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).

本报告描述了旨在研究 PDE3A 和 PDE3B 在调节心肌功能和能量稳态中的作用的研究。 心肌功能：在人和小鼠心脏中，cAMP 通过增加蛋白激酶 A (PKA) 诱导的参与细胞内 Ca2+ 循环和兴奋/收缩耦合的膜结合底物的磷酸化来刺激心肌收缩力。使用 PDE3A 和 PDE3B KO 小鼠，我们发现 PDE3A（而非 PDE3B）调节基础收缩力，并且抑制 PDE3A（而非 PDE3B）介导 PDE3 抑制剂米力农 (Primacor) 的正性肌力作用 (Circ Res 112:289-97， 2013）。 PDE3A 作为包含 AKAP18、PKA、SERCA2、受磷蛋白和 PP2A 的 SERCA2 调节复合物或信号体的组成部分，调节基础收缩性和 cAMP 介导的 Ca++ 摄取到 SR 中。正在进行的工作（Faiyaz Ahmad，研究员科学家）表明，在人类心脏中，PDE3A 与 SERCA2、PLB 和 AKAP18 一起定位在肌节 Z 线带上，并且类似的 AKAP18/SERCA2/PDE3A 含有信号体调节人类对 SERCA2 和 Ca++ 的摄取SR。在这些制剂中，信号体组装/形成通过磷酸化得到增强，重组蛋白实验表明 rPDE3A 可能直接与 rSERCA2 和 rAKAP18 相互作用。 人类心脏中表达三种 PDE3A 亚型：PDE3A1、PDE3A2 和 PDE3A3；它们具有相同的氨基酸序列，只是 N 末端区域长度不同。合作研究（PNAS，已提交修订版）表明，在 HEK293 细胞中，rPDE3A1 和 rPDE3A2 分别在不同的 14-3-3 结合位点被异丙肾上腺素和佛波酯差异磷酸化。 rPDE3A1 被 PKA 磷酸化和 rPDE3A2 被 PKC 磷酸化引起凝胶过滤色谱洗脱的变化，这与它们磷酸化衍生的掺入不同调节信号体的情况一致。 PDE3A1 和 PDE3A2 通过不同信号通路在替代位点选择性磷酸化，以及每种磷酸化的不同功能后果，表明它们可能在人类心肌中环核苷酸介导的信号传导中具有不同的作用。 关于 PDE3B，我们证明（Circ Res，修订版）靶向破坏 PDE3B（而非 PDE3A）可保护小鼠心脏免受体内和体外缺血再灌注 (I/R) 诱导的损伤，并显着减小梗塞面积。在诱导缺血之前，给小鼠施用 PDE3 抑制剂米力农，可以减少 WT 和 PDE3A KO 小鼠的梗塞面积，但没有进一步增加 PDE3B KO 小鼠的保护作用。 PDE3B 的缺失保护了 Langendorff 灌注的心脏免受 I/R 损伤，最有可能是通过增强 mitoKCa 通道的开放、减少 ROS 的产生以及减少 PDE3B KO 线粒体中 Ca2+ 诱导的线粒体通透性转换孔 (mPTP) 的开放。心脏保护机制可能涉及 PI3K/Akt/GSK-3 信号通路的激活和 cAMP/PKA 诱导的 ICEF（缺血诱导的小窝蛋白富集部分）的组装，其中各种心脏保护分子积累，导致功能性预处理在PDE3B KO心中。 ICEF 是有浮力的小窝样级分，通过不连续蔗糖梯度离心与粗线粒体级分分离。 ICEF 含有膜修复复合物的蛋白质成分（dysferlin、膜联蛋白 A2、caveolin-3 和 TRIM72）、钙信号蛋白以及与心脏保护免受 I/R 损伤相关的其他蛋白质。