EXPRESSION/REGULATION OF PHOSPHODIESTERASE 3 ISOFORMS

环核苷酸磷酸二酯酶(PDE)通过催化cAMP和cGMP的水解，调节细胞内环核苷酸的浓度及其介导的生物反应，包括免疫/炎症反应。了解PDE亚型[属于11个基因家族(PDE1-11)]的细胞调控将对靶向PDE治疗肺部疾病具有越来越重要的意义。虽然单个细胞通常含有几个PDE基因家族的代表，但对单个细胞中涉及不同PDE的细胞因子和生长因子调节的信号通路知之甚少。在小鼠FDCP2早幼粒细胞中，IL-4和IGF-1激活PDE3和PDE4，而IL-3仅激活PDE4。对肿瘤坏死因子α和JAK、PI3-K、PKC和MAPK激酶抑制剂的研究表明，IGF-1和IL-3都通过PI3-K依赖的信号激活PDE3和PDE4。在PI3-K下游，调节通路分化；依赖于MEK/MAPK的信号激活的是PDE4，而不是PDE3。因此，将野生型(Wt)、成分活性(CA)或非活性(Ki)形式的MEK和PKB永久地导入FDCP2细胞。对这些转基因细胞的研究表明，PDE4被依赖于MEK/MAPK的信号激活，而PDE3被磷酸化并被依赖于PKB的信号激活。重组小鼠(M)PDE3B在体外可被PKB磷酸化和激活；一个缺失共有的PKB磷酸化位点的MPDE3B突变体不被磷酸化或激活。在完整的FDCP2细胞和Sf21细胞裂解产物中的实验表明，尽管Ser272参与了PKB对MPDE3B的激活，Ser296参与了PKA对MPDE3B的激活，Ser421似乎在调节PKA和PKB对MPDE3B的激活中起着重要的作用。在表达WT PKB的FDCP2细胞中，促凋亡蛋白BAD被IGF-1磷酸化；8-BR-cAMP或PDE3抑制剂ciloamide阻止了磷酸化。这些和其他数据表明，PDE3B是PKB的下游靶点，如果不是底物的话，它可能作为PKB的效应器，调节cAMP池，至少部分地调节IGF-1和胰岛素对FDCP2细胞的生存/增殖和脂肪细胞的脂肪分解的影响。在MPDE3B基因5‘侧翼区已鉴定出两个启动子区域，位于翻译起始点上游4kb的远端启动子区域和近端TATA缺失区域。用不同的荧光素酶报告质粒载体转导3T3-L1成纤维细胞和分化3T3-L1脂肪细胞表明：(1)远端和近端启动子区域之间存在一个强烈的负调控区域；(2)Cre顺式元件和CREB蛋白可能在3T3-L1脂肪细胞分化过程中对PDE3B的诱导起着重要的调节作用。我们在协同实验中发现，肿瘤坏死因子α对脂肪细胞脂分解的刺激作用可能部分与下调3T3-L1脂肪细胞中PDE3B的表达有关。

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 eleven gene families(PDE1-11)] 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 murine FDCP2 promyeloid cells, IL-4 and IGF-1 activate PDE3 and PDE4, whereas IL-3 activates only PDE4. Studies with TNFalpha and inhibitors of JAK, PI3-K, PKC, and MAPK kinases indicate that both IGF-1 and IL-3 activate PDE3 and PDE4 via PI3-K-dependent signals. Downstream of PI3-K, regulatory pathways diverge; PDE4, but not PDE3, is activated by MEK/MAPK-dependent signals. FDCP2 cells were, therefore, permanently transfected with wild type (wt), constitutively active (CA), or kinase inactive (KI) forms of MEK and PKB. Studies with these transfected cells indicated that PDE4 was activated by MEK/MAPK-dependent signals, and that PDE3 was phosphorylated and activated by PKB-dependent signals. Recombinant mouse (M) PDE3B was phosphorylated and activated in vitro by PKB; a truncated MPDE3B mutant lacking consensus PKB phosphorylation sites was not phosphorylated or activated. Serine-alanine mutations were introduced into MPDE3B at S272(PKB consensus phosphorylation site),and at Ser296 and 421(PKA sites).Experiments in intact FDCP2 cells and Sf21 cell lysates indicated that although Ser 272 was involved in activation of MPDE3B by PKB,and Ser296 by PKA, Ser421 seemed to be important in regulation of activation of MPDE3B by both PKA and PKB.In FDCP2 cells expressing WT PKB, the proapoptotic protein BAD was phosphorylated in response to IGF-1; phosphorylation was blocked by 8-Br-cAMP or the PDE3 inhibitor cilostamide. These and other data suggest that PDE3B is a downstream target, if not substrate, of PKB and may function as an effector of PKB in regulation of cAMP pools that modulate, at least in part, effects of IGF-1 and insulin on survival/proliferation of FDCP2 cells and lipolysis in adipocytes. Two promoter regions in the 5'-flanking region of the MPDE3B gene have been identified,a distal promoter region located ~4kb upstream from the translation initiation site and a proximal TATA-less region.Transfection of 3T3-L1 fibroblasts and differentiating 3T3-L1 adipocytes with various luciferase reporter plasmid vectors suggested that:(1)a strong negative regulatory region was present between the distal and proximal promoter regions;(2)CRE cis-elements and CREB proteins might play a crucial regulatory role in the induction of PDE3B that occurs during differentiation of 3T3-L1 adipocytes.In collaborative experiments we found that the stimulatory effects of TNF alphaon lipolysis may in part be related to down-regulation of PDE3B expression in 3T3-L1 adipocytes.Whether downregulation of PDE3B is involved in the effects of TNF alphaon the development of insulin-resistance is not known.