Download for? of Immune Homeostasis: Molecules to Die FOXO Transcription

FOXO Transcription Factors as Regulators
of Immune Homeostasis: Molecules to Die
for?
This information is current as
of June 12, 2017.
Subscription
Permissions
Email Alerts
J Immunol 2003; 171:1623-1629; ;
doi: 10.4049/jimmunol.171.4.1623
http://www.jimmunol.org/content/171/4/1623
This article cites 42 articles, 25 of which you can access for free at:
http://www.jimmunol.org/content/171/4/1623.full#ref-list-1
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2003 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ by guest on June 12, 2017
References
Kim U. Birkenkamp and Paul J. Coffer
OF
THE
JOURNAL IMMUNOLOGY
BRIEF REVIEWS
FOXO Transcription Factors as Regulators of Immune
Homeostasis: Molecules to Die for?
Kim U. Birkenkamp and Paul J. Coffer1
R
serine-threonine kinase protein kinase B (PKB/c-Akt), has been
the subject of much attention as it appears to mediate many of the
cellular functions previously ascribed to PI3K (2). Critical in attenuation of PI3K signaling is dephosphorylation of the lipid products
generated by PI3K by phosphatase and tensin homolog deleted on
chromosome ten (PTEN), a phosphoinositide phosphatase.
The PI3K family contains various isoforms with diverse functions and has been subdivided into several classes (1). The class IA
PI3Ks are composed of a regulatory subunit and a catalytic subunit, of which currently four isoforms (p110␣,␤,␥,␦) have been
identified in mammalian cells. p110␣, p110␤, and p110␦, associate with a regulatory molecule which can include p85␣, p85␤, or
p55␥, all generated by alternative splicing of the same gene. The
specific functions of potential heterodimers of these catalytic and
regulatory subunits remain unclear, although this class of PI3K isoforms tend to be activated by receptors with intrinsic or associated
tyrosine kinase activity. In contrast, the class IB PI3Ks are regulated
by G-protein coupled receptors, and the sole catalytic subunit,
p110␥, associates with a p101 adapter protein.
The specific role of the different PI3K isoforms in immune
cell function have been analyzed using specific, targeted gene
deletions (3). Studies in mice that were deficient in all the class
IA regulatory subunits demonstrated that these animals have
impaired B cell development and reduced numbers of peripheral mature B cells. The B cells that are present show decreased
proliferative responses. Surprisingly, no apparent defects in T
cell activation were found in p85-deficient cells. However,
studies with mutants of the different catalytic subunits demonstrated that PI3K does play an important role in T cell proliferation and survival. p110␣ and p110␤ knockout mice were
found to be embryonic lethal thus hampering analysis of their
individual roles on immune function. But, p110␦ loss of function mutants demonstrated impaired T cell proliferation and
function, and a reduced number of T cells, with a more naive
phenotype. This suggests that T cells develop normally, but fail
to mature or survive in the periphery. p110␥ knockout mice
showed major defects in functions of innate immune cells, a
decreased number of splenic CD4⫹ T cells, proliferation of T
Department of Pulmonary Diseases, University Medical Center, Utrecht, The
Netherlands
2
Abbreviations used in this paper: PI3K, phosphatidylinositol 3-kinase; PKB, protein kinase B; FOXO, Forkhead box class O; FKHR, Forkhead in rhabdomyosarcoma; SGK,
serum- and glucocorticoid-induced kinase; NLS, nuclear localization signal; CDK, cyclindependent kinase; CKI, CDK inhibitor.
Received for publication April 8, 2003. Accepted for publication May 9, 2003.
The costs of publication of this article were defrayed in part by the payment of page charges.
This article must therefore be hereby marked advertisement in accordance with 18 U.S.C.
Section 1734 solely to indicate this fact.
1
Address correspondence and reprint requests to Dr. Paul J. Coffer, Department of Pulmonary Diseases, G03.550, University Medical Center, Heidelberglaan 100, 3584 CX
Utrecht, The Netherlands. E-mail address: [email protected]
Copyright © 2003 by The American Association of Immunologists, Inc.
0022-1767/03/$02.00
Downloaded from http://www.jimmunol.org/ by guest on June 12, 2017
egulation of phosphatidylinositol 3-kinase (PI3K) activity has been demonstrated to be critical for correct
lymphocyte function. The molecular targets of this
lipid kinase have been the subject of extensive research, and
many functional effects of PI3K activation are thought to be
mediated by the serine-threonine kinase protein kinase B (PKB/
c-akt). Genetic analyses in the nematode worm Caenorhabditis
elegans have identified a novel PI3K-regulated signaling pathway that regulates organism lifespan through inhibition of a
Forkhead (FOX) transcription factor, DAF-16. Recent studies
have subsequently revealed an evolutionarily conserved signaling module in higher eukaryotes in which PKB can directly phosphorylate and inactive a family of Forkhead box class O
(FOXO) transcription factors. Phosphorylation results in nuclear exclusion and inhibition of transcription. FOXO transcription factors have been found to play critical roles in regulation of proliferation, apoptosis and control of oxidative stress.
