Epac Activation Research Articles

Epac activation sensitizes rat sensory neurons through activation of Ras.

Guanine nucleotide exchange factors directly activated by cAMP (Epacs) have emerged as important signaling molecules mediating persistent hypersensitivity in animal models of inflammation, by augmenting the excitability of sensory neurons. Although Epacs activate numerous downstream signaling cascades, the intracellular signaling which mediates Epac-induced sensitization of capsaicin-sensitive sensory neurons remains unknown. Here, we demonstrate that selective activation of Epacs with 8-CPT-2'-O-Me-cAMP-AM (8CPT-AM) increases the number of action potentials (APs) generated by a ramp of depolarizing current and augments the evoked release of calcitonin gene-related peptide (CGRP) from isolated rat sensory neurons. Internal perfusion of capsaicin-sensitive sensory neurons with GDP-βS, substituted for GTP, blocks the ability of 8CPT-AM to increase AP firing, demonstrating that Epac-induced sensitization is G-protein dependent. Treatment with 8CPT-AM activates the small G-proteins Rap1 and Ras in cultures of sensory neurons. Inhibition of Rap1, by internal perfusion of a Rap1-neutralizing antibody or through a reduction in the expression of the protein using shRNA does not alter the Epac-induced enhancement of AP generation or CGRP release, despite the fact that in most other cell types, Epacs act as Rap-GEFs. In contrast, inhibition of Ras through expression of a dominant negative Ras (DN-Ras) or through internal perfusion of a Ras-neutralizing antibody blocks the increase in AP firing and attenuates the increase in the evoked release of CGRP induced by Epac activation. Thus, in this subpopulation of nociceptive sensory neurons, it is the novel interplay between Epacs and Ras, rather than the canonical Epacs and Rap1 pathway, that is critical for mediating Epac-induced sensitization.

Abstract 11879: Control of Fibroblast Function by Adrenergic/EPAC System and Potential Role in Atrial Remodeling

Introduction: Heart failure (HF) produces left atrial (LA)-selective fibrosis and atrial fibrillation. HF also causes adrenergic activation, which contributes to remodeling via a variety of pathways mediated by altered signaling molecules, including the exchange protein activated by cAMP (Epac). Hypothesis: To evaluate the effects of adrenergic receptor (AR) and Epac1 signaling on LA fibroblast (FB) function and its potential role in HF-induced atrial remodeling. Methods: HF was induced in dogs by ventricular tachypacing (VTP, 240 bpm) for various periods. mRNA and protein expression were measured in freshly isolated LA FBs by qPCR and immunoblot, respectively. Results: Epac1 expression decreased in LA FBs within 12 hrs (-3.9 fold) of VTP onset (Fig. A). The selective Epac activator, 8-pCPT (33 μM) reduced, whereas the Epac blocker ESI-09 (1 μM) enhanced, LA FB collagen expression (Fig. B). Norepinephrine (NE, 1 μM) decreased Epac1 expression, an effect blocked by prazosin, and increased FB collagen protein production (Fig. C). Isoproterenol (ISO) increased Epac1 expression (Fig. D), an effect antagonized by ICI (β2-AR antagonist, 2 μM), but not CGP (β1-AR antagonist, 2 μM). β2-AR activation with ISO decreased collagen expression (Fig. E), an effect mimicked by salbutamol (10 μM) and blocked by ICI but not CGP. Transforming growth factor (TGF) β1 (10 ng/ml), known to be activated in HF, suppressed Epac1 expression, an effect blocked by the Smad3 inhibitor SIS3 (Fig. F). Conclusions: AR activation has complex effects on FBs, with NE/α-AR-activation suppressing Epac1 and increasing collagen expression, and β2-ARs having opposite effects. HF reduces LA FB Epac1 expression, likely via increased NE release from adrenergic nerve endings, along with TGF β1 activation. Epac1 signaling reduces FB collagen expression, so Epac1 downregulation in HF contributes to the atrial profibrotic milieu and may be a novel target for prevention of profibrillatory atrial remodeling.

