Foxo1 and Foxo3 help Foxp3
Naganari Ohkura1, 2, , and Shimon Sakaguchi1, 2
1 Department of Experimental Immunology, World Premier International Immunology Frontier Research Center, Osaka University, Suita 565-0871, Japan
2 Department of Experimental Pathology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan
In this issue of Immunity, Kerdiles et al. (2010) report that Foxo transcription factors are essential for the development and function of Foxp3-expressing regulatory T (Treg) cells via controlling the expression of genes associated with Treg cell function.
Foxo transcription factors belong to the Forkhead box family of transcription factors characterized by a conserved winged helix DNA binding domain. In mammals, the Foxo subfamily is comprised of four members, Foxo1, Foxo3, Foxo4, and Foxo6. Foxo1 and Foxo3 are the main isoforms expressed in the immune system. They are important regulators of cell cycle progression, apoptosis, glucose metabolism, and stress resistance via integrating information of the presence of nutrients, growth factors, and stress signals. Recent studies have shown that Foxo transcription factors are also associated with lymphocyte functions such as gene recombination, homing, and cytokine receptor expression. Although Foxo transcription factors appear to play important roles in a variety of biological processes, the functions of Foxo1 and Foxo3 in T cells still remain obscure. In this issue of Immunity, Kerdiles et al. (2010) investigate autoimmunity resulting from T cell-specific deletion of Foxo1 and additional deletion of Foxo3. They conclude that Foxo transcription factors are essential for specifying the program of T cell differentiation especially into regulatory T (Treg) cells expressing the transcription factor Foxp3 (Figure 1).
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Foxo1 and Foxo3 Are Necessary for the Development and Function of Regulatory T cells
The PI3K-Akt pathway is an upstream signal for the Foxo family members. It induces phosphorylation of Foxo transcriptional factors and their nuclear export into the cytoplasm. Foxo1 and Foxo3 bind to the promoter regions of Foxp3 and CTLA-4 and other genes. Those factors are required for the adequate gene expression of Foxp3 and its target genes, such as CTLA-4, in Treg cells.
Foxo transcription factors can act as either transcriptional activators or repressors by forming different molecular complexes with different transcriptional modulators including β-catenin, STAT3, Runx3, Smad3, or Smad4. In addition, their function is tightly regulated by the upstream phosphoinositide 3-kinase (PI3K) and Akt pathway, which phosphorylates Foxo molecules and facilitates their nuclear export into the cytoplasm. After antigen or cytokine stimulation, Foxo transcription factors are rapidly phosphorylated and deactivated in a PI3K-dependent manner, whereas cytokine withdrawal elicits their dephosphorylation and activation. Foxo transcription factors are therefore the major downstream target of the PI3K-Akt signaling pathway. In immune cells, the PI3K pathway is activated by several stimuli via specific receptors, including the B cell antigen receptor (BCR), T cell antigen receptor (TCR), and cytokine and chemokine receptors. Thus Foxos may well play an essential part in immune cell functions.
Kerdiles et al. (2010) made mice with a T cell-specific deletion of Foxo1 by crossing Cd4-cre mice with Foxo1f/f mice. They found that mice with a T cell-specific deletion of Foxo1 harbor an expanded population of activated and/or memory CD4+CD44hi T cells and developed B cell autoimmunity as evidenced by B cell activation, hypergammaglobulinemia, and the production of autoantibodies. Young mice with conditional deletion of Foxo1 in CD4+ T cells exhibited a noticeable decrease in the proportion and the number of Foxp3+ Treg cells among thymic mature CD4 single-positive cells, whereas Foxo1-deficient Foxp3+ Treg cells expanded in the peripheral lymphoid organs. However, such Foxp3+ Treg cells were nonfunctional in vivo, suggesting that the autoimmunity associated with Foxo1 deficiency in CD4+ T cells could be attributed to impaired function of Foxp3+ Treg cells. Notably, whereas the Foxp3 expression was nearly normal in Foxo1-deficent Treg cells, their expression of Treg-associated genes, such as CD25 and CTLA-4, were substantially reduced. Indeed, by chromatin immunoprecipitation, Foxo1 was shown to bind to the upstream of the transcription initiation site of the Ctla4 gene, and the Foxo binding element was required for full expression of CTLA-4. Recently, Ouyang et al. (2010) also reported that mice with T cell-specific deletion of both Foxo1 and Foxo3 developed fatal systemic inflammatory disease due in part to functional defect in Foxp3+ Treg cells. They showed that Foxo1 and Foxo3 directly bind to the Foxp3 promoter region and transactivate its promoter activity in a Foxo1 binding sequence specific manner. Thus, these reports together indicate that Foxo family transcription factors are required for appropriate control of the expression of Foxp3 and its target genes and that impairment in this Foxo-dependent gene expression in Foxp3+ Treg cells hampers their function and thereby produces autoimmunity.
