Regulation of a rat VL30 element in human breast cancer cells in hypoxia and anoxia: role of HIF-1

Novel approaches to cancer gene therapy currently exploit tumour hypoxia to achieve transcriptional targeting using oxygen-regulated enhancer elements called hypoxia response elements. The activity of such elements in hypoxic cells is directly dependent on upregulation of the hypoxia-inducible transcription factor-1 However tumours also contain areas of anoxia, which may be considered a more tumour-selective transcriptional stimulus than hypoxia for targeting gene therapy to tumours. Another element, from the rat virus-like retrotransposon, VL30 (termed the ‘secondary anoxia response element’) has been reported to be more highly inducible in rat fibroblasts under anoxia than hypoxia. To investigate anoxia as a potential transcriptional target in human tumours, we have examined secondary anoxia response element inducibility in two human breast cancer cell lines, MCF-7 and T47D, under anoxia, hypoxia and normoxia. In both cell types, the trimerised secondary anoxia response element showed greater inducibility in anoxia than hypoxia (1% and 0.5% O2). The anoxic response of the secondary anoxia response element was shown to be dependent on hypoxia-inducible transcription factor-1 and the presence of a hypoxia-inducible transcription binding site consensus (5′-ACGTG-3′). Mutational analysis demonstrated that the base immediately 5′ to this modulates the anoxic/hypoxic induction of the secondary anoxia response element, such that TACGTG>GACGTG>>CACGTG. A similar correlation was found for erythropoietin, phosphoglycerate kinase 1, and aldolase hypoxia response elements, which contain these respective 5′ flanking bases

Functional and Transcriptional Induction of Aquaporin-1 Gene by Hypoxia; Analysis of Promoter and Role of Hif-1α

Aquaporin-1 (AQP1) is a water channel that is highly expressed in tissues with rapid O2 transport. It has been reported that this protein contributes to gas permeation (CO2, NO and O2) through the plasma membrane. We show that hypoxia increases Aqp1 mRNA and protein levels in tissues, namely mouse brain and lung, and in cultured cells, the 9L glioma cell line. Stopped-flow light-scattering experiments confirmed an increase in the water permeability of 9L cells exposed to hypoxia, supporting the view that hypoxic Aqp1 up-regulation has a functional role. To investigate the molecular mechanisms underlying this regulatory process, transcriptional regulation was studied by transient transfections of mouse endothelial cells with a 1297 bp 5′ proximal Aqp1 promoter-luciferase construct. Incubation in hypoxia produced a dose- and time-dependent induction of luciferase activity that was also obtained after treatments with hypoxia mimetics (DMOG and CoCl2) and by overexpressing stabilized mutated forms of HIF-1α. Single mutations or full deletions of the three putative HIF binding domains present in the Aqp1 promoter partially reduced its responsiveness to hypoxia, and transfection with Hif-1α siRNA decreased the in vitro hypoxia induction of Aqp1 mRNA and protein levels. Our results indicate that HIF-1α participates in the hypoxic induction of AQP1. However, we also demonstrate that the activation of Aqp1 promoter by hypoxia is complex and multifactorial and suggest that besides HIF-1α other transcription factors might contribute to this regulatory process. These data provide a conceptual framework to support future research on the involvement of AQP1 in a range of pathophysiological conditions, including edema, tumor growth, and respiratory diseases

Expression profiling of laser-microdissected intrapulmonary arteries in hypoxia-induced pulmonary hypertension

