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...

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Hypoxia-inducible factor-1 (HIF-1) controls the expression of a number of genes such as vascular endothelial growth factor and erythropoietin in low oxygen conditions. However, the molecular mechanisms that underlie the activation of the limiting subunit, HIF-1␣, are still poorly resolved. Results showing that endogenous HIF-1␣ migrated 12 kDa higher than in vitro translated protein led us to evaluate the possible role of phosphorylation on this phenomenon. We report here that HIF-1␣ is strongly phosphorylated in vivo and that phosphorylation is responsible for the marked differences in the migration pattern of HIF-1␣. In vitro, HIF-1␣ is phosphorylated by p42 and p44 mitogen-activated protein kinases (MAPKs) and not by p38 MAPK or c-Jun N-terminal kinase. Interestingly, p42/p44 MAPK stoichiometrically phosphorylate HIF-1␣ in vitro, as judged by a complete upper shift of HIF-1␣. More importantly, we demonstrate that activation of the p42/p44 MAPK pathway in quiescent cells induced the phosphorylation and shift of HIF-1␣, which was abrogated in presence of the MEK inhibitor, PD 98059. Finally, we found that in a vascular endothelial growth factor promoter mutated at sites previously shown to be MAPK-sensitive (SP1/AP2-88-66 site), p42/p44 MAPK activation is sufficient to promote the transcriptional activity of HIF-1. This interaction between HIF-1␣ and p42/p44 MAPK suggests a cooperation between hypoxic and growth factor signals that ultimately leads to the increase in HIF-1-mediated gene expression.The growth of new blood vessels in the adult is termed angiogenesis. Angiogenesis occurs in natural situations such as the female reproductive cycle, or in pathological situations such as wound healing, retinopathy, tumor proliferation, and metastasis. In the latter cases, these events share a common characteristic of occurring in a hypoxic environment.A major mediator of vasculogenesis and angiogenesis is vascular endothelial growth factor (VEGF) 1 (1, 2). In many cell types, hypoxia has been shown to promote VEGF expression (2-9). Induction of VEGF is a multistage process in which the hypoxia-inducible factor 1 (HIF-1) plays a key role in transcriptional activation (10). During hypoxia, HIF-1 is expressed, binds to DNA, and induces the transcription of VEGF mRNA. HIF-1 is composed of two subunits, HIF-1␣ and HIF-1␤ (11). Each subunit contains an N-terminal basic-helix-loop-helix domain. They also contain a PAS motif, which is found in a number of transcription factors including the Drosophila proteins Period, Single-minded, and Trachealess, and mammalian proteins such as the aryl hydrocarbon receptor (AHR) and the aryl hydrocarbon receptor nuclear translocator (ARNT). HIF-1␤ was identified as being the previously described ARNT protein (11). The basic domain is responsible for DNA binding, while the helix-loop-helix and PAS domains play a role in heterodimerization (12). C-terminal transactivation domains can be found on both HIF-1␣ and HIF-1␤ (13-16). HIF-1␣ and HIF-1␤ are constitutively expressed in cells. However...

Reactive oxygen species (ROS) are implicated in the pathophysiology of various diseases, including cancer. In this study, we show that JunD, a member of the AP-1 family of transcription factors, reduces tumor angiogenesis by limiting Ras-mediated production of ROS. Using junD-deficient cells, we demonstrate that JunD regulates genes involved in antioxidant defense, H2O2 production, and angiogenesis. The accumulation of H2O2 in junD-/- cells decreases the availability of FeII and reduces the activity of HIF prolyl hydroxylases (PHDs) that target hypoxia-inducible factors-alpha (HIFalpha) for degradation. Subsequently, HIF-alpha proteins accumulate and enhance the transcription of VEGF-A, a potent proangiogenic factor. Our study uncovers the mechanism by which JunD protects cells from oxidative stress and exerts an antiangiogenic effect. Furthermore, we provide new insights into the regulation of PHD activity, allowing immediate reactive adaptation to changes in O2 or iron levels in the cell.

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Protein kinase C zeta (zeta PKC) is critically involved in the control of a number of cell functions, including proliferation and nuclear factor kappa B (NF‐kappa B) activation. Previous studies indicate that zeta PKC is an important step downstream of Ras in the mitogenic cascade. The stimulation of Ras initiates a kinase cascade that culminates in the activation of MAP kinase (MAPK), which is required for cell growth. MAPK is activated by phosphorylation by another kinase named MAPK kinase (MEK), which is the substrate of a number of Ras‐activated serine/threonine kinases such as c‐Raf‐1 and B‐Raf. We show here that MAPK and MEK are activated in vivo by an active mutant of zeta PKC, and that a kinase‐defective dominant negative mutant of zeta PKC dramatically impairs the activation of both MEK and MAPK by serum and tumour necrosis factor (TNF alpha). The stimulation of other kinases, such as stress‐activated protein kinase (SAPK) or p70S6K, is shown here to be independent of zeta PKC. The importance of MEK/MAPK in the signalling mechanisms activated by zeta PKC was addressed by using the activation of a kappa B‐dependent promoter as a biological read‐out of zeta PKC.

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...