Exchange Protein Activated By cAMP Research Articles

Role of the adenylate cyclase/cyclic AMP pathway in oxytocin-induced lacrimal gland myoepithelial cells contraction

The aim of these studies was to investigate the involvement of the second messenger 3′,5′-cyclic adenosine monophosphate (cAMP) and its downstream effectors in oxytocin (OXT)-mediated lacrimal gland myoepithelial cell (MEC) contraction. Lacrimal gland MEC were isolated and propagated from alpha-smooth muscle actin (SMA)-GFP mice. RNA and protein samples were prepared to analyze G protein expression by RT-PCR and western blotting; respectively. Changes in intracellular cAMP concentration were measured using a competitive ELISA kit. To increase intracellular cAMP concentration, the following agents were used: forskolin (FKN, a direct activator of adenylate cyclase), 3-isobutyl-1-methylxanthine (IBMX, an inhibitor of the phosphodiesterase that hydrolyzes cAMP), or a cell permeant cAMP analog, dibutyryl (db)-cAMP. In addition, inhibitors and selective agonists were used to investigate the role of cAMP effector molecules, protein kinase A (PKA) and exchange protein activated by cAMP (EPAC) in OXT-induced MEC contraction. MEC contraction was monitored in real time and changes in cell size were quantified using ImageJ software. The adenylate cyclase coupling G proteins, Gαs, Gαo, and Gαi, are expressed in lacrimal gland MEC at both the mRNA and protein levels. OXT increased intracellular cAMP in a concentration-dependent manner. FKN, IBMX and db-cAMP significantly stimulated MEC contraction. Preincubation of cells with either Myr-PKI, a specific PKA inhibitor or ESI09, an EPAC inhibitor, resulted in almost complete inhibition of both FKN- as well as OXT-stimulated MEC contraction. Finally, direct activation of PKA or EPAC using selective agonists triggered MEC contraction. We conclude that cAMP agonists modulate lacrimal gland MEC contraction via PKA and EPAC activation which also play a major role in OXT induced MEC contraction.

Inhibition of exchange proteins directly activated by cAMP as a strategy for broad-spectrum antiviral development

The recent SARS-CoV-2 and mpox outbreaks have highlighted the need to expand our arsenal of broad-spectrum antiviral agents for future pandemic preparedness. Host-directed antivirals are an important tool to accomplish this as they typically offer protection against a broader range of viruses than direct-acting antivirals and have a lower susceptibility to viral mutations that cause drug resistance. In this study, we investigate the Exchange Protein Activated by cAMP (EPAC) as a target for broad-spectrum antiviral therapy. We find that the EPAC-selective inhibitor, ESI-09 provides robust protection against a variety of viruses, including SARS-CoV-2 and Vaccinia (VACV) - an orthopoxvirus from the same family as mpox. We show, using a series of immunofluorescence experiments, that ESI-09 remodels the actin cytoskeleton through Rac1/Cdc42 GTPases and the Arp2/3 complex, impairing internalization of viruses that use clathrin-mediated endocytosis (e.g. VSV) or micropinocytosis (e.g. VACV). Additionally, we find that ESI-09 disrupts syncytia formation and inhibits cell-to-cell transmission of viruses such as measles and VACV. When administered to immune-deficient mice in an intranasal challenge model, ESI-09 protects mice from lethal doses of VACV and prevents formation of pox lesions. Altogether, our finding show that EPAC antagonists such as ESI-09 are promising candidates for broad-spectrum antiviral therapy that can aid in the fight against ongoing and future viral outbreaks.

Serum Estradiol Level Correlates With Molecules Regulating Phrenic Motoneuron Plasticity