PKA Phosphorylation of NCLX Reverses Mitochondrial Calcium Overload and Depolarization, Promoting Survival of PINK1-Deficient Dopaminergic Neurons

SUMMARYMitochondrial Ca2+ overload is a critical, preceding event in neuronal damage encountered during neurodegenerative and ischemic insults. We found that loss of PTEN-induced putative kinase 1 (PINK1) function, implicated in Parkinson disease, inhibits the mitochondrial Na+/Ca2+ exchanger (NCLX), leading to impaired mitochondrial Ca2+ extrusion. NCLX activity was, however, fully rescued by activation of the protein kinase A (PKA) pathway. We further show that PKA rescues NCLX activity by phosphorylating serine 258, a putative regulatory NCLX site. Remarkably, a constitutively active phosphomimetic mutant of NCLX (NCLXS258D) prevents mitochondrial Ca2+ overload and mitochondrial depolarization in PINK1 knockout neurons, thereby enhancing neuronal survival. Our results identify an mitochondrial Ca2+ transport regulatory pathway that protects against mitochondrial Ca2+ overload. Because mitochondrial Ca2+ dyshomeostasis is a prominent feature of multiple disorders, the link between NCLX and PKA may offer a therapeutic target.

Exchange protein directly activated by cyclic AMP (EPAC) activation reverses neutrophil dysfunction induced by β2-agonists, corticosteroids, and critical illness

Neutrophils play a role in the pathogenesis of asthma, chronic obstructive pulmonary disease, and pulmonary infection. Impaired neutrophil phagocytosis predicts hospital-acquired infection. Despite this, remarkably few neutrophil-specific treatments exist. We sought to identify novel pathways for the restoration of effective neutrophil phagocytosis and to activate such pathways effectively in neutrophils from patients with impaired neutrophil phagocytosis. Blood neutrophils were isolated from healthy volunteers and patients with impaired neutrophil function. In healthy neutrophils phagocytic impairment was induced experimentally by using β2-agonists. Inhibitors and activators of cyclic AMP (cAMP)-dependent pathways were used to assess the influence on neutrophil phagocytosis invitro. β2-Agonists and corticosteroids inhibited neutrophil phagocytosis. Impairment of neutrophil phagocytosis by β2-agonists was associated with significantly reduced RhoA activity. Inhibition of protein kinase A(PKA) restored phagocytosis and RhoA activity, suggesting that cAMP signals through PKA to drive phagocytic impairment. However, cAMP can signal through effectors other than PKA, such as exchange protein directly activated by cyclic AMP (EPAC). An EPAC-activating analog of cAMP (8CPT-2Me-cAMP) reversed neutrophil dysfunction induced by β2-agonists or corticosteroids but did not increase RhoA activity. 8CPT-2Me-cAMP reversed phagocytic impairment induced by Rho kinase inhibition but was ineffective in the presence of Rap-1 GTPase inhibitors. 8CPT-2Me-cAMP restored function to neutrophils from patients with known acquired impairment of neutrophil phagocytosis. EPAC activation consistently reverses clinical and experimental impairment of neutrophil phagocytosis. EPAC signals through Rap-1 and bypasses RhoA. EPAC activation represents a novel potential means by which to reverse impaired neutrophil phagocytosis.

ID: 120: Involvement of cyclic AMP signaling pathway in T cell immunoglobulin and mucin domain(TIM)-3 expression by Jurkat T cells

T cell immunoglobulin and mucin domain-3 (TIM-3) plays inhibitory roles in CD4+ T cells leading to low production of interferon- γ . TIM-3 expression increases in T cells of subjects harboring a tumor but the underlying mechanisms remain elusive. Elevated PGE2 levels in tumor-bearing individuals prompted us to hypothesize that PGE2 signaling pathway may affect TIM-3 expression in T cells. We found that PGE2 enhanced TIM-3 protein levels in Jurkat T cells. Also, forskolin, PKA activator and Epac activator which activated PGE2 signaling pathway stimulated TIM-3 expression. Further, forskolin induced TIM-3 mRNA levels in Jurkat T cells, although we did not find the enhanced luciferase activity in response to forskolin using luciferase reporter vectors containing TIM-3 promoter. Our results suggest that cAMP signaling may up-regulate TIM-3 expression in T cells.

