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Sample records for activating factor receptor

  1. Selenoprotein W controls epidermal growth factor receptor surface expression, activation and degradation via receptor ubiquitination

    Epidermal growth factor (EGF) receptor (EGFR) is the founding member of the ErbB family of growth factor receptors that modulate a complex network of intracellular signaling pathways controlling growth, proliferation and differentiation. Selenoprotein W (SEPW1) is a diet-regulated, highly conserved...

  2. Receptor activator of nuclear factor kappa B ligand and osteoprotegerin levels in gingival crevicular fluid

    Fatemeh Sarlati; Mandana Sattari; Shilan Razzaghi; Malihe Nasiri

    2012-01-01

    Background: Osteoclastogenesis is coordinated by the interaction of three members of the tumor necrosis factor (TNF) superfamily: Osteoprotegerin (OPG)/receptor activator of nuclear factor kappa B ligand (RANKL)/receptor activator of nuclear factor kappa B (RANK). The aim of this study was to investigate RANKL and OPG levels, and their relative ratio in gingival crevicular fluid (GCF) of patients with chronic and aggressive periodontitis, as well as healthy controls. Materials and Methods:...

  3. Regulation of platelet activating factor receptor coupled phosphoinositide-specific phospholipase C activity

    Morrison, W.J.

    1988-01-01

    The major objectives of this study were two-fold. The first was to establish whether binding of platelet activating factor (PAF) to its receptor was integral to the stimulation of polyphosphoinositide-specific phospholipase C (PLC) in rabbit platelets. The second was to determine regulatory features of this receptor-coupled mechanism. ({sup 3}H)PAF binding demonstrated two binding sites, a high affinity site with a inhibitory constant (Ki) of 2.65 nM and a low affinity site with a Ki of 0.80 {mu}M. PAF receptor coupled activation of phosphoinositide-specific PLC was studied in platelets which were made refractory, by short term pretreatments, to either PAF or thrombin. Saponin-permeabilized rabbit platelets continue to regulate the mechanism(s) coupling PAF receptors to PLC stimulation. However, TRP{gamma}S and GDP{beta}S, which affect guanine nucleotide regulatory protein functions, were unable to modulate the PLC activity to any appreciable extent as compared to PAF. The possible involvement of protein kinase C (PKC) activation in regulating PAF-stimulated PLC activity was studied in rabbit platelets pretreated with staurosporine followed by pretreatments with PAF or phorbol 12-myristate 13-acetate (PMA).

  4. Therapy for acute pancreatitis with platelet-activating factor receptor antagonists

    Chen, Chong; Xia, Shi-hai; Chen, Hong; Li, Xiao-Hong

    2008-01-01

    Acute pancreatitis (AP) causes release of platelet-activating factor (PAF), which induces systemic effects that contribute to circulatory disturbances and multiple organ failure. PAF is a cell surface secretion of bioactive lipid, which could produce physiological and pathological effects by binding to its cell surface receptor called platelet-activating factor receptor (PAF-R). Studies showed that PAF participates in the occurrence and development of AP and administration of platelet-activat...

  5. Antisense epidermal growth factor receptor RNA transfection in human glioblastoma cells down-regulates telomerase activity and telomere length

    Tian, X-X; Pang, JC-S; J. Zheng; Chen, J; To, S S T; Ng, H-K

    2002-01-01

    Epidermal growth factor receptor is overexpressed and/or amplified in up to 50% of glioblastomas, suggesting an important role of this gene in glial tumorigenesis and progression. In the present study we demonstrated that epidermal growth factor receptor is involved in regulation of telomerase activity in glioblastoma. Antisense-epidermal growth factor receptor approach was used to inhibit epidermal growth factor receptor expression of glioblastoma U87MG cells. Telomerase activity in antisens...

  6. Defective lysosomal targeting of activated fibroblast growth factor receptor 3 in achondroplasia

    Cho, Jay Y.; Guo, Changsheng; Torello, Monica; Lunstrum, Gregory P.; Iwata, Tomoko; Deng, Chuxia; Horton, William A.

    2003-01-01

    Mutations of fibroblast growth factor receptor 3 (FGFR3) are responsible for achondroplasia (ACH) and related dwarfing conditions in humans. The pathogenesis involves constitutive activation of FGFR3, which inhibits proliferation and differentiation of growth plate chondrocytes. Here we report that activating mutations in FGFR3 increase the stability of the receptor. Our results suggest that the mutations disrupt c-Cbl-mediated ubiquitination that serves as a targeting signal for lysosomal de...

  7. Internalization mechanisms of the epidermal growth factor receptor after activation with different ligands.

    Lasse Henriksen

    Full Text Available The epidermal growth factor receptor (EGFR regulates normal growth and differentiation, but dysregulation of the receptor or one of the EGFR ligands is involved in the pathogenesis of many cancers. There are eight ligands for EGFR, however most of the research into trafficking of the receptor after ligand activation focuses on the effect of epidermal growth factor (EGF and transforming growth factor-α (TGF-α. For a long time it was believed that clathrin-mediated endocytosis was the major pathway for internalization of the receptor, but recent work suggests that different pathways exist. Here we show that clathrin ablation completely inhibits internalization of EGF- and TGF-α-stimulated receptor, however the inhibition of receptor internalization in cells treated with heparin-binding EGF-like growth factor (HB-EGF or betacellulin (BTC was only partial. In contrast, clathrin knockdown fully inhibits EGFR degradation after all ligands tested. Furthermore, inhibition of dynamin function blocked EGFR internalization after stimulation with all ligands. Knocking out a number of clathrin-independent dynamin-dependent pathways of internalization had no effect on the ligand-induced endocytosis of the EGFR. We suggest that EGF and TGF-α lead to EGFR endocytosis mainly via the clathrin-mediated pathway. Furthermore, we suggest that HB-EGF and BTC also lead to EGFR endocytosis via a clathrin-mediated pathway, but can additionally use an unidentified internalization pathway or better recruit the small amount of clathrin remaining after clathrin knockdown.

  8. Stimulation of glucose uptake by transforming growth factor β: evidence for the requirement of epidermal growth factor-receptor activation

    Transforming growth factor β (TGF-β), derived from human platelets, stimulates the uptake of 2-deoxyglucose by cultured cell monolayers 2- to 4-fold. Stimulation can be detected as early as 30 min with as little as 0.1 ng of TGF-β per ml and maximal effects can be obtained at 2 hr with 1 ng of the growth factor per ml. TGF-β-induced stimulation of sugar uptake is enhanced by the co-addition of platelet-derived growth factor (10 ng/ml) or epidermal growth factor (EGF, 1 ng/ml). The NR-6 variant of mouse 3T3 cells, which lack EGF receptors, is not stimulated by TGF-β. Antisera to EGF receptors that block 125I-labeled EGF binding also inhibit TGF-β stimulation of 2-deoxyglucose uptake, although 125I-labeled TGF-β binding remains unimpaired. In contrast, antisera to the EGF receptor, which do not block EGF binding, have no measurable effect on the TGF-β-stimulated uptake of 2-deoxyglucose. The authors confirm that the receptor for TGF-β is distinct from the receptor for EGF and the authors conclude that TGF-β stimulation of 2-deoxyglucose uptake requires the co-activation of the EGF receptor kinase system

  9. Internalization mechanisms of the epidermal growth factor receptor after activation with different ligands

    Henriksen, Lasse; Grandal, Michael Vibo; Knudsen, Stine Louise Jeppe; van Deurs, Bo; Grøvdal, Lene Melsæther

    2013-01-01

    after ligand activation focuses on the effect of epidermal growth factor (EGF) and transforming growth factor-α (TGF-α). For a long time it was believed that clathrin-mediated endocytosis was the major pathway for internalization of the receptor, but recent work suggests that different pathways exist....... Here we show that clathrin ablation completely inhibits internalization of EGF- and TGF-α-stimulated receptor, however the inhibition of receptor internalization in cells treated with heparin-binding EGF-like growth factor (HB-EGF) or betacellulin (BTC) was only partial. In contrast, clathrin knockdown...... fully inhibits EGFR degradation after all ligands tested. Furthermore, inhibition of dynamin function blocked EGFR internalization after stimulation with all ligands. Knocking out a number of clathrin-independent dynamin-dependent pathways of internalization had no effect on the ligand...

  10. Dimer formation of receptor activator of nuclear factor κB induces incomplete osteoclast formation

    Receptor activator of nuclear factor κB-ligand (RANKL) transduces a differentiation signal appropriate to osteoclasts likely through induction a receptor homotrimer; however, biological importance of RANK-trimerizarion is unknown. To address the signaling mechanism of the RANK receptor, we analyzed the effect of two different types of homodimer inducers RANK-TM-FKBP36v and hEpoR-RANK-TM on osteoclastogenesis. Dimerizing component FKBP36v or extracellular portion of human erythropoietin receptor (hEpoR) was fused to RANK lacking the extracellular domain, and the dimerization of this fusion protein was induced by addition of the chemical inducer of dimerization AP20187 or erythropoietin, respectively. Such treatment resulted in induction of TRAP-activity, a marker of osteoclast in a dose dependent manner, with an efficiency equivalent to that of induction by RANKL. However, dimerized-RANK-induced osteoclasts showed relatively low levels of multinucleation, pit forming activity, and expression of calcitonin receptor and cathepsin K, compared with osteoclasts which were induced in the presence of RANKL. As expression of nuclear factor of activated T cells 1 (NFATc1) was also reduced in dimerized-RANK-induced osteoclasts, RANK oligomerization by RANKL is a critical event to generate fully matured osteoclasts through upregulation of NFATc1

  11. TFIIB-directed transcriptional activation by the orphan nuclear receptor hepatocyte nuclear factor 4.

    Malik, S; Karathanasis, S K

    1996-01-01

    The orphan nuclear receptor hepatocyte nuclear factor 4 (HNF-4) is required for development and maintenance of the liver phenotype. HNF-4 activates several hepatocyte-specific genes, including the gene encoding apolipoprotein AI (apoAI), the major protein component of plasma high-density lipoprotein. The apoAI gene is activated by HNF-4 through a nuclear receptor binding element (site A) located in its liver-specific enhancer. To decipher the mechanism whereby HNF-4 enhances apoAI gene transc...

  12. Tyrosine kinase JAK1 is associated with the granulocyte-colony-stimulating factor receptor and both become tyrosine-phosphorylated after receptor activation.

    Nicholson, S. E.; Oates, A. C.; Harpur, A G; Ziemiecki, A; Wilks, A F; Layton, J E

    1994-01-01

    Granulocyte-colony-stimulating factor (G-CSF) stimulates the proliferation and differentiation of cells of the neutrophil lineage by interaction with a specific receptor. Early signal transduction events following G-CSF receptor activation were studied. We detected tyrosine phosphorylation of both the G-CSF receptor and the protein tyrosine kinase JAK1 following G-CSF binding to the human G-CSF receptor. In vitro, the kinase activity of JAK1 was increased by G-CSF stimulation. Coimmunoprecipi...

  13. A transcription factor active on the epidermal growth factor receptor gene

    The authors have developed an in vitro transcription system for the epidermal growth factor receptor (EGFR) oncogene by using nuclear extracts of A431 human epidermoid carcinoma cells, which overproduce EGFR. They found that a nuclear factor, termed EGFR-specific transcription factor (ETF), specifically stimulated EGFR transcription by 5- to 10-fold. In this report, ETF, purified by using sequence-specific oligonucleotide affinity chromatography, is shown by renaturing material eluted from a NaDodSO4/polyacrylamide gel to be a protein with a molecular mass of 120 kDa. ETF binds to the promoter region, as measured by DNase I footprinting and gel-mobility-shift assays, and specifically stimulates the transcription of the EGFR gene in a reconstituted in vitro transcription system. These results suggest that ETF could play a role in the overexpression of the cellular oncogene EGFR

  14. Nuclear Factor-κB: Activation and Regulation during Toll-like Receptor Signaling

    Ruaidhrí J. Carmody; Youhai H. Chen

    2007-01-01

    Toll-like receptors (TLRs) recognize distinct microbial components to initiate the innate and adaptive immune responses. TLR activation culminates in the expression of appropriate pro-inflammatory and immunomodulatory factors to meet pathogenic challenges. The transcription factor NF-κB is the master regulator of all TLR-induced responses and its activation is the pivotal event in TLR-mediated activation of the innate immune response. Many of the key molecular events required for TLR-induced NF-κB activation have been elucidated. However, much remain to be learned about the ability of TLRs to generate pathogen-specific responses using a limited number of transcription factors. This review will focus on our current understanding of NF-κB activation by TLRs and potential mechanisms for achieving a signal-specific response through NF-κB.

  15. Activated factor X signaling via protease-activated receptor 2 suppresses pro-inflammatory cytokine production from LPS-stimulated myeloid cells.

    Gleeson, Eimear M

    2013-07-19

    Vitamin K-dependent proteases generated in response to vascular injury and infection enable fibrin clot formation, but also trigger distinct immuno-regulatory signaling pathways on myeloid cells. Factor Xa, a protease crucial for blood coagulation, also induces protease-activated receptor-dependent cell signaling. Factor Xa can bind both monocytes and macrophages, but whether factor Xa-dependent signaling stimulates or suppresses myeloid cell cytokine production in response to Toll-like receptor activation is not known. In this study, exposure to factor Xa significantly impaired pro-inflammatory cytokine production from lipopolysaccharide-treated peripheral blood mononuclear cells, THP-1 monocytic cells and murine macrophages. Furthermore, factor Xa inhibited nuclear factor-kappa B activation in THP-1 reporter cells, requiring phosphatidylinositide 3-kinase activity for its anti-inflammatory effect. Active-site blockade, γ-carboxyglutamic acid domain truncation and a peptide mimic of the factor Xa inter-epidermal growth factor-like region prevented factor Xa inhibition of lipopolysaccharide-induced tumour necrosis factor-α release. In addition, factor Xa anti-inflammatory activity was markedly attenuated by the presence of an antagonist of protease-activated receptor 2, but not protease-activated receptor 1. The key role of protease-activated receptor 2 in eliciting factor Xa-dependent anti-inflammatory signaling on macrophages was further underscored by the inability of factor Xa to mediate inhibition of tumour necrosis factor-α and interleukin-6 release from murine bone marrow-derived protease-activated receptor 2-deficient macrophages. We also show for the first time that, in addition to protease-activated receptor 2, factor Xa requires a receptor-associated protein-sensitive low-density lipoprotein receptor to inhibit lipopolysaccharide-induced cytokine production. Collectively, this study supports a novel function for factor Xa as an endogenous, receptor

  16. Androgen regulation of epidermal growth factor receptor binding activity during fetal rabbit lung development.

    Klein, J M; Nielsen, H C

    1993-01-01

    Fetal lung development progresses in a sex-specific manner with male fetuses exhibiting delayed maturation. Androgens, both exogenous and endogenous, inhibit while epidermal growth factor (EGF) enhances fetal lung development. We hypothesized that one mechanism responsible for the delay in male fetal lung development is an androgen-induced delay in EGF receptor binding activity. We measured EGF binding in sex-specific fetal rabbit lung plasma membranes isolated from control fetuses (days 21, ...

  17. Combinatorial activation and repression by seven transcription factors specify Drosophila odorant receptor expression.

    Shadi Jafari

    Full Text Available The mechanism that specifies olfactory sensory neurons to express only one odorant receptor (OR from a large repertoire is critical for odor discrimination but poorly understood. Here, we describe the first comprehensive analysis of OR expression regulation in Drosophila. A systematic, RNAi-mediated knock down of most of the predicted transcription factors identified an essential function of acj6, E93, Fer1, onecut, sim, xbp1, and zf30c in the regulation of more than 30 ORs. These regulatory factors are differentially expressed in antennal sensory neuron classes and specifically required for the adult expression of ORs. A systematic analysis reveals not only that combinations of these seven factors are necessary for receptor gene expression but also a prominent role for transcriptional repression in preventing ectopic receptor expression. Such regulation is supported by bioinformatics and OR promoter analyses, which uncovered a common promoter structure with distal repressive and proximal activating regions. Thus, our data provide insight into how combinatorial activation and repression can allow a small number of transcription factors to specify a large repertoire of neuron classes in the olfactory system.

  18. Vascular Endothelial Growth Factor Receptor 3 Controls Neural Stem Cell Activation in Mice and Humans

    Jinah Han

    2015-02-01

    Full Text Available Neural stem cells (NSCs continuously produce new neurons within the adult mammalian hippocampus. NSCs are typically quiescent but activated to self-renew or differentiate into neural progenitor cells. The molecular mechanisms of NSC activation remain poorly understood. Here, we show that adult hippocampal NSCs express vascular endothelial growth factor receptor (VEGFR 3 and its ligand VEGF-C, which activates quiescent NSCs to enter the cell cycle and generate progenitor cells. Hippocampal NSC activation and neurogenesis are impaired by conditional deletion of Vegfr3 in NSCs. Functionally, this is associated with compromised NSC activation in response to VEGF-C and physical activity. In NSCs derived from human embryonic stem cells (hESCs, VEGF-C/VEGFR3 mediates intracellular activation of AKT and ERK pathways that control cell fate and proliferation. These findings identify VEGF-C/VEGFR3 signaling as a specific regulator of NSC activation and neurogenesis in mammals.

  19. Gastrin-Releasing Peptide Receptor Mediates Activation of the Epidermal Growth Factor Receptor in Lung Cancer Cells

    Sufi Mary Thomas

    2005-04-01

    Full Text Available Gastrin-releasing peptide receptor (GRPR and the epidermal growth factor receptor (EGFR are expressed in several cancers including non-small cell lung carcinoma (NSCLC. Here we demonstrate the activation of EGFR by the GRPR ligand, gastrin-releasing peptide (GRP, in NSCLC cells. GRP induced rapid activation of p44/42 MAPK in lung cancer cells through EGFR. GRP-mediated activation of MAPK in NSCLC cells was abrogated by pretreatment with the anti-EGFR-neutralizing antibody, C225. Pretreatment of NSCLC cells with neutralizing antibodies to the EGFR ligands, TGF-α or HB-EGF, also decreased GRP-mediated MAPK activation. On matrix metalloproteinase (MMP inhibition, GRP failed to activate MAPK in NSCLC cells. EGF and GRP both stimulated NSCLC proliferation, and inhibition of either EGFR or GRPR resulted in cell death. Combining a GRPR antagonist with the EGFR tyrosine kinase inhibitor, gefitinib, resulted in additive cytotoxic effects. Additive effects were seen at gefitinib concentrations from 1 to 18μM, encompassing the ID50 values of both gefitinib-sensitive and gefitinib-resistant NSCLC cell lines. Because a major effect of GRPR appears to be promoting the release of EGFR ligand, this study suggests that a greater inhibition of cell proliferation may occur by abrogating EGFR ligand release in consort with inhibition of EGFR.

  20. Therapy for acute pancreatitis with platelet-activating factor receptor antagonists

    Chong Chen; Shi-Hai Xia; Hong Chen; Xiao-Hong Li

    2008-01-01

    Acute pancreatitis (AP) causes release of plateletactivating factor (PAF),which induces systemic effects that contribute to circulatory disturbances and multiple organ failure.PAF is a cell surface secretion of bioactive lipid,which could produce physiological and pathological effects by binding to its cell surface receptor called platelet-activating factor receptor (PAF-R).Studies showed that PAF participates in the occurrence and development of AP and administration of platelet-activating factor receptor antagonists (PAF-RAs) could significantly reduce local and systemic events after AP.PAF has also been implicated as a key mediator in the progression of severe AP,which can lead to complications and unacceptably high mortality rates.Several classes of PAF-RA show PAFRAs significant local and systemic effects on reducing inflammatory changes.As a preventive treatment,PAF-RA could block a series of PAF-mediatedinflammatory injury and thus improve the prognosis of AR This review introduces the important role of PAF-RA in the treatment of AP.

  1. Platelet activating factor receptor blockade enhances recovery after multifocal brain ischemia

    Kochanek, P.M.; Dutka, A.J.; Kumaroo, K.K.; Hallenbech, J.M.

    1987-12-14

    The authors treated four anesthetized dogs with the platelet activating factor (PAF) receptor antagonist kadsurenone prior to 60 min of multifocal ischemia induced by air embolism, and measured neuronal recovery, blood flow and autologous /sup 111/In-labeled platelet accumulation for 4 h after ischemia. Four anesthetized animals with identical ischemia served as controls. Kadsurenone administered 5 min prior to ischemia and continuously throughout ischemia and recovery significantly enhanced recovery of cortical somatosensory evoked response (CSER) amplitude when compared to controls. They estimated platelet accumulation as /sup 111/In activity (cmp/g tissue) in the injured hemisphere minus that in the non-injured hemisphere. Kadsurenone treated animals did not exhibit significantly altered /sup 111/In labeled platelet accumulation when compared to controls. Beneficial effects of PAF receptor blockade other than those on platelet accumulation may be involved. 20 references, 1 figure.

  2. Platelet activating factor receptor blockade enhances recovery after multifocal brain ischemia

    The authors treated four anesthetized dogs with the platelet activating factor (PAF) receptor antagonist kadsurenone prior to 60 min of multifocal ischemia induced by air embolism, and measured neuronal recovery, blood flow and autologous 111In-labeled platelet accumulation for 4 h after ischemia. Four anesthetized animals with identical ischemia served as controls. Kadsurenone administered 5 min prior to ischemia and continuously throughout ischemia and recovery significantly enhanced recovery of cortical somatosensory evoked response (CSER) amplitude when compared to controls. They estimated platelet accumulation as 111In activity (cmp/g tissue) in the injured hemisphere minus that in the non-injured hemisphere. Kadsurenone treated animals did not exhibit significantly altered 111In labeled platelet accumulation when compared to controls. Beneficial effects of PAF receptor blockade other than those on platelet accumulation may be involved. 20 references, 1 figure

  3. Progesterone-induced blocking factor activates STAT6 via binding to a novel IL-4 receptor.

    Kozma, Noemi; Halasz, Melinda; Polgar, Beata; Poehlmann, Tobias G; Markert, Udo R; Palkovics, Tamas; Keszei, Marton; Par, Gabriella; Kiss, Katalin; Szeberenyi, Jozsef; Grama, Laszlo; Szekeres-Bartho, Julia

    2006-01-15

    Progesterone-induced blocking factor (PIBF) induces Th2-dominant cytokine production. Western blotting and EMSA revealed phosphorylation as well as nuclear translocation of STAT6 and inhibition of STAT4 phosphorylation in PIBF-treated cells. The silencing of STAT6 by small interfering RNA reduced the cytokine effects. Because the activation of the STAT6 pathway depends on the ligation of IL-4R, we tested the involvement of IL-4R in PIBF-induced STAT6 activation. Although PIBF does not bind to IL-4R, the blocking of the latter with an Ab abolished PIBF-induced STAT6 activation, whereas the blocking of the IL-13R had no effect. PIBF activated suppressor of cytokine signaling-3 and inhibited IL-12-induced suppressor of cytokine signaling-1 activation. The blocking of IL-4R counteracted all the described effects, suggesting that the PIBF receptor interacts with IL-4R alpha-chain, allowing PIBF to activate the STAT6 pathway. PIBF did not phosphorylate Jak3, suggesting that the gamma-chain is not needed for PIBF signaling. Confocal microscopic analysis revealed a colocalization and at 37 degrees C a cocapping of the FITC PIBF-activated PIBF receptor and PE anti-IL-4R-labeled IL-4R. After the digestion of the cells with phosphatidylinositol-specific phospholipase C, the STAT6-activating effect of PIBF was lost, whereas that of IL-4 remained unaltered. These data suggest the existence of a novel type of IL-4R composed of the IL-4R alpha-chain and the GPI-anchored PIBF receptor. PMID:16393965

  4. Multiple autophosphorylation sites of the epidermal growth factor receptor are essential for receptor kinase activity and internalization. Contrasting significance of tyrosine 992 in the native and truncated receptors

    Sorkin, A; Helin, K; Waters, C M; Carpenter, G; Beguinot, L

    1992-01-01

    similar to a kinase-negative receptor. Mutation of tyrosine residue Y992 alone in the context of full length EGF receptor, however, did not affect receptor internalization or kinase activity toward phospholipase C-gamma 1. These data indicate that tyrosine 992 is critical for substrate phosphorylation and...... internalization only in the context of the truncated receptor, and that minor autophosphorylation sites, such as Y992, may act as compensatory regulatory sties in the absence of the major EGF receptor autophosphorylation sites....

  5. Suppression of estrogen receptor transcriptional activity by connective tissue growth factor.

    Long Cheng

    Full Text Available Secreted growth factors have been shown to stimulate the transcriptional activity of estrogen receptors (ER that are responsible for many biological processes. However, whether these growth factors physically interact with ER remains unclear. Here, we show for the first time that connective tissue growth factor (CTGF physically and functionally associates with ER. CTGF interacted with ER both in vitro and in vivo. CTGF interacted with ER DNA-binding domain. ER interaction region in CTGF was mapped to the thrombospondin type I repeat, a cell attachment motif. Overexpression of CTGF inhibited ER transcriptional activity as well as the expression of estrogen-responsive genes, including pS2 and cathepsin D. Reduction of endogenous CTGF with CTGF small interfering RNA enhanced ER transcriptional activity. The interaction between CTGF and ER is required for the repression of estrogen-responsive transcription by CTGF. Moreover, CTGF reduced ER protein expression, whereas the CTGF mutant that did not repress ER transcriptional activity also did not alter ER protein levels. The results suggested the transcriptional regulation of estrogen signaling through interaction between CTGF and ER, and thus may provide a novel mechanism by which cross-talk between secreted growth factor and ER signaling pathways occurs.

  6. Integrin α1β1 Regulates Epidermal Growth Factor Receptor Activation by Controlling Peroxisome Proliferator-Activated Receptor γ-Dependent Caveolin-1 Expression ▿ # ‖

    Chen, Xiwu; Whiting, Carrie; Borza, Corina; Hu, Wen; Mont, Stacey; Bulus, Nada; Zhang, Ming-Zhi; Harris, Raymond C.; Zent, Roy; Pozzi, Ambra

    2010-01-01

    Integrin α1β1 negatively regulates the generation of profibrotic reactive oxygen species (ROS) by inhibiting epidermal growth factor receptor (EGFR) activation; however, the mechanism by which it does this is unknown. In this study, we show that caveolin-1 (Cav-1), a scaffolding protein that binds integrins and controls growth factor receptor signaling, participates in integrin α1β1-mediated EGFR activation. Integrin α1-null mesangial cells (MCs) have reduced Cav-1 levels, and reexpression of the integrin α1 subunit increases Cav-1 levels, decreases EGFR activation, and reduces ROS production. Downregulation of Cav-1 in wild-type MCs increases EGFR phosphorylation and ROS synthesis, while overexpression of Cav-1 in the integrin α1-null MCs decreases EGFR-mediated ROS production. We further show that integrin α1-null MCs have increased levels of activated extracellular signal-regulated kinase (ERK), which leads to reduced activation of peroxisome proliferator-activated receptor γ (PPARγ), a transcription factor that positively regulates Cav-1 expression. Moreover, activation of PPARγ or inhibition of ERK increases Cav-1 levels in the integrin α1-null MCs. Finally, we show that glomeruli of integrin α1-null mice have reduced levels of Cav-1 and activated PPARγ but increased levels of phosphorylated EGFR both at baseline and following injury. Thus, integrin α1β1 negatively regulates EGFR activation by positively controlling Cav-1 levels, and the ERK/PPARγ axis plays a key role in regulating integrin α1β1-dependent Cav-1 expression and consequent EGFR-mediated ROS production. PMID:20368353

  7. Integrin alpha1beta1 regulates epidermal growth factor receptor activation by controlling peroxisome proliferator-activated receptor gamma-dependent caveolin-1 expression.

    Chen, Xiwu; Whiting, Carrie; Borza, Corina; Hu, Wen; Mont, Stacey; Bulus, Nada; Zhang, Ming-Zhi; Harris, Raymond C; Zent, Roy; Pozzi, Ambra

    2010-06-01

    Integrin alpha1beta1 negatively regulates the generation of profibrotic reactive oxygen species (ROS) by inhibiting epidermal growth factor receptor (EGFR) activation; however, the mechanism by which it does this is unknown. In this study, we show that caveolin-1 (Cav-1), a scaffolding protein that binds integrins and controls growth factor receptor signaling, participates in integrin alpha1beta1-mediated EGFR activation. Integrin alpha1-null mesangial cells (MCs) have reduced Cav-1 levels, and reexpression of the integrin alpha1 subunit increases Cav-1 levels, decreases EGFR activation, and reduces ROS production. Downregulation of Cav-1 in wild-type MCs increases EGFR phosphorylation and ROS synthesis, while overexpression of Cav-1 in the integrin alpha1-null MCs decreases EGFR-mediated ROS production. We further show that integrin alpha1-null MCs have increased levels of activated extracellular signal-regulated kinase (ERK), which leads to reduced activation of peroxisome proliferator-activated receptor gamma (PPARgamma), a transcription factor that positively regulates Cav-1 expression. Moreover, activation of PPARgamma or inhibition of ERK increases Cav-1 levels in the integrin alpha1-null MCs. Finally, we show that glomeruli of integrin alpha1-null mice have reduced levels of Cav-1 and activated PPARgamma but increased levels of phosphorylated EGFR both at baseline and following injury. Thus, integrin alpha1beta1 negatively regulates EGFR activation by positively controlling Cav-1 levels, and the ERK/PPARgamma axis plays a key role in regulating integrin alpha1beta1-dependent Cav-1 expression and consequent EGFR-mediated ROS production. PMID:20368353

  8. Nuclear factor RIP140 modulates transcriptional activation by the estrogen receptor.

    Cavaillès, V; Dauvois, S; L'Horset, F; Lopez, G; Hoare, S.; Kushner, P J; Parker, M G

    1995-01-01

    A conserved region in the hormone-dependent activation domain AF2 of nuclear receptors plays an important role in transcriptional activation. We have characterized a novel nuclear protein, RIP140, that specifically interacts in vitro with this domain of the estrogen receptor. This interaction was increased by estrogen, but not by anti-estrogens and the in vitro binding capacity of mutant receptors correlates with their ability to stimulate transcription. RIP140 also interacts with estrogen re...

  9. Multimodular biosensors reveal a novel platform for activation of G proteins by growth factor receptors

    Midde, Krishna K.; Aznar, Nicolas; Laederich, Melanie B.; Ma, Gary S.; Kunkel, Maya T.; Newton, Alexandra C.; Ghosh, Pradipta

    2015-01-01

    Environmental cues are transmitted to the interior of the cell via a complex network of signaling hubs. Receptor tyrosine kinases (RTKs) and trimeric G proteins are two such major signaling hubs in eukaryotes. Conventionally, canonical signal transduction via trimeric G proteins is thought to be triggered exclusively by G protein-coupled receptors. Here we used molecular engineering to develop modular fluorescent biosensors that exploit the remarkable specificity of bimolecular recognition, i.e., of both G proteins and RTKs, and reveal the workings of a novel platform for activation of G proteins by RTKs in single living cells. Comprised of the unique modular makeup of guanidine exchange factor Gα-interacting vesicle-associated protein (GIV)/girdin, a guanidine exchange factor that links G proteins to a variety of RTKs, these biosensors provide direct evidence that RTK–GIV–Gαi ternary complexes are formed in living cells and that Gαi is transactivated within minutes after growth factor stimulation at the plasma membrane. Thus, GIV-derived biosensors provide a versatile strategy for visualizing, monitoring, and manipulating the dynamic association of Gαi with RTKs for noncanonical transactivation of G proteins in cells and illuminate a fundamental signaling event regulated by GIV during diverse cellular processes and pathophysiologic states. PMID:25713130

  10. Nuclear localization of platelet-activating factor receptor controls retinal neovascularization

    K Bhosle, Vikrant; Rivera, José Carlos; Zhou, Tianwei (Ellen); Omri, Samy; Sanchez, Melanie; Hamel, David; Zhu, Tang; Rouget, Raphael; Rabea, Areej Al; Hou, Xin; Lahaie, Isabelle; Ribeiro-da-Silva, Alfredo; Chemtob, Sylvain

    2016-01-01

    Platelet-activating factor (PAF) is a pleiotropic phospholipid with proinflammatory, procoagulant and angiogenic actions on the vasculature. We and others have reported the presence of PAF receptor (Ptafr) at intracellular sites such as the nucleus. However, mechanisms of localization and physiologic functions of intracellular Ptafr remain poorly understood. We hereby identify the importance of C-terminal motif of the receptor and uncover novel roles of Rab11a GTPase and importin-5 in nuclear translocation of Ptafr in primary human retinal microvascular endothelial cells. Nuclear localization of Ptafr is independent of exogenous PAF stimulation as well as intracellular PAF biosynthesis. Moreover, nuclear Ptafr is responsible for the upregulation of unique set of growth factors, including vascular endothelial growth factor, in vitro and ex vivo. We further corroborate the intracrine PAF signaling, resulting in angiogenesis in vivo, using Ptafr antagonists with distinct plasma membrane permeability. Collectively, our findings show that nuclear Ptafr translocates in an agonist-independent manner, and distinctive functions of Ptafr based on its cellular localization point to another dimension needed for pharmacologic selectivity of drugs. PMID:27462464

  11. Sequential expression of cyclooxygenase-2, glutamate receptor-2, and platelet activating factor receptor in rat hippocampal neurons after fluid percussion injury

    Zhiqiang Li; Qingming Shu; Lingzhi Li; Maolin Ge; Yongliang Zhang

    2014-01-01

    Traumatic brain injury causes gene expression changes in different brain regions. Occurrence and development of traumatic brain injury are closely related, involving expression of three factors, namely cyclooxygenase-2, glutamate receptor-2, and platelet activating factor receptor. However, little is known about the correlation of these three factors and brain neuronal injury. In this study, primary cultured rat hippocampal neurons were subjected to fluid percussion injury according to Scott’s method, with some modifications. RT-PCR and semi-quantitative immunocytochemical staining was used to measure the expression levels of cyclooxygenase-2, glutamate receptor-2, and platelet activating factor receptor. Our results found that cycloox-ygenase-2 expression were firstly increased post-injury, and then decreased. Both mRNA and protein expression levels reached peaks at 8 and 12 hours post-injury, respectively. Similar sequential changes in glutamate receptor 2 were observed, with highest levels mRNA and pro-tein expression at 8 and 12 hours post-injury respectively. On the contrary, the expressions of platelet activating factor receptor were firstly decreased post-injury, and then increased. Both mRNA and protein expression levels reached the lowest levels at 8 and 12 hours post-injury, respectively. Totally, our findings suggest that these three factors are involved in occurrence and development of hippocampal neuronal injury.

  12. Thrombospondin-1 modulates vascular endothelial growth factor activity at the receptor level

    Zhang, Xuefeng; Kazerounian, Shideh; Duquette, Mark; Perruzzi, Carole; Nagy, Janice A.; Dvorak, Harold F.; Parangi, Sareh; Lawler, Jack

    2009-01-01

    Vascular endothelial growth factor (VEGF) is a well-established stimulator of vascular permeability and angiogenesis, whereas thrombospondin-1 (TSP-1) is a potent angiogenic inhibitor. In this study, we have found that the TSP-1 receptors CD36 and β1 integrin associate with the VEGF receptor 2 (VEGFR2). The coclustering of receptors that regulate angiogenesis may provide the endothelial cell with a platform for integration of positive and negative signals in the plane of the membrane. Thus, t...

  13. Recurrent exposure to nicotine differentiates human bronchial epithelial cells via epidermal growth factor receptor activation

    Cigarette smoking is the major preventable cause of lung cancer in developed countries. Nicotine (3-(1-methyl-2-pyrrolidinyl)-pyridine) is one of the major alkaloids present in tobacco. Besides its addictive properties, its effects have been described in panoply of cell types. In fact, recent studies have shown that nicotine behaves as a tumor promoter in transformed epithelial cells. This research focuses on the effects of acute repetitive nicotine exposure on normal human bronchial epithelial cells (NHBE cells). Here we show that treatment of NHBE cells with recurrent doses of nicotine up to 500 μM triggered cell differentiation towards a neuronal-like phenotype: cells emitted filopodia and expressed neuronal markers such as neuronal cell adhesion molecule, neurofilament-M and the transcription factors neuronal N and Pax-3. We also demonstrate that nicotine treatment induced NF-kB translocation to the nucleus, phosphorylation of the epidermal growth factor receptor (EGFR), and accumulation of heparin binding-EGF in the extracellular medium. Moreover, addition of AG1478, an inhibitor of EGFR tyrosine phosphorylation, or cetuximab, a monoclonal antibody that precludes ligand binding to the same receptor, prevented cell differentiation by nicotine. Lastly, we show that differentiated cells increased their adhesion to the extracellular matrix and their protease activity. Given that several lung pathologies are strongly related to tobacco consumption, these results may help to better understand the damaging consequences of nicotine exposure

  14. Adenosine inhibits neutrophil vascular endothelial growth factor release and transendothelial migration via A2B receptor activation.

    Wakai, A

    2012-02-03

    The effects of adenosine on neutrophil (polymorphonuclear neutrophils; PMN)-directed changes in vascular permeability are poorly characterized. This study investigated whether adenosine modulates activated PMN vascular endothelial growth factor (vascular permeability factor; VEGF) release and transendothelial migration. PMN activated with tumour necrosis factor-alpha (TNF-alpha, 10 ng\\/mL) were incubated with adenosine and its receptor-specific analogues. Culture supernatants were assayed for VEGF. PMN transendothelial migration across human umbilical vein endothelial cell (HUVEC) monolayers was assessed in vitro. Adhesion molecule receptor expression was assessed flow cytometrically. Adenosine and some of its receptor-specific analogues dose-dependently inhibited activated PMN VEGF release. The rank order of potency was consistent with the affinity profile of human A2B receptors. The inhibitory effect of adenosine was reversed by 3,7-dimethyl-1-propargylxanthine, an A2 receptor antagonist. Adenosine (100 microM) or the A2B receptor agonist 5\\'-N-ethylcarboxamidoadenosine (NECA, 100 microM) significantly reduced PMN transendothelial migration. However, expression of activated PMN beta2 integrins and HUVEC ICAM-1 were not significantly altered by adenosine or NECA. Adenosine attenuates human PMN VEGF release and transendothelial migration via the A2B receptor. This provides a novel target for the modulation of PMN-directed vascular hyperpermeability in conditions such as the capillary leak syndrome.

  15. A novel signaling pathway of tissue kallikrein in promoting keratinocyte migration: Activation of proteinase-activated receptor 1 and epidermal growth factor receptor

    Biological functions of tissue kallikrein (TK, KLK1) are mainly mediated by kinin generation and subsequent kinin B2 receptor activation. In this study, we investigated the potential role of TK and its signaling pathways in cultured human keratinocyte migration and in a rat skin wound healing model. Herein, we show that TK promoted cell migration and proliferation in a concentration- and time-dependent manner. Inactive TK or kinin had no significant effect on cell migration. Interestingly, cell migration induced by active TK was not blocked by icatibant or L-NAME, indicating an event independent of kinin B2 receptor and nitric oxide formation. TK's stimulatory effect on cell migration was inhibited by small interfering RNA for proteinase-activated receptor 1 (PAR1), and by PAR1 inhibitor. TK-induced migration was associated with increased phosphorylation of epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase (ERK), which was blocked by inhibition of protein kinase C (PKC), Src, EGFR and ERK. TK-induced cell migration and EGFR phosphorylation were blocked by metalloproteinase (MMP) inhibitor, heparin, and antibodies against EGFR external domain, heparin-binding EGF-like growth factor (HB-EGF) and amphiregulin (AR). Local application of TK promoted skin wound healing in rats, whereas icatibant and EGFR inhibitor blocked TK's effect. Skin wound healing was further delayed by aprotinin and neutralizing TK antibody. This study demonstrates a novel role of TK in skin wound healing and uncovers new signaling pathways mediated by TK in promoting keratinocyte migration through activation of the PAR1-PKC-Src-MMP pathway and HB-EGF/AR shedding-dependent EGFR transactivation.

  16. Increased expression of protease-activated receptor 4 and Trefoil factor 2 in human colorectal cancer.

    Guoyu Yu

    Full Text Available Protease-activated receptor 4 (PAR4, a member of G-protein coupled receptors family, was recently reported to exhibit decreased expression in gastric cancer and esophageal squamous cancer, yet increased expression during the progression of prostate cancer. Trefoil factor 2 (TFF2, a small peptide constitutively expressed in the gastric mucosa, plays a protective role in restitution of gastric mucosa. Altered TFF2 expression was also related to the development of gastrointestinal cancer. TFF2 has been verified to promote cell migration via PAR4, but the roles of PAR4 and TFF2 in the progress of colorectal cancer are still unknown. In this study, the expression level of PAR4 and TFF2 in colorectal cancer tissues was measured using real-time PCR (n = 38, western blotting (n=38 and tissue microarrays (n = 66. The mRNA and protein expression levels of PAR4 and TFF2 were remarkably increased in colorectal cancer compared with matched noncancerous tissues, especially in positive lymph node and poorly differentiated cancers. The colorectal carcinoma cell LoVo showed an increased response to TFF2 as assessed by cell invasion upon PAR4 expression. However, after intervention of PAR4 expression, PAR4 positive colorectal carcinoma cell HT-29 was less responsive to TFF2 in cell invasion. Genomic bisulfite sequencing showed the hypomethylation of PAR4 promoter in colorectal cancer tissues and the hypermethylation in the normal mucosa that suggested the low methylation of promoter was correlated to the increased PAR4 expression. Taken together, the results demonstrated that the up-regulated expression of PAR4 and TFF2 frequently occurs in colorectal cancer tissues, and that overexpression of PAR4 may be resulted from promoter hypomethylation. While TFF2 promotes invasion activity of LoVo cells overexpressing PAR4, and this effect was significantly decreased when PAR4 was knockdowned in HT-29 cells. Our findings will be helpful in further investigations into the

  17. Receptor activator of nuclear factor-κB ligand and osteoprotegerin: maintaining the balance to prevent bone loss

    Trouvin, Anne-Priscille; Goëb, Vincent

    2010-01-01

    Bone remodeling requires a precise balance between resorption and formation. It is a complex process that involves numerous factors: hormones, growth factors, vitamins, and cytokines, and notably osteoprotegerin (OPG) and receptor activator for nuclear factor-κB (RANK) ligand. The signaling pathway OPG/RANK/RANKL is key to regulation for maintaining the balance between the activity of osteoblasts and osteoclasts in order to prevent bone loss and ensure a normal bone turnover. In this review, ...

  18. Fibroblast growth factor receptor 3 effects on proliferation and telomerase activity in sheep growth plate chondrocytes

    Smith Logan B

    2012-12-01

    Full Text Available Abstract Background Fibroblast growth factor receptor 3 (FGFR3 inhibits growth-plate chondrocyte proliferation and limits bone elongation. Gain-of-function FGFR3 mutations cause dwarfism, reduced telomerase activity and shorter telomeres in growth plate chondroyctes suggesting that FGFR3 reduces proliferative capacity, inhibits telomerase, and enhances senescence. Thyroid hormone (T3 plays a role in cellular maturation of growth plate chondrocytes and a known target of T3 is FGFR3. The present study addressed whether reduced FGFR3 expression enhanced telomerase activity, mRNA expression of telomerase reverse transcriptase (TERT and RNA component of telomerase (TR, and chondrocyte proliferation, and whether the stimulation of FGFR3 by T3 evoked the opposite response. Results Sheep growth-plate proliferative zone chondrocytes were cultured and transfected with siRNA to reduce FGFR3 expression; FGFR3 siRNA reduced chondrocyte FGFR3 mRNA and protein resulting in greater proliferation and increased TERT mRNA expression and telomerase activity (p 3 significantly enhanced FGFR3 mRNA and protein expression and reduced telomerase activity (p 3 at the growth plate may be partially mediated through the FGFR3 pathway. Conclusions The results suggest that FGFR3 inhibits chondrocyte proliferation by down-regulating TERT expression and reducing telomerase activity indicating an important role for telomerase in sustaining chondrocyte proliferative capacity during bone elongation.

  19. AP-2α suppresses skeletal myoblast proliferation and represses fibroblast growth factor receptor 1 promoter activity

    Skeletal muscle development is partly characterized by myoblast proliferation and subsequent differentiation into postmitotic muscle fibers. Developmental regulation of expression of the fibroblast growth factor receptor 1 (FGFR1) gene is required for normal myoblast proliferation and muscle formation. As a result, FGFR1 promoter activity is controlled by multiple transcriptional regulatory proteins during both proliferation and differentiation of myogenic cells. The transcription factor AP-2α is present in nuclei of skeletal muscle cells and suppresses myoblast proliferation in vitro. Since FGFR1 gene expression is tightly linked to myoblast proliferation versus differentiation, the FGFR1 promoter was examined for candidate AP-2α binding sites. Mutagenesis studies indicated that a candidate binding site located at - 1035 bp functioned as a repressor cis-regulatory element. Furthermore, mutation of this site alleviated AP-2α-mediated repression of FGFR1 promoter activity. Chromatin immunoprecipitation studies demonstrated that AP-2α interacted with the FGFR1 promoter in both proliferating myoblasts and differentiated myotubes. In total, these results indicate that AP-2α is a transcriptional repressor of FGFR1 gene expression during skeletal myogenesis.

  20. Downstream-of-FGFR Is a Fibroblast Growth Factor-Specific Scaffolding Protein and Recruits Corkscrew upon Receptor Activation

    Petit, Valérie; Nussbaumer, Ute; Dossenbach, Caroline; Affolter, Markus

    2004-01-01

    Fibroblast growth factor (FGF) receptor (FGFR) signaling controls the migration of glial, mesodermal, and tracheal cells in Drosophila melanogaster. Little is known about the molecular events linking receptor activation to cytoskeletal rearrangements during cell migration. We have performed a functional characterization of Downstream-of-FGFR (Dof), a putative adapter protein that acts specifically in FGFR signal transduction in Drosophila. By combining reverse genetic, cell culture, and bioch...

  1. Receptor activator of nuclear factor-κB ligand and osteoprotegerin: maintaining the balance to prevent bone loss

    Anne-Priscille Trouvin; Vincent Goëb

    2010-01-01

    Anne-Priscille Trouvin, Vincent GoëbDepartment of Rheumatology, Rouen University Hospital, Rouen, FranceAbstract: Bone remodeling requires a precise balance between resorption and formation. It is a complex process that involves numerous factors: hormones, growth factors, vitamins, and cytokines, and notably osteoprotegerin (OPG) and receptor activator for nuclear factor-κB (RANK) ligand. The signaling pathway OPG/RANK/RANKL is key to regulation for maintaining the balance ...

  2. Leukemia inhibitory factor promote trophoblast invasion via urokinase-type plasminogen activator receptor in preeclampsia.

    Zheng, Qin; Dai, Kuixing; Cui, Xinyuan; Yu, Ming; Yang, Xuesong; Yan, Bin; Liu, Shuai; Yan, Qiu

    2016-05-01

    Preeclampsia is a pregnancy-related syndrome which can cause perinatal mortality and morbidity. Inadequate invasion by trophoblast cells may lead to poor perfusion of the placenta, even result in preeclampsia. Understanding the molecular mechanisms underlying placentation facilitates the better intervention of preeclampsia. Urokinase-type plasminogen activator receptor (uPAR) is involved in the physiological and pathological processes. Leukemia inhibitory factor (LIF) is an important regulator in the establishment of pregnancy. However, the expression of uPAR in preeclamptic patients and its relationship with LIF remains unclear. In the current study, we found that the level of uPAR was relatively lower in the placentas from preeclamptic patients as compared with normal pregnant women. LIF promoted trophoblast cell outgrowth by upregulating uPAR in an explants culture, and LIF also enhanced migration and invasion potential through uPAR in trophoblast JAR and JEG-3 cell lines, and with increased gelatinolytic activities of matrix metalloproteinase 2 (MMP-2). The effect of LIF and uPAR on trophoblast migration and invasion was mediated by PI3K/AKT signaling pathway. Our data indicates the roles of LIF in promoting trophoblast migration and invasion through uPAR and suggest that abnormal expression of uPAR might be associated with the etiology of preeclampsia. PMID:27133045

  3. UV-B Radiation Induces Macrophage Migration Inhibitory Factor–Mediated Melanogenesis through Activation of Protease-Activated Receptor-2 and Stem Cell Factor in Keratinocytes

    Enomoto, Akiko; Yoshihisa, Yoko; Yamakoshi, Takako; Ur Rehman, Mati; Norisugi, Osamu; HARA Hiroshi; Matsunaga, Kenji; Makino, Teruhiko; Nishihira, Jun; Shimizu, Tadamichi

    2011-01-01

    UV radiation indirectly regulates melanogenesis in melanocytes through a paracrine regulatory mechanism involving keratinocytes. Protease-activated receptor (PAR)-2 activation induces melanosome transfer by increasing phagocytosis of melanosomes by keratinocytes. This study demonstrated that macrophage migration inhibitory factor (MIF) stimulated PAR-2 expression in human keratinocytes. In addition, we showed that MIF stimulated stem cell factor (SCF) release in keratinocytes; however, MIF ha...

  4. Recruitment of epidermal growth factor receptors into coated pits requires their activated tyrosine kinase

    1995-01-01

    EGF-receptor (EGF-R) tyrosine kinase is required for the down- regulation of activated EGF-R. However, controversy exists as to whether ligand-induced activation of the EGF-R tyrosine kinase is required for internalization or for lysosomal targeting. We have addressed this issue using a cell-free assay that selectively measures the recruitment of EGF-R into coated pits. Here we show that EGF bound to wild-type receptors is efficiently sequestered in coated pits. In contrast, sequestration of ...

  5. Search for a platelet-activating factor receptor in the Trypanosoma cruzi proteome: a potential target for Chagas disease chemotherapy

    Daniel Fábio Kawano

    2011-12-01

    Full Text Available Chagas disease (CD causes the highest burden of parasitic diseases in the Western Hemisphere and is therefore a priority for drug research and development. Platelet-activating factor (PAF causes the CD parasite Trypanosoma cruzi to differentiate, which suggests that the parasite may express PAF receptors. Here, we explored the T. cruzi proteome for PAF receptor-like proteins. From a total of 23,000 protein sequences, we identified 29 hypothetical proteins that are predicted to have seven transmembrane domains (TMDs, which is the main characteristic of the G protein-coupled receptors (GPCRs, including the PAF receptor. The TMDs of these sequences were independently aligned with domains from 25 animal PAF receptors and the sequences were analysed for conserved residues. The conservation score mean values for the TMDs of the hypothetical proteins ranged from 31.7-44.1%, which suggests that if the putative T. cruzi PAF receptor is among the sequences identified, the TMDs are not highly conserved. These results suggest that T. cruzi contains several GPCR-like proteins and that one of these GPCRs may be a PAF receptor. Future studies may further validate the PAF receptor as a target for CD chemotherapy.

  6. A novel epidermal growth factor receptor variant lacking multiple domains directly activates transcription and is overexpressed in tumors

    Piccione, EC; Lieu, TJ; Gentile, CF; Williams, TR; Connolly, AJ; Godwin, AK; Koong, AC; Wong, AJ

    2011-01-01

    The epidermal growth factor receptor (EGFR) is essential to multiple physiological and neoplastic processes via signaling by its tyrosine kinase domain and subsequent activation of transcription factors. EGFR overexpression and alteration, including point mutations and structural variants, contribute to oncogenesis in many tumor types. In this study, we identified an in-frame splice variant of the EGFR called mini-LEEK (mLEEK) that is more broadly expressed than the EGFR and is overexpressed ...

  7. Effect of peripheral benzodiazepine receptor ligands on lipopolysaccharide-induced tumor necrosis factor activity in thioglycolate-treated mice.

    Matsumoto, T.; Ogata, M.; Koga, K.; Shigematsu, A

    1994-01-01

    To investigate the effect of peripheral and central benzodiazepine receptor ligands on lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF) activity in mouse macrophages, three types of ligands, 4'-chlorodiazepam (pure peripheral), midazolam (mixed), and clonazepam (pure central), were compared. Midazolam and 4'-chlorodiazepam significantly suppressed LPS (1-microgram/ml)-induced TNF activity in thioglycolate-elicited mouse macrophages. In every concentration examined (0.001 to 100 mi...

  8. Artemin growth factor increases nicotinic cholinergic receptor subunit expression and activity in nociceptive sensory neurons

    Albers, Kathryn M.; Zhang, Xiu Lin; Diges, Charlotte M.; Schwartz, Erica S.; Yang, Charles I; Davis, Brian M.; Gold, Michael S.

    2014-01-01

    Background Artemin (Artn), a member of the glial cell line-derived growth factor (GDNF) family, supports the development and function of a subpopulation of peptidergic, TRPV1-positive sensory neurons. Artn (enovin, neublastin) is elevated in inflamed tissue and its injection in skin causes transient thermal hyperalgesia. A genome wide expression analysis of trigeminal ganglia of mice that overexpress Artn in the skin (ART-OE mice) showed elevation in nicotinic acetylcholine receptor (nAChR) s...

  9. Constitutive asymmetric dimerization drives oncogenic activation of epidermal growth factor receptor carboxyl-terminal deletion mutants

    Park, Angela K.J.; Francis, Joshua M; Park, Woong-Yang; Park, Joon-Oh; Cho, Jeonghee

    2015-01-01

    Genomic alterations targeting the Epidermal Growth Factor Receptor (EGFR) gene have been strongly associated with cancer pathogenesis. The clinical effectiveness of EGFR targeted therapies, including small molecules directed against the kinase domain such as gefitinib, erlotinib and afatinib, have been proven successful in treating non-small cell lung cancer patients with tumors harboring EGFR kinase domain mutations. Recent large-scale genomic studies in glioblastoma and lung cancer have ide...

  10. Peripheral activation of corticotropin-releasing factor receptor 2 inhibits food intake and alters meal structures in mice

    Wang, Lixin; Stengel, Andreas; Goebel, Miriam; Martinez, Vicente; Gourcerol, Guillaume; Rivier, Jean; Taché, Yvette

    2010-01-01

    The orexigenic effect of urocortins (Ucn 1, Ucn 2 and Ucn 3) through activation of corticotropin-releasing factor (CRF) receptors, has been well characterized after injection into the brain but not in the periphery. We examined the role of CRF receptor subtype 2 (CRF2) in the regulation of food intake using intraperitoneal (ip) injection of Ucns, the selective CRF2 antagonist, astressin2-B, and CRF2 knockout (−/−) mice. Meal structures were monitored using an automated episodic solid food int...

  11. Synergy between estrogen receptor α activation functions AF1 and AF2 mediated by transcription intermediary factor TIF2

    Benecke, Arndt; Chambon, Pierre; Gronemeyer, Hinrich

    2000-01-01

    The activation function AF2 in the ligand-binding domain of estrogen receptors ERα and ERβ signals through the recruitment of nuclear receptor coactivators. Recent evidence indicates that coactivators, such as the transcription intermediary factor TIF2, also bind to and transactivate the N-terminal AF1 function of the two ERs. We have generated TIF2 mutant proteins that are deficient in either AF1 or AF2 interaction and use these mutants to investigate the relative contribution of both AFs to...

  12. Dopamine receptor regulating factor, DRRF: A zinc finger transcription factor

    Hwang, Cheol Kyu; D'Souza, Ursula M.; Eisch, Amelia J.; Yajima, Shunsuke; Lammers, Claas-Hinrich; Yang, Young; Lee, Sang-Hyeon; Kim, Yong-Man; Nestler, Eric J.; Mouradian, M. Maral

    2001-01-01

    Dopamine receptor genes are under complex transcription control, determining their unique regional distribution in the brain. We describe here a zinc finger type transcription factor, designated dopamine receptor regulating factor (DRRF), which binds to GC and GT boxes in the D1A and D2 dopamine receptor promoters and effectively displaces Sp1 and Sp3 from these sequences. Consequently, DRRF can modulate the activity of these dopamine receptor promoters. Highest DRRF mRNA levels are found in ...

  13. Soluble perlecan domain i enhances vascular endothelial growth factor-165 activity and receptor phosphorylation in human bone marrow endothelial cells

    Gomes Ronald R

    2010-11-01

    Full Text Available Abstract Background Immobilized recombinant perlecan domain I (PlnDI binds and modulates the activity of heparin-binding growth factors, in vitro. However, activities for PlnDI, in solution, have not been reported. In this study, we assessed the ability of soluble forms to modulate vascular endothelial growth factor-165 (VEGF165 enhanced capillary tube-like formation, and VEGF receptor-2 phosphorylation of human bone marrow endothelial cells, in vitro. Results In solution, PlnDI binds VEGF165 in a heparan sulfate and pH dependent manner. Capillary tube-like formation is enhanced by exogenous PlnDI; however, PlnDI/VEGF165 mixtures combine to enhance formation beyond that stimulated by either PlnDI or VEGF165 alone. PlnDI also stimulates VEGF receptor-2 phosphorylation, and mixtures of PlnDI/VEGF165 reduce the time required for peak VEGF receptor-2 phosphorylation (Tyr-951, and increase Akt phosphorylation. PlnDI binds both immobilized neuropilin-1 and VEGF receptor-2, but has a greater affinity for neuropilin-1. PlnDI binding to neuropilin-1, but not to VEGF receptor-2 is dependent upon the heparan sulfate chains adorning PlnDI. Interestingly, the presence of VEGF165 but not VEGF121 significantly enhances PlnDI binding to Neuropilin-1 and VEGF receptor-2. Conclusions Our observations suggest soluble forms of PlnDI are biologically active. Moreover, PlnDI heparan sulfate chains alone or together with VEGF165 can enhance VEGFR-2 signaling and angiogenic events, in vitro. We propose PlnDI liberated during basement membrane or extracellular matrix turnover may have similar activities, in vivo.

  14. Epidermal growth factor receptor activation by diesel particles is mediated by tyrosine phosphatase inhibition

    Exposure to particulate matter (PM) is associated with increased cardiopulmonary morbidity and mortality. Diesel exhaust particles (DEP) are a major component of ambient PM and may contribute to PM-induced pulmonary inflammation. Proinflammatory signaling is mediated by phosphorylation-dependent signaling pathways whose activation is opposed by the activity of protein tyrosine phosphatases (PTPases) which thereby function to maintain signaling quiescence. PTPases contain an invariant catalytic cysteine that is susceptible to electrophilic attack. DEP contain electrophilic oxy-organic compounds that may contribute to the oxidant effects of PM. Therefore, we hypothesized that exposure to DEP impairs PTPase activity allowing for unopposed basal kinase activity. Here we report that exposure to 30 μg/cm2 DEP for 4 h induces differential activation of signaling in primary cultures of human airway epithelial cells (HAEC), a primary target cell in PM inhalation. In-gel kinase activity assay of HAEC exposed to DEPs of low (L-DEP), intermediate (I-DEP) or high (H-DEP) organic content showed differential activation of intracellular kinases. Exposure to these DEP also induced varying levels of phosphorylation of the receptor tyrosine kinase EGFR in a manner that requires EGFR kinase activity but does not involve receptor dimerization. We demonstrate that treatment with DEP results in an impairment of total and EGFR-directed PTPase activity in HAEC with a potency that is independent of the organic content of these particles. These data show that DEP-induced EGFR phosphorylation in HAEC is the result of a loss of PTPase activities which normally function to dephosphorylate EGFR in opposition to baseline EGFR kinase activity

  15. Prediction of inhibitory activity of epidermal growth factor receptor inhibitors using grid search-projection pursuit regression method.

    Hongying Du

    Full Text Available The epidermal growth factor receptor (EGFR protein tyrosine kinase (PTK is an important protein target for anti-tumor drug discovery. To identify potential EGFR inhibitors, we conducted a quantitative structure-activity relationship (QSAR study on the inhibitory activity of a series of quinazoline derivatives against EGFR tyrosine kinase. Two 2D-QSAR models were developed based on the best multi-linear regression (BMLR and grid-search assisted projection pursuit regression (GS-PPR methods. The results demonstrate that the inhibitory activity of quinazoline derivatives is strongly correlated with their polarizability, activation energy, mass distribution, connectivity, and branching information. Although the present investigation focused on EGFR, the approach provides a general avenue in the structure-based drug development of different protein receptor inhibitors.

  16. Tyrosine kinase activity of a chimeric insulin-like-growth-factor-1 receptor containing the insulin receptor C-terminal domain. Comparison with the tyrosine kinase activities of the insulin and insulin-like-growth-factor-1 receptors using a cell-free system.

    Mothe, I; Tartare, S; Kowalski-Chauvel, A; Kaliman, P; Van Obberghen, E; Ballotti, R

    1995-03-15

    In a previous study, we showed that a chimeric insulin-like-growth-factor-1 (IGF-1) receptor, with the beta subunit C-terminal part of the insulin receptor was more efficient in stimulating glycogen synthesis and p44mapk activity compared to the wild-type IFG-1 receptor [Tartare, S., Mothe, I., Kowalski-Chauvel, A., Breittmayer, J.-P., Ballotti, R. & Van Obberghen, E. (1994) J. Biol. Chem. 269, 11449-11455]. These data indicate that the receptor C-terminal domain plays an important role in the transmission of biological effects. To understand the molecular basis of the differences in receptor specificity, we studied the characteristics of insulin, IGF-1 and chimeric receptor tyrosine kinase activities in a cell-free system. We found that, compared to wild-type insulin and IGF-1 receptors, the chimeric receptor showed a decrease in (a) autophosphorylation, (b) tyrosine kinase activity towards insulin receptor substrate-1 and the insulin receptor-(1142-1158)-peptide, and (c) the ability to activate phosphatidylinositol 3-kinase. However, for all the effects measured in a cell-free system, the chimeric receptor displayed an increased response to IGF-1 compared to the native IGF-1 receptor. Concerning the cation dependence of the tyrosine kinase activity, we showed that, at 10 mM Mg2+, the ligand-stimulated phosphorylation of poly(Glu80Tyr20) by both insulin receptor and chimeric receptor was increased by Mn2+. Conversely at 50 mM Mg2+, the chimeric receptor behaved like the IGF-1 receptor, since the presence of Mn2+ decreased the stimulatory effect of IGF-1 on their kinase activity. Furthermore, the Km of the chimeric receptor for ATP was increased compared to the wild-type receptors. These data demonstrate that the replacement of the C-terminal tail of the IGF-1 receptor by that of the insulin receptor has changed the receptor characteristics studied in a cell-free system. Our findings indicate that the C-terminal domain of the insulin receptor beta subunit plays a

  17. Analysis of Activated Platelet-Derived Growth Factor β Receptor and Ras-MAP Kinase Pathway in Equine Sarcoid Fibroblasts

    Gennaro Altamura

    2013-01-01

    Full Text Available Equine sarcoids are skin tumours of fibroblastic origin affecting equids worldwide. Bovine papillomavirus type-1 (BPV-1 and, less commonly, type-2 are recognized as etiological factors of sarcoids. The transforming activity of BPV is related to the functions of its major oncoprotein E5 which binds to the platelet-derived growth factor β receptor (PDGFβR causing its phosphorylation and activation. In this study, we demonstrate, by coimmunoprecipitation and immunoblotting, that in equine sarcoid derived cell lines PDGFβR is phosphorylated and binds downstream molecules related to Ras-mitogen-activated protein kinase-ERK pathway thus resulting in Ras activation. Imatinib mesylate is a tyrosine kinase receptors inhibitor which selectively inhibits the activation of PDGFβR in the treatment of several human and animal cancers. Here we show that imatinib inhibits receptor phosphorylation, and cell viability assays demonstrate that this drug decreases sarcoid fibroblasts viability in a dose-dependent manner. This study contributes to a better understanding of the molecular mechanisms involved in the pathology of sarcoids and paves the way to a new therapeutic approach for the treatment of this common equine skin neoplasm.

  18. Nuclear factor of activated T cells negatively regulates expression of the tumor necrosis factor receptor-related 2 gene in T cells

    Kim, Woon-Ki; Sul, Ok-Ju; Kwak, Jung-Sook; Hur, Hye-Young; Latour, Anne M.; Koller, Beverly H.; Kwon, Byoung S.; Jeong, Choon-Soo

    2010-01-01

    Tumor necrosis factor receptor-related 2 (TR2, HVEM or TNFRSF-14) plays an important role in immune responses, however, the mechanisms regulating its expression are unclear. To understand the control of TR2 gene expression, we studied the upstream region of the gene. Gel supershift assays revealed inducible binding of nuclear factor of activated T cells (NFAT) to a putative NFAT site within the TR2 promoter. Furthermore, cotransfection of a dominant negative NFAT construct, or siRNA for NFAT,...

  19. Bronchial hyperreactivity, increased endotoxin lethality and melanocytic tumorigenesis in transgenic mice overexpressing platelet-activating factor receptor.

    Ishii, S.; Nagase, T; Tashiro, F; Ikuta, K. (Koichi); Sato, S.; Waga, I.; Kume, K.; Miyazaki, J; Shimizu, T

    1997-01-01

    Although platelet-activating factor (PAF) has been shown to exert pleiotropic effects on isolated cells or tissues, controversy still exists as to whether it plays significant pathophysiological roles in vivo. To answer this question, we established transgenic mice over-expressing a guinea-pig PAF receptor (PAFR). The transgenic mice showed a bronchial hyperreactivity to methacholine and an increased mortality when exposed to bacterial endotoxin. An aberrant melanogenesis and proliferative ab...

  20. Deficiency of the B Cell-Activating Factor Receptor Results in Limited CD169+ Macrophage Function during Viral Infection

    Xu, Haifeng C.; Huang, Jun; Khairnar, Vishal; Duhan, Vikas; Pandyra, Aleksandra A; Grusdat, Melanie; Shinde, Prashant; McIlwain, David R.; Maney, Sathish Kumar; Gommerman, Jennifer; Löhning, Max; Ohashi, Pamela S.; Mak, Tak W; Pieper, Kathrin; Sic, Heiko

    2015-01-01

    The B cell-activating factor (BAFF) is critical for B cell development and humoral immunity in mice and humans. While the role of BAFF in B cells has been widely described, its role in innate immunity remains unknown. Using BAFF receptor (BAFFR)-deficient mice, we characterized BAFFR-related innate and adaptive immune functions following infection with vesicular stomatitis virus (VSV) and lymphocytic choriomeningitis virus (LCMV). We identified a critical role for BAFFR signaling in the gener...

  1. Tumor necrosis factor alpha stimulates NMDA receptor activity in mouse cortical neurons resulting in ERK-dependent death

    Jara, Javier H.; Singh, Brij B.; Floden, Angela M.; Combs, Colin K.

    2007-01-01

    Multiple cytokines are secreted in the brain during pro-inflammatory conditions and likely affect neuron survival. Previously, we demonstrated that glutamate and tumor necrosis factor alpha (TNFα) kill neurons via activation of the N-methyl-d-aspartate (NMDA) and TNFα receptors, respectively. This report continues characterizing the signaling cross-talk pathway initiated during this inflammation-related mechanism of death. Stimulation of mouse cortical neuron cultures with TNFα results in a t...

  2. TRAP/SMCC/Mediator-Dependent Transcriptional Activation from DNA and Chromatin Templates by Orphan Nuclear Receptor Hepatocyte Nuclear Factor 4

    Malik, Sohail; Wallberg, Annika E.; Kang, Yun Kyoung; Roeder, Robert G.

    2002-01-01

    The orphan nuclear receptor hepatocyte nuclear factor 4 (HNF-4) regulates the expression of many liver-specific genes both during development and in the adult animal. Towards understanding the molecular mechanisms by which HNF-4 functions, we have established in vitro transcription systems that faithfully recapitulate HNF-4 activity. Here we have focused on the coactivator requirements for HNF-4, especially for the multicomponent TRAP/SMCC/Mediator complex that has emerged as the central regu...

  3. Estradiol repression of tumor necrosis factor-α transcription requires estrogen receptor activation function-2 and is enhanced by coactivators

    An, Jinping; Ribeiro, Ralff C. J.; Webb, Paul; Gustafsson, Jan-Åke; Kushner, Peter J.; Baxter, John D.; Leitman, Dale C.

    1999-01-01

    The tumor necrosis factor-α (TNF-α) promoter was used to explore the molecular mechanisms of estradiol (E2)-dependent repression of gene transcription. E2 inhibited basal activity and abolished TNF-α activation of the TNF-α promoter. The E2-inhibitory element was mapped to the −125 to −82 region of the TNF-α promoter, known as the TNF-responsive element (TNF-RE). An AP-1-like site in the TNF-RE is essential for repression activity. Estrogen receptor (ER) β is more potent than ERα at repressin...

  4. In situ autoradiography and ligand-dependent tyrosine kinase activity reveal insulin receptors and insulin-like growth factor I receptors in prepancreatic chicken embryos

    We previously reported specific cross-linking of 125I-labeled insulin and 125I-labeled insulin-like growth factor I (IGF-I) to the alpha subunit of their respective receptors in chicken embryos of 20 somites and older. To achieve adequate sensitivity and localize spatially the receptors in younger embryos, we adapted an autoradiographic technique using whole-mounted chicken blastoderms. Insulin receptors and IGF-I receptors were expressed and could be localized as early as gastrulation, before the first somite is formed. Relative density was analyzed by a computer-assisted image system, revealing overall slightly higher binding of IGF-I than of insulin. Structures rich in both types of receptors were predominantly of ectodermal origin: Hensen's node in gastrulating embryos and neural folds, neural tube and optic vesicles during neurulation. The signal transduction capability of the receptors in early organogenesis was assessed by their ability to phosphorylate the exogenous substrate poly(Glu80Tyr20). Ligand-dependent tyrosine phosphorylation was demonstrable with both insulin and IGF-I in glycoprotein-enriched preparations from embryos at days 2 through 6 of embryogenesis. There was a developmentally regulated change in ligand-dependent tyrosine kinase activity, with a sharp increase from day 2 to day 4, in contrast with a small increase in the ligand binding. Binding of 125I-labeled IGF-I was, with the solubilized receptors, severalfold higher than binding of 125I-labeled insulin. However, the insulin-dependent phosphorylation was as high as the IGF-I-dependent phosphorylation at each developmental stage

  5. Activation of corticotropin-releasing factor receptors in the rostral ventrolateral medulla is required for glucose-induced sympathoexcitation.

    Bardgett, Megan E; Sharpe, Amanda L; Toney, Glenn M

    2014-11-15

    Energy expenditure is determined by metabolic rate and diet-induced thermogenesis. Normally, energy expenditure increases due to neural mechanisms that sense plasma levels of ingested nutrients/hormones and reflexively increase sympathetic nerve activity (SNA). Here, we investigated neural mechanisms of glucose-driven sympathetic activation by determining contributions of neuronal activity in the hypothalamic paraventricular nucleus (PVN) and activation of corticotropin-releasing factor (CRF) receptors in the rostral ventrolateral medulla (RVLM). Glucose was infused intravenously (150 mg/kg, 10 min) in male rats to raise plasma glucose concentration to a physiological postprandial level. In conscious rats, glucose infusion activated CRF-containing PVN neurons and TH-containing RVLM neurons, as indexed by c-Fos immunofluorescence. In α-chloralose/urethane-anesthetized rats, glucose infusion increased lumbar and splanchnic SNA, which was nearly prevented by prior RVLM injection of the CRF receptor antagonist astressin (10 pmol/50 nl). This cannot be attributed to a nonspecific effect, as sciatic afferent stimulation increased SNA and ABP equivalently in astressin- and aCSF-injected rats. Glucose-stimulated sympathoexcitation was largely reversed during inhibition of PVN neuronal activity with the GABA-A receptor agonist muscimol (100 pmol/50 nl). The effects of astressin to prevent glucose-stimulated sympathetic activation appear to be specific to interruption of PVN drive to RVLM because RVLM injection of astressin prior to glucose infusion effectively prevented SNA from rising and prevented any fall of SNA in response to acute PVN inhibition with muscimol. These findings suggest that activation of SNA, and thus energy expenditure, by glucose is initiated by activation of CRF receptors in RVLM by descending inputs from PVN. PMID:25269482

  6. Structure of the active core of human stem cell factor and analysis of binding to its receptor Kit

    Jiang, Xuliang; Gurel, Ogan; Mendiaz, Elizabeth A.; Stearns, George W.; Clogston, Christi L.; Lu, Hsieng S; Osslund, Timothy D.; Syed, Rashid S.; Langley, Keith E.; Hendrickson, Wayne A

    2000-01-01

    Stem cell factor (SCF) is an early-acting hematopoietic cytokine that elicits multiple biological effects. SCF is dimeric and occurs in soluble and membrane-bound forms. It transduces signals by ligand- mediated dimerization of its receptor, Kit, which is a receptor tyrosine kinase related to the receptors for platelet-derived growth factor (PDGF), macrophage colony-stimulating factor, Flt-3 ligand and vascular endothelial growth factor (VEGF). All of these have extracellular ligand-binding p...

  7. Transcription factor network downstream of protease activated receptors (PARs modulating mouse bladder inflammation

    Hurst Robert E

    2007-08-01

    Full Text Available Abstract Background All four PARs are present in the urinary bladder, and their expression is altered during inflammation. In order to search for therapeutic targets other than the receptors themselves, we set forth to determine TFs downstream of PAR activation in the C57BL/6 urinary bladders. Methods For this purpose, we used a protein/DNA combo array containing 345 different TF consensus sequences. Next, the TF selected was validated by EMSA and IHC. As mast cells seem to play a fundamental role in bladder inflammation, we determined whether c-kit receptor deficient (Kitw/Kitw-v mice have an abrogated response to PAR stimulation. Finally, TFEB antibody was used for CHIP/Q-PCR assay and revealed up-regulation of genes known to be downstream of TFEB. Results TFEB, a member of the MiTF family of basic helix-loop-helix leucine zipper, was the only TF commonly up-regulated by all PAR-APs. IHC results confirm a correlation between inflammation and TFEB expression in C57BL/6 mice. In contrast, Kitw/Kitw-v mice did not exhibit inflammation in response to PAR activation. EMSA results confirmed the increased TFEB binding activity in C57BL/6 but not in Kitw/Kitw-v mice. Conclusion This is the first report describing the increased expression of TFEB in bladder inflammation in response to PAR activation. As TFEB belongs to a family of TFs essential for mast cell survival, our findings suggest that this molecule may influence the participation of mast cells in PAR-mediated inflammation and that targeting TFEB/MiTF activity may be a novel approach for the treatment of bladder inflammatory disorders.

  8. Platelet-activating factor (PAF) receptor binding antagonist activity of the methanol extracts and isolated flavonoids from Chromolaena odorata (L.) King and Robinson.

    Ling, Sui Kiong; Pisar, Mazura Md; Man, Salbiah

    2007-06-01

    The leaf, stem and root extracts of Chromolaena odorata were evaluated for their effect on platelet-activating factor (PAF) receptor binding on rabbit platelets using 3H-PAF as a ligand. The leaf extract demonstrated high PAF receptor binding inhibitory activity of 79.2+/-2.1% at 18.2 microg/ml. A total of eleven flavonoids were subsequently isolated from the active leaf extract and evaluated for their effects on PAF receptor binding. Eight of the flavonoids exhibited >50% inhibition on the binding activity at 18.2 microg/ml. These flavonoids were identified as eriodictyol 7,4'-dimethyl ether, quercetin 7,4'-methyl ether, naringenin 4'-methyl ether, kaempferol 4'-methyl ether, kaempferol 3-O-rutinoside, taxifolin 4'-methyl ether, taxifolin 7-methyl ether and quercetin 4'-methyl ether. Their IC50 values ranged from 19.5 to 62.1 microM. PMID:17541171

  9. pH dependence of ligand-induced human epidermal growth factor receptor activation investigated by molecular dynamics simulations.

    Dong, Jun; Zhang, Yonghui; Zhang, Zhiyong

    2016-06-01

    The activation of human epidermal growth factor receptor (hEGFR) involves a large conformational change in its soluble extracellular domains (sECD, residues 1-620), from a tethered to an extended conformation upon binding of ligands, such as EGF. It has been reported that this dynamic process is pH-dependent, that is, hEGFR can be activated by EGF at high pH to form an extended dimer but remains as an inactive monomer at low pH. In this paper, we perform all-atom molecular dynamics (MD) simulations starting from the tethered conformation of sECD:EGF complex, at pH 5.0 and 8.5, respectively. Simulation results indicate that sECD:EGF shows different dynamic properties between the two pHs, and the complex may have a higher tendency of activation at pH 8.5. Twenty residues, including 13 histidines, in sECD:EGF have different protonation states between the two pHs (calculated by the H++ server). The charge distribution at pH 8.5 is more favorable for forming an extended conformation toward the active state of sECD than that at pH 5.0. Our study may shed light on the mechanism of pH dependence of hEGFR activation. Graphical abstract pH dependence of ligand-induced human epidermal growth factor receptor activation. PMID:27179806

  10. Activation of the epidermal growth factor receptor (EGFR) by a novel metalloprotease pathway.

    Bergin, David A

    2008-11-14

    Neutrophil Elastase (NE) is a pro-inflammatory protease present at higher than normal levels in the lung during inflammatory disease. NE regulates IL-8 production from airway epithelial cells and can activate both EGFR and TLR4. TACE\\/ADAM17 has been reported to trans-activate EGFR in response to NE. Here, using 16HBE14o-human bronchial epithelial cells we demonstrate a new mechanism by which NE regulates both of these events. A high molecular weight soluble metalloprotease activity detectable only in supernatants from NE-treated cells by gelatin and casein zymography was confirmed to be meprin alpha by Western immunoblotting. In vitro studies demonstrated the ability of NE to activate meprin alpha, which in turn could release soluble TGFalpha and induce IL-8 production from 16HBE14o- cells. These effects were abrogated by actinonin, a specific meprin inhibitor. NE-induced IL-8 expression was also inhibited by meprin alpha siRNA. Immunoprecipitation studies detected EGFR\\/TLR4 complexes in NE-stimulated cells overexpressing these receptors. Confocal studies confirmed colocalization of EGFR and TLR4 in 16HBE14o- cells stimulated with meprin alpha. NFkappaB was also activated via MyD88 in these cells by meprin alpha. In bronchoalveolar lavage fluid from NE knock-out mice infected intra-tracheally with Pseudomonas aeruginosa meprin alpha was significantly decreased compared with control mice, and was significantly increased and correlated with NE activity, in bronchoalveolar lavage fluid from individuals with cystic fibrosis but not healthy controls. The data describe a previously unidentified lung metalloprotease meprin alpha, and its role in NE-induced EGFR and TLR4 activation and IL-8 production.

