TY - JOUR
T1 - The nuclear translocation of endostatin is mediated by its receptor nucleolin in endothelial cells
AU - Song, Nan
AU - Ding, Yanping
AU - Zhuo, Wei
AU - He, Ting
AU - Fu, Zhiguang
AU - Chen, Yang
AU - Song, Xiaomin
AU - Fu, Yan
AU - Luo, Yongzhang
N1 - Funding Information:
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.
PY - 2012/12
Y1 - 2012/12
N2 - 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 clathrindependent 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.
AB - 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 clathrindependent 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.
KW - Endostatin
KW - Integrin α5β1
KW - Nuclear translocation
KW - Nucleolin
UR - http://www.scopus.com/inward/record.url?scp=84875054866&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84875054866&partnerID=8YFLogxK
U2 - 10.1007/s10456-012-9284-y
DO - 10.1007/s10456-012-9284-y
M3 - Article
C2 - 22711211
AN - SCOPUS:84875054866
SN - 0969-6970
VL - 15
SP - 697
EP - 711
JO - Angiogenesis
JF - Angiogenesis
IS - 4
ER -