Editorial

9 pages, 275 KiB

Open AccessEditorial

Special Issue on “New Advances in Cyclic AMP Signalling”—An Editorial Overview

by Stephen John Yarwood

Cells 2020, 9(10), 2274; https://doi.org/10.3390/cells9102274 - 12 Oct 2020

Cited by 10 | Viewed by 2239

The cyclic nucleotides 3′,5′-adenosine monophosphate (cyclic AMP) signalling system underlies the control of many biological events and disease processes in man. Cyclic AMP is synthesised by adenylate cyclase (AC) enzymes in order to activate effector proteins and it is then degraded by phosphodiesterase [...] Read more.

The cyclic nucleotides 3′,5′-adenosine monophosphate (cyclic AMP) signalling system underlies the control of many biological events and disease processes in man. Cyclic AMP is synthesised by adenylate cyclase (AC) enzymes in order to activate effector proteins and it is then degraded by phosphodiesterase (PDE) enzymes. Research in recent years has identified a range of cell-type-specific cyclic AMP effector proteins, including protein kinase A (PKA), exchange factor directly activated by cyclic AMP (EPAC), cyclic AMP responsive ion channels (CICs), and the Popeye domain containing (POPDC) proteins, which participate in different signalling mechanisms. In addition, recent advances have revealed new mechanisms of action for cyclic AMP signalling, including new effectors and new levels of compartmentalization into nanodomains, involving AKAP proteins and targeted adenylate cyclase and phosphodiesterase enzymes. This Special Issue contains 21 papers that highlight advances in our current understanding of the biology of compartmentlised cyclic AMP signalling. This ranges from issues of pathogenesis and associated molecular pathways, functional assessment of novel nanodomains, to the development of novel tool molecules and new techniques for imaging cyclic AMP compartmentilisation. This editorial aims to summarise these papers within the wider context of cyclic AMP signalling. Full article

(This article belongs to the Special Issue New Advances in Cyclic AMP Signalling)

Research

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22 pages, 7848 KiB

Open AccessArticle

Effect of cAMP Signaling Regulation in Osteogenic Differentiation of Adipose-Derived Mesenchymal Stem Cells

by Sławomir Rumiński, Ilona Kalaszczyńska and Małgorzata Lewandowska-Szumieł

Cells 2020, 9(7), 1587; https://doi.org/10.3390/cells9071587 - 30 Jun 2020

Cited by 7 | Viewed by 3791

Abstract

The successful implementation of adipose-derived mesenchymal stem cells (ADSCs) in bone regeneration depends on efficient osteogenic differentiation. However, a literature survey and our own experience demonstrated that current differentiation methods are not effective enough. Since the differentiation of mesenchymal stem cells (MSCs) into [...] Read more.

The successful implementation of adipose-derived mesenchymal stem cells (ADSCs) in bone regeneration depends on efficient osteogenic differentiation. However, a literature survey and our own experience demonstrated that current differentiation methods are not effective enough. Since the differentiation of mesenchymal stem cells (MSCs) into osteoblasts and adipocytes can be regulated by cyclic adenosine monophosphate (cAMP) signaling, we investigated the effects of cAMP activator, forskolin, and inhibitor, SQ 22,536, on the early and late osteogenic differentiation of ADSCs cultured in spheroids or in a monolayer. Intracellular cAMP concentration, protein kinase A (PKA) activity, and inhibitor of DNA binding 2 (ID2) expression examination confirmed cAMP up- and downregulation. cAMP upregulation inhibited the cell cycle and protected ADSCs from osteogenic medium (OM)-induced apoptosis. Surprisingly, the upregulation of cAMP level at the early stages of osteogenic differentiation downregulated the expression of osteogenic markers RUNX2, Osterix, and IBSP, which was more significant in spheroids, and it is used for the more efficient commitment of ADSCs into preosteoblasts, according to the previously reported protocol. However, cAMP upregulation in a culture of ADSCs in spheroids resulted in significantly increased osteocalcin production and mineralization. Thus, undifferentiated and predifferentiated ADSCs respond differently to cAMP pathway stimulation in terms of osteogenesis, which might explain the ambiguous results from the literature. Full article

(This article belongs to the Special Issue New Advances in Cyclic AMP Signalling)

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17 pages, 2509 KiB

Open AccessArticle

Molecular Basis for Ser/Thr Specificity in PKA Signaling

by Matthias J. Knape, Maximilian Wallbott, Nicole C. G. Burghardt, Daniela Bertinetti, Jan Hornung, Sven H. Schmidt, Robin Lorenz and Friedrich W. Herberg

Cells 2020, 9(6), 1548; https://doi.org/10.3390/cells9061548 - 25 Jun 2020

Cited by 4 | Viewed by 3499

Abstract

cAMP-dependent protein kinase (PKA) is the major receptor of the second messenger cAMP and a prototype for Ser/Thr-specific protein kinases. Although PKA strongly prefers serine over threonine substrates, little is known about the molecular basis of this substrate specificity. We employ classical enzyme [...] Read more.

cAMP-dependent protein kinase (PKA) is the major receptor of the second messenger cAMP and a prototype for Ser/Thr-specific protein kinases. Although PKA strongly prefers serine over threonine substrates, little is known about the molecular basis of this substrate specificity. We employ classical enzyme kinetics and a surface plasmon resonance (SPR)-based method to analyze each step of the kinase reaction. In the absence of divalent metal ions and nucleotides, PKA binds serine (PKS) and threonine (PKT) substrates, derived from the heat-stable protein kinase inhibitor (PKI), with similar affinities. However, in the presence of metal ions and adenine nucleotides, the Michaelis complex for PKT is unstable. PKA phosphorylates PKT with a higher turnover due to a faster dissociation of the product complex. Thus, threonine substrates are not necessarily poor substrates of PKA. Mutation of the DFG+1 phenylalanine to β-branched amino acids increases the catalytic efficiency of PKA for a threonine peptide substrate up to 200-fold. The PKA Cα mutant F187V forms a stable Michaelis complex with PKT and shows no preference for serine versus threonine substrates. Disease-associated mutations of the DFG+1 position in other protein kinases underline the importance of substrate specificity for keeping signaling pathways segregated and precisely regulated. Full article

(This article belongs to the Special Issue New Advances in Cyclic AMP Signalling)

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28 pages, 3462 KiB

Open AccessArticle

The Cell Cycle Checkpoint System MAST(L)-ENSA/ARPP19-PP2A is Targeted by cAMP/PKA and cGMP/PKG in Anucleate Human Platelets

by Elena J. Kumm, Oliver Pagel, Stepan Gambaryan, Ulrich Walter, René P. Zahedi, Albert Smolenski and Kerstin Jurk

Cells 2020, 9(2), 472; https://doi.org/10.3390/cells9020472 - 18 Feb 2020

Cited by 16 | Viewed by 5004

Abstract

The cell cycle is controlled by microtubule-associated serine/threonine kinase-like (MASTL), which phosphorylates the cAMP-regulated phosphoproteins 19 (ARPP19) at S62 and 19e/α-endosulfine (ENSA) at S67and converts them into protein phosphatase 2A (PP2A) inhibitors. Based on initial proteomic data, we hypothesized that the MASTL-ENSA/ARPP19-PP2A pathway, [...] Read more.