  11. Tensile force on human macrophage cells promotes osteoclastogenesis through receptor activator of nuclear factor κB ligand induction.

    Kao, Chia-Tze; Huang, Tsui-Hsien; Fang, Hsin-Yuan; Chen, Yi-Wen; Chien, Chien-Fang; Shie, Ming-You; Yeh, Chia-Hung

    2016-07-01

    Little is known about the effects of tensile forces on osteoclastogenesis by human monocytes in the absence of mechanosensitive cells, including osteoblasts and fibroblasts. In this study we consider the effects of tensile force on osteoclastogenesis in human monocytes. The cells were treated with receptor activator of nuclear factor κB ligand (RANKL) to promote osteoclastogenesis. Then,expression and secretion of cathepsin K were examined. RANKL and the formation of osteoclasts during the osteoclast differentiation process under continual tensile stress were evaluated by Western blot. It was also found that -100 kPa or lower induces RANKL-enhanced tartrate-resistant acid phosphatase activity in a dose-dependent manner. Furthermore, an increased tensile force raises the expression and secretion of cathepsin K elevated by RANKL, and is concurrent with the increase of TNF-receptor-associated factor 6 induction and nuclear factor κB activation. Overall, the current report demonstrates that tensile force reinforces RANKL-induced osteoclastogenesis by retarding osteoclast differentiation. The tensile force is able to modify every cell through dose-dependent in vitro RANKL-mediated osteoclastogenesis, affecting the fusion of preosteoclasts and function of osteoclasts. However, tensile force increased TNF-receptor-associated factor 6 expression. These results are in vitro findings and were obtained under a condition of tensile force. The current results help us to better understand the cellular roles of human macrophage populations in osteoclastogenesis as well as in alveolar bone remodeling when there is tensile stress. PMID:26204845

  12. Human NCU-G1 can function as a transcription factor and as a nuclear receptor co-activator

    Bakke Oddmund

    2007-11-01

    Full Text Available Abstract Background Novel, uncharacterised proteins represent a challenge in biochemistry and molecular biology. In this report we present an initial functional characterization of human kidney predominant protein, NCU-G1. Results NCU-G1 was found to be a highly conserved nuclear protein rich in proline with a molecular weight of approximately 44 kDa. It is localized on chromosome 1 and consists of 6 exons. Analysis of the amino acid sequence revealed no known transcription activation domains or DNA binding regions, however, four nuclear receptor boxes (LXXLL, and four SH3-interaction motives in addition to numerous potential phosphorylation sites were found. Two nuclear export signals were identified, but no nuclear localization signal. In man, NCU-G1 was found to be widely expressed at the mRNA level with especially high levels detected in prostate, liver and kidney. Electrophoretic mobility shift analysis showed specific binding of NCU-G1 to an oligonucleotide representing the footprint 1 element of the human cellular retinol-binding protein 1 gene promoter. NCU-G1 was found to activate transcription from this promoter and required presence of the footprint 1 element. In transiently transfected Drosophila Schneider S2 cells, we demonstrated that NCU-G1 functions as a co-activator for ligand-activated PPAR-alpha, resulting in an increased expression of a CAT reporter gene under control of the peroxisome proliferator-activated receptor-alpha responsive acyl-CoA oxidase promoter. Conclusion We propose that NCU-G1 is a dual-function protein capable of functioning as a transcription factor as well as a nuclear receptor co-activator.

  13. Receptor activator of nuclear factor-κB ligand and osteoprotegerin: maintaining the balance to prevent bone loss

    Anne-Priscille Trouvin

    2010-11-01

    Full Text Available Anne-Priscille Trouvin, Vincent GoëbDepartment of Rheumatology, Rouen University Hospital, Rouen, FranceAbstract: Bone remodeling requires a precise balance between resorption and formation. It is a complex process that involves numerous factors: hormones, growth factors, vitamins, and cytokines, and notably osteoprotegerin (OPG and receptor activator for nuclear factor-κB (RANK ligand. The signaling pathway OPG/RANK/RANKL is key to regulation for maintaining the balance between the activity of osteoblasts and osteoclasts in order to prevent bone loss and ensure a normal bone turnover. In this review, the RANK/RANKL/OPG pathway is described. The multiple interactions of various factors (hormones, cytokines, growth factors, and vitamins with the OPG/RANK/RANKL pathway are also commented on. Finally, the effects of denosumab, a human monoclonal antibody that binds to RANKL and thereby inhibits the activation of osteoclasts, and of strontium ranelate are also described. Indeed, these two new drugs afford appreciable assistance in daily care practice, helping to prevent bone loss in patients with osteoporosis.Keywords: osteoprotegerin, OPG, RANK, RANKL, denosumab, strontium ranelate, osteoporosis

  14. Endoplasmic Reticulum Stress-Induced Activation of Activating Transcription Factor 6 Decreases Insulin Gene Expression via Up-Regulation of Orphan Nuclear Receptor Small Heterodimer Partner

    Seo, Hye-Young; Kim, Yong Deuk; Lee, Kyeong-Min; Min, Ae-Kyung; Kim, Mi-Kyung; Kim, Hye-Soon; Won, Kyu-Chang; Park, Joong-Yeol; Lee, Ki-Up; Choi, Hueng-Sik; Park, Keun-Gyu; Lee, In-Kyu

    2008-01-01

    The highly developed endoplasmic reticulum (ER) structure of pancreatic β-cells is a key factor in β-cell function. Here we examined whether ER stress-induced activation of activating transcription factor (ATF)-6 impairs insulin gene expression via up-regulation of the orphan nuclear receptor small heterodimer partner (SHP; NR0B2), which has been shown to play a role in β-cell dysfunction. We examined whether ER stress decreases insulin gene expression, and this process is mediated by ATF6. A...

  15. Striatal but not frontal cortical up-regulation of the epidermal growth factor receptor in rats exposed to immune activation in utero and cannabinoid treatment in adolescence.

    Idrizi, Rejhan; Malcolm, Peter; Weickert, Cynthia Shannon; Zavitsanou, Katerina; Suresh Sundram

    2016-06-30

    In utero maternal immune activation (MIA) and cannabinoid exposure during adolescence constitute environmental risk factors for schizophrenia. We investigated these risk factors alone and in combination ("two-hit") on epidermal growth factor receptor (EGFR) and neuregulin-1 receptor (ErbB4) levels in the rat brain. EGFR but not ErbB4 receptor protein levels were significantly increased in the nucleus accumbens and striatum of "two-hit" rats only, with no changes seen at the mRNA level. These findings support region specific EGF-system dysregulation as a plausible mechanism in this animal model of schizophrenia pathogenesis. PMID:27138815

  16. Activation of corticotropin releasing factor receptors up regulates collagen production by hepatic stellate cells via promoting p300 expression.

    Wang, Changzhen; Yang, Shan; Huang, Jingjing; Chen, Songlin; Li, Yuan; Li, Quanqiang

    2016-05-01

    Liver fibrosis is characterized with the over expression and excessive accumulation of extracellular matrix proteins, including collagens. The causative factors in the over production of collagens are not fully understood. This study aims to test a hypothesis that activation of corticotropin releasing factor receptors up regulates the expression of collagen in hepatic stellate cells. In this study, human hepatic stellate cell line, LX-2 cells were cultured. Expression of collagens by LX-2 cells was assessed by real time RT-PCR, Western blotting. The results showed that, upon exposure to urocortin in the culture, LX-2 cells (a human hepatic stellate cell line) increased the expression of collagen IV (Col4) markedly. The exposure to urocortin also enhanced the levels of pTip60, H3K9, RNA polymerase II and forkhead box protein 3 at the collagen promoter locus as well as increase in the expression of Col4 mRNA and protein in the cells. Blocking p300 efficiently suppressed the urocortin-induced Col4 expression in LX-2 cells and unveiled an apoptosis-inducing effect of urocortin. In conclusion, activation of CRF receptors is capable of enforcing the production of Col4 by LX-2 cells via up regulating the p300 pathway, which may contribute to the development of liver fibrosis. PMID:26756093

  17. Structural basis for activation of trimeric Gi proteins by multiple growth factor receptors via GIV/Girdin

    Lin, Changsheng; Ear, Jason; Midde, Krishna; Lopez-Sanchez, Inmaculada; Aznar, Nicolas; Garcia-Marcos, Mikel; Kufareva, Irina; Abagyan, Ruben; Ghosh, Pradipta

    2014-01-01

    A long-standing issue in the field of signal transduction is to understand the cross-talk between receptor tyrosine kinases (RTKs) and heterotrimeric G proteins, two major and distinct signaling hubs that control eukaryotic cell behavior. Although stimulation of many RTKs leads to activation of trimeric G proteins, the molecular mechanisms behind this phenomenon remain elusive. We discovered a unifying mechanism that allows GIV/Girdin, a bona fide metastasis-related protein and a guanine-nucleotide exchange factor (GEF) for Gαi, to serve as a direct platform for multiple RTKs to activate Gαi proteins. Using a combination of homology modeling, protein–protein interaction, and kinase assays, we demonstrate that a stretch of ∼110 amino acids within GIV C-terminus displays structural plasticity that allows folding into a SH2-like domain in the presence of phosphotyrosine ligands. Using protein–protein interaction assays, we demonstrated that both SH2 and GEF domains of GIV are required for the formation of a ligand-activated ternary complex between GIV, Gαi, and growth factor receptors and for activation of Gαi after growth factor stimulation. Expression of a SH2-deficient GIV mutant (Arg 1745→Leu) that cannot bind RTKs impaired all previously demonstrated functions of GIV—Akt enhancement, actin remodeling, and cell migration. The mechanistic and structural insights gained here shed light on the long-standing questions surrounding RTK/G protein cross-talk, set a novel paradigm, and characterize a unique pharmacological target for uncoupling GIV-dependent signaling downstream of multiple oncogenic RTKs. PMID:25187647

  18. Modulation of Voltage-Gated Sodium Channels by Activation of Tumor Necrosis Factor Receptor-1 and Receptor-2 in Small DRG Neurons of Rats

    M. Leo

    2015-01-01

    Full Text Available Tumor necrosis factor- (TNF- α is a proinflammatory cytokine involved in the development and maintenance of inflammatory and neuropathic pain. Its effects are mediated by two receptors, TNF receptor-1 (TNFR-1 and TNF receptor-2 (TNFR-2. These receptors play a crucial role in the sensitization of voltage-gated sodium channels (VGSCs, a key mechanism in the pathogenesis of chronic pain. Using the whole-cell patch-clamp technique, we examined the influence of TNFR-1 and TNFR-2 on VGSCs and TTX-resistant NaV1.8 channels in isolated rat dorsal root ganglion neurons by using selective TNFR agonists. The TNFR-1 agonist R32W (10 pg/mL caused an increase in the VGSC current (INa(V by 27.2 ± 5.1%, while the TNFR-2 agonist D145 (10 pg/mL increased the current by 44.9 ± 2.6%. This effect was dose dependent. Treating isolated NaV1.8 with R32W (100 pg/mL resulted in an increase in INaV(1.8 by 18.9 ± 1.6%, while treatment with D145 (100 pg/mL increased the current by 14.5 ± 3.7%. Based on the current-voltage relationship, 10 pg of R32W or D145 led to an increase in INa(V in a bell-shaped, voltage-dependent manner with a maximum effect at −30 mV. The effects of TNFR activation on VGSCs promote excitation in primary afferent neurons and this might explain the sensitization mechanisms associated with neuropathic and inflammatory pain.

  19. Antibodies to a soluble form of a tumor necrosis factor (TNF) receptor have TNF-like activity

    Engelmann, H; Holtmann, H; Brakebusch, C;

    1990-01-01

    Immunological cross-reactivity between tumor necrosis factor (TNF) binding proteins which are present in human urine (designated TBPI and TBPII) and two molecular species of the cell surface receptors for TNF is demonstrated. The two TNF receptors are shown to be immunologically distinct, to differ...... process which appears to involve clustering of these receptors, transduce the signal for response to TNF into the cell's interior....

  20. Nuclear epidermal growth factor receptor (EGFR) interacts with signal transducer and activator of transcription 5 (STAT5) in activating Aurora-A gene expression

    Hung, Liang-Yi; Tseng, Joseph T.; Lee, Yi-Chao; Xia, Weiya; Wang, Ying-Nai; Wu, Min-Li; Chuang, Yu-Hsuan; Lai, Chein-Hsien; Chang, Wen-Chang

    2008-01-01

    Loss of the maintenance of genetic material is a critical step leading to tumorigenesis. It was reported that overexpression of Aurora-A and the constitutive activation of the epidermal growth factor (EGF) receptor (EGFR) are implicated in chromosome instability. In this study, we examined that when cells treated with EGF result in centrosome amplification and microtubule disorder, which are critical for genetic instability. Interestingly, the expression of Aurora-A was also increased by EGF ...

  1. Homoerydictyl-7-O-β-D-glycosidc—A Receptor Antagonist of Platelet-activating Factor (PAF)

    GuanZengwei; WangYinye; YangXiuwei; CuiYuxin

    2001-01-01

    Homoeriodictyl-7-O-β-D-glycoside, a flavonoid compound isolated from the Chinese medic inalherb, viscum coloratura inhibited platelet aggregation induced by platelet-activating factor(PAF), but it had no inhibitory activity on adenosine diphosphate-induced platelet aggregation. In the present study, we intended to get an insight into the mechanism of its anti-PAF action. Using [3H]PAF receptor binding assay we found that the compound exhibited inhibitory activity. The inhibitory rate was 18.5%, 28.4%, 58.7%, 78% and 78%, respestively, at concentrations of 10-8, 10-7, 10-6, 10-5 and 10-4 mol.L-1, There was a visible dose-effect relationship as well as a correlation between different concentrations and their inhibitory rates (r=0.985, P<0.05) when the dose was equal to or less than 1×10-5 mol.L-1, and its IC50 was 8.0×10-7 mol.L-1. The inhibitory rate didn't increase with increasing concentration of the compound when it went beyond1×10-5 mol.L-1 indicating competitive inhibition of binding of [3H]PAF to PAF receptor reached saturation.

  2. Epidermal growth factor (EGF) receptor gene transcription

    The authors have studied in vitro transcription of the human epidermal growth factor (EGF) receptor proto-oncogene using nuclear extracts of A431 human epidermoid carcinoma cells, which overproduce the EGF receptor. With the in vitro system we found that Sp1 and other trans-acting factors bound to the EGF receptor promoter regions and are required for maximal expression. Fractionation showed that a DEAE-Sepharose fraction (BA) contained a novel factor, which specifically stimulated EGF receptor transcription 5- to 10-fold. The molecular mass of the native form of the factor is about 270-kDa based on its migration on Sephacryl S-300. This factor may activate transcription of the proto-oncogene through a weak or indirect interaction with the DNA template

  3. Neuroligin-1 induces neurite outgrowth through interaction with neurexin-1ß and activation of fibroblast growth factor receptor-1

    Gjørlund, Michelle D; Nielsen, Janne; Pankratova, Stanislava;

    2012-01-01

    Neurexin-1 (NRXN1) and neuroligin-1 (NLGN1) are synaptic cell adhesion molecules that connect pre- and postsynaptic neurons at synapses and mediate signaling across the synapse, which modulates synaptic activity and determines the properties of neuronal networks. Defects in the genes encoding NLGN1...... have been linked to cognitive diseases such as autism. The roles of both NRXN1 and NLGN1 during synaptogenesis have been studied extensively, but little is known about the role of these molecules in neuritogenesis, which eventually results in neuronal circuitry formation. The present study investigated...... the neuritogenic effect of NLGN1 in cultures of hippocampal neurons. Our results show that NLGN1, both in soluble and membrane-bound forms, induces neurite outgrowth that depends on the interaction with NRXN1ß and on activation of fibroblast growth factor receptor-1. In addition, we demonstrate that a...

  4. Risk Factors Associated with Serum Levels of the Inflammatory Biomarker Soluble Urokinase Plasminogen Activator Receptor in a General Population

    Haupt, Thomas H; Kallemose, Thomas; Ladelund, Steen;

    2014-01-01

    .001). An unhealthy diet and alcohol abstinence in men were also associated with higher suPAR levels. Physical activity in leisure time had a modest impact on suPAR levels in univariate analysis, but not in the final adjusted model. In conclusion, smoking and morbid obesity were strongly associated with higher serum...... suPAR levels in this general population. Diet and alcohol consumption also seemed to impact suPAR levels. Lifestyle changes are likely to affect suPAR since ex-smokers had suPAR levels comparable to those of never-smokers.......The soluble urokinase plasminogen activator receptor (suPAR) is a biomarker of mortality risk in various patient populations. However, little is known about the implications of lifestyle for suPAR levels in the general population. Lifestyle, demographic, and cardiovascular disease (CVD) risk factor...

  5. Dysfunctional epileptic neuronal circuits and dysmorphic dendritic spines are mitigated by platelet-activating factor receptor antagonism

    Musto, Alberto E.; Rosencrans, Robert F.; Walker, Chelsey P.; Bhattacharjee, Surjyadipta; Raulji, Chittalsinh M.; Belayev, Ludmila; Fang, Zhide; Gordon, William C.; Bazan, Nicolas G.

    2016-01-01

    Temporal lobe epilepsy or limbic epilepsy lacks effective therapies due to a void in understanding the cellular and molecular mechanisms that set in motion aberrant neuronal network formations during the course of limbic epileptogenesis (LE). Here we show in in vivo rodent models of LE that the phospholipid mediator platelet-activating factor (PAF) increases in LE and that PAF receptor (PAF-r) ablation mitigates its progression. Synthetic PAF-r antagonists, when administered intraperitoneally in LE, re-establish hippocampal dendritic spine density and prevent formation of dysmorphic dendritic spines. Concomitantly, hippocampal interictal spikes, aberrant oscillations, and neuronal hyper-excitability, evaluated 15–16 weeks after LE using multi-array silicon probe electrodes implanted in the dorsal hippocampus, are reduced in PAF-r antagonist-treated mice. We suggest that over-activation of PAF-r signaling induces aberrant neuronal plasticity in LE and leads to chronic dysfunctional neuronal circuitry that mediates epilepsy. PMID:27444269

  6. Peptides derived from specific interaction sites of the fibroblast growth factor 2 - FGF receptor complexes induce receptor activation and signaling

    Manfè, Valentina; Kochoyan, Artur; Bock, Elisabeth; Berezin, Vladimir

    2010-01-01

    J. Neurochem. (2010) 10.1111/j.1471-4159.2010.06718.x Abstract Basic fibroblast growth factor (FGF2, bFGF) is the most extensively studied member of the FGF family and is involved in neurogenesis, differentiation, neuroprotection, and synaptic plasticity in the CNS. FGF2 executes its pleiotropic...

  7. Anti-epidermal growth factor receptor monoclonal antibody cetuximab inhibits EGFR/HER-2 heterodimerization and activation.

    Patel, Dipa; Bassi, Rajiv; Hooper, Andrea; Prewett, Marie; Hicklin, Daniel J; Kang, Xiaoqiang

    2009-01-01

    Human carcinomas frequently express one or more members of the epidermal growth factor receptor family. Two family members, epidermal growth factor receptor (EGFR) and c-erbB2/neu (HER2), homodimerize or heterodimerize upon activation with ligand and trigger potent mechanisms of cellular proliferation, differentiation and migration. In this study, we examined the effect of the anti-EGFR monoclonal antibody Erbitux (cetuximab) on human tumor cells expressing both EGFR and HER2. Investigation of the effect of cetuximab on the activation of EGFR-EGFR, EGFR-HER2 and HER2-HER2 homodimers and heterodimers was conducted using the NCI-N87 human gastric carcinoma cell line. Treatment of NCI-N87 cells with cetuximab completely inhibited formation of EGFR-EGFR homodimers and EGFR-HER2 heterodimers. Activation of HER2-HER2 homodimers was not appreciably stimulated by exogenous ligand and was not inhibited by cetuximab treatment. Furthermore, cetuximab inhibited EGF-induced EGFR and HER2 phosphorylation in CAL27, NCI-H226 and NCI-N87 cells. The activation of downstream signaling molecules such as AKT, MAPK and STAT-3 were also inhibited by cetuximab in these cells. To examine the effect of cetuximab on the growth of tumors in vivo, athymic mice bearing established NCI-N87 or CAL27 xenografts were treated with cetuximab (1 mg, i.p., q3d). The growth of NCI-N87 and CAL27 tumors was significantly inhibited with cetuximab therapy compared to the control groups (p<0.0001 in both cases). In the CAL27 xenograft model, tumor growth inhibition by cetuximab treatment was similar to that by cetuximab and trastuzumab combination treatment. Immunohistological analysis of cetuximab-treated tumors showed a decrease in EGFR-HER2 signaling and reduced tumor cell proliferation. These results suggest that cetuximab may be useful in the treatment of carcinomas co-expressing EGFR and HER2. PMID:19082474

  8. Acyl-CoA esters antagonize the effects of ligands on peroxisome proliferator-activated receptor alpha conformation, DNA binding, and interaction with Co-factors

    Elholm, M; Dam, I; Jorgensen, C;

    2001-01-01

    The peroxisome proliferator-activated receptor alpha (PPARalpha) is a ligand-activated transcription factor and a key regulator of lipid homeostasis. Numerous fatty acids and eicosanoids serve as ligands and activators for PPARalpha. Here we demonstrate that S-hexadecyl-CoA, a nonhydrolyzable...

  9. Human lung-resident macrophages express CB1 and CB2 receptors whose activation inhibits the release of angiogenic and lymphangiogenic factors.

    Staiano, Rosaria I; Loffredo, Stefania; Borriello, Francesco; Iannotti, Fabio Arturo; Piscitelli, Fabiana; Orlando, Pierangelo; Secondo, Agnese; Granata, Francescopaolo; Lepore, Maria Teresa; Fiorelli, Alfonso; Varricchi, Gilda; Santini, Mario; Triggiani, Massimo; Di Marzo, Vincenzo; Marone, Gianni

    2016-04-01

    Macrophages are pivotal effector cells in immune responses and tissue remodeling by producing a wide spectrum of mediators, including angiogenic and lymphangiogenic factors. Activation of cannabinoid receptor types 1 and 2 has been suggested as a new strategy to modulate angiogenesis in vitro and in vivo. We investigated whether human lung-resident macrophages express a complete endocannabinoid system by assessing their production of endocannabinoids and expression of cannabinoid receptors. Unstimulated human lung macrophage produce 2-arachidonoylglycerol,N-arachidonoyl-ethanolamine,N-palmitoyl-ethanolamine, andN-oleoyl-ethanolamine. On LPS stimulation, human lung macrophages selectively synthesize 2-arachidonoylglycerol in a calcium-dependent manner. Human lung macrophages express cannabinoid receptor types 1 and 2, and their activation induces ERK1/2 phosphorylation and reactive oxygen species generation. Cannabinoid receptor activation by the specific synthetic agonists ACEA and JWH-133 (but not the endogenous agonist 2-arachidonoylglycerol) markedly inhibits LPS-induced production of vascular endothelial growth factor-A, vascular endothelial growth factor-C, and angiopoietins and modestly affects IL-6 secretion. No significant modulation of TNF-α or IL-8/CXCL8 release was observed. The production of vascular endothelial growth factor-A by human monocyte-derived macrophages is not modulated by activation of cannabinoid receptor types 1 and 2. Given the prominent role of macrophage-assisted vascular remodeling in many tumors, we identified the expression of cannabinoid receptors in lung cancer-associated macrophages. Our results demonstrate that cannabinoid receptor activation selectively inhibits the release of angiogenic and lymphangiogenic factors from human lung macrophage but not from monocyte-derived macrophages. Activation of cannabinoid receptors on tissue-resident macrophages might be a novel strategy to modulate macrophage-assisted vascular remodeling

  10. Suppression of piriform cortex activity in rat by corticotropin-releasing factor 1 and serotonin 2A/C receptors

    Chakravarthi Narla

    2015-05-01

    Full Text Available The piriform cortex (PC is richly innervated by Corticotropin-releasing factor (CRF and Serotonin (5-HT containing axons arising from central amygdala and Raphe nucleus. CRFR1 and 5-HT2A/2CRs have been shown to interact in manner where CRFR activation subsequently potentiates the activity of 5-HT2A/2CRs. The purpose of this study was to determine how the activation of CRFR1 and/or 5-HT2Rs modulates PC activity at both the circuit and cellular level. Voltage sensitive dye imaging showed that CRF acting through CRFR1 dampened activation of the layer II of PC and interneurons of endopiriform nucleus. Application of the selective 5-HT2A/CR agonist 2,5-dimethoxy-4-iodoamphetamine (DOI following CRFR1 activation potentiated this effect. Blocking the interaction between CRFR1 and 5-HT2R with a Tat-CRFR1-CT peptide abolished this potentiation. Application of forskolin did not mimic CRFR1 activity but instead blocked it, while a protein kinase A antagonist had no effect. However, activation and antagonism of protein kinase C (PKC either mimicked or blocked CRF modulation respectively. DOI had no effect when applied alone indicating that the prior activation of CRFR1 receptors was critical for DOI to show significant effects similar to CRF. Patch clamp recordings showed that both CRF and DOI reduced the synaptic responsiveness of layer II pyramidal neurons. CRF had highly variable effects on interneurons within layer III, both increasing and decreasing their excitability, but DOI had no effect on the excitability of this group of neurons. These data show that CRF and serotonin, acting through both CRFR1 and 5-HT2A/CRs, reduce the activation of the PC. This modulation may be an important blunting mechanism of stressor behaviours mediated through the olfactory cortex.

  11. Activation of insulin-like growth factor 1 receptor in patients with non-small cell lung cancer.

    Kim, Jin-Soo; Kim, Edward S; Liu, Diane; Lee, J Jack; Behrens, Carmen; Lippman, Scott M; Hong, Waun Ki; Wistuba, Ignacio I; Lee, Euni; Lee, Ho-Young

    2015-06-30

    According to previous reports demonstrating the implication of insulin-like growth factor receptor (IGF-1R) signaling in non-small cell lung cancer (NSCLC), in this study, the potential prognostic values of IGF-1R expression/activation were analyzed. The expression and activation of IGF-1R were evaluated in two tissue microarray (TMA) sets from NSCLC patients (N = 352 for TMA I, and N = 353 for TMA II). Alterations in IGF-1R protein or mRNA expression in NSCLC patients were evaluated using publicly available data from The Cancer Genome Atlas (TCGA). We found that membranous and cytoplasmic IGF-1R expressions were significantly associated with squamous cell carcinoma (SCC) in both of the TMAs. Analysis of the TCGA data revealed increased mRNA levels in NSCLC patients, which was significantly associated with reductions in overall survival (OS) (median survival 26.51 vs. 47.77 months, P = 0.017) and disease-free survival (median survival 17.44 vs. 37.65 months, P = 0.045) only in NSCLC patients with adenocarcinoma (ADC). These data suggest that IGF-1R is activated in patients with NSCLC, particularly those with SCC. IGF-1R mRNA expression is a potential prognostic factor in patients with NSCLC, especially those with ADC. Further studies are warranted to investigate the prognostic value of IGF-1R in NSCLC patients. PMID:25944691

  12. Activation of AMP-activated kinase modulates sensitivity of glioma cells against epidermal growth factor receptor inhibition.

    Hartel, Ines; Ronellenfitsch, Michael; Wanka, Christina; Wolking, Stefan; Steinbach, Joachim P; Rieger, Johannes

    2016-07-01

    The epidermal growth factor (EGFR) pathway is frequently activated in glioblastoma but the clinical efficacy of EGFR inhibitors in malignant glioma has been disappointing. The reasons for the failure of the mechanisms of resistance of these inhibitors are unclear, but may involve factors of the tumor microenvironment such as limited glucose availability and hypoxia. It was therefore examined whether glucose and oxygen influenced the response of glioma cells to EGFR inhibition. Decreased levels of glucose and oxygen led to resistance against the EGFR inhibitor PD153035, whereas high glucose amounts and normoxia sensitised glioma cells towards the inhibitor. Low levels of glucose and oxygen stimulated AMP-activated kinase (AMPK) in glioma cells. 2DG, an inhibitor of glycolysis, and the AMPK activator A769662 reduced glucose consumption, induced phosphorylation of AMPK and mimicked the effects of low glucose availability on the toxicity of PD153035. Similarly, 2DG reduced toxicity of imatinib in K562 leukemia cells. In contrast, inhibition of AMPK by compound C or by short-hairpin (sh)-mediated gene suppression increased cell death induced by the EGFR inhibitor and reverted the protective effects of 2DG and A769662. In conclusion, cytotoxicity of EGFR inhibition can be diminished by AMPK activation in glioma cells. These results may provide one explanation for the low activity of EGFR inhibitors in clinical trials and suggest antagonism of AMPK or of AMPK-regulated metabolic alterations as a promising approach to enhance their therapeutic efficacy. PMID:27121290

  13. Essential Role of Nuclear Factor (NF)-κB–Inducing Kinase and Inhibitor of κb (Iκb) Kinase α in Nf-κb Activation through Lymphotoxin β Receptor, but Not through Tumor Necrosis Factor Receptor I

    Matsushima, Akemi; Kaisho, Tsuneyasu; Rennert, Paul D.; Nakano, Hiroyasu; Kurosawa, Kyoko; Uchida, Daisuke; Takeda, Kiyoshi; Akira, Shizuo; Matsumoto, Mitsuru

    2001-01-01

    Both nuclear factor (NF)-κB–inducing kinase (NIK) and inhibitor of κB (IκB) kinase (IKK) have been implicated as essential components for NF-κB activation in response to many external stimuli. However, the exact roles of NIK and IKKα in cytokine signaling still remain controversial. With the use of in vivo mouse models, rather than with enforced gene-expression systems, we have investigated the role of NIK and IKKα in signaling through the type I tumor necrosis factor (TNF) receptor (TNFR-I) ...

  14. Activation of the human keratinocyte B1 bradykinin receptor induces expression and secretion of metalloproteases 2 and 9 by transactivation of epidermal growth factor receptor.

    Matus, Carola E; Ehrenfeld, Pamela; Pavicic, Francisca; González, Carlos B; Concha, Miguel; Bhoola, Kanti D; Burgos, Rafael A; Figueroa, Carlos D

    2016-09-01

    The B1 bradykinin receptor (BDKRB1) is a component of the kinin cascade localized in the human skin. Some of the effects produced by stimulation of BDKRB1 depend on transactivation of epidermal growth factor receptor (EGFR), but the mechanisms involved in this process have not been clarified yet. The primary purpose of this study was to determine the effect of a BDKRB1 agonist on wound healing in a mouse model and the migration and secretion of metalloproteases 2 and 9 from human HaCaT keratinocytes and delineate the signalling pathways that triggered their secretion. Although stimulation of BDKRB1 induces weak chemotactic migration of keratinocytes and wound closure in an in vitro scratch-wound assay, the BDKRB1 agonist improved wound closure in a mouse model. BDKRB1 stimulation triggers synthesis and secretion of both metalloproteases, effects that depend on the activity of EGFR and subsequent phosphorylation of ERK1/2 and p38 mitogen-activated protein kinases and PI3K/Akt. In the mouse model, immunoreactivity for both gelatinases was concentrated around wound borders. EGFR transactivation by BDKRB1 agonist involves Src kinases family and ADAM17. In addition to extracellular matrix degradation, metalloproteases 2 and 9 regulate cell migration and differentiation, cell functions that are associated with the role of BDKRB1 in keratinocyte differentiation. Considering that BDKRB1 is up-regulated by inflammation and/or by cytokines that are abundant in the inflammatory milieu, more stable BDKRB1 agonists may be of therapeutic value to modulate wound healing. PMID:27093919

  15. Mouse Balb/c3T3 cell mutant with low epidermal growth factor receptor activity: induction of stable anchorage-independent growth by transforming growth factor β

    A mutant clone (MO-5) was originally isolated as a clone resistant to Na+/K+ ionophoric antibiotic monensin from mouse Balb/c3T3 cells. MO-5 was found to show low receptor-endocytosis activity for epidermal growth factor (EGF):binding activity for EGF in MO-5 was less than one tenth of that in Balb/c3T3. Anchorage-independent growth of MO-5 was compared to that of Balb/c3T3 when assayed by colony formation capacity in soft agar. Coadministration of EGF and TGF-β efficiently enhanced anchorage-independent growth of normal rat kidney (NRK) cells, but neither factor alone was competent to promote the anchorage-independent growth. The frequency of colonies appearing in soft agar of MO-5 or Balb/c3T3 was significantly enhanced by TGF-β while EGF did not further enhance that of MO-5 or Balb/c3T3. Colonies of Balb/c3T3 formed in soft agar in the presence of TGF-β showed low colony formation capacity in soft agar in the absence of TGF-β. Colonies of MO-5 formed by TGF-β in soft agar, however, showed high colony formation capacity in soft agar in the absence of TGF-β. Pretreatment of MO-5 with TGF-β induced secretion of TGF-β-like activity from the cells, while the treatment of Balb/c3T3 did not induce the secretion of a significant amount of TGF-β-like activity. The loss of EGF-receptor activity in the stable expression and maintenance of the transformed phenotype in MO-5 is discussed

  16. Constitutive activation of fibroblast growth factor receptor 3 by the transmembrane domain point mutation found in achondroplasia.

    Webster, M K; Donoghue, D J

    1996-01-01

    Achondroplasia, the most common genetic form of dwarfism, is an autosomal dominant disorder whose underlying mechanism is a defect in the maturation of the cartilage growth plate of long bones. Achondroplasia has recently been shown to result from a Gly to Arg substitution in the transmembrane domain of the fibroblast growth factor receptor 3 (FGFR3), although the molecular consequences of this mutation have not been investigated. By substituting the transmembrane domain of the Neu receptor t...

  17. Inhibitory Activities of Epidermal Growth Factor Receptor Tyrosine Kinase-Targeted Dihydroxyisoflavone and Trihydroxydeoxybenzoin Derivatives on Sarcocystis neurona, Neospora caninum, and Cryptosporidium parvum Development

    Gargala, G.; Baishanbo, A.; Favennec, L; François, A; Ballet, J J; Rossignol, J.-F.

    2005-01-01

    Several gene sequences of parasitic protozoa belonging to protein kinase gene families and epidermal growth factor (EGF)-like peptides, which act via binding to receptor tyrosine kinases of the EGF receptor (EGFR) family, appear to mediate host-protozoan interactions. As a clue to EGFR protein tyrosine kinase (PTK) mediation and a novel approach for identifying anticoccidial agents, activities against Sarcocystis neurona, Neospora caninum, and Cryptosporidium parvum grown in BM and HCT-8 cell...

  18. Modulation of Transcriptional Activation and Coactivator Interaction by a Splicing Variation in the F Domain of Nuclear Receptor Hepatocyte Nuclear Factor 4α1

    Sladek, Frances M.; Ruse, Michael D.; Nepomuceno, Luviminda; Huang, Shih-Ming; Stallcup, Michael R.

    1999-01-01

    Transcription factors, such as nuclear receptors, often exist in various forms that are generated by highly conserved splicing events. Whereas the functional significance of these splicing variants is often not known, it is known that nuclear receptors activate transcription through interaction with coactivators. The parameters, other than ligands, that might modulate those interactions, however, are not well characterized, nor is the role of splicing variants. In this study, transient transf...

  19. Diversity of Interstitial Lung Fibroblasts Is Regulated by Platelet-Derived Growth Factor Receptor α Kinase Activity.

    Green, Jenna; Endale, Mehari; Auer, Herbert; Perl, Anne-Karina T

    2016-04-01

    Epithelial-mesenchymal cell interactions and factors that control normal lung development are key players in lung injury, repair, and fibrosis. A number of studies have investigated the roles and sources of epithelial progenitors during lung regeneration; such information, however, is limited in lung fibroblasts. Thus, understanding the origin, phenotype, and roles of fibroblast progenitors in lung development, repair, and regeneration helps address these limitations. Using a combination of platelet-derived growth factor receptor α-green fluorescent protein (PDGFRα-GFP) reporter mice, microarray, real-time polymerase chain reaction, flow cytometry, and immunofluorescence, we characterized two distinct interstitial resident fibroblasts, myo- and matrix fibroblasts, and identified a role for PDGFRα kinase activity in regulating their activation during lung regeneration. Transcriptional profiling of the two populations revealed a myo- and matrix fibroblast gene signature. Differences in proliferation, smooth muscle actin induction, and lipid content in the two subpopulations of PDGFRα-expressing fibroblasts during alveolar regeneration were observed. Although CD140α(+)CD29(+) cells behaved as myofibroblasts, CD140α(+)CD34(+) appeared as matrix and/or lipofibroblasts. Gain or loss of PDGFRα kinase activity using the inhibitor nilotinib and a dominant-active PDGFRα-D842V mutation revealed that PDGFRα was important for matrix fibroblast differentiation. We demonstrated that PDGFRα signaling promotes alveolar septation by regulating fibroblast activation and matrix fibroblast differentiation, whereas myofibroblast differentiation was largely PDGFRα independent. These studies provide evidence for the phenotypic and functional diversity as well as the extent of specificity of interstitial resident fibroblasts differentiation during regeneration after partial pneumonectomy. PMID:26414960

  20. Activation of tumor necrosis factor receptor 1 in airway smooth muscle: a potential pathway that modulates bronchial hyper-responsiveness in asthma?

    Panettieri Reynold A

    2000-07-01

    Full Text Available Abstract The cellular and molecular mechanisms that are involved in airway hyper-responsiveness are unclear. Current studies suggest that tumor necrosis factor (TNF-α, a cytokine that is produced in considerable quantities in asthmatic airways, may potentially be involved in the development of bronchial hyper-responsiveness by directly altering the contractile properties of the airway smooth muscle (ASM. The underlying mechanisms are not known, but growing evidence now suggests that most of the biologic effects of TNF-α on ASM are mediated by the p55 receptor or tumor necrosis factor receptor (TNFR1. In addition, activation of TNFR1 coupled to the tumor necrosis factor receptor-associated factor (TRAF2-nuclear factor-κB (NF-κB pathway alters calcium homeostasis in ASM, which appears to be a new potential mechanism underlying ASM hyper-responsiveness.

  1. Essential Role of Nuclear Factor (NF)-κB–Inducing Kinase and Inhibitor of κb (Iκb) Kinase α in Nf-κb Activation through Lymphotoxin β Receptor, but Not through Tumor Necrosis Factor Receptor I

    Matsushima, Akemi; Kaisho, Tsuneyasu; Rennert, Paul D.; Nakano, Hiroyasu; Kurosawa, Kyoko; Uchida, Daisuke; Takeda, Kiyoshi; Akira, Shizuo; Matsumoto, Mitsuru

    2001-01-01

    Both nuclear factor (NF)-κB–inducing kinase (NIK) and inhibitor of κB (IκB) kinase (IKK) have been implicated as essential components for NF-κB activation in response to many external stimuli. However, the exact roles of NIK and IKKα in cytokine signaling still remain controversial. With the use of in vivo mouse models, rather than with enforced gene-expression systems, we have investigated the role of NIK and IKKα in signaling through the type I tumor necrosis factor (TNF) receptor (TNFR-I) and the lymphotoxin β receptor (LTβR), a receptor essential for lymphoid organogenesis. TNF stimulation induced similar levels of phosphorylation and degradation of IκBα in embryonic fibroblasts from either wild-type or NIK-mutant mice. In contrast, LTβR stimulation induced NF-κB activation in wild-type mice, but the response was impaired in embryonic fibroblasts from NIK-mutant and IKKα-deficient mice. Consistent with the essential role of IKKα in LTβR signaling, we found that development of Peyer's patches was defective in IKKα-deficient mice. These results demonstrate that both NIK and IKKα are essential for the induction of NF-κB through LTβR, whereas the NIK–IKKα pathway is dispensable in TNFR-I signaling. PMID:11238593

  2. Essential role of nuclear factor (NF)-kappaB-inducing kinase and inhibitor of kappaB (IkappaB) kinase alpha in NF-kappaB activation through lymphotoxin beta receptor, but not through tumor necrosis factor receptor I.

    Matsushima, A; Kaisho, T; Rennert, P D; Nakano, H; Kurosawa, K; Uchida, D; Takeda, K; Akira, S; Matsumoto, M

    2001-03-01

    Both nuclear factor (NF)-kappaB-inducing kinase (NIK) and inhibitor of kappaB (IkappaB) kinase (IKK) have been implicated as essential components for NF-kappaB activation in response to many external stimuli. However, the exact roles of NIK and IKKalpha in cytokine signaling still remain controversial. With the use of in vivo mouse models, rather than with enforced gene-expression systems, we have investigated the role of NIK and IKKalpha in signaling through the type I tumor necrosis factor (TNF) receptor (TNFR-I) and the lymphotoxin beta receptor (LTbetaR), a receptor essential for lymphoid organogenesis. TNF stimulation induced similar levels of phosphorylation and degradation of IkappaBalpha in embryonic fibroblasts from either wild-type or NIK-mutant mice. In contrast, LTbetaR stimulation induced NF-kappaB activation in wild-type mice, but the response was impaired in embryonic fibroblasts from NIK-mutant and IKKalpha-deficient mice. Consistent with the essential role of IKKalpha in LTbetaR signaling, we found that development of Peyer's patches was defective in IKKalpha-deficient mice. These results demonstrate that both NIK and IKKalpha are essential for the induction of NF-kappaB through LTbetaR, whereas the NIK-IKKalpha pathway is dispensable in TNFR-I signaling. PMID:11238593

  3. Cigarette smoke and platelet-activating factor receptor dependent adhesion of Streptococcus pneumoniae to lower airway cells

    Grigg, Jonathan; Walters, Haydn; Sohal, Sukhwinder Singh;

    2012-01-01

    BACKGROUND: Exposure to cigarette smoke (CS) is associated with increased risk of pneumococcal infection. The mechanism for this association is unknown. We recently reported that the particulate matter from urban air simulates platelet-activating factor receptor (PAFR)-dependent adhesion of...... active-smokers by immunostaining. RESULTS: In A549 cells, CSE 1% increased pneumococcal adhesion (p<0.05 vs control), PAFR transcript level (p<0.01), and PAFR expression (p<0.01). Pneumococcal adhesion to A549 cells was attenuated by CV-3988 (p<0.001). CSE 1% stimulated pneumococcal adhesion to BEAS2-B...... cells and HBEpC (p<0.01 vs control). CSE 1% increased PAFR expression in BEAS2-B (p<0.01), and in HBEpC (p<0.05). Lung PAFR transcript level was increased in mice exposed to CS in vivo (p<0.05 vs room air). Active smokers (n=16) had an increased percentage of bronchial epithelium with PAFR...

  4. Eps15 is recruited to the plasma membrane upon epidermal growth factor receptor activation and localizes to components of the endocytic pathway during receptor internalization

    Torrisi, M R; Lotti, L V; Belleudi, F; Gradini, R; Salcini, A E; Confalonieri, S; Pelicci, P G; Di Fiore, P P

    1999-01-01

    Eps15 is a substrate for the tyrosine kinase of the epidermal growth factor receptor (EGFR) and is characterized by the presence of a novel protein:protein interaction domain, the EH domain. Eps15 also stably binds the clathrin adaptor protein complex AP-2. Previous work demonstrated an essential...

  5. The Epidermal Growth Factor Receptor Is a Regulator of Epidermal Complement Component Expression and Complement Activation

    Abu-Humaidan, Anas H A; Ananthoju, Nageshwar; Mohanty, Tirthankar;

    2014-01-01

    The complement system is activated in response to tissue injury. During wound healing, complement activation seems beneficial in acute wounds but may be detrimental in chronic wounds. We found that the epidermal expression of many complement components was only increased to a minor extent in skin...... wounds in vivo and in cultured keratinocytes after exposure to supernatant from stimulated mononuclear cells. In contrast, the epidermal expression of complement components was downregulated in ex vivo injured skin lacking the stimulation from infiltrating inflammatory cells but with intact injury...

  6. Peroxisome proliferator-activated receptor-gamma agonists suppress tissue factor overexpression in rat balloon injury model with paclitaxel infusion.

    Jun-Bean Park

    Full Text Available The role and underlying mechanisms of rosiglitazone, a peroxisome proliferator-activated receptor-gamma (PPAR-γ agonist, on myocardial infarction are poorly understood. We investigated the effects of this PPAR-γ agonist on the expression of tissue factor (TF, a primary molecule for thrombosis, and elucidated its underlying mechanisms. The PPAR-γ agonist inhibited TF expression in response to TNF-α in human umbilical vein endothelial cells, human monocytic leukemia cell line, and human umbilical arterial smooth muscle cells. The overexpression of TF was mediated by increased phosphorylation of mitogen-activated protein kinase (MAPK, which was blocked by the PPAR-γ agonist. The effective MAPK differed depending on each cell type. Luciferase and ChIP assays showed that transcription factor, activator protein-1 (AP-1, was a pivotal target of the PPAR-γ agonist to lower TF transcription. Intriguingly, two main drugs for drug-eluting stent, paclitaxel or rapamycin, significantly exaggerated thrombin-induced TF expression, which was also effectively blocked by the PPAR-γ agonist in all cell types. This PPAR-γ agonist did not impair TF pathway inhibitor (TFPI in three cell types. In rat balloon injury model (Sprague-Dawley rats, n = 10/group with continuous paclitaxel infusion, the PPAR-γ agonist attenuated TF expression by 70±5% (n = 4; P<0.0001 in injured vasculature. Taken together, rosiglitazone reduced TF expression in three critical cell types involved in vascular thrombus formation via MAPK and AP-1 inhibitions. Also, this PPAR-γ agonist reversed the paclitaxel-induced aggravation of TF expression, which suggests a possibility that the benefits might outweigh its risks in a group of patients with paclitaxel-eluting stent implanted.

  7. Synthetic Lethality Screen Identifies RPS6KA2 as Modifier of Epidermal Growth Factor Receptor Activity in Pancreatic Cancer

    Nada Milosevic

    2013-12-01

    Full Text Available Pancreatic cancer is characterized by a high degree of resistance to chemotherapy. Epidermal growth factor receptor (EGFR inhibition using the small-molecule inhibitor erlotinib was shown to provide a small survival benefit in a subgroup of patients. To identify kinases whose inhibition acts synergistically with erlotinib, we employed a kinome-wide small-interfering RNA (siRNA-based loss-of-function screen in the presence of erlotinib. Of 779 tested kinases, we identified several targets whose inhibition acted synergistically lethal with EGFR inhibition by erlotinib, among them the S6 kinase ribosomal protein S6 kinase 2 (RPS6KA2/ribosomal S6 kinase 3. Activated RPS6KA2 was expressed in approximately 40% of 123 human pancreatic cancer tissues. RPS6KA2 was shown to act downstream of EGFR/RAS/mitogen-activated protein kinase kinase (MEK/extracellular-signal regulated kinase (ERK signaling and was activated by EGF independently of the presence of KRAS mutations. Knockdown of RPS6KA2 by siRNA led to increased apoptosis only in the presence of erlotinib, whereas RPS6KA2 activation or overexpression rescued from erlotinib- and gemcitabine-induced apoptosis. This effect was at least in part mediated by downstream activation of ribosomal protein S6. Genetic as well as pharmacological inhibition of RPS6KA2 by the inhibitor BI-D1870 acted synergistically with erlotinib. By applying this synergistic lethality screen using a kinome-wide RNA interference-library approach, we identified RPS6KA2 as potential drug target whose inhibition synergistically enhanced the effect of erlotinib on tumor cell survival. This kinase therefore represents a promising drug candidate suitable for the development of novel inhibitors for pancreatic cancer therapy.

  8. Regulation of the ligand-dependent activation of the epidermal growth factor receptor by calmodulin

    Li, Hongbing; Panina, Svetlana; Kaur, Amandeep;

    2012-01-01

    Calmodulin (CaM) is the major component of calcium signaling pathways mediating the action of various effectors. Transient increases in the intracellular calcium level triggered by a variety of stimuli lead to the formation of Ca2+/CaM complexes, which interact with and activate target proteins. ...

  9. Transcription factor activity of estrogen receptor α activation upon nonylphenol or bisphenol A treatment enhances the in vitro proliferation, invasion, and migration of neuroblastoma cells

    Ma, Hongda; Yao, Yao; Wang, Changli; Zhang, Liyu; Cheng, Long; Wang, Yiren; Wang, Tao; Liang, Erguang; Jia, Hui; Ye, Qinong; Hou, Mingxiao; Feng, Fan

    2016-01-01

    Many kinds of endocrine-disrupting chemicals (EDCs), for example, the environmental estrogens bisphenol A and nonylphenol, may regulate the activity of estrogen receptor α (ERα) and therefore induce potential disruption of normal endocrine function. However, the involvement of EDCs in human cancers, especially in endocrine-related cancer neuroblastoma regulation, is not very clear. In this work, results showed that upon bisphenol A or nonylphenol treatment, the transcription factor activity of ERα was significantly increased in neuroblastoma cell line SH-SY5Y. Bisphenol A and nonylphenol could enhance ERα activity via recruiting it to the target gene promoter. Furthermore, treatment of bisphenol A and nonylphenol enhanced the in vitro proliferation, invasion, and migration ability of neuroblastoma cells. By investigating the role of EDC-induced ERα upregulation, our data extend the understanding of the function of EDCs and further suggest that ERα might be a potential therapeutic target in human neuroblastoma treatment. PMID:27366082

  10. Prognostic value of receptor activator of nuclear factor kappa-B (RANK marker in patients with breast cancer

    S. I. Zabroda

    2015-07-01

    Full Text Available Despite notable progress made in studying breast cancer (BC, the mechanisms of metastases, in view of the classification into molecular subtypes, in patients with BC remain to be fully uninvestigated, in the presence of a good prognosis in particular. To study novel diagnostic and predictive markers in a new way presents current problems in the pathology of BC. This investigation deals with the expression of osteoprotegerin (OPG in the tumor cells of patients with BC. It enrolled 83 patients with locally advanced BC (T2–4N0–3M0 who had been treated in 2003 to 2010. The inclusion criterion was a histologically verified diagnosis of invasive BC. To study the level of OPG, the investigators conducted an immunohistochemical study of biopsy sections according to the standard protocol. The mean expression of receptor activator of nuclear factor kappa-B (RANK in the BC cells was 18.7 %; itsmedian was 5 % (range, 0–90 %. The patients were divided into 2 groups according to the level of RANK expression: 1 high (higher than the median; 2 low (lower than the median. The high RANK group included 39 patients; the low RANK group comprised 44 patients. Analysis of the clinical and pathological characteristics of BC patients with regard RANK expression did not show any statistically significant differences in the presence or absence of affected regional lymph nodes, T category, and Ki-67 index. The analysis of clinical and pathomorphological and immunohistochemical characteristics in patients with breast cancer, taking into consideration RANK expression level, did not show any statistically significant differences with respect to presence or absence of affected regional lymph nodes, age, T category and Ki-67 index (р > 0.05. However, it revealed the following pattern: the high expression of RANK was more common in patients positive for estrogen and progesterone receptors than in those for negative receptors (p = 0.04.

  11. SRC-DEPENDENT PHOSPHORYLATION OF THE EPIDERMAL GROWTH FACTOR RECEPTOR ON TYROSINE 845 IS REQUIRED FOR ZINC-INDUCED RAS ACTIVATION

    Src-dependent Phosphorylation of the Epidermal Growth Factor Receptor on Tyrosine 845 Is Required for Zinc-induced Ras ActivationWeidong Wu 1 , Lee M. Graves 2 , Gordon N. Gill 3 , Sarah J. Parsons 4 , and James M. Samet 51 Center for Environmental Medicine and Lung Biolo...

  12. Influence of baicalin on the expression of receptor activator of nuclear factor-κB ligand and osteoprotegerin in human periodontal ligament cells

    2009-01-01

    Objective To study the effect of baicalin on the expression of receptor activator of nuclear factor-κB ligand(RANKL)and osteoprotegerin(OPG)in cultured human periodontal ligament(HPDL)cells.Methods Small interfering RNA(siRNA)eukaryotic expression vector targeted transforming growth factor βⅡ receptor(TGF-β RⅡ)was constructed and transfected into T cells.HPDL cells with T cells transfected with siRNA or not were placed in the culture medium that had been added with lipopolysaccharide(LPS)and baicalin.The ob...

  13. Tumor necrosis factor-α and lymphotoxin-α mediate myocardial ischemic injury via TNF receptor 1, but are cardioprotective when activating TNF receptor 2.

    Yanqing Zhang

    Full Text Available OBJECTIVE: This study determines the roles of tumor necrosis factor-α (TNFα and lymphotoxin-α (LTα in post-myocardial infarction (post-MI cardiac injury, and identifies the TNF receptor type responsible for TNFα- and LTα-mediated cardiac injury. METHODS AND RESULTS: Adult male wild type (WT, TNFα(-/-, LTα(-/-, TNFR1(-/-, and TNFR2(-/- mice were subjected to MI via coronary artery occlusion. Functional, histological, and biochemical analyses were performed 1 to 7 days post-MI. In WT mice, MI significantly increased both TNFα and LTα levels in plasma, but in distinct temporal manner. Plasma TNFα peaked 1 day after MI, and decreased toward baseline 3 days after MI. In contrast, plasma LTα became significantly increased 3 days post-MI, and remained elevated thereafter. TNFα deletion significantly improved cardiac function 3 days, but not 7 days, after MI. In contrast, LTα deletion had no effect upon cardiac dysfunction 3 days after MI, but improved cardiac function 7 days after MI. More importantly, knockout of TNFR1 and TNFR2 had opposite effects upon post-MI cardiac dysfunction, which was markedly attenuated by TNFR1 deletion (P<0.01 vs. WT, but exacerbated by TNFR2 deletion (P<0.05 vs. WT. CONCLUSION: Our study demonstrates that TNFα and LTα overproduction contribute to early and late cardiac dysfunction after MI, respectively. We provide clear evidence that both TNFα and LTα mediate post-MI cardiac dysfunction via TNFR1 stimulation, whereas TNFR2 activation is cardioprotective against ischemic injury. Simultaneous inhibition of TNFα and LTα or specific TNFR1 function blockade may represent superior cardioprotective approaches over general TNF activity suppression.

  14. Lack of Platelet-Activating Factor Receptor Attenuates Experimental Food Allergy but Not Its Metabolic Alterations regarding Adipokine Levels

    Batista, Nathália Vieira; Fonseca, Roberta Cristelli; Perez, Denise; Pereira, Rafaela Vaz Sousa; de Lima Alves, Juliana; Pinho, Vanessa; Faria, Ana Maria Caetano; Cara, Denise Carmona

    2016-01-01

    Platelet-activating factor (PAF) is known to be an important mediator of anaphylaxis. However, there is a lack of information in the literature about the role of PAF in food allergy. The aim of this work was to elucidate the participation of PAF during food allergy development and the consequent adipose tissue inflammation along with its alterations. Our data demonstrated that, both before oral challenge and after 7 days receiving ovalbumin (OVA) diet, OVA-sensitized mice lacking the PAF receptor (PAFR) showed a decreased level of anti-OVA IgE associated with attenuated allergic markers in comparison to wild type (WT) mice. Moreover, there was less body weight and adipose tissue loss in PAFR-deficient mice. However, some features of inflamed adipose tissue presented by sensitized PAFR-deficient and WT mice after oral challenge were similar, such as a higher rate of rolling leukocytes in this tissue and lower circulating levels of adipokines (resistin and adiponectin) in comparison to nonsensitized mice. Therefore, PAF signaling through PAFR is important for the allergic response to OVA but not for the adipokine alterations caused by this inflammatory process. Our work clarifies some effects of PAF during food allergy along with its role on the metabolic consequences of this inflammatory process. PMID:27314042

  15. P2X4 receptor in the dorsal horn partially contributes to brain-derived neurotrophic factor oversecretion and toll-like receptor-4 receptor activation associated with bone cancer pain.

    Jin, Xiao-Hong; Wang, Li-Na; Zuo, Jian-Ling; Yang, Jian-Ping; Liu, Si-Lan

    2014-12-01

    Previous studies have suggested that the microglial P2X7 purinoceptor is involved in the release of tumor necrosis factor-α (TNFα) following activation of toll-like receptor-4 (TLR4), which is associated with nociceptive behavior. In addition, this progress is evoked by the activation of the P2X4 purinoceptor (P2X4R). Although P2X4R is also localized within spinal microglia in the dorsal horn, little is known about its role in cancer-induced bone pain (CIBP), which is in some ways unique. With the present rat model of CIBP, we demonstrate a critical role of the microglial P2X4R in the enhanced nociceptive transmission, which is associated with TLR4 activation and secretion of brain-derived neurotrophic factor (BDNF) and TNFα in the dorsal horn. We assessed mechanical threshold and spontaneous pain of CIBP rats. Moreover, P2X4R small interfering RNA (siRNA) was administered intrathecally, and real-time PCR, Western blots, immunofluorescence histochemistry, and ELISA were used to detect the expression of P2X4R, TLR4, OX-42, phosphorylated-p38 MAPK (p-p38), BDNF, and TNFα. Compared with controls, intrathecal injection of P2X4R siRNA could prevent nociceptive behavior induced by ATP plus lipopolysaccharide and CIBP and reduce the expression of P2X4R, TLR4, p-p38, BDNF, and TNFα. In addition, the increase of BDNF protein in rat microglial cells depended on P2X4 receptor signaling, which is partially associated with TLR4 activation. The ability of microglial P2X4R to activate TLR4 in spinal cord leading to behavioral hypersensitivity and oversecretion of BDNF could provide an opportunity for the prevention and treatment of CIBP. PMID:24984884

  16. Antitumor activity of F90,an epidermal growth factor receptor tyrosine kinase inhibitor,on glioblastoma cell line SHG-44

    LIU Fang-jun; GUI Song-bai; LI Chu-zhong; SUN Ze-lin; ZHANG Ya-zhuo

    2008-01-01

    Background Over-expression of epidermal growth factor receptor (EGFR) is thought to be related to cell proliferation,invasion,metastasis,resistance to chemoradiotherapy and poor prognosis of various human cancers.Forty percent to fifty percent of glioblastoma multiforme (GBM) possess deregulated EGFR,which may contribute to the aggressive and refractory course of GBM.Therefore,blockade of EGFR signal transduction may be a promising treatment strategy for GBM.Methods MIT assay,cell growth curve assay and tumor xenograft model were used to evaluate the antitumor activity of F90 against SHG-44 in vitro and in vivo.Western blot assay was applied to evaluate the expression of p-EGFR,p-ERK1,p-JNK,p-P38,Bcl2 and P53 proteins.Results F90 inhibited the cell proliferation in a dose-dependent manner in vitro.The growth of SHG-44 tumor xenografts was suppressed by F90 at a high dose level (100 mg.kg-1.d-1).Phosphorylation of EGFR and activated downstream signaling proteins,such as ERK1,JNK and P38,were found to be depressed after incubation with F90 for 48 hours in vitro.Down-regulated Bcl2 protein and up-regulated P53 protein were also observed.Conclueions The results demonstrate that F90 is effective in inhibiting the proliferation of SHG-44 cells in vitro and tumor growth in vivo,suggesting that F90 may be a new therapeutic option for treatment of GBM.

  17. Dopamine receptor regulating factor, DRRF: a zinc finger transcription factor.

    Hwang, C K; D'Souza, U M; Eisch, A J; Yajima, S; Lammers, C H; Yang, Y; Lee, S H; Kim, Y M; Nestler, E J; Mouradian, M M

    2001-06-19

    Dopamine receptor genes are under complex transcription control, determining their unique regional distribution in the brain. We describe here a zinc finger type transcription factor, designated dopamine receptor regulating factor (DRRF), which binds to GC and GT boxes in the D1A and D2 dopamine receptor promoters and effectively displaces Sp1 and Sp3 from these sequences. Consequently, DRRF can modulate the activity of these dopamine receptor promoters. Highest DRRF mRNA levels are found in brain with a specific regional distribution including olfactory bulb and tubercle, nucleus accumbens, striatum, hippocampus, amygdala, and frontal cortex. Many of these brain regions also express abundant levels of various dopamine receptors. In vivo, DRRF itself can be regulated by manipulations of dopaminergic transmission. Mice treated with drugs that increase extracellular striatal dopamine levels (cocaine), block dopamine receptors (haloperidol), or destroy dopamine terminals (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) show significant alterations in DRRF mRNA. The latter observations provide a basis for dopamine receptor regulation after these manipulations. We conclude that DRRF is important for modulating dopaminergic transmission in the brain. PMID:11390978

  18. An active form of Vav1 induces migration of mammary epithelial cells by stimulating secretion of an epidermal growth factor receptor ligand

    Moores Sheri L

    2006-05-01

    Full Text Available Abstract Background Vav proteins are guanine nucleotide exchange factors (GEF for Rho family GTPases and are activated following engagement of membrane receptors. Overexpression of Vav proteins enhances lamellipodium and ruffle formation, migration, and cell spreading, and augments activation of many downstream signaling proteins like Rac, ERK and Akt. Vav proteins are composed of multiple structural domains that mediate their GEF function and binding interactions with many cellular proteins. In this report we examine the mechanisms responsible for stimulation of cell migration by an activated variant of Vav1 and identify the domains of Vav1 required for this activity. Results We found that expression of an active form of Vav1, Vav1Y3F, in MCF-10A mammary epithelial cells increases cell migration in the absence or presence of EGF. Vav1Y3F was also able to drive Rac1 activation and PAK and ERK phosphorylation in MCF-10A cells in the absence of EGF stimulation. Mutations in the Dbl homology, pleckstrin homology, or cysteine-rich domains of Vav1Y3F abolished Rac1 or ERK activation in the absence of EGF and blocked the migration-promoting activity of Vav1Y3F. In contrast, mutations in the SH2 and C-SH3 domains did not affect Rac activation by Vav1Y3F, but reduced the ability of Vav1Y3F to induce EGF-independent migration and constitutive ERK phosphorylation. EGF-independent migration of MCF-10A cells expressing Vav1Y3F was abolished by treatment of cells with an antibody that prevents ligand binding to the EGF receptor. In addition, conditioned media collected from Vav1Y3F expressing cells stimulated migration of parental MCF-10A cells. Lastly, treatment of cells with the EGF receptor inhibitory antibody blocked the Vav1Y3F-induced, EGF-independent stimulation of ERK phosphorylation, but had no effect on Rac1 activation or PAK phosphorylation. Conclusion Our results indicate that increased migration of active Vav1 expressing cells is dependent on

  19. SOCIAL DEFEAT STRESS ACTIVATES MEDIAL AMYGDALA CELLS THAT EXPRESS TYPE 2 CORTICOTROPIN-RELEASING FACTOR RECEPTOR mRNA

    FEKETE, É. M.; Y. Zhao; Li, C.; SABINO, V.; Vale, W.W.; ZORRILLA, E. P.

    2009-01-01

    Defeat is a social stressor involving subordination by a threatening conspecific. Type 2 corticotropin-releasing factor receptors (CRF2) are abundant in brain regions implicated in defeat responses and are putative stress-related molecules. The present study sought to determine whether neuroactivation and CRF2 expression co-occurred at brain region or cellular levels following acute defeat. Male “intruder” Wistar rats were placed into the cage of an aggressive “resident” Long-Evans rat (n=6)....

  20. Epidermal growth factor-like repeats of tenascin-C-induced constriction of cerebral arteries via activation of epidermal growth factor receptors in rats.

    Fujimoto, Masashi; Shiba, Masato; Kawakita, Fumihiro; Liu, Lei; Nakasaki, Asuka; Shimojo, Naoshi; Imanaka-Yoshida, Kyoko; Yoshida, Toshimichi; Suzuki, Hidenori

    2016-07-01

    Tenascin-C (TNC), one of matricellular proteins, has been suggested to be involved in cerebral vasospasm after aneurysmal subarachnoid hemorrhage. However, the mechanisms of how TNC constricts cerebral arteries remain unclear. The aim of this study was to examine if epidermal growth factor (EGF)-like repeats of TNC is involved in TNC-induced constriction of cerebral arteries in rats via EGF receptor (EGFR) activation. Two dosages of recombinant TNC (r-TNC) consisting of the EGF-like repeats was administered intracisternally to healthy rats, and its vasoconstrictor effects were evaluated by neurobehavioral tests and India-ink angiography at 24, 48, and 72 hours after the administration. Western blotting and immunohistochemistry were performed to explore the underlying mechanisms on constricted cerebral arteries after 24 hours. The effects of a selective EGFR tyrosine kinase inhibitor (AG1478) on r-TNC-induced vasoconstriction were evaluated by neurobehavioral tests, India-ink angiography and immunohistochemistry at 24 hours after the administration. A higher dosage of r-TNC induced cerebral arterial constriction more severely, which continued for 48 hours. The effects were associated with the activation of EGFR and extracellular signal-regulated kinase (ERK)1/2 in the smooth muscle cell layer of the constricted cerebral artery, while c-Jun N-terminal kinase and p38 were not activated. AG1478 blocked r-TNC-induced vasoconstrictive effects, as well as activation of EGFR and ERK1/2. These findings demonstrate that TNC induces constriction of cerebral arteries via activation of EGFR and ERK1/2. PMID:27086972

  1. Tumor necrosis factor (TNF) receptor shedding controls thresholds of innate immune activation that balance opposing TNF functions in infectious and inflammatory diseases

    Xanthoulea, Sofia; Pasparakis, Manolis; Kousteni, Stavroula;

    2004-01-01

    Tumor necrosis factor (TNF) is a potent cytokine exerting critical functions in the activation and regulation of immune and inflammatory responses. Due to its pleiotropic activities, the amplitude and duration of TNF function must be tightly regulated. One of the mechanisms that may have evolved to...... modulate TNF function is the proteolytic cleavage of its cell surface receptors. In humans, mutations affecting shedding of the p55TNF receptor (R) have been linked with the development of the TNFR-associated periodic syndromes, disorders characterized by recurrent fever attacks and localized inflammation....... Here we show that knock-in mice expressing a mutated nonsheddable p55TNFR develop Toll-like receptor-dependent innate immune hyperreactivity, which renders their immune system more efficient at controlling intracellular bacterial infections. Notably, gain of function for antibacterial host defenses...

  2. Platelet-activating factor receptor (PAF-R)-dependent pathways control tumour growth and tumour response to chemotherapy

    Phagocytosis of apoptotic cells by macrophages induces a suppressor phenotype. Previous data from our group suggested that this occurs via Platelet-activating factor receptor (PAF-R)-mediated pathways. In the present study, we investigated the impact of apoptotic cell inoculation or induction by a chemotherapeutic agent (dacarbazine, DTIC) on tumour growth, microenvironmental parameters and survival, and the effect of treatment with a PAF-R antagonist (WEB2170). These studies were performed in murine tumours: Ehrlich Ascitis Tumour (EAT) and B16F10 melanoma. Tumour growth was assessed by direct counting of EAT cells in the ascitis or by measuring the volume of the solid tumour. Parameters of the tumour microenvironment, such as the frequency of cells expressing cyclo-oxygenase-2 (COX-2), caspase-3 and galectin-3, and microvascular density, were determined by immunohistochemistry. Levels of vascular endothelium growth factor (VEGF) and prostaglandin E2 (PGE2) were determined by ELISA, and levels of nitric oxide (NO) by Griess reaction. PAF-R expression was analysed by immunohistochemistry and flow cytometry. Inoculation of apoptotic cells before EAT implantation stimulated tumour growth. This effect was reversed by in vivo pre-treatment with WEB2170. This treatment also reduced tumour growth and modified the microenvironment by reducing PGE2, VEGF and NO production. In B16F10 melanoma, WEB2170 alone or in association with DTIC significantly reduced tumour volume. Survival of the tumour-bearing mice was not affected by WEB2170 treatment but was significantly improved by the combination of DTIC with WEB2170. Tumour microenvironment elements were among the targets of the combination therapy since the relative frequency of COX-2 and galectin-3 positive cells and the microvascular density within the tumour mass were significantly reduced by treatment with WEB2170 or DTIC alone or in combination. Antibodies to PAF-R stained the cells from inside the tumour, but not the

  3. Platelet-activating factor receptor (PAF-R-dependent pathways control tumour growth and tumour response to chemotherapy

    Rohde Ciro BS

    2010-05-01

    Full Text Available Abstract Background Phagocytosis of apoptotic cells by macrophages induces a suppressor phenotype. Previous data from our group suggested that this occurs via Platelet-activating factor receptor (PAF-R-mediated pathways. In the present study, we investigated the impact of apoptotic cell inoculation or induction by a chemotherapeutic agent (dacarbazine, DTIC on tumour growth, microenvironmental parameters and survival, and the effect of treatment with a PAF-R antagonist (WEB2170. These studies were performed in murine tumours: Ehrlich Ascitis Tumour (EAT and B16F10 melanoma. Methods Tumour growth was assessed by direct counting of EAT cells in the ascitis or by measuring the volume of the solid tumour. Parameters of the tumour microenvironment, such as the frequency of cells expressing cyclo-oxygenase-2 (COX-2, caspase-3 and galectin-3, and microvascular density, were determined by immunohistochemistry. Levels of vascular endothelium growth factor (VEGF and prostaglandin E2 (PGE2 were determined by ELISA, and levels of nitric oxide (NO by Griess reaction. PAF-R expression was analysed by immunohistochemistry and flow cytometry. Results Inoculation of apoptotic cells before EAT implantation stimulated tumour growth. This effect was reversed by in vivo pre-treatment with WEB2170. This treatment also reduced tumour growth and modified the microenvironment by reducing PGE2, VEGF and NO production. In B16F10 melanoma, WEB2170 alone or in association with DTIC significantly reduced tumour volume. Survival of the tumour-bearing mice was not affected by WEB2170 treatment but was significantly improved by the combination of DTIC with WEB2170. Tumour microenvironment elements were among the targets of the combination therapy since the relative frequency of COX-2 and galectin-3 positive cells and the microvascular density within the tumour mass were significantly reduced by treatment with WEB2170 or DTIC alone or in combination. Antibodies to PAF-R stained

  4. An antagonistic activity of etizolam on platelet-activating factor (PAF). In vitro effects on platelet aggregation and PAF receptor binding.

    Mikashima, H; Takehara, S; Muramoto, Y; Khomaru, T; Terasawa, M; Tahara, T; Maruyama, Y

    1987-08-01

    The antagonistic effect of etizolam, an anti-anxiety drug, on platelet-activating factor (PAF) was investigated in rabbit platelets in vitro. Etizolam inhibited PAF-induced aggregation in a dose-dependent manner, with an IC50 of 3.8 microM, about one tenth that of triazolam (IC50 = 30 microM). At 300 microM, it inhibited both ADP and arachidonic acid-induced aggregation only slightly, while the other anti-anxiety drugs tested had no effect on PAF-induced aggregation even at this concentration. Etizolam and triazolam inhibited the specific binding of 3H-PAF to PAF receptor sites on washed rabbit platelets with IC50 values of 22 nM and 320 nM, respectively. Diazepam and estazolam were inactive even at 1 microM. These results indicate that etizolam is a specific antagonist of PAF. PMID:2890779

  5. Synthesis of [[sup 11]C] platelet-activating factor (PAF) analogs for in vivo imaging of PAF receptors

    Sasaki, T.; Ishiwata, K. (Tokyo Metropolitan Inst. of Gerontology, Itabishi (Japan)); Karasawa, K. (Teikyo Univ., Kanagawa (Japan). Faculty of Pharmaceutical Sciences) (and others)

    1993-10-01

    [1-O-hexadecyl-2-O-N], N-dimethylcarbamoyl-sn-glycero-3-phosphocholine[choline methyl-[sup 11]C] ([[sup 11]C])dimethylcarbamoyl-platelet-activating factor (PAF) and 1-O-hexadecyl-2-O-acetyl-sn-glycero-3-phosphocholine[choline methyl-[sup 11]C] ([[sup 11]C]C[sub 16]-PAF) were synthesized as follows. Each of non-labeled dimethylcarbamoyl-PAF and C[sub 16]-PAF was treated with sodium benzene thiolate to derive their desmethyl-precursors containing a dimethylphosphoethanolamine at sn-3. [sup 11]C-Labeled dimethylcarbamoyl-PAF and C[sub 16]-PAF were synthesized by methylation of the respective desmethyl-precursors using [[sup 11]C]CH[sub 3]I. The radiochemical yield of methylation in [[sup 11]C]dimethylcarbamoyl-PAF and [[sup 11]C]C[sub 16]-PAF was about 15 and 10% (decay corrected), respectively. To study the stability to enzymatic hydrolysis, [[sup 11]C]dimethylcarbamoyl-PAF or [[sup 11]C]C[sub 16]-PAF was incubated with mouse plasma at 37[sup o]C. [[sup 11]C]Dimethylcarbamoyl-PAF remained intact for 60 min. On the other hand, almost all the radioactivity of [[sup 11]C]C[sub 16]-PAF was converted into [[sup 11]C]C[sub 16]-lyso-PAF in 5 min. These observations indicate that [[sup 11]C]dimethylcarbamoyl-PAF can be a suitable probe for in vivo imaging of PAF receptors. (author).

  6. Hypoxia-inducible factor 1 mediates hypoxia-induced cardiomyocyte lipid accumulation by reducing the DNA binding activity of peroxisome proliferator-activated receptor α/retinoid X receptor

    In response to cellular hypoxia, cardiomyocytes adapt to consume less oxygen by shifting ATP production from mitochondrial fatty acid β-oxidation to glycolysis. The transcriptional activation of glucose transporters and glycolytic enzymes by hypoxia is mediated by hypoxia-inducible factor 1 (HIF-1). In this study, we examined whether HIF-1 was involved in the suppression of mitochondrial fatty acid β-oxidation in hypoxic cardiomyocytes. We showed that either hypoxia or adenovirus-mediated expression of a constitutively stable hybrid form (HIF-1α/VP16) suppressed mitochondrial fatty acid metabolism, as indicated by an accumulation of intracellular neutral lipid. Both treatments also reduced the mRNA levels of muscle carnitine palmitoyltransferase I which catalyzes the rate-limiting step in the mitochondrial import of fatty acids for β-oxidation. Furthermore, adenovirus-mediated expression of HIF-1α/VP16 in cardiomyocytes under normoxic conditions also mimicked the reduction in the DNA binding activity of peroxisome proliferator-activated receptor α (PPARα)/retinoid X receptor (RXR), in the presence or absence of a PPARα ligand. These results suggest that HIF-1 may be involved in hypoxia-induced suppression of fatty acid metabolism in cardiomyocytes by reducing the DNA binding activity of PPARα/RXR

  7. Identification of a region which directs the monocytic activity of the colony-stimulating factor 1 (macrophage colony-stimulating factor) receptor promoter and binds PEBP2/CBF (AML1).

    Zhang, D E; Fujioka, K.; Hetherington, C J; Shapiro, L H; CHEN, H. M.; Look, A T; Tenen, D. G.

    1994-01-01

    The receptor for the macrophage colony-stimulating factor (or colony-stimulating factor 1 [CSF-1]) is expressed from different promoters in monocytic cells and placental trophoblasts. We have demonstrated that the monocyte-specific expression of the CSF-1 receptor is regulated at the level of transcription by a tissue-specific promoter whose activity is stimulated by the monocyte/B-cell-specific transcription factor PU.1 (D.-E. Zhang, C.J. Hetherington, H.-M. Chen, and D.G. Tenen, Mol. Cell. ...

  8. Krüppel-like factor 8 is a novel androgen receptor co-activator in human prostate cancer

    Hong-jiang HE; Xue-feng GU; Wan-hai XU; De-jun YANG; Xiao-min WANG; Yu SU

    2013-01-01

    Aim: Krüppel-like factor 8 (KLF8) plays important roles in cell cycle and oncogenic transformation.On other hand,androgen receptor (AR) is crucial in development of both androgen-dependent and independent prostatic malignancies.The aim of this study is to investigate the role of KLF8 in prostate cancer (PCa) and the relationship between KLF8 and AR.Methods.: Eight human PCa cell lines,including androgen-dependent LNCap cells and androgen-independent 22Rv1 cells,as well as human PCa samples were studied.LNCap cells and 22Rv1 cells were transfected with plasmids encoding full-length wild-type KLF8 or KLF8 shRNA.The expression of KLF8 protein was detected using Western blotting or immunohistochemical staining.Cell proliferation in vitro was measured with MTT assay,and in vivo in a xenograft nude mouse model.Yeast two-hybrid screening,co-immunoprecipitation and pull down assays were used to examine the binding of KLF8 to AR.Luciferase reporter gene assay was used to measure the transcriptional activity of the genes targeted by AR.Results: In 133 human PCa samples,KLF8 protein staining was observed in 92.65% (63/68) of high-grade PCa,66.15% (43/65) of low-grade PCa,and 6.82% (3/44) of adjacent normal tissues.The expression of KLF8 was significantly associated with poorer overall survival.Overexpression of KLF8 enhanced the proliferation of both LNCap and 22Rv1 cells,while knockdown of endogenous KLF8 suppressed the proliferation.These manipulations exerted similar effects on the tumor volumes in the xenograft nude mouse model.Yeast two-hybrid screening revealed that KLF8 was a novel AR-interacting protein.With pull down assay and co-immunoprecipitation assay,we demonstrated that KLF8 bound directly to AR,and KLF8 enhanced AR target gene transcription.Conclusion: The results demonstrate that KLF8 is a novel AR transcriptional co-activator that is overexpressed in PCa and may play a role in progression of hormone-refractory PCa.

