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.

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.

Effects of tobacco smoke and benzo[a]pyrene on human endothelial cell and monocyte stress responses.

Smoking is an important risk factor for atherosclerosis. We compared tobacco smoke filtrate with benzo[a]pyrene (a prominent xenobiotic component of tobacco smoke) for the capacity to induce stress proteins and cause cell death in human monocytes and vascular endothelial cells, two cell types that are involved in the formation of atherosclerotic lesions. Exposure to freshly prepared filtrates of tobacco smoke induced in both monocytes and endothelial cells expression of the inducible heat shock protein (HSP)70 and heme oxygenase-1 (HO-1) and produced loss of mitochondrial membrane potential. Later, cell death by apoptosis or necrosis occurred depending on the concentration of tobacco smoke. These toxic effects could be prevented by the antioxidant N-acetylcysteine. In contrast, exposure of these cells to benzo[a]pyrene alone evoked neither stress proteins nor mitochondrial damage but did induce cell death by necrosis. Thus our results indicate that tobacco smoke rapidly induces complex oxidant-mediated stress responses in both vascular endothelial cells and circulating monocytes that are independent of the benzo[a]pyrene content of the smoke.

Metalloregulation of the tumor suppressor protein p53: zinc mediates the renaturation of p53 after exposure to metal chelators in vitro and in intact cells.

The tumor suppressor p53 is a transcription factor which binds DNA through a structurally complex domain stabilized by a zinc atom. Zinc chelation disrupts the architecture of this domain, inducing the protein to adopt an immunological phenotype identical to that of many mutant forms of p53. In this report, we used 65Zn to show that incorporation of zinc within the protein was required for folding in the 'wild-type' conformation capable of specific DNA-binding. Using a cellular assay, we show that addition of extracellular zinc at concentrations within the physiological range (5 microM) was required for renaturation and reactivation of wild-type p53. Among other divalent metals tested (Cd2+, Cu2+, Co2+, Fe2+ and Ni2+), only Co2+ at 125 microM had a similar effect. Recombinant metallothionein (MT), a metal chelator protein, was found to modulate p53 conformation in vitro. In cultured cells, overexpression of MT by transfection could modulate p53 transcriptional activity. Taken together, these results suggest that zinc binding plays a regulatory role in the control of p53 folding and DNA-binding activity.

Redox signalling and transition metals in the control of the p53 pathway.

The p53 tumour suppressor protein exerts multiple, antiproliferative effects in response to genotoxic exposures. Reactive oxygen intermediates (ROI) play several distinct roles in the p53 pathway. First, they are important activators of p53 through their capacity to induce DNA strand breaks. Second, they regulate the DNA-binding activity of p53 by modulating the redox status of a critical set of cysteines in the DNA-binding domain, which are also involved in the coordination of zinc. Third, they play a role in the signalling pathways regulated by p53, as several genes encoding redox effectors are transcriptionally controlled by p53. In this review, we summarize the evidence for the involvement of ROI at these three levels. Emphasis is placed on the role of metals and ROI as potential regulators of p53 protein conformation and functions, and on the putative toxicological consequences of such a regulation.

Metal ions as regulators of the conformation and function of the tumour suppressor protein p53: implications for carcinogenesis.

The p53 protein is a multi-function nuclear factor that is activated in response to multiple forms of stress and controls the proliferation, survival, DNA repair and differentiation of cells exposed to potentially genotoxic DNA damage. Loss of p53 function by mutation is a frequent event in human cancer, and is thought to result in the capacity of cells to acquire and accumulate oncogenic mutations during the progression of neoplasia. The p53 protein is a metal-binding transcription factor that is inactivated by metal chelation and by oxidation in vitro. In intact cells, p53 protein activity is crucially dependent on the availability of Zn ions and is impaired by exposure to Cd, a metal which readily substitutes for Zn in a number of transcription factors. Inactivation by Cd suppresses the p53-dependent responses to DNA damage. Overall, these findings indicate that regulation by metals plays an important role in the control of p53, and that perturbation of this control may explain the carcinogenic potential of several metal compounds.

