TAp73 Protein Stability Is Controlled by Histone Deacetylase 1 via Regulation of Hsp90 Chaperone Function

Histone deacetylases (HDACs) play important roles in fundamental cellular processes, and HDAC inhibitors are emerging as promising cancer therapeutics. p73, a member of the p53 family, plays a critical role in tumor suppression and neural development. Interestingly, p73 produces two classes of proteins with opposing functions: the full-length TAp73 and the N-terminally truncated ΔNp73. In the current study, we sought to characterize the potential regulation of p73 by HDACs and found that histone deacetylase 1 (HDAC1) is a key regulator of TAp73 protein stability. Specifically, we showed that HDAC1 inhibition by HDAC inhibitors or by siRNA shortened the half-life of TAp73 protein and subsequently decreased TAp73 expression under normal and DNA damage-induced conditions. Mechanistically, we found that HDAC1 knockdown resulted in hyperacetylation and inactivation of heat shock protein 90, which disrupted the interaction between heat shock protein 90 and TAp73 and thus promoted the proteasomal degradation of TAp73. Functionally, we found that down-regulation of TAp73 was required for the enhanced cell migration mediated by HDAC1 knockdown. Together, we uncover a novel regulation of TAp73 protein stability by HDAC1-heat shock protein 90 chaperone complex, and our data suggest that TAp73 is a critical downstream mediator of HDAC1-regulated cell migration.     

GLS2 is transcriptionally regulated by p73 and contributes to neuronal differentiation

The amino acid Glutamine is converted into Glutamate by a deamidation reaction catalyzed by the enzyme Glutaminase (GLS). Two isoforms of this enzyme have been described, and the GLS2 isoform is regulated by the tumor suppressor gene p53. Here, we show that the p53 family member TAp73 also drives the expression of GLS2. Specifically, we demonstrate that TAp73 regulates GLS2 during retinoic acid-induced terminal neuronal differentiation of neuroblastoma cells, and overexpression or inhibition of GLS2 modulates neuronal differentiation and intracellular levels of ATP. Moreover, inhibition of GLS activity, by removing Glutamine from the growth medium, impairs in vitro differentiation of cortical neurons. Finally, expression of GLS2 increases during mouse cerebellar development. Although, p73 is dispensable for the in vivo expression of GLS2, TAp73 loss affects GABA and Glutamate levels in cortical neurons. Together, these findings suggest a role for GLS2 acting, at least in part, downstream of p73 in neuronal differentiation and highlight a possible role of p73 in regulating neurotransmitter synthesis.     

p73-dependent expression of DAN during cisplatin-induced cell death and osteoblast differentiation

We previously reported that DAN, a founding member of the DAN family of secreted proteins, acts as an inhibitor of cell cycle progression and is closely involved in retinoic acid-induced neuroblastoma differentiation. In this study, we found that DAN as well as p73, the recently identified p53 family member, was up-regulated during osteoblast differentiation. Additionally, the expression of DAN was increased in response to cisplatin-induced cell death of neuroblastoma SH-SY5Y cells. Consistent with the previous reports, p73 was accumulated after the treatment with cisplatin. Intriguingly, we found a putative p53/p73-binding site in the 5'-upstream region of the human DAN gene. A luciferase reporter assay and an in vitro DNA-binding experiment revealed that this canonical p53/p73-binding site was a functional responsive element and was specific for p73. Our results suggest that there exists a functional association between DAN and p73 during osteoblast differentiation as well as cisplatin-induced cell death.     

