cAMP-dependent Signaling Pathway Research Articles

Soluble Adenylyl Cyclase Controls Mitochondria-dependent Apoptosis in Coronary Endothelial Cells

The cAMP signaling pathway plays an essential role in modulating the apoptotic response to various stress stimuli. Until now, it was attributed exclusively to the activity of the G-protein-responsive transmembrane adenylyl cyclase. In addition to transmembrane AC, mammalian cells possess a second source of cAMP, the ubiquitously expressed soluble adenylyl cyclase (sAC). However, the role of this cyclase in apoptosis was unknown. A mitochondrial localization of this cyclase has recently been demonstrated, which led us to the hypothesis that sAC may play a role in apoptosis through modulation of mitochondria-dependent apoptosis. To prove this hypothesis, apoptosis was induced by simulated in vitro ischemia or by acidosis, which is an important component of ischemia. Suppression of sAC activity with the selective inhibitor KH7 or sAC knockdown by small interfering RNA transfection abolished endothelial apoptosis. Furthermore, pharmacological inhibition or knockdown of protein kinase A, an important cAMP target, demonstrated a significant anti-apoptotic effect. Analysis of the underlying mechanisms revealed (i) the translocation of sAC to mitochondria under acidic stress and (ii) activation of the mitochondrial pathway of apoptosis, i.e. cytochrome c release and caspase-9 cleavage. sAC inhibition or knockdown abolished the activation of the mitochondrial pathway of apoptosis. Analysis of mitochondrial co-localization of Bcl-2 family proteins demonstrated sAC- and protein kinase A-dependent translocation of Bax to mitochondria. Taken together, these results suggest the important role of sAC in modulating the mitochondria-dependent pathway of apoptosis in endothelial cells.

Synergistic IL-10 induction by LPS and the ceramide-1-phosphate analog PCERA-1 is mediated by the cAMP and p38 MAP kinase pathways

Expression of the anti-inflammatory cytokine IL-10 can be induced either by TLR agonists such as lipopolysaccharide (LPS), or by various endogenous stimuli, in particular those acting via a cAMP-dependent signaling pathway. We have previously reported that the synthetic phospho-ceramide analogue-1 (PCERA-1) increases cAMP level and subsequently down-regulates production of TNFα and up-regulates production of IL-10 in LPS-stimulated macrophages. The objective of this study was to determine the mechanism of activity of PCERA-1 and the role of cAMP in LPS-induced IL-10 production. We show here that PCERA-1 induces IL-10 production in synergism with various TLR agonists in mouse RAW264.7 macrophages. Cooperativity is evident both at the mRNA and protein levels. IL-10 production by LPS and PCERA-1 is mediated by the cAMP pathway and by the p38 MAP kinase. Phosphorylation of p38 is cooperatively accomplished by LPS and PCERA-1 or other cAMP inducers. Furthermore, the activity of PCERA-1 can be partially mimicked by a cell-permeable analog of cAMP, and blocked by the protein kinase A (PKA) inhibitor H89. Finally, in the absence of PCERA-1, the residual IL-10 induction by LPS depends on the basal cAMP level as it can be largely elevated by the phosphodiesterase (PDE)-4 inhibitor rolipram. Our results thus indicate that IL-10 induction by LPS critically depends on basal cAMP level, and that a co-stimulus by a TLR agonist and a cAMP-elevating agent results in synergistic PKA-dependent and p38-dependent IL-10 production.

The cAMP-Dependent Signaling Pathway and Its Role in Conidial Germination, Growth, and Virulence of the Gray Mold Botrytis cinerea

