Ablation of a peptidyl prolyl isomerase Pin1 from p53-null ... - Nature

Oncogene (2007) 26, 3835–3845

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ORIGINAL ARTICLE

Ablation of a peptidyl prolyl isomerase Pin1 from p53-null mice accelerated thymic hyperplasia by increasing the level of the intracellular form of Notch1 K Takahashi1,4, H Akiyama1,2, K Shimazaki4, C Uchida1,5, H Akiyama-Okunuki4, M Tomita4, M Fukumoto3 and T Uchida1,2 1 Center for Interdisciplinary Research, Tohoku University, Sendai, Miyagi, Japan; 2Molecular Enzymology, Agricultural Science, Tohoku University, Sendai, Miyagi, Japan; 3Institute of Development, Aging and Cancer, Tohoku University, Sendai, Miyagi, Japan; 4 School of Pharmaceutical Sciences, Showa University, Tokyo, Japan and 5University Health Center, Ibaraki University, Mito, Ibaraki, Japan

Tumor suppressor p53 is essential for checkpoint control in response to a variety of genotoxic stresses. DNA damage leads to phosphorylation on the Ser/Thr-Pro motifs of p53, which facilitates interaction with Pin1, a pSer/pThr-Prospecific peptidyl prolyl isomerase. Pin1 is required for the timely activation of p53, resulting in apoptosis or cell cycle arrest. To investigate the physiological relationship between Pin1 and p53, we created Pin1
/
p53
/
mice. These p53-deficient mice spontaneously developed lymphomas, mainly of thymic origin, as well as generalized lymphoma infiltration into other organs, including the liver, kidneys and lungs. Ablation of Pin1, in addition to p53, accelerated the thymic hyperplasia, but the thymocytes in these Pin1
/
p53
/
mice did not infiltrate other organs. The thymocytes in 12-week-old Pin1
/
p53
/
mice were CD4
CD8
(double negative) and had significantly higher levels of the intracellular form of Notch1 (NIC) than the thymocytes of p53
/
or wild-type mice. Presenilin-1, a cleavage enzyme for NIC generation from full-length Notch1 was increased in the thymocytes of Pin1
/
p53
/
mice. Pin1 depletion also inhibited the degradation of NIC by proteasomes. These results suggest that both Pin1 and p53 control the normal proliferation and differentiation of thymocytes by regulating the NIC level. Oncogene (2007) 26, 3835–3845. doi:10.1038/sj.onc.1210153; published online 11 December 2006 Keywords: p53; Notch1; presenilin-1; thymocytes; hyperplasia

Introduction The p53 tumor suppressor gene plays a complex and critical role in maintaining genome integrity (Levine, Correspondence: Professor T Uchida, Molecular Enzymology, Graduate School of Agricultural Science, Tohoku University, 1-1 Amamiya, Tsutsumidori, Aoba, Sendai, Miyagi 981-8555, Japan. E-mail: [email protected] Received 8 May 2006; revised 21 August 2006; accepted 2 October 2006; published online 11 December 2006

1997; Vogelstein et al., 2000; Soussi, 2000; Slee et al., 2004). Loss of p53 function increases susceptibility to malignant transformation, and mutations in the p53 tumor suppressor gene are the most common genetic abnormalities in oncology, found in >50% of all human cancers (Lutzker and Levine, 1996). The level of active p53 in cells is rapidly elevated through acetylation and phosphorylation in response to DNA damage caused by various stresses, such as ultraviolet irradiation, DNA-damaging chemicals, hypoxia and activated oncogenes (Appella and Anderson, 2001). Transcriptional activity of the p53 gene is also physiologically elevated during organogenesis (Rogel et al., 1988). High levels of activated p53 then drive transcription of a variety of genes that mediate the protein’s biological functions in the decision to arrest cells to allow for DNA repair or to eliminate the cell by apoptosis (Soengas et al., 1999; Wahl and Carr, 2001). Although p53-deficient mice survive birth they rapidly succumb to tumors, predominantly of the thymic lymphoid lineages (Donehower et al., 1992; Jacks et al., 1994; Purdie et al., 1994). There have also been reports of high-frequency embryonic lethality derived from female-specific developmental abnormalities, namely neural tube-closure failure leading to exencephaly and subsequent anencephaly (Armstrong et al., 1995; Sah et al., 1995). The peptidyl-prolyl cis/trans isomerase (PPIase) Pin1 regulates factors involved in the control of cell growth and apoptosis (Lu et al., 1996; Shaw, 2002). Pin1 specifically associates with a phosphorylated serine or threonine residue followed by proline (pS/pT-P), and catalyses the cis–trans isomerization of the peptide bond. Isomerization modulates protein folding, biological activity, stability and subcellular localization of associated substrates (Lu et al., 1996; Shaw, 2002). Pin1 has been linked to cell cycle control and tumorigenesis. Mice lacking Pin1 display immature germ cells, mammary gland impairment and retinal dystrophy when they get old (Liou et al., 2002). Furthermore, serum depletion induces G0 arrest in Pin1
/
MEF (Fujimori et al., 1999).

