Role of Membrane Microdomains in Compartmentation of cAMP Signaling

Spatially restricting cAMP production to discrete subcellular locations permits selective regulation of specific functional responses. But exactly where and how cAMP signaling is confined is not fully understood. Different receptors and adenylyl cyclase isoforms responsible for cAMP production are not uniformly distributed between lipid raft and non-lipid raft domains of the plasma membrane. We sought to determine the role that these membrane domains play in organizing cAMP responses in HEK293 cells. The freely diffusible FRET-based biosensor Epac2-camps was used to measure global cAMP responses, while versions of the probe targeted to lipid raft (Epac2-MyrPalm) and non-raft (Epac2-CAAX) domains were used to monitor local cAMP production near the plasma membrane. Disruption of lipid rafts by cholesterol depletion selectively altered cAMP responses produced by raft-associated receptors. The results indicate that receptors associated with lipid raft as well as non-lipid raft domains can contribute to global cAMP responses. In addition, basal cAMP activity was found to be significantly higher in non-raft domains. This was supported by the fact that pharmacologic inhibition of adenylyl cyclase activity reduced basal cAMP activity detected by Epac2-CAAX but not Epac2-MyrPalm or Epac2-camps. Responses detected by Epac2-CAAX were also more sensitive to direct stimulation of adenylyl cyclase activity, but less sensitive to inhibition of phosphodiesterase activity. Quantitative modeling was used to demonstrate that differences in adenylyl cyclase and phosphodiesterase activities are necessary but not sufficient to explain compartmentation of cAMP associated with different microdomains of the plasma membrane.     

The mAKAP signaling complex: integration of cAMP, calcium, and MAP kinase signaling pathways

Following its production by adenylyl cyclases, the second messenger cAMP is in involved in pleiotrophic signal transduction. The effectors of cAMP include the cAMP-dependent protein kinase (PKA), the guanine nucleotide exchange factor Epac (exchange protein activated by cAMP), and cAMP-dependent ion channels. In turn, cAMP signaling is attenuated by phosphodiesterase-catalyzed degradation. The association of cAMP effectors and the enzymes that regulate cAMP concentration into signaling complexes helps to explain the differential signaling initiated by members of the G(s)-protein coupled receptor family. The signal transduction complex formed by the scaffold protein mAKAP (muscle A kinase-anchoring protein) at the nuclear envelope of both striated myocytes and neurons contains three cAMP-binding proteins, PKA, Epac1, and the phosphodiesterase PDE4D3. In addition, the mAKAP complex also contains components of the ERK5 MAP kinase signaling pathway, the calcium release channel ryanodine receptor and the phosphatases PP2A as well as calcineurin. Analysis of the mAKAP complex illustrates how a macromolecular complex can serve as a node in the intracellular signaling network of cardiac myocytes to integrate multiple cAMP signals with those of calcium and MAP kinases to regulate the hypertrophic actions of several hormones.     

Epac activation converts cAMP from a proliferative into a differentiation signal in PC12 cells

Elevation of the intracellular cAMP concentration ([cAMP]i) regulates metabolism, cell proliferation, and differentiation and plays roles in memory formation and neoplastic growth. cAMP mediates its effects mainly through activation of protein kinase A (PKA) as well as Epac1 and Epac2, exchange factors activating the small GTPases Rap1 and Rap2. However, how cAMP utilizes these effectors to induce distinct biological responses is unknown. We here studied the specific roles of PKA and Epac in neuroendocrine PC12 cells. In these cells, elevation of [cAMP]i activates extracellular signal-regulated kinase (ERK) 1/2 and induces low-degree neurite outgrowth. The present study showed that specific stimulation of PKA triggered ERK1/2 activation that was considerably more transient than that observed upon simultaneous activation of both PKA and Epac. Unexpectedly, the PKA-specific cAMP analog induced cell proliferation rather than neurite outgrowth. The proliferative signaling pathway activated by the PKA-specific cAMP analog involved activation of the epidermal growth factor receptor and ERK1/2. Activation of Epac appeared to extend the duration of PKA-dependent ERK1/2 activation and converted cAMP from a proliferative into an anti-proliferative, neurite outgrowth-promoting signal. Thus, the present study showed that the outcome of cAMP signaling can depend heavily on the set of cAMP effectors activated.     

