Evidence for a role of p38 kinase in hypoxia-inducible factor 1-independent induction of vascular endothelial growth factor expression by sodium arsenite

Recently we have demonstrated that sodium arsenite induces the expression of hypoxia-inducible factor 1alpha (HIF-1alpha) protein and vascular endothelial growth factor (VEGF) in OVCAR-3 human ovarian cancer cells. We now show that arsenic trioxide, an experimental anticancer drug, exerts the same effects. The involvement of phosphatidylinositol 3-kinase and mitogen-activated protein kinase (MAPK) pathways in the effects of sodium arsenite was investigated. By using kinase inhibitors in OVCAR-3 cells, both effects of sodium arsenite were found to be independent of phosphatidylinositol 3-kinase and p44/p42 MAPKS but were attenuated by inhibition of p38 MAPK. A role for p38 in the regulation of HIF-1alpha and VEGF expression was supported further by analysis of activation kinetics. Experiments in mouse fibroblast cell lines, lacking expression of c-Jun N-terminal kinases 1 and 2, suggested that these kinases are not required for induction of HIF-1alpha protein and VEGF mRNA. Unexpectedly, sodium arsenite did not activate a HIF-1-dependent reporter gene in OVCAR-3 cells, indicating that functional HIF-1 was not induced. In agreement with this hypothesis, up-regulation of VEGF mRNA was not reduced in HIF-1alpha(-/-) mouse fibroblast cell lines. Altogether, these data suggest that not HIF-1, but rather p38, mediates induction of VEGF mRNA expression by sodium arsenite.     

Induction of hypoxia-inducible-factor 1 by nitric oxide is mediated via the PI 3K pathway

Adaptation to hypoxic stress provokes activation of the hypoxia-inducible-factor-1 (HIF-1) which mediates gene expression of, e.g., erythropoietin or vascular endothelial growth factor. Detailed information on signaling pathways that stabilize HIF-1 is missing, but reactive oxygen species degrade the HIF-1 alpha subunit, whereas phosphorylation causes its stabilization. It was believed that hypoxia resembles the only HIF-1 inducer but recent evidence characterized other activators of HIF-1 such as nitric oxide (NO). Herein, we concentrated on NO-evoked HIF-1 induction as a heretofore unappreciated inflammatory response in association with massive NO formation. We demonstrated that S-nitrosoglutathione induces HIF-1 alpha accumulation and concomitant DNA binding. The response was attenuated by the kinase inhibitor genistein and blockers of phosphatidylinositol 3-kinase such as Ly 294002 or wortmannin. Whereas mitogen-activated protein kinases were not involved, we noticed phosphorylation/activation of Akt in correlation with HIF-1 alpha stabilization. NO appears to regulate HIF-1 alpha via the PI 3K/Akt pathway under normoxic conditions.     

Crosstalk of hypoxia-mediated signaling pathways in upregulating plasminogen activator inhibitor-1 expression in keloid fibroblasts

Keloids are skin fibrotic conditions characterized by an excess accumulation of extracellular matrix (ECM) components secondary to trauma or surgical injuries. Previous studies have shown that plasminogen activator inhibitor-1 (PAI-1) can be upregulated by hypoxia and may contribute to keloid pathogenesis. In this study we investigate the signaling mechanisms involved in hypoxia-mediated PAI-1 expression in keloid fibroblasts. Using Northern and Western blot analysis, transient transfections, and pharmacological agents, we demonstrate that hypoxia-induced upregulation of PAI-1 expression is mainly controlled by hypoxia inducible factors-1alpha (HIF-1alpha) and that hypoxia leads to a rapid and transient activation of phosphatidylinositol-3-kinase/Akt (PI3-K/Akt) and extracellular signal-regulated kinases 1/2 (ERK1/2). Treatment of cells with PI-3K/Akt inhibitor (LY294002) and tyrosine protein kinase inhibitor (genistein) significantly attenuated hypoxia-induced PAI-1 mRNA and protein expression as well as promoter activation, apparently via an inhibition of the hypoxia-induced stabilization of HIF-1alpha protein, attenuation of the steady-state level of HIF-1alpha mRNA, and its DNA-binding activity. Even though disruption of ERK1/2 signaling pathway by PD98059 abolished hypoxia-induced PAI-1 promoter activation and mRNA/protein expression in keloid fibroblasts, it did not inhibit the hypoxia-mediated stabilization of HIF-1alpha protein and the steady-state level of HIF-1alpha mRNA nor its DNA binding activity. Our findings suggest that a combination of several signaling pathways, including ERK1/2, PI3-K/Akt, and protein tyrosine kinases (PTKs), may contribute to the hypoxia-mediated induction of PAI-1 expression via activation of HIF-1alpha in keloid fibroblasts.     

