title: Expression of dominant-negative Fas-associated death domain blocks human keratinocyte apoptosis and vesication induced by sulfur mustard.
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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Georgetown University School of Medicine, Washington, D. C. 20007, USA. rosenthd@georgetown.edu
authors.forename: Dean S
authors.initials: DS
authors.lastname: Rosenthal
authors.forename: Alfredo
authors.initials: A
authors.lastname: Velena
authors.forename: Feng-Pai
authors.initials: FP
authors.lastname: Chou
authors.forename: Richard
authors.initials: R
authors.lastname: Schlegel
authors.forename: Radharaman
authors.initials: R
authors.lastname: Ray
authors.forename: Betty
authors.initials: B
authors.lastname: Benton
authors.forename: Dana
authors.initials: D
authors.lastname: Anderson
authors.forename: William J
authors.initials: WJ
authors.lastname: Smith
authors.forename: Cynthia M
authors.initials: CM
authors.lastname: Simbulan-Rosenthal
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D002352:Carrier Proteins / Q000378:metabolism / Q000502:physiology*; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D053200:Fas-Associated Death Domain Protein; D006801:Humans; D006868:Hydrolysis; D015603:Keratinocytes / Q000166:cytology*; D009151:Mustard Gas / Q000494:pharmacology*; D009842:Oligopeptides / Q000494:pharmacology; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D015853:Cysteine Proteinase Inhibitors; C505533:FADD protein, human; D053200:Fas-Associated Death Domain Protein; D009842:Oligopeptides; C099158:acetyl-aspartyl-glutamyl-valyl-aspartal; D011065:Poly(ADP-ribose) Polymerases; D020169:Caspases; D009151:Mustard Gas