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Blood First Edition Paper, prepublished online January 10, 2006; DOI 10.1182/blood-2005-07-2961
The angiogenesis function of nucleolin is mediated by vascular
endothelial growth factor and nonmuscle myosin
Short title: Function of nucleolin in angiogenesis
Yujie Huang, Hubing Shi, Hao Zhou, Xiaomin Song, Shaopeng Yuan, &
Yongzhang Luo*
Laboratory of Protein Chemistry, Department of Biological Sciences and
Biotechnology, Tsinghua University, Beijing 100084, P. R. China
Specific contribution of all authors:
YH performed research and wrote the paper; HS and HZ performed research and
analyzed data; XS and SY contributed vital analytical tools; YL designed research and
wrote the paper.
This study was supported by grants from the Major Program of National Science
Foundation of China (No. 30291000), the National Science Fund for
Distinguished Young Scholars in China (No. 30225014), and the National 863
Program in China (No. 2004AA2Z3802).
All authors declare no conflicting financial interest.
*Correspondence Author: Yongzhang Luo, Ph.D.
Laboratory of Protein Chemistry, Department of Biological Sciences and
Biotechnology, Tsinghua University, Beijing 100084, P. R. China Tel:
86-10-6277-2897, Fax: 86-10-6279-4691, E-mail: [email protected]
Word count: Abstract: 167
Text length: 3743
Appropriate scientific heading: Homeostasis, Thrombosis, and Vascular Biology
1
Copyright © 2006 American Society of Hematology
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ABSTRACT
Nucleolin, originally described as a nuclear protein, was recently found to be
expressed on the surface of endothelial cells during angiogenesis. However, the
functions of cell surface nucleolin in angiogenesis remain mysterious. Here we report
that upon endothelial cells adhering to extracellular matrix components, vascular
endothelial growth factor (VEGF) mobilizes nucleolin from nucleus to cell surface.
Functional blockage or down-regulation of the expression of cell surface nucleolin in
endothelial cells significantly inhibits the migration of endothelial cells and prevents
capillary-tubule formation. Moreover, nonmuscle myosin heavy chain 9 (MyH9), an
actin-based motor protein, is identified as a nucleolin-binding protein. Subsequent
studies reveal that MyH9 serves as a physical linker between nucleolin and
cytoskeleton thus modulating the translocation of nucleolin. Knocking down
endogenous MyH9, or specifically inhibiting myosin activity, or over-expressing
functional deficient MyH9 disrupts the organization of cell surface nucleolin and
inhibits its angiogenesis function. These studies indicate that VEGF, extracellular
matrix, and intracellular motor protein MyH9 are all essential for the novel function
of nucleolin in angiogenesis.
2
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INTRODUCTION
Angiogenesis,
the
generation
of
new
capillaries
from
preexisting
microvasculature, occurs in a variety of pathological processes, such as tumor growth
and metastasis and various inflammatory disorders 1, 2. Angiogenic vessels differ from
normal vessels in their morphological and molecular characteristics. Some unique
molecules have been identified specifically expressing on angiogenic vessels,
including some types of endothelial growth factor receptors, integrins, proteolytic
enzymes and extracellular matrix, as well as membrane proteins of unknown
functions
3, 4
. Recently, cell surface nucleolin was reported as a specific marker of
angiogenic endothelial cells 5.
Nucleolin has been described as a nucleolar protein of eukaryotic cells, involved
in regulation of cell proliferation, cytokinesis, replication, embryogenesis, and
nucleogenesis
6, 7
. Recently, a number of studies have shown that it can also be
expressed on cell surface and serve as a receptor for several ligands such as matrix
laminin-1, midkine, and lactoferrin
8-13
. However, little information about the
physiological relevance of cell surface nucleolin in angiogenesis is known except that
cell surface nucleolin specifically appears in tumor-induced angiogenic vessels rather
than mature vessels or capillaries 5. Here we report that the cell surface nucleolin is
essential for the migration and tubule formation of endothelial cells. During the
process of angiogenesis, the up-regulation of cell surface nucleolin attributes to the
shuttle of nucleolin from nucleus to cell membrane. VEGF, a core regulator in tumor
angiogenesis and stroma generation
14, 15
, can significantly stimulate the translocation
of nucleolin when endothelial cells adhere to extracellular matrix. Cell surface
3
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nucleolin was reported to associate with actin cytoskeleton 9. In this study, we found
that nonmuscle myosin heavy chain 9 (MyH9), an actin-based motor protein, provides
a linker between cell surface nucleolin and actin cytoskeleton and mediates its
function in angiogenesis. MyH9 is one isoform of class II myosin family which
mediates a variety of cellular processes, including protrusion, migration, and
modulation of cell locomotion
19, 20
. Recent studies showed that MyH9 plays an
important role in modulating T cell motility and tissue organization during embryo
development
21, 22
. The shuttle of nucleolin between nucleus and cell surface depends
on anchoring to MyH9, which appears essential for the angiogenesis function of cell
surface nucleolin.
