June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Involvement of the p38 mitogen-activated protein kinase pathway in Fuchs endothelial corneal dystrophy
Author Affiliations & Notes
  • Takako Onishi
    Biomedical Engineering, Doshisha University, Kyotanabe, Japan
  • Naoki Okumura
    Biomedical Engineering, Doshisha University, Kyotanabe, Japan
  • Keisuke Hashimoto
    Biomedical Engineering, Doshisha University, Kyotanabe, Japan
  • Theofilos Tourtas
    Ophthalmology, University of Erlangen-Nürnberg, Erlangen, Germany
  • Ursula Schlotzer-Schrehardt
    Ophthalmology, University of Erlangen-Nürnberg, Erlangen, Germany
  • Friedrich E Kruse
    Ophthalmology, University of Erlangen-Nürnberg, Erlangen, Germany
  • Noriko Koizumi
    Biomedical Engineering, Doshisha University, Kyotanabe, Japan
  • Footnotes
    Commercial Relationships   Takako Onishi, None; Naoki Okumura, JCR Pharmaceutical Co. (P), Senju Pharmaceutical Co. (P); Keisuke Hashimoto, None; Theofilos Tourtas, None; Ursula Schlotzer-Schrehardt, None; Friedrich Kruse, None; Noriko Koizumi, Doshisha University (P), Senju Pharmaceutical Co. (P)
  • Footnotes
    Support  Program for the Strategic Research Foundation at Private Universities from MEXT (Koizumi N and Okumura N).
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 3797. doi:
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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)

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Abstract

Purpose : 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.

Methods : 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.

Results : 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).

Conclusions : 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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