Correspondence to: Shaoqing Tang, Albert Einstein College of Medicine, Forchheimer Building G-42, 1300 Morris Park Ave., Bronx, NY 10461. Tel:(718) 430-2365 Fax:(718) 430-8989 E-mail:tang{at}aecom.yu.edu.
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Abstract |
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Migration, proliferation, and tube formation of endothelial cells are regulated by a protein kinase C isoenzyme PKC. A full-length cDNA encoding a novel 20-kD protein, whose expression was PKC
-dependent, was identified in endothelial cells, cloned, characterized, and designated as theta-associated protein (TAP) 20. Overexpression of TAP20 decreased cell adhesion and enhanced migration on vitronectin and tube formation in three-dimensional culture. An antiintegrin
vß5 antibody prevented these TAP20 effects. Overexpression of TAP20 also decreased focal adhesion formation in
vß3-deficient cells. The interaction between TAP20 and ß5 integrin cytoplasmic domain was demonstrated by protein coprecipitation and immunoblotting. Thus, the discovery of TAP20, which interacts with integrin ß5 and modulates cell adhesion, migration, and tube formation, further defines a possible pathway to angiogenesis dependent on PKC
.
Key Words:
TAP20, integrin, PKC, endothelial cells, migration
INTEGRIN receptors mediate the interactions between cells and extracellular matrix (ECM),1 including endothelial cell (EC) adhesion and migration, which are required events for angiogenesis (-actinin (
Integrin affinity modulation is a cell type and integrin-specific process; therefore, the interactions of integrins with the abundant and integrin-nonspecific proteins discussed above may not definitively explain integrin affinity regulation. Recently, several proteins interacting with specific integrin subunit cytoplasmic tails have been identified. ß3-endonexin, a novel 111-residue polypeptide that is capable of binding the cytoplasmic tail of the ß3 integrin subunit was identified by a yeast two-hybrid screening strategy (IIb. Transient coexpression of integrin receptor
IIbß3 and ß3-endonexin in CHO cells revealed that ß3-endonexin can modulate the affinity state of
IIbß3 in a manner that is structurally specific and subject to metabolic regulation. A ß2 integrin cytoplasmic domain binding protein, cytohesin-1, has been found to increase
Lß2-mediated cell adhesion (
IIb tail (
Modulation of integrin function, in particular that of the vitronectin (VN) receptors vß3 and
vß5, is an important process controlling angiogenesis. In vivo angiogenesis models have defined two distinct angiogenic pathways: a pathway using the
vß3 integrin that mediates angiogenesis induced by fibroblast growth factor 2 or by tumor necrosis factor
; and a pathway initiated by vascular endothelial growth factor (VEGF), transforming growth factor
, or PKC that is linked to integrin
vß5 (
, prevents EC migration, proliferation, and tube formation in vitro, thus raising the possibility that regulation of EC function by PKC
is accomplished by modulation of
vß5 function. One mechanism by which PKC
might regulate
vß5 function is by controlling the expression of one or more integrin regulatory proteins.
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Materials and Methods |
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Molecular Cloning
RNA display was performed using an RNAmap Kit (GenHunter) according to the manufacturer's instructions. The resulting PCR products were cloned into plasmid pBluescript for sequencing analysis. To screen the rat PC12 cDNA gt11 library (Clontech) for full-length theta-associated protein (TAP) 20, both the 5' and 3' insert screening amplimers of
gt11 as the forward primers and a TAP20 3' end sequence ACCATAAGAATGCAGACAAGA as the reverse primer were used for PCR reaction with pfu DNA polymerase (Stratagene). The PCR products were cloned into the pBluescript plasmid (Stratagene) and sequenced with T7 and T3 primers.
Plasmids and Cell Culture
To explore the function of TAP20 in mammalian cells, we constructed a TAP20 + green fluorescent protein (GFP) expression plasmid. The EGFP (enhanced GFP) gene from plasmid pEGFP-C1 (Clontech) including its 5' end cytomegalovirus (CMV) promoter to 3' end polyA signal region, was inserted into the EcoRI-KpnI restriction sites (non-multicloning sites) to create a GFP/pRc plasmid. TAP20 cDNA was then cloned into the HindIII-XbaI restriction sites in the plasmid GFP/pRc. Thus, in this vector the TAP20 and EGFP genes are controlled by separate CMV promoters. To localize TAP20 protein in the cell, we cloned the TAP20 cDNA into the BglII-SmaI restriction sites in plasmid pEGFP-C1 for expression of a GFP-TAP20 fusion protein in mammalian cells.
