Edurne Berra

HIF prolyl‐hydroxylase 2 is the key oxygen sensor setting low steady‐state levels of HIF‐1α in normoxia

Hypoxia‐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‐α subunits are targeted for destruction by prolyl hydroxylation, a specific modification 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 identified recently in mammals and shown to hydroxylate HIF‐α subunits. Here we show that specific ‘silencing’ of PHD2 with short interfering RNAs is sufficient to stabilize and activate HIF‐1α in normoxia in all the human cells investigated. ‘Silencing’ of PHD1 and PHD3 has no effect on the stability of HIF‐1α either in normoxia or upon re‐oxygenation of cells briefly 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‐1α in normoxia. Interestingly, PHD2 is upregulated by hypoxia, providing an HIF‐1‐dependent auto‐regulatory mechanism driven by the oxygen tension.

p42/p44 mitogen-activated protein kinases phosphorylate hypoxia-inducible factor 1alpha (HIF-1alpha) and enhance the transcriptional activity of HIF-1.

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-1α, are still poorly resolved. Results showing that endogenous HIF-1α migrated 12 kDa higher than in vitrotranslated protein led us to evaluate the possible role of phosphorylation on this phenomenon. We report here that HIF-1α is strongly phosphorylated in vivo and that phosphorylation is responsible for the marked differences in the migration pattern of HIF-1α. In vitro, HIF-1α 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-1α in vitro, as judged by a complete upper shift of HIF-1α. More importantly, we demonstrate that activation of the p42/p44 MAPK pathway in quiescent cells induced the phosphorylation and shift of HIF-1α, 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-1α 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.

Prolyl hydroxylase-1 negatively regulates IκB kinase-β, giving insight into hypoxia-induced NFκB activity

Hypoxia is a feature of the microenvironment of a growing tumor. The transcription factor NFκB is activated in hypoxia, an event that has significant implications for tumor progression. Here, we demonstrate that hypoxia activates NFκB through a pathway involving activation of IκB kinase-β (IKKβ) leading to phosphorylation-dependent degradation of IκBα and liberation of NFκB. Furthermore, through increasing the pool and/or activation potential of IKKβ, hypoxia amplifies cellular sensitivity to stimulation with TNFα. Within its activation loop, IKKβ contains an evolutionarily conserved LxxLAP consensus motif for hydroxylation by prolyl hydroxylases (PHDs). Mimicking hypoxia by treatment of cells with siRNA against PHD-1 or PHD-2 or the pan-prolyl hydroxylase inhibitor DMOG results in NFκB activation. Conversely, overexpression of PHD-1 decreases cytokine-stimulated NFκB reporter activity, further suggesting a repressive role for PHD-1 in controlling the activity of NFκB. Hypoxia increases both the expression and activity of IKKβ, and site-directed mutagenesis of the proline residue (P191A) of the putative IKKβ hydroxylation site results in a loss of hypoxic inducibility. Thus, we hypothesize that hypoxia releases repression of NFκB activity through decreased PHD-dependent hydroxylation of IKKβ, an event that may contribute to tumor development and progression through amplification of tumorigenic signaling pathways.

JunD Reduces Tumor Angiogenesis by Protecting Cells from Oxidative Stress

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.

MAP Kinases and Hypoxia in the Control of VEGF Expression

Vascular endothelial growth factor (VEGF), a potent cytokine secreted by virtually all cells plays a key role in tumor angiogenesis. Disruption of one VEGF allele in mice has revealed a dramatic lethal effect in early embryogenesis, suggesting a very tight regulation of this gene. This commentary reviews the mechanisms whereby VEGF mRNA is controlled within the tumor environment by hypoxia and the MAP kinase signaling cascades. Using hamster fibroblasts as a cellular model, we demonstrated that the Ras-mediated activation of p42/p44 MAP kinases exerts a prominent action at the transcriptional level. In normoxic conditions, p42/p44 MAPKs activate the VEGF promoter at the proximal (−88/−66) region where Sp1/AP-2 transcriptional factor complexes are recruited. At low O2 tension, the stabilized and nuclear hypoxia inducible factor-1α (HIF-1α) is directly phosphorylated by p42/p44 MAPKs, an action which enhances HIF-1-dependent transcriptional activition of VEGF. In addition, MAPKs activated under various cellular stresses (p38MAPK and JNK), contribute to the increased expression of this angiogenic growth and survival factor by stabilizing the VEGF mRNA.

