The syndecan-1 ectodomain regulates alphavbeta3 integrin activity in human mammary carcinoma cells - PubMed (original) (raw)
The syndecan-1 ectodomain regulates alphavbeta3 integrin activity in human mammary carcinoma cells
DeannaLee M Beauvais et al. J Cell Biol. 2004.
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.
Figures
Figure 1.
MDA-MB-231 human mammary carcinoma cell spreading on VN, but not FN, is disrupted by soluble, recombinant S1ED. Cells were plated on wells coated with 10 μg/ml VN or FN in plating medium alone or medium containing either 30 μg/ml mAb LM609, 25 μg/ml mAb 13, or 20 μM GST-mS1ED or -mS4ED. Cells were incubated at 37°C for 2 h, fixed, and stained with rhodamine-conjugated phalloidin. Bar, 50 μm.
Figure 2.
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.
Figure 3.
MDA-MB-435, but not MCF-7, human carcinoma cells display functional coupling of S1 and αvβ3 integrins on VN. Depicted are phalloidin-stained cells 2 h after plating on wells coated with 10 μg/ml VN (top half of panels) or FN (bottom half) in plating medium alone or medium containing either 30 μg/ml mAb LM609, 25 μg/ml mAb 13, or 20 μM GST-mS1ED or -mS4ED. Bar, 50 μm.
Figure 4.
Polyclonal S1ED antibodies disrupt αvβ3 integrin–dependent cell spreading on VN. MDA-MB-231 and -435 cells were plated on wells coated with 10 μg/ml VN (top half) or FN (bottom half) in plating medium alone or medium containing 10, 100, or 250 μg/ml of anti-mS1ED pAb. Cells were incubated at 37°C for 2 h, fixed, and stained with rhodamine-conjugated phalloidin. Bar, 50 μm.
Figure 5.
S1ED inhibitors disrupt αvβ3 integrin–dependent cell migration on VN. MDA-MB-231, MDA-MB-435, and MCF-7 cells in plating medium alone (black) or in plating medium containing either 20 μM GST-mS1ED (white) or 250 μg/ml mS1ED pAbs (gray) were seeded on polycarbonate filters coated with either 10 μg/ml VN (A) or FN (B) 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.
Figure 6.
Deletion of a region of the S1ED blocks αvβ3-mediated cell spreading. (A) Graphic representation of S1 expression constructs transfected into MDA-MB-231 cells and their relative expression levels as detected by FACS (mean fluorescent intensity). Asterisks indicate the HS attachment sites. (B) Cells transfected with empty vector (NEO) or S1 constructs were seeded in plating medium on wells coated with either anti-hS1 mAb B-B4 (NEO + inset, hS1, and insets of mS1) or anti-mS1 mAb 281.2 (all others). Where noted, cells were pretreated in suspension with 1 μg/ml mAb P5D2 for 15 min before plating. Cells were incubated at 37°C for 2 h, fixed, and stained with rhodamine-conjugated phalloidin. Bar, 50 μm.
Figure 7.
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.
Figure 8.
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.
References
- Barbareschi, M., P. Maisonneuve, D. Aldovini, M.G. Cangi, L. Pecciarini, F. Angelo Mauri, S. Veronese, O. Caffo, A. Lucenti, P.D. Palma, et al. 2003. High syndecan-1 expression in breast carcinoma is related to an aggressive phenotype and to poorer prognosis. Cancer. 98:474–483. - PubMed
- Beauvais, D.M., and A.C. Rapraeger. 2003. Syndecan-1-mediated cell spreading requires signaling by αvβ3 integrins in human breast carcinoma cells. Exp. Cell Res. 286:219–232. - PubMed
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