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Query: UNIPROT:P06889 (Mol)
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Expression of vascular endothelial growth factor (VEGF) is induced in cells exposed to hypoxia or ischemia. Neovascularization stimulated by VEGF occurs in several important clinical contexts, including myocardial ischemia, retinal disease, and tumor growth. Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic helix-loop-helix protein that activates transcription of the human erythropoietin gene in hypoxic cells. Here we demonstrate the involvement of HIF-1 in the activation of VEGF transcription. VEGF 5'-flanking sequences mediated transcriptional activation of reporter gene expression in hypoxic Hep3B cells. A 47-bp sequence located 985 to 939 bp 5' to the VEGF transcription initiation site mediated hypoxia-inducible reporter gene expression directed by a simian virus 40 promoter element that was otherwise minimally responsive to hypoxia. When reporters containing VEGF sequences, in the context of the native VEGF or heterologous simian virus 40 promoter, were cotransfected with expression vectors encoding HIF-1alpha and HIF-1beta (ARNT [aryl hydrocarbon receptor nuclear translocator]), reporter gene transcription was much greater in both hypoxic and nonhypoxic cells than in cells transfected with the reporter alone. A HIF-1 binding site was demonstrated in the 47-bp hypoxia response element, and a 3-bp substitution eliminated the ability of the element to bind HIF-1 and to activate transcription in response to hypoxia and/or recombinant HIF-1. Cotransfection of cells with an expression vector encoding a dominant negative form of HIF-1alpha inhibited the activation of reporter transcription in hypoxic cells in a dose-dependent manner. VEGF mRNA was not induced by hypoxia in mutant cells that do not express the HIF-1beta (ARNT) subunit. These findings implicate HIF-1 in the activation of VEGF transcription in hypoxic cells.
Mol Cell Biol 1996 Sep
PMID:Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. 875 16

Erythropoietin (Epo) is a glycoprotein hormone that is the primary regulator of red blood cell production. Epo production increases in response to tissue hypoxia. This increase occurs primarily at the transcriptional level. Hypoxia inducible factor (HIF-1) is a DNA binding protein that binds to a hypoxia inducible enhancer in the 3' flanking sequence of the Epo gene. HIF-1 is a heterodimer that consists of an alpha and beta subunit. HIF-1 DNA binding activity is induced in response to hypoxia. In order to determine if one or both HIF-1 subunits is capable of ligand binding, subsequently leading to Epo production we performed co-transactivation experiments. Transfections were performed in Hep 3B, an Epo producing human hepatoma cell line and Cos-7, a non-Epo producing monkey kidney cell line. Cells were co-transfected with the 38 bp Epo enhancer fragment bearing the HIF-1 binding motif, subcloned in the luciferase reporter plasmid and either the HIF-1alpha cDNA, HIF-1beta cDNA, HIF-1alpha and HIF-1beta cDNAs or pREP-4 respectively. Cells were incubated in an hypoxic (1%O2) or normoxic (21%O2) environment and assayed for luciferase activity. Epo levels were measured in the culture media from the transfected plates by an ELISA assay. Under hypoxic conditions Hep 3B cells transfected with the HIF-1alpha cDNA alone showed a 2.2 fold increase in luciferase activity, HIF-1beta showed a 3.4 fold increase and cells transfected with HIF-1 alpha and beta showed a 6. 9 fold increase in activity over cells transfected with pREP-4. The baseline luciferase activity in transfected 3B cells incubated in normoxia was very low. However, a similar fold increase in luciferase activity in cells transfected with both HIF-1alpha and beta was noted. Under normoxic or hypoxic conditions in Cos-7 cells, a 1.5 fold increase was obtained with the HIF-1alpha and beta constructs transfected independently and a 3.5 fold increase was noted in cells transfected with both constructs. Epo levels increased several fold in all Hep 3B cells that were incubated in hypoxic conditions. However, there was no additional increase in Epo levels in transfected Hep 3B cells. We therefore conclude that although the HIF-1alpha and beta subunits can independently co-transactivate the Epo enhancer, binding of both subunits and a hypoxic environment is necessary for maximal transactivation. Overexpression of the HIF-1 protein alone in normoxic or hypoxic conditions is insufficient for an increase in Epo secretion. Activation/inactivation and interaction of other tissue specific factors is necessary for an increase in Epo gene expression in response to hypoxia.
Blood Cells Mol Dis 1997 Aug
PMID:Co-transactivation of the 3' erythropoietin hypoxia inducible enhancer by the HIF-1 protein. 923 55

