Diaphanous-1 affects the nanoscale clustering and lateral diffusion of receptor for advanced glycation endproducts (RAGE)

The interactions between the cytoplasmic protein diaphanous-1 (Diaph1) and the receptor for advanced glycation endproducts (RAGE) drive the negative consequences of RAGE signaling in several disease processes. Reported in this work is how Diaph1 affects the nanoscale clustering and diffusion of RAGE measured using super-resolution stochastic optical reconstruction microscopy (STORM) and single particle tracking (SPT). Altering the Diaph1 binding site has a different impact on RAGE diffusion compared to when Diaph1 expression is reduced in HEK293 cells. In cells with reduced Diaph1 expression (RAGE-Diaph1^?/?), the average RAGE diffusion coefficient is increased by 35%. RAGE diffusion is known to be influenced by the dynamics of the actin cytoskeleton. Actin labeling shows that a reduced Diaph1 expression leads to cells with reduced filopodia density and length. In contrast, when two RAGE amino acids that interact with Diaph1 are mutated (RAGE_RQ/AA), the average RAGE diffusion coefficient is decreased by 16%. Since RAGE diffusion is slowed when the interaction between Diaph1 and RAGE is disrupted, the interaction of the two proteins results in faster RAGE diffusion. In both RAGE_RQ/AA and RAGE-Diaph1^?/? cells the number and size of RAGE clusters are decreased compared to cells expressing RAGE and native concentrations of Diaph1. This work shows that Diaph1 has a role in affecting RAGE clusters and diffusion.     

Cellular signalling of the receptor for advanced glycation end products (RAGE)

The receptor for advanced glycation end-product (RAGE) is the signal transduction receptor which senses a variety of signalling molecules including advanced glycation end products (AGEs), HMGB1, S100/calgranulins, β-amyloid, phosphatidylserine, C3a and advanced oxidation protein products (AOPPs). It is usually abnormally up-regulated and plays crucial roles during the development of many human diseases such as diabetes, cardiovascular diseases, osteoarthritis and cancer. RAGE regulates a number of cell processes of pivotal importance like inflammation, apoptosis, proliferation and autophagy. Therapeutic strategies to block RAGE may represent great therapeutic potentials and therefore it has been under extensive investigation during the last decade. Accordingly, there is a growing interest of unraveling the intracellular signalling pathways by which RAGE controls these disease-related processes. Early studies are mainly focused on inflammatory pathways involving the NFκB and the MAPK pathways. Nevertheless, many novel signalling pathways implicated in other cell processes, such as autophagy, have also recently been found to be activated upon RAGE stimulation and contribute to the detrimental effects of RAGE. In this review, we aim to provide a comprehensive summary of previous and recent studies relating to the complex molecular network of RAGE signalling, with a particular emphasis on RAGE transgenic mouse models.     

Structural basis for pattern recognition by the receptor for advanced glycation end products (RAGE)

The receptor for advanced glycated end products (RAGE) is a multiligand receptor that is implicated in the pathogenesis of various diseases, including diabetic complications, neurodegenerative disorders, and inflammatory responses. The ability of RAGE to recognize advanced glycated end products (AGEs) formed by nonenzymatic glycoxidation of cellular proteins places RAGE in the category of pattern recognition receptors. The structural mechanism of AGE recognition was an enigma due to the diversity of chemical structures found in AGE-modified proteins. Here, using NMR spectroscopy we showed that the immunoglobulin V-type domain of RAGE is responsible for recognizing various classes of AGEs. Three distinct surfaces of the V domain were identified to mediate AGE-V domain interactions. They are located in the positively charged areas of the V domain. The first interaction surface consists of strand C and loop CC ', the second interaction surface consists of strand C ', strand F, and loop FG, and the third interaction surface consists of strand A ' and loop EF. The secondary structure elements of the interaction surfaces exhibit significant flexibility on the ms-micros time scale. Despite highly specific AGE-V domain interactions, the binding affinity of AGEs for an isolated V domain is low, approximately 10 microm. Using in-cell fluorescence resonance energy transfer we show that RAGE is a constitutive oligomer on the plasma membrane. We propose that constitutive oligomerization of RAGE is responsible for recognizing patterns of AGE-modified proteins with affinities less than 100 nm.     

p38 inhibitor SB203580 sensitizes the resveratrol-induced apoptosis in human lung adenocarcinoma (A549) cells

