Nuclear/cytoplasmic shuttling of the transcription factor FoxO1 is regulated by neurotrophic factors

Nuclear/cytoplasmic shuttling of the transcription factor FoxO1 isregulated by neurotrophic factors

Lixia Gan,* Wenhua Zheng,* Jean-Guy Chabot,* Terry G. Unterman,�,� and Remi Quirion*

*Douglas Hospital Research Center, Department of Psychiatry, McGill University, Montreal, Quebec, Canada

�University of Illinois at Chicago, College of Medicine, Chicago, Illinois, USA

�Jesse Brown Department of Veterans Affairs Medical Center, Chicago, Illinois, USA

Abstract

FoxO1, a member of the FoxO subfamily of forkhead tran-

scription factors, is an important target for insulin and growth

factor signaling in the regulation of metabolism, cell cycle and

proliferation, and survival in peripheral tissues. However, its

role in the central nervous system is mostly unknown. In this

study, we examined the effect of neurotrophic factors on

nuclear/cytoplasmic shuttling of FoxO1. We showed that

insulin-like growth factor-1 (IGF-1) and nerve growth factor

(NGF) potently induced the nuclear exclusion of FoxO1-green

fluorescent protein (GFP) while neurotrophin (NT)-3 and NT-4

were much weaker and brain-derived neurotrophic factor

(BDNF) failed to induce FoxO1 translocation in PC12 cells.

FoxO1 translocation was inhibited by LY294002, a well-

established PI3K/Akt kinase inhibitor. Moreover, FoxO1 was

phosphorylated at Thr24 and Ser256 residues by the above

neurotrophic factors, with the exception of BDNF. Triple

mutant FoxO1, in which three Akt/PKB phosphorylation sites

(Thr24, Ser256 and Ser319) were mutated to alanine, resulted

in the complete nuclear targeting of the expressed FoxO1-

GFP fusion protein in the presence of the above neurotrophic

factors in both PC12 cells and cultured hippocampal and

cortical neurons. Taken together, these findings demonstrate

that neurotrophic factors are able to regulate nuclear/cyto-

plasmic shuttling of FoxO1 via the PI3K/Akt pathway in

neuronal cells.

Keywords: FoxO1, insulin-like growth factor-1, nerve growth

factor, neurotrophins, PC12 cells, trafficking.

J. Neurochem. (2005) 93, 1209–1219.

FoxO proteins are a subclass of the forkhead transcriptionfactors which now contain four mammalian members: FoxO1,FoxO3a, FoxO4 and FoxO6 (Kaufmann and Knochel 1996;Anderson et al. 1998; Biggs et al. 2001; Jacobs et al. 2003).The physiological importance of this subfamily of proteins hasbeen demonstrated in cell culture models and by loss-of-function and gain-of-function experiments in knockout andtransgenic mice, and includes effects on mammalian develop-ment and metabolic regulation (Castrillon et al. 2003; Nakaeet al. 2002; Hosaka et al. 2004). By regulating the expressionof functionally important target genes, FoxO proteins havebeen reported to play important roles in regulating cell cycle,apoptosis and survival, proliferation and differentiation,protection from oxidative stress, DNA repair and metabolismin a variety of cell types (Burgering and Kops 2002;Birkenkamp and Coffer 2003; Accili and Arden 2004).

Although sharing a high structural homology in their DNAbinding domains, each isoform of FoxO protein showsdifferent functions by knockout studies in mice. FoxO1

homozygous null mutants die before birth as a result ofincomplete vascular development and other embryonic defects(Hosaka et al. 2004) while FoxO1 heterozygote-null mutantsshow impaired pancreatic b-cell growth and reduced hepaticgluconeogenesis (Kitamura et al. 2002; Nakae et al. 2002).FoxO3a knockout mice have abnormal ovarian follicular

Received November 8, 2004; revised manuscript received January 21,2005; accepted January 24, 2005.Address correspondence and reprint requests to Remi Quirion PhD,

Douglas Hospital Research Center, 6875 LaSalle Boulevard, Verdun,H4H 1R3, Quebec, Canada. E-mail: [email protected] used: BDNF, brain-derived neurotrophic factor;

DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum;GFP, green fluorescent protein; IGF-1, insulin-like growth factor-1;MEK, mitogen-activated protein kinase kinase; NGF, nerve growthfactor; NT-3, neurotrophin-3; NT-4, neurotrophin-4; PBS, phosphate-buffered saline; PI3, phosphatidylinositide 3; PKB, protein kinase B;SDS–PAGE, sodium dodecyl sulfate – polyacrylamide gel electrophor-esis; SGK, serum- and glucocorticoid-inducible kinase.

Journal of Neurochemistry, 2005, 93, 1209–1219 doi:10.1111/j.1471-4159.2005.03108.x

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 1209–1219 1209

development, mild anemia and decreased glucose uptake inglucose-tolerance tests (Castrillon et al. 2003; Hosaka et al.2004). Interestingly, FoxO4 knockout mice do not show anyapparent phenotype (Hosaka et al. 2004). These resultsdemonstrate functional divergence among the isoforms.

