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 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 mumol/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 micrograms/mL) or serum-free medium. Results were recorded as cells/5 fields (400x). Then quiescent VSMCs were exposed to TSP-1 (20 micrograms/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 micrograms/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.
Gahtan
V
V
Yale University School of Medicine, Section of Vascular Surgery, New Haven, CT 06520, USA.
Wang
X J
XJ
Willis
A I
AI
Tuszynski
G P
GP
Sumpio
B E
BE
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000502:physiology*; D002417:Cattle; D015815:Cell Adhesion Molecules / Q000502:physiology; D002478:Cells, Cultured; D002633:Chemotaxis / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D051416:Focal Adhesion Protein-Tyrosine Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D011505:Protein-Tyrosine Kinases / Q000502:physiology; D019700:Thrombospondin 1 / Q000494:pharmacology*
D015815:Cell Adhesion Molecules; D019700:Thrombospondin 1; D011505:Protein-Tyrosine Kinases; D051416:Focal Adhesion Protein-Tyrosine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases