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Endocrinology Vol. 148, No. 5 2424-2434
Copyright © 2007 by The Endocrine Society

Nuclear Forkhead Box O1 Controls and Integrates Key Signaling Pathways in Hepatocytes

Mourad Naïmi, Nadine Gautier, Claire Chaussade, Angela M. Valverde, Domenico Accili and Emmanuel Van Obberghen

Institut National de la Santé et de la Recherche Médicale, U145, and Université de Nice-Sophia Antipolis, Faculté de Médecine, Institut de Génétique et Signalisation Moléculaire (IFR50) (M.N., N.G., C.C., E.V.O.), F-06107 Nice, France; Instituto de Investigaciones Biomédicas Alberto Sols Consejo Superior de Investigaciones Científicas C/Arturo Duperier 4 (A.M.V.), 28029 Madrid, Spain; and College of Physicians and Surgeons (D.A.), Columbia University, New York 10032

Address all correspondence and requests for reprints to: Emmanuel Van Obberghen, Institut National de la Santé et de la Recherche Médicale U145, Institut Federatif de Recherche 50, Faculté de Médecine, Avenue de Valombrose, F-06107 Nice, France. E-mail: vanobbeg{at}unice.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Insulin inhibits forkhead O class (FoxO) transcription factors, which down-regulate the expression of genes involved in metabolism, cell cycle arrest, and apoptosis. After being phosphorylated by protein kinase B (PKB) on S253 in its DNA-binding domain, Foxo1 is phosphorylated on T24 and additional sites, which overall triggers its nuclear exclusion. During this process, Foxo1 is thought to retain some transcriptional activity and signaling potential. To evaluate this Foxo1 action, we used a Foxo1-ADA mutant that is constitutively nuclear due to mutation of T24 and S316 to A and harbors a mutation of S253 to D. Adenoviral-mediated expression of Foxo1-ADA in hepatocytes activates PKB and MAPK pathways more than expression of wild-type or of a transactivation domain-deleted mutant ({Delta}256). PKB activation cannot be accounted for by a Foxo1-mediated increase in upstream signaling components such as insulin receptor substrate 1 or 2 or by Foxo1-mediated down-regulation of Tribbles homolog 3. In contrast, Foxo1-ADA increases p38 activity, and p38 is required for effects of Foxo1 on PKB, at least in part. We propose that Foxo1 turns on a feed-forward loop, relayed by p38 and acting to amplify both PKB activation and Foxo1 inhibition. To conclude, key signaling pathways are activated in hepatocytes through nuclear Foxo1.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FORKHEAD MEMBERS of the O class (FoxO) transcription factors consist of a family of four members, Foxo1, Foxo3a, Foxo4, and Foxo6, serving as transcriptional endpoints of several signaling cascades. In response to growth factors, activated protein kinase B (PKB) phosphorylates Foxo1, Foxo3a, and Foxo4 on conserved serine/threonine residues. It is generally believed that this leads to the retention of FoxO transcription factors in the cytoplasm, thereby reducing the expression of their target genes (1). FoxO transcription factors are involved in a wide range of events, including cell cycle progression, cell death and metabolism, DNA repair, protection against oxidative stress, and organismal longevity (1).

In Caenorhabditis elegans and Drosophila, deficiency in the insulin-like pathway requires FoxO nuclear localization to extend lifespan (2). In Drosophila, activation of the c-Jun-N-terminal kinase (JNK) homolog pathway protects against environmental insults and enhances longevity by counteracting insulin-like signaling and promoting Drosophila Foxo nuclear localization (3). In mammalian cells, FoxO factors are viewed as tumor suppressors, triggering cell death or cell cycle arrest. Interestingly, a phosphorylation-site mutant of Foxo1 (FKHR-AAA), in which the three Akt phosphorylation sites are altered, induces cell death, whereas a similar construct (FKHR-HRAAA) with a negative charge in the DNA-binding domain, which reduces its affinity for insulin-responsive elements, fails to do so (4). Moreover, in transgenic mice overexpressing FKHR-AAA in the liver, increased apoptosis is not observed (5). Taken together, these observations led us to hypothesize that the apoptotic properties of FKHR-AAA may, in part, reflect constitutive binding to insulin-responsive elements and the consequent inability to promote a survival signal otherwise transmitted by nuclear phosphorylated FoxO species. Notably, stress signals promote survival and relocalize FoxO factors to the nucleus without affecting their phosphorylation on PKB target sites (6).

Tribbles homolog 3 (TRB3) belongs to a family of pseudokinase proteins acting as scaffolds, but their exact mechanisms of action are unknown. Tribbles expression in Drosophila is restrained to a narrow developmental window to control the timing and location of mitoses during gastrulation.

In adult mammals and notably humans, TRB3 expression is physiologically restrained to the liver and is up-regulated in tumor cells (7). Some of the hepatic functions attributed to TRB3 such as the inhibition of PKB activity have been initially addressed in transformed cell lines that may not represent a physiological model to study metabolism (8, 9), and these findings are at odds with its involvement in tumorigenesis. Other studies conducted in primary hepatocytes have failed to prove either a regulation of PKB by TRB3 or a modulation of TRB3 levels by hormonal signals (10).

