Epac1 Expression Research Articles

Neuroprotective effect of omidenepag on excitotoxic retinal ganglion cell death regulating COX-2–EP2–cAMP–PKA/Epac pathway via Neuron–Glia interaction

Glutamate excitotoxicity is involved in retinal ganglion cell (RGC) death in various retinal degenerative diseases, including ischemia–reperfusion injury and glaucoma. Excitotoxic RGC death is caused by both direct damage to RGCs and indirect damage through neuroinflammation of retinal glial cells. Omidenepag (OMD), a novel E prostanoid receptor 2 (EP2) agonist, is a recently approved intraocular pressure-lowering drug. The second messenger of EP2 is cyclic adenosine monophosphate (cAMP), which activates protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac). In this study, we investigated the neuroprotective effects of OMD on excitotoxic RGC death by focusing on differences in cAMP downstream signaling from the perspective of glia–neuron interactions.We established a glutamate excitotoxicity model in vitro and NMDA intravitreal injection model in vivo. In vitro, rat primary RGCs were used in an RGC survival rate assay. MG5 cells (mouse microglial cell line) and A1 cells (astrocyte cell line) were used for immunocytochemistry and Western blotting to evaluate the expressions of COX-1/2, PKA, Epac1/2, pCREB, cleaved caspase-3, inflammatory cytokines, and neurotrophic factors. Mouse retinal specimens underwent hematoxylin and eosin staining, flat-mounted retina examination, and immunohistochemistry.OMD significantly suppressed excitotoxic RGC death, cleaved caspase-3 expression, and activated glia both in vitro and in vivo. Moreover, it inhibited Epac1 and inflammatory cytokine expression and promoted COX-2, pCREB, and neurotrophic factor expression.OMD may have neuroprotective effects through inhibition of the Epac pathway and promotion of the COX-2–EP2–cAMP–PKA pathway by modulating glia–neuron interaction.

Inhibition of CXCR2 as a therapeutic target for chronic post-surgical pain: Insights from animal and cell models.

Studies have shown that chemokines can stimulate the migration and activation of microglia to cause chronic post-surgical pain (CPSP). However, the involvement of C-X-C motif chemokine receptor 2 (CXCR2) as a new chemotactic factor in regulating CPSP and its underlying mechanism remains unclear. This study is to investigate the role of CXCR2 in the development of CPSP and reveal the underlying mechanism. A rat model of skin/muscle incision and retraction was established, and treated with or without SB225002 (a selective inhibitor of CXCR2). In addition, the primary microglia cells induced by lipopolysaccharide were applied as an in vitro model for CPSP and treated individually with si-negative control (NC), si-CXCR2, si-CXCR2+Interleukin (IL)-6 (an agonist of the janus kinase (JAK)/signal transducers and activators of transcription (STAT)3 signaling pathway), si-CXCR2+IL-6+si-NC, or si-CXCR2+IL-6+si-exchange protein 1 directly activated by cAMP (EPAC1). Results from the database analysis showed that CXCR2 and JAK/STAT3 signaling pathway-related genes, including JAK1, STAT3, and EPAC1, were mainly involved in the development of CPSP. Inhibition of CXCR2 expression not only inhibited the reduction of foot pain threshold in CPSP models but also led to a decreased expression of CXCR2 and the phosphorylation levels of JAK and STAT3 in both animal and cell models. Furthermore, inhibition of EPAC1 expression can hinder the regulatory function of CXCR2. This study indicated that the high expression of CXCR2 activates the JAK1/STAT3 signaling pathway, enhances EPAC1 activation in microglial cells, and exacerbates CPSP.

Upregulation of Epac1 Promotes Pericyte Loss by Inducing Mitochondrial Fission, Reactive Oxygen Species Production, and Apoptosis.

