Hexameric calgranulin C (S100A12) binds to the receptor for advanced glycated end products (RAGE) using symmetric hydrophobic target-binding patches

Calgranulin C (S100A12) is a member of the S100 family of proteins that undergoes a conformational change upon calcium binding allowing them to interact with target molecules and initiate biological responses; one such target is the receptor for advanced glycation products (RAGE). The RAGE-calgranulin C interaction mediates a pro-inflammatory response to cellular stress and can contribute to the pathogenesis of inflammatory lesions. The soluble extracellular part of RAGE (sRAGE) was shown to decrease the inflammation response possibly by scavenging RAGE-activating ligands. Here, by using high resolution NMR spectroscopy, we identified the sRAGE-calgranulin C interaction surface. Ca2+ binding creates two symmetric hydrophobic surfaces on Ca2+-calgranulin C that allow calgranulin C to bind to the C-type immunoglobulin domain of RAGE. Apo-calgranulin C also binds to sRAGE using a completely different surface and with substantially lower affinity, thus underscoring the role of Ca2+ binding to S100 proteins as a molecular switch. By using native gel electrophoresis, chromatography, and fluorescence spectroscopy, we established that sRAGE forms tetramers that bind to hexamers of Ca2+-calgranulin C. This arrangement creates a large platform for effectively transmitting RAGE-dependent signals from extracellular S100 proteins to the cytoplasmic signaling complexes.     

RAGE and its ligands in retinal disease

RAGE, the receptor for advanced glycation endproducts (AGEs), is a multiligand signal transduction receptor of the immunoglobulin superfamily of cell surface molecules that has been implicated in the pathogenesis of diabetic complications, neurodegenerative diseases, inflammatory disorders, and cancer. These diverse biologic disorders reflect the multiplicity of ligands capable of cellular interaction via RAGE that include, in addition to AGEs, amyloid-beta (Abeta) peptide, the S100/calgranulin family of proinflammatory cytokines, and amphoterin, a member of the High Mobility Group Box (HMGB) DNA-binding proteins. In the retina, RAGE expression is present in neural cells, the vasculature, and RPE cells, and it has also been detected in pathologic cellular retinal responses including epiretinal and neovascular membrane formation. Ligands for RAGE, in particular AGEs, have emerged as relevant to the pathogenesis of diabetic retinopathy and age-related macular disease. While the understanding of RAGE and its role in retinal dysfunction with aging, diabetes mellitus, and/or activation of pro-inflammatory pathways is less complete compared to other organ systems, increasing evidence indicates that RAGE can initiate and sustain significant cellular perturbations in the inner and outer retina. For these reasons, antagonism of RAGE interactions with its ligands may be a worthwhile therapeutic target in such seemingly disparate, visually threatening retinal diseases as diabetic retinopathy, age-related macular degeneration, and proliferative vitreoretinopathy.     

RAGE: The beneficial and deleterious effects by diverse mechanisms of actions

Receptor for advanced glycation endproducts (RAGE) is a transmembrane protein that belongs to the immunoglobulin superfamily. RAGE is expressed ubiquitously-high in lung and moderate to low in a wide range of cells-in a tightly regulated manner at various stages of development. RAGE is a pattern recognition receptor that binds to multiple ligands, including amphoterin, members of the S100/calgranulin family, the integrin Mac-1, and amyloid β-peptide (Aβ). RAGE-ligand engagement effects the activation of diverse cascades that initiate and stimulate chronic stress pathways and repair, depending on the ligand, environment, and developmental stage. Further, RAGE-ligand interaction and the consequent upregulation of RAGE through a positive feedback loop are often associated with various diseases, including vascular disease, diabetes, cancer, and neurodegenerative disease. It is unknown how RAGE mediates these events, but such phenomena appear to be linked to the inflammatory response. In this review, we summarize the findings on RAGE from published reports and ongoing studies. Also, the implication of RAGE in Alzheimer disease, the most common neurodegenerative disease in the elderly population, will be discussed, with a focus on Aβ-RAGE interactions with regard to signaling pathways and their impact on cellular activity.     