CCMP causes caspase-dependent apoptosis in mouse lymphoma cell lines

cCMP is a cyclic pyrimidine nucleotide which binds to and activates cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG). In S49 lymphoma cells, cAMP induces apoptosis via PKA. In our present study, we examined the effect of cCMP on apoptosis in S49 mouse lymphoma cells and in PKA-deficient S49kin−cells. These two cell lines also lack PKG, hyperpolarization-activated cyclic nucleotide-gated channels 2 and 4 (HCN2 and HCN4) as assessed by real-time PCR. The cell-permeable analog cCMP–AM induced PKA- and PKG-independent apoptosis in S49 cells. In contrast, exchange protein activated by cAMP (Epac) activation did not induce apoptosis. cCMP induced caspase-dependent apoptosis via the intrinsic pathway, led to cytochrome c release from mitochondria and also activated the ER stress pathway. On the contrary, the extrinsic apoptotic pathway was not involved. Autophagy was not detectable after treatment with cCMP–AM in both cell lines. cAMP–AM, cGMP–AM, cUMP–AM as well as the cyclic nucleotides lacking the acetoxymethylester (AM)-group had no effect. cCMP–AM altered gene expression of the apoptotic-relevant gene Gadd45α and the immediate early response genes cFos and Nr4A1 in S49 wild-type (wt) cells. In conclusion, cCMP induces apoptosis of S49 lymphoma cells, independently of hitherto known cCMP target proteins.

RACK1 and β-arrestin2 attenuate dimerization of PDE4 cAMP phosphodiesterase PDE4D5

PDE4 family cAMP-selective cyclic nucleotide phosphodiesterases are important in the regulation of cAMP abundance in numerous systems, and thereby play an important role in the regulation of PKA and EPAC activity and the phosphorylation of CREB. We have used the yeast 2-hybrid system to demonstrate recently that long PDE4 isoforms form homodimers, consistent with data obtained recently by structural studies. The long PDE4 isoform PDE4D5 interacts selectively with β-arrestin2, implicated in the regulation of G-protein-coupled receptors and other cell signaling components, and also with the β-propeller protein RACK1. In the present study, we use 2-hybrid approaches to demonstrate that RACK1 and β-arrestin2 inhibit the dimerization of PDE4D5. We also show that serine-to-alanine mutations at PKA and ERK1/2 phosphorylation sites on PDE4D5 detectably ablate dimerization. Conversely, phospho-mimic serine-to-aspartate mutations at the MK2 and oxidative stress kinase sites ablate dimerization. Analysis of PDE4D5 that is locked into the dimeric configuration by the formation of a trans disulfide bond between Ser261 and Ser602 shows that RACK1 interacts strongly with both the monomeric and dimeric forms, but that β-arrestin2 interacts exclusively with the monomeric form. This is consistent with the concept that β-arrestin2 can preferentially recruit the monomeric, or “open,” form of PDE4D5 to β2-adrenergic receptors, where it can regulate cAMP signaling.

Cilostazol Induces PGI2 Production via Activation of the Downstream Epac-1/Rap1 Signaling Cascade to Increase Intracellular Calcium by PLCε and to Activate p44/42 MAPK in Human Aortic Endothelial Cells.

BackgroundCilostazol, a selective phosphodiesterase 3 (PDE3) inhibitor, is known as an anti-platelet drug and acts directly on platelets. Cilostazol has been shown to exhibit vascular protection in ischemic diseases. Although vascular endothelium-derived prostaglandin I2 (PGI2) plays an important role in vascular protection, it is unknown whether cilostazol directly stimulates PGI2 synthesis in endothelial cells. Here, we elucidate the mechanism of cilostazol-induced PGI2 stimulation in endothelial cells.Methods and ResultsHuman aortic endothelial cells (HAECs) were stimulated with cilostazol and PGI2 accumulation in the culture media was measured. Cilostazol increased PGI2 synthesis via the arachidonic acid pathway. Cilostazol-induced intracellular calcium also promoted PGI2 synthesis via the inositol 1,4,5-trisphosphate receptor. Using RNAi, silencing of PDE3B abolished the induction effect of cilostazol on PGI2 synthesis and intracellular cAMP accumulation. Inhibition of the exchange protein, which was directly activated by cyclic AMP 1 (Epac-1) and its downstream signal the Ras-like small GTPase (Rap-1), abolished cilostazol-induced PGI2 synthesis, but this did not take place via protein kinase A (PKA). Inhibition of downstream signaling, such as mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K) γ, and phospholipase C (PLC) ε, suppressed cilostazol-induced PGI2 synthesis.ConclusionsThe PDE3/Epac-1/Rap-1 signaling pathway plays an important role in cilostazol-induced PGI2 synthesis. Namely, stimulation of HAECs with cilostazol induces intracellular calcium elevation via the Rap-1/PLCε/IP3 pathway, along with MAPK activation via direct activation by Epac-1/Rap-1 and indirect activation by Epac-1/Rap-1/PI3Kγ, resulting in synergistically induced PGI2 synthesis.

