Protein kinase A inhibitor proteins (PKIs) divert GPCR-Gαs-cAMP signaling toward EPAC and ERK activation and are involved in tumor growth.
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SUBMITTER: Hoy JJ
PROVIDER: S-EPMC7722164 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Background and aimsActivation of the cAMP/Epac signalling stabilises endothelial barrier function. Moreover, its activation is accompanied by an activation of PI3K/Akt and MEK/ERK signalling in diverse cell types but their impact on endothelial barrier function is largely unknown. Here the role of PI3K/Akt and MEK/ERK signalling in cAMP/Epac-mediated endothelial barrier stabilisation was analysed.MethodsEndothelial barrier function was analysed in cultured human umbilical vein endothelial cells (HUVECs) by measuring flux of albumin. A modified cAMP analogue 8-pCPT-2'-O-Me-cAMP (Epac agonist) was used to specifically activate cAMP/Epac signalling.ResultsEpac agonist reduces the basal and attenuates thrombin-induced endothelial hyperpermeability accompanied by an activation of PI3K/Akt and MEK/ERK signalling. The qPCR data demonstrate HUVECs express PI3Kα, PI3Kβ, and PI3Kγ but not PI3Kδ isoforms. The western blot data demonstrate Epac agonist activates PI3Kα and PI3Kβ isoforms. Inhibition of MEK/ERK but not PI3K/Akt pathway potentiates the endothelial barrier protective effects of cAMP/Epac signalling. Inhibition of MEK/ERK signalling in the presence of Epac agonist induces a reorganisation of actin cytoskeleton to the cell periphery, enhanced VE-cadherin localisation at cell-cell junctions, and dephosphorylation of myosin light chains (MLC) but not inhibition of RhoA/Rock signalling. Moreover, Epac agonist promotes endothelial cell (EC) survival via reduction in activities of pro-apoptotic caspases in a PI3K/Akt and MEK/ERK signalling-dependent manner.ConclusionOur data demonstrate that the Epac agonist simultaneously activates diverse signalling pathways in ECs, which may have differential effects on endothelial barrier function. It activates PI3K/Akt and MEK/ERK signalling which mainly govern its pro-survival effects on ECs. Inhibition of MEK/ERK but not PI3K/Akt signalling enhances barrier stabilising and barrier protective effects of cAMP/Epac activation.Chemical compounds used in this study8-pCPT-2'-O-Me-cAMP (PubChem CID: 9913268); Akt inhibitor VIII (PubChem CID: 10196499); AS-252424 (PubChem CID: 11630874); IC-87114 (PubChem CID: 9908783); PD 98059 (PubChem CID: 4713); PIK-75 (PubChem CID: 10275789); TGX-221 (PubChem CID: 9907093); Thrombin (PubChem CID: 90470996); U0126 (PubChem CID: 3006531); Wortmannin (PubChem CID: 312145).
| S-EPMC6855264 | biostudies-literature
Project description:This study explores the role of G-protein-coupled receptor-intracellular signaling in the development of P450-mediated insecticide resistance in mosquitoes, Culex quinquefasciatus, focusing on the essential function of the GPCRs and their downstream effectors of Gs alpha subunit protein (Gαs) and adenylyl cyclase (ACs) in P450-mediated insecticide resistance of Culex mosquitoes. Our RNAi-mediated functional study showed that knockdown of Gαs caused the decreased expression of the downstream effectors of ACs and PKAs in the GPCR signaling pathway and resistance P450 genes, whereas knockdown of ACs decreased the expression of PKAs and resistance P450 genes. Knockdown of either Gαs or ACs resulted in an increased susceptibility of mosquitoes to permethrin. These results add significantly to our understanding of the molecular basis of resistance P450 gene regulation through GPCR/Gαs/AC/cAMP-PKA signaling pathways in the insecticide resistance of mosquitoes. The temporal and spatial dynamic analyses of GPCRs, Gαs, ACs, PKAs, and P450s in two insecticide resistant mosquito strains revealed that all the GPCR signaling pathway components tested, namely GPCRs, Gαs, ACs and PKAs, were most highly expressed in the brain for both resistant strains, suggesting the role played by these genes in signaling transduction and regulation. The resistance P450 genes were mainly expressed in the brain, midgut and malpighian tubules (MTs), suggesting their critical function in the central nervous system and importance for detoxification. The temporal dynamics analysis for the gene expression showed a diverse expression profile during mosquito development, indicating their initially functional importance in response to exposure to insecticides during their life stages.