Transcriptional activities of FoxO proteins can be regula-ted by multiple mechanisms, including protein stability(Barthel et al. 2002; Matsuzaki et el. 2003; Plas andThompson 2003), subcellular distribution (Brunet et al.1999; Kops et al. 1999; Rena et al. 2001), modulation ofDNA binding (Guo et al. 1999; Zhang et al. 2002) andassociation with transcriptional co-regulators (activators orrepressors) (Nasrin et al. 2000; Schuur et al. 2001). Centralto the regulation is the nuclear/cytoplasmic shuttling thatregulates the intracellular FoxO distribution. Phosphorylationat three conserved Akt/PKB sites corresponding to Thr24,Ser256 and Ser319 in human FoxO1 (Rena et al. 1999) arecritical in regulating the shuttling of FoxO proteins (Brunetet al. 1999; Kops et al. 1999; Rena et al. 2001). Besides, theRas/Ral dependent pathway (De Ruiter et al. 2001), proteinkinase CK1 (Rena et al. 2002) and DYRK1A (dual-specif-icity tyrosine-phosphorylated and regulated kinase 1A)(Woods et al. 2001) also have been shown to phosphorylateFoxO proteins at other sites that are thought to be importantin the regulation of FoxO functions.

An extensive list of growth factors has been documentedas stimulating FoxOs phosphorylation including insulin (Guoet al. 1999; Hall et al. 2000; Nakae et al. 2001), insulin-likegrowth factor-1 (IGF-1) (Zheng et al. 2000; Linseman et al.2002), epidermal growth factor (EGF) (Jackson et al. 2000),erythropoietin (EPO) (Uddin et al. 2000; Mahmud et al.2002), stem cell factor (SCF) (Uddin et al. 2000; Mahmudet al. 2002), thrombopoietin (TPO) (Tanaka et al. 2001) andcytokines (Dijkers et al. 2002; Perez et al. 2002; Stahl et al.2002). Moreover, an earlier study in our laboratory hasshown that FoxO3a can be phosphorylated by neurotrophins(Zheng et al. 2002b). The neurotrophin family currentlyincludes four closely related members, namely nerve growthfactor (NGF), brain derived neurotrophic factor (BDNF),neurotrophin (NT)-3 and NT-4. They exert a wide variety ofneuronal functions on survival and differentiation (Lewinand Barde 1996; Huang and Reichardt 2001; Chao 2003).However, limited information is currently available on thepotential role of neurotrophins in regulating the nuclear/cytoplasmic shuttling of FoxO proteins.

In the present study, by introducing a FoxO1-GFP fusionprotein into cultured neuronal cells, we examined thecapacity of IGF-1 and a panel of neurotrophic factors tomodulate the nuclear/cytoplasmic shuttling of FoxO1. Ourdata show that IGF-1 and the neurotrophic factors NGF,NT-3 and NT-4 are able to promote the nuclear exclusion ofFoxO1. Furthermore, by using pharmacological inhibitors ofvarious protein kinases as well as a triple mutant of FoxO1,in which three Akt/PKB phosohorylation sites (Thr24,

Ser256 and Ser319) were mutated to alanine, we demonstra-ted that these effects are mediated by the PI3-kinase/Aktpathway.

Materials and methods

Materials

Human recombinant IGF-1, LY294002, PD98059 and K252a were

from Calbiochem (La Jolla, CA, USA), human recombinant NGF

from Upstate Biotechnology (Lake Placid, NY, USA), BDNF, NT-3

and NT-4 from Pepro Tec. (Rocky Hill, NJ, USA). LipofectAMINE-

2000 and all cell culture reagents were purchased from Invitrogen

(Carlsbad, CA, USA) or Fisher Scientific (Nepean, ON, Canada).

Anti-FKHR, anti-phospho-FKHR (Ser319) and anti-Akt1 were from

Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phospho-

FKHR (Ser319) was purchased from Upstate Biotechnology

(Dundee, Scotland, UK). Anti-phospho-FKHR (Thr24), anti-phos-

pho-FKHR (Ser256) and anti-phospho-Akt (Ser473) were from Cell

Signaling Technology (Woburn, MA, USA). Anti-b-actin and poly-

L-lysine were from Sigma (St Louis, MO, USA). Glass chamber

slides were from Nalge Nunc International Inc (Rochester, NY,

USA). NE-PERTM nuclear and cytoplasmic extraction reagents and

bicinchoninic acid (BCA) protein assay kit were from Pierce

(Rockford, IL, USA). Plasmids wild-type FoxO1-wt-GFP and triple

mutant FoxO1-tm-GFP were kindly provided by one of the

co-authors (TGU).

PC-12 cell culture

Cells were cultured and maintained as described before (Zheng

et al. 2002a) in 75-cm2 flasks in high-glucose Dulbecco’s modified

Eagle’s medium (Invitrogen) supplemented with 5% (v/v) fetal

bovine serum, 5% horse serum, 1% antibiotic–anti-mycotic, and

incubated at 37�C in 5% CO2 incubator. Cells were routinely

passaged at a 1 : 5 ratio every week.

FoxO1-GFP translocation studies

PC-12 cells were plated on 2-well glass chamber slides coated with

poly-L-lysine and fed with DMEM medium supplemented with 5%

FBS, 5% horse serum plus antibiotics in 5% CO2 at 37�C. The nextday, cells were transfected with 0.8 lg of plasmid DNA with

lipofectAMINE-2000 for 4–6 h. Then, media were removed.