Thymic hyperplasia in Pin1
/
p53
/
mice K Takahashi et al

3836

Recently, Pin1 was shown to be involved in controlling the accumulation and activity of p53 in cells exposed to DNA-damaging conditions (Wulf et al., 2002; Zacchi et al., 2002; Zheng et al., 2002). These reports indicated that p53 is one of the substrates of Pin1, and that the effects on p53 are the result of isomerization by Pin1. Therefore, Pin1 may play a critical role in integration of the checkpoint induced by DNA damage (Wulf et al., 2002; Zacchi et al., 2002; Zheng et al., 2002). These findings prompted us to study the physiological relationship between Pin1 and p53. Notch1 is a transmembrane receptor crucial for T-cell development (Maillard et al., 2003; Radtke et al., 2004.). The intracellular domain of Notch1 (NIC), which is the activated form, translocates from the membrane to the nucleus and induces transcription of genes downstream of Notch1, such as HES1 and cyclinD1 (Robey et al., 1996; Ronchini and Capobianco, 2001; Fowlkes and Robey, 2002). p53 downregulates presenilin-1, a component of the g-secretase complex required to cleave and consequently activate Notch1. Thus, p53 may negatively regulate Notch1 activation (Roperch et al., 1998; Amson et al., 2000; Pastorcic and Das, 2000). Notch1 is expressed at varying levels in developing and mature T cells, from hematopoietic progenitors to peripheral lymphocytes (Hasserjian et al., 1996). Both p53 and Notch1 are linked to lymphoid malignancies (Oswald et al., 2001). Pear et al. (1996) showed that transduction of bone marrow cells with NIC results in T-cell leukemia, and that Notch1 plays a role in lymphoid oncogenesis in mice. Overactivation of Notch1 may play a role in tumorigenesis resulting from a loss of functional p53. Here, in order to identify the physiological relationship between Pin1 and p53, we created Pin1
/
p53
/
mice and found that Pin1 deletion accelerated the thymic hyperplasia in p53
/
mice. NIC was significantly increased in the thymocytes of 12-week-old Pin1
/
p53
/
mice, and thus these thymocytes may cause hyperplasia by upregulating NIC. The presenilin-1 level also increased concomitantly with the NIC level in the thymocytes of Pin1
/
p53
/
mice. We also determined that Pin1 binds to NIC to induce degradation. These data indicate that both Pin1 and p53 are key regulators of thymocyte proliferation and act through Notch1 activation.

Results Generation of Pin1
/
p53
/
mice Mice carrying the null mutation for Pin1 were mated with mice heterozygous for a targeted mutation for p53. The progeny of these mice (Pin1 þ /
p53 þ /
) were viable and fertile, and were then interbred to generate double-null neonates (Pin1
/
p53
/
). Pin1 þ /
p53 þ /
mice produced in the initial mating appeared normal for 6 months, and were mated with each other. In the first 24 litters of this cross, 201 neonates were Oncogene

raised beyond weaning age. The frequencies of the genotypes obtained from these crosses are shown in Table 1. The birth rates of the p53-deficient mice were not consistent with Mendelian inheritance; the incidence of p53
/
was lower than that with p53 þ / þ and p53 þ /
, regardless of the genotype of the Pin1 allele. Of 14 Pin1 þ / þ p53
/
mice, there were nine males and five females, and of 10 Pin1
/
p53
/
mice, there were eight males and two females. In the absence of p53, there was a reduction in the number of females, with a proportion of female embryos developing exencephaly. The Pin1
/
p53
/
mice also died earlier than the p53
/
mice (Figure 1a). Kaplan–Meier survival analysis was performed and the median survival time (t1/2) was calculated. The median survival time represented the time at which half of the animals were either killed or died because of overt illness. p53
/
mice had t1/2 of 141 days (mean ¼ 148.8721.9 days), compared with 195 days (mean ¼ 209.7718.3 days) for Pin1
/
p53
/
mice. More than 90% of the p53
/
and Pin1
/
p53
/
mice developed lymphomas, predominantly involving the thymus. p53-deficiency frequently induced lymphomas in the mice (Table 2). The Pin1
/
p53
/
mice developed thymic hyperplasia earlier than the p53
/
mice, suggesting that Pin1 may act as a brake on pathogenesis in the absence of p53 (Figure 1a). The thymus of Pin1
/
p53
/
mouse of weaning age (12-weeks-old) is shown in Figures 1b. The picture indicates an enlarged thymus that occupies the thoracic cavity. The enlarged thymuses from Pin1
/
p53
/
mice covered the heart and lungs, but had not penetrated surrounding tissue. The Pin1
/
p53
/
mice with thymic hyperplasia died suddenly, due to the thymomatic tissue in the thoracic wall, along with massive air pockets in the stomach, pressing against the esophagus (Figure 1b). We then focused on and examined the thymic phenotypes of both 6- and 12week-old Pin1
/
p53
/
mice. Hyperplasia of the thymic medulla in Pin1
/
; p53
/
mice (6-week-old) At necropsy, the thymuses from the 6-week-old Pin1
/
p53
/
mice displayed complex architecture with medullary hyperplasia (Figure 2b). The sizes and appearances of the thymuses were indistinguishable among the genotypes (Figure 2a). The phenotypes of the Pin1 þ /
mice and wild-type mice were the same, so we showed the phenotypes of Pin1 þ /
as the control. The thymuses of the Pin1
/
p53
/
mice displayed a more thickened medulla than the other genotypes, which showed normal architecture with clearly distinguishable cortex and medulla, as in the thymus of the Pin1 þ /
mouse (Figure 2b). Pin1
/
p53
/
mice specifically displayed a prominent ‘starry sky’ pattern produced by tingible body macrophages – large macrophages in the germinal center that have phagocytosed debris from dividing lymphoma cells. Flow cytometry analysis of the CD4/CD8 expression patterns of thymocytes did not reveal any differences among

Thymic hyperplasia in Pin1
/
p53
/
mice K Takahashi et al

Survlvor (%)

10 4.98 6.25

~ 2 # 8 ~ 11

100 90 80 70 60 50 40 30 20 10 0

Pin1− /− p53−/− p53− /−

0

100

200

300

400

500

600

23 11.44 12.5

Days

# 9

16 7.96 6.25

~ 7

# 12

b

# 12

26 12.94 12.5

~ 14

# 26

48 23.88 25

~ 22

# 11

13 6.47 12.5

~ 2

Figure 1 Survival curves of Pin1
/
p53
/
, and p53
/
mice. (a) Kaplan–Meier survival analysis was performed and t1/2 calculated. The median survival time represents the time at which half of the animals were either killed or died because of overt illness. p53
/
mice had a t1/2 of 141 days (mean, 148.8721.9 days), compared with 195 days (mean, 209.7718.3 days) for Pin1
/
p53
/
mice. The Pin1
/
p53
/
mice succumbed earlier than the p53
/
mice. Red: Pin1
/
p53
/
; black: p53
/
. (b) Photo of a 12-week-old Pin1
/
p53
/
mouse thymus. Swelling at the breast was observed. Massively enlarged thymus in a 12-week-old Pin1
/
p53
/
mouse. The massively enlarged thymus occupied the majority of the mediastinum and thoracic cavities.

