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[Cancer Research 62, 1944-1947, April 1, 2002]
© 2002 American Association for Cancer Research

Advances in Brief

Endostatin Regulates Endothelial Cell Adhesion and Cytoskeletal Organization1

Johan Dixelius, Michael Cross, Taro Matsumoto, Takako Sasaki, Rupert Timpl and Lena Claesson-Welsh2

Department of Genetics and Pathology, Uppsala University, Rudbeck Laboratory, 751 85 Uppsala, Sweden [J. D., M. C., T. M., L. C-W.], and Max-Planck-Institute, Department of Protein Chemistry, Martinsried an München, D-82152 Germany [T. S., R. T.]


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Endostatin, an endogenous angiogenesis inhibitor, attenuates endothelial cell migration through an unknown mechanism. We show that endostatin induced tyrosine phosphorylation of focal adhesion kinase and paxillin, and promoted formation of focal adhesions and actin stress fibers, similar to fibroblast growth factor-2 (FGF-2). In cells cotreated with endostatin and FGF-2, focal adhesions and actin stress fibers were decreased, indicating that endostatin disturbs cell-matrix adhesion. Reduced tyrosine phosphorylation and cytoplasmic relocalization of ß-catenin in cells treated with FGF-2 and endostatin indicates that loosening of cell-cell adhesion is also disturbed by endostatin. These data provide a molecular basis both for the lack of effect of endostatin on the normal, quiescent vasculature, and its antagonistic effects on stimulated tumor vessels.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
Endostatin, the COOH-terminal part of the collagen XVIII {alpha}1-chain, is a potent angiogenesis inhibitor that has been shown to regress a range of model tumors in mice, either alone (1) or in combination with conventional therapy (2) . Its importance in regulation of vascular quiescence is highlighted by the fact that a single nucleotide polymorphism (D104N) in the endostatin domain of the collagen {alpha}1 XVIII gene predisposes for the development of prostatic adenocarcinoma (3) . The antitumor effect of endostatin is based on the fact that expansion of solid tumors is dependent on angiogenesis, the formation of blood capillaries, required to supply the tissue with oxygen and nutrients. Endostatin is a heparin-binding protein (4) ; it binds with low affinity to the heparin sulfate proteoglycans glypican-1 and glypican-4, and with high affinity to an as yet unidentified molecule on endothelial cells (5) . Endostatin has also been shown to associate with proteins such as {alpha}V and {alpha}5 integrins (6) , fibulin, and laminin-1 (7) . Several studies report inhibition of endothelial cell migration by endostatin (8) ; ectopic expression of endostatin in Caenorhabditis elegans leads to cell and axon migration defects (9) . Other types of motility responses, for example induced by the trimeric NC1 domain of collagen XVIII, are also inhibited by endostatin (10) . Migration requires continuous turnover of cell-cell and cell-matrix interactions (11) . Cell-matrix interactions via focal adhesions, sites of actin attachments, are regulated by a molecular machinery including focal adhesion kinase (FAK)3 and paxillin (reviewed in Ref. 12 ). Tyrosine phosphorylation of adherens junctions components, such as ß-catenin, leads to the loosening of cell-cell adhesions in endothelial cells (13) and has been correlated to migration (14) . A recent study showed that the cytoplasmatic localization of ß-catenin was a feature of the angiogenic endothelium, in contrast to the mature endothelium (15) . In this report, endostatin and the proangiogenic fibroblast growth factor-2 (FGF-2) are shown to affect actin, FAK, paxillin, and ß-catenin. Our data indicate that endostatin interferes with FGF-2-induced signal transduction leading to a block in endothelial cell motility.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Antibodies and Fluorescent Probes.
The antibodies and fluorescent probes used were: antiphosphoFAK (pY397) and antiphosphopaxillin (pY118) (BioSource International, Camarillo CA,); anti-FAK, antipaxillin and anti-ß-catenin (Transduction Laboratories, Lexington, KY); antiphosphotyrosine 4G10 (Upstate Biotechnology, Lake Placid, NY); antiphospho 42/44 Erk and anti-42/44 Erk (Cell Signaling Technology, Beverly, MA); antirabbit horseradish peroxidase and antimouse-horseradish peroxidase (Amersham-Pharmacia Biotech, Uppsala Sweden); and antimouse Alexa 488 F(ab')2, antirabbit Alexa 568 F(ab')2, rhodamin-phalloidin, and Hoechst 33342.