BACKGROUND: Chronic hypoxia influences gene expression in the lung resulting in pulmonary hypertension and vascular remodelling. For specific investigation of the vascular compartment, laser-microdissection of intrapulmonary arteries was combined with array profiling. METHODS AND RESULTS: Analysis was performed on mice subjected to 1, 7 and 21 days of hypoxia (FiO(2 )= 0.1) using nylon filters (1176 spots). Changes in the expression of 29, 38, and 42 genes were observed at day 1, 7, and 21, respectively. Genes were grouped into 5 different classes based on their time course of response. Gene regulation obtained by array analysis was confirmed by real-time PCR. Additionally, the expression of the growth mediators PDGF-B, TGF-β, TSP-1, SRF, FGF-2, TIE-2 receptor, and VEGF-R1 were determined by real-time PCR. At day 1, transcription modulators and ion-related proteins were predominantly regulated. However, at day 7 and 21 differential expression of matrix producing and degrading genes was observed, indicating ongoing structural alterations. Among the 21 genes upregulated at day 1, 15 genes were identified carrying potential hypoxia response elements (HREs) for hypoxia-induced transcription factors. Three differentially expressed genes (S100A4, CD36 and FKBP1a) were examined by immunohistochemistry confirming the regulation on protein level. While FKBP1a was restricted to the vessel adventitia, S100A4 and CD36 were localised in the vascular tunica media. CONCLUSION: Laser-microdissection and array profiling has revealed several new genes involved in lung vascular remodelling in response to hypoxia. Immunohistochemistry confirmed regulation of three proteins and specified their localisation in vascular smooth muscle cells and fibroblasts indicating involvement of different cells types in the remodelling process. The approach allows deeper insight into hypoxic regulatory pathways specifically in the vascular compartment of this complex organ

Oxygen-Independent Stabilization of Hypoxia Inducible Factor (HIF)-1 during RSV Infection

BACKGROUND: Hypoxia-inducible factor 1 (HIF)-1alpha is a transcription factor that functions as master regulator of mammalian oxygen homeostasis. In addition, recent studies identified a role for HIF-1alpha as transcriptional regulator during inflammation or infection. Based on studies showing that respiratory syncytial virus (RSV) is among the most potent biological stimuli to induce an inflammatory milieu, we hypothesized a role of HIF-1alpha as transcriptional regulator during infections with RSV. METHODOLOGY, PRINCIPAL FINDINGS: We gained first insight from immunohistocemical studies of RSV-infected human pulmonary epithelia that were stained for HIF-1alpha. These studies revealed that RSV-positive cells also stained for HIF-1alpha, suggesting concomitant HIF-activation during RSV infection. Similarly, Western blot analysis confirmed an approximately 8-fold increase in HIF-1alpha protein 24 h after RSV infection. In contrast, HIF-1alpha activation was abolished utilizing UV-treated RSV. Moreover, HIF-alpha-regulated genes (VEGF, CD73, FN-1, COX-2) were induced with RSV infection of wild-type cells. In contrast, HIF-1alpha dependent gene induction was abolished in pulmonary epithelia following siRNA mediated repression of HIF-1alpha. Measurements of the partial pressure of oxygen in the supernatants of RSV infected epithelia or controls revealed no differences in oxygen content, suggesting that HIF-1alpha activation is not caused by RSV associated hypoxia. Finally, studies of RSV pneumonitis in mice confirmed HIF-alpha-activation in a murine in vivo model. CONCLUSIONS/SIGNIFICANCE: Taking together, these studies suggest hypoxia-independent activation of HIF-1alpha during infection with RSV in vitro and in vivo

Redox cycling metals: Pedaling their roles in metabolism and their use in the development of novel therapeutics

Essential metals, such as iron and copper, play a critical role in a plethora of cellular processes including cell growth and proliferation. However, concomitantly, excess of these metal ions in the body can have deleterious effects due to their ability to generate cytotoxic reactive oxygen species (ROS). Thus, the human body has evolved a very well-orchestrated metabolic system that keeps tight control on the levels of these metal ions. Considering their very high proliferation rate, cancer cells require a high abundance of these metals compared to their normal counterparts. Interestingly, new anti-cancer agents that take advantage of the sensitivity of cancer cells to metal sequestration and their susceptibility to ROS have been developed. These ligands can avidly bind metal ions to form redox active metal complexes, which lead to generation of cytotoxic ROS. Furthermore, these agents also act as potent metastasis suppressors due to their ability to up-regulate the metastasis suppressor gene, N-myc downstream regulated gene 1. This review discusses the importance of iron and copper in the metabolism and progression of cancer, how they can be exploited to target tumors and the clinical translation of novel anti-cancer chemotherapeutics

The hypoxia-inducible factor-1 DNA recognition site is cAMP-responsive.

