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: 8344768
• 负责人: VINCENT MANGANIELLO
• 金额: $ 246.41万
• 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/8344768
• 关键词: 5' -AMP-activated protein kinase; Adipocytes; Adipose tissue; Adrenergic Agonists; Affect; Age; Agonist; Animal Model; Aorta; Apolipoprotein E; Arterial Fatty Streak; Atherosclerosis; Binding; Biogenesis; Biological; Biological Assay; Biological Models; Biological Process; Blood Platelets; Blood Vessels; Body Weight; Body fat; Brown Fat; Burn injury; CREB1 gene; Caenorhabditis elegans; Caloric Restriction; Cardiovascular Diseases; Cardiovascular system; Catalytic Domain; Cell Culture Techniques; Cells; Characteristics; Chemotaxis; Cholesterol; Chromosomes; Code; Complex; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Cyclic Nucleotides; Deposition; Development; Diabetes Mellitus; Diet; Disease; Endocrine; Enzymes; Exons; Family; Fatty acid glycerol esters; Female; Female infertility; Fertilization; Foam Cells; Gene Expression; Gene Expression Regulation; Gene Family; Genealogical Tree; Genes; Glucose; Glycerol; Hepatocyte; Homeostasis; Homologous Gene; Human; Hydrolysis; Incidence; Incubated; Infertility; Infiltration; Inflammation; Inflammatory Infiltrate; Inflammatory Response; Inhibitory Concentration 50; Injection of therapeutic agent; Insulin; Insulin Resistance; Insulin Signaling Pathway; Insulin-Like Growth Factor I; Interferon Type II; Interleukin-12; Interleukin-18; Knock-out; Knockout Mice; LDL-R knockout mouse; Laboratories; Ligands; Lipids; Lipolysis; Low Density Lipoprotein Receptor; Low-Density Lipoproteins; Lower Organism; Malignant Neoplasms; Membrane; Metabolic; Metabolism; Metals; Mitochondria; Mitochondrial Proteins; Modeling; Modification; Molecular; Monocyte Chemoattractant Protein-1; Morphology; Mus; Myocardial; Nematoda; Non-Insulin-Dependent Diabetes Mellitus; Obesity; Oocytes; Organ; Oxygen Consumption; PDE 3B; PKA inhibitor; Pathway interactions; Peroxisome Proliferator-Activated Receptors; Phosphorylation; Phosphorylation Site; Physiological; Plasma; Platelet aggregation; Play; Production; Protein Isoforms; Proteins; Recombinants; Regulation; Regulator Genes; Reporting; Research; Respiration; Resveratrol; Risk Factors; Role; Rolipram; Satiation; Second Messenger Systems; Sequence Homology; Signal Pathway; Signal Transduction; Signaling Molecule; Small Interfering RNA; Structure; Structure of beta Cell of islet; T-Lymphocyte; TNF gene; Techniques; Tissues; Triglycerides; Tumor Necrosis Factor-alpha; United States National Institutes of Health; Vascular Smooth Muscle; Xenopus oocyte; base; beta-Chemokines; cilostamide; cytokine; fatty acid oxidation; genetic manipulation; glycogenolysis; in vivo; inhibitor/antagonist; insulin secretion; insulin sensitivity; macrophage; monocyte chemoattractant protein 1 receptor; obesity treatment; oocyte maturation; phosphodiester; phosphoric diester hydrolase; response; second messenger; sterol esterase; sugar; therapeutic target; tool

Obesity is a major risk factor for type 2 diabetes and cardiovascular disease. White adipose tissue (WAT), a highly regulated and dynamic secretory organ, affects body fat and energy utilization through storage and turnover/hydrolysis of triglycerides. In addition, via production of endocrine factors, adipocytokines, and lipids, WAT regulates and integrates important physiological pathways, including satiety, energy utilization, glucose sensitivity, insulin sensitivity, and inflammation. WAT, however, also contributes to metabolic dysregulation that characterizes insulin resistance and obesity-related metabolic and cardiovascular complications. In white epididymal adipose tissue (EWAT) of PDE3B KO mice, cAMP/PKA- and AMP-activated protein kinase (AMPK)-signaling pathways are activated, the integration of which results in KO EWAT assuming phenotypic characteristics of brown adipose tissue (BAT), including alterations in morphology and increased expression of genes, such as PDRM16, LRP130, and PGC-1, that are important in differentiation of BAT and mitochondrial biogenesis. 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 PPAR, UCP-1, 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 KO EWAT. These findings 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 adipocytes, uncoupled EWAT mitochondrial respiration, increased oxygen consumption in vivo response to Beta-3 adrenergic receptor agonist stimulation, increased oxygen consumption in isolated BAT and EWAT fragments, increased fatty acid oxidation in PDE3B KO adipocytes and increased treadmill endurance. In cultured 3T3 L1 adipocytes, cilostamide (specific PDE3 inhibitor) activated PKA and AMPK, and cilostamide or siRNA knockdown of PDE3B markedly potentiated induction of UCP-1 by a Beta-3 adrenergic receptor agonist. These results suggest that PDE3B may regulate a cAMP-sensitive switch for WAT/BAT phenotypic conversion, regulating downstream effects of cAMP on cAMP/PKA- and AMPK-signaling, mitochondrial biogenesis and function, and energy dissipation. Understanding