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R J Flower - One of the best experts on this subject based on the ideXlab platform.
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Reverses the Inhibitory Actions of Dexamethasone on the Release of Adrenocorticotropin from Rat Pituitary Tissue in Vitro*
2013Co-Authors: An Antisense, R J Flower, A D Taylor, Hc Christian, J. F. Morris, Oligodeoxynucleotide Lipocortin, Jc BuckinghamAbstract:Our previous studies have demonstrated that Lipocortin 1 (LC1, also called annexin 1) is an important mediator of glucocorticoid action in the neuroendocrine system, particularly with regard to the powerful inhibitory actions of the steroids on the secretion of ACTH and its hypothalamic releasing hormones. In the present study, we have used an antisense oligodeoxynucleotide (ODN) unique to LC1 to investigate further the role of this protein in the regulatory effects of dexamethasone on ACTH release in vitro from rat anterior pituitary cells. Pituitary cells dispersed with collagenase retained their functional and morphological integrity in vitro and sequestered ODNs in a time-dependent manner from the incubation medium. LC1 was readily detected in the cells by Western blot analysis or by immunoprecipitation/autoradiography after preloading with 35 S-methionine/ cysteine; the bulk of the protein was contained within an intracellula
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Printed in U.S.A. Copyright © 1997 by The Endocrine Society Characterization and Localization of Lipocortin 1- Binding Sites on Rat Anterior Pituitary Cells by
2013Co-Authors: Fluorescence-activated Cell Analysis/sorting, R J Flower, A D Taylor, Hc Christian, J. F. Morris, Jc BuckinghamAbstract:Lipocortin 1 (LC1; also called annexin 1) is a 37-kDa member of the annexin superfamily of Ca 2 �- and phospholipid-binding proteins (1–3). Since its initial identification in extracts of conditioned medium from glucocorticoid-stimulated peritoneal macrophages (4), LC1 has been shown to exhibit a wide range of glucocorticoid-like activities and has been widely advocated as a mediator of these steroids, particularly in immune/inflammatory cells (2, 3) and the neuroendocrine system (2, 5). Our recent in vivo and in vitro studies have identified a significant role for LC1 in effecting the negative feedback action of the glucocorticoids in the hypothalamo-pituitary-adrenal axis at the levels of both the hypothalamus (5–7) and the anterior pituitary gland (5, 8). Further studies have shown that LC1 also contributes to the less well documented inhibitory actions of the glucocorticoids on the release of PRL (9), GH (10), and TSH (11). LC1 is expressed in abundance in the anterior pituitary gland where, as in several peripheral cell types (12, 13), both it
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annexin 1 Lipocortin 1 mediates the glucocorticoid inhibition of cyclic adenosine 3 5 monophosphate stimulated prolactin secretion
Endocrinology, 2000Co-Authors: A D Taylor, R J Flower, James G. Philip, C D John, Patricia O Cover, John Morris, Julia C. BuckinghamAbstract:Our previous studies have identified a role for annexin 1 (also called Lipocortin 1) in the regulatory actions of glucocorticoids (GCs) on the release of PRL from the rat anterior pituitary gland. In the present study we used antisense and immunoneutralization strategies to extend this work. Exposure of rat anterior pituitary tissue to corticosterone (1 nm) or dexamethasone (100 nm) in vitro induced 1) de novo annexin 1 synthesis and 2) translocation of the protein from intracellular to pericellular sites. Both responses were prevented by the inclusion in the medium of an annexin 1 antisense oligodeoxynucleotide (ODN; 50 nm), but not by the corresponding sense and scrambled ODN sequences. Unlike the GCs, 17β-estradiol, testosterone, and aldosterone (1 nm) had no effect on either the synthesis or the cellular disposition of annexin 1; moreover, none of the steroids or ODNs tested influenced the expression of annexin 5, a protein closely related to annexin 1. The increases in PRL release induced in vitro by...
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Localisation and semi-quantitative measurement of Lipocortin 1 in rat anterior pituitary cells by fluorescence-activated cell analysis/sorting and electron microscopy.
