Limbal Stem Cells Activate Autophagy to Recruit Ocular Master Regulator PAX6 in Cellular Stress Response to Ultraviolet A Radiation

Abstract

Purpose : PAX6, a pleiotropic factor for corneal homeostasis and regeneration, has recently been implicated in LSCs’ cellular response to oxidative corneal injury. Solar UVA radiation is the major cause of oxidative stress and tissue damage in the ocular surface. Previously, we reported that autophagy plays a cytoprotective role for irradiated LSCs by reducing oxidative stress. Here, we sought to investigate a potential crosstalk of PAX6 and autophagy in the antioxidant defense mechanism of LSCs after radiation insult.

Methods : LSCs were obtained from Atg7^f/f and Krt14-Cre;Atg7^f/f mice as well as human corneoscleral rims. Autophagy-deficient human LSCs were generated by pharmacological inhibition with 3MA or by siRNA-assisted knockdown of ATG7. LSCs were subjected to 20 J/cm² UVA radiation. Autophagic activity was monitored by autophagosomal CytoID staining. ROS were determined by CM-H₂DCFDA dye. Apoptotic cells were quantified by Cas3/7 staining. RT-qPCR and immunofluorescence (IF) were employed to assess PAX6 expression.

Results : Compared to non-irradiated controls, UVA radiation induced autophagy by increasing 30% and 38% of autophagosome-forming cells in Atg7^f/f and human LSC colonies, respectively (p<.05). In contrast, autophagic activity of autophagy-deficient LSCs (Krt14-Cre;Atg7^f/f, ATG7^Δ and 3MA treated LSCs) remained unaltered by irradiation. Conversely, UVA radiation remarkably increased the intracellular ROS in autophagy-deficient LSCs with negligible effect in autophagy-competent controls. The impact of UVA irradiation on PAX6 expression was studied by RT-qPCR and IF. A 68% and 53% down-regulation of PAX6 mRNA levels were observed in autophagy-competent and –deficient LSC groups, respectively, compared to non-irradiated controls (p<.05). After UVA, nuclear-to-cytoplasmic translocation of PAX6 was observed in 28% of autophagy-competent LSCs, in stark contrast to no cytoplasmic redistribution detected in autophagy-deficient counterparts. Apoptosis was significantly induced by UVA in autophagy-deficient LSCs, with a 17% increase of cas3/7⁺ cells. NAC pretreatment rescued UVA-induced apoptosis and prevented PAX6 redistribution in autophagy context.

Conclusions : Reduction of intracellular ROS after UVA irradiation contributes to stem cell survival. Autophagy and redistribution of PAX6 synergistically constitute LSCs’ antioxidant defense.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.