Role of Epac and protein kinase A in thyrotropin-induced gene expression in primary thyrocytes

cAMP pathway activation by thyrotropin (TSH) induces differentiation and gene expression in thyrocytes. We investigated which partners of the cAMP cascade regulate gene expression modulations: protein kinase A and/or the exchange proteins directly activated by cAMP (Epac). Human primary cultured thyrocytes were analysed by microarrays after treatment with the adenylate cyclase activator forskolin, the protein kinase A (PKA) activator 6-MB-cAMP and the Epac-selective cAMP analog 8-pCPT-2'-O-Me-cAMP (007) alone or combined with 6-MB-cAMP. Profiles were compared to those of TSH. Cultures treated with the adenylate cyclase- or the PKA activator alone or the latter combined with 007 had profiles similar to those induced by TSH. mRNA profiles of 007-treated cultures were highly distinct from TSH-treated cells, suggesting that TSH-modulated gene expressions are mainly modulated by cAMP and PKA and not through Epac in cultured human thyroid cells. To investigate whether the Epac-Rap-RapGAP pathway could play a potential role in thyroid tumorigenesis, the mRNA expressions of its constituent proteins were investigated in two malignant thyroid tumor types. Modulations of this pathway suggest an increased Rap pathway activity in these cancers independent from cAMP activation.     

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.     

The cross talk between protein kinase A- and RhoA-mediated signaling in cancer cells

The cross talk between cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) and RhoA-mediated signal transductions and the effect of this cross talk on biologic features of human prostate and gastric cancer cells were investigated. In the human gastric cancer cell line, SGC-7901, lysophosphatidic acid (LPA) increased RhoA activity in a dose-dependent manner. The cellular permeable cAMP analog, 8-chlorophenylthio-cAMP (CPT-cAMP), inhibited the LPA-induced RhoA activation and caused phosphorylation of RhoA at serine(188). Immunofluorescence microscopy, Western blotting, and green fluorescent protein (GFP)-tagged RhoA location assay in live cells revealed that RhoA was distributed in both the cytoplasm and nucleus of SGC-7901 cells. Treatment with LPA and/or CPT-cAMP did not induce obvious translocation of RhoA in the cells. The LPA treatment caused formation of F-actin in SGC-7901 cells, and CPT-cAMP inhibited the formation. In a modified Boyden chamber assay, LPA stimulated the migration of SGC-7901 cells, and CPT-cAMP dose-dependently inhibited the stimulating effect of LPA. In soft agar assay, LPA stimulated early proliferation of SGC-7901 cells, and CPT-cAMP significantly inhibited the growth of LPA-stimulated cells. In the prostate cancer cell line, PC-3, LPA caused morphologic changes from polygonal to round, and transfection with plasmid DNA encoding constitutively active RhoA(63L) caused a similar change. Treatment with CPT-cAMP inhibited the changes in both cases. However, in PC-3 cells transfected with a plasmid encoding mutant RhoA188A, LPA induced rounding, but CPT-cAMP could not prevent the change. Results of this experiment indicated that cAMP/PKA inhibited RhoA activation, and serine188 phosphorylation on RhoA was necessary for PKA to exert its inhibitory effect on RhoA activation. The cross talk between cAMP/PKA and RhoA-mediated signal transductions had significant affect on biologic features of gastric and prostate cancer cells, such as morphologic and cytoskeletal change, migration, and anchorage-independent growth. The results may be helpful in implementing novel therapeutic strategies for invasive and metastatic prostate and gastric cancers.     

Activated 3',5'-cyclic AMP-dependent protein kinase is sufficient to induce neuroendocrine-like differentiation of the LNCaP prostate tumor cell line

Neuroendocrine (NE) differentiation within prostate tumors is proposed to be a contributing factor in disease progression. However, the cellular origin and molecular mechanism controlling differentiation of prostatic NE cells are unresolved. The prostate tumor cell line, LNCaP, can reversibly acquire many NE characteristics in response to treatment with beta-adrenergic receptor agonists and activators of adenylate cyclase. In this study, we demonstrate that these treatments induce protein kinase A (PKA) activation in LNCaP cells and that ectopic expression of a constitutively activated form of the PKA catalytic subunit, CIalpha, results in acquisition of NE characteristics, including the extension of neuritic processes, cessation of mitotic activity, and production of neuron-specific enolase. Forskolin-, epinephrine-, and isoproterenol-dependent NE differentiation of LNCaP cells was significantly inhibited by expressing a dominant negative mutant of the PKA regulatory subunit, RIalpha. These results demonstrate that prostatic NE differentiation in response to these agents depends on PKA activation, and this signaling pathway may provide a therapeutic target for treating advanced forms of prostate cancer.     

