Aminosalicylic acid inhibits IkappaB kinase alpha phosphorylation of IkappaBalpha in mouse intestinal epithelial cells

Tumor necrosis factor alpha (TNFalpha)-stimulated nuclear factor (NF) kappaB activation plays a key role in the pathogenesis of inflammatory bowel disease (IBD). Phosphorylation of NFkappaB inhibitory protein (IkappaB) leading to its degradation and NFkappaB activation, is regulated by the multimeric IkappaB kinase complex, including IKKalpha and IKKbeta. We recently reported that 5-aminosalicylic acid (5-ASA) inhibits TNFalpha-regulated IkappaB degradation and NFkappaB activation. To determine the mechanism of 5-ASA inhibition of IkappaB degradation, we studied young adult mouse colon (YAMC) cells by immunodetection and in vitro kinase assays. We show 5-ASA inhibits TNFalpha-stimulated phosphorylation of IkappaBalpha in intact YAMC cells. Phosphorylation of a glutathione S-transferase-IkappaBalpha fusion protein by cellular extracts or immunoprecipitated IKKalpha isolated from cells treated with TNFalpha is inhibited by 5-ASA. Recombinant IKKalpha and IKKbeta autophosphorylation and their phosphorylation of glutathione S-transferase-IkappaBalpha are inhibited by 5-ASA. However, IKKalpha serine phosphorylation by its upstream kinase in either intact cells or cellular extracts is not blocked by 5-ASA. Surprisingly, immunodepletion of cellular extracts suggests IKKalpha is predominantly responsible for IkappaBalpha phosphorylation in intestinal epithelial cells. In summary, 5-ASA inhibits TNFalpha-stimulated IKKalpha kinase activity toward IkappaBalpha in intestinal epithelial cells. These findings suggest a novel role for 5-ASA in the management of IBD by disrupting TNFalpha activation of NFkappaB.     

Nuclear factor kappa B activation and regulation of cyclooxygenase type-2 expression in human amnion mesenchymal cells by interleukin-1beta

Interleukin-1beta (IL-1beta) has been shown in numerous studies to increase prostaglandin (PG) output by up-regulating the expression of cyclooxygenase-2 (COX-2), a rate-limiting enzyme in PG synthesis. In this study, we investigated the possible role of the nuclear factor kappa B (NFkappaB) in IL-1beta signaling, leading to the expression of COX-2 in human amnion cell culture. Fetal amnion was obtained following vaginal delivery and digested with collagenase, and the subepithelial (mesenchymal) cells were isolated. Cultures were characterized with antisera to keratin (epithelial cells) and vimentin (mesenchymal cells). Confluent cells were stimulated with human recombinant IL-1beta, and activation of NFkappaB was assessed by measuring changes in the inhibitory protein IkappaB (total IkappaB and phosphorylated IkappaB) using Western blot analysis as well as by nuclear binding of NFkappaB using an electrophoretic mobility shift assay. COX-2 protein levels were determined by Western blot analysis. After 5 min of stimulation with IL-1beta, phosphorylated IkappaB began to appear, 90% of which was degraded within 15 min. This was temporally associated with decreased total IkappaB and increased nuclear NFkappaB DNA-binding activity. In the IL-1beta-treated group, COX-2 protein began to increase after 6 h; this response was time-dependent, with a significant increase until 24 h after IL-1beta stimulation. When NFkappaB translocation was blocked by using SN50 (a cell-permeable inhibitory peptide of NFkappaB translocation), the synthesis of COX-2 protein was inhibited. These results suggest that NFkappaB is involved in the IL-1beta-induced COX-2 expression in the mesenchymal cells of human amnion.     

Saccharomyces boulardii produces a soluble anti-inflammatory factor that inhibits NF-kappaB-mediated IL-8 gene expression

Saccharomyces boulardii (Sb) is a non-pathogenic yeast that ameliorates intestinal injury and inflammation caused by a wide variety of enteric pathogens. We hypothesized that Sb may exert its probiotic effects by modulation of host cell signaling and pro-inflammatory gene expression. Human HT-29 colonocytes and THP-1 monocytes were stimulated with IL-1beta, TNFalpha or LPS in the presence or absence of Sb culture supernatant (SbS). IL-8 protein and mRNA levels were measured by ELISA and RT-PCR, respectively. The effect of SbS on IkappaB alpha degradation was studied by Western blotting and on NF-kappaB-DNA binding by EMSA. NF-kappaB-regulated gene expression was evaluated by transient transfection of THP-1 cells with a NF-kappaB-responsive luciferase reporter gene. SbS inhibited IL-8 protein production in IL-1beta or TNFalpha stimulated HT-29 cells (by 75% and 85%, respectively; P<0.001) and prevented IL-1beta-induced up-regulation of IL-8 mRNA. SbS also inhibited IL-8 production, prevented IkappaB alpha degradation, and reduced both NF-kappaB-DNA binding and NF-kappaB reporter gene up-regulation in IL-1beta or LPS-stimulated THP-1 cells. Purification and characterization studies indicate that the S. boulardii anti-inflammatory factor (SAIF) is small (<1 kDa), heat stable, and water soluble. The probiotic yeast Saccharomyces boulardii exerts an anti-inflammatory effect by producing a low molecular weight soluble factor that blocks NF-kappaB activation and NF-kappaB-mediated IL-8 gene expression in intestinal epithelial cells and monocytes. SAIF may mediate, at least in part, the beneficial effects of Saccharomyces boulardii in infectious and non-infectious human intestinal disease.     

