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The syndecan-1 ectodomain regulates alphavbeta3 integrin activity in human mammary carcinoma cells.

Beauvais DM, Burbach BJ, Rapraeger AC - J. Cell Biol. (2004)

Bottom Line: This paper demonstrates that the alpha(v)beta(3) integrin and syndecan-1 (S1) are functionally coupled.Coupling of the syndecan to alpha(v)beta(3) requires the S1 ectodomain (ED), as ectopic expression of glycosylphosphatidylinositol-linked S1ED enhances alpha(v)beta(3) recognition of vitronectin; and treatments that target this domain, including competition with recombinant S1ED protein or anti-S1ED antibodies, mutation of the S1ED, or down-regulation of S1 expression by small-interfering RNAs, disrupt alpha(v)beta(3)-dependent cell spreading and migration.Thus, S1 is likely to be a critical regulator of many cellular behaviors that depend on activated alpha(v)beta(3) integrins.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison, Madison, WI 53706, USA.

ABSTRACT
The alpha(v)beta(3) integrin participates in cell morphogenesis, growth factor signaling, and cell survival. Activation of the integrin is central to these processes and is influenced by specific ECM components, which engage both integrins and syndecans. This paper demonstrates that the alpha(v)beta(3) integrin and syndecan-1 (S1) are functionally coupled. The integrin is dependent on the syndecan to become activated and to mediate signals required for MDA-MB-231 and MDA-MB-435 human mammary carcinoma cell spreading on vitronectin or S1-specific antibody. Coupling of the syndecan to alpha(v)beta(3) requires the S1 ectodomain (ED), as ectopic expression of glycosylphosphatidylinositol-linked S1ED enhances alpha(v)beta(3) recognition of vitronectin; and treatments that target this domain, including competition with recombinant S1ED protein or anti-S1ED antibodies, mutation of the S1ED, or down-regulation of S1 expression by small-interfering RNAs, disrupt alpha(v)beta(3)-dependent cell spreading and migration. Thus, S1 is likely to be a critical regulator of many cellular behaviors that depend on activated alpha(v)beta(3) integrins.

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Down-regulation of S1 expression by siRNA disrupts cell spreading and migration on VN. (A) SiRNA targeting of hS1 mRNA. FACS analysis for (B and C) hS1 (mAb B-B4), (D) hS4 (mAb F94-8G3), and (E) mS1 (mAb 281.2) expression against IgG controls (black-filled histograms) in NEO- and GPI-mS1ED–expressing MDA-MB-231 cells 72 h after transfection with either lipid-vehicle alone (Vehicle or (V)) or 200 nM siRNA (RNAi or (R)). (F) NEOMDA-MB-231 cells and MDA-MB-231 cells expressing GPI-mS1ED, mS1Δ88-252, mS1Δ122-252, and mS1TDM were transfected with lipid-vehicle alone or 200 nM hS1-siRNA and seeded on wells coated with either 10 μg/ml VN or FN. Cells were incubated at 37°C for 2 h, fixed, and stained with rhodamine-conjugated phalloidin. Bar, 50 μm. (G and H) Lipid-vehicle (gray) or hS1-siRNA (black) transfected cells were also plated on polycarbonate filters coated with either 10 μg/ml VN (G) or FN (H) in a modified Boyden chamber. After 16 h, cells that migrated through the filter in response to 10% FBS in the lower chamber were quantified by colorimetric staining. The error bars represent the SEM from three independent experiments.
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fig8: Down-regulation of S1 expression by siRNA disrupts cell spreading and migration on VN. (A) SiRNA targeting of hS1 mRNA. FACS analysis for (B and C) hS1 (mAb B-B4), (D) hS4 (mAb F94-8G3), and (E) mS1 (mAb 281.2) expression against IgG controls (black-filled histograms) in NEO- and GPI-mS1ED–expressing MDA-MB-231 cells 72 h after transfection with either lipid-vehicle alone (Vehicle or (V)) or 200 nM siRNA (RNAi or (R)). (F) NEOMDA-MB-231 cells and MDA-MB-231 cells expressing GPI-mS1ED, mS1Δ88-252, mS1Δ122-252, and mS1TDM were transfected with lipid-vehicle alone or 200 nM hS1-siRNA and seeded on wells coated with either 10 μg/ml VN or FN. Cells were incubated at 37°C for 2 h, fixed, and stained with rhodamine-conjugated phalloidin. Bar, 50 μm. (G and H) Lipid-vehicle (gray) or hS1-siRNA (black) transfected cells were also plated on polycarbonate filters coated with either 10 μg/ml VN (G) or FN (H) in a modified Boyden chamber. After 16 h, cells that migrated through the filter in response to 10% FBS in the lower chamber were quantified by colorimetric staining. The error bars represent the SEM from three independent experiments.

Mentions: To test the activity of the mS1 mutants on matrix ligands, the expression of the endogenous hS1 needs to be blocked. Thus, cells expressing mS1 constructs were transfected with siRNA designed to specifically target hS1 (Fig. 8 A). Transfection with siRNA efficiently silences hS1 (>90% reduction) in both NEO vector-control cells (Fig. 8 B) and in cells expressing mS1 constructs (GPI-mS1ED provided as a representative result; Fig. 8 C). Importantly, hS1 siRNA affects neither mS1 expression (Fig. 8 E) nor the expression of hS4 in either NEO vector control or mS1-expressing cells (Fig. 8 D). In addition, hS1 siRNA has no effect on the expression levels of either αvβ3 or β1 integrins as determined by FACS (unpublished data).


