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J. Biol. Chem., Vol. 280, Issue 6, 4544-4552, February 11, 2005
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9
1*
¶
From the
Lung Biology Center, University of California San Francisco, San Francisco, California, 94143-2922 and Thoracic Disease Research Unit, Mayo Clinic College of Medicine, Rochester, Minnesota 55905
Received for publication, November 12, 2004
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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9 subunit die 6–12 days after birth from bilateral chylothoraces suggesting an underlying defect in lymphatic development. However, until now the mechanisms by which the integrin 9
1 modulates lymphangiogenesis have not been described. In this study we show that adhesion to and migration on the lymphangiogenic vascular endothelial growth factors (VEGF-C and -D) are 91-dependent. Mouse embryonic fibroblasts and human colon carcinoma cells (SW-480) transfected to express 91 adhered and/or migrated on both growth factors in a concentration-dependent fashion, and both adhesion and migration were abrogated by anti-91 function-blocking antibody. In SW-480 cells, which lack cognate receptors for VEGF-C and -D, both growth factors induced 91-dependent Erk and paxillin phosphorylation. Human microvascular endothelial cells, which express both 91 and VEGF-R3, also adhered to and migrated on both growth factors, and both responses were blocked by anti-91 antibody. Furthermore, in a solid phase binding assay recombinant VEGF-C and -D bound to purified 91 integrin in a dose- and cation-dependent fashion showing that VEGF-C and VEGF-D are ligands for the integrin 91. The interaction between 91 and VEGF-C and/or -D may begin to explain the abnormal lymphatic phenotype of the 9 knock-out mice. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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subunits and 18 subunits that form at least 25 different heterodimers (4). The integrin 9 subunit forms a single heterodimer with 1 and is expressed in epithelial cells, smooth and skeletal muscle, neutrophils, and a subset of endothelial cells (5, 6). In vitro, the principal demonstrated function of 91 is acceleration of cell migration, an effect that depends on unique sequences within the 9 cytoplasmic domain (7, 8).
In a previous study we inactivated the 9 subunit in mice to better understand the function of 91. In these mice lymph leaked into the pleural space (chylothorax), and the mice died 6–12 days after birth. This phenotype was an unexpected finding which indicated that lymph vessel development and/or function was abnormal (9). On gross inspection, the thoracic duct and peripheral lymphatic vessels were present, but their integrity was compromised, as evidenced by chylothoraces and edema of the thoracic dermis, skeletal muscle, and pleural surface. To date the molecular mechanisms underlying the role of 91 in lymphatic development and/or function remain unexplained.
The vascular endothelial growth factors (VEGF-C and -D)1 are important mediators of lymphatic development (10, 11). VEGF-C and -D constitute a subfamily of VEGF proteins characterized by 48% overall homology, receptor specificity (VEGF-R3), and highly homologous cysteine-rich C-terminal regions (11, 12). These growth factors are secreted as pro-proteins and after enzymatic cleavage to their mature form (13–15), signal through VEGF receptors 2 (VEGF-R2) and 3 (VEGF-R3), inducing angiogenesis and lymphangiogenesis, respectively (16–22). The importance of these VEGF proteins in lymphangiogenesis was demonstrated by their transgenic overexpression in skin, resulting in dermal lymphatic hyperplasia, which could be blocked by soluble VEGF-R3-Ig (23).
Therefore, in this study, we hypothesized that the lymphatic abnormality in 9 knock-out mice could be explained by an interaction between 91 and VEGF-C and/or -D. To address this question we used 9-transfected cell lines and primary microvascular endothelial cells to assess in vitro cell adhesion, migration, and receptor signaling and purified 91 and VEGF-C or -D protein for solid phase binding assays.
