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Journal ArticleDOI

Differential regulation of the rainbow trout ( Oncorhynchus mykiss ) MT-A gene by nuclear factor interleukin-6 and activator protein-1

17 Dec 2013-BMC Molecular Biology (BioMed Central)-Vol. 14, Iss: 1, pp 28-28

TL;DR: The involvement of NF-IL6 and AP1 in rtMT-A gene expression following exposure to oxidative stress and tumour promotion suggests an involvement of MT in protection during pathological processes such as inflammation and cancer.

AbstractPreviously we have identified a distal region of the rainbow trout (Oncorhynchus mykiss) metallothionein-A (rtMT-A) enhancer region, being essential for free radical activation of the rtMT-A gene. The distal promoter region included four activator protein 1 (AP1) cis-acting elements and a single nuclear factor interleukin-6 (NF-IL6) element. In the present study we used the rainbow trout hepatoma (RTH-149) cell line to further examine the involvement of NF-IL6 and AP1 in rtMT-A gene expression following exposure to oxidative stress and tumour promotion. Using enhancer deletion studies we observed strong paraquat (PQ)-induced rtMT-A activation via NF-IL6 while the AP1 cis-elements showed a weak but significant activation. In contrast to mammals the metal responsive elements were not activated by oxidative stress. Electrophoretic mobility shift assay (EMSA) mutation analysis revealed that the two most proximal AP1 elements, AP11,2, exhibited strong binding to the AP1 consensus sequence, while the more distal AP1 elements, AP13,4 were ineffective. Phorbol-12-myristate-13-acetate (PMA), a known tumor promoter, resulted in a robust induction of rtMT-A via the AP1 elements alone. To determine the conservation of regulatory functions we transfected human Hep G2 cells with the rtMT-A enhancer constructs and were able to demonstrate that the cis-elements were functionally conserved. The importance of NF-IL6 in regulation of teleost MT is supported by the conservation of these elements in MT genes from different teleosts. In addition, PMA and PQ injection of rainbow trout resulted in increased hepatic rtMT-A mRNA levels. These studies suggest that AP1 primarily is involved in PMA regulation of the rtMT-A gene while NF-IL6 is involved in free radical regulation. Taken together this study demonstrates the functionality of the NF-IL6 and AP-1 elements and suggests an involvement of MT in protection during pathological processes such as inflammation and cancer.

Topics: Rainbow trout (55%), Enhancer (55%), Gene expression (53%), Electrophoretic mobility shift assay (52%), Promoter (52%)

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Citations
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Journal ArticleDOI
TL;DR: The results of this study raise a possibility that a genetic variation of MT2A may be implicated in the etiology of laryngeal cancer in a Polish population.
Abstract: Metallothioneins are intracellular regulators of many biological mechanisms including differentiation, proliferation, angiogenesis and invasion, which are crucial processes in carcinogenesis. This study examines the association between three single-nucleotide polymorphisms at loci -5 A/G (rs28366003) and -209 A/G (rs1610216) in the core promoter region and at locus +838 C/G (rs10636) in 3'UTR region of the metallothionein 2A (MT2A) gene with squamous cell laryngeal cancer (SCLC) risk, as well as with tumor invasiveness according to tumor front grading (TFG). Genotyping was performed using the polymerase chain reaction-restriction fragment length polymorphism technique in 323 genetically unrelated individuals with SCLC and 418 randomly selected healthy volunteers. Only one SNP (rs28366003) was significantly related to laryngeal cancer in the study population. Compared with homozygous common allele carriers, heterozygous and homozygous for the G variant had significantly increased risk of SCLC [adjusted odds ratio (OR) = 2.90, 95 % confidence interval (CI) 1.53-5.21, p dominant 18 points) (OR = 3.76, 95 % CI 1.15-12.56, p = 0.03) and diffuse tumor growth (OR = 5.86, 95 % Cl 0.72-44.79, p = 0.08). The results of this study raise a possibility that a genetic variation of MT2A may be implicated in the etiology of laryngeal cancer in a Polish population.

13 citations


Cites background from "Differential regulation of the rain..."

