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Here we demonstrate that elevation of cyclic AMP (cAMP) levels in human umbilical vein endothelial cells (HUVECs) specifically attenuates ERK1,2 activation in response to either leptin or a soluble interleukin IL-6 receptor-alpha/IL-6 (sIL-6R alpha/IL-6) trans-signalling complex but not protein kinase C activator phorbol 12-myristate 13-acetate. The inhibitory effects of cAMP on sIL-6R alpha/IL-6-stimulated phosphorylation of ERK1,2 and STAT3 were abolished by either short interfering (si) RNA-mediated knockdown or genetic ablation of suppressor of cytokine signalling-3 (SOCS-3). The inhibitory effect of cAMP could not be reversed by inhibition of cAMP-dependent protein kinase (PKA) but was blocked by depletion of the alternative intracellular cAMP sensor exchange protein activated by cAMP 1 (Epac1), which is also required to observe SOCS-3 accumulation in response to cAMP. Interestingly, the ability of cAMP elevation to inhibit IL-6 signalling was blocked by ERK inhibition. Consistent with this observation, cAMP elevation in HUVECs produced a transient yet robust activation of ERK, and subsequent phosphorylation of transcription factor C/EBP beta, both of which were resistant to PKA inhibition. However, siRNA depletion and immunoblotting experiments revealed that neither Epac1 nor Epac2 contributed to the PKA-independent activation of ERK1,2 observed following cAMP elevation. Together, these observations suggest that while SOCS-3 induction and subsequent inhibition of cytokine-mediated phosphorylation of ERK1,2 and STAT3 in response to cAMP require Epac1 and a transient PKA-independent activation of the ERK pathway, these two events are controlled by distinct mechanisms. In addition, it reveals a novel Epac-dependent mechanism by which cAMP can specifically inhibit ERK in response to cytokine receptor activation.

Cell differentiation in Dictyostelium results in the formation of two cell types, stalk and spore cells. The stalk cells undergo programmed cell death, whereas spore cells retain viability. The current evidence suggests that stalk cell differentiation is induced by Differentiation Inducing Factor (DIF), while spore cell differentiation occurs in response to cAMP. We have discovered the first developmentally regulated Dictyostelium gene, the glycogen phosphorylase gene 2 (gp2) gene, that can be induced by both DIF-1 and cAMP, suggesting the possibility of a new group of developmentally regulated genes that have DIF-1 and cAMP dual responsiveness. The gp2 gene was found to be expressed in both prestalk/stalk cells and prespore/spore cells. The DIF-1 competence of the gp2 gene required uninterrupted development, whereas the cAMP-competence for the gene required only starvation. Both DIF-1 and cAMP induction of the gene could be inhibited by NH3, a factor that is thought to act as a developmental signal in Dictyostelium. Another developmental signal, adenosine, was found to repress the DIF-1 induction of the gp2 gene. Two introns in the gp2 gene were examined for their involvement in the regulation of the gene, but no regulatory function was detected. A model for the regulation of the gp2 gene during the development is proposed.

The Dictyostelium discoideum glycogen phosphorylase-1 (gp-1) exhibits a complex pattern of developmental expression in which differential temporal regulation of enzyme activity, protein levels and mRNA levels is observed. This pattern of expression implies that gp-1 regulation occurs at multiple levels, probably involving both transcriptional and post-transcriptional events. Post-translational control of gp-1 activity, in effect, actually regulates the protein from a developmental perspective. In this report we have examined several facets of this regulation. We show that addition of exogenous cAMP to cells in suspension culture caused changes in gp-1 enzyme activity and mRNA levels that are identical to those observed during normal development, suggesting that cAMP is involved in the regulation of gp-1. Exogenous cAMP could regulate gp-1 mRNA expression at concentrations as low as 1.0 microM. cAMP regulation of gp-1 mRNA appeared to occur through a mechanism that required intracellular cAMP signalling. We identified regions of the promoter necessary for gp-1 expression by using gp-1 promoter deletions to drive the expression of a luciferase reporter gene. Results of these experiments suggested that developmental and cAMP-mediated changes in gp-1 mRNA levels were the result of alterations in transcription. The promoter analysis also suggested that a vegetative specific element is located between -785 and -1894 nucleotides from the transcriptional start site. Elements necessary for maximal developmental and cAMP-mediated expression appear to be located between -1153 and -1894 nucleotides from the cap site. Sequence elements located between -180 and -1153 appear to be required for a basal level of late developmental expression.