In Botrytis cinerea, some components of the cAMP-dependent pathway, such as alpha subunits of heterotrimeric G proteins and the adenylate cyclase BAC, have been characterized and their impact on growth, conidiation, germination, and virulence has been demonstrated. Here, we describe the functions of more components of the cAMP cascade: the catalytic subunits BcPKA1 and BcPKA2 and the regulatory subunit BcPKAR of the cAMP-dependent protein kinase (PKA). Although Deltabcpka2 mutants showed no obvious phenotypes, growth and virulence were severely affected by deletion of both bcpka1 and bcpkaR. Similar to Deltabac, lesion development of Deltabcpka1 and DeltabcpkaR was slower than in controls and soft rot of leaves never occurred. In contrast to Deltabac, Deltabcpka1 and DeltabcpkaR mutants sporulated in planta, and growth rate, conidiation, and conidial germination were not impaired, indicating PKA-independent functions of cAMP. Unexpectedly, Deltabcpka1 and DeltabcpkaR showed identical phenotypes, suggesting the total loss of PKA activity in both mutants. The deletion of bcras2 encoding the fungal-specific Ras GTPase resulted in significantly delayed germination and decreased growth rates. Both effects could be partially restored by exogenous cAMP, suggesting that BcRAS2 activates the adenylate cyclase in addition to the Galpha subunits BCG1 and BCG3, thus influencing cAMP-dependent signal transduction.

Discovery of Pituitary Adenylate Cyclase‐Activating Polypeptide‐Regulated Genes through Microarray Analyses in Cell Culture and In Vivo

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an evolutionarily well conserved neuropeptide with multiple functions in the nervous, endocrine, and immune systems. PACAP provides neuroprotection from ischemia and toxin exposure, is anti-inflammatory in gastric inflammatory disease and sepsis, controls proliferative signaling pathways involved in neural cell transformation, and modulates glucohomeostasis. PACAP-based, disease-targeted therapeutics might thus be both effective and benign, enhancing homeostatic responses to behavioral, metabolic, oncogenic, and inflammatory stressors. PACAP signal transduction employs synergistic regulation of calcium and cyclic adenosine monophosphate (cAMP), and noncanonical activation of both calcium- and cAMP-dependent processes. Pharmacological activation of PACAP signaling should consequently have highly specific effects even in vivo. Here, a combined cellular biochemical, pharmacologic, transcriptomic, and bioinformatic approach to understanding PACAP signal transduction by identifying PACAP target genes with oligonucleotide- and cDNA-based microarray is described. Calcium- and cAMP-dependent PACAP signaling pathways for regulation of genes encoding proteins required for neuritogenesis, changes in cell morphology, and cell survival have been traced in PC12 cells. Pharmacological experiments have linked gene expression to cell physiological responses in this system, in which gene silencing can also be employed to confirm the functional significance of induction of specific transcripts. Differential transcriptional responses to metabolic, ischemic, and other stressors in wild type compared to PACAP-deficient mice establish in principle which PACAP-responsive transcripts in culture are PACAP-dependent in vivo. Bioinformatic approaches aid in creating a pipeline for identifying neuropeptide-regulated genes, validating their cellular functions, and defining their expression in the context of neuropeptide signaling physiology, required for discovery of new targets for drug action.

Excitability of small-diameter trigeminal ganglion neurons by 5-HT is mediated by enhancement of the tetrodotoxin-resistant sodium current due to the activation of 5-HT4 receptors and/or by the inhibition of the transient potassium current

The aims of the present study were to investigate whether the activation of the 5-HT receptor subtypes (5-HT4 and 5-HT3) acted significantly on the modification of the tetrodotoxin-resistant sodium current (INaR) in small-sized rat trigeminal ganglion (TG) neurons and whether the inhibition of the transient K+ current (IA) contributed to the excitability in those neurons. 5-HT applications in at concentrations ranging from 0.01–10 μM significantly increased the peak INaR. One micromolar 5-HT application caused the greatest increase in the peak INaR amplitude accompanied by a hyperpolarizing shift in the activation curve. A similar modification of INaR properties was also obtained via the application of the 5-HT4 receptor agonist, RS 67333, in concentrations ranging from 0.001–1 μM. The largest effects of 5-HT (1 μM) and RS 67333 (0.1 μM) on the modification of INaR were abolished by pretreatment with ICS 205–930 (a 5-HT3/4 receptor antagonist, 10 μM), which showed no significant effect on the baseline INaR. However, ICS 205–930 application at 30 μM caused a significant decrease in the baseline INaR. Phenylbiguanide (a 5-HT3 receptor agonist) did not significantly alter INaR properties when applied in concentrations ranging from 1 to 100 μM. The application of 0.1 μM RS 67333 decreased the transient K+ current (IA) by approximately 31%. The threshold for action potential generation was significantly lower after the application of 0.1 μM RS 67333. Furthermore, 0.1 μM RS 67333 application increased the number of action potentials and the resting membrane potential got more positive, but it decreased the duration of depolarization phase of action potential. In addition, neither the additional application of 1 μM 5-HT in the presence of 10 μM forskolin, a stimulator of adenylyl cyclase, nor the opposite applications of 5-HT and forskolin caused the enhancement of increased INaR, which indicates the presence of an ‘occluding effect.’ These results suggest that the 5-HT-induced modification of INaR is mediated by the activation of 5-HT4 receptors, involving a cAMP-dependent signaling pathway, and that the inhibition of IA following the application of a 5-HT4 receptor agonist also contributes to the increased number of action potentials.

