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R H Wenger

Publications and source records attributed to R H Wenger.

At least 19 recordsLinked to original sources
PubMed2006

Induction of activating transcription factor 3 by anoxia is independent of p53 and the hypoxic HIF signalling pathway.

Solid tumors often have an inadequate blood supply, which results in large regions that are subjected to hypoxic or anoxic stress. Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that regulates much of the transcriptional response of cells to hypoxia. Activating transcription factor 3 (ATF3) is another transcription factor that responds to a variety of stresses and is often upregulated in cancer. We investigated the regulation of ATF3 by oxygen deprivation. ATF3 induction occurred most robustly under anoxia, is common, and it is not dependent on presence of HIF-1 or p53, but is sensitive to the inhibition of c-Jun NH2-terminal kinase activation and the antioxidant N-acetylcystein. ATF3 could also be induced by desferrioxamine but not by the mitochondrial poison cyanide or the nonspecific 2-oxoglutarate dioxygenase inhibitor dimethyloxalylglycine. We also show that anoxic ATF3 mRNA is more stable than normoxic mRNA providing a mechanism for this induction. Thus, this study demonstrates that the regulation of ATF3 under anoxia is independent of 2-oxoglutarate dioxygenase, HIF-1 and p53, presumably involving multiple regulatory pathways.

Acetylcysteine↗
PubMed2004

[Oxygen homeostasis in tissues and cells: how hypoxia regulates gene expression].

Every cell of the body is capable of measuring the environmental oxygen concentration. When oxygen supply falls below oxygen demand of the cell (hypoxia), a series of genes will be activated whose products help the cell, the tissue as well as the whole organism in adapting to these altered oxygen concentrations. The discovery of hypoxia-inducible transcription factors (HIFs) allowed the molecular understanding of these processes.

Cell Hypoxia↗
PubMed2003

Oxygen-regulated expression of TGF-beta 3, a growth factor involved in trophoblast differentiation.

The transforming growth factor-beta 3 (TGF-beta 3) is involved in oxygen-dependent differentiation processes during placental development and pregnancy disorders. However, the importance of oxygen partial pressure for the regulation of TGF-beta 3 expression is presently unclear. We and others presented preliminary evidence that the hypoxia-inducible factor-1 (HIF-1) confers TGF-beta 3 transcription but it was unknown whether this occurred directly or indirectly. To analyze how HIF-1 regulates TGF-beta 3 gene transcription, we cloned and sequenced the mouse TGF-beta 3 promoter region. Multiple putative HIF-1 binding sites (HBSs) were identified, many of which co-localized with two G+C rich CpG islands 5' to the TGF-beta 3 transcription start site. A 6.8 kb fragment of the TGF-beta 3 promoter induced reporter gene expression under hypoxic conditions or when treated with an iron chelator known to stabilize and activate the HIF-1 alpha subunit. Deletion of a 2.4 kb fragment upstream of the distal CpG island abolished inducibility of reporter gene expression. Two HBSs (HBS1 and HBS6) that bound the HIF-1 protein could be identified within this 2.4 kb fragment. These results suggest that TGF-beta 3 gene expression is directly regulated by HIF-1.

Animals↗
PubMed2001

Mammalian PASKIN, a PAS-serine/threonine kinase related to bacterial oxygen sensors.

The PAS domain is a versatile protein fold found in many archaeal, bacterial, and plant proteins capable of sensing environmental changes in light intensity, oxygen concentration, and redox potentials. The oxygen sensor FixL from Rhizobium species contains a heme-bearing PAS domain and a histidine kinase domain that couples sensing to signaling. We identified a novel mammalian PAS protein (PASKIN) containing a domain architecture resembling FixL. PASKIN is encoded by an evolutionarily conserved single-copy gene which is ubiquitously expressed. The human PASKIN and mouse Paskin genes show a conserved intron-exon structure and share their promoter regions with another ubiquitously expressed gene that encodes a regulator of protein phosphatase-1. The 144-kDa PASKIN protein contains a PAS region homologous to the FixL PAS domain and a serine/threonine kinase domain which might be involved in signaling. Thus, PASKIN is likely to function as a mammalian PAS sensor protein.

Amino Acid Sequence↗
PubMed2001

Dissecting hypoxia-dependent and hypoxia-independent steps in the HIF-1alpha activation cascade: implications for HIF-1alpha gene therapy.

