Abstract
Purpose :
Recently we reported a cytoprotective role for autophagy and Nrf2-ARE pathway in physiological UVA-stressed limbal stem cells (LSC). The current study sought to identify the mechanistic underpinnings linking these two pathways.
Methods :
Krt14-Cre:Atg7f/f transgenic mice and global Nrf2-KO mice on C57BL/6 background were used to generate Atg7-/- and Nrf2-/- LSCs, respectively. 3-MA was used to inhibit autophagy in cultured Nrf2-/- LSCs for creating autophagy-deficient Nrf2-KO LSCs. UVA irradiation at a low dose (10 J/cm2) was applied to the cultured LSCs, pre-treated with either ROS-scanvenger NAC or drug vehicle. CM-H2DCFDA live staining and Caspase3/7 CytoEvent were used to detect intracellular ROS levels and cellular apoptosis, respectively. Autophagic activity was measured by CytoID live staining. Dual immunofluorescence was used to study the cellular kinetics of Nrf2 and p62/SQSTM1. A nuclear localization of Nrf2 was used as a surrogate readout for Nrf2-ARE activity.
Results :
Confocal microscopy using CM-H2DCFDA live staining did not detect changes of ROS level in 10 J/Cm2-irradiated Atg7f/f LSCs. In contrast, a 2-3 fold elevation in ROS level was observed in Atg7-/-, Nrf2-/- and autophagy-deficient Nrf2-/- LSCs (all p<.05). Compared to the perspective non-irradiated baseline, Caspase3(+) apoptotic cells was increased by 3.5% (p>.05), 21.7% (p<.05), 29.3% (p<.05), and 18.4% (p<.05) in Atg7f/f, Atg7-/-, Nrf2-/- and autophagy-deficient Nrf2-/-LSC at 24-hr post-irradiation. The radiation-induced apoptosis was successfully rescued by NAC pre-treatment. Dual immunofluorescence revealed a diffuse cytosolic distribution for Nrf2 and p62 in unstressed LSCs, with a low-degree co-localization. After irradiation, Nrf2 nuclear translocation was observed exclusively in Atg7f/f cells with distinct p62 perinuclear speckle formation. Such a Nrf2/p62 redistribution was not seen in any other irradiated groups.
Conclusions :
Our data collectively suggest that autophagy activation in LSCs under physiological UVA stress functions as a non-canonical antioxidant defense mechanism. The p62/SQSTM1-mediated enhancement of Nrf2-ARE response enables LSC to have a tighter control in cellular redox. New therapeutics targeting p62/SQSTM1 might thus supplement the current antioxidant-based treatment for corneal degenerative diseases associated chronic oxidative stress.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.