Multidrug resistance-associated protein 4 regulates cAMP-dependent signaling pathways and controls human and rat SMC proliferation

The second messengers cAMP and cGMP can be degraded by specific members of the phosphodiesterase superfamily or by active efflux transporters, namely the multidrug resistance-associated proteins (MRPs) MRP4 and MRP5. To determine the role of MRP4 and MRP5 in cell signaling, we studied arterial SMCs, in which the effects of cyclic nucleotide levels on SMC proliferation have been well established. We found that MRP4, but not MRP5, was upregulated during proliferation of isolated human coronary artery SMCs and following injury of rat carotid arteries in vivo. MRP4 inhibition significantly increased intracellular cAMP and cGMP levels and was sufficient to block proliferation and to prevent neointimal growth in injured rat carotid arteries. The antiproliferative effect of MRP4 inhibition was related to PKA/CREB pathway activation. Here we provide what we believe to be the first evidence that MRP4 acts as an independent endogenous regulator of intracellular cyclic nucleotide levels and as a mediator of cAMP-dependent signal transduction to the nucleus. We also identify MRP4 inhibition as a potentially new way of preventing abnormal VSMC proliferation.

ACTH Inhibits bTREK-1 K+ Channels through Multiple cAMP-dependent Signaling Pathways

Bovine adrenal zona fasciculata (AZF) cells express bTREK-1 K+ channels that set the resting membrane potential and function pivotally in the physiology of cortisol secretion. Inhibition of these K+ channels by adrenocorticotropic hormone (ACTH) or cAMP is coupled to depolarization and Ca2+ entry. The mechanism of ACTH and cAMP-mediated inhibition of bTREK-1 was explored in whole cell patch clamp recordings from AZF cells. Inhibition of bTREK-1 by ACTH and forskolin was not affected by the addition of both H-89 and PKI(6–22) amide to the pipette solution at concentrations that completely blocked activation of cAMP-dependent protein kinase (PKA) in these cells. The ACTH derivative, O-nitrophenyl, sulfenyl-adrenocorticotropin (NPS-ACTH), at concentrations that produced little or no activation of PKA, inhibited bTREK-1 by a Ca2+-independent mechanism. Northern blot analysis showed that bovine AZF cells robustly express mRNA for Epac2, a guanine nucleotide exchange protein activated by cAMP. The selective Epac activator, 8-pCPT-2′-O-Me-cAMP, applied intracellularly through the patch pipette, inhibited bTREK-1 (IC50 = 0.63 μM) at concentrations that did not activate PKA. Inhibition by this agent was unaffected by PKA inhibitors, including RpcAMPS, but was eliminated in the absence of hydrolyzable ATP. Culturing AZF cells in the presence of ACTH markedly reduced the expression of Epac2 mRNA. 8-pCPT-2′-O-Me-cAMP failed to inhibit bTREK-1 current in AZF cells that had been treated with ACTH for 3–4 d while inhibition by 8-br-cAMP was not affected. 8-pCPT-2′-O-Me-cAMP failed to inhibit bTREK-1 expressed in HEK293 cells, which express little or no Epac2. These findings demonstrate that, in addition to the well-described PKA-dependent TREK-1 inhibition, ACTH, NPS-ACTH, forskolin, and 8-pCPT-2′-O-Me-cAMP also inhibit these K+ channels by a PKA-independent signaling pathway. The convergent inhibition of bTREK-1 through parallel PKA- and Epac-dependent mechanisms may provide for failsafe membrane depolarization by ACTH.

