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The Journal of Immunology, 2006, 176: 4818-4825.
Copyright © 2006 by The American Association of Immunologists

Wip1 Phosphatase-Deficient Mice Exhibit Defective T Cell Maturation Due To Sustained p53 Activation1

Marco L. Schito2,*, Oleg N. Demidov*, Shin’ichi Saito*, Jonathan D. Ashwell{dagger} and Ettore Appella*

* Laboratory of Cell Biology and {dagger} Laboratory of Immune Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The PP2C phosphatase Wip1 dephosphorylates p38 and blocks UV-induced p53 activation in cultured human cells. Although the level of TCR-induced p38 MAPK activity is initially comparable between Wip1–/– and wild-type thymocytes, phosphatase-deficient cells failed to down-regulate p38 MAPK activity after 6 h. Analysis of young Wip1-deficient mice showed that they had fewer splenic T cells. Their thymi were smaller, contained significantly fewer cells, and failed to undergo age-dependent involution compared with wild-type animals. Analysis of thymocyte subset numbers by flow cytometry suggested that cell numbers starting at the double-negative (DN)4 stage are significantly reduced in Wip1-deficient mice, and p53 activity is elevated in cell-sorted DN4 and double-positive subpopulations. Although apoptosis and proliferation was normal in Wip1–/– DN4 cells, they appeared to be in cell cycle arrest. In contrast, a significantly higher percentage of apoptotic cells were found in the double-positive population, and down-regulation of thymocyte p38 MAPK activation by anti-CD3 was delayed. To examine the role of p38 MAPK in early thymic subpopulations, fetal thymic organ cultures cultured in the presence/absence of a p38 MAPK inhibitor did not correct the thymic phenotype. In contrast, the abnormal thymic phenotype of Wip1-deficient mice was reversed in the absence of p53. These data suggest that Wip1 down-regulates p53 activation in the thymus and is required for normal {alpha}beta T cell development.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
T cell development is defined by a timely and ordered set of phenotypic changes in the expression of multiple surface markers and receptors. Hemopoietic precursors enter the thymus as (CD4–CD8–) double-negative (DN)3 T cell progenitors and progress through CD44+CD25– (DN1), CD44+CD25+ (DN2), CD44–CD25+ (DN3), and CD44–CD25– (DN4) developmental stages (1). During the transition from the DN3 to the DN4 stage, thymocytes must first pass beta-selection to develop into {alpha}beta T cells (2, 3, 4). The transition from DN to CD4+CD8+ double positive (DP) depends on the expression of a functional pre-TCR composed of the invariant pre-TCR{alpha} coupled to the TCRbeta-chain (5). Thymocytes that express a pre-TCR are rescued from apoptosis, differentiate (up-regulate CD4 and CD8 coreceptor expression and rearrange the TCR{alpha}-chain), and proliferate (6, 7, 8). Thymocytes that successfully rearrange the TCR{alpha}-chain gene undergo positive or negative selection during the transition between the DP to the CD4+CD8– or CD4–CD8+ (single positive; SP) stage. It is believed that a balance between proapoptotic (Bim, Bad, Bax) and antiapoptotic (Bcl-2, Bcl-xL) Bcl-2 family members controls the fate of thymocytes. The particular signaling pathways involved are diverse and depend on the nature of the apoptotic signal and the stage of cell differentiation. Thus, maturation of thymocytes occurs through complex interactions that involve TCR signaling (9), cell-cell communication (10, 11), and soluble factors (12, 13).

The role of p38 MAPK is well established in the thymus. Using a p38 dominant-negative transgene under control of the proximal lck-promotor, it was demonstrated that activation of p38 MAPK was required for the earliest stage of DN differentiation (14). Furthermore, by using a constitutively active MAPK kinase (MKK)6 transgene that phosphorylates p38 in the absence of upstream stimulation, Diehl et al. (14) found that activation of p38 MAPK blocked thymocyte development at the DN3 stage. In addition, more thymocytes from constitutively active MKK6 transgenic mice were found to be cycling compared with nontransgenic littermates. Therefore, p38 MAPK needs to be active for cells to differentiate from the DN1 to DN3 stage, but kinase activity must be turned off for cells to differentiate to the DN4 stage. Thus, initial activation of p38 MAPK appears to be important for early thymocyte development, but continuous activation blocks the generation of T cells resulting in immunodeficiency.

The p53 tumor suppressor protein regulates cell cycle and apoptosis in response to DNA damaging events such as irradiation, but p53 may also play a role under more physiological conditions such as dsDNA breaks due to site-specific V(D)J recombination in T cell precursors (15). Interestingly, T cell development can, to some extent, be rescued by the simultaneous loss of p53 in mice, where T cell development is otherwise blocked (RAG–/–, SCID, CD3{gamma}–/–) (16, 17, 18, 19). Thus, down-regulation of p53 allows {alpha}beta T cells to proceed from the DN to DP stage.

