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.

环状核苷酸磷酸二酯酶（PDES）是cAMP和CGMP的细胞内浓度和生物学作用的关键调节剂。了解PDE同工型的细胞表达和调节[属于11个基因家族（PDES 1-11）]将对靶向特定PDE在治疗各种疾病（包括肺部疾病）中的重要性。在分化的人类脂肪细胞，鼠3T3-L1脂肪细胞或人类单核细胞衍生的巨噬细胞的分化过程中，PDE3B（或大幅增加）在活性中出现。我们的结果表明，在3T3-L1脂肪细胞分化过程中，cAMP（响应IBMX的积累）在调节PDE3B表达的调节中起着至关重要的作用，而PDE3B的诱导似乎与脂质的积累直接相关。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诱导的信号分子转移到细胞内膜上，PKB可以通过PKB激活PDE3B。此外，在细胞内膜的一部分PKB库中，与细胞内膜相关，在溶解的3T3-L1微体膜的凝胶过滤色谱期间与膜相关的PDE3B共同残留，以及与PDE3B的共发蛋白酶。后一种相互作用的结构决定因素位于PDE3B的N末端调节区域和PKB的pH域；来自N末端区域的富含脯氨酸的肽序列似乎抑制了PDE3B和PKB之间的相互作用。为了进一步检查PDE3同工型的功能作用，已经产生了具有PDE3A和PDE3B基因的靶向破坏的小鼠。雌性PDE3A KO小鼠是无菌的，很可能是因为卵母细胞中缺乏功能性PDE3A会导致cAMP增加，蛋白激酶A（PKA）的激活以及抑制成熟因子/地图激酶的抑制作用，对中等水平的进展，卵母细胞的成熟（分类），并散布（分别是卵子），并散布（分别）受精。与这一假设一致，用PKA抑制剂对PDE3A KO卵母细胞的处理可重新启动减数分裂的进展，并恢复体外受精的能力。 PDE3B KO小鼠似乎积累了较少的脂肪，并且表现出破坏胰岛素稳态机制和胰岛素抵抗的迹象。给予完整的小鼠β-3激动剂会导致血清胰岛素增加得多，但PDE3B KO小鼠的葡萄糖处置相对较少，这也表现出异常的i.p.胰岛素耐受性测试有关血糖减少。高胰岛素血糖夹表明，胰岛素在抑制KO小鼠的内源性葡萄糖产生方面的有效性要小得多。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。