EXPRESSION/REGULATION OF PHOSPHODIESTERASE 3 ISOFORMS

通过催化cAMP和cGMP的水解，环 核苷酸磷酸二酯酶(PDE)是关键的调节因子 介导的细胞内浓度和生物反应 通过环核苷酸，包括免疫/炎症反应。 了解PDE亚型的细胞调控[属于 到九个基因家族(PDE1-9)]将对 靶向特定的PDE治疗肺部疾病。虽然 单个细胞通常含有几个PDE基因的代表 家族，对细胞因子所涉及的信号通路知之甚少 以及生长因子对单个细胞中不同PDE的调节作用。在……里面 大鼠脂肪细胞胰岛素磷酸化(丝氨酸302)并激活 PDE3B通过磷脂酰肌醇3-激酶(PI3-K)，可能， 依赖PKB的信号。脂肪细胞胞浆提取液中 与胰岛素孵育，激活的PKB与激酶共纯化 使PDE3B磷酸化。在FDCP2造血细胞中，IL-4， IGF-1和胰岛素激活PDE3B和PDE4，而IL-3， GMCSF和PMA(佛波酯)只能激活PDE4。IL-4、IGF-1 IL-3通过PI3-K依赖信号激活PDE3B和PDE4；PMA 通过PKC激活PDE4。PI3-K和PKC、IL-4下游， IGF-1、IL-3和PMA通过MAP信号激活PDE4；IL-4 IGF-1通过MAP非依赖性信号激活PDE3B。至 阐明PKB在完整细胞中激活PDE3B中的作用 将野生型(F/B)基因分别转入FDCP2细胞。 活性(F/B*)和非活性(F/B-)PKB和对照载体 (F/V)在F/V细胞中，IGF-1增加PKB、PDE3B和PDE4 活动约为2折。然而，在F/B细胞中，IGF-1在一个 Wortmannin敏感的方式，增加PKB活性约。 10倍和PDE3B的磷酸化和活性(约4倍)， 但PDE4的增加程度与F/V细胞相同。在F/B*中 细胞，在没有IGF-1的情况下，PKB活性显著增加 )10倍)，PDE3B被磷酸化并激活 (3-4倍)，这些作用可被Wortmannin抑制。在F/B*中 细胞中，IGF-1对PKB和PKB几乎没有进一步的影响 PDE3B的激活/磷酸化。雷帕霉素(p70S6激酶 抑制物)和PD98059[丝裂原激活的激酶-1 (MEK-1)抑制剂]不影响F/B中PKB或PDE3B的活性。 细胞。F/B*细胞的胸腺嘧啶核苷掺入量高于F/V细胞 细胞，并被PDE3抑制剂抑制的程度更大 而不是PDE4抑制剂罗利普兰。这些结果表明， PDE3B，而不是PDE4，是下游的目标，如果不是底物的话 PKB和激活PDE3B可能调节cAMP池 至少部分调节PKB对增殖/存活的影响 FDCP2造血细胞。尽管PDE4被认为是 在大多数炎症/免疫细胞中以PDE亚型为主， 对cAMP有很高亲和力的PDE3和PDE4都是 存在于淋巴细胞和巨噬细胞中。逆转录-聚合酶链式反应 淋巴细胞和巨噬细胞RNA显示存在 PDE3B，而不是PDE3A，mRNA。在培养5-7天后洗脱 外周血单核细胞黏附在塑料表面并增加 体型惊人。形态变化伴随着一种 PDE3B mRNA和活性相对增加，GLUT 5转运体 和PDE1C mRNAs，以及PDE4活性的相对下降和 PDE4基因的表达。佛波醇与淋洗出的单核细胞孵育 四个小时的酯化与他们强烈的依从性和 PDE3B活性和mRNA(RT-PCR)相对于 PDE4，单核细胞无明显变化 形态学。重要的是要定义特定的PDE异构体 在肺泡巨噬细胞中存在，并确定细胞 炎症反应可以被PDE抑制剂改变。

By catalyzing hydrolysis of cAMP and cGMP, cyclic nucleotide phosphodiesterases (PDEs) are critical regulators of intracellular concentrations of, and biological responses mediated by, cyclic nucleotides, including immune/inflammatory responses. Understanding cellular regulation of PDE isoforms [which belong to nine gene families (PDE1-9)] will be of increasing importance for targeting specific PDEs in treating pulmonary disorders. Although individual cells usually contain representatives of several PDE gene families, little is known of signalling pathways involved in cytokine and growth factor regulation of different PDEs in a single cell. In rat adipocytes insulin phosphorylates (serine 302) and activates PDE3B via phosphatidylinositol 3-kinase (PI3-K) and perhaps, PKB-dependent signals. In cytosolic extracts from adipocytes incubated with insulin, activated PKB co-purified with the kinase that phosphorylated PDE3B. In FDCP2 hematopoietic cells, IL-4, IGF-1 and insulin activate PDE3B and PDE4, whereas IL-3, GMCSF and PMA (phorbol ester) only activate PDE4. IL-4, IGF-1 and IL-3 activate PDE3B and 4 by