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Enhancement of endothelial cell migration and in vitro tube formation by TAP20, a novel beta 5 integrin-modulating, PKC theta-dependent protein.

Tang S, Gao Y, Ware JA - J. Cell Biol. (1999)

Bottom Line: A full-length cDNA encoding a novel 20-kD protein, whose expression was PKCtheta-dependent, was identified in endothelial cells, cloned, characterized, and designated as theta-associated protein (TAP) 20.An antiintegrin alphavbeta5 antibody prevented these TAP20 effects.The interaction between TAP20 and beta5 integrin cytoplasmic domain was demonstrated by protein coprecipitation and immunoblotting.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Division, Department of Medicine, Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461, USA. tang@aecom.yu.edu

ABSTRACT
Migration, proliferation, and tube formation of endothelial cells are regulated by a protein kinase C isoenzyme PKCtheta. A full-length cDNA encoding a novel 20-kD protein, whose expression was PKCtheta-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 alphavbeta5 antibody prevented these TAP20 effects. Overexpression of TAP20 also decreased focal adhesion formation in alphavbeta3-deficient cells. The interaction between TAP20 and beta5 integrin cytoplasmic domain was demonstrated by protein coprecipitation and immunoblotting. Thus, the discovery of TAP20, which interacts with integrin beta5 and modulates cell adhesion, migration, and tube formation, further defines a possible pathway to angiogenesis dependent on PKCtheta.

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Inhibition of cell adhesion by TAP20. Transfected cells were sorted with GFP fluorescence and recultured for 1–2 d. Cells were briefly treated with trypsin, washed with PBS, and resuspended in M199 medium with 2% BSA. Cells (50,000/well) were allowed to adhere to a 96-well plate coated with LN, FN, or VN (A). Cells attached to VN were further investigated with monoclonal antiintegrin antibodies (10 μg/ml) as indicated: the anti-αvβ3 antibody, LM609; anti-αvβ5, P1F6, and as a control for integrin ligation, an anti–α2β1 integrin antibody, BHA2.1 (B). The attached EC were quantified by the dye staining method. Attached cells are expressed as the percentage of the seeded cells. The values are means from four separate experiments. Error bars indicate standard deviation; asterisk indicates P < 0.01, TAP20 transfected ECV (TAP20 + GFP) versus either wt or GFP alone transfectants, by t test.
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Figure 3: Inhibition of cell adhesion by TAP20. Transfected cells were sorted with GFP fluorescence and recultured for 1–2 d. Cells were briefly treated with trypsin, washed with PBS, and resuspended in M199 medium with 2% BSA. Cells (50,000/well) were allowed to adhere to a 96-well plate coated with LN, FN, or VN (A). Cells attached to VN were further investigated with monoclonal antiintegrin antibodies (10 μg/ml) as indicated: the anti-αvβ3 antibody, LM609; anti-αvβ5, P1F6, and as a control for integrin ligation, an anti–α2β1 integrin antibody, BHA2.1 (B). The attached EC were quantified by the dye staining method. Attached cells are expressed as the percentage of the seeded cells. The values are means from four separate experiments. Error bars indicate standard deviation; asterisk indicates P < 0.01, TAP20 transfected ECV (TAP20 + GFP) versus either wt or GFP alone transfectants, by t test.

Mentions: Transfected ECV cells were sorted by GFP fluorescence using a FACScan flow cytometer and recultured for 1–2 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 αvβ3 and/or αvβ5 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–α2β1 integrin (LN receptor) antibody, BHA2.1 was tested. The results obtained with the anti–VN receptor antibodies were compared with those with anti-α2β1 antibody (Fig. 3 B). The anti–α2β1 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 anti–VN receptor antibodies, the effects of the anti-αvβ3 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-αvβ5 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 αvβ3 integrin remains functional as a VN receptor, which can be blocked by the anti-αvβ3 antibody LM609. On the other hand, the VN receptor function of αvβ5 integrin was attenuated by TAP20 overexpression. Thus, blockage of αvβ5 in TAP20 transfectants by anti-αvβ5 antibody P1F6 did not further reduce cell adhesion on VN significantly, whereas the adhesion of the control cells with normal αvβ5 function was dramatically decreased by this anti-αvβ5 antibody.