  • ...MT promoter activity is regulated by six zinc-finger metalresponsive transcription factor (MTF-1) and a nuclear factor I (NF-I) protein [2]....

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Journal ArticleDOI
TL;DR: The primary fish gill cell culture system (FIGCS) is an in vitro technique which has the potential to replace animals in whole effluent toxicity tests and the increase in the expression of genes encoding mta, cyp1a1 and cyp3a27 in FIGCS is indicative of the presence of biologically active pollutants.
Abstract: The primary fish gill cell culture system (FIGCS) is an in vitro technique which has the potential to replace animals in whole effluent toxicity tests. In the current study FIGCS were transported into the field and exposed to filtered (0.2μm) river water for 24h from 4 sites, on 2 different sampling dates. Sites 1 and 2 are situated in an urban catchment (River Wandle, London, UK) with site 1 downstream of a sewage treatment work; site 3 is located in a suburban park (River Cray, Kent, UK), and site 4 is more rural (River Darent, Kent, UK). The change in transepithelial electrical resistance (TER), the expression of the metal responsive genes metallothionein A (mta) and B (mtb), cytochrome P450 1A1 (cyp1a1) and 3A27 (cyp3a27), involved in phase 1 metabolism, were assessed following exposure to sample water for 24h. TER was comparable between FIGCS exposed to 0.2μm filtered river water and those exposed to synthetic moderately soft water for 24h. During the first sampling time, there was an increase in mta, cyp1a1 and cyp3a27 gene expression in epithelium exposed to water from sites 1 and 2, and during the second sampling period an increase in cyp3a27 gene expression at sites 1 and 4. Urban river water is a complex mixture of contaminants (e.g., metals, pesticides, pharmaceuticals and polyaromatic hydrocarbons) and the increase in the expression of genes encoding mta, cyp1a1 and cyp3a27 in FIGCS is indicative of the presence of biologically active pollutants.

13 citations


Cites background from "Differential regulation of the rain..."

  • ...The distal region of the rainbow 319 trout mta promoter possess both activator protein 1 (AP1) and a nuclear factor interleukin-6 320 (NF-IL6) elements that have been shown to play a direct role in mta induction in response to 321 paraquat, an herbicide that induces ROS (Kling et al., 2013)....

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  • ...Some pharmaceuticals and personal care products 316 have been shown to affect reactive oxygen species(ROS) production in rainbow trout gonad 317 cell line (RTG-2) (Fernandez et al., 2013) and PAHs are known to induce free radical 318 production (e.g. Wells and Winn 1996; Zhu et al., 2014)....

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  • ...Effects of human 402 pharmaceuticals on cytotoxicity, EROD activity and ROS production in fish hepatocytes....

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  • ...In addition, FIGCS treated with 322 H2O2, that produces ROS, show an increase in mta and b gene expression, as well as other 323 antioxidant genes (Chung et al., 2005)....

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  • ...The antioxidant gene expression is attenuated by the 324 14 zinc chelator N,N,N’,N’-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), suggesting that the 325 response to ROS in FIGCS is in part mediated by an increase in intracellular zinc 326 concentrations (Chung et al., 2005)....

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Journal ArticleDOI
TL;DR: The findings suggest that MT2A gene variation rs28366003 may be implicated in the etiology of sinonasal inverted papilloma in a Polish population.
Abstract: Inverted papillomas are a unique group of locally aggressive benign epithelial neoplasms in the nasal cavity and paranasal sinuses arising from the Schneiderian mucosa. Metallothioneins are sulfhydryl-rich heavy metal-binding proteins required for metal toxicity protection and regulation of biological mechanisms including proliferation and invasion. The goal of this study was to identify three SNPs at loci −5 A/G (rs28366003) and −209 A/G (rs1610216) in the core promoter region and at locus +838 C/G (rs10636) in 3′UTR region of the MT2A gene with IP risk and with tumor invasiveness according to Krouse staging. Genotyping was performed using the PCR restriction fragment length polymorphism technique in 130 genetically unrelated IP individuals, and 418 randomly selected healthy volunteers. The presence of the rs28366003 SNP was significantly related to the risk of IP within the present population-based case-control study. Compared to homozygous common allele carriers, heterozygosity and homozygosity for the G variant had a significantly increased risk of IP (adjusted odds ratio [OR] = 7.71, 95 % confidence interval [CI]: 4.01–14.91, p dominant < 0.001). Moreover, risk allele carriers demonstrated higher Krouse stage (pT1 vs. pT2-4) (OR = 19.32; 95 % CI, 2.30–173.53; p < 0.0001), diffuse tumor growth (OR = 4.58; 95 % CI, 1.70–12.11; p = 0.0008), bone destruction (OR = 4.13; 95 % CI, 1.50–11.60; p = 0.003), and higher incidence of tumor recurrences (OR = 5.11; 95 % CI, 1.68–15.20; p = 0.001). The findings suggest that MT2A gene variation rs28366003 may be implicated in the etiology of sinonasal inverted papilloma in a Polish population.

