Subcellular localization and regulation of hypoxia-inducible factor-2alpha in vascular endothelial cells.

The hypoxia-inducible factors 1alpha (HIF-1alpha) and 2alpha (HIF-2alpha) have extensive structural homology and have been identified as transcription factors that mediate hypoxia-inducible gene expression through hypoxia-responsive element (HRE). They play critical roles not only in normal development, but also in tumor progression. Endothelial cells (EC) express both HIF-1alpha and -2alpha. In this study, we examined the subcellular localization of HIF-1alpha and -2alpha in bovine arterial EC (BAEC) by immunoblotting and immunocytostaining analysis and found that even under normoxic conditions, as with its heterodimeric partner ARNT, HIF-2alpha was stable, and was localized in the nucleus of BAEC differently than HIF-1alpha. HIF-2alpha might be regulated by a different mechanism than HIF-1alpha and might mediate the expression of some EC-specific genes under normoxic conditions. We further found that cardiovascular helix-loop-helix factor (CHF) 2, which had been identified as an ARNT-interacting protein, was expressed in BAEC and suppressed HRE-dependent gene expression both under normoxia and hypoxia. CHF2 might be one of the key regulators of HIF-2alpha-mediated gene expression in normoxic EC.

Takahashi R ，Kobayashi C ，Kondo Y ，Nakatani Y ，Kudo I ，Kunimoto M ，Imura N ，Hara S ... -  《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》

Nuclear localization of hypoxia-inducible factor-2alpha in bovine arterial endothelial cells.

Hypoxia-inducible factor (HIF)-2alpha is a recently identified hypoxia-inducible transcription factor abundantly expressed in vascular endothelial cells. As well as HIF-1alpha, HIF-2alpha forms a heterodimeric complex with the aryl hydrocarbon receptor nuclear translocator and upregulates hypoxia-inducible genes such as vascular endothelial growth factor. We found in this study that using green fluorescent protein (GFP) fusion constructs, the subcellular localization of HIF-2alpha was different from that of HIF-1alpha in bovine arterial endothelial cells (BAEC). HIF-1alpha was localized in the cytoplasm under normoxic cells and translocated from the cytoplasm into the nucleus in response to hypoxic induction. In contrast, HIF-2alpha was clearly localized in the nucleus of BAEC even under normoxic conditions. The regulation of HIF-2alpha might differ from that of HIF-1alpha in BAEC. We further showed that nuclear localization of HIF-2alpha was inhibited by either deletion or a single amino acid substitution within the C-terminal end of the protein. The amino acid sequence surrounding Lys737 and Arg738 functions as a nuclear localization signal of HIF-2alpha.

Hara S ，Kobayashi C ，Imura N 《-》

Insulin induces transcription of target genes through the hypoxia-inducible factor HIF-1alpha/ARNT.

Hypoxic stress induces the expression of genes associated with increased energy flux, including the glucose transporters Glut1 and Glut3, several glycolytic enzymes, nitric oxide synthase, tyrosine hydroxylase, erythropoietin and vascular endothelial growth factor (VEGF). Induction of these genes is mediated by a common basic helix-loop-helix-PAS transcription complex, the hypoxia-inducible factor-1alpha (HIF-1alpha)/aryl hydrocarbon nuclear translocator (ARNT). Insulin also induces some of these genes; however, the underlying mechanism is unestablished. We report here that insulin shares with hypoxia the ability to induce the HIF-1alpha/ARNT transcription complex in various cell types. This induction was demonstrated by electrophoretic mobility shift of the hypoxia response element (HRE), and abolished by specific antisera to HIF-1alpha and ARNT, and by transcription activation of HRE reporter vectors. Furthermore, basal and insulin-induced expression of Glut1, Glut3, aldolase A, phosphoglycerate kinase and VEGF was reduced in cells having a defective ARNT. Similarly, the insulin-induced activation of HRE reporter vectors and VEGF was impaired in these cells and was rescued by re-introduction of ARNT. Finally, insulin-like growth factor-I (IGF-I) also induced the HIF-1alpha/ARNT transcription complex. These observations establish a novel signal transduction pathway of insulin and IGF-I and broaden considerably the scope of activity of HIF-1alpha/ARNT.

Zelzer E ，Levy Y ，Kahana C ，Shilo BZ ，Rubinstein M ，Cohen B ... -  《-》

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》

Evidence for the functional activity of hypoxia-inducible transcription factors overexpressed in preeclamptic placentae.

Placentas from women with preeclampsia overexpress the hypoxia-inducible transcription factor proteins, HIF-1alpha and -2alpha (Rajakumar 2001, Biol Reprod 64; p499-506 and p1019-1020). As a first step in evaluating whether HIF-alpha overexpressed in preeclamptic placentae is capable of transactivation, we tested its ability to bind to the DNA hypoxia response element (HRE). Six pairs of normal and preeclamptic placentae obtained by cesarean section were investigated. Three biopsy sites per placenta were analyzed. We first confirmed HIF-1alpha protein overexpression in the preeclamptic placentae using Western analysis. The ratios of the arbitrary densitometry units for HIF-1alpha protein from the preeclamptic and normal placentae (PE/NP) in the three biopsy sites were: 1.9 +/- 0.3, 1.7 +/- 0.2 and 1.8 +/- 0.2, each p < 0.05 vs 1.0. (A ratio of >1.0 indicates that HIF-1alpha protein expression in placentas of women with PE exceeds that in placentas of NP women.) Conventional methods for extracting nuclear proteins and subsequent analysis by electrophoretic mobility shift assay were not suited for the frozen, archived samples (data not shown). Therefore, we employed DNA affinity chromatography using a biotinylated oligonucleotide representing the HRE of the erythropoietin gene coupled to streptavidin-coated Dynabeads. The HRE-bound proteins were then characterized by Western blot analysis. The PE/NP ratios of HRE-bound HIF-1alpha in the three biopsy sites from the six pairs of normal and preeclamptic placentae were 1.7 +/- 0.2, 2.1 +/- 0.4 and 2.4 +/- 0.5, each p < 0.05 vs 1.0. Having established DNA-binding potential at least in vitro, we subsequently analyzed three proteins that have been shown to be regulated by HIF-alpha as downstream, molecular markers of HIF-1alpha activity in vivo. VEGF receptor Flt-1 and Flk-1 play key roles in angiogenesis. Tyrosine hydroxylase is the rate-limiting enzyme in catecholamine synthesis. All three genes contain functional HRE in their promoter sequences. Total proteins were extracted from the same biopsy samples that were used for total and HRE-bound HIF-1alpha. Using specific antibodies we performed Western analysis and the levels of these three proteins were quantitated. The Flt-1 and tyrosine hydroxylase proteins were significantly higher, and Flk-1 significantly lower in placentae from preeclamptic compared to normal pregnancies. In summary, HIF-1alpha protein overexpressed in preeclamptic placentae is capable of binding to its DNA recognition sequence in vitro, and modulates gene expression in vivo.

Rajakumar A ，Brandon HM ，Daftary A ，Ness R ，Conrad KP ... -  《PLACENTA》