We found that 9-transfected cells and primary microvascular endothelial cells, which endogenously express 91, utilize 91 to adhere to and migrate on VEGF-C and -D. This effect was inhibited by the specific 91-blocking antibody Y9A2 and siRNA silencing of 9 protein expression. Furthermore, VEGF-C and -D directly bound to 91 in a solid phase protein binding assay and activated 91 signaling, as evidenced by Erk 1/2 and paxillin phosphorylation that was inhibited by anti-91 antibody. These novel findings therefore identify the growth factors VEGF-C and -D as ligands for 91 and provide a potential explanation for the abnormal lymphatic phenotype of the 9 knock-out mouse.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Cells and Cell Culture—9- and mock-transfected mouse embryonic fibroblasts (MEF) and SW-480 cells were made as described previously (8, 24). Cells were grown in Dulbecco's minimum essential medium (DMEM) supplemented with 10% fetal calf serum (HyClone), 1% penicillin/streptomycin (Sigma), and stable clones maintained with 2.5 µg/ml puromycin (Invitrogen, MEF) or 1 mg/ml G418 (Sigma, SW-480). Primary adult human dermal microvascular endothelial cells (HMVEC, Cambrex, East Rutherford, NJ) were grown in cell-specific growth factor-supplemented nutrient media (Cambrex, EBM-2). Schneider Drosophila S2 cells (Invitrogen) used for production of recombinant VEGF-C and -D proteins were grown in Schneider S2 media (Invitrogen) supplemented with 10% fetal calf serum and 1% penicillin/streptomycin at 27 °C and no CO2. Stable clones were maintained in 25 µg/ml blasticidin (Invitrogen).
Synthesis and Purification of VEGF-C and -D—The cDNAs encoding the fully processed and active forms of VEGF-C (GenBankTM accession number NM-005429) and VEGF-D (GenBankTM accession number NM-004469) were expressed in Schneider Drosophila S2 cells (Invitrogen). The following primers were used to introduce 5' NcoI and 3' XbaI restriction sites for cloning into the multiple cloning site of the insect expression vector pCoBlast/MT/BiP/V5-His (Invitrogen): 5' VEGF-D, 5'-gcagccatggtttgcggcaactttc-3'; 3' VEGF-D, 5'-cgatctagatcttctgataattgagtatg-3'; 5' VEGF-C, 5'-gcgaccatgggcacattataatacag-3'; and 3' VEGF-C, 5'-cggtctagaacgtctaataatggaatg-3'. S2 cells were transfected by calcium phosphate precipitation. Individual clones were isolated by limiting dilution in 25 µg/ml blasticidin and screened by Western blotting cell supernatants with anti-V5 antibody conjugated to horseradish peroxidase (V5-HRP, Invitrogen).
Transfected S2 cells were initially grown in 15-cm culture plates (Corning) and then transferred to 1-liter spinner flasks (Corning) for 5 days in reduced serum conditions. Protein secretion was induced with 500 µM CuSO4 (Sigma) for 5 days. The supernatant was collected following centrifugation (3000 x g for 10 min at 4 °C) and then filtered, dialyzed, and concentrated (Vivaflow 200, VivaScience Inc., Carlsbad, CA) in binding buffer (0.5 M NaCl, 50 mM Na2PO4, pH 8.0). Concentrated supernatant containing VEGF-C or -D was then applied to a Ni2+ column (ProBond, Invitrogen), and the column was washed with binding buffer containing 50 µM imidazole to remove contaminating proteins; the bound VEGF was eluted with buffer containing 300 µM imidazole. The eluents were then dialyzed against 150 mM NaCl and 20 mM Tris-HCl, pH 7.5. Purity was assessed by silver staining (GelCode SilverSNAP, Pierce).
Immunoprecipitation, SDS-PAGE, and Western Blot Analysis—For immunoprecipitation of VEGF-R3, HMVEC were grown in 6-well plates with full growth medium until 70% confluent and subsequently in basal medium with 0.1% fetal bovine serum for 4 h. VEGF-C or -D was then added to the cells for 5 min, and the cells were washed with PBS/sodium orthovanadate (NaVi 10 mM) and then lysed with buffer containing 20 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X, 0.5% sodium deoxycholate, 10% glycerol, 25 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 1 mM NaVi, and protease inhibitors (Complete Mini EDTA-free, Roche Applied Science). Lysates were centrifuged at 14,000 x g for 10 min at 4 °C and the precleared lysates immunoprecipitated with 2 µg of rabbit anti-VEGF-R3 antibody bound to 30 µl of protein A-Sepharose beads (Amersham Biosciences). The beads were washed five times with lysis buffer, resuspended in Laemmli sample buffer, boiled at 95 °C for 6 min, resolved on 8% SDS-PAGE under reducing conditions, and transferred to polyvinylidene difluoride membranes (Immobilon-P, Millipore, Billerica, MA). The membrane was blocked in 5% milk/Tris-buffered saline with 0.1% Tween (TBST) for 1 h at room temperature and then probed with anti-phosphotyrosine antibody.