This occurs through both activation and repression of target
gene expression by multiple mechanisms. Here the regulation
and function of these transcription factors is discussed with specific relevance to immune homeostasis. A greater understanding
of the regulation and function of this signaling pathway in lymphocytes may provide novel therapeutic opportunities for immune diseases.
Stimulation of cells by cytokines, growth factors, and hormones results in the phosphorylation of plasma membrane-associated inositol lipids and the subsequent initiation of a cascade
of events that can result in cell survival, division, or differentiation.
The molecular mechanisms by which inositol lipid phosphorylation results in dramatic changes in cellular homeostasis have been
the subject of intensive studies over the last decade. Pivotal in understanding the mechanisms regulating these processes was the discovery of the inositol lipid kinase, phosphatidylinositol 3-kinase
(PI3K)2 (1). PI3Ks phosphorylate the D3 position of the inositol
head group of the phosphoinositide lipid PtdIns(4,5)P2 to generate
PtdIns(3,4,5)P3. The generation of these phosphorylated lipid
products in the plasma membrane results in the relocalisation and
assembly of complexes of intracellular signaling molecules containing so-called PH-domains (1) (Fig. 1). One of these, the protein
1624
BRIEF REVIEWS: REGULATION AND FUNCTION OF FOXO TRANSCRIPTION FACTORS
cells was reduced and the thymocytes exhibited enhanced apoptosis. These data, together with the fact that mice ectopically
expressing an active PI3K variant show enhanced T cell viability
and reduced susceptibility to Fas-mediated apoptosis, demonstrate that PI3K plays a central role in lymphocyte survival, proliferation, and function.
While a role for PI3K in lymphocyte homeostasis is clear from
the studies outlined above, the importance of PKB has only recently been investigated. In transgenic mice that express a constitutively active variant of PKB (gagPKB) specifically in T cells, lymphocytic infiltration was observed in several organs, and mature T
cells were resistant to Fas-induced apoptosis (4). In another similar
study, mice developed thymomas at an early age, primarily due to
inappropriate cell cycle control (5). One mechanism by which
PI3K-PKB may regulate lymphocyte survival is through the transcriptional regulation of pro- or anti-apoptotic molecules. For example, in gagPKB transgenic mice, anti-apoptotic Bcl-XL protein
levels were increased in thymocytes and T cells (4). Indeed the hunt
for transcription factor targets which are directly regulated by PKB
has now become critical to understanding how this kinase can
modulate lymphocyte homeostasis.
Of worms and FOXes: a novel family of PKB-regulated transcription
factors
Initial investigations leading to identification of Forkhead transcription factors as key effectors of PI3K-PKB signaling began
in the nematode worm Caenorhabditis elegans. This soil
dwelling organism feeds on bacteria and usually lives for a
few days. However, when environmental conditions are adverse, worms can enter a so-called Dauer or “non-aging” larval stage where they can survive for months. This is regulated
by a neuroendocrine pathway which has been extensively
studied by genetic analysis (Fig. 1, inset). Regulation of a C.
elegans PI3K (age-1) signal transduction pathway was found to
be critical in modulation of diapause entry. Critically, mutations in
the Forkhead transcription factor DAF-16 completely suppress the
dauer arrest, metabolic shift and longevity observed in age-1 mutants (6, 7).
Members of the Forkhead superfamily are characterized by
the presence of a conserved 110 aa DNA binding domain,
called the “forkhead” domain or “winged helix” domain (8).
Recently, a new nomenclature for these factors has been
adopted and they are now denoted Forkhead box (FOX) factors. Within the larger family of FOX factors, a subfamily has
recently been identified with homology to DAF-16: the Forkhead box class O (FOXO) factors. Three mammalian proteins belong to this subfamily: Forkhead in rhabdomyosarcoma (FKHR), FKHR-like 1, and acute-lymphocyticleukemia-1 fused gene from chromosome X (AFX). FKHR is
now known as FOXO1, FKHR-like 1 is known as FOXO3a,
and AFX is known as FOXO4, respectively. These FOXO
proteins interact preferentially with a core consensus recognition motif 5⬘-TTGTTTAC-3⬘, although the residues flanking
this core contribute to the DNA binding specificity. Interestingly,
the FOXO factors were initially identified at chromosomal breakpoints in several human tumors. These genetic alterations of
FOXOs in human cancers strongly suggested that they may play a
role in the regulation of proliferation, survival or differentiation in
higher organisms.
Regulation of FOXO activity
Functional consequences of phosphorylation and localization of FOXOs. As discussed, mechanistic evidence sug-
gesting that FOXO isoforms are regulated by PKB was initially
obtained from genetic studies in the nematode worm C. elegans
(6, 7). Analysis of the DAF-16 sequence revealed that this transcription factor contains four putative consensus sequences for
PKB phosphorylation (RXRXXS/T), suggesting the possibility
Downloaded from http://www.jimmunol.org/ by guest on June 12, 2017
FIGURE 1. Mechanism of PKB activation. Activation of PI3K by receptor stimulation results in the production of PtdIns(3,4,5)P3 at the plasma membrane. PKB
translocates to the plasma membrane where it is subsequently phopshorylated by PDKs. Phoshorylated PKB is then released into the cytoplasm where it can
phosphorylate substrates, and subsequently migrate into the nucleus and phosphorylate FOXO transcription factors. Inset, this signaling pathway is conserved in C.
elegans where it regulates life span of the organism.
