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First published online 24 April 2007
doi: 10.1242/jcs.001586

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Force activates smooth muscle -actin promoter activity through the Rho signaling pathway

¹ CIHR Group in Matrix Dynamics, Faculty of Dentistry, University of Toronto, Canada
² St. Michael's Hospital Research Institute, Department of Surgery, University of Toronto, Canada

View larger version (41K): [in this window] [in a new window]   Fig. 1. Mechanical force increases RhoA activation. (A) Rat-2 cells were stretched with collagen or BSA-coated magnetite beads. RhoA activation was measured by rhotekin binding and total RhoA was measured by immunoblotting cell lysates. Rac and Cdc42 activity were measured as described in the Materials and Methods. NF, no force; +con, positive control with 5% serum addition; F and F-10', applied force for 10 minutes. (B) The ratio of active to total Rho was computed from densitometry measurements of immunoblots and are given as the mean ± s.d. compared with the no force ratio (designated as 1). NF, no force; F5, F10, F15, F20 and F30, applied force for 5, 10, 1 and 20 minutes, respectively; F10+BSA, applied force for10 minutes with BSA-coated beads; F10+4B4, applied force for 10 minutes in the presence of 4B4 antibody (1 µg/ml); F10+LB, applied force for 10 minutes after pre-treatment with latrunculin B (1 µM, 30 minutes before treatment). (C) Collagen-coated beads incubated with cells and treated with and without force (10 minutes). Cells were probed with rhotekin-binding domain (RBD)-GST and immunostained with anti-GST antibody.

View larger version (25K): [in this window] [in a new window]   Fig. 2. Force enhances phosphorylation of LIMK1 and cofilin. (A,B) Immunoblots of LIMK1 and phosphorylated LIMK (p-LIMK) in lysates of Rat-2 cells. The ratio of phosphorylated to total LIMK was computed from densitometry measurements of immunoblots. The data are mean ± s.d. compared with the no-force ratio (designated as 1). For cells treated or not with Y27632, the difference between force and no force at 5 minutes (A) or 15 minutes (B) is statistically significant (P<0.05). (C,D) Immunoblots of cofilin and phosphorylated cofilin (p-cofilin) in Rat-2 cells as measured for LIMK1.

View larger version (24K): [in this window] [in a new window]   Fig. 3. Phosphorylation of myosin light chain (MLC) after mechanical loading. (A) Immunoblots of phosphorylated MLC were prepared from Rat-2 cells treated with force. The ratio of phosphorylated to total MLC was computed from densitometry measurements of immunoblots. Data are given mean ± s.d. compared with the no-force ratio (designated as 1). (B) Positive control for phosphorylation of MLC. Cells were treated with ionomycin (2 µM) for 0, 15, 30 or 60 minutes (0', 15', 30' or 60', respectively). Cell lysates were immunoblotted for phosphorylated MLC (P-MLC).

View larger version (20K): [in this window] [in a new window]   Fig. 4. Force increases actin in collagen-bead-associated attachment complexes. (A) Bead-associated proteins from Rat-2 cells treated with force. Cells were incubated with collagen-coated beads. Cells were lysed in cytoskeletal stabilizing buffer containing phalloidin (1 µM). Soluble material was immunoblotted for GAPDH. Beads were removed magnetically and counted. Proteins from equal numbers of beads were immunoblotted for β-actin. (B) The relative change of bead-associated actin was computed from densitometry measurements of immunoblots. Data are given as the mean ± s.d. compared with the no-force ratio (designated as 1). Force increased the amount of actin filaments 2.5-fold above controls at 15 minutes (P<0.01). (C) Rhodamine-phalloidin staining of cells not subjected to force (NF) or subjected to force for 15 minutes (F15'). Notice the staining around beads in force-treated sample. (D) Fluorescence intensity around collagen beads was quantified in 4-µm circular regions of interest around beads with a CCD camera. The results are given as the mean ± s.d. fluorescence units and are derived from the counts of beads in 100 cells for each treatment in each experiment. (E) Cells were transiently transfected with vehicle (V), siRNA targeting cofilin (Co) or siRNA targeting GFP (G) for 1, 2 or 3 days. Immunoblots show that siRNA targeting cofilin suppresses cofilin protein by >80% after 2 days transfection.

View larger version (54K): [in this window] [in a new window]   Fig. 5. (A-D) MRTF translocation to the nucleus after force application. Representative images show NF (no force) and F (after 60 minutes of force). (Left panels, top rows) Endogenous MRTF A (red) in serum-reduced cells without force application was predominantly cytoplasmic. After force application endogenous MRTF A relocated to the nucleus. (Left panels, middle rows) Rat-2 cells transfected with MRTF-A–FLAG-tagged constructs for 20 hours, subjected to force and immunostained for FLAG. MRTF A (green) in serum-reduced cells without force application or with force application was predominantly nuclear. (Bottom left panels) Rat-2 cells transfected with MRTF-B–FLAG-tagged constructs for 20 hours. Nuclear translocation was never observed between 0 and 60 minutes. Cells were stained with DAPI for nuclear localization. (B) Percentage of cells in which transfected MRTF A was scored as predominantly cytoplasmic or predominantly nuclear after indicated times of force application. Results are given as the mean ± s.d. (C) Rat-2 cells transfected with MRTF A-FLAG (green). Blocking of ROCK with Y27632 (Y27632 F) and of actin filament assembly with latrunculin B (LB F) interfered with the nuclear translocation of MRTF A after force application. (D) Rat-2 cells transiently transfected with MRTF B-FLAG (green) were stimulated with serum for 30 minutes and show nuclear translocation of MRTF B.

View larger version (14K): [in this window] [in a new window]   Fig. 6. Force-induced SMA promoter activity. (A) Rat-2 cells were co-transfected with a construct containing a SMA-promoter–luciferase construct and a β-gal vector as a loading control. Cells were stimulated with force over a time course of 30 minutes to 4 hours. In some experiments, inhibitors were included as indicated. Promoter activities are shown as fold change compared with basal promoter activity and are adjusted for loading based on the β-gal-control loading vector. The results are given as the mean ± s.d. and are derived from four replicates. For experiments with wild-type MRTF A, cells were transfected with the SMA-promoter–luciferase construct and then, 5 hours prior to luciferase activity assay, with the constitutively active MRTF A plasmid. (B) Immunoblots for FLAG show expression of dominant-negative FLAG-tagged MRTF A in cells co-transfected with SMA promoter constructs and dominant-negative MRTF A-FLAG.

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