Purchase this article with an account.
Naoki Okumura, Ryuki Minamiyama, EunDuck P Kay, Satoshi Kawasaki, Robert D Young, Andrew J Quantock, Ursula Schlotzer-Schrehardt, Friedrich E Kruse, Shigeru Kinoshita, Noriko Koizumi; The involvement of transforming growth factor beta in endoplasmic reticulum stress of corneal endothelial cells in Fuchs’ endothelial corneal dystrophy. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2067.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
The pathogenesis of Fuchs’ endothelial corneal dystrophy (FECD) has yet to be elucidated. Here we demonstrate the involvement of transforming growth factor beta (TGF-β) signaling to induce endoplasmic reticulum (ER) stress in FECD corneal endothelial cells (CECs).
FECD-patient human CECs (HCECs) and normal donor-cornea HCECs were cultured and immortalized to produce iFECD and iHCEC cell lines, respectively. To determine extracellular matrix (ECM) protein production and ER colocalization, iFECD and iHCEC cells were immunostained by antibodies for collagen type-I and IV, fibronectin, and ER-marker protein disulfide isomerase. iFECD and iHCEC were assessed by transmission electron microscopy (TEM). To evaluate ER stress, molecular chaperone (GRP78) and ER stress-sensor (IRE1, PERK, and ATF6) expression was analyzed by western blotting of iFECD and iHCEC stimulated with or without TGF-β. To elucidate TGF-β signaling pathway involvement in apoptosis, cells were treated with TGF-β and inhibitors SB431542, A-83-01, and ALK5 inhibitor, and Annexin V-positive apoptotic cells were then evaluated by flow cytometry.
Immunocytochemistry showed that collagen type-I and IV and fibronectin were expressed higher in iFCED than in iHCEC, and that they localized in ER. TEM demonstrated that ER and mitochondria were dilated in iFECD, yet were in normal morphology in iHCEC. Expression of GRP78 was higher in iFECD than in iHCEC, and was markedly increased by TGF-β in iFECD in comparison to iHCEC. Likewise, TGF-β markedly increased the expression of IRE1, PERK, and ATF6 in iFECD in comparison to iHCEC. In iHCEC, the percentage of Annexin V-positive apoptotic cells was not increased in comparison to before TGF-β stimulation (12.3±0.5% and 11.1±0.6%, respectively). In contrast, TGF-β significantly increased Annexin V-positive cells in comparison to the control iFECD (29.9±1.5% and 19.4±1.4%, respectively). In addition, SB431542, A-83-01, and ALK5 inhibitor significantly suppressed the TGF-β induced apoptosis of iFECD.
Our findings suggest that ER stress caused by excessive production of ECM proteins is involved in cell loss in FECD, and that FECD cells have higher sensitivity towards TGF-β signaling. The TGF-β signaling pathway may be a potent therapeutic target for suppressing FECD CEC damage.
This PDF is available to Subscribers Only