Phosphodiesterase 3 Isoforms

环核苷酸磷酸二酯酶（PDEs）是cAMP和cGMP细胞内浓度和生物学效应的关键调节因子。了解PDE亚型[属于11个基因家族（PDEs 1-11）]的细胞表达和调控对于靶向特异性PDE治疗包括肺部疾病在内的各种疾病越来越重要。在IBMX、地塞米松（Dex）和胰岛素（ins）存在的情况下，在培养的人脂肪细胞或3T3-L1小鼠脂肪细胞分化过程中，膜相关PDE3B出现（或活性大幅增加）。IBMX是一种非特异性PDE抑制剂，可增加cAMP反应元件结合蛋白（CREB）的磷酸化，是PDE3B表达的主要调节因子。通过过表达KCREB （CREB的显性阴性版本），ibmx刺激的PDE3B mRNA和蛋白的诱导被抑制。小鼠PDE3B基因的5'侧区域包含两个启动子区域，一个远端区域（位于翻译起始位点上游约5kb处）包含典型的顺式CRE （cAMP Response Elements）序列，一个更近端的区域包含非典型CRE元件。CRE序列突变显著降低ibmx刺激的远端启动子活性；H89（一种PKA抑制剂）抑制ibmx刺激的近端启动子活性。染色质免疫沉淀试验表明，CREB、phospho-CREB和CBP/p300与近端启动子和远端启动子相关，并且在ibmx处理的细胞中，phospho-CREB与这两个启动子的相互作用增加。因此，顺式作用的CRE元件和CREB蛋白的激活似乎在PDE3B表达中起着至关重要的调节作用。

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. 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.