The pathogenic mechanisms behind the development of ischemic retinopathy are complex and poorly understood. This study investigates the involvement of exchange protein directly activated by cAMP (Epac)1 signaling in pericyte injury during ischemic retinopathy, including diabetic retinopathy, a disease that threatens vision. Mouse models of retinal ischemia-reperfusion injury and type 1 diabetes induced by streptozotocin were used to investigate the pathogenesis of these diseases. The roles of Epac1 signaling in the pathogenesis of ischemic retinopathy were determined by an Epac1 knockout mouse model. The cellular and molecular mechanisms of Epac1-mediated pericyte dysfunction in response to high glucose were investigated by specific modulation of Epac1 activity in primary human retinal pericytes using Epac1-specific RNA interference and a pharmacological inhibitor. Ischemic injury or diabetes-induced retinal capillary degeneration were associated with an increased expression of Epac1 in the mouse retinal vasculature, including both endothelial cells and pericytes. Genetic deletion of Epac1 protected ischemic injury-induced pericyte loss and capillary degeneration in the mouse retina. Furthermore, high glucose-induced Epac1 expression in retinal pericytes was accompanied by increased Drp1 phosphorylation, mitochondrial fission, reactive oxygen species production, and caspase 3 activation. Inhibition of Epac1 via RNA interference or pharmacological approaches blocked high glucose-mediated mitochondrial dysfunction and caspase 3 activation. Our study reveals an important role of Epac1 signaling in mitochondrial dynamics, reactive oxygen species production, and apoptosis in retinal pericytes and identifies Epac1 as a therapeutic target for treating ischemic retinopathy.

#4904 EPAC1- MEDIATED CAMP SIGNALLING PROMOTES CELLULAR ENERGY ADAPTATIONS IN PODOCYTES TO PROTECT FROM GLOMERULONEPHRITIS

Abstract Background and Aims Many efforts are made to identify new therapeutic targets to slow down, prevent or even reverse Chronic Kidney Disease (CKD) progression. One of the therapeutic approaches is activating the renoprotective cAMP pathway, especially by stimulation of its downstream effector, the protein kinase A (PKA). PKA was considered as the unique cAMP effector, however, the exchange factor directly activated by cAMP 1 (EPAC1) has been recently identified as a novel, PKA-independent, mediator of cAMP signalling. EPAC1 is a guanidine exchange factor that promotes the exchange of GDP for GTP regulating important cellular functions. Epac1 activation exerts a renoprotective effect during acute kidney injury, via maintenance of epithelial adhesion and protection from oxidative stress. However, the role of EPAC1 in CKD remains poorly understood. Here we aim to determine the role of EPAC1 in CKD progression. Method Nephrotoxic serum glomerulonephritis (NTS-GN) is induced in genetically modified mice with total and conditional EPAC1 deletion. Then isolated glomeruli from mice with conditional EPAC1 deletion in podocytes are analysed by RNA-sequencing. The main metabolic energy pathways are studied in podocytes in vitro under oxidative stress exposure in the presence or absence of an EPAC1 agonist. Results Following the induction of NTS-GN, mice with genetic deletion of EPAC1 show aggravated renal disease, characterized by increased proteinuria, glomerular damage, tissue inflammation and fibrosis compared to wild-type mice. Conversely, pharmacological activation of EPAC1, with the agonist 8-pCPT-2-OMe-cAMP, delays NTS-GN progression. Since in human and wild type mouse kidney tissues we observe EPAC1 expression in podocytes, mice with conditional deletion of EPAC1 in podocytes (Nphs2Cre:epac) are generated. Similar to the whole-body knockout, mice with EPAC1 deletion in podocytes show increased renal damage and worsened disease progression compared to control mice. RNA-sequencing analysis of glomeruli isolated from these mice show that gene expression of proteins linked to the pathway of glycolysis are abolished in early stage of NTS-GN (day 4). These data suggest that EPAC1-mediated activation of glycolysis in podocytes is essential to limit GN progression. This is substantiated by the in vitro experiments in podocytes, in which EPAC1 activation under oxidative stress promotes glycolysis with cellular energy production independently from mitochondrial respiration. The EPAC1-mediated glycolysis protected podocytes by increasing cell viability, decreasing LDH release and activating the AKT pathway. Conclusion Our results suggest a protective role of podocytes-derived EPAC1 against the development of GN through cellular energetic adaptations based on metabolic switch to glycolysis. Activating the cAMP-EPAC1 signalling axis could represent a therapeutic option to delay the development of CKD. Further investigations are needed to define its relevance in human CKD.

