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: 7969037
• 负责人: VINCENT MANGANIELLO
• 金额: $ 163.82万
• 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/7969037
• 关键词: 5' -AMP-activated protein kinase; ATP2A2; Acute; Adipocytes; Adipose tissue; Affect; Agonist; Animals; Antibodies; Arts; Biogenesis; Biological; Biological Models; Biological Process; Blood Vessels; Body Weight; Body fat; Brown Fat; CCL2 gene; Cardiac; Cardiovascular Diseases; Cardiovascular system; Caveolae; Cell Adhesion Molecules; Cell Culture Techniques; Cell membrane; Characteristics; Chemotaxis; Cholesterol; Complex; Confocal Microscopy; Cultured Cells; Cyclic AMP; Cyclic GMP; Cyclic Nucleotides; Data; Deposition; Development; Diabetes Mellitus; Diet; Down-Regulation; Endocrine; Endoplasmic Reticulum; Energy Metabolism; Enzymes; Fatty acid glycerol esters; Female; Fertilization; Gel Chromatography; Gene Expression; Gene Expression Regulation; Gene Family; Genes; Golgi Apparatus; Heart; Hepatic; Hepatocyte; Homeostasis; Hormones; Human; Hydrolysis; Infertility; Infiltration; Inflammation; Inflammatory; Inflammatory Response; Injection of therapeutic agent; Insulin; Insulin Resistance; Insulin-Like Growth Factor I; Interleukin-12; Interleukin-18; Knockout Mice; Laboratories; Ligands; Lipolysis; Location; Macromolecular Complexes; Macrophage Inflammatory Protein-1; Mediating; Membrane; Membrane Microdomains; Membrane Proteins; Metabolic; Metabolism; Microsomes; Mitochondria; Mitochondrial Proteins; Modification; Molecular; Molecular Chaperones; Morphology; Mus; Myocardial; Myocardium; Non-Insulin-Dependent Diabetes Mellitus; Obesity; Oocytes; Organ; Oxygen Consumption; Pathway interactions; Peripheral; Peroxisome Proliferator-Activated Receptors; Phosphorylation; Physiological; Physiology; Plasma; Platelet aggregation; Play; Production; Protein Isoforms; Protein Phosphatase 2A Regulatory Subunit PR53; Proteins; Regulation; Reporting; Research; Risk Factors; Role; Sarcoplasmic Reticulum; Satiation; Second Messenger Systems; Sepharose; Serum; Signal Pathway; Signaling Molecule; Signaling Protein; Small Interfering RNA; Stress; Structure; Structure of beta Cell of islet; TNF gene; Techniques; Triglycerides; United States National Institutes of Health; Vascularization; Xenopus oocyte; activating transcription factor 3; adenylate kinase; adiponectin; beta-Chemokines; blood glucose regulation; caveolin 1; cilostamide; diabetic; fatty acid oxidation; feeding; glucose production; glycogenolysis; homologous recombination; in vivo; inhibitor/antagonist; insulin secretion; insulin sensitivity; knock-down; macrophage; membrane activity; monocyte chemoattractant protein 1 receptor; obesity treatment; oocyte maturation; perilipin; perilipin A; phosphoric diester hydrolase; receptor; response; scaffold; second messenger; sterol esterase; sugar; tool; uptake

