Mechanisms of caspase-1 activation by P2X7 receptor-mediated K+ release
Mechanisms of caspase-1 activation by P2X7 receptor-mediated K+ release
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DOI：
10.1152/ajpcell.00494.2003
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发表时间：
2004-05-01
影响因子：
5.5
通讯作者：
Dubyak, GR
中科院分区：
文献类型：
--
作者：
Kahlenberg, JM;Dubyak, GR
在单核细胞和巨噬细胞的炎症激活过程中，caspase-1激活和IL-1 β加工的潜在机制尚未明确。在这里，我们描述了一种体外蛋白水解加工试验，允许比较半胱天冬酶-1调节成分在一个无细胞的系统，分别从IL-1 β分泌的混淆问题。对未受刺激的Bac 1小鼠巨噬细胞裂解物中IL-1 β和caspase-1的体外加工分析表明，IL-1 β的基础caspase-1活化和蛋白水解成熟速率缓慢。相反，在细胞溶解前用细胞外ATP（作为P2 X（7）受体的激活剂）或尼日利亚菌素短暂（5分钟）处理完整的巨噬细胞，明显加速了caspase-1和IL-1 β的体外加工。这种体外加工的加速严格依赖于完整细胞的细胞内K+的损失。通过裂解本身或裂解前K+损失诱导的体外caspase-1活化对用tyrphostin AG-126或溴烯醇内酯（一种Ca 2+非依赖性磷脂酶A的抑制剂）预处理完整巨噬细胞敏感（2）。加入重组ASC（一种先前鉴定的与caspase-1直接相关的衔接蛋白）也可加速未受刺激的巨噬细胞裂解液中Caspase-1的活化和IL-1 β的加工。这些数据表明，通过P2 X（7）核苷酸受体刺激增加的K+流出激活了小鼠巨噬细胞中的AG-126和溴烯醇内酯敏感性信号传导途径，从而在无细胞测定中稳定维持半胱天冬酶-1调节信号。
The mechanisms underlying caspase-1 activation and IL-1beta processing during inflammatory activation of monocytes and macrophages are not well defined. Here, we describe an in vitro proteolytic processing assay that allows for comparison of caspase-1 regulatory components in a cell-free system separately from the confounding issue of IL-1beta secretion. Analysis of in vitro IL-1beta and caspase-1 processing in lysates from unstimulated Bac1 murine macrophages indicated a slow rate of basal caspase-1 activation and proteolytic maturation of IL-1beta. In contrast, brief (5 min) treatment of intact macrophages with extracellular ATP (as an activator of the P2X(7) receptor) or nigericin before cell lysis markedly accelerated the in vitro processing of caspase-1 and IL-1beta. This acceleration of in vitro processing was strictly dependent on loss of intracellular K+ from the intact cells. The induction of in vitro caspase-1 activation by lysis per se or by K+ loss before lysis was sensitive to pretreatment of intact macrophages with the tyrphostin AG-126 or bromoenol lactone, an inhibitor of Ca2+-independent phospholipase A(2). Caspase-1 activation and IL-1beta processing in lysates from unstimulated macrophages were also accelerated by addition of recombinant ASC, a previously identified adapter protein that directly associates with caspase-1. These data indicate that increased K+ efflux via P2X(7) nucleotide receptor stimulation activates AG-126- and bromoenol lactone-sensitive signaling pathways in murine macrophages that result in stably maintained signals for caspase-1 regulation in cell-free assays.