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Fibulin-1 suppression of fibronectin-regulated cell adhesion and motility

¹ Medical University of South Carolina, Department of Cell Biology, 171 Ashley Avenue, Charleston, SC 29425-2204, USA
² Eotvos University, Department of Biological Physics, Budapest, Hungary

View larger version (35K): [in a new window]   Fig. 1. Fibulin-1 as an adhesion protein. The indicated cells (3.5x104 per well) were incubated in microtitre wells coated with varying concentrations of FN, fibulin-1 or BSA for 1 hour at 37°C, 5% CO2. After fixation the bound cells were stained with 0.25% crystal violet and the stain quantified by spectrophotometry after release with 1% SDS.

View larger version (16K): [in a new window]   Fig. 2. Effect of fibulin-1 on FN-mediated cell adhesion. (A) Microtitre wells (tissue-culture polystyrene) were coated with varying concentrations of FN, the unoccupied sites were blocked with BSA and the wells were incubated with either fibulin-1 or BSA (each at 100 µg ml–1). MDA MB231 cells (3.5x104 per well) were incubated with the coatings for 60 minutes at 37°C. The wells were rinsed, cells fixed with 10% neutral formalin and stained with 0.25% crystal violet. The stain was released with 1% SDS and quantified by spectrophotometry. (B) Microtitre wells (non-tissue-culture polystyrene) were coated with varying concentrations of FN and the adhesion of MDA MB231 cells evaluated as above.

View larger version (91K): [in a new window]   Fig. 3. Effect of fibulin-1 on FN-stimulated cell spreading. (A,B) The proportion of MDA MB231 cells that spread on tissue-culture polystyrene (A) or non-tissue-culture polystyrene (B) coated with FN or FN-fibulin-1 as a function of time. (C) Spreading of MDA MB231 cells transfected to express fibulin-1 and vector-transfected controls on FN substrata on tissue-culture polystyrene. (A-C) Unoccupied sites were blocked with BSA. Digital images of fields of cells containing 60-100 cells were collected at 2.5 minute intervals for 1 hour after seeding, and the number of cells spread in each successive image was quantified. (D,E) MDA MB231 cells transfected with empty vector (D) or fibulin-1 expression construct (E) were seeded onto FN coatings and allowed to attach for 40 minutes. The cells were then fixed, stained and photographed. Notice the spread and migratory morphology of the empty-vector-transfected cells as opposed to the rounded morphology of fibulin-1-transfected cells.

View larger version (23K): [in a new window]   Fig. 4. Cell type-specific inhibition of FN-stimulated haptotactic migration by fibulin-1. The undersurfaces of Transwell membranes (8 µm pores) were coated with FN (100 µg ml–1), unoccupied sites were blocked with BSA and were then incubated with fibulin-1 or BSA (100 µg ml–1 each). Cells (1x105) in sfDMEM were added to the upper chambers of inserts placed in wells containing sfDMEM plus fibulin-1 or BSA (50 µg ml–1 each). After 18 hours of incubation at 37°C, 5% CO2, the number of cells that had migrated to the undersurface of the membranes was determined. The plotted values are means ± the standard deviations of cell counts from five independent optical fields from triplicate experiments. The results shown were obtained using fibulin-1 isolated by monoclonal antibody 3A11 IgG-Sepharose. Similar findings were obtained using fibulin-1 isolated with a monoclonal antibody 5D12 which binds to an epitope distinct from that of 3A11 (data not shown).

View larger version (21K): [in a new window]   Fig. 5. Fibulin-1 inhibits the invasion of endocardial cushion mesenchymal cells into gels of type I collagen and fibronectin. Stage 18 chick endocardial cushion explants were placed onto the surface of gels of type I collagen (1 mg ml–1) containing FN (20 µg ml–1) plus or minus fibulin-1 (100 µg ml–1). The number of cells that had invaded into the gels (A) and the depth of invasion (B) were quantified after 50 hours. The plotted values in A are means ± the standard deviations from measurements made on four cushion explants. (A,B) The number of cells in optical planes extending through a depth of 400 µm of the gel in four areas underlying each explant was counted and summed. Asterisk (*) indicates that the value is significantly different from control (P<0.05).

