Ca2+ influx induced by protease-activated receptor-1 activates a feed-forward mechanism of TRPC1 expression via nuclear factor-κB activation in endothelial cells
Ca2+ influx induced by protease-activated receptor-1 activates a feed-forward mechanism of TRPC1 expression via nuclear factor-κB activation in endothelial cells
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DOI：
10.1074/jbc.m600722200
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发表时间：
2006-07-28
影响因子：
4.8
通讯作者：
Tiruppathi, Chinnaswamy
中科院分区：
文献类型：
--
作者：
Paria, Biman C.;Bair, Angela M.;Tiruppathi, Chinnaswamy
凝血酶激活蛋白酶激活受体-1诱导内皮细胞钙离子通过钙池操纵的阳离子通道TRPC 1内流我们研究了Ca 2+内流诱导的Ca 2+商店的耗竭信号TRPC 1在内皮细胞中的表达的作用。凝血酶和蛋白酶激活的受体-1-特异性激动剂肽诱导TRPC 1在人脐静脉内皮细胞中的表达，这是耦合到增强存储操作的Ca 2+内流和内皮通透性增加。为了阐明凝血酶诱导TRPC 1表达的机制，我们将含有多个核因子-κ B（NF-κ B）结合位点的TRPC 1-promoter-luciferase（TRPC 1-Pro-Luc）构建体转染内皮细胞。显性负性I κ B α突变体的共表达阻止了凝血酶诱导的TRPC 1表达的增加，表明NF-κ B活化在介导应答中的关键作用。使用TRPC 1启动子缺失突变体构建体，我们发现位于TRPC 1 5 '调控区-1623和-871之间的NF-κ B结合位点是凝血酶诱导TRPC 1表达所必需的。利用TRPC 1启动子特异性寡核苷酸的电泳迁移率变动分析鉴定了NF-κ B与NF-κ B共有位点的DNA结合活性位于该结构域。使用NF-κ B蛋白特异性抗体的超移位测定证明了p65同二聚体与TRPC 1启动子的结合。抑制钙库耗竭、缓冲细胞内钙或下调钙内流下游的蛋白激酶C α均阻断凝血酶诱导的NF-κ B活化和由此产生的内皮细胞TRPC 1表达。因此，通过TRPC 1的Ca 2+内流是负责TRPC 1表达的关键前馈途径。NF-κ B调节的TRPC 1表达可能是血管炎症的重要机制，因此是一种新的治疗靶点。
Thrombin activation of protease-activated receptor-1 induces Ca2+ influx through store-operated cation channel TRPC1 in endothelial cells. We examined the role of Ca2+ influx induced by the depletion of Ca2+ stores in signaling TRPC1 expression in endothelial cells. Both thrombin and a protease-activated receptor-1-specific agonist peptide induced TRPC1 expression in human umbilical vein endothelial cells, which was coupled to an augmented store-operated Ca2+ influx and increase in endothelial permeability. To delineate the mechanisms of thrombin-induced TRPC1 expression, we transfected in endothelial cells TRPC1-promoter-luciferase ( TRPC1-Pro-Luc) construct containing multiple nuclear factor-kappa B ( NF-kappa B) binding sites. Co-expression of dominant negative I kappa B alpha mutant prevented the thrombin-induced increase in TRPC1 expression, indicating the key role of NF-kappa B activation in mediating the response. Using TRPC1 promoter-deletion mutant constructs, we showed that NF-kappa B binding sites located between - 1623 and - 871 in the TRPC1 5'-regulatory region were required for thrombin-induced TRPC1 expression. Electrophoretic mobility shift assay utilizing TRPC1 promoter-specific oligonucleotides identified that the DNA binding activities of NF-kappa B to NF-kappa B consensus sites were located in this domain. Supershift assays using NF-kappa B protein-specific antibodies demonstrated the binding of p65 homodimer to the TRPC1 promoter. Inhibition of store Ca2+ depletion, buffering of intracellular Ca2+, or down-regulation of protein kinase C alpha downstream of Ca2+ influx all blocked thrombin-induced NF-kappa B activation and the resultant TRPC1 expression in endothelial cells. Thus, Ca2+ influx via TRPC1 is a critical feed-forward pathway responsible for TRPC1 expression. The NF-kappa B-regulated TRPC1 expression may be an essential mechanism of vascular inflammation and, hence, a novel therapeutic target.