The hypoxia-inducible factor-1 (HIF-1) was first described as a DNA binding activity that specifically recognizes an 8 bp hypoxia response element (HRE) known to be essential for oxygen-regulated erythropoietin gene expression. In electrophoretic mobility shift assays (EMSAs) HIF-1 DNA binding activity is only detectable in nuclear extracts of cells cultivated in a low oxygen atmosphere. In addition to HIF-1, a constitutive DNA binding activity also specifically binds the HIF-1 probe. Based on EMSAs using competitor oligonucleotides, specific antibodies and recombinant proteins, we previously reported that the constitutive HRE binding factor is composed of ATF-1 and CREB-1. Here we show that this site is functionally responsive to the cAMP agonist 8Br-cAMP in a dose-dependent manner under hypoxic but not under normoxic conditions. These results were confirmed by using the protein kinase A (PKA) activator Sp-cAMPS and the PKA inhibitor Rp-cAMPS: while Sp-cAMPS was synergistic with hypoxia on the HIF-1 DNA recognition site, the Rp-cAMPS isomer showed no effect. Our findings suggest that the PKA-signaling pathway is enhancing oxygen-dependent gene expression via the HRE

The transcription factors ATF-1 and CREB-1 bind constitutively to the hypoxia-inducible factor-1 (HIF-1) DNA recognition site.

The hypoxia-inducible factor-1 (HIF-1) was first described as a DNA binding activity that specifically recognizes an 8 bp motif known to be essential for hypoxia-inducible erythropoietin gene transcription. Subsequently HIF-1 activity has also been found in cell lines which do not express erythropoietin, suggesting that HIF-1 is part of a widespread oxygen sensing mechanism. In electrophoretic mobility shift assays HIF-1 DNA binding activity is only detectable in nuclear extracts of cells cultivated in a low oxygen atmosphere. In addition to HIF-1, a constitutive DNA binding activity also specifically binds the HIF1 probe. Here we report that CRE and AP1 oligonucleotides efficiently competed for binding of the HIF1 probe to this constitutive factor, whereas HIF-1 activity itself remained unaffected. Monoclonal antibodies raised against the CRE binding factors ATF-1 and CREB-1 supershifted the constitutive factors ATF-1 and CREB-1 supershifted the constitutive factor, while Jun and Fos family members, which constitute the AP-1 factor, were immunologically undetectable. Recombinant ATF-1 and CREB-1 proteins bound HIF1 probes either as homodimers or as heterodimers, indicating a new binding specificity for ATF-1/CREB-1. Finally, reporter gene assays in HeLa cells treated with either a cAMP analogue or a phorbol ester suggest that the PKA, but not the PKC signalling pathway is involved in oxygen sensing

Oxygen-regulated erythropoietin gene expression is dependent on a CpG methylation-free hypoxia-inducible factor-1 DNA-binding site.

The hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator involved in the expression of oxygen-regulated genes such as that for erythropoietin. Following exposure to low oxygen partial pressure (hypoxia), HIF-1 binds to an hypoxia-response element located 3' to the erythropoietin gene and confers activation of erythropoietin expression. The conserved core HIF-1 binding site (HBS) of the erythropoietin 3' enhancer (CGTG) contains a CpG dinucleotide known to be a potential target of cytosine methylation. We found that methylation of the HBS abolishes HIF-1 DNA binding as well as hypoxic reporter gene activation, suggesting that a methylation-free HBS is mandatory for HIF-1 function. The in vivo methylation pattern of the erythropoietin 3' HBS in various human cell lines and mouse organs was assessed by genomic Southern blotting using a methylation-sensitive restriction enzyme. Whereas this site was essentially methylation-free in the erythropoietin-producing cell line Hep3B, a direct correlation between erythropoietin protein expression and the degree of erythropoietin 3' HBS methylation was found in different HepG2 sublines. However, the finding that this site is partially methylation-free in human cell lines and mouse tissues that do not express erythropoietin suggests that there might be a general selective pressure to keep this site methylation-free, independent of erythropoietin expression

