S100B Increases Proliferation in PC12 Neuronal Cells and Reduces Their Responsiveness to Nerve Growth Factor via Akt Activation

S100B is a Ca2+-modulated protein of the EF-hand type expressed in high abundance in a restricted set of cell types including certain neuronal populations. S100B has been suggested to participate in cell cycle progression, and S100B levels are high in tumor cells, compared with normal parental cells. We expressed S100B in the neuronal cell line PC12, which normally does not express the protein, by the Tet-Off technique, and found the following: (i) proliferation was higher in S100B+ PC12 cells than in S100B- PC12 cells; (ii) nerve growth factor (NGF), which decreased the proliferation of S100B- PC12 cells, was less effective in the case of S100B+ PC12 cells; (iii) expression of S100B made PC12 cells resistant to the differentiating effect of NGF; and (iv) interruption of S100B expression did not result in an immediate restoration of PC12 cell sensitivity to the differentiating effect of NGF. Expression of S100B in PC12 cells resulted in activation of Akt; increased levels of p21WAF1, an inhibitor of cyclin-dependent kinase (cdk) 2 and a positive regulator of cdk4; increased p21WAF1-cyclin D1 complex formation; and increased phosphorylation of the retinoblastoma suppressor protein, Rb. These S100B-induced effects, as well as the reduced ability of S100B+ PC12 cells to respond to NGF, were dependent on Akt activation because they were remarkably reduced or abrogated in the presence of LY294002, an inhibitor of the Akt upstream kinase phosphatidylinositol 3-kinase. Thus, S100B might promote cell proliferation and interfere with NGF-induced PC12 cell differentiation by stimulating a p21WAF1/cyclin D1/cdk4/Rb/E2F pathway in an Akt-mediated manner

S100B Protein, A Damage-Associated Molecular Pattern Protein in the Brain and Heart, and Beyond

S100B belongs to a multigenic family of Ca2+-binding proteins of the EF-hand type and is expressed in high abundance in the brain. S100B interacts with target proteins within cells thereby altering their functions once secreted/released with the multiligand receptor RAGE. As an intracellular regulator, S100B affects protein phosphorylation, energy metabolism, the dynamics of cytoskeleton constituents (and hence, of cell shape and migration), Ca2+ homeostasis, and cell proliferation and differentiation. As an extracellular signal, at low, physiological concentrations, S100B protects neurons against apoptosis, stimulates neurite outgrowth and astrocyte proliferation, and negatively regulates astrocytic and microglial responses to neurotoxic agents, while at high doses S100B causes neuronal death and exhibits properties of a damage-associated molecular pattern protein. S100B also exerts effects outside the brain; as an intracellular regulator, S100B inhibits the postinfarction hypertrophic response in cardiomyocytes, while as an extracellular signal, (high) S100B causes cardiomyocyte death, activates endothelial cells, and stimulates vascular smooth muscle cell proliferation

An in vitro study of the mTORC1/2 inhibitor PP242 in glioblastoma multiforme

mTOR is a kinase complex involved in cell growth, proliferation, survival, metabolism and migration. The aberrant activation of mTOR has been previously demonstrated in glioblastoma multiforme (GBM), making it an interesting target for therapeutic approaches [1]. Unfortunately, the attempts to block mTOR activity made so far had disappointing clinical efficacy, as the mTOR inhibitor Rapamycin and analogs only target mTOR complex 1 (mTORC1) while mTOR actually exists in two distinct complexes, namely mTORC1 and mTOR complex 2 (mTORC2) that differ in terms of both regulation mechanisms and functions [2,3]. mTORC1 is inhibited by Rapamycin and acts as a downstream effector of the PTEN/PI3K/Akt pathway, linking growth factors, amino acids, ATP and O2 signals to protein translation, cell growth, proliferation and survival. Differently, mTORC2 is insensible to Rapamycin and acts as an upstream activator of Akt via phosphorylation of serine 473 [3]. To analyze the contribution of mTORC1/2 to GBM biology, we studied the in vitro effect of PP242, a novel mTORC1/2 inhibitor, on glioma cell lines of different malignancy degree, and compared it to the effect of Rapamycin and of the irreversible PI3K inhibitor, Wortmannin. Our results suggest that the inhibition of both mTOR complexes with PP242 induces sustained levels of autophagy that causes G0/G1 cell cycle arrest and a significantly reduction of cell viability, proliferation and migration. Additionally, we observed that administration of PP242 in U87MG cell line prevents stem cell growth, which results in the inhibition of neurospheres formation. This data confirms the pivotal role of mTOR in glioblastoma cells biology and expand upon this evidence suggesting a prominent role of the mTOR complex 2 in glioblastoma cell growth, migration and survival, and indicate that the mTORC2 might represent clinically valuable target in GMB

