Abstract
Purpose:
Single nucleotide polymorphism of high-temperature requirement factor A1 (HTRA1) has been shown strongly associated with increased risk for developing wet age-related macular degeneration (AMD). We have proved previously that amyloid β (Aβ) is a key component of drusen to trigger AMD. In this study, we investigated the effect of Aβ on expression and activity of HTRA1 in retinal pigment epithelial (RPE) cells.
Methods:
Primarily cultured human RPE cells were stimulated with 25 μM and 50 μM Aβ for 24 hours. HTRA1 mRNA expression and protein production in the supernatant were analyzed by real-time PCR and western blot. The binding between Aβ and HTRA1 was determined by co-immunoprecipitation. Activity of HTRA1 preincubated with/without 50 μM Aβ for 1 hour was measured by activity assay using β-casein substrate.
Results:
Aβ treatment induced a significant dose-dependent increase of HTRA1 mRNA expression and protein production in the supernatant of cultured human RPE cells. Aβ could efficiently bind to HTRA1 in vitro in co-immunoprecipiation analysis. In activity assay, HTRA1 preincubated with Aβ could more intensely degrade β-casein compared to HTRA1 preincubated without Aβ.
Conclusions:
These results suggest the mechanism that Aβ accumulated in drusen causes over-expression and enhanced activity of HTRA1 in subretinal space. This phenomenon might be an important process for the development of wet AMD.
Keywords: 695 retinal degenerations: cell biology