Epac1 participates in β1-adrenoreceptor autoantibody-mediated decreased autophagic flux in cardiomyocytes

Decreased autophagic flux in cardiomyocytes is an important mechanism by which the β1-adrenoreceptor (β1-AR) autoantibody (β1-AA) induces heart failure. A previous study found that β1-AA imparts its biological effects via the β1-AR/Gs/AC/cAMP/PKA canonical signaling pathway, but PKA inhibition does not completely reverse β1-AA-induced reduction in autophagy in myocardial tissues, suggesting that other signaling molecules participate in this process. This study confirmed that Epac1 upregulation is indeed involved β1-AA-induced decreased cardiomyocyte autophagy through CE3F4 pretreatment, Epac1 siRNA transfection, western blot and immunofluorescence methods. On this basis, we constructed β1-AR and β2-AR knockout mice, and used receptor knockout mice, β1-AR selective blocker (atenolol), and the β2-AR/Gi-biased agonist ICI 118551 to show that β1-AA upregulated Epac1 expression through β1-AR and β2-AR to inhibit autophagy, and biased activation of β2-AR/Gi signaling downregulated myocardial Epac1 expression to reverse β1-AA-induced myocardial autophagy inhibition. This study aimed to test the hypothesis that Epac1 acts as another effector downstream of cAMP on β1-AA-induced reduction in cardiomyocyte autophagy, and β1-AA upregulates myocardial Epac1 expression through β1-AR and β2-AR, and biased activation of the β2-AR/Gi signaling pathway can reverse β1-AA-induced myocardial autophagy inhibition. This study provides new ideas and therapeutic targets for the prevention and treatment of cardiovascular diseases related to dysregulated autophagy.

CAMP-Epac1 signaling is activated in DDAVP-induced endolymphatic hydrops of guinea pigs

ObjectiveTo explore whether Cyclic Adenosine Monophosphate (cAMP)-Epac1 signaling is activated in 1-Desamino-8-D-arginine-Vasopressin-induced Endolymphatic Hydrops (DDAVP-induced EH) and to provide new insight for further in-depth study of DDAVP-induced EH. MethodsEighteen healthy, red-eyed guinea pigs (36 ears) weighing 200–350 g were randomly divided into three groups: the control group, which received intraperitoneal injection of sterile saline (same volume as that in the other two groups) for 7 consecutive days; the DDAVP-7d group, which received intraperitoneal injection of 10 mg/mL/kg DDAVP for 7 consecutive days; and the DDAVP-14d group, which received intraperitoneal injection of 10 μg/mL/kg DDAVP for 14 consecutive days. After successful modeling, all animals were sacrificed, and cochlea tissues were collected to detect the mRNA and protein expression of the exchange protein directly activated by cAMP-1 and 2 (Epac1, Epac2), and Repressor Activator Protein-1 (Rap1) by Reverse Transcription (RT)-PCR and western blotting, respectively. ResultsCompared to the control group, the relative mRNA expression of Epac1, Epac2, Rap1A, and Rap1B in the cochlea tissue of the DDAVP-7d group was significantly higher (p < 0.05), while no significant difference in Rap1 GTPase activating protein (Rap1gap) mRNA expression was found between the two groups. The relative mRNA expression of Epac1, Rap1A, Rap1B, and Rap1gap in the cochlea tissue of the DDAVP-14d group was significantly higher than that of the control group (p < 0.05), while no significant difference in Epac2 mRNA expression was found between the DDAVP-14d and control groups. Comparison between the DDAVP-14d and DDAVP-7d groups showed that the DDAVP-14d group had significantly lower Epac2 and Rap1A (p < 0.05) and higher Rap1gap (p < 0.05) mRNA expression in the cochlea tissue than that of the DDAVP-7d group, while no significant differences in Epac1 and Rap1B mRNA expression were found between the two groups. Western blotting showed that Epac1 protein expression in the cochlea tissue was the highest in the DDAVP-14d group, followed by that in the DDAVP-7d group, and was the lowest in the control group, showing significant differences between groups (p < 0.05); Rap1 protein expression in the cochlea tissue was the highest in the DDAVP-7d group, followed by the DDAVP-14d group, and was the lowest in the control group, showing significant differences between groups (p < 0.05); no significant differences in Epac2 protein expression in the cochlea tissue were found among the three groups. ConclusionDDAVP upregulated Epac1 protein expression in the guinea pig cochlea, leading to activation of the inner ear cAMP-Epac1 signaling pathway. This may be an important mechanism by which DDAVP regulates endolymphatic metabolism to induce EH and affect inner ear function. Oxford Centre for Evidence-Based Medicine 2011 Levels of EvidenceLevel 5.

