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PubMed2001

Dual control of LIF expression and LIF receptor function regulate Stat3 activation at the onset of uterine receptivity and embryo implantation.

Leukemia inhibitory factor (LIF) expression in the uterus is essential for embryo implantation in mice. Here we describe the spatial and temporal regulation of LIF signaling in vivo by using tissues isolated from uteri on different days over the implantation period. During this time, LIF receptors are expressed predominantly in the luminal epithelium (LE) of the uterus. Isolated epithelium responds to LIF by phosphorylation and nuclear translocation of signal transducer and activator of transcription (Stat) 3, but not by an increase in mitogen-activated protein kinase levels. The related cytokines Il-6, ciliary neurotrophic factor, as well as epidermal growth factor, do not activate Stat3, although epidermal growth factor stimulates mitogen-activated protein kinase. In vivo Stat3 activation is induced by LIF alone, resulting in the localization of Stat3 specifically to the nuclei of the LE coinciding with the onset of uterine receptivity. The responsiveness of the LE to LIF is regulated temporally, with Stat activation being restricted to day 4 of pregnancy despite the presence of constant levels of LIF receptor throughout the preimplantation period. Uterine receptivity is therefore under dual control and is regulated by both the onset of LIF expression in the endometrial glands and the release from inhibition of receptor function in the LE.

Animals↗

PubMed2006

Dual control of quorum sensing by two TraM-type antiactivators in Agrobacterium tumefaciens octopine strain A6.

Agrobacterium tumefaciens wild-type strains have a unique quorum-sensing (QS)-dependent Ti plasmid conjugative transfer phenotype in which QS signaling is activated by corresponding conjugative opine inducers. Strain K588, with a nopaline-type chromosomal background harboring an octopine-type Ti plasmid, however, is a spontaneous mutant displaying a constitutive phenotype in QS. In this study, we show that a single amino acid mutation (L54P) in the QS antiactivator TraM encoded by the traM gene of Ti plasmid is responsible for the constitutive phenotype of strain K588. Introduction of the L54P point mutation to the TraM of wild-type strain A6 by allelic replacement, however, failed to generate the expected constitutive phenotype in this octopine-type strain. Intriguingly, the QS-constitutive phenotype appeared when the pTiA6 carrying the mutated traM was placed in the chromosomal background of the nopaline-type strain C58C1RS, suggesting an unknown inhibitory factor(s) encoded by the chromosomal background of strain A6 but not by C58C1RS. Low-stringency Southern blotting analysis showed that strain A6, but not strain C58 and its derivatives, contains a second traM homologue. The homologue, designated traM2, has 64% and 65% identities with traM at the DNA and peptide levels, respectively. Similar to TraM, TraM2 is a potent antiactivator that functions by blocking TraR, the QS activator, from specific binding to the tra gene promoters. Deletion of traM2 in strain A6 harboring the mutated traM confers a constitutive QS phenotype. The results demonstrate that the QS system in strain A6 is subjected to the dual control of TraM and TraM2.

Agrobacterium tumefaciens↗

PubMed1989

Dual control of the Bradyrhizobium japonicum symbiotic nitrogen fixation regulatory operon fixR nifA: analysis of cis- and trans-acting elements.

Aerobic expression of the fixR nifA operon in Bradyrhizobium japonicum was shown to depend on a cis-acting, promoter-upstream DNA sequence located between the -24/-12 promoter and position -86 relative to the transcription start site. An adenine at position -66 was essential for maximal expression. A chromosomal deletion of the upstream activator sequence (UAS) led to a symbiotically defective phenotype which was typical of nifA mutants. B. japonicum crude extracts contained a protein that bound to the UAS. By using chromosomally integrated fixR-lacZ fusions, the level of expression of the fixR nifA operon was found to be fivefold higher under reduced oxygen tension than under aerobiosis. This increase was due to autoactivation by the NifA protein and was partly independent of the UAS. Based on these data, we propose a model for the regulation of nitrogen fixation genes in B. japonicum that involves dual positive control of the fixR nifA operon. At high oxygen concentrations, the operon is expressed at a moderate level, subject to activation by the binding of a trans-acting factor to the UAS. Under such conditions, the nifA gene product is known to be inactive. At very low oxygen concentrations--a condition favorable to NifA activity--the NifA protein is the trans-acting factor which (i) enhances the level of fixR nifA expression (and hence its own synthesis) and (ii) activates other nif and fix genes.