Cadmium induces conformational modifications of wild-type p53 and suppresses p53 response to DNA damage in cultured cells.

The p53 tumor suppressor protein is a transcription factor that binds DNA in a sequence-specific manner through a protein domain stabilized by the coordination of zinc within a tetrahedral cluster of three cysteine residues and one histidine residue. We show that cadmium, a metal that binds thiols with high affinity and substitutes for zinc in the cysteinyl clusters of many proteins, inhibits the binding of recombinant, purified murine p53 to DNA. In human breast cancer MCF7 cells (expressing wild-type p53), exposure to cadmium (5-40 microM) disrupts native (wild-type) p53 conformation, inhibits DNA binding, and down-regulates transcriptional activation of a reporter gene. Cadmium at 10-30 microM impairs the p53 induction in response to DNA-damaging agents such as actinomycin D, methylmethane sulfonate, and hydrogen peroxide. Exposure to cadmium at 20 microM also suppresses the p53-dependent cell cycle arrest in G(1) and G(2)/M phases induced by gamma-irradiation. These observations indicate that cadmium at subtoxic levels impairs p53 function by inducing conformational changes in the wild-type protein. There is evidence that cadmium is carcinogenic to humans, in particular for lung and prostate, and cadmium is known to accumulate in several organs. This inhibition of p53 function could play a role in cadmium carcinogenicity.

Impairment of cultured cell proliferation and metallothionein expression by metal chelator NNN'N'-tetrakis-(2-pyridylmethyl)ethylene diamine.

Metallothioneins (MT) are a family of intracellular, cysteine-rich, zinc-binding proteins. Their expression is constitutive but can also be induced at the transcriptional level by various stimuli. In this study, we exposed HaCaT human keratinocytes to excess zinc (ZnCl2) or to zinc deprivation by the diffusible chelator NNN'N'-tetrakis(2-pyridylmethyl)ethylene diamine (TPEN), and to ultraviolet B (UVB) irradiation. We examined both cell proliferation and MT expression. Cell proliferation was maximally stimulated by 100 microM Zn2+ supply and was markedly inhibited by zinc deprivation or UVB irradiation. Zinc and UVB irradiation both increased MTI and/or MTII as detected by immunocytochemistry and enhanced the baseline level of MT-IIA mRNA, whereas TPEN treatment inhibited MT basal expression. Zinc partially prevented the concentration-dependent, UVB-induced decrease in cell proliferation. On the other hand, TPEN partially prevented the UVB-induced increase in MTIIA mRNA. These results suggest that zinc is involved in defense mechanisms of skin keratinocytes and in their stress-induced response.

S-phase-dependent action of cycloheximide in relieving chromatin-mediated general transcriptional repression.

Chromatin plays a major role in the tight regulation of gene expression and in constraining inappropriate gene activity. Replication-coupled chromatin assembly ensures maintenance of these functions of chromatin during S phase of the cell cycle. Thus treatment of cells with an inhibitor of translation, such as cycloheximide (CX), would be expected to have a dramatic effect on chromatin structure and function, essentially in S phase of the cell cycle, due to uncoupled DNA replication and chromatin assembly. In this work, we confirm this hypothesis and show that CX can induce a dramatic S-phase-dependent alteration in chromatin structure that is associated with general RNA polymerase II-dependent transcriptional activation. Using two specific RNA polymerase II-transcribed genes, we confirm the above conclusion and show that CX-mediated transcriptional activation is enhanced during the DNA replication phase of the cell cycle. Moreover, we show co-operation between an inhibitor of histone deacetylase and CX in inducing gene expression, which is again S-phase-dependent. The modest effect of CX in inducing the activity of a transiently transfected promoter shows that the presence of the promoter in an endogenous chromatin context is necessary in order to observe transcriptional activation. We therefore suggest that the uncoupled DNA replication and histone synthesis that occur after CX treatment induces a general modification of chromatin structure, and propose that this general disorganization of chromatin structure is responsible for a widespread activation of RNA polymerase II-mediated gene transcription.