Oncogene (2006) 25, 1903–1913 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc ORIGINAL ARTICLE Thioredoxin-interacting protein (txnip) is a glucocorticoid-regulated primary response gene involved in mediating glucocorticoid-induced apoptosis Z Wang, YP Rong, MH Malone, MC Davis, F Zhong and CW Distelhorst Departments of Medicine and Pharmacology, Comprehensive Cancer Center, Case Western Reserve University School of Medicine and University Hospitals of Cleveland, Cleveland, OH, USA Glucocorticoid hormones induce apoptosis in lymphoid Introduction cells.This process is transcriptionally regulated and requires de novo RNA/protein synthesis.However, the Glucocorticoid-induced apoptosis plays an important full spectrum of glucocorticoid-regulated genes mediating physiological role, contributing to maintenance of this cell death process is unknown.Through gene homeostasis in the immune system (Ashwell et al., expression profiling we discovered that the expression 2000; Jondal et al., 2004). As their ability to induce of thioredoxin-intereacting protein (txnip) mRNA is apoptosis in immature lymphocytes, glucocorticoids significantly induced by the glucocorticoid hormone dexa- (dexamethasone, prednisone) are among the most methasone not only in the murine T-cell lymphoma line effective agents for treatment of lymphoid malignancies WEHI7.2, but also in normal mouse thymocytes. This (Schmidt et al., 2004). However, their therapeutic use is result was confirmed by Northern blot analysis in multiple limited because of the development of glucocorticoid models of dexamethasone-induced apoptosis.The induc- resistance by lymphoma and leukemia cells. An im- tion of txnip mRNA by dexamethasone appears to be proved understanding of the fundamental mechanism of mediated through the glucocorticoid receptor as it is glucocorticoid-induced apoptosis will provide the means blocked in the presence of RU486, a glucocorticoid to decipher mechanisms of glucocorticoid resistance and receptor antagonist.Deletion and mutation analysis of to discover new therapeutic targets for use in treatment the txnip promoter identified a functional glucocorticoid of lymphoid malignancies. response element in the txnip promoter.Reporter assays Glucocorticoids suppress lymphocyte proliferation demonstrated that this glucocorticoid response element and survival by two fundamental processes. First, was necessary and sufficient for induction of txnip by glucocorticoids arrest proliferating lymphocytes in G1 dexamethasone.Expression of a GFP-TXNIP fusion phase of the cell cycle (Harmon et al., 1979). Second, protein was sufficient to induce apoptosis in WEHI7.2 glucocorticoids induce apoptosis (Wyllie, 1980). Gluco- cells, and repression of endogenous txnip by RNA corticoids initiate their biological function by binding to interference inhibited dexamethasone-induced apoptosis the ubiquitously expressed and cytoplasmically located in WEHI7.2 cells. Together, these findings indicate glucocorticoid receptor. The ligand/receptor complex that txnip is a novel glucocorticoid-induced primary enters the nucleus where it binds to glucocorticoid target gene involved in mediating glucocorticoid-induced response elements (GREs) in a dimeric state to activate apoptosis. or repress transcription of corresponding genes. This Oncogene (2006) 25, 1903–1913. doi:10.1038/sj.onc.1209218; process requires de novo RNA/protein synthesis (Cohen published online 21 November 2005 and Duke, 1984; Wyllie et al., 1984). The transactivation activity of the glucocorticoid receptor appears essential Keywords: glucocorticoid; apoptosis; lymphoma; for thymocyte apoptosis (Dieken and Miesfeld, 1992; dexamethasone-induced gene; thioredoxin-interacting Chapman et al., 1996; Ramdas and Harmon, 1998) and protein; txnip thymocytes from mice carrying a point mutation in one of the dimerization domains fail to undergo apoptosis in response to glucocorticoid treatment (Reichardt et al., 1998). Moreover, the trans-repression activity of the glucocorticoid receptor may also contribute to gluco- corticoid-induced apoptosis (Helmberg et al., 1995; Thompson, 1998). However, the identity of the target Correspondence: Dr CW Distelhorst, Comprehensive Cancer Center, genes whose trans-activation or trans-repression initi- Case Western Reserve University School of Medicine, WRB 3-133, ates glucocorticoid-induced apoptosis has been elusive. 