1999Co-Authors: Hc Christian, R J Flower, J F Morris, Jc BuckinghamAbstract:Lipocortin 1 (LC1, also called annexin 1), a Ca2(+)- and phospholipid-binding protein, is an important mediator of glucocorticoid action in the anterior pituitary gland. Previous studies based on immunoprecipitation and Western blot analysis suggest that LC1 is found intracellularly both in the cytoplasm and in association with membranes and also on the cell surface where it attaches to the membrane by a Ca2(+)-dependent mechanism. However, as yet it is unclear which anterior pituitary cell types express the protein. Accordingly, we have developed a method based on a combination of fluorescence activated cell (FAC) analysis/sorting and electron microscopy to detect and quantify intracellular LC1 in rat anterior pituitary cells and to identify the cell types in which it is expressed. In addition, we have measured cell surface LC1 and examined the influence of glucocorticoids on the cellular disposition of the protein. Anterior pituitary cells were dispersed with collagenase. For experiments measuring intracellular LC1, three cell fixation/permeabilisation methods were examined initially, i.e. (1) Zamboni's fluid (30 min) and Triton-X-100 (0.12%, 1 or 12 h); (2) paraformaldehyde (2%, 1 h) and Triton-X-100 (0.2%, 10 min); and (3) paraformaldehyde (0.2%, 15 min) and saponin (0.1%, 5 min). The protocol using paraformaldehyde/Triton-X-100 provided optimal preservation of cell ultrastructure and of LC1 immunoreactivity (ir-LC1) while also effectively permeabilising the cells; it was therefore used in subsequent studies. Using an anti-LC1 monoclonal antibody as a probe, 82+/-5% of the secretory cells in the heterogeneous anterior pituitary cell preparation were shown by FAC analysis to display specific fluorescence for intracellular ir-LC1. Morphological analysis and immunogold-histochemistry of cells separated by FAC sorting identified corticotrophs, lactotrophs, somatotrophs and gonadotrophs in the population displaying LC1 immunofluorescence. LC1 was also detected on the surface of anterior pituitary cells by FACS analysis. Incubation of anterior pituitary cells with dexamethasone or corticosterone (0.1 and 1.0 microM) prior to fixation and analysis produced a significant, concentration-dependent decrease in intracellular ir-LC1 and a concomitant increase in the amount of ir-LC1 detected on the surface of the cells; the effects of the two steroids were indistinguishable quantitatively. In conclusion, we report a novel method which permits (1) the detection and semi-quantitative measurement of intracellular and surface LC1 in anterior pituitary cells; and (2) the identification of the cell types in which the protein is found
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inhibitory effect of peptides derived from the n terminus of Lipocortin 1 on arachidonic acid release and proliferation in the a549 cell line identification of e q e y v as a crucial component
British Journal of Pharmacology, 1998Co-Authors: Jamie D. Croxtall, Qamrul G Choudhury, R J FlowerAbstract:1 The ability of the glucocorticoid-induced protein Lipocortin 1 (LC1) to inhibit arachidonic acid release and cell proliferation in A549 cells may be mimicked by a sequence taken from the N-terminal, LC113–25 (FIENEEQEYVQTV). We have now synthesized and tested for biological activity a library of 25 smaller peptides derived from this sequence. 2 Peptides were tested in two assays: A549 cells were prelabelled with tritiated arachidonic acid and thapsigargin (50 nM) and EGF (10 nM) used to stimulate the release of this fatty acid. Cell proliferation was determined by counting cell numbers following 3 day incubation with these peptides, or controls. 3 Many of the peptides were highly insoluble but could be more readily dissolved in aqueous solution in the presence of commercial liposomes or phosphatidyl serine (5 μM). Since neither of these agents alone had any effect on arachidonic acid release or cell proliferation, all peptides were tested in the presence of 5 μM phosphatidyl serine. Under these conditions LC113–25 was active in both assay systems with an IC40 of 40.7 and 57.0 μM respectively. 4 Deletion of amino acids from the C-terminus of the peptide progressively diminished (2–3 fold) the molar potency of LC113–25 in both assays: after the removal of Val22 biological activity was virtually undetectable or very weak (<30% of LC113–25). 5 Removal of amino acids from the N-terminus also lead to a progressive reduction (3–5 fold) in the molar potency of the peptides and biological activity became undetectable, or very weak, after the removal of Glu18. 6 All active peptides contained the core sequence EQEYV(Glu-Gln-Glu-Tyr-Val) which seems to represent a crucial component of the pharmacophore, although this sequence on its own was inactive and the shortest peptide with significant activity was LC118–25 (EQEYVQTV). 7 Methoxylation of Tyr21 abolished the ability of LC1 18–25 to inhibit cell proliferation and arachidonic acid release. A cyclized version of LC118–25 was also tested and found to be inactive. 8 LC118–25 (178 μM) inhibits cPLA2 activation in A549 cells as judged by a band-shift assay, whereas equimolar concentrations of an inactive peptide LC119–25 were without effect in this assay system. 9 Several possible mechanisms whereby these peptides act are discussed in the light of LC1 biology and of the effect of glucocorticoids on cell function. British Journal of Pharmacology (1998) 123, 975–983; doi:10.1038/sj.bjp.0701679
Julia C. Buckingham - One of the best experts on this subject based on the ideXlab platform.
