Vav1 Promotes T Cell Cycle Progression by Linking TCR/CD28 ...

The Journal of Immunology

Vav1 Promotes T Cell Cycle Progression by Linking TCR/CD28 Costimulation to FOXO1 and p27kip1 Expression1 Ce´line Charvet,2* Ann Janette Canonigo,* Ste´phane Be´cart,* Ulrich Maurer,3† Ana V. Miletic,‡ Wojciech Swat,‡ Marcel Deckert,§ and Amnon Altman4* Vav proteins play a critical role in T cell activation and proliferation by promoting cytoskeleton reorganization, transcription factor activation, and cytokine production. In this study, we investigated the role of Vav in T cell cycle progression. TCR/CD28stimulated Vav1ⴚ/ⴚ T cells displayed a cell cycle block at the G0-G1 stage, which accounted for their defective proliferation. This defect was associated with impaired TCR/CD28-induced phosphorylation of Akt and the Forkhead family transcription factor, FOXO1. The cytoplasmic localization of FOXO1 and its association with 14 –3-3␶ were also reduced in Vav1ⴚ/ⴚ T cells. Consistent with the important role of FOXO1 in p27kip1 transcription, stimulated Vav1ⴚ/ⴚ T cells failed to down-regulate the expression of p27kip1, explaining their G0-G1 arrest. These defects were more pronounced in Vav1/Vav3 double-deficient T cells, suggesting partial redundancy between Vav1 and Vav3. Importantly, IL-2-induced p27kip1 down-regulation and cyclin D3 up-regulation and FOXO1 phosphorylation were similar in Vav1ⴚ/ⴚ and wild-type T lymphoblasts, indicating that defective FOXO1 phosphorylation and p27kip1 and cyclin D3 expression do not result from deficient IL-2 signaling in the absence of Vav1. Thus, Vav1 is a critical regulator of a PI3K/Akt/FOXO1 pathway, which controls T cell cycle progression and proliferation. The Journal of Immunology, 2006, 177: 5024 –5031.

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ecognition of peptides presented by MHC molecules on the surface of APCs by the TCR induces quiescent T cells to enter the cell cycle. Progression of T cells through the cell cycle requires activation of signaling cascades that ultimately stimulate transcription factors and cytokines production. Secretion of IL-2 and interaction with its high affinity receptor (IL-2R), composed of the newly expressed ␣ subunit (IL-2R␣) and the constitutively expressed IL-2R␤ and IL-2R␥ subunits, provides the second signal for S phase entry and T cell expansion. The critical role of the Vav family of guanine exchange factors (GEFs)5 for Rho GTPases in T cell development, activation, and proliferation is well established (1, 2). This family consists of three mammalian members, Vav1, -2, and -3, which display different expression profiles, Vav1 expression being restricted to the hemopoietic lineage (1). Vav proteins are phosphorylated and activated upon engagement of different receptors, including TCR, costimulatory receptors (e.g., CD28), and cytokine receptors. Integration of the signals emanating from the TCR and costimulatory recep*Division of Cell Biology and †Division of Cellular Immunology, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037; ‡Department of Pathology and Immunology, Washington University School of Medicine and Siteman Cancer Center, St. Louis, MO 63110; and §Institut National de la Sante´ et de la Recherche Me´dicale U576, Hoˆpital de l’Archet I, 06202 Nice Cedex 3, France Received for publication April 17, 2006. Accepted for publication July 25, 2006. 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 This work was supported by National Institutes of Health Grant GM50819 (to A.A.). This is publication number 728 from La Jolla Institute for Allergy and Immunology. We thank the Philippe Foundation for financial support. 2 Current address: Laboratory of Transplantation Immunology and Nephrology, University Hospital Basel, CH-4031, Basel, Switzerland. 3 Current address: Institute for Molecular Medicine and Cell Research, Stefan Meier Strasse 17, 79104 Freiburg, Germany. 4 Address correspondence and reprint requests to Dr. Amnon Altman, 9420 Athena Circle, La Jolla, CA 92037. E-mail address: [email protected] 5 Abbreviations used in this paper: GEF, guanine nucleotide exchange factor; FOXO, Forkhead box class O; 7-AAD, 7-aminoactinomycin D.

Copyright © 2006 by The American Association of Immunologists, Inc.

tors induces translocation of Vav to the T cell membrane (3, 4) and formation of multisubunit signaling complexes comprising adapters like SLP-76 and LAT, and tyrosine kinases of the Syk, Src, and Tec families, ultimately leading to activation of different signaling pathways and proliferation. Vav1-deficient T lymphocytes show a severely impaired TCR signaling, characterized by defects in TCR-induced calcium flux, activation of MAPKs and transcription factors NF-␬B and NFAT (5), resulting in impaired IL-2 production and proliferation (6 – 8). These developmental and activation defects are more pronounced in vav1⫺/⫺vav2⫺/⫺vav3⫺/⫺ triple gene knockout mice, indicating a compensatory role for Vav3 in T cells (9). Vav proteins also regulate activation of the PI3K/Akt pathway in lymphocytes (10, 11). PI3K has been shown to be important in the control of cell cycle progression and apoptosis. Among the molecular targets of PI3K, the mammalian FOXO (Forkhead box, class O) family of Forkhead transcription factors plays a critical role in the regulation of proliferation and apoptosis. Three mammalian orthologues of Caenorhabditis elegans DAF-16, named FOXO1 (FKHR), FOXO4 (AFX), and FOXO3a (FKHR-L1) have been identified (12). Growth factor or antigenic stimulation induces phosphorylation of FOXO proteins by the serine/threonine kinase Akt on three consensus sites, resulting in their nuclear exclusion and sequestration in the cytosol by association with the chaperone protein 14-3-3 (13–15). Once in the nucleus, FOXO proteins are able to activate the transcription of their target genes implicated in cell cycle or apoptosis, including Bim, p27kip1, and FasL (16). Although FOXO1 deletion resulted in vascular defects and embryonic lethality (17), the recent characterization of FOXO3a-deficient mice revealed that FOXO3a plays a critical role in T cell tolerance by regulating the activation of NF-␬B (18). However, the precise functions and regulation of FOXO proteins during T cell activation remain unclear. Here, we investigated the mechanism through which Vav1 controls cell cycle progression of T lymphocytes, and provide novel evidence for the regulation of FOXO1 activity and p27kip1 expression 0022-1767/06/$02.00

