Nuclear factor kappa B (NF‐κB) is a key mediator of inflammation. Unchecked NF‐κB signalling can engender autoimmune pathologies and cancers. Here, we show that Tax1‐binding protein 1 (TAX1BP1) is a negative regulator of TNF‐α‐ and IL‐1β‐induced NF‐κB activation and that binding to mono‐ and polyubiquitin by a ubiquitin‐binding Zn finger domain in TAX1BP1 is needed for TRAF6 association and NF‐κB inhibition. Mice genetically knocked out for TAX1BP1 are born normal, but develop age‐dependent inflammatory cardiac valvulitis, die prematurely, and are hypersensitive to low doses of TNF‐α and IL‐1β. TAX1BP1−/− cells are more highly activated for NF‐κB than control cells when stimulated with TNF‐α or IL‐1β. Mechanistically, TAX1BP1 acts in NF‐κB signalling as an essential adaptor between A20 and its targets.

IntroductionThe development of novel therapies for sepsis depends on the understanding of the basic mechanisms of the disease. 1 The principal active agent involved in the pathogenesis of sepsis is bacterial lipopolysaccharide (LPS), an essential component of the surface of gram-negative bacteria. LPS exerts its toxic effects by potently activating macrophages and endothelial cells, and inducing the expression of inflammatory cytokines such as tumor necrosis factor ␣ (TNF␣) and interleukin 6 (IL-6). [2][3][4][5] Thus, elucidating how LPS signals through cell-surface receptors to induce inflammatory gene expression in humans is of major importance.Central to the recognition of LPS and also many other microbial products by the host is a family of transmembrane proteins that have leucine-rich repeats in their extracellular domains known as the toll-like receptors (TLRs). 6 LPS interacts with a heterologous receptor that contains TLR4 7,8 as well as CD14 9,10 and MD2. [11][12][13] As CD14 is a glycosyl phosphatidylinositol-anchored protein and MD2 is on the cell surface, transduction of the LPS signal across the membrane is mediated by TLR4. TLR4, as all TLR family members, contains a cytoplasmic domain that is homologous to a cytoplasmic domain found in the IL-1 receptor known as the Toll/IL-1 receptor (IL-1R) homology (TIR) domain that is essential for downstream signaling. [14][15][16] The presence of the TIR domain in both TLR and IL-1 receptor family members suggested that these receptors use an identical framework of signaling molecules to exert their downstream effects. This was supported by subsequent studies in mouse and human cell lines. Thus, IL-1R and TLR4 were shown to engage the TIR-containing cytosolic adaptor molecule myeloid differentiation protein 88 (MyD88) through homotypic interactions, [17][18][19] with subsequent recruitment of IL-1R-associated kinase (IRAK) and IRAK2, IRAK4, and TRAF6. 17,18,20,21 TRAF6 is thought to subsequently activate nuclear factor (NF)-B either through the IB kinase (IKK) complex and the kinases TAB-1 and TAK-1, 22 or through evolutionarily conserved signaling intermediate in Toll pathways (ECSIT) and mitogen-activated protein kinase/ERK kinase kinase 1 (MEKK-1). 23 The recent derivation of MyD88 Ϫ/Ϫ mice, however, challenged a universal role for MyD88 in LPS signaling. Although there was the expected complete ablation of IL-1 signaling, LPS still activated NF-B although the ability to induce TNF␣ from macrophages was lost. 24 In addition, LPS-induced NF-B activation and up-regulation of costimulatory molecules in bone marrowderived dendritic cells from these mice was not compromised. 25 To account for a MyD88-independent pathway of NF-B activation, a novel MyD88 homologue termed MyD88 adaptor-like (Mal) 26 or TIR domain-containing adaptor protein (TIRAP) 27 was described. This was shown to act as an adaptor protein specifically involved in TLR4 but not other TLRs or IL-1R-induced NF-B activation. 26,27 As Mal/TIRAP does not contain the death domain (DD) found in MyD88...

