Hypoxia-inducible Factor-1 DNA-binding Activity Research Articles

Distinct APE1 Activities Affect the Regulation of VEGF Transcription Under Hypoxic Conditions

Angiogenesis is essential for tumor growth. Vascular endothelial growth factor (VEGF), a crucial factor in tumor angiogenesis, has been reported to be transcriptionally regulated by hypoxia-inducible factor-1 (HIF-1). An 8-oxo-G or apurinic/apyrimidinic (AP) site, which is frequently associated with DNA damage, has been identified in the promoter region of VEGF. However, the detailed molecular mechanisms by which AP sites regulate VEGF gene transcription are largely unknown. The dual functional protein apurinic/apyrimidinic endonuclease 1 (APE1) is both the key enzyme in DNA base excision repair and the redox factor shown to regulate HIF-1 DNA-binding activity. In the present study, we tested the involvement of both the AP endonuclease and redox activity of APE1 in regulating HIF-1 DNA binding and VEGF transcription in HUVECs. By employing two APE1 activity-specific inhibitors and AP-site-containing reporter constructs, we confirmed that both activities of APE1 were involved in regulating VEGF expression under hypoxic conditions. Furthermore, we found that the interaction between APE1 and its downstream repair enzyme, DNA polymerase β, was compromised when the N-terminal structure of APE1 was distorted under oxidative conditions. Our data suggest that the DNA repair and redox activity of APE1 can play a collaborative role in regulating the transcriptional initiation of the AP-site-containing promoter.

Hypoxia-inducible factor 1 mediates hypoxia-enhanced synthesis of progesterone during luteinization of granulosa cells.

Hypoxia has been suggested to enhance progesterone (P4) synthesis in luteinizing granulosa cells (GCs), but the mechanism is unclear. The present study was designed to test the hypothesis that the hypoxia-induced increase in P4 synthesis during luteinization in bovine GCs is mediated by hypoxia-inducible factor 1 (HIF-1). GCs obtained from small antral follicles were cultured with 2 µg/ml insulin in combination with 10 µM forskolin for 24 h as a model of luteinizing GCs. To examine the influence of HIF-1 on P4 synthesis, we determined the effect of changes in protein expression of the α-subunit of HIF-1 (HIF1A) on P4 production and on the expression levels of StAR, P450scc, and 3β-HSD. CoCl2 (100 µM), a hypoxia-mimicking chemical, increased HIF-1α protein expression in luteinizing GCs. After the upregulation of HIF-1α, we observed an increase in P4 production and in the gene and protein expression levels of StAR in CoCl2-treated luteinizing GCs. In contrast, CoCl2 did not affect the expression of either P450scc or 3β-HSD. Echinomycin, a small-molecule inhibitor of HIF-1′s DNA-binding activity, attenuated the effects of CoCl2 and of low oxygen tension (10% O2) on P4 production and StAR expression in luteinizing GCs. Overall, these findings suggest that HIF-1 is one of the factors that upregulate P4 in GCs during luteinization.

Effects of Echinomycin on PCNA-Dependent Follicular Development in PMSG-Induced Sprague-Dawley Rats

Echinomycin (Ech) is a small-molecule inhibitor of hypoxia-inducible factor-1 DNA-binding activity, which plays a crucial role in the regulation of ovarian functions in mammals. The present study was designed to test the hypothesis that hypoxia-inducible factor (HIF)-1alpha-mediated proliferation cell nuclear antigen (PCNA) expressions contributed to the follicular development in the rat ovary primed by pregnant mare serum gonadotropin (PMSG). Through the histological examination, the decrease of growing and antral follicle numbers was found after Ech treatment both in control and PMSG treated groups. And then PCNA mRNA and protein expressions were found to significantly increase in the ovaries treated with PMSG, and the similar changes were found in HIF-1alpha mRNA and protein expressions, indicating PMSG-induced follicular development may be through HIF-1alpha/PCNA signaling. Furthermore, PCNA expression was found to significantly decrease in the ovaries after Ech treatment, while HIF-1alpha mRNA and protein expression was no obviously changes. Further analysis found the changes of PCNA expression were consistent with HIF-1 activity in the ovaries, further suggesting the regulatory roles in the follicular development. Taken together, these results demonstrated this HIF-1alpha-mediated PCNA expression is one of the important mechanisms regulating the ovarian follicular development in mammals. Keywords: HIF-1alpha; PCNA; echinomycin; HIF prolyl hyodroxylase acitvity; follicular development

