Macropinocytosis of the PDGF β-receptor promotes fibroblast transformation by H-RasG12V

PubMed Central

Schmees, C.; Villaseñor, R.; Zheng, W.; Ma, H.; Zerial, M.; Heldin, C.-H.; Hellberg, C.

2012-01-01

Receptor tyrosine kinase (RTK) signaling is frequently increased in tumor cells, sometimes as a result of decreased receptor down-regulation. The extent to which the endocytic trafficking routes can contribute to such RTK hyperactivation is unclear. Here, we show for the first time that fibroblast transformation by H-RasG12V induces the internalization of platelet-derived growth factor β-receptor (PDGFRβ) by macropinocytosis, enhancing its signaling activity and increasing anchorage-independent proliferation. H-RasG12V transformation and PDGFRβ activation were synergistic in stimulating phosphatidylinositol (PI) 3-kinase activity, leading to receptor macropinocytosis. PDGFRβ macropinocytosis was both necessary and sufficient for enhanced receptor activation. Blocking macropinocytosis by inhibition of PI 3-kinase prevented the increase in receptor activity in transformed cells. Conversely, increasing macropinocytosis by Rabankyrin-5 overexpression was sufficient to enhance PDGFRβ activation in nontransformed cells. Simultaneous stimulation with PDGF-BB and epidermal growth factor promoted macropinocytosis of both receptors and increased their activation in nontransformed cells. We propose that H-Ras transformation promotes tumor progression by enhancing growth factor receptor signaling as a result of increased receptor macropinocytosis. PMID:22573884

An Antagonistic Vascular Endothelial Growth Factor (VEGF) Variant Inhibits VEGF-Stimulated Receptor Autophosphorylation and Proliferation of Human Endothelial Cells

NASA Astrophysics Data System (ADS)

Siemeister, Gerhard; Schirner, Michael; Reusch, Petra; Barleon, Bernhard; Marme, Dieter; Martiny-Baron, Georg

1998-04-01

Vascular endothelial growth factor (VEGF) is a potent mitogen with a unique specificity for endothelial cells and a key mediator of aberrant endothelial cell proliferation and vascular permeability in a variety of human pathological situations, such as tumor angiogenesis, diabetic retinopathy, rheumatoid arthritis, or psoriasis. VEGF is a symmetric homodimeric molecule with two receptor binding interfaces lying on each pole of the molecule. Herein we report on the construction and recombinant expression of an asymmetric heterodimeric VEGF variant with an intact receptor binding interface at one pole and a mutant receptor binding interface at the second pole of the dimer. This VEGF variant binds to VEGF receptors but fails to induce receptor activation. In competition experiments, the heterodimeric VEGF variant antagonizes VEGF-stimulated receptor autophosphorylation and proliferation of endothelial cells. A 15-fold excess of the heterodimer was sufficient to inhibit VEGF-stimulated endothelial cell proliferation by 50%, and a 100-fold excess resulted in an almost complete inhibition. By using a rational approach that is based on the structure of VEGF, we have shown the feasibility to construct a VEGF variant that acts as an VEGF antagonist.

CD86 and beta2-adrenergic receptor signaling pathways, respectively, increase Oct-2 and OCA-B Expression and binding to the 3'-IgH enhancer in B cells.

PubMed

Podojil, Joseph R; Kin, Nicholas W; Sanders, Virginia M

2004-05-28

Stimulation of CD86 (formerly known as B7-2) and/or the beta2-adrenergic receptor on a CD40 ligand/interleukin-4-activated B cell increased the rate of mature IgG1 transcription. To identify the mechanism responsible for this effect, we determined whether CD86 and/or beta2-adrenergic receptor stimulation regulated transcription factor expression and binding to the 3'-IgH enhancer in vitro and in vivo. We showed that CD86 stimulation increased the nuclear localization of NF-kappaB1 (p50) and phosphorylated RelA (p65) and increased Oct-2 expression and binding to the 3'-IgH enhancer, in a protein kinase C-dependent manner. These effects were lost when CD86-deficient or NF-kappaB1-deficient B cells were used. CD86 stimulation also increased the level of IkappaB-alpha phosphorylation but in a protein kinase C-independent manner. Beta2-adrenergic receptor stimulation increased CREB phosphorylation, OCA-B expression, and OCA-B binding to the 3'-IgH enhancer in a protein kinase A-dependent manner, an effect lost when beta2-adrenergic receptor-deficient B cells were used. Also, the beta2-adrenergic receptor-induced increase in the level of mature IgG1 transcript was lost when OCA-B-deficient B cells were used. These data are the first to show that CD86 stimulation up-regulates the expression of the transcription factor Oct-2 in a protein kinase C- and NF-kappaB1-dependent manner, and that beta2-adrenergic receptor stimulation up-regulates the expression of the coactivator OCA-B in a protein kinase A-dependent manner to cooperate with Oct-2 binding to the 3'-IgH enhancer.

Two signaling molecules share a phosphotyrosine-containing binding site in the platelet-derived growth factor receptor.

PubMed

Nishimura, R; Li, W; Kashishian, A; Mondino, A; Zhou, M; Cooper, J; Schlessinger, J

1993-11-01

Autophosphorylation sites of growth factor receptors with tyrosine kinase activity function as specific binding sites for Src homology 2 (SH2) domains of signaling molecules. This interaction appears to be a crucial step in a mechanism by which receptor tyrosine kinases relay signals to downstream signaling pathways. Nck is a widely expressed protein consisting exclusively of SH2 and SH3 domains, the overexpression of which causes cell transformation. It has been shown that various growth factors stimulate the phosphorylation of Nck and its association with autophosphorylated growth factor receptors. A panel of platelet-derived growth factor (PDGF) receptor mutations at tyrosine residues has been used to identify the Nck binding site. Here we show that mutation at Tyr-751 of the PDGF beta-receptor eliminates Nck binding both in vitro and in living cells. Moreover, the Y751F PDGF receptor mutant failed to mediate PDGF-stimulated phosphorylation of Nck in intact cells. A phosphorylated Tyr-751 is also required for binding of phosphatidylinositol-3 kinase to the PDGF receptor. Hence, the SH2 domains of p85 and Nck share a binding site in the PDGF receptor. Competition experiments with different phosphopeptides derived from the PDGF receptor suggest that binding of Nck and p85 is influenced by different residues around Tyr-751. Thus, a single tyrosine autophosphorylation site is able to link the PDGF receptor to two distinct SH2 domain-containing signaling molecules.

IGF-II receptors and IGF-II-stimulated glucose transport in human fat cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sinha, M.K.; Buchanan, C.; Raineri-Maldonado, C.

1990-03-01

Insulin-like growth factor II (IGF-II) receptors have been described in rat but not in human adipocytes. In both species, IGF-II has been reported to stimulate glucose transport by interacting with the insulin receptor. In this study, we have unequivocally demonstrated the presence of IGF-II receptors in human adipocytes. 125I-labeled IGF-II specifically binds to intact adipocytes, membranes, and lectin-purified detergent solubilized extracts. Through the use of 0.5 mM disuccinimidyl suberate, 125I-IGF-II is cross-linked to a 260-kDa protein that is identified as the IGF-II receptor by displacement experiments with unlabeled IGF-II, IGF-I, and insulin and either by immunoprecipitation or by Western blotmore » analysis with mannose 6-phosphate receptor antibodies. The concentrations of IGF-II required for half-maximal and maximal stimulation of glucose transport in human adipocytes are 35 and 100 times more than that of insulin. The possibility of IGF-II stimulating glucose transport by interacting predominantly with the insulin receptor is suggested by the following: (1) the concentration of IGF-II that inhibits half of insulin binding is only 20 times more than that of insulin; (2) the lack of an additive effect of IGF-II and insulin for maximal stimulation of glucose transport; (3) the ability of monoclonal insulin receptor antibodies to decrease glucose transport stimulated by submaximal concentrations of both IGF-II and insulin; and (4) the ability of IGF-II to stimulate insulin receptor autophosphorylation albeit at a reduced potency when compared with insulin.« less

Epidermal growth factor receptor is required for estradiol-stimulated bovine satellite cell proliferation.

PubMed

Reiter, B C; Kamanga-Sollo, E; Pampusch, M S; White, M E; Dayton, W R

2014-07-01

The objective of this study was to assess the role of the epidermal growth factor receptor (EGFR) in estradiol-17β (E2)-stimulated proliferation of cultured bovine satellite cells (BSCs). Treatment of BSC cultures with AG1478 (a specific inhibitor of EGFR tyrosine kinase activity) suppresses E2-stimulated BSC proliferation (P < 0.05). In addition, E2-stimulated proliferation is completely suppressed (P < 0.05) in BSCs in which EGFR expression is silenced by treatment with EGFR small interfering RNA (siRNA). These results indicate that EGFR is required for E2 to stimulate proliferation in BSC cultures. Both AG1478 treatment and EGFR silencing also suppress proliferation stimulated by LR3-IGF-1 (an IGF1 analogue that binds normally to the insulin-like growth factor receptor (IGFR)-1 but has little or no affinity for IGF binding proteins) in cultured BSCs (P < 0.05). Even though EGFR siRNA treatment has no effect on IGFR-1β mRNA expression in cultured BSCs, IGFR-1β protein level is substantially reduced in BSCs treated with EGFR siRNA. These data suggest that EGFR silencing results in post-transcriptional modifications that result in decreased IGFR-1β protein levels. Although it is clear that functional EGFR is necessary for E2-stimulated proliferation of BSCs, the role of EGFR is not clear. Transactivation of EGFR may directly stimulate proliferation, or EGFR may function to maintain the level of IGFR-1β which is necessary for E2-stimulated proliferation. It also is possible that the role of EGFR in E2-stimulated BSC proliferation may involve both of these mechanisms. Copyright © 2014 Elsevier Inc. All rights reserved.

Identification of a nonsense mutation in the granulocyte-colony-stimulating factor receptor in severe congenital neutropenia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dong, F.; Loewenberg, B.; Hoefsloot, L.H.

Severe congenital neutropenia (Kostmann syndrome) is characterized by profound absolute neutropenia and a maturation arrest of marrow progenitor cells at the promyelocyte-myelocyte stage. Marrow cells from such patients frequently display a reduced responsiveness to granulocyte-colony-stimulating factor (G-CSF). G-CSF binds to and activates a specific receptor which transduces signals critical for the proliferation and maturation of granulocytic progenitor cells. Here the authors report the identification of a somatic point mutation in one allele of the G-CSF receptor gene in a patient with severe congenital neutropenia. The mutation results in a cytoplasmic truncation of the receptor. When expressed in murine myeloid cells,more » the mutant receptor transduced a strong growth signal but, in contrast to the wild-type G-CSF receptor, was defective in maturation induction. This mutant receptor chain may act in a dominant negative manner to block granulocytic maturation. 40 refs., figs., 2 tabs.« less

The receptor tyrosine kinase ERBB4 is expressed in skin keratinocytes and influences epidermal proliferation.

PubMed

Hoesl, Christine; Röhrl, Jennifer M; Schneider, Marlon R; Dahlhoff, Maik

2018-04-01

The epidermal growth factor receptor (EGFR) and associated receptors ERBB2 and ERBB3 are important for skin development and homeostasis. To date, ERBB4 could not be unambiguously identified in the epidermis. The aim of this study was to analyze the ERBB-receptor family with a special focus on ERBB4 in vitro in human keratinocytes and in vivo in human and murine epidermis. We compared the transcript levels of all ERBB-receptors and the seven EGFR-ligands in HaCaT and A431 cells. ERBB-receptor activity was analyzed after epidermal growth factor (EGF) stimulation by Western blot analysis. The location of the receptors was investigated by immunofluorescence in human keratinocytes and skin. Finally, we investigated the function of ERBB4 in the epidermis of skin-specific ERBB4-knockout mice. After EGF stimulation, all ligands were upregulated except for epigen. Expression levels of EGFR were unchanged, but all other ERBB-receptors were down-regulated after EGF stimulation, although all ERBB-receptors were phosphorylated. We detected ERBB4 at mRNA and protein levels in both human epidermal cell lines and in the basal layer of human and murine epidermis. Skin-specific ERBB4-knockout mice revealed a significantly reduced epidermal thickness with a decreased proliferation rate. ERBB4 is expressed in the basal layer of human epidermis and cultured keratinocytes as well as in murine epidermis. Moreover, ERBB4 is phosphorylated in HaCaT cells due to EGF stimulation, and its deletion in murine epidermis affects skin thickness by decreasing proliferation. ERBB4 is expressed in human keratinocytes and plays a role in murine skin homeostasis. Copyright © 2018 Elsevier B.V. All rights reserved.

Basic fibroblast growth factor accelerates matrix degradation via a neuro-endocrine pathway in human adult articular chondrocytes.

PubMed

Im, Hee-Jeong; Li, Xin; Muddasani, Prasuna; Kim, Gun-Hee; Davis, Francesca; Rangan, Jayanthi; Forsyth, Christopher B; Ellman, Michael; Thonar, Eugene J M A

2008-05-01

Pain-related neuropeptides released from synovial fibroblasts, such as substance P, have been implicated in joint destruction. Substance P-induced inflammatory processes are mediated via signaling through a G-protein-coupled receptor, that is, neurokinin-1 tachykinin receptor (NK(1)-R). We determined the pathophysiological link between substance P and its receptor in human adult articular cartilage homeostasis. We further examined if catabolic growth factors such as basic fibroblast growth factor (bFGF or FGF-2) or IL-1beta accelerate matrix degradation via a neural pathway upregulation of substance P and NK(1)-R. We show here that substance P stimulates the production of cartilage-degrading enzymes, such as matrix metalloproteinase-13 (MMP-13), and suppresses proteoglycan deposition in human adult articular chondrocytes via NK(1)-R. Furthermore, we have demonstrated that substance P negates proteoglycan stimulation promoted by bone morphogenetic protein-7, suggesting the dual role of substance P as both a pro-catabolic and anti-anabolic mediator of cartilage homeostasis. We report that bFGF-mediated stimulation of substance P and its receptor NK(1)-R is, in part, through an IL-1beta-dependent pathway. (c) 2007 Wiley-Liss, Inc.

Basic Fibroblast Growth Factor Accelerates Matrix Degradation Via a Neuro-Endocrine Pathway in Human Adult Articular Chondrocytes

PubMed Central

IM, HEE-JEONG; LI, XIN; MUDDASANI, PRASUNA; KIM, GUN-HEE; DAVIS, FRANCESCA; RANGAN, JAYANTHI; FORSYTH, CHRISTOPHER B.; ELLMAN, MICHAEL; THONAR, EUGENE JMA

2010-01-01

Pain-related neuropeptides released from synovial fibroblasts, such as substance P, have been implicated in joint destruction. Substance P-induced inflammatory processes are mediated via signaling through a G-protein-coupled receptor, that is, neurokinin-1 tachykinin receptor (NK1-R). We determined the pathophysiological link between substance P and its receptor in human adult articular cartilage homeostasis. We further examined if catabolic growth factors such as basic fibroblast growth factor (bFGF or FGF-2) or IL-1β accelerate matrix degradation via a neural pathway upregulation of substance P and NK1-R. We show here that substance P stimulates the production of cartilage-degrading enzymes, such as matrix metalloproteinase-13 (MMP-13), and suppresses proteoglycan deposition in human adult articular chondrocytes via NK1-R. Furthermore, we have demonstrated that substance P negates proteoglycan stimulation promoted by bone morphogenetic protein-7, suggesting the dual role of substance P as both a pro-catabolic and anti-anabolic mediator of cartilage homeostasis. We report that bFGF-mediated stimulation of substance P and its receptor NK1-R is, in part, through an IL-1β-dependent pathway. PMID:17960584

Harnessing tumor necrosis factor receptors to enhance antitumor activities of drugs.

PubMed

Muntané, Jordi

2011-10-17

Cancer is the second-leading cause of death in the U.S. behind heart disease and over stroke. The hallmarks of cancer comprise six biological capabilities acquired during the multistep development of human tumors. The inhibition of cell death pathways is one of these tumor characteristics which also include sustained proliferative signaling, evading growth suppressor signaling, replicative immortality, angiogenesis, and promotion of invasion and metastasis. Cell death is mediated through death receptor (DR) stimulation initiated by specific ligands that transmit signaling to the cell death machinery or through the participation of mitochondria. Cell death involving DR is mediated by the superfamily of tumor necrosis factor receptor (TNF-R) which includes TNF-R type I, CD95, DR3, TNF-related apoptosis-inducing ligand (TRAIL) receptor-1 (TRAIL-R1) and -2 (TRAIL-R2), DR6, ectodysplasin A (EDA) receptor (EDAR), and the nerve growth factor (NGF) receptor (NGFR). The expression of these receptors in healthy and tumor cells induces treatment side effects that limit the systemic administration of cell death-inducing therapies. The present review is focused on the different therapeutic strategies such as targeted antibodies or small molecules addressed to selective stimulated DR-mediated apoptosis or reduce cell proliferation in cancer cells.

The Influence of Thyroid-Stimulating Hormone and Thyroid-Stimulating Hormone Receptor Antibodies on Osteoclastogenesis

PubMed Central

Morshed, Syed; Latif, Rauf; Zaidi, Mone; Davies, Terry F.

2011-01-01

Background We have shown that thyroid-stimulating hormone (TSH) has a direct inhibitory effect on osteoclastic bone resorption and that TSH receptor (TSHR) null mice display osteoporosis. To determine the stage of osteoclast development at which TSH may exert its effect, we examined the influence of TSH and agonist TSHR antibodies (TSHR-Ab) on osteoclast differentiation from murine embryonic stem (ES) cells to gain insight into bone remodeling in hyperthyroid Graves' disease. Methods Osteoclast differentiation was initiated in murine ES cell cultures through exposure to macrophage colony stimulation factor, receptor activator of nuclear factor кB ligand, vitamin D, and dexamethasone. Results Tartrate resistant acid phosphatase (TRAP)-positive osteoclasts formed in ∼12 days. This coincided with the expected downregulation of known markers of self renewal and pluripotency (including Oct4, Sox2, and REX1). Both TSH and TSHR-Abs inhibited osteoclastogenesis as evidenced by decreased development of TRAP-positive cells (∼40%–50% reduction, p = 0.0047), and by decreased expression, in a concentration-dependent manner, of osteoclast differentiation markers (including the calcitonin receptor, TRAP, cathepsin K, matrix metallo-proteinase-9, and carbonic anhydrase II). Similar data were obtained using serum immunoglobulin-Gs (IgGs) from patients with hyperthyroid Graves' disease and known TSHR-Abs. TSHR stimulators inhibited tumor necrosis factor-alpha mRNA and protein expression, but increased the expression of osteoprotegerin (OPG), an antiosteoclastogenic human soluble receptor activator of nuclear factor кB ligand receptor. Neutralizing antibody to OPG reversed the inhibitory effect of TSH on osteoclast differentiation evidencing that the TSH effect was at least in part mediated by increased OPG. Conclusion These data establish ES-derived osteoclastogenesis as an effective model system to study the regulation of osteoclast differentiation in early development. The results support the observations that TSH has a bone protective action by negatively regulating osteoclastogenesis. Further, our results implicate TSHR-Abs in offering skeletal protection in hyperthyroid Graves' disease, even in the face of high thyroid hormone and low TSH levels. PMID:21745106

Signal transduction through the IL-4 and insulin receptor families.

PubMed

Wang, L M; Keegan, A; Frankel, M; Paul, W E; Pierce, J H

1995-07-01

Activation of tyrosine kinase-containing receptors and intracellular tyrosine kinases by ligand stimulation is known to be crucial for mediating initial and subsequent events involved in mitogenic signal transduction. Receptors for insulin and insulin-like growth factor 1 (IGF-1) contain cytoplasmic tyrosine kinase domains that undergo autophosphorylation upon ligand stimulation. Activation of these receptors also leads to pronounced and rapid tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) in cells of connective tissue origin. A related substrate, designated 4PS, is similarly phosphorylated by insulin and IGF-1 stimulation in many hematopoietic cell types. IRS-1 and 4PS possess a number of tyrosine phosphorylation sites that are within motifs that bind specific SH2-containing molecules known to be involved in mitogenic signaling such as PI-3 kinase, SHPTP-2 (Syp) and Grb-2. Thus, they appear to act as docking substrates for a variety of signaling molecules. The majority of hematopoietic cytokines bind to receptors that do not possess intrinsic kinase activity, and these receptors have been collectively termed as members of the hematopoietin receptor superfamily. Despite their lack of tyrosine kinase domains, stimulation of these receptors has been demonstrated to activate intracellular kinases leading to tyrosine phosphorylation of multiple substrates. Recent evidence has demonstrated that activation of different members of the Janus family of tyrosine kinases is involved in mediating tyrosine phosphorylation events by specific cytokines. Stimulation of the interleukin 4 (IL-4) receptor, a member of the hematopoietin receptor superfamily, is thought to result in activation of Jak1, Jak3, and/or Fes tyrosine kinases.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantification of growth factor signaling and pathway cross talk by live-cell imaging.

PubMed

Gross, Sean M; Rotwein, Peter

2017-03-01

Peptide growth factors stimulate cellular responses through activation of their transmembrane receptors. Multiple intracellular signaling cascades are engaged following growth factor-receptor binding, leading to short- and long-term biological effects. Each receptor-activated signaling pathway does not act in isolation but rather interacts at different levels with other pathways to shape signaling networks that are distinctive for each growth factor. To gain insights into the specifics of growth factor-regulated interactions among different signaling cascades, we developed a HeLa cell line stably expressing fluorescent live-cell imaging reporters that are readouts for two major growth factor-stimulated pathways, Ras-Raf-Mek-ERK and phosphatidylinositol (PI) 3-kinase-Akt. Incubation of cells with epidermal growth factor (EGF) resulted in rapid, robust, and sustained ERK signaling but shorter-term activation of Akt. In contrast, hepatocyte growth factor induced sustained Akt signaling but weak and short-lived ERK activity, and insulin-like growth factor-I stimulated strong long-term Akt responses but negligible ERK signaling. To address potential interactions between signaling pathways, we employed specific small-molecule inhibitors. In cells incubated with EGF or platelet-derived growth factor-AA, Raf activation and the subsequent stimulation of ERK reduced Akt signaling, whereas Mek inhibition, which blocked ERK activation, enhanced Akt and turned transient effects into sustained responses. Our results reveal that individual growth factors initiate signaling cascades that vary markedly in strength and duration and demonstrate in living cells the dramatic effects of cross talk from Raf and Mek to PI 3-kinase and Akt. Our data further indicate how specific growth factors can encode distinct cellular behaviors by promoting complex interactions among signaling pathways. Copyright © 2017 the American Physiological Society.

Specific Inhibitors of Platelet-Derived Growth Factor or Epidermal Growth Factor Receptor Tyrosine Kinase Reduce Pulmonary Fibrosis in Rats

PubMed Central

Rice, Annette B.; Moomaw, Cindy R.; Morgan, Daniel L.; Bonner, James C.

1999-01-01

The proliferation of myofibroblasts is a central feature of pulmonary fibrosis. In this study we have used tyrosine kinase inhibitors of the tyrphostin class to specifically block autophosphorylation of the platelet-derived growth factor receptor (PDGF-R) or epidermal growth factor receptor (EGF-R). AG1296 specifically inhibited autophosphorylation of PDGF-R and blocked PDGF-stimulated [3H]thymidine uptake by rat lung myofibroblasts in vitro. AG1478 was demonstrated as a selective blocker of EGF-R autophosphorylation and inhibited EGF-stimulated DNA synthesis in vitro. In a rat model of pulmonary fibrosis caused by intratracheal instillation of vanadium pentoxide (V2O5), intraperitoneal delivery of 50 mg/kg AG1296 or AG1478 in dimethylsulfoxide 1 hour before V2O5 instillation and again 2 days after instillation reduced the number of epithelial and mesenchymal cells incorporating bromodeoxyuridine (Brdu) by ∼50% at 3 and 6 days after instillation. V2O5 instillation increased lung hydroxyproline fivefold 15 days after instillation, and AG1296 was more than 90% effective in preventing the increase in hydroxyproline, whereas AG1478 caused a 50% to 60% decrease in V2O5-stimulated hydroxyproline accumulation. These data provide evidence that PDGF and EGF receptor ligands are potent mitogens for collagen-producing mesenchymal cells during pulmonary fibrogenesis, and targeting tyrosine kinase receptors could offer a strategy for the treatment of fibrotic lung diseases. PMID:10393853

Identification of SR1078, a synthetic agonist for the orphan nuclear receptors RORα and RORγ.

PubMed

Wang, Yongjun; Kumar, Naresh; Nuhant, Philippe; Cameron, Michael D; Istrate, Monica A; Roush, William R; Griffin, Patrick R; Burris, Thomas P

2010-11-19

The retinoic acid receptor-related receptors (RORs) are members of the nuclear receptor (NR) superfamily of transcription factors. Several NRs are still characterized as orphan receptors because ligands have not yet been identified for these proteins. Here, we describe the identification of a synthetic RORα/RORγ ligand, SR1078. SR1078 modulates the conformation of RORγ in a biochemical assay and activates RORα and RORγ driven transcription. Furthermore, SR1078 stimulates expression of endogenous ROR target genes in HepG2 cells that express both RORα and RORγ. Pharmacokinetic studies indicate that SR1078 displays reasonable exposure following injection into mice, and consistent with SR1078 functioning as a RORα/RORγ agonist, expression of two ROR target genes, glucose-6-phosphatase and fibroblast growth factor 21, were stimulated in the liver. Thus, we have identified the first synthetic RORα/γ agonist, and this compound can be utilized as a chemical tool to probe the function of these receptors both in vitro and in vivo.

Identification of a Synthetic Agonist for the Orphan Nuclear Receptors RORα and RORγ, SR1078

PubMed Central

Wang, Yongjun; Kumar, Naresh; Nuhant, Philippe; Cameron, Michael D.; Istrate, Monica A.; Roush, William R.; Griffin, Patrick R.; Burris, Thomas P.

2010-01-01

The retinoic acid receptor-related receptors (RORs) are members of the nuclear receptor (NR) superfamily of transcription factors. Several NRs are still characterized as orphan receptors since ligands have not yet been identified for these proteins. Here, we describe the identification of a synthetic RORα/RORγ ligand, SR1078. SR1078 modulates the conformation of RORγ in a biochemical assay and activates RORα and RORγ driven transcription. Furthermore, SR1078 stimulates expression of endogenous ROR target genes in HepG2 cells that express both RORα and RORγ. Pharmacokinetic studies indicate that SR1078 displays reasonable exposure following injection into mice and consistent with SR1078 functioning as a RORα/RORγ agonist, expression of two ROR target genes, glucose-6-phosphatase and fibroblast growth factor 21, were stimulated in the liver. Thus, we have identified the first synthetic RORα/γ agonist and this compound can be utilized as a chemical tool to probe the function of these receptors both in vitro and in vivo. PMID:20735016

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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 ormore » 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).« less

Chemiluminometric Immuno-Analysis of Innate Immune Response against Repetitive Bacterial Stimulations for the Same Mammalian Cells

PubMed Central

Jeon, Jin-Woo; Cho, Il-Hoon; Ha, Un-Hwan; Seo, Sung-Kyu; Paek, Se-Hwan

2014-01-01

For monitoring of human cellular response to repetitive bacterial stimulations (e.g., Pseudomonas aeruginosa in a lysate form), we devised a chemiluminescent immuno-analytical system for toll-like receptor 1 (TLR1) as marker present on cell surfaces (e.g., A549). Upon stimulation, TLR1 recognizes pathogen-associated molecular patterns of the infectious agent and are then up-regulated via activation of the nuclear factor-κB (NF-κB) pathway. In this study, the receptor density was quantified by employing an antibody specific to the target receptor and by producing a chemiluminometric signal from an enzyme labeled to the binder. The activated status was then switched back to normal down-regulated stage, by changing the culture medium to one containing animal serum. The major factors affecting activation were the stimulation dose of the bacterial lysate, stimulation timing during starvation, and up- and down-regulation time intervals. Reiterative TLR regulation switching up to three times was not affected by either antibody remained after immunoassay or enzyme substrate (e.g., hydrogen peroxide) in solution. This immuno-analysis for TLRs could be unique to acquire accumulated response of the human cells to repeated stimulations and, therefore, can eventually apply to persistency testing of the cellular regulation in screening of anti-inflammatory substances. PMID:25109895

Quantification of growth factor signaling and pathway cross talk by live-cell imaging

PubMed Central

Gross, Sean M.

2017-01-01

Peptide growth factors stimulate cellular responses through activation of their transmembrane receptors. Multiple intracellular signaling cascades are engaged following growth factor–receptor binding, leading to short- and long-term biological effects. Each receptor-activated signaling pathway does not act in isolation but rather interacts at different levels with other pathways to shape signaling networks that are distinctive for each growth factor. To gain insights into the specifics of growth factor-regulated interactions among different signaling cascades, we developed a HeLa cell line stably expressing fluorescent live-cell imaging reporters that are readouts for two major growth factor-stimulated pathways, Ras–Raf–Mek–ERK and phosphatidylinositol (PI) 3-kinase–Akt. Incubation of cells with epidermal growth factor (EGF) resulted in rapid, robust, and sustained ERK signaling but shorter-term activation of Akt. In contrast, hepatocyte growth factor induced sustained Akt signaling but weak and short-lived ERK activity, and insulin-like growth factor-I stimulated strong long-term Akt responses but negligible ERK signaling. To address potential interactions between signaling pathways, we employed specific small-molecule inhibitors. In cells incubated with EGF or platelet-derived growth factor-AA, Raf activation and the subsequent stimulation of ERK reduced Akt signaling, whereas Mek inhibition, which blocked ERK activation, enhanced Akt and turned transient effects into sustained responses. Our results reveal that individual growth factors initiate signaling cascades that vary markedly in strength and duration and demonstrate in living cells the dramatic effects of cross talk from Raf and Mek to PI 3-kinase and Akt. Our data further indicate how specific growth factors can encode distinct cellular behaviors by promoting complex interactions among signaling pathways. PMID:28100485

Gab-family adapter proteins act downstream of cytokine and growth factor receptors and T- and B-cell antigen receptors.

PubMed

Nishida, K; Yoshida, Y; Itoh, M; Fukada, T; Ohtani, T; Shirogane, T; Atsumi, T; Takahashi-Tezuka, M; Ishihara, K; Hibi, M; Hirano, T

1999-03-15

We previously found that the adapter protein Gab1 (110 kD) is tyrosine-phosphorylated and forms a complex with SHP-2 and PI-3 kinase upon stimulation through either the interleukin-3 receptor (IL-3R) or gp130, the common receptor subunit of IL-6-family cytokines. In this report, we identified another adapter molecule (100 kD) interacting with SHP-2 and PI-3 kinase in response to various stimuli. The molecule displays striking homology to Gab1 at the amino acid level; thus, we named it Gab2. It contains a PH domain, proline-rich sequences, and tyrosine residues that bind to SH2 domains when they are phosphorylated. Gab1 is phosphorylated on tyrosine upon stimulation through the thrombopoietin receptor (TPOR), stem cell factor receptor (SCFR), and T-cell and B-cell antigen receptors (TCR and BCR, respectively), in addition to IL-3R and gp130. Tyrosine phosphorylation of Gab2 was induced by stimulation through gp130, IL-2R, IL-3R, TPOR, SCFR, and TCR. Gab1 and Gab2 were shown to be substrates for SHP-2 in vitro. Overexpression of Gab2 enhanced the gp130 or Src-related kinases-mediated ERK2 activation as that of Gab1 did. These data indicate that Gab-family molecules act as adapters for transmitting various signals.

Inhibition of thrombin action ameliorates insulin resistance in type 2 diabetic db/db mice.

PubMed

Mihara, Masatomo; Aihara, Ken-ichi; Ikeda, Yasumasa; Yoshida, Sumiko; Kinouchi, Mizuho; Kurahashi, Kiyoe; Fujinaka, Yuichi; Akaike, Masashi; Matsumoto, Toshio

2010-02-01

The binding of thrombin to its receptor stimulates inflammatory cytokines including IL-6 and monocyte chemoattractant protein-1 (MCP-1); both are associated with the development of insulin resistance. Because increased adiposity enhanced the expression of coagulation factor VII that stimulates the coagulation pathway in adipose tissue, we tested whether the inhibition of thrombin action ameliorates insulin resistance in obese diabetic (Lpr(-/-):db/db) mice. The 4-wk administration of argatroban, a selective thrombin inhibitor, reduced fasting plasma glucose and ameliorated insulin resistance in these mice. It also reduced adipocyte size and macrophage infiltration into adipose tissue. The aberrant gene expression of MCP-1, IL-6, adiponectin, and factor VII and suppressed insulin receptor substrate-1-Akt signaling in adipose tissue of db/db mice were reversed by argatroban treatment. These results demonstrate that increased adiposity enhances the production of thrombin in adipose tissue by stimulating factor VII expression and suggest that increased thrombin activity in adipose tissue plays an important role in the development of insulin resistance via enhancing MCP-1 production, leading to macrophage infiltration and insulin receptor substrate-1-Akt pathway inactivation.

IRS-1: essential for insulin- and IL-4-stimulated mitogenesis in hematopoietic cells.

PubMed

Wang, L M; Myers, M G; Sun, X J; Aaronson, S A; White, M; Pierce, J H

1993-09-17

Although several interleukin-3 (IL-3)-dependent cell lines proliferate in response to IL-4 or insulin, the 32D line does not. Insulin and IL-4 sensitivity was restored to 32D cells by expression of IRS-1, the principal substrate of the insulin receptor. Although 32D cells possessed receptors for both factors, they lacked the IRS-1--related protein, 4PS, which becomes phosphorylated by tyrosine in insulin- or IL-4--responsive lines after stimulation. These results indicate that factors that bind unrelated receptors can use similar mitogenic signaling pathways in hematopoietic cells and that 4PS and IRS-1 are functionally similar proteins that are essential for insulin- and IL-4--induced proliferation.

Homodimeric cross-over structure of the human granulocyte colony-stimulating factor (GCSF) receptor signaling complex

PubMed Central

Tamada, Taro; Honjo, Eijiro; Maeda, Yoshitake; Okamoto, Tomoyuki; Ishibashi, Matsujiro; Tokunaga, Masao; Kuroki, Ryota

2006-01-01

A crystal structure of the signaling complex between human granulocyte colony-stimulating factor (GCSF) and a ligand binding region of GCSF receptor (GCSF-R), has been determined to 2.8 Å resolution. The GCSF:GCSF-R complex formed a 2:2 stoichiometry by means of a cross-over interaction between the Ig-like domains of GCSF-R and GCSF. The conformation of the complex is quite different from that between human GCSF and the cytokine receptor homologous domain of mouse GCSF-R, but similar to that of the IL-6/gp130 signaling complex. The Ig-like domain cross-over structure necessary for GCSF-R activation is consistent with previously reported thermodynamic and mutational analyses. PMID:16492764

Brain-derived neurotrophic factor and its receptors in Bergmann glia cells.

PubMed

Poblete-Naredo, Irais; Guillem, Alain M; Juárez, Claudia; Zepeda, Rossana C; Ramírez, Leticia; Caba, Mario; Hernández-Kelly, Luisa C; Aguilera, José; López-Bayghen, Esther; Ortega, Arturo

2011-12-01

Brain-derived neurotrophic factor is an abundant and widely distributed neurotrophin expressed in the Central Nervous System. It is critically involved in neuronal differentiation and survival. The expression of brain-derived neurotrophic factor and that of its catalytic active cognate receptor (TrkB) has been extensively studied in neuronal cells but their expression and function in glial cells is still controversial. Despite of this fact, brain-derived neurotrophic factor is released from astrocytes upon glutamate stimulation. A suitable model to study glia/neuronal interactions, in the context of glutamatergic synapses, is the well-characterized culture of chick cerebellar Bergmann glia cells. Using, this system, we show here that BDNF and its functional receptor are present in Bergmann glia and that BDNF stimulation is linked to the activation of the phosphatidyl-inositol 3 kinase/protein kinase C/mitogen-activated protein kinase/Activator Protein-1 signaling pathway. Accordingly, reverse transcription-polymerase chain reaction (RT-PCR) experiments predicted the expression of full-length and truncated TrkB isoforms. Our results suggest that Bergmann glia cells are able to express and respond to BDNF stimulation favoring the notion of their pivotal role in neuroprotection. Copyright © 2011 Elsevier B.V. All rights reserved.

Accelerate Genomic Aging in Congenital Neutropenia

DTIC Science & Technology

2015-08-01

for the markedly increased risk of transformation to myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) in patients with congenital...Hematopoietic stem cells Granulocyte colony-stimulating factor Granulocyte colony-stimulating factor receptor Acute myeloid leukemia Myelodysplastic... myeloid leukemia (AML) is perhaps the major clinical concern in patients with severe congenital neutropenia (SCN) and Shwachman-Diamond syndrome (SDS

Transcription control and neuronal differentiation by agents that activate the LXR nuclear receptor family.

PubMed

Schmidt, A; Vogel, R; Holloway, M K; Rutledge, S J; Friedman, O; Yang, Z; Rodan, G A; Friedman, E

1999-09-10

LXR and PPAR receptors belong to the nuclear receptor superfamily of transcriptional activating factors. Using ligand-dependent transcription assays, we found that 5-tetradecyloxy-2-furancarboxylic acid (TOFA) transactivates chimeric receptors composed of the glucocorticoid receptor DNA binding domain and the ligand binding regions of PPARalpha, PPARbeta (NUC-1) and LXRbeta (NER) receptors. In the same assays, ligands for PPARs (oleic acid, WY-14643 and L-631,033) and LXRs (hydroxycholesterols) maintain their respective receptor selectivity. TOFA and hydroxycholesterols also stimulate transcription from a minimal fibrinogen promoter that is under the control of AP-1 or NF-kappaB transcription factor binding sites. In addition to their effects on transcription, these LXRbeta activators induce neuronal differentiation in rat pheochromocytoma cells. TOFA and the natural LXR agonist, 22 (R)-hydroxycholesterol, stimulate neurite outgrowth in 55 and 28% of cells, respectively. No neurite outgrowth was induced by the related 22(S)-hydroxycholesterol, which does not activate the LXR family. These results suggest that the hydroxycholesterol signaling pathway has a complex effect on transcription that mediates the activity of TOFA and hydroxycholesterol on neuronal differentiation in pheochromocytoma cells.

Modeling hypertrophic IP3 transients in the cardiac myocyte.

PubMed

Cooling, Michael; Hunter, Peter; Crampin, Edmund J

2007-11-15

Cardiac hypertrophy is a known risk factor for heart disease, and at the cellular level is caused by a complex interaction of signal transduction pathways. The IP3-calcineurin pathway plays an important role in stimulating the transcription factor NFAT which binds to DNA cooperatively with other hypertrophic transcription factors. Using available kinetic data, we construct a mathematical model of the IP3 signal production system after stimulation by a hypertrophic alpha-adrenergic agonist (endothelin-1) in the mouse atrial cardiac myocyte. We use a global sensitivity analysis to identify key controlling parameters with respect to the resultant IP3 transient, including the phosphorylation of cell-membrane receptors, the ligand strength and binding kinetics to precoupled (with G(alpha)GDP) receptor, and the kinetics associated with precoupling the receptors. We show that the kinetics associated with the receptor system contribute to the behavior of the system to a great extent, with precoupled receptors driving the response to extracellular ligand. Finally, by reparameterizing for a second hypertrophic alpha-adrenergic agonist, angiotensin-II, we show that differences in key receptor kinetic and membrane density parameters are sufficient to explain different observed IP3 transients in essentially the same pathway.

Nuclear receptors in pancreatic tumor cells.

PubMed

Damaskos, Christos; Garmpis, Nikolaos; Karatzas, Theodore; Kostakis, Ioannis D; Nikolidakis, Lampros; Kostakis, Alkiviadis; Kouraklis, Gregory

2014-12-01

This review focuses on nuclear receptors expressed in pancreatic cancer. An extensive search of articles published up to March 2013 was conducted using the MEDLINE database. The key words used were "pancreatic cancer", "molecular receptors" and "growth factors". A total of 112 articles referred to pancreatic cancer, molecular receptors and/or growth factors were included. Receptors of growth factors, such as the epithelial growth factor receptor, insulin-like growth factor-1 receptor, vascular endothelial growth factor receptor and others, such as integrin α5β1, somatostatin receptors, the death receptor 5, claudin, notch receptors, mesothelin receptors, follicle-stimulating hormone receptors, the MUC1 receptor, the adrenomedullin receptor, the farnesoid X receptor, the transferrin receptor, sigma-2 receptors, the chemokine receptor CXCR4, the urokinase plasminogen activator receptor, the ephrine A2 receptor, the GRIA3 receptor, the RON receptor and the angiotensin II receptor AT-1 are expressed in pancreatic tumor cells. These molecules are implicated in tumor growth, apoptosis, angiogenesis, metastasis etc. After identifying the molecular receptors associated with the pancreatic cancer, many more target molecules playing important roles in tumor pathophysiology and senescence-associated signal transduction in cancer cells will be identified. This may have a significant influence on diagnosis, therapy and prognosis of pancreatic cancer. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Granulocyte-Colony Stimulating Factor Receptor, Tissue Factor, and VEGF-R Bound VEGF in Human Breast Cancer In Loco.

PubMed

Wojtukiewicz, Marek Z; Sierko, Ewa; Skalij, Piotr; Kamińska, Magda; Zimnoch, Lech; Brekken, Ralf A; Thorpe, Philip E

2016-01-01

Doxorubicin and docetaxel-based chemotherapy regimens used in breast cancer patients are associated with high risk of febrile neutropenia (FN). Granulocyte colony-stimulating factors (G-CSF) are recommended for both treating and preventing chemotherapy-induced neutropenia. Increased thrombosis incidence in G-CSF treated patients was reported; however, the underlying mechanisms remain unclear. The principal activator of blood coagulation in cancer is tissue factor (TF). It additionally contributes to cancer progression and stimulates angiogenesis. The main proangiogenic factor is vascular endothelial growth factor (VEGF). The aim of the study was to evaluate granulocyte-colony stimulating factor receptor (G-CSFR), tissue factor (TF) expression and vascular endothelial growth factor receptor (VEGF-R) bound VEGF in human breast cancer in loco. G-CSFR, TF and VEGFR bound VEGF (VEGF: VEGFR) were assessed in 28 breast cancer tissue samples. Immunohistochemical (IHC) methodologies according to ABC technique and double staining IHC procedure were employed utilizing antibodies against G-CSFR, TF and VEGF associated with VEGFR (VEGF: VEGFR). Expression of G-CSFR was demonstrated in 20 breast cancer tissue specimens (71%). In 6 cases (21%) the expression was strong (IRS 9-12). Strong expression of TF was observed in all investigated cases (100%). Moreover, expression of VEGF: VEGFR was visualized in cancer cells (IRS 5-8). No presence of G-CSFR, TF or VEGF: VEGFR was detected on healthy breast cells. Double staining IHC studies revealed co-localization of G-CSFR and TF, G-CSFR and VEGF: VEGFR, as well as TF and VEGF: VEGFR on breast cancer cells and ECs. The results of the study indicate that GCSFR, TF and VEGF: VEGFR expression as well as their co-expression might influence breast cancer biology, and may increase thromboembolic adverse events incidence.

Tumor Necrosis Factor α Stimulates Osteoclast Differentiation by a Mechanism Independent of the Odf/Rankl–Rank Interaction

PubMed Central

Kobayashi, Kanichiro; Takahashi, Naoyuki; Jimi, Eijiro; Udagawa, Nobuyuki; Takami, Masamichi; Kotake, Shigeru; Nakagawa, Nobuaki; Kinosaki, Masahiko; Yamaguchi, Kyoji; Shima, Nobuyuki; Yasuda, Hisataka; Morinaga, Tomonori; Higashio, Kanji; Martin, T. John; Suda, Tatsuo

2000-01-01

Osteoclast differentiation factor (ODF, also called RANKL/TRANCE/OPGL) stimulates the differentiation of osteoclast progenitors of the monocyte/macrophage lineage into osteoclasts in the presence of macrophage colony-stimulating factor (M-CSF, also called CSF-1). When mouse bone marrow cells were cultured with M-CSF, M-CSF–dependent bone marrow macrophages (M-BMMφ) appeared within 3 d. Tartrate-resistant acid phosphatase–positive osteoclasts were also formed when M-BMMφ were further cultured for 3 d with mouse tumor necrosis factor α (TNF-α) in the presence of M-CSF. Osteoclast formation induced by TNF-α was inhibited by the addition of respective antibodies against TNF receptor 1 (TNFR1) or TNFR2, but not by osteoclastogenesis inhibitory factor (OCIF, also called OPG, a decoy receptor of ODF/RANKL), nor the Fab fragment of anti–RANK (ODF/RANKL receptor) antibody. Experiments using M-BMMφ prepared from TNFR1- or TNFR2-deficient mice showed that both TNFR1- and TNFR2-induced signals were important for osteoclast formation induced by TNF-α. Osteoclasts induced by TNF-α formed resorption pits on dentine slices only in the presence of IL-1α. These results demonstrate that TNF-α stimulates osteoclast differentiation in the presence of M-CSF through a mechanism independent of the ODF/RANKL–RANK system. TNF-α together with IL-1α may play an important role in bone resorption of inflammatory bone diseases. PMID:10637272

Internalization Mechanisms of the Epidermal Growth Factor Receptor after Activation with Different Ligands

PubMed Central

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

2013-01-01

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. PMID:23472148

Stimulus-dependent regulation of nuclear Ca2+ signaling in cardiomyocytes: a role of neuronal calcium sensor-1.

PubMed

Nakao, Shu; Wakabayashi, Shigeo; Nakamura, Tomoe Y

2015-01-01

In cardiomyocytes, intracellular calcium (Ca2+) transients are elicited by electrical and receptor stimulations, leading to muscle contraction and gene expression, respectively. Although such elevations of Ca2+levels ([Ca2+]) also occur in the nucleus, the precise mechanism of nuclear [Ca2+] regulation during different kinds of stimuli, and its relationship with cytoplasmic [Ca2+] regulation are not fully understood. To address these issues, we used a new region-specific fluorescent protein-based Ca2+ indicator, GECO, together with the conventional probe Fluo-4 AM. We confirmed that nuclear Ca2+ transients were elicited by both electrical and receptor stimulations in neonatal mouse ventricular myocytes. Kinetic analysis revealed that electrical stimulation-elicited nuclear Ca2+ transients are slower than cytoplasmic Ca2+ transients, and chelating cytoplasmic Ca2+ abolished nuclear Ca2+ transients, suggesting that nuclear Ca2+ are mainly derived from the cytoplasm during electrical stimulation. On the other hand, receptor stimulation such as with insulin-like growth factor-1 (IGF-1) preferentially increased nuclear [Ca2+] compared to cytoplasmic [Ca2+]. Experiments using inhibitors revealed that electrical and receptor stimulation-elicited Ca2+ transients were mainly mediated by ryanodine receptors and inositol 1,4,5-trisphosphate receptors (IP3Rs), respectively, suggesting different mechanisms for the two signals. Furthermore, IGF-1-elicited nuclear Ca2+ transient amplitude was significantly lower in myocytes lacking neuronal Ca2+ sensor-1 (NCS-1), a Ca2+ binding protein implicated in IP3R-mediated pathway in the heart. Moreover, IGF-1 strengthened the interaction between NCS-1 and IP3R. These results suggest a novel mechanism for receptor stimulation-induced nuclear [Ca2+] regulation mediated by IP3R and NCS-1 that may further fine-tune cardiac Ca2+ signal regulation.

Activin A stimulates IkappaB-alpha/NFkappaB and RANK expression for osteoclast differentiation, but not AKT survival pathway in osteoclast precursors.

PubMed

Sugatani, T; Alvarez, U M; Hruska, K A

2003-09-01

Recent studies have reported that activin A enhances osteoclastogenesis in cultures of mouse bone marrow cells stimulated with receptor activator of nuclear factor-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). However, the exact mechanisms by which activin A functions during osteoclastogenesis are not clear. RANKL stimulation of RANK/TRAF6 signaling increases nuclear factor-kappaB (NFkappaB) nuclear translocation and activates the Akt/PKB cell survival pathway. Here we report that activin A alone activates IkappaB-alpha, and stimulates nuclear translocation of NFkappaB and receptor activator of nuclear factor-kappaB (RANK) expression for osteoclastogenesis, but not Akt/PKB survival signal transduction including BAD and mammalian target of rapamycin (mTOR) for survival in osteoclast precursors in vitro. Activin A alone failed to activate Akt, BAD, and mTOR by immunoblotting, and it also failed to prevent apoptosis in osteoclast precursors. While activin A activated IkappaB-alpha and induced nuclear translocation of phosphorylated-NFkappaB, and it also enhanced RANK expression in osteoclast precursors. Moreover, activin A enhanced RANKL- and M-CSF-stimulated nuclear translocation of NFkappaB. Our data suggest that activin A enhances osteoclastogenesis treated with RANKL and M-CSF via stimulation of RANK, thereby increasing the RANKL stimulation. Activin A alone activated the NFkappaB pathway, but not survival in osteoclast precursors in vitro, but it is, thus, insufficient as a sole stimulus to osteoclastogenesis. Copyright 2003 Wiley-Liss, Inc.

Adrenaline stimulates the proliferation and migration of mesenchymal stem cells towards the LPS-induced lung injury

PubMed Central

Wu, Xiaodan; Wang, Zhiming; Qian, Mengjia; Wang, Lingyan; Bai, Chunxue; Wang, Xiangdong

2014-01-01

Bone marrow-derived mesenchymal stem cells (BMSCs) could modulate inflammation in experimental lung injury. On the other hand, adrenergic receptor agonists could increase DNA synthesis of stem cells. Therefore, we investigated the therapeutic role of adrenaline-stimulated BMSCs on lipopolysaccharide (LPS)-induced lung injury. BMSCs were cultured with adrenergic receptor agonists or antagonists. Suspensions of lung cells or sliced lung tissue from animals with or without LPS-induced injury were co-cultured with BMSCs. LPS-stimulated alveolar macrophages were co-cultured with BMSCs (with adrenaline stimulation or not) in Transwell for 6 hrs. A preliminary animal experiment was conducted to validate the findings in ex vivo study. We found that adrenaline at 10 μM enhanced proliferation of BMSCs through both α- and β-adrenergic receptors. Adrenaline promoted the migration of BMSCs towards LPS-injured lung cells or lung tissue. Adrenaline-stimulated BMSCs decreased the inflammation of LPS-stimulated macrophages, probably through the expression and secretion of several paracrine factors. Adrenaline reduced the extent of injury in LPS-injured rats. Our data indicate that adrenaline-stimulated BMSCs might contribute to the prevention from acute lung injury through the activation of adrenergic receptors, promotion of proliferation and migration towards injured lung, and modulation of inflammation. PMID:24684532

Regulation of fibroblast growth factor receptor signalling and trafficking by Src and Eps8.

PubMed

Auciello, Giulio; Cunningham, Debbie L; Tatar, Tulin; Heath, John K; Rappoport, Joshua Z

2013-01-15

Fibroblast growth factor receptors (FGFRs) mediate a wide spectrum of cellular responses that are crucial for development and wound healing. However, aberrant FGFR activity leads to cancer. Activated growth factor receptors undergo stimulated endocytosis, but can continue to signal along the endocytic pathway. Endocytic trafficking controls the duration and intensity of signalling, and growth factor receptor signalling can lead to modifications of trafficking pathways. We have developed live-cell imaging methods for studying FGFR dynamics to investigate mechanisms that coordinate the interplay between receptor trafficking and signal transduction. Activated FGFR enters the cell following recruitment to pre-formed clathrin-coated pits (CCPs). However, FGFR activation stimulates clathrin-mediated endocytosis; FGF treatment increases the number of CCPs, including those undergoing endocytosis, and this effect is mediated by Src and its phosphorylation target Eps8. Eps8 interacts with the clathrin-mediated endocytosis machinery and depletion of Eps8 inhibits FGFR trafficking and immediate Erk signalling. Once internalized, FGFR passes through peripheral early endosomes en route to recycling and degredative compartments, through an Src- and Eps8-dependent mechanism. Thus Eps8 functions as a key coordinator in the interplay between FGFR signalling and trafficking. This work provides the first detailed mechanistic analysis of growth factor receptor clustering at the cell surface through signal transduction and endocytic trafficking. As we have characterised the Src target Eps8 as a key regulator of FGFR signalling and trafficking, and identified the early endocytic system as the site of Eps8-mediated effects, this work provides novel mechanistic insight into the reciprocal regulation of growth factor receptor signalling and trafficking.

Structural Basis for Activation of the Receptor Tyrosine Kinase KIT by Stem Cell Factor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yuzawa,S.; Opatowsky, Y.; Zhang, Z.

2007-01-01

Stem Cell Factor (SCF) initiates its multiple cellular responses by binding to the ectodomain of KIT, resulting in tyrosine kinase activation. We describe the crystal structure of the entire ectodomain of KIT before and after SCF stimulation. The structures show that KIT dimerization is driven by SCF binding whose sole role is to bring two KIT molecules together. Receptor dimerization is followed by conformational changes that enable lateral interactions between membrane proximal Ig-like domains D4 and D5 of two KIT molecules. Experiments with cultured cells show that KIT activation is compromised by point mutations in amino acids critical for D4-D4more » interaction. Moreover, a variety of oncogenic mutations are mapped to the D5-D5 interface. Since key hallmarks of KIT structures, ligand-induced receptor dimerization, and the critical residues in the D4-D4 interface, are conserved in other receptors, the mechanism of KIT stimulation unveiled in this report may apply for other receptor activation.« less

Granulocyte colony-stimulating factor enhances protection by anti-K1 capsular IgM antibody in murine Escherichia coli sepsis.

PubMed

Hustinx, W; Benaissa-Trouw, B; Van Kessel, K; Kuenen, J; Tavares, L; Kraaijeveld, K; Verhoef, J; Hoepelman, A

1997-12-01

Combined prophylactic treatment with recombinant murine granulocyte colony-stimulating factor (G-CSF) and a suboptimal dose of anti-K1 capsular IgM monoclonal antibody (MAb) significantly enhanced survival in an experimental mouse Escherichia coli O7:K1 peritonitis model compared with untreated animals (67% vs. 11% survival; P < 0.001) and with either treatment alone (67 vs. 29% and 27% survival, respectively; P < 0.01), which suggests synergism between these agents. Enhanced survival by combined treatment was associated with increased neutrophil counts in blood and peritoneal lavage fluid, lower systemic and higher levels of local tumour necrosis factor (TNF) and lower bacterial counts in blood cultures. Mouse neutrophils treated with G-CSF but not infected with E. coli showed enhanced phagocytic and respiratory burst capacity, down-regulation of L-selectin receptors and enhanced expression of Fc RII-III receptors but not of complement receptors.

Recent Advances of Colony-Stimulating Factor-1 Receptor (CSF-1R) Kinase and Its Inhibitors.

PubMed

El-Gamal, Mohammed I; Al-Ameen, Shahad K; Al-Koumi, Dania M; Hamad, Mawadda G; Jalal, Nouran A; Oh, Chang-Hyun

2018-01-17

Colony stimulation factor-1 receptor (CSF-1R), which is also known as FMS kinase, plays an important role in initiating inflammatory, cancer, and bone disorders when it is overstimulated by its ligand, CSF-1. Innate immunity, as well as macrophage differentiation and survival, are regulated by the stimulation of the CSF-1R. Another ligand, interlukin-34 (IL-34), was recently reported to activate the CSF-1R receptor in a different manner. The relationship between CSF-1R and microglia has been reviewed. Both CSF-1 antibodies and small molecule CSF-1R kinase inhibitors have now been tested in animal models and in humans. In this Perspective, we discuss the role of CSF-1 and IL-34 in producing cancer, bone disorders, and inflammation. We also review the newly discovered and improved small molecule kinase inhibitors and monoclonal antibodies that have shown potent activity toward CSF-1R, reported from 2012 until 2017.

Alkyl isothiocyanates suppress epidermal growth factor receptor kinase activity but augment tyrosine kinase activity.

PubMed

Nomura, Takahiro; Uehara, Yoshimasa; Kawajiri, Hiroo; Ryoyama, Kazuo; Yamori, Takao; Fuke, Yoko

2009-10-01

We have reported the in vitro and in vivo anticancer activities of 6-(methylsulfinyl)hexyl isothiocyanate (6-MITC) derived from a Japanese spice, wasabi. In order to obtain some clues about the mechanism of the anticancer activity, we have studied the effect of alkyl isothiocyanates (MITCs) on protein kinase activities. The anti-autophosphorylation activity of MITCs with respect to the epidermal growth factor (EGF)-stimulated receptor kinase of A431 epidermoid carcinoma cells was examined by incorporation of radioactive ATP into an acid-insoluble fraction. Their anti-phosphorylation activity with respect to the non-receptor protein kinase was analyzed by a standard SDS-PAGE method. All the tested MITCs interfered with the EGF-stimulated receptor kinase activity in a dose-dependent manner, although their effects were less than 1/10 of that of erbstatin in microg/ml. On the other hand, the MITCs did not interfere with non-receptor kinases (kinase A, kinase C, tyrosine kinase and calmodulin dependent kinase III), but enhanced non-receptor tyrosine kinase. A possible anticancer mechanism of MITCs may involve the suppression of EGF receptor kinase activity and augmentation of non-receptor PTK.

Anti-fibrotic efficacy of nintedanib in pulmonary fibrosis via the inhibition of fibrocyte activity.

PubMed

Sato, Seidai; Shinohara, Shintaro; Hayashi, Shinya; Morizumi, Shun; Abe, Shuichi; Okazaki, Hiroyasu; Chen, Yanjuan; Goto, Hisatsugu; Aono, Yoshinori; Ogawa, Hirohisa; Koyama, Kazuya; Nishimura, Haruka; Kawano, Hiroshi; Toyoda, Yuko; Uehara, Hisanori; Nishioka, Yasuhiko

2017-09-15

Nintedanib, a tyrosine kinase inhibitor that is specific for platelet-derived growth factor receptors (PDGFR), fibroblast growth factor receptors (FGFR), and vascular endothelial growth factor receptors (VEGFR), has recently been approved for idiopathic pulmonary fibrosis. Fibrocytes are bone marrow-derived progenitor cells that produce growth factors and contribute to fibrogenesis in the lungs. However, the effects of nintedanib on the functions of fibrocytes remain unclear. Human monocytes were isolated from the peripheral blood of healthy volunteers. The expression of growth factors and their receptors in fibrocytes was analyzed using ELISA and Western blotting. The effects of nintedanib on the ability of fibrocytes to stimulate lung fibroblasts were examined in terms of their proliferation. The direct effects of nintedanib on the differentiation and migration of fibrocytes were also assessed. We investigated whether nintedanib affected the accumulation of fibrocytes in mouse lungs treated with bleomycin. Human fibrocytes produced PDGF, FGF2, and VEGF-A. Nintedanib and specific inhibitors for each growth factor receptor significantly inhibited the proliferation of lung fibroblasts stimulated by the supernatant of fibrocytes. Nintedanib inhibited the migration and differentiation of fibrocytes induced by growth factors in vitro. The number of fibrocytes in the bleomycin-induced lung fibrosis model was reduced by the administration of nintedanib, and this was associated with anti-fibrotic effects. These results support the role of fibrocytes as producers of and responders to growth factors, and suggest that the anti-fibrotic effects of nintedanib are at least partly mediated by suppression of fibrocyte function.

Substance P - Neurokinin-1 Receptor Interaction Upregulates Monocyte Tissue Factor

PubMed Central

Khan, Mohammad M; Douglas, Steven D; Benton, Tami D

2011-01-01

Monocytes play an important role in hemostasis. In this study, the prothrombotic effects of the neuropeptide substance P (SP) on human monocytes through neurokinin-1 receptor (NK1-R) were characterized. SP upregulated monocyte tissue factor (TF), the major coagulation cascade stimulator, in a concentration and time dependent manner. Specific inhibition of NK1-R completely blocked TF expression. Monocytes stimulated by SP released cytokines and chemokines. When monocytes were stimulated with cytokines or chemokines, TF was expressed by the cytokines (GM-CSF, IFN-γ and TNF-α). Cytokines may play a major role in the mechanism of SP induced monocyte TF expression. NK1-R antagonists (NK1-RA) may have a role in developing novel therapeutic approaches to patients vulnerable to vaso-occlusive disorders. PMID:22115773

Ubiquitination of basal VEGFR2 regulates signal transduction and endothelial function

PubMed Central

Smith, Gina A.; Fearnley, Gareth W.; Abdul-Zani, Izma; Wheatcroft, Stephen B.; Tomlinson, Darren C.; Harrison, Michael A.

2017-01-01

ABSTRACT Cell surface receptors can undergo recycling or proteolysis but the cellular decision-making events that sort between these pathways remain poorly defined. Vascular endothelial growth factor A (VEGF-A) and vascular endothelial growth factor receptor 2 (VEGFR2) regulate signal transduction and angiogenesis, but how signaling and proteolysis is regulated is not well understood. Here, we provide evidence that a pathway requiring the E1 ubiquitin-activating enzyme UBA1 controls basal VEGFR2 levels, hence metering plasma membrane receptor availability for the VEGF-A-regulated endothelial cell response. VEGFR2 undergoes VEGF-A-independent constitutive degradation via a UBA1-dependent ubiquitin-linked pathway. Depletion of UBA1 increased VEGFR2 recycling from endosome-to-plasma membrane and decreased proteolysis. Increased membrane receptor availability after UBA1 depletion elevated VEGF-A-stimulated activation of key signaling enzymes such as PLCγ1 and ERK1/2. Although UBA1 depletion caused an overall decrease in endothelial cell proliferation, surviving cells showed greater VEGF-A-stimulated responses such as cell migration and tubulogenesis. Our study now suggests that a ubiquitin-linked pathway regulates the balance between receptor recycling and degradation which in turn impacts on the intensity and duration of VEGF-A-stimulated signal transduction and the endothelial response. PMID:28798148

Stimulation by thyroid-stimulating hormone and Grave's immunoglobulin G of vascular endothelial growth factor mRNA expression in human thyroid follicles in vitro and flt mRNA expression in the rat thyroid in vivo.

PubMed

Sato, K; Yamazaki, K; Shizume, K; Kanaji, Y; Obara, T; Ohsumi, K; Demura, H; Yamaguchi, S; Shibuya, M

1995-09-01

To elucidate the pathogenesis of thyroid gland hypervascularity in patients with Graves' disease, we studied the expression of mRNAs for vascular endothelial growth factor (VEGF) and its receptor, Flt family, using human thyroid follicles in vitro and thiouracil-fed rats in vivo. Human thyroid follicles, cultured in the absence of endothelial cells, secreted de novo-synthesized thyroid hormone in response to thyroid-stimulating hormone (TSH) and Graves' IgG. The thyroid follicles produced VEGF mRNA but not flt-1 mRNA. The expression of VEGF mRNA was enhanced by insulin, tumor-promoting phorbol ester, calcium ionophore, dibutyryl cAMP, TSH, and Graves' IgG. When rats were fed thiouracil for 4 wk, their serum levels of TSH were increased at day 3. VEGF mRNA was also increased on day 3, accompanied by an increase in flt family (flt-1 and KDR/ flk-1) mRNA expression. These in vitro and in vivo findings suggest that VEGF is produced by thyroid follicles in response to stimulators of TSH receptors, via the protein kinase A and C pathways. VEGF, a secretable angiogenesis factor, subsequently stimulates Flt receptors on endothelial cells in a paracrine manner, leading to their proliferation and producing hypervascularity of the thyroid gland, as seen in patients with Graves' disease.

Estradiol Is a Critical Mediator of Macrophage-Nerve Cross Talk in Peritoneal Endometriosis

PubMed Central

Greaves, Erin; Temp, Julia; Esnal-Zufiurre, Arantza; Mechsner, Sylvia; Horne, Andrew W.; Saunders, Philippa T.K.

2016-01-01

Endometriosis occurs in approximately 10% of women and is associated with persistent pelvic pain. It is defined by the presence of endometrial tissue (lesions) outside the uterus, most commonly on the peritoneum. Peripheral neuroinflammation, a process characterized by the infiltration of nerve fibers and macrophages into lesions, plays a pivotal role in endometriosis-associated pain. Our objective was to determine the role of estradiol (E2) in regulating the interaction between macrophages and nerves in peritoneal endometriosis. By using human tissues and a mouse model of endometriosis, we demonstrate that macrophages in lesions recovered from women and mice are immunopositive for estrogen receptor β, with up to 20% being estrogen receptor α positive. In mice, treatment with E2 increased the number of macrophages in lesions as well as concentrations of mRNAs encoded by Csf1, Nt3, and the tyrosine kinase neurotrophin receptor, TrkB. By using in vitro models, we determined that the treatment of rat dorsal root ganglia neurons with E2 increased mRNA concentrations of the chemokine C-C motif ligand 2 that stimulated migration of colony-stimulating factor 1–differentiated macrophages. Conversely, incubation of colony-stimulating factor 1 macrophages with E2 increased concentrations of brain-derived neurotrophic factor and neurotrophin 3, which stimulated neurite outgrowth from ganglia explants. In summary, we demonstrate a key role for E2 in stimulating macrophage-nerve interactions, providing novel evidence that endometriosis is an estrogen-dependent neuroinflammatory disorder. PMID:26073038

Neuropeptides activate human mast cell degranulation and chemokine production

PubMed Central

Kulka, Marianna; Sheen, Cecilia H; Tancowny, Brian P; Grammer, Leslie C; Schleimer, Robert P

2008-01-01

During neuronal-induced inflammation, mast cells may respond to stimuli such as neuropeptides in an FcεRI-independent manner. In this study, we characterized human mast cell responses to substance P (SP), nerve growth factor (NGF), calcitonin gene-related peptide (CGRP) and vasoactive intestinal polypeptide (VIP) and compared these responses to human mast cell responses to immunoglobulin E (IgE)/anti-IgE and compound 48/80. Primary cultured mast cells, generated from CD34+ progenitors in the presence of stem cell factor and interleukin-6 (IL-6), and human cultured mast cells (LAD2) were stimulated with these and other stimuli (gastrin, concanavalin A, radiocontrast media, and mannitol) and their degranulation and chemokine production was assessed. VIP and SP stimulated primary human mast cells and LAD cells to degranulate; gastrin, concanavalin A, radiocontrast media, mannitol, CGRP and NGF did not activate degranulation. While anti-IgE stimulation did not induce significant production of chemokines, stimulation with VIP, SP or compound 48/80 potently induced production of monocyte chemoattractant protein-1, inducible protein-10, monokine induced by interferon-γ (MIG), RANTES (regulated on activation, normal, T-cell expressed, and secreted) and IL-8. VIP, SP and compound 48/80 also activated release of tumour necrosis factor, IL-3 and granulocyte–macrophage colony-stimulating factor, but not IL-4, interferon-γ or eotaxin. Human mast cells expressed surface neurokinin 1 receptor (NK1R), NK2R, NK3R and VIP receptor type 2 (VPAC2) but not VPAC1 and activation of human mast cells by IgE/anti-IgE up-regulated expression of VPAC2, NK2R, and NK3R. These studies demonstrate the pattern of receptor expression and activation of mast cell by a host of G-protein coupled receptor ligands and suggest that SP and VIP activate a unique signalling pathway in human mast cells. These results are likely to have direct relevance to neuronally induced inflammatory diseases. PMID:17922833

Expression of platelet-derived growth factor and its receptors in proliferative disorders of fibroblastic origin.

PubMed

Smits, A; Funa, K; Vassbotn, F S; Beausang-Linder, M; af Ekenstam, F; Heldin, C H; Westermark, B; Nistér, M

1992-03-01

Platelet-derived growth factor (PDGF) is known to stimulate the proliferation of connective tissue-derived cells in vitro. Less is known about its functions in vivo, and the role of PDGF in the development of human tumors has not been clarified. The authors have investigated the occurrence of PDGF and PDGF receptors in a series of proliferative disorders of fibroblastic origin using immunohistochemical and in situ hybridization techniques. High expression of PDGF beta-receptor mRNA and protein was found in the malignant tumors, and also in some benign lesions, such as dermatofibroma. In all these cases, benign as well as malignant, the PDGF B-chain mRNA, and less clearly, the PDGF A-chain mRNA, were coexpressed with the beta-receptor. In contrast, high expression of PDGF alpha-receptor mRNA was only found in fully malignant lesions, i.e., malignant fibrous histiocytoma. These data indicate that an autocrine growth stimulation via the PDGF beta-receptor could occur in an early phase of tumorigenesis, and may be a necessary but insufficient event for the progression into fully malignant human connective tissue lesions.

Substance P reduces TNF-α-induced apoptosis in human tenocytes through NK-1 receptor stimulation.

PubMed

Backman, Ludvig J; Eriksson, Daniella E; Danielson, Patrik

2014-10-01

It has been hypothesised that an upregulation of the neuropeptide substance P (SP) and its preferred receptor, the neurokinin-1 receptor (NK-1 R), is a causative factor in inducing tenocyte hypercellularity, a characteristic of tendinosis, through both proliferative and antiapoptotic stimuli. We have demonstrated earlier that SP stimulates proliferation of human tenocytes in culture. The aim of this study was to investigate whether SP can mediate an antiapoptotic effect in tumour necrosis factor-α (TNF-α)-induced apoptosis of human tenocytes in vitro. A majority (approximately 75%) of tenocytes in culture were immunopositive for TNF Receptor-1 and TNF Receptor-2. Exposure of the cells to TNF-α significantly decreased cell viability, as shown with crystal violet staining. TNF-α furthermore significantly increased the amount of caspase-10 and caspase-3 mRNA, as well as both BID and cleaved-poly ADP ribosome polymerase (c-PARP) protein. Incubation of SP together with TNF-α resulted in a decreased amount of BID and c-PARP, and in a reduced lactate dehydrogenase release, as compared to incubation with TNF-α alone. The SP effect was blocked with a NK-1 R inhibitor. This study shows that SP, through stimulation of the NK-1 R, has the ability to reduce TNF-α-induced apoptosis of human tenocytes. Considering that SP has previously been shown to stimulate tenocyte proliferation, the study confirms SP as a potent regulator of cell-turnover in tendon tissue, capable of stimulating hypercellularity through different mechanisms. This gives further support for the theory that the upregulated amount of SP seen in tendinosis could contribute to hypercellularity. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Controlled and reversible induction of differentiation and activation of adult human hepatocytes by a biphasic culture technique

PubMed Central

Auth, Marcus K.H.; Boost, Kim A.; Leckel, Kerstin; Beecken, Wolf-Dietrich; Engl, Tobias; Jonas, Dietger; Oppermann, Elsie; Hilgard, Philip; Markus, Bernd H.; Bechstein, Wolf-Otto; Blaheta, Roman A.

2005-01-01

AIM: Clinical application of human hepatocytes (HC) is hampered by the progressive loss of growth and differentiation in vitro. The object of the study was to evaluate the effect of a biphasic culture technique on expression and activation of growth factor receptors and differentiation of human adult HC. METHODS: Isolated HC were sequentially cultured in a hormone enriched differentiation medium (DM) containing nicotinamide, insulin, transferrin, selenium, and dexame-thasone or activation medium (AM) containing hepatocyte growth factor (HGF), epidermal growth factor (EGF), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Expression, distribution and activation of the HC receptors (MET and EGFR) and the pattern of characteristic cytokeratin (CK) filaments were measured by fluorometry, confocal microscopy and Western blotting. RESULTS: In the biphasic culture system, HC underwent repeated cycles of activation (characterized by expression and activation of growth factor receptors) and re-differentiation (illustrated by distribution of typical filaments CK-18 but low or absent expression of CK-19). In AM increased expression of MET and EGFR was associated with receptor translocation into the cytoplasm and induction of atypical CK-19. In DM low expression of MET and EGFR was localized on the cell membrane and CK-19 was reduced. Receptor phosphorylation required embedding of HC in collagen type I gel. CONCLUSION: Control and reversible modulation of growth factor receptor activation of mature human HC can be accomplished in vitro, when defined signals from the extracellular matrix and sequential growth stimuli are provided. The biphasic technique helps overcome de-differentiation, which occurs during continuous stimulation by means of growth factors. PMID:15810072

Controlled and reversible induction of differentiation and activation of adult human hepatocytes by a biphasic culture technique.

PubMed

Auth, Marcus-K H; Boost, Kim A; Leckel, Kerstin; Beecken, Wolf-Dietrich; Engl, Tobias; Jonas, Dietger; Oppermann, Elsie; Hilgard, Philip; Markus, Bernd H; Bechstein, Wolf-Otto; Blaheta, Roman A

2005-04-14

Clinical application of human hepatocytes (HC) is hampered by the progressive loss of growth and differentiation in vitro. The object of the study was to evaluate the effect of a biphasic culture technique on expression and activation of growth factor receptors and differentiation of human adult HC. Isolated HC were sequentially cultured in a hormone enriched differentiation medium (DM) containing nicotinamide, insulin, transferrin, selenium, and dexame-thasone or activation medium (AM) containing hepatocyte growth factor (HGF), epidermal growth factor (EGF), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Expression, distribution and activation of the HC receptors (MET and EGFR) and the pattern of characteristic cytokeratin (CK) filaments were measured by fluorometry, confocal microscopy and Western blotting. In the biphasic culture system, HC underwent repeated cycles of activation (characterized by expression and activation of growth factor receptors) and re-differentiation (illustrated by distribution of typical filaments CK-18 but low or absent expression of CK-19). In AM increased expression of MET and EGFR was associated with receptor translocation into the cytoplasm and induction of atypical CK-19. In DM low expression of MET and EGFR was localized on the cell membrane and CK-19 was reduced. Receptor phosphorylation required embedding of HC in collagen type I gel. Control and reversible modulation of growth factor receptor activation of mature human HC can be accomplished in vitro, when defined signals from the extracellular matrix and sequential growth stimuli are provided. The biphasic technique helps overcome de-differentiation, which occurs during continuous stimulation by means of growth factors.

Identification of a new adapter protein that may link the common beta subunit of the receptor for granulocyte/macrophage colony-stimulating factor, interleukin (IL)-3, and IL-5 to phosphatidylinositol 3-kinase.

PubMed

Jücker, M; Feldman, R A

1995-11-17

Binding of human granulocyte/macrophage colony-stimulating factor (hGM-CSF) to its receptor induces the rapid activation of phosphatidylinositol-3 kinase (PI 3-kinase). As hGM-CSF receptor (hGMR) does not contain a consensus sequence for binding of PI 3-kinase, hGMR must use a distinct mechanism for its association with and activation of PI 3-kinase. Here, we describe the identification of a tyrosine-phosphorylated protein of 76-85 kDa (p80) that associates with the common beta subunit of hGMR and with the SH2 domains of the p85 subunit of PI 3-kinase in hGM-CSF-stimulated cells. Src/Yes and Lyn were tightly associated with the p80.PI 3-kinase complex, suggesting that p80 and other phosphotyrosyl proteins present in the complex were phosphorylated by Src family kinases. Tyrosine phosphorylation of p80 was only detected in hGM-CSF or human interleukin-3-stimulated cells, suggesting that activation of p80 might be specific for signaling via the common beta subunit. We postulate that p80 functions as an adapter protein that may participate in linking the hGM-CSF receptor to the PI 3-kinase signaling pathway.

Cellular localization of the activated EGFR determines its effect on cell growth in MDA-MB-468 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hyatt, Dustin C.; Ceresa, Brian P.

2008-11-01

The epidermal growth factor (EGF) receptor (EGFR) is a ubiquitously expressed receptor tyrosine kinase that regulates diverse cell functions that are dependent upon cell type, the presence of downstream effectors, and receptor density. In addition to activating biochemical pathways, ligand stimulation causes the EGFR to enter the cell via clathrin-coated pits. Endocytic trafficking influences receptor signaling by controlling the duration of EGFR phosphorylation and coordinating the receptor's association with downstream effectors. To better understand the individual contributions of cell surface and cytosolic EGFRs on cell physiology, we used EGF that was conjugated to 900 nm polystyrene beads (EGF-beads). EGF-beads canmore » stimulate the EGFR and retain the activated receptor at the plasma membrane. In MDA-MB-468 cells, a breast cancer cell line that over-expresses the EGFR, only internalized, activated EGFRs stimulate caspase-3 and induce cell death. Conversely, signaling cascades triggered from activated EGFR retained at the cell surface inhibit caspase-3 and promote cell proliferation. Thus, through endocytosis, the activated EGFR can differentially regulate cell growth in MDA-MB-468 cells.« less

Platelet-activating factor and group I metabotropic glutamate receptors interact for full development and maintenance of long-term potentiation in the rat medial vestibular nuclei.

PubMed

Grassi, S; Francescangeli, E; Goracci, G; Pettorossi, V E

1999-01-01

In rat brainstem slices, we investigated the interaction between platelet-activating factor and group I metabotropic glutamate receptors in mediating long-term potentiation within the medial vestibular nuclei. We analysed the N1 field potential wave evoked in the ventral portion of the medial vestibular nuclei by primary vestibular afferent stimulation. The group I metabotropic glutamate receptor antagonist, (R,S)-1-aminoindan-1,5-dicarboxylic acid, prevented long-term potentiation induced by a platelet-activating factor analogue [1-O-hexadecyl-2-O-(methylcarbamyl)-sn-glycero-3-phosphocholine], as well as the full development of potentiation, induced by high-frequency stimulation under the blocking agent for synaptosomal platelet-activating factor receptors (ginkolide B), at drug washout. However, potentiation directly induced by the group I glutamate metabotropic receptor agonist, (R,S)-3,5-dihydroxyphenylglycine, was reduced by ginkolide B. These findings suggest that platelet-activating factor, whether exogenous or released following potentiation induction, exerts its effect through presynaptic group I metabotropic glutamate receptors, mediating the increase of glutamate release. In addition, we found that this mechanism, which led to full potentiation through presynaptic group I metabotropic glutamate receptor activation, was inactivated soon after application of potentiation-inducing stimulus. In fact, the long-lasting block of the platelet-activating factor and metabotropic glutamate receptors prevented the full potentiation development and the induced potentiation progressively declined to null. Moreover, ginkolide B, given when high-frequency-dependent potentiation was established, only reduced it within 5 min after potentiation induction. We conclude that to fully develop vestibular long-term potentiation requires presynaptic events. Platelet-activating factor, released after the activation of postsynaptic mechanisms which induce potentiation, is necessary for coupling postsynaptic and presynaptic phenomena, through the activation of group I metabotropic glutamate receptors, and its action lasts only for a short period. If this coupling does not occur, a full and long-lasting potentiation cannot develop.

Nuclear Receptor Activity and Liver Cancer Lesion Progression

EPA Science Inventory

Nuclear receptors (NRs) are ligand-activated transcription factors that control diverse cellular processes. Chronic stimulation of some NRs is a non-genotoxic mechanism of rodent liver cancer with unclear relevance to humans. We explored this question using human CAR, PXR, PPARα,...

Effects of vitamin D(3)-binding protein-derived macrophage activating factor (GcMAF) on angiogenesis.

PubMed

Kanda, Shigeru; Mochizuki, Yasushi; Miyata, Yasuyoshi; Kanetake, Hiroshi; Yamamoto, Nobuto

2002-09-04

The vitamin D(3)-binding protein (Gc protein)-derived macrophage activating factor (GcMAF) activates tumoricidal macrophages against a variety of cancers indiscriminately. We investigated whether GcMAF also acts as an antiangiogenic factor on endothelial cells. The effects of GcMAF on angiogenic growth factor-induced cell proliferation, chemotaxis, and tube formation were examined in vitro by using cultured endothelial cells (murine IBE cells, porcine PAE cells, and human umbilical vein endothelial cells [HUVECs]) and in vivo by using a mouse cornea micropocket assay. Blocking monoclonal antibodies to CD36, a receptor for the antiangiogenic factor thrombospondin-1, which is also a possible receptor for GcMAF, were used to investigate the mechanism of GcMAF action. GcMAF inhibited the endothelial cell proliferation, chemotaxis, and tube formation that were all stimulated by fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor-A, or angiopoietin 2. FGF-2-induced neovascularization in murine cornea was also inhibited by GcMAF. Monoclonal antibodies against murine and human CD36 receptor blocked the antiangiogenic action of GcMAF on the angiogenic factor stimulation of endothelial cell chemotaxis. In addition to its ability to activate tumoricidal macrophages, GcMAF has direct antiangiogenic effects on endothelial cells independent of tissue origin. The antiangiogenic effects of GcMAF may be mediated through the CD36 receptor.

Specific growth stimulation of cultured smooth muscle cells from spontaneously hypertensive rats by platelet-derived growth factor A-chain homodimer.

PubMed Central

Resink, T J; Scott-Burden, T; Hahn, A W; Rouge, M; Hosang, M; Powell, J S; Bühler, F R

1990-01-01

Cultured vascular smooth muscle cells (VSMC)1 from spontaneously hypertensive rats (SHR) possess specific cell surface receptors for both homodimeric forms of platelet-derived growth factor (PDGF-AA and PDGF-BB), in contrast to cells from normotensive Wistar Kyoto (WKY) animals, which express receptors only for the B-chain form of PDGF. Stimulation of quiescent VSMC from SHR with PDGF-AA resulted in activation of S6-kinase and induction of phosphoinositide catabolism, as well as cellular proliferation when cultures were maintained for prolonged periods with daily supplementation of the growth factor. WKY-derived VSMC showed no response to PDGF-AA, which was consistent with their lack of specific receptors for this homodimer. The responsiveness of quiescent cells from SHR and WKY to the B-chain homodimer was similar. The enhanced growth responsiveness of SHR-derived cells to fetal calf serum, as compared with cells from their normotensive counterparts, may be accounted for in part by their expression of receptors for the AA homodimer of PDGF. PMID:1965150

Prostaglandin E2 produced by Entamoeba histolytica binds to EP4 receptors and stimulates interleukin-8 production in human colonic cells.

PubMed

Dey, Indranil; Chadee, Kris

2008-11-01

Entamoeba histolytica pathogenesis in the colon occurs in a stepwise fashion. It begins with colonization of the mucin layer, which is followed by stimulation of a proinflammatory response that causes nonspecific tissue damage that may facilitate parasite invasion of the underlying colonic mucosa. Unfortunately, the parasite and/or host factors that stimulate a proinflammatory response in the gut are poorly understood. In this study, we found that live E. histolytica or secretory or proteins (SP) and soluble ameba components (SAP) can markedly increase interleukin-8 (IL-8) mRNA expression and protein production in colonic epithelial cells. The IL-8-stimulating molecule produced by live amebae was identified as prostaglandin E(2) (PGE(2)) as trophozoites treated with cyclooxygenase inhibitors inhibited the biosynthesis of PGE(2) and eliminated IL-8 production induced by live parasites or ameba components. Moreover, using specific prostaglandin EP2 and EP4 receptor agonists and antagonists, we found that PGE(2) binds exclusively through EP4 receptors in colonic epithelial cells to stimulate IL-8 production. Silencing of EP4 receptors with EP4 small interfering RNA completely eliminated SP- and SAP-induced IL-8 production. These studies identified bioactive PGE(2) as a one of the major virulence factors produced by E. histolytica that can stimulate the potent neutrophil chemokine and activator IL-8, which can trigger an acute host inflammatory response. Thus, the induction of IL-8 production in response to E. histolytica-derived PGE(2) may be a mechanism that explains the initiation and amplification of acute inflammation associated with intestinal amebiasis.

Adrenaline stimulates the proliferation and migration of mesenchymal stem cells towards the LPS-induced lung injury.

PubMed

Wu, Xiaodan; Wang, Zhiming; Qian, Mengjia; Wang, Lingyan; Bai, Chunxue; Wang, Xiangdong

2014-08-01

Bone marrow-derived mesenchymal stem cells (BMSCs) could modulate inflammation in experimental lung injury. On the other hand, adrenergic receptor agonists could increase DNA synthesis of stem cells. Therefore, we investigated the therapeutic role of adrenaline-stimulated BMSCs on lipopolysaccharide (LPS)-induced lung injury. BMSCs were cultured with adrenergic receptor agonists or antagonists. Suspensions of lung cells or sliced lung tissue from animals with or without LPS-induced injury were co-cultured with BMSCs. LPS-stimulated alveolar macrophages were co-cultured with BMSCs (with adrenaline stimulation or not) in Transwell for 6 hrs. A preliminary animal experiment was conducted to validate the findings in ex vivo study. We found that adrenaline at 10 μM enhanced proliferation of BMSCs through both α- and β-adrenergic receptors. Adrenaline promoted the migration of BMSCs towards LPS-injured lung cells or lung tissue. Adrenaline-stimulated BMSCs decreased the inflammation of LPS-stimulated macrophages, probably through the expression and secretion of several paracrine factors. Adrenaline reduced the extent of injury in LPS-injured rats. Our data indicate that adrenaline-stimulated BMSCs might contribute to the prevention from acute lung injury through the activation of adrenergic receptors, promotion of proliferation and migration towards injured lung, and modulation of inflammation. © 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

A century old renin-angiotensin system still grows with endless possibilities: AT1 receptor signaling cascades in cardiovascular physiopathology.

PubMed

Balakumar, Pitchai; Jagadeesh, Gowraganahalli

2014-10-01

Ang II, the primary effector pleiotropic hormone of the renin-angiotensin system (RAS) cascade, mediates physiological control of blood pressure and electrolyte balance through its action on vascular tone, aldosterone secretion, renal sodium absorption, water intake, sympathetic activity and vasopressin release. It affects the function of most of the organs far beyond blood pressure control including heart, blood vessels, kidney and brain, thus, causing both beneficial and deleterious effects. However, the protective axis of the RAS composed of ACE2, Ang (1-7), alamandine, and Mas and MargD receptors might oppose some harmful effects of Ang II and might promote beneficial cardiovascular effects. Newly identified RAS family peptides, Ang A and angioprotectin, further extend the complexities in understanding the cardiovascular physiopathology of RAS. Most of the diverse actions of Ang II are mediated by AT1 receptors, which couple to classical Gq/11 protein and activate multiple downstream signals, including PKC, ERK1/2, Raf, tyrosine kinases, receptor tyrosine kinases (EGFR, PDGF, insulin receptor), nuclear factor κB and reactive oxygen species (ROS). Receptor activation via G12/13 stimulates Rho-kinase, which causes vascular contraction and hypertrophy. The AT1 receptor activation also stimulates G protein-independent signaling pathways such as β-arrestin-mediated MAPK activation and Src-JAK/STAT. AT1 receptor-mediated activation of NADPH oxidase releases ROS, resulting in the activation of pro-inflammatory transcription factors and stimulation of small G proteins such as Ras, Rac and RhoA. The components of the RAS and the major Ang II-induced signaling cascades of AT1 receptors are reviewed. Copyright © 2014 Elsevier Inc. All rights reserved.

Using Nuclear Receptor Activity to Stratify Hepatocarcinogens

EPA Science Inventory

Nuclear receptors (NR) are a superfamily of ligand-activated transcription factors that control a range of cellular processes. Persistent stimulation of some NR is a non-genotoxic mechanism of rodent liver cancer with unclear relevance to humans. Here we report on a systematic an...

LPS receptor CD14 participates in release of TNF-alpha in RAW 264.7 and peritoneal cells but not in kupffer cells.

PubMed

Lichtman, S N; Wang, J; Lemasters, J J

1998-07-01

Lipopolysaccharide (LPS) is a bacterial polymer that stimulates macrophages to release tumor necrosis factor-alpha (TNF-alpha). In macrophages (RAW 264.7 and peritoneal cells), LPS binds to the CD14 surface receptor as the first step toward signaling. Liver macrophages, Kupffer cells, are the most numerous fixed-tissue macrophage in the body. The presence of CD14 on Kupffer cells and its role in LPS stimulation of TNF-alpha were examined. TNF-alpha release by Kupffer cells after LPS stimulation was the same in the presence and absence of serum. RAW 264.7 and peritoneal cells, which utilize the CD14 receptor, released significantly less TNF-alpha after LPS stimulation in the absence of serum because of the absence of LPS-binding protein. Phosphatidylinositol-phospholipase C treatment, which cleaves the CD14 receptor, decreased LPS-stimulated TNF-alpha release by RAW 264.7 cells but not by Kupffer cells. Deacylated LPS (dLPS) competes with LPS at the CD14 receptor when incubated in a ratio of 100:1 (dLPS/LPS). Such competition blocked LPS-stimulated TNF-alpha release from RAW 264.7 cells but not from Kupffer cells. Western and fluorescence-activated cell sorter analysis directly demonstrated the presence of CD14 on RAW 264.7 cells and murine peritoneal cells but showed only minimal amounts of CD14 in murine Kupffer cells. LPS stimulation did not increase the amount of CD14 detectable on mouse Kupffer cells. CD14 expression is very low in Kupffer cells, and LPS-stimulated TNF-alpha release is independent of CD14 in these cells.

Role of G protein-coupled receptors (GPCR), matrix metalloproteinases 2 and 9 (MMP2 and MMP9), heparin-binding epidermal growth factor-like growth factor (hbEGF), epidermal growth factor receptor (EGFR), erbB2, and insulin-like growth factor 1 receptor (IGF-1R) in trenbolone acetate-stimulated bovine satellite cell proliferation.

PubMed

Thornton, K J; Kamange-Sollo, E; White, M E; Dayton, W R

2015-09-01

Implanting cattle with steroids significantly enhances feed efficiency, rate of gain, and muscle growth. However, the mechanisms responsible for these improvements in muscle growth have not been fully elucidated. Trenbolone acetate (TBA), a testosterone analog, has been shown to increase proliferation rate in bovine satellite cell (BSC) cultures. The classical genomic actions of testosterone have been well characterized; however, our results indicate that TBA may also initiate a quicker, nongenomic response that involves activation of G protein-coupled receptors (GPCR) resulting in activation of matrix metalloproteinases 2 and 9 (MMP2 and MMP9) that release membrane-bound heparin-binding epidermal growth factor-like growth factor (hbEGF), which then binds to and activates the epidermal growth factor receptor (EGFR) and/or erbB2. Furthermore, the EGFR has been shown to regulate expression of the IGF-1 receptor (IGF-1R), which is well known for its role in modulating muscle growth. To determine whether this nongenomic pathway is potentially involved in TBA-stimulated BSC proliferation, we analyzed the effects of treating BSC with guanosine 5'-O-2-thiodiphosphate (GDPβS), an inhibitor of all GPCR; a MMP2 and MMP9 inhibitor (MMPI); CRM19, a specific inhibitor of hbEGF; AG1478, a specific EGFR tyrosine kinase inhibitor; AG879, a specific erbB2 kinase inhibitor; and AG1024, an IGF-1R tyrosine kinase inhibitor on TBA-stimulated proliferation rate (H-thymidine incorporation). Assays were replicated at least 9 times for each inhibitor experiment using BSC cultures obtained from at least 3 different animals. Bovine satellite cell cultures were obtained from yearling steers that had no previous exposure to androgenic or estrogenic compounds. As expected, BSC cultures treated with 10 n TBA showed ( < 0.05) increased proliferation rate when compared with control cultures. Additionally, treatment with 5 ng hbEGF/mL stimulated proliferation in BSC cultures ( < 0.05). Treatment with GDPβS, MMPI, CRM197, AG1024, AG1478, and/or AG879 all suppressed ( < 0.05) TBA-induced increases in proliferation. These data indicate that TBA likely initiates a nongenomic response involving GPCR, MMP2 and MMP9, hbEGF, EGFR, erbB2, and IGF-1R, which may play a role in TBA-mediated increases in BSC proliferation.

Glutamate Stimulates Local Protein Synthesis in the Axons of Rat Cortical Neurons by Activating α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors and Metabotropic Glutamate Receptors.

PubMed

Hsu, Wei-Lun; Chung, Hui-Wen; Wu, Chih-Yueh; Wu, Huei-Ing; Lee, Yu-Tao; Chen, En-Chan; Fang, Weilun; Chang, Yen-Chung

2015-08-21

Glutamate is the principal excitatory neurotransmitter in the mammalian CNS. By analyzing the metabolic incorporation of azidohomoalanine, a methionine analogue, in newly synthesized proteins, we find that glutamate treatments up-regulate protein translation not only in intact rat cortical neurons in culture but also in the axons emitting from cortical neurons before making synapses with target cells. The process by which glutamate stimulates local translation in axons begins with the binding of glutamate to the ionotropic AMPA receptors and metabotropic glutamate receptor 1 and members of group 2 metabotropic glutamate receptors on the plasma membrane. Subsequently, the activated mammalian target of rapamycin (mTOR) signaling pathway and the rise in Ca(2+), resulting from Ca(2+) influxes through calcium-permeable AMPA receptors, voltage-gated Ca(2+) channels, and transient receptor potential canonical channels, in axons stimulate the local translation machinery. For comparison, the enhancement effects of brain-derived neurotrophic factor (BDNF) on the local protein synthesis in cortical axons were also studied. The results indicate that Ca(2+) influxes via transient receptor potential canonical channels and activated the mTOR pathway in axons also mediate BDNF stimulation to local protein synthesis. However, glutamate- and BDNF-induced enhancements of translation in axons exhibit different kinetics. Moreover, Ca(2+) and mTOR signaling appear to play roles carrying different weights, respectively, in transducing glutamate- and BDNF-induced enhancements of axonal translation. Thus, our results indicate that exposure to transient increases of glutamate and more lasting increases of BDNF would stimulate local protein synthesis in migrating axons en route to their targets in the developing brain. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Glutamate Stimulates Local Protein Synthesis in the Axons of Rat Cortical Neurons by Activating α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors and Metabotropic Glutamate Receptors*

PubMed Central

Hsu, Wei-Lun; Chung, Hui-Wen; Wu, Chih-Yueh; Wu, Huei-Ing; Lee, Yu-Tao; Chen, En-Chan; Fang, Weilun; Chang, Yen-Chung

2015-01-01

Glutamate is the principal excitatory neurotransmitter in the mammalian CNS. By analyzing the metabolic incorporation of azidohomoalanine, a methionine analogue, in newly synthesized proteins, we find that glutamate treatments up-regulate protein translation not only in intact rat cortical neurons in culture but also in the axons emitting from cortical neurons before making synapses with target cells. The process by which glutamate stimulates local translation in axons begins with the binding of glutamate to the ionotropic AMPA receptors and metabotropic glutamate receptor 1 and members of group 2 metabotropic glutamate receptors on the plasma membrane. Subsequently, the activated mammalian target of rapamycin (mTOR) signaling pathway and the rise in Ca2+, resulting from Ca2+ influxes through calcium-permeable AMPA receptors, voltage-gated Ca2+ channels, and transient receptor potential canonical channels, in axons stimulate the local translation machinery. For comparison, the enhancement effects of brain-derived neurotrophic factor (BDNF) on the local protein synthesis in cortical axons were also studied. The results indicate that Ca2+ influxes via transient receptor potential canonical channels and activated the mTOR pathway in axons also mediate BDNF stimulation to local protein synthesis. However, glutamate- and BDNF-induced enhancements of translation in axons exhibit different kinetics. Moreover, Ca2+ and mTOR signaling appear to play roles carrying different weights, respectively, in transducing glutamate- and BDNF-induced enhancements of axonal translation. Thus, our results indicate that exposure to transient increases of glutamate and more lasting increases of BDNF would stimulate local protein synthesis in migrating axons en route to their targets in the developing brain. PMID:26134564

Prostaglandin E2-stimulated prostanoid EP4 receptors induce prolonged de novo prostaglandin E2 synthesis through biphasic phosphorylation of extracellular signal-regulated kinases mediated by activation of protein kinase A in HCA-7 human colon cancer cells.

PubMed

Fujino, Hiromichi; Seira, Naofumi; Kurata, Naoki; Araki, Yumi; Nakamura, Hiroyuki; Regan, John W; Murayama, Toshihiko

2015-12-05

Approximately two decades have passed since E-type prostanoid 4 (EP4) receptors were cloned, and the signaling pathways mediated by these receptors have since been implicated in cancer development through the alliance of Gαi-protein/phosphatidylinositol 3-kinase (PI3K)/extracellular signal-regulated kinases (ERKs) activation. Although prostanoid EP4 receptors were initially identified as Gαs-coupled receptors, the specific/distinctive role(s) of prostanoid EP4 receptor-induced cAMP/protein kinase A (PKA) pathways in cancer development have not yet been elucidated in detail. We previously reported using HCA-7 human colon cancer cells that prostaglandin E2 (PGE2)-stimulated prostanoid EP4 receptors induced cyclooxygenase-2 (COX-2) as an initiating event in development of colon cancer. Moreover, this induction of COX-2 was mediated by transactivation of epidermal growth factor (EGF) receptors. However, direct activation of EGF receptors by EGF also induced similar amounts of COX-2 in this cell line. Thus, the emergence of unique role(s) for prostanoid EP4 receptors is expected by clarifying the different signaling mechanisms between PGE2-stimulated prostanoid EP4 receptors and EGF-stimulated EGF receptors to induce COX-2 and produce PGE2. We here demonstrated that prostanoid EP4 receptor activation by PGE2 in HCA-7 cells led to PKA-dependent re-activation of ERKs, which resulted in prolonged de novo synthesis of PGE2. Although EGF-stimulated EGF receptors in cells also induced COX-2 and the de novo synthesis of PGE2, the activation of this pathway was transient and not mediated by PKA. Therefore, the novel mechanism underlying prolonged de novo synthesis of PGE2 has provided an insight into the importance of prostanoid EP4 receptor-mediated Gαs-protein/cAMP/PKA pathway in development of colon cancer. Copyright © 2015 Elsevier B.V. All rights reserved.

