Inhibition of RAGE signaling through the intracellular delivery of inhibitor peptides by PEI cationization

The receptor for advanced glycation end products (RAGE) is a multi-ligand cell surface receptor and a member of the immunoglobulin superfamily. RAGE is involved in a wide range of inflammatory, degenerative and hyper-proliferative disorders which span over different organs by engaging diverse ligands, including advanced glycation end products, S100 family proteins, high-mobility group protein B1 (HMGB1) and amyloid beta. We previously demonstrated that the cytoplasmic domain of RAGE is phosphorylated upon the binding of ligands, enabling the recruitment of two distinct pairs of adaptor proteins, Toll-interleukin 1 receptor domain-containing adaptor protein (TIRAP) and myeloid differentiation protein 88 (MyD88). This engagement allows the activation of downstream effector molecules, and thereby mediates a wide variety of cellular processes, such as inflammatory responses, apoptotic cell death, migration and cell growth. Therefore, inhibition of the binding of TIRAP to RAGE may abrogate intracellular signaling from ligand-activated RAGE. In the present study, we developed inhibitor peptides for RAGE signaling (RAGE-I) by mimicking the phosphorylatable cytosolic domain of RAGE. RAGE-I was efficiently delivered into the cells by polyethylenimine (PEI) cationization. We demonstrated that RAGE-I specifically bound to TIRAP and abrogated the activation of Cdc42 induced by ligand-activated RAGE. Furthermore, we were able to reduce neuronal cell death induced by an excess amount of S100B and to inhibit the migration and invasion of glioma cells in vitro. Our results indicate that RAGE-I provides a powerful tool for therapeutics to block RAGE-mediated multiple signaling

Receptor for advanced glycation end products and its involvement in inflammatory diseases

The receptor for advanced glycation end products (RAGE) is a transmembrane receptor of the immunoglobulin superfamily, capable of binding a broad repertoire of ligands. RAGE-ligands interaction induces a series of signal transduction cascades and lead to the activation of transcription factor NF- B as well as increased expression of cytokines, chemokines, and adhesion molecules. These effects endow RAGE with the role in the signal transduction from pathogen substrates to cell activation during the onset and perpetuation of inflammation. RAGE signaling and downstream pathways have been implicated in a wide spectrum of inflammatory-related pathologic conditions such as arteriosclerosis, Alzheimer's disease, arthritis, acute respiratory failure, and sepsis. Despite the significant progress in other RAGE studies, the functional importance of the receptor in clinical situations and inflammatory diseases still remains to be fully realized. In this review, we will summarize current understandings and lines of evidence on the molecular mechanisms through which RAGE signaling contributes to the pathogenesis of the aforementioned inflammation-associated conditions

Advanced glycation end-products (AGEs) induce concerted changes in the osteoblastic expression of their receptor RAGE and in the activation of extracellular signal-regulated kinases (ERK)

An increase in the interaction between advanced glycation end-products (AGEs) and their receptor RAGE is believed to contribute to the pathogenesis of chronic complications of Diabetes mellitus, which can include bone alterations such as osteopenia. We have recently found that extracellular AGEs can directly regulate the growth and development of rat osteosarcoma UMR106 cells, and of mouse calvaria-derived MC3T3E1 osteoblasts throughout their successive developmental stages (proliferation, differentiation and mineralisation), possibly by the recognition of AGEs moieties by specific osteoblastic receptors which are present in both cell lines. In the present study we examined the possible expression of RAGE by UMR106 and MC3T3E1 osteoblastic cells, by immunoblot analysis. We also investigated whether short-, medium- or long-term exposure of osteoblasts to extracellular AGEs, could modify their affinity constant and maximal binding for AGEs (by 125I-AGE-BSA binding experiments), their expression of RAGE (by immunoblot analysis) and the activation status of the osteoblastic ERK 1/2 signal transduction mechanism (by immunoblot analysis for ERK and P-ERK). Our results show that both osteoblastic cell lines express readily detectable levels of RAGE. Short-term exposure of phenotypically mature osteoblastic UMR106 cells to AGEs decrease the cellular density of AGE-binding sites while increasing the affinity of these sites for AGEs. This culture condition also dose-dependently increased the expression of RAGE and the activation of ERK. In proliferating MC3T3E1 pre-osteoblasts, 24-72 h exposure to AGEs did not modify expression of RAGE, ERK activation or the cellular density of AGE-binding sites. However, it did change the affinity of these binding sites forAGEs, with both higher- and lower-affinity sites now being apparent. Medium-term ( 1 week) incubation of differentiated MC3T3E1 osteoblasts with AGEs, induced a simultaneous increase in RAGE expression and in the relative amount of P-ERK. Mineralising MC3T3E1 cultures grown for 3 weeks in the presence of extracellular AGEs showed a decrease both in RAGE and P-ERK expression. These results indicate that, in phenotypically mature osteoblastic cells, changes in ERK activation closely follow the AGEs-induced regulation of RAGE expression. Thus, the AGEs-induced biological effects that we have observed previously in osteoblasts, could be mediated by RAGE in the later stages of development, and mediated by other AGE receptors in the earlier pre-osteoblastic stage