The heterodimeric hypoxia-inducible factor (HIF)-1 is a master transcriptional regulator of oxygen homeostasis and a possible target for gene therapy of ischemic disease. Although the role of oxygen concentration in HIF-1a protein stabilization is well established, it is less clear whether and how oxygen-regulated mechanisms contribute to HIF-1a protein modifications, nuclear translocation, heterodimerization with the b-subunit, recruitment of cofactors, and gene trans-activation. Because the HIF-1a protein is proteolytically degraded under normoxic conditions, we established two HeLa Tet-Off cell lines (HT42 and HT43), which inducibly overexpress high levels of HIF-1a under normoxic conditions, allowing to distinguish hypoxia-dependent from hypoxia-independent activation mechanisms. Using these cells, we found that normoxically induced HIF-1a is localized to the nucleus, binds DNA, and trans-activates reporter and endogenous target genes. The levels of p53 expression remained unaffected. The MAP kinase inhibitor PD98059 attenuated HIF-1a protein modifications and trans-activation ability but not protein stabilization and DNA-binding activity. Because overexpressed HIF-1a is fully localized to the nucleus but displays only partial DNA-binding and trans-activation activity, mitogen-activated protein kinase-dependent phosphorylation might be required for full HIF-1 activation. HIF-1a protein was also overexpressed in vivo, following the transplantation of HT42 cells into nude mice, demonstrating the feasibility of HIF-1a gene transfer.

Animals↗
PubMed2001

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.

Base Sequence↗
PubMed2001

Hypoxia affects expression of circadian genes PER1 and CLOCK in mouse brain.

The key elements of circadian clockwork and oxygen homeostasis are the PAS protein family members PER and CLOCK and hypoxia-inducible factor 1alpha (HIF-1alpha). The PAS domain serves as an interface for protein-protein interactions. We asked whether a cross-talk exists between the PAS components of hypoxic and circadian pathways. We found several isoforms of PER1 protein that exhibit tissue-specific size differences. In the mouse brain, a predominantly nuclear 48 kDa isoform that followed a daily rhythm was observed. The 48 kDa form was found in the nuclear fractions derived from mouse liver, Swiss3T3 fibroblasts, and N2A neuroblastoma cells. In mouse kidney and human 293 kidney cells, a 55 kDa PER1 form was detected. CLOCK was observed as a predicted 100 kDa protein in rat-1 cells and in all analyzed mouse tissues including brain, liver, kidney, and spleen. In contrast to PER1, CLOCK protein expression was not rhythmic. Exposure to hypoxia led to increased PER1 and CLOCK protein levels in mice. Based on coimmunoprecipitation experiments that showed protein-protein interaction between PER1 and the alpha subunit of HIF-1, we suggest that these hypoxic effects may be modulated by HIF-1alpha.-Chilov, D., Hofer, T., Bauer, C., Wenger, R. H., Gassmann, M. Hypoxia affects expression of circadian genes PER1 and CLOCK in mouse brain.

3T3 Cells↗
PubMed2001

HIF-1 is expressed in normoxic tissue and displays an organ-specific regulation under systemic hypoxia.

Adaptation to hypoxia is regulated by hypoxia-inducible factor 1 (HIF-1), a heterodimeric transcription factor consisting of an oxygen-regulated alpha subunit and a constitutively expressed beta subunit. Although HIF-1 is regulated mainly by oxygen tension through the oxygen-dependent degradation of its alpha subunit, in vitro it can also be modulated by cytokines, hormones and genetic alterations. To investigate HIF-1 activation in vivo, we determined the spatial and temporal distribution of HIF-1 in healthy mice subjected to varying fractions of inspiratory oxygen. Immunohistochemical examination of brain, kidney, liver, heart, and skeletal muscle revealed that HIF-1alpha is present in mice kept under normoxic conditions and is further increased in response to systemic hypoxia. Moreover, immunoblot analysis showed that the kinetics of HIF-1alpha expression varies among different organs. In liver and kidney, HIF-1alpha reaches maximal levels after 1 h and gradually decreases to baseline levels after 4 h of continuous hypoxia. In the brain, however, HIF-1alpha is maximally expressed after 5 h and declines to basal levels by 12 h. Whereas HIF-1beta is constitutively expressed in brain and kidney nuclear extracts, its hepatic expression increases concomitantly with HIF-1alpha. Overall, HIF-1alpha expression in normoxic mice suggests that HIF-1 has an important role in tissue homeostasis.

Animals↗
PubMed2000

Nitric oxide prevents cardiovascular disease and determines survival in polyglobulic mice overexpressing erythropoietin.