One feature of the Foxo family transcription factors is that they have functional redundancy between their isoforms. Dejean et al. (2009) and Hosaka et al. (2004) reported that Foxo3-deficient mice showed no significant immunological abnormalities, such as spontaneous autoimmunity. Foxo3 deficiency did not alter the number or the proportion of activated or effector-memory T cells in the spleen and the lymph nodes. Moreover, T cells purified from Foxo3−/− mice showed no defect in proliferation and survival after in vitro stimulation. These results suggest that a loss of Foxo3 alone is not sufficient to elicit manifestations of T cell activation or autoimmunity. However, double-deficient mice produced by crossing Cd4-Cre Foxo1f/f mice with Foxo3-deficient mice developed severe systemic autoimmunity accompanying splenomegaly and lymphadenopathy (Kerdiles et al., 2010). Furthermore, retention of only one allele of either Foxo1 or Foxo3 in T cells was sufficient to prevent any major defects in Treg cell differentiation, contrasting with a marked reduction of thymic Treg cells in Foxo1−/−Foxo3−/− mice to approximately 50% of wild-type mice (Ouyang et al., 2010). These findings collectively indicate a redundancy in the function of Foxo1 and Foxo3 for the control of thymic Treg cell development and consequently for T cell homeostasis.
Transforming growth factor-β (TGF-β), which has pleiotropic effects, depending on cell types, induces Foxp3 expression in antigen-stimulated naive T cells. The phenotype of TGF-β-deficient or TGF-β receptor I-deficient mice closely resembles that of Foxp3-deficient mice; all these mutant strains develop lethal autoimmunity by 3–4 weeks of age. Kerdiles et al. (2010) showed that induction of TGF-β-induced Treg (iTreg) cells was highly impaired when T cells from tamoxifen-treated ER-Cre Foxo1f/f mice were stimulated by TGF-β, suggesting that Foxo1 is necessary for Foxp3 induction in iTreg cell differentiation. Moreover, TCR stimulation elicited an increase in the expression of T-bet, a key transcription factor for Th1 cell differentiation, in both wild-type and Foxo1-deficient T cells, whereas addition of TGF-β diminished T-bet expression in wild-type but not Foxo1-deficient T cells. These data indicate that Foxo1 is required for TGF-β-induced Treg cell differentiation, at least in part, via downregulation of T-bet. Harada et al. (2010) also showed that TGF-β-induced Foxp3 expression was impaired in CD4+ T cells from Cbl-b-deficient mice. Cbl-b-deficient T cells displayed augmented Foxo3 phosphorylation; further, forced expression of Foxo3 rescued their TGF-β-dependent Foxp3 expression. A Foxo3 binding motif is present in a proximal region of the Foxp3 promoter and was shown to be required for Foxp3 expression. Collectively, these studies have revealed that Foxo transcription factors promote the transcription of the Foxp3 gene in iTregs ([Kerdiles et al., 2010], [Harada et al., 2010] and [Ouyang et al., 2010]).
Regulation of Foxo transcriptional activity is mainly dependent on the phosphorylation of the Foxo proteins via PI3K-Akt pathway. Binding of growth factors to their receptors initiates PI3K and Akt activation, followed by Foxo phosphorylation, leading to inactivation of Foxos. The mammalian target of rapamycin (mTOR) is also one of the downstream targets of Akt. Recent studies ([Haxhinasto et al., 2008] and [Sauer et al., 2008]) have documented that mTOR forms the PI3K-Akt-mTOR axis in regulating Foxp3 expression. The involvement of mTOR in the differentiation of iTreg cells was further supported by the finding that T cells lacking mTOR kinase differentiate into iTreg cells by TCR stimulation alone in the absence of TGF-β. It is therefore plausible that PI3K-Akt signaling might suppress the Foxp3 expression via mTOR activation and Foxos inactivation.