Previously, we identified wild-type (WT) p53-inducible phosphatase, Wip1, an irradiation inducible type 2C phosphatase (PP2C), as a transcriptional target of p53 (20). PP2Cs have been associated with stress responses, sexual differentiation, and cell cycle control in a variety of organisms (21). We have generated mice deficient for Wip1, and although they appear to develop normally, males have defects in reproductive organs, and embryonic fibroblasts derived from these mice have multiple defects in cell cycle control (22). In addition, Wip1-deficient mice reportedly exhibit immunological defects in the peripheral lymphoid organs that result in an inability to control influenza infection, suggesting that this phosphatase may be involved in immune responses (22).

p38 MAPK was the first Wip1 substrate identified. Takekawa et al. (23) have shown that Wip1 inhibits p38 MAPK function by dephosphorylating Thr180. It has been suggested that Wip1 may mediate a negative feedback loop for p53, resulting in reduced p38 site-specific phosphorylation of p53 residues. More recently, Wip1 was found to regulate base excision repair through its dephosphorylation of the nuclear isoform of uracil DNA glycosylase (UNG2) in response to UV irradiation (24).

Our initial experiments determined that Wip1-deficient mice had reduced numbers of peripheral T cells that could not be attributed to reduced proliferation or enhanced apoptosis. Thus, we examined T cell development and found that all populations of cells in young Wip1–/– mice (<3 mo) were severely reduced in number, with the exception of the DN thymocyte population. The reduced number of cells appeared to be due to defects in the DN4 to DP transition during T cell development. Although p38 MAPK did not appear to play a role, thymic development appeared normal when Wip1-deficient mice were crossed onto a p53-null background. Our results suggest that Wip1 plays a role during the DN to DP transition in {alpha}beta T cell development by down regulating the p53 tumor suppressor.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

The generation of Wip1–/– and p53–/– mice has been described previously (22, 25). All mice were backcrossed at least five times on to a C57BL/6NCr background. Wip1–/– and p53+/– mice were mated to obtain Wip1–/– p53–/– double knockout animals. The breeders used were heterozygous for p53 to extend their survival, because the lack of this gene predisposes mice (>6 mo) to thymic lymphomas and early death. DNA (tail) samples were subjected to RT-PCR using the SuperScript preamplification system (Invitrogen Life Technologies) according to the manufacturer’s protocol. All experiments used age- and gender-matched mice. WT C57BL/6NCr control mice were purchased from Division of Cancer Treatment/National Cancer Institute (Frederick, MD). All mice were bred and housed in an Association for Assessment and Accreditation of Laboratory Animal Care-accredited facility under an approved National Institutes of Health animal study protocol and specific pathogen-free conditions.

Tissue culture and cell activation

Complete medium consisted of RPMI 1640 with glutamax (Invitrogen Life Technologies) supplemented with 10% FCS (BioWhittaker), 5.5 x 10–5 M 2-ME (Invitrogen Life Technologies), 100 U/ml penicillin, and 100 µg/ml streptomycin (Invitrogen Life Technologies). Thymi were isolated from mice, weighed, and pushed through a 70-micron membrane (BD Biosciences) in complete medium. The thymocytes were washed and suspended in complete medium at 10 x 106 cells/ml. Cells were cultured at 37°C with 5% CO2 at a density of 5 x 106 cells/ml in the presence/absence of plate-bound anti-CD3 (10 µg/ml) and anti-CD28 (5 µg/ml) (BD Biosciences).