The Journal of Immunology by Vav via the intermediates PI3K and Akt. Thus, Vav1⫺/⫺ T cells displayed a defect in the PI3K-dependent, Akt-mediated phosphorylation of FOXO1 and its nuclear export upon TCR/CD28 engagement and, in parallel, impaired stimulus-induced down-regulation of p27kip1. In addition, we provide evidence that these defects are early and directly result from impaired TCR/CD28 signaling rather than reflecting deficient IL-2 production by Vav1⫺/⫺ T cells. Together, our findings indicate that Vav1 acts as a critical inducer of cell cycle progression by controlling a TCR/CD28-stimulated pathway consisting of PI3K, Akt, FOXO1, and p27kip1.

Materials and Methods Mice, Abs, and reagents All mice were maintained under specific pathogen-free conditions in accordance with guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care International. Vav1⫺/⫺ mice were a gift from Dr. V. Tybulewicz (National Institute for Medical Research, Mill Hill, U.K.). Double knockout Vav1⫺/⫺Vav3⫺/⫺ mice have been described elsewhere (9). Six- to ten-week-old wild-type and Vav1⫺/⫺ C57BL/6 mice were used in all experiments. The mAbs specific for mouse CD3 (2C11) and mouse CD28 (37.51) were affinity-purified from culture supernatants of the respective hybridomas. Anti-FOXO1, anti-phospho-Thr24 FOXO1, anti-phospho-Ser256 FOXO1, anti-phospho-Ser473 Akt, anti-phosphoErk1/2, or anti-Erk1/2 polyclonal Abs, anti-cyclin D3 mAb, and the MEK inhibitor U0126 were obtained from Cell Signaling. Anti-p27kip1 mAb was purchased from BD Biosciences, rat anti-␣-tubulin (clone YL1/2) was purchased from Serotec, and anti-actin mAb was obtained from Stratagene. Polyclonal anti-lamin B Ab was purchased from Santa Cruz Biotechnology, and a neutralizing anti-mouse IL-2 Ab was obtained from eBioscience. LY294002 and wortmannin were obtained from Calbiochem, and recombinant human IL-2 was purchased from PeproTech.

Cell culture and stimulation Primary T cells were isolated from pooled spleen and lymph nodes and enriched to ⱖ90% purity using mouse T cell enrichment columns (R&D Systems). CD4⫹ T cells were purified by negative selection using a MACS system with rat anti-mouse CD8 and B220 Abs (BD Pharmingen) followed by incubation with goat anti-rat Ig-coated magnetic beads (Miltenyi Biotec) (19). T cells were cultured in RPMI 1640 medium (Invitrogen Life Technologies) supplemented with 10% FBS, 2 mM glutamine, 1 mM sodium pyruvate, 10 mM HEPES, I mM MEM nonessential amino acid solution (Invitrogen Life Technologies), and 100 U/ml each of penicillin G and streptomycin. For long stimulation, purified T cells were stimulated with plate-coated anti-CD3 plus anti-CD28 mAbs or with PMA plus ionomycin as indicated. For short stimulation (biochemical assays), T cells were suspended in serum-free medium and incubated for 30 min on ice with anti-CD3 mAb in the presence or absence of anti-CD28 mAb (20 ␮g/ml each), followed by cross-linking with mouse anti-hamster IgG (Pierce Biotechnology) for the indicated times at 37°C with gentle shaking. For IL-2 stimulation, total T cells from wild-type or Vav1⫺/⫺ mice were stimulated with PMA (20 ng/ml) plus ionomycin (0.5 ␮g/ml) for 7 days, split every second day and maintained with IL-2 (100 U/ml), to obtain a similar expression of CD25 in both cases. Cells were then washed three times with PBS, cultured in RPMI 1640 containing 0.5% FBS, and restimulated or not with IL-2 as indicated.

Proliferation and cell cycle analysis To measure [3H]TdR incorporation, purified T cells (3 ⫻ 105 cells/200 ␮l) in 96-well flat bottom tissue culture plates were stimulated in triplicate with the indicated concentrations of plate-coated anti-CD3 plus soluble antiCD28 (2.5 ␮g/ml) mAbs or with PMA (20 ng/ml) plus ionomycin (0.5 ␮g/ml) for 48 h. A total if 1 ␮Ci of [3H]TdR was added for the last 18 h of culture. Cells were then harvested and subjected to scintillation counting. To analyze cell division, T cells were labeled with CFSE (2.5 ␮M), stimulated as described above for 72 h and analyzed by flow cytometry. The cell cycle assay was performed using the FITC-BrdU/7-aminoactinomycin D (7-AAD) flow kit (BD Biosciences). Briefly, cells were pulsed with BrdU (10 ␮M) for 2 h, fixed, permeabilized, stained with FITCcoupled anti-BrdU Ab and 7-AAD for 20 min at room temperature, and analyzed by flow cytometry according to the manufacturer’s protocol.