The cell cycle is controlled by microtubule-associated serine/threonine kinase-like (MASTL), which phosphorylates the cAMP-regulated phosphoproteins 19 (ARPP19) at S62 and 19e/α-endosulfine (ENSA) at S67and converts them into protein phosphatase 2A (PP2A) inhibitors. Based on initial proteomic data, we hypothesized that the MASTL-ENSA/ARPP19-PP2A pathway, unknown until now in platelets, is regulated and functional in these anucleate cells. We detected ENSA, ARPP19 and various PP2A subunits (including seven different PP2A B-subunits) in proteomic studies of human platelets. ENSA-S109/ARPP19–S104 were efficiently phosphorylated in platelets treated with cAMP- (iloprost) and cGMP-elevating (NO donors/riociguat) agents. ENSA-S67/ARPP19-S62 phosphorylations increased following PP2A inhibition by okadaic acid (OA) in intact and lysed platelets indicating the presence of MASTL or a related protein kinase in human platelets. These data were validated with recombinant ENSA/ARPP19 and phospho-mutants using recombinant MASTL, protein kinase A and G. Both ARPP19 phosphorylation sites S62/S104 were dephosphorylated by platelet PP2A, but only S62-phosphorylated ARPP19 acted as PP2A inhibitor. Low-dose OA treatment of platelets caused PP2A inhibition, diminished thrombin-stimulated platelet aggregation and increased phosphorylation of distinct sites of VASP, Akt, p38 and ERK1/2 MAP kinases. In summary, our data establish the entire MASTL(like)–ENSA/ARPP19–PP2A pathway in human platelets and important interactions with the PKA, MAPK and PI3K/Akt systems. Full article

(This article belongs to the Special Issue New Advances in Cyclic AMP Signalling)

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25 pages, 7050 KiB

Open AccessArticle

A-Kinase Anchoring Proteins Diminish TGF-β₁/Cigarette Smoke-Induced Epithelial-To-Mesenchymal Transition

by Haoxiao Zuo, Marina Trombetta-Lima, Irene H. Heijink, Christina H. T. J. van der Veen, Laura Hesse, Klaas Nico Faber, Wilfred J. Poppinga, Harm Maarsingh, Viacheslav O. Nikolaev and Martina Schmidt

Cells 2020, 9(2), 356; https://doi.org/10.3390/cells9020356 - 03 Feb 2020

Cited by 16 | Viewed by 4011

Abstract

Epithelial-to-mesenchymal transition (EMT) plays a role in chronic obstructive pulmonary diseases (COPD). Cyclic adenosine monophosphate (cAMP) can inhibit transforming growth factor-β1 (TGF-β1) mediated EMT. Although compartmentalization via A-kinase anchoring proteins (AKAPs) is central to cAMP signaling, functional studies regarding their therapeutic value in [...] Read more.

Epithelial-to-mesenchymal transition (EMT) plays a role in chronic obstructive pulmonary diseases (COPD). Cyclic adenosine monophosphate (cAMP) can inhibit transforming growth factor-β1 (TGF-β1) mediated EMT. Although compartmentalization via A-kinase anchoring proteins (AKAPs) is central to cAMP signaling, functional studies regarding their therapeutic value in the lung EMT process are lacking. The human bronchial epithelial cell line (BEAS-2B) and primary human airway epithelial (pHAE) cells were exposed to TGF-β1. Epithelial (E-cadherin, ZO-1) and mesenchymal markers (collagen Ӏ, α-SMA, fibronectin) were analyzed (mRNA, protein). ELISA measured TGF-β1 release. TGF-β1-sensitive AKAPs Ezrin, AKAP95 and Yotiao were silenced while using siRNA. Cell migration was analyzed by wound healing assay, xCELLigence, Incucyte. Prior to TGF-β1, dibutyryl-cAMP (dbcAMP), fenoterol, rolipram, cilostamide, and forskolin were used to elevate intracellular cAMP. TGF-β1 induced morphological changes, decreased E-cadherin, but increased collagen Ӏ and cell migration, a process that was reversed by the inhibitor of δ/epsilon casein kinase I, PF-670462. TGF-β1 altered (mRNA, protein) expression of Ezrin, AKAP95, and Yotiao. St-Ht31, the AKAP antagonist, decreased E-cadherin (mRNA, protein), but counteracted TGF-β1-induced collagen Ӏ upregulation. Cigarette smoke (CS) increased TGF-β1 release, activated TGF signaling, augmented cell migration, and reduced E-cadherin expression, a process that was blocked by TGF-β1 neutralizing antibody. The silencing of Ezrin, AKAP95, and Yotiao diminished TGF-β1-induced collagen Ӏ expression, as well as TGF-β1-induced cell migration. Fenoterol, rolipram, and cilostamide, in AKAP silenced cells, pointed to distinct cAMP compartments. We conclude that Ezrin, AKAP95, and Yotiao promote TGF-β1-mediated EMT, linked to a TGF-β1 release by CS. AKAP members might define the ability of fenoterol, rolipram, and cilostamide to modulate the EMT process, and they might represent potential relevant targets in the treatment of COPD. Full article

(This article belongs to the Special Issue New Advances in Cyclic AMP Signalling)

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26 pages, 5714 KiB

Open AccessArticle

Cyclic AMP Regulates Key Features of Macrophages via PKA: Recruitment, Reprogramming and Efferocytosis

by Graziele L. Negreiros-Lima, Kátia M. Lima, Isabella Z. Moreira, Bruna Lorrayne O. Jardim, Juliana P. Vago, Izabela Galvão, Lívia Cristina R. Teixeira, Vanessa Pinho, Mauro M. Teixeira, Michelle A. Sugimoto and Lirlândia P. Sousa

Cells 2020, 9(1), 128; https://doi.org/10.3390/cells9010128 - 06 Jan 2020

Cited by 43 | Viewed by 7223

Abstract

Macrophages are central to inflammation resolution, an active process aimed at restoring tissue homeostasis following an inflammatory response. Here, the effects of db-cAMP on macrophage phenotype and function were investigated. Injection of db-cAMP into the pleural cavity of mice induced monocytes recruitment in [...] Read more.

