HIF-1 DNA Binding Activity Research Articles

Epstein-Barr virus latent membrane protein 1 induces synthesis of hypoxia-inducible factor 1 alpha.

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic helix-loop-helix transcription factor composed of HIF-1 alpha and HIF-1 beta that is the central regulator of responses to hypoxia. The specific binding of HIF-1 to the hypoxia-responsive element (HRE) induces the transcription of genes that respond to hypoxic conditions, including vascular endothelial growth factor (VEGF). Here we report that expression of HIF-1 alpha is increased in diverse Epstein-Barr virus (EBV)-infected type II and III cell lines, which express EBV latent membrane protein 1 (LMP1), the principal EBV oncoprotein, as well as other latency proteins, but not in the parental EBV-negative cell lines. We show first that transfection of an LMP1 expression plasmid into Ad-AH cells, an EBV-negative nasopharyngeal epithelial cell line, induces synthesis of HIF-1 alpha protein without increasing its stability or mRNA level. The mitogen-activated protein kinase (MAPK) kinase inhibitor PD98059 markedly reduces induction of HIF-1 alpha by LMP1. Catalase, an H(2)O(2) scavenger, strongly suppresses LMP1-induced production of H(2)O(2), which results in a decrease in the expression of HIF-1 alpha induced by LMP1. Inhibition of the NF-kappa B, c-jun N-terminal kinase, p38 MAPK, and phosphatidylinositol 3-kinase pathways did not affect HIF-1 alpha expression. Moreover, LMP1 induces HIF-1 DNA binding activity and upregulates HRE and VEGF promoter transcriptional activity. Finally, LMP1 increases the appearance of VEGF protein in extracellular fluids; induction of VEGF is suppressed by PD98059 or catalase. These results suggest that LMP1 increases HIF-1 activity through induction of HIF-1 alpha protein expression, which is controlled by p42/p44 MAPK activity and H(2)O(2). The ability of EBV, and specifically its major oncoprotein, LMP1, to induce HIF-1 alpha along with other invasiveness and angiogenic factors reported previously discloses additional oncogenic properties of this tumor virus.

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

Background 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. Methods 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. Results While HIF-1α mRNA expression was unchanged, HIF-1β was downregulated ( p < 0.05) in donor versus control lungs. Protein expression of both, HIF-1α and -β 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). Conclusions 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-1α mRNA expression did not affect HIF-1α protein levels. Endothelin-1 increases HIF-1α accumulation and activates HIF-1 transcription complex in vitro. Therefore, ET-1-mediated increased HIF-1α 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.

Hypoxia-inducible Factor 1 Activation by Aerobic Glycolysis Implicates the Warburg Effect in Carcinogenesis

Cancer cells display high rates of aerobic glycolysis, a phenomenon known historically as the Warburg effect. Lactate and pyruvate, the end products of glycolysis, are highly produced by cancer cells even in the presence of oxygen. Hypoxia-induced gene expression in cancer cells has been linked to malignant transformation. Here we provide evidence that lactate and pyruvate regulate hypoxia-inducible gene expression independently of hypoxia by stimulating the accumulation of hypoxia-inducible Factor 1alpha (HIF-1alpha). In human gliomas and other cancer cell lines, the accumulation of HIF-1alpha protein under aerobic conditions requires the metabolism of glucose to pyruvate that prevents the aerobic degradation of HIF-1alpha protein, activates HIF-1 DNA binding activity, and enhances the expression of several HIF-1-activated genes including erythropoietin, vascular endothelial growth factor, glucose transporter 3, and aldolase A. Our findings support a novel role for pyruvate in metabolic signaling and suggest a mechanism by which high rates of aerobic glycolysis can promote the malignant transformation and survival of cancer cells.

Phosphatidylinositol 3-Kinase/Akt Signaling Is Neither Required for Hypoxic Stabilization of HIF-1α nor Sufficient for HIF-1-dependent Target Gene Transcription

The serine/threonine kinase Akt/PKB and the oxygen-responsive transcription factor HIF-1 share the ability to induce such processes as angiogenesis, glucose uptake, and glycolysis. Akt activity and HIF-1 are both essential for development and implicated in tumor growth. Upon activation by products of phosphatidylinositol 3-kinase (PI3K), Akt phosphorylates downstream targets that stimulate growth and inhibit apoptosis. Previous reports suggest that Akt may achieve its effects on angiogenesis and glucose metabolism by stimulating HIF-1 activity. We report here that, whereas serum stimulation can induce a slight accumulation of HIF-1 alpha protein in a PI3K/Akt pathway-dependent fashion, hypoxia induces much higher levels of HIF-1 alpha protein and HIF-1 DNA binding activity independently of PI3K and mTOR activity. In addition, we find the effects of constitutively active Akt on HIF-1 activity are cell-type specific. High levels of Akt signaling can modestly increase HIF-1 alpha protein, but this increase does not affect HIF-1 target gene expression. Therefore, the PI3K/Akt pathway is not necessary for hypoxic induction of HIF-1 subunits or activity, and constitutively active Akt is not itself sufficient to induce HIF-1 activity.

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.

