Technical Reference #1822

1822.

Regulation of Vascular Endothelial Growth Factor-induced Endothelial Cell Migration by LIM Kinase 1-mediated Phosphorylation of Annexin 1 Maxime C. Coˆ te´; Jessie R. Lavoie; Franc¸ois Houle; Andre´e Poirier; Simon Rousseau; and Jacques Huot, Laval University, Journal of Biological Chemistry, 285(1822), (2010)
Link To Paper

Abstract:
In this study we obtained evidence indicating that annexin 1 is a new target of the p38/MAPKAP kinase-2 pathway and that it regulates endothelial cell migration in response to vascular endothelial growth factor (VEGF). These conclusions are supported by a series of substantiating experiments. First by two-dimensional gel electrophoresis and mass spectrometry we identified annexin 1 as a protein whose phosphorylation is induced by VEGF and is impaired by inhibiting p38. Second using in vitro kinase assays and in vivo phosphorylation assays we found that VEGF-mediated activation of LIM kinase 1 downstream of the p38 pathway triggers the phosphorylation of annexin 1. Third VEGF-induced cell migration and tube formation in Matrigel are inhibited following small interfering RNA-mediated knockdown of annexin 1. Fourth both processes are rescued in cells expressing an annexin 1 construct insensitive to the small interfering RNA knockdown. Finally the VEGF/annexin 1-mediated cell migration is impaired by inhibiting p38. We therefore conclude that phosphorylation of annexin 1 regulates the angiogenic effect that is associated with the activation of the p38/LIM kinase 1 axis by VEGF.

Keywords:
Actin; Annexin; Cell Migration; Endotheium; p38 MAPK; Angiogenesis; VEGF

Materials & Methods:
Live Cell Microscopy—HUVECs were electroporated with EGFPANXA1 wild-type and were plated on gelatin-coated 35-mm glass bottom microwell dishes (MatTek Corp. Ashland MA). Twenty-four h later the cells were made quiescent by serum starvation for 16 h. They were then placed in an incubator of the VivaView LCV110U. Images of green cells were taken every 4 min during 6 h using the time-lapse imaging VivaView LCV110U system equipped with a 403objective UPLSAPO40X (NA0.95WD0.18 mm). At the beginning cells were left untreated for 3 h before the addition of 5 ng/ml VEGF. Images were captured as 16-bit TIFF files and the supplemental Movie was created using Meta- Morph software.

Microscopic Technique
Live Cell Microscopy

Cell Type(s)
HUVECs