Attenuation of CD95-induced apoptosis by inorganic mercury: caspase-3 is not a direct target of low levels of Hg2+.
Exposure to environmental mercury may be a factor that contributes to idiosyncratic autoimmune disease. Studies have demonstrated that inorganic, ionic mercury (i.e., Hg2+) modulates several lymphocyte signal transduction pathways, which may be a mechanism whereby Hg2+ dysregulates the immune response. The CD95/Fas apoptotic signaling pathway, which is of critical importance in regulating peripheral tolerance, is disrupted by low and environmentally relevant concentrations of Hg2+. Activation of the cysteine protease caspase-3 is a critical component of both CD95-mediated and TNF-alpha-induced apoptosis. The present work demonstrates that Hg2+ selectively disrupts death receptor mediated caspase-3 activation, where CD95-mediated caspase-3 activation is impaired in Hg2+ treated cells; whereas TNF-alpha-induced caspase-3 activation is not. Using the fluorogenic caspase-3 substrate, Ac-DEVD-7-amino-4-methyl coumarin, to measure caspase-3 enzyme activity as well as Western blotting to track processing of the caspase-3 proenzyme, we have considered the potential direct and indirect effects of Hg2+ on caspase-3. At relatively high concentrations and in a cell-free system, Hg2+ is capable of targeting the active site cysteinyl of caspase-3 resulting in enzyme inhibition. However, at more environmentally relevant exposures, Hg2+ does not gain access in appreciable quantities to the intracellular compartment where caspase-3 resides. Collectively, these data establish that Hg2+ impairs CD95-mediated apoptosis by targeting a plasma membrane proximal signaling event. By measuring the cellular Hg2+ content following various exposure conditions, we have determined that a cellular Hg2+ burden of approximately 50 ng/10(6) cells is sufficient to impair CD95-mediated caspase-3 activation. The present study furthers an understanding of the mechanism whereby relatively low and non-cytotoxic concentrations of Hg2+ may disrupt peripheral tolerance leading to sustained autoimmune disease.
McCabe
Michael J
MJ
Department of Environmental Medicine, University of Rochester School of Medicine and Dentistry, Box EHSC, 525 Elmwood Ave., Rochester, NY 14642, USA. michael_mccabe@urmc.rochester.edu
Eckles
Kevin G
KG
Langdon
Margaret
M
Clarkson
Thomas W
TW
Whitekus
Michael J
MJ
Rosenspire
Allen J
AJ
D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D053148:Caspase 3; D020169:Caspases / Q000187:drug effects*; D003545:Cysteine / Q000378:metabolism; D006801:Humans; D007202:Indicators and Reagents; D019169:Jurkat Cells; D008628:Mercury / Q000032:analysis / Q000633:toxicity*; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D019014:fas Receptor / Q000187:drug effects* / Q000378:metabolism
D007202:Indicators and Reagents; D013438:Sulfhydryl Compounds; D014409:Tumor Necrosis Factor-alpha; D019014:fas Receptor; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D008628:Mercury; D003545:Cysteine