Thoracic vagal efferent nerve stimulation evokes substance P-induced early airway bronchonstriction and late proinflammatory and oxidative injury in the rat respiratory tract.

PubMed

Li, Ping-Chia; Li, Sheng-Chung; Lin, Yuan-Ju; Liang, Jin-Tung; Chien, Chiang-Ting; Shaw, Chen-Fu

2005-01-01

Electrical stimulation of efferent thoracic vagus nerve (TVN) evoked neurogenic inflammation in respiratory tract of atropine-treated rats by an undefined mechanism. We explored whether efferent TVN stimulation via substance P facilitates neurogenic inflammation via action of nuclear factor-kappaB (NF-kappaB) activation and reactive oxygen species (ROS) production. Our results showed that increased frequency of TVN stimulation concomitantly increased substance P-enhanced hypotension, and bronchoconstriction (increases in smooth muscle electromyographic activity and total pulmonary resistance). The enhanced SP release evoked the appearance of endothelial gap in silver-stained leaky venules, India-ink labeled extravasation, and accumulations of inflammatory cells in the respiratory tract, contributing to trachea plasma extravasation as well as increases in blood O (2)(-) and H(2)O(2) ROS amount. L-732138 (NK(1) receptor antagonist), SR-48968 (NK(2) receptor antagonist), dimethylthiourea (H(2)O(2) scavenger) or catechins (O (2)(-) and H(2)O(2) scavenger) pretreatment reduced efferent TVN stimulation-enhanced hypotension, bronchoconstriction, and plasma extravasation. Increased frequency of TVN stimulation significantly upregulated the expression of nuclear factor-kappaB (NF-kappaB) in nuclear protein and intercellular adhesion molecule-1 (ICAM-1) in total protein of the lower respiratory tract tissue. The upregulation of NF-kappaB and ICAM-1 was attenuated by NK receptor antagonist and antioxidants. In conclusion, TVN efferent stimulation increases substance P release to trigger NF-kappaB mediated ICAM-1 expression and O (2)(-) and H(2)O(2) ROS production in the respiratory tract.

Hematopoietic growth factors and human acute leukemia.

PubMed

Löwenberg, B; Touw, I

1988-10-22

The study of myelopoietic maturation arrest in acute myeloblastic leukemia (AML) has been eased by availability of the human recombinant hemopoietic growth factors, macrophage colony stimulating factor (M-CSF), granulocyte-(G-CSF), granulocyte-macrophage-(GM-CSF) and multilineage stimulating factor (IL-3). Nonphysiological expansion of the leukemic population is not due to escape from control by these factors. Proliferation in vitro of AML cells is dependent on the presence of one or several factors in most cases. The pattern of factor-dependency does not correlate with morphological criteria in individual cases, and may thus offer a new tool for classification of AML. Overproduction of undifferentiated cells is not due to abnormal expression of receptors for the stimulating factors acting at an immature level. Rather, autocrine secretion of early acting lymphokines maintains proliferation of the leukemic clone. When looking at causes of leukemic dysregulation, yet undefined inhibitors of differentiation probably are of equal importance as dysequilibrated stimulation by lymphokines.

Isolated dorsal root ganglion neurones inhibit receptor-dependent adenylyl cyclase activity in associated glial cells

PubMed Central

Ng, KY; Yeung, BHS; Wong, YH; Wise, H

2013-01-01

Background and Purpose Hyper-nociceptive PGE2 EP4 receptors and prostacyclin (IP) receptors are present in adult rat dorsal root ganglion (DRG) neurones and glial cells in culture. The present study has investigated the cell-specific expression of two other Gs-protein coupled hyper-nociceptive receptor systems: β-adrenoceptors and calcitonin gene-related peptide (CGRP) receptors in isolated DRG cells and has examined the influence of neurone–glial cell interactions in regulating adenylyl cyclase (AC) activity. Experimental Approach Agonist-stimulated AC activity was determined in mixed DRG cell cultures from adult rats and compared with activity in DRG neurone-enriched cell cultures and pure DRG glial cell cultures. Key Results Pharmacological analysis showed the presence of Gs-coupled β2-adrenoceptors and CGRP receptors, but not β1-adrenoceptors, in all three DRG cell preparations. Agonist-stimulated AC activity was weakest in DRG neurone-enriched cell cultures. DRG neurones inhibited IP receptor-stimulated glial cell AC activity by a process dependent on both cell–cell contact and neurone-derived soluble factors, but this is unlikely to involve purine or glutamine receptor activation. Conclusions and Implications Gs-coupled hyper-nociceptive receptors are readily expressed on DRG glial cells in isolated cell cultures and the activity of CGRP, EP4 and IP receptors, but not β2-adrenoceptors, in glial cells is inhibited by DRG neurones. Studies using isolated DRG cells should be aware that hyper-nociceptive ligands may stimulate receptors on glial cells in addition to neurones, and that variable numbers of neurones and glial cells will influence absolute measures of AC activity and affect downstream functional responses. PMID:22924655

Characterization of the growth of murine fibroblasts that express human insulin receptors. II. Interaction of insulin with other growth factors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Randazzo, P.A.; Jarett, L.

1990-09-01

The effects of insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and insulin on DNA synthesis were studied in murine fibroblasts transfected with an expression vector containing human insulin receptor cDNA (NIH 3T3/HIR) and the parental NIH 3T3 cells. In NIH 3T3/HIR cells, individual growth factors in serum-free medium stimulated DNA synthesis with the following relative efficacies: insulin greater than or equal to 10% fetal calf serum greater than PDGF greater than IGF-1 much greater than EGF. In comparison, the relative efficacies of these factors in stimulating DNA synthesis by NIH 3T3 cells were 10% fetalmore » calf serum greater than PDGF greater than EGF much greater than IGF-1 = insulin. In NIH 3T3/HIR cells, EGF was synergistic with 1-10 ng/ml insulin but not with 100 ng/ml insulin or more. Synergy of PDGF or IGF-1 with insulin was not detected. In the parental NIH 3T3 cells, insulin and IGF-1 were found to be synergistic with EGF (1 ng/ml), PDGF (100 ng/ml), and PDGF plus EGF. In NIH 3T3/HIR cells, the lack of interaction of insulin with other growth factors was also observed when the percentage of cells synthesizing DNA was examined. Despite insulin's inducing only 60% of NIH 3T3/HIR cells to incorporate thymidine, addition of PDGF, EGF, or PDGF plus EGF had no further effect. In contrast, combinations of growth factors resulted in 95% of the parental NIH 3T3 cells synthesizing DNA. The independence of insulin-stimulated DNA synthesis from other mitogens in the NIH 3T3/HIR cells is atypical for progression factor-stimulated DNA synthesis and is thought to be partly the result of insulin receptor expression in an inappropriate context or quantity.« less

Generalization of rapidly recurring seizures is suppressed in mice lacking glial cell line-derived neurotrophic factor family receptor alpha2.

PubMed

Nanobashvili, A; Kokaia, Z; Lindvall, O

2003-01-01

Recent experimental evidence indicates that neurotrophic factors play a role in the pathophysiology of epilepsy. The objective of this study was to explore whether signaling through one of the glial cell line-derived neurotrophic factor family receptors, GFRalpha2, influences the severity of kindling-evoked, rapidly recurring seizures and the subsequent development of permanent hyperexcitability. We applied the rapid kindling model to adult mice, using 40 threshold stimulations delivered with 5-min interval in the ventral hippocampus. Generalized seizures were fewer and developed later in response to kindling stimulations in mice lacking GFRalpha2. However, GFRalpha2 gene deletion did not influence the acquisition of the permanent abnormal excitability as assessed 4 weeks later. In situ hybridization revealed marked and dynamic changes of GFRalpha2 mRNA levels in several forebrain areas following the stimulus-evoked seizures. Our findings provide evidence that signaling through the GFRalpha2 receptor contributes to seizure generalization in rapid kindling.

IL-17s adopt a cystine knot fold: structure and activity of a novel cytokine, IL-17F, and implications for receptor binding

PubMed Central

Hymowitz, Sarah G.; Filvaroff, Ellen H.; Yin, JianPing; Lee, James; Cai, Liping; Risser, Philip; Maruoka, Miko; Mao, Weiguang; Foster, Jessica; Kelley, Robert F.; Pan, Guohua; Gurney, Austin L.; de Vos, Abraham M.; Starovasnik, Melissa A.

2001-01-01

The proinflammatory cytokine interleukin 17 (IL-17) is the founding member of a family of secreted proteins that elicit potent cellular responses. We report a novel human IL-17 homolog, IL-17F, and show that it is expressed by activated T cells, can stimulate production of other cytokines such as IL-6, IL-8 and granulocyte colony-stimulating factor, and can regulate cartilage matrix turnover. Unexpectedly, the crystal structure of IL-17F reveals that IL-17 family members adopt a monomer fold typical of cystine knot growth factors, despite lacking the disulfide responsible for defining the canonical ‘knot’ structure. IL-17F dimerizes in a parallel manner like neurotrophins, and features an unusually large cavity on its surface. Remarkably, this cavity is located in precisely the same position where nerve growth factor binds its high affinity receptor, TrkA, suggesting further parallels between IL-17s and neurotrophins with respect to receptor recognition. PMID:11574464

Inhibiting thyrotropin/insulin-like growth factor 1 receptor crosstalk to treat Graves' ophthalmopathy: studies in orbital fibroblasts in vitro.

PubMed

Place, Robert F; Krieger, Christine C; Neumann, Susanne; Gershengorn, Marvin C

2017-02-01

Crosstalk between thyrotropin (TSH) receptors and insulin-like growth factor 1 (IGF-1) receptors initiated by activation of TSH receptors could be important in the development of Graves' ophthalmopathy (GO). Specifically, TSH receptor activation alone is sufficient to stimulate hyaluronic acid (HA) secretion, a major component of GO, through both IGF-1 receptor-dependent and -independent pathways. Although an anti-IGF-1 receptor antibody is in clinical trials, its effectiveness depends on the relative importance of IGF-1 versus TSH receptor signalling in GO pathogenesis. TSH and IGF-1 receptor antagonists were used to probe TSH/IGF-1 receptor crosstalk in primary cultures of Graves' orbital fibroblasts (GOFs) following activation with monoclonal TSH receptor antibody, M22. Inhibition of HA secretion following TSH receptor stimulation was measured by modified HA elisa. TSH receptor antagonist, ANTAG3 (NCGC00242364), inhibited both IGF-1 receptor -dependent and -independent pathways at all doses of M22; whereas IGF-1 receptor antagonists linsitinib and 1H7 (inhibitory antibody) lost efficacy at high M22 doses. Combining TSH and IGF-1 receptor antagonists exhibited Loewe additivity within the IGF-1 receptor-dependent component of the M22 concentration-response. Similar effects were observed in GOFs activated by autoantibodies from GO patients' sera. Our data support TSH and IGF-1 receptors as therapeutic targets for GO, but reveal putative conditions for anti-IGF-1 receptor resistance. Combination treatments antagonizing both receptors yield additive effects by inhibiting crosstalk triggered by TSH receptor stimulatory antibodies. Combination therapy may be an effective strategy for dose reduction and/or compensate for any loss of anti-IGF-1 receptor efficacy. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Novel synergistic mechanism for sst2 somatostatin and TNFalpha receptors to induce apoptosis: crosstalk between NF-kappaB and JNK pathways.

PubMed

Guillermet-Guibert, J; Saint-Laurent, N; Davenne, L; Rochaix, P; Cuvillier, O; Culler, M D; Pradayrol, L; Buscail, L; Susini, C; Bousquet, C

2007-02-01

Somatostatin is a multifunctional hormone that modulates cell proliferation, differentiation and apoptosis. Mechanisms for somatostatin-induced apoptosis are at present mostly unsolved. Therefore, we investigated whether somatostatin receptor subtype 2 (sst2) induces apoptosis in the nontransformed murine fibroblastic NIH3T3 cells. Somatostatin receptor subtype 2 expression induced an executioner caspase-mediated apoptosis through a tyrosine phosphatase SHP-1 (Src homology domain phosphatase-1)-dependent stimulation of nuclear factor kappa B (NF-kappaB) activity and subsequent inhibition of the mitogen-activated protein kinase JNK. Tumor necrosis factor alpha (TNFalpha) stimulated both NF-kappaB and c-Jun NH2-terminal kinase (JNK) activities, which had opposite action on cell survival. Importantly, sst2 sensitized NIH3T3 cells to TNFalpha-induced apoptosis by (1) upregulating TNFalpha receptor protein expression, and sensitizing to TNFalpha-induced caspase-8 activation; (2) enhancing TNFalpha-mediated activation of NF-kappaB, resulting in JNK inhibition and subsequent executioner caspase activation and cell death. We have here unraveled a novel signaling mechanism for a G protein-coupled receptor, which directly triggers apoptosis and crosstalks with a death receptor to enhance death ligand-induced apoptosis.

Expression of platelet-derived growth factor and its receptors in proliferative disorders of fibroblastic origin.

PubMed Central

Smits, A.; Funa, K.; Vassbotn, F. S.; Beausang-Linder, M.; af Ekenstam, F.; Heldin, C. H.; Westermark, B.; Nistér, M.

1992-01-01

Platelet-derived growth factor (PDGF) is known to stimulate the proliferation of connective tissue-derived cells in vitro. Less is known about its functions in vivo, and the role of PDGF in the development of human tumors has not been clarified. The authors have investigated the occurrence of PDGF and PDGF receptors in a series of proliferative disorders of fibroblastic origin using immunohistochemical and in situ hybridization techniques. High expression of PDGF beta-receptor mRNA and protein was found in the malignant tumors, and also in some benign lesions, such as dermatofibroma. In all these cases, benign as well as malignant, the PDGF B-chain mRNA, and less clearly, the PDGF A-chain mRNA, were coexpressed with the beta-receptor. In contrast, high expression of PDGF alpha-receptor mRNA was only found in fully malignant lesions, i.e., malignant fibrous histiocytoma. These data indicate that an autocrine growth stimulation via the PDGF beta-receptor could occur in an early phase of tumorigenesis, and may be a necessary but insufficient event for the progression into fully malignant human connective tissue lesions. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:1372158

Proteinase-activated receptor 4 stimulation-induced epithelial-mesenchymal transition in alveolar epithelial cells

PubMed Central

Ando, Seijitsu; Otani, Hitomi; Yagi, Yasuhiro; Kawai, Kenzo; Araki, Hiromasa; Fukuhara, Shirou; Inagaki, Chiyoko

2007-01-01

Background Proteinase-activated receptors (PARs; PAR1–4) that can be activated by serine proteinases such as thrombin and neutrophil catepsin G are known to contribute to the pathogenesis of various pulmonary diseases including fibrosis. Among these PARs, especially PAR4, a newly identified subtype, is highly expressed in the lung. Here, we examined whether PAR4 stimulation plays a role in the formation of fibrotic response in the lung, through alveolar epithelial-mesenchymal transition (EMT) which contributes to the increase in myofibroblast population. Methods EMT was assessed by measuring the changes in each specific cell markers, E-cadherin for epithelial cell, α-smooth muscle actin (α-SMA) for myofibroblast, using primary cultured mouse alveolar epithelial cells and human lung carcinoma-derived alveolar epithelial cell line (A549 cells). Results Stimulation of PAR with thrombin (1 U/ml) or a synthetic PAR4 agonist peptide (AYPGKF-NH2, 100 μM) for 72 h induced morphological changes from cobblestone-like structure to elongated shape in primary cultured alveolar epithelial cells and A549 cells. In immunocytochemical analyses of these cells, such PAR4 stimulation decreased E-cadherin-like immunoreactivity and increased α-SMA-like immunoreactivity, as observed with a typical EMT-inducer, tumor growth factor-β (TGF-β). Western blot analyses of PAR4-stimulated A549 cells also showed similar changes in expression of these EMT-related marker proteins. Such PAR4-mediated changes were attenuated by inhibitors of epidermal growth factor receptor (EGFR) kinase and Src. PAR4-mediated morphological changes in primary cultured alveolar epithelial cells were reduced in the presence of these inhibitors. PAR4 stimulation increased tyrosine phosphorylated EGFR or tyrosine phosphorylated Src level in A549 cells, and the former response being inhibited by Src inhibitor. Conclusion PAR4 stimulation of alveolar epithelial cells induced epithelial-mesenchymal transition (EMT) as monitored by cell shapes, and epithelial or myofibroblast marker at least partly through EGFR transactivation via receptor-linked Src activation. PMID:17433115

Cross-talk between GPER and growth factor signaling.

PubMed

Lappano, Rosamaria; De Marco, Paola; De Francesco, Ernestina Marianna; Chimento, Adele; Pezzi, Vincenzo; Maggiolini, Marcello

2013-09-01

G protein-coupled receptors (GPCRs) and growth factor receptors mediate multiple physio-pathological responses to a diverse array of extracellular stimuli. In this regard, it has been largely demonstrated that GPCRs and growth factor receptors generate a multifaceted signaling network, which triggers relevant biological effects in normal and cancer cells. For instance, some GPCRs transactivate the epidermal growth factor receptor (EGFR), which stimulates diverse transduction pathways leading to gene expression changes, cell migration, survival and proliferation. Moreover, it has been reported that a functional interaction between growth factor receptors and steroid hormones like estrogens is involved in the growth of many types of tumors as well as in the resistance to endocrine therapy. This review highlights recent findings on the cross-talk between a member of the GPCR family, the G protein-coupled estrogen receptor 1 (GPER, formerly known as GPR30) and two main growth factor receptors like EGFR and insulin-like growth factor-I receptor (IGF-IR). The biological implications of the functional interaction between these important mediators of cell responses particularly in cancer are discussed. This article is part of a Special Issue entitled 'CSR 2013'. Copyright © 2013 Elsevier Ltd. All rights reserved.

Activation of adenosine A(3) receptors supports hematopoiesis-stimulating effects of granulocyte colony-stimulating factor in sublethally irradiated mice.

PubMed

Hofer, Michal; Pospísil, Milan; Sefc, Ludek; Dusek, Ladislav; Vacek, Antonín; Holá, Jirina; Hoferová, Zuzana; Streitová, Denisa

2010-08-01

Research areas of 'post-exposure treatment' and 'cytokines and growth factors' have top priority among studies aimed at radiological nuclear threat countermeasures. The experiments were aimed at testing the ability of N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA), an adenosine A(3) receptor agonist, to modulate hematopoiesis in sublethally irradiated mice, when administered alone or in a combination with granulocyte colony-stimulating factor (G-CSF) in a two-day post-irradiation treatment regimen. A complete analysis of hematopoiesis including determination of numbers of bone marrow hematopoietic progenitor and precursor cells, as well as of numbers of peripheral blood cells, was performed. The outcomes of the treatment were assessed at days 3 to 22 after irradiation. IB-MECA alone has been found to induce a significant elevation of numbers of bone marrow granulocyte-macrophage progenitor cells (GM-CFC) and peripheral blood neutrophils. IB-MECA given concomitantly with G-CSF increased significantly bone marrow GM-CFC and erythroid progenitor cells (BFU-E) in comparison with the controls and with animals administered each of the drugs alone. The findings suggest the ability of IB-MECA to stimulate hematopoiesis and to support the hematopoiesis-stimulating effects of G-CSF in sublethally irradiated mice.

Metabolic effects of growth factors and polycyclic aromatic hydrocarbons on cultured human placental cells of early and late gestation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guyda, H.J.

1991-03-01

The metabolic effects of epidermal growth factor (EGF), insulin, insulin-like growth factor-I (IGF-I), and IGF-II were determined on human placental cells in monolayer culture obtained from early gestation (less than 20 weeks) and late gestation (38-42 weeks). Parameters studied were uptake of aminoisobutyric acid (AIB), uptake of 3-O-methylglucose and (3H)thymidine incorporation into cell protein. Since benzo(alpha)pyrene (BP) inhibits EGF binding and autophosphorylation in cultured human placental cells, particularly in early gestation, we also studied the effect of benzo(alpha)pyrene and other polycyclic aromatic hydrocarbons (PAHs) on EGF-mediated AIB uptake. The metabolic effects of EGF, insulin, and the IGFs in cultured humanmore » placental cells varied with gestational age and the growth factor studied. All three classes of growth factors stimulated AIB uptake in both early and late gestation at concentrations from 10-100 micrograms/L, well within a physiological range. However, insulin stimulation of AIB uptake was maximal at a high concentration in both early and late gestation cells, suggesting an action via type 1 IGF receptors rather than via insulin receptors. EGF stimulated 3-O-methylglucose uptake only in term placental cells. No significant stimulation of (3H)thymidine incorporation by any of the growth factors tested was seen with either early or late gestation cells. The effect of PAHs on AIB uptake by cultured placental cells was variable. BP alone stimulated AIB uptake by both very early and late gestation cells and enhanced EGF-stimulated AIB uptake. alpha-naphthoflavone alone inhibited AIB uptake at all gestational ages and inhibited EGF-stimulated AIB uptake. beta-Naphthoflavone and 3-methylcholanthrene minimally inhibited AIB uptake by early gestation cells and did not modify EGF-stimulated uptake at any gestational period.« less

Endocannabinoids Stimulate Human Melanogenesis via Type-1 Cannabinoid Receptor*

PubMed Central

Pucci, Mariangela; Pasquariello, Nicoletta; Battista, Natalia; Di Tommaso, Monia; Rapino, Cinzia; Fezza, Filomena; Zuccolo, Michela; Jourdain, Roland; Finazzi Agrò, Alessandro; Breton, Lionel; Maccarrone, Mauro

2012-01-01

We show that a fully functional endocannabinoid system is present in primary human melanocytes (normal human epidermal melanocyte cells), including anandamide (AEA), 2-arachidonoylglycerol, the respective target receptors (CB1, CB2, and TRPV1), and their metabolic enzymes. We also show that at higher concentrations AEA induces normal human epidermal melanocyte apoptosis (∼3-fold over controls at 5 μm) through a TRPV1-mediated pathway that increases DNA fragmentation and p53 expression. However, at lower concentrations, AEA and other CB1-binding endocannabinoids dose-dependently stimulate melanin synthesis and enhance tyrosinase gene expression and activity (∼3- and ∼2-fold over controls at 1 μm). This CB1-dependent activity was fully abolished by the selective CB1 antagonist SR141716 or by RNA interference of the receptor. CB1 signaling engaged p38 and p42/44 mitogen-activated protein kinases, which in turn activated the cyclic AMP response element-binding protein and the microphthalmia-associated transcription factor. Silencing of tyrosinase or microphthalmia-associated transcription factor further demonstrated the involvement of these proteins in AEA-induced melanogenesis. In addition, CB1 activation did not engage the key regulator of skin pigmentation, cyclic AMP, showing a major difference compared with the regulation of melanogenesis by α-melanocyte-stimulating hormone through melanocortin 1 receptor. PMID:22431736

Inhibiting thyrotropin/insulin‐like growth factor 1 receptor crosstalk to treat Graves' ophthalmopathy: studies in orbital fibroblasts in vitro

PubMed Central

Place, Robert F; Neumann, Susanne; Gershengorn, Marvin C

2017-01-01

Background and Purpose Crosstalk between thyrotropin (TSH) receptors and insulin‐like growth factor 1 (IGF‐1) receptors initiated by activation of TSH receptors could be important in the development of Graves' ophthalmopathy (GO). Specifically, TSH receptor activation alone is sufficient to stimulate hyaluronic acid (HA) secretion, a major component of GO, through both IGF‐1 receptor‐dependent and ‐independent pathways. Although an anti‐IGF‐1 receptor antibody is in clinical trials, its effectiveness depends on the relative importance of IGF‐1 versus TSH receptor signalling in GO pathogenesis. Experimental Approach TSH and IGF‐1 receptor antagonists were used to probe TSH/IGF‐1 receptor crosstalk in primary cultures of Graves' orbital fibroblasts (GOFs) following activation with monoclonal TSH receptor antibody, M22. Inhibition of HA secretion following TSH receptor stimulation was measured by modified HA elisa. Key Results TSH receptor antagonist, ANTAG3 (NCGC00242364), inhibited both IGF‐1 receptor ‐dependent and ‐independent pathways at all doses of M22; whereas IGF‐1 receptor antagonists linsitinib and 1H7 (inhibitory antibody) lost efficacy at high M22 doses. Combining TSH and IGF‐1 receptor antagonists exhibited Loewe additivity within the IGF‐1 receptor‐dependent component of the M22 concentration‐response. Similar effects were observed in GOFs activated by autoantibodies from GO patients' sera. Conclusions and Implications Our data support TSH and IGF‐1 receptors as therapeutic targets for GO, but reveal putative conditions for anti‐IGF‐1 receptor resistance. Combination treatments antagonizing both receptors yield additive effects by inhibiting crosstalk triggered by TSH receptor stimulatory antibodies. Combination therapy may be an effective strategy for dose reduction and/or compensate for any loss of anti‐IGF‐1 receptor efficacy. PMID:27987211

Targeting the fibroblast growth factor receptors for the treatment of cancer.

PubMed

Lemieux, Steven M; Hadden, M Kyle

2013-06-01

Receptor tyrosine kinases (RTKs) are transmembrane proteins that play a critical role in stimulating signal transduction cascades to influence cell proliferation, growth, and differentiation and they have also been shown to promote angiogenesis when they are up-regulated or mutated. For this reason, their dysfunction has been implicated in the development of human cancer. Over the past decade, much attention has been devoted to developing inhibitors and antibodies against several classes of RTKs, including vascular endothelial growth factor receptors (VEGFRs), epidermal growth factor receptors (EGFRs), and platelet-derived growth factor receptors (PDGFRs). More recently, interest in the fibroblast growth factor receptor (FGFR) class of RTKs as a drug target for the treatment of cancer has emerged. Signaling through FGFRs is critical for normal cellular function and their dysregulation has been linked to various malignancies such as breast and prostate cancer. This review will focus on the current state of both small molecules and antibodies as FGFR inhibitors to provide insight into their development and future potential as anti-cancer agents.

(D-Phe/sup 12/)bombesin analogues: a new class of bombesin receptor antagonists

DOE Office of Scientific and Technical Information (OSTI.GOV)

Heinz-Erian, P.; Coy, D.H.; Tamura, M.

1987-03-01

Previous attempts to develop analogues of bombesin that function as specific receptor antagonists have been unsuccessful. Alteration of the histidine in luteinizing hormone releasing factor has resulted in analogues that function as competitive antagonists. In the present study the authors have used a similar strategy and altered the histidine in bombesin. (D-Phe/sup 12/)bombesin, (D-Phe/sup 12/,Leu/sup 14/)bombesin, and (Try/sup 4/, D-)je/sup 12/) bombesin did not stimulate amylase release from guinea pig pancreatic acini when present alone, but each analog inhibited bombesin-stimulated secretion. For each analog, detectable inhibition occurred at 1 ..mu..M and half-maximal inhibition at 4 ..mu..M. Each analog inhibited amylasemore » release by bombesin and other agonists that stimulate secretion by interacting with bombesin receptors. The analogues of bombesin did not alter stimulation by substance P or other agonists that interact with other receptors. The inhibition of the action of bombesin was competitive with Schild plots having slopes of 1.0. Each analog also inhibited binding of /sup 125/I-labeled (Try/sup 4/) bombesin but not /sup 125/I-labeled substance P. These results demonstrate that (D-Phe/sup 12/) analogues of bombesin function as bombesin receptor antagonists and are the only bombesin receptor antagonists that interact only with the bombesin receptor. Because of their specificity, these analogues may prove useful for defining the role of bombesin in various physiological or pathological processes.« less

A monoclonal antibody against PDGF B-chain inhibits PDGF-induced DNA synthesis in C3H fibroblasts and prevents binding of PDGF to its receptor.

PubMed

Vassbotn, F S; Langeland, N; Hagen, I; Holmsen, H

1990-09-01

A monoclonal antibody (MAb 6D11) against platelet-derived growth factor (PDGF) was studied. We found that the MAb 6D11 in concentrations equimolar to PDGF blocked the [3H]thymidine incorporation in C3H/10T1/2 C18 fibroblasts stimulated by PDGF B-B and PDGF A-B. This inhibition was overcome by high doses of PDGF. The [3H]thymidine incorporation stimulated by other growth factors (aFGF, bFGF and bombesin) was not inhibited by the antibody. The MAb 6D11 blocked receptor binding of PDGF B-B, but not PDGF A-A. These findings suggest that the MAb 6D11 abolishes PDGF-induced DNA synthesis by blocking PDGF receptor binding. In this communication we demonstrate an isoform-specific monoclonal antibody against PDGF.

The Fn14 cytoplasmic tail binds tumour-necrosis-factor-receptor-associated factors 1, 2, 3 and 5 and mediates nuclear factor-kappaB activation.

PubMed Central

Brown, Sharron A N; Richards, Christine M; Hanscom, Heather N; Feng, Sheau-Line Y; Winkles, Jeffrey A

2003-01-01

Fn14 is a growth-factor-inducible immediate-early-response gene encoding a 102-amino-acid type I transmembrane protein. The human Fn14 protein was recently identified as a cell-surface receptor for the tumour necrosis factor (TNF) superfamily member named TWEAK (TNF-like weak inducer of apoptosis). In the present paper, we report that the human TWEAK extracellular domain can also bind the murine Fn14 protein. Furthermore, site-specific mutagenesis and directed yeast two-hybrid interaction assays revealed that the TNFR-associated factor (TRAF) 1, 2, 3 and 5 adaptor molecules bind the murine Fn14 cytoplasmic tail at an overlapping, but non-identical, amino acid sequence motif. We also found that TWEAK treatment of quiescent NIH 3T3 cells stimulates inhibitory kappaBalpha phosphorylation and transcriptional activation of a nuclear factor-kappaB (NF-kappaB) enhancer/luciferase reporter construct. Fn14 overexpression in transiently transfected NIH 3T3 cells also promotes NF-kappaB activation, and this cellular response requires an intact TRAF binding site. These results indicate that Fn14 is a functional TWEAK receptor that can associate with four distinct TRAF family members and stimulate the NF-kappaB transcription factor signalling pathway. PMID:12529173

Crystallization of a 2:2 complex of granulocyte-colony stimulating factor (GCSF) with the ligand-binding region of the GCSF receptor

PubMed Central

Honjo, Eijiro; Tamada, Taro; Maeda, Yoshitake; Koshiba, Takumi; Matsukura, Yasuko; Okamoto, Tomoyuki; Ishibashi, Matsujiro; Tokunaga, Masao; Kuroki, Ryota

2005-01-01

The granulocyte-colony stimulating factor (GCSF) receptor receives signals for regulating the maturation, proliferation and differentiation of the precursor cells of neutrophilic granulocytes. The signalling complex composed of two GCSFs (GCSF, 19 kDa) and two GCSF receptors (GCSFR, 34 kDa) consisting of an Ig-like domain and a cytokine-receptor homologous (CRH) domain was crystallized. A crystal of the complex was grown in 1.0 M sodium formate and 0.1 M sodium acetate pH 4.6 and belongs to space group P41212 (or its enantiomorph P43212), with unit-cell parameters a = b = 110.1, c = 331.8 Å. Unfortunately, this crystal form did not diffract beyond 5 Å resolution. Since the heterogeneity of GCSF receptor appeared to prevent the growth of good-quality crystals, the GCSF receptor was fractionated by anion-exchange chromatography. Crystals of the GCSF–fractionated GCSF receptor complex were grown as a new crystal form in 0.2 M ammonium phosphate. This new crystal form diffracted to beyond 3.0 Å resolution and belonged to space group P3121 (or its enantiomorph P3221), with unit-cell parameters a = b = 134.8, c = 105.7 Å. PMID:16511159

Prostaglandin E₂ regulates cellular migration via induction of vascular endothelial growth factor receptor-1 in HCA-7 human colon cancer cells.

PubMed

Fujino, Hiromichi; Toyomura, Kaori; Chen, Xiao-bo; Regan, John W; Murayama, Toshihiko

2011-02-01

An important event in the development of tumors is angiogenesis, or the formation of new blood vessels. Angiogenesis is also known to be involved in tumor cell metastasis and is dependent upon the activity of the vascular endothelial growth factor (VEGF) signaling pathway. Studies of mice in which the EP3 prostanoid receptors have been genetically deleted have shown a role for these receptors in cancer growth and angiogenesis. In the present study, human colon cancer HCA-7 cells were used as a model system to understand the potential role of EP3 receptors in tumor cell migration. We now show that stimulation of HCA-7 cells with PGE₂ enhanced the up-regulation of VEGF receptor-1 (VEGFR-1) expression by a mechanism involving EP3 receptor-mediated activation of phosphatidylinositol 3-kinase and the extracellular signal-regulated kinases. Moreover, the PGE₂ stimulated increase in VEGFR-1 expression was accompanied by an increase in the cellular migration of HCA-7 cells. Given the known involvement of VEGFR-1 in cellular migration, our results suggest that EP3 receptors may contribute to tumor cell metastasis by increasing cellular migration through the up-regulation of VEGFR-1 signaling. Copyright © 2010 Elsevier Inc. All rights reserved.

Transformation-specific interaction of the bovine papillomavirus E5 oncoprotein with the platelet-derived growth factor receptor transmembrane domain and the epidermal growth factor receptor cytoplasmic domain.

PubMed Central

Cohen, B D; Goldstein, D J; Rutledge, L; Vass, W C; Lowy, D R; Schlegel, R; Schiller, J T

1993-01-01

The bovine papillomavirus E5 transforming protein appears to activate both the epidermal growth factor receptor (EGF-R) and the platelet-derived growth factor receptor (PDGF-R) by a ligand-independent mechanism. To further investigate the ability of E5 to activate receptors of different classes and to determine whether this stimulation occurs through the extracellular domain required for ligand activation, we constructed chimeric genes encoding PDGF-R and EGF-R by interchanging the extracellular, membrane, and cytoplasmic coding domains. Chimeras were transfected into NIH 3T3 and CHO(LR73) cells. All chimeras expressed stable protein which, upon addition of the appropriate ligand, could be activated as assayed by tyrosine autophosphorylation and biological transformation. Cotransfection of E5 with the wild-type and chimeric receptors resulted in the ligand-independent activation of receptors, provided that a receptor contained either the transmembrane domain of the PDGF-R or the cytoplasmic domain of the EGF-R. Chimeric receptors that contained both of these domains exhibited the highest level of E5-induced biochemical and biological stimulation. These results imply that E5 activates the PDGF-R and EGR-R by two distinct mechanisms, neither of which specifically involves the extracellular domain of the receptor. Consistent with the biochemical and biological activation data, coimmunoprecipitation studies demonstrated that E5 formed a complex with any chimera that contained a PDGF-R transmembrane domain or an EGF-R cytoplasmic domain, with those chimeras containing both domains demonstrating the greatest efficiency of complex formation. These results suggest that although different domains of the PDGF-R and EGF-R are required for E5 activation, both receptors are activated directly by formation of an E5-containing complex. Images PMID:8394451

Cross-talk between an activator of nuclear receptors-mediated transcription and the D1 dopamine receptor signaling pathway.

PubMed

Schmidt, Azriel; Vogel, Robert; Rutledge, Su Jane; Opas, Evan E; Rodan, Gideon A; Friedman, Eitan

2005-03-01

Nuclear receptors are transcription factors that usually interact, in a ligand-dependent manner, with specific DNA sequences located within promoters of target genes. The nuclear receptors can also be controlled in a ligand-independent manner via the action of membrane receptors and cellular signaling pathways. 5-Tetradecyloxy-2-furancarboxylic acid (TOFA) was shown to stimulate transcription from the MMTV promoter via chimeric receptors that consist of the DNA binding domain of GR and the ligand binding regions of the PPARbeta or LXRbeta nuclear receptors (GR/PPARbeta and GR/LXRbeta). TOFA and hydroxycholesterols also modulate transcription from NF-kappaB- and AP-1-controlled reporter genes and induce neurite differentiation in PC12 cells. In CV-1 cells that express D(1) dopamine receptors, D(1) dopamine receptor stimulation was found to inhibit TOFA-stimulated transcription from the MMTV promoter that is under the control of chimeric GR/PPARbeta and GR/LXRbeta receptors. Treatment with the D(1) dopamine receptor antagonist, SCH23390, prevented dopamine-mediated suppression of transcription, and by itself increased transcription controlled by GR/LXRbeta. Furthermore, combined treatment of CV-1 cells with TOFA and SCH23390 increased transcription controlled by the GR/LXRbeta chimeric receptor synergistically. The significance of this in vitro synergy was demonstrated in vivo, by the observation that SCH23390 (but not haloperidol)-mediated catalepsy in rats was potentiated by TOFA, thus showing that an agent that mimics the in vitro activities of compounds that activate members of the LXR and PPAR receptor families can influence D1 dopamine receptor elicited responses.

Application of an in silico liver model to determine nuclear receptor mediated pathways in liver cancer

EPA Science Inventory

Nuclear receptors (NRs) are ligand-activated transcription factors that control diverse cellular processes. Chronic stimulation of some NRs in rodents can result in increased incidence of liver tumors. These are generally thought to develop through a non-genotoxic mechanism with...

PGE2 released by primary sensory neurons modulates Toll-like receptor 4 activities through an EP4 receptor-dependent process.

PubMed

Tse, Kai-Hei; Chow, Kevin B S; Wise, Helen

2016-04-15

Exogenous prostaglandin E2 (PGE2) displays mixed regulatory properties with regard to inflammatory gene expression in dorsal root ganglion (DRG) cells. We show here that endogenously-produced nanomolar concentrations of PGE2, such as that generated in response to Toll-like receptor 4 (TLR4) stimulation, inhibits both cyclooxygenase-2 (COX-2) and tumour necrosis factor alpha (TNFα) mRNA expression in DRG cells in an EP4 receptor-dependent manner. DRG neurons appear to be the major source of PGE2 in the DRG and likely serve as both an autocrine and paracrine system for limiting over-activation of both DRG neurons and glial cells in response to TLR4 stimulation. Copyright © 2016 Elsevier B.V. All rights reserved.

Principles of antibody-mediated TNF receptor activation

PubMed Central

Wajant, H

2015-01-01

From the beginning of research on receptors of the tumor necrosis factor (TNF) receptor superfamily (TNFRSF), agonistic antibodies have been used to stimulate TNFRSF receptors in vitro and in vivo. Indeed, CD95, one of the first cloned TNFRSF receptors, was solely identified as the target of cell death-inducing antibodies. Early on, it became evident from in vitro studies that valency and Fcγ receptor (FcγR) binding of antibodies targeting TNFRSF receptors can be of crucial relevance for agonistic activity. TNFRSF receptor-specific antibodies of the IgM subclass and secondary cross-linked or aggregation prone dimeric antibodies typically display superior agonistic activity compared with dimeric antibodies. Likewise, anchoring of antibodies to cell surface-expressed FcγRs potentiate their ability to trigger TNFRSF receptor signaling. However, only recently has the relevance of oligomerization and FcγR binding for the in vivo activity of antibody-induced TNFRSF receptor activation been straightforwardly demonstrated in vivo. This review discusses the crucial role of oligomerization and/or FcγR binding for antibody-mediated TNFRSF receptor stimulation in light of current models of TNFRSF receptor activation and especially the overwhelming relevance of these issues for the rational development of therapeutic TNFRSF receptor-targeting antibodies. PMID:26292758

Proteinase-activated receptor-2 stimulates prostaglandin production in keratinocytes: analysis of prostaglandin receptors on human melanocytes and effects of PGE2 and PGF2alpha on melanocyte dendricity.

PubMed

Scott, Glynis; Leopardi, Sonya; Printup, Stacey; Malhi, Namrita; Seiberg, Miri; Lapoint, Randi

2004-05-01

Prostaglandins (PG) are key mediators of diverse functions in the skin and several reports suggest that PG mediate post-inflammatory pigmentary changes through modulation of melanocyte dendricity and melanin synthesis. The proteinase-activated receptor 2 (PAR-2) is important for skin pigmentation because activation of keratinocyte PAR-2 stimulates uptake of melanosomes through phagocytosis in a Rho-dependent manner. In this report, we show that activation of keratinocyte PAR-2 stimulates release of PGE(2) and PGF(2alpha) and that PGE(2) and PGF(2alpha) act as paracrine factors that stimulate melanocyte dendricity. We characterized the expression of the EP and FP receptors in human melanocytes and show that human melanocytes express EP1 and EP3, and the FP receptor, but not EP2 and EP4. Treatment of melanocytes with EP1 and EP3 receptor agonists resulted in increased melanocyte dendricity, indicating that both EP1 and EP3 receptor signaling contribute to PGE(2)-mediated melanocyte dendricity. Certain EP3 receptor subtypes have been shown to increase adenosine 3',5'-cyclic monophosphate (cAMP) through coupling to Gs, whereas EP1 is known to couple to Gq to activate phospholipase C with elevation in Ca(2+). The cAMP/protein kinase A system is known to modulate melanocyte dendrite formation through modulation of Rac and Rho activity. Neither PGF(2alpha) or PGE(2) elevated cAMP in human melanocytes showing that dendricity observed in response to PGE(2) and PGF(2alpha) is cAMP-independent. Our data suggest that PAR-2 mediates cutaneous pigmentation both through increased uptake of melanosomes by keratinocytes, as well as by release of PGE(2) and PGF(2alpha) that stimulate melanocyte dendricity through EP1, EP3, and FP receptors.

Impaired growth in Rabson-Mendenhall syndrome: lack of effect of growth hormone and insulin-like growth factor-I.

PubMed

Longo, N; Singh, R; Griffin, L D; Langley, S D; Parks, J S; Elsas, L J

1994-09-01

Mutations in the insulin receptor gene cause the severe insulin-resistant syndromes leprechaunism and Rabson-Mendenhall syndrome. There is no accepted therapy for these inherited conditions. Here we report the results of recombinant human GH (rhGH) and recombinant human insulin-like growth factor-I (rhIGF-I) treatment of a male patient, Atl-2, with Rabson-Mendenhall syndrome. The patient was small for gestational age, had premature dentition, absence of sc fat, acanthosis nigricans, fasting hypoglycemia and postprandial hyperglycemia, and extremely high concentrations of circulating insulin (up to 8500 microU/mL). Fibroblasts and lymphoblasts established from this patient had reduced insulin binding, which was 20-30% of the control value. Binding of epidermal growth factor, IGF-I, and GH to the patient's fibroblasts was normal. The growth of fibroblasts cultured from patient Atl-2 in vitro was intermediate between that of fibroblasts from patients with leprechaunism and control values. The patient's growth curve in vivo was far below the fifth percentile despite adequate nutrition. To stimulate growth, therapy with rhGH was initiated, the rationale being to stimulate hepatic IGF-I production and IGF-I receptor signaling, and bypass the inherited block in insulin receptor signaling. Therapy with rhGH (up to 0.5 mg/kg.week) did not improve growth and failed to increase the levels of circulating IGF-I and IGF-binding protein-3 over a 14-month period. As rhGH could not stimulate growth, rhIGF-I (up to 100 micrograms/kg.day) was given by daily sc injection. No increase in growth velocity was observed over a 14-month period. These results indicate that both GH and IGF-I fail to correct growth in a patient with severe inherited insulin resistance. The lack of efficacy of IGF-I treatment may be related to multiple factors, such as the poor metabolic state of the patient, the deficiency of serum carrier protein for IGF-I, an increased clearance of the growth factor, IGF-I resistance in target cells at a receptor or postreceptor level, or an inhibitory action of the mutant insulin receptors on IGF-I receptor signaling.

GPR3 Stimulates Aβ Production via Interactions with APP and β-Arrestin2

PubMed Central

Nelson, Christopher D.; Sheng, Morgan

2013-01-01

The orphan G protein-coupled receptor (GPCR) GPR3 enhances the processing of Amyloid Precursor Protein (APP) to the neurotoxic beta-amyloid (Aβ) peptide via incompletely understood mechanisms. Through overexpression and shRNA knockdown experiments in HEK293 cells, we show that β-arrestin2 (βarr2), a GPCR-interacting scaffold protein reported to bind γ-secretase, is an essential factor for GPR3-stimulated Aβ production. For a panel of GPR3 receptor mutants, the degree of stimulation of Aβ production correlates with receptor-β-arrestin binding and receptor trafficking to endocytic vesicles. However, GPR3’s recruitment of βarr2 cannot be the sole explanation, because interaction with βarr2 is common to most GPCRs, whereas GPR3 is relatively unique among GPCRs in enhancing Aβ production. In addition to β-arrestin, APP is present in a complex with GPR3 and stimulation of Aβ production by GPR3 mutants correlates with their level of APP binding. Importantly, among a broader selection of GPCRs, only GPR3 and prostaglandin E receptor 2 subtype EP2 (PTGER2; another GPCR that increases Aβ production) interact with APP, and PTGER2 does so in an agonist-stimulated manner. These data indicate that a subset of GPCRs, including GPR3 and PTGER2, can associate with APP when internalized via βarr2, and thereby promote the cleavage of APP to generate Aβ. PMID:24069330

β1-adrenergic receptors activate two distinct signaling pathways in striatal neurons

PubMed Central

Meitzen, John; Luoma, Jessie I.; Stern, Christopher M.; Mermelstein, Paul G.

2010-01-01

Monoamine action in the dorsal striatum and nucleus accumbens plays essential roles in striatal physiology. Although research often focuses on dopamine and its receptors, norepinephrine and adrenergic receptors are also crucial in regulating striatal function. While noradrenergic neurotransmission has been identified in the striatum, little is known regarding the signaling pathways activated by β-adrenergic receptors in this brain region. Using cultured striatal neurons, we characterized a novel signaling pathway by which activation of β1-adrenergic receptors leads to the rapid phosphorylation of cAMP Response Element Binding Protein (CREB), a transcription-factor implicated as a molecular switch underlying long-term changes in brain function. Norepinephrine-mediated CREB phosphorylation requires β1-adrenergic receptor stimulation of a receptor tyrosine kinase, ultimately leading to the activation of a Ras/Raf/MEK/MAPK/MSK signaling pathway. Activation of β1-adrenergic receptors also induces CRE-dependent transcription and increased c-fos expression. In addition, stimulation of β1-adrenergic receptors produces cAMP production, but surprisingly, β1-adrenergic receptor activation of adenylyl cyclase was not functionally linked to rapid CREB phosphorylation. These findings demonstrate that activation of β1-adrenergic receptors on striatal neurons can stimulate two distinct signaling pathways. These adrenergic actions can produce long-term changes in gene expression, as well as rapidly modulate cellular physiology. By elucidating the mechanisms by which norepinephrine and β1-adrenergic receptor activation affects striatal physiology, we provide the means to more fully understand the role of monoamines in modulating striatal function, specifically how norepinephrine and β1-adrenergic receptors may affect striatal physiology. PMID:21143600

Epidermal growth factor- and hepatocyte growth factor-receptor activity in serum-free cultures of human hepatocytes.

PubMed

Runge, D M; Runge, D; Dorko, K; Pisarov, L A; Leckel, K; Kostrubsky, V E; Thomas, D; Strom, S C; Michalopoulos, G K

1999-02-01

Serum-free primary cultures of hepatocytes are a useful tool to study factors triggering hepatocyte proliferation and regeneration. We have developed a chemically defined serum-free system that allows human hepatocyte proliferation in the presence of epidermal growth factor and hepatocyte growth factor. DNA synthesis and accumulation were determined by [3H]thymidine incorporation and fluorometry, respectively. Western blot analyses and co-immunoprecipitations were used to investigate the association of proteins involved in epidermal growth factor and hepatocyte growth factor activation and signaling: epidermal growth factor receptor, hepatocyte growth factor receptor (MET), urokinase-type plasminogen activator and its receptor, and a member of the signal transducer and activator of transcription family, STAT-3. Primary human hepatocytes proliferated under serum-free conditions in a chemically defined medium for up to 12 days. Epidermal growth factor-receptor and MET were present and functional, decreasing over time. MET, urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor co-precipitated to varying degrees during the culture period. STAT-3 co-precipitated with epidermal growth factor-receptor and MET to varying degrees. Proliferation of human hepatocytes can improve by modification of a chemically defined medium originally used for rat hepatocyte cultures. In these long-term cultures of human hepatocytes, hepatocyte growth factor and epidermal growth factor can stimulate growth and differentiation by interacting with their receptors and initiating downstream signaling. This involves complex formation of the receptors with other plasma membrane components for MET (urokinase-type plasminogen activator in context of its receptor) and activation of STAT-3 for both receptors.

Gene expression analysis of growth factor receptors in human chondrocytes in monolayer and 3D pellet cultures

PubMed Central

Witt, Anika; Salamon, Achim; Boy, Diana; Hansmann, Doris; Büttner, Andreas; Wree, Andreas; Bader, Rainer; Jonitz-Heincke, Anika

2017-01-01

The main goal of cartilage repair is to create functional tissue by enhancing the in vitro conditions to more physiological in vivo conditions. Chondrogenic growth factors play an important role in influencing cartilage homeostasis. Insulin-like growth factor (IGF)-1 and transforming growth factor (TGF)-β1 affect the expression of collagen type II (Col2) and glycosaminoglycans (GAGs) and, therefore, the targeted use of growth factors could make chondrogenic redifferentiation more efficient. In the present study, human chondrocytes were postmortally isolated from healthy articular cartilage and cultivated as monolayer or 3D pellet cultures either under normoxia or hypoxia and stimulated with IGF-1 and/or TGF-β1 to compare the impact of the different growth factors. The mRNA levels of the specific receptors (IGF1R, TGFBR1, TGFBR2) were analyzed at different time points. Moreover, gene expression rates of collagen type 1 and 2 in pellet cultures were observed over a period of 5 weeks. Additionally, hyaline-like Col2 protein and sulphated GAG (sGAG) levels were quantified. Stimulation with IGF-1 resulted in an enhanced expression of IGF1R and TGFBR2 whereas TGF-β1 stimulated TGFBR1 in the monolayer and pellet cultures. In monolayer, the differences reached levels of significance. This effect was more pronounced under hypoxic culture conditions. In pellet cultures, increased amounts of Col2 protein and sGAGs after incubation with TGF-β1 and/or IGF-1 were validated. In summary, constructing a gene expression profile regarding mRNA levels of specific growth factor receptors in monolayer cultures could be helpful for a targeted application of growth factors in cartilage tissue engineering. PMID:28534942

Negative feedback regulation of human platelets via autocrine activation of the platelet-derived growth factor alpha-receptor.

PubMed

Vassbotn, F S; Havnen, O K; Heldin, C H; Holmsen, H

1994-05-13

Human platelets contain platelet-derived growth factor (PDGF) in their alpha-granules which is released during platelet exocytosis. We show by immunoprecipitation and 125I-PDGF binding experiments that human platelets have functionally active PDGF alpha-receptors, but not beta-receptors. The PDGF alpha-receptor (PDGFR-alpha) was identified as a 170-kDa glycosylated protein-tyrosine kinase as found in other cell types. Stimulation of platelets with 0.1 unit/ml thrombin resulted in a significant increase (2-5-fold) of the tyrosine phosphorylation of the PDGFR-alpha, as determined by immunoprecipitation with phosphotyrosine antiserum as well as with PDGFR-alpha antiserum. The observed thrombin-induced autophosphorylation of the PDGFR-alpha was inhibited by the addition of a neutralizing monoclonal PDGF antibody. Thus, our results suggest that the platelet PDGFR-alpha is stimulated in an autocrine manner by PDGF secreted during platelet activation. Preincubation of platelets with PDGF inhibited thrombin-induced platelet aggregation and secretion of ATP + ADP and beta-hexosaminidase. Thrombin-induced platelet aggregation was also reversed when PDGF was added 30 s after thrombin stimulation. Inhibition of the autocrine PDGF pathway during platelet activation by the PDGF antibody led to a potentiation of thrombin-induced beta-hexosaminidase secretion. Thus, the PDGFR-alpha takes part in a negative feedback regulation during platelet activation. Our demonstration of PDGF alpha-receptors on human platelets and its inhibitory function during platelet activation identifies a new possible role of PDGF in the regulation of thrombosis.

The inability of phosphatidylinositol 3-kinase activation to stimulate GLUT4 translocation indicates additional signaling pathways are required for insulin-stimulated glucose uptake.

PubMed

Isakoff, S J; Taha, C; Rose, E; Marcusohn, J; Klip, A; Skolnik, E Y

1995-10-24

Recent experimental evidence has focused attention to the role of two molecules, insulin receptor substrate 1 (IRS-1) and phosphatidylinositol 3-kinase (PI3-kinase), in linking the insulin receptor to glucose uptake; IRS-1 knockout mice are insulin resistant, and pharmacological inhibitors of PI3-kinase block insulin-stimulated glucose uptake. To investigate the role of PI3-kinase and IRS-1 in insulin-stimulated glucose uptake we examined whether stimulation of insulin-sensitive cells with platelet-derived growth factor (PDGF) or with interleukin 4 (IL-4) stimulates glucose uptake; the activated PDGF receptor (PDGFR) directly binds and activates PI3-kinase, whereas the IL-4 receptor (IL-4R) activates PI3-kinase via IRS-1 or the IRS-1-related molecule 4PS. We found that stimulation of 3T3-L1 adipocytes with PDGF resulted in tyrosine phosphorylation of the PDGFR and activation of PI3-kinase in these cells. To examine whether IL-4 stimulates glucose uptake, L6 myoblasts were engineered to overexpress GLUT4 as well as both chains of the IL-4R (L6/IL-4R/GLUT4); when these L6/IL-4R/GLUT4 myoblasts were stimulated with IL-4, IRS-1 became tyrosine phosphorylated and associated with PI3-kinase. Although PDGF and IL-4 can activate PI3-kinase in the respective cell lines, they do not possess insulin's ability to stimulate glucose uptake and GLUT4 translocation to the plasma membrane. These findings indicate that activation of PI3-kinase is not sufficient to stimulate GLUT4 translocation to the plasma membrane. We postulate that activation of a second signaling pathway by insulin, distinct from PI3-kinase, is necessary for the stimulation of glucose uptake in insulin-sensitive cells.

Gab-family adapter molecules in signal transduction of cytokine and growth factor receptors, and T and B cell antigen receptors.

PubMed

Hibi, M; Hirano, T

2000-04-01

Gab1 and Gab2 (Grb2 associated binder 1 and 2) are scaffolding adapter molecules that display sequence similarity with Drosophila DOS (daughter of sevenless), which is a potential substrate for the protein tyrosine phosphatase, Corkscrew, Both Gab1 and Gab2, like DOS, have a pleckstrin homology domain and potential binding sites for SH2 and SH3 domains. Gab1 and Gab2 are phosphorylated on tyrosine upon the stimulation of various cytokines, growth factors, and antigen receptors, and interact with signaling molecules, such as Grb2, SHP-2, and PI-3 kinase. Overexpression of Gab1 or Gab2 mimics or enhances growth factor or cytokine-mediated biological processes and activates ERK MAP kinase. These data imply that Gab1 and Gab2 act downstream of a broad range of cytokine and growth factor receptors, as well as T and B antigen receptors, and link these receptors to ERK MAP kinase and biological actions.

Renal atrial natriuretic factor receptors in hamster cardiomyopathy.

PubMed

Mukaddam-Daher, S; Jankowski, M; Dam, T V; Quillen, E W; Gutkowska, J

1995-12-01

Hamsters with cardiomyopathy (CMO), an experimental model of congestive heart failure, display stimulated renin-angiotensin-aldosterone and enhanced sympathetic nervous activity, all factors that lead to sodium retention, volume expansion and subsequent elevation of plasma atrial natriuretic factor (ANF) by the cardiac atria. However, sodium and water retention persist in CMO, indicating hyporesponsiveness to endogenous ANF. These studies were undertaken to fully characterize renal ANF receptor subtypes in normal hamsters and to evaluate whether alterations in renal ANF receptors may contribute to renal resistance to ANF in cardiomyopathy. Transcripts of the guanylyl cyclase-A (GC-A) and guanylyl cyclase B (GC-B) receptors were detected by quantitative polymerase chain reaction (PCR) in renal cortex, and outer and inner medullas. Compared to normal controls, the cardiomyopathic hamster's GC-A mRNA was similar in cortex but significantly increased in outer and inner medulla. Levels of GC-B mRNA were not altered by the disease. On the other hand, competitive binding studies, autoradiography, and affinity cross-linking demonstrated the absence of functional GC-B receptors in the kidney glomeruli and inner medulla. Also, C-type natriuretic peptide (CNP), the natural ligand for the GC-B receptors, failed to stimulate glomerular production of its second messenger cGMP. In CMO, sodium and water excretion were significantly reduced despite elevated plasma ANF (50.5 +/- 11.1 vs. 309.4 +/- 32.6 pg/ml, P < 0.001). Competitive binding studies of renal glomerular ANF receptors revealed no change in total receptor density, Bmax (369.6 +/- 27.4 vs. 282.8 +/- 26.2 fmol/mg protein), nor in dissociation constant, Kd (647.4 +/- 79.4 vs. 648.5 +/- 22.9 pM). Also, ANF-C receptor density (254.3 +/- 24.8 vs. 233.8 +/- 23.5 fmol/mg protein), nor affinity were affected by heart failure. Inner medullary receptors were exclusively of the GC-A subtype with Bmax (153.2 +/- 26.4 vs. 134.5 +/- 21.2 fmol/mg protein) and Kd (395.7 +/- 148.0 vs. 285.8 +/- 45.0 pM) not altered by cardiomyopathy. The increase in ANF-stimulated glomerular cGMP production was similar in normal and CMO hamsters (94- vs. 75-fold). These results demonstrate that renal ANF receptors do not contribute to the attenuated renal responses to ANF in hamster cardiomyopathy.

Blocking of p38 and transforming growth factor β receptor pathways impairs the ability of tolerogenic dendritic cells to suppress murine arthritis.

PubMed

Gárate, David; Rojas-Colonelli, Nicole; Peña, Corina; Salazar, Lorena; Abello, Paula; Pesce, Bárbara; Aravena, Octavio; García-González, Paulina; Ribeiro, Carolina H; Molina, María C; Catalán, Diego; Aguillón, Juan C

2013-01-01

Dendritic cells (DCs) modulated with lipopolysaccharide (LPS) are able to reduce inflammation when therapeutically administered into mice with collagen-induced arthritis (CIA). The aim of this study was to uncover the mechanisms that define the tolerogenic effect of short-term LPS-modulated DCs on CIA. Bone marrow-derived DCs were stimulated for 4 hours with LPS and characterized for their expression of maturation markers and their cytokine secretion profiles. Stimulated cells were treated with SB203580 or SB431542 to inhibit the p38 or transforming growth factor β (TGFβ) receptor pathway, respectively, or were left unmodified and, on day 35 after CIA induction, were used to inoculate mice. Disease severity was evaluated clinically. CD4+ T cell populations were counted in the spleen and lymph nodes from inoculated or untreated mice with CIA. CD4+ splenic T cells were transferred from mice with CIA treated with LPS-stimulated DCs or from untreated mice with CIA into other mice with CIA on day 35 of arthritis. Treatment with LPS-stimulated DCs increased the numbers of interleukin-10 (IL-10)-secreting and TGFβ-secreting CD4+ T cells, but decreased the numbers of Th17 cells. Adoptive transfer of CD4+ T cells from treated mice with CIA reproduced the inhibition of active CIA accomplished with LPS-stimulated DCs. The therapeutic effect of LPS-stimulated DCs and their influence on T cell populations were abolished when the p38 and the TGFβ receptor pathways were inhibited. DCs modulated short-term (4 hours) with LPS are able to confer a sustained cure in mice with established arthritis by re-educating the CD4+ T cell populations. This effect is dependent on the p38 and the TGFβ receptor signaling pathways, which suggests the participation of IL-10 and TGFβ in the recovery of tolerance. Copyright © 2013 by the American College of Rheumatology.

Protein-tyrosine-phosphatase-mediated epidermal growth factor (EGF) receptor transinactivation and EGF receptor-independent stimulation of mitogen-activated protein kinase by bradykinin in A431 cells.

PubMed Central

Graness, A; Hanke, S; Boehmer, F D; Presek, P; Liebmann, C

2000-01-01

Transactivation of the epidermal growth factor (EGF) receptor (EGFR) has been proposed to represent an essential link between G-protein-coupled receptors and the mitogen-activated protein kinase (MAPK) pathway in various cell types. In the present work we report, in contrast, that in A431 cells bradykinin transinactivates the EGFR and stimulates MAPK activity independently of EGFR tyrosine phosphorylation. Both effects of bradykinin are mediated by a pertussis-toxin-insensitive G-protein. Three lines of evidence suggest the activation of a protein tyrosine phosphatase (PTP) by bradykinin: (i) treatment of A431 cells with bradykinin decreases both basal and EGF-induced EGFR tyrosine phosphorylation, (ii) this effect of bradykinin can be blocked by two different PTP inhibitors, and (iii) bradykinin significantly increased the PTP activity in total A431 cell lysates when measured in vitro. The transmembrane receptor PTP sigma was identified as a putative mediator of bradykinin-induced downregulation of EGFR autophosphorylation. Activation of MAPK in response to bradykinin was insensitive towards AG 1478, a specific inhibitor of EGFR tyrosine kinase, but was blocked by wortmannin or bisindolylmaleimide, inhibitors of phosphatidylinositol 3-kinase (PI3-K) and protein kinase C (PKC) respectively. These results also suggest that the bradykinin-induced activation of MAPK is independent of EGFR and indicate a pathway involving PI3-K and PKC. In addition, bradykinin evokes a rapid and transient increase in Src kinase activity. Although Src does not participate in bradykinin-induced stimulation of PTP activity, inhibition of Src by 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine leads to an increase in MAPK activation by bradykinin. Our results suggest that in A431 cells the G(q/11)-protein-coupled bradykinin B(2) receptor may stimulate PTP activity and thereby transinactivate the EGFR, and may simultaneously activate MAPK by an alternative signalling pathway which can bypass EGFR. PMID:10749673

Adolescent Stimulation of D2 Receptors Alters the Maturation of Dopamine-dependent Goal-Directed Behavior

PubMed Central

Naneix, Fabien; Marchand, Alain R; Pichon, Anaïs; Pape, Jean- Rémi; Coutureau, Etienne

2013-01-01

Adolescence is a period of high sensitivity to drugs and rewards, characterized by the immaturity of decision-making abilities. A chronic stimulation of reward systems during this period might constitute a factor of vulnerability to the development of psychiatric disorders. However, the long-term consequences of such an exposure have seldom been explored. Here, we investigate at the adult age the effects of chronic dopamine (DA) stimulation during adolescence on both the maturation of DA systems and the cognitive processes underlying goal-directed actions. We first demonstrate that chronic stimulation of D2 receptors by quinpirole during adolescence alters the development of DA systems. This treatment has particularly prominent effects on the mesocortical DA pathway where it decreases DA fibers density, DA concentration, and DA receptors expression. Furthermore, we show that quinpirole-treated rats exhibit specific impairments in instrumental goal-directed behavior, as they fail to adapt their action when action–outcome relationships change in a contingency degradation procedure. These results therefore highlight the vulnerability of DA system and prefrontal areas to prolonged stimulation during adolescence, and its potential long-term impact on cognitive functions. PMID:23443719

β-Adrenergic Receptor Stimulated Ncx1 Upregulation is Mediated via a CaMKII/AP-1 Signaling Pathway in Adult Cardiomyocytes

PubMed Central

Mani, Santhosh K.; Egan, Erin A.; Addy, Benjamin K.; Grimm, Michael; Kasiganesan, Harinath; Thiyagarajan, Thirumagal; Renaud, Ludivine; Brown, Joan Heller; Kern, Christine B.; Menick, Donald R.

2013-01-01

The Na+-Ca2+ exchanger gene (Ncx1) is upregulated in hypertrophy and is often found elevated in end-stage heart failure. Studies have shown that the change in its expression contributes to contractile dysfunction. β-adrenergic receptor (β-AR) signaling plays an important role in the regulation of calcium homeostasis in the cardiomyocyte but chronic activation in periods of cardiac stress contribute to heart failure by mechanisms which include Ncx1 upregulation. Here, using a Ca2+/Calmodulin-Dependent Protein Kinase II (CaMKIIδc) null mouse, we demonstrate that β-AR-stimulated Ncx1 upregulation is dependent on CaMKII. β-AR-stimulated Ncx1 expression is mediated by activator protein 1 (AP-1) factors and is independent of cAMP-response element-binding protein (CREB) activation. The MAP kinases (ERK1/2, JNK and p38) are not required for AP-1 factor activation. Chromatin immunoprecipitation demonstrates that β-AR stimulation activates the ordered recruitment of JunB homodimers which then are replaced by c-Jun homodimers binding to the proximal AP-1 elements of the endogenous Ncx1 promoter. In conclusion, this work has provided insight into the intracellular signaling pathways and transcription factors regulating Ncx1 gene expression in a chronically β-AR-stimulated heart. PMID:19945464

Epidermal growth factor–stimulated Akt phosphorylation requires clathrin or ErbB2 but not receptor endocytosis

PubMed Central

Garay, Camilo; Judge, Gurjeet; Lucarelli, Stefanie; Bautista, Stephen; Pandey, Rohan; Singh, Tanveer; Antonescu, Costin N.

2015-01-01

Epidermal growth factor (EGF) binding to its receptor (EGFR) activates several signaling intermediates, including Akt, leading to control of cell survival and metabolism. Concomitantly, ligand-bound EGFR is incorporated into clathrin-coated pits—membrane structures containing clathrin and other proteins—eventually leading to receptor internalization. Whether clathrin might regulate EGFR signaling at the plasma membrane before vesicle scission is poorly understood. We compared the effect of clathrin perturbation (preventing formation of, or receptor recruitment to, clathrin structures) to that of dynamin2 (allowing formation of clathrin structures but preventing EGFR internalization) under conditions in which EGFR endocytosis is clathrin dependent. Clathrin perturbation by siRNA gene silencing, with the clathrin inhibitor pitstop2, or knocksideways silencing inhibited EGF-simulated Gab1 and Akt phosphorylation in ARPE-19 cells. In contrast, perturbation of dynamin2 with inhibitors or by siRNA gene silencing did not affect EGF-stimulated Gab1 or Akt phosphorylation. EGF stimulation enriched Gab1 and phospho-Gab1 within clathrin structures. ARPE-19 cells have low ErbB2 expression, and overexpression and knockdown experiments revealed that robust ErbB2 expression bypassed the requirement for clathrin for EGF-stimulated Akt phosphorylation. Thus clathrin scaffolds may represent unique plasma membrane signaling microdomains required for signaling by certain receptors, a function that can be separated from vesicle formation. PMID:26246598

Identification of a Novel, Putative Rho-specific GDP/GTP Exchange Factor and a RhoA-binding Protein: Control of Neuronal Morphology

PubMed Central

Gebbink, Martijn F.B.G.; Kranenburg, Onno; Poland, Mieke; van Horck, Francis P.G.; Houssa, Brahim; Moolenaar, Wouter H.

1997-01-01

The small GTP-binding protein Rho has been implicated in the control of neuronal morphology. In N1E-115 neuronal cells, the Rho-inactivating C3 toxin stimulates neurite outgrowth and prevents actomyosin-based neurite retraction and cell rounding induced by lysophosphatidic acid (LPA), sphingosine-1-phosphate, or thrombin acting on their cognate G protein–coupled receptors. We have identified a novel putative GDP/GTP exchange factor, RhoGEF (190 kD), that interacts with both wild-type and activated RhoA, but not with Rac or Cdc42. RhoGEF, like activated RhoA, mimics receptor stimulation in inducing cell rounding and in preventing neurite outgrowth. Furthermore, we have identified a 116-kD protein, p116Rip, that interacts with both the GDP- and GTP-bound forms of RhoA in N1E-115 cells. Overexpression of p116Rip stimulates cell flattening and neurite outgrowth in a similar way to dominant-negative RhoA and C3 toxin. Cells overexpressing p116Rip fail to change their shape in response to LPA, as is observed after Rho inactivation. Our results indicate that (a) RhoGEF may link G protein–coupled receptors to RhoA activation and ensuing neurite retraction and cell rounding; and (b) p116Rip inhibits RhoA-stimulated contractility and promotes neurite outgrowth. PMID:9199174

Mechanism regulating nuclear calcium signaling.

PubMed

Malviya, Anant N; Klein, Christian

2006-01-01

Although the outer nuclear membrane is continuous with the endoplasmic reticulum, it is possible to isolate nuclei both intact and free from endoplasmic reticulum contaminants. The outer and the inner nuclear membranes can be purified free from cross-contamination. Evidence in support of autonomous regulation of nuclear calcium signaling relies upon the investigations with isolated nuclei. Mechanisms for generating calcium signaling in the nucleus have been identified. Two calcium transporting systems, an ATP-dependant nuclear Ca(2+)-ATPase and an IP4-mediated inositol 1,3,4,5-tetrakisphosphate receptor, are located on the outer nuclear membrane. Thus, ATP and IP4, depending on external free calcium concentrations, are responsible for filling the nuclear envelope calcium pool. The inositol 1,4,5-trisphosphate receptor is located on the inner nuclear membrane with its ligand binding domain facing toward the nucleoplasm. Likewise, the ryanodine receptor is located on the inner nuclear membrane and its ligand cADP-ribose is generated within the nucleus. A 120 kDa protein fragment of nuclear PLC-gamma1 is stimulated in vivo by epidermal growth factor nuclear signaling coincident with the time course of nuclear membrane epidermal growth factor receptor activation. Stimulated 120 kDa protein fragment interacts with PIKE, a nuclear GTPase, and together they form a complex with PI[3]kinase serving as a module for nuclear PI[3]K stimulation. Thus, the nucleus has its own IP(3) generating system.

Combinatorial Effects of the Glucocorticoid Receptor and Krüppel-Like Transcription Factor 15 on Bovine Herpesvirus 1 Transcription and Productive Infection.

PubMed

El-Mayet, Fouad S; Sawant, Laximan; Thunuguntla, Prasanth; Jones, Clinton

2017-11-01

Bovine herpesvirus 1 (BoHV-1), an important bovine pathogen, establishes lifelong latency in sensory neurons. Latently infected calves consistently reactivate from latency following a single intravenous injection of the synthetic corticosteroid dexamethasone. The immediate early transcription unit 1 (IEtu1) promoter, which drives bovine ICP0 (bICP0) and bICP4 expression, is stimulated by dexamethasone because it contains two glucocorticoid receptor (GR) response elements (GREs). Several Krüppel-like transcription factors (KLF), including KLF15, are induced during reactivation from latency, and they stimulate certain viral promoters and productive infection. In this study, we demonstrate that the GR and KLF15 were frequently expressed in the same trigeminal ganglion (TG) neuron during reactivation and cooperatively stimulated productive infection and IEtu1 GREs in mouse neuroblastoma cells (Neuro-2A). We further hypothesized that additional regions in the BoHV-1 genome are transactivated by the GR or stress-induced transcription factors. To test this hypothesis, BoHV-1 DNA fragments (less than 400 bp) containing potential GR and KLF binding sites were identified and examined for transcriptional activation by stress-induced transcription factors. Intergenic regions within the unique long 52 gene (UL52; a component of the DNA primase/helicase complex), bICP4, IEtu2, and the unique short region were stimulated by KLF15 and the GR. Chromatin immunoprecipitation studies revealed that the GR and KLF15 interacted with sequences within IEtu1 GREs and the UL52 fragment. Coimmunoprecipitation studies demonstrated that KLF15 and the GR were associated with each other in transfected cells. Since the GR stimulates KLF15 expression, we suggest that these two transcription factors form a feed-forward loop that stimulates viral gene expression and productive infection following stressful stimuli. IMPORTANCE Bovine herpesvirus 1 (BoHV-1) is an important viral pathogen that causes respiratory disease and suppresses immune responses in cattle; consequently, life-threatening bacterial pneumonia can occur. Following acute infection, BoHV-1 establishes lifelong latency in sensory neurons. Reactivation from latency is initiated by the synthetic corticosteroid dexamethasone. Dexamethasone stimulates lytic cycle viral gene expression in sensory neurons of calves latently infected with BoHV-1, culminating in virus shedding and transmission. Two stress-induced cellular transcription factors, Krüppel-like transcription factor 15 (KLF15) and the glucocorticoid receptor (GR), cooperate to stimulate productive infection and viral transcription. Additional studies demonstrated that KLF15 and the GR form a stable complex and that these stress-induced transcription factors bind to viral DNA sequences, which correlates with transcriptional activation. The ability of the GR and KLF15 to synergistically stimulate viral gene expression and productive infection may be critical for the ability of BoHV-1 to reactivate from latency following stressful stimuli. Copyright © 2017 American Society for Microbiology.

Combinatorial Effects of the Glucocorticoid Receptor and Krüppel-Like Transcription Factor 15 on Bovine Herpesvirus 1 Transcription and Productive Infection

PubMed Central

El-mayet, Fouad S.; Sawant, Laximan; Thunuguntla, Prasanth

2017-01-01

ABSTRACT Bovine herpesvirus 1 (BoHV-1), an important bovine pathogen, establishes lifelong latency in sensory neurons. Latently infected calves consistently reactivate from latency following a single intravenous injection of the synthetic corticosteroid dexamethasone. The immediate early transcription unit 1 (IEtu1) promoter, which drives bovine ICP0 (bICP0) and bICP4 expression, is stimulated by dexamethasone because it contains two glucocorticoid receptor (GR) response elements (GREs). Several Krüppel-like transcription factors (KLF), including KLF15, are induced during reactivation from latency, and they stimulate certain viral promoters and productive infection. In this study, we demonstrate that the GR and KLF15 were frequently expressed in the same trigeminal ganglion (TG) neuron during reactivation and cooperatively stimulated productive infection and IEtu1 GREs in mouse neuroblastoma cells (Neuro-2A). We further hypothesized that additional regions in the BoHV-1 genome are transactivated by the GR or stress-induced transcription factors. To test this hypothesis, BoHV-1 DNA fragments (less than 400 bp) containing potential GR and KLF binding sites were identified and examined for transcriptional activation by stress-induced transcription factors. Intergenic regions within the unique long 52 gene (UL52; a component of the DNA primase/helicase complex), bICP4, IEtu2, and the unique short region were stimulated by KLF15 and the GR. Chromatin immunoprecipitation studies revealed that the GR and KLF15 interacted with sequences within IEtu1 GREs and the UL52 fragment. Coimmunoprecipitation studies demonstrated that KLF15 and the GR were associated with each other in transfected cells. Since the GR stimulates KLF15 expression, we suggest that these two transcription factors form a feed-forward loop that stimulates viral gene expression and productive infection following stressful stimuli. IMPORTANCE Bovine herpesvirus 1 (BoHV-1) is an important viral pathogen that causes respiratory disease and suppresses immune responses in cattle; consequently, life-threatening bacterial pneumonia can occur. Following acute infection, BoHV-1 establishes lifelong latency in sensory neurons. Reactivation from latency is initiated by the synthetic corticosteroid dexamethasone. Dexamethasone stimulates lytic cycle viral gene expression in sensory neurons of calves latently infected with BoHV-1, culminating in virus shedding and transmission. Two stress-induced cellular transcription factors, Krüppel-like transcription factor 15 (KLF15) and the glucocorticoid receptor (GR), cooperate to stimulate productive infection and viral transcription. Additional studies demonstrated that KLF15 and the GR form a stable complex and that these stress-induced transcription factors bind to viral DNA sequences, which correlates with transcriptional activation. The ability of the GR and KLF15 to synergistically stimulate viral gene expression and productive infection may be critical for the ability of BoHV-1 to reactivate from latency following stressful stimuli. PMID:28794031

Are GnRH and FSH potentially damaging factors in the cardiovascular system?

PubMed

Poljak, Z; Hulin, I; Maruscakova, L; Mladosievicova, B

2018-04-02

In the physiological view the human cardiomyocytes express receptors of gonadotropin-releasing hormone and follicle-stimulating hormone. The local effects of these hormones in the heart are related also to some interstitial cells, such as endothelial cells with follicle-stimulating hormone receptors and immune cells with gonadotropin-releasing hormone receptors. The administration of androgen deprivation therapy in patients with prostate cancer is associated with increased incidence of cardiovascular complications. It is suggested that negative action of this therapy on cardiovascular system is due to the loss of testosterone but also levels of gonadotropin-releasing hormone and follicle-stimulating hormone are changed by therapy. In this article we review the literature to date with an emphasis on recent investigation focused on potential role of abnormal gonadotropin-releasing hormone and follicle-stimulating hormone levels induced by gonadotropin-releasing hormone agonists on the cardiovascular risk. These facts exacerbate the complexity of specific hormone and cell relationships within heart and vessels. Androgen deprivation therapy reveals the physiological relationships between hormones and specific tissues that are not part of the endocrine system.

Cloning and expression of porcine Colony Stimulating Factor-1 (CSF-1) and Colony Stimulating Factor-1 Receptor (CSF-1R) and analysis of the species specificity of stimulation by CSF-1 and Interleukin 34

PubMed Central

Gow, Deborah J.; Garceau, Valerie; Kapetanovic, Ronan; Sester, David P.; Fici, Greg J.; Shelly, John A.; Wilson, Thomas L.; Hume, David A.

2012-01-01

Macrophage Colony Stimulating Factor (CSF-1) controls the survival, differentiation and proliferation of cells of the mononuclear phagocyte system. A second ligand for the CSF-1R, Interleukin 34 (IL-34), has been described, but its physiological role is not yet known. The domestic pig provides an alternative to traditional rodent models for evaluating potential therapeutic applications of CSF-1R agonists and antagonists. To enable such studies, we cloned and expressed active pig CSF-1. To provide a bioassay, pig CSF-1R was expressed in the factor-dependent Ba/F3 cell line. On this transfected cell line, recombinant porcine CSF-1 and human CSF-1 had identical activity. Mouse CSF-1 does not interact with the human CSF-1 receptor but was active on pig. By contrast, porcine CSF-1 was active on mouse, human, cat and dog cells. IL-34 was previously shown to be species-specific, with mouse and human proteins demonstrating limited cross-species activity. The pig CSF-1R was equally responsive to both mouse and human IL-34. Based upon the published crystal structures of CSF-1/CSF-1R and IL34/CSF-1R complexes, we discuss the molecular basis for the species specificity. PMID:22974529

CRF1 receptor-deficiency increases cocaine reward.

PubMed

Contarino, Angelo; Kitchener, Pierre; Vallée, Monique; Papaleo, Francesco; Piazza, Pier-Vincenzo

2017-05-01

Stimulant drugs produce reward but also activate stress-responsive systems. The corticotropin-releasing factor (CRF) and the related hypothalamus-pituitary-adrenal (HPA) axis stress-responsive systems are activated by stimulant drugs. However, their role in stimulant drug-induced reward remains poorly understood. Herein, we report that CRF 1 receptor-deficient (CRF 1 -/-), but not wild-type, mice show conditioned place preference (CPP) responses to a relatively low cocaine dose (5 mg/kg, i.p.). Conversely, wild-type, but not CRF 1 -/-, mice display CPP responses to a relatively high cocaine dose (20 mg/kg, i.p.), indicating that CRF 1 receptor-deficiency alters the rewarding effects of cocaine. Acute pharmacological antagonism of the CRF 1 receptor by antalarmin also eliminates cocaine reward. Nevertheless, CRF 1 -/- mice display higher stereotypy responses to cocaine than wild-type mice. Despite the very low plasma corticosterone concentration, CRF 1 -/- mice show higher nuclear glucocorticoid receptor (GR) levels in the brain region of the hippocampus than wild-type mice. Full rescue of wild-type-like corticosterone and GR circadian rhythm and level in CRF 1 -/- mice by exogenous corticosterone does not affect CRF 1 receptor-dependent cocaine reward but induces stereotypy responses to cocaine. These results indicate a critical role for the CRF 1 receptor in cocaine reward, independently of the closely related HPA axis activity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Hepatocyte growth factor induces proliferation and differentiation of multipotent and erythroid hemopoietic progenitors.

PubMed

Galimi, F; Bagnara, G P; Bonsi, L; Cottone, E; Follenzi, A; Simeone, A; Comoglio, P M

1994-12-01

Hepatocyte growth factor (HGF) is a mesenchymal derived growth factor known to induce proliferation and "scattering" of epithelial and endothelial cells. Its receptor is the tyrosine kinase encoded by the c-MET protooncogene. Here we show that highly purified recombinant HGF stimulates hemopoietic progenitors to form colonies in vitro. In the presence of erythropoietin, picomolar concentrations of HGF induced the formation of erythroid burst-forming unit colonies from CD34-positive cells purified from human bone marrow, peripheral blood, or umbilical cord blood. The growth stimulatory activity was restricted to the erythroid lineage. HGF also stimulated the formation of multipotent CFU-GEMM colonies. This effect is synergized by stem cell factor, the ligand of the tyrosine kinase receptor encoded by the c-KIT protooncogene, which is active on early hemopoietic progenitors. By flow cytometry analysis, the receptor for HGF was found to be expressed on the cell surface in a fraction of CD34+ progenitors. Moreover, in situ hybridization experiments showed that HGF receptor mRNA is highly expressed in embryonic erythroid cells (megaloblasts). HGF mRNA was also found to be produced in the embryonal liver. These data show that HGF plays a direct role in the control of proliferation and differentiation of erythroid progenitors, and they suggest that it may be one of the long-sought mediators of paracrine interactions between stromal and hemopoietic cells within the hemopoietic microenvironment.

Insights into the TOR-S6K signaling pathway in maize (Zea mays L.). pathway activation by effector-receptor interaction.

PubMed

Garrocho-Villegas, Verónica; Aguilar C, Raúl; Sánchez de Jiménez, Estela

2013-12-23

The primordial TOR pathway, known to control growth and cell proliferation, has still not been fully described for plants. Nevertheless, in maize, an insulin-like growth factor (ZmIGF) peptide has been reported to stimulate this pathway. This research provides further insight into the TOR pathway in maize, using a biochemical approach in cultures of fast-growing (FG) and slow-growing (SG) calli, as a model system. Our results revealed that addition of either ZmIGF or insulin to SG calli stimulated DNA synthesis and increased the growth rate through cell proliferation and increased the rate of ribosomal protein (RP) synthesis by the selective mobilization of RP mRNAs into polysomes. Furthermore, analysis of the phosphorylation status of the main TOR and S6K kinases from the TOR pathway revealed stimulation by ZmIGF or insulin, whereas rapamycin inhibited its activation. Remarkably, a putative maize insulin-like receptor was recognized by a human insulin receptor antibody, as demonstrated by immunoprecipitation from membrane protein extracts of maize callus. Furthermore, competition experiments between ZmIGF and insulin for the receptor site on maize protoplasts suggested structural recognition of the putative receptor by either effector. These data were confirmed by confocal immunolocalization within the cell membrane of callus cells. Taken together, these data indicate that cell growth and cell proliferation in maize depend on the activation of the TOR-S6K pathway through the interaction of an insulin-like growth factor and its receptor. This evidence suggests that higher plants as well as metazoans have conserved this biochemical pathway to regulate their growth, supporting the conclusion that it is a highly evolved conserved pathway.

Platelet-derived growth factor receptor mediates activation of ras through different signaling pathways in different cell types.

PubMed Central

Satoh, T; Fantl, W J; Escobedo, J A; Williams, L T; Kaziro, Y

1993-01-01

A series of pieces of evidence have shown that Ras protein acts as a transducer of the platelet-derived growth factor (PDGF) receptor-mediated signaling pathway: (i) formation of Ras.GTP is detected immediately on PDGF stimulation, and (ii) a dominant inhibitory mutant Ras, as well as a neutralizing anti-Ras antibody, can interfere with PDGF-induced responses. On the other hand, several signal transducing molecules including phosphatidylinositol 3-kinase (PI3-K), GTPase-activating protein (GAP), and phospholipase C gamma (PLC gamma) bind directly to the PDGF receptor and become tyrosine phosphorylated. Recently, it was shown that specific phosphorylated tyrosines of the PDGF receptor are responsible for interaction between the receptor and each signaling molecule. However, the roles of these signaling molecules have not been elucidated, and it remains unclear which molecules are implicated in the Ras pathway. In this study, we measured Ras activation in cell lines expressing mutant PDGF receptors that are deficient in coupling with specific molecules. In fibroblast CHO cells, a mutant receptor (Y708F/Y719F [PI3-K-binding sites]) was unable to stimulate Ras, whereas another mutant (Y739F [the GAP-binding site]) could do so, suggesting an indispensable role of PI3-K or a protein that binds to the same sites as PI3-K for PDGF-stimulated Ras activation. By contrast, both of the above mutants were capable of stimulating Ras protein in a pro-B-cell line, BaF3. Furthermore, a mutant receptor (Y977F/Y989F [PLC gamma-binding sites]) could fully activate Ras, and the direct activation of protein kinase C and calcium mobilization had almost no effect on the GDP/GTP state of Ras in this cell line. These results suggest that, in the pro-B-cell transfectants, each of the above pathways (PI3-K, GAP, and PLC gamma) can be eliminated without a loss of Ras activation. It remains unclear whether another unknown essential pathway which regulates Ras protein exists within BaF3 cells. Therefore, it is likely that several different PDGF receptor-mediated signaling pathways function upstream of Ras, and the extent of the contribution of each pathway for the regulation of Ras may differ among different cell types. Images PMID:8388543

Profibrotic TGFβ responses require the cooperative action of PDGF and ErbB receptor tyrosine kinases.

PubMed

Andrianifahanana, Mahefatiana; Wilkes, Mark C; Gupta, Shiv K; Rahimi, Rod A; Repellin, Claire E; Edens, Maryanne; Wittenberger, Joshua; Yin, Xueqian; Maidl, Elizabeth; Becker, Jackson; Leof, Edward B

2013-11-01

Transforming growth factor β (TGFβ) has significant profibrotic activity both in vitro and in vivo. This reflects its capacity to stimulate fibrogenic mediators and induce the expression of other profibrotic cytokines such as platelet-derived growth factor (PDGF) and epidermal growth factor (EGF/ErbB) ligands. Here we address both the mechanisms by which TGFβ induced ErbB ligands and the physiological significance of inhibiting multiple TGFβ-regulated processes. The data document that ErbB ligand induction requires PDGF receptor (PDGFR) mediation and engages a positive autocrine/paracrine feedback loop via ErbB receptors. Whereas PDGFRs are essential for TGFβ-stimulated ErbB ligand up-regulation, TGFβ-specific signals are also required for ErbB receptor activation. Subsequent profibrotic responses are shown to involve the cooperative action of PDGF and ErbB signaling. Moreover, using a murine treatment model of bleomycin-induced pulmonary fibrosis we found that inhibition of TGFβ/PDGF and ErbB pathways with imatinib plus lapatinib, respectively, not only prevented myofibroblast gene expression to a greater extent than either drug alone, but also essentially stabilized gas exchange (oxygen saturation) as an overall measure of lung function. These observations provide important mechanistic insights into profibrotic TGFβ signaling and indicate that targeting multiple cytokines represents a possible strategy to ameliorate organ fibrosis dependent on TGFβ.

Modulation of type II TGF-β receptor degradation by integrin-linked kinase.

PubMed

Vi, Linda; Boo, Stellar; Sayedyahossein, Samar; Singh, Randeep K; McLean, Sarah; Di Guglielmo, Gianni M; Dagnino, Lina

2015-03-01

Cutaneous responses to injury, infection, and tumor formation involve the activation of resident dermal fibroblasts and subsequent transition to myofibroblasts. The key for induction of myofibroblast differentiation is the activation of transforming growth factor-β (TGF-β) receptors and stimulation of integrins and their associated proteins, including integrin-linked kinase (ILK). Cross-talk processes between TGF-β and ILK are crucial for myofibroblast formation, as ILK-deficient dermal fibroblasts exhibit impaired responses to TGF-β receptor stimulation. We now show that ILK associates with type II TGF-β receptors (TβRII) in ligand- and receptor kinase activity-independent manners. In cells with targeted Ilk gene inactivation, cellular levels of TβRII are decreased, through mechanisms that involve enhanced ubiquitination and proteasomal degradation. Partitioning of TGF-β receptors into membrane has been linked to proteasome-dependent receptor degradation. We found that interfering with membrane raft formation in ILK-deficient cells restored TβRII levels and signaling. These observations support a model whereby ILK functions in fibroblasts to direct TβRII away from degradative pathways during their differentiation into myofibroblasts.

Granulocyte macrophage-colony stimulating factor and interleukin-3 increase expression of type II tumour necrosis factor receptor, increasing susceptibility to tumour necrosis factor-induced apoptosis. Control of leukaemia cell life/death switching.

PubMed

Rae, C; MacEwan, D J

2004-12-01

Tumour necrosis factor (TNF) induces apoptosis in a range of cell types via its two receptors, TNFR1 and TNFR2. Here, we demonstrate that proliferation and TNFR2 expression was increased in human leukaemic TF-1 cells by granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-3 (IL-3), with TNFR1 expression unaffected. Consequently, they switch from a proliferative to a TNF-induced apoptotic phenotype. Raised TNFR2 expression and susceptibility to TNF-induced apoptosis was not a general effect of proliferation as IL-1beta and IFN-gamma both proliferated TF-1 cells with no effect on TNFR expression or apoptosis. Although raised TNFR2 expression correlated with the apoptotic phenotype, stimulation of apoptosis in GM-CSF-pretreated cells was mediated by TNFR1, with stimulation of TNFR2 alone insufficient to initiate cell death. However, TNFR2 did play a role in apoptotic and proliferative responses as they were blocked by the presence of an antagonistic TNFR2 antibody. Additionally, coincubation with cycloheximide blocked the mitotic effects of GM-CSF or IL-3, allowing only the apoptotic responses of TNF to persist. TNF life/death was also observed in K562, but not MOLT-4 and HL-60 human leukaemic cell types. These findings show a cooperative role of TNFR2 in the TNF life/death switching phenomenon.

Expression of CD73/ecto-5'-nucleotidase on human gingival fibroblasts and contribution to the inhibition of interleukin-1alpha-induced granulocyte-macrophage colony stimulating factor production.

PubMed

Nemoto, Eiji; Kunii, Ryotaro; Tada, Hiroyuki; Tsubahara, Taisuke; Ishihata, Hiroshi; Shimauchi, Hidetoshi

2004-02-01

CD73/5'-nucleotidase (5'-NT) is an ectoenzyme that participates in immune/inflammatory reactions. We examined the possible expression of CD73/5'-NT on human gingival fibroblasts (hGF), which are important to the immune/inflammatory system in periodontal tissue. We demonstrated that CD73/5'-NT was expressed on hGF by flow cytometry. We found that pre-treatment of hGF with 5'-AMP induced marked inhibition of granulocyte-macrophage colony-stimulating factor (GM-CSF) production from hGF upon stimulation with interleukin-1alpha (IL-1alpha) by enzyme-linked immunosorbent assay (ELISA). A specific inhibitor of 5'-NT, adenosine 5'-[alpha,beta-methylene] diphosphate blocked the inhibition of GM-CSF production, suggesting that adenosine converted from 5'-AMP acts on the inhibitory effects. The GM-CSF inhibition suggested that A3 receptor might be involved. The rank order of agonists was found to be (N6-benzyl-5'-N-ethylcarboxamidoadenosine) A3 receptor agonist > or = (2-chloroadenosine) non-selective agonist > (CGS-21680) A2A receptor agonist > adenosine > or = (N6-cyclohexyladenosine) A1 agonist. Further support for the main role of A3 receptor was the binding A3 antagonist [9-chloro-2-(2-furanyl)-5-([phenylacetyl]amino)[1,2,4]-triazolo[1,5-c]quinazdine] reversed the effect of adenosine, but no significant reverse was observed by A1 (1,3-dipropyl-8-cyclopentylxanthine), A2 [3,7-dimethyl-1-(2-propargyl)xanthine], A2A[8-(3-chlorostyryl)caffeine], and A2B (alloxazine) antagonists. The CD73/5'-NT expression was increased upon stimulation with gamma-interferon, but not other stimulants such as tumor necrosis factor-alpha, IL-4, lipopolysaccharide from Porphyromonas gingivalis and Escherichia coli, and fimbriae from P. gingivalis, and this increase was correlated with the enhanced GM-CSF inhibition by 5'-AMP but not adenosine. These findings suggested that CD73/5'-NT on hGF exerts an anti-inflammatory effects in periodontal disease by conversion from 5'-AMP to adenosine.

Dietary Fat Stimulates Pancreatic Cancer Growth and Promotes Fibrosis of the Tumor Microenvironment through the Cholecystokinin Receptor.

PubMed

Nadella, Sandeep; Burks, Julian; Al-Sabban, Abdulhameed; Inyang, Gloria; Wang, Juan; Tucker, Robin D; Zamanis, Marie E; Bukowski, William; Shivapurkar, Narayan; Smith, Jill P

2018-06-21

The gastrointestinal peptide cholecystokinin (CCK) is released from the duodenum in response to dietary fat to aid in digestion, and plasma CCK levels are elevated with the consumption of high fat diets. CCK is also a trophic peptide for the pancreas and has also been shown to stimulate growth of pancreatic cancer. In the current investigation, we studied the influence of a diet high in saturated fat on growth of pancreatic cancer in syngeneic murine models before the mice became obese to exclude the confounding factors associated with obesity. The high fat diet significantly increased growth and metastasis of pancreatic cancer compared to the control diet, and the stimulatory effect was blocked by the CCK-receptor antagonist proglumide. We then selectively knocked out the CCK receptor on the pancreatic cancer cells using CRISPR technology and showed that without CCK receptors, dietary fat was unable to stimulate cancer growth. Next we demonstrated that dietary fat failed to influence pancreatic cancer xenograft growth in genetically engineered CCK peptide knockout mice. The tumor associated fibrosis that is so prevalent in the pancreatic cancer microenvironment was significantly decreased with CCK receptor antagonist therapy since fibroblasts also have CCK receptors. The CCK receptor antagonist proglumide also altered tumor metalloprotease expression and increased tumor suppressor genes by a PCR array. Our studies confirm that a diet high in saturated fat promotes growth of pancreatic cancer and the action is mediated by the CCK- receptor pathway.

EphA2 is a functional receptor for the growth factor progranulin.

PubMed

Neill, Thomas; Buraschi, Simone; Goyal, Atul; Sharpe, Catherine; Natkanski, Elizabeth; Schaefer, Liliana; Morrione, Andrea; Iozzo, Renato V

2016-12-05

Although the growth factor progranulin was discovered more than two decades ago, the functional receptor remains elusive. Here, we discovered that EphA2, a member of the large family of Ephrin receptor tyrosine kinases, is a functional signaling receptor for progranulin. Recombinant progranulin bound with high affinity to EphA2 in both solid phase and solution. Interaction of progranulin with EphA2 caused prolonged activation of the receptor, downstream stimulation of mitogen-activated protein kinase and Akt, and promotion of capillary morphogenesis. Furthermore, we found an autoregulatory mechanism of progranulin whereby a feed-forward loop occurred in an EphA2-dependent manner that was independent of the endocytic receptor sortilin. The discovery of a functional signaling receptor for progranulin offers a new avenue for understanding the underlying mode of action of progranulin in cancer progression, tumor angiogenesis, and perhaps neurodegenerative diseases. © 2016 Neill et al.

EphA2 is a functional receptor for the growth factor progranulin

PubMed Central

Neill, Thomas; Goyal, Atul; Sharpe, Catherine

2016-01-01

Although the growth factor progranulin was discovered more than two decades ago, the functional receptor remains elusive. Here, we discovered that EphA2, a member of the large family of Ephrin receptor tyrosine kinases, is a functional signaling receptor for progranulin. Recombinant progranulin bound with high affinity to EphA2 in both solid phase and solution. Interaction of progranulin with EphA2 caused prolonged activation of the receptor, downstream stimulation of mitogen-activated protein kinase and Akt, and promotion of capillary morphogenesis. Furthermore, we found an autoregulatory mechanism of progranulin whereby a feed-forward loop occurred in an EphA2-dependent manner that was independent of the endocytic receptor sortilin. The discovery of a functional signaling receptor for progranulin offers a new avenue for understanding the underlying mode of action of progranulin in cancer progression, tumor angiogenesis, and perhaps neurodegenerative diseases. PMID:27903606

Interleukin 6 inhibits proliferation and, in cooperation with an epidermal growth factor receptor autocrine loop, increases migration of T47D breast cancer cells.

PubMed

Badache, A; Hynes, N E

2001-01-01

Interleukin (IL)-6, a multifunctional regulator of immune response, hematopoiesis, and acute phase reactions, has also been shown to regulate cancer cell proliferation. We have investigated IL-6 signaling pathways and cellular responses in the T47D breast carcinoma cell line. The IL-6-type cytokines, IL-6 and oncostatin M, simultaneously inhibited cell proliferation and increased cell migration. In T47D cells, IL-6 stimulated the activation of Janus-activated kinase 1 tyrosine kinase and signal transducers and activators of transcription (STAT) 1 and STAT3 transcription factors. Expression of dominant negative STAT3 in the cells strongly reduced IL-6-mediated growth inhibition but did not prevent IL-6-induced cell migration. IL-6 treatment led to activation of the mitogen-activated protein kinase (MAPK) and the phosphatidylinositol 3'-kinase (PI3K) pathways. Inhibition of MAPK or PI3K activity reversed IL-6- and oncostatin M-stimulated migration. Because cross-talk between cytokine receptors and members of the ErbB family of receptor tyrosine kinases has been described previously, we have examined their interaction in T47D cells. Down-regulation of ErbB receptor activity, through the use of specific pharmacological inhibitors or dominant negative receptor constructs, revealed that IL-6-induced MAPK activation was largely dependent on epidermal growth factor (EGF) receptor activity, but not on ErbB-2 activity. Using a monoclonal antibody that interferes with EGF receptor-ligand interaction, we have shown that in T47D cells, IL-6 cooperates with an EGF receptor autocrine activity loop for signaling through the MAPK and PI3K pathways and for cell migration. Both the tyrosine phosphatase SHP-2 and the multisubstrate docking molecule Gab1, which are potential links between IL-6 and the MAPK/PI3K pathways, were constitutively associated with the active EGF receptor. On IL-6 stimulation, SHP-2 and Gab1 were recruited to the gp130 subunit of the IL-6 receptor and tyrosine phosphorylated, allowing downstream signaling to the MAPK and PI3K pathways. Thus, in T47D breast carcinoma cells, IL-6 acts in synergy with EGF receptor autocrine activity to signal through the MAPK/PI3K pathways. Cooperation between IL-6 and the EGF receptor in T47D breast carcinoma cells illustrates how a combination of multiple stimuli, either exogenous or endogenous, may result in synergistic cellular responses.