The Journal of Immunology
FIGURE 2. Regulation and function of FOXO phosphorylation. A schematic representation of FOXO transcription factors and their potential sites of phosphorylation. The corresponding amino acids are indicated where
phosphorylation has been reported in vitro and/or in vivo.
NES, nuclear export sequence; Ral-dK, Ral GTPase – dependent kinase.
PKB-mediated phosphorylation of FOXOs occurs in the nucleus and creates docking sites for so-called 14-3-3 proteins. 143-3s are abundant, ubiquitously expressed molecules that preferentially bind to specific motifs containing phosphorylated
serine and threonine residues. In the nucleus, phosphorylated
FOXOs bind to 14-3-3 proteins immediately before they relocalise to the cytoplasm, and thus 14-3-3 proteins have been postulated to play a direct role in nuclear export (17). Once in the
cytoplasm, phosphorylated FOXOs remain complexed to 143-3 proteins preventing nuclear import. It has been proposed
that 14-3-3 proteins obscure the NLS or nuclear export signal
of the protein to which they bind, because they impart no specific information concerning subcellular targeting themselves
(17, 18). In this way, 14-3-3 proteins would affect subcellular
localization of their binding partners by sterically interfering
with the association of transport receptors inhibiting import receptor binding.
Recently it has been suggested that phosphorylation may also
regulate FOXO transcriptional activity through an alternative
mechanism. The phosphorylation of FOXOs has been proposed to directly inhibit DNA binding (19, 20). This might
imply that signaling by PKB would be primarily required to
release FOXOs from the DNA rather than to induce their cytoplasmic retention. In all three FOXO isoforms the NLS is
located in the DNA-binding domain. In FOXO1, part of this
putative NLS lies within the PKB phosphorylation motif, suggesting that phosphorylation contributes to nuclear exclusion
by masking the NLS (21). In addition, phosphorylation of
FOXO1 on the two CK1-specific sites has been proposed to
accelerate nuclear export and/or disrupt nuclear retention (15).
In summary it is likely that phosphorylation and 14-3-3 binding involves multiple events resulting in release of FOXOs from
DNA and subsequent nuclear-cytoplasmic shuttling.
Transcriptional regulation through multiple mechanisms: FOXO’s partners in crime. The interaction of
FOXOs with accessory proteins or cofactors might modulate
transcriptional activity as well as determine which target genes
are regulated by specific FOXOs (Fig. 3). A recent paper has
shed some light on the potential role of such cofactors in regulating FOXO activity (22). Using a comprehensive gene array
analysis, it was demonstrated that FOXOs can activate two different subsets of genes: (1) genes that require FOXO DNA
binding, and, intriguingly, (2) genes regulated independently of
FOXO DNA binding. For example, a down-regulation of Dtype cyclins was observed by a FOXO1 mutant that can no
longer bind DNA (22). This suggests that FOXOs interact with
other transcription factors, thereby modulating their activity.
Downloaded from http://www.jimmunol.org/ by guest on June 12, 2017
of direct phosphorylation of DAF-16 by PKB. Importantly,
three of these residues are conserved in the mammalian FOXO
isoforms (Fig. 2).
Although the concept that FOXO function could be mediated by direct PKB phosphorylation was proposed by C. elegans
researchers several years ago, it was work by the groups of Burgering and Greenberg (9, 10) that initially demonstrated this in
mammalian systems. Subsequently FOXOs have been found to
be phosphorylated in vivo on multiple threonine (T1, T2) and
serine residues (S1, S2, S3, S4, S5) (Fig. 2). Three of these phosphorylated residues, one N-terminal threonine and two C-terminal serines (T1, S1 and S2) lie within a consensus motif for
phosphorylation by PKB. Indeed, FOXO proteins can be phosphorylated by PKB on these sites, both in vitro and in vivo,
although with varying stoichiometry (9 –12).
Several reports have recently indicated that phosphorylation
of FOXOs may be more complex and involve additional sites
on these proteins. For example, the serum- and glucocorticoidinduced kinase (SGK) is also capable of inactivating FOXO3a
(13). SGKs are closely related to PKB and their activation is also
dependent upon PI3K-activity. Interestingly, SGK and PKB
display differences with respect to the efficacy with which they
phosphorylate the regulatory sites of FOXO3a. As the phosphorylation of each regulatory site of FOXO3a appears to be
critical for the efficient exclusion from the nucleus (see below),
it is likely that SGK and PKB cooperate in coordinately regulating FOXO transcription factors.
Recently, two further kinases have been identified that can
phosphorylate FOXOs on additional sites. Phosphorylation by
the dual-specificity tyrosine-phosphorylated and regulated kinase 1A (DYRK1A) decreases the ability of FOXO1 to stimulate transcription, and reduces the proportion in the nucleus
(14). In addition, PKB-catalyzed phosphorylation of the residue equivalent to S2 in FOXO1 creates a recognition motif for
casein kinase I (CK1), allowing it to phosphorylate S4 (Fig. 2).
