Vascular smooth muscle cells enhance immune/vascular interplay in a 3-cell model of vascular inflammation.

Atherosclerosis is a serious cardiovascular disease that is characterised by the development of atheroma, which are lipid-laden plaques that build up within arterial walls due to chronic inflammatory processes. These lesions are fundamentally attributed to a complex cellular crosstalk between vascular smooth muscle cells (VSMCs), vascular endothelial cells (VECs) and central immune cells, such as macrophages (Mɸs), which promote vascular inflammation. The presence of VSMCs exerts both positive and negative effects during atheroma development, which can be attributed to their phenotypic plasticity. Understanding the interactions between these key cell types during the development of vascular inflammation and atheroma will enhance the scope for new therapeutic interventions. This study aims to determine the importance of VSMCs for shaping the extracellular cytokine/chemokine profile and transcriptional responses of VECs (human coronary artery endothelial cells; HCAECs) to activated lipopolysaccharide (LPS)-stimulated THP1 Mɸs, in a 3-cell model of human vascular inflammation. It is evident that within the presence of VSMCs, enhanced cytokine production was associated with up-regulation of genes associated with vascular inflammation t. Results demonstrate that the presence of VSMCs in co-culture experiments enhanced cytokine production (including CXCL1/GROα, IL-6, IL-8 and CCL2/MCP1) and inflammatory gene expression (including genes involved in JAK/STAT, Jun and NFκB signalling) in HCAECs co-cultured with LPS-stimulated THP1 Mɸs. Our results highlight the importance of VSMCs in immune/endothelial cell interplay and indicate that 3-cell, rather than 2-cell co-culture, may be more appropriate for the study of cellular crosstalk between immune and vascular compartments in response to inflammatory and atherogenic stimuli.

Protein interaction, cytotoxic, transcriptomic and proteomic responses to structurally distinct EPAC1 activators in HUVECs.

The N-acylsulfonamide derivative, I942, represents the first non-cyclic nucleotide partial agonist of EPAC1. This was soon followed by the identification of the I942 analogues, PW0381, PW0521 and PWO577 and a series of benzofuran oxoacetic acid EPAC1 activators, SY006, SY007 and SY009. Protein interaction, cytotoxicity and EPAC1 activation assays applied here identify PWO577 and SY007 as being effective EPAC1 binders that are well tolerated in HUVECs at concentrations greater than 100 µM and up to 48 h incubation and are effective activators of transfected EPAC1 in U2OS cells. Using RNAseq in HUVECs we show that PWO577 and SY007 regulate approximately 11,000 shared genes, with only few differential gene changes being "off-target". The genes significantly regulated by both PWO577 and SY007 included a subset of genes normally associated with endothelial activation, including ICAM1, MMP1 and CCL2. Of these, only the expression of MMP1 was markedly increased at the protein level, as determined by LC-MS-based proteomics. Both PWO577 and SY007 suppressed IL-6-induced STAT3 activation and associated downstream gene expression, including inhibition of SOCS3, STAT3, IL6ST and JAK3 genes. Together these results demonstrate the utility of structurally distinct, specific and non-toxic EPAC1 activators. Future modifications will be aimed at eliminating the few noted off-target effects.

Identification and Characterization of an Affimer Affinity Reagent for the Detection of the cAMP Sensor, EPAC1.

An exchange protein directly activated by cAMP 1 (EPAC1) is an intracellular sensor for cAMP that is involved in a wide variety of cellular and physiological processes in health and disease. However, reagents are lacking to study its association with intracellular cAMP nanodomains. Here, we use non-antibody Affimer protein scaffolds to develop isoform-selective protein binders of EPAC1. Phage-display screens were carried out against purified, biotinylated human recombinant EPAC1ΔDEP protein (amino acids 149-811), which identified five potential EPAC1-selective Affimer binders. Dot blots and indirect ELISA assays were next used to identify Affimer 780A as the top EPAC1 binder. Mutagenesis studies further revealed a potential interaction site for 780A within the EPAC1 cyclic nucleotide binding domain (CNBD). In addition, 780A was shown to co-precipitate EPAC1 from transfected cells and co-localize with both wild-type EPAC1 and a mis-targeting mutant of EPAC1(K212R), predominantly in perinuclear and cytosolic regions of cells, respectively. As a novel EPAC1-selective binder, 780A therefore has the potential to be used in future studies to further understand compartmentalization of the cAMP-EPAC1 signaling system.

