Full-length RAGE Research Articles

Human intestine and placenta exhibit tissue-specific expression of RAGE isoforms

The receptor for advanced glycation end products (RAGE) is encoded by AGER, a gene that is subjected to tissue-specific alternative splicing. Splice variants of RAGE in intestine and placenta are unknown and contradictory data concerning RAGE protein expression in these tissues have been published. As a basis for future functional studies, we examined RAGE expression in small intestine, colon and placentas. PCR cloning revealed that full-length RAGE is the only RAGE transcript isoform expressed in placenta. In the small intestine, the major transcript isoform detected was RAGE_v1 encoding the C-terminally truncated soluble receptor. In the colon, both full-length RAGE as well as several splice variants were identified. Four antibodies were used to study protein expression by immunoblotting and were carefully validated. Appropriate controls were essential to avoid misinterpretation of bands caused by non-specific reactivity of antibodies. Only one of four antibodies tested detected full-length RAGE in placenta, whereas no RAGE-specific band was detected in intestinal tissues despite loading >30-fold more intestinal tissue than the positive control, human lung. RAGE expression levels in the placenta were 100-fold lower compared with human lung when analyzed by ELISA, and no significant differences in RAGE expression were detected between healthy placentas and placentas from women with preeclampsia, gestational diabetes mellitus, or fetal growth restriction. We conclude that healthy placental chorionic tissue expresses low levels of full-length RAGE, whereas expression of the tissue-specific intestinal isoforms is below the limit of detection. Low RAGE expression levels in combination with a lack of antibody validation may explain the conflicting published results on RAGE protein expression in intestine and placenta.

The Effect of HMGB1 Protein and Its Truncated Form on the Expression of RAGE Variants in Breast Cancer Cells

HMGB1 protein is a DNA binding nuclear protein. Its properties to bind different non-B DNA conformations and also to bend linear DNA implicate the protein in many essential cellular processes as DNA replication, repair, transcription, remodelling, etc. HMGB1 plays important role outside the cell as it is passively released from necrotic cells and actively from apoptotic ones and binds its specific receptor for advanced glycation end products RAGE. HMGB1/RAGE interactin is implicated in various diseases including cancer. Different soluble RAGE forms were reported whose functional role is to serve as a decoy for the ligands and in this way to block the signalling pathway. How the ligands regulate the production of RAGE variants is a subject of great scientific interest. We studied the effect of HMGB1 and its truncated form lacking the C- terminus on the expression of full-length (flRAGE) and soluble RAGE (sRAGE) in breast cancer cell lines: MCF7 represents a hormone dependent cancer with better prognosis and MDA-MB-231 – hormone independent with substantial invasive capacity. HMGB1 stimulates the total RAGE expression in both breast cancer cells but in MCF7 the ratio changes and is in favour of the membrane fraction. The absence of the C-tail of HMGB1 provokes comparable changes in RAGE production in MCF7 cells as the whole HMGB1 molecule. In MDA-MB- 231cell line the total amount of RAGE was slightly affected but it is entirely represented by the membrane form and the soluble one is in negligible amounts.

The Expression Level of RAGE Forms in Different Cancer Cell Lines

The receptor for advanced glycation end products (RAGE) is a cell surface multiligand receptor of the immunoglobulin superfamily. The interest of the researchers to RAGE was enhanced by the fact that it is expressed at low levels under normal physiology, but is upregulated in various pathological processes such as neuronal development, diabetes, inflammation, neurodegenerative disorders, cancer progression, cardiovascular disease (CVD), etc. The receptor can exist as full-length membrane bound and soluble form and it is considered that the balance between the amount of soluble and full-length RAGE might be a key factor for RAGE induced dysfunction. Different human cancer cell lines with various invasive potential were studied for the expression level of RAGE forms. Our finding shows that in less invasive cancers with better prognosis as MCF7 breast cancer and A549 (NSCLC – non small cell lung cancer) the membrane form of RAGE is the minor one and in A549 is even absent. Cancerous cells with great invasive potential and bad prognosis as MDA-MB-231, H1299 and HeLA expressed predominantly full-length receptor and sRAGE is poorly represented.