Transfected cells were re-fed with DMEM plus 2% serum for

2 days. The culture medium was replaced with serum-free DMEM

for 2 h before the designated treatment with IGF-1 or a neuro-

trophin. In experiments using kinase inhibitors, cells were pre-

incubated with either LY294002 (50 lM), wortmannin (0.5 lM) orPD98059 (50 lM) for 30 min. After treatment, cells were fixed with

4% paraformaldehyde in phosphate-buffered saline (PBS) and

rinsed in pre-warmed PBS prior to applying mounting solution

containing 4¢, 6-diamido-2-phenylindole-2 HCl (DAPI; Vector

Laboratories, Burlingame, CA, USA). GFP-tagged FoxO1 and

DAPI-stained nuclei were visualized and recorded with confocal

laser scanning microscope (Nikon PCM 2000, USA). For quanti-

tative analysis of FoxO1 distribution in the subcellular location, the

percentage of cells was used to represent the majority of

fluorescence in each compartment (nucleus or cytoplasm) of a

particular cell, and a total of 400 cells were counted per slide.

1210 L. Gan et al.

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 1209–1219

Hippocampal and cortical neuronal cultures

Hippocampal and cortical neurons were prepared from fetuses

(embryonic day 19) obtained from pregnant Sprague–Dawley rats

(Charles River Breeding Laboratories, St Constant, QC, Canada) as

previously described (Zheng et al. 2002a).Animal carewas according

to protocols and guidelines approved by McGill University Animal

Care Committee and the Canadian Council for Animal Care. Neurons

were plated at a density of 8 · 105 cells/well in two-well glass

chamber slides pre-coated with 0.1 mg/mL of poly-L-lysine under

serum-free conditions in neurobasal medium supplemented with 2%

B-27 (Invitrogen), 20 lM L-glutamine, 15 mM HEPES, 100 mM

sodium pyruvate (Sigma), 10% KCl and 1% antibiotic–anti-mycotic

(Invitrogen). On the third day after plating, the medium was replaced

with newmedium as above and transfectionwith FoxO1-GFP plasmid

was performed on the seventh day after plating. Cells were transfected

with 0.8 lg of plasmid DNA with lipofectAMINE-2000 for 4–6 h.

The media were then removed and transfected cells were re-fed with

B-27 supplemented neurobasal medium for 2 days. The culture

medium was replaced with B-27-free neurobasal medium for 3–16 h

before the designated treatment with IGF-1 or a neurotrophin.

Western blotting

Western blotting was performed according to protocols routinely

used in our laboratory. In brief, treated cells from different

experimental conditions were rinsed once with ice-cold PBS and

lysed in RIPA buffer [50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM

EDTA, 1% Igepal CA-630, 1% sodium dodecyl sulfate (SDS),

50 mM NaF, 1 mM NaVO3, 5 mM phenylmethysulfonyl fluoride,

10 lg/mL leupeptin (Sigma), and 50 lg/mL aprotinin (Sigma)].

Protein concentration was measured with a BCA protein assay kit

according to the manufacturer’s instructions. Samples with equal

amounts of proteins were denatured in stocked sample buffer with

final concentration of 62.5 mM Tris-HCl pH 6.8, 2% (w/v) SDS,

10% glycerol, 50 mM dithiothreitol and 0.1% (w/v) bromophenol

blue at 100�C for 5 min. Proteins were resolved by 4–20% gradient

Tris-Glycine SDS–poly acrylamide gel electrophoresis (PAGE), and

then transferred onto Hybond-C membranes. Membranes were

blocked with 6% (w/v) non-fat dried milk in Tris-bufferd saline,

then probed with primary antibody with a dilution of 1 : 500 to

1 : 2000 (see figure legends for details), and detected with second

antibodies at a dilution of 1 : 5000. Blots were stripped in stripping

buffer with final concentration of 62.5 mM Tris-HCl pH 6.8, 2%

(w/v) SDS and 100 mM b-mercaptoethanol at 50�C for 30 min, and

re-probed with the designated primary antibody to ensure equal

amounts of loaded protein samples.

Statistical analysis

Values are reported as means ± SEM from at least three different

experiments. Comparison between two experimental groups was

made using t-test. p < 0.05 was considered significant.

Results

Effect of different neurotrophic factors on the nuclear/

cytoplasmic distribution of FoxO1-GFP in PC12 cells

Previous studies have established that the nuclear/cytoplasmicdistribution of FoxO1 is modulated by IGF-1 and other growth

factors (Rena et al. 2001; Zhang et al. 2002). However, thereis no report so far regarding the neurotrophins and FoxO1trafficking. We have shown that serum deprivation promotesthe nuclear localization of FoxO1-wt-GFP fusion protein (GFPprotein tag alone is not affected by serum starvation, data notshown), whereas treatments with neurotrophic factors reversedthis process (Fig. 1a). Interestingly, different neurotrophicfactors show differential capacity to influence the subcellulardistribution of FoxO1 fusion protein, with IGF-1 and NGFbeing most effective, followed by NT-3 and NT-4, and BDNFbeing mostly inactive in PC12 cells.