31 15.42 12.5 Total Percent Expected percent

20 9.95 6.25

~ 8

Weaning age mice (n ¼ 201) of 24 litters were examined.

14 6.97 6.25

~ 5 ~ 15 # 16 # 12

# 9

pin1
/
;Wt pin1+/
;p53
/
pin1+/
;p53+/
pin1+/
;Wt Wt;p53
/
Wt;p53+/
Wt;Wt Genotypes

Table 1 Genotype frequency of offspring from interbreeding pin1+/
;p53+/
mice

pin1
/
;p53+/


pin1
/
;p53
/


3837

a

the genotypes (Figure 2c). In order to examine the sensitivities of the thymocytes to DNA damage, primary cultured thymocytes prepared from mice were irradiated with 5 Gy of g-radiation (Figure 2d). g-ray irradiation induced apoptosis in Pin1 þ /
and Pin1
/
thymocytes, indicated by DNA ladder formation and cleavage for caspase-3 activation, and p27Kip1 cleavage (cleaved p27Kip1 is produced by the caspases including caspase-3 (Loubat et al., 1999)). Interestingly, Cdk2 cleavage was specific to p53-expressing (Pin1 þ /
and Pin1
/
) thymocytes, and the level of cyclin A1 was significantly increased by g-ray irradiation in p53
/
and Pin1
/
p53
/
thymocytes. Rb is one of the substrates of the Cdks, and is also cleaved by p27Kip1 during apoptosis (Fattman et al., 1997; Katsuda et al., 2002). Although Rb was cleaved and was undetectable in the g-ray-irradiated Pin1 þ /
and Pin1
/
thymocytes, the hyper-phosphorylated form (Hyper-p-Rb) was detected in the g-ray-irradiated p53
/
and Pin1
/
p53
/
thymocytes. In the thymocytes of 6-week-old mice, the apoptotic response to DNA damage was initiated by p53-dependent activation of the caspases, via a pathway unrelated to Pin1. Thus, the thymocytes survived when disruption of p53 meant the caspases could not be activated in response to DNA damage. This survival of the g-ray-irradiated p53
/
and Pin1
/
p53
/
thymocytes may be dependent on activation of Cdk2/ cyclin A1 (Figure 2d). Oncogene

Thymic hyperplasia in Pin1
/
p53
/
mice K Takahashi et al

3838 Tumors of the deceased mice

Table 2 Wt;p53
/
(n ¼ 14)

Lymphoma Thymic Non-thymic Sarcoma

a

pin1+/
;p53
/
(n ¼ 13)

pin1
/
;Wt (n ¼ 16)

pin1
/
;p53
/
(n ¼ 10)

n

%

n

%

n

%

n

%

13 3 1

92.9 21.4 7.14

12 2 6

92.3 15.4 46.2

3 3 3

18.8 18.8 18.8

9 2 4

90 20 40

b

Pin1+/-;p53-/-

Pin1+/-

6 weeks

Pin1-/-;p53-/-

Pin1-/-

d

Pin1-/-;p53 -/-

Pin1+/-

Pin1 +/+/p53 IR (5Gy) - +

-/+/- +

+/-/- +

-/-/- +

DNA ladder

Thymus

Spleen 5mm

250

250

200

200

(mg)

(mg)

300

150

100

50

50

0

0

p27kip1

CF Pin1+/-p53+/+ Pin1-/-p53 +/+ Pin1+/-p53-/- Pin1-/-p53 -/-

4

10

CD 8

3

10

10

2

10

10

1

10

10

0

10

0

10

1

2

10 10

3

4 10

10 10

4

4

10

3

10

3

10

2

10

2

10

1

10

1

10

0 0

10

1

2

10 10

3

0 410

10 10

10 0

10

1

2

10 10

3

Rb

Pin1+/-;p53-/-

Pin1+/-;p53-/-

Pin1+/-

10

FL

Cyclin A1

Pin1+/-p53 +/+ Pin1-/-p53+/+ Pin1+/-p53-/- Pin1-/-p53-/-

Pin1+/-

FL CF

150

100

c

Caspase 3

Spleen

Thymus 300

4

10 10

10

4

Hyper-p-Rb Hypo-p-Rb


-Tubulin

3 2 1 0 0

10

1

2

10 10

3

4

10 10

CD 4

Figure 2 Thymus of 6-week-old Pin1
/
p53
/
mice. (a) Dissected thymuses and spleens from 6-week-old Pin1
/
p53
/
mice. The sizes and appearances of the thymuses were indistinguishable among the genotypes. The phenotypes of the Pin1 þ /
mice and wild-type mice were the same (data not shown). (b) Medullary hyperplasia in Pin1
/
p53
/
thymus. Hematoxylin- and eosin-stained sections of Pin1 þ /
p53 þ / þ (left) and Pin1
/
p53
/
(right) mice. Lower image: higher magnification image of boxed region. Thymus from Pin1
/
p53
/
(right) mice displayed only a slight ‘starry sky’ pattern, as produced by tingible body macrophage (arrows). (c) Immunofluorescence analysis of thymocyte-specific surface antigens in genetically defined 6-week-old mice. Single-cell suspensions were prepared from the thymus and stained with anti-CD4 PE and anti-CD8 fluorescein isothiocyanate (FITC). In each instance, 10 000 viable cells were collected and analysed on a FACScalibur unit (Becton-Dickinson). There were no significant differences among the genotypes examined. (d) Apoptosis induction by g-ray irradiation. Single-cell suspensions prepared from the thymus were treated with 5 Gy g-ray irradiation. T-cell apoptotic responses, including DNA ladder formation, caspase-3 activation and Rb cleavage were detected in p53-expressing (Pin1 þ /
p53 þ /
and Pin1
/
p53 þ /
) thymocytes, but not p53-deficient (Pin1 þ /
p53
/
and Pin1
/
p53
/
) cells. Cdk2 cleavage was induced specifically by p53-expressing (Pin1 þ /
and Pin1
/
) cells. Upregulation by g-ray irradiation of cyclin A1 was observed in p53-deficient (Pin1 þ /
p53
/
and Pin1
/
p53
/
) cells. Hyperphosphorylation of Rb was also induced by 5 Gy g-ray irradiation. Data are representative of multiple experiments.