Acute Intermittent Hypoxia (AIH) elicits phrenic long‐term facilitation (pLTF), an important model of respiratory motor plasticity. Although moderate AIH‐induced pLTF exhibits profound age‐dependent sexual dimorphism, little is known concerning the age or sex‐dependent changes in molecules regulating the Q or S pathways to phrenic motor facilitation. AIH elicits pLTF via distinct signaling pathways in phrenic motor neurons initiated by 5HT2A/2B(Q pathway) or 5HT7 & Adenosine 2A receptors (S pathway); these pathways interact via powerful crosstalk inhibition. Moderate AIH (mAIH)‐induced pLTF declines ~50% from 3 to 12 months of age in male rats (Zabka et al., 2001). Gonadectomy abolishes pLTF in young adult males, and it is restored by supplemental testosterone; pLTF is restored due to testosterone conversion into estrogen in the CNS (Zabka et al., 2006). Thus, CNS estrogen must impact keys molecules regulating the Q and/or S pathways to phrenic motor facilitation. Here, we tested the hypothesis that serum estradiol is correlated with keys molecules known to regulate the Q and S pathways in rats. We assessed the mRNA of molecules known to regulate pLTF in ventral cervical spinal homogenates containing the phrenic motor nucleus from young (3 month) and middle‐aged (12 month) male and female Sprague‐Dawley rats; mRNA levels were correlated with serum estradiol levels assessed via ELISA. Spinal cord tissues were preserved in “RNA Later” solution <10 min from harvest, and stored at ‐80°C until mRNA extraction. RT PCR was used to quantify mRNA levels for: serotonin 2A (Htr2a) & 2B (Htr2b) and adenosine 2A receptors (Adora2a); exchange protein activated by cAMP (EPAC); PKA catalytic subunit (Prkaa1); PKA regulatory subunit (Prkar1a); fractalkine (CX3CL1); phosphodiesterase type 4 (pde4b); NOX genes (gp91 and p47); and PKCδ. We found that serum estradiol was positively correlated with mRNA expression of EPAC (r2=0.28, p=0.002), pde4b mRNA (r2=0.15, p=0.029) and adora2a (r2=0.15, p=0.030), but not the other molecules studied. We conclude that serum estradiol may shift the Q‐S pathway balance, although we have not yet demonstrated a causal relationship. An important limitation of this study is that serum estradiol concentration may differ from that in the ventral cervical spinal cord. In future studies, it is important to test the significance in these neurochemical shifts in mechanisms giving rise to the age‐dependent sexual dimorphism in AIH‐induced pLTF.

Monoamine Oxidase-B Inhibitor Reduction in Pro-Inflammatory Cytokines Mediated by Inhibition of cAMP-PKA/EPAC Signaling.

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.

Regulation of P2X1 receptors by modulators of the cAMP effectors PKA and EPAC

P2X1 receptors are adenosine triphosphate (ATP)-gated cation channels that are functionally important for male fertility, bladder contraction, and platelet aggregation. The activity of P2X1 receptors is modulated by lipids and intracellular messengers such as cAMP, which can stimulate protein kinase A (PKA). Exchange protein activated by cAMP (EPAC) is another cAMP effector; however, its effect on P2X1 receptors has not yet been determined. Here, we demonstrate that P2X1 currents, recorded from human embryonic kidney (HEK) cells transiently transfected with P2X1 cDNA, were inhibited by the highly selective EPAC activator 007-AM. In contrast, EPAC activation enhanced P2X2 current amplitude. The PKA activator 6-MB-cAMP did not affect P2X1 currents, but inhibited P2X2 currents. The inhibitory effects of EPAC on P2X1 were prevented by triple mutation of residues 21 to 23 on the amino terminus of P2X1 subunits to the equivalent amino acids on P2X2 receptors. Double mutation of residues 21 and 22 and single mutation of residue 23 also protected P2X1 receptors from inhibition by EPAC activation. Finally, the inhibitory effects of EPAC on P2X1 were also prevented by NSC23766, an inhibitor of Rac1, a member of the Rho family of small GTPases. These data suggest that EPAC is an important regulator of P2X1 and P2X2 receptors.

Glucagon Activates mTOR1/2 Signaling via an EPAC/Rap1 Signaling Axis in Hepatocyte Cultures

