Exchange Proteins Directly Activated By cAMP Research Articles

Abstract 3075: Different roles of EPAC1 and EPAC2 in ovarian cancer: Implications for precision targeting in cancer therapy

Abstract There are two distinct functional isoforms of Exchange proteins directly activated by cAMP (EPACs), EPAC1 and EPAC2, which exhibit fundamental distinctions in structure, expression patterns, signaling specificity, and subcellular localization. This diversity suggests that EPAC1 and EPAC2 can operate independently, synergistically, or antagonistically, contributing to the wide range of reported functions and impacts in different cancers and studies. We compared the function of EPAC1 and EPAC2 in ovarian cancer using in vitro and in vivo models and further explored the underlying mechanism in the regulation of metabolism reprogramming. Selective inhibition of EPAC1 and EPAC2 markedly reduced proliferation in ovarian cancer cell lines, leading to cell death and cycle arrest. RNA-seq analysis indicated different downstream signaling pathways triggered by EPAC1 and EPAC2, with EPAC2 specifically linked to functions in energy metabolism. Treatment with an antagonist or knockdown of EPAC2 resulted in attenuation of both glycolysis and oxidative phosphorylation (OXPHOS), while EPAC1 did not induce such effects. Furthermore, inhibition of EPAC2 induced mitochondrial dysfunction, manifested by reduced ATP production, decreased mitochondrial membrane potential, and increased burden of reactive oxygen species (ROS). Our data also revealed that EPAC2 governs the MAPK pathway and mediates the mitochondrial unfolded protein response (UPRmt) in ovarian cancer cells. By delineating the distinctions between EPAC1 and EPAC2 within ovarian cancer cells, this project revised the prevailing understanding of EPACs and suggests the potential for precise targeting of EPAC isoforms as a viable strategy in cancer therapy. Citation Format: Zhengyin Gao, Wanjun Yuan, Leo T.O. Lee. Different roles of EPAC1 and EPAC2 in ovarian cancer: Implications for precision targeting in cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3075.

Activation of Exchange Proteins directly Activated by cAMP (EPAC) lengthens action potential duration by inhibiting potassium currents in atrial cardiomyocytes

IntroductionAtrial fibrillation (AF) is the most frequent sustained cardiac arrhythmia worldwide. Several mechanisms have been described in AF pathogenesis and sustainability such as dysregulation of calcium cycling and action potential (AP) duration. Effectors involved in these dysregulations are potential relevant therapeutic targets. Among them, the Exchange Proteins directly Activated by cAMP (EPAC) has been shown to increase arrhythmogenic activity in ventricular cardiomyocytes by lengthening of AP duration. However, the electrophysiological impact of EPAC at the atrial level remains to be established. ObjectiveTo evaluate the propensity of EPAC activation to induce atrial arrhythmogenic activity and by which mechanisms. MethodMyocytes from human right atrial appendages and wild type (WT) and EPAC1-/- mice atria were dissociated by enzymatic digestion. AP and potassium currents were recorded using the whole cell configuration of the patch clamp technique. EPAC proteins were pharmacologically activated by acute superfusion of 8-CPTAM (10μM), and the selective role of EPAC1 was evaluated by its selective inhibition by superfusion of AM-001 (20μM). ResultsIn mouse atrial myocytes, activation of EPAC by 8-CPTAM significantly increases AP duration at 90% of repolarization (APD90). Interestingly, this effect is higher in WT myocytes than in EPAC1-/-. Moreover, AM-001, which fails to correct AP lengthening in EPAC1-/- myocytes, reverses partially but significantly the effect of 8-CPTAM in WT myocytes. In human atrial myocytes, 8-CPTAM treatment lengthens APD90 by 32.3%. Additionally, in both WT mouse and human atrial myocytes, the treatment by 8-CPTAM induces an inhibition of both peak and sustained components of the K+ currents. Finally, effect of 8-CPTAM on K+ currents seems to be weaker in EPAC1-/- myocytes than in WT. ConclusionOur results show that EPAC activation lengthens AP duration in mouse and human atrial myocytes. Furthermore, inhibition of K+ currents participates in this AP duration modulation. Thus, these data suggest that EPAC proteins might be involved in atrial arrhythmogenic ectopic activity. Nevertheless, the role of each isoform of EPAC in these modulations and EPAC-induced regulation of other ionic currents remain to be specified.

Usefulness of circulating EPAC1 as biomarkers of therapeutic response to GLP-1 receptor agonists.