4
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MATERIALS AND METHODS
Antibodies, proteins, and chemicals
Polyclonal antibodies to nucleolin were from our lab storage. Plasma fibronectin was
purified from human blood by gelatin affinity column. Other antibodies, proteins, and
chemical were from Sigma-Aldrich (polyclonal antibodies of MyH9, anti-vinculin
mAb, ML7), Santa Cruz biotechnology, Inc. (TRITC, FITC-2nd antibodies to Ra, Mo
IgG, FITC-Anti-CD31), Dako (HRP-2nd antibodies to Ra, Mo IgG), Protgen, Inc.
(VEGF-165), respectively.
Identification of isolated proteins with MALDI-TOF mass spectrometry
The major band from co-immunoprecipitation was digested using sequencing grade
porcine-modified trypsin (Promega). The peptides produced were analyzed by
MALDI-TOF mass spectrometry using a Bruker Biflex linear time-of-flight
spectrometer (Bruker Franzen), equipped with a multiprobe SCOUT source,
ultra-nitrogen laser (337 nm), and a dual microchannel-plate detector. The
MALDI-TOF data was searched against Swiss-Prot protein data base for protein
identification.
Cell migration assay
Endothelial cells (HMECs or HUVECs, 2×104 cells per well) were seeded into the
upper chamber of collagen pre-coated transwell filter (8 µm pores, Costar) with
DMEM medium containing 10% FCS. The same culture medium and reagents were
added to the lower chamber. The endothelial cells were allowed to migrate for 4 h at
37 °C and 5% CO2. After fixed and stained with ethanol and Eosin, the cells migrated
5
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completely through the filter to the lower chamber were counted in three different
areas under the optical microscope, and the averaged number was obtained.
Tubule formation assay
The tubule formation assay was carried out as previously described
23, 24
. In brief,
twenty-four–well plates were coated with 100μl/well of Matrigel (Becton Dickinson
Labware, Bedford, MA). Endothelial cells on this matrix migrated and formed tubules
within 6 h of plating. ECV304 cells were seeded at 5×104 cells per well and incubated
for 6 h in complete medium with 10 ng/ml VEGF. Wells were then fixed and
visualized (at 100 x magnification), and tubule formation was assessed in 3 randomly
selected fields of view per well using the image analysis package, Scion Image (Scion,
Frederick, MA).
Cell invasion assay
ECV304 cells, transfected with MyH9ΔN-GFP or GFP, were seeded on 8 µm inserts
(costar) coated with Matrigel, using 5 ng/ml VEGF in 1% FBS as chemoattractant.
After a 24 h incubation at 37ºC, the invasive cells were fixed and observed and
quantitated under fluorescence microscope at 488/530 nm.
RNA interference
The pBS-U6 vector was used to express a short small interfering (siRNA) sequence
specific for MyH9 gene or nucleolin gene, and the nonsilencing pBS-U6 vector was
used as a control. The constructed plasmids were transfected into cells by lipofectin
(invitrogen). In GFP co-transfection, the GFP expression vector was combined with
the MyH9 RNAi vector at a ratio of 10:1. At 48 h after transfection, the inhibitory
6
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effect was detected by immunoblotting of MyH9.
Immunofluorescence and confocal microscopy
The cultured cells were fixed by 4% paraformaldehyde and then permeabilized with
PBS containing 0.2% Triton X-100. After washed twice with PBS, the cells were
blocked with PBS containing 10% normal goat serum. After treatment, the cells were
stained with different primary antibodies and FITC- or TRITC-conjugated secondary
antibodies. Actin filaments were directly stained by FITC-phalloidin (CHEMICON),
and nuclei were stained by DAPI. Confocal fluorescence imaging was performed on
an Olympus Fluoview laser scanning confocal imaging system (Olympus Inc.).
