Oncogene (2002) 21, 2455 ± 2465 ã 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc TAp63g (p51A) and dNp63a (p73L), two major isoforms of the p63 gene, exert opposite eects on the vascular endothelial growth factor (VEGF) gene expression Makoto Senoo1, Yasuko Matsumura1 and Sonoko Habu*,1 1Department of Immunology, Tokai University School of Medicine, Bouseidai, Isehara-city, Kanagawa 259-1193, Japan Tumor suppressor p53 has been shown to repress 1995). The VEGF mRNA is expressed predominantly expression of vascular endothelial growth factor in tumor cells and stromal cells, whereas the mRNA (VEGF), an endothelial cell-speci®c mitogen and a key for the VEGF receptors is expressed and upregulated mediator of tumor angiogenesis. The p63 gene, recently in tumor endothelial cells (Suzuki et al., 1996). This identi®ed as a p53-relative, encodes multiple isoforms expression pro®le hypothesizes the VEGF to be a with structural and functional similarities and dierences primary paracrine regulator of tumor angiogenesis. from p53. In this study, we show the evidence that the Several groups have shown that VEGF expression is two major isoforms of the p63 gene, TAp63g (p51A) and induced by exposing cultured cells to 1% O2, CoCl2 or dNp63a (p73L), represses and upregulates VEGF desferrioxamine, and induction can be blocked by expression, respectively, on transcription and protein treating hypoxic cells with cycloheximide (Gleadle et levels. Transient transfection assays show that a al., 1995; Goldberg and Schneider, 1994; Shweiki et al., hypoxia-inducible factor (HIF) 1 binding site within the 1992). Recent studies have demonstrated that hypoxia VEGF promoter region is responsible for both upregula- increases VEGF transcription in several dierent cell tion and repression by dNp63a and by TAp63g, types (Finkenzeller et al., 1995; Ikeda et al., 1995; Levy respectively, of the VEGF promoter activity. We also et al., 1995; Liu et al., 1995). Thus, transcriptional show that TAp63g targets HIF1a for promoting control of the VEGF mRNA might be one of the proteasomal degradation but that dNp63a targets major steps that regulate VEGF expression. HIF1a for stabilization. Mammalian two-hybrid assays Hypoxia-inducible factor 1 (HIF1) is a heterodimeric show that HIF1a-dependent transcription is repressed by transcription factor that regulates transcription of TAp63g as well as by p53, whereas it is upregulated by several genes involved in oxygen homeostasis (Wenger dNp63a in collaboration with a transcription coactivator and Gassmann, 1997; Semenza, 1998, 1999). HIF1 p300. Our data also show that dNp63a acts as a consists of two subunits, HIF1a and HIF1b (ARNT dominant-negative reagent toward both p53- and (aryl hydrocarbon receptor nuclear translocator)), TAp63g-mediated degradation of HIF1a and repression which belong to a subfamily of basic helix ± loop ± of HIF1a-dependent transcription. These results suggest helix ± PAS domain (bHLH ± PAS) transcription fac- that p63 is involved in the regulation of the VEGF gene tors (Wang et al., 1995). While hypoxia prevents expression and that modulation of VEGF expression by proteasomal degradation of HIF1a, the expression of TAp63g and dNp63a is closely correlated with their HIF1b is thought to be oxygen-independent (Huang et distinct roles on the regulation of HIF1a stability. al., 1998; Kallio et al., 1999; Salceda and Caro, 1997). Oncogene (2002) 21, 2455 ± 2465. DOI: 10.1038/sj/ The binding of HIF1a to the target DNA site, in onc/1205330 conjunction with HIF1b, leads to transcriptional activation of the downstream genes including the Keywords: p63; p51; p73L; p300; CBP; VEGF; HIF1a VEGF gene. The studies on HIF1a-de®cient mice have suggested a key role of HIF1a on the VEGF-mediated angiogenesis in response to hypoxia (Iyer et al., 1998; Kotch et al., 1999). In supporting this notion, both HIF1a and VEGF are overexpressed in a wide variety Introduction of human cancers (Folkman, 1997; Zong et al., 1999). The tumor suppressor p53 is suggested to have a Vascular endothelial growth factor (VEGF) has been negative role on VEGF expression (Bouvet et al., 1998; described as a key mediator of angiogenesis and Fontanini et al., 1998; Mukhopadhyay et al., 1995). At neovascularization (Martiny-Baron and Marme, the molecular level, it has been shown that p53 inhibits HIF1 activity by targeting the HIF1a subunit for ubiquitination and proteasomal degradation (Ravi et al., 2000). Conversely, both loss of p53 and forced *Correspondence: S Habu; E-mail: [email protected] Received 4 October 2001; revised 10 January 2002; accepted 11 expression of HIF1a in p53-expressing tumor cells have January 2002 been shown to augment VEGF expression in response p63 modulates VEGF expression M Senoo et al 2456 to hypoxia (Ravi et al., 2000). In this point of view, it is reasonable to consider p53 as a critical regulator of angiogenesis through regulating HIF1a stability. We and others have recently identi®ed a new p53 family member, termed p63/p51/p73L/p40/KET gene (Osada et al., 1998; Schmale