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Objectives To investigate the inhibitory effects of Kaempferol, a natural flavonol active compound, on pseudo-allergic reactions (in vivo and in vitro), particularly on the mechanism underlying its effect in human mast cells. Methods Compound 48/80 (C48/80)-induced immunoglobulin E (IgE)-independent passive cutaneous anaphylaxis (PCA) model and systemic anaphylaxis were applied to investigate the anti-allergic activity of Kaempferol. The degranulation assay, calcium imaging and the secretion of cytokines and chemokines were used to evaluate the inhibitory effect on mast cell activation. Western blot analysis was performed to investigate intracellular calcium fluctuation-related signalling pathways. Key findings Kaempferol dose-dependently attenuated C48/80-induced mice hind paw swelling, dye extravasation and skin mast cell degranulation, and rehabilitated the hypothermia, as well as reduced the serum concentrations of histamine, tryptase, tumour necrosis factor-alpha (TNF-α), interleukin-8 (IL-8) and monocyte chemo-attractant protein-1 (MCP-1). Furthermore, Kaempferol suppressed C48/80-triggered human MC degranulation and calcium fluctuations by inhibiting phospholipase Cγ (PLCγ) phosphorylation and subsequent cytokines synthesis pathways. Conclusions The inhibition of the process of PLCγ phosphorylation to Ca2+ mobilization represents a major strategy in Kaempferol-suppressed pseudo-allergic reactions. Thus, Kaempferol could be considered as a therapeutic drug candidate for non-IgE-mediated allergic reactions or inflammations. [ABSTRACT FROM AUTHOR]

Objectives: To investigate the inhibitory effects of Kaempferol, a natural flavonol active compound, on pseudo‐allergic reactions (in vivo and in vitro), particularly on the mechanism underlying its effect in human mast cells. Methods: Compound 48/80 (C48/80)‐induced immunoglobulin E (IgE)‐independent passive cutaneous anaphylaxis (PCA) model and systemic anaphylaxis were applied to investigate the anti‐allergic activity of Kaempferol. The degranulation assay, calcium imaging and the secretion of cytokines and chemokines were used to evaluate the inhibitory effect on mast cell activation. Western blot analysis was performed to investigate intracellular calcium fluctuation‐related signalling pathways. Key findings: Kaempferol dose‐dependently attenuated C48/80‐induced mice hind paw swelling, dye extravasation and skin mast cell degranulation, and rehabilitated the hypothermia, as well as reduced the serum concentrations of histamine, tryptase, tumour necrosis factor‐alpha (TNF‐α), interleukin‐8 (IL‐8) and monocyte chemo‐attractant protein‐1 (MCP‐1). Furthermore, Kaempferol suppressed C48/80‐triggered human MC degranulation and calcium fluctuations by inhibiting phospholipase Cγ (PLCγ) phosphorylation and subsequent cytokines synthesis pathways. Conclusions: The inhibition of the process of PLCγ phosphorylation to Ca2+ mobilization represents a major strategy in Kaempferol‐suppressed pseudo‐allergic reactions. Thus, Kaempferol could be considered as a therapeutic drug candidate for non‐IgE‐mediated allergic reactions or inflammations. [ABSTRACT FROM AUTHOR]

Vav1 is a hematopoietic-specific Rho GDP/GTP exchange factor and signaling adaptor. Although these activities are known to be stimulated by direct Vav1 phosphorylation, little information still exists regarding the regulatory layers that influence the overall Vav1 activation cycle. Using a collection of cell models and activation-mimetic Vav1 mutants, we show here that the dephosphorylated state of Vav1 in nonstimulated T cells requires the presence of a noncatalytic, phospholipase Cγ1–Slp76-mediated inhibitory pathway. Upon T cell stimulation, Vav1 becomes rapidly phosphorylated via the engagement of Lck and, to a much lesser extent, other Src family kinases and Zap70. In this process, Lck, Zap70 and the adaptor protein Lat contribute differently to the dynamics and amplitude of the Vav1 phosphorylated pool. Consistent with a multiphosphosite activation mechanism, the optimal stimulation of Vav1 can only be recapitulated by the combination of several activation-mimetic phosphosite mutants. The analysis of these mutants has also unveiled the presence of different Vav1 signaling competent states that are influenced by phosphosites present in the N- and C-terminal domains of the protein. [ABSTRACT FROM AUTHOR]

