Expression, Structure/function, Regulation, and Roles of PDE3 Isoforms

PDE3 同工型的表达、结构/功能、调节和作用

• 批准号: 8746564
• 负责人: VINCENT MANGANIELLO
• 金额: $ 240.57万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8746564
• 关键词: ANXA2 gene; ATP2A2; Adipocytes; Adipose tissue; Adverse effects; Agonist; Amino Acid Sequence; Aorta; Appearance; Binding; Binding Sites; Biological; Biological Models; Biological Process; Blood Vessels; Brown Fat; Calcium Signaling; Canada; Cardiac; Catalytic Domain; Caveolae; Caveolins; Cell Culture Techniques; Cell Cycle Progression; Cells; Centrifugation; Characteristics; Chronic; Cilostazol; Clinical; Clinical Trials; Complement; Complementary DNA; Complex; Coupling; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Cyclic Nucleotides; DYSF gene; Development; Diffuse; Disease; Electron Microscopy; Electrons; Endocrinology; Energy Metabolism; Exhibits; Female; Fertilization; Future; G2/M Transition; Gel Chromatography; Gene Family; Genes; Genetic; Glycogen Synthase Kinase 3; Goals; Health; Heart; Heart Transplantation; Heart failure; Homeostasis; Human; Hydrolysis; In Vitro; Incidence; Individual; Infarction; Inflammation; Injury; Insulin Resistance; Intermittent Claudication; Ischemia; Isoproterenol; Length; Location; Mediating; Membrane; Milrinone; Mitochondria; Mitogens; Modification; Molecular; Mus; Muscle; Myocardial; Myocardial Ischemia; Myocardium; N-terminal; National Heart, Lung, and Blood Institute; Obesity; Oxygen Consumption; Patients; Peripheral Vascular Diseases; Phenotype; Phorbol Esters; Phosphorylation; Physiological; Preparation; Production; Prostaglandins; Protein Isoforms; Protein Phosphatase 2A Regulatory Subunit PR53; Proteins; Recombinant Proteins; Recruitment Activity; Regulation; Repair Complex; Reperfusion Injury; Reperfusion Therapy; Report (account); Reporting; Research; Research Design; Resveratrol; Role; Route; Sarcomeres; Scientist; Signal Pathway; Signal Transduction; Signaling Protein; Site; Small Interfering RNA; Smooth Muscle Myocytes; Stem cells; Sterility; Structure; Sucrose; Techniques; Therapeutic; Therapeutic Effect; Transplant Recipients; Treatment Efficacy; Ventricular; Ventricular Arrhythmia; Very Long Chain Fatty Acid; Work; adverse outcome; caveolin-3; constriction; contraceptive target; cyclooxygenase 2; gene induction; in vivo; inflammatory marker; inhibitor/antagonist; mitochondrial permeability transition pore; mortality; novel; obesity treatment; oocyte maturation; phospholamban; phosphoric diester hydrolase; preconditioning; programs; protective effect; protein protein interaction; receptor; research study; response; restenosis; scaffold; therapeutic target; tool; uncoupling protein 1; uptake

This report describes studies designed to study roles of PDE3A and PDE3B in regulation of myocardial function and energy homeostasis. Myocardial function: In human and mouse heart, cAMP stimulates myocardial contractility by increasing protein kinase A (PKA)-induced phosphorylation of membrane-bound substrates involved in intracellular Ca2+ cycling and excitation/contraction coupling. Using PDE3A and PDE3B KO mice, we found that PDE3A, not PDE3B, regulates basal contractility, and that inhibition of PDE3A, not PDE3B, mediates the inotropic effects of the PDE3 inhibitor, milrinone (Primacor) (Circ Res 112:289-97, 2013). PDE3A regulates basal contractility and cAMP-mediated Ca++ uptake into the SR as a component of a SERCA2 regulatory complex or signalosome which contains