Flow cytometry, cell cycle, and sorting analysis

Thymocytes (5 x 105 to 1 x 106 cells) were suspended in staining buffer (PBS supplemented with 1% FCS) with the following Abs purchased from BD Pharmingen (BD Biosciences): CD4; CD8; B220; pan-NK; CD11c; CD3; {gamma}{delta}-TCR; beta-TCR; CD5; CD44; CD25; and Bcl-xL. All Abs used for flow cytometry were directly conjugated to FITC, PE, PerCP, or allophycocyanin with the exception of CD25, which was biotinylated for some experiments. Streptavidin conjugates were purchased from BD Biosciences. For intracellular Bcl-xL staining, cells were fixed and permeabilized with BD Cytofix/Cytoperm (BD Biosciences) using the manufacturer’s directions. Flow cytometry was performed using a FACScan and CellQuest software (BD Biosciences). For cell cycle analysis, thymocytes (1 x 106 cells) were washed, stained for cell surface markers, suspended in 70% ethanol, and fixed overnight at 4°C. The cells were then resuspended in staining solution containing either 50 µg/ml propidium iodide (PI) (Sigma-Aldrich) and 100 U/ml RNase A (Roche) or 7-amino actinomycin D (7-AAD). In experiments where cell cycle data was acquired from four-color stained samples, cells were stained for cell surface markers, fixed, and permeabilized with BD Cytofix/Cytoperm (BD Biosciences), then stained with 4',6'-diamidino-2-phenylindole (DAPI). Flow cytometry data was acquired using the LSRII cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star). For DN cell sorting experiments, thymocytes from five 4- to 6-wk-old Wip1–/– and WT mice were complement-depleted of CD4 and CD8 cells (Cedarlane Laboratories). Dead cells were removed by density gradient centrifugation using Histopaque-1083 (Sigma-Aldrich). The remaining live cells were stained with CD4, CD8, B220, CD3, pan-NK, and CD11c FITC-labeled Abs to gate these cells out and stained with CD44-allophycocyanin and CD25-PE Abs to sort the four DN cell populations using the FACSVantage SE sorter with DiVa option (BD Biosciences). To sort out DP and SP cells, thymocytes isolated from mice were stained with CD4-FITC and CD8-PE. Analysis was done using FACS DiVa software (BD Biosciences).

RNA purification and real-time RT-PCR

Total RNA was isolated from sorted cells using RNeasy Micro kit per manufacturer’s instructions (Qiagen). RNA concentration was determined using a nanodrop (Grace Scientific). Primers for Wip1 (forward, 5'-GCTAGAGGGAATATCCAGACTGTAGTGA-3' and reverse, 5'-AGTATTTGTTGAATTGGTTGGAATGAGGC-3') were designed using LightCycler Probe Design 2 software (Roche). Primers for GAPDH (forward, 5'-AATGTGTCCGTCGTGGATCTGA-3' and reverse, 5'-GATGCCTGCTTCACCACCTTCT-3') were designed using Primer Express software (PerkinElmer). One-step real-time RT-PCR was performed using the LightCycler RNA Master SYBR Green I kit according to the manufacturer’s instructions (Roche). LightCycler conditions were as follows: each run consists of reverse transcription at 61°C for 20 min, initial denaturation at 95°C for 30 s, followed by 45 cycles, denaturation; 15 s at 95°C, annealing; 12 s at 58°C, elongation; 30 s at 72°C, automatic measurement of the F2:F1 ratio. A melting curve was determined as another option by continuous heating from 40 to 95°C and measurement of F2:F1 ratio. Relative expression of the Wip1 gene was calculated using the delta-delta crossing point method (2–ddCP) after normalization to the endogenous control GAPDH RNA.

Apoptosis and cell proliferation

To determine the percentage of cells undergoing apoptosis, thymocytes were analyzed either directly after isolation or after incubation in complete medium for 24 h. Cells were washed and stained using either the annexin V-FITC apoptosis detection kit (Oncogene Research Products) with PI to stain dead cells or the annexin V-PE apoptosis detection kit (BD Biosciences) with 7-AAD to stain dead cells as directed by the manufacturer. In some cases, FITC-labeled CD8 and allophycocyanin-labeled CD4 Abs were also included with the annexin V-PE kit to examine thymocyte subpopulations. To determine apoptosis in the DN subpopulations, thymocytes were stained with CD4, CD8, B220, CD3, pan-NK, and CD11c FITC-labeled Abs to gate these cells out and stained with annexin V-PE, CD25-biotin, CD44-allophycocyanin, and streptavidin-PerCP. DN thymocyte subset proliferation was determined by measuring BrdU incorporation by flow cytometry. Six-week-old WT and Wip1–/– mice received injections i.p. with BrdU (Zymed Laboratories) and sacrificed 1 h later. Thymocytes were stained for surface markers (PE-CD3, -CD4, -CD8, -CD19, -CD11c; biotin-CD25; allophycocyanin-CD44; streptavidin-PerCP) and fixed and permeabilized with BD Cytofix/Cytoperm (BD Biosciences) using the manufacturer’s directions. The cells were resuspended in permeabilization wash buffer containing 300 µg/ml DNase (Sigma-Aldrich) and incubated for 60 min at 37°C, washed, and stained with anti-BrdU-FITC (BD Biosciences). Flow cytometry was performed using a FACScan with CellQuest software (BD Biosciences).