The role of brain somatostatin receptor 2 in the regulation of feeding and drinking behavior.

PubMed

Stengel, Andreas; Karasawa, Hiroshi; Taché, Yvette

2015-07-01

Somatostatin was discovered four decades ago as hypothalamic factor inhibiting growth hormone release. Subsequently, somatostatin was found to be widely distributed throughout the brain and to exert pleiotropic actions via interaction with five somatostatin receptors (sst1-5) that are also widely expressed throughout the brain. Interestingly, in contrast to the predominantly inhibitory actions of peripheral somatostatin, the activation of brain sst2 signaling by intracerebroventricular injection of stable somatostatin agonists potently stimulates food intake and independently, drinking behavior in rodents. The orexigenic response involves downstream orexin-1, neuropeptide Y1 and μ receptor signaling while the dipsogenic effect is mediated through the activation of the brain angiotensin 1 receptor. Brain sst2 activation is part of mechanisms underlying the stimulation of feeding and more prominently water intake in the dark phase and is able to counteract the anorexic response to visceral stressors. Published by Elsevier Inc.

Mobility of tethering factor EEA1 on endosomes is decreased upon stimulation of EGF receptor endocytosis in HeLa cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kosheverova, Vera V., E-mail: kosheverova_vera@incras.ru; Kamentseva, Rimma S., E-mail: rkamentseva@yandex.ru; St. Petersburg State University, 7-9, Universitetskaya nab, St. Petersburg, 199034

Tethering factor EEA1, mediating homotypic fusion of early endosomes, was shown to be localized in membrane-bound state both in serum-deprived and stimulated for EGF receptor endocytosis cells. However, it is not known whether dynamics behavior of EEA1 is affected by EGF stimulation. We investigated EEA1 cytosol-to-membrane exchange rate in interphase HeLa cells by FRAP analysis. The data obtained fitted two-states binding model, with the bulk of membrane-associated EEA1 protein represented by the mobile fraction both in serum-starved and EGF-stimulated cells. Fast recovery state had similar half-times in the two cases: about 1.6 s and 2.8 s, respectively. However, the recovery half-time ofmore » slowly cycled EEA1 fraction significantly increased in EGF-stimulated comparing to serum-starved cells (from 21 to 99 s). We suppose that the retardation of EEA1 fluorescence recovery upon EGF-stimulation may be due to the increase of activated Rab5 on endosomal membranes, the growth of the number of tethering events between EEA1-positive vesicles and their clustering. - Highlights: • EEA1 mobility was compared in serum-starved and EGF-stimulated interphase HeLa cells. • FRAP analysis revealed fast and slow components of EEA1 recovery in both cases. • Stimulation of EGFR endocytosis did not affect fast EEA1 turnover. • EGF stimulation significantly increased half-time of slowly exchanged EEA1 fraction.« less

Effects of macrophage colony-stimulating factor on macrophages and their related cell populations in the osteopetrosis mouse defective in production of functional macrophage colony-stimulating factor protein.

PubMed Central

Umeda, S.; Takahashi, K.; Shultz, L. D.; Naito, M.; Takagi, K.

1996-01-01

The development of macrophage populations in osteopetrosis (op) mutant mice defective in production of functional macrophage colony-stimulating factor (M-CSF) and the response of these cell populations to exogenous M-CSF were used to classify macrophages into four groups: 1) monocytes, monocyte-derived macrophages, and osteoclasts, 2) MOMA-1-positive macrophages, 3) ER-TR9-positive macrophages, and 4) immature tissue macrophages. Monocytes, monocyte-derived macrophages, osteoclasts in bone, microglia in brain, synovial A cells, and MOMA-1- or ER-TR9-positive macrophages were deficient in op/op mice. The former three populations expanded to normal levels in op/op mice after daily M-CSF administration, indicating that they are developed and differentiated due to the effect of M-CSF supplied humorally. In contrast, the other cells did not respond or very slightly responded to M-CSF, and their development seems due to either M-CSF produced in situ or expression of receptor for M-CSF. Macrophages present in tissues of the mutant mice were immature and appear to be regulated by either granulocyte/macrophage colony-stimulating factor and/or interleukin-3 produced in situ or receptor expression. Northern blot analysis revealed different expressions of GM-CSF and IL-3 mRNA in various tissues of the op/op mice. However, granulocyte/macrophage colony-stimulating factor and interleukin-3 in serum were not detected by enzyme-linked immunosorbent assay. The immature macrophages differentiated and matured into resident macrophages after M-CSF administration, and some of these cells proliferated in response to M-CSF. Images Figure 4 Figure 6 Figure 8 Figure 10 Figure 11 PMID:8701995

Involvement of suppressors of cytokine signaling in toll-like receptor-mediated block of dendritic cell differentiation.

PubMed

Bartz, Holger; Avalos, Nicole M; Baetz, Andrea; Heeg, Klaus; Dalpke, Alexander H

2006-12-15

Dendritic cells (DCs) are important sentinels within innate immunity, monitoring the presence of infectious microorganisms. They operate in 2 different maturation stages, with transition from immature to mature DCs being induced by activation of toll-like receptors (TLRs). However, TLRs are also expressed on precursor cells of DCs. Here we analyzed the effects of TLR stimulation during the process of granulocyte-macrophage-colony-stimulating factor (GM-CSF)-mediated in vitro generation of immature DCs from precursor cells. We show that TLR triggering deviated phenotypic and functional differentiation from CD14+ monocytes to CD1a+ DCs. Similar results were obtained when differentiation of murine myeloid DCs from bone marrow cells was analyzed. The inhibitory effects were independent of soluble factors. TLR stimulation in DC precursor cells induced proteins of the suppressor of cytokine signaling family (SOCS), which correlated with loss of sensitivity to GM-CSF. Overexpression of SOCS-1 abolished GM-CSF signal transduction. Moreover, forced SOCS-1 expression in DC precursors mimicked the inhibitory effects on DC generation observed for TLR stimulation. The results indicate that TLR stimulation during the period of DC generation interferes with and deviates DC differentiation and that these effects are mediated particularly by SOCS-1.

Insulin-Like Growth Factor (IGF) Binding Protein-2, Independently of IGF-1, Induces GLUT-4 Translocation and Glucose Uptake in 3T3-L1 Adipocytes

PubMed Central

Assefa, Biruhalem; Mahmoud, Ayman M.; Pfeiffer, Andreas F. H.; Birkenfeld, Andreas L.; Spranger, Joachim

2017-01-01

Insulin-like growth factor binding protein-2 (IGFBP-2) is the predominant IGF binding protein produced during adipogenesis and is known to increase the insulin-stimulated glucose uptake (GU) in myotubes. We investigated the IGFBP-2-induced changes in basal and insulin-stimulated GU in adipocytes and the underlying mechanisms. We further determined the role of insulin and IGF-1 receptors in mediating the IGFBP-2 and the impact of IGFBP-2 on the IGF-1-induced GU. Fully differentiated 3T3-L1 adipocytes were treated with IGFBP-2 in the presence and absence of insulin and IGF-1. Insulin, IGF-1, and IGFBP-2 induced a dose-dependent increase in GU. IGFBP-2 increased the insulin-induced GU after long-term incubation. The IGFBP-2-induced impact on GU was neither affected by insulin or IGF-1 receptor blockage nor by insulin receptor knockdown. IGFBP-2 significantly increased the phosphorylation of PI3K, Akt, AMPK, TBC1D1, and PKCζ/λ and induced GLUT-4 translocation. Moreover, inhibition of PI3K and AMPK significantly reduced IGFBP-2-stimulated GU. In conclusion, IGFBP-2 stimulates GU in 3T3-L1 adipocytes through activation of PI3K/Akt, AMPK/TBC1D1, and PI3K/PKCζ/λ/GLUT-4 signaling. The stimulatory effect of IGFBP-2 on GU is independent of its binding to IGF-1 and is possibly not mediated through the insulin or IGF-1 receptor. This study highlights the potential role of IGFBP-2 in glucose metabolism. PMID:29422987

Metaplasticity and behavior: how training and inflammation affect plastic potential within the spinal cord and recovery after injury

PubMed Central

Grau, James W.; Huie, J. Russell; Lee, Kuan H.; Hoy, Kevin C.; Huang, Yung-Jen; Turtle, Joel D.; Strain, Misty M.; Baumbauer, Kyle M.; Miranda, Rajesh M.; Hook, Michelle A.; Ferguson, Adam R.; Garraway, Sandra M.

2014-01-01

Research has shown that spinal circuits have the capacity to adapt in response to training, nociceptive stimulation and peripheral inflammation. These changes in neural function are mediated by physiological and neurochemical systems analogous to those that support plasticity within the hippocampus (e.g., long-term potentiation and the NMDA receptor). As observed in the hippocampus, engaging spinal circuits can have a lasting impact on plastic potential, enabling or inhibiting the capacity to learn. These effects are related to the concept of metaplasticity. Behavioral paradigms are described that induce metaplastic effects within the spinal cord. Uncontrollable/unpredictable stimulation, and peripheral inflammation, induce a form of maladaptive plasticity that inhibits spinal learning. Conversely, exposure to controllable or predictable stimulation engages a form of adaptive plasticity that counters these maladaptive effects and enables learning. Adaptive plasticity is tied to an up-regulation of brain derived neurotrophic factor (BDNF). Maladaptive plasticity is linked to processes that involve kappa opioids, the metabotropic glutamate (mGlu) receptor, glia, and the cytokine tumor necrosis factor (TNF). Uncontrollable nociceptive stimulation also impairs recovery after a spinal contusion injury and fosters the development of pain (allodynia). These adverse effects are related to an up-regulation of TNF and a down-regulation of BDNF and its receptor (TrkB). In the absence of injury, brain systems quell the sensitization of spinal circuits through descending serotonergic fibers and the serotonin 1A (5HT 1A) receptor. This protective effect is blocked by surgical anesthesia. Disconnected from the brain, intracellular Cl- concentrations increase (due to a down-regulation of the cotransporter KCC2), which causes GABA to have an excitatory effect. It is suggested that BDNF has a restorative effect because it up-regulates KCC2 and re-establishes GABA-mediated inhibition. PMID:25249941

Insulin-Like Growth Factor (IGF) Binding Protein-2, Independently of IGF-1, Induces GLUT-4 Translocation and Glucose Uptake in 3T3-L1 Adipocytes.

PubMed

Assefa, Biruhalem; Mahmoud, Ayman M; Pfeiffer, Andreas F H; Birkenfeld, Andreas L; Spranger, Joachim; Arafat, Ayman M

2017-01-01

Insulin-like growth factor binding protein-2 (IGFBP-2) is the predominant IGF binding protein produced during adipogenesis and is known to increase the insulin-stimulated glucose uptake (GU) in myotubes. We investigated the IGFBP-2-induced changes in basal and insulin-stimulated GU in adipocytes and the underlying mechanisms. We further determined the role of insulin and IGF-1 receptors in mediating the IGFBP-2 and the impact of IGFBP-2 on the IGF-1-induced GU. Fully differentiated 3T3-L1 adipocytes were treated with IGFBP-2 in the presence and absence of insulin and IGF-1. Insulin, IGF-1, and IGFBP-2 induced a dose-dependent increase in GU. IGFBP-2 increased the insulin-induced GU after long-term incubation. The IGFBP-2-induced impact on GU was neither affected by insulin or IGF-1 receptor blockage nor by insulin receptor knockdown. IGFBP-2 significantly increased the phosphorylation of PI3K, Akt, AMPK, TBC1D1, and PKC ζ / λ and induced GLUT-4 translocation. Moreover, inhibition of PI3K and AMPK significantly reduced IGFBP-2-stimulated GU. In conclusion, IGFBP-2 stimulates GU in 3T3-L1 adipocytes through activation of PI3K/Akt, AMPK/TBC1D1, and PI3K/PKC ζ / λ /GLUT-4 signaling. The stimulatory effect of IGFBP-2 on GU is independent of its binding to IGF-1 and is possibly not mediated through the insulin or IGF-1 receptor. This study highlights the potential role of IGFBP-2 in glucose metabolism.

Occupational levels of radiation exposure induce surface expression of interleukin-2 receptors in stimulated human peripheral blood lymphocytes.

PubMed

Xu, Y; Greenstock, C L; Trivedi, A; Mitchel, R E

1996-05-01

Interleukin-2 (IL-2) is a cytokine responsible for a variety of immune and non-immune stimulatory and regulatory functions, including the activation and stimulation of cytotoxic cells able to recognize and kill human tumour cells and T-cell proliferation and differentiation. We show that low doses of radiation, in the range commonly received by atomic radiation workers or as a result of minor medical diagnostic procedures (0.25 to 10 mGy), stimulate the expression of IL-2 receptors (IL-2R) on the surface of peripheral blood lymphocytes (PBL) taken from normal human donors. This stimulated surface expression after in vitro irradiation is an indirect effect, resulting from the secretion into the medium of a soluble factor from the irradiated cells. This factor can also stimulate IL-2R surface expression in unirradiated cells. Consequently, radiation stimulation of IL-2R expression in a large population of PBL shows a triggered-type response rather than being proportional to dose. These results demonstrate that normal human cells can respond to doses of radiation in the range of common occupational or medical exposures. The data also demonstrate a possible defence mechanism against environmental stress by which a radiation-exposed cell can use an indirect signalling mechanism to communicate with and influence the biological processes in an unexposed cell.

The interaction of protein-tyrosine phosphatase α (PTPα) and RACK1 protein enables insulin-like growth factor 1 (IGF-1)-stimulated Abl-dependent and -independent tyrosine phosphorylation of PTPα.

PubMed

Khanna, Ranvikram S; Le, Hoa T; Wang, Jing; Fung, Thomas C H; Pallen, Catherine J

2015-04-10

Protein tyrosine phosphatase α (PTPα) promotes integrin-stimulated cell migration in part through the role of Src-phosphorylated PTPα-Tyr(P)-789 in recruiting and localizing p130Cas to focal adhesions. The growth factor IGF-1 also stimulates PTPα-Tyr-789 phosphorylation to positively regulate cell movement. This is in contrast to integrin-induced PTPα phosphorylation, that induced by IGF-1 can occur in cells lacking Src family kinases (SFKs), indicating that an unknown kinase distinct from SFKs can target PTPα. We show that this IGF-1-stimulated tyrosine kinase is Abl. We found that PTPα binds to the scaffold protein RACK1 and that RACK1 coordinates the IGF-1 receptor, PTPα, and Abl in a complex to enable IGF-1-stimulated and Abl-dependent PTPα-Tyr-789 phosphorylation. In cells expressing SFKs, IGF-1-stimulated phosphorylation of PTPα is mediated by RACK1 but is Abl-independent. Furthermore, expressing the SFKs Src and Fyn in SFK-deficient cells switches IGF-1-induced PTPα phosphorylation to occur in an Abl-independent manner, suggesting that SFK activity dominantly regulates IGF-1/IGF-1 receptor signaling to PTPα. RACK1 is a molecular scaffold that integrates growth factor and integrin signaling, and our identification of PTPα as a RACK1 binding protein suggests that RACK1 may coordinate PTPα-Tyr-789 phosphorylation in these signaling networks to promote cell migration. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Receptor for macrophage colony-stimulating factor transduces a signal decreasing erythroid potential in the multipotent hematopoietic EML cell line.

PubMed

Pawlak, G; Grasset, M F; Arnaud, S; Blanchet, J P; Mouchiroud, G

2000-10-01

To test the hypothesis that hematopoietic growth factors may influence lineage choice in pluripotent progenitor cells, we investigated the effects of macrophage colony-stimulating factor (M-CSF) on erythroid and myeloid potentials of multipotent EML cells ectopically expressing M-CSF receptor (M-CSFR). EML cells are stem cell factor (SCF)-dependent murine cells that give rise spontaneously to pre-B cells, burst-forming unit erythroid (BFU-E), and colony-forming unit granulocyte macrophage (CFU-GM). We determined BFU-E and CFU-GM frequencies among EML cells transduced with murine M-CSFR, human M-CSFR, or chimeric receptors, and cultivated in the presence of SCF, M-CSF, or both growth factors. Effects of specific inhibitors of signaling molecules were investigated. EML cells transduced with murine M-CSFR proliferated in response to M-CSF but also exhibited a sharp and rapid decrease in BFU-E frequency associated with an increase in CFU-GM frequency. In contrast, EML cells expressing human M-CSFR proliferated in response to M-CSF without any changes in erythroid or myeloid potential. Using chimeric receptors between human and murine M-CSFR, we showed that the effects of M-CSF on EML cell differentiation potential are mediated by a large region in the intracellular domain of murine M-CSFR. Furthermore, phospholipase C (PLC) inhibitor U73122 interfered with the negative effects of ligand-activated murine M-CSFR on EML cell erythroid potential. We propose that signaling pathways activated by tyrosine kinase receptors may regulate erythroid potential and commitment decisions in multipotent progenitor cells and that PLC may play a key role in this process.

The SH2 and SH3 domains of mammalian Grb2 couple the EGF receptor to the Ras activator mSos1.

PubMed

Rozakis-Adcock, M; Fernley, R; Wade, J; Pawson, T; Bowtell, D

1993-05-06

Many tyrosine kinases, including the receptors for hormones such as epidermal growth factor (EGF), nerve growth factor and insulin, transmit intracellular signals through Ras proteins. Ligand binding to such receptors stimulates Ras guanine-nucleotide-exchange activity and increases the level of GTP-bound Ras, suggesting that these tyrosine kinases may activate a guanine-nucleotide releasing protein (GNRP). In Caenorhabditis elegans and Drosophila, genetic studies have shown that Ras activation by tyrosine kinases requires the protein Sem-5/drk, which contains a single Src-homology (SH) 2 domain and two flanking SH3 domains. Sem-5 is homologous to the mammalian protein Grb2, which binds the autophosphorylated EGF receptor and other phosphotyrosine-containing proteins such as Shc through its SH2 domain. Here we show that in rodent fibroblasts, the SH3 domains of Grb2 are bound to the proline-rich carboxy-terminal tail of mSos1, a protein homologous to Drosophila Sos. Sos is required for Ras signalling and contains a central domain related to known Ras-GNRPs. EGF stimulation induces binding of the Grb2-mSos1 complex to the autophosphorylated EGF receptor, and mSos1 phosphorylation. Grb2 therefore appears to link tyrosine kinases to a Ras-GNRP in mammalian cells.

Filamin A Modulates Kinase Activation and Intracellular Trafficking of Epidermal Growth Factor Receptors in Human Melanoma Cells

PubMed Central

Fiori, Jennifer L.; Zhu, Tie-Nian; O'Connell, Michael P.; Hoek, Keith S.; Indig, Fred E.; Frank, Brittany P.; Morris, Christa; Kole, Sutapa; Hasskamp, Joanne; Elias, George; Weeraratna, Ashani T.; Bernier, Michel

2009-01-01

The actin-binding protein filamin A (FLNa) affects the intracellular trafficking of various classes of receptors and has a potential role in oncogenesis. However, it is unclear whether FLNa regulates the signaling capacity and/or down-regulation of the activated epidermal growth factor receptor (EGFR). Here it is shown that partial knockdown of FLNa gene expression blocked ligand-induced EGFR responses in metastatic human melanomas. To gain greater insights into the role of FLNa in EGFR activation and intracellular sorting, we used M2 melanoma cells that lack endogenous FLNa and a subclone in which human FLNa cDNA has been stably reintroduced (M2A7 cells). Both tyrosine phosphorylation and ubiquitination of EGFR were significantly lower in epidermal growth factor (EGF)-stimulated M2 cells when compared with M2A7 cells. Moreover, the lack of FLNa interfered with EGFR interaction with the ubiquitin ligase c-Cbl. M2 cells exhibited marked resistance to EGF-induced receptor degradation, which was very active in M2A7 cells. Despite comparable rates of EGF-mediated receptor endocytosis, internalized EGFR colocalized with the lysosomal marker lysosome-associated membrane protein-1 in M2A7 cells but not M2 cells, in which EGFR was found to be sequestered in large vesicles and subsequently accumulated in punctated perinuclear structures after EGF stimulation. These results suggest the requirement of FLNa for efficient EGFR kinase activation and the sorting of endocytosed receptors into the degradation pathway. PMID:19213840

The Bile Acid Receptor TGR5 Does Not Interact with β-Arrestins or Traffic to Endosomes but Transmits Sustained Signals from Plasma Membrane Rafts*

PubMed Central

Jensen, Dane D.; Godfrey, Cody B.; Niklas, Christian; Canals, Meritxell; Kocan, Martina; Poole, Daniel P.; Murphy, Jane E.; Alemi, Farzad; Cottrell, Graeme S.; Korbmacher, Christoph; Lambert, Nevin A.; Bunnett, Nigel W.; Corvera, Carlos U.

2013-01-01

TGR5 is a G protein-coupled receptor that mediates bile acid (BA) effects on energy balance, inflammation, digestion, and sensation. The mechanisms and spatiotemporal control of TGR5 signaling are poorly understood. We investigated TGR5 signaling and trafficking in transfected HEK293 cells and colonocytes (NCM460) that endogenously express TGR5. BAs (deoxycholic acid (DCA), taurolithocholic acid) and the selective agonists oleanolic acid and 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide stimulated cAMP formation but did not induce TGR5 endocytosis or recruitment of β-arrestins, as assessed by confocal microscopy. DCA, taurolithocholic acid, and oleanolic acid did not stimulate TGR5 association with β-arrestin 1/2 or G protein-coupled receptor kinase (GRK) 2/5/6, as determined by bioluminescence resonance energy transfer. 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide stimulated a low level of TGR5 interaction with β-arrestin 2 and GRK2. DCA induced cAMP formation at the plasma membrane and cytosol, as determined using exchange factor directly regulated by cAMP (Epac2)-based reporters, but cAMP signals did not desensitize. AG1478, an inhibitor of epidermal growth factor receptor tyrosine kinase, the metalloprotease inhibitor batimastat, and methyl-β-cyclodextrin and filipin, which block lipid raft formation, prevented DCA stimulation of ERK1/2. Bioluminescence resonance energy transfer analysis revealed TGR5 and EGFR interactions that were blocked by disruption of lipid rafts. DCA stimulated TGR5 redistribution to plasma membrane microdomains, as localized by immunogold electron microscopy. Thus, TGR5 does not interact with β-arrestins, desensitize, or traffic to endosomes. TGR5 signals from plasma membrane rafts that facilitate EGFR interaction and transactivation. An understanding of the spatiotemporal control of TGR5 signaling provides insights into the actions of BAs and therapeutic TGR5 agonists/antagonists. PMID:23818521

Loss of EGF binding and cation transport response during differentiation of mouse neuroblastoma cells.

PubMed

Mummery, C L; van der Saag, P T; de Laat, S W

1983-01-01

Mouse neuroblastoma cells (clone N1E-115) differentiate in culture upon withdrawal of serum growth factors and acquire the characteristics of neurons. We have shown tht exponentially growing N1E-115 cells possess functional epidermal growth factor (EGF) receptors but that the capacity for binding EGF and for stimulation of DNA synthesis is lost as the cells differentiate. Furthermore, in exponentially growing cells, EGF induces a rapid increase in amiloride-sensitive Na+ influx, followed by stimulation of the (Na+-K+)ATPase, indicating that activation of the Na+/H+ exchange mechanism in N1E-115 cells [1] may be induced by EGF. The ionic response is also lost during differentiation, but we have shown that the stimulation of both Na+ and K+ influx is directly proportional to the number of occupied receptors in all cells whether exponentially growing or differentiating, thus only indirectly dependent on the external EGF concentration. The linearity of the relationships indicates that there is no rate-limiting step between EGF binding and the ionic response. Our data would suggest that as neuroblastoma cells differentiate and acquire neuronal properties, their ability to respond to mitogens, both biologically and in the activation of cation transport processes, progressively decreases owing to the loss of the appropriate receptors.

Rac-mediated Stimulation of Phospholipase Cγ2 Amplifies B Cell Receptor-induced Calcium Signaling*♦

PubMed Central

Walliser, Claudia; Tron, Kyrylo; Clauss, Karen; Gutman, Orit; Kobitski, Andrei Yu.; Retlich, Michael; Schade, Anja; Röcker, Carlheinz; Henis, Yoav I.; Nienhaus, G. Ulrich; Gierschik, Peter

2015-01-01

The Rho GTPase Rac is crucially involved in controlling multiple B cell functions, including those regulated by the B cell receptor (BCR) through increased cytosolic Ca2+. The underlying molecular mechanisms and their relevance to the functions of intact B cells have thus far remained unknown. We have previously shown that the activity of phospholipase Cγ2 (PLCγ2), a key constituent of the BCR signalosome, is stimulated by activated Rac through direct protein-protein interaction. Here, we use a Rac-resistant mutant of PLCγ2 to functionally reconstitute cultured PLCγ2-deficient DT40 B cells and to examine the effects of the Rac-PLCγ2 interaction on BCR-mediated changes of intracellular Ca2+ and regulation of Ca2+-regulated and nuclear-factor-of-activated-T-cell-regulated gene transcription at the level of single, intact B cells. The results show that the functional Rac-PLCγ2 interaction causes marked increases in the following: (i) sensitivity of B cells to BCR ligation; (ii) BCR-mediated Ca2+ release from intracellular stores; (iii) Ca2+ entry from the extracellular compartment; and (iv) nuclear translocation of the Ca2+-regulated nuclear factor of activated T cells. Hence, Rac-mediated stimulation of PLCγ2 activity serves to amplify B cell receptor-induced Ca2+ signaling. PMID:25903139

Glial cell line-derived neurotrophic factor (GDNF) induces neuritogenesis in the cochlear spiral ganglion via neural cell adhesion molecule (NCAM)

PubMed Central

Euteneuer, Sara; Yang, Kuo H.; Chavez, Eduardo; Leichtle, Anke; Loers, Gabriele; Olshansky, Adel; Pak, Kwang; Schachner, Melitta; Ryan, Allen F.

2013-01-01

Glial cell line-derived neurotrophic factor (GDNF) increases survival and neurite extension of spiral ganglion neurons (SGNs), the primary neurons of the auditory system, via yet unknown signaling mechanisms. In other cell types, signaling is achieved by the GPI-linked GDNF family receptor α1 (GFRα1) via recruitment of transmembrane receptors: Ret (re-arranged during transformation) and/or NCAM (neural cell adhesion molecule). Here we show that GDNF enhances neuritogenesis in organotypic cultures of spiral ganglia from 5-day-old rats and mice. Addition of GFRα1-Fc increases this effect. GDNF/GFRα1-Fc stimulation activates intracellular PI3K/Akt and MEK/Erk signaling cascades as detected by Western blot analysis of cultures prepared from rats at postnatal days 5 (P5, before the onset of hearing) and 20 (P20, after the onset of hearing). Both cascades mediate GDNF stimulation of neuritogenesis, since application of the Akt inhibitor Wortmannin or the Erk inhibitor U0126 abolished GDNF/GFRα1-Fc stimulated neuritogenesis in P5 rats. Since cultures of P5 NCAM-deficient mice failed to respond by neuritogenesis to GDNF/GFRα1-Fc, we conclude that NCAM serves as a receptor for GDNF signaling responsible for neuritogenesis in early postnatal spiral ganglion. PMID:23262364

Tumor necrosis factor receptor-1 can function through a G alpha q/11-beta-arrestin-1 signaling complex.

PubMed

Kawamata, Yuji; Imamura, Takeshi; Babendure, Jennie L; Lu, Juu-Chin; Yoshizaki, Takeshi; Olefsky, Jerrold M

2007-09-28

Tumor necrosis factor-alpha (TNFalpha) is a proinflammatory cytokine secreted from macrophages and adipocytes. It is well known that chronic TNFalpha exposure can lead to insulin resistance both in vitro and in vivo and that elevated blood levels of TNFalpha are observed in obese and/or diabetic individuals. TNFalpha has many acute biologic effects, mediated by a complex intracellular signaling pathway. In these studies we have identified new G-protein signaling components to this pathway in 3T3-L1 adipocytes. We found that beta-arrestin-1 is associated with TRAF2 (TNF receptor-associated factor 2), an adaptor protein of TNF receptors, and that TNFalpha acutely stimulates tyrosine phosphorylation of G alpha(q/11) with an increase in G alpha(q/11) activity. Small interfering RNA-mediated knockdown of beta-arrestin-1 inhibits TNFalpha-induced tyrosine phosphorylation of G alpha(q/11) by interruption of Src kinase activation. TNFalpha stimulates lipolysis in 3T3-L1 adipocytes, and beta-arrestin-1 knockdown blocks the effects of TNFalpha to stimulate ERK activation and glycerol release. TNFalpha also led to activation of JNK with increased expression of the proinflammatory gene, monocyte chemoattractant protein-1 and matrix metalloproteinase 3, and beta-arrestin-1 knockdown inhibited both of these effects. Taken together these results reveal novel elements of TNFalpha action; 1) the trimeric G-protein component G alpha(q/11) and the adapter protein beta-arrestin-1 can function as signaling molecules in the TNFalpha action cascade; 2) beta-arrestin-1 can couple TNFalpha stimulation to ERK activation and lipolysis; 3) beta-arrestin-1 and G alpha(q/11) can mediate TNFalpha-induced phosphatidylinositol 3-kinase activation and inflammatory gene expression.

Deficiency of a beta-arrestin-2 signal complex contributes to insulin resistance.

PubMed

Luan, Bing; Zhao, Jian; Wu, Haiya; Duan, Baoyu; Shu, Guangwen; Wang, Xiaoying; Li, Dangsheng; Jia, Weiping; Kang, Jiuhong; Pei, Gang

2009-02-26

Insulin resistance, a hallmark of type 2 diabetes, is a defect of insulin in stimulating insulin receptor signalling, which has become one of the most serious public health threats. Upon stimulation by insulin, insulin receptor recruits and phosphorylates insulin receptor substrate proteins, leading to activation of the phosphatidylinositol-3-OH kinase (PI(3)K)-Akt pathway. Activated Akt phosphorylates downstream kinases and transcription factors, thus mediating most of the metabolic actions of insulin. Beta-arrestins mediate biological functions of G-protein-coupled receptors by linking activated receptors with distinct sets of accessory and effecter proteins, thereby determining the specificity, efficiency and capacity of signals. Here we show that in diabetic mouse models, beta-arrestin-2 is severely downregulated. Knockdown of beta-arrestin-2 exacerbates insulin resistance, whereas administration of beta-arrestin-2 restores insulin sensitivity in mice. Further investigation reveals that insulin stimulates the formation of a new beta-arrestin-2 signal complex, in which beta-arrestin-2 scaffolds Akt and Src to insulin receptor. Loss or dysfunction of beta-arrestin-2 results in deficiency of this signal complex and disturbance of insulin signalling in vivo, thereby contributing to the development of insulin resistance and progression of type 2 diabetes. Our findings provide new insight into the molecular pathogenesis of insulin resistance, and implicate new preventive and therapeutic strategies against insulin resistance and type 2 diabetes.

MyD88 But Not TRIF Is Essential for Osteoclastogenesis Induced by Lipopolysaccharide, Diacyl Lipopeptide, and IL-1α

PubMed Central

Sato, Nobuaki; Takahashi, Naoyuki; Suda, Koji; Nakamura, Midori; Yamaki, Mariko; Ninomiya, Tadashi; Kobayashi, Yasuhiro; Takada, Haruhiko; Shibata, Kenichiro; Yamamoto, Masahiro; Takeda, Kiyoshi; Akira, Shizuo; Noguchi, Toshihide; Udagawa, Nobuyuki

2004-01-01

Myeloid differentiation factor 88 (MyD88) plays essential roles in the signaling of the Toll/interleukin (IL)-1 receptor family. Toll–IL-1 receptor domain-containing adaptor inducing interferon-β (TRIF)-mediated signals are involved in lipopolysaccharide (LPS)-induced MyD88-independent pathways. Using MyD88-deficient (MyD88−/−) mice and TRIF-deficient (TRIF−/−) mice, we examined roles of MyD88 and TRIF in osteoclast differentiation and function. LPS, diacyl lipopeptide, and IL-1α stimulated osteoclastogenesis in cocultures of osteoblasts and hemopoietic cells obtained from TRIF−/− mice, but not MyD88−/− mice. These factors stimulated receptor activator of nuclear factor-κB ligand mRNA expression in TRIF−/− osteoblasts, but not MyD88−/− osteoblasts. LPS stimulated IL-6 production in TRIF−/− osteoblasts, but not TRIF−/− macrophages. LPS and IL-1α enhanced the survival of TRIF−/− osteoclasts, but not MyD88−/− osteoclasts. Diacyl lipopeptide did not support the survival of osteoclasts because of the lack of Toll-like receptor (TLR)6 in osteoclasts. Macrophages expressed both TRIF and TRIF-related adaptor molecule (TRAM) mRNA, whereas osteoblasts and osteoclasts expressed only TRIF mRNA. Bone histomorphometry showed that MyD88−/− mice exhibited osteopenia with reduced bone resorption and formation. These results suggest that the MyD88-mediated signal is essential for the osteoclastogenesis and function induced by IL-1 and TLR ligands, and that MyD88 is physiologically involved in bone turnover. PMID:15353553

Increased abundance of aromatase and follicle stimulating hormone receptor mRNA and decreased insulin-like growth factor-2 receptor mRNA in small ovarian follicles of cattle selected for twin births

USDA-ARS?s Scientific Manuscript database

Cattle genetically selected for twin ovulations and births (Twinner) exhibit increased ovarian follicular development, increased ovulation rate, and greater blood and follicular fluid IGF 1 concentrations compared with contemporary cattle not selected for twins (Control). Experimental objectives wer...

A lactobacillus rhamnosus GG-derived soluble protein, p40, stimulates ligand release from intestinal epithelial cells to transactivate epidermal growth factor receptor

USDA-ARS?s Scientific Manuscript database

Protein p40, a Lactobacillus rhamnosus GG (LGG)-derived soluble protein, ameliorates intestinal injury and colitis, reduces apoptosis and preserves barrier function by activation of EGF receptor (EGFR) in intestinal epithelial cells. The aim of this study was to determine the mechanisms by which p40...

Role of the thrombin receptor in restenosis and atherosclerosis.

PubMed

Baykal, D; Schmedtje, J F; Runge, M S

1995-02-23

Thrombus generation is central to thrombosis at vascular lesion sites, including post-PCTA acute reocclusion and chronic restenosis. Thrombin stimulates platelet activation, monocyte and neutrophil chemotaxis, and endothelial production of prothrombotic factors. The varied physiologic effects of thrombin are due to the widespread presence of thrombin receptors in many cell types. The receptor is uniquely activated: thrombin binds to the receptor at the thrombin anion-binding exosite, the receptor ligand ("tethered ligand") apparently being a sequence of 6 amino acids (SFLLRN). Thus, peptides corresponding to the sequence of the tethered ligand can stimulate almost all functions of native thrombin itself. Several intracellular signaling pathways have been identified as important in the restenosis process: the G protein-related pathway, cyclic adenosine monophosphate (cAMP) mediator pathway, and tyrosine kinase activation pathway. In situ hybridization has demonstrated an increase in thrombin receptor mRNA throughout the period of neointimal and vascular lesion development. The mechanism of this increase is unknown, but may be mediated by multiple inflammatory modulators. Several strategies have been tested in animal models for inhibiting thrombin: (1) Hirudin not only prevents thrombin from cleaving fibrinogen, but also prevents thrombin receptor activation. (2) Thrombin receptor antagonist peptides block platelet aggregation effects of thrombin. (3) Mono- and polyclonal antibodies inhibit thrombin receptor activation. (4) Antisense oligonucleotides block thrombin receptor expression.

Differential routes of Ca2+ influx in Swiss 3T3 fibroblasts in response to receptor stimulation.

PubMed Central

Miyakawa, T; Kojima, M; Ui, M

1998-01-01

Ca2+ influx into cells in response to stimulation of various receptors was studied with Swiss 3T3 fibroblasts. The mechanisms involved were found to be so diverse that they were classified into four groups, Type I to IV. Type-I influx occurred, via pertussis toxin-susceptible G-proteins, immediately after internal Ca2+ mobilization by bradykinin, thrombin, endothelin, vasopressin or angiotensin II. Type-II influx induced by bombesin differed from Type I in its insusceptibility to pertussis toxin treatment. Ca2+ influx induced by prostaglandin E1, referred to as Type-III influx, was unique in that phospholipase C was apparently not activated without extracellular Ca2+, strongly suggesting that the Ca2+ influx preceded and was responsible for InsP3 generation and internal Ca2+ mobilization. More Ca2+ entered the cells more slowly via the Type-IV route opened by platelet-derived and other growth factors. These types of Ca2+ influx could be differentiated by their different susceptibilities to protein kinase C maximally activated by 1 h of exposure of cells to PMA, which inhibited phospholipase Cbeta coupled to receptors involved in Type-I and -II influx but did not inhibit growth-factor-receptor-coupled phospholipase Cgamma. Type-I and -II Ca2+ influxes, together with store-operated influx induced by thapsigargin, were not directly inhibited by exposure of cells to PMA, but Type-III and -IV influxes were completely inhibited. In addition, stimulation of receptors involved in Type-I and -IV Ca2+ influx, but not Type-II and -III influx, led to phospholipase A2 activation in the presence of extracellular Ca2+. Inhibition of Type-I and -IV Ca2+ influxes by their respective inhibitors, diltiazem and nifedipine, resulted in abolition of phospholipase A2 activation induced by the respective receptor agonists, in agreement with the notion that Ca2+ influx via these routes is responsible for receptor-mediated phospholipase A2 activation. PMID:9405282

Differential routes of Ca2+ influx in Swiss 3T3 fibroblasts in response to receptor stimulation.

PubMed

Miyakawa, T; Kojima, M; Ui, M

1998-01-01

Ca2+ influx into cells in response to stimulation of various receptors was studied with Swiss 3T3 fibroblasts. The mechanisms involved were found to be so diverse that they were classified into four groups, Type I to IV. Type-I influx occurred, via pertussis toxin-susceptible G-proteins, immediately after internal Ca2+ mobilization by bradykinin, thrombin, endothelin, vasopressin or angiotensin II. Type-II influx induced by bombesin differed from Type I in its insusceptibility to pertussis toxin treatment. Ca2+ influx induced by prostaglandin E1, referred to as Type-III influx, was unique in that phospholipase C was apparently not activated without extracellular Ca2+, strongly suggesting that the Ca2+ influx preceded and was responsible for InsP3 generation and internal Ca2+ mobilization. More Ca2+ entered the cells more slowly via the Type-IV route opened by platelet-derived and other growth factors. These types of Ca2+ influx could be differentiated by their different susceptibilities to protein kinase C maximally activated by 1 h of exposure of cells to PMA, which inhibited phospholipase Cbeta coupled to receptors involved in Type-I and -II influx but did not inhibit growth-factor-receptor-coupled phospholipase Cgamma. Type-I and -II Ca2+ influxes, together with store-operated influx induced by thapsigargin, were not directly inhibited by exposure of cells to PMA, but Type-III and -IV influxes were completely inhibited. In addition, stimulation of receptors involved in Type-I and -IV Ca2+ influx, but not Type-II and -III influx, led to phospholipase A2 activation in the presence of extracellular Ca2+. Inhibition of Type-I and -IV Ca2+ influxes by their respective inhibitors, diltiazem and nifedipine, resulted in abolition of phospholipase A2 activation induced by the respective receptor agonists, in agreement with the notion that Ca2+ influx via these routes is responsible for receptor-mediated phospholipase A2 activation.

Role of G protein-coupled estrogen receptor-1 in estradiol 17β-induced alterations in protein synthesis and protein degradation rates in fused bovine satellite cell cultures.

PubMed

Kamanga-Sollo, E; Thornton, K J; White, M E; Dayton, W R

2017-01-01

In feedlot steers, estradiol-17β (E2) and combined E2 and trenbolone acetate (a testosterone analog) implants enhance rate and efficiency of muscle growth; and, consequently, these compounds are widely used as growth promoters in several countries. Treatment with E2 stimulates protein synthesis rate and suppresses protein degradation rate in fused bovine satellite cell (BSC) cultures; however, the mechanisms involved in these effects are not known with certainty. Although the genomic effects of E2 mediated through the classical estrogen receptors have been characterized, recent studies indicate that binding of E2 to the G protein-coupled estrogen receptor (GPER)-1 mediates nongenomic effects of E2 on cellular function. Our current data show that inhibition of GPER-1, matrix metalloproteinases 2 and 9 (MMP2/9), or heparin binding epidermal growth factor-like growth factor (hbEGF) suppresses E2 stimulate protein synthesis rate in cultured BSCs (P < 0.001) suggesting that all of these are required in order for E2 to stimulate protein synthesis in these cultures. In contrast, inhibition of GPER-1, MMP2/9, or hbEGF has no effect on the ability of E2 to suppress protein degradation rates in fused BSC cultures indicating that these factors are not required in order for E2 to suppress protein degradation rate in these cells. Furthermore, treatment of fused BSC cultures with E2 increased (P < 0.05) pAKT levels indicating that the pAKT pathway may play a role in E2-stimulated effects on cultured BSC. In summary, our current data show that active GPER-1, MMP2/9, and hbEGF are necessary for E2-stimulated protein synthesis but not for E2-simulated suppression of protein degradation in cultured BSC. In addition, E2 treatment increases pAKT levels in cultured BSC. Copyright © 2016 Elsevier Inc. All rights reserved.

Dual agonist Surrobody™ simultaneously activates death receptors DR4 and DR5 to induce cancer cell death

PubMed Central

Milutinovic, Snezana; Kashyap, Arun K.; Yanagi, Teruki; Wimer, Carina; Zhou, Sihong; O' Neil, Ryann; Kurtzman, Aaron L.; Faynboym, Alexsandr; Xu, Li; Hannum, Charles H.; Diaz, Paul W.; Matsuzawa, Shu-ichi; Horowitz, Michael; Horowitz, Lawrence; Bhatt, Ramesh R.; Reed, John C.

2015-01-01

Death receptors of the Tumor Necrosis Factor (TNF) family are found on surface of most cancer cells and their activation typically kills cancer cells through the stimulation of the extrinsic apoptotic pathway. The endogenous ligand for death receptors-4 and -5 (DR4 and DR5) is Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand, TRAIL (Apo2L). Since most untransformed cells are not susceptible to TRAIL-induced apoptosis, death receptor activators have emerged as promising cancer therapeutic agents. One strategy to stimulate death receptors in cancer patients is to use soluble human recombinant TRAIL protein, but this agent has limitations of a short half-life and decoy receptor sequestration. Another strategy that attempted to evade decoy receptor sequestration and to provide improved pharmacokinetic properties was to generate DR4 or DR5 agonist antibodies. The resulting monoclonal agonist antibodies overcame the limitations of short half-life and avoided decoy receptor sequestration, but are limited by activating only one of the two death receptors. Here, we describe a DR4 and DR5 dual agonist produced using Surrobody™ technology that activates both DR4 and DR5 to induce apoptotic death of cancer cells in vitro and in vivo and also avoids decoy receptor sequestration. This fully human anti-DR4/DR5 Surrobody displays superior potency to DR4- and DR5-specific antibodies, even when combined with TRAIL-sensitizing pro-apoptotic agents. Moreover, cancer cells were less likely to acquire resistance to Surrobody than either anti-DR4 or anti-DR5 mono-specific antibodies. Taken together, Surrobody shows promising preclinical pro-apoptotic activity against cancer cells, meriting further exploration of its potential as a novel cancer therapeutic agent. PMID:26516157

Receptor-Targeted, Magneto-Mechanical Stimulation of Osteogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells

PubMed Central

Hu, Bin; El Haj, Alicia J; Dobson, Jon

2013-01-01

Mechanical cues are employed to promote stem cell differentiation and functional tissue formation in tissue engineering and regenerative medicine. We have developed a Magnetic Force Bioreactor (MFB) that delivers highly targeted local forces to cells at a pico-newton level, utilizing magnetic micro- and nano-particles to target cell surface receptors. In this study, we investigated the effects of magnetically targeting and actuating specific two mechanical-sensitive cell membrane receptors—platelet-derived growth factor receptor α (PDGFRα) and integrin ανβ3. It was found that a higher mineral-to-matrix ratio was obtained after three weeks of magneto-mechanical stimulation coupled with osteogenic medium culture by initially targeting PDGFRα compared with targeting integrin ανβ3 and non-treated controls. Moreover, different initiation sites caused a differentiated response profile when using a 2-day-lagged magneto-mechanical stimulation over culture periods of 7 and 12 days). However, both resulted in statistically higher osteogenic marker genes expression compared with immediate magneto-mechanical stimulation. These results provide insights into important parameters for designing appropriate protocols for ex vivo induced bone formation via magneto-mechanical actuation. PMID:24065106

Abnormal XPD-induced nuclear receptor transactivation in DNA repair disorders: trichothiodystrophy and xeroderma pigmentosum.

PubMed

Zhou, Xiaolong; Khan, Sikandar G; Tamura, Deborah; Ueda, Takahiro; Boyle, Jennifer; Compe, Emmanuel; Egly, Jean-Marc; DiGiovanna, John J; Kraemer, Kenneth H

2013-08-01

XPD (ERCC2) is a DNA helicase involved in nucleotide excision repair and in transcription as a structural bridge tying the transcription factor IIH (TFIIH) core with the cdk-activating kinase complex, which phosphorylates nuclear receptors. Mutations in XPD are associated with several different phenotypes, including trichothiodystrophy (TTD), with sulfur-deficient brittle hair, bone defects, and developmental abnormalities without skin cancer, xeroderma pigmentosum (XP), with pigmentary abnormalities and increased skin cancer, or XP/TTD with combined features, including skin cancer. We describe the varied clinical features and mutations in nine patients examined at the National Institutes of Health who were compound heterozygotes for XPD mutations but had different clinical phenotypes: four TTD, three XP, and two combined XP/TTD. We studied TFIIH-dependent transactivation by nuclear receptor for vitamin D (VDR) and thyroid in cells from these patients. The vitamin D stimulation ratio of CYP24 and osteopontin was associated with specific pairs of mutations (reduced in 5, elevated in 1) but not correlated with distinct clinical phenotypes. Thyroid receptor stimulation ratio for KLF9 was not significantly different from normal. XPD mutations frequently were associated with abnormal VDR stimulation in compound heterozygote patients with TTD, XP, or XP/TTD.

Insulin like growth factor 2 regulation of aryl hydrocarbon receptor in MCF-7 breast cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tomblin, Justin K.; Salisbury, Travis B., E-mail: salisburyt@marshall.edu

2014-01-17

Highlights: •IGF-2 stimulates concurrent increases in AHR and CCND1 expression. •IGF-2 promotes the binding of AHR to the endogenous cyclin D1 promoter. •AHR knockdown inhibits IGF-2 stimulated increases in CCND1 mRNA and protein. •AHR knockdown inhibits IGF-2 stimulated increases in MCF-7 proliferation. -- Abstract: Insulin like growth factor (IGF)-1 and IGF-2 stimulate normal growth, development and breast cancer cell proliferation. Cyclin D1 (CCND1) promotes cell cycle by inhibiting retinoblastoma protein (RB1). The aryl hydrocarbon receptor (AHR) is a major xenobiotic receptor that also regulates cell cycle. The purpose of this study was to investigate whether IGF-2 promotes MCF-7 breast cancermore » proliferation by inducing AHR. Western blot and quantitative real time PCR (Q-PCR) analysis revealed that IGF-2 induced an approximately 2-fold increase (P < .001) in the expression of AHR and CCND1. Chromatin immunoprecipitation (ChIP), followed by Q-PCR indicated that IGF-2 promoted (P < .001) a 7-fold increase in AHR binding on the CCND1 promoter. AHR knockdown significantly (P < .001) inhibited IGF-2 stimulated increases in CCND1 mRNA and protein. AHR knockdown cells were less (P < .001) responsive to the proliferative effects of IGF-2 than control cells. Collectively, our findings have revealed a new regulatory mechanism by which IGF-2 induction of AHR promotes the expression of CCND1 and the proliferation of MCF-7 cells. This previously uncharacterized pathway could be important for the proliferation of IGF responsive cancer cells that also express AHR.« less

Androgen Stimulates Growth of Mouse Preantral Follicles In Vitro: Interaction With Follicle-Stimulating Hormone and With Growth Factors of the TGFβ Superfamily

PubMed Central

Laird, Mhairi; Thomson, Kacie; Fenwick, Mark; Mora, Jocelyn; Hardy, Kate

2017-01-01

Androgens are essential for the normal function of mature antral follicles but also have a role in the early stages of follicle development. Polycystic ovary syndrome (PCOS), the most common cause of anovulatory infertility, is characterized by androgen excess and aberrant follicle development that includes accelerated early follicle growth. We have examined the effects of testosterone and dihydrotestosterone (DHT) on development of isolated mouse preantral follicles in culture with the specific aim of investigating interaction with follicle-stimulating hormone (FSH), the steroidogenic pathway, and growth factors of the TGFβ superfamily that are known to have a role in early follicle development. Both testosterone and DHT stimulated follicle growth and augmented FSH-induced growth and increased the incidence of antrum formation among the granulosa cell layers of these preantral follicles after 72 hours in culture. Effects of both androgens were reversed by the androgen receptor antagonist flutamide. FSH receptor expression was increased in response to both testosterone and DHT, as was that of Star, whereas Cyp11a1 was down-regulated. The key androgen-induced changes in the TGFβ signaling pathway were down-regulation of Amh, Bmp15, and their receptors. Inhibition of Alk6 (Bmpr1b), a putative partner for Amhr2 and Bmpr2, by dorsomorphin resulted in augmentation of androgen-stimulated growth and modification of androgen-induced gene expression. Our findings point to varied effects of androgen on preantral follicle growth and function, including interaction with FSH-activated growth and steroidogenesis, and, importantly, implicate the intrafollicular TGFβ system as a key mediator of androgen action. These findings provide insight into abnormal early follicle development in PCOS. PMID:28324051

Androgen Stimulates Growth of Mouse Preantral Follicles In Vitro: Interaction With Follicle-Stimulating Hormone and With Growth Factors of the TGFβ Superfamily.

PubMed

Laird, Mhairi; Thomson, Kacie; Fenwick, Mark; Mora, Jocelyn; Franks, Stephen; Hardy, Kate

2017-04-01

Androgens are essential for the normal function of mature antral follicles but also have a role in the early stages of follicle development. Polycystic ovary syndrome (PCOS), the most common cause of anovulatory infertility, is characterized by androgen excess and aberrant follicle development that includes accelerated early follicle growth. We have examined the effects of testosterone and dihydrotestosterone (DHT) on development of isolated mouse preantral follicles in culture with the specific aim of investigating interaction with follicle-stimulating hormone (FSH), the steroidogenic pathway, and growth factors of the TGFβ superfamily that are known to have a role in early follicle development. Both testosterone and DHT stimulated follicle growth and augmented FSH-induced growth and increased the incidence of antrum formation among the granulosa cell layers of these preantral follicles after 72 hours in culture. Effects of both androgens were reversed by the androgen receptor antagonist flutamide. FSH receptor expression was increased in response to both testosterone and DHT, as was that of Star, whereas Cyp11a1 was down-regulated. The key androgen-induced changes in the TGFβ signaling pathway were down-regulation of Amh, Bmp15, and their receptors. Inhibition of Alk6 (Bmpr1b), a putative partner for Amhr2 and Bmpr2, by dorsomorphin resulted in augmentation of androgen-stimulated growth and modification of androgen-induced gene expression. Our findings point to varied effects of androgen on preantral follicle growth and function, including interaction with FSH-activated growth and steroidogenesis, and, importantly, implicate the intrafollicular TGFβ system as a key mediator of androgen action. These findings provide insight into abnormal early follicle development in PCOS.

Prior stimulation of the endocannabinoid system prevents methamphetamine-induced dopaminergic neurotoxicity in the striatum through activation of CB2 receptors.

PubMed

Nader, Joëlle; Rapino, Cinzia; Gennequin, Benjamin; Chavant, Francois; Francheteau, Maureen; Makriyannis, Alexandros; Duranti, Andrea; Maccarrone, Mauro; Solinas, Marcello; Thiriet, Nathalie

2014-12-01

Methamphetamine toxicity is associated with cell death and loss of dopamine neuron terminals in the striatum similar to what is found in some neurodegenerative diseases. Conversely, the endocannabinoid system (ECS) has been suggested to be neuroprotective in the brain, and new pharmacological tools have been developed to increase their endogenous tone. In this study, we evaluated whether ECS stimulation could reduce the neurotoxicity of high doses of methamphetamine on the dopamine system. We found that methamphetamine alters the levels of the major endocannabinoids, anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) in the striatum, suggesting that the ECS participates in the brain responses to methamphetamine. Δ(9)-tetrahydrocannabinol (THC), a cannabis-derived agonist of both CB1 and CB2 cannabinoid receptors, or inhibitors of the main enzymes responsible for the degradation of AEA and 2-AG (URB597 and JZL184, respectively), blunted the decrease in striatal protein levels of tyrosine hydroxylase induced by methamphetamine. In addition, antagonists of CB2, but not of CB1, blocked the preventive effects of URB597 and JZL184, suggesting that only the former receptor subtype is engaged in neuroprotection exerted by ECS stimulation. Finally, we found that methamphetamine increases striatal levels of the cytokine tumor necrosis factor alpha, an effect that was blocked by ECS stimulation. Altogether, our results indicate that stimulation of ECS prior to the administration of an overdose of methamphetamine considerably reduces the neurotoxicity of the drug through CB2 receptor activation and highlight a protective function for the ECS against the toxicity induced by drugs and other external insults to the brain. This article is part of the Special Issue entitled 'CNS Stimulants'. Copyright © 2014 Elsevier Ltd. All rights reserved.

BDNF and AMPA receptors in the cNTS modulate the hyperglycemic reflex after local carotid body NaCN stimulation.

PubMed

Cuéllar, R; Montero, S; Luquín, S; García-Estrada, J; Melnikov, V; Virgen-Ortiz, A; Lemus, M; Pineda-Lemus, M; de Álvarez-Buylla, E

2017-07-01

The application of sodium cyanide (NaCN) to the carotid body receptors (CBR) (CBR stimulation) induces rapid blood hyperglycemia and an increase in brain glucose retention. The commissural nucleus tractus solitarius (cNTS) is an essential relay nucleus in this hyperglycemic reflex; it receives glutamatergic afferents (that also release brain derived neurotrophic factor, BDNF) from the nodose-petrosal ganglia that relays CBR information. Previous work showed that AMPA in NTS blocks hyperglycemia and brain glucose retention after CBR stimulation. In contrast, BDNF, which attenuates glutamatergic AMPA currents in NTS, enhances these glycemic responses. Here we investigated the combined effects of BDNF and AMPA (and their antagonists) in NTS on the glycemic responses to CBR stimulation. Microinjections of BDNF plus AMPA into the cNTS before CBR stimulation in anesthetized rats, induced blood hyperglycemia and an increase in brain arteriovenous (a-v) of blood glucose concentration difference, which we infer is due to increased brain glucose retention. By contrast, the microinjection of the TrkB antagonist K252a plus AMPA abolished the glycemic responses to CBR stimulation similar to what is observed after AMPA pretreatments. In BDNF plus AMPA microinjections preceding CBR stimulation, the number of c-fos immunoreactive cNTS neurons increased. In contrast, in the rats microinjected with K252a plus AMPA in NTS, before CBR stimulation, c-fos expression in cNTS decreased. The expression of AMPA receptors GluR2/3 did not change in any of the studied groups. These results indicate that BDNF in cNTS plays a key role in the modulation of the hyperglycemic reflex initiated by CBR stimulation. Copyright © 2017. Published by Elsevier B.V.

Cell-autonomous intracellular androgen receptor signaling drives the growth of human prostate cancer initiating cells.

PubMed

Vander Griend, Donald J; D'Antonio, Jason; Gurel, Bora; Antony, Lizamma; Demarzo, Angelo M; Isaacs, John T

2010-01-01

The lethality of prostate cancer is due to the continuous growth of cancer initiating cells (CICs) which are often stimulated by androgen receptor (AR) signaling. However, the underlying molecular mechanism(s) for such AR-mediated growth stimulation are not fully understood. Such mechanisms may involve cancer cell-dependent induction of tumor stromal cells to produce paracrine growth factors or could involve cancer cell autonomous autocrine and/or intracellular AR signaling pathways. We utilized clinical samples, animal models and a series of AR-positive human prostate cancer cell lines to evaluate AR-mediated growth stimulation of prostate CICs. The present studies document that stromal AR expression is not required for prostate cancer growth, since tumor stroma surrounding AR-positive human prostate cancer metastases (N = 127) are characteristically AR-negative. This lack of a requirement for AR expression in tumor stromal cells is also documented by the fact that human AR-positive prostate cancer cells grow equally well when xenografted in wild-type versus AR-null nude mice. AR-dependent growth stimulation was documented to involve secretion, extracellular binding, and signaling by autocrine growth factors. Orthotopic xenograft animal studies documented that the cellautonomous autocrine growth factors which stimulate prostate CIC growth are not the andromedins secreted by normal prostate stromal cells. Such cell autonomous and extracellular autocrine signaling is necessary but not sufficient for the optimal growth of prostate CICs based upon the response to anti-androgen plus/or minus preconditioned media. AR-induced growth stimulation of human prostate CICs requires AR-dependent intracellular pathways. The identification of such AR-dependent intracellular pathways offers new leads for the development of effective therapies for prostate cancer. (c) 2009 Wiley-Liss, Inc.

Expression of the Human Epidermal Growth Factor Receptor in a Murine T- Cell Hybridoma: A Transmembrane Protein Tyrosine Kinase Can Synergize with the T-Cell Antigen Receptor

DTIC Science & Technology

1992-01-01

protein kirases suchmembrane P17K. nln T ts ebaepoenkr sssc as the EGFR or platelet-derived growth factor re,,,ptor. For instance, the EGFR and TCR...stimulated with EGF (100 ng/mi, in PBS containing phosphatase inhibitors , lysed, and immunoprecip. 5 min, B) anti Thy- I antibody (07 1:50 dilution or...washed in PBS containing phosphatase inhibitors and lysed. Total cellular MX1- lysate was subjected to SDS.PAGE. Western-transferred, and im- Mxi

Granulocyte-macrophage colony-stimulating factor primes interleukin-13 production by macrophages via protease-activated receptor-2.

PubMed

Aoki, Manabu; Yamaguchi, Rui; Yamamoto, Takatoshi; Ishimaru, Yasuji; Ono, Tomomichi; Sakamoto, Arisa; Narahara, Shinji; Sugiuchi, Hiroyuki; Hirose, Eiji; Yamaguchi, Yasuo

2015-04-01

Chronic inflammation is often linked to the presence of type 2-polarized macrophages, which are induced by the T helper type 2 cytokines interleukin-4 and interleukin-13 (IL-13). IL-13 is a key mediator of tissue fibrosis caused by T helper type 2-based inflammation. Human neutrophil elastase (HNE) plays a pivotal role in the pathogenesis of pulmonary fibrosis. This study investigated the priming effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on IL-13 expression by macrophages stimulated with HNE. Adherent macrophages were obtained from primary cultures of human mononuclear cells. Expression of IL-13 mRNA and protein by GM-CSF-dependent macrophages was investigated after stimulation with HNE, using the polymerase chain reaction and enzyme-linked immunosorbent assay. GM-CSF had a priming effect on IL-13 mRNA and protein expression by macrophages stimulated with HNE, while this effect was not observed for various other cytokines. GM-CSF-dependent macrophages showed a significant increase in the expression of protease activated receptor-2 (PAR-2) mRNA and protein. The response of IL-13 mRNA to HNE was significantly decreased by pretreatment with alpha1-antitrypsin, a PAR-2 antibody (SAM11), or a PAR-2 antagonist (ENMD-1068). These findings suggest that stimulation with HNE can induce IL-13 production by macrophages, especially GM-CSF-dependent macrophages. Accordingly, neutrophil elastase may have a key role in fibrosis associated with chronic inflammation. Copyright © 2015 Elsevier Inc. All rights reserved.

Ghrelin-induced stimulation of colonic propulsion is dependent on hypothalamic neuropeptide Y1- and corticotrophin-releasing factor 1 receptor activation.

PubMed

Tebbe, J J; Mronga, S; Tebbe, C G; Ortmann, E; Arnold, R; Schäfer, M K-H

2005-09-01

Peptides participating in the hypothalamic control of feeding behaviour are also involved in the central autonomic control of gastrointestinal functions, such as secretion and motility. An anatomical interaction and functional relationship in the central nervous system between the feeding-related peptides neuropeptide Y and ghrelin is well documented. Furthermore, it has been shown that feeding-related peptides can influence digestive function via central corticotrophin-releasing factor (CRF) pathways. In the present study, we investigated the role of ghrelin in the central autonomic control of colonic motility. Furthermore, we addressed the hypothesis that ghrelin is involved in the hypothalamic control of colonic motor function, utilizing central neuropeptide Y receptors and hypothalamic CRF pathways. Ghrelin (0.03, 0.06 and 0.12 nmol) bilaterally microinjected into the paraventricular nucleus (PVN) induced a significant stimulation of colonic propulsion. In particular, the colonic transit time decreased from 312+/-7 min to 198+/-12 min. Microinjection of the neuropeptide Y1 receptor antagonist, BIBP-3226 (200 pmol), or the nonselective CRF receptor antagonist, astressin (30 pmol), into the PVN abolished the stimulatory effect of ghrelin injected into the PVN on colonic transit time, whereas pretreatment with the selective CRF2 receptor, antisauvagine-30 (28 pmol), failed to affect the effect of PVN-ghrelin injection on colonic propulsion. These results suggest that ghrelin can act as central modulator of gastrointestinal motor functions at the level of the PVN via neuropeptide Y1- and CRF1 receptor-dependent mechanisms.

Sema4D, the ligand for Plexin B1, suppresses c-Met activation and migration and promotes melanocyte survival and growth

PubMed Central

Soong, Joanne; Chen, Yulin; Shustef, Elina; Scott, Glynis

2011-01-01

Semaphorins are secreted and membrane bound proteins involved in neural pathfinding, organogenesis, and tumor progression, through Plexin and neuropilins receptors. We recently reported that Plexin B1, the Semaphorin 4D receptor, is a tumor suppressor protein for melanoma, in part, through inhibition of the oncogenic c-Met tyrosine kinase receptor. In this report we show that Sema4D is a protective paracrine factor for normal human melanocyte survival in response to ultraviolet irradiation, that it stimulates proliferation, and regulates the activity of the c-Met receptor. c-Met receptor signaling stimulates melanocyte migration, in part through down-regulation of the cell adhesion molecule E-cadherin. Sema4D suppressed activation of c-Met in response to its ligand hepatocyte growth factor (HGF), and partially blocked the suppressive effects of HGF on E-cadherin expression in melanocytes and HGF-dependent migration. These data demonstrate a role for Plexin B1 in maintenance of melanocyte survival and proliferation in the skin, and suggest that Semaphorin 4D and Plexin B1 act cooperatively with HGF and c-Met to regulate c-Met dependent effects in human melanocytes. Because our data show that Plexin B1 is profoundly down-regulated by UVB in melanocytes, loss of Plexin B1 may accentuate HGF dependent effects on melanocytes, including melanocyte migration. PMID:22189792

Sema4D, the ligand for Plexin B1, suppresses c-Met activation and migration and promotes melanocyte survival and growth.

PubMed

Soong, Joanne; Chen, Yulin; Shustef, Elina M; Scott, Glynis A

2012-04-01

Semaphorins are secreted and membrane-bound proteins involved in neural pathfinding, organogenesis, and tumor progression, through Plexin and neuropilin receptors. We recently reported that Plexin B1, the Semaphorin 4D (Sema4D) receptor, is a tumor-suppressor protein for melanoma, which functions, in part, through inhibition of the oncogenic c-Met tyrosine kinase receptor. In this report, we show that Sema4D is a protective paracrine factor for normal human melanocyte survival in response to UV irradiation, and that it stimulates proliferation and regulates the activity of the c-Met receptor. c-Met receptor signaling stimulates melanocyte migration, partly through downregulation of the cell adhesion molecule E-cadherin. Sema4D suppressed activation of c-Met in response to its ligand, hepatocyte growth factor (HGF), and partially blocked the suppressive effects of HGF on E-cadherin expression in melanocytes and HGF-dependent migration. These data demonstrate a role for Plexin B1 in maintenance of melanocyte survival and proliferation in the skin, and suggest that Sema4D and Plexin B1 act cooperatively with HGF and c-Met to regulate c-Met-dependent effects in human melanocytes. Because our data show that Plexin B1 is profoundly downregulated by UVB in melanocytes, loss of Plexin B1 may accentuate HGF-dependent effects on melanocytes, including melanocyte migration.

Iodine stimulates estrogen receptor singling and its systemic level is increased in surgical patients due to topical absorption.

PubMed

He, Shaohua; Wang, Bingchan; Lu, Xiyi; Miao, Suyu; Yang, Fengming; Zava, Theodore; Ding, Qiang; Zhang, Shijiang; Liu, Jiayin; Zava, David; Shi, Yuenian Eric

2018-01-02

Iodine is crucial for thyroid hormone production. However, recent epidemiologic studies have shown that breast cancer patients have an elevated risk of developing thyroid cancer and vice versa. A notable finding in this study is that iodine stimulated the transcriptional activity of estrogen receptor-α (ER-α) in breast cancer cells. Iodine stimulated expression of several ER-α regulated gene including PS2 , Cathepsin D , CyclinD1 , and PR both in vitro and in nude mice, which is consistent with its stimulation of both anchorage-dependent and -independent growth of ER-α positive breast cancer cells and the effect to dampen tumor shrinkage of MCF-7 xenograft in ovariectomized nude mice. Analyses of clinical urine samples from breast cancer patients undergoing surgery demonstrated that urinary iodine levels were significantly higher than that in controls; and this increased level is due to the antiseptic use of iodine during breast surgery. The present study indicates that excess iodine intake may be an unfavorable factor in breast cancer by stimulation of ER-α transcriptional activity.

Phosphatidylinositol 3-Kinase (PI3K) Activity Bound to Insulin-like Growth Factor-I (IGF-I) Receptor, which Is Continuously Sustained by IGF-I Stimulation, Is Required for IGF-I-induced Cell Proliferation*

PubMed Central

Fukushima, Toshiaki; Nakamura, Yusaku; Yamanaka, Daisuke; Shibano, Takashi; Chida, Kazuhiro; Minami, Shiro; Asano, Tomoichiro; Hakuno, Fumihiko; Takahashi, Shin-Ichiro

2012-01-01

Continuous stimulation of cells with insulin-like growth factors (IGFs) in G1 phase is a well established requirement for IGF-induced cell proliferation; however, the molecular components of this prolonged signaling pathway that is essential for cell cycle progression from G1 to S phase are unclear. IGF-I activates IGF-I receptor (IGF-IR) tyrosine kinase, followed by phosphorylation of substrates such as insulin receptor substrates (IRS) leading to binding of signaling molecules containing SH2 domains, including phosphatidylinositol 3-kinase (PI3K) to IRS and activation of the downstream signaling pathways. In this study, we found prolonged (>9 h) association of PI3K with IGF-IR induced by IGF-I stimulation. PI3K activity was present in this complex in thyrocytes and fibroblasts, although tyrosine phosphorylation of IRS was not yet evident after 9 h of IGF-I stimulation. IGF-I withdrawal in mid-G1 phase impaired the association of PI3K with IGF-IR and suppressed DNA synthesis the same as when PI3K inhibitor was added. Furthermore, we demonstrated that Tyr1316-X-X-Met of IGF-IR functioned as a PI3K binding sequence when this tyrosine is phosphorylated. We then analyzed IGF signaling and proliferation of IGF-IR−/− fibroblasts expressing exogenous mutant IGF-IR in which Tyr1316 was substituted with Phe (Y1316F). In these cells, IGF-I stimulation induced tyrosine phosphorylation of IGF-IR and IRS-1/2, but mutated IGF-IR failed to bind PI3K and to induce maximal phosphorylation of GSK3β and cell proliferation in response to IGF-I. Based on these results, we concluded that PI3K activity bound to IGF-IR, which is continuously sustained by IGF-I stimulation, is required for IGF-I-induced cell proliferation. PMID:22767591

Opioid agonists binding and responses in SH-SY5Y cells

NASA Technical Reports Server (NTRS)

Costa, E. M.; Hoffmann, B. B.; Loew, G. H.

1992-01-01

SH-SY5Y (human neuroblastoma) cultured cells, known to have mu-opioid receptors, have been used to assess and compare the ability of eight representative mu-selective compounds from diverse opioid families to recognize and activate these receptors. A wide range of receptor affinities spanning a factor of 10,000 was found between the highest affinity fentanyl analogs (Ki = 0.1nM) and the lowest affinity analog, meperidine (Ki = 1 microM). A similar range was found for inhibition of PGE1-stimulated cAMP accumulation with a rank order of activities that closely paralleled binding affinities. Maximum inhibition of cAMP accumulation by each compound was about 80%. Maximum stimulation of GTPase activity (approximately 50%) was also similar for all compounds except the lowest affinity meperidine. Both effects were naloxone reversible. These results provide further evidence that mu-receptors are coupled to inhibition of adenylate cyclase and that the SH-SY5Y cell line is a good system for assessment of mu-agonists functional responses.

Smad2-dependent glycosaminoglycan elongation in aortic valve interstitial cells enhances binding of LDL to proteoglycans.

PubMed

Osman, Narin; Grande-Allen, K Jane; Ballinger, Mandy L; Getachew, Robel; Marasco, Silvana; O'Brien, Kevin D; Little, Peter J

2013-01-01

Calcific aortic valve disease is a progressive condition that shares some common pathogenic features with atherosclerosis. Transforming growth factor-β1 is a recognized mediator of atherosclerosis and is expressed in aortic valve lesions. Transforming growth factorβ1 stimulates glycosaminoglycan elongation of proteoglycans that is associated with increased lipid binding. We investigated the presence of transforming growth factor-β1 and downstream signaling intermediates in diseased human aortic valves and the effects of activated transforming growth factor-β1 receptor signaling on aortic valve interstitial cell proteoglycan synthesis and lipid binding as a possible mechanism for the initiation of the early lesion of calcific aortic valve disease. Diseased human aortic valve leaflets demonstrated strong immunohistochemical staining for transforming growth factor-β1 and phosphorylated Smad2/3. In primary porcine aortic valve interstitial cells, Western blots showed that transforming growth factor-β1 stimulated phosphorylation in both the carboxy and linker regions of Smad2/3, which was inhibited by the transforming growth factor-β1 receptor inhibitor SB431542. Gel electrophoresis and size exclusion chromatography demonstrated that SB431542 decreased transforming growth factor-β1-mediated [(35)S]-sulfate incorporation into proteoglycans in a dose-dependent manner. Further, in proteoglycans derived from transforming growth factor-β1-treated valve interstitial cells, gel mobility shift assays demonstrated that inhibition of transforming growth factor-β1 receptor signaling resulted in decreased lipid binding. Classic transforming growth factor-β1 signaling is present in human aortic valves in vivo and contributes to the modification of proteoglycans expressed by valve interstitial cells in vitro. These findings suggest that transforming growth factor-β1 may promote increased low-density lipoprotein binding in the early phases of calcific aortic valve disease. Copyright © 2013 Elsevier Inc. All rights reserved.

Vascular Endothelial Growth Factor Receptor-2 Couples Cyclo-Oxygenase-2 with Pro-Angiogenic Actions of Leptin on Human Endothelial Cells

PubMed Central

Garonna, Elena; Botham, Kathleen M.; Birdsey, Graeme M.; Randi, Anna M.; Gonzalez-Perez, Ruben R.; Wheeler-Jones, Caroline P. D.

2011-01-01

Background The adipocyte-derived hormone leptin influences the behaviour of a wide range of cell types and is now recognised as a pro-angiogenic and pro-inflammatory factor. In the vasculature, these effects are mediated in part through its direct leptin receptor (ObRb)-driven actions on endothelial cells (ECs) but the mechanisms responsible for these activities have not been established. In this study we sought to more fully define the molecular links between inflammatory and angiogenic responses of leptin-stimulated human ECs. Methodology/Principal Findings Immunoblotting studies showed that leptin increased cyclo-oxygenase-2 (COX-2) expression (but not COX-1) in cultured human umbilical vein ECs (HUVEC) through pathways that depend upon activation of both p38 mitogen-activated protein kinase (p38MAPK) and Akt, and stimulated rapid phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2) on Tyr1175. Phosphorylation of VEGFR2, p38MAPK and Akt, and COX-2 induction in cells challenged with leptin were blocked by a specific leptin peptide receptor antagonist. Pharmacological inhibitors of COX-2, the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and p38MAPK abrogated leptin-induced EC proliferation (assessed by quantifying 5-bromo-2′-deoxyuridine incorporation, calcein fluorescence and propidium iodide staining), slowed the increased migration rate of leptin-stimulated cells (in vitro wound healing assay) and inhibited leptin-induced capillary-like tube formation by HUVEC on Matrigel. Inhibition of VEGFR2 tyrosine kinase activity reduced leptin-stimulated p38MAPK and Akt activation, COX-2 induction, and pro-angiogenic EC responses, and blockade of VEGFR2 or COX-2 activities abolished leptin-driven neo-angiogenesis in a chick chorioallantoic membrane vascularisation assay in vivo. Conclusions/Significance We conclude that a functional endothelial p38MAPK/Akt/COX-2 signalling axis is required for leptin's pro-angiogenic actions and that this is regulated upstream by ObRb-dependent activation of VEGFR2. These studies identify a new function for VEGFR2 as a mediator of leptin-stimulated COX-2 expression and angiogenesis and have implications for understanding leptin's regulation of the vasculature in both non-obese and obese individuals. PMID:21533119

Pathophysiological roles of P2 receptors in glial cells.

PubMed

Abbracchio, Maria P; Verderio, Claudia

2006-01-01

Extracellular nucleotides act through specific receptors on target cells: the seven ionotropic P2X and the eight G protein-coupled P2Y receptors. All these receptors are expressed by brain astroglia and microglia. In astrocytes, P2 receptors have been implicated in short-term calcium-dependent cell-cell communication. Upon mechanical stimulation or activation by other transmitters, astrocytes release ATP and respond to ATP with a propagating wave of intracellular calcium increases, allowing a homotypic astrocyte-astrocyte communication, as well as an heterotypic signalling which also involves neurons, oligodendrocytes and microglia. Astrocytic P2 receptors also mediate reactive astrogliosis, a reaction contributing to neuronal death in neurodegenerative diseases. Signalling leading to inflammatory astrogliosis involves induction of cyclo-oxygenase 2 through stimulation of ERK1,2 and of the transcriptional factors AP-1 and NF-kappaB. Microglia also express several P2 receptors linked to intracellular calcium increases. P2 receptor subtypes are differentially regulated by typical proinflammatory signals for these cells (e.g. lipopolysaccharide), suggesting specific roles in brain immune responses. Globally, these findings highlight the roles of P2 receptors in glial cell pathophysiology suggesting a contribution to neurodegenerative diseases characterized by excessive gliosis and neuro-inflammation. They also open up the possibility of modulating brain damage by ligands selectively targeting the specific P2 receptor subtypes involved in the gliotic response.

Epitope mapping of tsh receptor-blocking antibodies in Graves' disease that appear during pregnancy.

PubMed

Kung, A W; Lau, K S; Kohn, L D

2001-08-01

Spontaneous remission of Graves' disease during pregnancy is thought to be due to a reduction of thyroid-stimulating antibody activity. We suspected, however, that a broader change in TSH receptor antibody characteristics might play an important role in modulating disease activity during pregnancy. We measured TSH binding inhibitory Ig, thyroid-stimulating antibody, and thyroid stimulating-blocking antibody activities in 13 pregnant Graves' disease patients at first, second, and third trimesters and 4 months postpartum. To measure and epitope-map thyroid-stimulating antibody and thyroid stimulating-blocking antibody activities, we used CHO cells transfected with wild-type human TSH receptor or with several TSH receptor-LH/hCG receptor chimeras: Mc1+2, Mc2, and Mc4. These chimeric cells have their respective TSH receptor residues 9-165, 90-165, and 261-370 substituted with equivalent residues of the LH/hCG receptor. Overall thyroid-stimulating antibody decreased, whereas thyroid stimulating-blocking antibody increased progressively during pregnancy. TSH binding inhibitory Ig fluctuated in individual patients, but overall the activities remained statistically unchanged. Thyroid stimulating-blocking antibody appeared in subjects who were either negative for thyroid-stimulating antibody or whose thyroid-stimulating antibody activity increased or decreased during pregnancy. Epitope mapping showed that the thyroid-stimulating antibodies were mainly directed against residues 9-165 of the N-terminus of the TSH receptor extracellular domain. All thyroid stimulating-blocking antibodies had blocking activities against residues 261-370 of the C-terminus of the ectodomain. However, the majority of the thyroid stimulating-blocking antibodies had a hybrid conformational epitope directed against N-terminal residues 9-89 or 90-165 as well. Despite a change in the activity level, we did not observe any change in the epitope of either the stimulatory or blocking Abs as pregnancy advanced. In conclusion, a change in the specificity of TSH receptor antibody from stimulatory to blocking activity was observed during pregnancy, and the appearance of thyroid stimulating-blocking antibody may contribute to the remission of Graves' disease during pregnancy.

Cellular Decision Making by Non-Integrative Processing of TLR Inputs.

PubMed

Kellogg, Ryan A; Tian, Chengzhe; Etzrodt, Martin; Tay, Savaş

2017-04-04

Cells receive a multitude of signals from the environment, but how they process simultaneous signaling inputs is not well understood. Response to infection, for example, involves parallel activation of multiple Toll-like receptors (TLRs) that converge on the nuclear factor κB (NF-κB) pathway. Although we increasingly understand inflammatory responses for isolated signals, it is not clear how cells process multiple signals that co-occur in physiological settings. We therefore examined a bacterial infection scenario involving co-stimulation of TLR4 and TLR2. Independent stimulation of these receptors induced distinct NF-κB dynamic profiles, although surprisingly, under co-stimulation, single cells continued to show ligand-specific dynamic responses characteristic of TLR2 or TLR4 signaling rather than a mixed response, comprising a cellular decision that we term "non-integrative" processing. Iterating modeling and microfluidic experiments revealed that non-integrative processing occurred through interaction of switch-like NF-κB activation, receptor-specific processing timescales, cell-to-cell variability, and TLR cross-tolerance mediated by multilayer negative feedback. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Glutamatergic Receptor Activation in the Commisural Nucleus Tractus Solitarii (cNTS) Mediates Brain Glucose Retention (BGR) Response to Anoxic Carotid Chemoreceptor (CChr) Stimulation in Rats.

PubMed

Cuéllar, R; Montero, S; Luquín, S; García-Estrada, J; Dobrovinskaya, O; Melnikov, V; Lemus, M; de Álvarez-Buylla, E Roces

2015-01-01

Glutamate, released from central terminals of glossopharyngeal nerve, is a major excitatory neurotransmitter of commissural nucleus tractus solitarii (cNTS) afferent terminals, and brain derived neurotrophic factor (BDNF) has been shown to attenuate glutamatergic AMPA currents in NTS neurons. To test the hypothesis that AMPA contributes to glucose regulation in vivo modulating the hyperglycemic reflex with brain glucose retention (BGR), we microinjected AMPA and NBQX (AMPA antagonist) into the cNTS before carotid chemoreceptor stimulation in anesthetized normal Wistar rats, while hyperglycemic reflex an brain glucose retention (BGR) were analyzed. To investigate the underlying mechanisms, GluR2/3 receptor and c-Fos protein expressions in cNTS neurons were determined. We showed that AMPA in the cNTS before CChr stimulation inhibited BGR observed in aCSF group. In contrast, NBQX in similar conditions, did not modify the effects on glucose variables observed in aCSF control group. These experiments suggest that glutamatergic pathways, via AMPA receptors, in the cNTS may play a role in glucose homeostasis.

Association between GRB2/Sos and insulin receptor substrate 1 is not sufficient for activation of extracellular signal-regulated kinases by interleukin-4: implications for Ras activation by insulin.

PubMed

Pruett, W; Yuan, Y; Rose, E; Batzer, A G; Harada, N; Skolnik, E Y

1995-03-01

Insulin receptor substrate 1 (IRS-1) mediates the activation of a variety of signaling pathways by the insulin and insulin-like growth factor 1 receptors by serving as a docking protein for signaling molecules with SH2 domains. We and others have shown that in response to insulin stimulation IRS-1 binds GRB2/Sos and have proposed that this interaction is important in mediating Ras activation by the insulin receptor. Recently, it has been shown that the interleukin (IL)-4 receptor also phosphorylates IRS-1 and an IRS-1-related molecule, 4PS. Unlike insulin, however, IL-4 fails to activate Ras, extracellular signal-regulated kinases (ERKs), or mitogen-activated protein kinases. We have reconstituted the IL-4 receptor into an insulin-responsive L6 myoblast cell line and have shown that IRS-1 is tyrosine phosphorylated to similar degrees in response to insulin and IL-4 stimulation in this cell line. In agreement with previous findings, IL-4 failed to activate the ERKs in this cell line or to stimulate DNA synthesis, whereas the same responses were activated by insulin. Surprisingly, IL-4's failure to activate ERKs was not due to a failure to stimulate the association of tyrosine-phosphorylated IRS-1 with GRB2/Sos; the amounts of GRB2/Sos associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. Moreover, the amounts of phosphatidylinositol 3-kinase activity associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. In contrast to insulin, however, IL-4 failed to induce tyrosine phosphorylation of Shc or association of Shc with GRB2. Thus, ERK activation correlates with Shc tyrosine phosphorylation and formation of an Shc/GRB2 complex. Thus, ERK activation correlates with Shc tyrosine phosphorylation and formation of an Shc/GRB2 complex. Previous studies have indicated that activation of ERks in this cell line is dependent upon Ras since a dominant-negative Ras (Asn-17) blocks ERK activation by insulin. Our findings, taken in the context of previous work, suggest that binding of GRB2/Sos to Shc may be the predominant mechanism whereby insulin as well as cytokine receptors activate Ras.

In brown adipocytes, adrenergically induced β{sub 1}-/β{sub 3}-(G{sub s})-, α{sub 2}-(G{sub i})- and α{sub 1}-(G{sub q})-signalling to Erk1/2 activation is not mediated via EGF receptor transactivation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Yanling; Fälting, Johanna M.; Mattsson, Charlotte L.

2013-10-15

Brown adipose tissue is unusual in that the neurotransmitter norepinephrine influences cell destiny in ways generally associated with effects of classical growth factors: regulation of cell proliferation, of apoptosis, and progression of differentiation. The norepinephrine effects are mediated through G-protein-coupled receptors; further mediation of such stimulation to e.g. Erk1/2 activation is in cell biology in general accepted to occur through transactivation of the EGF receptor (by external or internal pathways). We have examined here the significance of such transactivation in brown adipocytes. Stimulation of mature brown adipocytes with cirazoline (α{sub 1}-adrenoceptor coupled via G{sub q}), clonidine (α{sub 2} via G{submore » i}) or CL316243 (β{sub 3} via G{sub s}) or via β{sub 1}-receptors significantly activated Erk1/2. Pretreatment with the EGF receptor kinase inhibitor AG1478 had, remarkably, no significant effect on Erk1/2 activation induced by any of these adrenergic agonists (although it fully abolished EGF-induced Erk1/2 activation), demonstrating absence of EGF receptor-mediated transactivation. Results with brown preadipocytes (cells in more proliferative states) were not qualitatively different. Joint stimulation of all adrenoceptors with norepinephrine did not result in synergism on Erk1/2 activation. AG1478 action on EGF-stimulated Erk1/2 phosphorylation showed a sharp concentration–response relationship (IC{sub 50} 0.3 µM); a minor apparent effect of AG1478 on norepinephrine-stimulated Erk1/2 phosphorylation showed nonspecific kinetics, implying caution in interpretation of partial effects of AG1478 as reported in other systems. Transactivation of the EGF receptor is clearly not a universal prerequisite for coupling of G-protein coupled receptors to Erk1/2 signalling cascades. - Highlights: • In brown adipocytes, norepinephrine regulates proliferation, apoptosis, differentiation. • EGF receptor transactivation is supposed to mediate GPCR-induced Erk1/2 activation. • α{sub 1}-, α{sub 2}-, β{sub 1}-, β{sub 3}-adrenoceptors all activate Erk1/2—but EGF receptor transactivation is not involved. • Adrenergic regulation of proliferation, apoptosis, differentiation must utilize cell-specific pathways in brown adipocytes. • EGF receptor transactivation is not universal in mediating GPCR-induced Erk1/2 activation.« less

Chloral hydrate-dependent reduction in the peptidoglycan-induced inflammatory macrophage response is associated with lower expression levels of toll-like receptor 2.

PubMed

Pan, Qingjun; Liu, Yuan; Zhu, Xuezhi; Liu, Huafeng

2014-05-01

The aim of this study was to investigate the effect and mechanism of action of chloral hydrate on the peptidoglycan (PGN)-induced inflammatory macrophage response. The effect of chloral hydrate on the production of tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) by murine peritoneal macrophages with PGN-stimulation was investigated. In addition, RAW264.7 cells transfected with a nuclear factor-κB (NF-κB) luciferase reporter plasmid stimulated by PGN were used to study the effect of chloral hydrate on the levels NF-κB activity. Flow cytometry and western blotting were performed to investigate the expression levels of toll-like receptor 2 (TLR2) in the treated RAW264.7 cells. It was identified that chloral hydrate reduced the levels of IL-6 and TNF-α produced by the peritoneal macrophages stimulated with PGN. The levels of NF-κB activity of the RAW264.7 cells stimulated by PGN decreased following treatment with chloral hydrate, which was associated with a reduction in the expression levels of TLR2 and reduced levels of TLR2 signal transduction. These data demonstrate that chloral hydrate reduced the magnitude of the PGN-induced inflammatory macrophage response associated with lower expression levels of TLR2.

Nuclear factor ETF specifically stimulates transcription from promoters without a TATA box.

PubMed

Kageyama, R; Merlino, G T; Pastan, I

1989-09-15

Transcription factor ETF stimulates the expression of the epidermal growth factor receptor (EGFR) gene which does not have a TATA box in the promoter region. Here, we show that ETF recognizes various GC-rich sequences including stretches of deoxycytidine or deoxyguanosine residues and GC boxes with similar affinities. ETF also binds to TATA boxes but with a lower affinity. ETF stimulated in vitro transcription from several promoters without TATA boxes but had little or no effect on TATA box-containing promoters even though they had strong ETF-binding sites. These inactive ETF-binding sites became functional when placed upstream of the EGFR promoter whose own ETF-binding sites were removed. Furthermore, when a TATA box was introduced into the EGFR promoter, the responsiveness to ETF was abolished. These results indicate that ETF is a specific transcription factor for promoters which do not contain TATA elements.

Selective inhibition of the platelet-derived growth factor signal transduction pathway by a protein-tyrosine kinase inhibitor of the 2-phenylaminopyrimidine class.

PubMed Central

Buchdunger, E; Zimmermann, J; Mett, H; Meyer, T; Müller, M; Regenass, U; Lydon, N B

1995-01-01

The platelet-derived growth factor (PDGF) receptor is a member of the transmembrane growth factor receptor protein family with intrinsic protein-tyrosine kinase activity. We describe a potent protein-tyrosine kinase inhibitor (CGP 53716) that shows selectivity for the PDGF receptor in vitro and in the cell. The compound shows selectivity for inhibition of PDGF-mediated events such as PDGF receptor autophosphorylation, cellular tyrosine phosphorylation, and c-fos mRNA induction in response to PDGF stimulation of intact cells. In contrast, ligand-induced autophosphorylation of the epidermal growth factor (EGF) receptor, insulin receptor, and the insulin-like growth factor I receptor, as well as c-fos mRNA expression induced by EGF, fibroblast growth factor, and phorbol ester, was insensitive to inhibition by CGP 53716. In antiproliferative assays, the compound was approximately 30-fold more potent in inhibiting PDGF-mediated growth of v-sis-transformed BALB/c 3T3 cells relative to inhibition of EGF-dependent BALB/Mk cells, interleukin-3-dependent FDC-P1 cells, and the T24 bladder carcinoma line. When tested in vivo using highly tumorigenic v-sis- and human c-sis-transformed BALB/c 3T3 cells, CGP 53716 showed antitumor activity at well-tolerated doses. In contrast, CGP 53716 did not show antitumor activity against xenografts of the A431 tumor, which overexpresses the EGF receptor. These findings suggest that CGP 53716 may have therapeutic potential for the treatment of diseases involving abnormal cellular proliferation induced by PDGF receptor activation. Images Fig. 1 Fig. 2 Fig. 3 PMID:7708684

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

PubMed Central

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 had no effect on the release of endothelin-1 or prostaglandin E2 in keratinocytes. In addition, MIF had no direct effect on melanin and tyrosinase synthesis in cultured human melanocytes. The effect of MIF on melanogenesis was also examined using a three-dimensional reconstituted human epidermal culture model, which is a novel, commercially available, cultured human epidermis containing functional melanocytes. Migration inhibitory factor induced an increase in melanin content in the epidermis after a 9-day culture period. Moreover, melanin synthesis induced by UV-B stimulation was significantly down-regulated by anti-MIF antibody treatment. An in vivo study showed that the back skin of MIF transgenic mice had a higher melanin content than that of wild-type mice after 12 weeks of UV-B exposure. Therefore, MIF-mediated melanogenesis occurs mainly through the activation of PAR-2 and SCF expression in keratinocytes after exposure to UV-B radiation. PMID:21281800

Regulation of cell growth by redox-mediated extracellular proteolysis of platelet-derived growth factor receptor beta.

PubMed

Okuyama, H; Shimahara, Y; Kawada, N; Seki, S; Kristensen, D B; Yoshizato, K; Uyama, N; Yamaoka, Y

2001-07-27

Redox-regulated processes are important elements in various cellular functions. Reducing agents, such as N-acetyl-l-cysteine (NAC), are known to regulate signal transduction and cell growth through their radical scavenging action. However, recent studies have shown that reactive oxygen species are not always involved in ligand-stimulated intracellular signaling. Here, we report a novel mechanism by which NAC blocks platelet-derived growth factor (PDGF)-induced signaling pathways in hepatic stellate cells, a fibrogenic player in the liver. Unlike in vascular smooth muscle cells, we found that reducing agents, including NAC, triggered extracellular proteolysis of PDGF receptor-beta, leading to desensitization of hepatic stellate cells toward PDGF-BB. This effect was mediated by secreted mature cathepsin B. In addition, type II transforming growth factor-beta receptor was also down-regulated. Furthermore, these events seemed to cause a dramatic improvement of rat liver fibrosis. These results indicated that redox processes impact the cell's response to growth factors by regulating the turnover of growth factor receptors and that "redox therapy" is promising for fibrosis-related disease.

Role of Gab1 in Heart, Placenta, and Skin Development and Growth Factor- and Cytokine-Induced Extracellular Signal-Regulated Kinase Mitogen-Activated Protein Kinase Activation

PubMed Central

Itoh, Motoyuki; Yoshida, Yuichi; Nishida, Keigo; Narimatsu, Masahiro; Hibi, Masahiko; Hirano, Toshio

2000-01-01

Gab1 is a member of the Gab/DOS (Daughter of Sevenless) family of adapter molecules, which contain a pleckstrin homology (PH) domain and potential binding sites for SH2 and SH3 domains. Gab1 is tyrosine phosphorylated upon stimulation of various cytokines, growth factors, and antigen receptors in cell lines and interacts with signaling molecules, such as SHP-2 and phosphatidylinositol 3-kinase, although its biological roles have not yet been established. To reveal the functions of Gab1 in vivo, we generated mice lacking Gab1 by gene targeting. Gab1-deficient embryos died in utero and displayed developmental defects in the heart, placenta, and skin, which were similar to phenotypes observed in mice lacking signals of the hepatocyte growth factor/scatter factor, platelet-derived growth factor, and epidermal growth factor pathways. Consistent with these observations, extracellular signal-regulated kinase mitogen-activated protein (ERK MAP) kinases were activated at much lower levels in cells from Gab1-deficient embryos in response to these growth factors or to stimulation of the cytokine receptor gp130. These results indicate that Gab1 is a common player in a broad range of growth factor and cytokine signaling pathways linking ERK MAP kinase activation. PMID:10779359

Epidermal growth factor receptor (EGFR) transactivation by estrogen via the G-protein-coupled receptor, GPR30: a novel signaling pathway with potential significance for breast cancer.

PubMed

Filardo, Edward J

2002-02-01

The biological and biochemical effects of estrogen have been ascribed to its known receptors, which function as ligand-inducible transcription factors. However, estrogen also triggers rapid activation of classical second messengers (cAMP, calcium, and inositol triphosphate) and stimulation of intracellular signaling cascades mitogen-activated protein kinase (MAP K), PI3K and eNOS. These latter events are commonly activated by membrane receptors that either possess intrinsic tyrosine kinase activity or couple to heterotrimeric G-proteins. We have shown that estrogen transactivates the epidermal growth factor receptor (EGFR) to MAP K signaling axis via the G-protein-coupled receptor (GPCR), GPR30, through the release of surface-bound proHB-EGF from estrogen receptor (ER)-negative human breast cancer cells [Molecular Endocrinology 14 (2000) 1649]. This finding is consistent with a growing body of evidence suggesting that transactivation of EGFRs by GPCRs is a recurrent theme in cell signaling. GPCR-mediated transactivation of EGFRs by estrogen provides a previously unappreciated mechanism of cross-talk between estrogen and serum growth factors, and explains prior data reporting the EGF-like effects of estrogen. This novel mechanism by which estrogen activates growth factor-dependent signaling and its implications for breast cancer biology are discussed further in this review.

Promoter-level expression clustering identifies time development of transcriptional regulatory cascades initiated by ErbB receptors in breast cancer cells.

PubMed

Mina, Marco; Magi, Shigeyuki; Jurman, Giuseppe; Itoh, Masayoshi; Kawaji, Hideya; Lassmann, Timo; Arner, Erik; Forrest, Alistair R R; Carninci, Piero; Hayashizaki, Yoshihide; Daub, Carsten O; Okada-Hatakeyama, Mariko; Furlanello, Cesare

2015-07-16

The analysis of CAGE (Cap Analysis of Gene Expression) time-course has been proposed by the FANTOM5 Consortium to extend the understanding of the sequence of events facilitating cell state transition at the level of promoter regulation. To identify the most prominent transcriptional regulations induced by growth factors in human breast cancer, we apply here the Complexity Invariant Dynamic Time Warping motif EnRichment (CIDER) analysis approach to the CAGE time-course datasets of MCF-7 cells stimulated by epidermal growth factor (EGF) or heregulin (HRG). We identify a multi-level cascade of regulations rooted by the Serum Response Factor (SRF) transcription factor, connecting the MAPK-mediated transduction of the HRG stimulus to the negative regulation of the MAPK pathway by the members of the DUSP family phosphatases. The finding confirms the known primary role of FOS and FOSL1, members of AP-1 family, in shaping gene expression in response to HRG induction. Moreover, we identify a new potential regulation of DUSP5 and RARA (known to antagonize the transcriptional regulation induced by the estrogen receptors) by the activity of the AP-1 complex, specific to HRG response. The results indicate that a divergence in AP-1 regulation determines cellular changes of breast cancer cells stimulated by ErbB receptors.

In vitro long-term culture of human primitive hematopoietic cells supported by murine stromal cell line MS-5.

PubMed

Nishi, N; Ishikawa, R; Inoue, H; Nishikawa, M; Yoneya, T; Kakeda, M; Tsumura, H; Ohashi, H; Mori, K J

1997-04-01

When Lin-CD34+CD38- cells from normal human cord blood were cocultured with MS-5, colony forming cells were maintained for over 8 weeks. Prevention of contact between MS-5 and Lin-CD34+CD38- cells by using a membrane filter was negligible for this activity, indicating that the activity of MS-5 on human primitive hematopoietic cells may be due to soluble factor(s) secreted from MS-5. We tried to purify this activity by a [3H]TdR incorporation assay. The activity was found in 150 kD fraction and was neutralized with anti-mSCF (stem cell factor) antibody. Another 20-30 kD fraction synergized with mSCF to stimulate the growth of Lin-CD34+CD38- cells but failed alone. This fraction supported the growth of the G-CSF (granulocyte-colony stimulating factor)-dependent cell line FD/GR3, FDC-P2 transfected with mG-CSF receptor cDNA. This synergy was canceled in the presence of soluble mG-CSF receptor. Addition of anti-mSCF antibody and soluble mG-CSF receptor to the culture completely abrogated the activity of MS-5-culture supernatant. These results indicate the activity of MS-5 on Lin-CD34+CD38- cells is due to synergistic effect of mSCF and mG-CSF.

