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Kenneth M Crawford, Brandon Farmer, Leah Frazier, Akhila Bethi; Endothelin-1 induces phosphorylation of ERK 1/2 and enhances cell migration in bovine corneal endothelial cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2024.
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The corneal endothelium forms a leaky barrier between the corneal stroma and aqueous humor and plays an important role in maintaining corneal hydration, thickness, and transparency. This function relies on maintaining an adequate cell density. Previous work in our lab has shown that Endothelin-1 (ET-1) enhances wound healing through cell proliferation in bovine corneal endothelial cells (BCEC). Since wound healing depends on both cell proliferation and cell migration, we explored whether ET-1 could directly promote cell migration. Moreover, the signaling pathway by which this occurs is not well understood. It was hypothesized that ET-1 induced proliferation involves the mitogen activating protein kinase (MAPK) pathway, which should result in phosphorylation of ERK1/2.
BCECs were isolated from bovine corneas and cultured in DMEM with 10% serum for two passages. Cell migration was examined in Calcein-AM loaded BCEC using the 96-well ChemoTx System. BCEC were treated with serum-free media (control), 0.1nM ET-1, 1nM ET-1, 10nM ET-1, or 10% serum. Cells were allowed to incubate for 48-h and fluorescence of migrated cells recorded. Confluent 2nd passage cells were serum starved for 24 h and then treated with 10nM ET-1 in serum free medium for 24 hours. Total cellular protein was isolated using RIPA buffer and quantified. Relative expression of ERK 1/2 and pERK1/2 proteins was determined by western blotting analysis.
10nM ET-1 stimulated a 13.5% increase in cell migration compared to serum free controls (p=0.003). Treatment of BCEC with 10 nM ET-1 for 15 min induced a 4.3 fold increase in the pERK1/2 (p < 0.001). This result will be supported by treatment of BCEC with a MAPK inhibitor (PD98059) which should block ET-1 induced phosphorylation of ERK1/2 and subsequent cell proliferation and wound healing.
ET-1 appears to enhance wound healing not only through cell proliferation, but also by stimulating cell migration. The wound healing action of ET-1 may be mediated through the MAP Kinase pathway.
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