Regulation of PRiMA-linked G 4 AChE by a cAMP-dependent signaling pathway in cultured rat pheochromocyoma PC12 cells

The catalytic subunit of acetylcholinesterase (AChE T) interacts with proline-rich membrane anchor (PRiMA) to form PRiMA-linked G 4 AChE on membrane surface for its cholinergic function. Cultured PC12 cells expressed the transcripts encoding AChE T and PRiMA I, but the expression of PRiMA II transcript was below detection. Upon the treatment of dibutyryl-cAMP (Bt 2-cAMP) and forskolin in cultured cells to stimulate the cAMP-dependent signaling pathway, the mRNA expressions of both AChE T and PRiMA I, as well as the enzymatic activity were up-regulated. More importantly, sucrose density gradient analysis revealed that both G 1 and G 4 AChE isoforms were increased in the Bt 2-cAMP-treated cultures. These results suggest that the regulation of PRiMA-linked G 4 AChE in terms of gene transcription and molecular assembly in the cultured PC12 cells could be mediated by a cAMP-dependent signaling mechanism.

Knockout of ATF1 leads to enhanced cardiac contractility and output

The transcriptional regulation by the CREB/CREM/ATF1 family factors presents an important cAMP‐dependent signalling pathway and it might contribute to the progression of heart failure. Activating transcription factor 1 (ATF1) is expressed ubiquitously, however, its role in the heart remains unknown. To elucidate the cardiac consequences of ATF1 ablation, we studied the left ventricular (LV) function in adult animals (14–18 weeks old). LV catheterization and analysis of pressure‐volume loops showed increased maximal LV pressure (LVPmax), stroke work (SW) and cardiac output (CO) of ATF1−/− mice.Experiments on isolated adult cardiomyocytes confirmed these findings. The cell shortening of ATF1−/− cells was increased (3.9±0.9★ vs. 1.5±0.2 in ATF1+/+, n=10–11), whereby the calcium transient (CaT) amplitude was not significantly altered (★p<0.05 vs. ATF1+/+).Hence, the presence of ATF1 is essential for a regular cardiomyocyte function, however, the increased contractility of ATF1−/− hearts cannot be explained by increased Ca2+ release from the sarcoplasmic reticulum. Supported by DFG (MU1376/10‐3)

A Constitutive Increase in Gamma Globin Gene Production during the Erythroid Differentiation of CD34+ Cells from Sickle Cell Disease Patients and Alterations in Cyclic Nucleotide Levels during This Differentiation.

