Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden.
PLoS One. 2013 May 30;8(5):e64897. doi: 10.1371/journal.pone.0064897. Print 2013.
Cells are exposed to several types of integrin stimuli, which generate responses generally referred to as "integrin signals", but the specific responses to different integrin stimuli are poorly defined. In this study, signals induced by integrin ligation during cell attachment, mechanical force from intracellular contraction, or cell stretching by external force were compared. The elevated phosphorylation levels of several proteins during the early phase of cell attachment and spreading of fibroblast cell lines were not affected by inhibition of ROCK and myosin II activity, i.e. the reactions occurred independently of intracellular contractile force acting on the adhesion sites. The contraction-independent phosphorylation sites included ERK1/2 T202/Y204, AKT S473, p130CAS Y410, and cofilin S3. In contrast to cell attachment, cyclic stretching of the adherent cells induced a robust phosphorylation only of ERK1/2 and the phosphorylation levels of the other investigated proteins were not or only moderately affected by stretching. No major differences between signaling via α5β1 or αvβ3 integrins were detected. The importance of mitochondrial ROS for the integrin-induced signaling pathways was investigated using rotenone, a specific inhibitor of complex I in the respiratory chain. While rotenone only moderately reduced ATP levels and hardly affected the signals induced by cyclic cell stretching, it abolished the activation of AKT and reduced the actin polymerization rate in response to attachment in both cell lines. In contrast, scavenging of extracellular ROS with catalase or the vitamin C analog Asc-2P did not significantly influence the attachment-derived signaling, but caused a selective and pronounced enhancement of ERK1/2 phosphorylation in response to stretching. In conclusion, the results showed that "integrin signals" are composed of separate sets of reactions triggered by different types of integrin stimulation. Mitochondrial ROS and extracellular ROS had specific and distinct effects on the integrin signals induced by cell attachment and mechanical stretching.
细胞暴露于几种类型的整合素刺激，这些刺激产生通常被称为“整合素信号”的反应，但对不同整合素刺激的具体反应尚不清楚。在这项研究中，比较了细胞附着过程中整合素交联、细胞内收缩产生的机械力或外力拉伸细胞时诱导的信号。在成纤维细胞系附着和铺展的早期阶段，几种蛋白质的磷酸化水平升高，但 ROCK 和肌球蛋白 II 活性的抑制并不影响这些反应，即这些反应独立于作用于黏附部位的细胞内收缩力。与细胞附着不同，黏附细胞的周期性拉伸仅诱导 ERK1/2 的强烈磷酸化，而其他研究的蛋白质的磷酸化水平不受或仅适度受拉伸影响。未检测到通过α5β1 或αvβ3 整合素信号传递的明显差异。使用鱼藤酮（呼吸链中复合物 I 的特异性抑制剂）研究了线粒体 ROS 对整合素诱导的信号通路的重要性。虽然鱼藤酮仅适度降低 ATP 水平，几乎不影响周期性细胞拉伸诱导的信号，但它可使 AKT 的激活和两种细胞系中对附着的肌动蛋白聚合速率的反应失活。相比之下，用过氧化氢酶或维生素 C 类似物 Asc-2P 清除细胞外 ROS 不会显著影响附着衍生的信号，但会选择性和显著增强对拉伸的 ERK1/2 磷酸化。总之，结果表明，“整合素信号”由不同类型的整合素刺激触发的独立反应组成。线粒体 ROS 和细胞外 ROS 对细胞附着和机械拉伸诱导的整合素信号有特定和不同的影响。