mechanisms for these changes in KO EWAT is important, since conversion of fat-storing EWAT to fat-burning BAT represents a potential strategy in treatment of obesity and diabetes. Apolipoprotein E knockout (apoE-/-) and low density lipoprotein receptor (LDL-R-/-) mice develop hypocholesterolmia and atherosclerosis, either spontaneously or under a high cholesterol diet, respectively. Inflammatory infiltrates in the atherosclerotic plaques contain cholesterol-laden macrophages (foam cells) and T lymphocytes. These and other inflammation-related cells are presumably responsible for the increased circulating levels of proinflammatory cytokines, interferon-gamma (INF-gamma), and tumor necrosis factor-α (TNF-α), as well as macrophage-derived interleukin (IL)-12 and IL-18, in apoE-/- mice.Interestingly, we found that targeted disruption of PDE3B was associated with decreased macrophage markers in epididymal white adipose tissue (EWAT). Moreover, chemokine (C-C motif) ligand 2 (CCL2)/monocyte chemotactic protein-1 (MCP-1) and its receptor CCR2, which play an important role in macrophage chemotaxis, were less highly expressed in EWAT of PDE3B-/- mice than WT mice. In addition, after lipopolysaccaride (LPS) injection, plasma levels of TNF-α, IL-12 and CCL2/MCP-1 were lower in PDE3B-/- mice than WT mice. To examine the possible effects of PDE3B on macrophage infiltration and atherosclerotic plaque formation, apoE-/-/PDE3B-/-, as well as LDL-R-/-/PDE3B--/- mice were generated. Compared to apoE-/- and LDL-R-/- mice, in the aorta of apoE-/-/PDE3B-/- (normal diet) and LDL-R-/-/PDE 3B-/- (Western diet high in fat for 5 months) mice, plaque formation was significantly reduced, respectively, suggesting a role for PDE3B in modulating the inflammatory response and suggesting that PDE3B signaling pathways might provide possible therapeutic targets to moderate atherosclerosis. In animal models, caloric restriction (CR) decreases the incidence of age-associated disorders such as cardiovascular disease, diabetes, and cancer. SIRT1 activators, i.e., resveratrol and SRT1720 (1000 fold more potent than resveratrol), mimic effects of CR in lower organisms and mice. In 3T3-L1 adipocytes, resveratrol and SRT1720 inhibited PDE activities in membrane (IC50, 40 and 7.5 uM respectively), and cytosolic fractions (IC50, 90 and 10 uM, respectively). Cilostamide (PDE3 inhibitor, 10 uM), resveratrol (12.5 uM), SRT-1720 (2 uM), and rolipram (PDE4 inhibitor, 30 uM), increased phosphorylation of Ser133-CREB, Ser431-LKB, Thr172-AMPK, Ser79-ACC as well as other unidentified PKA substrates. Rp-8-Br-cAMPs, a PKA inhibitor, blocked phosphorylation of these signaling molecules by cilostamide and rolipram. Rolipram had a relatively stronger effect on phosphorylation of PKA substrates and CREB than cilostamide, resveratrol and SRT1720, but had smaller effect on the phosphorylation of LKB, AMPK, and ACC, suggesting the presence of a distinct cAMP pool involved in activation of AMPK. In adipocytes incubated for 90 min with CL-316243 (B3-agonist), 10 uM resveratrol or 2 uM SRT1720 increased phosphorylation of Ser563 of hormone-sensitive lipase (S563-HSL), resulting in a significant increase in lipolysis (glycerol release). These and other results provide evidence that at least some effects of resveratrol and SRT-1720 may be related to their inhibition of PDEs and, thereby, alteration of intracellular cAMP concentrations. Mammalian PDE3 is known to play an important role in insulin signaling pathways and in platelets, cardiovascular tissues, adipocytes, and oocytes.Caenorhabditis elegans represents a unique model for genetic manipulation and thus can facilitate the identification of regulatory genes and characterization of their functions. We report here studies of expression and characterization of the C.elegans Phosphodiesterase3 (CEPDE3) gene, a homolog of the mammalian PDE3 family. The nematode PDE3 gene is present on chromosome II, spaning about 22.2 Kb, and encodes two different CEPDE3 isoforms. The CEPDE3 long form (LF) consists of 11 exons and codes for a 63.5 kDa protein; the short form (SF) has 8 exons and codes for a 54.2 kDa protein. Both CEPDE3 isoforms have the characteristic mammalian PDE Pfam and phosphodiester domains, and also contain the HD metal binding motif, which is unique for the PDE superfamily. Multiple sequence homology alignments of CEPDE3 with that of the human PDE families shows that C.elegans PDE3 is close to mammalian PDE3 on the PDE family tree. The predicted sequences of CEPDE3LF and SF isoforms show an overall 97 % homology between each other,with identical catalytic domains. PDE activity assays indicated that recombinant CEPDE3 long and short forms are markedly inhibited by cilostamide (a specific inhibitor of mammalian PDE3 ), but not by rolipram (a specific inhibitor of PDE4). The IC50 values for cilostamide and rolipram inhibition are similar for recombinant CEPDE3 long form and recombinant mammalian PDE3. The MS/MS sequence of purified recombinant CEPDE3LF contained several predicted phosphorylation sites.