AKAP18, PKA, SERCA2, phospholamban, and PP2A. Ongoing work (Faiyaz Ahmad, Staff Scientist) indicates that in human heart, PDE3A is localized with SERCA2, PLB and AKAP18 on sarcomere Z-line bands, and that a similar AKAP18/SERCA2/ PDE3A-containing signalosome regulates SERCA2 and Ca++ uptake in human SR. In these preparations, signalsome assembly/formation is enhanced by phosphorylation, and experiments with recombinant proteins indicate that rPDE3A may directly interact with rSERCA2 and rAKAP18. Three PDE3A isoforms, PDE3A1, PDE3A2 and PDE3A3 are expressed in human heart; they possess identical amino-acid sequences except for deletion of different lengths of the N-terminal region. Collaborative studies (PNAS, revision submitted) demonstrated that in HEK293 cells, rPDE3A1 and rPDE3A2 were differentially phosphorylated at distinct 14-3-3 binding sites by isoproterenol and phorbol ester, respectively. Phosphorylation of rPDE3A1 by PKA and of rPDE3A2 by PKC induced shifts in their elution on gel-filtration chromatography, consistent with their phosphorylation-dedendent incorporation into different regulatory signalosomes. Selective phosphorylation of PDE3A1 and PDE3A2 at alternative sites via different signaling pathways, together with different functional consequences of phosphorylation for each, suggest they are likely to have distinct roles in cyclic nucleotide-mediated signaling in human myocardium. With respect to PDE3B, we demonstrated (Circ Res, in revision) that targeted disruption of PDE3B, but not PDE3A, protected mouse hearts from ischemia reperfusion (I/R)-induced injury in vivo and in vitro, with significantly reduced infarct size. Administration of milrinone, a PDE3 inhibitor, to mice, prior to induction of ischemia, reduced infarct size in WT and PDE3A KO mice, but did not further increase protection in PDE3B KO mice. Deletion of PDE3B protected Langendorff-perfused hearts from I/R injury, most likely via enhanced opening of mitoKCa channels, less ROS production, and reduced Ca2+-induced opening of the mitochondrial permeability transition pore (mPTP) in PDE3B KO mitochondria. The mechanism(s) for cardioprotection may involve activation of PI3K/Akt/GSK-3 signaling pathways and cAMP/PKA-induced assembly of ICEF (Ischemia-induced caveolin-enriched fractions), in which various cardioprotective molecules accumulate, resulting in functional preconditioning in PDE3B KO hearts. ICEF are buoyant, caveolae-like fractions, separated from crude mitochondrial fractions by discontinuous sucrose gradient centrifugation. ICEF contain protein components of membrane repair complexes (dysferlin, annexin A2, caveolin-3 and TRIM72), calcium signaling proteins, and other proteins associated with cardioprotection from I/R injury. Protective effects associated with inhibition/deletion of PDE3B may reflect roles of PDE3A and PDE3B in regulating, at distinct subcellular sites, compartmentalization of specific cAMP-signaling pathways, since cryo-immunogold electron microscopy of ventricular muscle revealed that PDE3A was localized with SERCA on SR membranes, whereas PDE3B was localized with caveolin-3 on T-tubule membranes along the Z-line and within the sarcomere I-band, in regions where mitochondria are in close contact with both SR and T-tubules. Furthermore, in PDE3B KO hearts, ICEF, with their cardioprotective molecules, may be delivered to mitochondria via T-tubules, since analysis of heart electron micrographs demonstrated that contacts between T-tubules and mitochondria were