cAMP-GEFI; cAMP-GEFII; cAMP-regulated guanine nucleotide exchange factor; Exchange factor directly activated by cAMP; Exchange protein directly activated by cAMP (Epac); Exchange protein directly activated by cAMP 1 (Epac1); Exchange protein directly activated by cAMP 2 (Epac2); Rap guanine-nucleotide exchange factor (GEF); Rap guanine nucleotide exchange factor 3 (Rapgef3); Rap guanine nucleotide exchange factor 4 (Rapgef4); Rap1 guanine-nucleotide-exchange factor directly activated by cAMP
Eukaryotic cells respond to a wide range of extracellular signals such as growth factors, hormones, and neurotransmitters through the generation of intracellular second messengers. One of the most studied second messenger, cyclic adenosine 3′–5′-monophosphate (cAMP), regulates many cellular events, including secretion, differentiation, migration, and apoptosis. cAMP is produced from adenosine triphosphate (ATP) by adenylyl cyclase (AC) in response to Gαs-coupled G protein–coupled receptors (GPCRs) stimulation. Until 1998, it was believed that the biological action of cAMP was mainly mediated by the activation of the protein kinase A (PKA). However, several cellular effects of cAMP were shown to be insensitive to PKA, suggesting that the cellular action of cAMP may involve other effectors than PKA. A decade ago, the discovery of Epac proteins as novel cAMP sensors has broken the dogma surrounding cAMP and PKA (de Rooij et al. 1998; Kawasaki et al. 1998). Epac proteins are guanine nucleotide exchange factors (GEFs) and activate the Ras-like GTPases, Rap1 and Rap2, upon binding of cAMP. With the development of Epac-specific ligands and the recent generation of Epac knockout mice, numerous data in the literature have delineated the role of this multidomain protein Epac in a multitude of cellular events. Current evidence indicates that Epac is also involved in several diseases, including heart failure, diabetes, cancer, inflammation, and neurological disorders.
After a brief description of the structure of Epac isoforms and their downstream effectors, this review summarizes recent findings on their biological roles in different organs and pathophysiological situations.
Epac Proteins and Tissue Distribution
The comparison of the amino acid sequences of the Epac cAMP-binding domains with all other known cyclic-nucleotide-binding domains revealed that a glutamate residue was missing in Epac CNBD-B and not in the other cAMP-binding domains. This aminoacid residue interacts with the 2′-OH group of cAMP and is required for the high-affinity binding of cAMP to PKA. This observation led to the hypothesis that cAMP analogues that lacked the 2′-OH group might bind only to Epac. Based on this assumption, the first cAMP analogue missing the 2′-OH group and named 8-CPT or 8-pCPT-2′-O-Me-cAMP (8-(4-chloro-phenylthio)-2-O-methyladenosine -3′,5′-cyclic monophosphate) was synthesized in 2002. 8-CPT activates Epac and not PKA when used in the micromolar range. Additional chemical modifications of 8-CPT allowed the development of membrane permeable and phosphodiesterase-resistant Epac specific agonists. These compounds were named Sp-8CPT (Sp isomer of 8-CPT) and 8-CPT-AM (8-CPT-acetoxymethyl ester). Interestingly, 8-CPT and its derivatives are much more efficient in activating Epac1 than Epac2 (Lezoualc’h et al. 2016). The synthesis of such Epac selective ligands which do not activate the cAMP-PKA pathway has largely contributed to determine Epac biological action and its signaling (Schmidt et al. 2013; Bisserier et al. 2014; Parnell et al. 2015).
More recently, several selective inhibitors of Epac have been reported. A fluorescent-based high-throughput screening assay led to the identification of several competitive inhibitors of Epac. ESI (Epac specific inhibitor)-05 and ESI-07 show an Epac2 isoform-specific inhibition while other compounds, ESI-08 and ESI-09, prevent GEF activity of both Epac1 and Epac2 (Chen et al. 2014). In parallel, a noncompetitive Epac1 inhibitor named CE3F4 has been identified by probing Epac1 GEF activity towards Rap1 in vitro. This antagonistic activity is preferentially mediated by the (R)-enantiomer of CE3F4 and displays Epac1 isoform preference (Bisserier et al. 2014).