Matsumoto et al. (11) recently showed that overexpression of Foxo1-ADA or DNA-binding deficient Foxo1 (DBD-Foxo1-ADA) activates PKB. They attributed this effect to a reduced TRB3 expression induced by Foxo1 in primary hepatocytes. However, in these cells, endogenous Foxo1 small interfering RNA (siRNA) fails to up-regulate TRB3 levels. Consistent with the involvement of TRB3 Drosophila homolog in cell cycle regulation (12) and TRB3 overexpression in human tumors (7), this may indicate that in untransformed cells, the regulation of TRB3 expression does not solely depend on Foxo1.

A recent report provides evidence that Foxo1 regulates multiple metabolic pathways in the liver and may also modulate genes involved in cell signaling (5). Here we used primary cultures of rat hepatocytes to ectopically express wild-type (WT), dominant negative ({Delta}256), and constitutively nuclear Foxo1 mutant mimicking S253 phosphorylation (ADA). We examined the effects of these mutants on metabolic, mitogenic, and stress signaling pathways. Consistent with previous work, we found that all of these mutants were able to activate PKB, in both basal and insulin-stimulated conditions, and independently of IRS proteins. Moreover, TRB3 overexpression in primary hepatocytes did not affect Foxo1-induced PKB activation.

In addition to activating PKB, Foxo1 mutants increased the phosphorylation and activity of ERK, JNK, and p38. Neither ERK nor JNK were involved in PKB activation, but p38 inhibitors and expression of dominant negative p38 reduced Foxo1-mediated PKB activation. p38 inhibition reduced the assembly of mammalian Target of rapamycin complex 2 (mTorc2), the recently identified PKB S473 kinase. The phenomena we revealed in this study were all maximal with a constitutively nuclear Foxo1-ADA mutant.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Adenoviruses and adenoviral transduction
Adenoviruses encoding hemagglutinin (HA)-tagged WT, dominant negative ({Delta}256), and constitutively nuclear (ADA) mouse Foxo1 and LacZ were generated by the Cosmid cassettes and adenoviral DNA-Terminal Protein Complex method as described elsewhere (13). The S253 and S316 residues of mouse Foxo1 correspond to S256 and S319 in human Foxo1. Adenoviruses encoding HA-tagged TRB3 were kindly provided by P. Iynedjian (Geneva School of Medicine, Geneva, Switzerland). Adenoviruses encoding dominant negative p38 {alpha}-isoform were purchased from Cell Biolabs (San Diego, CA). Adenovirus production, amplification, and estimation of viral titer stocks (10) were done in HEK-293 cells. Unless specified differently, cells were transduced with a quantity of virus corresponding to 100 multiplicity of infection.

Hepatocyte preparation and culture conditions
The procedures that were followed in animal studies were reviewed and approved by our Institutional Animal Care Committee. WT and IRS-2 knockout immortalized neonatal mouse hepatocytes were obtained and cultured as described (14). Primary cultures of rat hepatocytes were isolated as described (15). Cells seeded on six-well collagen-coated plates were allowed to attach for 4 h before adenoviruses were added to the medium for 2 h. Medium was then replaced with serum-free Waymouth medium containing 0.2% (wt/vol) fatty-acid-free BSA, penicillin, streptomycin, and gentamicin for 15 h. Medium was finally replaced with Krebs-Ringer-bicarbonate buffer containing 0.2% (wt/vol) BSA for 2.5 h before cell lysis. Wortmannin and SP600125 were purchased from Sigma Aldrich (St. Louis, MO), rapamycin and SB202190 were from Calbiochem (San Diego, CA), and U0126 was from Cell Signaling Technology (Beverly, MA). Each inhibitor was added 1 h before lysis. For the 24-h rapamycin treatment, the medium was replaced after adenoviral transduction with serum-free Waymouth medium containing 10% (vol/vol) fetal bovine serum, penicillin, streptomycin, and gentamicin for 17 h and replaced for 24 h, with rapamycin added at this time or at 1 h before lysis. When indicated and unless specified differently, 100 nM insulin from Novo-Nordisk (Copenhagen, Denmark) was added for 5 min before lysis. Cells were washed with ice-cold PBS and then processed for protein isolation.

Immunoprecipitation and Western blot
Cells were scraped and incubated for 20 min in ice-cold lysis buffer (16). The lysates were centrifuged at 13,000 x g for 20 min at 4 C, and protein concentrations were determined by the BCA colorimetric assay (Interchim, Montluçon, France). Protein A Sepharose CL-4B (Uppsala, Sweden) was incubated with the indicated antibody or with the corresponding preimmune serum for 2 h at 4 C. Then, lysates corresponding to either 400 µg (IRS-1 immunoprecipitation) or 4 mg (Rictor immunoprecipitation) protein were added for 2 h at 4 C. The immunoprecipitates were washed three times with lysis buffer, and 50 µl of 4x Laemmli buffer was added. Immunoprecipitates or 100 µg total lysates were subjected to SDS-PAGE, transferred to Immobilon-P polyvinylidene difluoride membranes (Bedford, MA). Membranes were either presoaked in blocking buffer or directly incubated in blocking buffer containing antibodies, according to the manufacturer’s instructions.