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Printed in U.S.A. Copyright © 1999 by The Endocrine Society Lipocortin 1 (Annexin 1): A Candidate Paracrine Agent Localized in Pituitary Folliculo-Stellate Cells*
2013Co-Authors: Valerie Traverso, J F Morris, Helen C Christian, Julia C. BuckinghamAbstract:It is now well established that Lipocortin 1 (LC1) plays an important role as a mediator of early delayed glucocorticoid feedback action in the hypothalamo-hypophysial system. In both the hypothalamus and anterior pituitary gland, LC1 mimics some of the actions of glucocorticoids; moreover, glucocorticoids stimulate the synthesis of LC1 and cause the translocation of intracellular LC1 to the outer cell surface. The mechanism by which LC1 acts in these tissues is only partially understood, but may involve paracrine and/or autocrine actions. To address these possibilities we have investigated the localization of LC1 in the rat pituitary gland, using double labeling immunohistochemistry to identify the pituitary cell types that express LC1. At the light microscopic level LC1 was not detected in the endocrine cells in cryosections of the pituitary, but it was found in abundance in the GLUCOCORTICOID hormones and their synthetic analog
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annexin 1 Lipocortin 1 mediates the glucocorticoid inhibition of cyclic adenosine 3 5 monophosphate stimulated prolactin secretion
Endocrinology, 2000Co-Authors: A D Taylor, R J Flower, James G. Philip, C D John, Patricia O Cover, John Morris, Julia C. BuckinghamAbstract:Our previous studies have identified a role for annexin 1 (also called Lipocortin 1) in the regulatory actions of glucocorticoids (GCs) on the release of PRL from the rat anterior pituitary gland. In the present study we used antisense and immunoneutralization strategies to extend this work. Exposure of rat anterior pituitary tissue to corticosterone (1 nm) or dexamethasone (100 nm) in vitro induced 1) de novo annexin 1 synthesis and 2) translocation of the protein from intracellular to pericellular sites. Both responses were prevented by the inclusion in the medium of an annexin 1 antisense oligodeoxynucleotide (ODN; 50 nm), but not by the corresponding sense and scrambled ODN sequences. Unlike the GCs, 17β-estradiol, testosterone, and aldosterone (1 nm) had no effect on either the synthesis or the cellular disposition of annexin 1; moreover, none of the steroids or ODNs tested influenced the expression of annexin 5, a protein closely related to annexin 1. The increases in PRL release induced in vitro by...
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Lipocortin 1 annexin 1 a candidate paracrine agent localized in pituitary folliculo stellate cells
Endocrinology, 1999Co-Authors: Valerie Traverso, J F Morris, Helen C Christian, Julia C. BuckinghamAbstract:It is now well established that Lipocortin 1 (LC1) plays an important role as a mediator of early delayed glucocorticoid feedback action in the hypothalamo-hypophysial system. In both the hypothalamus and anterior pituitary gland, LC1 mimics some of the actions of glucocorticoids; moreover, glucocorticoids stimulate the synthesis of LC1 and cause the translocation of intracellular LC1 to the outer cell surface. The mechanism by which LC1 acts in these tissues is only partially understood, but may involve paracrine and/or autocrine actions. To address these possibilities we have investigated the localization of LC1 in the rat pituitary gland, using double labeling immunohistochemistry to identify the pituitary cell types that express LC1. At the light microscopic level LC1 was not detected in the endocrine cells in cryosections of the pituitary, but it was found in abundance in the surrounding folliculo-stellate (FS) cells. In the anterior and intermediate pituitary lobes, there was a near total colocaliza...
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Blockade of the classical pathway of protein secretion does not affect the cellular exportation of Lipocortin 1.
Regulatory peptides, 1998Co-Authors: James G. Philip, Roderick J. Flower, Julia C. BuckinghamAbstract:Abstract The mechanism by which Lipocortin 1 (LC1) is extruded from cells in the brain and periphery in response to a glucocorticoid challenge is unknown. This study examined the influence of three inhibitors of the classical endoplasmic reticulum–Golgi pathway of protein secretion on the dexamethasone-induced (0.1 μM, 2–3 h) cellular exportation of LC1 in vitro in brain (cortex, hippocampus, hypothalamus), anterior pituitary tissue and peritoneal macrophages. In all instances, the steroid-induced exportation of LC1 was unaffected by brefeldin A (1.4 μM), monensin (10 μM) and nocodazole (3.3 μM); however, these drugs readily blocked the release of corticotrophin from pituitary tissue. These data suggest that LC1 is exported by a mechanism distinct from the classical pathway of protein secretion.