Studies have demonstrated that the activation of soluble guanylate cyclase (sGC) in erythrocytic cell lines and in primary erythroid precursor culture causes an increase in gamma globin gene transcription and, consequently, an augmented production of fetal hemoglobin (HbF). The cyclic nucleotides, cGMP and cAMP, are second messengers that are responsible for mediating the signaling of various extracellular signals, including those of nitric oxide (NO), hormones and neurotransmitters and, probably, have an important role in SCD by regulating the production of HbF. The aim of this study was to compare gamma-globin gene production, in association with cGMP- and cAMP-dependent signaling pathways, during the erythroid differentiation of CD34+ cells from steady-state SCD patients and healthy controls. Hematopoietic CD34+ cells were separated from the peripheral blood of subjects by CD34+-positive cell magnetic separation and were then cultured on semi-solid medium for 7 days until reaching the erythroid colony forming unit (CFU-e) stage; these colonies were then differentiated in liquid-phase culture until day 13 when cells reached the orthochromatic erythroblast/ reticulocyte stage. Cell samples were collected at days 7, 10 and 13 of culture for flow cytometry, citospin, measurement of intracellular cGMP/cAMP levels and evaluation of the gene expressions of gamma globin and the sGC alpha and beta subunits by real-time RT-PCR. Genes for endothelial nitric oxide synthase (eNOS) and phosphodiesterase 3B (PDE3B) were also studied, since these genes are important for NO/cyclic nucleotide signaling pathways. Gamma globin gene expression was observed to be significantly higher in the cultures of SCD cells on the 13th day of erythroid differentiation compared to the same time point in control cells (2.95 ± 0.30 vs. 1.45 ± 0.17 rel expression, respectively, p<0.01, Mann-Whitney, n=6). The expressions of the sGC alpha and beta subunits, eNOS and PDE3B did not present any significant differences during control and SCD erythroid differentiation. Levels of cGMP were significantly decreased during erythropoiesis in the SCD cultures, compared to control cultures (day 7: 277 ± 53 vs. 639 ± 122 fmol/5x106cells (p<0.05); day 10: 117 ± 22 vs. 268 ± 118 fmol/5x106cells; day 13: 37 ± 6 vs. 93 ± 24 fmol/5x106cells (p<0.02), respectively). In contrast, levels of cAMP were significantly increased in the SCD cultures compared to the control cultures (day 7: 72.02 ± 14.85 vs. 24.99 ± 13.50 pmol/5x106cells (p<0.05); day 10: 36.34 ± 16.71 vs. 8.66 ± 4.59 pmol/5x106cells; day 13: 1.74 ± 1.09 vs. 0.68 ± 0.37 pmol/5x106cells, respectively). Results suggest that the hematopoietic stem cells of SCD patients are significantly and constitutively different to those of control stem cells, since under the same in vitro culture conditions, consistently higher levels of gamma globin were observed during sickle cell erythroid differentiation. Intracellular cGMP and cAMP levels were also significantly altered during SCD erythroid differentiation, providing further support to the theory that cyclic nucleotide signaling may regulate HbF production. The precise role of these signaling pathways in the mechanism studied and whether any cross talk between these pathways occurs remains to be determined.

The small heat shock-related protein, HSP20, is a cAMP-dependent protein kinase substrate that is involved in airway smooth muscle relaxation

Activation of the cAMP/cAMP-dependent PKA pathway leads to relaxation of airway smooth muscle (ASM). The purpose of this study was to examine the role of the small heat shock-related protein HSP20 in mediating PKA-dependent ASM relaxation. Human ASM cells were engineered to constitutively express a green fluorescent protein-PKA inhibitory fusion protein (PKI-GFP) or GFP alone. Activation of the cAMP-dependent signaling pathways by isoproterenol (ISO) or forskolin led to increases in the phosphorylation of HSP20 in GFP but not PKI-GFP cells. Forskolin treatment in GFP but not PKI-GFP cells led to a loss of central actin stress fibers and decreases in the number of focal adhesion complexes. This loss of stress fibers was associated with dephosphorylation of the actin-depolymerizing protein cofilin in GFP but not PKI-GFP cells. To confirm that phosphorylated HSP20 plays a role in PKA-induced ASM relaxation, intact strips of bovine ASM were precontracted with serotonin followed by ISO. Activation of the PKA pathway led to relaxation of bovine ASM, which was associated with phosphorylation of HSP20 and dephosphorylation of cofilin. Finally, treatment with phosphopeptide mimetics of HSP20 possessing a protein transduction domain partially relaxed precontracted bovine ASM strips. In summary, ISO-induced phosphorylation of HSP20 or synthetic phosphopeptide analogs of HSP20 decreases phosphorylation of cofilin and disrupts actin in ASM, suggesting that one possible mechanism by which HSP20 mediates ASM relaxation is via regulation of actin filament dynamics.