Antibodies
The antibodies to phosphorylated tyrosine residues (no. 05-321), IRS-1 (no. 06-248), IRS-1 phospho-Ser307 (no. 07-247), IRS-2 (no. 06-506), p85 {alpha}-subunit (no. 05-217), and ERK1/2 (no. 06-182) were from Upstate Biotechnology (Lake Placid, NY). Antibodies directed to the HA epitope (no. H6533) and ß-tubulin (no. T4026) were from Sigma Aldrich. The antibody directed to Foxo1 (no. sc-11350) was from Santa Cruz Biotechnology (Santa Cruz, CA). The antibody directed to TRB3 (no. ST1032) was from Calbiochem (San Diego, CA). The antibodies directed to Rictor used for immunoprecipitation and for Western blotting were, respectively, from Bethyl Laboratories (Montgomery, TX; no. BL2178) and Abnova Corp. (Heidelberg, Germany; no. 1F3). Other antibodies were purchased from Cell Signaling Technology (Beverly, MA).

RNA isolation and quantitative real-time PCR analysis
Total RNA was isolated by standard procedures, treated with DNase (Ambion, Austin, TX), and reverse transcribed using the reverse transcription system kit (Promega, Madison, WI). PCR were run in triplicate using SYBR Green I Master Mix Plus (Eurogentec, Seraing, Belgium) and quantitated using the ABI Prism 7700 sequence detection system (Applied Biosystems, Courtaboeuf, France) according to the manufacturer’s instructions. PCR primers for each gene were designed using Primer Express software (Applied Biosystems), and sequences used to quantify TRB3 and HPRT mRNA were as follows: rat TRB3 sense, 5'-CTC AAG TTG CGT CGA TTT GTC T-3', and antisense, 5'-CAC CAA CTT CGT CCT CTC ACA GT-3'; mouse/rat HPRT sense, 5'-TGA AAG ACT TGC TCG AGA TGT CA-3', and antisense, 5'-AAA GAA CTT ATA GCC CCC CTT GA-3'. Cycle threshold values were normalized to HPRT expression, and results are expressed as a fold change of mRNA compared with the LacZ condition, which was arbitrarily assigned a value of 1.

The siRNA experiments
Knockdown of endogenous Foxo1 was performed in primary rat hepatocytes using the JetPEI hepatocyte transfection reagent (Polyplus Transfection SA, Illkirch, France) and 100 nM of a nonspecific oligonucleotide duplex or a duplex of the following targeting oligonucleotides: Foxo1 sense, 5'-CCGCCAAACACCAGUCUAATT-3'; Foxo1 antisense, 5'-UUAGACUGGUGUUUGGCGGTT-3'. Cells were lysed and processed for Western blot analysis 48 h after transfection.

In vitro PKB kinase assays
The in vitro kinase activity of immunoprecipitated PKB toward recombinant glycogen synthase kinase 3 (GSK3) was determined in primary hepatocytes and IRS-2 knockout immortalized neonatal mouse hepatocytes with a nonradioactive Akt kinase assay kit (no. 9840; Cell Signaling Technology) according to the manufacturer’s instructions.

Glycogen synthesis
The effect of Foxo1-ADA on glycogen synthesis was assessed by measuring the incorporation of [3H]glucose into glycogen. Briefly, overnight starved primary hepatocytes in Krebs-Ringer-bicarbonate buffer containing 0.2% (wt/vol) BSA were added to 100 nM insulin, 10 mM cold glucose, and 0.2 µM [3H]glucose for 1 h. Cells were then scraped in 300 µl of a 30% (vol/vol) potassium hydroxide solution and heated at 100 C for 15 min, and glycogen was precipitated overnight at –20 C in a 200-µl 2% (vol/vol) Na2SO4 solution. After a wash in 66% (vol/vol) absolute ethanol, precipitated glycogen was dissolved in 300 µl water, and [3H]glucose incorporation was determined by scintillation counting.

Statistical analysis
Experiments performed at least three times are presented as mean values ± SEM, and P values were determined by unpaired Student’s t test. Results were considered significant for P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Foxo1 stimulates PKB through phosphatidylinositol 3-kinase (PI3K) and mTorc2, independently of TRB3
Ectopic expression of wild-type and Foxo1 mutants in primary cultures of rat hepatocytes increases the phosphorylation of PKB on threonine 308 (not shown) and serine 473 but does not alter PKB expression (Fig. 1Go, A and B). Consistent with PKB activation, we observed increased phosphorylation of wild-type and {Delta}256-Foxo1 on T24 (Fig. 1BGo), the PKB target site responsible for FoxO nuclear exclusion (17). In contrast, phosphorylation of Foxo1-ADA on T24 was not detected (Fig. 1BGo). Furthermore, the in vitro activity of immunoprecipitated PKB toward recombinant GSK3 protein was increased by Foxo1-ADA in basal and insulin-stimulated cells (Fig. 1CGo). Accordingly, in vivo glycogen synthesis was increased by Foxo1-ADA expression upon insulin treatment (Fig. 1DGo).