FGF-2 and Endostatin.
Recombinant human FGF-2 was from Boehringer Mannheim (Mannheim, Germany). Recombinant mouse endostatin purified from transfected human 293 cells (7) had endotoxin levels of 0.0005 endotoxin units/mg, as analyzed by the Limulus Amebocyte Lysate 82 27 34–63/3 Coatest (Chromogenix, Mölndal, Sweden).

Cell Culture.
BCE cells, a kind gift from Dr. R. Christofferson, Department of Medical Cell Biology, Uppsala University, were cultured on gelatin-coated dishes in DMEM (Life Technologies, Inc., Rockville, MD), 10% newborn calf serum, and 2 ng FGF-2/ml (complete medium). For microscopy and immunoblotting experiments, cells were seeded in complete medium, incubated for 24 h, washed, and incubated in DMEM/1% newborn calf serum (starvation medium) for 20–24 h before additional treatment.

Chemotaxis Assay.
Chemotaxis was assayed essentially as described (8) using a 48-well chemotaxis chamber (Neuro Probe, Cabin John, MD). The number of cells that had migrated to the lower surface of the membrane was counted in three fields using x400 magnification. Samples were analyzed in triplicate on three separate occasions.

Preparation of Cell Lysate, Immunoprecipitation, and Immunoblotting.
Starved cells were treated with 3 µg/ml endostatin, 35–50 ng/ml FGF-2, or both for 10 or 90 min and washed in TBS/100 µM Na3VO4. For anti-FAK blotting, the cells were lysed in NP40 lysis buffer [1% NP40, 20 mM HEPES (pH 7.5), 150 mM NaCl, and 10% glycerol]. Otherwise, the cells were lysed in radioimmunoprecipitation assay lysis buffer [1.5% Triton X-100, 1% deoxycholate, 0.1% SDS, 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.1 mM NaF]. The lysis buffers contained 2.5 mM EDTA, 10 kallikrein inhibitory units aprotinin/ml, 1 mM phenylmethylsulfonyl fluoride, and 100 µM Na3VO4. Lysates were clarified by centrifugation. Immunoprecipitation, SDS-PAGE, and immunoblotting were performed using standard procedures. Immunoreactive sites on the filters were detected by enhanced chemiluminescence. Quantification of scanned immunoblot films was made using the Adobe PhotoShop software.

Microscopy.
For treatment, starved cells on gelatin-coated eight-well microscope slides (Falcon; Becton Dickinson, Franklin Lakes, NJ) were treated with 1 µg/ml endostatin or 10 ng/ml FGF-2 for 10 and 90 min. For fixation, the cells were put on ice, washed in TBS twice, fixed for 15 min using Zn-fix (0.5% ZnCl and 0.5% ZnAcetate in TBS/0.2% Triton X-100), and washed in TBS. To stain, preparations were blocked for 1 h with TBS/10% FCS and incubated 1 h with the primary antibody in TBS/1% FCS, washed in TBS, incubated with secondary fluorescent antibody, and washed. Rhodamine-labeled phalloidin was added with the secondary antibody. Finally the slides were incubated with 1 µg/ml Hoechst 33342 for 5 min.

Photography.
The samples were examined using the x60 lens in a Nikon microscope, and pictures for each color were taken separately and merged.

Image Analysis.
A unix-based image analysis system was used for quantification. In brief, the area of the actin stress fiber staining was quantified for actin analysis. Paxillin was used as a marker for focal adhesions. The number of bright paxillin spots was quantified, including only objects >5 pixels. A minimum of 100 cells was analyzed per sample.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
Endostatin Inhibits Migration and Stimulates Formation of Signaling Complexes.
Recombinant mouse endostatin was purified to homogeneity using heparin affinity chromatography from the medium of transfected 293 embryonic fibroblasts (7) and shown to be free of endotoxin contamination. To confirm the biological activity of the recombinant endostatin, we tested its ability to inhibit endothelial cell migration. BCE cells were allowed to migrate in a modified Boyden chamber toward 10 ng/ml FGF-2 for 4 h, resulting in a 2-fold increase in migrating cells over the control condition. Addition of 0.1 µg/ml endostatin reduced the stimulated migration by 50%, whereas 1 µg/ml endostatin arrested migration at basal levels. The effect of endostatin was statistically significant at P < 0.02 (unpaired Student’s t test).

