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

Abstract

Background: Thrombospondin-1 (TSP-1), an extracellular matrix protein, induces vascular smooth muscle cell (VSMC) chemotaxis. We hypothesized that extracellular 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 μmol/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 μg/mL) or serum-free medium. Results were recorded as cells/5 fields (400×). Then quiescent VSMCs were exposed to TSP-1 (20 μg/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 μg/mL. PD98059 inhibited 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. (Surgery 1999;126:203-7.)