A model of full-length RAGE in complex with S100B

The receptor for advanced glycation end products (RAGE) is an immunoglobulin-type multiligand transmembrane protein expressed in numerous cell types, including the central nervous system cells. RAGE interaction with S100B, released during brain tissue damage, leads to RAGE upregulation and initialization of a spiral proinflammatory associated with different neural disorders. Here, we present the structural characterization of the hetero-oligomeric complex of the full-length RAGE with S100B, obtained by a combination of mass spectrometry-based methods and molecular modeling. We predict that RAGE functions as a tightly packed tetramer exposing a positively charged surface formed by V domains for S100B binding. Based on HDX results we demonstrate an allosteric coupling of the distal extracellular V domains and the transmembrane region, indicating a possible mechanism of signal transmission by RAGE across the membrane. Our model provides an insight into RAGE-ligand interactions, providing a basis for the rational design of the therapeutic modifiers of its activity.

Transforming Growth Factor-β1 and Receptor for Advanced Glycation End Products Gene Expression and Protein Levels in Adolescents with Type 1 iabetes Mellitus

Objective:Type 1 diabetes (T1D) mellitus is one of the most frequent autoimmune diseases in childhood. Chronic complications are the main causes of cardiovascular morbidity and mortality in T1D. Although interactions between advanced glycation end products (AGE) and their receptors (RAGE) and transforming growth factor-β1 (TGF-β1) are implicated in development and progression of diabetic microand macro-vascular complications, they also have important roles in immune system regulation.Methods:Blood samples were obtained from 156 adolescents with T1D and 80 apparently healthy controls. T1D patients diagnosed with any other autoimmune disease and receiving any kind of drugs except insulin therapy were excluded from this study. Exclusion criteria for controls were positive family history of T1D and drugs/supplements application. TGF-β1 and transmembrane full-length RAGE (flRAGE) messenger ribonucleic acid (mRNA) levels in peripheral blood mononuclear cells (PBMC) were obtained by quantitative polymerase chain reaction (qPCR) method. Circulating levels of biochemical markers, TGF-β1 and soluble RAGE (sRAGE) levels were also determined.Results:TGF-β1 and flRAGE mRNA levels were significantly higher in controls compared to patients (p<0.001, for both). However, TGF-β1 and sRAGE levels were higher in patients than controls (p<0.001, for both). There were significant independent associations of all mRNA and protein levels with T1D. TGF-β1 mRNA was the only marker independently negatively associated with urinary albumin excretion rate in T1D adolescents (p=0.005).Conclusion:Our results indicated gene expression downregulation of TGF-β1 and flRAGE in PBMC of T1D adolescents. TGF-β1 mRNA downregulation may be useful for predicting early elevation of urinary albumin excretion rate.

Elevated Plasma Level of Soluble Form of RAGE in Ischemic Stroke Patients with Dementia

The receptor for advanced glycation end products (RAGE) and its downstream pathways are involved in various inflammatory and immune responses. Importantly, there is soluble RAGE (sRAGE) that forms either by alternative splicing of RAGE messenger ribonucleic acid as the endogenous soluble form of RAGE (esRAGE) or by proteolytic cleavage of full-length RAGE protein. This study aimed to investigate the associations of the plasma levels of sRAGE and esRAGE in ischemic stroke (IS) patients with and without dementia. This cross-sectional study recruited patients with IS at a university medical center. Vascular dementia was defined as the scale of Clinical Dementia Ranking (CDR) ≥ 1. Standard enzyme-linked immunosorbent assay was used to measure the plasma concentration of sRAGE and esRAGE. From November 2014 to October 2015, a total of 172 IS patients (mean age: 72.1 ± 7.5 years, 64.5% male) were recruited, including 73 with CDR = 0, 63 with CDR = 0.5, and 36 with CDR ≥ 1. In univariate analysis, IS patients with dementia were older and had more diabetes mellitus, less atrial fibrillation, and higher post-stroke modified Rankin Scale scores than those without dementia. Plasma levels of sRAGE and esRAGE were significantly higher in IS patients with than those without dementia (1.44 ± 1.29 vs. 1.03 ± 0.48 and 0.39 ± 0.40 vs. 0.24 ± 0.13 ng/mL, both p < 0.01). Importantly, both parameters remained independent after adjustment for clinical variables (OR 2.683, p = 0.013 and OR 39.192, p = 0.006, respectively). In summary, plasma sRAGE and esRAGE were elevated in those with dementia compared with those without dementia among IS patients.

