Abnormal angiogenesis and responses to glucose and oxygen deprivation in mice lacking the protein ARNT.

The arylhydrocarbon-receptor nuclear translocator (ARNT) is a member of the basic-helix-loop-helix-PAS family of heterodimeric transcription factors which includes the arylhydrocarbon receptor (AHR), hypoxia-inducible factor-1alpha (HIF-1alpha) and the Drosophila single-minded protein (Sim). ARNT forms heterodimeric complexes with the arylhydrocarbon receptor, HIF-1alpha, Sim and the PAS protein Per. In response to environmental pollutants, AHR-ARNT heterodimers regulate genes involved in the metabolism of xenobiotics, whereas ARNT-HIF-1alpha heterodimers probably regulate those involved in the response to oxygen deprivation. By generating a targeted disruption of the Arnt locus in the mouse, we show here that Arnt-/- embryonic stem cells fail to activate genes that normally respond to low oxygen tension. Arnt-/- ES cells also failed to respond to a decrease in glucose concentration, indicating that ARNT is crucial in the response to hypoxia and to hypoglycaemia. Arnt-/- embryos were not viable past embryonic day 10.5 and showed defective angiogenesis of the yolk sac and branchial arches, stunted development and embryo wasting. The defect in blood vessel formation in Arnt-/- yolk sacs is similar to the angiogenic abnormalities reported for mice deficient in vascular endothelial growth factor or tissue factor. On the basis of these findings, we propose a model in which increasing tissue mass during organogenesis leads to the formation of hypoxic/nutrient-deprived cells, the subsequent activation of ARNT, and a concomitant increase in the expression of genes (including that encoding vascular endothelial growth factor) that promote vascularization of the developing yolk sac and solid tissues.

Maltepe E ，Schmidt JV ，Baunoch D ，Bradfield CA ，Simon MC ... -  《NATURE》

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》

Developmental expression of two members of a new class of transcription factors: II. Expression of aryl hydrocarbon receptor nuclear translocator in the C57BL/6N mouse embryo.

The aryl hydrocarbon receptor (AhR) and the AhR nuclear translocator protein (ARNT) are basic-helix-loop-helix (bHLH) proteins involved in transcriptional regulation. The AhR is a ligand-activated partner of the ARNT protein. Both proteins are required to transcriptionally regulate gene expression. ARNT must be complexed to AhR to permit binding to the regulatory DNA sequence. The AhR-ligand complex is known to mediate a range of biological responses, such as developmental toxicity, induction of cleft palate, and hydronephrosis. AhR and ARNT are expressed in human embryonic palatal cells and AhR was recently shown to have a specific developmental pattern of expression in the mouse embryo. In the present study, expression of ARNT is characterized in C57Bl/6N mouse embryos from gestation day (GD) 10-16 using immunohistochemistry and in situ hybridization. Af affinity purified antibody against human ARNT (1.1 micrograms/ml) was detected with an avidin-biotin-peroxidase complex. ARNT mRNA was localized with a 35S-RNA probe from pBM5/NEO-M1-1. Specific spatial and temporal patterns of ARNT expression emerged and mRNA and protein expression correlated. The GD 10-11 embryos showed highest levels of ARNT in neuroepithelial cells of the neural tube, visceral arches, otic and optic placodes, and preganglionic complexes. The heart also had significant expression of ARNT with strong nuclear localization. After GD11, expression in heart and brain declined. In GD 12-13 embryos expression was highest in the liver where expression increased from GD 12-16. At GD 15-16 the highest levels of ARNT occurred in adrenal gland and liver, although ARNT was also detected in submandibular gland, ectoderm, tongue, bone, and muscle. In all of these tissues ARNT was cytoplasmic as well as nuclear, except in some of the cortical adrenal cells in which ARNT was strongly cytoplasmic with little or no nuclear localization. These specific patterns of ARNT expression, which differ in certain tissues from the expression of AhR, suggest that ARNT may have additional roles in normal embryonic development.

Abbott BD ，Probst MR 《DEVELOPMENTAL DYNAMICS》

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》

Inhibitory PAS domain protein is a negative regulator of hypoxia-inducible gene expression.

Alteration of gene expression is a crucial component of adaptive responses to hypoxia. These responses are mediated by hypoxia-inducible transcription factors (HIFs). Here we describe an inhibitory PAS (Per/Arnt/Sim) domain protein, IPAS, which is a basic helix-loop-helix (bHLH)/PAS protein structurally related to HIFs. IPAS contains no endogenous transactivation function but demonstrates dominant negative regulation of HIF-mediated control of gene expression. Ectopic expression of IPAS in hepatoma cells selectively impairs induction of genes involved in adaptation to a hypoxic environment, notably the vascular endothelial growth factor (VEGF) gene, and results in retarded tumour growth and tumour vascular density in vivo. In mice, IPAS was predominantly expressed in Purkinje cells of the cerebellum and in corneal epithelium of the eye. Expression of IPAS in the cornea correlates with low levels of expression of the VEGF gene under hypoxic conditions. Application of an IPAS antisense oligonucleotide to the mouse cornea induced angiogenesis under normal oxygen conditions, and demonstrated hypoxia-dependent induction of VEGF gene expression in hypoxic corneal cells. These results indicate a previously unknown mechanism for negative regulation of angiogenesis and maintenance of an avascular phenotype.

Makino Y ，Cao R ，Svensson K ，Bertilsson G ，Asman M ，Tanaka H ，Cao Y ，Berkenstam A ，Poellinger L ... -  《NATURE》