Immunoblotting Cells were washed in ice-cold PBS, lysed in lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA (pH 8.0), 5 mM NaPiP, 1 mM

5025 Na3VO4, 20 mM NaPO4 (pH 7.6), 3 mM ␤-glycerophosphate, 10 mM NaF, 1% Triton X-100, and 10 ␮g/ml each aprotinin and leupeptin), and the lysates were collected after centrifugation at 13,000 ⫻ g for 10 min. Samples were subjected to SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with the indicated Abs. Signals were detected using the ECL system (Bio-Rad), and quantified by densitometry using the NIH Image 1.61 software.

Pull-down assay Purified total T cells (1 ⫻ 107 cells) were stimulated and lysed in 200 ␮l as described above. One-tenth of each lysate was saved and used as a loading control. Lysates were then incubated overnight with 5 ␮g of a GST-14-3-3␶ fusion protein at 4°C with gentle shaking. Glutathione sepharose beads (30 ␮g) were then added for 1 h. Pellets were washed three times with lysis buffer, dissolved in 1⫻ Laemmli buffer, subjected to SDSPAGE, transferred to nitrocellulose membranes, and immunoblotted with the indicated Abs.

Nuclear fractionation Purified total T cells (1 ⫻ 107) were washed with ice-cold PBS, resuspended in 100 ␮l of buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 1 mM DTT, and proteases inhibitors), and allowed to swell for 15 min on ice. Nonidet P-40 was then added to a final concentration of 0.5%, samples were quickly vortexed for 10 s, and centrifuged for 2 min (14,000 ⫻ g at 4°C). The supernatant was collected as the cytosolic fraction. Nuclear pellets were washed twice with buffer A (without Nonidet P-40), resuspended in 40 ␮l of buffer B (20 mM HEPES (pH 7.9), 400 mM NaCl, 1 mM EDTA, 1 mM DTT, and proteases inhibitors), vortexed for 10 s, and rocked for 30 min at 4°C. Samples were centrifuged for 10 min at 14,000 ⫻ g, and the supernatant was collected as the nuclear fraction. Samples were analyzed by SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted with the indicated Abs.

Real-time PCR Total RNA was isolated from resting or activated T cells and real-time PCR was performed as described (20). Sequences of primers used were: p27kip1 sense, 5⬘-CCCAAGCCTTCCGCCT-3⬘ and antisense, 5⬘-CTC CAAGTCCCGGGTTAGTTC-3⬘; L19 sense, 5⬘-GGAAAAAGAAGGTCT GGTTGGA-3⬘, and antisense 5⬘-TGATCTGCTGACGGGAGTTG-3⬘. p27kip1 mRNA expression was normalized to L19 mRNA expression.

Results Vav1-deficient T cells are blocked in the G0/G1 phase upon TCR/CD28 engagement We first confirmed the requirement of Vav1 for optimal T cell proliferation. Consistent with earlier reports (6, 9), the anti-CD3/ CD28-induced proliferation of Vav1⫺/⫺ primary T cells was decreased (Fig. 1A). As a control, PMA plus ionomycin stimulation induced a similar level of proliferation in both T cell types (Fig. 1B), ruling out intrinsic defects in the cell cycle machinery itself. To define more precisely this proliferation defect, we analyzed the division of CFSE-labeled T cells. Compared with wild-type T cells, the cycling of Vav1⫺/⫺ T cells was delayed but not totally abrogated (Fig. 1C). The residual proliferation of Vav1⫺/⫺ T cells most likely reflects a compensatory effect of Vav3 (see below). Last, staining with BrdU and 7-AAD allowed us to determine that Vav1⫺/⫺ T cells were blocked in the G0/G1 phase of the cell cycle and did not efficiently progress through the S phase (Fig. 1D). However, stimulated Vav1⫺/⫺ T cells did not show a substantial increase of cell death (represented by the fraction of cells in subG1) when compared with wild-type T cells (6.2% vs 4.5% cell death, respectively; Fig. 1D), indicating that the impaired expansion of Vav1⫺/⫺ T cells is due to a defect in cell cycle progression rather than to an increase in death rate. PI3K inhibition mimics the proliferation defect of Vav1-deficient T cells PI3K plays a key role in lymphocyte cell cycle progression (21) and has also been reported to mediate some functions of Vav in lymphocytes (10, 11, 22). Therefore, we investigated the role of

5026

FOXO1 REGULATION BY Vav1 IN T CELLS

FIGURE 2. PI3K inhibitors block T cell proliferation. A, Total T cells from Vav1⫹/⫹ or Vav1⫺/⫺ mice were stimulated as in Fig. 1C in the absence or presence of LY294002 (10 ␮M). CFSE-stained T cells were analyzed by flow cytometry. B, Vav1⫹/⫹ T cells were stimulated or not with anti-CD3 plus anti-CD28 mAbs (20 ␮g/ml each) for 5 min in the presence of indicated concentrations of LY294002. Whole cell lysates were resolved by SDS-PAGE and phospho-Ser473 Akt, total Akt, and phosphoErk1/2 were detected by immunoblotting.