The Astrocytic S100B Protein with Its Receptor RAGE Is Aberrantly Expressed in SOD1G93A Models, and Its Inhibition Decreases the Expression of Proinflammatory Genes.

Neuroinflammation is one of the major players in amyotrophic lateral sclerosis (ALS) pathogenesis, and astrocytes are significantly involved in this process. The astrocytic protein S100B can be released in pathological states activating the receptor for advanced glycation end products (RAGE). Different indications point to an aberrant expression of S100B and RAGE in ALS. In this work, we observed that S100B and RAGE are progressively and selectively upregulated in astrocytes of diseased rats with a tissue-specific timing pattern, correlated to the level of neurodegeneration. The expression of the full-length and soluble RAGE isoforms could also be linked to the degree of tissue damage. The mere presence of mutant SOD1 is able to increase the intracellular levels and release S100B from astrocytes, suggesting the possibility that an increased astrocytic S100B expression might be an early occurring event in the disease. Finally, our findings indicate that the protein may exert a proinflammatory role in ALS, since its inhibition in astrocytes derived from SOD1G93A mice limits the expression of reactivity-linked/proinflammatory genes. Thus, our results propose the S100B-RAGE axis as an effective contributor to the pathogenesis of the disease, suggesting its blockade as a rational target for a therapeutic intervention in ALS.

The Structure of the RAGE:S100A6 Complex Reveals a Unique Mode of Homodimerization for S100 Proteins

S100 proteins are calcium-dependent regulators ofhomeostatic processes. Upon cellular response to stress, and notably during tumorigenesis, they relocalize to the extracellular environment where they induce pro-inflammatory signals by activating the receptor for advanced glycation end products (RAGE), thereby facilitating tumor growth and metastasis. Despite its importance in sustaining inflammation, the structural basis for RAGE-S100 crosstalk is still unknown. Here we report two crystal structures of the RAGE:S100A6 complex encompassing a full-length RAGE ectodomain. The structures, in combination with a comprehensive interaction analysis, suggest that the primary S100A6 binding site is formed by the RAGE C1 domain. Complex formation with S100A6 induces a unique dimeric conformation of RAGE that appears suited for signal transduction and intracellular effector recruitment. Intriguingly, S100A6 adopts a dimeric conformation radically different from all known S100 dimers. We discuss the physiological relevance of this non-canonical homodimeric form invivo.

Regulation of RAGE splicing by hnRNP A1 and Tra2β-1 and its potential role in AD pathogenesis.

The receptor for advanced glycation end products (RAGE) gene expresses two major alternative splicing isoforms, full-length membrane-bound RAGE (mRAGE) and secretory RAGE (esRAGE). Both isoforms play important roles in Alzheimer's disease (AD) pathogenesis, either via interaction of mRAGE with β-amyloid peptide (Aβ) or inhibition of the mRAGE-activated signaling pathway. In the present study, we showed that heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) and Transformer2β-1 (Tra2β-1) were involved in the alternative splicing of mRAGE and esRAGE. Functionally, two factors had an antagonistic effect on the regulation. Glucose deprivation induced an increased ratio of mRAGE/esRAGE via up-regulation of hnRNP A1 and down-regulation of Tra2β-1. Moreover, the ratios of mRAGE/esRAGE and hnRNP A1/Tra2β-1 were increased in peripheral blood mononuclear cells from AD patients. The results provide a molecular basis for altered splicing of mRAGE and esRAGE in AD pathogenesis. The receptor for advanced glycation end products (RAGE) gene expresses two major alternative splicing isoforms, membrane-bound RAGE (mRAGE) and secretory RAGE (esRAGE). Both isoforms play important roles in Alzheimer's disease (AD) pathogenesis. Mechanism for imbalanced expression of these two isoforms in AD brain remains elusive. We proposed here a hypothetic model to illustrate that impaired glucose metabolism in AD brain may increase the expression of splicing protein hnRNP A1 and reduce Tra2β-1, which cause the imbalanced expression of mRAGE and esRAGE.