11 citations


Cites background from "Differential regulation of the rain..."

  • ...er region is played by the six zinc-finger-metal-responsive transcription factor (MTF-1) and short cis-acting DNA metal response elements (MREs) as transcriptional activators and a nuclear factor I (NF-I) protein as a downregulator of MT expression [11]....

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Journal ArticleDOI
TL;DR: Results obtained in this study suggest that -5 A/G SNP in MT2A gene may have an effect on allele-specific gene expression and accumulation of metal levels in laryngeal cancer.
Abstract: Metallothioneins (MTs) are low molecular weight, cysteine-rich heavy metal-binding proteins which participate in the mechanisms of Zn homeostasis, and protect against toxic metals. MTs contain metal-thiolate cluster groups and suppress metal toxicity by binding to them. The aim of this study was to determine the -5 A/G (rs28366003) single-nucleotide polymorphism (SNP) in the core promoter region of the MT2A gene and to investigate its effect on allele-specific gene expression and Cd, Zn and Cu content in squamous cell laryngeal cancer (SCC) and non-cancerous laryngeal mucosa (NCM) as a control. The MT2A promoter region -5 A/G SNP was determined by restriction fragment length polymorphism using 323 SCC and 116 NCM. MT2A gene analysis was performed by quantitative real-time PCR. The frequency of A allele carriage was 94.2% and 91.8% in SCC and NCM, respectively, while G allele carriage was detected in 5.8% and 8.2% of SCC and NCM samples, respectively. As a result, a significant association was identified between the -5 A/G SNP in the MT2A gene with mRNA expression in both groups. Metal levels were analyzed by flame atomic absorption spectrometry. The significant differences were identified between A/A and both the A/G and G/G genotypes, with regard to the concentration of the contaminating metal. The Spearman rank correlation results showed that the MT2A expression and Cd, Zn, Cu levels were negatively correlated. Results obtained in this study suggest that -5 A/G SNP in MT2A gene may have an effect on allele-specific gene expression and accumulation of metal levels in laryngeal cancer.

10 citations


Cites background from "Differential regulation of the rain..."

  • ...Furthermore, overexpression of MTF-1 overcomes NF-I-mediated repression of the MT promoter activity (Kling et al., 2013)....

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Journal ArticleDOI
TL;DR: Interestingly, Pl-Fra expression was induced by the different metals and the induction kinetics revealed its persistent expression during treatments, and its temporal and spatial behavior in response to the three metals was comparable to that of Pl-jun and Pl-MT.
Abstract: Lithium (Li), Nickel (Ni), and Zinc (Zn) are metals normally present in the seawater, although they can have adverse effects on the marine ecosystem at high concentrations by interfering with many biological processes. These metals are toxic for sea urchin embryos, affecting their morphology and developmental pathways. In particular, they perturb differently the correct organization of the embryonic axes (animal-vegetal, dorso-ventral): Li is a vegetalizing agent and Ni disrupts the dorso-ventral axis, while Zn has an animalizing effect. To deeply address the response of Paracentrotus lividus embryos to these metals, we studied the expression profiling of Pl-Fra transcription factor (TF), relating it to Pl-jun, a potential partner for AP-1 complex formation, and to Pl-MT, known to be an AP-1 target and to have a protective role against heavy metals. The AP-1 TFs are found throughout the animal kingdom and are involved in many cellular events, i.e. cell proliferation and differentiation, immune and stress responses, cancer growth. Here, we isolated the complete Pl-Fra cDNA and showed that Pl-Fra transcript, already present in the unfertilized eggs, was newly synthesized from the blastula stage, while its spatial distribution was mainly observed in skeletogenic cells, similarly to Pl-jun. Interestingly, Pl-Fra expression was induced by the different metals and the induction kinetics revealed its persistent expression during treatments. Moreover, its temporal and spatial behavior in response to the three metals was comparable to that of Pl-jun and Pl-MT. The understanding of AP-1 functions in invertebrates may provide new knowledge about the mechanisms of response to metal injuries, as well as it might lead to acknowledge the TFs as new type of biomarkers for the evaluation of hazards in polluted environment.

