Angiogenesis (2012) 15:697–711
DOI 10.1007/s10456-012-9284-y
ORIGINAL PAPER
The nuclear translocation of endostatin is mediated by its receptor
nucleolin in endothelial cells
Nan Song • Yanping Ding • Wei Zhuo •
Ting He • Zhiguang Fu • Yang Chen •
Xiaomin Song • Yan Fu • Yongzhang Luo
Received: 19 April 2012 / Accepted: 6 June 2012 / Published online: 19 June 2012
Ó Springer Science+Business Media B.V. 2012
Abstract Endostatin, the C-terminal fragment of colla-
gen XVIII, is a potent anti-angiogenic factor that signifi-
cantly 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 a5b1, two
widely accepted endostatin receptors, mediate this clathrin-
dependent uptake process, which also involves urokinase
plasminogen activator receptor (uPAR). Either mutagene-
sis study, fluorescence resonance energy transfer assay, or
Electronic supplementary material The online version of this
article (doi:10.1007/s10456-012-9284-y) contains supplementary
material, which is available to authorized users.
N. Song
Y. Ding
W. Zhuo
T. He
Z. Fu
Y. Chen

X. Song
Y. Fu
Y. Luo (&)
National Engineering Laboratory for Anti-tumor Protein
Therapeutics, School of Life Sciences, Tsinghua University,
Beijing 100084, China
e-mail: yluo@tsinghua.edu.cn
N. Song
Y. Ding
W. Zhuo
T. He
Z. Fu
Y. Chen

X. Song
Y. Fu
Y. Luo
Beijing Key Laboratory for Protein Therapeutics, School of Life
Sciences, Tsinghua University, Beijing 100084, China
N. Song
Y. Ding
W. Zhuo
T. He
Z. Fu
Y. Chen

X. Song
Y. Fu
Y. Luo
Cancer Biology Laboratory, School of Life Sciences,
Tsinghua University, Beijing 100084, China
fluorescence cell imaging demonstrates that nucleolin and
integrin a5b1 interact with uPAR simultaneously upon
endostatin stimulation. Blockade of uPAR decreases not
only the interaction between nucleolin and integrin a5b1,
but also the uptake process, suggesting that the nucleolin/
uPAR/integrin a5b1 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 a1b1 via the nuclear localization sequence. Taken
together, this study reveals the pathway by which endo-
statin 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.
Keywords Endostatin
Nuclear translocation

Nucleolin
Integrin a5b1
Introduction
Angiogenesis is an essential process of the onset and pro-
gression of various types of tumors, and thus predicted as a
potential target for the treatment of cancer [1]. Some
40 years after Dr. Folkman’s postulate, anti-angiogenic
therapy has been established as a fourth modality in cancer
therapeutics, joining surgery, chemotherapy, and radio-
therapy [2]. A handful of anti-angiogenic factors including
angiostatin, endostatin, tumstatin, canstatin, arresten, and
thrombospondin-1 and -2 [3–11], have been discovered and
developed as drug candidates. Among them, endostatin is
the first endogenous angiogenesis inhibitor to receive
approval for anticancer therapy in clinic [12]. Although the
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therapeutic effects of endostatin have been documented in
animal studies and clinical trials, several anti-angiogenic
activities of endostatin still have no molecular explanation.
The first mystery is related to the receptors of endo-
statin. Various proteins have been reported as binding
proteins of endostatin [13–17]. Nucleolin, which resides
mainly in the nucleolus, can translocate to the cell surface
when triggered by extracellular matrix and VEGF [18].
Only the surface nucleolin can mediate the anti-angiogenic
activity of endostatin [14]. Integrin a5b1, also the target for
other angiogenesis inhibitors, can interact with endostatin
and thus lead to the inhibition of the FAK/c-Raf/MEK1/2
[15]. Glypicans, heparan sulfate proteoglycans, serve as
low-affinity endostatin receptors, while few downstream
signaling has been investigated [17]. In addition, laminin-1,
MMP2, and tropomyosin have also been identified as the
endostatin associated proteins [19–21]. Although a series
of endostatin-binding proteins and their respective signal-
lings have been indicated, it remains obscure how these
proteins cooperate with each other to orchestrate the
function of endostatin and activate different pathways.
The second mystery is how endostatin translocates to the
nucleus. The majority of studies on endostatin focuses on
the signallings triggered extracellularly [16, 22–24]. For
instance, the binding of endostatin competes with interac-
tion between the extracellular matrix and integrins, and
associates with caveolin-1, and activates Src [16]. In fact,
endostatin can also be internalized, and translocated into
the nucleus with in minutes [14]. Moreover, the increased
uptake of endostatin induced by nystatin positively corre-
lates with its anti-angiogenic efficacy [25]. However, rel-
atively little attention has been paid to neither the
translocation mechanism, nor the role of the nuclear-
translocated endostatin.
In the present study, we demonstrated that the inter-
nalization of endostatin is clathrin-dependent, and medi-
ated by the integrin a5b1/nucleolin/uPAR receptor
complex. After the transportation of endostatin from cell
membrane to the early endosome, importin a1b1 recog-
nizes the nuclear localization signal (NLS) of nucleolin,
and further transports the endostatin/nucleolin cargo to the
nucleus. In summary, these results real how endostatin-
binding proteins cooperate to facilitate the endocytosis and
nuclear translocation of endostatin.
Materials and methods
Plasmids and construction
Mouse nucleolin (mNCL) (NM_005381) and human uPAR
(NM_002659) were subcloned into pCMV6-AN-GFP vec-
tor and pCMV6-AN-RFP (Origene), respectively. Human
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Angiogenesis (2012) 15:697–711
integrin a5 (NM_002205) was subcloned into pCMV6-AC-
RFP vector. Wild-type integrin a5 and nucleolin were
constructed into pCMV6 vector with HA or Myc tag,
respectively. Mutants of Integrin a5 and nucleolin were
constructed by the Quick Change Mutagenesis kit (Strata-
gene). All the plasmids were purified from Escherichia coli
using PowerPrepÒ Plasmid Purification Kits (Origene).