Differential induction of tumor necrosis factor alpha and manganese superoxide dismutase by endotoxin in human monocytes: role of protein tyrosine kinase, mitogen-activated protein kinase, and nuclear factor kappaB

A mutant Escherichia coli lipopolysaccharide (LPS) lacking myristoyl fatty acid markedly stimulates the activity of manganese superoxide dismutase (MnSOD) without inducing tumor necrosis factor alpha (TNFalpha) production by human monocytes (Tian et al., 1998, Am J Physiol 275:C740.), suggesting that induction of MnSOD and TNFalpha by LPS are regulated through different signal transduction pathways. The protein tyrosine kinase (PTK)/mitogen-activated protein kinase (MAPK) pathway plays an important role in the LPS-induced TNFalpha production. In the current study, we determined the effects of PTK inhibitors, genistein and herbimycin A, on the induction of MnSOD and TNFalpha in human monocytes. Genistein (10 microg/ml) and herbimycin A (1 microg/ml) markedly inhibited LPS-induced protein tyrosine phosphorylation, phosphorylation and nuclear translocation of MAPK (p42 ERK, extracellular signal-regulated kinase), and increases in the steady state level of TNFalpha mRNA as well as TNFalpha production. In contrast, at similar concentrations, genistein and herbimycin A had no effect on the LPS-induced activation of nuclear factor kappaB (NFkappaB) and induction of MnSOD (mRNA and enzyme activity) in human monocytes. In addition, inhibition of NFkappaB activation by gliotoxin and pyrrodiline dithiocarbamate, inhibited LPS induction of TNFalpha and MnSOD mRNAs. These results suggest that (1) while PTK and MAPK are essential for the production of TNFalpha, they are not necessary for the induction of MnSOD by LPS, and (2) while activation of NFkappaB alone is insufficient for the induction of TNFalpha mRNA by LPS, it is necessary for the induction of TNFalpha as well as MnSOD mRNAs.     

TAB1 modulates IL-1alpha mediated cytokine secretion but is dispensable for TAK1 activation

Biochemical evidence indicates that TGF-beta-activated kinase 1 (TAK1), a key modulator of the inflammatory response, exists in a complex with various adaptor proteins including the TAK1 binding protein 1 (TAB1). However, the physiological importance of TAB1 in TAK1 activation, and in the subsequent induction of proinflammatory mediators, remains unclear. In this study, a critical role for TAK1 in IL-1alpha or TNFalpha stimulated MAPK and NFkappaB activation was confirmed by inhibition of the nuclear accumulation of NFkappaB p65 and phosphorylated forms of c-Jun and p38 following siRNA mediated TAK1 silencing. These effects were associated with significant reductions in IL-1alpha stimulated levels of secreted IL-6, IL-8, MCP-1 and GM-CSF. In contrast, IL-1alpha or TNFalpha dependent cellular redistribution of NFkappaB p65 and phosphorylated c-Jun and p38 was not affected by 80% siRNA mediated knockdown of TAB1 protein levels. Interestingly, IL-6, IL-8 and GM-CSF release from TAB1 siRNA transfected cells was significantly reduced following IL-1alpha treatment, but was unchanged after TNFalpha stimulation, suggesting differential roles for TAB1 in IL-1alpha and TNFalpha signalling pathways. These findings may imply an as yet unidentified role for TAB1 in the inflammatory response, which is independent of the activation of classical TAK1 associated signalling cascades.     

