Phosphatidic Acid Is a Leukocyte Chemoattractant That Acts through S6 Kinase Signaling
Phosphatidic Acid Is a Leukocyte Chemoattractant That Acts through S6 Kinase Signaling
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DOI：
10.1074/jbc.m109.070524
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发表时间：
2010-05-21
影响因子：
4.8
通讯作者：
Gomez-Cambronero, Julian
中科院分区：
文献类型：
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作者：
Frondorf, Kathleen;Henkels, Karen M.;Gomez-Cambronero, Julian
磷脂酸（PA）是哺乳动物细胞中的一种多效性脂质第二信使。我们在这里报告，细胞外PA作为一个白细胞的化学引诱物，作为膜可溶性二油酰-PA（DOPA）elidoctin聚合和人类中性粒细胞和分化的前白血病HL-60细胞的趋化性。我们表明，这一机制涉及S6激酶（S6 K）信号酶。趋化性被S6 K抑制剂雷帕霉素和双吲哚马来酰亚胺以及使用双链RNA的S6 K1沉默抑制>90%。然而，它仅被mTOR siRNA中度抑制（类似于30%），表明S6 K存在mTOR非依赖性机制。外源性PA导致S6 K酶活性和Thr（421）/Ser（424）磷酸化的强烈时间和剂量依赖性增加，进一步支持PA/S6 K连接。我们还研究了细胞内PA的产生是否影响细胞迁移。磷脂酶D2（PLD 2）的过表达，并在较小程度上，PLD 1，导致升高的S6 K活性和化学运动，而PLD沉默是抑制性的。由于脂肪酶失活的PLD 2突变体K444 R和K758 R既不激活S6 K也不诱导趋化性，因此这种形式的细胞迁移需要细胞内PA。最后，我们证明了细胞外和细胞内PA之间的连接。使用增强的绿色荧光蛋白衍生的PA传感器（pEGFP-Spo 20 PABD），我们表明，外源PA或PA原位产生的细菌（链霉菌chromofuscus）PLD进入细胞，并积累在囊泡样胞质结构。总之，我们报告的发现PA作为一种白细胞趋化因子通过细胞进入和激活S6 K介导的细胞骨架肌动蛋白聚合和白细胞趋化性所需的这些细胞的免疫功能。
Phosphatidic acid (PA) is a pleiotropic lipid second messenger in mammalian cells. We report here that extracellular PA acts as a leukocyte chemoattractant, as membrane-soluble di-oleoyl-PA (DOPA) elicits actin polymerization and chemotaxis of human neutrophils and differentiated proleukemic HL-60 cells. We show that the mechanism for this involves the S6 kinase (S6K) signaling enzyme. Chemotaxis was inhibited >90% by the S6K inhibitors rapamycin and bisindolylmaleimide and by S6K1 silencing using double-stranded RNA. However, it was only moderately (similar to 30%) inhibited by mTOR siRNA, indicating the presence of an mTOR-independent mechanism for S6K. Exogenous PA led to robust time-and dose-dependent increases in S6K enzymatic activity and Thr(421)/Ser(424) phosphorylation, further supporting a PA/S6K connection. We also investigated whether intracellular PA production affects cell migration. Overexpression of phospholipase D2 (PLD2) and, to a lesser extent, PLD1, resulted in elevation of both S6K activity and chemokinesis, whereas PLD silencing was inhibitory. Because the lipase-inactive PLD2 mutants K444R and K758R neither activated S6K nor induced chemotaxis, intracellular PA is needed for this form of cell migration. Lastly, we demonstrated a connection between extracellular and intracellular PA. Using an enhanced green fluorescent protein-derived PA sensor (pEGFP-Spo20PABD), we showed that exogenous PA or PA generated in situ by bacterial (Streptomyces chromofuscus) PLD enters the cell and accumulates in vesicle-like cytoplasmic structures. In summary, we report the discovery of PA as a leukocyte chemoattractant via cell entry and activation of S6K to mediate the cytoskeletal actin polymerization and leukocyte chemotaxis required for the immune function of these cells.