Differential signaling of cyclic AMP: opposing effects of exchange protein directly activated by cyclic AMP and cAMP-dependent protein kinase on protein kinase B activation.

The recent discovery of Epac, a novel cAMP receptor protein, opens up a new dimension in studying cAMP-mediated cell signaling. It is conceivable that many of the cAMP functions previously attributed to cAMP-dependent protein kinase (PKA) are in fact also Epac-dependent. The finding of an additional intracellular cAMP receptor provides an opportunity to further dissect the divergent roles that cAMP exerts in different cell types. In this study, we probed cross-talk between cAMP signaling and the phosphatidylinositol 3-kinase/PKB pathways. Specifically, we examined the modulatory effects of cAMP on PKB activity by monitoring the specific roles that Epac and PKA play individually in regulating PKB activity. Our study suggests a complex regulatory scheme in which Epac and PKA mediate the opposing effects of cAMP on PKB regulation. Activation of Epac leads to a phosphatidylinositol 3-kinase-dependent PKB activation, while stimulation of PKA inhibits PKB activity. Furthermore, activation of PKB by Epac requires the proper subcellular targeting of Epac. The opposing effects of Epac and PKA on PKB activation provide a potential mechanism for the cell type-specific differential effects of cAMP. It is proposed that the net outcome of cAMP signaling is dependent upon the dynamic abundance and distribution of intracellular Epac and PKA.     

Cyclic AMP induces integrin-mediated cell adhesion through Epac and Rap1 upon stimulation of the beta 2-adrenergic receptor

cAMP controls many cellular processes mainly through the activation of protein kinase A (PKA). However, more recently PKA-independent pathways have been established through the exchange protein directly activated by cAMP (Epac), a guanine nucleotide exchange factor for the small GTPases Rap1 and Rap2. In this report, we show that cAMP can induce integrin-mediated cell adhesion through Epac and Rap1. Indeed, when Ovcar3 cells were treated with cAMP, cells adhered more rapidly to fibronectin. This cAMP effect was insensitive to the PKA inhibitor H-89. A similar increase was observed when the cells were transfected with Epac. Both the cAMP effect and the Epac effect on cell adhesion were abolished by the expression of Rap1-GTPase-activating protein, indicating the involvement of Rap1 in the signaling pathway. Importantly, a recently characterized cAMP analogue, 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate, which specifically activates Epac but not PKA, induced Rap-dependent cell adhesion. Finally, we demonstrate that external stimuli of cAMP signaling, i.e., isoproterenol, which activates the G alpha s-coupled beta 2-adrenergic receptor can induce integrin-mediated cell adhesion through the Epac-Rap1 pathway. From these results we conclude that cAMP mediates receptor-induced integrin-mediated cell adhesion to fibronectin through the Epac-Rap1 signaling pathway.     

Compartmentation of cAMP signaling in cardiac myocytes: a computational study

Receptor-mediated changes in cAMP production play an essential role in sympathetic and parasympathetic regulation of the electrical, mechanical, and metabolic activity of cardiac myocytes. However, responses to receptor activation cannot be easily ascribed to a uniform increase or decrease in cAMP activity throughout the entire cell. In this study, we used a computational approach to test the hypothesis that in cardiac ventricular myocytes the effects of beta(1)-adrenergic receptor (beta(1)AR) and M(2) muscarinic receptor (M(2)R) activation involve compartmentation of cAMP. A model consisting of two submembrane (caveolar and extracaveolar) microdomains and one bulk cytosolic domain was created using published information on the location of beta(1)ARs and M(2)Rs, as well as the location of stimulatory (G(s)) and inhibitory (G(i)) G-proteins, adenylyl cyclase isoforms inhibited (AC5/6) and stimulated (AC4/7) by G(i), and multiple phosphodiesterase isoforms (PDE2, PDE3, and PDE4). Results obtained with the model indicate that: 1), bulk basal cAMP can be high ( approximately 1 microM) and only modestly stimulated by beta(1)AR activation ( approximately 2 microM), but caveolar cAMP varies in a range more appropriate for regulation of protein kinase A ( approximately 100 nM to approximately 2 microM); 2), M(2)R activation strongly reduces the beta(1)AR-induced increases in caveolar cAMP, with less effect on bulk cAMP; and 3), during weak beta(1)AR stimulation, M(2)R activation not only reduces caveolar cAMP, but also produces a rebound increase in caveolar cAMP following termination of M(2)R activity. We conclude that compartmentation of cAMP can provide a quantitative explanation for several aspects of cardiac signaling.     