Protein kinase A (PKA) isoform RIIbeta mediates the synergistic killing effect of cAMP and glucocorticoid in acute lymphoblastic leukemia cells

Protein kinase A (PKA) or cAMP-dependent protein kinase (cAPK) mediates the synergistic effects of cAMP- and glucocorticoid (GC)-induced apoptosis in lymphoid cells. Using two human acute lymphoblastic leukemia cell (CEM) clones with respective GC-sensitive and GC-resistant phenotypes, we discovered that the PKA regulatory subunit isoform RII(beta) is preferentially expressed in the GC-sensitive clone C7-14 cells, whereas other intracellular cAMP receptors, including the exchange proteins directly activated by cAMP (Epac), are expressed at similar levels in both GC-sensitive and GC-resistant clones. High RII(beta) expression level in C7-14 cells is associated with elevated total PKA cellular activity and cAMP sensitivity, which consequently lead to an increased basal PKA activity. cAMP analogs that selectively activate type II PKA recapitulate the effects of forskolin of promoting apoptosis and antagonizing AKT/PKB activity in both GC-sensitive and GC-resistant clones, whereas type I PKA-selective agonists do not. Furthermore, down-regulation of RII(beta) leads to increased AKT/PKB activation and enhanced GC resistance in C7-14 cells. These results demonstrate that PKA RII(beta) is responsible for increased GC sensitivity, critical for cAMP-mediated synergistic cell killing in CEM cells, and may represent a novel therapeutic target for GC-resistant lymphoid malignancy.     

Opposing Roles of pka and epac in the cAMP-Dependent Regulation of Schwann Cell Proliferation and Differentiation

In Schwann cells (SCs), cyclic adenosine monophosphate (cAMP) not only induces differentiation into a myelinating SC-related phenotype, but also synergistically enhances the mitogenic action of growth factors such as neuregulin. To better understand the molecular mechanism by which cAMP exerts these apparently contradictory functions, we investigated the role of the two main effectors of cAMP, protein kinase A (PKA) and the exchange protein activated by cAMP (EPAC), on the proliferation and differentiation of both isolated and axon-related SCs. For these studies, a variety of PKA and EPAC agonists and antagonists were used, including pathway-selective analogs of cAMP and pharmacological inhibitors. Our studies indicated that the activity of PKA rather than EPAC was required for the adjuvant effect of cAMP on S-phase entry, whereas the activity of EPAC rather than PKA was required for SC differentiation and myelin formation. Even though selective EPAC activation had an overall anti-proliferative effect in SCs, it failed to drive the expression of Krox-20, a master regulator of myelination, and that of myelin-specific proteins and lipids, suggesting that EPAC activation was insufficient to drive a full differentiating response. Interestingly, inhibition of EPAC activity resulted in a drastic impairment of SC differentiation and myelin formation but not Krox-20 expression, which indicates an independent mechanism of Krox-20 regulation in response to cAMP. In conclusion, our data supports the idea that the outcome of cAMP signaling in SCs depends on the particular set of effectors activated. Whereas the mitogenic action of cAMP relies exclusively on PKA activity, the differentiating action of cAMP requires a PKA-independent (non-canonical) cAMP-specific pathway that is partially transduced by EPAC.     

cAMP-dependent protein kinase A and the dynamics of epithelial cell surface domains: moving membranes to keep in shape

Cyclic adenosine monophosphate (cAMP) and cAMP-dependent protein kinase A (PKA) are evolutionary conserved molecules with a well-established position in the complex network of signal transduction pathways. cAMP/PKA-mediated signaling pathways are implicated in many biological processes that cooperate in organ development including the motility, survival, proliferation and differentiation of epithelial cells. Cell surface polarity, here defined as the anisotropic organisation of cellular membranes, is a critical parameter for most of these processes. Changes in the activity of cAMP/PKA elicit a variety of effects on intracellular membrane dynamics, including membrane sorting and trafficking. One of the most intriguing aspects of cAMP/PKA signaling is its evolutionary conserved abundance on the one hand and its precise spatial-temporal actions on the other. Here, we review recent developments with regard to the role of cAMP/PKA in the regulation of intracellular membrane trafficking in relation to the dynamics of epithelial surface domains.     