Actions of cAMP on calcium sensitization in human detrusor smooth muscle contraction.

To clarify the effect of cAMP on the Ca(2+) -sensitized smooth muscle contraction in human detrusor, as well as the role of novel exchange protein directly activated by cAMP (Epac) in cAMP-mediated relaxation. All experimental protocols to record isometric tension force were performed using α-toxin-permeabilized human detrusor smooth muscle strips. The mechanisms of cAMP-mediated suppression of Ca(2+) sensitization activated by 10 μm carbachol (CCh) and 100 μm GTP were studied using a selective rho kinase (ROK) inhibitor, Y-27632, and a selective protein kinase C (PKC) inhibitor, GF-109203X. The relaxation mechanisms were further probed using a selective protein kinase A (PKA) activator, 6-Bnz-cAMP and a selective Epac activator, 8-pCPT-2'-O-Me-cAMP. We observed that CCh-induced Ca(2+) sensitization was inhibited by cAMP in a concentration-dependent manner. GF-109203X (10 μm) but not Y-27632 (10 μm) significantly enhanced the relaxation effect induced by cAMP (100 μm). 6-Bnz-cAMP (100 μm) predominantly decreased the tension force in comparison with 8-pCPT-2'-O-Me-cAMP (100 μm). We showed that cAMP predominantly inhibited the ROK pathway but not the PKC pathway. The PKA-dependent pathway is dominant, while Epac plays a minor role in human detrusor smooth muscle Ca(2+) sensitization.

CAMP-mediated secretion of brain-derived neurotrophic factor in developing airway smooth muscle

Moderate hyperoxic exposure in preterm infants contributes to subsequent airway dysfunction and to risk of developing recurrent wheeze and asthma. The regulatory mechanisms that can contribute to hyperoxia-induced airway dysfunction are still under investigation. Recent studies in mice show that hyperoxia increases brain-derived neurotrophic factor (BDNF), a growth factor that increases airway smooth muscle (ASM) proliferation and contractility. We assessed the mechanisms underlying effects of moderate hyperoxia (50% O2) on BDNF expression and secretion in developing human ASM. Hyperoxia increased BDNF secretion, but did not alter endogenous BDNF mRNA or intracellular protein levels. Exposure to hyperoxia significantly increased [Ca2+]i responses to histamine, an effect blunted by the BDNF chelator TrkB-Fc. Hyperoxia also increased ASM cAMP levels, associated with reduced PDE4 activity, but did not alter protein kinase A (PKA) activity or adenylyl cyclase mRNA levels. However, 50% O2 increased expression of Epac2, which is activated by cAMP and can regulate protein secretion. Silencing RNA studies indicated that Epac2, but not Epac1, is important for hyperoxia-induced BDNF secretion, while PKA inhibition did not influence BDNF secretion. In turn, BDNF had autocrine effects of enhancing ASM cAMP levels, an effect inhibited by TrkB and BDNF siRNAs. Together, these novel studies suggest that hyperoxia can modulate BDNF secretion, via cAMP-mediated Epac2 activation in ASM, resulting in a positive feedback effect of BDNF-mediated elevation in cAMP levels. The potential functional role of this pathway is to sustain BDNF secretion following hyperoxic stimulus, leading to enhanced ASM contractility and proliferation.

The cAMP-producing agonist beraprost inhibits human vascular smooth muscle cell migration via exchange protein directly activated by cAMP.

