Signaling and cross-talk by C5a and UDP in macrophages selectively use PLCβ3 to regulate intracellular free calcium
Signaling and cross-talk by C5a and UDP in macrophages selectively use PLCβ3 to regulate intracellular free calcium
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DOI：
10.1074/jbc.m800907200
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发表时间：
2008-06-20
影响因子：
4.8
通讯作者：
Seaman, William E.
中科院分区：
文献类型：
--
作者：
Roach, Tamara I. A.;Rebres, Robert A.;Seaman, William E.
对成纤维细胞、神经元和血小板的研究表明，来自不同 G 蛋白偶联受体 (GPCR) 的信号在细胞内游离 Ca2+ 升高中的整合。为了研究巨噬细胞中的信号整合，我们筛选了 RAW264.7 细胞和骨髓源性巨噬细胞 (BMDM)，了解它们对 GPCR 配体的 Ca2+ 反应。我们发现补体成分 5a (C5a) 与尿苷 5'-二磷酸 (UDP)、血小板激活因子 (PAF) 或溶血磷脂酸 (LPA) 组合可产生协同反应。 C5a 反应是 G α(i) 依赖性的，而 UDP、PAF 和 LPA 反应是 G α(q) 依赖性的。 C5a 和 UDP 之间的协同作用由 C5a 和 P2Y6 受体介导，需要双受体占据，并影响细胞内储存的 Ca2+ 的初始释放以及持续的 Ca2+ 水平。 C5a 和 UDP 协同生成肌醇 1,4,5-三磷酸，表明在激活磷脂酶 C (PLC) beta 方面存在协同作用。巨噬细胞表达三种 PLC beta 异构体（PLC beta 2、PLC beta 3 和 PLC beta 4）的转录物，但 GPCR 配体在 Ca2+ 信号传导中选择性地使用这些异构体。 C5a 主要使用 PLC beta 3，而 UDP 使用 PLC beta 3，但也使用 PLC beta 4。两种配体都不需要 PLC beta 2。C5a 和 UDP 之间的协同作用同样主要依赖于 PLC beta 3。重要的是，在 PLC beta 3 缺陷的 BMDM 中观察到的 Ca2+ 信号传导缺陷通过用 PLC beta 3 重构得到逆转。协同作用不需要磷脂酰肌醇 (PI) 3-激酶和蛋白激酶 C。与 Ca2+ 相比，C5a 激活的 PI 3 激酶被 UDP 抑制，巨胞饮作用也受到抑制，而巨胞饮作用依赖于 PI 3 激酶。因此，PLC beta 3 可能为抑制 Ca2+ 对炎症介质（包括 C5a、UDP、PAF 和 LPA）的反应提供选择性靶标。
Studies in fibroblasts, neurons, and platelets have demonstrated the integration of signals from different G protein-coupled receptors (GPCRs) in raising intracellular free Ca2+. To study signal integration in macrophages, we screened RAW264.7 cells and bone marrow-derived macrophages (BMDM) for their Ca2+ response to GPCR ligands. We found a synergistic response to complement component 5a (C5a) in combination with uridine 5'-diphosphate (UDP), platelet activating factor (PAF), or lysophosphatidic acid (LPA). The C5a response was G alpha(i)-dependent, whereas the UDP, PAF, and LPA responses were G alpha(q)-dependent. Synergy between C5a and UDP, mediated by the C5a and P2Y6 receptors, required dual receptor occupancy, and affected the initial release of Ca2+ from intracellular stores as well as sustained Ca2+ levels. C5a and UDP synergized in generating inositol 1,4,5-trisphosphate, suggesting synergy in activating phospholipase C (PLC) beta. Macrophages expressed transcripts for three PLC beta isoforms (PLC beta 2, PLC beta 3, and PLC beta 4), but GPCR ligands selectively used these isoforms in Ca2+ signaling. C5a predominantly used PLC beta 3, whereas UDP used PLC beta 3 but also PLC beta 4. Neither ligand required PLC beta 2. Synergy between C5a and UDP likewise depended primarily on PLC beta 3. Importantly, the Ca2+ signaling deficiency observed in PLC beta 3-deficient BMDM was reversed by re-constitution with PLC beta 3. Neither phosphatidylinositol (PI) 3-kinase nor protein kinase C was required for synergy. In contrast to Ca2+, PI 3-kinase activation by C5a was inhibited by UDP, as was macropinocytosis, which depends on PI 3-kinase. PLC beta 3 may thus provide a selective target for inhibiting Ca2+ responses to mediators of inflammation, including C5a, UDP, PAF, and LPA.