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.

Identification of A Novel Class of Benzofuran Oxoacetic Acid-Derived Ligands that Selectively Activate Cellular EPAC1.

Cyclic AMP promotes EPAC1 and EPAC2 activation through direct binding to a specific cyclic nucleotide-binding domain (CNBD) within each protein, leading to activation of Rap GTPases, which control multiple cell responses, including cell proliferation, adhesion, morphology, exocytosis, and gene expression. As a result, it has become apparent that directed activation of EPAC1 and EPAC2 with synthetic agonists may also be useful for the future treatment of diabetes and cardiovascular diseases. To identify new EPAC agonists we have developed a fluorescent-based, ultra-high-throughput screening (uHTS) assay that measures the displacement of binding of the fluorescent cAMP analogue, 8-NBD-cAMP to the EPAC1 CNBD. Triage of the output of an approximately 350,000 compound screens using this assay identified a benzofuran oxaloacetic acid EPAC1 binder (SY000) that displayed moderate potency using orthogonal assays (competition binding and microscale thermophoresis). We next generated a limited library of 91 analogues of SY000 and identified SY009, with modifications to the benzofuran ring associated with a 10-fold increase in potency towards EPAC1 over SY000 in binding assays. In vitro EPAC1 activity assays confirmed the agonist potential of these molecules in comparison with the known EPAC1 non-cyclic nucleotide (NCN) partial agonist, I942. Rap1 GTPase activation assays further demonstrated that SY009 selectively activates EPAC1 over EPAC2 in cells. SY009 therefore represents a novel class of NCN EPAC1 activators that selectively activate EPAC1 in cellulae.

Selective small-molecule EPAC activators.

The cellular signalling enzymes, EPAC1 and EPAC2, have emerged as key intracellular sensors of the secondary messenger cyclic 3',5'-adenosine monophosphate (cyclic adenosine monophosphate) alongside protein kinase A. Interest has been galvanised in recent years thanks to the emergence of these species as potential targets for new cardiovascular disease therapies, including vascular inflammation and insulin resistance in vascular endothelial cells. We herein summarise the current state-of-the-art in small-molecule EPAC activity modulators, including cyclic nucleotides, sulphonylureas, and N-acylsulphonamides.

Genome-Wide Mapping Defines a Role for C/EBPß and c-Jun in Non-Canonical Cyclic AMP Signalling.

The novel exchange protein activated by cyclic AMP (EPAC1) activator, I942, induces expression of the suppressor of cytokine signalling 3 (SOCS3) gene, thereby inhibiting interleukin 6 (IL6) inflammatory processes in human umbilical vein endothelial cells (HUVECs). Here we use RNA-SEQ and ChIP-SEQ to determine global gene responses to I942, in comparison with cyclic AMP production promoted by forskolin and rolipram (F/R). We found that I942 promoted significant changes in the RNA expression of 1413 genes, largely associated with microtubule stability and cell cycle progression, whereas F/R regulated 197 genes linked to endothelial cell function, including chemokine production and platelet aggregation. A further 108 genes were regulated by both treatments, including endothelial regulatory genes involved in purinergic signalling and cell junction organization. ChIP-SEQ demonstrated that F/R induced genome-wide recruitment of C/EBPß and c-Jun transcription factors, whereas I942 promoted recruitment of c-Jun to genes associated with IL6 signalling, with little effect on C/EBPß activation. Despite this, certain key inflammatory genes, including IL6, VEGF, CCL2/MCP1, VCAM1, SELE and ICAM1 were regulated by I942 without significant c-Jun recruitment, suggesting an additional, indirect mode of action for I942. In this regard, SOCS3 induction by I942 was found to require c-Jun and was associated with suppression of IL6-promoted ERK MAP kinase and AKT activity and induction of ICAM1. Pharmacological inhibition of ERK and AKT also potentiated ICAM1 induction by I942. We therefore propose that c-Jun activation by I942 regulates endothelial gene expression in HUVECs through direct mechanisms, involving recruitment of c-Jun or, as for ICAM1, through indirect regulation of tertiary regulators, including SOCS3.

