ΔNp73 Modulates Nerve Growth Factor-Mediated Neuronal Differentiation through Repression of TrkA

p73 belongs to the p53 family, which also includes p53 and p63. These proteins share a significant degree of sequence similarity, especially in the DNA binding domain (20, 26, 27). However, p73 is not a classic Knudson-type tumor suppressor despite its remarkable structural and functional similarities to p53. For example, p73-deficient mice lack a tumor phenotype. Instead, these mice exhibit abnormalities in neuronal development, such as hippocampal dysgenesis, hydrocephalus, loss of Cajal-Retzius neurons, and defects in pheromone sensory pathways (53). In addition, wild-type p73 is frequently up-regulated, but not inactivated, in human primary tumors, suggesting that p73 may promote, rather than suppress, tumor formation (42). These apparent contradictions of p73 activities might be due to the fact that p73 produces two classes of proteins, which are generated from two separate promoters and have distinct or even opposing functions. One, termed TAp73, is transcribed from the upstream promoter and contains an N-terminal activation domain with homology to that in p53. The other one, denoted ΔNp73, is transcribed from the downstream promoter in intron 3 and N-terminally truncated. Consequently, the TAp73 isoforms contain many p53-like properties, such as transactivation of a subset of p53 target genes necessary for induction of cell cycle arrest and apoptosis (4, 11, 15, 18, 24, 46, 49). In contrast, the ΔNp73 isoforms, because they lack the activation domain conserved in p53, are dominant negative towards TAp73 or p53 and thus are thought to have oncogenic potential (32). Consistent with this notion, ΔNp73 has been reported to be frequently up-regulated in human cancers, including breast cancer (54), lung cancer (45), and neuroblastoma (3). Moreover, overexpression of ΔNp73 promotes immortalization in primary cells and malignant transformation in NIH 3T3 fibroblasts (35, 43). In addition to its oncogenic potential, ΔNp73 plays a crucial role in neuronal survival through both p53-dependent and -independent mechanisms (29, 36, 37, 48). The underlying mechanism of these biological phenomena has been hypothesized as follows: ΔNp73, by forming a heterotetramer with TAp73 or competing with TAp73 or p53 to bind to the p53-responsive element (p53-RE), antagonizes TAp73 or p53 since it was thought not to contain an activation domain. However, we have reported recently that ΔNp73β, one of the ΔNp73 isoforms, is active in transactivation and suppression of cancer cell growth (30). In addition, ΔNp73α, the full-length ΔNp73, has been reported to specifically transactivate BTG2 in a p53-dependent manner in neuroblastoma cells but not in other cell types (4, 13). These findings suggest that ΔNp73 retains transcriptional activities, at least in some settings. On the other hand, these findings point out a possibility that in addition to antagonizing TAp73 and p53, ΔNp73 may regulate its own unique target genes for its biological activities. Cellular differentiation, a highly coordinated process, is tightly regulated by a group of key transcriptional factors (25, 33). Precise regulation of this process is very important to ensure the production of properly differentiated cells. The transcriptional activity of p53 plays an important role in several types of cellular differentiation, such as epithelial differentiation, myogenic differentiation, and neuronal differentiation (1, 6, 28). Like p53, TAp73 also plays a critical role in cellular differentiation. Overexpression of TAp73 is sufficient to induce differentiation in neuroblastoma cells (5) and platelet-derived growth factor withdrawal- or thyroid hormone-induced differentiation in oligodendrocyte precursor cells (2). In contrast, overexpression of ΔNp73 inhibits differentiation in oligodendrocyte precursor cells (2) and interferes with multiple developmental programs (23). Moreover, ΔNp73 is likely to be responsible for the neuronal defects in p73-deficient mice since it is the most predominant isoform in the developing mouse brain (37). Such observations support a model whereby ΔNp73 might regulate cellular differentiation or development at least in part by antagonizing other p53 family members. However, whether these observations reflect the role of endogenous ΔNp73 remains unclear. Nerve growth factor (NGF) plays a fundamental role in the neuronal development and maintenance of sympathetic and sensory neurons. The pheochromocytoma PC12 cell line is a well-established model for studying NGF-mediated neuronal differentiation (17). Upon NGF treatment, PC12 cells cease proliferation, exhibit somatic hypertrophy, and acquire neurites (47). The signal is initiated when NGF binds to its high-affinity receptor, TrkA, which is then phosphorylated and dimerized to activate its downstream cascades (22). Thus, the PC12 cell line is an ideal system with which to investigate the role of ΔNp73 in NGF-mediated neuronal differentiation. In this study, we show that overexpression of ΔNp73α or ΔNp73β inhibits, whereas knockdown of ΔNp73 promotes, NGF-mediated neuronal differentiation. Moreover, we show that endogenous ΔNp73 is gradually diminished in PC12 cells upon NGF treatment. To uncover the underlying mechanism, we show that ΔNp73, through binding to the p53-RE in the TrkA promoter, transrepresses TrkA expression, which then inhibits the NGF-mediated mitogen-activated protein kinase (MAPK) pathway. Conversely, we showed that the steady-state level of TrkA is increased in PC12 cells upon knockdown of ΔNp73. Finally, we showed that ΔNp73, by recruiting histone deacetylase 1 (HDAC1) and HDAC2 to the TrkA promoter, transcriptionally represses TrkA expression, which can be relieved by trichostatin A (TSA), an HDAC inhibitor.