Two β-strands of RAGE participate in the recognition and transport of amyloid-β peptide across the blood brain barrier

Amyloid-β (Aβ) peptide is central to the development of brain pathology in Alzheimer disease (AD) patients. Association with receptors for advanced glycation end-products (RAGE) enables the transport of Aβ peptide from circulating blood to human brain, and also causes the activation of the NF-κB signaling pathway. Here we show that two β-strands of RAGE participate in the interaction with Aβ peptide. Serial deletion analysis of the RAGE V domain indicates that the third and eighth β-strands are required for interaction with Aβ peptide. Site-directed mutagenesis of amino acids located in the third and eighth β-strands abolish the interaction of RAGE with Aβ peptide. Wild-type RAGE activates the NF-κB signaling pathway in response to Aβ peptide treatment, while a RAGE mutant defective in Aβ binding does not. Furthermore, use of peptide for the third β-strand or a RAGE monoclonal antibody that targets the RAGE–Aβ interaction interface inhibited transport of the Aβ peptide across the blood brain barrier in a mice model. These results provide information crucial to the development of RAGE-derived therapeutic reagents for Alzheimer disease.     

Augmenter of liver regeneration inhibits TGF-β1-induced renal tubular epithelial-to-mesenchymal transition via suppressing TβR II expression in vitro

Tubular epithelial-to-mesenchymal transition (EMT) plays a crucial role in the progression of renal tubular interstitial fibrosis (TIF), which subsequently leads to chronic kidney disease (CKD) and eventually, end-stage renal disease (ESRD). We propose that augmenter of liver regeneration (ALR), a member of the newly discovered ALR/Erv1 protein family shown to ameliorate hepatic fibrosis, plays a similar protective role in renal tubular cells and has potential as a new treatment option for CKD. Here, we showed that recombinant human ALR (rhALR) inhibits EMT in renal tubular cells by antagonizing activation of the transforming growth factor-β1 (TGF-β1) signaling pathway. Further investigation revealed that rhALR suppresses the expression of TGF-β receptor type II (TβR II) and significantly alleviates TGF-β1-induced phosphorylation of Smad2 and nuclear factor-κB (NF-κB). No apparent adverse effects were observed upon the addition of rhALR alone to cells. These findings collectively suggest that ALR plays a role in inhibiting progression of renal tubular EMT, supporting its potential utility as an effective antifibrotic strategy to reverse TIF in CKD.     

miR-627/HMGB1/NF-κB regulatory loop modulates TGF-β1-induced pulmonary fibrosis

Pulmonary fibrosis (PF) is a fibroproliferative disease that can eventually lead to fatal lung failure. It is characterized by abnormal proliferation of fibroblasts, dysregulated fibroblast differentiation to myofibroblast, and disorganized collagen and extracellular matrix production, deposition and degradation. There is still a lack of effective treatment strategies for PF. Extracellular high-mobility group box protein 1 (HMGB1) induces PF through NF-κB-mediated TGF-β1 release. Herein, we first validate the suppressive effect of HMGB1 knockdown on TGF-β1-induced α-smooth muscle actin (α-SMA) and collagen I protein expression. In PF, miRNAs exert different effects through targeting various downstream target messenger RNAs. We searched an online database for dysregulated miRNAs in PF tissues; among them, miR-627 was predicted by online tools to target HMGB1 to inhibit its expression. miR-627 overexpression could partially reverse TGF-β1-induced normal human lung fibroblast proliferation, as well as α-SMA and collagen I protein expression. miR-627 inhibition could partially reverse the suppressive effect of HMGB1 knockdown on TGF-β1-induced α-SMA and collagen I protein expression through direct binding to the 3′-untranslated region of HMGB1. Moreover, miR-627/HMGB1 affected TGF-β1 release through RAGE/NF-κB signaling; miR-627/HMGB1 and RAGE/NF-κB signaling formed a regulatory loop to modulate TGF-β1-induced PF in vitro. In conclusion, miR-627 may be a potential agent that targets HMGB1 to inhibit its expression, thereby improving TGF-β1-induced PF in vitro.     

