Polymorphism rs11200638 enhanced HtrA1 responsiveness and expression are associated with age-related macular degeneration

Abstract

Objectives: To investigate the role of polymorphism rs11200638 of high-temperature requirement factor A-1 (HtrA1) gene in the pathogenesis of age-related macular degeneration (AMD). Methods: Cultured adult retinal pigment epithelial cells (ARPE-19) expressing HtrA1 gene were treated with H2O2 or lipopolysaccharides (LPS) and analysed using western blot and quantitative polymerase chain reaction to illustrate the effects of oxidative and inflammatory stress on HtrA1 gene expression. Luciferase reporter plasmid driven by HtrA1 promoter with either normal allele G or risk allele A at SNP rs11200638 was transfected to ARPE-19 cells to investigate the effect of the G/A variation on HtrA1 promoter activity. The effects of HtrA1 overexpression on ARPE-19 cells were analysed with respect to percentage of cell proliferation inhibition and cell apoptosis. Results: HtrA1 expression was significantly increased with LPS or H2O2 stimulations (p < 0.05). In ARPE-19 cells, HtrA1 promoters (−1 to −2175 bp from translation starting point) with risk allele A or normal G at rs11200638 did not show statistically significant differences in their luciferase reporter expression (p = 0.054425173), however, both promoters showed a persistent trend of higher luciferase expressions after 100 ng/ml LPS treatment. The luciferase expression level was significantly greater in the promoter with risk A when compared to that with normal G. Overexpression of HtrA1 resulted in apoptosis of ARPE-19 cells with 53.8 ± 1.6% of proliferation inhibition (p < 0.01). Conclusions: Risk haplotype A at rs11200638 significantly increased the responsiveness of HtrA1 promoter to inflammation and subsequently enhanced HtrA1 expression. HtrA1 overexpression induced ARPE-19 apoptosis and growth inhibition, relevant to pathogenesis of AMD.