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Takako Onishi, Naoki Okumura, Ayaka Kusakabe, Miu Kitahara, Keisuke Hashimoto, Makiko Nakahara, Emi Ueda, Theofilos Tourtas, Ursula Schlötzer-Schrehardt, Friedrich E Kruse, Noriko Koizumi; p38 mitogen-activated protein kinase inhibitor suppresses apoptosis in a Fuchs endothelial corneal dystrophy cellular model. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5300.
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© ARVO (1962-2015); The Authors (2016-present)
We previously reported that transforming growth factor beta (TGF-β) signaling is involved in endoplasmic reticulum stress that causes progressive cell death in Fuchs endothelial corneal dystrophy (FECD)(ARVO 2015). p38 mitogen-activated protein kinase (p38 MAPK) is activated by stress stimuli and is involved in various cellular phenomena such as apoptosis, autophagy, and cell differentiation. This study investigated whether or not inhibition of p38 MAPK suppresses corneal endothelial cell (CEC) damage by using an FECD cellular model.
Human CECs derived from FECD patients were cultured and immortalized to produce an iFECD cellular model. iFECD cells were stimulated with TGF-β2 to induce cell damage. To evaluate the effect of p38MAPK inhibition on cell damage, iFECD cells stimulated with TGF-β2 were cultured with or without a selective inhibitor of p38 MAPK, SB203580 (10μM). Annexin V-positive apoptotic cells were evaluated by flow cytometry. Mitochondrial membrane potential (MMP) was determined by use of JC-1 dye via fluorescence microscopy and flow cytometry. To investigate activation of apoptotic signaling, cleavage of caspase 3 and poly (ADP-ribose) polymerase (PARP) was evaluated by western blotting. To confirm that the effect of SB203580 does not directly suppress the TGF-β signaling pathway, Smad2/3 phosphorylation in iFECD cells treated with SB203580 was evaluated by western blotting.
Flow cytometry demonstrated that TGF-β2 induced Annexin V-positive apoptotic cells in the iFECD cell model, whereas SB203580 significantly decreased TGF-β2-induced Annexin V-positive cells (32.8±0.8% and 13.0±0.3%, respectively)(p<0.01). JC-1 staining showed that MMP was decreased in TGF-β2-stimulated iFECD cells, but MMP was recovered by SB203580. Similarly, flow cytometry showed that the percentage of MMP depolarized cells was significantly suppressed by SB203580 in the TGF-β2-treated iFCED cells (35.9±0.3% and 18.5±1.6%, respectively)(p<0.01). Western blotting showed that TGF-β2 induced cleavage of caspase 3 and PARP, yet SB203580 suppressed that cleavage. SB203580 did not suppress TGF-β2-induced Smad2/3 phosphorylation.
Our findings indicate that inhibition of the p38 MAPK signaling pathway suppressed apoptosis in a TGF-β-induced FECD apoptotic cellular model, and that p38 MAPK might be a potential therapeutic target.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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