Oxygen-regulated expression of the RNA-binding proteins RBM3 and CIRP by a HIF-1-independent mechanism.

The transcriptional regulation of several dozen genes in response to low oxygen tension is mediated by hypoxia-inducible factor 1 (HIF-1), a heterodimeric protein composed of two subunits, HIF-1alpha and HIF-1beta. In the HIF-1alpha-deficient human leukemic cell line, Z-33, exposed to mild (8% O(2)) or severe (1% O(2)) hypoxia, we found significant upregulation of two related heterogenous nuclear ribonucleoproteins, RNA-binding motif protein 3 (RBM3) and cold inducible RNA-binding protein (CIRP), which are highly conserved cold stress proteins with RNA-binding properties. Hypoxia also induced upregulation of RBM3 and CIRP in the murine HIF-1beta-deficient cell line, Hepa-1 c4. In various HIF-1 competent cells, RBM3 and CIRP were induced by moderate hypothermia (32 degrees C) but hypothermia was ineffective in increasing HIF-1alpha or vascular endothelial growth factor (VEGF), a known HIF-1 target. In contrast, iron chelators induced VEGF but not RBM3 or CIRP. The RBM3 and CIRP mRNA increase after hypoxia was inhibited by actinomycin-D, and in vitro nuclear run-on assays demonstrated specific increases in RBM3 and CIRP mRNA after hypoxia, which suggests that regulation takes place at the level of gene transcription. Hypoxia-induced RBM3 or CIRP transcription was inhibited by the respiratory chain inhibitors NaN(3) and cyanide in a dose-dependent fashion. However, cells depleted of mitochondria were still able to upregulate RBM3 and CIRP in response to hypoxia. Thus, RBM3 and CIRP are adaptatively expressed in response to hypoxia by a mechanism that involves neither HIF-1 nor mitochondria.

Wellmann S ，Bührer C ，Moderegger E ，Zelmer A ，Kirschner R ，Koehne P ，Fujita J ，Seeger K ... -  《JOURNAL OF CELL SCIENCE》

Absolute requirement of aryl hydrocarbon receptor nuclear translocator protein for gene activation by hypoxia.

Hypoxia-inducible factor 1 (HIF-1) is a DNA-binding heterodimeric protein complex originally described in the transcriptional activation of the erythropoietin gene by hypoxia. This protein complex is composed of two subunits, HIF-1alpha and -1beta (aryl hydrocarbon receptor nuclear translocator, ARNT). In this study, we used ARNT-deficient cells, derived from the mouse hepatoma cell line Hepa1c1c7, to further characterize HIF-1 complex formation and its relationship with gene activation by hypoxia and desferrioxamine (Df). Gel shift assays revealed that ARNT is absolutely required for the formation of the HIF-1 DNA-binding complex. Results from RNase protection assays and Northern blots showed that the lack of functional HIF-1 complex completely abrogated the response to hypoxia of vascular endothelial growth factor (VEGF) and the glycolytic enzymes aldolase A (ALDA) and phosphoglycerate kinase 1 (PGK-1), genes known to be upregulated by low oxygen tension. Desferrioxamine induction of VEGF and PGK-1 genes was reduced in the ARNT-deficient cells, but at difference with hypoxia, it was not completely suppressed. These results suggest that Df is able to activate gene transcription through HIF-1-independent mechanisms. Exposure to hypoxia or Df did not induce any changes in HIF-1alpha and -1beta mRNA levels, suggesting that posttranscriptional mechanisms are involved in HIF-1 complex activation.

Salceda S ，Beck I ，Caro J 《ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS》

The redox protein thioredoxin-1 (Trx-1) increases hypoxia-inducible factor 1alpha protein expression: Trx-1 overexpression results in increased vascular endothelial growth factor production and enhanced tumor angiogenesis.

Hypoxia-inducible factor 1 (HIF-1), a heterodimer of HIF-1alpha and HIF-1beta subunits, is a transcriptional activator central to the cellular response to low oxygen that includes metabolic adaptation, angiogenesis, metastasis, and inhibited apoptosis. Thioredoxin-1 (Trx-1) is a small redox protein overexpressed in a number of human primary tumors. We have examined the effects of Trx-1 on HIF activity and the activation of downstream genes. Stable transfection of human breast carcinoma MCF-7 cells with human Trx-1 caused a significant increase in HIF-1alpha protein levels under both normoxic (20% oxygen) and hypoxic (1% oxygen) conditions. Trx-1 increased hypoxia-induced HIF-1 transactivation activity measured using a luciferase reporter under the control of the hypoxia response element. Changes in HIF-1alpha mRNA levels did not account for the changes observed at the protein level, and HIF-1beta protein levels did not change. Trx-1 transfection also caused a significant increase in the protein products of hypoxia-responsive genes, including vascular endothelial growth factor (VEGF) and nitric oxide synthase 2 in a number of different cell lines (MCF-7 human breast and HT29 human colon carcinomas and WEHI7.2 mouse lymphoma cells) under both normoxic and hypoxic conditions. The pattern of expression of the different isoforms of VEGF was not changed by Trx-1. Transfection of a redox-inactive Trx-1 (C32S/C35S) markedly decreased levels of HIF-1alpha protein, HIF-1 transactivating activity, and VEGF protein in MCF-7 cells compared with empty vector controls. In vivo studies using WEHI7.2 cells transfected with Trx-1 showed significantly increased tumor VEGF and angiogenesis. The results suggest that Trx-1 increases HIF-1alpha protein levels in cancer cells and increases VEGF production and tumor angiogenesis.