Epac Downstream Effectors
The Ras-like GTPases Rap1 and Rap2 are the direct effectors of Epac and couple Epac to its cellular effects. Since Epac mediates a plethora of biological effects in tissues, a multitude of effectors have been reported to act downstream of Epac-Rap signaling (Breckler et al. 2011). For instance, Epac activates the phospholipase Cε (PLCε) through the activation of Rap thereby stimulating the production of inositol-1,4,5-trisphosphate and subsequent release of Ca2+ from intracellular stores in various cell types including cardiomyocytes and neuroblastoma cells (Schmidt et al. 2013). Epac induced Ca2+ elevation can then activate various Ca2+ sensitive proteins such as the calmodulin-dependent protein kinase II (CaMKII) and the phosphatase calcineurin (Lezoualc’h et al. 2016). This cAMP-GEF also regulates the mitogen-activated protein kinase (MAPK), protein kinase C (PKC), phosphatidyl inositol 3 kinase, (PI3K) and various transcriptions factors (Breckler et al. 2011; Schmidt et al. 2013). Interestingly, few studies in the literature have reported Rap-independent effects of Epac. Indeed, Epac1 promotes activation of Rit, a close relative to Ras in a Rap-independent but Src-/TrkA-dependent manner. Epac can also activate the phospholipase D (PLD) via its GEF activity on R-Ras. Finally, Epac signals to the c-Jun N-terminal kinase (JNK) cascade through a mechanism that does not involve its Rap-specific GDP/GTP exchange (Schmidt et al. 2013).
Epac proteins are expressed in various subcellular compartments including the nucleus and plasma membrane where they exert their diverse biological functions either alone or in concert with PKA. The ability of Epac to trigger a specific signaling pathway depends on its cellular localization and molecular partners (Breckler et al. 2011). Indeed, specificity of Epac action is compartmentalized and is achieved by various scaffold proteins such as β-arrestin and A kinase anchoring proteins (AKAPs), which tether Epac signalosome at precise subcellular compartments and thereby permit and control specific cellular responses. Epac also interacts with cAMP phosphodiesterases (PDEs), which catalyze the hydrolysis of cAMP into 5-AMP and regulate the duration and intensity of Epac-cAMP signaling (Schmidt et al. 2013; Banerjee and Cheng 2015).
Role of Epac in the Cardiovascular System
Cyclic AMP is one of the most important second messenger in the heart because it regulates many physiological processes such as cardiac contractility, relaxation, and automaticity. It is a major second messenger involved in the sympathetic regulation of heart function. In human, Epac1 is the major Epac isoform expressed in the heart and is upregulated in heart failure, a major cause of mortality in developed countries (Métrich et al. 2010). Surprisingly, using KO mice of either Epac1 or Epac2 and double KO mice, it was reported that Epac gene ablation did not alter baseline cardiac function. However, Epac1 KO mice displayed an improved cardiac contractile function and were protected against cardiac hypertrophy and fibrosis in response to chronic adrenergic overdrive, a process leading to heart failure (Lezoualc’h et al. 2016). In addition, mice lacking Epac1 are cardioprotected against other forms of cardiac stress such as arrhythmogenic stress, pressure overload–induced cardiac dysfunction, and aging-induced cardiac dysfunction (Lezoualc’h et al. 2016). These results shed light on the therapeutic potential of the inhibition of Epac1 and the development of Epac1 inhibitors as new drugs to prevent heart failure. At the cellular and molecular levels, Epac signaling during cardiac stress is complex and involved various effector proteins such as the small GTPases Rap2, PLC, Rac, H-Ras, the Ca2+ sensitive proteins calcineurin and CaMKII, and their downstream prohypertrophic transcription factors such as nuclear factor of activated T cells (NFAT) and myocyte enhancer factor 2 (MEF2) (Lezoualc’h et al. 2016).
Because cAMP influences many facets of the biology of vascular smooth muscle cells (relaxation-contraction coupling, proliferation, migration) and vascular endothelial cells (proliferation, migration, cellular metabolism, and permeability), various studies are aimed at elucidating the role of Epac in this cellular process. Initial evidence for a role of Epac in the regulation of vasorelaxation came from the observation that the Epac agonist, 8-CPT, directly relaxes several types of vascular smooth muscle preparations (Lezoualc’h et al. 2016). Direct activation of Epac also inhibits mitogen-induced proliferation of cultured airway smooth muscle cells, suggesting that Epac activation may limit the remodeling observed in diseases of the airways such as asthma and chronic obstructive pulmonary disease (Dekkers et al. 2013). Interestingly, Epac promotes the migration of various types of vascular smooth muscle cells and facilitates the development of neointimal thickening, a process involved in vascular restenosis (Lezoualc’h et al. 2016). Of particular importance, the role of Epac in the regulation of cell migration is not restricted to vascular smooth muscle cells since several reports have shown that this guanine nucleotide exchange factor may influence this cellular process in various cell types, including tumor cell migration (Almahariq et al. 2016).
