Adenosine A2A receptor (A2AR) is a fine-tune regulator of the collagen1:collagen3 balance.
ABSTRACT: Adenosine is a potent endogenous anti-inflammatory and immunosuppressive metabolite that is a potent modulator of tissue repair. However, the adenosine A2A receptor (A2AR)-mediated promotion of collagen synthesis is detrimental in settings such as scarring and scleroderma. The signaling cascade from A2AR stimulation to increased collagen production is complex and obscure, not least because cAMP and its downstream molecules PKA and Epac1 have been reported to inhibit collagen production. We therefore examined A2AR-stimulated signaling for collagen production by normal human dermal fibroblasts (NHDF). Collagen1 (Col1) and collagen3 (Col3) content after A2AR activation by CGS21680 was studied by western blotting. Contribution of PKA and Epac was analyzed by the PKA inhibitor PKI and by knockdowns of the PKA-C?, -C?, -C?, Epac1, and Epac2. CGS21680 stimulates Col1 expression at significantly lower concentrations than those required to stimulate Col3 expression. A2AR stimulates Col1 expression by a PKA-dependent mechanism since PKA inhibition or PKA-C? and -C? knockdown prevents A2AR-mediated Col1 increase. In contrast, A2AR represses Col3 via PKA but stimulates both Col1 and Col3 via an Epac2-dependent mechanism. A2AR stimulation with CGS21680 at 0.1 ?M increased Col3 expression only upon PKA blockade. A2AR activation downstream signaling for Col1 and Col3 expression proceeds via two distinct pathways with varying sensitivity to cAMP activation; more highly cAMP-sensitive PKA activation stimulates Col1 expression, and less cAMP-sensitive Epac activation promotes both Col1 and Col3 expression. These observations may explain the dramatic change in Col1:Col3 ratio in hypertrophic and immature scars, where adenosine is present in higher concentrations than in normal skin.
Project description:Activation of adenosine A2A receptor (A2AR) promotes fibrosis and collagen synthesis. However, the underlying mechanism is still unclear, not least because cAMP, its principal effector, has been found to inhibit TGF?1-induced collagen synthesis. Here, we show that in primary normal human dermal fibroblasts, A2AR stimulation with CGS21680 elicits a modest cAMP increase (150 ± 12% of control; EC50 54.8 nM), which stimulates collagen1 (Col1) and collagen3 (Col3), but maximal cAMP resulting from direct activation of adenylyl cyclase by forskolin (15,689 ± 7038% of control; EC50 360.7 nM) inhibits Col1 and increases Col3. Similar to Col1 expression, fibroblast proliferation increased following physiological cAMP increases by CGS21680 but was inhibited by cAMP increases beyond the physiological range by forskolin. The A2AR-mediated increase of Col1 and Col3 was mediated by AKT, while Col3, but not Col1, expression was dependent on p38 and repressed by ERK. TGF?1 induced phosphorylation of Smad2/3 and increased Col3 expression, which was prevented by Smad3 depletion. In contrast, CGS21680 did not activate Smad2/3, and Smad2/3 knockdown did not prevent CGS21680-induced Col1 or Col3 increases. Our results indicate that cAMP is a concentration-dependent switch for collagen production via noncanonical, AKT-dependent, Smad2/3-independent signaling. These observations explain the paradoxical effects of cAMP on collagen expression.
Project description:BACKGROUND AND PURPOSE:Adenosine A2A receptor stimulation promotes the synthesis of collagen type I and type III (Col1 and Col3), mediators of fibrosis and scarring. The A2A receptor modulates collagen balance via cAMP/PKA/p38-MAPK/Akt pathways. Wnt signalling is important in fibrosis and the cAMP and Wnt pathways converge. Because the A2A receptor is Gs-linked and increases cAMP, we determined whether A2A receptors and Wnt signalling interact. EXPERIMENTAL APPROACH:Total ?-catenin, de-phosphorylated ?-catenin (canonical activation, de-phospho ?-catenin) and phosphorylated ?-catenin at Ser552 (non-canonical activation, p-Ser552 ?-catenin) levels were determined in primary human dermal fibroblasts, cytosol and nucleus, by western blot analysis and fluorescence microscopy, before and after stimulation by A2A receptor-selective agonist CGS21680, with/without A2A receptor-selective antagonist (SCH56261) pretreatment. ?-Catenin was knocked down by transfection with scrambled-siRNA or specific-siRNA, and Col1 and Col3 levels determined by western blots. KEY RESULTS:CGS21680 stimulation rapidly (15 min) increased cellular ?-catenin levels. Both de-phospho ?-catenin and p-Ser552 ?-catenin levels were also increased. CGS21680 stimulated the translocation of total de-phospho and p-Ser552 ?-catenin to the nucleus. A2A receptor-stimulation increased Col1 synthesis similarly in ?-catenin knockeddown and scrambled cells. However, ?-catenin knockdown abolished the increase in Col3 synthesis induced in A2A receptor-stimulated fibroblasts. CONCLUSIONS AND IMPLICATIONS:A2A receptor stimulation promotes Col3 synthesis via the activation of canonical and non-canonical ?-catenin, consistent with a role for A2A receptors in dermal fibrosis and scarring.