Enlarged thymuses in Pin1
/
; p53
/
mice (12-week-old) As shown in Figures 1b and c, 12-week-old Pin1
/
p53
/
mice displayed enlarged thymuses. The dissected thymuses are shown in Figure 3a. The phenotypes of the Pin1 þ /
mice and wild-type mice were the same, so we showed the phenotypes of Pin1 þ /
as the control. As the enlarged thymuses from Pin1
/
p53
/
mice covered the heart and lungs but did not penetrate surrounding tissue (Figure 1c), the dissected thymuses from Pin1
/
p53
/
mice were easily separated from the heart and lungs, and had a smooth surface (Figure 3a). On the other hand, p53
/
mice frequently displayed thymic lymphomas with penetration into surrounding tissue, and the dissected Oncogene

thymuses from p53
/
mice could not be separated from the heart and lungs (Figure 3a). p53-deficient thymocytes had permeated various tissues, including the lung (Figure 3b), heart, liver and kidney (data not shown), as in previous reports (Donehower et al., 1992; Jacks et al., 1994; Purdie et al., 1994). Thus, lymphoma was observed in p53
/
mice by approximately 3 months of age and marked swelling of the thymus with adhesion and invasion of the heart, lungs and thoracic wall were evident. In contrast, the thymocytes of Pin1
/
p53
/
mice did not penetrate other tissues, and splenic hyperplasia was not observed in these mice (Figure 3a). Histological examination revealed that 12-week-old Pin1
/
p53
/
mice displayed thymic

Thymic hyperplasia in Pin1
/
p53
/
mice K Takahashi et al

3839 Thymus

2500

a

12 weeks Pin1+/- Pin1-/- Pin1+/-;p53-/- Pin1+/-;p53-/-

2000

(mg)

1500

Thymus

1000 500 0 Pin1+/-p53+/+ Pin1-/-p53+/+ Pin1+/-p53-/- Pin1-/-p53-/-

Spleen

Spleen

1200 1000 5mm (mg)

800

Pin1+/-;p53-/-

b

600 600

Pin1-/-;p53-/-

200 0 Pin1+/-p53+/+ Pin1-/-p53+/+ Pin1+/-p53-/- Pin1-/-p53-/-

d

Pin1 p53

IR (5Gy) DNA ladder

-/-/-

+

Pin1 p53 IR (5Gy)

+/-/- +

-/-/-

+

kip1

p27

Cyclin A1

c CD 4

+/-/- +

Pin1+/-;p53-/104

104

3

10

103

102

102

101

101

100 100 101 102 103 104

Pin1-/-;p53-/Rb Caspase 3


-Tubulin

100 100 101 102 103 104 CD 8

Figure 3 Thymus of 12-week-old Pin1
/
p53
/
mice. (a) Dissected thymuses and spleens from 12-week-old Pin1
/
p53
/
mice. The thymuses of 12-week-old Pin1 þ /
p53
/
mice adhered to the heart and lungs, and it was difficult to isolate the thymus from the other tissues. The thymus of 12-week-old Pin1
/
p53
/
mice was enlarged but did not adhere to surrounding tissue. Pin1 þ /
p53
/
mice displayed enlarged spleens whereas Pin1
/
p53
/
mice had splenic atrophy. The phenotypes of the Pin1 þ /
mice and wild-type mice were the same (data not shown). (b) Histological characteristics of enlarged thymuses from 12-week-old Pin1 þ /
p53
/
(left) and Pin1
/
p53
/
(right) mice. The medulla and cortex were indistinguishable. In the photo of Pin1 þ /
p53
/
, thymocytes have penetrated the lung. Lower image: higher magnification image of boxed region. Thymus from Pin1
/
p53
/
(right) mice displayed prominent ‘starry sky’ pattern produced by tingible body macrophages (arrows). (c) Immunofluorescence analysis of thymocytes from 12-week-old Pin1 þ /
p53
/
(left) and Pin1
/
p53
/
(right) mice. Single-cell suspensions were prepared from the thymus and then stained with anti-CD4 PE (phycoerythrin) and anti-CD8 FITC. In each instance, 10 000 viable cells were collected and analysed on a FACScalibur unit (Becton-Dickinson). Thymocytes from Pin1
/
p53
/
were double-negative (CD4
CD8
) cells. (d) Effects of 5 Gy g-ray irradiation on thymocytes from 12-week-old Pin1
/
p53 mice. Single-cell suspensions prepared from the thymus were treated with 5 Gy g-ray irradiation. DNA ladder formation was spontaneous and was enhanced by 5 Gy g-ray irradiation in Pin1
/
p53
/
thymocytes. Biochemical T-cell apoptotic responses, including caspase-3 activation, were not induced by 5 Gy g-ray irradiation. These thymocytes displayed no induction of cyclin A1, which was observed in thymocytes from 6-week-old Pin1
/
p53 mice (Figure 2d). Interestingly, p27Kip2, a Cdk inhibitor and Rb were not detected in non-stimulated thymocytes from 12-week-old Pin1
/
p53 mice. Data are representative of multiple experiments.

hyperplasia, which appeared as sheets of relatively monotonous cells with scattered tingible body macrophages giving a ‘starry-sky’ appearance; cells also had scant cytoplasm, enlarged round nuclei, irregular nuclear contours, stippled chromatin, inconspicuous or single small nucleoli and numerous mitotic figures (Figure 3b). Flow cytometry revealed that most of the thymocytes in 12-week-old Pin1
/
p53
/
mice were immature double negative (DN; CD4
CD8
) cells (Figure 3c). These cells differed from those of the 6-week-old mice and displayed apoptosis in response to DNA damage; however, activation of caspase-3 and cleavage of Cdk2 were not induced. p27Kip1 and Rb expression was downregulated, independent of DNA damage.