Chromosome Deletion↗

PubMed2006

Esophageal-gastric relaxation reflex in rat: dual control of peripheral nitrergic and cholinergic transmission.

It has long been known that the esophageal distension produced by swallowing elicits a powerful proximal gastric relaxation. Gastroinhibitory control by the esophagus involves neural pathways from esophageal distension-sensitive neurons in the nucleus tractus solitarius centralis (cNTS) with connections to virtually all levels of the dorsal motor nucleus of the vagus (DMV). We have shown recently that cNTS responses are excitatory and primarily involve tyrosine hydroxylase-immunoreactive cells, whereas the DMV response involves both an alpha1 excitatory and an alpha2 inhibitory response. In the present study, using an esophageal balloon distension to evoke gastric relaxation (esophageal-gastric reflex, EGR), we investigated the peripheral pharmacological basis responsible for this reflex. Systemic administration of atropine methyl nitrate reduced the amplitude of the gastric relaxation to 52.0+/-4.4% of the original EGR, whereas NG-nitro-L-arginine methyl ester (L-NAME) reduced it to 26.3+/-7.2% of the original EGR. Concomitant administration of atropine methyl nitrate and L-NAME reduced the amplitude of the gastric relaxation to 4.0+/-2.5% of control. This reduction in the amplitude of induced EGR is quite comparable (4.3+/-2.6%) to that seen when the animal was pretreated with the nicotinic ganglionic blocker hexamethonium. In the presence of bethanechol, the amplitude of the esophageal distension-induced gastric relaxation was increased to 177.0+/-10.0% of control; administration of L-NAME reduced this amplitude to 19.9+/-9.5%. Our data provide a clear demonstration that the gastroinhibitory control by the esophagus is mediated via a dual vagal innervation consisting of inhibitory nitrergic and excitatory cholinergic transmission.

Animals↗

PubMed1990

Dual control of local blood flow by purines.

The potent and widespread vascular actions of purine nucleotides and nucleosides have long been recognized. A dual function for ATP in the regulation of vascular tone is considered. ATP acts as an excitatory cotransmitter with noradrenaline from sympathetic perivascular nerves, to cause vasoconstriction via P2X-purinoceptors located on vascular smooth muscle. In contrast, ATP can act via P2Y-purinoceptors located on vascular endothelial cells to release EDRF, which diffuses to the vascular smooth muscle and produces vasodilatation. The main source of intraluminal ATP is likely to be endothelial cells, and its release can be measured during conditions such as changes in flow and hypoxia, in amounts sufficient to activate endothelial P2Y-purinoceptors. In some vessels, ATP acts directly on P2Y-purinoceptors located in the vascular smooth muscle to produce vasodilatation; the possibility that the origins of this ATP are nonsympathetic purinergic or sensory-motor nerves is discussed. ATP can also be released during intravascular platelet aggregation and from intact and damaged vascular smooth muscle cells, and so may play a role in the complex physiological mechanisms controlling local vascular tone under normoxic conditions, during changes in blood flow and during vessel injury.

Adenosine Triphosphate↗

PubMed2004

N-methyl-d-aspartate receptor activation exerts a dual control on postnatal development of nucleus tractus solitarii neurons in vivo.

We have used a morphological approach to evaluate the role of NMDA receptors (NMDAR) in postnatal development of brainstem neurons in awake rats. Chronic NMDAR blockade was performed by placing drug-impregnated Elvax implants over the brainstem at the fifth postnatal day (P5). Compared with control, NMDAR blockade led to a transient increase in dendritic arbor area and filopodium density until P12 followed by a rapid decline in both parameters. Electron microscopy observations showed that these changes correlated with an increase in synapse density at P14 followed by a decrease in synapse density at P28 if chronic NMDAR blockade was maintained until P21. These results support the hypothesis that synapse formation does not require NMDAR activation. In addition, our data suggest a dual role for NMDAR in controlling the synapse number. Early in development NMDARs may be involved in controlling the rate of synapse elimination. Later on, they may subserve synapse stabilization. The physiological significance of these results is discussed.

