Hypomethylation of thrombospondin-1 promoter region is associated with reduced aqueous humor flow

Prolonged use of dexamethasone (DEX) elevates intraocular pressure (IOP) and increases the risk of developing glaucoma. In a previous study, we demonstrate that DEX stimulates the expression of thrombospondin-1 (THBS1) in primary human trabecular meshwork (hTM) cells, and that inhibiting THBS1 expression prevents DEX-induced elevation of IOP in mice. Therefore, we investigate the mechanism by which DEX regulates THBS1 expression. Treatment with the DNA methylation inhibitors, 5-azacytosine or 5-aza-2'-deoxycytidine, upregulates THBS1 protein levels in vitro and in vivo, reduces outflow facility in perfused mouse eyes, and elevates IOP in mice. In primary hTM cells, 7-day DEX treatment results in hypomethylation of the THBS1 promoter region and reduces transcript levels of 2 DNA methyltransferases (DNMTs), DNMT1 and DNMT3A. Taken together, we show that DEX reduces expression of DNMTs and DNA methylation of the THBS1 promoter region, supporting a critical role for THBS1 in DEX-induced outflow reduction. Overall design: To study the effect of dexamethasone (DEX) on DNA methylation in primary human trabecular meshwork (hTM) cells (n = 3 different cell strains, two male, one female), we treated hTM cells with 300 nM DEX or PBS (as vehicle-control) for 7 days, extracted the genomic DNAs and submitted to BGI (China) for whole genome bisulfite sequencing (WGBS).