The response to Glucagon-like peptide-1 receptor agonists (GLP-1RAs) is highly varia-ble among patients. Thus, the identification of predictive biomarkers of therapeutic response to GLP-1 RA could help us to optimize the use of this class of drugs. GLP-1RAs increase exchange proteins directly activated by cAMP (EPAC). The aim of the present study was to assess whether the increase of EPAC1 after GLP-1RAs treatment could be a biomarker of clinical response. After showing that GLP-1 (10ng/mL) significantly increased the expression of EPAC1 in human endo-thelial vascular cells (HUVEC), a pilot clinical study was planned. For this purpose 49 patients with type 2 diabetes who started treatment with liraglutide were included. EPAC1 concentration was determined by ELISA before and at one month of liraglutide treatment. We found that serum concentration of EPAC1 increased significantly after treatment with liraglutide. Only in those patients in whom EPAC1 increased (64%), a significant decrease in HbA1c, LDL-C, body mass index (BMI), and waist circumference was shown. This pilot study suggests that the increase of circulating EPAC1 after GLP-1RAs treatment could be a useful biomarker to predict clinical GLP1-RAs response.

652 EPAC-mTORC1 signaling regulates proliferation of primary melanoma cells and loss of dependence on EPAC signaling correlates with melanoma progression

Exchange Proteins directly Activated by cAMP (EPACs) belong to a family of RAP guanine nucleotide exchange factors (RAPGEF). EPAC1/2 (RAPGEF3/4) activate RAP1 and the alternative cAMP signaling pathway. We previously showed that the differential growth response of primary and metastatic melanoma cells to cAMP is mediated by EPAC. However, the mechanisms responsible for this differential response to EPAC signaling are not understood. In this study, we show that pharmacological inhibition or siRNA-mediated knockdown of EPAC selectively inhibits the growth and survival of primary melanoma cells by downregulation of cell cycle proteins and inhibiting the cell cycle progression independent of ERK1/2 phosphorylation.

How do maternal emotion and sleep conditions affect infant sleep: a prospective cohort study

BackgroundRecent studies suggest that the incidence of infant sleep disorder is related to maternal emotional and sleep conditions, but how they influence each other is not fully understood.MethodsA total of 513 pairs of parents and infants were enrolled in this prospective cohort study. Maternal emotional and sleep conditions were assessed using a self-rating depression scale, self-rating anxiety scale, and Pittsburgh Sleep Quality Index at the third trimester and within 3 months after delivery. Infant sleep was assessed by the Brief Screening Questionnaire for Infant Sleep Problems within 3 months after birth. Expression of the glucocorticoid receptor (GR), melatonin receptors (MR), exchange proteins directly activated by cAMP (EPAC) receptors, and dopamine receptor (DR) in the placenta was detected by immunohistochemistry. Methylation of the promoter regions for the GR (NR3C1 and NR3C2), MR (MTNR1A and MTNR1B), EPAC (RASGRF1 and RASGRF2), and DR (DRD1 and DRD2) genes was assessed by next generation sequencing-based bisulfite sequencing PCR.ResultsThe incidence of sleep disorders in infants 0–3 months of age in this cohort was 40.5%. Risk factors for infant sleep disorder were low education level of the father, depression of father, maternal postpartum depression, postpartum anxiety, postpartum sleep disorder, and maternal sleep disorder extend from the third trimester to postpartum. There was no difference in expression of placental DR, GR, MR, and EPAC between mothers whose infants were with and without sleep disorders. Methylation of MTNR1B was higher and expression of MR was lower in the placenta of mothers with sleep disorder in the third trimester than in mothers without sleep disorder. Level of NR3C2 methylation was lower and GR expression was higher in the placenta of mothers with sleep disorder extend from the third trimester to postpartum than in mothers without sleep disorder.ConclusionMaternal sleep disorders in the third trimester could lead to decreased MR expression by up-regulating MTNR1B methylation, and then resulting in elevated cortisol and increased GR expression by down-regulating NR3C2 methylation, which could increase the incidence of maternal postpartum sleep disorders, finally, the maternal postpartum sleep disorder could result in the high incidence of infant sleep disorder.

The Role of Neuropeptide-Stimulated cAMP-EPACs Signalling in Cancer Cells.