S100B's double life: Intracellular regulator and extracellular signal

AbstractThe Ca2+-binding protein of the EF-hand type, S100B, exerts both intracellular and extracellular functions. Recent studies have provided more detailed information concerning the mechanism(s) of action of S100B as an intracellular regulator and an extracellular signal. Indeed, intracellular S100B acts as a stimulator of cell proliferation and migration and an inhibitor of apoptosis and differentiation, which might have important implications during brain, cartilage and skeletal muscle development and repair, activation of astrocytes in the course of brain damage and neurodegenerative processes, and of cardiomyocyte remodeling after infarction, as well as in melanomagenesis and gliomagenesis. As an extracellular factor, S100B engages RAGE (receptor for advanced glycation end products) in a variety of cell types with different outcomes (i.e. beneficial or detrimental, pro-proliferative or pro-differentiative) depending on the concentration attained by the protein, the cell type and the microenvironment. Yet, RAGE might not be the sole S100B receptor, and S100B's ability to engage RAGE might be regulated by its interaction with other extracellular factors. Future studies using S100B transgenic and S100B null mice might shed more light on the functional role(s) of the protein

Inhibitory effects of agmatine on monoamine oxidase (MAO) activity: Reconciling the discrepancies

Abstract Agmatine has been functionally characterized as an important hormone and co-neurotransmitter in mammals. Given its ability in binding Imidazoline sites, a regolatory site of monoaminoxydase, it has been suggested to be involved in many neurological aspects. However, its inhibitory effect on this enzyme still remains an unanswered question. This present study is aimed to asses whether different experimental conditions could affect the agmatine action on monoaminoxydase activity. We demonstrate that the monoaminoxydase inhibition by agmatine is obtained under alkaline conditions and a long time of incubation. No inhibitiory action was found for shorter times of reaction at elevated pH, or at neutral condition and long time of incubation. No inhibition was also detected by substituting the monoamineoxydase substrate tyramine with kynuramine, however, while in these conditions a remarkable inhibition was shown by two aminoxydase inhibitors tranylcypromine and idazoxan. Herein, we discuss a mechanism model and the functional consequences of agmatine action on monoaminoxydase

Rat Brain Cortex Mitochondria Release Group II Secretory Phospholipase A2 under Reduced Membrane Potential

Activation of brain mitochondrial phospholipase(s) A(2) (PLA(2)) might contribute to cell damage and be involved in neurodegeneration. Despite the potential importance of the phenomenon, the number, identities, and properties of these enzymes are still unknown. Here, we demonstrate that isolated mitochondria from rat brain cortex, incubated in the absence of respiratory substrates, release a Ca(2+)-dependent PLA(2) having biochemical properties characteristic to secreted PLA(2) (sPLA(2)) and immunoreacting with the antibody raised against recombinant type IIA sPLA(2) (sPLA(2)-IIA). Under identical conditions, no release of fumarase in the extramitochondrial medium was observed. The release of sPLA(2) from mitochondria decreases when mitochondria are incubated in the presence of respiratory substrates such as ADP, malate, and pyruvate, which causes an increase of transmembrane potential determined by cytofluorimetric analysis using DiOC(6)(3) as a probe. The treatment of mitochondria with the uncoupler carbonyl cyanide 3-chlorophenylhydrazone slightly enhances sPLA(2) release. The increase of sPLA(2) specific activity after removal of mitochondrial outer membrane indicates that the enzyme is associated with mitoplasts. The mitochondrial localization of the enzyme has been confirmed by electron microscopy in U-251 astrocytoma cells and by confocal laser microscopy in the same cells and in PC-12 cells, where the structurally similar isoform type V-sPLA(2) has mainly nuclear localization. In addition to sPLA(2), mitochondria contain another phospholipase A(2) that is Ca(2+)-independent and sensitive to bromoenol lactone, associated with the outer mitochondrial membrane. We hypothesize that, under reduced respiratory rate, brain mitochondria release sPLA(2)-IIA that might contribute to cell damage

Oxidative stress-induced S100B accumulation in myoblasts converts myoblasts into brown preadipocytes via an NF-κB/YY1/MIR-133 axis and NF-κB/YY1/BMP7 axis

