Inhibitory Effects of Heavy Metals on Transcription Factor Sp1.

Gong, Peng; Ogra, Yasumitsu; Koizumi, Satoshi

doi:10.2486/indhealth.38.224

Cited by 14 publications

(13 citation statements)

References 15 publications

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“…Reaction mixtures were incubated at 25°C for 20 min and analyzed by native 5% polyacrylamide gel electrophoresis and autoradiography as described previously (16,37). A double-stranded oligonucleotide containing the sequence 5Ј-TTTTCTGCTGAGTCAAGGTCCG-3Ј was used as a probe in EMSA reactions (core StRE sequence is underlined).…”

Section: Methodsmentioning

confidence: 99%

“…IRPTCs were seeded (1 ϫ 10 6 cells/10-cm plate) and cultured for 48-72 h in complete medium and then treated with vehicle or 10 M heme in serum-free medium for 3 h. Whole cell extracts (WCEs) were prepared as described previously (16). The standard binding reaction mixture (12.5 l) contained 18 mM HEPES (pH 7.9), 80 mM KCl, 2 mM MgCl 2, 10 mM DTT, 10% glycerol, 0.2 mg/ml bovine serum albumin, 160 g/ml poly(dIdC), 20,000 counts per minute [␥-…”

Section: Methodsmentioning

confidence: 99%

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Heme activates the heme oxygenase-1 gene in renal epithelial cells by stabilizing Nrf2

Alam

Killeen

Gong

et al. 2003

American Journal of Physiology-Renal Physiology

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157

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The mechanism of heme oxygenase-1 gene ( ho-1) activation by heme in immortalized rat proximal tubular epithelial cells was examined. Analysis of the ho-1promoter identified the heme-responsive sequences as the stress-response element (StRE), multiple copies of which are present in two enhancer regions, E1 and E2. Electrophoretic mobility shift assays identified Nrf2, MafG, ATF3, and Jun and Fos family members as StRE-binding proteins; binding of Nrf2, MafG, and ATF3 was increased in response to heme. Dominant-negative mutants of Nrf2 and Maf, but not of c-Fos and c-Jun, inhibited basal and heme-induced expression of an E1-controlled luciferase gene. Heme did not affect the transcription activity of Nrf2, dimerization between Nrf2 and MafG, or the level of MafG, but did stimulate expression of Nrf2. Heme did not influence the level of Nrf2 mRNA but increased the half-life of Nrf2 protein from ∼10 min to nearly 110 min. These results indicate that heme promotes stabilization of Nrf2, leading to accumulation of Nrf2 · MafG dimers that bind to StREs to activate the ho-1 gene.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Heme activates the heme oxygenase-1 gene in renal epithelial cells by stabilizing Nrf2

Alam

Killeen

Gong

et al. 2003

American Journal of Physiology-Renal Physiology

Self Cite

157

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“…Reaction mixtures were incubated at 25°C for 20 min and analyzed by native 5% polyacrylamide gel electrophoresis and autoradiography as described previously (29,30). A double-stranded oligonucleotide containing the sequence 5Ј-GATCTTTTATGCTGAGTCATGGTTT-3Ј (core StRE underlined) was end-labeled by T4 polynucleotide kinase, purified on a 10% polyacrylamide gel, and used as probe in EMSA reactions.…”

Section: Methodsmentioning

confidence: 99%

Cobalt Induces Heme Oxygenase-1 Expression by a Hypoxia-inducible Factor-independent Mechanism in Chinese Hamster Ovary Cells