Endostatin Induces Activation of FAK and Phosphorylation of Paxillin.
Biochemical analysis confirmed the involvement of FAK and its substrate paxillin in endostatin action. BCE cells were treated for 10 or 90 min with endostatin, FGF-2, or both together. As shown in Fig. 1ACitation , treatment with either endostatin or FGF-2 increased the phosphorylation of FAK at Y397 [Y(p)397-FAK], which is a major autophosphorylation site in FAK and an indicator of activation of the FAK kinase. The level of Y(p)397-FAK was also increased in the FGF-2 and endostatin cotreated cells. Treatment with FGF-2, endostatin, or both together also resulted in the phosphorylation of paxillin (Fig. 1B)Citation , most likely as a consequence of FAK activation. In contrast, a number of other signaling molecules were not affected by endostatin using the conditions used here. Thus, the extracellular regulated kinases (Erk)-1 and -2 were neither affected by endostatin alone nor did endostatin modulate FGF-2-induced Erk 1/2 activation (Fig. 1C)Citation . Similarly, we found no indication for endostatin-induced effects on Pyk-2, Janus kinase-1 and -2, Tyrosine kinase-2, p130Cas (Crk-associated substrate), CasL, Vav, or c-Cbl (data not shown).



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  		Fig. 1. Endostatin treatment induces phosphorylation of FAK and paxillin. BCE cells in 1% serum were treated with endostatin (ES; 3 µg/ml), FGF-2 (50 ng/ml), or both for 10 or 90 min. A, immunoblotting of total cell lysates was performed using anti-(p)Y397-FAK (P-FAK), antiphosphotyrosine PY, and anti-FAK (FAK) antibodies, as indicated. B, lysates were immunoprecipitated with antipaxillin antibody and immunoblotted using anti (p)Y118-paxillin (P-paxillin) or antipaxillin (Paxillin) antibodies. Quantification of signal ratios in A (P-Fak/Fak) and B (P-paxillin/Paxillin) is indicated. C, total cell lysates were immunoblotted using antiphospho-Erk-1/2 (P-Erk) and anti-Erk-1/2 (Erk) antibodies.

 
Modulation of Actin Fibers and Focal Adhesions by Endostatin.
The possible involvement of FAK in endostatin action prompted an examination of the cytoskeletal organization and matrix interactions of endostatin-treated endothelial cells. BCE cells were treated with endostatin, FGF-2, or both together for 10 or 90 min. Actin stress fibers and focal adhesions were visualized using rhodamine-labeled phalloidin and immunostaining for paxillin, respectively. To quantify changes in actin stress fibers and the number of intensely stained focal adhesions, 100 cells for each treatment were photographed at random and analyzed by image analysis software. Actin stress fiber formation was increased transiently by endostatin and in a sustained manner by FGF-2 (red in Fig. 2ACitation and quantified in B). In contrast, treatment with both endostatin and FGF-2 together prevented stress fiber formation. Individual treatment with endostatin or FGF-2 increased the number of focal adhesions to the same extent and in a sustained manner (green in Fig. 2A and quantified in BCitation ). In contrast, cotreatment with FGF-2 and endostatin resulted in a transient rather than sustained increase in the number of focal adhesions. After 90 min of cotreatment, the number of focal adhesions was consistently below that in control cells.



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  		Fig. 2. Cotreatment with endostatin and FGF-2 blocks formation of actin stress fibers and focal adhesions. BCE cells starved in 1% serum were treated with endostatin (1 µg/ml), FGF-2 (10 ng/ml), or both for 10 or 90 min. To visualize focal adhesions, fixed preparations were stained with antipaxillin antibody and a secondary alexa-488-conjugated antibody (green). TRITC-conjugated phalloidin (red) was used to detect actin stress fibers, and Hoechst 33342 (blue) was used to visualize the nuclei. A minimum of 100 cells/treatment were photographed, and subjected to image analysis for quantification of actin fiber area per cell and number of intensely stained focal adhesions per cell. A, images of BCE cells treated as indicated. Bar, 10 µm. B, quantification of actin fiber formation (left) and number of focal adhesions (right).

