Differentiating ischemic from hemorrhagic stroke using plasma biomarkers: the S100B/RAGE pathway

Although neuroimaging is useful in differentiating ischemic (IS) from hemorrhagic (ICH) stroke in the Emergency Department, a wide-available rapid biochemical test would add advantages in the pre-hospital triage and management of stroke patients. Our aim was to examine the predictive value of a panel of blood-borne biomarkers to differentiate IS from ICH. Admission blood samples obtained within 24h from stroke symptoms onset were tested by ELISA for CRP, D-dimer, sRAGE, MMP9, S100B, BNP, NT-3, caspase-3, chimerin-II, secretagogin, cerebellin and NPY. The complete protocol was achieved in 915 patients (776 IS, 139 ICH). Among blood samples obtained <6 h from symptoms onset (n=337), S100B levels were increased in ICH (107.58 vs 58.70 pg/mL; p<0.001) whereas sRAGE levels were decreased (0.77 vs 1.02 ng/mL; p=0.009) as compared to IS. In this subset of patients S100B (OR 3.97 95% CI 1.82-8.68; p=0.001) and sRAGE (OR 0.22 95% CI 0.10-0.52; p<0.001) were independently associated with ICH. A regression tree was created by CART method showing good classification ability (AUC=0.762). Similar results were found for samples obtained within 3 h. In conclusion, a combination of biomarkers including those of the S100B/RAGE pathway seems promising to achieve a rapid biochemical diagnosis of IS versus ICH in the first hours from symptoms onset. This article is part of a Special Issue entitled: Translational Proteomics.     

Epicatechin gallate increases glutamate uptake and S100B secretion in C6 cell lineage

There is a current interest in dietary compounds, such as green tea polyphenols, that can favor protection against a variety of brain disorders, including Alzheimer’s disease, ischemia, and stroke. The objective of the present study was to investigate the effects of (^_)-epicatechin-3-gallate (ECG), one of three three major green tea antioxidants, on C6 lineage cells. Here, we evaluated cell morphology and integrity and specific astrocyte activities; glutamate uptake and secretion of S100B in the presence of 0.1, 1 and 10 μM ECG. During 6 h of incubation, cell morphology was altered only at 10 μM ECG; however, after 24 h of treatment, cells become stellate in the presence of all concentrations of ECG. Loss of cell integrity was observed after 24 h with 10 μM ECG and represented only 6% of cells, in contrast with 2% observed at basal conditions. ECG (1–10 μM) induced a decrease (about 36%) in glutamate uptake after 1 h of incubation. After 6 h, an opposite effect occurred and ECG induced a sustained increase in glutamate uptake of about 70% from 0.1 μM. In addition, a significant increase in S100B was observed at 1 μM ECG (36%) and 10 μM ECG (69%) after 1 h, in contrast to 6 h of treatment, where all doses of ECG induced a significant increase (about 60%) in S100B secretion. These data demonstrate that ECG induces a significant improvement in glutamate uptake and S100B secretion in C6 cells, indicating that ECG could contribute to the neuroprotective role of astroglial cells.     

