P2Y12 receptors in primary microglia activate nuclear factor of activated T-cell signaling to induce C-C chemokine 3 expression.

Microglia are widely accepted as surveillants in the central nervous system that are continually searching the local environment for signs of injury. Following an inflammatory situation, microglia alter their morphology, extend ramified processes, and undergo cell body hypertrophy. Extracellular nucleotides are recognized as a danger signal by microglia. ADP acting on P2Y12 receptors induce process extension of microglia thereby attracting microglia to the site of adenosine tri-phosphate/ADP leaking or release. However, the question whether ADP/P2Y12 receptor signaling directly stimulates the production or release of inducible factors such as cytokines remains unclear. In this study, we found that CC chemokine ligand 3 (CCL3) is induced by ADP-treated primary microglia. Pharmacological characterization using pertussis toxin, a P2Y12 receptor inhibitor, and a calcium chelator revealed that CCL3 induction was caused by P2Y12 receptor-mediated intracellular calcium elevation. Next, nuclear factor of activated T-cell dephosphorylation and nuclear translocalization were observed. Calcineurin, an inhibitor for nuclear factor of activated T cell, suppressed CCL3 induction. These data indicate that microglial P2Y12 receptors are utilized to trigger an acute inflammatory response in microglia via rapid CCL3 induction after ADP stimulation.

Quantifying MHC dextramer-induced NFAT activation in antigen-specific T cells as a functional response parameter.

MHC-multimers are reagents used for the detection and enumeration of antigen-specific T cells (ASTs). These reagents exploit the mechanism by which T cell receptors (TCR) on cytotoxic CD8 T cells recognize specific antigens in the context of a major histocompatibility complex (MHC) molecule during antigen presentation. MHC-multimers are fluorescently-labeled dextran polymers that carry MHC Class I molecules and peptide sequences that can be modified to represent specific cognate sequences of the antigen of interest with dextramers having a 10-fold multiplicity of the MHC/peptide combination within a single multimer. Since the binding of antigen-specific dextramers mimics antigen presentation to the TCR, the present study sought to determine whether this TCR engagement on the AST was sufficient to elicit a functional T cell response. The effect of binding of CMV specific dextramers on the activation of the NFAT signal transduction cascade was assessed in peripheral blood from bone marrow transplant recipients previously determined to be positive for CMV-ASTs (CASTs). NFAT activation was quantified by measuring nuclear translocation of NFAT1 in CD8+ CASTs and CD8+ non-CASTs by imaging flow cytometry. Our results demonstrate that an increase in the nuclear localization of NFAT1 was detectable in the CASTs following the CMV-dextramer binding and could be observed as early as 10min post-exposure. The NFAT1 activation correlated with a downstream functional response in the form of interferon gamma production. Sample preparation, temperature, and duration of dextramer exposure were important parameters affecting the dextramer-induced NFAT activation with 2h exposure in whole blood at room temperature being the optimal of the conditions tested. Intra- and inter-individual heterogeneity was observed with regards to the NFAT activation in the CASTs. Importantly, no effect of the dextramers was observed in the CD8+ non-CASTs, and therefore dextramer negative cell populations. Exposure to PMA/ionomycin following dextramer exposure resulted in a homogeneous NFAT activation in both the dextramer-positive but NFAT1 nonresponsive CAST and non-CAST cells. Thus, the data demonstrate that binding of antigen-specific dextramers to ASTs specifically results in activation of NFAT, that the NFAT activation correlates with a downstream functional response and that the response can be heterogeneous. This functional parameter may provide insight to the issue whether enumeration alone of ASTs is a sufficient parameter to assess an individual's immune status against a specific antigen.

Bone Morphogenetic Protein-2 (BMP-2) Activates NFATc1 Transcription Factor via an Autoregulatory Loop Involving Smad/Akt/Ca2+ Signaling.