AimsDuring restenosis, vascular smooth muscle cells (VSMCs) migrate from the vascular media to the developing neointima. Preventing VSMC migration is therefore a therapeutic target for restenosis. Drugs, such as prostacyclin analogues, that increase the intracellular concentration of cyclic adenosine monophosphate (cAMP) can inhibit VSMC migration, but the mechanisms via which this occurs are unknown. Two main downstream mediators of cAMP are protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac). This study has examined the effects of the prostacyclin analogue beraprost on VSMC migration and investigated the intracellular pathways involved.Methods and resultsIn a chemotaxis chamber, human saphenous vein VSMC migrated towards a platelet-derived growth-factor-BB (PDGF) chemogradient. Incubation with therapeutically relevant concentrations of cAMP-producing agonist beraprost significantly decreased PDGF-induced migration. Direct activation of either PKA or Epac inhibited migration whereas inhibition of PKA did not prevent the anti-migratory effect of beraprost. Direct activation of Epac also prevented hyperplasia in ex vivo serum-treated human veins. Using fluorescence resonance energy transfer, we demonstrated that beraprost activated Epac but not PKA. The mechanisms of this Epac-mediated effect involved activation of Rap1 with subsequent inhibition of RhoA. Cytoskeletal rearrangement at the leading edge of the cell was consequently inhibited. Interestingly, Epac1 was localized to the leading edge of migrating VSMC.ConclusionsThese results indicate that therapeutically relevant concentrations of beraprost can inhibit VSMC migration via a previously unknown mechanism involving the cAMP mediator Epac. This may provide a novel target that could blunt neointimal formation.

Salbutamol inhibits RhoA activation in normal but not in desensitized bronchial smooth muscle cells.

This study was aimed at investigating whether the β2 -adrenoceptor agonist, salbutamol, could modulate RhoA activation in normal and homologously desensitized bronchial smooth muscle cells (BSMC). Serum-starved BSMCs were stimulated with the Rho-activating compound calpeptin in the presence or absence of salbutamol, the Epac activator, 8-pCPT-2'-O-Me-cAMP, or the site-selective activator of cAMP-dependent protein kinase A (PKA), 6-Bnz-cAMP. Activated RhoA was assessed by immunocytochemical detection and by RhoA G-LISA assay. Stimulation with calpeptin caused translocation of RhoA from cytosol to plasma membrane, a condition required for the functional coupling of RhoA with its cellular targets. Pretreatment with salbutamol 10 μm for 15 min was found to block calpeptin-induced activation of RhoA in normal, but not in homologously desensitized cells. Pretreatment of calpeptin-stimulated BSMC with 8-pCPT-2'-O-Me-cAMP or 6-Bnz-cAMP could reproduce the effect of salbutamol. These findings demonstrated that salbutamol inhibits RhoA activation in human BSMC through β2 -adrenoceptor/Epac/PKA pathway. An important pharmacological implication of these finding is the possible contribution of RhoA pathway to the molecular mechanism involved in airway smooth muscle relaxation caused by acute/chronic exposure to β2-adrenoceptor agonists.

WS06.1 Stabilization of CFTR at the membrane by EPAC1 activation occurs through interaction with NHERF-1

Background CFTR-mediated ion transport is stimulated by increase in subcortical cAMP levels through protein kinase A (PKA) activation thus triggering CFTR phosphorylation and channel opening. However, besides PKA, exchange proteins (EPACs) are also directly activated by cAMP. EPACs are guanine nucleotide exchange factors (GEFs) for Rap family small GTPases and shift to the plasma membrane (PM) in response to cAMP. This suggests that activation of both CFTR and EPACs might be spatially and temporally coincident. Objectives We aimed at elucidating the impact of EPAC1 on CFTR trafficking and PM anchoring. Methods Protein biochemistry and imaging techniques. Results Our results show that in CFBE cells: i. EPAC1 co-localizes and is present in protein complexes involving wt- or F508del-CFTR and this interaction is mediated by NHERF1 but not ezrin. ii. EPAC activation leads to its increased association with CFTR and increased CFTR PM levels. iii. Increase in endogenous cAMP levels increases CFTR membrane levels. iv. EPAC1 knockdown leads to decreased of CFTR PM levels. Conclusion Our results establish that EPAC1 associates to CFTR in membrane complexes with NHERF-1 and enhances CFTR traffic to the PM, making it a novel potential therapeutic target for CF. These data indicate that cAMP signalling regulates CFTR through two distinct but complementary pathways – PKA and EPAC1. Grants from FCT (Portugal) through MCTES EXPL/BIM-MEC/1451/2013 grant and PEst-OE/BIA/UI4046/2011 BioFig centre grant and by 2012 ERS Romain Pauwels Research Award.