Images were captured using multiple photomultiplier tubes regulated by Fluoview 2.0
software (Olympus).
Fluorescence activated cell-sorting analysis
Cells were briefly trypsinized and resuspended in PBS supplemented with 0.1%
bovine serum albumin. Cells were incubated with anti-nucleolin polyclonal antibodies
for 30 min on ice followed by 30 min incubation on ice with anti-rabbit secondary
antibody conjugated to FITC. After the final wash, cells were directly analyzed using
a FACScan (Becton Dickinson, San Jose, CA).
7
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RESULTS
Distribution of nucleolin in vivo
The presence of cell surface nucleolin on angiogenic vessels, described by
Christian et al.5, led us to further assess systematically the distribution of cell surface
nucleolin in different tissues. Since the majority of nucleolin is located in nucleus in
all eukaryotic cells, in order to detect cell surface nucleolin, we injected anti-nucleolin
polyclonal antibodies (Ab) in to the tail vein of tumor-bearing or matrigel-plus mice.
Three h after injection, mice were sacrificed and their organs, heart, liver, kidney,
spleen, lung, matrigel plug, and tumor were removed for immunohistochemical
analysis. As shown in Figure 1A, the normal organs, heart, liver, kidney, spleen, and
lung bearing mature vessels showed negative staining for anti-nucleolin antibodies.
However, in the tumor or matrigel induced angiogenic vessels, anti-nucleolin Ab was
positive stained and colocalized with neo-vessels, which indicates that the cell surface
nucleolin selectively appears on tumor induced anigogenic vessels or other
inflammatory tissues but not mature vessels of normal organs. In order to exclude the
accumulation of antibody for nonspecific effects, Isotype Ab was employed as a
control (Figure 1B). Based on these observations, we propose that there must have
some factors stimulating the translocation of nucleolin to cell surface, and it is the cell
surface nucleolin which functionally contributes to angiogenesis.
Angiogenesis function of nucleolin
To elucidate the functions of cell surface nucleolin in angiogenesis, polyclonal
antibodies (pAb) against nucleolin were applied to neutralize cell surface nucleolin of
8
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endothelial cells in vitro. The binding specificity of the pAb was shown in Figure 2A.
Cell migration was measured by the transwell migration assay. It was observed that
the migration of human microvascular endothelial cells (HMECs) was inhibited when
cell surface nucleolin was functionally blocked. As shown in (Figure 2B), the
nucleolin Ab have significant inhibitory effect on endothelial cell’s migration, which
implies that cell surface nucleolin regulates cell-matrix interaction and cell motility.
Anti-CD71, transferin receptor on endothelial cell surface, was employed as a control.
However, anti-nucleolin Ab had only marginal effect on cell adhesion and cell
proliferation (Figure 2C and 2D). Apparent inhibition of cell migration caused by the
treatment of anti-nucleolin pAb may result in suppression of the network of
endothelial cell formation. Human umbilic vein endothelial cell line-ECV304 was
seeded on matrigel to assess its ability of forming tubule network. In consistent with
our expectation, soluble anti-nucleolin pAb significantly inhibits tubule formation
(see Figure 2G below). To further confirm this conclusion, RNA interference was
subjected to suppress the expression level of nucleolin in endothelial cells, which also
resulted in the reduction of the amount of cell surface nucleolin (Figure 2E). It was
observed that if nucleolin was down-regulated, cell migration and tubule formation
were apparently inhibited (Figure 2F and 2G).
Translocation of nucleolin is mediated by VEGF and extracellular matrix
Nucleolin is normally located in nucleolus or nucleus; however, during the
process of angiogenesis, it mysteriously appears on cell membrane of endothelial cells.
However, how nucleolin expresses on cell membrane remains unclear. We found that
9
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VEGF and extracellular matrix synergistically mediate the mobilization of nucleolin
from nucleus to cell surface.