Neuropeptides are autocrine and paracrine signalling factors and mainly bind to G protein-coupled receptors (GPCRs) to trigger intracellular secondary messenger release including adenosine 3′, 5′-cyclic monophosphate (cAMP), thus modulating cancer progress in different kind of tumours. As one of the downstream effectors of cAMP, exchange proteins directly activated by cAMP (EPACs) play dual roles in cancer proliferation and metastasis. More evidence about the relationship between neuropeptides and EPAC pathways have been proposed for their potential role in cancer development; hence, this review focuses on the role of neuropeptide/GPCR system modulation of cAMP/EPACs pathways in cancers. The correlated downstream pathways between neuropeptides and EPACs in cancer cell proliferation, migration, and metastasis is discussed to glimmer the direction of future research.

EPAC2: A new and promising protein for glioma pathogenesis and therapy.

Gliomas are prime brain cancers which are initiated by malignant modification of neural stem cells, progenitor cells and differentiated glial cells such as astrocyte, oligodendrocyte as well as ependymal cells. Exchange proteins directly activated by cAMP (EPACs) are crucial cyclic adenosine 3’,5’-monophosphate (cAMP)-determined signaling pathways. Cyclic AMP-intermediated signaling events were utilized to transduce protein kinase A (PKA) leading to the detection of EPACs or cAMP-guanine exchange factors (cAMP-GEFs). EPACs have been detected as crucial proteins associated with the pathogenesis of neurological disorders as well as numerous human diseases. EPAC proteins have two isoforms. These isoforms are EPAC1 and EPAC2. EPAC2 also known as Rap guanine nucleotide exchange factor 4 (RAPGEF4) is generally expression in all neurites. Higher EAPC2 levels was detected in the cortex, hippocampus as well as striatum of adult mouse brain. Activation as well as over-secretion of EPAC2 triggers apoptosis in neurons and EPAC-triggered apoptosis was intermediated via the modulation of Bcl-2 interacting member protein (BIM). EPAC2 secretory levels has proven to be more in low-grade clinical glioma than high-grade clinical glioma. This review therefore explores the effects of EPAC2/RAPGEF4 on the pathogenesis of glioma instead of EPAC1 because EPAC2 and not EPAC1 is predominately expressed in the brain. Therefore, EPAC2 is most likely to modulate glioma pathogenesis rather than EPAC1.

Synthesis and Biochemical Evaluation of Noncyclic Nucleotide Exchange Proteins Directly Activated by cAMP 1 (EPAC1) Regulators.

Exchange proteins directly activated by cAMP (EPAC) play a central role in various biological functions, and activation of the EPAC1 protein has shown potential benefits for the treatment of various human diseases. Herein, we report the synthesis and biochemical evaluation of a series of noncyclic nucleotide EPAC1 activators. Several potent EPAC1 binders were identified including 25g, 25q, 25n, 25u, 25e, and 25f, which promote EPAC1 guanine nucleotide exchange factor activity in vitro. These agonists can also activate EPAC1 protein in cells, where they exhibit excellent selectivity toward EPAC over protein kinase A and G protein-coupled receptors. Moreover, 25e, 25f, 25n, and 25u exhibited improved selectivity toward activation of EPAC1 over EPAC2 in cells. Of these, 25u was found to robustly inhibit IL-6-activated signal transducer and activator of transcription 3 (STAT3) and subsequent induction of the pro-inflammatory vascular cell adhesion molecule 1 (VCAM1) cell-adhesion protein. These novel EPAC1 activators may therefore act as useful pharmacological tools for elucidation of EPAC function and promising drug leads for the treatment of relevant human diseases.

CAMP/EPAC Signaling Enables ETV2 to Induce Endothelial Cells with High Angiogenesis Potential.

Although the generation of ETV2-induced endothelial cells (iECs) from human fibroblasts serves as a novel therapeutic strategy in regenerative medicine, the process is inefficient, resulting in incomplete iEC angiogenesis. Therefore, we employed chromatin immunoprecipitation (ChIP) sequencing and identified molecular mechanisms underlying ETV2-mediated endothelial transdifferentiation to efficiently produce iECs retaining appropriate functionality in long-term culture. We revealed that the majority of ETV2 targets in human fibroblasts are related to vasculature development and signaling transduction pathways, including Rap1 signaling. From a screening of signaling pathway modulators, we confirmed that forskolin facilitated efficient and rapid iEC reprogramming via activation of the cyclic AMP (cAMP)/exchange proteins directly activated by cAMP (EPAC)/RAP1 axis. The iECs obtained via cAMP signaling activation showed superior angiogenesis invivo as well as invitro. Moreover, these cells could form aligned endothelium along the vascular lumen exvivo when seeded into decellularized liver scaffold. Overall, our study provided evidence that the cAMP/EPAC/RAP1 axis is required for the efficient generation of iECs with angiogenesis potential.