Enhanced cGMP Interactor Rap Guanine Exchange Factor 4 (EPAC2) Expression and Activity in Degenerating Photoreceptors: A Neuroprotective Response?

The disease retinitis pigmentosa (RP) leads to photoreceptor degeneration by a yet undefined mechanism(s). In several RP mouse models (i.e., rd mice), a high cyclic GMP (cGMP) level within photoreceptors is detected, suggesting that cGMP plays a role in degeneration. The rap guanine exchange factor 4 (EPAC2) is activated by cyclic AMP (cAMP) and is an accepted cGMP-interacting protein. It is unclear whether and how cGMP interacts with EPAC2 in degenerating photoreceptors; we therefore investigated EPAC2 expression and interactions with cGMP and cAMP in retinas of the rd1 and rd10 models for retinal degeneration. EPAC2 expression in the photoreceptor layer increased significantly during rd1 and rd10 degeneration, and an increase in EPAC2 interactions with cGMP but not cAMP in the rd1 was also seen via a proximity ligation assay on histological sections. Retinal explant cultures revealed that pharmacological inhibition of the EPAC2 activity reduced the photoreceptor layer thickness in the rd10 retina, suggesting that EPAC2 inhibition promotes degeneration. Taken together, our results support the hypothesis that high degeneration-related cGMP leads to increased EPAC2 and cGMP interactions, inhibiting EPAC2. By inference, EPAC2 could have neuroprotective capacities that may be exploited in the future.

The expression and significance of Epac1 and Epac2 in the inner ear of guinea pigs.

To detect the expression of Epac1 and Epac2 in the inner ear of guinea pigs and its association with microcirculation in the inner ear. The temporal bones of 30 healthy red-eye guinea pigs (60 ears) weighing 200-350g were collected, then the surrounding bone wall of the cochlea was removed under a dissection microscope. Real-time quantitative PCR (RT-qPCR) and Western blot were used to detect mRNA and protein expression, respectively, of Epac1 and Epac2 in the inner ear and to compare their expression in heart, liver, kidney, intestine, and lung tissues. The specimens of the cochlea included the stria vascularis, basilar membrane, saccule, and utricles isolated under a microscope to detect the localization of Epac1 and Epac2 proteins in various parts of the inner ear through immunofluorescence staining. The RT-qPCR and Western blot results showed that Epac1 mRNA was universally expressed in the inner ear, heart, liver, kidneys, intestines, and lungs, and was highly expressed in the liver, kidneys, and intestines (p < 0.05 vs heart, liver, kidney, intestine; p > 0.05 vs lung). Epac2 mRNA was expressed in the inner ear and heart, but not in the liver, kidneys, intestines, or lungs (p < 0.05 vs Heart). Epac1 and Epac2 proteins were both expressed in the inner ear, heart, liver, kidneys, intestines, and lungs. The relative expression of Epac1 proteins in the inner ear was significantly different from the liver, kidneys, intestines, and lungs (p < 0.05). The relative expression of Epac2 proteins in the inner ear was significantly different from the liver, kidneys, and lungs (p < 0.05), but not from the heart (p = 0.127) or intestines (p = 0.274). Immunofluorescence staining observed under confocal microscopy indicated that Epac1 and Epac2 proteins were expressed in the stria vascularis, basilar membrane, saccule, and utricles of the inner ear. They were expressed in maginal cells, intermediate cells, and basal cells of the stria vascularis, and highly expressed in capillary endothelial cells. Epac1 and Epac2 mRNA and proteins were both expressed in the inner ear of guinea pigs and evenly expressed in the spiral ganglion, basilar membrane, saccule, and utricles. However, their expression in capillary endothelial cells of the stria vascularis was more obvious, suggesting that cyclic adenosine monophosphate-Epac1 signaling may play an important role in maintaining the function of the blood-labyrinth barrier and regulating the stability of microcirculation in the inner ear.

Dorsal root ganglia NR2B-mediated Epac1-Piezo2 signaling pathway contributes to mechanical allodynia of bone cancer pain.

