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Miles D Houslay - One of the best experts on this subject based on the ideXlab platform.
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disruption of the Cyclic AMP Phosphodiesterase 4 pde4 hsp20 complex attenuates the β agonist induced hypertrophic response in cardiac myocytes
Journal of Molecular and Cellular Cardiology, 2011Co-Authors: Yuan Yan Sin, Miles D Houslay, Frank Christian, Jonathan P Day, Allan J Dunlop, Helen V Edwards, Dave Adams, Manuela Zaccolo, George S BaillieAbstract:Abstract The small heat shock protein HSP20 is known to be cardioprotective during times of stress and the mechanism underlying its protective abilities depends on its phosphorylation on Ser16 by PKA (protein kinase A). Although the external stimuli that trigger Ser16 phosphorylation have been well studied, the events that modulate spatial and temporal control of this modification remain to be clarified. Here, we report that inhibition of cAMP Phosphodiesterase-4 (PDE4) induces the phosphorylation of HSP20 in resting cardiac myocytes and augments its phosphorylation by PKA following β-adrenergic stimulation. Moreover, using peptide array technology, in vitro binding studies, co-immunoprecipitation techniques and immunocytochemistry, we show that HSP20 binds directly to PDE4 within a region of the conserved catalytic domain. We also show that FRET-based, genetically-encoded cAMP reporters anchored to HSP20 exhibit a larger response to PDE4 inhibition compared to free cytosolic cAMP reporters, suggesting that the interaction with PDE4 is crucial in modulating the highly localised pool of cAMP to which HSP20 is exposed. Using information gleaned from peptide array analyses, we developed a cell-permeable peptide that serves to inhibit the interaction of PDE4 with HSP20. Disruption of the HSP20–PDE4 complex, using this peptide, suffices to induce phosphorylation of HSP20 by PKA and to protect against the hypertrophic response measured in neonatal cardiac myocytes following chronic β-adrenergic stimulation.
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Cyclic AMP Phosphodiesterase 4d pde4d tethers epac1 in a vascular endothelial cadherin ve cad based signaling complex and controls cAMP mediated vascular permeability
Journal of Biological Chemistry, 2010Co-Authors: Sarah Rampersad, Jeffrey D Ovens, Elaine Huston, Bibiana M Umana, Lindsay S Wilson, Stuart J Netherton, Martin J Lynch, George S Baillie, Miles D Houslay, Donald H MauriceAbstract:Vascular endothelial cell (VEC) permeability is largely dependent on the integrity of vascular endothelial cadherin (VE-cadherin or VE-Cad)-based intercellular adhesions. Activators of protein kinase A (PKA) or of exchange protein activated by cAMP (EPAC) reduce VEC permeability largely by stabilizing VE-Cad-based intercellular adhesions. Currently, little is known concerning the nature and composition of the signaling complexes that allow PKA or EPAC to regulate VE-Cad-based structures and through these actions control permeability. Using pharmacological, biochemical, and cell biological approaches we identified and determined the composition and functionality of a signaling complex that coordinates cAMP-mediated control of VE-Cad-based adhesions and VEC permeability. Thus, we report that PKA, EPAC1, and Cyclic nucleotide Phosphodiesterase 4D (PDE4D) enzymes integrate into VE-Cad-based signaling complexes in human arterial endothelial cells. Importantly, we show that protein-protein interactions between EPAC1 and PDE4D serve to foster their integration into VE-Cad-based complexes and allow robust local regulation of EPAC1-based stabilization of VE-Cad-based adhesions. Of potential translational importance, we mapped the EPAC1 peptide motif involved in binding PDE4D and show that a cell-permeable variant of this peptide antagonizes EPAC1-PDE4D binding and directly alters VEC permeability. Collectively, our data indicate that PDE4D regulates both the activity and subcellular localization of EPAC1 and identify a novel mechanism for regulated EPAC1 signaling in these cells.