A crucial developmental event in the cellular slime mold, Dictyostelium discoideum, is glycogen degradation. The enzyme that catalyzes this degradation, glycogen phosphorylase 2 (gp-2), is developmentally regulated and cAMP appears to be involved in this regulation. We have examined several aspects of the cAMP regulation of gp-2. We show that addition of exogenous cAMP to aggregation competent amoebae induced the appearance of gp-2 mRNA. The induction of gp-2 mRNA occurred within 1 and 1.5 h after the initial exposure to cAMP. Exposure to exogenous cAMP concentrations as low as 1.0 microM could induce gp-2 mRNA. We also examined the molecular mechanism through which cAMP induction of gp-2 occurs. Induction of gp-2 appears to result from a mechanism that does not require intracellular cAMP signaling, and may occur directly through a cAMP binding protein without the requirement of any intracellular signalling. We also examined the promoter region of the gp-2 gene for cis-acting elements that are involved in the cAMP regulation of gp-2. A series of deletions of the promoter were fused to a luciferase reporter gene and then analyzed for cAMP responsiveness. The results indicated that a region from -258 nucleotides to the transcriptional start site is sufficient for essentially full activity and appears to carry all necessary cis-acting sites for cAMP induction. Further deletion of 58 nucleotides from the 5' end, results in fivefold less activity in the presence of cAMP. Deletion of the next 104 nucleotides eliminates the cAMP response entirely.

We have recently reported the existence of two forms of glycogen phosphorylase (1,4-alpha-D-glucan: orthophosphate-alpha-glucosyltransferase; EC 2.4.1.1) in Dictyostelium discoideum. During development the activity of the glycogen phosphorylase b form decreased as the activity of the a form increased. The total phosphorylase activity remained constant. The physical and kinetic properties of the Dictyostelium enzyme were similar to those of the mammalian enzyme. In mammals, cAMP regulates the conversion of the two forms by a cAMP dependent protein kinase (cAMPdPK). We report here that if cAMP is added to a single cell suspension, the Dictyostelium phosphorylase activity becomes independent of 5'AMP and a 104 kd peptide appears. We also show the effect of several cAMP analogs on the phosphorylase activity in these single-cell suspensions. The cAMP analogs were selected on the basis of their affinities for the membrane-bound cAMP receptor or the cytoplasmic cAMPdPK. We found that relatively low levels, 100 microM, of cAMP or 2'd-cAMP added to aggregation-competent cells in shaking culture caused a loss of phosphorylase b activity and the appearance of phosphorylase a activity. The analog, 2'd-cAMP, has a high affinity for the cAMP receptor but a low affinity for the cAMPdPK. Two other analogs, Bt2-cAMP and 8-Br-cAMP, which have low affinities for the cAMP receptor but high affinities for the cAMPdPK, required high levels (500 microM) for 'b' to 'a' conversion. cDNAs to three cAMP-regulated genes--PL3, D11, and D3--were used as controls in the above experiments. In order to determine if intracellular levels of cAMP were involved in the regulation of phosphorylase activity, both the phosphorylase and the PL3, D11 and D3 mRNA levels were examined in cells suspended in a glucose/albumin mixture--a medium in which adenylate cyclase is inhibited. Under these conditions, neither gene regulation nor a change in the phosphorylase b to a activity occurred in response to added extra cellular cAMP. The results suggest that an intracellular increase in cAMP is involved in the regulation of the two forms of glycogen phosphorylase in Dictyostelium.

Cyclic AMP functions as the chemotactic signal during aggregation of amoebae of the cellular slime mold Dictyostelium discoideum. Evidence suggests that cAMP also acts as a regulatory molecule during Dictyostelium multicellular differentiation. We have used ultramicrotechniques and a sensitive radioimmunoassay to measure the levels of cAMP within the culmination stage individual. We show that there is a peak of cAMP at the culmination stage of development and that in the individual at this stage the molecule is localized in a gradient within the spore mass.

