Abstract
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.
Highlights
The manipulation of angiogenesis has potential therapeutic value for the treatment of a variety of diseases including cancer, arthritis, and cardiovascular disease [1, 2]
Together with our finding that c-Met/hepatocyte growth factor (HGF) receptor is not required for the inhibition of DNA synthesis by NK4, we propose that the association of NK4 with perlecan plays a key role in angiogenesis inhibition by NK4
Western immunoblot of fibronectin in deoxy- cells treated with NK4, knockdown of perlecan resulted in cholate-insoluble extracellular matrix (ECM) or conditioned medium showed effi- defective DNA synthesis induced either by bFGF or by VEGF
Summary
Materials—Recombinant NK4 was prepared as described elsewhere [12]. VEGF-A165 was obtained from R&D Systems. bFGF was a gift from Kaken Pharmaceutical. The cover glass was coated with 0.1% gelatin, 5 g/ml fibronectin (Sigma), 5 g/ml vitronectin (Sigma), 5 g/ml collagen type I (BD Biosciences), 5 g/ml laminin (Sigma), or 10 g/ml collagen type IV (Sigma) at room temperature for 5 h. G0-synchronized HUVECs were re-plated and cultured with MCDB131 medium supplemented with 5% FBS, 2 mM L-glutamine, 10 M BrdUrd with and without 20 ng/ml bFGF, VEGF, or HGF, and NK4 for 24 h. Immunostaining—For immunofluorescent staining, cells on cover glass were washed twice with PBS, fixed with 4% paraformaldehyde in PBS for 15 min, permeabilized with 0.2% Triton X-100 in PBS for 5 min, and blocked with 3% BSA in PBS for 1 h at room temperature. Biotinylated fibronectin was stained with Alexa Fluor 488-conjugated streptavidin (Invitrogen). Cells (2 ϫ 106) resuspended in 400 l of Opti-MEM (Invitrogen) were transfected with 20 g of each vector by electroporation at 240 V and 950 microfarads and recovered for 16 h in complete medium before each assay
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