Phosphodiesterase type 3 (PDE3) exists as two subtypes: PDE3A and PDE3B, each with distinct cellular and subcellular locations. We are examining the subcellular localization of adipocyte and myocardial PDE3 isoforms and their contributions to regulation of cAMP signaling pathways. Approximately 30% of total membrane PDE3 activity is associated with plasma membrane/caveolae (PM); 70%, with internal membrane (Golgi/ sarcoplasmic reticulum) fractions from failing human heart myocardium. Of the three PDE3A isoforms, PDE3A3 is the dominant PDE3A isoform in PM/caveolae fractions, while PDE3B and all isoforms of PDE3A (A1-3) are present in internal membrane fractions. PDE3A2 and PDE3A3 predominate in cytosolic fractions. Confocal microscopy indicates that both PDE3A and SERCA2 are detected in Z-band regions. PDE3A and SERCA2 were co-immunoprecipitated from human myocardial membranes using anti-PDE3A and anti-SERCA2 antibodies or cAMP-agarose beads; cilostamide, a specific PDE3 inhibitor, augmented the stimulatory effect of cAMP on Ca uptake into human myocardial microsomes. PDE3A and SERCA2 may be components of a multimolecular complex that regulates cAMP-induced Ca transients and uptake into cardiac sarcoplasmic reticulum. In adipocytes, PDE3B is an important regulatory effector of signaling pathways controlled by insulin and cAMP-increasing hormones. Stimulation of 3T3-L1 adipocytes with insulin or the B3-receptor agonist CL316243 (CL) demonstrated that insulin or CL preferentially phosphorylated /activated PDE3B associated with internal membranes (endoplasmic reticulum/golgi) or caveolae, respectively. siRNA-mediated knock-down (KD) of caveolin-1 (Cav-1) in 3T3-L1 adipocytes resulted in down-regulation of expression and activity of membrane- associated PDE3B. Insulin-induced activation of PDE3B was reduced, whereas CL-mediated activation was almost totally abolished. Cav-1 KD resulted also in inhibition of CL-stimulated phosphorylation of 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 signaling molecules thought to be involved in its activation. Insulin- and CL-induced macromolecular complexes were enriched in cholesterol, and contained certain common signaling proteins (14-3-3, PP2A, cav-1, perilipin) as well as ligand-specific proteins. Insulin- and CL-mediated macromolecular complex formation was significantly inhibited by cav-1 KD. These data 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. WAT, a highly regulated and dynamic secretory organ, affects body fat and energy utilization via storage and turnover/hydrolysis of triglycerides. In addition, via production of endocrine factors, WAT regulates and integrates important physiological pathways and homeostatic functions, including satiety, energy utilization, peripheral insulin sensitivity, glucose homeostasis, and systemic inflammatory responses. Obesity is a major risk factor for developing type 2 diabetes and cardiovascular disease. As obesity develops, there is a progressive increase in macrophages and inflammation-related effectors in WAT. Thus, WAT contributes not only to modulation of energy utilization and homeostasis, but also to metabolic dysregulation and inflammation that characterizes insulin resistance and obesity-related metabolic and cardiovascular complications. Aquirement of BAT characteristics by WAT, with enhanced intra-adipocyte FAO (fatty acid oxidation), represents a potential new strategy in treatment of obesity and diabetes. Although mechanisms for acute activation of PDE3B have been studied in isolated adipocytes and cultured cells, its role(s)in human and animal physiology, especially with regard to energy homeostatic mechanisms, is not well understood. To