An EPAC1/PDE1C-Signaling Axis Regulates Formation of Leading-Edge Protrusion in Polarized Human Arterial Vascular Smooth Muscle Cells

Pharmacological activation of protein kinase A (PKA) reduces migration of arterial smooth muscle cells (ASMCs), including those isolated from human arteries (HASMCs). However, when individual migration-associated cellular events, including the polarization of cells in the direction of movement or rearrangements of the actin cytoskeleton, are studied in isolation, these individual events can be either promoted or inhibited in response to PKA activation. While pharmacological inhibition or deficiency of exchange protein activated by cAMP-1 (EPAC1) reduces the overall migration of ASMCs, the impact of EPAC1 inhibition or deficiency, or of its activation, on individual migration-related events has not been investigated. Herein, we report that EPAC1 facilitates the formation of leading-edge protrusions (LEPs) in HASMCs, a critical early event in the cell polarization that underpins their migration. Thus, RNAi-mediated silencing, or the selective pharmacological inhibition, of EPAC1 decreased the formation of LEPs by these cells. Furthermore, we show that the ability of EPAC1 to promote LEP formation by migrating HASMCs is regulated by a phosphodiesterase 1C (PDE1C)-regulated “pool” of intracellular HASMC cAMP but not by those regulated by the more abundant PDE3 or PDE4 activities. Overall, our data are consistent with a role for EPAC1 in regulating the formation of LEPs by polarized HASMCs and show that PDE1C-mediated cAMP hydrolysis controls this localized event.

cAMP metabolism controls caspase-11 inflammasome activation and pyroptosis in sepsis

The ability of cytosolic lipopolysaccharide (LPS) to activate caspase-11–dependent nonclassical inflammasome is intricately controlled to avoid excessive inflammatory responses. However, very little is known about the regulatory role of various metabolic pathways in the control of caspase-11 activation. Here, we demonstrate that l-adrenaline can act on receptor ADRA2B to inhibit the activation of the caspase-11 inflammasome by cytosolic LPS or Escherichia coli infection in macrophages. l-adrenaline–induced cAMP production via the enzyme ADCY4 promotes protein kinase A (PKA) activation, which then blocks the caspase-11–mediated proteolytic maturation of interleukin-1β, gasdermin D (GSDMD) cleavage, and consequent DAMP release. Inhibition of PDE8A-mediated cAMP hydrolysis limits caspase-11 inflammasome activation and pyroptosis in macrophages. Consequently, pharmacological modulation of the ADRA2B-ADCY4-PDE8A-PKA axis, knockout of caspase-11 (Casp11−/−), or Gsdmd inactivation (GsdmdI105N/I105N) similarly protects against LPS-induced lethality in poly(I:C)-primed mice. Our results provide previously unidentified mechanistic insight into immune regulation by cAMP and represent a proof of concept that immunometabolism constitutes a potential therapeutic target in sepsis.

Abstract 049: Phosphodiesterase-3a Catalytic-domain Mutation and Hypertension

We recently discovered phosphodiesterase-3A (PDE3A) mutations causing a 50 mm Hg increase in blood pressure and stroke >50 years, as the first non-salt form of Mendelian genetic hypertension, autosomal-dominant hypertension with brachydactyly (HTNB). The mutations cause increased PDE3A phosphorylation and higher cAMP affinity. We now have found a completely different PDE3A mutation causing a similar syndrome in a New Zealand pedigree. The mutation resides in the enzyme’s catalytic domain, results in an arginine-to-cysteine substitution, and represents a more direct mechanism of PDE3A activation. For Michaelis-Menten kinetics of cAMP hydrolysis, we transfected HEK293 cells transiently expressing Flag-tagged versions of PDE3A1, PDE3A2, or PDE3A3 mutant vs. wildtype and stimulated with forskolin and phorbol-12-myristate-13-acetate (PMA) to enhance intrinsic phosphorylation. Vmax and Km (Michaelis constant) were calculated using GraphPad Prism software to reveal the maximum cAMP turnover rate at saturated substrate concentration and the affinity of cAMP to wildtype and mutated PDE3A1, PDE3A2 and PDE3A3. For PDE3A1 hydrolytic activity (triplicate), we observed: Vmax Km Wildtype 7.5 340 Wildtype+forskolin/PMA 7.2 203 Mutant 6.6 116 Mutant+forskolin/PMA 6.3 81 The dramatically lower Km of mutant PDE3A indicates a substantially greater affinity for cAMP consistent with gain-of-function. These data underscore the importance of PDE3A to high blood pressure by means of a different, novel genetic mechanism directly implicating the catalytic domain.

