PDEs Are Integrators of Different Signaling Pathways

Initially, it was thought that the different signaling pathways were organized as a linear sequence of steps functioning in parallel and that each extracellular signal would activate its own pathway. The last 10 yr of investigation of signaling have completely revolutionized this view. Signaling pathways are composed of nodes and branches, and different pathways are meshed into an intricate network of crosstalks. The concept of combinatorial signaling has been put forward to underscore the almost infinite number of second messenger combinations that control cell function. This organization of the signaling machinery accounts for the pleiotropic effects that hormones, neurotransmitters, and cytokines have on cell function. An increase in cAMP not only changes metabolic activities, but also regulates gene expression and differentiation, cell shape, and motility, as well as entry and exit from the cell cycle. These regulations are mediated by changes in virtually all the signaling pathways operating in the cell. Because PDEs are regulated by multiple second messengers, they function as a branching point in a signaling pathway and allow the integration of cyclic nucleotides pathway with other pathways. Here we provide the most relevant examples of this function.

3.2.1. PDE1 and Ca2+ Signaling

By virtue of their ability to be regulated by the Ca2+-calmodulin complex, PDE1s serve to integrate the Ca2+ signaling pathway with the cyclic nucleotide dependent pathway. In cells where a PDE1 is expressed, an increase in cytoplasmic Ca2+ causes an activation of the PDE hydrolytic activity, with a consequent decrease in intracellular cyclic nucleotide concentration. This, for instance, has been observed for the muscarinic cholinergic signaling pathway (Fig. 5) (Erneaux et al., 1985; Harden et al., 1985). Muscarinic receptors are coupled either to adenylyl cyclase inhibition through a Gi-protein or to phospholipase C (PLC) activation through Gq. In spite of the fact that two signaling pathways are activated, in both cases a decrease in intracellular cAMP is produced. In those cells where cholinergic agonists stimulate PLC, phosphoinositol (PI) turnover, and inositol tri-

Cyclic nucleotide-O!factory receptor gated Channels

Cyclic nucleotide-O!factory receptor gated Channels

Ctt *-calmodulin regulated PDE

Fig. 6. Mechanism of regulation of PDE1 in the olfactory neurons. Some olfactory neurons express receptors coupled to the cyclase ACIII through a unique G protein (Golg). The cyclic nucleotide gated cationic channels are sensitive to the intracellular cAMP concentration. PDE1C is localized in the cilia, where receptors and cyclases are located. The Ca2+ influx which follows the opening of the channel, activates PDE1C, causing the termination of the stimulus.

phosphate (IP3) production, the resulting increase in intracellular Ca2+ causes the activation of a CaM-PDE. The increase in activity of this enzyme, in turn, produces a net decrease in cAMP concentration, as observed in glyoma cell lines and in the thyroid. In many instances, as in smooth muscle cells, the increase in Ca2+ and the decrease in cAMP have synergistic effects in stimulating contraction.

The cyclic nucleotide signaling in the olfactory system is remarkable because of the time frame in which it operates. While in endocrine cells cAMP increases minutes after peptide hormone stimulation, the cyclic nucleotide spike develops in milliseconds in the olfactory epithelium (Boekhoff and Breer, 1992). It is believed that these rapid responses are possible because of the presence on the olfactory ciliae of cyclic nucleotide gated channels and CaM PDEs in close proximity to the olfactory receptors coupled to cyclases (Fig. 6) (Beavo, 1995; Juilfs et al., 1997). The local increase in cAMP opens a cyclic nucleotide gated cation channel causing an influx in Ca2+. In turn, the Ca2+ influx activates PDE1C, causing a rapid return of cyclic AMP to basal levels. This rapid and transient response is thought to be critical for the correct spatial and temporal perception of the olfactory cue, a discrimination particularly important in animals.