For immunoblotting of VEGF-C, VEGF-D, 91, Erk 1/2, and paxillin, proteins were resuspended in Laemmli sample buffer and resolved on 15, 10, or 8% SDS-PAGE before transfer to a polyvinylidene difluoride membrane. The membrane was probed with the appropriate specific primary antibodies, washed three times with TBST, and subsequently probed with horseradish peroxidase-conjugated secondary antibodies and developed using chemiluminescence (ECL, Amersham Biosciences).
For analysis of Erk 1/2 and paxillin phosphorylation, SW-480 cells were grown to 70–80% confluence in full growth media and then treated with 20 µg/ml of cycloheximide. Cells were trypsinized, kept in suspension for 2 h in 6-well plates coated with 1% BSA (Sigma). Subsequently, 1 x 106 cells/ml untreated or pretreated with relevant blocking antibodies were seeded into separate 6-well plates coated with either 1% BSA, VEGF-C or -D (3 µg/ml), or Tnfn3RAA (2.5 µg/ml). Tnfn3RAA is an 91-specific ligand, which is a recombinant form of the third fibronectin type 3 repeat of tenascin C in which the arginine-glycine-aspartic acid sequence is mutated to RAA, as described previously (25, 26). After 60 min, cells were washed with PBS/NaVi (1 mM) and then lysed with a buffer containing 20 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X, 25 mM NaF, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 1 mM NaVi, and protease inhibitors (complete mini EDTA-free, Roche Applied Science). Lysates were centrifuged at 14,000 g at 4 °C for 10 min, and Western blotting was performed as outlined above.
Flow Cytometry—Cultured cells were trypsinized, washed with PBS, blocked with normal goat serum at 4 °C for 10 min, incubated with primary antibody for 20 min at 4 °C and then with phycoerythrin-conjugated goat anti-mouse antibody. Labeled cells were suspended in PBS, and fluorescence was determined for 5,000 cells with a flow cytometer (FACSort, BD Biosciences). Primary mouse monoclonal antibody Y9A2 (Wang, 1996 (10 µg/ml)), was used to detect 91 and 9D9f9 (5 µg/ml, gift from Dr. K. Alitalo) to detect VEGF-R3.
Adhesion Assay—96-well non-tissue culture flat-bottomed microtiter plates (ICN, Linbro/Titertek, Aurora, OH) were coated with VEGF-C or -D at 4 °C overnight and then blocked with 1% BSA (Sigma) in DMEM for 30 min at 37 °C. After trypsinization cells were incubated with or without the 91-blocking antibody Y9A2 (20 µg/ml) for 30 min on ice, and 5 x 104 cells/well were seeded (8). Control wells were treated with 1% BSA in DMEM. The plates were centrifuged (top side up) at 10 x g for 5 min and incubated for 1 h (MEF) or 2 h (HMVEC) at 37 °C. Non-adherent cells were removed by centrifugation (top side down) for 5 min at 48 x g, and adherent cells were fixed and stained with 1% formaldehyde, 0.5% crystal violet, and 20% methanol for 30 min after which the wells were washed three times with PBS. Following solubilization in 2% Triton X-100, the number of adherent cells was evaluated by measuring absorbance at 595 nm in a microplate reader (Spectra-Max 190, Molecular Devices, Sunnyvale, CA).
