In addition to controlling a switch to glycolytic metabolism and induction of erythropoiesis and angiogenesis, hypoxia promotes the undifferentiated cell state in various stem and precursor cell populations. Here, we show that the latter process requires Notch signaling. Hypoxia blocks neuronal and myogenic differentiation in a Notch-dependent manner. Hypoxia activates Notch-responsive promoters and increases expression of Notch direct downstream genes. The Notch intracellular domain interacts with HIF-1alpha, a global regulator of oxygen homeostasis, and HIF-1alpha is recruited to Notch-responsive promoters upon Notch activation under hypoxic conditions. Taken together, these data provide molecular insights into how reduced oxygen levels control the cellular differentiation status and demonstrate a role for Notch in this process.

In neuroblastoma specimens, HIF-2alpha but not HIF-1alpha is strongly expressed in well-vascularized areas. In vitro, HIF-2alpha protein was stabilized at 5% O2 (resembling end capillary oxygen conditions) and, in contrast to the low HIF-1alpha activity at this oxygen level, actively transcribed genes like VEGF. Under hypoxia (1% O2), HIF-1alpha was transiently stabilized and primarily mediated acute responses, whereas HIF-2alpha protein gradually accumulated and governed prolonged hypoxic gene activation. Knockdown of HIF-2alpha reduced growth of neuroblastoma tumors in athymic mice. Furthermore, high HIF-2alpha protein levels were correlated with advanced clinical stage and high VEGF expression and predicted poor prognosis in a clinical neuroblastoma material. Our results demonstrate the relevance of HIF-2alpha in neuroblastoma progression and have general tumor biological implications.

Hypoxia-inducible factor 1 alpha (HIF-1 alpha) and the intracellular dioxin receptor mediate hypoxia and dioxin signalling, respectively. Both proteins are conditionally regulated basic helix-loop-helix (bHLH) transcription factors that, in addition to the bHLH motif, share a Per-Arnt-Sim (PAS) region of homology and form heterodimeric complexes with the common bHLH/PAS partner factor Arnt. Here we demonstrate that HIF-1 alpha required Arnt for DNA binding in vitro and functional activity in vivo. Both the bHLH and PAS motifs of Arnt were critical for dimerization with HIF-1 alpha. Strikingly, HIF-1 alpha exhibited very high affinity for Arnt in coimmunoprecipitation assays in vitro, resulting in competition with the ligand-activated dioxin receptor for recruitment of Arnt. Consistent with these observations, activation of HIF-1 alpha function in vivo or overexpression of HIF-1 alpha inhibited ligand-dependent induction of DNA binding activity by the dioxin receptor and dioxin receptor function on minimal reporter gene constructs. However, HIF-1 alpha- and dioxin receptor-mediated signalling pathways were not mutually exclusive, since activation of dioxin receptor function did not impair HIF-1 alpha-dependent induction of target gene expression. Both HIF-1 alpha and Arnt mRNAs were expressed constitutively in a large number of human tissues and cell lines, and these steady-state expression levels were not affected by exposure to hypoxia. Thus, HIF-1 alpha may be conditionally regulated by a mechanism that is distinct from induced expression levels, the prevalent model of activation of HIF-1 alpha function. Interestingly, we observed that HIF-1 alpha was associated with the molecular chaperone hsp90. Given the critical role of hsp90 for ligand binding activity and activation of the dioxin receptor, it is therefore possible that HIF-1 alpha is regulated by a similar mechanism, possibly by binding an as yet unknown class of ligands.

