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Phosphodiesterase 3 (PDE3) and PDE4 regulate levels of cyclic AMP, which are critical in various cell types involved in allergic airway inflammation. Although PDE4 inhibition attenuates allergic airway inflammation, reported side effects preclude its application as an antiasthma drug in humans. Case reports showed that enoximone, which is a smooth muscle relaxant that inhibits PDE3, is beneficial and lifesaving in status asthmaticus and is well tolerated. However, clinical observations also showed antiinflammatory effects of PDE3 inhibition. In this study, we investigated the role of PDE3 in a house dust mite-driven (HDM-driven) allergic airway inflammation (AAI) model that is characterized by T helper 2 cell activation, eosinophilia, and reduced mucosal barrier function. Compared with wild-type (WT) littermates, mice with a targeted deletion of the PDE3A or PDE3B gene showed significantly reduced HDM-driven AAI. Therapeutic intervention in WT mice showed that all hallmarks of HDM-driven AAI were abrogated by the PDE3 inhibitors enoximone and milrinone. Importantly, we found that enoximone also reduced the upregulation of the CD11b integrin on mouse and human eosinophils in vitro, which is crucial for their recruitment during allergic inflammation. This study provides evidence for a hitherto unknown antiinflammatory role of PDE3 inhibition in allergic airway inflammation and offers a potentially novel treatment approach.

Phosphodiesterase 3A (PDE3A), a member of the cGMP-inhibited cyclic nucleotide phosphodiesterase (PDE) family, plays important roles in oocyte maturation and vascular smooth muscle cell proliferation. However, the molecular mechanisms that regulate PDE3A gene expression remain largely unknown. In this study, we investigated the transcriptional regulation of PDE3A , and found that the splicing factor proline and glutamine rich (SFPQ) protein modulated PDE3A mRNA levels. Multiple transcription start sites (TSS1, 2, and 3) were identified within the first exon of PDE3A using 5'-rapid amplification of cDNA ends (RACE). Variable expression levels of three PDE3A variants were also observed in human tissues and HeLa cells. Several putative SFPQ-binding sites were identified upstream of the regulatory region of PDE3A -TSSs using chromatin immunoprecipitation sequencing (ChIP-seq). Serum-induced PDE3A expression was affected by increasing the amount of SFPQ binding to the upstream regulatory region of PDE3A In addition, transcription of PDE3A was lower in human cervical adenocarcinoma cells compared to normal cervical tissue. Furthermore, over-expression of PDE3A induced sensitivity to anti-cancer therapeutic agent, 6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (DNMDP), in HeLa cells. Taken together, these results suggest that SFPQ functions as a transcriptional activator of PDE3A, which is involved in the regulation of DNMDP sensitivity , offering a novel molecular target for the development of anticancer therapies., (©2017 The Author(s).)

We previously identified phosphodiesterase 3A (PDE3A) as a marker for interstitial cells of Cajal (ICC) in adult mouse gut. However, PDE3A expression and function during gut development and in ICC-derived gastrointestinal stromal tumors (GIST) remained unknown. Here we found that PDE3A was expressed throughout ICC development and that ICC density was halved in PDE3A-deficient mice. In the human imatinib-sensitive GIST882 cell line, the PDE3 inhibitor cilostazol halved cell viability (IC50 0.35 μM) and this effect synergized with imatinib (Chou-Talalay's CI50 0.15). Recently the compound 6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one, or DNMDP was found to be cytotoxic selectively for cells expressing both PDE3A and Schlafen12 (SLFN12) (de Waal L et al. Nat Chem Bio 2016), identifying a new, non-catalytic, role for PDE3A. 108 out of 117 (92%) of our human GIST samples displayed both PDE3A and SLFN12 immunoreactivity. GIST882 cells express both PDE3A and SLFN12 and DNMDP decreased their viability by 90%. Our results suggest a role for PDE3A during ICC development and open novel perspectives for PDE3A in targeted GIST therapy, on one hand by the synergism between imatinib and cilostazol, a PDE3 inhibitor already in clinical use for other indications, and, on the other hand, by the neomorphic, druggable, PDE3A-SLFN12 cytotoxic interplay.

Understanding mechanisms by which a population of beige adipocytes is increased in white adipose tissue (WAT) reflects a potential strategy in the fight against obesity and diabetes. Cyclic adenosine monophosphate (cAMP) is very important in the development of the beige phenotype and activation of its thermogenic program. To study effects of cyclic nucleotides on energy homeostatic mechanisms, mice were generated by targeted inactivation of cyclic nucleotide phosphodiesterase 3b (Pde3b) gene, which encodes PDE3B, an enzyme that catalyzes hydrolysis of cAMP and cGMP and is highly expressed in tissues that regulate energy homeostasis, including adipose tissue, liver, and pancreas. In epididymal white adipose tissue (eWAT) of PDE3B KO mice on a SvJ129 background, cAMP/protein kinase A (PKA) and AMP-activated protein kinase (AMPK) signaling pathways are activated, resulting in "browning" phenotype, with a smaller increases in body weight under high-fat diet, smaller fat deposits, increased β-oxidation of fatty acids (FAO) and oxygen consumption. Results reported here suggest that PDE3B and/or its downstream signaling partners might be important regulators of energy metabolism in adipose tissue, and potential therapeutic targets for treating obesity, diabetes and their associated metabolic disorders.

Oxidative stress plays a pivotal role in pathogenesis of cardiovascular diseases and diabetes; however, the roles of protein kinase A (PKA) and human phosphodiesterase 3A (hPDE3A) remain unknown. Here, we show that yeast expressing wild-type (WT) hPDE3A or K13R hPDE3A (putative ubiquitinylation site mutant) exhibited resistance or sensitivity to exogenous hydrogen peroxide (H 2 O 2 ), respectively. H 2 O 2 -stimulated ROS production was markedly increased in yeast expressing K13R hPDE3A (Oxidative stress Sensitive 1, OxiS1), compared with yeast expressing WT hPDE3A (Oxidative stress Resistant 1, OxiR1). In OxiR1, YAP1 and YAP1-dependent antioxidant genes were up-regulated, accompanied by a reduction in thioredoxin peroxidase. In OxiS1, expression of YAP1 and YAP1-dependent genes was impaired, and the thioredoxin system malfunctioned. H 2 O 2 increased cyclic adenosine monophosphate (cAMP)-hydrolyzing activity of WT hPDE3A, but not K13R hPDE3A, through PKA-dependent phosphorylation of hPDE3A, which was correlated with its ubiquitinylation. The changes in antioxidant gene expression did not directly correlate with differences in cAMP-PKA signaling. Despite differences in their capacities to hydrolyze cAMP, total cAMP levels among OxiR1, OxiS1, and mock were similar; PKA activity, however, was lower in OxiS1 than in OxiR1 or mock. During exposure to H 2 O 2 , however, Sch9p activity, a target of Rapamycin complex 1-regulated Rps6 kinase and negative-regulator of PKA, was rapidly reduced in OxiR1, and Tpk1p, a PKA catalytic subunit, was diffusely spread throughout the cytosol, with PKA activation. In OxiS1, Sch9p activity was unchanged during exposure to H 2 O 2 , consistent with reduced activation of PKA. These results suggest that, during oxidative stress, TOR-Sch9 signaling might regulate PKA activity, and that post-translational modifications of hPDE3A are critical in its regulation of cellular recovery from oxidative stress., (© 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

Activation of inflammation in white adipose tissue (WAT), includes infiltration/expansion of WAT macrophages, contributes pathogenesis of obesity, insulin resistance, and metabolic syndrome. The inflammasome comprises an intracellular sensor (NLR), caspase-1 and the adaptor ASC. Inflammasome activation leads to maturation of caspase-1 and processing of IL1β, contributing to many metabolic disorders and directing adipocytes to a more insulin-resistant phenotype. Ablation of PDE3B in WAT prevents inflammasome activation by reducing expression of NLRP3, caspase-1, ASC, AIM2, TNFα, IL1β and proinflammatory genes. Following IP injection of lipopolysaccharide (LPS), serum levels of IL1β and TNFα were reduced in PDE3B(-/-)mice compared to WT. Activation of signaling cascades, which mediate inflammasome responses, were modulated in PDE3B(-/-)mice WAT, including smad, NFAT, NFkB, and MAP kinases. Moreover, expression of chemokine CCL2, MCP-1 and its receptor CCR2, which play an important role in macrophage chemotaxis, were reduced in WAT of PDE3B(-/-)mice. In addition, atherosclerotic plaque formation was significantly reduced in the aorta of apoE(-/-)/PDE3B(-/-)and LDL-R(-/-)/PDE3B(-/-)mice compared to apoE(-/-)and LDL-R(-/-)mice, respectively. Obesity-induced changes in serum-cholesterol were blocked in PDE3B(-/-)mice. Collectively, these data establish a role for PDE3B in modulating inflammatory response, which may contribute to a reduced inflammatory state in adipose tissue.

