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. Methods: 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. | Jiang et al. Journal of Biomedical Science 2010 17 6 http content 17 1 6 I NSC The cost of publication in Journal of Biomedical Science is bourne by the National Science Council Taiwan. JOURNAL OF BIOMEDICAL SCIENCE RESEARCH Open Access Caldhesmon regulates the motility of vascular smooth muscle cells by modulating the actin cytoskeleton stability Qifeng Jiang1 2 Renjian Huang 1 2 Shaoxi Cai 1 and Chih-Lueh A Wang 2 Abstract Background 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. Methods 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 . Results 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
