Murphy-Ullrich J E, Lane T F, Pallero M A, Sage E H
Department of Pathology, University of Alabama at Birmingham 35294, USA.
J Cell Biochem. 1995 Feb;57(2):341-50. doi: 10.1002/jcb.240570218.
SPARC is a one of a group of extracellular matrix proteins that regulate cell adhesion through a loss of focal adhesion plaques from spread cells. We previously reported that SPARC reduced the number of bovine aortic endothelial (BAE) cells positive for focal adhesions [Murphy-Ullrich et al. (1991): J Cell Biol 115:1127-1136]. We have now characterized the effect of SPARC on the cytoskeleton of BAE cells. Addition of SPARC to spread BAE cells caused a dose-dependent loss of focal adhesion-positive cells, that was maximal at approximately 1 microgram/ml (0.03 microM). Consistent with the loss of adhesion plaques as detected by interference reflection microscopy, vinculin appeared diffuse and F-actin was redistributed to the periphery of cells incubated with SPARC. However, the distribution of the integrin alpha v beta 3 remained clustered in a plaque-like distribution. These data, and the observation that SPARC binds to BAE cells but not to the extracellular matrix, indicate that SPARC acts via interactions with cell surface molecules and not by steric/physical disruption of integrin-extracellular matrix ligands. To determine the region(s) of SPARC that mediate a loss of focal adhesions, we tested peptides from the four distinct regions of SPARC. The cationic, cysteine-rich peptide 2.1 (amino acids 54-73) and the Ca(2+)-binding EF-hand-containing peptide 4.2 (amino acids 254-273) were active in focal adhesion disassembly. Furthermore, antibodies specific for these regions neutralized the focal adhesion-labilizing activity of SPARC. These results are consistent with previous data showing that peptide 2.1 and 4.2 interact with BAE cell surface proteins and indicate that the loss of focal adhesions from endothelial cells exposed to SPARC is a receptor-mediated event.
SPARC是一组细胞外基质蛋白之一，它通过使铺展细胞的粘着斑丧失来调节细胞粘附。我们之前报道过，SPARC减少了粘着斑阳性的牛主动脉内皮（BAE）细胞的数量[Murphy-Ullrich等人（1991年）：《细胞生物学杂志》115:1127-1136]。我们现在已经对SPARC对BAE细胞细胞骨架的影响进行了表征。向铺展的BAE细胞中添加SPARC导致粘着斑阳性细胞呈剂量依赖性丧失，在约1微克/毫升（0.03微摩尔）时达到最大值。与干涉反射显微镜检测到的粘着斑丧失一致，纽蛋白出现弥散分布，而F-肌动蛋白重新分布到与SPARC一起孵育的细胞周边。然而，整合素αvβ3的分布仍呈斑块状聚集。这些数据，以及SPARC与BAE细胞结合但不与细胞外基质结合的观察结果，表明SPARC通过与细胞表面分子相互作用起作用，而不是通过对整合素-细胞外基质配体的空间/物理破坏。为了确定介导粘着斑丧失的SPARC区域，我们测试了来自SPARC四个不同区域的肽。富含阳离子和半胱氨酸的肽2.1（氨基酸54-73）和含Ca(2+)结合EF手的肽4.2（氨基酸254-273）在粘着斑解体中具有活性。此外，针对这些区域的特异性抗体中和了SPARC的粘着斑不稳定活性。这些结果与之前显示肽2.1和4.2与BAE细胞表面蛋白相互作用的数据一致，并表明暴露于SPARC的内皮细胞粘着斑的丧失是一个受体介导的事件。