The effects of nuclear factor-kappa B in pancreatic stellate cells on inflammation and fibrosis of chronic pancreatitis

The activation of pancreatic stellate cells (PSCs) plays a critical role in the progression of pancreatic fibrosis. Nuclear factor-kappa B ( NF-κB) is associated with chronic pancreatitis (CP). Previous evidence indicated that NF-κB in acinar cells played a double-edged role upon pancreatic injury, whereas NF-κB in inflammatory cells promoted the progression of CP. However, the effects of NF-κB in PSCs have not been studied. In the present study, using two CP models and RNAi strategy of p65 in cultured PSCs, we found that the macrophage infiltration and MCP-1 expression were increased, and the NF-κBp65 protein level was elevated. NF-κBp65 was co-expressed with PSCs. In vitro, TGF-β1 induced overexpression of the TGF-β receptor 1, phosphorylated TGF-β1–activated kinase 1 (p-TAK1) and NF-κB in the PSCs. Moreover, the concentration of MCP-1 in the supernatant of activated PSCs was elevated. The migration of BMDMs was promoted by the supernatant of activated PSCs. Further knockdown of NF-κBp65 in PSCs resulted in a decline of BMDM migration, accompanied by a lower production of MCP-1. These findings indicate that TGF-β1 can induce the activation of NF-κB pathway in PSCs by regulating p-TAK1, and the NF-κB pathway in PSCs may be a target of chronic inflammation and fibrosis.     

HMGB-1 induces IL-6 production in human synovial fibroblasts through c-Src, Akt and NF-κB pathways

High mobility group box chromosomal protein 1 (HMGB-1) is a widely studied, ubiquitous nuclear protein that is present in eukaryotic cells, and plays a crucial role in inflammatory response. However, the effects of HMGB-1 on human synovial fibroblasts are largely unknown. In this study, we investigated the intracellular signaling pathway involved in HMGB-1-induced IL-6 production in human synovial fibroblast cells. HMGB-1 caused concentration- and time-dependent increases in IL-6 production. HMGB-1-mediated IL-6 production was attenuated by receptor for advanced glycation end products (RAGE) monoclonal antibody (Ab) or siRNA. Pretreatment with c-Src inhibitor (PP2), Akt inhibitor and NF-κB inhibitor (pyrrolidine dithiocarbamate and L-1-tosylamido-2-phenylenylethyl chloromethyl ketone) also inhibited the potentiating action of HMGB-1. Stimulation of cells with HMGB-1 increased the c-Src and Akt phosphorylation. HMGB-1 increased the accumulation of p-p65 in the nucleus, as well as NF-κB luciferase activity. HMGB-1-mediated increase of NF-κB luciferase activity was inhibited by RAGE Ab, PP2 and Akt inhibitor or RAGE siRNA, or c-Src and Akt mutant. Our results suggest that HMGB-1-increased IL-6 production in human synovial fibroblasts via the RAGE receptor, c-Src, Akt, p65, and NF-κB signaling pathways.     

S100B alters neuronal survival and dendrite extension via RAGE-mediated NF-κB signaling

S100B is a soluble protein secreted by astrocytes that exerts pro-survival or pro-apoptotic effects depending on the concentration reached in the extracellular millieu. The S100B receptor termed RAGE (for receptor for advanced end glycation products) is highly expressed in the developing brain but is undetectable in normal adult brain. In this study, we show that RAGE expression is induced in cortical neurons of the ischemic penumbra. Increased RAGE expression was also observed in primary cortical neurons exposed to excitotoxic glutamate (EG). S100B exerts effects on survival pathways and neurite extension when the cortical neurons have been previously exposed to EG and these S100B effects were prevented by anti-RAGE blocking antibodies. Furthermore, nuclear factor kappa B (NF-κB) is activated by S100B in a dose- and RAGE-dependent manner and neuronal death induced by NF-κB inhibition was prevented by S100B that restored NF-κB activation levels. Together, these findings suggest that excitotoxic damage can induce RAGE expression in neurons from ischemic penumbra and demonstrate that cortical neurons respond to S100B through engagement of RAGE followed by activation of NF-κB signaling. In addition, basal NF-κB activity in neurons is crucial to modulate the extent of pro-survival or pro-death S100B effects.     

