Distinct roles of heterogeneous nuclear ribonuclear protein K and microRNA-16 in cyclooxygenase-2 RNA stability induced by S100b, a ligand of the receptor for advanced glycation end products

Advanced glycation end products play major roles in diabetic complications. They act via their receptor RAGE to induce inflammatory genes such as cyclooxygenase-2 (COX-2). We examined the molecular mechanisms by which the RAGE ligand, S100b, induces COX-2 in monocytes. S100b significantly increased COX-2 mRNA accumulation in THP-1 monocytes at 2 h via mRNA stability. This was further confirmed by showing that S100b increased stability of luciferase-COX-2 3'-UTR mRNA. Chromatin immunoprecipitation and RNA immunoprecipitation revealed that S100b decreased occupancy of the DNA/RNA-binding protein, heterogeneous nuclear ribonuclear protein K (hnRNPK), at the COX-2 promoter but simultaneously increased its binding to the COX-2 3'-UTR. S100b treatment promoted the translocation of nuclear hnRNPK to cytoplasm, whereas a cytoplasmic translocation-deficient hnRNPK mutant inhibited S100b-induced COX-2 mRNA stability. Small interfering RNA-mediated specific knockdown of hnRNPK blocked S100b-induced COX-2 mRNA stability, whereas on the other hand, overexpression of hnRNPK increased S100b-induced COX-2 mRNA stability. S100b promoted the release of entrapped COX-2 mRNA from cytoplasmic processing bodies, sites of mRNA degradation. Furthermore, S100b significantly down-regulated the expression of a key microRNA, miR-16, which can destabilize COX-2 mRNA by binding to its 3'-UTR. MiR-16 inhibitor oligonucleotides increased, whereas, conversely, miR-16 mimic oligonucleotides decreased COX-2 mRNA stability in monocytes, further supporting the inhibitory effects of miR-16. Interestingly, hnRNPK knockdown increased miR-16 binding to COX-2 3'-UTR, indicating a cross-talk between them. These new results demonstrate that diabetic stimuli can efficiently stabilize inflammatory genes via opposing actions of key RNA-binding proteins and miRs.     

Dimerumic acid attenuates receptor for advanced glycation endproducts signal to inhibit inflammation and diabetes mediated by Nrf2 activation and promotes methylglyoxal metabolism into d-lactic acid

This study was designed to evaluate the effects of dimerumic acid (DMA) on receptor for advanced glycation endproducts (RAGE) signal activation and THP-1 monocyte inflammation treated with S100b, a specific ligand of RAGE. We found that DMA inhibited inflammatory cytokine production via upregulation of nuclear factor-erythroid 2-related factor 2 (Nrf2) and alleviated oxidative stress through attenuation of p47phox translocation to the membrane of S100b-treated THP-1 monocytes. We found that DMA activated Nrf2 mediated by the p38 kinase pathway in THP-1 monocytes. However, anti-inflammatory activity of DMA was attenuated by Nrf2 siRNA treatment. In an animal model, methylglyoxal (MG; 200 mg/kg bw) was chosen to induce diabetes in Balb/C mice (6 weeks) in this work. The in vivo verification of anti-inflammation in peripheral blood mononuclear cells by DMA treatment was confirmed by tumor necrosis factor-α and interleukin-1β measurements. Oral glucose tolerance test, insulin tolerance test, hyperinsulinemia, and hyperglycemia were improved in MG-treated mice by DMA treatment and these effects were greater than those of silymarin and N-acetylcysteine. Furthermore, DMA increased hepatic glyoxalase mRNA and glutathione mediated by Nrf2 activation to metabolize MG into d-lactic acid, thereby reducing serum and hepatic AGE levels and suppressing inflammatory factor generation in MG-treated mice. However, DMA did not exert the antiglycation activity in MG–bovine serum albumin incubation. Taken together, the results indicate that DMA is a novel antioxidant and Nrf2 activator that lowers AGE levels and may prove to be an effective treatment for diabetes.     