Activation of the protein kinase mTOR (mechanistic target of rapamycin) in complex 1/2 (mTORC1/2) in the liver is repressed during fasting and rapidly stimulated in response to feeding. The effect of a meal on hepatic mTORC1/2 is largely mediated by an increase in plasma levels of nutrients (i.e. amino acids) and growth factors (i.e. insulin). By contrast, fasting is associated with elevated plasma levels of glucagon, which is conventionally viewed as playing a counter-regulatory role to insulin. However, glucagon and insulin secretion are both rapidly increased by consumption of a protein meal. In fact, amino acid-induced glucagon secretion is many times greater than that seen with fasting. Herein we investigated the impact of insulin and glucagon on mTORC1/2 activation in rat H4IIE and human HepG2 cell cultures. As expected, insulin enhanced phosphorylation of the mTORC1 substrates p70S6K1 and 4E-BP1. Surprisingly, the effect of glucagon on mTORC1 signaling was biphasic, wherein there was an acute increase in phosphorylation of p70S6K1 and 4E-BP1 over the first hour of exposure, followed by latent suppression. Notably, the stimulatory effect of glucagon on mTORC1 was not additive with insulin, suggesting a convergent signaling axis. Glucagon-induced mTORC1 activation was associated with enhanced cAMP levels and required activation of the glucagon receptor, PI3K/Akt, and exchange protein activated by cAMP (EPAC). An EPAC-specific cAMP analog as well as PKA inhibition enhanced activation of mTORC1/2. EPAC acts as the guanine nucleotide exchange factor for the small GTPase Rap1, and in H4IIE cultures, transient expression of Rap1 was sufficient to stimulate activation of mTORC1/2. Finally, we found that glucagon addition to culture medium was sufficient to promote Rap1-GTP loading in H4IIE cells. Overall, the findings support that glucagon elicits biphasic regulation of hepatic mTORC1/2 by an EPAC-dependent increase in Rap1-GTP which is followed by a repression of mTORC1.

Glucagon transiently stimulates mTORC1 by activation of an EPAC/Rap1 signaling axis

Activation of the protein kinase mechanistic target of rapamycin (mTOR) in both complexes 1 and 2 (mTORC1/2) in the liver is repressed during fasting and rapidly stimulated in response to a meal. The effect of feeding on hepatic mTORC1/2 is attributed to an increase in plasma levels of nutrients, such as amino acids, and insulin. By contrast, fasting is associated with elevated plasma levels of glucagon, which is conventionally viewed as having a counter-regulatory role to insulin. More recently an expanded role for glucagon action in post-prandial metabolism has been demonstrated. Herein we investigated the impact of insulin and glucagon on mTORC1/2 activation. In H4IIE and HepG2 cultures, insulin enhanced phosphorylation of the mTORC1 substrates S6K1 and 4E-BP1. Surprisingly, the effect of glucagon on mTORC1 was biphasic, wherein there was an acute increase in phosphorylation of S6K1 and 4E-BP1 over the first hour of exposure, followed by latent suppression. The transient stimulatory effect of glucagon on mTORC1 was not additive with insulin, suggesting convergent signaling. Glucagon enhanced cAMP levels and mTORC1 stimulation required activation of the glucagon receptor, PI3K/Akt, and exchange protein activated by cAMP (EPAC). EPAC acts as the guanine nucleotide exchange factor for the small GTPase Rap1. Rap1 expression enhanced S6K1 phosphorylation and glucagon addition to culture medium promoted Rap1-GTP loading. Signaling through mTORC1 acts to regulate protein synthesis and we found that glucagon promoted an EPAC-dependent increase in protein synthesis. Overall, the findings support that glucagon elicits acute activation of mTORC1/2 by an EPAC-dependent increase in Rap1-GTP.

Serotonin enhances depolarizing spontaneous fluctuations, excitability, and ongoing activity in isolated rat DRG neurons via 5-HT4 receptors and cAMP-dependent mechanisms

