Protease in Normal Human Epidermal Keratinocytes

ABSTRACT

An in vitro normal human epidermal keratinocytes (NHEK) model was used to study and to characterize the protease stimulated by the mustards 2-chloroethyl ethyl sulphide (CEES), 2-chloro-N-(2-chloroethyl)-N-methylethanamine hydrochloride (nitrogen mustard, HN₂), and Bis-2-chloroethyl sulfide (sulfur mustard, HD). the results obtained by using a chromozym (TRY) peptide substrate protease assay showed the optimum mustard concentration and time for protease stimulation to be about 200 äM CEES, 100 äM HN₂ or HD, and 16 hours. the mustard-stimulated protease was membrane-bound, and was inhibited by adding a Ca²⁺ chelator EGTA (2 mM), BAPTA AM (50 äM) or a serine protease inhibitor diisopropyl fluoro-phosphate DPP (1 mM), or a protein synthesis inhibitor cycloheximide (10 ä;M) in the extracellular medium. These results suggest that one of the mechanisms of mustard toxicity is via the stimulation of a trypsin/chymotrypsin like serine protease, which is dependent on Ca²⁺ and new protein synthesis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a mustard-stimulated 70-80 KDa protein band that was associated with protease activity which was inhibitable by EGTA, BAPTA, DFP or cycloheximide. This mustard-stimulated protein (protease) may serve as a diagnostic tool for mustard exposure as well as an assay for screening prospective antivesicant protease inhibitor drugs.