Amyloid-B Precursor-Like Protein APLP1 Is a Novel P53transcriptional Target Gene That Augments Neuroblastoma Cell Death Upon Genotoxic Stress
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Oncogene (2007) 26, 7302–7312 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc ORIGINAL ARTICLE Amyloid-b precursor-like protein APLP1 is a novel p53transcriptional target gene that augments neuroblastoma cell death upon genotoxic stress X Tang, M Milyavsky, N Goldfinger and V Rotter Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot, Israel The tumor suppressor p53is a key modulator of the predisposes cells to neoplastic transformation (Ryan cellular stress response, inducing cell-cycle arrest, apop- et al., 2001). tosis, senescence and cell differentiation. To evaluate p53 mediates many of its key functions largely by the further the molecular mechanism underlying p53function, transactivation or transrepression of its target genes the transcriptional profiles of proliferating and senescent (Bargonetti and Manfredi, 2002;Vousden and Lu, WI-38 cells, both wild-type p53 expressers and counter- 2002). Most tumor-derived mutants of p53 are defective parts with an inactivated p53, were compared by DNA in DNA binding and transactivation, supporting a microarray analysis. In particular, the amyloid-b pre- critical role for transactivation in p53’s ability to cursor-like protein 1 (APLP1) is induced in senescent cells suppress neoplasia. A number of p53 responsive genes in a p53-dependent manner. APLP1 was confirmed to be a have been identified and characterized to participate novel transcriptional target of p53by in vivo and in vitro downstream in different p53 cellular pathways, such as characterization of a p53responsive element found in the growth arrest, cellular differentiation or apoptosis. first intron of the APLP1 gene locus. APLP1 knockdown p53-dependent G1 arrest is primarily mediated by experiments demonstrate that APLP1 is required for the p21WAF1, a cyclin-dependent kinase inhibitor (el-Deiry proliferation of fibroblastic and epithelial cells. Moreover, et al., 1992). Many p53 target genes have been proposed depletion of APLP1 expression diminishes stress-induced to play roles in apoptosis, such as Bax, Bid and PUMA apoptosis of neural cells, whereas ectopic APLP1 expres- (p53-upregulated modulator of apoptosis), transmem- sion augments apoptosis. Based on these data, a mechanism brane proteins Perp, Fas and Killer/Dr5, and PIGs is proposed whereby p53-dependent induction of APLP1 is proteins. Bax, Bid and PUMA proteins induced by p53 involved in neural cell death, and which may exacerbate activate an intrinsic pathway to induce cell death. Fas neuronal cell loss in some acute or chronic neurodegenera- and Killer/Dr5 induce cell apoptosis through an tive disorders. extrinsic pathway. The PIGs, encode proteins that are Oncogene (2007) 26, 7302–7312;doi:10.1038/sj.onc.1210542; generally connected to the redox state of the cell, which published online 28 May 2007 may suggest that p53 activation affects the production of reactive oxygen species and causes cell death (Polyak Keywords: p53;APLP1;transcription;apoptosis;senes- et al., 1997). The diverse proapoptotic genes transacti- cence;neuroblastoma and Alzheimer’s disease vated by p53 may suggest that these genes may play a role in different contexts of p53-dependent apoptosis. Many acute or chronic neurodegenerative diseases such as ischemic stroke, Alzheimer’s disease and Parkinson’s disease result from degeneration and death Introduction of specific populations of neurons. Although the genetic and nongenetic factors that initiate these disorders differ The tumor suppressor p53 is a key modulator of the between the various diseases, a biochemical cascade of cellular stress response. In response to DNA damage, events executing the cell death process appears to be hypoxia, viral infection or oncogene activation, the shared (Culmsee and Mattson, 2005). Interestingly, protein is stabilized and activated. This leads to diverse activation of p53 has been observed frequently in acute biological effects, such as cell-cycle arrest, apoptosis, neuronal injury as well as in chronic neurodegenerative senescence, differentiation and anti-angiogenesis (Vousden disorders. Elevated levels of p53 messenger RNA and Lu, 2002). p53 is the most frequently altered (mRNA) and protein have been detected in brain tissue gene in human cancer (Hainaut et al., 1997). Absence derived from patients diagnosed with neurodegenerative of functional p53 allows cellular immortalization and diseases and from animal models with such disorders. This suggests a role for p53 in neuron death during neurodegeneration (Morrison and Kinoshita, 2000). Correspondence: Dr V Rotter, Department of Molecular Cell Biology, Alzheimer’s is a neurodegenerative disease character- The