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characterization and localization of Lipocortin 1 binding sites on rat anterior pituitary cells by fluorescence activated cell analysis sorting and electron microscopy
Endocrinology, 1997Co-Authors: Helen C Christian, R J Flower, A D Taylor, J. F. Morris, Julia C. BuckinghamAbstract:Lipocortin 1 (LC1) is an important mediator of glucocorticoid action in the anterior pituitary gland, where it appears to act via cell surface binding sites to suppress peptide release. We have exploited a combination of fluorescence-activated cell (FAC) analysis/sorting and electron microscopy to detect, characterize, and localize LC1-binding sites on the surface of dispersed rat anterior pituitary cells, using human recombinant LC1 (hu-r-LC1) as a probe. High affinity (Kd = 14 +/- 3 nM) hu-r-LC1-binding sites were detected on approximately 80% of anterior pituitary cells dispersed with collagenase. The binding characteristics of the ligand resembled those observed in leukocytes, in that it was saturable; concentration, Ca2+, and temperature dependent; and abolished by trypsin. Functional studies demonstrated an excellent correlation between the presence of the cell surface binding protein and the capacity of an anti-LC1 monoclonal antibody to abrogate the inhibitory actions of dexamethasone (10 nM) on the release of ACTH initiated in vitro by CRH-41 (1 nM). Morphological analysis of cells harvested by FAC sorting showed that 1) somatotrophs, corticotrophs, lactotrophs, thyrotrophs, and gonadotrophs were all included in the population expressing LC1 binding sites; and 2) the LC1-binding sites assume a punctate distribution across the cell surface. These data show that anterior pituitary cells express high affinity surface LC1-binding protein(s); they thus provide further evidence for a specific membrane mechanism of action of LC1 in regulating the endocrine function of the anterior pituitary.
Mauro Perretti - One of the best experts on this subject based on the ideXlab platform.
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Annexin 1 localisation in tissue eosinophils as detected by electron microscopy
Hindawi Limited, 2002Co-Authors: Sonia M. Oliani, Amilcar S. Damazo, Mauro PerrettiAbstract:Background: Human and rodent leukocytes express high levels of the glucocorticoid-inducible protein annexin 1 (ANXA1) (previously referred to as Lipocortin 1). Neutrophils and monocytes have abundant ANXA1 levels
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Lipocortin 1 reduces myocardial ischemia reperfusion injury by affecting local leukocyte recruitment
The FASEB Journal, 2000Co-Authors: Michele Damico, Roderick J. Flower, Egle Solito, Clara Di Filippo, Peter G Mclean, Sonia Maria Oliani, Mauro PerrettiAbstract:We assessed here the effect of the glucocorticoid-regulated protein Lipocortin 1 (LC1) in a model of rat myocardial ischemia reperfusion. Treatment of animals with human recombinant LC1 at the end of a 25-min ischemic period significantly reduced the extent of infarct size in the area at risk as measured 2 h later, with approximately 50% inhibition at the highest dose tested of 50 microg per rat (equivalent to 5.4 nmol/kg). The protective effect of LC1 was abolished by protein denaturation and not mimicked by the structurally related protein annexin V. A combination of electron and light microscopy techniques demonstrated the occurrence of the myocardial damage at the end of the reperfusion period, with loss of fiber organization. LC1 provided a partial and visible protection. The dose-dependent protection afforded by LC1 was paralleled by lower values of myeloperoxidase activity, tumor necrosis factor a, and macrophage inflammatory protein-1a. The functional link between migrated leukocytes and the myocardial damage was confirmed by electron and light microscopy, and a significantly lower number of extravasated leukocytes was counted in the group of rats treated with LC1 (50 microg). In conclusion, we demonstrate for the first time that LC1 reduces the leukocyte-dependent myocardial damage associated with an ischemia-reperfusion procedure.
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down regulation of microglial cyclo oxygenase 2 and inducible nitric oxide synthase expression by Lipocortin 1
British Journal of Pharmacology, 1999Co-Authors: Luisa Minghetti, Luca Parente, Mauro Perretti, Alessia Nicolini, Elisabetta Polazzi, Anita Greco, Giulio LeviAbstract:Activated microglial cells are believed to play an active role in most brain pathologies, during which they can contribute to host defence and repair but also to the establishment of tissue damage. These actions are largely mediated by microglial secretory products, among which are prostaglandins (PGs) and nitric oxide (NO). The anti-inflammatory protein, Lipocortin 1 (LC1) was reported to have neuroprotective action and to be induced by glucocorticoids in several brain structures, with a preferential expression in microglia. In this paper we tested whether the neuroprotective effect of LC1 could be explained by an inhibitory effect on microglial activation. We have previously shown that bacterial endotoxin (LPS) strongly stimulates PGE2 and NO production in rat primary microglial cultures, by inducing the expression of the key enzymes cyclo-oxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), respectively. Dexamethasone (DEX, 1–100 nM) and LC1-derived N-terminus peptide (peptide Ac2-26, 1–100 μg ml−1) dose-dependently inhibited the production of both PGE2 and NO from LPS-stimulated microglia. The inhibitory effects of DEX on NO and of the peptide on NO and PGE2 synthesis were partially abrogated by a specific antiserum, raised against the N-terminus of human LC1. The peptide Ac2-26 did not affect arachidonic acid release from control and LPS-stimulated microglial cultures. Western blot experiments showed that the LPS-induced expression of COX-2 and iNOS was effectively down-regulated by DEX (100 nM) and peptide Ac2-26 (100 μg ml−1). In conclusion, our findings support the hypothesis that LC1 may foster neuroprotection by limiting microglial activation, through autocrine and paracrine mechanisms. British Journal of Pharmacology (1999) 126, 1307–1314; doi:10.1038/sj.bjp.0702423