Endocrine fibroblast growth factor 23 (FGF23) is predominantly secreted by osteocytes and facilitates renal phosphate excretion. However, FGF23 is also present in cerebrospinal fluid. In chronic kidney disease, FGF23 serum levels are excessively elevated and associated with learning and memory deficits. Structural plasticity of the hippocampus such as formation of new synapses or an altered dendritic arborization comprises a cellular and morphological correlate of memory formation. Therefore, we hypothesize that FGF23 alters hippocampal neuron morphology and synapses. To address this, we prepared primary murine hippocampal cultures and incubated them with recombinant FGF23 alone or together with a soluble isoform of its co-receptor α-Klotho. Neuronal expression of a fluorescent reporter allowed for a detailed evaluation of the neuronal morphology by Sholl analysis. Additionally, we evaluated synaptic density, identified by stainings, for synaptic markers. We show an enhanced number of primary neurites combined with a reduced arborization, resulting in a less complex morphology of neurons treated with FGF23. Moreover, FGF23 enhances the synaptic density in a FGF-receptor (FGF-R) dependent manner. Finally, we addressed the corresponding signaling events downstream of FGF-R employing a combination of western blots and quantitative immunofluorescence. Interestingly, FGF23 induces phospholipase Cγ activity in primary hippocampal neurons. Co-application of soluble α-Klotho leads to activation of the Akt-pathway and modifies FGF23-impact on neuronal morphology and synaptic density. Compared with other FGFs, this alternative signaling pattern is a possible reason for differential effects of FGF23 on hippocampal neurons and may thereby contribute to learning and memory deficits in chronic kidney disease patients. [ABSTRACT FROM AUTHOR]

In vitro and in vivo imaging of protein tyrosine kinase activity requires minimally invasive, molecularly precise optical probes to provide spatiotemporal mechanistic information of dimerization and complex formation with downstream effectors. We present here a construct with genetically encoded, site-specifically incorporated, bioorthogonal reporter that can be selectively labelled with exogenous fluorogenic probes to monitor the structure and function of fibroblast growth factor receptor (FGFR). GyrB.FGFR1KD.TC contains a coumermycin-induced artificial dimerizer (GyrB), FGFR1 kinase domain (KD) and a tetracysteine (TC) motif that enables fluorescent labelling with biarsenical dyes FlAsH-EDT 2 and ReAsH-EDT 2 . We generated bimolecular system for time-resolved FRET (TR-FRET) studies, which pairs FlAsH-tagged GyrB.FGFR1KD.TC and N-terminal Src homology 2 (nSH2) domain of phospholipase Cγ (PLCγ), a downstream effector of FGFR1, fused to mTurquoise fluorescent protein (mTFP). We demonstrated phosphorylation-dependent TR-FRET readout of complex formation between mTFP.nSH2 and GyrB.FGFR1KD.TC. By further application of TR-FRET, we also demonstrated formation of the GyrB.FGFR1KD.TC homodimer by coumermycin-induced dimerization. Herein, we present a spectroscopic FRET approach to facilitate and propagate studies that would provide structural and functional insights for FGFR and other tyrosine kinases. [ABSTRACT FROM AUTHOR]

Abstract: We report a new step in the fertilization in Xenopus laevis which has been found to involve activation of Src tyrosine kinase to stimulate phospholipase C-γ (PLC-γ) which increases inositol 1,4,5-trisphosphate (IP3) to release intracellular calcium ([Ca] i ). Molecular species analysis and mass measurements suggested that sperm activate phospholipase D (PLD) to elevate phosphatidic acid (PA). We now report that PA mass increased 2.7 fold by 1min after insemination and inhibition of PA production by two methods inhibited activation of Src and PLCγ, increased [Ca] i and other fertilization events. As compared to 14 other lipids, PA specifically bound Xenopus Src but not PLCγ. Addition of synthetic PA activated egg Src (an action requiring intact lipid rafts) and PLCγ as well as doubling the amount of PLCγ in rafts. In the absence of elevated [Ca] i , PA addition elevated IP3 mass to levels equivalent to that induced by sperm (but twice that achieved by calcium ionophore). Finally, PA induced [Ca] i release that was blocked by an IP3 receptor inhibitor. As only PLD1b message was detected, and Western blotting did not detect PLD2, we suggest that sperm activate PLD1b to elevate PA which then binds to and activates Src leading to PLCγ stimulation, IP3 elevation and [Ca] i release. Due to these and other studies, PA may also play a role in membrane fusion events such as sperm–egg fusion, cortical granule exocytosis, the elevation of phosphatidylinositol 4,5-bisphosphate and the large, late increase in sn 1,2-diacylglycerol in fertilization. [Copyright &y& Elsevier]