To examine the effects of IGF-1 and NGF in greater detail,different doses of these factors were used on the intracellulartrafficking of FoxO1 fusion protein in PC12 cells. The nuclearexclusion of FoxO1 fusion protein induced by IGF-1 and NGFis dose-dependent (Figs 1b and c). During serum starvation,the GFP fusion protein is located predominantly in the nucleusof�73%of cells (control panel). Localization of fusion proteinin the nucleus decreased while that in the cytoplasm increasedwith increasing doses of IGF-1 or NGF. The maximal effect ofIGF-1 was seen at 10 nM and at 100 ng/mL for NGF.

We further compared the overall effectiveness of variousneurotrophic factors. Cytoplasmic localization of fusionprotein in cell populations increased from 27% (control) to90, 82, 48, 40 and 31% in cells treated with 10 nM IGF-1 or100 ng/mL NGF, NT-3, NT-4 or BDNF, respectively, in PC12cells. Statistical analysis revealed the significance of the effectsinduced by IGF-1, NGF, NT-3 and NT-4 under our assayconditions, whereas BDNF had no significant effect (Fig. 1d).

Nuclear export of FoxO1-wt-GFP by neurotrophic factors

is PI3-kinase dependent

We examined next the mechanism(s) mediating the effects ofneurotrophic factors on FoxO1 trafficking using kinaseinhibitors. As shown in Fig. 2 (i–iv), treatment withLY294002, an inhibitor known to interact irreversibly withthe catalytic subunit of PI3 kinase, blocked the nuclearexport signal induced by IGF-1, NGF, NT-3 and NT-4. Incontrast, treatment with PD98059, an inhibitor of theactivation of MAP kinase kinase (MAPKK, or MEK),showed no significant effect (Fig. 2, v–viii).

Akt/PKB is activated by IGF-1 and NGF

Several kinases downstream of the PI3 kinase have beenreported as being able to phosphorylate FoxO proteins;among them are Akt/PKB (Rena et al. 1999; Brunet et al.1999; Kops et al. 1999) and SGK (Brunet et al. 2001). Wethus determined whether neurotrophic factors were able toactivate them. As the full activation of both kinases requiresphosphorylation in their kinase domains (equivalent toThr308 in Akt/PKB and Thr246 in SGK) and their regulatoryC-terminal domains (equivalent to Ser473 in Akt/PKB andSer422 in SGK) (Kandel and Hay 1999), we thus determinedthe effects of neurotrophic factors on the phosphorylation of

FoxO1 trafficking regulated by neurotrophic factors 1211


Akt/PKB and SGK using anti-phospho-Akt (Ser473) andanti-phospho-SGK (Ser422) antibodies. As shown in Fig. 3(aand b), IGF-1 and NGF stimulated the phosphorylation ofAkt/PKB at Ser473 in a dose-dependent manner, while thephosphorylation at this residue by NT-3, NT-4 and BDNF didnot change significantly (Fig. 3c). Phosphorylation of SGKat Ser422 was not altered by treatments with any of thesetrophic factors (data not shown).

Phosphorylation of FoxO1 at Thr24 and Ser256 is

stimulated by neurotrophic factors and is blocked by the

PI3-kinase inhibitor

The phosphorylation of FoxO1 was reported to be respon-sible for its intracellular trafficking (Rena et al. 2001; Zhang

et al. 2002). Studies in our laboratory have previously shownthat FoxO3a can be phosphorylated by IGF-1 and neurotro-phins (Zheng et al. 2002a,b). As the three Akt/PKB phos-phorylation sites present in FoxO3a are known to beconserved in FoxO1, we tested whether neurotrophic factorscould also stimulate the phosphorylation of this transcriptionfactor. We have shown that IGF-1 and NGF dramaticallyinduced, in a concentration-dependent manner, the phos-phorylation of FoxO1 at Ser256 and Thr24 residues, with amaximal effect observed at 10 nM for IGF-1 (Fig. 4a) and30 ng/mL for NGF (Fig. 4b). Phosphorylation of FoxO1 atall three Akt/PKB sites (Thr24, Ser256 and Ser319) wascompared among the neurotrophic factors. As shown inFig. 4(c), while IGF-1 and NGF were most potent in

(b)

(a)

(c) (d)

Fig. 1 Cellular trafficking of FoxO1-wt-GFP fusion proteins in PC12

cells. (a). Neurotrophic factors modulate the nuclear/cytoplasmic dis-

tribution of FoxO1-wt-GFP fusion protein. PC12 cells were plated onto

poly-L-lysine coated slides, then transfected with FoxO1-wt-GFP

expression vector. Cells were quiescenced in serum-reduced (2%)

medium for 2 days and starved for 2 h in serum-free medium before

treatment with various neurotrophic factors. Cells were treated for 1 h,

then fixed and visualized with GFP fusion proteins (green) by laser

confocal microscopy. Subcellular distributions of FoxO1-wt-GFP

fusion protein were shown under the following conditions: (i) serum

starvation; (ii) IGF-1 (10 nM); (iii) NGF (100 ng/mL); (iv) NT-3

(100 ng/mL); (v) NT-4 (100 ng/mL) and (vi) BDNF (100 ng/mL). Scale

bar, 20 lm. (b, c). Dose-dependent effects of IGF-1 and NGF on the

nuclear exclusion of FoxO1-wt-GFP fusion protein. Subcellular distri-

bution of fusion protein was determined according to the majority of

fluorescence in the nucleus (solid bars) or cytoplasm (open bars). Four

hundred cells were counted per slide for each dose. Mean ± SEM was

from at least three separate experiments. Control represents baseline

conditions without treatment (serum starvation). (d) Cytoplasmic

localization of FoxO1-GFP fusion protein following treatment with dif-

ferent neurotrophic factors. Percentage of cells was defined as above.

