Ninjurin1, a target of p53, regulates p53 expression and p53-dependent cell survival, senescence, and radiation-induced mortality

Abstract

The tumor suppressor protein p53 plays a crucial role in coordinating cellular processes, such as cell cycle arrest, apoptosis, and senescence. The nerve injury-induced protein 1 (Ninjurin1, Ninj1) is a homophilic adhesion molecule and involved in nerve regeneration. Interestingly, Ninj1 is found to be overexpressed in human cancer, but its role in tumorigenesis is not clear. Here, we found that Ninj1 is transcriptionally regulated by p53 and can be induced by DNA damage in a p53-dependent manner. We also found that knockout or knockdown of Ninj1 increases p53 expression potentially through enhanced p53 mRNA translation. In addition, we found that Ninj1 deficiency suppresses cell proliferation but enhances apoptosis and premature senescence in a p53-dependent manner. Consistent with this, we found that mice heterozygous in ninj1 are hypersensitive to ionizing radiation-induced lethality, along with increased expression of p53 in thymus. Taken together, we provided evidence that Ninj1 is a p53 target and modulates p53 mRNA translation and p53-dependent premature senescence, cell proliferation, apoptosis, and radiation-induced mortality in vitro and in vivo. Thus, we postulate that as a membrane adhesion molecule, Ninj1 is an ideal target to regulate p53 activity via the p53-Ninj1 loop.

The tumor suppressor protein p53 plays a pivotal role in tumor suppression by regulating cell cycle arrest, apoptosis, and senescence (1). Under unstressed condition, p53 protein is maintained at a low level mostly through ubiquitination-mediated proteosomal degradation (2). In response to DNA damage and other stress signals, p53 is activated and functions as a transcription factor to induce its downstream targets for various cellular processes such as cell cycle arrest [p21, growth arrest and DNA-damage-inducible protein 45 (GADD45)] (3, 4), apoptosis [p53 up-regulated modulator of apoptosis protein (PUMA), insulin-like growth factor binding protein 3 (IGFBP3), DR5] (5–7), and senescence [plasminogen activator inhibitor-1 (PAI-1), differentially expressed in chondrocytes protein 1 (DEC1)] (8, 9). Although many p53 target genes related to various cellular processes have been identified, these are still insufficient to explain how p53 exerts its functions, in particular cellular senescence. Previously, many potential p53 target genes, including nerve injury-induced protein 1 (Ninjurin1, Ninj1), were identified by a DNA microarray study (10).

The ninj1 gene, which encodes a homophilic adhesion molecule and cell surface protein, was found to be highly induced following nerve injury in dorsal root ganglion (DRG) neurons and Schwann cells (11). Ninj1 protein is necessary for mediating homophilic adhesion and promoting neurite outgrowth of DRG neurons (11, 12), suggesting that Ninj1 plays a role in nerve regeneration. The ninj1 gene was also found to be up-regulated in myeloid cells associated with experimental allergic encephalomyelitis and active multiple sclerosis, which subsequently modulates the infiltration of inflammatory myeloid cells into central nerve system (13). Additionally, Ninj1 was found to be overexpressed in hepatocellular carcinoma (14) and acute lymphoblastic B-cell leukemia (15). Furthermore, Ninj1 was found to be induced by ionizing radiation in keratinocytes and dermal fibroblasts (16). These reports let us postulate that Ninj1 plays a role in cell survival and cell death. However, whether p53 directly regulates Ninj1 expression and whether Ninj1 has a role in the p53 pathway are not clear.

In this study, we found that Ninj1 is a target of p53 and can be induced by DNA damage in a p53-dependent manner. Most importantly, silencing of Ninj1 led to increased p53 mRNA translation, resulting in p53-dependent cellular senescence, apoptosis, and growth suppression. Moreover, mice heterozygous in ninj1 showed an increased radiosensitivity along with increased expression of p53 in thymus upon whole-body γ-irradiation. Our findings support the idea that Ninj1 plays a role in p53-mediated tumor suppression in addition to nerve regeneration.

Results

Ninj1 Is a p53 Target.