Macrophages are central to inflammation resolution, an active process aimed at restoring tissue homeostasis following an inflammatory response. Here, the effects of db-cAMP on macrophage phenotype and function were investigated. Injection of db-cAMP into the pleural cavity of mice induced monocytes recruitment in a manner dependent on PKA and CCR2/CCL2 pathways. Furthermore, db-cAMP promoted reprogramming of bone-marrow-derived macrophages to a M2 phenotype as seen by increased Arg-1/CD206/Ym-1 expression and IL-10 levels (M2 markers). Db-cAMP also showed a synergistic effect with IL-4 in inducing STAT-3 phosphorylation and Arg-1 expression. Importantly, db-cAMP prevented IFN-γ/LPS-induced macrophage polarization to M1-like as shown by increased Arg-1 associated to lower levels of M1 cytokines (TNF-α/IL-6) and p-STAT1. In vivo, db-cAMP reduced the number of M1 macrophages induced by LPS injection without changes in M2 and Mres numbers. Moreover, db-cAMP enhanced efferocytosis of apoptotic neutrophils in a PKA-dependent manner and increased the expression of Annexin A1 and CD36, two molecules associated with efferocytosis. Finally, inhibition of endogenous PKA during LPS-induced pleurisy impaired the physiological resolution of inflammation. Taken together, the results suggest that cAMP is involved in the major functions of macrophages, such as nonphlogistic recruitment, reprogramming and efferocytosis, all key processes for inflammation resolution. Full article

(This article belongs to the Special Issue New Advances in Cyclic AMP Signalling)

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18 pages, 2374 KiB

Open AccessArticle

Conformational States of Exchange Protein Directly Activated by cAMP (EPAC1) Revealed by Ensemble Modeling and Integrative Structural Biology

by Mark Andrew White, Tamara Tsalkova, Fang C. Mei and Xiaodong Cheng

Cells 2020, 9(1), 35; https://doi.org/10.3390/cells9010035 - 21 Dec 2019

Cited by 7 | Viewed by 2631

Abstract

Exchange proteins directly activated by cAMP (EPAC1 and EPAC2) are important allosteric regulators of cAMP-mediated signal transduction pathways. To understand the molecular mechanism of EPAC activation, we performed detailed Small-Angle X-ray Scattering (SAXS) analysis of EPAC1 in its apo (inactive), cAMP-bound, and effector [...] Read more.

Exchange proteins directly activated by cAMP (EPAC1 and EPAC2) are important allosteric regulators of cAMP-mediated signal transduction pathways. To understand the molecular mechanism of EPAC activation, we performed detailed Small-Angle X-ray Scattering (SAXS) analysis of EPAC1 in its apo (inactive), cAMP-bound, and effector (Rap1b)-bound states. Our study demonstrates that we can model the solution structures of EPAC1 in each state using ensemble analysis and homology models derived from the crystal structures of EPAC2. The N-terminal domain of EPAC1, which is not conserved between EPAC1 and EPAC2, appears folded and interacts specifically with another component of EPAC1 in each state. The apo-EPAC1 state is a dynamic mixture of a compact (Rg = 32.9 Å, 86%) and a more extended (Rg = 38.5 Å, 13%) conformation. The cAMP-bound form of EPAC1 in the absence of Rap1 forms a dimer in solution; but its molecular structure is still compatible with the active EPAC1 conformation of the ternary complex model with cAMP and Rap1. Herein, we show that SAXS can elucidate the conformational states of EPAC1 activation as it proceeds from the compact, inactive apo conformation through a previously unknown intermediate-state, to the extended cAMP-bound form, and then binds to its effector (Rap1b) in a ternary complex. Full article

(This article belongs to the Special Issue New Advances in Cyclic AMP Signalling)

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15 pages, 3504 KiB

Open AccessArticle

A Membrane Permeable Prodrug of S223 for Selective Epac2 Activation in Living Cells

by Yunjian Xu, Frank Schwede, Hans Wienk, Anders Tengholm and Holger Rehmann

Cells 2019, 8(12), 1589; https://doi.org/10.3390/cells8121589 - 06 Dec 2019

Cited by 4 | Viewed by 3153

Abstract

Signalling by cyclic adenosine monophosphate (cAMP) occurs via various effector proteins, notably protein kinase A and the guanine nucleotide exchange factors Epac1 and Epac2. These proteins are activated by cAMP binding to conserved cyclic nucleotide binding domains. The specific roles of the effector [...] Read more.

Signalling by cyclic adenosine monophosphate (cAMP) occurs via various effector proteins, notably protein kinase A and the guanine nucleotide exchange factors Epac1 and Epac2. These proteins are activated by cAMP binding to conserved cyclic nucleotide binding domains. The specific roles of the effector proteins in various processes in different types of cells are still not well defined, but investigations have been facilitated by the development of cyclic nucleotide analogues with distinct selectivity profiles towards a single effector protein. A remaining challenge in the development of such analogues is the poor membrane permeability of nucleotides, which limits their applicability in intact living cells. Here, we report the synthesis and characterisation of S223-AM, a cAMP analogue designed as an acetoxymethyl ester prodrug to overcome limitations of permeability. Using total internal reflection imaging with various fluorescent reporters, we show that S223-AM selectively activates Epac2, but not Epac1 or protein kinase A, in intact insulin-secreting β-cells, and that this effect was associated with pronounced activation of the small G-protein Rap. A comparison of the effects of different cAMP analogues in pancreatic islet cells deficient in Epac1 and Epac2 demonstrates that cAMP-dependent Rap activity at the β-cell plasma membrane is exclusively dependent on Epac2. With its excellent selectivity and permeability properties, S223-AM should get broad utility in investigations of cAMP effector involvement in many different types of cells. Full article

(This article belongs to the Special Issue New Advances in Cyclic AMP Signalling)

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10 pages, 1024 KiB

Open AccessCommunication

A Software Tool for High-Throughput Real-Time Measurement of Intensity-Based Ratio-Metric FRET

by Masoud Ramuz, Alveera Hasan, Lena Gruscheski, Ivan Diakonov, Nikoleta Pavlaki, Viacheslav O. Nikolaev, Sian Harding, Chris Dunsby and Julia Gorelik

Cells 2019, 8(12), 1541; https://doi.org/10.3390/cells8121541 - 29 Nov 2019

Cited by 7 | Viewed by 4350

Abstract

Förster resonance energy transfer (FRET) is increasingly used for non-invasive measurement of fluorescently tagged molecules in live cells. In this study, we have developed a freely available software tool MultiFRET, which, together with the use of a motorised microscope stage, allows multiple single [...] Read more.