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Promoting detachment of neutrophils adherent to murine postcapillary venules to control inflammation: Effect of Lipocortin 1
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Egle Solito, Roderick J. Flower, Françoise Russo-marie, Mauro PerrettiAbstract:In this study we investigated, using intravital microscopy, how neutrophil extravasation across mouse mesenteric postcapillary venules is inhibited by the glucocorticoid-regulated protein Lipocortin (LC; also termed annexin) 1. Intraperitoneal injection of 1 mg of zymosan into mice induced neutrophil rolling on the activated mesenteric endothelium followed by adhesion (maximal at 2 hr: 5–6 cells per 100-μm of vessel length) and emigration (maximal at 4 hr: 8–10 cells per high-powered field). Treatment of mice with human recombinant LC1 (2 mg/kg s.c.) or its mimetic peptide Ac2–26 (13 mg/kg s.c.) did not modify cell rolling but markedly reduced (≥50%) the degree of neutrophil adhesion and emigration (P < 0.05). Intravenous treatment with peptide Ac2–26 (13 mg/kg) or recombinant human LC1 (0.7–2 mg/kg) promoted detachment of neutrophils adherent to the endothelium 2 hr after zymosan administration, with adherent cells detaching within 4.12 ± 0.75 min and 2.36 ± 0.31 min, respectively (n = 20–25 cells). Recruitment of newly adherent cells to the endothelium was unaffected. The structurally related protein LC5 was inactive in this assay, whereas a chimeric molecule constructed from the N terminus of LC1 (49 aa) attached to the core region of LC5 produced cell detachment with kinetics similar to LC1. Removal of adherent neutrophils from activated postcapillary endothelium is a novel pharmacological action, and it is at this site where LC1 and its mimetics operate to down-regulate this aspect of the host inflammatory response.
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Lipocortin 1 and chemokine modulation of granulocyte and monocyte accumulation in experimental inflammation
General Pharmacology-the Vascular System, 1998Co-Authors: Mauro PerrettiAbstract:1. Migration of blood-derived leukocytes to tissue sites of inflammation is a hallmark of the response that the host organizes to counteract an insult or a trauma or an infection. A cascade of events is then activated to allow interaction between the leukocyte and the endothelium of postcapillary venule, and this cascade is finely regulated such that mechanisms of negative control are operating side by side with pathways that promote and sustain the extravasation process. Examples of both these positive and negative regulatory systems are discussed here. 2. In vivo accumulation of specific subtypes of leukocytes in response to application of selective chemokines operates through an indirect mechanism that includes the perivenular mast cell and, in particular, the mast cell-derived amines, such as histamine and serotonin. In fact, treatments of animals with (1) histamine H1 or serotonin antagonists or with (2) the mast cell stabilizer cromolyn or with (3) prior depletion of intact mast cells are maneuvers that successfully reduce eosinophil, neutrophil and monocyte extravasation in response to eotaxin, interleukin-8 or monocyte chemoattractant protein-1, respectively. A model in which histamine provides a P-selectin-dependent rolling phenomenon is then postulated. 3. The discovery that neutrophil-derived Lipocortin 1 acts as an autocrine mediator with an inhibitory action on the emigration (diapedesis) process confirms the growing body of experimental data that showed that exogenously administered Lipocortin 1 and Lipocortin 1 mimetics (peptide Ac2-26) potently inhibit neutrophil extravasation in response to different stimuli. Externalization of Lipocortin 1 on the plasma membrane of adherent neutrophils reduces their rate of passage through the endothelial gaps. Because cell-associated Lipocortin 1 levels are under the partial control of corticosterone (endogenous circulating glucocorticoid hormone in rodents) and dexamethasone (a synthetic glucocorticoid hormone with a potent anti-inflammatory profile), a model is proposed in which a balance between anti-inflammatory (Lipocortin 1, etc.) and pro-inflammatory (adhesion molecules, cytokines and chemokines) mediators explains the difference in the rate of leukocyte accumulation during the different stages of the host inflammatory response. 4. In conclusion, this review emphasizes the importance of in vivo experimental systems as a valid way of obtaining pertinent observations and reiterates the importance of negative regulatory mechanisms on the leukocyte extravasation process operating within the host.
Jamie D. Croxtall - One of the best experts on this subject based on the ideXlab platform.
