Figure 5.

RhoA (ras homolog family member A) and actomyosin contractility mediate feedback regulation of DDR1 (discoidin domain receptor-1) expression, phosphorylation, and clustering. A, Serum-starved wild-type (WT) vascular smooth muscle cells (VSMCs) were treated with combinations of Col-I (collagen-I), D1in1 (DDR1-in-1), ACT (Rho activator II), or C3 (C3 exoenzyme) for 24 h, immunoblotted (n=3), and (B) DDR1 expression was quantified. C, Serum-starved WT VSMCs were treated for 3 h with Col-I or ACT or 4 h with D1in1 or C3, immunoblotted (n=6), and (D) pDDR1/DDR1 ratio was quantified. E, WT VSMCs were treated for 3 h with ACT or 4 h with C3, Y27 (Y-27632 [Rho-associated protein kinase inhibitor]), or blebbistatin (Bleb), immunoblotted (n=4), and F, pDDR1/DDR1 ratio was quantified. DDR1 expression or pDDR1/DDR1 ratios were quantified and expressed relative to control (Ctrl). G, WT VSMCs seeded on uncoated glass or Col-I -coated glass-bottomed dishes and transfected with DDR1b-YFP (yellow fluorescent protein-tagged full length DDR1b) plasmid were imaged at 600x magnification. Scale bar represents 50 μm. H, WT VSMCs expressing DDR1b-YFP and Actin-mApple were plated on Col-I. Stress fibers were cut with a UV laser then imaged for 2 min. Representative images for Actin-mApple and DDR1b-YFP at the cut site where the actin stress fiber was cut and DDR1b-YFP at a Ctrl site are shown. The merged images on the right show the cut site (white arrow) and the Ctrl site (green arrow). Scale bars represent 10 μm (left images) or 50 μm (right merged images). I, DDR1b-YFP cluster fluorescence intensity in the vicinity of the cut was quantified and compared to the Ctrl region. Means were derived from 17 clusters for Ctrl and 16 clusters for cut quantified from 3 independent experiments. *P<0.05, **P<0.01, ***P<0.001, bars or points represent means±SEM. D–F, Statistical analysis was performed by 1-way ANOVA or (I) 2-way ANOVA with Bonferroni post hoc test.