Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044, China.
J Biomed Sci. 2010 Feb 3;17(1):6. doi: 10.1186/1423-0127-17-6.
Migration of vascular smooth muscle cells (SMCs) from the media to intima constitutes a critical step in the development of proliferative vascular diseases. To elucidate the regulatory mechanism of vacular SMC motility, the roles of caldesmon (CaD) and its phosphorylation were investigated.
We have performed Transwell migration assays, immunofluorescence microscopy, traction microscopy and cell rounding assays using A7r5 cells transfected with EGFP (control), EGFP-wtCaD or phosphomimetic CaD mutants, including EGFP-A1A2 (the two PAK sites Ser452 and Ser482 converted to Ala), EGFP-A3A4 (the two Erk sites Ser497 and Ser527 converted to Ala), EGFP-A1234 (both PAK- and Erk-sites converted to Ala) and EGFP-D1234 (both PAK- and Erk-sites converted to Asp).
We found that cells transfected with wtCaD, A1A2 or A3A4 mutants of CaD migrated at a rate approximately 50% more slowly than those EGFP-transfected cells. The migration activity for A1234 cells was only about 13% of control cells. Thus it seems both MAPK and PAK contribute to the motility of A7r5 cells and the effects are comparable and additive. The A1234 mutant also gave rise to highest strain energy and lowest rate of cell rounding. The migratory and contractile properties of these cells are consistent with stabilized actin cytoskeletal structures. Indeed, the A1234 mutant cells exhibited most robust stress fibers, whereas cells transfected with wtCaD or A3A4 (and A1A2) had moderately reinforced actin cytoskeleton. The control cells (transfected with EGFP alone) exhibited actin cytoskeleton that was similar to that in untransfected cells, and also migrated at about the same speed as the untransfected cells.
These results suggest that both the expression level and the level of MAPK- and/or PAK-mediated phosphorylation of CaD play key roles in regulating the cell motility by modulating the actin cytoskeleton stability in dedifferentiated vascular SMCs such as A7r5.
血管平滑肌细胞（SMC）从中膜迁移到内膜是增殖性血管疾病发展的关键步骤。为了阐明血管 SMC 运动的调节机制，研究了钙调蛋白（CaD）及其磷酸化的作用。
我们使用 A7r5 细胞进行了 Transwell 迁移测定、免疫荧光显微镜检查、牵引力显微镜检查和细胞圆化测定，这些细胞转染了 EGFP（对照）、wtCaD 或磷酸模拟 CaD 突变体，包括 EGFP-A1A2（两个 PAK 位点 Ser452 和 Ser482 转换为 Ala）、EGFP-A3A4（两个 Erk 位点 Ser497 和 Ser527 转换为 Ala）、EGFP-A1234（两个 PAK 和 Erk 位点均转换为 Ala）和 EGFP-D1234（两个 PAK 和 Erk 位点均转换为 Asp）。
我们发现，转染 wtCaD、CaD 的 A1A2 或 A3A4 突变体的细胞的迁移速度比转染 EGFP 的细胞慢约 50%。A1234 细胞的迁移活性仅为对照细胞的约 13%。因此，MAPK 和 PAK 似乎都有助于 A7r5 细胞的运动，并且作用相当且具有加性。A1234 突变体还导致最高的应变能和最低的细胞圆化率。这些细胞的迁移和收缩特性与稳定的肌动蛋白细胞骨架结构一致。事实上，A1234 突变体细胞表现出最强的应力纤维，而转染 wtCaD 或 A3A4（和 A1A2）的细胞则具有适度增强的肌动蛋白细胞骨架。对照细胞（单独转染 EGFP）的肌动蛋白细胞骨架与未转染细胞相似，并且迁移速度也与未转染细胞相似。
这些结果表明，CaD 的表达水平以及 MAPK 和/或 PAK 介导的磷酸化水平在调节去分化的血管 SMC（如 A7r5）中的细胞运动方面发挥着关键作用，通过调节肌动蛋白细胞骨架的稳定性。