Persisting mild hypothermia suppresses hypoxia-inducible factor-1α protein synthesis and hypoxia-inducible factor-1-mediated gene expression

The transcription factor hypoxia-inducible factor-1 (HIF-1) plays an essential role in regulating gene expression in response to hypoxia-ischemia. Ischemia causes the tissue not only to be hypoxic but also to be hypothermic because of the hypoperfusion under certain circumstances. On the other hand, the induced hypothermia is one of the most common therapeutic modalities to extend tolerance to hypoxia. Although hypoxia elicits a variety of cellular and systemic responses at different organizational levels in the body, little is known about how hypoxia-induced responses are affected by low temperature. We examined the influence of mild hypothermic conditions (28–32°C) on HIF-1 in both in vitro and in vivo settings. In vitro experiments adopting cultured cells elucidated that hypoxia-induced HIF-1 activation was resistant to 4-h exposure to the low temperature. In contrast, exposure to the low temperature as long as 24 h suppressed HIF-1 activation and the subsequent upregulation of HIF-1 target genes such as VEGF or GLUT-1. HIF-1α protein stability in the cell was not affected by hypothermic treatment. Furthermore, intracellular ATP content was reduced under 1% O2 conditions but was not largely affected by hypothermic treatment. The evidence indicates that reduction of oxygen consumption is not largely involved in suppression of HIF-1. In addition, we demonstrated that HIF-1 DNA-binding activity and HIF-1-dependent gene expressions induced under 10% O2 atmosphere in mouse brain were not influenced by treatment under 3-h hypothermic temperature but were inhibited under 5-h treatment. On the other hand, we indicated that warming ischemic legs of mice for 24 h preserved HIF-1 activity. In this report we describe for the first time that persisting low temperature significantly reduced HIF-1α neosynthesis under hypoxic conditions, leading to a decrease in gene expression for adaptation to hypoxia in both in vitro and in vivo settings.

Regulation of Vascularization by Hypoxia‐Inducible Factor 1

Vascularization and vascular remodeling represent critical adaptive responses to tissue hypoxia that are mediated by hypoxia-inducible factor 1 (HIF-1). In patients with peripheral arterial disease, these responses are impaired by aging and diabetes, leading to critical limb ischemia and amputation. Intramuscular injection of an adenovirus encoding a constitutively active form of the HIF-1alpha subunit (CA5) increases the recovery of blood flow following femoral artery ligation in a mouse model of age-dependent critical limb ischemia. Intradermal injection of a plasmid encoding CA5 promotes healing of cutaneous wounds in a mouse model of diabetes. In cancer, vascularization is required for tumors to grow beyond microscopic size, a process that involves HIF-1-dependent production of angiogenic growth factors. Daily treatment of prostate cancer xenograft-bearing mice with low-dose anthracycline (doxorubicin or daunorubicin) chemotherapy inhibits HIF-1 DNA-binding activity, HIF-1-dependent expression of angiogenic growth factors, mobilization of circulating angiogenic cells, and tumor vascularization, thereby arresting tumor growth.

Inhibitory effect of zinc on hypoxic HIF-1 activation in astrocytes

Hypoxia-inducible factor-1 (HIF-1) regulates the expression of neuroprotective genes such as erythropoietin (EPO). We investigated the mechanism by which zinc, an excitotoxin-like metal, regulates HIF-1 under hypoxic conditions in astrocytes. In hypoxic LN215 cells, HIF-1alpha stabilized and accumulated in the nucleus, resulting in an increase in its DNA-binding activity to the EPO enhancer. Zinc inhibited hypoxia-induced increases in HIF-1 DNA-binding activity and the HIF-1-dependent mRNA expression of EPO. Zinc did not affect hypoxic stabilization of HIF-1alpha. Nuclear migration of HIF-1alpha upon hypoxia was reduced by zinc. Complete blockade of hypoxia-induced assembly of HIF-1alpha-HIF-1beta complex was observed after treatment of zinc. These findings suggest that zinc hampers hypoxia-stimulated HIF-1 activation in astrocytes by inhibiting nuclear HIF-1alpha translocation and subsequently disrupting HIF-1 heterodimerization.

