Abstract
Purpose :
Oxidant-antioxidant imbalance plays an important role in the pathogenesis of Fuchs endothelial corneal dystrophy (FECD). AMP-activated protein kinase (AMPK) has been shown to confer anti-oxidant properties in part via the upregulation of nuclear factor erythroid 2-related factor 2 (NRF2), a transcription factor critical for anti-oxidant cellular defense. The role of AMPK in the corneal endothelium remains unknown. We hypothesized that AMPK activation induces NRF2-dependent anti-oxidant transcriptional programs critical for the suppression of DNA damage, thereby establishing a corneal endothelial protective phenotype.
Methods :
AMPK subunit expression in normal immortalized human corneal endothelial cells (HCECi) was examined by immunoblot analysis. HCECi were treated with varying doses, including 25, 100, 250, and 500 μM, of a small molecule, direct AMPK activator A-769662 or DMSO control for 24 hours. The effects of A-769662 in immortalized human Fuchs corneal endothelial cells (FECDi) were also investigated. Changes in mRNA levels of NRF2 as well as its target genes heme oxygenase 1 (HO-1) and NAD(P)H dehydrogenase quinone 1 (NQO1) were assessed by real-time quantitative PCR.
Results :
AMPK expression was detected by immunoblot in HCECi. NRF2 mRNA levels were signficantly increased in HCECi by 16.4 ± 4.8% (n=3; p<0.05) and 94.4 ± 20.0% (n=3; p<0.01) compared to control cells following 24 hour incubation with 100 and 500 μM A-769662. Levels of HO-1 mRNA were significantly upregulated by 44.1 ± 8.0% (n=3; p<0.01), 111.4 ± 30.0% (n=3; p<0.05), and 349.0 ± 91.6% (n=3; p<0.05) compared to control cells following incubation with 100, 250, and 500 μM A-769662. Similarly, NQO1 mRNA levels were dose-dependently upregulated by 17.6 ± 1.7% (n=3; p<0.001), 72.1 ± 10.0% (n=3; p<0.01), and 185.8 ± 20.0% (n=3; p<0.001) with 100, 250, and 500 μM A-769662. While incubation of FECDi for 24 hours with 250 μM A-769662 did not result in a significant induction in NRF2 mRNA levels compared to control cells, HO-1 and NQO1 mRNA levels were significantly upregulated by 3,084.0 ± 278.0% (n=3; p<0.001) and 67.0 ± 22.5% (n=3; p<0.05).
Conclusions :
Our data identify AMPK as an important regulator of anti-oxidant gene expression in normal and Fuchs human corneal endothelial cells, and suggest that pharmacological activation of AMPK may serve as a novel therapeutic approach for the establishment of a corneal endothelial protective phenotype.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.