| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Tumor Biology |
5ß1 and Caveolin-1, and Activates Src via a Tyrosyl Phosphatase-dependent Pathway in Human Endothelial Cells1
Departments of Pathology and Virology [S. A. W.], Molecular/Cancer Biology Laboratory and Ludwig Institute for Cancer Research [K. A.], and Departments of Pathology and Virology [J. K-O.], Haartman Institute and University of Helsinki, Biomedicum Helsinki and Helsinki University Hospital, FIN-00014 Helsinki, Finland
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
5ß1 integrin associated with actin stress fibers and its concomitant colocalization with the membrane anchor protein caveolin-1. Furthermore, endostatin could be coimmunoprecipitated with 5ß1 and caveolin-1 from endothelial cell extracts. Endostatin treatment induced phosphatase-dependent activation of caveolin-associated Src family kinases. The disassembly of actin stress fibers and focal adhesions by endostatin was found to occur via activation of Src and in a tyrosyl phosphatase-dependent manner. The endostatin-treated cells void of the focal adhesions had impaired ability to deposit fibronectin into their extracellular matrices and were unable to migrate in response to basic fibroblast growth factor in a wounding experiment. These results indicate that recombinant endostatin interacts with 5ß1 integrin and caveolin-1 at the endothelial cell surface. In addition, the antimigratory effect of endostatin involves phosphatase-dependent Src activation and impaired cell-matrix interactions. |
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Endostatin has been observed to associate with cell surface integrins, especially with 5ß1, the major fibronectin receptor in endothelial cells (6)
. Binding of integrins to extracellular ligands initiates a series of events including integrin clustering, association with cytoplasmic linker proteins, cytoskeletal reorganization, and downstream signaling (8, 9, 10, 11)
. The interaction between integrins and extracellular matrix proteins is essential for endothelial cell proliferation and angiogenesis (12)
. Integrin 5ß1 has been identified as one of the integrins involved in these processes (13)
.
Signaling through integrins requires physical interaction with other membrane proteins and subsequent association with cytoplasmic signal transduction proteins. Integrin 5ß1 has been found to specifically associate with caveolin-1, a transmembrane anchor protein, which couples integrins to cytoplasmic signaling cascades such as the Src pathway (14)
. Phosphorylation signals generated as a result of integrin ligation regulate cellular events such as the assembly and disassembly of focal adhesions, and reorganization of the actin cytoskeleton (9
, 15
, 16)
. Members of the Src family of membrane-associated tyrosine kinases are important regulators of these events; they associate directly with caveolin-1 (17)
and are regulated by tyrosine phosphorylation. A constitutively active variant of c-Src, v-Src, has been observed to induce disruption of the actin cytoskeleton, loss of focal adhesions, and impaired deposition of fibronectin matrix (18, 19, 20)
.
Fibronectin is deposited as a polymerized insoluble matrix by endothelial cells to provide support in cell migration and adhesion during angiogenesis (13 , 21) . The deposition is a cell-dependent event initiated with binding of soluble fibronectin to cell surface integrins in vinculin-containing focal adhesions (22 , 23) . Fibronectin deposition requires intact focal adhesions (24) , which in turn serve as anchors for actin stress fibers involved in cell locomotion.
The current study was carried out to identify molecular interactions of recombinant endostatin at the endothelial cell surface that could explain the decreased migration of endostatin-treated endothelial cells. We found that exogenous recombinant endostatin associates with caveolin-1 and integrin 5ß1 in microvascular endothelial cells. In addition, endostatin treatment initiates intracellular tyrosyl phosphatase-dependent signaling resulting in the activation of Src tyrosine kinase and the loss of actin stress fibers. Endostatin-induced disassembly of focal adhesions and actin stress fibers disturbs the deposition of the fibronectin matrix. These changes are likely to contribute to the antimigratory effect of endostatin.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
5ß1 integrin (clone JBS5), and goat polyclonal antibodies against 5ß1 integrin were from Chemicon Co. (Temecula, CA). Rabbit polyclonal and mouse monoclonal anticaveolin-1 antibodies, and mouse monoclonal antipaxillin antibodies were from Transduction Laboratories (Lexington, KY). Rabbit polyclonal antibodies against Tyr-416 phosphorylated Src was from New England Biolabs Inc. (Beverly, MA). Rabbit polyclonal antifibronectin and mouse monoclonal antivinculin antibodies were from Sigma.
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 were from passages 3–6. Unless indicated otherwise, the cells were washed twice and incubated in serum-free medium for at least 12 h before treatment with the various proteins or chemicals. All of the experiments were carried out under serum-free conditions.
Immunofluorescence.
