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Tumor Biology |
Cell Biology and Molecular/Cancer Biology Laboratories, Biomedicum Helsinki, and Haartman Institute, Departments of Virology and Pathology, University of Helsinki, FIN-00014 Helsinki, Finland [S. A. W., T. V., K. A., J. K-O.], and the Collagen Research Unit, Biocenter and Department of Medical Biochemistry, University of Oulu, FIN-90220 Oulu, Finland [M. R., T. P.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Antiangiogenesis is a promising field of cancer therapy, and several inhibitors of angiogenesis have been introduced (1 , 5) . Endostatin, the COOH-terminal Mr20,000 fragment of collagen XVIII, is able to inhibit angiogenesis and tumor growth in an endothelial cell-specific manner (6 , 7) . It is a potent inducer of endothelial cell apoptosis (8) , and it is also capable of inhibiting cell proliferation and migration (7 , 9) ; but the mechanisms behind these effects of endostatin are poorly understood thus far.
The adhesion of cells is crucial for their survival and organization. Cell adhesion is controlled by functional complexes of ECM components, transmembrane adhesion molecules, and cytoplasmic proteins (reviewed in Ref. 10 ). Focal adhesions represent such functional complexes, consisting of integrins, integral membrane proteoglycans, associated cytoplasmic proteins such as vinculin and paxillin, and several protein kinases. These proteins serve as anchors for actin stress fibers (11) . The assembly and disassembly of the focal adhesions is a dynamic process under complex regulation as the cell converts from the adhesive to the migratory phenotype.
The uPA system comprises uPA, PAI-1, and the cell surface uPAR (12) . All components of this system are constitutively expressed in endothelial cells and are up-regulated by various angiogenic growth factors, transforming growth factor-ß, and hypoxia (13, 14, 15, 16) . Through activation of plasminogen and its intrinsic proteolytic activity, uPA is able to degrade fibrin, laminin, fibronectin, proteoglycans, gelatin, and thrombospondin and activate members of other proteolytic systems and various growth factors (17) . Because of its wide spectrum of proteolytic activity, uPA has been identified as an important factor in angiogenic proteolysis (18) . The proteolytic activity of uPA is localized to the cell surface by glycosylphosphatidyl inositol-linked uPAR. The binding of uPA to its receptor increases the rate of plasmin formation, whereas the binding of PAI-1 to the receptor complex or soluble uPA inhibits plasminogen activation (19 , 20) . In addition to their role in targeted proteolysis, uPA, PAI-1, and uPAR play a role in cell adhesion and migration (21, 22, 23, 24) . Proteolytic activity dependent on uPA localizes at the leading edge of migrating endothelial cells forming tubular structures and, more specifically, to focal adhesions (25) .
We report here that endostatin interferes with the targeting of pericellular proteolysis by down-regulating the levels of secreted soluble uPA and PAI-1 and their complexes and by removing uPAR-associated uPA from the focal adhesions. This is accompanied by the disassembly of focal adhesion complexes and the reassembly of the actin cytoskeleton by disruption of stress fibers in response to endostatin treatment. These changes plausibly affect the adhesive, migratory, and apoptotic behavior of endothelial cells.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Culture.
HDMECs were purchased from Promocell (Heidelberg, Germany) and were
cultured in Endothelial Cell Growth Medium (Promocell) at 37°C
in a humidified 5% CO2 atmosphere. The cells
used for the experiments between were undergoing passages 3–6. Human
embryonic lung fibroblasts (CCL-137; American Type Culture Collection,
Rockville, MD) were cultured in Eagle’s minimal essential medium
supplemented with 10% FCS, 100 IU ml-1 of
penicillin, and 50 µg ml-1 of streptomycin.
The cells were washed twice with serum-free medium and incubated for at
least 6 h before treatment with the various chemicals. All
experiments were carried out under serum-free conditions.
Expression and Characterization of Recombinant Human Endostatin.