Collectively, the studies by Kerdiles et al. and others strongly support the notion that Foxo1 and Foxo3 have critical overlapping roles in the development of thymic-derived natural Treg and TGF-β-induced iTreg cells. Kerdiles et al. (2009) previously reported that Foxo1 regulates the homeostasis and life span of naive T cells. Yet it remains unclear how Foxos play different roles when they are in naive or Treg cells. Furthermore, several interacting partners with Foxos have been identified, whereas the mechanisms by which Foxos regulate their target genes in Treg cells still remain unknown. Recent studies have shown that a functional NFAT binding site lies in the Foxp3 enhancer region, with close proximity to the Smad3 binding motif (Tone et al., 2008), and the Runx transcription factors are critically involved in induction and suppressive function of Treg cells through the direct binding to the Foxp3 gene (Kitoh et al., 2009). Taken together, Treg development via Foxp3 induction is a complex event controlled by a variety of transcription factors, including Foxo family transcription factors. Further study is required to decipher the complex transcriptional network for regulating Foxp3 expression.
Dejean et al., 2009 A.S. Dejean, D.R. Beisner, I.L. Ch'en, Y.M. Kerdiles, A. Babour, K.C. Arden, D.H. Castrillon, R.A. DePinho and S.M. Hedrick, Nat. Immunol. 10 (2009), pp. 504–513. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (11)
Harada et al., 2010 Y. Harada, Y. Harada, C. Elly, G. Ying, J.H. Paik, R.A. DePinho and Y.C. Liu, J. Exp. Med. 207 (2010), pp. 1381–1391. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (5)
Haxhinasto et al., 2008 S. Haxhinasto, D. Mathis and C. Benoist, J. Exp. Med. 205 (2008), pp. 565–574. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (107)
Hosaka et al., 2004 T. Hosaka, W.H. Biggs 3rd, D. Tieu, A.D. Boyer, N.M. Varki, W.K. Cavenee and K.C. Arden, Proc. Natl. Acad. Sci. USA 101 (2004), pp. 2975–2980. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (203)
Kerdiles et al., 2010 Y.M. Kerdiles, E.L. Stone, D.L. Beisner, M.A. McGargill, I.L. Ch'en, C. Stockmann, C.D. Katayama and S.M. Hedrick, Immunity 33 (2010), pp. 890–904 this issue. Article | PDF (2169 K) | View Record in Scopus | Cited By in Scopus (0)
Kerdiles et al., 2009 Y.M. Kerdiles, D.R. Beisner, R. Tinoco, A.S. Dejean, D.H. Castrillon, R.A. DePinho and S.M. Hedrick, Nat. Immunol. 10 (2009), pp. 176–184. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (37)
Kitoh et al., 2009 A. Kitoh, M. Ono, Y. Naoe, N. Ohkura, T. Yamaguchi, H. Yaguchi, I. Kitabayashi, T. Tsukada, T. Nomura and Y. Miyachi et al., Immunity 31 (2009), pp. 609–620. Article | PDF (1706 K) | View Record in Scopus | Cited By in Scopus (16)
Ouyang et al., 2010 W. Ouyang, O. Beckett, Q. Ma, J.H. Paik, R.A. DePinho and M.O. Li, Nat. Immunol. 11 (2010), pp. 618–627. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (4)
Sauer et al., 2008 S. Sauer, L. Bruno, A. Hertweck, D. Finlay, M. Leleu, M. Spivakov, Z.A. Knight, B.S. Cobb, D. Cantrell and E. O'Connor et al., Proc. Natl. Acad. Sci. USA 105 (2008), pp. 7797–7802. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (97)
Tone et al., 2008 Y. Tone, K. Furuuchi, Y. Kojima, M.L. Tykocinski, M.I. Greene and M. Tone, Nat. Immunol. 9 (2008), pp. 194–202. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (104)
Volume 33, Issue 6, 14 December 2010, Pages 835-837