Cells cultured on glass coverslips were washed with PBS [170 mM NaCl and 10 mM sodium phosphate buffer (pH 7.4)] and fixed with 3% paraformaldehyde at 4°C for 10 min. When required, the cells were permeabilized with 0.2% Triton X-100 in PBS for 3 min. Nonspecific protein binding sites were then saturated with 5% BSA in PBS for 30 min. The cells were then washed with PBS, and incubated with polyclonal antibodies against fibronectin or monoclonal antibodies against vinculin and paxillin. Rhodamine-conjugated phalloidin (Sigma) was used for the staining of the actin cytoskeleton. Unbound proteins were removed by washing, followed by incubation with Texas Red- or fluorescein isothiocyanate-labeled secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 h. Hoechst fluorochrome 33258 (Sigma) was used for fluorescent labeling of the nuclei. 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.
Immunoprecipitation and Immunoblotting Analysis.
Cells were lysed with RIPA-DOC lysis buffer [50 mM Tris-HCl buffer (pH 8.0), containing 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, 10 mM EDTA, 10 µg/ml aprotinin, 1 µg/ml pepstatin A, 1 µg/ml aminoethylbenzene sulfonyl fluoride, 1 mM OV, and 10 mM sodium fluoride] and subjected to immunoprecipitation. Cell lysates were preabsorbed by incubation with preimmune serum and Gamma-bind Sepharose (Amersham Pharmacia, Uppsala, Sweden) at 4°C in an end-over mixer for 2 h. The beads were removed by centrifugation, and the supernatants were incubated with polyclonal antibodies against 5ß1 integrin or caveolin-1 on ice for 1 h. Gamma-bind Sepharose was then 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 RIPA-DOC buffer. The bound proteins were eluted with Laemmli sample buffer at 95°C and separated in 4–15% gradient PAGE in the presence of SDS (Bio-Rad, Hercules, CA) under reducing conditions and transferred onto nitrocellulose (Schleicher & Schuell, Dassel, Germany). Immunodetection of the proteins was performed essentially as described (25)
.
Metabolic Labeling.
Cells were seeded on plastic culture dishes in complete culture medium and allowed to attach for 1.5 h. After washing, the cells were incubated with endostatin for 3 h in serum-free medium containing [35S]methionine (80 µCi/ml, 1175 Ci/mmol; Amersham Pharmacia Biotech). Subsequently, aliquots of conditioned medium were subjected to immunoprecipitation with antibodies against fibronectin as described above. The eluted polypeptides were separated by 4–15% gradient PAGE in the presence of SDS under reducing conditions and visualized by autoradiography.
Src Family Kinase Autophosphorylation Assay.
HDMECs were treated with 50 nM endostatin for 50 min. The cells were subsequently lysed and subjected to immunoprecipitation with pan-Src antibodies (Src-2; Santa Cruz Biotechnology Inc., Santa Cruz, CA) as described above. The immunoprecipitates were washed three times with RIPA lysis buffer and twice with Src kinase buffer [20 mM HEPES (pH 7.35) and 3 mM MnCl2]. Autophosphorylation was initiated by adding 5 µCi of [
-32P]ATP (5000 Ci/mmol; Amersham Pharmacia Biotech) and carried out in 40 µl of kinase buffer at 30°C for 15 min. The reactions were stopped by adding 10 mM EDTA on ice. The pellets were then washed twice with lysis buffer followed by SDS-PAGE and autoradiography. Incorporated radioactivity was detected and quantitated with Fujifilm BAS-2500 Image Analyzer and MacBAS 2.5 program.
Isolation of the Extracellular Matrix.
Extracellular matrices were prepared according to Hedman et al. (26)
. Briefly, cells were seeded on plastic culture dishes in complete culture medium and allowed to attach for 1 h after which the medium was withdrawn and replaced with serum-free medium containing endostatin or various chemicals in concentrations indicated. After 4 h of incubation the cells were washed with PBS and treated three times with 0.5% sodium deoxycholate in 10 mM Tris-HCl buffer (pH 8.0) for 10 min. Subsequently, the plates were washed with PBS and allowed to dry. The resulting extracellular matrices were extracted in reducing Laemmli sample buffer, solubilized by boiling, and separated by SDS-PAGE. The proteins were then transferred onto nitrocellulose and detected by immunoblot analysis.
Wound-induced Migration Assay.
Confluent HDMECs on glass coverslips in complete culture medium were wounded with a sterile tip and washed with serum-free medium. The cells were treated with 50 ng/ml bFGF and with 50 nM endostatin or 50 nM endostatin in combination with OV immediately after wounding and again after 12 h. After 24 h of incubation the cells were fixed with paraformaldehyde and subjected to immunofluorescence analysis as described above. The migration distance was measured using an inverted phase contrast microscope and UltraView 4.0 software (Perkin-Elmer, Fremont, CA). The mean migration distances were calculated from three reference points on each coverslip by subtracting the distance between the migrating cell fronts measured after 24 h of incubation from that measured at the time of wounding. The results represent three independent experiments.