*p < 0.05; , nucleus; h, cytoplasm.

1212 L. Gan et al.


increasing FoxO1 phosphorylation at Thr24 and Ser256, NT-3and NT-4 were much less effective, and BDNF was inactive ata concentration up to 100 ng/mL. No significant changes wereobserved on the phosphorylation of the Ser319 residue(Figs 4a–c).

In order to directly observe the linkage between theactivity of the kinases and the phosphorylation of the FoxO1sites, specific kinase inhibitors were used. We have shownthat pretreatment with LY294002, but not that withPD98059, could significantly block the phosphorylation ofFoxO1 at both Thr24 and Ser256 sites induced by IGF-1 orNGF (Figs 5a and b), and by NT-3 and NT-4 (Figs 5c and d).As NT-3 receptor TrkC and NT-4 receptor TrkB are notexpressed in our PC12 cells, we tested whether TrkA isinvolved in the mediation of the effects of NT-3 and NT-4.For these experiments, a potent inhibitor of the Trk family ofreceptor tyrosine kinases, K252a, was used. A pretreatmentwith K252a markedly decreased the phosphorylation signalsat Thr24 and Ser256 (Figs 5c and d).

Subcellular levels of endogenous FoxO1 are altered by

IGF-1 treatment

To confirm the above trafficking results visualized withexogenously introduced tagged GFP fusion protein, weexamined endogenous FoxO1 levels in nuclear versuscytoplasmic compartments. PC12 cells were serum-starvedand then treated with 10 nM IGF-1 from 15 min to 4 h, andcytoplasmic and nuclear protein fractions were subsequentlyprepared with NE-PERTM nuclear and cytoplasmic extractionkit. FoxO1 levels in either compartment were analyzed by

western blot. As shown in Fig. 6, treatment with 10 nM IGF-1induced a remarkable shift of FoxO1 from the nucleus to thecytoplasm. Nuclear FoxO1 was noticeably decreased, whileits cytoplasmic counterpart markedly increased as early as by15 min post-treatment, with a maximal effect seen at 30 minand sustained up to 4 h.

Akt/PKB phosphorylation sites are critical for FoxO1

shuttling in PC12 cells and primary cultured neurons

To verify whether Akt/PKB is indeed the critical pathway forFoxO1 shuttling in neuronal cells, the three conserved Akt/PKB phosphorylation sites (Thr24, Ser256 and Ser319)within FoxO1 were specifically mutated into alanine. Triplemutant construct (FoxO1-tm-GFP) was then introduced intoPC12 cells and primary hippocampal and cortical neurons.As shown in Fig. 7, triple mutant fusion protein wasexclusively located in the nucleus in the absence (panel i)and in the presence of IGF-1, NGF, NT-3 and BDNF NT-4(panels ii–vi) in PC12 cells.

The trafficking of FoxO1 fusion protein was also observedin primary hippocampal and cortical neurons. FoxO1-wt-GFP was predominantly located in the cytoplasm in bothtypes of cultured neurons under conditions of starvation(Figs 8ai and bi) and following treatment with variousneurotrophic factors (not shown). Treatment with LY294002resulted in the complete retention of the FoxO1 fusionprotein in the nucleus (Figs 8aii and bii). This effect wasmimicked by the triple mutant construct, FoxO1-tm-GFP, inwhich the mutant protein was exclusively nuclear in bothhippocampal and cortical neurons (Figs 8aiii and biii).

Fig. 2 Nuclear export of FoxO1-wt-GFP induced by neurotrophic

factors is PI3-kinase dependent. PC12 cells were transfected with

FoxO1-wt-GFP expression vector, then quienscenced and starved

under serum-free conditions. Cells were then treated for 30 min with a

PI3-kinase inhibitor LY294002 (50 lM) or a MEK kinase inhibitor

PD98059 (50 lM), followed by treatments with neurotrophic factors for

1 h. (i–iv) Treatment with LY294002 (LY) resulted in the complete

nuclear retention of GFP fusion protein in the presence of any of the

trophic factors, while treatment with PD98059 (PD) (v–viii) did not alter

the distribution pattern of FoxO1-wt-GFP induced by the neurotrophic

factors tested. Scale bar, 20 lm.



Interestingly, a high concentration of IGF-1 (100 nM) couldnot reverse the nuclear location of the mutant protein(Figs 8aiv and biv).

