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.     

H-89 potentiates adipogenesis in 3T3-L1 cells by activating insulin signaling independently of protein kinase A

Among four kinds of protein kinase A (PKA) inhibitors tested, H-89 exhibited a unique action to remarkably enhance adipocyte differentiation of 3T3-L1 cells, whereas the other three PKA inhibitors, PKA inhibitor Fragment 14-22 (PKI), Rp-cAMP, and KT 5720, did not enhance adipocyte differentiation. H-85, which is an inactive form of H-89, exhibited a similar enhancing effect on adipocyte differentiation. H-89 also potentiated the phosphorylation of Akt and extracellular signal-regulated kinase (ERK) 1/2 in 3T3-L1 cells, which function as downstream signaling of insulin. Phosphoinositide 3-kinase (PI3K) inhibitor wortmannin and mitogen-activated protein kinase kinase (MEK) inhibitor PD 98059 suppressed both the H-89-induced promotion of adipocyte differentiation and the H-89-induced potentiation of phosphorylation of Akt and ERK1/2. Rho kinase inhibitor Y-27632 also promoted the phosphorylation of both Akt and ERK1/2 and enhanced adipocyte differentiation, although its effect was somewhat less than that of H-89. Even when cells were treated with a mixture of Y-27632 and H-89, the additive enhancing effects on both the insulin signaling and adipocyte differentiation were not detected. Therefore, it is suggested that the major possible mechanism whereby H-89 potentiates adipocyte differentiation of 3T3-L1 cells is activation of insulin signaling that is elicited mostly by inhibiting Rho/Rho kinase pathway.     

Activation of protein kinase A and exchange protein directly activated by cAMP promotes adipocyte differentiation of human mesenchymal stem cells

Human mesenchymal stem cells are primary multipotent cells capable of differentiating into several cell types including adipocytes when cultured under defined in vitro conditions. In the present study we investigated the role of cAMP signaling and its downstream effectors, protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac) in adipocyte conversion of human mesenchymal stem cells derived from adipose tissue (hMADS). We show that cAMP signaling involving the simultaneous activation of both PKA- and Epac-dependent signaling is critical for this process even in the presence of the strong adipogenic inducers insulin, dexamethasone, and rosiglitazone, thereby clearly distinguishing the hMADS cells from murine preadipocytes cell lines, where rosiglitazone together with dexamethasone and insulin strongly promotes adipocyte differentiation. We further show that prostaglandin I(2) (PGI(2)) may fully substitute for the cAMP-elevating agent isobutylmethylxanthine (IBMX). Moreover, selective activation of Epac-dependent signaling promoted adipocyte differentiation when the Rho-associated kinase (ROCK) was inhibited. Unlike the case for murine preadipocytes cell lines, long-chain fatty acids, like arachidonic acid, did not promote adipocyte differentiation of hMADS cells in the absence of a PPARγ agonist. However, prolonged treatment with the synthetic PPARδ agonist L165041 promoted adipocyte differentiation of hMADS cells in the presence of IBMX. Taken together our results emphasize the need for cAMP signaling in concert with treatment with a PPARγ or PPARδ agonist to secure efficient adipocyte differentiation of human hMADS mesenchymal stem cells.     

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.     

Epac proteins: multi-purpose cAMP targets

Epac1 and Epac2 are cAMP-dependent guanine-nucleotide-exchange factors for the small GTPases Rap1 and Rap2, and are known to be important mediators of cAMP signaling. The recent determination of the crystal structure of Epac2 has indicated a mechanism for the activation of the multi-domain Epac proteins. In addition, these proteins have been implicated in various cellular processes such as integrin-mediated cell adhesion and cell-cell junction formation, the control of insulin secretion and neurotransmitter release. In most of these processes, cAMP signaling through protein kinase A (PKA) is also involved, stressing the interconnectivity between Epac- and PKA-mediated signaling.     