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inhibitory effect of peptides derived from the n terminus of Lipocortin 1 on arachidonic acid release and proliferation in the a549 cell line identification of e q e y v as a crucial component
British Journal of Pharmacology, 1998Co-Authors: Jamie D. Croxtall, Qamrul G Choudhury, R J FlowerAbstract:1 The ability of the glucocorticoid-induced protein Lipocortin 1 (LC1) to inhibit arachidonic acid release and cell proliferation in A549 cells may be mimicked by a sequence taken from the N-terminal, LC113–25 (FIENEEQEYVQTV). We have now synthesized and tested for biological activity a library of 25 smaller peptides derived from this sequence. 2 Peptides were tested in two assays: A549 cells were prelabelled with tritiated arachidonic acid and thapsigargin (50 nM) and EGF (10 nM) used to stimulate the release of this fatty acid. Cell proliferation was determined by counting cell numbers following 3 day incubation with these peptides, or controls. 3 Many of the peptides were highly insoluble but could be more readily dissolved in aqueous solution in the presence of commercial liposomes or phosphatidyl serine (5 μM). Since neither of these agents alone had any effect on arachidonic acid release or cell proliferation, all peptides were tested in the presence of 5 μM phosphatidyl serine. Under these conditions LC113–25 was active in both assay systems with an IC40 of 40.7 and 57.0 μM respectively. 4 Deletion of amino acids from the C-terminus of the peptide progressively diminished (2–3 fold) the molar potency of LC113–25 in both assays: after the removal of Val22 biological activity was virtually undetectable or very weak (<30% of LC113–25). 5 Removal of amino acids from the N-terminus also lead to a progressive reduction (3–5 fold) in the molar potency of the peptides and biological activity became undetectable, or very weak, after the removal of Glu18. 6 All active peptides contained the core sequence EQEYV(Glu-Gln-Glu-Tyr-Val) which seems to represent a crucial component of the pharmacophore, although this sequence on its own was inactive and the shortest peptide with significant activity was LC118–25 (EQEYVQTV). 7 Methoxylation of Tyr21 abolished the ability of LC1 18–25 to inhibit cell proliferation and arachidonic acid release. A cyclized version of LC118–25 was also tested and found to be inactive. 8 LC118–25 (178 μM) inhibits cPLA2 activation in A549 cells as judged by a band-shift assay, whereas equimolar concentrations of an inactive peptide LC119–25 were without effect in this assay system. 9 Several possible mechanisms whereby these peptides act are discussed in the light of LC1 biology and of the effect of glucocorticoids on cell function. British Journal of Pharmacology (1998) 123, 975–983; doi:10.1038/sj.bjp.0701679
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Lipocortin 1 co-associates with cytokeratins 8 and 18 in A549 cells via the N-terminal domain
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1998Co-Authors: Jamie D. Croxtall, Hsi-yuan Yang, Bryan J. Smith, Chris Sutton, Bi Ing Chang, Guey Yueh Shi, Rod J. FlowerAbstract:Abstract An affinity chromatography strategy was used to search for proteins in A549 cells which interact with the N-terminus of Lipocortin 1 (annexin 1). Using the biologically active fragment Lc13–25 as the affinity ligand, two proteins of molecular weight (m.w.) 52 and 48 kDa were extracted. Affinity blots of these proteins bound iodinated Lc13–25. Partial tryptic digests of these proteins were analysed by matrix assisted laser desorption mass spectrometry and found to display fragmentation patterns with a strong similarity to those of cytokeratin 8 and 18 respectively. Subsequent blotting with a panel of specific cytokeratin antibodies strongly supported the idea that the two proteins were cytokeratin 8 and cytokeratin 18. Cytokeratin 8 was isolated from A549 cells in intermediate filament (IF) preparations which were also found to contain Lipocortin 1 as a potential intermediate filament associated protein (IFAP). This association persisted throughout cycles of IF assembly and disassembly. Dual-labelling immuno-histochemistry in A549 cells showed strong co-localization of Lipocortin 1 and cytokeratin 8. The implications of this finding are discussed in the light of the biological activity and possible function of Lipocortin 1.
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the co ordinate regulation of Lipocortin 1 cox 2 and cpla2 by il 1β in a549 cells
Advances in Experimental Medicine and Biology, 1997Co-Authors: S P Newman, Jamie D. Croxtall, Qam Choudhury, R J FlowerAbstract:In the A549 human lung adenocarcinoma cell line, generation of prostaglandin E2 (PGE2) is an important regulator of cell proliferation in vitro (1). Both epidermal growth factor (EGF) and the pro-inflammatory cytokine IL-1β up regulate cell proliferation by inducing PGE2 release. The PGE2 mediated increase in cell proliferation can be inhibited by dexamethasone. The inhibition of PGE2 release by the dexamethasone is mediated via the induction of Lipocortin 1 on the surface of A549 cells (1). Furthermore, we have shown the EGF simulated arachidonic acid release, PGE2 release and subsequent cell proliferation can all be repressed by the addition of fragments of Lipocortin-1 to the cells (2). These results imply that cell surface Lipocortin 1 can regulate arachidonic acid release, and when EGF simulated cells are treated with dexamethasone the increase in cell surface Lipocortin 1 is responsible for down-regulating arachidonic acid release, PGE2 release and cell proliferation.
