Structural and functional insights into RAGE activation by multimeric S100B.

Nervous system development and plasticity require regulation of cell proliferation, survival, neurite outgrowth and synapse formation by specific extracellular factors. The EF-hand protein S100B is highly expressed in human brain. In the extracellular space, it promotes neurite extension and neuron survival via the receptor RAGE (receptor for advanced glycation end products). The X-ray structure of human Ca(2+)-loaded S100B was determined at 1.9 A resolution. The structure revealed an octameric architecture of four homodimeric units arranged as two tetramers in a tight array. The presence of multimeric forms in human brain extracts was confirmed by size-exclusion experiments. Recombinant tetrameric, hexameric and octameric S100B were purified from Escherichia coli and characterised. Binding studies show that tetrameric S100B binds RAGE with higher affinity than dimeric S100B. Analytical ultracentrifugation studies imply that S100B tetramer binds two RAGE molecules via the V-domain. In line with these experiments, S100B tetramer caused stronger activation of cell growth than S100B dimer and promoted cell survival. The structural and the binding data suggest that tetrameric S100B triggers RAGE activation by receptor dimerisation.

Hypochlorite-modified albumin colocalizes with RAGE in the artery wall and promotes MCP-1 expression via the RAGE-Erk1/2 MAP-kinase pathway.

Signal transduction via the endothelial receptor for advanced glycation end products (RAGE) plays a key role in vascular inflammation. Recent observations have shown that the myeloperoxidase-H2O2-chloride system of activated phagocytes is highly up-regulated under inflammatory conditions where hypochlorous acid (HOCl) is formed as the major oxidant. Albumin, an in vivo carrier for myeloperoxidase is highly vulnerable to oxidation and a major representative of circulating advanced oxidized proteins during inflammatory diseases. Immunohistochemical studies performed in the present study revealed marked colocalization of HOCl-modified epitopes with RAGE and albumin in sections of human atheroma, mainly at the endothelial lining. We show that albumin modified with physiologically relevant concentrations of HOCl, added as reagent or generated by the myeloperoxidase-H2O2-chloride system, is a high affinity ligand for RAGE. Albumin, modified by HOCl in the absence of free amino acids/carbohydrates/lipids to exclude formation of AGE-like structures, induced a rapid, RAGE-dependent activation of extracellular signal-regulated kinase 1/2 and up-regulation of the proinflammatory mediator monocyte chemoattractant protein-1. Cellular activation could be blocked either by a specific polyclonal anti-RAGE IgG and/or a specific mitogen-activated protein-kinase kinase inhibitor. The present study demonstrates that HOCl-modified albumin acts as a ligand for RAGE and promotes RAGE-mediated inflammatory complications.

Proliferative stimulus of lung fibroblasts on lung cancer cells is impaired by the receptor for advanced glycation end-products.

The receptor for advanced glycation end-products (RAGE) is highly expressed in lung tissue, especially at the site of the alveolar epithelium, but its expression is reduced in lung carcinomas. Because epithelial-mesenchymal interactions are suggested to contribute to cancer progression, we investigated the RAGE-dependent impact of fibroblasts on tumor cell growth. Cocultivation of human lung cancer cells (H358) with lung fibroblasts (WI-38) improved their proliferation in monolayer and spheroid culture models, the number of H358 cells in the S/G2 cell cycle phase increased, and there was less spontaneous cell death. Overexpression of full-length human RAGE reduced the proliferative stimulus of fibroblasts as seen in monolayers (cell number, cell cycle), spheroid cultures (spheroid size), and in a colony-forming assay compared with mock-transfected cells. Comparable results were observed by culturing H358 cells with and without RAGE overexpression in the presence of conditioned medium taken from WI-38 cells, or in response to selected growth factors, such as basic fibroblast growth factor. Moreover, we clearly showed that the fibroblast-induced proliferation correlates with activation of the p42/44 mitogen-activated protein kinase, but not with Akt kinase activation. On the basis of lung cancer as an age-related disease, we additionally proved the impact of senescent WI-38 fibroblasts. Here, we show that senescent fibroblasts improve H358 cell proliferation to the same extent as do presenescent fibroblasts. From our data, we conclude that re-expression of RAGE in lung cancer cells impairs the proliferative stimulus mediated by fibroblasts. Therefore, lung cancer progression may be enhanced by the RAGE downregulation in human lung carcinomas.

Promotion of cell adherence and spreading: a novel function of RAGE, the highly selective differentiation marker of human alveolar epithelial type I cells.

The receptor for advanced glycation endproducts (RAGE) is expressed under pathological conditions in many tissues and has been assigned many functions. We demonstrate, in normal human lung tissue, the preferential and highly abundant expression of RAGE by quantitative polymerase chain reaction. In addition, RAGE expression, as a specific differentiation marker of alveolar epithelial type I cells (AT I cells), and its localization to the basolateral plasma membrane have been confirmed by means of newly raised monoclonal antibodies. The physiological function of RAGE on AT I cells has previously remained elusive. By using HEK293 cells transfected with cDNA encoding for full-length RAGE, we show that RAGE enhances the adherence of epithelial cells to collagen-coated surfaces and has a striking capacity for inducing cell spreading. The preferential binding of RAGE to collagen has been confirmed by assaying the binding of soluble RAGE to various substrates. RAGE might thus assist AT I cells to acquire a spreading morphology, thereby ensuring effective gas exchange and alveolar stability.