ST2 suppresses IL-6 production via the inhibition of I{kappa}B degradation induced by the LPS signal in THP-1 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Takezako, Naoki; Hayakawa, Morisada; Hayakawa, Hiroko

2006-03-10

LPS induces the production of inflammatory cytokines via the stimulation of Toll-like receptors. In this study, we demonstrated that a soluble secreted form of the ST2 gene product (ST2), a member of the interleukin-1 receptor family, suppressed the production of IL-6 in an LPS-stimulated human monocytic leukemia cell line, THP-1. Immunofluorescence confocal microscopy revealed the binding of ST2 to the surface of the THP-1 cells, in which ST2 led to decreased binding of nuclear factor-{kappa}B to the IL-6 promoter. Furthermore, the degradation of I{kappa}B in the cytoplasm after LPS stimulation was reduced by pretreatment with ST2. These results demonstrated thatmore » ST2 negatively regulates LPS-induced IL-6 production via the inhibition of I{kappa}B degradation in THP-1 cells.« less

Abnormal XPD-induced nuclear receptor transactivation in DNA repair disorders: trichothiodystrophy and xeroderma pigmentosum

PubMed Central

Zhou, Xiaolong; Khan, Sikandar G; Tamura, Deborah; Ueda, Takahiro; Boyle, Jennifer; Compe, Emmanuel; Egly, Jean-Marc; DiGiovanna, John J; Kraemer, Kenneth H

2013-01-01

XPD (ERCC2) is a DNA helicase involved in nucleotide excision repair and in transcription as a structural bridge tying the transcription factor IIH (TFIIH) core with the cdk-activating kinase complex, which phosphorylates nuclear receptors. Mutations in XPD are associated with several different phenotypes, including trichothiodystrophy (TTD), with sulfur-deficient brittle hair, bone defects, and developmental abnormalities without skin cancer, xeroderma pigmentosum (XP), with pigmentary abnormalities and increased skin cancer, or XP/TTD with combined features, including skin cancer. We describe the varied clinical features and mutations in nine patients examined at the National Institutes of Health who were compound heterozygotes for XPD mutations but had different clinical phenotypes: four TTD, three XP, and two combined XP/TTD. We studied TFIIH-dependent transactivation by nuclear receptor for vitamin D (VDR) and thyroid in cells from these patients. The vitamin D stimulation ratio of CYP24 and osteopontin was associated with specific pairs of mutations (reduced in 5, elevated in 1) but not correlated with distinct clinical phenotypes. Thyroid receptor stimulation ratio for KLF9 was not significantly different from normal. XPD mutations frequently were associated with abnormal VDR stimulation in compound heterozygote patients with TTD, XP, or XP/TTD. PMID:23232694

Bone marrow-derived mesenchymal stem cells from early diffuse systemic sclerosis exhibit a paracrine machinery and stimulate angiogenesis in vitro.

PubMed

Guiducci, Serena; Manetti, Mirko; Romano, Eloisa; Mazzanti, Benedetta; Ceccarelli, Claudia; Dal Pozzo, Simone; Milia, Anna Franca; Bellando-Randone, Silvia; Fiori, Ginevra; Conforti, Maria Letizia; Saccardi, Riccardo; Ibba-Manneschi, Lidia; Matucci-Cerinic, Marco

2011-11-01

To characterise bone marrow-derived mesenchymal stem cells (MSCs) from patients with systemic sclerosis (SSc) for the expression of factors implicated in MSC recruitment at sites of injury, angiogenesis and fibrosis. The study also analysed whether the production/release of bioactive mediators by MSCs were affected by stimulation with cytokines found upregulated in SSc serum and tissues, and whether MSCs could modulate dermal microvascular endothelial cell (MVEC) angiogenesis. MSCs obtained from five patients with early severe diffuse SSc (SSc-MSCs) and five healthy donors (H-MSCs) were stimulated with vascular endothelial growth factor (VEGF), transforming growth factor β (TGFβ) or stromal cell-derived factor-1 (SDF-1). Transcript and protein levels of SDF-1 and its receptor CXCR4, VEGF, TGFβ(1) and receptors TβRI and TβRII were evaluated by quantitative real-time PCR, western blotting and confocal microscopy. VEGF, SDF-1 and TGFβ(1) secretion in culture supernatant was measured by ELISA. MVEC capillary morphogenesis was performed on Matrigel with the addition of MSC-conditioned medium. In SSc-MSCs the basal expression of proangiogenic SDF-1/CXCR4 and VEGF was significantly increased compared with H-MSCs. SSc-MSCs constitutively released higher levels of SDF-1 and VEGF. SDF-1/CXCR4 were upregulated after VEGF stimulation and CXCR4 redistributed from the cytoplasm to the cell surface. VEGF was increased by SDF-1 challenge. VEGF, TGFβ and SDF-1 stimulation upregulated TGFβ(1), TβRI and TβRII in SSc-MSCs. TβRII redistributed from the cytoplasm to focal adhesion contacts. SSc-MSC-conditioned medium showed a greater proangiogenic effect on MVECs than H-MSCs. Experiments with blocking antibodies showed that MSC-derived cytokines were responsible for this potent proangiogenic effect. SSc-MSCs constitutively overexpress and release bioactive mediators/proangiogenic factors and potentiate dermal MVEC angiogenesis.

Epidermal growth factor induction of front–rear polarity and migration in keratinocytes is mediated by integrin-linked kinase and ELMO2

PubMed Central

Ho, Ernest; Dagnino, Lina

2012-01-01

Epidermal growth factor (EGF) is a potent chemotactic and mitogenic factor for epidermal keratinocytes, and these properties are central for normal epidermal regeneration after injury. The involvement of mitogen-activated protein kinases as mediators of the proliferative effects of EGF is well established. However, the molecular mechanisms that mediate motogenic responses to this growth factor are not clearly understood. An obligatory step for forward cell migration is the development of front–rear polarity and formation of lamellipodia at the leading edge. We show that stimulation of epidermal keratinocytes with EGF, but not with other growth factors, induces development of front–rear polarity and directional migration through a pathway that requires integrin-linked kinase (ILK), Engulfment and Cell Motility-2 (ELMO2), integrin β1, and Rac1. Furthermore, EGF induction of front–rear polarity and chemotaxis require the tyrosine kinase activity of the EGF receptor and are mediated by complexes containing active RhoG, ELMO2, and ILK. Our findings reveal a novel link between EGF receptor stimulation, ILK-containing complexes, and activation of small Rho GTPases necessary for acquisition of front–rear polarity and forward movement. PMID:22160594

Epidermal growth factor induction of front-rear polarity and migration in keratinocytes is mediated by integrin-linked kinase and ELMO2.

PubMed

Ho, Ernest; Dagnino, Lina

2012-02-01

Epidermal growth factor (EGF) is a potent chemotactic and mitogenic factor for epidermal keratinocytes, and these properties are central for normal epidermal regeneration after injury. The involvement of mitogen-activated protein kinases as mediators of the proliferative effects of EGF is well established. However, the molecular mechanisms that mediate motogenic responses to this growth factor are not clearly understood. An obligatory step for forward cell migration is the development of front-rear polarity and formation of lamellipodia at the leading edge. We show that stimulation of epidermal keratinocytes with EGF, but not with other growth factors, induces development of front-rear polarity and directional migration through a pathway that requires integrin-linked kinase (ILK), Engulfment and Cell Motility-2 (ELMO2), integrin β1, and Rac1. Furthermore, EGF induction of front-rear polarity and chemotaxis require the tyrosine kinase activity of the EGF receptor and are mediated by complexes containing active RhoG, ELMO2, and ILK. Our findings reveal a novel link between EGF receptor stimulation, ILK-containing complexes, and activation of small Rho GTPases necessary for acquisition of front-rear polarity and forward movement.

The biologic role of ganglioside in neuronal differentiation--effects of GM1 ganglioside on human neuroblastoma SH-SY5Y cells.

PubMed Central

Lee, M. C.; Lee, W. S.; Park, C. S.; Juhng, S. W.

1994-01-01

Human neuroblastoma SH-SY5Y cell is a cloned cell line which has many attractive features for the study of neuronal proliferation and neurite outgrowth, because it has receptors for insulin, IGF-I and PDGF. Gangliosides are sialic acid containing glycosphingolipids which form an integral part of the plasma membrane of many mammalian cells. They inhibit cell growth mediated by tyrosine kinase receptors and ligand-stimulated tyrosine kinase activity, and autophosphorylation of EGF(epidermal growth factor) and PDGF receptors. The experiment was designed to study the effects of GM1 ganglioside on growth of human neuroblastoma SH-SY5Y cells stimulated with trophic factor in vitro. The cells were plated in Eagle's minimum essential medium without serum. The number and morphologic change of SH-SY5Y cells were evaluated in the serum free medium added GM1 ganglioside with insulin or PDGF. SH-SY5Y cells were maintained for six days in serum-free medium, and then cultured for over two weeks in serum-free medium containing either insulin or PDGF. The effect of insulin on cell proliferation developed earlier and was more potent than that of PDGF. These proliferative effects were inhibited by GM1 ganglioside, and the cells showed prominent neurites outgrowth. These findings suggest that GM1 ganglioside inhibits the cell proliferation mediated by tyrosine kinase receptors and directly induces neuritogenesis as one of the neurotrophic factors. PMID:7986393

Mechanisms of integrin-vascular endothelial growth factor receptor cross-activation in angiogenesis.

PubMed

Mahabeleshwar, Ganapati H; Feng, Weiyi; Reddy, Kumar; Plow, Edward F; Byzova, Tatiana V

2007-09-14

The functional responses of endothelial cells are dependent on signaling from peptide growth factors and the cellular adhesion receptors, integrins. These include cell adhesion, migration, and proliferation, which, in turn, are essential for more complex processes such as formation of the endothelial tube network during angiogenesis. This study identifies the molecular requirements for the cross-activation between beta3 integrin and tyrosine kinase receptor 2 for vascular endothelial growth factor (VEGF) receptor (VEGFR-2) on endothelium. The relationship between VEGFR-2 and beta3 integrin appears to be synergistic, because VEGFR-2 activation induces beta3 integrin tyrosine phosphorylation, which, in turn, is crucial for VEGF-induced tyrosine phosphorylation of VEGFR-2. We demonstrate here that adhesion- and growth factor-induced beta3 integrin tyrosine phosphorylation are directly mediated by c-Src. VEGF-stimulated recruitment and activation of c-Src and subsequent beta3 integrin tyrosine phosphorylation are critical for interaction between VEGFR-2 and beta3 integrin. Moreover, c-Src mediates growth factor-induced beta3 integrin activation, ligand binding, beta3 integrin-dependent cell adhesion, directional migration of endothelial cells, and initiation of angiogenic programming in endothelial cells. Thus, the present study determines the molecular mechanisms and consequences of the synergism between 2 cell surface receptor systems, growth factor receptor and integrins, and opens new avenues for the development of pro- and antiangiogenic strategies.

UV-B radiation induces macrophage migration inhibitory factor-mediated melanogenesis through activation of protease-activated receptor-2 and stem cell factor in keratinocytes.

PubMed

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

2011-02-01

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 had no effect on the release of endothelin-1 or prostaglandin E2 in keratinocytes. In addition, MIF had no direct effect on melanin and tyrosinase synthesis in cultured human melanocytes. The effect of MIF on melanogenesis was also examined using a three-dimensional reconstituted human epidermal culture model, which is a novel, commercially available, cultured human epidermis containing functional melanocytes. Migration inhibitory factor induced an increase in melanin content in the epidermis after a 9-day culture period. Moreover, melanin synthesis induced by UV-B stimulation was significantly down-regulated by anti-MIF antibody treatment. An in vivo study showed that the back skin of MIF transgenic mice had a higher melanin content than that of wild-type mice after 12 weeks of UV-B exposure. Therefore, MIF-mediated melanogenesis occurs mainly through the activation of PAR-2 and SCF expression in keratinocytes after exposure to UV-B radiation. Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Regulation of insulin-like growth factor I receptors on vascular smooth muscle cells by growth factors and phorbol esters.

PubMed

Ververis, J J; Ku, L; Delafontaine, P

1993-06-01

Insulin-like growth factor I (IGF I) is an important mitogen for vascular smooth muscle cells. To characterize regulation of vascular IGF I receptors, we performed radioligand displacement experiments using rat aortic smooth muscle cells (RASMs). Serum deprivation for 48 hours caused a 40% decrease in IGF I receptor number. Exposure of quiescent RASMs to platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), or angiotensin II (Ang II) caused a 1.5-2.0-fold increase in IGF I receptors per cell. After FGF exposure, there was a marked increase in the mitogenic response to IGF I. IGF I downregulated its receptors in the presence of platelet-poor plasma. Stimulation of protein kinase C (PKC) by exposure of quiescent RASMs to phorbol 12-myristate 13-acetate caused a biphasic response in IGF I binding; there was a 42% decrease in receptor number at 45 minutes and a 238% increase at 24 hours. To determine the role of PKC in growth factor-induced regulation of IGF I receptors, we downregulated PKC by exposing RASMs to phorbol 12,13-dibutyrate (PDBu) for 48 hours. PDGF- and FGF- but not Ang II-mediated upregulation of IGF I receptors was completely inhibited in PDBu-treated cells. Thus, acute PKC activation by phorbol esters inhibits IGF I binding, whereas chronic PKC activation increases IGF I binding. PDGF and FGF but not Ang II regulate vascular IGF I receptors through a PKC-dependent pathway. These data provide new insights into the regulation of vascular smooth muscle cell IGF I receptors in vitro and are of potential importance in characterizing vascular proliferative responses in vivo.

Redox-regulated growth factor survival signaling.

PubMed

Woolley, John F; Corcoran, Aoife; Groeger, Gillian; Landry, William D; Cotter, Thomas G

2013-11-20

Once the thought of as unwanted byproducts of cellular respiration in eukaryotes, reactive oxygen species (ROS) have been shown to facilitate essential physiological roles. It is now understood that ROS are critical mediators of intracellular signaling. Control of signal transduction downstream of growth factor receptors by ROS is a complex process whose details are only recently coming to light. Indeed, recent evidence points to control of signal propagation by ROS at multiple levels in the typical cascade. Growth factor stimulation activates nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (Noxs) at the membrane, producing superoxide in the extracellular matrix, which is catalyzed to the membrane-permeable hydrogen peroxide (H2O2) that mediates intracellular signaling events. The potential for H2O2, however, to disrupt cellular functions by damaging proteins and nucleic acids demands that its levels are kept in check by receptor-associated peroxiredoxins. This interplay of Nox and peroxiredoxin activity moderates levels of H2O2 sufficiently to modify signaling partners locally. Among the best studied of these partners are redox-controlled phosphatases that are inactivated by H2O2. Phosphatases regulate signal propagation downstream of receptors, and thus their inactivation allows a further level of control. Transmission of information further downstream to targets such as transcription factors, themselves regulated by ROS, completes this pathway. Thus, signal propagation or attenuation can be dictated by ROS at multiple points. Given the complex nature of these processes, we envisage the emerging trends in the field of redox signaling in the context of growth factor stimulation.

Self-renewal of human embryonic stem cells requires insulin-like growth factor-1 receptor and ERBB2 receptor signaling

PubMed Central

Wang, Linlin; Schulz, Thomas C.; Sherrer, Eric S.; Dauphin, Derek S.; Shin, Soojung; Nelson, Angelique M.; Ware, Carol B.; Zhan, Mei; Song, Chao-Zhong; Chen, Xiaoji; Brimble, Sandii N.; McLean, Amanda; Galeano, Maria J.; Uhl, Elizabeth W.; D'Amour, Kevin A.; Chesnut, Jonathan D.; Rao, Mahendra S.

2007-01-01

Despite progress in developing defined conditions for human embryonic stem cell (hESC) cultures, little is known about the cell-surface receptors that are activated under conditions supportive of hESC self-renewal. A simultaneous interrogation of 42 receptor tyrosine kinases (RTKs) in hESCs following stimulation with mouse embryonic fibroblast (MEF) conditioned medium (CM) revealed rapid and prominent tyrosine phosphorylation of insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R); less prominent tyrosine phosphorylation of epidermal growth factor receptor (EGFR) family members, including ERBB2 and ERBB3; and trace phosphorylation of fibroblast growth factor receptors. Intense IGF1R and IR phosphorylation occurred in the absence of MEF conditioning (NCM) and was attributable to high concentrations of insulin in the proprietary KnockOut Serum Replacer (KSR). Inhibition of IGF1R using a blocking antibody or lentivirus-delivered shRNA reduced hESC self-renewal and promoted differentiation, while disruption of ERBB2 signaling with the selective inhibitor AG825 severely inhibited hESC proliferation and promoted apoptosis. A simple defined medium containing an IGF1 analog, heregulin-1β (a ligand for ERBB2/ERBB3), fibroblast growth factor-2 (FGF2), and activin A supported long-term growth of multiple hESC lines. These studies identify previously unappreciated RTKs that support hESC proliferation and self-renewal, and provide a rationally designed medium for the growth and maintenance of pluripotent hESCs. PMID:17761519

Activation of the protein tyrosine phosphatase SHP2 via the interleukin-6 signal transducing receptor protein gp130 requires tyrosine kinase Jak1 and limits acute-phase protein expression.

PubMed

Schaper, F; Gendo, C; Eck, M; Schmitz, J; Grimm, C; Anhuf, D; Kerr, I M; Heinrich, P C

1998-11-01

Stimulation of the interleukin-6 (IL-6) signalling pathway occurs via the IL-6 receptor-glycoprotein 130 (IL-6R-gp130) receptor complex and results in the regulation of acute-phase protein genes in liver cells. Ligand binding to the receptor complex leads to tyrosine phosphorylation and activation of Janus kinases (Jak), phosphorylation of the signal transducing subunit gp130, followed by recruitment and phosphorylation of the signal transducer and activator of transcription factors STAT3 and STAT1 and the src homology domain (SH2)-containing protein tyrosine phosphatase (SHP2). The tyrosine phosphorylated STAT factors dissociate from the receptor, dimerize and translocate to the nucleus where they bind to enhancer sequences of IL-6 target genes. Phosphorylated SHP2 is able to bind growth factor receptor bound protein (grb2) and thus might link the Jak/STAT pathway to the ras/raf/mitogen-activated protein kinase pathway. Here we present data on the dose-dependence, kinetics and kinase requirements for SHP2 phosphorylation after the activation of the signal transducer, gp130, of the IL-6-type family receptor complex. When human fibrosarcoma cell lines deficient in Jak1, Jak2 or tyrosine kinase 2 (Tyk2) were stimulated with IL-6-soluble IL-6R complexes it was found that only in Jak1-, but not in Jak 2- or Tyk2-deficient cells, SHP2 activation was greatly impaired. It is concluded that Jak1 is required for the tyrosine phosphorylation of SHP2. This phosphorylation depends on Tyr-759 in the cytoplasmatic domain of gp130, since a Tyr-759-->Phe exchange abrogates SHP2 activation and in turn leads to elevated and prolonged STAT3 and STAT1 activation as well as enhanced acute-phase protein gene induction. Therefore, SHP2 plays an important role in acute-phase gene regulation.

Prior stimulation of the endocannabinoid system prevents methamphetamine-induced dopaminergic neurotoxicity in the striatum through activation of CB2 receptors

PubMed Central

Nader, Joëlle; Rapino, Cinzia; Gennequin, Benjamin; Chavant, Francois; Francheteau, Maureen; Makriyannis, Alexandros; Duranti, Andrea; Maccarrone, Mauro; Solinas, Marcello; Thiriet, Nathalie

2016-01-01

Methamphetamine toxicity is associated with cell death and loss of dopamine neuron terminals in the striatum similar to what is found in some neurodegenerative diseases. Conversely, the endocannabinoid system (ECS) has been suggested to be neuroprotective in the brain, and new pharmacological tools have been developed to increase their endogenous tone. In this study, we evaluated whether ECS stimulation could reduce the neurotoxicity of high doses of methamphetamine on the dopamine system. We found that methamphetamine alters the levels of the major endocannabinoids, anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) in the striatum, suggesting that the ECS participates in the brain responses to methamphetamine. Δ9-tetrahydrocannabinol (THC), a cannabis-derived agonist of both CB1 and CB2 cannabinoid receptors, or inhibitors of the main enzymes responsible for the degradation of AEA and 2-AG (URB597 and JZL184, respectively), blunted the decrease in striatal protein levels of tyrosine hydroxylase induced by methamphetamine. In addition, antagonists of CB2, but not of CB1, blocked the preventive effects of URB597 and JZL184, suggesting that only the former receptor subtype is engaged in neuroprotection exerted by ECS stimulation. Finally, we found that methamphetamine increases striatal levels of the cytokine tumor necrosis factor alpha, an effect that was blocked by ECS stimulation. Altogether, our results indicate that stimulation of ECS prior to the administration of an overdose of meth-amphetamine considerably reduces the neurotoxicity of the drug through CB2 receptor activation and highlight a protective function for the ECS against the toxicity induced by drugs and other external insults to the brain. This article is part of the Special Issue entitled ‘CNS Stimulants’. PMID:24709540

Direct stimulation of angiotensin II type 2 receptor initiated after stroke ameliorates ischemic brain damage.

PubMed

Min, Li-Juan; Mogi, Masaki; Tsukuda, Kana; Jing, Fei; Ohshima, Kousei; Nakaoka, Hirotomo; Kan-No, Harumi; Wang, Xiao-Li; Chisaka, Toshiyuki; Bai, Hui-Yu; Iwanami, Jun; Horiuchi, Masatsugu

2014-08-01

Stroke is a leading cause of death and disability; however, meta-analysis of randomized controlled trials of blood pressure-lowering drugs in acute stroke has shown no definite evidence of a beneficial effect on functional outcome. Accumulating evidence suggests that angiotensin II type 1 receptor blockade with angiotensin II type 2 (AT2) receptor stimulation could contribute to protection against ischemic brain damage. We examined the possibility that direct AT2 receptor stimulation by compound 21 (C21) initiated even after stroke can prevent ischemic brain damage. Stroke was induced by middle cerebral artery (MCA) occlusion, and the area of cerebral infarction was measured by magnetic resonant imaging. C21 (10 µg/kg/day) treatment was initiated immediately after MCA occlusion by intraperitoneal injection followed by treatment with C21 once daily. We observed that ischemic area was enlarged in a time dependent fashion and decreased on day 5 after MCA occlusion. Treatment with C21 initiated after MCA occlusion significantly reduced the ischemic area, with improvement of neurological deficit in a time-dependent manner without affecting blood pressure. The decrease of cerebral blood flow after MCA occlusion was also ameliorated by C21 treatment. Moreover, treatment with C21 significantly attenuated superoxide anion production and expression of proinflammatory cytokines, monocyte chemoattractant protein 1, and tumor necrosis factor α. Interestingly, C21 administration significantly decreased blood-brain barrier permeability and cerebral edema on the ischemic side. These results provide new evidence that direct AT2 receptor stimulation with C21 is a novel therapeutic approach to prevent ischemic brain damage after acute stroke. © American Journal of Hypertension, Ltd 2014. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

G protein-coupled receptor 30 (GPR30) mediates gene expression changes and growth response to 17beta-estradiol and selective GPR30 ligand G-1 in ovarian cancer cells.

PubMed

Albanito, Lidia; Madeo, Antonio; Lappano, Rosamaria; Vivacqua, Adele; Rago, Vittoria; Carpino, Amalia; Oprea, Tudor I; Prossnitz, Eric R; Musti, Anna Maria; Andò, Sebastiano; Maggiolini, Marcello

2007-02-15

Estrogens play a crucial role in the development of ovarian tumors; however, the signal transduction pathways involved in hormone action are still poorly defined. The orphan G protein-coupled receptor 30 (GPR30) mediates the nongenomic signaling of 17beta-estradiol (E2) in a variety of estrogen-sensitive cancer cells through activation of the epidermal growth factor receptor (EGFR) pathway. Whether estrogen receptor alpha (ERalpha) also contributes to GPR30/EGFR signaling is less understood. Here, we show that, in ERalpha-positive BG-1 ovarian cancer cells, both E2 and the GPR30-selective ligand G-1 induced c-fos expression and estrogen-responsive element (ERE)-independent activity of a c-fos reporter gene, whereas only E2 stimulated an ERE-responsive reporter gene, indicating that GPR30 signaling does not activate ERalpha-mediated transcription. Similarly, both ligands up-regulated cyclin D1, cyclin E, and cyclin A, whereas only E2 enhanced progesterone receptor expression. Moreover, both GPR30 and ERalpha expression are required for c-fos stimulation and extracellular signal-regulated kinase (ERK) activation in response to either E2 or G-1. Inhibition of the EGFR transduction pathway inhibited c-fos stimulation and ERK activation by either ligand, suggesting that in ovarian cancer cells GPR30/EGFR signaling relays on ERalpha expression. Interestingly, we show that both GPR30 and ERalpha expression along with active EGFR signaling are required for E2-stimulated and G-1-stimulated proliferation of ovarian cancer cells. Because G-1 was able to induce both c-fos expression and proliferation in the ERalpha-negative/GPR30-positive SKBR3 breast cancer cells, the requirement for ERalpha expression in GPR30/EGFR signaling may depend on the specific cellular context of different tumor types.

GPR30: a seven-transmembrane-spanning estrogen receptor that triggers EGF release.

PubMed

Filardo, Edward J; Thomas, Peter

2005-10-01

Heterotrimeric G proteins and seven-transmembrane-spanning (7TM) receptors are implicated in rapid estrogen signaling. The orphan 7TM receptor GPR30 is linked to estrogen-mediated activation of adenylyl cyclase, release of epidermal growth factor (EGF)-related ligands, and specific estrogen binding. GPR30 acts independently of estrogen receptors, ERalpha and ERbeta, and probably functions as a heptahelical ER. 7TM receptors elicit signals that stimulate second messengers, and convey intracellular signals via EGF receptors. Identification of GPR30 as a Gs-coupled 7TM receptor that triggers release of heparin-binding EGF establishes its role in cell signaling cascades initiated by estrogens, and explains their capacity to activate second messengers and promote EGF-like effects. Thus, estrogen can signal by the same mechanism as various other hormones, through a specific 7TM receptor.

p75 Neurotrophin Receptor Signaling Activates Sterol Regulatory Element-binding Protein-2 in Hepatocyte Cells via p38 Mitogen-activated Protein Kinase and Caspase-3.

PubMed

Pham, Dan Duc; Do, Hai Thi; Bruelle, Céline; Kukkonen, Jyrki P; Eriksson, Ove; Mogollón, Isabel; Korhonen, Laura T; Arumäe, Urmas; Lindholm, Dan

2016-05-13

Nerve growth factor (NGF) influences the survival and differentiation of a specific population of neurons during development, but its role in non-neuronal cells has been less studied. We observed here that NGF and its pro-form, pro-NGF, are elevated in fatty livers from leptin-deficient mice compared with controls, concomitant with an increase in low density lipoprotein receptors (LDLRs). Stimulation of mouse primary hepatocytes with NGF or pro-NGF increased LDLR expression through the p75 neurotrophin receptor (p75NTR). Studies using Huh7 human hepatocyte cells showed that the neurotrophins activate the sterol regulatory element-binding protein-2 (SREBP2) that regulates genes involved in lipid metabolism. The mechanisms for this were related to stimulation of p38 mitogen-activated protein kinase (p38 MAPK) and activation of caspase-3 and SREBP2 cleavage following NGF and pro-NGF stimulations. Cell fractionation experiments showed that caspase-3 activity was increased particularly in the membrane fraction that harbors SREBP2 and caspase-2. Experiments showed further that caspase-2 interacts with pro-caspase-3 and that p38 MAPK reduced this interaction and caused caspase-3 activation. Because of the increased caspase-3 activity, the cells did not undergo cell death following p75NTR stimulation, possibly due to concomitant activation of nuclear factor-κB (NF-κB) pathway by the neurotrophins. These results identify a novel signaling pathway triggered by ligand-activated p75NTR that via p38 MAPK and caspase-3 mediate the activation of SREBP2. This pathway may regulate LDLRs and lipid uptake particularly after injury or during tissue inflammation accompanied by an increased production of growth factors, including NGF and pro-NGF. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Exposure of T lymphocytes to leflunomide but not to dexamethasone favors the production by monocytic cells of interleukin-1 receptor antagonist and the tissue-inhibitor of metalloproteinases-1 over that of interleukin-1beta and metalloproteinases.

PubMed

Déage, V; Burger, D; Dayer, J M

1998-12-01

On direct cell-cell contact, stimulated T lymphocytes potently trigger the production of pro-inflammatory factors such as interleukin-1beta (IL-1beta) and matrix metalloproteinases (MMP-1 and MMP-9), as well as anti-inflammatory factors such as IL-1 receptor antagonist (IL-1Ra) and the tissue inhibitor of metalloproteinases (TIMP-1) in peripheral blood monocytes and the monocytic cell line THP-1. Such mechanisms might play an important part in many inflammatory diseases where tissue destruction occurs. To assess whether anti-inflammatory agents such as dexamethasone (DEX) and leflunomide (LF) would affect contact-activation of monocytic cells, T lymphocytes were stimulated by PMA and PHA in the presence or absence of increasing concentrations of drug. LF and DEX (10- 4 M) inhibited the ability of stimulated T lymphocytes to activate monocytic cells by 66-97% and 43-70%, respectively, depending on the readout product. Upon contact with T lymphocytes stimulated in the presence of 10- 5 M LF, the molar ratio of IL-1Ra/IL-1beta and TIMP-1/MMP-1 produced by THP-1 cells was enhanced 3.6- and 1.9-fold, respectively, whereas it was enhanced only 1.3- and 1.4-fold upon contact with T lymphocytes stimulated in the presence of 10- 4 M DEX. Therefore, LF tends to favor the inhibition of pro-inflammatory and matrix-destructive factors over that of anti-inflammatory factors and metalloproteinase inhibitors, thus interfering with both inflammation and tissue destruction. These experiments indicate that LF and DEX have the potential to affect the capacity of stimulated T lymphocytes to activate, on direct cell-cell contact, monocytic cells. Furthermore, flow cytometric analysis revealed that surface molecules of T lymphocytes that were partially involved in contact-signaling of monocytes (i.e., CD69 and CD11) were not modulated by either LF or DEX, suggesting that factors which remain to be identified were mainly involved in the activation of monocytes on direct cell-cell contact.

Neuropeptide-stimulated cell migration in prostate cancer cells is mediated by RhoA kinase signaling and inhibited by neutral endopeptidase.

PubMed

Zheng, R; Iwase, A; Shen, R; Goodman, O B; Sugimoto, N; Takuwa, Y; Lerner, D J; Nanus, D M

2006-09-28

The neuropeptides bombesin and endothelin-1 stimulate prostate cancer (PC) cell migration and invasion (J Clin Invest, 2000; 106: 1399-1407). The intracellular signaling pathways that direct this cell movement are not well delineated. The monomeric GTPase RhoA is required for migration in several cell types including neutrophils, monocytes and fibroblasts. We demonstrate that bombesin-stimulated PC cell migration occurs via the heterotrimeric G-protein-coupled receptors (G-protein) G alpha 13 subunit leading to activation of RhoA, and Rho-associated coiled-coil forming protein kinase (ROCK). Using siRNA to suppress expression of the three known G-protein alpha-subunit-associated RhoA guanine nucleotide exchange factors (GEFs), we also show that two of these RhoA GEFs, PDZ-RhoGEF and leukemia-associated RhoGEF (LARG), link bombesin receptors to RhoA in a non-redundant manner in PC cells. We next show that focal adhesion kinase, which activates PDZ-RhoGEF and LARG, is required for bombesin-stimulated RhoA activation. Neutral endopeptidase (NEP) is expressed on normal prostate epithelium whereas loss of NEP expression contributes to PC progression. We also demonstrate that NEP inhibits neuropeptide activation of RhoA. Together, these results establish a contiguous signaling pathway from the bombesin receptor to ROCK in PC cells, and they implicate NEP as a major regulator of neuropeptide-stimulated RhoA in these cells. This work also identifies members of this signaling pathway as potential targets for rational pharmacologic manipulation of neuropeptide-stimulated migration of PC cells.

Hyperactivity and reactivity of peripheral blood neutrophils in chronic periodontitis.

PubMed

Matthews, J B; Wright, H J; Roberts, A; Cooper, P R; Chapple, I L C

2007-02-01

Some evidence exists that peripheral neutrophils from patients with chronic periodontitis generate higher levels of reactive oxygen species (ROS) after Fcgamma-receptor stimulation than those from healthy controls. We hypothesized that peripheral neutrophils in periodontitis also show both hyper-reactivity to plaque organisms and hyperactivity in terms of baseline, unstimulated generation and release of ROS. Peripheral neutrophils from chronic periodontitis patients and age/sex/smoking-matched healthy controls (18 pairs) were assayed for total ROS generation and extracellular ROS release, with and without stimulation (Fcgamma-receptor and Fusobacterium nucleatum), using luminol and isoluminol chemiluminescence. Assays were performed with and without priming with Escherichia coli lipopolysaccharide (LPS) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Phox gene expression (p22, p47, p67, gp91) was investigated using reverse transcription-polymerase chain reaction (RT-PCR). Neutrophils from patients produced higher mean levels of ROS in all assays. Total generation and extracellular release of ROS by patients' cells were significantly greater than those from controls after FcgammaR-stimulation, with (P = 0.023) and without (P < or = 0.023) priming with GM-CSF. Differences in unstimulated total ROS generation were not significant. By contrast, patients' cells demonstrated greater baseline, extracellular ROS release than those from controls (P = 0.004). This difference was maintained after priming with LPS (P = 0.028) but not GM-CSF (P = 0.217). Phox gene expression was similar in patient and control cells at baseline and stimulation with F. nucleatum (3 h) consistently reduced gp91(PHOX) transcripts. Our data demonstrate that peripheral neutrophils from periodontitis patients exhibit hyper-reactivity following stimulation (Fcgamma-receptor and F. nucleatum) and hyperactivity in terms of excess ROS release in the absence of exogenous stimulation. This hyperactive/-reactive neutrophil phenotype is not associated with elevated phox gene expression.

Mechanism of interleukin-13 production by granulocyte-macrophage colony-stimulating factor-dependent macrophages via protease-activated receptor-2.

PubMed

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

2015-06-01

Granulocyte-macrophage colony-stimulating factor (GM-CSF) promotes classically activated M1 macrophages. GM-CSF upregulates protease-activated receptor-2 (PAR-2) protein expression and activation of PAR-2 by human neutrophil elastase (HNE) regulates cytokine production. This study investigated the mechanism of PAR-2-mediated interleukin (IL)-13 production by GM-CSF-dependent macrophages stimulated with HNE. Adherent macrophages were obtained from primary cultures of human mononuclear cells. After stimulation with HNE to activate the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway, IL-13 mRNA and protein levels were assessed by the reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. PAR-2 protein was detected in GM-CSF-dependent macrophages by Western blotting. Unexpectedly, PD98059 (an ERK1 inhibitor) increased IL-13 production, even at higher concentrations. Interestingly, U0126 (an ERK1/2 inhibitor) reduced IL-13 production in a concentration-dependent manner. Neither SB203580 (a p38alpha/p38beta inhibitor) nor BIRB796 (a p38gamma/p38delta inhibitor) affected IL-13 production, while TMB-8 (a calcium chelator) diminished IL-13 production. Stimulation with HNE promoted the production of IL-13 (a Th2 cytokine) by GM-CSF-dependent M1 macrophages. PAR-2-mediated IL-13 production may be dependent on the Ca(2+)/ERK2 signaling pathway. Copyright © 2015 Elsevier Inc. All rights reserved.

The therapeutic potential of erythropoiesis-stimulating agents for tissue protection: a tale of two receptors.

PubMed

Brines, Michael

2010-01-01

Erythropoietin (EPO) is a well-known therapeutic protein employed widely in the treatment of anemia. Over the past decade, abundant evidence has shown that in addition to its systemic role in the regulation of plasma pO(2) by modulating erythrocyte numbers, EPO is also a cytoprotective molecule made locally in response to injury or metabolic stress. Many studies have shown beneficial effects of EPO administration in reducing damage caused by ischemia-reperfusion, trauma, cytotoxicity, infection and inflammation in a variety of organs and tissues. Notably, the receptor mediating the nonerythropoietic effects of EPO differs from the one responsible for hematopoiesis. The tissue-protective receptor exhibits a lower affinity for EPO and is a heteromer consisting of EPO receptor monomers in association with the common receptor that is also employed by granulocyte macrophage colony-stimulating factor, interleukin 3, and interleukin 5. This heteromeric receptor is expressed immediately following injury, whereas EPO production is delayed. Thus, early administration of EPO can dramatically reduce the deleterious components of the local inflammatory cascade. However, a high dose of EPO is required and this also stimulates the bone marrow to produce highly reactive platelets and activates the vascular endothelium into a prothrombotic state. To circumvent these undesirable effects, the EPO molecule has been successfully altered to selectively eliminate erythropoietic and prothrombotic potencies, while preserving tissue-protective activities. Very recently, small peptide mimetics have been developed that recapitulate the tissue-protective activities of EPO. Nonerythropoietic tissue-protective molecules hold high promise in a wide variety of acute and chronic diseases. Copyright (c) 2010 S. Karger AG, Basel.

Purinergic receptor ligands stimulate pro-opiomelanocortin gene expression in AtT-20 pituitary corticotroph cells.

PubMed

Zhao, L-F; Iwasaki, Y; Oki, Y; Tsugita, M; Taguchi, T; Nishiyama, M; Takao, T; Kambayashi, M; Hashimoto, K

2006-04-01

Although recent studies have suggested that purinergic receptors are expressed in the anterior pituitary gland, their involvement in the regulation of pituitary hormone gene expression is not completely understood. In the present study, we examined the expression of purinergic receptors and the effects of purinergic receptor ligands on pro-opiomelanocortin (POMC) gene expression, in AtT20 mouse corticotroph cells. We identified the expression of most of the purinergic receptor subtypes (A1, A2, P2X1, 3-7, P2Y1, 2, 4) mRNAs, analysed by the reverse transcriptase-polymerase chain reaction. We also found that adenosine and ATP, two representative and endogenous agonists of A1-3 and P2X/P2Y receptors, respectively, stimulated the 5'-promoter activity of the POMC gene in a dose- and time-related manner. When these ligands were simultaneously used with corticotrophin-releasing hormone (CRH), effects that were more than additive were observed, suggesting an enhancing role of these compounds in CRH-mediated adrenocorticotrophic hormone (ACTH) synthesis. These ligands also stimulated the expression of transcription factors involved in the regulation of the POMC gene, but did not enhance ACTH secretion. Finally, the positive effect of adenosine as well as CRH was completely inhibited by the protein kinase A inhibitor H89, whereas that of ATP was not influenced, indicating that different intracellular signalling pathways mediate these effects. Altogether, our results suggest a stimulatory role for these purinergic receptor ligands in the regulation of POMC gene expression in corticotroph cells. Because adenosine and ATP are known to be produced within the pituitary gland, it is possible they may be acting in an autocrine/paracrine fashion.

ErbB2 and EGFR are downmodulated during the differentiation of 3T3-L1 preadipocytes.

PubMed

Pagano, Eleonora; Calvo, Juan Carlos

2003-10-15

The expression of receptors belonging to the epidermal growth factor receptor subfamily has been largely studied these last years in epithelial cells mainly as involved in cell proliferation and malignant progression. Although much work has focused on the role of these growth factor receptors in the differentiation of a variety of tissues, there is little information in regards to normal stromal cells. We investigated erbB2 expression in the murine fibroblast cell line Swiss 3T3L1, which naturally or hormonally induced undergoes adipocyte differentiation. We found that the Swiss 3T3-L1 fibroblasts express erbB2, in addition to EGFR, and in a quantity comparable to or even greater than the breast cancer cell line T47D. Proliferating cells increased erbB2 and EGFR levels when reaching confluence up to 4- and 10-fold, respectively. This expression showed a significant decrease when growth-arrested cells were stimulated to differentiate with dexamethasone and isobutyl-methylxanthine. Differentiated cells presented a decreased expression of both erbB2 and EGFR regardless of whether the cells were hormonally or spontaneously differentiated. EGF stimulation of serum-starved cells increased erbB2 tyrosine phosphorylation and retarded erbB2 migration in SDS-PAGE, suggesting receptor association and activation. Heregulin-alpha1 and -beta1, two EGF related factors, had no effect on erbB2 or EGFR phosphorylation. Although 3T3-L1 cells expressed heregulin, its specific receptors, erbB3 and erbB4, were not found. This is the first time in which erbB2 is reported to be expressed in an adipocytic cell line which does not depend on non EGF family growth factors (thyroid hormone, growth hormone, etc.) to accomplish adipose differentiation. Since erbB2 and EGFR expression were downmodulated as differentiation progressed it is conceivable that a mechanism of switching from a mitogenic to a differentiating signaling pathway may be involved, through regulation of the expression of these growth factor receptors. Copyright 2003 Wiley-Liss, Inc.

A Pdx-1-Regulated Soluble Factor Activates Rat and Human Islet Cell Proliferation

PubMed Central

Hayes, Heather L.; Zhang, Lu; Becker, Thomas C.; Haldeman, Jonathan M.; Stephens, Samuel B.; Arlotto, Michelle; Moss, Larry G.; Newgard, Christopher B.

2016-01-01

The homeodomain transcription factor Pdx-1 has important roles in pancreas and islet development as well as in β-cell function and survival. We previously reported that Pdx-1 overexpression stimulates islet cell proliferation, but the mechanism remains unclear. Here, we demonstrate that overexpression of Pdx-1 triggers proliferation largely by a non-cell-autonomous mechanism mediated by soluble factors. Consistent with this idea, overexpression of Pdx-1 under the control of a β-cell-specific promoter (rat insulin promoter [RIP]) stimulates proliferation of both α and β cells, and overexpression of Pdx-1 in islets separated by a Transwell membrane from islets lacking Pdx-1 overexpression activates proliferation in the untreated islets. Microarray and gene ontology (GO) analysis identified inhibin beta-B (Inhbb), an activin subunit and member of the transforming growth factor β (TGF-β) superfamily, as a Pdx-1-responsive gene. Overexpression of Inhbb or addition of activin B stimulates rat islet cell and β-cell proliferation, and the activin receptors RIIA and RIIB are required for the full proliferative effects of Pdx-1 in rat islets. In human islets, Inhbb overexpression stimulates total islet cell proliferation and potentiates Pdx-1-stimulated proliferation of total islet cells and β cells. In sum, this study identifies a mechanism by which Pdx-1 induces a soluble factor that is sufficient to stimulate both rat and human islet cell proliferation. PMID:27620967

Measurement of Epidermal Growth Factor Receptor-Derived Signals Within Plasma Membrane Clathrin Structures.

PubMed

Lucarelli, Stefanie; Delos Santos, Ralph Christian; Antonescu, Costin N

2017-01-01

The epidermal growth factor (EGF) receptor (EGFR) is an important regulator of cell growth, proliferation, survival, migration, and metabolism. EGF binding to EGFR triggers the activation of the receptor's intrinsic kinase activity, in turn eliciting the recruitment of many secondary signaling proteins and activation of downstream signals, such as the activation of phosphatidylinositol-3-kinase (PI3K) and Akt, a process requiring the phosphorylation of Gab1. While the identity of many signals that can be activated by EGFR has been revealed, how the spatiotemporal organization of EGFR signaling within cells controls receptor outcome remains poorly understood. Upon EGF binding at the plasma membrane, EGFR is internalized by clathrin-mediated endocytosis following recruitment to clathrin-coated pits (CCPs). Further, plasma membrane CCPs, but not EGFR internalization, are required for EGF-stimulated Akt phosphorylation. Signaling intermediates such as phosphorylated Gab1, which lead to Akt phosphorylation, are enriched within CCPs upon EGF stimulation. These findings indicate that some plasma membrane CCPs also serve as signaling microdomains required for certain facets of EGFR signaling and are enriched in key EGFR signaling intermediates. Understanding how the spatiotemporal organization of EGFR signals within CCP microdomains controls receptor signaling outcome requires imaging methods that can systematically resolve and analyze the properties of CCPs, EGFR and key signaling intermediates. Here, we describe methods using total internal reflection fluorescence microscopy imaging and analysis to systematically study the enrichment of EGFR and key EGFR-derived signals within CCPs.

Essential roles of angiotensin II in vascular endothelial growth factor expression in sleep apnea syndrome.

PubMed

Takahashi, Susumu; Nakamura, Yutaka; Nishijima, Tsuguo; Sakurai, Shigeru; Inoue, Hiroshi

2005-09-01

Hypoxia-induced endothelial cell dysfunction has been implicated in increased cardiovascular disease associated with obstructive sleep apnea syndrome (OSAS). OSAS mediates hypertension by stimulating angiotensin II (Ang II) production. Hypoxia and Ang II are the major stimuli of vascular endothelial growth factor (VEGF), which is a potent angiogenic cytokine and also contributes to the atherogenic process itself. We observed serum Ang II and VEGF levels and peripheral blood mononuclear cell (PBMC) and neutrophil VEGF expression. Compared to controls, subjects with OSAS had significantly increased levels of serum Ang II and VEGF and VEGF mRNA expression in their leukocytes. To examine whether Ang II stimulates VEGF expression in OSAS, we treated PBMCs obtained from control subjects with Ang II and with an Ang II receptor type 1 (AT(1)) blocker, olmesartan. We observed an increased expression of VEGF in the Ang II-stimulated PBMCs and decreased in VEGF mRNA and protein expression in the PBMCs treated with olmesartan. These findings suggest that the Ang II-AT(1) receptors pathway potentially are involved in OSAS and VEGF-induced vascularity and that endothelial dysfunction might be linked to this change in Ang II activity within leukocytes of OSAS patients.

Molecular cloning of rock bream (Oplegnathus fasciatus) tumor necrosis factor-alpha and its effect on the respiratory burst activity of phagocytes.

PubMed

Kim, Min Sun; Hwang, Yoon Jung; Yoon, Ki Joon; Zenke, Kosuke; Nam, Yoon Kwon; Kim, Sung Koo; Kim, Ki Hong

2009-11-01

Rock bream (Oplegnathus fasciatus) tumor necrosis factor-alpha (rbTNF-alpha) gene was cloned, recombinantly produced, and the effect of the recombinant rbTNF-alpha on the respiratory burst activity of rock bream phagocytes was analyzed. Structurally, genomic DNA of rbTNF-alpha was comprised with four exons and three introns, and deduced amino acid sequence of its cDNA possessed the TNF family signature, a transmembrane domain, a protease cleavage site, and two cysteine residues, which are the typical characteristics of TNF-alpha gene in mammals and fish. The chemiluminescent (CL) response of rock bream phagocytes was significantly enhanced by pre-incubation with recombinant rbTNF-alpha, when opsonized zymosan was used as a stimulant of the respiratory burst. However, CL enhancing effect of the recombinant rbTNF-alpha was very weak when the respiratory burst activity of phagocytes was triggered with phorbol-12-myristate-13-acetate (PMA) instead of zymosan. These results suggest that rock bream TNF-alpha might have an ability to prime the respiratory burst activity of phagocytes against receptor-mediated phagocytosis inducing stimulants, such as zymosan, but have little ability against stimulants not accompanying receptor-mediated phagocytosis.

The Antimicrobial Peptide Lysozyme Is Induced after Multiple Trauma

PubMed Central

Klüter, Tim; Fitschen-Oestern, Stefanie; Lippross, Sebastian; Weuster, Matthias; Pufe, Thomas; Tohidnezhad, Mersedeh; Beyer, Andreas; Seekamp, Andreas; Varoga, Deike

2014-01-01

The antimicrobial peptide lysozyme is an important factor of innate immunity and exerts high potential of antibacterial activity. In the present study we evaluated the lysozyme expression in serum of multiple injured patients and subsequently analyzed their possible sources and signaling pathways. Expression of lysozyme was examined in blood samples of multiple trauma patients from the day of trauma until 14 days after trauma by ELISA. To investigate major sources of lysozyme, its expression and regulation in serum samples, different blood cells, and tissue samples were analysed by ELISA and real-time PCR. Neutrophils and hepatocytes were stimulated with cytokines and supernatant of Staphylococcus aureus. The present study demonstrates the induction and release of lysozyme in serum of multiple injured patients. The highest lysozyme expression of all tested cells and tissues was detected in neutrophils. Stimulation with trauma-related factors such as interleukin-6 and S. aureus induced lysozyme expression. Liver tissue samples of patients without trauma show little lysozyme expression compared to neutrophils. After stimulation with bacterial fragments, lysozyme expression of hepatocytes is upregulated significantly. Toll-like receptor 2, a classic receptor of Gram-positive bacterial protein, was detected as a possible target for lysozyme induction. PMID:25258475

Interdependence of Platelet-Derived Growth Factor and Estrogen-Signaling Pathways in Inducing Neonatal Rat Testicular Gonocytes Proliferation1

PubMed Central

Thuillier, Raphael; Mazer, Monty; Manku, Gurpreet; Boisvert, Annie; Wang, Yan; Culty, Martine

2010-01-01

We previously found that platelet-derived growth factor (PDGF) and 17beta-estradiol stimulate gonocyte proliferation in a dose-dependent, nonadditive manner. In the present study, we report that gonocytes express RAF1, MAP2K1, and MAPK1/3. Inhibition of RAF1 and MAP2K1/2, but not phosphoinositide-3-kinase, blocked PDGF-induced proliferation. AG-370, an inhibitor of PDGF receptor kinase activity, suppressed not only PDGF-induced proliferation but also that induced by 17beta-estradiol. In addition, RAF1 and MAP2K1/2 inhibitors blocked 17beta-estradiol-activated proliferation. The estrogen receptor antagonist ICI 182780 inhibited both the effects of 17beta-estradiol and PDGF. PDGF lost its stimulatory effect when steroid-depleted serum or no serum was used. Similarly, 17beta-estradiol did not induce gonocyte proliferation in the absence of PDGF. The xenoestrogens genistein, bisphenol A, and DES, but not coumestrol, stimulated gonocyte proliferation in a dose-dependent and PDGF-dependent manner similarly to 17beta-estradiol. Their effects were blocked by ICI 182780, suggesting that they act via the estrogen receptor. AG-370 blocked genistein and bisphenol A effects, demonstrating their requirement of PDGF receptor activation in a manner similar to 17beta-estradiol. These results demonstrate the interdependence of PDGF and estrogen pathways in stimulating in vitro gonocyte proliferation, suggesting that this critical step in gonocyte development might be regulated in vivo by the coordinated action of PDGF and estrogen. Thus, the inappropriate exposure of gonocytes to xenoestrogens might disrupt the crosstalk between the two pathways and potentially interfere with gonocyte development. PMID:20089883

Combined inhibition of EMMPRIN and epidermal growth factor receptor prevents the growth and migration of head and neck squamous cell carcinoma cells.

PubMed

Suzuki, Shinsuke; Ishikawa, Kazuo

2014-03-01

It has been reported that the epidermal growth factor receptor (EGFR) expression is associated with the extracellular matrix metalloproteinase inducer (EMMPRIN) in some solid tumors; however, the relationship of EMMPRIN with EGFR in head and neck cancers is not fully understood. To determine the relationship between EMMPRIN and EGFR in head and neck squamous cell carcinoma (HNSCC), HNSCC cells were stimulated with epidermal growth factor (EGF), a ligand of EGFR. EMMPRIN expression in HNSCC cells was upregulated by EGF. In addition, EGF stimulation induced HNSCC cell invasion and MMP-9 expression. This increase in invasion and MMP-9 expression was abrogated by downmodulation of EMMPRIN. Furthermore, to determine the effects of combined EMMPRIN and EGFR targeting in HNSCC, HNSCC cells were treated with an EMMPRIN function-blocking antibody and the EGFR inhibitor AG1478. This combined treatment resulted in greater inhibition of HNSCC cell proliferation and migration compared with the individual agents alone. These results suggest that EMMPRIN mediates EGFR-induced tumorigenicity and that combined targeting of EMMPRIN and EGFR may be an efficacious treatment approach.

Biosynthesis and intracellular transport of the receptor for platelet-derived growth factor.

PubMed Central

Claesson-Welsh, L; Rönnstrand, L; Heldin, C H

1987-01-01

The biosynthesis of the receptor for platelet-derived growth factor (PDGF) was examined in metabolically labeled human foreskin fibroblasts. The receptor was synthesized as a 145-kDa precursor, which, when incubated with endo-beta-N-acetylglucosaminidase H (endo H), underwent a 15-kDa decrease in molecular mass. This indicates that the size of the core protein is about 130 kDa and that the 145-kDa form represents a receptor precursor carrying high-mannose N-linked oligosaccharide groups. Within 15 min after synthesis, the receptor was converted to a 165-kDa form. This form was entirely resistant to endo H treatment and probably represents a receptor molecule that has undergone further posttranslational modification, including O-linked glycosylation. Subsequently, within 30 min, a molecule of 170 kDa--i.e., the size of the mature receptor--appeared. A slightly larger molecule, of 175 kDa, which could be immunoprecipitated from PDGF-stimulated 32P-labeled cells, probably represents a receptor further modified by autophosphorylation. The 170-kDa molecule had an isoelectric point of about 4.5. Addition of PDGF increased the turnover rate of the 170-kDa PDGF receptor. Images PMID:2827155

Ligand-independent activation of the oestrogen receptor by mutation of a conserved tyrosine.

PubMed Central

White, R; Sjöberg, M; Kalkhoven, E; Parker, M G

1997-01-01

The oestrogen receptor is a member of the nuclear receptor family of transcription factors which, on binding the steroid hormone 17beta-oestradiol, interacts with co-activator proteins and stimulates gene expression. Replacement of a single tyrosine in the hormone-binding domain generated activated forms of the receptor which stimulated transcription in the absence of hormone. This increased activation is related to a decrease in hydrophobicity and a reduction in size of the side chain of the amino acid with which the tyrosine is replaced. Ligand-independent, in common with ligand-dependent transcriptional activation, requires an amphipathic alpha-helix at the C-terminus of the ligand-binding domain which is essential for the interaction of the receptor with a number of potential co-activator proteins. In contrast to the wild-type protein, constitutively active receptors were able to bind both the receptor-interacting protein RIP-140 and the steroid receptor co-activator SRC-1 in a ligand-independent manner, although in the case of SRC-1 this was only evident when the receptors were prebound to DNA. We propose, therefore, that this tyrosine is required to maintain the receptor in a transcriptionally inactive state in the absence of hormone. Modification of this residue may generate a conformational change in the ligand-binding domain of the receptor to form an interacting surface which allows the recruitment of co-activators independent of hormone binding. This suggests that this tyrosine may be a target for a different signalling pathway which forms an alternative mechanism of activating oestrogen receptor-mediated transcription. PMID:9135157

DDB1 Stimulates Viral Transcription of Hepatitis B Virus via HBx-Independent Mechanisms.

PubMed

Kim, Woohyun; Lee, Sooyoung; Son, Yeongnam; Ko, Chunkyu; Ryu, Wang-Shick

2016-11-01

HBx, a small regulatory protein of hepatitis B virus (HBV), augments viral DNA replication by stimulating viral transcription. Among numerous reported HBx-binding proteins, DDB1 has drawn attention, because DDB1 acts as a substrate receptor of the Cul4-DDB1 ubiquitin E3 ligase. Previous work reported that the DDB1-HBx interaction is indispensable for HBx-stimulated viral DNA replication, suggesting that the Cul4-DDB1 ubiquitin E3 ligase might target cellular restriction factors for ubiquitination and proteasomal degradation. To gain further insight into the DDB1-HBx interaction, we generated HBx mutants deficient for DDB1 binding (i.e., R96A, L98A, and G99A) and examined whether they support HBx-stimulated viral DNA replication. In contrast to data from previous reports, our results showed that the HBx mutants deficient for DDB1 binding supported viral DNA replication to nearly wild-type levels, revealing that the DDB1-HBx interaction is largely dispensable for HBx-stimulated viral DNA replication. Instead, we found that DDB1 directly stimulates viral transcription regardless of HBx expression. Through an HBV infection study, importantly, we demonstrated that DDB1 stimulates viral transcription from covalently closed circular DNA, a physiological template for viral transcription. Overall, we concluded that DDB1 stimulates viral transcription via a mechanism that does not involve an interaction with HBx. DDB1 constitutes a cullin-based ubiquitin E3 ligase, where DDB1 serves as an adaptor linking the cullin scaffold to the substrate receptor. Previous findings that the DDB1-binding ability of HBx is essential for HBx-stimulated viral DNA replication led to the hypothesis that HBx could downregulate host restriction factors that limit HBV replication through the cullin ubiquitin E3 ligase that requires the DDB1-HBx interaction. Consistent with this hypothesis, recent work identified Smc5/6 as a host restriction factor that is regulated by the viral cullin ubiquitin E3 ligase. In contrast, here we found that the DDB1-HBx interaction is largely dispensable for HBx-stimulated viral DNA replication. Instead, our results clearly showed that DDB1, regardless of HBx expression, enhances viral transcription. Overall, besides its role in the viral cullin ubiquitin E3 ligase, DDB1 itself stimulates viral transcription via HBx-independent mechanisms. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Membrane and Integrative Nuclear Fibroblastic Growth Factor Receptor (FGFR) Regulation of FGF-23*

PubMed Central

Han, Xiaobin; Xiao, Zhousheng; Quarles, L. Darryl

2015-01-01

Fibroblastic growth factor receptor 1 (FGFR1) signaling pathways are implicated in the regulation of FGF-23 gene transcription, but the molecular pathways remain poorly defined. We used low molecular weight (LMW, 18 kDa) FGF-2 and high molecular weight (HMW) FGF-2 isoforms, which, respectively, activate cell surface FGF receptors and intranuclear FGFR1, to determine the roles of membrane FGFRs and integrative nuclear FGFR1 signaling (INFS) in the regulation of FGF-23 gene transcription in osteoblasts. We found that LMW-FGF-2 induced NFAT and Ets1 binding to conserved cis-elements in the proximal FGF-23 promoter and stimulated FGF-23 promoter activity through PLCγ/calcineurin/NFAT and MAPK pathways in SaOS-2 and MC3T3-E1 osteoblasts. In contrast, HMW-FGF-2 stimulated FGF-23 promoter activity in osteoblasts through a cAMP-dependent binding of FGFR1 and cAMP-response element-binding protein (CREB) to a conserved cAMP response element (CRE) contiguous with the NFAT binding site in the FGF-23 promoter. Mutagenesis of the NFAT and CRE binding sites, respectively, inhibited the effects of LMW-FGF-2 and HMW-FGF-23 to stimulate FGF-23 promoter activity. FGF-2 activation of both membrane FGFRs and INFS-dependent FGFR1 pathways may provide a means to integrate systemic and local regulation of FGF-23 transcription under diverse physiological and pathological conditions. PMID:25752607

Glucocorticoid- and Protein Kinase A–Dependent Transcriptome Regulation in Airway Smooth Muscle

PubMed Central

Misior, Anna M.; Deshpande, Deepak A.; Loza, Matthew J.; Pascual, Rodolfo M.; Hipp, Jason D.; Penn, Raymond B.

2009-01-01

Glucocorticoids (GCs) and protein kinase A (PKA)–activating agents (β-adrenergic receptor agonists) are mainstream asthma therapies based on their ability to prevent or reverse excessive airway smooth muscle (ASM) constriction. Their abilities to regulate another important feature of asthma—excessive ASM growth—are poorly understood. Recent studies have suggested that GCs render agents of inflammation such as IL-1β and TNF-α mitogenic to ASM, via suppression of (antimitogenic) induced cyclooxygenase-2–dependent PKA activity. To further explore the mechanistic basis of these observations, we assessed the effects of epidermal growth factor and IL-1β stimulation, and the modulatory effects of GC treatment and PKA inhibition, on the ASM transcriptome by microarray analysis. Results demonstrate that ASM stimulated with IL-1β, in a manner that is often cooperative with stimulation with epidermal growth factor, exhibit a profound capacity to function as immunomodulatory cells. Moreover, results implicate an important role for induced autocrine/paracrine factors (many whose regulation was minimally affected by GCs or PKA inhibition) as regulators of both airway inflammation and ASM growth. Induction of numerous chemokines, in conjunction with regulation of proteases and agents of extracellular matrix remodeling, is suggested as an important mechanism promoting upregulated G protein–coupled receptor signaling capable of stimulating ASM growth. Additional functional assays suggest that intracellular PKA plays a critical role in suppressing the promitogenic effects of induced autocrine factors in ASM. Finally, identification and comparison of GC- and PKA-sensitive genes in ASM provide insight into the complementary effects of β-agonist/GC combination therapies, and suggest specific genes as important targets for guiding the development of new generations of GCs and adjunct asthma therapies. PMID:19059887

Insight into Buffalo (Bubalus bubalis) RIG1 and MDA5 Receptors: A Comparative Study on dsRNA Recognition and In-Vitro Antiviral Response

PubMed Central

Singh, Manvender; Brahma, Biswajit; Maharana, Jitendra; Patra, Mahesh Chandra; Kumar, Sushil; Mishra, Purusottam; Saini, Megha; De, Bidhan Chandra; Mahanty, Sourav; Datta, Tirtha Kumar; De, Sachinandan

2014-01-01

RIG1 and MDA5 have emerged as important intracellular innate pattern recognition receptors that recognize viral RNA and mediate cellular signals controlling Type I interferon (IFN-I) response. Buffalo RIG1 and MDA5 genes were investigated to understand the mechanism of receptor induced antiviral response. Sequence analysis revealed that RIG1 and MDA5 maintain a domain arrangement that is common in mammals. Critical binding site residues of the receptors are evolutionary conserved among mammals. Molecular dynamics simulations suggested that RIG1 and MDA5 follow a similar, if not identical, dsRNA binding pattern that has been previously reported in human. Moreover, binding free energy calculation revealed that MDA5 had a greater affinity towards dsRNA compared to RIG1. Constitutive expressions of RLR genes were ubiquitous in different tissues without being specific to immune organs. Poly I:C stimulation induced elevated expressions of IFN-β and IFN-stimulated genes (ISGs) through interferon regulatory factors (IRFs) mediated pathway in buffalo foetal fibroblast cells. The present study provides crucial insights into the structure and function of RIG1 and MDA5 receptors in buffalo. PMID:24587036

Analgesic and Antineuropathic Drugs Acting Through Central Cholinergic Mechanisms

PubMed Central

Bartolini, Alessandro; Cesare Mannelli, Lorenzo Di; Ghelardini, Carla

2011-01-01

The role of muscarinic and nicotinic cholinergic receptors in analgesia and neuropathic pain relief is relatively unknown. This review describes how such drugs induce analgesia or alleviate neuropathic pain by acting on the central cholinergic system. Several pharmacological strategies are discussed which increase synthesis and release of acetylcholine (ACh) from cholinergic neurons. The effects of their acute and chronic administration are described. The pharmacological strategies which facilitate the physiological functions of the cholinergic system without altering the normal modulation of cholinergic signals are highlighted. It is proposed that full agonists of muscarinic or nicotinic receptors should be avoided. Their activation is too intense and un-physiological because neuronal signals are distorted when these receptors are constantly activated. Good results can be achieved by using agents that are able to a) increase ACh synthesis, b) partially inhibit cholinesterase activity c) selectively block the autoreceptor or heteroreceptor feedback mechanisms. Activation of M1 subtype muscarinic receptors induces analgesia. Chronic stimulation of nicotinic (N1) receptors has neuronal protective effects. Recent experimental results indicate a relationship between repeated cholinergic stimulation and neurotrophic activation of the glial derived neurotrophic factor (GDNF) family. At least 9 patents covering novel chemicals for cholinergic system modulation and pain control are discussed. PMID:21585331

MAP kinase-independent signaling in angiotensin II regulation of neuromodulation in SHR neurons.

PubMed

Yang, H; Raizada, M K

1998-09-01

Angiotensin II (Ang II), via its interaction with the angiotensin type 1 (AT1) receptor subtype, causes enhanced stimulation of norepinephrine (NE) neuromodulation. This involves increased transcription of NE transporter, tyrosine hydroxylase, and dopamine ss-hydroxylase genes in Wistar-Kyoto rat (WKY) brain neurons. AT1 receptor-mediated regulation of certain signaling events (such as activation of the Ras-Raf-1-mitogen activated protein (MAP) kinase signaling pathway, nuclear translocation of transcription factors such as Fos and Jun, and the interactions of these factors with AP-1 binding sites) is involved in this NE neuromodulation (Lu et al. J Cell Biol. 1996;135:1609-1617). The aim of this study was to compare the signal transduction mechanism of Ang II regulation of NE neuromodulation in WKY and spontaneously hypertensive rat (SHR) brain neurons, in view of the fact that AT1 receptor expression and Ang II stimulation of NE neuromodulation are higher in SHR neurons compared with WKY neurons. Despite this hyperactivity, Ang II stimulation of Ras, Raf-1, and MAP kinase activities was comparable between the neurons from WKY and SHR. Similarly, central injections of Ang II caused a comparable stimulation of MAP kinase in the hypothalamic and brain stem areas of adult WKY and SHR. Inhibition of MAP kinase by either an MAP kinase kinase inhibitor (PD98059) or an MAP kinase antisense oligonucleotide completely attenuated the stimulatory effects of Ang II on [3H]-NE uptake, NE transporter mRNA, and tyrosine hydroxylase mRNA levels in WKY neurons. These treatments resulted in only 43% to 50% inhibition of [3H]-NE uptake and NE transporter and tyrosine hydroxylase mRNAs in SHR neurons. Thus, Ang II stimulation of NE neuromodulation was completely blocked by MAP kinase inhibition in WKY neurons and only partially blocked in the SHR neurons. These observations suggest the presence of an additional signal transduction pathway involved in NE neuromodulation in SHR neurons that is independent of the MAP kinase pathway.

Enhancement of high glucose-induced PINK1 expression by melatonin stimulates neuronal cell survival: Involvement of MT2 /Akt/NF-κB pathway.

PubMed

Onphachanh, Xaykham; Lee, Hyun Jik; Lim, Jae Ryong; Jung, Young Hyun; Kim, Jun Sung; Chae, Chang Woo; Lee, Sei-Jung; Gabr, Amr Ahmed; Han, Ho Jae

2017-09-01

Hyperglycemia is a representative hallmark and risk factor for diabetes mellitus (DM) and is closely linked to DM-associated neuronal cell death. Previous investigators reported on a genome-wide association study and showed relationships between DM and melatonin receptor (MT), highlighting the role of MT signaling by assessing melatonin in DM. However, the role of MT signaling in DM pathogenesis is unclear. Therefore, we investigated the role of mitophagy regulators in high glucose-induced neuronal cell death and the effect of melatonin against high glucose-induced mitophagy regulators in neuronal cells. In our results, high glucose significantly increased PTEN-induced putative kinase 1 (PINK1) and LC-3B expressions; as well it decreased cytochrome c oxidase subunit 4 expression and Mitotracker™ fluorescence intensity. Silencing of PINK1 induced mitochondrial reactive oxygen species (ROS) accumulation and mitochondrial membrane potential impairment, increased expressions of cleaved caspases, and increased the number of annexin V-positive cells. In addition, high glucose-stimulated melatonin receptor 1B (MTNR1B) mRNA and PINK1 expressions were reversed by ROS scavenger N-acetyl cysteine pretreatment. Upregulation of PINK1 expression in neuronal cells is suppressed by pretreatment with MT 2 receptor-specific inhibitor 4-P-PDOT. We further showed melatonin stimulated Akt phosphorylation, which was followed by nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) phosphorylation and nuclear translocation. Silencing of PINK1 expression abolished melatonin-regulated mitochondrial ROS production, cleaved caspase-3 and caspase-9 expressions, and the number of annexin V-positive cells. In conclusion, we have demonstrated the melatonin stimulates PINK1 expression via an MT 2 /Akt/NF-κB pathway, and such stimulation is important for the prevention of neuronal cell apoptosis under high glucose conditions. © 2017 The Authors. Journal of Pineal Research Published by John Wiley & Sons Ltd.

Possible mechanism for preterm labor associated with bacterial infection. I. Stimulation of phosphoinositide metabolism by endotoxin in endometrial fibroblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khan, A.A.; Imai, A.; Tamaya, T.

Growing evidence suggests an association between intra-amniotic infection and premature initiation of parturition. We recently demonstrated that some factor(s) including endotoxin produced by the organism stimulates endogenous phospholipase A2 resulting in liberation of arachidonic acid and prostaglandin formation. The studies presented in this report were designated to evaluate the mechanism for endotoxin to stimulate phospholipase A2 using human endometrial fibroblasts. Exposure of the fibroblasts to endotoxin from Escherichia coli in the presence of ({sup 32}P) phosphate increased {sup 32}P-labeling of phosphatidic acid (PA) and phosphatidyl-inositol (PI) in a dose-dependent and a time-dependent manners. The PA labeling occurred without a measurablemore » lag time. These findings demonstrate that the endotoxin stimulates phosphoinositide metabolism in human endometrial fibroblasts by a receptor-mediated mechanism. Membrane phosphoinositide turnover stimulated by endotoxin results in cytosolic Ca{sup 2+} increment, liberation of arachidonic acid, which may be involved in the initiation of parturition.« less

Regulatory T cells protect mice against coxsackievirus-induced myocarditis through the transforming growth factor beta-coxsackie-adenovirus receptor pathway.

PubMed

Shi, Yu; Fukuoka, Masahiro; Li, Guohua; Liu, Youan; Chen, Manyin; Konviser, Michael; Chen, Xin; Opavsky, Mary Anne; Liu, Peter P

2010-06-22

Coxsackievirus B3 infection is an excellent model of human myocarditis and dilated cardiomyopathy. Cardiac injury is caused either by a direct cytopathic effect of the virus or through immune-mediated mechanisms. Regulatory T cells (Tregs) play an important role in the negative modulation of host immune responses and set the threshold of autoimmune activation. This study was designed to test the protective effects of Tregs and to determine the underlying mechanisms. Carboxyfluorescein diacetate succinimidyl ester-labeled Tregs or naïve CD4(+) T cells were injected intravenously once every 2 weeks 3 times into mice. The mice were then challenged with intraperitoneal coxsackievirus B3 immediately after the last cell transfer. Transfer of Tregs showed higher survival rates than transfer of CD4(+) T cells (P=0.0136) but not compared with the PBS injection group (P=0.0589). Interestingly, Tregs also significantly decreased virus titers and inflammatory scores in the heart. Transforming growth factor-beta and phosphorylated AKT were upregulated in Tregs-transferred mice and coxsackie-adenovirus receptor expression was decreased in the heart compared with control groups. Transforming growth factor-beta decreased coxsackie-adenovirus receptor expression and inhibited coxsackievirus B3 infection in HL-1 cells and neonatal cardiac myocytes. Splenocytes collected from Treg-, CD4(+) T-cell-, and PBS-treated mice proliferated equally when stimulated with heat-inactivated virus, whereas in the Treg group, the proliferation rate was reduced significantly when stimulated with noninfected heart tissue homogenate. Adoptive transfer of Tregs protected mice from coxsackievirus B3-induced myocarditis through the transforming growth factor beta-coxsackie-adenovirus receptor pathway and thus suppresses the immune response to cardiac tissue, maintaining the antiviral immune response.

Muscarinic cholinergic and alpha/sub 1/ adrenergic receptors in murine atria: phosphatidylinositol breakdown and receptor interaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Scherer, R.W.

Upon stimulation of muscarinic cholinergic receptors, there is a decrease in the force of contraction rate of firing in heart, while stimulation of ..cap alpha.. adrenergic receptors causes an increase in the force of contraction with no change in the heart rate. Yet both receptors stimulate the breakdown of phosphatidylinositol (PI). Therefore, the breakdown of PI was examined to determine how the process differed between the two receptor systems. Murine atria, prelabelled with (/sup 3/H)inositol, were stimulated with the muscarinic cholinergic agonists, carbamylcholine (CARB), and oxotremorine (OXO); and with the ..cap alpha.. adrenergic agonists, norepinephrine (NE) and phenylephrine (PE); eithermore » singly or in combination. Breakdown of PI was assessed by measurement of individual inositol phosphates by anion exchange chromatography. Binding of CARB to atrial muscarinic receptors was measured by competition with (/sup 3/H)quinuclidinyl benzilate.« less

Mutant protein of recombinant human granulocyte colony-stimulating factor for receptor binding assay.

PubMed

Watanabe, M; Fukamachi, H; Uzumaki, H; Kabaya, K; Tsumura, H; Ishikawa, M; Matsuki, S; Kusaka, M

1991-05-15

A new mutant protein of recombinant human granulocyte colony-stimulating factor (rhG-CSF) was produced for the studies on receptors for human G-CSF. The mutant protein [(Tyr1, Tyr3]rhG-CSF), the biological activity of which was almost equal to that of rhG-CSF, was prepared by the replacement of threonine-1 and leucine-3 of rhG-CSF with tyrosine. The radioiodinated preparation of the mutant protein showed high specific radioactivity and retained full biological activity for at least 3 weeks. The binding capacity of the radioiodinated ligand was compared with that of [35S]rhG-CSF. Both radiolabeled ligands showed specific binding to murine bone marrow cells. Unlabeled rhG-CSF and human G-CSF purified from the culture supernatant of the human bladder carcinoma cell line 5637 equally competed for the binding of labeled rhG-CSFs in a dose-dependent manner, demonstrating that the sugar moiety of human G-CSF made no contribution to the binding of human G-CSF to target cells. In contrast, all other colony-stimulating factors and lymphokines examined did not affect the binding. Scatchard analysis of the specific binding of both labeled ligands revealed a single class of binding site with an apparent dissociation constant (Kd) of 20-30 pM and 100-200 maximal binding sites per cell. These data indicate that the radioiodinated preparation of the mutant protein binds the same specific receptor with the same affinity as [35S]rhG-CSF. The labeled mutant protein also showed specific binding to human circulating neutrophils.(ABSTRACT TRUNCATED AT 250 WORDS)

Sigma receptor ligand N,N'-di-(ortho-tolyl)guanidine inhibits release of acetylcholine in the guinea pig ileum.

PubMed

Cambell, B G; Keana, J F; Weber, E

1991-11-26

The inhibition of stimulated contractions of the guinea pig ileum longitudinal muscle/myenteric plexus preparation by sigma receptor ligands has been previously described. In this study, the stimulated release of [3H]acetylcholine from cholinergic nerve terminals in this same preparation was monitored in the presence and absence of sigma receptor ligands. N,N'-Di-(orthotolyl)guanidine (DTG) and other compounds selective for the sigma receptor inhibited stimulated [3H]acetylcholine release. These results suggest that their inhibition of stimulated contractions in this preparation was mediated by inhibition of acetylcholine release.

The potential of IGF-1 and TGFbeta1 for promoting "adult" articular cartilage repair: an in vitro study.

PubMed

Davies, Lindsay C; Blain, Emma J; Gilbert, Sophie J; Caterson, Bruce; Duance, Victor C

2008-07-01

Research into articular cartilage repair, a tissue unable to spontaneously regenerate once injured, has focused on the generation of a biomechanically functional repair tissue with the characteristics of hyaline cartilage. This study was undertaken to provide insight into how to improve ex vivo chondrocyte amplification, without cellular dedifferentiation for cell-based methods of cartilage repair. We investigated the effects of insulin-like growth factor 1 (IGF-1) and transforming growth factor beta 1 (TGFbeta1) on cell proliferation and the de novo synthesis of sulfated glycosaminoglycans and collagen in chondrocytes isolated from skeletally mature bovine articular cartilage, whilst maintaining their chondrocytic phenotype. Here we demonstrate that mature differentiated chondrocytes respond to growth factor stimulation to promote de novo synthesis of matrix macromolecules. Additionally, chondrocytes stimulated with IGF-1 or TGFbeta1 induced receptor expression. We conclude that IGF-1 and TGFbeta1 in addition to autoregulatory effects have differential effects on each other when used in combination. This may be mediated by regulation of receptor expression or endogenous factors; these findings offer further options for improving strategies for repair of cartilage defects.

STC1 interference on calcitonin family of receptors signaling during osteoblastogenesis via adenylate cyclase inhibition.

PubMed

Terra, Silvia R; Cardoso, João Carlos R; Félix, Rute C; Martins, Leo Anderson M; Souza, Diogo Onofre G; Guma, Fatima C R; Canário, Adelino Vicente M; Schein, Vanessa

2015-03-05

Stanniocalcin 1 (STC1) and calcitonin gene-related peptide (CGRP) are involved in bone formation/remodeling. Here we investigate the effects of STC1 on functional heterodimer complex CALCRL/RAMP1, expression and activity during osteoblastogenesis. STC1 did not modify CALCRL and ramp1 gene expression during osteoblastogenesis when compared to controls. However, plasma membrane spatial distribution of CALCRL/RAMP1 was modified in 7-day pre-osteoblasts exposed to either CGRP or STC1, and both peptides induced CALCRL and RAMP1 assembly. CGRP, but not STC1 stimulated cAMP accumulation in 7-day osteoblasts and in CALCRL/RAMP1 transfected HEK293 cells. Furthermore, STC1 inhibited forskolin stimulated cAMP accumulation of HEK293 cells, but not in CALCRL/RAMP1 transfected HEK293 cells. However, STC1 inhibited cAMP accumulation in calcitonin receptor (CTR) HEK293 transfected cells stimulated by calcitonin. In conclusion, STC1 signals through inhibitory G-protein modulates CGRP receptor spatial localization during osteoblastogenesis and may function as a regulatory factor interacting with calcitonin peptide members during bone formation. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Antagonist interaction with the human 5-HT7 receptor mediates the rapid and potent inhibition of non-G-protein-stimulated adenylate cyclase activity: a novel GPCR effect

PubMed Central

Klein, MT; Teitler, M

2011-01-01

BACKGROUND AND PURPOSE The human 5-hydroxytryptamine7 (h5-HT7) receptor is Gs-coupled and stimulates the production of the intracellular signalling molecule cAMP. Previously, we reported a novel property of the h5-HT7 receptor: pseudo-irreversible antagonists irreversibly inhibit forskolin-stimulated (non-receptor-mediated) cAMP production. Herein, we sought to determine if competitive antagonists also affect forskolin-stimulated activity and if this effect is common among other Gs-coupled receptors. EXPERIMENTAL APPROACH Recombinant cell lines expressing h5-HT7 receptors or other receptors of interest were briefly exposed to antagonists; cAMP production was then stimulated by forskolin and quantified by an immunocompetitive assay. KEY RESULTS In human embryonic kidney 293 cells stably expressing h5-HT7 receptors, all competitive antagonists inhibited nearly 100% of forskolin-stimulated cAMP production. This effect was insensitive to pertussis toxin, that is, not Gi/o-mediated. Potency to inhibit forskolin-stimulated activity strongly correlated with h5-HT7 binding affinity (r2= 0.91), indicating that the antagonists acted through h5-HT7 receptors to inhibit forskolin. Potency and maximal effects of clozapine, a prototypical competitive h5-HT7 antagonist, were unaffected by varying forskolin concentration. Antagonist interaction with h5-HT6, human β1, β2, and β3 adrenoceptors did not inhibit forskolin's activity. CONCLUSIONS AND IMPLICATIONS The inhibition of adenylate cyclase, as measured by forskolin's activity, is an underlying property of antagonist interaction with h5-HT7 receptors; however, this is not a common property of other Gs-coupled receptors. This phenomenon may be involved in the roles played by h5-HT7 receptors in human physiology. Development of h5-HT7 antagonists that do not elicit this effect would aid in the elucidation of its mechanisms and shed light on its possible physiological relevance. PMID:21198551

Mechanisms of Disease: the first kiss-a crucial role for kisspeptin-1 and its receptor, G-protein-coupled receptor 54, in puberty and reproduction.

PubMed

Seminara, Stephanie B

2006-06-01

Although the hypothalamic secretion of gonadotropin-releasing hormone (GnRH) is the defining hormonal event of puberty, the physiologic mechanisms that drive secretion of GnRH at the time of sexual maturation have been difficult to identify. After puberty is initiated, the factors that modulate the frequency and amplitude of GnRH secretion in rapidly changing sex-steroid environments (i.e. the female menstrual cycle) also remain unknown. The discovery that, in both humans and mouse models, loss-of-function mutations in the gene that encodes G-protein-coupled receptor 54 result in phenotypes of hypogonadotropic hypogonadism with an absence of pubertal development has unearthed a novel pathway regulating GnRH secretion. Ligands for G-protein-coupled receptor 54 (KiSS-1R), including metastin (derived from the parent compound, kisspeptin-1) and metastin's C-terminal peptide fragments, have been shown to be powerful stimulants for GnRH release in vivo via their stimulation of G-protein-coupled receptor 54. This article reviews the discovery of the GPR54 gene, places it into the appropriate biological context, and explores the data from in vitro and in vivo studies that point to this ligand-receptor system as a major driver of GnRH secretion.

Growth stimulating antibody, as another predisposing factor of Graves' disease (GD): analysis using monoclonal TSH receptor antibodies derived from patients with GD.

PubMed

Ihara, Yoshiaki; Kanda, Yasunari; Seo, Marie; Watanabe, Yasuhiro; Akamizu, Takashi; Tanaka, Yuji

2012-01-01

TSH receptor antibody (TRAb) is clinically classified into thyroid stimulating antibody (TSAb) and thyroid-stimulation blocking antibody (TSBAb). Although the former is considered to cause Graves' disease (GD), its activity does not necessarily reflect hormone production and goiter size. Moreover, uptake of 99mTcO4(-), the best indicator for GD, is correlated with activity of TSH binding inhibitor immunoglobulin better than activity of TSAb. Because uptake of 99mTcO4(-) reflects thyroid volume, these observations suggest that there exist TRAb with thyrocyte growth stimulating activity (GSA) other than TSAb. In this study, we analyzed GSA of monoclonal TRAb established from patients with GD or idiopathic myxedema (IME). GSA was measured as the degree of FRTL-5 cell growth stimulated by each TRAb. The signaling pathways of the cell growth were pharmacologically analyzed. The cell growth stimulated by TSH was strongly suppressed by protein kinase A (PKA) inhibitor, but was not affected by extracellular signal regulated kinase kinase (MEK) inhibitor. Although TSAb from GD stimulated the cell growth, both inhibitors suppressed it. Surprisingly, the cell growth was also induced by TSBAb from GD and was only suppressed by MEK inhibitor. TSBAb from IME did not have GSA and attenuated the cell growth stimulated by TSH. We concluded that 1; in GD, not only TSAb but some TSBAb could stimulate thyrocyte growth. 2; TSBAb might be classified with respect to their effects on thyrocyte growth; i.e., thyrocyte growth stimulating antibody and thyrocyte growth-stimulation blocking antibody.

Anti-lipoteichoic acid antibodies enhance release of cytokines by monocytes sensitized with lipoteichoic acid.

PubMed Central

Mancuso, G; Tomasello, F; Ofek, I; Teti, G

1994-01-01

Lipoteichoic acid (LTA) from gram-positive bacteria can stimulate monocytes to produce cytokines. To ascertain whether aggregation of LTA receptors can contribute to this effect, human monocytes were sensitized with LTA from Streptococcus pyogenes, washed, and treated with anti-LTA antibodies. The addition of anti-LTA antibodies or F(ab')2 fragments markedly enhanced the aggregation of LTA receptors, as evidenced by indirect immunofluorescence and the release of tumor necrosis factor alpha and interleukin-1 beta. These findings suggest that aggregation of LTA receptors of monocytes is required for triggering marked cytokine responses. PMID:8132355

Comparisons of mRNA expression for insulin-like growth factor (IGF) type 2 receptor (IGF2R) and IGF-1 in small ovarian follicles between cattle selected and not selected for twin ovulations

USDA-ARS?s Scientific Manuscript database

Both IGF-1 and -2 stimulate ovarian follicular cell proliferation and antral follicle development. Actions of IGF-1 and -2 are mediated through the IGF type 1 receptor, whereas binding of IGF-2 to the IGF2R results in its degradation. Information on the role of IGF2R in regulating bovine follicula...

Minoxidil-induced hair growth is mediated by adenosine in cultured dermal papilla cells: possible involvement of sulfonylurea receptor 2B as a target of minoxidil.

PubMed

Li, M; Marubayashi, A; Nakaya, Y; Fukui, K; Arase, S

2001-12-01

The mechanism by which minoxidil, an adenosine-triphosphate-sensitive potassium channel opener, induces hypertrichosis remains to be elucidated. Minoxidil has been reported to stimulate the production of vascular endothelial growth factor, a possible promoter of hair growth, in cultured dermal papilla cells. The mechanism of production of vascular endothelial growth factor remains unclear, however. We hypothesize that adenosine serves as a mediator of vascular endothelial growth factor production. Minoxidil-induced increases in levels of intracellular Ca(2+) and vascular endothelial growth factor production in cultured dermal papilla cells were found to be inhibited by 8-sulfophenyl theophylline, a specific antagonist for adenosine receptors, suggesting that dermal papilla cells possess adenosine receptors and sulfonylurea receptors, the latter of which is a well-known target receptor for adenosine-triphosphate-sensitive potassium channel openers. The expression of sulfonylurea receptor 2B and of the adenosine A1, A2A, and A2B receptors was detected in dermal papilla cells by means of reverse transcription polymerase chain reaction analysis. In order to determine which of the adenosine receptor subtypes contribute to minoxidil-induced hair growth, the effects of subtype-specific antagonists for adenosine receptors were investigated. Significant inhibition in increase in intracellular calcium level by minoxidil or adenosine was observed as the result of pretreatment with 8-cyclopentyl-1,3-dipropylxanthine, an antagonist for adenosine A1 receptor, but not by 3,7-dimethyl-1-propargyl-xanthine, an antagonist for adenosine A2 receptor, whereas vascular endothelial growth factor production was blocked by both adenosine A1 and A2 receptor antagonists. These results indicate that the effect of minoxidil is mediated by adenosine, which triggers intracellular signal transduction via both adenosine A1 and A2 receptors, and that the expression of sulfonylurea receptor 2B in dermal papilla cells might play a role in the production of adenosine.

Identification and in vitro characterization of novel nanobodies against human granulocyte colony-stimulating factor receptor to provide inhibition of G-CSF function.

PubMed

Bakherad, Hamid; Gargari, Seyed Latif Mousavi; Sepehrizadeh, Zargham; Aghamollaei, Hossein; Taheri, Ramezan Ali; Torshabi, Maryam; Yazdi, Mojtaba Tabatabaei; Ebrahimizadeh, Walead; Setayesh, Neda

2017-09-01

It has been shown that Granulocyte colony-stimulating factor (G-CSF) has a higher expression in malignant tumors, and anti-G-CSF therapy considerably decreases tumor growth, tumor vascularization and metastasis. Thus, blocking the signaling pathway of G-CSF could be beneficial in cancer therapy. This study is aimed at designing and producing a monoclonal nanobody that could act as an antagonist of G-CSF receptor. Nanobodies are the antigen binding fragments of camelid single-chain antibodies, also known as VHH. These fragments have exceptional properties which makes them ideal for tumor imaging and therapeutic applications. We have used our previously built nanobody phage libraries to isolate specific nanobodies to the G-CSF receptor. After a series of cross-reactivity and affinity experiments, two unique nanobodies were selected for functional analysis. Proliferation assay, real-time PCR and immunofluorescence assays were used to characterize these nanobodies. Finally, VHH26 nanobody that was able to specifically bind G-CSF receptor (G-CSF-R) on the surface of NFS60 cells and efficiently block G-CSF-R downstream signaling pathway in a dose-dependent manner was selected. This nanobody could be further developed into a valuable tool in tumor therapy and it forms a basis for additional studies in preclinical animal models. Copyright © 2017. Published by Elsevier Masson SAS.

Reflex effects on components of synchronized renal sympathetic nerve activity.

PubMed

DiBona, G F; Jones, S Y

1998-09-01

The effects of peripheral thermal receptor stimulation (tail in hot water, n = 8, anesthetized) and cardiac baroreceptor stimulation (volume loading, n = 8, conscious) on components of synchronized renal sympathetic nerve activity (RSNA) were examined in rats. The peak height and peak frequency of synchronized RSNA were determined. The renal sympathoexcitatory response to peripheral thermal receptor stimulation was associated with an increase in the peak height. The renal sympathoinhibitory response to cardiac baroreceptor stimulation was associated with a decrease in the peak height. Although heart rate was significantly increased with peripheral thermal receptor stimulation and significantly decreased with cardiac baroreceptor stimulation, peak frequency was unchanged. As peak height reflects the number of active fibers, reflex increases and decreases in synchronized RSNA are mediated by parallel increases and decreases in the number of active renal nerve fibers rather than changes in the centrally based rhythm or peak frequency. The increase in the number of active renal nerve fibers produced by peripheral thermal receptor stimulation reflects the engagement of a unique group of silent renal sympathetic nerve fibers with a characteristic response pattern to stimulation of arterial baroreceptors, peripheral and central chemoreceptors, and peripheral thermal receptors.

Crk synergizes with epidermal growth factor for epithelial invasion and morphogenesis and is required for the met morphogenic program.

PubMed

Lamorte, Louie; Rodrigues, Sonia; Naujokas, Monica; Park, Morag

2002-10-04

Activation of the Met receptor tyrosine kinase through its ligand, hepatocyte growth factor, stimulates cell spreading, cell dispersal, and the inherent morphogenic program of various epithelial cell lines. Although both hepatocyte growth factor and epidermal growth factor (EGF) can activate downstream signaling pathways in Madin-Darby canine kidney epithelial cells, EGF fails to promote the breakdown of cell-cell junctional complexes and initiate an invasive morphogenic program. We have undertaken a strategy to identify signals that synergize with EGF in this process. We provide evidence that the overexpression of the CrkII adapter protein complements EGF-stimulated pathways to induce cell dispersal in two-dimensional cultures and cell invasion and branching morphogenesis in three-dimensional collagen gels. This finding correlates with the ability of CrkII to promote the breakdown of adherens junctions in stable cell lines and the ability of EGF to stimulate enhanced Rac activity in cells overexpressing CrkII. We have previously shown that the Gab1-docking protein is required for branching morphogenesis downstream of the Met receptor. Consistent with a role for CrkII in promoting EGF-dependent branching morphogenesis, the binding of Gab1 to CrkII is required for the branching morphogenic program downstream of Met. Together, our data support a role for the CrkII adapter protein in epithelial invasion and morphogenesis and underscores the importance of considering the synergistic actions of signaling pathways in cancer progression.

CSF-1R regulates non-small cell lung cancer cells dissemination through Wnt3a signaling.

PubMed

Yu, Yan Xia; Wu, Hai Jian; Tan, Bing Xu; Qiu, Chen; Liu, Hui Zhong

2017-01-01

Therapeutic antibodies targeting colony stimulating factor 1 receptor (CSF-1R) to block colony stimulating factor-1/colony stimulating factor 1 receptor (CSF-1/CSF-R) signaling axis have exhibit remarkable efficacy in the treatment of malignant tumor. Yet, little is known about the effects of intrinsic CSF-1R in human non-small-cell carcinoma (NSCLC). Here we demonstrated that NSCLC cell-intrinsic CSF-1R promoted cells growth and metastasis both in vitro and in vivo. CSF-1R knocked-down by transfecting with shRNA target CSF-1R suppressed NSCLC cells proliferation and tumor growth in nude mice. Conversely, ectopic expression of CSF-1R promoted cells proliferation and accelerated tumor growth. Mechanistically, the NSCLC CSF-1R modulated downstream effectors of phosphatidylinositol 3-kinase (PI3K) signaling. In addition, CSF-1R overexpression significantly enhanced NSCLC cells mobility, invasion and epithelial-mesenchymal transition (EMT) process, whereas silencing CSF-1R inhibits these phenotypes. Microarray analysis suggested that Wnt family member 3a (Wnt3a) function as a downstream factor of CSF-1R. On account of this, we future identified CSF-1R/Wnt3a a signaling pathway sustained NSCLC cells metastasis. Finally, in patients, CSF-1R and Wnt3a expression positively correlated with the of NSCLC patients. Our results identify NSCLC cell intrinsic functions of CSF-1R/Wnt3a axis in dissemination of NSCLC.

CSF-1R regulates non-small cell lung cancer cells dissemination through Wnt3a signaling

PubMed Central

Yu, Yan Xia; Wu, Hai Jian; Tan, Bing Xu; Qiu, Chen; Liu, Hui Zhong

2017-01-01

Therapeutic antibodies targeting colony stimulating factor 1 receptor (CSF-1R) to block colony stimulating factor-1/colony stimulating factor 1 receptor (CSF-1/CSF-R) signaling axis have exhibit remarkable efficacy in the treatment of malignant tumor. Yet, little is known about the effects of intrinsic CSF-1R in human non-small-cell carcinoma (NSCLC). Here we demonstrated that NSCLC cell-intrinsic CSF-1R promoted cells growth and metastasis both in vitro and in vivo. CSF-1R knocked-down by transfecting with shRNA target CSF-1R suppressed NSCLC cells proliferation and tumor growth in nude mice. Conversely, ectopic expression of CSF-1R promoted cells proliferation and accelerated tumor growth. Mechanistically, the NSCLC CSF-1R modulated downstream effectors of phosphatidylinositol 3-kinase (PI3K) signaling. In addition, CSF-1R overexpression significantly enhanced NSCLC cells mobility, invasion and epithelial-mesenchymal transition (EMT) process, whereas silencing CSF-1R inhibits these phenotypes. Microarray analysis suggested that Wnt family member 3a (Wnt3a) function as a downstream factor of CSF-1R. On account of this, we future identified CSF-1R/Wnt3a a signaling pathway sustained NSCLC cells metastasis. Finally, in patients, CSF-1R and Wnt3a expression positively correlated with the of NSCLC patients. Our results identify NSCLC cell intrinsic functions of CSF-1R/Wnt3a axis in dissemination of NSCLC. PMID:29218239

Brain-derived neurotrophic factor in the nucleus tractus solitarii modulates glucose homeostasis after carotid chemoreceptor stimulation in rats.

PubMed

Montero, Sergio; Cuéllar, Ricardo; Lemus, Mónica; Avalos, Reyes; Ramírez, Gladys; de Álvarez-Buylla, Elena Roces

2012-01-01

Neuronal systems, which regulate energy intake, energy expenditure and endogenous glucose production, sense and respond to input from hormonal related signals that convey information from body energy availability. Carotid chemoreceptors (CChr) function as sensors for circulating glucose levels and contribute to glycemic counterregulatory responses. Brain-derived neurotrophic factor (BDNF) that plays an important role in the endocrine system to regulate glucose metabolism could play a role in hyperglycemic glucose reflex with brain glucose retention (BGR) evoked by anoxic CChr stimulation. Infusing BDNF into the nucleus tractus solitarii (NTS) before CChr stimulation, showed that this neurotrophin increased arterial glucose and BGR. In contrast, BDNF receptor (TrkB) antagonist (K252a) infusions in NTS resulted in a decrease in both glucose variables.

Activation of Akt by Advanced Glycation End Products (AGEs): Involvement of IGF-1 Receptor and Caveolin-1

PubMed Central

Yang, Su-Jung; Chen, Chen-Yu; Chang, Geen-Dong; Wen, Hui-Chin; Chen, Ching-Yu; Chang, Shi-Chuan; Liao, Jyh-Fei; Chang, Chung-Ho

2013-01-01

Diabetes is characterized by chronic hyperglycemia, which in turn facilitates the formation of advanced glycation end products (AGEs). AGEs activate signaling proteins such as Src, Akt and ERK1/2. However, the mechanisms by which AGEs activate these kinases remain unclear. We examined the effect of AGEs on Akt activation in 3T3-L1 preadipocytes. Addition of AGEs to 3T3-L1 cells activated Akt in a dose- and time-dependent manner. The AGEs-stimulated Akt activation was blocked by a PI3-kinase inhibitor LY 294002, Src inhibitor PP2, an antioxidant NAC, superoxide scavenger Tiron, or nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase inhibitor DPI, suggesting the involvement of Src and NAD(P)H oxidase in the activation of PI3-kinase-Akt pathway by AGEs. AGEs-stimulated Src tyrosine phosphorylation was inhibited by NAC, suggesting that Src is downstream of NAD(P)H oxidase. The AGEs-stimulated Akt activity was sensitive to Insulin-like growth factor 1 receptor (IGF-1R) kinase inhibitor AG1024. Furthermore, AGEs induced phosphorylation of IGF-1 receptorβsubunit (IGF-1Rβ) on Tyr1135/1136, which was sensitive to PP2, indicating that AGEs stimulate Akt activity by transactivating IGF-1 receptor. In addition, the AGEs-stimulated Akt activation was attenuated by β-methylcyclodextrin that abolishes the structure of caveolae, and by lowering caveolin-1 (Cav-1) levels with siRNAs. Furthermore, addition of AGEs enhanced the interaction of phospho-Cav-1 with IGF-1Rβ and transfection of 3T3-L1 cells with Cav-1 Y14F mutants inhibited the activation of Akt by AGEs. These results suggest that AGEs activate NAD(P)H oxidase and Src which in turn phosphorylates IGF-1 receptor and Cav-1 leading to activation of IGF-1 receptor and the downstream Akt in 3T3-L1 cells. AGEs treatment promoted the differentiation of 3T3-L1 preadipocytes and addition of AG1024, LY 294002 or Akt inhibitor attenuated the promoting effect of AGEs on adipogenesis, suggesting that IGF-1 receptor, PI3-Kinase and Akt are involved in the facilitation of adipogenesis by AGEs. PMID:23472139

The GHR mutations related to individual’s dwarf

USDA-ARS?s Scientific Manuscript database

Growth hormone (GH) promotes body’s growth through binding with two receptors (GHRs) at the cell surface to interact with Janus kinase and signal transducers and activators of transcription, and then to stimulate metabolic effects and insulin-like growth factor (IGF) synthesis. However, the disorder...