Discussion

We have shown here that the subcellular distribution ofFoxO1 can be regulated by several neurotrophic factors, withIGF-1 and NGF being most potent, followed by NT-3 andNT-4, while BDNF was inactive in our PC12 cell culturemodel. The phosphorylation of FoxO1 was also increasedwith an identical order of potency in PC12 cells. Moreover,the inhibition of PI3-kinase or the mutation of Akt/PKBphosphorylation sites in FoxO1 resulted in the completenuclear retention of FoxO1 in the presence of trophic factorsin both PC12 cells and primary cultured hippocampal andcortical neurons. Taken together, these findings demonstratethat the nuclear/cytoplasmic shuttling of FoxO1 can beregulated by neurotrophic factors via the PI3K/PKB pathwayin neuronal cells.

IGF-1 is known to bind to three distinct receptors, namelythe IGF-I, IGF-II and insulin receptors (LeRoith et al. 1995;Dore et al. 1997; Kar et al. 1997). Among them, the IGF-Ireceptor shows highest affinity for IGF-1. The binding ofIGF-1 to this receptor activates its intrinsic receptor tyrosinekinase activity, which subsequently phosphorylates intra-cellular substrates such as the insulin receptor substrate-1(IRS-1) and Shc (LeRoith et al. 1995; Stewart and Rotwein

(a)

(b)

(c)

Fig. 4 FoxO1 is phosphorylated at Thr24 and Ser256 by neurotrophic

factors. PC12 cells were serum starved and treated with different

concentrations of IGF-1, NGF or other trophic factors for 1 h, then

lysed in RIPA buffer. Phosphorylation of FoxO1 was determined with

designated phosphorylated FoxO1 antibody. Blots were stripped and

re-probed with anti-FoxO1 antibody (anti-FKHR, 1 : 500 dilution) as

internal control. IGF-1 significantly induced the phosphorylation of

FoxO1 at Thr24 (anti-phospho-FKHR.Thr24, 1 : 1000 dilution) and

Ser256 (anti-phospho-FKHR.Ser256, 1 : 500 dilution) residues and in

a concentration-dependent manner (a), NGF shows a similar dose-

dependent effect on the phosphorylation at Thr24 and Ser256 (b),

whereas NT-3 and NT-4 were much less potent with BDNF being

inactive in PC12 cells (c). The phosphorylation of Ser319 (anti-phos-

pho-FKHR.Ser319, 1 : 500 dilution) residue was not altered by any of

the above trophic factors.

(a)

(b)

(c)

Fig. 3 Akt/PKB is activated by IGF-1 and NGF. Cultured PC12 cells

were serum starved and treated with different concentrations of IGF-1,

NGF or other trophic factors for 1 h, then lysed in RIPA buffer. Pro-

teins were loaded and separated on SDS–PAGE, and transferred for

western blotting with phospho-Akt (Ser473) rabbit monoclonal anti-

body (1 : 500 dilution). Blots were stripped and re-probed with anti-

Akt1/PKB antibody (1 : 1000 dilution) as an internal control. IGF-1 and

NGF induced the phosphorylation of Akt at the Ser473 residue in a

concentration-dependent manner (a, b), whereas NT-3, NT-4 and

BDNF did not show significant induction on phosphorylaton of Akt (c).

1214 L. Gan et al.


1996), leading to the activation of several signaling path-ways, including the Ras/Raf/MAPK and PI3-K/Akt path-ways (Vincent and Feldman 2002; Zheng et al. 2002a). Wehave shown here that FoxO1 is a downstream effector of thePI3K-Akt pathway induced by IGF-1 in PC12 cells, as wellas in primary hippocampal and cortical neurons. It is thusadding to our previous findings on the involvement of theforkhead family transcription factors, such as FoxO3a(Zheng et al. 2002a), in mediating the neuroprotectiveeffects of IGF-1.

Neurotrophins, currently including four closely relatedmembers (NGF, BDNF, NT-3 and NT-4), bind to twostructurally unrelated families of receptors, termed Trk andp75NTR. They display high affinity for the Trk receptors andlower affinity for p75 (Segal and Greenberg 1996; Kaplanand Miller 2000). Trk includes three family members, TrkA,TrkB and TrkC. NGF preferentially binds to TrkA, whileBDNF and NT-4/5 bind to TrkB, and NT-3 is a preferredligand of TrkC (Klein et al. 1989; Kaplan et al. 1991;Lamballe et al. 1991). All neurotrophins apparently bind top75NTR with equivalent affinities (Segal and Greenberg1996; Kaplan and Miller 2000). Following the binding totheir respective Trk receptors, the neurotrophins promotereceptor transphosphorylation, which subsequently recruitsan extensive class of adaptor proteins, including IRS-1/2,Shc, FRS-2, rAPS, SH2-B and CHK, to docking sites on the

(a) (b)

(c) (d)

Fig. 5 Phosphorylation of FoxO1 at Thr24 and Ser256 is blocked by

PI3-kinase inhibitor. PC12 cells were serum starved and pretreated for

30 min with PI3-kinase inhibitor (LY294002, 50 lM), MEK kinase

inhibitor (PD98059, 50 lM) or TrkA receptor tyrosine kinase inhibitor

(K252a, 0.2 l), then treated with the designated concentration of

trophic factors for 1 h. Phosphorylation of FoxO1 was determined with

anti-phospho-FKHR.Thr24 (1 : 1000 dilution) or anti-phospho-

FKHR.Ser256 (1 : 500 dilution). Parallel blots were probed with anti-

FKHR (1 : 500 dilution) to monitor equal loading. As shown in this

figure, pretreatment with LY294002, but not that with PD98059, could

significantly block the phosphorylation of FoxO1 at both Thr24 and

Ser256 sites induced by IGF-1 or NGF (a, b) and by NT-3 and NT-4

(c, d). Pretreatment with K252a could also markedly decrease the

phosphorylation at Thr24 and Ser256 induced by NT-3 and NT-4

(c, d).

