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Andrea S. DeSantis, Rita A. Hahn, Donald R. Gerecke, Kathy K. Svoboda, Marion K. Gordon; Inhibition of ERK Attenuates ADAM17 Activation in Nitrogen Mustard-Induced Corneal Injuries. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2044.
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Nitrogen Mustard (NM) exposure causes microbullae, with subsequent separation of the corneal epithelial-stromal junction. Separation of the cell layers is, in part, due to activation of ADAM17, which cleaves the anchoring complex component, collagen XVII. We hypothesized that NM-induced separation of cell layers is a result of ADAM17 activation by ERK, which leads to cleavage of collagen XVII. To test this hypothesis we determined whether ERK and ADAM17 had a direct interaction, and whether the interaction was attenuated by the addition of the MEK inhibitor, PD98059.
Corneas were dissected from rabbit eyes (PelFreez) and placed in air lifted organ cultures (Gordon et al., J. Ocul Pharmacol. Ther. 26:407-419, 2010). 100 nmoles of NM were applied onto central corneas for 0, 5 or 10 mins, then corneas were washed with medium or medium plus PD98059 and incubated for 10 min. Corneas were then frozen for analysis by light and immunofluorescence microscopy or by western blotting.
pERK and ectodomain of ADAM17 (i.e., activated ADAM17) were undetectable in sections of unexposed corneas by immunofluorescence. NM-exposed corneas showed signal along the basement membrane zone with both antibodies. The NM-exposed corneas given PD98059 treatment showed a decrease in pERK expression at all timepoints. Pull down assays were performed to examine the association between ERK and ADAM17. Western blots of immunoprecipitated ERK were probed with ERK, pERK and ADAM17 antibodies. Unexposed corneas contained ERK, but very little pERK and ADAM17 ectodomain, however after NM exposure both pERK and the ADAM17 ectodomain were abundant. Addition of PD98059 after NM exposure reduced the quantity of ectodomain ADAM17 immunoprecipitated in the pull down assay.
ADAM17 becomes associated with ERK after 5 and 10 minute NM exposures, which causes its activation. This leads to cleavage of collagen XVII and separation of the epithelial and stromal layers. The ERK inhibitor PD98059 inhibits generation of ADAM17 ectodomain when applied right after the timed NM exposures.
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