Pyrido[2,3-d]pyrimidin-5-ones: A Novel Class of Antiinflammatory Macrophage Colony-Stimulating Factor-1 Receptor Inhibitors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Hui; Hutta, Daniel A.; Rinker, James M.

A series of pyrido[2,3-d]pyrimidin-5-ones has been synthesized and evaluated as inhibitors of the kinase domain of macrophage colony-stimulating factor-1 receptor (FMS). FMS inhibitors may be useful in treating rheumatoid arthritis and other chronic inflammatory diseases. Structure-based optimization of the lead amide analogue 10 led to hydroxamate analogue 37, which possessed excellent potency and an improved pharmacokinetic profile. During the chronic phase of streptococcal cell wall-induced arthritis in rats, compound 37 (10, 3, and 1 mg/kg) was highly effective at reversing established joint swelling. In an adjuvant-induced arthritis model in rats, 37 prevented joint swelling partially at 10 mg/kg. In thismore » model, osteoclastogenesis and bone erosion were prevented by low doses (1 or 0.33 mg/kg) that had minimal impact on inflammation. These data underscore the potential of FMS inhibitors to prevent erosions and reduce symptoms in rheumatoid arthritis.« less

Stimulation of acid secretion and phosphoinositol production by rat parietal cell muscarinic M sub 2 receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pfeiffer, A.; Rochlitz, H.; Herz, A.

The muscarinic receptor system involved in hydrogen production by enriched rat gastric parietal cells was investigated. Muscarinic receptor density determined by (N-methyl-{sup 3}H)scopolamine binding was 8,100/cell. The receptor appeared to be of the M{sub 2} muscarinic receptor subtype, since it had a low affinity (K{sub d} 189 nM) for the M{sub 1} receptor antagonist pirenzepine compared with atropine. Receptor activation by carbachol rapidly augmented levels of polyphosphoinositides, indicating an activation of phospholipase C. The dose-response relations for the increase in inositol phosphates closely paralleled the binding of carbachol to muscarinic receptors. The inositol phosphate response was antagonized by pirenzepine withmore » a K{sub i} of 177 nM. the stimulation of inositol phosphate levels by carbachol correlated well with the stimulation of ({sup 14}C)aminopyrine uptake, determine as an index of acid secretion. The muscarinic agonists oxotremorine, pilocarpine, and bethanechol elicited partial increases in inositol phosphates at maximal drug concentrations, and these partial increases correlated with their ability to stimulate ({sup 14}C)aminopyrine uptake. These data indicate that inositolpolyphosphates may be a second messenger of M{sub 2} receptors stimulating acid secretion.« less

Non-Ligand-Induced Dimerization is Sufficient to Initiate the Signalling and Endocytosis of EGF Receptor.

PubMed

Kourouniotis, George; Wang, Yi; Pennock, Steven; Chen, Xinmei; Wang, Zhixiang

2016-07-25

The binding of epidermal growth factor (EGF) to EGF receptor (EGFR) stimulates cell mitogenesis and survival through various signalling cascades. EGF also stimulates rapid EGFR endocytosis and its eventual degradation in lysosomes. The immediate events induced by ligand binding include receptor dimerization, activation of intrinsic tyrosine kinase and autophosphorylation. However, in spite of intensified efforts, the results regarding the roles of these events in EGFR signalling and internalization is still very controversial. In this study, we constructed a chimeric EGFR by replacing its extracellular domain with leucine zipper (LZ) and tagged a green fluorescent protein (GFP) at its C-terminus. We showed that the chimeric LZ-EGFR-GFP was constitutively dimerized. The LZ-EGFR-GFP dimer autophosphorylated each of its five well-defined C-terminal tyrosine residues as the ligand-induced EGFR dimer does. Phosphorylated LZ-EGFR-GFP was localized to both the plasma membrane and endosomes, suggesting it is capable of endocytosis. We also showed that LZ-EGFR-GFP activated major signalling proteins including Src homology collagen-like (Shc), extracellular signal-regulated kinase (ERK) and Akt. Moreover, LZ-EGFR-GFP was able to stimulate cell proliferation. These results indicate that non-ligand induced dimerization is sufficient to activate EGFR and initiate cell signalling and EGFR endocytosis. We conclude that receptor dimerization is a critical event in EGF-induced cell signalling and EGFR endocytosis.

Role of the epidermal growth factor receptor in signaling strain-dependent activation of the brain natriuretic peptide gene.

PubMed

Anderson, Hope D I; Wang, Feng; Gardner, David G

2004-03-05

The epidermal growth factor receptor (EGFR) and ectoshedding of heparin-binding epidermal growth factor (HBEGF), an EGFR ligand, have been linked to the development of cardiac myocyte hypertrophy. However, the precise role that the liganded EGFR plays in the transcriptional activation of the gene program that accompanies hypertrophy remains undefined. Utilizing the human (h) BNP gene as a model of hypertrophy-dependent gene activation, we show that activation of the EGFR plays an important role in mediating mechanical strain-dependent stimulation of the hBNP promoter. Strain promotes endothelin (ET) generation through NAD(P)H oxidase-dependent production of reactive oxygen species. ET in turn induces metalloproteinase-mediated cleavage of pro-HBEGF and ectoshedding of HBEGF, which activates the EGFR and stimulates hBNP promoter activity. HBEGF also stimulates other phenotypic markers of hypertrophy including protein synthesis and sarcomeric assembly. The antioxidant N-acetylcysteine or the NAD(P)H oxidase inhibitor, apocynin, inhibited strain-dependent activation of the ET-1 promoter, HBEGF shedding, and hBNP promoter activation. The metalloproteinase inhibitor, GM-6001, prevented the induction of HBEGF ectoshedding and the hBNP promoter response to strain, suggesting a critical role for the metalloproteinase-dependent cleavage event in signaling the strain response. These findings suggest that metalloproteinase activity as an essential step in this pathway may prove to be a relevant therapeutic target in the management of cardiac hypertrophy.

Expression of the peroxisome proliferator-activated receptor γ (PPARγ) in human atherosclerosis and regulation in macrophages by colony stimulating factors and oxidized low density lipoprotein

PubMed Central

Ricote, Mercedes; Huang, Jannet; Fajas, Luis; Li, Andrew; Welch, John; Najib, Jamila; Witztum, Joseph L.; Auwerx, Johan; Palinski, Wulf; Glass, Christopher K.

1998-01-01

The peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-dependent transcription factor that has been demonstrated to regulate fat cell development and glucose homeostasis. PPARγ is also expressed in a subset of macrophages and negatively regulates the expression of several proinflammatory genes in response to natural and synthetic ligands. We here demonstrate that PPARγ is expressed in macrophage foam cells of human atherosclerotic lesions, in a pattern that is highly correlated with that of oxidation-specific epitopes. Oxidized low density lipoprotein (oxLDL) and macrophage colony-stimulating factor, which are known to be present in atherosclerotic lesions, stimulated PPARγ expression in primary macrophages and monocytic cell lines. PPARγ mRNA expression was also induced in primary macrophages and THP-1 monocytic leukemia cells by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). Inhibition of protein kinase C blocked the induction of PPARγ expression by TPA, but not by oxLDL, suggesting that more than one signaling pathway regulates PPARγ expression in macrophages. TPA induced the expression of PPARγ in RAW 264.7 macrophages by increasing transcription from the PPARγ1 and PPARγ3 promoters. In concert, these observations provide insights into the regulation of PPARγ expression in activated macrophages and raise the possibility that PPARγ ligands may influence the progression of atherosclerosis. PMID:9636198

Arctigenin suppresses receptor activator of nuclear factor κB ligand (RANKL)-mediated osteoclast differentiation in bone marrow-derived macrophages.

PubMed

Kim, A-Ram; Kim, Hyuk Soon; Lee, Jeong Min; Choi, Jung Ho; Kim, Se Na; Kim, Do Kyun; Kim, Ji Hyung; Mun, Se Hwan; Kim, Jie Wan; Jeon, Hyun Soo; Kim, Young Mi; Choi, Wahn Soo

2012-05-05

Osteoclasts, multinucleated bone-resorbing cells, are closely associated with bone diseases such as rheumatoid arthritis and osteoporosis. Osteoclasts are derived from hematopoietic precursor cells, and their differentiation is mediated by two cytokines, including macrophage colony stimulating factor and receptor activator of nuclear factor κB ligand (RANKL). Previous studies have shown that arctigenin exhibits an anti-inflammatory effect. However, the effect of arctigenin on osteoclast differentiation is yet to be elucidated. In this study, we found that arctigenin inhibited RANKL-mediated osteoclast differentiation in bone marrow macrophages in a dose-dependent manner and suppressed RANKL-mediated bone resorption. Additionally, the expression of typical marker proteins, such as NFATc1, c-Fos, TRAF6, c-Src, and cathepsin K, were significantly inhibited. Arctigenin inhibited the phosphorylation of Erk1/2, but not p38 and JNK, in a dose-dependent manner. Arctigenin also dramatically suppressed immunoreceptor tyrosine-based activation motif-mediated costimulatory signaling molecules, including Syk and PLCγ2, and Gab2. Notably, arctigenin inhibited the activation of Syk through RANKL stimulation. Furthermore, arctigenin prevented osteoclast differentiation in the calvarial bone of mice following stimulation with lipopolysaccharide. Our results show that arctigenin inhibits osteoclast differentiation in vitro and in vivo. Therefore, arctigenin may be useful for treating rheumatoid arthritis and osteoporosis. Copyright © 2012 Elsevier B.V. All rights reserved.

TRPC5-eNOS Axis Negatively Regulates ATP-Induced Cardiomyocyte Hypertrophy.

PubMed

Sunggip, Caroline; Shimoda, Kakeru; Oda, Sayaka; Tanaka, Tomohiro; Nishiyama, Kazuhiro; Mangmool, Supachoke; Nishimura, Akiyuki; Numaga-Tomita, Takuro; Nishida, Motohiro

2018-01-01

Cardiac hypertrophy, induced by neurohumoral factors, including angiotensin II and endothelin-1, is a major predisposing factor for heart failure. These ligands can induce hypertrophic growth of neonatal rat cardiomyocytes (NRCMs) mainly through Ca 2+ -dependent calcineurin/nuclear factor of activated T cell (NFAT) signaling pathways activated by diacylglycerol-activated transient receptor potential canonical 3 and 6 (TRPC3/6) heteromultimer channels. Although extracellular nucleotide, adenosine 5'-triphosphate (ATP), is also known as most potent Ca 2+ -mobilizing ligand that acts on purinergic receptors, ATP never induces cardiomyocyte hypertrophy. Here we show that ATP-induced production of nitric oxide (NO) negatively regulates hypertrophic signaling mediated by TRPC3/6 channels in NRCMs. Pharmacological inhibition of NO synthase (NOS) potentiated ATP-induced increases in NFAT activity, protein synthesis, and transcriptional activity of brain natriuretic peptide. ATP significantly increased NO production and protein kinase G (PKG) activity compared to angiotensin II and endothelin-1. We found that ATP-induced Ca 2+ signaling requires inositol 1,4,5-trisphosphate (IP 3 ) receptor activation. Interestingly, inhibition of TRPC5, but not TRPC6 attenuated ATP-induced activation of Ca 2+ /NFAT-dependent signaling. As inhibition of TRPC5 attenuates ATP-stimulated NOS activation, these results suggest that NO-cGMP-PKG axis activated by IP 3 -mediated TRPC5 channels underlies negative regulation of TRPC3/6-dependent hypertrophic signaling induced by ATP stimulation.

Effect of estradiol on the expression of angiogenic factors in epithelial ovarian cancer.

PubMed

Valladares, Macarena; Plaza-Parrochia, Francisca; Lépez, Macarena; López, Daniela; Gabler, Fernando; Gayan, Patricio; Selman, Alberto; Vega, Margarita; Romero, Carmen

2017-11-01

Ovarian cancer presents a high angiogenesis (formation of new blood vessels) regulated by pro-angiogenic factors, mainly vascular endothelial growth factor (VEGF) and nerve growth factor (NGF). An association between endogenous levels of estrogen and increased risk of developing ovarian cancer has been reported. Estrogen action is mediated by the binding to its specific receptors (ERα and ERβ), altered ERα/ERβ ratio may constitute a marker of ovarian carcinogenesis progression. To determine the effect of estradiol through ERα on the expression of NGF and VEGF in epithelial ovarian cancer (EOC). Levels of phosphorylated estrogen receptor alpha (pERα) were evaluated in well, moderate and poorly differentiated EOC samples (EOC-I, EOC-II, EOC-III). Additionally, ovarian cancer explants were stimulated with NGF (0, 10 and 100 ng/ml) and ERα, ERβ and pERα levels were detected. Finally, human ovarian surface epithelial (HOSE) and epithelial ovarian cancer (A2780) cell lines were stimulated with estradiol, where NGF and VEGF protein levels were evaluated. In tissues, ERs were detected being pERα levels significantly increased in EOC-III samples compared with EOC-I (p<0.05). Additionally, ovarian explants treated with NGF increased pERα levels meanwhile total ERα and ERβ levels did not change. Cell lines stimulated with estradiol revealed an increase of NGF and VEGF protein levels (p<0.05). Estradiol has a positive effect on pro-angiogenic factors such as NGF and VEGF expression in EOC, probably through the activation of ERα; generating a positive loop induced by NGF increasing pERα levels in epithelial ovarian cells.

Neuroendocrine immunomodulation network dysfunction in SAMP8 mice and PrP-hAβPPswe/PS1ΔE9 mice: potential mechanism underlying cognitive impairment

PubMed Central

Wang, Jian-hui; Cheng, Xiao-rui; Zhang, Xiao-rui; Wang, Tong-xing; Xu, Wen-jian; Li, Fei; Liu, Feng; Cheng, Jun-ping; Bo, Xiao-chen; Wang, Sheng-qi; Zhou, Wen-xia; Zhang, Yong-xiang

2016-01-01

Senescence-accelerated mouse prone 8 strain (SAMP8) and PrP-hAβPPswe/PS1ΔE9 (APP/PS1) mice are classic animal models of sporadic Alzheimer's disease and familial AD respectively. Our study showed that object recognition memory, spatial learning and memory, active and passive avoidance were deteriorated and neuroendocrine immunomodulation (NIM) network was imbalance in SAMP8 and APP/PS1 mice. SAMP8 and APP/PS1 mice had their own specific phenotype of cognition, neuroendocrine, immune and NIM molecular network. The endocrine hormone corticosterone, luteinizing hormone and follicle-stimulating hormone, chemotactic factor monocyte chemotactic protein-1, macrophage inflammatory protein-1β, regulated upon activation normal T cell expressed and secreted factor and eotaxin, pro-inflammatory factor interleukin-23, and the Th1 cell acting as cell immunity accounted for cognitive deficiencies in SAMP8 mice, while adrenocorticotropic hormone and gonadotropin-releasing hormone, colony stimulating factor granulocyte colony stimulating factor, and Th2 cell acting as humoral immunity in APP/PS1 mice. On the pathway level, chemokine signaling and T cell receptor signaling pathway played the key role in cognition impairments of two models, while cytokine-cytokine receptor interaction and natural killer cell mediated cytotoxicity were more important in cognitive deterioration of SAMP8 mice than APP/PS1 mice. This mechanisms of NIM network underlying cognitive impairment is significant for further understanding the pathogenesis of AD and can provide useful information for development of AD therapeutic drug. PMID:27049828

Toll-like Receptors and B-cell Receptors Synergize to Induce Immunoglobulin Class Switch DNA Recombination: Relevance to Microbial Antibody Responses

PubMed Central

Pone, Egest J.; Zan, Hong; Zhang, Jinsong; Al-Qahtani, Ahmed; Xu, Zhenming; Casali, Paolo

2011-01-01

Differentiation of naïve B cells, including immunoglobulin (Ig) class switch DNA recombination (CSR), is critical for the immune response and depends on the extensive integration of signals from the B cell receptor (BCR), tumor necrosis factor (TNF) receptor family members, Toll-like receptors (TLRs) and cytokine receptors. TLRs and BCR synergize to induce CSR in T cell-dependent and T cell-independent antibody responses to microbial pathogens. BCR triggering together with simultaneous endosomal TLR engagement leads to enhanced B cell differentiation and antibody responses. The requirement of both BCR and TLR engagement would ensure appropriate antigen-specific activation in an infection. Co-stimulation of TLRs and BCR likely plays a significant role in anti-microbial antibody responses to contain pathogen loads until the T cell-dependent antibody responses peak. Furthermore, the temporal sequence of different signals is also critical for optimal B cell responses, as exemplified by the activation of B cells by initial TLR engagement, leading to the upregulation of co-stimulatory CD80 and MHC-II receptors, which, in turn, result in more efficient interactions with T cells, thereby enhancing the germinal center (GC) reaction and antibody affinity maturation. Overall, BCR and TLR stimulation and the integration with signals from the pathogen or immune cells and their products, determine the ensuing B cell antibody response. PMID:20370617

Circadian control of p75 neurotrophin receptor leads to alternate activation of Nrf2 and c-Rel to reset energy metabolism in astrocytes via brain-derived neurotrophic factor.

PubMed

Ishii, Tetsuro; Warabi, Eiji; Mann, Giovanni E

2018-05-01

Circadian clock genes regulate energy metabolism partly through neurotrophins in the body. The low affinity neurotrophin receptor p75 NTR is a clock component directly regulated by the transcriptional factor Clock:Bmal1 complex. Brain-derived neurotrophic factor (BDNF) is expressed in the brain and plays a key role in coordinating metabolic interactions between neurons and astrocytes. BDNF transduces signals through TrkB and p75 NTR receptors. This review highlights a novel molecular mechanism by which BDNF via circadian control of p75 NTR leads to daily resetting of glucose and glycogen metabolism in brain astrocytes to accommodate their functional interaction with neurons. Astrocytes store glycogen as an energy reservoir to provide active neurons with the glycolytic metabolite lactate. Astrocytes predominantly express the truncated receptor TrkB.T1 which lacks an intracellular receptor tyrosine kinase domain. TrkB.T1 retains the capacity to regulate cell morphology through regulation of Rho GTPases. In contrast, p75 NTR mediates generation of the bioactive lipid ceramide upon stimulation with BDNF and inhibits PKA activation. As ceramide directly activates PKCζ, we discuss the importance of the TrkB.T1-p75 NTR -ceramide-PKCζ signaling axis in the stimulation of glycogen and lipid synthesis and activation of RhoA. Ceramide-PKCζ-casein kinase 2 signaling activates Nrf2 to support oxidative phosphorylation via upregulation of antioxidant enzymes. In the absence of p75 NTR , TrkB.T1 functionally interacts with adenosine A 2A R and dopamine D1R receptors to enhance cAMP-PKA signaling and activate Rac1 and NF-κB c-Rel, favoring glycogen hydrolysis, gluconeogenesis and aerobic glycolysis. Thus, diurnal changes in p75 NTR levels in astrocytes resets energy metabolism via BDNF to accommodate their metabolic interaction with neurons. Copyright © 2018 Elsevier Inc. All rights reserved.

Pattern of cytokine receptors expressed by human dendritic cells migrated from dermal explants.

PubMed Central

Larregina, A T; Morelli, A E; Kolkowski, E; Sanjuan, N; Barboza, M E; Fainboim, L

1997-01-01

Different reasons account for the lack of information about the expression of cytokine receptors on human dendritic cells (DC): (a) DC are a trace population; (b) the proteolytic treatment used to isolate DC may alter enzyme-sensitive epitopes; and (c) low numbers of receptors per cell. In the present work the expression of cytokine receptors was analysed by flow cytometry on the population of dermal DC (DDC) that spontaneously migrate from short-term culture dermal explants. DDC obtained after dermal culture were CD1alow, CD1b+, CD1c+, human leucocyte antigen (HLA)-DR+, CD11chigh, CD11b+ and CD32+. The DC lineage was confirmed by ultrastructural analysis. DDC expressed interleukin (IL)-1R type 1 (monoclonal antibody (mAb) hIL-1R1-M1; and 6B5); IL-1R type 2 (mAb hIL-1R2-M22); IL-2R alpha chain (mAb anti-Tac; and hIL-2R-M1) and IL-2R gamma chain (mAb 3B5; and AG14C). DDC did not stain for IL-2R beta chain using four mAbs recognizing two different epitopes of IL-2R beta (mAb 2R-B; Mik-beta 1; and CF1; Mik-beta 3, respectively). DDC were also positive for the cytokine binding chains (alpha chains) of IL-3R (mAb 9F5); IL-4R (mAb hIL-4R-M57; and S456C9); and IL-7R (mAb hIL-7R-M20; and R3434). DDC showed low levels of IL-6R alpha chain (mAb B-F19; B-R6; and B-E23) and its signal transducer gp130 (mAb A2; and B1). DDC strongly expressed interferon-gamma receptor (IFN-gamma R) (mAb GIR-208) and were negative for IL-8R (mAb B-G20; and B-F25). All DDC were highly positive for granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) alpha chain (mAb hGM-CSFR-M1; SC06; SC04, and 8G6) and to a lesser extent for the common beta chain of GM-CSFR, IL-3R and IL-5R (mAb 3D7). On the other hand, reactivity was not found for granulocyte colony-stimulating factor receptor (G-CSFR) (mAb hGCSFR-M1) nor macrophage colony-stimulating factor receptor (M-CSFR) (mAb 7-7A3-17) confirming the DC lineage of DDC. As previously reported for lymphoid DC, DDC expressed tumour necrosis factor receptort (TNFR) 75000 MW (mAb utr-1; hTNFR-M1; and MR2-1) but lacked TNFR 55000 MW (mAb htr-9; MR1-1; and MR1-2). In summary, DDC express receptors for a broad panel of cytokines, even receptors for cytokines whose effects on DC are still unknown (i.e. IL-2R alpha gamma; IL-6R alpha/gp 130; IL-7R alpha gamma). Images Figure 1 PMID:9227332

Interactions between IGFBP-3 and Nuclear Receptors in Prostate Cancer Apoptosis

DTIC Science & Technology

2008-01-01

manner. Tumor necrosis factor (TNF)- treatment also inhibited glucose transport to the same degree as IGFBP-3 and, in addition, increased IGFBP-3...mechanisms involved in its actions. In addition, we show here that the insulin-antagonistic effects of tumor necrosis factor (TNF)- are mediated in...adipocytes. J Biol Chem 265:12434–12443 14. Ranganathan S, Davidson MB 1996 Effect of tumor necrosis factor-a on basal and insulin-stimulated glucose

Rational Design of Potent Antagonists to the Human Growth Hormone Receptor

NASA Astrophysics Data System (ADS)

Fuh, Germaine; Cunningham, Brian C.; Fukunaga, Rikiro; Nagata, Shigekazu; Goeddel, David V.; Wells, James A.

1992-06-01

A hybrid receptor was constructed that contained the extracellular binding domain of the human growth hormone (hGH) receptor linked to the transmembrane and intracellular domains of the murine granulocyte colony-stimulating factor receptor. Addition of hGH to a myeloid leukemia cell line (FDC-P1) that expressed the hybrid receptor caused proliferation of these cells. The mechanism for signal transduction of the hybrid receptor required dimerization because monoclonal antibodies to the hGH receptor were agonists whereas their monovalent fragments were not. Receptor dimerization occurs sequentially-a receptor binds to site 1 on hGH, and then a second receptor molecule binds to site 2 on hGH. On the basis of this sequential mechanism, which may occur in many other cytokine receptors, inactive hGH analogs were designed that were potent antagonists to hGH-induced cell proliferation. Such antagonists could be useful for treating clinical conditions of hGH excess, such as acromegaly.

Substance P release and neurokinin 1 receptor activation in the rat spinal cord increase with the firing frequency of C-fibers.

PubMed

Adelson, D; Lao, L; Zhang, G; Kim, W; Marvizón, J C G

2009-06-30

Both the firing frequency of primary afferents and neurokinin 1 receptor (NK1R) internalization in dorsal horn neurons increase with the intensity of noxious stimulus. Accordingly, we studied how the pattern of firing of primary afferent influences NK1R internalization. In rat spinal cord slices, electrical stimulation of the dorsal root evoked NK1R internalization in lamina I neurons by inducing substance P release from primary afferents. The stimulation frequency had pronounced effects on NK1R internalization, which increased up to 100 Hz and then diminished abruptly at 200 Hz. Peptidase inhibitors increased NK1R internalization at frequencies below 30 Hz, indicating that peptidases limit the access of substance P to the receptor at moderate firing rates. NK1R internalization increased with number of pulses at all frequencies, but maximal internalization was substantially lower at 1-10 Hz than at 30 Hz. Pulses organized into bursts produced the same NK1R internalization as sustained 30 Hz stimulation. To determine whether substance P release induced at high stimulation frequencies was from C-fibers, we recorded compound action potentials in the sciatic nerve of anesthetized rats. We observed substantial NK1R internalization when stimulating at intensities evoking a C-elevation, but not at intensities evoking only an Adelta-elevation. Each pulse in trains at frequencies up to 100 Hz evoked a C-elevation, demonstrating that C-fibers can follow these high frequencies. C-elevation amplitudes declined progressively with increasing stimulation frequency, which was likely caused by a combination of factors including temporal dispersion. In conclusion, the instantaneous firing frequency in C-fibers determines the amount of substance P released by noxious stimuli.

Substance P release and neurokinin 1 receptor activation in the rat spinal cord increases with the firing frequency of C-fibers

PubMed Central

Adelson, David; Lao, Lijun; Zhang, Guohua; Kim, Woojae; Marvizón, Juan Carlos G.

2009-01-01

Both the firing frequency of primary afferents and neurokinin 1 receptor (NK1R) internalization in dorsal horn neurons increase with the intensity of noxious stimulus. Accordingly, we studied how the pattern of firing of primary afferent influences NK1R internalization. In rat spinal cord slices, electrical stimulation of the dorsal root evoked NK1R internalization in lamina I neurons by inducing substance P release from primary afferents. The stimulation frequency had pronounced effects on NK1R internalization, which increased up to 100 Hz and then diminished abruptly at 200 Hz. Peptidase inhibitors increased NK1R internalization at frequencies below 30 Hz, indicating that peptidases limit the access of substance P to the receptor at moderate firing rates. NK1R internalization increased with number of pulses at all frequencies, but maximal internalization was substantially lower at 1–10 Hz than at 30 Hz. Pulses organized into bursts produced the same NK1R internalization as sustained 30 Hz stimulation. To determine whether substance P release induced at high stimulation frequencies was from C-fibers, we recorded compound action potentials in the sciatic nerve of anesthetized rats. We observed substantial NK1R internalization when stimulating at intensities evoking a C-elevation, but not at intensities evoking only an Aδ-elevation. Each pulse in trains at frequencies up to 100 Hz evoked a C-elevation, demonstrating that C-fibers can follow these high frequencies. C-elevation amplitudes declined progressively with increasing stimulation frequency, which was likely caused by a combination of factors including temporal dispersion. In conclusion, the instantaneous firing frequency in C-fibers determines the amount of substance P released by noxious stimuli. PMID:19336248

Icariin and icaritin stimulate the proliferation of SKBr3 cells through the GPER1-mediated modulation of the EGFR-MAPK signaling pathway.

PubMed

Ma, Hai-Rong; Wang, Jie; Chen, Yiu-Fai; Chen, Hua; Wang, Wei-Shan; Aisa, Haji Akber

2014-06-01

Icariin (ICA) and icaritin (ICT), with a similar structure to genistein, are the important bioactive components of the genus Epimedium, and regulate many cellular processes. In the present study, using the estrogen receptor (ER)-negative breast cancer cell line, SKBr3, as a model, we examined the hypothesis that ICA and ICT at low concentrations stimulate SKBr3 cell proliferation in vitro through the functional membrane, G protein‑coupled estrogen receptor 1 (GPER1), mediated by the epithelial growth factor receptor (EGFR)‑mitogen-activated protein kinase (MAPK) signaling pathway. MTT assay revealed that ICA and ICT at doses of 1 nM to 1 µM markedly stimulated SKBr3 cell proliferation in a dose-dependent manner. The ICA- and ICT-stimulated cell growth was completely suppressed by the GPER1 antagonist, G-15, indicating that the ICA‑ and ICT-stimulated cell proliferation was mediated by GPER1 activation. Semi-quantitative RT-PCR analysis revealed that treatment with ICA and ICT enhanced the transcription of c-fos, a proliferation-related early gene. The ICA- and ICT-stimulated mRNA expression was markedly attenuated by G-15, AG-1478 (an EGFR antagonist) or PD98059 (a MAPK inhibitor). Our data also demonstrated that ICA and ICT increased the phosphorylation of ERK1/2. The ICA- and ICT-stimulated ERK1/2 phosphorylation was blocked by pre-treatment of the cells with G-15 and AG-1478 or PD 98059. Flow cytometric analysis confirmed that the ICA- and ICT-stimulated SKBr3 cell proliferation involved the GPER1-mediated modulation of the EGFR‑MAPK signaling pathway. To the best of our knowledge, our current findings demonstrate for the first time that ICA and ICT promote the progression of ER-negative breast cancer through the activation of membrane GPER1.

Interference by 2,3,7,8-tetrachlorodibenzo-p-dioxin with cultured mouse submandibular gland branching morphogenesis involves reduced epidermal growth factor receptor signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kiukkonen, Anu; Sahlberg, Carin; Partanen, Anna-Maija

2006-05-01

Toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to mouse embryonic teeth, sharing features of early development with salivary glands in common, involves enhanced apoptosis and depends on the expression of epidermal growth factor (EGF) receptor. EGF receptor signaling, on the other hand, is essential for salivary gland branching morphogenesis. To see if TCDD impairs salivary gland morphogenesis and if the impairment is associated with EGF receptor signaling, we cultured mouse (NMRI) E13 submandibular glands with TCDD or TCDD in combination with EGF or fibronectin (FN), both previously found to enhance branching morphogenesis. Explants were examined stereomicroscopically and processed to paraffin sections. TCDD exposuremore » impaired epithelial branching and cleft formation, resulting in enlarged buds. The glands were smaller than normal. EGF and FN alone concentration-dependently stimulated or inhibited branching morphogenesis but when co-administered with TCDD, failed to compensate for its effect. TCDD induced cytochrome P4501A1 expression in the glandular epithelium, indicating activation of the aryl hydrocarbon receptor. TCDD somewhat increased epithelial apoptosis as observed by terminal deoxynucleotidyl transferase (TdT)-mediated nick end-labeling method but the increase could not be correlated with morphological changes. The frequency of proliferating cells was not altered. Corresponding to the reduced cleft sites in TCDD-exposed explants, FN immunoreactivity in the epithelium was reduced. The results show that TCDD, comparably with EGF and FN at morphogenesis-inhibiting concentrations, impaired salivary gland branching morphogenesis in vitro. Together with the failure of EGF and FN at morphogenesis-stimulating concentrations to compensate for the effect of TCDD this implies that TCDD toxicity to developing salivary gland involves reduced EGF receptor signaling.« less

The insulin receptor substrate-1-related 4PS substrate but not the interleukin-2R gamma chain is involved in interleukin-13-mediated signal transduction.

PubMed

Wang, L M; Michieli, P; Lie, W R; Liu, F; Lee, C C; Minty, A; Sun, X J; Levine, A; White, M F; Pierce, J H

1995-12-01

Interleukin-13 (IL-13) induced a potent mitogenic response in IL-3-dependent TF-1 cells and DNA synthesis to a lesser extent in MO7E and FDC-P1 cells. IL-13 stimulation of these lines, like IL-4 and insulin-like growth factor-1 (IGF-1), resulted in tyrosine phosphorylation of a 170-kD substrate. The tyrosine-phosphorylated 170-kD substrate strongly associated with the 85-kD subunit of phosphoinositol-3 (PI-3) kinase and with Grb-2. Anti-4PS serum readily detected the 170-kD substrate in lysates from both TF-1 and FDC-P1 cells stimulated with IL-13 or IL-4. These data provide evidence that IL-13 induces tyrosine phosphorylation of the 4PS substrate, providing an essential interface between the IL-13 receptor and signaling molecules containing SH2 domains. IL-13 and IL-4 stimulation of murine L cell fibroblasts, which endogenously express the IL-4 receptor (IL-4R alpha) and lack expression of the IL-2 receptor gamma subunit (IL-2R gamma), resulted in tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1)/4PS. Enhanced tyrosine phosphorylation of IRS-1/4PS was observed in response to IL-4, but not IL-13 treatment of L cells transfected with the IL-2R gamma chain. These results indicate that IL-13 does not use the IL-2R gamma subunit in its receptor complex and that expression of IL-2R gamma enhances, but is not absolutely required for mediating IL-4-induced tyrosine phosphorylation of IRS-1/4PS.

Continuous 4-1BB co-stimulatory signals for the optimal expansion of tumor-infiltrating lymphocytes for adoptive T-cell therapy.

PubMed

Chacon, Jessica Ann; Pilon-Thomas, Shari; Sarnaik, Amod A; Radvanyi, Laszlo G

2013-09-01

Co-stimulation through members of the tumor necrosis factor receptor (TNFR) family appears to be critical for the generation of T cells with optimal effector-memory properties for adoptive cell therapy. Our work suggests that continuous 4-1BB/CD137 co-stimulation is required for the expansion of T cells with an optimal therapeutic profile and that the administration of 4-1BB agonists upon adoptive cell transfer further improves antitumor T-cell functions.

The IRS-1 signaling system.

PubMed

White, M F

1994-02-01

IRS-1 is a principal substrate of the insulin receptor tyrosine kinase. It undergoes multi-site tyrosine phosphorylation and mediates the insulin signal by associating with various signaling molecules containing Src homology 2 domains. Interleukin-4 also stimulates IRS-1 phosphorylation, and it is suspected that a few more growth factors or cytokines will be added to form a select group of receptors that utilize the IRS-1 signaling pathway. More IRS-1-like adapter molecules, such as 4PS (IRS-2), may remain to be found.

Clopidogrel inhibits angiogenesis of gastric ulcer healing via downregulation of vascular endothelial growth factor receptor 2.

PubMed

Luo, Jiing-Chyuan; Peng, Yen-Ling; Chen, Tseng-Shing; Huo, Teh-Ia; Hou, Ming-Chih; Huang, Hui-Chun; Lin, Han-Chieh; Lee, Fa-Yauh

2016-09-01

Although clopidogrel does not cause gastric mucosal injury, it does not prevent peptic ulcer recurrence in high-risk patients. We explored whether clopidogrel delays gastric ulcer healing via inhibiting angiogenesis and to elucidate the possible mechanisms. Gastric ulcers were induced in Sprague Dawley rats, and ulcer healing and angiogenesis of ulcer margin were compared between clopidogrel-treated rats and controls. The expressions of the proangiogenic growth factors and their receptors including basic fibroblast growth factor (bFGF), bFGF receptor (FGFR), vascular endothelial growth factor (VEGF), VEGFR1, VEGFR2, platelet-derived growth factor (PDGF)A, PDGFB, PDGFR A, PDGFR B, and phosphorylated form of mitogenic activated protein kinase pathways over the ulcer margin were compared via western blot and reverse transcription polymerase chain reaction. In vitro, human umbilical vein endothelial cells (HUVECs) were used to elucidate how clopidogrel inhibited growth factors-stimulated HUVEC proliferation. The ulcer sizes were significantly larger and the angiogenesis of ulcer margin was significantly diminished in the clopidogrel (2 and 10 mg/kg/d) treated groups. Ulcer induction markedly increased the expression of phosphorylated form of extracellular signal-regulated kinase (pERK), FGFR2, VEGF, VEGFR2, and PDGFRA when compared with those of normal mucosa. Clopidogrel treatment significantly decreased pERK, FGFR2, VEGF, VEGFR2, and PDGFRA expression at the ulcer margin when compared with those of the respective control group. In vitro, clopidogrel (10(-6)M) inhibited VEGF-stimulated (20 ng/mL) HUVEC proliferation, at least, via downregulation of VEGFR2 and pERK. Clopidogrel inhibits the angiogenesis of gastric ulcer healing at least partially by the inhibition of the VEGF-VEGFR2-ERK signal transduction pathway. Copyright © 2015. Published by Elsevier B.V.

Mechanism of action of a nanomolar potent, allosteric antagonist of the thyroid-stimulating hormone receptor

PubMed Central

van Koppen, Chris J; de Gooyer, Marcel E; Karstens, Willem-Jan; Plate, Ralf; Conti, Paolo GM; van Achterberg, Tanja AE; van Amstel, Monique GA; Brands, Jolanda HGM; Wat, Jesse; Berg, Rob JW; Lane, J Robert D; Miltenburg, Andre MM; Timmers, C Marco

2012-01-01

BACKGROUND AND PURPOSE Graves' disease (GD) is an autoimmune disease in which the thyroid is overactive, producing excessive amounts of thyroid hormones, caused by thyroid-stimulating hormone (TSH) receptor-stimulating immunoglobulins (TSIs). Many GD patients also suffer from thyroid eye disease (Graves' ophthalmopathy or GO), as TSIs also activate TSH receptors in orbital tissue. We recently developed low molecular weight (LMW) TSH receptor antagonists as a novel therapeutic strategy for the treatment of GD and GO. Here, we determined the molecular pharmacology of a prototypic, nanomolar potent LMW TSH receptor antagonist, Org 274179-0. EXPERIMENTAL APPROACH Using CHO cells heterogeneously expressing human TSH receptors and rat FRTL-5 cells endogenously expressing rat TSH receptors, we determined the potency and efficacy of Org 274179-0 at antagonizing TSH- and TSI-induced TSH receptor signalling and its cross-reactivity at related follicle-stimulating hormone and luteinizing hormone receptors. We analysed the allosteric mode of interaction of Org 274179-0 and determined whether it is an inverse agonist at five naturally occurring, constitutively active TSH receptor mutants. KEY RESULTS Nanomolar concentrations of Org 274179-0 completely inhibited TSH (and TSI)-mediated TSH receptor activation with little effect on the potency of TSH, in accordance with an allosteric mechanism of action. Conversely, increasing levels of TSH receptor stimulation only marginally reduced the antagonist potency of Org 274179-0. Org 274179-0 fully blocked the increased basal activity of all the constitutively active TSH receptor mutants tested with nanomolar potencies. CONCLUSIONS AND IMPLICATIONS Nanomolar potent TSH receptor antagonists like Org 274179-0 have therapeutic potential for the treatment of GD and GO. PMID:22014107

Granulocyte-colony stimulating factor and stem cell factor are the crucial factors in long-term culture of human primitive hematopoietic cells supported by a murine stromal cell line.

PubMed

Nishi, N; Ishikawa, R; Inoue, H; Nishikawa, M; Kakeda, M; Yoneya, T; Tsumura, H; Ohashi, H; Yamaguchi, Y; Motoki, K; Sudo, T; Mori, K J

1996-09-01

The findings that murine marrow stromal cell line MS-5 supported the proliferation of human lineage-negative (Lin-) CD34+CD38- bone marrow cells in long-term culture have been reported. In this study, we analyzed this proliferating activity of MS-5-conditioned medium (CM) on human primitive hematopoietic cells. When Lin-CD34+CD38- cells of normal human cord blood cells were co-cultured with MS-5, colony forming cells (CFCs) were maintained over 7 weeks in vitro. Prevention of contact between MS-5 and Lin-CD34+CD38- cells by using membrane filter (0.45 micron) was negligible for this activity. This indicated that the activity of MS-5 on human primitive hematopoietic cells is a soluble factor(s) secreted from MS-5, which is not induced by the contact between MS-5 and Lin-CD34+CD38- cells. We tried to purify this soluble activity. An active material with a molecular weight of about 150 kDa, determined by gel filtration chromatography, solely supported the growth of Lin-CD34+CD38- cells and Mo7e, a human megakaryocytic cell line. This activity not only reacted with anti-mouse stem cell factor (mSCF) antibody on Western blots, but it was also neutralized in the presence of anti-mSCF antibody. Another active material with a molecular weight of about 20-30 kDa synergized with mSCF to stimulate the growth of Lin-CD34+CD38- cells but failed to do so alone, although this synergy was inhibited in the presence of soluble mouse granulocyte-colony stimulating factor (mG-CSF) receptor, which is a chimeric protein consisting of the extracellular domain of mG-CSF receptor and the Fe region of human IgG1. In addition, the latter molecule supported the growth of the G-CSF dependent cell line FD/GR3, which is a murine myeloid leukemia cell line, FDC-P2, transfected with mG-CSF receptor cDNA. Adding of anti-mSCF antibody and soluble mG-CSF receptor to the culture completely abrogated the activity of MS-5-CM. Recombinant (r) mSCF and rmG-CSF had synergistic activity on the growth of Lin-CD34+CD38- cells. These results indicated that the activity on Lin-CD34+CD38- cells included in MS-5-CM is based upon the synergistic effects of mSCF and mG-CSF.

Muscimol increases acetylcholine release by directly stimulating adult striatal cholinergic interneurons.

PubMed

Login, I S; Pal, S N; Adams, D T; Gold, P E

1998-01-01

Because GabaA ligands increase acetylcholine (ACh) release from adult striatal slices, we hypothesized that activation of GabaA receptors on striatal cholinergic interneurons directly stimulates ACh secretion. Fractional [3H]ACh release was recorded during perifusion of acutely dissociated, [3H]choline-labeled, adult male rat striata. The GabaA agonist, muscimol, immediately stimulated release maximally approximately 300% with EC50 = approximately 1 microM. This action was enhanced by the allosteric GabaA receptor modulators, diazepam and secobarbital, and inhibited by the GabaA antagonist, bicuculline, by ligands for D2 or muscarinic cholinergic receptors or by low calcium buffer, tetrodotoxin or vesamicol. Membrane depolarization inversely regulated muscimol-stimulated secretion. Release of endogenous and newly synthesized ACh was stimulated in parallel by muscimol without changing choline release. Muscimol pretreatment inhibited release evoked by K+ depolarization or by receptor-mediated stimulation with glutamate. Thus, GabaA receptors on adult striatal cholinergic interneurons directly stimulate voltage- and calcium-dependent exocytosis of ACh stored in vesamicol-sensitive synaptic vesicles. The action depends on the state of membrane polarization and apparently depolarizes the membrane in turn. This functional assay demonstrates that excitatory GabaA actions are not limited to neonatal tissues. GabaA-stimulated ACh release may be prevented in situ by normal tonic dopaminergic and muscarinic input to cholinergic neurons.

Prolonged Stimulation of a Brainstem Raphe Region Attenuates Experimental Autoimmune Encephalomyelitis

PubMed Central

Madsen, Pernille M.; Sloley, Stephanie S.; Vitores, Alberto A.; Carballosa-Gautam, Melissa M.; Brambilla, Roberta; Hentall, Ian D.

2017-01-01

Multiple sclerosis (MS), a neuroinflammatory disease, has few treatment options, none entirely adequate. We studied whether prolonged electrical stimulation of a hindbrain region (the nucleus raphe magnus) can attenuate experimental autoimmune encephalomyelitis, a murine model of MS induced by MOG35-55 injection. Eight days after symptoms emerged, a wireless electrical stimulator with a connectorless protruding microelectrode was implanted cranially, and daily intermittent stimulation of awake, unrestrained mice began immediately. The thoracic spinal cord was analyzed for changes in histology (on day 29) and gene expression (on day 37), with a focus on myelination and cytokine production. Controls, with inactive implants, showed a phase of disease exacerbation on days 19–25 that stimulation for >16 days eliminated. Prolonged stimulation also reduced infiltrating immune cells and increased numbers of myelinated axons. It additionally lowered gene expression for some pro-inflammatory cytokines (interferon gamma and tumor necrosis factor) and for platelet-derived growth factor receptor alpha, a marker of oligodendrocyte precursors, while raising it for myelin basic protein. Restorative treatments for MS might profitably consider ways to stimulate the raphe magnus, directly or via its inputs, or to emulate its serotonergic and peptidergic output. PMID:28147248

Toll like Receptor 2 engagement on CD4+ T cells promotes TH9 differentiation and function.

PubMed

Karim, Ahmad Faisal; Reba, Scott M; Li, Qing; Boom, W Henry; Rojas, Roxana E

2017-09-01

We have recently demonstrated that mycobacterial ligands engage Toll like receptor 2 (TLR2) on CD4 + T cells and up-regulate T-cell receptor (TCR) triggered Th1 responses in vitro and in vivo. To better understand the role of T-cell expressed TLR2 on CD4 + T-cell differentiation and function, we conducted a gene expression analysis of murine naïve CD4 + T-cells stimulated in the presence or absence of TLR2 co-stimulation. Unexpectedly, naïve CD4 + T-cells co-stimulated via TLR2 showed a significant up-regulation of Il9 mRNA compared to cells co-stimulated via CD28. Under TH9 differentiation, we observed up-regulation of TH9 differentiation, evidenced by increases in both percent of IL-9 secreting cells and IL-9 in culture supernatants in the presence of TLR2 agonist both in polyclonal and Ag85B cognate peptide specific stimulations. Under non-polarizing conditions, TLR2 engagement on CD4 + T-cells had minimal effect on IL-9 secretion and TH9 differentiation, likely due to a prominent effect of TLR2 signaling on IFN-γ secretion and TH1 differentiation. We also report that, TLR2 signaling in CD4 + T cells increased expression of transcription factors BATF and PU.1, known to positively regulate TH9 differentiation. These results reveal a novel role of T-cell expressed TLR2 in enhancing the differentiation and function of TH9 T cells. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Extracellular Purines Promote the Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells to the Osteogenic and Adipogenic Lineages

PubMed Central

Zini, Roberta; Rossi, Lara; Salvestrini, Valentina; Ferrari, Davide; Manfredini, Rossella; Lemoli, Roberto M.

2013-01-01

Extracellular nucleotides are potent signaling molecules mediating cell-specific biological functions, mostly within the processes of tissue damage and repair and flogosis. We previously demonstrated that adenosine 5′-triphosphate (ATP) inhibits the proliferation of human bone marrow-derived mesenchymal stem cells (BM-hMSCs), while stimulating, in vitro and in vivo, their migration. Here, we investigated the effects of ATP on BM-hMSC differentiation capacity. Molecular analysis showed that ATP treatment modulated the expression of several genes governing adipogenic and osteoblastic (ie, WNT-pathway-related genes) differentiation of MSCs. Functional studies demonstrated that ATP, under specific culture conditions, stimulated adipogenesis by significantly increasing the lipid accumulation and the expression levels of the adipogenic master gene PPARγ (peroxisome proliferator-activated receptor-gamma). In addition, ATP stimulated osteogenic differentiation by promoting mineralization and expression of the osteoblast-related gene RUNX2 (runt-related transcription factor 2). Furthermore, we demonstrated that ATP stimulated adipogenesis via its triphosphate form, while osteogenic differentiation was induced by the nucleoside adenosine, resulting from ATP degradation induced by CD39 and CD73 ectonucleotidases expressed on the MSC membrane. The pharmacological profile of P2 purinergic receptors (P2Rs) suggests that adipogenic differentiation is mainly mediated by the engagement of P2Y1 and P2Y4 receptors, while stimulation of the P1R adenosine-specific subtype A2B is involved in adenosine-induced osteogenic differentiation. Thus, we provide new insights into molecular regulation of MSC differentiation. PMID:23259837

Cot/tpl2-MKK1/2-Erk1/2 controls mTORC1-mediated mRNA translation in Toll-like receptor-activated macrophages.

PubMed

López-Pelaéz, Marta; Fumagalli, Stefano; Sanz, Carlos; Herrero, Clara; Guerra, Susana; Fernandez, Margarita; Alemany, Susana

2012-08-01

Cot/tpl2 is the only MAP3K that activates MKK1/2-Erk1/2 in Toll-like receptor-activated macrophages. Here we show that Cot/tpl2 regulates RSK, S6 ribosomal protein, and 4E-BP phosphorylation after stimulation of bone marrow-derived macrophages with lipopolysaccharide (LPS), poly I:C, or zymosan. The dissociation of the 4E-BP-eIF4E complex, a key event in the cap-dependent mRNA translation initiation, is dramatically reduced in LPS-stimulated Cot/tpl2-knockout (KO) macrophages versus LPS-stimulated wild-type (Wt) macrophages. Accordingly, after LPS activation, increased cap-dependent translation is observed in Wt macrophages but not in Cot/tpl2 KO macrophages. In agreement with these data, Cot/tpl2 increases the polysomal recruitment of the 5´ TOP eEF1α and eEF2 mRNAs, as well as of inflammatory mediator gene-encoding mRNAs, such as tumor necrosis factor α (TNFα), interleukin-6 (IL-6), and KC in LPS-stimulated macrophages. In addition, Cot/tpl2 deficiency also reduces total TNFα, IL-6, and KC mRNA expression in LPS-stimulated macrophages, which is concomitant with a decrease in their mRNA half-lives. Macrophages require rapid fine control of translation to provide an accurate and not self-damaging response to host infection, and our data show that Cot/tpl2 controls inflammatory mediator gene-encoding mRNA translation in Toll-like receptor-activated macrophages.

Cancer Cell-derived Exosomes Induce Mitogen-activated Protein Kinase-dependent Monocyte Survival by Transport of Functional Receptor Tyrosine Kinases*

PubMed Central

Song, Xiao; Ding, Yanping; Liu, Gang; Yang, Xiao; Zhao, Ruifang; Zhang, Yinlong; Zhao, Xiao; Anderson, Gregory J.; Nie, Guangjun

2016-01-01

Tumor-associated macrophages (TAM) play pivotal roles in cancer initiation and progression. Monocytes, the precursors of TAMs, normally undergo spontaneous apoptosis within 2 days, but can subsist in the inflammatory tumor microenvironment for continuous survival and generation of sufficient TAMs. The mechanisms underlying tumor-driving monocyte survival remain obscure. Here we report that cancer cell-derived exosomes were crucial mediators for monocyte survival in the inflammatory niche. Analysis of the survival-promoting molecules in monocytes revealed that cancer cell-derived exosomes activated Ras and extracellular signal-regulated kinases in the mitogen-activated protein kinase (MAPK) pathway, resulting in the prevention of caspase cleavage. Phosphorylated receptor tyrosine kinases (RTKs), such as phosphorylated epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER-2), were abundantly expressed in cancer cell-derived exosomes. Knock-out of EGFR or/and HER-2, or alternatively, inhibitors against their phosphorylation significantly disturbed the exosome-mediated activation of the MAPK pathway, inhibition of caspase cleavage, and increase in survival rate in monocytes. Moreover, the deprived survival-stimulating activity of exosomes due to null expression of EGFR and HER-2 could be restored by activation of another RTK, insulin receptor. Overall, our study uncovered a mechanism of tumor-associated monocyte survival and demonstrated that cancer cell-derived exosomes can stimulate the MAPK pathway in monocytes through transport of functional RTKs, leading to inactivation of apoptosis-related caspases. This work provides insights into the long sought question on monocyte survival prior to formation of plentiful TAMs in the tumor microenvironment. PMID:26895960

[Significance of CRF and dopamine receptors in amygdala for reinforcing effects of opiates and opioids on self-stimulation of lateral hypothalamus in rats].

PubMed

Shabanov, P D; Lebedev, A A; Liubimov, A V; Kornilov, V A

2011-01-01

Bipolar electrodes were implanted in the lateral hypothalamus in a group of 44 Wistar male rats in order to study self-stimulation reaction in the Skinner box. Simultaneously, microcanules were implanted into the central nucleus of the amygdala to inject the drugs (1 microl per injection). The blockade of corticoliberin (CRF) receptors (astressin, 1 microg) or Na+influx currents (xycaine or lidocain 1 microg) by the intrastructural administration of drugs into the amygdala decreased self-stimulation reaction of the lateral hypothalamus in rats by 29-55%. The inhibition of D1 and D2 dopamine receptors in the amygdala with SCH23390 (1 microg) or sulpiride (1 microg) respectively, also reduced self-stimulation but to a lower degree. On the background of blockade of CRF (astressin) and dopamine (sulpiride) receptors as well as sodium influx ionic currents (lidocain) in the amygdala neurons, psychomotor stimulant amphetamine (1 mg/kg) and barbiturate sodium ethaminal (5 mg/kg) retained their psychoactivating effect on self-stimulation (+30-37%), while fentanyl (0.1 mg/kg) and leu-enkephaline (0.1 mg/kg) did not produce this effect. Fentanyl moderately activated self-stimulation only after the blockade of D1 dopamine receptors with SCH23390. After the blockade of CRF receptors, leu-enkephaline strengthened its depressant effect on self-stimulation reaction (-89%). Therefore, if the modulating action of amygdala on the hypothalamus is eliminated, the enhancing effects of opiates (fentanyl) and opioids (leu-encephaline) are blocked, but the effects of psychomotor stimulant amphetamine and barbiturate sodium ethaminal are retained.

TAM receptor-dependent regulation of SOCS3 and MAPKs contributes to pro-inflammatory cytokine downregulation following chronic NOD2 stimulation of human macrophages1

PubMed Central

Zheng, Shasha; Hedl, Matija; Abraham, Clara

2014-01-01

Microbial-induced cytokine regulation is critical to intestinal immune homeostasis. Acute stimulation of NOD2, the Crohn’s disease-associated sensor of bacterial peptidoglycan, induces cytokines. However, cytokines are attenuated after chronic NOD2 and pattern recognition receptor (PRR) stimulation of macrophages; similar attenuation is observed in intestinal macrophages. The role of Tyro3, Axl and Mer (TAM) receptors in regulating chronic PRR stimulation and NOD2-induced outcomes has not been examined. Moreover, TAM receptors have been relatively less investigated in human macrophages. Whereas TAM receptors did not downregulate acute NOD2-induced cytokines in primary human macrophages, they were essential for downregulating signaling and pro-inflammatory cytokine secretion after chronic NOD2 and TLR4 stimulation. Axl and Mer were similarly required in mice for cytokine downregulation after chronic NOD2 stimulation in vivo and in intestinal tissues. Consistently, TAM expression was increased in human intestinal myeloid-derived cells. Chronic NOD2 stimulation led to IL-10- and TGFβ-dependent TAM upregulation in human macrophages, which in turn, upregulated SOCS3 expression. Restoring SOCS3 expression under TAM knockdown conditions restored chronic NOD2-mediated pro-inflammatory cytokine downregulation. In contrast to the upregulated pro-inflammatory cytokines, attenuated IL-10 secretion was maintained in TAM-deficient macrophages upon chronic NOD2 stimulation. The level of MAPK activation in TAM-deficient macrophages after chronic NOD2 stimulation was insufficient to upregulate IL-10 secretion; however, full restoration of MAPK activation under these conditions restored c-Fos, c-Jun, MAFK and PU.1 binding to the IL-10 promoter and IL-10 secretion. Therefore, TAM receptors are critical for downregulating pro-inflammatory cytokines under the chronic NOD2 stimulation conditions observed in the intestinal environment. PMID:25567680

Smoking-associated lung cancer prevention by blockade of the beta-adrenergic receptor-mediated insulin-like growth factor receptor activation.

PubMed

Min, Hye-Young; Boo, Hye-Jin; Lee, Ho Jin; Jang, Hyun-Ji; Yun, Hye Jeong; Hwang, Su Jung; Smith, John Kendal; Lee, Hyo-Jong; Lee, Ho-Young

2016-10-25

Activation of receptor tyrosine kinases (RTKs) is associated with carcinogenesis, but its contribution to smoking-associated lung carcinogenesis is poorly understood. Here we show that a tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced insulin-like growth factor 1 receptor (IGF-1R) activation via β-adrenergic receptor (β-AR) is crucial for smoking-associated lung carcinogenesis. Treatment with NNK stimulated the IGF-1R signaling pathway in a time- and dose-dependent manner, which was suppressed by pharmacological or genomic blockade of β-AR and the downstream signaling including a Gβγ subunit of β-AR and phospholipase C (PLC). Consistently, β-AR agonists led to increased IGF-1R phosphorylation. The increase in IGF2 transcription via β-AR, signal transducer and activator of transcription 3 (STAT3), and nuclear factor-kappa B (NF-κB) was associated with NNK-induced IGF-1R activation. Finally, treatment with β-AR antagonists suppressed the acquisition of transformed phenotypes in lung epithelial cells and lung tumor formation in mice. These results suggest that blocking β-AR-mediated IGF-1R activation can be an effective strategy for lung cancer prevention in smokers.

TAM receptor-dependent regulation of SOCS3 and MAPKs contributes to proinflammatory cytokine downregulation following chronic NOD2 stimulation of human macrophages.

PubMed

Zheng, Shasha; Hedl, Matija; Abraham, Clara

2015-02-15

Microbial-induced cytokine regulation is critical to intestinal immune homeostasis. Acute stimulation of nucleotide-binding oligomerization domain 2 (NOD2), the Crohn's disease-associated sensor of bacterial peptidoglycan, induces cytokines. However, cytokines are attenuated after chronic NOD2 and pattern recognition receptor stimulation of macrophages; similar attenuation is observed in intestinal macrophages. The role of Tyro3, Axl, and Mer (TAM) receptors in regulating chronic pattern recognition receptor stimulation and NOD2-induced outcomes has not been examined. Moreover, TAM receptors have been relatively less investigated in human macrophages. Whereas TAM receptors did not downregulate acute NOD2-induced cytokines in primary human macrophages, they were essential for downregulating signaling and proinflammatory cytokine secretion after chronic NOD2 and TLR4 stimulation. Axl and Mer were similarly required in mice for cytokine downregulation after chronic NOD2 stimulation in vivo and in intestinal tissues. Consistently, TAM expression was increased in human intestinal myeloid-derived cells. Chronic NOD2 stimulation led to IL-10- and TGF-β-dependent TAM upregulation in human macrophages, which, in turn, upregulated suppressor of cytokine signaling 3 expression. Restoring suppressor of cytokine signaling 3 expression under TAM knockdown conditions restored chronic NOD2-mediated proinflammatory cytokine downregulation. In contrast to the upregulated proinflammatory cytokines, attenuated IL-10 secretion was maintained in TAM-deficient macrophages upon chronic NOD2 stimulation. The level of MAPK activation in TAM-deficient macrophages after chronic NOD2 stimulation was insufficient to upregulate IL-10 secretion; however, full restoration of MAPK activation under these conditions restored c-Fos, c-Jun, musculoaponeurotic fibrosarcoma oncogene homolog K, and PU.1 binding to the IL-10 promoter and IL-10 secretion. Therefore, TAM receptors are critical for downregulating proinflammatory cytokines under the chronic NOD2 stimulation conditions observed in the intestinal environment. Copyright © 2015 by The American Association of Immunologists, Inc.

DIRECT MODULATION OF THE PROTEIN KINASE A CATALYTIC SUBUNIT α BY GROWTH FACTOR RECEPTOR TYROSINE KINASES

PubMed Central

Caldwell, George B.; Howe, Alan K.; Nickl, Christian K.; Dostmann, Wolfgang R.; Ballif, Bryan A.; Deming, Paula B.

2011-01-01

The cyclic-AMP-dependent protein kinase A (PKA) regulates processes such as cell proliferation and migration following activation of growth factor receptor tyrosine kinases (RTKs), yet the signaling mechanisms that link PKA with growth factor receptors remain largely undefined. Here we report that RTKs can directly modulate the function of the catalytic subunit of PKA (PKA-C) through post-translational modification. In vitro kinase assays revealed that both the epidermal growth factor and platelet derived growth factor receptors (EGFR and PDGFR, respectively) tyrosine phosphorylate PKA-C. Mass spectrometry identified tyrosine 330 (Y330) as a receptor-mediated phosphorylation site and mutation of Y330 to phenylalanine (Y330F) all but abolished the RTK-mediated phosphorylation of PKA-C in vitro. Y330 resides within a conserved region at the C-terminal tail of PKA-C that allosterically regulates enzymatic activity. Therefore, the effect of phosphorylation at Y330 on the activity of PKA-C was investigated. The Km for a peptide substrate was markedly decreased when PKA-C subunits were tyrosine phosphorylated by the receptors as compared to un-phosphorylated controls. Importantly, tyrosine-phosphorylated PKA-C subunits were detected in cells stimulated with EGF, PDGF and FGF2 and in fibroblasts undergoing PDGF-mediated chemotaxis. These results demonstrate a direct, functional interaction between RTKs and PKA-C and identify tyrosine phosphorylation as a novel mechansim for regulating PKA activity. PMID:21866565

Identification of heparin-binding EGF-like growth factor as a target in intercellular regulation of epidermal basal cell growth by suprabasal retinoic acid receptors.

PubMed Central

Xiao, J H; Feng, X; Di, W; Peng, Z H; Li, L A; Chambon, P; Voorhees, J J

1999-01-01

The role of retinoic acid receptors (RARs) in intercellular regulation of cell growth was assessed by targeting a dominant-negative RARalpha mutant (dnRARalpha) to differentiated suprabasal cells of mouse epidermis. dnRARalpha lacks transcriptional activation but not DNA-binding and receptor dimerization functions. Analysis of transgenic mice revealed that dnRARalpha dose-dependently impaired induction of basal cell proliferation and epidermal hyperplasia by all-trans RA (tRA). dnRARalpha formed heterodimers with endogenous retinoid X receptor-alpha (RXRalpha) over RA response elements in competition with remaining endogenous RARgamma-RXRalpha heterodimers, and dose-dependently impaired retinoid-dependent gene transcription. To identify genes regulated by retinoid receptors and involved in cell growth control, we analyzed the retinoid effects on expression of the epidermal growth factor (EGF) receptor, EGF, transforming growth factor-alpha, heparin-binding EGF-like growth factor (HB-EGF) and amphiregulin genes. In normal epidermis, tRA rapidly and selectively induced expression of HB-EGF but not the others. This induction occurred exclusively in suprabasal cells. In transgenic epidermis, dnRARalpha dose-dependently inhibited tRA induction of suprabasal HB-EGF and subsequent basal cell hyperproliferation. Together, our observations suggest that retinoid receptor heterodimers located in differentiated suprabasal cells mediate retinoid induction of HB-EGF, which in turn stimulates basal cell growth via intercellular signaling. These events may underlie retinoid action in epidermal regeneration during wound healing. PMID:10075925

Ginkgolide B Reduces LOX-1 Expression by Inhibiting Akt Phosphorylation and Increasing Sirt1 Expression in Oxidized LDL-Stimulated Human Umbilical Vein Endothelial Cells

PubMed Central

Chen, Beidong; Li, Xingguang; Qi, Ruomei

2013-01-01

Oxidized low-density lipoprotein (ox-LDL) is an important risk factor in the development of atherosclerosis. LOX-1, a lectin-like receptor for ox-LDL, is present primarily on endothelial cells and upregulated by ox-LDL, tumor necrosis factor a, shear stress, and cytokines in atherosclerosis. Recent studies demonstrated that ginkgolide B, a platelet-activating factor receptor antagonist, has antiinflammatory and antioxidant effects on endothelial and nerve cells. The present study investigated the effects of ginkgolide B on LOX-1 expression and the possible mechanism of action. Our results showed that ginkgolide B inhibited LOX-1 and intercellular cell adhesion molecule-1 (ICAM-1) expression in ox-LDL-stimulated endothelial cells through a mechanism associated with the attenuation of Akt activation. Similar data were obtained by silencing Akt and LY294002. We also evaluated Sirt1 and nuclear factor erythroid 2-related factor 2 (Nrf2) expression. These molecules play a protective role in endothelial cell injury. The results showed that ginkgolide B increased Sirt1 expression in ox-LDL-treated cells. The inhibitory effects of ginkgolide B on LOX-1 and ICAM-1 expression were reduced in Sirt1 siRNA-transfected cells. Nrf2 expression was increased in ox-LDL-treated cells, and ginkgolide B downregulated Nrf2 expression. These results suggest that ginkgolide B reduces Nrf2 expression by inhibiting LOX-1 expression, consequently reducing oxidative stress injury in ox-LDL-stimulated cells. Altogether, these results indicate that the protective effect of ginkgolide B on endothelial cells may be attributable to a decrease in LOX-1 expression and an increase in Sirt1 expression in ox-LDL-stimulated endothelial cells, the mechanism of which is linked to the inhibition of Akt activation. Ginkgolide B may be a multiple-target drug that exerts protective effects in ox-LDL-treated human umbilical vein endothelial cells. PMID:24069345

Monocyte Tumor Necrosis Factor-α–Converting Enzyme Catalytic Activity and Substrate Shedding in Sepsis and Noninfectious Systemic Inflammation*

PubMed Central

O’Callaghan, David J. P.; O’Dea, Kieran P.; Scott, Alasdair J.; Takata, Masao

2015-01-01

Objectives: To determine the effect of severe sepsis on monocyte tumor necrosis factor-α–converting enzyme baseline and inducible activity profiles. Design: Observational clinical study. Setting: Mixed surgical/medical teaching hospital ICU. Patients: Sixteen patients with severe sepsis, 15 healthy volunteers, and eight critically ill patients with noninfectious systemic inflammatory response syndrome. Interventions: None. Measurements and Main Results: Monocyte expression of human leukocyte antigen-D-related peptide, sol-tumor necrosis factor production, tumor necrosis factor-α–converting enzyme expression and catalytic activity, tumor necrosis factor receptor 1 and 2 expression, and shedding at 48-hour intervals from day 0 to day 4, as well as p38-mitogen activated protein kinase expression. Compared with healthy volunteers, both sepsis and systemic inflammatory response syndrome patients’ monocytes expressed reduced levels of human leukocyte antigen-D-related peptide and released less sol-tumor necrosis factor on in vitro lipopolysaccharide stimulation, consistent with the term monocyte deactivation. However, patients with sepsis had substantially elevated levels of basal tumor necrosis factor-α–converting enzyme activity that were refractory to lipopolysaccharide stimulation and this was accompanied by similar changes in p38-mitogen activated protein kinase signaling. In patients with systemic inflammatory response syndrome, monocyte basal tumor necrosis factor-α–converting enzyme, and its induction by lipopolysaccharide, appeared similar to healthy controls. Changes in basal tumor necrosis factor-α–converting enzyme activity at day 0 for sepsis patients correlated with Acute Physiology and Chronic Health Evaluation II score and the attenuated tumor necrosis factor-α–converting enzyme response to lipopolysaccharide was associated with increased mortality. Similar changes in monocyte tumor necrosis factor-α–converting enzyme activity could be induced in healthy volunteer monocytes using an in vitro two-hit inflammation model. Patients with sepsis also displayed reduced shedding of monocyte tumor necrosis factor receptors upon stimulation with lipopolysaccharide. Conclusions: Monocyte tumor necrosis factor-α–converting enzyme catalytic activity appeared altered by sepsis and may result in reduced shedding of tumor necrosis factor receptors. Changes seemed specific to sepsis and correlated with illness severity. A better understanding of how tumor necrosis factor-α–converting enzyme function is altered during sepsis will enhance our understanding of sepsis pathophysiology, which will help in the assessment of patient inflammatory status and ultimately may provide new strategies to treat sepsis. PMID:25867908

Glutamate Signaling and Mitochnodrial Dysfunction in Models of Parkinson’s Disease

DTIC Science & Technology

2012-12-01

synaptic response; 3) antagonism of ionotropic glutamate receptors essentially eliminates the response to PPN stimulation, suggesting that nicotinic...systemic administration of drug, significantly lowers mitochondrial oxidant stress. Third, antagonizing glutamatergic NMDA receptors , but not...metabotropic glutamate receptors , diminishes oxidant stress in dopaminergic neurons; stimulating NMDA receptors raises stress levels. Fourth, blocking

Peptide hormones and lung cancer.

PubMed

Moody, T W

2006-03-01

Several peptide hormones have been identified which alter the proliferation of lung cancer. Small cell lung cancer (SCLC), which is a neuroendocrine cancer, produces and secretes gastrin releasing peptide (GRP), neurotensin (NT) and adrenomedullin (AM) as autocrine growth factors. GRP, NT and AM bind to G-protein coupled receptors causing phosphatidylinositol turnover or elevated cAMP in SCLC cells. Addition of GRP, NT or AM to SCLC cells causes altered expression of nuclear oncogenes, such as c-fos, and stimulation of growth. Antagonists have been developed for GRP, NT and AM receptors which function as cytostatic agents and inhibit SCLC growth. Growth factor antagonists, such as the NT1 receptor antagonist SR48692, facilitate the ability of chemotherapeutic drugs to kill lung cancer cells. It remains to be determined if GRP, NT and AM receptors will served as molecular targets, for development of new therapies for the treatment of SCLC patients. Non-small cell lung cancer (NSCLC) cells also have a high density of GRP, NT, AM and epidermal growth factor (EGF) receptors. Several NSCLC patients with EGF receptor mutations respond to gefitinib, a tyrosine kinase inhibitor. Gefitinib relieves NSCLC symptoms, maintaining stable disease in patients who are not eligible for systemic chemotherapy. It is important to develop new therapeutic approaches using translational research techniques for the treatment of lung cancer patients.

Estradiol coupling to human monocyte nitric oxide release is dependent on intracellular calcium transients: evidence for an estrogen surface receptor.

PubMed

Stefano, G B; Prevot, V; Beauvillain, J C; Fimiani, C; Welters, I; Cadet, P; Breton, C; Pestel, J; Salzet, M; Bilfinger, T V

1999-10-01

We tested the hypothesis that estrogen acutely stimulates constitutive NO synthase (cNOS) activity in human peripheral monocytes by acting on an estrogen surface receptor. NO release was measured in real time with an amperometric probe. 17beta-estradiol exposure to monocytes stimulated NO release within seconds in a concentration-dependent manner, whereas 17alpha-estradiol had no effect. 17beta-estradiol conjugated to BSA (E2-BSA) also stimulated NO release, suggesting mediation by a membrane surface receptor. Tamoxifen, an estrogen receptor inhibitor, antagonized the action of both 17beta-estradiol and E2-BSA, whereas ICI 182,780, a selective inhibitor of the nuclear estrogen receptor, had no effect. We further showed, using a dual emission microfluorometry in a calcium-free medium, that the 17beta-estradiol-stimulated release of monocyte NO was dependent on the initial stimulation of intracellular calcium transients in a tamoxifen-sensitive process. Leeching out the intracellular calcium stores abolished the effect of 17beta-estradiol on NO release. RT-PCR analysis of RNA obtained from the cells revealed a strong estrogen receptor-alpha amplification signal and a weak beta signal. Taken together, a physiological dose of estrogen acutely stimulates NO release from human monocytes via the activation of an estrogen surface receptor that is coupled to increases in intracellular calcium.

Altered (/sup 125/I)epidermal growth factor binding and receptor distribution in psoriasis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nanney, L.B.; Stoscheck, C.M.; Magid, M.

1986-03-01

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 normalmore » 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 (/sup 125/I)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.« less

Quantification of transcription factor-DNA binding affinity in a living cell

PubMed Central

Belikov, Sergey; Berg, Otto G.; Wrange, Örjan

2016-01-01

The apparent dissociation constant (Kd) for specific binding of glucocorticoid receptor (GR) and androgen receptor (AR) to DNA was determined in vivo in Xenopus oocytes. The total nuclear receptor concentration was quantified as specifically retained [3H]-hormone in manually isolated oocyte nuclei. DNA was introduced by nuclear microinjection of single stranded phagemid DNA, chromatin is then formed during second strand synthesis. The fraction of DNA sites occupied by the expressed receptor was determined by dimethylsulphate in vivo footprinting and used for calculation of the receptor-DNA binding affinity. The forkhead transcription factor FoxA1 enhanced the DNA binding by GR with an apparent Kd of ∼1 μM and dramatically stimulated DNA binding by AR with an apparent Kd of ∼0.13 μM at a composite androgen responsive DNA element containing one FoxA1 binding site and one palindromic hormone receptor binding site known to bind one receptor homodimer. FoxA1 exerted a weak constitutive- and strongly cooperative DNA binding together with AR but had a less prominent effect with GR, the difference reflecting the licensing function of FoxA1 at this androgen responsive DNA element. PMID:26657626

Role of CRF Receptor Signaling in Stress Vulnerability, Anxiety, and Depression

PubMed Central

Hauger, Richard L.; Risbrough, Victoria; Oakley, Robert H.; Olivares-Reyes, J. Alberto; Dautzenberg, Frank M.

2011-01-01

Markers of hyperactive central corticotropin releasing factor (CRF) systems and CRF-related single nucleotide polymorphisms (SNPs) have been identified in patients with anxiety and depressive disorders. Designing more effective antagonists may now be guided by data showing that small molecules bind to transmembrane domains. Specifically, CRF1 receptor antagonists have been developed as novel anxiolytic and antidepressant treatments. Because CRF1 receptors become rapidly desensitized by G protein-coupled receptor kinase (GRK) and β-arrestin mechanisms in the presence of high agonist concentrations, neuronal hypersecretion of synaptic CRF alone may be insufficient to account for excessive central CRF neurotransmission in stress-induced affective pathophysiology. In addition to desensitizing receptor function, GRK phosphorylation and β-arrestin binding can shift a G protein-coupled receptor (GPCR) to signal selectively via the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK-MAPK) or Akt pathways independent of G proteins. Also, Epac-dependent CRF1 receptor signaling via the ERK-MAPK pathway has been found to potentiate brain-derived neurotrophic factor (BDNF)-stimulated TrkB signaling. Thus, genetic or acquired abnormalities in GRK and β-arrestin function may be involved in the pathophysiology of stress-induced anxiety and depression. PMID:19906236

Phospholipase C-{delta}{sub 1} regulates interleukin-1{beta} and tumor necrosis factor-{alpha} mRNA expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chung, Eric; Jakinovich, Paul; Bae, Aekyung

Phospholipase C-{delta}{sub 1} (PLC{delta}{sub 1}) is a widely expressed highly active PLC isoform, modulated by Ca{sup 2+} that appears to operate downstream from receptor signaling and has been linked to regulation of cytokine production. Here we investigated whether PLC{delta}{sub 1} modulated expression of the pro-inflammatory cytokines interleukin-1{beta} (IL-1{beta}), tumor necrosis factor-{alpha} (TNF-{alpha}) and interleukin-6 (IL-6) in rat C6 glioma cells. Expression of PLC{delta}{sub 1} was specifically suppressed by small interfering RNA (siRNA) and the effects on cytokine mRNA expression, stimulated by the Toll-like receptor (TLR) agonist, lipopolysaccharide (LPS), were examined. Real-time polymerase chain reaction (RT-PCR) results showed that PLC{delta}{sub 1}more » knockdown enhanced expression IL-1{beta} and tumor necrosis factor-{alpha} (TNF-{alpha}) mRNA by at least 100 fold after 4 h of LPS stimulation compared to control siRNA treatment. PLC{delta}{sub 1} knock down caused persistently high Nf{kappa}b levels at 4 h of LPS stimulation compared to control siRNA-treated cells. PLC{delta}{sub 1} knockdown was also associated with elevated nuclear levels of c-Jun after 30 min of LPS stimulation, but did not affect LPS-stimulated p38 or p42/44 MAPK phosphorylation, normally associated with TLR activation of cytokine gene expression; rather, enhanced protein kinase C (PKC) phosphorylation of cellular proteins was observed in the absence of LPS stimulation. An inhibitor of PKC, bisindolylmaleimide II (BIM), reversed phosphorylation, prevented elevation of nuclear c-Jun levels, and inhibited LPS-induced increases of IL-1{beta} and TNF-{alpha} mRNA's induced by PLC{delta}{sub 1} knockdown. Our results show that loss of PLC{delta}{sub 1} enhances PKC/c-Jun signaling and up-modulates pro-inflammatory cytokine gene transcription in concert with the TLR-stimulated p38MAPK/Nf{kappa}b pathway. Our findings are consistent with the idea that PLC{delta}{sub 1} is a suppressor of PKC activity.« less

The Jak-STAT pathway stimulated by interferon alpha or interferon beta.

PubMed

Horvath, Curt M

2004-11-23

Type I interferons, such as interferon alpha and interferon beta (IFN-alpha and beta), signal through a Janus kinase (Jak) to signal transduction and activator of transcription (STAT) pathway to stimulate gene expression. In response to ligand binding, the receptors dimerize, Jaks phosphorylate STAT1 and STAT2, which then dimerize and interact with a third transcriptional regulator IFN regulatory factor 9 (IRF9) to stimulate gene expression. IFN-alpha is the main innate antiviral cytokine and is essential for effective immune response to viral infection. The animation shows activation of STAT-responsive gene expression in response to type I IFNs.

Dopamine D2High receptors stimulated by phencyclidines, lysergic acid diethylamide, salvinorin A, and modafinil.

PubMed

Seeman, Philip; Guan, Hong-Chang; Hirbec, Hélène

2009-08-01

Although it is commonly stated that phencyclidine is an antagonist at ionotropic glutamate receptors, there has been little measure of its potency on other receptors in brain tissue. Although we previously reported that phencyclidine stimulated cloned-dopamine D2Long and D2Short receptors, others reported that phencyclidine did not stimulate D2 receptors in homogenates of rat brain striatum. This study, therefore, examined whether phencyclidine and other hallucinogens and psychostimulants could stimulate the incorporation of [(35)S]GTP-gamma-S into D2 receptors in homogenates of rat brain striatum, using the same conditions as previously used to study the cloned D2 receptors. Using 10 microM dopamine to define 100% stimulation, phencyclidine elicited a maximum incorporation of 46% in rat striata, with a half-maximum concentration of 70 nM for phencyclidine, when compared with 80 nM for dopamine, 89 nM for salvinorin A (48 nM for D2Long), 105 nM for lysergic acid diethylamide (LSD), 120 nM for R-modafinil, 710 nM for dizocilpine, 1030 nM for ketamine, and >10,000 nM for S-modafinil. These compounds also inhibited the binding of the D2-selective ligand [(3)H]domperidone. The incorporation was inhibited by the presence of 200 microM guanylylimidodiphosphate and also by D2 blockade, using 10 microM S-sulpiride, but not by D1 blockade with 10 microM SCH23390. Hypertonic buffer containing 150 mM NaCl inhibited the stimulation by phencyclidine, which may explain negative results by others. It is concluded that phencyclidine and other psychostimulants and hallucinogens can stimulate dopamine D2 receptors at concentrations related to their behavioral actions.

Odontoblasts as sensory receptors: transient receptor potential channels, pannexin-1, and ionotropic ATP receptors mediate intercellular odontoblast-neuron signal transduction.

PubMed

Shibukawa, Yoshiyuki; Sato, Masaki; Kimura, Maki; Sobhan, Ubaidus; Shimada, Miyuki; Nishiyama, Akihiro; Kawaguchi, Aya; Soya, Manabu; Kuroda, Hidetaka; Katakura, Akira; Ichinohe, Tatsuya; Tazaki, Masakazu

2015-04-01

Various stimuli induce pain when applied to the surface of exposed dentin. However, the mechanisms underlying dentinal pain remain unclear. We investigated intercellular signal transduction between odontoblasts and trigeminal ganglion (TG) neurons following direct mechanical stimulation of odontoblasts. Mechanical stimulation of single odontoblasts increased the intracellular free calcium concentration ([Ca(2+)]i) by activating the mechanosensitive-transient receptor potential (TRP) channels TRPV1, TRPV2, TRPV4, and TRPA1, but not TRPM8 channels. In cocultures of odontoblasts and TG neurons, increases in [Ca(2+)]i were observed not only in mechanically stimulated odontoblasts, but also in neighboring odontoblasts and TG neurons. These increases in [Ca(2+)]i were abolished in the absence of extracellular Ca(2+) and in the presence of mechanosensitive TRP channel antagonists. A pannexin-1 (ATP-permeable channel) inhibitor and ATP-degrading enzyme abolished the increases in [Ca(2+)]i in neighboring odontoblasts and TG neurons, but not in the stimulated odontoblasts. G-protein-coupled P2Y nucleotide receptor antagonists also inhibited the increases in [Ca(2+)]i. An ionotropic ATP (P2X3) receptor antagonist inhibited the increase in [Ca(2+)]i in neighboring TG neurons, but not in stimulated or neighboring odontoblasts. During mechanical stimulation of single odontoblasts, a connexin-43 blocker did not have any effects on the [Ca(2+)]i responses observed in any of the cells. These results indicate that ATP, released from mechanically stimulated odontoblasts via pannexin-1 in response to TRP channel activation, transmits a signal to P2X3 receptors on TG neurons. We suggest that odontoblasts are sensory receptor cells and that ATP released from odontoblasts functions as a neurotransmitter in the sensory transduction sequence for dentinal pain.

Effects of different ligands on epidermal growth factor receptor (EGFR) nuclear translocation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Faria, Jerusa A.Q.A.; Andrade, Carolina de; Goes, Alfredo M.

The epidermal growth factor receptor (EGFR) is activated through binding to specific ligands and generates signals for proliferation, differentiation, migration, and cell survival. Recent data show the role of nuclear EGFR in tumors. Although many EGFR ligands are upregulated in cancers, little is known about their effects on EGFR nuclear translocation. We have compared the effects of six EGFR ligands (EGF, HB-EGF, TGF-α, β-Cellulin, amphiregulin, and epiregulin) on nuclear translocation of EGFR, receptor phosphorylation, migration, and proliferation. Cell fractionation and confocal immunofluorescence detected EGFR in the nucleus after EGF, HB-EGF, TGF-α and β-Cellulin stimulation in a dose-dependent manner. In contrast,more » amphiregulin and epiregulin did not generate nuclear translocation of EGFR. EGF, HB-EGF, TGF-α and β-Cellulin showed correlations between a higher rate of wound closure and increased phosphorylation of residues in the carboxy-terminus of EGFR, compared to amphiregulin and epiregulin. The data indicate that EGFR is translocated to the nucleus after stimulation with EGF, HB-EGF, TGF-α and β-Cellulin, and that these ligands are related to increased phosphorylation of EGFR tyrosine residues, inducing migration of SkHep-1 cells. - Highlights: • EGF, HB-EGF, TGF-α, β-Cellulin are involved in the EGFR nuclear translocation. • Amphiregulin and epiregulin did not promote nuclear translocation of EGFR. • EGF, HB-EGF, TGF-α and β-Cellulin have a role in SkHep-1 cells migration. • EGFR ligands associated with better prognosis don't stimulate EGFR translocation.« less

Peripheral corticotropin-releasing factor (CRF) induces stimulation of gastric contractions in freely moving conscious rats: role of CRF receptor types 1 and 2.

PubMed

Nozu, T; Tsuchiya, Y; Kumei, S; Takakusaki, K; Okumura, T

2013-02-01

Peripheral corticotrophin-releasing factor (CRF) plays an important role in stress-induced alterations of gastrointestinal motility. CRF injected peripherally inhibits gastric emptying, but its effect on gastric contractions has not been clarified in freely moving conscious rats. Intraluminal gastric pressure waves were measured in freely moving conscious non-fasted rats using the perfused manometric method. We assessed the area under the manometric trace as the motor index (MI), and compared this result with those obtained 1 h before and after drug administration. Subcutaneous injection (sc) of CRF (15 μg kg(-1)) increased the MI significantly. Pretreatment with intravenous astressin (100 μg kg(-1)), a non-selective CRF antagonist, blocked the sc CRF (15 μg kg(-1))-induced response, but astressin(2)-B (200 μg kg(-1), sc), a selective CRF receptor type 2 (CRF(2)) antagonist, enhanced the CRF-induced increase in MI significantly. Meanwhile urocortin 2 (15 μg kg(-1), sc), a selective CRF(2) agonist, did not alter the basal MI, but it inhibited the sc CRF (15 μg kg(-1))-induced stimulation of gastric contractions. The intraperitoneal injection of cortagine (30 μg kg(-1)), a selective CRF receptor type 1 (CRF(1)) agonist, mimicked the response induced by sc CRF. Peripheral CRF stimulates gastric contractions through CRF(1). CRF(2) activation inhibits the response induced by CRF, suggesting that CRF(2) may have a modulatory action to CRF(1) signaling in gastric motor activity. © 2012 Blackwell Publishing Ltd.

Activation of phosphatidylinositol-3 kinase by nerve growth factor involves indirect coupling of the trk proto-oncogene with src homology 2 domains.

PubMed

Ohmichi, M; Decker, S J; Saltiel, A R

1992-10-01

Growth factor receptor tyrosine kinases can form stable associations with intracellular proteins that contain src homology (SH) 2 domains, including the p85 regulatory subunit of phosphatidylinositol (PI)-3 kinase. The activation of this enzyme by growth factors is evaluated in PC12 pheochromocytoma cells and NIH 3T3 fibroblasts expressing the pp140c-trk nerve growth factor (NGF) receptor (3T3-c-trk). NGF causes the rapid stimulation of PI-3 kinase activity detected in anti-phosphotyrosine, but not in anti-trk, immunoprecipitates. This effect coincides with the tyrosine phosphorylation of two proteins, with molecular masses of of 100 kd and 110 kd, that coimmunoprecipitate with p85. Similar phosphorylation patterns are induced when an immobilized fusion protein containing the amino-terminal SH2 domain of p85 is used to precipitate tyrosine-phosphorylated proteins. Thus, although NGF produces the rapid activation of PI-3 kinase through a mechanism that involves tyrosine phosphorylation, there is no evidence for tyrosine phosphorylation of p85, or for its ligand-dependent association with the NGF receptor. Perhaps another phosphoprotein may link the NGF receptor to this enzyme.

Suboptimal T-cell receptor signaling compromises protein translation, ribosome biogenesis, and proliferation of mouse CD8 T cells.

PubMed

Tan, Thomas C J; Knight, John; Sbarrato, Thomas; Dudek, Kate; Willis, Anne E; Zamoyska, Rose

2017-07-25

Global transcriptomic and proteomic analyses of T cells have been rich sources of unbiased data for understanding T-cell activation. Lack of full concordance of these datasets has illustrated that important facets of T-cell activation are controlled at the level of translation. We undertook translatome analysis of CD8 T-cell activation, combining polysome profiling and microarray analysis. We revealed that altering T-cell receptor stimulation influenced recruitment of mRNAs to heavy polysomes and translation of subsets of genes. A major pathway that was compromised, when TCR signaling was suboptimal, was linked to ribosome biogenesis, a rate-limiting factor in both cell growth and proliferation. Defective TCR signaling affected transcription and processing of ribosomal RNA precursors, as well as the translation of specific ribosomal proteins and translation factors. Mechanistically, IL-2 production was compromised in weakly stimulated T cells, affecting the abundance of Myc protein, a known regulator of ribosome biogenesis. Consequently, weakly activated T cells showed impaired production of ribosomes and a failure to maintain proliferative capacity after stimulation. We demonstrate that primary T cells respond to various environmental cues by regulating ribosome biogenesis and mRNA translation at multiple levels to sustain proliferation and differentiation.

FGFR and PTEN signaling interact during lens development to regulate cell survival

PubMed Central

Chaffee, Blake R.; Hoang, Thanh V.; Leonard, Melissa R.; Bruney, Devin G.; Wagner, Brad D.; Dowd, Joseph Richard; Leone, Gustavo; Ostrowski, Michael C.; Robinson, Michael L.

2016-01-01

Lens epithelial cells express many receptor tyrosine kinases (RTKs) that stimulate PI3K-AKT and RAS-RAF-MEK-ERK intracellular signaling pathways. These pathways ultimately activate the phosphorylation of key cellular transcription factors and other proteins that control proliferation, survival, metabolism, and differentiation in virtually all cells. Among RTKs in the lens, only stimulation of fibroblast growth factor receptors (FGFRs) elicits a lens epithelial cell to fiber cell differentiation response in mammals. Moreover, although the lens expresses three different Fgfr genes, the isolated removal of Fgfr2 at the lens placode stage inhibits both lens cell survival and fiber cell differentiation. Phosphatase and tensin homolog (PTEN), commonly known as a tumor suppressor, inhibits ERK and AKT activation and initiates both apoptotic pathways, and cell cycle arrest. Here, we show that the combined deletion of Fgfr2 and Pten rescues the cell death phenotype associated with Fgfr2 loss alone. Additionally, Pten removal increased AKT and ERK activation, above the levels of controls, in the presence or absence of Fgfr2. However, isolated deletion of Pten failed to stimulate ectopic fiber cell differentiation, and the combined deletion of Pten and Fgfr2 failed to restore differentiation-specific Aquaporin0 and DnaseIIβ expression in the lens fiber cells. PMID:26764128

Granulocyte-macrophage and macrophage colony-stimulating factors differentially regulate alpha v integrin expression on cultured human macrophages.

PubMed

De Nichilo, M O; Burns, G F

1993-03-15

The colony-stimulating factors (CSFs) greatly influence mature macrophage function in vitro: macrophage (M)-CSF induces maturation of monocytes and enhances differentiated cell function; granulocyte-macrophage (GM)-CSF stimulates a variety of antimicrobial functions. In vivo M-CSF is thought to promote differentiation, and GM-CSF is thought to potentiate the inflammatory response. One mechanism by which these differential effects may be achieved is through the receptor-mediated interaction of macrophages with their extracellular matrix. Here we show that M-CSF induces specifically the expression of the alpha v beta 5 integrin receptor, whereas GM-CSF rapidly induces mRNA and surface expression of the alpha v beta 3 integrin. The M-CSF-treated cells acquire a flattened epitheloid phenotype, and on vitronectin the alpha v beta 5 is located in adhesion plaques. These cells do not bind collagen or laminin. In contrast, cells treated with GM-CSF adopt an elongated phenotype on a number of substrates, including collagen and laminin, and express alpha v beta 3 at the leading edge of cells on vitronectin. These results suggest that a primary means by which the CSFs exert their individual effects on mature cells may be through regulating integrin expression.

delta opioid receptors stimulate Akt-dependent phosphorylation of c-jun in T cells.

PubMed

Shahabi, Nahid A; McAllen, Kathy; Sharp, Burt M

2006-02-01

Activation of naive T cells markedly up-regulates the expression of delta opioid receptors (DORs). These receptors are bound by DOR peptides released by T cells, modulating T cell functions such as interleukin-2 production, cellular proliferation, and chemotaxis. Previous studies have shown that DOR agonists [e.g., [D-Ala(2)-D-Leu(5)]-enkephalin (DADLE)] modulate T cell antigen receptor signaling through mitogen-activated protein kinases (MAPKs; i.e., extracellular signal-regulated kinases 1 and 2) and that DORs directly induce phosphorylation of activating transcription factor-2 (implicated in cytokine gene transcription) and its association with the MAPK c-jun1 NH(2)-terminal kinase (JNK). Such observations suggest that DORs may induce the phosphorylation of c-jun. These experiments were performed to test this hypothesis and determine the potential roles of phosphoinositide 3-kinase (PI3K) and Akt (protein kinase B). DADLE (10(-10) to 10(-6) M) dose-dependently induced c-jun phosphorylation. This was blocked by pertussis toxin and the DOR-specific antagonist naltindole. Fluorescence flow cytometry showed that DADLE significantly stimulated c-jun phosphorylation by T cells. DADLE stimulated phosphorylation of membrane-associated Akt; wortmannin and LY294002 ([2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one]), specific inhibitors of PI3K, abolished the DADLE-induced phosphorylation of c-jun. Finally, inhibitors of Akt and JNK blocked DADLE-induced phosphorylation of c-jun. Thus, activated DORs directly stimulate c-jun phosphorylation through a PI3K-dependent pathway in T cells, apparently involving Akt. This implies that DORs activate JNK through a novel pathway dependent on PI3K and Akt, thereby regulating the function of activator protein-1 transcription complexes containing c-jun and other transcription partners.

The Structure of the GM-CSF Receptor Complex Reveals a Distinct Mode of Cytokine Receptor Activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hansen, Guido; Hercus, Timothy R.; McClure, Barbara J.

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 andmore » 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.« less

PGE2 signaling through the EP4 receptor on fibroblasts upregulates RANKL and stimulates osteolysis.

PubMed

Tsutsumi, Ryosuke; Xie, Chao; Wei, Xiaochao; Zhang, Minjie; Zhang, Xinping; Flick, Lisa M; Schwarz, Edward M; O'Keefe, Regis J

2009-10-01

Periprosthetic osteolysis is the most common cause of aseptic loosening in total joint arthroplasty. The role of inflammatory mediators such as prostaglandin E2 (PGE2) and osteoclast promoting factors including RANKL in the pathogenesis of osteolysis has been well characterized. However, the PGE2 receptor (EP1, EP2, or EP4), and cell type in which it is expressed, which is responsible for PGE2 induction of RANKL during wear debris-induced osteolysis, has yet to be elucidated. To address this, we used mice genetically deficient in these EP receptors to assess PGE2 and wear debris responses in vitro and in vivo. Wear debris-induced osteolysis and RANKL expression were observed at similar levels in WT, EP1(-/-), and EP2(-/-) mice, indicating that these receptors do not mediate PGE2 signals in this process. A conditional knockout approach was used to eliminate EP4 expression in FSP1(+) fibroblasts that are the predominant source of RANKL. In the absence of EP4, fibroblasts do not express RANKL after stimulation with particles or PGE2, nor do they exhibit high levels of osteoclasts and osteolysis. These results show that periprosthetic fibroblasts are important mediators of osteolysis through the expression of RANKL, which is induced after PGE2 signaling through the EP4 receptor.

Differential responses of EGFR-/AGT-expressing cells to the "combi-triazene" SMA41.

PubMed

Matheson, Stephanie L; McNamee, James P; Jean-Claude, Bertrand J

2003-01-01

Previous studies have demonstrated enhanced potency associated with the binary [DNA/epidermal growth factor receptor (EGFR)] targeting properties of SMA41 (a chimeric 3-(alkyl)-1,2,3-triazene linked to a 4-anilinoquinazoline backbone) in the A431 (epidermal carcinoma of the vulva) cell line. We now report on the dependence of its antiproliferative effects (e.g. DNA damage, cell survival) on the EGFR and the DNA repair protein O6-alkylguanine DNA alkyltransferase (AGT) contents of 12 solid tumor cell lines, two of which, NIH3T3 and NIH3T3 HER14 (engineered to overexpress EGFR), were isogenic. Receptor type specificity was determined using ELISA for competitive binding, as well as growth factor-stimulation assays. DNA damage was studied using single-cell microelectrophoresis (comet) assays, and levels of EGFR were determined by Western blotting. The effects of SMA41 on the cell cycle of NIH3T3 cells were investigated using univariate flow cytometry. Studies of receptor type specificity showed that SMA41: (a) preferentially inhibited the kinase activity of EGFR over those of Src, insulin receptor and protein kinase C (PKC, a serine/threonine kinase), (b) induced stronger inhibition of growth stimulated with EGF than of growth stimulated with platelet-derived growth factor (PDGF) or fetal bovine serum (FBS). Despite the EGFR specificity of SMA41, there was an absence of a linear correlation between the EGFR status of our solid tumor cell lines and levels of DNA damage induced by the alkylating component. Similarly, EGFR levels did not correlate with IC(50) values. The antiproliferative activities of SMA41 correlated more with the AGT status of these cells and paralleled those of the clinical triazene temozolomide (TEM). However, throughout the panel, tumor cell sensitivity to SMA41 was consistently stronger than to its closest analogue TEM. Experiments performed with the isogenic cells showed that SMA41 was capable of inducing twofold higher levels of DNA damage in the EGFR transfectant and delayed cell entry to G(2)/M in both cell types. When the cells were starved and growth-stimulated with FBS (conditions under which both cell types were growth-stimulated), in contrast to TEM, SMA41 and its hydrolytic metabolite SMA52 exhibited approximately nine- and threefold stronger inhibition of growth of the EGFR transfectant. These results suggest that, in addition to its ability to induce DNA damage and cell cycle perturbations, SMA41 is capable of selectively targeting the cells with a growth advantage conferred by EGFR transfection. When compared with the monoalkyltriazene prodrug TEM, its potency may be further enhanced by its ability to hydrolyze to another signal transduction inhibitor (SMA52) plus a DNA alkylating agent that may further contribute to chemosensitivity. Thus, our new "combi-targeting" strategy may well represent a tandem approach to selectively blocking receptor tyrosine kinase-mediated growth signaling while inducing significant levels of cytotoxic DNA lesions in refractory tumors.

Establishment and characterization of mouse bone marrow-derived mast cell hybridomas

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kawahara, Takeshi, E-mail: tkawafb@shinshu-u.ac.jp

2012-11-01

Interleukin (IL)-3-dependent mouse bone marrow-derived mast cells (BMMCs) are an important model for studying the function of mucosal-type mast cells. In the present study, BMMCs were successfully immortalized by cell fusion using a hypoxanthine-aminopterin-thymidine medium-sensitive variant of P815 mouse mastocytoma (P815-6TgR) as a partner cell line. The established mouse mast cell hybridomas (MMCHs) expressed {alpha}, {beta}, and {gamma} subunits of high-affinity immunoglobulin E (IgE) receptor (Fc{epsilon}RI) and possessed cytoplasmic granules devoid of or partially filled with electron-dense material. Four independent MMCH clones continuously proliferated without supplemental exogenous IL-3 and showed a degranulation response on stimulation with IgE+antigen. Furthermore, histamine synthesismore » and release by degranulation were confirmed in MMCH-D5, a MMCH clone that showed the strongest degranulation response. MMCH-D5 exhibited elevated levels of IL-3, IL-4, IL-13, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor (TNF)-{alpha}, and cyclooxygenase 2, and production of prostaglandin D{sub 2} and leukotriene C{sub 4} in response to IgE-induced stimulation. MMCH clones also expressed Toll-like receptors (TLRs) 1, 2, 4, and 6 and showed elevated levels of TNF-{alpha} expression in response to stimulation with TLR2 and TLR4 ligands. The MMCHs established using this method should be suitable for studies on Fc{epsilon}RI- and TLR-mediated effector functions of mast cells.« less

Cloning and expression analysis of a novel G-protein-coupled receptor selectively expressed on granulocytes.