To ensure that the changes in microvascular endothelial cells observed to be induced by recombinant endostatin were specific for this protein, myelin basic protein was included in all of the experiments as an unrelated peptide control (data not shown).
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
5ß1 Forms Clusters and Associates with Caveolin-1 in Endostatin-treated Cells.5ß1 and vß3 (6)
. To characterize the interaction between endostatin and 5ß1 integrin, we immunostained 5ß1 on HDMECs after the administration of 50 nM recombinant endostatin into the culture medium of serum-starved cells. We observed that endostatin treatment induced clustering of 5ß1 integrin at the HDMEC periphery (Fig. 1)
. The integrin clusters were associated laterally with bundles of actin stress fibers as visualized by coimmunostaining with rhodamine-phalloidin and monoclonal antibodies against 5ß1 integrin. Concomitant loss of actin stress fibers was seen in the endostatin-treated cells compared with untreated control cells with no apparent integrin clusters (Fig. 1)
. These effects were rapid, becoming visible after 30 min of incubation.
|
5ß1 integrin involved increased association with membrane adaptor proteins to enable intracellular signaling, we coimmunostained the endostatin-treated cells with antibodies against caveolin-1, a known integrin-associated transmembrane anchor protein (14
, 27)
. We observed that part of the clustered 5ß1 integrin colocalized with caveolin-1 at the cell periphery (Fig. 2)
. The clustering of 5ß1 integrin and its colocalization with caveolin-1 occurred in the same time scale, appearing after 30 min of incubation with endostatin and persisting up to 1 h of incubation (Fig. 2)
. After 2 h of incubation most of the 5ß1 integrin became redistributed, but some clustering and colocalization with caveolin-1 could still be seen (Fig. 2B)
.
|
5ß1 Integrin in Cultured Endothelial Cells.5ß1, and caveolin-1, coimmunoprecipitation analysis was carried out. Subconfluent cultures of HDMECs were treated with increasing concentrations of endostatin for 30 min and immunoprecipitated with polyclonal antibodies against integrin 5ß1 followed by immunoblotting with antibodies against endostatin and caveolin-1. This analysis revealed that endostatin coimmunoprecipitated with integrin 5ß1 from endostatin-treated cells (Fig. 3A)
. The interaction between caveolin and integrin 5ß1 was dependent on the concentration of endostatin administered to cells while the levels of integrin 5ß1 protein were equal (Fig. 3A
, bottom panel). Similar coimmunoprecipitation results were obtained with monoclonal antibodies against integrin 5ß1 (data not shown). No endostatin was coimmunoprecipitated with integrins vß3 or 4 (data not shown). Also, endostatin and integrin 5ß1 coimmunoprecipitated with caveolin-1 from extracts of endostatin-treated cells (Fig. 3B)
. Antibodies against caveolin-1 or integrin 5ß1 did not recognize recombinant human endostatin as confirmed by immunoblot analysis (data not shown).
|
60,000 Src family kinase in endostatin-treated cells when compared with control cells (Fig. 4A
, bottom panel).
|
60,000 phosphoprotein Src in the endostatin-treated cells (Fig. 4B)
. This increase was dependent on tyrosyl phosphatase activity as orthovanadate treatment inhibited the endostatin-induced increase in Tyr-416-phosphorylated Src (Fig. 4B)
. The levels of total Src protein associated with caveolin-1 were not affected by endostatin treatment (data not shown).
The endostatin-induced increase in Src phosphorylation became visible after 40 min and persisted up to 120 min of incubation (Fig. 4B)
. In vitro kinase analysis of caveolin-1 immunoprecipitates additionally confirmed that increased autophosphorylation of a Mr 60,000 protein was associated with caveolin-1 in endostatin-treated cells (Fig. 4C)
.
To additionally analyze the effect of endostatin on Src phosphorylation, immunofluorescence analysis with antibodies against Tyr-416-phosphorylated Src was carried out. Before the addition of recombinant endostatin, very low levels of Tyr-416-phosphorylated Src staining was visible in cytoplasmic plaque-like structures. After endostatin treatment, intense clusters of staining became apparent in the periphery of the cells, reaching maximal intensity after 60 min of incubation (Fig. 4D)
.
Both Phosphatase and Src Activity Are Required for the Disassembly of Focal Adhesions and Actin Cytoskeleton in Response to Endostatin.