Up-regulation of NFkappaB-responsive gene expression by DeltaNp73alpha in p53 null cells

Transactivation domain (TAD)-truncated p73, DeltaNp73, associates with p53, resulting in suppression of p53's functions. Using p53 null cell lines, we examined whether or not DeltaNp73 can regulate gene expression in a p53-independent manner. When DeltaNp73alpha was co-transfected with a luciferase reporter plasmid with various enhancer elements, NFkappaB-responsive luciferase gene expression was selectively up-regulated by DeltaNp73alpha, but not by other p73-isoforms with TAD and DeltaNp73beta. Deletion of the TAD endowed p73alpha with the ability to enhance the responsive gene's expression, but deletion of the N-terminal proline-rich domain (PRD) rendered the TAD-deleted p73alpha inactive. Considering the inability of DeltaNp73beta, which is the C-terminus-truncated form of DeltaNp73alpha, to function, these results indicate that both the PRD and C-terminus are necessary for DeltaNp73alpha to can activate NFkappaB-responsive luciferase expression. Over-expression of p53 suppressed the TAD-truncated p73alpha-mediated luciferase expression, suggesting that p53 interferes with the TAD-truncated p73alpha-mediated activation of NFkappaB. Inhibitors for NFkappaB activation reduced the TAD-truncated p73alpha-dependent NFkappaB-responsive gene expression, indicating that TAD-truncated p73alpha activates NFkappaB as does TNFalpha. In addition to the results obtained in the reporter gene assay, TAD-truncated p73alpha stimulated the translocation of NFkappaB to the nucleus and the expression of an endogenous NFkappaB-responsive gene, Bcl-XL. Taken together, these results demonstrate that TAD-truncated p73alpha can activate NFkappaB.     

TAp73调节肺腺癌细胞的血管生成能力依赖P53状态

TAp73是P53家族的一员,能够调节肿瘤的生成、侵袭和转移。但是,TAp73调节肿瘤血管生成的作用备受争议。本研究将外源TAp73转染至P53基因表达状态不同的两株肺腺癌细胞系H1299(P53-null)和A549(wt P53)中,观察TAp73对肿瘤血管生成的作用并探讨与P53基因的关系。首先,使用RT-PCR和Western印迹验证转染效率。细胞划痕实验表明,TAp73在A549细胞中促进细胞迁移,而在H1299细胞中抑制细胞迁移。体外HUVEC血管形成结果表明,TAp73在A549细胞中促进细胞血管形成,而在H1299细胞中抑制细胞血管形成。同时,血管生成抑制蛋白1（VASH1）的表达水平,也分别升高或降低。 本文研究结果表明,TAp73对肺腺癌细胞血管生成的作用依赖于P53基因的状态：在野生型P53基因存在时,TAp73促进血管生成,而在缺失P53基因的情况下,TAp73抑制血管生成。本研究对于TAp73作为肿瘤的潜在治疗靶点具有重要意义。     

Regulation of HSF1-responsive gene expression by N-terminal truncated form of p73alpha

DNp73 is a transactivation domain (TAD)-truncated form of p73. The ability of DNp73alpha to regulate gene expression was examined using reporter assays with luciferase gene constructs. Among various promoter-regulated reporter genes tested, heat shock factor (HSF)-responsive gene expression was selectively activated by DNp73alpha, but not by other p73-isoforms with TAD and DNp73beta. Deletion of TAD endowed p73alpha with the ability to activate HSF-responsive gene expression, but deletion of N-terminal proline-rich domain (PRD) rendered both DNp73alpha and the TAD-deleted p73alpha inactive. Considering the inability of DNp73beta, which is the C-terminus-truncated form of DNp73alpha, to function, these results indicate that both the PRD and C-terminus are necessary for DNp73alpha to be able to activate the HSF-dependent gene expression. In addition to the reporter gene expression, both DNp73alpha and TAD-deleted p73alpha activated the expression of an endogenous gene, hsp70, corresponding with an increase in the active form of HSF1. Taken together, these results demonstrate that TAD-truncated p73alpha can activate HSF-dependent gene expression via induction of active HSF1.     

A critical role of glucose-6-phosphate dehydrogenase in TAp73-mediated cell proliferation

The pentose phosphate pathway (PPP) provides ribose and NADPH that support biosynthesis and antioxidant defense. Our recent findings suggest that the p53-related protein TAp73 enhances the PPP flux. TAp73 stimulates the expression of glucose-6-phophate dehydrogenase (G6PD), the rate-limiting enzymes of the PPP. Through this regulation, TAp73 promotes the accumulation of macromolecules and increases cellular capability to withstand oxidative stresses. TAp73 also regulates other metabolic enzymes, and the relative importance of these targets in TAp73-mediated cell growth is not well understood. Here we show that, like in other cell lines, TAp73 is required for supporting proliferation and maintaining the expression of G6PD in the human lung cancer H1299 cells. Restoration of G6PD expression almost fully rescues the defects in cell growth caused by TAp73 knockdown, suggesting that G6PD is the major proliferative target of TAp73 in these cells. G6PD expression is elevated in various tumors, correlating with the upregulation of TAp73. These results indicate that TAp73 may function as an oncogene, and that G6PD is likely a focal point of regulation in oncogenic growth.     