Aberrant intracellular calcium levels and increased cAMP signaling contribute to the development of polycystic kidney disease (PKD). cAMP can be hydrolyzed by various phosphodiesterases (PDEs). To examine the role of cAMP hydrolysis and the most relevant PDEs in the pathogenesis of PKD, we examined cyst development in Pde1- or Pde3-knockout mice on the Pkd2(-/WS25) background (WS25 is an unstable Pkd2 allele). These PDEs were selected because of their importance in cross-talk between calcium and cyclic nucleotide signaling (PDE1), control of cell proliferation and cystic fibrosis transmembrane conductance regulator (CFTR) -driven fluid secretion (PDE3), and response to vasopressin V2 receptor activation (both). In Pkd2(-/WS25) mice, knockout of Pde1a, Pde1c, or Pde3a but not of Pde1b or Pde3b aggravated the development of PKD and was associated with higher levels of protein kinase A-phosphorylated (Ser133) cAMP-responsive binding protein (P-CREB), activating transcription factor-1, and CREB-induced CRE modulator proteins in kidney nuclear preparations. Immunostaining also revealed higher expression of P-CREB in Pkd2(-/) (WS25);Pde1a(-/-), Pkd2(-) (/WS25);Pde1c(-/-), and Pkd2(-/) (WS25);Pde3a(-/-) kidneys. The cystogenic effect of desmopressin administration was markedly enhanced in Pkd2(-/WS25);Pde3a(-/-) mice, despite PDE3 accounting for only a small fraction of renal cAMP PDE activity. These observations show that calcium- and calmodulin-dependent PDEs (PDE1A and PDE1C) and PDE3A modulate the development of PKD, possibly through the regulation of compartmentalized cAMP pools that control cell proliferation and CFTR-driven fluid secretion. Treatments capable of increasing the expression or activity of these PDEs may, therefore, retard the development of PKD., (Copyright © 2016 by the American Society of Nephrology.)

Pulmonary tuberculosis (TB) is characterized by oxidative stress and lung tissue destruction by matrix metalloproteinases (MMPs). The interplay between these distinct pathological processes and the implications for TB diagnosis and disease staging are poorly understood. Heme oxygenase-1 (HO-1) levels were previously shown to distinguish active from latent TB, as well as successfully treated Mycobacterium tuberculosis infection. MMP-1 expression is also associated with active TB. In this study, we measured plasma levels of these two important biomarkers in distinct TB cohorts from India and Brazil. Patients with active TB expressed either very high levels of HO-1 and low levels of MMP-1 or the converse. Moreover, TB patients with either high HO-1 or MMP-1 levels displayed distinct clinical presentations, as well as plasma inflammatory marker profiles. In contrast, in an exploratory North American study, inversely correlated expression of HO-1 and MMP-1 was not observed in patients with other nontuberculous lung diseases. To assess possible regulatory interactions in the biosynthesis of these two enzymes at the cellular level, we studied the expression of HO-1 and MMP-1 in M. tuberculosis-infected human and murine macrophages. We found that infection of macrophages with live virulent M. tuberculosis is required for robust induction of high levels of HO-1 but not MMP-1. In addition, we observed that CO, a product of M. tuberculosis-induced HO-1 activity, inhibits MMP-1 expression by suppressing c-Jun/AP-1 activation. These findings reveal a mechanistic link between oxidative stress and tissue remodeling that may find applicability in the clinical staging of TB patients., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Although inhibition of cyclic nucleotide phosphodiesterase type 3 (PDE3) has been reported to protect rodent heart against ischemia/reperfusion (I/R) injury, neither the specific PDE3 isoform involved nor the underlying mechanisms have been identified. Targeted disruption of PDE3 subfamily B (PDE3B), but not of PDE3 subfamily A (PDE3A), protected mouse heart from I/R injury in vivo and in vitro, with reduced infarct size and improved cardiac function. The cardioprotective effect in PDE3B(-/-) heart was reversed by blocking cAMP-dependent PKA and by paxilline, an inhibitor of mitochondrial calcium-activated K channels, the opening of which is potentiated by cAMP/PKA signaling. Compared with WT mitochondria, PDE3B(-/-) mitochondria were enriched in antiapoptotic Bcl-2, produced less reactive oxygen species, and more frequently contacted transverse tubules where PDE3B was localized with caveolin-3. Moreover, a PDE3B(-/-) mitochondrial fraction containing connexin-43 and caveolin-3 was more resistant to Ca(2+)-induced opening of the mitochondrial permeability transition pore. Proteomics analyses indicated that PDE3B(-/-) heart mitochondria fractions were enriched in buoyant ischemia-induced caveolin-3-enriched fractions (ICEFs) containing cardioprotective proteins. Accumulation of proteins into ICEFs was PKA dependent and was achieved by ischemic preconditioning or treatment of WT heart with the PDE3 inhibitor cilostamide. Taken together, these findings indicate that PDE3B deletion confers cardioprotective effects because of cAMP/PKA-induced preconditioning, which is associated with the accumulation of proteins with cardioprotective function in ICEFs. To our knowledge, our study is the first to define a role for PDE3B in cardioprotection against I/R injury and suggests PDE3B as a target for cardiovascular therapies.

Cyclic nucleotide phosphodiesterases (PDEs) regulate the intracellular concentrations and effects of adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP). The role of PDEs in malignant tumor cells is still uncertain. The role of PDEs, especially PDE2, in human malignant melanoma PMP cell line was examined in this study. In PMP cells, 8-bromo-cAMP, a cAMP analog, inhibited cell growth and invasion. However, 8-bromo-cGMP, a cGMP analog, had little or no effect. PDE2 and PDE4, but not PDE3, were expressed in PMP cells. Growth and invasion of PMP cells were inhibited by erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), a specific PDE2 inhibitor, but not by rolipram, a specific PDE4 inhibitor. Moreover, cell growth and invasion were inhibited by transfection of small interfering RNAs (siRNAs) specific for PDE2A and a catalytically-dead mutant of PDE2A. After treating cells with EHNA or rolipram, intracellular cAMP concentrations were increased. Growth and invasion were stimulated by PKA14-22, a PKA inhibitor, and inhibited by N(6)-benzoyl-c AMP, a PKA specific cAMP analog, whereas 8-(4-chlorophenylthio)-2'-O-methyl-cAMP, an Epac specific cAMP analog, did not. Invasion, but not growth, was stimulated by A-kinase anchor protein (AKAP) St-Ht31 inhibitory peptide. Based on these results, PDE2 appears to play an important role in growth and invasion of the human malignant melanoma PMP cell line. Selectively suppressing PDE2 might possibly inhibit growth and invasion of other malignant tumor cell lines., (Copyright © 2014. Published by Elsevier Inc.)

Cyclic nucleotide phosphodiesterases (PDEs) catalyse the hydrolysis of cyclic AMP and cyclic GMP, thereby regulating the intracellular concentrations of these cyclic nucleotides, their signalling pathways and, consequently, myriad biological responses in health and disease. Currently, a small number of PDE inhibitors are used clinically for treating the pathophysiological dysregulation of cyclic nucleotide signalling in several disorders, including erectile dysfunction, pulmonary hypertension, acute refractory cardiac failure, intermittent claudication and chronic obstructive pulmonary disease. However, pharmaceutical interest in PDEs has been reignited by the increasing understanding of the roles of individual PDEs in regulating the subcellular compartmentalization of specific cyclic nucleotide signalling pathways, by the structure-based design of novel specific inhibitors and by the development of more sophisticated strategies to target individual PDE variants.

