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First published online 23 May 2006
doi: 10.1242/jcs.02970

Journal of Cell Science 119, 2445-2456 (2006)
Published by The Company of Biologists 2006
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Syndecan-1 regulates {alpha}vß5 integrin activity in B82L fibroblasts

K. J. McQuade1,*, D. M. Beauvais2,*, B. J. Burbach2 and A. C. Rapraeger1,2,3,{ddagger}

1 Graduate Programs in Cellular and Molecular Biology, University of Wisconsin-Madison, Madison, WI 53706, USA
2 Graduate Programs in Molecular and Cellular Pharmacology, University of Wisconsin-Madison, Madison, WI 53706, USA
3 Graduate Programs in Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison, Madison, WI 53706, USA


Figure 1
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  		Fig. 1. Sdc1 mediates cell spreading in B82L fibroblasts. B82L cells were plated on nitrocellulose coated with 60 µg/ml non-immune mouse IgG (A,B), 60 µg/ml mAb 281.2 (C,D) or 200 µg/ml S4ED pAb (E,F). Fetal bovine serum (FBS) (10%) was added to cells plated in B, D and F. Cells were incubated for 2 hours then fixed with paraformaldehyde and stained with Rhodamine-conjugated phalloidin. Bar, 50 µm.

 

Figure 2
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  		Fig. 2. Vitronectin induces complete spreading of B82L fibroblasts. B82L cells were plated on wells co-coated with increasing amounts of VN and 60 µg/ml mAb 281.2, 150 µg/ml S4ED pAb or in the absence of antibody. Cells were allowed to spread 2 hours before fixation and labeling with Rhodamine-phalloidin. Bar, 50 µm.

 

Figure 3
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  		Fig. 3. siRNA blockade of ß5-subunit expression blocks syndecan-induced cell spreading. (A) Suspended cells are analyzed by flow-cytometry with antibodies capable of recognizing mouse ß1 (HMß1-1), ß3 (2C9.G2) or {alpha}v (H9.2B8) integrin subunits, mAb 281.2 specific for mouse Sdc1, or nonspecific IgG control (gray fill). Cells treated with ß5-integrin-specific or control siRNA are compared. (B) Representative western blot of lysates of cells treated with 0, 200, 400, 600 or 800 nM ß5-specific siRNA and probed for expression of ß5-integrin subunit. FAK expression levels are shown as a loading control. (C) Quantification (± s.e.) of relative ß5 integrin subunit expression from duplicate blots as described in (B). (D) B82L cells were plated on wells coated with 60 µg/ml mAb 281.2 and increasing amounts of VN after treatment with ß5-integrin-specific or control siRNA. Cells were allowed to spread 2 hours before fixation and labeling with Rhodamine-phalloidin. Bar, 50 µm.

 

Figure 4
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  		Fig. 4. Downregulation of mouse Sdc1 expression by siRNA blocks {alpha}vß5-dependent cell attachment and spreading on vitronectin. FACS or immunoblot analysis for (A,F) mouse Sdc1 (mAb 281.2), (B) {alpha}v integrin subunit (mAb H9.2B8), (C) mouse Sdc4 (mAb KY8.2), (D) human Sdc1 (mAb B-B4) and (E) FcRecto-hS1 chimera (FITC-conjugated hIgG) expression against isotype IgG controls (red fill) in vector NEO (A-C), human Sdc1 (D), FcRecto-hS1 (E) and GPI-mS1ED (F) expressing B82L cells 48 hours after transfection with either 600 nM control (Control) or mouse Sdc1-specific siRNA (siRNA). (G) B82LNEO empty vector-transfected control cells and B82L cells expressing human Sdc1, the FcRecto-hS1 or GPI-mS1ED chimera were transfected with control or mouse Sdc1-specific siRNA and seeded on wells coated with either 5 µg/ml VN alone or a mixed substratum of VN plus 60 µg/ml of antibody directed against mouse Sdc1 (281.2), human Sdc1 (B-B4) or the FcRecto-hS1 chimera (hIgG). Cells were incubated at 37°C for 2 hours, fixed and stained with Rhodamine-conjugated phalloidin. Bar, 50 µm.

 

Figure 5
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  		Fig. 5. GAG chains are not required for filopodial extension or complete B82L cell spreading. B82L cells were detached using EDTA and treated in suspension either without (A,C,F,I) or with a combination of heparinases I and III and chondroitin ABC lyases (B,D,G,J) for 2 hours before plating on 200 µg/ml HBD-FN (A,B), 60 µg/ml mAb 281.2 (C,D), 60 µg/ml plus 1 µg/ml VN (F,G) or 5 µg/ml VN (I,J). Quantification of treated (white bars) or untreated (gray bars) cell extension of filopodia on 60 µg/ml mAb 281.2 (E) or complete cell spreading on mAb 281.2 supplemented with 1 µg/ml VN or 3 µg/ml FN (H) or complete cell spreading on 5 µg/ml VN or 60 µg/ml FN (K) is also shown. Bar, 50 µm.

 

Figure 6
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  		Fig. 6. B82L-cell spreading on VN is blocked by recombinant S1ED. (A) B82L fibroblasts were plated on 5 µg/ml VN in the absence of other treatment, or in the presence of 30 µM GST, 1-30 µM GST-mS1ED or 30 µM GST-mS4ED (inset), then fixed and stained with Rhodamine-phalloidin for visualization. (B) Quantification of cell adhesion in triplicate samples (± s.e.) plated either with no additions, or concentrations of GST-mS1ED ranging from 0-30 µM. Bar, 50 µm.

 

Figure 7
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  		Fig. 7. Co-immunoprecipitation of ß5 integrin with Sdc1 requires the Sdc1 ectodomain. Western blots probed with rabbit polyclonal ß5 integrin (A,B), pan-syndecan or S1ED (C) antibody for detection of ß5 integrin and Sdc1, respectively, in immune complexes isolated after immunoprecipitation of full-length mouse Sdc1 (mAb 281.2), human Sdc1 constructs (mAb B-B4) and FcRecto-hS1 chimera (mAb 10.1) from pre-cleared B82L whole-cell lysates. In S1ED-competition experiments (A), 30 µM soluble GST, GST-mS1ED (with mAb B-B4) or GST-hS1ED (with mAb 281.2) was added to the reaction mixture. Provided as a reference is a methanol precipitation (MeOH) of 300 µg of whole-cell lysate. ß5 integrin immunoblotting reveals a 110 kDa band, under reduced conditions, detectable in the mouse Sdc1 and select human Sdc1 (hS1, pLTM, {Delta}cyto), but not FcRecto-hS1 chimera immunoprecipitates or immunoprecipitates isolated with antibody isotype control IgG: mouse IgG1 (mIgG) for mAbs B-B4 and 10.1 and rat IgG2A (rIgG) for mAb 281.2.

 

Figure 8
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  		Fig. 8. Model of Sdc1-regulated {alpha}vß5-integrin signaling.

 




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