Expression, Structure/function And Regulation Of Phospho

环核苷酸磷酸二酯酶（cyclicnucleotidephosphodiesterases，PDE）通过催化环磷酸腺苷（cAMP）和环磷酸鸟苷（cGMP）的水解，是环核苷酸在细胞内浓度和生物学效应的重要调节因子。了解PDE亚型[属于11个基因家族（PDE 1-11）]的细胞表达和调控对于靶向特定PDE治疗各种疾病（包括肺部疾病）将变得越来越重要。PDE 3B在培养的人脂肪细胞、鼠3 T3-L1脂肪细胞或人单核细胞衍生的巨噬细胞的分化过程中出现（或活性大大增加）。我们的研究结果表明，CREB蛋白的激活在调节3 T3-L1脂肪细胞分化过程中PDE 3B的表达起着至关重要的作用。在分化的人脂肪细胞或3 T3-L1脂肪细胞中，TNF α至少部分地通过下调PDE 3B表达来增加脂解，导致cAMP增加和脂肪酶敏感性脂肪酶的活化。TNF α还抑制胰岛素诱导的PDE 3B活化。尚不清楚TNF α对PDE 3B的作用是否参与TNF α诱导胰岛素抵抗。我们的研究结果还表明，胰岛素诱导的PDE 3B的激活是由PI 3-K-和PKB-依赖性信号介导的，PDE 3B是PKB的底物。小鼠PDE 3B中的丝氨酸273似乎分别在完整细胞和体外对胰岛素和PKB激活PDE 3B至关重要。此外，发现PKB的细胞内池的一部分与细胞内膜结合，在溶解的3 T3-L1微粒体膜的凝胶过滤色谱期间与膜结合的PDE 3B共洗脱，并与PDE 3B共免疫沉淀。后一种相互作用的结构决定因素位于PDE 3B的N-末端调控区;丝氨酸273附近的N-末端区域富含脯氨酸的肽序列似乎抑制PDE 3B和PKB之间的相互作用。为了进一步研究PDE 3同种型的功能作用，已经产生了PDE 3A和PDE 3B缺失小鼠。雌性PDE 3A小鼠是不育的，最有可能是因为卵母细胞中功能性PDE 3A的缺乏导致cAMP诱导的减数分裂进程和卵母细胞成熟的阻滞（以生殖囊泡破裂（GVBD）为标志）和受精失败。PDE 3B KO小鼠表现出胰岛素稳态机制破坏和胰岛素抵抗的迹象。向完整小鼠施用β-3激动剂导致PDE 3B KO小鼠的血清胰岛素增加更大，但葡萄糖处置更少，这也证明了关于血糖和血清游离脂肪酸减少的异常腹膜内胰岛素耐受性试验。研究正在进行中，以了解PDE 3A和PDE 3B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.