Rap1-mediated activation of extracellular signal-regulated kinases by cyclic AMP is dependent on the mode of Rap1 activation

Like other small G proteins of the Ras superfamily, Rap1 is activated by distinct guanine nucleotide exchange factors (GEFs) in response to different signals to elicit cellular responses. Activation of Rap1 by cyclic AMP (cAMP) can occur via cAMP-dependent protein kinase A (PKA)-independent and PKA-dependent mechanisms. PKA-independent activation of Rap1 by cAMP is mediated by direct binding of cAMP to Rap1-guanine nucleotide exchange factors (Rap1-GEFs) Epac1 (exchange protein directly activated by cAMP 1) and Epac2 (Epac1 and Epac2 are also called cAMP-GEFI and -GEFII). The availability of cAMP analogues that selectively activate Epacs, but not PKA, provides a specific tool to activate Rap1. It has been argued that the inability of these analogues to regulate extracellular signal-regulated kinases (ERKs) signaling despite activating Rap1 provides evidence that Rap1 is incapable of regulating ERKs. We confirm that the PKA-independent activation of Rap1 by Epac1 activates a perinuclear pool of Rap1 and that this does not result in ERK activation. However, we demonstrate that this inability to regulate ERKs is not a property of Rap1 but is rather a property of Epacs themselves. The addition of a membrane-targeting motif to Epac1 (Epac-CAAX) relocalizes Epac1 from its normal perinuclear locale to the plasma membrane. In this new locale it is capable of activating ERKs in a Rap1- and cAMP-dependent manner. Rap1 activation by Epac-CAAX, but not wild-type Epac, triggers its association with B-Raf. Therefore, we propose that its intracellular localization prevents Epac1 from activating ERKs. C3G (Crk SH3 domain Guanine nucleotide exchanger) is a Rap1 exchanger that is targeted to the plasma membrane upon activation. We show that C3G can be localized to the plasma membrane by cAMP/PKA, as can Rap1 when activated by cAMP/PKA. Using a small interfering RNA approach, we demonstrate that C3G is required for the activation of ERKs and Rap1 by cAMP/PKA. This activation requires the GTP-dependent association of Rap1 with B-Raf. These data demonstrate that B-Raf is a physiological target of Rap1, but its utilization as a Rap1 effector is GEF specific. We propose a model that specific GEFs activate distinct pools of Rap1 that are differentially coupled to downstream effectors.     

Radixin assembles cAMP effectors Epac and PKA into a functional cAMP compartment: role in cAMP-dependent cell proliferation

cAMP is an ubiquitous second messenger. Localized areas with high cAMP concentration, i.e. cAMP microdomains, provide an elegant mechanism to generate signaling specificity and transduction efficiency. However, the mechanisms underlying cAMP effector targeting into these compartments is still unclear. Here we report the identification of radixin as a scaffolding unit for both cAMP effectors, Epac and PKA. This complex localizes in a submembrane compartment where cAMP synthesis occurs. Compartment disruption by shRNA and dominant negative approaches negatively affects cAMP action. Inhibition can be rescued by expression of Rap1b, a substrate for both Epac1 and PKA, but only in its GTP-bound and phosphorylated state. We propose that radixin scaffolds both cAMP effectors in a functional cAMP-sensing compartment for efficient signal transduction, using Rap1 as a downstream signal integrator.     

