Characterization and Modulation of Proteins Involved in SM Vesication
Abstract
We defined the molecular events leading to SM vesication to develop medical countermeasures for exposure of military personnel and civilians. We blocked SM-induced toxicity using a genetic approach and are now adopting a chemical inhibitor-based strategy to block these pathways. We made significant headway in elucidating important pathways of SM-induced cell death in cultured human keratinocytes (KC) and in intact mouse and grafted human skin (Rosenthal et al, 2002) SM induces terminal differentiation markers as well as apoptosis in KC and involves activation of a death receptor pathway for apoptosis, in which Fas plays a role, as well as a calmodulin (CaM)/Bcl-2-mediated mitochondrial apoptotic pathway (Rosenthal et al 1998) Significantly, altering Fas/FADD pathways in human skin grafted onto nude mice reduces vesication and tissue injury in response to SM (Rosenthal et al 2003) We now tested whether CaM mediates the mitochondrial apoptotic pathway induced by SM RT-PCR and immunoblot analysis revealed rapid modulation in CaM expression following SM treatment. To delineate the potential role of CaM1 the predominant form expressed in KC, in the regulation of SM-induced apoptosis, retroviral vectors expressing CaM1 RNA in the antisense (AS) orientation were used to transduce and derive CaM1 AS cells, which were exposed to SM and subjected to immunoblot analysis for expression of apoptotic markers. Proteolytic activation of executioner" caspases-3, -6, -7, and the "upstream" caspase-9, as well as caspase-mediated PARP cleavage were inhibited by CaM1 AS expression. Consistent with a mitochondrial apoptotic pathway for CaM, CaM1 AS did not prevent Fas-induced apoptosis. Changes in pro- and anti-apoptotic Bcl-2 family proteins were examined to determine their role in CaM-mediated pathways. CaM1 AS upregulated anti-apoptotic protein Bcl-xL, and blocked Bcl-2 down-regulation.
Document Details
- Document Type
- Technical Report
- Publication Date
- May 01, 2005
- Accession Number
- ADA451753
Entities
People
- Dean S. Rosenthal
Organizations
- Georgetown University