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p62 sqstm1 and Cyclic AMP Phosphodiesterase 4a4 pde4a4 locate to a novel reversible protein aggregate with links to autophagy and proteasome degradation pathways
Cellular Signalling, 2010Co-Authors: Frank Christian, George S Baillie, Diana F Anthony, Suryakiran Vadrevu, Tracy Riddell, Jonathan P Day, Ruth Mcleod, David R Adams, Miles D HouslayAbstract:Chronic challenge of Cyclic AMP Phosphodiesterase-4A4 (PDE4A4) with certain PDE4 selective inhibitors causes it to reversibly form intracellular aggregates that are not membrane-encapsulated. These aggregates are neither stress granules (SGs) nor processing bodies (PBs) as they contain neither PABP-1 nor Dcp1a, respectively. However, the PDE4 inhibitor rolipram decreases arsenite-induced SGs and increases the amount of PBs, while arsenite challenge ablates rolipram-induced PDE4A4 aggregates. PDE4A4 aggregates are neither autophagic vesicles (autophagosomes) nor aggresomes, although microtubule disruptors ablate PDE4A4 aggregate formation. PDE4A4 constitutively co-immunoprecipitates with p62 protein (sequestosome1, SQSTM1), which locates to both PDE4A4 aggregates and autophagosomes in cells constitutively challenged with rolipram. The mTor inhibitor, rapamycin, activates autophagy, prevents PDE4A4 from forming intracellular aggregates and triggers the loss of bound p62 from PDE4A4. siRNA-mediated knockdown of p62 attenuates PDE4A4 aggregate formation. The p62-binding protein, light chain 3 (LC3), is not found in PDE4A4 aggregates. Blockade of proteasome activity and activation of autophagy with MG132 both increases the level of ubiquitinated proteins found associated with PDE4A4 and inhibits PDE4A4 aggregate formation. Activation of autophagy with either thapsigargin or ionomycin inhibits PDE4A4 aggregate formation. Inhibition of autophagy with either wortmannin or LY294002 activates PDE4A4 aggregate formation. The protein kinase C inhibitors, RO 320432 and GO 6983, and the ERK inhibitors UO 126 and PD 98059 all activated PDE4A4 aggregate formation, whilst roscovitine, thalidomide and the tyrosine kinase inhibitors, genistein and AG17, all inhibited this process. We suggest that the fate of p62-containing protein aggregates need not necessarily be terminal, through delivery to autophagic vesicles and aggresomes. Instead, we propose a novel regulatory mechanism where a sub-population of p62-containing protein aggregates would form in a rapid, reversible manner so as to sequester specific cargo away from their normal, functionally important site(s) within the cell. Thus an appropriate conformational change in the target protein would confer reversible recruitment into a sub-population of p62-containing protein aggregates and so provide a regulatory function by removing these cargo proteins from their functionally important site(s) in a cell.
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in cardiac myocytes cAMP elevation triggers the down regulation of transcripts and promoter activity for Cyclic AMP Phosphodiesterase 4a10 pde4a10
Cellular Signalling, 2008Co-Authors: Angela Mccahill, Martin J Lynch, George S Baillie, Lachlan Campbell, Theresa Mcsorley, Arvind Sood, Chen Yan, Miles D HouslayAbstract:Transcripts for the PDE4A10 Cyclic AMP Phosphodiesterase isoform are present in a wide variety of rat tissues including the heart. Sequence comparisons between the putative human and mouse promoters revealed a number of conserved regions including both an Sp1 and a CREB-binding site. The putative mouse PDE4A10 promoter was AMPlified from genomic DNA and sub-cloned into a luciferase reporter vector for investigation of activity in neonatal cardiac myocytes. Transfection with this construct identified a high level of luciferase expression in neonatal cardiac myocytes. Surprisingly, this activity was down-regulated by elevation of intracellular cAMP through a process involving PKA, but not EPAC, signalling. Such inhibition of the rodent PDE4A10 promoter activity in response to elevated cAMP levels is in contrast to the PDE4 promoters so far described. Site-directed mutagenesis revealed that the Sp1 binding site at promoter position -348 to -336 is responsible for the basal constitutive expression of murine PDE4A10. The conserved CREB-binding motif at position -370 to -363 also contributes to basal promoter activity but does not in itself confer cAMP inhibition upon the PDE4A10 promoter. EMSA analysis confirmed the authenticity of CREB and Sp1 binding sites. The transcriptional start site was identified to be an adenine residue at position -55 in the mouse PDE4A10 promoter. We present evidence that this novel down-regulation of PDE4A10 is mediated by the transcription factor ICER in a PKA dependent manner. The pool of cAMP in cardiac myocytes that down-regulates PDE4A10 is regulated by beta-adrenoceptor coupled adenylyl cyclase activity and via hydrolysis determined predominantly by the action of PDE4 (cAMP Phosphodiesterase-4) and not PDE3 (cAMP Phosphodiesterase-3). We suggest that increased cAMP may remodel cAMP-mediated signalling events by not only increasing the expression of specific PDE4 cAMP Phosphodiesterases but also by down-regulating specific isoforms, such as is shown here for PDE4A10 in cardiac myocytes.
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noradrenergic activity differentially regulates the expression of rolipram sensitive high affinity Cyclic AMP Phosphodiesterase pde4 in rat brain
Journal of Neurochemistry, 2002Co-Authors: Marco Conti, Miles D Houslay, Shakeel M Farooqui, Ming Chen, James M OdonnellAbstract:Ina previousstudy,it wasobservedthat the activityof rolipram-sensitive, low-Km,Cyclic AMPphos- phodiesterase (PDE4)wasdecreasedin vivowith dimin- ishednoradrenergic stimulation.Theresultsof the pres- entexperiments indicatedthat thereductionintheactivity maybeassociatedwithdown-regulation of PDE4protein. Immunoblotanalysis using PDE4-specific,subfamily- nonspecificantibody(K116)revealedfour majorbands ofPDE4in ratcerebralcortex;thosewithapparentmolec- ularmassesof 109 and 102kDaare variantsof PDE4A. Diminished noradrenergic activity,producedby intracere- broventricular infusionof 6-hydroxydopamine (6-OHDA) or chronic subcutaneousinfusion of propranolol,de- creasedthe intensitiesof the proteinbandsfor the 109- and 102-kDaPDE4Avariantsin rat cerebralcortex but notof the 98- or 91-kDaPDE4forms. 6-OHDA-induced noradrenergiclesioningalso decreasedthe content of 102-kDaPDE4Ain hippocAMPusas labeledby PDE4A- specific antibody(C-PDE4A).Enhancednoradrenergic stimulationup-regulatedPDE4in cerebralcortex. This wasindicatedby thefindingthat repeatedtreatmentwith desipramineincreasedthe intensityof the protein band for the 102-kDaPDE4but not for the other variantsof PDE4.Theseresultssuggestthat PDE4subtypesaredif- ferentiallyregulatedat the level of expression,as evi- dencedby an apparentchangein the amountof PDE4 protein, following changes in noradrenergicactivity. Theseobservationsare consistentwith the notion that PDE4s,especiallythe PDE4Avariantswith molecular massesof 109 and 102 kDa,play an importantrole in maintaining the homeostasisof the noradrenergicsignal transductionsystemin the brainand may be involved in the mediationof antidepressantactivity. KeyWords: Phosphodiesterase— Noradrenergicsystem — Immuno- blot analysis—6-Hydroxydopamine—Propranolol—De- sipramine. J. Neurochem.69, 2397—2404 (1997).
