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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 on α9-dependent enhancement of cell migration. (a) α9-expressing MEFs were transfected with siRNA-1, siRNA-2, siRNA-3, siRNA-4, and no siRNA control in 6-well plates. 24 h later, SSAT mRNA was assayed by quantitative PCR and expressed as percent basal level (normalized to no siRNA). (b) 50 μM BE-3-3-3 was used to induce the expression of SSAT 24 h after siRNA transfection, for 24 h. SSAT protein was detected by Western blot. Western blot for Crk was used as a control for equal protein loading. (c and d) α9-, α9α4-, and α9α5-expressing MEFs were transfected with siRNA-1, siRNA-2, siRNA-3, siRNA-4, and no siRNA; 24 h later, cells suspended in serum-free medium were used for migration assays without BE-3-3-3 (c) and with 50 μM BE-3-3-3 (d). The cells were assayed and counted as described in the Fig. 3 legend.
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fig6: Effects of siRNA knockdown on α9-dependent enhancement of cell migration. (a) α9-expressing MEFs were transfected with siRNA-1, siRNA-2, siRNA-3, siRNA-4, and no siRNA control in 6-well plates. 24 h later, SSAT mRNA was assayed by quantitative PCR and expressed as percent basal level (normalized to no siRNA). (b) 50 μM BE-3-3-3 was used to induce the expression of SSAT 24 h after siRNA transfection, for 24 h. SSAT protein was detected by Western blot. Western blot for Crk was used as a control for equal protein loading. (c and d) α9-, α9α4-, and α9α5-expressing MEFs were transfected with siRNA-1, siRNA-2, siRNA-3, siRNA-4, and no siRNA; 24 h later, cells suspended in serum-free medium were used for migration assays without BE-3-3-3 (c) and with 50 μM BE-3-3-3 (d). The cells were assayed and counted as described in the Fig. 3 legend.

Mentions: To further investigate the importance of SSAT for α9β1-dependent enhancement of cell migration, we used RNA interference to suppress the expression of SSAT in α9-expressing mouse embryonic fibroblasts (MEFs). MEFs were chosen because the availability of complete genomic sequence data in the mouse allows improved design of specific small interfering RNA (siRNA), and because mouse and human SSAT are virtually identical. Four 21-nucleotide siRNA segments directed against the middle (siRNA-1 and siRNA-3) or the 3′ end (siRNA-2 and siRNA-4) of the SSAT transcript were transfected into MEFs that had been stably transfected to express either wild-type α9, α9α4, or α9α5, and the cells were examined 24 h later (Young et al., 2001). Real-time PCR showed that SSAT mRNA was reduced by >75% by siRNA-2, siRNA-3, and siRNA-4, compared with untransfected cells or cells transfected with siRNA-1 (Fig. 6 a). Because endogenous SSAT cannot be detected by a polyclonal antibody against SSAT, BE-3-3-3 was used to induce the expression of SSAT 24 h after siRNA transfection. The SSAT protein level was decreased by siRNA-2, siRNA-3, and siRNA-4, compared with untransfected cells or cells transfected with siRNA-1 (Fig. 6 b). Transfection with siRNA-2, siRNA-3, and siRNA-4 decreased the cell migration on TNfn3RAA of cells expressing wild-type α9 to the level seen for cells transfected with the α9α5 chimera, but had no effect on the migration of cells expressing α9α4 or α9α5 (Fig. 6 c). The enhancement of α9-dependent cell migration by BE-3-3-3 was also decreased by siRNA transfection (Fig. 6 d). None of these treatments had any effect on cell adhesion on TNfn3RAA or on cell migration on fibronectin in any cell type (unpublished data). These results further support the concept that endogenous levels of SSAT play an important role in α9-dependent enhancement of cell migration.


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 on α9-dependent enhancement of cell migration. (a) α9-expressing MEFs were transfected with siRNA-1, siRNA-2, siRNA-3, siRNA-4, and no siRNA control in 6-well plates. 24 h later, SSAT mRNA was assayed by quantitative PCR and expressed as percent basal level (normalized to no siRNA). (b) 50 μM BE-3-3-3 was used to induce the expression of SSAT 24 h after siRNA transfection, for 24 h. SSAT protein was detected by Western blot. Western blot for Crk was used as a control for equal protein loading. (c and d) α9-, α9α4-, and α9α5-expressing MEFs were transfected with siRNA-1, siRNA-2, siRNA-3, siRNA-4, and no siRNA; 24 h later, cells suspended in serum-free medium were used for migration assays without BE-3-3-3 (c) and with 50 μM BE-3-3-3 (d). The cells were assayed and counted as described in the Fig. 3 legend.
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fig6: Effects of siRNA knockdown on α9-dependent enhancement of cell migration. (a) α9-expressing MEFs were transfected with siRNA-1, siRNA-2, siRNA-3, siRNA-4, and no siRNA control in 6-well plates. 24 h later, SSAT mRNA was assayed by quantitative PCR and expressed as percent basal level (normalized to no siRNA). (b) 50 μM BE-3-3-3 was used to induce the expression of SSAT 24 h after siRNA transfection, for 24 h. SSAT protein was detected by Western blot. Western blot for Crk was used as a control for equal protein loading. (c and d) α9-, α9α4-, and α9α5-expressing MEFs were transfected with siRNA-1, siRNA-2, siRNA-3, siRNA-4, and no siRNA; 24 h later, cells suspended in serum-free medium were used for migration assays without BE-3-3-3 (c) and with 50 μM BE-3-3-3 (d). The cells were assayed and counted as described in the Fig. 3 legend.
Mentions: To further investigate the importance of SSAT for α9β1-dependent enhancement of cell migration, we used RNA interference to suppress the expression of SSAT in α9-expressing mouse embryonic fibroblasts (MEFs). MEFs were chosen because the availability of complete genomic sequence data in the mouse allows improved design of specific small interfering RNA (siRNA), and because mouse and human SSAT are virtually identical. Four 21-nucleotide siRNA segments directed against the middle (siRNA-1 and siRNA-3) or the 3′ end (siRNA-2 and siRNA-4) of the SSAT transcript were transfected into MEFs that had been stably transfected to express either wild-type α9, α9α4, or α9α5, and the cells were examined 24 h later (Young et al., 2001). Real-time PCR showed that SSAT mRNA was reduced by >75% by siRNA-2, siRNA-3, and siRNA-4, compared with untransfected cells or cells transfected with siRNA-1 (Fig. 6 a). Because endogenous SSAT cannot be detected by a polyclonal antibody against SSAT, BE-3-3-3 was used to induce the expression of SSAT 24 h after siRNA transfection. The SSAT protein level was decreased by siRNA-2, siRNA-3, and siRNA-4, compared with untransfected cells or cells transfected with siRNA-1 (Fig. 6 b). Transfection with siRNA-2, siRNA-3, and siRNA-4 decreased the cell migration on TNfn3RAA of cells expressing wild-type α9 to the level seen for cells transfected with the α9α5 chimera, but had no effect on the migration of cells expressing α9α4 or α9α5 (Fig. 6 c). The enhancement of α9-dependent cell migration by BE-3-3-3 was also decreased by siRNA transfection (Fig. 6 d). None of these treatments had any effect on cell adhesion on TNfn3RAA or on cell migration on fibronectin in any cell type (unpublished data). These results further support the concept that endogenous levels of SSAT play an important role in α9-dependent enhancement of cell migration.

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