ThephosphorylatedS4inturn“primes”theCK1catalyzedphosphorylation of S5 (15).
So what are the functional consequences of FOX phosphorylation? Well, as is true for regulation of many signaling modules, the key theme is relocalization. Transport of proteins between the nuclear and cytoplasmic compartments is a highly
regulated process requiring both importin and exportin proteins. The importins and exportins bind to specific sequences in
their target proteins termed nuclear localization signal (NLS)
and nuclear export signal. Shuttling of FOXOs was shown to be
dependent on the small GTPase Ran and the export receptor
Crm1 and this is critical for the inhibition of FOXO transcriptional activity (16, 17).
1625
1626
BRIEF REVIEWS: REGULATION AND FUNCTION OF FOXO TRANSCRIPTION FACTORS
In support of this, several recent studies have demonstrated that
FOXOs can indeed interact with different binding partners.
For instance, it has been shown that FOXO1 can interact with
both the transcriptional coactivator p300/CREB-binding protein, an acetyl transferase that modulates the activity of its targets by acetylation, as well as the steroid receptor coactivator
protein (23). Furthermore, FOXO1 was shown to interact with
both steroid and nonsteroid nuclear receptors. FOXO1 differentially regulated the transactivation mediated by these different nuclear receptors, either as a coactivator or corepressor, depending on the receptor and cell type (24, 25). In
differentiating human endometrial stromal cells it was shown
that FOXO1 directly interacts and transcriptionally cooperates
with C/EBP␤, a transcription factor which has been shown to
play a role in differentiation (26). Finally, roles for FOXOs in
modulating HNF-4 and STAT3-mediated transcription have
very recently been reported (27, 28). Interestingly, FOXO1 increased STAT3- but not STAT5-mediated transcription, demonstrating specificity for the STAT transcription factor
isoforms.
The ability of the different FOXO isoforms to specifically
interact with transcriptional cofactors predicts distinct biological functions for these proteins (Fig. 3). This is born out
by the distinct subset of genes modulated by DNA-binding
deficient FOXO1 relative to wild-type FOXO1 (22). In fact
this mutant also had the ability to suppress tumor formation
in nude mice, suggesting that interaction of cofactors is in
fact critical in modulating the functional effects of FOXO
isoforms.
Functional consequences of FOXO activation
The PI3K/PKB/FOXO signaling module appears to be remarkably conserved during evolution. While the regulation and
function of FOXO activity has been resolved in detail through
genetic analysis in C. elegans, in mammalian cells FOXO function has only recently started to be explored.
Regulation of cell cycle progression. Cell cycle progression is a process that is tightly controlled by internal and exter-
Downloaded from http://www.jimmunol.org/ by guest on June 12, 2017
FIGURE 3. Potential mechanisms of transcriptional regulation by FOXOs.
nal signals. Cells require an extracellular proliferative signal directly after mitosis to keep on growing and dividing. When they
are faced with a lack of such a signal, depending on the cell-type,
the cell will either die or growth-arrest. The sequential events of
the cell cycle are coordinated by the cyclins and the cyclin-dependent kinases (CDKs). Two classes of proteins negatively
regulate progression through the cell cycle, the retinoblastoma
protein family and the CDK inhibitors (CKIs). CKIs associate
to and inhibit the activity of the cyclin/CDK complexes. It has
been reported recently that the FOXO-induced cell cycle arrest
depends on the transcriptional up-regulation and subsequent
increased protein levels of the CKI p27KIP1 (29, 30). In addition, FOXO-induced cell cycle arrest may also require a p27independent mechanism since conditional activation of
FOXOs leads to transcriptional repression and reduced protein
expression of the D-type cyclins resulting in cell cycle arrest
even in the absence of functional p27 (31).
The continuation of cell division at various stages of the cell
cycle involves the inactivation of at least one of three members
of the retinoblastoma family of nuclear pocket proteins. The
general mechanism by which this family exerts its effects is by
binding different members of the E2F transcription factors,
thereby inhibiting their activity. It has been shown that conditional activation of FOXO3a can result in an increase in mRNA
and protein levels of the retinoblastoma–like protein p130,
concomitant with induction of cell cycle arrest (32).
Furthermore, FOXOs also have an important role in the control of mammalian cell cycle completion (33). The mechanism
by which FOXO regulates cytokinesis, and transition from M
to G1, include transcriptional up-regulation of cyclin B and polo-like kinase genes in G2. These results propose an interesting
model whereby FOXO transcription factors must be inactivated for cells to initiate a program of division, but execution of
the mitotic program depends on down-regulation of PKB and
subsequent FOXO activation.