James M Odonnell - One of the best experts on this subject based on the ideXlab platform.
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diminished noradrenergic stimulation reduces the activity of rolipram sensitive high affinity Cyclic AMP Phosphodiesterase in rat cerebral cortex
Journal of Neurochemistry, 2002Co-Authors: James M OdonnellAbstract:The present study examined the in vivo regulation of rolipram-sensitive, high-affinity Cyclic AMP Phosphodiesterase (PDE4) in rat cerebral cortex. The hydrolysis of Cyclic AMP, formed by stimulation of beta-adrenergic receptors, was measured in cerebral cortical slices. Hydrolysis of Cyclic AMP formed under these conditions was inhibited by the PDE4-selective inhibitor rolipram but not by selective inhibitors of other PDE families. Intraventricular infusion of 6-hydroxydopamine (6-OHDA; 200 micrograms) decreased the rate constant of Cyclic AMP hydrolysis and increased the Cyclic AMP half-life 17 days, but not 1 or 7 days, following the treatment. A reduction in norepinephrine (NE) content occurred first; the NE level was reduced to 42, 24, and 6% of control at 1, 7, and 17 days after 6-OHDA infusion, respectively. This was followed by the development of supersensitivity of beta-adrenergic receptor-linked adenylyl cyclase, which occurred 7 days after the infusion. The reduction in PDE4 activity occurred last. When a higher dose of 6-OHDA (300 micrograms) was used, the reduction in the rate constant of Cyclic AMP hydrolysis occurred by 7 days; at this time NE content was depleted to 6% of control. Similar to 6-OHDA treatment, continuous blockade of beta-adrenergic receptors, produced by chronic propranolol infusion, decreased the rate constant of Cyclic AMP hydrolysis. Therefore, the current results indicate that diminished stimulation of beta-adrenergic receptors, either by loss of noradrenergic innervation or by receptor blockade, reduces the activity of PDE4. This suggests that PDE4 regulation may contribute in the homeostasis of the noradrenergic receptor-effector system in the brain.
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noradrenergic activity differentially regulates the expression of rolipram sensitive high affinity Cyclic AMP Phosphodiesterase pde4 in rat brain
Journal of Neurochemistry, 2002Co-Authors: Marco Conti, Miles D Houslay, Shakeel M Farooqui, Ming Chen, James M OdonnellAbstract:Ina previousstudy,it wasobservedthat the activityof rolipram-sensitive, low-Km,Cyclic AMPphos- phodiesterase (PDE4)wasdecreasedin vivowith dimin- ishednoradrenergic stimulation.Theresultsof the pres- entexperiments indicatedthat thereductionintheactivity maybeassociatedwithdown-regulation of PDE4protein. Immunoblotanalysis using PDE4-specific,subfamily- nonspecificantibody(K116)revealedfour majorbands ofPDE4in ratcerebralcortex;thosewithapparentmolec- ularmassesof 109 and 102kDaare variantsof PDE4A. Diminished noradrenergic activity,producedby intracere- broventricular infusionof 6-hydroxydopamine (6-OHDA) or chronic subcutaneousinfusion of propranolol,de- creasedthe intensitiesof the proteinbandsfor the 109- and 102-kDaPDE4Avariantsin rat cerebralcortex but notof the 98- or 91-kDaPDE4forms. 6-OHDA-induced noradrenergiclesioningalso decreasedthe content of 102-kDaPDE4Ain hippocAMPusas labeledby PDE4A- specific antibody(C-PDE4A).Enhancednoradrenergic stimulationup-regulatedPDE4in cerebralcortex. This wasindicatedby thefindingthat repeatedtreatmentwith desipramineincreasedthe intensityof the protein band for the 102-kDaPDE4but not for the other variantsof PDE4.Theseresultssuggestthat PDE4subtypesaredif- ferentiallyregulatedat the level of expression,as evi- dencedby an apparentchangein the amountof PDE4 protein, following changes in noradrenergicactivity. Theseobservationsare consistentwith the notion that PDE4s,especiallythe PDE4Avariantswith molecular massesof 109 and 102 kDa,play an importantrole in maintaining the homeostasisof the noradrenergicsignal transductionsystemin the brainand may be involved in the mediationof antidepressantactivity. KeyWords: Phosphodiesterase— Noradrenergicsystem — Immuno- blot analysis—6-Hydroxydopamine—Propranolol—De- sipramine. J. Neurochem.69, 2397—2404 (1997).