Gong¹,

Hu²,

Stewart³

et al. 2001

Journal of Biological Chemistry

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138

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We have shown previously that activation of the heme oxygenase-1 (ho-1) gene by hypoxia in aortic smooth muscle cells is mediated by hypoxia-inducible factor-1 (HIF-1). In mutant (Ka13) Chinese hamster ovary cells lacking HIF activity, accumulation of ho-1 mRNA in response to hypoxia and the hypoxia-mimetic CoCl 2 was similar to that observed in wild type (K1) cells. These results support the existence of HIF-dependent and HIFindependent mechanisms for ho-1 gene activation by hypoxia and CoCl 2 . In Ka13 cells, CoCl 2 stimulated expression of a luciferase reporter gene under the control of a 15-kilobase pair mouse ho-1 promoter (pHO15luc). Mutation analyses identified the cobalt-responsive sequences as the stress-response elements (StREs). In electrophoretic mobility shift assays, two specific StRE-protein complexes were observed using extracts from Ka13 cells. In response to cobalt, the level of the slower migrating complex X increased, whereas that of complex Y decreased, in a time-dependent manner. Members of the AP-1 superfamily of basic-leucine zipper factors bind to the StRE. Antibody supershift electrophoretic mobility shift assays did not detect Jun, Fos, or ATF/CREB proteins but identified Nrf2 and the small Maf protein, MafG, as components of complex X. Furthermore, dominant-negative mutants of Nrf2 and small Maf, but not of other bZIP factors, attenuated cobalt-mediated gene activation. Additional experiments demonstrated that induction by cobalt does not result from increased expression of MafG or regulated nuclear translocation of Nrf2 but is dependent on cellular oxidative stress. Unlike cobalt, hypoxia did not stimulate pHO15luc expression and did not increase StRE binding activity, indicating distinct mechanisms for ho-1 gene activation by cobalt and hypoxia in Chinese hamster ovary cells.The adaptive response to low oxygen tension (hypoxia) includes increased production of a select set of proteins involved in oxygen homeostasis at the cellular and systemic levels. These proteins include: erythropoietin (EPO), 1 which stimulates erythropoiesis and the oxygen carrying capacity of the blood; vascular endothelial growth factor (VEGF) and one of its receptors Flt-1, which promote angiogenesis and the delivery of oxygen carrying blood to hypoxic sites; and glucose transporter-1 (Glut-1) and various glycolytic enzymes that provide for increased energy production through glycolysis during periods of reduced energy production via oxidative phosphorylation (reviewed in Ref. 1). Hypoxia-dependent expression of these and other proteins is regulated by both transcriptional and post-transcriptional mechanisms.Transcriptional regulation of many hypoxia-responsive genes is critically dependent on a common cis-acting sequence, the hypoxia response element (HRE), and the trans-acting factor hypoxia-inducible factor-1 (HIF-1), a heterodimeric protein comprising ␣ and ␤ subunits. The ␣ subunit is unique to HIF-1, whereas the ␤ subunit is shared by other transcription factors, most notably the arylhydrocarbon recept...

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“…Interactions of these nonspecific complexes with molecular oxygen leads to the formation of reactive oxygen species such as H 2 O 2 , resulting in oxidative stress within the cell (23). In addition to oxidative stress, complexation of sulfhydryl groups with heavy metal cations results in reduced activity of sensitive enzymes (16). Previous studies identified several examples in which heavy metals were toxic to cellular processes, including the following.…”

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confidence: 99%

Whole-Genome Transcriptional Analysis of Heavy Metal Stresses in Caulobacter crescentus

et al. 2005

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The bacterium Caulobacter crescentus and related stalk bacterial species are known for their distinctive ability to live in low-nutrient environments, a characteristic of most heavy metal-contaminated sites. Caulobacter crescentus is a model organism for studying cell cycle regulation with well-developed genetics. We have identified the pathways responding to heavy-metal toxicity in C. crescentus to provide insights for the possible application of Caulobacter to environmental restoration. We exposed C. crescentus cells to four heavy metals (chromium, cadmium, selenium, and uranium) and analyzed genome-wide transcriptional activities postexposure using an Affymetrix GeneChip microarray. C. crescentus showed surprisingly high tolerance to uranium, a possible mechanism for which may be the formation of extracellular calcium-uranium-phosphate precipitates. The principal response to these metals was protection against oxidative stress (up-regulation of manganese-dependent superoxide dismutase sodA). Glutathione S-transferase, thioredoxin, glutaredoxins, and DNA repair enzymes responded most strongly to cadmium and chromate. The cadmium and chromium stress response also focused on reducing the intracellular metal concentration, with multiple efflux pumps employed to remove cadmium, while a sulfate transporter was down-regulated to reduce nonspecific uptake of chromium. Membrane proteins were also up-regulated in response to most of the metals tested. A two-component signal transduction system involved in the uranium response was identified. Several differentially regulated transcripts from regions previously not known to encode proteins were identified, demonstrating the advantage of evaluating the transcriptome by using whole-genome microarrays.Potentially hazardous levels of heavy metals have dispersed into subsurface sediment and groundwater in a number of metal-contaminated sites and represent a challenge for environmental restoration. Effective bioremediation of these sites requires knowledge of genetic pathways for resistance and biotransformation by component organisms within a microbial community. However, a comprehensive understanding of bacterial mechanisms of heavy metal toxicity and resistance has yet to be achieved. While many metals are essential to microbial function, heavy metals, i.e., most of those with a density above 5 g/cm 3 , have toxic effects on cellular metabolism (46). The majority of heavy metals are transition elements with incompletely filled d orbitals providing heavy metal cations which can form complex compounds with redox activity (46,70). Therefore, it is important to the health of the organism that the intracellular concentrations of heavy metal ions are tightly controlled. However, due to their structural and valence similarities to nontoxic metals, heavy metals are often transported into the cytoplasm through constitutively expressed nonspecific transport systems (46). As such, heavy metals invariably find their way into the cell. Once inside the cell, toxic effects of heavy metals...

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Inhibitory Effects of Heavy Metals on Transcription Factor Sp1.

Cited by 14 publications

References 15 publications

Heme activates the heme oxygenase-1 gene in renal epithelial cells by stabilizing Nrf2

Heme activates the heme oxygenase-1 gene in renal epithelial cells by stabilizing Nrf2

Cobalt Induces Heme Oxygenase-1 Expression by a Hypoxia-inducible Factor-independent Mechanism in Chinese Hamster Ovary Cells

Whole-Genome Transcriptional Analysis of Heavy Metal Stresses in Caulobacter crescentus

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