Prestress in the extracellular matrix sensitizes latent TGF-β1 for activation.
Bottom Line: Mechanically conditioned ECM served as a substrate to measure the efficacy of TGF-β1 activation after cell contraction or direct force application using magnetic microbeads.The release of active TGF-β1 was always higher from prestrained ECM as compared with unorganized and/or relaxed ECM.The finding that ECM prestrain regulates the bioavailability of TGF-β1 is important to understand the context of diseases that involve excessive ECM remodeling, such as fibrosis or cancer.
Affiliation: Laboratory of Tissue Repair and Regeneration, Matrix Dynamics Group, Faculty of Dentistry, University of Toronto, Toronto, Ontario M5S 3E2, Canada.Show MeSH
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Mentions: Because human LTBP-1 contains an RGD consensus site, it is conceivable that αv integrins participate in LTBP-1 incorporation into ECM fibrils and cell attachment to LTBP-1–coated substrates. To identify the integrin putatively mediating adhesion to LTBP-1, we generated microcontact printed islet arrays of purified LTBP-1–EGFP with typical focal adhesion features (10 × 4 × 1.5 µm). hDMfs adhered specifically to the printed LTBP-1 at sites of vinculin-positive focal adhesions when seeded for 4 h in serum-free medium (Fig. 5 A). Focal adhesions forming on LTBP-1 islets contained β1 integrin, to a lesser extent β3 integrin, and no integrin β5. Control prints using FN as a ligand demonstrated that all tested integrins were expressed in hDMfs and localized to focal adhesions (Fig. 5 B). Next, we seeded hDMfs onto fully LTBP-1–coated substrates for 4 h in the presence of specific integrin-blocking antibodies and different RGD peptides (Fig. 5 C). Focal adhesion formation and cell spreading were reduced by all integrin blocking antibodies in order from strongest to weakest: β1, β3, and β5 integrin. All competitive integrin-blocking peptides, but not controls, inhibited focal adhesion formation and cell spreading on LTBP-1 (Fig. 5 C). Strongest blocking was achieved with RGD as confirmed by quantifying the number of adherent hDMfs on LTBP-1–coated substrates (Fig. 5 D). These results indicate that hDMfs bind directly to the RGD sequence in LTBP-1 via integrins that possibly aid the active organization of LTBP-1 into ECM fibrils in a cell tension–dependent manner. Of the RGD-recognizing integrins expressed in hDMfs, β5 integrin seem to bind weakest by far.
Affiliation: Laboratory of Tissue Repair and Regeneration, Matrix Dynamics Group, Faculty of Dentistry, University of Toronto, Toronto, Ontario M5S 3E2, Canada.