Regulation of lymphocyte survival. The regulation of programmed cell death plays a critical role in the maintenance of
self-tolerance in the immune system. The number of T cells that
leave the thymus and enter the peripheral T cell pool is a minor
fraction of the number initially generated. Thymocytes that express receptors binding to self-peptide-complexes with high affinity, are negatively selected and die. The negative selection of
thymocytes is clearly mediated by apoptosis and the key mediators of this process are just starting to be identified. The innate
immune system must also be able to adapt to sudden changes in
the production of blood cells, such as during microbial infection. Therefore, the size of each hematopoietic subpopulation is
controlled by continually counterbalancing stem cell renewal
and differentiation with apoptotic cell death. A plethora of lineage specific survival factors result in PI3K/PKB-mediated phosphorylation and inhibition of FOXOs in these cell types. For
instance, IL-2 and IL-3 which play important roles in the proliferation and survival of lymphocytes inactivate FOXO3a via
PI3K/PKB-mediated phosphorylation (30, 34).
Although FOXO transcription factors have been shown to be
regulated by a variety of receptors present on immune cells,
Greenberg and coworkers (10) were the first to describe a role
for FOXO3a in the modulation of programmed cell death.
Here, a constitutively active FOXO3a mutant was found to induce apoptosis of Jurkat T cells in a Fas-dependent manner
(Fig. 4). However this appears to be a cell type-dependent effect
The Journal of Immunology
1627
because Fas-independent mechanisms of apoptosis have been
observed in other cell lines (35).
While Fas has been clearly implicated in regulating lymphocyte death, there are alternative pathways resulting in the same
cellular fate. In contrast to Fas, Bcl-2 regulates a separate pathway leading to T cell death (36). The Bcl-2 family of apoptotic
regulators consists of pro- and anti-apoptotic members whose
individual levels determine whether a cell will survive or die. An
abundance of pro-apoptotic Bcl-2 proteins results in a breakdown of mitochondrial integrity, leakage of cytochrome c into
the cytoplasm and caspase activation. The pro-apoptotic Bcl-2
family member Bim has been shown to be dramatically up-regulated by FOXO transcription factors (34, 35, 37). Withdrawal
of cytokines from survival factor-dependent lymphocyte cell
lines results in an up-regulation of Bim expression, concomitant
with induction of the apoptotic program (Fig. 4). Specific activation of FOXO3a alone was found to be sufficient to induce
Bim expression, and could recapitulate all known elements of the
apoptotic program normally induced by cytokine withdrawal (34,
35, 37). In the immune system Bim has been critically implicated
in modulating lymphocyte homeostasis. Bim⫺/⫺ mice succumb to
autoimmune kidney disease, accumulation of lymphoid and myeloid cells and perturbed T cell development (38). In addition,
lymphocytes were refractory to apoptotic stimuli and showed impaired negative selection. Because Bim is a direct target of FOXOs,
(34, 35, 37), this suggests that FOXOs play an important role in
regulating T cell selection through regulation of pro-apoptotic
Bcl-2 family members.
Oxidative stress and organismal lifespan. In C. elegans,
worms deficient in components of the PI3K-PKB pathway have
enhanced DAF-16 activity resulting in enhanced lifespan, or entry into the Dauer larval stage. These mutants
develop a resistance to cellular stress that may play a role in development of the observed phenotypes. FOXOs have recently
also been demonstrated to play a role in the stress response of
mammalian cells, thereby providing protection from oxidative
stress. Intracellular reactive oxygen species are thought to contribute to aging in a wide spectrum of organisms, although the
precise mechanism remains elusive. Following activation, lymphocytes produce increased levels of reactive oxygen species,
which may serve as an intracellular second messenger (39). Antioxidants can block MHC/Ag activation-induced death of
specific T cell hybridomas and thus a redox imbalance may play
a role in regulating programmed cell death.
It was recently shown that FOXO3a can protect quiescent
cells from oxidative stress by directly increasing expression of
manganese superoxide dismutase (MnSOD), an anti-oxidant
enzyme (40). Cells deficient in MnSOD were no longer protected from oxidant damage by ectopic expression of FOXO,
suggesting that this is a critical defense pathway. These results
are consistent with previous studies in C. elegans demonstrating
Downloaded from http://www.jimmunol.org/ by guest on June 12, 2017
FIGURE 4. FOXO can induce multiple pathways of caspase activation. FOXO can induce programmed cell death through both death receptor-dependent and
–independent pathways. Up-regulation of FasL results in cross-linking and activation of Fas (CD95), with subsequent caspase-8 activation and resultant apoptosis.
Direct transcriptional regulation of the pro-apoptotic Bcl-2 family member Bim, results in mitochondrial permeability and activation of the apoptosome. These
pathways may act independently or in unison in a cell type-specific fashion.
1628
BRIEF REVIEWS: REGULATION AND FUNCTION OF FOXO TRANSCRIPTION FACTORS
that life-extending mutations in the DAF-16 pathway requires
the presence of a gene that encodes a cytosolic catalase, which
degrades hydrogen peroxide (41).
The growth arrest and DNA damage response gene Gadd45a
was also shown to be a direct target of FOXO3a (42) indicating
that FOXOs can play a role in the resistance of cells to stress by
regulating DNA repair. It is suggested that under low stress conditions, FOXOs may promote DNA repair, whereas at higher
levels of cellular stress, programmed cell death is induced. The
ability to protect certain cells from damage suggests that, similar
to the nematode worm, the evolutionary conserved FOXO
transcription factors may also regulate life span in mammalian
cells.