Cell culture, transfection and RNAi
Human umbilical vein endothelial cells (HUVEC, CRL-
1730, ATCC), and dermal microvascular endothelial cells
(HMEC-1, lab stock), mouse tumor-associated endothelial
cells (MTEC, lab stock) were cultured with 5 % CO2 in
endothelial cell medium (ECM, Sciencell) as previously
described [14, 18, 26]. In brief, cells were grown to
approximately 60–70 % confluence and then treated with
recombinant human endostatin (rhEndostatin, E. coli,
Protgen Ltd.) as indicated.
Biotin-labeled endostatin was generated by the EZ-
LinkTM sulfo-NHS-LC biotinylation kit (Pierce) in PBS.
Dylight 649-labeled endostatin, bovine serum albumin
(BSA), and rabbit anti-nucleolin polyclonal antibody
were generated with Dylight-649 fluorescent labeling kit
(Pierce) according to the manufacturer’s instruction.
For RNAi, oligofection of siRNA duplexes was per-
formed using Oligofectamine (Invitrogen) according to the
manufacturer’s protocols. Briefly, HUVECs were trans-
fected twice (at 0 and 24 h) with 200 pmol of scramble
siRNA (or siRNA pool), or indicated siRNAs. After 24 h
had passed since the second oligofection, HUVECs were
lysed or tested in functional assays. The oligonucleotide
sequences were listed in Supplementary Table 1.
Preparation of nuclear extracts
To isolate nuclei, HUVECs were fractionated as described
in Stepanova et al. [27]. All manipulations were performed
on ice or at 4 °C. Briefly, the cells were homogenized in
lysis buffer containing 20 mM HEPES, pH 7.0, 2 mM
MgCl2, 10 mM KCl, 0.5 % NP40, and protease inhibitor
cocktail (Roche) by 40 strokes in a Dounce homogenizer.
The homogenates were then centrifuged at 1,5009g for
5 min. The nuclear pellets were washed and resuspended in
lysis buffer containing 0.5 M NaCl to extract nuclear
proteins. Nuclear extracts were prepared by further cen-
trifugation at 15,0009g for 10 min.
The nuclear and cytoplasmic extraction kit (Pierce) was
also used to isolate the cytoplasmic and nuclear protein
extracts in accordance with the instructions supplied by the
manufacturer. In brief, HUVECs in 10-cm dishes were
washed with ice-cold PBS, and harvested by centrifugation
(5009g). The cell pellets were suspended in 200 lL of
Angiogenesis (2012) 15:697–711
Cytoplasmic Extraction Reagent I (CER I) solution and
incubated for 10 min on ice, followed by adding 11 lL of
CER II solution and incubation for 5 min on ice. The cell
suspensions were centrifuged at 16,0009g for 5 min to
collect supernatant as the cytoplasmic fraction. The pellets
were then resuspended with 100 lL of Nuclear Extraction
Reagent (NER) for 30 min on ice. The cell suspension was
centrifuged at 16,0009g for 10 min at 4 °C to collect
supernatant as the nuclear fraction. Samples were nor-
malized using BCA assay.
Isolation of triton-soluble and -insoluble membrane
fractions
Triton-soluble and -insoluble fractions of cell lysate were
prepared as previously described [25]. In brief, cells were
harvested, resuspended in 0.5 mL of MBS [150 mM NaCl,
25 mM 2-(N-morpholino) ethanesulfonic acid, pH 6.5] con-
taining the protease inhibitor cocktail (Roche) and 1 % Triton
X-100, and incubated on ice for 30 min. The whole cell lysates
were separated into Triton-soluble and -insoluble fractions
using OptiPrep Density Gradient Medium (Sigma-Aldrich).
Sandwich ELISA
The microtiter plates were coated overnight with rabbit
anti-endostatin polyclonal antibody (lab stock) at a con-
centration of 4 lg/mL in PBS buffer. Following the
blocking and washing steps, the dilutions of nuclear frac-
tion extracted from biotin-endostatin-incubated HUVECs
were added and incubated at 37 °C for 1 h. Then HRP-
conjugated strepavidin were used. After washing, the
reaction is developed by adding 100 lL of a tetramethyl-
benzidine substrate solution. The plate is incubated 20 min
at 37 °C. The reaction is stopped by adding 1 M HCl. The
absorbance was measured at 450 nm with ELISA reader
(Varioskan Flash, Thermo Electron).
qRT-PCR
The total RNA was isolated using RNAeasy Kit (Qiagen),
and reverse transcribed using the First Strand cDNA Syn-
thesis Kit (Fermentas). qRT-PCR amplification was per-
formed using the SYBR Green qRT-PCR Master Mix kit
(Stratagene). Relative quantitation was performed using the
DD
Ct method. All primers used were listed in Supplemen-
tary Table 2. Independent experiments were performed in
triplicates.
Indirect immunofluorescence staining
HUVECs were incubated with 100 nM Dylight 649-labeled
endostatin for 30 min before washing and partial fixation
699
with 0.25 % PFA, then incubated (20 °C, 60 min) with
labeled antibodies (Dylight 649-anti-integrin a5 mAb,
Dylight 549-anti-uPAR mAb, and Dylight 488-anti-nucle-
olin pAb). After washing, cells were fixed with 3.7 % PFA.
Nuclei were counterstained with DAPI (0.5 g/mL). Stained
cells were mounted in ProLong Gold (Molecular Probes)
and analyzed using the Nikon A1 fluorescence microscope
using 63X/1.49 NA oil objectives. Images were captured
with NES-Elements AR 3.0 software.
For the internalization assay, HUVECs pretreated with
20 nM Dylight 649-endostatin were washed by PBS and
fixed with 3.7 % PFA, then incubated with Dylight
549-anti-EEA, and Dylight 488-anti-integrin a5 mAb or
anti-nucleolin pAb, respectively. Stained cells were
mounted and captured as previously described.
Immunoprecipitation and immunoblotting analysis
Following stimulations, HUVECs were lysed in Lysis
Buffer (25 mM Tris–HCl, pH 7.4, 130 mM NaCl, 0.1 %
Tween-20, 5 % glycerol, and protease inhibitors). Cell
debris were eliminated by centrifugation at 15,0009g at
4 °C for 10 min. The samples were subjected to immuno-
blot directly, or immunoprecipitation using indicated
antibody-conjugated agarose beads, respectively. Equal
amounts of protein were loaded per lane and separated by
12 % SDS-PAGE under reducing conditions. The protein
was transferred to PVDF membrane, and immunolabeled
with primary antibodies overnight at 4 °C. Subsequently,
membranes were washed and incubated with horseradish
peroxidase-conjugated secondary antibody for 1 h at room
temperature. After washing 3 times for 45 min, the mem-
branes were developed with an enhanced chemilumines-
cence system (Roche) according to the manufacturer’s
instructions.