Interleukin-2 induces extracellular matrix synthesis and TGF-β2 expression in retinal pigment epithelial cells

Macular fibrosis is a vital obstacle of vision acuity improvement of age-related macular degeneration patients. This study was to investigate the effects of interleukin 2 (IL-2) on epithelial-mesenchymal transition (EMT), extracellular matrix (ECM) synthesis and transforming growth factor β2 (TGF-β2) expression in retinal pigment epithelial (RPE) cells. 10 μg/L IL-2 was used to induce fibrosis in RPE cells for various times. Western blot was used to detect the EMT marker α-smooth muscle actin (α-SMA), ECM markers fibronectin (Fn) and type 1 collagen (COL-1), TGF-β2, and the activation of the JAK/STAT3 and NF-κB signaling pathway. Furthermore, JAK/STAT3 and NF-κB signaling pathways were specifically blocked by WP1066 or BAY11-7082, respectively, and the expression of α-SMA, COL-1, Fn and TGF-β2 protein were detected. Wound healing and Transwell assays were used to measure cell migration ability of IL-2 with or without WP1066 or BAY11-7082. After induction of IL-2, the expressions of Fn, COL-1, TGF-β2 protein were significantly increased, and this effect was correlated with IL-2 treatment duration, while α-SMA protein expression did not change significantly. Both WP1066 and BAY11-7082 could effectively downregulate the expression of Fn, COL-1 and TGF-β2 induced by IL-2. What's more, both NF-κB and JAK/STAT3 inhibitors could suppress the activation of the other signaling pathway. Additionally, JAK/STAT3 inhibitor WP1066 and NF-κB inhibitor BAY 11-7082 could obviously decrease RPE cells migration capability induced by IL-2. IL-2 promotes cell migration, ECM synthesis and TGF-β2 expression in RPE cells via JAK/STAT3 and NF-κB signaling pathways, which may play an important role in proliferative vitreoretinopathy.     

Liquiritin attenuates advanced glycation end products-induced endothelial dysfunction via RAGE/NF-κB pathway in human umbilical vein endothelial cells

Advanced glycation end products (AGEs)-induced vasculopathy, including oxidative stress, inflammation and apoptosis responses, contributes to the high morbidity and mortality of coronary artery diseases in diabetic patients. The present study was conducted to evaluate the protective activity of liquiritin (Liq) on AGEs-induced endothelial dysfunction and explore its underlying mechanisms. After pretreatment with Liq, a significant reduction in AGEs-induced apoptosis, as well as reactive oxygen species generation and malondialdehyde level in human umbilical vein endothelial cells (HUVECs) were observed via acridine orange/ethidium bromide fluorescence staining test. Notably, Liq also significantly increased AGEs-reduced superoxide dismutase activity. Furthermore, the pretreatment with receptor for advanced glycation end products (RAGE)-antibody or Liq remarkably down-regulated TGF-beta1 and RAGE protein expressions and significantly blocked NF-κB activation which were proved by immunocytochemistry or immunofluorescence assays. These results indicated that Liq held potential for the protection on AGEs-induced endothelial dysfunction via RAGE/NF-κB pathway in HUVECs and might be a promising agent for the treatment of vasculopathy in diabetic patients.     

Mindin deficiency alleviates renal fibrosis through inhibiting NF-κB and TGF-β/Smad pathways

Renal fibrosis acts as a clinical predictor in patients with chronic kidney disease and is characterized by excessive extracellular matrix (ECM) accumulation. Our previous study suggested that mindin can function as a mediator for liver steatosis pathogenesis. However, the role of mindin in renal fibrosis remains obscure. Here, tumour necrosis factor (TGF)-β-treated HK-2 cells and global mindin knockout mouse were induced with renal ischaemia reperfusion injury (IRI) to test the relationship between mindin and renal fibrosis. In vitro, mindin overexpression promoted p65—the hub subunit of the NF-κB signalling pathway—translocation from the cytoplasm into the nucleus, resulting in NF-κB pathway activation in TGF-β-treated HK-2 cells. Meanwhile, mindin activated the TGF-β/Smad pathway, thereby causing fibrotic-related protein expression in vitro. Mindin^−/− mice exhibited less kidney lesions than controls, with small renal tubular expansion, inflammatory cell infiltration, as well as collagen accumulation, following renal IRI. Mechanistically, mindin^−/− mice suppressed p65 translocation and deactivated NF-κB pathway. Simultaneously, mindin disruption inhibited the TGF-β/Smad pathway, alleviating the expression of ECM-related proteins. Hence, mindin may be a novel target of renal IRI in the treatment of renal fibrogenesis.     