Involvement of the receptor for advanced glycation end products in liver transplantation

Background and aim. Receptor for advanced glycation end products (RAGE) blockade by a soluble form of RAGE (sRAGE) appears to be protective against hepatocellular death and necrosis after I/R injury. Little is known about the role of the hepatic RAGE, its ligands, and the plasma levels of sRAGE in liver transplantation (LT).Material and methods. This was a prospective study on patients (n = 28) undergoing deceased donor LT. RAGE ligands [the N(epsilon)-carboxy-methyl-lysine (CML) adduct and the high-mobility group box 1 (HMGB1) protein] and sRAGE levels were measured in donors at the time of organ procurement, while in recipients they were tested before surgery (baseline), after graft reperfusion, and on day 1 and 7 posttransplantation. Donors and recipients liver biopsies were collected to assess the transcriptional expression of the full-length RAGE and of its truncated isoform, the endogenous secreted RAGE (esRAGE).Results. At baseline, CML levels were higher in LT recipients than in donors (p = 0.02), decreased immediately after graft reperfusion (p < 0.0001) and returned to baseline values on day 7. Baseline HMGB1 levels (3.8 ± 2.3 ng/mL) increased after graft reperfusion (39.9±18 ng/mL, p < 0.0001), and returned to baseline values within day 1, while circulating sRAGE decreased significantly on day 7 (p < 0.0001). The graft esRAGE mRNA expression was inversely associated with bilirubin on day 7 (β = -0.62, p = 0.005).Conclusions. Early on after LT, there is accumulation of CML and a rapid increase of HMGB1 concurrent with a remarkable decline in circulating sRAGE. The RAGE-ligand axis may also be involved in early graft dysfunction.

Neuroprotection by the PARP inhibitor PJ34 modulates cerebral and circulating RAGE levels in rats exposed to focal brain ischemia

The receptor for advanced glycation end products (RAGE) has a potential role as a damage-sensing molecule; however, to date, its involvement in the pathophysiology of stroke and its modulation following neuroprotective treatment are not completely understood. We have previously demonstrated that expression of distinct RAGE isoforms, recognized by different antibodies, is differentially modulated in the brain of rats subjected to focal cerebral ischemia. Here, we focus on the full-length membrane-bound RAGE isoform, showing that its expression is significantly elevated in the striatum, whereas it is reduced in the cortex of rats subjected to transient middle cerebral artery occlusion (MCAo). Notably, the reduction of cortical levels of full-length RAGE detected 24h after reperfusion is abolished by systemic administration of a neuroprotective dose of the poly(ADP-ribose) polymerase (PARP) inhibitor, N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-N,N-dimethylacetamide (PJ34). More interestingly, a significant reduction of plasma soluble RAGE (sRAGE) occurs 24h after reperfusion and this effect is reverted by a neuroprotective dose of PJ34. Soluble forms of RAGE, generated either by alternative splicing or by proteolysis of the full-length form, effectively bind advanced glycation end products, thereby competing with the cell surface full-length RAGE, thus providing a ‘decoy’ function that may counteract the adverse effects of receptor signaling in neurons and may possibly exert cytoprotective effects. Thus, our data confirm the important role of RAGE in ischemic cerebral damage and, more interestingly, suggest the potential use of sRAGE as a blood biomarker of stroke severity and of neuroprotective treatment efficacy.

Overexpression of RAGE Contributes to Cigarette Smoke-Induced Nitric Oxide Generation in COPD

Receptor for advanced glycation end products (RAGE), a multiple-ligands receptor, is implicated in chronic obstructive pulmonary disease (COPD). This study was designed to investigate the potential role of RAGE in nitric oxide (NO) generation, an endogenous marker of nitrosative stress in COPD. Lung tissues from COPD patients were used to describe the relationship between RAGE expression and NO level. RAGE expression was assessed by immunohistochemistry, western blot, and ELISA. Human bronchial epithelial cells (16HBE) were cultured with cigarette smoke extract (CSE). Neutralizing antibody against RAGE was used to detect the role of RAGE in CSE-induced NO generation by 16HBE cells. Compared with nonsmoker controls, overexpression of RAGE was significantly detected in COPD smokers (p<0.01), but not healthy smokers and nonsmokers with COPD, which was dominantly expressed at bronchiolar epithelia. Correlation analysis showed that RAGE in COPD smokers was positively related to NO level, smoking status, and lung function decline. In cultured 16HBE cells treated with CSE, soluble RAGE was reduced; however, full-length RAGE was enhanced significantly as the same trend as NO generation. Moreover, increased NO level and NO synthase activity, decreased total glutathione (a major cellular antioxidant), enhanced nuclear translocation of p65 (a key molecule of nuclear factor (NF)-κB) and release of NF-κB-dependent proinflammatory cytokines were all reversed by pretreatment of anti-RAGE antibody. These findings suggest that overexpression of RAGE contributes to CS-induced NO generation in COPD with involvement in NF-κB activation.