Abstract: In the previous paper, we reported that apolipoprotein (apo) A-I enhances generation of HDL-like lipoproteins in rat astrocytes to be accompanied with both increase in tyrosine phosphorylation of phospholipase Cγ (PL-Cγ) and PL-Cγ translocation to cytosolic lipid–protein particles (CLPP) fraction. In this paper, we studied the interaction between apoA-I and ATP-binding cassette transporter A1 (ABCA1) to relate with PL-Cγ function for generation of HDL-like lipoproteins in the apoA-I-stimulated astrocytes. ABCA1 co-migrated with exogenous apoA-I with apparent molecular weight over 260kDa on SDS-PAGE when rat astrocytes were treated with apoA-I and then with a cross-linker, BS3. The solubilized ABCA1 of rat astrocytes was associated with the apoA-I-immobilized Affi-Gel 15. An LXR agonist, To901317, increased the cellular level of ABCA1, association of apoA-I with ABCA1 and apoA-I-mediated lipid release in rat astrocytoma GA-1/Mock cells where ABCA1 expression at baseline is very low. PL-Cγ was co-isolated by apoA-I-immobilized Affi-Gel 15 and co-immunoprecipitated by anti-ABCA1 antibody along with ABCA1 from the solubilized membrane fraction of rat astrocytes. The SiRNA of ABCA1 suppressed not only the PL-Cγ binding to ABCA1 but also the tyrosine phosphorylation of PL-Cγ. A PL-C inhibitor, U73122, prevented generation of apoA-I-mediated HDL-like lipoproteins in rat astrocytes. To901317 increased the association of PL-Cγ with ABCA1 in GA-1/Mock cells dependently on the increase of cellular level of ABCA1 without changing that of PL-Cγ. These findings suggest that the exogenous apoA-I augments the interaction between PL-Cγ and ABCA1 to stimulate tyrosine phosphorylation and activation of PL-Cγ for generation of HDL-like lipoproteins in astrocytes. [Copyright &y& Elsevier]

In response to injury, airway epithelia utilize an epidermal growth factor (EGF) receptor (EGFR) signaling program to institute repair and restitution. Protein tyrosine phosphatases (PTPs) counterregulate EGFR autophosphorylation and downstream signaling. PTPμμ is highly expressed in lung epithelia and can be localized to intercellular junctions where its ectodomain homophilically interacts with PTPμμ ectodomain expressed on neighboring cells. We asked whether PTPμμ expression might be altered in response to epithelial injury and whether altered PTPμμ expression might influence EGFR signaling. In A549 cells, diverse injurious stimuli dramatically reduced PTPμμ protein expression. Under basal conditions, small interfering RNA (siRNA)-induced silencing of PTPμμ increased EGFR Y992 and Y1068 phosphorylation. In the presence of EGF, PTPμμ knockdown increased EGFR Y845, Y992, Y1045, Y1068, Y1086, and Y1173 but not Y1148 phosphorylation. Reduced PTPμμ expression increased EGF-stimulated phosphorylation of Y992, a docking site for phospholipase C (PLC)γγ1, activation of PLCγγ1 itself, and increased cell migration in both wounding and chemotaxis assays. In contrast, overexpression of PTPμμ decreased EGF-stimulated EGFR Y992 and Y1068 phosphorylation. Therefore, airway epithelial injury profoundly reduces PTPμμ expression, and PTPμμ depletion selectively increases phosphorylation of specific EGFR tyrosine residues, PLCγγ1 activation, and cell migration, providing a novel mechanism through which epithelial integrity may be restored. [ABSTRACT FROM AUTHOR]

Abstract: To activate the GTPase Rac in rat basophilic leukemia (RBL) cells and mouse bone marrow-derived mast cells (BMMC) a TAT fusion toxin of Bordetella dermonecrotic toxin (DNT-TAT) was constructed. The fusion toxin activated Rac1 and RhoA in vitro but only Rac in RBL cells and BMMC. DNT-TAT caused an increase in inositol phosphate formation, calcium mobilization, ERK activation and degranulation of mast cells. All these effects were inhibited by the Rho GTPase-inactivating Clostridium difficile toxin B and Clostridium sordellii lethal toxin. Also the calcium ionophore A23187 caused mast cell activation, including ERK phosphorylation, by processes involving an activation of Rac. The data indicate pleiotropic functions of Rac in mast cell activation. [Copyright &y& Elsevier]

Abstract: Alterations in neurotrophic signaling pathways may contribute to the changes in the mesolimbic dopamine system induced by chronic morphine exposure. In a rat model of morphine dependence, we previously identified increased levels of phospholipase C gamma-1 (PLCγ1) immunoreactivity specifically within the ventral tegmental area (VTA) following chronic morphine treatment. Using an antibody specific for the tyrosine-phosphorylated, activated form of PLCγ1, we now show that chronic morphine also significantly upregulates PLCγ1 activity in the VTA, as well as in the nucleus accumbens and hippocampus, regions which are also implicated in the reinforcing properties of morphine. In contrast, no increase in PLCγ1 activity was found in the substantia nigra or dorsal striatum. HSV-mediated overexpression of PLCγ1 in PC12 cells induced ERK activation via a mechanism dependent, in part, on both MAP-ERK kinase (MEK) and protein kinase C. PLCγ1 overexpression in the VTA similarly induced ERK activation in the VTA in vivo. As chronic morphine treatment has been shown to increase ERK activity within the VTA, the current results suggest that increased PLCγ1 activity may be an upstream mediator of this effect. [Copyright &y& Elsevier]