Abstract 445: Beraprost, a Prostacyclin Analogue, Inhibits Human Vascular Smooth Muscle Cell Migration via Activation of Exchange Protein Activated by cAMP (Epac)

Following vein grafting, endothelial damage initiates vascular smooth muscle cell (VSMC) migration to the intima leading to neointimal hyperplasia and restenosis. Inhibition of VSMC migration is therefore a therapeutic target to prevent restenosis. Previous studies have shown that cyclic adenosine monophosphate (cAMP) inhibits VSMC migration although the downstream mediators are unknown. This suggests that cyclic AMP-producing agonists, such as the prostacyclin analogue beraprost which binds to prostanoid receptors, could be used as a potential drug against neointimal formation. We hypothesised that prostacyclin analogues inhibit VSMC migration by activating specific cAMP downstream mediators. The aims of this project were to examine the role of cAMP mediators protein kinase A (PKA) and Exchange protein activated by cAMP (Epac) in this inhibitory effect. Saphenous veins were obtained from patients undergoing coronary artery bypass. VSMCs were primary cultured and migration measured using a chemotaxis chamber. Cell migration stimulated by PDGF was significantly inhibited by 68 ± 5% (n=6) in the presence of a therapeutically relevant concentration of beraprost (1 nM). To examine potential mechanisms of this inhibition, selective activators of PKA and Epac were used. Both Epac and PKA activators significantly inhibited migration towards PDGF. However, fluorescence resonance energy transfer using cells transfected with specific probes revealed that 1 nM beraprost only activates Epac and not PKA in VSMCs (n=4). The downstream mechanisms were further investigated. Pulldown assays demonstrated that beraprost-mediated stimulation of Epac produces subsequent activation of the monomeric G-protein Rap1 and ultimately inhibits RhoA-induced cytoskeleton rearrangement required for migration (n=3). Using siRNA to knockdown Rap1, the inhibition of PDGF-induced migration by beraprost in VSMC cells was shown to be dependent on the Rap1 pathway (n=3). In conclusion, the prostacyclin analogue beraprost inhibits VSMC migration via activation of Epac/Rap1. This may provide a new therapeutic target to prevent neointimal hyperplasia.

Phosphorylation of ezrin on Thr567 is required for the synergistic activation of cell spreading by EPAC1 and protein kinase A in HEK293T cells

Recent studies have demonstrated that the actin binding protein, ezrin, and the cAMP-sensor, EPAC1, cooperate to induce cell spreading in response to elevations in intracellular cAMP. To investigate the mechanisms underlying these effects we generated a model of EPAC1-dependent cell spreading based on the stable transfection of EPAC1 into HEK293T (HEK293T–EPAC1) cells. We found that direct activation of EPAC1 with the EPAC-selective analogue, 8-pCPT-2′-O-Me-cAMP (007), promoted cell spreading in these cells. In addition, co-activation of EPAC1 and PKA, with a combination of the adenylate cyclase activator, forskolin, and the cAMP phosphodiesterase inhibitor, rolipram, was found to synergistically enhance cell spreading, in association with cortical actin bundling and mobilisation of ezrin to the plasma membrane. PKA activation was also associated with phosphorylation of ezrin on Thr567, as detected by an electrophoretic band mobility shift during SDS-PAGE. Inhibition of PKA activity blocked ezrin phosphorylation and reduced the cell spreading response to cAMP elevation to levels induced by EPAC1-activation alone. Transfection of HEK293T–EPAC1 cells with inhibitory ezrin mutants lacking the key PKA phosphorylation site, ezrin-Thr567Ala, or the ability to associate with actin, ezrin-Arg579Ala, promoted cell arborisation and blocked the ability of EPAC1 and PKA to further promote cell spreading. The PKA phospho-mimetic mutants of ezrin, ezrin-Thr567Asp had no effect on EPAC1-driven cell spreading. Our results indicate that association of ezrin with the actin cytoskeleton and phosphorylation on Thr567 are required, but not sufficient, for PKA and EPAC1 to synergistically promote cell spreading following elevations in intracellular cAMP.

