Thrombospondin-1 regulation of smooth muscle cell chemotaxis is extracellular signal-regulated protein kinases 1/2 dependent

Background. Thrombospondin-1 (TSP-1), an extracellular matrix protein, induces vascular smooth muscle cell (VSMC) chemotaxis. We hypothesized that extracellular si,signal-regulated protein kinases 1/2 (ERK1/2), a pathway of the mitogen activated protein kinase (MAPK) family is important in TSP-1-induced VSMC chemotaxis. Methods. A,modified Boyden chamber was used to assess chemotaxis. First, a concentration curve was performed to determine the level for optimal TSP-1-induced; chemotaxis. Then quiescent VSMCs were preincubated (30 minutes) in serum-free medium, dimethyl sulfoxide (the inhibitor vehicle), or PD98059 (10 mu mol/L, an upstream inhibitor of ERK1/2). VSMCs(50, 000 cells/well) with the appropriate preincubation were placed in the top chamber. The bottom chamber contained TSP-1 (20 mu g/mL) or serum-free medium. Results were recorded as cells/5 fields (400x). Then quiescent VSMCs were exposed to TSP-1 (20 mu g/ml)for 0, 1, 5, 10, 30, 120, or 300 minutes. Platelet-derived growth factor (10 ng/mL) was the positive control for ERK1/2 activation. Western blot analysis was performed for activated ERK1/2. All comparisons were made by a paired t test (n = 3). Results. TSP-1-induced chemotaxis;peaks by a concentration of 20 mu g/mL. PD98059 inhibitPn TSP-1-induced chemotaxis (P < .05). ERK1/2 was activated by TSP-1-stimulated VSMCs. Conclusions. TSP-1-stimulated VSMCs activated ERK1/2. An ERK1/2 inhibitor abolished chemotaxis, suggesting the functional importance of MAPK in TSP-1-induced VSMC chemotaxis.