  9. Retention of biological activity following radioiodination of human interleukin 2: comparison with biosynthetically labeled growth factor in receptor binding assays

    Human interleukin 2 (IL-2) was radioiodinated by a modified form of the chloramine-T reaction. Comparison with biosynthetically radiolabeled IL-2 demonstrated that the iodinated molecule retained similar receptor binding characteristics and proliferation-inducing ability. The iodinated molecule also possessed the distinct advantages of a higher specific radioactivity and a reduced processing time for the assay samples. The majority of the iodine was incorporated at the tyrosine in position 45 of the polypeptide chain. Evidently, this residue is unimportant for the molecule's association with its receptor. The development of active radioiodinated IL-2 should facilitate routine measurement of the high-affinity IL-2 binding sites which mediate the physiological response to this lymphokine. (Auth.)

  10. Angiotensin II-induced Akt activation through the epidermal growth factor receptor in vascular smooth muscle cells is mediated by phospholipid metabolites derived by activation of phospholipase D.

    Li, Fang; Malik, Kafait U

    2005-03-01

    Angiotensin II (Ang II) activates cytosolic Ca(2+)-dependent phospholipase A(2) (cPLA(2)), phospholipase D (PLD), p38 mitogen-activated protein kinase (MAPK), epidermal growth factor receptor (EGFR) and Akt in vascular smooth muscle cells (VSMC). This study was conducted to investigate the relationship between Akt activation by Ang II and other signaling molecules in rat VSMC. Ang II-induced Akt phosphorylation was significantly reduced by the PLD inhibitor 1-butanol, but not by its inactive analog 2-butanol, and by brefeldin A, an inhibitor of the PLD cofactor ADP-ribosylation factor, and in cells infected with retrovirus containing PLD(2) siRNA or transfected with PLD(2) antisense but not control LacZ or sense oligonucleotide. Diacylglycerol kinase inhibitor II diminished Ang II-induced and diC8-phosphatidic acid (PA)-increased Akt phosphorylation, suggesting that PLD-dependent Akt activation is mediated by PA. Ang II-induced EGFR phosphorylation was inhibited by 1-butanol and PLD(2) siRNA and also by cPLA(2) siRNA. In addition, the inhibitor of arachidonic acid (AA) metabolism 5,8,11,14-eicosatetraynoic acid (ETYA) reduced both Ang II- and AA-induced EGFR transactivation. Furthermore, ETYA, cPLA(2) antisense, and cPLA(2) siRNA attenuated Ang II-elicited PLD activation. p38 MAPK inhibitor SB202190 [4-(4-flurophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole] reduced PLD activity and EGFR and Akt phosphorylation elicited by Ang II. Pyrrolidine-1, a cPLA(2) inhibitor, and cPLA(2) siRNA decreased p38 MAPK activity. These data indicate that Ang II-stimulated Akt activity is mediated by cPLA(2)-dependent, p38 MAPK regulated PLD(2) activation and EGFR transactivation. We propose the following scheme of the sequence of events leading to activation of Akt in VSMC by Ang II: Ang II-->cPLA(2)-->AA-->p38 MAPK-->PLD(2)-->PA-->EGFR-->Akt. PMID:15525798

  11. Activity Assay of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Triple-Negative Breast Cancer Cells Using Peptide-Conjugated Magnetic Beads

    Ghosh, Gargi; Yan, Xiaoliang; Kron, Stephen J.; Palecek, Sean P.

    2013-01-01

    Triple-negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer with limited treatment options. Epidermal growth factor receptor I (EGFR) has emerged as a promising target in TNBC. Limited success of the EGFR kinase inhibiting small molecules in clinical trials may be attributed in part to inaccuracy in identifying EGFR signatures in patient tumors. In light of the absence of a simple correlation between EGFR expression and its degree of activation, a simple and reliable ...

  12. Fermented milk containing Lactobacillus GG alleviated DSS-induced colitis in mice and activated epidermal growth factor receptor and Akt signaling in intestinal epithelial cells

    Kazutoyo Yoda

    2012-06-01

    Full Text Available Lactobacillus rhamnosus GG was assessed for its ability to alleviate DSS-induced colitis in mice and activate epidermal growth factor receptor and Akt signaling in intestinal epithelial cells. In this study mice were treated with DSS to induce colitis and they were given Lactobacillus GG fermented milk to assess the effect of probiotic on colitis. Lactobacillus GG fermented milk significantly reduced the colitis associated changes suggesting a protective effect against DSS induced colitis.

  13. Fermented milk containing Lactobacillus GG alleviated DSS-induced colitis in mice and activated epidermal growth factor receptor and Akt signaling in intestinal epithelial cells

    Yoda, Kazutoyo; He, Fang; Miyazawa, Kenji; Hiramatsu, Masaru

    2012-01-01

    Lactobacillus rhamnosus GG was assessed for its ability to alleviate DSS-induced colitis in mice and activate epidermal growth factor receptor and Akt signaling in intestinal epithelial cells. In this study mice were treated with DSS to induce colitis and they were given Lactobacillus GG fermented milk to assess the effect of probiotic on colitis. Lactobacillus GG fermented milk significantly reduced the colitis associated changes suggesting a protective effect against DSS induced colitis.Key...

  14. Orthovanadate-induced vasocontraction is mediated by the activation of Rho-kinase through Src-dependent transactivation of epidermal growth factor receptor

    Yayama, Katsutoshi; Sasahara, Tomoya; Ohba, Hisaaki; Funasaka, Ayaka; Okamoto, Hiroshi

    2014-01-01

    Orthovanadate (OVA), a protein tyrosine phosphatase (PTPase) inhibitor, exerts contractile effects on smooth muscle in a Rho-kinase-dependent manner, but the precise mechanisms are not elucidated. The aim of this study was to determine the potential roles of Src and epidermal growth factor receptor (EGFR) in the OVA-induced contraction of rat aortas and the phosphorylation of myosin phosphatase target subunit 1 (MYPT1; an index of Rho-kinase activity) in vascular smooth muscle cells (VSMCs). ...

  15. Esculetin attenuates receptor activator of nuclear factor kappa-B ligand-mediated osteoclast differentiation through c-Fos/nuclear factor of activated T-cells c1 signaling pathway

    Baek, Jong Min; Park, Sun-Hyang; Cheon, Yoon-Hee; Ahn, Sung-Jun [Department of Anatomy, School of Medicine, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Lee, Myeung Su [Division of Rheumatology, Department of Internal Medicine, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Imaging Science-based Lung and Bone Diseases Research Center, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Institute for Skeletal Disease, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Oh, Jaemin, E-mail: jmoh@wku.ac.kr [Department of Anatomy, School of Medicine, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Imaging Science-based Lung and Bone Diseases Research Center, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Institute for Skeletal Disease, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Kim, Ju-Young, E-mail: kimjy1014@gmail.com [Imaging Science-based Lung and Bone Diseases Research Center, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of)

    2015-05-29

    Esculetin exerts various biological effects on anti-oxidation, anti-tumors, and anti-inflammation. However, the involvement of esculetin in the bone metabolism process, particularly osteoclast differentiation has not yet been investigated. In the present study, we first confirmed the inhibitory effect of esculetin on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation. We then revealed the relationship between esculetin and the expression of osteoclast-specific molecules to elucidate its underlying mechanisms. Esculetin interfered with the expression of c-Fos and nuclear factor of activated T cell c1 (NFATc1) both at the mRNA and protein level with no involvement in osteoclast-associated early signaling pathways, suppressing the expression of various transcription factors exclusively expressed in osteoclasts such as tartrate-resistant acid phosphatase (Trap), osteoclast-associated receptor (Oscar), dendritic cell-specific transmembrane protein (Dcstamp), osteoclast stimulatory transmembrane protein (Ocstamp), cathepsin K, αvβ3 integrin, and calcitonin receptor (Ctr). Additionally, esculetin inhibited the formation of filamentous actin (F-actin) ring-positive osteoclasts during osteoclast differentiation. However, the development of F-actin structures and subsequent bone resorbing activity of mature osteoclasts, which are observed in osteoclast/osteoblast co-culture systems were not affected by esculetin. Taken together, our results indicate for the first time that esculetin inhibits RANKL-mediated osteoclastogenesis via direct suppression of c-Fos and NFATc1 expression and exerts an inhibitory effect on actin ring formation during osteoclastogenesis. - Highlights: • We first investigated the effects of esculetin on osteoclast differentiation and function. • Our data demonstrate for the first time that esculetin can suppress osteoclastogenesis in vitro. • Esculetin acts as an inhibitor of c-Fos and NFATc1 activation.

  16. Esculetin attenuates receptor activator of nuclear factor kappa-B ligand-mediated osteoclast differentiation through c-Fos/nuclear factor of activated T-cells c1 signaling pathway

    Esculetin exerts various biological effects on anti-oxidation, anti-tumors, and anti-inflammation. However, the involvement of esculetin in the bone metabolism process, particularly osteoclast differentiation has not yet been investigated. In the present study, we first confirmed the inhibitory effect of esculetin on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation. We then revealed the relationship between esculetin and the expression of osteoclast-specific molecules to elucidate its underlying mechanisms. Esculetin interfered with the expression of c-Fos and nuclear factor of activated T cell c1 (NFATc1) both at the mRNA and protein level with no involvement in osteoclast-associated early signaling pathways, suppressing the expression of various transcription factors exclusively expressed in osteoclasts such as tartrate-resistant acid phosphatase (Trap), osteoclast-associated receptor (Oscar), dendritic cell-specific transmembrane protein (Dcstamp), osteoclast stimulatory transmembrane protein (Ocstamp), cathepsin K, αvβ3 integrin, and calcitonin receptor (Ctr). Additionally, esculetin inhibited the formation of filamentous actin (F-actin) ring-positive osteoclasts during osteoclast differentiation. However, the development of F-actin structures and subsequent bone resorbing activity of mature osteoclasts, which are observed in osteoclast/osteoblast co-culture systems were not affected by esculetin. Taken together, our results indicate for the first time that esculetin inhibits RANKL-mediated osteoclastogenesis via direct suppression of c-Fos and NFATc1 expression and exerts an inhibitory effect on actin ring formation during osteoclastogenesis. - Highlights: • We first investigated the effects of esculetin on osteoclast differentiation and function. • Our data demonstrate for the first time that esculetin can suppress osteoclastogenesis in vitro. • Esculetin acts as an inhibitor of c-Fos and NFATc1 activation.

  17. Impaired coactivator activity of the Gly482 variant of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) on mitochondrial transcription factor A (Tfam) promoter

    Mitochondrial dysfunction may cause diabetes or insulin resistance. Peroxisome proliferation-activated receptor-γ (PPAR-γ) coactivator-1 α (PGC-1α) increases mitochondrial transcription factor A (Tfam) resulting in mitochondrial DNA content increase. An association between a single nucleotide polymorphism (SNP), G1444A(Gly482Ser), of PGC-1α coding region and insulin resistance has been reported in some ethnic groups. In this study, we investigated whether a change of glycine to serine at codon 482 of PGC-1α affected the Tfam promoter activity. The cDNA of PGC-1α variant bearing either glycine or serine at 482 codon was transfected into Chang human hepatocyte cells. The PGC-1α protein bearing glycine had impaired coactivator activity on Tfam promoter-mediated luciferase. We analyzed the PGC-1α genotype G1444A and mitochondrial DNA (mtDNA) copy number from 229 Korean leukocyte genomic DNAs. Subjects with Gly/Gly had a 20% lower amount of peripheral blood mtDNA than did subjects with Gly/Ser and Ser/Ser (p < 0.05). No correlation was observed between diabetic parameters and PGC-1α genotypes in Koreans. These results suggest that PGC-1α variants with Gly/Gly at 482nd amino acid may impair the Tfam transcription, a regulatory function of mitochondrial biogenesis, resulting in dysfunctional mtDNA replication

  18. Using in vivo electroporation to identify hepatic LDL receptor promoter elements and transcription factors mediating activation of transcription by T3

    Dayami Lopez

    2012-12-01

    Full Text Available The technique of in vivo electroporation was adapted to investigate the promoter elements and transcription factors mediating the rapid induction of hepatic LDL receptor expression in response to thyroid hormone. Direct comparisons between wild type and mutant promoter constructs were made within the same animal. It was demonstrated that both TREs at bp −612 and −156 were required for the l-triiodothyronine (T3 response. ChIP analysis showed that binding of TRβ1 to the −612 and −156 TREs was markedly stimulated by T3 in vivo. Introduction of siRNAs against TRβ1/RXRα with LDL receptor promoter-luciferase construct by in vivo electroporation demonstrated that these transcription factors play the major physiological role in the activation of hepatic LDL receptor transcription. The findings agree with those made by transfecting H4IIE cells in vitro thus validating this technique for in vivo studies of mechanisms of transcriptional regulation. The findings reported herein also indicated, for the first time, that PPARα and USF-2 were required for maximum transcriptional activation of the LDL receptor in response to T3 treatment.

  19. Reactivation of Desensitized Formyl Peptide Receptors by Platelet Activating Factor: A Novel Receptor Cross Talk Mechanism Regulating Neutrophil Superoxide Anion Production

    Forsman, Huamei; Önnheim, Karin; Andréasson, Emil; Christenson, Karin; Karlsson, Anna; Bylund, Johan; Dahlgren, Claes

    2013-01-01

    Neutrophils express different chemoattractant receptors of importance for guiding the cells from the blood stream to sites of inflammation. These receptors communicate with one another, a cross talk manifested as hierarchical, heterologous receptor desensitization. We describe a new receptor cross talk mechanism, by which desensitized formyl peptide receptors (FPRdes) can be reactivated. FPR desensitization is induced through binding of specific FPR agonists and is reached after a short perio...

  20. Polymeric nanoparticles conjugate a novel heptapeptide as an epidermal growth factor receptor-active targeting ligand for doxorubicin

    Liu CW

    2012-08-01

    Full Text Available Chia Wen Liu,1,2 Wen Jen Lin11Graduate Institute of Pharmaceutical Sciences, College of Medicine, National Taiwan University, Taipei; 2Drug Delivery Department, Biomedical Engineering Research Laboratories, Industrial Technology Research Institute, Hsinchu, TaiwanBackground: This study was performed to develop a functional poly(D,L-lactide-co-glycolide-poly(ethylene glycol (PLGA-PEG-bearing amino-active end group for peptide conjugation.Methods and results: PLGA was preactivated following by copolymerization with PEG diamine. The resulting amphiphilic PLGA-PEG copolymer bearing 97.0% of amino end groups had a critical micelle concentration of 3.0 × 10-8 mol/L, and the half-effective inhibition concentration (IC50 of the prepared PLGA-PEG nanoparticles was >100 mg/mL, which was much higher than that of PLGA nanoparticles (1.02 ± 0.37 mg/mL. The amphiphilic properties of PLGA-PEG spontaneously formed a core-shell conformation in the aqueous environment, and this special feature provided the amino group on the PEG chain scattered on the surface of PLGA-PEG nanoparticles for efficient peptide conjugation. The peptide-conjugated PLGA-PEG nanoparticles showed three-fold higher uptake than peptide-free PLGA-PEG nanoparticles in a SKOV3 cell line with high expression of epidermal growth factor receptor. Both peptide-conjugated and peptide-free PLGA-PEG nanoparticles were used as nanocarriers for delivery of doxorubicin. Although the rate of release of doxorubicin from both nanoparticles was similar, drug release at pH 4.0 (500 U lipase was faster than at pH 7.4. The IC50 of doxorubicin-loaded peptide-conjugated PLGA-PEG nanoparticles in SKOV3 cells (0.05 ± 0.03 µg/mL was much lower (by 62.4-fold than that of peptide-free PLGA-PEG nanoparticles (3.12 ± 1.44 µg/mL.Conclusion: This in vivo biodistribution study in SKOV3 tumor-bearing mice was further promising in that accumulation of doxorubicin in tumor tissue was in the order of peptide

  1. The activation of peroxisome proliferator-activated receptor γ is regulated by Krüppel-like transcription factors 6 & 9 under steatotic conditions

    Escalona-Nandez, Ivonne; Guerrero-Escalera, Dafne; Estanes-Hernández, Alma [Departamento de Gastroenterología, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Vasco de Quiroga 15 Sección XVI, Tlalpan, 14000, México, D.F. (Mexico); Ortíz-Ortega, Victor; Tovar, Armando R. [Departamento de Fisiología de la Nutrición, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Vasco de Quiroga 15 Sección XVI, Tlalpan, 14000, México, D.F. (Mexico); Pérez-Monter, Carlos, E-mail: carlos.perezm@incmnsz.mx [Departamento de Gastroenterología, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Vasco de Quiroga 15 Sección XVI, Tlalpan, 14000, México, D.F. (Mexico)

    2015-03-20

    Liver steatosis is characterised by lipid droplet deposition in hepatocytes that can leads to an inflammatory and fibrotic phenotype. Peroxisome proliferator-activated receptors (PPARs) play key roles in energetic homeostasis by regulating lipid metabolism in hepatic tissue. In adipose tissue PPARγ regulates the adipocyte differentiation by promoting the expression of lipid-associated genes. Within the liver PPARγ is up-regulated under steatotic conditions; however, which transcription factors participate in its expression is not completely understood. Krüppel-like transcription factors (KLFs) regulate various cellular mechanisms, such as cell proliferation and differentiation. KLFs are key components of adipogenesis by regulating the expression of PPARγ and other proteins such as the C-terminal enhancer binding protein (C/EBP). Here, we demonstrate that the transcript levels of Klf6, Klf9 and Pparγ are increased in response to a steatotic insult in vitro. Chromatin immunoprecipitation (ChIp) experiments showed that klf6 and klf9 are actively recruited to the Pparγ promoter region under these conditions. Accordingly, the loss-of-function experiments reduced cytoplasmic triglyceride accumulation. Here, we demonstrated that KLF6 and KLF9 proteins directly regulate PPARγ expression under steatotic conditions. - Highlights: • Palmitic acid promotes expression of KlF6 & KLF9 in HepG2 cells. • KLF6 and KLF9 promote the expression of PPARγ in response to palmitic acid. • Binding of KLF6 and KLF9 to the PPARγ promoter promotes steatosis in HepG2 cells. • KLF6 and KLF9 loss-of function diminishes the steatosis in HepG2 cells.

  2. The activation of peroxisome proliferator-activated receptor γ is regulated by Krüppel-like transcription factors 6 & 9 under steatotic conditions

    Liver steatosis is characterised by lipid droplet deposition in hepatocytes that can leads to an inflammatory and fibrotic phenotype. Peroxisome proliferator-activated receptors (PPARs) play key roles in energetic homeostasis by regulating lipid metabolism in hepatic tissue. In adipose tissue PPARγ regulates the adipocyte differentiation by promoting the expression of lipid-associated genes. Within the liver PPARγ is up-regulated under steatotic conditions; however, which transcription factors participate in its expression is not completely understood. Krüppel-like transcription factors (KLFs) regulate various cellular mechanisms, such as cell proliferation and differentiation. KLFs are key components of adipogenesis by regulating the expression of PPARγ and other proteins such as the C-terminal enhancer binding protein (C/EBP). Here, we demonstrate that the transcript levels of Klf6, Klf9 and Pparγ are increased in response to a steatotic insult in vitro. Chromatin immunoprecipitation (ChIp) experiments showed that klf6 and klf9 are actively recruited to the Pparγ promoter region under these conditions. Accordingly, the loss-of-function experiments reduced cytoplasmic triglyceride accumulation. Here, we demonstrated that KLF6 and KLF9 proteins directly regulate PPARγ expression under steatotic conditions. - Highlights: • Palmitic acid promotes expression of KlF6 & KLF9 in HepG2 cells. • KLF6 and KLF9 promote the expression of PPARγ in response to palmitic acid. • Binding of KLF6 and KLF9 to the PPARγ promoter promotes steatosis in HepG2 cells. • KLF6 and KLF9 loss-of function diminishes the steatosis in HepG2 cells

  3. Identification of Heparin-Binding EGF-Like Growth Factor (HB-EGF) as a Biomarker for Lysophosphatidic Acid Receptor Type 1 (LPA1) Activation in Human Breast and Prostate Cancers

    David, Marion; Sahay, Debashish; Mege, Florence; Descotes, Françoise; Leblanc, Raphaël; Ribeiro, Johnny; Clézardin, Philippe; Peyruchaud, Olivier

    2014-01-01

    Lysophosphatidic acid (LPA) is a natural bioactive lipid with growth factor-like functions due to activation of a series of six G protein-coupled receptors (LPA1–6). LPA receptor type 1 (LPA1) signaling influences the pathophysiology of many diseases including cancer, obesity, rheumatoid arthritis, as well as lung, liver and kidney fibrosis. Therefore, LPA1 is an attractive therapeutic target. However, most mammalian cells co-express multiple LPA receptors whose co-activation impairs the vali...

  4. ERK activation causes epilepsy by stimulating NMDA receptor activity

    Nateri, Abdolrahman S.; Raivich, Gennadij; Gebhardt, Christine; Da Costa, Clive; Naumann, Heike; Vreugdenhil, Martin; Makwana, Milan; Brandner, Sebastian; Adams, Ralf H.; Jefferys, John G. R.; Kann, Oliver; Behrens, Axel

    2007-01-01

    The ERK MAPK signalling pathway is a highly conserved kinase cascade linking transmembrane receptors to downstream effector mechanisms. To investigate the function of ERK in neurons, a constitutively active form of MEK1 (caMEK1) was conditionally expressed in the murine brain, which resulted in ERK activation and caused spontaneous epileptic seizures. ERK activation stimulated phosphorylation of eukaryotic translation initiation factor 4E (eIF4E) and augmented NMDA receptor 2B (NR2B) protein ...

  5. Activation of Human Toll-like Receptor 4 (TLR4)·Myeloid Differentiation Factor 2 (MD-2) by Hypoacylated Lipopolysaccharide from a Clinical Isolate of Burkholderia cenocepacia.

    Di Lorenzo, Flaviana; Kubik, Łukasz; Oblak, Alja; Lorè, Nicola Ivan; Cigana, Cristina; Lanzetta, Rosa; Parrilli, Michelangelo; Hamad, Mohamad A; De Soyza, Anthony; Silipo, Alba; Jerala, Roman; Bragonzi, Alessandra; Valvano, Miguel A; Martín-Santamaría, Sonsoles; Molinaro, Antonio

    2015-08-28

    Lung infection by Burkholderia species, in particular Burkholderia cenocepacia, accelerates tissue damage and increases post-lung transplant mortality in cystic fibrosis patients. Host-microbe interplay largely depends on interactions between pathogen-specific molecules and innate immune receptors such as Toll-like receptor 4 (TLR4), which recognizes the lipid A moiety of the bacterial lipopolysaccharide (LPS). The human TLR4·myeloid differentiation factor 2 (MD-2) LPS receptor complex is strongly activated by hexa-acylated lipid A and poorly activated by underacylated lipid A. Here, we report that B. cenocepacia LPS strongly activates human TLR4·MD-2 despite its lipid A having only five acyl chains. Furthermore, we show that aminoarabinose residues in lipid A contribute to TLR4-lipid A interactions, and experiments in a mouse model of LPS-induced endotoxic shock confirmed the proinflammatory potential of B. cenocepacia penta-acylated lipid A. Molecular modeling combined with mutagenesis of TLR4-MD-2 interactive surfaces suggests that longer acyl chains and the aminoarabinose residues in the B. cenocepacia lipid A allow exposure of the fifth acyl chain on the surface of MD-2 enabling interactions with TLR4 and its dimerization. Our results provide a molecular model for activation of the human TLR4·MD-2 complex by penta-acylated lipid A explaining the ability of hypoacylated B. cenocepacia LPS to promote proinflammatory responses associated with the severe pathogenicity of this opportunistic bacterium. PMID:26160169

  6. Effects of Somatic Mutations in the C-Terminus of Insulin-Like Growth Factor 1 Receptor on Activity and Signaling

    Barbara P. Craddock

    2012-01-01

    Full Text Available The insulin-like growth factor I receptor (IGF1R is overexpressed in several forms of human cancer, and it has emerged as an important target for anticancer drug design. Cancer genome sequencing efforts have recently identified three somatic mutations in IGF1R: A1374V, a deletion of S1278 in the C-terminal tail region of the receptor, and M1255I in the C-terminal lobe of the kinase catalytic domain. The possible effects of these mutations on IGF1R activity and biological function have not previously been tested. Here, we tested the effects of the mutations on the in vitro biochemical activity of IGF1R and on major IGF1R signaling pathways in mammalian cells. While the mutations do not affect the intrinsic tyrosine kinase activity of the receptor, we demonstrate that the basal (unstimulated levels of MAP kinase and Akt activation are increased in the mutants (relative to wild-type IGF1R. We hypothesize that the enhanced signaling potential of these mutants is due to changes in protein-protein interactions between the IGF1R C-terminus and cellular substrates or modulators.

  7. EGCG inhibits activation of the insulin-like growth factor-1 receptor in human colon cancer cells

    The IGF/IGF-1R system, which includes the IGF, IGF-1R, and IGFBPs proteins, plays an important role in the development and growth of colorectal cancer. We previously reported that in the HT29 human colon cancer cell line EGCG, the major biologically active component of green tea, inhibits activation of the RTKs EGFR, HER2, and HER3, and that this is associated with inhibition of multiple downstream signaling pathways. Since IGF-1R is also a RTK, in this study we examined the effects of EGCG on the activity of IGF/IGF-1R system in human colon cancer cells. We found that the colon cancer cell lines Caco2, HT29, SW837, and SW480 express high levels of the IGF-1R receptor, and that both SW837 and SW480 cells display constitutive activation of this receptor. Treatment of SW837 cells with 20 μg/ml of EGCG (the IC50 concentration for growth inhibition) caused within 6 h a decrease in the phosphorylated (i.e., activated) form of the IGF-1R protein. At 12 h, there was a decrease in the levels of both IGF-1 protein and mRNA and within 3-6 h there was an increase in the levels of both IGFBP-3 protein and mRNA. The increased expression of the latter protein was sustained for at least 48 h. When SW837 cells were treated with EGCG for a longer time, i.e., 96 h, a very low concentration (1.0 μg/ml) of EGCG also caused inhibition of activation of IGF-1R, a decrease in the IGF-1 protein, and an increase in the IGFBP-3 protein. EGCG also caused a decrease in the levels of mRNAs that encode MMPs-7 and -9, proteins that proteolyze IGFBP-3. In addition, treatment with EGCG caused a transient increase in the expression of TGF-β2, an inducer of IGFBP-3 expression. These findings expand the roles of EGCG as an inhibitor of critical RTKs involved in cell proliferation, providing further evidence that EGCG and related compounds may be useful in the chemoprevention or treatment of colorectal cancer

  8. Urokinase-type Plasminogen Activator-like Proteases in Teleosts Lack Genuine Receptor-binding Epidermal Growth Factor-like Domains*

    Bager, René; Kristensen, Thomas K.; Jensen, Jan K.; Szczur, Agnieszka; Christensen, Anni; Andersen, Lisbeth M.; Johansen, Jesper S.; Larsen, Niels; Baatrup, Erik; Huang, Mingdong; Ploug, Michael; Andreasen, Peter A.

    2012-01-01

    Plasminogen activation catalyzed by urokinase-type plasminogen activator (uPA) plays an important role in normal and pathological tissue remodeling processes. Since its discovery in the mid-1980s, the cell membrane-anchored urokinase-type plasminogen activator receptor (uPAR) has been believed to be central to the functions of uPA, as uPA-catalyzed plasminogen activation activity appeared to be confined to cell surfaces through the binding of uPA to uPAR. However, a functional uPAR has so far only been identified in mammals. We have now cloned, recombinantly produced, and characterized two zebrafish proteases, zfuPA-a and zfuPA-b, which by several criteria are the fish orthologs of mammalian uPA. Thus, both proteases catalyze the activation of fish plasminogen efficiently and both proteases are inhibited rapidly by plasminogen activator inhibitor-1 (PAI-1). But zfuPA-a differs from mammalian uPA by lacking the exon encoding the uPAR-binding epidermal growth factor-like domain; zfuPA-b differs from mammalian uPA by lacking two cysteines of the epidermal growth factor-like domain and a uPAR-binding sequence comparable with that found in mammalian uPA. Accordingly, no zfuPA-b binding activity could be found in fish white blood cells or fish cell lines. We therefore propose that the current consensus of uPA-catalyzed plasminogen activation taking place on cell surfaces, derived from observations with mammals, is too narrow. Fish uPAs appear incapable of receptor binding in the manner known from mammals and uPA-catalyzed plasminogen activation in fish may occur mainly in solution. Studies with nonmammalian vertebrate species are needed to obtain a comprehensive understanding of the mechanism of plasminogen activation. PMID:22733817

  9. Platelet-activating factor in Iberian pig spermatozoa: receptor expression and role as enhancer of the calcium-induced acrosome reaction.

    Bragado, M J; Gil, M C; Garcia-Marin, L J

    2011-12-01

    Platelet-activating factor (PAF) is a phospholipid involved in reproductive physiology. PAF receptor is expressed in some mammalian spermatozoa species where it plays a role in these germ-cell-specific processes. The aim of this study is to identify PAF receptor in Iberian pig spermatozoa and to evaluate PAF's effects on motility, viability and acrosome reaction. Semen samples from Iberian boars were used. PAF receptor identification was performed by Western blotting. Spermatozoa motility was analysed by computer-assisted sperm analysis system, whereas spermatozoa viability and acrosome reaction were evaluated by flow cytometry. Different PAF concentrations added to non-capacitating medium during 60 min have no effect on any spermatozoa motility parameter measured. Acrosome reaction was rapid and potently induced by 1 μm calcium ionophore A23187 showing an effect at 60 min and maximum at 240 min. PAF added to a capacitating medium is not able to induce spermatozoa acrosome reaction at any time studied. However, PAF, in the presence of A23187, significantly accelerates and enhances the calcium-induced acrosome reaction in a concentration-dependent manner in Iberian boar spermatozoa. Exogenous PAF does not affect at all spermatozoa viability, whereas slightly exacerbated the A23187-induced loss in viability. This work demonstrates that PAF receptor is expressed in Iberian pig spermatozoa and that its stimulation by PAF regulates the calcium-induced acrosome reaction. This work contributes to further elucidate the physiological regulation of the most relevant spermatozoa functions for successful fertilization: acrosome reaction. PMID:22023717

  10. Circulating levels of osteopontin, osteoprotegerin, total soluble receptor activator of nuclear factor-kappa B ligand, and high-sensitivity C-reactive protein in patients with active rheumatoid arthritis randomized to etanercept alone or in combination with methotrexate

    Sennels, H.; Sørensen, Steen; Østergaard, Mikkel;

    2008-01-01

    OBJECTIVE: To determine whether circulating levels of osteopontin (OPN), osteoprotegerin (OPG), total soluble receptor activator of nuclear factor-kappa B ligand (total sRANKL), and high-sensitivity C-reactive protein (hsCRP) change in patients with rheumatoid arthritis (RA) during...

  11. Hypoxia-inducible factor-1α increased the expression of peroxisome proliferator activated receptor α in lung cancer cell A549

    张惠兰; 张珍祥; 徐永健

    2004-01-01

    @@ Hypoxia plays a fundamental role in many pathologic processes. Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric basic helix-loop-helix-per-aryl hydrocarbon receptor ARNT-sim (PAS) domain protein, consisting of α and β subunits and is precisely regulated by cellular oxygen levels.1 The peroxisome proliferator-activated receptors (PPARs) are family nuclear hormone-binding proteins with increasing diverse functions as transcriptional regulators, owning three subtypes (α, β, and γ).2 PPARα plays a critical physiological role as lipid sensors and regulators of proliferation.3 Hypoxia can elicit up-regulation of PPAR-α expression.4 Herein, we report the results of an investigation on the correlation of HIF-1α and PPARα.

  12. ERas protein is overexpressed and binds to the activated platelet-derived growth factor β receptor in bovine urothelial tumour cells associated with papillomavirus infection.

    Russo, Valeria; Roperto, Franco; Esposito, Iolanda; Ceccarelli, Dora Maria; Zizzo, Nicola; Leonardi, Leonardo; Capparelli, Rosanna; Borzacchiello, Giuseppe; Roperto, Sante

    2016-06-01

    Embryonic stem cell-expressed Ras (ERas) encodes a constitutively active form of guanosine triphosphatase (GTPase) that binds to and activates phosphatidylinositol 3 kinase (PI3K), which in turn phosphorylates and activates downstream targets such as Akt. The current study evaluated ERas regulation and expression in papillomavirus-associated urothelial tumours in cattle grazing on lands rich in bracken fern. ERas was found upregulated and overexpressed by PCR, real time PCR and Western blot. Furthermore, protein overexpression was also confirmed by immunohistochemistry. ERas was found to interact physically and colocalise with the activated platelet derived growth factor β receptor (PDGFβR) by coimmunoprecipitation and laser scanning confocal investigations. Phosphorylation of Akt, a downstream effector both of ERas and PDGFβR, appeared to be increased in urothelial tumour cells. Altogether, these data indicate that ERas/PDGFβR complex could play a role in the pathogenesis of bovine papillomavirus-associated bladder neoplasia. PMID:27256024

  13. Effect of The Receptor Activator of Nuclear Factor кB and RANK Ligand on In Vitro Differentiation of Cord Blood CD133+ Hematopoietic Stem Cells to Osteoclasts

    Kalantari, Nasim; Abroun, Saeid; Soleimani, Masoud; Kaviani, Saeid; Azad, Mehdi; Eskandari, Fatemeh; Habibi, Hossein

    2016-01-01

    Objective Receptor activator of nuclear factor-kappa B ligand (RANKL) appears to be an osteoclast-activating factor, bearing an important role in the pathogenesis of multiple myeloma. Some studies demonstrated that U-266 myeloma cell line and primary myeloma cells expressed RANK and RANKL. It had been reported that the expression of myeloid and monocytoid markers was increased by co-culturing myeloma cells with hematopoietic stem cells (HSCs). This study also attempted to show the molecular mechanism of RANK and RANKL on differentiation capability of human cord blood HSC to osteoclast, as well as expression of calcitonin receptor (CTR) on cord blood HSC surface. Materials and Methods In this experimental study, CD133+ hematopoietic stem cells were isolated from umbilical cord blood and cultured in the presence of macrophage colony-stimulating factor (M-CSF) and RANKL. Osteoclast differentiation was characterized by using tartrate-resistant acid phosphatase (TRAP) staining, giemsa staining, immunophenotyping, and reverse transcription-polymerase chain reaction (RT-PCR) assay for specific genes. Results Hematopoietic stem cells expressed RANK before and after differentiation into osteoclast. Compared to control group, flow cytometric results showed an increased expression of RANK after differentiation. Expression of CTR mRNA showed TRAP reaction was positive in some differentiated cells, including osteoclast cells. Conclusion Presence of RANKL and M-CSF in bone marrow could induce HSCs differentiation into osteoclast. PMID:27602313

  14. UDP acts as a growth factor for vascular smooth muscle cells by activation of P2Y(6) receptors

    Hou, Mingyan; Harden, T Kendall; Kuhn, Cynthia M; Baldetorp, Bo; Lazarowski, Eduardo; Pendergast, William; Möller, Sebastian; Edvinsson, Lars; Erlinge, David

    2002-01-01

    Mitogenic effects of the extracellular nucleotides ATP and UTP are mediated by P2Y(1), P2Y(2), and P2Y(4) receptors. However, it has not been possible to examine the highly expressed UDP-sensitive P2Y(6) receptor because of the lack of stable, selective agonists. In rat aorta smooth muscle cells ...

  15. Dexamethasone effects on creatine kinase activity and insulin-like growth factor receptors in cultured muscle cells

    Whitson, Peggy A.; Stuart, Charles A.; Huls, M. H.; Sams, Clarence F.; Cintron, Nitza M.

    1989-01-01

    The effect of dexamethasone on the activity of creatine kinase (CK) and the insulin-like growth factor I (IGF-I) binding were investigated using skeletal- and cardiac-muscle-derived cultured cell lines (mouse, C2C12; rat, L6 and H9c2). It was found that, in skeletal muscle cells, dexamethasone treatment during differentiation of skeletal-muscle cells caused dose-dependent increases in CK activity and increases in the degree of myotube formation, whereas cardiac cells (H9c2) exhibited very low CK activity during culture or dexamethasone treatment. Results for IGF-I binding were similar in all three cell lines. The IGF-I binding to dexamethasone-treated cells (50 nM for 24 hr on the day prior to confluence) resulted in an increased number of available binding sites, with no effect on the binding affinities.

  16. Solubilization of high affinity corticotropin-releasing factor receptors from rat brain: Characterization of an active digitonin-solubilized receptor complex

    The binding characteristics of CRF receptors in rat frontal cerebral cortex membranes solubilized in 1% digitonin were determined. The binding of [125I]Tyro-ovine CRF ([125I]oCRF) to solubilized membrane proteins was dependent on incubation time, temperature, and protein concentration, was saturable and of high affinity, and was absent in boiled tissue. The solubilized receptors retained their high affinity for [125I] oCRF in the solubilized state, exhibiting a dissociation constant (KD) of approximately 200 pM, as determined by direct binding saturation isotherms. Solubilized CRF receptors maintained the rank order of potencies for various related and unrelated CRF peptides characteristic of the membrane CRF receptor: rat/human CRF congruent to ovine CRF congruent to Nle21,38-rat CRF greater than alpha-helical oCRF-(9-41) greater than oCRF-(7-41) much greater than vasoactive intestinal peptide, arginine vasopressin, or the substance-P antagonist. Furthermore, the absolute potencies (Ki values) for the various CRF-related peptides in solubilized receptors were almost identical to those observed in the membrane preparations, indicating that the CRF receptor retained its high affinity binding capacity in the digitonin-solubilized state. Chemical affinity cross-linking of digitonin-solubilized rat cortical membrane proteins revealed a specifically labeled protein with an apparent mol wt of 58,000 which was similar to the labeled protein in native membrane homogenates. Although solubilized CRF receptors retained their high affinity for agonists, their sensitivity for guanine nucleotide was lost. Size exclusion chromatography substantiated these results, demonstrating that in the presence or absence of guanine nucleotides, [125I]oCRF labeled the same size receptor complex

  17. Corticotropin-releasing factor receptor 1 activation during exposure to novelty stress protects against Alzheimer's disease-like cognitive decline in AβPP/PS1 mice.

    Scullion, Gillian A; Hewitt, Katherine N; Pardon, Marie-Christine

    2013-01-01

    A lifestyle rich in physical and mental activities protects against Alzheimer's disease (AD) but the underlying mechanisms are unclear. We have proposed that this is mediated by a stress response and have shown that repeated exposure to novelty stress, which induces physical and exploratory activities, delays the progression of AD-like pathology in the TASTPM mouse model. Here, we aimed to establish the role played by corticotrophin-releasing factor receptor 1 (CRFR1), a major component of the stress axis, in TASTPM's behavioral and neuroendocrine responses to novelty and related protective effects. We show that the stress response of TASTPM mice is altered with reduced CRFR1-mediated neuroendocrine and behavioral responses to novelty and a distinct profile of behavioral responses. Repeated novelty-induced CRFR1 activation, however, mediated the improved contextual fear memory and extinction performance of TASTPM mice and increased hippocampal and fronto-cortical levels of synaptophysin, a marker of synaptic density, and fronto-cortical levels of the post-synaptic marker PSD95. The N-methyl-D-aspartate receptor (NMDAR) is the major receptor for synaptic plasticity underlying learning and memory. Although novelty-induced NMDAR activation contributed to enhancement of fear memory and synaptophysin levels, antagonism of CRFR1 and NMDAR prevented the novelty-induced increase in hippocampal synaptophysin levels but reversed the other effects of CRFR1 inactivation, i.e., the enhancement of contextual fear extinction and fronto-cortical synaptophysin and PSD95 levels. These findings suggest a novel mechanism whereby a stimulating environment can delay AD symptoms through CRFR1 activation, facilitating NMDAR-mediated synaptic plasticity and synaptogenesis in a region-dependent manner, either directly, or indirectly, by modulating PSD95. PMID:23302658

  18. Screening of human vascular endothelial growth factor (VEGF)receptor Flt-1 domain and study on its biological activity

    马骊; 张智清; 王小宁; 孙大军; 周小明; 陈爱君; 姚立红

    2001-01-01

    Four human vascular endothelial growth factor receptor Flt-1 cDNA fragments containing extracellular domain loops 2,1-2,2-3 and 1-3 respectively were amplified from human placental cDNA library by PCR and used for screening ligand binding domains by yeast two-hybrid system.The result showed that,not only loop 1-3,but also the smaller fragment loop 2-3 could bind bo hVEGF165.Recombinant expression plasmids pPIC9K/Flt-1(1-3)and pPIC9K/Flt-1(2-3)were constructed and transformed to Pichia.pastoris host strain GS115,cultured in flasks,and expressed under the induction of 1% methanol.The expressed product existed in supernatant in the form of soluble molecules and contained more than 60% of total protein after being induced for 4d.After being purified by CM-Sepharose FF and Sephacryl S-100 chromatography,its purity reached above 90%.Biological assay in vitro showed that the binding capacity of expressed soluble Flt-1(2-3)to hVEGF165 and its nhibiting effect on the proliferation of human umbilical reins endothelial cells(HUVEC)stimulated with hVEGF165 were close to those of s Flt-1(1-3).Animal test showed that sFlt-1(2-3)could nhibit the formation of regenerate blood vessels stimulated with hVEGF165 significantly.

  19. Transcriptional Regulation of Urokinase-type Plasminogen Activator Receptor by Hypoxia-Inducible Factor 1 Is Crucial for Invasion of Pancreatic and Liver Cancer

    Peter Büchler

    2009-02-01

    Full Text Available Angioinvasion is critical for metastasis with urokinase-type plasminogen activator receptor (uPAR and tumor hypoxia-activated hypoxia-inducible factor 1 (HIF-1 as key players. Transcriptional control of uPAR expression by HIF has never been reported. The aim of the present study, therefore, was to test whether tumor hypoxia-induced HIF expression may be linked to transcriptional activation of uPAR and dependent angioinvasion. We used human pancreatic cancer cells and a model of parental and derived HIF-1β-deficient mouse liver cancer cell lines and performed Northern blot analysis, nuclear runoff assays, electrophoretic mobility shift assay, polymerase chain reaction-generated deletion mutants, luciferase assays, Matrigel invasion assays, and in vivo angioinvasion assays in the chorioallantoic membrane of fertilized chicken eggs. Urokinase-type plasminogen activator receptor promoter analysis resulted in four putative HIF binding sites. Hypoxia strongly induced de novo transcription of uPAR mRNA. With sequential deletion mutants of the uPAR promoter, it was possible to identify one HIF binding site causing a nearly 200-fold increase in luciferase activity. Hypoxia enhanced the number of invading tumor cells in vitro and in vivo. In contrast, HIF-1β-deficient cells failed to upregulate uPAR expression, to activate luciferase activity, and to invade on hypoxia. Taken together, we show for the first time that uPAR is under transcriptional control of HIF and that this is important for hypoxia-induced metastasis.

  20. Mechanism of FGF receptor dimerization and activation

    Sarabipour, Sarvenaz; Hristova, Kalina

    2016-01-01

    Fibroblast growth factors (fgfs) are widely believed to activate their receptors by mediating receptor dimerization. Here we show, however, that the FGF receptors form dimers in the absence of ligand, and that these unliganded dimers are phosphorylated. We further show that ligand binding triggers structural changes in the FGFR dimers, which increase FGFR phosphorylation. The observed effects due to the ligands fgf1 and fgf2 are very different. The fgf2-bound dimer structure ensures the smallest separation between the transmembrane (TM) domains and the highest possible phosphorylation, a conclusion that is supported by a strong correlation between TM helix separation in the dimer and kinase phosphorylation. The pathogenic A391E mutation in FGFR3 TM domain emulates the action of fgf2, trapping the FGFR3 dimer in its most active state. This study establishes the existence of multiple active ligand-bound states, and uncovers a novel molecular mechanism through which FGFR-linked pathologies can arise.

  1. Characterization of osteoprotegerin binding to glycosaminoglycans by surface plasmon resonance: Role in the interactions with receptor activator of nuclear factor κB ligand (RANKL) and RANK

    Osteoprotegerin (OPG) is a decoy receptor for receptor activator of nuclear factor κB ligand (RANKL), a key inducer of osteoclastogenesis via its receptor RANK. We previously showed that RANK, RANKL, and OPG are able to form a tertiary complex and that OPG must be also considered as a direct effector of osteoclast functions. As OPG contains a heparin-binding domain, the present study investigated the interactions between OPG and glycosaminoglycans (GAGs) by surface plasmon resonance and their involvement in the OPG functions. Kinetic data demonstrated that OPG binds to heparin with a high-affinity (K D: 0.28 nM) and that the pre-incubation of OPG with heparin inhibits in a dose-dependent manner the OPG binding to the complex RANK-RANKL. GAGs from different structure/origin (heparan sulfate, dermatan sulfate, and chondroitin sulfate) exert similar activity on OPG binding. The contribution of the sulfation pattern and the size of the oligosaccharide were determined in this inhibitory mechanism. The results demonstrated that sulfation is essential in the OPG-blocking function of GAGs since a totally desulfated heparin loses its capacity to bind and to block OPG binding to RANKL. Moreover, a decasaccharide is the minimal structure that totally inhibits the OPG binding to the complex RANK-RANKL. Western blot analysis performed in 293 cells surexpressing RANKL revealed that the pre-incubation of OPG with these GAGs strongly inhibits the OPG-induced decrease of membrane RANKL half-life. These data support an essential function of the related glycosaminoglycans heparin and heparan sulfate in the activity of the triad RANK-RANKL-OPG

  2. Response to platelet-activating factor in human platelets stored and aged in plasma. Decrease in aggregation, phosphoinositide turnover, and receptor affinity

    Human platelet concentrates were stored in polyolefin bags at 22 to 24 degrees C on a horizontal shaker for up to 8 days. At different intervals, aliquots of platelet-rich plasma (PRP) were removed aseptically and five variables, i.e., platelet counts, morphology, platelet-activating factor (PAF)-stimulated aggregation, phosphoinositide turnover, and [3H]PAF binding to platelet receptors, were studied. The number of platelets did not change during the 8 days of storage. Scanning electron microscopy of the platelets revealed a gradual morphologic change from biconcave flat discs to irregular, crenated forms. The PAF-induced aggregation of platelets declined with time of storage. A decrease to 50 percent of the Day 1 aggregatory response to PAF was evident on Day 2, and there was a further decline to about 20 percent by Day 6. Similarly, PAF receptor-coupled phosphoinositide turnover, as monitored by 32P incorporation into individual phosphoinositides, decreased dramatically with storage. After 2 to 3 days of storage, the phosphoinositide turnover was reduced to 50 percent of the original response, and it continued to decline to about 25 percent of original response by Day 5 or 6. The binding of [3H]PAF to washed human platelets indicated subtle changes between Days 2 and 4, which became more noticeable by Day 6. These results have raised the possibility of changes in the number of the receptors and/or their affinity for the ligand during storage. We conclude that although the number of platelets was maintained during storage for 8 days, a general deterioration of their responses to PAF occurred at the levels of cell surface receptor, transmembrane signaling (phosphoinositide turnover), and response (aggregation)

  3. Human circulating monocytes can express receptor activator of nuclear factor-kappaB ligand and differentiate into functional osteoclasts without exogenous stimulation.

    Seta, Noriyuki; Okazaki, Yuka; Kuwana, Masataka

    2008-07-01

    Osteoclast formation from mononuclear precursors is believed to require accessory cells expressing receptor activator of nuclear factor-kappaB ligand (RANKL). We recently identified a human cell population originated from circulating CD14(+) monocytes, called monocyte-derived multipotential cells (MOMCs), which can differentiate into several distinct mesenchymal cells, neuron and endothelial cells. This study was undertaken to examine whether MOMCs can differentiate into functional osteoclasts. MOMCs prepared from peripheral blood of healthy volunteers cultured on fibronectin for 7 days at high density (8 x 10(5) cells cm(-2)), but not at regular density (2 x 10(4) cells cm(-2)), resulted in the appearance of tartrate-resistant acid phosphatase-positive giant multi-nucleated cells forming actin ring without exogenous osteoclastogenic factors. A subset of these cells showed bone resorption capacity on dentine slices and expression of genes for cathepsin K and calcitonin receptor, characteristic of functional osteoclasts. Such osteoclast differentiation was not observed in high-density culture of circulating monocytes, macrophages or dendritic cells, or the high-density culture of MOMCs on type I collagen. Among cells of the monocyte lineage, untreated MOMCs exclusively showed gene and protein expression of RANKL. When osteoprotegerin/IgG1 Fc chimera was added to high-density MOMC cultures, osteoclast formation was completely inhibited by neutralizing the endogenous RANKL. These results indicate that human MOMCs derived from circulating monocytes can express RANKL and differentiate into functional osteoclasts without RANKL-expressing accessory cells. PMID:18301383

  4. Spongian diterpenoids inhibit androgen receptor activity

    Yang, Yu Chi; Labros G Meimetis; Tien, Amy H; Mawji, Nasrin R.; Carr, Gavin; Wang, Jun; Andersen, Raymond J.; Sadar, Marianne D.

    2013-01-01

    Androgen receptor (AR) is a ligand-activated transcription factor and a validated drug target for all stages of prostate cancer. Antiandrogens compete with physiological ligands for AR ligand-binding domain (LBD). High-throughput screening of a marine natural product library for small molecules that inhibit AR transcriptional activity yielded the furanoditerpenoid spongia-13(16),-14-dien-19-oic acid, designated terpene 1 (T1). Characterization of T1 and the structurally related semi-synthetic...

  5. Hepatitis C Virus Activates a Neuregulin-Driven Circuit to Modify Surface Expression of Growth Factor Receptors of the ErbB Family.

    Sabine Stindt

    Full Text Available Recently, the epidermal growth factor (EGF receptor (EGFR, a member of the ErbB receptor family, and its down-stream signalling have been identified as co-factors for HCV entry and replication. Since EGFR also functions as a heterodimer with other ErbB receptor family members, the subject of the present study was to investigate a possible viral interference with these cellular components. By using genotype 1b replicon cells as well as an infection-based system we found that while transcript and protein levels of EGFR and ErbB2 were up-regulated or unaffected, respectively, HCV induced a substantial reduction of ErbB3 and ErbB4 expression. Down-regulation of ErbB3 expression by HCV involves specificity protein (Sp1-mediated induction of Neuregulin (NRG1 expression as well as activation of Akt. Consistently, at transcript level disruption of ErbB3 expression by HCV can be prevented by knockdown of NRG1 or Sp1 expression, whereas reconstitution of ErbB3 protein levels requires inhibition of HCV-induced NRG1 expression and of Akt activity. Interestingly, the NRG1-mediated suppression of ErbB3 expression by HCV results in an enhanced expression of EGFR and ErbB2 on the cell surface, which can be mimicked by siRNA-mediated knockdown of ErbB3 expression. These data delineate a novel mechanism enabling HCV to sway the composition of the ErbB family members on the surface of its host cell by an NRG1-driven circuit and unravels a yet unknown cross-regulation between ErbB3 and the two other family members ErbB2 and EGFR. The shift of the receptor surface expression of the ErbB family towards enhanced expression of ErbB2 and EGFR triggered by HCV was found to promote viral RNA replication and infectivity. This suggests that HCV rearranges expression of ErbB family members to adapt the cellular environment to its requirements.

  6. Squamosamide derivative FLZ protects retinal pigment epithelium cells from oxidative stress through activation of epidermal growth factor receptor (EGFR)-AKT signaling.

    Cheng, Li-Bo; Chen, Chun-Ming; Zhong, Hong; Zhu, Li-Juan

    2014-01-01

    Reactive oxygen species (ROS)-mediated retinal pigment epithelium (RPE) cell apoptosis is attributed to age-related macular degeneration (AMD) pathogenesis. FLZ, a novel synthetic squamosamide derivative from a Chinese herb, Annona glabra, has displayed significant cyto-protective activity. In the current study, we explored the pro-survival effect of FLZ in oxidative stressed-RPE cells and studied the underlying signaling mechanisms. Our results showed that FLZ attenuated hydrogen peroxide (H2O2)-induced viability decrease and apoptosis in the RPE cell line (ARPE-19 cells) and in primary mouse RPE cells. Western blotting results showed that FLZ activated AKT signaling in RPE cells. The AKT-specific inhibitor, MK-2206, the phosphoinositide 3-kinase (PI3K)/AKT pan inhibitor, wortmannin, and AKT1-shRNA (short hairpin RNA) depletion almost abolished FLZ-mediated pro-survival/anti-apoptosis activity. We discovered that epidermal growth factor receptor (EGFR) trans-activation mediated FLZ-induced AKT activation and the pro-survival effect in RPE cells, and the anti-apoptosis effect of FLZ against H2O2 was inhibited by the EGFR inhibitor, PD153035, or by EGFR shRNA-knockdown. In conclusion, FLZ protects RPE cells from oxidative stress through activation of EGFR-AKT signaling, and our results suggest that FLZ might have therapeutic values for AMD. PMID:25329617

  7. Insulin-like growth factor 1 receptor and p38 mitogen-activated protein kinase signals inversely regulate signal transducer and activator of transcription 3 activity to control human dental pulp stem cell quiescence, propagation, and differentiation.

    Vandomme, Jerome; Touil, Yasmine; Ostyn, Pauline; Olejnik, Cecile; Flamenco, Pilar; El Machhour, Raja; Segard, Pascaline; Masselot, Bernadette; Bailliez, Yves; Formstecher, Pierre; Polakowska, Renata

    2014-04-15

    Dental pulp stem cells (DPSCs) remain quiescent until activated in response to severe dental pulp damage. Once activated, they exit quiescence and enter regenerative odontogenesis, producing reparative dentin. The factors and signaling molecules that control the quiescence/activation and commitment to differentiation of human DPSCs are not known. In this study, we determined that the inhibition of insulin-like growth factor 1 receptor (IGF-1R) and p38 mitogen-activated protein kinase (p38 MAPK) signaling commonly activates DPSCs and promotes their exit from the G0 phase of the cell cycle as well as from the pyronin Y(low) stem cell compartment. The inhibition of these two pathways, however, inversely determines DPSC fate. In contrast to p38 MAPK inhibitors, IGF-1R inhibitors enhance dental pulp cell sphere-forming capacity and reduce the cells' colony-forming capacity without inducing cell death. The inverse cellular changes initiated by IGF-1R and p38 MAPK inhibitors were accompanied by inverse changes in the levels of active signal transducer and activator of transcription 3 (STAT3) factor, inactive glycogen synthase kinase 3, and matrix extracellular phosphoglycoprotein, a marker of early odontoblast differentiation. Our data suggest that there is cross talk between the IGF-1R and p38 MAPK signaling pathways in DPSCs and that the signals provided by these pathways converge at STAT3 and inversely regulate its activity to maintain quiescence or to promote self-renewal and differentiation of the cells. We propose a working model that explains the possible interactions between IGF-1R and p38 MAPK at the molecular level and describes the cellular consequences of these interactions. This model may inspire further fundamental study and stimulate research on the clinical applications of DPSC in cellular therapy and tissue regeneration. PMID:24266654

  8. Insulin-Like Growth Factor 1 Receptor and p38 Mitogen-Activated Protein Kinase Signals Inversely Regulate Signal Transducer and Activator of Transcription 3 Activity to Control Human Dental Pulp Stem Cell Quiescence, Propagation, and Differentiation

    Vandomme, Jerome; Touil, Yasmine; Ostyn, Pauline; Olejnik, Cecile; Flamenco, Pilar; El Machhour, Raja; Segard, Pascaline; Masselot, Bernadette; Bailliez, Yves; Formstecher, Pierre

    2014-01-01

    Dental pulp stem cells (DPSCs) remain quiescent until activated in response to severe dental pulp damage. Once activated, they exit quiescence and enter regenerative odontogenesis, producing reparative dentin. The factors and signaling molecules that control the quiescence/activation and commitment to differentiation of human DPSCs are not known. In this study, we determined that the inhibition of insulin-like growth factor 1 receptor (IGF-1R) and p38 mitogen-activated protein kinase (p38 MAPK) signaling commonly activates DPSCs and promotes their exit from the G0 phase of the cell cycle as well as from the pyronin Ylow stem cell compartment. The inhibition of these two pathways, however, inversely determines DPSC fate. In contrast to p38 MAPK inhibitors, IGF-1R inhibitors enhance dental pulp cell sphere-forming capacity and reduce the cells' colony-forming capacity without inducing cell death. The inverse cellular changes initiated by IGF-1R and p38 MAPK inhibitors were accompanied by inverse changes in the levels of active signal transducer and activator of transcription 3 (STAT3) factor, inactive glycogen synthase kinase 3, and matrix extracellular phosphoglycoprotein, a marker of early odontoblast differentiation. Our data suggest that there is cross talk between the IGF-1R and p38 MAPK signaling pathways in DPSCs and that the signals provided by these pathways converge at STAT3 and inversely regulate its activity to maintain quiescence or to promote self-renewal and differentiation of the cells. We propose a working model that explains the possible interactions between IGF-1R and p38 MAPK at the molecular level and describes the cellular consequences of these interactions. This model may inspire further fundamental study and stimulate research on the clinical applications of DPSC in cellular therapy and tissue regeneration. PMID:24266654

  9. The Effect of Mutations on Drug Sensitivity and Kinase Activity of Fibroblast Growth Factor Receptors: A Combined Experimental and Theoretical Study

    Tom D. Bunney

    2015-03-01

    Full Text Available Fibroblast growth factor receptors (FGFRs are recognized therapeutic targets in cancer. We here describe insights underpinning the impact of mutations on FGFR1 and FGFR3 kinase activity and drug efficacy, using a combination of computational calculations and experimental approaches including cellular studies, X-ray crystallography and biophysical and biochemical measurements. Our findings reveal that some of the tested compounds, in particular TKI258, could provide therapeutic opportunity not only for patients with primary alterations in FGFR but also for acquired resistance due to the gatekeeper mutation. The accuracy of the computational methodologies applied here shows a potential for their wider application in studies of drug binding and in assessments of functional and mechanistic impacts of mutations, thus assisting efforts in precision medicine.

  10. Biological variation and reference intervals for circulating osteopontin, osteoprotegerin, total soluble receptor activator of nuclear factor kappa B ligand and high-sensitivity C-reactive protein

    Sennels, Henriette Pia; Jacobsen, S; Jensen, T; Hansen, M S; Østergaard, Mikkel; Nielsen, Hans Jørgen; Sørensen, Steen

    2007-01-01

    Objective. Monitoring inflammatory diseases and osteoclastogenesis with osteopontin (OPN), osteoprotegerin (OPG), total soluble receptor activator of nuclear factor kappa B ligand (total sRANKL) and high-sensitivity C-reactive protein (hsCRP) has recently attracted increased interest. The purpose...... of our study was to determine reference intervals, variability caused by sampling time, biological variation and stability after repeated freeze-thaw cycles of circulating levels of OPN, OPG, total sRANKL and hsCRP. Material and methods. Plasma OPN and plasma OPG concentrations were determined with...... sandwich ELISA; serum total sRANKL concentration was determined using a two-site sandwich ELISA; and hsCRP was analysed by turbidimetry in 300 Danish blood donors (183 M and 117 F) with a median age of 43 years (range 18-64 years). Variability due to biological variation and sampling time was studied in...

  11. Type III Transforming Growth FactorReceptor Drives Cardiac Hypertrophy Through β-Arrestin2-Dependent Activation of Calmodulin-Dependent Protein Kinase II.

    Lou, Jie; Zhao, Dan; Zhang, Ling-Ling; Song, Shu-Ying; Li, Yan-Chao; Sun, Fei; Ding, Xiao-Qing; Yu, Chang-Jiang; Li, Yuan-Yuan; Liu, Mei-Tong; Dong, Chang-Jiang; Ji, Yong; Li, Hongliang; Chu, Wenfeng; Zhang, Zhi-Ren

    2016-09-01

    The role of type III transforming growth factorreceptor (TβRIII) in the pathogenesis of heart diseases remains largely unclear. Here, we investigated the functional role and molecular mechanisms of TβRIII in the development of myocardial hypertrophy. Western blot and quantitative real time-polymerase chain reaction analyses revealed that the expression of TβRIII was significantly elevated in human cardiac hypertrophic samples. Consistently, TβRIII expression was substantially increased in transverse aortic constriction (TAC)- and isoproterenol-induced mouse cardiac hypertrophy in vivo and in isoproterenol-induced cardiomyocyte hypertrophy in vitro. Overexpression of TβRIII resulted in cardiomyocyte hypertrophy, whereas isoproterenol-induced cardiomyocyte hypertrophy was greatly attenuated by knockdown of TβRIII in vitro. Cardiac-specific transgenic expression of TβRIII independently led to cardiac hypertrophy in mice, which was further aggravated by isoproterenol and TAC treatment. Cardiac contractile function of the mice was not altered in TβRIII transgenic mice; however, TAC led to significantly decreased cardiac contractile function in TβRIII transgenic mice compared with control mice. Conversely, isoproterenol- and TAC-induced cardiac hypertrophy and TAC-induced cardiac contractile function impairment were partially reversed by suppression of TβRIII in vivo. Our data suggest that TβRIII mediates stress-induced cardiac hypertrophy through activation of Ca(2+)/calmodulin-dependent protein kinase II, which requires a physical interaction of β-arrestin2 with both TβRIII and calmodulin-dependent protein kinase II. Our findings indicate that stress-induced increase in TβRIII expression results in cardiac hypertrophy through β-arrestin2-dependent activation of calmodulin-dependent protein kinase II and that transforming growth factor-β and β-adrenergic receptor signaling are not involved in spontaneous cardiac hypertrophy in cardiac

  12. Different TBP-associated factors are required for mediating the stimulation of transcription in vitro by the acidic transactivator GAL-VP16 and the two nonacidic activation functions of the estrogen receptor.

    Brou, C; J. Wu; Ali, S; Scheer, E; Lang, C.; Davidson, I; P. Chambon; Tora, L

    1993-01-01

    The estrogen receptor (ER) contains two nonacidic transcriptional activation functions, AF-1 and AF-2 (formerly TAF-1 and TAF-2). In this study we show that AF-1 and AF-2 are able to stimulate transcription in vitro in a HeLa cell system when fused to the DNA binding domain of the yeast activator GAL4. We also demonstrate that a factor(s) required for the function of the ER AFs is chromatographically separable from a factor(s) necessary for the activity of the acidic activation domain of VP16...

  13. The vascular endothelial growth factor receptor inhibitor sunitinib causes a preeclampsia-like syndrome with activation of the endothelin system

    Kappers, Mariëtte H W; Smedts, Frank M M; Horn, Thomas;

    2011-01-01

    of endothelin 1, decreased nitric oxide (NO) bioavailability, and increased oxidative stress in the development of sunitinib-induced hypertension and renal toxicity. In rats on sunitinib, light and electron microscopic examination revealed marked glomerular endotheliosis, a characteristic...... rise could largely be prevented with the endothelin receptor antagonist macitentan (ΔBP: 12.3±1.5 mm Hg) and only mildly with Tempol, a superoxide dismutase mimetic (ΔBP: 25.9±2.3 mm Hg). Both compounds could not prevent the sunitinib-induced rise in serum creatinine or renal histological abnormalities...

  14. The vascular endothelial growth factor receptor inhibitor sunitinib causes a preeclampsia-like syndrome with activation of the endothelin system

    Kappers, Mariëtte H W; Smedts, Frank M M; Horn, Thomas;

    2011-01-01

    of endothelin 1, decreased nitric oxide (NO) bioavailability, and increased oxidative stress in the development of sunitinib-induced hypertension and renal toxicity. In rats on sunitinib, light and electron microscopic examination revealed marked glomerular endotheliosis, a characteristic...... rise could largely be prevented with the endothelin receptor antagonist macitentan (¿BP: 12.3±1.5 mm Hg) and only mildly with Tempol, a superoxide dismutase mimetic (¿BP: 25.9±2.3 mm Hg). Both compounds could not prevent the sunitinib-induced rise in serum creatinine or renal histological abnormalities...

  15. Proteolytic activation of proapoptotic kinase protein kinase Cδ by tumor necrosis factor α death receptor signaling in dopaminergic neurons during neuroinflammation

    Gordon Richard

    2012-04-01

    Full Text Available Abstract Background The mechanisms of progressive dopaminergic neuronal loss in Parkinson’s disease (PD remain poorly understood, largely due to the complex etiology and multifactorial nature of disease pathogenesis. Several lines of evidence from human studies and experimental models over the last decade have identified neuroinflammation as a potential pathophysiological mechanism contributing to disease progression. Tumor necrosis factor α (TNF has recently emerged as the primary neuroinflammatory mediator that can elicit dopaminergic cell death in PD. However, the signaling pathways by which TNF mediates dopaminergic cell death have not been completely elucidated. Methods In this study we used a dopaminergic neuronal cell model and recombinant TNF to characterize intracellular signaling pathways activated during TNF-induced dopaminergic neurotoxicity. Etanercept and neutralizing antibodies to tumor necrosis factor receptor 1 (TNFR1 were used to block TNF signaling. We confirmed the results from our mechanistic studies in primary embryonic mesencephalic cultures and in vivo using the stereotaxic lipopolysaccharide (LPS model of nigral dopaminergic degeneration. Results TNF signaling in dopaminergic neuronal cells triggered the activation of protein kinase Cδ (PKCδ, an isoform of the novel PKC family, by caspase-3 and caspase-8 dependent proteolytic cleavage. Both TNFR1 neutralizing antibodies and the soluble TNF receptor Etanercept blocked TNF-induced PKCδ proteolytic activation. Proteolytic activation of PKCδ was accompanied by translocation of the kinase to the nucleus. Notably, inhibition of PKCδ signaling by small interfering (siRNA or overexpression of a PKCδ cleavage-resistant mutant protected against TNF-induced dopaminergic neuronal cell death. Further, primary dopaminergic neurons obtained from PKCδ knockout (−/− mice were resistant to TNF toxicity. The proteolytic activation of PKCδ in the mouse substantia nigra in the

  16. Cyclooxygenase-2 transactivates the epidermal growth factor receptor through specific E-prostanoid receptors and Tumor Necrosis Factor-α converting enzyme

    Al-Salihi, Mazin A.; Ulmer, Scott C.; Doan, Thao; Nelson, Cory D.; Crotty, Tracy; Prescott, Stephen M.; Stafforini, Diana M.; Topham, Matthew K.

    2007-01-01

    Cyclooxygenase-2 is often highly expressed in epithelial malignancies and likely has an active role in tumor development. But how it promotes tumorigenesis is not clearly defined. Recent evidence suggests that this may involve transactivation of the epidermal growth factor receptor through E-prostanoid receptors, but reports differ about the mechanism by which this occurs. We found that E-prostanoid receptors 2–4, but not 1, transactivated the epidermal growth factor receptor. This required m...

  17. High-altitude hypoxia induces disorders of the brain-endocrine-immune network through activation of corticotropin-releasing factor and its type-1 receptors

    Xue-qun CHEN; Fan-ping KONG; Yang ZHAO; Ji-zeng DU

    2012-01-01

    High-altitude hypoxia can induce physiological dysfunction and mountain sickness,but the underlying mechanism is not fully understood.Corticotrophin-releasing factor (CRF) and CRF type-1 receptors (CRFR1) are members of the CRF family and the essential controllers of the physiological activity of the hypothalamo-pituitary-adrenal (HPA) axis and modulators of endocrine and behavioral activity in response to various stressors.We have previously found that high-altitude hypoxia induces disorders of the brain-endocrine-immune network through activation of CRF and CRFR1 in the brain and periphery that include activation of the HPA axis in a time-and dose-dependent manner,impaired or improved learning and memory,and anxiety-like behavioral change.Meanwhile,hypoxia induces dysfunctions of the hypothalamo-pituitary-endocrine and immune systems,including suppression of growth and development,as well as inhibition of reproductive,metabolic and immune functions.In contrast,the small mammals that live on the Qinghai-Tibet Plateau alpine meadow display low responsiveness to extreme high-altitudehypoxia challenge,suggesting well-acclimatized genes and a physiological strategy that developed during evolution through interact-ions between the genes and environment.All the findings provide evidence for understanding the neuroendocrine mechanisms of hypoxia-induced physiological dysfunction.This review extends these findings.

  18. A new analysis approach of epidermal growth factor receptor pathway activation patterns provides insights into cetuximab resistance mechanisms in head and neck cancer

    von der Heyde Silvia

    2012-05-01

    Full Text Available Abstract The pathways downstream of the epidermal growth factor receptor (EGFR have often been implicated to play crucial roles in the development and progression of various cancer types. Different authors have proposed models in cell lines in which they study the modes of pathway activities after perturbation experiments. It is prudent to believe that a better understanding of these pathway activation patterns might lead to novel treatment concepts for cancer patients or at least allow a better stratification of patient collectives into different risk groups or into groups that might respond to different treatments. Traditionally, such analyses focused on the individual players of the pathways. More recently in the field of systems biology, a plethora of approaches that take a more holistic view on the signaling pathways and their downstream transcriptional targets has been developed. Fertig et al. have recently developed a new method to identify patterns and biological process activity from transcriptomics data, and they demonstrate the utility of this methodology to analyze gene expression activity downstream of the EGFR in head and neck squamous cell carcinoma to study cetuximab resistance. Please see related article: http://www.biomedcentral.com/1471-2164/13/160

  19. The role of peroxisome proliferator-activated receptor-β/δ in epidermal growth factor-induced HaCaT cell proliferation

    Epidermal growth factor (EGF) has been shown to be a potent mitogen for epidermal cells both in vitro and in vivo, thus contributing to the development of an organism. It has recently become clear that peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) expression and activation is involved in the cell proliferation. However, little is known about the role of PPARβ/δ in EGF-induced proliferation of HaCaT keratinocytes. In this study, HaCaT cells were cultured in the presence and absence of EGF and we identified that EGF induced an increase of PPARβ/δ mRNA and protein level expression in time-dependent and dose-dependent manner, and AG1487, an EGF receptor (EGFR) special inhibitor, caused attenuation of PPARβ/δ protein expression. Electrophoretic mobility shift assay (EMSA) revealed that EGF significantly increased PPARβ/δ binding activity in HaCaT keratinocytes. Antisense phosphorothioate oligonucleotides (asODNs) against PPARβ/δ caused selectively inhibition of PPARβ/δ protein content induced by EGF and significantly attenuated EGF-mediated cell proliferation. Treatment of the cells with L165041, a specific synthetic ligand for PPARβ/δ, significantly enhanced EGF-mediated cell proliferation. Finally, c-Jun ablation inhibited PPARβ/δ up-regulation induced by EGF, and chromatin immunoprecipitation (ChIP) showed that c-Jun bound to the PPARβ/δ promoter and the binding increased in EGF-stimulated cells. These results demonstrate that EGF induces PPARβ/δ expression in a c-Jun-dependent manner and PPARβ/δ plays a vital role in EGF-stimulated proliferation of HaCaT cells

  20. Factors Associated with the Serum Myostatin Level in Patients Undergoing Peritoneal Dialysis: Potential Effects of Skeletal Muscle Mass and Vitamin D Receptor Activator Use.

    Yamada, Shunsuke; Tsuruya, Kazuhiko; Yoshida, Hisako; Tokumoto, Masanori; Ueki, Kenji; Ooboshi, Hiroaki; Kitazono, Takanari

    2016-07-01

    Myostatin is a member of the transforming growth factor-β family, which regulates synthesis and degradation of skeletal muscle proteins and is associated with the development of sarcopenia. It is up-regulated in the skeletal muscle of chronic kidney disease patients and is considered to be involved in the development of uremic sarcopenia. However, serum myostatin levels have rarely been determined, and the relationship between serum myostatin levels with clinical and metabolic factors remains unknown. This cross-sectional study investigated the association between serum myostatin level and clinical factors in 69 outpatients undergoing peritoneal dialysis. Serum myostatin level was determined by commercially available enzyme-linked immunosorbent assay (ELISA). Univariable and multivariable analysis were conducted to determine factors associated with serum myostatin levels. The factors included age, sex, diabetes mellitus, dialysis history, body mass index, residual kidney function, peritoneal dialysate volume, serum biochemistries, and the use of vitamin D receptor activators (VDRAs). Mean serum myostatin level was 7.59 ± 3.37 ng/mL. There was no association between serum myostatin level and residual kidney function. Serum myostatin levels were significantly and positively associated with lean body mass measured by the creatinine kinetic method and negatively associated with the use of VDRAs after adjustment for potential confounding factors. Our study indicated that serum myostatin levels are associated with skeletal muscle mass and are lower in patients treated with VDRAs. Further studies are necessary to determine the significance of measuring serum myostatin level in patients undergoing peritoneal dialysis. PMID:26895008

  1. Ligustrazine attenuates oxidative stress-induced activation of hepatic stellate cells by interrupting platelet-derived growth factorreceptor-mediated ERK and p38 pathways

    Hepatic fibrosis represents a frequent event following chronic insult to trigger wound healing reactions with accumulation of extracellular matrix (ECM) in the liver. Activation of hepatic stellate cells (HSCs) is the pivotal event during liver fibrogenesis. Compelling evidence indicates that oxidative stress is concomitant with liver fibrosis irrespective of the underlying etiology. Natural antioxidant ligustrazine exhibits potent antifibrotic activities, but the mechanisms are poorly understood. Our studies were to investigate the ligustrazine effects on HSC activation stimulated by hydrogen peroxide (H2O2), an in vitro model mimicking the oxidative stress in liver fibrogenesis, and to elucidate the possible mechanisms. Our results demonstrated that H2O2 at 5 μM significantly stimulated HSC proliferation and expression of marker genes of HSC activation; whereas ligustrazine dose-dependently suppressed proliferation and induced apoptosis in H2O2-activated HSCs, and attenuated expression of fibrotic marker genes. Mechanistic investigations revealed that ligustrazine reduced platelet-derived growth factorreceptor (PDGF-βR) expression and blocked the phosphorylation of extracellular regulated protein kinase (ERK) and p38 kinase, two downstream effectors of PDGF-βR. Further molecular evidence suggested that ligustrazine interruption of ERK and p38 pathways was dependent on the blockade of PDGF-βR and might be involved in ligustrazine reduction of fibrotic marker gene expression under H2O2 stimulation. Furthermore, ligustrazine modulated some proteins critical for HSC activation and ECM homeostasis in H2O2-stimulated HSCs. These data collectively indicated that ligustrazine could attenuate HSC activation caused by oxidative stress, providing novel insights into ligustrazine as a therapeutic option for hepatic fibrosis. Highlights: ► Ligustrazine inhibits oxidative stress-induced HSC activation. ► Ligustrazine reduces fibrotic marker genes in HSCs under

  2. An early granulocyte colony-stimulating factor treatment attenuates neuropathic pain through activation of mu opioid receptors on the injured nerve.

