Effect of ADAM17 on EMMPRIN Expression after Vesicant Injury to the Cornea

Abstract

In a cancer cell line expressing EMMPRIN and ADAM17, it has been shown that inhibiting EMMPRIN expression has no effect on ADAM17 levels, but inhibiting ADAM17 expression significantly inhibits EMMPRIN expression (Xu et al. Tumour Biol. 35:7575‐75‐86, 2014). To verify that this sequence of events was not a phenomenon peculiar to cancer cells, but instead represented a cell's normal response to injury, we examined whether ADAM17 was activated quickly in corneas injured by nitrogen mustard (NM), and whether inhibiting ADAM17 activation had any effect on EMMPRIN expression. Rabbit corneal organ cultures were exposed to topical NM for 0 min (i.e., immediately washed off), or 5 min, or 10 min. NM was washed off at the appointed times and the corneal proteins extracted for ADAM17 activity assays using the Innozyme TACE activity assay kit. This revealed that ADAM17 activity was turned on immediately by NM exposure. We then looked at EMMPRIN in exposed corneas, as well as in exposed corneas where ADAM17 activity was inhibited by hydroxamate NDH4417 (N‐octyl‐N‐hydroxy‐2‐[4‐hydroxy‐3‐methoxyphenyl acetamide). Western analyses showed that inhibiting ADAM17 in exposed corneas reduced EMMPRIN levels by 10 fold, as compared to NM‐exposed corneas not treated with the hydroxamate. Furthermore, reducing EMMPRIN resulted in reduced MMP‐9 activity. These data support the idea that, upon injury, ADAM17 activation impacts EMMPRIN expression, and thereby plays a key role in the downstream activation of matrix metalloproteinase, such as MMP9.Support or Funding InformationNIAMD U54AR055073 Counter ACT award and NIEHS P30ES005022 Center for Environmental Exposures and Disease