The following antibodies were used for immunoblotting:
anti-tGFP (2H8), anti-Histone H3 (D1H2), anti-HIF-1b/
ARNT (C15A11), anti-Myc-Tag (71D10), anti-HA-Tag
(C29F4), anti-GAPDH (D16H11), anti-beta-Tubulin (9F3),
anti-Lamin B1, anti-Caveolin-1, anti-Clathrin Heavy Chain
(D3C6) (Cell Signaling Technology), anti-Nucleolin
(ab70493), anti-Integrin alpha 5 [P1D6], anti-Integrin
beta 1 [BV7] (ab7168), anti-Importin beta (ab2811), anti-
uPAR (ab89932), anti-uPAR (ab103791), anti-KPNA2
(ab70160), anti-Glypican 1 (ab55971), anti-Transferrin
Receptor (ab84036) (Abcam), and anti-endostatin (NBP1-
40051) (Novus Biologicals).
The following antibodies were used for immunoprecip-
itation: anti-tGFP (2H8), anti-Myc-Tag (71D10), anti-
HA-Tag (C29F4), anti-GAPDH (D16H11) (Cell Signaling
Technology), anti-Nucleolin (ab70493), anti-Integrin alpha
5 [P1D6], anti-uPAR (ab3129), anti-KPNA2 (ab70160)
(Abcam).
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Fluorescence resonance energy transfer (FRET)
detection
HUVEC transfectants expressing integrin a5-RFP/uPAR-
GFP or nucleolin-RFP/uPAR-GFP were spun directly onto
poly-L-lysine coated glass slides (the Förster radius of the
GFP/RFP pair is 4.7 nm). FRET was performed on the
Nikon A1 confocal microscope with a 63X/1.49 NA oil
objectives. GFP and RFP were excited and emission sig-
nals were detected for indicated time points. Meanwhile,
the immuno-FRET analysis was performed. In brief, HU-
VECs pretreated with or without endostatin were labeled
with FITC-anti-integrin a5/TRITC-anti-uPAR, or FITC-
anti-nucleolin/TRITC-anti-uPAR (the Förster radius of the
FITC/TRITC pair is 5.5 nm). Then FRET was performed
as previously described. For analysis, only the cell mem-
brane was selected as the region of interest. Fluorescent
signal detected in the peri-nuclear region of the cell
(integrins in the Golgi) was not considered. FRET quanti-
fication was performed according to the manufacturer’s
protocol.
Statistical analysis
All data from individual experiments are represented as
means ± standard errors. Statistical significance of groups
was determined using two-tailed Student’s t tests. Differ-
ences were considered to be statistically significant at
P \ 0.05.
Results
Nuclear translocation of endostatin is mediated
by clathrin-dependent endocytosis
We firstly investigated the pattern of the nuclear translo-
cation of endostatin. To examine this question, we incu-
bated HUVECs with 100 nM Dylight-649-labeled
endostatin for 5, 10, 15, 30, or 60 min at 37 °C, respec-
tively. The cells were washed twice in PBS and then once
with mild acid to remove surface-bound endostatin, and
then fixed in methanol to unmask nuclear antigens.
Immunofluorescence cell imaging captured by confocal
microscopy indicated that the nuclear translocation of
endostatin was initiated within minutes and reached the
peak after 30 min (Fig. 1a). In addition, no immunofluo-
rescence signal was observed in the nucleus when incu-
bated with either Dylight-649-labeled BSA or rabbit anti-
nucleolin IgG, suggesting that the uptake of endostatin is
specific (Supplementary Fig. 1a). To assess translocation
using an entirely independent approach, we then prepared
the nuclear fraction of HUVECs that were incubated with
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biotin-labeled endostatin. HUVECs were lysed using
homogenization combined with limited lysis in a low-salt
buffer containing 0.5 % NP-40. We analyzed the amount
of endostatin and also the purity of the nuclear fraction by
SDS-PAGE and Western blotting analysis. As shown in
Fig. 1b, the trend of the nuclear translocated endostatin
resembled the previous result (Fig. 1a). Moreover, the
nuclear fraction is negatively stained for Rab4 (endosome
marker), LAMP1 (lysosome marker), and tubulin (cytosol
marker) as expected (Fig. 1b), suggesting the purity of the
fractionation. Similar results were also observed by sand-
wich ELISA (Fig. 1c). In addition, we also utilized the NE-
PER nuclear-cytoplasmic extraction kit (Pierce) to isolate
both the nuclear and cytosol fractions. Western blotting
analysis indicated a high purity fractionation, and also
showed the nuclear translocation of endostatin (Supple-
mentary Fig. 1b).
Since the nuclear translocation of endostatin should be
mediated by some uptake pathway(s), we then examined
the potential pathways by chemical inhibition. To investi-
gate whether the nuclear translocation of endostatin occurs
by endocytosis, we firstly tested for involvement of the
actin cytoskeleton, which has been implicated in mediating
endocytic pathways [28]. To this end, HUVECs were
pretreated with an actin polymerization inhibitor, cyto-
chalasin D, and assessed for the nuclear-translocated
endostatin by sandwich ELISA as described in Fig. 1c.
Cytocholasin D-treated HUVECs showed a significant
decrease in the nuclear translocation of endostatin com-
pared with the untreated cells (Fig. 2a). Well-documented
pathways of endocytosis include clathrin-dependent endo-
cytosis, caveolae-dependent endocytosis, clathrin- and
caveolae-independent endocytosis, and macropinocytosis
[29]. Since dynamin is required for both clathrin- and
caveolae-mediated endocytosis, we next examined the
effect of dynasore (dynamin inhibitor) on the nuclear
translocation of endostatin. As shown in Fig. 2a, pre-
treatment of HUVECs with dynasore dramatically
decreased the amount of the nuclear-translocated endo-
statin. In contrast, amiloride (macropinocytosis inhibitor)
did not show any significant difference with control group
(Fig. 2a). All these results demonstrate that the clathrin- or
caveolae-mediated endocytosis regulates the nuclear
translocation of endostatin.