In response to hypoxia the hypoxia-inducible factor-1 (HIF-1) mediates transcriptional activation of a network of genes encoding erythropoietin, vascular endothelial growth factor, and several glycolytic enzymes. HIF-1 consists of a heterodimer of two basic helix-loop-helix PAS (Per/Arnt/Sim) proteins, HIF-1alpha and Arnt. HIF-1alpha and Arnt mRNAs are constitutively expressed and were not altered upon exposure of HeLa or HepG2 cells to hypoxia, suggesting that the activity of the HIF-1alpha-Arnt complex may be regulated by some as yet unknown posttranscriptional mechanism. In support of this model, we demonstrate here that Arnt protein levels were not increased under conditions that induce an hypoxic response in HeLa and HepG2 cells. However, under identical conditions, HIF-1alpha protein levels were rapidly and dramatically up-regulated, as assessed by immunoblot analysis. In addition, HIF-1alpha acquired a new conformational state upon dimerization with Arnt, rendering HIF-1alpha more resistant to proteolytic digestion in vitro. Dimerization as such was not sufficient to elicit the conformational change in HIF-1alpha, since truncated forms of Arnt that are capable of dimerizing with HIF-1alpha did not induce this effect. Moreover, the high affinity DNA binding form of the HIF-1alpha-Arnt complex was only generated by forms of Arnt capable of eliciting the allosteric change in conformation. In conclusion, the combination of enhanced protein levels and allosteric change by dimerization defines a novel mechanism for modulation of transcription factor activity.

gene regulation ͉ hydroxylation ͉ signal transduction R educed oxygen levels (hypoxia) lead to a set of cellular adaptations, including increased angiogenesis and erythropoiesis and a switch to glycolytic metabolism. The cellular machinery that senses hypoxia is composed of several proteins. A critical component is the transcription factor hypoxiainducible factor 1␣ (HIF-1␣) (1). The level and activity of HIF-1␣ are controlled by oxygen-dependent prolyl (PHD) and asparaginyl factor-inhibiting HIF-1␣ (FIH-1)] hydroxylases. PHDs hydroxylate two proline residues in the degradation domain of HIF-1␣ in normoxia, which makes HIF-1␣ a substrate for the von Hippel-Lindau E3 ubiquitin ligase and proteasomal degradation. After stabilization in hypoxia, HIF-1␣ interacts with aryl hydrocarbon receptor nuclear translocator (ARNT) to bind to hypoxia response elements (HREs)

The dioxin/aryl hydrocarbon receptor (AhR) functions as a ligand-activated transcription factor regulating transcription of a battery of genes encoding xenobiotic metabolizing enzymes. Known receptor ligands are environmental pollutants including polycyclic aromatic hydrocarbons and polychlorinated dioxins. Loss-of-function (gene-disruption) studies in mice have demonstrated that the AhR is involved in toxic effects of dioxins but have not yielded unequivocal results concerning the physiological function of the receptor. Gain-of-function studies therefore were performed to unravel the biological functions of the AhR. A constitutively active AhR expressed in transgenic mice reduced the life span of the mice and induced tumors in the glandular part of the stomach, demonstrating the oncogenic potential of the AhR and implicating the receptor in regulation of cell proliferation.

Skin plays an essential role, mediated in part by its remarkable vascular plasticity, in adaptation to environmental stimuli. Certain vertebrates, such as amphibians, respond to hypoxia in part through the skin; but it is unknown whether this tissue can influence mammalian systemic adaptation to low oxygen levels. We have found that epidermal deletion of the hypoxia-responsive transcription factor HIF-1alpha inhibits renal erythropoietin (EPO) synthesis in response to hypoxia. Conversely, mice with an epidermal deletion of the von Hippel-Lindau (VHL) factor, a negative regulator of HIF, have increased EPO synthesis and polycythemia. We show that nitric oxide release induced by the HIF pathway acts on cutaneous vascular flow to increase systemic erythropoietin expression. These results demonstrate that in mice the skin is a critical mediator of systemic responses to environmental oxygen.

Premature senescence in vitro has been attributed to oxidative stress leading to a DNA damage response. In the absence of oxidative damage that occurs at atmospheric oxygen levels, proliferation of untransformed cells continues for extended periods of time. We have investigated the role of the hypoxia-inducible factor 1␣ (HIF1␣) transcription factor in preventing senescence in aerobic and hypoxic conditions. Using embryonic fibroblasts from a conditional HIF1␣ knockout mouse, we found that loss of HIF1␣ under aerobic conditions significantly accelerated the onset of cellular senescence, and decreased proliferation under hypoxia. Furthermore, we identify the macrophage migration inhibitory factor (MIF) as a crucial effector of HIF1␣ that delays senescence. Inhibition of MIF phenocopies loss of HIF1␣. Our findings highlight a novel role for HIF1␣ under aerobic conditions, and identify MIF as a target responsible for this function.Supplemental material is available at http://www.genesdev.org.