Modulation of cell proliferation and differentiation through substrate-dependent changes in fibronectin conformation

Abstract

Integrin-mediated cell adhesion to extracellular matrices provides signals essential for cell cycle progression and differentiation. We demonstrate that substrate-dependent changes in the conformation of adsorbed fibronectin (Fn) modulated integrin binding and controlled switching between proliferation and differentiation. Adsorption of Fn onto bacterial polystyrene (B), tissue culture polystyrene (T), and collagen (C) resulted in differences in Fn conformation as indicated by antibody binding. Using a biochemical method to quantify bound integrins in cultured cells, we found that differences in Fn conformation altered the quantity of bound alpha5 and beta1 integrin subunits but not alphav or beta3. C2C12 myoblasts grown on these Fn-coated substrates proliferated to different levels (B > T > C). Immunostaining for muscle-specific myosin revealed minimal differentiation on B, significant levels on T, and extensive differentiation on C. Differentiation required binding to the RGD cell binding site in Fn and was blocked by antibodies specific for this site. Switching between proliferation and differentiation was controlled by the levels of alpha5beta1 integrin bound to Fn, and differentiation was inhibited by anti-alpha5, but not anti-alphav, antibodies, suggesting distinct integrin-mediated signaling pathways. Control of cell proliferation and differentiation through conformational changes in extracellular matrix proteins represents a versatile mechanism to elicit specific cellular responses for biological and biotechnological applications.

Figure 1

Fn adsorption (mean ± SD; three separate experiments in duplicate) as a function of coating concentration for different substrates. Surfaces were coated with different concentrations of Fn for 30 min and blocked in 1% BSA for 30 min. Adsorption of ¹²⁵I-Fn increased linearly with coating concentration until saturation levels reached ∼10 μg/ml.

Figure 2

Antibody binding assay for Fn conformation. Relative fluorescent intensity (RFI, mean ± SD; n = 3) for antibody binding as a function of adsorbed Fn surface density is shown. Different anti-Fn antibodies were examined: HFN7.1, 3E1, 4B2, and Cappel polyclonal antibody. Antibody binding increased sigmoidally with the log of Fn surface density. Shifts in antibody binding profiles represent variations in binding efficiency and reflect differences in the conformation of Fn adsorbed to the different substrates.

Figure 3

Integrin binding analysis for IMR-90 fibroblasts plated on different Fn-coated substrates for 16 h under serum-free conditions. (A) Schematic diagram of cross-linking and extraction procedure. (1) Cells are plated on Fn-coated substrates. (2) Bound integrins are cross-linked to extracellular matrix using sulfo-BSOCOES. (3) Cellular components, including unbound integrins, are extracted using 0.1% SDS, leaving behind Fn and its cross-linked integrins. (4) Cross-linking is reversed, and integrins are recovered and quantified by Western blotting. (B) Representative Western blots for integrin subunits (α₃, α₅, α_v, β₁, and β₃). Blots show soluble fractions (+) and cross-linked fractions for C, T, and B. For β₁, soluble fractions for each substrate (sc, st, and sb) were used to normalize for cell numbers and show two bands: surface-expressed integrin (slow) and intracellular integrin precursor (fast).

Figure 4

Immunofluorescent staining for α₅ integrin subunit on IMR-90 cells cultured on different Fn-coated substrates for 16 h and cross-linked as in Figure 3. Cells were extracted with either 0.1% SDS (A) or 1% Triton X-100 (B) before staining using an antibody against the α₅ cytoplasmic domain. (C) Phase contrast micrographs of Triton X-100–extracted cells. All photographs are at the same magnification (600×) and exposure. More intense staining of the SDS-extracted cells reflects more complete removal of cytoplasmic proteins that can block antibody access to epitopes in the cytoplasmic domain of α₅.

Figure 5

C2C12 myoblast proliferation and differentiation on Fn-coated substrates. Phase contrast micrographs showing cell density and morphology at 16 h (A) and after 3 d (B) in culture (both at 40× magnification). (C) At 3 d, cultures were stained with MF20 for sarcomeric myosin (green) and ethidium homodimer for nuclei (red) (200× magnification). Levels of myogenic differentiation are quantified in Table 3.

Figure 6

Effect of α₅ and α_v integrin-specific blocking antibodies on C2C12 differentiation for Fn-coated substrates. Cells were plated as for Figure 5 and incubated for 3 d in the presence or absence of the antibodies. (A) Dose-dependent inhibition of myogenic differentiation on Fn-coated C by anti-α₅, but not anti-α_v, antibodies (mean ± SD; two separate experiments in duplicate). (B) Inhibition of myogenic differentiation for Fn-coated B, T, and C with saturating levels of anti-α₅, but not anti-α_v, antibodies (mean ± SD; two separate experiments in duplicate).

Figure 7

Effect of Fn-specific antibodies on C2C12 myogenic differentiation for Fn-coated substrates. Cells were plated as for Figure 5 and 6 and incubated for 3 d in the presence or absence of antibodies against Fn. The cells remained normally spread for all antibody concentrations reported. (A) Dose-dependent inhibition of C2C12 differentiation on Fn-coated C with adhesion-blocking polyclonal and HFN7.1 monoclonal antibodies (mean ± SD; two separate experiments in duplicate). (B) Comparison of inhibition by mAbs to different domains in Fn. Adhesion-blocking HFN7.1 inhibited differentiation (mean ± SD; two separate experiments in duplicate), whereas 3E1 and 4B2 (mean ± SD; n = 2), which bind to epitopes outside the cell binding domain, did not affect differentiation.