Abstract: Nuclear envelope assembly is an essential event in each cell cycle but the proteins and lipids involved in its regulation remain mostly unknown. Assembly involves membrane fusions but neither specific SNAREs nor Rab GTPases have been identified in its control. We report that a precursor membrane population (MV1) required for NE assembly has a unique lipid composition consisting prominently of poly-phosphatidylinositides. The lipid composition was determined by adapting HPLC electrospray ionisation tandem mass spectrometry to phosphoinositide analysis, revealing the capacity of this technique to document dynamic lipid transitions of functional importance in natural membrane populations. MV1 is >100-fold enriched in endogenous PLCγ and >25-fold enriched in the PLC substrate phosphatidylinositol bisphosphate (PtdInsP2) compared to the second membrane population, derived largely from endoplasmic reticulum (ER), that contributes most of the NE. During NE formation PLCγ becomes transiently phosphorylated at the tyrosine 783 site indicative of its activation. In addition specific inhibition of PLCγ blocks nuclear envelope formation. In vivo, PLCγ is concentrated on vesicles of similar size to purified MV1. These associate with nuclei during the period of NE formation and are distinct from ER membranes. The unprecedented concentration of PLCγ and its substrate PtdInsP2 in a subset of membranes that binds to only two regions of the nucleus, and activation of PLCγ by GTP during initial stages of NE formation provide a mechanism for temporal control of NE assembly and offer an explanation for how such a process of membrane fusion can be spatially regulated. [Copyright &y& Elsevier]

Abstract: EGF and TGF-α induce an equipotent stimulation of fibroblast migration and proliferation. In spite of their homologous structure and ligation by the same receptor (EGFR), we report that their respective motogenic activities are mediated by different signal transduction intermediates, with p70S6K participating in EGF signalling and phospholipase Cγ in TGF-α signalling. We additionally demonstrate that EGF and TGF-α motogenic activities may be resolved into two stages: (a) cell “activation” by a transient exposure to either cytokine, and (b) the subsequent “manifestation” of an enhanced migratory phenotype in the absence of cytokine. The cell activation and manifestation stages for each cytokine are mediated by distinct matrix-dependent mechanisms: motogenetic activation by EGF requires the concomitant functionality of EGFR and the hyaluronan receptor CD44, whereas activation by TGF-α requires EGFR and integrin αvβ3. Manifestation of elevated migration no longer requires the continued presence of exogenous cytokine and functional EGFR but does require the above mentioned matrix receptors, as well as their respective ligands, i.e., hyaluronan in the case of EGF, and vitronectin in the case of TGF-α. In contrast, the mitogenic activities of EGF and TGF-α are independent of CD44 and αvβ3 functionality. These results demonstrate clear qualitative differences between EGF and TGF-α pathways and highlight the importance of the extracellular matrix in regulating cytokine bioactivity. [Copyright &y& Elsevier]

Abstract: In the biogenesis of HDL by exogenous apolipoprotein (apo) A-I in rat astrocytes, apoA-I induced translocation of phospholipase Cγ (PLCγ) and PKCα to cytosolic lipid protein particle (CLPP) [Ito et al., 2004. J. Lipid Res. 45, 2269] and caused tyrosine-phosphorylation of PLCγ in CLPP in the initial 5min. It also induced translocation of caveolin-1 and newly synthesized cholesterol and phospholipid to CLPP, and increased cholesterol biosynthesis prior to the HDL biogenesis [Ito et al., 2002a. J. Biol. Chem. 277, 7929]. Cyclosporin A (CsA), an indirect inhibitor of protein phosphatase 2B (PP2B) and a potential inhibitor of ABC transporter A1 (ABCA1), suppressed all of these apoA-I-induced cellular events. CsA, however, did not affect the basal lipid release by the production of HDL with endogenous apoE, except for moderate decrease of its cholesterol content. Direct inhibitors of PP2B, inhibited only the release of lipids by apoA-I and had no effect on other apoA-I-induced events. CsA thus interferes with cellular cholesterol homeostasis independently of PP2B inhibition, perhaps by direct inhibition of ABCA1 reactivity to exogenous apoA-I, although PP2B may be involved in the lipid release step. CsA could therefore cause some neurological side effects by interfering with cellular cholesterol homeostasis in the brain. [Copyright &y& Elsevier]