The novel exchange protein activated by cyclic AMP 1 (EPAC1) agonist, I942, regulates inflammatory gene expression in human umbilical vascular endothelial cells (HUVECs).

Exchange protein activated by cyclic AMP (EPAC1) suppresses multiple inflammatory actions in vascular endothelial cells (VECs), partly due to its ability to induce expression of the suppressor of cytokine signalling 3 (SOCS3) gene, the protein product of which inhibits interleukin 6 (IL6) signalling through the JAK/STAT3 pathway. Here, for the first time, we use the non-cyclic nucleotide EPAC1 agonist, I942, to determine its actions on cellular EPAC1 activity and cyclic AMP-regulated gene expression in VECs. We demonstrate that I942 promotes EPAC1 and Rap1 activation in HEK293T cells and induces SOCS3 expression and suppresses IL6-stimulated JAK/STAT3 signalling in HUVECs. SOCS3 induction by I942 in HUVECs was blocked by the EPAC1 antagonist, ESI-09, and EPAC1 siRNA, but not by the broad-spectrum protein kinase A (PKA) inhibitor, H89, indicating that I942 regulates SOCS3 gene expression through EPAC1. RNA sequencing was carried out to further identify I942-regulated genes in HUVECs. This identified 425 I942-regulated genes that were also regulated by the EPAC1-selective cyclic AMP analogue, 007, and the cyclic AMP-elevating agents, forskolin and rolipram (F/R). The majority of genes identified were suppressed by I942, 007 and F/R treatment and many were involved in the control of key vascular functions, including the gene for the cell adhesion molecule, VCAM1. I942 and 007 also inhibited IL6-induced expression of VCAM1 at the protein level and blocked VCAM1-dependent monocyte adhesion to HUVECs. Overall, I942 represents the first non-cyclic nucleotide EPAC1 agonist in cells with the ability to suppress IL6 signalling and inflammatory gene expression in VECs.

Probe-Dependent Negative Allosteric Modulators of the Long-Chain Free Fatty Acid Receptor FFA4.

High-affinity and selective antagonists that are able to block the actions of both endogenous and synthetic agonists of G protein-coupled receptors are integral to analysis of receptor function and to support suggestions of therapeutic potential. Although there is great interest in the potential of free fatty acid receptor 4 (FFA4) as a novel therapeutic target for the treatment of type II diabetes, the broad distribution pattern of this receptor suggests it may play a range of roles beyond glucose homeostasis in different cells and tissues. To date, a single molecule, 4-methyl-N-9H-xanthen-9-yl-benzenesulfonamide (AH-7614), has been described as an FFA4 antagonist; however, its mechanism of antagonism remains unknown. We synthesized AH-7614 and a chemical derivative and demonstrated these to be negative allosteric modulators (NAMs) of FFA4. Although these NAMs did inhibit FFA4 signaling induced by a range of endogenous and synthetic agonists, clear agonist probe dependence in the nature of allosteric modulation was apparent. Although AH-7614 did not antagonize the second long-chain free fatty acid receptor, free fatty acid receptor 1, the simple chemical structure of AH-7614 containing features found in many anticancer drugs suggests that a novel close chemical analog of AH-7614 devoid of FFA4 activity, 4-methyl-N-(9H-xanthen-9-yl)benzamide (TUG-1387), will also provide a useful control compound for future studies assessing FFA4 function. Using TUG-1387 alongside AH-7614, we show that endogenous activation of FFA4 expressed by murine C3H10T1/2 mesenchymal stem cells is required for induced differentiation of these cells toward a more mature, adipocyte-like phenotype.

Identification of a Novel, Small Molecule Partial Agonist for the Cyclic AMP Sensor, EPAC1.