Endothelial barrier function restricts the passage of plasma proteins and circulating cells across endothelial cells, and its dysfunction may result in an increase in vascular permeability, thereby causing edema, inflammatory, or metastatic cell infiltration. Thus, the selective regulation of vascular permeability is critical for maintaining vascular integrity in homeostasis and disease. Epac1 is abundantly expressed in vascular endothelial cells and activation of Epac1–Rap1 signaling reduces vascular permeability by increasing junctional molecules such as vascular–endothelial cadherin at cell–cell contacts in a human umbilical vein endothelial cell (HUVEC) line (Parnell et al. 2015). Epac1 also induces reorganization of cortical actin, which supports junctional adhesion molecules thereby contributing to stabilize endothelial barrier function (Gloerich and Bos 2010). The effect of Epac on actin remodeling involves Rap, which can then regulate indirectly the activity of the Rho GTPase family such as RhoA and Rac (Métrich et al. 2010).
Other biological actions of cAMP have been assigned to Epac in the vasculature. Indeed, Epac1 has been identified as a modulator of interleukin (IL)-6 signaling in HUVECs, suggesting that this cAMP-binding protein may serve as a potential anti-inflammatory protein in vascular endothelial cells (Parnell et al. 2015). Of particular importance, the regulatory function of Epac1 on inflammation is strictly cell-type dependent. This is well illustrated in phagocytotic cells in which Epac reduces and/or increases the production of inflammatory mediators (Schmidt et al. 2013). Finally, also related to vascular function are recent works showing that Epac1-Rap1 pathway participates in regulating the bacterial transmigration across the vascular wall, a process which is considered to be an important step in the infectious process (Banerjee and Cheng 2015).
Biological Action of Epac in the Brain
Epac proteins regulate neuronal differentiation, neurite growth, and axon regeneration, suggesting that Epac plays a key role in the development and the maintenance of the central nervous system (Fig. 3). Several studies have revealed that Epac mediates the effects of pituitary adenylate cyclase-activating polypeptide-38 (PACAP-38) on neurite outgrowth in PC12 cells and human neuroblastoma SH-SY5Y cells (Laurent et al. 2012). The signaling pathway linking PACAP-38 receptor/Epac to neurite outgrowth of PC12 cells involves Rit, a Ras_like GTPase (Schmidt et al. 2013). Furthermore, Epac enhances neurite regeneration on adult spinal cord in vitro suggesting that Epac represents a target to induce axon regeneration after injury (Schmidt et al. 2013).
Growing evidence indicates that Epac proteins regulate both presynaptic and postsynaptic neurotransmission. It has been reported that Epac increases excitatory neurotransmission in the central nervous system and facilitates neurotransmitter release at the glutamatergic synapses of the rat brain calyx of Held and in the crayfish neuromuscular junction (Sugawara et al. 2016). In cultured mouse cerebral neurons, Epac activates neuronal excitability upon modulation of Ca2+-dependent K+-channels through Rap and p38 mitogen activated protein kinase (Schmidt et al. 2013). Yet, experiments performed in sensory neurons demonstrated that direct activation of Epac with 8-CPT sensitized pain receptors and augmented the sensitivity to mechanical pain. Further studies using Epac1 antisense oligonucleotide and Epac1-KO mice showed that Epac1 prevented prostaglandin E2-induced chronic hyperalgesia (Banerjee and Cheng 2015). Therefore, Epac1 may be a promising therapeutic target for the prevention and treatment of abnormal pain. Finally, recent studies showed that activation of Epac regulates various processes required for synaptic plasticity, learning, and memory such as long-term potentiation and long-term depression (Schmidt et al. 2013; Sugawara et al. 2016).
