Antimigratory effect of TK1-2 is mediated in part by interfering with integrin alpha2beta1.
The recombinant two kringle domain of human tissue-type plasminogen activator (TK1-2) has been shown to inhibit endothelial cell proliferation, angiogenesis, and tumor cell growth despite of sharing a low amino acid sequence homology with angiostatin. Here, we explored a possible inhibitory mechanism of action of TK1-2 by focusing on antimigratory effect. TK1-2 effectively inhibited endothelial cell migration induced by basic fibroblast growth factor or vascular endothelial growth factor in a dose-dependent manner and tube formation on Matrigel. It blocked basic fibroblast growth factor-induced or vascular endothelial growth factor-induced phosphorylation of extracellular signal-regulated kinase 1/2 and formation of actin stress fibers and focal adhesions. Interestingly, TK1-2 alone induced the weak phosphorylation of focal adhesion kinase, whereas it inhibited focal adhesion kinase phosphorylation induced by growth factors. When immobilized, TK1-2 promoted adhesion and spreading of endothelial cells compared with bovine serum albumin. However, treatment with anti-alpha(2)beta(1) blocking antibody markedly diminished endothelial cell adhesion to immobilized TK1-2 compared with anti-alpha(v)beta(3) or anti-alpha(5)beta(1) antibody. Pretreatment of soluble TK1-2 also altered the binding level of anti-alpha(2)beta(1) antibody to endothelial cells in fluorescence-activated cell sorting analysis. Indeed, a blocking antibody against integrin alpha(2)beta(1) or knocking down of integrin alpha(2) expression prevented the inhibitory effect of TK1-2 in cell migration. Therefore, these results suggest that TK1-2 inhibits endothelial cell migration through inhibition of signaling and cytoskeleton rearrangement in part by interfering with integrin alpha(2)beta(1).
Kim
Hyun-Kyung
HK
Cancer Research Institute and Department of Biomedical Science, College of Medicine, The Catholic University of Korea, Banpo-dong 505, Seocho-ku, Seoul 137-701, Korea.
Oh
Dae-Shik
DS
Lee
Sang-Bae
SB
Ha
Jung-Min
JM
Joe
Young Ae
YA
D000199:Actins / Q000378:metabolism; D000906:Antibodies; D002465:Cell Movement / Q000187:drug effects*; D002478:Cells, Cultured; D015536:Down-Regulation / Q000187:drug effects; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000378:metabolism; D022001:Focal Adhesions / Q000187:drug effects; D006801:Humans; D038982:Integrin alpha2beta1 / Q000378:metabolism*; D018919:Neovascularization, Physiologic / Q000187:drug effects; D010455:Peptides / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D015398:Signal Transduction / Q000187:drug effects; D022502:Stress Fibers / Q000187:drug effects; D010959:Tissue Plasminogen Activator / Q000737:chemistry*
D000199:Actins; D000906:Antibodies; D038982:Integrin alpha2beta1; D010455:Peptides; D051416:Focal Adhesion Protein-Tyrosine Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D010959:Tissue Plasminogen Activator