Thus we evaluated the relative contributions of these
two endocytosis pathways to the nuclear translocation of
endostatin. HUVECs were pre-treated with heparinase III
for 30 min to specifically reduce the raft-associated endo-
statin. Then cells were incubated with 100 nM endostatin,
lysed and fractionated into Triton-soluble non-raft and
Triton-insoluble raft fraction using a discontinuous Opti-
Prep gradient as previously described. As shown in Fig. 2b,
in the absence of heparinase III, endostatin was present in
Angiogenesis (2012) 15:697–711
Fig. 1 The nuclear translocation of endostatin. a HUVECs were
incubated with 100 nM Dylight-649 labeled endostatin at 37 °C for 5,
10, 15, 30, or 60 min, respectively. HUVECs were washed twice in
PBS and once with mild acid, and then fixed. Digital images were
captured by the Nikon A1 fluorescence microscope using 63X/1.49
NA oil objectives. Images were obtained with NIS-Elements AR 3.0
software. Nuclei were stained by DAPI. Scale bars: 10 lm.
b HUVECs were pretreated with 100 nM biotin-labeled endostatin
for indicated time. The nuclear fraction was extracted as described in
‘‘Materials and methods’’. Samples were normalized using BCA
protein assay. 10 lg of each cytosolic and nuclear extract sample was
analyzed by immunoblotting using indicated antibodies, respectively.
both fractions. By contrast, pre-incubation with heparinase
III remarkably decreased the localization of endostatin in
raft fraction (Fig. 2b). Intriguingly, although the membrane
distribution was significantly altered by heparinase III, the
nuclear translocation of endostatin was only slightly
interrupted (Fig. 2c), indicating that the caveolae/raft-
dependent endocytosis is not essential for this process.
Consistently, the knockdown of caveolin-1 did not disrupt
the nuclear translocation of endostatin either, while the
knockdown of clathrin markedly decreased the amount of
endostatin in both cytosol and nuclear fractions (Fig. 2d).
701
The HRP-conjugated secondary antibodies were used with chemilu-
minescent substrates for detection. Rab4, LAMP1, tubulin and lamin
were used as endosome, lysosome, cytosol, and nucleus marker,
respectively. c HUVECs were pretreated with biotin-labeled endo-
statin and fractioned as described in b. Sandwich ELISA that utilising
rabbit anti-endostatin antibody and HRP-conjugated Strepavidin was
performed to assess the amount of the nuclear translocated endostatin.
The absorbance was measured at 450 nm with ELISA reader
(Varioskan Flash, Thermo Electron). Data are shown as mean ± SD
of three independent experiments. P value indicates difference with
untreated control
All these observations demonstrate that the clathrin-
dependent endocytosis, but not the caveolae/raft-depen-
dent, is indispensable for the nuclear translocation of
endostatin.
Nucleolin, integrin a5b1, and uPAR mediate
the nuclear translocation of endostatin
We then investigated the associated proteins that contribute
to the clathrin-mediated endocytosis of endostatin. Endo-
statin can bind to several endothelial cell-surface proteins
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Fig. 2 Clathrin-dependent endocytosis mediates the nuclear translo-
cation of endostatin. a HUVECs were pretreated with 5 lM
cytochalasin D for 30 min, 80 lM dynasore for 30 min, or 3 mM
amiloride for 60 min at 37 °C, and then incubated with 100 nM
biotin-labeled endostatin for 60 min. HUVECs were fractioned as
described in Fig. 1b. Sandwich ELISA that utilising rabbit anti-
endostatin antibody and HRP-conjugated Strepavidin was performed
to assess the amount of the nuclear translocated endostatin. The
absorbance was measured at 450 nm with ELISA reader (Varioskan
Flash, Thermo Electron). Data are shown as mean ± SD of three
independent experiments. P value indicates difference with untreated
control. b HUVECs pretreated with heparinase III were incubated
with 100 nM endostatin at 37 °C for 30 min after which membrane
fractions were prepared using a 5/30/40 % OptiPrep gradient.
Aliquots of each fractions were analyzed by immunoblotting with
anti-endostatin antibody. Clathrin heavy chain (Clathrin HC) and
that are critical for angiogenesis [13–17, 19–21]. It binds
directly to integrin a5b1, and also interacts with aV inte-
grins [13]. Cell surface nucleolin, an endothelial marker for
angiogenic blood vessels, has been identified as a receptor
for endostatin as well [14]. Similar to many other endog-
enous angiogenesis inhibitors, endostatin has a high affinity
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Angiogenesis (2012) 15:697–711
transferrin receptor (Transferrin R) were used as loading controls
as well as non-raft markers. Caveolin-1 and glypican-1 were used as
loading controls and raft markers. c HUVECs were pretreated as
described in b, then lysed using NE-PER Nuclear and Cytoplasmic
Extraction Kit. Samples were normalized using BCA protein assay.
10 lg of each cytosolic and nuclear extract sample was analyzed by
immunoblotting using indicated antibodies, respectively. The HRP-
conjugated secondary antibodies were used with chemiluminescent
substrates for detection. Rab4, LAMP1, and histone H3 were used as
endosome, lysosome, and nucleus marker, respectively. d HUVECs
were transfected twice with scamble siRNA or siRNA against
caveolin-1 (si-caveolin), clathrin heavy chain (si-clathrin), respec-
tively. After incubation with 100 nM endostatin at 37 °C for 30 min,
cells were fractionated and analyzed by immunoblotting as described
in c
binding to heparin, and can bind to heparan sulfate prote-
oglycans such as glypican-1/4 [17]. Moreover, MMP2 is
also considered as an endostatin interacting partner [21].
We firstly used siRNA to knock-down the aforementioned
proteins. HUVECs transfected with scramble siRNA or
indicated siRNAs were treated with 100 nM endostatin for
Angiogenesis (2012) 15:697–711
Fig. 3 Integrin a5b1, nucleolin, and uPAR mediates the internaliza-
tion of endostatin. a HUVECs were transfected twice with indicated
siRNAs, and then treated with 100 nM endostatin for 30 min.