    Liao, Ming-Feng; Yeh, Shin-Rung; Lo, Ai-Lun; Chao, Po-Kuan; Lee, Yun-Lin; Hung, Yu-Hui; Lu, Kwok-Tung; Ro, Long-Sun

    2016-01-01

    Several studies have shown that the mu opioid receptor (MOR) located in the peripheral nerves can be activated after nerve injury and that it attenuates peripheral nociceptive signals to the spinal dorsal horn. Various cytokines and phosphorylated-p38 (p-p38) activation in the dorsal horn also play an important role in neuropathic pain development. Granulocyte-colony stimulating factor (GCSF) is a growth factor that can stimulate granulocyte formation and has been shown to exert an analgesic effect on neuropathic pain through recruiting opioid-containing leukocytes to the injured nerve. However, the underlying mechanisms are not well understood. Herein, the results of behavior tests in addition to MOR levels in the injured sciatic nerve and the levels of p-p38 and various cytokines in the spinal dorsal horn were studied in vehicle-treated or GCSF-treated chronic constriction injured (CCI) rats at different time points (i.e., 1, 3, and 7 days, respectively) after nerve injury. The results showed that a single early systemic GCSF treatment after nerve injury can up-regulate MORs in the injured nerve, which can decrease peripheral nociceptive signals. Thereafter, those changes suppress the pro-inflammatory cytokine IL-6 but enhance the anti-inflammatory cytokine IL-4, followed by decreases in p-p38 in the dorsal horn, and thus further attenuate neuropathic pain. PMID:27180600

  3. Role of Osteoprotegerin and Receptor Activator of Nuclear Factor-κB Ligand in Bone Loss Related to Advanced Chronic Obstructive Pulmonary Disease

    Ugay, Ludmila; Kochetkova, Evgenia; Nevzorova, Vera; Maistrovskaia, Yuliya

    2016-01-01

    Background: Osteoporosis is a common complication of chronic obstructive pulmonary disease (COPD). Recent clinical and biological researches have increasingly delineated the biomolecular pathways of bone metabolism regulation in COPD. We extended this work by examining the specific association and potential contribution of the osteoprotegerin (OPG)/receptor activator of nuclear factor-κB ligand (RANKL) axis to the pathogenesis of osteoporosis in advanced COPD. The aim of this study was to assess the relationships of serum OPG, RANKL, and tumor necrosis factor-alpha (TNF-α) with bone turnover in men with very severe COPD. Methods: Pulmonary function, T-score at the lumbar spine (LS) and femoral neck (FN), serum OPG, RANKL, soluble receptor of tumor necrosis factor-alpha-I and II (sTNFR-I, sTNFR-II), osteocalcin (OC), and β-CrossLaps (βCL) levels were measured in 45 men with very severe stage COPD and 36 male non-COPD volunteers. COPD patients and healthy controls were compared using an independent t-test and Mann–Whitney U-test. The Pearson coefficient was used to assess the relationships between variables. Results: OPG and OC were lower in male COPD patients than in control subjects whereas RANKL, serum βCL, TNF-α, and its receptors were higher. OPG directly correlated with forced expiratory volume in 1 s (FEV1) % predicted (r = 0.46, P < 0.005), OC (r = 0.34, P < 0.05), LS (r = 0.56, P < 0.001), and FN T-score (r = 0.47, P < 0.01). In contrast, serum RANKL inversely associated with LS and FN T-score (r = −0.62, P < 0.001 and r = −0.48, P < 0.001) but directly correlated with βCL (r = 0.48, P < 0.001). In addition, OPG was inversely correlated with RANKL (r = −0.39, P < 0.01), TNF-α (r = −0.56, P < 0.001), and sTNFR-I (r = −0.40, P < 0.01). Conclusion: Our results suggest that serum OPG and RANKL levels are inversely associated with bone loss in men with advanced stage COPD. PMID:27411457

  4. Antiosteoclastogenesis activity of a CO2 laser antagonizing receptor activator for nuclear factor kappaB ligand-induced osteoclast differentiation of murine macrophages

    Macrophage cells are the important effector cells in the immune reaction which are indispensable for osteoclastogenesis; their heterogeneity and plasticity renders macrophages a primer target for immune system modulation. In recent years, there have been very few studies about the effects of macrophage cells on laser treatment-regulated osteoclastogenesis. In this study, RAW 264.7 macrophage cells were treated with RANKL to regulate osteoclastogenesis. We used a CO2 laser as a model biostimulation to investigate the role of osteoclastogenic. We also evaluated cell viability, cell death and cathepsin K expression. The CO2 laser inhibited a receptor activator of the NF-ĸB ligand (RANKL)-induced formation of osteoclasts during the osteoclast differentiation process. It was also found that irradiation for two times reduced RANKL-enhanced TRAP activity in a dose-dependent manner. Furthermore, CO2 laser-treatment diminished the expression and secretion of cathepsin K elevated by RANKL and was concurrent with the inhibition of TRAF6 induction and NF-ĸB activation. The current report demonstrates that CO2 laser abrogated RANKL-induced osteoclastogenesis by retarding osteoclast differentiation. The CO2 laser can modulate every cell through dose-dependent in vitro RANKL-mediated osteoclastogenesis, such as the proliferation and fusion of preosteoclasts and the maturation of osteoclasts. Therefore, the current results serve as an improved explanation of the cellular roles of macrophage cell populations in osteoclastogenesis as well as in alveolar bone remodeling by CO2 laser-treatment. (letter)

  5. From pure compounds to complex exposure: Effects of dietary cadmium and lignans on estrogen, epidermal growth factor receptor, and mitogen activated protein kinase signaling in vivo.

    Ali, Imran; Hurmerinta, Teija; Nurmi, Tarja; Berglund, Marika; Rüegg, Joelle; Poutanen, Matti; Halldin, Krister; Mäkelä, Sari; Damdimopoulou, Pauliina

    2016-06-24

    Exposure to environmental endocrine active compounds correlates with altered susceptibility to disease in human populations. Chemical risk assessment is single compound based, although exposure often takes place as heterogeneous mixtures of man-made and natural substances within complex matrices like diet. Here we studied whether the effects of cadmium and enterolactone on endocrine endpoints in dietary exposure can be predicted based on pure compound effects. Ovariectomized estrogen reporter ERE-luciferase (ERE-luc) mice were maintained on diets that intrinsically contain increasing concentrations of cadmium and enterolactone precursors for three and 21 days. The activation of the ERE-luc, epidermal growth factor receptor (EGFR), mitogen activated protein kinase (MAPK)-ERK1/2, and classical estrogen responses were measured. Interactions between the diets and endogenous hormone were evaluated by challenging the animals with 17β-estradiol. Compared to animals on basal purified diet, mice consuming experimental diets were exposed to significantly higher levels of cadmium and enterolactone, yet the exposure remained comparable to typical human dietary intake. Surprisingly, we could not detect effects on endpoints regulated by pure enterolactone, such as ERE-luc activation. However, cadmium accumulation in the liver was accompanied with activation of EGFR and MAPK-ERK1/2 in line with our earlier CdCl2 studies. Further, attenuation of 17β-estradiol-induced ERE-luc response in liver by experimental diets was observed. Our findings indicate that the exposure context can have substantial effects on the activity of endocrine active compounds in vivo. Thus, whenever possible, a context that mimics human exposure should be tested along with pure compounds. PMID:27108949

  6. miR-17-5p targets the p300/CBP-associated factor and modulates androgen receptor transcriptional activity in cultured prostate cancer cells

    Androgen receptor (AR) signalling is critical to the initiation and progression of prostate cancer (PCa). Transcriptional activity of AR involves chromatin recruitment of co-activators, including the p300/CBP-associated factor (PCAF). Distinct miRNA expression profiles have been identified in PCa cells during the development and progression of the disease. Whether miRNAs regulate PCAF expression in PCa cells to regulate AR transcriptional activity is still unclear. Expression of PCAF was investigated in several PCa cell lines by qRT-PCR, Western blot, and immunocytochemistry. The effects of PCAF expression on AR-regulated transcriptional activity and cell growth in PCa cells were determined by chromatin immunoprecipitation, reporter gene construct analysis, and MTS assay. Targeting of PCAF by miR-17-5p was evaluated using the luciferase reporter assay. PCAF was upregulated in several PCa cell lines. Upregulation of PCAF promoted AR transcriptional activation and cell growth in cultured PCa cells. Expression of PCAF in PCa cells was associated with the downregulation of miR-17-5p. Targeting of the 3’-untranslated region of PCAF mRNA by miR-17-5p caused translational suppression and RNA degradation, and, consequently, modulation of AR transcriptional activity in PCa cells. PCAF is upregulated in cultured PCa cells, and upregulation of PCAF is associated with the downregulation of miR-17-5p. Targeting of PCAF by miR-17-5p modulates AR transcriptional activity and cell growth in cultured PCa cells

  7. Influence of baicalin on the expression of receptor activator of nuclear factor-κB ligand and osteoprotegerin in human periodontal ligament cells

    Yue Chen

    2009-01-01

    Objective To study the effect of baicalin on the expression of receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) in cultured human periodontal ligament (HPDL) cells. Methods Small interfering RNA (siRNA) eukaryotic expression vector targeted transforming growth factor βⅡ receptor (TGF-β RⅡ) was constructed and transfected into T cells. HPDL cells with T cells transfected with siRNA or not were placed in the culture medium that had been added with lipopolysaccharide (LPS) and baicalin. The obtained solution was divided into six groups according to the components (group Ⅰ: HPDL cells+LPS+T cells transfected with siRNA1+baicalin;group Ⅱ: HPDL cells+LPS+T cells transfected with siRNA1; group Ⅲ: HPDL cells+LPS+T cells+baicalin; group Ⅳ: HPDL cells+LPS+T cells; group Ⅴ: HPDL cells+baicalin; group Ⅵ: HPDL cells) and was cultured for 48 hours. RT-PCR was used to observe the effect of baicalin on the expression of OPG-RANKL in HPDL cells. Results The ratio of RANKL/OPG in group Ⅰ was lower than that in group Ⅱ (P<0.01) and higher than that in group Ⅲ (P<0.01); The ratio of RANKL/OPG in gronp Ⅲ was lower than that in group Ⅳ (P<0.01); the ratio of RANKL/ OPG in group Ⅳ was higher than that in group Ⅵ (P<0.01); the ratio of RANKL/OPG in group Ⅴ was lower than TGF-β signaling transduction plays an important role in the effect of baicalin on the RANKL/OPG ratio in HPDL cells.

  8. Epidermal growth factor receptor targeted molecularly therapies of cancers

    Epidermal growth factor receptor (EGFR) has been known to be a significant factor in the development and growth of many types of cancers. It is now accepted that the EGFR signal transduction net work plays an important role in multiple tumorigenic processes, contributing to cancer cell proliferation, angiogenesis, and metastasis, as well as protection from apoptosis. Recently, EGFR monoclonal antibodies (McAb) and epidermal growth factor receptor-tyrosine kinase (EGFR-TK) inhibitors have been validated as new treatment approach for those EGFR-positive cancers and have shown activity aginst advanced, chemofractory cancers in clinical trials. This article focuses on three EGFR targeted molecularly therapies of cancers. (authors)

  9. The poly-γ-d-glutamic acid capsule surrogate of the Bacillus anthracis capsule induces nitric oxide production via the platelet activating factor receptor signaling pathway.

    Lee, Hae-Ri; Jeon, Jun Ho; Park, Ok-Kyu; Chun, Jeong-Hoon; Park, Jungchan; Rhie, Gi-Eun

    2015-12-01

    The poly-γ-d-glutamic acid (PGA) capsule, a major virulence factor of Bacillus anthracis, confers protection of the bacillus from phagocytosis and allows its unimpeded growth in the host. PGA capsules released from B. anthracis are associated with lethal toxin in the blood of experimentally infected animals and enhance the cytotoxic effect of lethal toxin on macrophages. In addition, PGA capsule itself activates macrophages and dendritic cells to produce proinflammatory cytokine such as IL-1β, indicating multiple roles of PGA capsule in anthrax pathogenesis. Here we report that PGA capsule of Bacillus licheniformis, a surrogate of B. anthracis capsule, induces production of nitric oxide (NO) in RAW264.7 cells and bone marrow-derived macrophages. NO production was induced by PGA in a dose-dependent manner and was markedly reduced by inhibitors of inducible NO synthase (iNOS), suggesting iNOS-dependent production of NO. Induction of NO production by PGA was not observed in macrophages from TLR2-deficient mice and was also substantially inhibited in RAW264.7 cells by pretreatment of TLR2 blocking antibody. Subsequently, the downstream signaling events such as ERK, JNK and p38 of MAPK pathways as well as NF-κB activation were required for PGA-induced NO production. In addition, the induced NO production was significantly suppressed by treatment with antagonists of platelet activating factor receptor (PAFR) or PAFR siRNA, and mediated through PAFR/Jak2/STAT-1 signaling pathway. These findings suggest that PGA capsule induces NO production in macrophages by triggering both TLR2 and PAFR signaling pathways which lead to activation of NF-kB and STAT-1, respectively. PMID:26350415

  10. Astrocyte Mitogen Inhibitor Related to Epidermal Growth Factor Receptor

    Nieto-Sampedro, Manuel

    1988-06-01

    Epidermal growth factor (EGF) is a well-characterized polypeptide hormone with diverse biological activities, including stimulation of astrocyte division. A soluble astrocyte mitogen inhibitor, immunologically related to the EGF receptor, is present in rat brain. Injury to the brain causes a time-dependent reduction in the levels of this inhibitor and the concomitant appearance of EGF receptor on the astrocyte surface. Intracerebral injection of antibody capable of binding the inhibitor caused the appearance of numerous reactive astrocytes. EGF receptor-related inhibitors may play a key role in the control of glial cell division in both normal and injured brain.

  11. Platelet-derived growth factor receptor-β, carrying the activating mutation D849N, accelerates the establishment of B16 melanoma

    Platelet-derived growth factor (PDGF)-BB and PDGF receptor (PDGFR)-β are mainly expressed in the developing vasculature, where PDGF-BB is produced by endothelial cells and PDGFR-β is expressed by mural cells, including pericytes. PDGF-BB is produced by most types of solid tumors, and PDGF receptor signaling participates in various processes, including autocrine stimulation of tumor cell growth, recruitment of tumor stroma fibroblasts, and stimulation of tumor angiogenesis. Furthermore, PDGF-BB-producing tumors are characterized by increased pericyte abundance and accelerated tumor growth. Thus, there is a growing interest in the development of tumor treatment strategies by blocking PDGF/PDGFR function. We have recently generated a mouse model carrying an activated PDGFR-β by replacing the highly conserved aspartic acid residue (D) 849 in the activating loop with asparagine (N). This allowed us to investigate, in an orthotopic tumor model, the role of increased stromal PDGFR-β signaling in tumor-stroma interactions. B16 melanoma cells lacking PDGFR-β expression and either mock-transfected or engineered to express PDGF-BB, were injected alone or in combination with matrigel into mice carrying the activated PDGFR-β (D849N) and into wild type mice. The tumor growth rate was followed and the vessel status of tumors, i.e. total vessel area/tumor, average vessel surface and pericyte density of vessels, was analyzed after resection. Tumors grown in mice carrying an activated PDGFR-β were established earlier than those in wild-type mice. In this early phase, the total vessel area and the average vessel surface were higher in tumors grown in mice carrying the activated PDGFR-β (D849N) compared to wild-type mice, whereas we did not find a significant difference in the number of tumor vessels and the pericyte abundance around tumor vessels between wild type and mutant mice. At later phases of tumor progression, no significant difference in tumor growth rate was

  12. Platelet-derived growth factor receptor-β, carrying the activating mutation D849N, accelerates the establishment of B16 melanoma

    Suzuki Shioto

    2007-12-01

    Full Text Available Abstract Background Platelet-derived growth factor (PDGF-BB and PDGF receptor (PDGFR-β are mainly expressed in the developing vasculature, where PDGF-BB is produced by endothelial cells and PDGFR-β is expressed by mural cells, including pericytes. PDGF-BB is produced by most types of solid tumors, and PDGF receptor signaling participates in various processes, including autocrine stimulation of tumor cell growth, recruitment of tumor stroma fibroblasts, and stimulation of tumor angiogenesis. Furthermore, PDGF-BB-producing tumors are characterized by increased pericyte abundance and accelerated tumor growth. Thus, there is a growing interest in the development of tumor treatment strategies by blocking PDGF/PDGFR function. We have recently generated a mouse model carrying an activated PDGFR-β by replacing the highly conserved aspartic acid residue (D 849 in the activating loop with asparagine (N. This allowed us to investigate, in an orthotopic tumor model, the role of increased stromal PDGFR-β signaling in tumor-stroma interactions. Methods B16 melanoma cells lacking PDGFR-β expression and either mock-transfected or engineered to express PDGF-BB, were injected alone or in combination with matrigel into mice carrying the activated PDGFR-β (D849N and into wild type mice. The tumor growth rate was followed and the vessel status of tumors, i.e. total vessel area/tumor, average vessel surface and pericyte density of vessels, was analyzed after resection. Results Tumors grown in mice carrying an activated PDGFR-β were established earlier than those in wild-type mice. In this early phase, the total vessel area and the average vessel surface were higher in tumors grown in mice carrying the activated PDGFR-β (D849N compared to wild-type mice, whereas we did not find a significant difference in the number of tumor vessels and the pericyte abundance around tumor vessels between wild type and mutant mice. At later phases of tumor progression, no

  13. INHIBITION OF PROTEIN TYROSINE PHOSPHATASE ACTIVITY MEDIATES EPIDERMAL GROWTH FACTOR RECEPTOR SIGNALING IN HUMAN AIRWAY EPITHELIAL CELLS

    Epidemiological studies have implicated zinc in the toxicity of ambient particulate matter (PM) inhalation. We previously showed that exposure to metal-laden PM inhibits protein tyrosine phosphatase (PTP) activity in human primary bronchial epithelial cells (HAEC) and leads t...

  14. Effect of platelet-activating factor receptor antagonist, etizolam, on resolution of chronic subdural hematoma. A prospective study to investigate use as conservative therapy

    Inflammatory reaction is very important for formation of the neomembrane of chronic subdural hematoma (CSDH). The present study evaluated medical treatment with the platelet-activating factor receptor antagonist, etizolam, for the resolution of CSDH, and the factors indicating surgery or conservative therapy. Alternate patients were assigned to the etizolam group or control group without medical treatment. Patients in the etizolam group received 3.0 mg etizolam per day for 14 days. A total of 53 patients were followed up for at least 6 months. Univariate analysis of differences in demographic characteristics, clinical findings, and initial computed tomography (CT) findings, and multiple logistic regression analysis of the relationship between etizolam treatment and requirement for surgery using age, sex, low density of hematoma on CT, and paresis as confounders were performed. Etizolam treatment (adjusted odds ratio [OR] 0.156, 95% confidence interval [CI] 0.024-0.999, p=0.049) was negatively correlated with requirement for surgery. Low density of hematoma (adjusted OR 0.125, 95% CI 0.019-0.846, p=0.033) was found to be an independent negative predictor, and paresis as an initial symptom (adjusted OR 6.35, 95% CI 1.04-38.7, p=0.045) was an independent positive predictor of requirement for surgery. Etizolam administration can promote the resolution of CSDH, especially at the stage of hygroma appearing as low density on CT. Surgery is recommended if the patient presents with paresis. (author)

  15. Effect of platelet-activating factor receptor antagonist, etizolam, on resolution of chronic subdural hematoma--a prospective study to investigate use as conservative therapy.

    Hirashima, Yutaka; Kurimoto, Masanori; Nagai, Shoichi; Hori, Emiko; Origasa, Hideki; Endo, Shunro

    2005-12-01

    Inflammatory reaction is very important for formation of the neomembrane of chronic subdural hematoma (CSDH). The present study evaluated medical treatment with the platelet-activating factor receptor antagonist, etizolam, for the resolution of CSDH, and the factors indicating surgery or conservative therapy. Alternate patients were assigned to the etizolam group or control group without medical treatment. Patients in the etizolam group received 3.0 mg etizolam per day for 14 days. A total of 53 patients were followed up for at least 6 months. Univariate analysis of differences in demographic characteristics, clinical findings, and initial computed tomography (CT) findings, and multiple logistic regression analysis of the relationship between etizolam treatment and requirement for surgery using age, sex, low density of hematoma on CT, and paresis as confounders were performed. Etizolam treatment (adjusted odds ratio [OR] 0.156, 95% confidence interval [CI] 0.024-0.999, p = 0.049) was negatively correlated with requirement for surgery. Low density of hematoma (adjusted OR 0.125, 95% CI 0.019-0.846, p = 0.033) was found to be an independent negative predictor, and paresis as an initial symptom (adjusted OR 6.35, 95% CI 1.04-38.7, p = 0.045) was an independent positive predictor of requirement for surgery. Etizolam administration can promote the resolution of CSDH, especially at the stage of hygroma appearing as low density on CT. Surgery is recommended if the patient presents with paresis. PMID:16377949

  16. Activation of histamine H4 receptors decreases epithelial-to-mesenchymal transition progress by inhibiting transforming growth factor-β1 signalling pathway in non-small cell lung cancer.

    Cai, Wen-Ke; Hu, Jing; Li, Teng; Meng, Jing-Ru; Ma, Xue; Yin, Sun-Jun; Zhao, Can-Hu; He, Gong-Hao; Xu, Gui-Li

    2014-04-01

    Previous investigations found that epithelial-to-mesenchymal transition (EMT) was an important character of non-small cell lung cancer (NSCLC) and it was also suggested that histamine H4 receptors may have a role in preventing EMT progress in certain kind of tumours. However, the effect of H4 receptor activation on EMT progress of NSCLC and its potential mechanisms remain unclear. Therefore, we performed both in vitro and in vivo experiments to explore the effects of specific H4 receptor agonist 4-methylhistamine and antagonist JNJ7777120 on EMT progress. We showed the expression of H4 receptors in NSCLC and found that 4-methylhistamine increased the expression of the epithelial marker E-cadherin and decreased the expression of Vimentin, the mesenchymal marker, in both NSCLC cell lines and xenograft NSCLC tumours. Pretreatment with JNJ7777120 or H4 receptor gene silencing decreased while overexpression of H4 receptors facilitated this effect of 4-methylhistamine. Furthermore, we showed that down-regulation of cyclic adenosine monophosphate (cAMP) was the secondary signalling after H4 receptor activation, which in turn resulted in inactivation of transforming growth factor-β1 (TGF-β1) pathway and down-regulation of several important EMT inducing factors such as ZEB1, Snail and Slug. In conclusion, these findings revealed the anti-EMT effect of histamine H4 receptor activation in NSCLC, which provide novel insight into the development mechanism of NSCLC; and H4 receptors may be a new therapeutic target for NSCLC treatment. PMID:24447834

  17. Acquired resistance mechanisms to tyrosine kinase inhibitors in lung cancer with activating epidermal growth factor receptor mutation--diversity, ductility, and destiny.

    Suda, Kenichi; Mizuuchi, Hiroshi; Maehara, Yoshihiko; Mitsudomi, Tetsuya

    2012-12-01

    Lung cancers that harbor somatic activating mutations in the gene for the epidermal growth factor receptor (EGFR) depend on mutant EGFR for their proliferation and survival; therefore, lung cancer patients with EGFR mutations often dramatically respond to orally available EGFR tyrosine kinase inhibitors (TKIs). However, emergence of acquired resistance is virtually inevitable, thus limiting improvement in patient outcomes. To elucidate and overcome this acquired resistance, multidisciplinary basic and clinical investigational approaches have been applied, using in vitro cell line models or samples obtained from lung cancer patients treated with EGFR-TKIs. These efforts have revealed several acquired resistance mechanisms and candidates, including EGFR secondary mutations (T790M and other rare mutations), MET amplification, PTEN downregulation, CRKL amplification, high-level HGF expression, FAS-NFκB pathway activation, epithelial-mesenchymal transition, and conversion to small cell lung cancer. Interestingly, cancer cells harbor potential destiny and ductility together in acquiring resistance to EGFR-TKIs, as shown in in vitro acquired resistance models. Molecular mechanisms of "reversible EGFR-TKI tolerance" that occur in early phase EGFR-TKI exposure have been identified in cell line models. Furthermore, others have reported molecular markers that can predict response to EGFR-TKIs in clinical settings. Deeper understanding of acquired resistance mechanisms to EGFR-TKIs, followed by the development of molecular target drugs that can overcome the resistance, might turn this fatal disease into a chronic disorder. PMID:22736441

  18. DEPENDENCE OF PPAR LIGAND-INDUCED MAPK SIGNALING ON EPIDERMAL GROWTH FACTOR RECEPTOR TRANSACTIVATION HEPARIN-BINDING EGF CLEAVAGE MEDIATES ZINC-INDUCED EGF RECEPTOR PHOSPHORYLATION

    Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that function as ligand-activated transcription factors regulating lipid metabolism and homeostasis. In addition to their ability to regulate PPAR-mediated gene transcription, PPARalpha and gamma li...

  19. Activation of adenosine A3 receptors supports hematopoiesis-stimulating effects of granulocyte colony-stimulating factor in sublethally irradiated mice

    Hofer, Michal; Pospíšil, Milan; Šefc, L.; Dušek, L.; Vacek, Antonín; Holá, Jiřina; Hoferová, Zuzana; Štreitová, Denisa

    2010-01-01

    Roč. 86, č. 8 (2010), s. 649-656. ISSN 0955-3002 R&D Projects: GA ČR(CZ) GA305/08/0158 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : ionising radiation * hematopoiesis * adenosine A3 receptors Subject RIV: BO - Biophysics Impact factor: 1.861, year: 2010

  20. In vitro effects of buyang huanwu decoction and its ingredients on inhibiting the specific binding of 3H-platelet activating factor to its receptor in rabbits

    2007-01-01

    BACKGROUND: Pharmacologic action of traditional Chinese medicine compound is the comprehensive effect of various ingredients, and the interactions of various ingredients are closely correlated with the final effect. In order to reveal the compatibility mechanism of buyang huanwu decoction (BHD)'s prescription in treating and preventing ischemic cerebrovascular disease, we need to explore the effect and relation of ingredients in prescription except for considering the effect of each ingredient on the whole prescription.OBJECTIVE: To study the effect of BHD and its ingredients in the prescription on the specific binding of 3H-platelet activating factor (PAF) to its receptor (PAFR)in rabbits in vitro, and to analyze the action of each ingredient in the prescription.DESIGN: A decomposed recipe study based on orthogonal test.SETTING: Guangzhou University of Traditional Chinese Medicine.MATERIALS: Five healthy adult New Zealand rabbits of either gender were provided by the Experimental Animal Center of Guangzhou University of Traditional Chinese medicine. The prescription herbal pieces were purchased from Foshan Kangpu Pharmaceuticals Company and Jianmin Pharmaceuticals Company, and were appraised by Professor Yanchen Xu from College of Traditional Chinese Medicine, Guangzhou University of Traditional Chinese Medicine. 3H-PAF was supplied by Amersham Co.,Ltd.(Specific activity:6.475 TBq/mmol;batch number:200402); PAF standard by Biomol Co., Ltd.(batch number: P1318V).METHODS: This experiment was carried out in the Laboratory of Nuclear Medicine, Guangzhou University of Traditional Chinese Medicine between September and December 2004. ① The seven influencing factors were selected: such as Shenghuangqi , Dangguiwei, Chishao, Dilong, Taoren, Honghua, Chuanxiong. Each factor was divided into two levels, selected or not selected. The tests were arranged according to L8 (27) orthogonal test table. ②The specific binding of 3H-PAF to its receptors in rabbits was measured by

  1. Signal transduction by growth factor receptors: signaling in an instant

    Dengjel, Joern; Akimov, Vyacheslav; Blagoev, Blagoy; Andersen, Jens S

    2007-01-01

    mass spectrometry-based proteomics has allowed exciting views on the very early events in signal transduction. Activation profiles of regulated phosphorylation sites on epidermal growth factor receptor and downstream signal transducers showed different kinetics within the first ten seconds of...

  2. Osteoprotegerin, Soluble Receptor Activator of Nuclear Factor- κ B Ligand, and Subclinical Atherosclerosis in Children and Adolescents with Type 1 Diabetes Mellitus.

    Lambrinoudaki, Irene; Tsouvalas, Emmanouil; Vakaki, Marina; Kaparos, George; Stamatelopoulos, Kimon; Augoulea, Areti; Pliatsika, Paraskevi; Alexandrou, Andreas; Creatsa, Maria; Karavanaki, Kyriaki

    2013-01-01

    Aims. To evaluate carotid intima-media thickness (cIMT) and biomarkers of the osteoprotegerin/receptor activator of nuclear factor- κ B ligand (OPG/RANKL) system in type 1 diabetes (T1DM) children and adolescents and controls. Subjects and Methods. Fifty six T1DM patients (mean ± SD age: 12.0 ± 2.7 years, diabetes duration: 5.42 ± 2.87 years and HbA1c: 8.0 ± 1.5%) and 28 healthy matched controls, were studied with anthropometric and laboratory measurements, including serum OPG, soluble RANKL (sRANKL) and cIMT. Results. Anthropometric, laboratory, and cIMT measurements were similar between T1DM youngsters and controls. However patients with longer diabetes duration (>/7.0 years) had indicatively higher cIMT (cIMT = 0.49 vs 0.44 mm, P 0.072) and triglyceride levels than the rest of the patients (93.7 vs 64.6 mg/dl, P 0.025). Both in the total study population (β 0.418, P 0.027) and among T1DM patients separately (β 0.604, P 0.013), BMI was the only factor associated with cIMT. BMI was further associated with OPG in both groups (β -0.335, P 0.003 and β -0.356, P 0.008 respectively), while sRANKL levels were not associated with any factor. Conclusions. BMI was the strongest independent predictor of cIMT among the whole population, and especially in diabetics, suggesting a possible synergistic effect of diabetes and adiposity on atherosclerotic burden. BMI was overall strongly associated with circulating OPG, but the causes of this association remain unclear. PMID:24288529

  3. Osteoprotegerin, Soluble Receptor Activator of Nuclear Factor-κB Ligand, and Subclinical Atherosclerosis in Children and Adolescents with Type 1 Diabetes Mellitus

    Irene Lambrinoudaki

    2013-01-01

    Full Text Available Aims. To evaluate carotid intima-media thickness (cIMT and biomarkers of the osteoprotegerin/receptor activator of nuclear factor-κB ligand (OPG/RANKL system in type 1 diabetes (T1DM children and adolescents and controls. Subjects and Methods. Fifty six T1DM patients (mean ± SD age: 12.0 ± 2.7 years, diabetes duration: 5.42 ± 2.87 years and HbA1c: 8.0 ± 1.5% and 28 healthy matched controls, were studied with anthropometric and laboratory measurements, including serum OPG, soluble RANKL (sRANKL and cIMT. Results. Anthropometric, laboratory, and cIMT measurements were similar between T1DM youngsters and controls. However patients with longer diabetes duration (>/7.0 years had indicatively higher cIMT (cIMT = 0.49 vs 0.44 mm, P 0.072 and triglyceride levels than the rest of the patients (93.7 vs 64.6 mg/dl, P 0.025. Both in the total study population (β 0.418, P 0.027 and among T1DM patients separately (β 0.604, P 0.013, BMI was the only factor associated with cIMT. BMI was further associated with OPG in both groups (β −0.335, P 0.003 and β −0.356, P 0.008 respectively, while sRANKL levels were not associated with any factor. Conclusions. BMI was the strongest independent predictor of cIMT among the whole population, and especially in diabetics, suggesting a possible synergistic effect of diabetes and adiposity on atherosclerotic burden. BMI was overall strongly associated with circulating OPG, but the causes of this association remain unclear.

  4. Mechanism for the activation of glutamate receptors

    Scientists at the NIH have used a technique called cryo-electron microscopy to determine a molecular mechanism for the activation and desensitization of ionotropic glutamate receptors, a prominent class of neurotransmitter receptors in the brain and spina

  5. Angiotensin II-Induced Migration of Vascular Smooth Muscle Cells Is Mediated by p38 Mitogen-Activated Protein Kinase-Activated c-Src Through Spleen Tyrosine Kinase and Epidermal Growth Factor Receptor Transactivation

    Mugabe, Benon E.; Yaghini, Fariborz A.; Song, Chi Young; Buharalioglu, Cuneyt K.; Waters, Christopher M.; Malik, Kafait U.

    2010-01-01

    Angiotensin II (Ang II) stimulates protein synthesis by activating spleen tyrosine kinase (Syk) and DNA synthesis through epidermal growth factor receptor (EGFR) transactivation in vascular smooth muscle cells (VSMCs). This study was conducted to determine whether Syk mediates Ang II-induced migration of aortic VSMCs using a scratch wound approach. Treatment with Ang II (200 nM) for 24 h increased VSMC migration by 1.56 ± 0.14-fold. Ang II-induced VSMC migration and Syk phosphorylation as det...

  6. Ligustrazine attenuates oxidative stress-induced activation of hepatic stellate cells by interrupting platelet-derived growth factorreceptor-mediated ERK and p38 pathways

    Zhang, Feng [Department of Clinical Pharmacy, College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210029 (China); Ni, Chunyan [Department of Clinical Pharmacy, College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210029 (China); The First People' s Hospital of Changzhou, Changzhou 213003 (China); Kong, Desong; Zhang, Xiaoping; Zhu, Xiaojing; Chen, Li [Department of Clinical Pharmacy, College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210029 (China); Lu, Yin [Department of Clinical Pharmacy, College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210029 (China); Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing 210046 (China); National First-Class Key Discipline for Traditional Chinese Medicine of Nanjing University of Chinese Medicine, Nanjing 210046 (China); Zheng, Shizhong, E-mail: nytws@163.com [Department of Clinical Pharmacy, College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210029 (China); Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing 210046 (China); National First-Class Key Discipline for Traditional Chinese Medicine of Nanjing University of Chinese Medicine, Nanjing 210046 (China)

    2012-11-15

    Hepatic fibrosis represents a frequent event following chronic insult to trigger wound healing reactions with accumulation of extracellular matrix (ECM) in the liver. Activation of hepatic stellate cells (HSCs) is the pivotal event during liver fibrogenesis. Compelling evidence indicates that oxidative stress is concomitant with liver fibrosis irrespective of the underlying etiology. Natural antioxidant ligustrazine exhibits potent antifibrotic activities, but the mechanisms are poorly understood. Our studies were to investigate the ligustrazine effects on HSC activation stimulated by hydrogen peroxide (H{sub 2}O{sub 2}), an in vitro model mimicking the oxidative stress in liver fibrogenesis, and to elucidate the possible mechanisms. Our results demonstrated that H{sub 2}O{sub 2} at 5 μM significantly stimulated HSC proliferation and expression of marker genes of HSC activation; whereas ligustrazine dose-dependently suppressed proliferation and induced apoptosis in H{sub 2}O{sub 2}-activated HSCs, and attenuated expression of fibrotic marker genes. Mechanistic investigations revealed that ligustrazine reduced platelet-derived growth factorreceptor (PDGF-βR) expression and blocked the phosphorylation of extracellular regulated protein kinase (ERK) and p38 kinase, two downstream effectors of PDGF-βR. Further molecular evidence suggested that ligustrazine interruption of ERK and p38 pathways was dependent on the blockade of PDGF-βR and might be involved in ligustrazine reduction of fibrotic marker gene expression under H{sub 2}O{sub 2} stimulation. Furthermore, ligustrazine modulated some proteins critical for HSC activation and ECM homeostasis in H{sub 2}O{sub 2}-stimulated HSCs. These data collectively indicated that ligustrazine could attenuate HSC activation caused by oxidative stress, providing novel insights into ligustrazine as a therapeutic option for hepatic fibrosis. Highlights: ► Ligustrazine inhibits oxidative stress-induced HSC activation.

  7. Molecular Mechanisms Underlying the Antitumor Activity of 3-Aminopropanamide Irreversible Inhibitors of the Epidermal Growth Factor Receptor in Non–Small Cell Lung Cancer

    Elena Galvani

    2013-01-01

    Full Text Available Overcoming the emergence of acquired resistance to clinically approved epidermal growth factor receptor (EGFR inhibitors is a major challenge in the treatment of advanced non–small cell lung cancer (NSCLC. The aim of this study was to investigate the effects of a series of novel compounds affecting viability of NSCLC NCI-H1975 cells (carrying the EGFR T790M mutation. The inhibition of the autophosphorylation of EGFR occurred at nanomolar concentrations and both UPR1282 and UPR1268 caused a significant induction of apoptosis. Targeting of EGFR and downstream pathways was confirmed by a peptide substrate array, which highlighted the inhibition of other kinases involved in NSCLC cell aggressive behavior. Accordingly, the drugs inhibited migration (about 30% vs. control, which could be, in part, explained also by the increase of E-cadherin expression. Additionally, we observed a contraction of the volume of H1975 spheroids, associated with the reduction of the cancer stem–like cell hallmark CD133. The activity of UPR1282 was retained in H1975 xenograft models where it determined tumor shrinkage (P < .05 and resulted well tolerated compared to canertinib. Of note, the kinase activity profile of UPR1282 on xenograft tumor tissues showed overlapping results with respect to the activity in H1975 cells, unraveling the inhibition of kinases involved in pivotal proliferation and invasive signaling pathways. In conclusion, UPR1282 and UPR1268 are effective against various processes involved in malignancy transformation and progression and may be promising compounds for the future treatment of gefitinib-resistant NSCLCs.

  8. Role of nuclear factor of activated T-cells 5 in regulating hypertonic-mediated secretin receptor expression in kidney collecting duct cells.

    Chua, Oscar W H; Wong, Kenneth K L; Ko, Ben C; Chung, Sookja K; Chow, Billy K C; Lee, Leo T O

    2016-07-01

    A growing body of evidence suggests that secretin (SCT) is an important element in the osmoregulatory pathway. It is interesting to note that both SCT and its receptor (SCTR) gene are activated upon hyperosmolality in the kidney. However, the precise molecular mechanisms underlying the induction of the SCTR gene expression in response to changes in osmolality have yet to be clarified. Detailed DNA sequence analysis of the promoter regions of the SCTR gene reveals the presence of multiple osmotic response elements (ORE). The ORE is the binding site of a key osmosensitive transactivator, namely, the nuclear factor of activated T-cells 5 (NFAT5). SCTR and NFAT5 are co-expressed in the kidney cortex and medulla collecting duct cells. We therefore hypothesize that NFAT5 is responsible for modulating SCTR expression in hypertonic environments. In this study, we found hypertonicity stimulates the promoter activities and endogenous gene expression of SCTR in mouse kidney cortex collecting duct cells (M1) and inner medulla collecting duct cells (mIMCD3). The overexpression and silencing of NFAT5 further confirmed it to be responsible for the up-regulation of the SCTR gene under hypertonic conditions. A significant increase in the interaction between NFAT5 and the SCTR promoter was also observed following chromatin immunoprecipitation assay. In vivo, osmotic stress up-regulates the SCTR gene in the kidney cortex and medulla of wild-type mice, but does not do so in NFAT5(+/-) animals. Hence, this study provides comprehensive information on how NFAT5 regulates SCTR expression in different osmotic environments. PMID:27080132

  9. Production of two hemopoietic growth factors is differentially regulated in single T lymphocytes activated with an anti-T cell receptor antibody

    Kelso, A; Owens, T

    1988-01-01

    A method has been developed to measure the production by single activated T lymphocytes of two hemopoietic growth factors, granulocyte-macrophage CSF (GM-CSF) and multipotential CSF (multi-CSF or IL-3). When individual cells of the L3T4 (CD4)+ F23.1+ T cell clone E9.D4 were transferred...... by micromanipulation into wells coated with the monoclonal anti-T cell receptor antibody F23.1, up to 90% of cells produced CSF as detected by CSF-dependent hemopoietic cell lines. Production occurred in the absence of proliferation and did not require the addition of accessory cells or IL-2. Both the frequency of CSF...... in their total CSF titer with a mean value of about 0.05 U/cell. They also varied in their relative production of GM-CSF and multi-CSF. Thus, low producing cells secreted only GM-CSF whereas high producing cells also secreted multi-CSF. The failure of low producing cells to secrete multi-CSF was not genetically...

  10. Bleomycin-induced lung injury in the rat: Effects of the platelet-activating factor (PAF) receptor antagonist BN 52021 and platelet depletion

    Evans, T.W.; McAnulty, R.J.; Rogers, D.F.; Chung, K.F.; Barnes, P.J.; Laurent, G.J. (Brompton Hospital, London (England))

    1990-04-01

    Bleomycin is a highly effective antitumor agent, but pulmonary toxicity, characterized by an acute inflammatory reaction and associated pulmonary edema, limits clinical use of the drug. Platelets and platelet-activating factor (PAF), a membrane-derived phospholipid, have been implicated in the mechanisms that can mediate pulmonary microvascular injury. The authors sought to investigate the role of PAF in bleomycin-induce lung injury in the rat, using the PAF receptor antagonist BN 52021; and the role of platelets though the use of an anti-platelet antibody. Lung injury was induced by intratracheal bleomycin (1.5 mg) and assessed by measurements of lung wet weight and total pulmonary extravascular albumin space (TPEAS). Bleomycin caused a significant increase in both indices after 48 hr, compared with control animals. A single dose of BN 52021 significantly reduced the bleomycin-induced increase in lung weight, but not the rise in TPEAS. Increasing the dose of BN 52021 had no additional effect. Reducing circulating platelet numbers by approximately 75% had no effect on either the increase in lung weight or TPEAS, observed 48 hr after bleomycin. PAF may partially contribute to the acute inflammatory reaction seen after intratracheal bleomycin in rats.

  11. Skeletal changes in osteoprotegerin and receptor activator of nuclear factor-kappab ligand mRNA levels in primary hyperparathyroidism: effect of parathyroidectomy and association with bone metabolism

    Stilgren, L S; Rettmer, E; Eriksen, E F;

    2004-01-01

    The effect of parathyroid hormone (PTH) on the production of osteoprotegerin (OPG) and ligand of receptor activator of NF-kappaB (RANKL) in human bone is incompletely understood. Most in vitro studies indicate that PTH decreases OPG and increases RANKL production. In primary hyperparathyroidism (...

  12. Radiotherapy and receptor of epidermal growth factor

    The expression level of the receptor of the epidermal growth factor is in correlation with the tumor cells radiosensitivity. An overexpression of the E.G.F.R. is often present in the bronchi cancer, epidermoid carcinomas of the O.R.L. sphere, esophagus, uterine cervix, and anal duct but also in the rectum cancers and glioblastomas. At the clinical level, the E.G.F.R. expression is in correlation with an unfavourable prognosis after radiotherapy in numerous tumoral localizations. In the rectum cancers it is an independent prognosis factor found in multifactorial analysis: increase of the rate of nodes and local recurrence when the E.G.F.R. is over expressed. In the uterine cervix cancers, the survival is is negatively affected in multifactorial analysis by the E.G.F.R. membranes expression level. At the therapy level, the development of anti E.G.F.R. targeted therapies (tyrosine kinase inhibitors and monoclonal antibodies) opens a new therapy field at radio-sensitivity potentiality. The irradiation makes an activation of the E.G.F.R. way that would be partially responsible of the post irradiation tumoral repopulation. This activation leads the phosphorylation of the PI3 kinase ways and M.A.P. kinase ones, then the Akt protein one that acts an apoptotic modulator part. It has been shown that blocking the E.G.F.R. way acts on three levels: accumulation of ells in phase G1, reduction of the cell repair and increasing of apoptosis. he inhibition of post irradiation action of the E.G.F.R. signal way is a factor explaining the ionizing radiation - anti E.G.F.R. synergy. The preclinical data suggest that the E.G.F.R. blocking by the monoclonal antibodies is more important than the use of tyrosine kinase inhibitors. A first positive randomized study with the cetuximab, published in 2006 in the epidermoid carcinomas of the O.R.L. sphere lead to its authorization on the market with the radiotherapy for this localization. The use of cetuximab in other indication with or in

  13. Epidermal Growth Factor Receptor in Pancreatic Cancer

    Pancreatic cancer is the fourth leading cause of cancer related death. The difficulty in detecting pancreatic cancer at an early stage, aggressiveness and the lack of effective therapy all contribute to the high mortality. Epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein, which is expressed in normal human tissues. It is a member of the tyrosine kinase family of growth factors receptors and is encoded by proto-oncogenes. Several studies have demonstrated that EGFR is over-expressed in pancreatic cancer. Over-expression correlates with more advanced disease, poor survival and the presence of metastases. Therefore, inhibition of the EGFR signaling pathway is an attractive therapeutic target. Although several combinations of EGFR inhibitors with chemotherapy demonstrate inhibition of tumor-induced angiogenesis, tumor cell apoptosis and regression in xenograft models, these benefits remain to be confirmed. Multimodality treatment incorporating EGFR-inhibition is emerging as a novel strategy in the treatment of pancreatic cancer

  14. Reactive oxygen species are required for 5-HT-induced transactivation of neuronal platelet-derived growth factor and TrkB receptors, but not for ERK1/2 activation.

    Jeff S Kruk

    Full Text Available High concentrations of reactive oxygen species (ROS induce cellular damage, however at lower concentrations ROS act as intracellular second messengers. In this study, we demonstrate that serotonin (5-HT transactivates the platelet-derived growth factor (PDGF type β receptor as well as the TrkB receptor in neuronal cultures and SH-SY5Y cells, and that the transactivation of both receptors is ROS-dependent. Exogenous application of H₂O₂ induced the phosphorylation of these receptors in a dose-dependent fashion, similar to that observed with 5-HT. However the same concentrations of H₂O₂ failed to increase ERK1/2 phosphorylation. Yet, the NADPH oxidase inhibitors diphenyleneiodonium chloride and apocynin blocked both 5-HT-induced PDGFβ receptor phosphorylation and ERK1/2 phosphorylation. The increases in PDGFβ receptor and ERK1/2 phosphorylation were also dependent on protein kinase C activity, likely acting upstream of NADPH oxidase. Additionally, although the ROS scavenger N-acetyl-l-cysteine abrogated 5-HT-induced PDGFβ and TrkB receptor transactivation, it was unable to prevent 5-HT-induced ERK1/2 phosphorylation. Thus, the divergence point for 5-HT-induced receptor tyrosine kinase (RTK transactivation and ERK1/2 phosphorylation occurs at the level of NADPH oxidase in this system. The ability of 5-HT to induce the production of ROS resulting in transactivation of both PDGFβ and TrkB receptors may suggest that instead of a single GPCR to single RTK pathway, a less selective, more global RTK response to GPCR activation is occurring.

  15. Pharmacological actions of Y-24180, a new specific antagonist of platelet activating factor (PAF): II. Interactions with PAF and benzodiazepine receptors.

    Takehara, S; Mikashima, H; Muramoto, Y; Terasawa, M; Setoguchi, M; Tahara, T

    1990-12-01

    The inhibitory effect of Y-24180, 4-(2-chlorophenyl)-2-[2-(4-isobutylphenyl)ethyl]-6,9-dimethyl-6H-t hieno [3,2-f][1,2,4]triazolo [4,3-a][1,4]diazepine, on platelet activating factor (PAF)-induced platelet aggregation and the specific binding of 3H-PAF to platelets was compared with other thienodiazepine derivatives, WEB 2086 and etizolam. Y-24180 inhibited PAF-induced rabbit platelet aggregation in vitro (IC50 3.84 nM), but had little effect on adenosine diphosphate- or arachidonic acid-induced aggregation. WEB 2086 and etizolam also showed an inhibitory effect of PAF-induced aggregation (IC50 values are 456 and 6730 nM, respectively). In PAF-induced human platelet aggregation, Y-24180 (IC50 0.84 nM) was more potent than WEB 2086 (IC50 4.21 nM) and etizolam (IC50 998 nM). Y-24180, WEB 2086 and etizolam displaced 3H-PAF binding from the washed-platelets of rabbits with an IC50 value of 3.50, 9.35 and 29.5 nM, respectively. In rabbits, pretreatment with Y-24180 and WEB 2086 antagonized PAF-induced platelet aggregation dose-dependently. The significant inhibitory effect of Y-24180 (1 mg/kg, p.o.) lasted 72 hr after a single dose oral administration. WEB 2086 (10 mg/kg, p.o.) also antagonized the ex vivo response induced by PAF 1 hr after administration, but no significant effect was observed 3 hr after administration. Y-24180 displaced 3H-diazepam binding from the synaptosomal membranes of rat cerebral cortex with a Ki value of 3.68 microM. The affinity of Y-24180 for benzodiazepine(BZP) receptors was lower than those of WEB 2086 and etizolam and was about 1000 times lower than that for PAF receptors in platelets. PMID:1965554

  16. Insulin and insulin-like growth factor receptors and responses

    Insulin is a member of a family of structurally related hormones with diverse physiological functions. In humans, the best-characterized members of this family include insulin, insulin-like growth factor (IGF)-I, and IGF-II. Each of these three polypeptide hormones has its own distinct receptor. The structures of each of these receptors have now been deduced from analyses of isolated cDNA clones. To study further the responses mediated through these three different receptors, the authors have been studying cells expressing the proteins encoded by these three cDNAs. The isolated cDNAs have been transfected into Chinese hamster ovary (CHO) cells, and the resulting transfected cell lines have been characterized as to the ligand-binding activities and signal-transducing activities of the expressed proteins

  17. Phosphorylation of insulin-like growth factor I receptor by insulin receptor tyrosine kinase in intact cultured skeletal muscle cells

    The interaction between insulin and insulin-like growth factor I (IGF I) receptors was examined by determining the ability of each receptor type to phosphorylate tyrosine residues on the other receptor in intact L6 skeletal muscle cells. This was made possible through a sequential immunoprecipitation method with two different antibodies that effectively separated the phosphorylated insulin and IGF I receptors. After incubation of intact L6 cells with various concentrations of insulin or IGF I in the presence of [32P]-orthophosphate, insulin receptors were precipitated with one of two human polyclonal anti-insulin receptor antibodies (B2 or B9). Phosphorylated IGF I receptors remained in solution and were subsequently precipitated by anti-phosphotyrosine antibodies. The identifies of the insulin and IGF I receptor β-subunits in the two immunoprecipitates were confirmed by binding affinity, by phosphopeptide mapping after trypsin digestion, and by the distinct patterns of expression of the two receptors during differentiation. Stimulated phosphorylation of the β-subunit of the insulin receptor correlated with the occupancy of the β-subunit of the insulin receptor by either insulin or IGF I as determined by affinity cross-linking. Similarly, stimulation of phosphorylation of the β-subunit of the IGF I receptor by IGF I correlated with IGF I receptor occupancy. In contrast, insulin stimulated phosphorylation of the β-subunit of the IGF I receptor at hormone concentrations that were associated with significant occupancy of the insulin receptor but negligible IGF I receptor occupancy. These findings indicate that the IGF I receptor can be a substrate for the hormone-activated insulin receptor tyrosine kinase activity in intact L6 skeletal muscle cells

  18. Phosphorylation of insulin-like growth factor I receptor by insulin receptor tyrosine kinase in intact cultured skeletal muscle cells

    Beguinot, F.; Smith, R.J.; Kahn, C.R.; Maron, R.; Moses, A.C.; White, M.F.

    1988-05-03

    The interaction between insulin and insulin-like growth factor I (IGF I) receptors was examined by determining the ability of each receptor type to phosphorylate tyrosine residues on the other receptor in intact L6 skeletal muscle cells. This was made possible through a sequential immunoprecipitation method with two different antibodies that effectively separated the phosphorylated insulin and IGF I receptors. After incubation of intact L6 cells with various concentrations of insulin or IGF I in the presence of (/sup 32/P)-orthophosphate, insulin receptors were precipitated with one of two human polyclonal anti-insulin receptor antibodies (B2 or B9). Phosphorylated IGF I receptors remained in solution and were subsequently precipitated by anti-phosphotyrosine antibodies. The identifies of the insulin and IGF I receptor ..beta..-subunits in the two immunoprecipitates were confirmed by binding affinity, by phosphopeptide mapping after trypsin digestion, and by the distinct patterns of expression of the two receptors during differentiation. Stimulated phosphorylation of the ..beta..-subunit of the insulin receptor correlated with the occupancy of the ..beta..-subunit of the insulin receptor by either insulin or IGF I as determined by affinity cross-linking. Similarly, stimulation of phosphorylation of the ..beta..-subunit of the IGF I receptor by IGF I correlated with IGF I receptor occupancy. In contrast, insulin stimulated phosphorylation of the ..beta..-subunit of the IGF I receptor at hormone concentrations that were associated with significant occupancy of the insulin receptor but negligible IGF I receptor occupancy. These findings indicate that the IGF I receptor can be a substrate for the hormone-activated insulin receptor tyrosine kinase activity in intact L6 skeletal muscle cells.

  19. Direct interaction between surface β1,4-galactosyltransferase 1 and epidermal growth factor receptor (EGFR) inhibits EGFR activation in hepatocellular carcinoma

    Highlights: •β1,4GT1 interacts with EGFR both in vitro and in vivo. •β1,4GT1 co-localizes with EGFR on the cell surface. •β1,4GT1 inhibits 125I-EGF binding to EGFR. •β1,4GT1 inhibits EGF induced EGFR dimerization and phosphorylation. -- Abstract: Our previous studies showed that cell surface β1,4-galactosyltransferase 1 (β1,4GT1) negatively regulated cell survival through inhibition and modulation of the epidermal growth factor receptor (EGFR) signaling pathway in human hepatocellular carcinoma (HCC) SMMC-7721 cells. However, the underlying mechanism remains unclear. Here we demonstrated that β1,4-galactosyltransferase 1 (β1,4GT1) interacted with EGFR in vitro by GST pull-down analysis. Furthermore, we demonstrated that β1,4GT1 bound to EGFR in vivo by co-immunoprecipitation and determined the co-localization of β1,4GT1 and EGFR on the cell surface via confocal laser scanning microscopy analysis. Finally, using 125I-EGF binding experiments and Western blot analysis, we found that overexpression of β1,4GT1 inhibited 125I-EGF binding to EGFR, and consequently reduced the levels of EGFR dimerization and phosphorylation. In contrast, RNAi-mediated knockdown of β1,4GT1 increased the levels of EGFR dimerization and phosphorylation. These data suggest that cell surface β1,4GT1 interacts with EGFR and inhibits EGFR activation

  20. NMDA Receptor Activation by Spontaneous Glutamatergic Neurotransmission

    Espinosa, Felipe; Kavalali, Ege T.

    2009-01-01

    Under physiological conditions N-methyl-d-aspartate (NMDA) receptor activation requires coincidence of presynaptic glutamate release and postsynaptic depolarization due to the voltage-dependent block of these receptors by extracellular Mg2+. Therefore spontaneous neurotransmission in the absence of action potential firing is not expected to lead to significant NMDA receptor activation. Here we tested this assumption in layer IV neurons in neocortex at their resting membrane potential (approxi...

  1. A protease-activated receptor 2 agonist (AC-264613) suppresses interferon regulatory factor 5 and decreases interleukin-12p40 production by lipopolysaccharide-stimulated macrophages: Role of p53.

    Yamaguchi, Rui; Yamamoto, Takatoshi; Sakamoto, Arisa; Ishimaru, Yasuji; Narahara, Shinji; Sugiuchi, Hiroyuki; Yamaguchi, Yasuo

    2016-06-01

    The transcription factor interferon regulatory factor 5 (IRF5) has a key role in the production of interleukin (IL)-12 by macrophages. IRF5 is also a central mediator of toll-like receptor signaling and is a direct target of p53. Activation of protease-activated receptor 2 (PAR-2) upregulates p53 and suppresses apoptosis. This study investigated the influence of human neutrophil elastase (HNE) and PAR-2 agonists on expression of IRF5 and IL-12p40 by macrophages stimulated with lipopolysaccharide. Granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent macrophages showed upregulation of IRF5 expression, while HNE reduced expression of p53 and IRF5 in a concentration-dependent manner. HNE also caused a concentration-dependent decrease of IRF5 in macrophages transfected with small interfering RNA to silence p53, while silencing of β-arrestin 2 blunted the reduction of p53 or IRF5 by HNE. Incubation of macrophages with a PAR-2 agonist, AC-264613, caused a decrease of IRF5 expression and also significantly reduced p53 protein expression. HNE upregulated the expression of tumor necrosis factor receptor-associated factor 6 (TRAF6) and caused transactivation of TLR4, while AC-264613 did not promote TLR4 transactivation. In conclusion, the PAR-2 agonist AC-264613 attenuated IRF5-associated IL-12p40 production by macrophages. PMID:26833899

  2. Activation of microglial cells triggers a release of brain-derived neurotrophic factor (BDNF inducing their proliferation in an adenosine A2A receptor-dependent manner: A2A receptor blockade prevents BDNF release and proliferation of microglia

    Gomes Catarina

    2013-01-01

    Full Text Available Abstract Background Brain-derived neurotrophic factor (BDNF has been shown to control microglial responses in neuropathic pain. Since adenosine A2A receptors (A2ARs control neuroinflammation, as well as the production and function of BDNF, we tested to see if A2AR controls the microglia-dependent secretion of BDNF and the proliferation of microglial cells, a crucial event in neuroinflammation. Methods Murine N9 microglial cells were challenged with lipopolysaccharide (LPS, 100 ng/mL in the absence or in the presence of the A2AR antagonist, SCH58261 (50 nM, as well as other modulators of A2AR signaling. The BDNF cellular content and secretion were quantified by Western blotting and ELISA, A2AR density was probed by Western blotting and immunocytochemistry and cell proliferation was assessed by BrdU incorporation. Additionally, the A2AR modulation of LPS-driven cell proliferation was also tested in primary cultures of mouse microglia. Results LPS induced time-dependent changes of the intra- and extracellular levels of BDNF and increased microglial proliferation. The maximal LPS-induced BDNF release was time-coincident with an LPS-induced increase of the A2AR density. Notably, removing endogenous extracellular adenosine or blocking A2AR prevented the LPS-mediated increase of both BDNF secretion and proliferation, as well as exogenous BDNF-induced proliferation. Conclusions We conclude that A2AR activation plays a mandatory role controlling the release of BDNF from activated microglia, as well as the autocrine/paracrine proliferative role of BDNF.

  3. Biochemical and biological properties of the nerve growth factor receptor

    We have utilized a monoclonal antibody (192-IgG) to study the rat nerve growth factor receptor. After intraocular injection, 125I-192-IgG was retrogradely transported in sympathetic neuronal axons to the superior cervical ganglion. When the sciatic nerve was ligated to induce the accumulation of axonally transported materials, 192-IgG immunostaining was observed on both sides of the ligature, indicating that NGF receptors are transported in both orthograde and retrograde directions. By using 125I-NGF crosslinking and 192-IgG immunoprecipitation, we detected receptor molecules throughout the rat brain, thereby supporting the hypothesis that NGF is active in the central nervous system. We also discovered that sciatic nerve transection leads to a dramatic increase in the amount of NGF receptor found in the distal portion of the nerve. Immunostaining revealed that all Schwann cells in the distal axotomized nerve were expressing NGF receptors. We examined phosphorylation of NGF receptor in cultured sympathetic neurons and PC12 cells. We also examined pharmacological effects of 192-IgG. Systemic injection of 192-IgG into neonatal rats caused a permanent partial sympathectomy in a dose-dependent manner; a maximum of 50% of the cells were killed

  4. Penehyclidine ameliorates acute lung injury by inhibiting Toll-like receptor 2/4 expression and nuclear factor-κB activation

    WANG, NA; SU, YUE; CHE, XIANG-MING; ZHENG, HUI; SHI, ZHI-GUO

    2016-01-01

    The aim of the present study was to investigate the effect of penehyclidine (PHC) on endotoxin-induced acute lung injury (ALI), as well as to examine the mechanism underlying this effect. A total of 60 rats were randomly divided into five groups, including the control (saline), LPS and three LPS + PHC groups. ALI was induced in the rats by injection of 8 mg lipopolysaccharide (LPS)/kg body weight. The rats were then treated with or without PHC at 0.3, 1 or 3 mg/kg body weight 1 min following LPS injection. After 6 h, serum levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 were determined by ELISA. In addition, the mRNA expression levels of toll-like receptor (TLR)2 and TLR4 were examined by reverse transcription-quantitative polymerase chain reaction in the lung tissue samples, and nuclear factor (NF)-κB p65 protein expression levels were examined by western blot analysis. The results demonstrated that lung injury was ameliorated by treatment with PHC (1 and 3 mg/kg body weight) as compared with treatment with LPS alone. Injection of LPS significantly increased the mRNA expression levels of TLR2 and TLR4, as well as the protein expression levels of NF-κB p65 in the lung tissue samples. Serum levels of TNF-α and IL-6 were also upregulated by LPS injection. Treatment of the rats with PHC following LPS injection suppressed the LPS-induced increase in TLR2/4 mRNA and NF-κB p65 protein expression levels. PHC also inhibited the increase in TNF-α and IL-6 serum levels. In addition, PHC reduced LPS-induced ALI and decreased the serum levels of TNF-α and IL-6, possibly by downregulating TLR2/4 mRNA expression and inhibiting NF-κB activity, and consequently alleviating the inflammatory response. PMID:27168812

  5. Determination of the exact molecular requirements for type 1 angiotensin receptor epidermal growth factor receptor transactivation and cardiomyocyte hypertrophy.

    Smith, Nicola J; Chan, Hsiu-Wen; Qian, Hongwei; Bourne, Allison M; Hannan, Katherine M; Warner, Fiona J; Ritchie, Rebecca H; Pearson, Richard B; Hannan, Ross D; Thomas, Walter G

    2011-05-01

    Major interest surrounds how angiotensin II triggers cardiac hypertrophy via epidermal growth factor receptor transactivation. G protein-mediated transduction, angiotensin type 1 receptor phosphorylation at tyrosine 319, and β-arrestin-dependent scaffolding have been suggested, yet the mechanism remains controversial. We examined these pathways in the most reductionist model of cardiomyocyte growth, neonatal ventricular cardiomyocytes. Analysis with [(32)P]-labeled cardiomyocytes, wild-type and [Y319A] angiotensin type 1 receptor immunoprecipitation and phosphorimaging, phosphopeptide analysis, and antiphosphotyrosine blotting provided no evidence for tyrosine phosphorylation at Y319 or indeed of the receptor, and mutation of Y319 (to A/F) did not prevent either epidermal growth factor receptor transactivation in COS-7 cells or cardiomyocyte hypertrophy. Instead, we demonstrate that transactivation and cardiomyocyte hypertrophy are completely abrogated by loss of G-protein coupling, whereas a constitutively active angiotensin type 1 receptor mutant was sufficient to trigger transactivation and growth in the absence of ligand. These results were supported by the failure of the β-arrestin-biased ligand SII angiotensin II to transactivate epidermal growth factor receptor or promote hypertrophy, whereas a β-arrestin-uncoupled receptor retained these properties. We also found angiotensin II-mediated cardiomyocyte hypertrophy to be attenuated by a disintegrin and metalloprotease inhibition. Thus, G-protein coupling, and not Y319 phosphorylation or β-arrestin scaffolding, is required for epidermal growth factor receptor transactivation and cardiomyocyte hypertrophy via the angiotensin type 1 receptor. PMID:21383310

  6. Hormone activation of baculovirus expressed progesterone receptors.

    Elliston, J F; Beekman, J M; Tsai, S Y; O'Malley, B W; Tsai, M J

    1992-03-15

    Human and chicken progesterone receptors (A form) were overproduced in a baculovirus expression system. These recombinant progesterone receptors were full-length bound progesterone specifically and were recognized by monoclonal antibodies, AB52 and PR22, specific for human and chicken progesterone receptor, respectively. In gel retardation studies, binding of recombinant human and chicken progesterone receptors to their progesterone response element (PRE) was specific and was enhanced in the presence of progesterone. Binding of human progesterone receptor to the PRE was also enhanced in the presence of the antiprogestin, RU486, but very little effect was observed in the presence of estradiol, dexamethasone, testosterone, and vitamin D. In our cell-free transcription system, human progesterone receptor induced transcription in a receptor-dependent and hormone-activable manner. Receptor-stimulated transcription required the presence of the PRE in the test template and could be specifically inhibited by excess PRE oligonucleotides. Furthermore, chicken progesterone receptor also induced in vitro transcription in a hormone-activable manner. These results demonstrate that steroid receptors overexpressed in a baculovirus expression system are functional and exhibit steroid-responsive binding and transcription. These observations support our present understanding of the mechanism of steroid receptor-regulated gene expression and provide a technological format for studies of the role of hormone and antihormone in altering gene expression. PMID:1544902

  7. Peroxisome proliferator-activated receptors and biotransformation responses in relation to condition factor and contaminant burden in tilapia species from Ogun River, Nigeria.

    Adeogun, Aina O; Ibor, Oju R; Regoli, Francesco; Arukwe, Augustine

    2016-01-01

    A major development in fishery science has been the Fulton's condition factor (CF) as a reliable physiological index of fish growth and health status (Fulton 1902). As a general rule, CF-value greater than 1 (>1) should be regarded as an indicator for good growth and health. Therefore, exposure of fish to contaminants in the environment will be expected to produce a reduction in scope for growth, since energy for growth will be allocated to overcome stressful conditions. In the present study, we hypothesized that tilapia species from Ogun River (Nigeria) are experiencing severe contaminant-induced obesogen effects leading to high CF (≥2) in fish with pathological alterations. The environmental obesogen hypothesis has related the interaction between environmental pollutants and PPAR isoform activation In this respect, peroxisome proliferator-activated receptors (PPARs) and biotransformation responses in relation to contaminant burden were investigated in a total of 1074 specimens of Tilapias species (Tilapia guineensis, Sarotherodon galileaus and Oreochromis niloticus) collected from three areas with different degrees of anthropogenic contamination and from a putative control site along the Ogun River. Liver mRNA expression of cytochrome cyp1 isoforms (cyp1a, 1b and 1c) and PPAR isoforms (ppar-α, β and γ) were analyzed using validated real-time PCR. Fish were also analyzed for CF and muscle contaminant burden (aliphatic and polycyclic aromatic hydrocarbons, organochlorine pesticides, and polychlorinated biphenyls). A significant increase in mRNA expression of cyp1- and ppar isoforms was observed in fish from polluted areas, and these results paralleled data on PCBs and PAHs tissue concentrations. Further, cyp1 isoforms showed clear sex-related differences, with higher mRNA expression in male fish than in females. Principal component analysis revealed a relationship between cyp1 isoforms, ppar-α, β, PCBs and PAHs and these interactions may suggest a crosstalk

  8. Amphetamine elevates phosphorylation of eukaryotic initiation factor 2α (eIF2α) in the rat forebrain via activating dopamine D1 and D2 receptors.

    Xue, Bing; Fitzgerald, Cole A; Jin, Dao-Zhong; Mao, Li-Min; Wang, John Q

    2016-09-01

    Psychostimulants have an impact on protein synthesis, although underlying molecular mechanisms are unclear. Eukaryotic initiation factor 2α-subunit (eIF2α) is a key player in initiation of protein translation and is regulated by phosphorylation. While this factor is sensitive to changing synaptic input and is critical for synaptic plasticity, its sensitivity to stimulants is poorly understood. Here we systematically characterized responses of eIF2α to a systemic administration of the stimulant amphetamine (AMPH) in dopamine responsive regions of adult rat brains. Intraperitoneal injection of AMPH at 5mg/kg increased eIF2α phosphorylation at serine 51 in the striatum. This increase was transient. In the medial prefrontal cortex (mPFC), AMPH induced a relatively delayed phosphorylation of the factor. Pretreatment with a dopamine D1 receptor antagonist SCH23390 blocked the AMPH-stimulated eIF2α phosphorylation in both the striatum and mPFC. Similarly, a dopamine D2 receptor antagonist eticlopride reduced the effect of AMPH in the two regions. Two antagonists alone did not alter basal eIF2α phosphorylation. AMPH and two antagonists did not change the amount of total eIF2α proteins in both regions. These results demonstrate the sensitivity of eIF2α to stimulant exposure. AMPH possesses the ability to stimulate eIF2α phosphorylation in striatal and mPFC neurons in vivo in a D1 and D2 receptor-dependent manner. PMID:27338925

  9. Correlation of serum tumor necrosis factor-alpha, interleukin-4 and soluble interleukin-2 receptor levels with radiologic and clinical manifestations in active pulmonary tuberculosis.

    Levent Kart; Hakan Buyukoglan; Ishak O. Tekin; Remzi Altin; Zuhal Senturk; Inci Gulmez; Ramazan Demir; Mustafa Ozesmi

    2003-01-01

    The precise clinical manifestations of tuberculosis are likely to result from a complex interaction between the host and the pathogen. We took serum samples from a group of patients with a variety of clinical and radiological stages of pulmonary tuberculosis in order to characterize tumor necrosis factor-alpha (TNF-alpha), interleukin-4 (IL-4) and soluble interleukin-2 receptor (sIL-2R) response. We further evaluated whether the levels of TNF-alpha, IL-4 and soluble IL-2R are related with eac...

  10. Multikinase activity of fibroblast growth factor receptor (FGFR) inhibitors SU5402, PD173074, AZD1480, AZD4547 and BGJ398 compromises the use of small chemicals targeting FGFR catalytic activity for therapy of short-stature syndromes.

    Gudernova, Iva; Vesela, Iva; Balek, Lukas; Buchtova, Marcela; Dosedelova, Hana; Kunova, Michaela; Pivnicka, Jakub; Jelinkova, Iva; Roubalova, Lucie; Kozubik, Alois; Krejci, Pavel

    2016-01-01

    Activating mutations in the fibroblast growth factor receptor 3 (FGFR3) cause the most common genetic form of human dwarfism, achondroplasia (ACH). Small chemical inhibitors of FGFR tyrosine kinase activity are considered to be viable option for treating ACH, but little experimental evidence supports this claim. We evaluated five FGFR tyrosine kinase inhibitors (TKIs) (SU5402, PD173074, AZD1480, AZD4547 and BGJ398) for their activity against FGFR signaling in chondrocytes. All five TKIs strongly inhibited FGFR activation in cultured chondrocytes and limb rudiment cultures, completely relieving FGFR-mediated inhibition of chondrocyte proliferation and maturation. In contrast, TKI treatment of newborn mice did not improve skeletal growth and had lethal toxic effects on the liver, lungs and kidneys. In cell-free kinase assays as well as in vitro and in vivo cell assays, none of the tested TKIs demonstrated selectivity for FGFR3 over three other FGFR tyrosine kinases. In addition, the TKIs exhibited significant off-target activity when screened against a panel of 14 unrelated tyrosine kinases. This was most extensive in SU5402 and AZD1480, which inhibited DDR2, IGF1R, FLT3, TRKA, FLT4, ABL and JAK3 with efficiencies similar to or greater than those for FGFR. Low target specificity and toxicity of FGFR TKIs thus compromise their use for treatment of ACH. Conceptually, different avenues of therapeutic FGFR3 targeting should be investigated. PMID:26494904

  11. NMDA receptor activation by spontaneous glutamatergic neurotransmission.

    Espinosa, Felipe; Kavalali, Ege T

    2009-05-01

    Under physiological conditions N-methyl-D-aspartate (NMDA) receptor activation requires coincidence of presynaptic glutamate release and postsynaptic depolarization due to the voltage-dependent block of these receptors by extracellular Mg(2+). Therefore spontaneous neurotransmission in the absence of action potential firing is not expected to lead to significant NMDA receptor activation. Here we tested this assumption in layer IV neurons in neocortex at their resting membrane potential (approximately -67 mV). In long-duration stable recordings, we averaged a large number of miniature excitatory postsynaptic currents (mEPSCs, >100) before or after application of dl-2 amino 5-phosphonovaleric acid, a specific blocker of NMDA receptors. The difference between the two mEPSC waveforms showed that the NMDA current component comprises approximately 20% of the charge transfer during an average mEPSC detected at rest. Importantly, the contribution of the NMDA component was markedly enhanced at membrane potentials expected for the depolarized up states (approximately -50 mV) that cortical neurons show during slow oscillations in vivo. In addition, partial block of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor component of the mEPSCs did not cause a significant reduction in the NMDA component, indicating that potential AMPA receptor-driven local depolarizations did not drive NMDA receptor activity at rest. Collectively these results indicate that NMDA receptors significantly contribute to signaling at rest in the absence of dendritic depolarizations or concomitant AMPA receptor activity. PMID:19261712

  12. Profiling epidermal growth factor receptor and heregulin receptor 3 heteromerization using receptor tyrosine kinase heteromer investigation technology.

    Mohammed Akli Ayoub

    Full Text Available Heteromerization can play an important role in regulating the activation and/or signal transduction of most forms of receptors, including receptor tyrosine kinases (RTKs. The study of receptor heteromerization has evolved extensively with the emergence of resonance energy transfer based approaches such as bioluminescence resonance energy transfer (BRET. Here, we report an adaptation of our Receptor-Heteromer Investigation Technology (Receptor-HIT that has recently been published as the G protein-coupled receptor (GPCR Heteromer Identification Technology (GPCR-HIT. We now demonstrate the utility of this approach for investigating RTK heteromerization by examining the functional interaction between the epidermal growth factor (EGF receptor (EGFR; also known as erbB1/HER1 and heregulin (HRG receptor 3 (HER3; also known as erbB3 in live HEK293FT cells using recruitment of growth factor receptor-bound protein 2 (Grb2 to the activated receptors. We found that EGFR and HER3 heteromerize specifically as demonstrated by HRG inducing a BRET signal between EGFR/Rluc8 and Grb2/Venus only when HER3 was co-expressed. Similarly, EGF stimulation promoted a specific BRET signal between HER3/Rluc8 and Grb2/Venus only when EGFR was co-expressed. Both EGF and HRG effects on Grb2 interaction are dose-dependent, and specifically blocked by EGFR inhibitor AG-1478. Furthermore, truncation of HER3 to remove the putative Grb2 binding sites appears to abolish EGF-induced Grb2 recruitment to the EGFR-HER3 heteromer. Our results support the concept that EGFR interacts with Grb2 in both constitutive and EGF-dependent manners and this interaction is independent of HER3 co-expression. In contrast, HER3-Grb2 interaction requires the heteromerization between EGFR and HER3. These findings clearly indicate the importance of EGFR-HER3 heteromerization in HER3-mediated Grb2-dependent signaling pathways and supports the central role of HER3 in the diversity and regulation of HER

  13. Interactions of the ubiquitous octamer-binding transcription factor-1 with both the signal transducer and activator of transcription 5 and the glucocorticoid receptor mediate prolactin and glucocorticoid-induced β-casein gene expression in mammary epithelial cells.

    Qian, Xi; Zhao, Feng-Qi

    2013-03-01

    Regulation of milk protein gene expression by lactogenic hormones (prolactin and glucocorticoids) provides an attractive model for studying the mechanisms by which protein and steroid hormones synergistically regulate gene expression. β-Casein is one of the major milk proteins and its expression in mammary epithelial cells is stimulated by lactogenic hormones. The signal transducer and activator of transcription 5 and glucocorticoid receptor are essential downstream mediators of prolactin and glucocorticoid signaling, respectively. Previous studies have shown that mutating the octamer-binding site of the β-casein gene proximal promoter dramatically reduces the hormonal induction of the promoter activity. However, little is known about the underlying molecular mechanisms. In this report, we show that lactogenic hormones rapidly induce the binding of octamer-binding transcription factor-1 to the β-casein promoter and this induction is not mediated by either increasing the expression of octamer-binding transcription factor-1 or inducing its translocation to the nucleus. Rather, lactogenic hormones induce physical interactions between the octamer-binding transcription factor-1, signal transducer and activator of transcription 5, and glucocorticoid receptor to form a ternary complex, and these interactions enhance or stabilize the binding of these transcription factors to the promoter. Abolishing these interactions significantly reduces the hormonal induction of β-casein gene transcription. Thus, our study indicates that octamer-binding transcription factor-1 may serve as a master regulator that facilitates the DNA binding of both signal transducer and activator of transcription 5 and glucocorticoid receptor in hormone-induced β-casein expression, and defines a novel mechanism of regulation of tissue-specific gene expression by the ubiquitous octamer-binding transcription factor-1. PMID:23313770

  14. Multikinase activity of fibroblast growth factor receptor (FGFR) inhibitors SU5402, PD173074, AZD1480, AZD4547 and BGJ398 compromises the use of small chemicals targeting FGFR catalytic activity for therapy of short-stature syndromes

    Gudernová, I.; Veselá, Iva; Balek, L.; Buchtová, Marcela; Dosedělová, Hana; Kunová, M.; Pivnička, J.; Jelínková, I.; Roubalová, L.; Kozubík, Alois; Krejčí, P.

    2016-01-01

    Roč. 25, č. 1 (2016), s. 9-23. ISSN 0964-6906 R&D Projects: GA ČR(CZ) GA14-31540S Institutional support: RVO:67985904 ; RVO:68081707 Keywords : fibroblast growth factor receptor * tyrosine kinase domain * ERK MAP kinase Subject RIV: EA - Cell Biology Impact factor: 6.393, year: 2014

  15. The Complex of Ciliary Neurotrophic Factor-Ciliary Neurotrophic Factor Receptor α Up-Regulates Connexin43 and Intercellular Coupling in Astrocytes via the Janus Tyrosine Kinase/Signal Transducer and Activator of Transcription PathwayD⃞

    Ozog, Mark A.; Bernier, Suzanne M; Bates, Dave C.; Chatterjee, Bishwanath; Lo, Cecilia W.; Naus, Christian C.G.

    2004-01-01

    Cytokines regulate numerous cell processes, including connexin expression and gap junctional coupling. In this study, we examined the effect of ciliary neurotrophic factor (CNTF) on connexin43 (Cx43) expression and intercellular coupling in astrocytes. Murine cortical astrocytes matured in vitro were treated with CNTF (20 ng/ml), soluble ciliary neurotrophic factor receptor α (CNTFRα) (200 ng/ml), or CNTF-CNTFRα. Although CNTF and CNTFRα alone had no effect on Cx43 expression, the heterodimer...

  16. Activation of Epidermal Growth Factor Receptor/p38/Hypoxia-inducible Factor-1α Is Pivotal for Angiogenesis and Tumorigenesis of Malignantly Transformed Cells Induced by Hexavalent Chromium.

    Kim, Donghern; Dai, Jin; Park, Youn-Hee; Fai, Leonard Yenwong; Wang, Lei; Pratheeshkumar, Poyil; Son, Young-Ok; Kondo, Kazuya; Xu, Mei; Luo, Jia; Shi, Xianglin; Zhang, Zhuo

    2016-07-29

    Hexavalent chromium (Cr(VI))-containing compounds are well established environmental carcinogens. Most mechanistic investigations of Cr(VI)-induced carcinogenesis focus on oxidative stress and various cellular responses, leading to malignant cell transformation or the first stage of metal-induced carcinogenesis. The development of malignantly transformed cells into tumors that require angiogenesis is the second stage. This study focuses on the second stage, in particular, the role of EGF receptor (EGFR) signaling in angiogenesis and tumorigenesis of Cr(VI)-transformed cells. Our preliminary studies have shown that EGFR is constitutively activated in Cr(VI)-transformed cells, in lung tissue from Cr(VI)-exposed animals, and in lung tumor tissue from a non-smoking worker occupationally exposed to Cr(VI) for 19 years. Using in vitro and in vivo models, the present study has investigated the role of EGFR in angiogenesis of Cr(VI)-transformed cells. The results show that Cr(VI)-transformed cells are angiogenic. Hypoxia-inducible factor-1α, pro-angiogenic protein matrix metalloproteinase 1, and VEGF are all highly expressed in Cr(VI)-transformed cells, in lung tissue from animals exposed to Cr(VI), and in lung tumor tissue from a non-smoking worker occupationally exposed to Cr(VI) for 19 years. p38 MAPK is also activated in Cr(VI)-transformed cells and in human lung tumor tissue. Inhibition of EGFR reduces p38 MAPK, resulting in decreased expression of hypoxia-inducible factor-1α, metalloproteinase 1, and VEGF, leading to suppressions of angiogenesis and tumorigenesis. Overall, the present study has demonstrated that EGFR plays an important role in angiogenesis and tumorigenesis of Cr(VI)-transformed cells. PMID:27226640

  17. Functional and Physical Interactions among Saccharomyces cerevisiae α-Factor Receptors

    Gehret, Austin U.; Connelly, Sara M.; Dumont, Mark E.