Receptor for advanced glycation end products (RAGE) in vascular and inflammatory diseases

Historically, the receptor for advanced glycation end products (RAGE) was thought to exclusively play an important role under hyperglycemic conditions. However, more and more evidence suggests that RAGE in fact is an inflammation perpetuating multi-ligand receptor and participates actively in various vascular and inflammatory diseases even in normoglycaemic conditions. Various ligands include advanced glycation end products (AGEs), S100 proteins and amphoterins etc. Besides full-length RAGE, numerous truncated forms of the receptor have also been described including the well-characterized soluble RAGE (sRAGE). sRAGE has an ability to act as a decoy to avoid interaction of RAGE with its pro-inflammatory ligands. Ligand engagement of RAGE activates multiple signaling pathways and also forms a positive feedback loop for its own enhanced expression. This review will discuss the role of multi-ligand receptor i.e. RAGE in context to various vascular diseases, which have a pathophysiologically important inflammatory component in normoglycaemic conditions.

Effect of insulin on the soluble receptor for advanced glycation end products (RAGE)

The receptor for advanced glycation end products (RAGE) plays an important role in the pathogenesis of diabetic complications. RAGE transcript splicing generates a number of isoforms, including a full-length membrane-bound receptor and a soluble isoform, endogenous secretory RAGE (esRAGE). Soluble forms of the receptor (sRAGE) can also be formed by ectodomain shedding of the membrane-associated receptor. We have evaluated serum levels of sRAGE and esRAGE in Chinese patients with Type 1 diabetes and investigated the effect of insulin on the generation of esRAGE and sRAGE in vitro. Serum sRAGE and esRAGE were measured by ELISA. The in vitro effect of insulin was investigated by incubating THP-1 macrophages with insulin and RAGE isoforms in cell lysate and conditioned media determined. In patients with diabetes, both serum esRAGE and sRAGE were significantly higher than in age-matched healthy subjects without diabetes. In vitro, insulin increased esRAGE and total RAGE isoform expression in cell lysate on a western blot, and reverse transcription-polymerase chain reaction showed an increase in esRAGE and full-length RAGE mRNA. This was accompanied by an increase in esRAGE and sRAGE in cell conditioned media. Pretreatment of THP-1 cells with a general metalloproteinase inhibitor GM6001 significantly reduced the production of sRAGE, suggesting that insulin also increased the cleavage of full-length cell surface RAGE to form sRAGE. Chinese patients with Type 1 diabetes have higher serum levels of esRAGE and sRAGE. In vitro, insulin not only increases both full-length RAGE and esRAGE expression, but can also stimulate the shedding of sRAGE from the membrane-bound receptor.

Dynamics of Circulating Ligands and Soluble Receptor for Advanced Glycation End-Product in Liver Transplantation

