Induction of Neuronal Differentiation by p73 in a Neuroblastoma Cell Line

Barbara Tanno,Daniela Barcaroli,Antonio Costanzo,Margherita Annicchiarico-Petruzzelli,Vincenzo De Laurenzi,Gerry Melino,Marco Ranalli,Massimo Levrero,Maria Valeria Catani,Giuseppe Raschellá

doi:10.1074/jbc.275.20.15226

Abstract

The p53-related p73 and p63 genes encode proteins that share considerable structural and functional homology with p53. Despite similarities, their deletion in mice has different outcomes, implying that the three genes may play distinct roles in vivo. Here we show that endogenous p73 levels increase in neuroblastoma cells induced to differentiate by retinoic acid and that exogenously expressed p73, but not p53, is sufficient to induce both morphological (neurite outgrowth) and biochemical (expression of neurofilaments and neural cell adhesion molecule (N-CAM); down-regulation of N-MYC and up-regulation of pRB) markers of neuronal differentiation. This activity is shared, to different extents, by all p73 isoforms, whereas the transcriptionally inactive mutants of p73 isoforms are ineffective. Conversely, blockage of endogenous p73 isoforms with a dominant negative p73 results in the abrogation of retinoid-induced N-CAM promoter-driven transcription. Our results indicate that the p73 isoforms activate a pathway that is not shared by p53 and that is required for neuroblastoma cell differentiation in vitro.

Highlights

The p53-related p73 and p63 genes encode proteins that share considerable structural and functional homology with p53
Our results indicate that the p73 isoforms activate a pathway that is not shared by p53 and that is required for neuroblastoma cell differentiation in vitro
N1E-115 cells were transfected with 1.5 ␮g of p53 or the different p73 isoform expression vectors, together with 0.5 ␮g of a LacZ reporter or a GFP-spectrin fusion protein expression vector [18] in order to follow the fate of transfected cells (Fig. 2)

Summary

Introduction

The p53-related p73 and p63 genes encode proteins that share considerable structural and functional homology with p53. We show that endogenous p73 levels increase in neuroblastoma cells induced to differentiate by retinoic acid and that exogenously expressed p73, but not p53, is sufficient to induce both morphological (neurite outgrowth) and biochemical (expression of neurofilaments and neural cell adhesion molecule (N-CAM); down-regulation of N-MYC and up-regulation of pRB) markers of neuronal differentiation This activity is shared, to different extents, by all p73 isoforms, whereas the transcriptionally inactive mutants of p73 isoforms are ineffective. Neurons from p53 null mice and neurons from wild type mice cultured with p53 antisense oligonucleotides in vitro show accelerated spontaneous differentiation [14] This suggests the hypothesis that loss of the p53 heterodimeric partner of p73 may liberate more uncomplexed or homodimerized p73 and allow expression of its differentiation-inducing function. We have investigated the role of p73 in the differentiation of neuroblastoma cell lines, a well studied model of neuronal differentiation and death [15,16,17]

Methods

Results

Conclusion