Oxidative modification of IkappaB by monochloramine inhibits tumor necrosis factor alpha-induced NF-kappaB activation

We have previously reported that monochloramine (NH(2)Cl), a neutrophil-derived oxidant, inhibited tumor necrosis factor alpha (TNFalpha)-induced expression of cell adhesion molecules and nuclear factor-kappaB (NF-kappaB) activation (Free Radical Research 36 (2002) 845-852). Here, we studied the mechanism how NH(2)Cl inhibited TNFalpha-induced NF-kappaB activation, and compared the effects with taurine chloramine (Tau-NHCl). Pretreatment of Jurkat cells with NH(2)Cl at 70 microM resulted in suppression of TNFalpha-induced IkappaB phosphorylation and degradation, and inhibited NF-kappaB activation. In addition, a slow-moving IkappaB band appeared on SDS-PAGE. By contrast, Tau-NHCl for up to 200 microM had no effects. Interestingly, NH(2)Cl did not inhibit IkappaB kinase activation by TNFalpha. Protein phosphatase activity did not show apparent change. When recombinant IkappaB was oxidized by NH(2)Cl in vitro and phosphorylated by TNFalpha-stimulated Jurkat cell lysate, its phosphorylation occurred less effectively than non-oxidized IkappaB. In addition, when NF-kappaB-IkappaB complex was immunoprecipitated from NH(2)Cl-treated cells and phosphorylated in vitro by recombinant active IkappaB kinase, native IkappaB but not oxidized IkappaB was phosphorylated. Amino acid analysis of the in vitro oxidized IkappaB showed methionine oxidation to methionine sulfoxide. Although Tau-NHCl alone had little effects on TNFalpha-induced NF-kappaB activation, simultaneous presence of Tau-NHCl and ammonium ion significantly inhibited the NF-kappaB activation, probably through the conversion of Tau-NHCl to NH(2)Cl. These results indicated that NH(2)Cl inhibited TNFalpha-induced NF-kappaB activation through the oxidation of IkappaB, and that NH(2)Cl is physiologically more relevant than Tau-NHCl in modifying NF-kappaB-mediated cellular responses.     

Suppression of TNFalpha-mediated NFkappaB activity by myricetin and other flavonoids through downregulating the activity of IKK in ECV304 cells.

Flavonoids are a group of naturally-occurring phenolic compounds in the plant kingdom, and many flavonoids are found with vascular protective properties. Nevertheless how the protective response is exerted by flavonoids is not well characterized. In view of the nuclear factor-kappaB (NFkappaB) may play a central role in the initiation of atherosclerosis, prevention of the activation of NFkappaB represents an important role in protecting vascular injury. In this study, the effects of flavonoids on NFkappaB/inhibitor-kappaB (IkappaB) system in ECV304 cells activated with tumor necrosis factor-alpha (TNFalpha) were examined. We investigated the inhibitory action of six flavonoids on IkappaB kinase (IKK) activity, an enzyme recently found to phosphorylate critical serine residues of IkappaB for degradation. Of six flavonoids tested, myricetin was found to strongly inhibit IKK kinase activity, and prevent the degradation of IkappaBalpha and IkappaBbeta in activated endothelial cells. Furthermore, myricetin was also found to inhibit NFkappaB activity correlated with suppression of monocyte adhesion to ECV304 cells. Therefore we conclude that flavonoids may be of therapeutic value for vascular disease through down regulation of NFkappaB/IkappaB system.     

Ultraviolet light activates NFkappaB through translational inhibition of IkappaBalpha synthesis

UV light induces a delayed and prolonged (3-20 h) activation of NFkappaB when compared with the immediate and acute (10-90 min) activation of NFkappaB in response to tumor necrosis factor alpha treatment. In the early phase (3-12 h) of NFkappaB activation, UV light reduces inhibitor of NFkappaB (IkappaB) through an IkappaB kinase-independent, but polyubiquitin-dependent, pathway. However, the mechanism for the UV light-induced reduction of IkappaB and activation of NFkappaB is not known. In this report, we show that UV light down-regulates the total amount of IkappaB through decreasing IkappaB mRNA translation. Our data show that UV light inhibits translation of IkappaB in wild-type mouse embryo fibroblasts (MEF(S/S)) and that this inhibition is prevented in MEF(A/A) cells in which the phosphorylation site, Ser-51 in the eukaryotic translation initiation factor 2 alpha-subunit, is replaced with a non-phosphorylatable Ala (S51A). Our data also show that UV light-induced NFkappaB activation is delayed in MEF(A/A) cells and in an MCF-7 cell line that is stably transfected with a trans-dominant negative mutant protein kinase-like endoplasmic reticulum kinase (PERK). These results suggest that UV light-induced eukaryotic translation initiation factor 2 alpha-subunit phosphorylation translationally inhibits new IkappaB synthesis. Without a continuous supply of newly synthesized IkappaB, the existing IkappaB is degraded through a polyubiquitin-dependent proteasomal pathway leading to NFkappaB activation. Based upon our results, we propose a novel mechanism by which UV light regulates early phase NFkappaB activation by means of an ER-stress-induced translational inhibition pathway.