The galectin-3/RAGE dyad modulates vascular osteogenesis in atherosclerosis

Vascular calcification correlates with inflammation and plaque instability in a dual manner, depending on the spotty/granular (micro) or sheet-like/lamellated (macro) pattern of calcification. Modified lipoproteins trigger both inflammation and calcification via receptors for advanced lipoxidation/glycation endproducts (ALEs/AGEs). This study compared the roles of galectin-3 and receptor for AGEs (RAGE), two ALEs/AGEs-receptors with diverging effects on inflammation and bone metabolism, in the process of vascular calcification. We evaluated galectin-3 and RAGE expression/localization in 62 human carotid plaques and its relation to calcification pattern, plaque phenotype, and markers of inflammation and vascular osteogenesis; and the effect of galectin-3 ablation and/or exposure to an ALE/AGE on vascular smooth muscle cell (VSMC) osteogenic differentiation. While RAGE co-localized with inflammatory cells in unstable regions with microcalcification, galectin-3 was expressed also by VSMCs, especially in macrocalcified areas, where it co-localized with alkaline phosphatase. Expression of galectin-3 and osteogenic markers was higher in macrocalcified plaques, whereas the opposite occurred for RAGE and inflammatory markers. Galectin-3-deficient VSMCs exhibited defective osteogenic differentiation, as shown by altered expression of osteogenic transcription factors and proteins, blunted activation of pro-osteoblastogenic Wnt/β-catenin signalling and proliferation, enhanced apoptosis, and disorganized mineralization. These abnormalities were associated with RAGE up-regulation, but were only in part prevented by RAGE silencing, and were partially mimicked or exacerbated by treatment with an AGE/ALE. These data indicate a novel molecular mechanism by which galectin-3 and RAGE modulate in divergent ways, not only inflammation, but also vascular osteogenesis, by modulating Wnt/β-catenin signalling, and independently of ALEs/AGEs

