Abstract
DNA damaging agents up-regulate levels of the Fas receptor or its ligand, resulting in recruitment of Fas-associated death domain (FADD) and autocatalytic activation of caspase-8, consequently activating the executioner caspases-3, -6, and -7. We found that human epidermal keratinocytes exposed to a vesicating dose (300 microm) of sulfur mustard (SM) exhibit a dose-dependent increase in the levels of Fas receptor and Fas ligand. Immunoblot analysis revealed that the upstream caspases-8 and -9 are both activated in a time-dependent fashion, and caspase-8 is cleaved prior to caspase-9. These results are consistent with the activation of both death receptor (caspase-8) and mitochondrial (caspase-9) pathways by SM. Pretreatment of keratinocytes with a peptide inhibitor of caspase-3 (Ac-DEVD-CHO) suppressed SM-induced downstream markers of apoptosis. To further analyze the importance of the death receptor pathway in SM toxicity, we utilized Fas- or tumor necrosis factor receptor-neutralizing antibodies or constructs expressing a dominant-negative FADD (FADD-DN) to inhibit the recruitment of FADD to the death receptor complex and block the Fas/tumor necrosis factor receptor pathway following SM exposure. Keratinocytes pretreated with Fas-blocking antibody or stably expressing FADD-DN and exhibiting reduced levels of FADD signaling demonstrated markedly decreased caspase-3 activity when treated with SM. In addition, the processing of procaspases-3, -7, and -8 into their active forms was observed in SM-treated control keratinocytes, but not in FADD-DN cells. Blocking the death receptor complex by expression of FADD-DN additionally inhibited SM-induced internucleosomal DNA cleavage and caspase-6-mediated nuclear lamin cleavage. Significantly, we further found that altering the death receptor pathway by expressing FADD-DN in human skin grafted onto nude mice reduces vesication and tissue injury in response to SM. These results indicate that the death receptor pathway plays a pivotal role in SM-induced apoptosis and is therefore a target for therapeutic intervention to reduce SM injury.
Highlights
Sulfur mustard (bis-(2-chloroethyl) sulfide; SM),1 the vesicant agent used as recently as 1988/1989 in the Iraq/Iran conflict and implied to have been used in the Gulf War, induces vesication in human skin by its ability to cause cytotoxic, genotoxic, or a combination of both effects in the skin
We first determined whether SM induces expression of the Fas receptor or its ligand because enhanced expression of Fas or Fas and its ligand (FasL) has been shown to occur in cells exposed to DNA damaging agents, leading to activation of upstream caspase-8 and downstream apoptotic events such as caspase-3-mediated poly(ADP-ribose) polymerase (PARP) cleavage [19, 20]
Immunoblot analysis of extracts derived from keratinocytes exposed to different doses of SM revealed a dose-dependent increase in the levels of both Fas receptor and FasL in response to SM (Fig. 1A)
Summary
Sulfur mustard (bis-(2-chloroethyl) sulfide; SM), the vesicant agent used as recently as 1988/1989 in the Iraq/Iran conflict and implied to have been used in the Gulf War, induces vesication in human skin by its ability to cause cytotoxic, genotoxic, or a combination of both effects in the skin. Utilizing a combination of techniques including the stable expression of a dominant-negative inhibitor of Fas-associated death domain protein (FADD), we demonstrate a role for the Fas/TNF receptor family in mediating the response of human keratinocytes to SM. The abbreviations used are: SM, sulfur mustard; NHEK, normal human epidermal keratinocytes; FADD, Fas-associated death domain; DN, dominant-negative; PARP, poly(ADP-ribose) polymerase; FasL, Fas ligand; TNF, tumor necrosis factor; TNFR, tumor necrosis factor receptor; SFM, serum-free medium; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; AMC, aminomethylcoumarin; DFF, DNA fragmentation factor; FACS, fluorescence-activated cell sorter; PBS, phosphate-buffered saline. We have utilized the graft system to genetically engineer human keratinocytes prior to grafting to ectopically express a dominant-negative FADD and generate a human epidermis containing FADD-DN keratinocytes These human grafts were exposed to SM, and showed a reduced vesication response compared with control keratinocyte. Topical SM exposure of Fas-deficient mice in the current study indicates the viability of this strategy to suppress vesication by using inhibitors of the death receptor pathway
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