Mechanical allodynia is a painful perception of mechanical stimuli and one of the typical symptoms in bone cancer pain (BCP). Previous studies have revealed that mice and humans lacking mechanically activated Piezo2 channels do not sense mechanical stimuli. However, the underlying mechanism of Piezo2 in BCP has not been well established. The aim of the present study was to investigate whether exchange protein directly activated by cAMP 1 (Epac1) mediated Piezo2 signaling pathway may be responsible for the mechanical allodynia of BCP and whether N-methyl-D-aspartic acid (NMDA) receptor subunit 2B (NR2B) is involved in the pathway. In the present study, a BCP model was established in C3H/HeJ mice by intramedullary injection of osteosarcoma cells. The results of the mechanical allodynia test demonstrated a markedly decreased paw withdrawal mechanical threshold in BCP mice, accompanied by a significant increase in Epac1, NR2B proteins and Piezo2 mRNA expression levels in the ipsilateral dorsal root ganglion (DRG). Compared with the sham group, intrathecal Epac1 antisense oligodeoxynucleotides (Epac1-ASODN) effectively ameliorated the mechanical allodynia and decreased the expression levels of NR2B and Piezo2 in the tumor group. Pretreatment of naïve mice with a NR2B antagonist prevented the aggravation of mechanical allodynia and DRG Piezo2 levels induced by an Epac1 agonist. However, the NR2B agonist-induced increase in Piezo2 expression levels was not reversed by pretreatment with Epac1-ASODN. In conclusion, the results of the present study demonstrated that NR2B, which is a crucial downstream regulator of Epac1, may mediate the Epac1-Piezo2 pathway contributing to the development of the mechanical allodynia of BCP. The present study may enrich the theoretical knowledge of the mechanical allodynia of BCP and provide a potential analgesic strategy for clinical treatment.

Vitexin Mitigates Myocardial Ischemia/Reperfusion Injury in Rats by Regulating Mitochondrial Dysfunction via Epac1-Rap1 Signaling.

Revascularization is an effective therapy for rescuing myocardial tissue after ischemic events. However, the process of reperfusion can lead to more severe cardiomyocyte damage, called myocardial ischemia-reperfusion (I/R) injury (MIRI). We have previously shown that vitexin (VT) (a flavonoid compound derived from natural products) protects against MIRI; however, the exact mechanisms underpinning this effect require further elucidation. This study is aimed at elucidating the protective mechanism of VT in inhibiting ischemic myocardial mitochondrial dysfunction and reducing cardiomyocyte apoptosis by regulating Epac1-Rap1 signaling. Isolated rat hearts were subjected to MIRI in a Langendorff perfusion system, and H9c2 cells were subjected to hypoxia/reoxygenation (H/R) in vitro. Our analyses show that during I/R, Epac1 expression was upregulated, left ventricular dysfunction deteriorated, mitochondrial dynamics were disrupted, and both myocardial cells and tissues exhibited apoptosis. Furthermore, administration of 8-CPT (an Epac agonist) exacerbated cardiomyocyte injury and mitochondrial dysfunction. Interestingly, suppressing the function of Epac1 through VT or ESI-09 (an Epac inhibitor) treatment during I/R reduced the myocardial infarct size, cardiomyocyte apoptosis, and reactive oxygen species production; alleviated mitochondrial dysfunction by increasing mitochondrial membrane potential; elevated MFN2 expression; and inhibited Drp1 expression. To our knowledge, our results reveal, for the first time, the mechanisms underlying the protective effect of VT in the myocardium of rats with MIRI. Moreover, we provide a new target and theoretical basis for VT in the treatment of ischemic heart disease.

LSM12-EPAC1 defines a neuroprotective pathway that sustains the nucleocytoplasmic RAN gradient.

Nucleocytoplasmic transport (NCT) defects have been implicated in neurodegenerative diseases such as C9ORF72-associated amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD). Here, we identify a neuroprotective pathway of like-Sm protein 12 (LSM12) and exchange protein directly activated by cyclic AMP 1 (EPAC1) that sustains the nucleocytoplasmic RAN gradient and thereby suppresses NCT dysfunction by the C9ORF72-derived poly(glycine-arginine) protein. LSM12 depletion in human neuroblastoma cells aggravated poly(GR)-induced impairment of NCT and nuclear integrity while promoting the nuclear accumulation of poly(GR) granules. In fact, LSM12 posttranscriptionally up-regulated EPAC1 expression, whereas EPAC1 overexpression rescued the RAN gradient and NCT defects in LSM12-deleted cells. C9-ALS patient-derived neurons differentiated from induced pluripotent stem cells (C9-ALS iPSNs) displayed low expression of LSM12 and EPAC1. Lentiviral overexpression of LSM12 or EPAC1 indeed restored the RAN gradient, mitigated the pathogenic mislocalization of TDP-43, and suppressed caspase-3 activation for apoptosis in C9-ALS iPSNs. EPAC1 depletion biochemically dissociated RAN-importin β1 from the cytoplasmic nuclear pore complex, thereby dissipating the nucleocytoplasmic RAN gradient essential for NCT. These findings define the LSM12-EPAC1 pathway as an important suppressor of the NCT-related pathologies in C9-ALS/FTD.