HUVECs were fractioned as described in Fig. 1b. Sandwich ELISA
that utilising rabbit anti-endostatin antibody and HRP-conjugated
Strepavidin was performed to assess the amount of the nuclear
translocated endostatin. The absorbance was measured at 450 nm
with ELISA reader (Varioskan Flash, Thermo Electron). Data are
shown as mean ± SD of three independent experiments. P value
indicates difference with untreated control (s.c. siRNA). b HUVECs
were pre-incubated with 20 lg/mL indicated antibodies for 30 min,
following with a 30-min treatment of 100 nM endostatin. Cells were
60 min, then lysed and analyzed by sandwich ELISA.
Meanwhile, the RNAi efficiencies were verified by qRT-
PCR (Supplementary Fig. 2a). As shown in Fig. 3a, com-
pared with the control, siRNAs that targeted integrin a5
(ITGA5), integrin b1 (ITGB1), and nucleolin (NCL) all
remarkably abolished the nuclear translocation of endo-
statin. Similar results were also obtained by Western
blotting analysis (Supplementary Fig. 2b). To further
confirm this observation, HUVECs were pre-incubated
with anti-integrin a5, anti-integrin b1, anti-nucleolin neu-
tralizing antibody or isotype control IgG, respectively,
followed by a 60-min treatment of 100 nM endostatin.
Consistently, antibody neutralization of integrin a5b1 or
703
then lysed and subjected to immunoblotting analysis. c HUVECs
were treated with or without 100 nM endostatin for 30 min, lysed and
immunoprecipitated using anti-uPAR mAb or isotype control IgG,
respectively. The indicated proteins were detected by immunoblot-
ting. Live cell FRET was performed in HUVECs co-transfected with
uPAR-GFP and integrin a5-RFP (d) and those co-transfected with
nucleolin-RFP and uPAR-GFP (e) after the treatment of 100 nM
endostatin for 15 min. The mean efficiency was analyzed by Nikon
A1 confocal system and calculated by NIS-Elements AR 3.0 software.
Data are shown as mean ± SD of three independent experiments.
P value indicates difference with untreated control
nucleolin also efficiently disrupted the internalization of
endostatin (Fig. 3b). Similar results were also observed in
HMEC-1 and MTEC (Supplementary Fig. 2c–2d).
Nucleolin shuttles specific proteins from the cell surface
and the cytoplasm to the nucleus [27, 30–32]. To further
confirm the role of nucleolin in nuclear translocation of
endostatin, we next investigated the competitive effect
of other nucleolin ligands on the nuclear translocation of
endostatin. HUVECs were pre-incubated with either lac-
toferrin or uPA, both of which could translocate to the
nucleus in a nucleolin-dependent manner [27, 30]. The
cells were then incubated with 100 nM endostatin for an
additional hour at 37 °C. As shown in Supplementary
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Fig. 3, both lactoferrin and uPA reduced the nuclear
transport of endostatin, compared with cells pre-incubated
with BSA-containing medium alone. These observations
suggest that nucleolin act as a carrier for the nuclear
translocation of endostatin.
Since uPA could compete with endostatin in transloca-
tion, together with the report that uPAR and nucleolin form a
complex to mediate the nuclear transportation of uPA [27],
we then tested the contribution of uPAR to the nuclear
translocation of endostatin. Consistent with the report of
Keski-Oja and his colleagues [22], we also found
that endostatin could induce the redistribution of the uPAR/
uPA/PAI-1 complex from focal contacts (Supplementary
Fig. 4a). Intriguingly, an extensive co-localization of
endostatin and uPAR was observed at the same time (Sup-
plementary Fig. 4b). The clathrin-coated pits have been
largely documented to be responsible for the removal of
uPAR from the plasma membrane [33, 34], suggesting that
uPAR is involved in the clathrin-mediated endocytosis of
endostatin. As shown in Supplementary Fig. 4c, the endo-
cytosis of endostatin was indeed significantly interrupted by
blockade of surface uPAR using neutralizing uPAR anti-
body (mouse anti-uPAR, ab89932, abcam), soluble uPAR
(807-UK-100/CF, R&D Systems), or uPAR-target siRNA,
respectively. In addition, the in vitro immunoprecipitation
assay did not reveal any interaction between uPAR and
endostatin (Supplementary Fig. 4d). These results indicate
that uPAR, although does not have any physical interaction
with endostatin, contributes to the endocytosis of endostatin.
The uPAR/nucleolin/integrin a5b1 complex regulates
the transport of endostatin from membrane to the early
endosome
GAR domain in nucleolin is indispensable for the inter-
action between uPAR and nucleolin [27]. It has also been
documented that the uPAR directly interacts with integrin
a5b1, affecting their activation state and changing the
downstream intracellular signaling [35]. We then detected
whether endostatin could induce the interaction of uPAR
with integrin a5b1 or nucleolin. Immunoprecipitation assay
showed that after the treatment of endostatin, more integrin
a5b1 and nucleolin could be precipitated by anti-uPAR
antibody (Fig. 3c), indicating that uPAR interacts with
integrin a5b1 and nucleolin. Since FRET is widely used to
measure the intimate interactions between two molecules,
we thus measured the mean FRET efficiencies in live
HUVECs co-transfected with either integrin a5-RFP/
uPAR-GFP or nucleolin-RFP/uPAR-GFP, respectively.
The results clearly demonstrate the increasing interaction
between integrin a5/uPAR (Fig. 3d) and nucleolin/uPAR
(Fig. 3e) after 15-min endostatin treatment, respectively.
Consistent results were also observed by immune-FRET
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Angiogenesis (2012) 15:697–711
using FITC-anti-integrin a5/TRITC-anti-uPAR (Supple-
mentary Fig. 5a), or FITC-anti-nucleolin/TRITC-anti-
uPAR (Supplementary Fig. 5b).
To further confirm these results, we mapped the binding
sites that modulate the physical interactions. The NLTY
motif of integrin a5 appears to be critical to the binding of
uPAR [35], thus we generated two integrin a5 mutants
(ITGA5ALTA and ITGA5DNLTY) with HA-His tag as shown
in Fig. 4a. Co-immunoprecipitation analysis showed that
both integrin a5 mutants could not interact with uPAR,
with or without endostatin treatment (Fig. 4b).