Epac/Rap and PKA are novel mechanisms of ANP-induced Rac-mediated pulmonary endothelial barrier protection

Acute lung injury, sepsis, lung inflammation, and ventilator-induced lung injury are life-threatening conditions associated with lung vascular barrier dysfunction, which may lead to pulmonary edema. Increased levels of atrial natriuretic peptide (ANP) in lung circulation reported in these pathologies suggest its potential role in the modulation of lung injury. Besides well recognized physiological effects on vascular tone, plasma volume, and renal function, ANP may exhibit protective effects in models of lung vascular endothelial cell (EC) barrier dysfunction. However, the molecular mechanisms of ANP protective effects are not well understood. The recently described cAMP-dependent guanine nucleotide exchange factor (GEF) Epac activates small GTPase Rap1, which results in activation of small GTPase Rac-specific GEFs Tiam1 and Vav2 and Rac-mediated EC barrier protective responses. Our results show that ANP stimulated protein kinase A and the Epac/Rap1/Tiam/Vav/Rac cascade dramatically attenuated thrombin-induced pulmonary EC permeability and the disruption of EC monolayer integrity. Using pharmacological and molecular activation and inhibition of cAMP-and cGMP-dependent protein kinases (PKA and PKG), Epac, Rap1, Tiam1, Vav2, and Rac we linked ANP-mediated protective effects to the activation of Epac/Rap and PKA signaling cascades, which dramatically inhibited the Rho pathway of thrombin-induced EC hyper-permeability. These results suggest a novel mechanism of ANP protective effects against agonist-induced pulmonary EC barrier dysfunction via inhibition of Rho signaling by Epac/Rap1-Rac and PKA signaling cascades.     

Epac, in synergy with cAMP-dependent protein kinase (PKA), is required for cAMP-mediated mitogenesis

cAMP stimulates proliferation in many cell types. For many years, cAMP-dependent protein kinase (PKA) represented the only known cAMP effector. PKA, however, does not fully mimic the action of cAMP, indicating the existence of a PKA-independent component. Since cAMP-mediated activation of the G-protein Rap1 and its phosphorylation by PKA are strictly required for the effects of cAMP on mitogenesis, we hypothesized that the Rap1 activator Epac might represent the PKA-independent factor. Here we report that Epac acts synergistically with PKA in cAMP-mediated mitogenesis. We have generated a new dominant negative Epac mutant that revealed that activation of Epac is required for thyroid-stimulating hormone or cAMP stimulation of DNA synthesis. We demonstrate that Epac's action on cAMP-mediated activation of Rap1 and cAMP-mediated mitogenesis depends on the subcellular localization of Epac via its DEP domain. Disruption of the DEP-dependent subcellular targeting of Epac abolished cAMP-Epac-mediated Rap1 activation and thyroid-stimulating hormone-mediated cell proliferation, indicating that an Epac-Rap-PKA signaling unit is critical for the mitogenic action of cAMP.     

The cAMP-responsive Rap1 guanine nucleotide exchange factor, Epac, induces smooth muscle relaxation by down-regulation of RhoA activity

Agonist activation of the small GTPase, RhoA, and its effector Rho kinase leads to down-regulation of smooth muscle (SM) myosin light chain phosphatase activity, an increase in myosin light chain (RLC(20)) phosphorylation and force. Cyclic nucleotides can reverse this process. We report a new mechanism of cAMP-mediated relaxation through Epac, a GTP exchange factor for the small GTPase Rap1 resulting in an increase in Rap1 activity and suppression of RhoA activity. An Epac-selective cAMP analog, 8-pCPT-2'-O-Me-cAMP ("007"), significantly reduced agonist-induced contractile force, RLC(20), and myosin light chain phosphatase phosphorylation in both intact and permeabilized vascular, gut, and airway SMs independently of PKA and PKG. The vasodilator PGI(2) analog, cicaprost, increased Rap1 activity and decreased RhoA activity in intact SMs. Forskolin, phosphodiesterase inhibitor isobutylmethylxanthine, and isoproterenol also significantly increased Rap1-GTP in rat aortic SM cells. The PKA inhibitor H89 was without effect on the 007-induced increase in Rap1-GTP. Lysophosphatidic acid-induced RhoA activity was reduced by treatment with 007 in WT but not Rap1B null fibroblasts, consistent with Epac signaling through Rap1B to down-regulate RhoA activity. Isoproterenol-induced increase in Rap1 activity was inhibited by silencing Epac1 in rat aortic SM cells. Evidence is presented that cooperative cAMP activation of PKA and Epac contribute to relaxation of SM. Our findings demonstrate a cAMP-mediated signaling mechanism whereby activation of Epac results in a PKA-independent, Rap1-dependent Ca(2+) desensitization of force in SM through down-regulation of RhoA activity. Cyclic AMP inhibition of RhoA is mediated through activation of both Epac and PKA.     

