EXPRESSION/REGULATION OF PHOSPHODIESTERASE 3 ISOFORMS

磷酸二酯酶 3 异构体的表达/调节

• 批准号: 6432692
• 负责人: VINCENT MANGANIELLO
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6432692
• 关键词: 3'5' cyclic nucleotide phosphodiesterase; B lymphocyte; T lymphocyte; cell differentiation; cell type; enzyme activity; esterase inhibitor; gene targeting; in situ hybridization; inflammation; isozymes; laboratory mouse; laboratory rat; leukocyte activation /transformation; macrophage; natural killer cells; northern blottings; protein isoforms; transfection

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.

通过催化cAMP和CGMP的水解，环核苷酸磷酸二酯酶（PDES）是由环状核苷酸介导的细胞内浓度和生物学反应的关键调节剂，环环核苷酸，包括免疫/炎症反应，包括属于Pde soforms的细胞调节，属于PDE semoforms [属于PDE semofems [ （PDE1-11）]对靶向特定PDE在治疗肺部疾病中的重要性将增加。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PDES参与细胞因子和生长因子调节的信号传导途径鲜为人知。在鼠FDCP2 promyeloid细胞，IL-4和IGF-1激活PDE3和PDE4 ，而IL-3仅激活PDE4。 对JAK，PI3-K，PKC和MAPK激酶的TNFALPHA和抑制剂的研究表明，IGF-1和IL-3都通过PI3-K依赖性信号激活PDE3和PDE4。 PI3-K的下游，调节途径不同； PDE4而不是PDE3是由MEK/MAPK依赖性信号激活的。 因此，FDCP2细胞被野生型（WT）永久转染，组成型活性（CA）或激酶无活性（Ki）形式的MEK和PKB形式。 对这些转染细胞的研究表明，PDE4被MEK/MAPK依赖性信号激活，并且PDE3被磷酸化并被PKB依赖性信号激活。重组小鼠（M）PDE3B被PKB磷酸化并在体外激活；缺乏共识PKB磷酸化位点的截短的MPDE3B突变体未被磷酸化或激活。在S272（PKB共有磷酸化位点）和Ser296和421（PKA位点）中，将丝氨酸 - 丙氨酸突变引入了MPDE3B中。完整的FDCP2细胞和SF21细胞裂解物中的体现表明，尽管Ser272参与了MPDE3B的活性。 SER296由PKA，Ser421似乎在调节PKA和PKB.IN表达WT PKB的FDCP2细胞对MPDE3B激活中很重要，促凋亡蛋白不良因响应于IGF-1而磷酸化。磷酸化被8-BR-cAMP或PDE3抑制剂纤毛胺阻塞。 这些和其他数据表明，PDE3B是PKB的下游靶标，即使不是底物，并且可能是PKB的效应子，在调节营地池中，至少部分调节了IGF-1和胰岛素对生存/增殖的影响FDCP2细胞和脂肪细胞中的脂解。已经鉴定出MPDE3B基因5'Fanking区域的两个启动子区域，这是一个位于翻译起始位点上游的远端启动子区域，无tata-nose tata-note tata-note。3T3-L1的转染3T3-L1成纤维细胞和区分3T3-L1具有各种荧光素酶报道质粒载体的脂肪细胞表明：（1）远端和近端启动子区域之间存在强大的负调节区域；（2）CRE CIS元素和CREB蛋白可能在PDE3B诱导PDE3B中起关键的调节作用发生在3T3-L1脂肪细胞的分化过程中。在协作实验中，我们发现TNFαN脂解的刺激作用可能部分与3T3-L1脂肪细胞中PDE3B表达的下调有关。 TNF alphaon尚不清楚胰岛素抵抗的发展。