Bao Wenjie, Strömblad Staffan
Karolinska Institutet, Department of Microbiology, Pathology and Immunology. Huddinge University Hospital F46, SE-141 86 Huddinge. Sweden.
Biol Proced Online. 2002 Nov 11;4:81-87. doi: 10.1251/bpo37.
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 p21(CIP1) and p27(KIP1), 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.
细胞与细胞外基质（ECM）的附着会使整合素信号传导进入细胞，但部分信号反应也源于细胞铺展（3）。为了分析独立于细胞铺展的特定整合素信号传导介导的反应，我们开发了一种方法，即通过使用针对纤连蛋白（FN）受体整合素α5β1的固定化抗整合素单克隆抗体（mab）来激活整合素信号传导。将ECV 304细胞接种到FN、固定化的mab JBS5（抗整合素α5β1）或聚-L-赖氨酸（P-L-L）上，P-L-L介导非整合素依赖性附着。细胞在FN上附着并铺展，而在JBS5或P-L-L上的细胞附着但不铺展。重要的是，接种到FN或mab JBS5上会引发相同的整合素诱导反应，包括细胞周期蛋白依赖性激酶（Cdk2）抑制剂p21（CIP1）和p27（KIP1）的下调，而附着到P-L-L上则不会。我们得出结论，FN受体整合素α5β1的激活会诱导整合素信号传导，从而在不依赖细胞铺展的情况下调节Cdk2抑制剂，并提出了一种如何将整合素信号传导与细胞铺展的影响分开分析的方法。