Förster resonance energy transfer (FRET) is increasingly used for non-invasive measurement of fluorescently tagged molecules in live cells. In this study, we have developed a freely available software tool MultiFRET, which, together with the use of a motorised microscope stage, allows multiple single cells to be studied in one experiment. MultiFRET is a Java plugin for Micro-Manager software, which provides real-time calculations of ratio-metric signals during acquisition and can simultaneously record from multiple cells in the same experiment. It can also make other custom-determined live calculations that can be easily exported to Excel at the end of the experiment. It is flexible and can work with multiple spectral acquisition channels. We validated this software by comparing the output of MultiFRET to that of a previously established and well-documented method for live ratio-metric FRET experiments and found no significant difference between the data produced with the use of the new MultiFRET and other methods. In this validation, we used several cAMP FRET sensors and cell models: i) isolated adult cardiomyocytes from transgenic mice expressing the cytosolic epac1-camps and targeted pmEpac1 and Epac1-PLN sensors, ii) isolated neonatal mouse cardiomyocytes transfected with the AKAP79-CUTie sensor, and iii) human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) transfected with the Epac-S^H74 sensor. The MultiFRET plugin is an open source freely available package that can be used in a wide area of live cell imaging when live ratio-metric calculations are required. Full article

(This article belongs to the Special Issue New Advances in Cyclic AMP Signalling)

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12 pages, 3310 KiB

Open AccessArticle

An EPAC1/PDE1C-Signaling Axis Regulates Formation of Leading-Edge Protrusion in Polarized Human Arterial Vascular Smooth Muscle Cells

by Paulina Brzezinska and Donald H. Maurice

Cells 2019, 8(12), 1473; https://doi.org/10.3390/cells8121473 - 20 Nov 2019

Cited by 4 | Viewed by 2531

Abstract

Pharmacological activation of protein kinase A (PKA) reduces migration of arterial smooth muscle cells (ASMCs), including those isolated from human arteries (HASMCs). However, when individual migration-associated cellular events, including the polarization of cells in the direction of movement or rearrangements of the actin [...] Read more.

Pharmacological activation of protein kinase A (PKA) reduces migration of arterial smooth muscle cells (ASMCs), including those isolated from human arteries (HASMCs). However, when individual migration-associated cellular events, including the polarization of cells in the direction of movement or rearrangements of the actin cytoskeleton, are studied in isolation, these individual events can be either promoted or inhibited in response to PKA activation. While pharmacological inhibition or deficiency of exchange protein activated by cAMP-1 (EPAC1) reduces the overall migration of ASMCs, the impact of EPAC1 inhibition or deficiency, or of its activation, on individual migration-related events has not been investigated. Herein, we report that EPAC1 facilitates the formation of leading-edge protrusions (LEPs) in HASMCs, a critical early event in the cell polarization that underpins their migration. Thus, RNAi-mediated silencing, or the selective pharmacological inhibition, of EPAC1 decreased the formation of LEPs by these cells. Furthermore, we show that the ability of EPAC1 to promote LEP formation by migrating HASMCs is regulated by a phosphodiesterase 1C (PDE1C)-regulated “pool” of intracellular HASMC cAMP but not by those regulated by the more abundant PDE3 or PDE4 activities. Overall, our data are consistent with a role for EPAC1 in regulating the formation of LEPs by polarized HASMCs and show that PDE1C-mediated cAMP hydrolysis controls this localized event. Full article

(This article belongs to the Special Issue New Advances in Cyclic AMP Signalling)

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20 pages, 2411 KiB

Open AccessArticle

Identification of Novel Adenylyl Cyclase 5 (AC5) Signaling Networks in D₁ and D₂ Medium Spiny Neurons using Bimolecular Fluorescence Complementation Screening

by Trevor B. Doyle, Brian S. Muntean, Karin F. Ejendal, Michael P. Hayes, Monica Soto-Velasquez, Kirill A. Martemyanov, Carmen W. Dessauer, Chang-Deng Hu and Val J. Watts

Cells 2019, 8(11), 1468; https://doi.org/10.3390/cells8111468 - 19 Nov 2019

Cited by 13 | Viewed by 4108

Abstract

Adenylyl cyclase type 5 (AC5), as the principal isoform expressed in striatal medium spiny neurons (MSNs), is essential for the integration of both stimulatory and inhibitory midbrain signals that initiate from dopaminergic G protein-coupled receptor (GPCR) activation. The spatial and temporal control of [...] Read more.

Adenylyl cyclase type 5 (AC5), as the principal isoform expressed in striatal medium spiny neurons (MSNs), is essential for the integration of both stimulatory and inhibitory midbrain signals that initiate from dopaminergic G protein-coupled receptor (GPCR) activation. The spatial and temporal control of cAMP signaling is dependent upon the composition of local regulatory protein networks. However, there is little understanding of how adenylyl cyclase protein interaction networks adapt to the multifarious pressures of integrating acute versus chronic and inhibitory vs. stimulatory receptor signaling in striatal MSNs. Here, we presented the development of a novel bimolecular fluorescence complementation (BiFC)-based protein-protein interaction screening methodology to further identify and characterize elements important for homeostatic control of dopamine-modulated AC5 signaling in a neuronal model cell line and striatal MSNs. We identified two novel AC5 modulators: the protein phosphatase 2A (PP2A) catalytic subunit (PPP2CB) and the intracellular trafficking associated protein—NSF (N-ethylmaleimide-sensitive factor) attachment protein alpha (NAPA). The effects of genetic knockdown (KD) of each gene were evaluated in several cellular models, including D₁- and D₂-dopamine receptor-expressing MSNs from CAMPER mice. The knockdown of PPP2CB was associated with a reduction in acute and sensitized adenylyl cyclase activity, implicating PP2A is an important and persistent regulator of adenylyl cyclase activity. In contrast, the effects of NAPA knockdown were more nuanced and appeared to involve an activity-dependent protein interaction network. Taken together, these data represent a novel screening method and workflow for the identification and validation of adenylyl cyclase protein-protein interaction networks under diverse cAMP signaling paradigms. Full article

(This article belongs to the Special Issue New Advances in Cyclic AMP Signalling)

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18 pages, 2867 KiB

Open AccessArticle

Identification of A Novel Class of Benzofuran Oxoacetic Acid-Derived Ligands that Selectively Activate Cellular EPAC1

by Elizabeth M. Beck, Euan Parnell, Angela Cowley, Alison Porter, Jonathan Gillespie, John Robinson, Lindsay Robinson, Andrew D. Pannifer, Veronique Hamon, Philip Jones, Angus Morrison, Stuart McElroy, Martin Timmerman, Helma Rutjes, Pravin Mahajan, Jolanta Wiejak, Urszula Luchowska-Stańska, David Morgan, Graeme Barker, Holger Rehmann and Stephen J. Yarwood Show full author list Hide full author list

Cells 2019, 8(11), 1425; https://doi.org/10.3390/cells8111425 - 12 Nov 2019

Cited by 11 | Viewed by 4225

Abstract

Cyclic AMP promotes EPAC1 and EPAC2 activation through direct binding to a specific cyclic nucleotide-binding domain (CNBD) within each protein, leading to activation of Rap GTPases, which control multiple cell responses, including cell proliferation, adhesion, morphology, exocytosis, and gene expression. As a result, [...] Read more.

