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NF-Κb Inhibits T-Cell Activation-Induced, P73-Dependent Cell Death by Induction of MDM2

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NF-Κb Inhibits T-Cell Activation-Induced, P73-Dependent Cell Death by Induction of MDM2 NF-κB inhibits T-cell activation-induced, p73-dependent cell death by induction of MDM2 Valere Busuttila,1, Nathalie Droina,b,1, Laura McCormicka, Francesca Bernassolac, Eleonora Candic, Gerry Melinoc,d, and Douglas R. Greena,2 aDepartment of Immunology, St. Jude Children’s Research Hospital, Memphis, TN 38105; bHematopoiesis Unit, Institut National de la Santé et de la Recherche Médicale U1009, Institut de Recherche Intégrée en Cancérologie de Villejuif, Institut Gustave Roussy, 94805 Villejuif, France; cDepartment of Experimental Medicine and Biochemical Sciences, Biochemistry Laboratory, Istituto Dermopatico dell’Immacolata, Istituto di Ricovero e Cura a Carattere Scientifico, Università degli Studi di Roma “Tor Vergata,” 00133 Rome, Italy; and dMedical Research Council Toxicology Unit, University of Leicester, Leicester LE1 9HN, United Kingdom Edited by Tak Wah Mak, The Campbell Family Institute for Breast Cancer Research, Ontario Cancer Institute at Princess Margaret Hospital, University Health Network, Toronto, ON, Canada, and approved September 8, 2010 (received for review May 5, 2010) NF-κB is a key transcription factor involved in the regulation of T-cell have not been identified to date. Interestingly, NF-κB has been activation and proliferation upon engagement of the T-cell receptor shown to induce MDM2 expression in mouse embryonic fibro- (TCR). T cells that lack the IκB kinase (IKKβ) are unable to activate blasts (19), as an NF-κB–responsive element has been identified in NF-κB, and rapidly undergo apoptosis upon activation. NF-κBacti- intron 1 of MDM2. vation following T-cell receptor engagement induces the expression Here, we explore the role of NF-κB in the expression of MDM2 of Mdm2 through interaction with NF-κB sites in its P1 promoter, in activated T cells and its role in preventing p73-dependent, and enforced expression of Mdm2 protected T cells deficient for activation-induced apoptosis. We further identify BIM as a p73 NF-κB activation from activation-induced cell death. In T cells with target in T lymphocytes, required for this form of AICD. intact NF-κB signaling, ablation or pharmacologic inhibition of Mdm2 resulted in activation-induced apoptosis. Mdm2 coprecipi- Results tates with p73 in activated T cells, and apoptosis induced by inhibi- To generate T cells defective in NF-κB signaling, we bred −/− −/− tion of Mdm2 was p73-dependent. Further, Bim was identified as Ikkβ Tnfr1 animals to rescue the embryonic lethality seen −/− +/+ −/− a p73 target gene required for cell death induced by Mdm2 inhibi- in Ikkβ mice. Splenic T cells from 7-d-old Ikkβ Tnfr1 −/− −/− +/+ tion, and a p73-responsive element in intron 1 of Bim was charac- or Ikkβ Tnfr1 mice (hereafter referred to as Ikkβ or −/− terized. Our results demonstrate a pathway for survival of activated Ikkβ ) were stimulated with anti-CD3 plus anti-CD28, and as T cells through NF-κB–induced Mdm2, which blocks Bim-dependent expected (5), apoptosis was dramatically elevated in activated −/− apoptosis through binding and inhibition of p73. Ikkβ T cells (Fig. S1 A and B). Similarly, we found that Jurkat T cells, stably expressing an IκBαM superrepressor (20), un- apoptosis | T lymphocyte | Bim derwent apoptosis upon stimulation with anti-CD3, whereas cells stably transduced with a control vector did not (Fig. S1C). uring the course of an immune response, activated T lym- In mouse embryonic fibroblasts, NF-κB has been observed to Dphocytes undergo cell death by a variety of mechanisms (1). drive the expression of MDM2, which regulates p53 function (19). Such activation-induced cell death (AICD) can be mediated by Similarly, in both primary T cells (Fig. 1 A and C) and Jurkat cells expression of death ligands of the TNF family, including CD95 (Fig. 1 B and D), activation led to increased mRNA and protein −/− ligand (1) and TNF-related apoptosis-inducing ligand (2), and the expression of MDM2. Ikkβ T cells and Jurkat cells expressing engagement of their respective receptors. An independent mech- IκBαM did not display increased MDM2 following activation. anism of AICD involves the expression and stabilization of the We next asked if the expression of MDM2 participates in NF- proapoptotic BCL-2 protein, BIM (3). Another, less well charac- κB–dependent cell survival following T-cell activation. To ectop- IMMUNOLOGY terized mode of AICD in T cells involves engagement of the p53 ically express MDM2 in primary T cells, we optimized transduction family member p73 (4). Intriguingly, this mechanism of T-cell to achieve expression of GFP in almost 50% of primary cells (Fig. death appears to be