FIGURE 1. Vav1 is required for an optimal T cell cycle progression. A, Purified total T cells from wild-type (Vav1⫹/⫹; ⽧) or Vav1⫺/⫺ (u) mice were stimulated with the indicated concentrations of plate-coated antiCD3 plus soluble anti-CD28 (2.5 ␮g/ml) mAbs for 48 h. [3H]Thymidine was added for the final 18 h of culture, and proliferation was measured by tritium uptake. B, Proliferation of PMA (20 ng/ml) plus ionomycin (500 ng/ml)-stimulated T cells was analyzed as described in A. C, CFSE-labeled purified CD4⫹ T cells from Vav1⫹/⫹ or Vav1⫺/⫺ mice were left unstimulated or stimulated with anti-CD3 (5 ␮g/ml) plus anti-CD28 (2.5 ␮g/ml) mAbs or with PMA plus ionomycin for 72 h. Cell division was determined by flow cytometry analysis. D, Total T cells from Vav1⫹/⫹ or Vav1⫺/⫺ mice were stimulated with anti-CD3 (10 ␮g/ml) plus anti-CD28 (5 ␮g/ ml) mAbs, and cell cycle progression was determined by BrdU/7-AAD staining and flow cytometry analysis. The numbers indicate the percentages of cells at each stage of the cell cycle. The sub-G1 fraction represents apoptotic cells.

the PI3K/Akt pathway in cell division of wild-type vs Vav1⫺/⫺ T cells. Pharmacological inhibition of PI3K by LY294002 strongly inhibited the anti-CD3/CD28-induced proliferation of wild-type T cells, confirming that PI3K is required for T cell division (Fig. 2A, upper panels). Interestingly, the residual cycling of stimulated Vav1⫺/⫺ T cells was further inhibited by LY294002 (Fig. 2A, lower panels), suggesting residual PI3K activation in these cells. LY294002 treatment did not significantly increase the death of wild-type or Vav1⫺/⫺ T cells (data not shown). We confirmed the specificity of the PI3K inhibitor by demonstrating that it inhibited the inducible phosphorylation of Akt on Ser473 in a dose-dependent manner, but had no effect on Erk1/2 phosphorylation, at least up to a concentration of 5 ␮M (Fig. 2B). The significance of the apparent inhibition of Erk1/2 activation by 10 ␮M LY294002 is questionable since it was not observed in another experiment (Fig.

3E). Thus, it may reflect a lower loading of the gel in this lane. At any rate, this reduction in Erk activation was substantially weaker than the corresponding inhibition of Akt phosphorylation (ⱖ90%) by the same LY294002 concentration. These results indicate that the PI3K pathway is required for primary T cell proliferation and that its pharmacological inhibition partially mimics the proliferative defect of Vav1⫺/⫺ T cells. Impaired TCR/CD28-induced FOXO1 phosphorylation/inactivation in Vav1⫺/⫺ T cells Transcription factors of the Forkhead family constitute important targets of the PI3K/Akt pathway and are phosphorylated by Akt (13, 15). Therefore, we questioned whether Vav1 may play a role in regulating the activity of FOXO proteins. Within 5 min of stimulation, wild-type T cells displayed phosphorylation of FOXO1 on residues Thr24 and Ser256, two consensus Akt phosphorylation sites, and this phosphorylation declined after 15 and 30 min. However, FOXO1 phosphorylation was substantially reduced (by ⱖ70%) in stimulated Vav1⫺/⫺ T cells (Fig. 3A). TCR and CD28 signals have both been shown to contribute to Vav1 phosphorylation and activation (23). Therefore, to define more precisely the contribution of TCR and/or CD28 signals to FOXO1 phosphorylation, we stimulated T cells with different Ab combinations for 5 min. Although anti-CD3 stimulation induced strong FOXO1 phosphorylation on Thr24 and Ser256 in wild-type T cells, it induced reduced FOXO1 phosphorylation in Vav1⫺/⫺ T cells (Fig. 3B). Anti-CD28 stimulation alone did not induce FOXO phosphorylation, but it cooperated with anti-CD3 to further enhance FOXO1 phosphorylation on Thr24, but not on Ser256 (Fig. 3B). Therefore, in subsequent experiments we used anti-CD3/CD28 costimulation to accomplish maximal activation of FOXO1. PMA stimulation,


5027 FOXO1 is retained in the nucleus of activated Vav1⫺/⫺ T cells

FIGURE 3. Impaired FOXO1 phosphorylation in Vav1⫺/⫺ T cells. A, Total T cells from Vav1⫹/⫹ or Vav1⫺/⫺ mice were stimulated with anti-CD3 plus anti-CD28 mAbs (20 ␮g/ml each) for the indicated times, and the expression of Vav1, phospho-Thr24 FOXO1, phospho-Ser256 FOXO1, FOXO1, ZAP-70, phospho-Ser473 Akt, or Akt in cell lysates was analyzed by immunoblotting with the corresponding Abs. B, Lysates of Vav1⫹/⫹ or Vav1⫺/⫺ T cells stimulated for 5 min with anti-CD3 and/or anti-CD28 mAb or with PMA (100 ng/ml), were analyzed as in A. C, Purified CD4⫹ T cells were stimulated and similarly analyzed. D, Wildtype, Vav1⫺/⫺, and Vav1⫺/⫺/Vav3⫺/⫺ T cells were stimulated with for 5 min, and whole cell lysates were analyzed by immunoblotting with the indicated Abs as in C. E, Wild-type T cells were pretreated with LY294002 (10 ␮M) or U0126 (20 ␮M) for 1 h and stimulated as in D. Cell lystates were immunoblotted with the indicated Abs.