With respect to brain disorders, an Epac2 rare coding variant has been identified in human subjects diagnosed with autism suggesting a role of Epac2 in the pathophysiological mechanism of autism (Schmidt et al. 2013). In addition, single nucleotide polymorphisms in the genes encoding Epac1 and Epac2 have been associated with anxiety/depression and nicotine dependence, respectively (Laurent et al. 2012). Interestingly, in primary cortical neurons, Epac influences the processing of the amyloid precursor protein, a key gene involved in Alzheimer’s disease (Laurent et al. 2012). Epac is also linked to polyglutamine disorders such as Huntington’s disease (Schmidt et al. 2013).
Role of Epac in Pancreas
Cyclic AMP plays a critical role in the regulation of insulin secretion in pancreatic β-cells to maintain glucose homeostasis. Epac2 is the predominantly Epac isoform expressed in pancreatic β-cells and is required for the potentiation of glucose-induced secretion of insulin by glucagon-like peptide 1 and gastric inhibitory polypeptide (Sugawara et al. 2016) (Fig. 3). Epac2 induces insulin secretion by several mechanisms involving the regulation of intracellular calcium dynamics and direct interactions with proteins that directly facilitate the secretion of insulin granules (Almahariq et al. 2014; Sugawara et al. 2016). It is proposed that Epac2/Rap1 signaling increases the size of the readily releasable pool (RRP) and/or recruitment of insulin granules from the RRP (Sugawara et al. 2016). Various Epac-interacting proteins such as the small G protein Rab3 and secretory granule-associated proteins (Rim2, Piccolo) have been implicated in the Epac2-mediated potentiation of insulin secretion. Changes in the intracellular Ca2+ concentration play a pivotal role in the regulation of insulin granule exocytosis. In this line, Epac2 activation induces transient increases of Ca2+ associated with insulin exocytosis. The effect of Epac2 on Ca2+ mobilization is mediated by PLC and involves the inositol 1,4,5-trisphosphate receptor and ryanodine receptor in β-cells (Gloerich and Bos 2010). In addition, Epac2 also favours the closure of ATP-sensitive K+-channels, thereby inducing membrane depolarization and opening of voltage-dependent Ca2+-channels (VDCC) which facilitates insulin secretion through fusion of insulin granules with the plasma membrane (Schmidt et al. 2013).
Other Roles for Epac
Given the large distribution of Epac isoforms in the whole body, the biological action of Epac proteins is not limited to the aforementioned tissues. In the kidney, Epac1 has been implicated in the regulation of the Na+/H+ exchanger 3 activity and urea transport in proximal tubules and inner medullary collecting ducts, respectively. In addition, 8-CPT has been shown to protect the epithelial barrier function against hypoxia in vitro. Activation of Epac-Rap signaling also reduces renal failure in a mouse model for ischemia-reperfusion injury (Schmidt et al. 2013). Based on pharmacological and genetic studies, it is suggested that Epac1 plays a critical role in regulating adiposity and energy balance (Almahariq et al. 2014). Specifically, Epac1-KO mice show reduced concentrations of leptin in white adipose tissue and plasma and have enhanced hypothalamic leptin sensitivity. Leptin is an appetite-suppressing hormone derived from adipose tissue and plays a key role in the central regulation of satiety. Epac1-KO mice are more resistant to high-fat diet-induced obesity, hyperleptinemia, glucose intolerance, and insulin resistance (Almahariq et al. 2014).
Epac1 and Epac2 are novel effectors of the second messenger cAMP and act as specific guanine nucleotide exchange factors (GEFs) for the small G proteins, Rap1 and Rap2 of the Ras family. Upon binding of cAMP, Epac promotes the exchange of GDP for GTP, hence switching on the Rap GTPases. Epac functions in a PKA-independent manner and therefore represent a novel mechanism for regulating the specificity of cAMP cascade. Given their multidomain structure, Epac proteins have multiple binding partners and are interconnected with many signaling pathways into various subcellular compartments. Numerous studies using Epac specific ligands and genetically engineered mouse models have revealed the importance of these multidomain proteins in the control of key cellular functions, including cell division, migration, growth, and secretion. Recent reports now support the involvement of Epac proteins in the manifestation of diseases such as heart failure, tumor invasion, and inflammation, suggesting that Epac may represent attractive therapeutic targets for the treatment of various disorders.
PKA, PDEs, small G proteins, GEFs
F. Lezoualc’h was supported by grants from Institut National de la Santé et de la Recherche Médicale, Fondation pour la Recherche Médicale (Programme “Equipes FRM 2016”, DEQ20160334892), Fondation de France (00066331) and Université de Toulouse.