Cooperation of Epac1/Rap1/Akt and PKA in prostaglandin E(2) -induced proliferation of human umbilical cord blood derived mesenchymal stem cells: Involvement of c-Myc and VEGF expression

Prostaglandin E₂ (PGE₂) is well known to regulate cell functions through cAMP; however, the role of exchange protein directly activated by cAMP (Epac1) and protein kinase A (PKA) in modulating such functions is unknown in human umbilical cord blood‐derived mesenchymal stem cells (hUCB‐MSCs). Therefore, we investigated the relationship between Epac1 and PKA during PGE₂‐induced hUCB‐MSC proliferation and its related signaling pathways. PGE₂ increased cell proliferation, and E‐type prostaglandin (EP) 2 receptor mRNA expression level and activated cAMP generation, which were blocked by EP2 receptor selective antagonist AH 6809. PGE₂ increased Epac1 expression, Ras‐related protein 1 (Rap1) activation level, and Akt phosphorylation, which were inhibited by AH 6809, adenylyl cyclase inhibitor SQ 22536, and Epac1/Rap1‐specific siRNA. Also, PGE₂ increased PKA activity, which was inhibited by AH 6809, SQ 22536, and PKA inhibitor PKI. HUCB‐MSCs were incubated with the Epac agonist 8‐pCPT‐cAMP or the PKA agonist 6‐phe‐cAMP to examine whether Epac1/Rap1/Akt activation was independent of PKA activation. 8‐pCPT‐cAMP increased Akt phosphorylation but not PKA activity. 6‐Phe‐cAMP increased PKA activity, but not Akt phosphorylation. Additionally, an Akt inhibitor or PKA inhibitor (PKI) did not block the PGE₂‐induced increase in PKA activity or Akt phosphorylation, respectively. Moreover, PGE₂ increased glycogen synthase kinase (GSK)‐3β phosphorylation and nuclear translocation of active‐β‐catenin, which were inhibited by Akt inhibitor or/and PKI. PGE₂ increased c‐Myc and vascular endothelial growth factor (VEGF) expression levels, which were blocked by β‐catenin siRNA. In conclusion, PGE₂ stimulated hUCB‐MSC proliferation through β‐catenin‐mediated c‐Myc and VEGF expression via Epac/Rap1/Akt and PKA cooperation. J. Cell. Physiol. 227: 3756–3767, 2012. © 2012 Wiley Periodicals, Inc.     

Molecular determinants and feedback circuits regulating type 2 CRH receptor signal integration

In most target tissues, the adenylyl cyclase/cAMP/PKA, the extracellular signal regulated kinase and the protein kinase B/Akt are the main pathways employed by the type 2 corticotropin-releasing hormone receptor to mediate the biological actions of urocortins (Ucns) and CRH. To decipher the molecular determinants of CRH-R2 signaling, we studied the signaling pathways in HEK293 cells overexpressing recombinant human CRH-R2β receptors. Use of specific kinase inhibitors showed that the CRH-R2β cognate agonist, Ucn 2, activated extracellular signal regulated kinase in a phosphoinositide 3-kinase and cyclic adenosine monophosphate/PKA-dependent manner with contribution from Epac activation. Ucn 2 also induced PKA-dependent association between AKAP250 and CRH-R2β that appeared to be necessary for extracellular signal regulated kinase activation. PKB/Akt activation was also mediated via pertussis toxin-sensitive G-proteins and PI3-K activation but did not require cAMP/PKA, Epac or protein kinase C for optimal activation. Potential feedback mechanisms that target the CRH-R2β itself and modulate receptor trafficking and endocytosis were also investigated. Indeed, our results suggested that inhibition of either PKA or extracellular signal regulated kinase pathway accelerates CRH-R2β endocytosis. Furthermore, Ucn 2-activated extracellular signal regulated kinase appeared to target β-arrestin1 and modulate, through phosphorylation at Ser412, β-arrestin1 translocation to the plasma membrane and CRH-R2β internalization kinetics. Loss of this “negative feedback” mechanism through inhibition of the extracellular signal regulated kinase activity resulted in significant attenuation of Ucn 2-induced cAMP response, whereas Akt phosphorylation was not affected by altered receptor endocytosis. These findings reveal a complex interplay between the signaling molecules that allow “fine-tuning” of CRH-R2β functional responses and regulate signal integration.     

