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Ying-Ting Chen, Maria Laggner, Ursula Schmidt-Erfurth, Andreas Pollreisz; Unfolded protein response coordinates autophagy in maintaining proteostasis and clonal survival of limbal stem cells under UVA stress. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4240.
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© ARVO (1962-2015); The Authors (2016-present)
Unfolded protein response (UPR) and autophagy are two evolutionarily conserved pathways for maintaining protein homeostasis. Here we test the hypothesis whether an integrated induction of UPR and autophagy serves as an adaptive mechanism to increase clonal survival of limbal stem cells (LSC) under UVA-induced proteotoxic stress.
Low fluency UVA was applied to Krt14-Cre:Atg7f/f transgenic mice and ATG7-knockdown primary human LSC culture. Transcriptional regulators and effectors for UPR and autophagy pathways were profiled by qPCR arrays and immunoblotting. Intracellular ROS and proteotoxic stress were measured by CM-H2DCFDA live staining and by OxyIHC Oxidative Stress Detection Kit. Autophagic activity and apoptotic response were determined by CytoID staining and Caspase3/7 CytoEvent.
In autophagy-competent LSCs, UVA induced autophagy as evidenced by 35+/-5% increase of CytoID (+) cells in culture, and LC3II lipidation and 15% reduction of p62 in WB. In company, regulators and effectors of UPR in PERK arm were transcriptionally activated (EIF2AK3 1.27-fold, ATF4 1.85-fold, ATG12 2.05-fold, MAP1LC3B 1.21-fold, HMOX1 1.61-fold), while pro-apoptotic factors BAD, BAX and BID were down-regulated. After UVA, levels of intracellular ROS, oxidized protein and Caspase 3/7(+) cells remained unaltered. In autophagy-deficient LSCs, UVA failed to induce autophagy. WB probing p62 and ubiquitin demonstrated the conjugation of p62 with ubiquitinated polypeptides ranging from low (62 kD) to high molecular weight (>250 kD), suggestive ofmisfolded protein aggregates. ROS and protein oxidation were increased and correlated with caspase 3/7 apoptotic activity. At transcriptional level, EIF2AK3 and ATF4 of UPR remained upregulated (1.84-fold, 1.66-fold, respectively) while none of core autophagy or antioxidant genes was active. 2.08-fold decrease in BCL2 and 1.88-fold increase in BAX further supported the activation of apoptosis pathway.
Our data suggest that UPR might function as an ER stress sensor of oxidized protein aggregates and activate downstream cyto-protective autophagy through p62-ubiquitin adaptor protein. Modulators targeting the UPR-autophagy axis might be beneficial to prevent or treat UVA-associated LSC ocular surface diseases.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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