ECV304 cells (a human bladder carcinoma cell line, distributed by American Type Culture Collection) were cultured in M199 medium supplemented with 10% FBS and antibiotics (GIBCO BRL), MV3 cells were cultured in DMEM medium with 10% FBS and antibiotics, and human umbilical vein ECs (HUVEC) were cultured in M199 with 20% newborn calf serum, and 5% human serum, at 37°C in a humidified 5% CO2 atmosphere. Monolayers of cells were transfected with either control vector or TAP20 construct using lipofectin (GIBCO BRL) or GenePorter (Gene Therapy System) for HUVEC. 1 d after transfection of GFP-containing plasmids, cells were harvested and suspended in Hanks' buffer (137 mM NaCl, 5.4 mM KCl, 5.6 mM dextrose, 4.2 mM NaHCO3, 0.42 mM Na2HPO4, 0.44 mM KH2PO4, pH 7.4), and were sorted by GFP fluorescence using a FACScan flow cytometer (Becton Dickinson). The sorted cells were recultured in the complete medium. Expression of TAP20 was confirmed by Western analysis using the anti-TAP20 antibody. The sorted cells were tested in the cell adhesion, migration, and tube formation experiments.
Northern Transfer Analysis
Total RNA was prepared using Trizol (GIBCO BRL). For Northern transfer analysis, 20 µg of total RNA was subjected to electrophoresis on a 1.5% formaldehyde-agarose gel and transferred to Gene-Screen Plus membrane according to the manufacturer's recommendations. The blot was hybridized with random-primed TAP20 cDNA probes at 65°C for 3 h in Quik-Hyb solution (Stratagene), and was washed under high stringency conditions before autoradiography.
Immunoblotting
Immunoblotting was performed as follows: cell lysates were prepared by addition of 1 ml of lysis buffer (PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mg/ml PMSF, 30 ml/ml aprotinin) per 107 cells. Samples were run on a 15 or 10% SDS polyacrylamide gel and electrophoretically transferred to Immobilon-P membranes (Millipore). The membranes were then hybridized with indicated antibodies (anti-v polyclonal antibody; Santa Cruz Biotechnology), PKC
, ß1, and ß3 mAb (Transduction Laboratories), and anti-ß5 polyclonal antibody (Chemicon), in PBS containing 5% dry milk and detected via ECL (Amersham Pharmacia Biotech). The affinity-purified rabbit polyclonal anti-TAP20 antibody was produced by QCB Inc.
Adhesion Assay
Cell adhesion was performed as described previously (
Cell Migration Assay
The cell monolayer was briefly treated with trypsin to lift the cells, which were pelleted and washed once with PBS and resuspended in M199 medium containing 0.1% BSA to a final concentration of 500,000 cells/ml. Cells were added to 3 µm FALCON Cell Culture FluoroBlok Inserts (Becton Dickinson) at a density of 50,000 cells/insert. M199 (0.8 ml) with 10% FBS and VEGF (10 ng/ml) was used as a chemoattractant in the lower wells. The inserts were incubated for 4 h at 37°C. The membranes of the inserts were then mounted on glass slides and coverslips. Cells migrated through the FluoroBlok Inserts were quantitated by counting the fluorescent cells with a Nikon Optiphot-2 fluorescence microscope with a COHU video camera (COHU Electronics) and NIH Image software for Macintosh computer.
In Vitro Tube Formation
Matrix gel (MATRIGEL Basement Membrane matrix; Becton Dickinson) plates were prepared in 12-well plates following the manufacturer's instructions. After transfecting and sorting, HUVEC (80% confluent) were treated with trypsin, and 5 x 104 cells were seeded on the top of plates with complete M199 medium. The photographs were taken under a 100x light microscope, monitoring with a COHU video camera, and captured with a Scion 7 video card (Scion Image) in a PowerMac computer.
