REGULATORY MECHANISM BY PHOSPHOLIPASE D IN OXIDANT-STRESS INDUCED SURVIVAL SIGNALING

我们研究了磷脂酶D(PLD)在氧化应激中的意义。PLD激活在过氧化氢(H_2O_2)诱导的信号转导和细胞反应中的作用尚不完全清楚。我们提出的证据表明，在氧化应激下大鼠嗜铬细胞瘤PC12细胞中，钙离子酪氨酸激酶，PYK2需要PLD的激活来调节生存途径。PLD转磷脂酰化的阻断剂1-丁醇和小鼠PLD2K758R(PLD2KR)对H_2O_2诱导的PYK2的磷酸化均有抑制作用。此外，我们还发现PLD2与Pyk2和Src相关，并且需要PLD2的激活才能使H_2O_2增强Src与Pyk2的结合，从而导致Pyk2的完全激活。H_2O_2诱导的Akt和p70S6K的磷酸化依赖于磷脂酰肌醇3-激酶(PI3K)的活性，并可被正丁醇所阻断，但不能被叔丁醇所阻断。此外，…还降低了H_2O_2诱导的PI3K/Akt的激活PLD2KR或显性负性Pyk2DN的转染率较高。本研究首次证明，在H_20_2暴露的PC12细胞中，PLD2的激活参与了Src依赖的Pyk2的磷酸化，从而促进了Pyk2与激活的Src之间的复合体的形成，从而激活了生存信号通路PI3K/Akt/p70S6K。人前列腺癌PC3细胞对喜树碱(CPU)具有耐药性。为了阐明这种耐药的机制，我们研究了鞘氨醇激酶(SPHK)和1-磷酸鞘氨醇(S1P)受体在CPT耐药的PC3和敏感的LNCaP细胞中的参与。与LNCaP细胞相比，PC3细胞表现出更高的活性，同时SPHK1的蛋白和mRNA表达水平也更高，sip受体S1P1和S1P3的表达也更高。CPT诱导PC3细胞SPHK1/S1P信号转导上调是通过SPHK1酶和S1PL1/S1P3受体的共同作用实现的。这些结果有力地表明，SPHK1和S1P受体的高表达和上调对CPT诱导的PC3细胞的凋亡具有保护作用。较少

We examined implication of phospholipase D (PLD) in oxidative stress. The role of PLD activation in hydrogen peroxide (H_20_2)-induced signal transduction and cellular responses are not completely understood. We present evidence that Ca^<2+> tyrosine kinase, Pyk2 requires PLD activation to mediate survival pathways in rat pheochromocytoma PC12 cells under oxidative stress. The H_20_2-induced phosphorylation of Pyk2 was suppressed by 1-butanol, an inhibitor of transphosphatidylation by PLD, and also by transfection of catalytically negative mouse PLD2K758R (PLD2KR). Furthermore, we found that PLD2 was associated with Pyk2 and Src, and that activation of PLD2 was required for H_20_2-enhanced association of Src with Pyk2 leading to full activation of Pyk2. H_20_2-induced phosphorylation of Akt and p70S6K was dependent on phosphatidylinositol 3-kinase (PI3K) activity and was abolished by 1-butanol but not t-butanol. Furthermore, the PI3K/Akt activation in response to H_20_2 was reduced by … More transfection of either PLD2KR or the dominant negative Pyk2DN. This study is the first demonstration that PLD2 activation is implicated in Src-dependent phosphorylation of Pyk2 by promoting the complex formation between Pyk2 and activated Src in PC12 cells exposed to H_20_2, thereby resulting in activation of the survival signaling pathway PI3K/Akt/p70S6K.A human prostate cancer cell line PC3 is resistant to camptothecin (CPU). To elucidate the mechanism of this resistance, we have examined the involvement of sphingosine kinase (SPHK) and sphingosine 1-phosphate (S1P) receptor in CPT-resistant PC3 and -sensitive LNCaP cells. PC3 cells exhibited higher activity accompanied with higher expression levels of protein and mRNA of SPHK1, and also elevated expression of SiP receptors, S1P1 and S1P3, as compared with those of LNCaP cells. The treatment of PC3 cells with CPT was found to induce up-regulation of the SPHK1/S1P signaling by induction of both SPHK1 enzyme and S1PL1/S1P3 receptors. These findings strongly suggest that high expression and up-regulation of SPHK1 and S1P receptors protect PC3 cells from the apoptosis induced by CPT. Less