Stabilization Of Hypoxia-inducible Factor-1alpha Research Articles

The Akt1-eNOS axis illustrates the specificity of kinase-substrate relationships in vivo.

Akt1 is critical for many in vivo functions; however, the cell-specific substrates responsible remain to be defined. Here, we examine the importance of endothelial nitric oxide synthase (eNOS) as an Akt1 substrate by generating Akt1-deficient mice (Akt1(-/-) mice) carrying knock-in mutations (serine to aspartate or serine to alanine substitutions) of the critical Akt1 phosphorylation site on eNOS (serine 1176) that render the enzyme "constitutively active" or "less active." The eNOS mutations did not influence several phenotypes in Akt1(-/-) mice; however, the defective postnatal angiogenesis characteristic of Akt1(-/-) mice was rescued by crossing the Akt1(-/-) mice with mice carrying the constitutively active form of eNOS, but not by crossing with mice carrying the less active eNOS mutant. This genetic rescue resulted in the stabilization of hypoxia-inducible factor 1alpha (HIF-1alpha) and increased production of HIF-1alpha-responsive genes in vivo and in vitro. Thus, Akt1 regulates angiogenesis largely through phosphorylation of eNOS and NO-dependent signaling.

Oxidative Stress, Gene Expression, and Protein Changes Induced in the Human Placenta during Labor

Malperfusion of the placenta has been implicated as a cause of oxidative stress in complications of human pregnancy, leading to release of proinflammatory cytokines and anti-angiogenic factors into the maternal circulation. Uterine contractions during labor are known to be associated with intermittent utero-placental perfusion. We therefore tested whether oxidative stress, proinflammatory cytokines, and angiogenic regulators were increased in placentas subjected to short (<5 hours) and long (>15 hours) labor compared with nonlabored controls delivered by cesarean section. In addition, broader changes in gene transcripts were assessed by microarray analysis. Oxidative stress, activation of the nuclear factor-kappaB pathway, tumor necrosis factor-alpha and interleukin 1beta all increased in placental tissues after labor. Stabilization of hypoxia-inducible factor-1alpha and increased vascular endothelial growth factor soluble receptor-1 were also observed. By contrast, tissue levels of placenta growth factor decreased. Apoptosis was also activated in labored placentas. The magnitude of these changes related to the duration of labor. After labor, 55 gene transcripts were up-regulated and 35 down-regulated, and many of these changes were reflected at the protein level. In conclusion, labor is a powerful inducer of placental oxidative stress, inflammatory cytokines, and angiogenic regulators. Our findings are consistent with intermittent perfusion being the initiating cause. Placentas subjected to labor do not reflect the normal in vivo state at the molecular level.

Soluble nickel inhibits HIF‐prolyl‐hydroxylases creating persistent hypoxic signaling in A549 cells

Soluble nickel compounds are carcinogenic to humans although the mechanism by which they cause cancer remains unclear. One major consequence of exposure to nickel is the stabilization of hypoxia inducible factor-1alpha (HIF-1alpha), a protein known to be overexpressed in a variety of cancers. In this study, we report a persistent stabilization of HIF-1alpha by nickel chloride up to 72 h after the removal of nickel from the culture media. In addition, we show that the HIF-prolyl hydroxylases (PHD's) are inhibited when cells are exposed to nickel and that they remain repressed for up to 72 h after nickel is removed. We then show that nickel can inhibit purified HIF-PHD's 2 in vitro, through direct interference with the enzyme. Through theoretical calculations, we also demonstrate that nickel may be able to replace the iron in the active site of this enzyme, providing a plausible mechanism for the persistent inhibition of HIF-PHD's by nickel. The data presented suggest that nickel can interfere with HIF-PHD directly and does not inhibit the enzyme by simply depleting cellular factors, such as iron or ascorbic acid. Understanding the mechanisms by which nickel can inhibit HIF-PHD's and stabilize HIF-1alpha may be important in the treatment of cancer and ischemic diseases.

RSV Causes HIF-1α Stabilization via NO Release in Primary Bronchial Epithelial Cells

RSV infection is characterized by airway edema. Stabilization of hypoxia inducible factor-1alpha (HIF-1alpha) is important in both inflammation and edema formation. In this study we evaluated whether RSV induced release of nitric oxide (NO) by bronchial airway epithelial cells leading to the stabilization of HIF-1alpha and subsequent transcription of VEGF(165). Primary human bronchial epithelial cells (HBEpC) were used; cell supernatants were analyzed. Western blot analysis was used for the detection of HIF-1alpha. Bronchial airway epithelial monolayer permeability was assessed using electric cell-substrate impedance sensing (ECIS) in real time. There was increased stabilization of HIF-1alpha in RSV infected cells. Addition of an NO inhibitor blocked RSV mediated HIF-1alpha expression. Antagonism of NO also inhibited VEGF production and HBEpC monolayer permeability. Our results demonstrate that in HBEpC, RSV induced NO causes stabilization of HIF-1alpha in vitro.

Nitric oxide and reactive oxygen species exert opposing effects on the stability of hypoxia-inducible factor-1alpha (HIF-1alpha) in explants of human pial arteries.

Hypoxia induces angiogenesis, partly through stabilization of hypoxia-inducible factor-1alpha (HIF-1alpha), leading to transcription of pro-angiogenic factors. Here we examined the regulation of HIF-1alpha by hypoxia and nitric oxide (NO) in explants of human cerebrovascular smooth muscle cells. Cells were treated with NO donors under normoxic or hypoxic (2% O2) conditions, followed by analysis of HIF-1alpha protein levels. Treatment with the NO donor sodium nitroprusside reduced levels of HIF-1alpha, whereas NO donors, NOC-18 and S-nitrosoglutathione, increased HIF-1alpha levels. SIN-1, which releases both NO and superoxide (O2*-), reduced HIF-1alpha levels, suggesting that inhibitory NO donors may elicit effects through peroxynitrite (ONOO*-). O2*- generation by xanthine/xanthine oxidase also reduced HIF-1alpha levels, confirming an inhibitory role for reactive oxygen species (ROS). Furthermore, superoxide dismutase increased HIF-1alpha levels, and the NO scavenger carboxy-PTIO reversed HIF-1alpha stabilization by NO donors. Effects on HIF-1alpha levels correlated with vascular endothelial growth factor transcription but did not affect HIF-1alpha transcription, as measured by RT-PCR and luciferase-reporter assays. The results indicate that HIF-1alpha is stabilized by agents that produce NO and reduce ROS but destabilized by agents that increase ROS, including O2*- and ONOO*-. Thus we propose that the effect of NO on HIF-1alpha signaling is critically dependent on the form of NO and the physiological environment of the responding cell.