Focal Adhesion
24 h after transfection with GFP alone, TAP20 + GFP, or GFP-TAP20 plasmids, human MV3 (
Glutathione S-Transferase Coprecipitation
The TAP20 cDNA was inserted into BamHI and XhoI cloning sites in pGEX4T1 (Amersham Pharmacia Biotech). Cytoplasmic domains of integrin ß3 (amino acid sequence: KLLITIHDRKEFAKFEEERARAKWFTANNPLYKEATSTFTNITYRGT) or ß5 (amino acid sequence: KLLVTIHDRREFAKFQSERSRARYEMASNPLYRKPISTHTVDFTFNKFNKSYNGTVD) were synthesized by the PCR method using the pfu enzyme and inserted into the BamHI and XhoI cloning sites. Glutathione S-transferase (GST) or GST fusion proteins were expressed in Escherichia coli BL21 cells and immobilized on glutathione Sepharose beads (Amersham Pharmacia Biotech) following the manufacturer's instructions. To precipitate protein from EC lysate, the GST-TAP20 coated beads were incubated with the cell lysate in PBS for 20 h at 4°C. To precipitate TAP20 with GST-integrin tail fusion protein, the GST-TAP20 beads were first incubated in thrombin containing buffer for 16 h at room temperature to release the TAP20 protein. The TAP20-containing solution was then incubated with GST-integrin tail fusion protein beads for 20 h at 4°C. After three washes with PBS, the proteins bound to the beads were eluted in 10 mM glutathione buffer and analyzed by Western blotting. The antibodies used included: GST polyclonal antibody (Amersham Pharmacia Biotech), v polyclonal antibody (Santa Cruz Biotechnology), ß1 mAb and ß3 mAb (Transduction Laboratories), and ß5 polyclonal antibody (Chemicon).
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Results |
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Cloning of TAP20
We investigated the expression of genes regulated by PKC with the mRNA display method (
(PKC
-kn, a dominant negative inhibitor) or a constitutively active form of PKC
(PKC
-ca) were overexpressed (
0.8 kb whose expression depended upon the presence of active PKC
was identified. Northern transfer analysis showed that this 0.8-kb mRNA is highly expressed in PKC
-ca RCE but is dramatically suppressed in PKC
-kn RCE (Fig 1 B). The partial sequence of this gene obtained from differential display was used to screen a rat PC12 cDNA library by the PCR method. The cDNA generated from the PCR was cloned, and the nucleotide sequence was determined (Fig 1 A). The open reading frame encodes a novel protein of 175 residues with a calculated molecular mass of
20 kD. An antibody raised against the COOH terminus of this putative protein recognized a protein of 20 kD (Fig 1 C), as assessed by immunoblotting of an RCE cell lysate, which confirmed that the expression of this protein depended upon functional PKC
, as suggested by the Northern transfer analysis. In the PKC
kinase-negative RCE, TAP20 expression was significantly decreased at the protein level. This anti-TAP20 antibody also recognized a 20-kD band in TAP20 transfected human cells (Fig 1 C and Fig 2), suggesting that TAP20 can be expressed as a 20-kD, full-length protein in the cells. Accordingly, this novel PKC
-associated protein was designated TAP20. By searching the GeneBank database, we found that a partial 3' end sequence of TAP20 had been reported previously (
.
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Expression of TAP20 and TAP20 Fusion Proteins in Human Cell Lines
The immunoblotting with the anti-TAP20 antibody that recognizes an epitope at the COOH terminus of the protein suggests that TAP20 is expressed as a full-length protein in RCE. To explore the function of TAP20 in human cells, TAP20 cDNA was cloned into the pRc/CMV vector and a cDNA encoding GFP was inserted into a separate region of the same plasmid; thus, these two cDNAs were controlled by separate CMV promoters (Fig 2 A). 1 d after transfection, human ECV304 or HUVEC cells were sorted by GFP fluorescence using FACS®. Cells with GFP expression were recultured for 12 d before experiments. Cells were then harvested for cell adhesion, migration, and tube formation experiments, and also for the protein expression test (Fig 2B and Fig C). Blotting with anti-TAP20 antibody showed that the antibody does not detect antigen in the 20-kD range in wild-type (wt) or GFP transfected cells. The expression of TAP20 in unsorted TAP20 + GFP cells was 3070% of the sorted cells, depending on the transfection efficiency. Furthermore, the expression level of PKC, integrin
v, ß3, and ß5 was not changed in the TAP20 + GFP expressed ECV cells compared with the untransfected or GFP transfected cells (Fig 2 B).