which was used as a positive control, induced similar phosphorylation of FOXO1 in wild-type and Vav1⫺/⫺ T cells. A similar defect in FOXO1 phosphorylation on Thr24 was observed in purified Vav1⫺/⫺ CD4⫹ T cells (Fig. 3C). Thus, Vav1 is required for TCR/CD28-induced optimal FOXO1 phosphorylation and inactivation. Next, we investigated whether defective Akt phosphorylation/ activation in Vav1⫺/⫺ T cells could account for the impaired FOXO1 phosphorylation. TCR/CD28 costimulation induced substantial Akt phosphorylation on Ser473 in wild-type T cells, which was significantly reduced in Vav1⫺/⫺ T cells (Fig. 3, A and B). To determine whether the residual FOXO1 phosphorylation in Vav1⫺/⫺ T cells (Fig. 3, A-C) reflects the compensatory activity of Vav3, we also analyzed FOXO1 phosphorylation in Vav1/Vav3 double-deficient T cells (Fig. 3D). Indeed, the phosphorylation of FOXO1 was abrogated in the double-deficient T cells, indicating that Vav1 and Vav3 play a redundant role in FOXO1 phosphorylation. Last, pretreatment of wild-type T cells with the PI3K inhibitor, LY294002, strongly decreased the TCR/CD28-mediated phosphorylation of FOXO1 at both Ser256 and Thr24, whereas the MEK inhibitor, U0126, had no effect (Fig. 3E). These results indicate that Vav1 controls TCR/CD28-mediated FOXO1 phosphorylation through Akt.

Upon growth factor activation, phosphorylated FOXO proteins bind the chaperone protein 14-3-3, and shuttle from the nucleus to the cytosol, where they remain inactive (13). We thus investigated the localization of FOXO1 in anti-CD3/CD28-stimulated wildtype and Vav1⫺/⫺ T cells by nuclear fractionation. Upon CD3/ CD28 costimulation, an increased (⬃2.5-fold) fraction of FOXO1 was localized in the cytosol fraction of wild-type, but not Vav1⫺/⫺ T cells (Fig. 4A, four left lanes). When we evaluated the expression of FOXO1 in the nuclear fraction, we found in wild-type T cells two protein species of ⬃75 and ⬃62 kDa, which were recognized by both anti-FOXO1 (Fig. 4A, four right lanes) and anti-phosphoThr24 FOXO1 (data not shown) Abs, indicating that the ⬃62-kDa represents a bona fide processed form of FOXO1. However, we only detected the smaller, ⬃62-kDa protein in the nuclear fraction of Vav1⫺/⫺ T cells. Neither treatment with a pan-caspase inhibitor, nor addition of a cocktail of proteases inhibitors, could reverse this process, ruling out the possibility that this 62-kDa band was the result of a proteolytic cleavage as previously shown (24). Additional experiments will be required to determine precisely the mechanism responsible for this change in FOXO1 migration. Nevertheless, stimulation reduced the nuclear expression of both FOXO1 species in wild-type T cells by ⬃2-fold, but did not affect the level of the ⬃62-kDa species in Vav1⫺/⫺ T cells, a result consistent with the pattern of cytosolic FOXO1 expression. We also analyzed the sequestration of FOXO1 by 14-3-3 in wild-type and Vav1⫺/⫺ T cells by a pull-down assay. Whereas the

FIGURE 4. Reduced FOXO1 cytosolic translocation and 14-3-3 association in Vav1⫺/⫺ T cells. A, Purified T cells were stimulated with antiCD3 plus anti-CD28 mAbs for 5 min, and cytoplasmic or nuclear fraction were prepared and analyzed by SDS-PAGE and immunoblotting with the indicated Abs. Tubulin and lamin B expression served as a control for the relative purity of the cytosol and nuclear fractions, respectively. Numbers indicate the relative expression levels of FOXO1 as determined by densitometry. These results are representative of three similar experiments. B, T cells were stimulated as in A, and whole cell lysates were incubated with GST-14-3-3␶ (5 ␮g) and glutathione-Sepharose beads. An aliquot was saved as a control for protein loading. Complexes were separated by SDSPAGE and analyzed by immunoblotting with the indicated Abs.