PubMed

Yousefi, S; Cooper, P R; Potter, S L; Mueck, B; Jarai, G

2001-06-01

The migration of neutrophils into sites of acute and chronic inflammation is mediated by chemokines. We used degenerate-primer reverse transcriptase-polymerase chain reaction (RT-PCR) to analyze chemokine receptor expression in neutrophils and identify novel receptors. RNA was isolated from human peripheral blood neutrophils and from neutrophils that had been stimulated for 5 h with granulocyte-macrophage colony-stimulating factor or by coculturing with primary human bronchial epithelial cells. Amplification products were cloned, and clone redundancy was determined. Seven known G-protein-coupled receptors were identified among 38 clones-CCR1, CCR4, CXCR1, CXCR2, CXCR4, HM63, and FPR1-as well as a novel gene, EX33. The full-length EX33 clone was obtained, and an in silico approach was used to identify the putative murine homologue. The EX33 gene encodes a 396-amino-acid protein with limited sequence identity to known receptors. Expression studies of several known chemokine receptors and EX33 revealed that resting neutrophils expressed higher levels of CXCRs and EX33 compared with activated neutrophils. Northern blot experiments revealed that EX33 is expressed mainly in bone marrow, lung, and peripheral blood leukocytes. Using RT-PCR analysis, we showed more abundant expression of EX33 in neutrophils and eosinophils, in comparison with that in T- or B-lymphocytes, indicating cell-specific expression among leukocytes.

Roles of the locus coeruleus and adrenergic receptors in brain-mediated hypothalamic-pituitary-adrenal axis responses to intracerebroventricular alcohol.

PubMed

Selvage, Dan

2012-06-01

Alcohol activates the hypothalamic-pituitary-adrenal (HPA) axis through its actions in both the periphery and the central nervous system (CNS). The studies presented here were designed to test the CNS-specific noradrenergic mechanisms by which alcohol stimulates HPA activity in the male rat. We used an experimental paradigm in which a small, nontoxic amount (5 μl) of alcohol was slowly microinfused intracerebroventricularly (icv). Alcohol was administered icv to animals with lesions of the locus coeruleus (LC) or in animals pretreated with α- or β-adrenergic receptor antagonists. Hormonal HPA activation was determined by measuring secretion of the pituitary stress hormone adrenocorticotropin (ACTH). Neuronal activation was determined by quantification of the expression of the transcription factor c-fos (Fos). As expected, icv alcohol stimulated ACTH secretion from the pituitary and Fos expression in the paraventricular nucleus of the hypothalamus (PVN). Bilateral electrolytic LC lesions blocked the ability of icv alcohol to stimulate ACTH secretion. Pretreatment with icv propranolol increased basal ACTH secretion levels, but icv alcohol did not increase this effect. Propranolol also blunted icv alcohol-induced PVN Fos expression. A low dose of phenoxybenzamine, an α-adrenergic receptor antagonist, did not affect the ability of icv alcohol to stimulate ACTH release. However, a higher dose of the drug was able to block the ACTH response to icv alcohol. Despite this, phenoxybenzamine did not inhibit alcohol-induced Fos expression. Icv pretreatment with corynanthine, a selective α-1 adrenergic receptor antagonist, modestly raised basal ACTH levels and blocked the icv alcohol-induced secretion of this hormone. These results indicate that the LC and norepinephrine play important roles in HPA activation caused by icv alcohol administration, but that the specific adrenergic receptor subtypes involved in this phenomenon still need to be identified. Copyright © 2012 by the Research Society on Alcoholism.

The different roles of opioid receptors in the inhibitory effects induced by sacral dorsal root ganglion stimulation on nociceptive and nonnociceptive conditions in cats.

PubMed

Wang, Zhaoxia; Liao, Limin; Deng, Han; Li, Xing; Chen, Guoqing; Liao, Xiwen

2018-06-04

To examine the roles of opioid receptors in the inhibition of nociceptive and nonnociceptive bladder reflexes by sacral dorsal root ganglion (DRG) stimulation in cats. Hook electrodes were placed in the right S1 and S2 DRG of cats. The bladders were infused with physiologic saline or 0.25% acetic acid (AA). Naloxone (0.1, 0.3, and 1 mg/kg), an opioid receptor antagonist, was administered intravenously. S1 or S2 DRG stimulation was applied before and after administering the drug. Multiple cystometrograms were performed to determine the effects of DRG stimulation and opioid receptors on the micturition reflex under nociceptive and non-nociceptive conditions. AA significantly (P < 0.01) reduced bladder capacity (BC). DRG stimulation at threshold (T) and 1.5 T significantly increased BC of the saline control under nociceptive and non-nociceptive conditions. When saline was infused, naloxone (0.1-1 mg/kg) significantly (P < 0.01) reduced BC; however, naloxone did not change BC during AA irritation. During saline infusion, naloxone (0.3 and 1 mg/kg) partly blocked S1 DRG stimulation-induced inhibition but had only a slight effect on S2 DRG stimulation. During AA infusion, naloxone (0.3 and 1 mg/kg) only partially blocked S1 DRG stimulation at T intensity but not during 1.5 T stimulation. However, no doses of naloxone significantly affected S2 DRG stimulation. Opioid receptors play a role in sacral DRG stimulation on non-nociceptive condition but are not involved in the inhibitory effect of stimulation in nociceptive conditions. © 2018 Wiley Periodicals, Inc.

Oncostatin M promotes bone formation independently of resorption when signaling through leukemia inhibitory factor receptor in mice

PubMed Central

Walker, Emma C.; McGregor, Narelle E.; Poulton, Ingrid J.; Solano, Melissa; Pompolo, Sueli; Fernandes, Tania J.; Constable, Matthew J.; Nicholson, Geoff C.; Zhang, Jian-Guo; Nicola, Nicos A.; Gillespie, Matthew T.; Martin, T. John; Sims, Natalie A.

2010-01-01

Effective osteoporosis therapy requires agents that increase the amount and/or quality of bone. Any modification of osteoclast-mediated bone resorption by disease or drug treatment, however, elicits a parallel change in osteoblast-mediated bone formation because the processes are tightly coupled. Anabolic approaches now focus on uncoupling osteoblast action from osteoclast formation, for example, by inhibiting sclerostin, an inhibitor of bone formation that does not influence osteoclast differentiation. Here, we report that oncostatin M (OSM) is produced by osteoblasts and osteocytes in mouse bone and that it has distinct effects when acting through 2 different receptors, OSM receptor (OSMR) and leukemia inhibitory factor receptor (LIFR). Specifically, mouse OSM (mOSM) inhibited sclerostin production in a stromal cell line and in primary murine osteoblast cultures by acting through LIFR. In contrast, when acting through OSMR, mOSM stimulated RANKL production and osteoclast formation. A key role for OSMR in bone turnover was confirmed by the osteopetrotic phenotype of mice lacking OSMR. Furthermore, in contrast to the accepted model, in which mOSM acts only through OSMR, mOSM inhibited sclerostin expression in Osmr–/– osteoblasts and enhanced bone formation in vivo. These data reveal what we believe to be a novel pathway by which bone formation can be stimulated independently of bone resorption and provide new insights into OSMR and LIFR signaling that are relevant to other medical conditions, including cardiovascular and neurodegenerative diseases and cancer. PMID:20051625

Extrasynaptic N-methyl-D-aspartate (NMDA) receptor stimulation induces cytoplasmic translocation of the CDKL5 kinase and its proteasomal degradation.

PubMed

Rusconi, Laura; Kilstrup-Nielsen, Charlotte; Landsberger, Nicoletta

2011-10-21

Mutations in the X-linked gene cyclin-dependent kinase-like 5 (CDKL5) have been found in patients with epileptic encephalopathy characterized by early onset intractable epilepsy, including infantile spasms and other types of seizures, severe developmental delay, and often the development of Rett syndrome-like features. Despite its clear involvement in proper brain development, CDKL5 functions are still far from being understood. In this study, we analyzed the subcellular localization of the endogenous kinase in primary murine hippocampal neurons. CDKL5 was localized both in nucleus and cytoplasm and, conversely to proliferating cells, did not undergo constitutive shuttling between these compartments. Nevertheless, glutamate stimulation was able to induce the exit of the kinase from the nucleus and its subsequent accumulation in the perinuclear cytoplasm. Moreover, we found that sustained glutamate stimulation promoted CDKL5 proteasomal degradation. Both events were mediated by the specific activation of extrasynaptic pool of N-methyl-d-aspartate receptors. Proteasomal degradation was also induced by withdrawal of neurotrophic factors and hydrogen peroxide treatment, two different paradigms of cell death. Altogether, our results indicate that both subcellular localization and expression of CDKL5 are modulated by the activation of extrasynaptic N-methyl-D-aspartate receptors and suggest regulation of CDKL5 by cell death pathways.

The Toll-Like Receptor 2/6 Agonist, FSL-1 Lipopeptide, Therapeutically Mitigates Acute Radiation Syndrome.

PubMed

Kurkjian, Cathryn J; Guo, Hao; Montgomery, Nathan D; Cheng, Ning; Yuan, Hong; Merrill, Joseph R; Sempowski, Gregory D; Brickey, W June; Ting, Jenny P-Y

2017-12-11

Risks of radiation exposure from nuclear incidents and cancer radiotherapy are undeniable realities. These dangers urgently compel the development of agents for ameliorating radiation-induced injuries. Biologic pathways mediated by myeloid differentiation primary response gene 88 (MyD88), the common adaptor for toll-like receptor (TLR) and Interleukin-1 receptor signaling, are critical for radioprotection. Treating with agonists prior to radiation enhances survival by activating TLR signaling, whereas radiomitigating TLR-activating therapeutics given after exposure are less defined. We examine the radiomitigation capability of TLR agonists and identify one that is superior for its efficacy and reduced toxic consequences compared to other tested agonists. We demonstrate that the synthetic TLR2/6 ligand Fibroblast-stimulating lipopeptide (FSL-1) substantially prolongs survival in both male and female mice when administered 24 hours after radiation and shows MyD88-dependent function. FSL-1 treatment results in accelerated hematopoiesis in bone marrow, spleen and periphery, and augments systemic levels of hematopoiesis-stimulating factors. The ability of FSL-1 to stimulate hematopoiesis is critical, as hematopoietic dysfunction results from a range of ionizing radiation doses. The efficacy of a single FSL-1 dose for alleviating radiation injury while protecting against adverse effects reveals a viable radiation countermeasures agent.

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

PubMed Central

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

Identification of P2X3 and P2X7 Purinergic Receptors Activated by ATP in Rat Lacrimal Gland

PubMed Central

Vrouvlianis, Joanna; Scott, Rachel; Dartt, Darlene A.

2011-01-01

Purpose. To identify the type of purinergic receptors activated by adenosine triphosphate (ATP) in rat lacrimal gland and to determine their role in protein secretion. Methods. Purinergic receptors were identified by RT-PCR, Western blot analysis, and immunofluorescence techniques. Acini from rat lacrimal gland were isolated by collagenase digestion. Acini were incubated with the fluorescence indicator fura-2 tetra-acetoxylmethyl ester, and intracellular [Ca2+] ([Ca2+]i) was determined. Protein secretion was measured by fluorescence assay. Results. The authors previously showed that P2X7 receptors were functional in the lacrimal gland. In this study, they show that P2X1–4, and P2X6receptors were identified in the lacrimal gland by RT-PCR, Western blot, and immunofluorescence analyses. P2X5 receptors were not detected. ATP increased [Ca2+]i and protein secretion in a concentration-dependent manner. Removal of extracellular Ca2+ significantly reduced the ATP-stimulated increase in [Ca2+]i. Repeated applications of ATP caused desensitization of the [Ca2+]i response. Incubation with the P2X1 receptor inhibitor NF023 did not alter ATP-stimulated [Ca2+]i. Incubation with zinc, which potentiates P2X2 and P2X4 receptor responses, or lowering the pH to 6.8, which potentiates P2X2 receptor responses, did not alter the ATP-stimulated [Ca2+]i. P2X3 receptor inhibitors A-317491 and TNP-ATP significantly decreased ATP-stimulated [Ca2+]i and protein secretion, whereas the P2X3 receptor agonist α,β methylene ATP significantly increased them. The P2X7 receptor inhibitor A438079 had no effect on ATP-stimulated [Ca2+]i at 10−6 M but did have an effect at 10−4 M. Conclusions. Purinergic receptors P2X1–4 and P2X6 are present in the lacrimal gland. ATP uses P2X3 and P2X7 receptors to stimulate an increase in [Ca2+]i and protein secretion. PMID:21421865

α-Naphthoflavone Increases Lipid Accumulation in Mature Adipocytes and Enhances Adipocyte-Stimulated Endothelial Tube Formation.

PubMed

Wang, Mei-Lin; Lin, Shyh-Hsiang; Hou, Yuan-Yu; Chen, Yue-Hwa

2015-04-30

The aryl hydrocarbon receptor (AhR) is a ligand-activated factor that regulates biological effects associated with obesity. The AhR agonists, such as environmental contaminants 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and β-naphthoflavone (BNF), inhibit preadipocyte differentiation and interfere with the functions of adipose tissue, whereas the antagonist may have opposite or protective effects in obesity. This study investigated the effects of α-naphthoflavone (α-NF), an AhR antagonist, on adipogenesis- and angiogenesis-associated factors in mature adipocytes and on cross-talk of mature adipocytes with endothelial cells (ECs). Besides, the roles of the AhR on lipid accumulation and on secretion of vascular endothelial growth factor were also determined by introducing siRNA of AhR. Differentiated 3T3-L1 cells were treated with α-naphthoflavone (α-NF) (1-5 μM) for 16 h. Lipid accumulation and the expressions of AhR-associated factors in the cells were determined. The interaction between adipocytes and ECs was investigated by cultivating ECs with conditioned medium (CM) from α-NF-treated mature adipocytes, followed by the determination of endothelial tube formation. The results showed that α-NF significantly increased triglyceride (TG) accumulation in mature adipocytes, which was associated with increased expression of hormone-sensitive lipase (HSL), estrogen receptor (ER), as well as decreased expression of AhR, AhR nuclear translocator (ARNT), cytochrome P4501B1 (CYP1B1), and nuclear factor erythroid-2-related factor (NRF-2) proteins. In addition, CM stimulated formation of tube-like structures in ECs, and α-NF further enhanced such stimulation in association with modulated the secretions of various angiogenic mediators by mature adipocytes. Similarly, increased TG accumulation and vascular endothelial growth factor (VEGF) secretion were observed in AhR-knockout cells. In conclusion, α-NF increased TG accumulation in mature adipocytes and enhanced mature adipocyte-stimulated tube formation in ECs, suggesting that the AhR may suppress obesity-induced adverse effects, and α-NF abolished the protective effects of the AhR.

Induction of osteoblast differentiation by selective activation of kinase-mediated actions of the estrogen receptor.

PubMed

Kousteni, Stavroula; Almeida, Maria; Han, Li; Bellido, Teresita; Jilka, Robert L; Manolagas, Stavros C

2007-02-01

Estrogens control gene transcription by cis or trans interactions of the estrogen receptor (ER) with target DNA or via the activation of cytoplasmic kinases. We report that selective activation of kinase-mediated actions of the ER with 4-estren-3alpha,17beta-diol (estren) or an estradiol-dendrimer conjugate, each a synthetic compound that stimulates kinase-mediated ER actions 1,000 to 10,000 times more potently than direct DNA interactions, induced osteoblastic differentiation in established cell lines of uncommitted osteoblast precursors and primary cultures of osteoblast progenitors by stimulating Wnt and BMP-2 signaling in a kinase-dependent manner. In sharp contrast, 17beta-estradiol (E(2)) suppressed BMP-2-induced osteoblast progenitor commitment and differentiation. Consistent with the in vitro findings, estren, but not E(2), stimulated Wnt/beta-catenin-mediated transcription in T-cell factor-lacZ transgenic mice. Moreover, E(2) stimulated BMP signaling in mice in which ERalpha lacks DNA binding activity and classical estrogen response element-mediated transcription (ERalpha(NERKI/-)) but not in wild-type controls. This evidence reveals for the first time the existence of a large signalosome in which inputs from the ER, kinases, bone morphogenetic proteins, and Wnt signaling converge to induce differentiation of osteoblast precursors. ER can either induce it or repress it, depending on whether the activating ligand (and presumably the resulting conformation of the receptor protein) precludes or accommodates ERE-mediated transcription.

CD95 co-stimulation blocks activation of naive T cells by inhibiting T cell receptor signaling

PubMed Central

Lindquist, Jonathan A.; Arhel, Nathalie; Felder, Edward; Karl, Sabine; Haas, Tobias L.; Fulda, Simone; Walczak, Henning; Kirchhoff, Frank; Debatin, Klaus-Michael

2009-01-01

CD95 is a multifunctional receptor that induces cell death or proliferation depending on the signal, cell type, and cellular context. Here, we describe a thus far unknown function of CD95 as a silencer of T cell activation. Naive human T cells triggered by antigen-presenting cells expressing a membrane-bound form of CD95 ligand (CD95L) or stimulated by anti-CD3 and -CD28 antibodies in the presence of recombinant CD95L had reduced activation and proliferation, whereas preactivated, CD95-sensitive T cells underwent apoptosis. Triggering of CD95 during T cell priming interfered with proximal T cell receptor signaling by inhibiting the recruitment of ζ-chain–associated protein of 70 kD, phospholipase-γ, and protein kinase C-θ into lipid rafts, thereby preventing their mutual tyrosine protein phosphorylation. Subsequently, Ca2+ mobilization and nuclear translocation of transcription factors NFAT, AP1, and NF-κB were strongly reduced, leading to impaired cytokine secretion. CD95-mediated inhibition of proliferation in naive T cells could not be reverted by the addition of exogenous interleukin-2 and T cells primed by CD95 co-stimulation remained partially unresponsive upon secondary T cell stimulation. HIV infection induced CD95L expression in primary human antigeen-presenting cells, and thereby suppressed T cell activation, suggesting that CD95/CD95L-mediated silencing of T cell activation represents a novel mechanism of immune evasion. PMID:19487421

Angiogenic CXC chemokine expression during differentiation of human mesenchymal stem cells towards the osteoblastic lineage.

PubMed

Bischoff, D S; Zhu, J H; Makhijani, N S; Kumar, A; Yamaguchi, D T

2008-02-15

The potential role of ELR(+) CXC chemokines in early events in bone repair was studied using human mesenchymal stem cells (hMSCs). Inflammation, which occurs in the initial phase of tissue healing in general, is critical to bone repair. Release of cytokines from infiltrating immune cells and injured bone can lead to recruitment of MSCs to the region of repair. CXC chemokines bearing the Glu-Leu-Arg (ELR) motif are also released by inflammatory cells and serve as angiogenic factors stimulating chemotaxis and proliferation of endothelial cells. hMSCs, induced to differentiate with osteogenic medium (OGM) containing ascorbate, beta-glycerophosphate (beta-GP), and dexamethasone (DEX), showed an increase in mRNA and protein secretion of the ELR(+) CXC chemokines CXCL8 and CXCL1. CXCL8 mRNA half-life studies reveal an increase in mRNA stability upon OGM stimulation. Increased expression and secretion is a result of DEX in OGM and is dose-dependent. Inhibition of the glucocorticoid receptor with mifepristone only partially inhibits DEX-stimulated CXCL8 expression indicating both glucocorticoid receptor dependent and independent pathways. Treatment with signal transduction inhibitors demonstrate that this expression is due to activation of the ERK and p38 mitogen-activated protein kinase (MAPK) pathways and is mediated through the G(alphai)-coupled receptors. Angiogenesis assays demonstrate that OGM-stimulated conditioned media containing secreted CXCL8 and CXCL1 can induce angiogenesis of human microvascular endothelial cells in an in vitro Matrigel assay. Copyright 2007 Wiley-Liss, Inc.

Endothelial microparticle formation by angiotensin II is mediated via Ang II receptor type I/NADPH oxidase/ Rho kinase pathways targeted to lipid rafts.

PubMed

Burger, Dylan; Montezano, Augusto C; Nishigaki, Nobuhiro; He, Ying; Carter, Anthony; Touyz, Rhian M

2011-08-01

Circulating microparticles are increased in cardiovascular disease and may themselves promote oxidative stress and inflammation. Molecular mechanisms underlying their formation and signaling are unclear. We investigated the role of reactive oxygen species (ROS), Rho kinase, and lipid rafts in microparticle formation and examined their functional significance in endothelial cells (ECs). Microparticle formation from angiotensin II (Ang II)-stimulated ECs and apolipoprotein E(-/-) mice was assessed by annexin V or by CD144 staining and electron microscopy. Ang II promoted microparticle formation and increased EC O(2)(-) generation and Rho kinase activity. Ang II-stimulated effects were inhibited by irbesartan (Ang II receptor type I blocker) and fasudil (Rho kinase inhibitor). Methyl-β-cyclodextrin and nystatin, which disrupt lipid rafts/caveolae, blocked microparticle release. Functional responses, assessed in microparticle-stimulated ECs, revealed increased O(2)(-) production, enhanced vascular cell adhesion molecule/platelet-EC adhesion molecule expression, and augmented macrophage adhesion. Inhibition of epidermal growth factor receptor blocked the prooxidative and proinflammatory effects of microparticles. In vitro observations were confirmed in apolipoprotein E(-/-) mice, which displayed vascular inflammation and high levels of circulating endothelial microparticles, effects that were reduced by apocynin. We demonstrated direct actions of Ang II on endothelial microparticle release, mediated through NADPH oxidase, ROS, and Rho kinase targeted to lipid rafts. Microparticles themselves stimulated endothelial ROS formation and inflammatory responses. Our findings suggest a feedforward system whereby Ang II promotes EC injury through its own endothelial-derived microparticles.

Hyperactivity and reactivity of peripheral blood neutrophils in chronic periodontitis

PubMed Central

Matthews, J B; Wright, H J; Roberts, A; Cooper, P R; Chapple, I L C

2007-01-01

Some evidence exists that peripheral neutrophils from patients with chronic periodontitis generate higher levels of reactive oxygen species (ROS) after Fcγ-receptor stimulation than those from healthy controls. We hypothesized that peripheral neutrophils in periodontitis also show both hyper-reactivity to plaque organisms and hyperactivity in terms of baseline, unstimulated generation and release of ROS. Peripheral neutrophils from chronic periodontitis patients and age/sex/smoking-matched healthy controls (18 pairs) were assayed for total ROS generation and extracellular ROS release, with and without stimulation (Fcγ-receptor and Fusobacterium nucleatum), using luminol and isoluminol chemiluminescence. Assays were performed with and without priming with Escherichia coli lipopolysaccharide (LPS) and granulocyte–macrophage colony-stimulating factor (GM-CSF). Phox gene expression (p22, p47, p67, gp91) was investigated using reverse transcription–polymerase chain reaction (RT–PCR). Neutrophils from patients produced higher mean levels of ROS in all assays. Total generation and extracellular release of ROS by patients' cells were significantly greater than those from controls after FcγR-stimulation, with (P = 0·023) and without (P ≤ 0·023) priming with GM-CSF. Differences in unstimulated total ROS generation were not significant. By contrast, patients' cells demonstrated greater baseline, extracellular ROS release than those from controls (P = 0·004). This difference was maintained after priming with LPS (P = 0·028) but not GM-CSF (P = 0·217). Phox gene expression was similar in patient and control cells at baseline and stimulation with F. nucleatum (3 h) consistently reduced gp91PHOX transcripts. Our data demonstrate that peripheral neutrophils from periodontitis patients exhibit hyper-reactivity following stimulation (Fcγ-receptor and F. nucleatum) and hyperactivity in terms of excess ROS release in the absence of exogenous stimulation. This hyperactive/-reactive neutrophil phenotype is not associated with elevated phox gene expression. PMID:17223966

Investigation of the Fate of Type I Angiotensin Receptor after Biased Activation

PubMed Central

Szakadáti, Gyöngyi; Tóth, András D.; Oláh, Ilona; Erdélyi, László Sándor; Balla, Tamas; Várnai, Péter; Balla, András

2015-01-01

Biased agonism on the type I angiotensin receptor (AT1-R) can achieve different outcomes via activation of G protein–dependent and –independent cellular responses. In this study, we investigated whether the biased activation of AT1-R can lead to different regulation and intracellular processing of the receptor. We analyzed β-arrestin binding, endocytosis, and subsequent trafficking steps, such as early and late phases of recycling of AT1-R in human embryonic kidney 293 cells expressing wild-type or biased mutant receptors in response to different ligands. We used Renilla luciferase–tagged receptors and yellow fluorescent protein–tagged β-arrestin2, Rab5, Rab7, and Rab11 proteins in bioluminescence resonance energy transfer measurements to follow the fate of the receptor after stimulation. We found that not only is the signaling of the receptor different upon using selective ligands, but the fate within the cells is also determined by the type of the stimulation. β-arrestin binding and the internalization kinetics of the angiotensin II–stimulated AT1-R differed from those stimulated by the biased agonists. Similarly, angiotensin II–stimulated wild-type AT1-R showed differences compared with a biased mutant AT1-R (DRY/AAY AT1-R) with regards to β-arrestin binding and endocytosis. We found that the differences in the internalization kinetics of the receptor in response to biased agonist stimulation are due to the differences in plasma membrane phosphatidylinositol 4,5-bisphosphate depletion. Moreover, the stability of the β-arrestin binding is a major determinant of the later fate of the internalized AT1-R receptor. PMID:25804845

Intracrine prostaglandin E2 pro-tumoral actions in prostate epithelial cells originate from non-canonical pathways.

PubMed

Madrigal-Martínez, Antonio; Fernández-Martínez, Ana B; Lucio Cazaña, Francisco J

2018-04-01

Prostaglandin E 2 (PGE 2 ) increases cell proliferation and stimulates migratory and angiogenic abilities in prostate cancer cells. However, the effects of PGE 2 on non-transformed prostate epithelial cells are unknown, despite the fact that PGE 2 overproduction has been found in benign hyperplastic prostates. In the present work we studied the effects of PGE 2 in immortalized, non-malignant prostate epithelial RWPE-1 cells and found that PGE 2 increased cell proliferation, cell migration, and production of vascular endothelial growth factor-A, and activated in vitro angiogenesis. These actions involved a non-canonic intracrine mechanism in which the actual effector was intracellular PGE 2 (iPGE 2 ) instead of extracellular PGE 2 : inhibition of the prostaglandin uptake transporter (PGT) or antagonism of EP receptors prevented the effects of PGE 2 , which indicated that PGE 2 activity depended on its carrier-mediated translocation from the outside to the inside of cells and that EP receptors located intracellularly (iEP) mediated the effects of PGE 2 . iPGE 2 acted through transactivation of epidermal growth factor-receptor (EGFR) by iEP, leading to increased expression and activity of hypoxia-inducible factor-1α (HIF-1α). Interestingly, iPGE 2 also mediates the effects of PGE 2 on prostate cancer PC3 cells through the axis iPGE 2 -iEP receptors-EGFR-HIF-1α. Thus, this axis might be responsible for the growth-stimulating effects of PGE 2 on prostate epithelial cells, thereby contributing to prostate proliferative diseases associated with chronic inflammation. Since this PGT-dependent non-canonic intracrine mechanism of PGE 2 action operates in both benign and malignant prostate epithelial cells, PGT inhibitors should be tested as a novel therapeutic modality to treat prostate proliferative disease. © 2017 Wiley Periodicals, Inc.

Europium-labeled epidermal growth factor and neurotensin: novel probes for receptor-binding studies.

PubMed

Mazor, Ohad; Hillairet de Boisferon, Marc; Lombet, Alain; Gruaz-Guyon, Anne; Gayer, Batya; Skrzydelsky, Delphine; Kohen, Fortune; Forgez, Patricia; Scherz, Avigdor; Rostene, William; Salomon, Yoram

2002-02-01

We investigated the possibility of labeling two biologically active peptides, epidermal growth factor (EGF) and neurotensin (NT), with europium (Eu)-diethylenetriaminepentaacetic acid. More specifically, we tested them as probes in studying receptor binding using time-resolved fluorescence of Eu3+. The relatively simple synthesis yields ligands with acceptable binding characteristics similar to isotopically labeled derivatives. The binding affinity (Kd) of labeled Eu-EGF to human A431 epidermal carcinoid cells was 3.6 +/- 1.2 nM, similar to the reported Kd values of EGF, whereas the Kd of Eu-NT to human HT29 colon cancer cells (7.4 +/- 0.5 nM) or to Chinese hamster ovary (CHO) cells transfected with the high-affinity NT receptor (CHO-NT1) were about 10-fold higher than the Kd values of NT. The bioactivity of the Eu-labeled EGF as determined by stimulation of cultured murine D1 hematopoietic cell proliferation was nearly the same as that obtained with native EGF. The maximal stimulation of Ca2+ influx with NT and Eu-NT in CHO-NT1 cells was similar, but the respective K0.5 values were 20 pM and 1 nM, corresponding to differences in the binding affinities previously described. The results of these studies indicate that Eu labeling of peptide hormones and growth factor molecules ranging from 10(3) to 10(5) Da can be conveniently accomplished. Importantly, the Eu-labeled products are stable for approximately 2 years and are completely safe for laboratory use compared to the biohazardous radioligands. Thus, Eu-labeled peptides present an attractive alternative for commonly used radiolabeled ligands in biological studies in general and in receptor assays in particular.

Sustained activation of toll-like receptor 9 induces an invasive phenotype in lung fibroblasts: possible implications in idiopathic pulmonary fibrosis.

PubMed

Kirillov, Varvara; Siler, Jonathan T; Ramadass, Mahalakshmi; Ge, Lingyin; Davis, James; Grant, Geraldine; Nathan, Steven D; Jarai, Gabor; Trujillo, Glenda

2015-04-01

Idiopathic pulmonary fibrosis (IPF) is characterized by excessive scarring of the lung parenchyma, resulting in a steady decline of lung function and ultimately respiratory failure. The disease course of IPF is extremely variable, with some patients exhibiting stability of symptoms for prolonged periods of time, whereas others exhibit rapid progression and loss of lung function. Viral infections have been implicated in IPF and linked to disease severity; however, whether they directly contribute to progression is unclear. We previously classified patients as rapid and slow progressors on the basis of clinical features and expression of the pathogen recognition receptor, Toll-like receptor 9 (TLR9). Activation of TLR9 in vivo exacerbated IPF in mice and induced differentiation of myofibroblasts in vitro, but the mechanism of TLR9 up-regulation and progression of fibrosis are unknown. Herein, we investigate whether transforming growth factor (TGF)-β, a pleiotropic cytokine central to IPF pathogenesis, regulates TLR9 in lung myofibroblasts. Results showed induction of TLR9 expression by TGF-β in lung myofibroblasts and a distinct profibrotic myofibroblast phenotype driven by stimulation with the TLR9 agonist, CpG-DNA. Chronic TLR9 stimulation resulted in stably differentiated α-smooth muscle actin(+)/platelet-derived growth factor receptor α(+)/CD44(+)/matrix metalloproteinase-14(+)/matrix metalloproteinase-2(+) myofibroblasts, which secrete inflammatory cytokines, invade Matrigel toward platelet-derived growth factor, and resist hypoxia-induced apoptosis. These results suggest a mechanism by which TGF-β and TLR9 responses in myofibroblasts collaborate to drive rapid progression of IPF. Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Neurotensin is an autocrine trophic factor stimulated by androgen withdrawal in human prostate cancer.

PubMed Central

Sehgal, I; Powers, S; Huntley, B; Powis, G; Pittelkow, M; Maihle, N J

1994-01-01

After therapeutic hormone deprivation, prostate cancer cells often develop androgen-insensitive growth through mechanisms thus far undefined. Neuropeptides have been previously implicated as growth factors in some prostate cancers. Here, we demonstrate that androgen-sensitive LNCaP human prostate cancer cells produce and secrete neurotensin following androgen withdrawal. We show that while LNCaP cells express the neurotensin receptor, only androgen-deprived cells exhibit a growth response to exogenous neurotensin. We further demonstrate that androgen-stimulated cells may be refractory to exogenous neurotensin due to androgen induction of a metalloprotease active toward neurotensin. Thus, prostate cancer cells deprived of androgen develop an alternative autocrine growth mechanism involving neurotensin. Images PMID:8197117

Expression of receptors for putative anabolic growth factors in human intervertebral disc: implications for repair and regeneration of the disc.

PubMed

Le Maitre, Christine L; Richardson, Stephen M A; Baird, Pauline; Freemont, Anthony J; Hoyland, Judith A

2005-12-01

Low back pain (LBP) is a common, debilitating and economically important disorder. Current evidence implicates loss of intervertebral disc (IVD) matrix consequent upon 'degeneration' as a major cause of LBP. Degeneration of the IVD involves increases in degradative enzymes and decreases in the extracellular matrix (ECM) component in a process that is controlled by a range of cytokines and growth factors. Studies have suggested using anabolic growth factors to regenerate the normal matrix of the IVD, hence restoring disc height and reversing degenerative disc disease. However, for such therapies to be successful it is vital that the target cells (i.e. the disc cells) express the appropriate receptors. This immunohistochemical study has for the first time investigated the expression and localization of four potentially beneficial growth factor receptors (i.e. TGFbetaRII, BMPRII, FGFR3 and IGFRI) in non-degenerate and degenerate human IVDs. Receptor expression was quantified across regions of the normal and degenerate disc and showed that cells of the nucleus pulposus (NP) and inner annulus fibrosus (IAF) expressed significantly higher levels of the four growth factor receptors investigated. There were no significant differences between the four growth factor expression in non-degenerate and degenerate biopsies. However, expression of TGFbetaRII, FGFR3 and IGFRI, but not BMP RII, were observed in the ingrowing blood vessels that characterize part of the disease aetiology. In conclusion, this study has demonstrated the expression of the four growth factor receptors at similar levels in the chondrocyte-like cells of the NP and IAF in both non-degenerate and degenerate discs, implicating a role in normal disc homeostasis and suggesting that the application of these growth factors to the degenerate human IVD would stimulate matrix production. However, the expression of some of the growth factor receptors on ingrowing blood vessels might be problematic in a therapeutic approach. Copyright 2005 Pathological Society of Great Britain and Ireland.

Steroid receptor coactivators, HER-2 and HER-3 expression is stimulated by tamoxifen treatment in DMBA-induced breast cancer.

PubMed

Moi, Line L Haugan; Flågeng, Marianne Hauglid; Gjerde, Jennifer; Madsen, Andre; Røst, Therese Halvorsen; Gudbrandsen, Oddrun Anita; Lien, Ernst A; Mellgren, Gunnar

2012-06-15

Steroid receptor coactivators (SRCs) may modulate estrogen receptor (ER) activity and the response to endocrine treatment in breast cancer, in part through interaction with growth factor receptor signaling pathways. In the present study the effects of tamoxifen treatment on the expression of SRCs and human epidermal growth factor receptors (HERs) were examined in an animal model of ER positive breast cancer. Sprague-Dawley rats with DMBA-induced breast cancer were randomized to 14 days of oral tamoxifen 40 mg/kg bodyweight/day or vehicle only (controls). Tumors were measured throughout the study period. Blood samples and tumor tissue were collected at sacrifice and tamoxifen and its main metabolites were quantified using LC-MS/MS. The gene expression in tumor of SRC-1, SRC-2/transcription intermediary factor-2 (TIF-2), SRC-3/amplified in breast cancer 1 (AIB1), ER, HER-1, -2, -3 and HER-4, as well as the transcription factor Ets-2, was measured by real-time RT-PCR. Protein levels were further assessed by Western blotting. Tamoxifen and its main metabolites were detected at high concentrations in serum and accumulated in tumor tissue in up to tenfolds the concentration in serum. Mean tumor volume/rat decreased in the tamoxifen treated group, but continued to increase in controls. The mRNA expression levels of SRC-1 (P = 0.035), SRC-2/TIF-2 (P = 0.002), HER-2 (P = 0.035) and HER-3 (P = 0.006) were significantly higher in tamoxifen treated tumors compared to controls, and the results were confirmed at the protein level using Western blotting. SRC-3/AIB1 protein was also higher in tamoxifen treated tumors. SRC-1 and SRC-2/TIF-2 mRNA levels were positively correlated with each other and with HER-2 (P ≤ 0.001), and the HER-2 mRNA expression correlated with the levels of the other three HER family members (P < 0.05). Furthermore, SRC-3/AIB1 and HER-4 were positively correlated with each other and Ets-2 (P < 0.001). The expression of SRCs and HER-2 and -3 is stimulated by tamoxifen treatment in DMBA-induced breast cancer. Stimulation and positive correlation of coactivators and HERs may represent an early response to endocrine treatment. The role of SRCs and HER-2 and -3 should be further studied in order to evaluate their effects on response to long-term tamoxifen treatment.

Steroid receptor coactivators, HER-2 and HER-3 expression is stimulated by tamoxifen treatment in DMBA-induced breast cancer

PubMed Central

2012-01-01

Background Steroid receptor coactivators (SRCs) may modulate estrogen receptor (ER) activity and the response to endocrine treatment in breast cancer, in part through interaction with growth factor receptor signaling pathways. In the present study the effects of tamoxifen treatment on the expression of SRCs and human epidermal growth factor receptors (HERs) were examined in an animal model of ER positive breast cancer. Methods Sprague-Dawley rats with DMBA-induced breast cancer were randomized to 14 days of oral tamoxifen 40 mg/kg bodyweight/day or vehicle only (controls). Tumors were measured throughout the study period. Blood samples and tumor tissue were collected at sacrifice and tamoxifen and its main metabolites were quantified using LC-MS/MS. The gene expression in tumor of SRC-1, SRC-2/transcription intermediary factor-2 (TIF-2), SRC-3/amplified in breast cancer 1 (AIB1), ER, HER-1, -2, -3 and HER-4, as well as the transcription factor Ets-2, was measured by real-time RT-PCR. Protein levels were further assessed by Western blotting. Results Tamoxifen and its main metabolites were detected at high concentrations in serum and accumulated in tumor tissue in up to tenfolds the concentration in serum. Mean tumor volume/rat decreased in the tamoxifen treated group, but continued to increase in controls. The mRNA expression levels of SRC-1 (P = 0.035), SRC-2/TIF-2 (P = 0.002), HER-2 (P = 0.035) and HER-3 (P = 0.006) were significantly higher in tamoxifen treated tumors compared to controls, and the results were confirmed at the protein level using Western blotting. SRC-3/AIB1 protein was also higher in tamoxifen treated tumors. SRC-1 and SRC-2/TIF-2 mRNA levels were positively correlated with each other and with HER-2 (P ≤ 0.001), and the HER-2 mRNA expression correlated with the levels of the other three HER family members (P < 0.05). Furthermore, SRC-3/AIB1 and HER-4 were positively correlated with each other and Ets-2 (P < 0.001). Conclusions The expression of SRCs and HER-2 and -3 is stimulated by tamoxifen treatment in DMBA-induced breast cancer. Stimulation and positive correlation of coactivators and HERs may represent an early response to endocrine treatment. The role of SRCs and HER-2 and -3 should be further studied in order to evaluate their effects on response to long-term tamoxifen treatment. PMID:22703232

The role of protease-activated receptors PAR-1 and PAR-2 in the repair of 16HBE 14o(-) epithelial cell monolayers in vitro.

PubMed

Ewen, D; Clarke, S L; Smith, J R; Berger, C; Salmon, G; Trevethick, M; Shute, J K

2010-03-01

We recently reported that repair following mechanical wounding of epithelial cell layers in vitro is dependent on fibrin formation and the activity of locally expressed coagulation cascade proteins. Serine proteases of the coagulation cascade are an important group of protease-activated receptor (PAR) activators and PAR-1 to 4 are expressed by the normal bronchial epithelium. We tested the hypothesis that activation of PAR-1 and PAR-2 by coagulation cascade proteases stimulates epithelial repair via effects on fibrin formation. Using mechanically wounded 16HBE 14o(-) epithelial cell layers in culture, we investigated the effect of PAR-1 and PAR-2 agonist peptides, control partially scrambled peptides and PAR-neutralizing antibodies on the rate of repair and fibrin formation. Coagulation factors in culture supernatants were measured by immunoblot. RT-PCR was used to investigate PAR-1, PAR-2 and PGE2 receptor (EP-1 to EP-4) expression in this model and qRT-PCR to quantify responses to wounding. Additionally, we investigated the effect of exogenously added factor Xa (FXa) and neutrophil elastase and the influence of PGE2 and indomethacin on the repair response. PAR-1 and PAR-2 peptide agonists stimulated the rate of repair and enhanced the formation of a fibrin provisional matrix to support the repair process. Conversely, PAR-neutralizing antibodies inhibited repair. Under serum-free culture conditions, 16HBE 14o(-) cells expressed EP-2 and EP-3, but not EP-1 or EP-4, receptors. Wounding induced an increased expression of EP-3 but did not alter EP-2, PAR-1 or PAR-2 expression. In the absence of PAR agonists, there was no evidence for a role for PGE2 in fibrin formation or the repair process. Indomethacin attenuated fibrin formation in wounded cultures only in the presence of the PAR-2 peptide. FXa stimulated epithelial repair while neutrophil elastase reduced the levels of coagulation factors and inhibited repair. Locally expressed serine proteases of the coagulation cascade activate PAR-1 and PAR-2 to enhance fibrin formation and bronchial epithelial repair.

Recent advances in colony stimulating factor-1 receptor/c-FMS as an emerging target for various therapeutic implications.

PubMed

Kumari, Archana; Silakari, Om; Singh, Rajesh K

2018-07-01

Colony stimulating factor-1 (CSF-1) is one of the most common proinflammatory cytokine responsible for various inflammatory disorders. It has a remarkable role in the development and progression of osteoarthritis, cancer and other autoimmune disease conditions. The CSF-1 acts by binding to the receptor, called colony stimulating factor-1 receptor (CSF-1R) also known as c-FMS resulting in the cascade of signalling pathway causing cell proliferation and differentiation. Interleukin-34 (IL-34), recently identified as another ligand for CSF-IR, is a cytokine protein. Both, CSF-1 and IL-34, although two distinct cytokines, follow the similar signalling pathway on binding to the same receptor, CSF-1R. Like CSF-1, IL-34 promotes the differentiation and survival of monocyte, macrophages and osteoclasts. This CSF-1R/c-FMS is over expressed in many cancers and on tumour associated macrophages, consequently, have been exploited as a drug target for promising treatment for cancer and inflammatory diseases. Some CSF-1R/c-FMS inhibitors such as ABT-869, Imatinib, AG013736, JNJ-40346527, PLX3397, DCC-3014 and Ki20227 have been successfully used in these disease conditions. Many c-FMS inhibitors have been the candidates of clinical trials, but suffer from some side effects like cardiotoxicity, vomiting, swollen eyes, diarrhoea, etc. If selectivity of cFMS inhibition is achieved successfully, side effects can be overruled and this approach may become a novel therapy for treatment of various therapeutic interventions. Thus, successful targeting of c-FMS may result in multifunctional therapy. With this background of information, the present review focuses on the recent developments in the area of CSF-1R/c-FMS inhibitors with emphasis on crystal structure, mechanism of action and various therapeutic implications in which c-FMS plays a pivotal role. The review on structure activity relationship of various compounds acting as the inhibitors of c-FMS which gives the selection criteria for the development of novel molecules is also being presented. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Effects of GABA receptor antagonists on thresholds of P23H rat retinal ganglion cells to electrical stimulation of the retina

NASA Astrophysics Data System (ADS)

Jensen, Ralph J.; Rizzo, Joseph F., III

2011-06-01

An electronic retinal prosthesis may provide useful vision for patients suffering from retinitis pigmentosa (RP). In animal models of RP, the amount of current needed to activate retinal ganglion cells (RGCs) is higher than in normal, healthy retinas. In this study, we sought to reduce the stimulation thresholds of RGCs in a degenerate rat model (P23H-line 1) by blocking GABA receptor mediated inhibition in the retina. We examined the effects of TPMPA, a GABAC receptor antagonist, and SR95531, a GABAA receptor antagonist, on the electrically evoked responses of RGCs to biphasic current pulses delivered to the subretinal surface through a 400 µm diameter electrode. Both TPMPA and SR95531 reduced the stimulation thresholds of ON-center RGCs on average by 15% and 20% respectively. Co-application of the two GABA receptor antagonists had the greatest effect, on average reducing stimulation thresholds by 32%. In addition, co-application of the two GABA receptor antagonists increased the magnitude of the electrically evoked responses on average three-fold. Neither TPMPA nor SR95531, applied alone or in combination, had consistent effects on the stimulation thresholds of OFF-center RGCs. We suggest that the effects of the GABA receptor antagonists on ON-center RGCs may be attributable to blockage of GABA receptors on the axon terminals of ON bipolar cells.

Acetylcholine is released from taste cells, enhancing taste signalling

PubMed Central

Dando, Robin; Roper, Stephen D

2012-01-01

Acetylcholine (ACh), a candidate neurotransmitter that has been implicated in taste buds, elicits calcium mobilization in Receptor (Type II) taste cells. Using RT-PCR analysis and pharmacological interventions, we demonstrate that the muscarinic acetylcholine receptor M3 mediates these actions. Applying ACh enhanced both taste-evoked Ca2+ responses and taste-evoked afferent neurotransmitter (ATP) secretion from taste Receptor cells. Blocking muscarinic receptors depressed taste-evoked responses in Receptor cells, suggesting that ACh is normally released from taste cells during taste stimulation. ACh biosensors confirmed that, indeed, taste Receptor cells secrete acetylcholine during gustatory stimulation. Genetic deletion of muscarinic receptors resulted in significantly diminished ATP secretion from taste buds. The data demonstrate a new role for acetylcholine as a taste bud transmitter. Our results imply specifically that ACh is an autocrine transmitter secreted by taste Receptor cells during gustatory stimulation, enhancing taste-evoked responses and afferent transmitter secretion. PMID:22570381

Cell-line-specific stimulation of tumor cell aggressiveness by wound healing factors - a central role for STAT3.

PubMed

Ekblad, Lars; Lindgren, Gustaf; Persson, Emma; Kjellén, Elisabeth; Wennerberg, Johan

2013-01-25

Local recurrence is a major factor affecting survival after treatment for head and neck squamous cell carcinoma (HNSCC). It is possible that the normal processes involved in wound healing after surgical removal of a primary tumor can boost the regrowth of residual cancer cells, thereby contributing to the recurrent growth. In this work, we collected human wound fluids and used them to investigate the effect of wound healing factors on HNSCC cell lines in vitro. Wound fluids were collected from thyroidectomized patients diagnosed with benign disease and were included in assays of cell proliferation, migration, cell scattering, and invasion. The involvement of intracellular signaling pathways and membrane receptors were investigated by western blotting and the inclusion of specific inhibitors. One out of four cell lines was greatly stimulated in proliferation, migration, cell scattering, and invasion by the addition of wound fluid as compared with addition of fetal bovine or human serum. These effects were accompanied by a sharp increase in activation of signal transducer and activator of transcription 3 (STAT3). Inhibition of STAT3 activation abolished the wound fluid response, showing that STAT3 plays an important role in the wound healing response. Several of the observed phenotypic changes were epithelial-to-mesenchymal transition (EMT)-like, but the appropriate changes were not seen in any of the EMT markers investigated. The involvement of c-Met or epidermal growth factor receptor family members was excluded, while the interleukin-6 receptor was found to be partly responsible for the activation of STAT3. In conclusion, we found cell-line-specific effects of wound healing factors on HNSCC, setting the stage for therapy development and predictive opportunities.

Effect of endothelin-1 and endothelin receptor blockade on the release of microparticles.

PubMed

Jung, Christian; Lichtenauer, Michael; Wernly, Bernhard; Franz, Marcus; Goebel, Bjoern; Rafnsson, Arnar; Figulla, Hans-Reiner; Pernow, John

2016-08-01

Increased levels of endothelial cell microparticles (EMP) are known to reflect endothelial dysfunction (ED). In diabetes mellitus type 2 (T2DM), the expression of endothelin (ET)-1 is increased. As treatment with an ET-1 antagonist significantly inhibited atherosclerosis in animal models, we sought to investigate whether treatment with ET-1 antagonists affects EMP levels in vitro and in vivo in patients with T2DM. In vitro study: Human umbilical vein endothelial cells (HUVEC) were stimulated with ET-1 alone and ET-1 in combination with a dual ET-A and ET-B endothelin receptor blocker. In vivo study: Patients with T2DM were randomized to treatment with the ET receptor antagonist bosentan or placebo. After 4 weeks, the patients were re-examined and blood samples were obtained. EMP counts in supernatants and plasma samples were determined using flow cytometry. In vitro study: In supernatants of ET-1-stimulated HUVECs, the increased release of EMP was reduced significantly by co-incubation with an ET-1 receptor antagonist (e.g. CD31+/CD42b-EMP decreased from 37·1% ± 2·8 to 31·5% ± 2·8 SEM, P = 0·0078). In vivo study: No changes in EMP levels in blood samples of patients with T2DM were found after 4 weeks of bosentan treatment (n = 36, P = ns). Our in vitro results suggest that ET-1 stimulates the release of EMP from HUVECs via a receptor-dependent mechanism. Co-incubation with an endothelin receptor blocker abolished ET-1-dependent EMP release. However, treatment with bosentan for 4 weeks failed to alter EMP levels in patients with T2DM. Other factors seem to have influenced EMP release in patients with T2DM independent of ET-1 receptor-mediated mechanisms. © 2016 Stichting European Society for Clinical Investigation Journal Foundation.

Receptor density balances signal stimulation and attenuation in membrane-assembled complexes of bacterial chemotaxis signaling proteins

PubMed Central

Besschetnova, Tatiana Y.; Montefusco, David J.; Asinas, Abdalin E.; Shrout, Anthony L.; Antommattei, Frances M.; Weis, Robert M.

2008-01-01

All cells possess transmembrane signaling systems that function in the environment of the lipid bilayer. In the Escherichia coli chemotaxis pathway, the binding of attractants to a two-dimensional array of receptors and signaling proteins simultaneously inhibits an associated kinase and stimulates receptor methylation—a slower process that restores kinase activity. These two opposing effects lead to robust adaptation toward stimuli through a physical mechanism that is not understood. Here, we provide evidence of a counterbalancing influence exerted by receptor density on kinase stimulation and receptor methylation. Receptor signaling complexes were reconstituted over a range of defined surface concentrations by using a template-directed assembly method, and the kinase and receptor methylation activities were measured. Kinase activity and methylation rates were both found to vary significantly with surface concentration—yet in opposite ways: samples prepared at high surface densities stimulated kinase activity more effectively than low-density samples, whereas lower surface densities produced greater methylation rates than higher densities. FRET experiments demonstrated that the cooperative change in kinase activity coincided with a change in the arrangement of the membrane-associated receptor domains. The counterbalancing influence of density on receptor methylation and kinase stimulation leads naturally to a model for signal regulation that is compatible with the known logic of the E. coli pathway. Density-dependent mechanisms are likely to be general and may operate when two or more membrane-related processes are influenced differently by the two-dimensional concentration of pathway elements. PMID:18711126

IFN-beta1b augments glucocorticoid-induced suppression of tumor necrosis factor-alpha production by increasing the number of glucocorticoid receptors on a human monocytic cell line.

PubMed

Uitdehaag, B M; Hoekstra, K; Koper, J W; Polman, C H; Dijkstra, C D

2001-03-01

We studied the effect of recombinant interferon-beta1b (IFN-beta1b) on the sensitivity to glucocorticoids (GC) and on the number of GC receptors (GCR) in the human monocytic cell line THP-1. We found that IFN-beta1b augments the suppressive effect that dexamethasone has on the stimulated production of tumor necrosis factor-alpha (TNF-alpha), most likely related to the increased number of GCR observed after exposure to IFN-beta1b. This provides a possible clue to the mechanism of action of IFN-beta in multiple sclerosis.

A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging

PubMed Central

Poland, Simon P.; Krstajić, Nikola; Monypenny, James; Coelho, Simao; Tyndall, David; Walker, Richard J.; Devauges, Viviane; Richardson, Justin; Dutton, Neale; Barber, Paul; Li, David Day-Uei; Suhling, Klaus; Ng, Tony; Henderson, Robert K.; Ameer-Beg, Simon M.

2015-01-01

We demonstrate diffraction limited multiphoton imaging in a massively parallel, fully addressable time-resolved multi-beam multiphoton microscope capable of producing fluorescence lifetime images with sub-50ps temporal resolution. This imaging platform offers a significant improvement in acquisition speed over single-beam laser scanning FLIM by a factor of 64 without compromising in either the temporal or spatial resolutions of the system. We demonstrate FLIM acquisition at 500 ms with live cells expressing green fluorescent protein. The applicability of the technique to imaging protein-protein interactions in live cells is exemplified by observation of time-dependent FRET between the epidermal growth factor receptor (EGFR) and the adapter protein Grb2 following stimulation with the receptor ligand. Furthermore, ligand-dependent association of HER2-HER3 receptor tyrosine kinases was observed on a similar timescale and involved the internalisation and accumulation or receptor heterodimers within endosomes. These data demonstrate the broad applicability of this novel FLIM technique to the spatio-temporal dynamics of protein-protein interaction. PMID:25780724

Structural Basis of Intracellular TGF-β Signaling: Receptors and Smads.

PubMed

Chaikuad, Apirat; Bullock, Alex N

2016-11-01

Stimulation of the transforming growth factor β (TGF-β) family receptors activates an intracellular phosphorylation-dependent signaling cascade that culminates in Smad transcriptional activation and turnover. Structural studies have identified a number of allosteric mechanisms that control the localization, conformation, and oligomeric state of the receptors and Smads. Such mechanisms dictate the ordered binding of substrate and adaptor proteins that determine the directionality of the signaling process. Activation of the pathway has been illustrated by the various structures of the receptor-activated Smads (R-Smads) with SARA, Smad4, and YAP, respectively, whereas mechanisms of down-regulation have been elucidated by the structural complexes of FKBP12, Ski, and Smurf1. Interesting parallels have emerged between the R-Smads and the Forkhead-associated (FHA) and interferon regulatory factor (IRF)-associated domains, as well as the Hippo pathway. However, important questions remain as to the mechanism of Smad-independent signaling. Copyright © 2016 Cold Spring Harbor Laboratory Press; all rights reserved.

The Induction of Serine/Threonine Protein Phosphorylations by a PDGFR/TrkA Chimera in Stably Transfected PC12 Cells*

PubMed Central

Biarc, Jordane; Chalkley, Robert J.; Burlingame, A. L.; Bradshaw, Ralph A.

2012-01-01

Stably transfected PC12 cells expressing a chimeric receptor composed of the extracellular domain of the platelet-derived growth factor receptor BB and the transmembrane and intracellular domains of TrkA, the nerve growth factor receptor, were stimulated for 20 min with platelet-derived growth factor and the resulting phosphoproteome was determined from affinity purified tryptic peptides identified by tandem MS (MS/MS) analyses. The changes in the levels of individual phosphorylation sites in stimulated cells versus control were ascertained by the stable isotope labeling of amino acids in cell culture technique. A total of 2035 peptides (806 proteins) were indentified and quantified in both data sets. Of these, 424 phosphopeptides on 259 proteins were found to be up-regulated and 392 sites on 206 proteins were down-regulated (1.8-fold or more). Protein kinases and phosphatases, as well as sites in many proteins involved in G-protein signaling, were prominently represented in the up-regulated group and more than half of the kinase up-regulated phosphosites could be clustered into three sequence motifs; a similar distribution was also found for the down-regulated sites. A comparison of the up-regulated motif profile observed to that calculated from a previous study of the EGFR-induced phosphoproteome in human HeLa cells at the same time point showed a considerable amount of similarity, supporting the view that RTK signal transduction pathways and downstream modifications are likely to be extensively overlapping. PMID:22027198

The granulocyte-macrophage colony-stimulating factor promoter cis-acting element CLE0 mediates induction signals in T cells and is recognized by factors related to AP1 and NFAT.

PubMed Central

Masuda, E S; Tokumitsu, H; Tsuboi, A; Shlomai, J; Hung, P; Arai, K; Arai, N

1993-01-01

Expression of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene in T cells is activated by the combination of phorbol ester (phorbol myristate acetate) and calcium ionophore (A23187), which mimic antigen stimulation through the T-cell receptor. We have previously shown that a fragment containing bp -95 to +27 of the mouse GM-CSF promoter can confer inducibility to reporter genes in the human Jurkat T-cell line. Here we use an in vitro transcription system to demonstrate that a cis-acting element (positions -54 to -40), referred to as CLE0, is a target for the induction signals. We observed induction with templates containing intact CLE0 but not with templates with deleted or mutated CLE0. We also observed that two distinct signals were required for the stimulation through CLE0, since only extracts from cells treated with both phorbol myristate acetate and A23187 supported optimal induction. Stimulation probably was mediated by CLE0-binding proteins because depletion of these proteins specifically reduced GM-CSF transcription. One of the binding factors possessed biochemical and immunological features identical to those of the transcription factor AP1. Another factor resembled the T-cell-specific factor NFAT. The characteristics of these two factors are consistent with their involvement in GM-CSF induction. The presence of CLE0-like elements in the promoters of interleukin-3 (IL-3), IL-4, IL-5, GM-CSF, and NFAT sites in the IL-2 promoter suggests that the factors we detected, or related factors that recognize these sites, may account for the coordinate induction of these genes during T-cell activation. Images PMID:8246960

Differential effects of c-fms and c-kit ligands on the lineage development of the lymphohematopoietic cell line EML C1.

PubMed

Tsai, S

1996-01-01

The lymphohematopoietic progenitors represent < 0.01% of nucleated marrow cells. We have shown that murine lymphohematopoietic progenitors can be immortalized by a recombinant retroviral vector harboring a dominant-negative retinoic acid (RA) receptor. The immortalized progenitors proliferate as a stem-cell factor-dependent clonal line designated EML C1. The EML C1 cell line spontaneously generates prepro-B-lymphocytes and erythroid and myeloid progenitors. Upon stimulation with interleukin 7 and marrow stromal cells, the prepro-B-lymphocytes express recombination-activating gene 1 (RAG-1) and undergo D-J rearrangements of the immunoglobulin heavy-chain genes. With erythropoietin, the erythroid progenitors proliferate and differentiate into red cells. Generation of the common progenitors for neutrophils and macrophages [colony-forming units-granulocyte-macrophage (CFU-GM)] is suppressed in EML C1 cells but is inducible by high concentrations of RA. An additional block in neutrophil differentiation occurs at the promyelocyte stage, but this can also be overcome by high concentrations of RA. Although c-fms is homologous to c-kit, which encodes the receptor for stem-cell factor (SCF), EML C1 cells neither express c-fms nor respond to macrophage colony-stimulating factor (M-CSF), the ligand for c-fms. Transduction and expression of c-fms cDNA in EML C1 cells confers responsiveness to M-CSF. This finding indicates that c-kit and c-fms share substantially overlapping signal-transduction pathways. However, c-fms-transduced EML C1 cells (EML C1/c-fms cells) exhibit different development patterns when stimulated by SCF alone or by M-CSF alone. When stimulated by SCF alone, EML C1/c-fms cells show mostly erythroid and B-lymphoid development. When stimulated by M-CSF alone, development switches to mostly myeloid (neutrophil and macrophage) development. This observation suggests that c-kit and c-fms must have unique signal-transduction pathways in addition to the common ones.

Intra-accumbens baclofen, but not muscimol, increases second order instrumental responding for food reward in rats.

PubMed

Pulman, Kim G T; Somerville, Elizabeth M; Clifton, Peter G

2012-01-01

Stimulation of either GABA(A) or GABA(B) receptors within the nucleus accumbens shell strongly enhances food intake in rats. However the effects of subtype-selective stimulation of GABA receptors on instrumental responses for food reward are less well characterized. Here we contrast the effects of the GABA(A) receptor agonist muscimol and GABA(B) receptor agonist baclofen on instrumental responding for food using a second order reinforcement schedule. Bilateral intra-accumbens administration of baclofen (220-440 pmol) stimulated responding but a higher dose (660 pmol) induced stereotyped oral behaviour that interfered with responding. Baclofen (220-660 pmol) also stimulated intake of freely available chow. Muscimol (220-660 pmol) was without effect on responding for food on this schedule but did stimulate intake of freely available chow. Unilateral administration of either baclofen or muscimol (220 pmol) induced similar patterns of c-fos immunoreactivity in several hypothalamic sites but differed in its induction in the central nucleus of the amygdala. We conclude that stimulation of GABA(A) or GABA(B) receptors in the nucleus accumbens shell of rats produces clearly distinguishable effects on operant responding for food.

Substance P enhances tissue factor release from granulocyte-macrophage colony-stimulating factor-dependent macrophages via the p22phox/β-arrestin 2/Rho A signaling pathway.

PubMed

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

2016-03-01

Granulocyte-macrophage colony stimulating factor (GM-CSF) induces procoagulant activity of macrophages. Tissue factor (TF) is a membrane-bound glycoprotein and substance P (SP) is a pro-inflammatory neuropeptide involved in the formation of membrane blebs. This study investigated the role of SP in TF release by GM-CSF-dependent macrophages. SP significantly decreased TF levels in whole-cell lysates of GM-CSF-dependent macrophages. TF was detected in the culture supernatant by enzyme-linked immunosorbent assay after stimulation of macrophages by SP. Aprepitant (an SP/neurokinin 1 receptor antagonist) reduced TF release from macrophages stimulated with SP. Pretreatment of macrophages with a radical scavenger(pyrrolidinedithiocarbamate) also limited the decrease of TF in whole-cell lysates after stimulation with SP. A protein kinase C inhibitor (rottlerin) partially blocked this macrophage response to SP, while it was significantly inhibited by a ROCK inhibitor (Y-27632) or a dynamin inhibitor (dinasore). An Akt inhibitor (perifosine) also partially blocked this response. Furthermore, siRNA targeting p22phox, β-arrestin 2, or Rho A, blunted the release of TF from macrophages stimulated with SP. In other experiments, visceral adipocytes derived from cryopreserved preadipocytes were found to produce SP. In conclusion, SP enhances the release of TF from macrophages via the p22phox/β-arrestin 2/Rho A signaling pathway. Copyright © 2016 Elsevier Inc. All rights reserved.

Growth factor pleiotropy is controlled by a receptor Tyr/Ser motif that acts as a binary switch

PubMed Central

Guthridge, Mark A; Powell, Jason A; Barry, Emma F; Stomski, Frank C; McClure, Barbara J; Ramshaw, Hayley; Felquer, Fernando A; Dottore, Mara; Thomas, Daniel T; To, Bik; Begley, C Glenn; Lopez, Angel F

2006-01-01

Pleiotropism is a hallmark of cytokines and growth factors; yet, the underlying mechanisms are not clearly understood. We have identified a motif in the granulocyte macrophage-colony-stimulating factor receptor composed of a tyrosine and a serine residue that functions as a binary switch for the independent regulation of multiple biological activities. Signalling occurs either through Ser585 at lower cytokine concentrations, leading to cell survival only, or through Tyr577 at higher cytokine concentrations, leading to cell survival as well as proliferation, differentiation or functional activation. The phosphorylation of Ser585 and Tyr577 is mutually exclusive and occurs via a unidirectional mechanism that involves protein kinase A and tyrosine kinases, respectively, and is deregulated in at least some leukemias. We have identified similar Tyr/Ser motifs in other cell surface receptors, suggesting that such signalling switches may play important roles in generating specificity and pleiotropy in other biological systems. PMID:16437163

Endocrine resistance in breast cancer: new roles for ErbB3 and ErbB4.

PubMed

Sutherland, Robert L

2011-05-20

Endocrine resistance is a major limitation to the successful treatment of estrogen receptor-positive (ER(+)) breast cancer, and the EGFR (epidermal growth factor receptor) and ErbB-2 receptor tyrosine kinases are involved in this process. A recent study now implicates the other two ErbB family members, ErbB-3 and -4. Exposure of ER+ breast cancer cells to the pure antiestrogen, fulvestrant, increased levels of ErbB-3 or ErbB-4 and sensitivity to the growth-stimulatory effects of heregulin β1, a potent ligand for these receptors. Thus, the initial growth-inhibitory effects of fulvestrant appear compromised by cellular plasticity that allows rapid compensatory growth stimulation via ErbB-3/4. Further evaluation of pan-ErbB receptor inhibitors in endocrine-resistant disease appears warranted.

Prostaglandin E2 acts via bone marrow macrophages to block PTH-stimulated osteoblast differentiation in vitro

PubMed Central

Choudhary, Shilpa; Blackwell, Katherine; Voznesensky, Olga; Roy, Abhijit Deb; Pilbeam, Carol

2014-01-01

Intermittent PTH is the major anabolic therapy for osteoporosis while continuous PTH causes bone loss. PTH acts on the osteoblast (OB) lineage to regulate bone resorption and formation. PTH also induces cyclooxygenase-2 (COX-2), producing prostaglandin E2 (PGE2) that can act on both OBs and osteoclasts (OCs). Because intermittent PTH is more anabolic in Cox-2 knockout (KO) than wild type (WT) mice, we hypothesized COX-2 might contribute to the effects of continuous PTH by suppressing PTH-stimulated differentiation of mesenchymal stem cells into OBs. We compared effects of continuous PTH on bone marrow stromal cells (BMSCs) and primary OBs (POBs) from Cox-2 KO mice, mice with deletion of PGE2 receptors (Ptger4 and Ptger2 KO mice), and WT controls. PTH increased OB differentiation in BMSCs only in the absence of COX-2 expression or activity. In the absence of COX-2, PTH stimulated differentiation if added during the first week of culture. In Cox-2 KO BMSCs, PTH-stimulated differentiation was prevented by adding PGE2 to cultures. Co-culture of POBs with M-CSF-expanded bone marrow macrophages (BMMs) showed that the inhibition of PTH-stimulated OB differentiation required not only COX-2 or PGE2 but also BMMs. Sufficient PGE2 to mediate the inhibitory effect was made by either WT POBs or WT BMMs. The inhibitory effect mediated by COX-2/PGE2 was transferred by conditioned media from RANKL-treated BMMs and could be blocked by osteoprotegerin, which interferes with RANKL binding to its receptor on OC lineage cells. Deletion of Ptger4, but not Ptger2, in BMMs prevented the inhibition of PTH-stimulated OB differentiation. As expected, PGE2 also stimulated OB differentiation, but when given in combination with PTH, the stimulatory effects of both were abrogated. These data suggest that PGE2, acting via EP4R on BMMs committed to the OC lineage, stimulated secretion of a factor or factors that acted to suppress PTH-stimulated OB differentiation. This suppression of OB differentiation could contribute to the bone loss seen with continuous PTH in vivo. PMID:23639875

Linking diet to acne metabolomics, inflammation, and comedogenesis: an update.

PubMed

Melnik, Bodo C

2015-01-01

Acne vulgaris, an epidemic inflammatory skin disease of adolescence, is closely related to Western diet. Three major food classes that promote acne are: 1) hyperglycemic carbohydrates, 2) milk and dairy products, 3) saturated fats including trans-fats and deficient ω-3 polyunsaturated fatty acids (PUFAs). Diet-induced insulin/insulin-like growth factor (IGF-1)-signaling is superimposed on elevated IGF-1 levels during puberty, thereby unmasking the impact of aberrant nutrigenomics on sebaceous gland homeostasis. Western diet provides abundant branched-chain amino acids (BCAAs), glutamine, and palmitic acid. Insulin and IGF-1 suppress the activity of the metabolic transcription factor forkhead box O1 (FoxO1). Insulin, IGF-1, BCAAs, glutamine, and palmitate activate the nutrient-sensitive kinase mechanistic target of rapamycin complex 1 (mTORC1), the key regulator of anabolism and lipogenesis. FoxO1 is a negative coregulator of androgen receptor, peroxisome proliferator-activated receptor-γ (PPARγ), liver X receptor-α, and sterol response element binding protein-1c (SREBP-1c), crucial transcription factors of sebaceous lipogenesis. mTORC1 stimulates the expression of PPARγ and SREBP-1c, promoting sebum production. SREBP-1c upregulates stearoyl-CoA- and Δ6-desaturase, enhancing the proportion of monounsaturated fatty acids in sebum triglycerides. Diet-mediated aberrations in sebum quantity (hyperseborrhea) and composition (dysseborrhea) promote Propionibacterium acnes overgrowth and biofilm formation with overexpression of the virulence factor triglyceride lipase increasing follicular levels of free palmitate and oleate. Free palmitate functions as a "danger signal," stimulating toll-like receptor-2-mediated inflammasome activation with interleukin-1β release, Th17 differentiation, and interleukin-17-mediated keratinocyte proliferation. Oleate stimulates P. acnes adhesion, keratinocyte proliferation, and comedogenesis via interleukin-1α release. Thus, diet-induced metabolomic alterations promote the visible sebofollicular inflammasomopathy acne vulgaris. Nutrition therapy of acne has to increase FoxO1 and to attenuate mTORC1/SREBP-1c signaling. Patients should balance total calorie uptake and restrict refined carbohydrates, milk, dairy protein supplements, saturated fats, and trans-fats. A paleolithic-like diet enriched in vegetables and fish is recommended. Plant-derived mTORC1 inhibitors and ω-3-PUFAs are promising dietary supplements supporting nutrition therapy of acne vulgaris.

Linking diet to acne metabolomics, inflammation, and comedogenesis: an update

PubMed Central

Melnik, Bodo C

2015-01-01

Acne vulgaris, an epidemic inflammatory skin disease of adolescence, is closely related to Western diet. Three major food classes that promote acne are: 1) hyperglycemic carbohydrates, 2) milk and dairy products, 3) saturated fats including trans-fats and deficient ω-3 polyunsaturated fatty acids (PUFAs). Diet-induced insulin/insulin-like growth factor (IGF-1)-signaling is superimposed on elevated IGF-1 levels during puberty, thereby unmasking the impact of aberrant nutrigenomics on sebaceous gland homeostasis. Western diet provides abundant branched-chain amino acids (BCAAs), glutamine, and palmitic acid. Insulin and IGF-1 suppress the activity of the metabolic transcription factor forkhead box O1 (FoxO1). Insulin, IGF-1, BCAAs, glutamine, and palmitate activate the nutrient-sensitive kinase mechanistic target of rapamycin complex 1 (mTORC1), the key regulator of anabolism and lipogenesis. FoxO1 is a negative coregulator of androgen receptor, peroxisome proliferator-activated receptor-γ (PPARγ), liver X receptor-α, and sterol response element binding protein-1c (SREBP-1c), crucial transcription factors of sebaceous lipogenesis. mTORC1 stimulates the expression of PPARγ and SREBP-1c, promoting sebum production. SREBP-1c upregulates stearoyl-CoA- and Δ6-desaturase, enhancing the proportion of monounsaturated fatty acids in sebum triglycerides. Diet-mediated aberrations in sebum quantity (hyperseborrhea) and composition (dysseborrhea) promote Propionibacterium acnes overgrowth and biofilm formation with overexpression of the virulence factor triglyceride lipase increasing follicular levels of free palmitate and oleate. Free palmitate functions as a “danger signal,” stimulating toll-like receptor-2-mediated inflammasome activation with interleukin-1β release, Th17 differentiation, and interleukin-17-mediated keratinocyte proliferation. Oleate stimulates P. acnes adhesion, keratinocyte proliferation, and comedogenesis via interleukin-1α release. Thus, diet-induced metabolomic alterations promote the visible sebofollicular inflammasomopathy acne vulgaris. Nutrition therapy of acne has to increase FoxO1 and to attenuate mTORC1/SREBP-1c signaling. Patients should balance total calorie uptake and restrict refined carbohydrates, milk, dairy protein supplements, saturated fats, and trans-fats. A paleolithic-like diet enriched in vegetables and fish is recommended. Plant-derived mTORC1 inhibitors and ω-3-PUFAs are promising dietary supplements supporting nutrition therapy of acne vulgaris. PMID:26203267

Ethylene Regulates Levels of Ethylene Receptor/CTR1 Signaling Complexes in Arabidopsis thaliana

DOE PAGES

Shakeel, Samina N.; Gao, Zhiyong; Amir, Madiha; ...

2015-03-26

The plant hormone ethylene is perceived by a five-member family of receptors in Arabidopsis thaliana. The receptors function in conjunction with the Raf-like kinase CTR1 to negatively regulate ethylene signal transduction. CTR1 interacts with multiple members of the receptor family based on co-purification analysis, interacting more strongly with receptors containing a receiver domain. Levels of membrane-associated CTR1 vary in response to ethylene, doing so in a post-transcriptional manner that correlates with ethylene-mediated changes in levels of the ethylene receptors ERS1, ERS2, EIN4, and ETR2. Interactions between CTR1 and the receptor ETR1 protect ETR1 from ethylene-induced turnover. Kinetic and dose-response analysesmore » support a model in which two opposing factors control levels of the ethylene receptor/CTR1 complexes. Ethylene stimulates the production of new complexes largely through transcriptional induction of the receptors. However, ethylene also induces turnover of receptors, such that levels of ethylene receptor/CTR1 complexes decrease at higher ethylene concentrations. Lastly, we discuss implications of this model for ethylene signaling.« less

Ethylene Regulates Levels of Ethylene Receptor/CTR1 Signaling Complexes in Arabidopsis thaliana*

PubMed Central

Shakeel, Samina N.; Gao, Zhiyong; Amir, Madiha; Chen, Yi-Feng; Rai, Muneeza Iqbal; Haq, Noor Ul; Schaller, G. Eric

2015-01-01

The plant hormone ethylene is perceived by a five-member family of receptors in Arabidopsis thaliana. The receptors function in conjunction with the Raf-like kinase CTR1 to negatively regulate ethylene signal transduction. CTR1 interacts with multiple members of the receptor family based on co-purification analysis, interacting more strongly with receptors containing a receiver domain. Levels of membrane-associated CTR1 vary in response to ethylene, doing so in a post-transcriptional manner that correlates with ethylene-mediated changes in levels of the ethylene receptors ERS1, ERS2, EIN4, and ETR2. Interactions between CTR1 and the receptor ETR1 protect ETR1 from ethylene-induced turnover. Kinetic and dose-response analyses support a model in which two opposing factors control levels of the ethylene receptor/CTR1 complexes. Ethylene stimulates the production of new complexes largely through transcriptional induction of the receptors. However, ethylene also induces turnover of receptors, such that levels of ethylene receptor/CTR1 complexes decrease at higher ethylene concentrations. Implications of this model for ethylene signaling are discussed. PMID:25814663

High-mobility group (HMG) protein HMG-1 and TATA-binding protein-associated factor TAF(II)30 affect estrogen receptor-mediated transcriptional activation.

PubMed

Verrier, C S; Roodi, N; Yee, C J; Bailey, L R; Jensen, R A; Bustin, M; Parl, F F

1997-07-01

The estrogen receptor (ER) belongs to a family of ligand-inducible nuclear receptors that exert their effects by binding to cis-acting DNA elements in the regulatory region of target genes. The detailed mechanisms by which ER interacts with the estrogen response element (ERE) and affects transcription still remain to be elucidated. To study the ER-ERE interaction and transcription initiation, we employed purified recombinant ER expressed in both the baculovirus-Sf9 and his-tagged bacterial systems. The effect of high-mobility group (HMG) protein HMG-1 and purified recombinant TATA-binding protein-associated factor TAF(II)30 on ER-ERE binding and transcription initiation were assessed by electrophoretic mobility shift assay and in vitro transcription from an ERE-containing template (pERE2LovTATA), respectively. We find that purified, recombinant ER fails to bind to ERE in spite of high ligand-binding activity and electrophoretic and immunological properties identical to ER in MCF-7 breast cancer cells. HMG-1 interacts with ER and promotes ER-ERE binding in a concentration- and time-dependent manner. The effectiveness of HMG-1 to stimulate ER-ERE binding in the electrophoretic mobility shift assay depends on the sequence flanking the ERE consensus as well as the position of the latter in the oligonucleotide. We find that TAF(II)30 has no effect on ER-ERE binding either alone or in combination with ER and HMG-1. Although HMG-1 promotes ER-ERE binding, it fails to stimulate transcription initiation either in the presence or absence of hormone. In contrast, TAF(II)30, while not affecting ER-ERE binding, stimulates transcription initiation 20-fold in the presence of HMG-1. These results indicate that HMG-1 and TAF(II)30 act in sequence, the former acting to promote ER-ERE binding followed by the latter to stimulate transcription initiation.

Activation of histamine H4 receptor inhibits TNFα/IMD-0354-induced apoptosis in human salivary NS-SV-AC cells.

PubMed

Stegajev, Vasili; Kouri, Vesa-Petteri; Salem, Abdelhakim; Rozov, Stanislav; Stark, Holger; Nordström, Dan C E; Konttinen, Yrjö T

2014-12-01

Apoptosis is involved in the pathogenesis of Sjögren's syndrome (SS), an autoimmune disease affecting exocrine glands. Our recent studies revealed diminished histamine H4 receptor (H₄R) expression and impaired histamine transport in the salivary gland epithelial cells in SS. The aim was now to test if nanomolar histamine and high-affinity H₄R signaling affect apoptosis of human salivary gland epithelial cell. Simian virus 40-immortalized acinar NS-SV-AC cells were cultured in serum-free keratinocyte medium ± histamine H₄R agonist HST-10. Expression and internalization of H₄R were studied by immunofluorescence staining ± clathrin inhibitor methyl-β-cyclodextrin (MβCD). Apoptosis induced using tumor necrosis factor-α with nuclear factor-κB inhibitor IMD-0354 was studied using phase contrast microscopy, Western blot, flow cytometry and polymerase chain reaction (qRT-PCR). HST-10-stimulated H₄R internalization was inhibited by MβCD. Western blotting revealed diminished phosphorylated c-Jun N-terminal kinase JNK, but unchanged levels of phosphorylated extracellular signal regulated kinase pERK1/2 in H₄R-stimulated samples compared to controls. qRT-PCR showed up-regulated expression of anti-apoptotic B cell lymphoma-extra large/Bcl-xL mRNAs and proteins, whereas pro-apoptotic Bcl-2-associated X protein/BAX remained unchanged in H4R-stimulated samples. H₄R stimulation diminished cleavage of PARP and flow cytometry showed significant dose-dependent inhibitory effect of H₄R stimulation on apoptosis. As far as we know this is the first study showing inhibitory effect of H₄R activation on apoptosis of human salivary gland cells. Diminished H₄R-mediated activation may contribute to loss of immune tolerance in autoimmune diseases and in SS in particular.

Modeling invasive breast cancer: growth factors propel progression of HER2-positive premalignant lesions

PubMed Central

Pradeep, C-R; Zeisel, A; Köstler, WJ; Lauriola, M; Jacob-Hirsch, J; Haibe-Kains, B; Amariglio, N; Ben-Chetrit, N; Emde, A; Solomonov, I; Neufeld, G; Piccart, M; Sagi, I; Sotiriou, C; Rechavi, G; Domany, E; Desmedt, C; Yarden, Y

2013-01-01

The HER2/neu oncogene encodes a receptor-like tyrosine kinase whose overexpression in breast cancer predicts poor prognosis and resistance to conventional therapies. However, the mechanisms underlying aggressiveness of HER2 (human epidermal growth factor receptor 2)-overexpressing tumors remain incompletely understood. Because it assists epidermal growth factor (EGF) and neuregulin receptors, we overexpressed HER2 in MCF10A mammary cells and applied growth factors. HER2-overexpressing cells grown in extracellular matrix formed filled spheroids, which protruded outgrowths upon growth factor stimulation. Our transcriptome analyses imply a two-hit model for invasive growth: HER2-induced proliferation and evasion from anoikis generate filled structures, which are morphologically and transcriptionally analogous to preinvasive patients’ lesions. In the second hit, EGF escalates signaling and transcriptional responses leading to invasive growth. Consistent with clinical relevance, a gene expression signature based on the HER2/EGF-activated transcriptional program can predict poorer prognosis of a subgroup of HER2-overexpressing patients. In conclusion, the integration of a three-dimensional cellular model and clinical data attributes progression of HER2-overexpressing lesions to EGF-like growth factors acting in the context of the tumor's microenvironment. PMID:22139081

Effects of the noncompetitive N-methyl-d-aspartate receptor antagonists ketamine and MK-801 on pain-stimulated and pain-depressed behaviour in rats.

PubMed

Hillhouse, T M; Negus, S S

2016-09-01

Pain is a significant public health concern, and current pharmacological treatments have problematic side effects and limited effectiveness. N-methyl-d-aspartate (NMDA) glutamate receptor antagonists have emerged as one class of candidate treatments for pain because of the significant contribution of glutamate signalling in nociceptive processing. This study compared effects of the NMDA receptor antagonists ketamine and MK-801 in assays of pain-stimulated and pain-depressed behaviour in rats. The nonsteroidal anti-inflammatory drug ketoprofen was examined for comparison as a positive control. Intraperitoneal injection of dilute acid served as an acute visceral noxious stimulus to stimulate a stretching response or depress intracranial self-stimulation (ICSS) in male Sprague-Dawley rats. Ketamine (1.0-10.0 mg/kg) blocked acid-stimulated stretching but failed to block acid-induced depression of ICSS, whereas MK-801 (0.01-0.1 mg/kg) blocked both acid-stimulated stretching and acid-induced depression of ICSS. These doses of ketamine and MK-801 did not alter control ICSS in the absence of the noxious stimulus; however, higher doses of ketamine (10 mg/kg) and MK-801 (0.32 mg/kg) depressed all behaviour. Ketoprofen (1.0 mg/kg) blocked both acid-induced stimulation of stretching and depression of ICSS without altering control ICSS. These results support further consideration of NMDA receptor antagonists as analgesics; however, some NMDA receptor antagonists are more efficacious at attenuating pain-depressed behaviours. NMDA receptor antagonists produce dissociable effects on pain-depressed behaviour. Provides evidence that pain-depressed behaviours should be considered and evaluated when determining the antinociceptive effects of NMDA receptor antagonists. © 2016 European Pain Federation - EFIC®

Effects of the noncompetitive N-methyl-D-aspartate receptor antagonists ketamine and MK-801 on pain-stimulated and pain-depressed behaviour in rats

PubMed Central

Hillhouse, T.M.; Negus, S.S.

2017-01-01

Background Pain is a significant public health concern, and current pharmacological treatments have problematic side effects and limited effectiveness. N-methyl-D-aspartate (NMDA) glutamate receptor antagonists have emerged as one class of candidate treatments for pain because of the significant contribution of glutamate signalling in nociceptive processing. Methods This study compared effects of the NMDA receptor antagonists ketamine and MK-801 in assays of pain-stimulated and pain-depressed behaviour in rats. The nonsteroidal anti-inflammatory drug ketoprofen was examined for comparison as a positive control. Intraperitoneal injection of dilute acid served as an acute visceral noxious stimulus to stimulate a stretching response or depress intracranial self-stimulation (ICSS) in male Sprague–Dawley rats. Results Ketamine (1.0–10.0 mg/kg) blocked acid-stimulated stretching but failed to block acid-induced depression of ICSS, whereas MK-801 (0.01–0.1 mg/kg) blocked both acid-stimulated stretching and acid-induced depression of ICSS. These doses of ketamine and MK-801 did not alter control ICSS in the absence of the noxious stimulus; however, higher doses of ketamine (10 mg/kg) and MK-801 (0.32 mg/kg) depressed all behaviour. Ketoprofen (1.0 mg/kg) blocked both acid-induced stimulation of stretching and depression of ICSS without altering control ICSS. Conclusion These results support further consideration of NMDA receptor antagonists as analgesics; however, some NMDA receptor antagonists are more efficacious at attenuating pain-depressed behaviours. What does this study add? NMDA receptor antagonists produce dissociable effects on pain-depressed behaviour. Provides evidence that pain-depressed behaviours should be considered and evaluated when determining the antinociceptive effects of NMDA receptor antagonists. PMID:26914635

EVIDENCE THAT CA2+ SIGNALING AND TRANSCRIPTION FACTOR (CREB) ACTIVITIES STIMULATED BY POLYCHLORINATED BIPHENYLS ARE LOCALIZED TO DEVELOPING NEURONS.

EPA Science Inventory

Using a mixed culture of neonatal cortical cells, we have demonstrated that the polychlorinated biphenyl (PCB) mixture Aroclor 1254 (A1254) induces complex Ca2+i signals involving multiple receptors/channels (Inglefield and Shafer, J.Pharm.Exp.Ther. 295:105) and also activates/ p...

Nature of the stimulation of biogenesis of cholesterol in the liver by noradrenaline.

PubMed Central

George, R; Ramasarma, T

1977-01-01

1. Administration of noradrenaline increased the incorporation of [1-14C]acetate into hepatic sterols and the activity of liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase. 2. The stimulation was observed at short time-intervals with a maximum at 4h and was progressive with increasing concentrations of noradrenaline. 3. Protein synthesis de novo was a necessary factor for the effect. 4. The stimulatory effect was not mediated through the adrenergic receptors, but appears to involve a direct action of the hormone within the hepatocyte. PMID:68775

Klotho modulates FGF23-mediated NO synthesis and oxidative stress in human coronary artery endothelial cells.

PubMed

Richter, Beatrice; Haller, Jacqueline; Haffner, Dieter; Leifheit-Nestler, Maren

2016-09-01

Chronic kidney disease (CKD) is a state of Klotho deficiency and excess of the phosphaturic hormone fibroblast growth factor 23 (FGF23). Both dysregulations were shown to be associated with endothelial dysfunction in humans, but direct vascular effects of FGF23 remain largely elusive. In vitro experiments were performed to assess the effects of FGF23 (10 ng/mL) in relation to its co-receptor Klotho on nitric oxide (NO) synthesis and reactive oxygen species (ROS) formation and detoxification in human coronary artery endothelial cells (HCAEC). Membrane-bound Klotho is expressed in HCAEC, and FGF23 increases the expression of the Klotho shedding protease ADAM17, and consequently the secretion of soluble Klotho. FGF23 activates FGF receptor 1 and stimulates NO release via Akt-dependent activation of endothelial NO synthase (eNOS). Both FGF receptor (FGFR)-dependent ROS formation via activation of NADPH oxidase 2 (Nox2) as well as ROS degradation via superoxide dismutase 2 (SOD2) and catalase (CAT) is stimulated by FGF23. Pre-incubation with a Klotho inhibitor blunts the FGF23-stimulated Akt-eNOS activation and NO synthesis, and decreases ROS degradation by blocking SOD2 and CAT enzymes, whereas FGF23-stimulated ROS synthesis via Nox2 is unaffected, resulting in low NO bioavailability and increased oxidative stress. Our data indicate that in the presence of Klotho, FGF23 induces NO release in HCAEC and its stimulating effects on ROS production are counterbalanced by increased ROS degradation. In states of Klotho deficiency, e.g., CKD, FGF23-mediated NO synthesis is blunted and ROS formation overrules ROS degradation. Thus, FGF23 excess may primarily promote oxidative stress and thus endothelial dysfunction.

Colony Stimulating Factor-1 Receptor is a central component of the foreign body response to biomaterial implants in rodents and non-human primates

PubMed Central

Doloff, Joshua C.; Veiseh, Omid; Vegas, Arturo J.; Tam, Hok Hei; Farah, Shady; Ma, Minglin; Li, Jie; Bader, Andrew; Chiu, Alan; Sadraei, Atieh; Aresta-Dasilva, Stephanie; Griffin, Marissa; Jhunjhunwala, Siddharth; Webber, Matthew; Siebert, Sean; Tang, Katherine; Chen, Michael; Langan, Erin; Dholokia, Nimit; Thakrar, Raj; Qi, Meirigeng; Oberholzer, Jose; Greiner, Dale L.; Langer, Robert; Anderson, Daniel G.

2017-01-01

Host recognition and immune-mediated foreign body response (FBR) to biomaterials can compromise the performance of implanted medical devices. To identify key cell and cytokine targets, here we perform in-depth systems analysis of innate and adaptive immune system responses to implanted biomaterials in rodents and non-human primates. While macrophages are indispensable to the fibrotic cascade, surprisingly neutrophils and complement are not. Macrophages, via CXCL13, lead to downstream B cell recruitment, which further potentiated fibrosis, as confirmed by B cell knock out and CXCL13 neutralization. Interestingly, Colony Stimulating Factor-1 Receptor (CSF1R) is significantly increased following implantation of multiple biomaterial classes: ceramic, polymer, and hydrogel. Its inhibition, like macrophage depletion, leads to complete loss of fibrosis, but spares other macrophage functions such as wound healing, ROS production, and phagocytosis. Our results indicate targeting CSF1R may allow for a more selective method of fibrosis inhibition, and improve biomaterial biocompatibility without the need for broad immunosuppression. PMID:28319612

Complex interactions in EML cell stimulation by stem cell factor and IL-3.

PubMed

Ye, Zhi-jia; Gulcicek, Erol; Stone, Kathryn; Lam, Tukiet; Schulz, Vincent; Weissman, Sherman M

2011-03-22

Erythroid myeloid lymphoid (EML) cells are an established multipotent hematopoietic precursor cell line that can be maintained in medium including stem cell factor (SCF). EML cultures contain a heterogeneous mixture of cells, including a lineage-negative, CD34+ subset of cells that propagate rapidly in SCF and can clonally regenerate the mixed population. A second major subset of EML cells consists of lineage-negative. CD34- cells that can be propagated in IL-3 but grow slowly, if at all, in SCF, although they express the SCF receptor (c-kit). The response of these cells to IL-3 is stimulated synergistically by SCF, and we present evidence that both the synergy and the inhibition of c-kit responses may be mediated by direct interaction with IL-3 receptor. Further, the relative level of tyrosine phosphorylation of various substrates by either cytokine alone differs from that produced by the combination of the two cytokines, suggesting that cell signaling by the combination of the two cytokines differs from that produced by either alone.

Complex interactions in EML cell stimulation by stem cell factor and IL-3

PubMed Central

Ye, Zhi-jia; Gulcicek, Erol; Stone, Kathryn; Lam, Tukiet; Schulz, Vincent; Weissman, Sherman M.

2011-01-01

Erythroid myeloid lymphoid (EML) cells are an established multipotent hematopoietic precursor cell line that can be maintained in medium including stem cell factor (SCF). EML cultures contain a heterogeneous mixture of cells, including a lineage-negative, CD34+ subset of cells that propagate rapidly in SCF and can clonally regenerate the mixed population. A second major subset of EML cells consists of lineage-negative. CD34− cells that can be propagated in IL-3 but grow slowly, if at all, in SCF, although they express the SCF receptor (c-kit). The response of these cells to IL-3 is stimulated synergistically by SCF, and we present evidence that both the synergy and the inhibition of c-kit responses may be mediated by direct interaction with IL-3 receptor. Further, the relative level of tyrosine phosphorylation of various substrates by either cytokine alone differs from that produced by the combination of the two cytokines, suggesting that cell signaling by the combination of the two cytokines differs from that produced by either alone. PMID:21383156

Colony stimulating factor-1 receptor is a central component of the foreign body response to biomaterial implants in rodents and non-human primates

NASA Astrophysics Data System (ADS)

Doloff, Joshua C.; Veiseh, Omid; Vegas, Arturo J.; Tam, Hok Hei; Farah, Shady; Ma, Minglin; Li, Jie; Bader, Andrew; Chiu, Alan; Sadraei, Atieh; Aresta-Dasilva, Stephanie; Griffin, Marissa; Jhunjhunwala, Siddharth; Webber, Matthew; Siebert, Sean; Tang, Katherine; Chen, Michael; Langan, Erin; Dholokia, Nimit; Thakrar, Raj; Qi, Meirigeng; Oberholzer, Jose; Greiner, Dale L.; Langer, Robert; Anderson, Daniel G.

2017-06-01

Host recognition and immune-mediated foreign body response to biomaterials can compromise the performance of implanted medical devices. To identify key cell and cytokine targets, here we perform in-depth systems analysis of innate and adaptive immune system responses to implanted biomaterials in rodents and non-human primates. While macrophages are indispensable to the fibrotic cascade, surprisingly neutrophils and complement are not. Macrophages, via CXCL13, lead to downstream B cell recruitment, which further potentiated fibrosis, as confirmed by B cell knockout and CXCL13 neutralization. Interestingly, colony stimulating factor-1 receptor (CSF1R) is significantly increased following implantation of multiple biomaterial classes: ceramic, polymer and hydrogel. Its inhibition, like macrophage depletion, leads to complete loss of fibrosis, but spares other macrophage functions such as wound healing, reactive oxygen species production and phagocytosis. Our results indicate that targeting CSF1R may allow for a more selective method of fibrosis inhibition, and improve biomaterial biocompatibility without the need for broad immunosuppression.

Cyclic AMP differentiates two separate but interacting pathways of phosphoinositide hydrolysis in the DDT1-MF2 smooth muscle cell line.

PubMed

Schachter, J B; Wolfe, B B

1992-03-01

The activation of adenosine A1 receptors in DDT1-MF2 smooth muscle cells resulted in both the inhibition of agonist-stimulated cAMP accumulation and the potentiation of norepinephrine-stimulated phosphoinositide hydrolysis. Pharmacological analysis indicated the involvement of an A1 adenosine receptor subtype in both of these responses. In the absence of norepinephrine, the activation of the adenosine receptor did not directly stimulate phosphoinositide hydrolysis. The adenosine receptor-mediated augmentation of norepinephrine-stimulated phosphoinositide hydrolysis was pertussis toxin sensitive and was selectively antagonized by agents that mimicked cAMP (8-bromo-cAMP) or raised cellular cAMP levels (forskolin). This initially suggested that cAMP might partially regulate the magnitude of the phospholipase C response to norepinephrine and that adenosine agonists might enhance the phospholipase C response by reducing cAMP levels. However, neither the reduction of cellular cAMP levels by other agents nor the inhibition of cAMP-dependent protein kinase was sufficient to replicate the action of adenosine receptor activation on phosphoinositide hydrolysis. Thus, in the presence of norepinephrine, adenosine receptor agonists appear to stimulate phosphoinositide hydrolysis via a pathway that is separate from, but dependent upon, that of norepinephrine. This second pathway can be distinguished from that which is stimulated by norepinephrine on the basis of its sensitivity to inhibition by both cAMP and pertussis toxin.

Differential regulation of Smad3 and of the type II transforming growth factor-β receptor in mitosis: implications for signaling.

PubMed

Hirschhorn, Tal; Barizilay, Lior; Smorodinsky, Nechama I; Ehrlich, Marcelo

2012-01-01

The response to transforming growth factor-β (TGF-β) depends on cellular context. This context is changed in mitosis through selective inhibition of vesicle trafficking, reduction in cell volume and the activation of mitotic kinases. We hypothesized that these alterations in cell context may induce a differential regulation of Smads and TGF-β receptors. We tested this hypothesis in mesenchymal-like ovarian cancer cells, arrested (or not) in mitosis with 2-methoxyestradiol (2ME2). In mitosis, without TGF-β stimulation, Smad3 was phosphorylated at the C-terminus and linker regions and localized to the mitotic spindle. Phosphorylated Smad3 interacted with the negative regulators of Smad signaling, Smurf2 and Ski, and failed to induce a transcriptional response. Moreover, in cells arrested in mitosis, Smad3 levels were progressively reduced. These phosphorylations and reduction in the levels of Smad3 depended on ERK activation and Mps1 kinase activity, and were abrogated by increasing the volume of cells arrested in mitosis with hypotonic medium. Furthermore, an Mps1-dependent phosphorylation of GFP-Smad3 was also observed upon its over-expression in interphase cells, suggesting a mechanism of negative regulation which counters increases in Smad3 concentration. Arrest in mitosis also induced a block in the clathrin-mediated endocytosis of the type II TGF-β receptor (TβRII). Moreover, following the stimulation of mitotic cells with TGF-β, the proteasome-mediated attenuation of TGF-β receptor activity, the degradation and clearance of TβRII from the plasma membrane, and the clearance of the TGF-β ligand from the medium were compromised, and the C-terminus phosphorylation of Smad3 was prolonged. We propose that the reduction in Smad3 levels, its linker phosphorylation, and its association with negative regulators (observed in mitosis prior to ligand stimulation) represent a signal attenuating mechanism. This mechanism is balanced by the retention of active TGF-β receptors at the plasma membrane. Together, both mechanisms allow for a regulated cellular response to TGF-β stimuli in mitosis.

