Dexamethasone-induced Gene 2 (dig2) Is a Novel Pro-survival Stress Gene Induced Rapidly by Diverse Apoptotic Signals

Abstract

Glucocorticoid hormones induce apoptosis in lymphoid cells. This process requires de novo RNA/protein synthesis. Here we report the identification and cloning of a novel dexamethasone-induced gene designated dig2. Using Affymetrix oligonucleotide microarray analysis of ϳ10,000 genes and expressed sequence tags, we found that the expression of dig2 mRNA is significantly induced not only in the murine T cell lymphoma lines S49.A2 and WEHI7.2 but also in normal mouse thymocytes following dexamethasone treatment. This result was confirmed by Northern blot analysis. The induction of dig2 mRNA by dexamethasone appears to be mediated through the glucocorticoid receptor as it is blocked in the presence of RU486, a glucocorticoid receptor antagonist. Furthermore, we demonstrated that dig2 is a novel stress response gene, as its mRNA is induced in response to a variety of cellular stressors including thapsigargin, tunicamycin, and heat shock. In addition, the levels of dig2 mRNA were up-regulated after treatment with the apoptosis-inducing chemotherapeutic drug etoposide. Though the function of dig2 is unknown, dig2 appears to have a pro-survival function, as overexpression of dig2 reduces the sensitivity of WEHI7.2 cells to dexamethasone-induced apoptosis.Glucocorticosteriod hormones are potent inhibitors of T cell proliferation and inducers of thymocyte death (1, 2). Hence, glucocorticoids are frequently used as immunosuppressives to treat a broad range of autoimmune and inflammatory disorders and prevent graft rejection following bone marrow or organ transplantation. Also, glucocorticoids are effective agents for treatment of lymphomas and lymphoid leukemias, including acute lymphoblastic leukemia and chronic lymphocytic leukemia (3-5).Glucocorticoids suppress lymphocyte proliferation and survival by two fundamental processes. First, glucocorticoids arrest proliferating lymphocytes in the G 1 phase of the cell cycle (6). Second, glucocorticoids induce apoptosis in immature lymphocytes (7). The negative effects of glucocorticoids on lymphocyte proliferation and survival are mediated through the glucocorticoid receptor, a ligand-activated transcription factor that induces or represses transcription of individual genes and gene networks (8). The transactivation activity of the glucocorticoid receptor appears essential for glucocorticoid-induced cell death, although glucocorticoid-induced "death genes" have not yet been identified (reviewed in Ref.2).Recent developments in DNA microarray technology permit a large number of cellular transcripts to be analyzed in parallel fashion (9). Using this technology, we have analyzed gene expression profiles of dexamethasone (Dex) 1 -treated lymphocytes to identify genes that are potentially involved in mediating or regulating glucocorticoid-induced apoptosis. For this purpose, we have utilized three separate but related Dex-sensitive model systems, i.e. the S49.A2 murine T cell lymphoma line, the WEHI7.2 murine T cell lymphoma line, and primary murine thymocytes. Although there were ...

Tnfaip8 is an essential gene for the regulation of glucocorticoid-mediated apoptosis of thymocytes

“…Many of the dexamethasoneinduced genes found in this study have been previously reported in similar screens, including Tdag8, 18,19 Fkbp51, 19 Dig2 20 and Bim. 19,20 Overexpression of these genes changes susceptibility to apoptosis and also changes sensitivity to dexamethasone in a variety of systems. We, therefore, first addressed if the genes identified in this screen could regulate the survival of developing thymocytes.…”

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

“…Expression Analysis-Gene expression analysis using Affymetrix MG-U74A(v2) GeneChips was performed as previously described, with the following modifications (30,38). Metrics files were filtered to include only those genes whose expression were considered by the Affymetrix Microarray Analysis Suite 5.0 statistical algorithm to be "changed" in at least one time point in each of the three model systems (WEHI7.2, S49.A2, and thymus) analyzed.…”

“…Interestingly, several of these genes appear to mediate glucocorticoid resistance. These include the stress response gene dig2, whose expression protects against glucocorticoidinduced apoptosis (38); FKBP51, a known glucocorticoid-regulated component of the glucocorticoid receptor chaperone complex that can mediate glucocorticoid resistance by inhibiting cortisol binding (43)(44)(45); and the ABC transporter 2, which has been identified as a mediator of multidrug resistance (46,47). Also included in this list is bim, a pro-apoptotic member of the bcl-2 family that acts at the mitochondria to activate Bax and release cytochrome c, promoting critical and irreversible steps in the execution phase of the apoptotic process (48,49).…”

“…In an attempt to identify glucocorticoid-regulated genes that promote apoptosis, we have performed microarray expression analysis on three models of glucocorticoid-induced apoptosis (30,38). We have found that tdag8 (T-cell death-associated gene 8), a G protein-coupled receptor selectively expressed in lymphoid tissues (primarily thymus, spleen, and peripheral blood monocytes), is rapidly and markedly induced by glucocorticoids in all three model systems.…”

Apoptosis inhibition by Bcl-2 gives way to autophagy in glucocorticoid-treated lymphocytes

“…2,3 In addition to apoptosis-related genes, gene expression profiling has uncovered glucocorticoid regulation of genes involved in cellular metabolism, including genes that regulate glucose homeostasis and respond to ER stress. 2,[4][5][6][7] This is not surprising since it has been known for over forty years that glucocorticoids inhibit glucose uptake and glycolysis in thymocytes. 8,9 These welldocumented effects on lymphocyte metabolism suggested to us that glucocorticoids might induce macroautophagy (hereafter referred to as autophagy), as well as apoptosis.…”