Targeting RAGE prevents muscle wasting and prolongs survival in cancer cachexia

Background: Cachexia, a multifactorial syndrome affecting more than 50% of patients with advanced cancer and responsible for ~20% of cancer-associated deaths, is still a poorly understood process without a standard cure available. Skeletal muscle atrophy caused by systemic inflammation is a major clinical feature of cachexia, leading to weight loss, dampening patients' quality of life, and reducing patients' response to anticancer therapy. RAGE (receptor for advanced glycation end-products) is a multiligand receptor of the immunoglobulin superfamily and a mediator of muscle regeneration, inflammation, and cancer. Methods: By using murine models consisting in the injection of colon 26 murine adenocarcinoma (C26-ADK) or Lewis lung carcinoma (LLC) cells in BALB/c and C57BL/6 or Ager−/− (RAGE-null) mice, respectively, we investigated the involvement of RAGE signalling in the main features of cancer cachexia, including the inflammatory state. In vitro experiments were performed using myotubes derived from C2C12 myoblasts or primary myoblasts isolated from C57BL/6 wild type and Ager−/− mice treated with the RAGE ligand, S100B (S100 calcium-binding protein B), TNF (tumor necrosis factor)α±IFN (interferon) γ, and tumour cell- or masses-conditioned media to analyse hallmarks of muscle atrophy. Finally, muscles of wild type and Ager−/− mice were injected with TNFα/IFNγ or S100B in a tumour-free environment. Results: We demonstrate that RAGE is determinant to activate signalling pathways leading to muscle protein degradation in the presence of proinflammatory cytokines and/or tumour-derived cachexia-inducing factors. We identify the RAGE ligand, S100B, as a novel factor able to induce muscle atrophy per se via a p38 MAPK (p38 mitogen-activated protein kinase)/myogenin axis and STAT3 (signal transducer and activator of transcription 3)-dependent MyoD (myoblast determination protein 1) degradation. Lastly, we found that in cancer conditions, an increase in serum levels of tumour-derived S100B and HMGB1 (high mobility group box 1) occurs leading to chronic activation/overexpression of RAGE, which induces hallmarks of cancer cachexia (i.e. muscle wasting, systemic inflammation, and release of tumour-derived pro-cachectic factors). Absence of RAGE in mice translates into reduced serum levels of cachexia-inducing factors, delayed loss of muscle mass and strength, reduced tumour progression, and increased survival. Conclusions: RAGE is a molecular determinant in inducing the hallmarks of cancer cachexia, and molecular targeting of RAGE might represent a therapeutic strategy to prevent or counteract the cachectic syndrome

Sensory neuronal sensitisation occurs through HMGB-1/RAGE and TRPV1 in high glucose conditions

Many potential causes for painful diabetic neuropathy have been proposed including actions of cytokines and growth factors. High mobility group protein B1 (HMGB1) is a RAGE agonist, increased in diabetes, that contributes to pain by modulating peripheral inflammatory responses. HMGB1 enhances nociceptive behaviour in naïve animals through an unknown mechanism. We tested the hypothesis that HMGB1 causes pain through direct neuronal activation of RAGE and alteration of nociceptive neuronal responsiveness. HMGB1 and RAGE expression were increased in skin and primary sensory (DRG) neurons of diabetic rats at times when pain behaviour was enhanced. Agonist-evoked TRPV1-mediated calcium responses increased in cultured DRG neurons from diabetic rats and in neurons from naïve rats exposed to high glucose concentrations. HMGB1-mediated increases in TRPV1-evoked calcium responses in DRG neurons were RAGE and PKC-dependent, and this was blocked by co-administration of the growth factor splice variant, VEGF-A165b. Pain behaviour and DRG RAGE expression increases were blocked by VEGF-A 165 b treatment of diabetic rats in vivo. HMGB-1-RAGE activation sensitizes DRG neurons in vitro. VEGF-A165b blocks HMGB-1/RAGE DRG activation, which may contribute to its analgesic properties in vivo

Glycation End Products (RAGE) and Promotes Proliferation in ECV304 Cells via the c-Jun N-Terminal Kinases (JNK) Pathway Following Stimulation by Advanced Glycation End-Products In Vitro

Abstract: Hyperoside is a major active constituent in many medicinal plants which are traditionally used in Chinese medicines for their neuroprotective, anti-inflammatory and antioxidative effects. The molecular mechanisms underlying these effects are unknown. In this study, quiescent ECV304 cells were treated in vitro with advanced glycation end products (AGEs) in the presence or absence of hyperoside. The results demonstrated that AGEs induced c-Jun N-terminal kinases (JNK) activation and apoptosis in ECV304 cells. Hyperoside inhibited these effects and promoted ECV304 cell proliferation. Furthermore, hyperoside significantly inhibited RAGE expression in AGE-stimulated ECV304 cells, whereas knockdown of RAGE inhibited AGE-induced JNK activation. These results suggested that AGEs may promote JNK activation, leading to viability inhibition of ECV304 cells via the RAGE signaling pathway. These effects could be inhibited by hyperoside. Our findings suggest a novel role for hyperoside in the treatment and prevention of diabetes

