Attenuation of HIF-1 DNA-binding activity limits hypoxia-inducible endothelin-1 expression.

Hypoxia-inducible factors (HIFs) locate to HIF-binding sites (HBSs) within the hypoxia-response elements (HREs) of oxygen-regulated genes. Whereas HIF-1alpha is expressed ubiquitously, HIF-2alpha is found primarily in the endothelium, similar to endothelin-1 (ET-1) and fms-like tyrosine kinase 1 (Flt-1), the expression of which is controlled by HREs. We identified an unique sequence alteration in both ET-1 and Flt-1 HBSs not found in other HIF-1 target genes, implying that these HBSs might cause binding of HIF-2 rather than HIF-1. However, electrophoretic mobility shift assays showed HIF-1 and HIF-2 DNA complex formation with the unique ET-1 HBS to be about equal. Both DNA-binding and hypoxic activation of reporter genes using the ET-1 HBS was decreased compared with transferrin and erythropoietin HBSs. The Flt-1 HBS was non-functional when assayed in isolation, suggesting that additional factors are required for hypoxic up-regulation via the reported Flt-1 HRE. Interestingly, HIF-1 activity could be restored fully by point-mutating the ET-1 (but not the Flt-1) HBS, suggesting that the wild-type ET-1 HBS attenuated the full hypoxic response known from other oxygen-regulated genes. Such a mechanism might serve to limit the expression of this potent vasoconstrictor in hypoxia.

Camenisch G ，Stroka DM ，Gassmann M ，Wenger RH ... -  《PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY》

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》

Upregulated hypoxia-inducible factor-1 DNA binding activity to the vascular endothelial growth factor-A promoter mediates increased vascular permeability in donor lung grafts.

Transplantation-induced hypoxia results in enhanced vascular permeability and tissue vascular endothelial growth factor (VEGF) and endothelin-1 (ET-1) overexpression in donor lung grafts. Promoter studies have uncovered a hypoxia-inducible factor (HIF)-1 binding site (HBS) in 5'-flanking region of VEGF gene that regulates the hypoxia-induced expression of VEGF; and ET-1 potently stimulates VEGF-A production. We hypothesized that HIF-1 regulates VEGF-mediated vascular permeability in lung grafts. We studied the mRNA and protein expression of HIF-1 and its protein-binding capacity to the HBS of the VEGF gene in biopsies of preserved donor and control lungs, using real-time reverse transcription-polymerase chain reaction, Western blotting, and electrophoretic mobility shift assay. Wet-to-dry lung weight ratio was measured in donor and control lungs. While HIF-1 alpha mRNA expression was unchanged, HIF-1 beta was downregulated (p < 0.05) in donor versus control lungs. Protein expression of both, HIF-1 alpha and -beta was significantly upregulated in donor lung grafts. HIF-1 binding to the HBS of the VEGF promoter as well as tissue fluid content were increased in donor lung biopsies versus controls (p < 0.05). These data indicate that upregulated HIF-1 DNA binding activity to the HBS of VEGF-A most likely contributes to elevated VEGF levels in preserved lung grafts. Unchanged HIF-1 alpha mRNA expression did not affect HIF-1 alpha protein levels. Endothelin-1 increases HIF-1 alpha accumulation and activates HIF-1 transcription complex in vitro. Therefore, ET-1-mediated increased HIF-1 alpha protein stability most likely leads to transcriptional activation of VEGF during lung graft preservation. Targeting HIF might be of benefit to counteract edema formation in preserved lung grafts.

Abraham D ，Krenn K ，Seebacher G ，Paulus P ，Klepetko W ，Aharinejad S ... -  《ANNALS OF THORACIC SURGERY》

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 ... -  《-》

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 《-》