Estimating the magnitude of near-membrane PDE4 activity in living cells

Recent studies have demonstrated that functionally discrete pools of phosphodiesterase (PDE) activity regulate distinct cellular functions. While the importance of localized pools of enzyme activity has become apparent, few studies have estimated enzyme activity within discrete subcellular compartments. Here we present an approach to estimate near-membrane PDE activity. First, total PDE activity is measured using traditional PDE activity assays. Second, known cAMP concentrations are dialyzed into single cells and the spatial spread of cAMP is monitored using cyclic nucleotide-gated channels. Third, mathematical models are used to estimate the spatial distribution of PDE activity within cells. Using this three-tiered approach, we observed two pharmacologically distinct pools of PDE activity, a rolipram-sensitive pool and an 8-methoxymethyl IBMX (8MM-IBMX)-sensitive pool. We observed that the rolipram-sensitive PDE (PDE4) was primarily responsible for cAMP hydrolysis near the plasma membrane. Finally, we observed that PDE4 was capable of blunting cAMP levels near the plasma membrane even when 100 μM cAMP were introduced into the cell via a patch pipette. Two compartment models predict that PDE activity near the plasma membrane, near cyclic nucleotide-gated channels, was significantly lower than total cellular PDE activity and that a slow spatial spread of cAMP allowed PDE activity to effectively hydrolyze near-membrane cAMP. These results imply that cAMP levels near the plasma membrane are distinct from those in other subcellular compartments; PDE activity is not uniform within cells; and localized pools of AC and PDE activities are responsible for controlling cAMP levels within distinct subcellular compartments.

Abstract 054: Mutations In Pde3a Explain Mendelian Hypertension With Brachydactyly

We identified Mendelian type E brachydactyly (BDE) with hypertension in six different families from Turkey, America, France, Canada and South Africa. The two phenotypes, hypertension and BDE, invariably coincide. The blood pressure in affected individuals increases with increasing age, the mean arterial blood pressure at 50 years exceeds 150 mm Hg, resulting in stroke. Earlier we suggested that a chromosomal rearrangement on 12p could be responsible. We now used whole-genome sequencing and discovered six different so far unknown missense mutations in the gene encoding PDE3A. The mutations are not identical, but adjacent to each other. Each is responsible for an amino acid substitution in a conserved portion of the protein. The mutations were not present in any non-affected family members, in more than 200 additional controls, and not found in the “1000 genome” project. We performed in vitro cell transfections and found that mutated PDE3A showed gain-of-function with increased cAMP hydrolysis. Mesenchymal stromal cell-derived vascular smooth muscle cells and chondrocytes gave insight into the molecular pathogenesis; PDE3A and vasodilator-stimulated phosphoprotein (VASP) were differently phosphorylated and parathyroid hormone-related peptide (PTHrP) was dysregulated. Our preliminary results reveal that the mutated PDE3A enzymes show gain-of-function with increased cAMP hydrolysis and sensitivity to cGMP inhibition. We suggest that the mutations are responsible for both phenotypes. This Mendelian hypertension is the first that is not attributable to increased sodium reabsorption in the distal nephron.

PDE2-mediated cAMP hydrolysis accelerates cardiac fibroblast to myofibroblast conversion and is antagonized by exogenous activation of cGMP signaling pathways

Recent studies suggest that the signal molecules cAMP and cGMP have antifibrotic effects by negatively regulating pathways associated with fibroblast to myofibroblast (MyoCF) conversion. The phosphodiesterase 2 (PDE2) has the unique property to be stimulated by cGMP, which leads to a remarkable increase in cAMP hydrolysis and thus mediates a negative cross-talk between both pathways. PDE2 has been recently investigated in cardiomyocytes; here we specifically addressed its role in fibroblast conversion and cardiac fibrosis. PDE2 is abundantly expressed in both neonatal rat cardiac fibroblasts (CFs) and cardiomyocytes. The overexpression of PDE2 in CFs strongly reduced basal and isoprenaline-induced cAMP synthesis, and this decrease was sufficient to induce MyoCF conversion even in the absence of exogenous profibrotic stimuli. Functional stress-strain experiments with fibroblast-derived engineered connective tissue (ECT) demonstrated higher stiffness in ECTs overexpressing PDE2. In regard to cGMP, neither basal nor atrial natriuretic peptide-induced cGMP levels were affected by PDE2, whereas the response to nitric oxide donor sodium nitroprusside was slightly but significantly reduced. Interestingly, despite persistently depressed cAMP levels, both cGMP-elevating stimuli were able to completely prevent the PDE2-induced MyoCF phenotype, arguing for a double-tracked mechanism. In conclusion, PDE2 accelerates CF to MyoCF conversion, which leads to greater stiffness in ECTs. Atrial natriuretic peptide- and sodium nitroprusside-mediated cGMP synthesis completely reverses PDE2-induced fibroblast conversion. Thus PDE2 may augment cardiac remodeling, but this effect can also be overcome by enhanced cGMP. The redundant role of cAMP and cGMP as antifibrotic meditators may be viewed as a protective mechanism in heart failure.