Heparanase is a key player in renal fibrosis by regulating TGF-β expression and activity

Epithelial–mesenchymal transition (EMT) of tubular cells is one of the mechanisms which contribute to renal fibrosis and transforming growth factor-β (TGF-β) is one of the main triggers. Heparanase (HPSE) is an endo-β-D-glucuronidase that cleaves heparan-sulfate thus regulating the bioavailability of growth factors (FGF-2, TGF-β). HPSE controls FGF-2-induced EMT in tubular cells and is necessary for the development of diabetic nephropathy in mice.The aim of this study was to investigate whether HPSE can modulate the expression and the effects of TGF-β in tubular cells.First we proved that the lack of HPSE or its inhibition prevents the increased synthesis of TGF-β by tubular cells in response to pro-fibrotic stimuli such as FGF-2, advanced glycosylation end products (AGE) and albumin overload.Second, since TGF-β may derive from sources different from tubular cells, we investigated whether HPSE modulates tubular cell response to exogenous TGF-β. HPSE does not prevent EMT induced by TGF-β although it slows its onset; indeed in HPSE-silenced cells the acquisition of a mesenchymal phenotype does not develop as quickly as in wt cells. Additionally, TGF-β induces an autocrine loop to sustain its signal, whereas the lack of HPSE partially interferes with this autocrine loop.Overall these data confirm that HPSE is a key player in renal fibrosis since it interacts with the regulation and the effects of TGF-β. HPSE is needed for pathological TGF-β overexpression in response to pro-fibrotic factors. Furthermore, HPSE modulates TGF-β-induced EMT: the lack of HPSE delays tubular cell transdifferentiation, and impairs the TGF-β autocrine loop.     

Effect of atorvastatin on AGEs-induced injury of cerebral cortex via inhibiting NADPH oxidase -NF-κB pathway in ApoE−/− mice

Advanced glycation end products (AGEs) are a group of modified proteins and/or lipids with damaging potential. AGEs-RAGE pathway plays a critical role to induce neurodegenerative encephalopathy. Statins can reduce the expression of AGEs-induced AGEs receptor (RAGE) in the aorta. It is not clear whether statins have potential benefits on AGEs-induced cognitive impairment. In this study, the effects of atorvastatin (ATV) on inflammation and oxidation stress in the cerebral cortex were investigated, and the underlying mechanisms were explored. Apolipoprotein E (ApoE)^?/? male mice were divided into four groups: control, AGEs, AGEs?+?ALT711 (Alagebrium chloride) and AGEs?+?ATV. β-amyloid (Aβ) formation in the cerebral cortex was assessed through Congo red staining and the functional state of neurons was evaluated by Nissl’s staining. Immunostaining was performed to assess the accumulation of AGEs in the cerebral cortex. The expressions of mRNA and protein of RAGE, Nuclear factor kappa B (NF-κB) p65 and Nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase) p47phox were detected by real-time polymerase chain reaction (PCR) and western blot. There were significant increases in AGEs deposit, Aβ formation, and the expressions of RAGE, NF-κB p65, and NADPH oxidase p47phox, and a decrease Nissl body in AGEs group compared with control group. ALT711 group recovered above change compared with AGEs group. Atorvastatin reduced Aβ formation and suppressed AGEs-induced expressions of NF-κB p65 and NADPH oxidase p47phox. Atorvastatin has little effects on AGEs deposit and RAGE expressions. Atorvastatin alleviates AGEs-induced neuronal impairment by alleviating inflammation and oxidative stress via inhibiting NADPH oxidase-NF-κB pathway.

