Differential effects of selenium and knock-down of glutathione peroxidases on TNFα and flagellin inflammatory responses in gut epithelial cells.

Selenium (Se) is essential for human health. Despite evidence that Se intake affects inflammatory responses, the mechanisms by which Se and the selenoproteins modulate inflammatory signalling, especially in the gut, are not yet defined. The aim of this work was to assess effects of altered Se supply and knock-down of individual selenoproteins on NF-κB activation in gut epithelial cells. Caco-2 cells were stably transfected with gene constructs expressing luciferase linked either to three upstream NF-κB response elements and a TATA box or only a TATA box. TNFα and flagellin activated NF-κB-dependent luciferase activity and increased IL-8 expression. Se depletion decreased expression of glutathione peroxidase1 (GPX1) and selenoproteins H and W and increased TNFα-stimulated luciferase activity, endogenous IL-8 expression and reactive oxygen species (ROS) production. These effects were not mimicked by independent knock-down of either GPX1, selenoprotein H or W; indeed, GPX1 knock-down lowered TNFα-induced NF-κB activation and did not affect ROS levels. GPX4 knock-down decreased NF-κB activation by flagellin but not by TNFα. We hypothesise that Se depletion alters the pattern of expression of multiple selenoproteins that in turn increases ROS and modulates NF-κB activation in epithelial cells, but that the effect of GPX1 knock-down is ROS-independent.

The RNA-binding zinc-finger protein tristetraprolin regulates AU-rich mRNAs involved in breast cancer-related processes.

Tristetraprolin (TTP or ZFP36) is a tandem CCCH zinc-finger RNA-binding protein that regulates the stability of certain AU-rich element (ARE) mRNAs. Recent work suggests that TTP is deficient in cancer cells when compared with normal cell types. In this study we found that TTP expression was lower in invasive breast cancer cells (MDAMB231) compared with normal breast cell lines MCF12A and MCF-10. TTP targets were probed using a novel approach by expressing the C124R zinc-finger TTP mutant that functions as dominant negative and increases target mRNA expression. In contrast to wild-type TTP, C124R TTP was able to increase certain ARE-mRNA expressions in serum-stimulated breast cancer cells. Using an ARE-gene microarray, novel targets of TTP regulation were identified, namely, urokinase plasminogen activator (uPA), uPA receptor and matrix metalloproteinase-1, all known to have prominent roles in breast cancer invasion and metastasis. Expression of these targets was upregulated in tumorigenic types, particularly in highly invasive MDAMB231. The mRNA half-lives of these TTP-regulated genes were increased in TTP-knockout embryonic mouse fibroblasts, as assessed using real-time polymerase chain reaction, whereas forced restoration of TTP by transfection led to a reduction in their mRNA levels. RNA immunoprecipitation confirmed an association of TTP, but not C124R, with these target transcripts. Moreover, TTP reduced, whereas the mutant C124R TTP increased, the activity of reporter constructs fused to target ARE. As a result of TTP regulation, invasiveness of MDAMB231 cells was reduced. The data suggest that TTP, in a 3' untranslated region-and ARE-dependent manner, regulates an important subset of cancer-related genes that are involved in cellular growth, invasion and metastasis.

Two common single nucleotide polymorphisms in the gene encoding beta-carotene 15,15'-monoxygenase alter beta-carotene metabolism in female volunteers.

