Macrophage migration inhibitory factor activates hypoxia-inducible factor in a p53-dependent manner

Background

Macrophage migration inhibitory factor (MIF) is not only a cytokine which has a critical role in several inflammatory conditions but also has endocrine and enzymatic functions. MIF is identified as an intracellular signaling molecule and is implicated in the process of tumor progression, and also strongly enhances neovascularization. Overexpression of MIF has been observed in tumors from various organs. MIF is one of the genes induced by hypoxia in an hypoxia-inducible factor 1 (HIF-1)-dependent manner.

Methods/Principal Findings

The effect of MIF on HIF-1 activity was investigated in human breast cancer MCF-7 and MDA-MB-231 cells, and osteosarcoma Saos-2 cells. We demonstrate that intracellular overexpression or extracellular administration of MIF enhances activation of HIF-1 under hypoxic conditions in MCF-7 cells. Mutagenesis analysis of MIF and knockdown of 53 demonstrates that the activation is not dependent on redox activity of MIF but on wild-type p53. We also indicate that the MIF receptor CD74 is involved in HIF-1 activation by MIF at least when MIF is administrated extracellularly.

Conclusion/Significance

MIF regulates HIF-1 activity in a p53-dependent manner. In addition to MIF''s potent effects on the immune system, MIF is linked to fundamental processes conferring cell proliferation, cell survival, angiogenesis, and tumor invasiveness. This functional interdependence between MIF and HIF-1α protein stabilization and transactivation activity provide a molecular mechanism for promotion of tumorigenesis by MIF.     

FOXO3a-dependent up-regulation of Mxi1-0 promotes hypoxia-induced apoptosis in endothelial cells

Endothelial cell apoptosis induced by hypoxia is implicated in the pathogenesis of vascular diseases. However, the underlying mechanism is not clearly elucidated. In this study, we found that hypoxia increased Mxi1-0 expression, and the Mxi1-0 siRNA could inhibit caspase-8 activation and apoptosis in HUVECs induced by hypoxia. In addition, hypoxia induced FOXO3 activation, while Mxi1-0 expression and apoptosis were inhibited by transfection with FOXO3 siRNA. Using ChIP assay, we confirmed that FOXO3a binds to the Mxi1-0 promoter region. Furthermore, hypoxia treatment leads to remarkable production of reactive oxygen species (ROS), while ROS scavenger N-acetyl-L-cysteine (NAC) inhibits hypoxia-induced ROS production, apoptosis and FOXO3a-mediated Mxi1-0 up-regulation. Finally, we found that the HIF-1α siRNA inhibited hypoxia-induced HIF-1α expression and ROS production, as well as FOXO3a/Mxi1-0 activation and apoptosis in HUVECs. Taken together, this study identifies a HIF-1α/FOXO3a/Mxi1-0/caspase-8 signaling pathway in hypoxia-induced endothelial cell apoptosis. These data also indicate that HIF-1α-dependent ROS production is required for FOXO3a-mediated Mxi1-0 up-regulation and apoptosis in hypoxic endothelial cells.     

The MIF Antagonist ISO-1 Attenuates Corticosteroid-Insensitive Inflammation and Airways Hyperresponsiveness in an Ozone-Induced Model of COPD

Introduction

Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine associated with acute and chronic inflammatory disorders and corticosteroid insensitivity. Its expression in the airways of patients with chronic obstructive pulmonary disease (COPD), a relatively steroid insensitive inflammatory disease is unclear, however.

Methods

Sputum, bronchoalveolar lavage (BAL) macrophages and serum were obtained from non-smokers, smokers and COPD patients. To mimic oxidative stress-induced COPD, mice were exposed to ozone for six-weeks and treated with ISO-1, a MIF inhibitor, and/or dexamethasone before each exposure. BAL fluid and lung tissue were collected after the final exposure. Airway hyperresponsiveness (AHR) and lung function were measured using whole body plethysmography. HIF-1α binding to the Mif promoter was determined by Chromatin Immunoprecipitation assays.

Results

MIF levels in sputum and BAL macrophages from COPD patients were higher than those from non-smokers, with healthy smokers having intermediate levels. MIF expression correlated with that of HIF-1α in all patients groups and in ozone-exposed mice. BAL cell counts, cytokine mRNA and protein expression in lungs and BAL, including MIF, were elevated in ozone-exposed mice and had increased AHR. Dexamethasone had no effect on these parameters in the mouse but ISO-1 attenuated cell recruitment, cytokine release and AHR.

Conclusion

MIF and HIF-1α levels are elevated in COPD BAL macrophages and inhibition of MIF function blocks corticosteroid-insensitive lung inflammation and AHR. Inhibition of MIF may provide a novel anti-inflammatory approach in COPD.     

