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.     

Induction of leptin resistance by activation of cAMP-Epac signaling

Leptin regulates energy balance and glucose homeostasis. Shortly after leptin was identified, it was established that obesity is commonly associated with leptin resistance, though the molecular mechanisms remain to be identified. To explore potential mechanisms of leptin resistance, we employed organotypic brain slices to identify candidate signaling pathways that negatively regulate leptin sensitivity. We found that elevation of adenosine 3', 5'-monophosphate (cAMP) levels impairs multiple signaling cascades activated by leptin within the hypothalamus. Notably, this effect is independent of protein kinase A activation. In contrast, activation of Epac, a cAMP-regulated guanine nucleotide exchange factor for the small G protein Rap1, was sufficient to impair leptin signaling with concomitant induction of SOCS-3 expression. Epac activation also blunted leptin-induced depolarization of hypothalamic POMC neurons. Finally, central infusion of an Epac activator blunted the anorexigenic actions of leptin. Thus, activation of hypothalamic cAMP-Epac pathway is sufficient to induce multiple indices of leptin resistance.     

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.     

A transforming growth factor beta-induced Smad3/Smad4 complex directly activates protein kinase A

Transforming growth factor beta (TGFbeta) interacts with cell surface receptors to initiate a signaling cascade critical in regulating growth, differentiation, and development of many cell types. TGFbeta signaling involves activation of Smad proteins which directly regulate target gene expression. Here we show that Smad proteins also regulate gene expression by using a previously unrecognized pathway involving direct interaction with protein kinase A (PKA). PKA has numerous effects on growth, differentiation, and apoptosis, and activation of PKA is generally initiated by increased cellular cyclic AMP (cAMP). However, we found that TGFbeta activates PKA independent of increased cAMP, and our observations support the conclusion that there is formation of a complex between Smad proteins and the regulatory subunit of PKA, with release of the catalytic subunit from the PKA holoenzyme. We also found that the activation of PKA was required for TGFbeta activation of CREB, induction of p21(Cip1), and inhibition of cell growth. Taken together, these data indicate an important and previously unrecognized interaction between the TGFbeta and PKA signaling pathways.     

Globular adiponectin decreases leptin-induced tumor necrosis factor-alpha expression by murine macrophages: involvement of cAMP-PKA and MAPK pathways

Several lines of evidence have supported a link between obesity and inflammation. The present study investigated the capacity of leptin and globular adiponectin to affect tumor necrosis factor alpha (TNF-alpha) production in murine peritoneal macrophages. Leptin stimulated TNF-alpha production at mRNA as well as protein levels in a dose- and time-dependent manner. Intracellular cAMP concentration was increased and protein kinase A (PKA) was activated with the treatment of leptin, subsequently downstream MAPK signal proteins, ERK1/2 and p38, were phosphorylated. Specific inhibitors for the signal proteins, Rp cAMPS, H89, PD98059, and U0126, or SB203580, suppressed the signaling pathway and TNF-alpha expression. Although gAd partially increased cAMP concentration and PKA activity, it directly reduced leptin-induced ERK1/2 and p38 MAPK phosphorylation thus inhibiting TNF-alpha production. In conclusion, leptin promotes inflammation by stimulating TNF-alpha production, which is mediated by cAMP-PKA-ERK1/2 and p38 MAPK pathways. gAd inhibited leptin-induced TNF-alpha production through suppressing phosphorylation of ERK1/2 and p38 pathways.     

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.     

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.     

Epac-selective cAMP analog 8-pCPT-2'-O-Me-cAMP as a stimulus for Ca2+-induced Ca2+ release and exocytosis in pancreatic beta-cells

The second messenger cAMP exerts powerful stimulatory effects on Ca(2+) signaling and insulin secretion in pancreatic beta-cells. Previous studies of beta-cells focused on protein kinase A (PKA) as a downstream effector of cAMP action. However, it is now apparent that cAMP also exerts its effects by binding to cAMP-regulated guanine nucleotide exchange factors (Epac). Although one effector of Epac is the Ras-related G protein Rap1, it is not fully understood what the functional consequences of Epac-mediated signal transduction are at the cellular level. 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3'-5'-cyclic monophosphate (8-pCPT-2'-O-Me-cAMP) is a newly described cAMP analog, and it activates Epac but not PKA. Here we demonstrate that 8-pCPT-2'-O-Me-cAMP acts in human pancreatic beta-cells and INS-1 insulin-secreting cells to mobilize Ca(2+) from intracellular Ca(2+) stores via Epac-mediated Ca(2+)-induced Ca(2+) release (CICR). The cAMP-dependent increase of [Ca(2+)](i) that accompanies CICR is shown to be coupled to exocytosis. We propose that the interaction of cAMP and Epac to trigger CICR explains, at least in part, the blood glucose-lowering properties of an insulinotropic hormone (glucagon-like peptide-1, also known as GLP-1) now under investigation for use in the treatment of type-2 diabetes mellitus.     