Animals↗

PubMed1989

A quantitative comparison of dual control of a hormone response element by progestins and glucocorticoids in the same cell line.

Progesterone receptor-containing T47D human breast cancer cells are responsive to progestins but fail to respond to other steroid hormones, in particular dexamethasone, because they have no measurable levels of receptors for estrogens, androgens, or glucocorticoids. To quantitatively study dual responsiveness of the mouse mammary tumor virus (MMTV) promoter to progestins and glucocorticoids, we have stably transfected T47D cells with a glucocorticoid receptor (GR) expression vector. A cloned derivative (A1-2) was isolated that expresses a normal, full length GR, as assessed by steroid binding and Western immunoblot with a monoclonal anti-GR antibody. Moreover, GR is expressed at levels (80,000-100,000 molecules per cell) comparable to the high levels of endogenous progesterone receptor (200,000 molecules per cell). In A1-2 cells transiently transfected with an MMTV-chloramphenicol acetyl transferase reporter gene, induction by glucocorticoid was substantially greater (5-fold) than induction mediated by progestins. These results suggest that glucocorticoids may be the primary regulator of MMTV.

Breast Neoplasms↗

PubMed1999

Dual control by regulatory gene fdsR of the fds operon encoding the NAD+-linked formate dehydrogenase of Ralstonia eutropha.

The transcriptional regulator gene fdsR was identified 150 bp upstream of the divergently oriented fdsGBACD operon encoding the soluble, NAD+-linked formate dehydrogenase in the chemoautotrophic bacterium Ralstonia eutropha H16. Its deduced product, FdsR, displays a basal sequence similarity to the regulatory proteins of the LysR family. The carboxy-terminal domain of FdsR contains a short region that is conserved in formate dehydrogenases. Deletion of fdsR revealed a dual regulatory effect of FdsR on the fds operon by acting as transcriptional activator in the presence of formate or as repressor in the absence of formate. Studies with fdsR transcriptional fusions also suggested a negative autoregulation of the gene. A promoter structure resembling sigma70-dependent promoters from Escherichia coli was identified upstream of the fdsR transcriptional start site. FdsR purified to homogeneity after overexpression of fdsR in E. coli is a 130 kDa homotetramer binding to the fds control region located between the fdsR and fdsG genes. Formate significantly increased the binding affinity of FdsR for this region. Two FdsR binding sites characterized by the inverted-repeat structure ATANG-N10-CNTAT were identified. The regulatory pattern found in R. eutropha was also observed in the heterologous host E. coli and results from a novel mode of control of formate dehydrogenase genes.

Amino Acid Sequence↗

PubMed2006

Parallel mechanisms for resting nucleo-cytoplasmic shuttling and activity dependent translocation provide dual control of transcriptional regulators HDAC and NFAT in skeletal muscle fiber type plasticity.