To identify target genes regulated by p53, we performed a microarray assay using H1299 cells that can inducibly express p53. Many well-defined p53 target genes were identified, including p21, mouse double minute protein 2 (Mdm2), and Gadd45. In addition, we found that Ninj1 was induced by p53, consistent with a previous microarray study (10). To confirm the microarray study, H1299 cells were uninduced or induced to express p53, followed by Northern blot analysis. We found that the level of Ninj1 transcript was markedly increased by p53, along with increased expression of p21 (Fig. 1A). Moreover, we found that in response to treatment with camptothecin, the level of Ninj1 transcript was increased in HCT116, p53^+/−HCT116, MCF7, and LNCaP cells (Fig. 1B, compare lanes 3, 5, 11, and 13 with 4, 6, 12, and 14, respectively). These cells carry either one or two alleles of the wild-type p53 gene. As a positive control, p21 was also induced in these cells treated with camptothecin (Fig. 1B). By contrast, Ninj1 was not induced in p53^−/−HCT116 and mutant p53-containing T98G cells upon treatment with camptothecin (Fig. 1B, compare lanes 1 and 9 with 2 and 10, respectively). We also found that Ninj1 was induced in p21^−/−HCT116 cells treated with camptothecin (Fig. 1B, compare lane 7 with 8), suggesting that the induction of Ninj1 by p53 is not affected by loss of p21. Furthermore, quantitative RT-PCR showed that the level of Ninj1 mRNA was increased by threefold in MCF7 cells upon treatment with doxorubicin, whereas p21 mRNA, as a positive control, was increased by fourfold (Fig. 1C). Next, Western blot analysis was performed and showed that the level of Ninj1 protein was increased in MCF7 cells by ectopically expressed p53 (Fig. 1D) and upon treatment with doxorubicin and camptothecin (Fig. 1E, compare lanes 1 and 4 with 2–3 and 5–6, respectively). Mdm2, a well-defined p53 target, was measured as a control and found to be increased (Fig. 1 D and E). Similarly, the level of Ninj1 protein was increased in HepG2 cells, which carry wild-type p53, upon treatment with doxorubicin (Fig. 1F, compare lane 1 with 2 and 3). Furthermore, we found that upon treatment with doxorubicin, the level of Ninj1 protein on the cell membrane was increased in MCF7 cells (Fig. 1G) and HepG2 cells (Fig. S1). However, Claudin1, another cell adhesion molecule, was not increased by DNA damage (Fig. 1G and Fig. S1). Similarly, we showed that Ninj1 protein was found to be localized on the MCF7 cell membrane, and the intensity of Ninj1 staining was markedly increased upon treatment with doxorubicin and camptothecin (Fig. 1H).

Fig. 1.

Ninj1 is induced by p53. (A) Northern blots were prepared with total RNAs isolated from H1299 cells, which were uninduced (−) or induced (+) to express p53 for 24 h. Northern blot analysis was performed with ³²P-labeled cDNA probes derived from the Ninj1, p21, and GAPDH transcripts. (B) p53^−/−HCT116, p53^+/−HCT116, p53^+/+HCT116, p21^−/−HCT116, T98G, MCF7, and LNCaP cells were untreated (−) or treated (+) with camptothecin (300 nM) for 12 h, followed by Northern blot analysis. (C) Quantitative RT-PCR was performed with total RNAs purified from MCF7 cells, which were untreated (Ctrl) or treated with doxorubicin (DOX) (200 ng/mL) for 12 h. The levels of Ninj1 and p21 transcripts were normalized to the GAPDH transcript level. The experiment was performed in triplicate. Error bars indicate SD. *P < 0.05 by two-tailed t test. (D) The levels of Ninj1, p53, Mdm2, and Actin proteins were measured by Western blots analysis in MCF7 cells uninduced (−) or induced (+) to express p53 for 24 h. (E) MCF7 cells were untreated (Ctrl) or treated with doxorubicin (200 ng/mL) or camptothecin (200 nM) for 12 or 24 h and the levels of Ninj1, p53, Mdm2, and Actin proteins were measured by Western blot analysis. (F) HepG2 cells were treated with or without doxorubicin (200 ng/mL) for 12 or 24 h, and the levels of Ninj1, p53, and Actin proteins were measured by Western blot analysis. (G) MCF7 cells were treated with or without doxorubicin (200 ng/mL) for 12 h, and then cell membranes and nuclei were isolated. The levels of Ninj1 on the cell membrane (CM) and p53 in nucleus (NC) were measured by Western blot analysis. Claudin1 (Cldn1) and hnRNP c1/c2 proteins were measured as a cell membrane protein and a nuclear protein, respectively. (H) The subcellular localization of Ninj1 (green) was measured by immunofluorescence staining in MCF7 cells treated without or with doxorubicin (200 ng/mL) or camptothecin (200 nM) for 12 h. The nuclei were visualized using 4′, 6-diamidino-2-phenylindole (DAPI). CPT, camptothecin.