The key enzyme responsible for beta-carotene conversion into retinal is beta-carotene 15,15'-monoxygenase (BCMO1). Since it has been reported that the conversion of beta-carotene into vitamin A is highly variable in up to 45% of healthy individuals, we hypothesized that genetic polymorphisms in the BCMO1 gene could contribute to the occurrence of the poor converter phenotype. Here we describe the screening of the total open reading frame of the BCMO1 coding region that led to the identification of two common nonsynonymous single nucleotide polymorphisms (R267S: rs12934922; A379V: rs7501331) with variant allele frequencies of 42 and 24%, respectively. In vitro biochemical characterization of the recombinant 267S + 379V double mutant revealed a reduced catalytic activity of BCMO1 by 57% (P<0.001). Assessment of the responsiveness to a pharmacological dose of beta-carotene in female volunteers confirmed that carriers of both the 379V and 267S + 379V variant alleles had a reduced ability to convert beta-carotene, as indicated through reduced retinyl palmitate:beta-carotene ratios in the triglyceride-rich lipoprotein fraction [-32% (P=0.005) and -69% (P=0.001), respectively] and increased fasting beta-carotene concentrations [+160% (P=0.025) and +240% (P=0.041), respectively]. Our data show that there is genetic variability in beta-carotene metabolism and may provide an explanation for the molecular basis of the poor converter phenotype within the population.

Differential regulation of expression of cytosolic and mitochondrial thioredoxin reductase in rat liver and kidney.

Adequate supply of selenium (Se) is critical for synthesis of selenoproteins through selenocysteine insertion mechanism. To explore this process we investigated the expression of the cytosolic and mitochondrial isoenzymes of thioredoxin reductase (TrxR1 and TrxR2) in response to altered Se supply. Rats were fed diets containing different quantities of selenium and the levels of TrxR1 and TrxR2 protein and their corresponding mRNAs were determined in liver and kidney. Expression of the two isoenzymes was differentially affected, with TrxR1 being more sensitive to Se depletion than TrxR2 and greater changes in liver than kidney. In order to determine if the selenocysteine incorporation sequence (SECIS) element was critical in this response liver and kidney cell lines (H4 and NRK-52E) were transfected with reporter constructs in which expression of luciferase required read-through at a UGA codon and which contained either the TrxR1 or TrxR2 3'UTR, or a combination of the TrxR1 5' and 3'UTRs. Cell lines expressing constructs with the TrxR1 3'UTR demonstrated no response to restricted Se supply. In comparison the Se-deficient cells expressing constructs with the TrxR2 3'UTR showed considerably less luciferase activity than the Se-adequate cells. No disparity of response to Se supply was observed in the constructs containing the different TrxR1 5'UTR variants. The data show that there is a prioritisation of TrxR2 over TrxR1 during Se deficiency such that TrxR1 expression is more sensitive to Se supply than TrxR2 but this sensitivity of TrxR1 was not fully accounted for by TrxR1 5' or 3'UTR sequences when assessed using luciferase reporter constructs.

Evidence that a polymorphism within the 3'UTR of glutathione peroxidase 4 is functional and is associated with susceptibility to colorectal cancer.

Low selenium (Se) status has been associated with increased risk of colorectal cancer (CRC). Se is present as the amino acid selenocysteine in selenoproteins, such as the glutathione peroxidases. Se incorporation requires specific RNA structures in the 3' untranslated region (3'UTR) of the selenoprotein mRNAs. A single nucleotide polymorphism (SNP) occurs at nucleotide 718 (within the 3'UTR) in the glutathione peroxidase 4 gene. In the present study, Caco-2 cells were transfected with constructs in which type 1 iodothyronine deiodinase coding region was linked to the GPx4 3'UTR with either C or T variant at position 718. Higher reporter activity was observed in cells expressing the C variant compared to those expressing the T variant, under either Se-adequate or Se-deficient conditions. In addition, a disease association study was carried out in cohorts of patients with either adenomatous polyps, colorectal adenocarcinomas and in healthy controls. A higher proportion of individuals with CC genotype at the GPx4 T/C 718 SNP was present in the cancer group, but not in the polyp group, compared with the control group (P < 0.05). The present data demonstrate the functionality of the GPx4 T/C 718 SNP and suggest that T genotype is associated with lower risk of CRC.

Perinuclear localisation of cellular retinoic acid binding protein I mRNA.

