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Marion K Gordon, Andrea DeSantis-Rodrigues, Yokechen Chang, Rita A Hahn, Peihong Zhou, Ned D Heindel, Kathy K H Svoboda, Donald R Gerecke; Inhibiting ADAM17 after Ocular Mustard Exposure Reduces EMMPRIN Expression as well as MMP-9 Activation. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4922.
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© ARVO (1962-2015); The Authors (2016-present)
Recent evidence from a cancer cell line demonstrated that inhibiting ADAM17 with siRNA resulted in inhibition of expression of the matrix metalloproteinase inhibitor, EMMPRIN (Xu et al., Tumor Biol. 35:7575-7586, 2014). If this is true in normal tissues, it suggests that ADAM17 activity after injury may be factor involved in promoting EMMPRIN expression, and therefore activation of matrix metalloproteinases. Since MMP activation is a major cause of corneal injury after mustard exposure, we examined whether inhibiting ADAM17 would reduce EMMPRIN expression levels in corneas exposed to nitrogen mustard (NM).
For some experiments, rabbit corneal organ cultures were exposed to 10 µl of 10 mM NM for 0, 5, or 10 min, and then proteins were extracted and analyzed for ADAM17 activity using the Innozyme TACE Activity Assay kit. In addition, 10 µl of 10 mM NM was allowed to react on corneas for 2 hrs, followed by replacing the medium with either fresh medium or medium with an ADAM17 inhibitor. These applications were reapplied 3 times over the course of the following 22 hours. Proteins were extracted and analyzed for ADAM17 activity with the Innozyme kit and for EMMPRIN levels by Western blot analysis at 24 hr post exposure.
As compared to unexposed control corneas, ADAM17 was turned on slightly by applying NM and immediately washing it off (0 min exposure). Activity increased in the 5 min exposed sample, and became significantly elevated in the corneas exposed for 10 min. In the 24 hr experiments, corneas exposed to NM for 2h, then either treated 3 times over the course of 22 hr with medium or with an ADAM17 inhibitor, showed that inhibiting ADAM17 over the 22 hr reduced EMMPRIN expression levels 10 fold. The lower levels of EMMPRIN resulted in reduced levels of MMP-9 activity.
These data indicate that the inhibition of ADAM17 has the downstream effect of reducing EMMPRIN’s activity, and thereby reducing MMP-9 activation. Therefore, ADAM17 inhibitors should be considered as potential therapies for ocular mustard injury, where MMP activation is responsible for much of the tissue damage.
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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