p53 induces target gene expression via binding to specific DNA sequences in the promoter or intron. Thus, we searched for p53-responsive element (p53-RE) in the genomic locus of the Ninj1 gene and found one potential binding site at nucleotides −2573 to −2407 in the Ninj1 promoter (Fig. 2A). To determine whether the Ninj1 promoter is recognized by p53, MCF7 cells were mock-treated or treated with camptothecin to induce p53 expression, followed by chromatin immunoprecipitation (ChIP) assay. We found that endogenous p53 directly bound to the Ninj1, as well as p21, promoters (Fig. 2B, lanes 4 and 6) but not to the control GAPDH promoter (Fig. 2B, lane 3). Moreover, to test whether the p53-RE in the Ninj1 promoter is responsive to p53, we generated two luciferase reporters carrying either wild-type or mutant p53-RE under the control of the C-fos minimal promoter (Fig. 2C). We showed that the luciferase activity under the control of wild-type but not mutant p53-RE was markedly increased by wild-type p53, whereas mutant p53 was inert in both H1299 and MCF7 cells (Fig. 2D and Fig. S2). As a positive control, the luciferase activity under the control of the p21 promoter was increased by wild-type but not mutant p53 (Fig. 2D and Fig. S2). Our findings suggest that p53 transcriptionally regulates the Ninj1 gene by binding to the p53-RE in the promoter.

Fig. 2.

p53 induces Ninj1 expression through binding to the p53-RE in the Ninj1 promoter. (A) Schematic presentation of the Ninj1 and p21 promoters along with the locations of the p53-RE and the primers used for ChIP assay. (B) ChIP assay was performed with MCF7 cells untreated (−) or treated (+) with camptothecin (200 nM) for 12 h. The p53/DNA complexes were captured with anti-p53 (DO-1) and the binding of p53 to the p53-REs in the Ninj1 or p21 promoter was quantified by PCR. (C) Schematic presentation of luciferase constructs carrying wild-type (Ninj1-W) or mutant p53-RE (Ninj1-M) from the Ninj1 promoter. (D) The luciferase activity was measured from the luciferase constructs Ninj1-W or Ninj1-M in H1299 cells, which were transfected with an empty vector or pcDNA3 expressing wild-type p53 or mutant p53(R249S) for 24 h. The luciferase construct carrying the p21 promoter was used as positive control. The experiment was performed in triplicate. Error bars indicate SD. *P < 0.001 between pcDNA3 and p53; **P < 0.001 between p53 and p53(R249S) by two-tailed t test.

Lack of Ninj1 Up-Regulates p53 Expression Potentially Through Enhanced mRNA Translation.

To determine the biological function of Ninj1, we generated stable cell lines in which shRNA against Ninj1 can be inducibly expressed in RKO cells as described previously (17) and two clones were chosen for further analysis. We showed that upon induction of Ninj1 shRNA, the level of Ninj1 transcript was decreased in RKO cells (Fig. S3A). Next, colony-formation assay was performed and showed that Ninj1 knockdown suppressed cell proliferation in RKO cells regardless of treatment with doxorubicin (Fig. S3B). Because p53 is a potent growth suppressor, we reasoned that p53 may be involved in growth suppression induced by Ninj1 knockdown. To address this, the level of p53 protein was measured in RKO cells uninduced or induced to knock down Ninj1, along with treatment of doxorubicin or etoposide for various times. We found that the levels of p53 protein were markedly increased by Ninj1 knockdown upon DNA damage, concomitantly with increased expression of Mdm2 and p21 (Fig. S4 A–C). However, knockdown of Ninj1 had little, if any, effect on the expression of Integrin β4 and β-catenin (Fig. S4D). Conversely, ectopic expression of Ninj1 decreased the levels of p53 protein along with decreased expression of Mdm2 in RKO (Fig. S5).

