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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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Overexpression and ligation of S1 activates αvβ3 integrins and primes cells to spread on VN. (A) MDA-MB-231 cells transfected with empty vector (NEO), GPI-mS1ED, or mS1TDM were seeded on wells coated with 1, 3, or 10 μg/ml VN. Cells were incubated at 37°C for 2 h, fixed, and stained with rhodamine-conjugated phalloidin. Bar, 50 μm. (B–E) Suspended cells in which mS1 was clustered (mAb 281.2, B and E) or not clustered (mAb KY8.2, C and D) were fixed and labeled with mAb LM609 (B) or WOW1 mouse Fab (C–E) followed by an Alexa 488–conjugated secondary antibody and analyzed by FACS. As controls for WOW1 staining (C), suspended cells were incubated with plating medium alone (black-filled histogram), plating medium containing 1 mM MnCl2 (right-shifted histogram), or divalent cation-free PBS (left-shifted histogram) before fixation and staining. (F) Cells were seeded on wells coated with either mAb B-B4 or 281.2, 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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fig7: Overexpression and ligation of S1 activates αvβ3 integrins and primes cells to spread on VN. (A) MDA-MB-231 cells transfected with empty vector (NEO), GPI-mS1ED, or mS1TDM were seeded on wells coated with 1, 3, or 10 μg/ml VN. Cells were incubated at 37°C for 2 h, fixed, and stained with rhodamine-conjugated phalloidin. Bar, 50 μm. (B–E) Suspended cells in which mS1 was clustered (mAb 281.2, B and E) or not clustered (mAb KY8.2, C and D) were fixed and labeled with mAb LM609 (B) or WOW1 mouse Fab (C–E) followed by an Alexa 488–conjugated secondary antibody and analyzed by FACS. As controls for WOW1 staining (C), suspended cells were incubated with plating medium alone (black-filled histogram), plating medium containing 1 mM MnCl2 (right-shifted histogram), or divalent cation-free PBS (left-shifted histogram) before fixation and staining. (F) Cells were seeded on wells coated with either mAb B-B4 or 281.2, 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 if overexpression of S1 enhances VN recognition via the αvβ3 integrin, cell attachment and spreading were assessed on wells containing increasing concentrations of VN (1, 3, and 10 μg/ml; Fig. 7 A). NEO control cells largely fail to bind to wells coated with 1 μg/ml VN and display only modest spreading in response to 3 μg/ml. Full adhesion and spreading is not achieved until cells encounter high concentrations of VN (10 μg/ml). In contrast, cells overexpressing the S1ED (GPI-mS1ED) attach and spread on low VN concentrations and this response increases with higher concentrations of VN. However, this response is dependent on S1's engagement of the matrix as cells expressing mS1TDM (which lacks its HS chains) mimic the response of NEO control cells.


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

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

Overexpression and ligation of S1 activates αvβ3 integrins and primes cells to spread on VN. (A) MDA-MB-231 cells transfected with empty vector (NEO), GPI-mS1ED, or mS1TDM were seeded on wells coated with 1, 3, or 10 μg/ml VN. Cells were incubated at 37°C for 2 h, fixed, and stained with rhodamine-conjugated phalloidin. Bar, 50 μm. (B–E) Suspended cells in which mS1 was clustered (mAb 281.2, B and E) or not clustered (mAb KY8.2, C and D) were fixed and labeled with mAb LM609 (B) or WOW1 mouse Fab (C–E) followed by an Alexa 488–conjugated secondary antibody and analyzed by FACS. As controls for WOW1 staining (C), suspended cells were incubated with plating medium alone (black-filled histogram), plating medium containing 1 mM MnCl2 (right-shifted histogram), or divalent cation-free PBS (left-shifted histogram) before fixation and staining. (F) Cells were seeded on wells coated with either mAb B-B4 or 281.2, 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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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172512&req=5

fig7: Overexpression and ligation of S1 activates αvβ3 integrins and primes cells to spread on VN. (A) MDA-MB-231 cells transfected with empty vector (NEO), GPI-mS1ED, or mS1TDM were seeded on wells coated with 1, 3, or 10 μg/ml VN. Cells were incubated at 37°C for 2 h, fixed, and stained with rhodamine-conjugated phalloidin. Bar, 50 μm. (B–E) Suspended cells in which mS1 was clustered (mAb 281.2, B and E) or not clustered (mAb KY8.2, C and D) were fixed and labeled with mAb LM609 (B) or WOW1 mouse Fab (C–E) followed by an Alexa 488–conjugated secondary antibody and analyzed by FACS. As controls for WOW1 staining (C), suspended cells were incubated with plating medium alone (black-filled histogram), plating medium containing 1 mM MnCl2 (right-shifted histogram), or divalent cation-free PBS (left-shifted histogram) before fixation and staining. (F) Cells were seeded on wells coated with either mAb B-B4 or 281.2, 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 if overexpression of S1 enhances VN recognition via the αvβ3 integrin, cell attachment and spreading were assessed on wells containing increasing concentrations of VN (1, 3, and 10 μg/ml; Fig. 7 A). NEO control cells largely fail to bind to wells coated with 1 μg/ml VN and display only modest spreading in response to 3 μg/ml. Full adhesion and spreading is not achieved until cells encounter high concentrations of VN (10 μg/ml). In contrast, cells overexpressing the S1ED (GPI-mS1ED) attach and spread on low VN concentrations and this response increases with higher concentrations of VN. However, this response is dependent on S1's engagement of the matrix as cells expressing mS1TDM (which lacks its HS chains) mimic the response of NEO control cells.

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