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.     

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.     

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.     

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.     

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.     

Neuronal Cbl Controls Biosynthesis of Insulin-Like Peptides in Drosophila melanogaster

The Cbl family proteins function as both E3 ubiquitin ligases and adaptor proteins to regulate various cellular signaling events, including the insulin/insulin-like growth factor 1 (IGF1) and epidermal growth factor (EGF) pathways. These pathways play essential roles in growth, development, metabolism, and survival. Here we show that in Drosophila melanogaster, Drosophila Cbl (dCbl) regulates longevity and carbohydrate metabolism through downregulating the production of Drosophila insulin-like peptides (dILPs) in the brain. We found that dCbl was highly expressed in the brain and knockdown of the expression of dCbl specifically in neurons by RNA interference increased sensitivity to oxidative stress or starvation, decreased carbohydrate levels, and shortened life span. Insulin-producing neuron-specific knockdown of dCbl resulted in similar phenotypes. dCbl deficiency in either the brain or insulin-producing cells upregulated the expression of dilp genes, resulting in elevated activation of the dILP pathway, including phosphorylation of Drosophila Akt and Drosophila extracellular signal-regulated kinase (dERK). Genetic interaction analyses revealed that blocking Drosophila epidermal growth factor receptor (dEGFR)-dERK signaling in pan-neurons or insulin-producing cells by overexpressing a dominant-negative form of dEGFR abolished the effect of dCbl deficiency on the upregulation of dilp genes. Furthermore, knockdown of c-Cbl in INS-1 cells, a rat β-cell line, also increased insulin biosynthesis and glucose-stimulated secretion in an ERK-dependent manner. Collectively, these results suggest that neuronal dCbl regulates life span, stress responses, and metabolism by suppressing dILP production and the EGFR-ERK pathway mediates the dCbl action. Cbl suppression of insulin biosynthesis is evolutionarily conserved, raising the possibility that Cbl may similarly exert its physiological actions through regulating insulin production in β cells.     

Novel mechanisms of the regulation of protein kinase B in adipocytes; implications for protein kinase A, Epac, phosphodiesterases 3 and 4

Crosstalk between insulin and cAMP signalling pathways has a great impact on adipocyte metabolism. Whilst Protein kinase B (PKB) is a pivotal mediator of insulin action, in some cells regulation of PKB by cAMP has also been demonstrated. Here we provide evidence that, in a phosphatidyl inositol 3-kinase dependent manner, beta3-adrenergic stimulation (using CL316243) in adipocytes induces PKB phosphorylation in the absence of insulin and also potentiates insulin-induced phosphorylation of PKB. Interestingly, insulin- and CL316243-induced PKB phosphorylation was found to be inhibited by pools of cAMP controlled by PDE3B and PDE4 (mainly in the context of insulin), whereas a cAMP pool controlling protein kinase A appeared to mediate stimulation of PKB phosphorylation (mainly in the context of CL316243). Furthermore, an Epac (exchange protein directly activated by cAMP) agonist (8-pCPT-2'-O-Me-cAMP) mimicked the effect of the PDE inhibitors, giving evidence that Epac has an inhibitory effect on PKB phosphorylation in adipocytes. Further, we put the results obtained at the level of PKB in the context of possible downstream signalling components in the regulation of adipocyte metabolism. Thus, we found that overexpression of PKB induced lipogenesis in a PDE3B-dependent manner. Furthermore, overexpression or inhibition of PDE3B was associated with reduced or increased phosphorylation of the key lipogenic enzyme acetyl-CoA carboxylase (ACC), respectively. These PDE3B-dependent effects on ACC correlated with changes in lipogenesis. The Epac agonist, 8-pCPT-2'-O-Me-cAMP, mimicked the effect of PDE3B inhibition on ACC phosphorylation and lipogenesis.