    2012-01-01

    The α-factor receptor Ste2p is a G protein-coupled receptor (GPCR) expressed on the surface of MATa haploid cells of the yeast Saccharomyces cerevisiae. Binding of α-factor to Ste2p results in activation of a heterotrimeric G protein and of the pheromone response pathway. Functional interactions between α-factor receptors, such as dominant-negative effects and recessive behavior of constitutive and hypersensitive mutant receptors, have been reported previously. We show here that dominant-nega...

  18. Isolation of an additional member of the fibroblast growth factor receptor family, FGFR-3

    The fibroblast growth factors are a family of polypeptide growth factors involved in a variety of activities including mitogenesis, angiogenesis, and wound healing. Fibroblast growth factor receptors (FGFRs) have previously been identified in chicken, mouse, and human and have been shown to contain an extracellular domain with either two or three immunoglobulin-like domains, a transmembrane domain, and a cytoplasmic tyrosine kinase domain. The authors have isolated a human cDNA for another tyrosine kinase receptor that is highly homologous to the previously described FGFR. Expression of this receptor cDNA in COS cells directs the expression of a 125-kDa glycoprotein. They demonstrate that this cDNA encodes a biologically active receptor by showing that human acidic and basic fibroblast growth factors activate this receptor as measured by 45Ca2+ efflux assays. These data establish the existence of an additional member of the FGFR family that they have named FGFR-3

  19. Epidermal Growth Factor Receptor (EGFR) Crosstalks in Liver Cancer

    Hepatocarcinogenesis is a complex multistep process in which many different molecular pathways have been implicated. Hepatocellular carcinoma (HCC) is refractory to conventional chemotherapeutic agents, and the new targeted therapies are meeting with limited success. Interreceptor crosstalk and the positive feedback between different signaling systems are emerging as mechanisms of targeted therapy resistance. The identification of such interactions is therefore of particular relevance to improve therapeutic efficacy. Among the different signaling pathways activated in hepatocarcinogenesis the epidermal growth factor receptor (EGFR) system plays a prominent role, being recognized as a “signaling hub” where different extracellular growth and survival signals converge. EGFR can be transactivated in response to multiple heterologous ligands through the physical interaction with multiple receptors, the activity of intracellular kinases or the shedding of EGFR-ligands. In this article we review the crosstalk between the EGFR and other signaling pathways that could be relevant to liver cancer development and treatment

  20. Evidence that the modulator of the glucocorticoid-receptor complex is the endogenous molybdate factor.

    Bodine, P V; Litwack, G

    1988-01-01

    We have recently purified the modulator of the glucocorticoid-receptor complex from rat liver. Purified modulator inhibits glucocorticoid-receptor complex activation and stabilizes the steroid-binding ability of the unoccupied glucocorticoid receptor. Since these activities are shared by exogenous sodium molybdate, modulator appears to be the endogenous factor that sodium molybdate mimics. In this report, we present additional evidence for the mechanism of action of purified modulator. (i) Mo...

  1. Insulin-like growth factor I receptor dose not contribute to heat shock-induced activation of Akt and extracellular signal-regulated kinase (ERK) in mouse embryo fibroblasts

    We have investigated the role of insulin-like growth factor I receptor (IGF-IR) in heat shock-induced activation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3' kinase (PI3-K) pathways. We utilized mouse embryo fibroblasts (MEFs) devoid of endogenous IGF-LR (R-) and MEFs overexpressing human IGF-IR (WT) and examined the activation kinetics of extracellular signal-regulated kinase (ERK) and Akt following heat shock treatment. There were no differences in the kinetics or temperature dependence of activation of either ERK or Akt between the cell lines. As expected, heat shock failed to induce autophosphorylation of IGF-IR overexpressed in WT cells. Surprisingly, the autophosphorylation of endogenous epidermal growth factor receptor (EGFR), which is thought to play an important role in heat shock-induced activation of the MAPK and PI3-K pathways, was not observed in either WT or R-cells. These results suggest that neither IGF-IR nor EGFR contributes to the heat shock-induced activation of ERK and Akt in these cell lines. (author)

  2. CD44 function as a growth factor co-receptor

    Chen, L.

    2003-06-01

    CD44 splice variant proteins containing exon v6 encoded sequence have been implicated in tumour metastasis. The work presented in this thesis shows that CD44 isoforms containing exon v6 encoded sequences act as coreceptor for the c-Met receptor, a tyrosine kinase receptor that is involved in growth control and invasive growth. The c-Met receptor and its ligand hepatocyte growth factor (HGF) have also been implicated in tumour metastasis. My results show the cooperation between CD44, HGF and c-Met. A CD44 isoform containing variant exon v6 encoded sequences is strictly required for c-Met activation by HGF/SF in rat and human carcinoma cells. In a non-metastatic cell line BSp73AS cells which only expressed CD44 standard form, HGF can not activate c-Met. Upon transfection with the CD44 bearing v6 encoded sequences, the cells become HGF inducible. Antibodies against two CD44 exon v6-encoded epitopes inhibit autophosphorylation of c-Met. The CD44 isoform is required for the assembly of signalling complex containing at least HGF, c-Met and CD44 v6 bearing isoform. Furthermore, this growth factor co-receptor function could be a more general mechanism. I have investigated the involvement of CD44 isoforms in the signalling by the EGF receptor family. My results show that HB-EGF, EGF and Amphiregulin activate their receptors in a CD44 dependent manner. CD44 v6 specific antibodies can interfere with HB-EGF, EGF and Amphiregulin signalling both at Erk level and at receptor level. (orig.) [German] In der nicht metastasierenden Zelllinie Bsp73 AS, die ausschliesslich die CD44 Standardform exprimiert, fuehrt HGF nicht zur Aktivierung von c-Met. Durch Transfektion mit unterschiedlichen CD44 v6 enthaltenden Isoformen, werden die Zellen HGF-induzierbar. Antikoerper gegen zwei von CD44 Exon v6 kodierte Epitope verhindern die Autophosphorylierung von c-Met. Die CD44 Isoform wird zur Bildung eines Signalkomplexes benoetigt, der zumindest HGF, c-Met und CD44v6 tragende Isoformen

  3. Profiling Epidermal Growth Factor Receptor and Heregulin Receptor 3 Heteromerization Using Receptor Tyrosine Kinase Heteromer Investigation Technology

    Mohammed Akli Ayoub; Heng B See; Seeber, Ruth M.; Armstrong, Stephen P.; Pfleger, Kevin D.G.

    2013-01-01

    Heteromerization can play an important role in regulating the activation and/or signal transduction of most forms of receptors, including receptor tyrosine kinases (RTKs). The study of receptor heteromerization has evolved extensively with the emergence of resonance energy transfer based approaches such as bioluminescence resonance energy transfer (BRET). Here, we report an adaptation of our Receptor-Heteromer Investigation Technology (Receptor-HIT) that has recently been published as the G p...

  4. Peroxisome proliferator-activated receptors, metabolic syndrome and cardiovascular disease

    Azhar, Salman

    2010-01-01

    Metabolic syndrome (MetS) is a constellation of risk factors including insulin resistance, central obesity, dyslipidemia and hypertension that markedly increase the risk of Type 2 diabetes (T2DM) and cardiovascular disease (CVD). The peroxisome proliferators-activated receptor (PPAR) isotypes, PPARα, PPARδ/β and PPARγ are ligand-activated nuclear transcription factors, which modulate the expression of an array of genes that play a central role in regulating glucose, lipid and cholesterol meta...

  5. Injury-induced innate immune response in human skin mediated by transactivation of the epidermal growth factor receptor

    Sørensen, Ole E.; Thapa, Dharma R.; Roupé, K. Markus; Valore, Erika V.; Sjöbring, Ulf; Roberts, Alice A.; Schmidtchen, Artur; Ganz, Tomas

    2006-01-01

    We found that sterile wounding of human skin induced epidermal expression of the antimicrobial (poly)peptides human β-defensin–3, neutrophil gelatinase–associated lipocalin, and secretory leukocyte protease inhibitor through activation of the epidermal growth factor receptor. After skin wounding, the receptor was activated by heparin-binding epidermal growth factor that was released by a metalloprotease-dependent mechanism. Activation of the epidermal growth factor receptor generated antimicr...

  6. Androgen insensitivity syndrome: gonadal androgen receptor activity

    To determine whether abnormalities of the androgen receptor previously observed in skin fibroblasts from patients with androgen insensitivity syndrome also occur in the gonads of affected individuals, androgen receptor activity in the gonads of a patient with testicular feminization syndrome was investigated. Using conditions for optimal recovery of androgen receptor from human testes established by previous studies, we detected the presence of a high-affinity (dissociation constant . 3.2 X 10(-10) mol/L), low-capacity (4.2 X 10(-12) mol/mg DNA), androgen-binding protein when tritium-labeled R1881 was incubated at 4 degrees C with nuclear extracts from the gonads of control patients or from a patient with testicular feminization syndrome but not when incubated at 37 degrees C. Thus this patient has an androgen receptor with a temperature lability similar to that of receptors from normal persons

  7. Poxviral Protein A52 Stimulates p38 Mitogen-activated Protein Kinase (MAPK) Activation by Causing Tumor Necrosis Factor Receptor-associated Factor 6 (TRAF6) Self-association Leading to Transforming Growth Factor β-activated Kinase 1 (TAK1) Recruitment*

    Stack, Julianne; Hurst, Tara P.; Flannery, Sinead M.; Brennan, Kiva; Rupp, Sebastian; Oda, Shun-ichiro; Khan, Amir R.; Bowie, Andrew G.

    2013-01-01

    Vaccinia virus encodes a number of proteins that inhibit and manipulate innate immune signaling pathways that also have a role in virulence. These include A52, a protein shown to inhibit IL-1- and Toll-like receptor-stimulated NFκB activation, via interaction with interleukin-1 receptor-associated kinase 2 (IRAK2). Interestingly, A52 was also found to activate p38 MAPK and thus enhance Toll-like receptor-dependent IL-10 induction, which was TRAF6-dependent, but the manner in which A52 manipulates TRAF6 to stimulate p38 activation was unclear. Here, we show that A52 has a non-canonical TRAF6-binding motif that is essential for TRAF6 binding and p38 activation but dispensable for NFκB inhibition and IRAK2 interaction. Wild-type A52, but not a mutant defective in p38 activation and TRAF6 binding (F154A), caused TRAF6 oligomerization and subsequent TRAF6-TAK1 association. The crystal structure of A52 shows that it adopts a Bcl2-like fold and exists as a dimer in solution. Residue Met-65 was identified as being located in the A52 dimer interface, and consistent with that, A52-M65E was impaired in its ability to dimerize. A52-M65E although capable of interacting with TRAF6, was unable to cause either TRAF6 self-association, induce the TRAF6-TAK1 association, or activate p38 MAPK. The results suggest that an A52 dimer causes TRAF6 self-association, leading to TAK1 recruitment and p38 activation. This reveals a molecular mechanism whereby poxviruses manipulate TRAF6 to activate MAPKs (which can be proviral) without stimulating antiviral NFκB activation. PMID:24114841

  8. Are different stoichiometries feasible for complexes between lymphotoxin-alpha and tumor necrosis factor receptor 1?

    Mascarenhas Nahren; Kästner Johannes

    2012-01-01

    Abstract Background Tumor necrosis factors, TNF and lymphotoxin-α (LT), are cytokines that bind to two receptors, TNFR1 and TNFR2 (TNF-receptor 1 and 2) to trigger their signaling cascades. The exact mechanism of ligand-induced receptor activation is still unclear. It is generally assumed that three receptors bind to the homotrimeric ligand to trigger a signaling event. Recent evidence, though, has raised doubts if the ligand:receptor stoichiometry should indeed be 3:3 for ligand-induced cell...

  9. Peroxisome Proliferator-Activated Receptor and Vitamin D Receptor Signaling Pathways in Cancer Cells

    Peroxisome proliferator-activated receptors (PPARs) are members of the superfamily of nuclear hormone receptors, which respond to specific ligands such as polyunsaturated fatty acids by altering gene expression. Three subtypes of this receptor have been discovered, each evolving to achieve different biological functions. Like other nuclear receptors, the transcriptional activity of PPARs is affected not only by ligand-stimulation, but also by cross-talk with other molecules. For example, both PPARs and the RXRs are ligand-activated transcription factors that coordinately regulate gene expression. In addition, PPARs and vitamin D receptor (VDR) signaling pathways regulate a multitude of genes that are of importance for cellular functions including cell proliferation and cell differentiation. Interaction of the PPARs and VDR signaling pathways has been shown at the level of molecular cross-regulation of their transcription factor. A variety of ligands influencing the PPARs and VDR signaling pathways have been shown to reveal chemopreventive potential by mediating tumor suppressive activities in human cancers. Use of these compounds may represent a potential novel strategy to prevent cancers. This review summarizes the roles of the PPARs and the VDR in pathogenesis and progression of cancer

  10. Peroxisome Proliferator-Activated Receptor and Vitamin D Receptor Signaling Pathways in Cancer Cells

    Yasuko Kitagishi

    2013-10-01

    Full Text Available Peroxisome proliferator-activated receptors (PPARs are members of the superfamily of nuclear hormone receptors, which respond to specific ligands such as polyunsaturated fatty acids by altering gene expression. Three subtypes of this receptor have been discovered, each evolving to achieve different biological functions. Like other nuclear receptors, the transcriptional activity of PPARs is affected not only by ligand-stimulation, but also by cross-talk with other molecules. For example, both PPARs and the RXRs are ligand-activated transcription factors that coordinately regulate gene expression. In addition, PPARs and vitamin D receptor (VDR signaling pathways regulate a multitude of genes that are of importance for cellular functions including cell proliferation and cell differentiation. Interaction of the PPARs and VDR signaling pathways has been shown at the level of molecular cross-regulation of their transcription factor. A variety of ligands influencing the PPARs and VDR signaling pathways have been shown to reveal chemopreventive potential by mediating tumor suppressive activities in human cancers. Use of these compounds may represent a potential novel strategy to prevent cancers. This review summarizes the roles of the PPARs and the VDR in pathogenesis and progression of cancer.

  11. Peroxisome Proliferator-Activated Receptor and Vitamin D Receptor Signaling Pathways in Cancer Cells

    Matsuda, Satoru, E-mail: smatsuda@cc.nara-wu.ac.jp; Kitagishi, Yasuko [Department of Food Science and Nutrition, Nara Women’s University, Kita-Uoya Nishimachi, Nara 630-8506 (Japan)

    2013-10-21

    Peroxisome proliferator-activated receptors (PPARs) are members of the superfamily of nuclear hormone receptors, which respond to specific ligands such as polyunsaturated fatty acids by altering gene expression. Three subtypes of this receptor have been discovered, each evolving to achieve different biological functions. Like other nuclear receptors, the transcriptional activity of PPARs is affected not only by ligand-stimulation, but also by cross-talk with other molecules. For example, both PPARs and the RXRs are ligand-activated transcription factors that coordinately regulate gene expression. In addition, PPARs and vitamin D receptor (VDR) signaling pathways regulate a multitude of genes that are of importance for cellular functions including cell proliferation and cell differentiation. Interaction of the PPARs and VDR signaling pathways has been shown at the level of molecular cross-regulation of their transcription factor. A variety of ligands influencing the PPARs and VDR signaling pathways have been shown to reveal chemopreventive potential by mediating tumor suppressive activities in human cancers. Use of these compounds may represent a potential novel strategy to prevent cancers. This review summarizes the roles of the PPARs and the VDR in pathogenesis and progression of cancer.

  12. Development of a Quantitative PCR Assay for Detection of Human Insulin-Like Growth Factor Receptor and Insulin Receptor Isoforms.

    Flannery, Clare A; Rowzee, Anne M; Choe, Gina H; Saleh, Farrah L; Radford, Caitlin C; Taylor, Hugh S; Wood, Teresa L

    2016-04-01

    The biological activity of insulin and the insulin-like growth factor (IGF) ligands, IGF-I and IGF-II, is based in part on the relative abundance and distribution of their target receptors: the insulin receptor (IR) splice variants A (IR-A) and B (IR-B) and IGF 1 receptor (IGF-1R). However, the relative quantity of all three receptors in human tissues has never been measured together on the same scale. Due to the high homology between insulin receptor (IR)-A and IR-B proteins and lack of antibodies that discern the two IR splice variants, their mRNA sequence is the most reliable means of distinguishing between the receptors. Hence, highly specific primers for IR-A, IR-B, and IGF-1R mRNA were designed to accurately detect all three receptors by quantitative RT-PCR and enable direct quantification of relative receptor expression levels. A standard concentration curve of cDNA from each receptor was performed. Assay specificity was tested using competition assays and postamplification analysis by gel electrophoresis and cloning. Forward and reverse primer concentrations were optimized to ensure equal efficiencies across primer pairs. This assay enables a specific molecular signature of IGF/insulin signaling receptors to be assayed in different tissues, cell types, or cancers. PMID:26862994

  13. Biased Signaling of Protease-activated Receptors

    PeishenZhao; NigelWilliamBunnett

    2014-01-01

    In addition to their role in protein degradation and digestion, proteases can also function as hormone-like signaling molecules that regulate vital patho-physiological processes, including inflammation, hemostasis, pain and repair mechanisms. Certain proteases can signal to cells by cleaving protease-activated receptors (PARs), a family of four G protein-coupled receptors. PARs are expressed by almost all cell types, control important physiological and disease-relevant processes, and are an e...

  14. The Anti-sigma Factor RsiV Is a Bacterial Receptor for Lysozyme: Co-crystal Structure Determination and Demonstration That Binding of Lysozyme to RsiV Is Required for σV Activation.

    Hastie, Jessica L; Williams, Kyle B; Bohr, Lindsey L; Houtman, Jon C; Gakhar, Lokesh; Ellermeier, Craig D

    2016-09-01

    σ factors provide RNA polymerase with promoter specificity in bacteria. Some σ factors require activation in order to interact with RNA polymerase and transcribe target genes. The Extra-Cytoplasmic Function (ECF) σ factor, σV, is encoded by several Gram-positive bacteria and is specifically activated by lysozyme. This activation requires the proteolytic destruction of the anti-σ factor RsiV via a process of regulated intramembrane proteolysis (RIP). In many cases proteases that cleave at site-1 are thought to directly sense a signal and initiate the RIP process. We previously suggested binding of lysozyme to RsiV initiated the proteolytic destruction of RsiV and activation of σV. Here we determined the X-ray crystal structure of the RsiV-lysozyme complex at 2.3 Å which revealed that RsiV and lysozyme make extensive contacts. We constructed RsiV mutants with altered abilities to bind lysozyme. We find that mutants that are unable to bind lysozyme block site-1 cleavage of RsiV and σV activation in response to lysozyme. Taken together these data demonstrate that RsiV is a receptor for lysozyme and binding of RsiV to lysozyme is required for σV activation. In addition, the co-structure revealed that RsiV binds to the lysozyme active site pocket. We provide evidence that in addition to acting as a sensor for the presence of lysozyme, RsiV also inhibits lysozyme activity. Thus we have demonstrated that RsiV is a protein with multiple functions. RsiV inhibits σV activity in the absence of lysozyme, RsiV binds lysozyme triggering σV activation and RsiV inhibits the enzymatic activity of lysozyme. PMID:27602573

  15. Cheiradone: a vascular endothelial cell growth factor receptor antagonist

    Ahmed Nessar

    2008-01-01

    Full Text Available Abstract Background Angiogenesis, the growth of new blood vessels from the pre-existing vasculature is associated with physiological (for example wound healing and pathological conditions (tumour development. Vascular endothelial growth factor (VEGF, fibroblast growth factor-2 (FGF-2 and epidermal growth factor (EGF are the major angiogenic regulators. We have identified a natural product (cheiradone isolated from a Euphorbia species which inhibited in vivo and in vitro VEGF- stimulated angiogenesis but had no effect on FGF-2 or EGF activity. Two primary cultures, bovine aortic and human dermal endothelial cells were used in in vitro (proliferation, wound healing, invasion in Matrigel and tube formation and in vivo (the chick chorioallantoic membrane models of angiogenesis in the presence of growth factors and cheiradone. In all cases, the concentration of cheiradone which caused 50% inhibition (IC50 was determined. The effect of cheiradone on the binding of growth factors to their receptors was also investigated. Results Cheiradone inhibited all stages of VEGF-induced angiogenesis with IC50 values in the range 5.20–7.50 μM but did not inhibit FGF-2 or EGF-induced angiogenesis. It also inhibited VEGF binding to VEGF receptor-1 and 2 with IC50 values of 2.9 and 0.61 μM respectively. Conclusion Cheiradone inhibited VEGF-induced angiogenesis by binding to VEGF receptors -1 and -2 and may be a useful investigative tool to study the specific contribution of VEGF to angiogenesis and may have therapeutic potential.

  16. Toll-Like Receptor 2- and 6-Mediated Stimulation by Macrophage-Activating Lipopeptide 2 Induces Lipopolysaccharide (LPS) Cross Tolerance in Mice, Which Results in Protection from Tumor Necrosis Factor Alpha but in Only Partial Protection from Lethal LPS Doses

    Deiters, Ursula; Gumenscheimer, Marina; Galanos, Chris; Mühlradt, Peter F.

    2003-01-01

    Patients or experimental animals previously exposed to lipopolysaccharide (LPS) become tolerant to further LPS challenge. We investigated the potential of the macrophage-activating lipopeptide 2 (MALP-2) to induce in vivo cross tolerance to tumor necrosis factor alpha (TNF-α) and LPS. MALP-2-induced tolerance could be of practical interest, as MALP-2 proved much less pyrogenic in rabbits than LPS. Whereas LPS signals via Toll-like receptor 4 (TLR4), MALP-2 uses TLR2 and TLR6. LPS-mediated cyt...

  17. Urokinase-type plasminogen activator-like proteases in teleosts lack genuine receptor-binding epidermal growth factor-like domains

    Bager, René; Kristensen, Thomas K.; Jensen, Jan; Szczur, Agnieszka; Christensen, Anni; Andersen, Lisbeth; Johansen, Jesper Sanderhoff; Larsen, Niels; Baatrup, Erik; Huang, Mingdong; Ploug, Michael; Andreasen, Peter A.

    2012-01-01

    zebrafish proteases, zfuPA-a and zfuPA-b, which by several criteria are the fish orthologs of mammalian uPA. Thus, both proteases catalyze the activation of fish plasminogen efficiently and both proteases are inhibited rapidly by plasminogen activator inhibitor-1 (PAI-1). But zfuPA-a differs from mammalian...... uPA by lacking the exon encoding the uPAR-binding epidermal growth factor-like domain; zfuPA-b differs from mammalian uPA by lacking two cysteines of the epidermal growth factor-like domain and a uPAR-binding sequence comparable with that found in mammalian uPA. Accordingly, no zfuPA-b binding...

  18. Transcriptional activation of nuclear estrogen receptor and progesterone receptor and its regulation.

    Xin, Qi-Liang; Qiu, Jing-Tao; Cui, Sheng; Xia, Guo-Liang; Wang, Hai-Bin

    2016-08-25

    Estrogen receptor (ER) and progesterone receptor (PR) are two important members of steroid receptors family, an evolutionarily conserved family of transcription factors. Upon binding to their ligands, ER and PR enter cell nucleus to interact with specific DNA element in the context of chromatin to initiate the transcription of diverse target genes, which largely depends on the timely recruitment of a wide range of cofactors. Moreover, the interactions between steroid hormones and their respective receptors also trigger post-translational modifications on these receptors to fine-tune their transcriptional activities. Besides the well-known phosphorylation modifications on tyrosine and serine/threonine residues, recent studies have identified several other covalent modifications, such as ubiquitylation and sumoylation. These post-translational modifications of steroid receptors affect its stability, subcellular localization, and/or cofactor recruitment; eventually influence the duration and extent of transcriptional activation. This review is to focus on the recent research progress on the transcriptional activation of nuclear ER and PR as well as their physiological functions in early pregnancy, which may help us to better understand related female reproductive diseases. PMID:27546504

  19. Corticotropin-Releasing Factor in the Basolateral Amygdala Enhances Memory Consolidation via an Interaction with the β-Adrenoceptor-cAMP Pathway: Dependence on Glucocorticoid Receptor Activation

    Roozendaal, Benno; Schelling, Gustav; McGaugh, James L.

    2008-01-01

    Extensive evidence indicates that stress hormone effects on the consolidation of emotionally influenced memory involve noradrenergic activation of the basolateral complex of the amygdala (BLA). The present experiments examined whether corticotropin-releasing factor (CRF) modulates memory consolidation via an interaction with the β-adrenoceptor-adenosine 3′,5′-cyclic monophosphate (cAMP) system in the BLA. In a first experiment, male Sprague-Dawley rats received bilateral infusions of the CRF-...

  20. Epidermal growth factor receptor inhibition in lung cancer: status 2012.

    Hirsch, Fred R; Jänne, Pasi A; Eberhardt, Wilfried E; Cappuzzo, Federico; Thatcher, Nick; Pirker, Robert; Choy, Hak; Kim, Edward S; Paz-Ares, Luis; Gandara, David R; Wu, Yi-Long; Ahn, Myung-Ju; Mitsudomi, Tetsuya; Shepherd, Frances A; Mok, Tony S

    2013-03-01

    Lung cancer is the most common cause of cancer deaths. Most patients present with advanced-stage disease, and the prognosis is generally poor. However, with the understanding of lung cancer biology, and development of molecular targeted agents, there have been improvements in treatment outcomes for selected subsets of patients with non-small-cell lung cancer (NSCLC). Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have demonstrated significantly improved tumor responses and progression-free survival in subsets of patients with advanced NSCLC, particularly those with tumors harboring activating EGFR mutations. Testing for EGFR mutations is a standard procedure for identification of patients who will benefit from first-line EGFR TKIs. For patients with advanced NSCLC and no activating EGFR mutations (EGFR wild-type) or no other driving oncogenes such as ALK-gene rearrangement, chemotherapy is still the standard of care. A new generation of EGFR TKIs, targeting multiple receptors and with irreversible bindings to the receptors, are in clinical trials and have shown encouraging effects. Research on primary and acquired resistant mechanisms to EGFR TKIs are ongoing. Monoclonal antibodies (e.g. cetuximab), in combination with chemotherapy, have demonstrated improved outcomes, particularly for subsets of NSCLC patients, but further validations are needed. Novel monoclonal antibodies are combined with chemotherapy, and randomized comparative studies are ongoing. This review summarizes the current status of EGFR inhibitors in NSCLC in 2012 and some of the major challenges we are facing. PMID:23370315

  1. Identification of COUP-TFII Orphan Nuclear Receptor as a Retinoic Acid-Activated Receptor

    Kruse, Schoen W; Suino-Powell, Kelly; Zhou, X Edward; Kretschman, Jennifer E; Reynolds, Ross; Vonrhein, Clemens; Xu, Yong; Wang, Liliang; Tsai, Sophia Y; Tsai, Ming-Jer; Xu, H Eric [Baylor; (Van Andel); (Globel Phasing); (Grand Valley)

    2010-01-12

    The chicken ovalbumin upstream promoter-transcription factors (COUP-TFI and II) make up the most conserved subfamily of nuclear receptors that play key roles in angiogenesis, neuronal development, organogenesis, cell fate determination, and metabolic homeostasis. Although the biological functions of COUP-TFs have been studied extensively, little is known of their structural features or aspects of ligand regulation. Here we report the ligand-free 1.48 {angstrom} crystal structure of the human COUP-TFII ligand-binding domain. The structure reveals an autorepressed conformation of the receptor, where helix {alpha}10 is bent into the ligand-binding pocket and the activation function-2 helix is folded into the cofactor binding site, thus preventing the recruitment of coactivators. In contrast, in multiple cell lines, COUP-TFII exhibits constitutive transcriptional activity, which can be further potentiated by nuclear receptor coactivators. Mutations designed to disrupt cofactor binding, dimerization, and ligand binding, substantially reduce the COUP-TFII transcriptional activity. Importantly, retinoid acids are able to promote COUP-TFII to recruit coactivators and activate a COUP-TF reporter construct. Although the concentration needed is higher than the physiological levels of retinoic acids, these findings demonstrate that COUP-TFII is a ligand-regulated nuclear receptor, in which ligands activate the receptor by releasing it from the autorepressed conformation.

  2. Identification of ciliary neurotrophic factor (CNTF) residues essential for leukemia inhibitory factor receptor binding and generation of CNTF receptor antagonists.

    Di Marco, A; Gloaguen, I; Graziani, R; Paonessa, G; Saggio, I; Hudson, K R; Laufer, R

    1996-01-01

    Ciliary neurotrophic factor (CNTF) drives the sequential assembly of a receptor complex containing the ligand-specific alpha-receptor subunit (CNTFR alpha) and the signal transducers gp130 and leukemia inhibitory factor receptor-beta (LIFR). The D1 structural motif, located at the beginning of the D-helix of human CNTF, contains two amino acid residues, F152 and K155, which are conserved among all cytokines that signal through LIFR. The functional importance of these residues was assessed by ...

  3. Soluble and cell surface receptors for tumor necrosis factor

    Wallach, D; Engelmann, H; Nophar, Y;

    1991-01-01

    Tumor necrosis factor (TNF) initiates its multiple effects on cell function by binding at a high affinity to specific cell surface receptors. Two different molecular species of these receptors, which are expressed differentially in different cells, have been identified. The cDNAs of both receptors...... certain pathological situations. Release of the soluble receptors from the cells seems to occur by proteolytic cleavage of the cell surface forms and appears to be a way of down-regulating the cell response to TNF. Because of their ability to bind TNF, the soluble receptors exert an inhibitory effect on...

  4. NMDA receptor activity in neuropsychiatric disorders

    ShaheenELakhan

    2013-06-01

    Full Text Available N-Methyl-D-aspartate (NMDA receptors play a variety of physiologic roles and their proper signaling is essential for cellular homeostasis. Any disruption in this pathway, leading to either enhanced or decreased activity, may result in the manifestation of neuropsychiatric pathologies such as schizophrenia, mood disorders, substance induced psychosis, Huntington's disease, Alzheimer's disease, and neuropsychiatric systemic lupus erythematosus. Here, we explore the notion that the overlap in activity of at least one biochemical pathway, the NMDA receptor pathway, may be the link to understanding the overlap in psychotic symptoms between diseases. This review intends to present a broad overview of those neuropsychiatric disorders for which alternations in NMDA receptor activity is prominent thus suggesting that continued direction of pharmaceutical intervention to this pathway may present a viable option for managing symptoms.

  5. Mechanisms of Activation of Receptor Tyrosine Kinases: Monomers or Dimers

    Ichiro N. Maruyama

    2014-04-01

    Full Text Available Receptor tyrosine kinases (RTKs play essential roles in cellular processes, including metabolism, cell-cycle control, survival, proliferation, motility and differentiation. RTKs are all synthesized as single-pass transmembrane proteins and bind polypeptide ligands, mainly growth factors. It has long been thought that all RTKs, except for the insulin receptor (IR family, are activated by ligand-induced dimerization of the receptors. An increasing number of diverse studies, however, indicate that RTKs, previously thought to exist as monomers, are present as pre-formed, yet inactive, dimers prior to ligand binding. The non-covalently associated dimeric structures are reminiscent of those of the IR family, which has a disulfide-linked dimeric structure. Furthermore, recent progress in structural studies has provided insight into the underpinnings of conformational changes during the activation of RTKs. In this review, I discuss two mutually exclusive models for the mechanisms of activation of the epidermal growth factor receptor, the neurotrophin receptor and IR families, based on these new insights.

  6. Neurotrophin receptors expression and JNK pathway activation in human astrocytomas

    Neurotrophins are growth factors that regulate cell growth, differentiation and apoptosis in the nervous system. Their diverse actions are mediated through two different transmembrane – receptor signaling systems: Trk receptor tyrosine kinases (TrkA, TrkB, TrkC) and p75NTR neurotrophin receptor. Trk receptors promote cell survival and differentiation while p75NTR induces, in most cases, the activity of JNK-p53-Bax apoptosis pathway or suppresses intracellular survival signaling cascades. Robust Trk activation blocks p75NTR -induced apoptosis by suppressing the JNK-p53-Bax pathway. The aim of this exploratory study was to investigate the expression levels of neurotrophin receptors, Trks and p75NTR, and the activation of JNK pathway in human astrocytomas and in adjacent non-neoplastic brain tissue. Formalin-fixed paraffin-embedded serial sections from 33 supratentorial astrocytomas (5 diffuse fibrillary astrocytomas, WHO grade II; 6 anaplastic astrocytomas, WHO grade III; 22 glioblastomas multiforme, WHO grade IV) were immunostained following microwave pretreatment. Polyclonal antibodies against TrkA, TrkB, TrkC and monoclonal antibodies against p75NTR and phosphorylated forms of JNK (pJNK) and c-Jun (pc-Jun) were used. The labeling index (LI), defined as the percentage of positive (labeled) cells out of the total number of tumor cells counted, was determined. Moderate to strong, granular cytoplasmic immunoreactivity for TrkA, TrkB and TrkC receptors was detected in greater than or equal to 10% of tumor cells in the majority of tumors independently of grade; on the contrary, p75NTR receptor expression was found in a small percentage of tumor cells (~1%) in some tumors. The endothelium of tumor capillaries showed conspicuous immunoreactivity for TrkB receptor. Trk immunoreactivity seemed to be localized in some neurons and astrocytes in non-neoplastic tissue. Phosphorylated forms of JNK (pJNK) and c-Jun (pc-Jun) were significantly co-expressed in a tumor grade

  7. Identification of heparin-binding EGF-like growth factor (HB-EGF as a biomarker for lysophosphatidic acid receptor type 1 (LPA1 activation in human breast and prostate cancers.

    Marion David

    Full Text Available Lysophosphatidic acid (LPA is a natural bioactive lipid with growth factor-like functions due to activation of a series of six G protein-coupled receptors (LPA₁₋₆. LPA receptor type 1 (LPA₁ signaling influences the pathophysiology of many diseases including cancer, obesity, rheumatoid arthritis, as well as lung, liver and kidney fibrosis. Therefore, LPA₁ is an attractive therapeutic target. However, most mammalian cells co-express multiple LPA receptors whose co-activation impairs the validation of target inhibition in patients because of missing LPA receptor-specific biomarkers. LPA₁ is known to induce IL-6 and IL-8 secretion, as also do LPA₂ and LPA₃. In this work, we first determined the LPA induced early-gene expression profile in three unrelated human cancer cell lines expressing different patterns of LPA receptors (PC3: LPA₁,₂,₆; MDA-MB-231: LPA1,2; MCF-7: LPA₂,₆. Among the set of genes upregulated by LPA only in LPA₁-expressing cells, we validated by QPCR and ELISA that upregulation of heparin-binding EGF-like growth factor (HB-EGF was inhibited by LPA₁-₃ antagonists (Ki16425, Debio0719. Upregulation and downregulation of HB-EGF mRNA was confirmed in vitro in human MDA-B02 breast cancer cells stably overexpressing LPA₁ (MDA-B02/LPA₁ and downregulated for LPA₁ (MDA-B02/shLPA1, respectively. At a clinical level, we quantified the expression of LPA₁ and HB-EGF by QPCR in primary tumors of a cohort of 234 breast cancer patients and found a significantly higher expression of HB-EGF in breast tumors expressing high levels of LPA₁. We also generated human xenograph prostate tumors in mice injected with PC3 cells and found that a five-day treatment with Ki16425 significantly decreased both HB-EGF mRNA expression at the primary tumor site and circulating human HB-EGF concentrations in serum. All together our results demonstrate that HB-EGF is a new and relevant biomarker with potentially high value in

  8. A novel hydroxyfuroic acid compound as an insulin receptor activator – structure and activity relationship of a prenylindole moiety to insulin receptor activation

    Tsai Henry J

    2009-07-01

    Full Text Available Abstract Background Diabetes Mellitus is a chronic disease and many patients of which require frequent subcutaneous insulin injection to maintain proper blood glucose levels. Due to the inconvenience of insulin administration, an orally active insulin replacement has long been a prime target for many pharmaceutical companies. Demethylasterriquinone (DMAQ B1, extracted from tropical fungus, Pseudomassaria sp., has been reported to be an orally effective agent at lowering circulating glucose levels in diabetic (db/db mice; however, the cytotoxicity associated with the quinone moiety has not been addressed thus far. Methods A series of hydroxyfuroic acid compounds were synthesized and tested for their efficacies at activating human insulin receptor. Cytotoxicity to Chinese hamster ovary cells, selectivities over insulin-like growth factor-1 (IGF-1, epidermal growth factor (EGF, and fibroblast growth factor (FGF receptors were examined in this study. Result and Conclusion This study reports a new non-quinone DMAQ B1 derivative, a hydroxyfuroic acid compound (D-410639, which is 128 fold less cytotoxic as DMAQ B1 and as potent as compound 2, a DMAQ B1 synthetic derivative from Merck, at activating human insulin receptor. D-410639 has little activation potential on IGF-1 receptor but is a moderate inhibitor to EGF receptor. Structure and activity relationship of the prenylindole moiety to insulin receptor activation is discussed.

  9. Angiotensin II-induced migration of vascular smooth muscle cells is mediated by p38 mitogen-activated protein kinase-activated c-Src through spleen tyrosine kinase and epidermal growth factor receptor transactivation.

    Mugabe, Benon E; Yaghini, Fariborz A; Song, Chi Young; Buharalioglu, Cuneyt K; Waters, Christopher M; Malik, Kafait U

    2010-01-01

    Angiotensin II (Ang II) stimulates protein synthesis by activating spleen tyrosine kinase (Syk) and DNA synthesis through epidermal growth factor receptor (EGFR) transactivation in vascular smooth muscle cells (VSMCs). This study was conducted to determine whether Syk mediates Ang II-induced migration of aortic VSMCs using a scratch wound approach. Treatment with Ang II (200 nM) for 24 h increased VSMC migration by 1.56 +/- 0.14-fold. Ang II-induced VSMC migration and Syk phosphorylation as determined by Western blot analysis were minimized by the Syk inhibitor piceatannol (10 microM) and by transfecting VSMCs with dominant-negative but not wild-type Syk plasmid. Ang II-induced VSMC migration and Syk phosphorylation were attenuated by inhibitors of c-Src [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2)], p38 mitogen-activated protein kinase (MAPK) [4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole (SB202190)], and extracellular signal-regulated kinase (ERK) 1/2 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio) butadiene (U0126)]. SB202190 attenuated p38 MAPK and c-Src but not ERK1/2 phosphorylation, indicating that p38 MAPK acts upstream of c-Src and Syk. The c-Src inhibitor PP2 attenuated Syk and ERK1/2 phosphorylation, suggesting that c-Src acts upstream of Syk and ERK1/2. Ang II- and epidermal growth factor (EGF)-induced VSMC migration and EGFR phosphorylation were inhibited by the EGFR blocker 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478) (2 microM). Neither the Syk inhibitor piceatannol nor the dominant-negative Syk mutant altered EGF-induced cell migration or Ang II- and EGF-induced EGFR phosphorylation. The c-Src inhibitor PP2 diminished EGF-induced VSMC migration and EGFR, ERK1/2, and p38 MAPK phosphorylation. The ERK1/2 inhibitor U0126 (10 microM) attenuated EGF-induced cell migration and ERK1/2 but not EGFR phosphorylation. These data suggest that Ang II stimulates VSMC migration via p38 MAPK-activated c

  10. 多巴胺受体在可卡因诱导的转录因子CREB活化中的作用%Dopamine receptors oppositely regulate cocaine-induced transcription factor CREB activation

    刘怒云; 张琳; 王小宁; 张璐

    2006-01-01

    Objective To study the role of dopamine receptors in the regulation of the activity of transcription factor cAMP response element-binding protein (CREB) after cocaine treatment. Methods By using dopamine receptor antagonists SCH23390 and nafadotride, the activation of CREB by D1 and D3 dopamine receptors after cocaine treatment and role of extracellular signal-regulated kinase (ERK) in cocaine-induced CREB activation were examined by Western blotting, which was also employed for determination of the effect of SCH23390 and nafadotride on CREB activation. Results D1 receptor antagonist could inhibit cocaine-induced CREB activation, while D3 receptor antagonist enhanced cocaine-induced CREB activation. Dopamine receptor antagonists SCH23390 and nafadotride did not induce CREB activation. SL327, a MEK inhibitor, inhibited cocaine-induced CREB activation. Conclusion D1 and D3 dopamine receptors can oppositely regulate CREB activation after cocaine treatment and this regulation depends on ERK signaling pathway.%目的研究多巴胺受体在可卡因诱导的转录因子CREB磷酸化活化中的调控作用.方法采用D1和D3多巴胺受体抑制剂,应用Western blotting检测D1与D3多巴胺受体在可卡因诱导的cAMP反应元件结合蛋白(CREB)磷酸化活化中的作用及D1和D3多巴胺受体抑制剂本身对CREB磷酸化活化的影响,进一步应用Westernblotting检测细胞外信号调节激酶(ERK)在CREB磷酸化活化中的作用.结果 D1多巴胺受体抑制剂阻止可卡因诱导的CREB磷酸化活化,而D3多巴胺受体抑制剂促进可卡因诱导的CREB磷酸化活化,D1和D3多巴胺受体抑制剂本身不能诱导CREB磷酸化活化.MEK的特异性抑制剂SL327可以抑制可卡因诱导的CREB磷酸化活化.结论 D1和D3多巴胺受体对CREB的磷酸化活化起反式调控作用,并且这种反式调控作用依赖于ERK信号通路.

  11. Dietary and nutritional manipulation of the nuclear transcription factors peroxisome proliferator-activated receptor and sterol regulatory element-binding proteins as a tool for reversing the primary diseases of premature death and delaying aging.

    Kurtak, Karen A

    2014-04-01

    Evolution over 2.1 billion years has equipped us with a biochemical pathway that has the power to literally reverse the primary disease etiologies that have become the leading causes of death and aging in the developed world. Activation of the peroxisome proliferator-activated receptor (PPAR) pathway arrests inflammatory signaling throughout the body, reverses damage to tissues, reverses insulin resistance, and can even dissolve beta-amyloid plaque in the brain. It has played a critical role in the evolution of the metazoans and the successful migration of humans to all corners of the Earth. For two decades, various pharmaceuticals have been designed to activate the PPAR pathway but have consistently fallen short of expectations. There is nothing wrong with these drugs. The problem has been the standard "healthy" diet creating mixed signals that render the drugs ineffective. This article explores the ongoing dance between the two primary nuclear receptors that mediate gene regulation of fatty acids. It discusses their interaction with sirtuins and telomerase, optimization of their obligate heterodimers, and why manipulation of dietary and nutritional factors, like the ketogenic diet, is the most effective means of activation. These are effective tools that we can start implementing now to slow, and in some cases reverse, the diseases of aging. PMID:24713058

  12. Effects of alpha-calcitonin gene-related peptide on osteoprotegerin and receptor activator of nuclear factor-κB ligand expression in MG-63 osteoblast-like cells exposed to polyethylene particles

    Kauther Max D

    2010-11-01

    Full Text Available Abstract Background Recent studies demonstrated an impact of the nervous system on particle-induced osteolysis, the major cause of aseptic loosening of joint replacements. Methods In this study of MG-63 osteoblast-like cells we analyzed the influence of ultra-high molecular weight polyethylene (UHMWPE particles and the neurotransmitter alpha-calcitonin gene-related peptide (CGRP on the osteoprotegerin/receptor activator of nuclear factor-κB ligand/receptor activator of nuclear factorκB (OPG/RANKL/RANK system. MG-63 cells were stimulated by different UHMWPE particle concentrations (1:100, 1:500 and different doses of alpha-CGRP (10-7 M, 10-9 M, 10-11 M. RANKL and OPG mRNA expression and protein levels were measured by RT-PCR and Western blot. Results Increasing particle concentrations caused an up-regulation of RANKL after 72 hours. Alpha-CGRP showed a dose-independent depressive effect on particle-induced expression of RANKL mRNA in both cell-particle ratios. RANKL gene transcripts were significantly (P -7 M lead to an up-regulation of OPG protein. Conclusion In conclusion, a possible osteoprotective influence of the neurotransmitter alpha-CGRP on particle stimulated osteoblast-like cells could be shown. Alpha-CGRP might be important for bone metabolism under conditions of particle-induced osteolysis.

  13. Anxiolytic activity of adenosine receptor activation in mice.

    Jain, N; Kemp, N; Adeyemo, O; Buchanan, P; Stone, T W

    1995-10-01

    1. Purine analogues have been examined for anxiolytic- and anxiogenic-like activity in mice, by use of the elevated plus-maze. 2. The selective A1 receptor agonist, N6-cyclopentyladenosine (CPA) had marked anxiolytic-like activity at 10 and 50 microg kg(-1), with no effect on locomotor performance at these doses. 3. The A1 selective adenosine receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (CPX) had no significant effect on anxiety-related measures or locomotor behaviour, but blocked the anxiolytic-like activity of CPA. The hydrophilic xanthine, 8-(p-sulphophenyl) theophylline did not prevent anxiolysis by CPA. 4. Caffeine had anxiogenic-like activity at 30 mg kg(-1) which was prevented by CPA at 50 micro kg(-1). 5. The A2 receptor agonist, N6-[2-(3,5-dimethoxyphenyl)-2(2-methylphenyl)-ethyl]adenosine (DPMA) had no effect on anxiety behaviour but depressed locomotor activity at the highest dose tested of 1 mg kg(-1). The A2 receptor antagonist, 1,3-dimethyl-l-propargylxanthine (DMPX) had no effect on anxiety-related measures or locomotion and did not modify the anxiolytic-like activity of CPA. 6. Administration of DPMA in combination with anxiolytic doses of CPA prevented the anxiolytic-like activity of the latter. 7. The results suggest that the selective activation of central A1 adenosine receptors induces anxiolytic-like behaviour, while the activation of A2 sites causes locomotor depression and reduces the effects of A1 receptor activation. The absence of any effect of CPX alone suggests that the receptors involved in modulating behaviour in the elevated plus-maze in mice are not activated tonically by endogenous adenosine. PMID:8640355

  14. Hepatic Aryl Hydrocarbon Receptor Attenuates Fibroblast Growth Factor 21 Expression.

    Girer, Nathaniel G; Murray, Iain A; Omiecinski, Curtis J; Perdew, Gary H

    2016-07-15

    The Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor involved in many physiological processes. Several studies indicate that AHR is also involved in energy homeostasis. Fibroblast growth factor 21 (FGF21) is an important regulator of the fasting and feeding responses. When administered to various genetic and diet-induced mouse models of obesity, FGF21 can attenuate obesity-associated morbidities. Here, we explore the role of AHR in hepatic Fgf21 expression through the use of a conditional, hepatocyte-targeted AHR knock-out mouse model (Cre(Alb)Ahr(Fx/Fx)). Compared with the congenic parental strain (Ahr(Fx/Fx)), non-fasted Cre(Alb)Ahr(Fx/Fx) mice exhibit a 4-fold increase in hepatic Fgf21 expression, as well as elevated expression of the FGF21-target gene Igfbp1 Furthermore, in vivo agonist activation of AHR reduces hepatic Fgf21 expression during a fast. The Fgf21 promoter contains several putative dioxin response elements (DREs). Using EMSA, we demonstrate that the AHR-ARNT heterodimer binds to a specific DRE that overlaps binding sequences for peroxisome proliferator-activated receptor α (PPARα), carbohydrate response element-binding protein (ChREBP), and cAMP response element-binding protein, hepatocyte specific (CREBH). In addition, we reveal that agonist-activated AHR impairs PPARα-, ChREBP-, and CREBH-mediated promoter activity in Hepa-1 cells. Accordingly, agonist treatment in Hepa-1 cells ablates potent ER stress-driven Fgf21 expression, and pre-treatment with AHR antagonist blocks this effect. Finally, we show that pre-treatment of primary human hepatocytes with AHR agonist diminishes PPARα-, glucose-, and ER stress-driven induction of FGF21 expression, indicating the effect is not mouse-specific. Together, our data show that AHR contributes to hepatic energy homeostasis, partly through the regulation of FGF21 expression and signaling. PMID:27226639

  15. Oxygen dependency of epidermal growth factor receptor binding and DNA synthesis of rat hepatocytes

    Background/Aims: Changes in oxygen availability modulate replicative responses in several cell types, but the effects on hepatocyte replication remain unclear. We have studied the effects of transient nonlethal hypoxia on epidermal growth factor receptor binding and epidermal growth factor-induced DNA synthesis of rat hepatocytes. Methods: Lactate dehydrogenase activity in culture supernatant, intracellular adenosine triphosphate content, 125I-epidermal growth factor specific binding, epidermal growth factor receptor protein expression, and 3H-thymidine incorporation were compared between hepatocytes cultured in hypoxia and normoxia. Results: Hypoxia up to 3 h caused no significant increase in lactate dehydrogenase activity in the culture supernatant, while intracellular adenosine triphosphate content decreased time-dependently and was restored to normoxic levels by reoxygenation (nonlethal hypoxia). Concomitantly, 125I-epidermal growth factor specific binding to hepatocytes decreased time-dependently (to 54.1% of normoxia) and was restored to control levels by reoxygenation, although 125I-insulin specific binding was not affected. The decrease in 125I-epidermal growth factor specific binding was explained by the decrease in the number or available epidermal growth factor receptors (21.37±3.08 to 12.16±1.42 fmol/105 cells), while the dissociation constant of the receptor was not affected. The change in the number of available receptors was not considered to be due to receptor degradation-resynthesis, since immuno-detection of the epidermal growth factor receptor revealed that the receptor protein expression did not change during hypoxia and reoxygenation, and since neither actinomycin D nor cycloheximide affected the recovery of 125I-epidermal growth factor binding by reoxygenation. Inhibition of epidermal growth factor-induced DNA synthesis after hypoxia (to 75.4% of normoxia by 3 h hypoxia) paralleled the decrease in 125I-epidermal growth factor binding. (au)

  16. Evaluation of the fibroblast growth factor receptor 1 (FGFR1) in experimental autoimmune encephalomyelitis (EAE)

    Rajendran, Ranjithkumar

    2014-01-01

    Fibroblast growth factors (FGFs) exert diverse biological effects by binding and activation of specific fibroblast growth factor receptors (FGFRs). Recent studies on the function of FGF2 in MOG35-55-induced experimental autoimmune encephalitis (EAE) showed that systemic deletion of FGF2 leads to a more severe disease course, increased lymphocyte and macrophage infiltration and decreased remyelination. In the present study the in vivo function of the corresponding receptor Fgfr1 was characteri...

  17. CERAPP: Collaborative estrogen receptor activity prediction project

    Mansouri, Kamel; Abdelaziz, Ahmed; Rybacka, Aleksandra;

    2016-01-01

    Background: Humans are exposed to thousands of man-made chemicals in the environment. Some chemicals mimic natural endocrine hormones and, thus, have the potential to be endocrine disruptors. Most of these chemicals have never been tested for their ability to interact with the estrogen receptor (ER......). Risk assessors need tools to prioritize chemicals for evaluation in costly in vivo tests, for instance, within the U.S. EPA Endocrine Disruptor Screening Program. oBjectives: We describe a large-scale modeling project called CERAPP (Collaborative Estrogen Receptor Activity Prediction Project) and...

  18. Analysis of corkscrew signaling in the Drosophila epidermal growth factor receptor pathway during myogenesis.

    Johnson Hamlet, M R; Perkins, L A

    2001-01-01

    The Drosophila nonreceptor protein tyrosine phosphatase, Corkscrew (Csw), functions positively in multiple receptor tyrosine kinase (RTK) pathways, including signaling by the epidermal growth factor receptor (EGFR). Detailed phenotypic analyses of csw mutations have revealed that Csw activity is required in many of the same developmental processes that require EGFR function. However, it is still unclear where in the signaling hierarchy Csw functions relative to other proteins whose activities...

  19. Corticotropin-releasing factor receptors and stress-related alterations of gut motor function.

    Taché, Yvette; Bonaz, Bruno

    2007-01-01

    Over the past few decades, corticotropin-releasing factor (CRF) signaling pathways have been shown to be the main coordinators of the endocrine, behavioral, and immune responses to stress. Emerging evidence also links the activation of CRF receptors type 1 and type 2 with stress-related alterations of gut motor function. Here, we review the role of CRF receptors in both the brain and the gut as part of key mechanisms through which various stressors impact propulsive activity of the gastrointe...

  20. Mlc is a transcriptional activator with a key role in integrating cyclic AMP receptor protein and integration host factor regulation of leukotoxin RNA synthesis in Aggregatibacter actinomycetemcomitans

    Aggregatibacter actinomycetemcomitans, a periodontal pathogen, synthesizes leukotoxin (LtxA), a protein that helps the bacterium evade the host immune response. Transcription of the ltxA operon is induced during anaerobic growth. The cAMP receptor protein (CRP) indirectly increases ltxA expression...

  1. Anti-cancer activity of an osthole derivative, NBM-T-BMX-OS01: targeting vascular endothelial growth factor receptor signaling and angiogenesis.

    Yang, Hung-Yu; Hsu, Ya-Fen; Chiu, Pei-Ting; Ho, Shiau-Jing; Wang, Chi-Han; Chi, Chih-Chin; Huang, Yu-Han; Lee, Cheng-Feng; Li, Ying-Shiuan; Ou, George; Hsu, Ming-Jen

    2013-01-01

    Angiogenesis occurs during tissue growth, development and wound healing. It is also required for tumor progression and represents a rational target for therapeutic intervention. NBM-T-BMX-OS01 (BMX), derived from the semisynthesis of osthole, an active ingredient isolated from Chinese herb Cnidium monnieri (L.) Cuss., was recently shown to enhance learning and memory in rats. In this study, we characterized the anti-angiogenic activities of NBM-T-BMX-OS01 (BMX) in an effort to develop novel inhibitors to suppress angiogenesis and tumor growth. BMX inhibited vascular endothelial growth factor (VEGF)-induced proliferation, migration and endothelial tube formation in human umbilical endothelial cells (HUVECs). BMX also attenuated VEGF-induced microvessel sprouting from aortic rings ex vivo and reduced HCT116 colorectal cancer cells-induced angiogenesis in vivo. Moreover, BMX inhibited the phosphorylation of VEGFR2, FAK, Akt and ERK in HUVECs exposed to VEGF. BMX was also shown to inhibit HCT116 cell proliferation and to suppress the growth of subcutaneous xenografts of HCT116 cells in vivo. Taken together, this study provides evidence that BMX modulates vascular endothelial cell remodeling and leads to the inhibition of tumor angiogenesis. These results also support the role of BMX as a potential drug candidate and warrant the clinical development in the treatment of cancer. PMID:24312323

  2. AZD8931, an equipotent, reversible inhibitor of signaling by epidermal growth factor receptor (EGFR), HER2, and HER3: preclinical activity in HER2 non-amplified inflammatory breast cancer models

    Mu, Zhaomei; Klinowska, Teresa; Dong, Xiaoshen; Foster, Emily; Womack, Chris; Fernandez, Sandra V.; Cristofanilli, Massimo

    2014-01-01

    Introduction Epidermal growth factor receptor (EGFR) overexpression has been associated with prognostic and predictive value in inflammatory breast cancer (IBC). Epidermal growth factor receptor 2 (HER2) overexpression is observed at a higher rate in IBC compared with noninflammatory breast cancer. Current clinically available anti-HER2 therapies are effective only in patients with HER2 amplified breast cancer, including IBC. AZD8931 is a novel small-molecule equipotent inhibitor of EGFR, HER...

  3. Fatty acids activate a chimera of the clofibric acid-activated receptor and the glucocorticoid receptor.

    Göttlicher, M; Widmark, E; Q. Li; Gustafsson, J.A.

    1992-01-01

    Peroxisome proliferators such as clofibric acid, nafenopin, and WY-14,643 have been shown to activate PPAR (peroxisome proliferator-activated receptor), a member of the steroid nuclear receptor superfamily. We have cloned the cDNA from the rat that is homologous to that from the mouse [Issemann, I. & Green, S. (1990) Nature (London) 347, 645-650], which encodes a 97% similar protein with a particularly well-conserved putative ligand-binding domain. To search for physiologically occurring acti...

  4. Human Receptor Activation by Aroclor 1260, a Polychlorinated Biphenyl Mixture

    Wahlang, Banrida; Falkner, K. Cameron; Clair, Heather B.; Al-Eryani, Laila; Prough, Russell A.; States, J. Christopher; Coslo, Denise M.; Omiecinski, Curtis J.; Cave, Matthew C.

    2014-01-01

    Polychlorinated biphenyls (PCBs) are persistent environmental toxicants, present in 100% of U.S. adults and dose-dependently associated with obesity and non-alcoholic fatty liver disease (NAFLD). PCBs are predicted to interact with receptors previously implicated in xenobiotic/energy metabolism and NAFLD. These receptors include the aryl hydrocarbon receptor (AhR), pregnane xenobiotic receptor (PXR), constitutive androstane receptor (CAR), peroxisome proliferator-activated receptors (PPARs), ...

  5. Phosphoinositide 3-kinase/Akt Pathway Mediates Fip1-like1-platelet-derived Growth Factor Receptor α-induced Cell Infiltration and Activation: Possible Molecular Mechanism for the Malignant Phenotype of Chronic Eosinophilic Leukemia

    Bin Li

    2015-01-01

    Full Text Available The fip1-like1/platelet-derived growth factor receptor-α fusion gene (F/P is responsible for 14-60% cases of hypereosinophilia syndrome (HES, also known as F/P-positive chronic eosinophilic leukemia (F/P(+ CEL. The major pathogenesis of F/P(+ CEL is known to involve migration and activation of mast cells and eosinophils, leading to severe multi-organ dysfunction, but the mechanism was still unclear. Phosphoinositide 3-kinase (PI3K and serine-threonine protein kinase Akt have been reported to be targets of F/P in the F/P-promoted cell proliferation. They are extensively involved in the migration and adhesion of hematopoietic stem/progenitor cells, and also control cell invasion in some leukemias. The PI3K/Akt pathway is involved in eosinophil/neutrophil activation and infiltration; its possible role in regulating F/P induced cytotoxicity and upregulation of A4-integrin in eosinophils, and inducing eosinophil activation through controlling F/P-induced Nuclear factor-kB activity. Akt was recently shown to be stimulated by F/P, synergistically with stem cell factor, resulting in the induction of MCs migration and excessive activation. PI3K/Akt pathway is also a principal mediator of interleukin-5 (IL-5-induced signal transduction promoting eosinophil trafficking and degranulation, whereas IL-5 is a necessary cytokine for F/P-mediated CEL development. We, therefore, propose the hypothesis that the PI3K/Akt pathway might be vital downstream of F/P to induce target cell activation and tissue infiltration, resulting in the malignant phenotype seen in F/P(+ CEL.

  6. Secreted phospholipase A2-IIA-induced a phenotype of activated microglia in BV-2 cells requires epidermal growth factor receptor transactivation and proHB-EGF shedding

    Martín Rubén

    2012-07-01

    Full Text Available Abstract Background Activation of microglia, the primary component of the innate immune response in the brain, is a hallmark of neuroinflammation in neurodegenerative disorders, including Alzheimer’s disease (AD and other pathological conditions such as stroke or CNS infection. In response to a variety of insults, microglial cells produce high levels of inflammatory cytokines that are often involved in neuronal injury, and play an important role in the recognition, engulfment, and clearance of apoptotic cells and/or invading microbes. Secreted phospholipase A2-IIA (sPLA2-IIA, an enzyme that interacts with cells involved in the systemic immune/inflammatory response, has been found up-regulated in the cerebrospinal fluid and brain of AD patients. However, despite several approaches, its functions in mediating CNS inflammation remain unknown. In the present study, the role of sPLA2-IIA was examined by investigating its direct effects on microglial cells. Methods Primary and immortalized microglial cells were stimulated by sPLA2-IIA in order to characterize the cytokine-like actions of the phospholipase. The hallmarks of activated microglia analyzed include: mitogenic response, phagocytic capabilities and induction of inflammatory mediators. In addition, we studied several of the potential molecular mechanisms involved in those events. Results The direct exposure of microglial cells to sPLA2-IIA stimulated, in a time- and dose-dependent manner, their phagocytic and proliferative capabilities. sPLA2-IIA also triggered the synthesis of the inflammatory proteins COX-2 and TNFα. In addition, EGFR phosphorylation and shedding of the membrane-anchored heparin-binding EGF-like growth factor (pro-HB-EGF ectodomain, as well as a rapid activation/phosphorylation of the classical survival proteins ERK, P70S6K and rS6 were induced upon sPLA2-IIA treatment. We further demonstrated that the presence of an EGFR inhibitor (AG1478, a matrix metalloproteinase

  7. RELAXIN ACTIVATES PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR GAMMA

    Singh, Sudhir; Bennett, Robert G

    2009-01-01

    Relaxin is a polypeptide hormone that triggers multiple signaling pathways through its receptor RXFP1. Many of relaxin’s functions, including vascular and antifibrotic effects, are similar to those induced by activation of PPARγ. In this study, we tested the hypothesis that relaxin signaling through RXFP1 would activate PPARγ activity. In cells overexpressing RXFP1 (HEK-RXFP1), relaxin increased transcriptional activity through a PPAR response element (PPRE) in a concentration-dependent manne...

  8. The role of the ubiquitination–proteasome pathway in breast cancer: Ubiquitin mediated degradation of growth factor receptors in the pathogenesis and treatment of cancer

    Aberrant activity of growth factor receptors has been implicated in the pathogenesis of a wide variety of malignancies. The negative regulation of signaling by growth factor receptors is mediated in large part by the ubiquitination, internalization, and degradation of the activated receptor. Over the past few years, considerable insight into the mechanisms that control receptor downregulation has been gained. There are also data suggesting that mutations that lead to inhibition of downregulation of growth factor receptors could play a role in the pathogenesis of cancer. Therapies directed at enhancing the degradation of growth factor receptors offer a promising approach to the treatment of malignancies

  9. Ubiquitin-specific Protease 20 Regulates the Reciprocal Functions of β-Arrestin2 in Toll-like Receptor 4-promoted Nuclear Factor κB (NFκB) Activation.

    Jean-Charles, Pierre-Yves; Zhang, Lisheng; Wu, Jiao-Hui; Han, Sang-Oh; Brian, Leigh; Freedman, Neil J; Shenoy, Sudha K

    2016-04-01

    Toll-like receptor 4 (TLR4) promotes vascular inflammatory disorders such as neointimal hyperplasia and atherosclerosis. TLR4 triggers NFκB signaling through the ubiquitin ligase TRAF6 (tumor necrosis factor receptor-associated factor 6). TRAF6 activity can be impeded by deubiquitinating enzymes like ubiquitin-specific protease 20 (USP20), which can reverse TRAF6 autoubiquitination, and by association with the multifunctional adaptor protein β-arrestin2. Although β-arrestin2 effects on TRAF6 suggest an anti-inflammatory role, physiologic β-arrestin2 promotes inflammation in atherosclerosis and neointimal hyperplasia. We hypothesized that anti- and proinflammatory dimensions of β-arrestin2 activity could be dictated by β-arrestin2's ubiquitination status, which has been linked with its ability to scaffold and localize activated ERK1/2 to signalosomes. With purified proteins and in intact cells, our protein interaction studies showed that TRAF6/USP20 association and subsequent USP20-mediated TRAF6 deubiquitination were β-arrestin2-dependent. Generation of transgenic mice with smooth muscle cell-specific expression of either USP20 or its catalytically inactive mutant revealed anti-inflammatory effects of USP20in vivoandin vitro Carotid endothelial denudation showed that antagonizing smooth muscle cell USP20 activity increased NFκB activation and neointimal hyperplasia. We found that β-arrestin2 ubiquitination was promoted by TLR4 and reversed by USP20. The association of USP20 with β-arrestin2 was augmented when β-arrestin2 ubiquitination was prevented and reduced when β-arrestin2 ubiquitination was rendered constitutive. Constitutive β-arrestin2 ubiquitination also augmented NFκB activation. We infer that pro- and anti-inflammatory activities of β-arrestin2 are determined by β-arrestin2 ubiquitination and that changes in USP20 expression and/or activity can therefore regulate inflammatory responses, at least in part, by defining the ubiquitination

  10. Signal transduction by the platelet-derived growth factor receptor

    The mitogenic effects of platelet-derived growth factor (PDGF) are mediated by the PDGF receptor. The mouse PDGF receptor was recently purified on the basis of its ability to become tyrosine phosphorylated in response to the A-B human platelet form of PDGF, and the receptor amino acid sequence was determined from a full-length cDNA clone. Both the human and mouse receptor cDNA sequences have been expressed in Chinese hamster ovary fibroblast (CHO) cells that normally lack PDGF receptors. This paper summarizes recent results using this system to study signal transduction by the PDGF receptor. Some of the findings show that the KI domain of the PDGF receptor plays an important role in the stimulation of DNA synthesis by PDGF. Surprisingly, the kinase insert region is not essential for PDGF stimulation of PtdIns turnover, pH change, increase in cellular calcium, and receptor autophosphorylation. In addition, PDGF stimulates a conformational change in the receptor

  11. Neural cell adhesion molecule differentially interacts with isoforms of the fibroblast growth factor receptor

    Christensen, Claus; Berezin, Vladimir; Bock, Elisabeth

    2011-01-01

    The fibroblast growth factor receptor (FGFR) can be activated through direct interactions with various fibroblast growth factors or through a number of cell adhesion molecules, including the neural cell adhesion molecule (NCAM). We produced recombinant proteins comprising the ligand...... the expression pattern of various FGFR isoforms determines the cell context-specific effects of NCAM signaling through FGFR....

  12. Interdependent epidermal growth factor receptor signalling and trafficking.

    Jones, Sylwia; Rappoport, Joshua Z

    2014-06-01

    Epidermal growth factor (EGF) receptor (EGFR) signalling regulates diverse cellular functions, promoting cell proliferation, differentiation, migration, cell growth and survival. EGFR signalling is critical during embryogenesis, in particular in epithelial development, and disruption of the EGFR gene results in epithelial immaturity and perinatal death. EGFR signalling also functions during wound healing responses through accelerating wound re-epithelialisation, inducing cell migration, proliferation and angiogenesis. Upregulation of EGFR signalling is often observed in carcinomas and has been shown to promote uncontrolled cell proliferation and metastasis. Therefore aberrant EGFR signalling is a common target for anticancer therapies. Various reports indicate that EGFR signalling primarily occurs at the plasma membrane and EGFR degradation following endocytosis greatly attenuates signalling. Other studies argue that EGFR internalisation is essential for complete activation of downstream signalling cascades and that endosomes can serve as signalling platforms. The aim of this review is to discuss current understanding of intersection between EGFR signalling and trafficking. PMID:24681003

  13. Role of the fibroblast growth factor receptor axis in cholangiocarcinoma.

    Ang, Celina

    2015-07-01

    Advanced cholangiocarcinoma (CCA) is a highly lethal disease with limited therapeutic options beyond cytotoxic chemotherapy. Molecular profiling of CCA has provided insights into the pathogenesis of this disease and identified potential therapeutic targets. The fibroblast growth factor receptor (FGFR) axis is important for maintaining tissue homeostasis. Aberrations in FGFR activity have been implicated in the development and progression of CCA and other malignancies, which has generated significant interest in exploring FGFR's therapeutic potential. FGFR2 fusion events are present in up to 17% of intrahepatic CCAs and appear to predict sensitivity to FGFR inhibitors even after progression on chemotherapy. These observations have led to a clinical trial evaluating FGFR inhibition in patients with CCA enriched for FGFR alterations. This review summarizes current knowledge about the role of the FGFR pathway in cholangiocarcinogenesis and ongoing work in developing FGFR-directed therapies as an antineoplastic strategy for CCA. PMID:25678238

  14. Retraction: "Concurrent inhibition of NF-κB, cyclooxygenase-2, and epidermal growth factor receptor leads to greater anti-tumor activity in pancreatic cancer" by Ali et al.

    2016-08-01

    The above article, published online on March 8, 2010 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the journal Editor in Chief, Gary S. Stein, and Wiley Periodicals, Inc. The retraction has been agreed following an investigation from Wayne State University involving the first author and the corresponding author that found Figures 2A, 4, 6A, and 6C to be inappropriately manipulated. REFERENCE Ali S, Banerjee S, Schaffert JM, El-Rayes BF, Philip PA, Sarkar FH. 2010. Concurrent inhibition of NF-κB, cyclooxygenase-2, and epidermal growth factor receptor leads to greater anti-tumor activity in pancreatic cancer. J Cell Biochem 110:171-181; doi: 10.1002/jcb.22523. PMID:27301888

  15. Kallikrein Promotes Inflammation in Human Dental Pulp Cells Via Protease-Activated Receptor-1.

    Hayama, Tomomi; Kamio, Naoto; Okabe, Tatsu; Muromachi, Koichiro; Matsushima, Kiyoshi

    2016-07-01

    Plasma kallikrein (KLKB1), a serine protease, cleaves high-molecular weight kininogen to produce bradykinin, a potent vasodilator and pro-inflammatory peptide. In addition, KLKB1 activates plasminogen and other leukocyte and blood coagulation factors and processes pro-enkephalin, prorenin, and C3. KLKB1 has also been shown to cleave protease-activated receptors in vascular smooth muscle cells to regulate the expression of epidermal growth factor receptor. In this study, we investigated KLKB1-dependent inflammation and activation of protease-activated receptor-1 in human dental pulp cells. These cells responded to KLKB1 stimulation by increasing intracellular Ca(2+) , upregulating cyclooxygenase-2, and secreting prostaglandin E2 . Remarkably, SCH79797, an antagonist of protease-activated receptor-1, blocked these effects. Thus, these data indicate that KLKB1 induces inflammatory reactions in human dental tissues via protease-activated receptor 1. J. Cell. Biochem. 117: 1522-1528, 2016. © 2015 Wiley Periodicals, Inc. PMID:26566265

  16. Anxiolytic activity of adenosine receptor activation in mice.

    Jain, N; Kemp, N; Adeyemo, O; Buchanan, P.; Stone, T W

    1995-01-01

    1. Purine analogues have been examined for anxiolytic- and anxiogenic-like activity in mice, by use of the elevated plus-maze. 2. The selective A1 receptor agonist, N6-cyclopentyladenosine (CPA) had marked anxiolytic-like activity at 10 and 50 microg kg(-1), with no effect on locomotor performance at these doses. 3. The A1 selective adenosine receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (CPX) had no significant effect on anxiety-related measures or locomotor behaviour, but blocked ...

  17. The eukaryotic translation initiation factor 3f (eIF3f) interacts physically with the alpha 1B-adrenergic receptor and stimulates adrenoceptor activity

    Gutiérrez-Fernández, Mario Javier; Higareda-Mendoza, Ana Edith; Gómez-Correa, César Adrián; Pardo-Galván, Marco Aurelio

    2015-01-01

    Background eIF3f is a multifunctional protein capable of interacting with proteins involved in different cellular processes, such as protein synthesis, DNA repair, and viral mRNA edition. In human cells, eIF3f is related to cell cycle and proliferation, and its deregulation compromises cell viability. Results We here report that, in native conditions, eIF3f physically interacts with the alpha 1B-adrenergic receptor, a plasma membrane protein considered as a proto-oncogene, and involved in vas...

  18. Interaction of the αβ dimers of the insulin-like growth factor I receptor required for receptor autophosphorylation

    The authors have recently found that association of the two αβ dimers of the insulin-like growth factor I (IGF I) receptor is required for formation of a high-affinity binding site for IGF I. To determine the structural requirements for IGF I activated kinase activity, they have examined the effect of dissociation of the two αβ dimers of the IGF I receptor on β subunit autophosphorylation. The αβ dimers formed after treatment with 2 mM dithiothreitol (DTT) at pH 8.75 for 5 min were separated from IGF I receptor remaining as tetramers after DTT treatment by fast protein liquid chromatography on a Superose 6 gel filtration column. Purification of the αβ dimers was confirmed by Western blot analysis using 125I-labeled αIR-3, a monoclonal antibody to the IGF I receptor. Autophosphorylation of the IGF I receptor (αβ)2 tetramer, treated without DTT or remaining after DTT treatment, is stimulated 1.6-2.9-fold by IGF I. In contrast, autophosporylation of the αβ dimers incubated in the presence or absence of IGF I (100 ng/mL) does not occur. Both IGF I receptor dimers and tetramers exhibit similar kinase activities using the synthetic substrate Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly, indicating that the failure to detect autophosphorylation of the IGF I receptor dimers does not result from inactivation of the kinase by DTT treatment. They conclude that autophosphorylation of the IGF I receptor depends upon the interaction of the two αβ dimers

  19. Acetylcholine receptor-inducing factor from chicken brain increases the level of mRNA encoding the receptor α subunit

    A 42-kDa glycoprotein isolated from chicken brain, referred to as acetylcholine receptor-inducing activity (ARIA), that stimulates the rate of incorporation of acetylcholine receptors into the surface of chicken myotubes may play a role in the nerve-induced accumulation of receptors at developing neuromuscular synapses. Using nuclease-protection assays, the authors have found that ARIA causes a 2- to 16-fold increase in the level of mRNA encoding the α subunit of the receptor, with little or no change in the levels of γ- and δ-subunit messengers. ARIA also increases the amount of a putative nuclear precursor of α-subunit mRNA, consistent with an activation of gene transcription. These results suggest that the concentration of α subunit may limit the rate of biosynthesis of the acetylcholine receptors in chicken myotubes. They also indicate that neuronal factors can regulate the expression of receptor subunit genes in a selective manner. Tetrodotoxin, 8-bromo-cAMP, and forskolin also increase the amount of α-subunit mRNA, with little change in the amount of γ- and δ-subunit mRNAs. Unlike ARIA, however, these agents have little effect on the concentration of the α-subunit nuclear precursor

  20. The Orphan Nuclear Receptor ERRγ Regulates Hepatic CB1 Receptor-Mediated Fibroblast Growth Factor 21 Gene Expression

    Jung, Yoon Seok; Lee, Ji-Min; Kim, Don-Kyu; Lee, Yong-Soo; Kim, Ki-Sun; Kim, Yong-Hoon; Kim, Jina; Lee, Myung-Shik; Lee, In-Kyu; Kim, Seong Heon; Cho, Sung Jin; Jeong, Won-Il; Lee, Chul-Ho; Harris, Robert A.; Choi, Hueng-Sik

    2016-01-01

    Background Fibroblast growth factor 21 (FGF21), a stress inducible hepatokine, is synthesized in the liver and plays important roles in glucose and lipid metabolism. However, the mechanism of hepatic cannabinoid type 1 (CB1) receptor-mediated induction of FGF21 gene expression is largely unknown. Results Activation of the hepatic CB1 receptor by arachidonyl-2’-chloroethylamide (ACEA), a CB1 receptor selective agonist, significantly increased FGF21 gene expression. Overexpression of estrogen-related receptor (ERR) γ increased FGF21 gene expression and secretion both in hepatocytes and mice, whereas knockdown of ERRγ decreased ACEA-mediated FGF21 gene expression and secretion. Moreover, ERRγ, but not ERRα and ERRβ, induced FGF21 gene promoter activity. In addition, deletion and mutation analysis of the FGF21 promoter identified a putative ERRγ-binding motif (AGGTGC, a near-consensus response element). A chromatin immunoprecipitation assay revealed direct binding of ERRγ to the FGF21 gene promoter. Finally, GSK5182, an ERRγ inverse agonist, significantly inhibited hepatic CB1 receptor-mediated FGF21 gene expression and secretion. Conclusion Based on our data, we conclude that ERRγ plays a key role in hepatic CB1 receptor-mediated induction of FGF21 gene expression and secretion. PMID:27455076

  1. ATAR, a novel tumor necrosis factor receptor family member, signals through TRAF2 and TRAF5.

    Hsu, H; Solovyev, I; Colombero, A; Elliott, R; Kelley, M; Boyle, W J

    1997-05-23

    Members of tumor necrosis factor receptor (TNFR) family signal largely through interactions with death domain proteins and TRAF proteins. Here we report the identification of a novel TNFR family member ATAR. Human and mouse ATAR contain 283 and 276 amino acids, respectively, making them the shortest known members of the TNFR superfamily. The receptor is expressed mainly in spleen, thymus, bone marrow, lung, and small intestine. The intracellular domains of human and mouse ATAR share only 25% identity, yet both interact with TRAF5 and TRAF2. This TRAF interaction domain resides at the C-terminal 20 amino acids. Like most other TRAF-interacting receptors, overexpression of ATAR activates the transcription factor NF-kappaB. Co-expression of ATAR with TRAF5, but not TRAF2, results in synergistic activation of NF-kappaB, suggesting potentially different roles for TRAF2 and TRAF5 in post-receptor signaling. PMID:9153189

  2. Tumor epidermal growth factor receptor molecular imaging research

    Because of the importance of epidermal growth factor signaling pathway in oncogenesis, maintenance, and progression of different types of tumors, there are great significance that non-invasive monitoring of epidermal growth factor receptor (EGFR) in the diagnosis and the judge of therapeutic efficacy. The studys of radioactive tracers for EGFR have provided a good basis for the molecular imaging of EGFR. (authors)

  3. Epidermal Growth Factor Receptor in Prostate Cancer Derived Exosomes

    Geetanjali Kharmate; Elham Hosseini-Beheshti; Josselin Caradec; Mei Yieng Chin; Tomlinson Guns, Emma S.

