Integration of oxygen signaling at the consensus HRE Wenger, R H; Stiehl, D P; Camenisch, G Wenger, R H; Stiehl, D P; Camenisch, G. Integration of oxygen signaling at the consensus HRE. Sci. STKE 2005STKE , 2005 Integration of oxygen signaling at the consensus HRE AbstractThe hypoxia-inducible factor 1 (HIF-1) was initially identified as a transcription factor that regulated erythropoietin gene expression in response to a decrease in oxygen availability in kidney tissue. Subsequently, a family of oxygen-dependent protein hydroxylases was found to regulate the abundance and activity of three oxygen-sensitive HIFalpha subunits, which, as part of the HIF heterodimer, regulated the transcription of at least 70 different effector genes. In addition to responding to a decrease in tissue oxygenation, HIF is proactively induced, even under normoxic conditions, in response to stimuli that lead to cell growth, ultimately leading to higher oxygen consumption. The growing cell thus profits from an anticipatory increase in HIF-dependent target gene expression. Growth stimuli-activated signaling pathways that influence the abundance and activity of HIFs include pathways in which kinases are activated and pathways in which reactive oxygen species are liberated. These pathways signal to the HIF protein hydroxylases, as well as to HIF itself, by means of covalent or redox modifications and protein-protein interactions. The final point of integration of all of these pathways is the hypoxia-response element (HRE) of effector genes. Here, we provide comprehensive compilations of the known growth stimuli that promote increases in HIF abundance, of protein-protein interactions involving HIF, and of the known HIF effector genes. The consensus HRE derived from a comparison of the HREs of these HIF effectors will be useful for identification of novel HIF target genes, design of oxygen-regulated gene therapy, and prediction of effects of future drugs targeting the HIF system. GlossOxygen availability regulates many physiological and pathophysiological processes, including embryonic development, high-altitude adaptation, wound healing, inflammation, ischemic diseases and cancer. Central to the understanding of these processes is the elucidation of the molecular mechanisms by which cells react and adapt to insufficient oxygen supply (hypoxia). The last few years brought a wealth of novel insights into these processes. Four oxygen-sensing protein hydroxylases have been discovered which regulate the abundance and activity of three hypoxia-inducible transcription factors (HIFs) and thereby the activity of at least 70 effector genes involved in hypoxic adaptation. In addition to its reactive nature in response to a decrease in tissue oxygenation, it became evident that HIFs are also proactively induced, even under normoxic conditions, in response to growth stimuli which ultimately lead to higher oxygen consumption. The growing cell thus profits from an anticipatory increase in HIF-dependent target gene expression. Growth stimuli-activated s...

Prolyl 4-hydroxylase domain (PHD) proteins are oxygen-dependent enzymes that hydroxylate hypoxia-inducible transcription factor (HIF) ␣-subunits, leading to their subsequent ubiquitination and degradation. Paradoxically, the expression of two family members (PHD2 and PHD3) is induced in hypoxic cell culture despite the reduced availability of the oxygen co-substrate, and it has been suggested that they become functionally relevant following re-oxygenation to rapidly terminate the HIF response. Here we show that PHDs are also induced in hypoxic mice in vivo, albeit in a tissue-specific manner. As demonstrated under chronically hypoxic conditions in vitro, PHD2 and PHD3 show a transient maximum but remain upregulated over more than 10 days, suggesting a feedback down-regulation of HIF-1␣ which then levels off at a novel set point. Indeed, hypoxic induction of PHD2 and PHD3 is paralleled by the attenuation of endogenous HIF-1␣. Using an engineered oxygen-sensitive reporter gene in a cellular background lacking endogenous HIF-1␣ and hence inducible PHD expression, we could show that increased exogenous PHD levels can compensate for a wide range of hypoxic conditions. Similar data were obtained in a reconstituted cellfree system in vitro. In summary, these results suggest that due to their high O 2 K m values, PHDs have optimal oxygensensing properties under all physiologically relevant oxygen concentrations; increased PHDs play a functional role even under oxygen-deprived conditions, allowing the HIF system to adapt to a novel oxygen threshold and to respond to another hypoxic insult. Furthermore, such an autoregulatory oxygen-sensing system would explain how a single mechanism works in a wide variety of differently oxygenated tissues.

IntroductionThe hypoxia-inducible factor 1 (HIF-1) is an ubiquitously expressed transcriptional master regulator of many genes regulating mammalian oxygen homeostasis. 1 Among others, the corresponding gene products are involved in erythropoiesis, iron metabolism, angiogenesis, control of blood flow, glucose uptake and glycolysis, pH regulation, and cell-cycle control. 2 HIF-1 is a ␣ 1 ␤ 1 heterodimer specifically recognizing the HIF-binding site within cis-regulatory hypoxia response elements. 3 Under normoxic conditions, the von Hippel-Lindau tumor suppressor protein (pVHL) targets the HIF-1␣ subunit for rapid ubiquitination and proteasomal degradation. 4 Binding of the pVHL tumor suppressor protein requires the modification of HIF-1␣ by prolyl-4-hydroxylation at prolines 402 and 564 of human HIF-1␣. [5][6][7][8] A family of 3 oxygen-and iron-dependent prolyl-4-hydroxylases called PHD1, PHD2, PHD3, or HPH3, HPH2, HPH1, respectively, has been shown to hydroxylate HIF␣. 9,10 A fourth member, called PH-4, regulates HIF-1␣ in overexpression conditions only. 11 Thus, limited oxygen supply prevents HIF␣ hydroxylation and degradation. 12 This unusual mechanism of protein regulation provides the basis for the very rapid HIF-1␣ response to hypoxia. 13 In addition to protein stability, oxygen-dependent C-terminal asparagine hydroxylation of HIF-1␣ by factor inhibiting HIF (FIH) prevents transcriptional cofactor recruitment, thereby fine-tuning HIF-1 activity following a further decrease in oxygen availability. 14,15 Among the HIF-1 targets are the genes encoding transferrin, transferrin receptor, heme oxygenase-1, and ceruloplasmin, which coordinately regulate iron metabolism. [16][17][18][19][20] Increased iron uptake, release from the liver, plasma transport, and uptake in the bone marrow are essential to sustain the erythropoietic function of erythropoietin, the prototype HIF-1 target. Ceruloplasmin is a multicopper plasma protein containing ferroxidase activity necessary for Fe 3ϩ saturation of transferrin. 21 Hereditary aceruloplasminemia in humans as well as targeted deletion of the ceruloplasmin gene (Cp) in mice results in iron metabolism disorders characterized by anemia, hepatic iron overload, and neurodegeneration, demonstrating a tight connection between copper and iron metabolism. [22][23][24][25][26] Iron deficiency has been known for more than a decade to induce erythropoietin gene expression and HIF-1␣ protein stabilization. 27 Nowadays, these results are most likely explained by inactivation of the iron-dependent protein hydroxylases PHD1 to 3 and FIH. 12 Iron deficiency also results in mRNA induction of ceruloplasmin by HIF-1-dependent promoter activation and subsequent transcriptional up-regulation of the Cp gene. 20 Materials and methods Cell lines and cell cultureAll cell lines were cultured in Dulbecco modified Eagle medium (high glucose) as described previously. 29 Oxygen partial pressures in the hypoxic workstation (InVivO 2 -400; Ruskinn Technology, Leeds, United Kingdom) or in the incubator (M...