Bone remodeling is controlled by dual actions of osteoclasts (OCs) and osteoblasts (OBs). The calcium-sensitive nuclear factor of activated T cells (NFAT) c1 transcription factor, as an OC signature gene, regulates differentiation of OCs downstream of bone morphogenetic protein-2 (BMP-2)-stimulated osteoblast-coded factors. To analyze a functional link between BMP-2 and NFATc1, we analyzed bones from OB-specific BMP-2 knock-out mice for NFATc1 expression by immunohistochemical staining and found significant reduction in NFATc1 expression. This indicated a requirement of BMP-2 for NFATc1 expression in OBs. We showed that BMP-2, via the receptor-specific Smad pathway, regulates expression of NFATc1 in OBs. Phosphatidylinositol 3-kinase/Akt signaling acting downstream of BMP-2 also drives NFATc1 expression and transcriptional activation. Under the basal condition, NFATc1 is phosphorylated. Activation of NFAT requires dephosphorylation by the calcium-dependent serine/threonine phosphatase calcineurin. We examined the role of calcium in BMP-2-stimulated regulation of NFATc1 in osteoblasts. 1,2Bis(2aminophenoxy)ethaneN,N,N',N'-tetraacetic acid acetoxymethyl ester, an inhibitor of intracellular calcium abundance, blocked BMP-2-induced transcription of NFATc1. Interestingly, BMP-2 induced calcium release from intracellular stores and increased calcineurin phosphatase activity, resulting in NFATc1 nuclear translocation. Cyclosporin A, which inhibits calcineurin upstream of NFATc1, blocked BMP-2-induced NFATc1 mRNA and protein expression. Expression of NFATc1 directly increased its transcription and VIVIT peptide, an inhibitor of NFATc1, suppressed BMP-2-stimulated NFATc1 transcription, confirming its autoregulation. Together, these data show a role of NFATc1 downstream of BMP-2 in mouse bone development and provide novel evidence for the presence of a cross-talk among Smad, phosphatidylinositol 3-kinase/Akt, and Ca(2+) signaling for BMP-2-induced NFATc1 expression through an autoregulatory loop.

Stalk-dependent and Stalk-independent Signaling by the Adhesion G Protein-coupled Receptors GPR56 (ADGRG1) and BAI1 (ADGRB1).

The adhesion G protein-coupled receptors (aGPCRs) are a large yet poorly understood family of seven-transmembrane proteins. A defining characteristic of the aGPCR family is the conserved GAIN domain, which has autoproteolytic activity and can cleave the receptors near the first transmembrane domain. Several aGPCRs, including ADGRB1 (BAI1 or B1) and ADGRG1 (GPR56 or G1), have been found to exhibit significantly increased constitutive activity when truncated to mimic GAIN domain cleavage (ΔNT). Recent reports have suggested that the new N-terminal stalk, which is revealed by GAIN domain cleavage, can directly activate aGPCRs as a tethered agonist. We tested this hypothesis in studies on two distinct aGPCRs, B1 and G1, by engineering mutant receptors lacking the entire NT including the stalk (B1- and G1-SL, with "SL" indicating "stalkless"). These receptors were evaluated in a battery of signaling assays and compared with full-length wild-type and cleavage-mimicking (ΔNT) forms of the two receptors. We found that B1-SL, in multiple assays, exhibited robust signaling activity, suggesting that the membrane-proximal stalk region is not necessary for its activation. For G1, however, the results were mixed, with the SL mutant exhibiting robust activity in several signaling assays (including TGFα shedding, activation of NFAT luciferase, and ß-arrestin recruitment) but reduced activity relative to ΔNT in a distinct assay (activation of SRF luciferase). These data support a model in which the activation of certain pathways downstream of aGPCRs is stalk-dependent, whereas signaling to other pathways is stalk-independent.

Exposure to nerve growth factor worsens nephrotoxic effect induced by Cyclosporine A in HK-2 cells.