Cellular adaptation to hypoxia involves regulation of specific genes such as vascular endothelial growth factor (VEGF), erythropoietin (EPO) and hypoxia inducible factor (HIF)-1 . In this study, we have evaluated the protective effect of picroliv (a purified iridoid glycoside fraction from roots of Picrorhiza kurrooa with hepatoprotective, anti-inflammatory and antioxidant properties) against hypoxic injury by examining lactate dehydrogenase (LDH) release in Hep 3B and Glioma cells. The expression of hypoxia regulated genes, VEGF and HIF-1 was studied in human umbilical vein endothelial cells (HUVEC), Hep 3B and Glioma cells. Picroliv reduced the cellular damage caused by hypoxia as revealed by a significant reduction in LDH release compared to untreated control. The expression of VEGF and HIF-1 subunits (HIF-1alpha and HIF-1beta) was enhanced by treatment with picroliv during normoxia and hypoxia in HUVEC and Hep 3B cells and on reoxygenation the expression of these genes was significantly reduced as revealed by mRNA analysis using RT-PCR. Simultaneous treatment with picroliv during hypoxia inhibited VEGF and HIF-1 expression in Glioma cells whereas the expression was not reduced by picroliv treatment during reoxygenation as evidenced by both RT-PCR and Northern hybridization. VEGF expression as revealed by immunofluorescence studies correlates well with the regulations observed in the mRNA expression. We have also examined the kinase activity of tyrosine phosphorylated proteins and protein kinase C (PKC) in Glioma cells treated with picroliv during hypoxia/reoxygenation. A selective inhibition of protein tyrosine kinase activity leading to tyrosine dephosphorylation of several proteins including 80 kd protein, and a reduction in PKC was seen in cells treated with picroliv and hypoxia. These findings suggest that picroliv may act as a protective agent against hypoxia/reoxygenation induced injuries, and the underlying mechanism may involve a novel signal transduction pathway.
Mol Cell Biochem 1999 Apr
PMID:Picroliv -- a natural product protects cells and regulates the gene expression during hypoxia/reoxygenation. 1039 Nov 50

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.
Mol Cell Biol Res Commun 1999 Aug
PMID:Nuclear localization of hypoxia-inducible factor-2alpha in bovine arterial endothelial cells. 1054 35

Vascular endothelial growth factor (VEGF) is a potent mediator of endothelial barrier dysfunction, and is upregulated during ischemia in many organs. Because ventilated pulmonary ischemia causes a marked increase in pulmonary vascular permeability, we hypothesized that VEGF would increase during ischemic lung injury. To test this hypothesis, we measured VEGF expression by Northern and Western blot analysis in isolated ferret lungs after 45 (n = 12) or 180 (n = 12) min of ventilated (95% or 0% O(2)) ischemia. Pulmonary vascular permeability, assessed by measurement of osmotic reflection coefficient for albumin (sigma(alb)), was evaluated in the same lungs, as was expression of the transcription factor, hypoxia-inducible factor (HIF)-1alpha. Distribution of VEGF as a function of ischemic time and oxygen tension was also evaluated by immunohistochemical staining in separate groups of lungs (n = 3). VEGF messenger RNA (mRNA) increased 3-fold by 180 min of ventilated ischemia, independent of oxygen tension. VEGF protein increased in parallel to mRNA. Immunohistochemical staining demonstrated the appearance of VEGF protein along alveolar septae after 180 min of hyperoxic ischemia, and after 45 or 180 min of hypoxic ischemia. sigma(alb) was not altered by 45 min of hyperoxic ischemia (0.69+/-0.09 versus 0.50+/-0.12, respectively), but decreased significantly after 180 min of hyperoxic ischemia and after 45 and 180 min of hypoxic ischemia (0.20+/-0.03, 0.26+/-0.08, and 0.23+/-0.03, respectively; P<0.05). HIF-1alpha mRNA increased during both hyperoxic and hypoxic ischemia, but HIF-1alpha protein increased only during hypoxic ischemia. These results implicate VEGF as a potential mediator of increased pulmonary vascular permeability in this model of acute lung injury.
Am J Respir Cell Mol Biol 2000 Mar
PMID:Oxygen-independent upregulation of vascular endothelial growth factor and vascular barrier dysfunction during ventilated pulmonary ischemia in isolated ferret lungs. 1069 63