The syndecan-1 ectodomain regulates alphavbeta3 integrin activity in human mammary carcinoma cells.

Beauvais DM, Burbach BJ, Rapraeger AC - J. Cell Biol. (2004)

Down-regulation of S1 expression by siRNA disrupts cell spreading and migration on VN. (A) SiRNA targeting of hS1 mRNA. FACS analysis for (B and C) hS1 (mAb B-B4), (D) hS4 (mAb F94-8G3), and (E) mS1 (mAb 281.2) expression against IgG controls (black-filled histograms) in NEO- and GPI-mS1ED–expressing MDA-MB-231 cells 72 h after transfection with either lipid-vehicle alone (Vehicle or (V)) or 200 nM siRNA (RNAi or (R)). (F) NEOMDA-MB-231 cells and MDA-MB-231 cells expressing GPI-mS1ED, mS1Δ88-252, mS1Δ122-252, and mS1TDM were transfected with lipid-vehicle alone or 200 nM hS1-siRNA and seeded on wells coated with either 10 μg/ml VN or FN. Cells were incubated at 37°C for 2 h, fixed, and stained with rhodamine-conjugated phalloidin. Bar, 50 μm. (G and H) Lipid-vehicle (gray) or hS1-siRNA (black) transfected cells were also plated on polycarbonate filters coated with either 10 μg/ml VN (G) or FN (H) in a modified Boyden chamber. After 16 h, cells that migrated through the filter in response to 10% FBS in the lower chamber were quantified by colorimetric staining. The error bars represent the SEM from three independent experiments.
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Related In: Results  -  Collection

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fig8: Down-regulation of S1 expression by siRNA disrupts cell spreading and migration on VN. (A) SiRNA targeting of hS1 mRNA. FACS analysis for (B and C) hS1 (mAb B-B4), (D) hS4 (mAb F94-8G3), and (E) mS1 (mAb 281.2) expression against IgG controls (black-filled histograms) in NEO- and GPI-mS1ED–expressing MDA-MB-231 cells 72 h after transfection with either lipid-vehicle alone (Vehicle or (V)) or 200 nM siRNA (RNAi or (R)). (F) NEOMDA-MB-231 cells and MDA-MB-231 cells expressing GPI-mS1ED, mS1Δ88-252, mS1Δ122-252, and mS1TDM were transfected with lipid-vehicle alone or 200 nM hS1-siRNA and seeded on wells coated with either 10 μg/ml VN or FN. Cells were incubated at 37°C for 2 h, fixed, and stained with rhodamine-conjugated phalloidin. Bar, 50 μm. (G and H) Lipid-vehicle (gray) or hS1-siRNA (black) transfected cells were also plated on polycarbonate filters coated with either 10 μg/ml VN (G) or FN (H) in a modified Boyden chamber. After 16 h, cells that migrated through the filter in response to 10% FBS in the lower chamber were quantified by colorimetric staining. The error bars represent the SEM from three independent experiments.
Mentions: To test the activity of the mS1 mutants on matrix ligands, the expression of the endogenous hS1 needs to be blocked. Thus, cells expressing mS1 constructs were transfected with siRNA designed to specifically target hS1 (Fig. 8 A). Transfection with siRNA efficiently silences hS1 (>90% reduction) in both NEO vector-control cells (Fig. 8 B) and in cells expressing mS1 constructs (GPI-mS1ED provided as a representative result; Fig. 8 C). Importantly, hS1 siRNA affects neither mS1 expression (Fig. 8 E) nor the expression of hS4 in either NEO vector control or mS1-expressing cells (Fig. 8 D). In addition, hS1 siRNA has no effect on the expression levels of either αvβ3 or β1 integrins as determined by FACS (unpublished data).

Bottom Line: This paper demonstrates that the alpha(v)beta(3) integrin and syndecan-1 (S1) are functionally coupled.Coupling of the syndecan to alpha(v)beta(3) requires the S1 ectodomain (ED), as ectopic expression of glycosylphosphatidylinositol-linked S1ED enhances alpha(v)beta(3) recognition of vitronectin; and treatments that target this domain, including competition with recombinant S1ED protein or anti-S1ED antibodies, mutation of the S1ED, or down-regulation of S1 expression by small-interfering RNAs, disrupt alpha(v)beta(3)-dependent cell spreading and migration.Thus, S1 is likely to be a critical regulator of many cellular behaviors that depend on activated alpha(v)beta(3) integrins.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison, Madison, WI 53706, USA.

ABSTRACT
The alpha(v)beta(3) integrin participates in cell morphogenesis, growth factor signaling, and cell survival. Activation of the integrin is central to these processes and is influenced by specific ECM components, which engage both integrins and syndecans. This paper demonstrates that the alpha(v)beta(3) integrin and syndecan-1 (S1) are functionally coupled. The integrin is dependent on the syndecan to become activated and to mediate signals required for MDA-MB-231 and MDA-MB-435 human mammary carcinoma cell spreading on vitronectin or S1-specific antibody. Coupling of the syndecan to alpha(v)beta(3) requires the S1 ectodomain (ED), as ectopic expression of glycosylphosphatidylinositol-linked S1ED enhances alpha(v)beta(3) recognition of vitronectin; and treatments that target this domain, including competition with recombinant S1ED protein or anti-S1ED antibodies, mutation of the S1ED, or down-regulation of S1 expression by small-interfering RNAs, disrupt alpha(v)beta(3)-dependent cell spreading and migration. Thus, S1 is likely to be a critical regulator of many cellular behaviors that depend on activated alpha(v)beta(3) integrins.

Show MeSH
Related in: MedlinePlus