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mobilizing Lipocortin 1 in adherent human leukocytes downregulates their transmigration
Nature Medicine, 1996Co-Authors: Mauro Perretti, Jamie D. Croxtall, Robert Hannon, Samantha K Wheller, Nicolas J Goulding, Roderick J. FlowerAbstract:Polymorphonuclear leukocyte (PMN) migration into sites of inflammation is fundamental to the host defense response. Activation of endothelial cells and PMNs increases the expression or activation of adhesion molecules, culminating in rolling and subsequent adherence of these cells to the vascular wall. Further activation of adherent PMNs, possibly by endothelial cell ligands, leads, within a few minutes, to extravasation itself. This process is not clearly understood, but adhesion molecules or related proteins, as well as endogenous chemokines, may play an important role. The anti-inflammatory glucocorticoids delay extravasation, which implies that an inhibitory regulatory system exists. Resting PMNs contain abundant cytoplasmic Lipocortin 1 (LC1, also called annexin I)', and the activity profile of this protein suggests that it could reduce PMN responsiveness. To investigate this we have assessed neutrophil transmigration both in vivo and in vitro and examined the content and subcellular distribution of LC1 in PMNs by fluorescence-activated cell-sorting (FACS) analysis, western blotting and confocal microscopy. We report that LC1 is mobilized and externalized following PMN adhesion to endothelial monolayers in vitro or to venular endothelium in vivo and that the end point of this process is a negative regulation of PMN transendothelial passage.
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Lipocortin 1 and the control of cpla2 activity in a549 cells glucocorticoids block egf stimulation of cpla2 phosphorylation
Biochemical Pharmacology, 1996Co-Authors: Jamie D. Croxtall, Qam Choudhury, Simon Newman, R J FlowerAbstract:Epidermal growth factor (EGF) rapidly stimulates the release of arachidonic acid in A549 cells by a mechanism that is sensitive to pertussis toxin [1]. We show that EGF treatment of A549 cells stimulates phosphorylation of cytosolic phospholipase A2 (cPLA2) through a mechanism that is similarly inhibited by pertussis toxin. The level of cPLA2 expression is, apparently, not changed during this period. Pretreatment of cells with dexamethasone (10-100 nM) for 3 hr prevents this activation of cPLA2 by EFG, without changing the level of cPLA21 expression. The effect of dexamethasone is reversed in the presence of the neutralizing antiLipocortin Mab 1A but not by the nonneutralizing antiLipocortin 1 control Mab 1B. This strongly suggests that Lipocortin 1 mediates the effect of dexamethasone by inhibiting activation of cPLA2. This concept is supported by the fact that a peptide Lc13-25 (10-200 micrograms/mL), derived from the N-terminus of Lipocortin 1, also inhibits activation of cPLA2 by EGF in these cells.
A D Taylor - One of the best experts on this subject based on the ideXlab platform.
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Reverses the Inhibitory Actions of Dexamethasone on the Release of Adrenocorticotropin from Rat Pituitary Tissue in Vitro*
2013Co-Authors: An Antisense, R J Flower, A D Taylor, Hc Christian, J. F. Morris, Oligodeoxynucleotide Lipocortin, Jc BuckinghamAbstract:Our previous studies have demonstrated that Lipocortin 1 (LC1, also called annexin 1) is an important mediator of glucocorticoid action in the neuroendocrine system, particularly with regard to the powerful inhibitory actions of the steroids on the secretion of ACTH and its hypothalamic releasing hormones. In the present study, we have used an antisense oligodeoxynucleotide (ODN) unique to LC1 to investigate further the role of this protein in the regulatory effects of dexamethasone on ACTH release in vitro from rat anterior pituitary cells. Pituitary cells dispersed with collagenase retained their functional and morphological integrity in vitro and sequestered ODNs in a time-dependent manner from the incubation medium. LC1 was readily detected in the cells by Western blot analysis or by immunoprecipitation/autoradiography after preloading with 35 S-methionine/ cysteine; the bulk of the protein was contained within an intracellula
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Printed in U.S.A. Copyright © 1997 by The Endocrine Society Characterization and Localization of Lipocortin 1- Binding Sites on Rat Anterior Pituitary Cells by
2013Co-Authors: Fluorescence-activated Cell Analysis/sorting, R J Flower, A D Taylor, Hc Christian, J. F. Morris, Jc BuckinghamAbstract:Lipocortin 1 (LC1; also called annexin 1) is a 37-kDa member of the annexin superfamily of Ca 2 �- and phospholipid-binding proteins (1–3). Since its initial identification in extracts of conditioned medium from glucocorticoid-stimulated peritoneal macrophages (4), LC1 has been shown to exhibit a wide range of glucocorticoid-like activities and has been widely advocated as a mediator of these steroids, particularly in immune/inflammatory cells (2, 3) and the neuroendocrine system (2, 5). Our recent in vivo and in vitro studies have identified a significant role for LC1 in effecting the negative feedback action of the glucocorticoids in the hypothalamo-pituitary-adrenal axis at the levels of both the hypothalamus (5–7) and the anterior pituitary gland (5, 8). Further studies have shown that LC1 also contributes to the less well documented inhibitory actions of the glucocorticoids on the release of PRL (9), GH (10), and TSH (11). LC1 is expressed in abundance in the anterior pituitary gland where, as in several peripheral cell types (12, 13), both it