3.2.2. PDE2 and Integration of cGMP and cAMP Regulated Pathways

The two classes of PDEs that are sensitive to cGMP (PDE2 and PDE3) integrate the cyclic GMP and cAMP signaling pathway. In several cells, ligands signal through an increase in intracellular cGMP concentration and also indirectly through changes in cAMP levels. This is, for instance, the case for the atrial natriuretic peptide (ANP), a peptide released by the cardiac atrium, which plays an important function in electrolyte homeostasis. It is established that the major effects of ANP on natriuresis are mediated by the inhibition of aldosterone secretion. In the glo-merulosa cells of the adrenal cortex, ANP binds to and activates a membrane-bound guanylyl cyclase, thus stimulating cGMP production (Fig. 7). It has been shown that the increase in cGMP causes an activation of the cGS-PDE (PDE2) abundantly expressed in the adrenal glomerulosa cells (MacFarland et al., 1991). This cGS-PDE activation causes a decrease in cAMP, inhibiting signaling through this pathway. This type of crosstalk has been proposed to mediate the antagonistic effects of ANP on ACTH-mediated aldosterone release. A similar signaling pathway is thought to control epinephrine release by chromaffin cells of the adrenal medulla (Whalin et al., 1991).

Conversely in other cells such as platelets or smooth muscle cells, an increase in cGMP is associated with a concomitant increase in cAMP (Maurice and Haslam, 1990). In general, cells in which cGMP and cAMP have synergstic effects express a cGI-PDE (PDE3), an enzyme that hydrolyzes predominantly cAMP. Although cGMP can be hydrolyzed by PDE3, the velocity of the reaction is one order of magnitude lower than that for cAMP, so that cGMP, in essence, functions as an inhibitor. An increase in cGMP concentration in these cells therefore blocks cAMP hydrolysis, thus promoting its accumulation. This mechanism is thought to play a role in nitric oxide (NO) signaling and in the control of the vascular smooth muscle tone. In platelets, aggregation is prevented by both an increase in cGMP or cAMP.

3.2.3. PDE3 Role in Insulin and Growth Factor Action

In adipocytes, lipolysis is regulated by agents that activate adenylyl cyclase and that increase intracellu-lar cAMP (i.e., catecholamines and glucagon). The

Ctt *-calmodulin regulated PDE

Lipolysis Anp

Fig. 7. Regulation of PDE2 by the atrial natriuretic peptide in the adrenal glomerulosa cells. The ANP binding guanylyl cyclase on the plasma membrane is shown on the left. ANP binding stimulates cGMP production and activation of the cGMP-stimulated PDE2. This in turn decreases cAMP levels, thus inhibiting the cAMP-stimulated steroidogenesis.

Fig. 7. Regulation of PDE2 by the atrial natriuretic peptide in the adrenal glomerulosa cells. The ANP binding guanylyl cyclase on the plasma membrane is shown on the left. ANP binding stimulates cGMP production and activation of the cGMP-stimulated PDE2. This in turn decreases cAMP levels, thus inhibiting the cAMP-stimulated steroidogenesis.

increase in cAMP activates the hormone sensitive lipase via a PKA-mediated phosphorylation. In a similar fashion, an activation of the cyclic nucleotide dependent pathway in hepatocytes stimulates glyco-genolysis via a PKA-dependent activation of glyco-gen phosphorylase. Insulin is an important physiological regulator of both of these metabolic functions. This hormone inhibits lipolysis and glycogenolysis via suppression of the activity of the two enzymes. Although the exact mechanism of insulin action is still a matter of debate, it is generally accepted that an insulin-dependent decrease in cAMP levels mediates these antilipolytic and antiglycogenolytic effects on these cells. In 1970 Loten and Sneyd first reported that insulin activates cAMP hydrolysis in intact adipocytes. That this increased cAMP hydrolysis is important for insulin action has been confirmed by experiments with nonhydrolyzable cAMP analogs, as insulin does not block lipolysis activated by these analogs (Beavo, 1995). It is now clear that the activity of a PDE3 abundantly expressed in the adipose cells is the primary target for this activation. Degerman et al., 1997 have charted most of the steps of the signaling pathway impinging on this PDE3 in adipocytes (Fig. 8). The PDE3 activation is dependent on the insulin activation of phosphatidylinositol 3'-kinase (PI3K). Downstream of the PI3 kinase, a kinase activity that is activated by insulin and that phosphorylates PDE3B has been identified. On the basis of biochemi cal observations, as well as experiments with reconstitution systems, this kinase is most likely the recently discovered PKB-AKT. Finally, leptin, a recently discovered hormone that controls food intake, also signals through the PI3 kinases-PDE3 pathway. In P cells of the pancreas, leptin causes the activation of PDE3B, which leads to a marked inhibition of insulin secretion stimulated by the glucagon-like peptide-1.