Interaction of the RAGE cytoplasmic domain with diaphanous-1 is required for ligand-stimulated cellular migration through activation of Rac1 and Cdc42

Cellular migration is a fundamental process linked to diverse pathological states such as diabetes and its complications, atherosclerosis, inflammation, and cancer. The receptor for advanced glycation end products (RAGE) is a multiligand cell surface macromolecule which binds distinct ligands that accumulate in these settings. RAGE-ligand interaction evokes central changes in key biological properties of cells, including proliferation, generation of inflammatory mediators, and migration. Although RAGE-dependent signal transduction is critically dependent on its short cytoplasmic domain, to date the proximate mechanism by which this RAGE domain engages and stimulates cytoplasmic signaling pathways has yet to be identified. Here we show that the RAGE cytoplasmic domain interacts with Diaphanous-1 (Dia-1) both in vitro and in vivo. We employed the human RAGE cytoplasmic domain as "bait" in the yeast two-hybrid assay and identified the formin homology (FH1) domain of Dia-1 as a potential binding partner of this RAGE domain. Immunoprecipitation studies revealed that the RAGE cytoplasmic domain interacts with the FH1 domain of Dia-1. Down-regulation of Dia-1 expression by RNA interference blocks RAGE-mediated activation of Rac-1 and Cdc42 and, in parallel, RAGE ligand-stimulated cellular migration. Taken together, these findings indicate that the interaction of the RAGE cytoplasmic domain with Dia-1 is required to transduce extracellular environmental cues evoked by binding of RAGE ligands to their cell surface receptor, a chief consequence of which is Rac-1 and Cdc42 activation and cellular migration. Because RAGE and Dia-1 are implicated in the regulation of inflammatory, vascular, and transformed cell migration, these findings highlight this interaction as a novel target for therapeutic intervention in inflammation, atherosclerosis, diabetes, and cancer.     

Receptor for AGE (RAGE): weaving tangled webs within the inflammatory response

The family of RAGE ligands, including Advanced Glycation Endproducts (AGEs), S100/calgranulins, High Mobility Group Box-1 (HMGB1) and amyloid beta peptide (Abeta) and beta-sheet fibrils are highly enriched in immune and inflammatory foci. In parallel, upregulation of Receptor for AGE (RAGE) is noted in diverse forms of inflammation and autoimmunity, based on experiments examining human tissues as well as animal models. Indeed, prior to the demonstration that S100/calgranulins were signal transduction ligands of RAGE, these molecules were considered "biomarkers" of disease and disease activity in disorders such as colitis and arthritis. Premiere roles for RAGE in advancing cellular migration implicate this receptor in targeting immune cells to vulnerable foci. Once engaged, ligand-RAGE interaction in inflammatory and vascular cells amplifies upregulation of inflammatory cytokines, adhesion molecules and matrix metalloproteinases (MMPs). Discerning the primal versus chronic injury-provoking roles for this ligand-receptor interaction is a challenge in delineating the functions of the ligand/RAGE axis. As RAGE is expressed by many of the key cell types linked integrally to the immune response, we propose that the sites and time course of ligand-RAGE stimulation determine the phenotype produced by this axis. Ultimately, drawing the fine line between antagonism versus stimulation of the receptor in health and disease will depend on the full characterization of RAGE in repair versus injury.     

Association of advanced glycation end products with A549 cells, a human pulmonary epithelial cell line, is mediated by a receptor distinct from the scavenger receptor family and RAGE

Cellular interactions with advanced glycation end products (AGE)-modified proteins are known to induce several biological responses, not only endocytic uptake and degradation, but also the induction of cytokines and growth factors, combined responses that may be linked to the development of diabetic vascular complications. In this study we demonstrate that A549 cells, a human pulmonary epithelial cell line, possess a specific binding site for AGE-modified bovine serum albumin (AGE-BSA) (K(d) = 27.8 nM), and additionally for EN-RAGE (extracellular newly identified RAGE binding protein) (K(d) = 118 nM). Western blot and RT-PCR analysis showed that RAGE (receptor for AGE) is highly expressed on A549 cells, while the expression of other known AGE-receptors such as galectin-3 and SR-A (class A scavenger receptor), are below the level of detection. The binding of (125)I-AGE-BSA to these cells is inhibited by unlabeled AGE-BSA, but not by EN-RAGE. In contrast, the binding of (125)I-EN-RAGE is significantly inhibited by unlabeled EN-RAGE and soluble RAGE, but not by AGE-BSA. Our results indicate that A549 cells possess at least two binding sites, one specific for EN-RAGE and the other specific for AGE-BSA. The latter receptor on A549 cells is distinct from the scavenger receptor family and RAGE.     