Cyclic AMP promotes EPAC1 and EPAC2 activation through direct binding to a specific cyclic nucleotide-binding domain (CNBD) within each protein, leading to activation of Rap GTPases, which control multiple cell responses, including cell proliferation, adhesion, morphology, exocytosis, and gene expression. As a result, it has become apparent that directed activation of EPAC1 and EPAC2 with synthetic agonists may also be useful for the future treatment of diabetes and cardiovascular diseases. To identify new EPAC agonists we have developed a fluorescent-based, ultra-high-throughput screening (uHTS) assay that measures the displacement of binding of the fluorescent cAMP analogue, 8-NBD-cAMP to the EPAC1 CNBD. Triage of the output of an approximately 350,000 compound screens using this assay identified a benzofuran oxaloacetic acid EPAC1 binder (SY000) that displayed moderate potency using orthogonal assays (competition binding and microscale thermophoresis). We next generated a limited library of 91 analogues of SY000 and identified SY009, with modifications to the benzofuran ring associated with a 10-fold increase in potency towards EPAC1 over SY000 in binding assays. In vitro EPAC1 activity assays confirmed the agonist potential of these molecules in comparison with the known EPAC1 non-cyclic nucleotide (NCN) partial agonist, I942. Rap1 GTPase activation assays further demonstrated that SY009 selectively activates EPAC1 over EPAC2 in cells. SY009 therefore represents a novel class of NCN EPAC1 activators that selectively activate EPAC1 in cellulae. Full article

(This article belongs to the Special Issue New Advances in Cyclic AMP Signalling)

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20 pages, 10195 KiB

Open AccessArticle

Genome-Wide Mapping Defines a Role for C/EBPβ and c-Jun in Non-Canonical Cyclic AMP Signalling

by Jolanta Wiejak, Boy van Basten, Graham Hamilton and Stephen J. Yarwood

Cells 2019, 8(10), 1253; https://doi.org/10.3390/cells8101253 - 14 Oct 2019

Cited by 5 | Viewed by 3991

Abstract

The novel exchange protein activated by cyclic AMP (EPAC1) activator, I942, induces expression of the suppressor of cytokine signalling 3 (SOCS3) gene, thereby inhibiting interleukin 6 (IL6) inflammatory processes in human umbilical vein endothelial cells (HUVECs). Here we use RNA-SEQ and ChIP-SEQ to [...] Read more.

The novel exchange protein activated by cyclic AMP (EPAC1) activator, I942, induces expression of the suppressor of cytokine signalling 3 (SOCS3) gene, thereby inhibiting interleukin 6 (IL6) inflammatory processes in human umbilical vein endothelial cells (HUVECs). Here we use RNA-SEQ and ChIP-SEQ to determine global gene responses to I942, in comparison with cyclic AMP production promoted by forskolin and rolipram (F/R). We found that I942 promoted significant changes in the RNA expression of 1413 genes, largely associated with microtubule stability and cell cycle progression, whereas F/R regulated 197 genes linked to endothelial cell function, including chemokine production and platelet aggregation. A further 108 genes were regulated by both treatments, including endothelial regulatory genes involved in purinergic signalling and cell junction organization. ChIP-SEQ demonstrated that F/R induced genome-wide recruitment of C/EBPβ and c-Jun transcription factors, whereas I942 promoted recruitment of c-Jun to genes associated with IL6 signalling, with little effect on C/EBPβ activation. Despite this, certain key inflammatory genes, including IL6, VEGF, CCL2/MCP1, VCAM1, SELE and ICAM1 were regulated by I942 without significant c-Jun recruitment, suggesting an additional, indirect mode of action for I942. In this regard, SOCS3 induction by I942 was found to require c-Jun and was associated with suppression of IL6-promoted ERK MAP kinase and AKT activity and induction of ICAM1. Pharmacological inhibition of ERK and AKT also potentiated ICAM1 induction by I942. We therefore propose that c-Jun activation by I942 regulates endothelial gene expression in HUVECs through direct mechanisms, involving recruitment of c-Jun or, as for ICAM1, through indirect regulation of tertiary regulators, including SOCS3. Full article

(This article belongs to the Special Issue New Advances in Cyclic AMP Signalling)

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17 pages, 4663 KiB

Open AccessArticle

Gγ and Gα Identity Dictate a G-Protein Heterotrimer Plasma Membrane Targeting

by Paweł Mystek, Beata Rysiewicz, Jan Gregrowicz, Marta Dziedzicka-Wasylewska and Agnieszka Polit

Cells 2019, 8(10), 1246; https://doi.org/10.3390/cells8101246 - 13 Oct 2019

Cited by 9 | Viewed by 5098

Abstract

Heterotrimeric G-proteins along with G-protein-coupled receptors (GPCRs) regulate many biochemical functions by relaying the information from the plasma membrane to the inside of the cell. The lipid modifications of Gα and Gγ subunits, together with the charged regions on the membrane interaction surface, [...] Read more.

Heterotrimeric G-proteins along with G-protein-coupled receptors (GPCRs) regulate many biochemical functions by relaying the information from the plasma membrane to the inside of the cell. The lipid modifications of Gα and Gγ subunits, together with the charged regions on the membrane interaction surface, provide a peculiar pattern for various heterotrimeric complexes. In a previous study, we found that Gαs and Gαi₃ prefer different types of membrane-anchor and subclass-specific lipid domains. In the present report, we examine the role of distinct Gγ subunits in the membrane localization and spatiotemporal dynamics of Gαs and Gαi₃ heterotrimers. We characterized lateral diffusion and G-protein subunit interactions in living cells using fluorescence recovery after photobleaching (FRAP) microscopy and fluorescence resonance energy transfer (FRET) detected by fluorescence lifetime imaging microscopy (FLIM), respectively. The interaction of Gγ subunits with specific lipids was confirmed, and thus the modulation of heterotrimeric G-protein localization. However, the Gα subunit also modulates trimer localization, and so the membrane distribution of heterotrimeric G-proteins is not dependent on Gγ only. Full article

(This article belongs to the Special Issue New Advances in Cyclic AMP Signalling)

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25 pages, 5902 KiB

Open AccessArticle

Proteomic and Transcriptomic Profiling Identifies Early Developmentally Regulated Proteins in Dictyostelium Discoideum

by Óscar González-Velasco, Javier De Las Rivas and Jesus Lacal

Cells 2019, 8(10), 1187; https://doi.org/10.3390/cells8101187 - 01 Oct 2019

Cited by 8 | Viewed by 5351

Abstract

Cyclic AMP acts as a secondary messenger involving different cellular functions in eukaryotes. Here, proteomic and transcriptomic profiling has been combined to identify novel early developmentally regulated proteins in eukaryote cells. These proteomic and transcriptomic experiments were performed in Dictyostelium discoideum given the [...] Read more.