Differential Regulation of Smad3 and of the Type II Transforming Growth Factor-β Receptor in Mitosis: Implications for Signaling

PubMed Central

Hirschhorn, Tal; Barizilay, Lior; Smorodinsky, Nechama I.; Ehrlich, Marcelo

2012-01-01

The response to transforming growth factor-β (TGF-β) depends on cellular context. This context is changed in mitosis through selective inhibition of vesicle trafficking, reduction in cell volume and the activation of mitotic kinases. We hypothesized that these alterations in cell context may induce a differential regulation of Smads and TGF-β receptors. We tested this hypothesis in mesenchymal-like ovarian cancer cells, arrested (or not) in mitosis with 2-methoxyestradiol (2ME2). In mitosis, without TGF-β stimulation, Smad3 was phosphorylated at the C-terminus and linker regions and localized to the mitotic spindle. Phosphorylated Smad3 interacted with the negative regulators of Smad signaling, Smurf2 and Ski, and failed to induce a transcriptional response. Moreover, in cells arrested in mitosis, Smad3 levels were progressively reduced. These phosphorylations and reduction in the levels of Smad3 depended on ERK activation and Mps1 kinase activity, and were abrogated by increasing the volume of cells arrested in mitosis with hypotonic medium. Furthermore, an Mps1-dependent phosphorylation of GFP-Smad3 was also observed upon its over-expression in interphase cells, suggesting a mechanism of negative regulation which counters increases in Smad3 concentration. Arrest in mitosis also induced a block in the clathrin-mediated endocytosis of the type II TGF-β receptor (TβRII). Moreover, following the stimulation of mitotic cells with TGF-β, the proteasome-mediated attenuation of TGF-β receptor activity, the degradation and clearance of TβRII from the plasma membrane, and the clearance of the TGF-β ligand from the medium were compromised, and the C-terminus phosphorylation of Smad3 was prolonged. We propose that the reduction in Smad3 levels, its linker phosphorylation, and its association with negative regulators (observed in mitosis prior to ligand stimulation) represent a signal attenuating mechanism. This mechanism is balanced by the retention of active TGF-β receptors at the plasma membrane. Together, both mechanisms allow for a regulated cellular response to TGF-β stimuli in mitosis. PMID:22927969

Proteomic Analysis of the Epidermal Growth Factor Receptor (EGFR) Interactome and Post-translational Modifications Associated with Receptor Endocytosis in Response to EGF and Stress*

PubMed Central

Tong, Jiefei; Taylor, Paul; Moran, Michael F.

2014-01-01

Aberrant expression, activation, and stabilization of epidermal growth factor receptor (EGFR) are causally associated with several human cancers. Post-translational modifications and protein-protein interactions directly modulate the signaling and trafficking of the EGFR. Activated EGFR is internalized by endocytosis and then either recycled back to the cell surface or degraded in the lysosome. EGFR internalization and recycling also occur in response to stresses that activate p38 MAP kinase. Mass spectrometry was applied to comprehensively analyze the phosphorylation, ubiquitination, and protein-protein interactions of wild type and endocytosis-defective EGFR variants before and after internalization in response to EGF ligand and stress. Prior to internalization, EGF-stimulated EGFR accumulated ubiquitin at 7 K residues and phosphorylation at 7 Y sites and at S1104. Following internalization, these modifications diminished and there was an accumulation of S/T phosphorylations. EGFR internalization and many but not all of the EGF-induced S/T phosphorylations were also stimulated by anisomycin-induced cell stress, which was not associated with receptor ubiquitination or elevated Y phosphorylation. EGFR protein interactions were dramatically modulated by ligand, internalization, and stress. In response to EGF, different E3 ubiquitin ligases became maximally associated with EGFR before (CBL, HUWE1, and UBR4) or after (ITCH) internalization, whereas CBLB was distinctively most highly EGFR associated following anisomycin treatment. Adaptin subunits of AP-1 and AP-2 clathrin adaptor complexes also became EGFR associated in response to EGF and anisomycin stress. Mutations preventing EGFR phosphorylation at Y998 or in the S1039 region abolished or greatly reduced EGFR interactions with AP-2 and AP-1, and impaired receptor trafficking. These results provide new insight into spatial, temporal, and mechanistic aspects of EGFR regulation. PMID:24797263

Cancer Cell-derived Exosomes Induce Mitogen-activated Protein Kinase-dependent Monocyte Survival by Transport of Functional Receptor Tyrosine Kinases.

PubMed

Song, Xiao; Ding, Yanping; Liu, Gang; Yang, Xiao; Zhao, Ruifang; Zhang, Yinlong; Zhao, Xiao; Anderson, Gregory J; Nie, Guangjun

2016-04-15

Tumor-associated macrophages (TAM) play pivotal roles in cancer initiation and progression. Monocytes, the precursors of TAMs, normally undergo spontaneous apoptosis within 2 days, but can subsist in the inflammatory tumor microenvironment for continuous survival and generation of sufficient TAMs. The mechanisms underlying tumor-driving monocyte survival remain obscure. Here we report that cancer cell-derived exosomes were crucial mediators for monocyte survival in the inflammatory niche. Analysis of the survival-promoting molecules in monocytes revealed that cancer cell-derived exosomes activated Ras and extracellular signal-regulated kinases in the mitogen-activated protein kinase (MAPK) pathway, resulting in the prevention of caspase cleavage. Phosphorylated receptor tyrosine kinases (RTKs), such as phosphorylated epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER-2), were abundantly expressed in cancer cell-derived exosomes. Knock-out of EGFR or/and HER-2, or alternatively, inhibitors against their phosphorylation significantly disturbed the exosome-mediated activation of the MAPK pathway, inhibition of caspase cleavage, and increase in survival rate in monocytes. Moreover, the deprived survival-stimulating activity of exosomes due to null expression of EGFR and HER-2 could be restored by activation of another RTK, insulin receptor. Overall, our study uncovered a mechanism of tumor-associated monocyte survival and demonstrated that cancer cell-derived exosomes can stimulate the MAPK pathway in monocytes through transport of functional RTKs, leading to inactivation of apoptosis-related caspases. This work provides insights into the long sought question on monocyte survival prior to formation of plentiful TAMs in the tumor microenvironment. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Increased expression of both insulin receptor substrates 1 and 2 confers increased sensitivity to IGF-1 stimulated cell migration.

PubMed

de Blaquière, Gail E; May, Felicity E B; Westley, Bruce R

2009-06-01

Insulin-like growth factors (IGFs) are thought to promote tumour progression and metastasis in part by stimulating cell migration. Insulin receptor substrate-1 (IRS-1) and IRS-2 are multisite docking proteins positioned immediately downstream from the type I IGF and insulin receptors. IRS-2 but not IRS-1 has been reported to be involved in the migratory response of breast cancer cells to IGFs. The purpose of this investigation was to determine if IRS-1 is involved in, and to assess the contributions of IRS-1 and IRS-2 to, the migratory response of breast cancer cells to IGFs. The expression of IRS-1 and IRS-2 varied considerably between ten breast cancer cell lines. Oestrogen increases expression of the type I IGF receptor, IRS-1 and IRS-2 in MCF-7 and ZR-75 cells. Oestrogens may control the sensitivity of breast cancer cells to IGFs by regulating the expression of components of the IGF signal transduction pathway. The migratory response to a range of IGF-1 concentrations was measured in MCF-7 and MDA-MB-231 breast cancer cells in which IRS-1 and IRS-2 levels were modulated using a doxycycline-inducible expression system. Induction of both IRS-1 and IRS-2 expression increased the sensitivity of the migratory response to IGF-1 but did not increase the magnitude of the response stimulated at higher concentrations of IGF-1. Knockdown of IRS-1, IRS-2 and the type I IGF receptor in MCF-7 and MDA-MB-2231 cells decreased sensitivity to IGF-1. We conclude that both IRS-1 and IRS-2 control the migratory response of breast cancer cells to IGF-1 and may, therefore, be key molecules in determining breast cancer spread.

Mechanical stretch augments insulin-induced vascular smooth muscle cell proliferation by insulin-like growth factor-1 receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Gang; Department of Anesthesiology, First Affiliated Hospital of China Medical University, Shenyang; Hitomi, Hirofumi, E-mail: hitomi@kms.ac.jp

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,more » 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.« less

Isolation and purification of an early pregnancy factor-like molecule from culture supernatants obtained from lymphocytes of pregnant women: II. Identification of the molecule as a Fc-receptor-like molecule: a preliminary report.

PubMed

Aranha, C; Bordekar, A; Shahani, S

1998-11-01

Early pregnancy factor (EPF)-like activity from culture supernatants obtained from stimulated lymphocytes of pregnant women was characterized and identified. The enzyme-linked immunosorbent assay depending on the presence of "Fc" receptors on bovine spermatozoa was used to identify the EPF-like molecule purified by gel filtration and reverse-phase high-performance liquid chromatography. The results indicated that the crude lymphocyte culture supernatant, the EPF-positive G IV fraction obtained on gel filtration, and the EPF-positive reverse-phase high-performance liquid chromatography protein readily bound with the different concentrations of aggregated human gamma-globulin in a manner similar to that in which the standard control of aggregated human gamma-globulin binds to the bovine spermatozoa. EPF-like activity synthesized and secreted by lymphocytes during pregnancy may be a Fc-receptor-like molecule.

CSF1R mutations in hereditary diffuse leukoencephalopathy with spheroids are loss of function

NASA Astrophysics Data System (ADS)

Pridans, Clare; Sauter, Kristin A.; Baer, Kristin; Kissel, Holger; Hume, David A.

2013-10-01

Hereditary diffuse leukoencephalopathy with spheroids (HDLS) in humans is a rare autosomal dominant disease characterized by giant neuroaxonal swellings (spheroids) within the CNS white matter. Symptoms are variable and can include personality and behavioural changes. Patients with this disease have mutations in the protein kinase domain of the colony-stimulating factor 1 receptor (CSF1R) which is a tyrosine kinase receptor essential for microglia development. We investigated the effects of these mutations on Csf1r signalling using a factor dependent cell line. Corresponding mutant forms of murine Csf1r were expressed on the cell surface at normal levels, and bound CSF1, but were not able to sustain cell proliferation. Since Csf1r signaling requires receptor dimerization initiated by CSF1 binding, the data suggest a mechanism for phenotypic dominance of the mutant allele in HDLS.

Insulin release: the receptor hypothesis.

PubMed

Malaisse, Willy J

2014-07-01

It is currently believed that the stimulation of insulin release by nutrient secretagogues reflects their capacity to act as fuel in pancreatic islet beta cells. In this review, it is proposed that such a fuel concept is not incompatible with a receptor hypothesis postulating the participation of cell-surface receptors in the recognition of selected nutrients as insulinotropic agents. Pursuant to this, attention is drawn to such matters as the anomeric specificity of the beta cell secretory response to D-glucose and its perturbation in diabetes mellitus, the insulinotropic action of artificial sweeteners, the possible role of bitter taste receptors in the stimulation of insulin secretion by L-glucose pentaacetate, the recently documented presence of cell-surface sweet taste receptors in insulin-producing cells, the multimodal signalling process resulting from the activation of these latter receptors, and the presence in beta cells of a sweet taste receptor mediating the fructose-induced potentiation of glucose-stimulated insulin secretion.

Stimulation of cell proliferation by histamine H2 receptors in dimethylhdrazine-induced adenocarcinomata.

PubMed

Tutton, P J; Barkla, D H

1978-03-01

Cell proliferation in dimethylhydrazine-induced colonic carcinomata was stimulated by histamine and by the histamine H2 receptor agonist dimaprit and inhibited by the histamine H2 receptor antagonists Metiamide and Cimetidine but not by the histamine H1 receptor antagonist Mepyramine. In contrast histamine had no effect on colonic crypt cell proliferation in normal or dimethylhydrazine-treated rats.

DARPP chocolate: a caffeinated morsel of striatal signaling.

PubMed

Bastia, Elena; Schwarzschild, Michael A

2003-01-14

The psychomotor stimulant effects of caffeine, the most widely consumed psychoactive substance, are mediated through its antagonism of extracellular adenosine receptors in the basal ganglia. In the absence of caffeine, adenosine stimulates inhibitory striatopallidal neurons that suppress motor activity by binding to A2A receptors, thereby activating a cyclic adenosine 3',5'-monophosphate (cAMP) and protein kinase A signaling pathway. Bastia and Schwarzschild discuss recent research implicating DARRP-32 (dopamine- and cAMP-regulated phosphoprotein of 32 kilodaltons) as an attractive mediator of the sustained psychomotor stimulant effect seen with low doses of caffeine. They highlight the role of postsynaptic A2A receptor blockade, but leave open the possibility that antagonism of presynaptic or postsynaptic A1 receptors also contributes to DARPP-32-dependent psychomotor stimulation by caffeine.

Interaction of medullary P2 and glutamate receptors mediates the vasodilation in the hindlimb of rat.

PubMed

Korim, Willian Seiji; Ferreira-Neto, Marcos L; Pedrino, Gustavo R; Pilowsky, Paul M; Cravo, Sergio L

2012-12-01

In the nucleus tractus solitarii (NTS) of rats, blockade of extracellular ATP breakdown to adenosine reduces arterial blood pressure (AP) increases that follow stimulation of the hypothalamic defense area (HDA). The effects of ATP on NTS P2 receptors, during stimulation of the HDA, are still unclear. The aim of this study was to determine whether activation of P2 receptors in the NTS mediates cardiovascular responses to HDA stimulation. Further investigation was taken to establish if changes in hindlimb vascular conductance (HVC) elicited by electrical stimulation of the HDA, or activation of P2 receptors in the NTS, are relayed in the rostral ventrolateral medulla (RVLM); and if those responses depend on glutamate release by ATP acting on presynaptic terminals. In anesthetized and paralyzed rats, electrical stimulation of the HDA increased AP and HVC. Blockade of P2 or glutamate receptors in the NTS, with bilateral microinjections of suramin (10 mM) or kynurenate (50 mM) reduced only the evoked increase in HVC by 75 % or more. Similar results were obtained with the blockade combining both antagonists. Blockade of P2 and glutamate receptors in the RVLM also reduced the increases in HVC to stimulation of the HDA by up to 75 %. Bilateral microinjections of kynurenate in the RVLM abolished changes in AP and HVC to injections of the P2 receptor agonist α,β-methylene ATP (20 mM) into the NTS. The findings suggest that HDA-NTS-RVLM pathways in control of HVC are mediated by activation of P2 and glutamate receptors in the brainstem in alerting-defense reactions.

Regulation of mGlu4 metabotropic glutamate receptor signaling by type-2 G-protein coupled receptor kinase (GRK2).

PubMed

Iacovelli, L; Capobianco, L; Iula, M; Di Giorgi Gerevini, V; Picascia, A; Blahos, J; Melchiorri, D; Nicoletti, F; De Blasi, A

2004-05-01

We examined the role of G-protein coupled receptor kinase-2 (GRK2) in the homologous desensitization of mGlu4 metabotropic glutamate receptors transiently expressed in human embryonic kidney (HEK) 293 cells. Receptor activation with the agonist l-2-amino-4-phosphonobutanoate (l-AP4) stimulated at least two distinct signaling pathways: inhibition of cAMP formation and activation of the mitogen-activated protein kinase (MAPK) pathway [assessed by Western blot analysis of phosphorylated extracellular signal-regulated kinase (ERK) 1 and 2]. Activation of both pathways was attenuated by pertussis toxin. Overexpression of GRK2 (but not GRK4) largely attenuated the stimulation of the MAPK pathway by l-AP4, whereas it slightly potentiated the inhibition of FSK-stimulated cAMP formation. Transfection with a kinase-dead mutant of GRK2 (GRK2-K220R) or with the C-terminal fragment of GRK2 also reduced the mGlu4-mediated stimulation of MAPK, suggesting that GRK2 binds to the Gbetagamma subunits to inhibit signal propagation toward the MAPK pathway. This was confirmed by the evidence that GRK2 coimmunoprecipitated with Gbetagamma subunits in an agonist-dependent manner. Finally, neither GRK2 nor its kinase-dead mutant had any effect on agonist-induced mGlu4 receptor internalization in HEK293 cells transiently transfected with GFP-tagged receptors. Agonist-dependent internalization was instead abolished by a negative-dominant mutant of dynamin, which also reduced the stimulation of MAPK pathway by l-AP4. We speculate that GRK2 acts as a "switch molecule" by inhibiting the mGlu4 receptor-mediated stimulation of MAPK and therefore directing the signal propagation toward the inhibition of adenylyl cyclase.

Linking γ-aminobutyric acid A receptor to epidermal growth factor receptor pathways activation in human prostate cancer.

PubMed

Wu, Weijuan; Yang, Qing; Fung, Kar-Ming; Humphreys, Mitchell R; Brame, Lacy S; Cao, Amy; Fang, Yu-Ting; Shih, Pin-Tsen; Kropp, Bradley P; Lin, Hsueh-Kung

2014-03-05

Neuroendocrine (NE) differentiation has been attributed to the progression of castration-resistant prostate cancer (CRPC). Growth factor pathways including the epidermal growth factor receptor (EGFR) signaling have been implicated in the development of NE features and progression to a castration-resistant phenotype. However, upstream molecules that regulate the growth factor pathway remain largely unknown. Using androgen-insensitive bone metastasis PC-3 cells and androgen-sensitive lymph node metastasis LNCaP cells derived from human prostate cancer (PCa) patients, we demonstrated that γ-aminobutyric acid A receptor (GABA(A)R) ligand (GABA) and agonist (isoguvacine) stimulate cell proliferation, enhance EGF family members expression, and activate EGFR and a downstream signaling molecule, Src, in both PC-3 and LNCaP cells. Inclusion of a GABA(A)R antagonist, picrotoxin, or an EGFR tyrosine kinase inhibitor, Gefitinib (ZD1839 or Iressa), blocked isoguvacine and GABA-stimulated cell growth, trans-phospohorylation of EGFR, and tyrosyl phosphorylation of Src in both PCa cell lines. Spatial distributions of GABAAR α₁ and phosphorylated Src (Tyr416) were studied in human prostate tissues by immunohistochemistry. In contrast to extremely low or absence of GABA(A)R α₁-positive immunoreactivity in normal prostate epithelium, elevated GABA(A)R α₁ immunoreactivity was detected in prostate carcinomatous glands. Similarly, immunoreactivity of phospho-Src (Tyr416) was specifically localized and limited to the nucleoli of all invasive prostate carcinoma cells, but negative in normal tissues. Strong GABAAR α₁ immunoreactivity was spatially adjacent to the neoplastic glands where strong phospho-Src (Tyr416)-positive immunoreactivity was demonstrated, but not in adjacent to normal glands. These results suggest that the GABA signaling is linked to the EGFR pathway and may work through autocrine or paracine mechanism to promote CRPC progression. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Cannabinoid CB1 receptor facilitation of substance P release in the rat spinal cord, measured as neurokinin 1 receptor internalization

PubMed Central

Zhang, Guohua; Chen, Wenling; Lao, Lijun; Marvizón, Juan Carlos G.

2010-01-01

The contribution of CB1 receptors in the spinal cord to cannabinoid analgesia is still unclear. The objective of this study was to investigate the effect of CB1 receptors on substance P release from primary afferent terminals in the spinal cord. Substance P release was measured as NK1 receptor internalization in lamina I neurons. It was induced in spinal cord slices by dorsal root stimulation and in live rats by a noxious stimulus. In spinal cord slices, the CB1 receptor antagonists AM251, AM281 and rimonabant partially but potently inhibited NK1 receptor internalization induced by electrical stimulation of the dorsal root. This was due to an inhibition of substance P release and not of NK1 receptor internalization itself, because AM251 and AM281 did not inhibit NK1 receptor internalization induced by exogenous substance P. The CB1 receptor agonist ACEA increased NK1 receptor internalization evoked by dorsal root stimulation. The effects of AM251 and ACEA cancelled each other. In vivo, AM251 injected intrathecally decreased NK1 receptor internalization in spinal segments L5 and L6 induced by noxious hind paw clamp. Intrathecal AM251 also produced analgesia to radiant heat stimulation of the paw. The inhibition by AM251 of NK1 receptor internalization was reversed by antagonists of μ-opioid and GABAB receptors. This indicates that CB1 receptors facilitate substance P release by inhibiting the release of GABA and opioids next to primary afferent terminals, producing disinhibition. This results in a pronociceptive effect of CB1 receptors in the spinal cord. PMID:20074214

Hypoxia-inducing factors as master regulators of stemness properties and altered metabolism of cancer- and metastasis-initiating cells

PubMed Central

Mimeault, Murielle; Batra, Surinder K

2013-01-01

Accumulating lines of experimental evidence have revealed that hypoxia-inducible factors, HIF-1α and HIF-2α, are key regulators of the adaptation of cancer- and metastasis-initiating cells and their differentiated progenies to oxygen and nutrient deprivation during cancer progression under normoxic and hypoxic conditions. Particularly, the sustained stimulation of epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF-1R), stem cell factor (SCF) receptor KIT, transforming growth factor-β receptors (TGF-βRs) and Notch and their downstream signalling elements such as phosphatidylinositol 3′-kinase (PI3K)/Akt/molecular target of rapamycin (mTOR) may lead to an enhanced activity of HIFs. Moreover, the up-regulation of HIFs in cancer cells may also occur in the hypoxic intratumoral regions formed within primary and secondary neoplasms as well as in leukaemic cells and metastatic prostate and breast cancer cells homing in the hypoxic endosteal niche of bone marrow. The activated HIFs may induce the expression of numerous gene products such as induced pluripotency-associated transcription factors (Oct-3/4, Nanog and Sox-2), glycolysis- and epithelial-mesenchymal transition (EMT) programme-associated molecules, including CXC chemokine receptor 4 (CXCR4), snail and twist, microRNAs and angiogenic factors such as vascular endothelial growth factor (VEGF). These gene products in turn can play critical roles for high self-renewal ability, survival, altered energy metabolism, invasion and metastases of cancer cells, angiogenic switch and treatment resistance. Consequently, the targeting of HIF signalling network and altered metabolic pathways represents new promising strategies to eradicate the total mass of cancer cells and improve the efficacy of current therapies against aggressive and metastatic cancers and prevent disease relapse. PMID:23301832

Trigeminal Medullary Dorsal Horn Neurons Activated by Nasal Stimulation Coexpress AMPA, NMDA, and NK1 Receptors

PubMed Central

McCulloch, P. F.; DiNovo, K. M.; Westerhaus, D. J.; Vizinas, T. A.; Peevey, J. F.; Lach, M. A.; Czarnocki, P.

2013-01-01

Afferent information initiating the cardiorespiratory responses during nasal stimulation projects from the nasal passages to neurons within the trigeminal medullary dorsal horn (MDH) via the anterior ethmoidal nerve (AEN). Central AEN terminals are thought to release glutamate to activate the MDH neurons. This study was designed to determine which neurotransmitter receptors (AMPA, kainate, or NMDA glutamate receptor subtypes or the Substance P receptor NK1) are expressed by these activated MDH neurons. Fos was used as a neuronal marker of activated neurons, and immunohistochemistry combined with epifluorescent microscopy was used to determine which neurotransmitter receptor subunits were coexpressed by activated MDH neurons. Results indicate that, during nasal stimulation with ammonia vapors in urethane-anesthetized Sprague-Dawley rats, activated neurons within the superficial MDH coexpress the AMPA glutamate receptor subunits GluA1 (95.8%) and GluA2/3 (88.2%), the NMDA glutamate receptor subunits GluN1 (89.1%) and GluN2A (41.4%), and NK1 receptors (64.0%). It is therefore likely that during nasal stimulation the central terminals of the AEN release glutamate and substance P that then produces activation of these MDH neurons. The involvement of AMPA and NMDA receptors may mediate fast and slow neurotransmission, respectively, while NK1 receptor involvement may indicate activation of a nociceptive pathway. PMID:24967301

Evolution of specificity in cartilaginous fish glycoprotein hormones and receptors.

PubMed

Buechi, Hanna B; Bridgham, Jamie T

2017-05-15

Glycoprotein hormones (GpH) interact very specifically with their receptors to mediate hypothalamic-pituitary-peripheral gland endocrine signaling. Vertebrates typically have three functionally distinct GpH endocrine signaling complexes: follicle-stimulating hormone, luteinizing hormone, and thyroid-stimulating hormone, and their receptors. Each hormone consists of a common α subunit bound to one of three different β subunits. Individual hormone subunits and receptors are present in genomes of early metazoans, and a subset of hormone subunits and receptors has been recently characterized in sea lamprey. However, it remains unclear when the full complement of hormone and receptor protein families first appeared, and when specificity of interactions between GpH hormones and receptors first evolved. Here we present phylogenetic analyses showing that the elephant shark (Callorhinchus milii) genome contains sequences representing the current diversity of all hormone subunits and receptors in these co-evolving protein families. We examined specificity of hormone and receptor interactions using functional assays testing reporter gene activation by elephant shark follicle-stimulating hormone, luteinizing hormone, and thyroid-stimulating hormone receptors. We show highly specific, dose-responsive hormone interactions for all three complexes. Our results suggest that co-evolution of specificity between proteins in these endocrine signaling complexes occurred prior to the divergence of Chondrichthyes from the chordate lineage. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Cyclic guanosine monophosphate responses to atrial natriuretic factor, brain natriuretic peptide, but not C-type natriuretic peptide, and the characterization of their receptors in rat medullary thick ascending limb.

PubMed

Luk, J K; Wong, E F; Sun, A; Wong, N L

1994-12-01

The effects of atrial natriuretic factor (ANF), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) on renal medullary thick ascending limb (mTAL) have not been fully understood. The aim of this study is to examine the second-messenger responses of rat mTAL to ANF, BNP, and CNP. Characterizations of the ANF, BNP, and CNP receptors in mTAL were also performed by radioligand studies. Results showed that ANF and BNP were both capable of eliciting cyclic guanosine monophosphate (cGMP) responses in mTAL. Conversely, no cGMP response was observed upon stimulation by CNP in mTAL. The presence of ANF receptors was demonstrated by radioligand studies. One receptor site was found, and the Kd and maximum binding capacity were 4.0 +/- 0.45 nmol/L and 277.8 +/- 47.7 fmol/mg protein, respectively. BNP receptors were also found in mTAL, and ANF and BNP were sharing the same receptor. On the contrary, no CNP receptor could be shown by radioligand studies. These results suggest that guanylyl cyclase-coupled receptors (atrial natriuretic peptide receptor-A [ANPR-A]) specific for ANF and BNP are present in rat mTAL, while those for CNP (ANPR-B) are absent. ANF and BNP but not CNP act on mTAL to control water excretion.

Endothelin-1 (ET-1) stimulates carboxy terminal Smad2 phosphorylation in vascular endothelial cells by a mechanism dependent on ET receptors and de novo protein synthesis.

PubMed

Sharifat, Narges; Mohammad Zadeh, Ghorban; Ghaffari, Mohammad-Ali; Dayati, Parisa; Kamato, Danielle; Little, Peter J; Babaahmadi-Rezaei, Hossein

2017-01-01

G protein-coupled receptor (GPCR) agonists through their receptors can transactivate protein tyrosine kinase receptors such as epidermal growth factor receptor and serine/threonine kinase receptors most notably transforming growth factor (TGF)-β receptor (TβRI). This signalling mechanism represents a major expansion in the cellular outcomes attributable to GPCR signalling. This study addressed the role and mechanisms involved in GPCR agonist, endothelin-1 (ET-1)-mediated transactivation of the TβRI in bovine aortic endothelial cells (BAECs). The in-vitro model used BAECs. Signalling intermediate phospho-Smad2 in the carboxy terminal was detected and quantified by Western blotting. ET-1 treatment of BAECs resulted in a time and concentration-dependent increase in pSmad2C. Peak phosphorylation was evident with 100 nm treatment of ET-1 at 4-6 h. TβRI antagonist, SB431542 inhibited ET-1-mediated pSmad2C. In the presence of bosentan, a mixed ET A and ET B receptor antagonist ET-1-mediated pSmad2C levels were inhibited. The ET-mediated pSmad2C was blocked by the protein synthesis inhibitor, cycloheximide. In BAECs, ET-1 via the ETB receptor is involved in transactivation of the TβRI. The transactivation-dependent response is dependent upon de novo protein synthesis. © 2016 Royal Pharmaceutical Society.

Metaplasticity within the spinal cord: Evidence brain-derived neurotrophic factor (BDNF), tumor necrosis factor (TNF), and alterations in GABA function (ionic plasticity) modulate pain and the capacity to learn.

PubMed

Grau, James W; Huang, Yung-Jen

2018-04-07

Evidence is reviewed that behavioral training and neural injury can engage metaplastic processes that regulate adaptive potential. This issue is explored within a model system that examines how training affects the capacity to learn within the lower (lumbosacral) spinal cord. Response-contingent (controllable) stimulation applied caudal to a spinal transection induces a behavioral modification indicative of learning. This behavioral change is not observed in animals that receive stimulation in an uncontrollable manner. Exposure to uncontrollable stimulation also engages a process that disables spinal learning for 24-48 h. Controllable stimulation has the opposite effect; it engages a process that enables learning and prevents/reverses the learning deficit induced by uncontrollable stimulation. These observations suggest that a learning episode can impact the capacity to learn in future situations, providing an example of behavioral metaplasticity. The protective/restorative effect of controllable stimulation has been linked to an up-regulation of brain-derived neurotrophic factor (BDNF). The disruption of learning has been linked to the sensitization of pain (nociceptive) circuits, which is enabled by a reduction in GABA-dependent inhibition. After spinal cord injury (SCI), the co-transporter (KCC2) that regulates the outward flow of Cl - is down-regulated. This causes the intracellular concentration of Cl - to increase, reducing (and potentially reversing) the inward flow of Cl - through the GABA-A receptor. The shift in GABA function (ionic plasticity) increases neural excitability caudal to injury and sets the stage for nociceptive sensitization. The injury-induced shift in KCC2 is related to the loss of descending serotonergic (5HT) fibers that regulate plasticity within the spinal cord dorsal horn through the 5HT-1A receptor. Evidence is presented that these alterations in spinal plasticity impact pain in a brain-dependent task (place conditioning). The findings suggest that ionic plasticity can affect learning potential, shifting a neural circuit from dampened/hard-wired to excitable/plastic. Copyright © 2018 Elsevier Inc. All rights reserved.

Tyrosine dephosphorylation enhances the therapeutic target activity of epidermal growth factor receptor (EGFR) by disrupting its interaction with estrogen receptor (ER).

PubMed

Ma, Shao; Yin, Ning; Qi, Xiaomei; Pfister, Sandra L; Zhang, Mei-Jie; Ma, Rong; Chen, Guan

2015-05-30

Protein-protein interactions can increase or decrease its therapeutic target activity and the determining factors involved, however, are largely unknown. Here, we report that tyrosine-dephosphorylation of epidermal growth factor receptor (EGFR) increases its therapeutic target activity by disrupting its interaction with estrogen receptor (ER). Protein tyrosine phosphatase H1 (PTPH1) dephosphorylates the tyrosine kinase EGFR, disrupts its interaction with the nuclear receptor ER, and increases breast cancer sensitivity to small molecule tyrosine kinase inhibitors (TKIs). These effects require PTPH1 catalytic activity and its interaction with EGFR, suggesting that the phosphatase may increase the sensitivity by dephosphorylating EGFR leading to its dissociation with ER. Consistent with this notion, a nuclear-localization defective ER has a higher EGFR-binding activity and confers the resistance to TKI-induced growth inhibition. Additional analysis show that PTPH1 stabilizes EGFR, stimulates the membranous EGFR accumulation, and enhances the growth-inhibitory activity of a combination therapy of TKIs with an anti-estrogen. Since EGFR and ER both are substrates for PTPH1 in vitro and in intact cells, these results indicate that an inhibitory EGFR-ER protein complex can be switched off through a competitive enzyme-substrate binding. Our results would have important implications for the treatment of breast cancer with targeted therapeutics.

Signal transduction by VEGF receptors in regulation of angiogenesis and lymphangiogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shibuya, Masabumi; Claesson-Welsh, Lena

2006-03-10

The VEGF/VPF (vascular endothelial growth factor/vascular permeability factor) ligands and receptors are crucial regulators of vasculogenesis, angiogenesis, lymphangiogenesis and vascular permeability in vertebrates. VEGF-A, the prototype VEGF ligand, binds and activates two tyrosine kinase receptors: VEGFR1 (Flt-1) and VEGFR2 (KDR/Flk-1). VEGFR1, which occurs in transmembrane and soluble forms, negatively regulates vasculogenesis and angiogenesis during early embryogenesis, but it also acts as a positive regulator of angiogenesis and inflammatory responses, playing a role in several human diseases such as rheumatoid arthritis and cancer. The soluble VEGFR1 is overexpressed in placenta in preeclampsia patients. VEGFR2 has critical functions in physiological and pathologicalmore » angiogenesis through distinct signal transduction pathways regulating proliferation and migration of endothelial cells. VEGFR3, a receptor for the lymphatic growth factors VEGF-C and VEGF-D, but not for VEGF-A, regulates vascular and lymphatic endothelial cell function during embryogenesis. Loss-of-function variants of VEGFR3 have been identified in lymphedema. Formation of tumor lymphatics may be stimulated by tumor-produced VEGF-C, allowing increased spread of tumor metastases through the lymphatics. Mapping the signaling system of these important receptors may provide the knowledge necessary to suppress specific signaling pathways in major human diseases.« less

An extension of hypotheses regarding rapid-acting, treatment-refractory, and conventional antidepressant activity of dextromethorphan and dextrorphan.

PubMed

Lauterbach, Edward C

2012-06-01

It was previously hypothesized that dextromethorphan (DM) and dextrorphan (DX) may possess antidepressant properties, including rapid and conventional onsets of action and utility in treatment-refractory depression, based on pharmacodynamic similarities to ketamine. These similarities included sigma-1 (σ(1)) agonist and NMDA antagonist properties, calcium channel blockade, muscarinic binding, serotonin transporter (5HTT) inhibition, and μ receptor potentiation. Here, six specific hypotheses are developed in light of additional mechanisms and evidence. Comparable potencies to ketamine for DM and DX are detailed for σ(1) (DX>DM>ketamine), NMDA PCP site (DX>ketamine>DM), and muscarinic (DX>ketamine>DM) receptors, 5HTT (DM>DX≫ketamine), and NMDA antagonist potentiation of μ receptor stimulation (DM>ketamine). Rapid acting antidepressant properties of DM include NMDA high-affinity site, NMDR-2A, and functional NMDR-2B receptor antagonism, σ(1) stimulation, putative mTOR activation (by σ(1) stimulation, μ potentiation, and 5HTT inhibition), putative AMPA receptor trafficking (by mTOR activation, PCP antagonism, σ(1) stimulation, μ potentiation, and 5HTT inhibition), and dendritogenesis, spinogenesis, synaptogenesis, and neuronal survival by NMDA antagonism and σ(1) and mTOR signaling. Those for dextrorphan include NMDA high-affinity site and NMDR-2A antagonism, σ(1) stimulation, putative mTOR activation (by σ(1) stimulation and ß adrenoreceptor stimulation), putative AMPA receptor trafficking (by mTOR activation, PCP antagonism, σ(1) stimulation, ß stimulation, and μ antagonism), and dendritogenesis, spinogenesis, synaptogenesis, and neuronal survival by NMDA antagonism and σ(1) and mTOR signaling. Conventional antidepressant properties for dextromethorphan and dextrorphan include 5HTT and norepinephrine transporter inhibition, σ(1) stimulation, NMDA and PCP antagonism, and possible serotonin 5HT1b/d receptor stimulation. Additional properties for dextromethorphan include possible presynaptic α(2) adrenoreceptor antagonism or postsynaptic α(2) stimulation and, for dextrorphan, ß stimulation and possible muscarinic and μ antagonism. Treatment-refractory depression properties include increased serotonin and norepinephrine availability, PCP, NMDR-2B, presynaptic alpha-2 antagonism, and the multiplicity of other antidepressant receptor mechanisms. Suggestions for clinical trials are provided for oral high-dose dextromethorphan and Nuedexta (dextromethorphan combined with quinidine to block metabolism to dextrorphan, thereby increasing dextromethorphan plasma concentrations). Suggestions include exclusionary criteria, oral dosing, observation periods, dose-response approaches, and safety and tolerability are considered. Although oral dextromethorphan may be somewhat more likely to show efficacy through complementary antidepressant mechanisms of dextrorphan, a clinical trial will be more logistically complex than one of Nuedexta due to high doses and plasma level variability. Clinical trials may increase our therapeutic armamentarium and our pharmacological understanding of treatment-refractory depression and antidepressant onset of action. Copyright © 2012 Elsevier Ltd. All rights reserved.

Neurotrophin/receptor expression in urinary bladder of mice with overexpression of NGF in urothelium.

PubMed

Girard, Beatrice M; Malley, Susan E; Vizzard, Margaret A

2011-02-01

Urothelium-specific overexpression of nerve growth factor (NGF) in the urinary bladder of transgenic mice stimulates neuronal sprouting in the urinary bladder, produces increased voiding frequency, and results in increased referred somatic hypersensitivity. Additional NGF-mediated pleiotropic changes might contribute to the increased voiding frequency and pelvic hypersensitivity observed in these transgenic mice, such as modulation of other growth factor/receptor systems. Chronic overexpression of NGF in the urothelium was achieved through the use of a highly urothelium-specific uroplakin II promoter. In the present study, we examined NGF, brain-derived neurotrophic factor (BDNF), and associated receptor [p75(NTR), tyrosine kinase (Trk)A, TrkB] transcript and protein expression in urothelium and detrusor smooth muscle of NGF-overexpressing (OE) and littermate wild-type mice, using real-time quantitative reverse transcription-polymerase chain reaction, ELISAs, and semiquantitation of immunohistochemistry. We focused on these growth factor/receptors given the established roles of NGF/TrkA, NGF/p75(NTR), and BDNF/TrkB systems in bladder function. Increased voiding frequency in NGF-OE mice was confirmed by examining urination patterns. BDNF, TrkA, and TrkB protein expression was significantly (P ≤ 0.01) reduced and p75(NTR) protein expression was significantly (P ≤ 0.01) increased in urinary bladder of NGF-OE mice. The NGF-OE-induced changes in neurotrophic factor/receptor expression in urinary bladder may represent compensatory changes to reduce voiding frequency in the NGF-OE mouse.

The G Protein-Coupled Bile Acid Receptor, TGR5, Stimulates Gallbladder Filling

PubMed Central

Li, Tingting; Holmstrom, Sam R.; Kir, Serkan; Umetani, Michihisa; Schmidt, Daniel R.

2011-01-01

TGR5 is a G protein-coupled bile acid receptor present in brown adipose tissue and intestine, where its agonism increases energy expenditure and lowers blood glucose. Thus, it is an attractive drug target for treating human metabolic disease. However, TGR5 is also highly expressed in gallbladder, where its functions are less well characterized. Here, we demonstrate that TGR5 stimulates the filling of the gallbladder with bile. Gallbladder volume was increased in wild-type but not Tgr5−/− mice by administration of either the naturally occurring TGR5 agonist, lithocholic acid, or the synthetic TGR5 agonist, INT-777. These effects were independent of fibroblast growth factor 15, an enteric hormone previously shown to stimulate gallbladder filling. Ex vivo analyses using gallbladder tissue showed that TGR5 activation increased cAMP concentrations and caused smooth muscle relaxation in a TGR5-dependent manner. These data reveal a novel, gallbladder-intrinsic mechanism for regulating gallbladder contractility. They further suggest that TGR5 agonists should be assessed for effects on human gallbladder as they are developed for treating metabolic disease. PMID:21454404

Physiologic regulation of atrial natriuretic peptide receptors in rat renal glomeruli.

PubMed Central

Ballermann, B J; Hoover, R L; Karnovsky, M J; Brenner, B M

1985-01-01

Isolated rat renal glomeruli and cultured glomerular mesangial and epithelial cells were examined for atrial natriuretic peptide (ANP) receptors, and for ANP-stimulated cyclic guanosine monophosphate (cGMP) generation. In glomeruli from normal rats, human (1-28) 125I-ANP bound to a single population of high affinity receptors with a mean equilibrium dissociation constant of 0.46 nM. Human (1-28) ANP markedly stimulated cGMP generation, but not cAMP generation in normal rat glomeruli. Analogues of ANP that bound to the glomerular ANP receptor with high affinity stimulated cGMP accumulation, whereas the (13-28) ANP fragment, which failed to bind to the receptor, was devoid of functional activity. Cell surface receptors for ANP were expressed on cultured glomerular mesangial but not epithelial cells, and appreciable ANP-stimulated cGMP accumulation was elicited only in mesangial cells. Approximately 12,000 ANP receptor sites were present per mesangial cell, with an average value for the equilibrium dissociation constant of 0.22 nM. Feeding of a low-salt diet to rats for 2 wk resulted in marked up regulation of the glomerular ANP receptor density to a mean of 426 fmol/mg protein, compared with 116 fmol/mg in rats given a high-salt diet. A modest reduction in the affinity of glomerular ANP receptors was also observed in rats fed the low-salt diet. ANP-stimulated cGMP generation in glomeruli did not change with alterations in salt intake. We conclude that high salt feeding in the rat results in reduced glomerular ANP receptor density relative to values in salt restricted rats. Furthermore, the mesangial cell is a principal target for ANP binding in the glomerulus. Images PMID:3001139

Role of Corticotropin-releasing Factor Signaling in Stress-related Alterations of Colonic Motility and Hyperalgesia

PubMed Central

Taché, Yvette; Million, Mulugeta

2015-01-01

The corticotropin-releasing factor (CRF) signaling systems encompass CRF and the structurally related peptide urocortin (Ucn) 1, 2, and 3 along with 2 G-protein coupled receptors, CRF1 and CRF2. CRF binds with high and moderate affinity to CRF1 and CRF2 receptors, respectively while Ucn1 is a high-affinity agonist at both receptors, and Ucn2 and Ucn3 are selective CRF2 agonists. The CRF systems are expressed in both the brain and the colon at the gene and protein levels. Experimental studies established that the activation of CRF1 pathway in the brain or the colon recaptures cardinal features of diarrhea predominant irritable bowel syndrome (IBS) (stimulation of colonic motility, activation of mast cells and serotonin, defecation/watery diarrhea, and visceral hyperalgesia). Conversely, selective CRF1 antagonists or CRF1/CRF2 antagonists, abolished or reduced exogenous CRF and stress-induced stimulation of colonic motility, defecation, diarrhea and colonic mast cell activation and visceral hyperalgesia to colorectal distention. By contrast, the CRF2 signaling in the colon dampened the CRF1 mediated stimulation of colonic motor function and visceral hyperalgesia. These data provide a conceptual framework that sustained activation of the CRF1 system at central and/or peripheral sites may be one of the underlying basis of IBS-diarrhea symptoms. While targeting these mechanisms by CRF1 antagonists provided a relevant novel therapeutic venue, so far these promising preclinical data have not translated into therapeutic use of CRF1 antagonists. Whether the existing or newly developed CRF1 antagonists will progress to therapeutic benefits for stress-sensitive diseases including IBS for a subset of patients is still a work in progress. PMID:25611064

Actin-Sorting Nexin 27 (SNX27)-Retromer Complex Mediates Rapid Parathyroid Hormone Receptor Recycling*

PubMed Central

McGarvey, Jennifer C.; Xiao, Kunhong; Bowman, Shanna L.; Mamonova, Tatyana; Zhang, Qiangmin; Bisello, Alessandro; Sneddon, W. Bruce; Ardura, Juan A.; Jean-Alphonse, Frederic; Vilardaga, Jean-Pierre; Puthenveedu, Manojkumar A.; Friedman, Peter A.

2016-01-01

The G protein-coupled parathyroid hormone receptor (PTHR) regulates mineral-ion homeostasis and bone remodeling. Upon parathyroid hormone (PTH) stimulation, the PTHR internalizes into early endosomes and subsequently traffics to the retromer complex, a sorting platform on early endosomes that promotes recycling of surface receptors. The C terminus of the PTHR contains a type I PDZ ligand that binds PDZ domain-containing proteins. Mass spectrometry identified sorting nexin 27 (SNX27) in isolated endosomes as a PTHR binding partner. PTH treatment enriched endosomal PTHR. SNX27 contains a PDZ domain and serves as a cargo selector for the retromer complex. VPS26, VPS29, and VPS35 retromer subunits were isolated with PTHR in endosomes from cells stimulated with PTH. Molecular dynamics and protein binding studies establish that PTHR and SNX27 interactions depend on the PDZ recognition motif in PTHR and the PDZ domain of SNX27. Depletion of either SNX27 or VPS35 or actin depolymerization decreased the rate of PTHR recycling following agonist stimulation. Mutating the PDZ ligand of PTHR abolished the interaction with SNX27 but did not affect the overall rate of recycling, suggesting that PTHR may directly engage the retromer complex. Coimmunoprecipitation and overlay experiments show that both intact and mutated PTHR bind retromer through the VPS26 protomer and sequentially assemble a ternary complex with PTHR and SNX27. SNX27-independent recycling may involve N-ethylmaleimide-sensitive factor, which binds both PDZ intact and mutant PTHRs. We conclude that PTHR recycles rapidly through at least two pathways, one involving the ASRT complex of actin, SNX27, and retromer and another possibly involving N-ethylmaleimide-sensitive factor. PMID:27008860

HIV-1 evades innate immune recognition through specific cofactor recruitment

NASA Astrophysics Data System (ADS)

Rasaiyaah, Jane; Tan, Choon Ping; Fletcher, Adam J.; Price, Amanda J.; Blondeau, Caroline; Hilditch, Laura; Jacques, David A.; Selwood, David L.; James, Leo C.; Noursadeghi, Mahdad; Towers, Greg J.

2013-11-01

Human immunodeficiency virus (HIV)-1 is able to replicate in primary human macrophages without stimulating innate immunity despite reverse transcription of genomic RNA into double-stranded DNA, an activity that might be expected to trigger innate pattern recognition receptors. We reasoned that if correctly orchestrated HIV-1 uncoating and nuclear entry is important for evasion of innate sensors then manipulation of specific interactions between HIV-1 capsid and host factors that putatively regulate these processes should trigger pattern recognition receptors and stimulate type 1 interferon (IFN) secretion. Here we show that HIV-1 capsid mutants N74D and P90A, which are impaired for interaction with cofactors cleavage and polyadenylation specificity factor subunit 6 (CPSF6) and cyclophilins (Nup358 and CypA), respectively, cannot replicate in primary human monocyte-derived macrophages because they trigger innate sensors leading to nuclear translocation of NF-κB and IRF3, the production of soluble type 1 IFN and induction of an antiviral state. Depletion of CPSF6 with short hairpin RNA expression allows wild-type virus to trigger innate sensors and IFN production. In each case, suppressed replication is rescued by IFN-receptor blockade, demonstrating a role for IFN in restriction. IFN production is dependent on viral reverse transcription but not integration, indicating that a viral reverse transcription product comprises the HIV-1 pathogen-associated molecular pattern. Finally, we show that we can pharmacologically induce wild-type HIV-1 infection to stimulate IFN secretion and an antiviral state using a non-immunosuppressive cyclosporine analogue. We conclude that HIV-1 has evolved to use CPSF6 and cyclophilins to cloak its replication, allowing evasion of innate immune sensors and induction of a cell-autonomous innate immune response in primary human macrophages.

MicroRNA-155 silencing enhances inflammatory response and lipid uptake in oxidized low-density lipoprotein-stimulated human THP-1 macrophages.

PubMed

Huang, Ri-sheng; Hu, Guan-qiong; Lin, Bin; Lin, Zhi-yi; Sun, Cheng-chao

2010-12-01

It has been proposed that the inflammatory response of monocytes/macrophages induced by oxidized low-density lipoprotein (oxLDL) is a key event in the pathogenesis of atherosclerosis. MicroRNA-155 (miR-155) is an important regulator of the immune system and has been shown to be involved in acute inflammatory response. However, the function of miR-155 in oxLDL-stimulated inflammation and atherosclerosis remains unclear. Here, we show that the exposure of human THP-1 macrophages to oxLDL led to a marked up-regulation of miR-155 in a dose-dependent manner. Silencing of endogenous miR-155 in THP-1 cells using locked nucleic acid-modified antisense oligonucleotides significantly enhanced oxLDL-induced lipid uptake, up-regulated the expression of scavenger receptors (lectinlike oxidized LDL receptor-1, cluster of differentiation 36 [CD36], and CD68), and promoted the release of several cytokines including interleukin (IL)-6, -8, and tumor necrosis factor α (TNF-α). Luciferase reporter assay showed that targeting miR-155 promoted nuclear factor-kappa B (NF-κB) nuclear translocation and potentiated the NF-κB-driven transcription activity. Moreover, miR-155 knockdown resulted in a marked increase in the protein amount of myeloid differentiation primary response gene 88 (MyD88), an important adapter protein used by Toll-like receptors to activate the NF-κB pathway. Our data demonstrate that miR-155 serves as a negative feedback regulator in oxLDL-stimulated THP-1 inflammatory responses and lipid uptake and thus might have potential therapeutic implications in atherosclerosis.

The SRC homology 2 domain of Rin1 mediates its binding to the epidermal growth factor receptor and regulates receptor endocytosis.

PubMed

Barbieri, M Alejandro; Kong, Chen; Chen, Pin-I; Horazdovsky, Bruce F; Stahl, Philip D

2003-08-22

Activated epidermal growth factor receptors (EGFRs) recruit intracellular proteins that mediate receptor signaling and endocytic trafficking. Rin1, a multifunctional protein, has been shown to regulate EGFR internalization (1). Here we show that EGF stimulation induces a specific, rapid, and transient membrane recruitment of Rin1 and that recruitment is dependent on the Src homology 2 (SH2) domain of Rin1. Immunoprecipitation of EGFR is accompanied by co-immunoprecipitation of Rin1 in a time- and ligand-dependent manner. Association of Rin1 and specifically the SH2 domain of Rin1 with the EGFR was dependent on tyrosine phosphorylation of the intracellular domain of the EGFR. The recruitment of Rin1, observed by light microscopy, indicated that although initially cytosolic, Rin1 was recruited to both plasma membrane and endosomes following EGF addition. Moreover, the expression of the SH2 domain of Rin1 substantially impaired the internalization of EGF without affecting internalization of transferrin. Finally, we found that Rin1 co-immunoprecipitated with a number of tyrosine kinase receptors but not with cargo endocytic receptors. These results indicate that Rin1 provides a link via its SH2 domain between activated tyrosine kinase receptors and the endocytic pathway through the recruitment and activation of Rab5a.

Targeted gene expression profiling in European sea bass (Dicentrarchus labrax, L.) follicles from primary growth to late vitellogenesis.

PubMed

García-López, Angel; Sánchez-Amaya, María Isabel; Prat, Francisco

2011-11-01

A real-time PCR-based gene expression survey was performed on isolated European sea bass follicles from primary growth to late vitellogenesis. Expression levels of 18 transcripts with demonstrated relevance during oogenesis, encoding gonadotropin, thyrotropin, estrogen, androgen, and vitellogenin receptors, steroidogenesis-related as well as growth and transcription factors were measured. Primary oocytes showed high mRNA levels of insulin-like growth factors 1 and 2, bone morphogenetic protein 4, estrogen receptor 2b, androgen receptor b, and SRY-box containing gene 17 together with low transcript amounts of gonadotropin receptors. Follicles at the lipid vesicles stage (i.e., the beginning of the secondary growth phase) showed elevated mRNA amounts of follicle stimulating hormone receptor (fshr) and anti-Mullerian hormone. Early-to-mid vitellogenic follicles showed high mRNA levels of fshr and cytochrome P450, family 19, subfamily A, polypeptide 1a while mid-to-late vitellogenic follicles expressed increasing transcript amounts of luteinizing hormone/choriogonadotropin receptor, steroidogenic acute regulatory protein, and estrogen receptors 1 and 2a. The molecular data presented here may serve as a solid base for future studies focused on unraveling the specific mechanisms orchestrating follicular development in teleost fish. Copyright © 2011 Elsevier Inc. All rights reserved.

Recombinant granulocyte colony-stimulating factor administered enterally to neonates is not absorbed.

PubMed

Calhoun, Darlene A; Maheshwari, Akhil; Christensen, Robert D

2003-08-01

Granulocyte colony-stimulating factor (G-CSF) is present in liquids swallowed by the fetus and neonate; specifically, amniotic fluid, colostrum, and human milk. The swallowed G-CSF has local effects on enteric cells, which express the G-CSF receptor. However, some portion of the G-CSF ingested by the fetus and neonate might be absorbed into the circulation and have systemic actions, such as stimulating neutrophil production. To assess this possibility we sought to determine if circulating G-CSF concentrations of neonates increase after enteral administration of recombinant human granulocyte colony-stimulating factor (rhG-CSF). This was a single-center, prospective, blinded, randomized, 2 x 2 crossover study, with each infant receiving 1 dose of rhG-CSF (100 microg/kg) and 1 dose of placebo. Plasma G-CSF concentrations were measured at 2 and 4 hours after administration of the test solution. No significant change in plasma G-CSF concentration was observed after the enteral administration of rhG-CSF. On this basis, we conclude that orally administered rhG-CSF is not absorbed in significant quantities, and we speculate that the G-CSF swallowed by the fetus and neonate has local but not systemic effects.

The importance of neuronal growth factors in the ovary.

PubMed

Streiter, S; Fisch, B; Sabbah, B; Ao, A; Abir, R

2016-01-01

The neurotrophin family consists of nerve growth factor (NGF), neurotrophin 3 (NT3) and neurotrophin 4/5 (NT4/5), in addition to brain-derived neurotrophic factor (BDNF) and the neuronal growth factors, glial cell line-derived neurotrophic factor (GDNF) and vasointestinal peptide (VIP). Although there are a few literature reviews, mainly of animal studies, on the importance of neurotrophins in the ovary, we aimed to provide a complete review of neurotrophins as well as neuronal growth factors and their important roles in normal and pathological processes in the ovary. Follicular assembly is probably stimulated by complementary effects of NGF, NT4/5 and BDNF and their receptors. The neurotrophins, GDNF and VIP and their receptors have all been identified in preantral and antral follicles of mammalian species, including humans. Transgenic mice with mutations in the genes encoding for Ngf, Nt4/5 and Bdnf and their tropomyosin-related kinase β receptor showed a reduction in preantral follicles and an abnormal ovarian morphology, whereas NGF, NT3, GDNF and VIP increased the in vitro activation of primordial follicles in rats and goats. Additionally, NGF, NT3 and GDNF promoted follicular cell proliferation; NGF, BDNF and VIP were shown to be involved in ovulation; VIP inhibited follicular apoptosis; NT4/5, BDNF and GDNF promoted oocyte maturation and NGF, NT3 and VIP stimulated steroidogenesis. NGF may also exert a stimulatory effect in ovarian cancer and polycystic ovarian syndrome (PCOS). Low levels of NGF and BDNF in follicular fluid may be associated with diminished ovarian reserve and high levels with endometriosis. More knowledge of the roles of neuronal growth factors in the ovary has important implications for the development of new therapeutic drugs (such as anti-NGF agents) for ovarian cancer and PCOS as well as various infertility problems, warranting further research. © The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

The chemokine CXCL16 induces migration and invasion of glial precursor cells via its receptor CXCR6.

PubMed

Hattermann, Kirsten; Ludwig, Andreas; Gieselmann, Volkmar; Held-Feindt, Janka; Mentlein, Rolf

2008-09-01

Chemokines are implicated in developmental and inflammatory processes in the brain. The transmembrane chemokine CXCL16 is produced in brain endothelial and reactive astroglial cells and released by shedding. Its receptor CXCR6 is detected during brain development highest at postnatal day 6, found in glial precursor cells differentiated from neural stem cells and in an A2B5-positive glial precursor cell line. Their stimulation by soluble CXCL16 induces the PI3-kinase/Akt and Erk pathways resulting in the activation of the transcription factor AP-1. As biological responses, soluble CXCL16 upregulates its own receptor, increases cell proliferation, stimulates cell migration in wound-healing and in spheroid confrontation assays. Invasion of CXCR6-positive glial cells into CXCL16-expressing spheroids can be blocked by sheddase inhibitors and CXCL16-antibody. Since CXCL16 is induced by cytokines at sites of inflammation, neurodegeneration, ischemia and malignant transformation, it should attract CXCR6-positive glial precursor cells, enhance their invasion and proliferation and thus favor astrogliosis.

TSHR intronic polymorphisms (rs179247 and rs12885526) and their role in the susceptibility of the Brazilian population to Graves' disease and Graves' ophthalmopathy.

PubMed

Bufalo, N E; Dos Santos, R B; Marcello, M A; Piai, R P; Secolin, R; Romaldini, J H; Ward, L S

2015-05-01

Intronic thyroid-stimulating hormone receptor polymorphisms have been associated with the risk for both Graves' disease and Graves' ophthalmopathy, but results have been inconsistent among different populations. We aimed to investigate the influence of thyroid-stimulating hormone receptor intronic polymorphisms in a large well-characterized population of GD patients. We studied 279 Graves' disease patients (231 females and 48 males, 39.80 ± 11.69 years old), including 144 with Graves' ophthalmopathy, matched to 296 healthy control individuals. Thyroid-stimulating hormone receptor genotypes of rs179247 and rs12885526 were determined by Real Time PCR TaqMan(®) SNP Genotyping. A multivariate analysis showed that the inheritance of the thyroid-stimulating hormone receptor AA genotype for rs179247 increased the risk for Graves' disease (OR = 2.821; 95 % CI 1.595-4.990; p = 0.0004), whereas the thyroid-stimulating hormone receptor GG genotype for rs12885526 increased the risk for Graves' ophthalmopathy (OR = 2.940; 95 % CI 1.320-6.548; p = 0.0083). Individuals with Graves' ophthalmopathy also presented lower mean thyrotropin receptor antibodies levels (96.3 ± 143.9 U/L) than individuals without Graves' ophthalmopathy (98.3 ± 201.9 U/L). We did not find any association between the investigated polymorphisms and patients clinical features or outcome. We demonstrate that thyroid-stimulating hormone receptor intronic polymorphisms are associated with the susceptibility to Graves' disease and Graves' ophthalmopathy in the Brazilian population, but do not appear to influence the disease course.

Transcription factor specificity protein 1 modulates TGFβ1/Smad signaling to negatively regulate SIGIRR expression by human M1 macrophages stimulated with substance P.

PubMed

Yamaguchi, Rui; Sakamoto, Arisa; Yamaguchi, Reona; Haraguchi, Misa; Narahara, Shinji; Sugiuchi, Hiroyuki; Yamaguchi, Yasuo

2018-08-01

The stimuli inducing expression of single immunoglobulin IL-1-related receptor (SIGIRR) and the relevant regulatory mechanisms are not well defined. Transforming growth factor β1 (TGFβ1) delays internalization of neurokinin-1 receptor (NK1R) and subsequently enhances cellular signaling. This study investigated the effect of TGFβ1 on SIGIRR protein production by human M1 macrophages in response to stimulation with substance P (SP). SP caused upregulation of SIGIRR expression in a concentration-dependent manner, whereas aprepitant (an NK1R inhibitor) blunted this response. Silencing p38γMAPK or TAK-1 partially attenuated the response to SP stimulation, while TGFβ1/2/3 siRNA dramatically diminished it. SP induced much greater SIGIRR protein production than either lipopolysaccharide (a TLR4 agonist) or resiquimod (a TLR7/8 agonist). Unexpectedly, silencing of transcription factor specificity protein 1 (Sp1) led to significant upregulation of SIGIRR expression after SP stimulation, while KLF2 siRNA only partially enhanced it and Fli-1 siRNA reduced it. SP also upregulated TGFβ1 expression, along with a corresponding increase of SIGIRR protein, whereas silencing TGFβ1/2/3 blunted these responses. Sp1 siRNA or mithramycin (a gene-selective Sp1 inhibitor) significantly enhanced the expression of TGFβ1 and SIGIRR by macrophages after SP stimulation. Importantly, this effect of Sp1 siRNA on TGFβ1 and SIGIRR was blunted by siRNA for Smad2, Smad3, or Smad4, but not by TAK-1 siRNA. Next, we investigated the influence of transcription factor cross-talk on SIGIRR expression in response to SP. Co-transfection of macrophages with Sp1 siRNA and C/EBPβ or TIF1β siRNA attenuated the upregulation of SIGIRR by SP, while a combination of Sp1 siRNA and Fli-1 siRNA dramatically diminished it. In conclusion, TGFβ1 may be an intermediary between SP/NK1R activation and SIGIRR expression in Sp1 siRNA-transfected macrophages. In addition, Sp1 modulates TGFβ1/Smad signaling and negatively regulates SIGIRR protein production by macrophages after SP stimulation. Copyright © 2018 Elsevier Ltd. All rights reserved.

Alpha 2-adrenergic receptor stimulation of phospholipase A2 and of adenylate cyclase in transfected Chinese hamster ovary cells is mediated by different mechanisms

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jones, S.B.; Halenda, S.P.; Bylund, D.B.

1991-02-01

The effect of alpha 2-adrenergic receptor activation on adenylate cyclase activity in Chinese hamster ovary cells stably transfected with the alpha 2A-adrenergic receptor gene is biphasic. At lower concentrations of epinephrine forskolin-stimulated cyclic AMP production is inhibited, but at higher concentrations the inhibition is reversed. Both of these effects are blocked by the alpha 2 antagonist yohimbine but not by the alpha 1 antagonist prazosin. Pretreatment with pertussis toxin attenuates inhibition at lower concentrations of epinephrine and greatly potentiates forskolin-stimulated cyclic AMP production at higher concentrations of epinephrine. alpha 2-Adrenergic receptor stimulation also causes arachidonic acid mobilization, presumably via phospholipasemore » A2. This effect is blocked by yohimbine, quinacrine, removal of extracellular Ca2+, and pretreatment with pertussis toxin. Quinacrine and removal of extracellular Ca2+, in contrast, have no effect on the enhanced forskolin-stimulated cyclic AMP production. Thus, it appears that the alpha 2-adrenergic receptor in these cells can simultaneously activate distinct signal transduction systems; inhibition of adenylate cyclase and stimulation of phospholipase A2, both via G1, and potentiation of cyclic AMP production by a different (pertussis toxin-insensitive) mechanism.« less

Adenosine transiently modulates stimulated dopamine release in the caudate putamen via A1 receptors

PubMed Central

Ross, Ashley E.; Venton, B. Jill

2014-01-01

Adenosine modulates dopamine in the brain via A1 and A2A receptors, but that modulation has only been characterized on a slow time scale. Recent studies have characterized a rapid signaling mode of adenosine that suggests a possible rapid modulatory role. Here, fast-scan cyclic voltammetry was used to characterize the extent to which transient adenosine changes modulate stimulated dopamine release (5 pulses at 60 Hz) in rat caudate putamen brain slices. Exogenous adenosine was applied and dopamine concentration monitored. Adenosine only modulated dopamine when it was applied 2 or 5 s before stimulation. Longer time intervals and bath application of 5 µM adenosine did not decrease dopamine release. Mechanical stimulation of endogenous adenosine 2s before dopamine stimulation also decreased stimulated dopamine release by 41 ± 7 %, similar to the 54 ± 6 % decrease in dopamine after exogenous adenosine application. Dopamine inhibition by transient adenosine was recovered within 10 minutes. The A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) blocked the dopamine modulation, whereas dopamine modulation was unaffected by the A2A receptor antagonist SCH 442416. Thus, transient adenosine changes can transiently modulate phasic dopamine release via A1 receptors. These data demonstrate that adenosine has a rapid, but transient, modulatory role in the brain. PMID:25219576

STK-1, the human homolog of Flk-2/Flt-3, is selectively expressed in CD34+ human bone marrow cells and is involved in the proliferation of early progenitor/stem cells.

PubMed Central

Small, D; Levenstein, M; Kim, E; Carow, C; Amin, S; Rockwell, P; Witte, L; Burrow, C; Ratajczak, M Z; Gewirtz, A M

1994-01-01

We cloned the cDNA for stem cell tyrosine kinase 1 (STK-1), the human homolog of murine Flk-2/Flt-3, from a CD34+ hematopoietic stem cell-enriched library and investigated its expression in subsets of normal human bone marrow. The cDNA encodes a protein of 993 aa with 85% identity and 92% similarity to Flk-2/Flt-3. STK-1 is a member of the type III receptor tyrosine kinase family that includes KIT (steel factor receptor), FMS (colony-stimulating factor 1R), and platelet-derived growth factor receptor. STK-1 expression in human blood and marrow is restricted to CD34+ cells, a population greatly enriched for stem/progenitor cells. Anti-STK-1 antiserum recognizes polypeptides of 160 and 130 kDa in several STK-1-expressing cell lines and in 3T3 cells transfected with a STK-1 expression vector. Antisense oligonucleotides directed against STK-1 sequences inhibited hematopoietic colony formation, most strongly in long-term bone marrow cultures. These data suggest that STK-1 may function as a growth factor receptor on hematopoietic stem and/or progenitor cells. Images Fig. 2 Fig. 3 Fig. 4 PMID:7507245

Number of junctional acetylcholine receptors: control by neural and muscular influences in the rat.

PubMed

Andreose, J S; Fumagalli, G; Lømo, T

1995-03-01

1. The number of acetylcholine receptors (AChRs) per neuromuscular junction in soleus muscles of adult rats was estimated from counts of 125I-alpha-bungarotoxin binding sites. The muscles were either denervated, denervated and electrically stimulated, paralysed by botulinum toxin (BoTX), or paralysed by tetrodotoxin (TTX). 2. After denervation, the number of junctional AChRs was normal after 18 days and then fell to 54 and 35% of normal after 33 and 57 days, respectively. 3. Direct high frequency muscle stimulation (100 Hz) maintained a normal number of junctional AChRs for at least 2 months when the stimulation started on the day of denervation. When the stimulation was started progressively later, the effect of the stimulation on AChR number disappeared within about a week. The disappearance was gradual and appeared to affect all the muscle fibres equally. 4. Stimulation at 100 Hz, starting on the day of denervation and stopping after 18 days, did not prevent the endplates from losing AChRs during the subsequent 15 days without stimulation. Thus 100 Hz stimulation and innervation are not equivalent in their effects on junctional AChR number. 5. Direct low frequency muscle stimulation from the day of denervation did not maintain a normal number of junctional AChRs, as the number of AChRs fell to 70 and 62% of normal after 33 days of stimulation at 20 and 10 Hz, respectively. 6. Endplates paralysed by BoTX or TTX for 33 days lost about as many junctional AChRs (54 and 55%) as endplates denervated for 33 days (46%). Direct stimulation at 100 Hz during the last 15 days of BoTX treatment reduced but did not prevent this AChR loss (36% loss at 33 days). 7. The results show that when motor nerve terminals in rat soleus muscles are removed by axotomy, they leave a 'trace' which, in conjunction with appropriate muscle stimulation, can maintain a normal number of AChRs in the postsynaptic region. In non-stimulated muscles the trace responsible for this maintenance disappears within about a week. In stimulated muscles it persists for at least 2 months. From indirect evidence it appears that the trace is a factor, or the postsynaptic effect of a factor, released by impulse activity in the nerve, and that its degradation after denervation is accelerated by the acute effects of nerve degeneration.

IL-29 Enhances CXCL10 Production in TNF-α-stimulated Human Oral Epithelial Cells.

PubMed

Hosokawa, Yoshitaka; Hosokawa, Ikuko; Shindo, Satoru; Ozaki, Kazumi; Matsuo, Takashi

2017-08-01

Interleukin-29 (IL-29) is a cytokine belonging to the Type III interferon family. It was recently detected in the gingival crevicular fluid of periodontitis patients. However, the role of IL-29 in the pathogenesis of periodontal disease remains unknown. The aim of this study was to examine the effects of IL-29 on C-X-C motif chemokine ligand 10 (CXCL10) production in human oral epithelial cells. We measured CXCL10 production in TR146 cells, which is a human oral epithelial cell line, using an enzyme-linked immunosorbent assay. We used a Western blot analysis to detect IL-29 receptor expression and the phosphorylation levels of signal transduction molecules, including p38 mitogen-activated protein kinases (MAPK), signal transducer and activator of transcription 3 (STAT3), and nuclear factor (NF)- κB p65, in the TR146 cells. The TR146 cells expressed the IL-29 receptor. IL-29 induced CXCL10 production in the TR146 cells. IL-29 significantly enhanced CXCL10 production in tumor necrosis factor (TNF)-α-stimulated TR146 cells. The p38 MAPK, STAT3, and NF-κB pathways were found to be related to the IL-29-induced enhancement of CXCL10 production in TNF-α-stimulated TR146 cells. IL-29 promotes T helper 1-cell accumulation in periodontal lesions by inducing CXCL10 production in oral epithelial cells.

Expression of granulocyte colony-stimulating factor receptor correlates with prognosis in oral and mesopharyngeal carcinoma.

PubMed

Tsuzuki, H; Fujieda, S; Sunaga, H; Noda, I; Saito, H

1998-02-15

Granulocyte colony-stimulating factor receptors (G-CSFRs) have been observed on the surface of not only hematopoietic cells but also several cancer cells. The stimulation of G-CSF has been demonstrated to induce proliferation and activation of G-CSFR-positive cells. In this study, we investigated the expression of G-CSFR on the surface of tumor cells and G-CSF production in oral and mesopharyngeal squamous cell carcinoma (SCC) by an immunohistochemical approach. Of 58 oral and mesopharyngeal SCCs, 31 cases (53.4%) and 36 cases (62.1%) were positive for G-CSFR and G-CSF, respectively. There was no association between G-CSFR expression and G-CSF staining. In the group positive for G-CSFR expression, relapse was significantly more likely after primary treatment (P = 0.0069), whereas there was no association between G-CSFR expression and age, sex, tumor size, lymph node metastasis, and clinical stage. Also, the G-CSFR-positive groups had a significantly lower disease-free and overall survival rate than the G-CSFR-negative groups (P = 0.0172 and 0.0188, respectively). However, none of the clinical markers correlated significantly with G-CSF staining, nor did the status of G-CSF production influence the overall survival. The results imply that assessment of G-CSFR may prove valuable in selecting patients with oral and mesopharyngeal SCC for aggressive therapy.

Regulation of vacuolar H{sup +}-ATPase in microglia by RANKL

DOE Office of Scientific and Technical Information (OSTI.GOV)

Serrano, Eric M.; Ricofort, Ryan D.; Zuo, Jian

2009-11-06

Vacuolar H{sup +}-ATPases (V-ATPases) are large electrogenic proton pumps composed of numerous subunits that play vital housekeeping roles in the acidification of compartments of the endocytic pathway. Additionally, V-ATPases play specialized roles in certain cell types, a capacity that is linked to cell type selective expression of isoforms of some of the subunits. We detected low levels of the a3 isoform of the a-subunit in mouse brain extracts. Examination of various brain-derived cell types by immunoblotting showed a3 was expressed in the N9 microglia cell line and in primary microglia, but not in other cell types. The expression of a3more » in osteoclasts requires stimulation by Receptor Activator of Nuclear Factor {kappa}B-ligand (RANKL). We found that Receptor Activator of Nuclear Factor {kappa}B (RANK) was expressed by microglia. Stimulation of microglia with RANKL triggered increased expression of a3. V-ATPases in microglia were shown to bind microfilaments, and stimulation with RANKL increased the proportion of V-ATPase associated with the detergent-insoluble cytoskeletal fraction and with actin. In summary, microglia express the a3-subunit of V-ATPase. The expression of a3 and the interaction between V-ATPases and microfilaments was modulated by RANKL. These data suggest a novel molecular pathway for regulating microglia.« less

Modulation of Immunoregulatory Properties of Mesenchymal Stromal Cells by Toll-Like Receptors: Potential Applications on GVHD

PubMed Central

2016-01-01

In the last decade, the immunomodulatory properties of mesenchymal stromal cells (MSCs) have attracted a lot of attention, due to their potential applicability in the treatment of graft-versus-host disease (GVHD), a condition frequently associated with opportunistic infections. The present review addresses how Pathogen-Associated Molecular Patterns (PAMPS) modulate the immunosuppressive phenotype of human MSCs by signaling through Toll-like receptors (TLRs). Overall, we observed that regardless of the source tissue, human MSCs express TLR2, TLR3, TLR4, and TLR9. Stimulation of distinct TLRs on MSCs elicits distinct inflammatory signaling pathways, differentially influencing the expression of inflammatory factors and the ability of MSCs to suppress the proliferation of immune system cells. The capacity to enhance the immunosuppressive phenotype of MSCs through TLRs stimulation might be properly elucidated in order to improve the MSC-based immunotherapy against GVHD. PMID:27738438

The Expression of Leptin, Estrogen Receptors, and Vitellogenin mRNAs in Migrating Female Chum Salmon, Oncorhynchus keta: The Effects of Hypo-osmotic Environmental Changes

PubMed Central

Choi, Young Jae; Kim, Na Na; Shin, Hyun Suk; Choi, Cheol Young

2014-01-01

Leptin plays an important role in energy homeostasis and reproductive function in fish, especially in reproduction. Migrating fish, such as salmonoids, are affected by external environmental factors, and salinity changes are a particularly important influence on spawning migrations. The aim of this study was to test whether changes in salinity affect the expression of leptin, estrogen receptors (ERs), and vitellogenin (VTG) in chum salmon (Oncorhynchus keta). The expression and activity of leptin, the expression of ERs and VTG, and the levels of estradiol-17β and cortisol increased after the fish were transferred to FW, demonstrating that changes in salinity stimulate the HPG axis in migrating female chum salmon. These findings reveal details about the role of elevated leptin levels and sex steroid hormones in stimulating sexual maturation and reproduction in response to salinity changes in chum salmon. PMID:25049977

Dihydrotestosterone inhibits hair growth in mice by inhibiting insulin-like growth factor-I production in dermal papillae.

PubMed

Zhao, Juan; Harada, Naoaki; Okajima, Kenji

2011-10-01

We demonstrated that insulin-like growth factor-I (IGF-I) production in dermal papillae was increased and hair growth was promoted after sensory neuron stimulation in mice. Although the androgen metabolite dihydrotestosterone (DHT) inhibits hair growth by negatively modulating growth-regulatory effects of dermal papillae, relationship between androgen metabolism and IGF-I production in dermal papillae is not fully understood. We examined whether DHT inhibits IGF-I production by inhibiting sensory neuron stimulation, thereby preventing hair growth in mice. Effect of DHT on sensory neuron stimulation was examined using cultured dorsal root ganglion (DRG) neurons isolated from mice. DHT inhibits calcitonin gene-related peptide (CGRP) release from cultured DRG neurons. The non-steroidal androgen-receptor antagonist flutamide reversed DHT-induced inhibition of CGRP release. Dermal levels of IGF-I and IGF-I mRNA, and the number of IGF-I-positive fibroblasts around hair follicles were increased at 6h after CGRP administration. DHT administration for 3weeks decreased dermal levels of CGRP, IGF-I, and IGF-I mRNA in mice. Immunohistochemical expression of IGF-I and the number of proliferating cells in hair follicles were decreased and hair re-growth was inhibited in animals administered DHT. Co-administration of flutamide and CGRP reversed these changes induced by DHT administration. These observations suggest that DHT may decrease IGF-I production in dermal papillae by inhibiting sensory neuron stimulation through interaction with the androgen receptor, thereby inhibiting hair growth in mice. Copyright © 2011 Elsevier Ltd. All rights reserved.

Vasodilator-stimulated phosphoprotein promotes activation of hepatic stellate cells by regulating Rab11-dependent plasma membrane targeting of transforming growth factor beta receptors.

PubMed

Tu, Kangsheng; Li, Jiachu; Verma, Vikas K; Liu, Chunsheng; Billadeau, Daniel D; Lamprecht, Georg; Xiang, Xiaoyu; Guo, Luyang; Dhanasekaran, Renumathy; Roberts, Lewis R; Shah, Vijay H; Kang, Ningling

2015-01-01

Liver microenvironment is a critical determinant for development and progression of liver metastasis. Under transforming growth factor beta (TGF-β) stimulation, hepatic stellate cells (HSCs), which are liver-specific pericytes, transdifferentiate into tumor-associated myofibroblasts that promote tumor implantation (TI) and growth in the liver. However, the regulation of this HSC activation process remains poorly understood. In this study, we tested whether vasodilator-stimulated phosphoprotein (VASP) of HSCs regulated the TGF-β-mediated HSC activation process and tumor growth. In both an experimental liver metastasis mouse model and cancer patients, colorectal cancer cells reaching liver sinusoids induced up-regulation of VASP and alpha-smooth muscle actin (α-SMA) in adjacent HSCs. VASP knockdown in HSCs inhibited TGF-β-mediated myofibroblastic activation of HSCs, TI, and growth in mice. Mechanistically, VASP formed protein complexes with TGF-β receptor II (TβRII) and Rab11, a Ras-like small GTPase and key regulator of recycling endosomes. VASP knockdown impaired Rab11 activity and Rab11-dependent targeting of TβRII to the plasma membrane, thereby desensitizing HSCs to TGF-β1 stimulation. Our study demonstrates a requirement of VASP for TGF-β-mediated HSC activation in the tumor microenvironment by regulating Rab11-dependent recycling of TβRII to the plasma membrane. VASP and its effector, Rab11, in the tumor microenvironment thus present therapeutic targets for reducing TI and metastatic growth in the liver. © 2014 by the American Association for the Study of Liver Diseases.

Direct Activation of NADPH Oxidase 2 by 2-Deoxyribose-1-Phosphate Triggers Nuclear Factor Kappa B-Dependent Angiogenesis

PubMed Central

Vara, Dina; Watt, Joanna M.; Fortunato, Tiago M.; Mellor, Harry; Burgess, Matthew; Wicks, Kate; Mace, Kimberly; Reeksting, Shaun; Lubben, Anneke; Wheeler-Jones, Caroline P.D.

2018-01-01

Abstract Aims: Deoxyribose-1-phosphate (dRP) is a proangiogenic paracrine stimulus released by cancer cells, platelets, and macrophages and acting on endothelial cells. The objective of this study was to clarify how dRP stimulates angiogenic responses in human endothelial cells. Results: Live cell imaging, electron paramagnetic resonance, pull-down of dRP-interacting proteins, followed by immunoblotting, gene silencing of different NADPH oxidases (NOXs), and their regulatory cosubunits by small interfering RNA (siRNA) transfection, and experiments with inhibitors of the sugar transporter glucose transporter 1 (GLUT1) were utilized to demonstrate that dRP acts intracellularly by directly activating the endothelial NOX2 complex, but not NOX4. Increased reactive oxygen species generation in response to NOX2 activity leads to redox-dependent activation of the transcription factor nuclear factor kappa B (NF-κB), which, in turn, induces vascular endothelial growth factor receptor 2 (VEGFR2) upregulation. Using endothelial tube formation assays, gene silencing by siRNA, and antibody-based receptor inhibition, we demonstrate that the activation of NF-κB and VEGFR2 is necessary for the angiogenic responses elicited by dRP. The upregulation of VEGFR2 and NOX2-dependent stimulation of angiogenesis by dRP were confirmed in excisional wound and Matrigel plug vascularization assays in vivo using NOX2−/− mice. Innovation: For the first time, we demonstrate that dRP acts intracellularly and stimulates superoxide anion generation by direct binding and activation of the NOX2 enzymatic complex. Conclusions: This study describes a novel molecular mechanism underlying the proangiogenic activity of dRP, which involves the sequential activation of NOX2 and NF-κB and upregulation of VEGFR2. Antioxid. Redox Signal. 28, 110–130. PMID:28793782

TRAF2 multitasking in TNF receptor-induced signaling to NF-κB, MAP kinases and cell death.

PubMed

Borghi, Alice; Verstrepen, Lynn; Beyaert, Rudi

2016-09-15

Tumor Necrosis Factor (TNF) is a potent inflammatory cytokine that exerts its functions through the activation of two distinct receptors, TNFR1 and TNFR2. Both receptors can activate canonical NF-κB and JNK MAP kinase signaling, while TNFR2 can also activate non-canonical NF-κB signaling, leading to numerous changes in gene expression that drive inflammation, cell proliferation and cell survival. On the other hand, TNFR1 also activates signaling pathways leading to cell death by either apoptosis or necroptosis, depending on the cellular context. A key player in TNFR1- and TNFR2-induced signaling is the RING finger protein TRAF2, which is recruited to both receptors upon their stimulation. TRAF2 exerts multiple receptor-specific functions but also mediates cross-talk between TNFR1 and TNFR2, dictating the outcome of TNF stimulation. In this review, we provide an overview of the positive and negative regulatory role of TRAF2 in different TNFR1 and TNFR2 signaling pathways. We discuss the underlying molecular mechanism of action, distinguishing between TRAF2 scaffold and E3 ubiquitin ligase functions, and the regulation of TRAF2 by specific post-translational modifications. Finally, we elaborate on some possible strategies to modulate TRAF2 function in the context of therapeutic targeting in autoimmunity and cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

Chronic changes in pituitary adenylate cyclase-activating polypeptide and related receptors in response to repeated chemical dural stimulation in rats.

PubMed

Han, Xun; Ran, Ye; Su, Min; Liu, Yinglu; Tang, Wenjing; Dong, Zhao; Yu, Shengyuan

2017-01-01

Background Preclinical experimental studies revealed an acute alteration of pituitary adenylate cyclase-activating polypeptide in response to a single activation of the trigeminovascular system, which suggests a potential role of pituitary adenylate cyclase-activating polypeptide in the pathogenesis of migraine. However, changes in pituitary adenylate cyclase-activating polypeptide after repeated migraine-like attacks in chronic migraine are not clear. Therefore, the present study investigated chronic changes in pituitary adenylate cyclase-activating polypeptide and related receptors in response to repeated chemical dural stimulations in the rat. Methods A rat model of chronic migraine was established by repeated chemical dural stimulations using an inflammatory soup for a different numbers of days. The pituitary adenylate cyclase-activating polypeptide levels were quantified in plasma, the trigeminal ganglia, and the trigeminal nucleus caudalis using radioimmunoassay and Western blotting in trigeminal ganglia and trigeminal nucleus caudalis tissues. Western blot analysis and real-time polymerase chain reaction were used to measure the protein and mRNA expression of pituitary adenylate cyclase-activating polypeptide-related receptors (PAC1, VPAC1, and VPAC2) in the trigeminal ganglia and trigeminal nucleus caudalis to identify changes associated with repetitive applications of chemical dural stimulations. Results All rats exhibited significantly decreased periorbital nociceptive thresholds to repeated inflammatory soup stimulations. Radioimmunoassay and Western blot analysis demonstrated significantly decreased pituitary adenylate cyclase-activating polypeptide levels in plasma and trigeminal ganglia after repetitive chronic inflammatory soup stimulation. Protein and mRNA analyses of pituitary adenylate cyclase-activating polypeptide-related receptors demonstrated significantly increased PAC1 receptor protein and mRNA expression in the trigeminal ganglia, but not in the trigeminal nucleus caudalis, and no significant differences were found in the expression of the VPAC1 and VPAC2 receptors. Conclusions This study demonstrated the chronic alteration of pituitary adenylate cyclase-activating polypeptide and related receptors in response to repeated chemical dural stimulation in the rat, which suggests the crucial involvement of pituitary adenylate cyclase-activating polypeptide in the development of migraine. The selective increase in pituitary adenylate cyclase-activating polypeptide-related receptors suggests that the PAC1 receptor pathway is a novel target for the treatment of migraine.

Inhibition of the CSF-1 receptor sensitizes ovarian cancer cells to cisplatin.

PubMed

Yu, Rong; Jin, Hao; Jin, Congcong; Huang, Xuefeng; Lin, Jinju; Teng, Yili

2018-03-01

Ovarian cancer is one of the most common female malignancies, and cisplatin-based chemotherapy is routinely used in locally advanced ovarian cancer patients. Acquired or de novo cisplatin resistance remains the barrier to patient survival, and the mechanisms of cisplatin resistance are still not well understood. In the current study, we found that colony-stimulating-factor-1 receptor (CSF-1R) was upregulated in cisplatin-resistant SK-OV-3 and CaoV-3 cells. Colony-stimulating-factor-1 receptor knockdown suppressed proliferation and enhanced apoptosis in cisplatin-resistant SK-OV-3 and CaoV-3 cells. However, CSF-1R overexpression had inverse effects. While parental SK-OV-3 and CaoV-3 cells were more resistant to cisplatin after CSF-1R overexpression, CSF-1R knockdown in SK-OV-3 and CaoV-3 cells promoted cisplatin sensitivity. Overexpression and knockdown studies also showed that CSF-1R significantly promoted active AKT and ERK1/2 signalling pathways in cisplatin-resistant cells. Furthermore, a combination of cisplatin and CSF-1R inhibitor effectively inhibited tumour growth in xenografts. Taken together, our results provide the first evidence that CSF-1R inhibition can sensitize cisplatin-refractory ovarian cancer cells. This study may help to increase understanding of the molecular mechanisms underlying cisplatin resistance in tumours. Copyright © 2018 John Wiley & Sons, Ltd.

Nckβ Adapter Regulates Actin Polymerization in NIH 3T3 Fibroblasts in Response to Platelet-Derived Growth Factor bb

PubMed Central

Chen, Min; She, Hongyun; Kim, Airie; Woodley, David T.; Li, Wei

2000-01-01

The SH3-SH3-SH3-SH2 adapter Nck represents a two-gene family that includes Nckα (Nck) and Nckβ (Grb4/Nck2), and it links receptor tyrosine kinases to intracellular signaling networks. The function of these mammalian Nck genes has not been established. We report here a specific role for Nckβ in platelet-derived growth factor (PDGF)-induced actin polymerization in NIH 3T3 cells. Overexpression of Nckβ but not Nckα blocks PDGF-stimulated membrane ruffling and formation of lamellipoda. Mutation in either the SH2 or the middle SH3 domain of Nckβ abolishes its interfering effect. Nckβ binds at Tyr-1009 in human PDGF receptor β (PDGFR-β) which is different from Nckα's binding site, Tyr-751, and does not compete with phosphatidylinositol-3 kinase for binding to PDGFR. Microinjection of an anti-Nckβ but not an anti-Nckα antibody inhibits PDGF-stimulated actin polymerization. Constitutively membrane-bound Nckβ but not Nckα blocks Rac1-L62-induced membrane ruffling and formation of lamellipodia, suggesting that Nckβ acts in parallel to or downstream of Rac1. This is the first report of Nckβ's role in receptor tyrosine kinase signaling to the actin cytoskeleton. PMID:11027258

The epidermal growth factor receptor (EGF-R) is present on the basolateral, but not the apical, surface of enterocytes in the human gastrointestinal tract.

PubMed Central

Playford, R J; Hanby, A M; Gschmeissner, S; Peiffer, L P; Wright, N A; McGarrity, T

1996-01-01

BACKGROUND: While it is clear that luminal epidermal growth factor (EGF) stimulates repair of the damaged bowel, its significance in maintaining normal gut growth remains uncertain. If EGF is important in maintaining normal gut growth, the EGF receptor (EGF-R) should be present on the apical (luminal) surface in addition to the basolateral surface. AIMS/SUBJECTS/METHODS: This study examined the distribution of the EGF-R in the epithelium throughout the human gastro-intestinal tract using immunohistochemistry, electron microscopy, and western blotting of brush border preparations. RESULTS: Immunostaining of the oesophagus showed circumferential EGF-R positivity in the cells of the basal portions of the stratified squamous epithelium but surface cells were EGF-R negative. In the normal stomach, small intestine, and colon, immunostaining localised the receptor to the basolateral surface with the apical membranes being consistently negative. EGF-R positivity within the small intestine appeared to be almost entirely restricted to the proliferative (crypt) region. Western blotting demonstrated a 170 kDa protein in whole tissue homogenates but not in the brush border vesicle preparations. CONCLUSIONS: As the EGF-R is located only on the basolateral surfaces in the normal adult gastrointestinal tract, the major role of luminal EGF is probably to stimulate repair rather than to maintain normal gut growth. Images Figure 1 Figure 2 Figure 3 PMID:8977341

An antigen receptor-driven, interleukin 2-independent pathway for proliferation of murine cytolytic T lymphocyte clones

PubMed Central

1986-01-01

Proliferation of T lymphocytes can be induced by IL-2, either through an autocrine pathway in which the responding cell produces its own IL-2 or through an exocrine pathway in which IL-2 secreted by Th stimulates proliferation of IL-2-dependent CTL. However, proliferation of at least some CTL clones, such as CTL L3 and CTL dB45, also can be induced by stimulation of the antigen receptor in the absence of IL-2. Stimulation of these cloned CTL with T cell-depleted allogeneic spleen cells, allogeneic tumor cells, or immobilized mAb reactive with the T cell antigen receptor (TCR) induced thymidine incorporation, entry into cell cycle, and secretion of macrophage activating factor, but these stimuli did not induce the secretion of IL-2. Several observations indicated that such proliferation of cloned CTL induced by stimulation of the TCR was independent of IL-2; IL-2 could not be detected in supernatants from stimulated CTL cells. mAbs reactive with the murine IL-2-R efficiently blocked IL-2-mediated thymidine incorporation in cloned CTL and Th, but had no inhibitory effect on TCR-driven thymidine incorporation in the CTL clones. TCR-driven thymidine incorporation in cloned Th L2 cells was profoundly inhibited by these antibodies, indicating the operation of an IL-2-mediated autocrine pathway for proliferation in this cloned Th. When antibodies to the TCR were used to stimulate cloned CTL and Th, IFN-gamma mRNA was easily shown in the cloned CTL and Th. Although IL-2 mRNA could be detected in the cloned Th, it was never observed in the cloned CTL. These findings provide evidence for the existence of a TCR-mediated, IL-2-independent pathway for induction of cellular proliferation in cloned murine CTL. PMID:3486939

A potential oncogenic activity of platelet-derived growth factor d in prostate cancer progression.

PubMed

Ustach, Carolyn V; Taube, Marcus E; Hurst, Newton J; Bhagat, Sunita; Bonfil, R Daniel; Cher, Michael L; Schuger, Lucia; Kim, Hyeong-Reh Choi

2004-03-01

The platelet-derived growth factor (PDGF) proteins are potent stimulators of cell proliferation/transformation and play a major role in cell-cell communication. For over two decades, PDGFs were thought to exist as three dimeric polypeptides (the homodimers AA and BB and the heterodimer AB). Recently, however, the PDGF C and D chains were discovered in a BLAST search of the expressed sequence tag databases. The PDGF CC and DD dimers have a unique two-domain structure with an NH(2)-terminal CUB (compliment subcomponents C1r/C1s, Uegf, and Bmp1) domain and a COOH-terminal PDGF/vascular endothelial growth factor domain. Whereas secreted PDGF AA, BB, and AB readily activate their cell surface receptors, it was suggested that extracellular proteolytic removal of the CUB domain is required for the PDGF/vascular endothelial growth factor domain of PDGF CC and DD to activate PDGF receptors. In the present study, we examined the processing of latent PDGF D into its active form and the effects of PDGF D expression on prostate cancer progression. We show that LNCaP cells auto-activate latent PDGF DD into the active PDGF domain, which can induce phosphorylation of the beta-PDGF receptor and stimulates LNCaP cell proliferation in an autocrine manner. Additionally, LNCaP-PDGF D-conditioned medium induces migration of the prostate fibroblast cell line 1532-FTX, indicating LNCaP-processed PDGF DD is active in a paracrine manner as well. In a severe combined immunodeficient mouse model, PDGF DD expression accelerates early onset of prostate tumor growth and drastically enhances prostate carcinoma cell interaction with surrounding stromal cells. These demonstrate a potential oncogenic activity of PDGF DD in the development and/or progression of prostate cancer.

A Potential Oncogenic Activity of Platelet-Derived Growth Factor D in Prostate Cancer Progression

PubMed Central

Ustach, Carolyn V.; Taube, Marcus E.; Hurst, Newton J.; Bhagat, Sunita; Bonfil, R. Daniel; Cher, Michael L.; Schuger, Lucia; Kim, Hyeong-Reh Choi

2014-01-01

The platelet-derived growth factor (PDGF) proteins are potent stimulators of cell proliferation/transformation and play a major role in cell-cell communication. For over two decades, PDGFs were thought to exist as three dimeric polypeptides (the homodimers AA and BB and the heterodimer AB). Recently, however, the PDGF C and D chains were discovered in a BLAST search of the expressed sequence tag databases. The PDGF CC and DD dimers have a unique two-domain structure with an NH2-terminal CUB (compliment subcomponents C1r/C1s, Uegf, and Bmp1) domain and a COOH-terminal PDGF/vascular endothelial growth factor domain. Whereas secreted PDGF AA, BB, and AB readily activate their cell surface receptors, it was suggested that extracellular proteolytic removal of the CUB domain is required for the PDGF/vascular endothelial growth factor domain of PDGF CC and DD to activate PDGF receptors. In the present study, we examined the processing of latent PDGF D into its active form and the effects of PDGF D expression on prostate cancer progression. We show that LNCaP cells auto-activate latent PDGF DD into the active PDGF domain, which can induce phosphorylation of the β-PDGF receptor and stimulates LNCaP cell proliferation in an autocrine manner. Additionally, LNCaP-PDGF D-conditioned medium induces migration of the prostate fibroblast cell line 1532-FTX, indicating LNCaP-processed PDGF DD is active in a paracrine manner as well. In a severe combined immunodeficient mouse model, PDGF DD expression accelerates early onset of prostate tumor growth and drastically enhances prostate carcinoma cell interaction with surrounding stromal cells. These demonstrate a potential oncogenic activity of PDGF DD in the development and/or progression of prostate cancer. PMID:14996732

αPIX Is a Trafficking Regulator that Balances Recycling and Degradation of the Epidermal Growth Factor Receptor

PubMed Central

Kortüm, Fanny; Harms, Frederike Leonie; Hennighausen, Natascha; Rosenberger, Georg

2015-01-01

Endosomal sorting is an essential control mechanism for signaling through the epidermal growth factor receptor (EGFR). We report here that the guanine nucleotide exchange factor αPIX, which modulates the activity of Rho-GTPases, is a potent bimodal regulator of EGFR trafficking. αPIX interacts with the E3 ubiquitin ligase c-Cbl, an enzyme that attaches ubiquitin to EGFR, thereby labelling this tyrosine kinase receptor for lysosomal degradation. We show that EGF stimulation induces αPIX::c-Cbl complex formation. Simultaneously, αPIX and c-Cbl protein levels decrease, which depends on both αPIX binding to c-Cbl and c-Cbl ubiquitin ligase activity. Through interaction αPIX sequesters c-Cbl from EGFR and this results in reduced EGFR ubiquitination and decreased EGFR degradation upon EGF treatment. However, quantitatively more decisive for cellular EGFR distribution than impaired EGFR degradation is a strong stimulating effect of αPIX on EGFR recycling to the cell surface. This function depends on the GIT binding domain of αPIX but not on interaction with c-Cbl or αPIX exchange activity. In summary, our data demonstrate a previously unappreciated function of αPIX as a strong promoter of EGFR recycling. We suggest that the novel recycling regulator αPIX and the degradation factor c-Cbl closely cooperate in the regulation of EGFR trafficking: uncomplexed αPIX and c-Cbl mediate a positive and a negative feedback on EGFR signaling, respectively; αPIX::c-Cbl complex formation, however, results in mutual inhibition, which may reflect a stable condition in the homeostasis of EGF-induced signal flow. PMID:26177020

Enhancing Proprioceptive Input to Motoneurons Differentially Affects Expression of Neurotrophin 3 and Brain-Derived Neurotrophic Factor in Rat Hoffmann-Reflex Circuitry

PubMed Central

Gajewska-Woźniak, Olga; Skup, Małgorzata; Kasicki, Stefan; Ziemlińska, Ewelina; Czarkowska-Bauch, Julita

2013-01-01

The importance of neurotrophin 3 (NT-3) for motor control prompted us to ask the question whether direct electrical stimulation of low-threshold muscle afferents, strengthening the proprioceptive signaling, could effectively increase the endogenous pool of this neurotrophin and its receptor TrkC in the Hoffmann-reflex (H-reflex) circuitry. The effects were compared with those of brain-derived neurotrophic factor (BDNF) and its TrkB receptor. Continuous bursts of stimuli were delivered unilaterally for seven days, 80 min daily, by means of a cuff-electrode implanted over the tibial nerve in awake rats. The H-reflex was recorded in the soleus muscle to control the strength of stimulation. Stimulation aimed at activation of Ia fibers produced a strong increase of NT-3 protein, measured with ELISA, in the lumbar L3-6 segments of the spinal cord and in the soleus muscle. This stimulation exerted much weaker effect on BDNF protein level which slightly increased only in L3-6 segments of the spinal cord. Increased protein level of NT-3 and BDNF corresponded to the changes of NT-3 mRNA and BDNF mRNA expression in L3-6 segments but not in the soleus muscle. We disclosed tissue-specificity of TrkC mRNA and TrkB mRNA responses. In the spinal cord TrkC and TrkB transcripts tended to decrease, whereas in the soleus muscle TrkB mRNA decreased and TrkC mRNA expression strongly increased, suggesting that stimulation of Ia fibers leads to sensitization of the soleus muscle to NT-3 signaling. The possibility of increasing NT-3/TrkC signaling in the neuromuscular system, with minor effects on BDNF/TrkB signaling, by means of low-threshold electrical stimulation of peripheral nerves, which in humans might be applied in non-invasive way, offers an attractive therapeutic tool. PMID:23776573

Enhancing proprioceptive input to motoneurons differentially affects expression of neurotrophin 3 and brain-derived neurotrophic factor in rat hoffmann-reflex circuitry.

PubMed

Gajewska-Woźniak, Olga; Skup, Małgorzata; Kasicki, Stefan; Ziemlińska, Ewelina; Czarkowska-Bauch, Julita

2013-01-01

The importance of neurotrophin 3 (NT-3) for motor control prompted us to ask the question whether direct electrical stimulation of low-threshold muscle afferents, strengthening the proprioceptive signaling, could effectively increase the endogenous pool of this neurotrophin and its receptor TrkC in the Hoffmann-reflex (H-reflex) circuitry. The effects were compared with those of brain-derived neurotrophic factor (BDNF) and its TrkB receptor. Continuous bursts of stimuli were delivered unilaterally for seven days, 80 min daily, by means of a cuff-electrode implanted over the tibial nerve in awake rats. The H-reflex was recorded in the soleus muscle to control the strength of stimulation. Stimulation aimed at activation of Ia fibers produced a strong increase of NT-3 protein, measured with ELISA, in the lumbar L3-6 segments of the spinal cord and in the soleus muscle. This stimulation exerted much weaker effect on BDNF protein level which slightly increased only in L3-6 segments of the spinal cord. Increased protein level of NT-3 and BDNF corresponded to the changes of NT-3 mRNA and BDNF mRNA expression in L3-6 segments but not in the soleus muscle. We disclosed tissue-specificity of TrkC mRNA and TrkB mRNA responses. In the spinal cord TrkC and TrkB transcripts tended to decrease, whereas in the soleus muscle TrkB mRNA decreased and TrkC mRNA expression strongly increased, suggesting that stimulation of Ia fibers leads to sensitization of the soleus muscle to NT-3 signaling. The possibility of increasing NT-3/TrkC signaling in the neuromuscular system, with minor effects on BDNF/TrkB signaling, by means of low-threshold electrical stimulation of peripheral nerves, which in humans might be applied in non-invasive way, offers an attractive therapeutic tool.

miR-155 functions downstream of angiotensin II receptor subtype 1 and calcineurin to regulate cardiac hypertrophy.