Nitric oxide (NO) induces vasodilatatory, antiaggregatory, and antiproliferative effects in vitro. To delineate potential beneficial effects of NO in preventing vascular disease in vivo, we generated transgenic mice overexpressing human erythropoietin. These animals induce polyglobulia known to be associated with a high incidence of vascular disease. Despite hematocrit levels of 80%, adult transgenic mice did not develop hypertension or thromboembolism. Endothelial NO synthase levels, NO-mediated endothelium-dependent relaxation and circulating and vascular tissue NO levels were markedly increased. Administration of the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) led to vasoconstriction of peripheral resistance vessels, hypertension, and death of transgenic mice, whereas wild-type siblings developed hypertension but did not show increased mortality. L-NAME-treated polyglobulic mice revealed acute left ventricular dilatation and vascular engorgement associated with pulmonary congestion and hemorrhage. In conclusion, we here unequivocally demonstrate that endothelial NO maintains normotension, prevents cardiovascular dysfunction, and critically determines survival in vivo under conditions of increased hematocrit.

Animals↗
PubMed2000

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.

Animals↗
PubMed2000

Epolones induce erythropoietin expression via hypoxia-inducible factor-1 alpha activation.

Induction of erythropoietin (Epo) expression under hypoxic conditions is mediated by the heterodimeric hypoxia-inducible factor (HIF)-1. Following binding to the 3' hypoxia-response element (HRE) of the Epo gene, HIF-1 markedly enhances Epo transcription. To facilitate the search for HIF-1 (ant)agonists, a hypoxia-reporter cell line (termed HRCHO5) was constructed containing a stably integrated luciferase gene under the control of triplicated heterologous HREs. Among various agents tested, we identified a class of substances called epolones, which induced HRE-dependent reporter gene activity in HRCHO5 cells. Epolones are fungal products known to induce Epo expression in hepatoma cells. We found that epolones (optimal concentration 4-8 micromol/L) potently induce HIF-1 alpha protein accumulation and nuclear translocation as well as HIF-1 DNA binding and reporter gene transactivation. Interestingly, the activity of a compound related to the fungal epolones, ciclopirox olamine (CPX), was blocked after addition of ferrous iron. This suggests that CPX might interfere with the putative heme oxygen sensor, as has been proposed for the iron chelator deferoxamine mesylate (DFX). However, about 10-fold higher concentrations of DFX (50-100 micromol/L) than CPX were required to maximally induce reporter gene activity in HRCHO5 cells. Moreover, structural, functional, and spectrophotometric data imply a chelator:iron stoichiometry of 1:1 for DFX but 3:1 for CPX. Because the iron concentration in the cell culture medium was determined to be 16 micromol/L, DFX but not CPX function can be explained by complete chelation of medium iron. These results suggest that the lipophilic epolones might induce HIF-1 alpha by intracellular iron chelation. (Blood. 2000;96:1558-1565)

Animals↗
PubMed2000

Regulation of the hypoxia-inducible factor-1 alpha. ARNT is not necessary for hypoxic induction of HIF-1 alpha in the nucleus.

Hypoxia-inducible factor-1 (HIF-1) is a master regulator of mammalian oxygen homeostasis. HIF-1 consists of two subunits, HIF-1 alpha and the aryl hydrocarbon receptor nuclear translocator (ARNT). Whereas hypoxia prevents ubiquitination and proteasomal degradation of HIF-1 alpha, ARNT expression is thought to be oxygen-independent. We and others previously showed that ARNT is indispensable for HIF-1 DNA-binding and transactivation function. To examine the requirement of ARNT for accumulation and nuclear translocation of HIF-1 alpha in hypoxia, we used ARNT-mutant mouse hepatoma and ARNT-deficient embryonic stem cells. As shown by immunofluorescence, HIF-1 alpha accumulation in the nucleus of hypoxic cells did not require ARNT, demonstrating that nuclear translocation is intrinsic to HIF-1 alpha. During biochemical separation, both HIF-1 alpha and ARNT tend to leak from the nuclei in the absence of the corresponding subunit, suggesting that heterodimerization is required for stable association within the nuclear compartment. Nuclear stabilization of the heterodimer might also explain the hypoxically increased total cellular ARNT levels observed in some of the cell lines examined.

Animals↗
PubMed2000

Hypoxia-regulated gene expression in fetal wound regeneration and adult wound repair.