Figure 1
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  		FIG. 1. Foxo1 stimulates PKB activity. A, Quantification of total or S473-phosphorylated PKB assessed by Western blots (W.B.) in primary cultures of hepatocytes infected with LacZ, WT, {Delta}256, or ADA Foxo1 expression vectors (n = 3). Results were normalized by assigning a value of 1 to the LacZ condition.*, Statistically different from the control LacZ condition; **, statistical difference between Foxo1-infected conditions; N.S., not significant. B, Representative Western blot showing the relative level of endogenous and ectopic Foxo1, Foxo1 T24 phosphorylation, total PKB, and S473-phosphorylated PKB in primary cultures of hepatocytes not infected ({emptyset}) or infected with LacZ, WT, {Delta}256, or ADA Foxo1 expression vectors. The {Delta}256 form of Foxo1 lacks the epitope recognized by the Foxo1 antibody. C, Western blots showing the in vitro kinase activity of immunoprecipitated PKB from primary cultures of hepatocytes infected with LacZ or ADA Foxo1 expression vectors and left untreated or stimulated for 5 min with 100 nM insulin before lysis (n = 3). D, Quantification of insulin-stimulated glycogen synthesis in primary cultures of hepatocytes infected with LacZ or ADA Foxo1 expression vectors (n = 3). Results were normalized by assigning a value of 1 to the LacZ condition.*, Statistically different from the control LacZ condition.

 
Consistent with the idea that Foxo1 uses components of the insulin signaling pathway to activate PKB, wortmannin treatment abolished its effects (Fig. 2AGo). However, addition of insulin to Foxo1-ADA-infected cells still significantly increased PKB phosphorylation. Also, PKB phosphorylation-mediated Foxo1-ADA was increased by a short (1-h) rapamycin treatment, compared with Foxo1-ADA-untreated cells. In agreement with mTorc2 being the genuine PKB S473 kinase (18), a 24-h rapamycin treatment, which has recently been shown to inhibit both PKB and mTorc2, blunted the phosphorylation of PKB S473 induced by Foxo1-ADA (19). The physiological role of endogenous Foxo1 in the regulation of PKB activity was confirmed by Foxo1 siRNA, which reduced insulin-stimulated phosphorylation of PKB on S473 (Fig. 2Go, B and C).


Figure 2
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  		FIG. 2. Foxo1 stimulates PKB through PI3K and mTorc2. A, Upper panel, quantification of the effect of different treatments on PKB Ser473 phosphorylation in primary cultures of hepatocytes infected with LacZ or ADA Foxo1 expression vectors (n = 4). Results were normalized by assigning a value of 1 to the untreated LacZ condition.*, Statistical difference between LacZ and ADA when subjected to the same treatment; **, statistical difference between treatments; N.S., not significant. Lower panel, a representative Western blot (W.B.) is shown. B, Western blot showing the relative level of endogenous Foxo1 and S473-phosphorylated PKB in primary cultures of hepatocytes transfected with control or Foxo1 siRNA in basal and insulin-stimulated conditions. C, Quantification of the effect of Foxo1 siRNA on endogenous Foxo1 expression and PKB Ser473 phosphorylation in primary cultures of hepatocytes in insulin-stimulated conditions (n = 3). Results were normalized by assigning a value of 1 to the control condition.*, Statistically different from the control condition.

 
Ectopic expression of WT and Foxo1 mutants decreased the mRNA and endogenous protein levels of TRB3 (Fig. 3Go, A and B). Using adenoviral expression of TRB3 in primary rat hepatocytes, Iynedjian (10) failed to provide evidence for a role of TRB3 as an inhibitor of insulin-mediated PKB activation. Consistent with this, we found in the same cells that coexpression of TRB3 with Foxo1-ADA did not affect PKB phosphorylation, indicating that Foxo1 does not activate PKB by down-regulating TRB3 (Fig. 3CGo).


Figure 3
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  		FIG. 3. Foxo1 stimulates PKB independently of TRB3. A, Quantification of TRB3 mRNA level assessed by quantitative RT-PCR in primary cultures of hepatocytes infected with LacZ, WT, {Delta}256, or ADA Foxo1 expression vectors (n = 3). Results were normalized by assigning a value of 1 to the LacZ condition.*, Statistically different from the control LacZ condition. B, Representative Western blot showing the relative level of endogenous TRB3 in primary cultures of hepatocytes infected with LacZ ({emptyset}) or increasing amount of ADA Foxo1 expression vector (n = 3). C, Representative Western blot showing the relative levels of total PKB and of S473-phosphorylated PKB in primary cultures of hepatocytes infected with LacZ or Foxo1-ADA or coinfected with both Foxo1-ADA and TRB3 expression vectors (n = 3).

 
In summary, wild-type, {Delta}256, and ADA Foxo1 proteins are able to activate PKB in hepatocytes through PI3K and mTorc2, albeit with different potencies, and independently of TRB3.

