Regulation of anti-inflammatory gene expression in vascular endothelial cells by EPAC1

Suppressor of cytokine signalling 3 (SOCS3) is a potent inhibitor of pro-inflammatory pathways involved in atherogenesis and the development of neo-intimal hyperplasia (NIH), which contributes to the in-stent re-stenosis responsible for the failure of percutaneous coronary intervention (PCI) procedures. We have shown that cyclic AMP sensor EPAC1 triggers induction of the SOCS3 gene in vascular endothelial cells (VECs), thereby attenuating interleukin 6 (IL-6)-mediated pro-inflammatory signalling. We propose that EPAC1 localisation to the nuclear pore controls cyclic AMP-mediated activation of a C/EBPβ/c-Jun transcriptional complex, leading to SOCS3 induction and suppression of pro-inflammatory signalling. Future work in this area will involve an integrated approach to determine the wider significance of the EPAC1-C/EBPβ/c-Jun pathway in controlling human VEC function and identify new therapeutic targets for management of chronic inflammation in vascular settings

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

The role of c-Jun in controlling the EPAC1-dependent induction of the SOCS3 gene in HUVECs

The cyclic AMP sensor, EPAC1, activates AP1-mediated transcription in HUVECs. Correspondingly, induction of the SOCS3 minimal promoter by EPAC1 requires a single AP1 site that constitutively binds phosphorylated (Ser63) c-Jun in DNA-pull-down assays. c-Jun (Ser63) becomes further phosphorylated following cyclic AMP stimulation and specific activation of protein kinase A (PKA), but not through selective activation of EPAC1. Moreover, despite a requirement for c-Jun for SOCS3 induction in fibroblasts, phospho-null c-Jun (Ser63/73Ala) had little effect on SOCS3 induction by cyclic AMP in HUVECs. AP1 activation and SOCS3 induction by EPAC1 in HUVECs therefore occur independently of c-Jun phosphorylation on Ser63