Insulin and hypoxia share common target genes but not the hypoxia-inducible factor-1alpha

Both hypoxia and insulin induce common target genes, including vascular endothelial growth factors and several glycolytic enzymes. However, these two signals eventually trigger quite different metabolic pathways. Hypoxia induces glycolysis, resulting in anaerobic ATP production, while insulin increases glycolysis for energy storage. Hypoxia-induced gene expression is mediated by the hypoxia-inducible factor-1 (HIF-1) that consists of HIF-1alpha and the aromatic hydrocarbon nuclear translocator (Arnt). Hypoxia-induced gene expression is initiated by the stabilization of the HIF-1alpha subunit. Here we investigated whether insulin-induced gene expression also requires stabilization of HIF-1alpha. Our results indicate that hypoxia but not insulin stabilizes HIF-1alpha protein levels, whereas both insulin- and hypoxia-induced gene expression require the presence of the Arnt protein. Insulin treatment fails to inactivate proline hydroxylation of HIF-1alpha, which triggers recruitment of the von Hippel-Lindau protein and oxygen-dependent degradation of HIF-1alpha. Insulin-induced gene expression is inhibited by the presence of the phosphoinositide (PI) 3-kinase inhibitor LY294002 and the dominant negative mutant of the p85 subunit of PI 3-kinase, whereas hypoxia-induced gene expression is not. Pyrrolidine dithiocarbamate, a scavenger of H2O2, reduces insulin-induced gene expression but not hypoxia-induced gene expression. Although both hypoxia and insulin induce the expression of common target genes through a hypoxia-responsive element- and Arnt-dependent mechanism, insulin cannot stabilize the HIF-1alpha protein. We believe that insulin activates other putative partner proteins for Arnt in PI 3-kinase- and H2O2-dependent pathways.     

Hypoxia-induced nucleophosmin protects cell death through inhibition of p53

Nucleophosmin (NPM) is a multifunctional protein that is overexpressed in actively proliferating cells and cancer cells. Here we report that this proliferation-promoting protein is strongly induced in response to hypoxia in human normal and cancer cells. Up-regulation of NPM is hypoxia-inducible factor-1 (HIF-1)-dependent. The NPM promoter encodes a functional HIF-1-responsive element that can be activated by hypoxia or forced expression of HIF-1alpha. Suppression of NPM expression by small interfering RNA targeting NPM increases hypoxia-induced apoptosis, whereas overexpression of NPM protects against hypoxic cell death of wild-type but not p53-null cells. Moreover, NPM inhibits hypoxia-induced p53 phosphorylation at Ser-15 and interacts with p53 in hypoxic cells. Thus, this study not only demonstrates hypoxia regulation of a proliferation-promoting protein but also suggests that hypoxia-driven cancer progression may require increased expression of NPM to suppress p53 activation and maintain cell survival.     

Two transactivation domains of hypoxia-inducible factor-1alpha regulated by the MEK-1/p42/p44 MAPK pathway

At a low-oxygen tension, cells increase the expression of several genes (such as erythropoietin, the vascular endothelial growth factor, and glycolytic enzymes) in order to adapt to hypoxic stress. A common transactivator, named the hypoxia-inducible factor 1 (HIF-1) activates these genes. HIF-1 is a heterodimeric transactivator that is composed of alpha and beta subunits. HIF-1 activity is primarily determined by the hypoxia-induced stabilization of the alpha subunit, whereas the HIF-1beta subunit is expressed constitutively. Our previous observation implied that the MEK-1/p42/p44 MAPK pathway is involved in the hypoxia-induced transactivation ability, but not in the stabilization and DNA binding of HIF-1alpha. In this paper, we dissected the transactivation domain of HIF-1alpha in more detail, and tested the correlation between specific domains of HIF-1alpha and specific signaling pathways. We designed several fusion proteins that contain deletion mutants of HIF-1alpha that is linked to the DNA binding domain of the yeast protein Gal4. By using the Gal4-driven reporter system, we tested the transactivation activities of the Gal4/HIF-1alpha fusion proteins in Hep3B cells. Our findings suggest that tyrosine kinases, the MEK-1/p42/p44 MAPK pathway, but not the PI-3 kinase/Akt pathway, are involved in the hypoxia-induced transactivation of HIF-1alpha. We have shown that the functional transactivation activities are located at both 522-649 and 650-822 amino acids of HIF-1alpha. Treatment of PD98059, a MEK-1 inhibitor, blocked the hypoxia-induced transactivation abilities of both the 522-649 and 650-822 amino acids of the C-terminal half of HIF-1alpha. This implies that the MEK-1/p42/p44 MAPK signaling pathway cannot distinguish between the two hypoxia-induced transactivation domains.     