Recombinant human endostatin was expressed and purified as described
(27)
. A fragment of human collagen XVIII that corresponds
to mouse endostatin sequences (6)
was cloned to the vector
pQE-31 (Qiagen, Santa Clarita, CA) and expressed as an
NH2-terminal His-tagged protein in
Escherichia coli strain M15, according to the
manufacturer’s protocol (Qiagen). Bacterial pellets were lysed in 6
M guanidine HCl, 0.5 M
NaCl, 10 mM ß-mercaptoethanol, and 20
mM Tris-HCl (pH 7.9) by freeze-thaw and then
centrifuged. The supernatant was lightly sonicated and applied
to a ProBond column (Invitrogen, San Diego, CA) that had been
preequilibrated with 8 M urea, 0.5
M NaCl, and 20 mM Tris-HCl
(pH 7.9). Bound protein was eluted by an imidazole gradient from 0 to
0.5 M in the equilibrium buffer. Endostatin
fractions were pooled and refolded in vitro, first by
dialyzing overnight at 4°C against 4 M urea,
0.1 M NaCl, 1 mM/0.1
mM reduced/oxidized glutathione, and 20
mM Tris-HCl (pH 7.9), then dialyzed for 6 h
against 1 M urea, 0.1 M
NaCl, 0.1 mM/0.01 mM
reduced/oxidized glutathione, 20 mM Tris-HCl (pH
7.9), and finally dialyzed overnight against PBS [170
mM NaCl and 10 mm sodium phosphate buffer (pH
6.9)]. Refolded soluble endostatin was separated by centrifugation and
applied to a HiTrap SP cation-exchange column (Amersham Pharmacia,
Piscataway, NJ). Endostatin fractions were eluted by a NaCl gradient
from 0.1 M to 1.5 M in PBS
(pH 6.9), pooled and dialyzed against 0.1 M NaCl
and 20 mM Tris-HCl (pH 7.4), and applied to a
heparin-Sepharose CL-6B column (Amersham Pharmacia, Piscataway,
NJ). Bound endostatin was eluted by a NaCl gradient from 0.1
M to 2 M in 20
mM Tris-HCl (pH 7.4). Endostatin fractions were
pooled and dialyzed against PBS (pH 7.4) and passed through a Polymyxin
agarose column using PBS buffer. Purified endostatin was concentrated
by ultrafiltration to 0.5–1.5 mg/ml and stored at -20°C until use.
Far UV circular dichroism spectrum was recorded on an AVIV Associates
(Lakewood, NJ) model 62DS spectrometer. Buffer conditions in the
circular dichroism analysis were 10 mM potassium
phosphate (pH 8.0), and cells of 1 mm path length were used. A 5-s time
constant and a 1.0 nm bandwidth was used during data acquisition over a
wavelength range of 184 to 260 nm; three spectra were collected for
protein or buffer and were averaged. Buffer spectra were subtracted
from the protein spectra. No endotoxin was found in the purified
fractions. The biological activity of endostatin was confirmed in a
three-dimensional collagen tube formation assay where endostatin
effectively inhibited growth factor-induced endothelial cell tube
formation (data not shown). The concentrations used in this study are
in agreement with amounts used commonly in cell culture, and possible
differences can be explained by the different sources of endostatin
(9)
.
Casein Zymography.
Zymography and reverse zymography were used to identify the molecular
forms of uPA and PAI (28)
. Polypeptides of conditioned
media or acid eluates were separated under nonreducing conditions in
gradient polyacrylamide gels (4–15%) in the presence of SDS (Bio-Rad,
Hercules, CA). SDS was removed by washing the gels with PBS (pH
7.4)/Triton X-100 (2.5%) 4 x 15 min. For reverse
zymography, reducing conditions were used, and uPA (2 IU/ml) was added
to the final wash. The gels were then placed on caseinolysis gels
containing plasminogen and casein in 1.2% agarose and incubated at
37°C until the lytic bands correlating to the PA-activity became
visible. A lysis resistant band indicated the migration of PAI.
Analysis of Membrane-bound PA Activity.
Analysis of membrane-bound PA activity was performed essentially as
described (29)
. Endothelial cells were washed with 0.2%
BSA in PBS (pH 7.4) and treated with 50 mM glycine-HCl
buffer (pH 3.0), containing 0.1 M NaCl at room temperature
for 10 min. The buffer was collected, immediately neutralized, and
subjected to casein zymography.
To test whether the cell surface accumulation could be prevented by blocking uPAR, blocking mAbs against uPAR were added to cells at a concentration of 30 µg/ml before the addition of 40 nM endostatin. Mouse IgG was used as a negative control. The treatment was carried out for 4 h, after which the cell surface proteins were collected with acid elution, as described above. The eluates were subsequently subjected to casein zymography to reveal the eluted PAs.
Metabolic Labeling and Immunoprecipitation.
Serum-starved cells were incubated with endostatin for 4 h in
[35S]methionine (80 µCi/ml; Amersham
Pharmacia Biotech, Uppsala, Sweden) in methionine-free medium.