Background and aim: The ligand-receptor for advanced glycation end products (RAGE) pathway is involved in liver injury and animal testing have highlighted that the RAGE blockade attenuates the extent of the liver injury. A truncated isoform of the RAGE (endogenous secreted RAGE, esRAGE), consisting of the extracellular ligand-binding domain only, is detectable as circulating soluble form (sRAGE) in the blood. These latter, acting as decoy, can contribute to the removal of circulating RAGE- ligands. Objective of this study is understand the hepatic expression of both RAGE isoforms (full-length and truncated), and kinetics of the RAGE-ligands and sRAGE plasma levels in patients undergoing liver transplantation (LT) and compared them with healthy donors. Methods: We prospectively included twenty four adult LT recipients (52.7 ± 9.2 years) of primary whole-size grafts by deceased donors (60.4 ± 17.9 years). On both LT recipients and donors, we measured the transcriptional expression of hepatic tissue RAGE and esRAGE, the plasma levels of the RAGE-ligands - N(epsilon)-carboxymethyllysine (CML) and high-mobility group protein 1 (HMGB-1) - and circulating sRAGE. In LT recipient CML, HMGB-1 and sRAGE plasma levels were measured before LT, after graft reperfusion, 1, 7, 30 and 90 days after LT. Results: In LT recipients the hepatic RAGE and esRAGE mRNA levels were higher than in healthy donors (p< 0.01 and p=0.02 respectively). The tissue full-length RAGE inversely correlated with antithrombin III (β= - 0.58, p = 0.013) and cholinesterase plasma levels (β= -0.717, p= 0.0018) and tended to directly correlate with MELD score (β= 0.422, p= 0.063). Basal plasma levels CML, but not sRAGE and HMGB-1, were higher in LT recipients than in healthy donors (p< 0.05). At follow-up CML levels decreased after graft reperfusion (p = 0.0001) while returned progressively to basal values during the follow-p. Instead, HMGB-1, increased after graft reperfusion (p = 0.0001) and returned suddenly to basal values one day after LT. The basal HMGB-1 correlated directly with MELD score (β= 0.448, p= 0.05). The plasma levels of sRAGE did not change significantly soon after LT while decreased seven days after LT (p = 0.0001) and remained constantly low during follow-up. Conclusions: The high levels of RAGE and esRAGE tissue expression in the end-stage liver failure highlight the involvement of the RAGEligand axis in liver injury. The circulating CML accumulation and the rapid increase of HMGB-1 followed by a dramatic decline of the protective sRAGE could have deleterious consequences on graft survival and long term outcomes in LT recipients.

Induction of RAGE Shedding by Activation of G Protein-Coupled Receptors

The multiligand Receptor for Advanced Glycation End products (RAGE) is involved in various pathophysiological processes, including diabetic inflammatory conditions and Alzheimes disease. Full-length RAGE, a cell surface-located type I membrane protein, can proteolytically be converted by metalloproteinases ADAM10 and MMP9 into a soluble RAGE form. Moreover, administration of recombinant soluble RAGE suppresses activation of cell surface-located RAGE by trapping RAGE ligands. Therefore stimulation of RAGE shedding might have a therapeutic value regarding inflammatory diseases. We aimed to investigate whether RAGE shedding is inducible via ligand-induced activation of G protein-coupled receptors (GPCRs). We chose three different GPCRs coupled to distinct signaling cascades: the V2 vasopressin receptor (V2R) activating adenylyl cyclase, the oxytocin receptor (OTR) linked to phospholipase Cβ, and the PACAP receptor (subtype PAC1) coupled to adenylyl cyclase, phospholipase Cβ, calcium signaling and MAP kinases. We generated HEK cell lines stably coexpressing an individual GPCR and full-length RAGE and then investigated GPCR ligand-induced activation of RAGE shedding. We found metalloproteinase-mediated RAGE shedding on the cell surface to be inducible via ligand-specific activation of all analyzed GPCRs. By using specific inhibitors we have identified Ca2+ signaling, PKCα/PKCβI, CaMKII, PI3 kinases and MAP kinases to be involved in PAC1 receptor-induced RAGE shedding. We detected an induction of calcium signaling in all our cell lines coexpressing RAGE and different GPCRs after agonist treatment. However, we did not disclose a contribution of adenylyl cyclase in RAGE shedding induction. Furthermore, by using a selective metalloproteinase inhibitor and siRNA-mediated knock-down approaches, we show that ADAM10 and/or MMP9 are playing important roles in constitutive and PACAP-induced RAGE shedding. We also found that treatment of mice with PACAP increases the amount of soluble RAGE in the mouse lung. Our findings suggest that pharmacological stimulation of RAGE shedding might open alternative treatment strategies for Alzheimeŕs disease and diabetes-induced inflammation.