Retinoids are important metabolic and developmental regulators that act through nuclear receptors. The cellular retinoic acid binding protein CRABPI has been suggested to play a role in trafficking of retinoic acid but its exact functions and subcellular localisation remain unclear. Here we show that in CHO cells both exogenous CRABPI transcripts and tagged CRABPI protein have a perinuclear distribution that depends upon the 3'UTR of the mRNA. The CRABPI 3'UTR conferred perinuclear localisation on globin reporter transcripts. Deletion analysis indicated that the first 123nt of CRABPI 3'UTR are necessary for localisation of both CRABPI mRNA and protein. We propose that CRABPI mRNA is localised by a signal within its 3'UTR and that this partly determines the distribution of CRABPI protein.

Isolation and identification of a protein binding to the localization element of Metallothionein-1 mRNA.

mRNA localization provides a mechanism for localized protein synthesis. mRNAs encoding certain proteins, including c-MYC, c-FOS, MT-1 (Metallothionein-1) and vimentin, are localized around the nuclei of mammalian cells and are associated with the cytoskeleton. Targeting of these mRNAs to the perinuclear cytoplasm is mediated by elements within their 3'-UTRs (3'-untranslated regions), but many of the trans-acting proteins remain unidentified. UV cross-linking assays using radiolabelled transcripts indicated that a protein of approx. 50 kDa (from the Chinese-hamster ovary cell extracts) bound to the MT-1 3'-UTR sequence. Competition experiments using unlabelled mutant 3'-UTR RNAs revealed that the binding of this protein is specific to localization-positive mutants. Isolation of a 50 kDa protein was achieved by an RNA affinity-based method in which biotinylated MT-1 3'-UTR RNA was anchored to paramagnetic beads. Bound proteins were eluted and analysed by SDS/PAGE. The 50 kDa protein was extracted from the gel, subjected to trypsin digestion and identified by matrix-assisted laser-desorption/ionization-time-of-flight mass spectrometry as eukaryote elongation factor 1alpha.

No change in apoptosis in skeletal muscle exposed acutely or chronically to alcohol.

The pathogenic mechanisms responsible for the deleterious changes in ethanol-exposed skeletal muscle are unknown, although apoptosis may be a causal process. We therefore investigated the responses of skeletal muscle to acute or chronic ethanol exposure in male Wistar rats. In acute studies, rats were dosed with ethanol (75 mmol (3.46 g)/kg BW) and killed after either 2.5 or 6 hours. In chronic studies, rats were fed ethanol as 35% of total dietary energy for 6 weeks. Apoptosis was determined by either DNA fragmentation or TUNEL (terminal deoxynucleotidyl transferase mediated dUTP nick end labelling) assays. The results showed that apoptosis was not increased in the ethanol-exposed muscle in both acute and chronic studies compared to appropriate controls.

Alcohol increases c-myc mRNA and protein in skeletal and cardiac muscle.

The pathogenic mechanisms responsible for alcohol-induced muscle disease are unknown, although it is possible that increased proto-oncogene expression may be the causative process. Therefore, we investigated the responses of skeletal muscle c-myc protein and mRNA to a standard acute ethanol dosage regimen (75 mmol/kg/body weight [BW]) for 2.5 to 24 hours. Comparative studies were made on the heart. Acute ethanol administration in vivo led to an increase in c-myc proto-oncogene mRNA in rat skeletal and cardiac muscle. The changes in c-myc mRNA were mirrored by increases in the c-myc protein as demonstrated by immunohistochemistry. The changes in the c-myc protein were localized to the myonuclei, with no corresponding changes seen in the interstitial cell nuclei. This is the first report of altered proto-oncogene expression in muscle in response to ethanol. Increased c-myc mRNA and protein may reflect adaptive changes, a stress response, or another uncharacterized cellular adaptation.

Ethanol and protein metabolism.

