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。在培养的3 T3-L1脂肪细胞或培养的人单核细胞衍生的巨噬细胞的分化过程中出现PDE 3B（或活性大大增加）。为了研究PDE 3B表达的调控，分离小鼠（M）PDE 3B基因5 '侧翼区的基因组片段;鉴定了两个启动子区，一个远端启动子位于上游约4 kb，一个近端区域位于翻译起始ATG密码子上游约500 bp。用一系列含有荧光素酶基因的报告质粒转染3 T3-L1成纤维细胞和分化中的3 T3-L1脂肪细胞，所述荧光素酶基因与MPDE 3B基因的5 '-UTR的不同片段偶联，表明远端区域诱导的启动子活性比近端区域高约50倍。用远端和近端启动子转染表明：（1）在远端和近端启动子区之间存在强负调控区;（2）用含有远端启动子CRE元件的构建体转染显著增加了分化脂肪细胞中的启动子活性。3 T3-L1脂肪细胞的分化由含有地塞米松、异丁基甲基黄嘌呤和胰岛素的培养基诱导。PDE 3B的诱导被M显著增强（推测其增加cAMP，导致CREB蛋白的磷酸化/活化），并且与脂质蓄积分离，这在DI存在下更明显。综上所述，这些结果表明CREB蛋白的活化可能在3 T3-L1分化脂肪细胞中MPDE 3B的表达中起关键作用。对3 T3-L1脂肪细胞和FDCP 2细胞的研究表明，胰岛素和IGF-1诱导的PDE 3B磷酸化/活化是通过PI 3-K和PKB依赖性信号介导的，并且PDE 3B最有可能是PKB的直接底物。MPDE 3B含有PKB的共有磷酸化位点（Ser 273）; Ser 273 Ala突变后，PKB在体外不激活MPDE 3B。为了研究3 T3-L1细胞中胰岛素对微粒体MPDE 3B的激活，我们检查了参与PDE 3B激活的胰岛素信号通路组分的亚细胞分布。在3 T3-L1脂肪细胞中，PDE 3B主要但不完全与细胞内膜（LDM和HDM部分）相关。与胰岛素孵育导致IRS-1的酪氨酸磷酸化和与细胞内膜相关的PI 3-K的活化。此外，还发现PKB的细胞内库的一部分与细胞内膜相关，在AcA 34上的尺寸排阻色谱期间与PDE 3B共洗脱，并与MPDE 3B共免疫沉淀。这种相互作用的结构决定因素位于MPDE 3的N-末端部分。这些结果表明，激活膜相关的PDE 3B的信号通过IRS/PI 3-K被引导至细胞内膜，其中PI 3-K的激活和磷酸肌醇的产生导致PDK、PKB以及因此PDE 3B的激活。为了进一步检查PDE 3B同种型的功能作用，产生了无效PDE 3A和PDE 3B小鼠。随着PDE 3B缺失小鼠年龄的增长，雄性小鼠显示出异常的葡萄糖和胰岛素耐量试验。来自PDE 3B缺失小鼠的脂肪细胞比来自WT动物的脂肪细胞小，并且它们对脂解刺激（包括异丙肾上腺素和腺苷脱氨酶）的反应较低。研究正在进行中，以了解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 3T3-L1 adipocytes or cultured human monocyte-derived macrophages. To study regulation of PDE3B expression, a genomic fragment of the 5'-flanking region of the mouse(M) PDE3B gene was isolated; two promoter regions were identified, a distal promoter located ~4 kb upstream, and a proximal region, ~500 bp upstream of the translation initiation ATG codon. Transfection of 3T3-L1 fibroblasts and differentiating 3T3-L1 adipocytes with a series of reporter plasmids containing the luciferase gene coupled to different fragments of the 5'-UTR of the MPDE3B gene indicated that the distal region induced about 50-fold higher promoter activity than did the proximal region. Transfection with distal and proximal promoters indicated that (1) a strong negative regulatory region was present between the distal and proximal promoter regions; and (2) transfection with constructs containing CRE elements of the distal promoter markedly increased promoter activity in differentiating adipocytes. Differentiation of 3T3-L1 adipocytes is induced by medium containing DMI (dexamethasone, isobutylmethylxanthine and insulin). Induction of PDE3B was markedly enhanced by M (which presumably increased cAMP, leading to phosphorylation/activation of CREB proteins)and was dissociated from lipid accumulation, which was more pronounced in the presence of DI. Taken together, these results suggest that activation of CREB proteins might play a crucial role in the expression of MPDE3B in 3T3-L1 differentiating adipocytes. Studies with 3T3-L1 adipocytes and FDCP2 cells indicated that insulin- and IGF-1-induced phosphorylation/activation of PDE3B was mediated via PI3-K- and PKB-dependent signals, and that PDE3B is most likely a direct substrate of PKB. MPDE3B contains a consensus phosphorylation site (Ser 273) for PKB; after mutation of Ser 273 Ala, PKB does not activate MPDE3B in vitro. To study activation of microsomal MPDE3B by insulin in 3T3-L1 cells, we examined the subcellular distribution of components of the insulin-signaling pathway involved in activation of PDE3B. In 3T3-L1 adipocytes, PDE3B is primarily, but not exclusively, associated with intracellular membranes (LDM and HDM fractions). Incubation with insulin results in tyrosine phosphorylation of IRS-1 and activation of PI3-K associated with intracellular membranes. In addition, a portion of the intracellular pool of PKB is also found in association with intracellular membranes, co-elutes with PDE3B during size exclusion chromatography on AcA34, and co-immunoprecipitates with MPDE3B. The structural determinants for this later interaction reside in the N-terminal portion of MPDE3. These results suggest that signals to activate membrane-associated PDE3B are directed via IRS/PI3-K to intracellular membranes where activation of PI3-K and production of phosphoinositides results in activation of PDK, PKB and, consequently, PDE3B. To further examine the functional role of PDE3B isoforms, null PDE3A and PDE3B mice were generated. As PDE3B null mice age, the males demonstrate abnormal glucose and insulin tolerance tests. Adipocytes from PDE3B null mice are smaller than those from WT animals, and they are less responsive to lipolytic stimuli, including isoproterenol and adenosine deaminase. Studies are in progress to understand the role of PDE3B in these and other phenotypic changes.