Because the PI3K pathway is shared by many growth factor signaling pathways, it is likely that many growth related signals branch out of the tyro-sine-Ras-MAP kinase cascade to regulate cAMP levels via a PDE3 phosphorylation. This is probably important for regulation of cell replication, as cAMP exerts control over several checkpoints of the cell cycle. In support of this hypothesis, a signaling pathway involving AKT and a PDE3 activation has been charted for the IGF1 activation of resumption of meio-sis in the Xenopus laevis oocyte (Sadler, 1991; Andersen et al., 1998).

3.2.4. Regulation of PDE7 in T Lymphocytes

Antigen stimulation of inflammatory cells involves both activation of stimulatory pathways as well as removal of inhibitory constraints. For instance, activation of peripheral T cells is mediated by occupancy of both the T-cell receptor-CD3 complex and the CD28 costimulatory receptors. When both receptors are occupied, T cells are stimulated to produce inter-

Cd3 Signaling Pathway

Fig. 8. Regulation of PDE3 by insulin. The insulin receptor (IR) tyrosine kinase is activated by insulin binding. The autophosphor-ylation of the receptor recruits the phosphatidylinositol 3'-kinase (PI3K), which increases the local accumulation of phosphatidyl-inositol polyphosphate. This in turn activates two lipid-dependent kinases (not shown) and recruits PKB/akt to the membrane. The phosphorylated and activated PKB/akt phosphorylates PDE3B, causing its activation. The increase in PDE activity decreases cAMP, thus blocking the hormone-sensitive lipase activation.

Fig. 8. Regulation of PDE3 by insulin. The insulin receptor (IR) tyrosine kinase is activated by insulin binding. The autophosphor-ylation of the receptor recruits the phosphatidylinositol 3'-kinase (PI3K), which increases the local accumulation of phosphatidyl-inositol polyphosphate. This in turn activates two lipid-dependent kinases (not shown) and recruits PKB/akt to the membrane. The phosphorylated and activated PKB/akt phosphorylates PDE3B, causing its activation. The increase in PDE activity decreases cAMP, thus blocking the hormone-sensitive lipase activation.

leukin-2 (IL-2) and to proliferate. The stimulatory limb of the pathway involves the activation of the mitogen-activated protein (MAP) kinase pathway and translocation of the nuclear factor of activated T cell (NFAT) to the nucleus. There is ample evidence that the MAP kinase pathway is inhibited by activation of the cAMP-dependent pathway and PKA. NFAT translocation is prevented by PKA phosphorylation. Furthermore, phosphorylation of raf by PKA prevents signaling through the MAP kinase pathway. Several reports have suggested that PDE activation may follow T-cell activation. PDE7 was recently discovered by using a yeast complementation screening. This PDE is expressed in skeletal muscle and in T cells. Recently, it has been reported that T-cell receptor activation causes an induction of PDE7 and that selectively blocking this induction with an antisense oligonucleotide prevents T-cell activation (Li et al., 1999). It is then probable that T-cell activation, among other signals, involves the relief of the cAMP inhibition through activation of PDEs.

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