This article represents the proceedings of a workshop at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Carol C. Cunningham and Victor R. Preedy. The presentations were (1) Ribosomal content, ribosomal localization and the levels of ribosomal protein mRNA and rRNA in rat skeletal muscle exposed to ethanol, by Alistair G. Paice, John E. Hesketh, Timothy J. Peters, and Victor R. Preedy; (2) Altered hepatic mitochondrial ribosome structure after chronic ethanol administration, by Vinood B. Patel and Carol C. Cunningham; (3) Clinical aspects of hepatic protein metabolism and alcohol, by Elena Volpi; and (4) Effects of oral intake of alanine plus glutamine on ethanol metabolism and ethanol-related depression in motor activity, by Kazunori Mawatari, H. Masaki, M. Mori, and Kunio Torii.

Perinuclear mRNA localisation by vimentin 3'-untranslated region requires a 100 nucleotide sequence and intermediate filaments.

The role of the vimentin 3'-untranslated region (3'-UTR) in mRNA localisation was studied in cells transfected with a reporter sequence linked to subregions of the 3'-UTR. In situ hybridisation showed that nucleotides 37-137, including a previously identified protein-binding domain, were sufficient to localise transcripts to perinuclear cytoplasm. Transfection of two SW13 cell lines that do and do not express vimentin showed that perinuclear localisation due to either the vimentin or c-myc 3'-UTR requires intermediate filaments. The data suggest that both a specific protein-binding region of the vimentin 3'-UTR and intermediate filaments themselves are required to determine the site of vimentin synthesis.

Influence of metallothionein-1 localization on its function.

Metallothioneins (MTs) have a major role to play in metal metabolism, and may also protect DNA against oxidative damage. MT protein has been found localized in the nucleus during S-phase. The mRNA encoding the MT-1 isoform has a perinuclear localization, and is associated with the cytoskeleton; this targeting, due to signals within the 3'-untranslated region (3'-UTR), facilitates nuclear localization of MT-1 during S-phase [Levadoux, Mahon, Beattie, Wallace and Hesketh (1999) J. Biol. Chem. 274, 34961-34966]. Using cells transfected with MT gene constructs differing in their 3'-UTRs, the role of MT protein in the nucleus has been studied. Chinese hamster ovary cells were transfected with either the full MT gene (MTMT cells) or with the MT 5'-UTR and coding region linked to the 3'-UTR of glutathione peroxidase (MTGSH cells). Cell survival following exposure to oxidative stress and chemical agents was higher in cells expressing the native MT gene than in cells where MT localization was disrupted, or in untransfected cells. Also, MTMT cells showed less DNA damage than MTGSH cells in response to either hydrogen peroxide or mutagen. After exposure to UV light or mutagen, MTMT cells showed less apoptosis than MTGSH cells, as assessed by DNA fragmentation and flow cytometry. The data indicate that the perinuclear localization of MT mRNA is important for the function of MT in a protective role against DNA damage and apoptosis induced by external stress.

Nuclear import of metallothionein requires its mRNA to be associated with the perinuclear cytoskeleton.

The influence of mRNA localization on metallothionein-1 protein distribution was studied by immunocytochemistry. We used Chinese hamster ovary cells that had been transfected with either a native metallothionein-1 gene construct or metallothionein-1 5'-untranslated region and coding sequences linked to the 3'-untranslated region from glutathione peroxidase. The change in the 3'-untranslated region caused the delocalization of the mRNA with a loss of the perinuclear localization and association with the cytoskeleton. Clones were selected which expressed similar levels of metallothionein-1 protein, as assessed by radioimmunoassay. The results showed that loss of metallothionein-1 mRNA localization was associated with a loss of metallothionein-1 protein localization, most notably with a lack of metallothionein-1 protein in the nucleus of synchronized cells which were beginning to synthesize DNA. This indicates that the association of metallothionein-1 mRNA with the cytoskeleton around the nucleus is essential for efficient shuttling of the protein into the nucleus during the G(1) to S phase transition. This is the first demonstration of a physiological role for perinuclear mRNA localization and we propose that such localization may be important for a wide range of nuclear proteins, including those that shuttle between nucleus and cytoplasm in a cell cycle dependent manner.

Localisation of a reporter transcript by the c-myc 3'-UTR is linked to translation.