Protein sample preparation, immunoprecipitation, kinase reactions, Western blotting, and immunodetection

p38 MAPK activity in the total thymocyte population was measured by using a p38 MAPK Assay Kit (Cell Signaling Technology; catalog no. 9820) according to the manufacturer’s instructions. Cells were rinsed with PBS then lysed with lysis buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerolphosphate, 1 mM NaVO4, 1 µg/ml leupeptin, 1 mM PMSF). After centrifugation for 20 min at 15,000 x g at 4°C, the supernatant was stored at –80°C. Protein content was measured using the BCA Protein Assay (Pierce). Phosphorylated p38 MAPK was immunoprecipitated with immobilized anti-phospho-p38 mAb (Thr180/Tyr182) on agarose hydrazide beads. Beads were washed twice with lysis buffer and twice with kinase buffer (25 mM Tris (pH 7.5), 5 mM beta-glycerolphosphate, 2 mM DTT, 0.1 mM Na3VO4, 10 mM MgCl2). Kinase reactions were performed for 30 min at 30°C in 50 µl of the kinase buffer supplemented with 200 µM ATP and 2 µg of activating transcription factor (ATF)-2 fusion protein. Activity was detected using phospho-ATF2 (Thr71) (rabbit polyclonal; Cell Signaling Technology) and p38 MAPK (rabbit polyclonal; Cell Signaling Technology) Abs. To detect cell signaling molecules in sorted and unsorted thymocyte populations, 1 x 106 cells were prepared in sample buffer, and the proteins were separated by SDS-PAGE. Specific Abs against p53 (CM-5; NovoCastra), phospho-p53Ser15 (rabbit polyclonal; Cell Signaling Technology), p21Waf1 (Ab-4; Oncogene Research Products), and {alpha}-tubulin (Ab-1; Oncogene Research Products) were used for Western blotting and were detected with HRP-conjugated anti-mouse or anti-rabbit (Jackson ImmunoResearch Laboratories) Abs.

Fetal thymic organ cultures (FTOC)

Timed pregnancies were initiated for WT and Wip1–/– mice, and, at day 15, fetal thymi were isolated and cultured on 0.8 µM nucleopore membranes (Whatman) in 12-well plates with complete medium supplemented with 100 µM nonessential amino acids (Invitrogen Life Technologies), 1 mM sodium pyruvate (Invitrogen Life Technologies), and 10 mM HEPES (Invitrogen Life Technologies). In some cultures, the p38 MAPK inhibitor, SB203580 (Calbiochem), was added for a final concentration of 10 µM. FTOC were cultured for 3 days, and then analyzed by flow cytometry as described above.

Histology

Thymic tissues were fixed in 10% buffered formaldehyde, sectioned, and stained with H&E (American HistoLabs).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Young Wip1–/– mice have significantly lower thymic cell numbers

Wip1-deficient mice have small thymi, immunological defects in the peripheral lymphoid organs, and are more susceptible to influenza infection (22). Although the number of peripheral T cells in Wip1–/– mice was reported to be normal (22), that study was performed in old animals (>8 mo). We have repeatedly observed substantially lower splenic T cell numbers in younger animals (Table I). However, T cell numbers in the periphery approaches that of WT animals as the mice age, suggesting that homeostatic proliferation may eventually reconstitute normal T cell numbers.


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  		Table I. Reduced numbers of splenic T cells in young Wip1-deficient micea

 
To determine whether the lower number of T cells in the spleen is due to abnormal T cell development, we examined thymocyte ontogeny. The lack of Wip1 expression resulted in a 50–75% reduction in thymocyte numbers (Table II). Although the thymi of 8-wk-old Wip1-deficient mice appeared structurally normal, the medulla was small, suggesting that there are fewer mature T cells (Fig. 1, A and B). In older Wip1–/– animals (>3 mo of age), the thymus appeared normal in size and was not histologically different from thymi from age-matched WT mice (Fig. 1, C and D). Thus, there appears to be a defect in early T cell development in young Wip1–/– mice that is not evident in older animals.


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  		Table II. Reduction of thymic cellularity during the first 6 mo of age in Wip1-deficient mice

 

Figure 1
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  		FIGURE 1. Thymi from young Wip1-deficient mice have smaller medulla compared with WT animals. Thymic sections of eight (A and B) and 24 (C and D)-wk-old WT (A and C) and Wip1–/– (B and D) mice were stained with H & E. Results are representative fields observed in sections of thymus from three individual WT and Wip1–/– mice examined. Images were examined at x100 total magnification and digitally captured using a SPOT camera (Diagnostic Instruments). M, Medulla; Cx, cortex; bar, 100 µm.

