Expression, Structure/function And Regulation Of Phospho

Phospho的表达、结构/功能和调控

• 批准号: 6809653
• 负责人: VINCENT MANGANIELLO
• 金额: --
• 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/6809653
• 关键词: 3'5' cyclic nucleotide phosphodiesterase; 3T3 cells; B lymphocyte; T lymphocyte; adipocytes; cAMP response element binding protein; cell differentiation; cell line; enzyme activity; esterase inhibitor; gene targeting; genetically modified animals; human tissue; insulin; insulinlike growth factor; isozymes; laboratory mouse; leukocyte activation /transformation; macrophage; nucleotide metabolism; protein kinase; protein structure function

Cyclic nucleotide phosphodiesterases (PDEs) are critical regulators of intracellular concentrations and biological effects of cAMP and cGMP. Understanding cellular expression and regulation of PDE isoforms [which belong to eleven gene families (PDEs 1-11)] will be of increasing importance for targeting specific PDEs in treating various diseases, including pulmonary disorders. PDE3B appears (or greatly increases in activity) during differentiation of cultured human adipocytes, murine 3T3-L1 adipocytes, or human monocyte-derived macrophages. Our results suggest that cAMP (accumulating in response to IBMX) plays a crucial role in regulation of PDE3B expression during differentiation of 3T3-L1 adipocytes, and induction of PDE3B does not seem to be directly linked to accumulation of lipid. Our results also indicate that insulin-induced activation of PDE3B is mediated by PI3-K- and PKB-dependent signals, and may be related to IRS-1 induced translocation of signaling molecules to intracellular membranes, where PDE3B may be activated by PKB. In addition a portion of the intracellular pool of PKB is found in association with intracellular membranes, co-elutes with membrane-associated PDE3B during gel filtration chromatography of solubilized 3T3-L1 microsomal membranes, and co-immunoprecipitates with PDE3B. The structural determinants for this latter interaction reside in the N-terminal regulatory region of PDE3B and PH-domain of PKB; proline-rich peptide sequences from the N-terminal region seem to inhibit interactions between PDE3B and PKB. To further examine functional roles of PDE3 isoforms, mice with targeted disruptions of PDE3A and PDE3B genes have been generated. Female PDE3A KO mice are sterile, most likely because the absence of functional PDE3A in oocytes leads to increased cAMP, activation of Protein kinase A (PKA), and inhibition of Maturation-promoting factor/ MAP-kinase signals that are important in meiotic progression , oocyte maturation (marked by germinal vesicle breakdown (GVBD)), and subsequent fertilization. Consistent with this hypothesis, treatment of PDE3A KO oocytes with PKA inhibitors reinitiates meiotic progression, and restores competency for fertilization in vitro. PDE3B KO mice seem to accumulate less fat and exhibit signs of disruption of insulin homeostatic mechanisms and insulin resistance. Administration of a Beta-3 agonist to intact mice results in a much larger increase in serum insulin but relatively less glucose disposal in PDE3B KO mice, which also demonstrate aberrant i.p. insulin tolerance tests with respect to reduction in blood glucose. Hyperinsulinemic-euglycemic clamps indicate that insulin is much less effective in inhibiting endogenous glucose production in KO mice. Studies are ongoing to understand the roles of PDE3A and PDE3B in these and other phenotypic changes.

环核苷酸磷酸二酯酶 (PDE) 是 cAMP 和 cGMP 的细胞内浓度和生物效应的关键调节剂。了解 PDE 亚型 [属于 11 个基因家族 (PDE 1-11)] 的细胞表达和调节对于靶向特定 PDE 治疗各种疾病（包括肺部疾病）将变得越来越重要。 PDE3B 在培养的人脂肪细胞、鼠 3T3-L1 脂肪细胞或人单核细胞来源的巨噬细胞的分化过程中出现（或活性大大增加）。我们的结果表明，cAMP（响应 IBMX 而积累）在 3T3-L1 脂肪细胞分化过程中 PDE3B 表达的调节中起着至关重要的作用，并且 PDE3B 的诱导似乎与脂质积累没有直接关系。我们的结果还表明，胰岛素诱导的 PDE3B 激活是由 PI3-K 和 PKB 依赖性信号介导的，并且可能与 IRS-1 诱导的信号分子易位至细胞内膜有关，其中 PDE3B 可能被 PKB 激活。此外，还发现细胞内 PKB 池的一部分与细胞内膜相关，在溶解的 3T3-L1 微粒体膜的凝胶过滤层析过程中与膜相关的 PDE3B 共洗脱，并与 PDE3B 共免疫沉淀。后一种相互作用的结构决定因素位于 PDE3B 的 N 末端调节区和 PKB 的 PH 结构域； N 末端区域富含脯氨酸的肽序列似乎抑制 PDE3B 和 PKB 之间的相互作用。为了进一步研究 PDE3 亚型的功能作用，我们培育了 PDE3A 和 PDE3B 基因有针对性破坏的小鼠。雌性 PDE3A KO 小鼠是不育的，很可能是因为卵母细胞中功能性 PDE3A 的缺失导致 cAMP 增加、蛋白激酶 A (PKA) 激活以及对减数分裂进程中重要的成熟促进因子/MAP 激酶信号的抑制，卵母细胞成熟（以生泡破裂 (GVBD) 为标志）和随后的受精。与这一假设一致，用 PKA 抑制剂处理 PDE3A KO 卵母细胞可重新启动减数分裂进程，并恢复体外受精的能力。 PDE3B KO 小鼠似乎积累的脂肪较少，并表现出胰岛素稳态机制破坏和胰岛素抵抗的迹象。对完整小鼠施用 Beta-3 激动剂会导致 PDE3B KO 小鼠血清胰岛素大幅增加，但葡萄糖处理相对较少，这也证明了腹腔注射异常。与降低血糖相关的胰岛素耐量试验。高胰岛素-正常血糖钳夹表明胰岛素在抑制 KO 小鼠内源性葡萄糖产生方面的效果要差得多。正在进行研究以了解 PDE3A 和 PDE3B 在这些和其他表型变化中的作用。