Migration Assay—The undersurfaces of 12-well, 8-µM Transwell plates (Corning Costar, Cambridge, MA) were coated with the relevant ligand (VEGF-C or -D) or 1% BSA as a binding control at 4 °C overnight. Wells were then washed with PBS and blocked with 1% BSA in DMEM or PBS for 60 min at 37 °C. After trypsinization, 5 x 104 cells in DMEM were incubated with or without Y9A2 and/or MAZ51 for 30 min on ice and then seeded into the top chamber of the Transwell (8). 1% fetal calf serum was added to the bottom well to serve as a chemoattractant, and the plates were incubated at 37 °C for 3 h. Cells that migrated and adhered to the bottom surface of the Transwell membrane were fixed with DiffQuik fixative (Pierce), and the non-adherent cells were gently removed from the top with a Q-tip. After air drying, the membranes were stained with DiffQuik (Pierce), washed in water, and air-dried, and then the membranes were cut from the Transwell and mounted onto glass slides. Cells were counted in 10 high power (x25) fields for each condition.
RNA Silencing of 91—HMVEC were grown in 10-cm dishes with full growth media (Cambrex) until 70% confluent. Cells were then washed twice with serum-free media and transfected with siRNA (20 µM, Ambion, catalog no. 86946) targeted to exon 4 of the 9 integrin using siPort Amine (Ambion) in Opti-MEM media. Cell media were replaced with full growth media 6 h after transfection and daily thereafter. Transfection efficiency was assessed by flow cytometry analysis of 91 expression 48–72 h after transfection and compared with mock and untransfected cells of the same passage. Cell adhesion assays using VEGF-C or VEGF-D as substrate were performed using transfected cells in which 91 was successfully silenced and compared with untransfected and transfected cells in which 91 was not silenced.
Production of Non-blocking Monoclonal Antibody to 91—A non-blocking antibody to 91 was produced for detection of integrin bound to the VEGF protein in binding assays. Murine L2 cells (ATCC) transfected with human 9 were injected into mice every 14 days for a total of three injections. 3 days after the final injection, lymphocytes were harvested and spin-combined with SP2/0 myeloma cells as described previously (27). After addition of selection media (RPMI 1640, 10% fetal calf serum, 1% penicillin/streptomycin, 1% sodium pyruvate, L-glutamine), single clones were selected, and the supernatants were screened by differential flow cytometry of mock- and 9-transfected cells. Antibody was purified by gradient anion exchange.
Metabolic Labeling of Cells and Immunoprecipitation—To determine whether A9A1 was able to immunoprecipitate the 91 integrin efficiently, 9-transfected SW-480 cells were grown for 4 h in methionine-free DMEM supplemented with 1% penicillin/streptomycin and then incubated with 0.5 mCi of 35S for 24 h. Lysates of cells labeled with [35S]methionine were then precleared with protein A-Sepharose for 1 h at 4 °C. The precleared supernatant was incubated with 20 µg of the primary antibody A9A1 at 4 °C overnight. The beads and bound protein were washed four times with buffer containing 100 mM Tris, 300 mM NaCl, 1% Triton X-100, 0.1% SDS and then boiled in reducing Laemmli's buffer at 95 °C for 6 min. Protein samples were separated by 8% SDS-PAGE, and autoradiography was performed after 1 week at –80 °C.
Immunoaffinity Purification of Human Integrin 91—9-Transfected SW-480 cells (24) were lysed with buffer containing 100 mM N-octylglucosylpyranoside (ICN), 150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 1 mM MgCl2 and CaCl2, 1 mM NaVi, 1 mM phenylmethylsulfonyl fluoride overnight at 4 °C. After centrifugation (12,000 x g, 15 min) and filtering, lysate containing 91 was applied to an 9 antibody (A9A1) column. The column was washed with wash buffer (20 mM N-octylglucosylpyranoside, 150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 2 mM MgCl2 and 0.1 mM CaCl2) and a second time with wash buffer containing 500 mM NaCl. Bound integrin was eluted in 150 mM NaCl at pH 3.0 into microcentrifuge tubes containing Tris, pH 9.0, to bring the final pH to 7.4. Eluents were analyzed for 91 by Western blotting with the rabbit polyclonal antibody 1057 (5).