Then we generated Myc-tagged full-sized, N- and C-ter-
minus deleted mouse nucleolin truncation mutants (mNLWT,
mNLD1–303 and mNLD307–707) as shown in Fig. 4c. Co-
immunoprecipitation analysis showed that the endostatin-
induced interaction between uPAR and mNLD307–707 was
dramatically disrupted, while only slight influence was
observed in the mNLD1–303 group, suggesting the uPAR-
binding region resides in the C-terminus (Fig. 4d). Another
two mutants (mNLD469–707 and mNLD647–707) were thus
constructed (Fig. 4c). Neither of these mutants showed
increased interaction with uPAR after endostatin treatment,
indicating that the C-terminal glycine-arginine-rich domain
(GAR domain, 646–707 aa) of nucleolin is required for the
physical interaction between uPAR and nucleolin (Fig. 4d).
Taken together, both nucleolin and integrin a5b1 can interact
with uPAR, and they together are involved in the endocytosis
of endostatin.
Since both integrin a5b1 and nucleolin could interact
with uPAR, together with our observation that blockade of
either integrin a5b1 or nucleolin could dramatically inter-
fere with endostatin internalization, we therefore proposed
that nucleolin together with integrin a5b1 simultaneously
mediate this endocytic process, with uPAR serving as an
adaptor to facilitate the formation of the complex.
We first examined the interactions among uPAR, inte-
grin a5b1, and nucleolin. After endostatin treatment, triple
staining of non-permeabilized HUVECs clearly demon-
strated pronounced co-localization of uPAR, nucleolin and
integrin a5 (Fig. 5a). The co-immunoprecipitation assay
also showed the endostatin-induced interaction between
nucleolin and integrin a5 (Fig. 5b). Retrospectively, inte-
grin a5 mutants that could not interact with uPAR, could
not bind with nucleolin (Fig. 4b). Vice versa, the truncated
nucleolin mutants, which have defects in the association
with uPAR, could not interact with integrin a5b1 either
(Fig. 4d). Moreover, blockade of uPAR by either siRNA or
blocking antibody markedly down-regulated the endo-
statin-induced interaction between nucleolin and integrin
a5b1 in both co-immunoprecipitation assay (Fig. 5c) and
confocal fluorescence analysis (Supplementary Fig. 5c-d).
These results demonstrate that endostatin induces the for-
mation of the nucleolin/uPAR/integrin a5b1 complex.
Angiogenesis (2012) 15:697–711 705

Fig. 4 The interaction between

uPAR and nucleolin or integrin
a5. HMEC-1 cells were
transfected with a pCMV6
vectors encoding HA-tagged
wild-type integrin a5 (ITGA5wt)
and mutants (ITGA5ALTA and
ITAG5DNLTY), or c pCMV6
vectors encoding Myc-tagged
nucleolin mutants, respectively.
After incubation with 100 nM
endostatin, cells were lysed and
immunoprecipitated with
agarose-conjugated anti-HA-
MAb, anti-Myc-Mab, or anti-
uPAR-Mab to detect the binding
pattern between integrin a5/
uPAR (b), or nucleolin/uPAR
(d), respectively. The
immunoprecipitates were
analyzed by immunoblotting
using indicated antibodies

uPAR, though cannot directly interact with endostatin
(Supplementary Fig. 4d), modulates the association
between nucleolin and integrin a5b1.
Since both nucleolin and integrin a5b1 undergo endo-
cytosis via clathrin pathway to the early endosome [25, 36],
we thus detected the co-localization of these proteins with
early endosome by immunostaining. As shown in Supple-
mentary Fig. 6a, both nucleolin and integrin a5b1 were
found to colocalize with early endosome marker EEA1 and
Dylight-649-labeled endostatin. We then asked whether
the binding pattern of endostatin would be changed as a
result of acidification in early endosomes. To verify this
hypothesis, we employed the in vitro immunoprecipitation
assay in different pH ranges. As shown in Supplementary
Fig. 6b, the interaction between endostatin and nucleolin
was stable over a pH range of 5.5–7.4, while the interaction
between endostatin and integrin a5b1 was dramatically
decreased over the same pH range. Furthermore, based on
current knowledge, integrin a5b1 does not show any
nuclear distribution, while nucleolin functions as a nucle-
ocytoplasmic shuttle protein, suggesting that nucleolin,
rather than integrin a5b1, plays an essential role in the
nuclear translocation of endostatin. We therefore focused
on the transporting/shuttling function of nucleolin and the
translocation-associated proteins in the following study.
Importin a1b1 mediates the nuclear translocation
of endostatin
In our previous study, we have identified a series of binding
proteins for nucleolin by immunoprecipitation and liquid
chromatography mass spectrometry (unpublished data).
We have found that importin b1, a key member that
mediates nucleocytoplasmic transport [37, 38], was one of
the nucleolin-associated proteins. In addition, a nuclear
localization signal (NLS), the recognition site for impor-
tins, lies within nucleolin [39]. We thus hypothesized that
importins play a crucial role in the nuclear translocation
process.
Initially, the expression profile of importins was exam-
ined in HUVECs. As shown in Supplementary Fig. 7a, the
mRNAs of importin a1, a3, a5, a7, and b1 were detected.
Then we knocked down these expressed importins, and
investigated their impact on the nuclear translocation of
endostatin. The knockdown efficiencies were shown in
Supplementary Fig. 7b. Remarkably, siRNA that targets

123
706 Angiogenesis (2012) 15:697–711

Fig. 5 The interaction between

integrin a5 and nucleolin is
mediated by uPAR. a HUVECs
were incubated in the presence
of 100 nM endostatin at 37 °C
for 0 or 15 min before further
incubation (20 °C, 60 min) with
Dylight 649-labeled anti-
integrin a5 mAb, Dylight 549
anti-uPAR mAb, and Dylight
488 anti-nucleolin pAb (1:100),
respectively. After fixation in
3.7 % PFA, the digital images
were captured by the Nikon A1
fluorescence microscope using
63X/1.49 NA oil objectives.
Images were obtained with NIS-
Elements AR 3.0 software.
