Regulation of Vascular Endothelial Growth Factor-induced Endothelial Cell Migration by LIM Kinase 1-mediated Phosphorylation of Annexin 1

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 twodimensional 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 RNAmediated 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.

Annexin-1-mediated Endothelial Cell Migration and Angiogenesis Are Regulated by Vascular Endothelial Growth Factor (VEGF)-induced Inhibition of miR-196a Expression

“…The mechanisms that underly the role of ANXA1 in regulating cell migration and angiogenesis are still ill defined. In endothelial cells, we found that phosphorylation of ANXA1 is required to trigger cell migration (7). Moreover, EGF-induced phosphorylation of ANXA1, triggers its colocalization with F-actin in the lamellipodia (23).…”

“…ANXA1 is a calcium-and phospholipid-binding protein involved in regulating a broad range of cellular events, including migration in different types of cells such as myoblasts, leukemia, and breast cancer lines and endothelial cells (7,26,47,48). Moreover, a recent study showed that ANXA1 Ϫ/Ϫ mice are unable to heal the damage associated with indomethacin-induced gastric ulcers (30).…”

“…In this context, there is a growing body of evidence indicating that ANXA1 may interact with motile cytoskeletal proteins such as tubulin and actin (23)(24)(25). Along these lines, we recently reported that phosphorylation of ANXA1 by LIM kinase downstream of the p38 MAP kinase pathway is an important regulator of endothelial cell migration in response to VEGF (7). Moreover, the cell invasion potential of intestinal epithelial cell line SKCO-15 is increased by 2-fold in the presence of extracellularly added full-length rANXA1 (26).…”

“…Ligand binding to VEGFR-2 triggers its oligomerization, which activates its tyrosine kinase activity leading to phosphorylation of specific tyrosine residues located within its cytoplasmic domain. In particular, VEGFR-2-mediated endothelial actin remodeling and cell migration are triggered by the autophosphorylation of the receptor at Tyr-1214, upstream of the activation of the p38 MAP kinase pathway (7,11,12). Cell migration downstream of VEGFR-2 has also been shown to be mediated through Gab1, which acts as an adaptor for Grb2, PI3-kinase and the tyrosine phosphatase SHP2 (13,14).…”

“…Members of the VEGF family contain six structurally related proteins: VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and placenta-derived growth factor (PIGF) (7,8). Human VEGF-A monomers exist as 5 different isoforms, of which VEGF165, hereafter referred to as VEGF, is the most abundant and biological active form (6).…”