Regulatory T cell-derived adenosine induces dendritic cell migration through the Epac-Rap1 pathway.

Dendritic cells (DC) are one target for immune suppression by regulatory T cells (Treg), because their interaction results in reduced T cell stimulatory capacity and secretion of inhibitory cytokines in DC. We show that DC in the presence of Treg are more mobile as compared with cocultures with conventional CD4(+) T cells and form DC-Treg aggregates within 2 h of culture. The migration of DC was specifically directed toward Treg, as Treg, but not CD4(+) T cells, attracted DC in Boyden chambers. Treg deficient for the ectonucleotidase CD39 were unable to attract DC. Likewise, addition of antagonists for A2A adenosine receptors abolished the formation of DC-Treg clusters, indicating a role for adenosine in guiding DC-Treg interactions. Analysis of the signal transduction events in DC after contact to Treg revealed increased levels of cAMP, followed by activation of Epac1 and the GTPase Rap1. Subsequently activated Rap1 localized to the subcortical actin cytoskeleton in DC, providing a means by which directed locomotion of DC toward Treg is facilitated. In aggregate, these data show that Treg degrade ATP to adenosine via CD39, attracting DC by activating Epac1-Rap1-dependent pathways. As a consequence, DC-Treg clusters are formed and DC are rendered less stimulatory. This adenosine-mediated attraction of DC may therefore act as one mechanism by which Treg regulate the induction of immune responses by DC.

Regulation of connexin 43 and microRNA expression via β2-adrenoceptor signaling in 1321N1 astrocytoma cells.

Connexin 43 (Cx43) is the main gap junction protein in astrocytes and exerts the same effects on growth inhibition in astrocytoma and glioma as microRNA-146a (miR-146a) in glioma. β2-adrenergic receptor (AR) signaling modulates Cx43 expression in myocytes via components downstream of protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac). However, it remains to be elucidated how expression of Cx43 is modulated in astrocytes. In the present study, 1321N1 astrocytoma cells were treated with β2-AR signaling agents in order to evaluate the expression of Cx43 and miRNAs. RNA and protein were extracted from the cells for use in reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. The results revealed that clenbuterol increased miR-146a level and upregulated Cx43 expression via cAMP/PKA at the mRNA and protein level. Pre-inhibition of adenyl cyclase decreased expression of Cx43 and miR-146a. PKA activation and overexpression of miR-146a in A-1321N1 cells increased the expression of Cx43. β2-AR stimulation and 6Bnz, a PKA activator, suppressed oncomiRs miR-155 and miR-27a, while 8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate, an Epac activator, increased their levels. The current findings demonstrated that β2-AR signaling has growth inhibitory effects via modulation of the cAMP/PKA pathway in A-1321N1 cells through increasing the expression level of Cx43 and miR-146a as well as decreasing miR-155 and miR-27a levels. Thus, stimulation of the β2-AR and PKA signaling pathway may be a useful approach for astrocytoma therapy.

Alcohol‐induced decreases in brain endothelial barrier integrity rescued by sphingosine‐1‐phosphate or 8‐pCPT‐2'‐O‐Me‐cAMP

Alcohol has previously been shown to increase blood-brain-barrier (BBB) permeability. We hypothesized that alcohol increases BBB permeability by disrupting the junctional proteins, VE-cadherin and claudin-5, and that barrier enhancers such as sphingosine-1-phosphate (S1P) and 8-pCPT-2'-O-Me-cAMP (CPT) can restore barrier function. We also hypothesized that p38 MAP kinase mediates alcohol-induced BBB dysfunction. Primary human brain microvascular endothelial cells (HBMEC) were used to model the BBB in vitro. Transendothelial electrical resistance (TER) served as an index of barrier function. Protein localization was determined by immunofluorescence microscopy. Alcohol (50-100 mM) rapidly and significantly decreased TER. S1P (1 μM) or CPT (100 μM) significantly enhanced barrier function. Application of S1P or CPT after alcohol (75 mM) significantly decreased the time for TER to recover to baseline levels, compared to alcohol alone. Pretreating HMBEC with the p38 MAP kinase inhibitor SB208530 (20 μM) did not affect the alcohol-induced decrease in TER. Immunofluorescence data showed localization of VE-cadherin and claudin-5 at intercellular junctions, which was disrupted by alcohol treatment. Treatment with S1P after alcohol restored junctional integrity of VE-cadherin and claudin-5. Our results demonstrate that alcohol weakens brain endothelial barrier integrity, and that S1P and CPT can enhance barrier function. In addition, our data shows that S1P can restore the integrity of intercellular junctions. These data suggest S1P or activation of Epac1 may be useful to ameliorate BBB dysfunction. Supported by NIH HL098215, HL070752, and GM097270.