White frames indicate areas
magnified. Scale bars: 5 lm
(normal); 100 lm (Meg.).
b Reciprocal co-
immunoprecipitation study was
performed. HUVECs were
treated with indicated doses of
endostatin for 15 min, and then
lysed. Either nucleolin or
integrin a5 was
immunoprecipitated with mouse
monoclonal anti-nucleolin or
with rabbit polyclonal anti-
integrin a5, respectively. The
indicated proteins were detected
by immunoblotting. c HUVECs
cells pretreated with 20 lg/mL
neutralizing uPAR antibody or
uPAR-targeting siRNA were
incubated with 100 nM
endostatin at 37 °C for 30 min.
Reciprocal co-
immunoprecipitation was then
performed as described in b

either importin a1 or b1 dramatically inhibited the trans-
location of endostatin (Fig. 6a). Consistently, after the
knockdown of importin a1, endostatin was found pre-
dominantly in the cytoplasm rather than the nucleus
(Supplementary Fig. 7c).
Then we verified that this importin-dependent translo-
cation is indeed mediated by nucleolin. After endostatin
treatment, anti-importin a1 antibody could specifically co-
immunoprecipitate importin b1 and nucleolin (Fig. 6b).
Similarly, anti-nucleolin antibody could also effectively
co-immunoprecipitate both importin a1 and importin b1
(Fig. 6b). To further validate this result, we generated
vectors encoding either the wild-type nucleolin (mNCLwt)
or an NLS-deficient mutant (mNCLmut), as shown in
Supplementary Fig. 8. Immunoprecipitation assay showed
that only mNCLwt could interact with importin a1 and

123
Angiogenesis (2012) 15:697–711
Fig. 6 Importin a1b1 regulates the nuclear translocation of endo-
statin. a HUVECs were transfected twice with indicated siRNAs, and
then treated with 100 nM endostatin for 30 min. The amount of
endostatin in both cytosol and nuclear fractions as well as the whole
cell lysate (WCL) was detected. IMPA, importin a; IMPB, importin
b. b Reciprocal co-immunoprecipitation study was performed.
HUVECs were treated with indicated doses of endostatin, and then
lysed. Nucleolin or importin a1 was immunoprecipitated with
anti-nucleolin or anti-importin a1 mAb, respectively. c HUVECs
importin b1 (Fig. 6c), demonstrating a specific interaction
between nucleolin and importins.
We then examined the role of the importin/nucleolin
complex in the nuclear translocation of endostatin. To
avoid the interference of endogenous nucleolin, HMEC-1
was co-transfected with siRNA pool targeting human nu-
cleolin and the vector encoding wild-type or mutant mouse
nucleolin. As shown in Fig. 6d, compared to the mNCLwt
group, less endostatin could translocate to the nucleus in
the mNCLmut group. In conclusion, these results demon-
strate that the importin a1b1/nucleolin complex is involved
in the nuclear translocation of endostatin.
Discussion
We have shown here that the nuclear translocation of
endostatin by endothelial cells is facilitated through the cell
surface nucleolin/uPAR/integrin a5b1 complex and the
association of importin a1b1. We therefore propose a
working model based on our present results as shown in
Fig. 7: upon the binding of endostatin, nucleolin, integrin
a5b1 and uPAR form a co-receptor complex to mediate the
707
transfected with mock vector (mNCLwt or mNCLmut) were treated
with or without 100 nM endostatin for 30 min, and then lysed. Co-
immunoprecipitated importin a1 and b1 with anti-nucleolin mAb was
detected. The indicated proteins were detected by immunoblotting.
d HUVECs co-transfected with mNCLwt or mNCLmut were further
transfected with scramble or hNCL siRNA pool, respectively. After
treatment with 100 nM endostatin for 30 min, HUVECs were
fractionated and analyzed
transportation of endostatin from membrane to the early
endosome in a clathrin-dependent manner. Thereafter,
integrin a5b1 and uPAR are recycled back to the cell
surface or degraded, while nucleolin, still binding with
endostatin, undergoes nuclear translocation. Via the NLS
of nucleolin, importin a1b1 is responsible for the recog-
nition and nuclear transport of the nucleolin-endostatin
complex.
Endostatin signaling
The mechanism of actions of endostatin has been exten-
sively investigated over the last dozen years, while contro-
versial reports still remain. One mystery is that why an
N-terminal modified E. coli-expressed endostatin show
good clinical responses in China, whereas the clinical trials
of the P. pastoris-expressed endostatin were terminated at
phase II in the USA [40]. The key reason is that both the
N-terminal integrity and correct refolding are very critical
for the biological functions of endostatin [41]. It turns out
that the N-terminal of endostatin expressed by P. pastoris, is
truncated, thus loses its zinc binding capacity with much
reduced stability and biological activities [41]. Furthermore,
123
708
Fig. 7 Model for the
internalization and nuclear
translocation of endostatin in
endothelial cells. Upon the
binding of endostatin on the
surface of endothelial cells,
nucleolin, integrin a5b1 and
uPAR form a receptor complex,
and mediate the clathrin-
dependent internalization of
endostatin. Once endocytosed,
integrin a5b1 and uPAR are
then recycled back to the cell
surface or degraded, while
nucleolin, association with the
adaptor importin a1b1,
transports endostatin to the
nucleus
in a parallel study performed by the Folkman laboratory,
E. coli-expressed endostatin is at least twice as potent as
P. pastoris-expressed endostatin in animal tumor models
(personal communication). Therefore, we used E. coli-
expressed endostatin, which has been proved to be an intact
molecule with full activity after correct refolding.
Association of endostatin inhibits the proliferation and
migration of endothelial cells due to the negative impact on
several well-known pathways [2, 42]. For instance, endo-
statin induces cell cycle arrest by down-regulating cyclin
D1, MAPK and c-Myc [42, 43]. Endostatin also interferes
with the anti-apoptotic signal induced via PI3K/Akt [15].
