Integrin-mediated mechanotransduction requires its dynamic interaction with specific extracellular matrix (ECM) ligands - PubMed (original) (raw)

Integrin-mediated mechanotransduction requires its dynamic interaction with specific extracellular matrix (ECM) ligands

S Jalali et al. Proc Natl Acad Sci U S A. 2001.

Abstract

The aim of this study is to elucidate the role of integrins in transducing fluid shear stress into intracellular signals in vascular endothelial cells, a fundamental process in vascular biology. We demonstrated that shear stress activates specific integrins in endothelial cells plated on substrates containing the cognate extracellular matrix (ECM) ligands. The shear stress-induced mechanotransduction, as manifested by integrin-Shc association, was abolished when new integrin-ECM ligand interactions were prevented by either blocking the integrin-binding sites of ECM ligands or conjugating the integrins to immobilized antibodies. Our results indicate that the dynamic formation of new connections between integrins and their specific ECM ligands is critical in relaying the signals induced by shear stress to intracellular pathways.

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Figures

Figure 1

Figure 1

Shear stress increases integrin occupancy on VN and FN. Glass slides were coated with in FN (5 μg/cm2;A) and VN (0.5 μg/cm2;B). HUVECs were plated on these coated slides in the absence of serum for 2 h and then exposed to shear stress (12 dyn/cm2) for 5 and 30 min or kept as static controls. The cells were fixed with 3% paraformaldehyde in PBS for 30 min, permeabilized in 0.2% Triton X-100-PBS for 5 min, and blocked with 10% normal goat serum. (A) The occupied β1 integrin was revealed by immunostaining using HUTS-21 mAb. (B) The occupied β3 integrin was revealed by LIBS-6 mAb. These mAbs were recognized by FITC-conjugated goat anti-mouse IgG (1:100).

Figure 2

Figure 2

Shear-induced integrin–Shc association is ECM-dependent. Glass slides were coated with FN (5 μg/cm2), CL (6 μg/cm2), LM (5 μg/cm2), or VN (0.5 μg/cm2). HUVECs were seeded on the slides in the absence of serum, and exposed to shear stress (12 dyn/cm2) for 30 min or kept as static controls. (A) The cell lysates were subjected to immunoprecipitation (IP) with LM609 anti-αvβ3, followed by immunoblotting (IB) with anti-Shc pAb. Each pair of lanes represents static control (C) and sheared (S) samples of cells plated on the ECM indicated. αvβ3–Shc association is demonstrated by coimmunoprecipitation in the sheared, but not static, HUVECs on FN and VN. (B) α6β1–Shc association is demonstrated by coimmunoprecipitation in the sheared HUVECs on LM only. Gels shown are representatives of three independent experiments.

Figure 3

Figure 3

Dynamic interaction with matrix proteins is essential for shear-induced αvβ3–Shc association. (A) HUVECs were allowed to adhere to FN-coated slides for 2 h in the absence of serum and then subjected to the following treatments: Lanes 1 and 2, untreated samples; lanes 3 and 4, incubated with 20 μg of the nonblocking 11E5; lanes 5 and 6, incubated with 3B8, which blocks the α5β1, but not the αvβ3, binding sites; lanes 7 and 8, incubated with 16G3, which blocks both the α5β1 and αvβ3 binding sites. HUVECs were either sheared (S) or kept as static control (C). The cell lysates were subjected to IP with LM609, and the immunoprecipitated αvβ3 was subjected to IB with an anti-Shc pAb. (B) Experiments were similar to Fig. 3_A_, except that anti-α5β1 mAb 1950 was used for IP. (C) Experiments were similar to Fig. 3_A_, except that HUVECs were seeded on VN-coated slides and incubated with either the nonblocking antibody 443 (lanes 1 and 2) or antibody 661 (lanes 3 and 4), which blocks the αvβ3 binding sites of VN. Gels are representative of three separate experiments.

Figure 4

Figure 4

Shear-induced αvβ3–Shc association and JNK activation were inhibited when HUVECs were seeded on slides coated with LM609 anti-αvβ3 mAb. The slides for lanes 1 and 2 were coated with FN (5 μg/cm2) and those for lanes 3 and 4 were coated with FG (5 μg/cm2). The slides for the remaining lanes were first coated with an anti-mouse IgG followed by LM609. HUVECs were seeded on the slides and allowed to adhere for 2 h (lanes 1–6) or 30 min (lane 7). (A) The seeded HUVECs were kept under static condition (C) or subjected to shear stress (S). Lane 7 (Ad) served as a positive adhesion control to show that αvβ3–Shc association occurred in static HUVECs at 30 min after onset of adhesion, but not at 2 h (lane 6). The cell lysates were subjected to IP with LM609, followed by IB with an anti-Shc pAb. (B) HUVECs were kept as static controls (C), subjected to shear stress (S), or exposed to UV irradiation of 80 J/m2 for 5 min (UV, lane 8). The cells were lysed and JNK kinase activity was assayed by using GST-c-Jun as the substrate. Gels are representative of three separate experiments.

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