Signaling angiogenesis via p42/p44 MAP kinase and hypoxia

Angiogenesis is associated with a number of pathological situations. In this study, we have focused our attention on the role of p42/p44 MAP (mitogen-activated protein) kinases and hypoxia in the control of angiogenesis. We demonstrate that p42/p44 MAP kinases play a pivotal role in angiogenesis by exerting a determinant action at three levels: i) persistent activation of p42/p44 MAP kinases abrogates apoptosis; ii) p42/p44 MAP kinase activity is critical for controlling proliferation and growth arrest of confluent endothelial cells; and iii) p42/p44 MAP kinases promote VEGF (vascular endothelial growth factor) expression by activating its transcription via recruitment of the AP-2/Sp1 (activator protein-2) complex on the proximal region (-88/-66) of the VEGF promoter and by direct phosphorylation of hypoxia-inducible factor 1 alpha (HIF-1 alpha). HIF-1 alpha plays a crucial role in the control of HIF-1 activity, which mediates hypoxia-induced VEGF expression. We show that oxygen-regulated HIF-1 alpha protein levels are not affected by intracellular localization (nucleus versus cytoplasm). Finally, we propose a model which suggests an autoregulatory feedback mechanism controlling HIF-1 alpha and therefore HIF-1-dependent gene expression.

PHDs overactivation during chronic hypoxia “desensitizes” HIFα and protects cells from necrosis

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 O2 homeostasis. In well oxygenated cells, PHDs hydroxylate the HIFα subunits, thereby targeting them for proteasomal degradation. In contrast, acute hypoxia inhibits PHDs, leading to HIFα stabilisation. However, here we show that chronic hypoxia induces HIF1/2α“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 O2 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 HIFα 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.

Hypoxia‐inducible factor‐1α (HIF‐1α) escapes O2‐driven proteasomal degradation irrespective of its subcellular localization: nucleus or cytoplasm

Eukaryotic cells sense oxygen and adapt to hypoxia by regulating a number of genes. Hypoxia‐inducible factor 1 (HIF‐1) is the ‘master’ in this pleiotypic response. HIF‐1 comprises two members of the basic helix–loop–helix transcription factor family, HIF‐1α and HIF‐1β. The HIF‐1α protein is subject to drastic O2‐dependent proteasomal control. However, the signalling components regulating the ‘switch’ for ‘escaping’ proteasomal degradation under hypoxia are still largely unknown. The rapid nuclear translocation of HIF‐1α could represent an efficient way to escape from this degradation. We therefore asked, where in the cell is HIF‐1α degraded? To address this question, we trapped HIF‐1α either in the cytoplasm, by fusing HIF‐1α to the cytoplasmic domain of the Na+‐H+ exchanger (NHE‐1), or in the nucleus, by treatment with leptomycin B. Surprisingly, we found that HIF‐1α is stabilized by hypoxia and undergoes O2‐dependent proteasomal degradation with an identical half‐life (5–8 min) in both cellular compartments. Therefore, HIF‐1α entry into the nucleus is not, as proposed, a key event that controls its stability. This result markedly contrasts with the mechanism that controls p53 degradation via MDM2.

Hypoxia-inducible factor 1α is a new target of microphthalmia-associated transcription factor (MITF) in melanoma cells

In melanocytes and melanoma cells α-melanocyte stimulating hormone (α-MSH), via the cAMP pathway, elicits a large array of biological responses that control melanocyte differentiation and influence melanoma development or susceptibility. In this work, we show that cAMP transcriptionally activates Hif1a gene in a melanocyte cell–specific manner and increases the expression of a functional hypoxia-inducible factor 1α (HIF1α) protein resulting in a stimulation of Vegf expression. Interestingly, we report that the melanocyte-specific transcription factor, microphthalmia-associated transcription factor (MITF), binds to the Hif1a promoter and strongly stimulates its transcriptional activity. Further, MITF “silencing” abrogates the cAMP effect on Hif1a expression, and overexpression of MITF in human melanoma cells is sufficient to stimulate HIF1A mRNA. Our data demonstrate that Hif1a is a new MITF target gene and that MITF mediates the cAMP stimulation of Hif1a in melanocytes and melanoma cells. Importantly, we provide results demonstrating that HIF1 plays a pro-survival role in this cell system. We therefore conclude that the α-MSH/cAMP pathway, using MITF as a signal transducer and HIF1α as a target, might contribute to melanoma progression.

HIF‐1‐dependent transcriptional activity is required for oxygen‐mediated HIF‐1α degradation

Hypoxia‐inducible factor‐1α (HIF‐1α) plays a central role in oxygen homeostasis. In normoxia, HIF‐1α is a short lived protein, whereas hypoxia rapidly increases HIF‐1α protein levels by relaxing its ubiquitin–proteasome‐dependent degradation. In this study, we show that the p42/p44 MAP kinase cascade, known to phosphorylate HIF‐1α, does not modulate the degradation/stabilization profile of HIF‐1α. However, we present evidence that the rate of HIF‐1α degradation depends on the duration of hypoxic stress. We demonstrate that degradation of HIF‐1α is suppressed by: (i) inhibiting general transcription with actinomycin D or (ii) specifically blocking HIF‐1‐dependent transcriptional activity. In keeping with these findings, we postulate that HIF‐1α is targetted to the proteasome via a HIF‐1α proteasome targetting factor (HPTF) which expression is directly under the control of HIF‐1‐mediated transcriptional activity. Although HPTF is not yet molecularly identified, it is clearly distinct from the von Hippel–Lindau protein (pVHL).