The Cyclic AMP Signalling System as a Regulator of Preadipocyte Differentiation

A study was carried out on the fibroblastic 3T3-F442A preadipocyte cell system to investigate the role of the adenylate cyclase signalling system in modulating their adipocyte differentiation. Three areas were investigated: 1) The expression of heterotrimeric guanine nucleotide binding protein (G-protein) subunits during individual stages of cellular differentiation. 2) The role of cyclic AMP in modulating preadipocyte differentiation. 3) Interactions occurring between the cyclic AMP and MAP kinase signalling cascades during the differentiation process. The differentiation of 31.3-F442A preadipocytes was shown, under serum-free conditions, to be divided into at least two stages. The first stage is dependent on the presence of growth hormone (GH) which primes cells to the action of other differentiative agents. The second, maturation, stage involved a combination of insulin, EGF and T3 which acted on GH-primed cells to promote terminal differentiation. Terminal differentiation was determined by two criteria; morphologically by the accumulation of Oil Red O-positive triacylglycerides in the cell cytoplasm and biochemically, by the emergence of the specific activity of an adipocyte-specific enzyme marker, alpha-glycerophosphate dehydrogenase. A range of specific anti-peptide antisera were generated and used to quantify specific changes in the expression of stimulatory- and inhibitory-G-proteins during the two-stages of 3T3-F442A preadipocyte differentiation. Stimulatory-G-proteins (GS) couple adenylate cyclase to positive-acting extracellular stimuli, whereas inhibitory-G-proteins (GO mediate the actions of negative-acting stimuli. Undifferentiated 3T3-F442A preadipocytes were found to express detectable levels of the GS alpha-subunits, Gsalpha42, Gsalpha44, and the Gi alpha-subunits, Gi2alpha and Gi3alpha in their cell membranes, together with the 36 kDa beta-suhunit (Gbeta36) which is subunit common to both GS and Gi. During the GH-priming stage of adipocyte differentiation significant increases in protein expression were observed for GSalpha42, GSalpha44. Gi2alpha and Gbeta36, but not Gi3alpha. Increased levels of GSalpha44 and Gbeta36 were sustained during the first two days of maturation and then fell, in terminally differentiated cells, to levels observed in undifferentiated 3T3-F442A fibroblasts. Levels of Gi2alpha were also sustained during the initial stages of maturation, but then fell, together with Gi3alpha, to levels significantly lower then those found in undifferentiated fibroblasts. In contrast, levels of GSalpha44 were maintained at a significantly elevated level in terminally differentiated cells when compared to undifferentiated fibroblasts. Changes in GSalpha subunit expression were also observed in non-differentiating control cultures, in the absence of differentiative agents. This G-protein may play a role in modulating preadipocyte differentiation. Indeed, treatment of cell with cholera toxin, which constitutively activates GS and adenylate cyclase, dramatically inhibited differentiation of 3T3-F442A cells by ~90%. Adenylate cyclase catalyses the formation of the intracellular second messenger cyclic AMP which then hinds to and activates protein kinase A. Direct activation of adenylate cyclase with forskolin (50muM) or treatment with the cyclic AMP analogue CPT-cyclic AMP (0.25mM) was found to potently inhibit the adipose conversion of 3T3-F442A preadipocytes induced with foetal calf serum and insulin (FCS/insulin; ~90% inhibition) or with a serum-free hormonally defined medium (DDM; ~70% inhibition). In contrast, treatment of cells with the cyclic AMP phosphodiesterase inhibitor, IBMX, or with low concentrations of CPT-cyclic AMP (10nM) or forskolin (10nM) potentiated cellular differentiation induced with FCS/insulin (~80-99% increase) or DDM (~30-40% increase). Both IBMX and 10nM forskolin induced small and relatively transient increases in intracellular cyclic AMP (~8 and ~3 fold maximal increase), whereas those induced by 50muM forskolin were much larger and more prolonged (~120 fold maximal increase). This suggests that the differential effects of cyclic AMP on the adipose conversion of 3T3-F442A cells could he attributable to interactions occurring at different stages of the differentiation program. Indeed, inclusion of 10nM forskolin or IBMX during the GH-priming stage of differentiation synergistically enhanced GH-promoted differentiation (~90% and ~130% increase, respectively). In contrast, when included during the maturation stage, these agents were found to inhibit terminal differentiation (~60% and ~70% inhibition, respectively). A potential mechanism underlying the effects of cyclic AMP on cell growth is through functional interplay with the MAP kinase signalling cascade. MAP kinases were potently activated in 3T3-F442A preadipocytes by the differentiative factors GH (~5 fold activation), insulin (~5 fold activation) and EGF (~20 fold activation). Antisense depletion of MAP kinase was found to severely retard the differentiation of 3T3-F442A cells with serum or DDM by approximately 95%, demonstrating that adipose conversion of these cells displays an overall requirement for MAP kinases

Genomic analysis of the role of transcription factor C/EBPδ in the regulation of cell behaviour on nanometric grooves

C/EBPδ is a tumour suppressor transcription factor that induces gene expression involved in suppressing cell migration. Here we investigate whether C/EBPδ-dependent gene expression also affects cell responses to nanometric topology. We found that ablation of the C/EBPδ gene in mouse embryonal fibroblasts (MEFs) decreased cell size, adhesion and cytoskeleton spreading on 240 nm and 540 nm nanometric grooves. ChIP-SEQ and cDNA microarray analyses demonstrated that many binding sites for C/EBPδ, and the closely related C/EBPβ, exist throughout the mouse genome and control the upregulation or downregulation of many adjacent genes. We also identified a group of C/EBPδ-dependent, trans-regulated genes, whose promoters contained no C/EBPδ binding sites and yet their activity was regulated in a C/EBPδ-dependent manner. These genes include signalling molecules (e.g. SOCS3), cytoskeletal components (Tubb2, Krt16 and Krt20) and cytoskeletal regulators (ArhGEF33 and Rnd3) and are possibly regulated by cis-regulated diffusible mediators, such as IL6. Of particular note, SOCS3 was shown to be absolutely required for efficient cell spreading and contact guidance on 240 nm and 540 nm nanometric grooves. C/EBPδ is therefore involved in the complex regulation of multiple genes, including cytoskeletal components and signalling mediators, which influence the nature of cell interactions with nanometric topology

Genome-Wide Mapping Defines a Role for C/EBPβ 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/EBPβ and c-Jun transcription factors, whereas I942 promoted recruitment of c-Jun to genes associated with IL6 signalling, with little effect on C/EBPβ 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

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