PDE4 inhibitor, roflumilast protects cardiomyocytes against NO-induced apoptosis via activation of PKA and Epac dual pathways

Myocyte apoptosis plays an important role in myocardial infarction and cAMP is crucial in the regulation of myocyte apoptosis. Phosphodiesterase-4 (PDE4) inhibitor blocks the hydrolysis of cAMP via inhibition of PDE4 and is attractive candidate for novel anti-inflammatory drugs. However, its function in cardiovascular diseases and cardiomyocyte apoptosis is unclear. Therefore, we investigated whether roflumilast, a PDE4 inhibitor, exerts protective effect against NO-induced apoptosis in both of H9c2 cells and neonatal rat cardiomyocytes (NRCMs), focusing on cAMP downstream molecules such as protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac). According to our data, intracellular cAMP was increased by roflumilast treatment in H9c2 cells and NRCMs. Roflumilast inhibited SNP-induced apoptosis and this effect was reversed by PKA specific inhibitor H-89 and KT-5720. In addition, PKA specific activator N(6)-benzoyladenosine 3',5-cyclic monophosphate (N(6)Bz-cAMP) mimicked the effects of roflumilast. CREB phosphorylation by roflumilast was also inhibited by H-89, indicating that roflumilast protects SNP-induced apoptosis via PKA-dependent pathway. Roflumilast increased Epac1/GTP-Rap1 and the protective effect was abolished by Epac1 siRNA transfection, demonstrating that Epac signaling was also involved in this protective response. In support, Epac specific activator 8-(4-chlrorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8CPT-2Me-cAMP) protected SNP-induced apoptosis. PI3K/Akt inhibitor LY294002 blocked roflumilast-induced Akt phosphorylation and protective effect. Furthermore, inhibition of Epac1 with siRNA had no effect on roflumilast-induced CREB phosphorylation, whereas inhibited Akt phosphorylation, implicating that Akt phosphorylation was regulated by Epac pathway. In addition, it was also observed that rolipram and cilomilast exert similar effects as roflumilast. In summary, our data indicate that roflumilast protects NO-induced apoptosis via both cAMP-PKA/CREB and Epac/Akt-dependent pathway. Our study suggests a possibility of PDE4 inhibitor roflumilast as a potential therapeutic agent against myocardial ischemia/reperfusion (I/R) injury.     

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.     

Constitutively active protein kinase A qualitatively mimics the effects of follicle-stimulating hormone on granulosa cell differentiation

Activation of the protein kinase A (PKA) signaling system is necessary for FSH-induced granulosa cell differentiation, but it is not known whether activation of PKA is sufficient to account for the complex pattern of gene expression that occurs during this process. We addressed this question by infecting granulosa cells with a lentiviral vector that directs the expression of a constitutively active mutant of PKA (PKA-CQR) and compared the cellular responses to PKA-CQR with cells stimulated by FSH. Expression of PKA-CQR in undifferentiated granulosa cells resulted in the induction of both estrogen and progesterone production in the absence of cAMP. The stimulatory effects of both PKA-CQR and FSH on estrogen and progesterone production were suppressed by the PKA inhibitor H-89 and were mimicked by PKA-selective cAMP agonists. mRNA levels for P450scc and 3beta-HSD were induced to a similar extent by FSH and PKA-CQR, whereas mRNA levels for P450arom and the LHr were induced to a greater extent by FSH. Microarray analysis of gene expression profiles revealed that the majority of genes appeared to be comparably regulated by FSH and PKA-CQR but that some genes appear to be induced to a greater extent by FSH than by PKA-CQR. These results indicate that the PKA signaling pathway is sufficient to account for the induction of most genes (as identified by microarray analysis), including those of the progesterone biosynthetic pathway during granulosa cell differentiation. However, optimal induction of aromatase, the LHr, and other genes by FSH appears to require activation of additional signaling pathways.     