Inhibitors of cyclic nucleotide phosphodiesterase (PDE) PDE3A have inotropic actions in human myocardium, but their long-term use increases mortality in patients with heart failure. Two isoforms in cardiac myocytes, PDE3A1 and PDE3A2, have identical amino acid sequences except for a unique N-terminal extension in PDE3A1. We expressed FLAG-tagged PDE3A1 and PDE3A2 in HEK293 cells and examined their regulation by PKA- and PKC-mediated phosphorylation. PDE3A1, which is localized to intracellular membranes, and PDE3A2, which is cytosolic, were phosphorylated at different sites within their common sequence. Exposure to isoproterenol led to phosphorylation of PDE3A1 at the 14-3-3-binding site S312, whereas exposure to PMA led to phosphorylation of PDE3A2 at an alternative 14-3-3-binding site, S428. PDE3A2 activity was stimulated by phosphorylation at S428, whereas PDE3A1 activity was not affected by phosphorylation at either site. Phosphorylation of PDE3A1 by PKA and of PDE3A2 by PKC led to shifts in elution on gel-filtration chromatography consistent with increased interactions with other proteins, and 2D electrophoresis of coimmunoprecipitated proteins revealed that the two isoforms have distinct protein interactomes. A similar pattern of differential phosphorylation of endogenous PDE3A1 and PDE3A2 at S312 and S428 is observed in human myocardium. The selective phosphorylation of PDE3A1 and PDE3A2 at alternative sites through different signaling pathways, along with the different functional consequences of phosphorylation for each isoform, suggest they are likely to have distinct roles in cyclic nucleotide-mediated signaling in human myocardium, and raise the possibility that isoform-selective inhibition may allow inotropic responses without an increase in mortality.

The superfamily of cyclic nucleotide phosphodiesterases is comprised of 11 gene families. By hydrolyzing cAMP and cGMP, PDEs are major determinants in the regulation of intracellular concentrations of cyclic nucleotides and cyclic nucleotide-dependent signaling pathways. Two PDE3 subfamilies, PDE3A and PDE3B, have been described. PDE3A and PDE3B hydrolyze cAMP and cGMP with high affinity in a mutually competitive manner and are regulators of a number of important cAMP- and cGMP-mediated processes. PDE3B is relatively more highly expressed in cells of importance for the regulation of energy homeostasis, including adipocytes, hepatocytes, and pancreatic β-cells, whereas PDE3A is more highly expressed in heart, platelets, vascular smooth muscle cells, and oocytes. Major advances have been made in understanding the different physiological impacts and biochemical basis for recruitment and subcellular localizations of different PDEs and PDE-containing macromolecular signaling complexes or signalosomes. In these discrete compartments, PDEs control cyclic nucleotide levels and regulate specific physiological processes as components of individual signalosomes which are tethered at specific locations and which contain PDEs together with cyclic nucleotide-dependent protein kinases (PKA and PKG), adenylyl cyclases, Epacs (guanine nucleotide exchange proteins activated by cAMP), phosphoprotein phosphatases, A-Kinase anchoring proteins (AKAPs), and pathway-specific regulators and effectors. This article highlights the identification of different PDE3A- and PDE3B-containing signalosomes in specialized subcellular compartments, which can increase the specificity and efficiency of intracellular signaling and be involved in the regulation of different cAMP-mediated metabolic processes., (© Georg Thieme Verlag KG Stuttgart · New York.)

Cyclic nucleotide phosphodiesterase 3 (PDE3) is an important regulator of cyclic adenosine monophosphate (cAMP) signaling within the cardiovascular system. In this study, we examined the role of PDE3A and PDE3B isoforms in regulation of growth of cultured vascular smooth muscle cells (VSMCs) and the mechanisms by which they may affect signaling pathways that mediate mitogen-induced VSMC proliferation. Serum- and PDGF-induced DNA synthesis in VSMCs grown from aortas of PDE3A-deficient (3A-KO) mice was markedly less than that in VSMCs from PDE3A wild type (3A-WT) and PDE3B-deficient (3B-KO) mice. The reduced growth response was accompanied by significantly less phosphorylation of extracellular signal-regulated kinase (ERK) in 3A-KO VSMCs, most likely due to a combination of greater site-specific inhibitory phosphorylation of Raf-1(Ser-²⁵⁹) by protein kinase A (PKA) and enhanced dephosphorylation of ERKs due to elevated mitogen-activated protein kinase phosphatase 1 (MKP-1). Furthermore, 3A-KO VSMCs, compared with 3A-WT, exhibited higher basal PKA activity and cAMP response element-binding protein (CREB) phosphorylation, higher levels of p53 and p53 phosphorylation, and elevated p21 protein together with lower levels of Cyclin-D1 and retinoblastoma (Rb) protein and Rb phosphorylation. Adenoviral overexpression of inactive CREB partially restored growth effects of serum in 3A-KO VSMCs. In contrast, exposure of 3A-WT VSMCs to VP16 CREB (active CREB) was associated with inhibition of serum-induced DNA synthesis similar to that in untreated 3A-KO VSMCs. Transfection of 3A-KO VSMCs with p53 siRNA reduced p21 and MKP-1 levels and completely restored growth without affecting amounts of Cyclin-D1 and Rb phosphorylation. We conclude that PDE3A regulates VSMC growth via two complementary pathways, i.e. PKA-catalyzed inhibitory phosphorylation of Raf-1 with resulting inhibition of MAPK signaling and PKA/CREB-mediated induction of p21, leading to G₀/G₁ cell cycle arrest, as well as by increased accumulation of p53, which induces MKP-1, p21, and WIP1, leading to inhibition of G₁ to S cell cycle progression.

As shown by transgenic mouse models and by using phosphodiesterase 3 (PDE3) inhibitors, PDE3B has an important role in the regulation of insulin secretion in pancreatic β-cells. However, very little is known about the regulation of the enzyme. Here, we show that PDE3B is activated in response to high glucose, insulin and cAMP elevation in rat pancreatic islets and INS-1 (832/13) cells. Activation by glucose was not affected by the presence of diazoxide. PDE3B activation was coupled to an increase as well as a decrease in total phosphorylation of the enzyme. In addition to PDE3B, several other PDEs were detected in human pancreatic islets: PDE1, PDE3, PDE4C, PDE7A, PDE8A and PDE10A. We conclude that PDE3B is activated in response to agents relevant for β-cell function and that activation is linked to increased as well as decreased phosphorylation of the enzyme. Moreover, we conclude that several PDEs are present in human pancreatic islets.

In adipocytes, PDE3B (phosphodiesterase 3B) is an important regulatory effector in signalling pathways controlled by insulin and cAMP-increasing hormones. Stimulation of 3T3-L1 adipocytes with insulin or the beta3-adrenergic receptor agonist CL316243 (termed CL) indicated that insulin preferentially phosphorylated/activated PDE3B associated with internal membranes (endoplasmic reticulum/Golgi), whereas CL preferentially phosphorylated/activated PDE3B associated with caveolae. siRNA (small interfering RNA)-mediated KD (knockdown) of CAV-1 (caveolin-1) in 3T3-L1 adipocytes resulted in down-regulation of expression of membrane-associated PDE3B. Insulin-induced activation of PDE3B was reduced, whereas CL-mediated activation was almost totally abolished. Similar results were obtained in adipocytes from Cav-1-deficient mice. siRNA-mediated KD of CAV-1 in 3T3-L1 adipocytes also resulted in inhibition of CL-stimulated phosphorylation of HSL (hormone-sensitive lipase) and perilipin A, and of lipolysis. Superose 6 gel-filtration chromatography of solubilized membrane proteins from adipocytes stimulated with insulin or CL demonstrated the reversible assembly of distinct macromolecular complexes that contained 32P-phosphorylated PDE3B and signalling molecules thought to be involved in its activation. Insulin- and CL-induced macromolecular complexes were enriched in cholesterol, and contained certain common signalling proteins [14-3-3, PP2A (protein phosphatase 2A) and cav-1]. The complexes present in insulin-stimulated cells contained tyrosine-phosphorylated IRS-1 (insulin receptor substrate 1) and its downstream signalling proteins, whereas CL-activated complexes contained beta3-adrenergic receptor, PKA-RII [PKA (cAMP-dependent protein kinase)-regulatory subunit] and HSL. Insulin- and CL-mediated macromolecular complex formation was significantly inhibited by CAV-1 KD. These results suggest that cav-1 acts as a molecular chaperone or scaffolding molecule in cholesterol-rich lipid rafts that may be necessary for the proper stabilization and activation of PDE3B in response to CL and insulin.