Screening of a carefully selected library of 5,195 small molecules identified 34 hit compounds that interact with the regulatory cyclic nucleotide-binding domain (CNB) of the cAMP sensor, EPAC1. Two of these hits (I942 and I178) were selected for their robust and reproducible inhibitory effects within the primary screening assay. Follow-up characterisation by ligand observed nuclear magnetic resonance (NMR) revealed direct interaction of I942 and I178 with EPAC1 and EPAC2-CNBs in vitro. Moreover, in vitro guanine nucleotide exchange factor (GEF) assays revealed that I942 and, to a lesser extent, I178 had partial agonist properties towards EPAC1, leading to activation of EPAC1, in the absence of cAMP, and inhibition of GEF activity in the presence of cAMP. In contrast, there was very little agonist action of I942 towards EPAC2 or protein kinase A (PKA). To our knowledge, this is the first observation of non-cyclic-nucleotide small molecules with agonist properties towards EPAC1. Furthermore, the isoform selective agonist nature of these compounds highlights the potential for the development of small molecule tools that selectively up-regulate EPAC1 activity.

The cyclic AMP phosphodiesterase 4D5 (PDE4D5)/receptor for activated C-kinase 1 (RACK1) signalling complex as a sensor of the extracellular nano-environment.

The cyclic AMP and protein kinase C (PKC) signalling pathways regulate a wide range of cellular processes that require tight control, including cell proliferation and differentiation, metabolism and inflammation. The identification of a protein complex formed by receptor for activated C kinase 1 (RACK1), a scaffold protein for protein kinase C (PKC), and the cyclic AMP-specific phosphodiesterase, PDE4D5, demonstrates a potential mechanism for crosstalk between these two signalling routes. Indeed, RACK1-bound PDE4D5 is activated by PKCα, providing a route through which the PKC pathway can control cellular cyclic AMP levels. Although RACK1 does not appear to affect the intracellular localisation of PDE4D5, it does afford structural stability, providing protection against denaturation, and increases the susceptibility of PDE4D5 to inhibition by cyclic AMP-elevating pharmaceuticals, such as rolipram. In addition, RACK1 can recruit PDE4D5 and PKC to intracellular protein complexes that control diverse cellular functions, including activated G protein-coupled receptors (GPCRs) and integrins clustered at focal adhesions. Through its ability to regulate local cyclic AMP levels in the vicinity of these multimeric receptor complexes, the RACK1/PDE4D5 signalling unit therefore has the potential to modify the quality of incoming signals from diverse extracellular cues, ranging from neurotransmitters and hormones to nanometric topology. Indeed, PDE4D5 and RACK1 have been found to form a tertiary complex with integrin-activated focal adhesion kinase (FAK), which localises to cellular focal adhesion sites. This supports PDE4D5 and RACK1 as potential regulators of cell adhesion, spreading and migration through the non-classical exchange protein activated by cyclic AMP (EPAC1)/Rap1 signalling route.

The Potential of a Novel Class of EPAC-Selective Agonists to Combat Cardiovascular Inflammation.

The cyclic 3',5'-adenosine monophosphate (cAMP) sensor enzyme, EPAC1, is a candidate drug target in vascular endothelial cells (VECs) due to its ability to attenuate proinflammatory cytokine signalling normally associated with cardiovascular diseases (CVDs), including atherosclerosis. This is through the EPAC1-dependent induction of the suppressor of cytokine signalling gene, SOCS3, which targets inflammatory signalling proteins for ubiquitinylation and destruction by the proteosome. Given this important role for the EPAC1/SOCS3 signalling axis, we have used high throughput screening (HTS) to identify small molecule EPAC1 regulators and have recently isolated the first known non-cyclic nucleotide (NCN) EPAC1 agonist, I942. I942 therefore represents the first in class, isoform selective EPAC1 activator, with the potential to suppress pro-inflammatory cytokine signalling with a reduced risk of side effects associated with general cAMP-elevating agents that activate multiple response pathways. The development of augmented I942 analogues may therefore provide improved research tools to validate EPAC1 as a potential therapeutic target for the treatment of chronic inflammation associated with deadly CVDs.

Nanotopography controls cell cycle changes involved with skeletal stem cell self-renewal and multipotency.

