Regulatory mechanisms and roles of phospholipase D (PLD) in cellular responses

我们发现了人类磷脂酶D1 （hPLD1）的选择性剪接异构体。此外，对大鼠PLD1和PLD2的整个编码区进行了分离和测序。rPLD1的一种更短的选择性剪接形式（rPLD1b）也被鉴定出来。这些基因在大鼠和小鼠的染色体位置通过FISH测定。PLD1和PLD2基因分别定位于大鼠染色体2q23.3-34近端和染色体10q23.3-24近端。在细胞分化或凋亡过程中，大鼠嗜铬细胞瘤PC12细胞、人白血病HL-60细胞和大鼠嗜碱性白血病RBL-2H3细胞中PLD1和PLD2 mrna的表达发生了显著变化。在乙醇或丁醇存在的情况下，PLD通过转磷脂酰化反应以牺牲磷脂酸生成磷脂酰醇，抑制了响应生长信号的转录因子c-jun和c-fos的表达，提示PLD密切参与生长信号转导。在HL-60细胞和PC12细胞的分化中研究PLD的调控机制。磷脂酰肌醇3-激酶（PI-3K）抑制剂、wortmannin和LY294002显示PI-3K位于HL-60分化细胞的受体和PLD之间。此外，一种酪氨酸激酶可能在PI-3K的下游起作用。在PC12细胞中，钙敏感的酪氨酸激酶可能参与了碳甾醇诱导的PLD激活。过氧化氢（H_2O_2）能显著刺激PC12细胞的PLD活性。一种未知的酪氨酸激酶似乎也在h_2o_2刺激的PC12细胞中起激活PLD的作用。我们目前正致力于确定参与这些过程的酪氨酸激酶，并计划使用反义寡核苷酸和转染PLD基因的细胞来进一步研究PLD在细胞反应中的作用。

We have found the alternatively spliced isoform of human phospholipase D1 (hPLD1). Moreover, the entire coding regions of rat PLD1 and PLD2 were isolated and sequenced. A shorter alternatively spliced form of rPLD1 (rPLD1b) was also identified. Chromosomal locations of these genes were determined in the rat and mouse by FISH.The PLD1 and PLD2 genes were localized to rat Chromosome 2q23.3-34 proximal end and Chromosome 10q23.3-24 proximal end, respectively. During cell differentiation or apoptotic processes, the expression of PLD1 and PLD2 mRNAs were drastically changed in rat phochromocytoma PC12 cells, human leukemic HL-60 cells and rat basophilic leukemia RBL-2H3 cells. In the presence of ethanol or butanol which is utilized for transphosphatidylation reaction of PLD to produce phosphatidylalcohol at the expense of phosphatidic acid, the expression of transcription factors, c-jun and c-fos in response to growth signal was inhibited, suggesting that PLD is closely involved in growth signal transduction. The regulatory mechanism of PLD was investigated in differentiated HL-60 cells and PC12 cells. Inhibitors of phosphatidylinositol 3-kinase (PI-3K), wortmannin and LY294002 revealed that PI-3K is located between receptor and PLD in differentiated HL-60 cells. Moreover, a kind of tyrosine kinase may work at the downstream of PI-3K.In PC12 cells, possible involvement of a calcium-sensitive tyrosine kinase in carbachol-induced PLD activation was suggested. Hydrogen peroxide (H_2O_2) greatly stimulated PLD activity in PC12 cells. An unidentified tyrosine kinase is also appeared to function to activate PLD in H_2O_2-stimulated PC12 cells. We are currently working on to identify the tyrosine kinase involved in these processes and also planning to use antisense oligonucleotides and cells transfected with PLD genes to further investigate the roles of PLD in cellular responses.