Project description:We have demonstrated that chronic stimulation of the prostaglandin E2-cAMP-dependent protein kinase A (PKA) signal pathway plays a critical role in intimal cushion formation in perinatal ductus arteriosus (DA) through promoting synthesis of hyaluronan. We hypothesized that Epac, a newly identified effector of cAMP, may play a role in intimal cushion formation (ICF) in the DA distinct from that of PKA. In the present study, we found that the levels of Epac1 and Epac2 mRNAs were significantly up-regulated in the rat DA during the perinatal period. A specific EP4 agonist, ONO-AE1-329, increased Rap1 activity in the presence of a PKA inhibitor, PKI-(14-22)-amide, in DA smooth muscle cells. 8-pCPT-2'-O-Me-cAMP (O-Me-cAMP), a cAMP analog selective to Epac activator, promoted migration of DA smooth muscle cells (SMC) in a dose-dependent manner. Adenovirus-mediated Epac1 or Epac2 gene transfer further enhanced O-Me-cAMP-induced cell migration, although the effect of Epac1 overexpression on cell migration was stronger than that of Epac2. In addition, transfection of small interfering RNAs for Epac1, but not Epac2, significantly inhibited serum-mediated migration of DA SMCs. In the presence of O-Me-cAMP, actin stress fibers were well organized with enhanced focal adhesion, and cell shape was widely expanded. Adenovirus-mediated Epac1, but not Epac2 gene transfer, induced prominent ICF in the rat DA explants when compared with those with green fluorescent protein gene transfer. The thickness of intimal cushion became significantly greater (1.98-fold) in Epac1-overexpressed DA. O-Me-cAMP did not change hyaluronan production, although it decreased proliferation of DA SMCs. The present study demonstrated that Epac, especially Epac1, plays an important role in promoting SMC migration and thereby ICF in the rat DA.
Project description:cAMP is a universal second messenger regulating a plethora of processes in the kidney. Two downstream effectors of cAMP are PKA and exchange protein directly activated by cAMP (Epac), which, unlike PKA, is often linked to elevation of [Ca2+]i. While both Epac isoforms (Epac1 and Epac2) are expressed along the nephron, their relevance in the kidney remains obscure. We combined ratiometric calcium imaging with quantitative immunoblotting, immunofluorescent confocal microscopy, and balance studies in mice lacking Epac1 or Epac2 to determine the role of Epac in renal water-solute handling. Epac1-/- and Epac2-/- mice developed polyuria despite elevated arginine vasopressin levels. We did not detect major deficiencies in arginine vasopressin [Ca2+]i signaling in split-opened collecting ducts or decreases in aquaporin water channel type 2 levels. Instead, sodium-hydrogen exchanger type 3 levels in the proximal tubule were dramatically reduced in Epac1-/- and Epac2-/- mice. Water deprivation revealed persisting polyuria, impaired urinary concentration ability, and augmented urinary excretion of Na+ and urea in both mutant mice. In summary, we report a nonredundant contribution of Epac isoforms to renal function. Deletion of Epac1 and Epac2 decreases sodium-hydrogen exchanger type 3 expression in the proximal tubule, leading to polyuria and osmotic diuresis.-Cherezova, A., Tomilin, V., Buncha, V., Zaika, O., Ortiz, P. A., Mei, F., Cheng, X., Mamenko, M., Pochynyuk, O. Urinary concentrating defect in mice lacking Epac1 or Epac2.
Project description: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 ? 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.
Project description:Intracellular cAMP and serotonin are important modulators of anxiety and depression. Fluoxetine, a selective serotonin reuptake inhibitor (SSRI) also known as Prozac, is widely used against depression, potentially by activating cAMP response element-binding protein (CREB) and increasing brain-derived neurotrophic factor (BDNF) through protein kinase A (PKA). However, the role of Epac1 and Epac2 (Rap guanine nucleotide exchange factors, RAPGEF3 and RAPGEF4, respectively) as potential downstream targets of SSRI/cAMP in mood regulations is not yet clear. Here, we investigated the phenotypes of Epac1 (Epac1(-/-)) or Epac2 (Epac2(-/-)) knockout mice by comparing them with their wild-type counterparts. Surprisingly, Epac2(-/-) mice exhibited a wide range of mood disorders, including anxiety and depression with learning and memory deficits in contextual and cued fear-conditioning tests without affecting Epac1 expression or PKA activity. Interestingly, rs17746510, one of the three single-nucleotide polymorphisms (SNPs) in RAPGEF4 associated with cognitive decline in Chinese Alzheimer's disease (AD) patients, was significantly correlated with apathy and mood disturbance, whereas no significant association was observed between RAPGEF3 SNPs and the risk of AD or neuropsychiatric inventory scores. To further determine the detailed role of Epac2 in SSRI/serotonin/cAMP-involved mood disorders, we treated Epac2(-/-) mice with a SSRI, Prozac. The alteration in open field behavior and impaired hippocampal cell proliferation in Epac2(-/-) mice were alleviated by Prozac. Taken together, Epac2 gene polymorphism is a putative risk factor for mood disorders in AD patients in part by affecting the hippocampal neurogenesis.
