Differential regulation of the rainbow trout ( Oncorhynchus mykiss ) MT-A gene by nuclear factor interleukin-6 and activator protein-1
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.
Abstract: Previously 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.
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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.
19 citations
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...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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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.
14 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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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.
13 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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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.
12 citations
TL;DR: Results indicate that neither INVX nor S25-7 is upregulated upon immune system activation; thus, teleosts have evolved a system of immune regulation that is different than that found in mammals.
Abstract: Major histocompatibility complex (MHC) class II-associated invariant chain is a chaperone responsible for targeting the MHC class II dimer to the endocytic pathway, thus enabling the loading of exogenous antigens onto the MHC class II receptor. In the current study, in vivo and in vitro methods were used to investigate the regulation of the rainbow trout invariant chain proteins S25-7 and INVX, upon immune system activation. Whole rainbow trout and the macrophage/monocyte-like cell line RTS11 were treated with PMA at concentrations shown to induce IL-1β transcripts and homotypic aggregation of RTS11. S25-7 transcript levels remained unchanged in the gill, spleen, and liver and were found to be significantly decreased in head kidney beginning 24 h post-stimulation. Meanwhile, INVX transcript levels remained unchanged in all tissues studied. Both S25-7 and INVX proteins were produced in gill and spleen tissues but their expression was unaffected by immune system stimulation. Surprisingly, neither INVX nor S25-7 protein was detected in the secondary immune organ, the head kidney. Analysis of RTS11 cultures demonstrated that both INVX and S25-7 transcript levels significantly increased at 96 h and 120 h following PMA stimulation before returning to control levels at 168 h. Meanwhile, at the protein level in RTS11, S25-7 remained unchanged while INVX had a significant decrease at 168 h post-stimulation. These results indicate that neither INVX nor S25-7 is upregulated upon immune system activation; thus, teleosts have evolved a system of immune regulation that is different than that found in mammals.
11 citations
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TL;DR: The distal part of the 5'-flanking region of the rainbow trout metallothionein-A promoter was sequenced in order to identify cis-acting regulatory elements and was found to be functional in promoting gene transcription following exposure of RTH-149 cells to hydrogen peroxide.
Abstract: In the present study, the distal part of the 5′-flanking region of the rainbow trout metallothionein-A promoter was sequenced in order to identify cis-acting regulatory elements Analysis of this sequence combined with that previously reported for the 5′-flanking region directly proximal to the start of transcription revealed several putative regulatory sequences In total, six metal-responsive elements (MREs) were identified; these sequences were organised into two clusters, one containing two copies of MRE and located close to the predicted TATA box sequence, and a second consisting of four MREs and lying 500–700 bp upstream from the start of transcription In addition, the 5′-flanking region contained sequences sharing high similarity with the activator protein 1 consensus sequence as well as one nuclear-factor-interleukin-6-responsive element Functional analysis of the promoter was performed by introducing deletion mutants of the 5′-flanking region into the vector pGL-2, directly upstream from the luciferase reporter gene Both MRE clusters were needed for maximal metal inducibility in both rainbow trout hepatoma (RTH-149) and human hepatoblastoma (Hep G2) cell lines Furthermore, the distal region was found to be functional in promoting gene transcription following exposure of RTH-149 cells to hydrogen peroxide
84 citations
"Differential regulation of the rain..." refers background in this paper
...Furthermore, it has been shown that MT is induced by ROS inducing agents such as paraquat (PQ), hydrogen peroxide (H2O2) and glutathione depleting agents such as diethylmaleate [1,4,7]....
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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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...Functional analysis of the rtMT-A promoter has indicated that free radicals regulate the rtMT-A gene via a region containing multiple copies of AP1 elements and one NF-IL6 element [7]....
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...Sequencing of several teleost MT enhancer regions have revealed the presence of distally located nuclear factor interleukin 6 (NF-IL6) and AP1 elements, inferring that these elements are involved in a conserved function [7,11-13]....
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TL;DR: The finding that the inhibitor isoform antagonizes activation of the native TSG-6 promoter by IL-1 and TNF-alpha suggests that NF-IL6 has a physiologic role in these cytokine responses.