Foxo1 activates ERK, JNK, and p38
It has been recently reported that, in addition to PKB, both the ERK and p38 kinases phosphorylate Foxo1 and regulate its activity (20). As part of a newly identified regulatory loop, we found that in turn, ectopic expression of WT and Foxo1 mutants in primary cultures of rat hepatocytes increased the phosphorylation of ERK1/2, JNK1/2, and p38 without affecting their respective expression levels (Fig. 4Go, A and B).


Figure 4
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  		FIG. 4. Foxo1 activates ERK, JNK, and p38. A, Quantification of total or phosphorylated ERK, JNK, and p38 assessed by Western blots (W.B.) in primary cultures of hepatocytes infected with LacZ, WT, {Delta}256, or ADA Foxo1 expression vectors (n = 3). Results were normalized by assigning a value of 1 to the LacZ condition.*, Statistically different from the control LacZ condition; **, statistical difference between Foxo1-infected conditions. B, Representative Western blots showing the levels of total or phosphorylated ERK, JNK, and p38 in primary cultures of hepatocytes infected with LacZ, WT, {Delta}256, or ADA Foxo1 expression vectors. C and D, Western blots showing the respective level of activation of p38 or ERK upstream kinases in primary cultures of hepatocytes infected with LacZ or Foxo1-ADA expression vectors (n = 3).

 
Another recent study showed that multiple pathways can lead to p38 activation in addition to the canonical MAPK kinase (MEK) MKK3/6 (MEK 3/6)-p38 pathway (21). Consistent with this notion, in comparison with the LacZ control, Foxo1-ADA did not increase the phosphorylation of p38 and ERK1/2 respective upstream kinases, MKK3/MKK6 and MEK1/2 (Fig. 4CGo and D).

Foxo1 effects on IRS proteins
As described previously (22), we found that WT and mutant Foxo1 increased IRS-2 expression, up to 5-fold for the Foxo1-ADA mutant (Fig. 5Go, A and B). Because up-regulation of IRS-2 may enhance insulin signaling (23, 24), we ruled out IRS-2 involvement in PKB activation using immortalized neonatal hepatocytes isolated from WT mice and mice with the IRS-2 gene deleted. Foxo1-ADA still significantly increased PKB phosphorylation in IRS-2 knockout cells, compared with the LacZ control, with no compensatory effects on IRS-1 (Fig. 5Go, C and D). Foxo1-ADA also increased the in vitro activity of PKB immunoprecipitated from basal and insulin-stimulated IRS-2 knockout hepatocytes (Fig. 5EGo). In rat hepatocytes, Foxo1-ADA increased IRS-1 expression but did not enhance its tyrosine phosphorylation or association with p85{alpha} (Fig. 6Go, A–C). This may be due to the concomitant increase in IRS-1 serine phosphorylation induced by Foxo1-ADA (Fig. 6Go, B and C), because serine phosphorylation of IRSs is thought to reduce IRS-1/p85{alpha} association and hence insulin action (25). These results suggest that neither IRS-1 nor IRS-2 is involved in Foxo1-mediated PKB activation.


Figure 5
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  		FIG. 5. Foxo1-mediated activation of PKB in IRS-2–/– hepatocytes. A, Quantification of IRS-2 protein level assessed by Western blots in primary cultures of hepatocytes infected with LacZ, WT, {Delta}256, or ADA Foxo1 expression vectors (n = 4). Results were normalized by assigning a value of 1 to the LacZ condition.*, Statistically different from the control LacZ condition; **, statistical difference between Foxo1-infected conditions. B, Representative Western blot showing the level of IRS-2 in primary cultures of hepatocytes infected with LacZ, WT, {Delta}256, or ADA Foxo1 expression vectors. C, Quantification of S473-phosphorylated PKB and of IRS-1 protein level from Western blots (W.B.) of WT or IRS-2–/– immortalized mouse hepatocytes infected with LacZ or ADA Foxo1 expression vectors (n = 3). Results were normalized by assigning a value of 1 to the LacZ condition in IRS-2 WT cells.*, Statistically different from the LacZ condition in the same cell line; **, statistical difference between Foxo1-infected conditions. D, Representative Western blot showing the relative levels of endogenous and ectopic Foxo1, IRS-1, IRS-2, and total and phosphorylated PKB in WT or IRS-2–/– immortalized mouse hepatocytes infected with LacZ or Foxo1-ADA expression vectors. E, Western blots showing the in vitro kinase activity of immunoprecipitated PKB from IRS-2–/– immortalized mouse hepatocytes infected with LacZ or ADA Foxo1 expression vectors and left untreated or stimulated for 5 min with 100 nM insulin before lysis (n = 3).

 

Figure 6
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  		FIG. 6. Foxo1-mediated activation of PKB is independent of IRS-1. A, Upper panel, immunoprecipitation (IP) with IRS-1 or preimmune (PI) serum from rat hepatocyte primary culture lysates (400 µg). Cells were infected with LacZ or Foxo1-ADA expression vectors and left untreated or stimulated for 5 min with 100 nM insulin before lysis. Immunoprecipitates were analyzed as described (n = 2). Lower panel, Western blot (WB) analysis of 100 µg protein of the corresponding total lysates. B, Western blot of total lysates from primary cultures of hepatocytes infected with LacZ or Foxo1-ADA expression vectors showing the level of total and S636-phosphorylated IRS-1 in basal and insulin-stimulated conditions (n = 3). C, Quantification of total and S307-, S636-, and S612-phosphorylated IRS-1 levels from Western blots for primary cultures of hepatocytes infected with LacZ or Foxo1-ADA expression vectors (n = 3). Results were normalized by assigning a value of 1 to the LacZ condition.*, Statistically different from the control LacZ condition.