Project description:EPAC1 and EPAC2, two isoforms of exchange proteins directly activated by cAMP (EPAC), respond to the second messenger cAMP and regulate a wide variety of intracellular processes under physiological and pathophysiological circumstances. Herein, we report the chemical design, synthesis, and pharmacological characterization of three different scaffolds (diarylsulfones, N,N-diarylamines, and arylsulfonamides) as highly potent and selective antagonists of EPAC2. Several selective EPAC2 antagonists have been identified including 20i (HJC0350), which has an apparent IC(50) of 0.3 ?M for competing with 8-NBD-cAMP binding of EPAC2 and is about 133-fold more potent than cAMP. Compounds 1 (ESI-05), 14c (HJC0338), and 20i, selected from each series, have exhibited no inhibition of EPAC1-mediated Rap1-GDP exchange activity at 25 ?M, indicating that they are EPAC2-specific antagonists. Moreover, live-cell imaging studies using EPAC1, EPAC2, or PKA FRET sensor also demonstrate that 20i functions as an EPAC2 specific antagonist.
Project description:Exchange proteins directly activated by cAMP (Epac1 and Epac2) have been recently recognized as key players in ?-adrenergic-dependent cardiac arrhythmias. Whereas Epac1 overexpression can lead to cardiac hypertrophy and Epac2 activation can be arrhythmogenic, it is unknown whether distinct subcellular distribution of Epac1 vs. Epac2 contributes to differential functional effects. Here, we characterized and used a novel fluorescent cAMP derivate Epac ligand 8-[Pharos-575]-2'-O-methyladenosine-3',5'-cyclic monophosphate (?-O-Me-cAMP) in mice lacking either one or both isoforms (Epac1-KO, Epac2-KO, or double knockout, DKO) to assess isoform localization and function. Fluorescence of ?-O-Me-cAMP was enhanced by binding to Epac. Unlike several Epac-specific antibodies tested, ?-O-Me-cAMP exhibited dramatically reduced signals in DKO myocytes. In WT, the apparent binding affinity (Kd = 10.2 ± 0.8 µM) is comparable to that of cAMP and nonfluorescent Epac-selective agonist 8-(4-chlorophenylthio)-2-O-methyladenosine-3'-,5'-cyclicmonophosphate (OMe-CPT). ?-O-Me-cAMP readily entered intact myocytes, but did not activate PKA and its binding was competitively inhibited by OMe-CPT, confirming its Epac specificity. ?-O-Me-cAMP is a weak partial agonist for purified Epac, but functioned as an antagonist for four Epac signaling pathways in myocytes. Epac2 and Epac1 were differentially concentrated along T tubules and around the nucleus, respectively. Epac1-KO abolished OMe-CPT-induced nuclear CaMKII activation and export of transcriptional regulator histone deacetylase 5. In conclusion, Epac1 is localized and functionally involved in nuclear signaling, whereas Epac2 is located at the T tubules and regulates arrhythmogenic sarcoplasmic reticulum Ca leak.
Project description:The major physiological effects of cAMP in mammalian cells are transduced by two ubiquitously expressed intracellular cAMP receptors, protein kinase A (PKA) and exchange protein directly activated by cAMP (EPAC), as well as cyclic nucleotide-gated ion channels in certain tissues. Although a large number of PKA inhibitors are available, there are no reported EPAC-specific antagonists, despite extensive research efforts. Here we report the identification and characterization of noncyclic nucleotide EPAC antagonists that are exclusively specific for the EPAC2 isoform. These EAPC2-specific antagonists, designated as ESI-05 and ESI-07, inhibit Rap1 activation mediated by EAPC2, but not EPAC1, with high potency in vitro. Moreover, ESI-05 and ESI-07 are capable of suppressing the cAMP-mediated activation of EPAC2, but not EPAC1 and PKA, as monitored in living cells through the use of EPAC- and PKA-based FRET reporters, or by the use of Rap1-GTP pull-down assays. Deuterium exchange mass spectroscopy analysis further reveals that EPAC2-specific inhibitors exert their isoform selectivity through a unique mechanism by binding to a previously undescribed allosteric site: the interface of the two cAMP binding domains, which is not present in the EPAC1 isoform. Isoform-specific EPAC pharmacological probes are highly desired and will be valuable tools for dissecting the biological functions of EPAC proteins and their roles in various disease states.
Project description: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.