We analyzed the quantitative change of cell surface nucleolin under stimulation
of VEGF. HMECs, cultured on poly-L-lysine (PLL) or fibronectin, were stimulated
by VEGF. After 12 h, cells were harvested and their surface-expressed nucleolin was
quantified by FACS. It was observed that under the stimulation of VEGF, the amount
of nucleolin on membrane of HMECs, cultured on fibronectin, significantly increased;
however, when cultured on poly-lysine, the amount of cell surface nucleolin was only
marginally elevated (Figure 3A). To further confirm the results, HMECs, grown on
fibronectin coated plates, were treated by VEGF (10 ng/ml) for different times: 0 h, 1
h, 2 h, 6 h, and 6 h then re-starved for 2 h. The 5 groups of HMECs were biotinlayted,
and their membrane proteins were completely labeled by biotins. Then, HMECs were
lysed and membrane proteins were pulled down by strepavidion conjugated agarose.
The fractions of cell lysate and membrane proteins were subjected to immnoblotting
by anti-nucleolin Ab. During the treatment by VEGF, the quantity of nucleolin
expressed on cell membrane increased gradually, and without stimulation of VEGF,
the cell surface nucleolin quickly decreased (Figure 3B). The quantitative change of
cell surface nucleolin under stimulation of VEGF was also confirmed by
immunofluorescence (Figure 3C). In this process, the quantity of total nucleolin did
not change, which suggests that the cell surface nucleolin come from the nucleolin
originally located in nucleus, and VEGF does not enhance the expression level of
nucleolin but mobilizes it from nucleus to cell surface. In order to prove that the
10
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streptavidin sharply separates biotinylated from non-biotinylated proteins, and to
exclude the traces of nucleolar proteins, we tested typical cell membrane protein of
CD31, cytoplasmic proteinof Cytochrome C, and nuclear protein of Histone-H1, in
both streptavidin pull-down fraction and supernatant. It was observed that only cell
membrane protein of CD31 could be pull-down by streptavidin. To capture the
process of nucleolin mobilization, the cells were trypsinized from plates, and then
replated on fibronectin or poly-L-lysine coated coverslips. At different time intervals,
0 h, 6 h, or 12 h, the cells were fixed and permeabilized to analyze the distribution of
nucleolin. In HMECs grown on fibronectin, nucleolin translocated gradually from
nucleus into cytoplasm and cell surface under stimulation of VEGF, while most of
nucleolin remained in nucleolus in HMECs grown on PLL (Figure 3D).
During the mobilization of nucleolin, stress fibers and focal adhesion complex
formed simultaneously (Figure 3E). Other matrix proteins such as collagen and
laminin also exhibit similar effect as fibronectin (data not shown), which indicates
that the mobilization of nucleolin is not only regulated by VEGF signaling but is also
correlated with reorganization of cytoskeleton modulated by ECM.
MyH9 provides an anchorage for nucleolin
To
further
investigate
co-immunoprecipitation
(co-IP)
how
was
nucleolin
applied
to
modulates
search
cell
for
the
motility,
potential
nucleolin-binding proteins. Finally, a 220 kD protein was obtained from HMECs as a
nucleolin-binding
protein
(Figure
4A).
Matrix
assisted
laser
desorption
ionization-time of flight (MALDI-TOF) identified it as nonmuscle myosin heavy
11
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chain 9 (MyH9), a motor protein expressed in endothelial cells. The identification was
further confirmed by immunoblotting (Figure 4B.). There may be some nucleolin
from nucleus appearing in the whole cell lysate. To exclude the possibility that MyH9
also binds to nucleolin from nucleus, we extracted nucleus from cells, then used both
the nuclear fraction and extra-nuclear fraction to perform co-immunoprecipitation. We
found that MyH9 can bind to nucleolin from cell surface and cytoplasm but not from
nucleus (Figure 4B). Interestingly, the interaction between nucleolin and MyH9 in the
cells grown on fibronectin is more pronounced than grown on PLL (Figure 4A and
4B), suggesting an essential role of matrix proteins in mediating the nucleolin-MyH9
interaction. To explain this phenomenon, the distribution of MyH9 in HMECs grown
on fibronectin was investigated. MyH9 was observed as stress fibers, along the
plasma membrane, and in membrane ruffles which co-localizes with actin filaments.
In contrast, MyH9 in the cells grown on PLL did not exhibit any stress fibers but
formed centrally located punctuate filaments (Figure 4C). In order to reveal the
relationship between MyH9-nucleolin interactions and angiogenesis, the distributions
of both nucleolin and MyH9 were studied under different conditions. ECV304 cells
were cultured for 12 h on matrigel supplemented with 10 ng/ml VEGF or PLL without
any supplement. Then nucleolin and MyH9 were stained by immnuofluorescence.