Cyclic AMP acts as a secondary messenger involving different cellular functions in eukaryotes. Here, proteomic and transcriptomic profiling has been combined to identify novel early developmentally regulated proteins in eukaryote cells. These proteomic and transcriptomic experiments were performed in Dictyostelium discoideum given the unique advantages that this organism offers as a eukaryotic model for cell motility and as a nonmammalian model of human disease. By comparing whole-cell proteome analysis of developed (cAMP-pulsed) wild-type AX2 cells and an independent transcriptomic analysis of developed wild-type AX4 cells, our results show that up to 70% of the identified proteins overlap in the two independent studies. Among them, we have found 26 proteins previously related to cAMP signaling and identified 110 novel proteins involved in calcium signaling, adhesion, actin cytoskeleton, the ubiquitin-proteasome pathway, metabolism, and proteins that previously lacked any annotation. Our study validates previous findings, mostly for the canonical cAMP-pathway, and also generates further insight into the complexity of the transcriptomic changes during early development. This article also compares proteomic data between parental and cells lacking glkA, a GSK-3 kinase implicated in substrate adhesion and chemotaxis in Dictyostelium. This analysis reveals a set of proteins that show differences in expression in the two strains as well as overlapping protein level changes independent of GlkA. Full article

(This article belongs to the Special Issue New Advances in Cyclic AMP Signalling)

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16 pages, 3312 KiB

Open AccessArticle

Regulation of I_Ks Potassium Current by Isoproterenol in Adult Cardiomyocytes Requires Type 9 Adenylyl Cyclase

by Yong Li, Thomas Hof, Tanya A. Baldwin, Lei Chen, Robert S. Kass and Carmen W. Dessauer

Cells 2019, 8(9), 981; https://doi.org/10.3390/cells8090981 - 27 Aug 2019

Cited by 15 | Viewed by 4008

Abstract

The subunits KCNQ1 and KCNE1 generate the slowly activating, delayed rectifier potassium current, I_Ks, that responds to sympathetic stimulation and is critical for human cardiac repolarization. The A-kinase anchoring protein Yotiao facilitates macromolecular complex formation between I_Ks and protein kinase [...] Read more.

The subunits KCNQ1 and KCNE1 generate the slowly activating, delayed rectifier potassium current, I_Ks, that responds to sympathetic stimulation and is critical for human cardiac repolarization. The A-kinase anchoring protein Yotiao facilitates macromolecular complex formation between I_Ks and protein kinase A (PKA) to regulate phosphorylation of KCNQ1 and I_Ks currents following beta-adrenergic stimulation. We have previously shown that adenylyl cyclase Type 9 (AC9) is associated with a KCNQ1-Yotiao-PKA complex and facilitates isoproterenol-stimulated phosphorylation of KCNQ1 in an immortalized cell line. However, requirement for AC9 in sympathetic control of I_Ks in the heart was unknown. Using a transgenic mouse strain expressing the KCNQ1-KCNE1 subunits of I_Ks, we show that AC9 is the only adenylyl cyclase (AC) isoform associated with the KCNQ1-KCNE1-Yotiao complex in the heart. Deletion of AC9 resulted in the loss of isoproterenol-stimulated KCNQ1 phosphorylation in vivo, even though AC9 represents less than 3% of total cardiac AC activity. Importantly, a significant reduction of isoproterenol-stimulated I_Ks currents was also observed in adult cardiomyocytes from I_Ks-expressing AC9KO mice. AC9 and Yotiao co-localize with N-cadherin, a marker of intercalated disks and cell–cell junctions, in neonatal and adult cardiomyocytes, respectively. In conclusion, AC9 is necessary for sympathetic regulation of PKA phosphorylation of KCNQ1 in vivo and for functional regulation of I_Ks in adult cardiomyocytes. Full article

(This article belongs to the Special Issue New Advances in Cyclic AMP Signalling)

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Review

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17 pages, 1068 KiB

Open AccessReview

Phosphodiesterase 4B: Master Regulator of Brain Signaling

by Amy J. Tibbo and George S. Baillie

Cells 2020, 9(5), 1254; https://doi.org/10.3390/cells9051254 - 19 May 2020

Cited by 33 | Viewed by 6048

Abstract

Phosphodiesterases (PDEs) are the only superfamily of enzymes that have the ability to break down cyclic nucleotides and, as such, they have a pivotal role in neurological disease and brain development. PDEs have a modular structure that allows targeting of individual isoforms to [...] Read more.

Phosphodiesterases (PDEs) are the only superfamily of enzymes that have the ability to break down cyclic nucleotides and, as such, they have a pivotal role in neurological disease and brain development. PDEs have a modular structure that allows targeting of individual isoforms to discrete brain locations and it is often the location of a PDE that shapes its cellular function. Many of the eleven different families of PDEs have been associated with specific diseases. However, we evaluate the evidence, which suggests the activity from a sub-family of the PDE4 family, namely PDE4B, underpins a range of important functions in the brain that positions the PDE4B enzymes as a therapeutic target for a diverse collection of indications, such as, schizophrenia, neuroinflammation, and cognitive function. Full article

(This article belongs to the Special Issue New Advances in Cyclic AMP Signalling)

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14 pages, 1273 KiB

Open AccessReview

Hedgehog and Gpr161: Regulating cAMP Signaling in the Primary Cilium

by Philipp Tschaikner, Florian Enzler, Omar Torres-Quesada, Pia Aanstad and Eduard Stefan

Cells 2020, 9(1), 118; https://doi.org/10.3390/cells9010118 - 03 Jan 2020

Cited by 27 | Viewed by 6394

Abstract

Compartmentalization of diverse types of signaling molecules contributes to the precise coordination of signal propagation. The primary cilium fulfills this function by acting as a spatiotemporally confined sensory signaling platform. For the integrity of ciliary signaling, it is mandatory that the ciliary signaling [...] Read more.

