Inhibition of I kappa B-alpha phosphorylation and degradation and subsequent NF-kappa B activation by glutathione peroxidase overexpression

We report here that both kappa B-dependent transactivation of a reporter gene and NF-kappa B activation in response to tumor necrosis factor (TNF alpha) or H2O2 treatments are deficient in human T47D cell transfectants that overexpress seleno-glutathione peroxidase (GSHPx). These cells feature low reactive oxygen species (ROS) levels and decreased intracellular ROS burst in response to TNF alpha treatment. Decreased ROS levels and NF-kappa B activation were likely to result from GSHPx increment since these phenomena were no longer observed when GSHPx activity was reduced by selenium depletion. The cellular contents of the two NF-kappa B subunits (p65 and p50) and of the inhibitory subunit I kappa B-alpha were unaffected by GSHPx overexpression, suggesting that increased GSHPx activity interfered with the activation, but not the synthesis or stability, of Nf-kappa B. Nuclear translocation of NF-kappa B as well as I kappa B-alpha degradation were inhabited in GSHPx-overexpressing cells exposed to oxidative stress. Moreover, in control T47D cells exposed to TNF alpha, a time correlation was observed between elevated ROS levels and I kappa B- alpha degradation. We also show that, in growing T47D cells, GSHPx overexpression altered the isoform composition of I kappa B-alpha, leading to the accumulation of the more basic isoform of this protein. GSHPx overexpression also abolished the TNF alpha-mediated transient accumulation of the acidic and highly phosphorylated I kappa B-alpha isoform. These results suggest that intracellular ROS are key elements that regulate the phosphorylation of I kappa B-alpha, a phenomenon that precedes and controls the degradation of this protein, and then NF- kappa B activation.     

A T cell nuclear factor resembling NF-AT binds to an NF-kappa B site and to the conserved lymphokine promoter sequence "cytokine-1".

Nuclear extracts from a nontransformed murine T lymphocyte clone contained two inducible factors that bound to a nuclear factor kappa B (NF-kappa B) site. One factor was NF-kappa B, and the other was differentiated from NF-kappa B by its mobility in the electrophoretic mobility shift assay and its lack of sensitivity to protein kinase C depletion. Competition and methylation interference assays showed that the binding site for the novel factor was limited to nucleotides in the 3' half of the kappa B site. This part of the kappa B site resembled sequences in the binding site for a second inducible nuclear factor of T cells, NF-AT, as well as a conserved sequence found in several lymphokine genes, termed "cytokine-1" (CK-1). Competition and methylation interference analysis showed that both NF-AT and CK-1 sequences bound a factor similar to the novel kappa B-binding factor and that binding involved a four-nucleotide sequence (TTCC) that the kappa B, CK-1, and NF-AT sites have in common. The complexes that form with each site have characteristics of NF-AT: they are induced upon T cell receptor stimulation, are sensitive to protein synthesis inhibitors and cyclosporin A, and are not sensitive to protein kinase C depletion. Thus, a factor or factors similar to NF-AT can bind to three distinct promoter sequences which occur commonly in several T cell activation genes. These results raise the possibility that related factors binding to kappa B, CK-1, and NF-AT sequences could play a role in the coordinate induction of T cell activation genes. In addition, our results suggest that kappa B and CK-1 sites represent potential cyclosporin-sensitive promoter elements by virtue of their ability to bind an NF-AT-like factor.     

RelA/p65 is a molecular target for the immunosuppressive action of protein kinase A.

Stimulation of the protein kinase A (PKA) signalling pathway exerts an inhibitory effect on the proliferation of numerous cells, including T lymphocytes. In CD4+ T helper cells, stimulation of PKA leads to suppression of interleukin 2 (IL-2) induction, while induction of the genes coding for the lymphokines IL-4 and IL-5 is enhanced. We show that the differential effect of PKA activity on induction of the IL-2 and IL-4 genes is mediated through their promoters. One major target of the suppressive effect of PKA is the kappa B site in the IL-2 promoter. A kappa B site is missing in the IL-4 promoter. Mutations preventing factor binding to the IL-2 kappa B site result in a loss of PKA-mediated suppression of IL-2 promoter activity. Furthermore, activation of the PKA signalling pathway impairs the inducible activity of multiple kappa B sites of the IL-2 promoter, but not of other factor binding sites. The reduction in activity of kappa B sites in activated and PKA-stimulated T cells is accompanied by changes in the concentration and DNA binding of Rel/NF-kappa B factors. Stimulation of the PKA pathway in Jurkat T cells with the PKA activator forskolin leads to an increase in synthesis of c-Rel and p105/p50, while synthesis of p65/RelA remains unchanged. However, nuclear translocation and DNA binding of p65 is distinctly impaired, probably due to a retarded degradation of I kappa B-alpha. In a similar way, stimulation of the PKA signalling pathway inhibits nuclear translocation of p65 and generation of nuclear kappa B complexes in peripheral T lymphocytes from murine lymph nodes. These results indicate that PKA-mediated suppression of NF-kappa B activity plays an important role in the control of activation of peripheral T lymphocytes.