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Takako Onishi, Naoki Okumura, Keisuke Hashimoto, Theofilos Tourtas, Ursula Schlotzer-Schrehardt, Friedrich E Kruse, Noriko Koizumi; Involvement of the p38 mitogen-activated protein kinase pathway in Fuchs endothelial corneal dystrophy. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3797.
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© ARVO (1962-2015); The Authors (2016-present)
Endoplasmic reticulum (ER) stress is reported to be involved in the pathophysiology of Fuchs endothelial corneal dystrophy (FECD) (Engler C, et al). We reported that inhibition of p38 mitogen-activated protein kinase (MAPK) suppressed the death of corneal endothelial cells (CECs) (Onishi, et al, ARVO 2016). Signaling by p38 MAPK is associated with various cellular activities, such as cell proliferation, differentiation, migration, and apoptosis. In this study, we investigated whether p38 MAPK signaling is involved in the pathogenesis of FECD and the feasibility of this pathway as a therapeutic target for FECD.
CECs were isolated from donor corneas and FECD-patient corneas, then cultured and immortalized as cell models. Immortalized human CECs (iHCECs) were treated with ER stress inducer thapsigargin (TG, 10 μM) and proteasome inhibitor MG132 (20 uM) to induce ER-stress-mediated apoptosis. Moreover, immortalized FECD cells (iFECD) were treated with TGF-β2 (10 ng/mL) to induce cell death. Expression of PERK (one of the ER stress sensors responsible for ER mediated apoptosis), p38 MAPK, ATF4, and CHOP (ER stress transducer to intrinsic pathway), and caspase 3 (apoptosis executing caspase) was evaluated by western blotting. siRNA was used to knockdown PERK. The percentage of apoptotic cells was determined by Annexin V staining using flow cytometry.
Western blotting showed that TG and MG132 activated PERK, p38 MAPK, ATF4, CHOP, and caspase 3. Knock down of PERK suppressed p38 MAPK, ATF4, CHOP, and caspase 3, suggesting phosphorylation of p38 MAPK was induced by PERK. SB203580 did not alter MG132 mediated PERK activation, but downregulated CHOP and cleavage of caspase 3. TGF-β2 also activated PERK, p38 MAPK, ATF4, CHOP, and caspase 3 in iFECD. Likewise, SB203580 suppressed CHOP and cleavage of caspase 3. Flow cytometry showed that TGF-β2 significantly upregulated Annexin V-positive apoptotic cells in iFECD. However, SB203580 suppressed TGF-β2-induced Annexin V-positive apoptotic cells in comparison to the TGF-β2 stimulated cells (17.1±0.1% and 45.5±1.0%, respectively) (p<0.01).
The findings of this study show that ER stress activated the p38 MAPK signaling pathway in CECs, and induced CHOP to activate apoptosis executer molecule caspase 3. p38 MAPK signaling may be a potential therapeutic target of 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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