p53, MDM2, and the Ubiquitin-Proteasome System in Glaucomatous Lamina Cribrosa Cells

Objectives: Lamina cribrosa (LC) cells play an integral role in extracellular-matrix remodelling and fibrosis in human glaucoma. LC cells bear similarities to myofibroblasts that adopt an apoptotic-resistant, proliferative phenotype, a process linked to dysregulation of tumour-suppressor-gene p53 pathways, including ubiquitin-proteasomal degradation via murine-double-minute-2 (MDM2). Here, we investigate p53 and MDM2 in glaucomatous LC cells. Methods: Primary human LC cells were isolated from glaucomatous donor eyes (GLC) and age-matched normal controls (NLC)(n=3 donors/group). LC cells were cultured under standard conditions +/- 48hours treatment with p53-MDM2-interaction inhibitor RG-7112. Markers of p53-MDM2, fibrosis, and apoptosis were analysed by real-time polymerase-chain-reaction (qRT-PCR), western blotting, and immunofluorescence. Cellular proliferation and viability were assessed using colorimetric methyl-thiazolyl-tetrazolium salt assays (MTS/MTT). Results: In GLC versus NLC cells, protein expression of p53 was significantly decreased (p<0.05), MDM2 was significantly increased, and immunofluorescence showed reduced p53 and increased MDM2 expression in GLC nuclei. RG-7112 treatment significantly increased p53 and significantly decreased MDM2 gene and protein expression. GLC cells had significantly increased protein expression of COL1A1 and αSMA, and significantly decreased BAX and caspase-3 protein expression, and significantly increased proliferation after 96 hours. RG-7112 treatment significantly decreased COL1A1 and αSMA and significantly increased BAX and caspase-3 gene expression, and significantly decreased proliferation in GLC cells. MTT-assay showed equivocal cellular viability in NLC/GLC cells with/without RG- 7112-treatment. Conclusion: Our data suggests that proliferation and ubiquitin-proteasomal pathway is significantly dysregulated in GLC cells with MDM2 led p53 protein degradation negatively impacting its protective role. Targeting the p53-MDM2-ubiquitin- proteasomal pathway warrants further clinical investigation.