These down-regulation effects were probably mediated by
E-selectin that confers endostatin sensitivity to non-
responsive endothelial cells [44]. An intriguing discovery
is that endostatin is exclusively internalized by endothelial
cells [25], suggesting that cell-surface binding is not the
only mechanism by which endostatin could influence the
angiogenic activity. It is worth noting that endostatin is a
Zn(II)-binding protein [45, 46]. A 27-amino-acid synthetic
peptide corresponding to the Zn(II)-binding domain of
endostatin is responsible for its anti-angiogenic activity
[47], also suggesting that the Zn(II)-binding capacity is
indispensable for its function. Moreover, titration analysis
has shown that the Zn(II) dissociation constant of endo-
statin is about 9 nM, indicating a very strong Zn(II)-bind-
ing capacity [45]. Thus, the Zn(II) homeostasis in the
nucleus may be significantly disturbed by the translocated
endostatin. Considering that Zn(II) participates in inter-
molecular interactions of various transcription factors and
regulators [48], it is plausible that the competition of Zn(II)
by the nuclear-translocated endostatin could result in the
transcription inactivation. This hypothesis may explain that
how endostatin could regulate 12 % of the gene expression
in endothelial cells.
123
Angiogenesis (2012) 15:697–711
Nucleolin: an ‘‘all the way’’ vehicle for nuclear
translocation of endostatin
Surface nucleolin, undergoes heavy N-glycosylation, is a
multifunctional protein [31, 49]. It interacts with many
surface proteins including uPAR, P-selectin, and ErbB
proteins, and exists in a 500 kDa complex implicated in
tumorigenesis, inflammation, and bacterial infections [50].
Intriguingly, Dumler et al. have demonstrated that surface
nucleolin co-localizes with uPAR on the cell surface and is
rapidly internalized [51]. Cines and his colleagues have
also mentioned that nucleolin, interacting with uPA/uPAR
complex, regulates the nuclear translocation of scuPA [27].
Together with the knowledge that nucleolin can function as
a shuttle protein to internalize various substrates including
virus, scuPA, and even DNA aptamers [31, 32, 52], we also
propose that the surface nucleolin is a multifunctional
regulator. It could fulfill different requirements of cell
actions by changing its binding partners. It is of note that
nucleolin could interact with matrix laminin-1 [53], which
is also an endostatin associated protein [19]. Thus it is
plausible that these three proteins may also form a complex
that is involved in the regulation of the uptake of endo-
statin. Regarding the interaction between nucleolin and
endostatin with nucleus, we did not observe the positive
result (data not shown). This phenomenon may attribute to
the competition of other nucleolin binding factors includ-
ing the pre-rRNA substrate [54]. Moreover, Shi et al. has
also demonstrated that endostatin could modulate the
phosphorylation status of nucleolin within nucleus. Both
the ligand-binding and the phosphorylation may trigger the
conformational change of nucleolin, which further interfere
with its interaction of endostatin.
In addition, the expression of nucleolin on the cell sur-
face is also an intriguing phenomenon. To elucidate the
Angiogenesis (2012) 15:697–711
reason why the cell surface translocation of nucleolin is
specific for activated but not quiescent endothelial cells, we
investigated the relationship between the phosphorylation
state of nucleolin and its subcellular localization. In a
separate study in our group, Hsc70 is involved in the
membrane translocation of nucleolin (Ding et al., submit-
ted). Together with the current study, these findings will
help us understand the whole regulatory mechanism of
nucleolin on the anti-angiogenic effects of endostatin.
uPAR and integrin a5b1 in the nuclear translocation
With regard to the role of uPAR in endocytosis, Stepano-
va’s work has shown that uPAR serves as a scaffold protein
for the uPA/uPAR/nucleolin complex, and also triggers the
endocytosis by the classical mechanism. In our model,
uPAR serves as a similar scaffolding factor, because after
the knock-down of uPAR, there is a significant decrease in
the interaction between integrin a5b1 and nucleolin. Con-
sidering the engagement of uPAR in lateral interactions
with integrin a5b1 and nucleolin on the cell surface, we
have proposed a novel working model that the internali-
zation of endostatin is mediated by integrin a5b1/uPAR/
nucleolin complex. This model also explains the effect of
endostatin on the plasminogen activation. Endostatin
treatment triggers the endocytosis of integrin a5b1/uPAR/
nucleolin complex, thus removes uPAR from focal adhe-
sions, and causes a rapid disassembly of focal adhesions,
finally disrupts the uPA/uPAR complex formation. In
addition, LDL receptor-related protein-1 (LRP-1) has been
reported to facilitate the nuclear translocation of several
factors including uPA and midkine [55], both of which are
nucleolin-associated ligands. Thus it is conceivable that
LRP-1 is also involved in the translocation of endostatin. In
the current study, we utilized both neutralizing antibody
and siRNA to block the cell surface LRP-1, and indeed
found the decreased uptake of endostatin (data not shown),
suggesting the nucleolin/uPAR/LRP-1 may modulate a
series of nuclear translocation processes. Considering that
surface nucleolin is normally observed in the proliferative
cells including tumor cells, the nucleolin/uPAR/LRP-1
might function as a regulator for the cell status transition.
Integrin a5b1 has been well documented as one key
mediator for the anti-angiogenic function of endostatin [13,
15, 16, 56]. Several biochemistry studies also support the
physical interaction between integrin a5b1 and endostatin.
Kalluri and his colleagues have reported that endostatin
competes for fibronectin binding to integrin a5b1, and
inhibits sustained phosphorylation of Raf and MEK1/2
[15]. Aside from the competition for the ECM binding, we
have previously mentioned that integrin a5b1-targeting
siRNA could interfere with the internalization of endo-
statin, indicating the crucial role of integrin a5b1 in the
709
endocytosis process. Since the endocytosis of both integrin
and endostatin is clathrin-dependent, it is plausible that
integrin could mediate the internalization of endostatin.
Furthermore, it has been reported that clathrin-dependent
endocytosis of integrin a5b1 induces focal adhesion dis-
assembly. Consistently, endostatin treatment could also
trigger the disassembly of focal adhesions and actin stress.
These observations also provide a convincing evident to
support the working model we proposed.
In summary, this study identifies a novel pathway by
which endostatin translocates to the nucleus in collabora-
tion with nucleolin. This discovery establishes a new
platform for understanding the detailed function of endo-
statin, and sheds light on the molecular mechanisms by
which endostatin, a single endogenous protein, modulates
the orchestration of gene expression in endothelial cells.
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 Sci-
ence and Technology Major Project (No. 2009ZX09103-703 and
No. 2009ZX09306-002), the General Programs of the National Nat-
ural 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.
Conflict of interest The authors declare no competing financial
interests.
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