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inhibition of Cyclic AMP Phosphodiesterase pde4 reverses memory deficits associated with nmda receptor antagonism
Neuropsychopharmacology, 2000Co-Authors: Hanting Zhang, Alicia M Crissman, Nandakumar R Dorairaj, Judson L Chandler, James M OdonnellAbstract:Rolipram, a selective inhibitor of type 4 Cyclic AMP Phosphodiesterase (PDE4), completely reversed the amnesic effects of MK-801 on working and reference memory (F[4,64] = 11.10; p <.0001 and F[4,64] = 2.53; p <.05, respectively) at doses of 0.01-0.1 mg/kg in the radial-arm maze task. Similar antagonism by rolipram of the effects of MK-801 was observed on inhibitory avoidance behavior (F[3,35] = 190.8; p <.0001). In vitro evidence suggests that an increase in cAMP concentrations may mediate the observed behavioral effects of rolipram. In the absence of PDE4 inhibition, NMDA did not increase cAMP concentrations in primary cultures of rat cerebral cortical neurons. However, when PDE4 was inhibited with rolipram, NMDA markedly elevated cAMP. These observations suggest that PDE4 is an integral component of the NMDA receptor-mediated signal transduction pathway involved in memory processes. Inhibitors of PDE4 may act on this pathway to produce their effects on memory and may represent a new class of cognitive enhancers.
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antidepressant like effects of rolipram and other inhibitors of Cyclic adenosine monophosphate Phosphodiesterase on behavior maintained by differential reinforcement of low response rate
Journal of Pharmacology and Experimental Therapeutics, 1993Co-Authors: James M OdonnellAbstract:Four inhibitors of Cyclic AMP Phosphodiesterase (PDE), rolipram, Ro 20-1724, ICI 63,197 and CP 76,593, reduced response rates and increased reinforcement rates of rats under a differential reinforcement of low rate 72-sec schedule for water reinforcement in a dose-dependent manner. These actions of the PDE inhibitors are similar to those reported for proven antidepressant drugs. Administration of forskolin, which increases Cyclic AMP levels by activation of the catalytic subunit of adenylyl cyclase, either i.p. or i.c.v. did not mimic the effects of the PDE inhibitors. The behavioral effects of the PDE inhibitors were not antagonized by the beta adrenergic antagonist propranolol, suggesting that these drugs were not altering behavior via an increase in norepinephrine release and an indirect stimulation of beta adrenergic receptors. 6-Hydroxydopamine-induced lesions of noradrenergic neurons increased sensitivity to rolipram. This suggests that alterations in PDE activity with a concomitant increase in sensitivity to PDE inhibitors may occur subsequent to functional denervation.
George S Baillie - One of the best experts on this subject based on the ideXlab platform.
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heterozygous mutations in Cyclic AMP Phosphodiesterase 4d pde4d and protein kinase a pka provide new insights into the molecular pathology of acrodysostosis
Cellular Signalling, 2014Co-Authors: Tadashi Kaname, Young Bae Sohn, George S Baillie, Changseok Ki, Nobuo Mastuura, Norio Niikawa, Gen Nishimura, Ken Ichi Yamamura, Tohru Ohta, Ah Ra KoAbstract:Acrodysostosis without hormone resistance is a rare skeletal disorder characterized by brachydactyly, nasal hypoplasia, mental retardation and occasionally developmental delay. Recently, loss-of-function mutations in the gene encoding cAMP-hydrolyzing Phosphodiesterase-4D (PDE4D) have been reported to cause this rare condition but the pathomechanism has not been fully elucidated. To understand the pathogenetic mechanism of PDE4D mutations, we conducted 3D modeling studies to predict changes in the binding efficacy of cAMP to the catalytic pocket in PDE4D mutants. Our results indicated diminished enzyme activity in the two mutants we analyzed (Gly673Asp and Ile678Thr; based on PDE4D4 residue numbering). Ectopic expression of PDE4D mutants in HEK293 cells demonstrated this reduction in activity, which was identified by increased cAMP levels. However, the cells from an acrodysostosis patient showed low cAMP accumulation, which resulted in a decrease in the phosphorylated cAMP Response Element-Binding Protein (pCREB)/CREB ratio. The reason for this discrepancy was due to a compensatory increase in expression levels of PDE4A and PDE4B isoforms, which accounted for the paradoxical decrease in cAMP levels in the patient cells expressing mutant isoforms with a lowered PDE4D activity. Skeletal radiographs of 10-week-old knockout (KO) rats showed that the distal part of the forelimb was shorter than in wild-type (WT) rats and that all the metacarpals and phalanges were also shorter in KO, as the name acrodysostosis implies. Like the G-protein α-stimulatory subunit and PRKAR1A, PDE4D critically regulates the cAMP signal transduction pathway and influences bone formation in a way that activity-compromising PDE4D mutations can result in skeletal dysplasia. We propose that specific inhibitory PDE4D mutations can lead to the molecular pathology of acrodysostosis without hormone resistance but that the pathological phenotype may well be dependent on an over-compensatory induction of other PDE4 isoforms that can be expected to be targeted to different signaling complexes and exert distinct effects on compartmentalized cAMP signaling.