Hypoxia induced HIF-1 accumulation and VEGF expression in gastric epithelial mucosa cells: Involvement of ERK1/2 and PI3K/Akt

Hypoxia is a common environmental stress that influences signaling pathways and cells function, which through initiating intracellular signaling pathways and hence leading to the activation of the transcription factor hypoxia-inducible factor-1 (HIF-1). In this study, we initially confirm that hypoxia activates HIF-1alpha protein expression in a time-dependent manner with a maximum reached at 60 min in vitro and 4h in vivo in gastric mucosa epithelial cells. The expression of HIF-1alpha is correlated with the activation of HIF-1 DNA binding and transcriptional activity. Hypoxia dose not affect HIF-1alpha mRNA transcription but regulates HIF-1alpha protein expression through a translation-dependent pathway to regulate protein synthesis. Hypoxia could induce phosphorylation of Akt, MAPK (ERK), and target of p70S6K1. PI3K and MAPK inhibitor, LY294002 and U0126 could inhibit hypoxia-induced HIF-1 and VEGF expression. We also investigated the role of reactive oxygen species (ROS) involved in HIF-1 and VEGF expression Exogenous addition of H2O2 was sufficient to activate Akt and ERK, scavengers of H2O2 significantly inhibited hypoxia-induced Akt and ERK, and subsequent HIF-lax expression and transcriptional activity. In conclusion, our data suggested that hypoxia- PI3K signaling through Akt and ERK kinases regulated ROS-dependent, hypoxia- induced HIF-1 activation and VEGF expression in gastric mucosa epithelial cells.

Synergystic induction of HIF-1α transcriptional activity by hypoxia and lipopolysaccharide in macrophages

Hypoxia Inducible Factor-1 (HIF-1) is activated by a variety of stimuli, including inflammatory mediators. In this report we investigated the role that bacterial lipopolysaccharide (LPS) and hypoxia play in the regulation of HIF-1-dependent gene expression in macrophages. We report that murine macrophages stimulated with low concentrations of LPS (1-10 ng/ml) expressed significantly higher levels of inducible nitric oxide synthase (iNOS) mRNA when cultured under hypoxic compared to normoxic conditions. Functional studies of the iNOS promoter demonstrated that the synergistic interaction between LPS and hypoxia was mediated, at least in part, by the NFκB and the HIF-1 binding sites. In addition, transient transfection experiments using a Hypoxia Response Element (HRE)-containing plasmid showed that LPS and hypoxia synergistically induced HIF-1-dependent transcriptional activity. Interestingly, LPS did not significantly affect HIF-1α protein levels or HIF-1 DNA binding activity relative to hypoxic induction. HIF-1α, but not HIF-2α, was critical for the synergistic induction of HRE-dependent transcriptional activity in macrophages, as indicated by experiments using siRNA targeting HIF-1α or HIF-2α. Addition of ROS-scavengers completely abrogated the synergistic induction of HIF-1 transcriptional activity by LPS and hypoxia, but neither inhibited HIF-1 transcriptional activity induced by hypoxia alone nor affected HIF-1α protein levels or HIF-1 DNA binding induced by hypoxia alone or hypoxia plus LPS. Taken together, our results demonstrate that LPS and hypoxia act synergistically to induce HIF-1α-transcriptional activity and they emphasize the existence of a cross talk between hypoxic and non-hypoxic signaling pathways in the regulation of macrophages gene expression.

