Oxidized low density lipoprotein inhibits interleukin-12 production in lipopolysaccharide-activated mouse macrophages via direct interactions between peroxisome proliferator-activated receptor-gamma and nuclear factor-kappa B

Lipopolysaccharide (LPS) increases the production of interleukin-12 (IL-12) from mouse macrophages via a kappaB site within the IL-12 p40 promoter. In this study, we found that oxidized low density lipoprotein (oxLDL) inhibited this LPS-stimulated production of IL-12 in a dose-dependent manner while native LDL did not. OxLDL inhibited p40 promoter activation in monocytic RAW264.7 cells transiently transfected with p40 promoter/reporter constructs, and the repressive effect mapped to a region in the p40 promoter containing a binding site for nuclear factor-kappaB (NF-kappaB) (p40-kappaB). Activation of macrophages by LPS in the presence of oxLDL resulted in markedly reduced binding to the kappaB site, as demonstrated by the electrophoretic mobility shift assays. In contrast, native LDL did not inhibit the IL-12 p40 promoter activation and NF-kappaB binding to the kappaB sites, suggesting that oxidative modification of LDL was crucial for the inhibition of NF-kappaB-mediated IL-12 production. 9-Hydroxyoctadecadienoic acid, a major oxidized lipid component of oxLDL, significantly inhibited IL-12 production in LPS-stimulated mouse macrophages and also suppressed NF-kappaB-mediated activation in IL-12 p40 promoter. The NF-kappaB components p50 and p65 directly bound peroxisome proliferator-activated receptor-gamma (PPAR-gamma) in vitro. In cotransfections of CV-1 and HeLa cells, PPAR-gamma inhibited the NF-kappaB transactivation in an oxLDL-dependent manner. From these results, we propose that oxLDL-mediated suppression of the IL-12 production from LPS-activated mouse macrophages may, at least in part, involve both inhibition of the NF-kappaB-DNA interactions and physical interactions between NF-kappaB and PPAR-gamma.     

The atypical orphan nuclear receptor DAX-1 interacts with orphan nuclear receptor Nur77 and represses its transactivation

DAX-1 (dosage-sensitive sex reversal adrenal hypoplasia congenital critical region on the X chromosome, gene 1) (NROB1) is an atypical member of the nuclear receptor family, which lacks the classical zinc finger DNA binding domain and acts as a coregulator of a number of nuclear receptors. In this study, we have found that DAX-1 is a novel coregulator of the orphan nuclear receptor Nur77 (NR4A1). We demonstrate that DAX-1 represses the Nur77 transactivation by transient transfection assays. Specific interaction between Nur77 and DAX-1 was detected by coimmunoprecipitation, yeast two-hybrid, and glutathione-S-transferase pull-down assays. The ligand binding domain of DAX-1 and the activation function-2 domain of Nur77 were determined as the direct interaction domains between DAX-1 and Nur77. In vitro competition binding assay showed that DAX-1 repressed Nur77 transactivation through the competition with steroid receptor coactivator-1 for the binding of Nur77. Moreover, DAX-1 repressed Nur77- and LH-dependent increase of cytochrome P450 protein 17 promoter activity in transient transfection assays. Furthermore, Nur77-mediated transactivation was significantly increased by down-regulation of DAX-1 expression with DAX-1 small interfering RNA in testicular Leydig cell line, K28. LH treatment induced a transient increase in Nur77 mRNA, whereas LH repressed DAX-1 expression in a time- and dose-dependent manner in K28 cells. In addition, immunohistochemical analysis showed the expression of Nur77 in mouse testicular Leydig cells. These results suggest that DAX-1 acts as a novel coregulator of the orphan nuclear receptor Nur77, and that the DAX-1 may play a key role in the regulation of Nur77-mediated steroidogenesis in testicular Leydig cells.     

Estrogen receptor inhibits interleukin-6 gene expression by disruption of nuclear factor kappaB transactivation

The estrogen receptor (ER) suppresses interleukin-6 (IL-6) gene expression through interaction with nuclear factor kappaB (NF-kappaB) in a hormone-dependent manner. Classic ER binding to DNA is not required and the mechanism of repression is unclear. Previously reported studies suggest that the interference of NF-kappaB binding to DNA by ER may play an important role. An alternative model for repression would be the disruption of NF-kappaB transactivation. In the present study, gel shift assays were used to examine the binding of RelA and p50 dimers to the IL-6 promoter in the presence of ER. The effect of ER on NF-kappaB transactivation was studied independent of NF-kappaB binding to DNA using the mammalian one-hybrid system. ER had little effect on the binding of homodimers or heterodimers of RelA and p50 to the IL-6 promoter. In transfection experiments, both ERalpha and ERbeta inhibited NF-kappaB-mediated expression in a hormone dependent manner with repression also dependent upon dimerization of RelA with p50. Mutant ER that is unable to transactivate failed to repress NF-kappaB expression, but deletion of the N-terminal portion of the receptor had no effect. Taken together, these results suggest that the disruption of NF-kappaB-mediated transactivation plays a significant role in ER inhibition of IL-6 gene expression.     

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.     

Human macrophage inflammatory protein-3alpha/CCL20/LARC/Exodus/SCYA20 is transcriptionally upregulated by tumor necrosis factor-alpha via a non-standard NF-kappaB site.

The 5'-flanking sequences of the human macrophage inflammatory protein-3alpha/CCL20 gene were cloned and transfected into G-361 human melanoma cells in a luciferase reporter construct. Tumor necrosis factor-alpha (TNF-alpha) treatment stimulated luciferase expression, and promoter truncations demonstrated that TNF-alpha inducibility is conferred by a region between nt -111 and -77, which contains a non-standard nuclear factor-kappaB (NF-kappaB) binding site. The requirement for NF-kappaB was demonstrated as follows: (i) mutations in this NF-kappaB site abrogated TNF-alpha responsiveness; (ii) TNF-alpha activated a construct containing two copies of the CCL20 NF-kappaB binding site; (iii) overexpression of NF-kappaB p65 activated the CCL20 promoter; (iv) NF-kappaB from nuclear extracts of TNF-alpha-stimulated cells bound specifically to this NF-kappaB site.