EXPRESSION/REGULATION OF PHOSPHODIESTERASE 3 ISOFORMS

环核苷酸磷酸二酯酶（cyclicnucleotidephosphodiesterases，PDE）是环核苷酸在细胞内的重要调节因子，通过催化cAMP和cGMP的水解，调节环核苷酸在细胞内的浓度，调节环核苷酸介导的生物学反应，包括免疫/炎症反应，了解PDE亚型（属于11个基因家族（PDE 1 -11））的细胞调节对于靶向PDEs治疗肺部疾病具有重要意义。尽管单个细胞通常包含几个PDE基因家族的代表，但对单个细胞中不同PDE的细胞因子和生长因子调节所涉及的信号通路知之甚少。在鼠FDCP 2早幼粒细胞中，IL-4和IGF-1激活PDE 3和PDE 4，而IL-3仅激活PDE 4。 TNF α和JAK、PI 3-K、PKC和MAPK激酶抑制剂的研究表明，IGF-1和IL-3均通过PI 3-K依赖性信号激活PDE 3和PDE 4。在PI 3-K的下游，调节通路发散; PDE 4而不是PDE 3被MEK/MAPK依赖性信号激活。 因此，用MEK和PKB的野生型（wt）、组成型活性（CA）或激酶失活（KI）形式永久转染FDCP 2细胞。 对这些转染细胞的研究表明，PDE 4被MEK/MAPK依赖性信号激活，而PDE 3被磷酸化并被PKB依赖性信号激活。重组小鼠（M）PDE 3B在体外被PKB磷酸化和活化;缺乏共有PKB磷酸化位点的截短MPDE 3B突变体不被磷酸化或活化。在S272处将丝氨酸-丙氨酸突变引入MPDE 3B中（PKB共有磷酸化位点），以及Ser 296和421在完整FDCP 2细胞和Sf 21细胞裂解物中的实验表明，尽管Ser 272参与PKB对MPDE 3B的激活，Ser 296参与PKA对MPDE 3B的激活，在表达WT PKB的FDCP 2细胞中，促凋亡蛋白BAD被IGF-1磷酸化;磷酸化被8-Br-cAMP或PDE 3抑制剂西洛酰胺阻断。 这些和其他数据表明，如果不是底物，PDE 3B是PKB的下游靶标，并且可能在调节cAMP池中作为PKB的效应物起作用，所述cAMP池至少部分地调节IGF-1和胰岛素对FDCP 2细胞的存活/增殖和脂肪细胞中的脂解的影响。在MPDE 3B基因5 '端侧翼区发现了两个启动子区，一个位于翻译起始位点上游约4kb的远端启动子区和一个近端无TATA区，用不同的荧光素酶报告质粒转染3 T3-L1成纤维细胞和分化中的3 T3-L1脂肪细胞表明：（1）在远端和近端启动子区之间存在一个强负调控区;（2）CRE顺式元件和CREB蛋白可能在3 T3-L1脂肪细胞分化过程中对PDE 3B的诱导起重要的调节作用。PDE 3B的下调是否涉及TNF α对胰岛素抵抗发展的影响尚不清楚。

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.