Activity of recombinant trypsin isoforms on human proteinase-activated receptors (PAR):: mesotrypsin cannot activate epithelial PAR-1,-2, but weakly activates brain PAR-1
Activity of recombinant trypsin isoforms on human proteinase-activated receptors (PAR):: mesotrypsin cannot activate epithelial PAR-1,-2, but weakly activates brain PAR-1
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DOI：
10.1038/sj.bjp.0706410
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发表时间：
2005-12-01
影响因子：
7.3
通讯作者：
Reiser, G
中科院分区：
文献类型：
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作者：
Grishina, Z;Ostrowska, E;Reiser, G
胰蛋白酶样丝氨酸蛋白酶通过蛋白酶激活受体（PARs）的蛋白水解切割触发许多组织中的信号转导途径。PAR-1、PAR-2和PAR-4是胰蛋白酶底物，因为胞外N末端的胰蛋白酶裂解产生受体活化。在这里，三种人胰蛋白酶异构体的能力在PAR上测试了作为重组蛋白的胰蛋白酶（阳离子胰蛋白酶、阴离子胰蛋白酶和中胰蛋白酶（胰蛋白酶IV））。（人支气管上皮）、A549（人肺上皮）和HEK（人胚肾）-293细胞，其表达功能性PAR-1和PAR-2。即使在高浓度下，人中胰蛋白酶也未能诱导人上皮细胞中PAR介导的Ca 2+反应。此外，中胰蛋白酶没有影响PAR激活的幅度，随后加入牛胰蛋白酶。在HBE细胞中，其像A549细胞一样表达高PAR-2水平，PAR-I水平可忽略不计（< 11%），在约：5 mM处观察到阳离子和阴离子胰蛋白酶的半数最大反应。在上皮细胞中，中胰蛋白酶不激活PAR-2或PAR-1，而阴离子和阳离子胰蛋白酶都是相当的激活剂。3我们还研究了表达PAR-1和一些PAR-3，但不表达PAR-2的人星形细胞瘤1321 N1细胞。高浓度（> 100 nm）的中胰蛋白酶产生相对弱的Ca 2+信号，显然是通过PAR-1活化。在60 nm中胰蛋白酶和10-20 nm阳离子和阴离子胰蛋白酶处观察到半最大响应。4使用PAR-2-AP的脱敏测定，我们证实阳离子和阴离子胰蛋白酶亚型主要通过PAR-2活化引起HBE细胞中[Ca 2 +]（i）升高。在1321 N1细胞中，用凝血酶受体激动剂肽对PAR-1进行脱敏，证明所有三种重组胰蛋白酶亚型都通过PAR-1起作用。因此，人阳离子和阴离子胰蛋白酶对PAR的活性与牛胰蛋白酶相当。与阳离子和阴离子胰蛋白酶相反，中胰蛋白酶（胰蛋白酶IV）不能激活或禁用人上皮细胞中的PAR，表明受体不是这种同工酶的底物。另一方面，中胰蛋白酶激活人星形细胞瘤细胞中的PAR-1。这可能在人类大脑的保护/变性或可塑性过程中发挥作用。
1 Trypsin-like serine proteinases trigger signal transduction pathways through proteolytic cleavage of proteinase-activated receptors (PARs) in many tissues. Three members, PAR-1, PAR-2 and PAR-4, are trypsin substrates, as trypsinolytic cleavage of the extracellular N terminus produces receptor activation. Here, the ability of the three human pancreatic trypsin isoforms (cationic trypsin, anionic trypsin and mesotrypsin (trypsin IV)) as recombinant proteins was tested on PARs.2 Using fura 2 [Ca2+], measurements, we analyzed three human epithelial cell lines, HBE (human bronchial epithelial), A549 (human pulmonary epithelial) and HEK (human embryonic kidney)-293 cells, which express functional PAR-1 and PAR-2. Human mesotrypsin failed to induce a PAR-mediated Ca2+ response in human epithelial cells even at high concentrations. In addition, mesotrypsin did not affect the magnitude of PAR activation by subsequently added bovine trypsin. In HBE cells, which like A549 cells express high PAR-2 levels with negligible PAR-I levels (< 11%), halfmaximal responses were seen for both cationic and anionic trypsins at about :5 rim. In the epithelial cells, mesotrypsin did not activate PAR-2 or PAR-1, whereas both anionic and cationic trypsins were comparable activators.3 We also investigated human astrocytoma 1321N1 cells, which express PAR-1 and some PAR-3, but no PAR-2. High concentrations (> 100 nm) of mesotrypsin produced a relatively weak Ca2+ signal, apparently through PAR-1 activation. Half-maximal responses were observed at 60 nm mesotrypsin, and at 10-20 nm cationic and anionic trypsins.4 Using a desensitization assay with PAR-2-AP, we confirmed that both cationic and anionic trypsin isoforms cause [Ca2+](i) elevation in HBE cells mainly through PAR-2 activation. Desensitization of PAR-1 with thrombin receptor agonist peptide in 1321N1 cells demonstrated that all three recombinant trypsin isoforms act through PAR-1.5 Thus, the activity of human cationic and anionic trypsins on PARs was comparable to that of bovine pancreatic trypsin. Mesotrypsin (trypsin IV), in contrast to cationic and anionic trypsin, cannot activate or disable PARs in human epithelial cells, demonstrating that the receptors are no substrates for this isoenzyme. On the other hand, mesotrypsin activates PAR-1 in human astrocytoma cells. This might play a role in protection/degeneration or plasticity processes in the human brain.