Vascular endothelial growth factor gene expression in the human breast cancer cell line MX-1 is controlled by O 2 availability in vitro and in vivo

The vascular endothelial growth factor (VEGF) plays an important role in angiogenesis. Mediated by the hypoxia-inducible transcription factor HIF-1alpha/beta, a reduction in O2 tension (pO2) leads to increased VEGF gene expression in nonmalignant tissues. In tumor cells VEGF mRNA levels are often constitutively elevated. We examined pO2-dependent VEGF mRNA expression and VEGF protein formation in the human breast cancer cell line MX-1 in vitro and in vivo. For in vitro study MX-1 cultures were grown on dishes with a gas-permeable bottom to expose the cells to defined O2 concentrations (from 95% to 0%) for 4 h. Northern blot analysis showed significant VEGF mRNA in MX-1 cultures under normoxic conditions which was further increased by hypoxia. The amount of secreted VEGF was also elevated in hypoxic cultures. Western blot analysis revealed a correlation between the severity of hypoxia and HIF-1alpha protein amounts in the nucleus. Furthermore, DNA-binding activity of HIF-1 could be demonstrated by gel-shift assays. For in vivo study immunodeficient nude mice bearing MX-1 tumor transplants were exposed to inspiratory hypoxia (10% O2). Northern blot and immunohistochemical analyses of MX-1 tumor transplants showed that VEGF mRNA and VEGF protein levels were increased in mice 17 h after the induction of inspiratory hypoxia. Thus, pO2-dependence of VEGF gene expression can be maintained in cancer cells, even in vivo, which may be relevant in regard to therapeutic attempts to inhibit tumor angiogenesis by increasing tumor oxygenation.

Age-dependent Defect in Vascular Endothelial Growth Factor Expression Is Associated with Reduced Hypoxia-inducible Factor 1 Activity

Previous studies have indicated that advanced age is associated with impaired angiogenesis in part because of reduced levels of vascular endothelial growth factor (VEGF) expression. To investigate potential mechanisms responsible for this age-dependent defect in VEGF expression, aortic smooth muscle cells isolated from young rabbits (ages 6-8 months) or old rabbits (ages 4-5 years) were exposed to normoxic (21% oxygen) or hypoxic (0.1% oxygen) conditions. Hypoxia-induced VEGF expression was significantly lower in old versus young cells. VEGF mRNA stability in hypoxic conditions was similar in both young and old cells. However, transient transfection with a luciferase reporter gene that was transcriptionally regulated by the VEGF promoter revealed a significant defect in VEGF up-regulation following hypoxia in old versus young cells (a 43 versus 117% increase in luciferase activity, p < 0.05); this difference was not seen when a deletion construct lacking the hypoxia-inducible 1 (HIF-1) binding site was used. Moreover, although HIF-1 alpha-mRNA expression was shown to be similar in young and old smooth muscle cells, HIF-1 alpha protein and DNA binding activity were significantly reduced in old versus young smooth muscle cells that were exposed to hypoxia. We propose that age-dependent reduction in hypoxia-induced VEGF expression results from reduced HIF-1 activity and may explain the previously described age-dependent impairment of angiogenesis in response to ischemia.

Differential signaling pathways of HO-1 gene expression in pulmonary and systemic vascular cells.

Heme oxygenase-1 (HO-1) is induced by oxidative stress and plays an important role in cellular protection against oxidant injury. Increasing evidence also suggests that HO-1 is markedly modulated by hypoxia in vitro and in vivo. Our group has previously demonstrated that the transcription factor hypoxia-inducible factor (HIF)-1 mediates hypoxia-induced HO-1 gene transcription and expression in systemic (aortic) vascular smooth muscle (AoVSM) cells (P. J. Lee, B. -H. Jiang, B. Y. Chin, N. V. Iyer, J. Alam, G. L. Semenza, and A. M. K. Choi. J. Biol. Chem. 272: 5375-5381, 1997). Because the pulmonary circulation is an important target of hypoxia, this study investigated whether HO-1 gene expression in pulmonary arterial vascular smooth muscle was differentially regulated by hypoxia in comparison to AoVSM cells. Interestingly, hypoxia neither induced HO-1 gene expression nor increased HIF-1 DNA binding activity in pulmonary arterial vascular smooth muscle cells. Conversely, pulmonary arterial endothelial cells (PAECs) demonstrated a marked induction of HO-1 gene expression after hypoxia. Electrophoretic mobility shift assays detected an increase in activator protein-1 rather than in HIF-1 DNA binding activity in nuclear extracts of hypoxic PAECs. Analyses of the promoter and 5'-flanking regions of the HO-1 gene were performed by transiently transfecting PAECs with either the hypoxia response element (HIF-1 binding site) or the HO-1 gene distal enhancer element (AB1) linked to a chloramphenicol acetyltransferase reporter gene. Increased chloramphenicol acetyltransferase activity was observed only in transfectants containing the AB1 distal enhancer, and mutational analysis of this enhancer suggested that the activator protein-1 regulatory element was critical for hypoxia-induced HO-1 gene transcription. Collectively, our data demonstrate that the molecular regulation of HO-1 gene transcription during hypoxia differs between the systemic and pulmonary circulations and also provide evidence that hypoxia-induced HO-1 gene expression in PAECs and AoVSM cells is regulated through two discrete signaling pathways.

Perspectives on Oxygen Sensing

Perspectives on Oxygen Sensing

Co-Transactivation of the 3′ Erythropoietin Hypoxia Inducible Enhancer by the HIF-1 Protein

ABSTRACT: 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 α and β 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-1α cDNA, HIF-1β cDNA, HIF-1α and HIF-1β 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-1α cDNA alone showed a 2.2 fold increase in luciferase activity, HIF-1β showed a 3.4 fold increase and cells transfected with HIF-1 α and β 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-1α and β was noted. Under normoxic or hypoxic conditions in Cos-7 cells, a 1.5 fold increase was obtained with the HIF-1α and β 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-1α and β 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.