Mice depleted for Exchange Proteins Directly Activated by cAMP (Epac) exhibit irregular liver regeneration in response to partial hepatectomy

The exchange proteins directly activated by cAMP 1 and 2 (Epac1 and Epac2) are expressed in a cell specific manner in the liver, but their biological functions in this tissue are poorly understood. The current study was undertaken to begin to determine the potential roles of Epac1 and Epac2 in liver physiology and disease. Male C57BL/6J mice in which expression of Epac1 and/or Epac2 are deleted, were subjected to partial hepatectomy and the regenerating liver was analyzed with regard to lipid accumulation, cell replication and protein expression. In response to partial hepatectomy, deletion of Epac1 and/or Epac2 led to increased hepatocyte proliferation 36 h post surgery, and the transient steatosis observed in wild type mice was virtually absent in mice lacking both Epac1 and Epac2. The expression of the protein cytochrome P4504a14, which is implicated in hepatic steatosis and fibrosis, was substantially reduced upon deletion of Epac1/2, while a number of factors involved in lipid metabolism were significantly decreased. Moreover, the number of Küpffer cells was affected, and Epac2 expression was increased in the liver of wild type mice in response to partial hepatectomy, further supporting a role for these proteins in liver function. This study establishes hepatic phenotypic abnormalities in mice deleted for Epac1/2 for the first time, and introduces Epac1/2 as regulators of hepatocyte proliferation and lipid accumulation in the regenerative process.

Dysfunctional EAT thickness may promote maladaptive heart remodeling in CVD patients through the ST2-IL33 system, directly related to EPAC protein expression

Dysfunctional epicardial adipose tissue (EAT) secretome can influence the heart’s stretch response. However, the molecular mechanisms are still poorly understood. The aim of this study was to clarify how dysfunctional EAT promotes maladaptive heart remodeling in cardiovascular disease (CVD) through ST2 production associated with exchange protein directly activated by cAMP (EPAC) proteins. A series of 55 CVD males were enrolled and their EAT thickness, LV mass and volumes were measured by echocardiography. Blood, plasma and EAT biopsies were collected for molecular and proteomic assays. Taking EAT thickness as a continuous variable there was a direct correlation between the ST2 cardiac stretch mediator and EAT thickness (r = 0.54, p < 0.01) and an inverse relation between the ST2 gene and IL-33 expression (r −0.50, p < 0.01). In the CVD population EPAC2 expression directly correlated with the ST2 gene (r = 0.74, p < 0.0001) causing an ST2/IL-33 system local (p < 0.001) and systemic (sST2 = 57.33 ± 3.22 and IL-33 = 0.53 ± 017 pg/mL; p < 0.0001) protein imbalance associated with maladaptive remodeling. This indicated that dysfunctional EAT is a source of both EPAC and ST2 protein and an EPAC2 isoform seems involved in ST2 production in adipose tissue. Both EPAC2 and ST2 expression were directly related to maladaptive heart remodeling indices, suggesting EAT measurements could be useful in the early assessment of CVD complications.

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.

PDE4 and Epac1 Synergistically Promote Rectal Carcinoma via the cAMP Pathway.