Compartmentalization of diverse types of signaling molecules contributes to the precise coordination of signal propagation. The primary cilium fulfills this function by acting as a spatiotemporally confined sensory signaling platform. For the integrity of ciliary signaling, it is mandatory that the ciliary signaling pathways are constantly attuned by alterations in both oscillating small molecules and the presence or absence of their sensor/effector proteins. In this context, ciliary G protein-coupled receptor (GPCR) pathways participate in coordinating the mobilization of the diffusible second messenger molecule 3′,5′-cyclic adenosine monophosphate (cAMP). cAMP fluxes in the cilium are primarily sensed by protein kinase A (PKA) complexes, which are essential for the basal repression of Hedgehog (Hh) signaling. Here, we describe the dynamic properties of underlying signaling circuits, as well as strategies for second messenger compartmentalization. As an example, we summarize how receptor-guided cAMP-effector pathways control the off state of Hh signaling. We discuss the evidence that a macromolecular, ciliary-localized signaling complex, composed of the orphan GPCR Gpr161 and type I PKA holoenzymes, is involved in antagonizing Hh functions. Finally, we outline how ciliary cAMP-linked receptor pathways and cAMP-sensing signalosomes may become targets for more efficient combinatory therapy approaches to counteract dysregulation of Hh signaling. Full article

(This article belongs to the Special Issue New Advances in Cyclic AMP Signalling)

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22 pages, 2283 KiB

Open AccessFeature PaperReview

The Role of Cyclic AMP Signaling in Cardiac Fibrosis

by Marion Delaunay, Halima Osman, Simon Kaiser and Dario Diviani

Cells 2020, 9(1), 69; https://doi.org/10.3390/cells9010069 - 26 Dec 2019

Cited by 45 | Viewed by 10029

Abstract

Myocardial stress and injury invariably promote remodeling of the cardiac tissue, which is associated with cardiomyocyte death and development of fibrosis. The fibrotic process is initially triggered by the differentiation of resident cardiac fibroblasts into myofibroblasts. These activated fibroblasts display increased proliferative capacity [...] Read more.

Myocardial stress and injury invariably promote remodeling of the cardiac tissue, which is associated with cardiomyocyte death and development of fibrosis. The fibrotic process is initially triggered by the differentiation of resident cardiac fibroblasts into myofibroblasts. These activated fibroblasts display increased proliferative capacity and secrete large amounts of extracellular matrix. Uncontrolled myofibroblast activation can thus promote heart stiffness, cardiac dysfunction, arrhythmias, and progression to heart failure. Despite the well-established role of myofibroblasts in mediating cardiac disease, our current knowledge on how signaling pathways promoting fibrosis are regulated and coordinated in this cell type is largely incomplete. In this respect, cyclic adenosine monophosphate (cAMP) signaling acts as a major modulator of fibrotic responses activated in fibroblasts of injured or stressed hearts. In particular, accumulating evidence now suggests that upstream cAMP modulators including G protein-coupled receptors, adenylyl cyclases (ACs), and phosphodiesterases (PDEs); downstream cAMP effectors such as protein kinase A (PKA) and the guanine nucleotide exchange factor Epac; and cAMP signaling organizers such as A-kinase anchoring proteins (AKAPs) modulate a variety of fundamental cellular processes involved in myocardial fibrosis including myofibroblast differentiation, proliferation, collagen secretion, and invasiveness. The current review will discuss recent advances highlighting the role of cAMP and AKAP-mediated signaling in regulating pathophysiological responses controlling cardiac fibrosis. Full article

(This article belongs to the Special Issue New Advances in Cyclic AMP Signalling)

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23 pages, 2292 KiB

Open AccessReview

The Role of the Popeye Domain Containing Gene Family in Organ Homeostasis

by Johanna Ndamwena Amunjela, Alexander H. Swan and Thomas Brand

Cells 2019, 8(12), 1594; https://doi.org/10.3390/cells8121594 - 07 Dec 2019

Cited by 18 | Viewed by 3943

Abstract

The Popeye domain containing (POPDC) gene family consists of POPDC1 (also known as BVES), POPDC2 and POPDC3 and encodes a novel class of cyclic adenosine monophosphate (cAMP) effector proteins. Despite first reports of their isolation and initial characterization at the protein level [...] Read more.

The Popeye domain containing (POPDC) gene family consists of POPDC1 (also known as BVES), POPDC2 and POPDC3 and encodes a novel class of cyclic adenosine monophosphate (cAMP) effector proteins. Despite first reports of their isolation and initial characterization at the protein level dating back 20 years, only recently major advances in defining their biological functions and disease association have been made. Loss-of-function experiments in mice and zebrafish established an important role in skeletal muscle regeneration, heart rhythm control and stress signaling. Patients suffering from muscular dystrophy and atrioventricular block were found to carry missense and nonsense mutations in either of the three POPDC genes, which suggests an important function in the control of striated muscle homeostasis. However, POPDC genes are also expressed in a number of epithelial cells and function as tumor suppressor genes involved in the control of epithelial structure, tight junction formation and signaling. Suppression of POPDC genes enhances tumor cell proliferation, migration, invasion and metastasis in a variety of human cancers, thus promoting a malignant phenotype. Moreover, downregulation of POPDC1 and POPDC3 expression in different cancer types has been associated with poor prognosis. However, high POPDC3 expression has also been correlated to poor clinical prognosis in head and neck squamous cell carcinoma, suggesting that POPDC3 potentially plays different roles in the progression of different types of cancer. Interestingly, a gain of POPDC1 function in tumor cells inhibits cell proliferation, migration and invasion thereby reducing malignancy. Furthermore, POPDC proteins have been implicated in the control of cell cycle genes and epidermal growth factor and Wnt signaling. Work in tumor cell lines suggest that cyclic nucleotide binding may also be important in epithelial cells. Thus, POPDC proteins have a prominent role in tissue homeostasis and cellular signaling in both epithelia and striated muscle. Full article

(This article belongs to the Special Issue New Advances in Cyclic AMP Signalling)

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18 pages, 3822 KiB

Open AccessReview

The Epac1 Protein: Pharmacological Modulators, Cardiac Signalosome and Pathophysiology

by Marion Bouvet, Jean-Paul Blondeau and Frank Lezoualc’h

Cells 2019, 8(12), 1543; https://doi.org/10.3390/cells8121543 - 29 Nov 2019

Cited by 19 | Viewed by 5419

Abstract

The second messenger 3′,5′-cyclic adenosine monophosphate (cAMP) is one of the most important signalling molecules in the heart as it regulates many physiological and pathophysiological processes. In addition to the classical protein kinase A (PKA) signalling route, the exchange proteins directly activated by [...] Read more.

