Abstract
Purpose :
Primary open angle glaucoma (POAG) is characterized by a loss of retinal ganglion cells and structural changes at the optic nerve head (ONH). Fibrosis in the form of extracellular matrix (ECM) deposition occurs at the lamina cribrosa (LC) region of the ONH and glaucomatous LC cells exhibit an increase in ECM and profibrotic factors, along with dysregulation of cellular growth and responses. Genome wide association studies have identified several single nucleotide polymorphisms within the 9p21 gene locus that are associated with glaucoma risk. The cyclin D Kinase inhibitor (CDKN)2B gene is located within this locus and encodes a key cell cycle regulator involved in inhibiting the Retinoblastoma (Rb) signalling pathway. Alterations in this pathway are typically associated with dysregulation of the cell cycle. In particular, gene silencing of CDKN2B by promoter hypermethylation is observed in several cancers and has been reported in fibrotic diseases. The aim of this study is to investigate if CDKN2B is regulated by promoter methylation in the LC in POAG.
Methods :
LC cells were cultured from age-matched control and confirmed glaucomatous donors (GLC). Promoter methylation was analysed using a methylated DNA immunoprecipitation (MeDIP) assay. Gene expression was quantified using quantitative real-time PCR. GLC cells were treated with the methylation inhibitor 5-azacytidine (0.3 µM) for 24 hours.
Results :
The promoter region of CDKN2B was found to be significantly methylated in GLC cells (P<0.05) compared to controls following MeDIP analysis. Expression of CDKN2B was decreased in GLC cells compared to controls, in concurrence with increased promoter methylation observed in GLC cells. Treatment of GLC cells with 5-azacytidine upregulated CDKN2B and decreased expression of the ECM gene collagen (COL)1A1. Additionally, gene expression of Rb pathway components cyclin (CCN)D1, cyclin D kinase (CDK)4, CDK6 and the transcription factor E2F1 were found to be increased in GLC cells compared to controls.
Conclusions :
These results show that the promoter of CDKN2B is methylated in the LC in POAG, resulting in downregulation of a key cell cycle regulator within the Rb pathway. Coupled with an increase in expression of the other main players in this pathway, this may explain, at least in part, dysregulated cell cycle control observed in fibrotic GLC cells.
This is a 2021 ARVO Annual Meeting abstract.