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，通过甘油三酸酯的存储和周转/水解影响人体脂肪和能量利用。另外，通过生产内分泌因子，脂肪细胞因子和脂质，WAT可以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还导致代谢失调，其特征在于胰岛素抵抗以及与肥胖相关的代谢和心血管并发症。 在PDE3B KO小鼠的白色附子脂肪组织（EWAT）中LRP130和PGC-1，对于分化BAT和线粒体生物发生很重要。在KO EWAT中，需要对基因表达，转录调节剂和能量耗散和脂肪酸氧化所需的线粒体蛋白的表达进行调节，例如PPAR，UCP -1，CIDEA，CIDEA和其他参与选举转运和脂肪酸酸 - 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 EWAT中显着升高。 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 PDE3B KO脂肪细胞中的脂肪酸氧化并增加了跑步机。在培养的3T3 L1脂肪细胞中，cilostamide（特异性PDE3抑制剂）激活了PKA和AMPK，以及PDE3B的Cilostamide或Cilostamide或siRNA敲低，通过BetA-3肾上腺素能受体激动剂明显增强了UCP-1的诱导。这些结果表明，PDE3B可以调节营地敏感的开关，以进行WAT/BAT表型转换，从而调节营地对CAMP/PKA和AMPK信号，线粒体生物发生和功能以及能量耗散的下游影响。理解KO EWAT中这些变化的机制很重要，因为将脂肪的ewat转化为燃烧脂肪的蝙蝠代表了治疗肥胖和糖尿病的潜在策略。 载脂蛋白E基因敲除（APOE-/ - ）和低密度脂蛋白受体（LDL-R - / - ）小鼠分别自发地或在高胆固醇饮食下形成降胆固醇和动脉粥样硬化。动脉粥样硬化斑块中的炎性浸润含有胆固醇巨噬细胞（泡沫细胞）和T淋巴细胞。这些和其他与炎症相关的细胞可能是导致促炎性细胞因子，干扰素 - γ（INF-GAMMA）和肿瘤坏死因子-α（TNF-α）的循环水平增加的原因，以及巨噬细胞衍生的白介素（IL）（IL） - 12和IL-18，apoe-int tocultion intery-tote。与附睾白脂肪组织（EWAT）中巨噬细胞标记的降低有关。此外，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在巨噬细胞趋化性中起重要作用，在巨噬细胞趋化性中起重要作用，在PDE3b的EWAT中低表达PDE3b - / - 小鼠。另外，在脂多多亚赛（LPS）注射后，PDE3B - / - 小鼠中TNF-α，IL-12和CCL2/MCP-1的血浆水平低于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-/ - / - / - / - / - / - / - / - 小鼠。与apoE-/ - / - / - / - / - /pde3b - / - （正常饮食）和ldl-r-/ - / - / - / - /pde 3b - / - （脂肪高5个月的西部饮食）小鼠相比，与ApoE-/ - 和LDL-R - / - 小鼠相比可能为中度动脉粥样硬化提供可能的治疗靶标。 在动物模型中，热量限制（CR）降低了与年龄相关的疾病（例如心血管疾病，糖尿病和癌症）的发生率。 SIRT1激活剂，即白藜芦醇和SRT1720（比白藜芦醇高1000倍），模仿CR在较低生物体和小鼠中的效果。在3T3-L1脂肪细胞中，白藜芦醇和SRT1720抑制了膜中的PDE活性（分别为IC50、40和7.5 UM），以及胞质分数（分别IC50、90和10 UM）。 Cilostamide（PDE3抑制剂，10 UM），白藜芦醇（12.5 UM），SRT-1720（2 UM）和Rolipram（PDE4抑制剂，30 UM），SER133-CREB的磷酸化增加，Ser133-Creb，Ser431-LKB，Ser431-LKB，Thr172-lk，Thr172-pampk，Ser79-ser79-Acc，以及其他Indimifiened Pk. PKA抑制剂RP-8-BR训练营通过草cilostamide和rolipram阻断了这些信号分子的磷酸化。与cilostamide，白藜芦醇和SRT1720相比，Rolipram对PKA底物和CREB的磷酸化的作用相对较大，但对LKB，AMPK和ACC的磷酸化的影响较小，这表明参与AMPK激活的cAMP池的存在。在与CL-316243（B3-agenist）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，从而导致Lipolsy spision（Glycerol释放）显着增加。这些结果和其他结果提供了证据，表明白藜芦醇和SRT-1720的某些影响可能与它们抑制PDE有关，从而改变细胞内cAMP浓度。 众所周知，哺乳动物PDE3在胰岛素信号通路和血小板，心血管组织，脂肪细胞和卵母细胞中起重要作用。Caenorhabditiselegans秀丽隐杆线虫代表了遗传操纵的独特模型，因此可以促进其功能调节基因和功能的识别。我们在这里报告了乳腺癌磷酸二酯酶的表达和表征的研究，哺乳动物PDE3家族的同源物。线虫PDE3基因存在于II染色体上，约为22.2 kb，并编码两个不同的CEPDE3同工型。 CEPDE3长形式（LF）由11个外显子和63.5 kDa蛋白的代码组成；短形式（SF）具有8个外显子和54.2 kDa蛋白的代码。两种CEPDE3同工型都有特征性的哺乳动物PDE PFAM和磷酸二酯域，还包含HD金属结合基序，这对于PDE超家族是独特的。 CEPDE3与人类PDE家族的多个序列同源分析表明，C.Elegans PDE3接近PDE家族树上的哺乳动物PDE3。 CEPDE3LF和SF同工型的预测序列彼此之间的总体同源性为97％，具有相同的催化域。 PDE活性分析表明，重组CEPDE3长和短形式明显地抑制了cilostamide（一种特定的哺乳动物PDE3抑制剂），而不是由rolipram（PDE4的特定抑制剂）抑制。对于重组CEPDE3长形式和重组哺乳动物PDE3的Cilostamide和Rolipram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。