Abstract: Red blood cell development is primarily controlled by erythropoietin (EPO). Several studies have revealed the importance of EPO-R Y343 and Y479 for erythroid cell growth, differentiation, and survival. In order to isolate critical signaling proteins that bind to EPO-R, we initiated a Cloning of Ligand Target (COLT) screen using a murine embryonic day 16 phage library and a biotinylated EPO-R Y343 phosphopeptide. One of the clones isolated encodes Phospholipase C (PLC)γ1. PLCγ1 is rapidly tyrosine phosphorylated upon EPO stimulation and associates with EPO-R in an SH2-domain-dependent manner. Although PLCγ1 bound EPO-R Y343, Y401, Y429, Y431, and Y479 in the COLT screen, PLCγ1 required Y479 for association with EPO-R in Ba/F3-EPO-R cells. Studies have identified EPO-R Y479 as important for ERK activation. Since PI3-kinase binds EPO-R Y479, one group has suggested that ERK activation downstream of PI3-kinase accounts for the importance of this residue in EPO signaling. However, we show that inhibition of PI3-kinase does not abolish ERK activation. Furthermore, we demonstrate interaction of PLCγ1 with Grb2 and SOS2. Hence, we have identified a novel adapter function for PLCγ1 in EPO signaling in which recruitment of PLCγ1 to EPO-R may lead to activation of the ERK pathway. [Copyright &y& Elsevier]

Chronic infection with cagA-positive Helicobacter pylon is associated with the development of atrophic gastritis, peptic ulcers, and gastric adenocarcinoma. The cagA gene product CagA is injectecd into gastric epithelial cells, where it undergoes tyrosine phosphorylation by Src family kinases. Translocated CagA disturbs cellular functions by physically interacting with and deregulating intracellular signaling transducers through both tyrosine phosphorylation-dependent and -independent mechanisms. To gain further insights into the pathophysiological activities of CagA in gastric epithelial cells, we executed a genome-wide screening of CagA-responsive genes by using DNA microarray and identified nuclear factor of activated T cells (NEAT) transcription factors whose binding sites were overrepresented in the promoter regions of CagA-activated genes. Results of reporter assays confirmed that CagA was capable of activating NFAT in a manner independent of CagA phosphorylation. Expression of CagA in gastric epithelial cells provoked translocation of NFATc3, a member of the NFAT family, from the cytoplasm to the nucleus and activated an NFAT-regulated gene, p21WAF1/CiP1. CagA-mediated NFAT activation was abolished by inhibiting calcineurin or phospholipase Cγ activity. Furthermore, treatment of cells with H. pylori VacA (vacuolating toxin), which inhibits NFAT activity in T lymphocytes, counteracted the ability of CagA to activate NFAT in gastric epithelial cells. These findings indicate that the two major H. pylori virulence factors inversely control NFAT activity. Considering the pleiotropic roles of NFAT in cell growth and differentiation, deregulation of NFAT, either positively or negatively, depending on the relative exposure of cells to CagA and VacA, may contribute to the various disease outcomes caused by H. pylori infection. [ABSTRACT FROM AUTHOR]

The high affinity IgE receptor (FcεRI) usually exists as a tetramer composed of αβγ2 subunits. The COOH-tail of β and γ subunits contains consensus sequence termed ‘immunoreceptor tyrosine-based activation motif’ (ITAM). Tyrosine phosphorylated ITAM interacts with signaling proteins that contain the Src homology domain, forming a main amplifying and signaling route for FcεRI. Unlike the COOH-tail, the functional role of NH2-tail of β subunit in the signaling of FcεRI is not clear because it lacks the ITAM sequences. To study the roles of NH2-tail of β subunit, the cDNA library of RBL-2H3 cells was screened by yeast two-hybrid assay, and the NH2-tail of the β subunit was found to interact with phospholipase Cγ2 (PLCγ2) but not with PLCγ1. Since both PLCγ1 and PLCγ2 are expressed in RBL-2H3 cells and they possess identical cellular functions, the functional meaning of the protein–protein interaction between PLCγ2 and NH2-tail of β subunit was studied by comparing the regulatory pathways that control the FcεRI-mediated tyrosine phosphorylation of the two enzymes. Our study shows that PI3-kinase and PMA-sensitive PKCs were required exclusively for the FcεRI-mediated tyrosine phosphorylation of PLCγ1. Also the FcεRI-mediated tyrosine phosphorylation of PLCγ1 was more sensitive to the inhibitors of Src and Syk kinases. These results therefore suggest that PLCγ1 is involved in dynamic regulation of protein kinase C activity and inositol triphosphate levels in response to cellular needs. In contrast, PLCγ2, through continuous interaction with the NH2-tail of β subunit, co-localizes with FcεRI in the same signaling domain, and maintains the basal cellular PLC activity. [Copyright &y& Elsevier]