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mitotic activation of the disc1 inducible Cyclic AMP Phosphodiesterase 4d9 pde4d9 through multi site phosphorylation influences cell cycle progression
Cellular Signalling, 2014Co-Authors: Catherine L Sheppard, Diana F Anthony, Louisa C Y Lee, Elaine V Hill, David Henderson, Daniel M Houslay, Krishna C Yalla, Lynne S Cairns, Allan J Dunlop, George S BaillieAbstract:In Rat-1 cells, the dramatic decrease in the levels of both intracellular Cyclic 3′5′ adenosine monophosphate (Cyclic AMP; cAMP) and in the activity of cAMP-activated protein kinase A (PKA) observed in mitosis was paralleled by a profound increase in cAMP hydrolyzing Phosphodiesterase-4 (PDE4) activity. The decrease in PKA activity, which occurs during mitosis, was attributable to PDE4 activation as the PDE4 selective inhibitor, rolipram, but not the Phosphodiesterase-3 (PDE3) inhibitor, cilostamide, specifically ablated this cell cycle-dependent effect. PDE4 inhibition caused Rat-1 cells to move from S phase into G2/M more rapidly, to transit through G2/M more quickly and to remain in G1 for a longer period. Inhibition of PDE3 elicited no observable effects on cell cycle dynamics. Selective immunopurification of each of the four PDE4 sub-families identified PDE4D as being selectively activated in mitosis. Subsequent analysis uncovered PDE4D9, an isoform whose expression can be regulated by Disrupted-In-Schizophrenia 1 (DISC1)/activating transcription factor 4 (ATF4) complex, as the sole PDE4 species activated during mitosis in Rat-1 cells. PDE4D9 becomes activated in mitosis through dual phosphorylation at Ser585 and Ser245, involving the combined action of ERK and an unidentified ‘switch’ kinase that has previously been shown to be activated by H2O2. Additionally, in mitosis, PDE4D9 also becomes phosphorylated at Ser67 and Ser81, through the action of MK2 (MAPKAPK2) and AMP kinase (AMPK), respectively. The multisite phosphorylation of PDE4D9 by all four of these protein kinases leads to decreased mobility (band-shift) of PDE4D9 on SDS-PAGE. PDE4D9 is predominantly concentrated in the perinuclear region of Rat-1 cells but with a fraction distributed asymmetrically at the cell margins. Our investigations demonstrate that the diminished levels of cAMP and PKA activity that characterise mitosis are due to enhanced cAMP degradation by PDE4D9. PDE4D9, was found to locate primarily not only in the perinuclear region of Rat-1 cells but also at the cell margins. We propose that the sequestration of PDE4D9 in a specific complex together with AMPK, ERK, MK2 and the H2O2-activatable ‘switch’ kinase allows for its selective multi-site phosphorylation, activation and regulation in mitosis.
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disruption of the Cyclic AMP Phosphodiesterase 4 pde4 hsp20 complex attenuates the β agonist induced hypertrophic response in cardiac myocytes
Journal of Molecular and Cellular Cardiology, 2011Co-Authors: Yuan Yan Sin, Miles D Houslay, Frank Christian, Jonathan P Day, Allan J Dunlop, Helen V Edwards, Dave Adams, Manuela Zaccolo, George S BaillieAbstract:Abstract The small heat shock protein HSP20 is known to be cardioprotective during times of stress and the mechanism underlying its protective abilities depends on its phosphorylation on Ser16 by PKA (protein kinase A). Although the external stimuli that trigger Ser16 phosphorylation have been well studied, the events that modulate spatial and temporal control of this modification remain to be clarified. Here, we report that inhibition of cAMP Phosphodiesterase-4 (PDE4) induces the phosphorylation of HSP20 in resting cardiac myocytes and augments its phosphorylation by PKA following β-adrenergic stimulation. Moreover, using peptide array technology, in vitro binding studies, co-immunoprecipitation techniques and immunocytochemistry, we show that HSP20 binds directly to PDE4 within a region of the conserved catalytic domain. We also show that FRET-based, genetically-encoded cAMP reporters anchored to HSP20 exhibit a larger response to PDE4 inhibition compared to free cytosolic cAMP reporters, suggesting that the interaction with PDE4 is crucial in modulating the highly localised pool of cAMP to which HSP20 is exposed. Using information gleaned from peptide array analyses, we developed a cell-permeable peptide that serves to inhibit the interaction of PDE4 with HSP20. Disruption of the HSP20–PDE4 complex, using this peptide, suffices to induce phosphorylation of HSP20 by PKA and to protect against the hypertrophic response measured in neonatal cardiac myocytes following chronic β-adrenergic stimulation.