 
Cytoplasmic Localization and Tyrosine Phosphorylation of ß-Catenin.
The effects of endostatin on the cytoskeletal arrangement and on focal adhesions indicated that cell-matrix interactions were affected by endostatin. We hypothesized that cell-cell interactions could also be disrupted by endostatin, because regulation of both cell-matrix and cell-cell interactions are critical in cell motility. This reasoning was promoted by our finding that ß-catenin, which is localized at cell-cell junctions in cadherin complexes, coprecipitated with paxillin in the FGF-2-treated endothelial cells (Fig. 3)Citation . In contrast, endostatin treatment reduced the complex formation below that seen in untreated cells. Cotreatment with FGF-2 and endostatin resulted in a delay in complex formation that was seen after 90 min only. The protein level of ß-catenin in the total cell lysate was not affected by the different treatments.



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  		Fig. 3. Endostatin transiently blocks FGF-2-induced complex-formation between ß-catenin and paxillin. BCE cells starved in 1% serum were treated with endostatin (3 µg/ml), FGF-2 (50 ng/ml), or both for 10 or 90 min. Lysates were immunoprecipitated with antipaxillin antibody and immunoblotted using anti-ß-catenin or antipaxillin antibodies, as indicated. Total cell lysates were immunoblotted with anti-ß-catenin antibody (bottom panel).

 
Next, the subcellular localization and phosphorylation status of ß-catenin were examined. BCE cells were treated with endostatin, FGF-2, or both. Microscopic analysis of untreated control cells (Fig. 4A)Citation revealed that ß-catenin was localized predominantly at the cell-cell contacts. Treatment with endostatin did not affect the localization of ß-catenin, whereas FGF-2 treatment shifted ß-catenin to the cytoplasm. Cotreatment with FGF-2 and endostatin blocked the increase in cytoplasmatic ß-catenin at 10 min, but at 90 min, cells treated with FGF-2 alone and the cotreated cells contained similar levels of cytoplasmatic ß-catenin. Immunoblotting with antiphosphotyrosine antibodies on immunoprecipitated ß-catenin revealed that treatment with either endostatin or FGF-2 induced transient tyrosine phosphorylation of ß-catenin at 10 min, which was attenuated at 90 min (Fig. 4B)Citation . In contrast, treatment with endostatin and FGF-2 together resulted in a small increase at 90 min only. These data show that endostatin affects both the subcellular localization and tyrosine phosphorylation status of ß-catenin, with implications for the regulation of cell adhesion. In accordance, FGF-2 treatment led to increased permeability of [3H]inulin through confluent monolayers of BCE cells in a transwell assay, an effect that was counteracted by cotreatment with endostatin (data not shown).



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  		Fig. 4. Subcellular localization and tyrosine phosphorylation of ß-catenin in response to treatment with endostatin and FGF-2. A, BCE cells starved in 1% serum were treated with either endostatin, FGF-2, or both together for 10 or 90 min. Immunostaining for ß-catenin is shown in red, whereas the nuclei were stained blue using Hoechst 33342. Bar, 10 µm. B, BCE cells starved in 1% serum were treated with endostatin (3 µg/ml), FGF-2 (50 ng/ml), or both for 10 or 90 min. Immunoprecipitation was performed using an anti-ß-catenin antibody followed by immunoblotting with antiphosphotyrosine (PY) or anti-ß-catenin antibodies.

 

   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
Migration of endothelial cells is an important step in the angiogenic response (16) , but the molecular machinery guiding directed motility of endothelial cells is incompletely understood. Migration of tissue culture cells toward growth factors is known to require activation of a phosphatidylinositol 3'-kinase signaling pathway leading to activation of the monomeric GTPase Rac, which regulates formation of membrane edge ruffles, lammelopodia. The related GTPase Rho on the other hand, regulates actin stress fiber formation. Inhibition of Rac and Rho function by expression of dominant-negative versions of these molecules attenuates chemotaxis (reviewed in Ref. 17 ). Using BCE cells and human dermal microvascular cells we have not been able to detect any modulatory effect of endostatin on growth factor-stimulated phosphatidylinositol 3'-kinase, Rac, or Rho activities (data not shown). In accordance with our data, Wickstrom et al. (18) showed that endostatin treatment attenuated focal adhesions in human dermal microvascular endothelial cells. These cells display abundant focal adhesions in the absence of stimulation probably because of endogenous production of growth factors (data not shown); therefore, the agonistic effect of endostatin is concealed in this cell model.