Immunoaffinity Purification of Human Integrin v6—Secreted v6 integrin was collected from conditioned media of Chinese hamster ovary cells stably transfected with truncated forms of each subunit of the integrin as described previously (28). The integrin was purified using immunoaffinity chromatography on columns with the v6 antibody R6G9 covalently cross-linked to protein A-Sepharose beads. Bound proteins were eluted with 100 mM glycine, pH 3, into 0.05 volume of 1 M Na2PO4, pH 8 (29).
Binding Assay—Direct binding of VEGF-C and -D to 91 was assessed by a solid phase binding assay in non-tissue-coated 96-well microtiter plates (Nunc ImmunoPlate, Naperville, IL). Recombinant VEGF-C or -D (5 µg/ml) was attached to the plates, and purified 91 was added for 2 h at room temperature in the presence or absence of 10 mM EDTA. Following five washes with PBS/1% BSA/0.05% Tween, the extent of 91 binding was detected using the A9A1 antibody (20 µg/ml, 1 h at 37 °C). After incubation with biotinylated rat anti-mouse antibody (1:250, 1 h, room temperature), streptavidin-horseradish peroxidase was added for 20 min at room temperature followed by 3,3',5,5'-tetramethylbenzidine substrate solution (Pharmingen). Absorbance was then measured at 450 nm with a microplate reader (Molecular Devices). The functional integrity of the purified 91 integrin was assessed in a similar assay using the 91-specific ligand Tnfn3RAA (5 µg/ml) as described previously (25, 26).
Statistical Methods—For statistical analysis of adhesion and migration assays comparisons were made by analysis of variance with a post-hoc Tukey's test. Statistical significance was assumed at p 
0.05 with respect to a two-tailed probability distribution. Data are presented as mean values ± S.E. unless otherwise stated from at least three separate experiments.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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23–24 kDa). In addition, immunoblotting with VEGF-C- and -D-specific antibodies (right panels) showed that the V5-tagged proteins were indeed VEGF-C and -D. The purity of each recombinant protein was determined by silver staining of 15% SDS-polyacrylamide gels as shown in Fig. 1B. VEGF-C and -D-V5 induced phosphorylation of their cognate receptor, VEGF-R3. Fig. 1C shows the expected VEGF-R3 isoforms and confirms the activity of the purified VEGF proteins.
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91-Dependent—Adhesion assays were performed using 9- and mock-transfected MEF and SW-480 cells that do not express VEGF-R2 or -R3, (11, 30) (data not shown). Flow cytometry with the anti-91 antibody Y9A2 showed robust expression of 91 in both 9-transfected cell types and no expression in mock transfectants (Fig. 2A). 9-Transfected MEF demonstrated a concentration-dependent adhesion to both VEGF-C and -D (Fig. 2, B and C). This effect was abolished by the 91-blocking antibody Y9A2 (p < 0.05). In contrast, mock-transfected MEF did not adhere to either VEGF protein above background levels of attachment to BSA-coated wells. Similarly, 91-dependent adhesion on both VEGF-D (Fig. 2D) and VEGF-C (data not shown) was demonstrated in SW-480 cells.
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91 Integrin—We have shown previously that in SW-480 cells, activation of the 91 integrin by established ligands results in phosphorylation of Erk 1/2 and paxillin. To assess whether binding of VEGF-C or -D similarly results in 91-induced signaling, phosphorylation of these proteins was compared in mock- and 9-transfected SW-480 cells in the presence or absence of the 91-blocking antibody. Fig. 3A shows that after 15 min of 9 transfectants binding to adherent Tnfn3RAA (lanes 3 and 4), VEGF-C (lanes 5 and 6) or VEGF-D (lanes 7 and 8), Erk 1/2 phosphorylation was increased in an 9-dependent fashion. Similar to 1% BSA (lane 1), this response to VEGF was not seen in mock-transfected cells (lane 2). Paxillin phosphorylation was also induced after 15 min in 9 transfectants and was inhibited by the 91-blocking antibody (Fig. 3B).