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Cyclic AMP Phosphodiesterase 4d pde4d tethers epac1 in a vascular endothelial cadherin ve cad based signaling complex and controls cAMP mediated vascular permeability
Journal of Biological Chemistry, 2010Co-Authors: Sarah Rampersad, Jeffrey D Ovens, Elaine Huston, Bibiana M Umana, Lindsay S Wilson, Stuart J Netherton, Martin J Lynch, George S Baillie, Miles D Houslay, Donald H MauriceAbstract:Vascular endothelial cell (VEC) permeability is largely dependent on the integrity of vascular endothelial cadherin (VE-cadherin or VE-Cad)-based intercellular adhesions. Activators of protein kinase A (PKA) or of exchange protein activated by cAMP (EPAC) reduce VEC permeability largely by stabilizing VE-Cad-based intercellular adhesions. Currently, little is known concerning the nature and composition of the signaling complexes that allow PKA or EPAC to regulate VE-Cad-based structures and through these actions control permeability. Using pharmacological, biochemical, and cell biological approaches we identified and determined the composition and functionality of a signaling complex that coordinates cAMP-mediated control of VE-Cad-based adhesions and VEC permeability. Thus, we report that PKA, EPAC1, and Cyclic nucleotide Phosphodiesterase 4D (PDE4D) enzymes integrate into VE-Cad-based signaling complexes in human arterial endothelial cells. Importantly, we show that protein-protein interactions between EPAC1 and PDE4D serve to foster their integration into VE-Cad-based complexes and allow robust local regulation of EPAC1-based stabilization of VE-Cad-based adhesions. Of potential translational importance, we mapped the EPAC1 peptide motif involved in binding PDE4D and show that a cell-permeable variant of this peptide antagonizes EPAC1-PDE4D binding and directly alters VEC permeability. Collectively, our data indicate that PDE4D regulates both the activity and subcellular localization of EPAC1 and identify a novel mechanism for regulated EPAC1 signaling in these cells.
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p62 sqstm1 and Cyclic AMP Phosphodiesterase 4a4 pde4a4 locate to a novel reversible protein aggregate with links to autophagy and proteasome degradation pathways
Cellular Signalling, 2010Co-Authors: Frank Christian, George S Baillie, Diana F Anthony, Suryakiran Vadrevu, Tracy Riddell, Jonathan P Day, Ruth Mcleod, David R Adams, Miles D HouslayAbstract:Chronic challenge of Cyclic AMP Phosphodiesterase-4A4 (PDE4A4) with certain PDE4 selective inhibitors causes it to reversibly form intracellular aggregates that are not membrane-encapsulated. These aggregates are neither stress granules (SGs) nor processing bodies (PBs) as they contain neither PABP-1 nor Dcp1a, respectively. However, the PDE4 inhibitor rolipram decreases arsenite-induced SGs and increases the amount of PBs, while arsenite challenge ablates rolipram-induced PDE4A4 aggregates. PDE4A4 aggregates are neither autophagic vesicles (autophagosomes) nor aggresomes, although microtubule disruptors ablate PDE4A4 aggregate formation. PDE4A4 constitutively co-immunoprecipitates with p62 protein (sequestosome1, SQSTM1), which locates to both PDE4A4 aggregates and autophagosomes in cells constitutively challenged with rolipram. The mTor inhibitor, rapamycin, activates autophagy, prevents PDE4A4 from forming intracellular aggregates and triggers the loss of bound p62 from PDE4A4. siRNA-mediated knockdown of p62 attenuates PDE4A4 aggregate formation. The p62-binding protein, light chain 3 (LC3), is not found in PDE4A4 aggregates. Blockade of proteasome activity and activation of autophagy with MG132 both increases the level of ubiquitinated proteins found associated with PDE4A4 and inhibits PDE4A4 aggregate formation. Activation of autophagy with either thapsigargin or ionomycin inhibits PDE4A4 aggregate formation. Inhibition of autophagy with either wortmannin or LY294002 activates PDE4A4 aggregate formation. The protein kinase C inhibitors, RO 320432 and GO 6983, and the ERK inhibitors UO 126 and PD 98059 all activated PDE4A4 aggregate formation, whilst roscovitine, thalidomide and the tyrosine kinase inhibitors, genistein and AG17, all inhibited this process. We suggest that the fate of p62-containing protein aggregates need not necessarily be terminal, through delivery to autophagic vesicles and aggresomes. Instead, we propose a novel regulatory mechanism where a sub-population of p62-containing protein aggregates would form in a rapid, reversible manner so as to sequester specific cargo away from their normal, functionally important site(s) within the cell. Thus an appropriate conformational change in the target protein would confer reversible recruitment into a sub-population of p62-containing protein aggregates and so provide a regulatory function by removing these cargo proteins from their functionally important site(s) in a cell.
Donald H Maurice - One of the best experts on this subject based on the ideXlab platform.
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Cyclic AMP Phosphodiesterase 4d pde4d tethers epac1 in a vascular endothelial cadherin ve cad based signaling complex and controls cAMP mediated vascular permeability
Journal of Biological Chemistry, 2010Co-Authors: Sarah Rampersad, Jeffrey D Ovens, Elaine Huston, Bibiana M Umana, Lindsay S Wilson, Stuart J Netherton, Martin J Lynch, George S Baillie, Miles D Houslay, Donald H MauriceAbstract:Vascular endothelial cell (VEC) permeability is largely dependent on the integrity of vascular endothelial cadherin (VE-cadherin or VE-Cad)-based intercellular adhesions. Activators of protein kinase A (PKA) or of exchange protein activated by cAMP (EPAC) reduce VEC permeability largely by stabilizing VE-Cad-based intercellular adhesions. Currently, little is known concerning the nature and composition of the signaling complexes that allow PKA or EPAC to regulate VE-Cad-based structures and through these actions control permeability. Using pharmacological, biochemical, and cell biological approaches we identified and determined the composition and functionality of a signaling complex that coordinates cAMP-mediated control of VE-Cad-based adhesions and VEC permeability. Thus, we report that PKA, EPAC1, and Cyclic nucleotide Phosphodiesterase 4D (PDE4D) enzymes integrate into VE-Cad-based signaling complexes in human arterial endothelial cells. Importantly, we show that protein-protein interactions between EPAC1 and PDE4D serve to foster their integration into VE-Cad-based complexes and allow robust local regulation of EPAC1-based stabilization of VE-Cad-based adhesions. Of potential translational importance, we mapped the EPAC1 peptide motif involved in binding PDE4D and show that a cell-permeable variant of this peptide antagonizes EPAC1-PDE4D binding and directly alters VEC permeability. Collectively, our data indicate that PDE4D regulates both the activity and subcellular localization of EPAC1 and identify a novel mechanism for regulated EPAC1 signaling in these cells.