Objective To assess the expression levels of exchange protein 1 directly activated by cAMP (Epac1) and phosphodiesterase 4 (PDE4) in rectal carcinoma, and their associations with clinicopathological indexes. In addition, the associations of PDE4 and Epac1 with A-kinase anchor protein 95, connexin 43, cyclin D1, and cyclin E1 were evaluated. Methods The PV-9000 two-step immunohistochemistry method was used to determine protein expression in 44 rectal carcinoma tissue samples and 16 paracarcinoma tissue specimens. Results The positive rate of PDE4 protein expression in rectal carcinoma tissues was higher than that of paracarcinoma tissues (59.09% vs. 12.5%, P < 0.05). Similar findings were obtained for Epac1 (55% vs. 6.25%, P < 0.05). No significant associations of PDE4 and Epac1 with degree of differentiation, histological type, and lymph node metastasis were found in rectal carcinoma (P > 0.05). Correlations between PDE4 and Epac1, PDE4 and Cx43, PDE4 and cyclin E1, and Epac1 and Cx43 were observed (all P < 0.05). There was no correlation between the other protein pairs examined (P > 0.05). Conclusion PDE4 and Epac1 expression levels are increased in rectal carcinoma tissues, suggesting that the two proteins may be involved in the development of this malignancy. Meanwhile, correlations between PDE4 and Epac1, PDE4 and Cx43, PDE4 and cyclin E1, and Epac1 and Cx43 suggested synergistic effects of these proteins in promoting rectal carcinoma.

Exchange protein activated by cyclic-adenosine monophosphate (Epac) regulates atrial fibroblast function and controls cardiac remodelling.

Heart failure (HF) produces left atrial (LA)-selective fibrosis and promotes atrial fibrillation. HF also causes adrenergic activation, which contributes to remodelling via a variety of signalling molecules, including the exchange protein activated by cAMP (Epac). Here, we evaluate the effects of Epac1-signalling on LA fibroblast (FB) function and its potential role in HF-related atrial remodelling. HF was induced in adult male mongrel dogs by ventricular tachypacing (VTP). Epac1-expression decreased in LA-FBs within 12 h (-3.9-fold) of VTP onset. The selective Epac activator, 8-pCPT (50 µM) reduced, whereas the Epac blocker ESI-09 (1 µM) enhanced, collagen expression in LA-FBs. Norepinephrine (1 µM) decreased Epac1-expression, an effect blocked by prazosin, and increased FB collagen production. The β-adrenoceptor (AR) agonist isoproterenol increased Epac1 expression, an effect antagonized by ICI (β2-AR-blocker), but not by CGP (β1-AR-blocker). β-AR-activation with isoproterenol decreased collagen expression, an effect mimicked by the β2-AR-agonist salbutamol and blocked by the Epac1-antagonist ESI-09. Transforming growth factor-β1, known to be activated in HF, suppressed Epac1 expression, an effect blocked by the Smad3-inhibitor SIS3. To evaluate effects on atrial fibrosis in vivo, mice subjected to myocardial infarction (MI) received the Epac-activator Sp-8-pCPT or vehicle for 2 weeks post-MI; Sp-8-pCPT diminished LA fibrosis and attenuated cardiac dysfunction. HF reduces LA-FB Epac1 expression. Adrenergic activation has complex effects on FBs, with α-AR-activation suppressing Epac1-expression and increasing collagen expression, and β2-AR-activation having opposite effects. Epac1-activation reduces cardiac dysfunction and LA fibrosis post-MI. Thus, Epac1 signalling may be a novel target for the prevention of profibrillatory cardiac remodelling.

Inhibition of Epac2 Attenuates Neural Cell Apoptosis and Improves Neurological Deficits in a Rat Model of Traumatic Brain Injury.

Traumatic brain injury (TBI) is a major cause of mortality and disability worldwide. TBI-induced neuronal apoptosis is one of the main contributors to the secondary injury process. The aim of this study is to investigate the involvement of Exchange protein directly activated by cAMP 2 (Epac2) on TBI. We found that the expression level of Epac2 surrounding the injured area of brain in rats of TBI model was significantly increased at 12 h after TBI. The role of Epac2 in TBI was further explored by using a selective Epac2 antagonist ESI-05 to decrease the Epac2 expression. We discovered that inhibition of Epac2 could improve the neurological impairment and attenuate brain edema following TBI. The Epac2 inhibition effectively reduced neuronal cell death and P38 MAPK signaling pathway may be involved in this process. Our results suggest that inhibition of Epac2 may be a potential therapy for TBI by reducing the neural cell death, alleviating brain edema and improving neurologic deficits.