evaluate these functions, we introduced a targeted disruption in the murine PDE3B gene by homologous recombination (in SvJ129 background). In PDE3B KO mice, epididymal WAT (EWAT) assumed some phenotypic characteristics of BAT, including changes in morphology (increased vascularization and mitochondria number), and activation of cAMP/PKA and AMP-activated protein kinase (AMPK)-signaling pathways, as well as increased mitochondrial biogenesis and expression of genes important in differentiation of BAT, including PDRM16 and LRP130. In KO EWAT, there is coordinate regulation of expression of genes, transcriptional regulators, and mitochondrial proteins required for energy dissipation and fatty acid oxidation, such as PGC-1, PPAR, UCP-1 and its regulator CIDEA, and other mitochondrial proteins involved in election transport and fatty acid -oxidation. UCP-1, a marker for brown adipose tissue (BAT) usually not present in EWAT, is markedly elevated in EWAT from KO mice. Furthermore, in EWAT from PDE3B KO mice, phospho-LKB1, AMP kinase 1 subunits, and AMP kinase enzymatic activity were increased, as were phosphorylated substrates of AMPK, including phospho-ACC and HSL, and serum adiponectin in KO mice. Alterations in cAMP/PKA- and AMP kinase-signaling pathways were reflected in alterations in lipolysis and FAO, in that the antilipolytic actions of insulin were inhibited and FAO was increased in isolated adipocytes from KO mice. These findings most likely contribute to several phenotypic characteristics of PDE3B KO mice, including a smaller increase in body weight in response to high fat diets, smaller gonadal fat deposits and smaller adipocytes, increased oxygen consumption in vivo in response to 3 agonist stimulation, increased oxygen consumption in isolated brown and white adipose tissue fragments, and increased fatty acid oxidation in adipocytes from PDE3B KO mice. Taken together, these surprising results suggested an entirely new role for PDE3B regarding mitochondrial function, energy dissipation, adipocyte metabolism, and perhaps, that PDE3B may regulate a molecular switch for WAT/BAT phenotypic conversion, and thereby could play an important role in regulation of energy metabolism. Interestingly, we found that targeted disruption of PDE3B was associated with decreased expression of inflammatory effectors and macrophage markers in epididymal white adipose tissue (EWAT), including MCP-1, macrophage inflammatory protein-1 (MIP1/Ccl3), Ccr2 and Ccr5, Adam8, F4/80 (Emr1), activating transcription factor 3 (Atf3) (a stress-inducible transcriptional factor), as well as cell adhesion molecules. Moreover, chemokine (C-C motif) ligand 2 (CCL2) and its receptor CCR2, which play an important role in macrophage chemotaxis, were less highly expressed in EWAT of PDE3B-/- mice than WT mice. Accumulation of macrophages in WT EWAT, associated with high fat feeding, was reduced in KO EWAT, consistent with reduced proinflammatory molecules in KO EWAT. In addition, after lipopolysaccaride (LPS) injection, plasma levels of TNF-, IL-12 and IL-18 were lower in PDE3B-/- mice than WT mice. Although, as we previously reported (J. Clin. Investig. 116:3240-3251, 2006), PDE3B seems to be important in regulating certain cAMP-signaling pathways, including lipolysis, insulin- induced anti-lipolysis, and cAMP-mediated insulin secretion, PDE3B KO mice also show signs of systemic insulin resistance, most likely due to dysregulation of hepatic glucose production. They are, however, lean and not diabetic, perhaps because, in PDE3B KO mice, the presence of good BAT in EWAT depots compensates for insulin resistance, and apparently protects WAT from infiltration/accumulation of inflammatory effectors.