5028

FOXO1 REGULATION BY Vav1 IN T CELLS

recombinant GST-14-3-3␶ protein bound a substantial amount of FOXO1 from lysates of wild-type T cells, it did not associate with any detectable amount of FOXO1 from stimulated Vav1⫺/⫺ T cells (Fig. 4B). Given that the association of 14-3-3 with FOXO1 depends on phosphorylation of the latter, this result is consistent with the phosphorylation defect of FOXO1 in Vav1⫺/⫺ T cells (Fig. 3). Together, our data indicate that Vav1 controls the cytosolic vs nuclear localization of FOXO1 and its association with the chaperone protein 14-3-3. Impaired early p27kip1 down-regulation in stimulated Vav1⫺/⫺ T cells Since the inhibitor of cyclin-dependent kinases, p27kip1, is a target of FOXO proteins (25, 26), we questioned whether the Vav1 mutation affected the expression of p27kip1. Upon anti-CD3/CD28 costimulation, the high basal expression of p27kip1 markedly decreased after 24 and 48 h of stimulation in wild-type T cells, whereas only minimal down-regulation of p27kip1 protein level was observed in stimulated Vav1⫺/⫺ T cells (Fig. 5A). PMA plus ionomycin stimulation partially overcame the effect of the Vav1 mutation and induced some decrease in p27kip1 level, albeit not to the same extent as in wild-type T cells. In contrast to p27kip1, the stimulus-induced up-regulation of cyclin D3 expression was not significantly affected by Vav1 deficiency (Fig. 5A). We also analyzed p27kip1 mRNA expression and found that in wild-type T cells, mRNA expression decreased as early as 1 h after anti-CD3/ CD28 costimulation (Fig. 5B). However, no decrease in p27kip1 mRNA expression was observed in Vav1⫺/⫺ T cells up to 6 h after stimulation. Only after 6 h of stimulation with PMA plus ionomycin did the Vav1⫺/⫺ T cells display reduced p27kip1 mRNA expression, which, however, was still higher than in wild-type T cells (Fig. 5B). Although IL-2 can down-regulate p27kip1 transcription via its high affinity receptor (27), the early (1 h) reduction in p27kip1 mRNA expression in wild-type T cells (Fig. 5B) suggested that this reduction is not secondary to IL-2. Consistent with this, we could not detect IL-2 in the supernatant of wild-type T cells after 1 h of TCR/CD28 stimulation (data not shown). We also measured the expression of p27kip1 mRNA in the presence or the absence of a neutralizing anti-IL-2 Ab, and found that addition of the Ab did not reverse p27kip1 mRNA down-regulation observed after 1 h of TCR/CD28 stimulation (Fig. 5C). Moreover, the fraction of CD25⫹ (IL-2-R␣-chain) wild-type T cells was at the basal level after 1 h of stimulation and started to increase after 3 h, but there was no significant increase in the proportion of CD25⫹ Vav1⫺/⫺ T cells up to 18 h following stimulation (Fig. 5D). However, both wild-type and Vav1⫺/⫺ T cells expressed high and similar levels of CD25 upon PMA plus ionomycin stimulation (Fig. 5D). Therefore, the early reduction of p27kip1 mRNA expression observed 1 h after anti-TCR/CD28 stimulation of wild-type T cells (Fig. 5B) does not result from IL-2 signaling via the high affinity IL-2R. Thus, Vav1 is required for an optimal TCR/CD28-induced transcriptional down-regulation of p27kip1. Vav1 is not required for IL-2-dependent FOXO1 phosphorylation and p27kip1 down-regulation IL-2 induces phosphorylation of Vav1 on tyrosine, and Vav1⫺/⫺ T cells show a defect in IL-2 production (5, 28). Therefore, to determine more precisely the contribution of IL-2 to the regulation of FOXO1, we first compared the TCR/CD28-induced proliferation of wild-type and Vav1⫺/⫺ T cells in the absence or presence of IL-2. As shown earlier (29), addition of IL-2 only partially rescued the proliferation of Vav1⫺/⫺ T cells; thus, even in the presence of exogenous IL-2, the proliferation of Vav1⫺/⫺ T cells was still

FIGURE 5. Impaired p27kip1 down-regulation in Vav1⫺/⫺ T cells. A, Total T cells from Vav1⫹/⫹ or Vav1⫺/⫺ mice were stimulated for 24 or 48 h with plate-coated anti-CD3 (5 ␮g/ml) plus soluble anti-CD28 (2.5 ␮g/ml) mAbs, or with PMA plus ionomycin as a positive control. Whole lysates were separated by SDS-PAGE and analyzed by immunoblotting with anti-Vav1, anti-cyclin D3, anti-p27kip1, or anti-actin Abs. Numbers indicate the relative expression levels of p27kip1 as determined by densitometry. These results are representative of four similar experiments. B, Vav1⫹/⫹ ( ) or Vav1⫺/⫺ (■) T cells were stimulated as in A for the indicated times. Purified cDNAs were prepared and subjected to real-time PCR using mouse p27kip1- or L19-specific primers. Normalized p27kip1 expression is shown. C, Vav1⫹/⫹ or Vav1⫺/⫺ T cells were stimulated for 1 h as in A, in the presence or the absence of a neutralizing anti-IL-2 Ab (50 ␮g/ml) and real-time PCR was performed as in B. D, Vav1⫹/⫹ or Vav1⫺/⫺ T cells were stimulated as in B for the indicated time points. Washed cells were stained with APC-conjugated anti-mouse CD25 Ab, and the percentage of CD25⫹ T cells was determined by flow cytometry.

reduced by ⬃40% when compared with wild-type T cells (Fig. 6A). To determine whether the IL-2R pathway signals properly in the absence of Vav1, we analyzed p27kip1 and cyclin D3 expression upon IL-2 stimulation. We stimulated primary T cells from wild-type and Vav1⫺/⫺ mice with PMA/ionomycin plus IL-2 for 7 days to allow a high and similar expression of CD25 in both groups (data not shown). We then withdrew IL-2 and restimulated the cells with IL-2 (Fig. 6B). Wild-type and Vav1⫺/⫺ T lymphoblasts showed a similar pattern of decreased p27kip1 expression and increased cyclin D3 expression upon IL-2 stimulation (Fig. 6B). Last, we determined whether IL-2-induced FOXO1 phosphorylation was dependent on Vav1. We found that FOXO1 phosphorylation peaked 5 min after IL-2 addition and declined thereafter (Fig. 6C). As an additional control for proper IL-2 stimulation, we


5029

FIGURE 6. Effects of IL-2 on T cell proliferation, FOXO1 phosphorylation and p27kip1 expression. A, Vav1⫹/⫹ (■) or Vav1⫺/⫺ (䊐) T cells were stimulated with anti-CD3 plus anti-CD28 mAbs for 48 h in the presence or absence of IL-2 (50 U/ml). [3H]TdR was added for the last 12 h of culture and proliferation was determined as in Fig. 1A. B, Primary T cells were stimulated with PMA (20 ng/ml)/ ionomycin (0.5 ␮g/ml), plus IL-2 (100 U/ml) for 7 days. Lymphoblasts were then washed three times in RPMI 0.5% FCS, maintained in the absence of IL-2 for 15 h, and restimulated or not with IL-2 (200 U/ml) for 24 h. The control (Ctrl) group represents lymphoblasts not starved and kept in culture medium. Whole cell lysates were separated by SDSPAGE and analyzed by immunoblotting with antiVav1, anti-p27kip1, anti-cyclin D3, or anti-ZAP-70 Abs. C, Whole cell lysates of cytokine-starved and IL-2 (500 U/ml)-stimulated lymphoblasts wild-type T cells were separated by SDS-PAGE and immunoblotted with the indicated Abs. D, Vav1⫹/⫹ or Vav1⫺/⫺ lymphoblasts were stimulated with IL-2 and immunoblotting with indicated Abs was determined. E, Preactivated Vav1⫹/⫹ or Vav1⫺/⫺ T cells prepared as in B were restimulated with anti-CD3 plus anti-CD28 mAbs for the indicated times, and lysates were immunoblotted with indicated Abs.