10900 Euclid Avenue, Cleveland, Ohio 44106-7285, USA. E-mail: [email protected] We have used oligonucleotide microarrays to identify Received 9 June 2005; revised 29 September 2005; accepted 29 September glucocorticoid-regulated genes in three separate but 2005; published online 21 November 2005 related model systems: the S49.A2 and WEHI7.2 murine txnip induction by dexamethasone Z Wang et al 1904 T-cell lymphoma lines, and primary murine thymocytes (Wang et al., 2003a, b; Malone et al., 2004). Dexa- methasone (Dex) induces apoptosis in all three systems, although with different kinetics. Primary thymocytes undergo apoptosis within 8–12 h of exposure to Dex, whereas in S49.A2 and WEHI7.2 cells apoptosis is delayed for over 24 h (Wang et al., 2003a, b). Through oligonucleotide microarray analysis, we have identified three apoptosis-related genes induced by Dex in all three model systems: bim, dig2, and tdag8 (Wang et al., 2003a, b; Malone et al., 2004). Now, in the present study, which is also based on microarray findings, we show that the gene encoding thioredoxin-interacting protein (TXNIP), a natural inhibitor of the antioxidant protein thioredoxin, is rapidly induced by Dex in each of the model systems. This induction is rapid and mediated by a GRE in the gene promoter. The induction of txnip Figure 1 Identification of txnip as a Dex-regulated gene by by Dex is intriguing because of extensive evidence that microarray analysis. Oligonucleotide microarray analysis was an alteration in redox state contributes to Dex-induced performed as described in Materials and methods. (a) The number of genes induced or repressed by Dex in each cell type and in apoptosis (Tome et al., 2001; Tonomura et al., 2003). In common among the three model systems tested is shown. 2 611 this regard, we show that expression of a GFP-TXNIP probes were excluded from this analysis since they were defective fusion protein is sufficient to induce apoptosis in on the GeneChips used in the gene expression profile of S49.A2 WEHI7.2 cells and that repression of txnip expression cells. (b) Analysis of the expression pattern in WEHI7.2 cells and primary thymocytes of the 2 611 functional probes excluded in by RNA interference significantly inhibits Dex-induced panel a. The number of probes either induced or repressed, and in apoptosis. These findings indicate that txnip is a novel common among these two cell types are indicated. (c) Signal glucocorticoid receptor transcriptional target involved intensity of the probe corresponding to txnip in WEHI7.2 cells in mediating Dex-induced apoptosis. treated with vehicle (Con) or 1 mM Dex. (d) Signal intensity of the probe corresponding to txnip in primary thymocytes treated with either vehicle (Con) or 1 mM Dex. Results Microarray analysis Malone et al., 2004). From the 2611 genes or expressed Oligonucleotide microarray analysis was employed to sequenced tags excluded from this initial analysis, we identify glucocorticoid-regulated genes that are poten- sought to identify additional Dex-regulated genes tially involved in mediating or regulating glucocorticoid- common to two model systems, WEHI7.2 cells and induced apoptosis in two murine T-cell lymphoma cell primary murine thymocytes, using arrays in which the lines, S49.A2 and WEHI7.2 as well as in primary murine defective probes had been corrected by Affymetrix. The thymocytes. Dex, a synthetic glucocorticoid, induces expression of a total of 55 genes in WEHI7.2 and 11 apoptosis in all three model systems, as reported genes in primary thymocytes were changed significantly. previously (Wang et al., 2003a, b). The gene expression Five genes were similarly regulated by Dex in both profiles of the S49.A2 and WEHI7.2 lines and of WEHI7.2 cells and the primary thymocytes (Figure 1b). primary murine thymocytes treated with or without Only one of them, T-cell death associated gene 51 was 1 mM Dex from 2 to 24 h were determined using downregulated by Dex. It has been suggested this gene Affymetrix GeneChips. There are 12 500 genes or may be associated with Fas and T-cell receptor expressed sequenced tags represented on the arrays activation-induced apoptosis although its functional employed in these experiments. Affymetrix reported that role is not entirely clear (Park et al., 1996; Qian et al., probes for 2611 genes or expressed sequenced tags were 1997; Wang et al., 1998; Rho et al., 2001; Oberg et al., defective on the early batch of MG-U74A(v2) Gene- 2004). Of the other four Dex-induced genes, one encodes Chips which we had used for the expression profiles of alpha mannosidase 1, one encodes TXNIP, and two are the S49.A2 cell line. However, these 2611 defective ESTs. The most prominently induced gene within this probes were corrected in the GeneChips, which we used group of five was txnip, which was induced B3–4-fold at subsequently for the expression profiles of WEHI7.2 every time point analysed after Dex treatment (Figure 1c cells and primary thymocytes. Since we initially sought and d). As the strength of its induction and its predicted to identify Dex-regulated genes common to all three function in redox regulation,