Phosphodiesterase 4 activity uniquely regulates ciliary cAMP-dependent 3T3-L1 adipogenesis.

Recent evidence indicates that the presence of a primary cilium (PC), and of selective cAMP signaling within this smallest of organelles, promotes adipogenic differentiation of 3T3-L1 preadipocytes incubated in media supplemented with either a natural (docosahexaenoic acid, DHA), or a synthetic (TUG-891), free fatty acid receptor 4 (FFAR4) agonist. Indeed, in this earlier work, activation of ciliary FFAR4 in 3T3-L1 cells was correlated with selective increases in PC cAMP and adipogenesis in these cells. However, this study was silent on the role of local PC cAMP phosphodiesterases (PDEs)-mediated events in regulating these adipogenic responses and on the identity of cAMP PDEs that could regulate the "pool" of ciliary cAMP accessed by FFAR4 agonists. In this context, we have identified the PDEs expressed by 3T3-L1 preadipocytes and showed that of these, only PDE4 inhibition promotes FFAR4-mediated adipogenesis. We propose that this work will identify more selective therapeutic targets through which to control adipogenesis, and perhaps the differentiation of other stem cells in which ciliary cAMP is critical.

Phosphodiesterase 1C integrates store-operated calcium entry and cAMP signaling in leading-edge protrusions of migrating human arterial myocytes.

In addition to maintaining cellular ER Ca2+ stores, store-operated Ca2+ entry (SOCE) regulates several Ca2+-sensitive cellular enzymes, including certain adenylyl cyclases (ADCYs), enzymes that synthesize the secondary messenger cyclic AMP (cAMP). Ca2+, acting with calmodulin, can also increase the activity of PDE1-family phosphodiesterases (PDEs), which cleave the phosphodiester bond of cAMP. Surprisingly, SOCE-regulated cAMP signaling has not been studied in cells expressing both Ca2+-sensitive enzymes. Here, we report that depletion of ER Ca2+ activates PDE1C in human arterial smooth muscle cells (HASMCs). Inhibiting the activation of PDE1C reduced the magnitude of both SOCE and subsequent Ca2+/calmodulin-mediated activation of ADCY8 in these cells. Because inhibiting or silencing Ca2+-insensitive PDEs had no such effects, these data identify PDE1C-mediated hydrolysis of cAMP as a novel and important link between SOCE and its activation of ADCY8. Functionally, we showed that PDE1C regulated the formation of leading-edge protrusions in HASMCs, a critical early event in cell migration. Indeed, we found that PDE1C populated the tips of newly forming leading-edge protrusions in polarized HASMCs, and co-localized with ADCY8, the Ca2+ release activated Ca2+ channel subunit, Orai1, the cAMP-effector, protein kinase A, and an A-kinase anchoring protein, AKAP79. Because this polarization could allow PDE1C to control cAMP signaling in a hyper-localized manner, we suggest that PDE1C-selective therapeutic agents could offer increased spatial specificity in HASMCs over agents that regulate cAMP globally in cells. Similarly, such agents could also prove useful in regulating crosstalk between Ca2+/cAMP signaling in other cells in which dysregulated migration contributes to human pathology, including certain cancers.

Distinct phosphodiesterase 5A-containing compartments allow selective regulation of cGMP-dependent signalling in human arterial smooth muscle cells.

Cyclic GMP (cGMP) translates and integrates much of the information encoded by nitric oxide (NO·) and several natriuretic peptides, including the atrial natriuretic peptide (ANP). Previously, we reported that integration of a cGMP-specific cyclic nucleotide phosphodiesterase, namely phosphodiesterase 5A (PDE5A), into a protein kinase G (PKG)- and inositol-1,4,5-trisphosphate receptor (IP3R)-containing endoplasmic reticulum (ER) signalosome allows localized control of PDE5A activity and of PKG-dependent inhibition of IP3-mediated release of ER Ca2+ in human platelets. Herein, we report that PDE5A integrates into an analogous signalosome in human arterial smooth muscle cells (HASMC), wherein it regulates muscarinic agonist-dependent Ca2+ release and is activated selectively by PKG-dependent phosphorylation. In addition, we report that PDE5A also regulates HASMC functions via events independent of PKG, but rather through actions coordinated by competitive cGMP-mediated inhibition of cAMP hydrolysis by the so-called cGMP-inhibited cAMP PDE, namely phosphodiesterase 3A (PDE3A). Indeed, we show that ANP increases both cGMP and cAMP levels in HASMC and promotes phosphorylation of vasodilator-stimulated phospho-protein (VASP) at each the PKG and PKA phospho-acceptor sites. Since selective inhibition of PDE5 decreased DNA synthesis and chemotaxis of HASMC, and that PDE3A knockdown obviated these effects, our findings are consistent with a role for a PDE5A-PDE3A-PKA axis in their regulation. Our findings provide insight into the existence of distinct "pools" of PDE5A in HASMC and support the idea that these discrete compartments regulate distinct cGMP-dependent events. As a corollary, we suggest that it may be possible to target these distinct PDE5A-regulated pools and in so-doing differentially impact selected cGMP-regulated functions in these cells.