increased in PDE3B KO hearts, compared to WT. PDE3 inhibitors (e.g. cilostazol (Pletal)) are in common use for treating intermittent claudication, a peripheral vascular disease, although earlier clinical trials with heart failure subjects demonstrated that chronic inhibition of PDE3 with milrinone (which increased contractility) increased the incidence of ventricular arrhythmias and mortality. Existing PDE3 inhibitors, however, have little selectivity for PDE3A versus PDE3B isoforms, whose catalytic domains are similar, and no selectivity for individual PDE3A isoforms, which possess identical catalytic domains. Isoform-selective targeting may increase contractility in failing hearts without increasing mortality, thus providing a novel route for developing therapeutics. Blocking the integration of PDE3A isoforms into different signalosomes, either by blocking PDE3A phosphorylation or blocking its interactions with constituents of the signalosomes, may be a another way of targeting PDE3A in a specific microdomain to produce inotropic actions without the adverse consequences that accompany diffuse increases in intracellular cAMP. Our findings also suggest that inhibition of cardiac PDE3B, not PDE3A, might account for reported cardioprotective effects of cilostazol from experimental I/R injury. Furthermore, PDE3B-selective inhibitors might provide benefit in heart transplant patients and heart failure patients, by limiting I/R damage. In this regard, a current collaborative project with Dr. Peter Backx (U Toronto, Canada) will study the clinical course and pathophysiological sequellae of transaortic constriction in WT and PDE3A KO and PDE3B KO mice, and effects of milrinone in these groups. PDE3B regulates energy homeostasis: PDE3B regulates energy metabolism (J Clin Invest 116:3240-3251, 2006), and recent studies (Endocrinology 154:3152-67, 2013) indicate that, in PDE3B KO mice (C57Bl6 background), white adipose tissue (WAT) assumes phenotypic characteristics of brown adipose tissue (BAT). The WAT/BAT phenotypic conversion was markedly enhaced by the Beta3 receptor agonist CL316243, and mediated, perhaps, by cAMP-induced differentiation of prostaglandin-responsive progenitor cells in KO WAT stromal vascular fractions into functional brown adipocytes. The appearance of BAT-like characteristics was accompanied by an increase in oxygen consumption and induction of genes involved in BAT recruitment (cyclooxygenase-2 (COX-2) and elongation of very long chain fatty acids 3 (Elovl3)), and its thermogenic program (PGC-1, uncoupling protein 1 (Ucp1)). Unpublished studies indicate that in SvJ129 PDE3B KO mice, WAT also assumes phenotypic characteristics of BAT, without administration of CL316243 and without induction of COX-2, suggesting critical influences of genetic background on development of the BAT phenotype. In SvJ129 PDE3B KO WAT expression of pro-inflammatory markers is reduced, compared to WT, as are components of the NLRP3 inflammasome (activation of the NLRP3 inflammasome may be related to insulin resistance and obesity-related inflammation).These studies are important, since reducing inflammation in WAT and inducing WAT to assume characteristics of BAT is viewed as potential treatment for obesity and related disorders. Furthermore, these studies with PDE3B KO mice complement our collaborative study (J. Chung, NHLBI) which demonstrated that beneficial/therapeutic effects of resveratrol on energy metabolism may be mediated by inhibition of PDEs, including PDE3 and PDE4 (Cell 148:421-433, 2012).