Background: Agents that target pro-inflammatory cytokines may be useful in pulmonary sarcoidosis., Objective: To determine effectiveness of a non-selective cyclic nucleotide phosphodiesterase (PDE) inhibitor, pentoxifylline (POF)., Design: Randomized, double-blind, placebo-controlled trial,, Setting: Clinical Research Center, National Institutes of Health., Patients: 27 patients with biopsy-confirmed pulmonary sarcoidosis receiving prednisone., Intervention: Placebo or POF (1200-2000 mg/day) for 10 months, as prednisone was tapered., Measurements: Primary endpoints: sustained improvement in two or more pulmonary function parameters, or a combination of one pulmonary function parameter and dyspnea., Results: Except for one patient, primary endpoints were not reached in POF-treated patients. Therefore, a post hoc analysis was performed. The observed relative risk reduction for flares associated with POF treatment was 54.9% (95% CI 0.21, 0.89) and the absolute risk reduction was 50.6% (95% CI 0.22, 0.80). Compared to placebo treatment, in the POF group, the mean prednisone dose was lower at 8 and 10 months (p = 0.007 and 0.01 respectively), and there was a trend towards less prednisone usage over the entire study period (p = 0.053), as determined by cumulative change analysis., Conclusions: Although our exploratory post hoc analysis suggested that POF reduced flares and had steroid-sparing effects, given the study limitations, definitive conclusions cannot be drawn regarding the efficacy of POF in pulmonary sarcoidosis. In addition, gastrointestinal side-effects, at the doses used, would seem to limit the use of POF in treating pulmonary sarcoidosis. Overall, however, this trial may provide a basis for using more specific, better-tolerated, PDE inhibitors in future clinical trials.

ADP-ribosylation factors (ARFs) have crucial roles in vesicular trafficking. Brefeldin A-inhibited guanine nucleotide-exchange proteins (BIG)1 and BIG2 catalyze the activation of class I ARFs by accelerating replacement of bound GDP with GTP. Several additional and differing actions of BIG1 and BIG2 have been described. These include the presence in BIG2 of 3 A kinase-anchoring protein (AKAP) domains, one of which is identical in BIG1. Proteins that contain AKAP sequences act as scaffolds for the assembly of PKA with other enzymes, substrates, and regulators in complexes that constitute molecular machines for the reception, transduction, and integration of signals from cAMP or other sources, which are initiated, propagated, and transmitted by chemical, electrical, or mechanical means. Specific depletion of HeLa cell PDE3A with small interfering RNA significantly decreased membrane-associated BIG1 and BIG2, which by confocal immunofluorescence microscopy were widely dispersed from an initial perinuclear Golgi concentration. Concurrently, activated ARF1-GTP was significantly decreased. Selective inhibition of PDE3A by 1-h incubation of cells with cilostamide similarly decreased membrane-associated BIG1. We suggest that decreasing PDE3A allowed cAMP to accumulate in microdomains where its enzymatic activity limited cAMP concentration. There, cAMP-activated PKA phosphorylated BIG1 and BIG2 (AKAPs for assembly of PKA, PDE3A, and other molecules), which decreased their GEP activity and thereby amounts of activated ARF1-GTP. Thus, PDE3A in these BIG1 and BIG2 AKAP complexes may contribute to the regulation of ARF function via limitation of cAMP effects with spatial and temporal specificity.

Cyclic nucleotide phosphodiesterases (PDEs) are important regulators of signal transduction processes mediated by cAMP and cGMP. One PDE family member, PDE3B, plays an important role in the regulation of a variety of metabolic processes such as lipolysis and insulin secretion. In this study, the cellular localization and the role of PDE3B in the regulation of triglyceride, cholesterol and glucose metabolism in hepatocytes were investigated. PDE3B was identified in caveolae, specific regions in the plasma membrane, and smooth endoplasmic reticulum. In caveolin-1 knock out mice, which lack caveolae, the amount of PDE3B protein and activity were reduced indicating a role of caveolin-1/caveolae in the stabilization of enzyme protein. Hepatocytes from PDE3B knock out mice displayed increased glucose, triglyceride and cholesterol levels, which was associated with increased expression of gluconeogenic and lipogenic genes/enzymes including, phosphoenolpyruvate carboxykinase, peroxisome proliferator-activated receptor gamma, sterol regulatory element-binding protein 1c and hydroxyl-3-methylglutaryl coenzyme A reductase. In conclusion, hepatocyte PDE3B is localized in caveolae and smooth endoplasmic reticulum and plays important roles in the regulation of glucose, triglyceride and cholesterol metabolism. Dysregulation of PDE3B could have a role in the development of fatty liver, a condition highly relevant in the context of type 2 diabetes.

Phosphodiesterase type 3 (PDE3) is an important regulator of cAMP-mediated responses within the cardiovascular system. PDE3 exists as two subtypes: PDE3A and PDE3B, with distinct cellular and subcellular locations. Due to the lack of subtype-specific pharmacological tools, the definitive role of each subtype in regulating cardiovascular function has not been determined. In this study, we investigated platelet and cardiac function, using PDE3A and PDE3B gene knockout (KO) mice. Platelet-rich-plasma was prepared from the blood of KO and age-matched wild-type (WT) mice. PGE1 (1 microg/mL) almost completely inhibited aggregation of platelets from WT, PDE3A KO and PDE3B KO mice. In platelets from WT mice, cilostamide (100 microM), a selective PDE3 inhibitor, blocked collagen- and ADP-induced aggregation. In contrast, cilostamide had no effect on aggregation of platelets from PDE3A KO mice. In PDE3B KO mice, inhibition of collagen- and ADP-induced platelet aggregation was similar to that in WT mice. The resting intra-platelet cAMP concentration in platelets from PDE3A KO mice was twice that in the WT platelets. After PGE1 (0.1 microg/mL) stimulation, intra-cellular cAMP concentration was increased significantly more in platelets from PDE3A KO mice compared to WT mice. In vivo, PDE3A KO mice were protected against collagen/epinephrine-induced pulmonary thrombosis and death, while no such protection was observed in PDE3B KO mice. The heart rate of PDE3A KO mice was significantly higher, compared with age-matched WT mice, while that of PDE3B KO mice was similar to WT. There was no difference in cardiac contractility between PDE3A or PDE3B KO mice. Heart rate and contractility were increased in a similar dose-dependent fashion by isoproterenol in both types of KO mice. Cilostamide increased heart rate and contractility in WT and PDE3B KO but not in PDE3A KO mice. Compared to WT and PDE3B KO mice, cyclic AMP-PDE activity in membrane fractions prepared from the hearts of PDE3A KO mice was lower and not inhibited by cilostamide. The data suggest that PDE3A is the main subtype of PDE3 expressed in platelets and cardiac ventricular myocytes, and is responsible for the functional changes caused by PDE3 inhibition.

cAMP signaling is important for the regulation of insulin secretion in pancreatic beta-cells. The level of intracellular cAMP is controlled through its production by adenylyl cyclases and its breakdown by cyclic nucleotide phosphodiesterases (PDEs). We have previously shown that PDE3B is involved in the regulation of nutrient-stimulated insulin secretion. Here, aiming at getting deeper functional insights, we have examined the role of PDE3B in the two phases of insulin secretion as well as its localization in the beta-cell. Depolarization-induced insulin secretion was assessed and in models where PDE3B was overexpressed [islets from transgenic RIP-PDE3B/7 mice and adenovirally (AdPDE3B) infected INS-1 (832/13) cells], the first phase of insulin secretion, occurring in response to stimulation with high K(+) for 5 min, was significantly reduced ( approximately 25% compared to controls). In contrast, in islets from PDE3B(-/-) mice the response to high K(+) was increased. Further, stimulation of isolated beta-cells from RIP-PDE3B/7 islets, using successive trains of voltage-clamped depolarizations, resulted in reduced Ca(2+)-triggered first phase exocytotic response as well as reduced granule mobilization-dependent second phase, compared to wild-type beta-cells. Using sub-cellular fractionation, confocal microscopy and transmission electron microscopy of isolated mouse islets and INS-1 (832/13) cells, we show that endogenous and overexpressed PDE3B is localized to insulin granules and plasma membrane. We conclude that PDE3B, through hydrolysis of cAMP in pools regulated by Ca(2+), plays a regulatory role in depolarization-induced insulin secretion and that the enzyme is associated with the exocytotic machinery in beta-cells.

