Darren E. Richard

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.

Induction of Hypoxia-inducible Factor-1α by Transcriptional and Translational Mechanisms

Hypoxia-inducible factor-1 (HIF-1) regulates the transcription of many genes induced by low oxygen conditions. Recent studies have demonstrated that non-hypoxic stimuli can also activate HIF-1 in a cell-specific manner. Here, we define two key mechanisms that are implicated in increasing the active subunit of the HIF-1 complex, HIF-1α, following the stimulation of vascular smooth muscle cells (VSMC) with angiotensin II (Ang II). We show that, in contrast to hypoxia, the induction of HIF-1α by Ang II in VSMC is dependent on active transcription and ongoing translation. We demonstrate that stimulation of VSMC by Ang II strongly increases HIF-1α gene expression. The activation of diacylglycerol-sensitive protein kinase C (PKC) plays a major role in the increase of HIF-1α gene transcription. We also demonstrate that Ang II relies on ongoing translation to maintain elevated HIF-1α protein levels. Ang II increases HIF-1α translation by a reactive oxygen species (ROS)-dependent activation of the phosphatidylinositol 3-kinase pathway, which acts on the 5′-untranslated region of HIF-1α mRNA. These results establish that the non-hypoxic induction of the HIF-1 transcription factor via vasoactive hormones (Ang II and thrombin) is triggered by a dual mechanism,i.e. a PKC-mediated transcriptional action and a ROS-dependent increase in HIF-1α protein expression. Elucidation of these signaling pathways that up-regulate the vascular endothelial growth factor (VEGF) could have a strong impact on different aspects of vascular biology.

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.

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.

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).

Identification of Alternative Spliced Variants of Human Hypoxia-inducible Factor-1α

Mammalian cells are able to sense oxygen and regulate a number of genes in response to hypoxia. The transcription factor Hypoxia Inducible Factor-1 (HIF-1) was identified as an important key component of the hypoxia signaling pathway. HIF-1 is a heterodimer composed of two members of the basic helix-loop-helix transcription factor superfamily containing a PAS (PER-ARNT-SIM) domain: HIF-1α and HIF-1β/ARNT. During the cloning by reverse transcriptase-polymerase chain reaction of the human HIF-1α subunit, we isolated two cDNA clones which corresponded to alternative splicing of the HIF-1α gene. Polymerase chain reaction analysis and sequencing revealed that both clones possessed three additional base pairs between exons 1 and 2. Also, one of them lacked 127 base pairs corresponding to exon 14. We demonstrate that the mRNA of this truncated form is expressed in several human cells lines and human skin but apparently not in rodents. When transfected in HEK 293 cells, the corresponding 736 amino acid protein (HIF-1α736) is regulated by hypoxia in a similar manner as the full-length HIF-1α (HIF-1αFL). In luciferase transfection assays, both recombinant proteins HIF-1α736 and HIF-1αFL dimerize with HIF-1β/ARNT and activate the VEGF promoter upon hypoxia. However, the shorter HIF-1α isoform is 3-fold less active than HIF-1αFL, a result consistent with the lack of the C-terminal transactivation domain. As expected, this small isoform can compete with the endogenous and transfected full-length HIF-1α. Altogether, these results suggest that the HIF-1α736isoform modulates gene expression upon hypoxia.

Angiogenesis and G-protein-coupled receptors: signals that bridge the gap

Angiogenesis is a mechanism that has repercussions in a number of physiological and pathological situations. Vascular endothelial growth factor and basic fibroblast growth factor have understandably received enormous research coverage for being the major mediators of new blood vessel growth, often overshadowing other agonist that also have strong angiogenic potential. We wish to put the spotlight on GPCR agonists that undoubtedly have their word to say on the subject of angiogenesis. In this short review, we will discuss our findings along with the work from other groups on the mechanisms by which GPCR agonists, like thrombin and angiotensin II, control a number of angiogenic signals. A complete understanding of these mechanisms could, by the design of new therapeutic strategies, have a strong impact in clinical oncology.