     

Loganin alleviates testicular damage and germ cell apoptosis induced by AGEs upon diabetes mellitus by suppressing the RAGE/p38MAPK/NF-κB pathway

Diabetes mellitus (DM) damages male reproduction at multiple levels, such as endocrine secretion, spermatogenesis and penile erection. We herein investigated the protective effects and mechanism of loganin targeting the advanced glycation end products (AGEs)/receptor for AGEs (RAGE)/p38 mitogen-activated protein kinase (p38MAPK)/NF-κB signalling pathway. Loganin relieved the general DM symptoms and decreased the blood glucose level of KK-Ay DM mice. Haematoxylin-eosin staining demonstrated that loganin ameliorated testicular histology and function and enhanced the activities of testis-specific markers lactate dehydrogenase (LDH), acid phosphatase (ACP) and gamma-glutamyl transferase (γ-GT). Loganin also showed evident anti-oxidative stress, anti-apoptotic and anti-inflammatory effects on DM-induced reproductive damage by restoring glutathione (GSH) level and superoxide dismutase (SOD) activity, as well as reducing reactive oxygen species (ROS) level and Bax/Bcl-2 ratio in vivo and in vitro. Western blotting exhibited that loganin significantly inhibited the AGEs/RAGE/p38MAPK/NF-κB signalling pathway. Acridine orange and ethidium bromide staining (AOEB) and Western blotting showed that loganin in combination with inhibitors of RAGE, p38MAPK and NF-κB exerted stronger anti-apoptotic effects on AGE-induced GC-2 cell damage compared with loganin alone. In conclusion, loganin can protect against DM-induced reproductive damage, probably by suppressing the AGEs/RAGE/p38MAPK/NF-κB pathway.     

Glycated albumin activates NADPH oxidase in rat mesangial cells through up-regulation of p47phox

Glycated albumin, an early-glycation Amadori-modified protein, stimulates transforming growth factor-β (TGF-β) expression and increases the production of the extracellular matrix proteins in mesangial cells, contributing to the pathogenesis of diabetic nephropathy. Glycated albumin has been shown to increase NADPH oxidase-dependent superoxide formation in mesangial cells. However, the mechanisms are not well understood. Therefore, in the present studies, we determined the mechanisms by which glycated albumin activates NADPH oxidase in primary rat mesangial cells and its contribution to glycated albumin-induced TGF-β expression and extracellular matrix protein production. Our data showed that glycated albumin treatment stimulated NADPH oxidase activity and increased the formation of superoxide formation in rat mesangial cells. Moreover, glycated albumin treatment stimulated the expression and phosphorylation of p47phox, one of the cytosolic regulatory subunits of the NADPH oxidase. However, the levels of other NADPH oxidase subunits including Nox1, Nox2, Nox4, p22phox, and p67phox were not altered by glycated albumin. Moreover, siRNA-mediated knockdown of p47phox inhibited glycated albumin-induced NADPH oxidase activity and superoxide formation. Glycated albumin-induced TGF-β expression and extracellular matrix production (fibronectin) was also inhibited by p47phox knock down. Taken together, these data suggest that up-regulation of p47phox is involved in glycated albumin-mediated activation of NADPH oxidase, leading to glycated albumin-induced expression of TGF-β and extracellular matrix proteins in mesangial cells and contributing to the development of diabetic nephropathy.     

LncRNA-ATB promotes TGF-β-induced glioma cells invasion through NF-κB and P38/MAPK pathway

Glioma constitutes the most aggressive primary intracranial malignancy in adults. We previously showed that long noncoding RNA activated by TGF-β (lncRNA-ATB) promoted the glioma cells invasion. However, whether lncRNA-ATB is involved in TGF-β-mediated invasion of glioma cells remains unknown. In this study, quantitative real-time polymerase chain reaction and western blot analysis were used for detecting the mRNA and protein expression of related genes, respectively. Transwell assay was performed to assess the impact of lncRNA-ATB on TGF-β-induced glioma cells migration and invasion. Immunofluorescence staining was utilized to characterize related protein distribution. Results showed that TGF-β upregulated lncRNA-ATB expression in glioma LN-18 and U251 cells. Overexpression of lncRNA-ATB activated nuclear factor-κB (NF-κB) pathway and promoted P65 translocation into the nucleus, thus facilitated glioma cells invasion stimulated by TGF-β. Similarly, lncRNA-ATB markedly enhanced TGF-β-mediated invasion of glioma cells through activation P38 mitogen-activated protein kinase (P38/MAPK) pathway. Moreover, both the NF-κB selected inhibitor pyrrolidinedithiocarbamate ammonium and P38/MAPK specific inhibitor SB203580 partly reversed lncRNA-ATB induced glioma cells invasion mediated by TGF-β. Collectively, this study revealed that lncRNA-ATB promotes TGF-β-induced glioma cell invasion through NF-κB and P38/MAPK pathway and established a detailed framework for understanding the way how lncRNA-ATB performs its function in TGF-β-mediated glioma invasion.     

