF. One interpretation is that lacritin transiently suppresses autophagy, as is the case with several growth factors in other cell systems. An alternative novel hypothesis is that lacritin might be rapidly accelerating autophagy to rid stressed cells of damaged proteins. Here we address this question.
Methods: :
Retroviral pBabe-puro-mCherry-EGFP-LC3B (Addgene) was expanded in HEK293T cells, and used to transduce HCE cells (Riken). An mCherry-EGFP-LC3B stable line ‘LC3-RG’ was developed by puromycin selection. LC3-RG cells grown on glass cover slips, were sensitized overnight with IFNG and then treated with TNF in the presence of either 10 nM lacritin or inactive C25 lacritin for 10, 30, 60 minutes, or 24 hours. After washing, cells were fixed, rinsed, mounted and examined in a Zeiss LMS 700 confocal microscope. Images were processed using Fiji software object counter to examine punctate size and area. In biochemical studies, INFG sensitized HCE cells were treated with 10 nM lacritin without or with leupeptin, and then lysed and blotted for LC3.
Results: :
LC3 is a key functional player in the formation of autophagic isolation membranes and autophagosomes (AP), the latter of which fuse with lysosomes (autophagolysosome (APL) to acidify and digest its contents. Acidification negates GFP, but not mCherry. Using this readout, we observe that lacritin rapidly triggers a conversion of AP into APL, as early as 10 min after addition. Autophagic flux was further elevated at 60 min but was reduced, though still significantly elevated, at 24 hrs. LC3 blotting of leupeptin treated cells confirmed this observation.
Conclusions: :
Lacritin signaling appears to rapidly accelerate autophagic flux in a manner that increases APL’s in relation AP’s. This presumably rids stressed cells of damaged proteins, possibly via p62 and Alfy that are also lacritin responsive.
Keywords: cornea: tears/tear film/dry eye