Expression, Structure/function And Regulation Of Phospho

环核苷酸磷酸二酯酶（PDEs）通过催化cAMP和cGMP的水解，是环核苷酸胞内浓度和生物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。PDE3B在培养的3T3-L1脂肪细胞或培养的人单核细胞源性巨噬细胞分化过程中出现（或活性大幅增加）。为了研究PDE3B基因的表达调控，我们分离了小鼠PDE3B基因5'-侧翼区域的基因组片段(M)；两个启动子区域被鉴定出来，一个远端启动子位于上游约4kb，一个近端启动子位于翻译起始ATG密码子上游约500bp。转染3T3-L1成纤维细胞，用一系列含有荧光素酶基因的报告质粒与MPDE3B基因的5'-UTR的不同片段偶联，分化3T3-L1脂肪细胞，表明远端区域诱导的启动子活性比近端区域高约50倍。转染远端和近端启动子表明：(1)在远端和近端启动子区域之间存在一个强的负调控区；(2)转染含有远端启动子CRE元素的构建物可显著提高脂肪细胞分化启动子的活性。用含有DMI（地塞米松、异丁基甲基黄嘌呤和胰岛素）的培养基诱导3T3-L1脂肪细胞分化。M显著增强了PDE3B的诱导（可能增加了cAMP，导致CREB蛋白的磷酸化/激活），并与脂质积累分离，这在DI存在时更为明显。综上所述，这些结果表明CREB蛋白的激活可能在3T3-L1分化脂肪细胞中MPDE3B的表达中起关键作用。对3T3-L1脂肪细胞和FDCP2细胞的研究表明，胰岛素和igf -1诱导的PDE3B磷酸化/激活是通过PI3-K和PKB依赖性信号介导的，PDE3B很可能是PKB的直接底物。MPDE3B包含PKB的一致磷酸化位点（Ser 273）；在Ser 273 α突变后，PKB在体外不激活MPDE3B。为了研究胰岛素在3T3-L1细胞中对微粒体MPDE3B的激活，我们检测了参与PDE3B激活的胰岛素信号通路组分的亚细胞分布。在3T3-L1脂肪细胞中，PDE3B主要与细胞膜（LDM和HDM部分）相关，但并非完全相关。胰岛素孵育导致IRS-1的酪氨酸磷酸化和与细胞膜相关的PI3-K的激活。此外，还发现PKB的一部分胞内池与细胞膜相关，在AcA34上的大小隔离层析中与PDE3B共洗脱，并与MPDE3B共免疫沉淀。这种后期相互作用的结构决定因素位于MPDE3的n端部分。这些结果表明，激活膜相关PDE3B的信号通过IRS/PI3-K定向到细胞膜内，在细胞膜内，PI3-K的激活和磷酸肌苷的产生导致PDK、PKB和PDE3B的激活。为了进一步研究PDE3B亚型的功能作用，我们产生了PDE3A和PDE3B空小鼠。随着PDE3B缺失小鼠年龄的增长，雄性小鼠表现出异常的葡萄糖和胰岛素耐量试验。PDE3B缺失小鼠的脂肪细胞比WT动物的脂肪细胞小，并且它们对溶脂刺激（包括异丙肾上腺素和腺苷脱氨酶）的反应较弱。研究正在进行中，以了解PDE3B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.