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Spermidine/spermine N1-acetyltransferase specifically binds to the integrin alpha9 subunit cytoplasmic domain and enhances cell migration.

Chen C, Young BA, Coleman CS, Pegg AE, Sheppard D - J. Cell Biol. (2004)

Bottom Line: Overexpression of SSAT increased alpha9beta1-mediated migration, and small interfering RNA knockdown of SSAT inhibited this migration without affecting cell adhesion or migration that was mediated by other integrin cytoplasmic domains.The enzyme activity of SSAT is critical for this effect, because a catalytically inactive version did not enhance migration.We conclude that SSAT directly binds to the alpha9 cytoplasmic domain and mediates alpha9-dependent enhancement of cell migration, presumably by localized effects on acetylation of polyamines or of unidentified substrates.

View Article: PubMed Central - PubMed

Affiliation: Lung Biology Center, Department of Medicine, University of California-San Francisco, San Francisco, CA 94110, USA.

ABSTRACT
The integrin alpha9beta1 is expressed on migrating cells, such as leukocytes, and binds to multiple ligands that are present at sites of tissue injury and inflammation. alpha9beta1, like the structurally related integrin alpha4beta1, mediates accelerated cell migration, an effect that depends on the alpha9 cytoplasmic domain. alpha4beta1 enhances migration through reversible binding to the adapter protein, paxillin, but alpha9beta1-dependent migration is paxillin independent. Using yeast two-hybrid screening, we identified the polyamine catabolizing enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) as a specific binding partner of the alpha9 cytoplasmic domain. Overexpression of SSAT increased alpha9beta1-mediated migration, and small interfering RNA knockdown of SSAT inhibited this migration without affecting cell adhesion or migration that was mediated by other integrin cytoplasmic domains. The enzyme activity of SSAT is critical for this effect, because a catalytically inactive version did not enhance migration. We conclude that SSAT directly binds to the alpha9 cytoplasmic domain and mediates alpha9-dependent enhancement of cell migration, presumably by localized effects on acetylation of polyamines or of unidentified substrates.

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Effects of siRNA knockdown and BE-3-3-3 on endogenous α9-dependent HMVEC migration. (a) Flow cytometric evaluation of cell surface expression of α9 integrin on HMVEC. Open peaks represent unstained cells, and shaded peaks represent cells stained with the anti-α9β1 antibody Y9A2. (b) HMVEC were transfected with siRNA-A, siRNA-B, and no siRNA control in 6-well plates. 24 h later, SSAT mRNA was assayed by quantitative PCR and expressed as percent basal levels (normalized to no siRNA). (c) 50 μM BE-3-3-3 was used to induce the expression of SSAT 24 h after siRNA transfection, for 24 h. SSAT protein concentration was assessed by Western blot. Western blot for Crk was used as a control for equal protein loading. (d and e) HMVEC were transfected with siRNA-A, siRNA-B, and no siRNA, and 24 h later, cells were or were not treated with BE-3-3-3 (50 μM) for 24 h. After treatment with BE-3-3-3, cells were suspended in serum-free medium and were used for migration assays on 5 μg/ml TNfn3RAA (d) or on 5 μg/ml fibronectin (FN; e), as described in the Fig. 3 legend. All data represent the means (±SEM) of triplicate experiments.
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fig8: Effects of siRNA knockdown and BE-3-3-3 on endogenous α9-dependent HMVEC migration. (a) Flow cytometric evaluation of cell surface expression of α9 integrin on HMVEC. Open peaks represent unstained cells, and shaded peaks represent cells stained with the anti-α9β1 antibody Y9A2. (b) HMVEC were transfected with siRNA-A, siRNA-B, and no siRNA control in 6-well plates. 24 h later, SSAT mRNA was assayed by quantitative PCR and expressed as percent basal levels (normalized to no siRNA). (c) 50 μM BE-3-3-3 was used to induce the expression of SSAT 24 h after siRNA transfection, for 24 h. SSAT protein concentration was assessed by Western blot. Western blot for Crk was used as a control for equal protein loading. (d and e) HMVEC were transfected with siRNA-A, siRNA-B, and no siRNA, and 24 h later, cells were or were not treated with BE-3-3-3 (50 μM) for 24 h. After treatment with BE-3-3-3, cells were suspended in serum-free medium and were used for migration assays on 5 μg/ml TNfn3RAA (d) or on 5 μg/ml fibronectin (FN; e), as described in the Fig. 3 legend. All data represent the means (±SEM) of triplicate experiments.

Mentions: To further investigate the importance of SSAT for endogenous α9β1-dependent enhancement of cell migration in untransfected cells, we tested the expression of integrin α9β1 on the cell surface of human microvascular endothelial cells (HMVEC) by flow cytometry (Fig. 8 a). The expression of SSAT in these cells was induced by BE-3-3-3 and inhibited by siRNA directed against human SSAT. Two siRNAs were evaluated, and only one (siRNA-A) consistently inhibited SSAT mRNA concentrations in untreated cells (Fig. 8 b) and dramatically reduced SSAT protein levels in BE-3-3-3–treated cells (Fig. 8 c). Induction of SSAT increased HMVEC migration on the α9β1-specific ligand, TNfn3RAA. Inhibition of SSAT by functional siRNA specifically decreased cell migration on TNfn3RAA (Fig. 8 d). Neither BE-3-3-3 nor siRNA against human SSAT had any effect on cell migration on the α9β1-irrelevant ligand, plasma fibronectin (Fig. 8 e). These results demonstrate that the expression level of SSAT specifically affects α9β1-dependent cell migration in cells that endogenously express α9β1.


