Abstract
Glucocorticoid hormones, including dexamethasone, induce apoptosis in lymphocytes and consequently are used clinically as chemotherapeutic agents in many hematologic malignancies. Dexamethasone also induces autophagy in lymphocytes, although the mechanism is not fully elucidated. Through gene expression analysis, we found that dexamethasone induces the expression of a gene encoding a stress response protein variously referred to as Dig2, RTP801, or REDD1. This protein is reported to inhibit mammalian target of rapamycin (mTOR) signaling. Because autophagy is one outcome of mTOR inhibition, we investigated the hypothesis that Dig2/RTP801/REDD1 elevation contributes to autophagy induction in dexamethasone-treated lymphocytes. In support of this hypothesis, RNAi-mediated suppression of Dig2/RTP801/REDD1 reduces mTOR inhibition and autophagy in glucocorticoid-treated lymphocytes. We observed similar results in Dig2/Rtp801/Redd1 knock-out murine thymocytes treated with dexamethasone. Dig2/RTP801/REDD1 knockdown also leads to increased levels of dexamethasone-induced cell death, suggesting that Dig2/RTP801/REDD1-mediated autophagy promotes cell survival. Collectively, these findings demonstrate for the first time that elevation of Dig2/RTP801/REDD1 contributes to the induction of autophagy.
Highlights
Introduction of Lentiviral shRNAA mouse pLKO.1 lentiviral vector set encoding five different shRNAs against Dig2/ RTP801/REDD1 (RMM4532-NM_029083) were purchased from Open Biosystems
Because autophagy is one outcome of mammalian target of rapamycin (mTOR) inhibition, we investigated the hypothesis that Dig2/RTP801/REDD1 elevation contributes to autophagy induction in dexamethasone-treated lymphocytes
B, Bcl-2-positive WEHI7.2 cells were transiently transfected with nontargeting control siRNA (NT) and Dig2/RTP801/REDD1 siRNA (KD), followed by treatment with vehicle (0.1% ethanol) or 1 M dexamethasone for 4 h. mTOR activity was assessed by immunoblotting for Thr-389-phosphorylated S6 kinase (S6K) and total S6K
Summary
Introduction of Lentiviral shRNAA mouse pLKO.1 lentiviral vector set encoding five different shRNAs against Dig2/ RTP801/REDD1 (RMM4532-NM_029083) were purchased from Open Biosystems. In this report we investigated the hypothesis that Dig2/ RTP801/REDD1 elevation following dexamethasone treatment contributes to induction of autophagy in thymocytes and murine lymphoma lines. Knocking down Dig2/ RTP801/REDD1 significantly reduced the percentage of cells displaying a punctate GFP-LC3 pattern following dexamethasone treatment, in both the presence and the absence of lysosomal protease inhibitors (Fig. 2, B and C).
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