S100/RAGE在实验性自身免疫性重症肌无力中对T细胞的作用及其机制研究

目的:探讨RAGE及其配体S100B在实验性自身免疫性重症肌无力(EAMG)中对T细胞的作用及其相关机制。方法:选取体重160-180g的Lewis大鼠,并将其随机分为EAMG模型组和CFA对照组。EAMG模型组大鼠通过尾根部注入200μL含有R-ACh R97-116肽段的免疫乳剂,并于初始免疫后的第30天尾根部追加免疫一次;CFA对照组大鼠为免疫乳剂中不含有R-ACh R97-116肽段。采用流式细胞术检测和比较两组大鼠CD4+T细胞上RAGE的表达情况;ELISA方法检测淋巴细胞培养上清中IFN-γ、IL-4、IL-17、TGF-β、IL-6和S100B的表达水平;采用S100B体外干预淋巴细胞,进一步检测S100B对EAMG大鼠T细胞增殖、亚型分布、细胞因子分泌的影响。结果:在疾病发生的晚期时相(初次免疫后第45天),EAMG组淋巴细胞CD4+T细胞上RAGE的表达明显高于CFA组(P0.001),血清中RAGE的配体S100B的表达也高于CFA组(P0.001);体外加入S100B干预能促进T淋巴细胞的增殖,与未干预组相比较差异显著(P0.05);S100B刺激后,Th1细胞和Th17细胞的百分比进一步增高,Th2细胞和Treg细胞百分比下降(PTh10.05,PTh170.05,PTh20.05,PTreg0.05),IFN-γ和IL-17的表达上调(PIFN-γ0.05,PIL-170.05),而IL-4和TGF-β表达下降(PTGF-β0.01,PIL-40.05),Th17细胞的调控因子IL-6表达升高(PIL-60.05)。结论:RAGE及其配体S100B参与EAMG的发病过程,在晚期时相中表现出明显的致病性;RAGE与S100B相互作用可以上调T细胞的致病性作用,加重四种辅助性T细胞之间的网络失衡。     

S100A8 and S100A9 activate MAP kinase and NF-kappaB signaling pathways and trigger translocation of RAGE in human prostate cancer cells

S100 proteins, a multigenic family of calcium-binding proteins, have been linked to human pathologies in recent years. Deregulated expression of S100 proteins, including S100A8 and S100A9, was reported in association with neoplastic disorders. In a previous study, we identified enhanced expression of S100A8 and S100A9 in human prostate cancer. To investigate potential functional implications of S100A8 and S100A9 in prostate cancer, we examined the influence of over-expressed and of purified recombinant S100A8 and S100A9 proteins in different prostate epithelial cell lines. S100A8 and S100A9 were secreted by prostate cancer cells, a finding which prompted us to analyze a possible function as extracellular ligands. S100A8/A9 induced the activation of NF-kappaB and an increased phosphorylation of p38 and p44/42 MAP kinases. In addition, extracellular S100A8/A9 stimulated migration of benign prostatic cells in vitro. Furthermore, in immunofluorescence experiments, we found a strong speckled co-localization of intracellular S100A8/A9 with RAGE after stimulating cells with recombinant S100A8/A9 protein or by increasing cytosolic Ca2+ levels. In summary, our findings show that S100A8 and S100A9 are linked to the activation of important features of prostate cancer cells.     

Resveratrol increases glutamate uptake and glutamine synthetase activity in C6 glioma cells

Resveratrol, a phytoalexin found mainly in grapes, is a promising natural product with anti-cancer and cardio-protective activities. Here, we investigated, in C6 glioma cells, the effect of resveratrol on some specific parameters of astrocyte activity (glutamate uptake, glutamine synthetase and secretion of S100B, a neurotrophic cytokine) commonly associated with the protective role of these cells. Cell proliferation was significantly decreased by 8% and 26%, following 24h of treatment with 100 and 250 microM resveratrol. Extracellular S100B increased after 48 h of resveratrol exposure. Short-term resveratrol exposure (from 1 to 100 microM) induced a linear increase in glutamate uptake (up to 50% at 100 microM resveratrol) and in glutamine synthetase activity. Changes in these glial activities can contribute to the protective role of astrocytes in brain injury conditions, reinforcing the putative use of this compound in the therapeutic arsenal against neurodegenerative diseases and ischemic disorders.     

The Zn2+ and Ca2+‐binding S100B and S100A1 proteins: beyond the myths

The S100 genes encode a conserved group of 21 vertebrate‐specific EF‐hand calcium‐binding proteins. Since their discovery in 1965, S100 proteins have remained enigmatic in terms of their cellular functions. In this review, we summarize the calcium‐ and zinc‐binding properties of the dimeric S100B and S100A1 proteins and highlight data that shed new light on the extracellular and intracellular regulation and functions of S100B. We point out that S100B and S100A1 homodimers are not functionally interchangeable and that in a S100A1/S100B heterodimer, S100A1 acts as a negative regulator for the ability of S100B to bind Zn²⁺. The Ca²⁺ and Zn²⁺‐dependent interactions of S100B with a wide array of proteins form the basis of its activities and have led to the derivation of some initial rules for S100B recognition of protein targets. However, recent findings have strongly suggested that these rules need to be revisited. Here, we describe a new consensus S100B binding motif present in intracellular and extracellular vertebrate‐specific proteins and propose a new model for stable interactions of S100B dimers with full‐length target proteins. A chaperone‐associated function for intracellular S100B in adaptive cellular stress responses is also discussed. This review may help guide future studies on the functions of S100 proteins in general.     