S100B is increased in Parkinson’s disease and ablation protects against MPTP-induced toxicity through the RAGE and TNF-α pathway

Peer reviewedPublisher PD

RAGE Signaling in Skeletal Biology

PURPOSE OF REVIEW: The receptor for advanced glycation end products (RAGE) and several of its ligands have been implicated in the onset and progression of pathologies associated with aging, chronic inflammation, and cellular stress. In particular, the role of RAGE and its ligands in bone tissue during both physiological and pathological conditions has been investigated. However, the extent to which RAGE signaling regulates bone homeostasis and disease onset remains unclear. Further, RAGE effects in the different bone cells and whether these effects are cell-type specific is unknown. The objective of the current review is to describe the literature over RAGE signaling in skeletal biology as well as discuss the clinical potential of RAGE as a diagnostic and/or therapeutic target in bone disease. RECENT FINDINGS: The role of RAGE and its ligands during skeletal homeostasis, tissue repair, and disease onset/progression is beginning to be uncovered. For example, detrimental effects of the RAGE ligands, advanced glycation end products (AGEs), have been identified for osteoblast viability/activity, while others have observed that low level AGE exposure stimulates osteoblast autophagy, which subsequently promotes viability and function. Similar findings have been reported with HMGB1, another RAGE ligand, in which high levels of the ligand are associated with osteoblast/osteocyte apoptosis, whereas low level/short-term administration stimulates osteoblast differentiation/bone formation and promotes fracture healing. Additionally, elevated levels of several RAGE ligands (AGEs, HMGB1, S100 proteins) induce osteoblast/osteocyte apoptosis and stimulate cytokine production, which is associated with increased osteoclast differentiation/activity. Conversely, direct RAGE-ligand exposure in osteoclasts may have inhibitory effects. These observations support a conclusion that elevated bone resorption observed in conditions of high circulating ligands and RAGE expression are due to actions on osteoblasts/osteocytes rather than direct actions on osteoclasts, although additional work is required to substantiate the observations. Recent studies have demonstrated that RAGE and its ligands play an important physiological role in the regulation of skeletal development, homeostasis, and repair/regeneration. Conversely, elevated levels of RAGE and its ligands are clearly related with various diseases associated with increased bone loss and fragility. However, despite the recent advancements in the field, many questions regarding RAGE and its ligands in skeletal biology remain unanswered

Identification of a functional interaction of HMGB1 with Receptor for Advanced Glycation End-products in a model of neuropathic pain

Recent studies indicate that the release of high mobility group box 1 (HMGB1) following nerve injury may play a central role in the pathogenesis of neuropathic pain. HMGB1 is known to influence cellular responses within the nervous system via two distinct receptor families; the Receptor for Advanced Glycation End-products (RAGE) and Toll-like receptors (TLRs). The degree to which HMGB1 activates a receptor is thought to be dependent upon the oxidative state of the ligand, resulting in the functional isoforms of all-thiol HMGB1 (at-HMGB1) acting through RAGE, and disufide HMGB1 (ds-HMGB1) interacting with TLR4. Though it is known that dorsal root ganglia (DRG) sensory neurons exposed to HMGB1 and TLR4 agonists can influence excitation, the degree to which at-HMGB1 signaling through neuronal RAGE contributes to neuropathic pain is unknown. Here we demonstrate that at-HMGB1 activation of nociceptive neurons is dependent on RAGE and not TLR4. To distinguish the possible role of RAGE on neuropathic pain, we characterized the changes in RAGE mRNA expression up to one month after tibial nerve injury (TNI). RAGE mRNA expression in lumbar dorsal root ganglion (DRG) is substantially increased by post-injury day (PID) 28 when compared with sham injured rodents. Protein expression at PID28 confirms this injury-induced event in the DRG. Moreover, a single exposure to monoclonal antibody to RAGE (RAGE Ab) failed to abrogate pain behavior at PID 7, 14 and 21. However, RAGE Ab administration produced reversal of mechanical hyperalgesia on PID28. Thus, at-HMGB1 activation through RAGE may be responsible for sensory neuron sensitization and mechanical hyperalgesia associated with chronic neuropathic pain states