To investigate the TAP20 effect on focal adhesion and to further characterize TAP20 distribution in the cells, either the TAP20 + GFP plasmid or the fusion protein GFP-TAP20 plasmid was transfected into MV3 cells, a ß3 integrindeficient cell line. The transfection efficiency varied from 2040% in different experiments. 2 d after transfection, the cells were harvested and tested for TAP20 expression. Similar to ECV cells, the anti-TAP20 antibody did not detect antigen in the 20-kD range in nontransfected or GFP transfected MV3 cells, whereas a 20-kD band in the TAP20 + GFP transfected cells and an 45-kD band in the GFP-TAP20 transfected cells were recognized, which indicated the expression of TAP20 and GFP-TAP20 fusion protein, respectively (Fig 2 C).
TAP20 Reduces EC Adhesion on VN
Transfected ECV cells were sorted by GFP fluorescence using a FACScan flow cytometer and recultured for 12 d. Adhesion of TAP20 + GFP expressing cells to VN, but not to FN and LN, was significantly attenuated (Fig 3 A) compared with that of either group of control cells, i.e., the wt cells or the cells expressing GFP only (a mean of 33.7 ± 5.1% of the added TAP20 + GFP cells adhered to VN, in contrast to 73.5 ± 7.5% [wt] and 75.0 ± 6.3% [GFP] of the control cells, P < 0.01). This result indicates that TAP20 affects cell adhesion by modulating the VN receptors, i.e., the vß3 and/or
vß5 integrins. To further investigate the TAP20 effect on VN receptors, antiintegrin antibodies were added to cells on VN-coated plates to block function of the VN receptors. As a control to rule out nonspecific effects of integrin ligation, an anti
2ß1 integrin (LN receptor) antibody, BHA2.1 was tested. The results obtained with the antiVN receptor antibodies were compared with those with anti-
2ß1 antibody (Fig 3 B). The anti
2ß1 integrin antibody BHA2.1 did not significantly affect cell adhesion on VN (comparing the BHA2.1 group of bars on Fig 3 B with the VN group of bars on Fig 3 A). Furthermore, adhesion of TAP20 + GFP cells on VN was reduced in the presence of BHA2.1 (31.6 ± 7.7% of added cells) to an extent similar to that with cells on VN plates without BHA2.1 antibody (Fig 3 A; VN group of bars). In the experiments with the antiVN receptor antibodies, the effects of the anti-
vß3 antibody LM609 were compared with those of the control antibody BHA2.1. The degree of adhesion in the BHA2.1 group was used as basal (100%) for comparison with that of the LM609 and P1F6 groups in Fig 3 B. LM609 caused a 50.3% reduction of adhesion of TAP20 + GFP cells, and also caused a 39.4% reduction in the wt cells and a 43.4% reduction in GFP cells. The difference in the magnitudes of the reductions between BHA2.1 and LM609 was not statistically significant among the three groups (P > 0.5). In contrast, the anti-
vß5 antibody P1F6 compared with the control antibody BHA2.1 caused only a 7.4% reduction of adhesion of the TAP20 + GFP cells, but caused significant reduction in both groups of the control cells (38.0 [wt] and 45.6% [GFP]). The magnitude of the reductions between BHA2.1 and P1F6 was significantly less in the TAP20 + GFP cells than that seen in either group of the control cells (P < 0.01). These observations suggest that in the TAP20 transfectants, the
vß3 integrin remains functional as a VN receptor, which can be blocked by the anti-
vß3 antibody LM609. On the other hand, the VN receptor function of
vß5 integrin was attenuated by TAP20 overexpression. Thus, blockage of
vß5 in TAP20 transfectants by anti-
vß5 antibody P1F6 did not further reduce cell adhesion on VN significantly, whereas the adhesion of the control cells with normal
vß5 function was dramatically decreased by this anti-
vß5 antibody.