We have observed previously that focal adhesions and actin stress fibers are disassembled in endostatin-treated HDMECs (4)
. Similar changes occur in cells treated with agents decreasing tyrosyl phosphorylation (15)
. To investigate whether the effects of endostatin on actin stress fibers and focal adhesions involve tyrosyl dephosphorylation, we administered the phosphatase inhibitor orthovanadate to HDMECs in combination with endostatin for 1 h. Whereas in untreated control cells distinct focal adhesions and a network of actin stress fibers were observed, in cells treated with endostatin the vinculin staining was restricted to cell-cell junctions and actin was present as cortical bundles with no evident stress fibers (Fig. 5A)
. In cells treated with endostatin in combination with orthovanadate, as well as in cells treated with orthovanadate alone, focal adhesions and stress fibers were present, and cell morphology resembled that of the untreated control cells (Fig. 5A)
. The endostatin-induced clustering of 5ß1 integrin and its increased colocalization with caveolin-1 were not affected with orthovanadate treatment (not shown).
|
. The endostatin-treated cells as well as the cells treated with 100 µM PP1 alone displayed vinculin staining restricted to cell-cell junctions and cortical actin bundles with no stress fibers present (Fig. 5B)
When treated with endostatin in combination with PP1 and endostatin, the cells displayed cytoskeletal morphology comparable with untreated control cells with distinct stress fibers and cytoplasmic focal adhesion complexes (Fig. 5B)
.
Deposition of Fibronectin Matrix Is Altered in Endostatin-treated Cells.
The assembly of insoluble fibronectin matrix fibrils occurs at specific assembly sites located in focal adhesions (22
, 24) . This process is dependent on the binding of soluble secreted fibronectin to integrin 5ß1 (23
, 31)
. As endostatin both associates with integrin 5ß1 and disassembles focal adhesions, we sought to investigate the effect of endostatin on the deposition of the fibronectin matrix. Cells suspended in complete culture medium were allowed to attach onto glass coverslips for 1.5 h. The medium was then removed, and after extensive washing, endostatin was added in serum-free medium. After 3 h, the cells were fixed and stained with antibodies against vinculin and fibronectin. Immunofluorescence analysis revealed that the untreated control cells had formed distinct focal adhesions and produced a fibrillar network of fibronectin matrix underneath the cells. In endostatin-treated cells the vinculin staining was diffusely distributed with no visible cytoplasmic focal adhesions. The levels of fibronectin deposited underneath the endostatin-treated cells were decreased and it formed no apparent fibrillar structures (Fig. 6A)
.
|
, indicating that tyrosyl phosphatase activity is required for endostatin-induced modulation of the fibronectin matrix assembly. The role of Src kinase activity in the altered deposition of fibronectin was also assayed. When endostatin was added in combination with the Src inhibitor PP1 (100 µM), the cells formed numerous focal adhesion structures and a fibrillar fibronectin matrix, indicating that Src kinase activity is involved in the endostatin-induced altered deposition of the fibronectin matrix (Fig. 6A)
. To more quantitatively determine the effect of endostatin on fibronectin matrix deposition, preparations of deoxycholate-insoluble matrices were prepared as described under "Materials and Methods." Immunoblot analysis of the matrices with antibodies against fibronectin indicated that the levels of deposited fibronectin were decreased in cells treated with 50 nM endostatin or 100 µM PP1 alone. In contrast, in cells treated with endostatin in combination with orthovanadate or orthovanadate alone the levels of fibronectin were comparable with that of untreated control cells. Also, in cells treated with endostatin together with PP1, the level of deposited fibronectin was higher than in cells treated with either of the chemicals alone (Fig. 6B)
.
To determine possible alterations in the levels of soluble secreted fibronectin in endostatin-treated HDMEC cultures, cells in complete culture medium were plated on plastic culture dishes and allowed to attach for 1.5 h. The cells were then metabolically labeled, and increasing concentrations of endostatin were added. Aliquots of conditioned medium were collected after 3 h and subjected to immunoprecipitation. In addition, the cell layers were trypsinized, and the cells were counted. Analysis of the amount of fibronectin precipitated per cell revealed that endostatin treatment enhanced the accumulation of soluble fibronectin to the medium in a dose-dependent manner (Fig. 6C)
.
Endostatin Inhibits bFGF-stimulated Endothelial Cell Migration with Concomitant Loss of Focal Adhesions and Impaired Deposition of Fibronectin Matrix.
