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Fawzia Bardag-Gorce, Andrew Makalinao, Richard Hoft, Amanda Laporte, Jeremy Stark, Imara Meepe, Joan Oliva, Yutaka Niihara; Activation of proteasome by inhibiting autophagy in corneal epithelia cells with limbal stem cell deficiency. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2601.
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© ARVO (1962-2015); The Authors (2016-present)
Autophagy and the ubiquitin proteasome pathway (UPP) are the two most important pathways of cellular protein degradation. It is well established that when UPP is defective, autophagy is stimulated to compensate for UPP failure. Our previous study on rabbit corneal epithelial cells with limbal stem cell deficiency (LSCD) has shown that autophagy was activated when UPP function is defective. However, damaged and unwanted proteins, especially keratin K4 and K13 aggregates, still accumulate, possibly contributing to corneal epithelial cell (CEC) dysfunction. The goal of the present study was to modulate autophagy and UPP activity with a focus on damaged keratin clearance.
In experiment A, rabbits with surgically-induced LSCD were used to quantify the elements of both pathways in corneal epithelial cells. In experiment B, a mechanistic study was performed using rabbit oral mucosa epithelial cells (OMECS) because, similar to conjunctival epithelial cells, they are rich in keratin K4 and K13. OMECS were isolated, cultured and treated with proteasome inhibitor, and with chloroquine to inhibit autophagy.
Experiment A: Morphologic analysis of corneal tissue sections showed that both pathways stained positive in normal corneal epithelium. Western blot analysis showed that while constitutive proteasome beta subunits (B1, B2 and B5) were decreased, autophagy biomarker such as ATG5 and MAPLC3B were significantly increased in LSCD-CEC. However, despite autophagy up regulation, modified keratins K4 and K13 still deposited and accumulated in LSCD-CEC without clearance. Experiment B: The mechanistic study of proteasome inhibition in OMECS, also showed a significant increase in autophagy biomarkers (ATG12, ATG5 and MAPLC3) confirming our observation that when UPP is defective, autophagy is stimulated. In addition when autophagy was inhibited by treating OMECS with chloroquine, the results showed not only an increase in proteasome chymotrypsin-like activity but also a significant decrease in unmodified K4 and K13 levels with no keratin high molecular weight deposition.
These results indicated that inhibition of autophagy was efficient in activating the proteasome pathway, which may stimulate protein degradation to improve the health and function of CEC, and alleviate the symptoms of LSCD. Supported by Emmaus Life Sciences.
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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