Folic Acid Represses Hypoxia-Induced Inflammation in THP-1 Cells through Inhibition of the PI3K/Akt/HIF-1α Pathway

Though hypoxia has been implicated as a cause of inflammation, the underlying mechanism is not well understood. Folic acid has been shown to provide protection against oxidative stress and inflammation in patients with cardiovascular disease and various models approximating insult to tissue via inflammation. It has been reported that hypoxia-induced inflammation is associated with oxidative stress, upregulation of hypoxia-inducible factor 1-alpha (HIF-1α), and production of pro-inflammatory molecules. Whether folic acid protects human monocytic cells (THP-1 cells) against hypoxia-induced damage, however, remains unknown. We used THP-1 cells to establish a hypoxia-induced cellular injury model. Pretreating THP-1 cells with folic acid attenuated hypoxia-induced inflammatory responses, including a decrease in protein and mRNA levels of interleukin (IL)-1β and tumor necrosis factor-alpha (TNF-α), coupled with increased levels of IL-10. Folic acid also reduced hypoxia-induced Akt phosphorylation and decreased nuclear accumulation of HIF-1α protein. Both {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (a selective inhibitor of phosphatidyl inositol-3 kinase, PI3K) and KC7F2 (a HIF-1α inhibitor) reduced levels of hypoxia-induced inflammatory cytokines. We also found that insulin (an Akt activator) and dimethyloxallyl glycine (DMOG, a HIF-1α activator) induced over-expression of inflammatory cytokines, which could be blocked by folic acid. Taken together, these findings demonstrate how folic acid attenuates the hypoxia-induced inflammatory responses of THP-1 cells through inhibition of the PI3K/Akt/HIF-1α pathway.     

Understanding hypoxia-induced gene expression in early development: in vitro and in vivo analysis of hypoxia-inducible factor 1-regulated zebra fish insulin-like growth factor binding protein 1 gene expression

Insulin-like growth factor binding protein 1 (IGFBP-1) is a hypoxia-inducible gene that plays an important role in regulating embryonic growth and development under hypoxic stress. The molecular mechanisms underlying hypoxia-induced IGFBP-1 gene expression in the embryonic tissues are not well understood. Here we report that the hypoxia-inducible factor 1 (HIF-1) pathway is established in early embryogenesis and mediates hypoxia-induced IGFBP-1 expression. Hypoxia increased the HIF-1 activity, and HIF-1alpha overexpression or CoCl2 treatment resulted in elevated IGFBP-1 expression in zebra fish embryos. Although the zebra fish IGFBP-1 promoter contains 13 consensus hypoxia response elements (HREs), deletion and mutational analysis revealed that only the HRE positioned at -1090/-1086 is required for the hypoxia and HIF-1 induction. Further experiments revealed that there is an HIF-1 ancillary sequence (HAS) adjacent only to the functional HRE. Mutation of this HAS greatly reduced the responsiveness of the IGFBP-1 promoter to hypoxia and HIF-1. The HAS does not directly bind to HIF-1 or affect the binding of the HRE to HIF-1. The HAS is bound to a nuclear protein(s), and this HAS binding activity is reduced by hypoxia. These results suggest that HIF-1 mediates hypoxia-induced IGFBP-1 gene expression in early development by selectively interacting with the -1090/-1086 HRE and its adjacent HAS.     

A hypoxia-induced decrease of either MICA/B or Hsp70 on the membrane of tumor cells mediates immune escape from NK cells

Recent findings suggest that hypoxia of the tumor microenvironment contributes to immune escape from natural killer (NK) cell-mediated cytotoxicity. Heat shock protein 70 (Hsp70) and the stress-regulated major histocompatibility class I chain-related protein A and B (MICA/B) both serve as ligands for activated NK cells when expressed on the cell surface of tumor cells. Herein, we studied the effects of hypoxia and hypoxia-inducible factor-1α (HIF-1α) on the membrane expression of these NK cell ligands in H1339 with high and MDA-MB-231 tumor cells with low basal HIF-1α levels and its consequences on NK cell-mediated cytotoxicity. We could show that a hypoxia-induced decrease in the membrane expression of MICA/B and Hsp70 on H1339 and MDA-MB-231 cells, respectively, is associated with a reduced sensitivity to NK cell-mediated lysis. A knockdown of HIF-1α revealed that the decreased surface expression of MICA/B under hypoxia is dependent on HIF-1α in H1339 cells with high basal HIF-1α levels. Hypoxia and HIF-1α did not affect the MICA/B expression in MDA-MB-231 cells but reduced the Hsp70 membrane expression which in turn also impaired NK cell recognition. Furthermore, we could show that the hypoxia-induced decrease in membrane Hsp70 is independent of HIF-1α in MDA-MB-231. Our data indicate that hypoxia-induced downregulation of both NK cell ligands MICA/B and Hsp70 impairs NK cell-mediated cytotoxicity, whereby only MICA/B appears to be regulated by HIF-1α.     

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.