Dissecting the mechanism of Epac activation via hydrogen-deuterium exchange FT-IR and structural modeling

Exchange proteins directly activated by cAMP (Epac) make up a family of cAMP binding domain-containing proteins that play important roles in mediating the effects of cAMP through the activation of downstream small GTPases, Ras-proximate proteins. To delineate the mechanism of Epac activation, we probed the conformation and structural dynamics of Epac using amide hydrogen-deuterium (H-D) exchange coupled with Fourier transform infrared spectroscopy (FT-IR) and structural modeling. Our studies show that unlike that of cAMP-dependent protein kinase (PKA), the classic intracellular cAMP receptor, binding of cAMP to Epac does not induce significant changes in overall secondary structure and structural dynamics, as measured by FT-IR and the rate of H-D exchange, respectively. These results suggest that Epac activation does not involve significant changes in the amount of exposed surface areas as in the case of PKA activation, and conformational changes induced by cAMP in Epac are most likely confined to small local regions. Homology modeling and comparative structural analyses of the CBDs of Epac and PKA lead us to propose a model of Epac activation. On the basis of our model, Epac activation by cAMP employs the same underlying structural principal utilized by PKA, although the detailed structural and conformational changes associated with Epac and PKA activation are significantly different. In addition, we predict that during Epac activation the first beta-strand of the switchboard switches its conformation to a alpha-helix, which folds back to the beta-barrel core of the CBD and interacts directly with cAMP to form the base of the cAMP-binding pocket.     

PKA and Epac1 regulate endothelial integrity and migration through parallel and independent pathways

The vascular endothelium provides a semi-permeable barrier, which restricts the passage of fluid, macromolecules and cells to the surrounding tissues. Cyclic AMP promotes endothelial barrier function and protects the endothelium against pro-inflammatory mediators. This study analyzed the relative contribution of two cAMP targets, PKA and Epac1, to the control of endothelial barrier function and endothelial cell migration. Real-time recording of transendothelial electrical resistance showed that activation of either PKA or Epac1 with specific cAMP analogues increases endothelial barrier function and promotes endothelial cell migration. In addition, reduction of Epac1 expression showed that Epac1 and PKA control endothelial integrity and cell motility by two independent and complementary signaling pathways. We demonstrate that integrin-mediated adhesion is required for PKA, but not Epac1-Rap1-driven stimulation of endothelial barrier function. In contrast, both PKA- and Epac1-stimulated endothelial cell migration requires integrin function. These data show that activation of Epac1 and PKA by cAMP results in the stimulation of two parallel, independent signaling pathways that positively regulate endothelial integrity and cell migration, which is important for recovery after endothelial damage and for restoration of compromised endothelial barrier function.     

Beta-adrenergic- and muscarinic receptor-induced changes in cAMP activity in adult cardiac myocytes detected with FRET-based biosensor

