NFAT transcription factors are highly phosphorylated proteins that are regulated by the calcium-dependent phosphatase calcineurin. We show by mass spectrometry that NFAT1 is phosphorylated on fourteen conserved phosphoserine residues in its regulatory domain, thirteen of which are dephosphorylated upon stimulation. Dephosphorylation of all thirteen residues is required to mask a nuclear export signal (NES), cause full exposure of a nuclear localization signal (NLS), and promote transcriptional activity. An inducible phosphorylation site in the transactivation domain contributes to transcriptional activity. Our data suggest that dephosphorylation promotes NFAT1 activation by increasing the probability of an active conformation, in a manner analogous to that by which depolarization increases the open probability of voltage-gated ion channels. This conformational switch paradigm may explain modification-induced functional changes in other heavily phosphorylated proteins.

In resting cells, the NFAT1 transcription factor is kept inactive in the cytoplasm by phosphorylation on multiple serine residues. These phosphorylated residues are primarily contained within two types of serine-rich motifs, the SRR-1 and SP motifs, which are conserved within the NFAT family. Several different kinases have been proposed to regulate NFAT, but no single candidate displays the specificity required to fully phosphorylate both types of motifs; thus, the identity of the kinase that regulates NFAT activity remains unclear. Here we show that the NFAT1 serine motifs are regulated by distinct kinases that must coordinate to control NFAT1 activation. CK1 phosphorylates only the SRR-1 motif, the primary region required for NFAT1 nuclear import. CK1 exists with NFAT1 in a high-molecular-weight complex in resting T cells but dissociates upon activation. GSK3 does not phosphorylate the SRR-1 region but can target the NFAT1 SP-2 motif, and it synergizes with CK1 to regulate NFAT1 nuclear export. We identify a conserved docking site for CK1 in NFAT proteins and show that mutation of this site disrupts NFAT1-CK1 interaction and causes constitutive nuclear localization of NFAT1. The CK1 docking motif is present in proteins of the Wnt, Hedgehog, and circadian-rhythm pathways, which also integrate the activities of CK1 and GSK3.

p300 and cAMP response element–binding protein (CREB)–binding protein (CBP) are members of a family of coactivators involved in the regulation of transcription and chromatin. We show that transcription factors of the nuclear factor of activated T cells (NFAT) family bind p300/CBP and recruit histone acetyltransferase activity from T cell nuclear extracts. The NH2-terminal transactivation domain of NFAT1 and the phospho-CREB- and E1A-binding sites of p300/CBP are involved in the interaction. The viral oncoprotein E1A inhibits NFAT-dependent transactivation in a p300-dependent manner. Recruitment of the coactivators p300/CBP by the transactivation domains of NFAT proteins is likely to play a critical role in NFAT-dependent gene expression during the immune response.

BackgroundToll-like receptors (TLRs) are transmembrane pattern-recognition receptors of the innate immune system recognizing diverse pathogen-derived and tissue damage-related ligands. It has been suggested that TLR signaling contributes to the pathogenesis of age-related, neurodegenerative diseases, including Alzheimer’s disease (AD). AD is associated to oligomers of the amyloid β peptide (Aβo) that cause intracellular Ca2+ dishomeostasis and neuron cell death in rat hippocampal neurons. Here we assessed the interplay between inflammation and Aβo in long-term cultures of rat hippocampal neurons, an in vitro model of neuron aging and/or senescence.MethodsCa2+ imaging and immunofluorescence against annexin V and TLR4 were applied in short- and long-term cultures of rat hippocampal neurons to test the effects of TLR4-agonist LPS and Aβo on cytosolic [Ca2+] and on apoptosis as well as on expression of TLR4.ResultsLPS increases cytosolic [Ca2+] and promotes apoptosis in rat hippocampal neurons in long-term culture considered aged and/or senescent neurons, but not in short-term cultured neurons considered young neurons. TLR4 antagonist CAY10614 prevents both effects. TLR4 expression in rat hippocampal neurons is significantly larger in aged hippocampal cultures. Treatment of aged hippocampal cultures with Aβo increases TLR4 expression and enhances LPS-induced Ca2+ responses and neuron cell death.ConclusionsAging and amyloid β oligomers, the neurotoxin involved in Alzheimer’s disease, enhance TLR4 expression as well as LPS-induced Ca2+ responses and neuron cell death in rat hippocampal neurons aged in vitro.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-017-0802-0) contains supplementary material, which is available to authorized users.

