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

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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.

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Acknowledgments

This work was supported in part by the National High Technology Research and Development Program of China (No. 2007AA02Z155 and No. 2008AA02Z136), the National Science and Technology Major Project (No. 2009ZX09103-703 and No. 2009ZX09306-002), the General Programs of the National Natural Science Foundation of China (No. 81071742, No. 81171998, and No. 81171999). We greatly thank the members of the Luo lab for their insightful discussions and suggestions on this manuscript. Special appreciation is extended to Bipo Sun for her contribution as the lab manager.

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The authors declare no competing financial interests.

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Authors and Affiliations

  1. National Engineering Laboratory for Anti-tumor Protein Therapeutics, School of Life Sciences, Tsinghua University, Beijing, 100084, China

    Nan Song, Yanping Ding, Wei Zhuo, Ting He, Zhiguang Fu, Yang Chen, Xiaomin Song, Yan Fu & Yongzhang Luo

  2. Beijing Key Laboratory for Protein Therapeutics, School of Life Sciences, Tsinghua University, Beijing, 100084, China

    Nan Song, Yanping Ding, Wei Zhuo, Ting He, Zhiguang Fu, Yang Chen, Xiaomin Song, Yan Fu & Yongzhang Luo

  3. Cancer Biology Laboratory, School of Life Sciences, Tsinghua University, Beijing, 100084, China

    Nan Song, Yanping Ding, Wei Zhuo, Ting He, Zhiguang Fu, Yang Chen, Xiaomin Song, Yan Fu & Yongzhang Luo

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  1. Nan Song
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  2. Yanping Ding
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  3. Wei Zhuo
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Correspondence to Yongzhang Luo.

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Song, N., Ding, Y., Zhuo, W. et al. The nuclear translocation of endostatin is mediated by its receptor nucleolin in endothelial cells. Angiogenesis 15, 697–711 (2012). https://doi.org/10.1007/s10456-012-9284-y

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