ECV304 grown on matrigel containing VEGF, spread their cell shape and formed
network or tubule structure; moreover, nucleolin colocalizes with MyH9 during tubule
formation (Figure 4D). However, ECV304 grown in PLL, nucleolin stays only in
nucleus and MyH9 stays in cytoplasm, respectively (Figure 4E). Based on these
12
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observations, we propose that the matrix-induced conformational change of MyH9
facilitates the MyH9-nucleolin interaction, which contributes to the movement of
nucleolin and subsequent angiogenesis. Since the structure of Myosin II consists of a
globular head that can bind to actin filament and has ATPase activity, a neck domain
that binds to myosin light chains or calmodulin, and a unique c-terminal tail domain
of variable length that can either promote multimerization or bind to different cellular
proteins
19-20
, we propose that the c-terminal of MyH9 is the potential
nucleolin-binding domain. To verify this hypothesis, we fused the c-terminal coil-coil
domain of MyH9 with GFP (MyH9ΔN-GFP) and transfected it into HMECs; Then
co-IP was performed to detect whether there is any interaction between the c-terminal
of MyH9 and nucleolin. As shown in Figure 4F, the MyH9ΔN-GFP was pulled
down by anti-nucleolin Ab, which suggests that the c-terminal of MyH9 is indeed the
binding site for nucleolin. Moreover, MyH9ΔN provides a useful framework to
investigate functional contribution of the interaction between MyH9 and nucleolin
due to the lack of the N-terminal motor domain of MyH9.
MyH9 modulates the angiogenesis function of nucleolin
Different strategies were performed in the current study to elucidate the potential
biological relevance of the association between MyH9 and cell surface nucleolin: 1)
knocking down endogenous expression of MyH9 by RNAi (efficiency was shown in
Figure 5A); 2) inhibiting the activity of MyH9 by ML-7, a specific myosin light
chain kinase inhibitor; 3) over-expressing nucleolin-binding domain of MyH9 (MyH9
ΔN), which is deficient in motor function in endothelial cells.
13
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HMECs were co-transfected with MyH9–RNAi and GFP vectors, and then the
cell surface nucleolin was stained. We observed that the organization of nucleolin was
disrupted and aggregated on cell surface in those GFP positive cells (Figure 5B), and
similar phenomenon was also observed in the ML7 treated cells (Figure 5C).
Moreover, the amount of cell surface nucleolin had 10-20% reduction when MyH9
was suppressed (Figure 5D). We propose that MyH9 is critical for the organization of
nucleolin on cell surface and may also play a potential role in regulating the recycling
of cell surface nucleolin. The following migration assay showed that suppressing
MyH9 with RNAi and ML7 significantly inhibited the migration of HMECs (Figure
5E, and 5F). Based on these observations, we propose that the shuttling trait of
nucleolin, translocating between cell surface and nucleus, is critical for angiogenesis;
furthermore, MyH9 not only provides an anchorage but also mediates the shuttle of
nucleolin.
Retrospect that MyH9ΔN is the binding site for nucleolin (see Figure 4F above)
but lacks of the N-terminal motor domain, we next over-expressed MyH9ΔN in
endothelial cells to form functional deficient complex with endogenous nucleolin.
Then, its effect on angiogenesis was evaluated. The over-expressed MyH9ΔN
significantly suppressed the migration of HMECs (Figure 5G) and invasive ability of
those cells was totally impaired (Figure 5H). In another endothelial cell line –
ECV304, the tubule formation assay showed that similar with blockage of cell surface
nucleolin, over-expression of MyH9ΔN or suppressing MyH9 led ECV304 unable to
form network structure (Figure 5I).