Fenvalerate inhibits progesterone production through cAMP-dependent signal pathway

Fenvalerate is a widely used synthetic pyrethroid insecticide and is known to impede the male reproductive function. However, the mechanisms remain to be elucidated. In this study, mouse Leydig tumor cells (MLTC-1) were used to investigate the effects of fenvalerate on progesterone production. Fenvalerate treatment inhibited progesterone secretion induced by human chorionic gonadotropin (hCG), cholera toxin (CT) or forskolin and decreased cAMP levels induced by hCG, but not by CT or forskolin, which suggested a repaired site on the upstream components of G protein or G protein per se by fenvalerate in the cAMP-mediated signal pathway. Furthermore, the addition of cAMP analog, 8-Br-cAMP, could not reverse fenvalerate-suppressed progesterone synthesis, indicating that fenvalerate interfered with the downstream molecules of cAMP. In addition, fenvalerate decreased steroidogenic acute regulatory protein (StAR) mRNA and protein levels, and also profoundly inhibited the activity of P450 side chain cleavage enzyme (P450scc) which was consistent with the decreased expression of P450scc mRNA and protein in MLTC-1 cells. These results suggested that fenvalerate might inhibit progesterone production by attenuating cAMP generation and inhibiting StAR expression and P450scc activity.

Phosphatidylinositol 3-kinase is involved in prostaglandin E2-mediated murine duodenal bicarbonate secretion

Prostaglandin E(2) (PGE(2)) plays an important role in the regulation of duodenal bicarbonate (HCO(3)(-)) secretion, but its signaling pathway(s) are not fully understood. In the present study, we investigated the signaling pathways involved in PGE(2)-mediated duodenal HCO(3)(-) secretion. Murine duodenal mucosal HCO(3)(-) secretion was examined in vitro in Ussing chambers by pH-stat titration in the presence of a variety of signal transduction modulators. Phosphatidylinositol 3-kinase (PI3K) activity was measured by immunoprecipitation of PI3K and ELISA, and Akt phosphorylation was measured by Western analysis with anti-phospho-Akt and anti-Akt antibodies. PGE(2)-stimulated duodenal HCO(3)(-) secretion was reduced by the cAMP-dependent signaling pathway inhibitors MDL-12330A and KT-5720 by 23% and 20%, respectively; the Ca(2+)-influx inhibitor verapamil by 26%; and the calmodulin antagonist W-13 by 24%; whereas the PI3K inhibitors wortmannin and LY-294002 reduced PGE(2)-stimulated HCO(3)(-) secretion by 51% and 47%, respectively. Neither the MAPK inhibitor PD-98059 nor the tyrosine kinase inhibitor genistein altered PGE(2)-stimulated HCO(3)(-) secretion. PGE(2) application caused a rapid and concentration-dependent increase in duodenal mucosal PI3K activity and Akt phosphorylation. These results demonstrated that PGE(2) activates PI3K in duodenal mucosa and stimulates duodenal HCO(3)(-) secretion via cAMP-, Ca(2+)-, and PI3K-dependent signaling pathways.

Epigenetic Reversion of Breast Carcinoma Phenotype Is Accompanied by Changes in DNA Sequestration as Measured by AluI Restriction Enzyme

The importance of microenvironment and context in regulation of tissue-specific genes is well established. DNA exposure to or the sequestration from nucleases detects differences in higher order chromatin structure in intact cells without disturbing cellular or tissue architecture. To investigate the relationship between chromatin organization and tumor phenotype, we used an established three-dimensional assay in which normal and malignant human breast cells can be easily distinguished by the morphology of the structures they make (acinus-like versus tumor-like, respectively). We show that these phenotypes can be distinguished also by sensitivity to AluI digestion in which the malignant cells resist digestion relative to nonmalignant cells. Treatment of T4-2 breast cancer cells in three-dimensional culture with cAMP analogs or a phosphatidylinositol 3-kinase inhibitor not only reverted their phenotype from nonpolar to polar acinar-like structures but also enhanced chromatin sensitivity to AluI. By using different cAMP analogs, we show that cAMP-induced phenotypic reversion, polarization, and shift in DNA organization act through a cAMP-dependent protein-kinase A-coupled signaling pathway. Importantly, inhibitory antibody to fibronectin produced the same effect. These experiments underscore the concept that modifying the tumor microenvironment can alter the organization of tumor cells and demonstrate that architecture and global chromatin organization are coupled and highly plastic.