IL-22 is induced by S100/calgranulin and impairs cholesterol efflux in macrophages by downregulating ABCG1

S100A8/9 and S100A12 are emerging biomarkers for disease activity of autoimmune and cardiovascular diseases. We demonstrated previously that S100A12 accelerates atherosclerosis accompanied by large cholesterol deposits in atherosclerotic lesions of apoE-null mice. The objective of this study was to ascertain whether S100/calgranulin influences cholesterol homeostasis in macrophages. Peritoneal macrophages from transgenic mice expressing human S100A8/9 and S100A12 in myeloid cells [human bacterial artificial chromosome (hBAC)/S100] have increased lipid content and reduced ABCG1 expression and [³H]cholesterol efflux compared with WT littermates. This was associated with a 6-fold increase in plasma interleukin (IL)-22 and increased IL-22 mRNA in splenic T cells. These findings are mediated by the receptor for advanced glycation endproducts (RAGE), because hBAC/S100 mice lacking RAGE had normal IL-22 expression and normal cholesterol efflux. In vitro, recombinant IL-22 reduced ABCG1 expression and [³H]cholesterol efflux in THP-1 macrophages, while recombinant S100A12 had no effect on ABCG1 expression. In conclusion, S100/calgranulin has no direct effect on cholesterol efflux in macrophages, but rather promotes the secretion of IL-22, which then directly reduces cholesterol efflux in macrophages by decreasing the expression of ABCG1.     

Human S100A12: a novel key player in inflammation?

S100A12 is a member of the S100 family of EF-hand calcium-binding proteins. Human S100A12 is predominantly expressed and secreted by neutrophil granulocytes and, therefore, has been assigned to the S100 protein subfamily of calgranulins or myeloid-related proteins. Intracellular S100A12 exists as an anti-parallel homodimer and upon calcium-dependent activation interacts with target proteins to regulate cellular functions. Extracellular S100A12 exists majorily as homodimer and hexamer, respectively, and shows cytokine-like characteristics. It is part of the innate immune response and linked to certain autoimmune reactions. Human S100A12 is markedly overexpressed in inflammatory compartments, and elevated serum levels of S100A12 are found in patients suffering from various inflammatory, neurodegenerative, metabolic, and neoplastic disorders. In this regard, interaction of calcium-activated S100A12 with the multiligand receptor for advanced glycation endproducts (RAGE) and its soluble form (sRAGE) plays a central pathogenetic role. Recent clinical evidence suggests a high potential of S100A12 as a sensitive and specific diagnostic marker of localized inflammatory processes.     

The association of receptor of advanced glycated end products and inflammatory mediators contributes to endothelial dysfunction in a prospective study of acute kidney injury patients with sepsis

The pathogenesis of acute kidney injury (AKI) occurring due to sepsis is incompletely understood. Endothelial activation, defined as up-regulation of adhesion molecules by proinflammatory cytokines, may be central to the development of sepsis-induced AKI. Our aim was to determine levels of circulating adhesion molecules endothelial (E)-selectin, intercellular adhesion molecule (ICAM), and vascular cell adhesion molecule (VCAM), inflammatory mediators; tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β), vasoactive mediators; endothelin-1 (ET-1) and nitric oxide (NO), soluble receptor for advanced glycated end products (sRAGE) and serum fetuin-A in septic AKI patients before and after antibiotic therapy. Nineteen AKI patients with sepsis and fifteen healthy controls were enrolled in this prospective study. Results revealed that 12 weeks of therapy caused amelioration of endothelial and inflammatory injuries as well as renal function markers. Moreover, the positive correlations between levels of RAGE and E-selectin (r=0.88), ET-1 (r=0.90), and TNF-α (r=0.94) and negative with NO (r=-0.75-0.95) suggest that possible interaction of RAGE and inflammation may contribute to endothelial dysfunction in septic AKI patients.     

Transamidation by transglutaminase 2 transforms S100A11 calgranulin into a procatabolic cytokine for chondrocytes

In osteoarthritis (OA), low-grade joint inflammation promotes altered chondrocyte differentiation and cartilage catabolism. S100/calgranulins share conserved calcium-binding EF-hand domains, associate noncovalently as homodimers and heterodimers, and are secreted and bind receptor for advanced glycation end products (RAGE). Chondrocyte RAGE expression and S100A11 release are stimulated by IL-1beta in vitro and increase in OA cartilage in situ. Exogenous S100A11 stimulates chondrocyte hypertrophic differentiation. Moreover, S100A11 is covalently cross-linked by transamidation catalyzed by transglutaminase 2 (TG2), itself an inflammation-regulated and redox stress-inducible mediator of chondrocyte hypertrophic differentiation. In this study, we researched mouse femoral head articular cartilage explants and knee chondrocytes, and a soluble recombinant double point mutant (K3R/Q102N) of S100A11 TG2 transamidation substrate sites. Both TG2 and RAGE knockout cartilage explants retained IL-1beta responsiveness. The K3R/Q102N mutant of S100A11 retained the capacity to bind to RAGE and chondrocytes but lost the capacity to signal via the p38 MAPK pathway or induce chondrocyte hypertrophy and glycosaminoglycans release. S100A11 failed to induce hypertrophy, glycosaminoglycan release, and appearance of the aggrecanase neoepitope NITEGE in both RAGE and TG2 knockout cartilages. We conclude that transamidation by TG2 transforms S100A11 into a covalently bonded homodimer that acquires the capacity to signal through the p38 MAPK pathway, accelerate chondrocyte hypertrophy and matrix catabolism, and thereby couple inflammation with chondrocyte activation to potentially promote OA progression.     

