Autophagy Regulates Redox Balance and Maintains Stemness of Limbal Stem Cells under UVA-induced Oxidative Stress

Abstract

 

Purpose

 

Limbal stem cells (LSC) are tissue reservoir cells for maintaining corneal homeostasis at resting state and repairing tissue upon UVA-induced oxidative damage. Autophagy is a lysosomal system for removal of damaged organelles. Recently, autophagy has been implicated in the redox homeostasis of adult stem cells in several types of tissues. Here, we aim to investigate the role of autophagy in LSC’s stress response to UVA-induced reactive oxygen species (ROS).

 

Methods

 

LC3-GFP:Krt14-Cre:Atg7^f/f transgenic mice were used as a LSC-specific autophagy-deficient model. Mouse eyes were subjected to UVA irradiation (0 - 80 J/cm²) and LSCs were enriched by rapid adhesion for RT-qPCR, immunofluorescence, flow cytometry and colony forming efficiency (CFE) assay.

 

Results

 

UVA at 10 J/cm² irradiance significantly activated autophagy in Atg7^f/f LSCs as evidenced by formation of cytoplasmic LC3-GFP punctae observed in 34 .2 + 3.4 % of irradiated cells versus 4.7 + 2.0 % in non-irradiated controls (p < 0.0001). Concomitantly, western blot (WB) analysis revealed a shift of LC3-I to LC3-II indicating autophagosome formation. In contrast, in response to UVA Atg7-deficient LSCs showed a diffuse, cytoplasmic LC3-GFP distribution without LC3-I to LC3-II conversion in WB. CM-H₂DCFDA live staining detected a 35 % increase of intracellular ROS in irriadiated Atg7-deficient LSCs compared to Atg7-sufficient counterparts (n = 4, p < 0.05). Furthermore, immunohistochemistry revealed oxidized proteins in 65 % of irradiated LSC colonies deficient in Atg7 versus 15 % in Atg7-sufficient controls. Such a discrepancy was compensated by pre-treating Atg7-deficient LSCs with an antioxidant mixture, i.e. N-acetylcystein and α-tocopherol, for 16 hours. UVA induced ROS up-regulated antioxidant master regulator Nrf2 and its downstream HO-1 effector in Atg7-suficient LSCs at transcriptional level, but not in Atg7-deficient counterparts (n = 6, p < 0.05). Lastly, compared to the non-irradiated Atg7-suficient and -deficient LSCs, CFE of irradiated LSC had a 45 % decrease in Atg7-suficient and a > 90 % decrease in Atg7-deficient groups (n = 3, p < 0.05).

 

Conclusions

 

Collectively, these data revealed a pivotal role of autophagy in reducing ROS for stem cell survival and maintaining stemness in vitro under UVA stress. In part, this cytoprotective effect might attribute to autophagy-activated Nrf2-HO-1 anti-oxidant pathway.