该报告描述了旨在研究PDE3A和PDE3B在调节心肌功能和能量稳态调节中的作用的研究。 心肌功能：在人和小鼠心脏中，cAMP通过增加蛋白激酶A（PKA）诱导的膜结合底物的磷酸化而刺激心肌收缩力，这些磷酸化参与了细胞内CA2+循环和激发/兴奋/收缩/收缩偶联的磷酸化。使用PDE3A和PDE3B KO小鼠，我们发现PDE3A而不是PDE3B调节基础收缩性，并且抑制PDE3A而非PDE3B，介导了PDE3抑制剂，Milrinone（Prirrinone）（Prirrinone）（Prirrinone）（Res Res 112：289-97-97-97，2013-97，2013-97，2013-97，2013-97，2013-97，2013-97，2013-97，2013年，2013年，2013年，2013年，2013年，pde3b。 PDE3A作为SERCA2调节络合物或信号体的组成部分，调节基础收缩性，并cAMP介导的Ca ++摄取，其中包含AKAP18，PKA，SERCA2，Phospholamban和PP2A。正在进行的工作（Faiyaz Ahmad，参谋科学家）指出，在人类心脏中，PDE3A在肌肉节Z线频段上与SERCA2，PLB和AKAP18一起定位，并且类似的AKAP18/ SERCA2/ PDE3A的信号体调节Serca2和CA ++ uptake in HumanS sre sre sre sr。在这些制剂中，通过磷酸化增强了信号组装/形成，重组蛋白的实验表明RPDE3A可能直接与Rserca2和Rakap18相互作用。 三种PDE3A同工型，PDE3A1，PDE3A2和PDE3A3在人心脏中表达；除了缺失N末端区域的不同长度外，它们具有相同的氨基酸序列。协作研究（PNAS，修订提交）表明，在HEK293细胞中，RPDE3A1和RPDE3A2分别在不同的14-3-3结合位点通过异丙肾上腺素和Phorbol酯在不同的14-3-3结合位点进行差异化。 PKA对RPDE3A1的磷酸化和PKC对RPDE3A2的磷酸化诱导了其在凝胶滤清色谱上洗脱的变化，这与它们在不同调节信号体中的磷酸化 - 近端结合一致。 PDE3A1和PDE3A2在替代位点通过不同的信号通路的选择性磷酸化以及每个磷酸化的功能后果，表明它们可能在人体心肌中的环状核苷酸介导的信号中具有不同的作用。 关于PDE3B，我们证明了（Cirs Res，在修订中），这些靶向PDE3B而非PDE3A的破坏，而不是PDE3A，受保护的小鼠心脏免受缺血性再灌注（I/R）诱导的体内损伤，并在体外造成的损伤，并显着降低了基部尺寸。在诱导缺血之前，米尔林酮是PDE3抑制剂，对小鼠的给药，WT和PDE3A KO小鼠的梗塞大小降低，但没有进一步增加PDE3B KO小鼠的保护。 PDE3B的删除保护了Langendorff的心脏不受I/R损伤，最有可能通过增强Mitokca通道的开放，较少的ROS产生和减少Ca2+诱导的线粒体渗透性过渡孔（MPTP）在PDE3B KO MITOCHOCHRIA中的开放。 The mechanism(s) for cardioprotection may involve activation of PI3K/Akt/GSK-3 signaling pathways and cAMP/PKA-induced assembly of ICEF (Ischemia-induced caveolin-enriched fractions), in which various cardioprotective molecules accumulate, resulting in functional preconditioning in PDE3B KO hearts. ICEF是浮力的，类似小窝的馏分，通过不连续的蔗糖梯度离心与粗线粒体分数分离。 ICEF包含膜修复复合物（Dysferlin，Annexin A2，Caveolin-3和Trim72），钙信号蛋白以及其他与I/R损伤的心脏保护相关的蛋白质的蛋白质成分。 Protective effects associated with inhibition/deletion of PDE3B may reflect roles of PDE3A and PDE3B in regulating, at distinct subcellular sites, compartmentalization of specific cAMP-signaling pathways, since cryo-immunogold electron microscopy of ventricular muscle revealed that PDE3A was localized with SERCA on SR membranes, whereas PDE3B was localized with Caveolin-3沿Z线和肌膜I波段内的T纤维膜上的Caveolin-3，在线粒体与SR和T-pubules密切接触的地区。此外，在PDE3B KO心脏中，ICEF及其心脏保护分子可以通过t小管传递到线粒体，因为对心脏电子显微照片的分析表明，与WT相比，PDE3B KO HERVE中T纤维和线粒体之间的接触增加了。 PDE3抑制剂（例如西洛舍唑（植物））常用用于治疗间歇性laudication，一种一种是一种外周血管疾病，尽管较早的心力衰竭受试者的临床试验早期的临床试验表明，慢性抑制PDE3对米兰酮（增加收缩性）增加了心脏心理和持续性的发病率。但是，现有的PDE3抑制剂对PDE3A与PDE3B同工型的选择性很小，它们的催化域是相似的，并且对具有相同催化域的单个PDE3A同工型没有选择性。同工型选择性靶向可能会增加心脏失败的收缩力而不会增加死亡率，从而为开发治疗剂提供了新的途径。通过阻断PDE3A磷酸化或阻止其与信号体成分的相互作用，将PDE3A同工型的整合到不同的信号体中，可能是另一种方法，它靶向特定微域中的PDE3A以产生不利影响的不利影响，而无需伴随着众多的野营群体，而无限型群众群体会产生异常的影响。我们的发现还表明，对心脏PDE3B而非PDE3A的抑制作用可能是据报道，据报道，cilostazol对实验I/R损伤的心脏保护作用。此外，PDE3B选择性抑制剂可能会通过限制I/R损伤来为心脏移植患者和心力衰竭患者提供益处。在这方面，与Peter Backx博士（加拿大U多伦多）进行了当前的合作项目，将研究WT和PDE3A KO和PDE3B KO小鼠的跨股体收缩的临床病程和病理生理后遗症，以及这些组中米尔林酮的作用。 PDE3B调节能量稳态：PDE3B调节能量代谢（J Clin Invest 116：3240-3251，2006）和最近的研究（内分泌学154：3152-67，2013） （蝙蝠）。 WAT/BAT表型转换通过beta3受体激动剂CL316243显着增强，并可能是通过cAMP诱导的ko wat Stromal血管级分中前列腺素反应性祖细胞分化为功能性棕色脂肪细胞中的介导的。类似蝙蝠的特征的出现伴随着氧气消耗和诱导蝙蝠募集的基因（环氧酶-2（Cox-2）（COX-2）（COX-2）以及非常长的链脂肪酸3（ELOVL3））及其热源程序（PGC-1，UNCLING蛋白1（UCPP1））。未发表的研究表明，在SVJ129 PDE3B KO小鼠中，WAT还假定BAT的表型特征，而无需施用CL316243，并且没有COX-2的诱导，这表明遗传背景对BAT表型的发展的关键影响。与WT相比，在SVJ129中，促炎标记的表达降低了，NLRP3炎性体的组成部分（NLRP3炎性体的激活可能与胰岛素抵抗和肥胖相关的炎症）有关，这些研究是重要的，因为这些研究是降低的，因为在WAT中的特征与肥胖相关，因此对WAT的特征降低了，因为对WAT的特征是重要的。和相关疾病。此外，这些对PDE3B KO小鼠的研究补充了我们的协作研究（J. Chung，NHLBI），这些研究表明，白藜芦醇对能量代谢的有益/治疗作用可能是通过PDE抑制PDE3和PDE4（包括PDE4和PDE4）（Cell 148：148：421-11-4333，2012）介导的。