also observed increased phosphorylation of Erk1/2 (lower panels). We then compared the IL-2-induced phosphorylation of FOXO1 in wild-type vs Vav1⫺/⫺ T cells and found a very similar increase in FOXO1 phosphorylation in both cell types (Fig. 6D). Since the experiments demonstrating reduced anti-CD3/CD28-induced FOXO1 phosphorylation were conducted using fresh primary T cells (Figs. 3 and 4), whereas the effect of IL-2 on FOXO1 phosphorylation was assessed on restimulated T cells, we also examined the effect of anti-CD3/CD28 costimulation on FOXO1 phosphorylation in preactivated and restimulated T cells. Under these conditions, we still observed defective FOXO1 phosphorylation in Vav1⫺/⫺ T cells (Fig. 6E), indicating that the difference between TCR/CD28 and IL-2 stimulation is not related to a difference in the activation status of the cells. Altogether, these results clearly indicate that, in contrast to TCR/CD28 costimulation, the IL-2-induced phosphorylation and inactivation of FOXO1, as well as IL2-dependent regulation of p27kip1 and cyclin D3 expression, does not require Vav1.

Discussion The FOXO subfamily of Forkhead transcription factors has been implicated in the regulation of cell cycle and apoptosis (30). Although the role of FOXO proteins has been extensively analyzed in

fibroblasts and neuronal cells, more recent work has extended this analysis to the immune system, where FOXO proteins also appear to regulate cell cycle progression and apoptosis of T and B lymphocytes (16). However, the pathways that link lymphocyte Ag receptors to FOXO inactivation and to the expression of FOXO target genes remain largely unexplored. In this study, we report three novel findings regarding the regulation of FOXO1 in T cells: 1) the activity of FOXO1 is tightly regulated upon TCR/CD28 engagement; 2) Vav1 is an important component in TCR/CD28 signaling pathways leading to phosphorylation and inactivation of FOXO1, resulting in down-regulation of the cell cycle inhibitor p27kip1 and S phase entry of stimulated T cells; and 3) in contrast to TCR/CD28 signals, IL-2 signals, which also inactivate FOXO1 and induce activated T cells to proliferate, are independent of Vav1. We confirmed the impaired proliferation of Vav1⫺/⫺ T cells previously demonstrated by others (6 –9). A more detailed analysis showed that while wild-type T cells transit efficiently from G0/G1 phase to the S phase, Vav1⫺/⫺ remained blocked in the G0/G1 phase, consistent with previous studies in B cells (31). Pharmacological PI3K inhibition completely inhibited TCR/CD28-mediated wild-type T cell division and, thus, mimicked Vav1 deficiency

5030 (Fig. 2). However, Vav1⫺/⫺ T cells showed a residual proliferation, most likely due to compensation by Vav3, as recently reported (9). Thus, deregulation of the PI3K pathway could explain the cell cycle progression defect observed in the absence of Vav1. Among several targets of the PI3K pathway, transcription factors of the FOXO family play an emerging role in the control of cell cycle progression and apoptosis in the immune system and, therefore, constituted a logical target for further analysis in our system (12). Upon growth factor or Ag receptor stimulation, FOXO proteins become phosphorylated on three consensus sites for Akt, inhibiting their activity as transcription factors and, thus, allowing optimal cell proliferation (13–15). Interestingly, FOXO1 was strongly phosphorylated at both Thr24 and Ser256 in wildtype T cells upon anti-CD3⫹/⫺CD28 activation. However, in Vav1⫺/⫺ T cells, TCR/CD28-mediated FOXO1 phosphorylation was strongly impaired, but not completely abolished (Fig. 3). The residual FOXO1 phosphorylation was abrogated in Vav1⫺/⫺Vav3⫺/⫺ T cells indicating that both proteins control FOXO1 regulation and, subsequently, optimal T cell expansion as recently described (9). Consistent with a defect of FOXO1 phosphorylation, TCR/CD28-induced Akt activation was also impaired in the absence of Vav1. However, PMA stimulation could increase both Akt and FOXO1 phosphorylation, and restore the proliferation of Vav1⫺/⫺ T cells when combined with ionomycin (Figs. 1 and 3). PMA stimulation induces strong activation of the Ras pathway, and PI3K constitutes a direct target of Ras (32). Thus, it is possible that PMA stimulation bypasses Vav1 deficiency by increasing Akt and FOXO1 phosphorylation through the Ras pathway. Our results indicate that Vav proteins control FOXO1, most likely via small GTPases of the Rho family, which in turn activate the PI3K/Akt pathway. However, we cannot rule out the possibility that the inhibitory effects of the Vav1 mutation on the phosphorylation of FOXO1 could be partially independent of Akt, reflecting some phosphorylation of FOXO1 by other Ser/Thr kinase(s). Unfortunately, attempts to rescue the defective FOXO1 phosphorylation in Vav1⫺/⫺ T cells by retroviral transduction of Vav1 were not successful, reflecting the low transduction efficiency of these poorly proliferating T cells (data not shown); for the same reason, we did not attempt to retrovirally transduce these cells with constitutively active versions of Rho family small GTPases or Akt. Reversible FOXO phosphorylation regulates its localization between the nucleus and the cytoplasm and, hence, its activity (33). We found that the cytosolic level of FOXO1 was increased in stimulated wild-type, but not in Vav1⫺/⫺, T cells (Fig. 4). Consistent with previous reports (33), we also demonstrated that FOXO1 was associated with 14-3-3␶ in activated wild-type, but not Vav1⫺/⫺, T cells, reinforcing the important role of Vav1 in the regulation of FOXO activity and localization in T cells. FOXO protein overexpression induces either cell cycle arrest or apoptosis in several cell types by transcriptionally regulating their targets, including the cyclin-dependent kinase inhibitor p27kip1 (25), and proteins that regulate apoptosis, e.g., the Bcl-2 family member, Bim (34), and FasL (13). Because we observed a severe arrest of Vav1⫺/⫺ T cells at the G0/G1 phase, we considered the possibility that deregulation of p27kip1 is involved in this defect. Indeed, p27kip1 mRNA and protein were not down-regulated in Vav1⫺/⫺ T cells upon stimulation with anti-CD3/CD28 Abs, whereas they were clearly reduced in wild-type T cells (Fig. 5), a finding consistent with an earlier study (29). Importantly, we could rule out an effect of IL-2 in p27kip1 transcriptional regulation at early time points of TCR/CD28 stimulation because: 1) a neutralizing anti-IL2 Ab did not reverse TCR/CD28-induced p27kip1 mRNA down-regulation observed after 1 h of activation with anti-