Phospholipase C (PLC)γ and phospholipase D (PLD) play pivotal roles in the signal transduction required for various cellular responses, including cell proliferation and differentiation. Dendritic cells (DCs), which are professional antigen-presenting cells, can be generated from human monocytes by stimulating the cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4). We investigated whether PLCγ and PLD expression levels can be changed during the differentiation of the human monocytes into DCs. The enzymatic activity and protein level of PLC γ1 were significantly increased in the human monocyte-derived DCs by GM-CSF/IL-4, but the protein levels of PLC γ2 were unaltered. Moreover, the enzymatic activity and protein level of PLD1b and PLD2 were up-regulated during the differentiation of human monocytes to DCs, but those of PLD1a were not changed. A higher phagocytic activity of DCs was found to be correlated with the up-regulations of PLCγ1 and PLD, and the phagocytic activity of DCs was inhibited by a PLC-specific inhibitor (U73122) and by a phosphatidic acid acceptor (n-butanol), but to be increased by phosphatidic acid. Thus, suggesting that PLC and PLD participate in the process. This study suggests that the up-regulations of PLCγ1 and PLD are accompanied by the differentiation of monocytes into DCs, which results in increased phagocytic activity. [Copyright &y& Elsevier]

The ErbB/HER protein-tyrosine kinases, which include the epidermal growth factor receptor, consist of a growth-factor-binding ectodomain, a single transmembrane segment, an intracellular protein-tyrosine kinase catalytic domain, and a tyrosine-containing cytoplasmic tail. The genes for the four members of this family, ErbB1–ErbB4, are found on different human chromosomes. Null mutations of any of the ErbB family members result in embryonic lethality. ErbB1 and ErbB2 are overexpressed in a wide variety of tumors including breast, colorectal, ovarian, and non-small cell lung cancers. The structures of the ectodomains of the ErbB receptors in their active and inactive conformation have shed light on the mechanism of receptor activation. The extracellular component of the ErbB proteins consists of domains I–IV. The activating growth factor, which binds to domains I and III, selects and stabilizes a conformation that allows a dimerization arm to extend from domain II to interact with an ErbB dimer partner. As a result of dimerization, protein kinase activation, trans-autophosphorylation, and initiation of signaling occur. The conversion of the inactive to active receptor involves a major rotation of the ectodomain. The ErbB receptors are targets for anticancer drugs. Two strategies for blocking the action of these proteins include antibodies directed against the ectodomain and drugs that inhibit protein-tyrosine kinase activity. A reversible ATP competitive inhibitor of ErbB1 (ZD1839, or Iressa) and an ErbB1 ectodomain directed antibody (IMC-C225, or Erbitux) have been approved for the treatment of non-small cell lung cancer and colorectal cancer, respectively. An ErbB2/HER2 ectodomain directed antibody (trastuzumab, or Herceptin) has also been approved for the treatment of breast cancer. Current research promises to produce additional agents based upon these approaches. [Copyright &y& Elsevier]

Xenopus oocytes expressing fibroblast growth factor receptor 1 (FGFR1) were used as a biological model system to analyse the signal transduction pathways that are triggered by fibroblast growth factor 1 (FGF1). Germinal vesicle breakdown (GVBD) and phosphorylation of extracellular signal-regulated protein kinase 2 (ERK2) occured 15 h after FGF1 addition. These events were Ras-dependent as they were blocked by a Ras dominant negative form. The Ras activity was promoted by three upstream effectors, growth factor-bound protein 2 (Grb2), phosphatidylinositol 3-kinase (PI3K) and Src cytoplasmic kinase. Ras activation was inhibited by a Grb2 dominant negative form (P49L), by PI3K inhibitors, including wortmannin, LY294002, the N-SH2 domain of p85α PI3K and by the SH2 domain of Src. Src activation induced by FGF1 was blocked by the SH2 domain of Src and PP2, a specific inhibitor of Src. The Grb2 adaptor was recruited by the upstream Src homology 2/α-collagen-related (Shc) effector, as the SH2-Shc domain prevented the GVBD and the ERK2 phosphorylation induced by FGF1. The importance of another signalling pathway involving phospholipase Cγ (PLCγ) was also investigated. The use of the PLCγ inhibitory peptide, neomycin and the calcium chelator BAPTA-AM on oocytes expressing FGFR1 or the stimulation by PDGF-BB of oocytes expressing PDGFR-FGFR1 mutated on the PLCγ binding site, prevented GVBD and ERK2 phosphorylation. This study shows that the transduction cascade induced by the FGFR1–FGF1 interaction in Xenopus oocytes represents the sum of Ras-dependent and PLCγ-dependent pathways. It emphasizes the role played by PI3K and Src and their connections with the Ras cascade in the FGFR1 signal transduction. [ABSTRACT FROM AUTHOR]

Phosphorylation on tyrosine and turnover of polyphosphoinositide metabolism are rapidly stimulated after fertilization. However, the interconnection between these pathways remains to be determined. In the present paper it is demonstrated that eggs of two different sea urchin species contain tyrosine phosphorylated proteins with calcium-sensitive phospholipase C activity. We have investigated whether phospholipase Cγ (PLCγ), characteristic of tyrosine kinase receptors, could be responsible for this activity. Western blot and immunocytochemistry performed with antibodies directed against PLCγ revealed the presence of this protein in cortical regions. It was also observed that PLCγ displayed calcium-sensitive activity. The present results suggest that PLCγ may be part of the cascade of events leading to the calcium signal responsible for egg activation at fertilization. [ABSTRACT FROM AUTHOR]

