Glucocorticoid-induced Leucine Zipper (GILZ) Promotes the Nuclear Exclusion of FOXO3 in a Crm1-dependent Manner □ S Received for publication, September 25, 2009, and in revised form, December 3, 2009 Published, JBC Papers in Press, December 14, 2009, DOI 10.1074/jbc.M109.068346 Perle Latre ´ de Late ´ ‡ , Aure ´ lie Pe ´ pin ‡ , Hind Assaf-Vandecasteele ‡ , Christophe Espinasse ‡ , Vale ´ rie Nicolas § , Marie-Liesse Asselin-Labat ‡1 , Jacques Bertoglio ‡ , Marc Pallardy ‡ , and Armelle Biola-Vidamment ‡2 From the ‡ Universite ´ Paris Sud, INSERM UMR-S 749, Faculte ´ de Pharmacie, and INSERM, Universite ´ Paris-Sud 11, 92296 Cha ˆtenay-Malabry, France and the § Plate-Forme Imagerie Cellulaire, IFR141-ITFM, Universite ´ Paris-Sud 11, 92296 Cha ˆtenay-Malabry, France GILZ (glucocorticoid-induced leucine zipper) is an ubiqui- tous protein whose expression is induced by glucocorticoids in lymphoid cells. We previously showed that GILZ expression is rapidly induced upon interleukin 2 deprivation in T-cells, pro- tecting cells from apoptosis induced by forkhead box subgroup O3 (FOXO3). The aim of this work is to elucidate the molecular mechanism of FOXO factor inhibition by GILZ. We show in the myeloid cell line HL-60 and the lymphoid CTLL-2 T-cell line that GILZ down-regulates the expression of p27 KIP1 and Bim, two FOXO targets involved in cell cycle regulation and apopto- sis, respectively. GILZ inhibits FOXO1, FOXO3, and FOXO4 transcriptional activities measured with natural or synthetic FOXO-responsive promoters in HL-60 cells. This inhibitory effect is independent of protein kinase B and IB kinase phos- phorylation sites. GILZ does not hinder FOXO3 DNA-binding activity and does not physically interact with FOXO3. However, using fluorescence microscopy, we observe that GILZ expres- sion provokes a Crm-1-dependent nuclear exclusion of FOXO3 leading to its relocalization to the cytoplasm. Moreover, GILZ exclusive cytoplasmic localization is a prerequisite for FOXO3 inhibition and relocalization. We propose that GILZ is a general inhibitor of FOXO factors acting through an original mecha- nism by preventing them from reaching target genes within the nucleus. Forkhead box subgroup O1 (FOXO1 or FKHR), FOXO3a (FKHRL1), FOXO4 (AFX), and FOXO6 constitute the mam- malian FOXO family of transcription factors and achieve important functions in the regulation of genes involved in cell cycle regulation, apoptosis, DNA repair, stress response, energy metabolism, and control of lifespan (for review, see Ref. 1). These highly related members are ubiquitously expressed in all mammalian tissues, interact with the same core consensus DNA sequence, and display overlapping patterns of transcrip- tional activities (2). Interest about FOXO factors in the hema- topoietic system is increasing due to their role in regulation of immune responses. In vitro, FOXO3 has been shown to partici- pate in cytokine withdrawal-induced apoptosis of lymphocytes through up-regulation of Bim (3) or Puma (4). Moreover, the Fas Ligand gene has been described as a downstream target of FOXO3 in Jurkat T-lymphocytes (5). Pink1 was recently described as an anti-apoptotic FOXO3 target gene whose induction upon growth factor deprivation paradoxically pro- longs lymphocyte survival (6). In vivo, FOXO3 appears to be predominant in peripheral lymphoid organs and to regulate lymphoid and myeloid homeostasis. Indeed, mice bearing a mutated FOXO3 allele presented spontaneous T-cell activation and a multisystemic inflammatory syndrome associated with lymphadenopathy (7). Moreover, adult mice with conditional deletion of FOXO1, FOXO3a, and FOXO4 showed hematopoi- etic stem cells with increased cell cycling and apoptosis and defective long term repopulating activity in the bone marrow (8). Somatic disruption of the three FOXO genes in mice resulted in thymic lymphomagenesis (9). Nuclear import of FOXO factors follows stress signals such as oxidative stress or growth factor deprivation, whereas nuclear export results from interaction with the exportin Crm1 (chromosomal region maintenance) and Ran-GTP and from the phosphorylation by the serine/threonine kinase Akt (also called protein kinase B (PKB)), 3 generating two binding sites for the 14-3-3 family of proteins. These post-translational modifi- cations also impair DNA binding (5) and promote proteasomal degradation (10). FOXO factors have been recently shown to be regulated by Akt-independent pathways such as phosphory- lation, acetylation, or interaction with numerous signaling molecules, suggesting that multiple mechanisms can regulate FOXO transcriptional activity. Indeed, IB kinase (IKK) has been shown to interact with and to phosphorylate FOXO3a at Ser-644, promoting nuclear exclusion and proteasomal degra- dation independently of PKB phosphorylation (11). Glucocorticoid-induced leucine zipper (GILZ) is a ubiqui- tous 17-kDa protein belonging to the TSC-22 family of proteins characterized by the presence of common domains termed the TSC box and leucine zipper. GILZ has been described as a reg- ulator of gene transcription through protein-protein interac- tions resulting in inhibition of AP-1 (12) and NF-B (13, 14) transcriptional activities, thereby regulating transduction path- ways essential to inflammation and immune response. GILZ □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2. 1 Present address: The Walter and Eliza Hall Institute of Medical Research, Victorian Breast Cancer Research Consortium, 1G Royal Parade, Parkville, VIC 3050, Australia. 2 To whom correspondence should be addressed: Faculte ´ de Pharmacie, 5 rue Jean-Baptiste Cle ´ ment, 92296 Cha ˆ tenay-Malabry, France. Tel.: 33-1-4683- 5980; Fax: 33-1-4683-5496; E-mail: armelle.biola-vidamment@u-psud.fr. 3 The abbreviations used are: PKB, protein kinase B; GILZ, glucocorticoid-in- duced leucine zipper; IL, interleukin; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; EGFP, enhanced green fluorescent protein; IRS, insulin response sequence; MLP, major late pro- moter; N/C, nuclear/cytoplasmic ratio; NES, nuclear export signal. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 8, pp. 5594 –5605, February 19, 2010 © 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. 5594 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 8 • FEBRUARY 19, 2010 by guest on June 7, 2020 http://www.jbc.org/ Downloaded from