Use of an immobilized monoclonal antibody to examine integrin alpha5beta1 signaling independent of cell spreading.
Use of an immobilized monoclonal antibody to examine integrin alpha5beta1 signaling independent of cell spreading.
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DOI：
10.1251/bpo37
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发表时间：
2002-11-11
影响因子：
6.4
通讯作者：
Stromblad, Staffan
中科院分区：
文献类型：
--
作者：
Bao, Wenjie;Stromblad, Staffan
细胞附着到细胞外基质(ECM)使整合素信号进入细胞，但部分信号反应也源于细胞扩散(3)。为了分析与细胞扩散无关的整合素信号介导的特异性反应，我们开发了一种通过使用针对纤维连接蛋白(FN)受体整合素α5beta1的固定化抗整合素单抗(MAb)来进行整合素信号转导的方法。将ECV304细胞接种于FN或固定化单抗JBS5(抗整合素α5β1)或介导非整合素依赖性黏附的多聚L赖氨酸(P-L-L)上。细胞在FN上贴壁和铺展，而在JBS5和P-L-L上贴壁但不铺展。重要的是，在FN或单抗JBS5上电镀产生了相同的整合素诱导的反应，包括下调细胞周期蛋白依赖性蛋白激酶(CDK2)抑制剂p21CIP1和p27KIP1的表达，而附着在P-L-L上则没有。我们得出结论，FN受体整合素α5beta1的参与诱导了整合素信号调节CDK2抑制物，而不依赖于细胞扩散，并提出了一种如何独立于细胞扩散的影响来分析整合素信号的方法。
Cell attachment to the extracellular matrix (ECM) engages integrin signaling into the cell, but part of the signaling response also stem from cell spreading (3). To analyze specific integrin signaling-mediated responses independent of cell spreading, we developed a method engaging integrin signaling by use of an immobilized anti-integrin monoclonal antibody (mab) directed against the fibronectin (FN) receptor integrin alpha5beta1. ECV 304 cells were plated onto FN or immobilized mab JBS5 (anti-integrin alpha5beta1) or onto poly-L-lysin (P-L-L), which mediates integrin-independent attachment. Cells attached and spread on FN, while cells on JBS5 or P-L-L attached but did not spread. Importantly, plating onto FN or mab JBS5 gave rise to identical integrin-induced responses, including a down-regulation of the cyclin-dependent kinase (Cdk2) inhibitors p21CIP1 and p27KIP1, while attachment to P-L-L did not. We conclude that engagement of the FN-receptor integrin alpha5beta1 induces integrin signaling regulating the Cdk2-inhibitors independent of cell spreading and present a method for how integrin signaling can be analyzed separate from the effects of cell spreading.