PAX6KD attenuates the uncurbed cell cycle response in ATG7KD LSCs following UVA irradiation.

<p>(A) Representative images of PCNA immunofluorescence in ATG7KD, PAX6KD and ATG7/PAX6 KD colonies following UVA irradiation. Scale bar, 100 μm. (B) Quantification of cells expressing PCNA in ATG7KD, PAX6KD or double KD LSCs. *<i>p</i> < .05, **<i>p</i> < .01.</p

Autophagy mediates cell cycle response by regulating nucleocytoplasmic transport of PAX6 in limbal stem cells under ultraviolet-A stress

<div><p>Limbal stem cells (LSC) account for homeostasis and regeneration of corneal epithelium. Solar ultraviolet A (UVA) is the major source causing oxidative damage in the ocular surface. Autophagy, a lysosomal degradation mechanism, is essential for physiologic function and stress defense of stem cells. PAX6, a master transcription factor governing corneal homeostasis by regulating cell cycle and cell fate of LSC, responds to oxidative stress by nucleocytoplasmic shuttling. Impaired autophagy and deregulated PAX6 have been reported in oxidative stress-related ocular surface disorders. We hypothesize a functional role for autophagy and PAX6 in LSC’s stress response to UVA. Therefore, human LSC colonies were irradiated with a sub-lethal dose of UVA and autophagic activity and intracellular reactive oxygen species (ROS) were measured by CYTO-ID assay and CM-H₂DCFDA live staining, respectively. Following UVA irradiation, the percentage of autophagic cells significantly increased in LSC colonies while intracellular ROS levels remained unaffected. siRNA-mediated knockdown (KD) of <i>ATG7</i> abolished UVA-induced autophagy and led to an excessive accumulation of ROS. Upon UVA exposure, LSCs displayed nuclear-to-cytoplasmic translocation of PAX6, while ATG7KD or antioxidant pretreatment largely attenuated the intracellular trafficking event. Immunofluorescence showing downregulation of proliferative marker PCNA and induction of cell cycle regulator p21 indicates cell cycle arrest in UVA-irradiated LSC. Abolishing autophagy, adenoviral-assisted restoration of nuclear PAX6 or antioxidant pretreatment abrogated the UVA-induced cell cycle arrest. Adenoviral expression of an ectopic PAX gene, PAX7, did not affect UVA cell cycle response. Furthermore, knocking down PAX6 attenuated the cell cycle progression of irradiated ATG7KD LSC by de-repressing p21 expression. Collectively, our data suggest a crosstalk between autophagy and PAX6 in regulating cell cycle response of ocular progenitors under UVA stress. Autophagy deficiency leads to impaired intracellular trafficking of PAX6, perturbed redox balance and uncurbed cell cycle progression in UVA-stressed LSCs. The coupling of autophagic machinery and PAX6 in cell cycle regulation represents an attractive therapeutic target for hyperproliferative ocular surface disorders associated with solar radiation.</p></div

Autophagy mediates PCNA expression and PAX6 cyto-localization in UVA-irradiated LSC colonies.

<p>(A) Dual immunofluorescent staining against PCNA and PAX6 in UVA-irradiated LSC colonies with or without adenoviral overexpression of <i>PAX6</i>. Scale bar, 100 μm. (B) Quantification of PAX6⁺PCNA⁺ cells. *<i>p</i> < .05, **<i>p</i> < .01, ***<i>p</i> < .001.</p

Model of autophagy-mediated regulation of PAX6 cyto-localization and cell cycle in UVA-stressed LSCs.

<p>In autophagy-competent LSCs, UVA activates autophagy by inducing ROS. In return, autophagic activity stabilizes intracellular ROS at physiological level. Furthermore, autophagy regulates cell cycle by facilitating cytoplasmic export of nuclear PAX6, which normally represses p21 in PCNA⁺, proliferative LSCs. In lack of functional autophagy, UVA induces excess levels of ROS, PAX6 is retained in the nucleus and the p21-mediated cell cycle response is hampered.</p

Autophagy mediates nuclear export of PAX6 in response to UVA-elicited oxidative stress.

<p>(A) Representative immunofluorescent images depicting subcellular localization of PAX6 in controls and H₂O₂-treated LSCs. Scale bar, 50 μm. (B) Quantification of cells expressing nuclear PAX6 after H₂O₂ treatment. (C) Representative micrographs of PAX6 subcellular localization in LSCs exposed to UVA with or without antioxidant pretreatment. Arrowheads indicate nuclear localization of PAX6, asterisks denote cytoplasmic distribution of PAX6. Scale bar, 100 μm. (D) Percentage of LSCs expressing nuclear PAX6. *<i>p</i> < .05, **<i>p</i> < .01.</p

PAX6KD restores UVA-induced, p21-mediated cell cycle arrest in ATG7KD LSC.

<p>(A) Immunofluorescence of p21 in LSC colonies with KD of ATG7, PAX6 or both. Scale bar, 100 μm. (B) Quantification of p21-expressing cells in ATG7KD, PAX6KD or ATG7/PAX6 double KD LSCs. *<i>p</i> < .05, **<i>p</i> < .01.</p

Validation of ATG7KD as an <i>in vitro</i> model of autophagy-deficient human LSCs.

<p>(A) Knockdown efficiency of <i>ATG7</i> verified by qPCR analysis. (B) Western blot analysis of ATG7 expression in SCR and ATG7KD LSC colonies. (C) ATG7 expression in SCR and ATG7KD LSCs assessed by immunofluorescence. Scale bar, 50 μm. (D) Representative micrographs of autophagosome staining by CYTO-ID assay. Arrowheads indicate autophagosomal vesicles, asterisks denote diffuse pattern of autophagic components. Scale bar, 50 μm. (E) Quantification of autophagosomes in SCR and ATG7KD LSCs in response to rapamycin treatment. *<i>p</i> < .05, **<i>p</i> < .01.</p

Autophagy is activated during LSC’s stress response to UVA.

<p>(A) Representative images of autophagosomes in ATG7KD LSCs under UVA stress. Arrowheads show autophagic cells, asterisks indicate absence of autophagosomes. Scale bar, 50 μm. (B) Quantification of cells with autophagic activity in response to UVA. (C) Representative western blot image of autophagic flux in UVA-irradiated LSC colonies in absence and presence of BafA1, with or without UVA. LC3B-I and II were detected by immunoblotting at indicated time points. (D) Densitometric analysis of LC3B-II expression normalized to GAPDH. n = 3, *<i>p</i> < .05.</p

Autophagy regulates intracellular ROS levels in LSCs under UVA stress.

<p>(A) Intracellular ROS determined by CM-H₂DCFDA staining of SCR and ATG7KD LSCs, with or without UVA and antioxidant pretreatment. Scale bar, 100 μm. (B) Quantification of ROS levels by mean fluorescence intensity. *<i>p</i> < .05, **<i>p</i> < .01.</p

Autophagy mediates p21 induction in response to UVA via nuclear export of PAX6.

<p>(A) Micrographs of p21 and PAX6 cyto-localization in irradiated SCR and ATG7KD LSCs with or without PAX6 overexpression. Arrowheads denote PAX6⁺ p21^- nuclei, asterisks indicate cells expressing nuclear p21 and cytoplasmic PAX6. Scale bar, 50 μm. (B) Quantification of p21⁺ nuclei. *<i>p</i> < .05, ***<i>p</i> < .001.</p