Muscles of sarcopenic people show hypotrophic myofibers and infiltration with adipose and, at later stages, fibrotic tissue. The origin of infiltrating adipocytes resides in fibro-adipogenic precursors, nonmyogenic mesenchymal progenitor cells, and satellite cells, the adult stem cells of skeletal muscles. Myoblasts and brown adipocytes share a common Myf5+ progenitor cell, and cell fate decision depends on levels of BMP7, a TGF-β family member; high BMP7 levels cause Myf5+ progenitor cells to differentiate in brown adipocytes. When expressed at relatively high levels as observed in myoblasts from sarcopenic humans, intracellular S100B, a Ca2+-binding protein of the EF-hand type (1), exerts anti-myogenic effects that are reversed by S100B knockdown (2,3). We show that ROS-activated NF-κB induces accumulation of S100B that causes myoblasts to convert into brown preadipocytes via 1) an NF-κB/YY1 axis that negatively regulates the promyogenic and anti-brown adipogenic miR-133 with consequent accumulation of the pro-brown adipogenic transcription factor, PRDM16, and 2) an NF-κB/YY1/BMP7 axis with resultant BMP7 autocrine activity. Also, culturing L6C8 (S100b-overexpressing) myoblasts (2) in adipocyte differentiation medium causes NF-κB-dependent upregulation of S100B expression, which precedes and is required for lipid droplet formation. Lastly, S100B knockdown in myoblast-derived brown adipocytes reconvert them into fusion competent myoblasts. Thus, S100B is a major molecular determinant of cell fate decision of proliferating myoblasts; while modulating myoblast differentiation (2,3), at high levels S100B promotes myoblast-brown adipocyte transition, which might have pathophysiological implications in sarcopenia.This work was supported by grants from MIUR FIRB RBFR12BUMH_003 and Fondazione CRP 2016.0136.021

The many faces of S100B protein: when an extracellular factor inactivates its own receptor and activates another one

The Ca2+-binding protein of the EF­hand type, S100B, is an intracellular regulator and an extracellular signal. Within cells S100B interacts with several proteins thereby regulating energy metabolism, Ca2+ homeostasis, protein phosphorylation and degradation, and cell locomotion, proliferation and differentiation. Once secreted/released, S100B exerts autocrine and paracrine effects on responsive cells by engaging the receptor for advanced glycation end products. However, recent evidence suggests that S100B might also activate basic fibroblast growth factor receptor 1 via prior binding to basic fibroblast growth factor

Nuclear lipid microdomains regulate daunorubicin resistance in hepatoma cells

Daunorubicin is an anticancer drug, and cholesterol is involved in cancer progression, but their relationship has not been defined. In this study, we developed a novel experimental model that utilizes daunorubicin, cholesterol, and daunorubicin plus cholesterol in the same cells (H35) to search for the role of nuclear lipid microdomains, rich in cholesterol and sphingomyelin, in drug resistance. We find that the daunorubicin induces perturbation of nuclear lipid microdomains, localized in the inner nuclear membrane, where active chromatin is anchored. As changes of sphingomyelin species in nuclear lipid microdomains depend on neutral sphingomyelinase activity, we extended our studies to investigate whether the enzyme is modulated by daunorubicin. Indeed the drug stimulated the sphingomyelinase activity that induced reduction of saturated long chain fatty acid sphingomyelin species in nuclear lipid microdomains. Incubation of untreated-drug cells with high levels of cholesterol resulted in the inhibition of sphingomyelinase activity with increased saturated fatty acid sphingomyelin species. In daunodubicin-treated cells, incubation with cholesterol reversed the action of the drug by acting via neutral sphingomyelinase. In conclusion, we suggest that cholesterol and sphingomyelin-forming nuclear lipid microdomains are involved in the drug resistance

Acute Delta Hepatitis in Italy spanning three decades (1991–2019): Evidence for the effectiveness of the hepatitis B vaccination campaign

Updated incidence data of acute Delta virus hepatitis (HDV) are lacking worldwide. Our aim was to evaluate incidence of and risk factors for acute HDV in Italy after the introduction of the compulsory vaccination against hepatitis B virus (HBV) in 1991. Data were obtained from the National Surveillance System of acute viral hepatitis (SEIEVA). Independent predictors of HDV were assessed by logistic-regression analysis. The incidence of acute HDV per 1-million population declined from 3.2 cases in 1987 to 0.04 in 2019, parallel to that of acute HBV per 100,000 from 10.0 to 0.39 cases during the same period. The median age of cases increased from 27 years in the decade 1991-1999 to 44 years in the decade 2010-2019 (p < .001). Over the same period, the male/female ratio decreased from 3.8 to 2.1, the proportion of coinfections increased from 55% to 75% (p = .003) and that of HBsAg positive acute hepatitis tested for by IgM anti-HDV linearly decreased from 50.1% to 34.1% (p < .001). People born abroad accounted for 24.6% of cases in 2004-2010 and 32.1% in 2011-2019. In the period 2010-2019, risky sexual behaviour (O.R. 4.2; 95%CI: 1.4-12.8) was the sole independent predictor of acute HDV; conversely intravenous drug use was no longer associated (O.R. 1.25; 95%CI: 0.15-10.22) with this. In conclusion, HBV vaccination was an effective measure to control acute HDV. Intravenous drug use is no longer an efficient mode of HDV spread. Testing for IgM-anti HDV is a grey area requiring alert. Acute HDV in foreigners should be monitored in the years to come