-Adrenergic receptor activation regulates cardiac myocyte function through the stimulation of cAMP production and subsequent activation of protein kinase A (PKA). Furthermore, muscarinic receptor activation inhibits as well as facilitates these cAMP-dependent effects. However, it has not always been possible to correlate the muscarinic responses with the direct measurement of changes in cellular cAMP activity. Genetically encoded biosensors have recently been developed, making it possible to monitor real-time changes in cAMP and PKA activity at the single cell level. One such biosensor consists of the regulatory and catalytic subunits of PKA labeled with cyan and yellow fluorescent proteins, respectively. Changes in cAMP activity affecting the association of these labeled PKA subunits can be detected as changes in fluorescence resonance energy transfer. In the present study, an adenovirus-based approach was developed to express this recombinant protein complex in adult cardiac myocytes and use it to monitor changes in cAMP activity produced by -adrenergic and muscarinic receptor activation. The biosensor expressed with the use of this system is able to detect changes in cAMP activity produced by physiologically relevant levels of -adrenergic receptor activation without disrupting normal functional responses. It was also possible to directly demonstrate the complex temporal pattern of inhibitory and stimulatory changes in cAMP activity produced by muscarinic receptor activation in these cells. The adenovirus-based approach we have developed should facilitate the use of this biosensor in studying cAMP and PKA-dependent signaling mechanisms in a wide variety of cell types. adenovirus; protein kinase A; phosphodiesterase; L-type Ca²⁺ channel     

cAMP effector mechanisms. Novel twists for an 'old' signaling system

Cyclic AMP (cAMP) has traditionally been thought to act exclusively through cAMP-dependent protein kinase (cAPK, PKA), but a growing number of cAMP effects are not attributable to general activation of cAPK. At present, cAMP is known also to directly regulate ion channels and the ubiquitous Rap guanine exchange factors Epac 1 and 2. Adding to the sophistication of cAMP signaling is the fact that (1) the cAPK holoenzyme is incompletely dissociated even at saturating cAMP, the level of free R subunit of cAPK being able to regulate the maximal activity of cAPK, (2) cAPK activity can be modulated by oxidative glutathionylation, and (3) cAPK is anchored close to relevant substrates, other signaling enzymes, and local compartments of cAMP. Finally, we will demonstrate an example of fine-tuning of cAMP signaling through synergistic induction of neurite extensions by cAPK and Epac.     

Conformational analysis of Epac activation using amide hydrogen/deuterium exchange mass spectrometry

Exchange proteins directly activated by cAMP (Epac) play important roles in mediating the effects of cAMP through the activation of downstream small GTPases, Rap. To delineate the mechanism of Epac activation, we probed the conformation and structural dynamics of Epac using amide hydrogen/deuterium exchange and structural modeling. Our studies show that cAMP induces significant conformational changes that lead to a spatial rearrangement of the regulatory components of Epac and allows the exposure of the catalytic core for effector binding without imposing significant conformational change on the catalytic core. Homology modeling and comparative structural analyses of the cAMP binding domains of Epac and cAMP-dependent protein kinase (PKA) lead to a model of Epac activation, in which Epac and PKA activation by cAMP employs the same underlying principle, although the detailed structural and conformational changes associated with Epac and PKA activation are significantly different.     

Cyclic AMP-dependent protein kinase and Epac mediate cyclic AMP responses in pancreatic acini

The pancreatic acinar cell has several phenotypic responses to cAMP agonists. At physiological concentrations of the muscarinic agonist carbachol (1 microM) or the CCK analog caerulein (100 pM), ligands that increase cytosolic Ca(2+), cAMP acts synergistically to enhance secretion. Supraphysiological concentrations of carbachol (1 mM) or caerulein (100 nM) suppress secretion and cause intracellular zymogen activation; cAMP enhances both zymogen activation and reverses the suppression of secretion. In addition to stimulating cAMP-dependent protein kinase (PKA), recent studies using cAMP analogs that lack a PKA response have shown that cAMP can also act through the cAMP-binding protein, Epac (exchange protein directly activated by cyclic AMP). The roles of PKA and Epac in cAMP responses were examined in isolated pancreatic acini. The activation of both cAMP-dependent pathways or the selective activation of Epac was found to enhance amylase secretion induced by physiological and supraphysiological concentrations of the muscarinic agonist carbachol. Similarly, activation of both PKA or the specific activation of Epac enhanced carbachol-induced activation of trypsinogen and chymotrypsinogen. Disorganization of the apical actin cytoskeleton has been linked to the decreased secretion observed with supraphysiological concentrations of carbachol and caerulein. Although stimulation of PKA and Epac or Epac alone could largely overcome the decreased secretion observed with either supraphysiological carbachol or caerulein, stimulation of cAMP pathways did not reduce the disorganization of the apical cytoskeleton. These studies demonstrate that PKA and Epac pathways are coupled to both secretion and zymogen activation in the pancreatic acinar cell.     