Perspectives
References
1. Katso, R., K. Okkenhaug, K. Ahmadi, S. White, J. Timms, and M. D. Waterfield.
2001. Cellular function of phosphoinositide 3-kinases: implications for development,
immunity, homeostasis, and cancer. Annu. Rev. Cell Dev. Biol. 17:615.
2. Coffer, P. J., J. Jin, and J. R. Woodgett. 1998. Protein kinase B (c-Akt): a multifunctional mediator of phosphatidylinositol 3-kinase activation. Biochem J. 335:1.
3. Sasaki, T., A. Suzuki, J. Sasaki, and J. M. Penninger. 2002. Phosphoinositide 3-kinases in immunity: lessons from knockout mice. J. Biochem. 131:495.
4. Jones, R. G., M. Parsons, M. Bonnard, V. S. Chan, W. C. Yeh, et al. 2000. Protein
kinase B regulates T lymphocyte survival, nuclear factor ␬B activation, and Bcl-XL
levels in vivo. J. Exp. Med. 191:1721.
5. Malstrom, S., E. Tili, D. Kappes, J. D. Ceci, and P. N. Tsichlis. 2001. Tumor induction by an Lck-MyrAkt transgene is delayed by mechanisms controlling the size of the
thymus. Proc. Natl. Acad. Sci. USA 98:14967.
6. Lin, K., J. B. Dorman, A. Rodan, and C. Kenyon. 1997. daf-16: an HNF-3/forkhead
family member that can function to double the life-span of Caenorhabditis elegans.
Science 278:1319.
7. Ogg, S., S. Paradis, S. Gottlieb, G. I. Patterson, L. Lee, H. A. Tissenbaum, and
G. Ruvkun. 1997. The fork head transcription factor DAF-16 transduces insulin-like
metabolic and longevity signals in C. elegans. Nature 389:994.
8. Kaufmann, E., and W. Knochel. 1996. Five years on the wings of fork head. Mech.
Dev. 57:3.
9. Kops, G. J. P. L., N. D. de Ruiter, A. M. M. De Vries-Smits, D. R. Powell, J. L. Bos,
and B. M. T. Burgering. 1999. Direct control of the forkhead transcription factor AFX
by protein kinase B. Nature 398:630.
10. Brunet, A., A. Bonni, M. J. Zigmond, M. Z. Lin, P. Juo, L. S. Hu, M. J. Anderson,
K. C. Arden, J. Blenis, and M. E. Greenberg. 1999. Akt promotes cell survival by
phosphorylating and inhibiting a forkhead transcription factor. Cell. 96:857.
11. Rena, G., S. Guo, S. C. Cichy, T. G. Unterman, and P. Cohen. 1999. Phosphorylation of the transcription factor forkhead family member FKHR by protein kinase B.
J. Biol. Chem. 274:17179.
12. Tang, E. D., G. Nunez, F. G. Barr, and K.-L. Guan. 1999. Negative regulation of the
forkhead transcription factor FKHR by Akt. J. Biol. Chem. 274:16741.
13. Brunet, A., J. Park, H. Tran, L. S. Hu, B. A. Hemmings, and M. E. Greenberg. 2001.
Protein kinase SGK mediates survival signals by phosphorylating the transcription
factor FKHRL1 (FOXO3a). Mol. Cell Biol. 21:952.
14. Woods, Y. L., G. Rena, N. Morrice, A. Barthel, W. Becker, S. Guo, T. G. Unterman,
and P. Cohen. 2001. The kinase DYRK1A phosphorylates the transcription factor
FKHR at Ser329 in vitro, a novel in vivo phosphorylation site. Biochem. J. 355:597.
15. Rena, G., Y. L. Woods, A. R. Prescott, M. Peggie, T. G. Unterman, M. R. Williams,
and P. Cohen. 2002. Two novel phosphorylation sites on FKHR that are critical for its
nuclear exclusion. EMBO J. 21:2263.
16. Brownawell, A. M., G. J. P. L. Kops, I. G. Macara, and B. M. T. Burgering. 2001.
Inhibition of nuclear import by protein kinase B (Akt) regulates the subcellular distribution and activity of the forkhead transcription factor AFX. Mol. Cell Biol.
21:3534.
17. Brunet, A., F. Kanai, J. Stehn, J. Xu, D. Sarbassova, J. V. Frangioni, S. N. Dalal,
J. A. DeCaprio, M. E. Greenberg, and M. B. Yaffe. 2002. 14-3-3 transits to the nucleus and participates in dynamic nucleocytoplasmic transport. J. Cell Biol. 156:817.
18. Muslin, A. J., and H. Xing. 2000. 14-3-3 proteins: regulation of subcellular localization by molecular interference. Cell Signal. 12:703.
19. Cahill, C. M., G. Tzivion, N. Nasrin, S. Ogg, J. Dore, G. Ruvkun, and
M. Alexander-Bridges. 2001. Phosphatidylinositol 3-kinase signaling inhibits
DAF-16 DNA binding and function via 14-3-3-dependent and 14-3-3-independent
pathways. J. Biol. Chem. 276:13402.