Ongoing activity in nociceptors, a driver of spontaneous pain, can be generated in dorsal root ganglion neurons in the absence of sensory generator potentials if one or more of three neurophysiological alterations occur – prolonged depolarization of resting membrane potential (RMP), hyperpolarization of action potential (AP) threshold, and/or increased amplitude of depolarizing spontaneous fluctuations of membrane potential (DSFs) to bridge the gap between RMP and AP threshold. Previous work showed that acute, sustained exposure to serotonin (5-HT) hyperpolarized AP threshold and potentiated DSFs, leading to ongoing activity if a separate source of maintained depolarization was present. Cellular signaling pathways that increase DSF amplitude and promote ongoing activity acutely in nociceptors are not known for any neuromodulator. Here, isolated DRG neurons from male rats were used to define the pathway by which low concentrations of 5-HT enhance DSFs, hyperpolarize AP threshold, and promote ongoing activity. A selective 5-HT4 receptor antagonist blocked these 5-HT-induced hyperexcitable effects, while a selective 5-HT4 agonist mimicked the effects of 5-HT. Inhibition of cAMP effectors, protein kinase A (PKA) and exchange protein activated by cAMP (EPAC), attenuated 5-HT's hyperexcitable effects, but a blocker of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels had no significant effect. 5-HT4-dependent PKA activation was specific to DRG neurons that bind isolectin B4 (a nonpeptidergic nociceptor marker). 5-HT's effects on AP threshold, DSFs, and ongoing activity were mimicked by a cAMP analog. Sustained exposure to 5-HT promotes ongoing activity in nonpeptidergic nociceptors through the Gs-coupled 5-HT4 receptor and downstream cAMP signaling involving both PKA and EPAC.

The Parkinson's disease–associated kinase LRRK2 regulates genes required for cell adhesion, polarization, and chemotaxis in activated murine macrophages

Leucine-rich repeat kinase 2 (LRRK2) encodes a complex protein that includes kinase and GTPase domains. Genome-wide association studies have identified dominant LRRK2 alleles that predispose their carriers to late-onset idiotypic Parkinson's disease (PD) and also to autoimmune disorders such as Crohn's disease. Considerable evidence indicates that PD initiation and progression involve activation of innate immune functions in microglia, which are brain-resident macrophages. Here we asked whether LRRK2 modifies inflammatory signaling and how this modification might contribute to PD and Crohn's disease. We used RNA-Seq–based high-resolution transcriptomics to compare gene expression in activated primary macrophages derived from WT and Lrrk2 knockout mice. Remarkably, expression of a single gene, Rap guanine nucleotide exchange factor 3 (Rapgef3), was strongly up-regulated in the absence of LRRK2 and down-regulated in its presence. We observed similar regulation of Rapgef3 expression in cells treated with a highly specific inhibitor of LRRK2 protein kinase activity. Rapgef3 encodes an exchange protein, activated by cAMP 1 (EPAC-1), a guanine nucleotide exchange factor that activates the small GTPase Rap-1. Rap-1 mediates cell adhesion, polarization, and directional motility, and our results indicate that LRRK2 modulates chemotaxis of microglia and macrophages. Dominant PD-associated LRRK2 alleles may suppress EPAC-1 activity, further restricting motility and preventing efficient migration of microglia to sites of neuronal damage. Functional analysis in vivo in a subclinical infection model also indicated that Lrrk2 subtly modifies the inflammatory response. These results indicate that LRRK2 modulates the expression of genes involved in murine immune cell chemotaxis.

EPAC Negatively Regulates Myelination via Controlling Proliferation of Oligodendrocyte Precursor Cells.

Increasing evidence suggests that a cyclic adenosine monophosphate (cAMP)-dependent intracellular signal drives the process of myelination. Yet, the signal transduction underlying the action of cAMP on central nervous system myelination remains undefined. In the present work, we sought to determine the role of EPAC (exchange protein activated by cAMP), a downstream effector of cAMP, in the development of the myelin sheath using EPAC1 and EPAC2 double-knockout (EPACdKO) mice. The results showed an age-dependent regulatory effect of EPAC1 and EPAC2 on myelin development, as their deficiency caused more myelin sheaths in postnatal early but not late adult mice. Knockout of EPAC promoted the proliferation of oligodendrocyte precursor cells and had diverse effects on myelin-related transcription factors, which in turn increased the expression of myelin-related proteins. These results indicate that EPAC proteins are negative regulators of myelination and may be promising targets for the treatment of myelin-related diseases.

Mechanism of Action of an EPAC1-Selective Competitive Partial Agonist.