Subsequently, aliquots of conditioned media were subjected to
immunoprecipitation. Samples were preabsorbed by incubation with mouse
preimmune serum and protein A-Sepharose beads at 4°C in an end-over
mixer for 2 h. The beads were removed by centrifugation and the
supernatants were incubated with antibodies against uPA or PAI-1 on ice
for 30 min. 
-bind Sepharose (Amersham Pharmacia, Uppsala,
Sweden) was added, and the samples were incubated in an end-over mixer
at 4°C for 1 h. The beads were subsequently collected by
centrifugation and washed three times with Triton lysis buffer [50
mM Tris-HCl buffer (pH 8.0) containing 150 mM
NaCl, 1% Triton X-100, 0.02% sodium azide, 10 mM EDTA, 10
µg/ml aprotinin, 1 µg/ml pepstatin A, and 1 µg/ml
aminoethylbenzene sulfonyl fluoride; Calbiochem, San Diego, CA). The
bound proteins were eluted with Laemmli sample buffer. The eluted
polypeptides were separated by 4–15% gradient PAGE in the presence of
SDS under nonreducing conditions and visualized by autoradiography.
Incorporated radioactivity was detected and quantitated with the Fuji
film BAS-2500 Image Analyzer and the MacBAS 2.5 computer
program. The quantified radioactive intensities were normalized
with the background intensities of each lane to account for possible
differences in sample loading.
Immunofluorescence.
The cells were cultured on glass coverslips and allowed to grow until
subconfluency. After the treatments, the cells were washed with PBS and
fixed with 3% paraformaldehyde at 4°C for 10 min. The cells were
then treated with 5% BSA for 30 min, washed with PBS (pH 7.4), and
incubated with polyclonal antibodies against uPA or with monoclonal
antibodies against vinculin or paxillin. TRITC-conjugated phalloidin
(Sigma Chemical Co.) was used for the staining of the actin
cytoskeleton. Unbound proteins were removed by washing with subsequent
incubation with Cy3- or FITC-labeled secondary antibodies (Jackson
ImmunoResearch Laboratories, West Grove, PA), respectively, for 1 h. For the immunolocalization of uPAR, cells were cultured on glass
coverslips and treated with endostatin and then washed with cold PBS
(pH 7.4). Cells were then incubated with antibodies against uPAR at
4°C for 20 min, washed with PBS, and fixed with 3% paraformaldehyde.
Subsequently, the cells were washed with PBS and incubated with
Cy3-labeled secondary antibodies for 1 h. The coverslips were then
washed and mounted on glass slides using Vectashield (Vector
Laboratories, Burlingame, CA). The fluorescent images were obtained
using an epifluorescent microscope.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The differences in uPA/PAI-1 levels became visible at
6 h, reaching a maximum within 12 h (Fig. 1B)
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. The effect of
endostatin reached a maximum in 12 h (Fig. 1B)
. No
change in the caseinolytic activity was observed when endostatin was
added to the media aliquots after their collection or when incubated on
caseinolysis gels together with purified human uPA, indicating that
endostatin itself does not affect uPA activity (data not shown).
To confirm the results obtained by zymography, HDMECs were
metabolically labeled with [35S]methionine for
4 h in the presence of endostatin as described in "Materials and
Methods." Conditioned medium was then collected and subjected to
immunoprecipitation with anti-uPA and anti-PAI-1 antibodies (Fig. 1C)
. Quantitation of the incorporated radioactivity of the
precipitated bands indicated that the levels of secreted uPA and PAI
were lower in the endostatin-treated cells than in the control cells
(Fig. 1C)
. Aliquots of conditioned media of endostatin
treated cells were then subjected to ELISA-analysis. A similar but
slightly less prominent down-regulation of uPA and PAI-1 after
endostatin-treatment was observed in this
assay.4
To assess whether the decrease observed in uPA and PAI-1 levels was caused by transcriptional regulation, Northern blotting analysis was performed on endostatin-treated cells. No changes in the mRNA levels of uPA or PAI-1 were observed within 6–12 h of endostatin treatment (data not shown).
Redistribution of Cell Surface-associated uPA and uPAR by
Endostatin.
The localization of proteolytic activity to distinct compartments of
the cell surface is crucial for the effective degradation of the
surrounding matrix (12
, 30)
. PA activity is localized to
focal adhesions through its cell surface receptor uPAR. To analyze the
effect of endostatin on the cell surface localization of uPA, the cells
were treated with endostatin for 3 h and fixed with 3%
paraformaldehyde. The fixed cells were stained with specific antibodies
against uPA. Control cells showed a distinct localization of uPA in
focal adhesions, which was confirmed with double staining for vinculin.