与抑制/删除 PDE3B 相关的保护作用可能反映了 PDE3A 和 PDE3B 在不同亚细胞位点调节特定 cAMP 信号通路区室化的作用，因为心室肌的冷冻免疫金电子显微镜显示 PDE3A 与 SR 上的 SERCA 一起定位膜上，而 PDE3B 与 Caveolin-3 一起定位在 T 管膜上，沿着 Z 线并位于肌节 I 带，位于线粒体与 SR 和 T 管紧密接触的区域。此外，在 PDE3B KO 心脏中，ICEF 及其心脏保护分子可能通过 T 管递送至线粒体，因为心脏电子显微照片的分析表明，与 WT 相比，PDE3B KO 心脏中 T 管和线粒体之间的接触增加。 PDE3 抑制剂（例如西洛他唑（Pletal））通常用于治疗间歇性跛行（一种周围血管疾病），尽管早期对心力衰竭受试者的临床试验表明，用米力农（增加收缩力）长期抑制 PDE3 会增加室性心律失常的发生率和死亡率。然而，现有的 PDE3 抑制剂对 PDE3A 与 PDE3B 同工型的选择性很小，其催化结构域相似，并且对具有相同催化结构域的各个 PDE3A 同工型没有选择性。同工型选择性靶向可能会增加衰竭心脏的收缩力而不增加死亡率，从而为开发治疗方法提供了一条新途径。通过阻断 PDE3A 磷酸化或阻断其与信号体成分的相互作用，阻断 PDE3A 异构体整合到不同的信号体中，可能是另一种靶向特定微域中的 PDE3A 的方法，以产生正性肌力作用，而不会产生伴随扩散性增加的不良后果。细胞内cAMP。我们的研究结果还表明，抑制心脏 PDE3B（而不是 PDE3A）可能是报道的西洛他唑对实验性 I/R 损伤的心脏保护作用的原因。此外，PDE3B 选择性抑制剂可能通过限制 I/R 损伤而为心脏移植患者和心力衰竭患者带来益处。在这方面，目前与 Peter Backx 博士（加拿大多伦多大学）的一个合作项目将研究 WT 和 PDE3A KO 和 PDE3B KO 小鼠经主动脉缩窄的临床过程和病理生理学后遗症，以及米力农在这些组中的作用。 PDE3B 调节能量稳态：PDE3B 调节能量代谢（J Clin Invest 116:3240-3251, 2006），最近的研究（Endocrinology 154:3152-67, 2013）表明，在 PDE3B KO 小鼠（C57Bl6 背景）中，白色脂肪组织(WAT) 呈现棕色脂肪的表型特征组织（最佳可行技术）。 Beta3 受体激动剂 CL316243 显着增强了 WAT/BAT 表型转化，并且可能是通过 cAMP 诱导 KO WAT 基质血管组分中的前列腺素反应性祖细胞分化为功能性棕色脂肪细胞来介导的。 BAT 样特征的出现伴随着耗氧量的增加和参与 BAT 募集的基因的诱导（环氧合酶-2 (COX-2) 和超长链脂肪酸 3 (Elovl3)）及其产热程序（PGC-1，解偶联蛋白 1 (Ucp1)）。未发表的研究表明，在 SvJ129 PDE3B KO 小鼠中，在不给予 CL316243 且不诱导 COX-2 的情况下，WAT 也呈现 BAT 的表型特征，这表明遗传背景对 BAT 表型的发展具有关键影响。与 WT 相比，在 SvJ129 PDE3B KO WAT 中，促炎标记物的表达降低，NLRP3 炎症小体的成分也降低（NLRP3 炎症小体的激活可能与胰岛素抵抗和肥胖相关炎症有关）。这些研究很重要，因为减少 WAT 中的炎症并诱导 WAT 呈现 BAT 的特征被视为肥胖和相关疾病的潜在治疗方法。此外，这些使用 PDE3B KO 小鼠的研究补充了我们的合作研究（J. Chung，NHLBI），该研究证明白藜芦醇对能量代谢的有益/治疗作用可能是通过抑制 PDE 介导的，包括 PDE3 和 PDE4 (Cell 148:421-433) ，2012）。