Treatment of endothelial cells with vascular endothelial growth factor is known to lead to tyrosine phosphorylation of VE-cadherin as well as ß-catenin and other components in the adherens junction complexes (13) . We did not observe tyrosine phosphorylation of VE-cadherin in response to endostatin or FGF-2 (data not shown), but tyrosine phosphorylation of ß-catenin was readily demonstrated. The kinase mediating tyrosine phosphorylation of ß-catenin has not been identified. Members of the Src family of cytoplasmatic kinases are potential candidates, but there are no indications for activation of Src by endostatin (data not shown). Complex formation between ß-catenin and paxillin in response to growth factor treatment (Fig. 3)Citation has not been reported previously; it did not correlate with the tyrosine phosphorylation status of ß-catenin. A recent report (19) implicates ß-catenin as a mediator of endostatin-induced cell cycle arrest. These data infer that endostatin promotes translocation of ß-catenin to the nucleus; such an effect was not evident in our study.

The stimulatory effect of individual treatment with either endostatin or FGF-2 on stress fibers/focal adhesions was not seen in cells receiving the two factors simultaneously, suggesting that the two agents activate distinct pathways to induce these responses. The distinct FGF-2- and endostatin-induced pathways may integrate at a specific point to evoke an effective down-regulation of the signal. Endostatin may induce this effect by activation of a specific receptor or by direct interference with cell-matrix adhesion. In an in vivo situation, the entire vascular bed would be exposed to circulating endostatin, but its antiangiogenic effect would only become evident in areas of high growth factor stimulation, such as those present in the tumor endothelium. For the first time, we provide a plausible mechanism to explain the clinical trial data in which antiangiogenic effects of endostatin are observed in the absence of systemic toxicity.


   ACKNOWLEDGMENTS
 
Expert advice on image analysis was provided by Kenneth Wester, Department of Genetics and Pathology, Uppsala University.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by grants from the Swedish Cancer Foundation (project no. 3820-B99-04XBC), the Novo Nordisk Foundation, the Pharmacia Corp., and by the Swedish Science Council. Back

2 To whom requests for reprints should be addressed, at Department of Genetics and Pathology, Uppsala University, Rudbeck Laboratory, Dag Hammarskjoldsv 20, 751 85 Uppsala, Sweden. Phone: 46-18-471-43-63; Fax: 46-18-55-89-31; E-mail: Lena.Welsh{at}genpat.uu.se Back

3 The abbreviations used are: FAK, focal adhesion kinase; FGF, fibroblast growth factor; Erk, extracellular regulated kinase; BCE, bovine capillary endothelial; TBS, Tris-buffered saline; VE-cadherin, vascular endothelial cadherin. Back

Received 12/27/01. Accepted 2/11/02.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

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PNAS, May 25, 2004; 101(21): 8005 - 8010.
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S. A. Wickstrom, K. Alitalo, and J. Keski-Oja
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E. Kikuchi, S. Menendez, M. Ohori, C. Cordon-Cardo, N. Kasahara, and B. H. Bochner
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D. J. Panka and J. W. Mier
Canstatin Inhibits Akt Activation and Induces Fas-dependent Apoptosis in Endothelial Cells
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S. A. Wickstrom, K. Alitalo, and J. Keski-Oja
Endostatin Associates with Lipid Rafts and Induces Reorganization of the Actin Cytoskeleton via Down-regulation of RhoA Activity
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P. Nyberg, P. Heikkila, T. Sorsa, J. Luostarinen, R. Heljasvaara, U.-H. Stenman, T. Pihlajaniemi, and T. Salo
Endostatin Inhibits Human Tongue Carcinoma Cell Invasion and Intravasation and Blocks the Activation of Matrix Metalloprotease-2, -9, and -13
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A. Sudhakar, H. Sugimoto, C. Yang, J. Lively, M. Zeisberg, and R. Kalluri
Human tumstatin and human endostatin exhibit distinct antiangiogenic activities mediated by alpha vbeta 3 and alpha 5beta 1 integrins
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N. Ortega and Z. Werb
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