To test the genetic capacity of the perinatal lung to respond to O(2) shifts that coincide with the first respiratory movements, rat fetal alveolar type II (fATII) epithelial cells were cultured at fetal distal lung PO(2) (23 Torr) and then exposed to postnatal (23 --> 76 Torr; mild hyperoxic shift), moderate (23 --> 152 Torr; moderate hyperoxic shift), or severe (23 --> 722 Torr; severe hyperoxic shift) oxygenation. Nuclear abundance and consensus binding characteristics of hypoxia-inducible factor (HIF)-1alpha and nuclear factor (NF)-kappaB (Rel A/p65) plus glutathione biosynthetic capacity were determined. Maximal HIF-1alpha activation at 23 Torr was sustained over the postnatal shift in (Delta) PO(2) and was elevated in vivo throughout late gestation. NF-kappaB was activated by the acute postnatal DeltaPO(2) in fATII cells, becoming maximal with moderate and severe oxygenation in vitro and within 6 h of birth in vivo, declining thereafter. fATII cell and whole lung glutathione and GSH-to-GSSG ratio increased fourfold with a postnatal DeltaPO(2) and were matched by threefold activity increases in gamma-glutamylcysteine synthetase and glutathione synthase. GSH concentration depletion by L-buthionine-(S, R)-sulfoximine abrogated both HIF-1alpha and NF-kappaB activation, with HIF-1alpha showing a heightened sensitivity to GSH concentration. We conclude that O(2)-linked genetic regulation in perinatal lung epithelium is responsive to developmental changes in glutathione biosynthetic capacity.
Am J Physiol Lung Cell Mol Physiol 2000 Mar
PMID:O(2)-evoked regulation of HIF-1alpha and NF-kappaB in perinatal lung epithelium requires glutathione biosynthesis. 1071 May 21

Tumor progression occurs as a result of the clonal selection of cells in which somatic mutations have activated oncogenes or inactivated tumor suppressor genes leading to increased proliferation and/or survival within the hypoxic tumor microenvironment. Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that mediates adaptive responses to reduced O2 availability, including angiogenesis and glycolysis. Expression of the O2-regulated HIF-1alpha subunit and HIF-1 transcriptional activity are increased dramatically in hypoxic cells. Recent studies indicate that many common tumor-specific genetic alterations also lead to increased HIF-1alpha expression and/or activity. Thus, genetic and physiologic alterations within tumors act synergistically to increase HIF-1 transcriptional activity, which appears to play a critical role in the development of invasive and metastatic properties that define the lethal cancer phenotype.
Crit Rev Biochem Mol Biol 2000
PMID:Hypoxia, clonal selection, and the role of HIF-1 in tumor progression. 1082 78

Little is known about the molecular mechanisms that control adrenomedullin (AM) production in human cancers. We demonstrate here that the expression of AM mRNA in a variety of human tumor cell lines is highly induced in a time-dependent manner by reduced oxygen tension (1% O2) or exposure to hypoxia mimetics such as desferrioxamine mesylate (DFX) or CoCl2. This AM expression seems to be under hypoxia-inducible factor-1 (HIF-1) transcriptional regulation, since HIF-1alpha and HIF-1beta knockout mouse cell lines had an ablated or greatly reduced hypoxia AM mRNA induction. Similarly, inhibition or enhancement of HIF-1 activity in human tumor cells showed an analogous modulation of AM mRNA. Under hypoxic conditions, immunohistochemical analysis of tumor cell lines revealed elevated levels of AM and HIF-1alpha as compared with normoxia, and we also found an increase of immunoreactive AM in the conditioned medium of tumor cells analyzed by RIA. AM mRNA stabilization was shown to be partially responsible for the hypoxic up-regulated expression of AM. In addition, we have identified several putative hypoxia response elements (HREs) in the human AM gene, and reporter studies with selected HREs were capable of enhancing luciferase expression after exposure to DFX. Furthermore, transient coexpression of HIF-1alpha resulted in an augmented transactivation of the reporter gene after DFX treatment. Given that most solid human tumors have focal hypoxic areas and that AM functions as a mitogen, angiogenic factor, and apoptosis-survival factor, our findings implicate the HIF-1/AM link as a possible promotion mechanism of carcinogenesis.
Mol Endocrinol 2000 Jun
PMID:Hypoxia-inducible factor-1 (HIF-1) up-regulates adrenomedullin expression in human tumor cell lines during oxygen deprivation: a possible promotion mechanism of carcinogenesis. 1084 87