    2016-01-01

    Exosomes proteins and microRNAs have gained much attention as diagnostic tools and biomarker potential in various malignancies including prostate cancer (PCa). However, the role of exosomes and membrane-associated receptors, particularly epidermal growth factor receptor (EGFR) as mediators of cell proliferation and invasion in PCa progression remains unexplored. EGFR is frequently overexpressed and has been associated with aggressive forms of PCa. While PCa cells and tissues express EGFR, it ...

  4. Activation of Penile Proadipogenic Peroxisome Proliferator-Activated Receptor with an Estrogen: Interaction with Estrogen Receptor Alpha during Postnatal Development

    Mahmoud M. Mansour

    2008-01-01

    Full Text Available Exposure to the estrogen receptor alpha (ER ligand diethylstilbesterol (DES between neonatal days 2 to 12 induces penile adipogenesis and adult infertility in rats. The objective of this study was to investigate the in vivo interaction between DES-activated ER and the proadipogenic transcription factor peroxisome proliferator-activated receptor gamma (PPAR. Transcripts for PPARs , , and and 1a splice variant were detected in Sprague-Dawley normal rat penis with PPAR predominating. In addition, PPAR1b and PPAR2 were newly induced by DES. The PPAR transcripts were significantly upregulated with DES and reduced by antiestrogen ICI 182, 780. At the cellular level, PPAR protein was detected in urethral transitional epithelium and stromal, endothelial, neuronal, and smooth muscular cells. Treatment with DES activated ER and induced adipocyte differentiation in corpus cavernosum penis. Those adipocytes exhibited strong nuclear PPAR expression. These results suggest a biological overlap between PPAR and ER and highlight a mechanism for endocrine disruption.

  5. Activated HER-receptors in predicting outcome of ER-positive breast cancer patients treated with adjuvant endocrine therapy

    Larsen, Mathilde Skaarup; Bjerre, Karsten; Lykkesfeldt, Anne Elisabeth; Giobbie-Hurder, Anita; Lænkholm, Anne Vibeke; Henriksen, Katrine L; Ejlertsen, Bent Laursen; Rasmussen, Birgitte

    2012-01-01

    The four human epidermal growth factor receptors (HER1-4) are involved in growth stimulation and may play a role in endocrine resistance. The receptors form dimers, leading to activation by mutual phosphorylation. Our purpose was to explore the role of the activated receptors (pHER1, pHER2, pHER3...

  6. RNA Interference of Interferon Regulatory Factor-1 Gene Expression in THP-1 Cell Line Leads to Toll-Like Receptor-4 Overexpression/Activation As Well As Up-modulation of Annexin-II

    Christos I. Maratheftis

    2007-12-01

    Full Text Available Interferon regulatory factor-1 (IRF-1 is a candidate transcription factor for the regulation of the Toll-like receptor-4 (TLR-4 gene. Using a small interfering RNAbased (siRNA process to silence IRF-1 gene expression in the leukemic monocytic cell line THP-1, we investigated whether such a modulation would alter TLR-4 expression and activation status in these cells. The siIRF-1 cells expressed elevated levels of TLR-4 mRNA and protein compared to controls by 90% and 77%, respectively. ICAM.1 protein expression and apoptosis levels were increased by 8.35- and 4.25-fold, respectively. The siIRF-1 cells overexpressed Bax mRNA compared to controls. Proteomic analysis revealed upmodulation of the Annexin-II protein in siIRF-1 THP-1 cells. Myelodysplastic syndrome (MDS patients with an absence of full-length IRF-1 mRNA also overexpressed Annexin-II. It is plausible that this overexpression may lead to the activation of TLR-4 contributing to the increased apoptosis characterizing MDS.

  7. Relaxin Family Peptide Receptor 1 (RXFP1) Activation Stimulates the Peroxisome Proliferator-Activated Receptor Gamma

    Singh, Sudhir; Bennett, Robert G

    2009-01-01

    Relaxin (Rlx) has antifibrotic effects in a number of tissues. Many of these effects are similar to those induced by the activators of peroxisome proliferator-activated receptor γ (PPARγ), raising the possibility that a mechanism for Rlx’s antifibrotic effects may involve activation of the PPARγ pathway. This study investigates the effect of Rlx on PPARs and their mechanism of upregulation. It shows that Rlx stimulates ligand-independent PPAR activation in a dose-dependent manner. The combine...

  8. Activated human neutrophils release hepatocyte growth factor/scatter factor.

    McCourt, M

    2012-02-03

    BACKGROUND: Hepatocyte growth factor or scatter factor (HGF\\/SF) is a pleiotropic cytokine that has potent angiogenic properties. We have previously demonstrated that neutrophils (PMN) are directly angiogenic by releasing vascular endothelial growth factor (VEGF). We hypothesized that the acute inflammatory response can stimulate PMN to release HGF. AIMS: To examine the effects of inflammatory mediators on PMN HGF release and the effect of recombinant human HGF (rhHGF) on PMN adhesion receptor expression and PMN VEGF release. METHODS: In the first experiment, PMN were isolated from healthy volunteers and stimulated with tumour necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS), interleukin-8 (IL-8), and formyl methionyl-leucyl-phenylalanine (fMLP). Culture supernatants were assayed for HGF using ELISA. In the second experiment, PMN were lysed to measure total HGF release and HGF expression in the PMN was detected by Western immunoblotting. Finally, PMN were stimulated with rhHGF. PMN CD 11a, CD 11b, and CD 18 receptor expression and VEGF release was measured using flow cytometry and ELISA respectively. RESULTS: TNF-alpha, LPS and fMLP stimulation resulted in significantly increased release of PMN HGF (755+\\/-216, 484+\\/-221 and 565+\\/-278 pg\\/ml, respectively) compared to controls (118+\\/-42 pg\\/ml). IL-8 had no effect. Total HGF release following cell lysis and Western blot suggests that HGF is released from intracellular stores. Recombinant human HGF did not alter PMN adhesion receptor expression and had no effect on PMN VEGF release. CONCLUSIONS: This study demonstrates that pro-inflammatory mediators can stimulate HGF release from a PMN intracellular store and that activated PMN in addition to secreting VEGF have further angiogenic potential by releasing HGF.

  9. Monoclonal antibodies to the human insulin receptor that activate glucose transport but not insulin receptor kinase activity

    Forsayeth, J.R.; Caro, J.F.; Sinha, M.K.; Maddux, B.A.; Goldfine, I.D.

    1987-05-01

    Three mouse monoclonal antibodies were produced that reacted with the ..cap alpha.. subunit of the human insulin receptor. All three both immunoprecipitated /sup 125/I-labeled insulin receptors from IM-9 lymphocytes and competitively inhibited /sup 125/I-labeled insulin binding to its receptor. Unlike insulin, the antibodies failed to stimulate receptor autophosphorylation in both intact IM-9 lymphocytes and purified human placental insulin receptors. Moreover, unlike insulin, the antibodies failed to stimulate receptor-mediated phosphorylation of exogenous substrates. However, like insulin, two of the three antibodies stimulated glucose transport in isolated human adipocytes. One antibody, on a molar basis, was as potent as insulin. These studies indicate, therefore, that monoclonal antibodies to the insulin receptor can mimic a major function of insulin without activating receptor kinase activity. They also raise the possibility that certain actions of insulin such as stimulation of glucose transport may not require the activation of receptor kinase activity.

  10. Monoclonal antibodies to the human insulin receptor that activate glucose transport but not insulin receptor kinase activity

    Three mouse monoclonal antibodies were produced that reacted with the α subunit of the human insulin receptor. All three both immunoprecipitated 125I-labeled insulin receptors from IM-9 lymphocytes and competitively inhibited 125I-labeled insulin binding to its receptor. Unlike insulin, the antibodies failed to stimulate receptor autophosphorylation in both intact IM-9 lymphocytes and purified human placental insulin receptors. Moreover, unlike insulin, the antibodies failed to stimulate receptor-mediated phosphorylation of exogenous substrates. However, like insulin, two of the three antibodies stimulated glucose transport in isolated human adipocytes. One antibody, on a molar basis, was as potent as insulin. These studies indicate, therefore, that monoclonal antibodies to the insulin receptor can mimic a major function of insulin without activating receptor kinase activity. They also raise the possibility that certain actions of insulin such as stimulation of glucose transport may not require the activation of receptor kinase activity

  11. Expression of Human Vascular Endothelial Growth Factor Receptor Flt-1 Extracellular Domain 1-3 Loop cDNA in Pichia pastoris, Purification of the Expressed Product and Detection of Its Biological Activity

    2001-01-01

    Objective To express human vascular endothelial growth factor receptor Flt-1 extracellular domain 1-3 loop cDNA in Pichia. Pastroris, and to purify the expressed product and detect its biological activity. Methods By inserting human Flt-1 (1-3 loop) cDNA coding 316 amino acid residues into Pichia pastoris expression vector pPIC9K containing AOX1 promoter and the sequences of α secreting signal peptides, a recombinant expression plasmid pPIC9K/Flt-1 (1-3) was constructed and transformed to yeast host strain GS115, then His+ Muts phenotype transformant was screened out and cultured in flasks, and Flt-1 (1-3) was expressed under the induction of 1% methanol. Results SDS-PAGE showed that after being induced with 1% methanol for 4d, the expressed product existed in supernatant in the form of soluble molecule and contained 60% of total protein expressed. Western blot showed good antigenicity and specificity of expressed product. After being purified by CM-Sepharose FF and Sephacryl S-100 chromatography, the purity of the expressed product reached above 90%. Biological assay proved that the expressed product could bind to hVEGF165 and inhibit the proliferation of HUVEC stimulated by hVEGF165. Conclusion Human vascular endothelial growth factor receptor Flt-1 extracellular domain 1-3 loop was successfully expressed. The study lays a foundation for further application of the expressed product in the treatment of vasoformation related diseases, such as tumor and diabetic retinopathy.

  12. Exercise reduces adipose tissue via cannabinoid receptor type 1 which is regulated by peroxisome proliferator-activated receptor-delta

    Yan, Zhen Cheng; Liu, Dao Yan; Zhang, Li Li;

    2007-01-01

    Obesity is one major cardiovascular risk factor. We tested effects of endurance exercise on cannabinoid receptor type 1 (CB1) and peroxisome proliferator-activated receptor-delta (PPAR-delta)-dependent pathways in adipose tissue. Male Wistar rats were randomly assigned to standard laboratory chow...... or a high-fat diet without and with regular endurance exercise. Exercise in rats on high-fat diet significantly reduced visceral fat mass, blood pressure, and adipocyte size (each p...

  13. Activation of Nuclear Receptors RAR, RXR, and LXR Does Not Reduce Cuprizone-Induced Demyelination in Mice

    Davina Kruczek; Tim Clarner; Cordian Beyer; Markus Kipp; Jörg Mey

    2015-01-01

    Experiments with animal models of multiple sclerosis have shown that the expression of retinoid X receptors (RXR) increases during demyelination and that RXR is involved in the regulation of remyelination. After ligand binding RXRs form heterodimeric transcription factors with other nuclear receptor (NR) families including the retinoic acid receptors (RAR) and liver X receptors (LXR). We tested whether activation of these nuclear receptor complexes reduces pathological demyelination using the...

  14. The DEAD-Box Protein DP103 (Ddx20 or Gemin-3) Represses Orphan Nuclear Receptor Activity via SUMO Modification

    Lee, Martin B.; Lebedeva, Lioudmila A.; Suzawa, Miyuki; Wadekar, Subhagya A.; Desclozeaux, Marion; Ingraham, Holly A.

    2005-01-01

    Structural analysis of nuclear receptor subfamily V orphan nuclear receptors suggests that ligand-independent mechanisms must regulate this subclass of receptors. Here, we report that steroidogenic factor 1 (SF-1) and liver receptor homolog 1 are repressed via posttranslational SUMO modification at conserved lysines within the hinge domain. Indeed, mutating these lysines or adding the SUMO isopeptidase SENP1 dramatically increased both native and Gal4-chimera receptor activities. The mechanis...

  15. INHIBITION OF PHOSPHATASE ACTIVITY MEDIATES EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR) SIGNALING IN HUMAN AIRWAY EPITHELIAL CELLS (HAEC) EXPOSED TO ZN2+

    A number of studies have implicated zinc in the toxicity of ambient particulate matter (PM) inhalation. We previously showed that exposure to metal-laden particulate matter inhibits protein tyrosine phosphatase activity in HAEC and leads to Src-dependent activation of EGFR sign...

  16. The proteosome inhibitor MG132 attenuates Retinoic Acid Receptor trans-activation and enhances trans-repression of Nuclear Factor κB. Potential relevance to chemo-preventive interventions with retinoids

    Rosier Randy N

    2004-03-01

    Full Text Available Abstract Background Nuclear factor kappa B (NFκB is a pro-malignant transcription factor with reciprocal effects on pro-metastatic and anti-metastatic gene expression. Interestingly, NFκB blockade results in the reciprocal induction of retinoic acid receptors (RARs. Given the established property of RARs as negative regulators of malignant progression, we postulated that reciprocal interactions between NFκB and RARs constitute a signaling module in metastatic gene expression and malignant progression. Using Line 1 tumor cells as a model for signal regulation of metastatic gene expression, we investigated the reciprocal interactions between NFκB and RARs in response to the pan-RAR agonist, all-trans retinoic acid (at-RA and the pan-RAR antagonist, AGN193109. Results At-RA [0.1–1 μM] dose-dependently activated RAR and coordinately trans-repressed NFκB, while AGN193109 [1–10 μM] dose-dependently antagonized the effects of at-RA. At-RA and AGN193109 reciprocally regulate pro-metastatic matrix metalloprotease 9 (MMP 9 and its endogenous inhibitor, the tissue inhibitor of metalloprotease 1 (TIMP 1, in a manner consistent with the putative roles of NFκB and RAR in malignant progression. Activation of RAR concurs with its ubiquitination and proteosomal degradation. Accordingly, the proteosome inhibitor, MG132 [5 μM], blocked RAR degradation, quelled RAR trans-activation and enhanced RAR trans-repression of NFκB. Conclusion We conclude that reciprocal interactions between NFκB and RARs constitute a signaling module in metastatic gene expression and malignant progression and propose that the dissociative effect of proteosome inhibitors could be harnessed towards enhancing the anticancer activity of retinoids.

  17. Altered [125I]epidermal growth factor binding and receptor distribution in psoriasis

    Stimulation of growth and differentiation of human epidermis by epidermal growth factor (EGF) is mediated by its binding to specific receptors. Whether EGF receptors primarily mediate cell division or differentiation in hyperproliferative disease such as psoriasis vulgaris is unclear. To study the pathogenesis of psoriasis, 4-mm2 punch biopsy specimens of normal, uninvolved, and involved psoriatic skin were assayed for EGF receptors by autoradiographic, immunohistochemical, and biochemical methods. Using autoradiographic and immunohistochemical methods, basal keratinocytes were found to contain the greatest number of EGF binding sites and immunoreactive receptors as compared to the upper layers of the epidermis in both normal epidermis and psoriatic skin. No EGF receptor differences between normal and psoriatic epidermis were observed in this layer. In the upper layers of the epidermis, a 2-fold increase in EGF binding capacity was observed in psoriatic skin as compared with normal thin or thick skin. Biochemical methods indicated that [125I]EGF binding was increased in psoriatic epidermis as compared with similar thickness normal epidermis when measured on a protein basis. Epidermal growth factor was shown to increase phosphorylation of the EGF receptor in skin. EGF receptors retained in the nonmitotic stratum spinosum and parakeratotic stratum corneum may reflect the incomplete, abnormal differentiation that occurs in active psoriatic lesions. Alternatively, retained EGF receptors may play a direct role in inhibiting cellular differentiation in the suprabasal layers

  18. Protease activated receptors (PARS) mediation in gyroxin biological activity

    Gyroxin is a serine protease enzyme from the South American rattlesnake (Crotalus durissus terrificus) venom; it is only partially characterized and has multiple activities. Gyroxin induces blood coagulation, blood pressure decrease and a neurotoxic behavior named barrel rotation. The mechanisms involved in this neurotoxic activity are not known. Whereas gyroxin is a member of enzymes with high potential to become a new drug with clinical applications such as thrombin, batroxobin, ancrod, tripsyn and kalicrein, it is important to find out how gyroxin works. The analysis on agarose gel electrophoresis and circular dichroism confirmed the molecules' integrity and purity. The gyroxin intravenous administration in mice proved its neurotoxicity (barrel rotation). In vivo studies employing intravital microscopy proved that gyroxin induces vasodilation with the participation of protease activated receptors (PARs), nitric oxide and Na+K+ATPase. The leukocytes' adherence and rolling counting indicated that gyroxin has no pro inflammatory activity. Gyroxin induced platelet aggregation, which was blocked by inhibitors of PAR1 and PAR4 receptors (SCH 79797 and tcY-NH2, respectively). Finally, it was proved that the gyroxin temporarily alter the permeability of the blood brain barrier (BBB). Our study has shown that both the protease-activated receptors and nitric oxide are mediators involved in the biological activities of gyroxin. (author)

  19. Inhibition of protein tyrosine phosphatase activity mediates epidermal growth factor receptor signaling in human airway epithelial cells exposed to Zn2+

    Epidemiological studies have implicated zinc (Zn2+) in the toxicity of ambient particulate matter (PM) inhalation. We previously showed that exposure to metal-laden PM inhibits protein tyrosine phosphatase (PTP) activity in human primary bronchial epithelial cells (HAEC) and leads to Src-dependent activation of EGFR signaling in B82 and A431 cells. In order to elucidate the mechanism of Zn2+-induced EGFR activation in HAEC, we treated HAEC with 500 μM ZnSO4 for 5-20 min and measured the state of activation of EGFR, c-Src and PTPs. Western blots revealed that exposure to Zn2+ results in increased phosphorylation at both trans- and autophosphorylation sites in the EGFR. Zn2+-mediated EGFR phosphorylation did not require ligand binding and was ablated by the EGFR kinase inhibitor PD153035, but not by the Src kinase inhibitor PP2. Src activity was inhibited by Zn2+ treatment of HAEC, consistent with Src-independent EGFR transactivation in HAEC exposed to Zn2+. The rate of exogenous EGFR dephosphorylation in lysates of HAEC exposed to Zn2+ or V4+ was significantly diminished. Moreover, exposure of HAEC to Zn2+ also resulted in a significant impairment of dephosphorylation of endogenous EGFR. These data show that Zn2+-induced activation of EGFR in HAEC involves a loss of PTP activities whose function is to dephosphorylate EGFR in opposition to baseline EGFR kinase activity. These findings also suggest that there are marked cell-type-specific differences in the mechanism of EGFR activation induced by Zn2+ exposure

  20. Activation of Cumulus Cell SMAD2/3 and Epidermal Growth Factor Receptor Pathways Are Involved in Porcine Oocyte-Cumulus Cell Expansion and Steroidogenesis

    Nagyová, Eva; Camaioni, A.; Scsuková, S.; Mlynarčíková, A.; Procházka, Radek; Němcová, Lucie; Salustri, A.

    2011-01-01

    Roč. 78, č. 6 (2011), s. 391-402. ISSN 1040-452X R&D Projects: GA ČR GA523/08/0111 Institutional research plan: CEZ:AV0Z50450515 Keywords : MURAL GRANULOSA-CELLS * IN-VITRO MATURATION * PREOVULATORY OVARIAN -FOLLICLES Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.532, year: 2011

  1. Elevated epidermal growth factor (EGF) receptor bindings in dog lung tumors

    The objective of this study is to measure and characterize epidermal growth factor (EGF) receptor binding in dog Pu induced in lung-tumor tissue and to compare then with normal lung tissue in dogs. Epidermal growth factor and its receptors have been shown to play an important role in the proliferation and differentiation of eukaryotic cells. It was therefore hypothesized that inappropriate activation of EGF receptor plays a role in oncogenesis. Microsomal preparations prepared from normal and lung-tumor tissue from beagle dogs were used in a radioreceptor assay, using 125I-EGF as a ligand. Specific EGF receptor bindings were determined in the presence of excess unlabeled EGF. They have examined EGF receptor bindings in 10 lung-tumor samples obtained from seven dogs at necropsy. Epidermal growth factor receptor bindings were significantly higher in eight of the lung-tumor samples compared with both normal lung tissue from control dogs and normal lung tissue from dogs from which tumors were obtained. Their results suggest that the high EGF receptor bindings observed in lung-tumor tissue may play a role in the oncogenesis in dogs

  2. Expression of tumor necrosis factor related apoptosis inducing ligand receptor in glioblastoma

    Dongling Gao; Zhongwei Zhao; Hongxin Zhang; Lan Zhang; Kuisheng Chen; Yunhan Zhang

    2008-01-01

    BACKGROUND: Receptors for tumor necrosis factor related apoptosis inducing ligand (TRAIL) include death receptor 4, death receptor 5, decoy receptor 1, and decoy receptor 2. Activation of death receptor 4 and 5 selectively kills tumor cells.OBJECTIVE: To detect TRAIL receptor expression in glioblastoma by immunohistochemistry and RT-PCR and to compare this expression to that in normal brain tissue.DESIGN: Observational analysis.SETTING: Department of Pathology, the First Affiliated Hospital of Zhengzhou University; Henan Tumor Pathology Key Laboratory.PARTICIPANTS: Twenty-five patients (17 males and 8 females) who received glioblastoma resection were selected from the Fifth Affiliated Hospital of Zhengzhou University, between September 2003 to June 2004. All glioblastoma samples were diagnosed pathologically. Twenty patients (12 males and 8 females) with craniocerebral injury who received normal brain tissue resection were selected in the same time period. There were no significant differences in sex and age between glioblastoma patients or between craniocerebral injury patients (P>0.05). All patients and appropriate relatives provided informed consent, and this study was approved by the local research ethics committee.METHODS: Polyclonal antibody against TRAIL receptors and an immunohistochemical kit (batch number: 200502) were purchased from Boster Company, Wuhan. Immunohistochemistry: Expression of death receptor 4, death receptor 5, decoy receptor 1, and decoy receptor 2 were observed in both glioblastoma and normal brain tissue. The experiment was performed according to the kit instructions, and positive staining was brown-yellow. Assessment: There were no positive signals (-); weakly positive signals, positive cells75% (++++). Evaluation: Expression levels of TRAIL receptors were estimated in both normal brain tissue and glioblastoma. Expression of decoy receptor 1 and decoy receptor 2 mRNA in glioblastoma were detected by reverse transcription polymerase

  3. Design and Synthesis of Benzimidazoles As Novel Corticotropin-Releasing Factor 1 Receptor Antagonists.

    Mochizuki, Michiyo; Kori, Masakuni; Kobayashi, Katsumi; Yano, Takahiko; Sako, Yuu; Tanaka, Maiko; Kanzaki, Naoyuki; Gyorkos, Albert C; Corrette, Christopher P; Cho, Suk Young; Pratt, Scott A; Aso, Kazuyoshi

    2016-03-24

    Benzazole derivatives with a flexible aryl group bonded through a one-atom linker as a new scaffold for a corticotropin-releasing factor 1 (CRF1) receptor antagonist were designed, synthesized, and evaluated. We expected that structural diversity could be expanded beyond that of reported CRF1 receptor antagonists. In a structure-activity relationship study, 4-chloro-N(2)-(4-chloro-2-methoxy-6-methylphenyl)-1-methyl-N(7),N(7)-dipropyl-1H-benzimidazole-2,7-diamine 29g had the most potent binding activity against a human CRF1 receptor and the antagonistic activity (IC50 = 9.5 and 88 nM, respectively) without concerns regarding cytotoxicity at 30 μM. Potent CRF1 receptor-binding activity in brain in an ex vivo test and suppression of stress-induced activation of the hypothalamus-pituitary-adrenocortical (HPA) axis were also observed at 138 μmol/kg of compound 29g after oral administration in mice. Thus, the newly designed benzimidazole 29g showed in vivo CRF1 receptor antagonistic activity and good brain penetration, indicating that it is a promising lead for CRF1 receptor antagonist drug discovery research. PMID:26901666

  4. An antagonist of the platelet-activating factor receptor inhibits adherence of both nontypeable Haemophilus influenzae and Streptococcus pneumoniae to cultured human bronchial epithelial cells exposed to cigarette smoke

    Shukla SD

    2016-07-01

    Full Text Available Shakti D Shukla,1,* Rory L Fairbairn,1,* David A Gell,1 Roger D Latham,1 Sukhwinder S Sohal,1,2 Eugene H Walters,1 Ronan F O’Toole11Breathe Well Centre, School of Medicine, Faculty of Health, University of Tasmania, Hobart, TAS, Australia; 2School of Health Sciences, Faculty of Health, University of Tasmania, Launceston, TAS, Australia*These authors contributed equally to this workBackground: COPD is emerging as the third largest cause of human mortality worldwide after heart disease and stroke. Tobacco smoking, the primary risk factor for the development of COPD, induces increased expression of platelet-activating factor receptor (PAFr in the lung epithelium. Nontypeable Haemophilus influenzae (NTHi and Streptococcus pneumoniae adhere to PAFr on the luminal surface of human respiratory tract epithelial cells.Objective: To investigate PAFr as a potential drug target for the prevention of infections caused by the main bacterial drivers of acute exacerbations in COPD patients, NTHi and S. pneumoniae.Methods: Human bronchial epithelial BEAS-2B cells were exposed to cigarette smoke extract (CSE. PAFr expression levels were determined using immunocytochemistry and quantitative polymerase chain reaction. The epithelial cells were challenged with either NTHi or S. pneumoniae labeled with fluorescein isothiocyanate, and bacterial adhesion was measured using immunofluorescence. The effect of a well-evaluated antagonist of PAFr, WEB-2086, on binding of the bacterial pathogens to BEAS-2B cells was then assessed. In silico studies of the tertiary structure of PAFr and the binding pocket for PAF and its antagonist WEB-2086 were undertaken.Results: PAFr expression by bronchial epithelial cells was upregulated by CSE, and significantly associated with increased bacterial adhesion. WEB-2086 reduced the epithelial adhesion by both NTHi and S. pneumoniae to levels observed for non-CSE-exposed cells. Furthermore, it was nontoxic toward the bronchial epithelial

  5. Activation of Protease-Activated Receptor 2 Induces VEGF Independently of HIF-1

    Rasmussen, Jeppe Grøndahl; Riis, Simone Elkjær; Frøbert, Ole; Yang, Sufang; Kastrup, Jens; Zachar, Vladimir; Simonsen, Ulf; Fink, Trine

    2012-01-01

    Background Human adipose stem cells (hASCs) can promote angiogenesis through secretion of proangiogenic factors such as vascular endothelial growth factor (VEGF). In other cell types, it has been shown that induction of VEGF is mediated by both protease activated receptor 2 (PAR2) and hypoxia inducible factor 1(HIF-1). The present study hypothesized that PAR2 stimulation through activation of kinase signaling cascades lead to induction of HIF-1 and secretion of VEGF. Methodology/Principal Findings Immunohistochemistry revealed the expression of PAR2 receptors on the surface of hASCs. Blocking the PAR2 receptors with a specific antibody prior to trypsin treatment showed these receptors are involved in trypsin-evoked increase in VEGF secretion from hASCs. Blocking with specific kinase inhibitors suggested that that activation of MEK/ERK and PI3-kinase/Akt pathways are involved in trypsin-eveoked induction of VEGF. The effect of the trypsin treatment on the transcription of VEGF peaked at 6 hours after the treatment and was comparable to the activation observed after keeping hASCs for 24 hours at 1% oxygen. In contrast to hypoxia, trypsin alone failed to induce HIF-1 measured with ELISA, while the combination of trypsin and hypoxia had an additive effect on both VEGF transcription and secretion, results which were confirmed by Western blot. Conclusion In hASCs trypsin and hypoxia induce VEGF expression through separate pathways. PMID:23049945

  6. The role of the ubiquitination–proteasome pathway in breast cancer: Ubiquitin mediated degradation of growth factor receptors in the pathogenesis and treatment of cancer

    Lipkowitz, Stan

    2002-01-01

    Aberrant activity of growth factor receptors has been implicated in the pathogenesis of a wide variety of malignancies. The negative regulation of signaling by growth factor receptors is mediated in large part by the ubiquitination, internalization, and degradation of the activated receptor. Over the past few years, considerable insight into the mechanisms that control receptor downregulation has been gained. There are also data suggesting that mutations that lead to inhibition of downregulat...

  7. Quantifying Agonist Activity at G Protein-coupled Receptors

    Ehlert, Frederick J.; Suga, Hinako; Griffin, Michael T.

    2011-01-01

    When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell or tissue. This process can be analyzed at the level of a single receptor, a population of receptors, or a downstream response. Here we describe how to analyze the downstream response to obtain an estimate of the agonist affinity constant for the active state of single receptors.

  8. Regulation of Epidermal Growth Factor Receptor Trafficking by Lysine Deacetylase HDAC6

    Lissanu Deribe, Yonathan; Wild, Philipp; Chandrashaker, Akhila;

    2009-01-01

    Binding of epidermal growth factor (EGF) to its receptor leads to receptor dimerization, assembly of protein complexes, and activation of signaling networks that control key cellular responses. Despite their fundamental role in cell biology, little is known about protein complexes associated with...... the EGF receptor (EGFR) before growth factor stimulation. We used a modified membrane yeast two-hybrid system together with bioinformatics to identify 87 candidate proteins interacting with the ligand-unoccupied EGFR. Among them was histone deacetylase 6 (HDAC6), a cytoplasmic lysine deacetylase......, which we found negatively regulated EGFR endocytosis and degradation by controlling the acetylation status of alpha-tubulin and, subsequently, receptor trafficking along microtubules. A negative feedback loop consisting of EGFR-mediated phosphorylation of HDAC6 Tyr(570) resulted in reduced deacetylase...

  9. Asymmetric Receptor Contact is Required for Tyrosine Autophosphorylation of Fibroblast Growth Factor Receptor in Living Cells

    Bae, J.; Boggon, T; Tomé, F; Mandiyan, V; Lax, I; Schlessinge, J

    2010-01-01

    Tyrosine autophosphorylation of receptor tyrosine kinases plays a critical role in regulation of kinase activity and in recruitment and activation of intracellular signaling pathways. Autophosphorylation is mediated by a sequential and precisely ordered intermolecular (trans) reaction. In this report we present structural and biochemical experiments demonstrating that formation of an asymmetric dimer between activated FGFR1 kinase domains is required for transphosphorylation of FGFR1 in FGF-stimulated cells. Transphosphorylation is mediated by specific asymmetric contacts between the N-lobe of one kinase molecule, which serves as an active enzyme, and specific docking sites on the C-lobe of a second kinase molecule, which serves a substrate. Pathological loss-of-function mutations or oncogenic activating mutations in this interface may hinder or facilitate asymmetric dimer formation and transphosphorylation, respectively. The experiments presented in this report provide the molecular basis underlying the control of transphosphorylation of FGF receptors and other receptor tyrosine kinases.

  10. Human epidermal growth factor receptor residue covalently cross-linked to epidermal growth factor

    An epidermal growth factor (EGF) receptor monoclonal antibody (mAb), mAb LA22, was used to analyze the covalent coupling of human EGF receptors to mouse EGF by the amine-reactive cross-linking agent disuccinimidyl suberate. A soluble Mr 105,000 truncated form of the receptor secreted by A-431 epidermoid carcinoma cells and consisting of the ligand-binding extracellular domain was cross-linked to 125I-labeled EGF. Digestion of this complex with an endoproteinase that specifically cleaves at the COOH side of glutamyl residue released a single radiolabeled glycosylated fragment of Mr18,000 that reacted with mAb LA22. The receptor residue(s) involved in the covalent coupling of rat 125I-labeled transforming growth factor α was similarly localized to this region of the receptor. This receptor interval, which included two glycosylated asparaginyl residues at positions 328 and 337, contained but three amino acid residues that were potentially reactive with disuccinimidyl suberate: Lys-332, Lys-333, and Lys-336. These results indicated that disuccinimidyl suberate cross-linked the NH2 group of EGF residue Asn-1 to the human EGF receptor residue Lys-336. The results further suggest that EGF and transforming growth factor α, two members of the EGF family of peptide growth factors, interact with closely apposed or identical features of the receptor

  11. C-type lectin receptor dectin-3 mediates trehalose 6,6'-dimycolate (TDM)-induced Mincle expression through CARD9/Bcl10/MALT1-dependent nuclear factor (NF)-κB activation.

    Zhao, Xue-Qiang; Zhu, Le-Le; Chang, Qing; Jiang, Changying; You, Yun; Luo, Tianming; Jia, Xin-Ming; Lin, Xin

    2014-10-24

    Previous studies indicate that both Dectin-3 (also called MCL or Clec4d) and Mincle (also called Clec4e), two C-type lectin receptors, can recognize trehalose 6,6'-dimycolate (TDM), a cell wall component from mycobacteria, and induce potent innate immune responses. Interestingly, stimulation of Dectin-3 by TDM can also induce Mincle expression, which may enhance the host innate immune system to sense Mycobacterium infection. However, the mechanism by which Dectin-3 induces Mincle expression is not fully defined. Here, we show that TDM-induced Mincle expression is dependent on Dectin-3-mediated NF-κB, but not nuclear factor of activated T-cells (NFAT), activation, and Dectin-3 induces NF-κB activation through the CARD9-BCL10-MALT1 complex. We found that bone marrow-derived macrophages from Dectin-3-deficient mice were severely defective in the induction of Mincle expression in response to TDM stimulation. This defect is correlated with the failure of TDM-induced NF-κB activation in Dectin-3-deficient bone marrow-derived macrophages. Consistently, inhibition of NF-κB, but not NFAT, impaired TDM-induced Mincle expression, whereas NF-κB, but not NFAT, binds to the Mincle promoter. Dectin-3-mediated NF-κB activation is dependent on the CARD9-Bcl10-MALT1 complex. Finally, mice deficient for Dectin-3 or CARD9 produced much less proinflammatory cytokines and keyhole limpet hemocyanin (KLH)-specific antibodies after immunization with an adjuvant containing TDM. Overall, this study provides the mechanism by which Dectin-3 induces Mincle expression in response to Mycobacterium infection, which will have significant impact to improve adjuvant and design vaccine for antimicrobial infection. PMID:25202022

  12. Gemfibrozil, a lipid lowering drug, inhibits the activation of primary human microglia via peroxisome proliferator-activated receptor β

    Jana, Malabendu; Pahan, Kalipada

    2012-01-01

    Microglial activation participates in the pathogenesis of various neuroinflammatory and neurodegenerative diseases. However, mechanisms by which microglial activation could be controlled are poorly understood. Peroxisome proliferator-activated receptors (PPAR) are transcription factors belonging to the nuclear receptor super family with diverse effect. This study underlines the importance of PPARβ/δ in mediating the anti-inflammatory effect of gemfibrozil, an FDA-approved lipid-lowering drug,...

  13. Kit receptor dimerization is driven by bivalent binding of stem cell factor.

    Lemmon, M A; Pinchasi, D; Zhou, M; Lax, I; Schlessinger, J

    1997-03-01

    Most growth factors and cytokines activate their receptors by inducing dimerization upon binding. We have studied binding of the dimeric cytokine stem cell factor (SCF) to the extracellular domain of its receptor Kit, which is a receptor tyrosine kinase similar to the receptors for platelet-derived growth factor and colony-stimulating factor-1. Calorimetric studies show that one SCF dimer binds simultaneously to two molecules of the Kit extracellular domain. Gel filtration and other methods show that this results in Kit dimerization. It has been proposed that SCF-induced Kit dimerization proceeds via a conformational change that exposes a key receptor dimerization site in the fourth of the five immunoglobulin (Ig)-like domains in Kit. We show that a form of Kit containing just the first three Ig domains (Kit-123) binds to SCF with precisely the same thermodynamic parameters as does Kit-12345. Analytical ultracentrifugation, light scattering, and gel filtration show that Kit-123 dimerizes upon SCF binding in a manner indistinguishable from that seen with Kit-12345. These data argue that the fourth Ig-like domain of Kit is not required for SCF-induced receptor dimerization and provide additional support for a model in which bivalent binding of the SCF dimer provides the driving force for Kit dimerization. PMID:9045650

  14. Molecular cloning and expression of an additional epidermal growth factor receptor-related gene

    Epidermal growth factor (EGF), transforming growth factor α (TGF-α), and amphiregulin are structurally and functionally related growth regulatory proteins. These secreted polypeptides all bind to the 170-kDa cell-surface EGF receptor, activating its intrinsic kinase activity. However, amphiregulin exhibits different activities than EGF and TGF-α in a number of biological assays. Amphiregulin only partially competes with EGF for binding EGF receptor, and amphiregulin does not induce anchorage-independent growth of normal rat kidney cells (NRK) in the presence of TGF-β. Amphiregulin also appears to abrogate the stimulatory effect of TGF-α on the growth of several aggressive epithelial carcinomas that over-express EGF receptor. These findings suggest that amphiregulin may interact with a separate receptor in certain cell types. Here the authors report the cloning of another member of the human EGF receptor (HER) family of receptor tyrosine kinases, which were named HER3/ERRB3. The cDNA was isolated from a human carcinoma cell line, and its 6-kilobase transcript was identified in various human tissues. They have generated peptide-specific antisera that recognizes the 160-kDa HER3 protein when transiently expressed in COS cells. These reagents will allow us to determine whether HER3 binds amphiregulin or other growth regulatory proteins and what role HER3 protein plays in the regulation of cell growth

  15. Advances in Variations of Estrogen Receptor, Progesterone Receptor and Human Epidermal Growth Factor Receptor-2 Status in Metastatic Breast Cancer

    Yuan Yuan; Zhang Lili

    2013-01-01

    Chemotherapy, endocrine therapy and molecular targeted therapy are vital means in the treatment of metastatic breast cancer (MBC), whose reasonable and standard applications are of great importance to prolong patients’ survival and improve the quality of life. The expressions of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER-2) present signiifcant differences between primary and metastatic breast cancer. However, these differences may affect the selection of MBC patients for therapeutic strategies and judgment on the prognosis. Hence, the relevant researches on variations of hormone receptors and HER-2 in primary and metastatic breast cancer, discordant causes of ER, PR and HER-2 expression in primary and metastatic lesions and clinical value of biopsy to the metastases are reviewed in the study.

  16. Design, synthesis, anti-tumor activity, and molecular modeling of quinazoline and pyrido[2,3-d]pyrimidine derivatives targeting epidermal growth factor receptor.

    Hou, Ju; Wan, Shanhe; Wang, Guangfa; Zhang, Tingting; Li, Zhonghuang; Tian, Yuanxin; Yu, Yonghuan; Wu, Xiaoyun; Zhang, Jiajie

    2016-08-01

    Three series of novel quinazoline and pyrido[2,3-d]pyrimidine derivatives were designed, synthesized and evaluated for their ability to inhibit EGFR tyrosine kinase and a panel of five human cancer cell lines (MCF-7, A549, BT-474, SK-BR-3, and MDA-MB-231). Bioassay results indicated that five of these prepared compounds (12c-12e and 13c-13d) exhibited remarkably higher inhibitory activities against EGFR and SK-BR-3 cell line. Compounds 12c and 12e displayed the most potent EGFR inhibitory activity (IC50 = 2.97 nM and 3.58 nM, respectively) and good anti-proliferative effect against SK-BR-3 cell with the IC50 values of 3.10 μM and 5.87 μM, respectively. Furthermore, molecular docking and molecular dynamics simulation studies verified that compound 12c and 12e shared similar binding pattern with gefitinib in the binding pocket of EGFR. MM-GBSA binding free energy revealed that the compound 12c and 12e have almost the same inhibitory activity against EGFR as gefitinib, and that the dominating effect of van der Waals interactions drives the binding process. PMID:27132165

  17. Tyrosine kinase signalling in breast cancer: Epidermal growth factor receptor - convergence point for signal integration and diversification

    Cross-communication between different signalling systems is critical for the integration of multiple and changing environmental influences on individual cells. The epidermal growth factor receptor (EGFR) has been identified as a key element in the complex signalling network that is utilized by various classes of cell-surface receptors. This nonclassical mode of signalling system cross-talk, in distinction to receptor activation induced by cognate ligands, has been termed 'signal transactivation'. With the EGFR as the convergence point and distribution focus, this scenario may involve signals emitted by other members of the tyrosine kinase family, cytokine receptors, ion channels, G-protein-coupled receptors and integrins

  18. Cannabinoids go nuclear: evidence for activation of peroxisome proliferator-activated receptors

    O'Sullivan, S E

    2007-01-01

    Cannabinoids act at two classical cannabinoid receptors (CB1 and CB2), a 7TM orphan receptor and the transmitter-gated channel transient receptor potential vanilloid type-1 receptor. Recent evidence also points to cannabinoids acting at members of the nuclear receptor family, peroxisome proliferator-activated receptors (PPARs, with three subtypes α, β (δ) and γ), which regulate cell differentiation and lipid metabolism. Much evidence now suggests that endocannabinoids are natural activators o...

  19. Roles of activin receptor-like kinase 7 signaling and its target, peroxisome proliferator-activated receptor γ, in lean and obese adipocytes

    Yogosawa, Satomi; Izumi, Tetsuro

    2013-01-01

    We recently discovered a novel signaling pathway involving activin receptor-like kinase 7 (ALK7), one of the type I transforming growth factorreceptors. ALK7 and activated Smads 2, 3, and 4 inhibit the master regulators of adipogenesis, CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ) specifically in differentiated adipocytes, but surprisingly increase both the adipocyte size and lipid content by suppressing lipolysis. Here, we show that, a...

  20. Platelet-derived growth factor mimics phorbol diester action on epidermal growth factor receptor phosphorylation at threonine-654

    Addition of platelet-derived growth factor (PDGF) to quiescent WI-38 human fetal lung fibroblasts mimics the effect of tumor-promoting phorbol diesters to inhibit the high-affinity binding of 125I-labeled epidermal growth factor (125I-EGF). PDGF, like phorbol diesters, was found to increase the phosphorylation state of EGF receptors immunoprecipitated from intact fibroblasts that were labeled to equilibrium with [32P]phosphate. Phosphoamino acid analysis of the EGF receptors indicated that both PDGF and phorbol diesters increased the level of [32P]phosphoserine and [32P]phosphothreonine. Phosphopeptide mapping of the EGF receptor demonstrated that PDGF increased the phosphorylation of several sites and induced the phosphorylation of a site that was not observed to be phosphorylated on EGF receptors isolated from control cells. This latter phosphorylation site on the EGF receptor was identified as threonine-654. These results are consistent with the hypothesis that increases in diacylglycerol and Ca2+ levels caused by addition of PDGF to fibroblasts activate protein kinase C and that this kinase, at least in part, mediates the effect of PDGF on the phosphorylation of the EGF receptor. The data further suggest that protein kinase C may play an important role in the regulation of cellular metabolism and proliferation by PDGF

  1. The ontogeny of epidermal growth factor receptors during mouse development

    In an attempt to understand the role(s) of epidermal growth factor (EGF) in vivo during murine development, we have examined the 125I-EGF binding characteristics of EGF-receptors in membrane preparations of tissues from the 12th day of gestation to parturition. Using autoradiography, the earliest time that we could detect EGF-receptors was on trophoblast cells cultured for 3 days as blastocyst outgrowths. Trophoblast eventually forms a large portion of the placenta, where EGF-receptors have long been recognized. We measured the number and affinity of EGF-receptors on tissues dissected from conceptuses from the 12th day of gestation in order to identify a stage when tissues may be most sensitive to EGF. Whereas the number of EGF receptors increases during gestation for all tissues examined, the affinity of the receptors declines for carcass and placenta and remains relatively unchanged for brain and liver. This suggests that EGF may function differently throughout development. Our hypothesis is that EGF (or its embryonic equivalent) initially stimulates proliferation in embryonic cells and then stimulates differentiation as the tissues mature. In the adult, its main role could be to stimulate tissue repair after damage

  2. Fluoxetine-induced transactivation of the platelet-derived growth factor type β receptor reveals a novel heterologous desensitization process.

    Kruk, Jeff S; Vasefi, Maryam S; Gondora, Nyasha; Ahmed, Nawaz; Heikkila, John J; Beazely, Michael A

    2015-03-01

    Many G protein-coupled receptors (GPCRs), including serotonin (5-HT) receptors promote the activity of receptor tyrosine kinases (RTKs) via intracellular signaling pathways in a process termed transactivation. Although transactivation pathways are commonly initiated by a GPCR, a recent report demonstrated that serotonin-selective reuptake inhibitors (SSRIs) were able to block 5-HT-induced transactivation of the platelet-derived growth factor (PDGF) type β receptor. We show that a 45 min pretreatment of SH-SY5Y cells with the SSRI fluoxetine indeed blocked 5-HT-induced transactivation of the PDGFβ receptor. However, upon further examination, we discovered that during the pretreatment period, fluoxetine itself was transiently transactivating the PDGFβ receptor via 5-HT2 receptor activation. After 45min, the increase in PDGFβ receptor phosphorylation induced by fluoxetine had returned to baseline, but a subsequent transactivating stimulus (5-HT) failed to "re-transactivate" the PDGFβ receptor. We further demonstrate that 45min, but not 3h, 5-HT pretreatment blocks dopamine-induced PDGFβ receptor transactivation. This did not involve changes in PDGF receptor function, since ligand (PDGF)-induced PDGFβ receptor activation was not inhibited by 5-HT pretreatment. To our knowledge this is the first demonstration of the heterologous desensitization of an RTK transactivation pathway and reveals a previously unknown short-term "blackout" period where no additional transactivation signaling is possible. PMID:25702926

  3. The neural cell adhesion molecule binds to fibroblast growth factor receptor 2

    Christensen, Claus; Lauridsen, Jes B; Berezin, Vladimir; Bock, Elisabeth; Kiselyov, Vladislav V

    2006-01-01

    The neural cell adhesion molecule (NCAM) can bind to and activate fibroblast growth factor receptor 1 (FGFR1). However, there are four major FGFR isoforms (FGFR1-FGFR4), and it is not known whether NCAM also interacts directly with the other three FGFR isoforms. In this study, we show by surface...

  4. Brain-derived neurotrophic factor in human subjects with function-altering melanocortin-4 receptor variants

    In rodents, hypothalamic brain-derived neurotrophic factor (BDNF) expression appears to be regulated by melanocortin-4 receptor (MC4R) activity. The impact of MC4R genetic variation on circulating BDNF in humans is unknown. The objective of this study is to compare BDNF concentrations of subjects wi...

  5. Model for growth hormone receptor activation based on subunit rotation within a receptor dimer

    Brown, Richard J.; Adams, Julian J.; Pelekanos, Rebecca A.; Wan, Yu; McKinstry, William J.; Palethorpe, Kathryn; Seeber, Ruth M.; Monks, Thea A.; Eidne, Karin A.; Parker, Michael W.; Waters, Michael J. (UWA); (St. Vincent); (Queensland)

    2010-07-13

    Growth hormone is believed to activate the growth hormone receptor (GHR) by dimerizing two identical receptor subunits, leading to activation of JAK2 kinase associated with the cytoplasmic domain. However, we have reported previously that dimerization alone is insufficient to activate full-length GHR. By comparing the crystal structure of the liganded and unliganded human GHR extracellular domain, we show here that there is no substantial change in its conformation on ligand binding. However, the receptor can be activated by rotation without ligand by inserting a defined number of alanine residues within the transmembrane domain. Fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET) and coimmunoprecipitation studies suggest that receptor subunits undergo specific transmembrane interactions independent of hormone binding. We propose an activation mechanism involving a relative rotation of subunits within a dimeric receptor as a result of asymmetric placement of the receptor-binding sites on the ligand.

  6. Dynamic correlation networks in human peroxisome proliferator-activated receptor-γ nuclear receptor protein.

    Fidelak, Jeremy; Ferrer, Silvia; Oberlin, Michael; Moras, Dino; Dejaegere, Annick; Stote, Roland H

    2010-10-01

    Peroxisome proliferator-activated receptor-γ nuclear receptor (PPAR-γ) belongs to the superfamily of nuclear receptor proteins that function as ligand-dependent transcription factors and plays a specific physiological role as a regulator of lipid metabolism. A number of experimental studies have suggested that allostery plays an important role in the functioning of PPAR-γ. Here we use normal-mode analysis of PPAR-γ to characterize a network of dynamically coupled amino acids that link physiologically relevant binding surfaces such as the ligand-dependent activation domain AF-2 with the ligand binding site and the heterodimer interface. Multiple calculations were done in both the presence and absence of the agonist rosiglitazone, and the differences in dynamics were characterized. The global dynamics of the ligand binding domain were affected by the ligand, and in particular, changes to the network of dynamically correlated amino acids were observed with only small changes in conformation. These results suggest that changes in dynamic couplings can be functionally significant with respect to the transmission of allosteric signals. PMID:20496064

  7. Activation of the chicken gonadotropin-inhibitory hormone receptor reduces gonadotropin releasing hormone receptor signaling.

    Shimizu, Mamiko; Bédécarrats, Grégoy Y

    2010-06-01

    Gonadotropin-inhibitory hormone (GnIH) is a hypothalamic peptide from the RFamide peptide family that has been identified in multiple avian species. Although GnIH has clearly been shown to reduce LH release from the anterior pituitary gland, its mechanism of action remains to be determined. The overall objectives of this study were (1) to characterize the GnIH receptor (GnIH-R) signaling pathway, (2) to evaluate potential interactions with gonadotropin releasing hormone type III receptor (GnRH-R-III) signaling, and (3) to determine the molecular mechanisms by which GnIH and GnRH regulate pituitary gonadotrope function during a reproductive cycle in the chicken. Using real-time PCR, we showed that in the chicken pituitary gland, GnIH-R mRNA levels fluctuate in an opposite manner to GnRH-R-III, with higher and lower levels observed during inactive and active reproductive stages, respectively. We demonstrated that the chicken GnIH-R signals by inhibiting adenylyl cyclase cAMP production, most likely by coupling to G(alphai). We also showed that this inhibition is sufficient to significantly reduce GnRH-induced cAMP responsive element (CRE) activation in a dose-dependent manner, and that the ratio of GnRH/GnIH receptors is a significant factor. We propose that in avian species, sexual maturation is characterized by a change in GnIH/GnRH receptor ratio, resulting in a switch in pituitary sensitivity from inhibitory (involving GnIH) to stimulatory (involving GnRH). In turn, decreasing GnIH-R signaling, combined with increasing GnRH-R-III signaling, results in significant increases in CRE activation, possibly initiating gonadotropin synthesis. PMID:20350548

  8. The diminished expression of proangiogenic growth factors and their receptors in gastric ulcers of cirrhotic patients.

    Jiing-Chyuan Luo

    Full Text Available OBJECTIVES: The pathogenesis of the higher occurrence of peptic ulcer disease in cirrhotic patients is complex. Platelets can stimulate angiogenesis and promote gastric ulcer healing. We compared the expressions of proangiogenic growth factors and their receptors in the gastric ulcer margin between cirrhotic patients with thrombocytopenia and those of non-cirrhotic patients to elucidate possible mechanisms. METHODS: Eligible cirrhotic patients (n = 55 and non-cirrhotic patients (n = 55 who had gastric ulcers were enrolled. Mucosa from the gastric ulcer margin and non-ulcer areas were sampled and the mRNA expressions of the proangiogenic growth factors (vascular endothelial growth factor [VEGF], platelet derived growth factor [PDGF], basic fibroblast growth factor [bFGF] and their receptors (VEGFR1, VEGFR2, PDGFRA, PDGFRB, FGFR1, FGFR2 were measured and compared. Platelet count and the expressions of these growth factors and their receptors were correlated with each other. RESULTS: The two groups were comparable in terms of gender, ulcer size and infection rate of Helicobacter pylori. However, the cirrhotic group were younger in age, had a lower platelet count than those in the non-cirrhotic group (p0.5, p<0.001. CONCLUSIONS: Our findings implied that diminished activity of proangiogenic factors and their receptors may contribute to the pathogenesis of gastric ulcers in cirrhotic patients.

  9. p53-independent activation of the hdm2-P2 promoter through multiple transcription factor response elements results in elevated hdm2 expression in estrogen receptor alpha-positive breast cancer cells.

    Phelps, Monika; Darley, Matthew; Primrose, John N; Blaydes, Jeremy P

    2003-05-15

    The negative-regulatory feedback loop between p53 and hdm2 forms part of a finely balanced regulatory network of proteins that controls cell cycle progression and commitment to apoptosis. Expression of hdm2, and its mouse orthologue mdm2, is known to be induced by p53, but recent evidence has demonstrated mdm2 expression can also be regulated via p53-independent pathways. However the p53 independent mechanisms that control transcription of the human hdm2 gene have not been studied. Differential levels of hdm2 mRNA and protein expression have been reported in several types of human malignancy, including breast cancers in which hdm2 expression correlates with positive estrogen receptor alpha (ERalpha) status. Experimental models have demonstrated that hdm2 overexpression can promote breast cancer development. Here, we show that the elevated level of hdm2 protein in ERalpha(+ve) breast cancer cell lines such as MCF-7 and T47D is because of transcription from the p53-inducible P2 promoter of hdm2. The P2 promoter is inactive in ERalpha(-ve) cell lines such as SKBr3. Hdm2-P2 promoter activity in T47D cells is independent of p53, as well as of known regulators of the mouse mdm2-P2 promoter, including ERalpha and ras-raf-mitogen-activated protein/extracellular signal-regulated kinase (MEK) mitogen-activated protein kinase (MAPK) signaling. We show that hdm2-P2 activity in T47D cells is dependent on the integrity of both an evolutionarily conserved composite binding site for AP1 and ETS family transcription factors (AP1-ETS) and a nonconserved upstream (nnGGGGC)(5) repeat sequence. Lack of hdm2-P2 activity in ERalpha(-ve) cells is shown to be a consequence of reduced transcriptional activation through the AP1-ETS element. Overexpression of ETS2 in SKBr3 cells reconstitutes AP1-ETS element-dependent hdm2-P2 promoter activity, resulting in increased levels of hdm2 protein in the cells. Our findings support the hypothesis that the elevated levels of hdm2 expression reported

  10. Quantitative structure-activity relationships of selective antagonists of glucagon receptor using QuaSAR descriptors.

    Manoj Kumar, Palanivelu; Karthikeyan, Chandrabose; Hari Narayana Moorthy, Narayana Subbiah; Trivedi, Piyush

    2006-11-01

    In the present paper, quantitative structure activity relationship (QSAR) approach was applied to understand the affinity and selectivity of a novel series of triaryl imidazole derivatives towards glucagon receptor. Statistically significant and highly predictive QSARs were derived for glucagon receptor inhibition by triaryl imidazoles using QuaSAR descriptors of molecular operating environment (MOE) employing computer-assisted multiple regression procedure. The generated QSAR models revealed that factors related to hydrophobicity, molecular shape and geometry predominantly influences glucagon receptor binding affinity of the triaryl imidazoles indicating the relevance of shape specific steric interactions between the molecule and the receptor. Further, QSAR models formulated for selective inhibition of glucagon receptor over p38 mitogen activated protein (MAP) kinase of the compounds in the series highlights that the same structural features, which influence the glucagon receptor affinity, also contribute to their selective inhibition. PMID:17077558

  11. The Structure of the GM-CSF Receptor Complex Reveals a Distinct Mode of Cytokine Receptor Activation

    Hansen, Guido; Hercus, Timothy R.; McClure, Barbara J.; Stomski, Frank C.; Dottore, Mara; Powell, Jason; Ramshaw, Hayley; Woodcock, Joanna M.; Xu, Yibin; Guthridge, Mark; McKinstry, William J.; Lopez, Angel F.; Parker, Michael W. (SVIMR-A); (Hanson)

    2008-08-11

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine that controls the production and function of blood cells, is deregulated in clinical conditions such as rheumatoid arthritis and leukemia, yet offers therapeutic value for other diseases. Its receptors are heterodimers consisting of a ligand-specific {alpha} subunit and a {beta}c subunit that is shared with the interleukin (IL)-3 and IL-5 receptors. How signaling is initiated remains an enigma. We report here the crystal structure of the human GM-CSF/GM-CSF receptor ternary complex and its assembly into an unexpected dodecamer or higher-order complex. Importantly, mutagenesis of the GM-CSF receptor at the dodecamer interface and functional studies reveal that dodecamer formation is required for receptor activation and signaling. This unusual form of receptor assembly likely applies also to IL-3 and IL-5 receptors, providing a structural basis for understanding their mechanism of activation and for the development of therapeutics.

  12. Thyroid hormone receptor β mutants: Dominant negative regulators of peroxisome proliferator-activated receptor γ action

    Araki, Osamu; Ying, Hao; Furuya, Fumihiko; Zhu, Xuguang; Cheng, Sheue-yann

    2005-01-01

    Thyroid hormone (T3) and peroxisome proliferators have overlapping metabolic effects in the maintenance of lipid homeostasis. Their actions are mediated by their respective receptors: thyroid hormone receptors (TR) and peroxisome proliferator-activated receptors (PPAR). We recently found that a dominantly negative TRβ mutant (PV) that causes a genetic disease, resistance to thyroid hormone, acts to repress the ligand (troglitazone)-mediated transcriptional activity of PPARγ in cultured thyroi...

  13. Distribution of corticotropin-releasing factor receptors in primate brain

    The distribution and properties of receptors for corticotropin-releasing factor (CRF) were analyzed in the brain of cynomolgus monkeys. Binding of [125I]tyrosine-labeled ovine CRF to frontal cortex and amygdala membrane-rich fractions was saturable, specific, and time- and temperature-dependent, reaching equilibrium in 30 min at 230C. Scatchard analysis of the binding data indicated one class of high-affinity sites with a K/sub d/ of 1 nM and a concentration of 125 fmol/mg. As in the rat pituitary and brain, CRF receptors in monkey cerebral cortex and amygdala were coupled to adenylate cyclase. Autoradiographic analysis of specific CRF binding in brain sections revealed that the receptors were widely distributed in the cerebral cortex and limbic system. Receptor density was highest in the pars tuberalis of the pituitary and throughout the cerebral cortex, specifically in the prefrontal, frontal, orbital, cingulate, insular, and temporal areas, and in the cerebellar cortex. A low binding density was present in the superior colliculus, locus coeruleus, substantia gelatinosa, preoptic area, septal area, and bed nucleus of the stria terminalis. These data demonstrate that receptors for CRF are present within the primate brain at areas related to the central control of visceral function and behavior, suggesting that brain CRF may serve as a neurotransmitter in the coordination of endocrine and neural mechanisms involved in the response to stress

  14. Vascular oxidative stress upregulates angiotensin II type I receptors via mechanisms involving nuclear factor kappa B

    Bhatt, Siddhartha R.; Lokhandwala, Mustafa F.; Banday, Anees Ahmad

    2014-01-01

    The association of oxidative stress with hypertension is well known. However, a causal role of oxidative stress in hypertension is unclear. Vascular angiotensin II type 1 receptor (AT1R) upregulation is a prominent contributor to pathogenesis of hypertension. However, the mechanisms causing this upregulation are unknown. Oxidative stress is an important regulator of protein expression via activation of transcription factors such as nuclear factor kappa B (NFκB). The present study was carried ...

  15. The insulin receptor C-terminus is involved in regulation of the receptor kinase activity.

    Kaliman, P; Baron, V; Alengrin, F; Takata, Y; Webster, N J; Olefsky, J M; Van Obberghen, E

    1993-09-21

    During the insulin receptor activation process, ligand binding and autophosphorylation induce two distinct conformational changes in the C-terminal domain of the receptor beta-subunit. To analyze the role of this domain and the involvement of the C-terminal autophosphorylation sites (Tyr1316 and Tyr1322) in receptor activation, we used (i) antipeptide antibodies against three different C-terminal sequences (1270-1281, 1294-1317, and 1309-1326) and (ii) an insulin receptor mutant (Y/F2) where Tyr1316 and Tyr1322 have been replaced by Phe. We show that the autophosphorylation-induced C-terminal conformational change is preserved in the Y/F2 receptor, indicating that this change is not induced by phosphorylation of the C-terminal sites but most likely by phosphorylation of the major sites in the kinase domain (Tyr1146, Tyr1150, and Tyr1151). Binding of antipeptide antibodies to the C-terminal domain modulated (activated or inhibited) both mutant and wild-type receptor-mediated phosphorylation of poly(Glu/Tyr). In contrast to the wild-type receptor, Y/F2 exhibited the same C-terminal configuration before and after insulin binding, evidencing that mutation of Tyr1316 and Tyr1322 introduced conformational changes in the C-terminus. Finally, the mutant receptor was 2-fold more active than the wild-type receptor for poly(Glu/Tyr) phosphorylation. In conclusion, the whole C-terminal region of the insulin receptor beta-subunit is likely to exert a regulatory influence on the receptor kinase activity. Perturbations of the C-terminal region, such as binding of antipeptides or mutation of Tyr1316 and Tyr1322, provoke alterations at the receptor kinase level, leading to activation or inhibition of the enzymic activity. PMID:7690586

  16. The WSXWS motif in cytokine receptors is a molecular switch involved in receptor activation

    Dagil, Robert; Knudsen, Maiken J.; Olsen, Johan Gotthardt;

    2012-01-01

    The prolactin receptor (PRLR) is activated by binding of prolactin in a 2:1 complex, but the activation mechanism is poorly understood. PRLR has a conserved WSXWS motif generic to cytokine class I receptors. We have determined the nuclear magnetic resonance solution structure of the membrane prox...

  17. P2X4-receptor mediated synthesis and release of brain-derived neurotrophic factor in microglia is dependent on calcium and p38-mitogen-activated protein kinase activation

    Trang, Tuan; Beggs, Simon; Wan, Xiang; Salter, Michael W

    2009-01-01

    Microglia in the dorsal horn of the spinal cord are increasingly recognized as being crucial in the pathogenesis of pain hypersensitivity following injury to a peripheral nerve. It is known that P2X4 purinoceptors (P2X4Rs) cause the release of brain-derived neurotrophic factor (BDNF) from microglia, which is necessary for maintaining pain hypersensitivity after nerve injury. However, there is a critical gap in understanding how activation of microglial P2X4Rs leads to the release of BDNF. Her...

  18. Farnesoid X Receptor Inhibits the Transcriptional Activity of Carbohydrate Response Element Binding Protein in Human Hepatocytes

    Caron, Sandrine; Huaman Samanez, Carolina; Dehondt, Hélène; Ploton, Maheul; Briand, Olivier; Lien, Fleur; Dorchies, Emilie; Dumont, Julie; Postic, Catherine; Cariou, Bertrand; Lefebvre, Philippe; Staels, Bart

    2013-01-01

    The glucose-activated transcription factor carbohydrate response element binding protein (ChREBP) induces the expression of hepatic glycolytic and lipogenic genes. The farnesoid X receptor (FXR) is a nuclear bile acid receptor controlling bile acid, lipid, and glucose homeostasis. FXR negatively regulates hepatic glycolysis and lipogenesis in mouse liver. The aim of this study was to determine whether FXR regulates the transcriptional activity of ChREBP in human hepatocytes and to unravel the...

  19. Platelet-derived growth factor receptors in the human central nervous system : autoradiographic distribution and receptor densities in multiple sclerosis

    De Keyser, J; Wilczak, N

    1997-01-01

    Platelet derived growth factor (PDGF) receptors were studied in postmortem adult human brain and cervical spinal cord using autoradiography with human recombinant I-125-PDGF-BB. PDGF-BB binds to the three different dimers of PDGF receptors (alpha alpha, alpha beta and beta beta) PDGF receptors were

  20. Antipeptide antibody that specifically inhibits insulin receptor autophosphorylation and protein kinase activity

    Two site-specific antibodies that immunoprecipitate the human insulin receptor have been prepared by immunizing rabbits with chemically synthesized peptides derived from the cDNA-predicted amino acid sequence of the β subunit of the proreceptor. Antibodies to the carboxyl terminus (AbP5) and to a domain around tyrosine-960 (AbP4) specifically recognize the β subunit of the receptor on immunoblots. Both antibodies immunoprecipitated 125I-labeled insulin-receptor complexes and the autophosphorylated receptor. Although neither antibody inhibited insulin binding to the receptor, both insulin-dependent autophosphorylation and exogenous substrate phosphorylation were inhibited by AbP4. Inhibition by AbP4 was dependent upon the phosphorylation state of the receptor; it was not detected when the receptor was autophosphorylated prior to addition of AbP4. AbP4 did not inhibit activity of the related epidermal growth factor (EGF)-receptor tyrosine protein kinase nor did it inhibit the activity of cAMP-dependent kinase or protein kinase C. The observation that an antibody directed to residues 952-967 of the proreceptor neutralizes the protein kinase activity of the β subunit suggest that this region may play a critical role in the function of the hormone-dependent, protein tyrosine-specific kinase activity of the insulin receptor

  1. Transcriptional regulation of the hepatocyte growth factor gene by the nuclear receptors chicken ovalbumin upstream promoter transcription factor and estrogen receptor.

    Jiang, J G; Bell, A; Liu, Y; Zarnegar, R

    1997-02-14

    Hepatocyte growth factor (HGF) is a multifunctional cytokine that controls the growth and differentiation of various tissues. Previously, we described the existence of a negative cis-acting regulatory element(s) within the -1- to -0.7-kilobase pair (kb) portion of the 5'-flanking region of the mouse HGF promoter. In the present study, we show that the repressor element is located at position -872 to -860 base pairs and comprises an imperfect estrogen-responsive element 5'-AGGTCAGAAAGACCA-3'. We demonstrate that chicken ovalbumin upstream promoter transcription factor (COUP-TF), a nuclear orphan receptor belonging to the steroid/thyroid hormone receptor superfamily, through binding to this site effectively silences the transcriptional activity of the HGF promoter. We show that estrogen receptor, on the other hand, relieves the repressive action of COUP-TF, resulting in the induction of the HGF promoter. Using mice transgenic for either 2.7 or 0.7 kb of the HGF promoter region linked to the chloramphenicol acetyltransferase reporter gene, we found that injection of estradiol stimulates HGF promoter activity in tissues such as the mammary gland and ovary of mice harboring 2.7 but not 0.7 kb of the mouse HGF promoter region. Potential involvement of the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors in the regulation of HGF gene expression is also discussed. PMID:9020096

  2. The adipogenic acetyltransferase Tip60 targets activation function 1 of peroxisome proliferator-activated receptor gamma

    van Beekum, Olivier; Brenkman, Arjan B; Grøntved, Lars;

    2008-01-01

    proteins with which this nuclear receptor interacts under specific conditions. Here we identify the HIV-1 Tat-interacting protein 60 (Tip60) as a novel positive regulator of PPARgamma transcriptional activity. Using tandem mass spectrometry, we found that PPARgamma and the acetyltransferase Tip60 interact......-mediated reduction of Tip60 protein impairs differentiation of 3T3-L1 preadipocytes. Taken together, these findings qualify the acetyltransferase Tip60 as a novel adipogenic factor....

  3. Ciliary Neurotrophic Factor Receptor Regulation of Adult Forebrain Neurogenesis

    Lee, Nancy; Batt, Myra K.; Cronier, Brigitte A.; Jackson, Michele C.; Bruno Garza, Jennifer L; Trinh, Dennis S.; Mason, Carter O.; Spearry, Rachel P.; Bhattacharya, Shayon; Robitz, Rachel; Nakafuku, Masato; MacLennan, A. John

    2013-01-01

    Appropriately targeted manipulation of endogenous neural stem progenitor (NSP) cells may contribute to therapies for trauma, stroke, and neurodegenerative disease. A prerequisite to such therapies is a better understanding of the mechanisms regulating adult NSP cells in vivo. Indirect data suggest that endogenous ciliary neurotrophic factor (CNTF) receptor signaling may inhibit neuronal differentiation of NSP cells. We challenged subventricular zone (SVZ) cells in vivo with low concentrations...

  4. Epidermal growth factor receptor expression in canine transitional cell carcinoma

    HANAZONO, Kiwamu; Fukumoto, Shinya; KAWAMURA, Yoshio; ENDO, Yoshifumi; Kadosawa, Tsuyoshi; IWANO, Hidetomo; UCHIDE, Tsuyoshi

    2014-01-01

    Transitional cell carcinoma (TCC), a urinary bladder tumor with high mortality, is encountered commonly in dogs. Whereas overexpression of epidermal growth factor receptor (EGFR) is associated with development of human urinary bladder cancer, information on EGFR expression in canine TCC is lacking. In this study, EGFR protein and mRNA expression in canine normal bladder (n=5), polypoid cystitis (n=5) and TCC (n=25) were examined by immunohistochemistry and real-time polymerase chain reaction....

  5. The epidermal growth factor receptor (EGFR in head and neck cancer: its role and treatment implications

    Azria David

    2006-05-01

    Full Text Available Abstract Epidermal growth factor receptor (EGFR is a member of the ErbB family of receptors. Its stimulation by endogenous ligands, EGF or transforming growth factor-alpha (TGF-α results in activation of intracellular tyrosine kinase, therefore, cell cycle progression. High levels of EGFR expression are correlated with poor prognosis and resistance to radiation therapy in a variety of cancers, mostly in squamous-cell carcinoma of the head and neck (SCCHN. Blocking the EGFR by a monoclonal antibody results in inhibition of the stimulation of the receptor, therefore, in inhibition of cell proliferation, enhanced apoptosis, and reduced angiogenesis, invasiveness and metastases. The EGFR is a prime target for new anticancer therapy in SCCHN, and other agents in development include small molecular tyrosine kinase inhibitors and antisense therapies.

  6. The epidermal growth factor receptor (EGFR) in head and neck cancer: its role and treatment implications

    Epidermal growth factor receptor (EGFR) is a member of the ErbB family of receptors. Its stimulation by endogenous ligands, EGF or transforming growth factor-alpha (TGF-α) results in activation of intracellular tyrosine kinase, therefore, cell cycle progression. High levels of EGFR expression are correlated with poor prognosis and resistance to radiation therapy in a variety of cancers, mostly in squamous-cell carcinoma of the head and neck (SCCHN). Blocking the EGFR by a monoclonal antibody results in inhibition of the stimulation of the receptor, therefore, in inhibition of cell proliferation, enhanced apoptosis, and reduced angiogenesis, invasiveness and metastases. The EGFR is a prime target for new anticancer therapy in SCCHN, and other agents in development include small molecular tyrosine kinase inhibitors and antisense therapies

  7. Activation of glucocorticoid receptors increases 5-HT2A receptor levels

    Trajkovska, Viktorija; Kirkegaard, Lisbeth; Krey, Gesa; Marcussen, Anders Bue; Thomsen, Morten Skøtt; Chourbaji, Sabine; Brandwein, Christiane; Ridder, Stephanie; Halldin, Christer; Gass, Peter; Knudsen, Gitte M; Aznar, Susana

    2009-01-01

    Major depression is associated with both dysregulation of the hypothalamic pituitary adrenal axis and serotonergic deficiency, not the least of the 5-HT2A receptor. However, how these phenomena are linked to each other, and whether a low 5-HT2A receptor level is a state or a trait marker of...... depression is unknown. In mice with altered glucocorticoid receptor (GR) expression we investigated 5-HT2A receptor levels by Western blot and 3H-MDL100907 receptor binding. Serotonin fibre density was analyzed by stereological quantification of serotonin transporter immunopositive fibers. To establish an...... effect of GR activation on 5-HT2A levels, mature organotypic hippocampal cultures were exposed to corticosterone with or without GR antagonist mifepristone and mineralocorticoid receptor (MR) antagonist spironolactone. In GR under-expressing mice, hippocampal 5-HT2A receptor protein levels were decreased...

  8. Altered expression of epidermal growth factor receptor and estrogen receptor in MCF-7 cells after single and repeated radiation exposures

    This study focuses on the characterization of expression modulation of two critical growth regulatory genes, estrogen receptor and epidermal growth factor-receptor, in malignant mammary epithelial cells in response to single and repeated ionizing radiation exposures. MCF-7 cells were used for single radiation exposure (2-50 Gy) experiments and MCF-IR-3 cells, generated by exposure to cumulative doses of 60 Gy in 2 Gy fractions, respectively, were used to study the effects of repeated exposures. Steady-state messenger ribonucleic acid levels for estrogen receptor, epidermal growth factor-receptor, and transforming growth factor-α were determined by ribonucleic acid protection experiments. Estrogen receptor and epidermal growth factor-receptor protein expression was quantitated by competitive binding studies with 3H-estradiol and 125I-EGF. MCF-IR-3 cells showed a permanent three-fold down-regulation of the estrogen receptor messenger ribonucleic acid and protein, while epidermal growth factor-receptor was upregulated about nine-fold. Epidermal growth factor-receptor was substantially up-regulated in MCF-7 cells, at both the mRNA and protein levels, within 24 h of a single 2 Gy exposures, while there was a two-fold concomitant increase in transforming growth factor-α messenger ribonucleic acid expression. A decrease in estrogen receptor messenger ribonucleic acid and protein was suggested only after higher doses of single radiation exposures. The inverse expression of estrogen receptor and epidermal growth factor-receptor established for estrogen receptor-positive malignant mammary epithelial cells is maintained in MCF-7 cells after single and repeated exposures, suggesting that radiation acts through common regulatory circuits and may modulate the cellular phenotype. 40 refs., 2 figs., 2 tabs

  9. Cell death sensitization of leukemia cells by opioid receptor activation

    Friesen, Claudia; Roscher, Mareike; Hormann, Inis; Fichtner, Iduna; Alt, Andreas; Hilger, Ralf A.; Debatin, Klaus-Michael; Miltner, Erich

    2013-01-01

    Cyclic AMP (cAMP) regulates a number of cellular processes and modulates cell death induction. cAMP levels are altered upon stimulation of specific G-protein-coupled receptors inhibiting or activating adenylyl cyclases. Opioid receptor stimulation can activate inhibitory Gi-proteins which in turn block adenylyl cyclase activity reducing cAMP. Opioids such as D,L-methadone induce cell death in leukemia cells. However, the mechanism how opioids trigger apoptosis and activate caspases in leukemia cells is not understood. In this study, we demonstrate that downregulation of cAMP induced by opioid receptor activation using the opioid D,L-methadone kills and sensitizes leukemia cells for doxorubicin treatment. Enhancing cAMP levels by blocking opioid-receptor signaling strongly reduced D,L-methadone-induced apoptosis, caspase activation and doxorubicin-sensitivity. Induction of cell death in leukemia cells by activation of opioid receptors using the opioid D,L-methadone depends on critical levels of opioid receptor expression on the cell surface. Doxorubicin increased opioid receptor expression in leukemia cells. In addition, the opioid D,L-methadone increased doxorubicin uptake and decreased doxorubicin efflux in leukemia cells, suggesting that the opioid D,L-methadone as well as doxorubicin mutually increase their cytotoxic potential. Furthermore, we found that opioid receptor activation using D,L-methadone alone or in addition to doxorubicin inhibits tumor growth significantly in vivo. These results demonstrate that opioid receptor activation via triggering the downregulation of cAMP induces apoptosis, activates caspases and sensitizes leukemia cells for doxorubicin treatment. Hence, opioid receptor activation seems to be a promising strategy to improve anticancer therapies. PMID:23633472

  10. Dynamic changes in the expression of growth factor receptors in the myocardium microvascular endothelium after murine myocardial infarction

    WANG Xin-hong; ZHANG Guo-ping; JIN Hui-ming; CHEN Si-feng

    2007-01-01

    Background After myocardial infarction, specific growth factors promote cardiac angiogenisis, leading to a therapeutic effect. Although this effect is mediated by specific receptors in the endothelium of the cardiac microvasculature, few studies have investigated dynamic changes in their expression. We explored this phenomenon in a murine model.Methods We observed the mRNA expression of receptors by specific angiogenesis gene microarray at day 3 and day 7after infarction. The vascular endothelial growth factor (VEGF) receptor Fik-1 was observed at the protein level at day 3and day 7 by immunohistochemistry. The dynamic expression of fibroblast growth factor receptor-1 (FGFR-1) mRNA in the border zone and the noninfarcted zone at day 3, day 7, day 14, and day 42 was investigated by real-time PCR.Statistical significance was analyzed with SPSS 10.0 software using one-way analysis of variance (ANOVA).Results Three days after infarction, 9 receptors in the border zone and 7 receptors in the noninfarcted zone were down-regulated. Two receptors in the infarct edge and 5 receptors in the distant myocardium were up-regulated. However,at day 7, 11 receptors in the border zone were up-regulated, and only one was down-regulated. In the border zone, Fik-1levels decreased at day 3 but increased significantly at day 7. Real-time PCR showed that FGFR-1 mRNA decreased markedly in the border zone at day 3 but increased afterward for at least 6 weeks. In the early stage (3 days) after infarction, the expression of receptors had decreased to some extent. However, at day 7, receptor expression was active and had moved from the distant noninfarcted zone to the border zone as a part of the acute repair process.Conclusion Selecting the proper growth factors to target receptors with protective activity, and determining appropriate therapeutic timing may be important to the success of therapeutic angiogenesis.