Advanced Glycation End Product (AGE)-Receptor for AGE (RAGE) Signaling and Up-regulation of Egr-1 in Hypoxic Macrophages

Receptor for advanced glycation end product (RAGE)-dependent signaling has been implicated in ischemia/reperfusion injury in the heart, lung, liver, and brain. Because macrophages contribute to vascular perturbation and tissue injury in hypoxic settings, we tested the hypothesis that RAGE regulates early growth response-1 (Egr-1) expression in hypoxia-exposed macrophages. Molecular analysis, including silencing of RAGE, or blockade of RAGE with sRAGE (the extracellular ligand-binding domain of RAGE), anti-RAGE IgG, or anti-AGE IgG in THP-1 cells, and genetic deletion of RAGE in peritoneal macrophages, revealed that hypoxia-induced up-regulation of Egr-1 is mediated by RAGE signaling. In addition, the observation of increased cellular release of RAGE ligand AGEs in hypoxic THP-1 cells suggests that recruitment of RAGE in hypoxia is stimulated by rapid production of RAGE ligands in this setting. Finally, we show that mDia-1, previously shown to interact with the RAGE cytoplasmic domain, is essential for hypoxia-stimulated regulation of Egr-1, at least in part through protein kinase C βII, ERK1/2, and c-Jun NH₂-terminal kinase signaling triggered by RAGE ligands. Our findings highlight a novel mechanism by which an extracellular signal initiated by RAGE ligand AGEs regulates Egr-1 in a manner requiring mDia-1.     

Advanced glycation end products impair function of late endothelial progenitor cells through effects on protein kinase Akt and cyclooxygenase-2

Endothelial progenitor cells (EPCs) exhibit impaired function in the context of diabetes, and advanced glycation end products (AGEs), which accumulate in diabetes, may contribute to this. In the present study, we investigated the mechanism by which AGEs impair late EPC function. EPCs from human umbilical cord blood were isolated, and incubated with AGE-modified albumin (AGE-albumin) at different concentrations found physiologically in plasma. Apoptosis, migration, and tube formation assays were used to evaluate EPC function including capacity for vasculogenesis, and expression of the receptor for AGEs (RAGE), Akt, endothelial nitric oxide synthase (eNOS), and cycloxygenase-2 (COX-2) were determined. Anti-RAGE antibody was used to block RAGE function. AGE-albumin concentration-dependently enhanced apoptosis and depressed migration and tube formation, but did not affect proliferation, of late EPCs. High AGE-albumin increased RAGE mRNA and protein expression, and decreased Akt and COX-2 protein expression, whilst having no effect on eNOS mRNA or protein in these cells. These effects were inhibited by co-incubation with anti-RAGE antibody. These results suggest that RAGE mediates the AGE-induced impairment of late EPC function, through down-regulation of Akt and COX-2 in these cells.     

N(epsilon)-(carboxymethyl)lysine adducts of proteins are ligands for receptor for advanced glycation end products that activate cell signaling pathways and modulate gene expression.

Recent studies suggested that interruption of the interaction of advanced glycation end products (AGEs), with the signal-transducing receptor receptor for AGE (RAGE), by administration of the soluble, extracellular ligand-binding domain of RAGE, reversed vascular hyperpermeability and suppressed accelerated atherosclerosis in diabetic rodents. Since the precise molecular target of soluble RAGE in those settings was not elucidated, we tested the hypothesis that predominant specific AGEs within the tissues in disorders such as diabetes and renal failure, N(epsilon)-(carboxymethyl)lysine (CML) adducts, are ligands of RAGE. We demonstrate here that physiologically relevant CML modifications of proteins engage cellular RAGE, thereby activating key cell signaling pathways such as NF-kappaB and modulating gene expression. Thus, CML-RAGE interaction triggers processes intimately linked to accelerated vascular and inflammatory complications that typify disorders in which inflammation is an established component.     

Cyclooxygenase-2 expression in lipopolysaccharide-stimulated human monocytes is modulated by cyclic AMP, prostaglandin E(2), and nonsteroidal anti-inflammatory drugs

