Expression, Structure/function And Regulation Of Phospho

环核苷酸磷酸二酯酶(PDE)通过催化cAMP和cGMP的水解，调节环核苷酸的胞内浓度和生物效应。了解PDE异构体[属于11个基因家族(PDEs1-11)]的细胞表达和调控，对于靶向PDEs治疗各种疾病，包括肺部疾病，将具有越来越重要的意义。PDE3B在培养的人脂肪细胞、小鼠3T3-L1脂肪细胞或人单核细胞来源的巨噬细胞分化过程中出现(或活性显著增加)。我们的结果表明，CREB蛋白的激活在3T3-L1脂肪细胞分化过程中对PDE3B表达的调控起着至关重要的作用。在分化的人类脂肪细胞或3T3-L1脂肪细胞中，肿瘤坏死因子α至少部分地通过下调PDE3B的表达来增加脂肪分解，导致cAMP增加和激素敏感脂肪酶的激活。肿瘤坏死因子α也抑制胰岛素诱导的PDE3B的激活。目前尚不清楚肿瘤坏死因子α对PDE3B的影响是否参与了肿瘤坏死因子α诱导的胰岛素抵抗。我们的结果还表明，胰岛素诱导的PDE3B的激活是由依赖于PI3-K和PKB的信号介导的，并且PDE3B是PKB的底物。小鼠PDE3B中的丝氨酸273似乎是胰岛素和PKB分别在完整细胞和体外激活PDE3B的关键。此外，在溶解的3T3-L1微粒体膜的凝胶过滤层析过程中，发现部分PKB与细胞内膜、与膜相关的PDE3B共洗脱，以及与PDE3B共免疫沉淀物。后一种相互作用的结构决定因素位于PDE3B的N-末端调节区；来自N-末端靠近丝氨酸273的富含Pro的多肽序列似乎抑制了PDE3B与PKB之间的相互作用。为了进一步研究PDE3亚型的功能作用，我们建立了PDE3A和PDE3B缺失小鼠。雌性PDE3A小鼠是不育的，最可能的原因是卵母细胞中缺乏功能性PDE3A导致cAMP诱导的减数分裂进程和卵母细胞成熟(以生发泡破裂(GVBD)为标志)，以及受精失败。PDE3B KO小鼠表现出胰岛素稳态机制被破坏和胰岛素抵抗的迹象。正常小鼠服用Beta-3激动剂后，PDE3B KO小鼠的血清胰岛素水平显著升高，但葡萄糖处置减少，这也显示出异常的I.P.关于降低血糖和血清游离脂肪酸的胰岛素耐量试验。研究正在进行中，以了解PDE3A和PDE3B在这些和其他表型变化中的作用。

By catalyzing hydrolysis of cAMP and cGMP, cyclic nucleotide phosphodiesterases (PDEs) are critical regulators of intracellular concentrations and biological effects of cyclic nucleotides. 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 activation of CREB proteins plays a crucial role in regulation of PDE3B expression during differentiation of 3T3-L1 adipocytes. In differentiated human adipocytes or 3T3-L1 adipocytes, TNFalpha increases lipolysis, at least in part, by downregulation of PDE3B expression, leading to increases in cAMP and activation of hormone-sensitive lipase. TNFalpha also inhibits insulin-induced activation of PDE3B. It is not known if effects of TNFalpha on PDE3B are involved in induction of insulin-resistance by TNFalpha. Our results also indicate that insulin-induced activation of PDE3B is mediated by PI3-K- and PKB-dependent signals, and that PDE3B is a substrate of PKB. Serine 273 in murine PDE3B seems to be critical for activation of PDE3B by insulin and PKB in intact cells and in vitro, respectively. 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; proline-rich peptide sequences from the N-terminal region near serine 273 seem to inhibit the interaction between PDE3B and PKB. To further examine functional roles of PDE3 isoforms, PDE3A and PDE3B null mice have been generated. Female PDE3A mice are sterile, most likely because the absence of functional PDE3A in oocytes leads to a cAMP-induced block in meiotic progression and oocyte maturation (marked by germinal vesicle breakdown (GVBD)), and failure of fertilization. PDE3B KO mice 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 less glucose disposal in PDE3B KO mice, which also demonstrate aberrant i.p. insulin tolerance tests with respect to reduction in blood glucose and serum free fatty acids. Studies are ongoing to understand the roles of PDE3A and PDE3B in these and other phenotypic changes.