Expression, Structure/function And Regulation Of Phospho

环核苷酸磷酸二酯酶(PDE)是cAMP和cGMP细胞内浓度和生物学效应的重要调节因子。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，cAMP(对IBMX的反应而积累)在3T3-L1脂肪细胞分化过程中对PDE3B的表达起着关键的调节作用，而PDE3B的诱导似乎与脂质的积累没有直接联系。我们的结果还表明，胰岛素诱导的PDE3B的激活是由PI3-K和PKB依赖的信号介导的，可能与IRS-1诱导的信号分子移位到细胞膜有关，其中PDE3B可能被PKB激活。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端调控区和PKB的PH域；来自N-端区的富含Pro的多肽序列似乎抑制了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，已经产生了PDE3A和PDE3B基因定向中断的小鼠。雌性PDE3A KO小鼠是不育的，最可能的原因是卵母细胞中功能PDE3A的缺失导致cAMP增加，蛋白激酶A(PKA)激活，成熟促进因子/MAP-激酶信号受到抑制，这些信号在减数分裂进程、卵母细胞成熟(以生发泡破裂(GVBD)为标志)和随后的受精过程中至关重要。与这一假设一致，用PKA抑制剂处理PDE3A KO卵母细胞可以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，并显示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有较大幅度的增加，但葡萄糖处置相对较少，这也显示出异常的I.P.与血糖降低有关的胰岛素耐量试验。高胰岛素-正常血糖钳夹试验表明，胰岛素抑制KO小鼠内源性葡萄糖产生的效果要差得多。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。

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.