Using human blood monocytes (for determination of cyclooxygenase-2 (COX-2) mRNA by RT-PCR) and human whole blood (for prostanoid determination), the present study investigates the influence of the second messenger cAMP on lipopolysaccharide (LPS)-induced COX-2 expression with particular emphasis on the role of prostaglandin E(2) (PGE(2)) in this process. Elevation of intracellular cAMP with a cell-permeable cAMP analogue (dibutyryl cAMP), an adenylyl cyclase activator (cholera toxin), or a phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine) substantially enhanced LPS-induced PGE(2) formation and COX-2 mRNA expression, but did not modify COX-2 enzyme activity. Moreover, up-regulation of LPS-induced COX-2 expression was caused by PGE(2), butaprost (selective agonist of the adenylyl cyclase-coupled EP(2) receptor) and 11-deoxy PGE(1) (EP(2)/EP(4) agonist), whereas sulprostone (EP(3)/EP(1) agonist) left COX-2 expression unaltered. Abrogation of LPS-induced PGE(2) synthesis with the selective COX-2 inhibitor NS-398 caused a decrease in COX-2 mRNA levels that was restored by exogenous PGE(2) and mimicked by S(+)-flurbiprofen and ketoprofen. Overall, these results indicate a modulatory role of cAMP in the regulation of COX-2 expression. PGE(2), a cAMP-elevating final product of the COX-2 pathway, may autoregulate COX-2 expression in human monocytes via a positive feedback mechanism.     

Sphingosine-1-phosphate enhances IL-1{beta}-induced COX-2 expression in mouse intestinal subepithelial myofibroblasts

Intestinal subepithelial myofibroblasts (SEMFs) is a specific population of cells involved in intestinal inflammation and carcinogenesis via an elaborate network of cytokines, chemokines and other inflammatory factors, including PGE(2). Sphingosine-1-phosphate (S1P) has been implicated as an important mediator of inflammation and cancer and in certain cell types increases cyclooxygenase-2 (COX-2) expression. In the present study, we aimed to assess involvement of S1P in COX-2 expression by SEMFs. Primary SEMFs were obtained from C57BL/6J mouse and their identity was verified by fluorescent staining of specific marker proteins. Expression of S1P receptors 1, 2, 3 and sphingosine kinases 1 and 2 in SEMFs were determined by RT-PCR analysis. COX-2 expression and PGE(2) production were assayed by Western blotting and ELISA, respectively. COX-2 mRNA stability was assayed by Northern blotting. S1P produced dose-dependent increase in COX-2 expression, resulting in increased PGE(2) release from SEMFs. Using specific inhibitors, we show that actions of p38, ERK, IKK, and PKC were involved in S1P-induced COX-2 expression. On the other hand, p38 and PKC had lesser roles in IL-1beta-induced COX-2 expression. Inhibition of sphingosine kinase to block S1P production did not affect IL-1beta-induced COX-2 expression, but S1P amplified IL-1beta-induced p38 activation and COX-2 expression. PKC inhibition blocked S1P amplified COX-2 expression. S1P addition increased COX-2 mRNA stability. In SEMFs, S1P amplifies IL-1beta-induced COX-2 expression through increased mRNA stability. These observations point to involvement of S1P in activation of SEMFs that may contribute to intestinal inflammation and carcinogenesis.     

RAGE and its ligands in retinal disease

RAGE, the receptor for advanced glycation endproducts (AGEs), is a multiligand signal transduction receptor of the immunoglobulin superfamily of cell surface molecules that has been implicated in the pathogenesis of diabetic complications, neurodegenerative diseases, inflammatory disorders, and cancer. These diverse biologic disorders reflect the multiplicity of ligands capable of cellular interaction via RAGE that include, in addition to AGEs, amyloid-beta (Abeta) peptide, the S100/calgranulin family of proinflammatory cytokines, and amphoterin, a member of the High Mobility Group Box (HMGB) DNA-binding proteins. In the retina, RAGE expression is present in neural cells, the vasculature, and RPE cells, and it has also been detected in pathologic cellular retinal responses including epiretinal and neovascular membrane formation. Ligands for RAGE, in particular AGEs, have emerged as relevant to the pathogenesis of diabetic retinopathy and age-related macular disease. While the understanding of RAGE and its role in retinal dysfunction with aging, diabetes mellitus, and/or activation of pro-inflammatory pathways is less complete compared to other organ systems, increasing evidence indicates that RAGE can initiate and sustain significant cellular perturbations in the inner and outer retina. For these reasons, antagonism of RAGE interactions with its ligands may be a worthwhile therapeutic target in such seemingly disparate, visually threatening retinal diseases as diabetic retinopathy, age-related macular degeneration, and proliferative vitreoretinopathy.     