and Bamberger, 1997; Senoo et al., 1998; Trink et al., 1998; Yang et al., 1998). This gene, referred to as the p63 gene hereafter, encodes multiple isoforms with structural and func- tional similarities and dierences from p53. For example, TAp63g(p51A), an isoform harboring a transactivation domain at its amino-terminus, stimu- lates several previously identi®ed p53 target gene promoters such as p21waf1/cip1, mdm2 and bax (Dohn et al., 2001; Shimada et al., 1999; Osada et al., 1998; Yang et al., 1998). It is also suggested that TAp63g induces apoptosis and growth suppression in a manner similar to p53 (Osada et al., 1998; Yang et al., 1998). The p63 gene encodes another major isoform called dNp63a (p73L), an amino terminal-deleted isoform, Figure 1 Repression by TAp63g and activation by dNp63a of which can potentially work as a dominant negative VEGF promoter activity. MEF and H1299 cells were transiently reagent toward both TAp63g and p53 (Yang et al., transfected with 0.2 mg of VEGF ± Luc reporter and 1.0 mgof 1998; Senoo et al., 2001a,b). Very recently, we and pCMV ± p53, pCMV ± TAp63g, pCMV ± dNp63a or the control others have shown that the expression of TAp63g is expression vector pcDNA3. Forty-eight hours after transfection, cells were assayed for luciferase activity. Data shown are frequently lacked and, in turn, dNp63a is more mean+s.d. from triplicate analysis dominantly expressed in a variety of tumors compared to TAp63g (Crook et al., 2000; Hibi et al., 2000; Nylander et al., 2000; Park et al., 2000; Senoo et al., 2001a). Unlike p53, the p63 gene is not frequently or dNp63a expression vector into wild-type mouse mutated in human cancers (Nishi et al., 1999; Park et embryonic ®broblast (MEF) cells and human non- al., 2000; Sunahara et al., 1999). From these small cell lung carcinoma-derived H1299 cells. As characteristics of p63, it is not unreasonable to shown in Figure 1, VEGF ± Luc reporter activity was speculate that TAp63g and dNp63a may play tumor decreased by more than 60% in the p53-transfected suppressive and oncogenic properties, respectively. MEF and H1299 cells as compared to the vector- In this study, we examined the possible roles of transfected cells. Although less signi®cant, VEGF ± Luc TAp63g and dNp63a in the regulation of VEGF reporter activity was also decreased by approximately expression. We show the evidence that VEGF expres- 40% in both TAp63g-transfected MEF and H1299 sion was repressed by TAp63g, whereas it was cells. In contrast, VEGF ± Luc reporter activity was conversely upregulated by dNp63a. We also show that surprisingly upregulated by more than 155% of the these dual functions of the two p63 isoforms were control in both dNp63a-transfected MEF and H1299 closely correlated with their distinct functions in the cells. The repression by TAp63g and activation by regulation of HIF1a stability. These data point to two dNp63a of the VEGF ± Luc reporter activity were in a distinct regulatory VEGF pathways by the p63 gene manner of dose-dependent (data not shown). H1299 products: a possible tumor suppressor signal by cells harbor a homozygous deletion at the p53 gene TAp63g suppressing VEGF, and an oncogenic one by locus (Mitsudomi et al., 1992) and does not express dNp63a enhancing VEGF production. detectable amounts of endogenous p63 and p73 (Dohn et al., 2001). Therefore, we speculate that transcrip- tional activation of the VEGF ± Luc reporter by dNp63a was not due to the dominant negative eect Results of dNp63a toward either endogenous p53 or transacti- vating versions of p73 and p63. Rather, we prefer to Repression by TAp63g and activation by dNp63a of interpret that dNp63a may modulate some cellular VEGF expression factor(s) that are important in the regulation of VEGF Since p53 is suggested to repress VEGF expression promoter activity (see below). through inactivation of the VEGF gene promoter To determine whether TAp63g and dNp63a sub- (Mukhopadhyay et al., 1995), we examined potential stantially represses and activates endogenous VEGF eects of TAp63g (p51A) and dNp63a (p73L), the two expression, we established stable H1299 cell lines which major isoforms of the p53-related p63 gene, on the inducibly express p53, TAp63g and dNp63a by Cre- VEGF promoter activity (Figure 1). We cotransfected loxP system upon infection with Cre-producing VEGF ± Luc bearing the promoter region 71217 to adenovirus AxCANCre (Figure 2a). These cell lines +370 (Shima et al., 1996) with wild-type p53, TAp63g showed equivalent cell growth and produced an Oncogene p63 modulates VEGF expression M Senoo et al 2457 VEGF protein expression level was conversely upregu- lated by 113% of the control level in dNp63a- expressing cells (Figure 2b). This increase was relatively moderate but was statistically signi®cant (P50.05, t-test). Consistent with the transient transfection experiments using VEGF ± Luc reporter (Figure 1), these results suggest that TAp63g and dNp63a have negative and positive roles on endogenous VEGF expression, respectively, even under normoxic condi- tion.