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Prestress in the extracellular matrix sensitizes latent TGF-β1 for activation.

Klingberg F, Chow ML, Koehler A, Boo S, Buscemi L, Quinn TM, Costell M, Alman BA, Genot E, Hinz B - J. Cell Biol. (2014)

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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Tissue Repair and Regeneration, Matrix Dynamics Group, Faculty of Dentistry, University of Toronto, Toronto, Ontario M5S 3E2, Canada.

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Integrins mediate binding of myofibroblasts to LTBP-1. (A and B) Arrays of an islet with typical focal adhesion features (10 × 4 × 1.5 µm) were microcontact printed using purified LTBP-1–EGFP (A) or plasma FN as a control (B). After attachment for 4 h to microcontact printed arrays in the absence of serum, hDMfs were immunostained for the printed protein (red, false colored for LTBP-1–EGFP), vinculin, and integrin subunits β1, β3, and β5 (green). Insets show higher magnification views of select focal adhesion. (C) hDMfs were seeded onto LTBP-1–EGFP-coated substrates in the presence of blocking antibodies or cyclic peptides against integrin subunits β1, β3, and β5 or the RGD site. Focal adhesion formation was visualized with vinculin (green), and nuclei were stained with DAPI (blue). (D) The number of adherent hDMfs on LTBP-1–EGFP-coated substrates was quantified by image analysis. Graph shows mean values and SDs from at least three independent experiments (***, P ≤ 0.005; two-tailed paired t test). con, control. Bars: (main images) 20 µm; (insets) 5 µm.
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fig5: Integrins mediate binding of myofibroblasts to LTBP-1. (A and B) Arrays of an islet with typical focal adhesion features (10 × 4 × 1.5 µm) were microcontact printed using purified LTBP-1–EGFP (A) or plasma FN as a control (B). After attachment for 4 h to microcontact printed arrays in the absence of serum, hDMfs were immunostained for the printed protein (red, false colored for LTBP-1–EGFP), vinculin, and integrin subunits β1, β3, and β5 (green). Insets show higher magnification views of select focal adhesion. (C) hDMfs were seeded onto LTBP-1–EGFP-coated substrates in the presence of blocking antibodies or cyclic peptides against integrin subunits β1, β3, and β5 or the RGD site. Focal adhesion formation was visualized with vinculin (green), and nuclei were stained with DAPI (blue). (D) The number of adherent hDMfs on LTBP-1–EGFP-coated substrates was quantified by image analysis. Graph shows mean values and SDs from at least three independent experiments (***, P ≤ 0.005; two-tailed paired t test). con, control. Bars: (main images) 20 µm; (insets) 5 µm.

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.


Prestress in the extracellular matrix sensitizes latent TGF-β1 for activation.

Klingberg F, Chow ML, Koehler A, Boo S, Buscemi L, Quinn TM, Costell M, Alman BA, Genot E, Hinz B - J. Cell Biol. (2014)

Integrins mediate binding of myofibroblasts to LTBP-1. (A and B) Arrays of an islet with typical focal adhesion features (10 × 4 × 1.5 µm) were microcontact printed using purified LTBP-1–EGFP (A) or plasma FN as a control (B). After attachment for 4 h to microcontact printed arrays in the absence of serum, hDMfs were immunostained for the printed protein (red, false colored for LTBP-1–EGFP), vinculin, and integrin subunits β1, β3, and β5 (green). Insets show higher magnification views of select focal adhesion. (C) hDMfs were seeded onto LTBP-1–EGFP-coated substrates in the presence of blocking antibodies or cyclic peptides against integrin subunits β1, β3, and β5 or the RGD site. Focal adhesion formation was visualized with vinculin (green), and nuclei were stained with DAPI (blue). (D) The number of adherent hDMfs on LTBP-1–EGFP-coated substrates was quantified by image analysis. Graph shows mean values and SDs from at least three independent experiments (***, P ≤ 0.005; two-tailed paired t test). con, control. Bars: (main images) 20 µm; (insets) 5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4210443&req=5

fig5: Integrins mediate binding of myofibroblasts to LTBP-1. (A and B) Arrays of an islet with typical focal adhesion features (10 × 4 × 1.5 µm) were microcontact printed using purified LTBP-1–EGFP (A) or plasma FN as a control (B). After attachment for 4 h to microcontact printed arrays in the absence of serum, hDMfs were immunostained for the printed protein (red, false colored for LTBP-1–EGFP), vinculin, and integrin subunits β1, β3, and β5 (green). Insets show higher magnification views of select focal adhesion. (C) hDMfs were seeded onto LTBP-1–EGFP-coated substrates in the presence of blocking antibodies or cyclic peptides against integrin subunits β1, β3, and β5 or the RGD site. Focal adhesion formation was visualized with vinculin (green), and nuclei were stained with DAPI (blue). (D) The number of adherent hDMfs on LTBP-1–EGFP-coated substrates was quantified by image analysis. Graph shows mean values and SDs from at least three independent experiments (***, P ≤ 0.005; two-tailed paired t test). con, control. Bars: (main images) 20 µm; (insets) 5 µm.
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.

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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Tissue Repair and Regeneration, Matrix Dynamics Group, Faculty of Dentistry, University of Toronto, Toronto, Ontario M5S 3E2, Canada.

Show MeSH
Related in: MedlinePlus