Stimulation of the NF-B pathway often causes p65-p50 and p50-p50 dimers to be simultaneously present in the cell nucleus. A natural polymorphism at nucleotide ؊863 in the human TNF promoter (encoding tumor necrosis factor [TNF]) region provides an opportunity to dissect the functional interaction of p65-p50 and p50-p50 at a single NF-B binding site. We found that this site normally binds both p65-p50 and p50-p50, but a single base change specifically inhibits p50-p50 binding. Reporter gene analysis in COS-7 cells expressing both p65-p50 and p50-p50 shows that the ability to bind p50-p50 reduces the enhancer effect of this NF-B site. Using an adenoviral reporter assay, we found that the variant which binds p50-p50 results in a reduction of lipopolysaccharide-inducible gene expression in primary human monocytes. This finding adds to a growing body of experimental evidence that p50-p50 can inhibit the transactivating effects of p65-p50 and illustrates the potential for genetic modulation of inflammatory gene regulation in humans by subtle nucleotide changes that alter the relative binding affinities of different forms of the NF-B complex.The NF-B/Rel family of transcription factors is involved in many physiological processes, including regulation of a wide range of inflammatory mediators (2). Inflammation is a critical component of host defense, but it is also responsible for many of the clinical symptoms of infection and injury and can be fatal if elicited in excess. This raises the fundamental question of how the level of response to NF-B is optimized across a large number of different inflammatory genes. Often this may involve functional interactions between NF-B and other transcription factors (24). This paper considers another mechanism, which has received relatively little attention, namely, through variation in the composition of NF-B dimers that bind to a specific regulatory site. The canonical form of NF-B is a heterodimer comprising a p65 subunit, containing both a DNA binding domain and a domain that is essential for transcriptional activation, plus a p50 subunit which has a DNA binding domain but no activation domain. The biological role of p50 was initially thought to relate solely to its DNA binding properties within the active p65-p50 complex, but more recent evidence suggests that p50-p50 homodimers are capable of acting as transcriptional repressors. For example, artificial overexpression of p50 acts to suppress the transactivating effects of p65 at some NF-B sites (15,17), and this mechanism has been implicated in the downregulation of major histocompatibility complex expression (16) and in viral postinduction repression of the beta interferon gene (23). Since the optimal binding sequences for p65 and p50 are similar but not identical (12,15,23), it is possible that NF-B-induced responses might be fine-tuned by minor sequence variations that alter the relative binding of p65-p50 and p50-p50 to different regulatory elements. To examine this question, we have investigated the functional properties o...

IL-10 is a potent anti-inflammatory cytokine and inhibitor of TNF-α production. The molecular pathways by which IL-10 inhibits TNF-α production are obscure, with diverse mechanisms having been published. In this study, a new approach has been taken for the study of human cells. Adenovirus was used to deliver TNF-α promoter-based luciferase reporter genes to primary human monocytic cells. The reporter genes were highly responsive to macrophage activation and appeared to mirror the behavior of the endogenous TNF-α gene. When added, either with or after the stimulus, IL-10 required the 3′ untranslated region of the TNF-α gene to inhibit luciferase mRNA and protein expression, indicating a posttranscriptional mechanism. However, if macrophages were incubated with IL-10 before activation, inhibition of gene expression was also mediated by the 5′ promoter, suggesting a transcriptional mechanism. To our knowledge, this is the first time that a dual mechanism for IL-10 function has been demonstrated. Studies to elucidate the mechanisms underlying the inhibition of TNF-α production addressed the effect of IL-10 on the activation of p38 mitogen-activated protein kinase and NF-κB. However, these studies could demonstrate no requirement for the inhibition of p38 mitogen-activated protein kinase or NF-κB activation as potential mechanisms. Overall, these results may explain the diversity previously ascribed to the complex mechanisms of IL-10 anti-inflammatory activity.

A double-row reconstruction of the supraspinatus tendon insertion may provide a more reliable construct than a single-row repair and could be used as an alternative to open reconstruction for the treatment of isolated tears.