Because p53 is an apoptosis inducer, we postulate that Ninj1 may mediate p53-dependent apoptosis. To test this, we measured the protein level of PUMA, a p53 target and a potent apoptosis inducer, in RKO cells uninduced or induced to knock down Ninj1. We found that Ninj1 knockdown increased the levels of p53 and PUMA proteins upon treatment with doxorubicin (Fig. S6A) and, consequently, enhanced DNA damage-induced apoptosis (5.5% vs. 15.0%) (Fig. S6B).

To verify the regulation of p53 by Ninj1, we generated ninj1-deficient mice by deleting exon 2 in the ninj1 gene, which contains most of the coding region (Fig. S7A). ninj1^+/− and ninj1^−/− mice were identified by genotyping using PCR (Fig. S7 B and C). Primary mouse embryonic fibroblasts (MEFs) were isolated from ninj1^+/+, ninj1^+/−, and ninj1^−/− embryos. We would like to mention that we were unable to detect mouse Ninj1 protein because of antibody limitation. Thus, the genetic status of the ninj1 gene in ninj1^+/+ and ninj1^−/− MEFs at passage 4 (Fig. 3A) and in ninj1^+/+, ninj1^+/−, and ninj1^−/− MEFs at passage 3 (Fig. 3C) was confirmed by RT-PCR. Importantly, we found that the level of p53 was increased in ninj1^−/− MEFs at passage 4 compared with that in ninj1^+/+ MEFs (Fig. 3B). Similarly, upon treatment with doxorubicin, the level of p53 was increased in ninj1^−/− MEFs at passage 3 and to a lesser extent in ninj1^+/− MEFs, compared with that in ninj1^+/+ MEFs (Fig. 3D, compare lanes 1 and 4 with lanes 2–3 and 5–6, respectively). Consistently, the levels of p53 protein at both the basal and DNA-damage conditions were markedly increased in immortalized ninj1^−/− MEFs at passage 11, compared with that in ninj1^+/+ MEFs (Fig. S8A, compare lanes 1 and 3 with 2 and 4, respectively). To confirm that Ninj1 modulates p53 expression, ninj1 was transiently knocked down in primary MEFs at passages 10 and 13 with two sets of siRNAs against ninj1 along with a scramble siRNA (Fig. 3E and Fig. S8B). Similarly, upon knockdown of ninj1, the level of p53 protein was markedly increased regardless of the passage of MEFs (Fig. 3F and Fig. S8C, compare lanes 1 and 4 with lanes 2–3 and 5–6, respectively).

Fig. 3.

Lack of Ninj1 enhances p53 expression potentially through mRNA translation. (A) Total RNA was isolated from ninj1^+/+ and ninj1^−/− MEFs at passage 4 and the levels of ninj1 and Gapdh transcripts were measured by RT-PCR. (B) The levels of p53 and Actin in ninj1^+/+ and ninj1^−/− MEFs at passage 4 were measured by Western blot analysis. (C) Total RNA was isolated from ninj1^+/+, ninj1^+/−, and ninj1^−/− MEFs at passage 3, and the levels of ninj1 and Gapdh transcripts were measured by RT-PCR. (D) ninj1^+/+, ninj1^+/−, and ninj1^−/− MEFs at passage 3 were treated without (Ctrl) or with doxorubicin (DOX) (200 ng/mL) for 24 h, and the levels of p53 and Actin were analyzed by Western blot analysis. (E) Total RNA was isolated from MEFs (passage 10) transiently transfected with scrambled siRNA (SCR) (50 nM) or siRNA (50 nM) against ninj1 (siNinj1-1 or siNinj1-2) for 48 h, followed by treatment without (Ctrl) or with doxorubicin (200 ng/mL) for 18 h. The levels of ninj1 and actin transcripts were measured by RT-PCR. The siRNA sequence targeting two separate regions in the ninj1 gene is described in Table S2. (F) Experiment was performed as described in E, and the levels of p53 and Actin were analyzed by Western blot analysis. (G) ³⁵S metabolic-labeling assay was performed with ninj1^+/+ and ninj1^−/− MEFs at passage 4 as described in Materials and Methods. The level of newly synthesized p53 protein was measured and quantified by densitometric analysis. NS indicates nonspecific band. (H) The experiment was performed as in G except that RKO cells uninduced (−) or induced (+) to express shRNA against Ninj1 for 3 d were used.

