Angioinhibitory action of NK4 involves impaired extracellular assembly of fibronectin mediated by perlecan-NK4 association.
NK4, a fragment of hepatocyte growth factor (HGF), exerts bifunctional action as a competitive antagonist against HGF and its receptor c-Met and an angiogenesis inhibitor. Here we studied the anti-angiogenic mechanism of NK4. In cultured human endothelial cells, NK4 inhibited DNA synthesis induced not only by HGF but also by either basic fibroblast growth factor or vascular endothelial growth factor. Even if c-Met expression was diminished by small interference RNA, NK4 inhibited basic fibroblast growth factor-induced DNA synthesis, indicating that anti-angiogenic action of NK4 is c-Met-independent. Affinity purification with NK4-immobilized beads revealed that NK4 binds to perlecan. Consistent with this, NK4 colocalized with perlecan in endothelial cells. Perlecan is a multidomain heparan sulfate proteoglycan that interacts with basement membrane components such as fibronectin. NK4 inhibited extracellular assembly of fibronectin, by which fibronectin-dependent endothelial cell spreading was inhibited by NK4. Knockdown of perlecan expression by small interference RNA significantly abrogated the inhibitory effect of NK4 on fibronectin assembly and cell spreading. In NK4-treated endothelial cells, tyrosine phosphorylation of focal adhesion kinase and Rac activation were reduced, whereas overexpression of activated Rac recovered the DNA synthesis in NK4-treated endothelial cells. These results indicate that the association between NK4 and perlecan impairs fibronectin assembly, thereby inhibiting anchorage-dependent signaling. The identified mechanism for angiostatic action provides further proof of significance for NK4 in the treatment of cancer and potentially for vascular regulation as well.
Sakai
Katsuya
K
Kringle Pharma Joint Research Division for Regenerative Drug Discovery, Center for Advanced Science and Innovation, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871.
Nakamura
Takahiro
T
Division of Tumor Dynamics and Regulation, Cancer Research Institute, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-0934, Japan.
Matsumoto
Kunio
K
Division of Tumor Dynamics and Regulation, Cancer Research Institute, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-0934, Japan.
Nakamura
Toshikazu
T
Kringle Pharma Joint Research Division for Regenerative Drug Discovery, Center for Advanced Science and Innovation, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871.
D003094:Collagen / Q000737:chemistry; D004730:Endothelium, Vascular / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D005353:Fibronectins / Q000737:chemistry*; D005786:Gene Expression Regulation; D019812:Heparan Sulfate Proteoglycans / Q000378:metabolism*; D017228:Hepatocyte Growth Factor / Q000378:metabolism*; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods; D008954:Models, Biological; D009389:Neovascularization, Pathologic; D019859:Proto-Oncogene Proteins c-met / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D014471:Umbilical Veins / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
D005353:Fibronectins; C066855:HGF protein, human; D019812:Heparan Sulfate Proteoglycans; D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A; C071927:perlecan; D017228:Hepatocyte Growth Factor; D003094:Collagen; D019859:Proto-Oncogene Proteins c-met