Fig. 6 Endogenous subcellular concentration of FoxO1 is modified in

response to IGF-1 in PC12 cells. PC12 cells were serum starved and

treated with 10 nM of IGF-1 for different time periods. Cytoplasmic and

nuclear proteins were separately extracted with NE-PERTM nuclear

and cytoplasmic extraction kit as described in methods. The FoxO1

levels were determined by western blotting. Samples loaded as

duplicate on the same gel were probed with anti-b-actin (1 : 2000

dilution) to monitor even loading. Nuclear levels of FoxO1 were

noticeably decreased as early as by 15 min post-treatment, while

cytoplasmic FoxO1 levels were markedly increased. FoxO1 redistri-

bution reached a stable level at 30 min post IGF-1 treatment, and

remained unchanged for up to 4 h.

Fig. 7 Nuclear export induced by neurotrophic factors is blocked by

mutation of PKB phosphorylation sites on FoxO1 in PC12 cells. PC12

cells were prepared and plated onto poly-L-lysine coated slides, then

transfected with triple mutant expression vector (FoxO1-tm-GFP), in

which three Akt/PKB phosphorylation sites (Thr24, Ser256 and

Ser319) within FoxO1 were mutated to alanine. Cells were starved

and treated with neurotrophic factors for 1 h. Triple mutant FoxO1-tm-

GFP fusion proteins were exclusively located in the nucleus in the

absence (a) and presence of IGF-1, NGF, NT-3, NT-4 and BDNF (b–f)

in PC12 cells, demonstrating the critical importance of this pathway in

the regulation of FoxO1 shuttling. Scale bar, 20 lm.



receptor, leading to the stimulation of multiple signalingcascades, including Ras/Raf/MAPK and PI3K/Akt, throughwhich neurotrophins induce their wide range of effects(Kaplan and Miller 2000).

In the present study, we have shown that neurotrophinsdemonstrate different effectiveness in regulating the nuclear/cytoplamic shuttling of FoxO1 in PC12 cells. This differencemay come from the differential expression of receptors inPC12 cells, which are known to preferentially express TrkAand p75NTR (Zheng et al. 2002b). Moreover, shuttlingeffectiveness of FoxO1 is well correlated with the abilityof the neurotrophins to phosphorylate FoxO1. Among theneurotrophins, NGF potently phosphorylated FoxO1. Thisresult is consistent with our previous study on FoxO3a(Zheng et al. 2002b) and a recent publication on theeffectiveness of BDNF to modulate FoxO3a shuttling inSH-SY5Y cells known to express TrkB receptors (Zhu et al.2004). The phosphorylation of FoxO proteins (FoxO1,FoxO3a and FoxO4) in PC12 cells was reported to bemediated through the high-affinity TrkA receptor without theparticipation of p75NTR as phosphorylation was induced onlyin TrkA- but not p75NTR-expressing cells (Zheng et al.2002b). The minor effect of NT-3 and NT-4 in inducing bothnuclear/cytoplasmic shuttling of FoxO1 and its phosphory-lation in PC12 cells is likely related to TrkA receptors. Insupport of this hypothesis, it is known that (i) NT-3 is a weakagonist ligand for TrkA receptor (Belliveau et al. 1997); (ii)NT-4, although not a direct ligand for TrkA, could bind to thep75NTR receptor (Kaplan and Miller 2000) which can directlyinteract with the TrkA receptor (Bibel et al. 1999; Jung et al.2003), and that interaction of p75NTR with TrkA can activate

the PI3K/Akt pathway (Epa et al. 2004); (iii) the inhibitionof TrkA with K252a markedly decreased signals at twoFoxO1 phosphorylation sites, Thr24 and Ser256, which arecritical for FoxO1 shuttling (Figs 5c and d). As a directactivation of Akt and its related kinase SGK by NT-3 andNT-4 was not observed by western blot assay, we cannotexclude the possibility that other kinases downstream fromPI3 kinase may be contributing to the phosphorylation ofFoxO1. Moreover, western blotting might not be sensitiveenough to detect modest changes in Akt activity (Park et al.1999).

We have also tested the abilities of neurotrophic factors toregulate FoxO1 shuttling on hippocampal and corticalneurons (TrkB+ TrKC+p75+). Shuttling effects of thesetrophic factors were difficult to observe because of thealready predominantly cytoplasmic localization of FoxO1-wt-GFP proteins under basal conditions in primary culturedneurons, even after a brief B27 starvation (data not shown). Itwould thus appear that basal FoxO1 phosphorylation levelsmay already be high enough in primary neurons to retainFoxO1 into the cytoplasm.

