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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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S1 adhesion–mediated cell spreading correlates with αvβ3 integrin expression and activity. (A) FACS analysis of αvβ3 integrin expression (mAb LM609) in human mammary carcinoma cells against an IgG isotype control. (B) Depicted on split panels are phalloidin-stained MDA-MB-435 (top) and MCF-7 (bottom) cells 2 h after plating on wells coated with mAb B-B4 in plating medium alone or medium containing either 30 μg/ml mAb LM609, 1 μg/ml mAb 13, or 20 μM GST-mS1ED. Bar, 50 μm. (C) Untreated MDA-MB-435, MDA-MB-231, and MCF-7 cells (first and last columns) and cells pretreated in suspension with 1 μg/ml mAb 13 (middle columns) were seeded on wells coated with either mAb B-B4 or COL I in plating medium alone (first, second, and last columns) or medium containing 20 μM GST-mS1ED (third column). Cells were incubated at 37°C for 2 h, fixed, permeabilized, and stained with WOW1 and an Alexa 488–conjugated secondary antibody. Panel insets are corresponding phase-contrast pictures. Bar, 20 μm.
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fig2: S1 adhesion–mediated cell spreading correlates with αvβ3 integrin expression and activity. (A) FACS analysis of αvβ3 integrin expression (mAb LM609) in human mammary carcinoma cells against an IgG isotype control. (B) Depicted on split panels are phalloidin-stained MDA-MB-435 (top) and MCF-7 (bottom) cells 2 h after plating on wells coated with mAb B-B4 in plating medium alone or medium containing either 30 μg/ml mAb LM609, 1 μg/ml mAb 13, or 20 μM GST-mS1ED. Bar, 50 μm. (C) Untreated MDA-MB-435, MDA-MB-231, and MCF-7 cells (first and last columns) and cells pretreated in suspension with 1 μg/ml mAb 13 (middle columns) were seeded on wells coated with either mAb B-B4 or COL I in plating medium alone (first, second, and last columns) or medium containing 20 μM GST-mS1ED (third column). Cells were incubated at 37°C for 2 h, fixed, permeabilized, and stained with WOW1 and an Alexa 488–conjugated secondary antibody. Panel insets are corresponding phase-contrast pictures. Bar, 20 μm.

Mentions: To test whether or not the αvβ3 integrin's dependence on S1 extends to other carcinoma cells, we screened a panel of human carcinoma cells with mAb LM609 using FACS analysis. MDA-MB-435 cells exhibit higher αvβ3 expression than MDA-MB-231 cells, whereas MCF-7 cells are negative for this integrin (Fig. 2 A). To test the collaboration between S1 and αvβ3 integrin when cells are adherent via S1, MDA-MB-435 and MCF-7 cells were plated on wells coated with hS1-specific mAb B-B4. Although αvβ3-positive MDA-MB-435 cells spread on this antibody substratum, their spreading is blocked (Beauvais and Rapraeger, 2003) by treatment with either mAb LM609 (added either before plating or 30 min after plating, when cells have already begun to spread) or GST-mS1ED protein (which is not recognized by mAb B-B4). MCF-7 (Fig. 2 B) and T47D (unpublished data) cells, which are αvβ3 negative, bind to mAb B-B4 but fail to spread.


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

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

S1 adhesion–mediated cell spreading correlates with αvβ3 integrin expression and activity. (A) FACS analysis of αvβ3 integrin expression (mAb LM609) in human mammary carcinoma cells against an IgG isotype control. (B) Depicted on split panels are phalloidin-stained MDA-MB-435 (top) and MCF-7 (bottom) cells 2 h after plating on wells coated with mAb B-B4 in plating medium alone or medium containing either 30 μg/ml mAb LM609, 1 μg/ml mAb 13, or 20 μM GST-mS1ED. Bar, 50 μm. (C) Untreated MDA-MB-435, MDA-MB-231, and MCF-7 cells (first and last columns) and cells pretreated in suspension with 1 μg/ml mAb 13 (middle columns) were seeded on wells coated with either mAb B-B4 or COL I in plating medium alone (first, second, and last columns) or medium containing 20 μM GST-mS1ED (third column). Cells were incubated at 37°C for 2 h, fixed, permeabilized, and stained with WOW1 and an Alexa 488–conjugated secondary antibody. Panel insets are corresponding phase-contrast pictures. Bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172512&req=5

fig2: S1 adhesion–mediated cell spreading correlates with αvβ3 integrin expression and activity. (A) FACS analysis of αvβ3 integrin expression (mAb LM609) in human mammary carcinoma cells against an IgG isotype control. (B) Depicted on split panels are phalloidin-stained MDA-MB-435 (top) and MCF-7 (bottom) cells 2 h after plating on wells coated with mAb B-B4 in plating medium alone or medium containing either 30 μg/ml mAb LM609, 1 μg/ml mAb 13, or 20 μM GST-mS1ED. Bar, 50 μm. (C) Untreated MDA-MB-435, MDA-MB-231, and MCF-7 cells (first and last columns) and cells pretreated in suspension with 1 μg/ml mAb 13 (middle columns) were seeded on wells coated with either mAb B-B4 or COL I in plating medium alone (first, second, and last columns) or medium containing 20 μM GST-mS1ED (third column). Cells were incubated at 37°C for 2 h, fixed, permeabilized, and stained with WOW1 and an Alexa 488–conjugated secondary antibody. Panel insets are corresponding phase-contrast pictures. Bar, 20 μm.
Mentions: To test whether or not the αvβ3 integrin's dependence on S1 extends to other carcinoma cells, we screened a panel of human carcinoma cells with mAb LM609 using FACS analysis. MDA-MB-435 cells exhibit higher αvβ3 expression than MDA-MB-231 cells, whereas MCF-7 cells are negative for this integrin (Fig. 2 A). To test the collaboration between S1 and αvβ3 integrin when cells are adherent via S1, MDA-MB-435 and MCF-7 cells were plated on wells coated with hS1-specific mAb B-B4. Although αvβ3-positive MDA-MB-435 cells spread on this antibody substratum, their spreading is blocked (Beauvais and Rapraeger, 2003) by treatment with either mAb LM609 (added either before plating or 30 min after plating, when cells have already begun to spread) or GST-mS1ED protein (which is not recognized by mAb B-B4). MCF-7 (Fig. 2 B) and T47D (unpublished data) cells, which are αvβ3 negative, bind to mAb B-B4 but fail to spread.

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