Endostatin has been shown to inhibit endothelial cell migration (2
, 3)
. To correlate our observations with this effect, we introduced an artificial wound to confluent cultures of HDMECs using a sterile tip. The cells were stimulated to migrate using the angiogenic growth factor bFGF (25 ng/ml) and incubated with endostatin under serum-free conditions, as indicated. The treatments were repeated after 12 h. After 24 h of incubation, the cells were fixed and stained with antibodies against paxillin to visualize focal adhesions, and antibodies against fibronectin to visualize the fibrillar matrix. Staining of nuclei was used to mark the cells migrating on the coverslips. Measurement of the migration distance together with immunofluorescence analysis revealed that the untreated control cells migrated readily across the wound, were rich in focal adhesions at the leading cell edge, and produced a fibrillar fibronectin matrix (Fig. 7)
. The endostatin-treated cells remained immobile at the edges of the wound, lacked focal adhesions, and were unable to assemble a provisional fibronectin matrix (Fig. 7)
.
|
. Increasing the concentration of orthovanadate up to 500 µM did not notably increase the migration of the endostatin-treated cells (not shown). |
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
5ß1 integrin with subsequent increased colocalization with caveolin-1. In addition, caveolin-1 and exogenous endostatin were recovered from cell extracts and subjected to immunoprecipitation with antibodies against 5ß1 integrin and vice versa, additionally indicating colocalization. Studies on the intracellular effects of endostatin treatment revealed that endostatin enhanced Src kinase activity and increased the level of phosphorylated Src associated with caveolin-1 in a phosphatase-dependent manner. Endostatin treatment induced disassembly of focal adhesions and actin stress fibers, and disturbed the deposition of the fibronectin matrix through a mechanism involving Src and tyrosyl phosphatase activity. In addition, wound-induced endothelial cell migration studies indicated that the lack of focal adhesions and fibronectin matrix is associated with the antimigratory effect of endostatin.
Integrin 5ß1 is an important regulator of tumor angiogenesis in vivo, and blocking this integrin by specific antibodies or peptides inhibits bFGF-induced angiogenesis in vitro (13)
. Furthermore, inhibition of integrin 5ß1 function with specific blocking antibodies results in the loss of endothelial cell focal adhesions and actin stress fibers (32)
, resembling the effect of endostatin treatment. Endostatin has been shown to associate with integrin 5ß1 in an RGD-independent manner (6)
. Our observations additionally elucidate the interaction between endostatin and molecules involved in cell-matrix interactions at the plasma membrane. Endostatin treatment induced the clustering of stress fiber-associated integrin 5ß1 on the cell surface in a manner not resembling the pattern of integrin activation when occupied by an immobilized ligand. The endostatin-induced integrin clusters did not appear to colocalize with vinculin or paxillin in focal adhesions, but directly with lateral actin stress fibers.
Additional investigation of the interaction revealed colocalization of integrin 5ß1 and the transmembrane anchor protein caveolin-1 in response to endostatin treatment. In addition, endostatin, through an undefined mechanism, associated with these two membrane-bound proteins. The presence of caveolin-1 in a complex with endostatin and integrin 5ß1 suggest a role for caveolae, liquid-ordered domains involved in membrane trafficking, in the effects of endostatin. However, the interaction between integrin 5ß1 and caveolin-1 has been suggested to occur independently of lipid rafts (14)
as integrins are generally considered not to localize in caveolae (33)
. Other authors suggest that caveolae serve as temporary scaffolding domains for signaling units consisting of integrins, caveolin-1, and Src kinases, and these structures are disassembled in response to integrin activation and recruitment to focal adhesions (16)
. In the light of these findings, our observations imply that endostatin treatment inhibits the formation of focal adhesions and instead, leads to the capping of 5ß1 integrins on the cell surface together with increased or stabilized association with caveolin-1. This interaction then results in the observed activation of the associated signaling molecules. The recruitment of integrin-endostatin complexes to caveolin-containing structures could also occur via a coreceptor for endostatin, such as a heparan sulfate proteoglycan (5
, 7)
.
Protein phosphorylation and dephosphorylation serve as molecular switches in the control of endothelial cell migration and adhesion. The migration-associated assembly and disassembly of focal adhesions and actin stress fibers is regulated through their phosphorylation status (15)
. Important regulators of these processes are the Src family kinases that are activated by integrin ligand binding and regulate the turnover of the focal adhesions. Our results indicate that endostatin acts as a soluble agonist, not an antagonist, for integrin 5ß1, and endostatin treatment leads to increased activation of Mr 60,000 Src associated with caveolin-1. However, these events may occur independently of each other. The activation of Src was inhibited by OV, suggesting phosphatase-dependent activation of the Mr 60,000 Src. Interestingly, the increase in Src activity was relatively sustained, persisting for up to 120 min, which is in contrast with the transient induction of Src phosphorylation by integrins ligated by physiological, immobilized substrates (34)
. This observation, together with the finding that endostatin-induced disassembly of focal adhesions and actin stress fibers was antagonized by the Src inhibitor PP1, which alone is capable of inducing disassembly of the cytoskeleton, suggests that endostatin binding to the cell surface leads to aberrant and perturbed signaling, resulting in excessive turnover of focal adhesions and actin stress fibers. Analogously, a similar absence of focal adhesions and actin stress fibers can be observed in cells transformed with the constitutively active v-Src (35
, 36) . Here it should also be noted that various protein tyrosine phosphatases function in the disassembly of focal adhesions in multiple cell systems (37, 38, 39, 40, 41)
.