| S-EPMC5613228 | biostudies-literature
Project description:Epac1 is a guanine nucleotide exchange factor for Rap1 that is activated by direct binding of cAMP. In vitro studies suggest that cAMP relieves the interaction between the regulatory and catalytic domains of Epac. Here, we monitor Epac1 activation in vivo by using a CFP-Epac-YFP fusion construct. When expressed in mammalian cells, CFP-Epac-YFP shows significant fluorescence resonance energy transfer (FRET). FRET rapidly decreases in response to the cAMP-raising agents, whereas it fully recovers after addition of cAMP-lowering agonists. Thus, by undergoing a cAMP-induced conformational change, CFP-Epac-YFP serves as a highly sensitive cAMP indicator in vivo. When compared with a protein kinase A (PKA)-based sensor, Epac-based cAMP probes show an extended dynamic range and a better signal-to-noise ratio; furthermore, as a single polypeptide, CFP-Epac-YFP does not suffer from the technical problems encountered with multisubunit PKA-based sensors. These properties make Epac-based FRET probes the preferred indicators for monitoring cAMP levels in vivo.
| S-EPMC1299185 | biostudies-literature
Project description:Background and purposeH2 O2 is widely understood to regulate intracellular signalling. In airway epithelia, H2 O2 stimulates anion secretion primarily by activating an autocrine PGE2 signalling pathway via EP4 and EP1 receptors to initiate cytic fibrosis transmembrane regulator (CFTR)-mediated Cl(-) secretion. This study investigated signalling downstream of the receptors activated by H2 O2 .Experimental approachAnion secretion by differentiated bronchial epithelial cells was measured in Ussing chambers during stimulation with H2 O2 , an EP4 receptor agonist or β2 -adrenoceptor agonist in the presence and absence of inhibitors of ACs and downstream effectors. Intracellular calcium ([Ca(2+) ]I ) changes were followed by microscopy using fura-2-loaded cells and PKA activation followed by FRET microscopy.Key resultsTransmembrane adenylyl cyclase (tmAC) and soluble AC (sAC) were both necessary for H2 O2 and EP4 receptor-mediated CFTR activation in bronchial epithelia. H2 O2 and EP4 receptor agonist stimulated tmAC to increase exchange protein activated by cAMP (Epac) activity that drives PLC activation to raise [Ca(2+) ]i via Ca(2+) store release (and not entry). Increased [Ca(2+) ]i led to sAC activation and further increases in CFTR activity. Stimulation of sAC did not depend on changes in [HCO3 (-) ]. Ca(2+) -activated apical KCa 1.1 channels and cAMP-activated basolateral KV 7.1 channels contributed to H2 O2 -stimulated anion currents. A similar Epac-mediated pathway was seen following β2 -adrenoceptor or forskolin stimulation.Conclusions and implicationsH2 O2 initiated a complex signalling cascade that used direct stimulation of tmACs by Gαs followed by Epac-mediated Ca(2+) crosstalk to activate sAC. The Epac-mediated Ca(2+) signal constituted a positive feedback loop that amplified CFTR anion secretion following stimulation of tmAC by a variety of stimuli.
| S-EPMC4280976 | biostudies-literature
Project description:G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR)-mediated increases in the second messenger cyclic adenosine monophosphate (cAMP) activate the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase (ERK), and in neuroendocrine cells, this pathway leads to cAMP-dependent neuritogenesis mediated through Rap1 and B-Raf. We found that the Rap guanine nucleotide exchange factor Rapgef2 was enriched from primary bovine neuroendocrine cells by cAMP-agarose affinity chromatography and that it was specifically eluted by cAMP. With loss-of-function experiments in the rat neuronal cell line Neuroscreen-1 (NS-1) and gain-of-function experiments in human embryonic kidney 293T cells, we demonstrated that Rapgef2 connected GPCR-dependent activation of adenylate cyclase and increased cAMP concentration with the activation of ERK in neurons and endocrine cells. Furthermore, knockdown of Rapgef2 blocked cAMP- and ERK-dependent neuritogenesis. Our data are consistent with a pathway involving the cAMP-mediated activation of Rapgef2, which then stimulates Rap1, leading to increases in B-Raf, MEK, and ERK activity.
| S-EPMC3932028 | biostudies-literature
Project description:The well-known second messenger cyclic adenosine monophosphate (cAMP) regulates the morphology and physiology of neurons and thus higher cognitive brain functions. The discovery of exchange protein activated by cAMP (Epac) as a guanine nucleotide exchange factor for Rap GTPases has shed light on protein kinase A (PKA)-independent functions of cAMP signaling in neural tissues. Studies of cAMP-Epac-mediated signaling in neurons under normal and disease conditions also revealed its diverse contributions to neurodevelopment, synaptic remodeling, and neurotransmitter release, as well as learning, memory, and emotion. In this mini-review, the various roles of Epac isoforms, including Epac1 and Epac2, highly expressed in neural tissues are summarized, and controversies or issues are highlighted that need to be resolved to uncover the critical functions of Epac in neural tissues and the potential for a new therapeutic target of mental disorders. [BMB Reports 2021; 54(3): 149-156].