14
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DISSCUSION
Nucleolin was originally described as a nucleolar protein involved in regulation of
ribosome biogenesis cell proliferation and growth, cytokinesis, replication,
embryogenesis, and nucleogenesis
6, 7
. Recently, a number of studies showed that
under some circumstance, nucleolin can also express on cell surface
8-10
. The cell
surface nucleolin has been described as a receptor for several ligands such as
laminin-1, midkine, and lactoferrin 11-13. Ruoslahti and his colleagues reported that the
cell surface nucleolin serves as a marker of endothelial cells in angiogenic blood
vessels5. However, unique roles of nucleolin in angiogenesis and what mobilizes
nucleolin, originally located in nucleus, to cell surface remain mysterious. Our studies
reveal that without cell surface nucleolin, endothelial cells partly lost their motility
and can not form tubule structure. In the adult, angiogenesis occurs in regenerating
tissue and inflammatory conditions, which activates the expression of some unique
molecules in endothelial cells 4. For instance, the levels of tyrosine kinase receptors
for VEGF in endothelial cells elevate during tumorigenesis, integrin αvβ3 and integrin
α5β1 are selectively expressed in angiogenic vasculature
25-27
. These marker proteins
are functionally important in the angiogenic process. Previous studies showed that
surface nucleolin could interact with laminin and internalize midkine. More recently,
our group found that cell surface nucleolin binds directly to collagen components (To
be published). The N-terminus of NL, composed of highly acidic stretches 7, is the
proposed binding site for ligands rich in basic amino acids, such as midkine, bFGF,
and heparan-binding domain of matrix proteins. Based on those studies and our
15
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results, we propose that the cell surface nucleolin serves as an adhesion molecule
modulating cell-matrix interaction, and internalizes potential stimulators of
endothelial cells, as well as triggers their downstream signaling during angiogenesis.
Normally, nucleolin totally stays at nucleus in endothelial cells at quiescent state
(Figure 4E) 5, 9. However, during tumorigenesis or other inflammatory conditions, the
conditional expression of cell surface nucleolin can be observed in angiogenic vessels.
As far as known, during the process of tumorigenesis, VEGF, a core regulator in
tumor angiogenesis and stroma generation
14, 15
, is released to increase endothelial
cells motility, and vascular permeability, thus to promote angiogenesis
16, 17
. In
sprouting vasculature, extravagated plasma proteins from permeable vasculature
construct extravascular matrix surrounding endothelial cells
18
. This circumstance is
consistent with our observation: VEGF significantly stimulates the translocation of
nucleolin in endothelial cells synergistically with extracellular matrix attachment, and
therefore nucleolin specifically appears on cell surface during angiogenesis. Under
stimulation of VEGF, a series of cellular events occur, including activation of several
kinases 28, and extracellular matrix induces cells to remodel their actin cytoskeletons29.
Since the cellular localization of nucleolin correlates with the phosphorylation of its
N-terminus and the organization of the cytoskeleton
9
, we propose that the
phosphorylation of nucleolin associated with the formation of myosin stress filaments
results in subsequent mobilization of nucleolin.
In this work, a proteomic approach led us to identify MyH9, the carboxyl domain
of which specifically binds to nucleolin and serves as an adaptor between nucleolin
16
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and actin cytoskeleton, in consistent with previous study on the association between
cell surface nucleolin and actin9. Our studies demonstrate that MyH9 provides an
anchorage to cell surface nucleolin, which is critical for the angiogenesis function of
nucleolin. Subsequent investigation on the relationship between nucleolin and MyH9
provides a reasonable explanation on how cell surface nucleolin carries out functions
as an adhesion molecule. Nonmuscle myosin is responsible for the formation of
intracellular contractility and tension by interacting with actin cytoskeleton
30
. The
fact that the interaction between a motor protein and nucleolin occurs in endothelial
cells at angiogenic conditions suggests that this phenomenon have an important
functional role in cell migration. Anchoring to actin filaments via MyH9, through this
unique connection, cell surface nucleolin can transmit information about the physical
state of the ECM into cells and altering cytoskeleton dynamics. Actually, the accurate
state and structure of nucleolin in cell surface is still unclear. We found that the
carboxyl terminal of surface-exposed nucleolin is located intracellularlly and the
N-terminus exposes extracellularlly (data not shown), which might indicate that
nucleolin is a transmembrane protein; meanwhile, intracellular nucleolin translocates
to cell surface dependent on the interaction with cytoplasmic MyH9. Recently, the
similar mechanism was found in integrins. Stromblad and his colleagues reported that
integrins facilitate cellular remodeling and filopodia elongation depending on myosin
10, which anchors to the β subunit of integrins 31. Our studies provide evidence for
the connection between angiogenesis function of nucleolin and the contractile forces
generated by the cell myosin system. In addition, the interaction between them is
17
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apparently regulated by surrounding environments of endothelial cells. In conclusion,
cell surface nucleolin, associated with extracellular growth factors and intracellular
motor system, plays a critical role in the modulation of angiogenesis.