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Cyclic AMP mediated regulation of vascular smooth muscle cell Cyclic AMP Phosphodiesterase activity
British Journal of Pharmacology, 1997Co-Authors: R J Rose, Hanguan Liu, D Palmer, Donald H MauriceAbstract:1. Rat cultured aortic vascular smooth muscle cells (VSMC) express both Cyclic GMP-inhibited Cyclic AMP Phosphodiesterase (PDE3) and Ro 20-1724-inhibited Cyclic AMP Phosphodiesterase (PDE4) activities. By utilizing either cilostamide, a PDE3-selective inhibitor, or Ro 20-1724, a PDE4-selective inhibitor, PDE3 and PDE4 activities were shown to account for 15% and 55% of total VSMC Cyclic AMP Phosphodiesterase (PDE) activity. 2. Treatment of VSMC with either forskolin or 8-bromo-Cyclic AMP caused significant concentration- and time-dependent increases in total cellular Cyclic AMP PDE activity. Using cilostamide or Ro 20-1724, we demonstrated that both PDE3 and PDE4 activities were increased following forskolin or 8-bromo-Cyclic AMP treatment, with a relatively larger effect observed on PDE3 activity. The increase in Cyclic AMP PDE activity induced by forskolin or 8-bromo-Cyclic AMP was inhibited by actinomycin D or cycloheximide, demonstrating that new mRNA synthesis and protein synthesis were required. An analogue of forskolin which does not activate adenylyl cyclase (1,9-dideoxyforskolin) or an analogue of Cyclic GMP (8-bromo-Cyclic GMP) did not affect total Cyclic AMP PDE activity. 3. Incubation of VSMC with 8-bromo-Cyclic AMP for 16 h caused a marked rightward shift in the concentration-response curves for both isoprenaline- and forskolin-mediated activation of adenylyl cyclase. A role for up-regulated Cyclic AMP PDE activity in this reduced potency is supported by our observation that Cyclic AMP PDE inhibitors (IBMX, cilostamide or Ro 20-1724) partially normalized the effects of isoprenaline or forskolin in treated cells to those in untreated cells. 4. We conclude that VSMC Cyclic AMP PDE activity is increased following long-term elevation of Cyclic AMP and that increases in PDE3 and PDE4 activities account for more than 70% of this effect. Furthermore, we conclude that increases in Cyclic AMP PDE activity contribute to the reduced potency of isoprenaline or forskolin in treated VSMC. These results have implications for long-term use of Cyclic AMP PDE inhibitors as therapeutic agents.
John E Souness - One of the best experts on this subject based on the ideXlab platform.
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evidence that Cyclic AMP Phosphodiesterase inhibitors suppress tnfα generation from human monocytes by interacting with a low affinity Phosphodiesterase 4 conformer
British Journal of Pharmacology, 1996Co-Authors: John E Souness, Miriam Griffin, Christopher Maslen, Karen Ebsworth, Lisa C Scott, Kenneth Pollock, Palfreyman Malcolm Norman, Jananders KarlssonAbstract:1. We have investigated the inhibitory effects of RP 73401 (piclamilast) and rolipram against human monocyte Cyclic AMP-specific Phosphodiesterase (PDE4) in relation to their effects on prostaglandin (PG)E2-induced Cyclic AMP accumulation and lipopolysaccharide (LPS)-induced TNF alpha production and TNF alpha mRNA expression. 2. PDE4 was found to be the predominant PDE isoenzyme in the cytosolic fraction of human monocytes. Cyclic GMP-inhibited PDE (PDE3) was also detected in the cytosolic and particulate fractions. Reverse transcription polymerase chain reaction (RT-PCR) of human monocyte poly (A+) mRNA revealed AMPlified products corresponding to PDE4 subtypes A and B of which the former was most highly expressed. A faint band corresponding in size to PDE4D was also observed. 3. RP 73401 was a potent inhibitor of cytosolic PDE4 (IC50: 1.5 +/- 0.6 nM, n = 3). (+/-)-Rolipram (IC50: 313 +/- 6.7 nM, n = 3) was at least 200 fold less potent than RP 73401. R-(-)-rolipram was approximately 3 fold more potent than S-(+)-rolipram against cytosolic PDE4. 4. RP 73401 (IC50: 9.2 +/- 2.1 nM, n = 6) was over 50 fold more potent than (+/-)-rolipram (IC50: 503 +/- 134 nM, n = 6) ) in potentiating PGE2-induced Cyclic AMP accumulation. R-(-)-rolipram (IC50: 289 +/- 121 nM, n = 5) was 4.7 fold more potent than its S-(+)-enantiomer (IC50: 1356 +/- 314 nM, n = 5). A strong and highly-significant, linear correlation (r = 0.95, P < 0.01, n = 13) was observed between the inhibitory potencies of a range of structurally distinct PDE4 inhibitors against monocyte PDE4 and their ED50 values in enhancing monocyte Cyclic AMP accumulation. A poorer, though still significant, linear correlation (r = 0.67, P < 0.01, n = 13) was observed between the potencies of the same compounds in potentiating PGE2-induced monocyte Cyclic AMP accumulation and their abilities to displace [3H]-rolipram binding to brain membranes. 