Inflammation: a novel mechanism for the transport of extracellular nucleotide-induced arachidonic acid by S100A8/A9 for transcellular metabolism

Extracellular nucleotides cause neutrophil degranulation by activating the purinergic receptor subtype P2Y. However, the molecular mechanism involved in the signal pathway remains unknown. A hypothetical scheme suggesting that leukotriene(s) and leukotriene receptor(s) activation is required for extracellular nucleotide-mediated neutrophil degranulation is presented here. Subsequent to the extracellular nucleotide binding to its receptors, intracellular arachidonic acid (AA) levels are elevated. Although AA is a known substrate of the lipoxygenase pathway mediated by 5-lipoxygenase, excess AA could form a complex with S100A8/A9 for transport to the extracellular milieu. Extracellular availability of the S100A8/A9+AA complex could potentially be used for transcellular metabolism by resting and/or activated leukocytes (PMN, MN), vascular endothelium and smooth muscle cells at the inflammatory foci. Once imported into the resting and/or activated leukocytes, AA derived from the S100A8/A9+AA complex could serve as a substrate in the 5-lipoxygenase-mediated leukotriene pathway. Essentially, in addition to extracellular nucleotide-induced leukotrienes, AA derived from the S100A8/A9+AA complex could also be utilized for the synthesis of inflammatory mediators such as leukotriene B(4)(LTB(4)), which in turn could trigger leukocyte degranulation, as well as cellular damage to vascular endothelium and smooth muscle cells, thereby exacerbating inflammation.     

RAGE: a single receptor for several ligands and different cellular responses: the case of certain S100 proteins

The S100 protein family comprises at least 25 members which, with the exception of S100G, act as Ca2+-sensor proteins that participate in Ca2+ signal transduction by interacting with target proteins thereby modifying their activities. S100 proteins are expressed in vertebrates exclusively, display a cell-specific distribution, and regulate a large variety of intracellular activities. Some S100 proteins are released by a non-classical pathway and exert regulatory effects on several cell types. The receptor for advanced glycation end products (RAGE) has been shown to transduce extracellular effects of S100B, S100A4, S100A6, S100A11, S100A12, S100A13 and S100P. However, some S100 proteins can signal by engaging RAGE as well as non-RAGE receptors. Immune cells (i.e., monocytes/macrophages/microglia, neutrophils and lymphocytes), activated endothelial and vascular smooth muscle cells, neurons, astrocytes, chondrocytes and pancreatic tumor cells are the cell types reported to respond to certain S100 proteins via RAGE engagement. In general, relatively high concentrations of S100 proteins are required for activation of RAGE in responsive cells. S100B is unique in that it can engage RAGE in neurons at low and high concentrations with trophic and toxic effects, respectively, and S100A4 stimulates matrix metalloproteinase 13 release from chondrocytes at nanomolar doses in a RAGE-mediated manner. Oligomerization of S100 proteins under the non-reducing, high-Ca2+ conditions found extracellularly appears to play a relevant role in RAGE activation, and binding of at least S100A12 and S100B results in RAGE oligomerization. Thus, S100/RAGE interactions might have important consequences during development and in tissue homeostasis as well as in inflammatory, degenerative and tumor processes.     

Lack of lymphatic vessel phenotype in LYVE‐1/CD44 double knockout mice

The receptor for advanced-glycation-end-products (RAGE) has been implicated as a pro-inflammatory factor in chronic inflammatory conditions such as diabetes mellitus and rheumatoid arthritis. The aim of this study was to investigate the inhibitory effect of the soluble-RAGE (sRAGE), the extracellular domain of RAGE, on RAGE expression and NF-κB translocation in human-salivary gland-cell-lines (HSG). Cells were stimulated with agonist S100A4, fusion protein of RAGE encompassing the extracellular domain of RAGE (ex-RAGE), ex-RAGE followed by S100A4, or S100A4 followed by ex-RAGE. Our study indicates that RAGE expression was highest at 150 µg/µl of S100A4 and efficiently down-regulated by 1.8-fold (P < 0.05) when ex-RAGE was incubated prior to agonist S100A4. RAGE protein was also consistently down-regulated by 20–40% with pre-incubation of ex-RAGE. More importantly, nuclear translocation of p65 and p52 of NF-κB by S100A4 was inhibited in the presence of ex-RAGE, confirming anti-inflammatory function of ex-RAGE. In conclusion, ex-RAGE down-regulates RAGE expression and inhibits p65 and p52 activation in HSG, providing evidence that ex-RAGE functions as a “decoy” to RAGE–ligand interaction and thus potentially dampening inflammatory conditions. J. Cell. Physiol. 221: 430–434, 2009. © 2009 Wiley-Liss, Inc.     