肥胖是 2 型糖尿病和心血管疾病的主要危险因素。白色脂肪组织（WAT）是一种高度调节和动态的分泌器官，通过甘油三酯的储存和周转/水解影响身体脂肪和能量利用。此外，通过产生内分泌因子、脂肪细胞因子和脂质，WAT 调节和整合重要的生理途径，包括饱腹感、能量利用、葡萄糖敏感性、胰岛素敏感性和炎症。然而，WAT 也会导致代谢失调，从而导致胰岛素抵抗以及肥胖相关的代谢和心血管并发症。 在 PDE3B KO 小鼠的白色附睾脂肪组织 (EWAT) 中，cAMP/PKA 和 AMP 激活蛋白激酶 (AMPK) 信号通路被激活，其整合导致 KO EWAT 呈现棕色脂肪组织 (BAT) 的表型特征，包括形态的改变和基因表达的增加，例如 PDRM16、LRP130 和 PGC-1，这些基因对于分化来说很重要BAT 和线粒体生物发生。在KO EWAT中，对能量耗散和脂肪酸氧化所需的基因、转录调节因子和线粒体蛋白的表达进行协调调节，例如PPAR、UCP-1、CIDEA以及其他参与选举运输和脂肪酸的线粒体蛋白——氧化。 UCP-1 是一种棕色脂肪组织 (BAT) 标记物，通常在 EWAT 中不存在，但在 KO EWAT 中显着升高。 这些发现有助于 PDE3B KO 小鼠的几个表型特征，包括对高脂肪饮食的体重增加较小、性腺脂肪沉积和脂肪细胞较小、EWAT 线粒体呼吸不偶联、对 Beta-3 肾上腺素受体的体内耗氧量增加激动剂刺激、分离的 BAT 和 EWAT 片段中的耗氧量增加、PDE3B KO 脂肪细胞中的脂肪酸氧化增加以及跑步机耐力增加。在培养的 3T3 L1 脂肪细胞中，西洛酰胺（特异性 PDE3 抑制剂）激活 PKA 和 AMPK，而西洛酰胺或 PDE3B 的 siRNA 敲低可显着增强 Beta-3 肾上腺素受体激动剂对 UCP-1 的诱导。这些结果表明，PDE3B 可能调节 WAT/BAT 表型转换的 cAMP 敏感开关，调节 cAMP 对 cAMP/PKA 和 AMPK 信号传导、线粒体生物发生和功能以及能量耗散的下游影响。了解 KO EWAT 这些变化的机制非常重要，因为将脂肪储存 EWAT 转化为脂肪燃烧 BAT 代表了治疗肥胖和糖尿病的潜在策略。 载脂蛋白 E 敲除 (apoE-/-) 和低密度脂蛋白受体 (LDL-R-/-) 小鼠分别自发或在高胆固醇饮食下出现低胆固醇血症和动脉粥样硬化。动脉粥样硬化斑块中的炎症浸润含有富含胆固醇的巨噬细胞（泡沫细胞）和 T 淋巴细胞。这些细胞和其他炎症相关细胞可能导致促炎细胞因子、干扰素-γ (INF-γ)、肿瘤坏死因子-α (TNF-α) 以及巨噬细胞衍生的白细胞介素 (IL) 循环水平升高。 -12 和 IL-18，在 apoE-/- 小鼠中。有趣的是，我们发现 PDE3B 的靶向破坏与附睾白色脂肪组织中巨噬细胞标记物的减少有关（EWAT）。此外，在巨噬细胞趋化中起重要作用的趋化因子（C-C基序）配体2（CCL2）/单核细胞趋化蛋白-1（MCP-1）及其受体CCR2在PDE3B-/-小鼠的EWAT中的表达水平低于PDE3B-/-小鼠WT小鼠。此外，注射脂多糖（LPS）后，PDE3B-/-小鼠的血浆TNF-α、IL-12和CCL2/MCP-1水平低于WT小鼠。 为了检查 PDE3B 对巨噬细胞浸润和动脉粥样硬化斑块形成的可能影响，生成了 apoE-/-/PDE3B-/- 以及 LDL-R-/-/PDE3B--/- 小鼠。