Receptor for AGEs (RAGE) blockade may exert its renoprotective effects in patients with diabetic nephropathy via induction of the angiotensin II type 2 (AT2) receptor

The receptor for AGEs (RAGE) contributes to the development and progression of diabetic nephropathy. In this study, we examined whether the protective effects of RAGE blockade are exerted via modulation of the renal angiotensin II type 2 (AT2) receptor. Control and streptozotocin diabetic mice, wild-type or deficient in the AT2 receptor (At2 knockout [KO]) or RAGE (Rage KO), were studied for 24weeks. Adenoviral overexpression of full-length Rage in primary rat mesangial cells was also used to determine the effects on AT2 production. With diabetes, Rage-deficient mice had less albuminuria, and an attenuation of hyperfiltration and glomerulosclerosis as compared with diabetic wild-type and At2 KO mice. Renal gene and protein expression of RAGE was elevated with diabetes. Diabetic Rage KO mice had a greater increase in renal AT2 receptor protein than was seen in diabetic wild-type mice. Diabetes-induced increases in renal cytosolic and mitochondrial superoxide generation were prevented in diabetic Rage KO mice, but enhanced in all At2 KO mice. Adenoviral overexpression of RAGE or AGE treatment decreased cell surface AT2 expression, in association with increasing superoxide generation; both were reversed using antioxidants N-acetylcysteine and apocynin, and soluble RAGE in primary mesangial cells. RAGE appears to be a common and key modulator of AT2 receptor expression, a finding that would implicate a newly defined RAGE-AT2 axis in the development and progression of diabetic nephropathy.

Carboxylated N‐glycans on RAGE promote S100A12 binding and signaling

The receptor for advanced glycation end products (RAGE) is a signaling receptor protein of the immunoglobulin superfamily implicated in multiple pathologies. It binds a diverse repertoire of ligands, but the structural basis for the interaction of different ligands is not well understood. We earlier showed that carboxylated glycans on the V-domain of RAGE promote the binding of HMGB1 and S100A8/A9. Here we study the role of these glycans on the binding and intracellular signaling mediated by another RAGE ligand, S100A12. S100A12 binds carboxylated glycans, and a subpopulation of RAGE enriched for carboxylated glycans shows more than 10-fold higher binding potential for S100A12 than total RAGE. When expressed in mammalian cells, RAGE is modified by complex glycans predominantly at the first glycosylation site (N25IT) that retains S100A12 binding. Glycosylation of RAGE and maximum binding sites for S100A12 on RAGE are also cell type dependent. Carboxylated glycan-enriched population of RAGE forms higher order multimeric complexes with S100A12, and this ability to multimerize is reduced upon deglycosylation or by using non-glycosylated sRAGE expressed in E. coli. mAbGB3.1, an antibody against carboxylated glycans, blocks S100A12-mediated NF-kappaB signaling in HeLa cells expressing full-length RAGE. These results demonstrate that carboxylated N-glycans on RAGE enhance binding potential and promote receptor clustering and subsequent signaling events following oligomeric S100A12 binding.

Proteomic studies on receptor for advanced glycation end product variants in idiopathic pulmonary fibrosis and chronic obstructive pulmonary disease

Proteomic screening revealed declined levels of the receptor for advanced glycation end products (RAGE) in human idiopathic pulmonary fibrosis (IPF). This study was undertaken to investigate the different RAGE isoforms in two lung diseases with destruction of the lung parenchyma, i.e. IPF and chronic obstructive pulmonary disease (COPD). RAGE was analyzed by 2-DE, MS and Western blotting using lung tissues from non-smokers, smokers, patients with IPF, COPD and α-1-antitrypsin deficiency (AAT) and by ELISA from the bronchoalveolar lavage fluid samples. RAGE, detected by 2-DE in the control lung, was confirmed to be glycosylated, soluble, C-truncated RAGE with characteristics indicative of the presence of endogenous secretory RAGE (esRAGE). Further studies revealed a decrease of the full length-RAGE (FL-RAGE) and its C-terminal processed variant (cRAGE) in the lung tissues of IPF and COPD patients but not in AAT. The esRAGE level was reduced in IPF but was unchanged in COPD. This study shows an involvement of the three RAGE variants (FL-RAGE, cRAGE, esRAGE) in IPF. The decline of FL-RAGE and cRAGE, but not esRAGE, in COPD lungs is evidence of involvement of specific RAGE variants also in this disease.

Studies on the mechanism of soluble-RAGE generation from full-length RAGE

Studies on the mechanism of soluble-RAGE generation from full-length RAGE