Fetal skin wounds heal scarlessly while adult wounds scar. Fetal wound healing occurs in a physiologically hypoxic environment whereas in adult wound healing, cells have to acutely adapt to hypoxia caused by locally impaired blood supply. We examined the expression of hypoxia-inducible factor 1 (HIF-1), a potent transcriptional regulator of oxygen-dependent genes such as vascular endothelial growth factor (VEGF), and transforming growth factor-beta (TGF-beta), a potentially HIF-1-regulated scarring cytokine, on fetal and adult responses to wounding. Incisional skin wounds were created in four sheep fetuses (twins served as controls) and two ewes at 100 days of gestation (term = 150 days). Fetal and adult wounds as well as non-wounded control tissues were harvested 2 days post-wounding. Intraoperative arterial blood gas analyses and invasive subcutaneous pO2 measurements revealed that the fetuses were indeed hypoxic while the mothers were normoxic. Expression patterns of HIF-1alpha were investigated by Western blot analyses. HIF-1alpha expression in fetal wounds and fetal control skin was similar, whereas HIF-1alpha was only detected in adult wounds but not in adult control skin. Exposure of cultured fetal and adult dermal fibroblasts to hypoxia (1% O2) showed a marked induction of VEGF mRNA. In contrast, exposure of these cell types to hypoxia did not significantly affect TGF-beta1 mRNA expression in comparison to their normoxic controls. The presence of HIF-1alpha in fetal but not in adult normal skin indicates that HIF-1alpha might be involved in fetal skin development. Conversely, the upregulation of HIF-1alpha in adult but not early fetal wound repair might represent a pathway in the pathogenesis of scarring, since several growth factors overexpressed in, and associated, with scarring are hypoxia-inducible. Further studies need to be performed in order to identify hypoxia-regulated HIF-1alpha target genes involved in the pathogenesis of scarring.

Age Factors↗
PubMed2000

Effects of hypobaric hypoxia on vascular endothelial growth factor and the acute phase response in subjects who are susceptible to high-altitude pulmonary oedema.

In order to investigate whether vascular endothelial growth factor (VEGF) and inflammatory pathways are activated during acute hypobaric hypoxia in subjects who are susceptible to high-altitude pulmonary oedema (HAPE-S), seven HAPE-S and five control subjects were exposed to simulated altitude corresponding to 4000 m in a hypobaric chamber for 1 day. Peripheral venous blood was taken at 450 m (Zürich level) and at 4000 m, and levels of erythropoietin (EPO), VEGF, interleukin-6 (IL-6) and the acute-phase proteins complement C3 (C3), alpha1-antitrypsin (alpha1AT), transferrin (Tf) and C-reactive protein (CRP) were measured. Peripheral arterial oxygen saturation (SaO2) was recorded. Chest radiography was performed before and immediately after the experiment. EPO increased during altitude exposure, correlating with SaO2, in both groups (r = -0.86, P < 0.001). Venous serum VEGF did not show any elevation despite a marked decrease in SaO2 in the HAPE-S subjects [mean (SD) HAPE-S: 69.6 (9.1)%; controls: 78.7 (5.2)%]. C3 and alpha1AT levels increased in HAPE-S during hypobaric hypoxia [from 0.94 (0.11) g/l to 1.07 (0.13) g/l, and from 1.16 (0.08) g/l to 1.49 (0.27) g/l, respectively; P < 0.05], but remained within the clinical reference ranges. No significant elevations of IL-6, Tf or CRP were observed in either group. The post-exposure chest radiography revealed no signs of oedema. We conclude that VEGF is not up-regulated in HAPE-S and thus does not seem to increase critically pulmonary vascular permeability during the 1st day at high altitude. Furthermore, our data provide evidence against a clinically relevant inflammation in the initial phase of exposure to hypoxia in HAPE-S, although C3 and alpha1AT are mildly induced.

Acute-Phase Proteins↗
PubMed2000

Mammalian oxygen sensing, signalling and gene regulation.

Oxygen is essential to the life of all aerobic organisms. Virtually every cell type is able to sense a limited oxygen supply (hypoxia) and specifically to induce a set of oxygen-regulated genes. This review summarizes current concepts of mammalian oxygen-sensing and signal-transduction pathways. Since the discovery of the hypoxia-inducible factors (HIFs), a great deal of progress has been made in our comprehension of how hypoxia induces the expression of oxygen-regulated genes. The alpha subunit of the heterodimeric transcription factors HIF-1, 2 and 3 is unstable under normoxia but is rapidly stabilized upon exposure to hypoxic conditions. Following heterodimerization with the constitutively expressed beta subunit, HIFs activate the transcription of an increasing number of genes involved in maintaining oxygen homeostasis at the cellular, local and systemic levels.

Animals↗
PubMed2000

Genetically modified mouse models in studies on cutaneous wound healing.

Genetically modified mice have become an invaluable tool in modern biomedical and basic research. This review provides an overview of knockout and transgenic mice studied with regard to their cutaneous wound healing properties. In addition, several gene transfer studies are briefly introduced, which have further highlighted our knowledge on individual gene function in wound healing.

Animals↗