Abstract: Neurotrophic signaling pathways have been implicated in the maintenance of the mesolimbic dopamine system, as well as in changes in this system induced by chronic morphine exposure. We found that many of these signaling pathway proteins are expressed at appreciable levels within the ventral tegmental area (VTA) and related regions, although with substantial regional variation. Moreover, phospholipase Cγ1 (PLCγ1) was significantly and specifically up-regulated within the VTA by 30% following chronic exposure to morphine. PLCγ1 mRNA expression is enriched in dopaminergic neurons within the VTA ; however, the up-regulation of PLCγ1 in this region was not seen at the mRNA level. In contrast to PLCγ1, insulin receptor substrate (IRS)-2, a protein involved in phosphatidylinositol 3-kinase signaling, and another putative IRS-like protein were significantly down-regulated within the VTA by 49 and 45%, respectively. Levels of several proteins within the Ras-ERK pathway were not altered. Regulation of neurotrophic factor signaling proteins may play a role in morphine-induced plasticity within the mesolimbic dopamine system. [ABSTRACT FROM AUTHOR]

Background: Vascular endothelial growth factors (VEGFs) are potential therapeutic agents for treatment of ischemic diseases. Their angiogenic effects are mainly mediated through VEGF receptor 2 (VEGFR2)., Methods: Receptor binding, signaling, and biological efficacy of several VEGFR2 ligands were compared to determine their characteristics regarding angiogenic activity and vascular permeability., Results: Tested VEGFR2 ligands induced receptor tyrosine phosphorylation with different efficacy depending on their binding affinities. However, the tyrosine phosphorylation pattern and the activation of the major downstream signaling pathways were comparable. The maximal angiogenic effect stimulated by different VEGFR2 ligands was dependent on their ability to bind to co-receptor Neuropilin (Nrp), which was shown to form complexes with VEGFR2. The ability of these VEGFR2 ligands to induce vascular permeability was dependent on their concentration and VEGFR2 affinity, but not on Nrp binding., Conclusions: VEGFR2 activation alone is sufficient for inducing endothelial cell proliferation, formation of tube-like structures and vascular permeability. The level of VEGFR2 activation is dependent on the binding properties of the ligand used. However, closely similar activation pattern of the receptor kinase domain is seen with all VEGFR2 ligands. Nrp binding strengthens the angiogenic potency without increasing vascular permeability., General Significance: This study sheds light on how different structurally closely related VEGFR2 ligands bind to and signal via VEGFR2/Nrp complex to induce angiogenesis and vascular permeability. The knowledge of this study could be used for designing VEGFR2/Nrp ligands with improved therapeutic properties., (© 2013.)