ACKNOWLEDGEMENTS
We thank Ying Li for critical reading of the manuscript and helpful advice, Yang Shi
for the generously gift of pBS-U6 plasmid, and our group members for helpful
discussion.
18
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traffic, and signal transduction. Science. 1998; 279:527-533.
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Jacobelli J, Chmura SA, Buxton DB, Davis MM, Krummel MF. A single class II myosin
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5:531-538.
22. Conti MA, Even-Ram S, Liu C, Yamada KM, Adelstein RS. Defects in cell adhesion and the
visceral endoderm following ablation of nonmuscle myosin heavy chain II-A in mice. J. Biol.
Chem. 2004; 279:41263-4126.
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Staton CA, Brown NJ, Rodgers GR, Corke KP, Tazzyman S, Underwood JC, Lewis CE.
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Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nat Med. 2003;
9:669-676.
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FIGURE LEGENDS
Figure 1.
The distribution of cell surface nucleolin in different tissues
(A), purified anti-nucleolin Ab was injected intravenously to mice bearing S180
exnograft tumors or matrigel plugs. Various normal organs: heart, kidney, liver, lung,
spleen with tumors and matrigel plugs were removed 6 h after injection for
immunohistochemical
assay.
Cell
surface
nucleolin
was
visualized
by
TRITC-secondary antibody against primary injected antibodies (red). The endothelial
cells of blood vessels were stained with FITC-Anti-CD31 (green), and nuclei were
counterstained with DAPI (blue). Anti-nucleolin Ab specifically accumulated at
angiogenic vessels of tumors or matrigel plugs but not appeared in normal organs. (B),
nonspecific isotype Ab was injected as a control. Bar, 50 μm.
Figure 2.
Nucleolin mediates cell migration and tubule formation
The binding specificity of employed anti-nucleolin Ab was shown in (A). Indicated
concentrations of Ab were incubated with HMECs to neutralize cell surface nucleolin
and the assay of cell adhesion (B), cell migration (C), and cell proliferation (D) were
performed. Purified rabbit IgG served as a positive control. Neutralizing cell surface
nucleolin significantly inhibited cell migration but only marginally affected cell
adhesion and unaffected cell proliferation. Anti-CD71 was employed in cell
migrantion assay as a control. (E), the efficiency of RNAi was detected by
immunoblotting, and the reduction of cell surface nucleolin was quantified by FACS.
(F), knocking down the endogenous nucleolin in HMECs led to apparent reduction of
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cell migration. (G), tubule formation of ECV304 on growth factors supplemented
matrigel in response to 20 μg/ml anti-nucleolin Abs or interfered with their
endogenous expression of nucleolin. Bar, 100 μm. *P < 0.05; ***P < 0.01.
Figure 3.
Translocation of nucleolin is mediated by VEGF and extracellular matrix
(A), cell surface nucleolin of HMECs was determined by FACS as described in
Materials and Methods. For the histograms: NC, negative control; Isotype Ab, isotye
Ab control; ST, serum starved cells; Matrix, HMECs grown on coated matrix
protein-fibronectin; VEGF, HMECs were cultured with serum-free medium
containing 10 ng/ml VEGF. To HMECs grown on fibronectin, VEGF significantly
enhanced the surface expression of nucleolin, but the two factors only have marginal
effect. (B), HMECs grown on fibronectin were stimulated by 10 ng/ml VEGF for 0 h,
1 h, 6 h, 12 h, and then re-starved by serum free medium (SF). The membrane
proteins were biotinlayted and pulled down by strepavidion conjugated agarose;
meanwhile, total proteins were from whole cell lysate. Both fractions were subjected
to immunoblotting by anti-nucleolin Abs. The amount of nucleolin in cell membrane
(nucleolin-CM) increased following VEGF stimulation and decreased without
stimulation; however, during the process, the amount of nucleolin in whole cells
(nucleolin-total) did not change. β-actin as a loading control. (C), cell membrane
protein: CD31, cytoplasmic protein: Cytochrome C, and nuclear protein: Histone-H1,
were detected in both streptavidin pull-down fraction and supernatant, to prove that
the streptavidin matrix sharply separates biotinylated from non-biotinylated proteins,
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and to exclude the traces of nucleolar proteins. (D), after HMECs were plated on
immolilized fibronectin (FN) or poly-L-lysine (PLL), the distribution of nucleolin
(green) was visualized by immunofluorescence at different time. Bar, 10 μm. (E), cell
surface nucleolin (green) and nuclei (blue) in HMECs grown on immobilized FN or
PLL were stained. Cells were not permeabilized here to visualize cell surface proteins.
