The role of G proteins in thromospondin-1-induced vascular smooth muscle cell migration.
OBJECTIVE
Thrombospondin-1 (TSP-1), which is a matricellular glycoprotein associated with chemotaxis of vascular smooth muscle cells (VSMCs), is relevant to the development of arterial lesions. Evidence suggests that TSP-1 receptors are linked to guanosine triphosphate-binding proteins (G proteins). The purpose of this study was to determine the role of G proteins in TSP-1-induced VSMC chemotaxis and whether this pathway was associated with extracellular signal-regulated kinase 1/2 (ERK) or p38 kinase activation (downstream pathways associated with VSMC chemotaxis).


METHODS
In all studies, quiescent VSMCs were preincubated either with serum-free medium, cholera toxin, pertussis toxin, forskolin, or 3-isobutyl-1-methylxanthine. Using a microchemotaxis chamber, preincubated VSMCs were exposed to TSP-1 or serum-free medium. Migrated VSMCs were recorded as cells/5 fields (400x) and analyzed by paired t-test. To evaluate the effect of G proteins on TSP-1-induced ERK or p38 activation, preincubated VSMCs were exposed to serum-free medium or TSP-1 and analyzed by Western immunoblotting. For measurement of intracellular cyclic adenosine monophosphate (cAMP) levels, enzyme-linked immunosorbant assay was performed on preincubated VSMCs exposed to serum-free medium or TSP-1.


RESULTS
Although pertussis toxin attenuated TSP-1-induced chemotaxis, cholera toxin abolished TSP-1-induced chemotaxis. Cholera toxin, but not pertussis toxin, inhibited both ERK and p38 activation. The cAMP stimulators forskolin and IBMX abolished TSP-1-induced chemotaxis and ERK and p38 activation. Although no changes were observed in cAMP levels in VSMCs treated with serum-free medium, TSP-1, or pertussis toxin, cholera toxin alone significantly increased cAMP levels.


CONCLUSIONS
G(s) protein signaling inhibits TSP-1-induced VSMC chemotaxis by increasing the levels of cAMP. G(i) signaling is involved in the mechanism of TSP-1 stimulated chemotaxis and warrants additional study. Agents that increase cAMP levels may be beneficial in reducing TSP-1-induced chemotaxis in response to vascular injury.
Yale University School of Medicine, Section of Vascular Surgery, New Haven, CT, USA.
Shoichi
S
Fuse
Nowokere
N
Esemuede
Miho
M
Yamaguchi
Kristopher G
KG
Maier
Susan M
SM
Nesselroth
Bauer E
BE
Sumpio
Vivian
V
Gahtan
D000818:Animals; D002417:Cattle; D002465:Cell Movement / Q000502:physiology*; D002478:Cells, Cultured; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000502:physiology*; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000502:physiology*; D006801:Humans; D009131:Muscle, Smooth, Vascular / Q000502:physiology; D032389:Myocytes, Smooth Muscle / Q000502:physiology*; D019700:Thrombospondin 1 / Q000502:physiology*
D019700:Thrombospondin 1; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D019205:GTP-Binding Protein alpha Subunits, Gs