RAGE Up-Regulation Differently Affects Cell Proliferation and Migration in Pancreatic Cancer Cells.

The receptor for advanced glycation end products (RAGE) contributes to many cellular aspects of pancreatic cancer including cell proliferation, migration, and survival. Studies have shown that RAGE activation by its ligands promotes pancreatic tumor growth by stimulating both cell proliferation and migration. In this study, we investigated the effect of RAGE up-regulation on the proliferation and migration of the human pancreatic cancer Panc-1 cell-line. We show that moderate overexpression of RAGE in Panc-1 cells results in increased cell proliferation, but decreased cell migration. The observed cellular changes were confirmed to be RAGE-specific and reversible by using RAGE-specific siRNAs and the small molecule RAGE inhibitor FPS-ZM1. At the molecular level, we show that RAGE up-regulation was associated with decreased activity of FAK, Akt, Erk1/2, and NF-κB signaling pathways and greatly reduced levels of α2 and ß1 integrin expression, which is in agreement with the observed decreases in cell migration. We also demonstrate that RAGE up-regulation changes the expression of key molecular markers of epithelial-to-mesenchymal transition (EMT). Our results suggest that in the absence of stimulation by external ligands, RAGE up-regulation can differently modulate cell proliferation and migration in pancreatic cancer cells and regulates partly EMT.

The Trp triad within the V-domain of the receptor for advanced glycation end products modulates folding, stability and ligand binding.

The receptor for advanced glycation end products (RAGE) recognizes damage-associated molecular patterns (DAMPs) and plays a critical role for the innate immune response and sterile tissue inflammation. RAGE overexpression is associated with diabetic complications, neurodegenerative diseases and certain cancers. Yet, the molecular mechanism of ligand recognition by RAGE is insufficiently understood to rationalize the binding of diverse ligands. The N-terminal V-type Ig-domain of RAGE contains a triad of tryptophan residue; Trp51, Trp61 and Trp72. The role of these three Trp residues for domain folding, stability and binding of the RAGE ligand S100B was investigated through site-directed mutagenesis, UV/VIS, CD and fluorescence spectrometry, protein-protein interaction studies, and X-ray crystallography. The data show that the Trp triad stabilizes the folded V-domain by maintaining a short helix in the structure. Mutation of any Trp residue increases the structural plasticity of the domain. Residues Trp61 and Trp72 are involved in the binding of S100B, yet they are not strictly required for S100B binding. The crystal structure of the RAGE-derived peptide W72 in complex with S100B showed that Trp72 is deeply buried in a hydrophobic depression on the S100B surface. The studies suggest that multiple binding modes between RAGE and S100B exist and point toward a not previously recognized role of the Trp residues for RAGE-ligand binding. The Trp triad of the V-domain appears to be a suitable target for novel RAGE inhibitors, either in the form of monoclonal antibodies targeting this epitope, or small organic molecules.

Structural insights into the binding of the human receptor for advanced glycation end products (RAGE) by S100B, as revealed by an S100B-RAGE-derived peptide complex.

S100B is a damage-associated molecular pattern protein that, when released into the extracellular milieu, triggers initiation of the inflammatory response through the receptor for advanced glycation end products (RAGE). Recognition of S100B is accomplished via the amino-terminal variable immunoglobulin domain (V-domain) of RAGE. To gain insights into this interaction, a complex between S100B and a 15-amino-acid peptide derived from residues 54-68 of the V-domain was crystallized. The X-ray crystal structure was solved to 2.55 Å resolution. There are two dimers of S100B and one peptide in the asymmetric unit. The binding interface of this peptide is compared with that found in the complex between S100B and the 12-amino-acid CapZ-derived peptide TRTK-12. This comparison reveals that although the peptides adopt completely different backbone structures, the residues buried at the interface interact with S100B in similar regions to form stable complexes. The binding affinities of S100B for the intact wild-type V-domain and a W61A V-domain mutant were determined to be 2.7 ± 0.5 and 1.3 ± 0.7 µM, respectively, using fluorescence titration experiments. These observations lead to a model whereby conformational flexibility in the RAGE receptor allows the adoption of a binding conformation for interaction with the stable hydrophobic groove on the surface of S100B.

The receptor for advanced glycation end products influences the expression of its S100 protein ligands in melanoma tumors.