  11. Angiotensin II (AT1) Receptor Blockade Reduces Vascular Tissue Factor in Angiotensin II-Induced Cardiac Vasculopathy

    Dominik N Müller; Mervaala, Eero M A; Dechend, Ralf; Fiebeler, Anette; Park, Joon-Keun; Schmidt, Folke; Theuer, Jürgen; Breu, Volker; Mackman, Nigel; Luther, Thomas; Schneider, Wolfgang; Gulba, Dietrich; Ganten, Detlev; Haller, Hermann; Luft, Friedrich C.

    2000-01-01

    Tissue factor (TF), a main initiator of clotting, is up-regulated in vasculopathy. We tested the hypothesis that chronic in vivo angiotensin (ANG) II receptor AT1 receptor blockade inhibits TF expression in a model of ANG II-induced cardiac vasculopathy. Furthermore, we explored the mechanisms by examining transcription factor activation and analyzing the TF promoter. Untreated transgenic rats overexpressing the human renin and angiotensinogen genes (dTGR) feature hypertension and severe left...

  12. Comparison of the activation kinetics of the M3 acetylcholine receptor and a constitutively active mutant receptor in living cells.

    Hoffmann, Carsten; Nuber, Susanne; Zabel, Ulrike; Ziegler, Nicole; Winkler, Christiane; Hein, Peter; Berlot, Catherine H; Bünemann, Moritz; Lohse, Martin J

    2012-08-01

    Activation of G-protein-coupled receptors is the first step of the signaling cascade triggered by binding of an agonist. Here we compare the activation kinetics of the G(q)-coupled M(3) acetylcholine receptor (M(3)-AChR) with that of a constitutively active mutant receptor (M(3)-AChR-N514Y) using M(3)-AChR constructs that report receptor activation by changes in the fluorescence resonance energy transfer (FRET) signal. We observed a leftward shift in the concentration-dependent FRET response for acetylcholine and carbachol with M(3)-AChR-N514Y. Consistent with this result, at submaximal agonist concentrations, the activation kinetics of M(3)-AChR-N514Y were significantly faster, whereas at maximal agonist concentrations the kinetics of receptor activation were identical. Receptor deactivation was significantly faster with carbachol than with acetylcholine and was significantly delayed by the N514Y mutation. Receptor-G-protein interaction was measured by FRET between M(3)-AChR-yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP)-Gγ(2). Agonist-induced receptor-G-protein coupling was of a time scale similar to that of receptor activation. As observed for receptor deactivation, receptor-G-protein dissociation was slower for acetylcholine than that for carbachol. Acetylcholine-stimulated increases in receptor-G-protein coupling of M(3)-AChR-N514Y reached only 12% of that of M(3)-AChR and thus cannot be kinetically analyzed. G-protein activation was measured using YFP-tagged Gα(q) and CFP-tagged Gγ(2). Activation of G(q) was significantly slower than receptor activation and indistinguishable for the two agonists. However, G(q) deactivation was significantly prolonged for acetylcholine compared with that for carbachol. Consistent with decreased agonist-stimulated coupling to G(q), agonist-stimulated G(q) activation by M(3)-AChR-N514Y was not detected. Taken together, these results indicate that the N514Y mutation produces constitutive activation of M(3

  13. FTZ-Factor1 and Fushi tarazu interact via conserved nuclear receptor and coactivator motifs

    Schwartz, Carol J.E.; Sampson, Heidi M.; Hlousek, Daniela; Percival-Smith, Anthony; Copeland, John W.R.; Simmonds, Andrew J.; Krause, Henry M.

    2001-01-01

    To activate transcription, most nuclear receptor proteins require coactivators that bind to their ligand-binding domains (LBDs). The Drosophila FTZ-Factor1 (FTZ-F1) protein is a conserved member of the nuclear receptor superfamily, but was previously thought to lack an AF2 motif, a motif that is required for ligand and coactivator binding. Here we show that FTZ-F1 does have an AF2 motif and that it is required to bind a coactivator, the homeodomain-containing protein Fushi tarazu (FTZ). We al...

  14. Soluble TL1A is sufficient for activation of death receptor 3.

    Bittner, Sebastian; Knoll, Gertrud; Füllsack, Simone; Kurz, Maria; Wajant, Harald; Ehrenschwender, Martin

    2016-01-01

    Death receptor 3 (DR3) is a typical member of the tumor necrosis factor receptor family, and was initially identified as a T-cell co-stimulatory molecule. However, further studies revealed a more complex and partly dichotomous role for DR3 and its ligand TL1A under (patho)physiological conditions. TL1A and DR3 are not only a driving force in the development of autoimmune and inflammatory diseases, but also play an important role in counteracting these processes through an increase in the number of regulatory T cells. Ligands of the tumor necrosis factor family typically occur in two forms, membrane-bound and soluble, that can differ strikingly with respect to their efficacy in activating their corresponding receptor(s). Ligand-based approaches to activate the TL1A-DR3 pathway therefore require understanding of the molecular prerequisites of TL1A-based DR3 activation. To date, this has not been addressed. Here, we show that recombinant soluble trimeric TL1A is fully sufficient to strongly activate DR3-associated pro- and anti-apoptotic signaling pathways. In contrast to the TRAIL death receptors, which are much better activated by soluble TRAIL upon secondary ligand oligomerization, but similarly to the death receptor tumor necrosis factor receptor 1, DR3 is efficiently activated by soluble TL1A trimers. Additionally, we have measured the affinity of TL1A-DR3 interaction in a cell-based system, and demonstrated TL1A-induced DR3 internalization. Identification of DR3 as a tumor necrosis factor receptor that responds to soluble ligand trimers without further oligomerization provides a basis for therapeutic exploitation of the TL1A-DR3 pathway. PMID:26509650

  15. The Orphan Nuclear Receptor TR4 Is a Vitamin A-activated Nuclear Receptor

    Zhou, X. Edward; Suino-Powell, Kelly M.; Xu, Yong; Chan, Cee-Wah; Tanabe, Osamu; Kruse, Schoen W.; Reynolds, Ross; Engel, James Douglas; Xu, H. Eric (Michigan-Med); (Van Andel)

    2015-11-30

    Testicular receptors 2 and 4 (TR2/4) constitute a subgroup of orphan nuclear receptors that play important roles in spermatogenesis, lipid and lipoprotein regulation, and the development of the central nervous system. Currently, little is known about the structural features and the ligand regulation of these receptors. Here we report the crystal structure of the ligand-free TR4 ligand binding domain, which reveals an autorepressed conformation. The ligand binding pocket of TR4 is filled by the C-terminal half of helix 10, and the cofactor binding site is occupied by the AF-2 helix, thus preventing ligand-independent activation of the receptor. However, TR4 exhibits constitutive transcriptional activity on multiple promoters, which can be further potentiated by nuclear receptor coactivators. Mutations designed to disrupt cofactor binding, dimerization, or ligand binding substantially reduce the transcriptional activity of this receptor. Importantly, both retinol and retinoic acid are able to promote TR4 to recruit coactivators and to activate a TR4-regulated reporter. These findings demonstrate that TR4 is a ligand-regulated nuclear receptor and suggest that retinoids might have a much wider regulatory role via activation of orphan receptors such as TR4.

  16. The C Terminus of the Saccharomyces cerevisiae α-Factor Receptor Contributes to the Formation of Preactivation Complexes with Its Cognate G Protein

    Dosil, Mercedes; Schandel, Kimberly A.; Gupta, Ekta; Jenness, Duane D.; Konopka, James B.

    2000-01-01

    Binding of the α-factor pheromone to its G-protein-coupled receptor (encoded by STE2) activates the mating pathway in MATa yeast cells. To investigate whether specific interactions between the receptor and the G protein occur prior to ligand binding, we analyzed dominant-negative mutant receptors that compete with wild-type receptors for G proteins, and we analyzed the ability of receptors to suppress the constitutive signaling activity of mutant Gα subunits in an α-factor-independent manner....

  17. A third distinct tumor necrosis factor receptor of orthopoxviruses.

    Loparev, V N; Parsons, J M; Knight, J C; Panus, J F; Ray, C A; Buller, R M; Pickup, D J; Esposito, J J

    1998-03-31

    Cowpox virus Brighton red strain (CPV) contains a gene, crmD, which encodes a 320-aa tumor necrosis factor receptor (TNFR) of 44% and 22% identity, respectively, to the CPV TNFR-like proteins, cytokine response modifiers (crm) CrmB and CrmC. The crmD gene was interrupted in three other cowpox strains examined and absent in various other orthopoxviruses; however, four strains of ectromelia virus (ECT) examined contained an intact crmD (97% identity to CPV crmD) and lacked cognates of crmB and crmC. The protein, CrmD, contains a transport signal; a 151-aa cysteine-rich region with 21 cysteines that align with human TNFRII ligand-binding region cysteines; and C-terminal region sequences that are highly diverged from cellular TNFR C-terminal region sequences involved in signal transduction. Bacterial maltose-binding proteins containing the CPV or ECT CrmD cysteine-rich region bound TNF and lymphotoxin-alpha (LTalpha) and blocked their in vitro cytolytic activity. Secreted viral CrmD bound TNF and LTalpha and was detectable after the early stage of replication, using nonreducing conditions, as 60- to 70-kDa predominant and 90- to 250-kDa minor disulfide-linked complexes that were able to be reduced to a 46-kDa form and deglycosylated to a 38-kDa protein. Cells infected with CPV produced extremely low amounts of CrmD compared with ECT. Possessing up to three TNFRs, including CrmD, which is secreted as disulfide-linked complexes in varied amounts by CPV and ECT, likely enhances the dynamics of the immune modulating mechanisms of orthopoxviruses. PMID:9520445

  18. Upregulation of epidermal growth factor receptor 4 in ora leukoplakia

    Hiroshi Kobayashi; Kenichi Kumagai; Akito Gotoh; Takanori Eguchi; Hiroyuki Yamada; Yoshiki Hamada; Satsuki Suzuki; Ryuji Suzuki

    2013-01-01

    In the present study, we investigate the expression profile of the epidermal growth factor receptor family, which comprises EGFR/ ErbB 1, HER2/ErbB2, HER3/ErbB3 and HER4/ErbB4 in oral leukoplakia (LP), The expression of four epidermal growth factor receptor (EGFR) family genes and their ligands were measured in LP tissues from 14 patients and compared with levels in 10 patients with oral lichen planus (OLP) and normal oral mucosa (NOM) from 14 healthy donors by real-time polymerase chain reaction (PCR) and immunohistochemistry. Synchronous mRNA coexpression of ErbB1, ErbB2, ErbB3 and ErbB4 was detected in LP lesions. Out of the receptors, only ErbB4 mRNA and protein was more highly expressed in LP compared with NOM tissues. These were strongly expressed by epithelial keratinocytes in LP lesions, as shown by immunohistochemistry. Regarding the ligands, the mRNA of Neuregulin2 and 4 were more highly expressed in OLP compared with NOM tissues. Therefore, enhanced ErbB4 on the keratinocytes and synchronous modulation of EGFR family genes may contribute to the pathogenesis and carcinogenesis of LP.

  19. Are different stoichiometries feasible for complexes between lymphotoxin-alpha and tumor necrosis factor receptor 1?

    Mascarenhas Nahren

    2012-05-01

    Full Text Available Abstract Background Tumor necrosis factors, TNF and lymphotoxin-α (LT, are cytokines that bind to two receptors, TNFR1 and TNFR2 (TNF-receptor 1 and 2 to trigger their signaling cascades. The exact mechanism of ligand-induced receptor activation is still unclear. It is generally assumed that three receptors bind to the homotrimeric ligand to trigger a signaling event. Recent evidence, though, has raised doubts if the ligand:receptor stoichiometry should indeed be 3:3 for ligand-induced cellular response. We used molecular dynamics simulations, elastic network models, as well as MM/PBSA to analyze this question. Results Applying MM/PBSA methodology to different stoichiometric complexes of human LT-(TNFR1n=1,2,3 the free energy of binding in these complexes has been estimated by single-trajectory and separate-trajectory methods. Simulation studies rationalized the favorable binding energy in the LT-(TNFR11 complex, as evaluated from single-trajectory analysis to be an outcome of the interaction of cysteine-rich domain 4 (CRD4 and the ligand. Elastic network models (ENMs help to associate the difference in the global fluctuation of the receptors in these complexes. Functionally relevant transformation associated with these complexes reveal the difference in the dynamics of the receptor when free and in complex with LT. Conclusions MM/PBSA predicts complexes with a ligand-receptor molar ratio of 3:1 and 3:2 to be energetically favorable. The high affinity associated with LT-(TNFR11 is due to the interaction between the CRD4 domain with LT. The global dynamics ascertained from ENMs have highlighted the differential dynamics of the receptor in different states.

  20. The insulin receptor activation process involves localized conformational changes.

    Baron, V; Kaliman, P; Gautier, N; Van Obberghen, E

    1992-11-15

    The molecular process by which insulin binding to the receptor alpha-subunit induces activation of the receptor beta-subunit with ensuing substrate phosphorylation remains unclear. In this study, we aimed at approaching this molecular mechanism of signal transduction and at delineating the cytoplasmic domains implied in this process. To do this, we used antipeptide antibodies to the following sequences of the receptor beta-subunit: (i) positions 962-972 in the juxtamembrane domain, (ii) positions 1247-1261 at the end of the kinase domain, and (iii) positions 1294-1317 and (iv) positions 1309-1326, both in the receptor C terminus. We have previously shown that insulin binding to its receptor induces a conformational change in the beta-subunit C terminus. Here, we demonstrate that receptor autophosphorylation induces an additional conformational change. This process appears to be distinct from the one produced by ligand binding and can be detected in at least three different beta-subunit regions: the juxtamembrane domain, the kinase domain, and the C terminus. Hence, the cytoplasmic part of the receptor beta-subunit appears to undergo an extended conformational change upon autophosphorylation. By contrast, the insulin-induced change does not affect the juxtamembrane domain 962-972 nor the kinase domain 1247-1261 and may be limited to the receptor C terminus. Further, we show that the hormone-dependent conformational change is maintained in a kinase-deficient receptor due to a mutation at lysine 1018. Therefore, during receptor activation, the ligand-induced change could precede ATP binding and receptor autophosphorylation. We propose that insulin binding leads to a transient receptor form that may allow ATP binding and, subsequently, autophosphorylation. The second conformational change could unmask substrate-binding sites and stabilize the receptor in an active conformation. PMID:1331080

  1. Selective effects of ligands on vitamin D3 receptor- and retinoid X receptor-mediated gene activation in vivo.

    Lemon, B D; Freedman, L P

    1996-01-01

    Steroid/nuclear hormone receptors are ligand-regulated transcription f factors that play key roles in cell regulation, differentiation, and oncogenesis. Many nuclear receptors, including the human 1,25-dihydroxyvitamin D3 receptor (VDR), bind cooperatively to DNA either as homodimers or as heterodimers with the 9-cis retinoic acid (RA) receptor (retinoid X-receptor [RXR]). We have previously reported that the ligands for VDR and RXR can differentially modulate the affinity of the receptors' i...

  2. UVB increases urokinase-type plasminogen activator receptor (uPAR) expression.

    Marschall, C; Lengyel, E; Nobutoh, T; Braungart, E; Douwes, K; Simon, A; Magdolen, V; Reuning, U; Degitz, K

    1999-07-01

    Keratinocytes synthesize and secrete urokinase-type plasminogen activator, which binds to its specific receptor on keratinocytes. When bound to urokinase-type plasminogen activator receptor, urokinase-type plasminogen activator proteolytically converts surface bound plasminogen to plasmin, which in turn cleaves many extracellular components leading to pericellular proteolysis. The activation of the urokinase system has been observed during re-epithelialization of skin wounds and in lesions of the autoimmune blistering skin disease pemphigus. As pemphigus is photoinducible, we investigated the effect of ultraviolet B on urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor expression in the epidermal keratinocyte cell line A431. Ultraviolet B increased cellular and secreted urokinase-type plasminogen activator protein (enzyme-linked immunosorbent assay) and urokinase-type plasminogen activator receptor cell surface expression (flow cytometry) 24 h postirradiation. Northern blot analysis indicated that ultraviolet B increased urokinase-type plasminogen activator receptor mRNA. Compared with a more rapid mRNA induction by epidermal growth factor (maximal after 4 h) the ultraviolet B response was maximal after 24 h and prolonged up to 36 h. The mRNA induction was not dependent on protein synthesis as judged by cycloheximide incubation. Ultraviolet B did not influence urokinase-type plasminogen activator receptor mRNA stability (actinomycin D incubation). A transiently transfected chloramphenicol acetyltransferase-reporter construct containing a -398/+51 urokinase-type plasminogen activator receptor promoter fragment was activated when cells were exposed to ultraviolet B. This induction was almost completely abolished by mutating a -182/-176 AP-1 binding sequence. Ultraviolet B increased the binding capacity at this AP-1 motif in electrophoretic mobility shift assays. These data identify a distinct transcriptional mechanism by which

  3. Evaluation of single nucleotide polymorphisms of Pro12Ala in peroxisome proliferator-activated receptor-γ and Gly308Ala in tumor necrosis factor-α genes in obese Asian Indians: a population-based study

    Namita Bhagat

    2010-10-01

    Full Text Available Namita Bhagat1,2, Mukta Agrawal1, Kalpana Luthra3, Naval K Vikram4, Anoop Misra4, Rajeev Gupta21Department of Home Science, University of Rajasthan, Jaipur, Rajasthan, India; 2Department of Medicine, Fortis Escorts Hospital, Jaipur, Rajasthan, India; 3Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India; 4Department of Medicine, All India Institute of Medical Sciences, New Delhi, IndiaBackground: A population-based case control study was performed to determine the associations of Pro12Ala polymorphism in peroxisome proliferator-activated receptor-γ (PPARG and Gly308Ala polymorphism in tumor necrosis factor-α (TNFA genes in obese subjects.Patients and methods: Of 1,400 eligible subjects, ≥20 years, we recruited only 1,127. For extreme phenotype case-control design, we evaluated 201 subjects with body mass index (BMI ≥30 kg/m2 (Group 1 and 143 with BMI <20 kg/m2 (Group 2. Clinical, anthropometric, biochemical, and nutritional details and polymorphisms were estimated.Results: In Group 1, the dietary intake of calories and fats was higher, physical activity was lower, and prevalence of truncal obesity, hypertension, high total cholesterol, low high-density lipoprotein cholesterol, and diabetes was greater than in Group 2. There were no homozygous polymorphisms of either gene. Heterozygous Pro12Ala polymorphism in PPARG was found in 15 (7.5% subjects in Group 1 and 3 (2.1% subjects in Group 2 (P = 0.028, and heterozygous Gly308Ala polymorphism in TNFA was found in 19 (9.5% in Group 1 and 7 (4.9% in Group 2 (P = 0.115. Presence of heterozygous polymorphism in PPARG and TNFA-predicted obesity with univariate odds ratio ([OR], 95% confidence intervals of 2.25 (1.32–3.84, P = 0.003 and 1.48 (1.10–1.99, P = 0.009 and with multivariate OR 1.74 (1.03–2.93, P = 0.038 and 1.46 (1.05–2.03, P = 0.024, respectively. The addition of dietary and physical activity variables did not result in significant change

  4. Phosphorylation of receptors for insulin and insulin-like growth factor I

    Jacobs, S.; Cuatrecasas, P.

    1986-01-15

    The phosphorylation of receptors for insulin and insulin-like growth factor I was studied by phosphoamino acid analysis and tryptic phosphopeptide maps in an attempt to determine if protein kinase C is involved in their phosphorylation in response to insulin and insulin-like growth factor I, respectively. Two cell lines were utilized, Hep G2 and IM-9 cells. sn-1,2-Dioctanoylglycerol and 12-O-tetradecanoylphorbol 13-acetate (TPA), agents known to activate protein kinase C, stimulated the phosphorylation of the ..beta.. subunits of both receptors, as did their hormones. In unstimulated cells, phosphorylation of the insulin receptor occurred on seryl and to a lesser extent on threonyl residues. TPA stimulated seryl and threonyl phosphorylation that resulted in the appearance of four major phosphoserine-containing phosphopeptides which were not detected in the basal state and an increase in phosphorylation of a phosphothreonine-containing peptide which was present in the basal state. Insulin treatment resulted in the appearance of three major phosphotyrosine-containing tryptic peptides. In IM-9 cells, insulin also increased the phosphoserine and possibly the phosphothreonine content of the ..beta.. subunit. In both cells, the major phosphoserine-containing peptides that were stimulated by TPA were not detected following treatment with insulin. Very similar results, including similar peptide maps, were obtained for the insulin-like growth factor I receptor from cells treated with TPA and insulin-like growth factor I. Although not entirely conclusive, these results suggest that the insulin- and insulin-like growth factor I-stimulated phosphorylation of their receptors does not result from activation of protein kinase C.

  5. Sprouty2 acts at the Cbl/CIN85 interface to inhibit epidermal growth factor receptor downregulation

    Haglund, Kaisa; Schmidt, Mirko H. H.; Wong, Esther Sook Miin; Guy, Graeme R.; Dikic, Ivan

    2005-01-01

    The ubiquitin ligase Cbl mediates ubiquitination of activated receptor tyrosine kinases (RTKs) and interacts with endocytic scaffold complexes, including CIN85/endophilins, to facilitate RTK endocytosis and degradation. Several mechanisms regulate the functions of Cbl to ensure the fine-tuning of RTK signalling and cellular homeostasis. One regulatory mechanism involves the binding of Cbl to Sprouty2, which sequesters Cbl away from activated epidermal growth factor receptors (EGFRs). Here, we...

  6. Redox-dependent regulation of epidermal growth factor receptor signaling.

    Heppner, David E; van der Vliet, Albert

    2016-08-01

    Tyrosine phosphorylation-dependent cell signaling represents a unique feature of multicellular organisms, and is important in regulation of cell differentiation and specialized cell functions. Multicellular organisms also contain a diverse family of NADPH oxidases (NOXs) that have been closely linked with tyrosine kinase-based cell signaling and regulate tyrosine phosphorylation via reversible oxidation of cysteine residues that are highly conserved within many proteins involved in this signaling pathway. An example of redox-regulated tyrosine kinase signaling involves the epidermal growth factor receptor (EGFR), a widely studied receptor system with diverse functions in normal cell biology as well as pathologies associated with oxidative stress such as cancer. The purpose of this Graphical Redox Review is to highlight recently emerged concepts with respect to NOX-dependent regulation of this important signaling pathway. PMID:26722841

  7. Epidermal growth factor receptor in primary human lung cancer

    Cell membranes were prepared from 12 human lung cancers for the study of the expression of epidermal growth factor receptors (EGFR). EGFR concentration was estimated by ligand binding studies using 125I-radiolabeled EGF. The dissociation constants of the high affinity sites were identical, 1.48 nmol and 1.1 nmol in cancer and normal lung tissues, the EGFR contents were higher in lung cancer tissues (range: 2.25 to 19.39 pmol·g-1 membrane protein) than that in normal tissues from the same patients (range: 0.72 to 7.43 pmol·g-1 membrane protein). These results suggest that EGF and its receptor may play a role in the regulatory mechanisms in the control of lung cellular growth and tumor promotion

  8. Activation of the unliganded estrogen receptor by EGF involves the MAP kinase pathway and direct phosphorylation.

    Bunone, G; Briand, P A; Miksicek, R J; Picard, D.

    1996-01-01

    The estrogen receptor (ER) can be activated as a transcription factor either by binding of cognate estrogenic ligand or, indirectly, by a variety of other extracellular signals. As a first step towards elucidating the mechanism of 'steroid-independent activation' of the ER by the epidermal growth factor (EGF), we have mapped the ER target domain and determined the signaling pathway. We show that the N-terminal transcriptional activation function AF-1, but not the C-terminal AF-2, is necessary...

  9. Immunohistochemical detection of gastrin and motilin peptides, their receptors, VIP receptors and caspase activity from the abomasal wall of cattle

    Özcan, Aycan

    2012-01-01

    Objective The aim of this study was to examine the possible effects of gastrin, motilin peptides, their receptors and vasoactive intestinal polypeptide (VIP) receptors on the occurrence of abomasal displacement (AD). A decreased amount of stimulating factors (motilin, motilin receptors) accompanied by an increased amount of inhibiting factors (gastrin, gastrin receptors, VIP receptors) in the abomasal wall could be a cause of the hypo- or atony of the abomasum prior to the abomasal displac...

  10. Interaction of chemokines with their receptors--from initial chemokine binding to receptor activating steps

    Thiele, Stefanie; Rosenkilde, Mette Marie

    2014-01-01

    The human chemokine system comprises 19 seven-transmembrane helix (7TM) receptors and 45 endogenous chemokines that often interact with each other in a promiscuous manner. Due to the chemokine system's primary function in leukocyte migration, it has a central role in immune homeostasis and...... interactions possibly occur, resulting in a multi-step process, as recently proposed for other 7TM receptors. Overall, the N-terminus of chemokine receptors is pivotal for binding of all chemokines. During receptor activation, differences between the two major chemokine subgroups occur, as CC-chemokines mainly...

  11. Oxidative Stress Stimulates Testicular Orphan Receptor 4 through Forkhead Transcription Factor Forkhead Box O3a

    Li, Gonghui; Lee, Yi-Fen; Liu, Su; Cai, Yi; Xie, Shaozhen; Liu, Ning-Chun; Bao, Bo-Ying; Chen, Zhaodian; Chang, Chawnshang

    2008-01-01

    Early studies reveal that testicular orphan nuclear receptor 4 (TR4) modulates signaling pathways that control various cell functions. However, how TR4 activity is regulated without the involvement of specific ligand(s) remains unclear. Here we identify a daf-16 family protein-binding element (DBE; 5′-TGTTTAC-3′) in the TR4 promoter that can be recognized by the forkhead transcriptional factor FOXO3a, a key stress-responsive factor, through which TR4 gene expression is activated. The interact...

  12. Chronic hyperammonemia induces tonic activation of NMDA receptors in cerebellum.

    ElMlili, Nisrin; Boix, Jordi; Ahabrach, Hanan; Rodrigo, Regina; Errami, Mohammed; Felipo, Vicente

    2010-02-01

    Reduced function of the glutamate--nitric oxide (NO)--cGMP pathway is responsible for some cognitive alterations in rats with hyperammonemia and hepatic encephalopathy. Hyperammonemia impairs the pathway in cerebellum by increasing neuronal nitric oxide synthase (nNOS) phosphorylation in Ser847 by calcium-calmodulin-dependent protein kinase II (CaMKII), reducing nNOS activity, and by reducing nNOS amount in synaptic membranes, which reduces its activation following NMDA receptors activation. The reason for increased CaMKII activity in hyperammonemia remains unknown. We hypothesized that it would be as a result of increased tonic activation of NMDA receptors. The aims of this work were to assess: (i) whether tonic NMDA activation receptors is increased in cerebellum in chronic hyperammonemia in vivo; and (ii) whether this tonic activation is responsible for increased CaMKII activity and reduced activity of nNOS and of the glutamate--NO--cGMP pathway. Blocking NMDA receptors with MK-801 increases cGMP and NO metabolites in cerebellum in vivo and in slices from hyperammonemic rats. This is because of reduced phosphorylation and activity of CaMKII, leading to normalization of nNOS phosphorylation and activity. MK-801 also increases nNOS in synaptic membranes and reduces it in cytosol. This indicates that hyperammonemia increases tonic activation of NMDA receptors leading to reduced activity of nNOS and of the glutamate--NO--cGMP pathway. PMID:20002515

  13. Neuroplastin-55 binds to and signals through the fibroblast growth factor receptor

    Owczarek, Sylwia; Kiryushko, Darya; Hald Larsen, Marianne;

    2010-01-01

    3), whereas Np55 lacks the Ig1 module. Of these two isoforms, only Np65 is involved in homophilic interactions resulting in cell adhesion, whereas the role of Np55 is poorly understood. The present study reports for the first time the crystal structure of the ectodomain of Np55 at 1.95-A resolution...... and demonstrates that Np55 binds to and activates the fibroblast growth factor receptor 1 (FGFR1). Furthermore, we identify a sequence motif in the Ig2 module of Np55 interacting with FGFR1 and show that a synthetic peptide encompassing this motif, termed narpin, binds to and activates FGFR1. We show...... Np55-induced signaling may be involved in synaptic plasticity in vivo. Owczarek, S., Kiryushko, D., Larsen, M. H., Kastrup, J. S., Gajhede, M., Sandi, C., Berezin, V., Bock, E., Soroka, V. Neuroplastin-55 binds to and signals through the fibroblast growth factor receptor....

  14. Lipid-soluble smoke particles upregulate vascular smooth muscle ETB receptors via activation of mitogen-activating protein kinases and NF-kappaB pathways

    Xu, Cang-Bao; Zheng, Jian-Pu; Zhang, Wei;

    2008-01-01

    , dexamethasone abolished the DSP-induced upregulation of ET(B) receptor-mediated contraction. In conclusion, upregulation of ET(B) receptors by DSP in arterial smooth muscle cells involves activation of mitogen-activating protein kinases (ERK1/2 and p38) and the downstream transcriptional factor NF...

  15. Redefining the concept of protease-activated receptors: cathepsin S evokes itch via activation of Mrgprs

    Reddy, Vemuri B.; Sun, Shuohao; Azimi, Ehsan; Elmariah, Sarina B.; Dong, Xinzhong; Lerner, Ethan A.

    2015-01-01

    Sensory neurons expressing Mas-related G protein coupled receptors (Mrgprs) mediate histamine-independent itch. We show that the cysteine protease cathepsin S activates MrgprC11 and evokes receptor-dependent scratching in mice. In contrast to its activation of conventional protease-activated receptors, cathepsin S mediated activation of MrgprC11 did not involve the generation of a tethered ligand. We demonstrate further that different cysteine proteases selectively activate specific mouse and...

  16. Peroxisome Proliferator-Activated Receptors (PPARs) as Potential Inducers of Antineoplastic Effects in CNS Tumors

    Lars Tatenhorst; Eric Hahnen; Heneka, Michael T

    2008-01-01

    The peroxisome proliferator-activated receptors (PPARs) are ligand-inducible transcription factors which belong to the superfamily of nuclear hormone receptors. In recent years it turned out that natural as well as synthetic PPAR agonists exhibit profound antineoplastic as well as redifferentiation effects in tumors of the central nervous system (CNS). The molecular understanding of the underlying mechanisms is still emerging, with partially controverse findings reported by a number of studie...

  17. Human receptor activation by aroclor 1260, a polychlorinated biphenyl mixture.

    Wahlang, Banrida; Falkner, K Cameron; Clair, Heather B; Al-Eryani, Laila; Prough, Russell A; States, J Christopher; Coslo, Denise M; Omiecinski, Curtis J; Cave, Matthew C

    2014-08-01

    Polychlorinated biphenyls (PCBs) are persistent environmental toxicants, present in 100% of U.S. adults and dose-dependently associated with obesity and non-alcoholic fatty liver disease (NAFLD). PCBs are predicted to interact with receptors previously implicated in xenobiotic/energy metabolism and NAFLD. These receptors include the aryl hydrocarbon receptor (AhR), pregnane xenobiotic receptor (PXR), constitutive androstane receptor (CAR), peroxisome proliferator-activated receptors (PPARs), liver-X-receptor (LXRα), and farnesoid-X-receptor (FXR). This study evaluates Aroclor 1260, a PCB mixture with congener composition mimicking that of human adipose tissue, and selected congeners, as potential ligands for these receptors utilizing human hepatoma-derived (HepG2) and primate-derived (COS-1) cell lines, and primary human hepatocytes. Aroclor 1260 (20 μg/ml) activated AhR, and PCB 126, a minor component, was a potent inducer. Aroclor 1260 activated PXR in a simple concentration-dependent manner at concentrations ≥10 μg/ml. Among the congeners tested, PCBs 138, 149, 151, 174, 183, 187, and 196 activated PXR. Aroclor 1260 activated CAR2 and CAR3 variants at lower concentrations and antagonize CAR2 activation by the CAR agonist, CITCO, at higher concentrations (≥20 μg/ml). Additionally, Aroclor 1260 induced CYP2B6 in primary hepatocytes. At subtoxic doses, Aroclor 1260 did not activate LXR or FXR and had no effect on LXR- or FXR-dependent induction by the agonists T0901317 or GW4064, respectively. Aroclor 1260 (20 μg/ml) suppressed PPARα activation by the agonist nafenopin, although none of the congeners tested demonstrated significant inhibition. The results suggest that Aroclor 1260 is a human AhR, PXR and CAR3 agonist, a mixed agonist/antagonist for CAR2, and an antagonist for human PPARα. PMID:24812009

  18. A third distinct tumor necrosis factor receptor of orthopoxviruses

    Loparev, Vladimir N.; Parsons, Joseph M.; Knight, Janice C.; Panus, Joanne Fanelli; Ray, Caroline A.; Buller, R. Mark L.; Pickup, David J.; Esposito, Joseph J.

    1998-01-01

    Cowpox virus Brighton red strain (CPV) contains a gene, crmD, which encodes a 320-aa tumor necrosis factor receptor (TNFR) of 44% and 22% identity, respectively, to the CPV TNFR-like proteins, cytokine response modifiers (crm) CrmB and CrmC. The crmD gene was interrupted in three other cowpox strains examined and absent in various other orthopoxviruses; however, four strains of ectromelia virus (ECT) examined contained an intact crmD (97% identity to CPV crmD) and lacked cognates of crmB and ...

  19. Epidermal growth factor and its receptors in human pancreatic carcinoma

    The role of epidermal growth factor (EGF) in oncogenesis and progression of malignant tumors is a subject of vast interest. In this study, radioimmunoassay and radioreceptor assay of EGF were established. EGF contents in malignant and benign pancreatic tumors, in normal pancreas tissue, and in culture media of a human pancreatic carcinoma cell line were determined. EGF receptor binding studies were performed. It was shown that EGF contents in pancreatic carcinomas were significantly higher than those in normal pancreas or benign pancreatic tumors. EGF was also detected in the culture medium of a pancreatic carcinoma cell line. The binding of 125I-EGF to the pancreatic carcinoma cells was time and temperature dependent, reversible, competitive, and specific. Scatchard analysis showed that the dissociation constant of EGF receptor was 2.1 X 10(-9) M, number of binding sites was 1.3 X 10(5) cell. These results indicate that there is an over-expression of EGF/EGF receptors in pancreatic carcinomas, and that an autocrine regulatory mechanism may exist in the growth-promoting effect of EGF on tumor cells

  20. Epidermal growth factor receptor subunit locations determined in hydrated cells with environmental scanning electron microscopy

    Peckys, Diana B.; Jean-Pierre Baudoin; Magdalena Eder; Ulf Werner; Niels de Jonge

    2013-01-01

    Imaging single epidermal growth factor receptors (EGFR) in intact cells is presently limited by the available microscopy methods. Environmental scanning electron microscopy (ESEM) of whole cells in hydrated state in combination with specific labeling with gold nanoparticles was used to localize activated EGFRs in the plasma membranes of COS7 and A549 cells. The use of a scanning transmission electron microscopy (STEM) detector yielded a spatial resolution of 3 nm, sufficient to identify the l...

  1. Expression of epidermal growth factor receptor is an independent prognostic factor for esophageal squamous cell carcinoma

    Wang, Qifeng; Zhu, Hongxia; Xiao, Zefen; Zhang, Wencheng; Liu, Xiao; Zhang, Xun; He, Jie; Kelin SUN; Wang, Lvhua; Xu, Ningzhi

    2013-01-01

    Background The overall survival of patients with esophageal squamous cell carcinoma (ESCC) remains poor. Prognostic predictions in ESCC are usually based on histological assessment of tumor invasion and lymph node metastasis, but a biomarker with better predictive accuracy could be more useful. Because overexpression of epidermal growth factor receptor (EGFR) has been associated with poor prognosis, this study investigated whether EGFR is an independent prognostic factor for overall survival ...

  2. Cloning of human tumor necrosis factor (TNF) receptor cDNA and expression of recombinant soluble TNF-binding protein

    The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNFα with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extracellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNFα with an affinity of 2.5 x 10-9 M. This binding can be competitively inhibited with unlabeled TNFα or lymphotoxin (TNFβ)

  3. Signaling by Tyrosine Kinases Negatively Regulates the Interaction between Transcription Factors and SMRT (Silencing Mediator of Retinoic Acid and Thyroid Hormone Receptor) Corepressor

    Hong, Suk-Hyun; Wong, Chi-Wai; Privalsky, Martin L.

    1998-01-01

    Nuclear hormone receptors are hormone-regulated transcription factors that bind to specific sites on DNA and modulate the expression of adjacent target genes. Many nuclear hormone receptors display bimodal transcriptional properties; thyroid hormone receptors, for example, typically repress target gene expression in the absence of hormone, but activate target gene expression in the presence of hormone. The ability to repress is closely linked to the ability of the apo-receptor to physically b...

  4. A peptide antagonist of the ErbB1 receptor inhibits receptor activation, tumor cell growth and migration in vitro and xenograft tumor growth in vivo

    Xu, Ruodan; Povlsen, Gro Klitgaard; Soroka, Vladislav;

    2010-01-01

    The epidermal growth factor family of receptor tyrosine kinases (ErbBs) plays essential roles in tumorigenesis and cancer disease progression, and therefore has become an attractive target for structure-based drug design. ErbB receptors are activated by ligand-induced homo- and heterodimerization...... lung cancer cell line A549. The Inherbin3 peptide may be a useful tool for investigating the mechanisms of ErbB receptor homo- and heterodimerization. Moreover, the here described biological effects of Inherbin3 suggest that peptide-based targeting of ErbB receptor dimerization is a promising anti...

  5. Effect of montelukast on platelet activating factor- and tachykinin induced mucus secretion in the rat

    Groneberg David A

    2008-02-01

    Full Text Available Abstract Background Platelet activating factor and tachykinins (substance P, neurokinin A, neurokinin B are important mediators contributing to increased airway secretion in the context of different types of respiratory diseases including acute and chronic asthma. Leukotriene receptor antagonists are recommended as add-on therapy for this disease. The cys-leukotriene-1 receptor antagonist montelukast has been used in clinical asthma therapy during the last years. Besides its inhibitory action on bronchoconstriction, only little is known about its effects on airway secretions. Therefore, the aim of this study was to evaluate the effects of montelukast on platelet activating factor- and tachykinin induced tracheal secretory activity. Methods The effects of montelukast on platelet activating factor- and tachykinin induced tracheal secretory activity in the rat were assessed by quantification of secreted 35SO4 labelled mucus macromolecules using the modified Ussing chamber technique. Results Platelet activating factor potently stimulated airway secretion, which was completely inhibited by the platelet activating factor receptor antagonist WEB 2086 and montelukast. In contrast, montelukast had no effect on tachykinin induced tracheal secretory activity. Conclusion Cys-leukotriene-1 receptor antagonism by montelukast reverses the secretagogue properties of platelet activating factor to the same degree as the specific platelet activating factor antagonist WEB 2086 but has no influence on treacheal secretion elicited by tachykinins. These results suggest a role of montelukast in the signal transduction pathway of platelet activating factor induced secretory activity of the airways and may further explain the beneficial properties of cys-leukotriene-1 receptor antagonists.

  6. EXPRESSION OF GROWTH-FACTORS AND GROWTH-FACTOR RECEPTORS IN NORMAL AND TUMOROUS HUMAN THYROID TISSUES

    van der Laan, B.F.A.M.; FREEMAN, JL; ASA, SL

    1995-01-01

    A number of growth factors have been implicated as stimuli of thyroid cell proliferation; overexpression of these growth factors and/or their receptors may play a role in the growth of thyroid tumors. To determine if immunohistochemical detection of growth factors and/or their receptors correlates w

  7. The insulin-like growth factor 1 receptor causes acquired resistance to erlotinib in lung cancer cells with the wild-type epidermal growth factor receptor.

    Suda, Kenichi; Mizuuchi, Hiroshi; Sato, Katsuaki; Takemoto, Toshiki; Iwasaki, Takuya; Mitsudomi, Tetsuya

    2014-08-15

    Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) therapy often provides a dramatic response in lung cancer patients with EGFR mutations. In addition, moderate clinical efficacy of the EGFR-TKI, erlotinib, has been shown in lung cancer patients with the wild-type EGFR. Numerous molecular mechanisms that cause acquired resistance to EGFR-TKIs have been identified in lung cancers with the EGFR mutations; however, few have been reported in lung cancers with the wild-type EGFR. We used H358 lung adenocarcinoma cells lacking EGFR mutations that showed modest sensitivity to erlotinib. The H358 cells acquired resistance to erlotinib via chronic exposure to the drug. The H358 erlotinib-resistant (ER) cells do not have a secondary EGFR mutation, neither MET gene amplification nor PTEN downregulation; these have been identified in lung cancers with the EGFR mutations. From comprehensive screening of receptor tyrosine kinase phosphorylation, we observed increased phosphorylation of insulin-like growth factor 1 receptor (IGF1R) in H358ER cells compared with parental H358 cells. H358ER cells responded to combined therapy with erlotinib and NVP-AEW541, an IGF1R-TKI. Our results indicate that IGF1R activation is a molecular mechanism that confers acquired resistance to erlotinib in lung cancers with the wild-type EGFR. PMID:24458568

  8. Psychosocial factors underlying physical activity

    Ji Cheng-Ye

    2007-09-01

    of physical activity on academic achievement and other factors beyond physical health; barriers of not having enough time and having too many assignments perceived to hinder frequent physical activity; and parental approval. More rigorous research on psychosocial determinants with close-ended items developed from these open-ended data and with larger sample sizes of students is necessary. Research with parents and school staff will be needed to understand the perceptions of these stakeholder groups key to creating the students' social environment.

  9. Tonic activation of presynaptic GABAB receptors on rat pallidosubthalamic terminals

    Lei CHEN; Wing-ho YUNG

    2005-01-01

    Aim: The subthalamic nucleus plays a critical role in the regulation of movement,and abnormal activity of its neurons is associated with some basal ganglia motor symptoms. We examined the presence of functional presynaptic GABAB receptors on pallidosubthalamic terminals and tested whether they were tonically active in the in vitro subthalamic slices. Methods: Whole-cell patch-clamp recordings were applied to acutely prepared rat subthalamic nucleus slices. The effects of specific GABAB agonist and antagonist on action potential-independent inhibitory postsynapfic currents (IPSCs), as well as holding current, were examined.Results: Superfusion of baclofen, a GABAB receptor agonist, significantly reduced the frequency of GABAA receptor-mediated miniature IPSCs (mIPSCs), in a Cd2+-sensitive manner, with no effect on the amplitude, indicating presynaptic inhibition on GABA release. In addition, baclofen induced a weak outward current only in a minority of subthalamic neurons. Both the pre- and post-synaptic effects of baclofen were prevented by the specific GABAB receptor antagonist,CGP55845. Furthermore, CGP55845 alone increased the frequency of mIPSCs,but had no effect on the holding current. Conclusion: These findings suggest the functional dominance of presynaptic GABAB receptors on the pallidosubthalamic terminals over the postsynaptic GABAB receptors on subthalamic neurons.Furthermore, the presynaptic, but not the postsynaptic, GABAB receptors are tonically active, suggesting that the presynaptic GABAB receptors in the subthalamic nucleus are potential therapeutic target for the treatment of Parkinson disease.

  10. Nanoconjugation prolongs endosomal signaling of the epidermal growth factor receptor and enhances apoptosis

    Wu, L.; Xu, F.; Reinhard, B. M.

    2016-07-01

    It is becoming increasingly clear that intracellular signaling can be subject to strict spatial control. As the covalent attachment of a signaling ligand to a nanoparticle (NP) impacts ligand-receptor binding, uptake, and trafficking, nanoconjugation provides new opportunities for manipulating intracellular signaling in a controlled fashion. To establish the effect of nanoconjugation on epidermal growth factor (EGF) mediated signaling, we investigate here the intracellular fate of nanoconjugated EGF (NP-EGF) and its bound receptor (EGFR) by quantitative correlated darkfield/fluorescence microscopy and density-based endosomal fractionation. We demonstrate that nanoconjugation prolongs the dwell time of phosphorylated receptors in the early endosomes and that the retention of activated EGFR in the early endosomes is accompanied by an EGF mediated apoptosis at effective concentrations that do not induce apoptosis in the case of free EGF. Overall, these findings indicate nanoconjugation as a rational strategy for modifying signaling that acts by modulating the temporo-spatial distribution of the activated EGF-EGFR ligand-receptor complex.It is becoming increasingly clear that intracellular signaling can be subject to strict spatial control. As the covalent attachment of a signaling ligand to a nanoparticle (NP) impacts ligand-receptor binding, uptake, and trafficking, nanoconjugation provides new opportunities for manipulating intracellular signaling in a controlled fashion. To establish the effect of nanoconjugation on epidermal growth factor (EGF) mediated signaling, we investigate here the intracellular fate of nanoconjugated EGF (NP-EGF) and its bound receptor (EGFR) by quantitative correlated darkfield/fluorescence microscopy and density-based endosomal fractionation. We demonstrate that nanoconjugation prolongs the dwell time of phosphorylated receptors in the early endosomes and that the retention of activated EGFR in the early endosomes is accompanied by an EGF

  11. Inhibition of epidermal growth factor receptor biosynthesis caused by the src oncogene product, pp60v-src

    The authors have previously shown that an intracellular mechanism down regulates epidermal growth factor (EGF) receptor levels in rodent fibroblasts transformed by the src oncogene. They now report that this down regulation is due to an inhibition of EGF receptor biosynthesis. With Rat-1 (R1) cell infected with a temperature-sensitive src mutant, we found that 125I-labeled EGF binding to cells began to decrease soon after the activation of pp60v-src by shift down to the permissive temperature for transformation. This effect of src on EGF receptors was reversible. Pulse-chase studies with [35S]methionine-labeled cells revealed that the tyrosine protein kinase activity of pp60v-src had little if any effect on EGF receptor degradation rate. By contrast, the expression of pp60v-src caused a large reduction in the apparent rate of EGF receptor biosynthesis

  12. Class A scavenger receptor promotes osteoclast differentiation via the enhanced expression of receptor activator of NF-κB (RANK)

    Osteoclasts originate from bone marrow monocyte/macrophage lineage cells, and their differentiation depends on macrophage colony-stimulating factor (M-CSF) and receptor activator nuclear factor kappa B (RANK) ligand. Class A scavenger receptor (SR-A) is one of the principal functional molecules of macrophages, and its level of expression declines during osteoclast differentiation. To investigate the role of SR-A in osteoclastogenesis, we examined pathological changes in femoral bone and the expression levels of osteoclastogenesis-related molecules in SR-A-/- mice. The femoral osseous density of SR-A-/- mice was higher than that of SR-A+/+ mice, and the number of multinucleated osteoclasts was significantly decreased. An in vitro differentiation assay revealed that the differentiation of multinucleated osteoclasts from bone marrow-derived progenitor cells is impaired in SR-A-/- mice. Elimination of SR-A did not alter the expression level of the M-CSF receptor, c-fms; however, the expression levels of RANK and RANK-related osteoclast-differentiation molecules such as nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1) and microphthalmia-associated transcription factor (MITF) significantly decreased. Furthermore, acetylated low-density lipoprotein (AcLDL), an SR-A ligand, significantly increased the expression level of RANK and MITF during osteoclast differentiation. These data indicate that SR-A promotes osteoclastogenesis via augmentation of the expression level of RANK and its related molecules.

  13. Class A scavenger receptor promotes osteoclast differentiation via the enhanced expression of receptor activator of NF-{kappa}B (RANK)

    Takemura, Kenichi [Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556 (Japan); Department of Orthopaedic and Neuro-Musculoskeletal Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto (Japan); Sakashita, Naomi; Fujiwara, Yukio; Komohara, Yoshihiro; Lei, XiaoFeng; Ohnishi, Koji [Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556 (Japan); Suzuki, Hiroshi [National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido (Japan); Kodama, Tatsuhiko [Department of Molecular Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo (Japan); Mizuta, Hiroshi [Department of Orthopaedic and Neuro-Musculoskeletal Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto (Japan); Takeya, Motohiro, E-mail: takeya@kumamoto-u.ac.jp [Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556 (Japan)

    2010-01-22

    Osteoclasts originate from bone marrow monocyte/macrophage lineage cells, and their differentiation depends on macrophage colony-stimulating factor (M-CSF) and receptor activator nuclear factor kappa B (RANK) ligand. Class A scavenger receptor (SR-A) is one of the principal functional molecules of macrophages, and its level of expression declines during osteoclast differentiation. To investigate the role of SR-A in osteoclastogenesis, we examined pathological changes in femoral bone and the expression levels of osteoclastogenesis-related molecules in SR-A{sup -/-} mice. The femoral osseous density of SR-A{sup -/-} mice was higher than that of SR-A{sup +/+} mice, and the number of multinucleated osteoclasts was significantly decreased. An in vitro differentiation assay revealed that the differentiation of multinucleated osteoclasts from bone marrow-derived progenitor cells is impaired in SR-A{sup -/-} mice. Elimination of SR-A did not alter the expression level of the M-CSF receptor, c-fms; however, the expression levels of RANK and RANK-related osteoclast-differentiation molecules such as nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1) and microphthalmia-associated transcription factor (MITF) significantly decreased. Furthermore, acetylated low-density lipoprotein (AcLDL), an SR-A ligand, significantly increased the expression level of RANK and MITF during osteoclast differentiation. These data indicate that SR-A promotes osteoclastogenesis via augmentation of the expression level of RANK and its related molecules.

  14. Helix 11 Dynamics is Critical for Constitutive Androstane Receptor Activity

    Wright, Edward; Busby, Scott A.; Wisecarver, Sarah; Vincent, Jeremy; Griffin, Patrick R.; Fernandez, Elias J.

    2011-01-01

    The constitutive androstane receptor (CAR) transactivation can occur in the absence of exogenous ligand and this activity is enhanced by agonists TCPOBOP and meclizine. We use biophysical and cell-based assays to show that increased activity of CAR(TCPOBOP) relative to CAR(meclizine) corresponds to a higher affinity of CAR(TCPOBOP) for the steroid receptor coactivator-1. Additionally, steady-state fluorescence spectra suggest conformational differences between CAR(TCPOBOP):RXR and CAR(meclizi...

  15. Tumor necrosis factor receptor superfamily costimulation couples T cell receptor signal strength to thymic regulatory T cell differentiation

    Mahmud, Shawn A.; Manlove, Luke S.; Schmitz, Heather M.; Xing, Yan; Wang, Yanyan; Owen, David L.; Schenkel, Jason M.; Boomer, Jonathan S; Jonathan M Green; Yagita, Hideo; Chi, Hongbo; Hogquist, Kristin A.; Farrar, Michael A.

    2014-01-01

    Regulatory T (Treg) cells express tumor necrosis factor receptor superfamily (TNFRSF) members, but their role in thymic Treg development is undefined. We demonstrate that Treg progenitors highly express the TNFRSF members GITR, OX40, and TNFR2. Expression of these receptors correlates directly with T cell receptor (TCR) signal strength, and requires CD28 and the kinase TAK1. Neutralizing TNFSF ligands markedly reduced Treg development. Conversely, TNFRSF agonists enhanced Treg differentiation...

  16. Cognitive disorder and changes in cholinergic receptors, N-methyl-D aspartate receptors, neural cell adhesion molecule, and brain-derived neurotrophic factor following brain injury

    Weiliang Zhao; Dezhi Kang; Yuanxiang Lin

    2008-01-01

    BACKGROUND: Learning and memory damage is one of the most permanent and the severest symptoms of traumatic brain injury; it can seriously influence the normal life and work of patients. Some research has demonstrated that cognitive disorder is closely related to nicotine cholinergic receptors, N-methyl-D aspartate receptors, neural cell adhesion molecule, and brain-derived neurotrophic factor. OBJECTIVE: To summarize the cognitive disorder and changes in nicotine cholinergic receptors, N-methyl-D aspartate receptors, neural cell adhesion molecule, and brain-derived neurotrophic factor following brain injury. RETRIEVAL STRATEGY: A computer-based online search was conducted in PUBMED for English language publications containing the key words "brain injured, cognitive handicap, acetylcholine, N-methyl-D aspartate receptors, neural cell adhesion molecule, brain-derived neurotrophic factor" from January 2000 to December 2007. There were 44 papers in total. Inclusion criteria: ① articles about changes in nicotine cholinergic receptors, N-methyl-D aspartate receptors, neural cell adhesion molecule, and brain-derived neurotrophic factor following brain injury; ② articles in the same researching circle published in authoritative journals or recently published. Exclusion criteria: duplicated articles.LITERATURE EVALUATION: References were mainly derived from research on changes in these four factors following brain injury. The 20 included papers were clinical or basic experimental studies. DATA SYNTHESIS: After craniocerebral injury, changes in these four factors in brain were similar to those during recovery from cognitive disorder, to a certain degree. Some data have indicated that activation of nicotine cholinergic receptors, N-methyl-D aspartate receptors, neural cell adhesion molecule, and brain-derived neurotrophic factor could greatly improve cognitive disorder following brain injury. However, there are still a lot of questions remaining; for example, how do these

  17. Differential Requirement of the Extracellular Domain in Activation of Class B G Protein-coupled Receptors.

    Zhao, Li-Hua; Yin, Yanting; Yang, Dehua; Liu, Bo; Hou, Li; Wang, Xiaoxi; Pal, Kuntal; Jiang, Yi; Feng, Yang; Cai, Xiaoqing; Dai, Antao; Liu, Mingyao; Wang, Ming-Wei; Melcher, Karsten; Xu, H Eric

    2016-07-15

    G protein-coupled receptors (GPCRs) from the secretin-like (class B) family are key players in hormonal homeostasis and are important drug targets for the treatment of metabolic disorders and neuronal diseases. They consist of a large N-terminal extracellular domain (ECD) and a transmembrane domain (TMD) with the GPCR signature of seven transmembrane helices. Class B GPCRs are activated by peptide hormones with their C termini bound to the receptor ECD and their N termini bound to the TMD. It is thought that the ECD functions as an affinity trap to bind and localize the hormone to the receptor. This in turn would allow the hormone N terminus to insert into the TMD and induce conformational changes of the TMD to activate downstream signaling. In contrast to this prevailing model, we demonstrate that human class B GPCRs vary widely in their requirement of the ECD for activation. In one group, represented by corticotrophin-releasing factor receptor 1 (CRF1R), parathyroid hormone receptor (PTH1R), and pituitary adenylate cyclase activating polypeptide type 1 receptor (PAC1R), the ECD requirement for high affinity hormone binding can be bypassed by induced proximity and mass action effects, whereas in the other group, represented by glucagon receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R), the ECD is required for signaling even when the hormone is covalently linked to the TMD. Furthermore, the activation of GLP-1R by small molecules that interact with the intracellular side of the receptor is dependent on the presence of its ECD, suggesting a direct role of the ECD in GLP-1R activation. PMID:27226600

  18. Results With Accelerated Partial Breast Irradiation in Terms of Estrogen Receptor, Progesterone Receptor, and Human Growth Factor Receptor 2 Status

    Purpose: To report our results with accelerated partial breast irradiation (APBI) in terms of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER-2/neu) status. Methods and Materials: Between February 2003 and June 2009, 209 women with early-stage breast carcinomas were treated with APBI using multicatheter, MammoSite, or Contura brachytherapy to 34 Gy in 10 fractions twice daily over 5-7 days. Three patient groups were defined by receptor status: Group 1: ER or PR (+) and HER-2/neu (-) (n = 180), Group 2: ER and PR (-) and HER-2/neu (+) (n = 10), and Group 3: ER, PR, and HER-2/neu (-) (triple negative breast cancer, n = 19). Median follow-up was 22 months. Results: Group 3 patients had significantly higher Scarff-Bloom-Richardson scores (p < 0.001). The 3-year ipsilateral breast tumor control rates for Groups 1, 2, and 3 were 99%, 100%, and 100%, respectively (p = 0.15). Group 3 patients tended to experience relapse in distant sites earlier than did non-Group 3 patients. The 3-year relapse-free survival rates for Groups 1, 2, and 3 were 100%, 100%, and 81%, respectively (p = 0.046). The 3-year cause-specific and overall survival rates for Groups 1, 2, and 3 were 100%, 100%, and 89%, respectively (p = 0.002). Conclusions: Triple negative breast cancer patients typically have high-grade tumors with significantly worse relapse-free, cause-specific, and overall survival. Longer follow-up will help to determine whether these patients also have a higher risk of ipsilateral breast tumor relapse.

  19. P2X7 receptor antagonist activity of the anti-allergic agent oxatomide.

    Yoshida, Kazuki; Ito, Masaaki; Matsuoka, Isao

    2015-11-15

    Activation of the P2X7 receptor by extracellular ATP is associated with various immune responses including allergic inflammation. Anti-allergic agents, such as H1-antihistamines, are known to inhibit the effects of different chemical mediators such as acetylcholine and platelet-activating factor. Therefore, we hypothesized that some anti-allergic agents might affect P2X7 receptor function. Using N18TG2 and J774 cells, which express functional P2X7 receptors, the effects of several anti-allergic agents on P2X7 receptor function were investigated by monitoring the ATP-induced increase in intracellular Ca(2+) concentrations ([Ca(2+)]i). Among the various agents tested, oxatomide significantly inhibited P2X7 receptor-mediated [Ca(2+)]i elevation in a concentration-dependent manner without affecting the P2Y2 receptor-mediated response in both N18TG2 and J774 cells. Consistently, oxatomide inhibited P2X7 receptor-mediated membrane current and downstream responses such as mitogen-activated protein kinase activation, inflammation-related gene induction, and cell death. In addition, oxatomide inhibited P2X7 receptor-mediated degranulation in mouse bone marrow-derived mast cells. Whole cell patch clamp analyses in HEK293 cells expressing human, mouse, and rat P2X7 receptors revealed that the inhibitory effect of oxatomide on ATP-induced current was most prominent for the human P2X7 receptor and almost non-existent for the rat P2X7 receptor. The potent inhibitory effects of oxatomide on human P2X7 receptor-mediated function were confirmed in RPMI8226 human B cell-like myeloma cells, which endogenously express the P2X7 receptor. Our results demonstrated that the antihistamine oxatomide also acts as a P2X7 receptor antagonist. Future studies should thus evaluate whether P2X7 receptor antagonism contributes to the anti-allergic effects of oxatomide. PMID:26463039

  20. In situ detection of TGF betas, TGF beta receptor II mRNA and telomerase activity in rat cholangiocarcinogenesis

    Lu, Jian-ping; Mao, Jian-Qun; Li, Ming-Sheng; Lu, Shi-Lun; Hu, Xi-Qi; Zhu, Shi-Neng; Nomura, Shintaro

    2003-01-01

    AIM: Initial report on the in situ examination of the mRNA expression of transforming growth factor betas (TGFβs), TGFβ type II receptor (TβRII) and telomerase activity in the experimental rat liver tissue during cholangiocarcinogenesis.

  1. Functional domains of the human orphan receptor ARP-1/COUP-TFII involved in active repression and transrepression.

    Achatz, G; Hölzl, B; Speckmayer, R; Hauser, C; Sandhofer, F; Paulweber, B.

    1997-01-01

    The orphan receptor ARP-1/COUP-TFII, a member of the chicken ovalbumin upstream promoter transcription factor (COUP-TF) subfamily of nuclear receptors, strongly represses transcriptional activity of numerous genes, including several apolipoprotein-encoding genes. Recently it has been demonstrated that the mechanism by which COUP-TFs reduce transcriptional activity involves active repression and transrepression. To map the domains of ARP-1/COUP-TFII required for repressor activity, a detailed ...

  2. Peroxisome Proliferator-Activated Receptors in Female Reproduction and Fertility

    Carta, Gaspare; Artini, Paolo Giovanni

    2016-01-01

    Reproductive functions may be altered by the exposure to a multitude of endogenous and exogenous agents, drug or environmental pollutants, which are known to affect gene transcription through the peroxisome proliferator-activated receptors (PPARs) activation. PPARs act as ligand activated transcription factors and regulate metabolic processes such as lipid and glucose metabolism, energy homeostasis, inflammation, and cell proliferation and differentiation. All PPARs isotypes are expressed along the hypothalamic-pituitary-gonadal axis and are strictly involved in reproductive functions. Since female fertility and energy metabolism are tightly interconnected, the research on female infertility points towards the exploration of potential PPARs activating/antagonizing compounds, mainly belonging to the class of thiazolidinediones (TZDs) and fibrates, as useful agents for the maintenance of metabolic homeostasis in women with ovarian dysfunctions. In the present review, we discuss the recent evidence about PPARs expression in the hypothalamic-pituitary-gonadal axis and their involvement in female reproduction. Finally, the therapeutic potential of their manipulation through several drugs is also discussed.

  3. Insulin-like growth factor (IGF)-I obliterates the pregnancy-associated protection against mammary carcinogenesis in rats: evidence that IGF-I enhances cancer progression through estrogen receptoractivation via the mitogen-activated protein kinase pathway

    Pregnancy protects against breast cancer development in humans and rats. Parous rats have persistently reduced circulating levels of growth hormone, which may affect the activity of the growth hormone/insulin-like growth factor (IGF)-I axis. We investigated the effects of IGF-I on parity-associated protection against mammary cancer. Three groups of rats were evaluated in the present study: IGF-I-treated parous rats; parous rats that did not receive IGF-I treatment; and age-matched virgin animals, which also did not receive IGF-I treatment. Approximately 60 days after N-methyl-N-nitrosourea injection, IGF-I treatment was discontinued and all of the animal groups were implanted with a silastic capsule containing 17β-estradiol and progesterone. The 17β-estradiol plus progesterone treatment continued for 135 days, after which the animals were killed. IGF-I treatment of parous rats increased mammary tumor incidence to 83%, as compared with 16% in parous rats treated with 17β-estradiol plus progesterone only. Tumor incidence and average number of tumors per animal did not differ between IGF-I-treated parous rats and age-matched virgin rats. At the time of N-methyl-N-nitrosourea exposure, DNA content was lowest but the α-lactalbumin concentration highest in the mammary glands of untreated parous rats in comparison with age-matched virgin and IGF-I-treated parous rats. The protein levels of estrogen receptor-α in the mammary gland was significantly higher in the age-matched virgin animals than in untreated parous and IGF-I-treated parous rats. Phosphorylation (activation) of the extracellular signal-regulated kinase-1/2 (ERK1/2) and expression of the progesterone receptor were both increased in IGF-I-treated parous rats, as compared with those in untreated parous and age-matched virgin rats. Expressions of cyclin D1 and transforming growth factor-β3 in the mammary gland were lower in the age-matched virgin rats than in the untreated parous and IGF-I-treated parous

  4. Heterodimeric interaction between retinoid X receptor alpha and orphan nuclear receptor OR1 reveals dimerization-induced activation as a novel mechanism of nuclear receptor activation.

    Wiebel, F F; Gustafsson, J.A.

    1997-01-01

    OR1 is a member of the steroid/thyroid hormone nuclear receptor superfamily which has been described to mediate transcriptional responses to retinoids and oxysterols. On a DR4 response element, an OR1 heterodimer with the nuclear receptor retinoid X receptor alpha (RXR alpha) has been described to convey transcriptional activation in both the absence and presence of the RXR ligand 9-cis retinoic acid, the mechanisms of which have remained unclear. Here, we dissect the effects of RXR alpha and...

  5. A quantitative method to assess extrasynaptic NMDA receptor function in the protective effect of synaptic activity against neurotoxicity

    Bading Hilmar

    2008-01-01

    Full Text Available Abstract Background Extrasynaptic NMDA receptors couple to a CREB shut-off pathway and cause cell death, whereas synaptic NMDA receptors and nuclear calcium signaling promote CREB-mediated transcription and neuronal survival. The distribution of NMDA receptors (synaptic versus extrasynaptic may be an important parameter that determines the susceptibility of neurons to toxic insults. Changes in receptor surface expression towards more extrasynaptic NMDA receptors may lead to neurodegeneration, whereas a reduction of extrasynaptic NMDA receptors may render neurons more resistant to death. A quantitative assessment of extrasynaptic NMDA receptors in individual neurons is needed in order to investigate the role of NMDA receptor distribution in neuronal survival and death. Results Here we refined and verified a protocol previously used to isolate the effects of extrasynaptic NMDA receptors using the NMDA receptor open channel blocker, MK-801. Using this method we investigated the possibility that the known neuroprotective shield built up in hippocampal neurons after a period of action potential bursting and stimulation of synaptic NMDA receptors is due to signal-induced trafficking of extrasynaptic NMDA receptors or a reduction in extrasynaptic NMDA receptor function. We found that extrasynaptic NMDA receptor-mediated calcium responses and whole cell currents recorded under voltage clamp were surprisingly invariable and did not change even after prolonged (16 to 24 hours periods of bursting and synaptic NMDA receptor activation. Averaging a large number of calcium imaging traces yielded a small (6% reduction of extrasynaptic NMDA receptor-mediated responses in hippocampal neurons that were pretreated with prolonged bursting. Conclusion The slight reduction in extrasynaptic NMDA receptor function following action potential bursting and synaptic NMDA receptor stimulation could contribute to but is unlikely to fully account for activity

  6. Novel Bioluminescent Binding Assays for Ligand–Receptor Interaction Studies of the Fibroblast Growth Factor Family

    Song, Ge; Shao, Xiao-Xia; Wu, Qing-Ping; Xu, Zeng-Guang; Liu, Ya-Li; Guo, Zhan-Yun

    2016-01-01

    We recently developed novel bioluminescent binding assays for several protein/peptide hormones to study their interactions with receptors using the so far brightest NanoLuc reporter. To validate the novel bioluminescent binding assay using a variety of protein/peptide hormones, in the present work we applied it to the fibroblast growth factor (FGF) family using the prototype member FGF2 as an example. A fully active recombinant FGF2 retaining a unique exposed cysteine (Cys) residue was chemically conjugated with an engineered NanoLuc carrying a unique exposed Cys residue at the C-terminus via formation of an intermolecular disulfide linkage. The NanoLuc-conjugated FGF2 (FGF2-Luc) retained high binding affinity to the overexpressed FGFR1 and the endogenous FGF receptor with the calculated dissociation constants of 161 ± 21 pM (n = 3) and 25 ± 4 pM (n = 3), respectively. In competition binding assays using FGF2-Luc as a tracer, receptor-binding potencies of wild-type or mutant FGF2s were accurately quantified. Thus, FGF2-Luc represents a novel non-radioactive tracer for the quantitative measurement of ligand–receptor interactions in the FGF family. These data suggest that the novel bioluminescent binding assay can be applied to a variety of protein/peptide hormones for ligand–receptor interaction studies. PMID:27414797

  7. The Role of Leukemia Inhibitory Factor Receptor Signaling in Skeletal Muscle Growth, Injury and Disease.

    Hunt, Liam C; White, Jason

    2016-01-01

    Cytokines are an incredibly diverse group of secreted proteins with equally diverse functions. The actions of cytokines are mediated by the unique and sometimes overlapping receptors to which the soluble ligands bind. Classified within the interleukin-6 family of cytokines are leukemia inhibitory factor (LIF), oncostatin-M (OSM), cardiotrophin-1 (CT-1) and ciliary neurotrophic factor (CNTF). These cytokines all bind to the leukemia inhibitory factor receptor (LIFR) and gp130, and in some cases an additional receptor subunit, leading to activation of downstream kinases and transcriptional activators. LIFR is expressed on a broad range of cell types and can generate pleiotropic effects. In the context of skeletal muscle physiology, these cytokines have been shown to exert effects on motor neurons, inflammatory and muscle cells. From isolated cells through to whole organisms, manipulations of LIFR signaling cytokines have a wide range of outcomes influencing muscle cell growth, myogenic differentiation, response to exercise, metabolism, neural innervation and recruitment of inflammatory cells to sites of muscle injury. This article will discuss the shared and distinct processes that LIFR cytokines regulate in a variety of experimental models with the common theme of skeletal muscle physiology. PMID:27003396

  8. Retinoic Acid-mediated Nuclear Receptor Activation and Hepatocyte Proliferation

    Bushue, Nathan; Wan, Yu-Jui Yvonne

    2016-01-01

    Due to their well-known differentiation and apoptosis-inducing abilities, retinoic acid (RA) and its analogs have strong anti-cancer efficacy in human cancers. However, in vivo RA is a liver mitogen. While speculation has persisted that RA-mediated signaling is likely involved in hepatocyte proliferation during liver regeneration, direct evidence is still required. Findings in support of this proposition include observations that a release of retinyl palmitate (the precursor of RA) occurs in liver stellate cells following liver injury. Nevertheless, the biological action of this released vitamin A is virtually unknown. More likely is that the released vitamin A is converted to RA, the biological form, and then bound to a specific receptor (retinoid x receptor; RXRα), which is most abundantly expressed in the liver. Considering the mitogenic effects of RA, the RA-activated RXRα would likely then influence hepatocyte proliferation and liver tissue repair. At present, the mechanism by which RA stimulates hepatocyte proliferation is largely unknown. This review summarizes the activation of nuclear receptors (peroxisome proliferator activated receptor-α, pregnane x receptor, constitutive androstane receptor, and farnesoid x receptor) in an RXRα dependent manner to induce hepatocyte proliferation, providing a link between RA and its proliferative role.

  9. Environmental polycyclic aromatic hydrocarbons affect androgen receptor activation in vitro

    Vinggaard, Anne Marie; Hnida, Christina; Larsen, John Christian

    2000-01-01

    of certain PAHs to activate the Ah receptor was assessed in H4IIE liver cancer cells, stably transfected with a luciferase reporter gene system. The positive control 2, 3,7, 8-tetrachlorodibenzodioxin (TCDD) caused a 13-14-fold induction of luciferase activity reaching maximum activity at 0.1 nM. DB...

  10. Environmental polycyclic aromatic hydrocarbons affect androgen receptor activation in vitro

    Vinggaard, Anne Marie; Hnida, Christina; Larsen, John Christian

    of certain PAHs to activate the Ah receptor was assessed in H4IIE liver cancer cells, stably transfected with a luciferase reporter gene system. The positive control 2, 3,7, 8-tetrachlorodibenzodioxin (TCDD) caused a 13-14-fold induction of luciferase activity reaching maximum activity at 0.1 nM. DB...

  11. Key facts and hot spots on tumor necrosis factor receptor-associated periodic syndrome.

    Rigante, Donato; Lopalco, Giuseppe; Vitale, Antonio; Lucherini, Orso Maria; De Clemente, Caterina; Caso, Francesco; Emmi, Giacomo; Costa, Luisa; Silvestri, Elena; Andreozzi, Laura; Iannone, Florenzo; Galeazzi, Mauro; Cantarini, Luca

    2014-09-01

    Tumor necrosis factor receptor-associated periodic syndrome (TRAPS), formerly known as familial Hibernian fever, is the most common autosomal dominant autoinflammatory disease, resulting from mutations in the TNFRSF1A gene, encoding the 55-kD tumor necrosis factor receptor. The pathophysiologic mechanism of TRAPS remains ambiguous and only partially explained. The onset age of the syndrome is variable and the clinical scenery is characterized by recurrent episodes of high-grade fever that typically lasts 1-3 weeks, associated with migrating myalgia, pseudocellulitis, diffuse abdominal pain, appendicitis-like findings, ocular inflammatory signs, and risk of long-term amyloidosis. Fever episodes are responsive to high-dose corticosteroids, but different classes of drugs have been reported to be ineffective. The use of etanercept is unable to control systemic inflammation, while interleukin-1 blockade has been shown as effective in the control of disease activity in many patients reported so far. PMID:24935411

  12. Modulation of β-catenin signaling by glucagon receptor activation.

    Jiyuan Ke

    Full Text Available The glucagon receptor (GCGR is a member of the class B G protein-coupled receptor family. Activation of GCGR by glucagon leads to increased glucose production by the liver. Thus, glucagon is a key component of glucose homeostasis by counteracting the effect of insulin. In this report, we found that in addition to activation of the classic cAMP/protein kinase A (PKA pathway, activation of GCGR also induced β-catenin stabilization and activated β-catenin-mediated transcription. Activation of β-catenin signaling was PKA-dependent, consistent with previous reports on the parathyroid hormone receptor type 1 (PTH1R and glucagon-like peptide 1 (GLP-1R receptors. Since low-density-lipoprotein receptor-related protein 5 (Lrp5 is an essential co-receptor required for Wnt protein mediated β-catenin signaling, we examined the role of Lrp5 in glucagon-induced β-catenin signaling. Cotransfection with Lrp5 enhanced the glucagon-induced β-catenin stabilization and TCF promoter-mediated transcription. Inhibiting Lrp5/6 function using Dickkopf-1(DKK1 or by expression of the Lrp5 extracellular domain blocked glucagon-induced β-catenin signaling. Furthermore, we showed that Lrp5 physically interacted with GCGR by immunoprecipitation and bioluminescence resonance energy transfer assays. Together, these results reveal an unexpected crosstalk between glucagon and β-catenin signaling, and may help to explain the metabolic phenotypes of Lrp5/6 mutations.

  13. Changing the insulin receptor to possess insulin-like growth factor I ligand specificity

    To examine the role of the N-terminal part of the insulin-like growth factor I (IGF-I) receptor and insulin receptor in determining ligand specificity, the authors prepared an expression vector encoding a hybrid receptor where exon 1 (encoding the signal peptide and seven amino acids of the α-subunit), exon 2, and exon 3 of the insulin receptor were replaced with the corresponding IGF-I receptor cDNA (938 nucleotides). To allow direct quantitative comparison of the binding capabilities of this hybrid receptor with those of the human IGF-I receptor and the insulin receptor, all three receptors were expressed in baby hamster kidney (BHK) cells as soluble molecules and partially purified before characterization. The hybrid IGF-I/insulin receptor bound IGF-I with an affinity comparable to that of the wild-type IGF-I receptor. In contrast, the hybrid receptor no longer displayed high-affinity binding of insulin. These results directly demonstrate that it is possible to change the specificity of the insulin receptor to that of the IGF-I receptor and, furthermore, that the binding specificity for IGF-I is encoded within the nucleotide sequence from 135 to 938 of the IGF-I receptor cDNA. Since the hybrid receptor only bound insulin with low affinity, the insulin binding region is likely to be located within exons 2 and 3 of the insulin receptor

  14. Cloning, constitutive activity and expression profiling of two receptors related to relaxin receptors in Drosophila melanogaster.

    Van Hiel, Matthias B; Vandersmissen, Hans Peter; Proost, Paul; Vanden Broeck, Jozef

    2015-06-01

    Leucine-rich repeat containing G protein-coupled receptors (LGRs) comprise a cluster of transmembrane proteins, characterized by the presence of a large N-terminal extracellular domain. This receptor group can be classified into three subtypes. Belonging to the subtype C LGRs are the mammalian relaxin receptors LGR7 (RXFP1) and LGR8 (RXFP2), which mediate important reproductive and other processes. We identified two related receptors in the genome of the fruit fly and cloned their open reading frames into an expression vector. Interestingly, dLGR3 demonstrated constitutive activity at very low doses of transfected plasmid, whereas dLGR4 did not show any basal activity. Both receptors exhibited a similar expression pattern during development, with relatively high transcript levels during the first larval stage. In addition, both receptors displayed higher expression in male adult flies as compared to female flies. Analysis of the tissue distribution of both receptor transcripts revealed a high expression of dLGR3 in the female fat body, while the expression of dLGR4 peaked in the midgut of both the wandering and adult stage. PMID:25064813

  15. DMPD: Receptor tyrosine kinases and the regulation of macrophage activation. [Dynamic Macrophage Pathway CSML Database

    Full Text Available 14726496 Receptor tyrosine kinases and the regulation of macrophage activation. Cor...(.csml) Show Receptor tyrosine kinases and the regulation of macrophage activation. PubmedID 14726496 Title Receptor tyrosine kinases

  16. Nuclear receptor corepressor-dependent repression of peroxisome-proliferator-activated receptor delta-mediated transactivation

    Krogsdam, Anne-M; Nielsen, Curt A F; Neve, Søren;

    2002-01-01

    delta-RXR alpha heterodimer bound to an acyl-CoA oxidase (ACO)-type peroxisome-proliferator response element recruited a glutathione S-transferase-NCoR fusion protein in a ligand-independent manner. Contrasting with most other nuclear receptors, PPAR delta was found to interact equally well......The nuclear receptor corepressor (NCoR) was isolated as a peroxisome-proliferator-activated receptor (PPAR) delta interacting protein using the yeast two-hybrid system. NCoR interacted strongly with the ligand-binding domain of PPAR delta, whereas interactions with the ligand-binding domains...

  17. Epidermal Growth Factor Receptor in Prostate Cancer Derived Exosomes.

    Geetanjali Kharmate

    Full Text Available Exosomes proteins and microRNAs have gained much attention as diagnostic tools and biomarker potential in various malignancies including prostate cancer (PCa. However, the role of exosomes and membrane-associated receptors, particularly epidermal growth factor receptor (EGFR as mediators of cell proliferation and invasion in PCa progression remains unexplored. EGFR is frequently overexpressed and has been associated with aggressive forms of PCa. While PCa cells and tissues express EGFR, it is unknown whether exosomes derived from PCa cells or PCa patient serum contains EGFR. The aim of this study was to detect and characterize EGFR in exosomes derived from PCa cells, LNCaP xenograft and PCa patient serum. Exosomes were isolated from conditioned media of different PCa cell lines; LNCaP xenograft serum as well as patient plasma/serum by differential centrifugation and ultracentrifugation on a sucrose density gradient. Exosomes were confirmed by electron microscopy, expression of exosomal markers and NanoSight™ analysis. EGFR expression was determined by western blot analysis and ELISA. This study demonstrates that exosomes may easily be derived from PCa cell lines, serum obtained from PCa xenograft bearing mice and clinical samples derived from PCa patients. Presence of exosomal EGFR in PCa patient exosomes may present a novel approach for measuring of the disease state. Our work will allow to build on this finding for future understanding of PCa exosomes and their potential role in PCa progression and as minimal invasive biomarkers for PCa.