Interaction of S100A13 with C2 domain of receptor for advanced glycation end products (RAGE)

S100A13 is involved in several key biological functions like angiogenesis, tumor formation and cell apoptosis. It is a homodimeric protein that belongs to the S100 protein family. S100A13 is co-expressed with acidic fibroblast growth factor (FGF1) and interleukin-1α which are key angiogenesis inducers. The S100 proteins have been shown to be involved in several cellular functions such as calcium homeostasis, cell growth and differentiation dynamic of cytoskeleton. Its biological functions are mainly mediated through the receptor for advanced glycation end products (RAGE) signaling. RAGE is involved in inflammatory processes and is associated with diabetic complications, tumor outgrowth, and neurodegenerative disorders. RAGE induces cellular signaling upon binding of different ligands, such as S100 proteins, glycated proteins, and HMGB1. RAGE signaling is complex, and it depends on the cell type and concentration of the ligand. Molecular level interactions of RAGE and S100 proteins are useful to understand the RAGE signaling diversity. In this report we focus on the molecular level interactions of S100A13 and RAGE C2 domain. The binding between RAGE C2 and S100A13 is moderately strong (K_d ~ 1.3 μM). We have solved the solution structure of the S100A13–RAGE C2 complex and pronounce the interface regions in S100A13–RAGE C2 complex which are helpful for drug development of RAGE induced diseases.     

Circular RNA circMAN2B2 facilitates glioma progression by regulating the miR-1205/S100A8 axis

The aim of this study was to research the mechanism of circMAN2B2 in the development of glioma. In our study, we found that circMAN2B2 has a higher expression in glioma tissues and cells, which was negatively related to the overall survival of glioma patients. The cell counting kit-8 assay, 5-ethynyl-2′-deoxyuridine labeling assay, transwell assay, and the nude mice assay indicated that knockdown of circMAN2B2 inhibited the cell proliferation, invasion, migration and decreased tumor size. In terms of mechanism, knockdown of circMAN2B2 increased the expression of miR-1205. Moreover, circMAN2B2 regulated S100A8 expression by inhibiting miR-1205. We also showed that knockdown of S100A8 inhibited cell proliferation, invasion, and migration. Increasing S100A8 expression rescued the effect of si-circMAN2B2. In conclusion, circMAN2B2 could improve cell proliferation, invasion, and migration of the glioma by inhibiting miR-1205 and promoting the expression of S100A8.     

S100A6 is a negative regulator of the induction of cardiac genes by trophic stimuli in cultured rat myocytes

S100A6 (calcyclin), a member of the S100 family of EF-hand Ca2+ binding proteins, has been implicated in the regulation of cell growth and proliferation. We have previously shown that S100B, another member of the S100 family, is induced postinfarction and limits the hypertrophic response of surviving cardiac myocytes. We presently report that S100A6 expression is also increased in the periinfarct zone of rat heart postinfarction and in cultured neonatal rat myocytes by treatment with several trophic agents, including platelet-derived growth factor (PDGF), the alpha1-adrenergic agonist phenylephrine (PE), and angiotensin II (AII). Cotransfection of S100A6 in cultured neonatal rat cardiac myocytes inhibits induction of the cardiac fetal gene promoters skeletal alpha-actin (skACT) and beta-myosin heavy chain (beta-MHC) by PDGF, PE, AII, and the prostaglandin F2alpha (PGF2alpha), induction of the S100B promoter by PE, and induction of the alpha-MHC promoter by triiodothyronine (T3). By contrast, S100B cotransfection selectively inhibited only PE induction of skACT and beta-MHC promoters. Fluorescence microscopy demonstrated overlapping intracellular distribution of S100B and S100A6 in transfected myocytes and in postinfarct myocardium but heterodimerization of the two proteins could not be detected by co-immunoprecipitation. We conclude that S100A6 may function as a global negative modulator of differentiated cardiac gene expression comparable to its putative role in cell cycle progression of dividing cells.     