Glucose Activates TORC2-Gad8 Protein via Positive Regulation of the cAMP/cAMP-dependent Protein Kinase A (PKA) Pathway and Negative Regulation of the Pmk1 Protein-Mitogen-activated Protein Kinase Pathway

The target of rapamycin (TOR) kinase belongs to the highly conserved eukaryotic family of phosphatidylinositol 3-kinase-related kinases. TOR proteins are found at the core of two evolutionary conserved complexes, known as TORC1 and TORC2. In fission yeast, TORC2 is dispensable for proliferation under optimal growth conditions but is required for starvation and stress responses. TORC2 has been implicated in a wide variety of functions; however, the signals that regulate TORC2 activity have so far remained obscure. TORC2 has one known direct substrate, the AGC kinase Gad8, which is related to AKT in human cells. Gad8 is phosphorylated by TORC2 at Ser-546 (equivalent to AKT Ser-473), leading to its activation. Here, we show that glucose is necessary and sufficient to induce Gad8 Ser-546 phosphorylation in vivo and Gad8 kinase activity in vitro. The glucose signal that activates TORC2-Gad8 is mediated via the cAMP/PKA pathway, a major glucose-sensing pathway. By contrast, Pmk1, similar to human extracellular signal-regulated kinases and a major stress-induced mitogen activated protein kinase (MAPK) in fission yeast, inhibits TORC2-dependent Gad8 phosphorylation and activation. Inhibition of TORC2-Gad8 also occurs in response to ionic or osmotic stress, in a manner dependent on the cAMP/PKA and Pmk1-MAPK signaling pathways. Our findings highlight the significance of glucose availability in regulation of TORC2-Gad8 and indicate a novel link between the cAMP/PKA, Pmk1/MAPK, and TORC2-Gad8 signaling.     

Sphingosine 1-phosphate induces MKP-1 expression via p38 MAPK- and CREB-mediated pathways in airway smooth muscle cells

Sphingosine 1-phosphate (S1P), a bioactive sphingolipid elevated in asthmatic airways, is increasingly recognized as playing an important role in respiratory disease. S1P activates receptor-mediated signaling to modulate diverse cellular functions and promote airway inflammation. Although many of the stimulatory pathways activated by S1P have been delineated, especially mitogen-activated protein kinases (MAPK), the question of whether S1P exerts negative feedback control on its own signaling cascade via upregulation of phosphatases remains unexplored. We show that S1P rapidly and robustly upregulates mRNA and protein expression of the MAPK deactivator-MAPK phosphatase 1 (MKP-1). Utilizing the pivotal airway structural cell, airway smooth muscle (ASM), we confirm that S1P activates all members of the MAPK family and, in part, S1P upregulates MKP-1 expression in a p38 MAPK-dependent manner. MKP-1 is a cAMP response element binding (CREB) protein-responsive gene and here, we reveal for the first time that an adenylate cyclase/PKA/CREB-mediated pathway also contributes to S1P-induced MKP-1. Thus, by increasing MKP-1 expression via parallel p38 MAPK- and CREB-mediated pathways, S1P temporally regulates MAPK signaling pathways by upregulating the negative feedback controller MKP-1. This limits the extent and duration of pro-inflammatory MAPK signaling and represses cytokine secretion in ASM cells. Taken together, our results demonstrate that S1P stimulates both kinases and the phosphatase MKP-1 to control inflammation in ASM cells and may provide a greater understanding of the molecular mechanisms responsible for the pro-asthmatic functions induced by the potent bioactive sphingolipid S1P in the lung.     

Critical role of cAMP-GEFII--Rim2 complex in incretin-potentiated insulin secretion

Incretins such as glucagon-like peptide-1 and gastric inhibitory polypeptide/glucose-dependent insulinotropic peptide are known to potentiate insulin secretion mainly through a cAMP/protein kinase A (PKA) signaling pathway in pancreatic beta-cells, but the mechanism is not clear. We recently found that the cAMP-binding protein cAMP-GEFII (or Epac 2), interacting with Rim2, a target of the small G protein Rab3, mediates cAMP-dependent, PKA-independent exocytosis in a reconstituted system. In the present study, we investigated the role of the cAMP-GEFII--Rim2 pathway in incretin-potentiated insulin secretion in native pancreatic beta-cells. Treatment of pancreatic islets with antisense oligodeoxynucleotides (ODNs) against cAMP-GEFII alone or with the PKA inhibitor H-89 alone inhibited incretin-potentiated insulin secretion approximately 50%, while a combination of antisense ODNs and H-89 inhibited the secretion approximately 80-90%. The effect of cAMP-GEFII on insulin secretion is mediated by Rim2 and depends on intracellular calcium as well as on cAMP. Treatment of the islets with antisense ODNs attenuated both the first and second phases of insulin secretion potentiated by the cAMP analog 8-bromo-cAMP. These results indicate that the PKA-independent mechanism involving the cAMP-GEFII--Rim2 pathway is critical in the potentiation of insulin secretion by incretins.     