Furthermore, the increase in cyclin A1 observed in the thymocytes of 6-week-old p53-deficient mice was not detected in the 12-week-old mice (Figure 3d). These findings suggest that the immature thymocytes did not disperse widely through different tissues during thymic hyperplasia and that these cells clearly differed from mature cells in their sensitivity to DNA damage. Activation of Notch1 in Pin1
/
p53
/
thymic hyperplasia Flow cytometry revealed that the immature lymphocytes resided in the thymuses of the Pin1
/
p53
/
mice. Because regulation of Notch1 activity is essential in the Oncogene

Thymic hyperplasia in Pin1
/
p53
/
mice K Takahashi et al

3840

expression of NIC lacking the PEST domain (NICv1744) with a myc-tag, was transiently transfected into COS7, because NIC with the PEST domain is too unstable to be detected by immunoblot analysis (Kopan et al., 1996; Schroeter et al., 1998). First, to investigate the association between Pin1 and NIC, we attempted a pull-down assay using gluthatione S-transferase fusion Pin1 (GSTPin1). As shown in Figure 4b, the associated NICv1744 with GST-Pin1, but not GST, was specifically detected. Next, to investigate the effects of Pin1 on the stability of NIC, we attempted co-transfection into COS7 cells of the myc-tagged NICv1744 expression-vector and Pin1 expression-vector (Figure 4c). The induction of Pin1expression reduced the NICv1744 detected by the anti-myc antibody and anti-NIC antibody, suggesting that Pin1 might induce degradation of NIC (Figure 4c). NIC degradation is known to occur via a ubiquitin-proteasome pathway (Lai, 2002). In the presence of the proteasome inhibitor MG132, accumulation of NICv1744 was observed (Figure 4d). Induction of Pin1 decreased NIC in a manner identical to that shown in Figure 4c, and thus the effects of MG132 were against Pin1. The addition of MG132 increased the NIC level under conditions of Pin1 overexpression, and thus ubiquitinproteasome-mediated degradation of NIC might be dependent on the PPIase activity of Pin1 (Figure 4d).

maturation of thymocytes (Maillard et al., 2003; Radtke et al., 2004.) and overactivation of Notch1 may play a role in tumorigenesis resulting from a loss of functional p53 (Oswald et al., 2001), we speculated that Notch1 activation might be irregular in the thymocytes of Pin1
/
p53
/
mice. The activated form of Notch1 is the intracellular form (NIC) produced by limited cleavage of full length Notch1 (Fowlkes and Robey, 2002). The level of NIC in the thymocytes of Pin1
/
p53
/
mice was significantly higher than in the other mice, and concomitantly with NIC, Pin1
/
p53
/
thymocytes displayed the highest levels of presenilin-1 (a component of the g-secretase complex essential for cleavage and consequent activation of Notch1) among the Pin1 þ /
, Pin1
/
Pin1 þ /
p53
/
and Pin1
/
p53
/
thymocytes (Figure 4a). The increased NIC levels in the Pin1
/
p53
/
thymocytes remained functional, as its transcriptional target molecules, hes1 and cyclinD1 transcripts, were also upregulated (data not shown). Thus, NIC is effectively produced from full-length Notch1 by presenilin-1 upregulated by a deficiency in both Pin1 and p53. The results of NIC overexpression in the thymocytes of Pin1
/
p53
/
mice suggested that Pin1 may affect the stability of NIC. We therefore analysed NIC stability in Pin1 expression-induced cells. A commercially available NIC expression-vector, which induces

a

b

Thymocytes Pin1 +/p53 +/-

-/+/-

+/-/-

-/-/-

Pull down

Notch IC

Input

NIC GST GST-Pin1 NICD

Presenilin 1

myc


-Tubulin

c

d

NIC Pin1 Flag Pin1 Cleaved Notch1
-tubulin lacZ

-

+

Pin1

MG132 (10 µM)

-

-

+

+

-

+

-

+

Flag Pin1 Cleaved Notch1
-tubulin lacZ

Figure 4 Notch1 intracellular domain (NIC) accumulation in Pin1
/
p53
/
thymocytes. (a) Increase in NIC and presenilin-1 in Pin1
/
p53
/
thymocytes. NP40 lysates from single-cell suspensions were used for immunoblot analysis. (b) Pull-down assay for interaction between Pin1 and NIC using glutathione-sepharose beads (Invitrogen) carrying the recombinant GST-Pin1 (or GST) and cell lysates from COS-7 transfected with myc-tagged NIC-expression vector (NICV1744). Captured NIC was detected using anti-myc antibody. (c) Effects of Pin1 on NIC stability. The effects of Pin1 on NIC accumulation was determined by immunoblot analysis of cell lysates from COS-7 cells co-transfected with pFlag-CMV2/Pin1 and NICV1744. NIC was detected by using anti-myc antibody and the polyclonal antibody specific to murine NIC (Cell signaling). Transfection efficiency was indicated by the expression level of the co-transfected LacZ. (d) Pin1-dependent proteasome-mediated degradation of NIC. We treated the co-transfected COS-7 cells with MG132, a peptidyl aldehyde inhibitor, and prepared lysates. NIC was detected using a polyclonal antibody specific for murine NIC (Cell ignaling). Data are representative of multiple experiments. Transfection efficiency was indicated by the expression level of the co-transfected LacZ. Oncogene