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TAP20 Inhibits Focal Adhesion Formation
The results of adhesion with the integrin-blocking antibodies suggested that TAP20 affects cell function through the vß5 integrin. To further clarify the role of TAP20 on
vß5 integrin, we next examined the effect of TAP20 on focal adhesion formation in MV3 cells (
vß3-negative human melanoma cells and therefore have the
vß5 integrin as the principal surface receptor for VN. Thus, when the cells are cultured on VN-coated plates, the focal adhesion formation is caused chiefly by the interaction of
vß5 integrin with VN. We first investigated the cellular localization of TAP20. After 16 h of adhesion to VN, MV3 cells expressing either GFP alone (Fig 4 A, panels a and b) or TAP20 + GFP (Fig 4 A, panels c and d) were stained with anti-TAP20 antibody that was visualized with a Cy-3conjugated secondary antibody (Fig 4 A, panels b and d). The strong staining in Fig 4 A, panel d, suggests that TAP20 exists in transfected cells as a full-length protein. The TAP20 fluorescence was observed diffusely in the cytoplasm and the nucleus, which was also demonstrated by GFP fluorescence of the cells expressing the GFP-TAP20 fusion protein (Fig 4 B, panel a). When these cells were stained with antifocal adhesion component antibodies (antivinculin shown in Fig 4 B, panel b) to visualize the focal adhesions, no strong colocalization of GFP-TAP20 with focal adhesions was observed, indicating a possibility that TAP20 might dissociate from or interrupt focal adhesion during the cell adhesion process. To characterize the effect of TAP20 on focal adhesions, MV3 cells were transfected with either control GFP plasmid or TAP + GFP plasmid and plated on VN-coated chamber slides. GFP transfectants formed numerous focal adhesions, as demonstrated by antibodies against vinculin, paxillin, or FAK (Fig 4 C). In TAP20 + GFP transfected cells, the number of focal adhesions was significantly reduced by 6070% (Fig 4 D), compared with those in wt or GFP alone transfected MV3 cells.
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TAP20 Enhances EC Migration on VN and Tube Formation on Matrix Gel
Next, we monitored the effects of TAP20 on cell migration with a modified Boyden chamber assay (Fig 5 A). Transfected HUVEC cells were sorted by GFP fluorescence using a FACScan flow cytometer and recultured for 12 d. 50,000 cells were seeded on each chamber. There was no significant difference in migration on an FN-coated membrane between the untransfected cells, GFP sorted GFP-expressing cells, and TAP20 + GFP expressing cells. Migration of cells on a VN-coated plate was markedly enhanced by expression of TAP20 (640.4 ± 78.8 [TAP20] versus 395.2 ± 36.2 [wt] and 377.2 ± 65.6 [GFP]). This enhancement (TAP20 versus the controls) could not be blocked by the control anti-2ß1 antibody, BHA2.1, or by the anti-
vß3 antibody, LM609, but it could be blocked by the anti-
vß3 antibody, P1F6 (Fig 5 B).
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Since TAP20 modulated cell adhesion and migration, we asked whether overexpressing TAP20 would alter the ability of cells to form tubes on matrix gel. When the GFP-sorted HUVEC cells were cultured in matrix gel, a three-dimensional matrix, tube formation by TAP20 + GFP transfectants was significantly enhanced (Fig 6). By 6 h, tube structures were observed in the TAP20 + GFP transfectants, but not in the control GFP cells. The number of tubes formed by TAP20 + GFP transfectants appeared to be dramatically increased at all timepoints compared with that by the control cells.
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TAP20 Interacts with the Cytoplasmic Domain of ß5 Integrin
The experimental evidence above suggested that the effects of TAP20 on cells were mediated by the vß5 integrin VN receptor. Immunoblotting with antiß5 integrin antibody showed that the expression of the ß5 integrin in TAP20 transfectants was similar to that in the control cells (Fig 2 B). Therefore, we asked whether the effects of TAP20 could result from direct interaction with the
vß5 integrin. We used a GST-TAP20 fusion protein purified with glutathione Sepharose beads to precipitate proteins from cell lysate, and probed proteins bound to the complexes with the antibodies against
v, ß1, ß3, and ß5 integrin subunits. GST alone did not precipitate any protein recognized by these antibodies. Only the ß5 integrin subunit was precipitated by the GST-TAP20 fusion protein (Fig 7 A). To further confirm the TAP20ß5 integrin interaction, we used GST fusion proteins containing the cytoplasmic tail of either the ß3 or ß5 integrin subunit to precipitate TAP20 protein from lysate of ECV cells transfected with TAP20 + GFP. Immunoblotting with anti-TAP20 antibody demonstrated that TAP20 protein could coprecipitate with the GSTß5 tail fusion protein (Fig 7 B). To rule out an indirect interaction between the GST-TAP20 protein and the ß5 integrin complex, or between the GSTß5 tail and the TAP20 protein complex, we then used the GST-integrin tail fusion proteins to precipitate purified TAP20 protein. The GST-TAP20 fusion protein was first purified with glutathione Sepharose beads, and then was digested with thrombin. After incubation of the GSTintegrin tailcoated glutathione Sepharose beads with TAP20 released by thrombin, GSTß5 tail beads were able to precipitate TAP20, as shown in immunoblotting with TAP20 antibody (Fig 7 C).