Regulation of melanosome movement by MAP kinase

Our objectives were to further characterize the signaling pathways in melatonin-induced aggregation in Xenopus melanophores, specifically to investigate a possible role of mitogen-activated protein kinase (MAPK). By Western blotting we found that melatonin activates MAPK, which precedes melanosome aggregation measured in a microplate reader. Activation of MAPK, tyrosine phosphorylation of a previously described 280-kDa protein, and melanosome aggregation are sensitive to PD98059, a selective inhibitor of MAPK kinase. The MAPK activation is also decreased by the adenylate cyclase stimulant forskolin. In summary, we found that MAPK is activated during melatonin-induced melanosome aggregation. Activation was decreased by an inhibitor of MAPK kinase, and by forskolin. In addition to inhibition of cyclic adenosine 3',5'-monophosphate (cAMP), reduction in protein kinase A activity (PKA), and activation of protein phosphatase 2A, we suggest that melatonin receptors activate the MAPK cascade and tyrosine phosphorylation of the 280-kDa protein. Although the cAMP/PKA signaling pathway is the most prominent, our data suggest that simultaneous activation of the MAPK cascade is of importance to obtain a completely aggregated state. This new regulatory mechanism of organelle transport by the MAPK cascade might be important in other eukaryotic cells.     

Dual contradictory roles of cAMP signaling pathways in hydroxyl radical production in the rat striatum

Studies have suggested that cAMP signaling pathways may be associated with the production of reactive oxygen species. In this study, we examined how modifications in cAMP signaling affected the production of hydroxyl radicals in rat striatum using microdialysis to measure extracellular 2,3-dihydroxybenzoic acid (2,3-DHBA), which is a hydroxyl radical adduct of salicylate. Up to 50 nmol of the cell-permeative cAMP mimetic 8-bromo-cAMP (8-Br-cAMP) increased 2,3-DHBA in a dose-dependent manner (there was no additional increase in 2,3-DHBA at 100 nmol). Another cAMP mimetic, dibutyryl cAMP (db-cAMP), caused a nonsignificant increase in 2,3-DHBA at 50 nmol and a significant decrease at 100 nmol. Up to 20 nmol of forskolin, which is a direct activator of adenylyl cyclase, increased 2,3-DHBA, similar to the effect of 8-Br-cAMP; however, forskolin resulted in a much greater increase in 2,3-DHBA. A potent inhibitor of protein kinase A (PKA), H89 (500 μM), potentiated the 8-Br-cAMP- and forskolin-induced increases in 2,3-DHBA and antagonized the inhibitory effect of 100 nmol of db-cAMP. Interestingly, the administration of 100 nmol of 8-bromo-cGMP alone or in combination with H89 had no significant effect on 2,3-DHBA levels. Doses of 100 nmol of a preferential PKA activator (6-phenyl-cAMP) or a preferential PKA inhibitor (8-bromoadenosine-3',5'-cyclic monophosphorothionate, Rp-isomer; Rp-8-Br-cAMPS), which also inhibits the cAMP-mediated activation of Epac (the exchange protein directly activated by cAMP), suppressed or enhanced, respectively, the formation of 2,3-DHBA. Up to 100 nmol of 8-(4-chlorophenylthio)-2'-O-methyladenosine-cAMP, which is a selective activator of Epac, dose-dependently stimulated the formation of 2,3-DHBA. These findings suggest that cAMP signaling plays contradictory roles (stimulation and inhibition) in the production of hydroxyl radicals in rat striatum by differential actions of Epac and PKA. These roles might contribute to the production of hydroxyl radicals concomitant with cAMP in carbon monoxide poisoning, because the formation of 2,3-DHBA was potentiated by the PKA inhibitor H89 and suppressed by Rp-8-Br-cAMPS, which inhibits PKA and Epac.