Abstract
Purpose :
Primary open-angle glaucoma (POAG) is an age-related fibrotic condition and a leading cause of irreversible blindness worldwide. POAG-related damage is initiated within the lamina cribrosa (LC) region of the optic nerve head, driven by the pathological activation of resident LC cells. LC cells bear striking similarities to proliferative, apoptotic-resistant myofibroblasts known to be responsible for organ fibrosis. Myofibroblast dysregulation is linked to targeted proteasomal degradation of p53 by the E3 ubiquitin-protein ligase MDM2 (mouse-double-minute-2) thus negating p53’s important regulatory role in cell-cycle/apoptosis. This project aims to evaluate the role of p53, MDM2, and the ubiquitin-proteasomal pathway in glaucomatous LC cells.
Methods :
Primary human normal LC (NLC) and glaucoma LC (GLC) cells (n=3 donors) were cultured under standard conditions and treated for 48 hours with RG-7112 (p53-MDM2 interaction inhibitor, Abcam). The p53-MDM2 ubiquitin-proteasomal pathway was analysed by real-time polymerase chain reaction (qRT-PCR) for gene expression and protein levels via western blotting.
Results :
MDM2 gene expression levels were significantly elevated (p=0.006) in GLC cells (1.00 ±0.12) versus NLC cells (0.89 ±0.08). p53-MDM2 inhibitor RG-7112 treatment caused a further significant (p=0.001) increase in MDM2 transcription levels in GLC cells (1.17 ±0.04). p53 transcription levels were equivocal between GLC cells (0.88 ±0.09) and NLC cells (0.87 ±0.08), with RG-7112 treatment leading to a significant increase (p=0.028) in p53 transcription levels in GLC cells (0.95 ±0.06). Western blot analysis showed significant decreased protein expression levels of p53 (0.76 ±0.09)(p=0.047) and increased protein expression of MDM2 (1.57 ±0.33)(p=0.042) in GLC cells compared to NLC. Interestingly, p53-MDM2 inhibitor RG-7112 treatment increased p53 (1.14 ±0.43)(p=0.267) and decreased MDM2 protein expression levels (0.92 ±0.25)(p=0.06) in GLC cells.
Conclusions :
Our data suggests the ubiquitin-proteasomal pathway is significantly dysregulated in GLC cells with MDM2 led p53 protein degradation negatively impacting its key role as “guardian of the genome”. Targeting the p53 ubiquitin-proteasomal pathway in lamina cribrosa fibrosis may lead to future novel therapeutic interventions.
This is a 2021 ARVO Annual Meeting abstract.