Manipulation of host signalling pathways by anthrax toxins

Infectious microbes face an unwelcoming environment in their mammalian hosts, which have evolved elaborate multicelluar systems for recognition and elimination of invading pathogens. A common strategy used by pathogenic bacteria to establish infection is to secrete protein factors that block intracellular signalling pathways essential for host defence. Some of these proteins also act as toxins, directly causing pathology associated with disease. Bacillus anthracis, the bacterium that causes anthrax, secretes two plasmid-encoded enzymes, LF (lethal factor) and EF (oedema factor), that are delivered into host cells by a third bacterial protein, PA (protective antigen). The two toxins act on a variety of cell types, disabling the immune system and inevitably killing the host. LF is an extraordinarily selective metalloproteinase that site-specifically cleaves MKKs (mitogen-activated protein kinase kinases). Cleavage of MKKs by LF prevents them from activating their downstream MAPK (mitogen-activated protein kinase) substrates by disrupting a critical docking interaction. Blockade of MAPK signalling functionally impairs cells of both the innate and adaptive immune systems and induces cell death in macrophages. EF is an adenylate cyclase that is activated by calmodulin through a non-canonical mechanism. EF causes sustained and potent activation of host cAMP-dependent signalling pathways, which disables phagocytes. Here I review recent progress in elucidating the mechanisms by which LF and EF influence host signalling and thereby contribute to disease.

Cardiomyocyte‐specific inactivation of transcription factor CREB in mice

The transcription factor cAMP response element (CRE)-binding protein (CREB, Creb1) plays a critical role in regulating gene expression in response to activation of the cAMP-dependent signaling pathway, which is implicated in the pathophysiology of heart failure. Using the Cre-loxP system, we generated mice with a cardiomyocyte-specific inactivation of CREB and studied in this model whether CREB is critical for cardiac function. CREB-deficient mice were viable and displayed neither changes in cardiac morphology nor alterations of basal or isoproterenol-stimulated left ventricular function in vivo or of important cardiac regulatory proteins. Since CREB was proposed as a negative regulator of cardiomyocyte apoptosis by enhancing the expression of the antiapoptotic protein Bcl-2, we analyzed the fragmentation of DNA, the activity of caspases 3/7 and the expression of Bcl-2 and did not observe any differences between CREB-deficient and CREB-normal hearts. Our results suggest that the presence of CREB is not critical for normal cardiac function in mice.

Multisite phosphorylation of adipocyte and hepatocyte phosphodiesterase 3B

Phosphodiesterase 3B (PDE3B) is an important component of insulin and cAMP-dependent signalling pathways. In order to study phosphorylation of PDE3B, we have used an adenoviral system to express recombinant flag-tagged PDE3B in primary rat adipocytes and H4IIE hepatoma cells. Phosphorylation of PDE3B after treatment of cells with insulin, cAMP-increasing agents, or the phosphatase inhibitor, calyculin A was analyzed by two-dimensional tryptic phosphopeptide mapping and mass spectrometry. We found that PDE3B is multisite phosphorylated in adipocytes and H4IIE hepatoma cells in response to all these stimuli. Several sites were identified; serine (S)273, S296, S421, S424/5, S474 and S536 were phosphorylated in adipocyte as well as H4IIE hepatoma cells whereas S277 and S507 were phosphorylated in hepatoma cells only. Several of the sites were phosphorylated by insulin as well as cAMP-increasing hormones indicating integration of the two signalling pathways upstream of PDE3B, maybe at the level of protein kinase B.

Activation of cAMP-Dependent Signaling Pathway Induces Mouse Organic Anion Transporting Polypeptide 2 Expression