In culture isolated bone marrow mesenchymal stem cells (more precisely termed skeletal stem cells, SSCs) spontaneously differentiate into fibroblasts, preventing the growth of large numbers of multipotent SSCs for use in regenerative medicine. However, the mechanisms that regulate the expansion of SSCs, while maintaining multipotency and preventing fibroblastic differentiation are poorly understood. Major hurdles to understanding how the maintenance of SSCs is regulated are (a) SSCs isolated from bone marrow are heterogeneous populations with different proliferative characteristics and (b) a lack of tools to investigate SSC number expansion and multipotency. Here, a nanotopographical surface is used as a tool that permits SSC proliferation while maintaining multipotency. It is demonstrated that retention of SSC phenotype in culture requires adjustments to the cell cycle that are linked to changes in the activation of the mitogen activated protein kinases. This demonstrates that biomaterials can offer cross-SSC culture tools and that the biological processes that determine whether SSCs retain multipotency or differentiate into fibroblasts are subtle, in terms of biochemical control, but are profound in terms of determining cell fate.

Metformin suppresses adipogenesis through both AMP-activated protein kinase (AMPK)-dependent and AMPK-independent mechanisms.

People with Type 2 diabetes mellitus (T2DM) have reduced bone mineral density and an increased risk of fractures due to altered mesenchymal stem cell (MSC) differentiation in the bone marrow. This leads to a shift in the balance of differentiation away from bone formation (osteogenesis) in favour of fat cell development (adipogenesis). The commonly used anti-diabetic drug, metformin, activates the osteogenic transcription factor Runt-related transcription factor 2 (Runx2), which may suppress adipogenesis, leading to improved bone health. Here we investigate the involvement of the metabolic enzyme, AMP-activated protein kinase (AMPK), in these protective actions of metformin. The anti-adipogenic actions of metformin were observed in multipotent C3H10T1/2 MSCs, in which metformin exerted reciprocal control over the activities of Runx2 and the adipogenic transcription factor, PPARγ, leading to suppression of adipogenesis. These effects appeared to be independent of AMPK activation but rather through the suppression of the mTOR/p70S6K signalling pathway. Basal AMPK and mTOR/p70S6K activity did appear to be required for adipogenesis, as demonstrated by the use of the AMPK inhibitor, compound C. This observation was further supported by using AMPK knockout mouse embryo fibroblasts (MEFs) where adipogenesis, as assessed by reduced lipid accumulation and expression of the adipogeneic transcription factor, C/EBPß, was found to display an absolute requirement for AMPK. Further activation of AMPK in wild type MEFS, with either metformin or the AMPK-specific activator, A769662, was also associated with suppression of adipogenesis. It appears, therefore, that basal AMPK activity is required for adipogenesis and that metformin can inhibit adipogenesis through AMPK-dependent or -independent mechanisms, depending on the cellular context.

Dynamic Surfaces for the Study of Mesenchymal Stem Cell Growth through Adhesion Regulation.

Out of their niche environment, adult stem cells, such as mesenchymal stem cells (MSCs), spontaneously differentiate. This makes both studying these important regenerative cells and growing large numbers of stem cells for clinical use challenging. Traditional cell culture techniques have fallen short of meeting this challenge, but materials science offers hope. In this study, we have used emerging rules of managing adhesion/cytoskeletal balance to prolong MSC cultures by fabricating controllable nanoscale cell interfaces using immobilized peptides that may be enzymatically activated to change their function. The surfaces can be altered (activated) at will to tip adhesion/cytoskeletal balance and initiate differentiation, hence better informing biological mechanisms of stem cell growth. Tools that are able to investigate the stem cell phenotype are important. While large phenotypical differences, such as the difference between an adipocyte and an osteoblast, are now better understood, the far more subtle differences between fibroblasts and MSCs are much harder to dissect. The development of technologies able to dynamically navigate small differences in adhesion are critical in the race to provide regenerative strategies using stem cells.

Epac1 links prostaglandin E2 to ß-catenin-dependent transcription during epithelial-to-mesenchymal transition.

In epithelial cells, ß-catenin is localized at cell-cell junctions where it stabilizes adherens junctions. When these junctions are disrupted, ß-catenin can translocate to the nucleus where it functions as a transcriptional cofactor. Recent research has indicated that PGE2 enhances the nuclear function of ß-catenin through cyclic AMP. Here, we aim to study the role of the cyclic AMP effector Epac in ß-catenin activation by PGE2 in non-small cell lung carcinoma cells. We show that PGE2 induces a down-regulation of E-cadherin, promotes cell migration and enhances ß-catenin translocation to the nucleus. This results in ß-catenin-dependent gene transcription. We also observed increased expression of Epac1. Inhibition of Epac1 activity using the CE3F4 compound or Epac1 siRNA abolished the effects of PGE2 on ß-catenin. Further, we observed that Epac1 and ß-catenin associate together. Expression of an Epac1 mutant with a deletion in the nuclear pore localization sequence prevents this association. Furthermore, the scaffold protein Ezrin was shown to be required to link Epac1 to ß-catenin. This study indicates a novel role for Epac1 in PGE2-induced EMT and subsequent activation of ß-catenin.