Endostatin-treated cells, on the other hand, exhibited a diffuse
staining pattern. The effect appeared in parallel with the loss of
vinculin staining from the focal adhesions at 1 h, but no
relocalization of uPA into the recovered focal adhesions was observed
within 6 h of treatment (Fig. 2A)
. Cells incubated with phosphoinositol-specific
phospholipase-C (Sigma Chemical Co.) before immunostaining showed no
positive staining with antibodies against uPA, confirming the
specificity of the staining for uPA-occupied uPAR.
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To investigate the observation further, we carried out experiments
where endothelial cells were incubated with increasing concentrations
of endostatin. After 3 h of incubation, the cell
surface-associated proteins were eluted with a mild acid treatment and
analyzed with casein zymography. These experiments revealed that
endostatin increased the amounts of uPA/PAI-1 complexes at the cell
surface in a dose-dependent manner, as detected by acid elution (Fig. 3A)
. Only negligible levels of free uPA were detected under
these conditions.
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Disruption of Focal Adhesions and the Actin Stress Fibers by
Endostatin.
The redistribution of uPA and vinculin suggested that endostatin has an
effect on focal adhesions, which are functional complexes involved in
cell adhesion and in migration on the pericellular matrix
(11)
. HDMECs grown on glass coverslips displayed prominent
focal adhesions, as visualized both by anti-vinculin and anti-paxillin
immunofluorescent staining. The focal adhesions appeared
characteristically as short streaks in the basal surface of the
cytoplasm. Treatment of spread cells with 40 nM endostatin
(800 ng/ml) for 1 h caused a dramatic reduction in the number of
cells containing such a staining pattern. The endostatin-treated cells
displayed vinculin staining restricted to cell-cell junctions and weak
paxillin staining distributed diffusely throughout the cytoplasm. The
majority of the cells were devoid of the cytoplasmic streaks (Fig. 4A)
. The disruption of focal adhesions had occurred already
after 1 h of treatment and persisted for at least 3 h of
incubation, whereas evidence of focal adhesion recovery was observed
after 6 h of incubation (Fig. 2)
.
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. Previously
it has been observed that the effects of endostatin are endothelial
cell-specific (6)
. To determine whether the
endostatin-induced alterations of the cytoskeletal structure are
limited to endothelial cells, human embryonic lung fibroblasts were
treated with 40 nM endostatin for 1 h. No
changes in the distribution of focal adhesions were observed in cells
stained with antibodies against vinculin or paxillin. Accordingly, no
changes in the distribution of actin were observed with
TRITC-phalloidin staining (Fig. 4B)
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DISCUSSION |
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Proteolytic activity is a prerequisite for endothelial cell sprouting and tube formation (4 , 31) . Culturing endothelial cells in type I collagen or stimulation with angiogenic growth factors such as basic fibroblast growth factor have been observed to increase the secretion of both uPA and PAI-1 (32) . For efficient proteolysis, proteases are targeted to distinct locations at the cell surface and, in particular, changes in uPA and PAI-1 secretion and localization can induce changes in cell migration and adhesion (33, 34, 35) . Our current results indicate that endostatin modulates both the amount and the localization of uPA and PAI-1 in endothelial cell cultures. We found that the levels of uPA and PAI-1 were decreased in the conditioned media of endothelial cells after endostatin treatment. Whether the observed down-regulation of secreted proteolytic activity is an independent phenomenon or a consequence of focal and cytoskeletal reorganization is not known yet (see Ref. 36 ).
Microvascular endothelial cells stained with antibodies against uPA and uPAR showed a strong signal in focal adhesions, whereas the cells treated with endostatin displayed a diffuse staining pattern. At the same time, the uPA/PAI-1 complexes isolated from the cell surface by acid treatment were increased in a dose-dependent manner. However, no significant increase in the levels of uPA or PAI-1 was detected in the immunoprecipitates of cell lysates or ECM preparations after the metabolic labeling of endostatin-treated cells, indicating that the increase of cell surface uPA/PAI-1 complexes is not attributable to intracellular or matrix accumulation of these proteins. The finding that up-regulation of acid-eluted uPA/PAI-1 complexes could be inhibited by blocking the cell surface receptor uPAR further elucidates this effect. The relocalization uPA and uPAR may be a consequence of the focal adhesion disassembly.