Abstract: Tumor necrosis factor alpha (TNF-alpha) and interleukin-1 (IL-1) activate transcription of the TSG-6 gene in normal human fibroblasts through a promoter region (-165 to -58) that encompasses an AP-1 and a NF-IL6 site. We show by deletion analysis and substitution mutagenesis that both sites are necessary for activation by TNF-alpha. Activation by IL-1 requires the NF-IL6 site and is enhanced by the AP-1 site. These results suggest that the NF-IL6 and AP-1 family transcription factors functionally cooperate to mediate TNF-alpha and IL-1 signals. Consistent with this possibility, IL-1 and TNF-alpha markedly increase the binding of Fos and Jun to the AP-1 site, and NF-IL6 activates the native TSG-6 promoter. Activation by NF-IL6 requires an intact NF-IL6 site and is modulated by the ratio of activator to inhibitor NF-IL6 isoforms that are translated from different in-frame AUGs. However, the inhibitor isoform can also bind to the AP-1 site and repress AP-1 site-mediated transcription. The finding that the inhibitor isoform antagonizes activation of the native TSG-6 promoter by IL-1 and TNF-alpha suggests that NF-IL6 has a physiologic role in these cytokine responses. Thus, the functionally distinct NF-IL6 isoforms cooperate with Fos and Jun to positively and negatively regulate the native TSG-6 promoter by TNF-alpha and IL-1.
84 citations
"Differential regulation of the rain..." refers background in this paper
...NF-IL6 is rapidly activated in response to cytokines and oxidative stress [42]....
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TL;DR: It is found that both species of fish express MRE-specific binding activities that are immunologically similar to mouse MTF-1, and this data demonstrate the conservation of binding specificity, binding properties, and regulation of M TF-1 in fishes.
Abstract: The metal response element (MRE)-binding transcription factor-1, MTF-1, is a zinc-responsive protein that controls transcription of metallothionein (MT) genes in many cell types. In addition, MTF-1 is also hypothesized to regulate transcription of a battery of genes involved in the defense against oxidative stress. Manipulating the Zn concentration in the low μM range reversibly modulates the DNA-binding activity of the mammalian MTF-1; this effect is inhibited at low temperature. This report examines the presence and binding properties of MTF-1 in cell lines derived from warm- and cold-water fishes (zebrafish and trout, respectively). We found that both species of fish express MRE-specific binding activities that are immunologically similar to mouse MTF-1. MTF-1-binding from the cells of both species of fish was activated when cells were treated with Zn but not with Cd. Zebrafish cells contained a single isoform of MTF-1 with binding properties similar to mammalian MTF-1. Trout cells, on the other hand, contained two isoforms of MTF-1: MTF-1H and MTF-1L. Zn reversibly modulated MTF-1H binding in a temperature-dependent manner. Similarly, Zn reversibly modulated MTF-1L binding, but, in contrast, such modulation occurred readily at 4°C. This data demonstrate the conservation of binding specificity, binding properties, and regulation of MTF-1 in fishes.
60 citations
"Differential regulation of the rain..." refers background in this paper
...Characterization of MTF-1, from zebrafish and rainbow trout has revealed a high conservation with regard to binding specificity and properties [36]....
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TL;DR: Results support the hypothesis that constitutive MT levels affect the sensitivity of mammalian cells to Cd and oxidative stress and suggest that MT does not protect against MNNG-induced cytotoxicity.
57 citations
"Differential regulation of the rain..." refers background in this paper
...Rainbow trout cells respond to ROS exposure by induction of MT, which result in cellular protection from ROS toxicity [3,4,6]....
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...The role of metallothionein (MT) as a free radical scavenger has been well documented in vitro [3], in cell lines [4], and at the organism level [5,6]....
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TL;DR: The results confirmed the stronger antioxidant power of MTs from aquatic organisms compared with the rabbit MT-II, and mussel MT-10 was more active in DPPH scavenging than fish MT-A, as well as confirming the higher stability of the Cd-mercapto complex.
51 citations
"Differential regulation of the rain..." refers background in this paper
...However, a study on the interaction between MT and free radicals indicate that rtMT-A and sea mussel (Mytilus galloprovincialis) MT10 has a higher ROS scavenging capacity than rabbit MT [32]....
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