To explore the underlying mechanism by which Ninj1 regulates p53 expression, RT-PCR was performed and showed that the level of p53 transcript was not altered in MEFs upon knockout of Ninj1 and in RKO cells upon knockdown of Ninj1 (Fig. S9 A and B). Moreover, we found that Ninj1 knockdown did not increase, but rather decreased, the half-life of p53 protein in RKO cells (Fig. S9 C and D). These data let us postulate that Ninj1 regulates p53 mRNA translation. To test this, the level of newly synthesized p53 protein in ninj1^+/+ and ninj1^−/− MEFs was measured by ³⁵S-metabolic labeling. Indeed, we found that loss of Ninj1 led to 1.7-fold increase in the level of newly synthesized p53 in MEFs at passage 4 and 1.8-fold at passage 11 (Fig. 3G and Fig. S9E). Similarly, upon knockdown of Ninj1 in RKO cells, the level of newly synthesized p53 protein was increased by 2.2-fold (Fig. 3H). Together, these data suggest that Ninj1 deficiency promotes p53 mRNA translation.

Ninj1 Deficiency Enhances Cellular Senescence and Radiosensitivity in a p53-Dependent Manner.

p53 is known to play a critical role in cellular senescence (1). Thus, to explore the biological significance of Ninj1-mediated p53 expression, we measured cellular senescence in primary ninj1^+/+ and ninj1^−/− MEFs at various passages. We found that from passage 5 to 7, lack of Ninj1 markedly increased the number of senescence-associated β-galactosidase (SA-β-gal)–positive cells under a nonstress condition (Fig. 4A, Upper). Quantitative analysis indicated that the SA-β-gal–positive cells were increased from 13.9% in ninj1^+/+ MEFs to 36.2% in ninj1^−/− MEFs at passage 5, from 21.7 to 40.3% at passage 6, and from 32.7 to 62.72% at passage 7 (Fig. 4A, Lower). We also found that lack of Ninj1 sensitized MEFs at passage 3 to DNA damage-induced premature senescence (50% in ninj1^+/+ MEFs vs. 82% in ninj1^−/− MEFs) (Fig. 4B). Consistent with this, we showed that the level of p53 protein along with p21, PAI-1, and p130 was significantly increased by lack of Ninj1 in MEFs at passage 4 at both the basal and stress conditions (Fig. 4C, compare lanes 1 and 3 with 2 and 4, respectively). We would like to mention that p21, PAI-1, and p130 are well-defined markers for p53-dependent cellular senescence (8, 18).

Fig. 4.

Lack of Ninj1 promotes cellular senescence and radiosensitivity. (A, Upper) ninj1^+/+ and ninj1^−/− MEFs at passage 5–7 were analyzed by SA-β-gal assay. (A, Lower) Quantification of SA-β-gal–positive cells as shown A, Upper. Error bars indicate SD. *P < 0.0001 by two-tailed t test. (B, Upper) ninj1^+/+ and ninj1^−/− MEFs at passage 3 were untreated (Ctrl) or treated with doxorubicin (DOX) (50 ng/mL) for 3 d, followed by SA-β-gal assay as described in Materials and Methods. (B, Lower) Quantification of SA-β-gal–positive cells as shown in B, Upper. Error bars indicate SD. *P < 0.0001 by two-tailed t test. (C) ninj1^+/+ and ninj1^−/− MEFs at passage 4 were untreated (Ctrl) or treated with doxorubicin (200 ng/mL) for 24 h, and the levels of p53, p21, p130, PAI-1, and Actin were measured by Western blot analysis. (D) Kaplan–Meier survival analysis of ninj1^+/+ and ninj1^+/− mice after 8 Gy of whole-body γ-ray. P = 0.043 by log-rank test. (E) ninj1^+/+ and ninj1^+/− mice were irradiated with 8 Gy and then killed 3 h later. Thymus was isolated, and the levels of p53 and Actin were measured by Western blot analysis.