Overexpression of EPAC2 reduces the invasion of glioma cells via MMP-2.

Exchange proteins directly activated by cAMP (EPACs) are crucial cyclic adenosine 3′,5′-monophosphate- determined signaling pathway intercessors, which are associated with the pathogenesis of neurological disorders and numerous human diseases. To the best of our knowledge, the role of EPAC2 signaling via matrix metalloproteinase 2 (MMP-2) in the pathogenesis of glioma has not been studied. Therefore, the present study focused on the role of EPAC2 in glioma, and assessed the invasiveness of human glioma cell lines following EPAC2 overexpression. Expression levels of EPAC2 in normal brain tissues and clinical glioma specimens were detected by western blotting. An EPAC2 overexpression vector was transfected into U251 and U87 cell lines to increase the expression levels of EPAC2. Expression levels of MMP-2 were detected by western blotting, and the invasive abilities of glioma cells were detected by a Transwell assay. EPAC2 was relatively highly expressed in normal brain tissue, while EPAC2 expression was significantly decreased in clinical glioma specimens (P<0.01). In vitro transfection of EPAC2 overexpression vector significantly reduced the MMP-2 protein levels of glioma cells, and, at the same time, the invasive cell number was significantly decreased in a Transwell assay. The present study demonstrated that MMP-2 regulation via EPAC2 overexpression is a novel promising therapeutic route in malignant types of glioma.

Inhibition of EPAC2 Attenuates Intracerebral Hemorrhage-Induced Secondary Brain Injury via the p38/BIM/Caspase-3 Pathway.

Exchange proteins directly activated by cAMP (EPACs) are critical cAMP-dependent signaling pathway intermediaries that have been implicated in the pathogenesis of several human diseases, particularly neurological disorders. However, their pathogenic role in secondary brain injury (SBI) induced by intracranial hemorrhage (ICH) is unknown. The aim of this study was to examine the effects of EPAC2 on ICH-induced SBI and its underlying mechanisms. An in vivo ICH model was established in Sprague-Dawley rats by autologous blood injection. In addition, rat primary cortical neuronal cultures were exposed to oxyhemoglobin to simulate ICH in vitro. The function of EPAC2 in SBI induced by ICH was studied using the EPAC2-specific inhibitor ESI-05. In this study, we found that EPAC2 protein expression was significantly increased in the ICH models in vitro and in vivo. Furthermore, EPAC2 activation was inhibited by ESI-05 under ICH conditions. Inhibition of EPAC2 decreased the apoptosis rate of nerve cells in the cortex accompanied by a corresponding decrease in the protein expression of phosphorylated p38, Bcl-2-like protein 11 (BIM), and caspase-3. In summary, this study showed that inhibition of EPAC2 activation by ESI-05 suppressed SBI induced by ICH via the p38/BIM/caspase-3 signaling pathway.

Intracellular cAMP Sensor EPAC: Physiology, Pathophysiology, and Therapeutics Development.

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.

Structure-activity relationships of 2-substituted phenyl-N-phenyl-2-oxoacetohydrazonoyl cyanides as novel antagonists of exchange proteins directly activated by cAMP (EPACs)

Exchange proteins directly activated by cAMP (EPACs) are critical cAMP-dependent signaling pathway mediators that play important roles in cancer, diabetes, heart failure, inflammations, infections, neurological disorders and other human diseases. EPAC specific modulators are urgently needed to explore EPAC’s physiological function, mechanism of action and therapeutic applications. On the basis of a previously identified EPAC specific inhibitor hit ESI-09, herein we have designed and synthesized a novel series of 2-substituted phenyl-N-phenyl-2-oxoacetohydrazonoyl cyanides as potent EPAC inhibitors. Compound 31 (ZL0524) has been discovered as the most potent EPAC inhibitor with IC50 values of 3.6 µM and 1.2 µM against EPAC1 and EPAC2, respectively. Molecular docking of 31 onto an active EPAC2 structure predicts that 31 occupies the hydrophobic pocket in cAMP binding domain (CBD) and also opens up new space leading to the solvent region. These findings provide inspirations for discovering next generation of EPAC inhibitors.