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91-Dependent Cell Adhesion Is Demonstrable in Primary Endothelial Cells—To verify the biological significance of the results obtained with the transfected cell lines and to understand the role of 91 in adhesion to VEGF-C and -D in the presence of their cognate receptor VEGF-R3, HMVEC were also studied. HMVEC were found to express both VEGF-R3 and 91 as measured by flow cytometry (Fig. 4, A and B). Essentially, all of the cells expressed VEGF-R3 (Fig. 4A), belying their lymphatic origin, whereas the expression of 91 varied from 40–55% (Fig. 4B) depending on the lot purchased and decreased with increasing passage number. As a result, all experiments were performed between passages 3 and 7. Like 9-transfected cell lines, HMVEC demonstrated concentration-dependent adhesion to both VEGF-C (Fig. 4C) and VEGF-D (Fig. 4D). Adhesion to both substrates was inhibited by the 91-blocking antibody Y9A2 (p < 0.05) to the levels measured in wells coated with 1% BSA, demonstrating that the presence of VEGF-R3 is not sufficient to mediate cell adhesion to VEGF-C or -D.
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91 was silenced using siRNA transfection (Fig. 5). The effectiveness of siRNA directed against exon 4 of 9(9 siRNA) was assessed by measuring 9 expression on the cell surface by flow cytometry (Fig. 5A). The expression of 9 in these cells was compared against cells transfected with mock siRNA and cells not treated with siRNA (No siRNA). Adhesion assays were then performed using these cell populations. Fig. 5B shows that knock-down of 9 in HMVEC cells significantly inhibits cell adhesion on both VEGF-C and VEGF-D. Mock siRNA cells adhered to VEGF-C or VEGF-D substrate to the same degree as non-transfected HMVEC.
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91-Dependent—We have shown previously that one of the principal functions of 91 is enhancement of cell migration. Because migration of lymphatic endothelial cells into mesenchyme enriched for VEGF-C (48) and/or VEGF-D is potentially an important step in lymphangiogenesis, we next sought to determine the role of 91 in migration on VEGF-C and -D. 9-Transfected MEF migrated on VEGF-C (Fig. 6A) and -D (Fig. 6B) in a concentration-dependent manner, whereas mock-transfected cells did not migrate on any concentration above values seen for migration on BSA. This effect depended on ligation of 91, because migration on VEGF-C or -D was inhibited by the 91-blocking antibody. Similar results were obtained for HMVEC, which also demonstrated concentration-dependent migration on VEGF-C (Fig. 6C) and VEGF-D (Fig. 6D) that was inhibited by the 91-blocking antibody.
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91 to HMVEC cell migration, cells were treated with the 91-blocking antibody and/or the VEGF-R3-blocking chemical MAZ51 (49). Fig. 6E shows that blocking either 91 or VEGF-R3 significantly inhibits cell migration. These results suggest that both 91 and VEGF-R3 are required for maximal cell migration on immobilized VEGF.
Production of Non-blocking 91 Antibody—A mouse anti-human monoclonal antibody (A9A1) was produced to serve as a detection antibody for 91 in VEGF-C or -D solid phase binding assays. Flow cytometry using the antibody A9A1 shows detection of the 91 integrin on 9-transfected MEF to a similar level as that of Y9A2, an established 91 integrin antibody (Fig. 7A). To further characterize its specificity, A9A1 was used to immunoprecipitate lysates of [35S]methionine-labeled 9-transfected SW-480 cells. Fig. 7B shows an autoradiograph of immunoprecipitated protein samples separated by 8% SDS-PAGE where the 9 and 1 bands of the heterodimer are clearly seen. Fig. 7C shows that in contrast to Y9A2, a blocking antibody to 91, A9A1 did not inhibit adhesion of 9-transfected MEF to the 91-specific ligand Tnfn3RAA. This antibody was thus suitable for use in 91-VEGF binding assays.