Adenylate cyclase activity and endogenous cyclic AMP levels were measured using a highly sensitive radioimmunoassay and protein binding assay during 24 h of development of Dictyostelium discoideum. Adenylate cyclase activity was not detected until the aggregation stage of development (10 h) when a sudden peak of activity was found. The enzyme was active at all subsequent stages, although a slow decline in activity was observed. Similarly, cyclic AMP levels were not detectable through the first 7 h of development and then showed a sudden peak at aggregation. Following aggregation the cyclic AMP levels decreased to approximately 1/2 the peak value and maintained that level throughout the remainder of the developmental cycle. Adenylate cyclase had a narrow range of substrate saturation with a maximum velocity at 1 to 4 mM ATP at both the aggregation stage (10 h) and the sorocarp stage (24 h). At levels of ATP higher than 6 mM the enzyme from both stages was strongly inhibited. No activity was observed in the absence of Mg2+ or dithiothreitol. The activity from 10-, 14-, and 20-h stages was found bound to a 25,000 x g pellet fraction. The sudden appearance of adenylate cyclase and its product cyclic AMP at aggregation provides additional evidence of a role for this nucleotide in chemotaxis, and the retention of enzyme activity and nucleotide level during the subsequent stages may reflect a further function of cyclic AMP during formation of the two cell types.

During the developmental cycle of Dictyostelium discoideum cyclic AMP functions as both a chemotactic signal for aggregation and a regulatory molecule during later events of differentiation. Morphological and biochemical data suggest that cAMP may direct cells during morphogenesis and differentiation. We utilized microtechniques to determine the stage- and cell-specific levels of the cAMP-dependent protein kinase, the probable intracellular cAMP receptor. Kinase activity was low and non-cAMP-dependent in amoebae and early aggregates but increased and became cAMP-dependent in aggregates after the formation of tight cell contacts. Maximum kinase activity and cAMP dependency occurred during the slug and culmination stages. The only differential distribution of the kinase within a single stage occurred during culmination when the activity in the stalks was approximately one-fourth of that in the prespore mass. Preliminary evidence indicates that this difference is not due to an inhibitor. In all other stages tested cAMP-dependent protein kinase activity was equal in prespore and prestalk cells.

A key step in the cellular differentiation of Dictyostelium is the degradation of glycogen to provide the precursors for synthesis of the structural end products of development. We have found that the enzyme that initiates this degradative pathway, glycogen phosphorylase (1,4-alpha-D-glucan:orthophosphate alpha-glucosyltransferase; EC 2.4.1.1), is developmentally regulated and exists as two forms. During the time course of development, a previously undescribed activity, the "b" form, decreases, while that of the "a" form increases. The "b" form is inactive unless 5'AMP is included in the reaction mixture. The two forms differ in their elution from DE52 cellulose, affinity constants, thermal stability, affinity for 5'AMP Sepharose, subunit molecular weight, and peptide maps. In crude extracts, anti-a antiserum stains a 104-kD protein that is associated with phosphorylase "a" activity and appears late in development, while anti-b antiserum stains a 92-kD protein that is associated with phosphorylase "b" activity and is present throughout development. We have also demonstrated in vitro phosphorylation of the "b" form by an endogenous protein kinase and a corresponding loss of 5'AMP dependence. If intact cells were exposed to exogenous cAMP, "b" activity decreased and was replaced by "a" activity, as well as the 104-kD protein band on SDS-PAGE. In order to determine if the two forms of the enzyme are different gene products, we screened lambda gt11 expression libraries with antibodies against the purified "a" and "b" forms. Three clones were found to be overlapping by Southern analysis. A yeast glycogen phosphorylase cDNA clone (gpy) and a human muscle glycogen phosphorylase clone (HM-11) cross-hybridized with the Dictyostelium inserts, and gpy shared a few common restriction fragments with the Dictyostelium clones on genomic blots. Northern analysis of Dictyostelium total RNA showed that the Dictyostelium inserts and gpy recognize an mRNA of 3.2 kb, while on poly A-enriched RNA, the yeast clone detects preferentially a 3.6-kb message.