Mechanotransduction and intracellular signaling mechanisms of stretch-induced remodeling in endothelial cells.
Mechanotransduction and intracellular signaling mechanisms of stretch-induced remodeling in endothelial cells.
复制标题
内皮细胞拉伸诱导重塑的机械转导和细胞内信号传导机制。
作者：
M. Sokabe;K. Naruse;S. Sai;T. Yamada;K. Kawakami;M. Inoue;K. Murase;M. Miyazu
我们研究了牵张诱导的人脐静脉内皮细胞(HUVECs)细胞重塑的信号机制。将新鲜分离的人脐静脉内皮细胞培养在弹性硅膜上，进行单轴循环拉伸(长度为20%，频率为1 Hz)。细胞在牵张开始后15min就开始改变形态，大部分细胞在1h内垂直于牵张轴排列，这种重塑依赖于细胞内钙离子浓度([Ca+]i)通过钙离子通透性拉伸激活(SA)通道的增加。在重塑过程中，观察到应力纤维和局灶性粘连的广泛重排，这可能接近细胞内信号级联的最后一步。这一事件是依赖于[Ca~(2+)]i的，提示存在一个依赖于Ca~(2+)的中间级联反应，将[Ca~(2+)]_i与细胞骨架重排和局部粘连联系起来。我们发现，一些蛋白质，包括pp125FAK(粘着斑激酶)和帕西林，在循环拉伸过程中被酪氨酸磷酸化，这种作用依赖于Ca(2+)。这种酪氨酸磷酸化的抑制阻止了细胞骨架的拉伸依赖重排和局部粘连以及重塑。最后发现，在拉伸过程中，能够磷酸化pp125FAK的酪氨酸激酶src以[Ca~(2+)]i依赖的方式被激活。所有上述分子事件都是钙依赖的，这导致我们提出了信号级联：SA通道激活-->；[Ca2+]i增加-gt；src激活-->；蛋白质酪氨酸磷酸化-->；细胞骨架重排和局部黏附-->；细胞重塑。
We investigated the signaling mechanism of stretch-induced cell remodeling in human umbilical vein endothelial cells (HUVECs). Freshly dissociated HUVECs were cultured on an elastic silicon membrane and subjected to uniaxial cyclic stretch (20% in length, 1 Hz). The cells started to change their morphology as early as 15 min after stretch onset, and most cells eventually aligned perpendicularly to the stretch axis within 1 h. This remodeling was dependent on the increase in intracellular calcium concentration ([Ca2+]i) via a Ca(2+)-permeable stretch-activated (SA) channel. During the process of remodeling, extensive rearrangement of stress fibers and focal adhesions was observed, which may be close to the final step in the intracellular signaling cascade. This event was [Ca2+]i-dependent, suggesting the existence of a Ca(2+)-dependent intermediate cascade that links [Ca2+]i to the rearrangement of cytoskeletons and focal adhesions. We found that some proteins, including pp125FAK (focal adhesion kinase) and paxillin, were tyrosine phosphorylated during cyclic stretch in a Ca(2+)-dependent manner. Inhibition of this tyrosine phosphorylation prohibited the stretch-dependent rearrangement of cytoskeletons and focal adhesions as well as the remodeling. Finally the tyrosine kinase src, which could phosphorylate pp125FAK, was found to be activated in a [Ca2+]i-dependent way during stretch. All of the above molecular events were consistently Ca(2+)-dependent, which led us to propose the signaling cascade: SA channel activation-->[Ca2+]i increase-->src activation-->protein tyrosine phosphorylation-->rearrangement of cytoskeletons and focal adhesions-->cell remodeling.