| S-EPMC8016657 | biostudies-literature
Project description:It has now been over 10 years since efforts to completely understand the signalling actions of cAMP (3'-5'-cyclic adenosine monophosphate) led to the discovery of exchange protein directly activated by cAMP (EPAC) proteins. In the current review we will highlight important advances in the understanding of EPAC structure and function and demonstrate that EPAC proteins mediate multiple actions of cAMP in cells, revealing future targets for pharmaceutical intervention. It has been known for some time that drugs that elevate intracellular cAMP levels have proven therapeutic benefit for diseases ranging from depression to inflammation. The challenge now is to determine which of these positive actions of cAMP involve activation of EPAC-regulated signal transduction pathways. EPACs are specific guanine nucleotide exchange factors for the Ras GTPase homologues, Rap1 and Rap2, which they activate independently of the classical routes for cAMP signalling, cyclic nucleotide-gated ion channels and protein kinase A. Rather, EPAC activation is triggered by internal conformational changes induced by direct interaction with cAMP. Leading from this has been the development of EPAC-specific agonists, which has helped to delineate numerous cellular actions of cAMP that rely on subsequent activation of EPAC. These include regulation of exocytosis and the control of cell adhesion, growth, division and differentiation. Recent work also implicates EPAC in the regulation of anti-inflammatory signalling in the vascular endothelium, namely negative regulation of pro-inflammatory cytokine signalling and positive support of barrier function. Further elucidation of these important signalling mechanisms will no doubt support the development of the next generation of anti-inflammatory drugs.
| S-EPMC2795248 | biostudies-literature
Project description:Mucosal epithelial cell integrity is an important component of innate immunity and it protects the host from an environment rich in microorganisms. Virulence factors from Gram-negative bacteria [e.g. lipopolysaccharide (LPS)] induce significant pro-inflammatory cytokine expression. Monoamine oxidase (MAO) inhibitors reduce cytokine expression in a variety of inflammatory models and may therefore have therapeutic potential for a number of inflammatory diseases. We tested the anti-inflammatory therapeutic potential of a recently developed reversible MAO-B inhibitor (RG0216) with reduced transport across the blood-brain barrier. In an epithelial cell culture model, RG0216 significantly decreased LPS-induced interleukin (IL)-6 and IL-1β gene and protein expression and was as effective as equimolar concentrations of deprenyl (an existing irreversible MAO-B inhibitor). Hydrogen peroxide and modulating dopamine receptor signaling had no effect on cytokine expression. We showed that LPS-induced expression of IL-6 and IL-1β was cAMP dependent, that IL-6 and IL-1β expression were induced by direct cAMP activation (forskolin) and that RG0216 and deprenyl effectively reduced cAMP-mediated cytokine expression. Targeted protein kinase A (PKA) and Exchange Protein Activated by cAMP (EPAC) activation regulated IL-6 and IL-1β expression, albeit in different ways, but both cytokines were effectively decreased with RG0216. RG0216 reduction of LPS-induced cytokine expression occurred by acting downstream of the cAMP-PKA/EPAC signaling cascade. This represents a novel mechanism by which MAO-B selective inhibitors regulate LPS-induced IL-6 and IL-1β expression.
| S-EPMC8635787 | biostudies-literature
Project description:Fibrosis, the result of excess deposition of extracellular matrix (ECM), in particular collagen, leads to scarring and loss of function in tissues that include the heart, lung, kidney and liver. The second messenger cAMP can inhibit the formation and extent of ECM during this late phase of inflammation, but the mechanisms for these actions of cAMP and of agents that elevate tissue cAMP levels are not well understood. In this article, we review the fibrotic process and focus on two recently recognized aspects of actions of cAMP and its effector Epac (Exchange protein activated by cAMP): (a) blunting of epithelial-mesenchymal transformation (EMT) and (b) down-regulation of Epac expression by profibrotic agents (e.g. TGF-?, angiotensin II), which may promote tissue fibrosis by decreasing Epac-mediated antifibrotic actions. Pharmacological approaches that raise cAMP or blunt the decrease in Epac expression by profibrotic agents may thus be strategies to block or perhaps reverse tissue fibrosis. LINKED ARTICLES This article is part of a themed section on Novel cAMP Signalling Paradigms. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.166.issue-2.
| S-EPMC3417479 | biostudies-literature
Project description:This review focuses on one family of the known cAMP receptors, the exchange proteins directly activated by cAMP (EPACs), also known as the cAMP-regulated guanine nucleotide exchange factors (cAMP-GEFs). Although EPAC proteins are fairly new additions to the growing list of cAMP effectors, and relatively "young" in the cAMP discovery timeline, the significance of an EPAC presence in different cell systems is extraordinary. The study of EPACs has considerably expanded the diversity and adaptive nature of cAMP signaling associated with numerous physiological and pathophysiological responses. This review comprehensively covers EPAC protein functions at the molecular, cellular, physiological, and pathophysiological levels; and in turn, the applications of employing EPAC-based biosensors as detection tools for dissecting cAMP signaling and the implications for targeting EPAC proteins for therapeutic development are also discussed.
| S-EPMC6050347 | biostudies-literature