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Discussion We generated Pin1
/
p53
/
mice and demonstrated that combined Pin1 and p53 ablation (Pin1
/
p53
/
) results in a reduction in the number of developed females as well as obvious enlargement of the thymus, with lymphoma. Furthermore, the Pin1
/
p53
/
mice had shorter life spans than the p53
/
mice. p53
/
females are known to display exencephaly (Armstrong et al., 1995; Sah et al., 1995), and in the present study Pin1
/
p53
/
female embryos displayed exencephaly to a greater extent than p53
/
female embryos. Thus, Pin1 may prevent p53-deficient mice from developing female-specific exencephaly. In the female embryonic lineage, dosage compensation is achieved by ‘X inactivation’ around the time of gastrulation, when intense embryonic cellular proliferation and apoptosis induce embryonic development (Lyon, 1961; Rastan, 1994). It has also been shown that the inactivated X chromosome replicates late in the S phase (Takagi, 1974), and cells lacking p53 have been shown to be defective in damage-induced G1/S checkpoint arrest (Baker et al., 1990; Lin et al., 1992). As (Cranston et al., 1997) discussed in a report on female lethality in Msh2
/
p53
/
, dysregulation of damageinduced arrest checkpoint control could result in cells being unable to arrest and repair damage introduced into the late-replicating inactive X chromosome. In DNA-damaged cells, Pin1 may cooperate with the various DNA repair molecules, including p53 and Msh2. In the present study, the DNA repair process in the late-replicating inactive X chromosome was not complete owing to a lack of both Pin1 and p53, thereby resulting in a high rate of female embryonic exencephaly. Thymic hyperplasia in Pin1
/
p53
/
mice Although both p53-null and double-null mice showed enlarged thymuses, by approximately 6 months of age p53-null mice displayed marked swelling of the thymus with penetration into the heart and thoracic wall, and double-null mice showed an enlarged thymus in the thorax without penetration into adjoining tissue. p53null mice almost always die by 4 months of age from various tumors, predominantly lymphomas. It is clear that within the thymus, p53 is essential for the elimination, via apoptosis, of DNA-damaged cells, and it appears that loss of apoptotic pathways is critical for tumor formation within this cell type (Donehower et al., 1992; Jacks et al., 1994; Purdie et al., 1994). In the present study, we focused on the thymic phenotypes of 6-week-old and 12-week-old Pin1
/
p53
/
mice. Six-week-old mice are generally used in immunofluorescence analysis of thymocyte-specific surface antigens. Although no differences among the examined genotypes were observed with respect to expression of CD4 and CD8 (Figure 2c), 6-week-old Pin1
/
p53
/
mice displayed medullary hyperplasia with a prominent ‘starry sky’ pattern, produced by tingible body macrophages – large macrophages in the

germinal center that have phagocytosed debris from dividing lymphoma cells. The thymuses of 6-week-old p53
/
mice did not display the ‘starry sky’ appearance, indicating that the Pin1-deficiency quickened tumorigenesis in the p53
/
mice. The 12-week-old Pin1
/
p53
/
mice displayed significantly enlarged thymuses (Figures 1b and 3a), that also had numerous ‘starry sky’ patterns with large tingible body macrophages. In thymic structure, the medulla and cortex were not distinguishable. Flow cytometry revealed that DN thymocytes occupied the enlarged thymuses. Thus, the enlarged thymuses of Pin1
/
p53
/
mice might be derived from hyperplasia of immature CD4
/CD8
, thymocytes. These 12-week-old thymuses also had smooth surfaces and did not penetrate the heart and lungs. On the other hand, Pin1 þ /
p53
/
and p53
/
thymocytes did infiltrate various tissues. Thus, the penetration of p53-deficient thymocytes into other tissue may be dependent on Pin1, which would explain the difference in spleen size between Pin1 þ /
p53
/
and Pin1
/
p53
/
mice. Pin1 may in fact inhibit thymocyte (thymic lymphoma)-mobility. Thymic hyperplasia was associated with increased T-lymphocyte proliferation. p27 deficiency induces multiple organ hyperplasia (Fero et al., 1996; Kiyokawa et al., 1996; Nakayama et al., 1996). p27 and Rb probably function on the same regulatory pathway. In late embryogenesis, p27 deletion caused an ovulatory defect and female sterility. Pin1 deletion showed these phenotypes. Taken together, decrease of p27 and increasing phosphorylated Rb levels in the Pin1 and p53 double knock out (KO) mice shown in Figure 2d are the important mechanism underlying the phenotypes. We examined cell death and cell proliferation of the thymus and spleen by TdT-mediated dUTP nick end labeling (TUNEL) and Ki67 staining, respectively. There are more cells with TUNEL- and Ki67-positive in the double KO mouse thymus than in the wild-type mouse thymus, but there was no difference between the double KO mouse spleen and the wild-type mouse spleen (Supplementary Figure). These results show that the thymus of double KO mouse is unique, because it contains more apoptotic cells as well as proliferating cells than the thymus of the wild-type mouse does. These results suggest that the mechanism underlying thymus hyperplasia is complicated, so we tried to search for another molecule that is related to thymus hyperplasia. Regulated Notch1 activity is essential for the maturation of thymocytes (Maillard et al., 2003; Radtke et al., 2004). Notch1 expression in the thymus is developmentally regulated, with expression highest in immature, DN (CD4
CD8
) cells, decreased in DN cells, and increased in mature single-positive populations (Hasserjian et al., 1996). Notch1 has been implicated in both differentiation and cell death and its expression is developmentally regulated in the thymus, suggesting that Notch1 may play a critical role in thymic development. Overactivation of Notch1 may play a role in tumorigenesis resulting from a loss of functional p53 (Beverly et al., 2005). In addition, NIC is abundant in human T-cell acute lymphoblastic leukemia, and a high Oncogene

