² Deceased during the course of this workHypoxia-inducible factor (HIF), a transcriptional complex conserved from Caenorhabditis elegans to vertebrates, plays a pivotal role in cellular adaptation to low oxygen availability. In normoxia, the HIF-a subunits are targeted for destruction by prolyl hydroxylation, a speci®c modi®cation that provides recognition for the E3 ubiquitin ligase complex containing the von Hippel±Lindau tumour suppressor protein (pVHL). Three HIF prolyl-hydroxylases (PHD1, 2 and 3) were identi®ed recently in mammals and shown to hydroxylate HIF-a subunits. Here we show that speci®c`silencing' of PHD2 with short interfering RNAs is suf®cient to stabilize and activate HIF-1a in normoxia in all the human cells investigated.`Silencing' of PHD1 and PHD3 has no effect on the stability of HIF-1a either in normoxia or upon re-oxygenation of cells brie¯y exposed to hypoxia. We therefore conclude that, in vivo, PHDs have distinct assigned functions, PHD2 being the critical oxygen sensor setting the low steady-state levels of HIF-1a in normoxia. Interestingly, PHD2 is upregulated by hypoxia, providing an HIF-1-dependent auto-regulatory mechanism driven by the oxygen tension. Keywords: angiogenesis/HIF prolyl-hydroxylases/ hypoxia signalling/oxygen sensor/small interfering RNA IntroductionAll organisms possess mechanisms to maintain oxygen homeostasis, which are essential for survival. The hypoxia-inducible factor-1 (HIF-1), conserved during evolution from worms to¯ies to vertebrates, is central to adaptation to low oxygen availability. HIF-1 in turn regulates transcription of many genes involved in cellular and systemic responses to hypoxia, including breathing, vasodilation, anaerobic metabolism, erythropoiesis and angiogenesis. Therefore, hif represents a`master' gene in oxygen homeostasis during embryonic development and postnatal life in both physiological and pathophysiological processes such as tumour growth and metastasis (for a review, see Semenza, 1998).HIF-1 is a heterodimer consisting of one of three a-subunits (HIF-1a, HIF-2a or HIF-3a) and the b-subunit (HIF-1b, also called aryl hydrocarbon nuclear translocator, or ARNT) (Wang et al., 1995;Ema et al., 1997;Tian et al., 1997;Gu et al., 1998). HIF-1b is a constitutive nuclear protein, which also participates in the cellular response to environmental toxins such as aryl hydrocarbons, whereas HIF-a is speci®c to the response to hypoxia (Hoffman et al., 1991). Although oxygen availability regulates multiple steps on HIF-1 transcriptional activation, the dominant control mechanism occurs through oxygen-dependent proteolysis of HIF-a (Huang et al., 1996). The most extensively studied isoform of the a-subunits is the ubiquitous HIF-1a.In normoxia, HIF-1a is constitutively synthesized and sent to destruction by the ubiquitin±proteasome pathway (half-life <5 min) (Salceda and Caro, 1997;Huang et al., 1998;Kallio et al., 1999). This process is mediated by the speci®c binding of pVHL, the product of the von Hippel± Lindau tumour suppressor gene, which...

No abstract

Cell adaptation to changes in oxygen (O2) availability is controlled by two subfamilies of O2-dependent enzymes: the hypoxia inducible factor (HIF)-prolyl and asparaginyl hydroxylases [prolyl hydroxylases domain (PHDs) and factor inhibiting HIF (FIH)]. These oxygen sensors regulate the activity of the HIF, a transcriptional complex central in O 2 homeostasis. In well oxygenated cells, PHDs hydroxylate the HIF␣ subunits, thereby targeting them for proteasomal degradation. In contrast, acute hypoxia inhibits PHDs, leading to HIF␣ stabilisation. However, here we show that chronic hypoxia induces HIF1/2␣''desensitization'' in cellulo and in mice. At the basis of this general adaptative mechanism, we demonstrate that chronic hypoxia not only increases the pool of PHDs but also overactivates the three PHD isoforms. This overactivation appears to be mediated by an increase in intracellular O 2 availability consequent to the inhibition of mitochondrial respiration. By using in cellulo and in vivo siRNA, we found that the PHDs are the key enzymes triggering HIF␣ desensitization, a feedback mechanism required to protect cells against necrotic cell death and thus to adapt them across a chronic hypoxia. Hence, PHDs serve as dual enzymes, for which inactivation and later overactivation is necessary for cell survival in acute or chronic hypoxia, respectively.cell survival ͉ oxygen sensing T he transcriptional complex hypoxia inducible factor (HIF) plays a central role in the maintenance of oxygen (O 2 ) homeostasis, which is essential for cell survival (1). HIF is tightly regulated in an O 2 -dependent manner by hydroxylation of one of the three HIF␣ subunits (HIF1␣, HIF2␣, and HIF3␣) (2, 3). In well oxygenated cells (normoxia), the hydroxylation of two proline residues (P 402 and P 564 in human HIF1␣) by the HIF-prolyl hydroxylases [prolyl hydroxylases domains (PHDs)] allows the specific recognition and polyubiquitination by the von Hippel-Lindau protein (pVHL) E3-ligase complex, leading to proteasomal degradation (4). Moreover, the hydroxylation of an asparagine residue (N 803 in human HIF1␣) by the factor inhibiting HIF (FIH) prevents binding of the coactivator p300/CBP and hence blocks HIF transcriptional activity (5). In contrast, restricted O 2 availability, by relaxing HIF␣ hydroxylation, results in HIF␣ stabilization and activation of the HIF transcriptional complex. Like FIH, the PHDs belong to the super family of iron-and 2-oxoglutarate-dependent dioxygenases, which, by using O 2 as a cosubstrate, provide the molecular basis for their O 2 -sensing function (6). In mammalian cells, three PHDs isoforms have been identified (PHD1, PHD2, and PHD3) and shown to hydroxylate HIF1␣ in cellulo depending on their relative abundance (7). Nevertheless, we report that PHD2 has a dominant role, as it is the rate-limiting enzyme that sets the low steady-state level of HIF1␣ in normoxia (8).In line with our previous work, we sought to look for HIF␣ regulation during long-term hypoxia. Contrary to acute hypoxia, we observed that chr...