5. RP 73401 (IC50: 6.9 +/- 3.3 nM, n = 5) was 71 fold more potent than (+/-)-rolipram (IC50: 490 +/- 260 nM, n = 4) in inhibiting LPS-induced TNF alpha release from monocytes. R-(-)-rolipram (IC50: 397 +/- 178 nM, n = 3) was 5.2-fold more potent than its S-(+)- enantiomer (IC50: 2067 +/- 659 nM, n = 3). As with Cyclic AMP, accumulation a closer, linear correlation existed between the potency of structurally distinct compounds in suppressing TNF alpha with PDE4 inhibition (r = 0.93, P < 0.01, n = 13) than with displacement of [3H]-rolipram binding (r = 0.65, P < 0.01, n = 13). 6. RP 73401 (IC50: 2 nM) was 180 fold more potent than rolipram (IC50: 360 nM) in suppressing LPS (10 ng ml-1)-induced TNF alpha mRNA. 7. The results demonstrate that RP 73401 is a very potent inhibitor of TNF alpha release from human monocytes suggesting that it may have therapeutic potential in the many pathological conditions associated with over-production of this pro-inflammatory cytokine. Furthermore, PDE inhibitor actions on functional responses are better correlated with inhibition of PDE4 catalytic activity than displacement of [3H]-rolipram from its high-affinity binding site, suggesting that the native PDE4 in human monocytes exists predominantly in a 'low-affinity' state.
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inhibition of pig aortic smooth muscle cell dna synthesis by selective type iii and type iv Cyclic AMP Phosphodiesterase inhibitors
Biochemical Pharmacology, 1992Co-Authors: John E Souness, Giles A Hassall, David P ParrottAbstract:Abstract Foetal calf serum (FCS) and platelet-derived growth factor (PDGF)-stimulated incorporation of [ 3 H]thymidine into pig aortic smooth muscle cell (ASMC) DNA was decreased by agents that either stimulated the synthesis (forskolin) or inhibited the breakdown (3-isobutyl-1-methylxanthine, IBMX) of cAMP. FCS-stimulated incorporation of [ 3 H]thymidine into DNA was also reduced by selective inhibitors of cAMP-specific Phosphodiesterase (PDE IV) (Ro-20-1724, rolipram) and cGMP-inhibited cAMP PDE (PDE III) (SKF however, in the presence of a threshold concentration of SK&F 94836 (20 μM), the potency of rolipram increased ( IC 25 = 4 μM ), suggesting synergy in the actions of PDE III and PDE IV inhibitors. SK&F 94836 and rolipram elicited 30% and 37%, respectively, reductions in FCS-induced ASMC proliferation and potentiated the inhibitory actions of forskolin. PDE III and PDE IV inhibitors alone, exerted minimal effects on ASMC cAMP levels after a short term (10 min) or long-term (2 or 24 hr) exposure, but enhanced forskolin-induced accumulation of cAMP. ASMC spontaneously released cAMP into the extracellular medium, a process that was increased by forskolin. PDE III and PDE IV inhibitors had no effect alone on cAMP extrusion but enhanced the effect of forskolin. Exposure of ASMC to forskolin or SK&F 94836 for 15 min increased the activity ratio (AR) of cAMP-dependent protein kinase from 0.05 to 0.17 and 0.23, respectively. Ro-20-1724, alone, did not affect cAMP-dependent protein kinase but enhanced the stimulatory effect of forskolin ( AR = 0.37) and SK&F 94836 ( AR = 0.27). Agents that increased cGMP synthesis (glycerol trinitrate, atrial natriuretic factor) or decreased its hydrolysis by selectively inhibiting cGMP-specific PDE (PDE V) (zaprinast) exerted no effects on FCS- or PDGF-stimulated [ 3 H]thymidine incorporation into DNA either alone or in combinatlon. The cytosolic fraction of pig ASMC contained four Cyclic nucleotide PDEs which were categorized as PDE V, Ca 2+ /calmodulin-stimulated PDE (PDE I), PDE III and PDE IV. PDE I and III activities were also associated with the particulate fraction. The results demonstrate that inhibitors of PDEs III and IV alone or in combination with forskolin, reduce ASMC DNA synthesis and proliferation, through an action likely to involve elevation of intracellular cAMP. In contrast, inhibition of cGMP hydrolysing PDE subtypes (I and V) exerted no effect on DNA synthesis in this cell type.