View larger version (42K): [in a new window]   Fig. 6. Two-dimensional cell migration analysis. (A) Average velocity of MDA MB231cells on FN or FN-fibulin-1 substrata. Each value that is plotted represents an average velocity calculated from the displacement of 20 cell centroids during a 5 hour period. Shown are data from two and three independent experiments of cell migration on FN and FN-fibulin-1 substrata, respectively. Lines represent linear fits of all points in all experiments for each substratum. (B) Average distance that a cell travelled during a given time on FN versus FN-fibulin-1 substratum. Each plotted value represents an average of the distance travelled for 20 cells for various times beginning 1 hour after seeding onto FN or FN-fibulin-1. Shown are data from two and three independent experiments of cell migration on FN and FN-fibulin-1 substrata, respectively. (C) Average distance that MDA MB231 cells transfected with either empty vector or fibulin-1 expression construct travel as a function of time on FN substratum. Each plotted value represents an average of the distance travelled by 20 cells for various times after seeding onto FN. (D) Quantification by ELISA of the amount of fibulin-1 secreted into the medium by MDA MB231 cells transfected with fibulin-1 expression construct.

View larger version (30K): [in a new window]   Fig. 7. 4-Integrins are not required for fibulin-1 inhibition of FN-stimulated haptotactic migration. (A) Evaluation of the effect of fibulin-1 on haptotactic migration of CHO cells engineered to express 4ß1. Analysis of expression of the integrin 4 subunit in various cultured lines by RT-PCR (B) and immunoblotting (C). The arrowheads in C indicate the intact 150 kDa and 180 kDa forms of the 4 polypeptide typically observed under non-reducing conditions.

View larger version (86K): [in a new window]   Fig. 8. Fibulin-1 does not bind to cell surface proteins and does not effect cell surface ß1 integrin binding to FN. Radiolabelled MDA MB231 cell surface proteins were bound to magnetic beads conjugated with fibulin-1, FN or BSA. Before incubation with cells, FN-beads were incubated with either fibulin-1 (FN + fibulin-1) or BSA (FN + BSA). (A) Autoradiograph and (B) immunoblot analysis using anti-ß1-integrin cytoplasmic domain antibody.

View larger version (14K): [in a new window]   Fig. 9. The binding of integrin 5ß1 to FN is not significantly altered by fibulin-1. Binding of 5ß1 integrin to FN and FN-fibulin-1 immobilized on tissue-culture polystyrene (A) and non-tissue-culture polystyrene (B) as measured by ELISA. The data shown has been corrected for non-specific integrin binding to BSA.

View larger version (14K): [in a new window]   Fig. 10. Glycosaminoglycan sulfation is not required for fibulin-1 inhibition of FN-stimulated haptotactic migration. MDA MB231 cells were cultured in the absence or presence of chlorate (20 mM) for 18 hours in low-sulfate HAMS/F12 medium and 10% dialysed BCS. (A) Chlorate treatment inhibited 98% of the normal amount of [35S]-SO4 incorporated into proteoglycans in the MDA MB231 cell layer. (B) Fibulin-1 inhibited the FN-stimulated haptotactic migration of both the control and chlorate-treated MDA MB231 cells to a similar extent.

View larger version (57K): [in a new window]   Fig. 11. Fibulin-1 reduces phosphorylation of MHC and ERK. (A) Immunoprecipitation analysis was used to assess [32P]-phosphate incorporation into MHC by MDA MB231 cells cultured for 30 minutes on surfaces of FN or FN-fibulin-1. (B) Immunoblotting was used to study the effect of fibulin-1 on the kinetics of ERK1/2 phosphorylation. The blot shown in B was re-probed with antibodies to non-phosphorylated ERK (C). The data shown in A-C represent at least three experiments each.

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