The 3'-untranslated region of c-myc mRNA contains a perinuclear localisation signal which is sufficient to target beta-globin coding sequences. The link between perinuclear mRNA localisation and translation has been investigated using cells transfected with chimeric gene constructs in which globin reporter sequences were linked to the c-myc 3'-untranslated region and the iron-responsive element from ferritin mRNA. Iron supplementation of the medium promoted translation of the chimeric mRNA as assessed by its presence in polysomes; in situ hybridisation showed that the mRNA was localised around the nucleus. Treatment with the iron chelator desferrioxamine for 16 h prevented both translation and mRNA localisation. In controls where the expressed mRNA lacked the iron-responsive element desferrioxamine had no effect upon localisation. In contrast, arrest of on-going global translation by puromycin treatment had no effect on mRNA localisation. The data suggest that if initiation of translation of a mRNA containing the c-myc localisation signal is prevented in some way then localisation does not occur, whereas once the mRNA has been localised further translation is not required to maintain mRNA localisation.

Competition between the signal sequence and a 3'UTR localisation signal during redirection of beta-globin mRNA to the endoplasmic reticulum: implications for biotechnology.

Secretion of an intracellular protein from a cell factory requires as a first step the redirection of the mRNA for synthesis of the protein on the endoplasmic reticulum. The feasibility of retargeting a mRNA coding for an intracellular protein to the endoplasmic reticulum was investigated using Ltk- fibroblasts stably transfected with gene constructs in which rabbit beta-globin coding region and 5'UTR was linked to albumin signal sequence and different 3'untranslated regions. Globin transcripts with the native globin 3'untranslated region or with the 3'untranslated region of c-myc are present in free/cytoskeletal-bound polysomes. The addition of the signal sequence from rat albumin redirects both these globin transcripts to membrane-bound polysomes but the presence of the c-myc 3'UTR reduces the extent of redirection. Globin transcripts with both the signal sequence and 3'untranslated region from the albumin gene are efficiently redirected to membrane-bound polysomes. The results suggest competition between 5' and 3' localising signals. The addition of the signal sequence does not destabilise the mRNA nor affect translational efficiency. It is concluded that it is possible to retarget an mRNA to the endoplasmic reticulum while maintaining stability and translational capacity. This has important implications for the development of vectors to promote secretion of intracellular proteins from cell factories.

Thyroid stimulating hormone and selenium supply interact to regulate selenoenzyme gene expression in thyroid cells (FRTL-5) in culture.

In the absence of a sodium selenite supplement, FRTL-5 cells showed a reduced activity of cytosolic glutathione peroxidase (cGSH-Px), a marker of selenium status, indicating the cells were Se-deficient. Se-deficient cells showed a 65% reduction in cGSH-Px mRNA abundance but little change in abundance of either phospholipid hydroperoxide glutathione peroxidase or type 1 deiodinase (IDI) mRNA. In Se-replete cells increased thyroid stimulating hormone (TSH) caused a small decrease in IDI abundance but in Se-deficient cells TSH caused a large increase. The results indicate an interaction between TSH and Se status in the regulation of thyroid selenoenzyme synthesis.

Regulatory signals in messenger RNA: determinants of nutrient-gene interaction and metabolic compartmentation.

Nutrition has marked influences on gene expression and an understanding of the interaction between nutrients and gene expression is important in order to provide a basis for determining the nutritional requirements on an individual basis. The effects of nutrition can be exerted at many stages between transcription of the genetic sequence and production of a functional protein. This review focuses on the role of post-transcriptional control, particularly mRNA stability, translation and localization, in the interactions of nutrients with gene expression. The effects of both macronutrients and micronutrients on regulation of gene expression by post-transcriptional mechanisms are presented and the post-transcriptional regulation of specific genes of nutritional relevance (glucose transporters, transferrin, selenoenzymes, metallothionein, lipoproteins) is described in detail. The function of the regulatory signals in the untranslated regions of the mRNA is highlighted in relation to control of mRNA stability, translation and localization and the importance of these mRNA regions to regulation by nutrients is illustrated by reference to specific examples. The localization of mRNA by signals in the untranslated regions and its function in the spatial organization of protein synthesis is described; the potential of such mechanisms to play a key part in nutrient channelling and metabolic compartmentation is discussed. It is concluded that nutrients can influence gene expression through control of the regulatory signals in these untranslated regions and that the post-transcriptional regulation of gene expression by these mechanisms may influence nutritional requirements. It is emphasized that in studies of nutritional control of gene expression it is important not to focus only on regulation through gene promoters but also to consider the possibility of post-transcriptional control.

