Phosphodiesterase 3 Isoforms

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。在IBMX、地塞米松(Dex)和胰岛素(INS)存在下，培养的人脂肪细胞或3T3-L1小鼠脂肪细胞在分化过程中出现膜相关PDE3B(或活性显著增加)。IBMX是一种非特异性的PDE抑制剂，可增加CREB(cAMP反应元件结合蛋白)的磷酸化，是PDE3B表达的主要调节因子。IBMX刺激的PDE3B mRNA和蛋白的诱导可被CREB的显性阴性版本KCREB的过度表达所抑制。小鼠PDE3B基因5‘侧翼区包含两个启动子区域，远端区域(位于翻译起始点上游~5kb)包含一个规范的顺式作用Cre(cAMP反应元件)序列，较近的区域包含非典型的Cre元件。Cre序列突变显著降低IBMX刺激的远端启动子活性；PKA抑制剂H89抑制IBMX刺激的近端启动子活性。染色质免疫沉淀分析表明，在IBMX处理的细胞中，CREB、磷酸化CREB和CBP/p300与近端和远端启动子结合，并且磷酸化CREB与这两个启动子的相互作用增强。因此，顺式作用的Cre元件和CREB蛋白的激活似乎在PDE3B的表达中起着至关重要的调节作用。 我们的结果还表明，胰岛素介导的膜相关PDE3B的激活可能涉及包含关键效应分子(如PKB)的信号复合体的形成，PKB在PDE3B活性的调节中起重要作用。部分内源性PKB池(PKB2多于PKB1)与胞膜结合，在凝胶过滤层析过程中与内源性PDE3B共洗脱，并与PDE3B共免疫沉淀。磷酸化的PKB似乎比非磷酸化的PKB更有效地与PDE3B结合。Wortmannin可阻断PDE3B和PDE3B的磷酸化/活化，从而阻断胰岛素诱导的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B基因定向中断的小鼠。雌性(非雄性)PDE3A KO小鼠是不育的，最可能的原因是卵母细胞PDE3A的缺失导致cAMP增加，以及在减数分裂、卵母细胞成熟和随后的受精过程中至关重要的信号中断。最近的结果表明，在PDE3A KO卵母细胞中，PKA的催化和调节亚基不能移位到核可能是抑制卵母细胞成熟的重要原因。PDE3B KO小鼠似乎积累的脂肪较少，并显示出胰岛素抵抗和胰岛素调节稳态机制被破坏的迹象。胰岛素抑制PDE3B卵母细胞中儿茶酚胺刺激的脂解反应，但不抑制PDE3B KO卵母细胞的脂解反应。在分离的胰岛中，葡萄糖和cAMP升高剂对PDE3B KO胰岛的胰岛素分泌的刺激作用大于WT胰岛。此外，PDE3B KO小鼠附睾脂肪组织表现出一些棕色脂肪组织的表型特征，包括PDE3B KO脂肪细胞UCP-1表达增加和脂肪酸氧化增加。与WT小鼠相比，PDE3B KO组正常小鼠对Beta-3受体激动剂的反应增加了更大程度的O2消耗。

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. Membrane-associated PDE3B appears (or greatly increases in activity) during differentiation of cultured human adipocytes or 3T3-L1 murine adipocytes in the presence of IBMX, dexamethasone (Dex), and insulin (ins). IBMX, a non-specific PDE inhibitor which increases CREB (cAMP Response Element Binding protein) phosphorylation, was the predominant regulator of PDE3B expression. IBMX-stimulated induction of PDE3B mRNA and protein was inhibited by overexpression of KCREB, a dominant-negative version of CREB. The 5'-flanking region of the murine PDE3B gene contains two promoter regions, a distal region (located ~5kb upstream of the translation initiation site) which include a canonical cis-acting CRE (cAMP Response Elements) sequence, and a more proximal region which includes atypical CRE elements. Mutation of CRE sequences markedly reduced IBMX-stimulated distal promoter activity; H89 (a PKA inhibitor)inhibited IBMX-stimulated proximal promoter activity. Chromatin immunoprecipitation assays indicated that CREB, phospho-CREB, and CBP/p300 associated with the proximal and distal promoters and that the interaction of phospho-CREB with both promoters was increased in IBMX-treated cells. Thus, cis-acting CRE elements and activation of CREB proteins seem to play a crucial regulatory role in PDE3B expression. Our results also indicate that insulin-mediated activation of membrane-associated PDE3B may involve formation of a signaling complex containing critical effector molecules, such as PKB, important in regulation of PDE3B activity. A portion of the intracellular pool of endogenous PKB (PKB2 more than PKB1) is found in association with intracellular membranes, co-elutes with endogenous PDE3B during gel filtration chromatography, and co-immunoprecipitates with PDE3B. Phosphorylated PKB seems to associate more effectively with PDE3B than non-phosphorylated PKB. The insulin-induced interactions between PDE3B and PKB are blocked by wortmannin, which blocks phosphorylation/activation of both PKB and PDE3B. To further examine the functional role of PDE3 isoforms, mice with targeted disruptions of PDE3A and B genes have been generated. Female (not male) PDE3A KO mice are sterile, most likely because the absence of oocyte PDE3A leads to increased cAMP and disruption of signals important in progression of meiosis, oocyte maturation, and subsequent fertilization. Recent results suggest that, in PDE3A KO oocytes, the failure of catalytic and regulatory subunits of PKA to translocate to the nucleus may be important in the inhibition of oocyte maturation. PDE3B KO mice seem to accumulate less fat and exhibit signs of insulin resistance and disruption of insulin-regulated homeostatic mechanisms. Insulin inhibits catecholamine-stimulated lipolysis in PDE3B WT but not PDE3B KO oocytes. In isolated pancreatic islets, glucose and cAMP-elevating agents stimulated insulin-secretion to a greater extent in PDE3B KO islets than WT islets. In addition, epididymal adipose tissue in PDE3B KO mice exhibit some phenotypic characteristics of brown adipose tissue, including increased expression of UCP-1 and increased fatty acid oxidation in PDE3B KO adipocytes. O2 consumption by intact mice in response to a Beta-3 receptor agonist was increaed to a greater extent in PDE3B KO than in WT mice.