The transcription factor nuclear factor-kappa B (NF-kappaB) is a crucial regulator of many physiological and patho-physiological processes, including control of the adaptive and innate immune responses, inflammation, proliferation, tumorigenesis, and apoptosis. Thus, the tight regulation of NF-kappaB activity within a cell is extremely important. The central mechanism of NF-kappaB regulation is the signal-induced proteolytic degradation of a family of cytoplasmic inhibitors of NF-kappaB, the IkappaBs. However, with the discovery of an IkappaB-independent noncanonical or "alternative" pathway of NF-kappaB activation, the importance of other regulatory mechanisms responsible for the fine-tuning of NF-kappaB became clear. Post-translational modification, especially phosphorylation, of the Rel proteins, of which dimeric NF-kappaB is composed, are such alternative regulatory mechanisms. The best analyzed example is RelA phosphorylation, which takes place at specific amino acids resulting in distinct functional changes of this gene regulatory protein. The interaction of NF-kappaB with other proteins such as glucocorticoid receptors is very important for the regulation of NF-kappaB activity. Recently, exciting new concepts of IkappaB-independent NF-kappaB control like dimer exchange and nucleolar sequestration of RelA have been described, indicating that many aspects of NF-kappaB control are waiting to be discovered.

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Ulcerative colitis and Crohn's disease are the two entities of chronic inflammatory bowel diseases (IBD). One of the main pathogenic mechanisms is probably a dysregulated immune response triggered by products of the enteric bacterial flora. The goal of this study was to evaluate the effects of the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 on inflammatory responses using the DSS-induced experimental colitis model in mice reflecting human IBD. We found that SB203580 improved the clinical score, ameliorates the histological alterations, and reduces the mRNA levels of proinflammatory cytokines. In addition to p38 kinase activity, the "classical" and the "alternative" NF-kappaB pathways were also strongly activated during colitis induction. All three pathways were drastically down-regulated by SB203580 treatment. An analysis of the molecular basis of NF-kappaB activation revealed that Rip-like interacting caspase-like apoptosis-regulatory protein kinase (RICK), a key component of a pathway leading to NF-kappaB induction, is also strongly inhibited by SB203580. Since RICK is an effector kinase of NOD2, an intracellular receptor of bacterial peptidoglycan, these results support the notion that NOD signaling could play a pivotal role in the IBD pathogenesis. Thus, RICK could represent a novel target for future therapies in human IBD.

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.

RelB is an unusual member of the NF-〉 transcription factor family that acts as both a transcriptional activator as well as a repressor of NF-〉-dependent gene expression. Although RelB promotes gene expression when it associates with p50/NF-〉1 or p52/NF-〉2, the precise molecular mechanisms through which it represses NF-〉 remain unclear. To examine this inhibitory function in more detail, we employed reporter gene assays and found that RelB represses at the level of RelA. Furthermore, electrophoretic mobility shift analysis revealed that in vitro translated RelB impaired the DNA binding activity of RelA and that overexpressed RelB significantly reduced tumor necrosis factor-␣-induced RelA activity in murine embryonic fibroblasts. Intriguingly, this inhibitory effect was due to the formation of RelA⅐RelB heterodimers that were unable to bind to B sites in vitro strongly suggesting that these newly described NF-〉 dimers cannot bind DNA. Expression pattern analysis revealed that RelA⅐RelB heterodimers appeared at relatively low levels in both lymphoid and non-lymphoid cells. However, the presence of these complexes increased following stimulation with phorbolesters or lipopolysaccharide or by overexpression of constitutively active IK⌲␤. Functional characterization of RelA⅐RelB heterodimers in NIH3T3 murine embryonic fibroblasts revealed that they are not regulated by I〉 proteins and are located in both the cytoplasm and the nucleus. Taken together, our findings demonstrate that sequestration of RelA in transcriptionally inactive RelA⅐RelB complexes provides a molecular mechanism that may explain the repressive role of RelB on NF-〉-dependent gene expression.

Nuclear factor-B (NF-B) is the main target of antiinflammatory therapies in human chronic inflammatory bowel diseases (IBD), Crohn disease, and ulcerative colitis. This study investigates the molecular anti-inflammatory mechanisms of SB203580, an inhibitor of the mitogen-activated protein kinase p38. The murine trinitrobenzene sulfonic acid (TNBS)-induced colitis was used as an established model of human Crohn disease. Here we show that SB203580 improved the clinical condition, reduced intestinal inflammation, and suppressed mRNA levels of pro-inflammatory cytokines elevated upon induction of colitis. Besides p38 kinase activity, the "classical" I B-dependent NF-B pathway was strongly up-regulated during colitis induction, whereas the "alternative" was not. SB203580 treatment resulted in a drastic down-regulation of p38 and NF-B activity. The molecular analysis of NF-B activation revealed that Rip-like interacting caspase-like apoptosis-regulatory protein kinase (RICK), a key component of a pathway leading to NF-B induction, is also strongly inhibited by SB203580. In contrast, SB203580 had no effect on the colitis-induced activation of other potential NF-Bactivating kinases such as protein kinase C (PKC ), mixed lineage kinase 3, and the oncogene product Cot/ TPL2. Thus, the inhibitory effect of SB203580 on NF-B activation is to a large extent mediated by RICK inhibition. RICK is the effector kinase of the intracellular receptor of bacterial peptidoglycan NOD. Because bacterial products are suggested to be the key pathogenic agents triggering IBD, inhibition of the NOD/RICK pathway may serve as a novel target of future therapies in human IBD.Crohn disease is characterized by chronic intestinal inflammation frequently relapsing with clinical manifestations including diarrhea, blood in the stool, abdominal pain, and weight loss. The etiology of Crohn disease is so far unknown (1) but epidemiological and linkage studies suggest a genetic predisposition of the patient and the involvement of environmental factors (1, 2). Furthermore, several observations strongly implicate a pathogenic role of the intestinal flora during the initiation process of the immunological dysregulation (3-5).

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Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) exerts potent cytotoxic activity against transformed keratinocytes, whereas primary keratinocytes are relatively resistant. In several cell types, inhibition of the proteasome sensitizes for TRAIL-induced apoptosis by interference with NF-B activation. Here we describe a novel intracellular mechanism of TRAIL resistance in primary cells and how this resistance is removed by proteasome inhibitors independent of NF-B in primary human keratinocytes. This sensitization was not mediated at the receptor-proximal level of TRAIL DISC formation or caspase 8 activation but further downstream. Activation of caspase 3 was critical, as it only occurred when mitochondrial apoptotic pathways were activated, as reflected by Smac/DIABLO, HtrA2, and cytochrome c release. Smac/DIABLO and HtrA2 are needed to release the X-linked inhibitor-of-apoptosis protein (XIAP)-mediated block of full caspase 3 maturation. XIAP can effectively block caspase 3 maturation and, intriguingly, is highly expressed in primary but not in transformed keratinocytes. Ectopic XIAP expression in transformed keratinocytes resulted in increased resistance to TRAIL. Our data suggest that breaking of this resistance via proteasome inhibitors, which are potential anticancer drugs, may sensitize certain primary cells to TRAIL-induced apoptosis and could thereby complicate the clinical applicability of a combination of TRAIL receptor agonists with proteasome inhibitors.