Skeletal muscle fibers exhibit plasticity of their physiological and biochemical properties in response to the firing pattern from the innervating motor neuron. In particular, the gene expression pattern generally characteristic of a slow twitch fiber can be induced in a fast twitch fiber by chronic slow fiber type electrical stimulation. We have studied the nucleo-cytoplasmic distribution of two transcriptional regulators of slow fiber type genes, HDAC4 and NFATc1, both in response to slow fiber type stimulation and in resting conditions using cultured fast twitch skeletal muscle fibers. HDAC4 is present in both cytoplasm and nuclei of resting fibers, and moves out of the nuclei in response to slow fiber type stimulation. The stimulation-dependent nuclear efflux of HDAC4 requires activation of nuclear CaMKII, which phosphorylates nuclear HDAC4 and thus allows its exit of the nucleus. In unstimulated resting fibers, a balance of nuclear efflux and influx of HDAC4 establishes the resting level of nuclear HDAC4. However, the nuclear efflux of HDAC4 in resting fibers does not involve CaMKII. Slow fiber type stimulation also causes NFATc1 translocation from the cytoplasm into muscle fiber nuclei following dephosphorylation by calcineurin (CaN) activated by the elevated cytosolic Ca2+ accompanying fiber stimulation. In resting fibers, NFATc1 exhibits balanced shuttling between cytoplasm and nucleus, but during this shuttling NFATc1 influx does not require CaN and NFATc1 efflux does not require the kinases involved in removing nuclear NFATc1 following prior activity. Thus different enzymes are responsible for HDAC4 nuclear efflux in resting and active fibers, and different pathways mediate NFATc1 nuclear influx and efflux in resting and active fibers. Such dual mechanisms for resting shuttling and active movements provide the potential for the resting level and the rate of translocation during fiber stimulation to be controlled independently for both of the transcriptional regulators HDAC4 and NFATc1.

Animals↗

PubMed2000

Dual control of dorsal raphe serotonergic neurons by GABA(B) receptors. Electrophysiological and microdialysis studies.

We assessed the role of GABA(B) receptors in the control of serotonergic (5-HT) neurons of the dorsal raphe nucleus (DRN) by using microdialysis in vivo and intra- and extracellular recording in vitro in the rat. The GABA(B) agonist R(+)baclofen (but not the inactive S(-)enantiomer) enhanced the 5-HT output in the DRN (4. 7-fold at 15 mg/kg s.c.) and, to a much lesser extent, striatum of unanesthetized rats. Phaclofen (2 mg/kg s.c.) antagonized the effects of 6 mg/kg R(+)baclofen in dorsal striatum. Using dual-probe microdialysis, R(+)baclofen (0.1-100 microM) applied in the DRN enhanced the local 5-HT output (4.5-fold at 100 microM) but decreased that in striatum at 100 microM. At concentrations higher than 100 microM there was a moderate decrement in the elevation of 5-HT in the DRN. In midbrain slices, bath R(+)baclofen exerted a biphasic effect on DRN 5-HT neurons. Consistent with a reduced striatal 5-HT release when infused in the DRN, R(+)baclofen (0.1-30 microM) induced an outward current in 5-HT neurons (IC(50) = 1.4 microM). Lower R(+)baclofen concentrations (0.01-1 microM) preferentially reduced GABAergic inhibitory postsynaptic currents induced by N-methyl-D-aspartate (20 microM) in 5-HT neurons (IC(50) = 72 nM). Using extracellular recordings, R(+)baclofen (300 nM) enhanced the ability of NMDA to induce firing in a subpopulation of serotonergic neurons. These results are consistent with a preferential activation by a low concentration of R(+)baclofen of presynaptic GABA(B) receptors on GABAergic afferents that could disinhibit 5-HT neurons and increase 5-HT release.

Animals↗

PubMed1987

Dual control mechanism for heme oxygenase: tin(IV)-protoporphyrin potently inhibits enzyme activity while markedly increasing content of enzyme protein in liver.

Tin(IV)-protoporphyrin (Sn-protoporphyrin) potently inhibits heme degradation to bile pigments in vitro and in vivo, a property that confers upon this synthetic compound the ability to suppress a variety of experimentally induced and naturally occurring forms of jaundice in animals and humans. Utilizing rat liver heme oxygenase purified to homogeneity together with appropriate immunoquantitation techniques, we have demonstrated that Sn-protoporphyrin possesses the additional property of potently inducing the synthesis of heme oxygenase protein in liver cells while, concurrently, completely inhibiting the activity of the newly formed enzyme. Substitution of tin for the central iron atom of heme thus leads to the formation of a synthetic heme analogue that regulates heme oxygenase by a dual mechanism, which involves competitive inhibition of the enzyme for the natural substrate heme and simultaneous enhancement of new enzyme synthesis. Cobaltic(III)-protoporphyrin (Co-protoporphyrin) also inhibits heme oxygenase activity in vitro, but unlike Sn-protoporphyrin it greatly enhances the activity of the enzyme in the whole animal. Co-protoporphyrin also acts as an in vivo inhibitor of heme oxygenase; however, its inducing effect on heme oxygenase synthesis is so pronounced as to prevail in vivo over its inhibitory effect on the enzyme. These studies show that certain synthetic heme analogues possess the ability to simultaneously inhibit as well as induce the enzyme heme oxygenase in liver. The net balance between these two actions, as reflected in the rate of heme oxidation activity in the whole animal, appears to be influenced by the nature of the central metal atom of the synthetic metalloporphyrin.

