Proteasomes as a therapeutic target for abnormal rabbit corneal epithelium

Abstract

Purpose: The present study investigates two proteasome populations in corneal epithelium after surgically induced limbal stem cell deficiency (LSCD) in an experimental rabbit model.

Methods: As a result of LSCD, the increased inflammatory cells on the ocular surface caused a relative increase in the formation of immunoproteasomes (IPR) and to a decrease in the formation of 26S proteasome or constitutive proteasome (CPR) leading to a reduction in the rate of ubiquitinated protein clearance.

Results: We found that LSCD was associated with a significant reduction in CPR enzyme activities in corneal epithelium. Using healthy and diseased LSCD corneal epithelial cells, IPR specific subunit B5i levels were measured and compared to CPR B5 subunits levels. The results showed that the ratio B5i/B5 was higher in diseased epithelium compared to normal epithelium, which indicated that the diseased corneal epithelium had approximately twice as many IPRs compared to CPRs. Histological staining of normal rabbit eye revealed that the levels of IPR were higher in the conjunctival and limbal tissue compared to corneal epithelium. Since the limbal barrier is absent in our model of LSCD, it is possible that the cornea surface was invaded by conjunctival cells with a higher amount of IPR. K13, a marker of conjunctival cells, was also found in diseased corneal epithelium. Western blot analysis of oral mucosa epithelial cells treated with CPR inhibitor PS-341 showed that an increase in K13 levels compared to untreated cells. These results support the idea that reduced CPR activities is associated with K13 accumulation in abnormal corneal epithelium. Our previous studies showed that the administration of PS341 in experimental animals caused CPR inhibition for 24hrs followed by a rebound in the activity at 72hrs. These results suggest that CPR activation may increase the clearance of damaged or altered proteins including K13, in abnormal corneal epithelium.

Conclusions: We believe that intentional activation of CPR would be beneficial for treatment of abnormal corneal epithelium associated with LSCD