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annexin 1 Lipocortin 1 mediates the glucocorticoid inhibition of cyclic adenosine 3 5 monophosphate stimulated prolactin secretion
Endocrinology, 2000Co-Authors: A D Taylor, R J Flower, James G. Philip, C D John, Patricia O Cover, John Morris, Julia C. BuckinghamAbstract:Our previous studies have identified a role for annexin 1 (also called Lipocortin 1) in the regulatory actions of glucocorticoids (GCs) on the release of PRL from the rat anterior pituitary gland. In the present study we used antisense and immunoneutralization strategies to extend this work. Exposure of rat anterior pituitary tissue to corticosterone (1 nm) or dexamethasone (100 nm) in vitro induced 1) de novo annexin 1 synthesis and 2) translocation of the protein from intracellular to pericellular sites. Both responses were prevented by the inclusion in the medium of an annexin 1 antisense oligodeoxynucleotide (ODN; 50 nm), but not by the corresponding sense and scrambled ODN sequences. Unlike the GCs, 17β-estradiol, testosterone, and aldosterone (1 nm) had no effect on either the synthesis or the cellular disposition of annexin 1; moreover, none of the steroids or ODNs tested influenced the expression of annexin 5, a protein closely related to annexin 1. The increases in PRL release induced in vitro by...
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characterization and localization of Lipocortin 1 binding sites on rat anterior pituitary cells by fluorescence activated cell analysis sorting and electron microscopy
Endocrinology, 1997Co-Authors: Helen C Christian, R J Flower, A D Taylor, J. F. Morris, Julia C. BuckinghamAbstract:Lipocortin 1 (LC1) is an important mediator of glucocorticoid action in the anterior pituitary gland, where it appears to act via cell surface binding sites to suppress peptide release. We have exploited a combination of fluorescence-activated cell (FAC) analysis/sorting and electron microscopy to detect, characterize, and localize LC1-binding sites on the surface of dispersed rat anterior pituitary cells, using human recombinant LC1 (hu-r-LC1) as a probe. High affinity (Kd = 14 +/- 3 nM) hu-r-LC1-binding sites were detected on approximately 80% of anterior pituitary cells dispersed with collagenase. The binding characteristics of the ligand resembled those observed in leukocytes, in that it was saturable; concentration, Ca2+, and temperature dependent; and abolished by trypsin. Functional studies demonstrated an excellent correlation between the presence of the cell surface binding protein and the capacity of an anti-LC1 monoclonal antibody to abrogate the inhibitory actions of dexamethasone (10 nM) on the release of ACTH initiated in vitro by CRH-41 (1 nM). Morphological analysis of cells harvested by FAC sorting showed that 1) somatotrophs, corticotrophs, lactotrophs, thyrotrophs, and gonadotrophs were all included in the population expressing LC1 binding sites; and 2) the LC1-binding sites assume a punctate distribution across the cell surface. These data show that anterior pituitary cells express high affinity surface LC1-binding protein(s); they thus provide further evidence for a specific membrane mechanism of action of LC1 in regulating the endocrine function of the anterior pituitary.
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An antisense oligodeoxynucleotide to Lipocortin 1 reverses the inhibitory actions of dexamethasone on the release of adrenocorticotropin from rat pituitary tissue in vitro.
Endocrinology, 1997Co-Authors: A D Taylor, Rod J. Flower, J F Morris, Helen C Christian, Jc BuckinghamAbstract:Our previous studies have demonstrated that Lipocortin 1 (LC1, also called annexin 1) is an important mediator of glucocorticoid action in the neuroendocrine system, particularly with regard to the powerful inhibitory actions of the steroids on the secretion of ACTH and its hypothalamic releasing hormones. In the present study, we have used an antisense oligodeoxynucleotide (ODN) unique to LC1 to investigate further the role of this protein in the regulatory effects of dexamethasone on ACTH release in vitro from rat anterior pituitary cells. Pituitary cells dispersed with collagenase retained their functional and morphological integrity in vitro and sequestered ODNs in a time-dependent manner from the incubation medium. LC1 was readily detected in the cells by Western blot analysis or by immunoprecipitation/autoradiography after preloading with 35S-methionine/cysteine; the bulk of the protein was contained within an intracellular pool but a small amount was attached to the outer cell surface (pericellular...