Sulfur Mustard Induces Markers of Terminal Differentiation and Apoptosis in Keratinocytes Via a Ca2+-Calmodulin and Caspase-Dependent Pathway

Abstract

Sulfur mustard (SM) induces vesication via poorly understood pathways. The blisters that are formed result primarily from the detachment of the epidermis from the dermis at the level of the basement membrane. In addition, there is toxicity to the basal cells, although no careful study has been performed to determine the precise mode of cell death biochemically. We describe here two potential mechanisms by which SM causes basal cell death and detachment: namely, induction of terminal differentiation and apoptosis. In the presence of 100 microM SM, terminal differentiation was rapidly induced in primary human keratinocytes that included the expression of the differentiation-specific markers K1 and K10 and the cross-linking of the cornified envelope precursor protein involucrin. The expression of the attachment protein, fibronectin, was also reduced in a time- and dose-dependent fashion. Features common to both differentiation and apoptosis were also induced in 100 microM SM, including the rapid induction of p53 and the reduction of Bcl-2. At higher concentrations of SM (i.e., 300 microM), formation of the characteristic nucleosome-sized DNA ladders, TUNEL-positive staining of cells, activation of the cysteine protease caspase-3/apopain, and cleavage of the death substrate poly(ADP-ribose) polymerase, were observed both in vivo and in vitro. Both the differentiation and the apoptotic processes appeared to be calmodulin dependent, because the calmodulin inhibitor W-7 blocked the expression of the differentiation-specific markers, as well as the apoptotic response, in a concentration-dependent fashion. In addition, the intracellular Ca2+ chelator, BAPTA-AM, blocked the differentiation response and attenuated the apoptotic response. These results suggest a strategy for designing inhibitors of SM vesication via the Ca2+-calmodulin or caspase-3/PARP pathway.