PubMed

Yang, Yong; Zhou, Yong; Cao, Zheng; Tong, Xin Zhu; Xie, Hua Qiang; Luo, Tao; Hua, Xian Ping; Wang, Han Qin

2016-09-01

Cardiac hypertrophy is characterized by maladaptive tissue remodeling that may lead to heart failure or sudden death. MicroRNAs (miRs) are negative regulators of angiotensin II and the angiotensin II receptor subtype 1 (AGTR 1 ), which are two components involved in cardiac hypertrophy. In the present study, the interaction between angiotensin II receptor subtype 1 (AGTR 1 ) signaling and miR-155 was investigated. Rat H9C2 (2-1) cardiomyocytes were transfected with miR-155 analogues or inhibitors, then stimulated with angiotensin II to induce cardiac hypertrophy. miR-155 expression was revealed to be altered following transfection with chemically-modified miR-155 analogues and inhibitors in rat cardiomyocytes. In cell cardiac hypertrophy models, the cell surface area, AGTR 1 , atrial natriuretic peptide and myosin heavy chain-β mRNA expression levels were revealed to be lower in cells stimulated with miR-155 analogue-transfected cells treated with angiotensin II compared with cells stimulated with angiotensin alone (P<0.05), as determined using reverse transcription-polymerase chain reaction (PCR), quantitative PCR and western blot analyses. Furthermore, calcineurin mRNA and protein, intracellular free calcium and nuclear factor of activated T-cells-4 proteins were downregulated in miR-155 analogue-transfected cells treated with angiotensin II, as compared with cells stimulated with angiotensin II alone (P<0.05). In conclusion, the current study indicates that miR-155 may improve cardiac hypertrophy by downregulating AGTR 1 and suppressing the calcium signaling pathways activated by AGTR 1 .

Bovine gallbladder muscularis: Source of a myogenic receptor for cholecystokinin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schjoldager, B.; Shaw, M.J.; Powers, S.P.

1988-03-01

Despite being a classic target for the gastrointestinal peptide hormone, cholecystokinin (CCK), the gallbladder CCK receptor is not well characterized. Pharmacological studies of small species suggest that CCK action can be mediated by direct myogenic or by both myogenic and neurogenic receptors. To prepare for the biochemical characterization of a gallbladder CCK receptor and to define the subtype of the receptor being studied. The authors have performed autoradiographic localization and pharmacological characterization of CCK receptors on bovine gallbladder. Autoradiography demonstrated high-affinity specific CCK-binding sites only on the muscularis. CCK-8 stimulated tonic contraction of longitudinal strips of gallbladder muscularis in amore » concentration-dependent manner. Antagonism at the cholinergic receptor with 1{mu}M atropine or axonal transmission with 1{mu}M tetrodotoxin did not modify CCK-induced contraction, supporting a direct myogenic effect of this hormone. Optimal electrical field stimulation to elicit a neuronal response resulted in muscle strip relaxation, which was abolished with adrenergic blockade. Although acetylcholine administration stimulated contraction, electrical field stimulation did not, even in the presence of phentolamine, propranolol, and/or CCK. Thus, in bovine gallbladder muscularis, there is evidence for a functional CCK receptor only on smooth muscle cells. Demonstration of a single, high-affinity specific CCK-binding site on an enriched plasma membrane preparation of bovine gallbladder muscularis is consistent with this representing a myogenic CCK receptor.« less

Mechanical stress-induced interleukin-1beta expression through adenosine triphosphate/P2X7 receptor activation in human periodontal ligament cells.

PubMed

Kanjanamekanant, K; Luckprom, P; Pavasant, P

2013-04-01

Mechanical stress is an important factor in maintaining homeostasis of the periodontium. Interleukin-1beta (IL-1β) and adenosine triphosphate (ATP) are considered potent inflammatory mediators. In macrophages, ATP-activated P2X7 receptor is involved in IL-1β processing and release. Our previous works demonstrated mechanical stress-induced expression of osteopontin and RANKL through the ATP/P2Y1 receptor in human periodontal ligament (HPDL) cells. This study was designed to examine the effect of mechanical stress on IL-1β expression in HPDL cells, as well as the mechanism and involvement of ATP and the P2 purinergic receptor. Cultured HPDL cells were treated with continuous compressive loading. IL-1β expression was analyzed at both mRNA and protein levels, using RT-PCR and ELISA, respectively. Cell viability was examined using the MTT assay. ATP was also used to stimulate HPDL cells. Inhibitors, antagonists and the small interfering RNA (siRNA) technique were used to investigate the role of ATP and the specific P2 subtypes responsible for IL-1β induction along with the intracellular mechanism. Mechanical stress could up-regulate IL-1β expression through the release of ATP in HPDL cells. ATP alone was also capable of increasing IL-1β expression. The induction of IL-1β was markedly inhibited by inhibitors and by siRNA targeting the P2X7 receptor. ATP-stimulated IL-1β expression was also diminished by intracellular calcium inhibitors. Our work clearly indicates the capability of HPDL cells to respond directly to mechanical stimulation. The results signified the important roles of ATP/P2 purinergic receptors, as well as intracellular calcium signaling, in mechanical stress-induced inflammation via up-regulation of the proinflammatory cytokine, IL-1β, in HPDL cells. © 2012 John Wiley & Sons A/S.

Farnesoid X receptor induces Takeda G-protein receptor 5 cross-talk to regulate bile acid synthesis and hepatic metabolism.

PubMed

Pathak, Preeti; Liu, Hailiang; Boehme, Shannon; Xie, Cen; Krausz, Kristopher W; Gonzalez, Frank; Chiang, John Y L

2017-06-30

The bile acid-activated receptors, nuclear farnesoid X receptor (FXR) and the membrane Takeda G-protein receptor 5 (TGR5), are known to improve glucose and insulin sensitivity in obese and diabetic mice. However, the metabolic roles of these two receptors and the underlying mechanisms are incompletely understood. Here, we studied the effects of the dual FXR and TGR5 agonist INT-767 on hepatic bile acid synthesis and intestinal secretion of glucagon-like peptide-1 (GLP-1) in wild-type, Fxr -/- , and Tgr5 -/- mice. INT-767 efficaciously stimulated intracellular Ca 2+ levels, cAMP activity, and GLP-1 secretion and improved glucose and lipid metabolism more than did the FXR-selective obeticholic acid and TGR5-selective INT-777 agonists. Interestingly, INT-767 reduced expression of the genes in the classic bile acid synthesis pathway but induced those in the alternative pathway, which is consistent with decreased taurocholic acid and increased tauromuricholic acids in bile. Furthermore, FXR activation induced expression of FXR target genes, including fibroblast growth factor 15, and unexpectedly Tgr5 and prohormone convertase 1/3 gene expression in the ileum. We identified an FXR-responsive element on the Tgr5 gene promoter. Fxr -/- and Tgr5 -/- mice exhibited reduced GLP-1 secretion, which was stimulated by INT-767 in the Tgr5 -/- mice but not in the Fxr -/- mice. Our findings uncovered a novel mechanism in which INT-767 activation of FXR induces Tgr5 gene expression and increases Ca 2+ levels and cAMP activity to stimulate GLP-1 secretion and improve hepatic glucose and lipid metabolism in high-fat diet-induced obese mice. Activation of both FXR and TGR5 may therefore represent an effective therapy for managing hepatic steatosis, obesity, and diabetes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Grb2 mediates semaphorin-4D-dependent RhoA inactivation.

PubMed

Sun, Tianliang; Krishnan, Rameshkumar; Swiercz, Jakub M

2012-08-01

Signaling through the semaphorin 4D (Sema4D) receptor plexin-B1 is modulated by its interaction with tyrosine kinases ErbB-2 and Met. In cells expressing the plexin-B1-ErbB-2 receptor complex, ligand stimulation results in the activation of small GTPase RhoA and stimulation of cellular migration. By contrast, in cells expressing plexin-B1 and Met, ligand stimulation results in an association with the RhoGTPase-activating protein p190 RhoGAP and subsequent RhoA inactivation--a process that involves the tyrosine phosphorylation of plexin-B1 by Met. Inactivation of RhoA is necessary for Sema4D-mediated inhibition of cellular migration. It is, however, unknown how plexin-B1 phosphorylation regulates RhoGAP interaction and activity. Here we show that the activation of plexin-B1 by Sema4D and its subsequent tyrosine phosphorylation by Met creates a docking site for the SH2 domain of growth factor receptor bound-2 (Grb2). Grb2 is thereby recruited into the plexin-B1 receptor complex and, through its SH3 domain, interacts with p190 RhoGAP and mediates RhoA deactivation. Phosphorylation of plexin-B1 by Met and the recruitment of Grb2 have no effect on the R-RasGAP activity of plexin-B1, but are required for Sema4D-induced, RhoA-dependent antimigratory effects of Sema4D on breast cancer cells. These data show Grb2 as a direct link between plexin and p190-RhoGAP-mediated downstream signaling.

Basigin-2 Is a Cell Surface Receptor for Soluble Basigin Ligand*S⃞

PubMed Central

Belton, Robert J.; Chen, Li; Mesquita, Fernando S.; Nowak, Romana A.

2008-01-01

The metastatic spread of a tumor is dependent upon the ability of the tumor to stimulate surrounding stromal cells to express enzymes required for tissue remodeling. The immunoglobulin superfamily protein basigin (EMMPRIN/CD147) is a cell surface glycoprotein expressed by tumor cells that stimulates matrix metalloproteinase and vascular endothelial growth factor expression in stromal cells. The ability of basigin to stimulate expression of molecules involved in tissue remodeling and angiogenesis makes basigin a potential target for the development of strategies to block metastasis. However, the identity of the cell surface receptor for basigin remains controversial. The goal of this study was to determine the identity of the receptor for basigin. Using a novel recombinant basigin protein (rBSG) corresponding to the extracellular domain of basigin, it was demonstrated that the native, nonglycosylated rBSG protein forms dimers in solution. Furthermore, rBSG binds to the surface of uterine fibroblasts, activates the ERK1/2 signaling pathway, and induces expression of matrix metalloproteinases 1, 2, and 3. Proteins that interact with rBSG were isolated using a biotin label transfer technique and sequenced by matrix-assisted laser desorption ionization tandem mass spectrophotometry. The results demonstrate that rBSG interacts with basigin expressed on the surface of fibroblasts and is subsequently internalized. During internalization, rBSG associates with a novel form of human basigin (basigin-3). It was concluded that cell surface basigin functions as a membrane receptor for soluble basigin and this homophilic interaction is not dependent upon glycosylation of the basigin ligand. PMID:18434307

Adhesion-Dependent Redistribution of MAP Kinase and MEK Promotes Muscarinic Receptor-Mediated Signaling to the Nucleus

PubMed Central

Slack, Barbara E.; Siniaia, Marina S.

2008-01-01

The mitogen-activated protein kinases (MAPKs) are activated by extracellular signals, and translocate to the nucleus where they modulate transcription. Integrin-mediated cell adhesion to extracellular matrix (ECM) proteins is required for efficient transmission of MAPK-based signals initiated by growth factors. However, the modulation of G protein-coupled receptor (GPCR) signaling by adhesion is less well understood. In the present study we assessed the impact of cell adhesion on MAPK activation by muscarinic M3 receptors. The muscarinic agonist carbachol more efficiently promoted stress fiber formation and tyrosine phosphorylation of focal adhesion-associated proteins in M3 receptor-expressing cells adherent to fibronectin or collagen type I, as compared to polylysine. Overall MAPK activation was robust in cells adherent to all three substrata. However, total levels of MAPK and mitogen-activated protein kinase kinase (MEK) in the nucleus were significantly greater in cells adherent to ECM proteins for 2.5 hours, and levels of activated MAPK and MEK in the nuclei of these cells were higher following carbachol stimulation, relative to levels in cells adherent to polylysine. MEK inhibitors did not prevent adhesion-dependent translocation of MAPK and MEK to the nucleus, and increased nuclear phospho-MEK levels in carbachol-stimulated cells. The results suggest that adhesion of cells to ECM triggers the redistribution of MAPK and MEK to the nucleus, possibly as a result of the cytoskeletal rearrangements that accompany cell spreading. This may represent a mechanism for priming the nucleus with MEK and MAPK, leading to more rapid and pronounced increases in intranuclear phospho-MAPK upon GPCR stimulation. PMID:15779001

Extrasynaptic N-Methyl-d-aspartate (NMDA) Receptor Stimulation Induces Cytoplasmic Translocation of the CDKL5 Kinase and Its Proteasomal Degradation*

PubMed Central

Rusconi, Laura; Kilstrup-Nielsen, Charlotte; Landsberger, Nicoletta

2011-01-01

Mutations in the X-linked gene cyclin-dependent kinase-like 5 (CDKL5) have been found in patients with epileptic encephalopathy characterized by early onset intractable epilepsy, including infantile spasms and other types of seizures, severe developmental delay, and often the development of Rett syndrome-like features. Despite its clear involvement in proper brain development, CDKL5 functions are still far from being understood. In this study, we analyzed the subcellular localization of the endogenous kinase in primary murine hippocampal neurons. CDKL5 was localized both in nucleus and cytoplasm and, conversely to proliferating cells, did not undergo constitutive shuttling between these compartments. Nevertheless, glutamate stimulation was able to induce the exit of the kinase from the nucleus and its subsequent accumulation in the perinuclear cytoplasm. Moreover, we found that sustained glutamate stimulation promoted CDKL5 proteasomal degradation. Both events were mediated by the specific activation of extrasynaptic pool of N-methyl-d-aspartate receptors. Proteasomal degradation was also induced by withdrawal of neurotrophic factors and hydrogen peroxide treatment, two different paradigms of cell death. Altogether, our results indicate that both subcellular localization and expression of CDKL5 are modulated by the activation of extrasynaptic N-methyl-d-aspartate receptors and suggest regulation of CDKL5 by cell death pathways. PMID:21832092

Role of GPER on proliferation, migration and invasion in ligand-independent manner in human ovarian cancer cell line SKOV3.

PubMed

Yan, Yan; Jiang, Xueli; Zhao, Ying; Wen, Haixia; Liu, Guoyi

2015-12-01

G protein-coupled estrogen receptor (GPER) is identified as a critical estrogen receptor, in addition to the classical estrogen receptors ERα and ERβ. In ERα-negative ovarian cancer cells, our previous studies have found that estrogen stimulated cell proliferation and metastasis via GPER. However, the ligand-independent function of GPER in ovarian cancer cells is still not clear. Herein, we describe that GPER has a co-expression with ERα and ERβ, which are first determined in SKOV3 ovarian cancer cell line. In the absence of estrogen, GPER depletion by specific siRNA inhibits the proliferation, migration and invasion of SKOV3 cells. Whereas abrogation of ERα or ERβ by specific antagonist MPP and PHTPP has the opposite effects for stimulation of cell growth. Markedly, GPER knockdown attenuates MPP or PHTPP-induced cell proliferation, migration and invasion. Furthermore, GPER modulates protein expression of the cell cycle critical components, c-fos and cyclin D1 and factors for cancer cell invasion and metastasis, matrix metalloproteinase 2 (MMP-2) and MMP-9. These findings establish that GPER ligand-independently stimulates the proliferation, migration and invasion of SKOV3 cells. Knockdown of GPER attenuates the progression of ovarian cancer that caused by functional loss of ERα or ERβ. Targeting GPER provides new aspect as a potential therapeutic strategy in ovarian cancer. Copyright © 2015 John Wiley & Sons, Ltd.

Chemokine receptor binding and signal transduction in native cells of the central nervous system.

PubMed

Davis, Christopher N; Chen, Shuzhen; Boehme, Stefen A; Bacon, Kevin B; Harrison, Jeffrey K

2003-04-01

Chemokine receptors belong to the superfamily of seven-transmembrane-spanning, G-protein-coupled receptors, and their expression by central nervous system cells is clearly documented. As this gene family has become the target of novel therapeutic development, the analysis of these receptors requires radioligand binding techniques as well as methods that entail assessing receptor stimulation of signal transduction pathways. Herein, we describe specific protocols for measuring radiolabeled chemokine binding to their cognate receptors on cultured glial cells as well as to receptors expressed in heterologous cell systems. Multiple downstream signaling pathways, including intracellular calcium influx and receptor-dependent kinase activation, are associated with chemokine receptor stimulation. Protocols for measuring these signaling events in chemokine-receptor-expressing cells are also presented.

Agonist-induced internalization of the platelet-activating factor receptor is dependent on arrestins but independent of G-protein activation. Role of the C terminus and the (D/N)PXXY motif.

PubMed

Chen, Zhangguo; Dupré, Denis J; Le Gouill, Christian; Rola-Pleszczynski, Marek; Stanková, Jana

2002-03-01

As with most G-protein-coupled receptors, repeated agonist stimulation of the platelet-activating factor receptor (PAFR) results in its desensitization, sequestration, and internalization. In this report, we show that agonist-induced PAFR internalization is independent of G-protein activation but is dependent on arrestins and involves the interaction of arrestins with a limited region of the PAFR C terminus. In cotransfected COS-7 cells, both arrestin-2 and arrestin-3 could be coimmunoprecipitated with PAFR, and agonist stimulation of PAFR induced the translocation of both arrestin-2 and arrestin-3. Furthermore, coexpression of arrestin-2 with PAFR potentiated receptor internalization, whereas agonist-induced PAFR internalization was inhibited by a dominant negative mutant of arrestin-2. The coexpression of a minigene encoding the C-terminal segment of the receptor abolished PAF-induced arrestin translocation and inhibited PAFR internalization. Using C terminus deletion mutants, we determined that the association of arrestin-2 with the receptor was dependent on the region between threonine 305 and valine 330 because arrestin-2 could be immunoprecipitated with the mutant PAFRstop330 but not PAFRstop305. Consistently, stop330 could mediate agonist-induced arrestin-2 translocation, whereas stop305 could not. Two other deletion mutants with slightly longer regions of the C terminus, PAFRstop311 and PAFRstop317, also failed to induce arrestin-2 translocation. Finally, the PAFR mutant Y293A, containing a single substitution in the putative internalization motif DPXXY in the seventh transmembrane domain (which we had shown to be able to internalize but not to couple to G-proteins) could efficiently induce arrestin translocation. Taken together, our results indicate that ligand-induced PAFR internalization is dependent on arrestins, that PAFR can associate with both arrestin-2 and -3, and that their translocation involves interaction with the region of residues 318-330 in the PAFR C terminus but is independent of G-protein activation.

Alternative pathway regulation by factor H modulates Streptococcus pneumoniae induced proinflammatory cytokine responses by decreasing C5a receptor crosstalk.

PubMed

van der Maten, Erika; de Bont, Cynthia M; de Groot, Ronald; de Jonge, Marien I; Langereis, Jeroen D; van der Flier, Michiel

2016-12-01

Bacterial pathogens not only stimulate innate immune receptors, but also activate the complement system. Crosstalk between complement C5a receptor (C5aR) and other innate immune receptors is known to enhance the proinflammatory cytokine response. An important determinant of the magnitude of complement activation is the activity of the alternative pathway, which serves as an amplification mechanism for complement activation. Both alternative pathway activity as well as plasma levels of factor H, a key inhibitor of the alternative pathway, show large variation within the human population. Here, we studied the effect of factor H-mediated regulation of the alternative pathway on bacterial-induced proinflammatory cytokine responses. We used the human pathogen Streptococcus pneumoniae as a model stimulus to induce proinflammatory cytokine responses in human peripheral blood mononuclear cells. Serum containing active complement enhanced pneumococcal induced proinflammatory cytokine production through C5a release and C5aR crosstalk. We found that inhibition of the alternative pathway by factor H, with a concentration equivalent to a high physiological level, strongly reduced C5a levels and decreased proinflammatory cytokine production in human peripheral blood mononuclear cells. This suggests that variation in alternative pathway activity due to variation in factor H plasma levels affects individual cytokine responses during infection. Copyright © 2016 Elsevier Ltd. All rights reserved.

Hypoxia-inducible factor-1α in vascular smooth muscle regulates blood pressure homeostasis through a peroxisome proliferator-activated receptor-γ-angiotensin II receptor type 1 axis.

PubMed

Huang, Yan; Di Lorenzo, Annarita; Jiang, Weidong; Cantalupo, Anna; Sessa, William C; Giordano, Frank J

2013-09-01

Hypertension is a major worldwide health issue for which only a small proportion of cases have a known mechanistic pathogenesis. Of the defined causes, none have been directly linked to heightened vasoconstrictor responsiveness, despite the fact that vasomotor tone in resistance vessels is a fundamental determinant of blood pressure. Here, we reported a previously undescribed role for smooth muscle hypoxia-inducible factor-1α (HIF-1α) in controlling blood pressure homeostasis. The lack of HIF-1α in smooth muscle caused hypertension in vivo and hyperresponsiveness of resistance vessels to angiotensin II stimulation ex vivo. These data correlated with an increased expression of angiotensin II receptor type I in the vasculature. Specifically, we show that HIF-1α, through peroxisome proliferator-activated receptor-γ, reciprocally defined angiotensin II receptor type I levels in the vessel wall. Indeed, pharmacological blockade of angiotensin II receptor type I by telmisartan abolished the hypertensive phenotype in smooth muscle cell-HIF-1α-KO mice. These data revealed a determinant role of a smooth muscle HIF-1α/peroxisome proliferator-activated receptor-γ/angiotensin II receptor type I axis in controlling vasomotor responsiveness and highlighted an important pathway, the alterations of which may be critical in a variety of hypertensive-based clinical settings.

GRB2 Interaction with the Ecotropic Murine Leukemia Virus Receptor, mCAT-1, Controls Virus Entry and Is Stimulated by Virus Binding

PubMed Central

Chen, Zeming; Kolokoltsov, Andrey A.; Wang, Jia; Adhikary, Shramika; Lorinczi, Marta; Elferink, Lisa A.

2012-01-01

For retroviruses such as HIV-1 and murine leukemia virus (MLV), active receptor recruitment and trafficking occur during viral entry. However, the underlying mechanisms and cellular factors involved in the process are largely uncharacterized. The viral receptor for ecotropic MLV (eMLV), a classical model for retrovirus infection mechanisms and pathogenesis, is mouse cationic amino acid transporter 1 (mCAT-1). Growth factor receptor-bound protein 2 (GRB2) is an adaptor protein that has been shown to couple cell surface receptors, such as epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor, to intracellular signaling events. Here we examined if GRB2 could also play a role in controlling infection by retroviruses by affecting receptor function. The GRB2 RNA interference (RNAi)-mediated suppression of endogenous GRB2 resulted in a consistent and significant reduction of virus binding and membrane fusion. The binding between eMLV and cells promoted increased GRB2–mCAT-1 interactions, as detected by immunoprecipitation. Consistently, the increased colocalization of GRB2 and mCAT-1 signals was detected by confocal microscopy. This association was time dependent and paralleled the kinetics of cell-virus membrane fusion. Interestingly, unlike the canonical binding pattern seen for GRB2 and growth factor receptors, GRB2–mCAT-1 binding does not depend on the GRB2-SH2 domain-mediated recognition of tyrosine phosphorylation on the receptor. The inhibition of endogenous GRB2 led to a reduction in surface levels of mCAT-1, which was detected by immunoprecipitation and by a direct binding assay using a recombinant MLV envelope protein receptor binding domain (RBD). Consistent with this observation, the expression of a dominant negative GRB2 mutant (R86K) resulted in the sequestration of mCAT-1 from the cell surface into intracellular vesicles. Taken together, these findings suggest a novel role for GRB2 in ecotropic MLV entry and infection by facilitating mCAT-1 trafficking. PMID:22090132

GRB2 interaction with the ecotropic murine leukemia virus receptor, mCAT-1, controls virus entry and is stimulated by virus binding.

PubMed

Chen, Zeming; Kolokoltsov, Andrey A; Wang, Jia; Adhikary, Shramika; Lorinczi, Marta; Elferink, Lisa A; Davey, Robert A

2012-02-01

For retroviruses such as HIV-1 and murine leukemia virus (MLV), active receptor recruitment and trafficking occur during viral entry. However, the underlying mechanisms and cellular factors involved in the process are largely uncharacterized. The viral receptor for ecotropic MLV (eMLV), a classical model for retrovirus infection mechanisms and pathogenesis, is mouse cationic amino acid transporter 1 (mCAT-1). Growth factor receptor-bound protein 2 (GRB2) is an adaptor protein that has been shown to couple cell surface receptors, such as epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor, to intracellular signaling events. Here we examined if GRB2 could also play a role in controlling infection by retroviruses by affecting receptor function. The GRB2 RNA interference (RNAi)-mediated suppression of endogenous GRB2 resulted in a consistent and significant reduction of virus binding and membrane fusion. The binding between eMLV and cells promoted increased GRB2-mCAT-1 interactions, as detected by immunoprecipitation. Consistently, the increased colocalization of GRB2 and mCAT-1 signals was detected by confocal microscopy. This association was time dependent and paralleled the kinetics of cell-virus membrane fusion. Interestingly, unlike the canonical binding pattern seen for GRB2 and growth factor receptors, GRB2-mCAT-1 binding does not depend on the GRB2-SH2 domain-mediated recognition of tyrosine phosphorylation on the receptor. The inhibition of endogenous GRB2 led to a reduction in surface levels of mCAT-1, which was detected by immunoprecipitation and by a direct binding assay using a recombinant MLV envelope protein receptor binding domain (RBD). Consistent with this observation, the expression of a dominant negative GRB2 mutant (R86K) resulted in the sequestration of mCAT-1 from the cell surface into intracellular vesicles. Taken together, these findings suggest a novel role for GRB2 in ecotropic MLV entry and infection by facilitating mCAT-1 trafficking.

Rupatadine inhibits inflammatory mediator release from human laboratory of allergic diseases 2 cultured mast cells stimulated by platelet-activating factor.

PubMed

Alevizos, Michail; Karagkouni, Anna; Vasiadi, Magdalini; Sismanopoulos, Nikolaos; Makris, Michael; Kalogeromitros, Dimitrios; Theoharides, Theoharis C

2013-12-01

Mast cells are involved in allergy and inflammation by the secretion of multiple mediators, including histamine, cytokines, and platelet-activating factor (PAF), in response to different triggers, including emotional stress. PAF has been associated with allergic inflammation, but there are no clinically available PAF inhibitors. To investigate whether PAF could stimulate human mast cell mediator release and whether rupatadine (RUP), a dual histamine-1 and PAF receptor antagonist, could inhibit the effect of PAF on human mast cells. Laboratory of allergic diseases 2 cultured mast cells were stimulated with PAF (0.001, 0.01, and 0.1 μmol/L) and substance P (1 μmol/L) with or without pretreatment with RUP (2.5 and 25 μmol/L), which was added 10 minutes before stimulation. Release of β-hexosaminidase was measured in supernatant fluid by spectrophotoscopy, and histamine, interleukin-8, and tumor necrosis factor were measured by enzyme-linked immunosorbent assay. PAF stimulated a statistically significant release of histamine, interleukin-8, and tumor necrosis factor (0.001-0.1 μmol/L) that was comparable to that stimulated by substance P. Pretreatment with RUP (25 μmol/L) for 10 minutes inhibited this effect. In contrast, pretreatment of laboratory of allergic diseases 2 cells with diphenhydramine (25 μmol/L) did not inhibit mediator release, suggesting that the effect of RUP was not due to its antihistaminic effect. PAF stimulates human mast cell release of proinflammatory mediators that is inhibited by RUP. This action endows RUP with additional properties in treating allergic inflammation. Copyright © 2013 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Tumor necrosis factor-alpha stimulation of calcitonin gene-related peptide expression and secretion from rat trigeminal ganglion neurons.

PubMed

Bowen, Elizabeth J; Schmidt, Thomas W; Firm, Christina S; Russo, Andrew F; Durham, Paul L

2006-01-01

Expression of the neuropeptide calcitonin gene-related peptide (CGRP) in trigeminal ganglion is implicated in neurovascular headaches and temporomandibular joint disorders. Elevation of cytokines contributes to the pathology of these diseases. However, a connection between cytokines and CGRP gene expression in trigeminal ganglion nerves has not been established. We have focused on the effects of the cytokine tumor necrosis factor-alpha (TNF-alpha). TNFR1 receptors were found on the majority of CGRP-containing rat trigeminal ganglion neurons. Treatment of cultures with TNF-alpha stimulated CGRP secretion. In addition, the intracellular signaling intermediate from the TNFR1 receptor, ceramide, caused a similar increase in CGRP release. TNF-alpha caused a coordinate increase in CGRP promoter activity. TNF-alpha treatment activated the transcription factor NF-kappaB, as well as the Jun N-terminal kinase (JNK) and p38 mitogen-activated protein (MAP) kinase pathways. The importance of TNF-alpha induction of MAP kinase pathways was demonstrated by inhibiting MAP kinases with pharmacological reagents and gene transfer with an adenoviral vector encoding MAP kinase phosphatase-1 (MKP-1). We propose that selective and regulated inhibition of MAP kinases in trigeminal neurons may be therapeutically beneficial for inflammatory disorders involving elevated CGRP levels.

Tumor necrosis factor-α stimulation of calcitonin gene-related peptide expression and secretion from rat trigeminal ganglion neurons

PubMed Central

Bowen, Elizabeth J.; Schmidt, Thomas W.; Firm, Christina S.; Russo, Andrew F.; Durham, Paul L.

2006-01-01

Expression of the neuropeptide calcitonin gene-related peptide (CGRP) in trigeminal ganglion is implicated in neurovascular headaches and temporomandibular joint disorders. Elevation of cytokines contributes to the pathology of these diseases. However, a connection between cytokines and CGRP gene expression in trigeminal ganglion nerves has not been established. We have focused on the effects of the cytokine tumor necrosis factorα (TNFα). TNFR1 receptors were found on the majority of CGRP-containing rat trigeminal ganglion neurons. Treatment of cultures with TNFα stimulated CGRP secretion. In addition, the intracellular signaling intermediate from the TNFR1 receptor, ceramide, caused a similar increase in CGRP release. TNFα caused a coordinate increase in CGRP promoter activity. TNFα treatment activated the transcription factor NF-κB, as well as the Jun N-terminal kinase (JNK) and p38 mitogen-activated protein (MAP) kinase pathways. The importance of TNFα induction of MAP kinase pathways was demonstrated by inhibiting MAP kinases with pharmacological reagents and gene transfer with an adenoviral vector encoding MAP kinase phosphatase-1 (MKP-1). We propose that selective and regulated inhibition of MAP kinases in trigeminal neurons may be therapeutically beneficial for inflammatory disorders involving elevated CGRP levels. PMID:16277606

Role of insulin receptor and insulin signaling on αPS2CβPS integrins' lateral diffusion.

PubMed

Mainali, Dipak; Syed, Aleem; Arora, Neha; Smith, Emily A

2014-12-01

Integrins are ubiquitous transmembrane receptors with adhesion and signaling properties. The influence of insulin receptor and insulin signaling on αPS2CβPS integrins' lateral diffusion was studied using single particle tracking in S2 cells before and after reducing the insulin receptor expression or insulin stimulation. Insulin signaling was monitored by Western blotting for phospho-Akt expression. The expression of the insulin receptor was reduced using RNA interference (RNAi). After insulin receptor RNAi, four significant changes were measured in integrin diffusion properties: (1) there was a 24% increase in the mobile integrin population, (2) 14% of the increase was represented by integrins with Brownian diffusion, (3) for integrins that reside in confined zones of diffusion, there was a 45% increase in the diameter of the confined zone, and (4) there was a 29% increase in the duration integrins spend in confined zones of diffusion. In contrast to reduced expression of the insulin receptor, which alters integrin diffusion properties, insulin stimulation alone or insulin stimulation under conditions of reduced insulin receptor expression have minimal effects on altering the measured integrin diffusion properties. The differences in integrin diffusion measured after insulin receptor RNAi in the presence or absence of insulin stimulation may be the result of other insulin signaling pathways that are activated at reduced insulin receptor conditions. No change in the average integrin diffusion coefficient was measured for any conditions included in this study.

GSK3 Protein Positively Regulates Type I Insulin-like Growth Factor Receptor through Forkhead Transcription Factors FOXO1/3/4

PubMed Central

Huo, Xiaodong; Liu, Shu; Shao, Ting; Hua, Hui; Kong, Qingbin; Wang, Jiao; Luo, Ting; Jiang, Yangfu

2014-01-01

Glycogen synthase kinase-3 (GSK3) has either tumor-suppressive roles or pro-tumor roles in different types of human tumors. A number of GSK3 targets in diverse signaling pathways have been uncovered, such as tuberous sclerosis complex subunit 2 and β-catenin. The O subfamily of forkhead/winged helix transcription factors (FOXO) is known as tumor suppressors that induce apoptosis. In this study, we find that FOXO binds to type I insulin-like growth factor receptor (IGF-IR) promoter and stimulates its transcription. GSK3 positively regulates the transactivation activity of FOXO and stimulates IGF-IR expression. Although kinase-dead GSK3β cannot up-regulate IGF-IR, the constitutively active GSK3β induces IGF-IR expression in a FOXO-dependent manner. Serum starvation or Akt inhibition leads to an increase in IGF-IR expression, which could be blunted by GSK3 inhibition. GSK3β knockdown or GSK3 inhibitor suppresses IGF-I-induced IGF-IR, Akt, and ERK1/2 phosphorylation. Moreover, knockdown of GSK3β or FOXO1/3/4 leads to a decrease in cellular proliferation and abrogates IGF-I-induced hepatoma cell proliferation. These results suggest that GSK3 and FOXO may positively regulate IGF-I signaling and hepatoma cell proliferation. PMID:25053419

Thalidomide suppressed IL-1beta while enhancing TNF-alpha and IL-10, when cells in whole blood were stimulated with lipopolysaccharide.

PubMed

Shannon, Edward; Noveck, Robert; Sandoval, Felipe; Kamath, Burde

2008-01-01

Thalidomide is used to treat erythema nodosum leprosum (ENL). The events that precipitate this inflammatory reaction, which may occur in multibacillary leprosy patients, and the mechanism by which thalidomide arrest ENL, are not known. Thalidomide's ability to inhibit tumor necrosis factor alpha (TNF-alpha) in vitro has been proposed as a partial explanation of its effective treatment of ENL. In in vitro assays, thalidomide can enhance or suppress TNF-alpha. This is dependent on the stimulant used to evoke TNF-alpha; the procedure used to isolate the mononuclear cells from blood, and the predominant mononuclear cell type in the culture. To avoid artifacts that may occur during isolation of mononuclear cells from blood, we stimulated normal human blood with LPS and evaluated the effect of thalidomide and dexamethasone on TNF-alpha, and other inflammatory cytokines and biomarkers. Thalidomide suppressed interleukin 1 beta (IL-1beta) (p = 0.007), and it enhanced TNF-alpha (p = 0.007) and interleukin 10 (IL-10) (p = 0.031). Dexamethasone enhanced IL-10 (p = 0.013) and suppressed IL-1beta, TNF-alpha, interleukin 6 (IL-6), and interleukin 8 (IL-8) (p = 0.013). The two drugs did not suppress: C-reactive protein (CRP), Ig-superfamily cell-adhesion molecule 1 (ICAM 1), tumor necrosis factor receptor 1 (TNFR1), tumor necrosis factor receptor 2 (TNFR2), or amyloid A. In vitro and in vivo evidence is accumulating that TNF-alpha is not the primary cytokine targeted by thalidomide in ENL and other inflammatory conditions.

β-Nicotinamide adenine dinucleotide acts at prejunctional adenosine A1 receptors to suppress inhibitory musculomotor neurotransmission in guinea pig colon and human jejunum

PubMed Central

Wang, Guo-Du; Wang, Xi-Yu; Liu, Sumei; Xia, Yun; Zou, Fei; Qu, Meihua; Needleman, Bradley J.; Mikami, Dean J.

2015-01-01

Intracellular microelectrodes were used to record neurogenic inhibitory junction potentials in the intestinal circular muscle coat. Electrical field stimulation was used to stimulate intramural neurons and evoke contraction of the smooth musculature. Exposure to β-nicotinamide adenine dinucleotide (β-NAD) did not alter smooth muscle membrane potential in guinea pig colon or human jejunum. ATP, ADP, β-NAD, and adenosine, as well as the purinergic P2Y1 receptor antagonists MRS 2179 and MRS 2500 and the adenosine A1 receptor agonist 2-chloro-N6-cyclopentyladenosine, each suppressed inhibitory junction potentials in guinea pig and human preparations. β-NAD suppressed contractile force of twitch-like contractions evoked by electrical field stimulation in guinea pig and human preparations. P2Y1 receptor antagonists did not reverse this action. Stimulation of adenosine A1 receptors with 2-chloro-N6-cyclopentyladenosine suppressed the force of twitch contractions evoked by electrical field stimulation in like manner to the action of β-NAD. Blockade of adenosine A1 receptors with 8-cyclopentyl-1,3-dipropylxanthine suppressed the inhibitory action of β-NAD on the force of electrically evoked contractions. The results do not support an inhibitory neurotransmitter role for β-NAD at intestinal neuromuscular junctions. The data suggest that β-NAD is a ligand for the adenosine A1 receptor subtype expressed by neurons in the enteric nervous system. The influence of β-NAD on intestinal motility emerges from adenosine A1 receptor-mediated suppression of neurotransmitter release at inhibitory neuromuscular junctions. PMID:25813057

Prostaglandin E(2) and insulin-like growth factor I interact to enhance proliferation of theca externa cells from chicken prehierarchical follicles.

PubMed

Jia, Yudong; Lin, Jinxing; Mi, Yuling; Zhang, Caiqiao

2013-10-01

The interactive effect of insulin-like growth factor I (IGF-I) and prostaglandin E2 (PGE2) on the proliferation of theca externa cells (TECs) was investigated in the prehierarchical small yellow follicles of laying hens. IGF-I manifested a proliferating effect like PGE2 on TECs, but this stimulating effect was restrained by AG1024 (IGF-IR inhibitor), KP372-1 (PKB/AKT inhibitor) or NS398 (COX-2 inhibitor). AG1024, KP372-1 or NS398 abolished IGF-I-stimulated COX-2 expression and PGE2 production. Meanwhile, KP372-1, NS398 or AG1024 depressed the PGE2-stimulated expression of COX-2 and IGF-IR mRNA. Therefore, the IGF-I receptor pathway up-regulates COX-2 expression and PGE2 synthesis via PKB signaling cascade, and then PGE2 stimulates IGF-IR mRNA expression to promote TEC proliferation in an autocrine pattern. Overall, the reciprocal stimulation of intracellular PGE2 and IGF-I may enhance TEC proliferation and facilitate the development of chicken prehierarchical follicles. Copyright © 2013 Elsevier Inc. All rights reserved.

Two different factors act separately or together to specify functionally distinct activities at a single transcriptional enhancer.

PubMed Central

DeFranco, D; Yamamoto, K R

1986-01-01

The expression of genes fused downstream of the Moloney murine sarcoma virus (MoMSV) long terminal repeat is stimulated by glucocorticoids. We mapped the glucocorticoid response element that conferred this hormonal regulation and found that it is a hormone-dependent transcriptional enhancer, designated Sg; it resides within DNA fragments that also carry a previously described enhancer element (B. Levinson, G. Khoury, G. Vande Woude, and P. Gruss, Nature [London] 295:568-572, 1982), here termed Sa, whose activity is independent of the hormone. Nuclease footprinting revealed that purified glucocorticoid receptor bound at multiple discrete sites within and at the borders of the tandemly repeated sequence motif that defines Sa. The Sa and Sg activities stimulated the apparent efficiency of cognate or heterologous promoter utilization, individually providing modest enhancement and in concert yielding higher levels of activity. A deletion mutant lacking most of the tandem repeat but retaining a single receptor footprint sequence lost Sa activity but still conferred Sg activity. The two enhancer components could also be distinguished physiologically: both were operative within cultured rat fibroblasts, but only Sg activity was detectable in rat exocrine pancreas cells. Therefore, the sequence determinants of Sa and Sg activity may be interdigitated, and when both components are active, the receptor and a putative Sa factor can apparently bind and act simultaneously. We concluded that MoMSV enhancer activity is effected by at least two distinct binding factors, suggesting that combinatorial regulation of promoter function can be mediated even from a single genetic element. Images PMID:3023887

Occupation of low-affinity cholecystokinin (CCK) receptors by CCK activates signal transduction and stimulates amylase secretion in pancreatic acinar cells.

PubMed

Vinayek, R; Patto, R J; Menozzi, D; Gregory, J; Mrozinski, J E; Jensen, R T; Gardner, J D

1993-03-10

Based on the effects of monensin on binding of 125I-CCK-8 and its lack of effect on CCK-8-stimulated amylase secretion we previously proposed that pancreatic acinar cells possess three classes of CCK receptors: high-affinity receptors, low-affinity receptors and very low-affinity receptors [1]. In the present study we treated pancreatic acini with carbachol to induce a complete loss of high-affinity CCK receptors and then examined the action of CCK-8 on inositol trisphosphate IP3(1,4,5), cytosolic calcium and amylase secretion in an effort to confirm and extend our previous hypothesis. We found that first incubating pancreatic acini with 10 mM carbachol decreased binding of 125I-CCK-8 measured during a second incubation by causing a complete loss of high-affinity CCK receptors with no change in the low-affinity CCK receptors. Carbachol treatment of acini, however, did not alter the action of CCK-8 on IP3(1,4,5), cytosolic calcium or amylase secretion or the action of CCK-JMV-180 on amylase secretion or on the supramaximal inhibition of amylase secretion caused by CCK-8. The present findings support our previous hypothesis that pancreatic acinar cells possess three classes of CCK receptors and suggest that high-affinity CCK receptors do not mediate the action of CCK-8 on enzyme secretion, that low-affinity CCK receptors may mediate the action of CCK on cytosolic calcium that does not involve IP3(1,4,5) and produce the upstroke of the dose-response curve for CCK-8-stimulated amylase secretion and that very low-affinity CCK receptors mediate the actions of CCK on IP3(1,4,5) and cytosolic calcium and produce the downstroke of the dose-response curve for CCK-8-stimulated amylase secretion. Moreover, CCK-JMV-180 is a full agonist for stimulating amylase secretion by acting at low-affinity CCK receptors and is an antagonist at very low-affinity CCK receptors.

Dehydroepiandrosterone Activation of G-protein-coupled Estrogen Receptor Rapidly Stimulates MicroRNA-21 Transcription in Human Hepatocellular Carcinoma Cells*

PubMed Central

Teng, Yun; Radde, Brandie N.; Litchfield, Lacey M.; Ivanova, Margarita M.; Prough, Russell A.; Clark, Barbara J.; Doll, Mark A.; Hein, David W.; Klinge, Carolyn M.

2015-01-01

Little is known about the regulation of the oncomiR miR-21 in liver. Dehydroepiandrosterone (DHEA) regulates gene expression as a ligand for a G-protein-coupled receptor and as a precursor for steroids that activate nuclear receptor signaling. We report that 10 nm DHEA increases primary miR-21 (pri-miR-21) transcription and mature miR-21 expression in HepG2 cells in a biphasic manner with an initial peak at 1 h followed by a second, sustained response from 3–12 h. DHEA also increased miR-21 in primary human hepatocytes and Hep3B cells. siRNA, antibody, and inhibitor studies suggest that the rapid DHEA-mediated increase in miR-21 involves a G-protein-coupled estrogen receptor (GPER/GPR30), estrogen receptor α-36 (ERα36), epidermal growth factor receptor-dependent, pertussis toxin-sensitive pathway requiring activation of c-Src, ERK1/2, and PI3K. GPER antagonist G-15 attenuated DHEA- and BSA-conjugated DHEA-stimulated pri-miR-21 transcription. Like DHEA, GPER agonists G-1 and fulvestrant increased pri-miR-21 in a GPER- and ERα36-dependent manner. DHEA, like G-1, increased GPER and ERα36 mRNA and protein levels. DHEA increased ERK1/2 and c-Src phosphorylation in a GPER-responsive manner. DHEA increased c-Jun, but not c-Fos, protein expression after 2 h. DHEA increased androgen receptor, c-Fos, and c-Jun recruitment to the miR-21 promoter. These results suggest that physiological concentrations of DHEA activate a GPER intracellular signaling cascade that increases pri-miR-21 transcription mediated at least in part by AP-1 and androgen receptor miR-21 promoter interaction. PMID:25969534

Apomorphine, dopamine and phenylethylamine reduce the proportion of phosphorylated insulin receptor substrate 1.

PubMed

Chiarenza, A; Scarselli, M; Novi, F; Lempereur, L; Bernardini, R; Corsini, G U; Maggio, R

2001-12-14

We tested the ability of dopamine, apomorphine, phenylethylamine and pergolide to inhibit the proliferation of fetal calf serum-stimulated human breast cancer (MCF)-7 cells. While the first three compounds were able to block the proliferation of MCF-7 cells, pergolide failed to do so (up to 100 microM). The inhibitory effect of dopamine, apomorphine and phenylethylamine was also evident in serum-starved insulin-stimulated MCF-7 cells. Apomorphine also inhibited the proliferation of the human oestrogen receptor-negative breast cancer (MDA-MB231) and prostate carcinoma (LNCaP) cell lines. In a second set of experiments, we measured the ability of dopamine, apomorphine, phenylethylamine and pergolide to inhibit the phosphorylation (or increase the dephosphorylation) of the insulin receptor substrate (IRS)-1, a major intracellular substrate of the insulin-like growth factor (IGF)-1 receptor. Dopamine, apomorphine and phenylethylamine all reduced to zero the level of phosphorylated IRS-1 with potencies ranging between 0.01 and 1 microM. Finally, we found that fibroblasts from IRS-1 null (-/-) mice were less sensitive to the anti-proliferative effect of apomorphine compared to fibroblasts from wild type-mice, suggesting that the inhibition of IRS-1 phosphorylation by apomorphine is an important aspect of the activity of this compound.

Effect of naringin on gp120-induced injury mediated by P2X7 receptors in rat primary cultured microglia

PubMed Central

Liu, Chenglong; Deng, Zeyu; Liu, Yang; Chen, Guoqiao; Liu, Baoyun

2017-01-01

Human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein 120 has been shown to activate microglia, causing release of inflammatory and toxic factors. The P2X7 receptor, primarily expressed on microglia, is closely associated with inflammation. Naringin, a plant bioflavonoid, has anti-inflammatory and anti-oxidative properties. We hypothesized that P2X7 receptor mediated gp120-induced injury in primary cultured microglia, and that naringin would have a protective effect. We showed that HIV-1 gp120 peptide (V3 loop, fragment 308–331) appeared to induce apoptosis of primary cultured microglia. However, there was a decrease of microglia apoptosis in gp120+naringin group compared with gp120 group. Using qPCR, Western blot, and immunofluorescence, we showed that gp120 stimulated expression of P2X7 mRNA and receptor protein, and this stimulation was inhibited by naringin. Treatment with gp120 increased concentrations of eATP, TNFα and IL-1β, and these effects were inhibited by naringin. Taken together, these results suggested that gp120 contributed to microglial cell injury and neurotoxic activity by up-regulating expression of P2X7, in a naringin-protective manner. PMID:28832643

Effect of naringin on gp120-induced injury mediated by P2X7 receptors in rat primary cultured microglia.

PubMed

Chen, Qiang; Wu, Hui; Tao, Jia; Liu, Chenglong; Deng, Zeyu; Liu, Yang; Chen, Guoqiao; Liu, Baoyun; Xu, Changshui

2017-01-01

Human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein 120 has been shown to activate microglia, causing release of inflammatory and toxic factors. The P2X7 receptor, primarily expressed on microglia, is closely associated with inflammation. Naringin, a plant bioflavonoid, has anti-inflammatory and anti-oxidative properties. We hypothesized that P2X7 receptor mediated gp120-induced injury in primary cultured microglia, and that naringin would have a protective effect. We showed that HIV-1 gp120 peptide (V3 loop, fragment 308-331) appeared to induce apoptosis of primary cultured microglia. However, there was a decrease of microglia apoptosis in gp120+naringin group compared with gp120 group. Using qPCR, Western blot, and immunofluorescence, we showed that gp120 stimulated expression of P2X7 mRNA and receptor protein, and this stimulation was inhibited by naringin. Treatment with gp120 increased concentrations of eATP, TNFα and IL-1β, and these effects were inhibited by naringin. Taken together, these results suggested that gp120 contributed to microglial cell injury and neurotoxic activity by up-regulating expression of P2X7, in a naringin-protective manner.

A multi-parametric imaging investigation of the response of C6 glioma xenografts to MLN0518 (tandutinib) treatment.

PubMed

Boult, Jessica K R; Terkelsen, Jennifer; Walker-Samuel, Simon; Bradley, Daniel P; Robinson, Simon P

2013-01-01

Angiogenesis, the development of new blood vessels, is essential for tumour growth; this process is stimulated by the secretion of numerous growth factors including platelet derived growth factor (PDGF). PDGF signalling, through its receptor platelet derived growth factor receptor (PDGFR), is involved in vessel maturation, stimulation of angiogenesis and upregulation of other angiogenic factors, including vascular endothelial growth factor (VEGF). PDGFR is a promising target for anti-cancer therapy because it is expressed on both tumour cells and stromal cells associated with the vasculature. MLN0518 (tandutinib) is a potent inhibitor of type III receptor tyrosine kinases that demonstrates activity against PDGFRα/β, FLT3 and c-KIT. In this study a multi-parametric MRI and histopathological approach was used to interrogate changes in vascular haemodynamics, structural response and hypoxia in C6 glioma xenografts in response to treatment with MLN0518. The doubling time of tumours in mice treated with MLN0518 was significantly longer than tumours in vehicle treated mice. The perfused vessel area, number of alpha smooth muscle actin positive vessels and hypoxic area in MLN0518 treated tumours were also significantly lower after 10 days treatment. These changes were not accompanied by alterations in vessel calibre or fractional blood volume as assessed using susceptibility contrast MRI. Histological assessment of vessel size and total perfused area did not demonstrate any change with treatment. Intrinsic susceptibility MRI did not reveal any difference in baseline R2* or carbogen-induced change in R2*. Dynamic contrast-enhanced MRI revealed anti-vascular effects of MLN0518 following 3 days treatment. Hypoxia confers chemo- and radio-resistance, and alongside PDGF, is implicated in evasive resistance to agents targeted against VEGF signalling. PDGFR antagonists may improve potency and efficacy of other therapeutics in combination. This study highlights the challenges of identifying appropriate quantitative imaging response biomarkers in heterogeneous models, particularly considering the multifaceted roles of angiogenic growth factors.

Cholecystokinin receptor antagonism by peptidergic and non-peptidergic agents in rat pancreas.

PubMed Central

Dembinski, A; Jaworek, J; Konturek, P K; Konturek, S J; Warzecha, Z

1989-01-01

1. Graded doses of bombesin infused I.V. into conscious rats with chronic pancreatic fistulae induced a dose-dependent stimulation of protein secretion, similar to that obtained with caerulein. This stimulation does not appear to be mediated by cholecystokinin (CCK) receptors because peptidergic (CR-1409) and non-peptidergic (L-364718) CCK antagonists failed to affect protein secretion at a dose range which caused almost complete suppression of caerulein-induced pancreatic secretion. 2. Studies in vitro on isolated rat pancreatic acini revealed that caerulein, pentagastrin and bombesin all showed the same efficacy in their ability to stimulate amylase release. In contrast, CCK antagonists competitively inhibited amylase release induced by caerulein and pentagastrin but not by bombesin or urecholine, indicating that the latter two agents act directly on acinar cells via receptors which are separate from those involved in stimulation induced by caerulein and pentagastrin. 3. DNA synthesis, measured by the incorporation of [3H]thymidine into DNA, was significantly stimulated by caerulein, soybean trypsin inhibitor (FOY 305), pentagastrin and by bombesin in a dose-dependent manner. CCK receptor antagonists prevented stimulation of DNA synthesis induced by caerulein, FOY 305 and pentagastrin but not by bombesin. 4. This study indicates that bombesin strongly stimulates pancreatic enzyme secretion, with an efficacy similar to that of caerulein, and also exerts a potent growth-promoting action on the pancreas, both effects appearing to be mediated by mechanisms independent of the CCK receptors. PMID:2614728

Hippocampal low-frequency stimulation inhibits afterdischarge and increases GABA (A) receptor expression in amygdala-kindled pharmacoresistant epileptic rats.

PubMed

Wu, Guofeng; Wang, Likun; Hong, Zhen; Ren, Siying; Zhou, Feng

2017-08-01

The purpose of the present study was to observe the effects of hippocampal low-frequency stimulation (Hip-LFS) on amygdala afterdischarge and GABA (A) receptor expression in pharmacoresistant epileptic (PRE) rats. A total of 110 healthy adult male Wistar rats were used to generate a model of epilepsy by chronic stimulation of the amygdala. Sixteen PRE rats were selected from 70 amygdala-kindled rats by testing their response to Phenytoin and Phenobarbital, and they were randomly assigned to a pharmacoresistant stimulation group (PRS group, 8 rats) or a pharmacoresistant control group (PRC group, 8 rats). A stimulation electrode was implanted into the hippocampus of all of the rats. Hip-LFS was administered twice per day in the PRS group for two weeks. Simultaneously, amygdala stimulus-induced seizures and afterdischarge were recorded. After the hippocampal stimulation was terminated, the brain tissues were obtained to determine the GABA (A) receptors by a method of immumohistochemistry and a real-time polymerase chain reaction. The stages and duration of the amygdala stimulus-induced epileptic seizures were decreased in the PRS group. The afterdischarge threshold was increased and the duration as well as the afterdischarge frequency was decreased. Simultaneously, the GABA (A) expression was significantly increased in the PRS group. Hip-LFS may inhibit amygdala stimulus-induced epileptic seizures and up-regulate GABA (A) receptor expression in PRE rats. The antiepileptic effects of hippocampal stimulation may be partly achieved by increasing the GABA (A) receptor.

Distinct Roles of Opioid and Dopamine Systems in Lateral Hypothalamic Intracranial Self-Stimulation.

PubMed

Ide, Soichiro; Takahashi, Takehiro; Takamatsu, Yukio; Uhl, George R; Niki, Hiroaki; Sora, Ichiro; Ikeda, Kazutaka

2017-05-01

Opioid and dopamine systems play crucial roles in reward. Similarities and differences in the neural mechanisms of reward that are mediated by these 2 systems have remained largely unknown. Thus, in the present study, we investigated the differences in reward function in both µ-opioid receptor knockout mice and dopamine transporter knockout mice, important molecules in the opioid and dopamine systems. Mice were implanted with electrodes into the right lateral hypothalamus (l hour). Mice were then trained to put their muzzle into the hole in the head-dipping chamber for intracranial electrical stimulation, and the influences of gene knockout were assessed. Significant differences are observed between opioid and dopamine systems in reward function. µ-Opioid receptor knockout mice exhibited enhanced intracranial electrical stimulation, which induced dopamine release. They also exhibited greater motility under conditions of "despair" in both the tail suspension test and water wheel test. In contrast, dopamine transporter knockout mice maintained intracranial electrical stimulation responding even when more active efforts were required to obtain the reward. The absence of µ-opioid receptor or dopamine transporter did not lead to the absence of intracranial electrical stimulation responsiveness but rather differentially altered it. The present results in µ-opioid receptor knockout mice are consistent with the suppressive involvement of µ-opioid receptors in both positive incentive motivation associated with intracranial electrical stimulation and negative incentive motivation associated with depressive states. In contrast, the results in dopamine transporter knockout mice are consistent with the involvement of dopamine transporters in positive incentive motivation, especially its persistence. Differences in intracranial electrical stimulation in µ-opioid receptor and dopamine transporter knockout mice underscore the multidimensional nature of reward. © The Author 2016. Published by Oxford University Press on behalf of CINP.

Functional subcellular distribution of β1- and β2-adrenergic receptors in rat ventricular cardiac myocytes

PubMed Central

Cros, Caroline; Brette, Fabien

2013-01-01

β-adrenergic stimulation is a key regulator of cardiac function. The localization of major cardiac adrenergic receptors (β1 and β2) has been investigated using biochemical and biophysical approaches and has led to contradictory results. This study investigates the functional subcellular localization of β1- and β2-adrenergic receptors in rat ventricular myocytes using a physiological approach. Ventricular myocytes were isolated from the hearts of rat and detubulated using formamide. Physiological cardiac function was measured as Ca2+ transient using Fura-2-AM and cell shortening. Selective activation of β1- and β2-adrenergic receptors was induced with isoproterenol (0.1 μmol/L) and ICI-118,551 (0.1 μmol/L); and with salbutamol (10 μmol/L) and atenolol (1 μmol/L), respectively. β1- and β2-adrenergic stimulations induced a significant increase in Ca2+ transient amplitude and cell shortening in intact rat ventricular myocytes (i.e., surface sarcolemma and t-tubules) and in detubulated cells (depleted from t-tubules, surface sarcolemma only). Both β1- and β2-adrenergic receptors stimulation caused a greater effect on Ca2+ transient and cell shortening in detubulated myocytes than in control myocytes. Quantitative analysis indicates that β1-adrenergic stimulation is ∼3 times more effective at surface sarcolemma compared to t-tubules, whereas β2- adrenergic stimulation occurs almost exclusively at surface sarcolemma (∼100 times more effective). These physiological data demonstrate that in rat ventricular myocytes, β1-adrenergic receptors are functionally present at surface sarcolemma and t-tubules, while β2-adrenergic receptors stimulation occurs only at surface sarcolemma of cardiac cells. PMID:24303124

Overexpression of cellular repressor of E1A-stimulated genes inhibits TNF-{alpha}-induced apoptosis via NF-{kappa}B in mesenchymal stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Peng, Cheng-Fei; Cardiovascular Research Institute and Department of Cardiology, Shenyang Northern Hospital, Shenyang; Han, Ya-Ling, E-mail: hanyaling53@gmail.com

2011-03-25

Research highlights: {yields} CREG protected MSCs from tumor necrosis factor-{alpha} (TNF-{alpha}) induced apoptosis. {yields} CREG inhibits the phosphorylation of I{kappa}B{alpha} and prevents the activation of NF-{kappa}B. {yields} CREG inhibits NF-{kappa}B nuclear translocation and pro-apoptosis protein transcription. {yields} CREG anti-apoptotic effect involves inhibition of the death receptor pathway. {yields} p53 is downregulated by CREG via NF-{kappa}B pathway under TNF-{alpha} stimulation. -- Abstract: Bone marrow-derived mesenchymal stem cells (MSCs) show great potential for therapeutic repair after myocardial infarction. However, poor viability of transplanted MSCs in the ischemic heart has limited their use. Cellular repressor of E1A-stimulated genes (CREG) has been identified asmore » a potent inhibitor of apoptosis. This study therefore aimed to determine if rat bone marrow MSCs transfected with CREG-were able to effectively resist apoptosis induced by inflammatory mediators, and to demonstrate the mechanism of CREG action. Apoptosis was determined by flow cytometric and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assays. The pathways mediating these apoptotic effects were investigated by Western blotting. Overexpression of CREG markedly protected MSCs from tumor necrosis factor-{alpha} (TNF-{alpha}) induced apoptosis by 50% after 10 h, through inhibition of the death-receptor-mediated apoptotic pathway, leading to attenuation of caspase-8 and caspase-3. Moreover, CREG resisted the serine phosphorylation of I{kappa}B{alpha} and prevented the nuclear translocation of the transcription factor nuclear factor-{kappa}B (NF-{kappa}B) under TNF-{alpha} stimulation. Treatment of cells with the NF-{kappa}B inhibitor pyrrolidine dithiocarbamate (PDTC) significantly increased the transcription of pro-apoptosis proteins (p53 and Fas) by NF-{kappa}B, and attenuated the anti-apoptotic effects of CREG on MSCs. The results of this study indicate that CREG acts as a novel and potent survival factor in MSCs, and may therefore be a useful therapeutic adjunct for transplanting MSCs into the damaged heart after myocardial infarction.« less

Once-Monthly Continuous Erythropoietin Receptor Activator (C.E.R.A.) in Patients with Hemodialysis-Dependent Chronic Kidney Disease: Pooled Data from Phase III Trials.

PubMed

Locatelli, Francesco; Choukroun, Gabriel; Truman, Matt; Wiggenhauser, Alfons; Fliser, Danilo

2016-04-01

Erythropoiesis-stimulating agents and iron are commonly used in patients with chronic kidney disease with the aim of correcting anemia and maintaining stable hemoglobin levels. We analyzed pooled data from 13 studies with similar designs included in the Umbrella Continuous Erythropoietin Receptor Activator (C.E.R.A.) program to investigate the effects of continuous erythropoiesis receptor activator in clinically relevant subgroups of patients with chronic kidney disease and to determine whether the efficacy and safety outcomes demonstrated in the overall chronic kidney disease population are maintained in specific subgroups. Data from 13 Phase III trials set up with similar design were retrospectively pooled for this analysis. Patients with chronic kidney disease who had previously been receiving epoetin or darbepoetin were switched to continuous erythropoiesis receptor activator once-monthly after a 4- to 8-week screening period. Patients entered a 16-week continuous erythropoiesis receptor activator dose-titration period followed by an 8-week evaluation period. In total, 2060 patients were included in the analysis. Subgroups were defined based on: hemoglobin target range [lower (10.0-12.0 g/dL)/upper (10.5-13.0 g/dL)], gender (female/male), age (<65/≥65), baseline N-terminal pro-B-type natriuretic peptide levels (<5000/≥5000), cardiovascular risk factors (diabetes/cardiac/vascular/none). Across all subgroups analyzed, switching from shorter-acting erythropoiesis-stimulating agents to continuous erythropoiesis receptor activator once-monthly maintained stable hemoglobin concentrations in a high proportion of patients (78%), with only moderate hemoglobin fluctuations and a low number of dose changes. The safety profile across subgroups was as expected based on pre-existing risk factors; observed increases in adverse events were attributable to underlying risk factors rather than study drug. This retrospective analysis of 13 trials showed that continuous erythropoiesis receptor activator once-monthly maintained stable hemoglobin levels across a number of clinically relevant patient subgroups, including those with higher inherent cardiovascular risk. The safety profile was consistent with that previously established in the chronic kidney disease population. CLINICALTRIALS. NCT00413894/NCT00545571/NCT00517413/NCT00560404/NCT00882713/NCT00550680/NCT00576303/NCT00660023/NCT00717821/NCT00642850/NCT00605293/NCT00661505/NCT00699348. F. Hoffmann-La Roche Ltd, Basel, Switzerland.

Calcitonin gene-related peptide stimulates proliferation of human endothelial cells.

PubMed Central

Haegerstrand, A; Dalsgaard, C J; Jonzon, B; Larsson, O; Nilsson, J

1990-01-01

The effects of the vasoactive perivascular neuropeptides calcitonin gene-related peptide (CGRP), neurokinin A (NKA), neuropeptide Y (NPY), and vasoactive intestinal polypeptide (VIP) on proliferation of cultured human umbilical vein endothelial cells (HUVECs) were investigated. CGRP was shown to increase both cell number and DNA synthesis, whereas NKA, NPY, and VIP were ineffective. 125I-labeled CGRP was shown to bind to HUVECs and this binding was displaced by addition of unlabeled CGRP, suggesting the existence of specific CGRP receptors. The effect of CGRP on formation of adenosine 3',5'-cyclic monophosphate (cAMP) and inositol phosphates (InsP), two intracellular messengers known to be involved in regulation of cell proliferation, was investigated. CGRP stimulated cAMP formation but was without effect on the formation of InsP. Proliferation, as well as cAMP formation, was also stimulated by cholera toxin. Basic fibroblast growth factor stimulated growth without affecting cAMP or InsP formation, whereas thrombin, which increased InsP formation, did not stimulate proliferation. We thus suggest that CGRP may act as a local factor stimulating proliferation of endothelial cells; that the mechanism of action is associated with cAMP formation; and that this effect of CGRP may be important for formation of new vessels during physiological and pathophysiological events such as ischemia, inflammation, and wound healing. PMID:2159144

Calcitonin gene-related peptide stimulates proliferation of human endothelial cells.

PubMed

Haegerstrand, A; Dalsgaard, C J; Jonzon, B; Larsson, O; Nilsson, J

1990-05-01

The effects of the vasoactive perivascular neuropeptides calcitonin gene-related peptide (CGRP), neurokinin A (NKA), neuropeptide Y (NPY), and vasoactive intestinal polypeptide (VIP) on proliferation of cultured human umbilical vein endothelial cells (HUVECs) were investigated. CGRP was shown to increase both cell number and DNA synthesis, whereas NKA, NPY, and VIP were ineffective. 125I-labeled CGRP was shown to bind to HUVECs and this binding was displaced by addition of unlabeled CGRP, suggesting the existence of specific CGRP receptors. The effect of CGRP on formation of adenosine 3',5'-cyclic monophosphate (cAMP) and inositol phosphates (InsP), two intracellular messengers known to be involved in regulation of cell proliferation, was investigated. CGRP stimulated cAMP formation but was without effect on the formation of InsP. Proliferation, as well as cAMP formation, was also stimulated by cholera toxin. Basic fibroblast growth factor stimulated growth without affecting cAMP or InsP formation, whereas thrombin, which increased InsP formation, did not stimulate proliferation. We thus suggest that CGRP may act as a local factor stimulating proliferation of endothelial cells; that the mechanism of action is associated with cAMP formation; and that this effect of CGRP may be important for formation of new vessels during physiological and pathophysiological events such as ischemia, inflammation, and wound healing.

Coordinate regulation of estrogen-mediated fibronectin matrix assembly and epidermal growth factor receptor transactivation by the G protein-coupled receptor, GPR30.

PubMed

Quinn, Jeffrey A; Graeber, C Thomas; Frackelton, A Raymond; Kim, Minsoo; Schwarzbauer, Jean E; Filardo, Edward J

2009-07-01

Estrogen promotes changes in cytoskeletal architecture not easily attributed to the biological action of estrogen receptors, ERalpha and ERbeta. The Gs protein-coupled transmembrane receptor, GPR30, is linked to specific estrogen binding and rapid estrogen-mediated release of heparin-bound epidermal growth factor. Using marker rescue and dominant interfering mutant strategies, we show that estrogen action via GPR30 promotes fibronectin (FN) matrix assembly by human breast cancer cells. Stimulation with 17beta-estradiol or the ER antagonist, ICI 182, 780, results in the recruitment of FN-engaged integrin alpha5beta1 conformers to fibrillar adhesions and the synthesis of FN fibrils. Concurrent with this cellular response, GPR30 promotes the formation of Src-dependent, Shc-integrin alpha5beta1 complexes. Function-blocking antibodies directed against integrin alpha5beta1 or soluble Arg-Gly-Asp peptide fragments derived from FN specifically inhibited GPR30-mediated epidermal growth factor receptor transactivation. Estrogen-mediated FN matrix assembly and epidermal growth factor receptor transactivation were similarly disrupted in integrin beta1-deficient GE11 cells, whereas reintroduction of integrin beta1 into GE11 cells restored these responses. Mutant Shc (317Y/F) blocked GPR30-induced FN matrix assembly and tyrosyl phosphorylation of erbB1. Interestingly, relative to recombinant wild-type Shc, 317Y/F Shc was more readily retained in GPR30-induced integrin alpha5beta1 complexes, yet this mutant did not prevent endogenous Shc-integrin alpha5beta1 complex formation. Our results suggest that GPR30 coordinates estrogen-mediated FN matrix assembly and growth factor release in human breast cancer cells via a Shc-dependent signaling mechanism that activates integrin alpha5beta1.

Muscle and the physiology of locomotion. [in zero gravity

NASA Technical Reports Server (NTRS)

Rambaut, P. C.; Nicogossian, A. E.; Pool, S. L.

1983-01-01

NASA's past, current, and planned research on muscle deterioration at zero gravity and development of countermeasures are reviewed; Soviet studies are discussed as well. A definition of muscle mass and strength regulation factors, and improved measurement methods of muscle atrophy are needed. Investigations of tissue growth factors and their receptors, endogenous and exogenous anabolic protein synthesis stimulation, and a potential neurotropic factor are among the projects in progress or planned. At present, vigorous physical exercise during spaceflight is recommended as the most effective countermeasure against skeletal muscle atrophy.

Paracrine control of vascularization and neurogenesis by neurotrophins.

PubMed

Emanueli, Costanza; Schratzberger, Peter; Kirchmair, Rudolf; Madeddu, Paolo

2003-10-01

The neuronal system plays a fundamental role in the maturation of primitive embryonic vascular network by providing a paracrine template for blood vessel branching and arterial differentiation. Furthermore, postnatal vascular and neural regeneration cooperate in the healing of damaged tissue. Neurogenesis continues in adulthood although confined to specific brain regions. Following ischaemic insult, neural staminal cells contribute towards the healing process through the stimulation of neurogenesis and vasculogenesis. Evidence indicates that nerves and blood vessels exert a reciprocal control of their own growth by paracrine mechanisms. For instance, guidance factors, including vascular endothelial growth factor A (VEGF-A) and semaphorins, which share the ability of binding neuropilin receptors, play a pivotal role in the tridimensional growth pattern of arterial vessels and nerves. Animal models and clinical studies have demonstrated a role of VEGF-A in the pathogenesis of ischaemic and diabetic neuropathies. Further, supplementation with VEGF-A ameliorates neuronal recovery by exerting protective effects on nerves and stimulating reparative neovascularization. Human tissue kallikrein, a recently discovered angiogenic and arteriogenic factor, accelerates neuronal recovery by stimulating the growth of vasa nervorum. Conversely, the neurotrophin nerve growth factor, known to regulate neuronal survival and differentiation, is now regarded as a stimulator of angiogenesis and arteriogenesis. These results indicate that angiogenesis and neurogenesis are paracrinally regulated by growth factors released by endothelial cells and neurons. Supplementation of these growth factors, alone or in combination, could benefit the treatment of ischaemic diseases and neuropathies.

Soluble and insoluble immune complexes activate human neutrophil NADPH oxidase by distinct Fc gamma receptor-specific mechanisms.

PubMed

Crockett-Torabi, E; Fantone, J C

1990-11-01

Signal transduction initiated by interaction of immune complexes (IC) with Fc gamma RII and Fc gamma RIII receptors on human neutrophils was studied by investigating the capacity of well-defined complexes to stimulate O2- generation in neutrophils. IC consisting of polyclonal rabbit antibody to human albumin were prepared at equivalence (insoluble complexes) and at five times Ag excess (soluble complexes). Stimulation of human neutrophils with soluble and insoluble IC caused a dose-dependent activation of the respiratory burst and O2- generation. Incubation of neutrophils with cytochalasin B significantly enhanced O2- generation in neutrophils stimulated with soluble IC. In contrast, cytochalasin B treatment had a minimal effect on O2- generation in neutrophils stimulated with insoluble IC. Treatment of neutrophils with PGE1 or pertussis toxin (PTx) significantly inhibited O2- generation by soluble IC-stimulated neutrophils. However, neither PGE1 nor PTx treatment significantly altered O2- generation in neutrophils stimulated with insoluble complexes. Although O2- generation induced by soluble IC was significantly inhibited by mAb against both Fc gamma RII and Fc gamma RIII receptor, insoluble IC stimulation of neutrophil O2- generation was significantly diminished only by mAb against Fc gamma RIII receptor. Cross-linking of either Fc gamma RII or Fc gamma RIII receptors on neutrophil surfaces induced O2- generation, and this activation was inhibited by both PGE1 and PTx treatment. These findings indicate that soluble and insoluble ICs induce O2- production in human neutrophils through distinct mechanisms. Soluble IC induce activation of neutrophils through a PTx- and PGE1-sensitive pathway that is dependent upon both Fc gamma RII and Fc gamma RIII receptors. Although insoluble IC induce O2- production through a PTx and PGE1 insensitive pathway mediated primarily through Fc gamma RIII receptor.

Retrograde transport of the transmembrane estrogen receptor, G-protein-coupled-receptor-30 (GPR30/GPER) from the plasma membrane towards the nucleus.

PubMed

Cheng, Shi-Bin; Graeber, Carl T; Quinn, Jeffrey A; Filardo, Edward J

2011-08-01

G-protein-coupled receptor 30 (GPR30/GPER) belongs to the seven transmembrane receptor (7TMR) superfamily, the most common class of surface receptor with approximately 800 known members. GPER promotes estrogen binding and rapid signaling via membrane-associated enzymes resulting in increased cAMP and release of heparan bound epidermal growth factor (proHB-EGF) from breast cancer cells. However, GPER is predominately localized intracellularly in breast cancer cells with minor amounts of receptor on the cell surface, an observation that has caused some controversy regarding its potential role as a plasma membrane estrogen receptor. Using the widely employed approach of tracking recombinant 7TMRs by surface labeling live cells, we have begun to characterize and compare the endocytic fate of GPER to other similarly labeled 7TMRs. Upon ectopic expression in human embryonic kidney HEK-293 cells, functional GPER is generated as these cells acquire the capacity to stimulate cAMP and activate cyclic AMP responsive binding protein in response to estradiol-17 beta stimulation. GPER is detectable on the cell surface by immunofluorescent analysis using HA-specific antibodies, albeit the bulk of the receptor is located intracellularly. Like β1AR (beta 1 adrenergic receptor) and CXCR4 (C-X-C chemokine receptor 4), GPER exits the plasma membrane via clathrin-coated pits and enters early endosomes. Interestingly, GPER has a destination that is uncommon among 7TMRs, as it accumulates in a perinuclear compartment. Like many 7TMRs (approximately one-third), GPER trafficking from the plasma membrane is constitutive (occurs in the absence of agonist). However, its route of intracellular trafficking is highly unusual, as 7TMRs typically recycle to the plasma membrane (e.g. β1AR) or are degraded in lysosomes (e.g. CXCR4). The accumulation of GPER in the perinuclear space and its possible significance for attenuating estrogen action via this newly recognized membrane estrogen receptor is discussed herein. Published by Elsevier Inc.

Grave's disease 1835-2002.

PubMed

Weetman, A P

2003-01-01

This brief review describes the history of Graves' disease, starting with the original descriptions by Parry, Graves and von Basedow. The true aetiology of the disorder was uncovered in the 1950s and 1960s, based on the search for a novel thyroid stimulator which turned out to be an immunoglobulin G autoantibody. Assays for these thyroid stimulatory antibodies have been continually refined and their epitopes on the thyroid stimulating hormone receptor are increasingly well characterized. We also understand far more about the genetic and environmental susceptibility factors that predispose to disease, and even thyroid-associated ophthalmopathy has now been better defined as primarily a T-cell-mediated disease resulting from cytokine stimulation of orbital fibroblasts. These advances should improve treatment options for Graves' disease in the foreseeable future.

Corn silk induced cyclooxygenase-2 in murine macrophages.

PubMed

Kim, Kyung A; Shin, Hyun-Hee; Choi, Sang Kyu; Choi, Hye-Seon

2005-10-01

Stimulation of murine macrophages with corn silk induced cyclooxygenase (COX)-2 with secretion of PGE2. Expression of COX-2 was inhibited by pyrolidine dithiocarbamate (PDTC), and increased DNA binding by nuclear factor kappa B (NF-kappaB), indicating that COX-2 induction proceeds also via the NF-kappaB signaling pathway. A specific inhibitor of COX-2 decreased the expression level of inducible nitric oxide synthase (iNOS) stimulated by corn silk. PGE2 elevated the expression level of iNOS, probably via EP2 and EP4 receptors on the surface of the macrophages.

The insulin and IGF1 receptor kinase domains are functional dimers in the activated state

NASA Astrophysics Data System (ADS)

Cabail, M. Zulema; Li, Shiqing; Lemmon, Eric; Bowen, Mark E.; Hubbard, Stevan R.; Miller, W. Todd

2015-03-01

The insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R) are highly related receptor tyrosine kinases with a disulfide-linked homodimeric architecture. Ligand binding to the receptor ectodomain triggers tyrosine autophosphorylation of the cytoplasmic domains, which stimulates catalytic activity and creates recruitment sites for downstream signalling proteins. Whether the two phosphorylated tyrosine kinase domains within the receptor dimer function independently or cooperatively to phosphorylate protein substrates is not known. Here we provide crystallographic, biophysical and biochemical evidence demonstrating that the phosphorylated kinase domains of IR and IGF1R form a specific dimeric arrangement involving an exchange of the juxtamembrane region proximal to the kinase domain. In this dimer, the active position of α-helix C in the kinase N lobe is stabilized, which promotes downstream substrate phosphorylation. These studies afford a novel strategy for the design of small-molecule IR agonists as potential therapeutic agents for type 2 diabetes.

Cigarette toxin 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induces experimental pancreatitis through α7 nicotinic acetylcholine receptors (nAChRs) in mice

PubMed Central

Alahmari, A. A.; Sreekumar, B.; Patel, V.; Ashat, M.; Alexandre, M.; Uduman, A. K.; Akinbiyi, E. O.; Ceplenski, A.; Shugrue, C. A.; Kolodecik, T. R.; Messenger, S. W.; Groblewski, G. E.; Gorelick, F. S.

2018-01-01

Clinical studies have shown that cigarette smoking is a dose-dependent and independent risk factor for acute pancreatitis. Cigarette smoke contains nicotine which can be converted to the potent receptor ligand and toxin, NNK [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone]. Previously, we have shown that NNK induces premature activation of pancreatic zymogens in rats, an initiating event in pancreatitis, and this activation is prevented by pharmacologic inhibition of nicotinic acetylcholine receptors (nAChR). In this study, we determined whether NNK mediates pancreatitis through the α7 isoform of nAChR using α7nAChR knockout mice. PCR analysis confirmed expression of non-neuronal α7nAChR in C57BL/6 (WT) mouse and human acinar cells. NNK treatment stimulated trypsinogen activation in acini from WT but not α7nAChR-/- mice. NNK also stimulated trypsinogen activation in human acini. To further confirm these findings, WT and α7nAChR-/- mice were treated with NNK in vivo and markers of pancreatitis were measured. As observed in acini NNK treatment induced trypsinogen activation in WT but not α7nAChR-/- mice. NNK also induced other markers of pancreatitis including pancreatic edema, vacuolization and pyknotic nuclei in WT but not α7nAChR-/- animals. NNK treatment led to increased neutrophil infiltration, a marker of inflammation, in WT mice and to a significantly lesser extent in α7nAChR-/- mice. We also examined downstream targets of α7nAChR activation and found that calcium and PKC activation are involved down stream of NNK stimulation of α7nAChR. In this study we used genetic deletion of the α7nAChR to confirm our previous inhibitor studies that demonstrated NNK stimulates pancreatitis by activating this receptor. Lastly, we demonstrate that NNK can also stimulate zymogen activation in human acinar cells and thus may play a role in human disease. PMID:29870540

Electrical Stimulation Decreases Coupling Efficiency Between Beta-Adrenergic Receptors and Cyclic AMP Production in Cultured Muscle Cells

NASA Technical Reports Server (NTRS)

Young, R. B.; Bridge, K. Y.

1999-01-01

Electrical stimulation of skeletal muscle cells in culture is an effective way to simulate the effects of muscle contraction and its effects on gene expression in muscle cells. Expression of the beta-adrenergic receptor and its coupling to cyclic AMP synthesis are important components of the signaling system that controls muscle atrophy and hypertrophy, and the goal of this project was to determine if electrical stimulation altered the beta-adrenergic response in muscle cells. Chicken skeletal muscle cells that had been grown for seven days in culture were subjected to electrical stimulation for an additional two days at a pulse frequency of 0.5 pulses/sec and a pulse duration of 200 msec. At the end of this two-day stimulation period, beta-adrenergic receptor population was measured by the binding of tritium-labeled CGP-12177 to muscle cells, and coupling to cAMP synthesis was measured by Radioimmunoassay (RIA) after treating the cells for 10 min with the potent (beta)AR agonist, isoproterenol. The number of beta adrenergic receptors and the basal levels of intracellular cyclic AMP were not affected by electrical stimulation. However, the ability of these cells to synthesize cyclic AMP was reduced by approximately 50%. Thus, an enhanced level of contraction reduces the coupling efficiency of beta-adrenergic receptors for cyclic AMP production.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jope, R.S.; Casebolt, T.L.; Johnson, G.V.

Cortical slices from rat brain were used to study carbachol-stimulated inositol phospholipid hydrolysis. Omission of calcium during incubation of slices with (/sup 3/H)inositol increased its incorporation into receptor-coupled phospholipids. Carbachol-stimulated hydrolysis of (/sup 3/H)inositol phospholipids in slices was dose-dependent, was affected by the concentrations of calcium and lithium present and resulted in the accumulation of mostly (/sup 3/H)inositol-1-phosphate. Incubation of slices with N-ethylmaleimide or a phorbol ester reduced the response to carbachol. Membranes prepared from cortical slices labeled with (/sup 3/H)inositol retained the receptor-stimulated inositol phospholipid hydrolysis reaction. The basal rate of inositol phospholipid hydrolysis was higher than in slicesmore » and addition of carbachol further stimulated the process. Addition of GTP stimulated inositol phospholipid hydrolysis, suggesting the presence of a guanine nucleotide-binding protein coupled to phospholipase C. Carbachol and GTP-stimulated inositol phospholipid hydrolysis in membranes was detectable following a 3 min assay period. In contrast to slices, increased levels of inositol bisphosphate and inositol trisphosphate were detected following incubation of membranes with carbachol. These results demonstrate that agonist-responsive receptors are present in cortical membranes, that the receptors may be coupled to phosphatidylinositol 4, 5-bisphosphate, rather than phosphatidylinositol, hydrolysis and that a guanine nucleotide-binding protein may mediate the coupling of receptor activation to inositol phospholipid hydrolysis in brain.« less

Activation of Brain Somatostatin Signaling Suppresses CRF Receptor-Mediated Stress Response.

PubMed

Stengel, Andreas; Taché, Yvette F

2017-01-01

Corticotropin-releasing factor (CRF) is the hallmark brain peptide triggering the response to stress and mediates-in addition to the stimulation of the hypothalamus-pituitary-adrenal (HPA) axis-other hormonal, behavioral, autonomic and visceral components. Earlier reports indicate that somatostatin-28 injected intracerebroventricularly counteracts the acute stress-induced ACTH and catecholamine release. Mounting evidence now supports that activation of brain somatostatin signaling exerts a broader anti-stress effect by blunting the endocrine, autonomic, behavioral (with a focus on food intake) and visceral gastrointestinal motor responses through the involvement of distinct somatostatin receptor subtypes.

Probing receptor-ligand interactions by sedimentation equilibrium

NASA Astrophysics Data System (ADS)

Philo, John S.

1997-05-01

While sedimentation equilibrium is most commonly used to characterize the molecular weight and state of association of single proteins, this technique is also a very powerful tool for probing the interactions between two or more different proteins, and can characterize both the binding stoichiometry and the equilibrium constants. To resolve the complex binding interactions that can occur in such systems, it is crucial to globally fit data from many experiments to a common binding model, including samples made with different mixing ratios and a wide range of total concentration. It is often also essential to constrain the parameters during fitting so that the fits correctly reproduce the molar ratio of proteins used in making each sample. We have applied this methodology to probe mechanisms of receptor activation for a number of hematopoietic receptors and their cognate ligands, using receptor extracellular domains expressed as soluble proteins. Such data can potentially help in the design of improved or new protein therapeutics, as well as in efforts to create small- molecular mimetics of protein hormones through structure- based drug design. Sedimentation equilibrium has shown that stem cell factor, erythropoietin, and granulocyte-colony stimulating factor can each dimerize their respective receptors in solution, but the mechanism of ligand-induced receptor dimerization for these three systems are strikingly different.

ATP mediates flow-induced NO production in thick ascending limbs

PubMed Central

Hong, Nancy J.; Garvin, Jeffrey L.

2012-01-01

Mechanical stimulation caused by increasing flow induces nucleotide release from many cells. Luminal flow and extracellular ATP stimulate production of nitric oxide (NO) in thick ascending limbs. However, the factors that mediate flow-induced NO production are unknown. We hypothesized that luminal flow stimulates thick ascending limb NO production via ATP. We measured NO in isolated, perfused rat thick ascending limbs using the fluorescent dye DAF FM. The rate of increase in dye fluorescence reflects NO accumulation. Increasing luminal flow from 0 to 20 nl/min stimulated NO production from 17 ± 16 to 130 ± 37 arbitrary units (AU)/min (P < 0.02). Increasing flow from 0 to 20 nl/min raised ATP release from 4 ± 1 to 21 ± 6 AU/min (P < 0.04). Hexokinase (10 U/ml) plus glucose, which consumes ATP, completely prevented the measured increase in ATP. Luminal flow did not increase NO production in the presence of luminal and basolateral hexokinase (10 U/ml). When flow was increased with the ATPase apyrase in both luminal and basolateral solutions (5 U/ml), NO levels did not change significantly. The P2 receptor antagonist suramin (300 μmol/l) reduced flow-induced NO production by 83 ± 25% (P < 0.03) when added to both and basolateral sides. Luminal hexokinase decreased flow-induced NO production from 205.6 ± 85.6 to 36.6 ± 118.6 AU/min (P < 0.02). Basolateral hexokinase also reduced flow-induced NO production. The P2X receptor-selective antagonist NF023 (200 μmol/l) prevented flow-induced NO production when added to the basolateral side but not the luminal side. We conclude that ATP mediates flow-induced NO production in the thick ascending limb likely via activation of P2Y receptors in the luminal and P2X receptors in the basolateral membrane. PMID:22496412

Opposing Effects of α2- and β-Adrenergic Receptor Stimulation on Quiescent Neural Precursor Cell Activity and Adult Hippocampal Neurogenesis

PubMed Central

Prosper, Boris W.; Marathe, Swanand; Husain, Basma F. A.; Kernie, Steven G.; Bartlett, Perry F.; Vaidya, Vidita A.

2014-01-01

Norepinephrine regulates latent neural stem cell activity and adult hippocampal neurogenesis, and has an important role in modulating hippocampal functions such as learning, memory and mood. Adult hippocampal neurogenesis is a multi-stage process, spanning from the activation and proliferation of hippocampal stem cells, to their differentiation into neurons. However, the stage-specific effects of noradrenergic receptors in regulating adult hippocampal neurogenesis remain poorly understood. In this study, we used transgenic Nestin-GFP mice and neurosphere assays to show that modulation of α2- and β-adrenergic receptor activity directly affects Nestin-GFP/GFAP-positive precursor cell population albeit in an opposing fashion. While selective stimulation of α2-adrenergic receptors decreases precursor cell activation, proliferation and immature neuron number, stimulation of β-adrenergic receptors activates the quiescent precursor pool and enhances their proliferation in the adult hippocampus. Furthermore, our data indicate no major role for α1-adrenergic receptors, as we did not observe any change in either the activation and proliferation of hippocampal precursors following selective stimulation or blockade of α1-adrenergic receptors. Taken together, our data suggest that under physiological as well as under conditions that lead to enhanced norepinephrine release, the balance between α2- and β-adrenergic receptor activity regulates precursor cell activity and hippocampal neurogenesis. PMID:24922313

Low-frequency transcranial magnetic stimulation is beneficial for enhancing synaptic plasticity in the aging brain.

PubMed

Zhang, Zhan-Chi; Luan, Feng; Xie, Chun-Yan; Geng, Dan-Dan; Wang, Yan-Yong; Ma, Jun

2015-06-01

In the aging brain, cognitive function gradually declines and causes a progressive reduction in the structural and functional plasticity of the hippocampus. Transcranial magnetic stimulation is an emerging and novel neurological and psychiatric tool used to investigate the neurobiology of cognitive function. Recent studies have demonstrated that low-frequency transcranial magnetic stimulation (≤1 Hz) ameliorates synaptic plasticity and spatial cognitive deficits in learning-impaired mice. However, the mechanisms by which this treatment improves these deficits during normal aging are still unknown. Therefore, the current study investigated the effects of transcranial magnetic stimulation on the brain-derived neurotrophic factor signal pathway, synaptic protein markers, and spatial memory behavior in the hippocampus of normal aged mice. The study also investigated the downstream regulator, Fyn kinase, and the downstream effectors, synaptophysin and growth-associated protein 43 (both synaptic markers), to determine the possible mechanisms by which transcranial magnetic stimulation regulates cognitive capacity. Transcranial magnetic stimulation with low intensity (110% average resting motor threshold intensity, 1 Hz) increased mRNA and protein levels of brain-derived neurotrophic factor, tropomyosin receptor kinase B, and Fyn in the hippocampus of aged mice. The treatment also upregulated the mRNA and protein expression of synaptophysin and growth-associated protein 43 in the hippocampus of these mice. In conclusion, brain-derived neurotrophic factor signaling may play an important role in sustaining and regulating structural synaptic plasticity induced by transcranial magnetic stimulation in the hippocampus of aging mice, and Fyn may be critical during this regulation. These responses may change the structural plasticity of the aging hippocampus, thereby improving cognitive function.

Endothelial cells of extremely premature infants display impaired immune response after proinflammatory stimulation.

PubMed

Wisgrill, Lukas; Muck, Martina; Wessely, Isabelle; Berger, Angelika; Spittler, Andreas; Förster-Waldl, Elisabeth; Sadeghi, Kambis

2018-01-01

BackgroundEndothelial cells (ECs) exert immunological functions such as production of proinflammatory cytokines/chemokines as well as facilitation of extravasation of immune cells into infected tissue. Limited data are available on the functionality of ECs from extremely preterm neonates during infection. Accordingly, the aim of our study was to investigate the immune response of premature ECs after proinflammatory stimulation.MethodsCell adhesion receptors' expression and function, nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NFκB) signaling, and chemokine production were analyzed in umbilical cord ECs from extremely preterm and term neonates after proinflammatory stimulation.ResultsP-selectin and E-selectin surface expression as well as NFκB signaling were lower after lipopolysaccharide (LPS) stimulation in premature ECs. Preterm ECs exhibited lower, but significant, cell-adhesive functions after LPS stimulation compared with term ECs. CCL2/CXCL8 chemokine secretion was significantly upregulated after proinflammatory stimulation in both groups. CXCL10 production was significantly increased in term but not in preterm ECs upon stimulation with tumor necrosis factor compared with unstimulated ECs.ConclusionExtremely premature ECs showed partly reduced expression levels and function of cell adhesion molecules. Both NFκB signaling and chemokine/cytokine production were reduced in premature ECs. The diminished endothelial proinflammatory immune response might result in impaired infection control of preterm newborns rendering them prone to severe infection.

Muscarinic Stimulation Facilitates Sarcoplasmic Reticulum Ca Release by Modulating Ryanodine Receptor 2 Phosphorylation Through Protein Kinase G and Ca/Calmodulin-Dependent Protein Kinase II.

PubMed

Ho, Hsiang-Ting; Belevych, Andriy E; Liu, Bin; Bonilla, Ingrid M; Radwański, Przemysław B; Kubasov, Igor V; Valdivia, Héctor H; Schober, Karsten; Carnes, Cynthia A; Györke, Sándor

2016-11-01

Although the effects and the underlying mechanism of sympathetic stimulation on cardiac Ca handling are relatively well established both in health and disease, the modes of action and mechanisms of parasympathetic modulation are poorly defined. Here, we demonstrate that parasympathetic stimulation initiates a novel mode of excitation-contraction coupling that enhances the efficiency of cardiac sarcoplasmic reticulum Ca store utilization. This efficient mode of excitation-contraction coupling involves reciprocal changes in the phosphorylation of ryanodine receptor 2 at Ser-2808 and Ser-2814. Specifically, Ser-2808 phosphorylation was mediated by muscarinic receptor subtype 2 and activation of PKG (protein kinase G), whereas dephosphorylation of Ser-2814 involved activation of muscarinic receptor subtype 3 and decreased reactive oxygen species-dependent activation of CaMKII (Ca/calmodulin-dependent protein kinase II). The overall effect of these changes in phosphorylation of ryanodine receptor 2 is an increase in systolic Ca release at the low sarcoplasmic reticulum Ca content and a paradoxical reduction in aberrant Ca leak. Accordingly, cholinergic stimulation of cardiomyocytes isolated from failing hearts improved Ca cycling efficiency by restoring altered ryanodine receptor 2 phosphorylation balance. © 2016 American Heart Association, Inc.

SRP RNA provides the physiologically essential GTPase activation function in cotranslational protein targeting

PubMed Central

Siu, Fai Y.; Spanggord, Richard J.; Doudna, Jennifer A.

2007-01-01

The signal recognition particle (SRP) cotranslationally targets proteins to cell membranes by coordinated binding and release of ribosome-associated nascent polypeptides and a membrane-associated SRP receptor. GTP uptake and hydrolysis by the SRP-receptor complex govern this targeting cycle. Because no GTPase-activating proteins (GAPs) are known for the SRP and SRP receptor GTPases, however, it has been unclear whether and how GTP hydrolysis is stimulated during protein trafficking in vivo. Using both biochemical and genetic experiments, we show here that SRP RNA enhances GTPase activity of the SRP–receptor complex above a critical threshold required for cell viability. Furthermore, this stimulation is a property of the SRP RNA tetraloop. SRP RNA tetraloop mutants that confer defective growth phenotypes can assemble into SRP–receptor complexes, but fail to stimulate GTP hydrolysis in these complexes in vitro. Tethered hydroxyl radical probing data reveal that specific positioning of the RNA tetraloop within the SRP–receptor complex is required to stimulate GTPase activity to a level sufficient to support cell growth. These results explain why no external GAP is needed and why the phylogenetically conserved SRP RNA tetraloop is required in vivo. PMID:17164479

Activation of NOX2 by the stimulation of ionotropic and metabotropic glutamate receptors contributes to glutamate neurotoxicity in vivo through the production of reactive oxygen species and calpain activation.

PubMed

Guemez-Gamboa, Alicia; Estrada-Sánchez, Ana María; Montiel, Teresa; Páramo, Blanca; Massieu, Lourdes; Morán, Julio

2011-11-01

Prolonged activation of glutamate receptors leads to excitotoxicity. Several processes such as reactive oxygen species (ROS) production and activation of the calcium-dependent protease, calpain, contribute to glutamate-induced damage. It has been suggested that the ROS-producing enzyme, NADPH oxidase (NOX), plays a role in excitotoxicity. Studies have reported NOX activation after NMDA receptor stimulation during excitotoxic damage, but the role of non-NMDA and metabotropic receptors is unknown. We evaluated the roles of different glutamate receptor subtypes on NOX activation and neuronal death induced by the intrastriatal administration of glutamate in mice. In wild-type mice, NOX2 immunoreactivity in neurons and microglia was stimulated by glutamate administration, and it progressively increased as microglia became activated; calpain activity was also induced. By contrast, mice lacking NOX2 were less vulnerable to excitotoxicity, and there was reduced ROS production and protein nitrosylation, microglial reactivity, and calpain activation. These results suggest that NOX2 is stimulated by glutamate in neurons and reactive microglia through the activation of ionotropic and metabotropic receptors. Neuronal damage involves ROS production by NOX2, which, in turn, contributes to calpain activation.

Muscarinic acetylcholine receptor subtype 4 is essential for cholinergic stimulation of duodenal bicarbonate secretion in mice - relationship to D cell/somatostatin.

PubMed

Takeuchi, K; Kita, K; Takahashi, K; Aihara, E; Hayashi, S

2015-06-01

We investigated the roles of muscarinic (M) acetylcholine receptor subtype in the cholinergic stimulation of duodenal HCO3(-) secretion using knockout (KO) mice. Wild-type and M1-M5 KO C57BL/6J mice were used. The duodenal mucosa was mounted on an Ussing chamber, and HCO3(-) secretion was measured at pH 7.0 using a pH-stat method in vitro. Carbachol (CCh) or other agents were added to the serosal side. CCh dose-dependently stimulated HCO3(-) secretion in wild-type mice, and this effect was completely inhibited in the presence of atropine. The HCO3(-) response to CCh in wild-type mice was also inhibited by pirenzepine (M1 antagonist), 4DAMP (M3 antagonist), and tropicamide (M4 antagonist), but not by methoctramine (M2 antagonist). CCh stimulated HCO3(-) secretion in M2 and M5 KO animals as effectively as in WT mice; however, this stimulatory effect was significantly attenuated in M1, M3, and M4 KO mice. The decrease observed in the CCh-stimulated HCO3(-) response in M4 KO mice was reversed by the co-application of CYN154806, a somatostatin receptor type 2 (SST2) antagonist. Octreotide (a somatostatin analogue) decreased the basal and CCh-stimulated secretion of HCO3(-) in wild-type mice. The co-localized expression of somatostatin and M4 receptors was confirmed immunohistologically in the duodenum. We concluded that the duodenal HCO3(-) response to CCh was directly mediated by M1/M3 receptors and indirectly modified by M4 receptors. The activation of M4 receptors was assumed to inhibit the release of somatostatin from D cells and potentiate the HCO3(-) response by removing the negative influence of somatostatin via the activation of SST2 receptors.