 
Young Wip1-deficient mice have lower numbers of DP and SP cells and are unable to down-regulate TCR-induced p38 MAPK activity

To clarify the mechanism for the reduced thymic cellularity of Wip1-deficient mice, thymocytes were analyzed for the expression of CD4 and CD8 surface markers by flow cytometry. The percentage of CD4 and CD8 SP as well as DP cells were not affected. However, we consistently observed that Wip1-deficient mice expressed higher percentages of DN cells compared with their WT counterparts (Fig. 2A). When the number of cells were taken into account, Wip1-deficient mice had significantly lower numbers of DP and SP thymocytes, whereas DN numbers were near normal (Fig. 2B). Therefore, Wip1-deficient mice either have a defect in the DN to DP thymocyte transition and/or more DP cells undergo apoptosis.


Figure 2
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  		FIGURE 2. Thymocytes from Wip1-deficient mice have significantly lower numbers of DP and SP cells and fail to regulate p38 MAPK. A, Thymocytes from 4-wk-old animals were analyzed for CD4 and CD8 surface expression using flow cytometry, and the percent positive of each subpopulation is depicted. Similar results were obtained for mice at 6, 8, and 12 wk of age. B, The total number of each thymocyte population from 4- to 6-wk-old WT and Wip1–/– mice was calculated from the percentage of cells that was determined by flow cytometry. The numbers of CD3+ cells expressing the beta-TCR vs the {gamma}{delta}-TCR were also determined. Similar results were obtained with 8-wk-old mice. C, To determine the level of p38 MAPK activation, thymocytes were cultured with plate-bound anti-CD3 and anti-CD28 Abs for the indicated times. Cells were lysed, protein levels were determined, and kinase activity was assessed. Numbers within FACS panels represent the percentage of cells in the respective quadrants. Each bar represents the mean with SD whiskers of three individual animals. The data are representative of three or more separate experiments. The * indicate significant differences (p < 0.05) between WT and Wip1-deficient cells.

 
To determine whether all thymocytes were affected, CD3+ cells were analyzed for the expression of the {alpha}beta- or {gamma}{delta}-TCR. The number of beta-TCR+ cells was significantly reduced in the Wip1-deficient animals, whereas the number of {gamma}{delta}-TCR+ cells was not significantly different (Fig. 2B) when compared with WT mice. This result indicates that Wip1 plays a role in {alpha}beta but not {gamma}{delta} T cell development in the thymus.

Wip1 acts as a phosphatase that regulates p38 MAPK activity in vitro. To determine whether Wip1 alters p38 MAPK signaling in mature TCR-expressing thymocytes, total thymocytes from Wip1-deficient and WT mice were stimulated with plate-bound anti-CD3/CD28, and MAPK activity was followed over time. TCR-stimulation induced p38 MAPK activity within the first 30 min. Increased MAPK activity was sustained for over 3 h in both WT and Wip1–/– thymocytes. However, whereas WT cells began to down-regulate p38 MAPK activity at 6 h and had little or no activity by 18 h, Wip1-deficient thymocytes still expressed high levels of p38 MAPK activity even up to 18 h after TCR-stimulation (Fig. 2C). This indicates that the TCR-induced activation of Wip1–/– thymocytes are unable to down-regulate p38 MAPK once activated, consistent with it being a substrate for Wip1.

DP thymocytes from Wip1–/– mice undergo spontaneous apoptosis and have cell cycle abnormalities

We can directly compare both DP and SP thymocytes from Wip1–/– and WT mice because these populations constitute the majority of the cells and are in similar proportions in both WT and knockout mice. However, compared with WT mice, Wip1–/– mice contain a higher percentage of larger thymocytes, suggesting that more cells were in cycle (Fig. 3A). To evaluate this more closely, thymocytes were subjected to cell cycle analysis by flow cytometry to determine whether the reduced cellularity of the thymus could be accounted for by alterations in cell cycle progression. Freshly isolated total thymocytes, as well as the DP and DN subpopulations, from Wip1–/– mice consistently had more cells in S/G2/M than WT animals (Fig. 3B) despite the fact that DP cells do not proliferate.


Figure 3
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  		FIGURE 3. DP thymocytes from Wip1–/– mice have altered cell cycle progression and undergo a higher rate of apoptosis. A, Thymocyte size and granularity from 6- to 8-wk-old Wip1-deficient mice were compared with that of age-matched WT control cells as determined by flow cytometric analysis of forward and side scatter. Values indicate the percentage of larger thymocytes in the boxed gate. B, Total thymocytes, as well as the DP and DN subset, were subjected to cell cycle analysis using flow cytometry. Values indicate the percentage thymocytes in cell cycle. C, Thymocytes were stained after incubation in medium for 24 h to determine apoptosis (AnnexinV+) and necrosis (7-AAD+ or PI+). The increase in apoptosis was confined to the CD4+CD8+ population, which had lower numbers of cells that expressed Bcl-xL by intracellular staining. {blacksquare} represent WT, and {square} represent Wip1–/– mice. Each bar represents the mean with SD whiskers of three individual animals. The data are representative of two or more separate experiments. The * indicate significant differences (p < 0.05) between WT and Wip1-deficient cells.

