title: TAp73 isoforms antagonize Notch signalling in SH-SY5Y neuroblastomas and in primary neurones.
abstract: p73, like Notch, has been implicated in neurodevelopment and in the maintenance of the mature central nervous system. In this study, by the use of reporter-gene assays, we demonstrate that C-promoter binding factor-1 (CBF-1)-dependent gene transcription driven by the Notch-1 intracellular domain (N1(ICD)) is potently antagonized by exogenously expressed transactivating (TA) p73 splice variants in SH-SY5Y neuroblastomas and in primary neurones. Time course analysis indicated that the inhibitory effects of TAp73 are direct and are not mediated via the product of a downstream target gene. We found that endogenous TAp73 stabilized by either c-Abl or cisplatin treatment also potently antagonized N1(ICD)/CBF-1-dependent gene transcription. Furthermore, western blotting revealed that exogenous TAp73 suppressed endogenous hairy and enhancer of split-1 (HES-1) protein levels and antagonized the increase in HES-1 protein induced by exogenous N1(ICD) expression. Evidence of a direct physical interaction between N1(ICD) and TAp73alpha was demonstrated by co-immunoprecipitation. Using Notch deletion constructs, we demonstrate that TAp73alpha binds the N1(ICD) in a region C-terminal of aa 2094. Interestingly, DeltaNp73alpha and TAp73alpha(R292H) also co-purified with N1(ICD), but neither inhibited N1(ICD)/CBF-1-dependent transcription. This suggests that an intact transactivation (TA) domain and the ability to bind DNA are necessary for TAp73 to antagonize Notch signalling. Finally we found that TAp73alpha reversed the N1(ICD)-mediated repression of retinoic acid-induced differentiation of SH-SY5Y neuroblastomas, providing functional evidence for an inhibitory effect of TAp73alpha on notch signalling. Collectively, these findings may have ramifications for neurodevelopment, neurodegeneration and oncogenesis.
authors.affiliation: King's College London, MRC Centre for Neurodegenerative Research, Institute of Psychiatry, London, UK.
authors.forename: Claudie
authors.initials: C
authors.lastname: Hooper
authors.forename: Mahvash
authors.initials: M
authors.lastname: Tavassoli
authors.forename: J Paul
authors.initials: JP
authors.lastname: Chapple
authors.forename: Dafe
authors.initials: D
authors.lastname: Uwanogho
authors.forename: Richard
authors.initials: R
authors.lastname: Goodyear
authors.forename: Gerry
authors.initials: G
authors.lastname: Melino
authors.forename: Simon
authors.initials: S
authors.lastname: Lovestone
authors.forename: Richard
authors.initials: R
authors.lastname: Killick
mesh_terms: D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics; D015153:Blotting, Western; D001932:Brain Neoplasms / Q000503:physiopathology*; D018797:Cell Cycle Proteins / Q000235:genetics; D002454:Cell Differentiation / Q000187:drug effects; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000502:physiology*; D006801:Humans; D047468:Immunoprecipitation; D008156:Luciferases / Q000378:metabolism; D018613:Microscopy, Confocal; D009447:Neuroblastoma / Q000503:physiopathology*; D009474:Neurons / Q000502:physiology*; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000502:physiology*; D051881:Receptor, Notch1 / Q000502:physiology*; D012097:Repressor Proteins / Q000235:genetics; D015398:Signal Transduction / Q000502:physiology; D014162:Transfection; D014212:Tretinoin / Q000037:antagonists & inhibitors / Q000494:pharmacology; D025521:Tumor Suppressor Proteins / Q000096:biosynthesis / Q000502:physiology*
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D008156:Luciferases; D009687:Nuclear Proteins; D051881:Receptor, Notch1; D012097:Repressor Proteins; D014212:Tretinoin; D025521:Tumor Suppressor Proteins; C485631:HEY1 protein, human; C077478:HES5 protein, human