Fibronectin protects from excessive liver fibrosis by modulating the availability of and responsiveness of stellate cells to active TGF-β

Fibrotic tissue in the liver is mainly composed of collagen. Fibronectin, which is also present in fibrotic matrices, is required for collagen matrix assembly in vitro. It also modulates the amount of growth factors and their release from the matrix. We therefore examined the effects of the absence of fibronectin on the development of fibrosis in mice.Conditional deletion of fibronectin in the liver using the Mx promoter to drive cre expression resulted in increased collagen production and hence a more pronounced fibrosis in response to dimethylnitrosamine in mice. Exclusive deletion of fibronectin in hepatocytes or normalization of circulating fibronectin in Mx-cKO mice did not affect the development of fibrosis suggesting a role for fibronectin production by other liver cell types. The boosted fibrosis in fibronectin-deficient mice was associated with enhanced stellate cell activation and proliferation, elevated concentrations of active TGF-β, and increased TGF-β-mediated signaling.In vitro experiments revealed that collagen-type-I production by fibronectin-deficient hepatic stellate cells stimulated with TGF-β was more pronounced, and was associated with augmented Smad3-mediated signaling. Interfering with TGF-β signaling using SB431542 normalized collagen-type-I production in fibronectin-deficient hepatic stellate cells. Furthermore, precoating culture plates with fibronectin, but not collagen, or providing fibronectin fibrils unable to interact with RGD binding integrins via the RGD domain significantly diminished the amount of active TGF-β in fibronectin-deficient stellate cells and normalized collagen-type-I production in response to TGF-β stimulation. Thus, excessive stellate cell activation and production of collagen results from increased active TGF-β and TGF-β signaling in the absence of fibronectin.In conclusion, our data indicate that fibronectin controls the availability of active TGF-β in the injured liver, which impacts the severity of the resulting fibrosis. We therefore propose a novel role for locally produced fibronectin in protecting the liver from an excessive TGF-β-mediated response.     

Nifedipine inhibits advanced glycation end products (AGEs) and their receptor (RAGE) interaction-mediated proximal tubular cell injury via peroxisome proliferator-activated receptor-gamma activation

There is a growing body of evidence that advanced glycation end products (AGEs) and their receptor (RAGE) interaction evokes oxidative stress generation and subsequently elicits inflammatory and fibrogenic reactions, thereby contributing to the development and progression of diabetic nephropathy. We have previously found that nifedipine, a calcium-channel blocker (CCB), inhibits the AGE-induced mesangial cell damage in vitro. However, effects of nifedipine on proximal tubular cell injury remain unknown. We examined here whether and how nifedipine blocked the AGE-induced tubular cell damage. Nifedipine, but not amlodipine, a control CCB, inhibited the AGE-induced up-regulation of RAGE mRNA levels in tubular cells, which was prevented by the simultaneous treatment of GW9662, an inhibitor of peroxisome proliferator-activated receptor-γ (PPARγ). GW9662 treatment alone was found to increase RAGE mRNA levels in tubular cells. Further, nifedipine inhibited the AGE-induced reactive oxygen species generation, NF-κB activation and increases in intercellular adhesion molecule-1 and transforming growth factor-beta gene expression in tubular cells, all of which were blocked by GW9662. Our present study provides a unique beneficial aspect of nifedipine on diabetic nephropathy; it could work as an anti-oxidative and anti-inflammatory agent against AGEs in tubular cells by suppressing RAGE expression via PPARγ activation.