 
To determine whether Wip1–/– thymocytes were more prone to undergo apoptosis, thymocytes were stained for annexin V and PI or 7-AAD, and examined by flow cytometry. On average, the percentage of cells undergoing spontaneous apoptosis was 2-fold higher in freshly isolated Wip1–/– thymocytes (4.3 ± 0.1%) compared with WT (2.4 ± 0.4%) control cells. The difference in apoptosis in Wip1-deficient compared with WT thymocytes was in the DP population, whereas the DN thymocytes of WT and Wip1–/– mice had similar levels of apoptosis (annexin positive, 7-AAD negative) (Fig. 3C). The increase in the number of Wip1–/– DP thymocytes undergoing apoptosis correlated with a reduced number of DP cells expressing the antiapoptotic factor Bcl-xL (Fig. 3C).

A block from the DN to DP transition is due to a block in thymocyte maturation at the DN4 stage of development

To determine which subpopulations within the DN compartment expresses Wip1, thymocytes from WT mice were sorted by flow cytometry and isolated based on surface marker expression. In WT mice, Wip1 mRNA expression was dramatically increased at the DN3 and DN4 developmental stages with lower levels present in DP and SP cells (Fig. 4A). Further analyses by flow cytometry of the DN subpopulations identified consistent defects in the absence of Wip1. A greater percentage of Wip1–/– thymocytes were observed at the DN3 stage and a lower percentage at the DN4 stage when compared with age-matched WT animals (Fig. 4B). When cell numbers were taken into account, there were no differences in the number of DN3 cells, but there were lower numbers of DN4 cells (Fig. 4C). This suggests that thymic {alpha}beta T cell development has a partial defect at the DN4 stage in Wip1-deficient mice.


Figure 4
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  		FIGURE 4. Lack of Wip1 expression affects early thymocyte development. A, FACS-sorted DN thymocytes from WT mice were analyzed for Wip1 expression by real-time RT-PCR and evaluated in reference to GAPDH. B, Thymocytes from 4- to 6-wk-old animals were used to determine the phenotype of the DN population with respect to CD44 and CD25 expression. Numbers within FACS panels represent the percentage of cells in the respective quadrants. C, The number of thymocytes in each of the DN subpopulation was calculated based on the number of DN cells. D, Levels of p53, phospho-p53Ser15, p21Waf1, and {alpha}-tubulin were detected in FACS-sorted DN4 and DP thymocyte subsets from WT and Wip1-deficient mice by Western blotting. E, The number of DN4 apoptotic cells (annexin V), the number of cells (DN3 and DN4) in cycle (DAPI) and proliferating (BrdU) were determined by flow cytometry. F, DN3 to DP thymic development of day 15 fetal thymi cultured for 3 days in the presence and absence of the p38 MAPK inhibitor SB203580 (10 µM). Cell subset numbers were based on the percentage of cells, as determined by flow cytometry, and number of cells isolated after culture. {blacksquare} represent WT, and {square} represent Wip1–/– mice. Each bar represents the mean with SD whiskers of three individual animals. The data are representative of two or more separate experiments. The * indicate significant differences (p < 0.05) between WT and Wip1-deficient cells.

 
To determine why fewer DN4 cells were present in mice that lack Wip1, we assessed the levels of p53-related biochemical markers (Fig. 4D). Thymocyte DN4 and DP subsets from both WT and Wip1-deficient mice were purified by FACS sorting. Higher levels of total p53, phospho-p53Ser15, and p21Waf1 were found in Wip1–/– DN4 and DP population. This also correlates with an increase in the number of Wip1–/– DN4 cells that appear to be in cell cycle (Fig. 4E), suggesting that lower numbers of DN4 cells in Wip1–/– mice may be the result of increased p53 activity, which blocks the cell cycle, without affecting cell proliferation or apoptosis (Fig. 4E).

To determine whether elevated levels of p38 activity within the DN subpopulations are responsible for the reduced numbers of DP and SP cells, FTOC were used to determine whether blocking p38 MAPK can correct the thymic phenotype. Thymi from day 15 fetuses were cultured for 3 days in the presence (10 µM) and absence of the p38 MAPK inhibitor SB203580 (Fig. 4F). The numbers of DN3 and DN4 cells were not affected, regardless of whether the inhibitor was present. Although treatment with SB203580 did reduce DP cell numbers from WT thymi, it had no effect on Wip1-deficient thymi. Thus, p38 MAPK is not responsible for the phenotype observed in Wip1–/– mice.