The second messenger 3′,5′-cyclic adenosine monophosphate (cAMP) is one of the most important signalling molecules in the heart as it regulates many physiological and pathophysiological processes. In addition to the classical protein kinase A (PKA) signalling route, the exchange proteins directly activated by cAMP (Epac) mediate the intracellular functions of cAMP and are now emerging as a new key cAMP effector in cardiac pathophysiology. In this review, we provide a perspective on recent advances in the discovery of new chemical entities targeting the Epac1 isoform and illustrate their use to study the Epac1 signalosome and functional characterisation in cardiac cells. We summarize the role of Epac1 in different subcompartments of the cardiomyocyte and discuss how cAMP–Epac1 specific signalling networks may contribute to the development of cardiac diseases. We also highlight ongoing work on the therapeutic potential of Epac1-selective small molecules for the treatment of cardiac disorders. Full article

(This article belongs to the Special Issue New Advances in Cyclic AMP Signalling)

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17 pages, 1384 KiB

Open AccessReview

Targeting Cyclic AMP Signalling in Hepatocellular Carcinoma

by Mara Massimi, Federica Ragusa, Silvia Cardarelli and Mauro Giorgi

Cells 2019, 8(12), 1511; https://doi.org/10.3390/cells8121511 - 25 Nov 2019

Cited by 29 | Viewed by 8221

Abstract

Hepatocellular carcinoma (HCC) is a major healthcare problem worldwide, representing one of the leading causes of cancer mortality. Since there are currently no predictive biomarkers for early stage diagnosis, HCC is detected only in advanced stages and most patients die within one year, [...] Read more.

Hepatocellular carcinoma (HCC) is a major healthcare problem worldwide, representing one of the leading causes of cancer mortality. Since there are currently no predictive biomarkers for early stage diagnosis, HCC is detected only in advanced stages and most patients die within one year, as radical tumour resection is generally performed late during the disease. The development of alternative therapeutic approaches to HCC remains one of the most challenging areas of cancer. This review focuses on the relevance of cAMP signalling in the development of hepatocellular carcinoma and identifies the modulation of this second messenger as a new strategy for the control of tumour growth. In addition, because the cAMP pathway is controlled by phosphodiesterases (PDEs), targeting these enzymes using PDE inhibitors is becoming an attractive and promising tool for the control of HCC. Among them, based on current preclinical and clinical findings, PDE4-specific inhibitors remarkably demonstrate therapeutic potential in the management of cancer outcomes, especially as adjuvants to standard therapies. However, more preclinical studies are warranted to ascertain their efficacy during the different stages of hepatocyte transformation and in the treatment of established HCC. Full article

(This article belongs to the Special Issue New Advances in Cyclic AMP Signalling)

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20 pages, 2040 KiB

Open AccessReview

Recent Advances in EPAC-Targeted Therapies: A Biophysical Perspective

by Alveena Ahmed, Stephen Boulton, Hongzhao Shao, Madoka Akimoto, Amarnath Natarajan, Xiaodong Cheng and Giuseppe Melacini

Cells 2019, 8(11), 1462; https://doi.org/10.3390/cells8111462 - 19 Nov 2019

Cited by 14 | Viewed by 4171

Abstract

The universal second messenger cAMP regulates diverse intracellular processes by interacting with ubiquitously expressed proteins, such as Protein Kinase A (PKA) and the Exchange Protein directly Activated by cAMP (EPAC). EPAC is implicated in multiple pathologies, thus several EPAC-specific inhibitors have been identified [...] Read more.

The universal second messenger cAMP regulates diverse intracellular processes by interacting with ubiquitously expressed proteins, such as Protein Kinase A (PKA) and the Exchange Protein directly Activated by cAMP (EPAC). EPAC is implicated in multiple pathologies, thus several EPAC-specific inhibitors have been identified in recent years. However, the mechanisms and molecular interactions underlying the EPAC inhibition elicited by such compounds are still poorly understood. Additionally, being hydrophobic low molecular weight species, EPAC-specific inhibitors are prone to forming colloidal aggregates, which result in non-specific aggregation-based inhibition (ABI) in aqueous systems. Here, we review from a biophysical perspective the molecular basis of the specific and non-specific interactions of two EPAC antagonists—CE3F4R, a non-competitive inhibitor, and ESI-09, a competitive inhibitor of EPAC. Additionally, we discuss the value of common ABI attenuators (e.g., TX and HSA) to reduce false positives at the expense of introducing false negatives when screening aggregation-prone compounds. We hope this review provides the EPAC community effective criteria to evaluate similar compounds, aiding in the optimization of existing drug leads, and informing the development of the next generation of EPAC-specific inhibitors. Full article

(This article belongs to the Special Issue New Advances in Cyclic AMP Signalling)

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28 pages, 1603 KiB

Open AccessReview

Ending Restenosis: Inhibition of Vascular Smooth Muscle Cell Proliferation by cAMP

by Sarah A. Smith, Andrew C. Newby and Mark Bond

Cells 2019, 8(11), 1447; https://doi.org/10.3390/cells8111447 - 16 Nov 2019

Cited by 37 | Viewed by 6246

Abstract

Increased vascular smooth muscle cell (VSMC) proliferation contributes towards restenosis after angioplasty, vein graft intimal thickening and atherogenesis. The second messenger 3′ 5′ cyclic adenosine monophosphate (cAMP) plays an important role in maintaining VSMC quiescence in healthy vessels and repressing VSMC proliferation during [...] Read more.

Increased vascular smooth muscle cell (VSMC) proliferation contributes towards restenosis after angioplasty, vein graft intimal thickening and atherogenesis. The second messenger 3′ 5′ cyclic adenosine monophosphate (cAMP) plays an important role in maintaining VSMC quiescence in healthy vessels and repressing VSMC proliferation during resolution of vascular injury. Although the anti-mitogenic properties of cAMP in VSMC have been recognised for many years, it is only recently that we gained a detailed understanding of the underlying signalling mechanisms. Stimuli that elevate cAMP in VSMC inhibit G₁-S phase cell cycle progression by inhibiting expression of cyclins and preventing S-Phase Kinase Associated Protein-2 (Skp2-mediated degradation of cyclin-dependent kinase inhibitors. Early studies implicated inhibition of MAPK signalling, although this does not fully explain the anti-mitogenic effects of cAMP. The cAMP effectors, Protein Kinase A (PKA) and Exchange Protein Activated by cAMP (EPAC) act together to inhibit VSMC proliferation by inducing Cyclic-AMP Response Element Binding protein (CREB) activity and inhibiting members of the RhoGTPases, which results in remodelling of the actin cytoskeleton. Cyclic-AMP induced actin remodelling controls proliferation by modulating the activity of Serum Response Factor (SRF) and TEA Domain Transcription Factors (TEAD), which regulate expression of genes required for proliferation. Here we review recent research characterising these mechanisms, highlighting novel drug targets that may allow the anti-mitogenic properties of cAMP to be harnessed therapeutically to limit restenosis. Full article

(This article belongs to the Special Issue New Advances in Cyclic AMP Signalling)

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