The nuclear/cytoplasmic shuttling of FoxO1 is regulatedby multiple mechanisms, including inhibiting nuclear import(14-3-3-dependent) and promoting nuclear export (14-3-3-independent) ones (Van Der Heide et al. 2004; Zhao et al.2004). Regarding the first one, the phosphorylation of FoxO1at Thr24 and Ser256 residues is critical. Phosphorylation atthese two sites functions co-operatively to bind 14-3-3proteins, while single phosphorylation at either the Thr24 orSer256 residue is not sufficient (Zhao et al. 2004). Bindingof 14-3-3 proteins to FoxO1 and 14-3-3 dimerization mask

(a)

(b)

Fig. 8 Subcellular localization of FoxO1

wild-type or triple mutant fusion protein in

cultured primary hippocampal and cortical

neurons. Primary hippocampal and cortical

neurons were prepared and maintained as

described in Materials and methods. (a, b)

The subcellular distribution of FoxO1-

wt-GFP was mostly cytoplasmic in both

kinds of cultured neurons (i). Treatment with

LY294002 resulted in the complete retent-

ion of FoxO1-wt-GFP fusion protein into the

nucleus (ii). This effect was mimicked by

the triple mutant fusion construct, FoxO1-

tm-GFP, in both hippocampal and cortical

neurons (iii). A high concentration of IGF-1

(100 nM) could not reverse the location of

the mutant protein from the nucleus into the

cytoplasm (iv) demonstrating the critical

importance of Akt/PKB phosphorylation

sites in mediating the trafficking effects

induced by neurotrophic factors. Scale bar,

20 lm.

1216 L. Gan et al.


two nuclear localization signals located in the region of theDNA-binding domain, therefore impairing DNA binding,limiting interaction with other nuclear transcription factor,and inhibiting FoxO1 transport back into the nucleus. Ourdata show that neurotrophic factors stimulate the phosphory-lation of both Thr24 and Ser256, strongly supporting theconcept that FoxO1/14-3-3 binding mechanism likely playsan important role in the regulation of FoxO1 trafficking inneuronal cells.

Besides inhibiting its nuclear import, the shuttling ofFoxO1 is also regulated by promoting nuclear exclusion(Nakae et al. 2000; Rena et al. 2001, 2002). Rena et al.(2002) found that the phosphorylation of Ser319 by PKBwas followed by the phosphorylation of Ser322 and thenSer325 by CK1. Moreover, we have recently shown that thecluster of phosphorylation sites at Ser319, Ser322, Ser325and Ser329 (Woods et al. 2001) enhances nuclear exportmediated by nearby nuclear export signal (NES) via Ran- andCrm-1 nuclear export proteins-dependent mechanisms (Zhaoet al. 2004). In the present study, we did not observesignificant change in the phosphorylation of the Ser319residue following treatments with neurotrophic factorsincluding IGF-1. This might result from its failure torecognize proteins containing multiple phosphorylated resi-dues near Ser319 as the synthetic peptides used to generatethis antibody only included the phospho-Ser319 site. Furtherstudies are in progress to clarify this point.

FoxO proteins serve multiple important functions fromdevelopment in mouse to lifespan control in Caenorhab-ditis elegans and Drosophila melanoganster (Lin et al.1997; Ogg et al. 1997; Hwangbo et al. 2004) and theregulation of the nuclear/cytoplasmic shuttling of FoxOtranscription factors is important to modulate their biolo-gical functions (Van Der Heide et al. 2004). In severalcultured neuronal cell models, the FoxO family memberFoxO3a is implicated in the regulation of cell apoptosis(Brunet et al. 1999; Gilley et al. 2003; Leinninger et al.2004). FoxO1 was reported to regulate FasL expression inthe hippocampal CA1 region in transient forebrain ische-mia in gerbils (Kawano et al. 2002). FasL is the prototypeof a large family of death ligands which can initiate anextrinsic apoptotic pathway by binding to correspondingdeath receptors (Strasser et al. 2000; Heidenreich 2003).FoxO1 was also recently reported as regulating theintrinsic apoptotic pathway by inducing the expression ofthe pro-apoptotic protein Bim in experimental seizures andin the hippocampi of patients with intractable temporallobe epilepsy (Shinoda et al. 2004). It is well known thatIGF-1 and NGF are trophic factors essential for growthand survival on different CNS and PNS tissues (Doreet al. 1997; Zheng et al. 2002a,b). Therefore, the potenteffects of IGF-1 and NGF on FoxO1 shuttling via thePI3K/Akt cascade provide a new mechanism by which

these neurotrophic factors can promote neuronal survivalunder ischemic or epileptic conditions.

In summary, we have shown that nuclear/cytoplasmicshuttling of FoxO1 is regulated by neurotrophic factors suchas IGF-1 and NGF via the PI3-kinase/PKB pathway in PC12cells and primary neuronal cultures. Because the regulationof the nuclear/cytoplasmic distribution of FoxO proteins iscritical to their biological functions, the shuttling of FoxOtranscription factors by neurotrophic factors provides amechanism by which they can prevent neuronal apoptosisand promote cellular integrity.

Acknowledgements

This research was supported by the Canadian Institute of Health

Research (RQ) and partly by grants from the National Institute of

Health (RO1 DK41430) (TGU) and the Department of Veterans

Affairs Merit Review Program (TGU).

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Nuclear/cytoplasmic shuttling of the transcription factor FoxO1 is regulated by neurotrophic factors

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