Fractionation of 3T3-L1 adipocyte membranes revealed that PDE3B (phosphodiesterase 3B) was associated with PM (plasma membrane) and ER (endoplasmic reticulum)/Golgi fractions, that insulin-induced phosphorylation/activation of PDE3B was greater in internal membranes than PM fractions, and that there was no significant translocation of PDE3B between membrane fractions. Insulin also induced formation of large macromolecular complexes, separated during gel filtration (Superose 6 columns) of solubilized membranes, which apparently contain phosphorylated/activated PDE3B and signalling molecules potentially involved in its activation by insulin, e.g. IRS-1 (insulin receptor substrate-1), IRS-2, PI3K p85 [p85-subunit of PI3K (phosphoinositide 3-kinase)], PKB (protein kinase B), HSP-90 (heat-shock protein 90) and 14-3-3. Expression of full-length recombinant FLAG-tagged murine (M) PDE3B and M3BDelta604 (MPDE3B lacking N-terminal 604 amino acids) indicated that the N-terminal region of MPDE3B was necessary for insulin-induced activation and recruitment of PDE3B. siRNA (small interfering RNA) knock-down of PDE3B indicated that PDE3B was not required for formation of insulin-induced complexes. Wortmannin inhibited insulin-induced assembly of macromolecular complexes, as well as phosphorylation/activation of PKB and PDE3B, and their co-immunoprecipitation. Another PI3K inhibitor, LY294002, and the tyrosine kinase inhibitor, Genistein, also inhibited insulin-induced activation of PDE3B and its co-immunoprecipitation with PKB. Confocal microscopy indicated co-localization of PDE3B and PKB. Recombinant MPDE3B co-immunoprecipitated, and co-eluted during Superose 12 chromatography, to a greater extent with recombinant pPKB (phosphorylated/activated PKB) than dephospho-PKB or p-DeltaPKB [pPKB lacking its PH domain (pleckstrin homology domain)]. Truncated recombinant MPDE3B proteins and pPKB did not efficiently co-immunoprecipitate, suggesting that structural determinants for their interaction reside in, or are regulated by, the N-terminal portion of MPDE3B. Recruitment of PDE3B in macromolecular complexes may be critical for regulation of specific cAMP pools and signalling pathways by insulin, e.g. lipolysis.

Regulatory CD4+ T cells (Tr cells), the development of which is critically dependent on X-linked transcription factor Foxp3 (forkhead box P3), prevent self-destructive immune responses. Despite its important role, molecular and functional features conferred by Foxp3 to Tr precursor cells remain unknown. It has been suggested that Foxp3 expression is required for both survival of Tr precursors as well as their inability to produce interleukin (IL)-2 and independently proliferate after T-cell-receptor engagement, raising the possibility that such 'anergy' and Tr suppressive capacity are intimately linked. Here we show, by dissociating Foxp3-dependent features from those induced by the signals preceding and promoting its expression in mice, that the latter signals include several functional and transcriptional hallmarks of Tr cells. Although its function is required for Tr cell suppressor activity, Foxp3 to a large extent amplifies and fixes pre-established molecular features of Tr cells, including anergy and dependence on paracrine IL-2. Furthermore, Foxp3 solidifies Tr cell lineage stability through modification of cell surface and signalling molecules, resulting in adaptation to the signals required to induce and maintain Tr cells. This adaptation includes Foxp3-dependent repression of cyclic nucleotide phosphodiesterase 3B, affecting genes responsible for Tr cell homeostasis.

Cyclic nucleotide phosphodiesterase 3B (PDE3B) has been suggested to be critical for mediating insulin/IGF-1 inhibition of cAMP signaling in adipocytes, liver, and pancreatic beta cells. In Pde3b-KO adipocytes we found decreased adipocyte size, unchanged insulin-stimulated phosphorylation of protein kinase B and activation of glucose uptake, enhanced catecholamine-stimulated lipolysis and insulin-stimulated lipogenesis, and blocked insulin inhibition of catecholamine-stimulated lipolysis. Glucose, alone or in combination with glucagon-like peptide-1, increased insulin secretion more in isolated pancreatic KO islets, although islet size and morphology and immunoreactive insulin and glucagon levels were unchanged. The beta(3)-adrenergic agonist CL 316,243 (CL) increased lipolysis and serum insulin more in KO mice, but blood glucose reduction was less in CL-treated KO mice. Insulin resistance was observed in KO mice, with liver an important site of alterations in insulin-sensitive glucose production. In KO mice, liver triglyceride and cAMP contents were increased, and the liver content and phosphorylation states of several insulin signaling, gluconeogenic, and inflammation- and stress-related components were altered. Thus, PDE3B may be important in regulating certain cAMP signaling pathways, including lipolysis, insulin-induced antilipolysis, and cAMP-mediated insulin secretion. Altered expression and/or regulation of PDE3B may contribute to metabolic dysregulation, including systemic insulin resistance.

Incubation of 3T3-L1 preadipocytes with isobutylmethylxanthine (IBMX), dexamethasone, and insulin, alone or in combination, demonstrated that IBMX, which increased cAMP-response element-binding protein (CREB) phosphorylation, was the predominant regulator of Pde3b expression. Real time PCR and immunoblotting indicated that in 3T3-L1 preadipocytes, IBMX-stimulated induction of Pde3b mRNA and protein was markedly inhibited by dominant-negative CREB proteins. By transfecting preadipocytes, differentiating preadipocytes, and HEK293A cells with luciferase reporter vectors containing different fragments of the 5'-flanking region of the Pde3b gene, we identified a distal promoter that contained canonical cis-acting cAMP-response elements (CRE) and a proximal, GC-rich promoter region, which contained atypical CRE. Mutation of the CRE sequences dramatically reduced distal promoter activity; H89 inhibited IBMX-stimulated CREB phosphorylation and proximal and distal promoter activities. Distal promoter activity was stimulated by IBMX and phorbol ester (PMA) in Raw264.7 monocytes, but only by IBMX in 3T3-L1 preadipocytes. Chromatin immunoprecipitation analyses with specific antibodies against CREB, phospho-CREB, and CBP/p300 (CREB-binding protein) showed that these proteins associated with both distal and proximal promoters and that interaction of phospho-CREB, the active form of CREB, with both Pde3b promoter regions was increased in IBMX-treated preadipocytes. These results indicate that CRE in distal and proximal promoter regions and activation of CREB proteins play a crucial role in transcriptional regulation of Pde3b expression during preadipocyte differentiation.

Inadequate islet adaptation to insulin resistance leads to glucose intolerance and type 2 diabetes. Here we investigate whether beta-cell cAMP is crucial for islet adaptation and prevention of glucose intolerance in mice. Mice with a beta-cell-specific, 2-fold overexpression of the cAMP-degrading enzyme phosphodiesterase 3B (RIP-PDE3B/2 mice) were metabolically challenged with a high-fat diet. We found that RIP-PDE3B/2 mice early and rapidly develop glucose intolerance and insulin resistance, as compared with wild-type littermates, after 2 months of high-fat feeding. This was evident from advanced fasting hyperinsulinemia and early development of hyper-glycemia, in spite of hyperinsulinemia, as well as impaired capacity of insulin to suppress plasma glucose in an insulin tolerance test. In vitro analyses of insulin-stimulated lipogenesis in adipocytes and glucose uptake in skeletal muscle did not reveal reduced insulin sensitivity in these tissues. Significant steatosis was noted in livers from high-fat-fed wild-type and RIP-PDE3B/2 mice and liver triacyl-glycerol content was 3-fold higher than in wild-type mice fed a control diet. Histochemical analysis revealed severe islet perturbations, such as centrally located alpha-cells and reduced immunostaining for insulin and GLUT2 in islets from RIP-PDE3B/2 mice. Additionally, in vitro experiments revealed that the insulin secretory response to glucagon-like peptide-1 stimulation was markedly reduced in islets from high-fat-fed RIP-PDE3B/2 mice. We conclude that accurate regulation of beta-cell cAMP is necessary for adequate islet adaptation to a perturbed metabolic environment and protective for the development of glucose intolerance and insulin resistance.

The antiobesity and antidiabetic effects of the beta3-adrenergic agonists were investigated on nonobese type 2 diabetic MKR mice after injection with a beta3-adrenergic agonist, CL-316243. An intact response to acute CL-316243 treatment was observed in MKR mice. Chronic intraperitoneal CL-316243 treatment of MKR mice reduced blood glucose and serum insulin levels. Hyperinsulinemic euglycemic clamps exhibited improvement of the whole body insulin sensitivity and glucose homeostasis concurrently with enhanced insulin action in liver and adipose tissue. Treating MKR mice with CL-316243 significantly lowered serum and hepatic lipid levels, in part due to increased whole body triglyceride clearance and fatty acid oxidation in adipocytes. A significant reduction in total body fat content and epididymal fat weight was observed along with enhanced metabolic rate in both wild-type and MKR mice after treatment. These data demonstrate that beta3-adrenergic activation improves the diabetic state of nonobese diabetic MKR mice by potentiation of free fatty acid oxidation by adipose tissue, suggesting a potential therapeutic role for beta3-adrenergic agonists in nonobese diabetic subjects.

Phosphodiesterase (PDE) 3 has been characterized in isolated rat submandibular acini. PDE3 activity was detected in homogenates of isolated rat submandibular acini; little or no PDE3 activity was found in ducts. About 62% of PDE3 activity in the acini was recovered in the supernatant fractions; 38% in particulate fractions. In the acini, but not ducts, PDE3A mRNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR). The PDE3-specific inhibitor, cilostamide, increased the ratio of apomucin mRNA/18s rRNA, as quantified by real-time RT-PCR. Our results indicate that PDE3A may be important in regulating cAMP pools that control acini functions.