Nerve growth factor is a neurotrophin that promotes cell growth, differentiation, survival and death through two different receptors: TrkA(NTR) and p75(NTR). Nerve growth factor serum concentrations increase during many inflammatory and autoimmune diseases, glomerulonephritis, chronic kidney disease, end-stage renal disease and, particularly, in renal transplant. Considering that nerve growth factor exerts beneficial effects in the treatment of major central and peripheral neurodegenerative diseases, skin and corneal ulcers, we asked whether nerve growth factor could also exert a role in Cyclosporine A-induced graft nephrotoxicity. Our hypothesis was raised from basic evidence indicating that Cyclosporine A-inhibition of calcineurin-NFAT pathway increases nerve growth factor expression levels. Therefore, we investigated the involvement of nerve growth factor and its receptors in the damage exerted by Cyclosporine A in tubular renal cells, HK-2. Our results showed that in HK-2 cells combined treatment with Cyclosporine A + nerve growth factor induced a significant reduction in cell vitality concomitant with a down-regulation of Cyclin D1 and up-regulation of p21 levels respect to cells treated with Cyclosporine A alone. Moreover functional experiments showed that the co-treatment significantly up-regulated human p21promoter activity by involvement of the Sp1 transcription factor, whose nuclear content was negatively regulated by activated NFATc1. In addition we observed that the combined exposure to Cyclosporine A + nerve growth factor promoted an up-regulation of p75 (NTR) and its target genes, p53 and BAD leading to the activation of intrinsic apoptosis. Finally, the chemical inhibition of p75(NTR) down-regulated the intrinsic apoptotic signal. We describe two new mechanisms by which nerve growth factor promotes growth arrest and apoptosis in tubular renal cells exposed to Cyclosporine A.

A selective antagonist reveals a potential role of G protein-coupled receptor 55 in platelet and endothelial cell function.

The G protein-coupled receptor 55 (GPR55) is a lysophosphatidylinositol (LPI) receptor that is also responsive to certain cannabinoids. Although GPR55 has been implicated in several (patho)physiologic functions, its role remains enigmatic owing mainly to the lack of selective GPR55 antagonists. Here we show that the compound CID16020046 ((4-[4-(3-hydroxyphenyl)-3-(4-methylphenyl)-6-oxo-1H,4H,5H,6H-pyrrolo[3,4-c]pyrazol-5-yl] benzoic acid) is a selective GPR55 antagonist. In yeast cells expressing human GPR55, CID16020046 antagonized agonist-induced receptor activation. In human embryonic kidney (HEK293) cells stably expressing human GPR55, the compound behaved as an antagonist on LPI-mediated Ca²âº release and extracellular signal-regulated kinases activation, but not in HEK293 cells expressing cannabinoid receptor 1 or 2 (CB1 or CB2). CID16020046 concentration dependently inhibited LPI-induced activation of nuclear factor of activated T-cells (NFAT), nuclear factor κ of activated B cells (NF-κB) and serum response element, translocation of NFAT and NF-κB, and GPR55 internalization. It reduced LPI-induced wound healing in primary human lung microvascular endothelial cells and reversed LPI-inhibited platelet aggregation, suggesting a novel role for GPR55 in platelet and endothelial cell function. CID16020046 is therefore a valuable tool to study GPR55-mediated mechanisms in primary cells and tissues.

NFATc1 induction in peripheral T and B lymphocytes.

NFAT transcription factors control the proliferation and survival of peripheral lymphocytes. We have reported previously that the short isoform NFATc1/αA whose generation is induced by immune receptor stimulation supports the proliferation and inhibits the activation-induced cell death of peripheral T and B cells. We will show in this study that in novel bacterial artificial chromosome transgenic mice that express EGFP under the control of entire Nfatc1 locus the Nfatc1/Egfp transgene is expressed as early as in double-negative thymocytes and in nonstimulated peripheral T and B cells. Upon immune receptor stimulation, Nfatc1/Egfp expression is elevated in B, Th1, and Th2 cells, but only weakly in T regulatory, Th9, and Th17 cells in vitro whose generation is affected by TGFß. In naive lymphocytes, persistent immune receptor signals led to a 3-5 increase in NFATc1/αA RNA levels during primary and secondary stimulation, but a much stronger induction was observed at the protein level. Whereas anti-CD3(+)CD28 stimulation of primary T cells induces both NFATc1/αA and their proliferation and survival, anti-IgM stimulation of B cells induces NFATc1/αA and proliferation, but activation-induced cell death after 3-d incubation in vitro. The anti-IgM-mediated activation-induced cell death induction of B cells in vitro is suppressed by anti-CD40-, LPS-, and CpG-mediated signals. In addition to inducing NF-κB factors, together with anti-IgM, these signals also support the generation of NFATc1/αA. According to these data and the architecture of its promoter region, the Nfatc1 gene resembles a primary response gene whose induction is affected at the posttranscriptional level.

