Maspin Regulates Endothelial Cell Adhesion and Migration through an Integrin Signaling Pathway

Li Qin,Ming Zhang

doi:10.1074/jbc.m110.131045

Abstract

Maspin has been identified as a potent angiogenesis inhibitor. However, the molecular mechanism responsible for its anti-angiogenic property is unclear. In this study, we examined the effect of maspin on endothelial cell (EC) adhesion and migration in a cell culture system. We found that maspin was expressed in blood vessels ECs and human umbilical vein endothelial cells (HUVECs). Maspin significantly enhanced HUVEC cell adhesion to various matrix proteins. This effect was dependent on the activation of integrin β(1), which subsequently led to distribution pattern changes of vinculin and F-actin. These results indicated that maspin affects cell adhesion and cytoskeleton reorganization through an integrin signal transduction pathway. Analysis of HUVECs following maspin treatment revealed increased integrin-linked kinase activities and phosphorylated FAK levels, consistent with increased cell adhesion. Interestingly, when HUVECs were induced to migrate by migration stimulatory factor bFGF, active Rac1 and cdc42 small GTPase levels were decreased dramatically at 30 min following maspin treatment. Using phosphorylated FAK at Tyr(397) as an indicator of focal adhesion disassembly, maspin-treated HUVECs had elevated FAK phosphorylation compared with the mock treated control. The results were a reduction in focal adhesion disassembly and the retardation in EC migration. This study uncovers a mechanism by which maspin exerts its effect on EC adhesion and migration through an integrin signal transduction pathway.

Highlights

Our laboratory identified maspin as a potent angiogenesis inhibitor [1]
Secreted maspin may act on the surface of endothelial cell (EC) to regulate cell adhesion, in a manner similar to mammary epithelialderived maspin that acts on the cell surface to modulate adhesion to the laminin 1-rich matrix [2, 10]
We detected the expression of maspin in ECs and smooth muscle cells in vessels of embryonic yolk sac tissue and cultured human umbilical vein endothelial cells (HUVECs) (Fig. 1)

Summary

EXPERIMENTAL PROCEDURES

Cell Culture and Reagents—Human umbilical vein endothelial cells (HUVEC) were cultured in endothelial basic medium, and harvested when cells reached 70 – 80% confluent. For the nocodazole-treated group, serum-starved HUVECs were first incubated with 10 nM nocodazole for 4 h and treated with 0.5 ␮M GST-Maspin or GST, and fixed with 4% paraformaldehyde at 0, 15, 30, 60, and 120 min. Immunoprecipitation and Western Blot Analysis—HUVEC cells were serum-starved for 4 h, detached with 0.05% trypsin, and incubated with GST-Maspin or GST for the indicated time at 37 °C with 5% CO2. After washing out the drug with PBS, cells were incubated with 0.5 ␮M GST-Maspin or GST, and fixed with 4% paraformaldehyde at 0-, 15-, 30-, 60-, and 120-min time points, followed by permeabilization with 0.5% Triton X-100. For Western blot analysis, starved cells were first treated with 10 nM nocodazole for 4 h and incubated with 0.5 ␮M GST-Maspin or GST for the indicated times as described above. Cells were lysed with RIPA buffer and extracts were separated in 10% SDS-PAGE gel to determine the level of Tyr(P)397-FAK

RESULTS

HUVECs can phosphorylate more

FAK antibody and blotted with

DISCUSSION

Li Qin and Ming Zhang