Weizmann Institute of Science, Rehovot 76100, Israel. E-mail: [email protected] ized by senile plaques containing amyloid-b peptide 1À42 Received 19 October 2006;revised 16 March 2007;accepted 18 April (Ab ) deposits, and these deposits are considered to be 2007;published online 28 May 2007 a major cause of the progressive degeneration and death APLP1 is a direct transcriptional target of p53 X Tang et al 7303 of neurons (Mattson, 2004). Neurotoxic Ab1À42 is a the bona fide p53 target gene p21waf1, as judged by our proteolytic product from amyloid precursor protein cluster analysis. (APP), which has two paralogues, namely amyloid-b To confirm that the induction of APLP1 gene precursor-like protein 1 (APLP1) and protein 2 (APLP2). expression in senescent cells is dependent on p53, the APP and APLP2 are expressed in tissues through- levels of APLP1 mRNA in proliferating and senescent out the body, while APLP1 expression appears to be WI-38 cells, as well as in their GSE56-infected counter- limited to the brain (Wasco et al., 1992). Knockout mice parts, were compared by quantitative reverse transcrip- studies have revealed that the functions of APP and tion (qRT)–PCR. As shown in Figure 1b, the level of its two paralogues are redundant (Heber et al., 2000). APLP1 mRNA is significantly induced in wild-type Indeed, both APLP1 and APLP2 have been found to senescent cells (p30V), but not in senescent cells where accumulate in the senile plaques of Alzheimer’s patients’ p53 has been inactivated (p30G). This is in contrast to the brains (Bayer et al., 1997;McNamara et al., 1998). The situation for APP and APLP2, two other members of the specific contribution of APLP1 and APLP2 to the APP family, which exhibit slightly increased mRNA pathogenesis of Alzheimer’s remains unclear. Notably, expression during replicative senescence, but in a p53- in mice overexpressing the Ab1À42, elevated p53 levels independent manner. Immunoblot analysis revealed were observed in those neurons associated with DNA that the APLP1 protein level is induced in senescent strand breaks (LaFerla et al., 1996;de la Monte et al., cells, and that this induction is absent in GSE56- 1997). These results raise the possibility that there are infected cells (Figure 1c). The basal level of APLP1, functional connections between p53 and APP or its different from p21Waf1, is not strongly affected by paralogues and neural cell death. p53. It is noteworthy that APLP1 protein undergoes In this work, the transcriptional profiles were post-translational glycosylation (Eggert et al., 2004), compared of senescent and proliferating WI-38 human giving rise to several bands, as was detected in this primary fibroblasts with their counterparts where p53 study. Western blot and RT–PCR analyses were used had been inactivated by DNA microarray analysis. to confirm that GSE56 infection abrogates p53 activity; Specific genes regulated by p53 during senescence were indeed massive p53 protein stabilization was observed, identified, in particular the APLP1. Various assays but p53 transactivation activity was eliminated com- demonstrated that APLP1 is transactivated directly by pletely, as indicated by reduced RNA and protein p53. In light of the putative role of APLP1 in expression of p21Waf1 (Figures 1b and c). Alzheimer’s disease, it was tested whether APLP1 Together, these experiments indicate that APLP1 influences neural cell death. Ectopic APLP1 expression expression is induced in a p53-dependent manner during was found to enhance neuroblastoma cell death, replicative senescence. whereas its depletion diminished cell death upon genotoxic stress. These results identify a novel p53 target gene that potentially influences neural cell death. APLP1 is induced by genotoxic stress in a p53-dependent manner It is well accepted that activated p53 induces the expression of specific target genes (el-Deiry, 1998). p53 Results can be activated by various stimuli, therefore it was interesting to examine whether genotoxic stress, the Induction of the APLP1 in senescent cells is p53 archetypal p53 inducer, results in elevated APLP1 dependent expression. To that end, APLP1 expression was Normal human primary fibroblasts undergo replicative examined in wild type (infected with control virus) and senescence after a limited number of cell divisions. p53 inactive (GSE56-infected) proliferating WI-38 cells Endogenous p53 can be inactivated using the GSE56 following exposure to doxorubicin (Dox). An increase in polypeptide that works by a negative-dominant mechan- APLP1 mRNA levels is observed only in control cells ism (Ossovskaya et al., 1996). This approach was taken following Dox exposure, and not in the GSE56-infected to inactivate p53 and was found to extend effectively the cells. A similar mRNA pattern is observed for p21Waf1 replicative life span of WI-38