Previous studies have indicated that intact focal adhesions and actin stress fibers are required for fibronectin matrix deposition (24) . We observed that endostatin-treated endothelial cells lacked focal adhesions and actin stress fibers, and were unable to assemble fibronectin matrix fibers. In addition, increased levels of soluble secreted fibronectin were found in the conditioned medium of endostatin-treated cells suggesting that the cells are unable to deposit the secreted soluble fibronectin into insoluble matrix fibers. Inhibition of the endostatin-induced focal adhesion disassembly and loss of fibronectin in cells treated with the phosphatase inhibitor orthovanadate or the Src kinase inhibitor PP1 established an association between these two events. This suggests that the Src-mediated disassembly of focal adhesions by endostatin results in altered fibronectin deposition and subsequent accumulation of soluble fibronectin in the culture medium. The disturbed deposition of fibronectin also occurs in v-Src transformed cells (20) . However, the contribution of outside-in signaling from the deficient fibronectin matrix to the disassembly of the cytoskeletal structures cannot be excluded on the basis of our results. Interestingly, the fibronectin matrix has been described to play an important role in angiogenesis in vivo (13) , and agents disrupting the fibronectin matrix have been observed to inhibit angiogenesis, tumor growth, and metastasis (42 , 43) , implying that these observations could correlate with the biological functions of endostatin.
To study the functional significance of the endostatin-induced disassembly of focal adhesions and decreased assembly of the fibronectin matrix, we carried out a bFGF-induced wound migration assay. In agreement with previous observations (2) , we found that endostatin-treated cells stimulated with bFGF were unable to migrate across a wound made to the culture monolayer. In addition, the cells located directly on the edges of the wound lacked focal adhesions on the leading edge and were unable to deposit a provisional fibronectin matrix. This was in marked contrast to the untreated control cells, which had focal adhesions and fibronectin matrix fibers, and which migrated readily across the wound. The finding that impaired migration of endostatin-treated endothelial cells could partly be restored by adding the phosphatase inhibitor orthovanadate, supports the hypothesis that the disassembly of focal adhesions together with decreased deposition of fibronectin contribute to the antimigratory effect of endostatin. However, additional mechanisms are likely to be involved in the endostatin-induced inhibition of endothelial cell migration, because even high doses of orthovanadate were not able to restore the migration of endostatin-treated cells to the level of untreated control cells. One putative mechanism is increased apoptosis, which has been shown to occur in endothelial cells in response to treatment with antagonists of integrins and also endostatin (44 , 45) .
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 Supported by the Academy of Finland, Sigrid Juselius Foundation, Biocentrum Helsinki, Helsinki University Hospital Fund, Novo Nordisk Foundation, Finnish Cancer Foundation, Finnish Medical Foundation, and the University of Helsinki. 
2 To whom requests for reprints should be addressed, at Laboratory of Cell Biology, Biomedicum Helsinki, POB 63 (Haartmaninkatu 8), FIN-00014, University of Helsinki, Finland. Phone 358-9-191-25566; Fax: 358-9-191-25573; E-mail: jorma.keski-oja{at}helsinki.fi
3 The abbreviations used are: bFGF, basic fibroblast growth factor; HDMEC, human dermal microvascular endothelial cell; Tyr-416, tyrosine 416; OV, sodium orthovanadate; RIPA, radioimmunoprecipitation assay.