Because p53 is known to be critical for radiosensitivity (19), we explored whether loss of Ninj1 predisposes mice to radiation-induced lethality upon whole-body γ-irradiation. We would like to mention that Ninj1-null mice were short-lived, potentially because of hydrocephalus (Fig. S10). Thus, ninj1^+/− mice (n = 11), which did not exhibit any detectable abnormality by 1 y of age, along with ninj1^+/+ mice (n = 9), were irradiated with 8 Gy of whole-body γ-ray and then monitored for radiation-associated mortality for 22 d. We found that ninj1^+/− mice were more sensitive to radiation-induced death than ninj1^+/+ mice (Fig. 4D). The median survival time was 13 d for ninj1^+/− mice vs. 19 d for ninj1^+/+ mice. The difference in median survival between ninj1^+/− and ninj1^+/+ mice was statistically significant (log-rank test, P = 0.043). Consistent with this, we found that the level of p53 protein in the thymus was higher in ninj1^+/− mice than in ninj1^+/+ mice upon exposure to 8 Gy of γ-ray (Fig. 4E, compare lanes 1 and 3 with 2 and 4, respectively). In addition, we found that the level of ninj1 mRNA in thymus was increased by γ-irradiation compared with unirradiated control thymus (Fig. S11), consistent with the observation that ninj1 can be induced by DNA damage (Fig. 1B). These data suggest that Ninj1 deficiency enhances ionizing radiation-induced p53 expression and lethality in mice.

To further explore the involvement of p53 in Ninj1-mediated biological function, we determined whether cellular senescence induced by loss of Ninj1 (Fig. 4 A–C) requires p53. To test this, we generated Ninj1 and p53 double-knockout MEFs. We showed that ninj1 transcript and p53 protein were absent in ninj1^−/−;p53^−/− MEFs regardless of doxorubicin treatment (Fig. 5 A and B, lanes 3 and 6). We also showed that loss of Ninj1 increased the number of SA-β-gal–positive cells in ninj1^−/− MEFs at passage 5 compared with that in ninj1^+/+ MEFs (Fig. 5 C and D), consistent with the observation in Fig. 4A. Most importantly, cellular senescence induced by loss of Ninj1 was abrogated by p53 knockout regardless of treatment with doxorubicin (Fig. 5 C and D). Consistent with this, the increased levels of senescence markers p130 and p21 by loss of Ninj1 were mitigated by p53 knockout regardless of treatment with doxorubicin (Fig. 5B, compare lanes 2 and 5 with 3 and 6, respectively). These data suggest that Ninj1 regulates cellular senescence in a p53-dependent manner.

Fig. 5.

Lack of Ninj1 enhances cellular senescence in a p53-dependent manner. (A) The levels of ninj1 and Gapdh mRNAs in ninj1^+/+, ninj1^−/−, and ninj1^−/−;p53^−/− MEFs at passage 4 were measured by RT-PCR. (B) ninj1^+/+, ninj1^−/−, and ninj1^−/−;p53^−/− MEFs at passage 5 were untreated (Ctrl) or treated with doxorubicin (DOX) (200 ng/mL) for 18 h, and the levels of p53, p21, p130, and Actin were measured by Western blot analysis. (C) ninj1^+/+, ninj1^−/−, and ninj1^−/−;p53^−/− MEFs at passage 5 were untreated (Ctrl) or treated with doxorubicin (50 ng/mL) for 3 d, followed by SA-β-gal assay. (D) Quantification of SA-β-gal–positive cells as shown in C. Error bars indicate SD. *P < 0.0001 between ninj1^+/+ and ninj1^−/−; **P < 0.0001 between ninj1^−/− and ninj1^−/−;p53^−/− by two-tailed t test. (E) Model for Ninj1 to regulate p53-dependent cellular senescence, cell survival, and radiosensitivity.

