EXPRESSION/REGULATION OF PHOSPHODIESTERASE 3 ISOFORMS

环核苷酸磷酸二酯酶（PDEs）通过催化cAMP和cGMP的水解，是环核苷酸介导的细胞内浓度和生物反应（包括免疫/炎症反应）的关键调节剂。了解PDE亚型的细胞调控[属于11个基因家族（PDE1-11）]对于靶向特异性PDE治疗肺部疾病将越来越重要。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控所涉及的信号通路知之甚少。在小鼠FDCP2早髓样细胞中，IL-4和IGF-1激活PDE3和PDE4，而IL-3仅激活PDE4。对TNFalpha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依赖性信号激活PDE3和PDE4。PI3-K下游，调控通路分化；PDE4，而不是PDE3，由MEK/ mapk依赖性信号激活。因此，FDCP2细胞被永久转染野生型（wt）、组成型活性（CA）或激酶失活（KI）形式的MEK和PKB。对这些转染细胞的研究表明，PDE4被MEK/ mapk依赖的信号激活，PDE3被pkb依赖的信号磷酸化和激活。重组小鼠(M) PDE3B在体外被PKB磷酸化和激活；缺少一致的PKB磷酸化位点的截断的MPDE3B突变体未被磷酸化或激活。将丝氨酸-丙氨酸突变引入MPDE3B的S272（PKB一致磷酸化位点）、Ser296和421（PKA位点）。在完整FDCP2细胞和Sf21细胞裂解物中进行的实验表明，尽管Ser 272参与了PKB对MPDE3B的激活，Ser296参与了PKA对MPDE3B的激活，但Ser421似乎在PKA和PKB对MPDE3B的激活的调节中都很重要。在表达WT PKB的FDCP2细胞中，促凋亡蛋白BAD响应IGF-1被磷酸化；磷酸化被8-Br-cAMP或PDE3抑制剂西洛胺阻断。这些和其他数据表明，PDE3B是PKB的下游靶点，如果不是底物，并且可能作为PKB的效应物调节cAMP池，至少部分调节IGF-1和胰岛素对FDCP2细胞存活/增殖和脂肪细胞脂肪分解的影响。在MPDE3B基因的5'侧区域已经鉴定出两个启动子区域，远端启动子区域位于翻译起始位点上游约4kb处，近端启动子区域位于TATA-less区域。转染3T3-L1成纤维细胞和用各种荧光素酶报告质粒载体分化3T3-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的负调控区；(2)CRE顺式元件和CREB蛋白可能在3T3-L1脂肪细胞分化过程中诱导PDE3B发挥重要调控作用。在合作实验中，我们发现TNF α脂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。PDE3B的下调是否参与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.