Dual effect of echinomycin on hypoxia‐inducible factor‐1 activity under normoxic and hypoxic conditions

Hypoxia-inducible factor-1 (HIF-1) is now recognized as a possible target for cancer treatment. This transcription factor is responsible for the overexpression of several genes favouring cancer cell survival and inducing neo-angiogenesis. Echinomycin has recently been described to inhibit HIF-1 DNA binding and transcriptional activity. In this work, it is shown that echinomycin strongly inhibits the activity of HIF-1 under hypoxic conditions, and also interferes with the activity of other transcription factors. These results demonstrate the lack of specificity of this molecule. Moreover, it is demonstrated that echinomycin induces an increase in HIF-1 activity under normoxic conditions, parallel to an increase in the expression of HIF-1 target genes. This effect is caused by an increase in HIF-1alpha protein level, resulting from an increase in the transcription of the HIF-1A gene in the presence of a low concentration of echinomycin. Transfection experiments with HIF-1alpha promoter constructs revealed the presence of an Sp1 binding element responsive to echinomycin. Furthermore, echinomycin enhanced Sp1 activity, as measured by the use of a specific reporter system. These findings show, for the first time, that echinomycin has a dual effect on HIF-1 activity under normoxic and hypoxic conditions, demonstrating that this molecule cannot be used in cancer treatment.

Adenosine A2a receptor-mediated, normoxic induction of HIF-1 through PKC and PI-3K-dependent pathways in macrophages

Adenosine released by cells in injurious or hypoxic environments has tissue-protecting and anti-inflammatory effects, which are also a result of modulation of macrophage functions, such as vascular endothelial growth factor (VEGF) production. As VEGF is a well-known target of hypoxia-inducible factor 1 (HIF-1), we hypothesized that adenosine may activate HIF-1 directly. Our studies using subtype-specific adenosine receptor agonists and antagonists showed that by activating the A(2A) receptor, adenosine treatment induced HIF-1 DNA-binding activity, nuclear accumulation, and transactivation capacity in J774A.1 mouse macrophages. Increased HIF-1 levels were also found in adenosine-treated mouse peritoneal macrophages. The HIF-1 activation induced by the A(2A) receptor-specific agonist CGS21680 required the PI-3K and protein kinase C pathways but was not mediated by changes in iron levels. Investigation of the molecular basis of HIF-1 activation revealed the involvement of transcriptional and to a larger extent, translational mechanisms. HIF-1 induction triggered the expression of HIF-1 target genes involved in cell survival (aldolase, phosphoglycerate kinase) and VEGF but did not induce inflammation-related genes regulated by HIF-1, such as TNF-alpha or CXCR4. Our results show that the formation of adenosine and induction of HIF-1, two events which occur in response to hypoxia, are linked directly and suggest that HIF-1 activation through A(2A) receptors may contribute to the anti-inflammatory and tissue-protecting activity of adenosine.

The hypoxia-inducible factor HIF-1 promotes intramyocardial expression of VEGF in infants with congenital cardiac defects

The response to hypoxia is primarily mediated by the transcription factor hypoxia-inducible factor-1 (HIF-1) which leads to the induction of a variety of adaptive gene products including vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS). This study was designed to test the hypothesis that HIF-1 and its target genes would be upregulated in the ventricular myocardium of infants with cyanotic congenital cardiac defects. 14 infants with cyanotic (n = 7) or acyanotic cardiac defects (n = 7) were investigated. Samples from the right ventricular myocardium taken immediately after aortic clamping were studied for protein expression and DNA-binding activity. Protein levels of HIF-1alpha were significantly elevated in patients with cyanotic compared to acyanotic congenital heart disease and inversely correlated with the degree of hypoxemia. This response was accompanied by significantly enhanced HIF-1 DNA binding activity. Furthermore, protein levels of VEGF and eNOS were significantly higher in the myocardium of cyanotic than of acyanotic infants. To test the potential involvement of upstream regulatory pathways, activation of MAP kinases was determined. Intramyocardial levels of phosphorylated p38 MAP kinase, but not of ERK1/2 were significantly higher in infants with cyanotic compared to those with acyanotic congenital heart disease and inversely correlated to hypoxemia. These findings show that chronic hypoxemia is associated with the induction and stabilization of the transcription factor HIF-1 as well as its target genes VEGF and eNOS in the myocardium of infants with cyanotic cardiac defects. Thus, stabilization of HIF-1 and induction of the adaptive hypoxia response could particularly participate in myocardial remodeling in children with congenital cardiac defects and chronic hypoxemia.