Long-term evidence has confirmed the involvement of an inflammatory component in neurodegenerative disorders including Alzheimer’s disease (AD). This view is supported, in part, by data suggesting that selected non-steroidal anti-inflammatory drugs (NSAIDs) provide protection. Additionally, molecular players of the innate immune system have recently been proposed to contribute to these diseases. Toll-like receptors (TLRs) are transmembrane pattern-recognition receptors of the innate immune system that recognize different pathogen-derived and tissue damage-related ligands. TLR4 mediated signaling has been reported to contribute to the pathogenesis of age-related neurodegenerative diseases, including AD. Although the pathophysiology of AD is not clear, soluble aggregates (oligomers) of the amyloid β peptide (Aβo) have been proven to be key players in the pathology of AD. Among others, Aβo promote Ca 2+ entry and mitochondrial Ca 2+ overload leading to cell death in neurons. TLR4 has recently been found to be involved in AD but the mechanisms are unclear. Our group recently reported that lipopolysaccharide (LPS), a TLR4 receptor agonist, increases cytosolic Ca 2+ concentration leading to apoptosis. Strikingly, this effect was only observed in long-term cultured primary neurons considered a model of aging neurons, but not in short-term cultured neurons resembling young neurons. These effects were significantly prevented by pharmacological blockade of TLR4 receptor signaling. Moreover, TLR4 expression in rat hippocampal neurons increased significantly in aged neurons in vitro . Therefore, molecular patterns associated with infection and/or brain cell damage may activate TLR4 and Ca 2+ signaling, an effect exacerbated during neuronal aging. Here, we briefly review the data regarding the involvement of TLR4 in AD.

Please cite this article as: Francisco, V., Figueirinha, A., Neves, B.M., García-Rodríguez, C., Lopes, M.C., Cruz, M.T., Batista, M.T., Cymbopogon citratus as source of new and safe anti-inflammatory drugs: bio-guided assay using lipopolysaccharide-stimulated macrophages, Journal of Ethnopharmacology (2010Ethnopharmacology ( ), doi:10.1016Ethnopharmacology ( /j.jep.2010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Page 1 of 30A c c e p t e d M a n u s c r i p t 1 Graphical abstractIn this report it was demonstrated that a lipid-and essential oil-free infusion of Cymbopogon citratus leaves (Cy), as well its polyphenols, have anti-inflammatory properties through inhibition of proinflammatory signaling pathways and nitric oxide production in lipopolysaccharide-stimulated macrophages. These evidences support the use of Cymbopogon citratus in traditional medicine and indicate that it could be a natural source of new and safe anti-inflammatory drugs. TitleCymbopogon citratus as source of new and safe anti-inflammatory drugs: bio-guided assay using lipopolysaccharide-stimulated macrophages Aim of the study: The aim of this study is to explore the anti-inflammatory properties of Cymbopogon citratus leaves and their polyphenol-rich fractions (PFs), as well its mechanism of action in murine macrophages. Materials and Methods:A lipid-and essential oil-free infusion of Cy leaves was prepared (Cy extract) and fractionated by column chromatography. Anti-inflammatory properties of Cy extract (1.115 mg/ml) and its PFs, namely phenolic acids (530 µg/ml), flavonoids (97.5 µg/ml) and tannins (78 µg/ml), were investigated using lipopolysaccharide (LPS)-stimulated Raw 264.7 macrophages as in vitro model. As inflammatory parameters, nitric oxide (NO) production was evaluated by Griess reaction, as well as effects on cyclooxygenase (COX-2), inducible NO synthase (iNOS) expression and on intracellular signaling pathways activation, which were analyzed by Western blot using specific antibodies. Page 3 of 30A c c e p t e d M a n u s c r i p t Conclusions: Our data provide evidence that support the usage of Cymbopogon citratus leaves extract in traditional medicine, and suggest that Cy, in particular its polyphenolic compounds, could constitute a natural source of a new and safe anti-inflammatory drugs.

Exposure of aortic valve interstitial cells to viral and Gram-negative bacteria molecular patterns induces distinct and long-term TLR-mediated pro-inflammatory and pro-osteogenic responses that might be relevant to the pathogenesis of degenerative aortic stenosis.

Given that TLRs and sphingosine-1-phosphate (S1P) are key players in inflammation, we explored the potential interplay between TLRs and S1P in the adhesion/inflammatory pathways in primary human endothelial cells. As determined by Western blot and flow cytometry, cells treated with LPS (a TLR4 ligand) and S1P showed significantly enhanced expression of adhesion molecules such as ICAM-1 and E-selectin compared with the effect of either ligand alone. Cell-type differences on E-selectin upregulation were observed. In contrast, no cooperation effect on ICAM-1 or E-selectin was observed with a TLR2/TLR1 ligand. Consistent with an increase in adhesion molecule expression, endothelial cell treatment with LPS plus S1P significantly enhanced adhesion of PBMCs under shear stress conditions compared with the effect of either ligand alone and exhibited comparable levels of cell adhesion strength as those after TNF-α treatment. Moreover, LPS and S1P cooperated to increase the expression of proinflammatory molecules such as IL-6, cyclooxygenase-2, and prostacyclin, as determined by ELISA and Western blot. The analysis of signaling pathways revealed the synergistic phosphorylation of ERK upon LPS plus S1P treatment of HUVEC and human aortic endothelial cells and cell-type differences on p38 and NF-κB activation. Moreover, pharmacological and small interfering RNA experiments disclosed the involvement of S1P1/3 and NF-κB in the cooperation effect and that cell origin determines the S1P receptors and signaling routes involved. Sphingosine kinase activity induction upon LPS plus S1P treatment suggests S1P– Sphingosine kinase axis involvement. In summary, LPS and S1P cooperate to increase proinflammatory molecules in endothelial cells and, in turn, to augment leukocyte adhesion, thus exacerbating S1P-mediated proadhesive/proinflammatory properties.