Discussion

p53 plays a critical role in growth suppression, and disruption of balanced p53 regulation would lead to tumor development. Thus, the p53 activity needs to be finely tuned in normal tissues. To tightly control the p53 pathway with complex downstream networks, various positive or negative autoregulatory feedback loops are involved in the regulation of the p53 pathway (20), including p53-Mdm2 (21), p53-RNPC1 (22), p53-Wig-1 (23), and p53-Ninj1 in this study. Specifically, we found that p53 can transcriptionally up-regulate Ninj1 by directly binding to the p53-RE in the promoter. In addition, we showed that silencing of Ninj1 enhances p53 mRNA translation, leading to increased p53 expression. Furthermore, we showed that Ninj1 deficiency suppresses cell proliferation but enhances apoptosis and premature cellular senescence in a p53-dependent manner. Finally, we showed that mice deficient in Ninj1 are hypersensitive to ionizing radiation-induced lethality along with increased expression of p53 in thymus. Together, we uncovered a feedback loop between p53 and Ninj1, and our data suggest that Ninj1 plays a role in p53-dependent cell proliferation, premature senescence, and radiosensitivity in vitro and in vivo (Fig. 5E).

Numerous observations suggest that cell–cell or cell–matrix adhesion through adhesion molecules, such as integrins and cadherins, regulates cell survival, tumor invasion, and metastasis (24, 25). Interestingly, the ability of p53 to regulate cell survival in response to DNA damage is also regulated by cell–cell interaction via adhesion molecules (26). In addition, focal adhesion kinase, an integrin- and growth factor-associated tyrosine kinase, is known to promote cell survival through enhancing p53 degradation (27). Although p53 regulates a diverse array of target genes (28), including the gene encoding integrin α5 (29), it is still not clear whether p53 directly regulates the gene encoding a cadherin. Here, we showed that Ninj1, a cell adhesion molecule, is regulated by p53, which, in turn, regulates cell proliferation and premature senescence through modulating p53 expression. Nevertheless, the mechanism by which Ninj1 regulates p53 mRNA translation is not clear but worth further investigation. One possibility is via mechanistic target of rapamycin (mTOR) kinase, which can be activated by growth signals and regulates mRNA translation via assembly of eukaryotic translation initiation factor (eIF) complex (30). For example, inhibition of mTOR decreases p53 translation (31) whereas knockout of protein regulated in development and DNA damage response 1, a stress-induced inhibitor of mTOR1, increases p53 translation (32). The other possibility is that Ninj1-mediated signals may modulate the assembly of translation initiation complex such as eIF4F complex (4E+4G+4A), eIF4B, and poly(A)-binding protein (33). For example, RNPC1, also called Rbm38, is a p53 target, which, in turn, represses p53 mRNA translation by preventing eIF4E from binding to p53 mRNA (22).

Ninj1 is known to be highly expressed in hepatocellular carcinoma (14) and acute lymphoblastic leukemia (15), suggesting that Ninj1 has an oncogenic potential. In this study, we showed that lack of Ninj1 leads to increased expression of p53 and subsequently enhances cellular senescence, growth suppression, and radiation-induced lethality in mice. Considering that p53 plays a critical role in growth suppression and radiation-induced hematopoietic injury (19, 34), our findings indicate that Ninj1 exerts its oncogenic function by repressing p53-dependent growth suppression. Overall, we postulate that as a membrane adhesion molecule, Ninj1 is an ideal target to regulate p53 activity via the p53-Ninj1 loop for cancer therapy.

Materials and Methods

Plasmid constructs, cell line generation with tetracycline-regulated inducible system, ninj1 knockout mouse generation, MEF isolation, γ-irradiation, Northern and Western blot analyses, ChIP assay, luciferase assay, RT-PCR, SA-β-gal assay, ³⁵S metabolic-labeling assay, colony-formation assay, cell fractionation, immunofluorescence analysis, DNA histogram analysis, and statistical analysis are available in SI Materials and Methods. Primers for RT-PCR and ninj1 genotyping are listed in Table S1. siRNA sequences for mouse ninj1 knockdown are listed in Table S2.