Enhancement of endothelial cell migration and in vitro tube formation by TAP20, a novel beta 5 integrin-modulating, PKC theta-dependent protein.

Tang S, Gao Y, Ware JA - J. Cell Biol. (1999)

Inhibition of cell adhesion by TAP20. Transfected cells were sorted with GFP fluorescence and recultured for 1–2 d. Cells were briefly treated with trypsin, washed with PBS, and resuspended in M199 medium with 2% BSA. Cells (50,000/well) were allowed to adhere to a 96-well plate coated with LN, FN, or VN (A). Cells attached to VN were further investigated with monoclonal antiintegrin antibodies (10 μg/ml) as indicated: the anti-αvβ3 antibody, LM609; anti-αvβ5, P1F6, and as a control for integrin ligation, an anti–α2β1 integrin antibody, BHA2.1 (B). The attached EC were quantified by the dye staining method. Attached cells are expressed as the percentage of the seeded cells. The values are means from four separate experiments. Error bars indicate standard deviation; asterisk indicates P < 0.01, TAP20 transfected ECV (TAP20 + GFP) versus either wt or GFP alone transfectants, by t test.
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Related In: Results  -  Collection

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Figure 3: Inhibition of cell adhesion by TAP20. Transfected cells were sorted with GFP fluorescence and recultured for 1–2 d. Cells were briefly treated with trypsin, washed with PBS, and resuspended in M199 medium with 2% BSA. Cells (50,000/well) were allowed to adhere to a 96-well plate coated with LN, FN, or VN (A). Cells attached to VN were further investigated with monoclonal antiintegrin antibodies (10 μg/ml) as indicated: the anti-αvβ3 antibody, LM609; anti-αvβ5, P1F6, and as a control for integrin ligation, an anti–α2β1 integrin antibody, BHA2.1 (B). The attached EC were quantified by the dye staining method. Attached cells are expressed as the percentage of the seeded cells. The values are means from four separate experiments. Error bars indicate standard deviation; asterisk indicates P < 0.01, TAP20 transfected ECV (TAP20 + GFP) versus either wt or GFP alone transfectants, by t test.
Mentions: Transfected ECV cells were sorted by GFP fluorescence using a FACScan flow cytometer and recultured for 1–2 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 αvβ3 and/or αvβ5 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–α2β1 integrin (LN receptor) antibody, BHA2.1 was tested. The results obtained with the anti–VN receptor antibodies were compared with those with anti-α2β1 antibody (Fig. 3 B). The anti–α2β1 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 anti–VN receptor antibodies, the effects of the anti-αvβ3 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-αvβ5 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 αvβ3 integrin remains functional as a VN receptor, which can be blocked by the anti-αvβ3 antibody LM609. On the other hand, the VN receptor function of αvβ5 integrin was attenuated by TAP20 overexpression. Thus, blockage of αvβ5 in TAP20 transfectants by anti-αvβ5 antibody P1F6 did not further reduce cell adhesion on VN significantly, whereas the adhesion of the control cells with normal αvβ5 function was dramatically decreased by this anti-αvβ5 antibody.

Bottom Line: A full-length cDNA encoding a novel 20-kD protein, whose expression was PKCtheta-dependent, was identified in endothelial cells, cloned, characterized, and designated as theta-associated protein (TAP) 20.An antiintegrin alphavbeta5 antibody prevented these TAP20 effects.The interaction between TAP20 and beta5 integrin cytoplasmic domain was demonstrated by protein coprecipitation and immunoblotting.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Division, Department of Medicine, Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461, USA. tang@aecom.yu.edu

ABSTRACT
Migration, proliferation, and tube formation of endothelial cells are regulated by a protein kinase C isoenzyme PKCtheta. A full-length cDNA encoding a novel 20-kD protein, whose expression was PKCtheta-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 alphavbeta5 antibody prevented these TAP20 effects. Overexpression of TAP20 also decreased focal adhesion formation in alphavbeta3-deficient cells. The interaction between TAP20 and beta5 integrin cytoplasmic domain was demonstrated by protein coprecipitation and immunoblotting. Thus, the discovery of TAP20, which interacts with integrin beta5 and modulates cell adhesion, migration, and tube formation, further defines a possible pathway to angiogenesis dependent on PKCtheta.

Show MeSH
Related in: MedlinePlus