Welsh SJ ，Bellamy WT ，Briehl MM ，Powis G ... -  《CANCER RESEARCH》

Baltic salmon (Salmo salar) yolk-sac fry mortality is associated with disturbances in the function of hypoxia-inducible transcription factor (HIF-1alpha) and consecutive gene expression.

Baltic salmon (Salmo salar) suffer from abnormally high yolk-sac fry mortality designated as M74-syndrome. In 1990s, 25-80% of salmon females, which ascended rivers to spawn, produced yolk-sac fry suffering from the syndrome. Symptoms of M74-affected fry include neurological disturbances, impaired vascular development and abnormal haemorrhages. The latter symptoms are observed in mammalian embryos if the function of hypoxia inducible transcription factor (HIF-1alpha), its dimerization partner aryl hydrocarbon nuclear translocator (ARNT) or target gene vascular endothelial growth factor (VEGF) is disturbed. To study the possible involvement of HIF-1alpha and its target gene VEGF in the development of the syndrome, we collected healthy and M74-affected wild Baltic salmon yolk-sac fry and analyzed HIF-1alpha mRNA and protein expression, HIF-1alpha DNA-binding, target gene VEGF protein expression, and blood vessel density in both groups at different stages of yolk-sac fry development. In addition, since Baltic salmon females contain organochlorine contaminants, which have been suggested to be the cause of M74 syndrome via the aryl hydrocarbon receptor (AhR)-dependent gene expression pathway, we studied AhR protein expression, AhR DNA-binding and target gene CYP1A protein expression. Since the parents of both healthy and M74-affected wild fry will have experienced the organochlorine load from the Baltic Sea, hatchery-reared fry were included in the studies as an additional control. The results show that the vascular defects observed in fry suffering from M74 are associated with reduced DNA-binding activity of HIF-1alpha and subsequent downregulation of its target gene vascular endothelial growth factor (VEGF). In addition, also AhR function is decreased in diseased fry making it unlikely that symptoms of M74-affected fry would be caused by an upregulation of xenobiotically induced AhR-dependent gene expression pathway.

Vuori KA ，Soitamo A ，Vuorinen PJ ，Nikinmaa M ... -  《AQUATIC TOXICOLOGY》

Divergent expression patterns for hypoxia-inducible factor-1beta and aryl hydrocarbon receptor nuclear transporter-2 in developing kidney.

The hypoxia-inducible factors (HIF) are alpha/beta heterodimeric transcription factors of the basic helix-loop-helix-Per-Arnt-Sim (bHLH-PAS) superfamily and are chiefly responsible for cellular adaptation to oxygen deprivation. HIF function relies on the stabilization of the alpha subunit. When oxygen tension falls, HIF-alpha subunits translocate to the nucleus and, upon dimerization with HIF-beta, activate transcription of target genes, including vascular endothelial growth factor, vascular endothelial growth factor receptor-1 and -2, and WT-1, which are vital for kidney development. HIF-beta subunits are stable regardless of oxygen concentration and constitutively translocate to the nucleus. It was shown previously that HIF-1beta protein expression is nearly ubiquitous in newborn kidney and that HIF-1beta dimerizes with either HIF-1alpha or -2alpha. Here it is shown that aryl hydrocarbon receptor nuclear transporter-2 (ARNT2/HIF-2beta) also heterodimerized with HIF-1alpha and -2alpha. ARNT2/HIF-2beta protein was highly expressed in newborn kidney but decreased significantly with age, whereas HIF-1beta levels remained relatively constant. By immunohistochemical analysis, widespread expression of HIF-1beta was observed in developing and mature kidneys. ARNT2/HIF-2beta protein distribution was restricted to distal segments of developing nephrons and in mature kidney was confined specifically to thick ascending limb of Henle's loop. The data presented here suggest that ARNT2/HIF-2beta is required at high levels during nephrogenesis in distal tubules and later exclusively in thick ascending limb. Furthermore, Hypoxyprobe-1 and lotus lectin co-localization studies showed that developing proximal convoluted tubules were the most severely hypoxic nephron segment in immature kidney. Because HIF-2beta protein was not abundantly expressed in this segment, it may not be engaged in mediating responses to severe hypoxia. The differential distribution patterns for HIF-1beta and -2beta suggest divergent roles during kidney development for these highly related bHLH-PAS proteins.

Freeburg PB ，Abrahamson DR 《JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY》