Receptor for advanced glycation end-products promotes premature senescence of proximal tubular epithelial cells via activation of endoplasmic reticulum stress-dependent p21 signaling

Premature senescence is a key process in the progression of diabetic nephropathy (DN). In our study, we hypothesized that receptors for advanced glycation end-products (RAGE) mediate endoplasmic reticulum (ER) stress to induce premature senescence via p21 signaling activation in diabetic nephropathy. Here, we demonstrated that elevated expression of RAGE, ER stress marker glucose-regulated protein 78 (GRP78), and cell-cycle regulator p21 was all positively correlated with enhanced senescence-associated-β-galactosidase (SA-β-gal) activity in DN patients. In addition, the fraction of SA-β-gal or cells in the G₀G₁ phase were enhanced in cultured mouse proximal tubular epithelial cells (PTECs) and the expression of RAGE, GRP78 and p21 was up-regulated by advanced glycation end-products (AGEs) in a dose- and time-dependent manner. Interestingly, ER stress inducers or RAGE overexpression mimicked AGEs induced-premature senescence, and this was significantly suppressed by p21 gene silencing. However, RAGE blocking successfully attenuated AGEs-induced ER stress and p21 expression, as well as premature senescence. Moreover, ER stress inducers directly caused p21 activation, premature senescence, and also enhanced RAGE expression by positive feedback. These observations suggest that RAGE promotes premature senescence of PTECs by activation of ER stress-dependent p21 signaling.     

Lack of the receptor for advanced glycation end-products attenuates E. coli pneumonia in mice

Background

The receptor for advanced glycation end-products (RAGE) has been suggested to modulate lung injury in models of acute pulmonary inflammation. To study this further, model systems utilizing wild type and RAGE knockout (KO) mice were used to determine the role of RAGE signaling in lipopolysaccharide (LPS) and E. coli induced acute pulmonary inflammation. The effect of intraperitoneal (i.p.) and intratracheal (i.t.) administration of mouse soluble RAGE on E. coli injury was also investigated.

Methodology/Principal Findings

C57BL/6 wild type and RAGE KO mice received an i.t. instillation of LPS, E. coli, or vehicle control. Some groups also received i.p. or i.t. administration of mouse soluble RAGE. After 24 hours, the role of RAGE expression on inflammation was assessed by comparing responses in wild type and RAGE KO. RAGE protein levels decreased in wild type lung homogenates after treatment with either LPS or bacteria. In addition, soluble RAGE and HMGB1 increased in the BALF after E. coli instillation. RAGE KO mice challenged with LPS had the same degree of inflammation as wild type mice. However, when challenged with E. coli, RAGE KO mice had significantly less inflammation when compared to wild type mice. Most cytokine levels were lower in the BALF of RAGE KO mice compared to wild type mice after E. coli injury, while only monocyte chemotactic protein-1, MCP-1, was lower after LPS challenge. Neither i.p. nor i.t. administration of mouse soluble RAGE attenuated the severity of E. coli injury in wild type mice.

Conclusions/Significance

Lack of RAGE in the lung does not protect against LPS induced acute pulmonary inflammation, but attenuates injury following live E. coli challenge. These findings suggest that RAGE mediates responses to E. coli-associated pathogen-associated molecular pattern molecules other than LPS or other bacterial specific signaling responses. Soluble RAGE treatment had no effect on inflammation.     

Conditionally induced RAGE expression by proximal airway epithelial cells in transgenic mice causes lung inflammation

Background

Receptors for advanced glycation end-products (RAGE) are multiligand cell-surface receptors expressed abundantly by distal pulmonary epithelium. Our lab has discovered RAGE-mediated effects in the orchestration of lung inflammation induced by tobacco smoke and environmental pollutants; however, the specific contribution of RAGE to the progression of proximal airway inflammation is still inadequately characterized.

Methods and results

We generated a Tet-inducible transgenic mouse that conditionally overexpressed RAGE using the club cell (Clara) secretory protein (CCSP) promoter expressed by club (Clara) cells localized to the proximal airway. RAGE was induced for 40 days from weaning (20 days of age) until sacrifice date at 60 days. Immunohistochemistry, immunoblotting, and qPCR revealed significant RAGE up-regulation when compared to non-transgenic controls; however, H&E staining revealed no detectible morphological abnormalities and apoptosis was not enhanced during the 40 days of augmentation. Freshly procured bronchoalveolar lavage fluid (BALF) from CCSP-RAGE TG mice had significantly more total leukocytes and PMNs compared to age-matched control littermates. Furthermore, CCSP-RAGE TG mice expressed significantly more tumor necrosis factor alpha (TNF-α), interleukin 7 (IL-7), and interleukin 14 (IL-14) in whole lung homogenates compared to controls.

Conclusions

These data support the concept that RAGE up-regulation specifically in lung airways may function in the progression of proximal airway inflammation.     

Identification of pyridinoline,a collagen crosslink,as a novel intrinsic ligand for the receptor for advanced glycation end-products (RAGE)

Advanced glycation end-products (AGEs) elicit inflammatory responses via the receptor for AGEs (RAGE) and participate in the pathogenesis of diabetic complications. An earlier study showed that 3-hydroxypyridinium (3-HP), a common moiety of toxic AGEs such as glyceraldehyde-derived pyridinium (GLAP) and GA-pyridine, is essential for the interaction with RAGE. However, the physiological significance of 3-HP recognition by RAGE remains unclear. We hypothesized that pyridinoline (Pyr), a collagen crosslink containing the 3-HP moiety, could have agonist activity with RAGE. To test this hypothesis, we purified Pyr from bovine achilles tendons and examined its cytotoxicity to rat neuronal PC12 cells. Pyr elicited toxicity to PC12 cells in a concentration-dependent manner, and this effect was attenuated in the presence of either the anti-RAGE antibody or the soluble form of RAGE. Moreover, surface plasmon resonance-based analysis showed specific binding of Pyr to RAGE. These data indicate that Pyr is an intrinsic ligand for RAGE.

Abbreviations: AGEs: advanced glycation end-products; RAGE: receptor for advanced glycation end-products; DAMPs: damage-associated molecular patterns; PRR: pattern recognition receptor; TLR: toll-like receptor; GLAP: glyceraldehyde-derived pyridinium; 3-HP: 3-hydroxypyridinium; Pyr: pyridinoline; HFBA: heptafluorobutyric acid; GST: glutathione S-transferase; SPR: surface plasmon resonance; ECM: extracellular matrix; EMT: epithelial to mesenchymal transition     

RAGE signaling contributes to neuroinflammation in infantile neuronal ceroid lipofuscinosis

Palmitoyl-protein thioesterase-1 (PPT1) deficiency causes infantile neuronal ceroid lipofuscinosis (INCL), a devastating childhood neurodegenerative storage disorder. We previously reported that neuronal apoptosis in INCL is mediated by endoplasmic reticulum-stress. ER-stress disrupts Ca²⁺-homeostasis and stimulates the expression of Ca²⁺-binding proteins. We report here that in the PPT1-deficient human and mouse brain the levels of S100B, a Ca²⁺-binding protein, and its receptor, RAGE (receptor for advanced glycation end-products) are elevated. We further demonstrate that activation of RAGE signaling in astroglial cells mediates pro-inflammatory cytokine production, which is inhibited by SiRNA-mediated suppression of RAGE expression. We propose that RAGE signaling contributes to neuroinflammation in INCL.     

Age dependent accumulation patterns of advanced glycation end product receptor (RAGE) ligands and binding intensities between RAGE and its ligands differ in the liver,kidney, and skeletal muscle

Background

Much evidence indicates receptor for advanced glycation end products (RAGE) related inflammation play essential roles during aging. However, the majority of studies have focused on advanced glycation end products (AGEs) and not on other RAGE ligands. In the present study, the authors evaluated whether the accumulation of RAGE ligands and binding intensities between RAGE and its ligands differ in kidney, liver, and skeletal muscle during aging.

Results

In C57BL/6 N mice aged 12 weeks, 12 months, and 22 months, ligands accumulation, binding intensities between RAGE and its ligands, activated macrophage infiltration, M1/M2 macrophage expression, glyoxalase-1expression, and signal pathways related to inflammation were evaluated. The RAGE ligands age-associated accumulation patterns were found to be organ dependent. Binding intensities between RAGE and its ligands in kidney and liver increased with age, but those in skeletal muscle were unchanged. Infiltration of activated macrophages in kidney and liver increased with age, but infiltration in the skeletal muscle was unchanged. M1 expression increased and M2 and glyoxalase-1 expression decreased with age in kidney and liver, but their expressions in skeletal muscle were not changed.

Conclusion

These findings indicate patterns of RAGE ligands accumulation, RAGE/ligands binding intensities, or inflammation markers changes during aging are organs dependent.

     

An Advanced Glycation End Product (AGE)-Receptor for AGEs (RAGE) Axis Restores Adipogenic Potential of Senescent Preadipocytes through Modulation of p53 Protein Function

The impaired adipogenic potential of senescent preadipocytes is a hallmark of adipose aging and aging-related adipose dysfunction. Although advanced glycation end products (AGEs) derived from both foods and endogenous nonenzymatic glycation and AGE-associated signaling pathways are known to play a key role in aging and its related diseases, the role of AGEs in adipose aging remains elusive. We show a novel pro-adipogenic function of AGEs in replicative senescent preadipocytes and mouse embryonic fibroblasts, as well as primary preadipocytes isolated from aged mice. Using glycated bovine serum albumin (BSA) as a model protein of AGEs, we found that glycated BSA restores the impaired adipogenic potential of senescent preadipocytes in vitro and ex vivo. However, glycated BSA showed no effect on adipogenesis in nonsenescent preadipocytes. The AGE-induced receptor for AGE (RAGE) expression is required for the pro-adipogenic function of AGEs in senescent preadipocytes. RAGE is required for impairment of p53 expression and p53 function in regulating p21 expression in senescent preadipocytes. We also observed a direct binding between RAGE and p53 in senescent preadipocytes. Taken together, our findings reveal a novel pro-adipogenic function of the AGE-RAGE axis in p53-regulated adipogenesis of senescent preadipocytes, providing new insights into aging-dependent adiposity by diet-driven and/or endogenous glycated proteins.     

RAGE modulates myocardial injury consequent to LAD infarction via impact on JNK and STAT signaling in a murine model

The receptor for advanced glycation end-products (RAGE) has been implicated in the pathogenesis of ischemia-reperfusion (I/R) injury in the isolated perfused heart. To test the hypothesis that RAGE-dependent mechanisms modulated responses to I/R in a murine model of transient occlusion and reperfusion of the left anterior descending coronary artery (LAD), we subjected male homozygous RAGE(-/-) mice and their wild-type age-matched littermates to 30 min of occlusion of the LAD followed by reperfusion. At 48 h of reperfusion, hematoxylin and eosin staining revealed significantly larger infarct size in wild-type versus RAGE(-/-) mice. Contractile function, as evaluated by echocardiography 48 h after reperfusion, revealed that fractional shortening was significantly higher in RAGE(-/-) versus wild-type mice. Plasma levels of creatine kinase were markedly decreased in RAGE(-/-) versus wild-type animals. Integral to the impact of RAGE deletion on diminished myocardial damage after infarction was significantly decreased apoptosis in the heart, as assessed by TUNEL staining, release of cytochrome c, and caspase-3 activity. Experiments investigating the impact of RAGE on early signaling pathways influencing myocardial ischemic injury revealed attenuation of JNK and STAT5 phosphorylation in RAGE(-/-) mouse hearts versus robust activation observed in wild-type mice upon ischemia and reperfusion. Solidifying the link to RAGE, these experiments revealed that infarction stimulated the rapid production of advanced glycation end-products in the heart. Thus, we tested the effect of ligand decoy soluble RAGE (sRAGE). Administration of sRAGE protected the myocardium from ischemic damage, similar to the effects observed in RAGE(-/-) mouse hearts. Taken together, these data implicate RAGE and its ligands in the pathogenesis of I/R injury and identify JNK and STAT signal transduction as central downstream effector pathways of the ligand-RAGE axis in the heart subjected to I/R injury.     

Targeting of Receptor for Advanced Glycation End Products Suppresses Cyst Growth in Polycystic Kidney Disease

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited renal disorder. Although a myriad of research groups have attempted to identify a new therapeutic target for ADPKD, no drug has worked well in clinical trials. Our research group has focused on the receptor for advanced glycation end products (RAGE) gene as a novel target for ADPKD. This gene is involved in inflammation and cell proliferation. We have already confirmed that blocking RAGE function attenuates cyst growth in vitro. Based on this previous investigation, our group examined the effect of RAGE on cyst enlargement in vivo. PC2R mice, a severe ADPKD mouse model that we generated, were utilized. An adenovirus containing anti-RAGE shRNA was injected intravenously into this model. We observed that RAGE gene knockdown resulted in loss of kidney weight and volume. Additionally, the cystic area that originated from different nephron segments decreased in size because of down-regulation of the RAGE gene. Blood urea nitrogen and creatinine values tended to be lower after inhibiting RAGE. Based on these results, we confirmed that the RAGE gene could be an effective target for ADPKD treatment.     

Timosaponin AIII attenuates inflammatory injury in AGEs-induced osteoblast and alloxan-induced diabetic osteoporosis zebrafish by modulating the RAGE/MAPK signaling pathways

BackgroundAdvanced glycation end products (AGEs) deposition causes inflammatory injury in osteoblasts and contributes to diabetic osteoporosis. The receptor for advanced glycation end product/mitogen-activated protein kinase pathway (RAGE/MAPK) signaling pathway is closely linked to the pathogenesis of diabetic osteoporosis. Timosaponin AIII, a steroidal saponin isolated from Anemarrhena asphodeloides Bunge (Asparagaceae), shows anti-inflammatory and anti-osteoporosis effects.PurposeThe present study was aimed to investigate the therapeutic effects of timosaponin AIII on diabetic osteoporosis and whether its effect is dependent on protecting osteoblasts against AGEs-induced injury via RAGE/MAPK signaling suppression.MethodsAn alloxan-induced diabetic osteoporosis zebrafish model was applied to investigate the effects of timosaponin AIII in vivo, and alendronate was used as a positive control. Moreover, related mechanisms were explored in primary rat osteoblasts. Molecular docking was applied to investigate the interactions between timosaponin AIII and RAGE.ResultsTimosaponin AIII treatment reversed alloxan-induced reduction in the mineralized area of the larvae head skeleton, accompanied by a decreased level of triglyceride and total cholesterol in the zebrafish. Additionally, AGEs significantly influenced RAGE expression, alkaline phosphatase activity, interleukin 1β expression, interleukin 6 expression, and tumor necrosis factor-α expression, and increased cell apoptosis. Timosaponin AIII significantly downregulated AGEs-induced interleukin 1β, interleukin 6, and tumor necrosis factor-α levels, and upregulated alkaline phosphatase and osteocalcin levels. Timosaponin AIII also significantly reduced the expression of RAGE and had additive effects on downstream P38, extracellular signal-regulated kinase and c-Jun N-terminal kinase in AGEs-induced osteoblast. Molecular docking predicted that hydrogen and hydrophobic interactions occurred between timosaponin AIII and RAGE.ConclusionThese data clarified that timosaponin AIII attenuates diabetic osteoporosis via a novel mechanism involved suppressing the RAGE/MAPK signaling pathway. Our finding highlights the potential value of timosaponin AIII as an anti-diabetic osteoporosis agent.     

The Role of the Receptor for Advanced Glycation End-Products in a Murine Model of Silicosis

Background

The role of the receptor for advanced glycation end-products (RAGE) has been shown to differ in two different mouse models of asbestos and bleomycin induced pulmonary fibrosis. RAGE knockout (KO) mice get worse fibrosis when challenged with asbestos, whereas in the bleomycin model they are largely protected against fibrosis. In the current study the role of RAGE in a mouse model of silica induced pulmonary fibrosis was investigated.

Methodology/Principal Findings

Wild type (WT) and RAGE KO mice received a single intratracheal (i.t.) instillation of silica in saline or saline alone as vehicle control. Fourteen days after treatment mice were subjected to a lung mechanistic study and the lungs were lavaged and inflammatory cells, protein and TGF-β levels in lavage fluid determined. Lungs were subsequently either fixed for histology or excised for biochemical assessment of fibrosis and determination of RAGE protein- and mRNA levels. There was no difference in the inflammatory response or degree of fibrosis (hydroxyproline levels) in the lungs between WT and RAGE KO mice after silica injury. However, histologically the fibrotic lesions in the RAGE KO mice had a more diffuse alveolar septal fibrosis compared to the nodular fibrosis in WT mice. Furthermore, RAGE KO mice had a significantly higher histologic score, a measure of affected areas of the lung, compared to WT silica treated mice. A lung mechanistic study revealed a significant decrease in lung function after silica compared to control, but no difference between WT and RAGE KO. While a dose response study showed similar degrees of fibrosis after silica treatment in the two strains, the RAGE KO mice had some differences in the inflammatory response compared to WT mice.

Conclusions/Significance

Aside from the difference in the fibrotic pattern, these studies showed no indicators of RAGE having an effect on the severity of pulmonary fibrosis following silica injury.     

The expression of the receptor for advanced glycation end-products (RAGE) in RA-FLS is induced by IL-17 via Act-1

Introduction

The receptor for advanced glycation end-products (RAGE) has been implicated in the pathogenesis of arthritis. We conducted this study to determine the effect of interleukin (IL)-17 on the expression and production of RAGE in fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA). The role of nuclear factor-κB (NF-κB) activator 1 (Act1) in IL-17-induced RAGE expression in RA-FLS was also evaluated.

Methods

RAGE expression in synovial tissues was assessed by immunohistochemical staining. RAGE mRNA production was determined by real-time polymerase chain reaction. Act-1 short hairpin RNA (shRNA) was produced and treated to evaluate the role of Act-1 on RAGE production.

Results

RAGE, IL-17, and Act-1 expression increased in RA synovium compared to osteoarthritis synovium. RAGE expression and production increased by IL-17 and IL-1β (*P <0.05 vs. untreated cells) treatment but not by tumor necrosis factor (TNF)-α in RA-FLS. The combined stimuli of both IL-17 and IL-1β significantly increased RAGE production compared to a single stimulus with IL-17 or IL-1β alone (P <0.05 vs. 10 ng/ml IL-17). Act-1 shRNA added to the RA-FLS culture supernatant completely suppressed the enhanced production of RAGE induced by IL-17.

Conclusions

RAGE was overexpressed in RA synovial tissues, and RAGE production was stimulated by IL-17 and IL-1β. Act-1 contributed to the stimulatory effect of IL-17 on RAGE production, suggesting a possible inhibitory target for RA treatment.     

Soluble Forms and Ligands of the Receptor for Advanced Glycation End-Products in Patients with Acute Respiratory Distress Syndrome: An Observational Prospective Study

Background

The main soluble form of the receptor for advanced glycation end-products (sRAGE) is elevated during acute respiratory distress syndrome (ARDS). However other RAGE isoforms and multiple ligands have been poorly reported in the clinical setting, and their respective contribution to RAGE activation during ARDS remains unclear. Our goal was therefore to describe main RAGE isoforms and ligands levels during ARDS.

Methods

30 ARDS patients and 30 mechanically ventilated controls were prospectively included in this monocenter observational study. Arterial, superior vena cava and alveolar fluid levels of sRAGE, endogenous-secretory RAGE (esRAGE), high mobility group box-1 protein (HMGB1), S100A12 and advanced glycation end-products (AGEs) were measured in duplicate ELISA on day 0, day 3 and day 6. In patients with ARDS, baseline lung morphology was assessed with computed tomography.

Results

ARDS patients had higher arterial, central venous and alveolar levels of sRAGE, HMGB1 and S100A12, but lower levels of esRAGE and AGEs, than controls. Baseline arterial sRAGE, HMGB1 and S100A12 were correlated with nonfocal ARDS (AUC 0.79, 0.65 and 0.63, respectively). Baseline arterial sRAGE, esRAGE, S100A12 and AGEs were associated with severity as assessed by PaO₂/FiO₂.

Conclusions

This is the first kinetics study of levels of RAGE main isoforms and ligands during ARDS. Elevated sRAGE, HMGB1 and S100A12, with decreased esRAGE and AGEs, were found to distinguish patients with ARDS from those without. Our findings should prompt future studies aimed at elucidating RAGE/HMGB1/S100A12 axis involvement in ARDS.

Trial Registration

clinicaltrials.gov Identifier: NCT01270295.     

Venestatin from parasitic helminths interferes with receptor for advanced glycation end products (RAGE)-mediated immune responses to promote larval migration

