Inhibition of NF-kappaB DNA binding and nitric oxide induction in human T cells and lung adenocarcinoma cells by selenite treatment

Abstract

NF-kappaB is a major transcription factor consisting of 50(p50)- and 65(p65)-kDa proteins that controls the expression of various genes, among which are those encoding cytokines, cell adhesion molecules, and inducible NO synthase (iNOS). After initial activation of NF-kappaB, which involves release and proteolysis of a bound inhibitor, essential cysteine residues are maintained in the active reduced state through the action of thioredoxin and thioredoxin reductase. In the present study, activation of NF-kappaB in human T cells and lung adenocarcinoma cells was induced by recombinant human tumor necrosis factor alpha or bacterial lipopolysaccharide. After lipopolysaccharide activation, nuclear extracts were treated with increasing concentrations of selenite, and the effects on DNA-binding activity of NF-kappaB were examined. Binding of NF-kappaB to nuclear responsive elements was decreased progressively by increasing selenite levels and, at 7 microM selenite, DNA-binding activity was completely inhibited. Selenite inhibition was reversed by addition of a dithiol, DTT. Proportional inhibition of iNOS activity as measured by decreased NO products in the medium (NO2- and NO3-) resulted from selenite addition to cell suspensions. This loss of iNOS activity was due to decreased synthesis of NO synthase protein. Selenium at low essential levels (nM) is required for synthesis of redox active selenoenzymes such as glutathione peroxidases and thioredoxin reductase, but in higher toxic levels (>5-10 microM) selenite can react with essential thiol groups on enzymes to form RS-Se-SR adducts with resultant inhibition of enzyme activity. Inhibition of NF-kappaB activity by selenite is presumed to be the result of adduct formation with the essential thiols of this transcription factor.

Figure 1

Effects of sodium selenite on NF-κB DNA binding activity with Jurkat T cell nuclear extracts. (A) Nuclear extracts prepared from human Jurkat T cells preincubated with 0.1 nM human TNF-α at 37°C for 20 min were examined for NF-κB activation by electrophoretic mobility shift assay after treatments with various concentrations of selenite. Lanes: 1, 0 selenite; 2, 0 selenite with wild-type NF-κB consensus oligonucleotide; 3, 0 selenite with mutant NF-κB consensus oligonucleotide; 4, 1 μM selenite; 5, 3 μM selenite; 6, 5 μM selenite; 7, 7 μM selenite; 8, 10 μM selenite; 9, 10 μM selenite with 1 mM DTT; and 10, 10 μM selenite with 2 mM DTT. (B) Quantitative representation of A. The resulting autoradiograms of NF-κB-DNA complex were quantitated by PhosphoImager analysis.

Figure 2

Effect of DTT on NF-κB DNA binding. Nuclear extracts prepared from human Jurkat T cells preincubated with 0.1 nM human TNF-α at 37°C for 20 min were examined for NF-κB activation by electrophoretic mobility shift assay in the presence of various concentrations of DTT as indicated.

Figure 3

Selenite-mediated inhibition of NF-κB DNA binding and NO production. The human Jurkat T cells, JPX9, were preincubated overnight with bacterial LPS at 75 ng/ml and then treated with indicated concentrations of sodium selenite for 3 h. For analysis of NF-κB activation (•), nuclear extracts were prepared from untreated and LPS-treated cells, and NF-κB DNA binding was determined by electrophoretic mobility shift assays. The resulting autoradiograms of NF-κB-DNA complex were quantitated by PhosphorImager. For analysis of NO production (▪), the final products of NO in vivo (sum of both NO₂⁻ and NO₃⁻) in the culture media of untreated and LPS-treated cells were measured as described in Materials and Methods. ←LPS→ indicates the cultured cells that were retreated with bacterial LPS.

Figure 4

Selenite-mediated inhibition of iNOS synthesis. Crude extracts were prepared from human Jurkat T cells treated with the indicated concentrations of selenite as described in the legend of Fig. 3 and immunoblotted with a polyclonal antibody against human iNOS, NOS2 (Santa Cruz Biotechnology). ←LPS→ indicates the cells that were treated with bacterial LPS.