The hypoxia-inducible factor HIF-1 promotes intramyocardial expression of VEGF in infants with congenital cardiac defects

The response to hypoxia is primarily mediated by the transcription factor hypoxia-inducible factor-1 (HIF-1) which leads to the induction of a variety of adaptive gene products including vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS). This study was designed to test the hypothesis that HIF-1 and its target genes would be upregulated in the ventricular myocardium of infants with cyanotic congenital cardiac defects. 14 infants with cyanotic (n = 7) or acyanotic cardiac defects (n = 7) were investigated. Samples from the right ventricular myocardium taken immediately after aortic clamping were studied for protein expression and DNA-binding activity. Protein levels of HIF-1α were significantly elevated in patients with cyanotic compared to acyanotic congenital heart disease and inversely correlated with the degree of hypoxemia. This response was accompanied by significantly enhanced HIF-1 DNA binding activity. Furthermore, protein levels of VEGF and eNOS were significantly higher in the myocardium of cyanotic than of acyanotic infants. To test the potential involvement of upstream regulatory pathways, activation of MAP kinases was determined. Intramyocardial levels of phosphorylated p38 MAP kinase, but not of ERK1/2 were significantly higher in infants with cyanotic compared to those with acyanotic congenital heart disease and inversely correlated to hypoxemia. These findings show that chronic hypoxemia is associated with the induction and stabilization of the transcription factor HIF-1 as well as its target genes VEGF and eNOS in the myocardium of infants with cyanotic cardiac defects. Thus, stabilization of HIF-1 and induction of the adaptive hypoxia response could particularly participate in myocardial remodeling in children with congenital cardiac defects and chronic hypoxemia.

IL-4 enhances hypoxia induced HIF-1α protein levels in human transformed intestinal cells

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that mediates the adaptive response to hypoxia. Increasing evidence suggests a crucial role for HIF-1 in immune reactions. Here we investigated the effect of the Th2 type cytokines IL-4 and IL-10 on HIF-1α mediated response in normoxia (21% O 2) and hypoxia (1% O 2). Incubation of human transformed intestinal cells (HT-29) with IL-4 significantly increased HIF-1α protein levels during hypoxia but not during normoxia. Mechanisms involved are IL-4 induced up-regulation of HIF-1α gene transcription and the PI3K signaling pathway. The increase in hypoxia-induced accumulation of HIF-1α protein after IL-4 treatment did not result in up-regulation of HIF-1 DNA-binding activity or HIF-1 dependent gene expression. IL-10 did not affect HIF-1α protein levels.

LPS Increases Hepatic HIF-1α Protein and Expression of the HIF-1-Dependent Gene Aldolase A in Rats

Cellular adaptation to hypoxia is mediated in part by the transcription factor hypoxia-inducible factor 1 (HIF-1). Accumulating data suggest that pro-inflammatory mediators can up-regulate HIF-1alpha protein expression and HIF-1 DNA-binding activity in the absence of hypoxia. Accordingly, we investigated HIF-1 mediated signaling in endotoxemic rats. We studied three groups of male Sprague Dawley rats. Controls (N = 5) were injected i.p. with saline. Endotoxemic rats (N = 9) received a sublethal dose of lipopolysaccaride (Escherichia coli; 5 mg/kg, i.p.). A third group of rats (N = 5) received the HIF-1 stabilizing agent CoCl(2) (14 mg/kg, i.p.) at T = 0 h and T = 16 h. At T = 18 h, liver microvascular perfusion was measured using laser Doppler flowmetry and hepatic tissue samples were obtained. RNA was isolated and mRNA levels of the HIF-1 dependent genes aldolase A and vascular endothelial growth factor (VEGF) were determined using quantitative real-time RT-PCR. HIF-1alpha content was estimated by immunoprecipitation followed by Western blotting. HIF-1alpha increased in hepatic tissue after treatment with LPS or CoCl(2). LPS markedly increased hepatic expression of aldolase A, but failed to alter expression of VEGF. CoCl(2) increased aldolase A and VEGF mRNA expression. Although hepatic microvascular perfusion was comparable in saline- and LPS-treated rats, hepatic microvascular blood flow and aldolase A expression were significantly inversely correlated among endotoxemic rats (r = 0.773; P = 0.003). Increased expression of aldolase A in endotoxemic rats is mediated by both hypoxia-dependent and hypoxia-independent mechanisms.