Overexpression of exchange protein directly activated by cAMP-1 (EPAC1) attenuates bladder cancer cell migration

Exchange protein directly activated by cAMP (EPAC) is a mediator of a cAMP signaling pathway that is independent of protein kinase A. EPAC has two isoforms (EPAC1 and EPAC2) and is a cAMP-dependent guanine nucleotide exchange factor for the small GTPases, Rap1 and Rap2. Recent studies suggest that EPAC1 has both positive and negative influences on cancer and is involved in cell proliferation, apoptosis, migration and metastasis. We report that EPAC1 and EPAC2 expression levels were significantly lower in bladder cancer tissue than in normal bladder tissue. In addition, bladder cancer cell lines showed reduced EPAC1 mRNA expression. Furthermore, EPAC1 overexpression in bladder cancer cell lines induced morphologic changes and markedly suppressed cell migration without affecting cell viability. The overexpressed EPAC1 preferentially localized at cell-cell interfaces. In conclusion, reduced EPAC1 expression in bladder tumors and poor migration of EPAC1-overexpressing cells implicate EPAC1 as an inhibitor of bladder cancer cell migration.

Upregulation of Epac-1 in Hepatic Stellate Cells by Prostaglandin E2 in Liver Fibrosis Is Associated with Reduced Fibrogenesis.

Exchange protein activated by cAMP (Epac-1) is an important signaling mechanism for cAMP-mediated effects, yet factors that change Epac-1 levels are unknown. Such factors are relevant because it has been postulated that Epac-1 directly affects fibrogenesis. Prostaglandin E2 (PGE2) is a well-known cAMP activator, and we therefore studied the effects of this cyclo-oxygenase product on Epac-1 expression and on fibrogenesis within the liver. Liver fibrosis was induced by 8 weeks carbon tetrachloride (CCL4) administration to mice. In the last 2 weeks, mice received vehicle, PGE2, the cyclo-oxygenase-2 inhibitor niflumic acid (NFA), or PGE2 coupled to cell-specific carriers to hepatocytes, Kupffer cells, or hepatic stellate cells (HSC). Results showed antifibrotic effects of PGE2 and profibrotic effects of NFA in CCL4 mice. Western blot analysis revealed reduced Epac-1 protein expression in fibrotic livers of mice and humans compared with healthy livers. PGE2 administration to fibrotic mice completely restored intrahepatic Epac-1 levels and also led to reduced Rho kinase activity, a downstream target of Epac-1. Cell-specific delivery of PGE2 to either hepatocytes, Kupffer cells, or HSC identified the latter cell as the key player in the observed effects on Epac-1 and Rho kinase. No significant alterations in protein kinase A expressions were found. In primary isolated HSC, PGE2 elicited Rap1 translocation reflecting Epac-1 activation, and Epac-1 agonists attenuated platelet-derived growth factor-induced proliferation and migration of these cells. These studies demonstrate that PGE2 enhances Epac-1 activity in HSC, which is associated with significant changes in (myo)fibroblast activities in vitro and in vivo. Therefore, Epac-1 is a potential target for antifibrotic drugs.

Epac1, PDE4, and PKC protein expression and their correlation with AKAP95 and Cx43 in esophagus cancer tissues.

BackgroundThis study examined the expression of exchange protein directly activated by cAMP1 (Epac1), PDE4, and PKC in esophageal cancer tissues, and analyzed the association of each protein with the pathological parameters of the samples.MethodsEpac1, PDE4, and PKC protein expression was evaluated by PV‐9000 two‐step immunohistochemical techniques in 51 esophageal cancer specimens and 10 para‐carcinoma tissues.ResultsThe positive expression rates of Epac1 and PKC in esophageal cancer tissues (62.7% and 68.6%, respectively) were higher compared to those in para‐carcinoma tissues (20% and 20%, respectively) (P < 0.05). The positive expression rate of PDE4 in esophageal cancer tissues (54.1%) was higher than in para‐carcinoma tissues (30%), (P > 0.05). Epac1, PDE4, and PKC protein expression levels were not associated with the extent of tumor differentiation and/or lymph node metastasis (P > 0.05). Epac1 protein expression levels correlated with PDE4, PKC, and AKAP95 protein expression levels. In addition, there was a correlation between PKC and Cx43 protein levels (P < 0.05).ConclusionThe expression rates of Epac1, PDE4, and PKC protein in esophageal cancer tissues were significantly higher compared to the rates in para‐carcinoma tissues, suggesting an association between these proteins and the development and progression of esophageal cancer. The correlations between these proteins also revealed that they may exert a synergistic effect during the development of esophageal cancer.