8 citations


References
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Journal ArticleDOI
TL;DR: This protocol provides an overview of the comparative CT method for quantitative gene expression studies and various examples to present quantitative gene Expression data using this method.
Abstract: Two different methods of presenting quantitative gene expression exist: absolute and relative quantification. Absolute quantification calculates the copy number of the gene usually by relating the PCR signal to a standard curve. Relative gene expression presents the data of the gene of interest relative to some calibrator or internal control gene. A widely used method to present relative gene expression is the comparative C(T) method also referred to as the 2 (-DeltaDeltaC(T)) method. This protocol provides an overview of the comparative C(T) method for quantitative gene expression studies. Also presented here are various examples to present quantitative gene expression data using this method.

17,260 citations


"Differential regulation of the rain..." refers methods in this paper

  • ...Relative gene-expression was determined by using the ΔΔCt method [49]....

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Journal ArticleDOI
23 Aug 2002-Science
Abstract: The compact genome of Fugu rubripes has been sequenced to over 95% coverage, and more than 80% of the assembly is in multigene-sized scaffolds. In this 365-megabase vertebrate genome, repetitive DNA accounts for less than one-sixth of the sequence, and gene loci occupy about one-third of the genome. As with the human genome, gene loci are not evenly distributed, but are clustered into sparse and dense regions. Some “giant” genes were observed that had average coding sequence sizes but were spread over genomic lengths significantly larger than those of their human orthologs. Although three-quarters of predicted human proteins have a strong match toFugu, approximately a quarter of the human proteins had highly diverged from or had no pufferfish homologs, highlighting the extent of protein evolution in the 450 million years since teleosts and mammals diverged. Conserved linkages between Fugu and human genes indicate the preservation of chromosomal segments from the common vertebrate ancestor, but with considerable scrambling of gene order.

1,395 citations


"Differential regulation of the rain..." refers background in this paper

  • ...Sequencing of the Fugu genome allowed determination of gene similarity between human and teleost genomes [18]....

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Journal ArticleDOI
TL;DR: The data suggest that the sequence, 5'-puGTGACNNNGC-3' 3'-pyCACTGNNNCG-5' where N is any nucleotide, represents the core sequence of the ARE required for transcriptional activation by phenolic antioxidants and metabolizable planar aromatic compounds.
Abstract: We have characterized further the antioxidant responsive element (ARE) identified in the 5'-flanking region of the rat glutathione S-transferase Ya subunit gene and the NAD(P)H:quinone reductase gene by mutational and deletion analyses. Our data suggest that the sequence, 5'-puGTGACNNNGC-3' 3'-pyCACTGNNNCG-5' where N is any nucleotide, represents the core sequence of the ARE required for transcriptional activation by phenolic antioxidants and metabolizable planar aromatic compounds (e.g. beta-naphthoflavone and 3-methylcholanthrene). We also have found that the ARE is responsive to hydrogen peroxide and phenolic antioxidants that undergo redox cycling. These latter data suggest that the ARE is responsive to reactive oxygen species and thus may represent part of a signal transduction pathway that allow eukaryotic cells to sense and respond to oxidative stress.