Thymocyte abnormalities of Wip1–/– mice are not observed for mice that are also deficient for p53

Knowing that p53 needs to be suppressed for T cells to proceed from the DN to DP stage (16, 17, 18, 19) and that p53 levels are higher in the absence of Wip1, we attempted to correct the T cell developmental defects in Wip1–/– mice by removing p53. The thymic abnormalities observed in mice that lack Wip1 were reversed when the animals were bred onto a p53-null background. Not only did the thymic cellularity (T cell numbers, percentage of DN thymocytes, and their phenotype (CD44 and CD25 expression)) from Wip1-deficient mice return to WT levels, but WT levels of apoptosis (primarily in the DP population) and cell cycle progression were restored as well (Fig. 5). This suggests that the block in T cell development from DN to DP in Wip1-deficient thymocytes results primarily from the inability to down-regulate p53.


Figure 5
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  		FIGURE 5. Thymic abnormalities of Wip1–/– mice are rescued in the absence of p53. A, Representative thymi from 4- to 6-wk-old WT, Wip1–/–, and Wip1–/– p53–/– double knockout mice were isolated and photographed to show size differences. Thymocytes from all mice were subjected to cell cycle analysis using PI (B), counted using trypan blue exclusion (C), and analyzed by flow cytometry to determine the following: the number of CD3+ cells that express the {alpha}beta-TCR vs the {gamma}{delta}-TCR (D); the percentage of cells that were CD4–CD8– (E); the percentage of cells undergoing apoptosis after culturing in medium for 18–24 h (F); and the phenotype of the DN population with respect to CD44 and CD25 expression (G). Each bar represents the mean with SD whiskers of three individual animals. The data are representative of three separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we have defined the role of Wip1 phosphatase in the control of p53 activity during murine thymic {alpha}beta T cell development, and p38 MAPK regulation after TCR stimulation. Wip1 mRNA is induced at the DN3 and DN4 stage with lower levels present in the DP and SP stage of thymocyte development. Although Wip1 can dephosphorylate p38 MAPK, the kinase activity was not different between WT and Wip1–/– thymocytes when the cells were cultured in medium alone. It remains possible that Wip1 phosphatase may be induced, as part of a feed-back mechanism due to p38 MAPK activation, because there was a delay in turning off kinase activity in TCR-stimulated Wip1-deficient thymocytes (Fig. 2C). Regardless, these data suggest that TCR activation may use Wip1 to down-regulate T cell activation and supports p38 MAPK as a substrate for Wip1 in TCR-stimulated thymocytes.

Early T cell development is, in part, controlled by the activity of p38 MAPK (26, 27). At the earliest DN stages, DN1 to DN3, p38 MAPK needs to be active, but is down-regulated as the cells progress to the DN4 stage (14). The lower number of DN4 cells isolated from Wip1–/– mice suggests that some cells are able to down-regulate p38 MAPK, likely by alternative phosphatases that may be less efficient. To determine whether p38 MAPK overexpression is playing a role in thymocyte development in Wip1–/– mice, FTOC from both WT and Wip1-deficient mice were assayed for thymocyte development in the presence and absence of SB203580, a specific p38 MAPK inhibitor. The inhibitor did not affect T cell development nor did it restore DP cell numbers, suggesting that Wip1 does not play a role in early T cell development through p38 MAPK.

p53 activity has been shown to affect T cell development in the thymus. Several studies have shown that thymocyte differentiation in some T cell-deficient mice (SCID, RAG1/2–/–, CD3{epsilon}–/–, and, to some degree, CD3{gamma}–/–) can be rescued in the absence of p53 (16, 17, 18, 19). Normally, these cells are blocked at the DN3 stage, but thymocytes will progress from a DN to a DP phenotype if the mice are bred to a p53-deficient background. Furthermore, the presence of a functional pre-TCR was shown to down-regulate phospho-p38 MAPK and phospho-p53Ser15 and that cytoplasmic deletion mutants of the pre-TCR{alpha}-chain failed to suppress this activity (28). Previous observations have shown that p53 activity is elevated in embryo fibroblasts derived from Wip1-deficient mice (29). Our results show that p53 activity is increased in DN4 and DP thymocyte subsets, which suggests that the defect in thymic development of Wip1–/– mice arose from the inability to down-regulate p53. Although the signaling pathways required to inactivate p53 from the pre-TCR are currently not known (18), our data suggests that Wip1 may play a role because its expression is highly up-regulated at the DN3 and DN4 stage.

p53 can control the fate of cells either through apoptosis or cell cycle arrest. Although the numbers of apoptotic DN4 cells were not significantly different in either WT or Wip1-deficient mice, there was a significant increase in the number of Wip1–/– DN4 thymocytes in cell cycle (Fig. 4E), suggesting that Wip1–/– DN4 thymocytes were proliferating more than WT cells. However, BrdU incorporation in the DN4 subset was not different in the absence of Wip1 (Fig. 4E), demonstrating that the higher p53 activity in the DN4 cells negatively affects the transition to DP via cell cycle arrest. However, cells that express both CD4 and CD8 are more susceptible to apoptosis, presumably due to higher p53 levels and/or the lower number of Bcl-xL DP thymocytes, suggesting that Wip1 plays different roles depending on the maturation state of the thymocyte.