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91 Integrin—To determine whether 91 directly binds to VEGF-C and/or -D, solid phase binding assays were performed using purified VEGF-C or -D and 91 purified from 9-transfected SW-480 cells (24) using an A9A1 affinity column. Fig. 8, A and B, shows a Coomassie stain (reduced, 8% SDS-PAGE) and immunoblot of eluted 91 (9 160 kDa, 1 130 kDa). This purified integrin was still capable of binding to the 91-specific ligand Tnfn3RAA, an effect that was inhibited by 10 mM EDTA (Fig. 8C).
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91—Solid phase binding assays demonstrated robust concentration-dependent adhesion of 91 to VEGF-C and -D, both at 5 µg/ml (Fig. 9, A and B). In both cases, binding was completely inhibited by chelating divalent cations (EDTA, 10 mM) as expected for authentic integrin-ligand interactions. The irrelevant integrin v6 (28) showed no binding to either VEGF-C or -D.
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DISCUSSION |
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9-transfected cells (lacking VEGF receptors) adhere and migrate on fully processed VEGF-C and -D. These cellular responses require functional 91 because they are inhibited by a blocking antibody. These results alone suggest that an interaction between 91 and the VEGF-C and -D proteins can affect cell behavior in the absence of VEGF-R3, the cognate receptor of the growth factors. Importantly, 91was also shown to mediate adhesion and migration on VEGF-C and -D in primary human endothelial cells that co-express 91 and VEGF-R3. Inhibition of migration by the 91 antibody and/or by blocking VEGF-R3 suggests that VEGF-R3 is not sufficient to mediate maximal migration on VEGF-C or -D. Solid phase binding assays of purified VEGF-C or -D and 91 verified that these cell functions are a result of VEGF-C and -D binding 91 in an integrin-specific and cation-dependent manner. Moreover, binding of VEGF-C or -D to 91 leads to induction of downstream signals as demonstrated by 91-dependent Erk 1/2 and paxillin phosphorylation. The finding that growth factors (VEGF-C and -D) bind to an integrin (91) highlights a novel and important mechanism of integrin-growth factor interaction.
A number of previous studies have described cooperative interactions between integrins and growth factors (reviewed in Refs. 31 and 32). Growth factor ligation of its cognate receptor has been shown to lead to close physical association with integrins. For example, ligation of the platelet-derived growth factor receptor not only allowed its co-immunoprecipitation with the v3 integrin but also potentiated v3-dependent cell migration (33). In addition, input from VEGF-R2, stimulated by VEGF-A, has been shown to activate the v integrins v3 and v5 and the 1 integrins 51 and 21 (34).
Activated integrins have also been shown to modulate growth factor protein and receptor expression and signaling. For example, integrin 64 increases VEGF-A translation in breast carcinoma cells through inactivation of the translational repressor 4E-BP1 (35). In addition, integrin-matrix interactions modulate expression of VEGF and fibroblast growth factor receptor 1 and 2 expression on HMVEC (36). The functional importance of growth factor/integrin interactions has also been demonstrated in vivo where blocking antibodies to v3 and v5 integrins inhibit basic fibroblast growth factor- and VEGF-induced angiogenesis, respectively (37). Finally, mice lacking the integrin v5 are specifically protected from the increases in vascular permeability induced by VEGF-A, again presumably through cross-talk with VEGF-R2 (38).
In the current study, we describe a novel mechanism of growth factor-integrin interaction, direct ligation of an integrin by a growth factor. This interaction thus provides a mechanism for VEGF-C and -D to affect directly cell behavior (i.e. cell adhesion and migration) even in the absence of their cognate receptor, VEGF-R3. Furthermore, even in cells expressing the cognate receptor, ligation of the integrin 91 is required for stable cell adhesion to these growth factors and substantially enhances cell migration across them. Our results do not exclude the possibility that 91 may modulate cell function by activating another receptor capable of binding VEGF or inhibiting a VEGF-R3 suppressor. However, taken together these results strongly suggest that VEGF-C and -D directly bind to 91. We speculate that simultaneous binding of both 91 and VEGF-R3 by VEGF-C and/or -D, which is present at high concentration in the extracellular matrix, results in co-clustering of both receptors, facilitating cooperative interactions between the two receptors as demonstrated by our cell migration data. The specific binding sites on VEGF-C and -D and their relationship to the binding sites on other 91 ligands, such as tenascin-C and osteopontin, are as yet undetermined. Because of the lack of a conserved binding sequence for known 91 ligands, determination of this site for VEGF-C or -D would be difficult. Because the VEGF family members share a VEGF homology domain our results suggests that other VEGF family member proteins may also interact with 91. It is also clear that in addition to integrins, other cell surface proteins can modulate VEGF responses, such as the other VEGF receptors (39) and the neuropilins (40). The potential interactions between these modulating proteins and 91 remain to be determined.