5HT7 Receptor Induced Phrenic Motor Facilitation Requires EPAC and mTORC1, But Not PKA Activity

Serotonin type‐7 (5HT7) receptors in the cervical spinal cord elicit complex effects on respiratory motor activity. For example, intermittent 5HT7 receptor activation gives rise to long‐lasting phrenic motor facilitation (pMF); on the other hand, 5HT7 receptor activation constrains competing serotonin type‐2 induced pMF via protein kinase A (PKA)‐dependent “cross‐talk inhibition.” We hypothesized that divergent signaling pathways give rise to these distinct 5HT7 receptor actions. Specifically, whereas PKA regulates cross‐talk inhibition of 5HT2‐dependent pMF, a different cyclic AMP signaling pathway that requires exchange protein activated by cyclic AMP (EPAC) underlies 5HT7‐dependent pMF. Anesthetized, paralyzed and ventilated rats receiving intermittent 5HT7 receptor agonist injections (3x5uL; AS‐19) into the C4 intrathecal space expressed pMF for at least 90 minutes. This 5HT7‐induced pMF was abolished by pretreatment with a selective EPAC inhibitor (ESI‐05), but not a selective PKA inhibitor (KT‐5720). Further, an intrathecal EPAC activator (8‐pCPT‐2′‐O‐Me‐cAMP) is sufficient to elicit pMF. mTORC1 inhibition (rapamycin) abolished both 5HT7‐ and EPAC‐induced pMF. Thus, spinal 5HT7 receptors elicit pMF by a non‐canonical EPAC‐AKT‐mTORC1 pathway, and constrain alternate pathways to spinal respiratory motor plasticity via PKA signaling.

Novel Epac fluorescent ligand reveals distinct Epac1 vs. Epac2 distribution and function in cardiomyocytes

Exchange proteins directly activated by cAMP (Epac1 and Epac2) have been recently recognized as key players in β-adrenergic-dependent cardiac arrhythmias. Whereas Epac1 overexpression can lead to cardiac hypertrophy and Epac2 activation can be arrhythmogenic, it is unknown whether distinct subcellular distribution of Epac1 vs. Epac2 contributes to differential functional effects. Here, we characterized and used a novel fluorescent cAMP derivate Epac ligand 8-[Pharos-575]-2'-O-methyladenosine-3',5'-cyclic monophosphate (Φ-O-Me-cAMP) in mice lacking either one or both isoforms (Epac1-KO, Epac2-KO, or double knockout, DKO) to assess isoform localization and function. Fluorescence of Φ-O-Me-cAMP was enhanced by binding to Epac. Unlike several Epac-specific antibodies tested, Φ-O-Me-cAMP exhibited dramatically reduced signals in DKO myocytes. In WT, the apparent binding affinity (Kd = 10.2 ± 0.8 µM) is comparable to that of cAMP and nonfluorescent Epac-selective agonist 8-(4-chlorophenylthio)-2-O-methyladenosine-3'-,5'-cyclicmonophosphate (OMe-CPT). Φ-O-Me-cAMP readily entered intact myocytes, but did not activate PKA and its binding was competitively inhibited by OMe-CPT, confirming its Epac specificity. Φ-O-Me-cAMP is a weak partial agonist for purified Epac, but functioned as an antagonist for four Epac signaling pathways in myocytes. Epac2 and Epac1 were differentially concentrated along T tubules and around the nucleus, respectively. Epac1-KO abolished OMe-CPT-induced nuclear CaMKII activation and export of transcriptional regulator histone deacetylase 5. In conclusion, Epac1 is localized and functionally involved in nuclear signaling, whereas Epac2 is located at the T tubules and regulates arrhythmogenic sarcoplasmic reticulum Ca leak.