Bar, 10 μm. (F), during the translocation of nucleolin, stress fibers and focal adhesion
formed simultaneously. Actin filaments were stained by FITC-phalloidin (green) and
focal adhesion complex were visualized by indirect immunostaining of vinculin
(green). Nuclei were counterstained with DAPI (blue). Bar, 10 μm.
Figure 4.
Nucleolin anchors to intracellular MyH9 during angiogenesis
(A), cell-free lysate was prepared from HMECs grown on immobilized FN or PLL.
co-IP was performed with anti-nucleolin Ab and purified rabbit IgG as a control. The
arrows indicate the precipitated protein band which was identified as MyH9 by
MALDI-TOF analysis. The SDS-PAGE gel was silver stained and finger printing map
was indicated. (B), co-IP was performed as described above, and the precipitated
proteins were then blotted (IB) with antibodies against MyH9 or nucleolin. The
nucleus of HMECs grown on immobilized FN, were extracted; then both nuclear and
extra-nuclear fraction were lysed to perform co-IP with the same condition. (C),
distribution of actin filament (green) and MyH9 (red) in HMECs grown on
immobilized FN or PLL were analyzed. Bar, 10 μm. (D), upon attached to matrix and
stimulated by growth factors, endothelial cells form tubule structure; meanwhile,
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nucleolin (red) translocated to extra-nuclei and colocolized with MyH9 (green).
Nucleolin and MyH9 were stained by indirect immunofluorescence. Bar, 10 μm. (E),
under normal condition without matrix and growth factors supplements, nucleolin
(green) stayed in nuclei and MyH9 (red) located in cytosol. The nuclei (blue) were
counterstained with DAPI. Bar, 10 μm.
Figure 5.
MyH9 is essential for the angiogenesis function of nucleolin
(A), MyH9-RNAi suppressed the expression of MyH9. After transfected with control
or MyH9 siRNA expression vectors, HMECs were lysed and subjected to
immunoblotting analysis with anti-MyH9 Ab, and anti-βactin as a sample loading
control. (B), MyH9-RNAi diminished cell surface nucleolin. After co-transfected with
GFP and MyH9 siRNA expression vectors, HMECs were fixed without permeabilized
to visualize cell surface nucleolin (red). Arrows indicate GFP negative cells, and stars
show GFP positive cells in which expression of MyH9 was inhibited. Bar, 20 μm. (C),
without permeabilized the ML7 treated HMECs, cell nucleolin (green) were stained
and the nuclei (blue) were counterstained with DAPI. PBS was added as a control.
Bar, 10 μm. (D), the amount of cell surface nucleolin of HMECs was determined by
FACS as described in Materials and Methods. For the histograms: NC, negative
control; Isotype Ab, isotye Ab control; ST, serum starved; VM, cultured on matrigel
supplemented with 10 ng/ml VEGF; MyH9i, RNAi against MyH9; ML7, treated cells
with 5 μM ML7. Suppression the endogenous MyH9 led to reduction of cell surface
nucleolin. HMECs were subjected to the following treatment: ML7 treated (E);
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down-regulated MyH9 by MyH9 RNAi (F); over-expressed MyH9ΔN-GFP (G), and
then the migration of HMECs was analyzed as described in Methods. ***P < 0.01.
(H), over-expressed MyH9ΔN-GFP in ECV304, and then cells invasive assay were
performed, cell invasion was significantly inhibited by over-expressed deficient
MyH9, over-expressed GFP as a control. Bar, 50 μm. (I), ECV304 were subjected to
the following treatment: ML7 treated; down-regulated MyH9 by MyH9 RNAi;
over-expressed MyH9ΔN-GFP, and then the tubule formation of each group was
analyzed as described in Materials and Methods. Bar, 100 μm. ***P < 0.01.
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FIGURES
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Prepublished online January 10, 2006;
doi:10.1182/blood-2005-07-2961
The angiogenesis function of nucleolin is mediated by vascular endothelial
growth factor and nonmuscle myosin
Yujie Huang, Hubing Shi, Hao Zhou, Xiaomin Song, Shaopeng Yuan and Yongzhang Luo
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