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Okuda Hirokazu, Naoki Okumura, Miu Kitahara, Keisuke Hashimoto, Noriko Koizumi; Activation of an intrinsic pathway by endoplasmic reticulum stress in corneal endothelial cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1435.
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© ARVO (1962-2015); The Authors (2016-present)
The involvement of endoplasmic reticulum (ER) stress and functional alteration of mitochondria is suggested to play an important role in Fuchs endothelial corneal dystrophy (FECD), yet the pathophysiology is currently not well elucidated. The purpose of this present study was to investigate how ER stress and mitochondria are related to cell death in corneal endothelium.
Immortalized human corneal endothelial cells (iHCECs) established from donor corneas were treated with ER stress inducer thapsigargin (TG, 10 μM) and proteasome inhibitor MG132 (20 uM) to induce ER stress. The expression levels of ER-stress sensor proteins (i.e., ATF6α, PERK, and IRE1α), CHOP (transcription factor), and apoptosis-involved mitochondrial proteins (bcl-2, caspase 9, and caspase 3) were evaluated by western blotting. Mitochondrial membrane potential (MMP) was determined by JC-1 staining, and siRNA was use to knock down CHOP.
Western blotting showed that TG phosphorylated PERK, cleaved ATF6α and IRE1α in a time dependent manner. TG suppressed MMP, downregulated bcl-2 expression, increased the release of cytochrome c to cytoplasm, and cleaved caspase 9 resulting in caspase 3 cleavage, thus suggesting an ER-stress-activated intrinsic pathway. The knock down of CHOP counteracted TG mediated-MMP decrease and caspase 9 cleavage. MG132 induced unfolded protein, and MG132 activated ER stress sensors, as with TG. Flow cytometry showed that MG132 significantly increased the percentages of MMP demoralized cells in comparison to control cells (21.2±0.9% and 5.6±0.5%, respectively) (p<0.01). In addition, MG132 increased the cleavage of caspase 9 and 3.
The findings of this study indicate that three ER stress sensors, i.e., ATF6α, PERK, and IRE1α, are conserved in corneal endothelium and are activated by ER stress. Moreover, CHOP was found to be expressed by ER-stress-sensor activation and transduced ER stress to mitochondria, thus resulting in intrinsic pathway activation. ER stress mediated apoptosis by an intrinsic pathway might play a central role in cell death in FECD.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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