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Ying-Ting Chen, Andreas Pollreisz, Supawadee Sukseree, Leopold Eckhart, Florian Gruber, Ursula Schmidt-Erfurth; The Roles of Autophagy in Limbal Stem Cells for Post-ultraviolet Corneal Regeneration. Invest. Ophthalmol. Vis. Sci. 2014;55(13):500.
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Autophagy is a cellular program for the lysosomal degradation of damaged organelles, protein aggregates and bulk cytoplasm. Critical roles of autophagy have been reported for various epithelia exposed to cellular stress caused by UV radiation and implicated in stem cell-mediated tissue regeneration. In current study we aim to establish an in vivo model for determining the role(s) of autophagy for limbal stem cells’ (LSC) stress response.
Mice carrying a floxed alleles of the essential autophagy-related gene Atg7 were crossed with mice expressing the Cre recombinase under the control of the basal progenitor’s Krt14 promoter. This led to selective deletion of Atg7 in the basal progenitors at the limbus. UVA at the dose of 100J/cm2 was applied to Atg7f/f Krt14-Cre mice and Atg7f/f controls. RNA and protein lysate of enzymatically isolated corneal epithelia were collected for qPCR and WB. Rapid adhesion with FN was used to enrich Krt14-expressing basal progenitors for determining the number of basal cells and the roles of autophagy for LSC during tissue repair.
Before UVA exposure, lissamine green staining revealed that the barrier function of corneal epithelium in Atg7f/f Krt14-Cre mice was not compromised and histology revealed no change in microarchitecture of stratified corneal epithelium. WB of Atg7f/f controls expressed basal expression of autophagy-associated LC3-I and LC3-II proteins, while absent in Cre mice. Six hours after UVA exposure, WB analysis revealed accumulation of autophagy-associated LC3-II protein in the corneas of Atg7f/f controls with a LC3 I to LC3 II isoform shift, in contrast to negative signals in Atg7f/f Krt14-Cre mice. In organotypic culture for 24 hours, Atg7f/f Krt14-Cre corneas revealed punctate corneal erosions while the controls regained the ocular surface integrity. LSC transcriptional profile by qPCR in controls mice revealed upregulated Atf3 and Ho-1anti-oxidant genes, while the Cre mice demonstrated downregulated p16INK4A (n=3, all p < 0.05)
The current study established Atg7f/f Krt14-Cre mice as a model suitable for characterizing the roles of autophagy in the LSC. In response to UVA radiation stress, autophagy might promote LSC’s anti-oxidative defence and be associated with p16-mediated cell cycle regulation.
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