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Discussion |
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In this study, we identified a cDNA that encodes a novel 20-kD, 176amino acid protein in RCE. Several observations in this study suggest that TAP20 is involved in integrin function in intact cells: (a) expression of TAP20 is dependent on PKC activity, which is required by RCE for migration and proliferation (
ß3-endonexin (
Our data show that overexpression of TAP20 in ECV cells affects cell adhesion and migration on VN, but not on other ECM proteins such as FN and LN, indicating that TAP20 regulates the function of the v integrin family. Furthermore, TAP20 effects were inhibited by a specific
vß5 integrinblocking antibody P1F6, not by an anti-
vß3 antibody LM609, indicating that TAP20 may specifically interact with the
vß5 integrin. A GST-TAP20 fusion protein coprecipitated specifically with detergent-solubilized ß5 integrin from ECV cells, and thus the binding of TAP20 to ß5 integrin does not depend on the existence of
v. A GSTß5 tail fusion protein coprecipitated specifically with TAP20 protein, indicating that the TAP20 binding site is in the cytoplasmic domain of ß5 integrin. Unlike ß3-endonexin, which binds the ß3 integrin and increases the
IIbß3 receptor affinity when overexpressed in CHO cells (
vß5-based adhesion and focal adhesion formation. Therefore, the effect of TAP20 is to decrease the affinity of its associated integrin receptor
vß5 for its ligand VN. These effects also differ from those of the
vß5 blocking antibody P1F6, which can prevent cell migration and angiogenesis initiated by VEGF or PKC activation (
vß5 signaling and thus modulating the functions of the
vß5 integrin, indicating a different integrin regulating mechanism. Formation of focal adhesions requires ECM-integrin ligation and integrin clustering. The decrease in focal adhesions in the TAP20-overexpressing cells could result from the attenuation of ECMß5 integrin ligation by TAP20. On the other hand, TAP20 may interfere with the interaction between ß5 integrin and the cytoskeleton, which is required for focal adhesion formation. Perhaps as a result of decreased binding to VN, TAP20 enhances EC migration and in vitro tube formation. It is also possible that, despite negative modulation of ß5 integrin function, TAP20 may upregulate recruitment of other molecules needed for migration, for instance.
This study demonstrates that TAP20 requires enzymatically active PKC for its transcription. It is very likely that TAP20 expression is PKC isoenzyme specific. Direct interaction of TAP20 with the cytoplasmic tail of the ß5 integrin subunit is at least one of the pathways by which PKC
might modulate EC migration and tube formation. Therefore, controlling TAP20 expression presents a mechanism, alternative to direct protein phosphorylation (
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Footnotes |
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1 Abbreviations used in this paper: EC, endothelial cell(s); ECM, extracellular matrix; EGFP, enhanced GFP; FAK, focal adhesion kinase; FN, fibronectin; HUVEC, human umbilical vein EC(s); GFP, green fluorescent protein; GST, glutathione S-transferase; LN, laminin; PKC, protein kinase C; RCE, rat capillary endothelial cell(s); TAP, theta-associated protein; VEGF, vascular endothelial growth factor; VN, vitronectin; wt, wild-type.
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Acknowledgements |
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We thank S. Zhou for technical assistance.
This work was supported by grants from the National Heart, Lung, and Blood Institute to J.A. Ware.
Submitted: 6 April 1999
Revised: 19 October 1999
Accepted: 22 October 1999
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References |
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