The exchange protein activated by cAMP (EPAC) is a promising drug target for a wide disease range, from neurodegeneration and infections to cancer and cardiovascular conditions. A novel partial agonist of the EPAC isoform 1 (EPAC1), I942, was recently discovered, but its mechanism of action remains poorly understood. Here, we utilize NMR spectroscopy to map the I942-EPAC1 interactions at atomic resolution and propose a mechanism for I942 partial agonism. We found that I942 interacts with the phosphate binding cassette (PBC) and base binding region (BBR) of EPAC1, similar to cyclic adenosine monophosphate (cAMP). These results not only reveal the molecular basis for the I942 vs cAMP mimicry and competition, but also suggest that the partial agonism of I942 arises from its ability to stabilize an inhibition-incompetent activation intermediate distinct from both active and inactive EPAC1 states. The mechanism of action of I942 may facilitate drug design for EPAC-related diseases.

Angiotensin-(1-7) Attenuates Protein O-GlcNAcylation in the Retina by EPAC/Rap1-Dependent Inhibition of O-GlcNAc Transferase.

Purpose O-GlcNAcylation of cellular proteins contributes to the pathophysiology of diabetes and evidence supports a role for augmented O-GlcNAcylation in diabetic retinopathy. The aim of this study was to investigate the impact of the renin-angiotensin system on retinal protein O-GlcNAcylation.MethodsMice fed a high-fat diet were treated chronically with the angiotensin-converting enzyme inhibitor captopril or captopril plus the angiotensin-(1–7) Mas receptor antagonist A779. Western blotting and quantitative polymerase chain reaction were used to analyze retinal homogenates. Similar analyses were performed on lysates from human MIO-M1 retinal Müller cell cultures exposed to media supplemented with angiotensin-(1–7). Culture conditions were manipulated to influence the hexosamine biosynthetic pathway and/or signaling downstream of the Mas receptor.ResultsIn the retina of mice fed a high-fat diet, captopril attenuated protein O-GlcNAcylation in a manner dependent on Mas receptor activation. In MIO-M1 cells, angiotensin-(1–7) or adenylate cyclase activation were sufficient to enhance cyclic AMP (cAMP) levels and inhibit O-GlcNAcylation. The repressive effect of cAMP on O-GlcNAcylation was dependent on exchange protein activated by cAMP (EPAC), but not protein kinase A, and was recapitulated by a constitutively active variant of the small GTPase Rap1. We provide evidence that cAMP and angiotensin-(1–7) act to suppress O-GlcNAcylation by inhibition of O-GlcNAc transferase (OGT) activity. In cells exposed to an O-GlcNAcase inhibitor or hyperglycemic culture conditions, mitochondrial superoxide levels were elevated; however, angiotensin-(1–7) signaling prevented the effect.ConclusionsAngiotensin-(1–7) inhibits retinal protein O-GlcNAcylation via an EPAC/Rap1/OGT signaling axis.

EPAC1 and EPAC2 promote nociceptor hyperactivity associated with chronic pain after spinal cord injury

Chronic pain following spinal cord injury (SCI) is associated with electrical hyperactivity (spontaneous and evoked) in primary nociceptors. Cyclic adenosine monophosphate (cAMP) signaling is an important contributor to nociceptor excitability, and knockdown of the cAMP effector, exchange protein activated by cAMP (EPAC), has been shown to relieve pain-like responses in several chronic pain models. To examine potentially distinct roles of each EPAC isoform (EPAC1 and 2) in maintaining chronic pain, we used rat and mouse models of contusive spinal cord injury (SCI). Pharmacological inhibition of EPAC1 or 2 in a rat SCI model was sufficient to reverse SCI-induced nociceptor hyperactivity, indicating that EPAC1 and 2 signaling activity are complementary, with both required to maintain hyperactivity. However, EPAC activation was not sufficient to induce similar hyperactivity in nociceptors from naïve rats, and we observed no change in EPAC protein expression after SCI. In the mouse SCI model, inhibition of both EPAC isoforms through a combination of pharmacological inhibition and genetic deletion was required to reverse SCI-induced nociceptor hyperactivity. This was consistent with our finding that neither EPAC1−/− nor EPAC2−/− mice were protected against SCI-induced chronic pain as assessed with an operant mechanical conflict test. Thus, EPAC1 and 2 activity may play a redundant role in mouse nociceptors, although no corresponding change in EPAC protein expression levels was detected after SCI. Despite some differences between these species, our data demonstrate a fundamental role for both EPAC1 and EPAC2 in mechanisms maintaining nociceptor hyperactivity and chronic pain after SCI.

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

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.

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

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.

