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J. Biol. Chem., Vol. 278, Issue 10, 8531-8540, March 7, 2003

This Article Full Text Full Text (PDF) All Versions of this Article: 278/10/8531    most recent M209549200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Rosenthal, D. S. Articles by Simbulan-Rosenthal, C. M. Search for Related Content PubMed PubMed Citation Articles by Rosenthal, D. S. Articles by Simbulan-Rosenthal, C. M. Social Bookmarking                      What's this?

Expression of Dominant-negative Fas-associated Death Domain Blocks Human Keratinocyte Apoptosis and Vesication Induced by Sulfur Mustard^*

Dean S. Rosenthal^§, Alfredo Velena, Feng-Pai Chou, Richard Schlegel^¶, Radharaman Ray, Betty Benton, Dana Anderson, William J. Smith, and Cynthia M. Simbulan-Rosenthal

From the Departments of  Biochemistry and Molecular Biology and ^¶ Pathology, Georgetown University School of Medicine, Washington, D. C. 20007 and the  United States Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, Maryland 21010

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 µM) 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.

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