Synergistic effects of CoCl2 and ROCK inhibition on mesenchymal stem cell differentiation into neuron-like cells

Bone-marrow-derived mesenchymal stem cells (MSCs) constitute an interesting cellular source to promote brain regeneration after neurodegenerative diseases. Recently, several studies suggested that oxygen-dependent gene expression is of crucial importance in governing the essential steps of neurogenesis such as cell proliferation, survival and differentiation. In this context, we analysed the effect of the HIF-1 (hypoxia inducible factor-1) activation-mimicking agent CoCl(2) on MSCs. CoCl(2) treatment increased the expression of the anti-proliferative gene BTG2/PC3 and decreased cyclin D1 expression. Expression of HIF-1alpha and its target genes EPO, VEGF and p21 was also upregulated. These changes were followed by inhibition of cell proliferation and morphological changes resulting in neuron-like cells, which had increased neuronal marker expression and responded to neurotransmitters. Echinomycin, a molecule inhibiting HIF-1 DNA-binding activity, blocked the CoCl(2) effect on MSCs. Additionally, by using Y-27632, we demonstrated that Rho kinase (ROCK) inhibition potentiated CoCl(2)-induced MSC differentiation in particular into dopaminergic neuron-like cells as attested by its effect on tyrosine hydroxylase expression. Altogether, these results support the ability of MSCs to differentiate into neuron-like cells in response to CoCl(2), an effect that might act, in part, through HIF-1 activation and cell-cycle arrest, and which is potentiated by inhibition of ROCK.

Echinomycin, a Small-Molecule Inhibitor of Hypoxia-Inducible Factor-1 DNA-Binding Activity

The identification of small molecules that inhibit the sequence-specific binding of transcription factors to DNA is an attractive approach for regulation of gene expression. Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that controls genes involved in glycolysis, angiogenesis, migration, and invasion, all of which are important for tumor progression and metastasis. To identify inhibitors of HIF-1 DNA-binding activity, we expressed truncated HIF-1alpha and HIF-1beta proteins containing the basic-helix-loop-helix and PAS domains. Expressed recombinant HIF-1alpha and HIF-1beta proteins induced a specific DNA-binding activity to a double-stranded oligonucleotide containing a canonical hypoxia-responsive element (HRE). One hundred twenty-eight compounds previously identified in a HIF-1-targeted cell-based high-throughput screen of the National Cancer Institute 140,000 small-molecule library were tested in a 96-well plate ELISA for inhibition of HIF-1 DNA-binding activity. One of the most potent compounds identified, echinomycin (NSC-13502), a small-molecule known to bind DNA in a sequence-specific fashion, was further investigated. Electrophoretic mobility shift assay experiments showed that NSC-13502 inhibited binding of HIF-1alpha and HIF-1beta proteins to a HRE sequence but not binding of the corresponding proteins to activator protein-1 (AP-1) or nuclear factor-kappaB (NF-kappaB) consensus sequences. Interestingly, chromatin immunoprecipitation experiments showed that NSC-13502 specifically inhibited binding of HIF-1 to the HRE sequence contained in the vascular endothelial growth factor (VEGF) promoter but not binding of AP-1 or NF-kappaB to promoter regions of corresponding target genes. Accordingly, NSC-13502 inhibited hypoxic induction of luciferase in U251-HRE cells and VEGF mRNA expression in U251 cells. Our results indicate that it is possible to identify small molecules that inhibit HIF-1 DNA binding to endogenous promoters.