To determine whether p53 overexpression is responsible for the phenotype described for Wip1-deficient mice, we bred the mice onto a p53-deficient background. Interestingly, the thymic developmental defects in mice that lack Wip1 were corrected in the absence of p53 (Fig. 5). This strongly supports the hypothesis that p53-induced Wip1 acts as a negative feedback loop that down-regulates p53 activity (23, 30). The importance of p53 in T cell development has been largely ignored because p53-deficient mice do not appear to have any early thymic defects. Although T cell development has not been addressed in p53-transgenic mice, it would be interesting to see whether the abnormalities observed in Wip1-deficient mice are also recapitulated in mice that overexpress p53. Nevertheless, our results suggest that Wip1 down-regulates p53 activity, which plays a role in the DN to DP transition during {alpha}beta T cell development.

p53 has been shown to regulate other down-stream cell signaling pathways that are known to affect thymic development. Recently, p53 was found to negatively regulate thymic Notch1 activation (31), which was shown to be required for beta-selection (32) and the DN to DP transition (33) of thymocytes. If the above observations are true, then Notch1 activation and the expression of downstream targets such as HES1 in Wip1–/– thymocyte DN4 subpopulations and the percentage of CD8 SP cells should be significantly reduced. Although we have not determined HES1 protein levels in thymocyte subpopulations, we did not see reduced percentages restricted to the CD8 SP population in Wip1-deficient mice, suggesting that Notch signaling is not significantly compromised. Interestingly, Wnt signaling through beta-catenin was also shown to be negatively regulated by p53 (34). Thus, the possibility exists that the Wnt pathway may be altered in Wip1-deficient mice. It has been observed that p53 down-regulates beta-catenin at the level of transcription (35) and through a proteosome-mediated mechanism involving glycogen synthase kinase-3beta activity (34, 36, 37). Interestingly, beta-catenin-deficient mice were also found to have defective T cell development at the beta-selection checkpoint (38), suggesting that the thymic phenotype of Wip1–/– mice may be due to altered Wnt signaling resulting from elevated p53 activity. Currently, we are pursuing both of these options to determine whether the expression levels correlate with the phenotype of Wip1–/– mice.

Although we cannot rule out the involvement of thymic epithelial cells in the Wip1 phenotype, the biochemical and in vitro biological differences observed support the premise that Wip1 directly affects thymic T cells. The finding that Wip1-deficient mice are relatively resistant to the development of certain types of breast cancer (29) have far-reaching implications. Because Wip1 is amplified in a number of human cancers, including breast tumors (30), the phosphatase could be a new target for treating breast cancer patients (39). Wip1 is also overexpressed in neuroblastomas and could also be a potential target for therapy (40). Neuroblastomas are one of the most common cancers in children accounting for ~15% of all cancer-related pediatric deaths in the United States (41) and about half of all cases result in death. Because most neuroblastoma patients are young infants with a developing immune system, treatments that would inhibit Wip1 may have immunological complications. The severity and extent that Wip1 inhibition could affect thymic development in these young patients would likely be dependent on patient age and length of treatment. Therefore, appropriate precautions must be taken into account to identify any long-term immunological risks that may be associated with inhibiting Wip1 in pediatric patients.


   Acknowledgments
 
We are indebted to C. Sommers and B. J. Taylor for their help with flow cytometry, sorting, and insightful discussion of the data. We are grateful to A. Cheever for evaluating histological samples and M. Vacchio for setting up the FTOC.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 the Intramural Research Program of the National Institutes of Health, Center for Cancer Research, National Cancer Institute. Back

2 Address correspondence and reprint requests to Dr. Marco L. Schito, National Cancer Institute, National Institutes of Health, Building 37, Room 2140, Bethesda, MD 20892. E-mail address: schitom{at}mail.nih.gov Back

3 Abbreviations used in this paper: DN, double negative; DP, double positive; SP, single positive; WT, wild type; PI, propidium iodide; 7-AAD, 7-amino actinomycin D; FTOC, fetal thymic organ culture. Back

Received for publication June 23, 2005. Accepted for publication February 2, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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