The lectin ArtinM binds to mast cells inducing cell activation and mediator release.

Mast cells are inflammatory cells that respond to signals of innate and adaptive immunity with immediate and delayed release of mediators. ArtinM, a lectin from Artocarpus integrifolia with immunomodulatory activities, is able to induce mast cell activation, but the mechanisms remain unknown. This study sought to further investigate the effects of the lectin on mast cells. We showed that ArtinM binds to mast cells, possibly to the high affinity receptor for immunoglobulin E (IgE) - FcεRI - and/or to IgE bound to FcεRI. Binding of the lectin resulted in protein tyrosine phosphorylation and release of the pre- and newly-formed mediators, ß-hexosaminidase and LTB(4) by mast cells, activities that were potentiated in the presence of IgE. ArtinM also induced the activation of the transcription factors NFκB and NFAT, resulting in expression of some of their target genes such as IL-4 and TNF-α. In view of the established significance of mast cells in many immunological and inflammatory reactions, a better understanding of the mechanisms involved in mast cell activation by ArtinM is crucial to the pharmacological application of the lectin.

Repression of NFAT3 transcriptional activity by estrogen receptors.

Nuclear factor of activated T cells 3 (NFAT3) activities have been implicated in many biological processes, such as breast cancer, cardiac hypertrophy, learning and memory, and adipocyte differentiation. However, how protein factors regulate NFAT3 transcriptional activity is poorly understood. Here, we report that regardless of estrogen, overexpression of estrogen receptor alpha and beta (ERalpha and ERbeta) suppresses NFAT3 transcriptional activity, whereas knockdown of endogenous ERalpha and ERbeta enhances the activity. Estrogen further enhances ER inhibition of NFAT3-dependent transcription. ERalpha and ERbeta interact with NFAT3 independently of the NFAT agonists phorbol myristate acetate (PMA) and ionomycin, and ERalpha is recruited to an NFAT3 target gene promoter. Phosphorylation of ERalpha at different sites differentially affects ERalpha modulation of NFAT3 transcriptional activity. These results suggest that ER may play a critical role in regulation of NFAT3 transcriptional activity.

Down syndrome candidate region 1 isoform 1 mediates angiogenesis through the calcineurin-NFAT pathway.

Down syndrome candidate region 1 (DSCR1) is one of more than 50 genes located in a region of chromosome 21 that has been implicated in Down syndrome. DSCR1 can be expressed as four isoforms, one of which, isoform 4 (DSCR1-4), has recently been found to be strongly induced by vascular endothelial growth factor A (VEGF-A(165)) and to provide a negative feedback loop that inhibits VEGF-A(165)-induced endothelial cell proliferation in vitro and angiogenesis in vivo. We report here that another DSCR1 isoform, DSCR1-1L, is also up-regulated by VEGF-A(165) in cultured endothelial cells and is strongly expressed in several types of pathologic angiogenesis in vivo. In contrast to DSCR1-4, the overexpression of DSCR1-1L induced the proliferation and activation of the transcription factor NFAT in cultured endothelial cells and promoted angiogenesis in Matrigel assays in vivo, even in the absence of VEGF-A. Similarly, small interfering RNAs specific for DSCR1-1L and DSCR1-4 had opposing inhibitory and stimulatory effects, respectively, on these same functions. DSCR1-4 is thought to inhibit angiogenesis by inactivating calcineurin, thereby preventing activation and nuclear translocation of NFAT, a key transcription factor. In contrast, DSCR1-1L, regulated by a different promoter than DSCR1-4, activates NFAT and its proangiogenic activity is inhibited by cyclosporin, an inhibitor of calcineurin. In sum, DSCR1-1L, unlike DSCR1-4, potently activates angiogenesis and could be an attractive target for antiangiogenesis therapy.