S100B protein stimulates microglia migration via RAGE-dependent up-regulation of chemokine expression and release

The Ca(2+)-binding protein of the EF-hand type, S100B, is abundantly expressed in and secreted by astrocytes, and release of S100B from damaged astrocytes occurs during the course of acute and chronic brain disorders. Thus, the concept has emerged that S100B might act an unconventional cytokine or a damage-associated molecular pattern protein playing a role in the pathophysiology of neurodegenerative disorders and inflammatory brain diseases. S100B proinflammatory effects require relatively high concentrations of the protein, whereas at physiological concentrations S100B exerts trophic effects on neurons. Most if not all of the extracellular (trophic and toxic) effects of S100B in the brain are mediated by the engagement of RAGE (receptor for advanced glycation end products). We show here that high S100B stimulates murine microglia migration in Boyden chambers via RAGE-dependent activation of Src kinase, Ras, PI3K, MEK/ERK1/2, RhoA/ROCK, Rac1/JNK/AP-1, Rac1/NF-κB, and, to a lesser extent, p38 MAPK. Recruitment of the adaptor protein, diaphanous-1, a member of the formin protein family, is also required for S100B/RAGE-induced migration of microglia. The S100B/RAGE-dependent activation of diaphanous-1/Rac1/JNK/AP-1, Ras/Rac1/NF-κB and Src/Ras/PI3K/RhoA/diaphanous-1 results in the up-regulation of expression of the chemokines, CCL3, CCL5, and CXCL12, whose release and activity are required for S100B to stimulate microglia migration. Lastly, RAGE engagement by S100B in microglia results in up-regulation of the chemokine receptors, CCR1 and CCR5. These results suggests that S100B might participate in the pathophysiology of brain inflammatory disorders via RAGE-dependent regulation of several inflammation-related events including activation and migration of microglia.     

Substitution of methionine 63 or 83 in S100A9 and cysteine 42 in S100A8 abrogate the antifungal activities of S100A8/A9: potential role for oxidative regulation

S100A8 and S100A9 and their heterocomplex calprotectin (S100A8/A9) are abundant cytosolic constituents in human neutrophils previously shown to possess antifungal activity. This study was designed to investigate mechanisms involved in the modulation of the antifungal properties of S100A8/A9. S100A8, S100A9 and site-directed mutants of both proteins were tested for their antifungal effect against Candida albicans in microplate dilution assays. Whereas S100A8 alone did not inhibit fungal growth, S100A9 by itself had a moderate antifungal effect. Combining both proteins had the strongest effect. Supporting a potential role for oxidation in S100A8/A9, substitution of methionine 63 or 83 of S100A9 resulted in the loss of antifungal activity. Additionally, the substitution to alanine of cysteine 42 of S100A8 also caused a loss of S100A8's ability to enhance S100A9's antifungal effect. Overall, our data indicate that both S100A8 and S100A9 are required for their fully active antifungal effect and that oxidation regulates S100A8/A9 antifungal activity through mechanisms that remain to be elucidated and evaluated. Finally, together with our previous work describing the oxidation-sensitive anti-inflammatory effects of S100A8/A9, we propose that S100A8/A9 exerts an anti-inflammatory activity in healthy state and that conditions associated with oxidative stress activate the antifungal activity of S100A8/A9.     