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level of NIC is associated with the attendant pathogenesis (Weng et al., 2004). We examined the level of NIC in the thymus of Pin1
/
p53
/
mice, and found that the expression level of NIC was significantly higher in the cells from the enlarged thymuses of Pin1
/
p53
/
mice. It was reported that thymoma in p53null mice is produced through Notch1 activation, and that p53 might regulate NIC production in thymocytes (Laws and Osborne, 2004). Moreover, Notch1 is cleaved by presenilin1, a component of g-secretase, and this generated NIC moves into the nucleus to form a transcriptional complex (Lai, 2002). p53 has been shown to downregulate presenilin-1 transcription and thereby inactivate thymic Notch1 signal (Roperch et al., 1998; Amson et al., 2000; Pastorcic and Das, 2000). The extreme elevation of NIC levels in the Pin1
/
p53
/
thymocytes in the present study may have been caused in part by an increase in presenilin-1, as shown in Figure 4a. NIC contains several pS/pT-P sites that can associate with the WW domain. We suspect that NIC is a substrate of Pin1. The results of our pull-down assay showed that Pin1 associates with NIC, whereas cotransfection analysis indicated that Pin1 induces degradation of NIC. Taken together, p53 may act as a regulator for presenilin-1 transcription and Pin1 may act as the inducing factor for degradation of NIC and as a downregulator of presenilin-1. In the present study, transfection of a constitutively active Notch1 (including NICv1744) construct was stable, owing to the presence of a PEST sequence involved in the degradation of NIC by a ubiquitin-proteasome (Kopan et al., 1996; Schroeter et al., 1998). NIC differs from intact NIC, and thus more studies are required to conclusively define the function of Pin1 on NIC stability. The lack of migration of thymocytes to the periphery in NIC-overexpressing mice resembled that observed in the Pin1
/
p53
/
mice (Robey et al., 1996). Thus, our results suggest that in these mice, enlargement of the thymus might be caused by transactivation of Notch1, and that activation of Notch1 might be regulated by both p53 and Pin1. p53-dependent apoptosis of thymocytes in response to g-ray irradiation Several reports have indicated that Pin1 is associated with phosphorylated p53 during the response to DNA damage (Wulf et al., 2002; Zacchi et al., 2002; Zheng et al., 2002). In order to examine the effects of Pin1 and p53 deficiency on the sensitivity of thymocytes to DNA damage, primary cultured thymocytes were irradiated with 5 Gy of g-ray irradiation. Both Pin1 þ /
p53 þ /
and Pin1
/
p53 þ /
thymocytes showed apoptotic responses, including DNA ladder formation, caspase-3 activation, and fragmentation of RB and p27Kip1 (Fattman et al., 1997; Loubat et al., 1999; Katsuda et al., 2002). In contrast, Pin1 þ /
p53
/
and Pin1
/
p53
/
thymocytes showed lower sensitivities to g-rays, and caspase-3 activation and fragmentation of its substrate molecules were not observed. These results indicate that p53, but not Pin1, in thymocytes is required for apoptosis in response to g-ray irradiation. Oncogene

g-ray irradiation significantly increased the level of cyclin A1 in the p53-deficient cells, that is, the Pin1 þ /
p53
/
and Pin1
/
p53
/
thymocytes. These results differ from those of Mu¨ller-Tidow et al. (2004), who reported that DNA-damaging stimuli activated p53, which in turn upregulated cyclin A1. Thus, the relationship between cyclin A1 and p53 remains to be clarified. Although the physiological role of cyclin A1 in somatic cells remains unknown, cyclin A1 is expected to play a role in the maintenance of genome stability (Mu¨ller-Tidow et al., 2004). In the present study, the endogenous substrate of Cdks, Rb, was hyperphosphorylated in p53-deficient (Pin1 þ /
p53
/
and Pin1
/
p53
/
) cells. When the DNA in the p53deficient thymocytes was damaged, phosphorylation of Rb may have been induced by the cyclinA1/Cdk2 complex, which is required for the cell cycle arrest necessary for DNA repair. Our results show that the Pin1-deficiency enhanced the proliferation of the p53deficient thymocytes, as Pin1
/
p53
/
mice displayed enlarged thymuses earlier than p53
/
and Pin1 þ /
p53
/
mice. Interestingly, the thymocytes of 12week-old Pin1
/
p53
/
mice differed from those of the 6-week-old mice in response to irradiation. The thymocytes from the12-week-old mice displayed DNA ladder before irradiation, and irradiation induced more apoptotic responses. However, the induction of caspase-3 activation was not one of them, and this apoptotic reaction might be independent of both caspase-3 and p53. The relationship between NIC accumulation and spontaneous apoptosis observed in the thymocytes of 12-week-old Pin1
/
p53
/
mice accorded with our expectations. Notch1 signaling regulates apoptosis during thymocyte maturation by preventing death by neglect and/or negative selection in cells destined to die (Deftos et al., 1998; Jehn et al., 1999). In many studies of the antiapoptotic effects of Notch1 in thymocytes or T-cell lines, constitutively active Notch1 (including NICv1744) constructs have been used, as the products are stable owing to the absence of ubiquitin-proteasomemediated degradation. NIC lacking the PEST domain found at the C-terminal end of intact NIC can localize to the nucleus in readily detectable amounts, and exhibit biological properties, such as transforming activity (Capobianco et al., 1997). However, this is different from intact NIC, which reaches the nucleus only in extremely small amounts and can only be detected indirectly through its effects (Schroeter et al., 1998; Struhl and Adachi, 1998). In the present study, the thymuses of the 12-week-old Pin1
/
p53
/
mice were occupied by immature, DN cells, suggesting that intact NIC might play a role in inducing apoptosis in the immature, DN thymocytes. In conclusion, we have found that Pin1
/
p53
/
female embryos more frequently displayed exencephaly than p53
/
female embryos, and that Pin1
/
p53
/
mice display thymic hyperplasia earlier than Pin1-deficient and p53-deficient mice. We also identified NIC accumulation and an increase in presenilin-1 in enlarged thymuses of Pin1
/
p53
/
mice. These

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findings point to a physiological role for the Pin1-p53 control of Notch1 signaling.