Hypoxia-induced DNp73 stabilization regulates Vegf-A expression and tumor angiogenesis similar to TAp73

P73, the homolog of p53, exists in 2 major forms: either as a pro-apoptotic TAp73 or an amino-terminally truncated DNp73, the latter lacking the first transactivation domain. While TAp73s tumor suppressive functions have been established, DNp73 is an anti-apoptotic protein conferring chemoresistance and is associated with poor survival. However, both forms are variably overexpressed in many human cancers. In this context, we have recently demonstrated that TAp73 is stabilized by hypoxia, a tumor-relevant condition that is associated with cell survival, via HIF-1α-mediated suppression of Siah1 E3 ligase that degrades TAp73. Consequently, hypoxic signals lead to TAp73-mediated activation of several angiogenic genes and blood vessel formation, thereby supporting tumorigenesis. We show here that, similar to TAp73, DNp73 is stabilized by hypoxia in a HIF-1α-dependent manner, which otherwise is degraded by Siah1. Moreover, DNp73 is capable of inducing the expression of Vegf-A, the prototypic angiogenic gene, and loss of DNp73 expression results in reduction in tumor vasculature and size. These data therefore indicate a common mode of regulation for both p73 forms by hypoxia, resulting in the promotion of angiogenesis and tumor growth, highlighting common functionality of these antagonistic proteins under specific physiological contexts.     

p53 directly suppresses BNIP3 expression to protect against hypoxia-induced cell death

Hypoxia stabilizes the tumour suppressor p53, allowing it to function primarily as a transrepressor; however, the function of p53 during hypoxia remains unclear. In this study, we showed that p53 suppressed BNIP3 expression by directly binding to the p53-response element motif and recruiting corepressor mSin3a to the BNIP3 promoter. The DNA-binding site of p53 must remain intact for the protein to suppress the BNIP3 promoter. In addition, taking advantage of zebrafish as an in vivo model, we confirmed that zebrafish nip3a, a homologous gene of mammalian BNIP3, was indeed induced by hypoxia and p53 mutation/knockdown enhanced nip3a expression under hypoxia resulted in cell death enhancement in p53 mutant embryos. Furthermore, p53 protected against hypoxia-induced cell death mediated by p53 suppression of BNIP3 as illustrated by p53 knockdown/loss assays in both human cell lines and zebrafish model, which is in contrast to the traditional pro-apoptotic role of p53. Our results suggest a novel function of p53 in hypoxia-induced cell death, leading to the development of new treatments for ischaemic heart disease and cerebral stroke.     

Upregulation of BNIP3 promotes apoptosis of lung cancer cells that were induced by p53

It has been shown that p53 induces cell apoptosis and the Bcl-2 family plays key roles in this process. However, the molecular mechanism of p53 apoptotic pathway is still unclear. Here, we show that overexpression of exogenous wild-type p53 induced apoptosis in lung cancer cells and high metastasis potential cells had a faster rate of apoptosis than low metastasis potential cells. The expression of pro-apoptotic gene BNIP3 was increased significantly both in Anip973 and 95D cell lines which have high metastasis ability, but not AGZY83-a or little increased in 95C cell lines which possess low metastasis ability. Overexpression of BNIP3 increases apoptotic rate induced by p53 in AGZY83-a cells. Blocking the expression of BNIP3 by siRNA in Anip973 cells decreased apoptotic rate mediated by p53. Taken together, these data suggest that high level expression of BNIP3 mediated rapid apoptosis that was triggered by p53 in lung cancer cells.