Differential Lysyl oxidase like 1 expression in pseudoexfoliation glaucoma is orchestrated via DNA methylation.

Pseudoexfoliation syndrome (PXF) is the most common cause of secondary open angle glaucoma worldwide. Single nucleotide polymorphisms (SNPs) in the gene Lysyl oxidase like 1 (LOXL1) are strongly associated with the development of pseudoexfoliation glaucoma (PXFG). However, these SNPs are also present in 50-80% of the general population, suggestive of other factors being involved in the pathogenesis of PXFG. In this study, we aimed to investigate the influence of epigenetic regulation, specifically DNA methylation, on LOXL1 expression in PXFG using human tenons fibroblasts (HTFs), aqueous humour and serum samples from donors with and without PXFG. LOXL1 expression in HTFs was measured by qPCR and Western Blotting and LOXL1 concentration in aqueous humour was determined by ELISA. Global DNA methylation levels were quantified using an ELISA for 5-methylcytosine. MeDIP assays assessed the methylation status of the LOXL1 promoter region. Expression of methylation-associated enzymes (DNMT1, DNMT3a and MeCP2) were determined by qPCR and inhibited by 0.3 µM 5-azacytidine (5-aza). Results showed that LOXL1 expression was significantly decreased in PXFG HTFs compared with Control HTFs at gene (Fold change 0.37 ± 0.05, P < 0.01) level and showed a decrease, when measured at the protein level (Fold change 0.65 ± 0.42, P = 0.22), however this was not found to be significant. LOXL1 concentration was increased in the aqueous of PXFG patients compared with Controls (2.76 ± 0.78 vs. 1.79 ± 0.33 ng/ml, P < 0.01). Increased global methylation (56.07% ± 4.87% vs. 32.39% ± 4.29%, P < 0.01) was observed in PXFG HTFs compared with Control HTFs, as was expression of methylation-associated enzymes (DNMT1 1.58 ± 0.30, P < 0.05, DNMT3a 1.89 ± 0.24, P < 0.05, MeCP2 1.63 ± 0.30, P < 0.01). Methylation-associated enzymes were also increased when measured at protein level (DNMT1 5.70 ± 2.64, P = 0.04, DNMT3a 1.79 ± 1.55, P = 0.42, MeCP2 1.64 ± 1.33, P = 0.45). LOXL1 promoter methylation was increased in patients with PXFG compared to Control patients in both blood (3.98 ± 2.24, 2.10 ± 1.29, P < 0.05) and HTF cells (37.31 ± 22.0, 8.66 ± 10.40, P < 0.01). Treatment of PXFG HTFs with in 5-azacytidine increased LOXL1 expression when compared with untreated PXFG HTFs (Fold change 2.26 ± 0.67, P < 0.05). These data demonstrate that LOXL1 expression is altered in PXFG via DNA methylation and that reversal of these epigenetic changes may represent future potential therapeutic targets in the management of PXFG.

Lysyl Oxidase Like 1: Biological roles and regulation.

Lysyl Oxidase Like 1 (LOXL1) is a gene that encodes for the LOXL1 enzyme. This enzyme is required for elastin biogenesis and collagen cross-linking, polymerising tropoelastin monomers into elastin polymers. Its main role is in elastin homeostasis and matrix remodelling during injury, fibrosis and cancer development. Because of its vast range of biological functions, abnormalities in LOXL1 underlie many disease processes. Decreased LOXL1 expression is observed in disorders of elastin such as Cutis Laxa and increased expression is reported in fibrotic disease such as Idiopathic Pulmonary Fibrosis. LOXL1 is also downregulated in the lamina cribrosa in pseudoexfoliation glaucoma and genetic variants in the LOXL1 gene have been linked with an increased risk of developing pseudoexfoliation glaucoma and pseudoexfoliation syndrome. However the two major risk alleles are reversed in certain ethnic groups and are present in a large proportion of the normal population, implying complex genetic and environmental regulation is involved in disease pathogenesis. It also appears that the non-coding variants in intron 1 of LOXL1 may be involved in the regulation of LOXL1 expression. Gene alteration may occur via a number of epigenetic and post translational mechanisms such as DNA methylation, long non-coding RNAs and microRNAs. These may represent future therapeutic targets for disease. Environmental factors such as hypoxia, oxidative stress and ultraviolet radiation exposure alter LOXL1 expression, and it is likely a combination of these genetic and environmental factors that influence disease development and progression. In this review, we discuss LOXL1 properties, biological roles and regulation in detail with a focus on pseudoexfoliation syndrome and glaucoma.