Materials and methods Mice Our study was approved by the Showa and Tohoku University Animal use and care committee. Pin1-deficient mice were generated according to our previous report (Fujimori et al., 1999). p53-null mice were first generated by Livingstone et al. (1992), and were provided by DuPont Central & Research Development (Wilmington, DE, USA). Both mouse lines were maintained on a C57BL/6 background. p53 þ /
mice were mated with Pin1 þ /
mice to obtain Pin1 þ /
p53 þ /
offspring, which were intercrossed. Mice carrying the nine possible genotypes (Pin1 þ / þ p53 þ / þ , Pin1 þ / þ p53 þ /
, Pin1 þ / þ p53
/
, Pin1 þ /
p53 þ / þ , Pin1 þ /
p53 þ /
, Pin1 þ /
p53
/
, Pin1
/
p53 þ / þ , Pin1
/
p53 þ /
and Pin1
/
p53
/
) were generated. To genotype the Pin1 locus, the primers WILD1.2A (50 -AAG GGA TTA GAA GCA AGA TTC G-30 ), 2L (50 -AGC ACC CGA TCC TGT TCT GCA A-30 ) and Start2 (50 -CAG AGG CCA CTT GTG TA-30 ) were used (Fujimori et al., 1999). To genotype the p53 locus, the primers x7 (50 -TAT ACT CAG AGC CGG CCT-30 ), x6.5 (50 -ACA GCG TGG TGG TAC CTT AT-30 ) and Neo19 (50 CAT TCA GGA CAT AGC GTT GG-30 ) were used (Livingstone et al., 1992). Kaplan–Meier survival analysis was performed using Stats direct (http://www.statsdirect.com/, StatsDirect, Cheshire, UK). All investigations were conducted according to the principles of the Declaration of Helsinki. Gross and histological analysis For histological analysis, dissected tissue was fixed in 4% paraformaldehyde and then dehydrated through ascending concentrations of ethanol, cleared in xylene, and embedded in paraffin wax. Sections micrometer thickness were cut and then stained with hematoxylin and eosin, dehydrated and cleared. They were then viewed using an Olympus AH3 microscope (Olympus, Tokyo, Japan).

the Bradford method (protein assay; Bio-Rad, Hercules, CA, USA). Total lysates were subjected to sodium dodecyl sulfate– polyacrylamide gel electrophoresis (SDS–PAGE) and then transferred to membranes (Immobilon-P, Millipore, Billerica, MA, USA). The membranes were probed with specific antibodies against caspase-3 (Cell Signaling, Beverly, MA, USA), Cdk2, Pin1, presenilin-1 (Santa Cruz, Santa Cruz, CA, USA), p27Kip1, Rb (BD Biosciences Pharmingen, San Diego, CA, USA) or a-tubulin (Sigma-Aldrich, St Louis, MO, USA). NIC was specifically detected with rabbit anti-cleaved Notch1 antibody (Val1744, Cell Signaling). Proteins were visualized using the ECL-plus system (Invitrogen, Carlsbad, CA, USA). Determination of association between Pin1 and NIC Pin1 was produced as N-terminal GST fusion protein, as described (Zacchi et al., 2002; Zheng et al., 2002). The murine NIC expression vector (NICV1744) used for transfection was constructed using a cDNA coding for murine NIC followed by a 30 hexameric myc epitope (Kopan et al., 1996; Schroeter et al., 1998). The interaction of Pin1 and NIC was determined by the GST pull-down assay using glutathione-sepharose beads (Invitrogen) carrying the recombinant GST-Pin1 and the cell lysates. NIC in the input samples and those captured by the glutathione beads were detected by immunoblot analysis. The effect of Pin1 on NIC accumulation was determined by immunoblot analysis of the lysates from the co-transfected COS-7 cells containing pFlag-CMV2/Pin1 and NICV1744 (Akiyama et al., 2005). To determine the effects of Pin1 on the ubiquitin–proteasome-mediated degradation of NIC, we treated the co-transfected COS-7 cells with MG132, a peptidyl aldehyde inhibitor, and prepared lysates from the cells. Immunoblot analysis was performed using the monoclonal anti-myc antibody and the polyclonal antibody specific to cleaved Notch1 (Cell Signaling), and horseradish peroxidase conjugated anti-mouse or anti-rabbit IgG (Southern Biotechnology, Birmingham, AL, USA), followed by visualization using the ECL-plus system (Invitrogen). Acknowledgements

Flow cytometry Single-cell suspensions were prepared from the thymuses and then analysed for expression of TCRa/b, CD3, CD4 or CD8 using flow cytometry. Monoclonal antibodies to TCRa/b, CD3, CD4 and CD8 labeled with fluorescein isothiocyanate or biotin purchased from commercial sources (Becton-Dickinson, Mountain View, CA, USA; Pharmingen, San Diego, CA, USA). Studies were performed using a FACScalibur unit (Becton-Dickinson) and analysed using Cell Quest (BectonDickinson). Immunoblot analysis Total lysates from single-cell suspensions of thymocytes were prepared with NP40 lysis buffer (Haapaja¨rvi et al., 1995). The protein concentration of the total lysates was determined by

We thank T Tachikawa, RW Shin and K Takeuchi for supporting the analysis of the histopathology of the mice. We thank A Kondoh and M Suzuki for taking care of the mice. K Fukuchi, T Hasegawa, K Ikeda, D Sato, Y Sano, K Saito, NH Choi-Miura and S Arata for their technical help, and L Ostroff for editorial assistance. This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas (TU), Grant-in-Aid for Scientific Research (TU), and a Special Research Grant-in-Aid for Development of Characteristic Education (KT) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, a Showa University Grant-in-Aid for Innovative Collaborative Research Projects (KT) and the Center for Interdisciplinary Research Tohoku University for Specially Promoted Research (TU).

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

Oncogene