The mouse gene for hypoxia-inducible factor-1alpha. Genomic organization, expression and characterization of an alternative first exon and 5' flanking sequence

The ubiquitously expressed hypoxia-inducible factor-1 (HIF-1) is involved in expression of a large number of oxygen-regulated genes. HIF-1 is a heterodimer consisting of an alpha and a beta subunit, both belonging to the basic-helix-loop-helix Per-aryl hydrocarbon receptor nuclear translocator-Sim (PAS) family of transcription factors. Whereas HIF-1alpha is a novel member of this family, HIF-1beta is identical to the aryl hydrocarbon receptor nuclear translocator, previously recognized to be involved in xenobiotic metabolism. cDNA cloning revealed that mouse HIF-1alpha can be expressed as two mRNA isoforms containing alternative 5' untranslated regions and two different predicted translational start sites. We cloned and characterized 20.5 kb of the mouse HIF-1alpha gene (Hif1a) containing exon II-XV. The two alternative first exons, I.1 and I.2, are separated from exon II by approximately 24 kb and 17 kb, respectively. We also sequenced Hif1a exon I.1 and flanking regions, and mapped a single exon I.1 transcription initiation site. Reverse transcription PCR analysis of total RNA derived from normoxic and hypoxic mouse hepatoma and fibroblast cell lines suggested that the two alternative mRNA isoforms are constitutively coexpressed in these cells, and that two different promoters drive transcription of HIF-1alpha. A minimal exon I.1 promoter was identified which moderately activated heterologous gene expression, indicating that additional cis-elements are required for efficient HIF-1alpha transcription in vivo

Efficient translation of mouse hypoxia-inducible factor-1alpha under normoxic and hypoxic conditions.

The heterodimeric hypoxia-inducible factor-1 (HIF-1), consisting of the subunits HIF-1alpha and HIF-1beta/ARNT, is a master transcriptional regulator of oxygen homeostasis. Under hypoxic conditions, HIF-1alpha levels very rapidly increase, mostly due to protein stabilization. However, translational regulation of HIF-1alpha has not been directly analyzed so far. Mouse HIF-1alpha exists as two mRNA isoforms (termed mHIF-1alphaI.1 and mHIF-1alphaI. 2) containing structurally different 5'-termini which might modulate translation initiation. Whereas the in vitro translation efficiency of these two mRNA isoforms was about equal, the mHIF-1alphaI.2 5'-untranslated region (5'-UTR) conferred significantly higher in vivo luciferase reporter gene activity than the mHIF-1alphaI.1 5'-UTR. Similar corresponding luciferase mRNA levels indicate translational rather than transcriptional alterations. Reporter gene expression was not affected upon exposure of transiently transfected cells to hypoxia (1% oxygen). Direct assessment of translational regulation by polysomal profile analysis of HeLaS3 cells showed that HIF-1alpha (and to a lower extent ARNT) mRNA was found mainly in the translationally active polyribosomal fractions under both normoxic and hypoxic conditions. In contrast, the association of mRNAs for beta-actin and ribosomal protein L28 with the polyribosomal fractions was substantially reduced under hypoxic conditions, suggesting decreased overall protein synthesis. Thus, efficient translation of mouse HIF-1alpha in a situation where the general translation efficiency is reduced represents a prerequisite for the very rapid accumulation of HIF-1alpha protein upon exposure to hypoxia