Trapidil, a platelet-derived growth factor antagonist, was originally developed as a vasodilator and anti-platelet agent and has been used to treat patients with ischemic coronary heart, liver, and kidney disease. In this study, we investigated the effects of trapidil on osteoclastogenesis and elucidated the possible mechanism of action of trapidil. Trapidil strongly inhibited osteoclast formation in co-cultures of bone marrow cells and osteoblasts without affecting receptor activator of NF-κB ligand (RANKL) or osteoprotegerin expression in osteoblasts. In addition, trapidil suppressed RANKL-induced osteoclast formation from osteoclast precursors. Trapidil reduced RANKL-induced expression of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), a master transcription factor for osteoclastogenesis, without affecting the expression of c-Fos that functions as a key upstream activator of NFATc1 during osteoclastogenesis. Ectopic expression of a constitutively active form of NFATc1 reversed the anti-osteoclastogenic effect of trapidil, indicating that NFATc1 is a critical target of the anti-osteoclastogenic action of trapidil. RANKL-induced calcium oscillation and Pim-1 expression, which are required for NFATc1 induction and osteoclastogenesis, were abrogated by trapidil. Consistent with the in vitro results, trapidil had a potent inhibitory effect on osteoclast formation and bone resorption induced by interleukin-1 in an animal model. Taken together, our data demonstrate that trapidil abrogates RANKL-induced calcium oscillation and Pim-1 expression required for NFATc1 induction, thereby inhibiting osteoclastogenesis., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Recently, some endocannabinoids were reported to show anti-inflammatory and anti-allergic activities. In this respect, various arachidonoyl endocannabinoids were screened for the inhibition of allergic response in IgE-activated RBL-2H3 cells. Among arachidonoyl endocannabinoids with a low cytotoxicity, only NA-5HT remarkably inhibited the release of β-hexosaminidase (IC(50), 13.58 μM), a marker of degranulation, and tumor necrosis factor-α (IC(50), 12.52 μM), a pro-inflammatory cytokine, in IgE-activated RBL-2H3 cells. Additionally, NA-5HT markedly suppressed the formation of prostaglandin D(2) (PGD(2)) with IC(50) value of 1.27 μM and leukotriene B(4) (LTB(4)) with IC(50) value of 1.20 μM, and slightly LTC4. When effect of NA-5HT on early stage of FcεRI cascade was investigated, it significantly inhibited phosphorylation of Syk, but not Lyn. Furthermore, NA-5HT suppressed phosphorylation of PLCγ1/2 and PKCδ, related to degranulation process, as well as phosphorylation of LAT, ERK1/2, p38, JNK, Gab2, PI3K and Akt, implicated in the expression of pro-inflammatory cytokines. Relative to its effect on the late stage, NA-5HT slightly reduced phosphorylation of 5-lipoxygenase (5-LO) and cyclooxygenase-2 (COX-2). Additionally, NA-5HT significantly reduced the level of p40(phox), and partially inhibited the expression of p47(phox) and p67(phox). From these results, it is suggested that NA-5HT expresses anti-allergic action by suppressing the activation of Syk, LAT, p38, JNK, PI3K and Akt, as well as the expression of ERK1/2 and NADPH oxidase subunits. Further, a strong inhibition of PGD(2) or LTB(4) biosynthesis by NA-5HT may be an additional mechanism for its anti-allergic action. Such anti-allergic actions of NA-5HT may contribute to further information about its biological functions., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: Aggregation of FcεRI activates a cascade of signaling events leading to mast cell activation, followed by inhibitory signals that turn off the activating signals. However, the overall view of negative signals in mast cells is still incomplete. Although AMP-activated protein kinase (AMPK), which is generally known as a regulator of energy metabolism, is also associated with anti-inflammation, little is known about the role of AMPK in mast cells., Objectives: We investigated the role of AMPK and its regulatory mechanism in mast cells., Method: The roles of AMPK in FcεRI-dependent activation of bone marrow-derived mast cells (BMMCs) were evaluated by using chemical agents, small interfering RNAs (siRNAs), or adenovirus that modulated the activity or expression of AMPK signaling components. In addition, AMPKα2(-/-) mice were used to verify the role of AMPK in anaphylactic models., Results: FcεRI signaling and associated effector functions in BMMCs were suppressed by the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR) and were conversely augmented by siRNA knockdown of AMPKα2 or liver kinase B1 (LKB1), an upstream kinase of AMPK. Furthermore, AMPKα2 deficiency led to increased FcεRI-mediated BMMC activation and anaphylaxis that were insensitive to AICAR, whereas enforced expression of AMPKα2 in AMPKα2(-/-) BMMCs reversed the hypersensitive FcεRI signaling to normal levels. Pharmacologic inhibition or siRNA knockdown of Fyn mimicked AMPK activation, suggesting that Fyn counterregulates the LKB1-AMPK axis. Mechanistically, Fyn controlled AMPK activity by regulating LKB1 localization., Conclusions: The Fyn-regulated LKB1-AMPK axis acts as a novel inhibitory module for mast cell activation, which points to AMPK activators as therapeutic drugs for allergic diseases., (Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Brain-derived neurotrophic factor (BDNF), a critical neurotrophin, regulates many neuronal aspects including cell differentiation, cell survival, neurotransmission, and synaptic plasticity in the central nervous system (CNS). Though BDNF has two types of receptors, high affinity tropomyosin-related kinase (Trk)B and low affinity p75 receptors, BDNF positively exerts its biological effects on neurons via activation of TrkB and of resultant intracellular signaling cascades including mitogen-activated protein kinase/extracellular signal-regulated protein kinase, phospholipase Cγ, and phosphoinositide 3-kinase pathways. Notably, it is possible that alteration in the expression and/or function of BDNF in the CNS is involved in the pathophysiology of various brain diseases such as stroke, Parkinson's disease, Alzheimer's disease, and mental disorders. On the other hand, glucocorticoids, stress-induced steroid hormones, also putatively contribute to the pathophysiology of depression. Interestingly, in addition to the reduction in BDNF levels due to increased glucocorticoid exposure, current reports demonstrate possible interactions between glucocorticoids and BDNF-mediated neuronal functions. Other steroid hormones, such as estrogen, are involved in not only sexual differentiation in the brain, but also numerous neuronal events including cell survival and synaptic plasticity. Furthermore, it is well known that estrogen plays a role in the pathophysiology of Parkinson's disease, Alzheimer's disease, and mental illness, while serving to regulate BDNF expression and/or function. Here, we present a broad overview of the current knowledge concerning the association between BDNF expression/function and steroid hormones (glucocorticoids and estrogen).