EPAC1 promotes adaptive responses in human arterial endothelial cells subjected to low levels of laminar fluid shear stress: Implications in flow-related endothelial dysfunction.

Blood flow-associated fluid shear stress (FSS) dynamically regulates the endothelium's ability to control arterial structure and function. While arterial endothelial cells (AEC) subjected to high levels of laminar FSS express a phenotype resistant to vascular insults, those exposed to low levels of laminar FSS, or to the FSS associated with oscillatory blood flow, are less resilient. Despite numerous reports highlighting how the cAMP-signaling system controls proliferation, migration and permeability of human AECs (HAECs), its role in coordinating HAEC responses to FSS has received scant attention. Herein we show that the cAMP effector EPAC1 is required for HAECs to align and elongate in the direction of flow, and for the induction of several anti-atherogenic and anti-thrombotic genes associated with these events. Of potential therapeutic importance, EPAC1 is shown to play a dominant role the in response of HAECs to low levels of laminar FSS, such as would be found within atherosclerosis-prone areas of the vasculature. Moreover, we show that EPAC1 promotes these HAEC responses to flow by regulating Vascular Endothelial Growth Factor Receptor-2 and Akt activation, within a VE-cadherin (VECAD)/PECAM1-based mechanosensor. We submit that these findings are consistent with the novel proposition that promoting EPAC1-signaling represents a novel means through which to promote expression of an adaptive phenotype in HAECs exposed to non-adaptive FSS-encoded signals as a consequence of vascular disease.

Cyclic nucleotide phosphodiesterases (PDEs): coincidence detectors acting to spatially and temporally integrate cyclic nucleotide and non-cyclic nucleotide signals.

The cyclic nucleotide second messengers cAMP and cGMP each affect virtually all cellular processes. Although these hydrophilic small molecules readily diffuse throughout cells, it is remarkable that their ability to activate their multiple intracellular effectors is spatially and temporally selective. Studies have identified a critical role for compartmentation of the enzymes which hydrolyse and metabolically inactivate these second messengers, the PDEs (cyclic nucleotide phosphodiesterases), in this specificity. In the present article, we describe several examples from our work in which compartmentation of selected cAMP- or cGMP-hydrolysing PDEs co-ordinate selective activation of cyclic nucleotide effectors, and, as a result, selectively affect cellular functions. It is our belief that therapeutic strategies aimed at targeting PDEs within these compartments will allow greater selectivity than those directed at inhibiting these enzymes throughout the cells.

Cyclic AMP phosphodiesterase 4D (PDE4D) Tethers EPAC1 in a vascular endothelial cadherin (VE-Cad)-based signaling complex and controls cAMP-mediated vascular permeability.

Vascular endothelial cell (VEC) permeability is largely dependent on the integrity of vascular endothelial cadherin (VE-cadherin or VE-Cad)-based intercellular adhesions. Activators of protein kinase A (PKA) or of exchange protein activated by cAMP (EPAC) reduce VEC permeability largely by stabilizing VE-Cad-based intercellular adhesions. Currently, little is known concerning the nature and composition of the signaling complexes that allow PKA or EPAC to regulate VE-Cad-based structures and through these actions control permeability. Using pharmacological, biochemical, and cell biological approaches we identified and determined the composition and functionality of a signaling complex that coordinates cAMP-mediated control of VE-Cad-based adhesions and VEC permeability. Thus, we report that PKA, EPAC1, and cyclic nucleotide phosphodiesterase 4D (PDE4D) enzymes integrate into VE-Cad-based signaling complexes in human arterial endothelial cells. Importantly, we show that protein-protein interactions between EPAC1 and PDE4D serve to foster their integration into VE-Cad-based complexes and allow robust local regulation of EPAC1-based stabilization of VE-Cad-based adhesions. Of potential translational importance, we mapped the EPAC1 peptide motif involved in binding PDE4D and show that a cell-permeable variant of this peptide antagonizes EPAC1-PDE4D binding and directly alters VEC permeability. Collectively, our data indicate that PDE4D regulates both the activity and subcellular localization of EPAC1 and identify a novel mechanism for regulated EPAC1 signaling in these cells.