Parasitic helminths can reside in humans owing to their ability to disrupt host protective immunity. Receptor for advanced glycation end products (RAGE), which is highly expressed in host skin, mediates inflammatory responses by regulating the expression of pro-inflammatory cytokines and endothelial adhesion molecules. In this study, we evaluated the effects of venestatin, an EF-hand Ca²⁺-binding protein secreted by the parasitic helminth Strongyloides venezuelensis, on RAGE activity and immune responses. Our results demonstrated that venestatin bound to RAGE and downregulated the host immune response. Recombinant venestatin predominantly bound to the RAGE C1 domain in a Ca²⁺-dependent manner. Recombinant venestatin effectively alleviated RAGE-mediated inflammation, including footpad edema in mice, and pneumonia induced by an exogenous RAGE ligand. Infection experiments using S. venezuelensis larvae and venestatin silencing via RNA interference revealed that endogenous venestatin promoted larval migration from the skin to the lungs in a RAGE-dependent manner. Moreover, endogenous venestatin suppressed macrophage and neutrophil accumulation around larvae. Although the invasion of larvae upregulated the abundance of RAGE ligands in host skin tissues, mRNA expression levels of tumor necrosis factor-α, cyclooxygenase-2, endothelial adhesion molecules vascular cell adhesion protein-1, intracellular adhesion molecule-1, and E-selectin were suppressed by endogenous venestatin. Taken together, our results indicate that venestatin suppressed RAGE-mediated immune responses in host skin induced by helminthic infection, thereby promoting larval migration. The anti-inflammatory mechanism of venestatin may be targeted for the development of anthelminthics and immunosuppressive agents for the treatment of RAGE-mediated inflammatory diseases.     

SIRT1 participates in the response to methylglyoxal-dependent glycative stress in mouse oocytes and ovary

Methylglyoxal (MG), a highly reactive dicarbonyl derived from metabolic processes, is the most powerful precursor of advanced glycation end products (AGEs). Glycative stress has been recently associated with ovarian dysfunctions in aging and PCOS syndrome. We have investigated the role of the NAD⁺-dependent Class III deacetylase SIRT1 in the adaptive response to MG in mouse oocytes and ovary. In mouse oocytes, MG induced up-expression of glyoxalase 1 (Glo1) and glyoxalase 2 (Glo2) genes, components of the main MG detoxification system, whereas inhibition of SIRT1 by Ex527 or sirtinol reduced this response. In addition, the inhibition of SIRT1 worsened the effects of MG on oocyte maturation rates, while SIRT1 activation by resveratrol counteracted MG insult. Ovaries from female mice receiving 100 mg/kg MG by gastric administration for 28 days (MG mice) exhibited increased levels of SIRT1 along with over-expression of catalase, superoxide dismutase 2, SIRT3, PGC1α and mtTFA. Similar levels of MG-derived AGEs were observed in the ovaries from MG and control groups, along with enhanced protein expression of glyoxalase 1 in MG mice. Oocytes ovulated by MG mice exhibited atypical meiotic spindles, a condition predisposing to embryo aneuploidy. Our results from mouse oocytes revealed for the first time that SIRT1 could modulate MG scavenging by promoting expression of glyoxalases. The finding that up-regulation of glyoxalase 1 is associated with that of components of a SIRT1 functional network in the ovaries of MG mice provides strong evidence that SIRT1 participates in the response to methylglyoxal-dependent glycative stress in the female gonad.     

The role of the receptor for advanced glycation end-products in lung fibrosis

The pathogenesis of pulmonary fibrosis remains unclear. The receptor for advanced glycation end-products (RAGE) is a multi-ligand receptor known to be involved in the process of fibrotic change in several organs, such as peritoneal fibrosis and kidney fibrosis. The aim of this study was to examine the contribution of RAGE during the acute inflammation and chronic fibrotic phases of lung injury induced by intratracheal instillation of bleomycin in mice. Bleomycin-induced lung fibrosis was evaluated in wild-type and RAGE-deficient (RAGE-/-) mice. Bleomycin administration to wild-type mice caused an initial pneumonitis that evolved into fibrosis. While RAGE-/- mice developed a similar early inflammatory response, the mice were largely protected from the late fibrotic effects of bleomycin. The protection afforded by RAGE deficiency was accompanied by reduced pulmonary levels of the potent RAGE-inducible profibrotic cytokines transforming growth factor (TGF)-beta and PDGF. In addition, bleomycin administration induced high mobility group box 1 (HMGB-1) production, one of the ligands of RAGE, from inflammatory cells that accumulated within the air space. Coculture with HMGB-1 induced epithelial-mesenchymal transition (EMT) in alveolar type II epithelial cells from wild-type mice. However, alveolar type II epithelial cells derived from RAGE-/- mice did not respond to HMGB-1 treatment, such that the RAGE/HMGB-1 axis may play an important role in EMT. Also, bleomycin administration induced profibrotic cytokines TGF-beta and PDGF only in wild-type mouse lungs. Our results suggested that RAGE contributes to bleomycin-induced lung fibrosis through EMT and profibrotic cytokine production. Thus, RAGE may be a new therapeutic target for pulmonary fibrosis.