20. Zhang, X., L. Gan, H. Pan, S. Guo, X. He, S. T. Olson, A. Mesecar, S. Adam, and
T. G. Unterman. 2002. Phosphorylation of serine 256 suppresses transactivation by
FKHR (FOXO1) by multiple mechanisms. J. Biol. Chem. 277:45276.
21. Rena, G., A. R. Prescott, S. Guo, P. Cohen, and T. G. Unterman. 2001. Roles of the
forkhead in rhabdomyosarcoma (FKHR) phosphorylation sites in regulating 14-3-3
binding, transactivation and nuclear targeting. Biochem J. 354:605.
22. Ramaswany, S., N. Nakamura, I. Sansal, L. Bergeron, and W. R. Sellers. 2002. A novel
mechanism of gene regulation and tunor suppression by the transcription factor
FKHR. Cancer Cell. 2:81.
23. Nasrin, N., S. Ogg, C. M. Cahill, W. Biggs, S. Nui, et al. 2000. DAF-16 recruits the
CREB-binding protein coactivator complex to the insulin-like growth factor binding
protein 1 promoter in HepG2 cells. Proc. Natl. Acad. Sci. USA 97:10412.
24. Schuur, E. R., A. V. Loktev, M. Sharma, Z. Sun, R. A. Roth, and R. J. Weigel. 2001.
Ligand-dependent interaction of estrogen receptor-␣ with members of the forkhead
transcription factor family. J. Biol. Chem. 276:33554.
Downloaded from http://www.jimmunol.org/ by guest on June 12, 2017
In conclusion, activation of FOXOs causes growth suppression
in a variety of cell types, while in cells of the immune system,
programmed cell death is often induced. These different effects
can most probably be ascribed to the fact that FOXO activation
results in cell type-specific gene regulation, which can be regulated by interaction of FOXOs with other transcription factors
or accessory proteins (Fig. 3). What is then the benefit for the
organism of this type of regulation? In the immune system lymphocytes undergo a process of selection where large number of
cells must be eliminated. Cells of the innate immune system are
produced daily in relatively large numbers in the bone marrow,
and thus they must also be readily eliminated. In cell systems in
which a rapid turnover of cells is required for the well being of
the organism, the stress-induced activation of FOXOs results in
cell death. In contrast, in cells that are not continuously produced, but rather are still required by the organism, stress-induced FOXO activity results in cell cycle arrest, letting them
survive the adverse conditions. These quiescent cells are kept
alive through the FOXO-mediated resistance to stress, for example by up-regulation of oxidant scavenging proteins. This is
similar to observations made in C. elegans where, in the absence
of nutrients, DAF-16 is activated, resulting in Dauer formation, and longevity as opposed to programmed cell death. This
method of regulation allows a single transcription factor family,
FOXO, to have distinct functional effects depending of the cellular context.
It is also interesting to note that the various FOXO isoforms
can have distinct biological effects within a single cell type. Ectopic expression of FOXO1 in Jurkat T cells induces a block in
proliferation, while in the same cells, FOXO3a induces apoptosis (29). Sellers and coworkers (22) have also made an interesting observation concerning the ability of FOXO transcription
factors to induce either cell cycle arrest or programmed cell
death. In their analysis of genes regulated by FOXO1, vs a mutant unable to bind DNA, the mutant transcription factor was
found to retain the ability to both inhibit cell cycle progression,
and suppress tumor formation. However, the same FOXO1
DNA-binding mutant was unable to induce apoptosis in cells
normally responsive to FOXO1 ectopic expression. Taken together these data suggest that the mechanism by which FOXOs
induce a block in proliferation is distinct from that inducing cell
death. The latter function apparently requires DNA-binding,
while the former doesn’t. Thus modulating the ability of
FOXO to bind DNA might provide the possibility to switch on
a genetic program inducing cell cycle arrest on one hand, or cell
death on another.
The broad expression of FOXO transcription factors in cells
of the immune system suggests that they could be critical in
modulating immune homeostasis and lymphocyte selection.
That the functions of this family of transcription factors relies
heavily on cell-type specific interactions with cofactors, suggests
that it might be possible to generate cell-type specific FOXO
inhibitors or activators. While this is an attractive idea, we will
have to await further research on this new family of Forkhead
proteins to determine whether their functions can be exploited
therapeutically.
The Journal of Immunology
33. Alvarez, B., C. Martinez-A, B. M. T. Burgering, and A. C. Carrera. 2001. Forkhead
transcription factors contribute to execution of the mitotic programme in mammals.
Nature 413:744.
34. Stahl, M., P. F. Dijkers, G. J. P. L. Kops, S. M. A. Lens, P. J. Coffer,
B. M. T. Burgering, and R. H. Medema. 2002. The forkhead transcription factor
FoxO regulates transcription of p27kip1 and Bim in response to IL-2. J. Immunol.
168:5024.
35. Dijkers, P. F., K. U. Birkenkamp, E. W.-F Lam, N. S. B. Thomas, J.-W. J. Lammers,
L. Koenderman, and P. J. Coffer. 2002. FKHR-L1 can act as a critical effector of cell
death induced by cytokine withdrawal: protein kinase B-enhanced cell survival
through maintenance of mitochondrial integrity. J. Cell Biol. 156:531.