 
P38 is involved in Foxo1-mediated PKB activation
Because JNK and p38 have been shown to enhance PKB activation (26), we asked whether Foxo1-induced PKB stimulation involved crosstalk between MAPK and PKB (Fig. 7Go). Using specific chemical inhibitors, we found that neither ERK1/2 nor JNK1/2 appears to be involved in PKB activation (Fig. 7Go, A and B). ERK1/2 blockade by the MEK1/2 inhibitor U0126 was confirmed by the decrease in ERK1/2 phosphorylation. The JNK1/2 ATP competitor SP600125 did not modify JNK1/2 phosphorylation (not shown) but reduced its activity as judged by the phosphorylation of c-Jun (Fig. 7BGo).


Figure 7
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  		FIG. 7. p38 is involved in Foxo1-mediated PKB activation. A, Quantification of the effect of different treatments on PKB Ser473 phosphorylation in primary cultures of hepatocytes infected with LacZ or ADA Foxo1 expression vectors (n = 3). Results were normalized by assigning a value of 1 to the untreated LacZ condition.*, Statistical difference between LacZ and ADA when subjected to the same treatment. B, Representative Western blots showing the relative levels of total and phosphorylated PKB and ERK and of phosphorylated c-Jun upon different treatments in primary cultures of hepatocytes infected with LacZ or ADA Foxo1 expression vectors. C, Quantification of phosphorylated PKB or p38 from Western blot for primary cultures of hepatocytes infected with LacZ or Foxo1-ADA or coinfected with Foxo1-ADA and either LacZ or p38 dominant negative (p38 DN) expression vectors (n = 3). Results were normalized by assigning a value of 1 to the LacZ condition.*, Statistically different from the control LacZ condition; **, statistical difference between ADA-infected conditions. D, Representative Western blot showing the relative levels of Foxo1-ADA, phosphorylated PKB and p38, and endogenous and ectopic total p38 in primary cultures of hepatocytes infected with LacZ or Foxo1-ADA or coinfected with Foxo1-ADA and either LacZ or p38 dominant negative (p38 DN) expression vectors. DMSO, Dimethylsulfoxide.

 
In contrast, we found that p38 inhibition with SB202190 reduced Foxo1-mediated PKB activation (Fig. 7Go, A and B). Coexpression of a nonphosphorylatable dominant negative p38 {alpha}-isoform together with Foxo1-ADA inhibited endogenous p38 and PKB phosphorylation (Fig. 7Go, C and D). Finally, we investigated whether mTorc2 was involved in Foxo1- and p38-mediated PKB activation. As shown on Fig. 8AGo, Foxo1-ADA increased the expression level of Rictor in primary hepatocytes. Moreover, mTor association with Rictor is increased by Foxo1-ADA and reduced by p38 inhibition with SB202190 (Fig. 8BGo).


Figure 8
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  		FIG. 8. Involvement of p38 in mTorc2 assembly. A, Quantification of Rictor protein level assessed by Western blots (W.B.) in primary cultures of hepatocytes infected with LacZ or ADA Foxo1 expression vectors (n = 4). Results were normalized by assigning a value of 1 to the LacZ condition.*, Statistically different from the control LacZ condition. B, Upper panel, immunoprecipitation with Rictor or nonimmune serum from rat hepatocyte primary culture lysates (4 mg). Cells were infected with LacZ or Foxo1-ADA expression vectors and left untreated or stimulated for 30 min with 20 µM SB202190 before lysis. Immunoprecipitates were analyzed as described (n = 2). Lower panel, Western blot analysis of 100 µg protein of the corresponding total lysates.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Given their role in a large array of signaling events, FoxO factors are likely to participate in the coordinated response of cells to changes in environmental conditions. Consistent with this view, we found that ectopic Foxo1 expression in hepatocytes activates PKB, ERK, JNK, and p38, without affecting their expression level (Figs. 1Go, A and B, and 4Go, A and B). Increased basal activity of ERK, JNK, and p38 is thought to participate in insulin resistance (27). Constitutive activation of these kinases increases IRS-1 and IRS-2 protein levels but reduces PKB activation (28). In rat hepatocytes ectopically expressing Foxo1, we also found an increase in IRS-1 and IRS-2 protein levels and in IRS-1 serine phosphorylation on sites previously associated with reduced insulin action (Figs. 5Go, A and B, and 6Go, A–C). However, upon Foxo1-ADA expression, we did not detect a substantial decrease in IRS-1 tyrosine phosphorylation or in IRS-1 association to p85{alpha} (Fig. 6AGo). Recent data (29, 30) and ours here presented suggest that the concept of insulin resistance induced by IRS-1 serine phosphorylation should be viewed with caution when mechanisms leading to increased insulin signaling act downstream of IRS molecules. This is apparent, for example, from our findings showing that PKB activity was increased by Foxo1 despite increased serine phosphorylation of IRS-1. At first glance, Foxo1-mediated up-regulation of IRS-2 appeared as an attractive mechanism by which it could enhance hepatic insulin signaling (24). However, we excluded this as being the sole possibility in Foxo1-ADA-infected hepatocytes that were isolated from IRS-2 knockout mice (Fig. 5Go, C–E).