Animals↗

PubMed1994

Chronic desipramine treatment influences D1 stimulation and D2 inhibition (dual control) of adenylate cyclase by dopamine in rat striatum.

The response of adenylate cyclase to GTP and to dopamine (DA) was investigated in striatal membranes from desipramine (DMI)- or saline-treated rats. DMI (15 mg/kg) or saline was injected i.p. once a day for 3 weeks. In saline-treated control membranes, GTP exerted a biphasic effect on basal and DA-stimulated enzyme activity; peak levels of stimulation by DA plus GTP were observed at 1 microM GTP. On the other hand, peak levels moved to the right in the GTP dose response curve in DMI-treated membranes. Therefore, D2 inhibition might be attenuated, while the D2 specific agonist, PPHT, was not observed to cause inhibition of adenylate cyclase. Furthermore, D1 stimulation of adenylate cyclase via D1 specific agonist SKF was attenuated in DMI-treated membranes. It seems, therefore, that chronic treatment of rat striatum with DMI exerts a dual influence, that is, a lessening of both D1 stimulation and D2 inhibition of adenylate cyclase, and alters specifically the overall process of the adenylate cyclase system.

Adenylyl Cyclase Inhibitors↗

PubMed1996

Dual control of human interleukin-2 and interferon-gamma gene expression by histamine: activation and suppression.

Histamine is considered to be an activator of cells with suppressive capacity. In agreement with this concept, we show that histamine elicits a strong inhibition of the induced expression of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) genes. However, our experiments reveal a novel property of histamine: early in the induction process, it strongly stimulates expression of these two genes in cultured human peripheral blood mononuclear cells (PBMC). The histamine-mediated superinduction of IL-2 mRNA is seen also in a Th cell line, showing that such cells respond directly to histamine. In the course of mitogenic induction, a 20-fold stimulation by histamine is converted into an equally strong inhibition. The response of a PBMC population to histamine thus undergoes a remarkable change following T cell activation. The dual effect of histamine can be blocked by the H2 histamine receptor antagonist cimetidine, while the early activation by histamine is mimicked by the H2 agonist impromidine, showing that both activation and inhibition of IL-2 and IFN-gamma gene expression by histamine are exerted via this receptor. These results support the concept that histamine, released during an immune response, exerts opposite regulatory effects by first activating cells able to express the IL-2 and IFN-gamma genes and only then suppressive cells that become responsive to histamine more slowly, but once activated shut off the expression of these genes.

Cells, Cultured↗

PubMed1990

Dual control of DNA synthesis by alpha- and beta-adrenergic mechanisms in normoxic and hypoxic neonatal rat brain.

To examine how catecholamines influence cell replication in the developing brain, we examined regional [3H]thymidine incorporation into DNA after acute challenge with an alpha-adrenergic blocking agent (phenoxybenzamine) or a beta-blocker (propranolol). Phenoxybenzamine inhibited DNA synthesis in 1-day-old rat pups but the effect was less pronounced at 8 days; regional differences corresponded to transient expression of alpha-receptors and their subsequent maturational decline. Propranolol given at 1 day of age exerted a regionally selective, promotional effect on DNA synthesis; in contrast, at 8 days, propranolol inhibited DNA synthesis in all brain regions. Propranolol, but not phenoxybenzamine, also exacerbated the reduction in DNA synthesis caused by neonatal hypoxia, and again the effect was limited to the 1-day-old group. These results indicate that catecholamines exert a dual action on DNA synthesis; the effects are dependent upon maturational profiles of specific receptor populations which are either transiently expressed or which couple to cell replication only during a critical period.