FOXO1 REGULATION BY Vav1 IN T CELLS CD3/CD28 Abs (data not shown); 2) no IL-2 secretion was detected in the supernatant of wild-type T cells upon 1 h of stimulation (data not shown); and 3) the IL-2R␣-chain (CD25) was not yet up-regulated in wild-type T cells after 1 h of stimulation (Fig. 5). Our results are consistent with previous reports showing that CD28 activation promoted cell cycle progression by down-regulating p27kip1 independently of IL-2 (35). However, Vav1 deficiency did not significantly affect cyclin D3 expression in response to anti-CD3/CD28 stimulation. This finding suggests that additional pathways control the expression of cyclin D3 in T cells, and/or that the compensatory function of Vav3 is sufficient to fully up-regulate cyclin D3 in the absence of Vav1. In quiescent fibroblasts, cyclin D3/CDK4 complex can effectively associate with p27kip1 (36). Therefore, one potential explanation for the defective proliferation of Vav1⫺/⫺ T cells despite their intact cyclin D3 expression is that p27kip1, which is highly present in Vav1⫺/⫺ T cells, might act by inhibiting the cyclin D3/CDK4 complex present in G1 phase and, thus, prevent their proliferation. Several reports showed that in T and B cells, IL-2 and IL3, respectively, regulate FOXO protein activation, p27kip1 expression and, thus, proliferation (25, 27). We found that IL-2 partially rescued the Vav1⫺/⫺ T cell proliferation defect and potentiated wildtype T cell proliferation (Fig. 6). IL-2 stimulation of wild-type and Vav1⫺/⫺ lymphoblasts expressing similar levels of CD25 (data not shown) induced a severe and similar reduction of p27kip1 expression in both wild-type and Vav1⫺/⫺ T cells, consistent with previous studies (27), as well as a strong increase of cyclin D3 expression. This explains the higher rate of cellular proliferation of the Vav1⫺/⫺ T cells in the presence of exogenous IL-2. Last, our finding that the IL-2-induced phosphorylation of FOXO1 remained intact in the absence of Vav1 (Fig. 6) indicates that, in contrast to the TCR/CD28 signaling pathway, the IL-2R pathway regulating FOXO1 activity, p27kip1, and cyclin D3 expression, is Vav1 independent. In summary, we describe a novel functional link between Vav1, FOXO1, and p27kip1, which plays an important role in regulating TCR/CD28-induced cell cycle progression of T lymphocytes. In the absence of Vav1, T cells are arrested in G0/G1 phase, correlating with a defect in FOXO1 phosphorylation, its cytosolic relocalization and association with 14-3-3␶ and a high level of p27kip1 expression. The regulation of FOXO protein activity seems to follow a common pattern, i.e., phosphorylation/inactivation by Akt. Thus, it will be interesting to determine whether Vav1 is also required for the regulation of FOXO4 and FOXO3a upon T cell activation. Moreover, the recent characterization of FOXO3a-deficient mice revealed that these mice develop an autoinflammatory disease and that their T cells are hyperactivated, produce higher levels of IL-2 and Th1 and Th2 cytokines, a result of constitutively elevated NF-␬B activity (18). Because Vav1⫺/⫺ T cells display a defect in NF-␬B activation (5), T cell proliferation, IL-2 secretion (6 – 8), and Th2 cytokines (19), it will be interesting to determine whether FOXO proteins constitute a link between Vav1, NF-␬B, and IL-2 in the development of Th cells and the generation of autoimmune diseases.

Acknowledgments We thank Nathalie Droin for help with real-time PCR, Samuel Connell for assistance with flow cytometry, Svetlana Lebedeva for animal breeding, and all members of the Division of Cell Biology (La Jolla Institute for Allergy and Immunology, La Jolla, CA) for helpful advice.

Disclosures The authors have no financial conflict of interest.


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