Elevated cyclic-AMP represses expression of exchange protein activated by cAMP (EPAC1) by inhibiting YAP-TEAD activity and HDAC-mediated histone deacetylation

Ligand-induced activation of Exchange Protein Activated by cAMP-1 (EPAC1) is implicated in numerous physiological and pathological processes, including cardiac fibrosis where changes in EPAC1 expression have been detected. However, little is known about how EPAC1 expression is regulated. Therefore, we investigated regulation of EPAC1 expression by cAMP in cardiac fibroblasts.Elevation of cAMP using forskolin, cAMP-analogues or adenosine A2B-receptor activation significantly reduced EPAC1 mRNA and protein levels and inhibited formation of F-actin stress fibres. Inhibition of actin polymerisation with cytochalasin-D, latrunculin-B or the ROCK inhibitor, Y-27632, mimicked effects of cAMP on EPAC1 mRNA and protein levels. Elevated cAMP also inhibited activity of an EPAC1 promoter-reporter gene, which contained a consensus binding element for TEAD, which is a target for inhibition by cAMP. Inhibition of TEAD activity using siRNA-silencing of its co-factors YAP and TAZ, expression of dominant-negative TEAD or treatment with YAP-TEAD inhibitors, significantly inhibited EPAC1 expression. However, whereas expression of constitutively-active YAP completely reversed forskolin inhibition of EPAC1-promoter activity it did not rescue EPAC1 mRNA levels. Chromatin-immunoprecipitation detected a significant reduction in histone3-lysine27-acetylation at the EPAC1 proximal promoter in response to forskolin stimulation. HDAC1/3 inhibition partially reversed forskolin inhibition of EPAC1 expression, which was completely rescued by simultaneously expressing constitutively active YAP.Taken together, these data demonstrate that cAMP downregulates EPAC1 gene expression via disrupting the actin cytoskeleton, which inhibits YAP/TAZ-TEAD activity in concert with HDAC-mediated histone deacetylation at the EPAC1 proximal promoter. This represents a novel negative feedback mechanism controlling EPAC1 levels in response to cAMP elevation.

Soluble adenylyl cyclase-mediated cAMP signaling and the putative role of PKA and EPAC in cerebral mitochondrial function.

Mitochondria produce the bulk of the ATP in most cells, including brain cells. Regulating this complex machinery to match the energetic needs of the cell is a complicated process that we have yet to understand in its entirety. In this context, 3',5'-cyclic AMP (cAMP) has been suggested to play a seminal role in signaling-metabolism coupling and regulation of mitochondrial ATP production. In cells, cAMP signals may affect mitochondria from the cytosolic side but more recently, a cAMP signal produced within the matrix of mitochondria by soluble adenylyl cyclase (sAC) has been suggested to regulate respiration and thus ATP production. However, little is known about these processes in brain mitochondria, and the effectors of the cAMP signal generated within the matrix are not completely clear since both protein kinase A (PKA) and exchange protein activated by cAMP 1 (EPAC1) have been suggested to be involved. Here, we review the current knowledge and relate it to brain mitochondria. Further, based on measurements of respiration, membrane potential, and ATP production in isolated mouse brain cortical mitochondria we show that inhibitors of sAC, PKA, or EPAC affect mitochondrial function in distinct ways. In conclusion, we suggest that brain mitochondria do regulate their function via sAC-mediated cAMP signals and that both PKA and EPAC could be involved downstream of sAC. Finally, due to the role of faulty mitochondrial function in a range of neurological diseases, we expect that the function of sAC-cAMP-PKA/EPAC signaling in brain mitochondria will likely attract further attention.