Conflicting results exist as to whether TRB3 is modulated during the fasting-feeding transition and acts to dampen insulin signaling and PKB activation in the liver. One possibility is that these discrepancies may be attributable to the use of transformed cellular systems (8, 9). Iynedjian (10) conducted studies in primary hepatocytes that failed to prove either a regulation of PKB by TRB3 or a modulation of TRB3 levels by hormonal signals.

Matsumoto et al. (11) showed that Foxo1-ADA promotes PKB phosphorylation and reduces TRB3 expression in primary hepatocytes in a DNA-binding-independent manner. However, in these cells, endogenous Foxo1 siRNA fails to up-regulate TRB3 levels (11). The notion that TRB3 is up-regulated in human tumors is at odds with its presumed functions on PKB. Ectopic expression of TRB3 in our conditions did not affect PKB stimulation induced by Foxo1 (Fig. 3CGo).

We found that inhibition of p38, but not of ERK or JNK, reduces PKB activation induced by Foxo1 expression (Fig. 7Go). In agreement with a recent report (20), our results suggest that the stress kinase p38 may also modulate FoxO phosphorylation through PKB activation. The search for the hypothetical phosphoinositide-dependent kinase-2, which phosphorylates PKB on serine 473, has led to the implication of several kinases such as p38 and JNK (26). However, more recent efforts have identified mTorc2 as the most likely genuine phosphoinositide-dependent kinase-2 (18). Very recent reports have shown that at least three different mTorc2 complexes are able to activate PKB, one of which may be regulated by stress signals (31, 32). Our findings suggest that Foxo1 and p38 activate PKB by up-regulating Rictor expression and increasing mTorc2 assembly (Fig. 8Go). Also, two recent reports have suggested that Rictor phosphorylation may control mTorc2 assembly (33, 34). Our results strongly suggest that Rictor may be a substrate of p38.

PKB phosphorylation on S473 by mTorc2 has been shown to be required for Foxo1 T24 phosphorylation but not for most other effects downstream of PKB including GSK3 or Foxo1 S253 phosphorylation (33, 35, 36). Although we do find an increase in the in vitro PKB kinase activity toward a GSK3-peptide in Foxo1-ADA-infected primary hepatocytes, the level of S473-phosphorylated PKB did not fully correlate with its ability to phosphorylate GSK3 (Fig. 1CGo). Compared with unstimulated Foxo1-ADA-infected cells, insulin-treated control conditions exhibited an increase in PKB phosphorylation on S473 but displayed reduced T308 phosphorylation and kinase activity. In contrast, in transformed hepatocytes, PKB phosphorylation on S473 mirrored that of T308 and its activity (Fig. 5EGo).

One possible explanation suggested by Guertin et al. (33) may be that in primary cells, PKB phosphorylation on T308 is less dependent on its previous phosphorylation on S473 than in transformed cells. This may indicate that PKB activity in tumor cells may be more sensitive to mTorc2 inhibition than in normal cells.

Another interesting conclusion that can be drawn from our experiments in primary hepatocytes is that Foxo1-ADA seems to increase the level of basal and insulin-stimulated phosphorylation of PKB on T308 relative to that of S473, compared with insulin-stimulated control conditions. Assuming that most of insulin’s metabolic effects depend solely on T308, this may be indicative of an unexpected insulin-sensitizing effect of Foxo1-ADA. Our findings that Foxo1-ADA increases insulin-stimulated glycogenesis are consistent with this view (Fig. 1DGo).

Remarkably, p38 activation in the liver is increased during fasting (37) and in diabetic mice models (38). cAMP response element binding protein phosphorylation and peroxisome proliferator-activated receptor-{gamma} coactivator 1{alpha} expression, which are necessary to turn on the gluconeogenic program, have been shown to be dependent on p38 stimulation (37). In addition, it has been reported that p38-mediated phosphorylation of CCAAT/enhancer binding protein {alpha} increases phosphoenolpyruvate carboxykinase transcription in the liver (38). During fasting, low insulin levels and activated p38 may partially stimulate PKB, which in turn is able to phosphorylate Foxo1 on S253 but not to exclude it from the nucleus. Consequently, Foxo1 and p38 may together activate the gluconeogenic and/or prosurvival program. When feeding and the subsequent rise in circulating insulin level occur, both p38 and PKB may act together to switch off Foxo1 through its phosphorylation on T24. Given our finding that Foxo1 is able to activate both PKB and MAPK, the benefit of this could be that during periods of starvation, a positive feed-forward loop shifts Foxo1 activity to enhance survival and gluconeogenesis, until PKB activation reaches a threshold driving Foxo1 nuclear exclusion. Such a scenario may also protect against unrestrained PKB activity.