Rodent Oatp2 is a hepatic uptake transporter for such compounds as cardiac glycosides. In the present study, we found that fasting resulted in a 2-fold induction of Oatp2 expression in liver of mice. Because the cAMP-protein kinase A (PKA) signaling pathway is activated during fasting, the role of this pathway in Oatp2 induction during fasting was examined. In Hepa-1c1c7 cells, adenylyl cyclase activator forskolin as well as two cellular membrane-permeable cAMP analogs, dibutyryl cAMP and 8-bromo-cAMP, induced Oatp2 mRNA expression in a time- and dose-dependent manner. These three chemicals induced reporter gene activity in cells transfected with a luciferase reporter gene construct containing a 7.6-kilobase (kb) 5'-flanking region of mouse Oatp2. Transient transfection of cells with 5'-deletion constructs derived from the 7.6-kb Oatp2 promoter reporter gene construct, as well as 7.6-kb constructs in which a consensus cAMP response element (CRE) half-site CGTCA (-1808/-1804 bp) was mutated or deleted, confirms that this CRE site was required for the induction of luciferase activity by forskolin. Luciferase activity driven by the Oatp2 promoter containing this CRE site was induced in cells cotransfected with a plasmid encoding the protein kinase A catalytic subunit. Cotransfection of cells with a plasmid encoding the dominant-negative CRE binding protein (CREB) completely abolished the inducibility of the reporter gene activity by forskolin. In conclusion, induction of Oatp2 expression in liver of fasted mice may be caused by activation of the cAMP-dependent signaling pathway, with the CRE site (-1808/-1804) and CREB being the cis- and trans-acting factors mediating the induction, respectively.

Insulin‐induced formation of macromolecular complexes involved in activation of Phosphodiesterase3B (PDE3B) in 3T3‐L1 adipocytes

PDE3B is an important regulator of several insulin- and cAMP-dependent signaling pathways. Fractionation of membranes from adipocytes revealed that PDE3B was associated with plasma membrane (PM) and internal membrane (IM) fractions, and that insulin-induced phosphorylation/activation of PDE3B was greater in IM than PM fractions. Insulin increased tyrosine phosphorylation of IRS-1, and activated IRS-1-associated PI3-K and PKB in both membrane and cytosolic fractions. Insulin also induced formation of large macromolecular complexes (LMCs), separated during gel filtration (Superose 6) of solubilized membranes, which apparently contain phosphorylated/activated PDE3B and several signaling molecules potentially involved in its activation by insulin, e.g. IRS-1, PI3-K, PKB, HSP-90, and 14-3-3. Expression of full length recombinant Flag-tagged murine (M)PDE3B (M3B) and M3BD604(M3B lacking N-terminal 604 amino acids) indicated that N-terminal region of M3B (which contains sites phosphorylated by insulin) was necessary for insulin-induced activation and recruitment of PDE3B into LMCs. M3BD604 was not activated by insulin, was not recruited into LMCs, and did not interfere with recruitment of endogenous PDE3B. siRNA knockdown of PDE3B indicated that PDE3B was not required for formation of insulin-induced LMCs. Wortmannin inhibited insulin-induced LMCs, as well as insulin-induced phosphorylation/activation of PKB and PDE3B, their co-immunoprecipitation, and interactions during Superose 6 chromatography. Insulin-stimulated complex formation of PDE3B and other signaling molecules may be critical for down stream events regulating adipocyte metabolism.

PACAP/PAC1 autocrine system promotes proliferation and astrogenesis in neural progenitor cells

The Pituitary adenylate cyclase-activating peptide (PACAP) ligand/type 1 receptor (PAC1) system regulates neurogenesis and gliogenesis. It has been well established that the PACAP/PAC1 system induces differentiation of neural progenitor cells (NPCs) through the Gs-mediated cAMP-dependent signaling pathway. However, it is unknown whether this ligand/receptor system has a function in proliferation of NPCs. In this study, we identified that PACAP and PAC1 were highly expressed and co-localized in NPCs of mouse cortex at embryonic day 14.5 (E14.5) and found that the PACAP/PAC1 system potentiated growth factor-induced proliferation of mouse cortical NPCs at E14.5 via Gq-, but not Gs-, mediated PLC/IP3-dependent signaling pathway in an autocrine manner. Moreover, PAC1 activation induced elongation of cellular processes and a stellate morphology in astrocytes that had the bromodeoxyuridine (BrdU)-incorporating ability of NPCs. Consistent with this notion, we determined that the most BrdU positive NPCs differentiated to astrocytes through PAC1 signaling. These results suggest that the PACAP/PAC1 system may play a dual role in neural/glial progenitor cells not only differentiation but also proliferation in the cortical astrocyte lineage via Ca2+-dependent signaling pathways through PAC1.