Animals↗

PubMed2000

Dual control of melanogenesis and melanoma growth: overview molecular to clinical level and the reverse.

Utilizing increased melanin pigmentation and accentuated melanogenesis seen in malignant melanoma, we newly developed melanoma-selective boron neutron capture therapy (BNCT) after designing and synthesizing the 10B-DOPA analogue, 10B-p-boronophenylalanine (10B-BPA). After multi-disciplined and extensive basic and pre-clinical investigations, we successfully treated 18 cases of human melanoma. Recently, we found that accentuated synthesis of melanin monomers, richest within coated vesicles (CV) in melanoma cells, plays a critical role in attracting 10B-BPA through chemical complex formation of monomers and 10B-BPA. CV are indeed BPA-localizing organelles. This led us to the new clinical endeavor that BPA may possess the potential ability to suppress melanin polymer formation through 'melanin monomer trapping' out of the melanogenic pathway which is highly regulated by the function of CV in pigment cells. It was soon found that melanin polymer formation can be suppressed by BPA at the chemical and cellular levels, then at the clinical level. Our discovery, that single molecule 10B-BPA possesses the dual nature of eradication of melanoma with BNCT and suppression of melanin hyperpigmentation, resulted from pursuing bilateral feedback at each stage from pure science to clinical application and vice versa. A further example of bilateral feedback is the development of gene-transfer applied BNCT (gBNCT). This also has its roots in clinical hurdles faced in treating amelanotic melanomas by 10B-BPA BNCT. The transfer of tyrosinase and melanin monomer synthesis-related genes into target cancer cells has produced more effective BNCT and may lead to gBNCT for non-melanoma cancers.

Animals↗

PubMed2004

The ytkD (mutTA) gene of Bacillus subtilis encodes a functional antimutator 8-Oxo-(dGTP/GTP)ase and is under dual control of sigma A and sigma F RNA polymerases.

The regulation of expression of ytkD, a gene that encodes the first functional antimutator 8-oxo-dGTPase activity of B. subtilis, was studied here. A ytkD-lacZ fusion integrated into the ytkD locus of wild-type B. subtilis 168 revealed that this gene is expressed during both vegetative growth and early stages of sporulation. In agreement with this result, ytkD mRNAs were detected by both Northern blotting and reverse transcription-PCR during both developmental stages. These results suggested that ytkD is transcribed by the sequential action of RNA polymerases containing the sigma factors sigma(A) and sigma(F), respectively. In agreement with this suggestion, the spore-associated expression was almost completely abolished in a sigF genetic background but not in a B. subtilis strain lacking a functional sigG gene. Primer extension analysis mapped transcriptional start sites on mRNA samples isolated from vegetative and early sporulating cells of B. subtilis. Inspection of the sequences lying upstream of the transcription start sites revealed the existence of typical sigma(A)- and sigma(F)-type promoters. These results support the conclusion that ytkD expression is subjected to dual regulation and suggest that the antimutator activity of YtkD is required not only during vegetative growth but also during the early sporulation stages and/or germination of B. subtilis. While ytkD expression obeyed a dual pattern of temporal expression, specific stress induction of the transcription of this gene does not appear to occur, since neither oxidative damage (following either treatment with paraquat or hydrogen peroxide) nor mitomycin C treatment or sigma(B) general stress inducers (sodium chloride, ethanol, or heat) affected the levels of the gene product produced.

Amino Acid Sequence↗

PubMed1981

Dual control of the gua operon of Escherichia coli K12 by adenine and guanine nucleotides.

