Glucocorticoid-Induced Apoptosis of Thymocytes: Requirement of Proteasome-Dependent Mitochondrial Activity

Abstract

Thymocytes undergo negative and positive selection during development in the thymus. During this selection process, the majority of thymocytes are eliminated by apoptosis through signaling via TCR or die by neglect, possibly mediated through glucocorticoids. In this study, we report that thymocytes require molecular oxygen to undergo apoptosis induced by dexamethasone (DEX), a synthetic glucocorticoid, and treatment with N-acetyl-l-cysteine (NAC), a thiol antioxidant, inhibits thymocyte apoptosis in vivo as well as ex vivo. We detected elevated intracellular levels of hydrogen peroxide (H₂O₂) during DEX-induced apoptosis, which is reduced by NAC treatment, indicating that the elevated levels of intracellular H₂O₂ are proapoptotic. We also show that loss of mitochondrial membrane potential, cytochrome c release, as well as caspase-3 activation induced by DEX are attenuated by NAC treatment. We identified the production site for H₂O₂ as the ubiquinone cycle at complex III of mitochondria by using various inhibitors of the mitochondrial electron transport chain, and we show that the cell death events mediated by mitochondria are also significantly reduced when the inhibitors were used. Through inhibition of the proteasome, we also show that the production of H₂O₂ and the cell death events mediated by mitochondria are regulated by proteosomal activities in DEX-induced thymocyte apoptosis. We conclude that in DEX-treated thymocytes, the increased production of H₂O₂ originates from mitochondria and is proapoptotic for cell death mediated by mitochondria. We also conclude that all the apoptotic events mediated by mitochondria are regulated by proteasomes.