Endothelin-1 (Et-1) is a vasoconstrictor peptide that plays an important role in the pathophysiology of hypertension, myocardial ischemia, and other diseases. We examined the mechanism of regulation the Et-1 mRNA expression in human microvascular endothelial cells (HMEC-1) in response to hypoxia and cobalt. To determine whether the 5'-flanking region of Et-1 gene mediate transcriptional responses to cellular hypoxia, we constructed reporter plasmids in which Et-1 5'-flanking sequences of Et-1 gene were fused to luciferase coding sequences. Constructs, which contain native Et-1 sequence 5'-AACGTGCA-3', located between -118 and -125 in the opposite orientation as the transcriptional unit, mediate transcriptional response to hypoxia and cobalt. This responsiveness was inhibited by genistein, a tyrosine kinase selective inhibitor. Both hypoxia and cobalt induced binding of HIF-1 (hypoxia inducible-1 factor) to this Et-1 hypoxia responsive element in gel shift assays. Mutation in this sequence eliminated both the hypoxia-induced HIF-1 binding and luciferase expression. Using the supershift assay we have shown that this hypoxia responsive element binds HIF-1alpha and HIF-1beta proteins. Interestingly, genistein only slightly affected HIF-1 binding. These results indicate that the Et-1 gene contains HIF-1 binding hypoxia responsive elements which mediate transcriptional responses to hypoxia and cobalt in microvascular endothelial cells. Genistein appears to inhibit this response by affecting the transcriptional activity of the HIF-1 complex, without significantly affecting its DNA-binding properties.
Mol Cell Biochem 2000 May
PMID:Regulation of endothelin-1 gene expression in human microvascular endothelial cells by hypoxia and cobalt: role of hypoxia responsive element. 1093 28

Oxygen is crucial to aerobic metabolism, but excesses of oxygen or reactive oxygen species (ROS) can injure cells. This minireview addresses two transcription factors that regulate several cellular responses to oxygen tension. Hypoxia inducible factor-1 (HIF-1) is a heterodimeric protein activated by hypoxia. Levels of HIF-1 are regulated by removal of the HIF-1alpha subunit through ubiquination and proteasomal destruction under normoxic conditions. Hypoxia inhibits the ubiquination of HIF-1alpha, preventing its destruction and allowing it to bind to hypoxia-responsive elements in gene promoter, enhancer, and intronic sequences. HIF-1 induces the expression of the hypoxia responsive genes vascular endothelial growth factor and erythropoietin. Its dysregulation has been implicated in von Hippel-Lindau disease. Nuclear factor kappaB (NFkappaB) is a family of pleotropic, dimeric transcription factors, and has a complex pattern of regulation. Under normoxic conditions, NFkappaB is bound to one of several inhibitory proteins (e.g., IkappaB) that prevent its nuclear translocation. Hyperoxia or elevations of ROS cause the ubiquination and destruction of the inhibitory proteins, freeing NFkappaB and allowing it to bind to target gene promoters. Hyperoxia in cell and animal models and acute lung injury in humans induce the expression of multiple proinflammatory cytokines through NFkappaB-dependent mechanisms. Although HIF-1 and NFkappaB respond to changes in pO(2), the precise nature of the oxygen sensing and transduction pathways is unclear in both cases. Both heme-protein and redox-sensitive mechanisms have been proposed. Improved understanding of oxygen-sensitive gene regulation may suggest targeted therapies for human disease.
Mol Genet Metab
PMID:Oxygen regulation of gene expression: a study in opposites. 1100 29


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