36. Strasser, A., A. W. Harris, D. C. Huang, P. H. Krammer, and S. Cory. 1995. Bcl-2
and Fas/APO-1 regulate distinct pathways to lymphocyte apoptosis. EMBO J.
14:6136.
37. Dijkers, P. F., R. H. Medema, J.-W. J. Lammers, L. Koenderman, and P. J. Coffer.
2000. Expression of the pro-apoptotic Bcl-2 family member Bim is regulated by the
forkhead transcription factor FKHR-L1. Current Biol. 10:1201.
38. Bouillet, P., D. Metcalf, D. C. S. Huang, D. M. Tarlinton, T. W. H. Kay, F. Köntgen,
J. M. Adams, and A. Strasser. 1999. Proapoptotic Bcl-2 relative Bim required for certain apoptotic responses, leukocyte homeostasis, and to preclude autoimmunity. Science 286:1735.
39. Buttke, T. M., and P. A. Sandstrom. 1994. Oxidative stress as a mediator of apoptosis.
Immunol. Today. 15:7.
40. Kops, G. J. P. L., T. B. Dansen, P. E. Polderman, I. Saarloos, K. W. A. Wirtz,
P. J. Coffer, T.-T. Huang, J. L. Bos, R. H. Medema, and B. M. T. Burgering. 2002.
Forkhead transcription factor FOXO3a protects quiescent cells from oxidative stress.
Nature 419:316.
41. Taub, J., J. F. Lau, C. Ma, J. H. Hahn, R. Hoque, J. Rothblatt, and M. Chalfie. 1999.
A cytosolic catalase is needed to extend adult lifespan in C. elegans daf-C and clk-1
mutants. Nature 399:162.
42. Tran, H., A. Brunet, J. M. Grenier, S. R. Datta, A. J. Fornace, Jr., P. S. DiStefano,
L. W. Chiang, and M. E. Greenberg. 2002. DNA repair pathway stimulated by the
forkhead transcription factor FOXO3a through the Gadd45 protein. Science 296:530.
Downloaded from http://www.jimmunol.org/ by guest on June 12, 2017
25. Zhao, H. H., R. E. Herrera, E. Coronado-Heinsohn, M. C. Yang,
J. H. Ludes-Meyers, K. J. Seybold-Tilson, Z. Nawaz, D. Yee, F. G. Barr, S. G. Diab,
et al. 2001. Forkhead homologue in rhabdomyosarcoma functions as a bifunctional
nuclear receptor-interacting protein with both coactivator and corepressor functions.
J. Biol. Chem. 276:27907.
26. Christian, M., X. Zhang, T. Schneider-Merck, T. G. Unterman, B. Gellersen,
J. O. White, and J. J. Brosens. 2002. Cyclic AMP-induced forkhead transcription
factor, FKHR, cooperates with CCAAT/enhancer-binding protein ␤ in differentiating human endometrial stromal cells. J. Biol. Chem. 277:20825.
27. Kortylewski, M., F. Feld, K.-D. Krüger, G. Bahrenberg, R. A. Roth, H.-G. Joost,
P. C. Heinrich, I. Behrmann, and A. Barthel. 2002. Akt modulates STAT-3-mediated
gene expression through a FKHR (FOXO1a)-dependent mechanism. J. Biol. Chem.
278:5242.
28. Hirota, K., H. Daitoku, H. Matsuzaki, N. Araya, K. Yamagata, S. Asada, T. Sugaya,
and A. Fukamizu. 2003. HNF-4 is a novel downstream target of insulin via FKHR as
a signal-regulated transcriptional inhibitor. J. Biol. Chem. 278:13056.
29. Medema, R. H., G. J. P. L. Kops, J. L. Bos, and B. M. T. Burgering. 2000. AFX-like
forkhead transcription factors mediate cell-cycle regulation by Ras and PKB through
p27kip1. Nature 404:782.
30. Dijkers, P. F., R. H. Medema, C. Pals, L. Banerji, N. S. B. Thomas, E. W.-F. Lam,
B. M. T. Burgering, J. A. M. Raaijmakers, J.-W. J. Lammers, L. Koenderman, and
P. J. Coffer. 2000. Forkhead transcription factor FKHR-L1 modulates cytokine-dependent transcriptional regulation of p27KIP1. Mol. Cell Biol. 20:9138.
31. Schmidt, M., S. Fernandez de Mattos, A. van der Horst, R. Klompmaker,
G. J. P. L. Kops, E. W.-F. Lam, B. M. T. Burgering, and R. H. Medema. 2002. Cell
cycle inhibition by FoxO forkhead transcription factors involves downregulation of
cyclin D. Mol. Cell Biol. 22:7842.
32. Kops, G. J. P. L., R. H. Medema, J. Glassford, M. A. G. Essers, P. F. Dijkers,
P. J. Coffer, E. W.-F. Lam, and B. M. T. Burgering. 2002. Control of cell cycle exit
and entry by protein kinase B-regulated forkhead transcription factors. Mol. Cell Biol.
22:2025.
1629