The requirement of Ras and Rap1 for the activation of ERKs by cAMP, PACAP, and KCl in cerebellar granule cells

In cerebellar granule cells, the mitogen-activated protein kinase (MAPK) or extracellular signal-regulated kinase (ERK) cascade mediates multiple functions, including proliferation, differentiation, and survival. In these cells, ERKs are activated by diverse stimuli, including cyclic adenosine monophosphate (cAMP), pituitary adenylate cyclase activating protein (PACAP), depolarization induced by elevated extracellular potassium (KCl), and the neurotrophin brain-derived neurotrophic factor. Extensive studies in neuronal cell lines have implicated the small G proteins Ras and Rap1 in the activation of ERKs by cAMP, PACAP, and KCl. However, the requirement of Ras and Rap1 in these pathways in cerebellar granule cells has not been addressed. In this study, we utilize multiple biochemical assays to determine the mechanisms of action and requirement of Ras and Rap1 in cultured cerebellar granule cells. We show that both Ras and Rap1 can be activated by cAMP or PACAP via protein kinase (PKA)-dependent mechanisms. KCl activation of Ras also required PKA. Using both adenoviral and transgenic approaches, we show that Ras plays a major role in ERK activation by cAMP, PACAP, and KCl, while Rap1 also mediates activation of a selective membrane-associated pool of ERKs. Furthermore, Rap1, but not Ras, activation by PKA appears to require the action of Src family kinases.     

Coordinated regulation of Rap1 and thyroid differentiation by cyclic AMP and protein kinase A

Originally identified as an antagonist of Ras action, Rap1 exhibits many Ras-independent effects, including a role in signaling pathways initiated by cyclic AMP (cAMP). Since cAMP is a critical mediator of the effects of thyrotropin (TSH) on cell proliferation and differentiation, we examined the regulation of Rap1 by TSH in a continuous line of rat thyroid-like cells. Both cAMP and protein kinase A (PKA) contribute to the regulation of Rap1 activity and signaling by TSH. TSH activates Rap1 through a cAMP-mediated and PKA-independent mechanism. TSH phosphorylates Rap1 in a PKA-dependent manner. Interference with PKA activity blocked phosphorylation but not the activation of Rap1. Rather, PKA inhibitors prolonged Rap1 activation, as did expression of a Rap1A mutant lacking a PKA phosphorylation site. These results indicate that PKA elicits negative feedback regulation on cAMP-stimulated Rap1 activity in some cells. The dual regulation of Rap1 by cAMP and PKA extends to downstream effectors. The ability of TSH to stimulate Akt phosphorylation was markedly enhanced by the expression of activated Rap1A and was repressed in cells expressing a putative dominant-negative Rap1A mutant. Although the expression of activated Rap1A was sufficient to stimulate wortmannin-sensitive Akt phosphorylation, TSH further increased Akt phosphorylation in a phosphatidylinositol 3-kinase- and PKA-dependent manner. The ability of TSH to phosphorylate Akt was impaired in cells expressing a Rap1A mutant that could be activated but not phosphorylated. These findings indicate that dual signals, Rap1 activation and phosphorylation, contribute to TSH-stimulated Akt phosphorylation. Rap1 plays an essential role in cAMP-regulated differentiation. TSH effects on thyroid-specific gene expression, but not its effects on proliferation, were markedly enhanced in cells expressing activated Rap1A and repressed in cells expressing a dominant-negative Rap1A mutant. These findings reveal complex regulation of Rap1 by cAMP including PKA-independent activation and PKA-dependent negative feedback regulation. Both signals appear to be required for TSH signaling to Akt.     

Regulation of nuclear PKA revealed by spatiotemporal manipulation of cyclic AMP

Understanding how specific cyclic AMP (cAMP) signals are organized and relayed to their effectors in different compartments of the cell to achieve functional specificity requires molecular tools that allow precise manipulation of cAMP in these compartments. Here we characterize a new method using bicarbonate-activatable and genetically targetable soluble adenylyl cyclase to control the location, kinetics and magnitude of the cAMP signal. Using this live-cell cAMP manipulation in conjunction with fluorescence imaging and mechanistic modeling, we uncovered the activation of a resident pool of protein kinase A (PKA) holoenzyme in the nuclei of HEK-293 cells, modifying the existing dogma of cAMP-PKA signaling in the nucleus. Furthermore, we show that phosphodiesterases and A-kinase anchoring proteins (AKAPs) are critical in shaping nuclear PKA responses. Collectively, our data suggest a new model in which AKAP-localized phosphodiesterases tune an activation threshold for nuclear PKA holoenzyme, thereby converting spatially distinct second messenger signals to temporally controlled nuclear kinase activity.     

Cooperative interactions of protein kinase C and cAMP-dependent protein kinase systems in human promyelocytic leukemia HL60 cells

Interactions of protein kinase C (PKC) and cAMP-dependent protein kinase (PKA) systems were investigated in HL60 cells. It was found that the differentiating effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) were potentiated by dibutyryl cAMP (dbcAMP) or prostaglandin E2 (PGE2). In addition, dbcAMP or PGE2 inhibited TPA-induced binding of PKC to plasma membrane, leading to decreased protein phosphorylation, and promoted subsequent redistribution of enzyme to the nuclear membrane region. The findings are consistent with the hypothesis that PKC and PKA systems regulate cooperatively the phenotypical differentiation of leukemic cells.     

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.