Based on our recent findings that resveratrol, a natural plant polyphenol found in red grape skins as well as other food products, induces apoptosis via a caspase-independent intrinsic pathway in human lung adenocarcinoma cells, this study is designed to explore whether SB203580, a p38 inhibitor, potentiates the resveratrol-induced apoptosis of human lung adenocarcinoma (A549) cells. We found that pretreatment with SB203580 enhanced the resveratrol-induced apoptosis by accelerating the intrinsic apoptotic pathway including Bax activation, loss of mitochondrial membrane potential, and activation of both caspase-9 and -3. Although treatment with resveratrol alone did not induce caspase-8 activation, cotreatment with both SB203580 and resveratrol not only enhanced FasL cleavage but also activated caspase-8, indicating that the extrinsic apoptotic pathway may be involved in the synergistic effect. Collectively, we for the first time demonstrate that SB203580 synergistically enhances the resveratrol-induced apoptosis by accelerating Bax-mediated intrinsic pathway and initiating extrinsic pathway, suggesting a possible alternative therapeutic strategy for human lung cancer.     

One-way cross-talk between p38(MAPK) and p42/44(MAPK). Inhibition of p38(MAPK) induces low density lipoprotein receptor expression through activation of the p42/44(MAPK) cascade.

In this paper, we report that SB202190 alone, a specific inhibitor of p38(MAPK), induces low density lipoprotein (LDL) receptor expression (6-8-fold) in a sterol-sensitive manner in HepG2 cells. Consistent with this finding, selective activation of the p38(MAPK) signaling pathway by expression of MKK6b(E), a constitutive activator of p38(MAPK), significantly reduced LDL receptor promoter activity. Expression of the p38(MAPK) alpha-isoform had a similar effect, whereas expression of the p38(MAPK) betaII-isoform had no significant effect on LDL receptor promoter activity. SB202190-dependent increase in LDL receptor expression was accompanied by induction of p42/44(MAPK), and inhibition of this pathway completely prevented SB202190-induced LDL receptor expression, suggesting that p38(MAPK) negatively regulates the p42/44(MAPK) cascade and the responses mediated by this kinase. Cross-talk between these kinases appears to be one-way because modulation of p42/44(MAPK) activity did not affect p38(MAPK) activation by a variety of stress inducers. Taken together, these findings reveal a hitherto unrecognized one-way communication that exists between p38(MAPK) and p42/44(MAPK) and provide the first evidence that through the p42/44(MAPK) signaling cascade, the p38(MAPK) alpha-isoform negatively regulates LDL receptor expression, thus representing a novel mechanism of fine tuning cellular levels of cholesterol in response to a diverse set of environmental cues.     

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.     

Developmental expression of the receptor for advanced glycation end-products (RAGE) and its response to hyperoxia in the neonatal rat lung

Background  

The receptor for advanced glycation end products (mRAGE) is associated with pathology in most tissues, while its soluble form (sRAGE) acts as a decoy receptor. The adult lung is unique in that it expresses high amounts of RAGE under normal conditions while other tissues express low amounts normally and up-regulate RAGE during pathologic processes. We sought to determine the regulation of the soluble and membrane isoforms of RAGE in the developing lung, and its expression under hyperoxic conditions in the neonatal lung.     

The receptor for advanced glycation end-products (RAGE) directly binds to ERK by a D-domain-like docking site

The receptor for advanced glycation end-products (RAGE)-mediated cellular activation through the mitogen-activated protein kinase (MAPK) cascade, activation of NF-κB and Rho family small G-proteins, cdc42/Rac, is implicated in the pathogenesis of inflammatory disorders and tumor growth/metastasis. However, the precise molecular mechanisms for the initiation of cell signaling by RAGE remain to be elucidated. In this study, proteins which directly bind to the cytoplasmic C-terminus of RAGE were purified from rat lung extracts using an affinity chromatography technique and identified to be extracellular signal-regulated protein kinase-1 and -2 (ERK-1/2). Their interactions were confirmed by immunoprecipitation of ERK-1/2 from RAGE-expressing HT1080 cell extracts with anti-RAGE antibody. Furthermore, the augmentation of kinase activity of RAGE-bound ERK upon the stimulation of cells with amphoterin was demonstrated by determining the phosphorylation level of myelin basic protein, an ERK substrate. In vitro binding studies using a series of C-terminal deletion mutants of human RAGE revealed the importance of the membrane-proximal cytoplasmic region of RAGE for the direct ERK–RAGE interaction. This region contained a sequence similar to the D-domain, a ERK docking site which is conserved in some ERK substrates including MAPK-interacting kinase-1/2, mitogen- and stress-activated protein kinase-1, and ribosomal S6 kinase. These data suggest that ERK may play a role in RAGE signaling through direct interaction with RAGE.     