  18. Purification of rat intestinal receptor for intrinsic factor-vitamin B12 complex

    The intrinsic factor (IF) in a rat gastric mucosal extract was bound efficiently to vitamin B12-sepharose without significant change in its nature to produce IF-vitamin B12-sepharose. The purification of the intestinal receptor for the IF-vitamin B12 complex was performed by the affinity chromatography using the IF-vitamin B12-sepharose as the affinity adsorbent. As a result of admixing the gastric mucosal extract sample with B12-sepharose while stirring for 4 hours, the adsorption was performed without any break through. Further, it was recognized that the B12-bound protein purified by the affinity chromatography using B12-sepharose was not much changed as compared with that before purification. Furthermore, it was recognized that IF-B12-sepharose was able to be made by binding IF with B12-sepharose which was made by coupling B12 with the market-available AH-sepharose. The IF-B12-sepharose was washed with buffer solution, and then was loaded with the small intestine mucosal extract. Thereafter, the receptor was eluted by making di-valent cation inert with the buffer solution. After the removal of EDTA in the eluted solution by dialysis, the activity of the receptor was measured. 48.5% of the receptor activity loaded was recovered by the elution with EDTA. The specific activity of the receptor represented by the final amount of B12 (pg)/the amount of protein (mg) in the purified substance was 335 folds of the original activity. (Iwakiri, K.)

  19. Monitoring leptin activity using the chicken leptin receptor.

    Hen, Gideon; Yosefi, Sera; Ronin, Ana; Einat, Paz; Rosenblum, Charles I; Denver, Robert J; Friedman-Einat, Miriam

    2008-05-01

    We report on the construction of a leptin bioassay based on the activation of chicken leptin receptor in cultured cells. A human embryonic kidney (HEK)-293 cell line, stably transfected with the full-length cDNA of chicken leptin receptor together with a STAT3-responsive reporter gene specifically responded to recombinant human and Xenopus leptins. The observed higher sensitivity of chicken leptin receptor to the former is in agreement with the degree of sequence similarity among these species (about 60 and 38% identical amino acids between humans and chickens, and between humans and Xenopus respectively). The specific activation of signal transduction through the chicken leptin receptor, shown here for the first time, suggests that the transition of Gln269 (implicated in the Gln-to-Pro Zucker fatty mutation in rats) to Glu in chickens does not impair its activity. Analysis of leptin-like activity in human serum samples of obese and lean subjects coincided well with leptin levels determined by RIA. Serum samples of pre- and post partum cows showed a tight correlation with the degree of adiposity. However, specific activation of the chicken leptin receptor in this assay was not observed with serum samples from broiler or layer chickens (representing fat and lean phenotypes respectively) or with those from turkey. Similar leptin receptor activation profiles were observed with cells transfected with human leptin receptor. Further work is needed to determine whether the lack of leptin-like activity in the chicken serum samples is due to a lack of leptin in this species or simply to a serum level of leptin that is below the detection threshold. PMID:18434362

  20. Lufaxin, a novel factor Xa inhibitor from the salivary gland of the sand fly lutzomyia longipalpis, blocks protease-activated receptor 2 activation and inhibits inflammation and thrombosis in vivo

    Collin, N.; Assumpção, T.C.F.; Mizurini, D.M.; Gilmore, D.; Dutra-Oliveira, A.; Kotsyfakis, Michalis; Sa-Nunes, A.; Teixeira, C.; Ribeiro, J.M.C.; Monteiro, R.Q.; Valenzuela, J. G.; Francischetti, I.M.B.

    2012-01-01

    Roč. 32, č. 9 (2012), s. 2185-2198. ISSN 1079-5642 R&D Projects: GA ČR GAP502/12/2409 Institutional support: RVO:60077344 Keywords : hematophagy * leishmaniasis * microcirculation * thrombosis * vector biology Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 6.338, year: 2012 http://atvb.ahajournals.org/content/32/9/2185

  1. Tumor-promoting phorbol diesters cause the phosphorylation of epidermal growth factor receptors in normal human fibroblasts at threonine-654.

    Davis, R. J.; Czech, M P

    1985-01-01

    The effect of tumor-promoting phorbol diesters to potentiate the action of epidermal growth factor (EGF) on cell proliferation is associated with phosphorylation of EGF receptors, acute depression of EGF binding, and inhibition of EGF receptor tyrosine kinase activity. In the present studies, normal human fibroblasts and A431 carcinoma cells were labeled with [32P]phosphate and treated with and without 10 nM 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA). The EGF receptors then were ...

  2. Flavonoids with M1 Muscarinic Acetylcholine Receptor Binding Activity

    Meyyammai Swaminathan

    2014-06-01

    Full Text Available Muscarinic acetylcholine receptor-active compounds have potential for the treatment of Alzheimer’s disease. In this study, a series of natural and synthetic flavones and flavonols was assayed in vitro for their ability to inhibit radioligand binding at human cloned M1 muscarinic receptors. Several compounds were found to possess competitive binding affinity (Ki = 40–110 µM, comparable to that of acetylcholine (Ki = 59 µM. Despite the fact that these compounds lack a positively-charged ammonium group under physiological conditions, molecular modelling studies suggested that they bind to the orthosteric site of the receptor, mainly through non-polar interactions.

  3. Peroxisome proliferator-activated receptor alpha polymorphisms and postprandial lipemia in healthy men

    Peroxisome proliferator-activated receptor alpha (PPARA) is a ligand-dependent transcription factor that plays a key role in lipid and glucose homeostasis. This study evaluated whether variants of PPARA are associated with postprandial lipemia. Subjects were given a single fat load comprised of 60% ...

  4. Deletion of protease-activated receptor 2 prolongs survival of scrapie-inoculated mice

    Matěj, R.; Olejár, Tomáš; Janoušková, O.; Holada, K.

    2012-01-01

    Roč. 93, č. 9 (2012), s. 2057-2061. ISSN 0022-1317 Institutional support: RVO:67985823 Keywords : protease-activated receptor (PAR2) * scrapie * neurodegenerative disorders Subject RIV: FN - Epidemiology, Contagious Disease s ; Clinical Immunology Impact factor: 3.127, year: 2012

  5. Expression of protease-activated receptor 1 and 2 and anti-tubulogenic activity of protease-activated receptor 1 in human endothelial colony-forming cells.

    Tiago M Fortunato

    Full Text Available Endothelial colony-forming cells (ECFCs are obtained from the culture of human peripheral blood mononuclear cell (hPBMNC fractions and are characterised by high proliferative and pro-vasculogenic potential, which makes them of great interest for cell therapy. Here, we describe the detection of protease-activated receptor (PAR 1 and 2 amongst the surface proteins expressed in ECFCs. Both receptors are functionally coupled to extracellular signal-regulated kinase (ERK 1 and 2, which become activated and phosphorylated in response to selective PAR1- or PAR2-activating peptides. Specific stimulation of PAR1, but not PAR2, significantly inhibits capillary-like tube formation by ECFCs in vitro, suggesting that tubulogenesis is negatively regulated by proteases able to stimulate PAR1 (e.g. thrombin. The activation of ERKs is not involved in the regulation of tubulogenesis in vitro, as suggested by use of the MEK inhibitor PD98059 and by the fact that PAR2 stimulation activates ERKs without affecting capillary tube formation. Both qPCR and immunoblotting showed a significant downregulation of vascular endothelial growth factor 2 (VEGFR2 in response to PAR1 stimulation. Moreover, the addition of VEGF (50-100 ng/ml but not basic Fibroblast Growth Factor (FGF (25-100 ng/ml rescued tube formation by ECFCs treated with PAR1-activating peptide. Therefore, we propose that reduction of VEGF responsiveness resulting from down-regulation of VEGFR2 is underlying the anti-tubulogenic effect of PAR1 activation. Although the role of PAR2 remains elusive, this study sheds new light on the regulation of the vasculogenic activity of ECFCs and suggests a potential link between adult vasculogenesis and the coagulation cascade.

  6. Identical splicing of aberrant epidermal growth factor receptor transcripts from amplified rearranged genes in human glioblastomas.

    Sugawa, N; Ekstrand, A J; James, C D; Collins, V P

    1990-01-01

    The epidermal growth factor receptor gene has been found to be amplified and rearranged in human glioblastomas in vivo. Here we present the sequence across a splice junction of aberrant epidermal growth factor receptor transcripts derived from corresponding and uniquely rearranged genes that are coamplified and coexpressed with non-rearranged epidermal growth factor receptor genes in six primary human glioblastomas. Each of these six tumors contains aberrant transcripts derived from identical...

  7. Canine lymphocyte activating factor (LAF)

    The immune response of an animal is the sum of the result of the interaction of various cells mainly through soluble mediators. It is not enough to look at specific cell populations, it is also necessary to study the interactions between purified cell population. The effect of one subpopulation on another is via soluble mediators. We have been studying one (of several) such mediators in its relation to radiation effects on the immune response. Lymphocyte activating factor (LAF) is defined functionally as a potentiator of the response of thymocytes to phytohemagglutinin (PHA) or concanavalin (con-A). It can also elicit response of unstimulated subpopulations separated from the thymus. It is a product of adherent populations, presumably macrophages. It has been shown to be produced by human, rabbit, and mouse cells, but has not been reported in the dog. It also was shown to be present in higher concentrations in irradiated mice than in comparable unirradiated mice. We have shown that LAF is produced by plastic-adherent populations derived from peripheral blood. Currently we are working to determine the lymphocyte subpopulations with which LAF interacts

  8. Activation of microglial cells triggers a release of brain-derived neurotrophic factor (BDNF) inducing their proliferation in an adenosine A2A receptor-dependent manner: A2A receptor blockade prevents BDNF release and proliferation of microglia

    Gomes Catarina; Ferreira Raquel; George Jimmy; Sanches Rui; Rodrigues Diana I; Gonçalves Nélio; Cunha Rodrigo A

    2013-01-01

    Abstract Background Brain-derived neurotrophic factor (BDNF) has been shown to control microglial responses in neuropathic pain. Since adenosine A2A receptors (A2ARs) control neuroinflammation, as well as the production and function of BDNF, we tested to see if A2AR controls the microglia-dependent secretion of BDNF and the proliferation of microglial cells, a crucial event in neuroinflammation. Methods Murine N9 microglial cells were challenged with lipopolysaccharide (LPS, 100 ng/mL) in the...

  9. Identification of bioactive compounds from flowers of black elder (Sambucus nigra L.) that activate the human peroxisome proliferator-activated receptor (PPAR) gamma

    Christensen, Kathrine B; Petersen, Rasmus K; Kristiansen, Karsten;

    2010-01-01

    Obesity is one of the predisposing factors for the development of overt Type 2 diabetes (T2D). T2D is caused by a combination of insulin resistance and beta-cell failure and can be treated with insulin sensitizing drugs that target the nuclear receptor peroxisome proliferator-activated receptor (...

  10. Stabilization of the bioactivity of tumor necrosis factor by its soluble receptors

    Aderka, D; Engelmann, H; Maor, Y; Brakebusch, C; Wallach, D

    1992-01-01

    The receptors for tumor necrosis factor (TNF) exist in cell-associated as well as soluble forms, both binding specifically to TNF. Since the soluble forms of TNF receptors (sTNF-Rs) can compete with the cell-associated TNF receptors for TNF, it was suggested that they function as inhibitors of TN...

  11. Mechanical stretch augments insulin-induced vascular smooth muscle cell proliferation by insulin-like growth factor-1 receptor

    Insulin resistance and hypertension have been implicated in the pathogenesis of cardiovascular disease; however, little is known about the roles of insulin and mechanical force in vascular smooth muscle cell (VSMC) remodeling. We investigated the contribution of mechanical stretch to insulin-induced VSMC proliferation. Thymidine incorporation was stimulated by insulin in stretched VSMCs, but not in un-stretched VSMCs. Insulin increased 2-deoxy-glucose incorporation in both stretched and un-stretched VSMCs. Mechanical stretch augmented insulin-induced extracellular signal-regulated kinase (ERK) and Akt phosphorylation. Inhibitors of epidermal growth factor (EGF) receptor tyrosine kinase and Src attenuated insulin-induced ERK and Akt phosphorylation, as well as thymidine incorporation, whereas 2-deoxy-glucose incorporation was not affected by these inhibitors. Moreover, stretch augmented insulin-like growth factor (IGF)-1 receptor expression, although it did not alter the expression of insulin receptor and insulin receptor substrate-1. Insulin-induced ERK and Akt activation, and thymidine incorporation were inhibited by siRNA for the IGF-1 receptor. Mechanical stretch augments insulin-induced VSMC proliferation via upregulation of IGF-1 receptor, and downstream Src/EGF receptor-mediated ERK and Akt activation. Similar to in vitro experiment, IGF-1 receptor expression was also augmented in hypertensive rats. These results provide a basis for clarifying the molecular mechanisms of vascular remodeling in hypertensive patients with hyperinsulinemia. -- Highlights: → Mechanical stretch augments insulin-induced VSMC proliferation via IGF-1 receptor. → Src/EGFR-mediated ERK and Akt phosphorylation are augmented in stretched VSMCs. → Similar to in vitro experiment, IGF-1 receptor is increased in hypertensive rats. → Results provide possible mechanisms of vascular remodeling in hypertension with DM.

  12. Mechanical stretch augments insulin-induced vascular smooth muscle cell proliferation by insulin-like growth factor-1 receptor

    Liu, Gang [Department of Pharmacology, Faculty of Medicine, Kagawa University, Kagawa (Japan); Department of Anesthesiology, First Affiliated Hospital of China Medical University, Shenyang (China); Hitomi, Hirofumi, E-mail: hitomi@kms.ac.jp [Department of Pharmacology, Faculty of Medicine, Kagawa University, Kagawa (Japan); Hosomi, Naohisa [Department of Cardiorenal and Cerebrovascular Medicine, Faculty of Medicine, Kagawa University, Kagawa (Japan); Lei, Bai; Nakano, Daisuke [Department of Pharmacology, Faculty of Medicine, Kagawa University, Kagawa (Japan); Deguchi, Kazushi; Mori, Hirohito; Masaki, Tsutomu [Department of Gastroenterology and Neurology, Faculty of Medicine, Kagawa University, Kagawa (Japan); Ma, Hong [Department of Anesthesiology, First Affiliated Hospital of China Medical University, Shenyang (China); Griendling, Kathy K. [Department of Medicine, Division of Cardiology, Emory University School of Medicine, Atlanta, GA (United States); Nishiyama, Akira [Department of Pharmacology, Faculty of Medicine, Kagawa University, Kagawa (Japan)

    2011-10-15

    Insulin resistance and hypertension have been implicated in the pathogenesis of cardiovascular disease; however, little is known about the roles of insulin and mechanical force in vascular smooth muscle cell (VSMC) remodeling. We investigated the contribution of mechanical stretch to insulin-induced VSMC proliferation. Thymidine incorporation was stimulated by insulin in stretched VSMCs, but not in un-stretched VSMCs. Insulin increased 2-deoxy-glucose incorporation in both stretched and un-stretched VSMCs. Mechanical stretch augmented insulin-induced extracellular signal-regulated kinase (ERK) and Akt phosphorylation. Inhibitors of epidermal growth factor (EGF) receptor tyrosine kinase and Src attenuated insulin-induced ERK and Akt phosphorylation, as well as thymidine incorporation, whereas 2-deoxy-glucose incorporation was not affected by these inhibitors. Moreover, stretch augmented insulin-like growth factor (IGF)-1 receptor expression, although it did not alter the expression of insulin receptor and insulin receptor substrate-1. Insulin-induced ERK and Akt activation, and thymidine incorporation were inhibited by siRNA for the IGF-1 receptor. Mechanical stretch augments insulin-induced VSMC proliferation via upregulation of IGF-1 receptor, and downstream Src/EGF receptor-mediated ERK and Akt activation. Similar to in vitro experiment, IGF-1 receptor expression was also augmented in hypertensive rats. These results provide a basis for clarifying the molecular mechanisms of vascular remodeling in hypertensive patients with hyperinsulinemia. -- Highlights: {yields} Mechanical stretch augments insulin-induced VSMC proliferation via IGF-1 receptor. {yields} Src/EGFR-mediated ERK and Akt phosphorylation are augmented in stretched VSMCs. {yields} Similar to in vitro experiment, IGF-1 receptor is increased in hypertensive rats. {yields} Results provide possible mechanisms of vascular remodeling in hypertension with DM.

  13. GATA Factor-G-Protein-Coupled Receptor Circuit Suppresses Hematopoiesis.

    Gao, Xin; Wu, Tongyu; Johnson, Kirby D; Lahvic, Jamie L; Ranheim, Erik A; Zon, Leonard I; Bresnick, Emery H

    2016-03-01

    Hematopoietic stem cells (HSCs) originate from hemogenic endothelium within the aorta-gonad-mesonephros (AGM) region of the mammalian embryo. The relationship between genetic circuits controlling stem cell genesis and multi-potency is not understood. A Gata2 cis element (+9.5) enhances Gata2 expression in the AGM and induces the endothelial to HSC transition. We demonstrated that GATA-2 rescued hematopoiesis in +9.5(-/-) AGMs. As G-protein-coupled receptors (GPCRs) are the most common targets for FDA-approved drugs, we analyzed the GPCR gene ensemble to identify GATA-2-regulated GPCRs. Of the 20 GATA-2-activated GPCR genes, four were GATA-1-activated, and only Gpr65 expression resembled Gata2. Contrasting with the paradigm in which GATA-2-activated genes promote hematopoietic stem and progenitor cell genesis/function, our mouse and zebrafish studies indicated that GPR65 suppressed hematopoiesis. GPR65 established repressive chromatin at the +9.5 site, restricted occupancy by the activator Scl/TAL1, and repressed Gata2 transcription. Thus, a Gata2 cis element creates a GATA-2-GPCR circuit that limits positive regulators that promote hematopoiesis. PMID:26905203

  14. GATA Factor-G-Protein-Coupled Receptor Circuit Suppresses Hematopoiesis

    Xin Gao

    2016-03-01

    Full Text Available Hematopoietic stem cells (HSCs originate from hemogenic endothelium within the aorta-gonad-mesonephros (AGM region of the mammalian embryo. The relationship between genetic circuits controlling stem cell genesis and multi-potency is not understood. A Gata2 cis element (+9.5 enhances Gata2 expression in the AGM and induces the endothelial to HSC transition. We demonstrated that GATA-2 rescued hematopoiesis in +9.5−/− AGMs. As G-protein-coupled receptors (GPCRs are the most common targets for FDA-approved drugs, we analyzed the GPCR gene ensemble to identify GATA-2-regulated GPCRs. Of the 20 GATA-2-activated GPCR genes, four were GATA-1-activated, and only Gpr65 expression resembled Gata2. Contrasting with the paradigm in which GATA-2-activated genes promote hematopoietic stem and progenitor cell genesis/function, our mouse and zebrafish studies indicated that GPR65 suppressed hematopoiesis. GPR65 established repressive chromatin at the +9.5 site, restricted occupancy by the activator Scl/TAL1, and repressed Gata2 transcription. Thus, a Gata2 cis element creates a GATA-2-GPCR circuit that limits positive regulators that promote hematopoiesis.

  15. Arsenite and insulin exhibit opposing effects on epidermal growth factor receptor and keratinocyte proliferative potential

    Previous work has suggested that arsenic exposure contributes to skin carcinogenesis by preserving the proliferative potential of human epidermal keratinocytes, thereby slowing the exit of putative target stem cells into the differentiation pathway. To find a molecular basis for this action, present work has explored the influence of arsenite on keratinocyte responses to epidermal growth factor (EGF). The ability of cultured keratinocytes to found colonies upon passaging several days after confluence was preserved by arsenite and EGF in an additive fashion, but neither was effective when the receptor tyrosine kinase activity was inhibited. Arsenite prevented the loss of EGF receptor protein and phosphorylation of tyrosine 1173, preserving its capability to signal. The level of nuclear β-catenin was higher in cells treated with arsenite and EGF in parallel to elevated colony forming ability, and expression of a dominant negative β-catenin suppressed the increase in both colony forming ability and yield of putative stem cells induced by arsenite and EGF. As judged by expression of three genes regulated by β-catenin, this transcription factor had substantially higher activity in the arsenite/EGF-treated cells. Trivalent antimony exhibited the same effects as arsenite. A novel finding is that insulin in the medium induced the loss of EGF receptor protein, which was largely prevented by arsenite exposure

  16. Differential role of tumor necrosis factor receptors in mouse brain inflammatory responses in cryolesion brain injury

    Quintana, Albert; Giralt, Mercedes; Rojas, Santiago;

    2005-01-01

    Tumor necrosis factor-alpha (TNF-alpha) is one of the mediators dramatically increased after traumatic brain injury that leads to the activation, proliferation, and hypertrophy of mononuclear, phagocytic cells and gliosis. Eventually, TNF-alpha can induce both apoptosis and necrosis via intracell......Tumor necrosis factor-alpha (TNF-alpha) is one of the mediators dramatically increased after traumatic brain injury that leads to the activation, proliferation, and hypertrophy of mononuclear, phagocytic cells and gliosis. Eventually, TNF-alpha can induce both apoptosis and necrosis via...... intracellular signaling. This cytokine exerts its functions via interaction with two receptors: type-1 receptor (TNFR1) and type-2 receptor (TNFR2). In this work, the inflammatory response after a freeze injury (cryolesion) in the cortex was studied in wild-type (WT) animals and in mice lacking TNFR1 (TNFR1 KO...... affected by TNFR1 deficiency. Overall, these results suggest that TNFR1 is involved in the early establishment of the inflammatory response and that its deficiency causes a decreased inflammatory response and tissue damage following brain injury....

  17. High-affinity prorenin binding to cardiac man-6-P/IGF-II receptors precedes proteolytic activation to renin

    Saris, Jasper; Derkx, Frans; Bruin, René; Dekkers, Dick; Lamers, Jos; Saxena, Pramod Ranjan; Schalekamp, Maarten; Danser, Jan

    2001-01-01

    textabstractMannose-6-phosphate (man-6-P)/insulin-like growth factor-II (man-6-P/IgF-II) receptors are involved in the activation of recombinant human prorenin by cardiomyocytes. To investigate the kinetics of this process, the nature of activation, the existence of other prorenin receptors, and binding of native prorenin, neonatal rat cardiomyocytes were incubated with recombinant, renal, or amniotic fluid prorenin with or without man-6-P. Intact and activated prorenin were measured in cell ...

  18. Potentiation of nerve growth factor-induced neurite outgrowth by fluvoxamine: role of sigma-1 receptors, IP3 receptors and cellular signaling pathways.

    Tomoko Nishimura

    Full Text Available BACKGROUND: Selective serotonin reuptake inhibitors (SSRIs have been widely used and are a major therapeutic advance in psychopharmacology. However, their pharmacology is quite heterogeneous. The SSRI fluvoxamine, with sigma-1 receptor agonism, is shown to potentiate nerve-growth factor (NGF-induced neurite outgrowth in PC 12 cells. However, the precise cellular and molecular mechanisms underlying potentiation by fluvoxamine are not fully understood. In this study, we examined the roles of cellular signaling pathways in the potentiation of NGF-induced neurite outgrowth by fluvoxamine and sigma-1 receptor agonists. METHODS AND FINDINGS: The effects of three SSRIs (fluvoxamine, sertraline, paroxetine and three sigma-1 receptor agonists (SA4503, 4-phenyl-1-(4-phenylbutyl piperidine (PPBP, and dehydroepiandrosterone (DHEA-sulfate on NGF-induced neurite outgrowth in PC12 cells were examined. Also examined were the effects of the sigma-1 receptor antagonist NE-100, inositol 1,4,5-triphosphate (IP(3 receptor antagonist, and specific inhibitors of signaling pathways in the potentiation of NGF-induced neurite outgrowth by selective sigma-1 receptor agonist SA4503. Fluvoxamine (but not sertraline or paroxetine and the sigma-1 receptor agonists SA4503, PPBP, and DHEA-sulfate significantly potentiated NGF-induced neurite outgrowth in PC12 cells in a concentration-dependent manner. The potentiation by fluvoxamine and the three sigma-1 receptor agonists was blocked by co-administration of the selective sigma-1 receptor antagonist NE-100, suggesting that sigma-1 receptors play a role in blocking the enhancement of NGF-induced neurite outgrowth. Moreover, the potentiation by SA4503 was blocked by co-administration of the IP(3 receptor antagonist xestospongin C. In addition, the specific inhibitors of phospholipase C (PLC-gamma, phosphatidylinositol 3-kinase (PI3K, p38MAPK, c-Jun N-terminal kinase (JNK, and the Ras/Raf/mitogen-activated protein kinase (MAPK

  19. Transcriptional regulation of the human TR2 orphan receptor gene by nuclear factor 1-A

    The human testicular receptor 2 (TR2), a member of the nuclear hormone receptor superfamily, has no identified ligand yet. Previous evidence demonstrated that a 63 bp DNA fragment, named the promoter activating cis-element (PACE), has been identified as a positive regulatory region in the 5' promoter region of the human TR2 gene. In the present report, the human nuclear factor 1-A (NF1-A) was identified as a transcriptional activator to recognize the center of the PACE, called the PACE-C. NF1-A could bind to the 18 bp PACE-C region, and enhance about 13- to 17-fold of the luciferase reporter gene activity via the PACE-C in dose-dependent and orientation-independent manners. This transcriptional activation was further confirmed by real-time RT-PCR assay. In conclusion, our results indicated that NF1-A transcription factor plays an important role in the transcriptional activation of the TR2 gene expression via the PACE-C in the minimal promoter region

  20. Neurotrophic factors and their receptors in human sensory neuropathies.

    Anand, Praveen

    2004-01-01

    Neurotrophic factors may play key roles in pathophysiological mechanisms of human neuropathies. Nerve growth factor (NGF) is trophic to small-diameter sensory fibers and regulates nociception. This review focuses on sensory dysfunction and the potential of neurotrophic treatments. Genetic neuropathy. Mutations of the NGF high-affinity receptor tyrosine kinase A (Trk A) have been found in congenital insensitivity to pain and anhidrosis; these are likely to be partial loss-of-function mutations, as axon-reflex vasodilatation and sweating can be elicited albeit reduced, suggesting rhNGF could restore nociception in some patients. Leprous neuropathy. Decreased NGF in leprosy skin may explain cutaneous hypoalgesia even with inflammation and rhNGF may restore sensation, as spared nerve fibers show Trk A-staining. Diabetic neuropathy. NGF is depleted in early human diabetic neuropathy skin, in correlation with dysfunction of nociceptor fibers. We proposed rhNGF prophylaxis may prevent diabetic foot ulceration. Clinical trials have been disappointed, probably related to difficulty delivering adequate doses and need for multiple trophic factors. NGF and glial cell line-derived neurotrophic factor (GDNF) are both produced by basal keratinocytes and neurotrophin (NT-3) by suprabasal keratinocytes: relative mRNA expression was significantly lower in early diabetic neuropathy skin compared to controls, for NGF (P 0.05). Posttranslational modifications of mature and pro-NGF may also affect bioactivity and immunoreactivity. A 53 kD band that could correspond to a prepro-NGF-like molecule was reduced in diabetic skin. Traumatic neuropathy and pain. While NGF levels are acutely reduced in injured nerve trunks, neuropathic patients with chronic skin hyperalgesia and allodynia show marked local increases of NGF levels; here anti-NGF agents may provide analgesia. Physiological combinations of NGF, NT-3 and GDNF, to mimic a 'surrogate target organ', may provide a novel 'homeostatic

  1. Involvement of Toll-like receptor 2 and epidermal growth factor receptor signaling in epithelial expression of airway remodeling factors.

    Homma, Tetsuya; Kato, Atsushi; Sakashita, Masafumi; Norton, James E; Suh, Lydia A; Carter, Roderick G; Schleimer, Robert P

    2015-04-01

    Staphylococcus aureus (SA) colonization and infection is common, and may promote allergic or inflammatory airway diseases, such as asthma, cystic fibrosis, and chronic rhinosinusitis by interacting with airway epithelial cells. Airway epithelial cells not only comprise a physical barrier, but also play key roles in immune, inflammatory, repair, and remodeling responses upon encounters with pathogens. To elucidate the impact of SA on epithelial-mediated remodeling of allergic airways, we tested the hypothesis that SA can enhance the remodeling process. Normal human bronchial epithelial (NHBE) cells were stimulated with heat-killed SA (HKSA) or transforming growth factor (TGF) α. Cell extracts were collected to measure mRNA (real-time RT-PCR) and signaling molecules (Western blot); supernatants were collected to measure protein (ELISA) after 24 hours of stimulation. Epidermal growth factor receptor (EGFR) signaling inhibition experiments were performed using a specific EGFR kinase inhibitor (AG1478) and TGF-α was blocked with an anti-TGF-α antibody. HKSA induced both mRNA and protein for TGF-α and matrix metalloproteinase (MMP) 1 from NHBE cells by a Toll-like receptor 2-dependent mechanism. Recombinant human TGF-α also induced mRNA and protein for MMP-1 from NHBE cells; anti-TGF-α antibody inhibited HKSA-induced MMP-1, suggesting that endogenous TGF-α mediates the MMP-1 induction by HKSA. HKSA-induced MMP-1 expression was suppressed when a specific EGFR kinase inhibitor was added, suggesting that EGFR signaling was mediating the HKSA-induced MMP-1 release. Exposure or colonization by SA in the airway may enhance the remodeling of tissue through a TGF-α-dependent induction of MMP-1 expression, and may thereby promote remodeling in airway diseases in which SA is implicated, such as asthma and chronic rhinosinusitis. PMID:25180535

  2. Structural mechanism of glutamate receptor activation and desensitization.

    Meyerson, Joel R; Kumar, Janesh; Chittori, Sagar; Rao, Prashant; Pierson, Jason; Bartesaghi, Alberto; Mayer, Mark L; Subramaniam, Sriram

    2014-10-16

    Ionotropic glutamate receptors are ligand-gated ion channels that mediate excitatory synaptic transmission in the vertebrate brain. To gain a better understanding of how structural changes gate ion flux across the membrane, we trapped rat AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) and kainate receptor subtypes in their major functional states and analysed the resulting structures using cryo-electron microscopy. We show that transition to the active state involves a 'corkscrew' motion of the receptor assembly, driven by closure of the ligand-binding domain. Desensitization is accompanied by disruption of the amino-terminal domain tetramer in AMPA, but not kainate, receptors with a two-fold to four-fold symmetry transition in the ligand-binding domains in both subtypes. The 7.6 Å structure of a desensitized kainate receptor shows how these changes accommodate channel closing. These findings integrate previous physiological, biochemical and structural analyses of glutamate receptors and provide a molecular explanation for key steps in receptor gating. PMID:25119039

  3. Conditional, genetic disruption of ciliary neurotrophic factor receptors reveals a role in adult motor neuron survival

    Lee, Nancy; Robitz, Rachel; Zurbrugg, Rebekah J; Karpman, Adam M; Mahler, Ashley M.; Cronier, Samantha A.; Vesey, Rachel; Spearry, Rachel P.; Zolotukhin, Sergei; MacLennan, A. John

    2008-01-01

    Indirect evidence suggests that endogenous ciliary neurotrophic factor (CNTF) receptor signaling can promote motor neuron (MN) survival in the adult. If so, proper targeting of this signaling may selectively counteract the effects of adult MN diseases. However, direct evidence for CNTF receptor involvement in adult MN survival is lacking, presumably because the unconditional blockade of the mouse CNTF receptor in vivo [through genetic disruption of the essential CNTF receptor α (CNTFRα) gene]...

  4. Receptors for B cell stimulatory factor 2. Quantitation, specificity, distribution, and regulation of their expression

    Taga, T.; Kawanishi, Y.; Hardy, R.R.; Hirano, T.; Kishimoto, T.

    1987-10-01

    B cell stimulatory factor 2 receptors (BSF-2-R) were studied using radioiodinated recombinant BSF-2 with a specific activity of 6.16 X 10(13) cpm/g. Kinetic studies showed that binding of /sup 125/I-BSF-2 to CESS cells reached maximum level within 150 min at 0 degrees C. There was a single class of receptors with high affinity (Kd 3.4 X 10(-10) M) on CESS, and the number of receptors was 2700 per cell. Binding of /sup 125/I-BSF-2 to CESS was competitively inhibited by unlabeled BSF-2 but not by IL-1, IL-2, IFN-beta, IFN-gamma, and G-CSF, indicating the presence of the receptors specific for BSF-2. EBV-transformed B lymphoblastoid cell lines (CESS, SKW6-CL4, LCL13, and LCL14) expressed BSF-2-R, whereas Burkitt's lines did not. EBV or EBNA2 did not induce the expression of the receptors on Burkitt's cells. The plasma cell lines (ARH-77 and U266) expressed BSF-2-R, fitting the function of BSF-2 as plasma cell growth factor. Several other cell lines, the histiocytic line U937, the promyelocytic line HL60, the astrocytoma line U373 and the glioblastoma line SK-MG-4, in which BSF-2 was inducible with IL-1 or TPA, displayed BSF-2-R with Kd in the range of 1.3-6.4 X 10(-10) M, suggesting the autocrine mechanism in BSF-2 function. The four T cell lines (CEM, HSB, Jurkat, and OM 1) did not express a detectable number of receptors, but normal resting T cells expressed 100-1000 receptors per cell. BSF-2-R were not present on normal resting B cells but expressed on activated B cells with a Kd of 3.6-5.0 X 10(-10) M, fitting the function of BSF-2, which acts on B cells at the final maturation stage to induce immunoglobulin production.

  5. Endothelial Cells Promote Pigmentation through Endothelin Receptor B Activation.

    Regazzetti, Claire; De Donatis, Gian Marco; Ghorbel, Houda Hammami; Cardot-Leccia, Nathalie; Ambrosetti, Damien; Bahadoran, Philippe; Chignon-Sicard, Bérengère; Lacour, Jean-Philippe; Ballotti, Robert; Mahns, Andre; Passeron, Thierry

    2015-12-01

    Findings of increased vascularization in melasma lesions and hyperpigmentation in acquired bilateral telangiectatic macules suggested a link between pigmentation and vascularization. Using high-magnification digital epiluminescence dermatoscopy, laser confocal microscopy, and histological examination, we showed that benign vascular lesions of the skin have restricted but significant hyperpigmentation compared with the surrounding skin. We then studied the role of microvascular endothelial cells in regulating skin pigmentation using an in vitro co-culture model using endothelial cells and melanocytes. These experiments showed that endothelin 1 released by microvascular endothelial cells induces increased melanogenesis signaling, characterized by microphthalmia-associated transcription factor phosphorylation, and increased tyrosinase and dopachrome tautomerase levels. Immunostaining for endothelin 1 in vascular lesions confirmed the increased expression on the basal layer of the epidermis above small vessels compared with perilesional skin. Endothelin acts through the activation of endothelin receptor B and the mitogen-activated protein kinase, extracellular signal-regulated kinase (ERK)1/2, and p38, to induce melanogenesis. Finally, culturing of reconstructed skin with microvascular endothelial cells led to increased skin pigmentation that could be prevented by inhibiting EDNRB. Taken together these results demonstrated the role of underlying microvascularization in skin pigmentation, a finding that could open new fields of research for regulating physiological pigmentation and for treating pigmentation disorders such as melasma. PMID:26308584

  6. A Peroxisome Proliferator-Activated ReceptorActivator Induces Renal CYP2C23 Activity and Protects from Angiotensin II-Induced Renal Injury

    Muller, Dominik N.; Theuer, Juergen; Shagdarsuren, Erdenechimeg; Kaergel, Eva; Honeck, Horst; Park, Joon-Keun; Markovic , Marija; Barbosa-Sicard, Eduardo; Dechend, Ralf; Wellner, Maren; Kirsch, Torsten; Fiebeler, Anette; Rothe, Michael; Haller, Hermann; Luft, Friedrich C.

    2004-01-01

    Cytochrome P450 (CYP)-dependent arachidonic acid (AA) metabolites are involved in the regulation of renal vascular tone and salt excretion. The epoxygenation product 11,12-epoxyeicosatrienoic acid (EET) is anti-inflammatory and inhibits nuclear factor-κB activation. We tested the hypothesis that the peroxisome proliferator-activated receptor-α-activator fenofibrate (Feno) induces CYP isoforms, AA hydroxylation, and epoxygenation activity, and protects against inflammatory organ damage. Double...

  7. Abelson tyrosine kinase links PDGFbeta receptor activation to cytoskeletal regulation of NMDA receptors in CA1 hippocampal neurons

    Beazely Michael A

    2008-12-01

    Full Text Available Abstract Background We have previously demonstrated that PDGF receptor activation indirectly inhibits N-methyl-D-aspartate (NMDA currents by modifying the cytoskeleton. PDGF receptor ligand is also neuroprotective in hippocampal slices and cultured neurons. PDGF receptors are tyrosine kinases that control a variety of signal transduction pathways including those mediated by PLCγ. In fibroblasts Src and another non-receptor tyrosine kinase, Abelson kinase (Abl, control PDGF receptor regulation of cytoskeletal dynamics. The mechanism whereby PDGF receptor regulates cytoskeletal dynamics in central neurons remains poorly understood. Results Intracellular applications of active Abl, but not heat-inactivated Abl, decreased NMDA-evoked currents in isolated hippocampal neurons. This mimics the effects of PDGF receptor activation in these neurons. The Abl kinase inhibitor, STI571, blocked the inhibition of NMDA currents by Abl. We demonstrate that PDGF receptors can activate Abl kinase in hippocampal neurons via mechanisms similar to those observed previously in fibroblasts. Furthermore, PDGFβ receptor activation alters the subcellular localization of Abl. Abl kinase is linked to actin cytoskeletal dynamics in many systems. We show that the inhibition of NMDA receptor currents by Abl kinase is blocked by the inclusion of the Rho kinase inhibitor, Y-27632, and that activation of Abl correlates with an increase in ROCK tyrosine phosphorylation. Conclusion This study demonstrates that PDGFβ receptors act via an interaction with Abl kinase and Rho kinase to regulated cytoskeletal regulation of NMDA receptor channels in CA1 pyramidal neurons.

  8. Atrial natriuretic factor R1 receptor from bovine adrenal zona glomerulosa: Purification, characterization, and modulation by amiloride

    The atrial natriuretic factor (ANF) R1 receptor from bovine adrenal zona glomerulosa was solubilized with Triton X-100 and purified 13,000-fold, to apparent homogeneity, by sequential affinity chromatography on ANF-agarose and steric exclusion high-performance liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of the purified receptor preparation in the absence or presence of dithiothreitol revealed a single protein band of Mr 130,000. Affinity cross-linking of 125I-ANF to the purified receptor resulted in the labeling of the Mr 130,000 band. The purified receptor bound ANF with a specific activity of 6.8 nmol/mg of protein, corresponding to a stoichiometry of 0.9 mol of ANF bound/mol of Mr 130,000 polypeptide. Starting with 500 g of adrenal zona glomerulosa tissue, the authors obtained more than 500 pmol of purified receptor with an overall yield of 9%. The purified receptor showed a typical ANF-R1 pharmacological specificity similar to that of the membrane-bound receptor. The homogeneous Mr 130,000 receptor protein displayed high guanylate cyclase activity [1.4 μmol of cyclic GMP formed min-1 (mg of protein)-1] which was not stimulated by ANF. This finding supports the notion that the ANF binding and the guanylate cyclase activities are intrinsic components of the same polypeptide. Finally, the purified ANF-R1 receptor retained its sensitivity to modulation by amiloride, suggesting the presence of an allosteric binding site for amiloride on the receptor protein

  9. Identification of a novel immunoreceptor tyrosine-based activation motif-containing molecule, STAM2, by mass spectrometry and its involvement in growth factor and cytokine receptor signaling pathways

    Pandey, A; Fernandez, M M; Steen, H;

    2000-01-01

    molecule containing a Src homology 3 domain as well as an immunoreceptor tyrosine-based activation motif (ITAM). This molecule is 55% identical to a previously isolated molecule designated signal transducing adaptor molecule (STAM) that was identified as an interleukin (IL)-2-induced phosphoprotein and is...... therefore designated STAM2. Tyrosine phosphorylation of STAM2 is induced by growth factors such as epidermal growth factor and platelet-derived growth factor as well as by cytokines like IL-3. Several of the deletion mutants tested except the one containing only the amino-terminal region underwent tyrosine...... phosphorylation upon growth factor stimulation, implying that STAM2 is phosphorylated on several tyrosine residues. STAM2 is downstream of the Jak family of kinases since coexpression of STAM2 with Jak1 or Jak2 but not an unrelated Tec family kinase, Etk, resulted in its tyrosine phosphorylation. In contrast to...

  10. Cloning and expression of cDNAs for two distinct murine tumor necrosis factor receptors demonstrate one receptor is species specific

    Complementary DNA clones encoding two distinct tumor necrosis factor receptors were isolated from a mouse macrophage cNDA library. The cDNA for murine tumor necrosis factor receptor type 1 (mTNF-R1) predicts a mature polypeptide of 425 amino acids that is 64% identical to its human counterpart, whereas the cDNA of murine tumor necrosis factor receptor type 2 (mTNF-R2) predicts a mature protein of 452 amino acids that is 62% identical to human tumor necrosis factor receptor type 2. The two murine tumor necrosis factor receptors have limited sequence homology (∼20% identity) in their extracellular regions but no apparent similarity in their cytoplasmic portions. Northern (RNA) analysis indicates a single 2.6-kilobase (kb) transcript for mTNF-R1; a 3.6-kb and a more predominant 4.5-kb transcript are observed for mTNF-R2. A human cell line transfected with either mTNF-R1 or mTNF-R2 expression vectors specifically bound 125I-labeled recombinant murine tumor necrosis factor α (TNF-α). Although mTNF-R1 had a similar affinity for both recombinant murine TNF-α and human TNF-α, mRNF-R2 showed strong specificity for recombinant murine TNF-α. This result suggest that the various activities of human tumor necrosis factor α reported in mice or in murine cell lines are probably mediated by mTNF-R1

  11. Epidermis-type lipoxygenase 3 regulates adipocyte differentiation and peroxisome proliferator-activated receptor gamma activity

    Hallenborg, Philip; Jørgensen, Claus; Petersen, Rasmus K;

    2010-01-01

    The nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR gamma) is essential for adipogenesis. Although several fatty acids and their derivatives are known to bind and activate PPAR gamma, the nature of the endogenous ligand(s) promoting the early stages of adipocyte differenti...

  12. Molecular Recognition of Corticotropin releasing Factor by Its G protein-coupled Receptor CRFR1

    Pioszak, Augen A.; Parker, Naomi R.; Suino-Powell, Kelly; Xu, H. Eric (Van Andel)

    2009-01-15

    The bimolecular interaction between corticotropin-releasing factor (CRF), a neuropeptide, and its type 1 receptor (CRFR1), a class B G-protein-coupled receptor (GPCR), is crucial for activation of the hypothalamic-pituitary-adrenal axis in response to stress, and has been a target of intense drug design for the treatment of anxiety, depression, and related disorders. As a class B GPCR, CRFR1 contains an N-terminal extracellular domain (ECD) that provides the primary ligand binding determinants. Here we present three crystal structures of the human CRFR1 ECD, one in a ligand-free form and two in distinct CRF-bound states. The CRFR1 ECD adopts the alpha-beta-betaalpha fold observed for other class B GPCR ECDs, but the N-terminal alpha-helix is significantly shorter and does not contact CRF. CRF adopts a continuous alpha-helix that docks in a hydrophobic surface of the ECD that is distinct from the peptide-binding site of other class B GPCRs, thereby providing a basis for the specificity of ligand recognition between CRFR1 and other class B GPCRs. The binding of CRF is accompanied by clamp-like conformational changes of two loops of the receptor that anchor the CRF C terminus, including the C-terminal amide group. These structural studies provide a molecular framework for understanding peptide binding and specificity by the CRF receptors as well as a template for designing potent and selective CRFR1 antagonists for therapeutic applications.

  13. Improved Insulin Resistance and Lipid Metabolism by Cinnamon Extract through Activation of Peroxisome Proliferator-Activated Receptors

    Xiaoyan Sheng; Yuebo Zhang; Zhenwei Gong; Cheng Huang; Ying Qin Zang

    2008-01-01

    Peroxisome proliferator-activated receptors (PPARs) are transcriptional factors involved in the regulation of insulin resistance and adipogenesis. Cinnamon, a widely used spice in food preparation and traditional antidiabetic remedy, is found to activate PPARγ and α, resulting in improved insulin resistance, reduced fasted glucose, FFA, LDL-c, and AST levels in high-caloric diet-induced obesity (DIO) and db/db mice in its water extract form. In vitro studies demonstrate that cinnamon increase...

  14. Tumor-Suppressive Activity of Lunatic Fringe in Prostate through Differential Modulation of Notch Receptor Activation

    Shubing Zhang

    2014-02-01

    Full Text Available Elevated Notch ligand and receptor expression has been associated with aggressive forms of prostate cancer, suggesting a role for Notch signaling in regulation of prostate tumor initiation and progression. Here, we report a critical role for Lunatic Fringe (Lfng, which encodes an O-fucosylpeptide 3-ß-N-acetylglucosaminyltransferase known to modify epidermal growth factor repeats of Notch receptor proteins, in regulation of prostate epithelial differentiation and proliferation, as well as in prostate tumor suppression. Deletion of Lfng in mice caused altered Notch activation in the prostate, associated with elevated accumulation of Notch1, Notch2, and Notch4 intracellular domains, decreased levels of the putative Notch3 intracellular fragment, as well as increased expression of Hes1, Hes5, and Hey2. Loss of Lfng resulted in expansion of the basal layer, increased proliferation of both luminal and basal cells, and ultimately, prostatic intraepithelial neoplasia. The Lfng-null prostate showed down-regulation of prostatic tumor suppressor gene NKX3.1 and increased androgen receptor expression. Interestingly, expression of LFNG and NKX3.1 were positively correlated in publically available human prostate cancer data sets. Knockdown of LFNG in DU-145 prostate cancer cells led to expansion of CD44+CD24− and CD49f+CD24− stem/progenitor-like cell population associated with enhanced prostatosphere-forming capacity. Taken together, these data revealed a tumor-suppressive role for Lfng in the prostate through differential regulation of Notch signaling.

  15. 15-Deoxy-Δ12,14-prostaglandin J2 and thiazolidinediones transactivate epidermal growth factor and platelet-derived growth factor receptors in vascular smooth muscle cells

    Proliferation of vascular smooth muscle cells (VSMCs) is induced by various mitogens through activation of extracellular signal-regulated protein kinase (ERK) pathway. We recently reported that peroxisome proliferator-activated receptor (PPAR)γ activators such as 15-deoxy-Δ12,14-prostaglandin J2 (15-d-PGJ2) and thiazolidinediones (TZDs) activated MEK/ERK pathway through phosphatidylinositol 3-kinase (PI3-K) and induced proliferation of VSMCs. However, the precise mechanisms of PPARγ activators-induced activation of PI3-K/ERK pathway have not been determined. We examined whether transactivation of growth factor receptor is involved in this process. Stimulation of VSMCs with 15-d-PGJ2 or TZDs for 15 min induced phosphorylation of ERK1/2 and Akt. 15-d-PGJ2- or TZDs-induced phosphorylation of ERK1/2 and Akt was inhibited by AG1478, an inhibitor of epidermal growth factor receptor (EGF-R) as well as AG1295, an inhibitor of platelet derived growth factor receptor (PDGF-R). 15-d-PGJ2-induced phosphorylation of both EGF-R and PDGF-R. GM6001, a matrix metalloproteinase inhibitor, and PP2, a Src family protein kinase inhibitor, suppressed 15-d-PGJ2- and TZDs-induced phosphorylation of EGF-R and PDGFβ-R as well as activation of ERK1/2 and Akt. PDGFβ-R was co-immunoprecipitated with EGF-R, regardless of the presence or absence of 15-d-PGJ2. These data suggest that 15-d-PGJ2 and TZDs activate PI3-K/ERK pathway through Src family kinase- and matrix metalloproteinase-dependent transactivation of EGF-R and PDGF-R. Both receptors seemed to associate constitutively. This novel signaling mechanisms may contribute to diverse biological functions of PPARγ activators

  16. Nature and regulation of the receptors for insulin-like growth factors

    Two subtypes of IGF receptors have been identified. Type I IGF receptors have a Mr greater than 300,000 and are composed of disulfide-linked 130,000-dalton (alpha) and approximately 90,000-dalton (beta) subunits. Type I receptors preferentially bind IGF-I but also bind IGF-II and, more weakly, insulin. Type II IGF receptors consist of a 250,000-dalton protein that contains internal disulfide bonds but is not linked to other membrane components. Type II receptors bind IGF-II with higher affinity than IGF-I. They do not interact with even very high concentrations of insulin. Type I IGF receptors and insulin receptors are homologous structures. Type II IGF receptors do not appear to be homologous to type I receptors. Type II receptors do not appear to be downregulated. Insulin acutely upregulates type II IGF receptors in intact rat adipose cells by effecting a redistribution of receptors cycling between a large intracellular pool and the plasma membrane. Insulin and the IGFs elicit the same biological responses, either by cross-reacting with one of the receptors for the heterologous ligand or by concurrent activation of convergent effector pathways by binding to the homologous receptor. Which mechanism is utilized appears to depend more on the tissue than on the biological response. Insulin desensitizes rat hepatoma cells to the actions of insulin and IGFs, mediated by both insulin and IGF receptors, by mechanisms distal to hormone binding and possibly common to IGF and insulin effector pathways

  17. A Premature Termination of Human Epidermal Growth Factor Receptor Transcription in Escherichia coli

    Jihene Elloumi-Mseddi

    2014-01-01

    Full Text Available Our success in producing an active epidermal growth factor receptor (EGFR tyrosine kinase in Escherichia coli encouraged us to express the full-length receptor in the same host. Despite its large size, we were successful at producing the full-length EGFR protein fused to glutathione S-transferase (GST that was detected by Western blot analysis. Moreover, we obtained a majoritarian truncated GST-EGFR form detectable by gel electrophoresis and Western blot. This truncated protein was purified and confirmed by MALDI-TOF/TOF analysis to belong to the N-terminal extracellular region of the EGFR fused to GST. Northern blot analysis showed two transcripts suggesting the occurrence of a transcriptional arrest.

  18. Characterization of human interleukin-3 receptors on a multi-factor-dependent cell line

    Recombinant human interleukin-3 (hIL-3) was radioiodinated by Bolton-Hunter method with maintenance of biological activity. Using 125I-hIL-3, hIL-3 receptors were characterized on a multi-factor-dependent cell line TF-1. Equilibrium binding studies revealed the existence of a single class of binding sites (667 +/- 306 sites/cell) with a Kd of 173 +/- 25 pM. Affinity labeling of TF-1 cells with 125I-IL-3 yielded two bands of 150 kDa and 85 kDa, implying molecular weights of 135 kDa and 70 kDa for the hIL-3 receptors

  19. New GABA amides activating GABA A-receptors

    Peter Raster; Andreas Späth; Svetlana Bultakova; Pau Gorostiza; Burkhard König; Piotr Bregestovski

    2013-01-01

    We have prepared a series of new and some literature-reported GABA-amides and determined their effect on the activation of GABAA-receptors expressed in CHO cells. Special attention was paid to the purification of the target compounds to remove even traces of GABA contaminations, which may arise from deprotection steps in the synthesis. GABA-amides were previously reported to be partial, full or superagonists. In our hands these compounds were not able to activate GABAA-receptor channels in wh...

  20. Glutamate receptor antagonists and growth factors modulate dentate granule cell neurogenesis in organotypic, rat hippocampal slice cultures

    Poulsen, Frantz Rom; Blaabjerg, Morten; Montero, Maria;

    2005-01-01

    Generation of dentate granule cells and its modulation by glutamate receptor antagonists, growth factors and pilocarpine-induced seizure-like activity was investigated in rat hippocampal slice cultures derived from 1-week-old rats and grown for 2 weeks. Focussing on the dentate granule cell layer...

  1. Development of real-time reverse transcription polymerase chain reaction assays to quantify insulin-like growth factor receptor and insulin receptor expression in equine tissue

    Stephen B. Hughes

    2013-03-01

    Full Text Available The insulin-like growth factor system (insulin-like growth factor 1, insulin-like growth factor 2, insulin-like growth factor 1 receptor, insulin-like growth factor 2 receptor and six insulin-like growth factor-binding proteins and insulin are essential to muscle metabolism and most aspects of male and female reproduction. Insulin-like growth factor and insulin play important roles in the regulation of cell growth, differentiation and the maintenance of cell differentiation in mammals. In order to better understand the local factors that regulate equine physiology, such as muscle metabolism and reproduction (e.g., germ cell development and fertilisation, real-time reverse transcription polymerase chain reaction assays for quantification of equine insulin-like growth factor 1 receptor and insulin receptor messenger ribonucleic acid were developed. The assays were sensitive: 192 copies/µLand 891 copies/µL for insulin-like growth factor 1 receptor, messenger ribonucleic acid and insulin receptor respectively (95%limit of detection, and efficient: 1.01 for the insulin-like growth factor 1 receptor assay and 0.95 for the insulin receptor assay. The assays had a broad linear range of detection (seven logs for insulin-like growth factor 1 receptor and six logs for insulin receptor. This allowed for analysis of very small amounts of messenger ribonucleic acid. Low concentrations of both insulin-like growth factor 1 receptor and insulin receptor messenger ribonucleic acid were detected in endometrium, lung and spleen samples, whilst high concentrations were detected in heart, muscle and kidney samples, this was most likely due to the high level of glucose metabolism and glucose utilisation by these tissues. The assays developed for insulin-like growth factor 1 receptor and insulin receptor messenger ribonucleic acid expression have been shown to work on equine tissue and will contribute to the understanding of insulin and insulin-like growth factor 1

  2. Correlation between hormone dependency and the regulation of epidermal growth factor receptor by tumor promoters in human mammary carcinoma cells

    The effects of the tumor promoter phorbol 12-tetradecanoate 13-acetate (TPA) on the epidermal growth factor (EGF) receptor levels were investigated in hormone-dependent (MCF-7, T-47-D, and ZR-75-1) and hormone-independent (MDA-MB-231, HBL-100, and BT-20) human mammary carcinoma cell lines. In the absence of TPA, hormone-independent cell lines contained high concentrations of low-affinity EGF receptors, whereas hormone-dependent cell lines exhibited low concentrations of high-affinity receptors. TPA causes a change of the receptor from a high- to the low-affinity state in hormone-dependent cell lines, as well as in the hormone-independent HBL-100, whereas the affinity remained unchanged in MDA-MB-231 and BT-20 cells. Tumor promoters such as TPA or teleocidin inhibited the proliferation of these cell lines at concentrations above 10 μM with the exception of the T-47-D cells. Evaluation of different TPA analogs indicated a positive correlation between the growth-inhibitory effects and their ability to stimulate the subcellular redistribution of protein kinase C activity in MCF-7 cells. These data suggest a protein kinase C-mediated down-regulation of the progesterone receptor concentration and of the EGF receptor affinity, which is supposed to mediate the mitogenic response. Furthermore, these results support the hypothesis that the tumor-derived growth factors induced by estradiol act via the EGF receptor in hormone-dependent mammary carcinoma cells

  3. Epidermal growth factor receptor cooperates with Src family kinases in acquired resistance to cetuximab.

    Wheeler, Deric L; Iida, Mari; Kruser, Tim J; Nechrebecki, Meghan M; Dunn, Emily F; Armstrong, Eric A; Huang, Shyhmin; Harari, Paul M

    2009-04-01

    The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that plays a major role in oncogenesis. Cetuximab is an EGFR-blocking antibody that is FDA approved for use in patients with metastatic colorectal cancer (mCRC) and head and neck squamous cell carcinoma (HNSCC). Although cetuximab has shown strong clinical benefit for a subset of cancer patients, most become refractory to cetuximab therapy. We reported that cetuximab-resistant NSCLC line NCI-H226 cells have increased steady-state expression and activity of EGFR secondary to altered trafficking/degradation and this increase in EGFR expression and activity lead to hyper-activation of HER3 and down stream signals to survival. We now present data that Src family kinases (SFKs) are highly activated in cetuximab-resistant cells and enhance EGFR activation of HER3 and PI(3)K/Akt. Studies using the Src kinase inhibitor dasatinib decreased HER3 and PI(3)K/Akt activity. In addition, cetuximab-resistant cells were resensitized to cetuximab when treated with dasatinib. These results indicate that SFKs and EGFR cooperate in acquired resistance to cetuximab and suggest a rationale for clinical strategies that investigate combinatorial therapy directed at both the EGFR and SFKs in patients with acquired resistance to cetuximab. PMID:19276677

  4. Calcium-sensing receptor activation depresses synaptic transmission

    Phillips, Cecilia G.; Harnett, Mark T.; Chen, Wenyan; Smith, Stephen M.

    2008-01-01

    At excitatory synapses, decreases in cleft [Ca] arising from activity-dependent transmembrane Ca flux reduce the probability of subsequent transmitter release. Intense neural activity, induced by physiological and pathological stimuli, disturb the external microenvironment reducing extracellular [Ca] ([Ca]o) and thus may impair neurotransmission. Increases in [Ca]o activate the extracellular calcium sensing receptor (CaSR) which in turn inhibits non-selective cation channels (NSCC) at the maj...

  5. Kallikrein activates bradykinin B2 receptors in absence of kininogen.

    Biyashev, Dauren; Tan, Fulong; Chen, Zhenlong; Zhang, Kai; Deddish, Peter A; Erdös, Ervin G; Hecquet, Claudie

    2006-03-01

    Kallikreins cleave plasma kininogens to release the bioactive peptides bradykinin (BK) or kallidin (Lys-BK). These peptides then activate widely disseminated B2 receptors with consequences that may be either noxious or beneficial. We used cultured cells to show that kallikrein can bypass kinin release to activate BK B2 receptors directly. To exclude intermediate kinin release or kininogen uptake from the cultured medium, we cultured and maintained cells in medium entirely free of animal proteins. We compared the responses of stably transfected Chinese hamster ovary (CHO) cells that express human B2 receptors (CHO B2) and cells that coexpress angiotensin I-converting enzyme (ACE) as well (CHO AB). We found that BK (1 nM or more) and tissue kallikrein (1-10 nM) both significantly increased release of arachidonic acid beyond unstimulated baseline level. An enzyme-linked immunoassay for kinin established that kallikrein did not release a kinin from CHO cells. We confirmed the absence of kininogen mRNA with RT-PCR to rule out kininogen synthesis by CHO cells. We next tested an ACE inhibitor for enhanced BK receptor activation in the absence of kinin release and synthesized an ACE-resistant BK analog as a control for these experiments. Enalaprilat (1 microM) potentiated kallikrein (100 nM) in CHO AB cells but was ineffective in CHO B2 cells that do not bear ACE. We concluded that kallikrein activated B2 receptors without releasing a kinin. Furthermore, inhibition of ACE enhanced the receptor activation by kallikrein, an action that may contribute to the manifold therapeutic effects of ACE inhibitors. PMID:16272198

  6. Allosteric activation mechanism of the cys-loop receptors

    Yong-chang CHANG; Wen WU; Jian-liang ZHANG; Yao HUANG

    2009-01-01

    Binding of a neurotransmitter to its ionotropic receptor opens a distantly located ion channel, a process termed allosteric activation. Here we review recent advances in the molecular mechanism by which the cys-loop receptors are activated with emphasis on the best studied nicotinic acetylcholine receptors (nAChRs). With a combination of affinity labeling, mutagenesis, electrophysiology, kinetic modeling, electron microscopy (EM), and crystal structure analysis, the allosteric activation mechanism is emerging. Specifically, the binding domain and gating domain are interconnected by an allosteric activation network. Agonist binding induces conformational changes, resulting in the rotation of a β sheet of amino-terminal domain and outward movement of loop 2, loop F, and cys-loop, which are coupled to the M2-M3 linker to pull the channel to open. However, there are still some controversies about the movement of the channel-lining domain M2. Nine angstrom resolution EM structure of a nAChR imaged in the open state suggests that channel opening is the result of rotation of the M2 domain. In contrast, recent crystal structures of bacterial homologues of the cys-loop receptor family in apparently open state have implied an M2 tilting model with pore dilation and quaternary twist of the whole pentameric receptor. An elegant study of the nAChR using protonation scanning of M2 domain supports a similar pore dilation activation mechanism with minimal rotation of M2. This remains to be validated with other approaches including high resolution structure determination of the mammalian cys-loop receptors in the open state.

  7. The role of tumour necrosis factor alpha and soluble tumour necrosis factor alpha receptors in the symptomatology of schizophrenia.

    Turhan, Levent; Batmaz, Sedat; Kocbiyik, Sibel; Soygur, Arif Haldun

    2016-07-01

    Background Immunological mechanisms may be responsible for the development and maintenance of schizophrenia symptoms. Aim The aim of this study is to measure tumour necrosis factor-alpha (TNF-α), soluble tumour necrosis factor-alpha receptor I (sTNF-αRI), and soluble tumour necrosis factor-alpha receptor II (sTNF-αRII) levels in patients with schizophrenia and healthy individuals, and to determine their relationship with the symptoms of schizophrenia. Methods Serum TNF-α, sTNF-αRI and sTNF-αRII levels were measured. The Positive and Negative Syndrome Scale (PANSS) was administered for patients with schizophrenia (n = 35), and the results were compared with healthy controls (n = 30). Hierarchical regression analyses were undertaken to predict the levels of TNF-α, sTNF-αRI and sTNF-αRII. Results No significant difference was observed in TNF-α levels, but sTNF-αRI and sTNF-αRII levels were lower in patients with schizophrenia. Serum sTNF-αRI and sTNF-αRII levels were found to be negatively correlated with the negative subscale score of the PANSS, and sTNF-αRI levels were also negatively correlated with the total score of the PANSS. Smoking, gender, body mass index were not correlated with TNF-α and sTNF-α receptor levels. Conclusions These results suggest that there may be a change in anti-inflammatory response in patients with schizophrenia due to sTNF-αRI and sTNF-αRII levels. The study also supports low levels of TNF activity in schizophrenia patients with negative symptoms. PMID:26754110

  8. Reduced expression of epidermal growth factor receptor related protein in gastric cancer

    Moon, W. S.; Tarnawski, A S; Chai, J.; Yang, J. T.; Majumdar, A.P.N.

    2005-01-01

    Objectives: The recently cloned epidermal growth factor receptor related protein (ERRP) has been proposed to be a negative regulator of the epidermal growth factor receptor (EGFR). Because of the causal involvement of EGFR and its ligands in gastric cancer growth, we investigated expression of ERRP and cell proliferation in human gastric cancer.

  9. Localization of the gene for the ciliary neutrotrophic factor receptor (CNTFR) to human chromosome 9

    Donaldson, D.H.; Jones, C.; Patterson, D. (Eleanor Roosevelt Institute, Denver, CO (United States) Univ. of Colorado Health Science Center, Denver, CO (United States)); Britt, D.E.; Jackson, C.L. (Brown Univ., Providence, RI (United States))

    1993-09-01

    Ciliary neurotrophic factor (CNTF) has recently been found to be important for the survival of motor neurons and has shown activity in animal models of amyotrophic lateral sclerosis (ALS). CNTF therefore holds promise as a treatment for ALS, and it and its receptor (CNTFR) are candidates for a gene involved in familial ALS. The CNTFR gene was mapped to chromosome 9 by PCR on a panel of human/CHO somatic cell hybrids and localized to 9p13 by PCR on a panel of radiation hybrids. 18 ref., 1 fig., 2 tabs.

  10. All-Atom Structural Models of the Transmembrane Domains of Insulin and Type 1 Insulin-Like Growth Factor Receptors

    Mohammadiarani, Hossein; Vashisth, Harish

    2016-01-01

    The receptor tyrosine kinase superfamily comprises many cell-surface receptors including the insulin receptor (IR) and type 1 insulin-like growth factor receptor (IGF1R) that are constitutively homodimeric transmembrane glycoproteins. Therefore, these receptors require ligand-triggered domain rearrangements rather than receptor dimerization for activation. Specifically, binding of peptide ligands to receptor ectodomains transduces signals across the transmembrane domains for trans-autophosphorylation in cytoplasmic kinase domains. The molecular details of these processes are poorly understood in part due to the absence of structures of full-length receptors. Using MD simulations and enhanced conformational sampling algorithms, we present all-atom structural models of peptides containing 51 residues from the transmembrane and juxtamembrane regions of IR and IGF1R. In our models, the transmembrane regions of both receptors adopt helical conformations with kinks at Pro961 (IR) and Pro941 (IGF1R), but the C-terminal residues corresponding to the juxtamembrane region of each receptor adopt unfolded and flexible conformations in IR as opposed to a helix in IGF1R. We also observe that the N-terminal residues in IR form a kinked-helix sitting at the membrane–solvent interface, while homologous residues in IGF1R are unfolded and flexible. These conformational differences result in a larger tilt-angle of the membrane-embedded helix in IGF1R in comparison to IR to compensate for interactions with water molecules at the membrane–solvent interfaces. Our metastable/stable states for the transmembrane domain of IR, observed in a lipid bilayer, are consistent with a known NMR structure of this domain determined in detergent micelles, and similar states in IGF1R are consistent with a previously reported model of the dimerized transmembrane domains of IGF1R. Our all-atom structural models suggest potentially unique structural organization of kinase domains in each receptor. PMID

  11. Cytokines and growth factors modify the upregulation of contractile endothelin ET(A) and ET(B) receptors in rat cerebral arteries after organ culture

    Ahnstedt, H; Stenman, E; Cao, L; Henriksson, M; Edvinsson, L

    2012-01-01

    Experimental cerebral ischaemia and organ culture of cerebral arteries induce an increased endothelin ET(B) receptor-mediated contraction. The aim of this study was to examine whether cytokines and growth factors, known to be activated in ischaemia, can influence the expression and function of...... endothelin receptors after organ culture....

  12. Inhibitory effects of sigma-2 receptor agonists on T lymphocyte activation

    MANUELFRESNO

    2013-03-01

    Full Text Available Sigma (σ receptor ligands are essentially known for their effects on the nervous system although recent studies have shown their potential effects modulating some other pathophysiological processes as cell proliferation, cancer and the immune response. Here, we have analyzed the actions of σ-1 and σ-2 receptors ligands on T cell activation. Our results show that treatment of Jurkat T cells with σ-2 agonists decreased the induction of the expression of Interleukin (IL-2, Tumor necrosis factor (TNF-α and Cyclooxygenase (COX-2 by activated T cells in a dose–dependent manner. These effects take place at the transcriptional level since σ-2 agonists BD-737 and CB-184 diminished the activity of the promoters of those genes. Those immunosuppressive effects could be attributable to interference with transcription factor activation. Induced transcription mediated by Nuclear factor (NF-κB or Nuclear Factor of Activated T cells (NFAT was inhibited by σ-2 agonists. These effects seem to be specific for σ-2 agonists as no significant effects on T cell activation by σ-1 ligands PRE-084 and BD-1063 were found. Our results provide new insights into the immunomodulatory actions of σ ligands and describe a new property of σ-2 agonists, through inhibition of activation of transcription factors as NFAT by which these compounds are regulating gene expression. This may have important consequences on the possible therapeutic use of those compounds.

  13. Nerve Growth Factor Is Regulated by Toll-Like Receptor 2 in Human Intervertebral Discs.

    Krock, Emerson; Currie, J Brooke; Weber, Michael H; Ouellet, Jean A; Stone, Laura S; Rosenzweig, Derek H; Haglund, Lisbet

    2016-02-12

    Nerve growth factor (NGF) contributes to the development of chronic pain associated with degenerative connective tissue pathologies, such as intervertebral disc degeneration and osteoarthritis. However, surprisingly little is known about the regulation of NGF in these conditions. Toll-like receptors (TLR) are pattern recognition receptors classically associated with innate immunity but more recently were found to be activated by endogenous alarmins such as fragmented extracellular matrix proteins found in degenerating discs or cartilage. In this study we investigated if TLR activation regulates NGF and which signaling mechanisms control this response in intervertebral discs. TLR2 agonists, TLR4 agonists, or IL-1β (control) treatment increased NGF, brain-derived neurotrophic factor (BDNF), and IL-1β gene expression in human disc cells isolated from healthy, pain-free organ donors. However, only TLR2 activation or IL-1β treatment increased NGF protein secretion. TLR2 activation increased p38, ERK1/2, and p65 activity and increased p65 translocation to the cell nucleus. JNK activity was not affected by TLR2 activation. Inhibition of NF-κB, and to a lesser extent p38, but not ERK1/2 activity, blocked TLR2-driven NGF up-regulation at both the transcript and protein levels. These results provide a novel mechanism of NGF regulation in the intervertebral disc and potentially other pathogenic connective tissues. TLR2 and NF-κB signaling are known to increase cytokines and proteases, which accelerate matrix degradation. Therefore, TLR2 or NF-κB inhibition may both attenuate chronic pain and slow the degenerative progress in vivo. PMID:26668319

  14. Production of neutralizing monoclonal antibody against human vascular endothelial growth factor receptor

    Rong LI; Dong-sheng XIONG; Xiao-feng SHAO; Jia LIU; Yuan-fu XU; Yuan-sheng XU; Han-zhi LIU; Zhen-ping ZHU; Chun-zheng YANG

    2004-01-01

    AIM: To prepare neutralizing monoclonal antibody (mAb) against extracellular immunoglobulin (Ig)-like domainⅢ of vascular endothelial growth factor receptor KDR and study its biological activity. METHODS: Soluble KDR Ig domain Ⅲ (KDR-Ⅲ) fusion protein was expressed in E Coli and purified from the bacterial periplasmic extracts via an affinity chromatography. Monoclonal antibodies against KDR-Ⅲ were prepared by hybridoma technique. ELISA and FACS analysis were used to identify its specificity. Immunoprecipitation and [3H]-thymidine incorporation assay were also used to detect the activity of anti-KDR mAb blocking the phosphorylation of KDR tyrosine kinase receptor and the influence on vascular endothelial growth factor-induced mitogenesis of human endothelial ceils.RESULTS: A monoclonal antibody, Ycom1D3 (IgG1), was generated from a mouse immunized with the recombinant KDR-Ⅲ protein. Ycom1D3 bound specifically to both the soluble KDR-Ⅲ and the cell-surface expressed KDR. Ycom1D3 effectively blocked VEGF/KDR interaction and inhibited VEGF-stimulated KDR activation in human endothelial cells. Furthermore, the antibody efficiently neutralized VEGF-induced mitogenesis of human endothelial cells. CONCLUSION: Our results suggest that the anti-KDR mAb, Ycom1D3, has potential applications in the treatment of cancer and other diseases where pathological angiogenesis is involved.

  15. Peroxisome proliferator-activated receptoractivation and excess energy burning in hepatocarcinogenesis.

    Misra, Parimal; Reddy, Janardan K

    2014-03-01

    Peroxisome proliferator-activated receptor-α (PPARα) modulates the activities of all three interlinked hepatic fatty acid oxidation systems, namely mitochondrial and peroxisomal β-oxidation and microsomal ω-oxidation pathways. Hyperactivation of PPARα, by both exogenous and endogenous activators up-regulates hepatic fatty acid oxidation resulting in excess energy burning in liver contributing to the development of liver cancer in rodents. Sustained PPARα signaling disproportionately increases H2O2-generating fatty acid metabolizing enzymes as compared to H2O2-degrading enzymes in liver leading to enhanced generation of DNA damaging reactive oxygen species, progressive endoplasmic reticulum stress and inflammation. These alterations also contribute to increased liver cell proliferation with changes in apoptosis. Thus, reactive oxygen species, oxidative stress and hepatocellular proliferation are likely the main contributing factors in the pathogenesis of hepatocarcinogenesis, mediated by sustained PPARα activation-related energy burning in liver. Furthermore, the transcriptional co-activator Med1, a key subunit of the Mediator complex, is essential for PPARα signaling in that both PPARα-null and Med1-null hepatocytes are unresponsive to PPARα activators and fail to give rise to liver tumors when chronically exposed to PPARα activators. PMID:24291192

  16. Development of a Selective Modulator of Aryl Hydrocarbon (Ah) Receptor Activity that Exhibits Anti-Inflammatory Properties

    Murray, Iain A.; KRISHNEGOWDA, GOWDAHALLI; DiNatale, Brett C.; Flaveny, Colin; Chiaro, Chris; Lin, Jyh-Ming; Arun K. Sharma; Amin, Shantu; Perdew, Gary H.

    2010-01-01

    The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin. However, the role of the AHR in normal physiology is still an area of intense investigation. For example, this receptor plays an important role in certain immune responses. We have previously determined that the AHR can mediate repression of acute-phase genes in the liver. For this observation to be therapeutically useful, selective activation of th...

  17. Interaction of the C-terminal acidic domain of the insulin receptor with histone modulates the receptor kinase activity.

    Baron, V; Kaliman, P; Alengrin, F; Van Obberghen, E

    1995-04-01

    In this study, we investigated the role of the insulin receptor domain 1270-1280, an acid-rich sequence located in the receptor C-terminus. Antipeptide IgG raised against this sequence were obtained and used to analyze their effect on receptor function. Antipeptide IgG inhibited receptor autophosphorylation at Tyr1146, Tyr1150 and Tyr1151. These sites are known to be key modulators of the receptor activity. Autophosphorylation at other sites may also have been inhibited. The antipeptide antibody decreased the receptor kinase activity measured with poly(Glu80Tyr20) and a synthetic peptide corresponding to the proreceptor sequence 1142-1158. We provide evidence that the effect of the antibody on substrate phosphorylation may result from the control of the phosphorylation level of the receptor. Concerning the action of the antipeptide IgG on the receptor kinase activity, histone did not behave similarly to poly(Glu80Tyr20). The antibody recognizing sequence 1270-1280 competed with histone for an overlapping binding site. Histone also modulated insulin receptor autophosphorylation, supporting the idea that interference with domain 1270-1280 alters the receptor kinase. Our data suggest that the acidic region including residues 1270-1280 of the insulin receptor C-terminus is involved in the following events: (a) receptor binding with histone, an exogenous substrate of the receptor kinase, and (b) the regulation of receptor autophosphorylation and kinase activity. Based on these observations, we would like to propose that this insulin receptor domain could interact with cellular proteins modulating the receptor kinase. PMID:7744039

  18. Psychosocial factors underlying physical activity

    Ji Cheng-Ye; Middlestadt Susan E; Zhang Juan

    2007-01-01

    Abstract Background Given the increasing importance of obesity in China, prevention interventions encouraging physical activity by middle school students are needed. The purpose of this study is to illustrate how a rapid elicitation method can be used to identify salient consequences, referents, and circumstances about physical activity as perceived by middle school students and to provide suggestions for interventions and quantitative research. Method A theory-based qualitative study using a...

  19. Sphingosine-1-phosphate receptor 1 reporter mice reveal receptor activation sites in vivo

    Kono, Mari; Tucker, Ana E.; Tran, Jennifer; Bergner, Jennifer B.; Turner, Ewa M; Proia, Richard L.

    2014-01-01

    Activation of the GPCR sphingosine-1-phosphate receptor 1 (S1P1) by sphingosine-1-phosphate (S1P) regulates key physiological processes. S1P1 activation also has been implicated in pathologic processes, including autoimmunity and inflammation; however, the in vivo sites of S1P1 activation under normal and disease conditions are unclear. Here, we describe the development of a mouse model that allows in vivo evaluation of S1P1 activation. These mice, known as S1P1 GFP signaling mice, produce a ...

  20. The aryl hydrocarbon receptor and glucocorticoid receptor interact to activate human metallothionein 2A

    Although the aryl hydrocarbon receptor (AHR) and glucocorticoid receptor (GR) play essential roles in mammalian development, stress responses, and other physiological events, crosstalk between these receptors has been the subject of much debate. Metallothioneins are classic glucocorticoid-inducible genes that were reported to increase upon treatment with AHR agonists in rodent tissues and cultured human cells. In this study, the mechanism of human metallothionein 2A (MT2A) gene transcription activation by AHR was investigated. Cotreatment with 3-methylcholanthrene and dexamethasone, agonists of AHR and GR respectively, synergistically increased MT2A mRNA levels in HepG2 cells. MT2A induction was suppressed by RNA interference against AHR or GR. Coimmunoprecipitation experiments revealed a physical interaction between AHR and GR proteins. Moreover, chromatin immunoprecipitation assays indicated that AHR was recruited to the glucocorticoid response element in the MT2A promoter. Thus, we provide a novel mechanism whereby AHR modulates expression of human MT2A via the glucocorticoid response element and protein–protein interactions with GR. - Highlights: • Aryl hydrocarbon receptor forms a complex with glucocorticoid receptor in cells. • Human metallothionein gene is regulated by the AHR and GR interaction. • AHR–GR complex binds to glucocorticoid response element in metallothionein gene. • We demonstrated a novel transcriptional mechanism via AHR and GR interaction