Hypoxia-inducible factor 1: regulation by hypoxic and non-hypoxic activators

Oxygen availability is crucial for cellular metabolism. Hypoxia-inducible factor 1 (HIF-1) is the major oxygen homeostasis regulator. Under normoxic conditions, HIF-1 is rapidly degraded by the proteasome. However, under hypoxic conditions, HIF-1 is stabilized and permits the activation of genes essential to cellular adaptation to low oxygen conditions. These genes include the vascular endothelial growth factor (VEGF), erythropoietin and glucose transporter-1. There is increasing evidence showing that HIF-1 is also implicated in biological functions requiring its activation under normoxic conditions. Amongst others, growth factors and vascular hormones are implicated in this normoxic activation. In this review, we will focus on differences between hypoxic and non-hypoxic induction and activation of HIF-1. We will also discuss the biological functions of HIF-1 associated with these two induction pathways. The clear understanding of both HIF-1 activation mechanisms could have a major impact in cancer and vascular disease.     

LPS-induced Toll-like receptor 4 signalling triggers cross-talk of apoptosis signal-regulating kinase 1 (ASK1) and HIF-1alpha protein

Toll-like receptor 4 (TLR4) is required for recognition of lipopolysaccharide (LPS) of Gram-negative bacteria and induction of the innate immune response to them. Nevertheless, the involvement of some crucial pathways in TLR4 signalling is poorly understood. Here, we report that LPS-induced TLR4 signalling triggers cross talk of HIF-1alpha and ASK1 in THP-1 human myeloid monocytic leukaemia cells. Both pathways are activated via redox-dependent mechanism associated with tyrosine kinase/phospholipase C-1gamma-mediated activation of protein kinase C alpha/beta, which are known to activate NADPH oxidase and the production of reactive oxygen species that activate both HIF-1alpha and ASK1. ASK1 contributes to the stabilisation of HIF-1alpha, most likely via activation of p38 MAP kinase.     

Raising the Bar: How HIF-1 Helps Determine Tumor Radiosensitivity

Through a poorly understood mechanism, tumors respond to radiation by secreting cytokines which inhibit endothelial cell apoptosis, thereby limiting treatment response by minimizing vessel damage. We have recently discovered that this pathway is governed by a major angiogenesis regulator, hypoxia-inducible factor-1 (HIF-1). We uncovered dual mechanisms initiated by radiation that both simultaneously lead to HIF-1 activation: 1) reoxygenation-induced stabilization of the HIF-1 dimer through free radical intermediates, and 2) reoxygenation-mediated depolymerization of hypoxia-induced translational suppressors known as stress granules. These findings have implications both for understanding the basic science of hypoxic signaling in tumors, and for discovering novel methods of enhancing conventional anti-tumor therapeutics in the clinic. In this article, we will highlight the apparent importance of free radical species in protecting tumor vasculature, stress granules in regulating hypoxic gene expression, and HIF-1 in regulating tumor sensitivity to ionizing radiation. The potential therapeutic utility of these findings will also be explored, with emphasis placed on putative targets in these pathways which may enhance tumor radiotherapy.