Induction of ID2 Expression by Hypoxia-inducible Factor-1

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. Hypoxiainduced 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.In solid tumors the supply of oxygen and nutrients to some areas is often deprived because of a high rate of cellular proliferation that outpaces the rate of angiogenesis, and structurally abnormal vascularization leading to inadequate intratumoral blood circulation (1). In the resulting hypoxic microenvironment, cancer cells undergo adaptive changes that allow them to survive and even proliferate under hypoxic conditions (2). Mammalian cells, including cancer cells, adapt to hypoxia primarily through a transcriptional response pathway mediated by the hypoxia-inducible factors HIF 1 -1 and HIF-2, which consist of an ␣-subunit (HIF-1␣ or HIF-2␣) and the constitutively expressed transcription factor ARNT/HIF-1␤ (3). At normoxia the ␣-subunit is hydroxylated at critical proline residues by Fe(II)-and O 2 -dependent prolyl hydroxylases (4, 5) and proteasomally degraded via interaction with the von Hippel-Lindau tumor suppressor protein, pVHL (6, 7). In addition to regulation of stability, hydroxylation of an asparagine residue within the ␣-subunit negatively interferes with the function of the transactivation region of HIF under normoxic conditions (8). Hypoxia abrogates both proline and asparagine hydroxylation (4, 8), leading to stable and functional HIFs that can bind regulatory HIF binding sites (HBSs) of target genes involved in maintaining homeostasis, for instance erythropoiet...

Regulation of the Unfolded Protein Response by eIF2Bδ Isoforms

“…We elected to assess UPR activity in a panel predominantly consisting of neuroblastoma cells because the hypoxic downregulation of Id-1, an event dependent on UPR activation, does not occur in several neuroblastoma cell lines (3,28,29). However, our assessment of UPR activity and eIF2B␦ isoform expression was limited to a relatively small number of cell lines.…”

“…The up-regulation of ATF-3 was responsible for the transcriptional repression of Id-1. However, previous reports suggested that Id-1 was not down-regulated in the neuroblastoma cells SK-N-BE(2) and SH-5YSY when rendered hypoxic (28,29). We confirmed this observation and noted that, despite a high basal phosphorylation of eIF2␣, ATF-4, ATF-3, and CHOP were not-up-regulated in response to thapsigargin (a calcium ionophore that permeabilizes the ER membrane) or tunicamycin (which leads to the glycosylation of ER proteins) or when rendered hypoxic.…”

Hypoxic Regulation of Id-1 and Activation of the Unfolded Protein Response Are Aberrant in Neuroblastoma

“…1F). Because Id-2 has been demonstrated to be a HIF-1 target in neuroblastoma cells (37), we sought to determine whether Id-1 is also a HIF-1 target. We constructed a lentivirus expressing a shRNA directed against HIF-1␣.…”

Neuroprotective effect of ultra-low doses of antibodies against S100 protein in neuroblastoma culture during oxygen and glucose deprivation

“…Hypoxia belongs to factors capable to induce in vitro cell differentiation in neuroblastoma cultures [5,7]. Oxygen deficiency directly affects activity of genes responsible for differentiation of neuroblasts [12,14]. The factors stimulating differentiation of neuroblasts exhibit also protective properties during hypoxia and other adverse exposures [7].…”