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Elucidation of the mechanism of vascular cell sensing and gene responses to mechanical stresses

阐明血管细胞感知和基因对机械应力的反应机制

基本信息

批准号：
11308035
负责人：
ANDO Joji
金额：
$ 17.86万
依托单位：
University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A).
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11308035/
关键词：
endothelial cells intracellular signal transduction calcium signaling purinoceptor mechano-sensitive channel calcium ion channel adenosine triphosphate fluid shear stress 血行力学因子遺伝子転写因子チロシンキナーゼ FAK

项目摘要

We investigated signal transduction leading to shear stress responsive element-mediated transcription. Bovine endothelial cells were transfected with luciferase reporter vectors containing tandem repeats of TRE, CRE or NFkB element, and exposed to a shear stress of 15 dynes/cm^2. Transcriptional activity via each element began to increase significantly at 3 h after the onset of shear stress and increased further with time, peaking at 12h. These responses to shear stress were significantly suppressed by the treatment of cells with a tyrosine kinase inhibitor (Herbimycin A, 1 mM). Shear stress induced tyrosine phosphorylation of proteins of around 30, 45-65, and 120 kDa including focal adhesion kinase (FAK) in endothelial cells. Overexpression of FRNK (FAK-related non-kinase), which acts as an inhibitor of FAK, markedly inhibited the shear stress-induced increase in transcription via TRE, CRE or NFkB element. These results suggest that tyrosine kinase and FAK signaling are critical for s … More hear stress induction of TRE-, CRE- and NFkB element-mediated transcription in endothelial cells.We also studied molecular mechanism of Ca^<2+> signaling, including the ion channels responsible for the shear stress-induced Ca^<2+> responses in endothelial cells. Human umbilical vein ECs (HUVECs) loaded with the Ca^<2+> indicator lndo-1/AM were exposed to laminar flow of Hanks' balanced salt solution (HBSS) at various concentrations of ATP and changes in intraceliular Ca^<2+> concentrations ([Ca^<2+>]i) were monitored by confocal laser scanning microscopy. A stepwise increase in flow rate elicited a corresponding stepwise-increase in [Ca^<2+>]i at 250 nmol/L ATP.The flow rate-dependent increase in [Ca^<2+>]i disappeared after the chelation of extracellular Ca^<2+> with EGTA.Antisense oligonucleotides designed to knockout P2X4 purinoceptor expression abolished the flow-induced Ca^<2+> influx. Human embryonic kidney 293 cells showed no Ca^<2+> response to flow at 2μmol/L ATP, but when transfected with P2X4 cDNA they began to show flow-rate-dependent Ca^<2+> influx. P2X4 purinoceptors may have a "shear-transducer" property by which shear stress is perceived directly or indirectly and transmitted into the cell interior via Ca^<2+> signaling. Less

我们研究了导致剪切应力响应元件介导转录的信号转导。用含有TRE、CRE或NFkB元素串联重复序列的荧光素酶报告载体转染牛内皮细胞，并暴露于15 dynes/cm^2的剪切应力下。各元件的转录活性在剪切胁迫开始后3 h开始显著增加，并随着时间的推移进一步增加，在12h达到峰值。这些对剪切应力的反应被酪氨酸激酶抑制剂（Herbimycin a, 1 mM）处理的细胞显著抑制。剪切应力诱导内皮细胞约30,45 -65和120kda蛋白酪氨酸磷酸化，包括局灶黏附激酶（FAK）。作为FAK抑制剂的FRNK （FAK相关非激酶）的过表达可显著抑制剪切应力诱导的通过TRE、CRE或NFkB元件的转录增加。这些结果表明，酪氨酸激酶和FAK信号在应激诱导内皮细胞中TRE-、CRE-和NFkB元件介导的转录中起着至关重要的作用。我们还研究了Ca^<2+>信号的分子机制，包括内皮细胞中剪应力诱导Ca^<2+>反应的离子通道。将负载Ca^<2+>指示剂indo -1/AM的人脐静脉内皮细胞（HUVECs）暴露于不同ATP浓度的Hanks平衡盐溶液（HBSS）的层流中，通过共聚焦激光扫描显微镜监测细胞内Ca^<2+>浓度（[Ca^<2+>]i）的变化。当ATP浓度为250 nmol/L时，流速的逐步增加引起[Ca^<2+>]i相应的逐步增加。细胞外Ca^<2+>与EGTA螯合后，[Ca^<2+>]i的流速依赖性增加消失。设计用于敲除P2X4嘌呤受体表达的反义寡核苷酸可消除血流诱导的Ca^<2+>内流。人胚胎肾293细胞对2μmol/L ATP流没有Ca^<2+>反应，但转染P2X4 cDNA后，细胞开始出现流速依赖的Ca^<2+>内流。P2X4嘌呤受体可能具有“剪切换能器”特性，通过这种特性可以直接或间接地感知剪切应力，并通过Ca^<2+>信号传导到细胞内部。少