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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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Modulation of cell migration by TAP20. Transfected HUVEC cells were sorted with GFP fluorescence and recultured for 1–2 d. Cells were briefly treated with trypsin and suspended in medium containing 0.1% BSA, with addition of antibodies as indicated: anti-α2β1, BHA2.1; anti-αvβ3, LM609; and anti-αvβ3, P1F6. Cells (50,000/0.1 ml) were seeded in coated FluoroBlok Insert precoated with either FN (A) or VN (B). Cells were incubated for 4 h at 37°C. After fixation, migrated cells were visualized under a fluorescence microscope. Cells that had migrated through the membrane of FluoroBlok Inserts were quantitated by counting the fluorescent cells in two random nonoverlapped 100× view fields. (A) Data are expressed as the mean ± S.D. (error bars) of four separate experiments. Asterisk indicates P < 0.01, TAP20 transfectants (TAP + GFP) versus GFP alone transfected cells by t test.
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Figure 5: Modulation of cell migration by TAP20. Transfected HUVEC cells were sorted with GFP fluorescence and recultured for 1–2 d. Cells were briefly treated with trypsin and suspended in medium containing 0.1% BSA, with addition of antibodies as indicated: anti-α2β1, BHA2.1; anti-αvβ3, LM609; and anti-αvβ3, P1F6. Cells (50,000/0.1 ml) were seeded in coated FluoroBlok Insert precoated with either FN (A) or VN (B). Cells were incubated for 4 h at 37°C. After fixation, migrated cells were visualized under a fluorescence microscope. Cells that had migrated through the membrane of FluoroBlok Inserts were quantitated by counting the fluorescent cells in two random nonoverlapped 100× view fields. (A) Data are expressed as the mean ± S.D. (error bars) of four separate experiments. Asterisk indicates P < 0.01, TAP20 transfectants (TAP + GFP) versus GFP alone transfected cells by t test.

Mentions: 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 1–2 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-α2β1 antibody, BHA2.1, or by the anti-αvβ3 antibody, LM609, but it could be blocked by the anti-αvβ3 antibody, P1F6 (Fig. 5 B).


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)

Modulation of cell migration by TAP20. Transfected HUVEC cells were sorted with GFP fluorescence and recultured for 1–2 d. Cells were briefly treated with trypsin and suspended in medium containing 0.1% BSA, with addition of antibodies as indicated: anti-α2β1, BHA2.1; anti-αvβ3, LM609; and anti-αvβ3, P1F6. Cells (50,000/0.1 ml) were seeded in coated FluoroBlok Insert precoated with either FN (A) or VN (B). Cells were incubated for 4 h at 37°C. After fixation, migrated cells were visualized under a fluorescence microscope. Cells that had migrated through the membrane of FluoroBlok Inserts were quantitated by counting the fluorescent cells in two random nonoverlapped 100× view fields. (A) Data are expressed as the mean ± S.D. (error bars) of four separate experiments. Asterisk indicates P < 0.01, TAP20 transfectants (TAP + GFP) versus GFP alone transfected cells by t test.
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Related In: Results  -  Collection

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Figure 5: Modulation of cell migration by TAP20. Transfected HUVEC cells were sorted with GFP fluorescence and recultured for 1–2 d. Cells were briefly treated with trypsin and suspended in medium containing 0.1% BSA, with addition of antibodies as indicated: anti-α2β1, BHA2.1; anti-αvβ3, LM609; and anti-αvβ3, P1F6. Cells (50,000/0.1 ml) were seeded in coated FluoroBlok Insert precoated with either FN (A) or VN (B). Cells were incubated for 4 h at 37°C. After fixation, migrated cells were visualized under a fluorescence microscope. Cells that had migrated through the membrane of FluoroBlok Inserts were quantitated by counting the fluorescent cells in two random nonoverlapped 100× view fields. (A) Data are expressed as the mean ± S.D. (error bars) of four separate experiments. Asterisk indicates P < 0.01, TAP20 transfectants (TAP + GFP) versus GFP alone transfected cells by t test.
Mentions: 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 1–2 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-α2β1 antibody, BHA2.1, or by the anti-αvβ3 antibody, LM609, but it could be blocked by the anti-αvβ3 antibody, P1F6 (Fig. 5 B).

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