Our data suggest that the phosphorylation of PKB on S473 and of Foxo1 on T24 are tightly coupled and are part of the same regulatory loop. In our experimental conditions, WT and {Delta}256-Foxo1 activate PKB to a similar degree, whereas the constitutively nuclear ADA mutant was more potent (Fig. 1BGo). The stronger action of the ADA mutant compared with WT and {Delta}256-Foxo1 may reflect the fact that once PKB is activated, WT Foxo1 and {Delta}256-Foxo1 become phosphorylated on both S253 and T24 and are excluded from the nucleus (Fig. 1CGo) (17).

Foxo1 transactivating potential involves protein-protein interactions or post-translational modifications that may be specific for WT, {Delta}256, or ADA-Foxo1 proteins. Our finding that {Delta}256 Foxo1 also activates PKB may indicate that the transactivation domain of Foxo1 is not required and that the shared effects of Foxo1 mutants may imply an active repression of gene transcription at specific promoters (4). On the other hand, the lesser effects of this mutant compared with Foxo1-ADA may indicate a requirement for the transactivation domain. As recently shown for the phosphoenolpyruvate carboxykinase promoter (39), it should be noted that endogenous and phosphorylated Foxo1 still interacts with IRS sequences, and the same may hold true for Foxo1-ADA, either directly or through protein-protein interactions. Interestingly, it has been shown that when Foxo1 is mainly cytosolic and phosphorylated, it interacts with and inhibits Tuberous Sclerosis Complex 2, promoting mTorc1 activation (40). This may be relevant to our study because some authors have suggested a competition between raptor and rictor for mTor binding (41, 42). Hence, the cytosolic localization of WT and {Delta}256-Foxo1 in our model may explain their reduced ability to activate mTorc2 and PKB.

Given the fact that FoxO transcription factors are involved in a gamut of important biological programs, it is not surprising that mechanisms have been built in to thwart their activity. The circuitries we have revealed here are likely to participate in the coordinated cellular responses to changes in FoxO functions.

Our data provide only a glimpse into the regulation of the FoxO transcription factors, because it involves not only phosphorylation but also acetylation and ubiquitination. The Foxo1-ADA mutant behaves as if it were phosphorylated on S253. Similar to acetylation of the FoxO factors, which acts as a switch controlling their apoptotic and survival properties (43, 44), increases their phosphorylation on S253, and may reduce their binding to DNA (45, 46), we propose that insulin-mediated phosphorylation of Foxo1 on S253 stimulates its potential to activate PKB and the MAPK pathways. However, additional studies are needed to evaluate the role of phosphorylation at this site.

A major challenge is to put the regulation loops we have identified in the broader network of the interplay between FoxO and the pathways it regulates and by which it is in turn modulated. Identification of alterations in these mutually regulating interactions is likely to contribute to a better understanding of several human disease conditions. In addition to their well-established metabolic functions, our data suggest that FoxO factors may be involved in tumorigenesis. Indeed, FoxO fusion transcripts found in both leukemias and alveolar rhabdomyosarcomas always possess disruptions in their DNA-binding domain, and they have been shown to activate the oncogenic potential of the translocations (47). Finally, as already shown for another Forkhead member (48), our results with Foxo1-ADA may explain the permissive role of insulin on liver regeneration because PKB (49), ERK (50), and JNK activation (51) and increased caveolin1 expression (52, 53) have been involved in this process.

Together, our data show that several major signaling pathways can be activated in hepatocytes through nuclear Foxo1. Dampening of these pathways would take place only with Foxo1 phosphorylation on T24. Similar to the dual role of Foxo1 in controlling hepatic metabolism, our data argue for a dual role of Foxo1 in death and survival control. Such a scenario reveals an unexpected role for Foxo1 in amplifying metabolic, survival, mitogenic, and stress signals and suggests the existence of multiple feedback loops by which Foxo1 integrates and controls these pathways.

In conclusion, Foxo1 appears to regulate multiple aspects of liver physiology, including development, metabolism, survival, proliferation, and maybe regeneration.


   Acknowledgments
 
We thank P. Iynedjian (Geneva School of Medicine, Switzerland) for providing us with TRB3-encoding adenoviruses and I. Mothe-Satney for scientific support.


   Footnotes
 
Our research was supported by Institut National de la Santé et de la Recherche Médicale, Région PACA, Université de Nice Sophia-Antipolis, and by grants from the European Community [FP6 EUGENE 2 (LSHM-CT-2004-512013)] and Institut de Recherche Servier (Suresnes, France).

Disclosure Statement: The authors have nothing to disclose.

First Published Online February 15, 2007

Abbreviations: FoxO, Forkhead members of the O class; GSK3, glycogen synthase kinase 3; HA, hemagglutinin; JNK, c-Jun-N-terminal kinase; MEK, MAPK kinase; MKK3, MEK3; mTorc2, mammalian Target of rapamycin complex 2; PI3K, phosphatidylinositol 3-kinase; PKB, protein kinase B; siRNA, small interfering RNA; TRB3, Tribbles homolog 3; WT, wild type.

Received October 20, 2006.

Accepted for publication February 5, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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