The 3' untranslated region plays a role in the targeting of metallothionein-I mRNA to the perinuclear cytoplasm and cytoskeletal-bound polysomes.

The mechanism of localisation of metallothionein-I (MT-I) mRNA was studied in transfected cells by in situ hybridisation and cell fractionation. Hepatoma cells were transfected with the 5'-untranslated region and coding region of the beta-globin gene alone or linked to either the beta-globin 3'-untranslated region (3'-UTR) or the MT-I 3'-UTR. The wild-type beta-globin mRNA and the beta-globin mRNA lacking its native 3'-UTR were present in free and cytoskeletal-bound polysomes to a similar extent and showed no localisation. Chimaeric globin-metallothionein transcripts were significantly enriched in cytoskeletal-bound polysomes and were localised in the perinuclear cytoplasm. Chimaeric globin-metallothionein and wild-type globin transcripts were of similar stability. Chinese Hamster Ovary cells were transfected with constructs in which the MT-I 5'-untranslated region and coding sequences were linked to either the endogenous 3'-UTR or the glutathione peroxidase 3'-UTR. Wild-type MT-I transcripts were localised in the perinuclear cytoplasm but the chimaeric MT-I-glutathione peroxidase transcripts showed no distinct localisation. The results indicate that the 3'-UTR of MT-I mRNA contains a localisation signal which promotes both the association of the mRNA with the cytoskeleton and its perinuclear localisation.

Evidence for a localisation signal in the 3'-untranslated region from vimentin messenger RNA.

There is increasing evidence that some mRNAs are localised in eukaryotic somatic cells, but it is unclear what proportion of mRNAs are localised and whether this sorting involves 3'-untranslated sequences. The presence of a localisation signal within the 3'-untranslated region of vimentin mRNA was investigated by studying mRNA distribution in fibroblasts transfected with beta-globin and hybrid globin-vimentin gene constructs. In cells transfected with constructs containing either a fragment of the rabbit beta-globin gene containing both coding sequences and 3'untranslated region or the beta-globin coding sequences alone in situ hybridisation showed that beta-globin mRNA was distributed throughout the cytoplasm without any evident localisation. In contrast, in cells transfected with globin coding sequences linked to the vimentin 3'-untranslated region there was a strong perinuclear localisation of the hybrid mRNA. The results show that loss of its endogenous 3'-untranslated region does not affect distribution of beta-globin mRNA whereas the vimentin 3'-untranslated region causes an altered localisation of beta-globin mRNA. We conclude that the vimentin 3'-untranslated region contains a localisation signal which can direct reporter sequences to the perinuclear cytoplasm.

Evidence for a localization signal in the 3'untranslated region of myosin heavy chain messenger RNA.

Localization signals in the 3'untranslated region (3'UTR) of myosin heavy chain mRNA were investigated using hybrid gene constructs. In myoblasts transfected with constructs containing either both coding sequences and 3'UTR of the rabbit beta-globin gene or the beta-globin coding sequences alone in situ hybridization showed that globin transcripts were distributed throughout the cytoplasm with no localization. In contrast, in myoblasts transfected with beta-globin coding sequences linked to the myosin heavy chain 3'UTR there was strong perinuclear localization of the hybrid mRNA; this was maintained in myotubes. We conclude that myosin heavy chain 3'UTR contains a localization signal.