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.

The future of EPAC-targeted therapies: agonism versus antagonism.

Pharmaceutical manipulation of cAMP levels exerts beneficial effects through the regulation of the exchange protein activated by cAMP (EPAC) and protein kinase A (PKA) signalling routes. Recent attention has turned to the specific regulation of EPAC isoforms (EPAC1 and EPAC2) as a more targeted approach to cAMP-based therapies. For example, EPAC2-selective agonists could promote insulin secretion from pancreatic ß cells, whereas EPAC1-selective agonists may be useful in the treatment of vascular inflammation. By contrast, EPAC1 and EPAC2 antagonists could both be useful in the treatment of heart failure. Here we discuss whether the best way forward is to design EPAC-selective agonists or antagonists and the current strategies being used to develop isoform-selective, small-molecule regulators of EPAC1 and EPAC2 activity.

The cAMP sensors, EPAC1 and EPAC2, display distinct subcellular distributions despite sharing a common nuclear pore localisation signal.

We have identified a conserved nuclear pore localisation signal (NPLS; amino acids 764-838 of EPAC1) in the catalytic domains of the cAMP-sensors, EPAC1 and EPAC2A. Consequently, EPAC1 is mainly localised to the nuclear pore complex in HEK293T cells where it becomes activated following stimulation with cAMP. In contrast, structural models indicate that the cAMP-binding domain of EPAC2A (CNBD1) blocks access to the conserved NPLS in EPAC2A, reducing its ability to interact with nuclear binding sites. Consequently, a naturally occurring EPAC2 isoform, EPAC2B, which lacks CNBD1 is enriched in nuclear fractions, similar to EPAC1. Structural differences in EPAC isoforms may therefore determine their intracellular location and their response to elevations in intracellular cAMP.

Serine-727 phosphorylation activates hypothalamic STAT-3 independently from tyrosine-705 phosphorylation.

Transcriptional activity of signal transducer and activator of transcription-3 (STAT-3) is a key element in the central regulation of appetite and energy homeostasis. Activation of hypothalamic STAT-3 has been attributed to cytokine-promoted phosphorylation at tyrosine-705 (Tyr-705). In nonhypothalamic cells, STAT-3 is also phosphorylated at serine-727 (Ser-727), but the functional significance of Ser-727 in the regulation of hypothalamic STAT-3 is not known. We used 2 hypothalamic cell lines and analyzed the effects of various hormones on STAT-3-dependent reporter gene activity and observed that IFN-γ, epidermal growth factor (EGF), and bradykinin (BK) induce similar STAT-3 reporter activation. EGF and BK solely increased Ser-727 and IFN-γ increased Tyr-705 phosphorylation of STAT-3. Specific inhibition of ERK-1/2 activity blocked EGF- and BK-induced STAT-3 activation and Ser-727 phosphorylation. BK-induced ERK-1/2 activation occurred via EGF receptor transactivation. Consequently, the BK-mediated effects on STAT-3 were blocked by a specific EGF receptor antagonist. Next, we analyzed the effects of IFN-γ and EGF on the expression of the STAT-3-dependent genes thyroliberin-releasing hormone and suppressors of cytokine signaling-3. EGF but not IFN-γ enhanced thyroliberin-releasing hormone expression via STAT-3. With regard to suppressors of cytokine signaling-3, we observed prolonged expression induced by IFN-γ and a transient effect of EGF that required coactivation of the activator protein-1. Thus, EGF-promoted Ser-727 phosphorylation by ERK-1/2 is not only sufficient to fully activate hypothalamic STAT-3, but, in terms of targeted genes and required cofactors, entails distinct modes of STAT-3 actions compared with IFN-γ-induced Tyr-705 phosphorylation.