Role of microRNA-214-targeting phosphatase and tensin homolog in advanced glycation end product-induced apoptosis delay in monocytes

Advanced glycation end products (AGEs) delay spontaneous apoptosis of monocytes and contribute to the development of inflammatory responses. However, the mechanism by which AGEs affect monocyte apoptosis is unclear. We studied the role of microRNA-214 (miR-214) and its target gene in AGE-induced monocytic apoptosis delay. Using microRNA (miRNA) microarray and stem-loop, quantitative RT-PCR assay, we studied genome-wide miRNA expression in THP-1 cells treated with or without AGEs. Significant upregulation of miR-214 was consistently observed in THP-1 and human monocytes treated with various AGEs, and AGE-induced monocytic miR-214 upregulation was likely through activation of receptor for AGEs. A striking increase in miR-214 was also detected in monocytes from patients with chronic renal failure. Luciferase reporter assay showed that miR-214 specifically binds to the phosphatase and tensin homolog (PTEN) mRNA 3'-untranslated region, implicating PTEN as a target gene of miR-214. PTEN expression is inversely correlated with miR-214 level in monocytes. Compared with normal monocytes, AGE-treated monocytes and monocytes from chronic renal failure patients exhibited lower PTEN levels and delayed apoptosis. Overexpression of pre-miR-214 led to impaired PTEN expression and delayed apoptosis of THP-1 cells, whereas knockdown of miR-214 level largely abolished AGE-induced cell survival. Our findings define a new role for miR-214-targeting PTEN in AGE-induced monocyte survival.     

Advanced Glycation End Products Induce Peroxisome Proliferator-Activated Receptor γ Down-Regulation-Related Inflammatory Signals in Human Chondrocytes via Toll-Like Receptor-4 and Receptor for Advanced Glycation End Products

Accumulation of advanced glycation end products (AGEs) in joints is important in the development of cartilage destruction and damage in age-related osteoarthritis (OA). The aim of this study was to investigate the roles of peroxisome proliferator-activated receptor γ (PPARγ), toll-like receptor 4 (TLR4), and receptor for AGEs (RAGE) in AGEs-induced inflammatory signalings in human OA chondrocytes. Human articular chondrocytes were isolated and cultured. The productions of metalloproteinase-13 and interleukin-6 were quantified using the specific ELISA kits. The expressions of related signaling proteins were determined by Western blotting. Our results showed that AGEs enhanced the productions of interleukin-6 and metalloproteinase-13 and the expressions of cyclooxygenase-2 and high-mobility group protein B1 and resulted in the reduction of collagen II expression in human OA chondrocytes. AGEs could also activate nuclear factor (NF)-κB activation. Stimulation of human OA chondrocytes with AGEs significantly induced the up-regulation of TLR4 and RAGE expressions and the down-regulation of PPARγ expression in a time- and concentration-dependent manner. Neutralizing antibodies of TLR4 and RAGE effectively reversed the AGEs-induced inflammatory signalings and PPARγ down-regulation. PPARγ agonist pioglitazone could also reverse the AGEs-increased inflammatory signalings. Specific inhibitors for p38 mitogen-activated protein kinases, c-Jun N-terminal kinase and NF-κB suppressed AGEs-induced PPARγ down-regulation and reduction of collagen II expression. Taken together, these findings suggest that AGEs induce PPARγ down-regulation-mediated inflammatory signalings and reduction of collagen II expression in human OA chondrocytes via TLR4 and RAGE, which may play a crucial role in the development of osteoarthritis pathogenesis induced by AGEs accumulation.     

Telmisartan inhibits advanced glycation end products (AGEs)-elicited endothelial cell injury by suppressing AGE receptor (RAGE) expression via peroxisome proliferator-activated receptor-gammaactivation

Advanced glycation end products (AGEs)-their receptor (RAGE) axis plays a central role in the pathogenesis of diabetic microangiopathy. Since the pathophysiological crosstalk between the AGEs-RAGE system and angiotensin II has also been associated with diabetic microangiopathy, we examined here whether and how telmisartan, a unique angiotensin II type 1 receptor blocker (ARB) with peroxisome proliferator-activated receptor-gamma (PPAR-gamma)-modulating activity, could inhibit the AGEs-elicited endothelial cell injury by suppressing RAGE expression in vitro. Telmisartan suppressed RAGE expression at both mRNA and protein levels in human cultured microvascular endothelial cells (ECs), which were prevented by GW9662, an inhibitor of PPAR-gamma. Further, telmisartan was found to inhibit up-regulation of mRNA levels for monocyte chemoattractant protein-1, intercellular adhesion molecule-1 and vascular endothelial growth factor in AGEs-exposed ECs. These results suggest that telmisartan inhibits the AGEs-elicited EC injury by down-regulating RAGE expression via PPAR-gamma activation. Our present study provides a unique beneficial aspect of telmisartan. Specifically, it could work as an anti-inflammatory agent against AGEs via PPAR-gamma activation and may play a protective role against diabetic microangiopathy.     