Structure-Activity Relationship Studies of Substituted 2-(Isoxazol-3-yl)-2-oxo-N'-phenyl-acetohydrazonoyl Cyanide Analogues: Identification of Potent Exchange Proteins Directly Activated by cAMP (EPAC) Antagonists.

Exchange proteins directly activated by cAMP (EPAC) as guanine nucleotide exchange factors mediate the effects of the pivotal second messenger cAMP, thereby regulating a wide variety of intracellular physiological and pathophysiological processes. A series of novel 2-(isoxazol-3-yl)-2-oxo-N'-phenyl-acetohydrazonoyl cyanide EPAC antagonists was synthesized and evaluated in an effort to optimize properties of the previously identified high-throughput (HTS) hit 1 (ESI-09). Structure-activity relationship (SAR) analysis led to the discovery of several more active EPAC antagonists (e.g., 22 (HJC0726), 35 (NY0123), and 47 (NY0173)) with low micromolar inhibitory activity. These inhibitors may serve as valuable pharmacological probes to facilitate our efforts in elucidating the biological functions of EPAC and developing potential novel therapeutics against human diseases. Our SAR results have also revealed that further modification at the 3-, 4-, and 5-positions of the phenyl ring as well as the 5-position of the isoxazole moiety may allow for the development of more potent EPAC antagonists.

Biochemical and pharmacological characterizations of ESI-09 based EPAC inhibitors: defining the ESI-09 "therapeutic window".

The cAMP signaling cascade is one of the most frequently targeted pathways for the development of pharmaceutics. A plethora of recent genetic and pharmacological studies suggest that exchange proteins directly activated by cAMP (EPACs) are implicated in multiple pathologies. Selective EPAC inhibitors have been recently developed. One specific inhibitor, ESI-09, has been shown to block EPAC activity and functions, as well as to recapitulate genetic phenotypes of EPAC knockout mice when applied in vivo. However, a recent study raised concern that ESI-09 might act as a non-specific protein denaturant. Herein, we present a detailed biochemical and pharmacological characterization, as well as a structure-activity relationship (SAR) analysis of ESI-09. Our studies show that ESI-09 dose-dependently inhibits activity of both EPAC1 and EPAC2 with apparent IC50 values well below the concentrations shown to induce “protein denaturation”. Moreover, the ESI-09's action towards EPAC proteins is highly sensitive to minor modifications of the 3-chlorophenyl moiety. Taken together, these results demonstrate that ESI-09 indeed acts as an EPAC specific antagonist and does not significantly destabilize/denature proteins at pharmacological effective concentrations. This conclusion is further supported by NMR data showing that ESI-09 induces residue-dependent chemical shift changes at low concentrations, while preserving well dispersed peaks.

Recent Advances in the Discovery of Small Molecules Targeting Exchange Proteins Directly Activated by cAMP (EPAC)

3',5'-Cyclic adenosine monophosphate (cAMP) is a pivotal second messenger that regulates numerous biological processes under physiological and pathological conditions, including cancer, diabetes, heart failure, inflammation, and neurological disorders. In the past, all effects of cAMP were initially believed to be mediated by protein kinase A (PKA) and cyclic nucleotide-regulated ion channels. Since the discovery of exchange proteins directly activated by cyclic adenosine 5'-monophosphate (EPACs) in 1998, accumulating evidence has demonstrated that the net cellular effects of cAMP are also regulated by EPAC. The pursuit of the biological functions of EPAC has benefited from the development and applications of a growing number of pharmacological probes targeting EPACs. In this review, we seek to provide a concise update on recent advances in the development of chemical entities including various membrane-permeable analogues of cAMP and newly discovered EPAC-specific ligands from high throughput assays and hit-to-lead optimizations.

Identification and Characterization of Small Molecules as Potent and Specific EPAC2 Antagonists

EPAC1 and EPAC2, two isoforms of exchange proteins directly activated by cAMP (EPAC), respond to the second messenger cAMP and regulate a wide variety of intracellular processes under physiological and pathophysiological circumstances. Herein, we report the chemical design, synthesis, and pharmacological characterization of three different scaffolds (diarylsulfones, N,N-diarylamines, and arylsulfonamides) as highly potent and selective antagonists of EPAC2. Several selective EPAC2 antagonists have been identified including 20i (HJC0350), which has an apparent IC(50) of 0.3 μM for competing with 8-NBD-cAMP binding of EPAC2 and is about 133-fold more potent than cAMP. Compounds 1 (ESI-05), 14c (HJC0338), and 20i, selected from each series, have exhibited no inhibition of EPAC1-mediated Rap1-GDP exchange activity at 25 μM, indicating that they are EPAC2-specific antagonists. Moreover, live-cell imaging studies using EPAC1, EPAC2, or PKA FRET sensor also demonstrate that 20i functions as an EPAC2 specific antagonist.