The gua operon of Escherichia coli K12 comprises structural genes for the two enzymes, IMP dehydrogenase and GMP synthetase, required for the biosynthesis of GMP from IMP. The specific activities of these enzymes were measured in various purine auxotrophs. GuaA and guaB mutants (guanine-specific) were depressed under conditions of growth limitation by guanine but were repressed by excess guanine. This suggests that formation of the enzymes is normally controlled by a guanine nucleotide. Derepression of the operon in purine-starved pur mutants depended on the type of mutant and on whether adenine or guanine was provided. A purA strain (adenine-specific) and strains with early blocks in purine biosynthesis (purF and purD) did not derepress. PurE or purC strains [5'-phosphoribosyl-5-aminoimidazole (AIR)-accumulating] derepressed only 4-fold. The operon was repressed in purH strains [5'-phosphoribosyl-5-amino-4-imidazolecarboxamide (AICAR)-accumulating] grown with limiting guanine or hypoxanthine, but derepressed by growth with limiting adenine. Two mutants (purA guaA and purA guaB) which can neither synthesize AMP and GMP de novo, nor interconvert them, were isolated. The specific activity of IMP dehydrogenase in one of these strains grown with different concentrations of guanine and adenine revealed that adenine induces tha gua operon whereas guanine represses it. Intracellular purine nucleotide pools wee measured in a purH mutant repressed (guanine-grown) and derepressed (adenine-grown) for IMP dehydrogenase. The guanylate pool was similar under the two growth conditions; however the adenylate pool of the adenine-grown bacteria was two to three times greater than that of the guanine-grown cells. A dual mechanism for regulating expression of the gua operon, involving induction by AMP and repression by GMP, is proposed.

Adenine↗

PubMed1997

Dual control of glut1 glucose transporter gene expression by hypoxia and by inhibition of oxidative phosphorylation.

glut1 gene expression and glucose transport are stimulated in a variety of cells and tissues in response to hypoxia. glut1 is also up-regulated by inhibitors of oxidative phosphorylation (such as azide) in the presence of oxygen. Here, we test the hypothesis that hypoxia stimulates glut1 gene expression independent of its inhibitory effect on oxidative phosphorylation. We examined the effect of cobalt chloride, a known stimulator of genes responsive to reduced oxygen concentration per se, on GLUT1 expression under normoxic conditions and compared the results with the response to azide. Exposure of a rat liver cell line (Clone 9) to 250 microM cobalt chloride increases GLUT1 mRNA content, which becomes evident at 2 h, reaches a maximal value of approximately 12-fold at 8 h, and remains elevated at approximately 8-fold at 24 h. GLUT1 mRNA was the only GLUT isoform expressed in control cells and in cells exposed to cobalt chloride or azide. The induction of GLUT1 mRNA by cobalt chloride is associated with a approximately 10-fold stimulation of cytochalasin B-inhibitable 3-O-methyl-D-glucose transport at 24 h. In contrast to the rapid decrease in cell ATP levels and the stimulation of glucose transport in response to azide, cell ATP content and glucose transport remained unaltered during the initial 1-h period of exposure to cobalt chloride. The effect of cobalt chloride on GLUT1 mRNA content is mimicked by Ni(II) or Mn(II) but not by Fe(II). Employing actinomycin D, we found no increase in the approximately 1.5-h half-life of GLUT1 mRNA in cobalt chloride-treated cells, suggesting that the effect of cobalt chloride on GLUT1 mRNA content is largely mediated at the transcriptional level; in contrast, GLUT1 mRNA half-life increased to >8 h in azide-treated cells. In transient transfections we found that approximately 6 kilobase pairs (kbp) of 5'-flanking region of the rat glut1 promoter confers both cobalt chloride- and azide-inducibility to a reporter gene. Deletion of approximately 2, 500 base pairs (bp) from the 5' end of the approximately 6-kbp DNA fragment results in a reduction of the response to cobalt chloride and a complete loss of the response to azide. A 666-bp DNA segment located approximately 6.0 kbp upstream of the transcription start site was found to be necessary for the increase in reporter gene expression in response to azide, whereas a 480-bp segment located at approximately -3.5 kbp mediated the response to cobalt chloride. The 480-bp segment is highly homologous to the previously reported mouse glut1 enhancer and contains several potential regulatory elements, including a hypoxia-inducible element; an additional hypoxia-inducible element is present in the 666-bp segment. Our results suggest that glut1 gene expression is regulated in a dual fashion by hypoxia per se and in response to inhibition of oxidative phosphorylation.

3T3 Cells↗