磷酸二酯酶3型（PDE3）以两种亚型的形式存在：PDE3A和PDE3B，每种都有不同的细胞和亚细胞位置。我们正在研究脂肪细胞和心肌PDE3同工型的亚细胞定位及其对cAMP信号通路调节的贡献。总膜PDE3活性的大约30％与质膜/小洞（PM）有关； 70％，内膜（高尔基/肌质网）因衰竭人心心肌而构成。在三个PDE3A同工型中，PDE3A3是PM/Caveolae级分中的主要PDE3A同工型，而PDE3B和所有PDE3A（A1-3）的所有同工型都存在于内部膜级分中。 PDE3A2和PDE3A3在胞质分数中占主导地位。共聚焦显微镜表明在Z波段中都检测到PDE3A和SERCA2。 PDE3A和SERCA2是使用抗PDE3A和抗凝集抗体或cAMP-琼脂糖珠从人体心肌膜中共免疫沉淀的； cilostamide是一种特定的PDE3抑制剂，增强了cAMP对人体心肌微粒体的CA摄取的刺激作用。 PDE3A和SERCA2可能是多细分子复合物的组成部分，可调节cAMP诱导的Ca瞬变并摄取进入心脏肌浆网。 在脂肪细胞中，PDE3B是由胰岛素和营地增添激素控制的信号通路的重要调节效应。用胰岛素或B3受体激动剂CL316243（CL）刺激3T3-L1脂肪细胞表明，胰岛素或CL优先磷酸化 /激活的PDE3B与内膜内膜（内质网 /高尔基）或Caveolae分别相关。 3T3-L1脂肪细胞中小窝蛋白1（CAV-1）的siRNA介导的敲除（KD）导致膜相关PDE3B的表达和活性下调。胰岛素诱导的PDE3B激活减少了，而CL介导的激活几乎被完全废除。 CAV-1 KD也导致了Cl刺激的激素敏感脂肪酶和钙蛋白A的磷酸化以及脂解的磷酸化。用胰岛素或CL刺激的脂肪细胞的溶解膜蛋白的Superose 6凝胶过滤色谱法证明了可逆的不同的大分子复合物的可逆组装，这些分子复合物的可逆组装包含32p磷酸化的PDE3B和被认为参与其活化的信号分子。胰岛素和CL诱导的大分子复合物富含胆固醇，并包含某些常见的信号蛋白（14-3-3，pp2a，cav-1，Perilipin）以及配体特异性蛋白。胰岛素和Cl介导的大分子复合物形成被CAV-1 KD显着抑制。这些数据表明，CAV-1充当富含胆固醇的脂质筏中的分子伴侣或脚手架分子，这对于响应CL和胰岛素而适当稳定和激活可能是必要的。 WAT是一种高度调节和动态的分泌器官，通过储存和周转/水解甘油三酸酯影响体内脂肪和能量利用。另外，通过生产内分泌因子，WAT调节和整合重要的生理途径和稳态功能，包括饱腹感，能量利用，周围胰岛素敏感性，葡萄糖稳态和全身性炎症反应。 肥胖是发展2型糖尿病和心血管疾病的主要危险因素。随着肥胖的发展，巨噬细胞和炎症相关的效应子在WAT中逐渐增加。 因此，WAT不仅有助于能量利用和稳态的调节，还有助于代谢失调和炎症，这表征了胰岛素抵抗和与肥胖相关的代谢和心血管并发症的特征。 WAT的蝙蝠特性含水，具有增强的脂肪细胞内粮农组织（脂肪酸氧化），代表了治疗肥胖症和糖尿病治疗的一种潜在的新策略。 尽管已经在孤立的脂肪细胞和培养细胞中研究了PDE3B急性激活的机制，但尚不清楚其在人类和动物生理中的作用，尤其是在能量稳态机制方面的作用。 为了评估这些功能，我们通过同源重组（在SVJ129背景）中引入了鼠PDE3B基因的靶向破坏。 In PDE3B KO mice, epididymal WAT (EWAT) assumed some phenotypic characteristics of BAT, including changes in morphology (increased vascularization and mitochondria number), and activation of cAMP/PKA and AMP-activated protein kinase (AMPK)-signaling pathways, as well as increased mitochondrial biogenesis and expression of genes important in differentiation of BAT, including PDRM16和LRP130。在KO EWAT中，需要调节基因表达，转录调节剂和所需的线粒体蛋白，需要用于耗散和脂肪酸氧化，例如PGC-1，PPAR，UCP-1及其调节剂CIDEA及其调节剂CIDEA，以及其他参与选举运输和脂肪酸酸 - 氧化氧化的线粒体蛋白。 UCP-1是通常在ewat中不存在的棕色脂肪组织（BAT）的标记，在KO小鼠的EWAT中显着升高。此外，在PDE3B KO小鼠的EWAT中，磷酸-LKB1，AMP激酶1亚基和AMP激酶酶活性增加，包括磷酸化和HSL的AMPK的磷酸化底物也增加了KO小鼠中的AMPK的磷酸化底物。 CAMP/PKA和AMP激酶信号途径的改变反映在脂解和FAO的变化中，因为胰岛素的抗溶液作用被抑制，并且在KO小鼠的分离脂肪细胞中增加了FAO。 这些发现很可能有助于PDE3B KO小鼠的几种表型特征，包括响应高脂肪饮食的体重增加较小，较小的性腺脂肪沉积物和较小的脂肪细胞，体内增加的氧气消耗量增加，响应3个激动剂的氧气，以及增加脂肪的脂肪量增加的氧气刺激，并增加脂肪的氧气含量，使其增加脂肪的氧气，而脂肪含量增加，脂肪含量会增加脂肪的脂肪含量。 PDE3B KO小鼠。综上所述，这些令人惊讶的结果表明，PDE3B在线粒体功能，能量耗散，脂肪细胞代谢方面具有全新的作用，也许PDE3B可能会调节WAT/BAT表型转换的分子开关，从而在能量代谢的调节中起重要作用。 有趣的是，我们发现PDE3B的靶向破坏与附睾白脂肪组织（EWAT）中炎症效应子和巨噬细胞标记的表达降低有关，包括MCP-1，巨噬细胞炎症蛋白-1（MIP1/CCL3），CCR2和CCR2和CCR5和CCR5，ADAM8，F4，F4，F4/80（EMR1）（EMR1）（EMR1）（EMR1），AICTICATIAN（EMR 1）应力诱导的转录因子）以及细胞粘附分子。此外，在巨噬细胞趋化性中起重要作用的趋化因子（C-C基序）配体2（CCL2）及其受体CCR2在PDE3B - / - 小鼠的EWAT中的表达不如WT小鼠。巨噬细胞在WT EWAT中的积累与高脂肪进食有关，在KO EWAT中降低，与KO EWAT中促炎分子的降低一致。另外，在脂孢菌（LPS）注射后，PDE3B - / - 小鼠中TNF-，IL-12和IL-18的血浆水平低于WT小鼠。 尽管正如我们先前报道的那样（J. Clin。 生产。但是，它们是瘦而不是糖尿病患者，也许是因为在PDE3B KO小鼠中，EWAT仓库中存在良好的蝙蝠可以补偿胰岛素抵抗，并且显然可以保护WAT免受炎症效应子的浸润/积累。