Normal vessel development requires the coordinated function of both cellular and extracellular regulatory and effector proteins to ensure an optimal milieu for correct vessel morphogenesis and function (41). The molecular regulators of angiogenesis include the VEGF family of proteins and receptors (VEGF-R1, -R2, -R3), acidic and basic fibroblast growth factors, angiopoietins and Tie receptors, ephrins, integrins, and matrix metalloproteases (10, 42). The molecules that regulate lymphangiogenesis have not been as well characterized. However, it is clear that VEGF-C and -D are key modulators of lymphatic development and function (21, 22), although their relative roles remain to be determined (19). These growth factors induce lymphatic endothelial cell growth, survival, and migration in vitro (16) and lymphangiogenesis in vivo (43), mediated at least in part through their activation of VEGF-R3. In addition, loss of function mutations in VEGF-R3 have been described in a significant subgroup of patients with congenital human lymphedema (44).
The 91 integrin has now been described to interact with a relatively large number of ligands including tenascin C, osteopontin, vascular cell adhesion molecule-1, coagulation factor XIII, and several members of the ADAMs family of transmembrane metalloproteinases (7, 24, 45–47). The biological significance of 91 interactions with most of these ligands remains to be determined. In contrast, we now show that the ligands VEGF-C and -D play a role in endothelial cell adhesion and migration, key cellular functions required for lymphangiogenesis. The most dramatic phenotypic feature of 9 null mice is the presence of bilateral chylothoraces, suggesting a functional defect in the collecting lymphatics of the thorax. Subtle edema and the accumulation of lymphocytes around some peripheral lymphatics provide further support for a role of this integrin in lymphangiogenesis (9). The phenotype of VEGF-D null mice is yet to be published; however, it is clear from E10.5 VEGF-C heterozygote mice that VEGF-C is expressed in mesenchymal cells surrounding the jugular vein toward which endothelial cells that already have a lymphatic differentiation (Prox-1-positive) must migrate (48). Given the specialized role that 91 plays in accelerated cell migration (8), we speculate that abnormal migration on VEGF-C and/or -D might be responsible for the functional defects that occur in 9 null mice. However, the fact that lymphatic vessels are formed in these mice suggests that either the presence of VEGF-R3 or some other unidentified receptor(s) can partially compensate for the loss of 91.
In summary, the findings of this study describe a novel integrin-growth factor interaction whereby the lymphangiogenic proteins VEGF-C and -D bind the integrin 91. Although the precise mechanisms by which binding of VEGF-C and/or -D to 91 contributes to lymphatic development remain to be fully elucidated, these data strongly support a role for this interaction in explaining the lymphatic defects in 9 knock-out mice.
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FOOTNOTES |
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¶ To whom correspondence should be addressed: Lung Biology Center, University of California, San Francisco, Box 2922, San Francisco, CA 94143-2922. Tel.: 415-514-4270; Fax: 415-514-4278; E-mail:deans{at}itsa.ucsf.edu.
1 The abbreviations used are: VEGF, vascular endothelial growth factor; VEGF-R, vascular endothelial growth factor receptor; MEF, mouse embryonic fibroblasts; HMVEC, human microvascular endothelial cells; BSA, bovine serum albumin; Tnfn3RAA, 9-specific ligand, recombinant third fibronectin repeat of tenascin C in which RGD is mutated to RAA; siRNA, small interfering RNA; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; NaVi, sodium orthovanadate; Erk, extracellular signal-regulated kinase.
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ACKNOWLEDGMENTS |
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