Spermidine/spermine N1-acetyltransferase specifically binds to the integrin alpha9 subunit cytoplasmic domain and enhances cell migration.

Chen C, Young BA, Coleman CS, Pegg AE, Sheppard D - J. Cell Biol. (2004)

Effects of siRNA knockdown and BE-3-3-3 on endogenous α9-dependent HMVEC migration. (a) Flow cytometric evaluation of cell surface expression of α9 integrin on HMVEC. Open peaks represent unstained cells, and shaded peaks represent cells stained with the anti-α9β1 antibody Y9A2. (b) HMVEC were transfected with siRNA-A, siRNA-B, and no siRNA control in 6-well plates. 24 h later, SSAT mRNA was assayed by quantitative PCR and expressed as percent basal levels (normalized to no siRNA). (c) 50 μM BE-3-3-3 was used to induce the expression of SSAT 24 h after siRNA transfection, for 24 h. SSAT protein concentration was assessed by Western blot. Western blot for Crk was used as a control for equal protein loading. (d and e) HMVEC were transfected with siRNA-A, siRNA-B, and no siRNA, and 24 h later, cells were or were not treated with BE-3-3-3 (50 μM) for 24 h. After treatment with BE-3-3-3, cells were suspended in serum-free medium and were used for migration assays on 5 μg/ml TNfn3RAA (d) or on 5 μg/ml fibronectin (FN; e), as described in the Fig. 3 legend. All data represent the means (±SEM) of triplicate experiments.
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fig8: Effects of siRNA knockdown and BE-3-3-3 on endogenous α9-dependent HMVEC migration. (a) Flow cytometric evaluation of cell surface expression of α9 integrin on HMVEC. Open peaks represent unstained cells, and shaded peaks represent cells stained with the anti-α9β1 antibody Y9A2. (b) HMVEC were transfected with siRNA-A, siRNA-B, and no siRNA control in 6-well plates. 24 h later, SSAT mRNA was assayed by quantitative PCR and expressed as percent basal levels (normalized to no siRNA). (c) 50 μM BE-3-3-3 was used to induce the expression of SSAT 24 h after siRNA transfection, for 24 h. SSAT protein concentration was assessed by Western blot. Western blot for Crk was used as a control for equal protein loading. (d and e) HMVEC were transfected with siRNA-A, siRNA-B, and no siRNA, and 24 h later, cells were or were not treated with BE-3-3-3 (50 μM) for 24 h. After treatment with BE-3-3-3, cells were suspended in serum-free medium and were used for migration assays on 5 μg/ml TNfn3RAA (d) or on 5 μg/ml fibronectin (FN; e), as described in the Fig. 3 legend. All data represent the means (±SEM) of triplicate experiments.
Mentions: To further investigate the importance of SSAT for endogenous α9β1-dependent enhancement of cell migration in untransfected cells, we tested the expression of integrin α9β1 on the cell surface of human microvascular endothelial cells (HMVEC) by flow cytometry (Fig. 8 a). The expression of SSAT in these cells was induced by BE-3-3-3 and inhibited by siRNA directed against human SSAT. Two siRNAs were evaluated, and only one (siRNA-A) consistently inhibited SSAT mRNA concentrations in untreated cells (Fig. 8 b) and dramatically reduced SSAT protein levels in BE-3-3-3–treated cells (Fig. 8 c). Induction of SSAT increased HMVEC migration on the α9β1-specific ligand, TNfn3RAA. Inhibition of SSAT by functional siRNA specifically decreased cell migration on TNfn3RAA (Fig. 8 d). Neither BE-3-3-3 nor siRNA against human SSAT had any effect on cell migration on the α9β1-irrelevant ligand, plasma fibronectin (Fig. 8 e). These results demonstrate that the expression level of SSAT specifically affects α9β1-dependent cell migration in cells that endogenously express α9β1.

Bottom Line: Overexpression of SSAT increased alpha9beta1-mediated migration, and small interfering RNA knockdown of SSAT inhibited this migration without affecting cell adhesion or migration that was mediated by other integrin cytoplasmic domains.The enzyme activity of SSAT is critical for this effect, because a catalytically inactive version did not enhance migration.We conclude that SSAT directly binds to the alpha9 cytoplasmic domain and mediates alpha9-dependent enhancement of cell migration, presumably by localized effects on acetylation of polyamines or of unidentified substrates.

View Article: PubMed Central - PubMed

Affiliation: Lung Biology Center, Department of Medicine, University of California-San Francisco, San Francisco, CA 94110, USA.

ABSTRACT
The integrin alpha9beta1 is expressed on migrating cells, such as leukocytes, and binds to multiple ligands that are present at sites of tissue injury and inflammation. alpha9beta1, like the structurally related integrin alpha4beta1, mediates accelerated cell migration, an effect that depends on the alpha9 cytoplasmic domain. alpha4beta1 enhances migration through reversible binding to the adapter protein, paxillin, but alpha9beta1-dependent migration is paxillin independent. Using yeast two-hybrid screening, we identified the polyamine catabolizing enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) as a specific binding partner of the alpha9 cytoplasmic domain. Overexpression of SSAT increased alpha9beta1-mediated migration, and small interfering RNA knockdown of SSAT inhibited this migration without affecting cell adhesion or migration that was mediated by other integrin cytoplasmic domains. The enzyme activity of SSAT is critical for this effect, because a catalytically inactive version did not enhance migration. We conclude that SSAT directly binds to the alpha9 cytoplasmic domain and mediates alpha9-dependent enhancement of cell migration, presumably by localized effects on acetylation of polyamines or of unidentified substrates.

Show MeSH
Related in: MedlinePlus