Three isoforms of PDE3 (cGMP-inhibited) cyclic nucleotide phosphodiesterase regulate cAMP content in different intracellular compartments of cardiac myocytes in response to different signals. We characterized the catalytic activity and inhibitor sensitivity of these isoforms by using recombinant proteins. We determined their contribution to cAMP hydrolysis in cytosolic and microsomal fractions of human myocardium at 0.1 and 1.0 microm cAMP in the absence and presence of Ca(2+)/calmodulin. We examined the effects of cGMP on cAMP hydrolysis in these fractions. PDE3A-136, PDE3A-118, and PDE3A-94 have similar K(m) and k(cat) values for cAMP and are equal in their sensitivities to inhibition by cGMP and cilostazol. In microsomes, PDE3A-136, PDE3A-118, and PDE3A-94 comprise the majority of cAMP hydrolytic activity under all conditions. In cytosolic fractions, PDE3A-118 and PDE3A-94 comprise >50% of the cAMP hydrolytic activity at 0.1 microm cAMP, in the absence of Ca(2+)/calmodulin. At 1.0 microm cAMP, in the presence of Ca(2+)/calmodulin, activation of Ca(2+)/calmodulin-activated (PDE1) and other non-PDE3 phosphodiesterases reduces their contribution to <20% of cAMP hydrolytic activity. cGMP inhibits cAMP hydrolysis in microsomal fractions by inhibiting PDE3 and in cytosolic fractions by inhibiting both PDE3 and PDE1. These findings indicate that the contribution of PDE3 isoforms to the regulation of cAMP hydrolysis in intracellular compartments of human myocardium and the effects of PDE3 inhibition on cAMP hydrolysis in these compartments are highly dependent on intracellular [Ca(2+)] and [cAMP], which are lower in failing hearts than in normal hearts. cGMP may amplify cAMP-mediated signaling in intracellular compartments of human myocardium by PDE3-dependent and PDE3-independent mechanisms.

We describe techniques for identifying functional promoter elements in the 5'-flanking region of the murine cyclic nucleotide phosphodiesterase 3B (mPDE3B) gene. The 5'-flanking region of the mPDE3B gene was cloned and sequenced, and putative transcription factor binding sites were identified with computational tools. A series of reporter plasmids containing the luciferase gene fused to different fragments of the 5'-flanking region of the mPDE3B gene was constructed and used to transfect 3T3-L1 fibroblasts or differentiating adipocytes. Reporter gene assays showed that there are two promoter regions in the 5'-flanking region in the mPDE3B gene: a distal region located approx 4 kb upstream of the translation initiation site that contains cAMP-response element (CRE) cis-acting elements, and a proximal region that is GC rich and lacks TATA sequences. The distal promoter region induced much higher luciferase activity than did the proximal one. Mutation of the CRE sequences or reversal of the orientation of the CRE-containing region abolished promoter activity of the distal region. Electrophoretic mobility shift assay analysis indicated that binding to CRE elements was greater in nuclear extracts from differentiating adipocytes than from fibroblasts. Mapping of transcription initiation sites suggested that the distal promoter region might function as an enhancer, whereas the proximal promoter drives transcription of the mPDE3B gene.

To construct the recombinant adenovirus vector containing the cDNA for recombinant mouse cyclic nucleotide phosphodiesterase 3B (mPDE3B), the cDNA for mPDE3B was subcloned into pACCMV.pLpA. Subsequently, this recombinant plasmid, pACCMV.mPDE3B, was cotransfected with pJM17 plasmid containing the adenoviral genome into 293 human embryonic kidney cells, and the replication-deficient adenovirus AdCMV.mPDE3B was generated via homologous recombination. Large-scale preparation of adenovirus yielded 10(11)-10(13) viral particles/mL and could be quantitated by real-time polymerase chain reaction using iCycler (Bio-Rad). Efficiency of gene transfer was assessed by infecting FDCP2 or H4IIE cells with a recombinant adenovirus expressing beta-galactosidase (beta-gal); greater than 75% of cells were infected. Expression of mPDE3B in H4IIE hepatoma cells, FDCP2 hematopoietic cells, and beta-cells from isolated pancreatic islets was detected by Western blot analysis. In lysates from FDCP2 cells and H4IIE hepatoma cells infected with recombinant adenoviral mPDE3B constructs, mPDE3B activity was increased 10- to 30-fold compared with the activity in lysates from cells infected with beta-gal adenovirus. Stimulation of FDCP2 cells infected with mPDE3B adenovirus with insulin (100 nM, 10 min) resulted in an approx 1.7-fold increase in endogenous PDE3B and recombinant wild-type PDE3B activities. Infection of rat pancreatic islets resulted in a 5- to 10-fold increase in PDE3B expression and activity and subsequent blunting of insulin secretion. Thus, adenovirus-mediated gene transfer is effective for studying expression and regulation of recombinant PDE3 in insulin-responsive cells as well as insulin-secreting cells.

The second messenger cAMP mediates potentiation of glucose-stimulated insulin release. Use of inhibitors of cAMP-hydrolyzing phosphodiesterase (PDE) 3 and overexpression of PDE3B in vitro have demonstrated a regulatory role for this enzyme in insulin secretion. In this work, the physiological significance of PDE3B-mediated degradation of cAMP for the regulation of insulin secretion in vivo and glucose homeostasis was investigated in transgenic mice overexpressing PDE3B in pancreatic beta-cells. A 2-fold overexpression of PDE3B protein and activity blunted the insulin response to intravenous glucose, resulting in reduced glucose disposal. The effects were "dose"-dependent because mice overexpressing PDE3B 7-fold failed to increase insulin in response to glucose and hence exhibited pronounced glucose intolerance. Also, the insulin secretory response to intravenous glucagon-like peptide 1 was reduced in vivo. Similarly, islets stimulated in vitro exhibited reduced insulin secretory capacity in response to glucose and glucagon-like peptide 1. Perifusion experiments revealed that the reduction specifically affected the first phase of glucose-stimulated insulin secretion. Furthermore, morphological examinations demonstrated deranged islet cytoarchitecture. In conclusion, these results are consistent with an essential role for PDE3B in cAMP-mediated regulation of insulin release and glucose homeostasis.

Pituitary adenylate cyclase-activating peptide (PACAP) is a neuropeptide that exerts its effects throughout the body by elevating the intracellular amounts of cAMP. In adipocytes, an increased amount of cAMP is associated with increased lipolysis. In this work we evaluated the effects of PACAP38 on triglyceride metabolism in primary rat adipocytes. Stimulation of adipocytes with PACAP (0.1-100 nm) resulted in stimulation of lipolysis to the same extent as isoproterenol. Lipolysis was blocked by 25 microm of the protein kinase A inhibitor H-89 and potentiated in the presence of 10 microm OPC3911, a phosphodiesterase 3 inhibitor. In addition, PACAP38 induced activation of protein kinase A. Insulin efficiently inhibited PACAP38-induced lipolysis in a phosphatidyl inositol 3-kinase and phosphodiesterase 3-dependent manner. Interestingly, we also found that PACAP38, as well as isoproterenol, induced potentiation of lipogenesis in the presence of insulin. These results show that PACAP38 and isoproterenol mediate catabolic as well as anabolic effects in adipocytes, depending on the concentration of insulin present. We speculate that in the early postprandial state and during fasting, when insulin levels are low, PACAP and beta-adrenergic catecholamines induce lipolysis, whereas when higher levels of insulin are present, these agents potentiate the anabolic effect of insulin, i.e. storage of triglycerides.

Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that HOSM-1 cells, an osteosarcoma cell line established from human mandible, expressed mRNA for osteoblastic markers, such as alkaline phosphatase, osteonectin, osteocalcin and parathyroid hormone receptor, thus exhibiting an osteoblastic phenotype. We have investigated a possible role of cyclic nucleotide phosphodiesterases (PDEs) in osteosarcoma cells. RT-PCR analysis revealed that HOSM-1 cells expressed mRNA for PDE4A, 4B and 4C. In addition, rolipram, a specific inhibitor of PDE4, inhibited HOSM-1 cell proliferation. The finding that PDE4 is involved in proliferation of osteosarcoma cells suggests the possibility that PDE4 may be a new target for antitumor therapy.