PI3-K-dependent signals; PMA activates PDE4 via PKC. Downstream of PI3-K and PKC, IL-4, IGF-1, IL3 and PMA activate PDE4 by MAP kinase signals; IL-4 and IGF-1 activate PDE3B by MAP kinase-independent signals. To elucidate the role of PKB in activation of PDE3B in intact cells, FDCP2 cells were transfected with wild-type (F/B), constituitively active (F/B*) and kinase-inactive (F/B-)PKB and control vector (F/V). In F/V cells, IGF-1 increased PKB, PDE3B, and PDE4 activities approx. 2-fold. In F/B cells, however, IGF-1, in a wortmannin-sensitive manner, increased PKB activity approx. 10-fold and PDE3B phosphorylation and activity (approx. 4-fold), but increased PDE4 to the same extent as in F/V cells. In F/B* cells, in the absence of IGF-1, PKB activity was markedly increased (approx. 10-fold) and PDE3B was phosphorylated and activated (3-4 fold); these effects were inhibited by wortmannin. In F/B* cells, IGF-1 had little or no further effect on PKB and activation/phosphorylation of PDE3B. Rapamycin (p70S6 kinase inhibitor) and PD98059 [Mitogen-activated kinase kinase-1 (MEK-1) inhibitor] did not affect PKB or PDE3B activities in F/B* cells. Thymidine incorporation was greater in F/B* cells than in F/V cells and was inhibited to a much greater extent by PDE3 inhibitors than by rolipram, a PDE4 inhibitor. These results suggest that PDE3B, not PDE4, is a downstream target, if not substrate, for PKB and that activated PDE3B may regulate cAMP pools that modulate, at least in part, effects of PKB on proliferation/survival of FDCP2 hematopoietic cells. Although PDE4 is thought to be the predominant PDE isoform in most inflammatory/immune cells, PDE3 and PDE4, which exhibit a high affinity for cAMP, are both present in lymphocytes and macrophages. RT-PCR amplification of lymphocyte and macrophage RNA demonstrated the presence of PDE3B, not PDE3A, mRNA. After 5-7 days in culture elutriated peripheral human monocytes adhere to plastic surfaces and increase dramatically in size. The morphologic change is accompanied by a relative increase in PDE3B mRNA and activity, Glut 5 transporter and PDE1C mRNAs and a relative decrease in PDE4 activity and PDE4 mRNA. Incubation of elutriated monocytes with phorbol ester for four hours was associated with their strong adherence and an increase in PDE3B activity and mRNA (RT-PCR) relative to that of PDE4, without a marked change in mononuclear cell morphology. It will be important to define specific PDE isoforms present in alveolar macrophages and to determine if cellular inflammatory responses can be altered by PDE inhibitors.