与apoE-/-和LDL-R-/-小鼠相比，apoE-/-/PDE3B-/-（正常饮食）和LDL-R-/-/PDE 3B-/-（西方饮食中高含量饮食）的主动脉中脂肪5个月）小鼠中，斑块形成分别显着减少，表明PDE3B在调节炎症反应中发挥作用，并表明PDE3B信号通路可能为缓解炎症反应提供可能的治疗靶点。动脉粥样硬化。 在动物模型中，热量限制（CR）可降低心血管疾病、糖尿病和癌症等与年龄相关的疾病的发生率。 SIRT1 激活剂，即白藜芦醇和 SRT1720（比白藜芦醇强 1000 倍），在低等生物和小鼠中模拟 CR 的作用。在 3T3-L1 脂肪细胞中，白藜芦醇和 SRT1720 抑制膜中的 PDE 活性（IC50 分别为 40 uM 和 7.5 uM）和胞质部分（IC50 分别为 90 uM 和 10 uM）。西洛酰胺（PDE3 抑制剂，10 uM）、白藜芦醇（12.5 uM）、SRT-1720（2 uM）和咯利普兰（PDE4 抑制剂，30 uM），增加 Ser133-CREB、Ser431-LKB、Thr172-AMPK、Ser79- 的磷酸化ACC 以及其他未鉴定的 PKA 底物。 Rp-8-Br-cAMPs 是一种 PKA 抑制剂，可阻断西洛酰胺和咯利普兰对这些信号分子的磷酸化。咯利普兰对 PKA 底物和 CREB ​​磷酸化的影响比西洛酰胺、白藜芦醇和 SRT1720 相对更强，但对 LKB、AMPK 和 ACC 磷酸化的影响较小，表明存在参与 AMPK 激活的独特 cAMP 库。在与 CL-316243（B3 激动剂）一起孵育 90 分钟的脂肪细胞中，10 uM 白藜芦醇或 2 uM SRT1720 增加了激素敏感脂肪酶 (S563-HSL) Ser563 的磷酸化，导致脂肪分解（甘油释放）显着增加。这些和其他结果提供了证据，表明白藜芦醇和 SRT-1720 的至少一些作用可能与它们对 PDE 的抑制有关，从而改变细胞内 cAMP 浓度。 已知哺乳动物 PDE3 在胰岛素信号通路以及血小板、心血管组织、脂肪细胞和卵母细胞中发挥重要作用。秀丽隐杆线虫代表了遗传操作的独特模型，因此可以促进调节基因的识别及其功能的表征。我们在这里报告了秀丽隐杆线虫磷酸二酯酶 3 (CEPDE3) 基因的表达和特征研究，该基因是哺乳动物 PDE3 家族的同源物。线虫 PDE3 基因存在于 II 号染色体上，跨度约 22.2 Kb，编码两种不同的 CEPDE3 亚型。 CEPDE3 长形式 (LF) 由 11 个外显子组成，编码 63.5 kDa 的蛋白质；短形式 (SF) 有 8 个外显子，编码 54.2 kDa 的蛋白质。两种 CEPDE3 亚型都具有哺乳动物 PDE Pfam 和磷酸二酯结构域的特征，并且还包含 PDE 超家族独有的 HD 金属结合基序。 CEPDE3与人类PDE家族的多重序列同源性比对表明，线虫PDE3在PDE家族树上与哺乳动物PDE3接近。 CEPDE3LF 和 SF 亚型的预测序列显示彼此之间总体同源性为 97%，具有相同的催化结构域。 PDE活性测定表明，重组CEPDE3长形式和短形式均被西洛酰胺（哺乳动物PDE3的特异性抑制剂）显着抑制，但咯利普兰（PDE4的特异性抑制剂）不被抑制。对于重组 CEPDE3 长型和重组哺乳动物 PDE3，西洛酰胺和咯利普兰抑制的 IC50 值相似。纯化的重组 CEPDE3LF 的 MS/MS 序列包含几个预测的磷酸化位点。