The nuclear translocation of endostatin is mediated by its receptor nucleolin in endothelial cells

Abstract

Endostatin, the C-terminal fragment of collagen XVIII, is a potent anti-angiogenic factor that significantly modulates the gene expression pattern in endothelial cells. Upon cell surface binding, endostatin can not only function extracellularly, but also translocate to the nucleus within minutes. However, the mechanism by which this occurs is partially understood. Here we systematically investigated the nuclear translocation mechanism of endostatin. By chemical inhibition and RNA interference, we firstly observed that clathrin-mediated endocytosis, but not caveolae-dependent endocytosis or macropinocytosis, is essential for the nuclear translocation of endostatin. We then indentified that nucleolin and integrin α5β1, two widely accepted endostatin receptors, mediate this clathrin-dependent uptake process, which also involves urokinase plasminogen activator receptor (uPAR). Either mutagenesis study, fluorescence resonance energy transfer assay, or fluorescence cell imaging demonstrates that nucleolin and integrin α5β1 interact with uPAR simultaneously upon endostatin stimulation. Blockade of uPAR decreases not only the interaction between nucleolin and integrin α5β1, but also the uptake process, suggesting that the nucleolin/uPAR/integrin α5β1 complex facilitates the internalization of endostatin. After endocytosis, nucleolin further regulates the nuclear transport of endostatin. RNA interference and mutational analysis revealed that the nuclear translocation of endostatin involves the association of nucleolin with importin α1β1 via the nuclear localization sequence. Taken together, this study reveals the pathway by which endostatin translocates to the nucleus and the importance of nucleolin in this process, providing a new perspective for the functional investigation of the nuclear-translocated endostatin in endothelial cells.

Endostatin Prevents Dietary-Induced Obesity by Inhibiting Adipogenesis and Angiogenesis

“…Nucleolin, which is mainly located in the nuclei, can translocate to the cell surface when endothelial cells are stimulated by extracellular matrix and VEGF (32). When endostatin binds to cell surface nucleolin, both can be internalized and then translocated to the nuclei of endothelial cells (20,29,33). Here we found that adipogenesis stimulated nucleolin expression on the cell surface of differentiating 3T3-L1s at days 2, 4, and 6 of differentiation (Supplementary Fig.…”

Role of Serum Levels of Angiogenic Cytokines in Assessment of Angiogenesis after Stem Cell Therapy of Diabetic Patients with Critical Limb Ischemia

“…Endostatin is cleaved from collagen XVIII, a core protein of heparan sulfate proteoglycan in vascular basement membranes, by proteases and metalloproteases such as elastase, matricillin, or cathepsin L that are produced by endothelial cells as a reaction to induced angiogenesis (3,8). Endostatin inhibits VEGF-induced endothelial progenitor cell (EPC) synthesis but also appears to interfere directly with endothelial nitric oxide synthase (eNOS) and targets upstream of its signaling pathway (23,29). The positive correlation between the number of injected CD34 + cells and endostatin levels could reflect this theory.…”

Pharmacokinetics of PEGylated recombinant human endostatin (M2ES) in rats

“…It has been widely reported that endostatin has potent inhibitory effects on endothelial cell proliferation, migration, and tube formation [2,3] . Nucleolin, integrins, caveolin and clathrin have been demonstrated to mediate the anti-tumor effects of endostatin [4][5][6] . Except in endothelial cells, endostatin also has anti-tumor lymphangiogenic and anti-lymphatic metastasis functions [7][8][9] , further supporting the conclusion that endostatin is a potent anti-tumor drug with the potential ability to restrict tumor progression and metastasis [10] .…”