Sulfur mustard-stimulated proteases and their inhibitors in a cultured normal human epidermal keratinocytes model: A potential approach for anti-vesicant drug development

In this study, we observed that SM stimulates several proteases and the epidermal-dermal attachment protein laminin-5 is one of the substrates. We propose that following SM exposure of the skin, laminin-5 degradation causes the detachment of the epidermis from the dermis and, therefore, vesication. We utilized gelatin zymography, Western blotting, immuno-fluorescence staining, and real-time polymerase chain reaction (RT-PCR) analyses to study the SM-stimulated proteases and laminin-5 degradation in NHEK. Two major protease bands (64kDa and 72kDa) were observed by zymography in SM-exposed cells. Addition of serine protease inhibitor (aprotinin, 100μM), or the metalloprotease inhibitor (amastatin, 100μM) to NHEK cultures prior to SM exposure decreased the SM-stimulated protease bands seen by zymography. These inhibitors completely or partially prevented SM-induced laminin-5 γ2 degradation as seen by Western blotting as well as immuno-fluorescence staining. Our results from Western blotting and RT-PCR studies also indicated that the membrane-type matrix metalloproteinase-1 (MT-MM-1) may be involved in SM-induced skin blistering. To summarize, our results in the NHEK model indicate the following: (a) SM stimulates multiple proteases including serine protease(s), and metalloproteases; (b) SM decreases the level of laminin-5 γ2, which is prevented by either a serine protease inhibitor or a metalloprotease inhibitor and (c) MT-MMP-1 maybe one of the proteases that is involved in...

Source: Toxicology Reports - March 15, 2016 Category: Toxicology Source Type: research