The activation of phosphodiesterase 3B (PDE3B) reduces free fatty acid output from adipocytes. A reduced PDE3B gene expression could lead to insulin resistance. To determine whether there are polymorphisms associated with type 2 diabetes in PDE3B gene promoter, this 5(') flanking region was isolated. The transcription initiation site was located 206bp upstream from the translation start site. Sequences of 2kb of the 5(') flanking region for 24 type 2 diabetic Japanese subjects were initially analyzed using PCR direct sequencing, and the regions including the identified polymorphisms were then examined. In 98 controls and 98 type 2 diabetic subjects, -1947T>C, -567G>A, -465G>T, -458T>C, and -1727&#95;-1726insTCAATT were found. Only -465G>T and this insertion had more than 5% frequencies. Since a complete linkage disequilibrium existed between them, -465G>T was further analyzed, along with a previously identified +1389G>A in the coding region, in a total of 200 controls and 207 type 2 diabetic subjects. These allele frequencies were not significantly different between these two groups (controls vs. cases; -465G>T, 12.0% vs. 10.1%, P=0.435; +1389G>A, 30.3% vs. 33.3%, P=0.408). These genotype distributions were not significantly different between these two groups. The T/T genotype at -465 was rare although this frequency could be higher in type 2 diabetes (4/207 subjects) than controls (0/200 subjects). The linkage disequilibrium existed between -465G>T and +1389G>A, and the estimated haplotype frequencies defined by these SNPs were not significantly different between the cases and controls. Thus, the identified polymorphisms are unlikely to have major effects on susceptibility to Japanese type 2 diabetes.

We have evaluated effects of a phosphodiesterase (PDE) 4 inhibitor on retinoic acid-increased alkaline phosphatase activity in the mouse fibroblastic C3H10T1/2 clone 8 (10T1/2) cell line. 10T1/2 cells were cultured in minimum essential medium (MEM) and 10% fetal bovine serum with or without 1 microM retinoic acid and/or the PDE 4 inhibitor, rolipram, and harvested at specific intervals before measurement of alkaline phosphatase activity, cAMP production in response to parathyroid hormone, osteocalcin synthesis and expression, and phosphodiesterase activity. Retinoic acid-increased alkaline phosphatase activity, and slightly enhanced cAMP production in response to parathyroid hormone. However, it did not affect osteocalcin synthesis and expression. In the presence of retinoic acid, PDE 4 activity was not changed. A PDE 4 inhibitor, rolipram, and cAMP analog, 8-bromo-cAMP dramatically increased retinoic acid's ability to induce alkaline phosphatase activity. This is the first report that PDE 4 may be involved in regulation of retinoic acid-increased alkaline phosphatase activity.

Little is known concerning the expression, distribution and function of phosphodiesterase (PDE) 3s in malignant tumor cells, including human malignant melanoma HMG and osteosarcoma HOSM-1 cells. PDE3 activity was detected in homogenates of HMG cells; however, much less activity was found in HOSM-1 cells. In HMG cells, most of the PDE3 activity was in the particulate fraction. PDE3A and 3B mRNAs were detected by RT-PCR in RNA from HMG cells only. The nucleotide sequences of the fragments were identical to those of human PDE3A and 3B. The PDE3-specific inhibitors, trequinsin and cilostamide, did not inhibit the proliferation of HMG or HOSM-1 cells. Although two PDE3 isoforms may be expressed in human malignant melanoma cells, their functional importance is not known.

PDE3A cyclic nucleotide phosphodiesterases regulate cAMP- and cGMP-mediated intracellular signaling in cardiac myocytes. We used antibodies to different regions of PDE3A to demonstrate the presence of three PDE3A isoforms in these cells. These isoforms, whose apparent molecular weights are 136,000, 118,000, and 94,000 ("PDE3A-136," "PDE3A-118," and "PDE3A-94"), are identical save for the deletion of different lengths of N-terminal sequence containing two membrane-association domains and sites for phosphorylation/activation by protein kinase B ("PK-B") and protein kinase A ("PK-A"). PDE3A-136 contains both membrane-association domains and the PK-B and PK-A sites. PDE3A-118 contains only the downstream membrane-association domain and the PK-A sites. PDE3A-94 lacks both membrane localization domains and the PK-B and PK-A sites. The three isoforms are translated from two mRNAs derived from the PDE3A1 gene: PDE3A-136 is translated from PDE3A1 mRNA, whereas PDE3A-118 and PDE3A-94 are translated from PDE3A2 mRNA. Experiments involving in vitro transcription/translation indicate that PDE3A-118 and PDE3A-94 may be translated from different AUGs in PDE3A2 mRNA. These findings suggest that alternative transcriptional and post-transcriptional processing of the PDE3A gene results in the generation of two mRNAs and three protein isoforms in cardiac myocytes that differ with respect to intracellular localization and may be regulated through different signaling pathways.

Cyclic AMP potentiates glucose-stimulated insulin release and mediates the stimulatory effects of hormones such as glucagon-like peptide 1 (GLP-1) on pancreatic beta-cells. By inhibition of cAMP-degrading phosphodiesterase (PDE) and, in particular, selective inhibition of PDE3 activity, stimulatory effects on insulin secretion have been observed. Molecular and functional information on beta-cell PDE3 is, however, scarce. To provide such information, we have studied the specific effects of the PDE3B isoform by adenovirus-mediated overexpression. In rat islets and rat insulinoma cells, approximate 10-fold overexpression of PDE3B was accompanied by a 6-8-fold increase in membrane-associated PDE3B activity. The cAMP concentration was significantly lowered in transduced cells (INS-1(832/13)), and insulin secretion in response to stimulation with high glucose (11.1 mm) was reduced by 40% (islets) and 50% (INS-1). Further, the ability of GLP-1 (100 nm) to augment glucose-stimulated insulin secretion was inhibited by approximately 30% (islets) and 70% (INS-1). Accordingly, when stimulating with cAMP, a substantial decrease (65%) in exocytotic capacity was demonstrated in patch-clamped single beta-cells. In untransduced insulinoma cells, application of the PDE3-selective inhibitor OPC3911 (10 microm) was shown to increase glucose-stimulated insulin release as well as cAMP-enhanced exocytosis. The findings suggest a significant role of PDE3B as an important regulator of insulin secretory processes.

Tumor necrosis factor-alpha (TNF-alpha) stimulates lipolysis in human adipocytes. However, the mechanisms regulating this process are largely unknown. We demonstrate that TNF-alpha increases lipolysis in differentiated human adipocytes by activation of mitogen-activated protein kinase kinase (MEK), extracellular signal-related kinase (ERK), and elevation of intracellular cAMP. TNF-alpha activated ERK and increased lipolysis; these effects were inhibited by two specific MEK inhibitors, PD98059 and U0126. TNF-alpha treatment caused an electrophoretic shift of perilipin from 65 to 67 kDa, consistent with perilipin hyperphosphorylation by activated cAMP-dependent protein kinase A (PKA). Coincubation with TNF-alpha and MEK inhibitors caused perilipin to migrate as a single 65-kDa band. Consistent with the hypothesis that TNF-alpha induces perilipin hyperphosphorylation by activating PKA, TNF-alpha increased intracellular cAMP approximately 1.7-fold, and the increase was abrogated by PD98059. Furthermore, H89, a specific PKA inhibitor, blocked TNF-alpha-induced lipolysis and the electrophoretic shift of perilipin, suggesting a role for PKA in TNF-alpha-induced lipolysis. Finally, TNF-alpha decreased the expression of cyclic-nucleotide phosphodiesterase 3B (PDE3B) by approximately 50%, delineating a mechanism by which TNF-alpha could increase intracellular cAMP. Cotreatment with PD98059 restored PDE3B expression. These studies suggest that in human adipocytes, TNF-alpha stimulates lipolysis through activation of MEK-ERK and subsequent increase in intracellular cAMP.

Phosphodiesterase (PDE) 3s have been characterized in human squamous cell carcinoma KB cells. PDE3 activity was detected in homogenates of KB cells. PDE3A and 3B mRNAs were detected by RT-PCR in RNA from KB cells; the nucleotide sequences of the fragments were identical to those of human PDE3A and 3B. Immunoblotting with anti-PDE3 antibodies detected both PDE3A- and 3B-immunoreactive proteins in KB cells. The PDE3-specific inhibitor, cilostamide, inhibited the proliferation of KB cells. Our results indicate that PDE3s may be important regulators of the growth of KB cells. Therefore, PDE3 inhibitors may be potential new drugs for antiproliferative therapies in squamous cell carcinoma in the head and neck.