Advanced Glycation End-Products Induce Apoptosis in Pancreatic Islet Endothelial Cells via NF-κB-Activated Cyclooxygenase-2/Prostaglandin E2 Up-Regulation

Microvascular complications eventually affect nearly all patients with diabetes. Advanced glycation end-products (AGEs) resulting from hyperglycemia are a complex and heterogeneous group of compounds that accumulate in the plasma and tissues in diabetic patients. They are responsible for both endothelial dysfunction and diabetic vasculopathy. The aim of this study was to investigate the cytotoxicity of AGEs on pancreatic islet microvascular endothelial cells. The mechanism underlying the apoptotic effect of AGEs in pancreatic islet endothelial cell line MS1 was explored. The results showed that AGEs significantly decreased MS1 cell viability and induced MS1 cell apoptosis in a dose-dependent manner. AGEs dose-dependently increased the expressions of cleaved caspase-3, and cleaved poly (ADP-ribose) polymerase in MS1 cells. Treatment of MS1 cells with AGEs also resulted in increased nuclear factor (NF)-κB-p65 phosphorylation and cyclooxygenase (COX)-2 expression. However, AGEs did not affect the expressions of endoplasmic reticulum (ER) stress-related molecules in MS1 cells. Pretreatment with NS398 (a COX-2 inhibitor) to inhibit prostaglandin E₂ (PGE₂) production reversed the induction of cleaved caspase-3, cleaved PARP, and MS1 cell viability. Moreover, AGEs significantly increased the receptor for AGEs (RAGE) protein expression in MS1 cells, which could be reversed by RAGE neutralizing antibody. RAGE Neutralizing antibody could also reverse the induction of cleaved caspase-3 and cleaved PARP and decreased cell viability induced by AGEs. These results implicate the involvement of NF-κB-activated COX-2/PGE₂ up-regulation in AGEs/RAGE-induced islet endothelial cell apoptosis and cytotoxicity. These findings may provide insight into the pathological processes within the pancreatic islet microvasculature induced by AGEs accumulation.     

Suppression of NF-κB and NF-κB regulated oxidative stress and neuroinflammation by BAY 11-7082 (IκB phosphorylation inhibitor) in experimental diabetic neuropathy

Inflammation is an emerging patho-mechanism of diabetes and its complications. NF-κB pathway is one of the central machinery initiating and propagating inflammatory responses. The present study envisaged the involvement of NF-κB inflammatory cascade in the pathophysiology of diabetic neuropathy using BAY 11-7082, an IκB phosphorylation inhibitor. Streptozotocin was used to induce diabetes in Sprauge Dawley rats. BAY 11-7082 (1 &; 3 mg/kg) was administered to diabetic rats for 14 days starting from the end of six weeks post diabetic induction. Diabetic rats developed deficits in nerve functions and altered nociceptive parameters and also showed elevated expression of NF-κB (p65), IκB and p-IκB along with increased levels of IL-6 &; TNF-α and inducible enzymes (COX-2 and iNOS). Furthermore, there was an increase in oxidative stress and decrease in Nrf2/HO-1 expression. We observed that BAY 11-7082 alleviated abnormal sensory responses and deficits in nerve functions. BAY 11-7082 also ameliorated the increase in expression of NF-κB, IκB and p-IκB. BAY 11-7082 curbed down the levels of IL-6, TNF-α, COX-2 and iNOS in the sciatic nerve. Lowering of lipid peroxidation and improvement in GSH levels was also seen along with increased expression of Nrf2/HO-1. Thus it can be concluded that NF-κB expression and downstream expression of proinflammatory mediators are prominent features of nerve damage leading to inflammation and oxidative stress and BAY 11-7082 was able to ameliorate experimental diabetic neuropathy by modulating neuroinflammation and improving antioxidant defence.