Angiotensin II Regulates Vascular Endothelial Growth Factor via Hypoxia-Inducible Factor-1α Induction and Redox Mechanisms in the Kidney

Angiotensin II (AngII) is a cytokine that participates in renal damage. Vascular endothelial growth factor (VEGF) is constitutively expressed in the kidney and is involved in the progression of renal disease. The aim of this work was to investigate the relation between AngII and VEGF and the mechanisms involved in its regulation in the kidney. We have observed that in cultured tubuloepithelial cells (NRK52E cell line) AngII increased VEGF gene expression and promoter activation. Hypoxia-inducible factor-1 (HIF-1) is one of the main VEGF gene activators. AngII induces HIF-1alpha protein production and increases HIF-1 DNA-binding activity. An antisense HIF-1alpha oligodeoxynucleotide inhibited AngII-induced VEGF overexpression. The reactive oxygen species act as second messengers in kidney damage caused by AngII and mediate the induction of HIF-1 by cytokines. In tubuloepithelial cells, VEGF up-regulation and HIF-1alpha induction due to AngII were significantly diminished by antioxidants, suggesting a redox-mediated mechanism. Infusion of AngII into mice caused renal VEGF overexpression, HIF-1 activation, and oxidative stress. In summary, these data show that AngII in vivo and in vitro up-regulates renal VEGF expression by a mechanism that involves HIF-1 activation and oxidative stress.

Hypoxia-inducible Factor-1-dependent Overexpression of Myeloid Cell Factor-1 Protects Hypoxic Cells against tert-Butyl Hydroperoxide-induced Apoptosis

Increased levels of Mcl-1 (myeloid cell factor-1) have been reported in several cancers, suggesting an important role played by Mcl-1 in cancer cell survival. Mcl-1 is an anti-apoptotic protein shown to delay or block apoptosis. In this work, using semiquantitative immunofluorescence, real-time PCR, and RNase protection assay, an increase in Mcl-1 expression was detected in hepatoma HepG2 cells incubated under hypoxia or in the presence of cobalt chloride. Through analysis of the Mcl-1 promoter sequence, a putative HIF-1 (hypoxiainducible factor-1) binding site was identified. A Mcl-1 promoter fragment containing this hypoxia-responsive element was able to bind HIF-1 in vitro. It also induced hypoxia-dependent transcription of a luciferase reporter gene, which was suppressed by anti-HIF-1alpha short interfering RNA. Finally, overexpression of Mcl-1 protected HepG2 cells against apoptosis induced by tert-butyl hydroperoxide as shown by inhibition of caspase-3 activation and DNA fragmentation. All these data suggest a potential anti-apoptotic role of HIF-1 that could protect cells against apoptosis under hypoxia by overexpression of the Mcl-1 protein.

Thyroid hormone induces erythropoietin gene expression through augmented accumulation of hypoxia-inducible factor-1.

Oxygen is of vital importance for the metabolism and function of all cells in the human body. Hypoxia, the reduction of oxygen supply, results in adaptationally appropriate alterations in gene expression through the activation of hypoxia-inducible factor 1 (HIF-1) to overcome any shortage of oxygen. Thyroid hormones are required for normal function of nearly all tissues, with major effects on oxygen consumption and metabolic rate. Thyroid hormones have been found to augment the oxygen capacity of the blood by increasing the production of erythropoietin (EPO) and to improve perfusion by vasodilation through the augmented expression of adrenomedullin (ADM). Because the hypoxic expression of both genes depends on HIF-1, we studied the influence of thyroid hormone on HIF-1 activation in the human hepatoma cell line HepG2 under normoxic and hypoxic conditions. We found that thyroid hormones increased HIF-1alpha protein accumulation by increasing HIF-1alpha protein synthesis rather than attenuating its proteasomal degradation. HIF-1alpha expression directly correlated with augmented HIF-1 DNA binding and transcriptional activity of luciferase reporter plasmids, whereas HIF-1beta levels remained unaffected. Knocking down HIF-1alpha by short interfering RNA (siRNA) clearly demonstrated that thyroid hormone-induced target gene expression required the presence of HIF-1. Although an increased association of the two known coactivators of HIF-1, p300 and SRC-1, was found, thyroid hormone did not affect the activity of the isolated COOH-terminal transactivating domain of HIF-1alpha. Increased synthesis of HIF-1alpha may contribute to the adaptive response of increased oxygen demand under hyperthyroid conditions.

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.