Structural analyses of a constitutively active mutant of exchange protein directly activated by cAMP.

Exchange proteins directly activated by cAMP (EPACs) are important allosteric regulators of cAMP-mediated signal transduction pathways. To understand the molecular mechanism of EPAC activation, we have combined site-directed mutagenesis, X-ray crystallography, and peptide amide hydrogen/deuterium exchange mass spectrometry (DXMS) to probe the structural and conformational dynamics of EPAC2-F435G, a constitutively active EPAC2 mutant. Our study demonstrates that conformational dynamics plays a critical role in cAMP-induced EPAC activation. A glycine mutation at 435 position shifts the equilibrium of conformational dynamics towards the extended active conformation.

A Fluorescence-Based High-Throughput Assay for the Discovery of Exchange Protein Directly Activated by Cyclic AMP (EPAC) Antagonists

BackgroundThe discovery, more than ten years ago, of exchange proteins directly activated by cAMP (EPAC) as a new family of intracellular cAMP receptors revolutionized the cAMP signaling research field. Extensive studies have revealed that the cAMP signaling network is much more complex and dynamic as many cAMP-related cellular processes, previously thought to be controlled by protein kinase A, are found to be also mediated by EPAC proteins. Although there have been many important discoveries in the roles of EPACs greater understanding of their physiological function in cAMP-mediated signaling is impeded by the absence of EPAC-specific antagonist.Methodology/Principal FindingsTo overcome this deficit, we have developed a fluorescence-based high throughput assay for screening EPAC specific antagonists. Our assay is highly reproducible and simple to perform using the “mix and measure” format. A pilot screening using the NCI-DTP diversity set library led to the identification of small chemical compounds capable of specifically inhibiting cAMP-induced EPAC activation while not affecting PKA activity.Conclusions/SignificanceOur study establishes a robust high throughput screening assay that can be effectively applied for the discovery of EPAC-specific antagonists, which may provide valuable pharmacological tools for elucidating the biological functions of EPAC and for promoting an understanding of disease mechanisms related to EPAC/cAMP signaling.

Modulation of Cytokine Production by Cyclic Adenosine Monophosphate Analogs in Human Leukocytes

Cyclic adenosine monophosphate (cAMP) is a well-known second messenger that operates through different signaling molecules, including protein kinase A (PKA) and guanine exchange proteins directly activated by cAMP (EPAC). Cell-permeable cAMP analogs such as 8-(4-chloro-phenyl-thio)-cAMP (8-pCPT-cAMP) modulate cytokine secretion by different leukocyte subsets, including T cells and monocytes. Since cAMP-modulating drugs such as phosphodiesterase inhibitors are being tested in inflammatory disorders such as asthma and chronic obstructive lung disease, it is important to obtain more insight into the regulation of cytokine production by cAMP. To address the signaling molecules involved in cAMP-mediated modulation of cytokine production, we used cAMP derivatives such as N(6)-benzoyladenosine-cAMP (6-Bnz-cAMP) and 8-pCPT-2-O-methyl cAMP (8-pCPT-2'-O-Me-cAMP), which selectively activate either PKA or EPAC, respectively. We show that in T cells, 6-Bnz-cAMP exerts similar globally inhibiting effects on cytokine secretion as 8-pCPT-cAMP, indicating that these effects are mediated by PKA. On the contrary, 8-pCPT-2'-O-Me-cAMP specifically inhibits the production of interleukin-10 (IL-10) in lipopolysaccharide-activated T-cell-depleted peripheral blood mononuclear cells, whereas the production of IL-1β, tumor necrosis factor α, and IL-12 is not or hardly affected. Inhibition by 8-pCPT-2'-O-Me-cAMP of IL-10 production was confirmed using purified monocytes. Further, in B cells 6-Bnz-cAMP, but not 8-pCPT-2'-O-Me-cAMP, stimulated IL-10 production. In conclusion, cAMP stimulates IL-10 production via PKA in activated B cells, but inhibits IL-10 production in activated monocytes through EPAC. We speculate that selective effects of PKA and EPAC on cytokine production in leukocyte subsets open up therapeutic possibilities using selective activators or inhibitors of EPAC or PKA.