A recent preliminary (unpublished) study showed that phosphodiesterase (PDE) 3A and 3B are expressed in rat submandibular glands. Here, PDE3 activity was detected in homogenates of rat submandibular gland acinar epithelial (SMIE) cells, but not rat A5 (epithelial duct) cells. Most of the PDE3 activity in SMIE cells was recovered in the particulate fraction. Only PDE3B mRNA was detected by reverse transcription-polymerase chain reaction in RNA from SMIE cells. The nucleotide sequence of the fragment was identical to the sequence of rat PDE3B. The PDE3 specific inhibitor, OPC3689 (10 and 50 microM), inhibited the growth of SMIE cells (19 and 63%), but not A5 cells. As the submandibular gland contains many types of cells, these results indicate that PDE3B may regulate a cAMP pool that is important in submandibular gland acinar epithelial cell function.

Phosphodiesterase (PDE) 3s have been characterized in human neoplastic submandibular gland intercalated duct HSG cells. There have been no reports on PDE3 in malignant salivary gland cells. PDE3 activity was detected in homogenates of HSG cells. About 75% of PDE3 activity in HSG cells was recovered in supernatant fractions and 25% in particulate fractions. PDE3A and 3B mRNAs were detected by reverse transcription-polymerase chain reaction in RNA from HSG cells. The nucleotide sequences of the fragments were identical to those of human PDE3A and 3B. The PDE3-specific inhibitor, cilostamide, inhibited the growth of HSG cells. Our results indicate that PDE3s may be important in the growth of HSG cells. PDE3 thus appears to be a potential new target for antiproliferative therapies.

We have identified a new cyclic-nucleotide phosphodiesterase isoform, PDE3A, and cloned its cDNA from cultured aortic myocytes. The nucleotide sequence of its coding region is similar to that of the previously cloned myocardial isoform except for the absence of the initial 300-400 nt that are present in the latter, as confirmed by reverse-transcriptase-mediated PCR, 5' rapid amplification of cDNA ends and a ribonuclease protection assay. Expression in Spodoptera frugiperda (Sf9) cells yields a protein with catalytic activity and inhibitor sensitivity typical of the PDE3 family. The recombinant protein's molecular mass of approx. 131 kDa is compatible with translation from an ATG sequence corresponding to nt 436-438 of the myocardial PDE3A coding region. Antibodies against residues 424-460 (nt 1270-1380) and 1125-1141 (nt 3373-3423) of the myocardial isoform react with an approx. 118 kDa band in Western blots of homogenates of human aortic myocytes, whereas antibodies against residues 29-42 (nt 85-126) do not react with any bands in these homogenates. Our results suggest that a vascular smooth-muscle isoform ('PDE3A2') is a product of the same gene as the longer myocardial ('PDE3A1') and the shorter placental ('PDE3A3') isoforms and is generated pre-translationally in a manner that results in the absence of the 145 N-terminal amino acids of PDE3A1.

Subcellular localization of cyclic nucleotide phosphodiesterases (PDEs) may be important in compartmentalization of cAMP/cGMP signaling responses. In 3T3-L1 adipocytes, mouse (M) PDE3B was associated with the endoplasmic reticulum (ER) as indicated by its immunofluorescent colocalization with the ER protein BiP and subcellular fractionation studies. In transfected NIH 3006 or COS-7 cells, recombinant wild-type PDE3A and PDE3B isoforms were both found almost exclusively in the ER. The N-terminal portion of PDE3 can be arbitrarily divided into region 1 (aa 1-300), which contains a large hydrophobic domain with six predicted transmembrane helices, followed by region 2 (aa 301-500) containing a smaller hydrophobic domain (of approximately 50 aa). To investigate the role of regions 1 and 2 in membrane association, we examined the subcellular localization of a series of catalytically active, Flag-tagged N-terminal-truncated human (H) PDE3A and MPDE3B recombinants, as well as a series of fragments from regions 1 and 2 of MPDE3B synthesized as enhanced green fluorescent (EGFP) fusion proteins in COS-7 cells. In COS-7 cells, the localization of a mutant HPDE3A, lacking the first 189 amino acids (aa) and therefore four of the six predicted transmembrane helices (H3A-Delta189), was virtually identical to that of the wild type. M3B-Delta302 (lacking region 1) and H3A-Delta397 (lacking region 1 as well as part of region 2) retained, to different degrees, the ability to associate with membranes, albeit less efficiently than H3A-Delta189. Proteins that lacked both regions 1 and 2, H3A-Delta510 and M3B-Delta604, did not associate with membranes. Consistent with these findings, region 1 EGFP-MPDE3B fusion proteins colocalized with the ER, whereas region 2 EGFP fusion proteins were diffusely distributed. Thus, some portion of the N-terminal hydrophobic domain in region 1 plus a second domain in region 2 are important for efficient membrane association/targeting of PDE3.

Wild-type (F/B), constitutively active (F/B*), and three kinase-inactive (F/Ba-, F/Bb-, F/Bc-) forms of Akt/protein kinase B (PKB) were permanently overexpressed in FDCP2 cells. In the absence of insulin-like growth factor-1 (IGF-1), activities of PKB, cyclic nucleotide phosphodiesterase 3B (PDE3B), and PDE4 were similar in nontransfected FDCP2 cells, mock-transfected (F/V) cells, and F/B and F/B- cells. In F/V cells, IGF-1 increased PKB, PDE3B, and PDE4 activities approximately 2-fold. In F/B cells, IGF-1, in a wortmannin-sensitive manner, increased PKB activity approximately 10-fold and PDE3B phosphorylation and activity ( approximately 4-fold), but increased PDE4 to the same extent as in F/V cells. In F/B* cells, in the absence of IGF-1, PKB activity was markedly increased ( approximately 10-fold) and PDE3B was phosphorylated and activated (3- to 4-fold); wortmannin inhibited these effects. In F/B* cells, IGF-1 had little further effect on PKB and activation/phosphorylation of PDE3B. In F/B- cells, IGF-1 activated PDE4, not PDE3B, suggesting that kinase-inactive PKB behaved as a dominant negative with respect to PDE3B activation. Thymidine incorporation was greater in F/B* cells than in F/V cells and was inhibited to a greater extent by PDE3 inhibitors than by rolipram, a PDE4 inhibitor. In F/B cells, IGF-1-induced phosphorylation of the apoptotic protein BAD was inhibited by the PDE3 inhibitor cilostamide. Activated PKB phosphorylated and activated rPDE3B in vitro. These results suggest that PDE3B, not PDE4, is a target of PKB and that activated PDE3B may regulate cAMP pools that modulate effects of PKB on thymidine incorporation and BAD phosphorylation in FDCP2 cells.

The N-terminal portion of phosphodiesterase (PDE) 3 was arbitrarily divided into region 1 (amino acids 1-300), which contains a large hydrophobic domain with six predicted transmembrane helices, and region 2 (amino acids 301-500), with a smaller hydrophobic domain ( approximately 50 residues). To analyze these regions, full-length human (H)PDE3A and mouse (M)PDE3B and a series of N-terminal truncated mutants were synthesized in Sf9 cells. Activities of HPDE3A, H3A-Delta189, MPDE3B, and M3B-Delta196, which retained all or part of the hydrophobic domain in region 1, were recovered almost entirely in particulate fractions. H3A-Delta321 and M3B-Delta302, containing region 2, were recovered essentially equally in particulate and cytosolic fractions. H3A-Delta397 and H3A-Delta457, lacking both hydrophobic domains, were predominantly cytosolic. H3A-Delta510 and M3B-Delta604, lacking both regions 1 and 2, were virtually completely cytosolic. M3B-Delta196 eluted as a large aggregated complex during gel filtration. With removal of greater amounts of N-terminal sequence, aggregation of PDE3 decreased, and H3A-Delta607, H3A-Delta721, and M3B-Delta604 eluted as dimers. Truncated HPDE3A proteins were more sensitive than full-length HPDE3A to inhibition by lixazinone. These results suggest that the hydrophobic domains in regions 1 and 2 contain structural determinants important for association of PDE3 with intracellular membranes, as well for self-association or aggregation during gel filtration and sensitivity to a specific inhibitor.

Phosphatidylinositol 3-kinase mediates several actions of insulin including its antilipolytic effect. This effect is elicited by the insulin-stimulated serine phosphorylation and activation of cGMP-inhibited phosphodiesterase (PDE3B). In human adipocytes, we found that insulin differentially stimulated phosphatidylinositol 3-kinase activity; the lipid kinase activity was associated with IRS-1, whereas the serine kinase activity was associated with the insulin receptor and phosphorylated a number of proteins including p85, p110, and a 135-kDa protein identified as PDE3B. PDE3B phosphorylation was associated with enzyme activation, thus initiating the antilipolytic effect of insulin. These results show a novel pathway for intracellular signaling through the insulin receptor leading to the serine phosphorylation of key proteins involved in insulin action.