EGF mediates protection against Fas-induced apoptosis by depleting and oxidizing intracellular GSH stocks.
Several pieces of evidence have demonstrated the importance of reduction/oxidation (redox) signaling in biological processes, including sensitivity toward apoptosis. In parallel, it was recently reported that growth factors induce the generation of reactive oxygen species (ROS). Therefore, we tested the hypothesis that the anti-apoptotic effect of epidermal growth factor (EGF) was mediated by changes in the redox state of hepatocytes through changes in GSH stocks. Isolated mouse hepatocytes were cultured and exposed to anti-Fas stimulation in order to induce apoptosis. Cell death by apoptosis was assessed by Hoechst 33258 staining and by measuring caspase-3 proteolysis activity. Cell treatment with EGF significantly decreased total (GSx) and reduced (GSH) glutathione levels in the presence and the absence of anti-Fas. Furthermore, glutathione reductase activity was lower in EGF-treated cultures (by 28%) as compared to untreated cultures which lead to a significant decline in GSH/GSx ratio. These effects were found to be EGF-receptor tyrosine kinase activity dependent. Co-stimulation of cells with anti-Fas and EGF attenuated caspase-3 activation and cell death by apoptosis by 70%. GSH monoethylester (GSHmee) significantly attenuated the effect of EGF on GSH and GSH/GSx ratio. It caused an increase in caspase-3 activation and in the percentage of apoptotic cells in anti-Fas + EGF-treated cells, thus resulting in a 53% decline in the protective effect of EGF. In conclusion, EGF induces a significant and specific depletion and oxidization of intracellular GSH, paralleled by a protection against Fas-induced apoptosis. GSH repenishment partly counteracted these effects suggesting that GSH depletion contributed to the protective effect of EGF against caspase-3 activation and cell death by apoptosis.
Musallam
Lina
L
Centre de recherche, Centre hospitalier de l'Université de Montréal-Hôpital Saint-Luc, and Déartement de pharmacologie, Université de Montréal, Québec, Canada.
Ethier
Chantal
C
Haddad
Pierre Selim
PS
Bilodeau
Marc
M
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000378:metabolism*; D066246:ErbB Receptors / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism*; D005980:Glutathione Reductase; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D010084:Oxidation-Reduction; D013880:Thioredoxin-Disulfide Reductase; D019014:fas Receptor / Q000378:metabolism*
D004791:Enzyme Inhibitors; D019014:fas Receptor; D004815:Epidermal Growth Factor; D009247:NADH, NADPH Oxidoreductases; D005980:Glutathione Reductase; D013880:Thioredoxin-Disulfide Reductase; C424443:Txnrd3 protein, mouse; D066246:ErbB Receptors; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases; D005978:Glutathione