Purpose: Ubiquitin proteasome pathway (UPP) failure has been reported in case of rabbit Limbal Stem Cell Deficiency (LSCD). In the present study, we examined the effect of LSCD on autophagy in comparison to LSCD's effect on UPP. Methods: Rabbits with surgically induced LSCD were used. Corneal epithelial cells (CEC) were collected to measure the levels of autophagy and UPP biomarkers. Rabbit oral epithelial cells were isolated, cultured and treated with autophagy and proteasome inhibitors. Results: Immunofluorescent staining showed that both pathways were positive in normal corneal epithelium. However, while proteasome subunits were decreased in LSCD-CEC, autophagy biomarker MAPLC3B and ATG12-ATG5 complex were significantly increased in LSCD-CEC, compared to healthy CEC. These results indicate that LSCD-induced impairment of UPP may cause a compensatory stimulation of autophagy. However, despite autophagy up regulation, damaged and unwanted proteins, such as modified keratin K4 and K13 aggregates, still deposited and accumulated in LSCD-CEC without clearance, possibly contributing to CEC haziness. Proteasome inhibition in cultured oral epithelial cell sheet caused an increase in the expression of MAPLC3B and ATG12-ATG5 complex, supporting the observation that when UPP is failing, autophagy is stimulated. When autophagy was inhibited with chloroquine, proteasome activity was significantly increased compared to untreated cells. In addition, there a slight decrease in unmodified K4 and K13 expression levels with no keratin high molecular weight deposition when autophagy was inhibited. Conclusions: We report that inhibition of autophagy leads to an activation in proteasome activity, which may stimulate protein degradation to alleviate the symptoms of LSCD.
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