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...

The angiopoietin family of secreted factors is functionally defined by the C-terminal fibrinogen (FBN)-like domain, which mediates binding to the Tie2 receptor and thereby facilitates a cascade of events ultimately regulating blood vessel formation. By screening expressed sequence tag data bases for homologies to a consensus FBN-like motive, we have identified ANGPTL3, a liver-specific, secreted factor consisting of an N-terminal coiled-coil domain and the C-terminal FBN-like domain. Co-immunoprecipitation experiments, however, failed to detect binding of ANGPTL3 to the Tie2 receptor. A molecular model of the FBN-like domain of ANGPTL3 was generated and predicted potential binding to integrins. This hypothesis was experimentally confirmed by the finding that recombinant ANGPTL3 bound to ␣ v ␤ 3 and induced integrin ␣ v ␤ 3 -dependent haptotactic endothelial cell adhesion and migration and stimulated signal transduction pathways characteristic for integrin activation, including phosphorylation of Akt, mitogen-activated protein kinase, and focal adhesion kinase. When tested in the rat corneal assay, ANGPTL3 strongly induced angiogenesis with comparable magnitude as observed for vascular endothelial growth factor-A. Moreover, the C-terminal FBN-like domain alone was sufficient to induce endothelial cell adhesion and in vivo angiogenesis. Taken together, our data demonstrate that ANGPTL3 is the first member of the angiopoietin-like family of secreted factors binding to integrin ␣ v ␤ 3 and suggest a possible role in the regulation of angiogenesis.

IntroductionWhen O 2 delivery is impaired, the resulting hypoxia activates homeostatic mechanisms at the systemic and cellular level. 1 This response involves changes in gene expression mediated by hypoxiainducible factor-1 (HIF-1), the master transcription factor of oxygen-regulated genes. HIF-1 is a heterodimeric protein comprising the oxygen-sensitive ␣-subunit (HIF-1␣, or the more cell-typespecifically expressed HIF-2␣) and the oxygen-insensitive ␤-subunit. 2 Oxygen-regulated gene expression involves binding of HIF to cis-regulatory hypoxia response elements (HREs) of HIF target genes such as erythropoietin or vascular endothelial growth factor. 3 The molecular basis for the hypoxia-induced stability and activity of HIF-1␣ and HIF-2␣ is the O 2 -dependent hydroxylation of distinct prolyl residues. [4][5][6] A family of oxygen-, iron-and 2-oxoglutarate-dependent prolyl-4-hydroxylases has been described recently to hydroxylate the oxygen-labile ␣ subunits of HIF-1 and HIF-2. 5,7,8 This family consists of 3 members called prolyl-4-hydroxylase domain (PHD) 1, PHD2, PHD3, or HIF prolyl hydroxylase (HPH) 3, HPH2, and HPH1, respectively. 4,5 Following prolyl-4-hydroxylation of the critical prolyl residues under normoxic conditions, the ubiquitin ligase von Hippel-Lindau tumor suppressor protein (pVHL) recognizes HIF␣ subunits and targets them for rapid ubiquitination and proteasomal degradation. 9 Binding of pVHL strictly requires prior modification of human HIF-1␣ and HIF-2␣ by prolyl-4-hydroxylation at prolines 402 and 564 or prolines 405 or 531, respectively. 10,11 Limited oxygen supply prevents HIF␣ hydroxylation and degradation. 12 In addition to protein stability, oxygen-dependent C-terminal asparagine hydroxylation of HIF␣ by factor-inhibiting HIF (FIH) prevents transcriptional cofactor recruitment, thereby fine-tuning HIF-1 activity after a further decrease in oxygen availability. 13 Most interestingly, in addition to HIF␣, ankyrin repeats present in IB and NF-B family members have recently been described to be hydroxylated by FIH, demonstrating that hydroxylation is not restricted to the HIF signaling pathway. 14 Besides similarities in the hydroxylation reaction in vitro, the 3 PHDs differ in their ability to hydroxylate HIF-1␣ in vivo and in their organ-specific expression pattern. [15][16][17] Moreover, the phenotypes of PHD knock-out mice demonstrate divergent roles of the 3 PHDs during embryonic development. 18 These data indicate that under physiologic conditions, PHD1, PHD2, and PHD3 mediate different, probably even HIF-independent, oxygen-regulated signal transduction pathways.By searching for novel targets of PHD3 using yeast 2-hybrid technology, we identified activating transcription factor-4 (ATF-4) as a novel interaction partner, and we found that PHD3 confers oxygen-dependent ATF-4 protein stability in a pVHL-independent manner. ATF-4-deficient mice are severely anemic during fetal development, apparently because of an impairment in definitive hematopoiesis. 19 In addition, overexpression of A...

No abstract

The hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator involved in the expression of oxygen-regulated genes such as that for erythropoietin. Following exposure to low oxygen partial pressure (hypoxia), HIF-1 binds to an hypoxia-response element located 3′ to the erythropoietin gene and confers activation of erythropoietin expression. The conserved core HIF-1 binding site (HBS) of the erythropoietin 3′ enhancer (CGTG) contains a CpG dinucleotide known to be a potential target of cytosine methylation. We found that methylation of the HBS abolishes HIF-1 DNA binding as well as hypoxic reporter gene activation, suggesting that a methylation-free HBS is mandatory for HIF-1 function. The in vivo methylation pattern of the erythropoietin 3′ HBS in various human cell lines and mouse organs was assessed by genomic Southern blotting using a methylation-sensitive restriction enzyme. Whereas this site was essentially methylation-free in the erythropoietin-producing cell line Hep3B, a direct correlation between erythropoietin protein expression and the degree of erythropoietin 3′ HBS methylation was found in different HepG2 sublines. However, the finding that this site is partially methylation-free in human cell lines and mouse tissues that do not express erythropoietin suggests that there might be a general selective pressure to keep this site methylation-free, independent of erythropoietin expression.

Here we present the Transcription Factor Encyclopedia (TFe), a new web-based compendium of mini review articles on transcription factors (TFs) that is founded on the principles of open access and collaboration. Our consortium of over 100 researchers has collectively contributed over 130 mini review articles on pertinent human, mouse and rat TFs. Notable features of the TFe website include a high-quality PDF generator and web API for programmatic data retrieval. TFe aims to rapidly educate scientists about the TFs they encounter through the delivery of succinct summaries written and vetted by experts in the field. TFe is available at http://www.cisreg.ca/tfe.