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.

Endoplasmic reticulum stress-induced cell death mediated by the proteasome

“…22 Pro-apoptotic functions of the proteasome have been described in several instances including the constitutive death of neutrophils as well as NGF withdrawal, DNA damage, glucocorticoid treatment and reduced extracellular potassium. [23][24][25][26][27] Additionally, the proteasome has been implicated in the early stages of wallerian degeneration after axotomy. 28 In response to DNA damage, the large Bcl-2 homology domain-only protein Mule/ARF-BP1 was shown to ubiquitylate Mcl-1 -an anti-apoptotic Bcl-2 family member -thereby causing its degradation via the proteasome.…”

The Glucocorticoid-induced Gene tdag8 Encodes a Pro-apoptotic G Protein-coupled Receptor Whose Activation Promotes Glucocorticoid-induced Apoptosis

“…17 and 18). These include activation of the proteasome (19,20), generation of reactive oxygen species (21)(22)(23), and an increase in intracellular calcium (9,24,25). How these pathways signal the commitment of the lymphocyte to apoptosis is unclear.…”

Adrenalectomy in mice does not prevent loss of intestinal lymphocytes after exercise

“…Lymphocyte loss and/or apoptosis have been observed in other situations characterized by extreme physiological stress, including burn injury (15), electrical shock (48), and chronic restraint stress (42), and may reflect release of stress hormones, in particular glucocorticoids (GCs). GCs trigger apoptosis in lymphocytes by acting on an intracellular pathway via the mitochondria (2), which results in the accumulation of mitochondrial derived cytochrome c within the cytosol, loss of mitochondrial membrane integrity, externalization of phosphatidylserine at cell membranes, and DNA fragmentation (27,45).…”

“…Lymphocyte loss and/or apoptosis have been observed in other situations characterized by extreme physiological stress, including burn injury (15), electrical shock (48), and chronic restraint stress (42), and may reflect release of stress hormones, in particular glucocorticoids (GCs). GCs trigger apoptosis in lymphocytes by acting on an intracellular pathway via the mitochondria (2), which results in the accumulation of mitochondrial derived cytochrome c within the cytosol, loss of mitochondrial membrane integrity, externalization of phosphatidylserine at cell membranes, and DNA fragmentation (27,45).The intestinal intraepithelial lymphocytes (IELs) are a unique population of T cells that function as a first line of defense against exogenous pathogens, many of which develop independently of the thymus gland [i.e., T cells with the ␥␦ form of the T cell receptor (TCR)] (35). IELs play an important role in protection from epithelial tumor cells through their function as cytolytic agents (37).…”