Impairment of the Ubiquitin-Proteasome Pathway Alters Gene Expression in the EGFR Pathway during Lens Regeneration in Rabbit

Abstract

Purpose : The object of this study is to investigate the role of UPP in lens regeneration and wound-healing.

Methods : Thirty six 4-week-old New Zealand albino rabbits were randomly divided into two groups (Group A: capsulorhexis of 2.0±0.5 mm, Group B: capsulorhexis of 6.0±0.5 mm). The surgeries were performed in the left eyes, and the right eyes were used as baseline control. The intracellular ubiquitinated proteins are relative abundant, therefore the ubiquitinated capacity of the UPP was presented as the amount of ubiquitinated proteins probed with Western Blot at day 3 post-op, when there was few of regenerated proteins. The I¹²⁵-labeled protein degradation assay was used to determine the capability of the UPP at day 7 post-op, when the regenerated proteins were enriched and the degradation ability of the UPP could be determined in a more direct way. A mutant crystalline T5P-γC, which is a known substrate of the UPP, was used as degradation substrate. The regenerated lens cells were collected at day 1, 3, and 7 post-op respectively, and the temporal alteration in gene expression of growth and inflammation related genes was defined with the QuantiGene Plex (QGP) 2.0 system. The target genes of the QGP included EGF, FGF10, FGF2, HBEGF, IL-A, IL-1β, IL-6, KGF, PDGF-A, TGF α, TGF-β1, TGF-β2, TGF-β3, and TNF, the levels of which were normalized with the gene level of GAPDH. The differences between groups were spastically analyzed with the ANOVO variance analysis. The morphology of the regenerated lenses was evaluated with slit lamp photography.

Results : The decreasing in the ubiquitinated ability and degradation capacity of the UPP was validated with the ubiquitinated assay and protein degradation assay. Subsequent alteration in the EGFR pathway was found with the QGP, including temporal elevation in the levels of IL-1β, EGF and HBEGF. The decline of the UPP and elevation of these genes were more dramatic in the Group B. There was no temporal alteration detected in the other target genes. Compared to the relative transparent regenerated lenses in the Group A, the regenerated lens fibers in the Group B were disorganized and opaque.

Conclusions : Our results indicate that the impairment of the UPP in the regenerated lens cells may lead to the alteration in the EGFR pathway, disrupting the process of lens regeneration.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.