Metastatic potential of B16-F10 melanoma cells is enhanced by extracellular S100A4 derived from RAW264.7 macrophages

S100A4, synthesized and secreted from both tumor and stroma cells, modulates an aggressive tumor phenotype in various cancers by intracellular and extracellular interactions which are not completely understood. Because of the high content of tumor-associated macrophages in melanoma, here, a syngeneic model (coculture of mouse B16-F10 melanoma cells (Mel) and RAW264.7 macrophages (M?); administration (i.v.) of Mel and M?/Mel in NMRI nu/nu mice) was used to investigate synthesis and secretion of (a) S100A4, (b) S100A4-mediated signaling and activation of NFκB, and (c) S100A4-mediated modulation of Mel invasiveness in vitro (transwell assay, transwell matrigel assay) and in vivo (metastatic lung colonization), respectively. In this model substantial S100A4 synthesis and secretion is demonstrated in M?. Macrophage-derived S100A4 promotes Mel invasiveness in a paracrine manner in vitro, which is further substantiated in control experiments using recombinant human S100A4 and Mel stably transfected with mouse S100A4. Moreover, the participation of S100A4-mediated signaling, e.g., via the receptor for advanced glycation endproducts (RAGE), resulting in activation of NFκB was demonstrated in all experimental settings. Finally, we demonstrated that interaction of macrophage-derived S100A4 with Mel results in increased metastatic lung colonization in vivo.     

S100B(betabeta) inhibits the protein kinase C-dependent phosphorylation of a peptide derived from p53 in a Ca2+-dependent manner.

S100B(betabeta) is a dimeric Ca2+-binding protein that is known to inhibit the protein kinase C (PKC)-dependent phosphorylation of several proteins. To further characterize this inhibition, we synthesized peptides based on the PKC phosphorylation domains of p53 (residues 367-388), neuromodulin (residues 37-53), and the regulatory domain of PKC (residues 19-31), and tested them as substrates for PKC. All three peptides were shown to be good substrates for the catalytic domain of PKC. As for full-length p53 (Baudier J, Delphin C, Grunwald D, Khochbin S, Lawrence JJ. 1992. Proc Natl Acad Sci USA 89:11627-11631), S100B(betabeta) binds the p53 peptide and inhibits its PKC-dependent phosphorylation (IC50 = 10 +/- 7 microM) in a Ca2+-dependent manner. Similarly, phosphorylation of the neuromodulin peptide and the PKC regulatory domain peptide were inhibited by S100B(betabeta) in the presence of Ca2+ (IC50 = 17 +/- 5 microM; IC50 = 1 +/- 0.5 microM, respectively). At a minimum, the C-terminal EF-hand Ca2+-binding domain (residues 61-72) of each S100beta subunit must be saturated to inhibit phosphorylation of the p53 peptide as determined by comparing the Ca2+ dependence of inhibition ([Ca]IC50 = 29.3 +/- 17.6 microM) to the dissociation of Ca2+ from the C-terminal EF-hand Ca2+-binding domain of S100B(betabeta).     

Identification of a dimeric intermediate in the unfolding pathway for the calcium-binding protein S100B

The S100 proteins comprise 25 calcium-signalling members of the EF-hand protein family. Unlike typical EF-hand signalling proteins such as calmodulin and troponin-C, the S100 proteins are dimeric, forming both homo- and heterodimers in vivo. One member of this family, S100B, is a homodimeric protein shown to control the assembly of several cytoskeletal proteins and regulate phosphorylation events in a calcium-sensitive manner. Calcium binding to S100B causes a conformational change involving movement of helix III in the second calcium-binding site (EF2) that exposes a hydrophobic surface enabling interactions with other proteins such as tubulin and Ndr kinase. In several S100 proteins, calcium binding also stabilizes dimerization compared to the calcium-free states. In this work, we have examined the guanidine hydrochloride (GuHCl)-induced unfolding of dimeric calcium-free S100B. A series of tryptophan substitutions near the dimer interface and the EF2 calcium-binding site were studied by fluorescence spectroscopy and showed biphasic unfolding curves. The presence of a plateau near 1.5 M GuHCl showed the presence of an intermediate that had a greater exposed hydrophobic surface area compared to the native dimer based on increased 4,4-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid fluorescence. Furthermore, ¹H-¹⁵N heteronuclear single quantum coherence analyses as a function of GuHCl showed significant chemical shift changes in regions near the EF1 calcium-binding loop and between the linker and C-terminus of helix IV. Together these observations show that calcium-free S100B unfolds via a dimeric intermediate.