Molecular control of oocyte meiotic arrest and resumption

Mammalian oocytes within Graafian follicles are arrested at prophase I by factors from surrounding follicle cells, and resume meiosis after an LH surge from the pituitary. The maintenance of meiotic arrest requires high levels of cAMP, resulting from G-protein-coupled receptor (GPR) 3 and/or GPR12 activation of adenylyl cyclase within the oocyte. Recent studies indicate that natriuretic peptide precursor C (NPPC), acting via its cognate receptor NPR2, increases cGMP levels in granulosa cells; the cGMP then diffuses into oocytes and inhibits phosphodiesterase 3A activity and cAMP hydrolysis. Meiotic resumption is induced by LH via the generation of epidermal growth factor (EGF)-like growth factors in mural granulosa cells that activate EGF receptors in cumulus cells. However, the exact mechanisms underlying the actions of these growth factors on oocyte maturation are unclear. Herein we summarise the regulatory functions of NPPC and NPR2 in maintaining oocyte meiotic arrest and discuss the possibility that LH could stimulate meiotic resumption by decreasing NPPC content and NPR2 activity.

Functional Proteomic Profiling of Phosphodiesterases Using SeraFILE Separations Platform

Functional proteomic profiling can help identify targets for disease diagnosis and therapy. Available methods are limited by the inability to profile many functional properties measured by enzymes kinetics. The functional proteomic profiling approach proposed here seeks to overcome such limitations. It begins with surface-based proteome separations of tissue/cell-line extracts, using SeraFILE, a proprietary protein separations platform. Enzyme kinetic properties of resulting subproteomes are then characterized, and the data integrated into proteomic profiles. As a model, SeraFILE-derived subproteomes of cyclic nucleotide-hydrolyzing phosphodiesterases (PDEs) from bovine brain homogenate (BBH) and rat brain homogenate (RBH) were characterized for cAMP hydrolysis activity in the presence (challenge condition) and absence of cGMP. Functional profiles of RBH and BBH were compiled from the enzyme activity response to the challenge condition in each of the respective subproteomes. Intersample analysis showed that comparable profiles differed in only a few data points, and that distinctive subproteomes can be generated from comparable tissue samples from different animals. These results demonstrate that the proposed methods provide a means to simplify intersample differences, and to localize proteins attributable to sample-specific responses. It can be potentially applied for disease and nondisease sample comparison in biomarker discovery and drug discovery profiling.

Thrombin regulates intracellular cyclic AMP concentration in human platelets through phosphorylation/activation of phosphodiesterase 3A

Thrombin-induced cyclic AMP (cAMP) reduction potentates several steps in platelet activation, including Ca++ mobilization, cytoskeletal reorganization, and fibrinogen receptor conformation. We now reinvestigate the signaling pathways by which intracellular cAMP content is controlled after platelet activation by thrombin. When washed human platelets were stimulated with thrombin, cAMP-dependent phosphodiesterase (PDE3A) activity was significantly increased. A nonselective PDE inhibitor, 3-isobutyl-1-methylxanthine (IBMX), and the PDE3 selective inhibitors milrinone and cilostazol each suppressed thrombin-induced cAMP-dependent PDE responses, but not 2 different PDE2 inhibitors. Selective inhibition of PDE3A resulted in reversal of thrombin-induced cAMP reduction, indicating that thrombin activated PDE3A. In synergy with inhibition of adenylate cyclase by thrombin, activated PDE3A accelerates cAMP hydrolysis and maximally reduces the cAMP content. Thrombin-induced PDE3A activation was diminished concomitantly with dephosphorylation of PDE3A by protein phosphatase 1 (PP1). An Akt inhibitor blocked PDE3A activation and constrained thrombin-induced cAMP reduction. A P2Y12 inhibitor also reduced thrombin-induced cAMP reduction. The combination of both reversed cAMP decrease by thrombin. Thrombin-mediated phosphorylated PDE3A was isolated by liquid chromatography, detected by a monoclonal antibody against Akt-phosphorylated substrate, and verified by immunoprecipitation study. The predominant isoform phosphorylated by Akt was the 136-kDa species. We suggest that activation/phosphorylation of PDE3A via Akt signaling pathway participates in regulating cAMP during thrombin activation of platelets.