Received 5/ 8/02. Accepted 7/26/02.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
5ß1 with the central cell-binding domain of fibronectin. Am. J. Pathol., 156: 1345-1362, 2000.5ß1-mediated signaling pathways by fibronectin’s cell adhesion and matrix assembly domains. J. Cell Biol., 141: 241-253, 1998.vß3-mediated endothelial cell migration and angiogenesis by integrin 5ß1 and protein kinase A. J. Biol. Chem., 275: 33920-33928, 2000.vß3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell, 79: 1157-1164, 1994.[Medline]
This article has been cited by other articles:
|
|
 |
|
  S. P. Tabruyn, S. Memet, P. Ave, C. Verhaeghe, K. H. Mayo, I. Struman, J. A. Martial, and A. W. Griffioen NF-{kappa}B activation in endothelial cells is critical for the activity of angiostatic agents Mol. Cancer Ther., September 1, 2009; 8(9): 2645 - 2654. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A G Richter, S McKeown, S Rathinam, L Harper, P Rajesh, D F McAuley, R Heljasvaara, and D R Thickett Soluble endostatin is a novel inhibitor of epithelial repair in idiopathic pulmonary fibrosis Thorax, February 1, 2009; 64(2): 156 - 161. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Silva, G. D'Amico, K. M. Hodivala-Dilke, and L. E. Reynolds Integrins: The Keys to Unlocking Angiogenesis Arterioscler Thromb Vasc Biol, October 1, 2008; 28(10): 1703 - 1713. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Serini, L. Napione, M. Arese, and F. Bussolino Besides adhesion: new perspectives of integrin functions in angiogenesis Cardiovasc Res, May 1, 2008; 78(2): 213 - 222. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T.-Y. Lee, R. M. Tjin Tham Sjin, S. Movahedi, B. Ahmed, E. A. Pravda, K.-M. Lo, S. D. Gillies, J. Folkman, and K. Javaherian Linking Antibody Fc Domain to Endostatin Significantly Improves Endostatin Half-life and Efficacy Clin. Cancer Res., March 1, 2008; 14(5): 1487 - 1493. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Yokoyama, G. Sedgewick, and S. Ramakrishnan Endostatin Binding to Ovarian Cancer Cells Inhibits Peritoneal Attachment and Dissemination Cancer Res., November 15, 2007; 67(22): 10813 - 10822. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Schaffhauser, T. Veikkola, K. Strittmatter, H. Antoniadis, K. Alitalo, and G. Christofori Moderate antiangiogenic activity by local, transgenic expression of endostatin in Rip1Tag2 transgenic mice J. Leukoc. Biol., October 1, 2006; 80(4): 669 - 676. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N.-Q.-N. Nguyen, S. P. Tabruyn, L. Lins, M. Lion, A. M. Cornet, F. Lair, F. Rentier-Delrue, R. Brasseur, J. A. Martial, and I. Struman Prolactin/growth hormone-derived antiangiogenic peptides highlight a potential role of tilted peptides in angiogenesis PNAS, September 26, 2006; 103(39): 14319 - 14324. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. H. Kulke, E. K. Bergsland, D. P. Ryan, P. C. Enzinger, T. J. Lynch, A. X. Zhu, J. A. Meyerhardt, J. V. Heymach, W. E. Fogler, C. Sidor, et al. Phase II Study of Recombinant Human Endostatin in Patients With Advanced Neuroendocrine Tumors J. Clin. Oncol., August 1, 2006; 24(22): 3555 - 3561. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. A. Khan, J. M. Melero-Martin, X. Wu, S. Paruchuri, E. Boscolo, J. B. Mulliken, and J. Bischoff Endothelial progenitor cells from infantile hemangioma and umbilical cord blood display unique cellular responses to endostatin Blood, August 1, 2006; 108(3): 915 - 921. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Monaghan-Benson and P. J. McKeown-Longo Urokinase-type Plasminogen Activator Receptor Regulates a Novel Pathway of Fibronectin Matrix Assembly Requiring Src-dependent Transactivation of Epidermal Growth Factor Receptor J. Biol. Chem., April 7, 2006; 281(14): 9450 - 9459. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Ou-Yang, K.-L. Lan, C.-T. Chen, J.-C. Liu, C.-L. Weng, C.-K. Chou, X. Xie, J.-Y. Hung, Y. Wei, G. N. Hortobagyi, et al. Endostatin-Cytosine Deaminase Fusion Protein Suppresses Tumor Growth by Targeting Neovascular Endothelial Cells Cancer Res., January 1, 2006; 66(1): 378 - 384. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Mehta and A. B. Malik Signaling Mechanisms Regulating Endothelial Permeability Physiol Rev, January 1, 2006; 86(1): 279 - 367. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Celik, O. Surucu, C. Dietz, J. V. Heymach, J. Force, I. Hoschele, C. M. Becker, J. Folkman, and O. Kisker Therapeutic Efficacy of Endostatin Exhibits a Biphasic Dose-Response Curve Cancer Res., December 1, 2005; 65(23): 11044 - 11050. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Baba, Y. Yonemitsu, T. Nakano, M. Onimaru, M. Miyazaki, Y. Ikeda, S. Sumiyoshi, Y. Ueda, M. Hasegawa, I. Yoshino, et al. Cytoplasmic Expression and Extracellular Deposition of an Antiangiogenic Factor, Pigment Epithelium-Derived Factor, in Human Atherosclerotic Plaques Arterioscler Thromb Vasc Biol, September 1, 2005; 25(9): 1938 - 1944. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Nyberg, L. Xie, and R. Kalluri Endogenous Inhibitors of Angiogenesis Cancer Res., May 15, 2005; 65(10): 3967 - 3979. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. M. Tjin Tham Sjin, R. Satchi-Fainaro, A. E. Birsner, V.M. S. Ramanujam, J. Folkman, and K. Javaherian A 27-Amino-Acid Synthetic Peptide Corresponding to the NH2-Terminal Zinc-Binding Domain of Endostatin Is Responsible for Its Antitumor Activity Cancer Res., May 1, 2005; 65(9): 3656 - 3663. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. M. Short, A. Derrien, R. P. Narsimhan, J. Lawler, D. E. Ingber, and B. R. Zetter Inhibition of endothelial cell migration by thrombospondin-1 type-1 repeats is mediated by {beta}1 integrins J. Cell Biol., February 14, 2005; 168(4): 643 - 653. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Y. Zhang, E. G. Teggatz, A.-P. Zou, W. B. Campbell, and P.-L. Li Endostatin uncouples NO and Ca2+ response to bradykinin through enhanced O2-{middle dot} production in the intact coronary endothelium Am J Physiol Heart Circ Physiol, February 1, 2005; 288(2): H686 - H694. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. K. Skovseth, M. J. T. Veuger, D. R. Sorensen, P. M. De Angelis, and G. Haraldsen Endostatin dramatically inhibits endothelial cell migration, vascular morphogenesis, and perivascular cell recruitment in vivo Blood, February 1, 2005; 105(3): 1044 - 1051. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M.A. GRANT and R. KALLUR Structural Basis for the Functions of Endogenous Angiogenesis Inhibitors Cold Spring Harb Symp Quant Biol, January 1, 2005; 70(0): 399 - 417. [Abstract] [PDF]
|
|
|
|
|
|
A.-K. Olsson, I. Johansson, H. Akerud, B. Einarsson, R. Christofferson, T. Sasaki, R. Timpl, and L. Claesson-Welsh The Minimal Active Domain of Endostatin Is a Heparin-Binding Motif that Mediates Inhibition of Tumor Vascularization Cancer Res., December 15, 2004; 64(24): 9012 - 9017. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. NAVARRO, B. ANAND-APTE, and M.-O. PARAT A role for caveolae in cell migration FASEB J, December 1, 2004; 18(15): 1801 - 1811. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. M. Mazzanti, A. Tandle, D. Lorang, N. Costouros, D. Roberts, G. Bevilacqua, and S. K. Libutti Early Genetic Mechanisms Underlying the Inhibitory Effects of Endostatin and Fumagillin on Human Endothelial Cells Genome Res., August 1, 2004; 14(8): 1585 - 1593. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Bix, J. Fu, E. M. Gonzalez, L. Macro, A. Barker, S. Campbell, M. M. Zutter, S. A. Santoro, J. K. Kim, M. Hook, et al. Endorepellin causes endothelial cell disassembly of actin cytoskeleton and focal adhesions through {alpha}2{beta}1 integrin J. Cell Biol., July 5, 2004; 166(1): 97 - 109. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-H. Ahn, J.-S. Kim, H.-K. Yu, H.-J. Lee, and Y. Yoon A Truncated Kringle Domain of Human Apolipoprotein(a) Inhibits the Activation of Extracellular Signal-regulated Kinase 1 and 2 through a Tyrosine Phosphatase-dependent Pathway J. Biol. Chem., May 21, 2004; 279(21): 21808 - 21814. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. A. Wickstrom, K. Alitalo, and J. Keski-Oja An Endostatin-derived Peptide Interacts with Integrins and Regulates Actin Cytoskeleton and Migration of Endothelial Cells J. Biol. Chem., May 7, 2004; 279(19): 20178 - 20185. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Citrin, A. K. Lee, T. Scott, M. Sproull, C. Menard, P. J. Tofilon, and K. Camphausen In vivo tumor imaging in mice with near-infrared labeled endostatin Mol. Cancer Ther., April 1, 2004; 3(4): 481 - 488. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Ricard-Blum, O. Feraud, H. Lortat-Jacob, A. Rencurosi, N. Fukai, F. Dkhissi, D. Vittet, A. Imberty, B. R. Olsen, and M. van der Rest Characterization of Endostatin Binding to Heparin and Heparan Sulfate by Surface Plasmon Resonance and Molecular Modeling: ROLE OF DIVALENT CATIONS J. Biol. Chem., January 23, 2004; 279(4): 2927 - 2936. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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 J. Biol. Chem., September 26, 2003; 278(39): 37895 - 37901. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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 PNAS, April 15, 2003; 100(8): 4766 - 4771. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