Recent studies have suggested that the receptor for advanced glycation end products (RAGE) participates in melanoma progression by promoting tumor growth. However, the mechanisms of RAGE activation in melanoma tumors are not clearly understood. To get deeper insights into these mechanisms, we transfected a melanoma cell line, which was established from a human melanoma primary tumor, with RAGE, and studied the effect of RAGE overexpression on cell proliferation and migration in vitro. We observed that overexpression of RAGE in these cells not only resulted in significantly increased migration rates compared to control cells, but also in decreased proliferation rates (Meghnani et al., 2014). In the present study, we compared the growth of xenograft tumors established from RAGE overexpressing WM115 cells, to that of control cells. We observed that when implanted in mice, RAGE overexpressing cells generated tumors faster than control cells. Analysis of protein tumor extracts showed increased levels of the RAGE ligands S100B, S100A2, S100A4, S100A6 and S100A10 in RAGE overexpressing tumors compared to control tumors. We show that the tumor growth was significantly reduced when the mice were treated with anti-RAGE antibodies, suggesting that RAGE, and probably several S100 proteins, were involved in tumor growth. We further demonstrate that the anti-RAGE antibody treatment significantly enhanced the efficacy of the alkylating drug dacarbazine in reducing the growth rate of RAGE overexpressing tumors.

Calorimetric investigation of diclofenac drug binding to a panel of moderately glycated serum albumins.

Glycation alters the drug binding properties of serum proteins and could affect free drug concentrations in diabetic patients with elevated glycation levels. We investigated the effect of bovine serum albumin glycation by eight physiologically relevant glycation reagents (glucose, ribose, carboxymethyllysine, acetoin, methylglyoxal, glyceraldehyde, diacetyl and glycolaldehyde) on diclofenac drug binding. We used this non-steroidal anti-inflammatory drug diclofenac as a paradigm for acidic drugs with high serum binding and because of its potential cardiovascular risks in diabetic patients. Isothermal titration calorimetry showed that glycation reduced the binding affinity Ka of serum albumin and diclofenac 2 to 6-fold by reducing structural rigidity of albumin. Glycation affected the number of drug binding sites in a glycation reagent dependent manner and lead to a 25% decrease for most reagent, expect for ribose, with decreased by 60% and for the CML-modification, increased the number of binding sites by 60%. Using isothermal titration calorimetry and differential scanning calorimetry we derived the complete thermodynamic characterization of diclofenac binding to all glycated BSA samples. Our results suggest that glycation in diabetic patients could significantly alter the pharmacokinetics of the widely used over-the-counter NSDAI drug diclofenac and with possibly negative implications for patients.

Interaction between glycated serum albumin and AGE-receptors depends on structural changes and the glycation reagent.

Physiologically relevant reactive carbonyl compounds vary greatly in their glycation reactivity and the resulting advanced glycation endproducts (AGE) are likely to have distinct structural and biological properties. We characterized a panel of twenty AGE-BSA preparations in terms of (i) their biophysical properties, (ii) their binding to the receptor for advanced glycation endproducts (RAGE) and galectin-3, and (iii) their effects on cellular proliferation. We could establish correlations between lysine glycation and changes in secondary structure. Circular dichroism and differential scanning calorimetry experiments showed that glycation causes albumin to adopt folding properties of a molten globule. Binding studies between AGE-albumin and RAGE or galectin-3 indicate that binding to the isolated receptor domains was weak. Only AGE compounds derived by glycation with ribose were able to bind tightly (K(d) < 10 µM) to both AGE receptors. Cell based assays using an engineered melanoma cell line demonstrated correlations between the extent of (i) lysine side chain modification, (ii) ß-sheet content and (iii) albumin multimerization with stimulation of cell proliferation. However, in addition to structural properties of the protein, the chemical structures of the AGE-modifications were important for receptor binding and biological activity as well.

Moderate glycation of serum albumin affects folding, stability, and ligand binding.

BACKGROUND: Serum protein glycation and formation of advanced glycation endproducts (AGE) correlates with diabetic complications. Highly AGE-modified albumin is frequently used to study the biochemical and cellular activities of AGE-proteins. However, moderately modified albumin samples are expected to be (patho)physiologically more relevant for diabetes research. We produced a panel of nine moderately modified albumin samples and characterized these in terms of side chain modifications, secondary structure, folding stability, and spectroscopic properties. METHODS: A panel of nine albumin samples modified with glucose, methylglyoxal, glyoxylic acid and carboxymethyl lysine was characterized in terms of side chain modifications, thermal folding stability, secondary structure, aggregation, surface charge, and ligand binding. The analytical tools employed included chemical analysis, biochemical and immunochemical assays for side chain modifications, near UV circular dichroism, differential scanning calorimetry, analytical size exclusion and ion exchange HPLC, and a ligand binding assay. CONCLUSION: Moderate glycation and AGE modification of serum albumin causes structural changes that depend on the chemical reactivity of the modifying reagent and the concentration used for in-vitro glycation. In general, the α-helical content is decreased and thermal unfolding behavior is altered. However, moderate glycation does not cause aggregation or formation of amyloid structures as previous reported for highly modified albumin. A structural characterization of in vitro produced AGE-proteins will be useful to correctly interpret the pathophysiological significance of AGE products in diabetes.