title: Induction of ID2 expression by hypoxia-inducible factor-1: a role in dedifferentiation of hypoxic neuroblastoma cells.
abstract: ID (inhibitor of differentiation/DNA binding) proteins, frequently deregulated in advanced human malignancies, can participate in multiple fundamental traits of cancer, such as block of differentiation, increased proliferation, tissue invasiveness, and angiogenesis. We have previously demonstrated that hypoxia decreases expression of neuronal marker genes in neuroblastoma, but induces genes expressed in the neural crest, such as ID2. Because of its involvement in normal neural crest development and its ability to inhibit proneuronal bHLH proteins, the hypoxic induction of ID2 was of particular interest. Here we report fast induction kinetics of ID2 expression in hypoxic neuroblastoma cells. The up-regulation of ID2 was abolished by addition of actinomycin D, implicating a hypoxia-driven transcriptional mechanism. Analyzing the ID2 promoter revealed several potential binding sites for hypoxia-inducible factors. Subsequent electrophoretic mobility shift and chromatin immunoprecipitation assays demonstrated two functional HIF-1 binding sites within ID2 gene regulatory sequences located at -725 and -1893 relative to the transcriptional initiation point. In transfection assays, DNA constructs of the ID2 promoter, including the functional HIF-1 binding sites, induced luciferase reporter activity in a HIF-1-specific manner. These observations demonstrate that ID2 is actively engaged by hypoxia and represents a novel HIF-1 target. Hypoxia-induced ID2 expression could play a significant role in the previously observed dedifferentiation of hypoxic neuroblastoma cells, which in a clinical setting could lead to less mature and more aggressive tumors.
authors.affiliation: Department of Laboratory Medicine, Division of Molecular Medicine, Lund University, University Hospital MAS, S-205 02 Malmö, Sweden.
authors.forename: Tobias
authors.initials: T
authors.lastname: Löfstedt
authors.forename: Annika
authors.initials: A
authors.lastname: Jögi
authors.forename: Mikael
authors.initials: M
authors.lastname: Sigvardsson
authors.forename: Katarina
authors.initials: K
authors.lastname: Gradin
authors.forename: Lorenz
authors.initials: L
authors.lastname: Poellinger
authors.forename: Sven
authors.initials: S
authors.lastname: Påhlman
authors.forename: Håkan
authors.initials: H
authors.lastname: Axelson
mesh_terms: D024506:5' Flanking Region / Q000502:physiology; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001943:Breast Neoplasms; D004268:DNA-Binding Proteins / Q000235:genetics*; D005786:Gene Expression Regulation / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D000860:Hypoxia / Q000235:genetics / Q000503:physiopathology*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D051798:Inhibitor of Differentiation Protein 1; D051797:Inhibitor of Differentiation Protein 2; D008156:Luciferases / Q000235:genetics; D016296:Mutagenesis; D009447:Neuroblastoma; D016716:PC12 Cells; D011401:Promoter Regions, Genetic / Q000502:physiology; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D012097:Repressor Proteins / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism*; D015533:Transcriptional Activation / Q000502:physiology; D015854:Up-Regulation
chemical_list: D004268:DNA-Binding Proteins; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; C497453:ID1 protein, human; C497454:ID1 protein, rat; C497450:ID2 protein, human; C497451:Id2 protein, rat; D051798:Inhibitor of Differentiation Protein 1; D051797:Inhibitor of Differentiation Protein 2; D012333:RNA, Messenger; D012097:Repressor Proteins; D014157:Transcription Factors; D008156:Luciferases