Neuronal AKAP150 coordinates PKA and Epac-mediated PKB/Akt phosphorylation

In diverse neuronal processes ranging from neuronal survival to synaptic plasticity cyclic adenosine monophosphate (cAMP)-dependent signaling is tightly connected with the protein kinase B (PKB)/Akt pathway but the precise nature of this connection remains unknown. In the current study we investigated the effect of two mainstream pathways initiated by cAMP, cAMP-dependent protein kinase (PKA) and exchange proteins directly activated by cAMP (Epac1 and Epac2) on PKB/Akt phosphorylation in primary cortical neurons and HT-4 cells. We demonstrate that PKA activation leads to a reduction of PKB/Akt phosphorylation, whereas activation of Epac has the opposite effect. This effect of Epac on PKB/Akt phosphorylation was mediated by Rap activation. The increase in PKB/Akt phosphorylation after Epac activation could be blocked by pretreatment with Epac2 siRNA and to a somewhat smaller extent by Epac1 siRNA. PKA, PKB/Akt and Epac were all shown to establish complexes with neuronal A-kinase anchoring protein150 (AKAP150). Interestingly, activation of Epac increased phosphorylation of PKB/Akt complexed to AKAP150. From experiments using PKA-binding deficient AKAP150 and peptides disrupting PKA anchoring to AKAPs, we conclude that AKAP150 acts as a key regulator in the two cAMP pathways to control PKB/Akt phosphorylation.     

Adenosine A2A receptor (A2AR) is a fine-tune regulator of the collagen1:collagen3 balance

Adenosine is a potent endogenous anti-inflammatory and immunosuppressive metabolite that is a potent modulator of tissue repair. However, the adenosine A_2A receptor (A_2AR)-mediated promotion of collagen synthesis is detrimental in settings such as scarring and scleroderma. The signaling cascade from A_2AR 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 A_2AR-stimulated signaling for collagen production by normal human dermal fibroblasts (NHDF). Collagen1 (Col1) and collagen3 (Col3) content after A_2AR 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-Cα, -Cβ, -Cγ, Epac1, and Epac2. CGS21680 stimulates Col1 expression at significantly lower concentrations than those required to stimulate Col3 expression. A_2AR stimulates Col1 expression by a PKA-dependent mechanism since PKA inhibition or PKA-Cα and -Cβ knockdown prevents A_2AR-mediated Col1 increase. In contrast, A_2AR represses Col3 via PKA but stimulates both Col1 and Col3 via an Epac2-dependent mechanism. A_2AR stimulation with CGS21680 at 0.1 μM increased Col3 expression only upon PKA blockade. A_2AR 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.     

Cyclic AMP (cAMP)-mediated stimulation of adipocyte differentiation requires the synergistic action of Epac- and cAMP-dependent protein kinase-dependent processes

Cyclic AMP (cAMP)-dependent processes are pivotal during the early stages of adipocyte differentiation. We show that exchange protein directly activated by cAMP (Epac), which functions as a guanine nucleotide exchange factor for the Ras-like GTPases Rap1 and Rap2, was required for cAMP-dependent stimulation of adipocyte differentiation. Epac, working via Rap, acted synergistically with cAMP-dependent protein kinase (protein kinase A [PKA]) to promote adipogenesis. The major role of PKA was to down-regulate Rho and Rho-kinase activity, rather than to enhance CREB phosphorylation. Suppression of Rho-kinase impaired proadipogenic insulin/insulin-like growth factor 1 signaling, which was restored by activation of Epac. This interplay between PKA and Epac-mediated processes not only provides novel insight into the initiation and tuning of adipocyte differentiation, but also demonstrates a new mechanism of cAMP signaling whereby cAMP uses both PKA and Epac to achieve an appropriate cellular response.