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.

Many of the biological functions attributed to cell surface proteoglycans are dependent on the interaction with extracellular mediators through their heparan sulphate (HS) moieties and the participation of their core proteins in signaling events. A class of recently identified inflammatory mediators is secreted cyclophilins, which are mostly known as cyclosporin A-binding proteins. We previously demonstrated that cyclophilin B (CyPB) triggers chemotaxis and integrin-mediated adhesion of T lymphocytes mainly of the CD4+/CD45RO+ phenotype. These activities are related to interactions with two types of binding sites, CD147 and cell surface HS. Here, we demonstrate that CyPB-mediated adhesion of CD4+/CD45RO+ T cells is related to p44/42 mitogen-activated protein kinase (MAPK) activation by a mechanism involving CD147 and HS proteoglycans (HSPG). Although HSPG core proteins are represented by syndecan-1, -2, -4, CD44v3 and betaglycan in CD4+/CD45RO+ T cells, we found that only syndecan-1 is physically associated with CD147. The intensity of the heterocomplex increased in response to CyPB, suggesting a transient enhancement and/or stabilization in the association of CD147 to syndecan-1. Pretreatment with anti-syndecan-1 antibodies or knockdown of syndecan-1 expression by RNA interference dramatically reduced CyPB-induced p44/p42 MAPK activation and consequent migration and adhesion, supporting the model in which syndecan-1 serves as a binding subunit to form the fully active receptor of CyPB. Altogether, our findings provide a novel example of a soluble mediator in which a member of the syndecan family plays a critical role in efficient interaction with signaling receptors and initiation of cellular responses.

Many of the biological functions of heparan sulfate (HS) proteoglycans can be attributed to specialized structures within HS moieties, which are thought to modulate binding and function of various effector proteins. Cyclophilin B (CyPB), which was initially identified as a cyclosporin A-binding protein, triggers migration and integrin-mediated adhesion of peripheral blood T lymphocytes by a mechanism dependent on interaction with cell surface HS. Here we determined the structural features of HS that are responsible for the specific binding of CyPB. In addition to the involvement of 2-O, 6-O, and N-sulfate groups, we also demonstrated that binding of CyPB was dependent on the presence of N-unsubstituted glucosamine residues (GlcNH 2 ), which have been reported to be precursors for sulfation by 3-Osulfotransferases-3 (3-OST-3). Interestingly, 3-OST-3B isoform was found to be the main 3-OST isoenzyme expressed in peripheral blood T lymphocytes and Jurkat T cells. Moreover, downregulation of the expression of 3-OST-3 by RNA interference potently reduced CyPB binding and consequent activation of p44/42 mitogen-activated protein kinases. Altogether, our results strongly support the hypothesis that 3-O-sulfation of GlcNH 2 residues could be a key modification that provides specialized HS structures for CyPB binding to responsive cells. Given that 3-O-sulfation of GlcNH 2 -containing HS by 3-OST-3 also provides binding sites for glycoprotein gD of herpes simplex virus type I, these findings suggest an intriguing structural linkage between the HS sequences involved in CyPB binding and viral infection. Heparan sulfate (HS)4 proteoglycans on the cell surface or in the extracellular matrix are involved in developmental, regeneratory, and inflammatory processes, as a consequence of their interactions with multiple proteins. These interactions are mediated mainly via the HS moieties of the proteoglycans, which bind to growth factors, cytokines, matrix components, enzymes, and enzyme inhibitors and thereby regulate tissue distribution, biological availability, and activity of the proteins. Characterization of heparin/HS oligosaccharides with high affinity to proteins such as antithrombin and growth factors has led to the identification of specialized protein binding domains with structural features exhibiting varying degrees of specificity (1-3). The structural distinctions in these functional heparin/HS domains are derived from enzymatic modifications in the Golgi apparatus of the nascent polymer composed of alternating D-glucuronic acid (GlcUA) and N-acetylated D-glucosamine (GlcNAc) units. The nonsulfated precursor is first subject to partial N-deacetylation/N-sulfation of GlcNAc residues, which leads to the occurrence of consecutively N-sulfated regions, regions that escape modification and remains N-acetylated, and regions of alternating N-acetylated and N-sulfated disaccharide units. Sometimes, the N-deacetylation/N-sulfation reaction is partial, giving rise to N-unsubstituted glucosamine units (GlcNH 2 ). The further mo...