Expression, purification and fluorine-18 radiolabeling of recombinant S100 proteins--potential probes for molecular imaging of receptor for advanced glycation endproducts (RAGE) in vivo

Data concerning the pathophysiological role of the interaction of circulating S100 proteins, a multigenic family of Ca(2+)-modulated proteins, with the receptor for advanced glycation endproducts (RAGE) in cardiovascular diseases, inflammatory processes, and tumorigenesis in vivo are scarce. One reason is the shortage of suitable radiotracer methods. We report a novel methodology using recombinant human S100A1, S100B, and S100A12 as potential probes for molecular imaging of this interaction. Therefore, human S100 proteins were cloned as GST fusion proteins in the bacterial expression vector pGEX-6P-1 and expressed in E. coli strain BL21. Purified recombinant human S100 proteins were radiolabeled with the positron emitter fluorine-18 ((18)F) by conjugation with N-succinimidyl-4-[(18)F]fluorobenzoate ([(18)F]SFB). The radiolabeled recombinant S100 proteins ((18)F-S100) were used in biodistribution experiments and small animal positron emission tomography (PET) studies in rats. The tissue-specific distribution of (18)F-S100 proteins in vivo correlated well with the anatomical localization of RAGE, e.g., in lungs and in the vascular system. These findings indicate circulating S100A1, S100B, and S100A12 proteins to be ligands for RAGE in rats in vivo. The approach allows the use of small animal PET and provides novel probes to delineate functional expression of RAGE under normal and pathophysiological conditions in rodent models of disease.     

Glypican-3 (GPC3) inhibits metastasis development promoting dormancy in breast cancer cells by p38 MAPK pathway activation

GPC3 is a proteoglycan involved in the control of proliferation and survival, which has been linked to several tumor types. In this respect, we previously demonstrated that normal breast tissues exhibit high levels of GPC3, while its expression is diminished in tumors. However, the role of the GPC3 downregulation in breast cancer progression and its molecular and cellular operational machineries are not fully understood.In this study we showed that GPC3 reverts the epithelial-to-mesenchymal transition (EMT) underwent by mammary tumor cells, blocks metastatic spread and induces dormancy at secondary site. Using genetically modified murine breast cancer cell sublines, we demonstrated that the phospho-Erk/phospho-p38 ratio is lower in GPC3 reexpressing cells, while p21, p27 and SOX2 levels are higher, suggesting a dormant phenotype. In vivo metastasis assays confirmed that GPC3 reexpressing cells reduce their metastatic ability. Interestingly, the presence of dormant cells was evidenced in the lungs of inoculated mice. Dormant cells could reactivate their proliferative capacity, remain viable as well as tumorigenic, but they reentered in dormancy upon reaching secondary site. We also proved that GPC3 inhibits metastasis through p38 pathway activation. The in vivo inhibition of p38 induced an increase in cell invasion of GPC3 reexpressing orthotropic tumors as well as in spontaneous and experimental metastatic dissemination.In conclusion, our study shows that GPC3 returns mesenchymal-like breast cancer cells to an epithelial phenotype, impairs in vivo metastasis and induces tumor dormancy through p38 MAPK signaling activation. These results help to identify genetic determinants of dormancy and suggest the translational potential of research focusing in GPC3.     

S100 protein translocation in response to extracellular S100 is mediated by receptor for advanced glycation endproducts in human endothelial cells

The extracellular functions of S100 proteins have attracted more attention in recent years. S100 proteins are a group of calcium-binding proteins which exhibit cell- and tissue-specific expression, and different expression levels of members from this family have been observed in various pathological conditions. The reported extracellular functions of S100 proteins include the ability to enhance neurite outgrowth, involvement in inflammation, and motility of tumour cells. In our previous study, we reported translocation of S100A13 in response to the elevated intracellular calcium levels induced by angiotensin II. In order to investigate potential effects of extracellular S100A13, recombinant S100A13 was used here to stimulate human endothelial cells. Addition of extracellular S100A13 to the cells resulted in both endogenous protein translocation and protein uptake from the extracellular space. To test specificity of this effect, addition of various other S100 proteins was also performed. Interestingly, translocation of specific S100 proteins was only observed when the cells were stimulated with the same extracellular S100 protein. Since the receptor for advanced glycation end products (RAGE) is a putative cell surface receptor for S100 proteins and is involved in various signal transduction pathways, we next investigated the interaction between the receptor and extracellular S100 proteins. We show here that NF-kappaB which is a downstream regulator in RAGE-mediated transduction pathways can be activated by addition of extracellular S100 proteins, and translocation of S100 proteins was inhibited by soluble RAGE. These experiments suggest a common cell surface receptor for S100 proteins on endothelial cells even though intracellular translocation induced by extracellular S100 proteins is specific.