Structural basis for ligand recognition and activation of RAGE

The receptor for advanced glycation end products (RAGE) is a pattern recognition receptor involved in?inflammatory processes and is associated with diabetic complications, tumor outgrowth, and neurodegenerative disorders. RAGE induces cellular signaling events upon binding of a variety of ligands, such as glycated proteins, amyloid-β, HMGB1, and S100 proteins. The X-ray crystal structure of the VC1 ligand-binding region of the human RAGE ectodomain was determined at 1.85?? resolution. The VC1 ligand-binding surface was mapped onto the structure from titrations with S100B monitored by heteronuclear NMR spectroscopy. These NMR chemical shift perturbations were used as input for restrained docking calculations to generate a model for the VC1-S100B complex. Together, the arrangement of VC1 molecules in the crystal and complementary biochemical studies suggest a role for self-association in RAGE function. Our results enhance understanding of the functional outcomes of S100 protein binding to RAGE and provide insight into mechanistic models for how the receptor is activated.     

Thioredoxin interacting protein (TXNIP) induces inflammation through chromatin modification in retinal capillary endothelial cells under diabetic conditions

Chronic hyperglycemia and activation of receptor for advanced glycation end products (RAGE) are known risk factors for microvascular disease development in diabetic retinopathy. Thioredoxin‐interacting protein (TXNIP), an endogenous inhibitor of antioxidant thioredoxin (TRX), plays a causative role in diabetes and its vascular complications. Herein we investigate whether HG and RAGE induce inflammation in rat retinal endothelial cells (EC) under diabetic conditions in culture through TXNIP activation and whether epigenetic mechanisms play a role in inflammatory gene expression. We show that RAGE activation by its ligand S100B or HG treatment of retinal EC induces the expression of TXNIP and inflammatory genes such as Cox2, VEGF‐A, and ICAM1. TXNIP silencing by siRNA impedes RAGE and HG effects while stable over‐expression of a cDNA for human TXNIP in EC elevates inflammation. p38 MAPK‐NF‐κB signaling pathway and histone H3 lysine (K) nine modifications are involved in TXNIP‐induced inflammation. Chromatin immunoprecipitation (ChIP) assays reveal that TXNIP over‐expression in EC abolishes H3K9 tri‐methylation, a marker for gene inactivation, and increases H3K9 acetylation, an indicator of gene induction, at proximal Cox2 promoter bearing the NF‐κB‐binding site. These findings have important implications toward understanding the molecular mechanisms of ocular inflammation and endothelial dysfunction in diabetic retinopathy. J. Cell. Physiol. 221: 262–272, 2009. © 2009 Wiley‐Liss, Inc     

Chemotactic activity of S100A7 (Psoriasin) is mediated by the receptor for advanced glycation end products and potentiates inflammation with highly homologous but functionally distinct S100A15

Human S100A7 (psoriasin) is overexpressed in inflammatory diseases. The recently discovered, co-evolved hS100A15 is almost identical in sequence and up-regulated with hS100A7 during cutaneous inflammation. The functional role of these closely related proteins for inflammation remains undefined. By generating specific Abs, we demonstrate that hS100A7 and hS100A15 proteins are differentially expressed by specific cell types in the skin. Although highly homologous, both proteins are chemoattractants with distinct chemotactic activity for leukocyte subsets. We define RAGE (receptor for advanced glycation end products) as the hS100A7 receptor, whereas hS100A15 functions through a Gi protein-coupled receptor. hS100A7-RAGE binding, signaling, and chemotaxis are zinc-dependent in vitro, reflecting the previously reported zinc-mediated changes in the hS100A7 dimer structure. When combined, hS100A7 and hS100A15 potentiate inflammation in vivo. Thus, proinflammatory synergism in disease may be driven by the diverse biology of these almost identical proteins that have just recently evolved. The identified S100A7 interaction with RAGE may provide a novel therapeutic target for inflammation.