Metazoans rapidly respond to changes in oxygen availability by regulating gene expression. The transcription factor hypoxiainducible-factor (HIF), which controls the expression of several genes, 'senses' the oxygen concentration indirectly through the hydroxylation of two proline residues that earmarks the HIF-α subunits for proteasomal degradation. We review the expression, regulation and function of the HIF prolyl hydroxylases or prolyl hydroxylases domain proteins, which are genuine oxygen sensors.

Background-The hypoxia-inducible transcription factor (HIF) subunits are destabilized via the O 2 -dependent prolyl hydroxylase domain proteins (PHD1, PHD2, and PHD3). We investigated whether inhibition of PHDs via upregulating HIF might promote postischemic neovascularization. Methods and Results-Mice with right femoral artery ligation were treated, by in vivo electrotransfer, with plasmids encoding for an irrelevant short hairpin RNA (shRNA) (shCON [control]) or specific shRNAs directed against HIF-1␣ (shHIF-1␣), PHD1 (shPHD1), PHD2 (shPHD2), and PHD3 (shPHD3). The silencing of PHDs induced a specific and transient downregulation of their respective mRNA and protein levels at day 2 after ischemia and, as expected, upregulated HIF-1␣. As a consequence, 2 key hypoxia-inducible proangiogenic actors, vascular endothelial growth factor-A and endothelial nitric oxide synthase, were upregulated at the mRNA and protein levels. In addition, monocyte chemotactic protein-1 mRNA levels and infiltration of Mac-3-positive macrophages were enhanced in ischemic leg of mice treated with shPHD2 and shPHD3. Furthermore, activation of HIF-1␣-related pathways was associated with changes in postischemic neovascularization. At day 14, silencing of PHD2 and PHD3 increased vessel density by 2.2-and 2.6-fold, capillary density by 1.8-and 2.1-fold, and foot perfusion by 1.2-and 1.4-fold, respectively, compared with shCON (PϽ0.001). shPHD1 displayed a lower proangiogenic effect. Of interest, coadministration of shHIF-1␣ with shPHD3 abrogated shPHD3-related effects, suggesting that activation of endogenous HIF-1-dependent pathways mediated the proangiogenic effects of PHD silencing. Conclusions-We demonstrated that a direct inhibition of PHDs, and more particularly PHD3, promoted therapeutic revascularization. Furthermore, we showed that activation of the HIF-1 signaling pathway is required to promote this revascularization. (Circulation. 2009;120:50-59.)

All organisms respond to changes in their environment by activating complex signaling cascades. The "hypoxia-signaling cascade" is activated in response to low oxygen availability and this activation is central to maintaining oxygen homeostasis and hence to survival. By regulating the transcriptional complex hypoxia-inducible factor, hypoxia is associated with several physiopathological processes. Several strategies, based on the targeting of the hypoxia-signaling cascade, have been developed to treat these pathologies. Our review summarize different aspects of the hypoxic pathway.

SummaryMicrobes regulate a large panel of intracellular signalling events that can promote inflammation and/or enhance tumour progression. Indeed, it has been shown that infection of human intestinal cells with the Afa/Dr diffusely adhering E. coli C1845 strain induces expression of pro-angiogenic and pro-inflammatory genes. Here, we demonstrate that exposure of cryptic-like intestinal epithelial cells to C1845 bacteria induces HIF-1a protein levels. This effect depends on the binding of F1845 adhesin to the membrane-associated DAF receptor that initiates signalling cascades promoting translational mechanisms. Indeed, inhibition of MAPK and PI-3K decreases HIF-1a protein levels and blocks C1845-induced phosphorylation of the ribosomal S6 protein. Using RNA interference we show that bacteria-induced HIF-1a regulates the expression of IL-8, VEGF and Twist1, thereby pointing to a role for HIF-1 in angiogenesis and inflammation. In addition, infection correlates with a loss of E-cadherin and cytokeratin 18 and a rise in fibronectin, suggesting that bacteria may induce an epithelial to mesenchymal transition-like phenotype. Since HIF-1a silencing results in reversion of bacteria-induced EMT markers, we speculate that HIF-1a plays a key role linking bacterial infection to angiogenesis, inflammation and some aspects of cancer initiation.

Epidermal wound repair is a complex process involving the fine orchestrated regulation of crucial cell functions, such as proliferation, adhesion and migration. Using an in vitro model that recapitulates central aspects of epidermal wound healing, we demonstrate that the transcription factor HIF1 is strongly stimulated in keratinocyte cultures submitted to mechanical injury. Signals generated by scratch wounding stabilise the HIF1α protein, which requires activation of the PI3K pathway independently of oxygen availability. We further show that upregulation of HIF1α plays an essential role in keratinocyte migration during the in vitro healing process, because HIF1α inhibition dramatically delays the wound closure. In this context, we demonstrate that HIF1 controls the expression of laminin-332, one of the major epithelial cell adhesion ligands involved in cell migration and invasion. Indeed, silencing of HIF1α abrogates injury-induced laminin-332 expression, and we provide evidence that HIF1 directly regulates the promoter activity of the laminin α3 chain. Our results suggest that HIF1 contributes to keratinocyte migration and thus to the re-epithelialisation process by regulating laminin-332.