1,356 citations


Journal ArticleDOI
TL;DR: It is shown that Nrf2 controls the expression of a group of electrophile- and oxidative stress-inducible proteins and activities, which includes heme oxygenase-1, A170, peroxiredoxin MSP23, and cystine membrane transport (system xc −) activity.
Abstract: Electrophiles and reactive oxygen species have been implicated in the pathogenesis of many diseases. Transcription factor Nrf2 was recently identified as a general regulator of one defense mechanism against such havoc. Nrf2 regulates the inducible expression of a group of detoxication enzymes, such as glutathione S-transferase and NAD(P)H:quinone oxidoreductase, via antioxidant response elements. Using peritoneal macrophages from Nrf2-deficient mice, we show here that Nrf2 also controls the expression of a group of electrophile- and oxidative stress-inducible proteins and activities, which includes heme oxygenase-1, A170, peroxiredoxin MSP23, and cystine membrane transport (system xc−) activity. The response to electrophilic and reactive oxygen species-producing agents was profoundly impaired in Nrf2-deficient cells. The lack of induction of system xc− activity resulted in the minimum level of intracellular glutathione, and Nrf2-deficient cells were more sensitive to toxic electrophiles. Several stress agents induced the DNA binding activity of Nrf2 in the nucleus without increasing its mRNA level. Thus Nrf2 regulates a wide-ranging metabolic response to oxidative stress.

1,288 citations


"Differential regulation of the rain..." refers background in this paper

  • ...However, it has been indicated that ARE driven gene expression can occur in the absence of oxidative stress through the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf-2) [26]....

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  • ...The USF/ARE composite transcription factor has also been identified in a number of other terrestrial vertebrates including chicken [21]....

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  • ...While free radical regulation of mammalian MT genes seem to be mediated via USF/ARE and MRE cis-acting elements [20], teleost MT genes may be regulated via conserved clusters of cis-acting elements sharing high homology to the NF-IL6 and the AP1 consensus core sequences [7,11,12]....

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  • ...The ARE cis-acting element has also been identified and characterized in enhancer regions from metabolizing enzymes participating in the phase II drug response and is activated by electrophilic xenobiotics and H2O2 [23]....

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  • ...However, AP1 has been shown to bind to ARE in the NADP (H): quinone oxidoreductase hNQO1 gene enhancer resulting in activation of this gene [28,29]....

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Journal ArticleDOI
TL;DR: It is reported here that Jun, Fos, Fra, and NRF nuclear transcription factors bind to the hARE, indicating that hARE-mediated expression of the NQO1 gene and its induction by xenobiotics and antioxidants are mediated by Nrf1 and Nrf2.
Abstract: Twenty-four base pairs of the human antioxidant response element (hARE) are required for high basal transcription of the NAD(P)H:quinone oxidoreductase1 (NQO1) gene and its induction in response to xenobiotics and antioxidants. hARE is a unique cis-element that contains one perfect and one imperfect AP1 element arranged as inverse repeats separated by 3 bp, followed by a “GC” box. We report here that Jun, Fos, Fra, and Nrf nuclear transcription factors bind to the hARE. Overexpression of cDNA derived combinations of the nuclear proteins Jun and Fos or Jun and Fra1 repressed hARE-mediated chloramphenicol acetyltransferase (CAT) gene expression in transfected human hepatoblastoma (Hep-G2) cells. Further experiments suggested that this repression was due to overexpression of c-Fos and Fra1, but not due to Jun proteins. The Jun (c-Jun, Jun-B, and Jun-D) proteins in all the possible combinations were more or less ineffective in repression or upregulation of hARE-mediated gene expression. Interestingly, overexpression of Nrf1 and Nrf2 individually in Hep-G2 and monkey kidney (COS1) cells significantly increased CAT gene expression from reporter plasmid hARE-thymidine kinase-CAT in transfected cells that were inducible by β-naphthoflavone and tert-butyl hydroquinone. These results indicated that hARE-mediated expression of the NQO1 gene and its induction by xenobiotics and antioxidants are mediated by Nrf1 and Nrf2. The hARE-mediated basal expression, however, is repressed by overexpression of c-Fos and Fra1.

999 citations


"Differential regulation of the rain..." refers background in this paper

  • ...However, AP1 has been shown to bind to ARE in the NADP (H): quinone oxidoreductase hNQO1 gene enhancer resulting in activation of this gene [28,29]....

    [...]