inhibited by the function of the transcription S2C). We introduced a construct for transient expression of −/− factor NF-κB, as T cells expressing a nondegradable mutant of its MDM2 in Ikkβ T cells before activation with anti-CD3 plus inhibitor, IκB, undergo AICD in a p73-dependent manner (5). anti-CD28 (Fig. 1E and Fig. S2A). Although activation of Ikkβ-null Upon ligation of the T-cell receptor (TCR) and coreceptors, T cells resulted in extensive cell death, enforced expression of T cells activate NF-κB via the IκB kinase (IKK) complex, com- MDM2 inhibited apoptosis. posed of IKKα,IKKβ, and IKKγ/NEMO (6). T cells deficient in Similarly, we stably enforced MDM2 expression in Jurkat cells NF-κB components (7) or lacking IKKβ (8, 9) develop normally with or without IκBαM superrepressor (Fig. S2B). Although acti- provided TNFR1 is disrupted (10), but display defective pro- vation of Jurkat cells with anti-CD3 resulted in apoptosis in cells liferation (11) and accumulation of memory T cells (12, 13). expressing IκBαM superrepressor, this cell death was inhibited by The p53 family of proteins includes p53, p63, and p73, and forms MDM2, but not by stable transduction with the control vector of these proteins produced by alternative splicing and/or alterna- (Fig. 1F). tive translation-start sites (14). A critical regulator of p53 is the E3- ubiquitin ligase, murine double minute-2 (MDM2), which binds directly to the NH2 terminus of p53 in the nucleus, inhibiting its Author contributions: V.B., N.D., and D.R.G. designed research; V.B. and N.D. performed transcriptional activity (15). The MDM2–p53 complex shuttles research; V.B., N.D., L. M., F.B., and E.C. contributed new reagents/analytic tools; V.B., from the nucleus to the cytoplasm, where MDM2 targets p53 for N.D., G.M., and D.R.G. analyzed data; and V.B., N.D., G.M., and D.R.G. wrote the paper. proteasome-dependent degradation. MDM2 also binds to p73 and The authors declare no conflict of interest. inhibits its transcriptional activity, but does not target p73 for This article is a PNAS Direct Submission. degradation (16). 1V.B. and N.D. contributed equally to this work. Expression of murine and human MDM2 is under the control of 2To whom correspondence should be addressed. E-mail: [email protected]. two promoters, P1 and P2 (17, 18). Whereas P2 is driven by p53 This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (17), transcription factors responsible for P1-mediated expression 1073/pnas.1006163107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1006163107 PNAS | October 19, 2010 | vol. 107 | no. 42 | 18061–18066 Downloaded by guest on September 25, 2021 12000 3000 Human Mdm2 A Ikk Tnfr1 -/- Mdm2 B LXSN-Jurkat -/- Ikk Tnfr1 I BM-Jurkat A Mdm2 promoter 1 -CD3/CD28 GFP 2000 8000 -CD3 0 1h B2 GFP 2h 1000 4000 B1 GFP B1 B2 GFP mRNA expression (relative to L32) (relative expression mRNA 0 to PPIA) (relative expression mRNA 0 pGlow Time (h): 0 1 2 3 12 18 24 Time (h): 0 0.5 1 2 4 8 12 24 2000 6000 10000 14000 18000 22000 26000 C D GFP mRNA expression (relative) Ikk Ikk -/- LXSN-Jurkat I BM-Jurkat Cold Probe: + Tnfr1 -/- Tnfr1 -/- 150 B -CD3 (h):0 0.5 1 2 3 4 3 114 100 Human 81 MDM2 MDM2 75 61 Actin 37 LDH 36 B1 0 24 0 24 0 0.5 1 2 4 0 0.5 1 2 4 Time (h) -CD3/CD28 Time (hours) -CD3 Cold Probe: + C -CD3 (h): 0 0.25 0.5 1 2 4 2 E 70 Ikk -/- Tnfr1 -/- pcDNA F 60 LXSN-pCEP4 Ikk -/- Tnfr1 -/- MDM2 LXSN-MDM2 60 50 I BM-pCEP4 50 I BM-MDM2 40 40 B2 30 30 20 Cold Probe - - - - - + - - - - - + 20 D Anti-RelA/p65 - - - - + - - - - - + - Apoptotic cells (%) Apoptotic cells (%) 10 10 Anti-p50 - - - + - - - - - + - - - CD3/CD28 -CD3 Anti-c-Rel - - + - - - - - + - - - 0 0 Anti-RelB - + - - - - - + - - - - Time (h): 0 12 24 36 48 Time (h): 0 24 48 72 − − − − Fig. 1. Impaired Mdm2 up-regulation in primary Ikkβ / Tnfr1 / and IκBM- Jurkat T cells upon TCR activation. (A) Total mRNA was isolated from anti- − − − − − − mCD3/anti-mCD28 activated Ikkβ+/+Tnfr1 / and Ikkβ / Tnfr1 / lymphocytes Non and assessed by real-time RT-PCR for Mdm2 mRNA expression. Absolute specific mRNA values were determined, normalized to L32, and reported as arbitrary 0 -CD3 B2 units. (B) Total mRNA was isolated from anti-hCD3 (OKT3) activated LXSN- κ and I BM-Jurkat T cells and assessed by real-time RT-PCR for human Mdm2 140 Human Mdm2 100 human I B mRNA expression. Absolute mRNA values were determined, normalized to E prom B1 − − 120 prom B Cyclophylin (PPIA), and reported as arbitrary units. (C) Ikkβ+/+Tnfr1 / and − − − − 80 Ikkβ / Tnfr1 / lymphocytes were activated for 24 h with anti-mCD3/anti- 100 mCD28. MDM2 and Actin protein expression was detected by immuno- 80 60 blotting. (D) LXSN- and IκBM-Jurkat T cells were activated with anti-hCD3 60 (OKT3) for the indicated times. Human MDM2 and lactate dehydrogenase 40 − − β / 40 protein expression were assessed by Western blotting.
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