Interestingly, the regulation of plasminogen activation is modulated by another antiangiogenic molecule, angiostatin, which has displayed a synergistic effect with endostatin (37 , 38) . Another important family of proteinases, the MMPs, has also been assessed by us as a possible target for endostatin. No changes in gelatinase A (MMP-2) activation were detected by gelatin zymography assays of conditioned media in response to endostatin treatment. The proteolytic processing of membrane-type-1 MMP and its localization on the cell surface, phenomena linked with efficient proteolysis (39) , were also unaffected.4
Cell migration in angiogenesis involves concerted assembly and disassembly of focal adhesions in the polarized endothelial cells. The process is thought to require parallel signaling through integrins and transmembrane proteoglycans (40) , both putative binding sites for endostatin (41 , 27) . Our results indicate that endostatin interferes with this system by disassembling focal adhesions as visualized by immunofluorescence staining with antibodies against two major components of the adhesion complex, vinculin and paxillin. The effect was rapid, already visible 1 h after endostatin administration, and persisted for up to 6 h. The reversibility of the observation suggests the cytoskeletal changes to be an independent phenomenon rather than a sign of apoptotic behavior.
Changes in cell-substratum interactions in adherent and motile cells are transduced through the focal adhesions into the actin cytoskeleton, which is responsible for the mechanical forces generated within the cell. Intact focal adhesions are required for actin stress fiber formation and anchorage. We observed that endostatin induced a marked rearrangement of the endothelial actin network, because the stress fibers were no longer detectable in contrast with their well-organized pattern of in control cells. This finding supports the view that the absence of paxillin and vinculin staining in focal adhesions is caused by the disassembly of the entire complexes rather than by failure to recruit these proteins to existing adhesion structures. The cellular mechanisms behind these changes remain unclear at present. It should be noted that endostatin affects cell survival, proliferation, and migration (7 , 8 , 9) , all events controlled in part by cell-substratum interactions. Interference with focal adhesion and actin cytoskeleton assembly as well as targeted proteolysis provide plausible mechanisms for these effects.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by the Academy of
Finland, the Sigrid Juselius Foundation, Biocentrum Helsinki, the
Helsinki University Hospital Fund, the Novo Nordisk Foundation, the
Finnish Cancer Foundation, the Emil Aaltonen Foundation, the Farmos
Research and Science Foundation, and the University of Helsinki. 
2 To whom requests for reprints should be
addressed, at Biomedicum Helsinki, Laboratory of Cell Biology, PL 63
(Haartmaninkatu 8), FIN-00014, University of Helsinki, Finland. Phone:
358-9-191-25566; Fax: 358-9-191-25573; Email: Jorma.Keski-Oja{at}Helsinki.fi
3 The abbreviations used are: ECM, extracellular
matrix; uPA, urokinase-type plasminogen activator; PAI-1, plasminogen
activator inhibitor, type 1; uPAR, urokinase-type plasminogen activator
receptor; HDMEC, human dermal microvascular endothelial cell; mAB,
monoclonal antibody; TRITC, rhodamine isothiocyanate; PA, plasminogen
activator; MMP, matrix metalloproteinase.
4 S. Wickström, T. Veikkola, K. Alitalo, and
J. Keski-Oja, unpublished observations.
Received 10/30/00. Accepted 7/ 3/01.
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98,000, corresponding to the uPA/PAI-1
complex. Reverse zymography (right) shows
lysis-resistant bands representing PAI-1. Note the dose-dependent
decrease of both uPA/PAI-1 complexes and PAI-1 in endostatin-treated
cultures. B, time-dependency analysis. Cells were
treated with endostatin (40 nM; 800 ng/ml), and aliquots of
the conditioned media were collected after the indicated incubation
periods, with subsequent zymography (uPA/PAI-1) and reverse zymography
for PAI-1. C, immunoprecipitation analysis. Cells were
labeled with [35S]methionine during incubation with 40
nM endostatin (800 ng/ml) for 4 h. The radiolabeled
proteins from the conditioned media were then immunoprecipitated using
antibodies against uPA or PAI-1, separated by 4–15% gradient PAGE in
the presence of SDS, and visualized by autoradiography. The migration
of molecular weight markers (kDa) is shown on the left.
Arrowhead, migration of the uPA/PAI-1 complex.
Graph below the autoradiograph illustrates the
incorporated radioactivities corresponding to precipitated uPA and
PAI-1. The bands were quantitated by Phospho-Imager analysis and
plotted as relative protein levels. The quantitative analysis revealed
that the levels of uPA and PAI-1 were about 60 and 50% lower,
respectively, in endostatin-treated cells than in control cells.