Phosphodiesterase 3B (PDE3B) antagonizes the anti-angiogenic actions of PKA in human and murine endothelial cells

Recent reports show that protein kinase A (PKA), but not exchange protein activated by cAMP (EPAC), acts in a cell autonomous manner to constitutively reduce the angiogenic sprouting capacity of murine and human endothelial cells. Specificity in the cellular actions of individual cAMP-effectors can be achieved when a cyclic nucleotide phosphodiesterase (PDE) enzyme acts locally to control the “pool” of cAMP that activates the cAMP-effector. Here, we examined whether PDEs coordinate the actions of PKA during endothelial cell sprouting. Inhibiting each of the cAMP-hydrolyzing PDEs expressed in human endothelial cells revealed that phosphodiesterase 3 (PDE3) inhibition with cilostamide reduced angiogenic sprouting in vitro, while inhibitors of PDE2 and PDE4 family enzymes had no such effect. Identifying a critical role for PDE3B in the anti-angiogenic effects of cilostamide, silencing this PDE3 variant, but not PDE3A, markedly impaired sprouting. Importantly, using both in vitro and ex vivo models of angiogenesis, we show the hypo-sprouting phenotype induced by PDE3 inhibition or PDE3B silencing was reversed by PKA inhibition. Examination of the individual cellular events required for sprouting revealed that PDE3B and PKA each regulated angiogenic sprouting by controlling the invasive capacity of endothelial cells, more specifically, by regulating podosome rosette biogenesis and matrix degradation. In support of the idea that PDE3B acts to inhibit angiogenic sprouting by limiting PKA-mediated reductions in active cdc42, the effects of PDE3B and/or PKA on angiogenic sprouting were negated in cells with reduced cdc42 expression or activity. Since PDE3B and PKA were co-localized in a perinuclear region in human ECs, could be co-immunoprecipitated from lysates of these cells, and silencing PDE3B activated the perinuclear pool of PKA in these cells, we conclude that PDE3B-mediated hydrolysis of cAMP acts to limit the anti-angiogenic potential of PKA in ECs.

Exchange-protein activated by cAMP (EPAC) regulates L-type calcium channel in atrial fibrillation of heart failure model.

Both atrial fibrillation (AF) and heart failure (HF) are increasingly prevalent and related to high hospitalization rate and mortality. AF is a cause as well as a consequence of HF, with complicated interactions resulting in impairment of cardiac systolic and diastolic function. Conversely, the complex structural and neurohormonal alterations in HF contribute to the occurrence and development of AF. However, the molecular mechanism remains unclear. This study aims to explore the effect of Exchange-protein activated by cAMP 1 (EPAC1) on AF in isoproterenol (ISO)-induced HF and the potential molecular mechanism. Mice and cultured isolated adult cardiomyocytes were treated with ISO and or not EPAC1 inhibitor CE3F4. Programmed electrical stimulation (PES) was performed to induce AF. EPAC1 expression was determined by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) and Western blot. Cellular electrophysiology was examined by whole cell patch clamp. Both mRNA and protein levels of EPAC1 were upregulated in HF mice. ISO increased the AF susceptibility, and the negative effect was deteriorated by CE3F4. ISO mediated high AF susceptibility of HF via prolonging action potential and exciting L-type calcium channel (LTCC). These could also be reversed by CE3F4 treatment. EPAC1 increased the AF susceptibility in ISO-induced HF mouse model via alternating LTCC.

The cAMP effectors, Rap2b and EPAC, are involved in the regulation of the development of the Coxiella burnetii containing vacuole by altering the fusogenic capacity of the vacuole.

Cyclic Adenosine 3′,5′-monophosphate (cAMP) is a key second messenger known to directly regulate not only the protein kinase A (PKA) activity but also other important molecules such as the exchange protein activated by cAMP (EPAC), which is as a guanine nucleotide exchange factor (GEF) of the low molecular weight GTPase, Rap2. Coxiella burnetii is a Gram negative bacterium that survives and grows in a large Coxiella replicative vacuole (CRV), which displays lysosomal and autophagic features. In this report, we present evidence that both, EPAC and its downstream effector Rap2b, were recruited to the CRV. The transient over-expression of the Rap2b wt protein, but not its inactive mutant Rap2b ΔAAX, markedly inhibited the development of the large CRV. Additionally, Rap2b wtinhibited the fusion of early Coxiella phagosomes with the fully developed CRV, indicating that homotypic fusion events are altered in the presence of high levels of Rap2b wt. Likewise, the fusion of endosome/lysosomal compartments (heterotypic fusions) with the large CRV was also affected by the over-expression of this GTPase. Interestingly, cell overexpression of Rap2b wt markedly decreased the levels of the v-SNARE, Vamp7, suggesting that this down-regulation impairs the homotypic and heterotypic fusions events of the Coxiella vacuole.