Therapeutic ultrasound bypasses canonical syndecan-4 signaling to activate rac1

Abstract

The application of pulsed, low intensity ultrasound is emerging as a potent therapy for the treatment of complex bone fractures and tissue damage. Ultrasonic stimuli accelerate fracture healing by up to 40% and enhance tendon and ligament healing by promoting cell proliferation, migration, and matrix synthesis through an unresolved mechanism. Ultrasound treatment also induces closure of nonunion fractures, at a success rate (85% of cases) similar to that of surgical intervention (68-96%) while avoiding the complications associated with surgery. The regulation of cell adhesion necessary for wound healing depends on cooperative engagement of the extracellular matrix receptors, integrin and syndecan, as exemplified by the wound healing defects observed in syndecan- and integrin-knock-out mice. This report distinguishes the influence of ultrasound on signals downstream of the prototypic fibronectin receptors, alpha(5)beta(1) integrin and syndecan-4, which cooperate to regulate Rac1 and RhoA. Ultrasonic stimulation fails to activate integrins or induce cell spreading on poor, electrostatic ligands. By contrast, ultrasound treatment overcomes the necessity of engagement or expression of syndecan-4 during the process of focal adhesion formation, which normally requires simultaneous engagement of both receptors. Ultrasound exerts an influence downstream of syndecan-4 and PKCalpha to specifically activate Rac1, itself a critical regulator of tissue repair, and to a lesser extent RhoA. The ability of ultrasound to bypass syndecan-4 signaling, which is known to facilitate efficient tissue repair, explains the reduction in healing times observed in ultrasound-treated patients. By substituting for one of the key axes of adhesion-dependent signaling, ultrasound therapy has considerable potential as a clinical technique.

FIGURE 1.

Ultrasound stimulates focal adhesion formation independently of syndecan-4 engagement. A, schematic representation of the ultrasound wave form. B, wild-type MEFs were spread on 50K for 2 h before stimulating with syndecan-4 ligand (H/0) for 40 min either with (closed bars) or without (open bars) ultrasound for 20 min. Fixed cells were stained for paxillin (green) and vinculin (red). Bar, 10 μm. C, 400 cells/condition were scored for focal adhesion formation. Total focal adhesion area (D) or total focal adhesion-integrated density (E) of 25-35 cells/condition was measured using image J software. F, average focal adhesion length of 20 cells/condition. G, focal adhesion area of MEFs stimulated with a range of ultrasound intensities. H, focal adhesion area of MEFs stimulated with ultrasound for a range of durations. Error bars, S.E. Significance values are as follows: ^*, p < 0.05; ^**, p < 0.01; ^***, p < 0.001. Images and analyses are representative of experiments performed on at least three separate occasions.

FIGURE 2.

Ultrasound stimulates focal adhesion formation independently of syndecan-4 expression. Syndecan-4^-/- MEFs were spread on 50K for 2 h before stimulating with syndecan-4 ligand (H/0) for 40 min either with (closed bars) or without (open bars) ultrasound for 20 min. A, representative cells stained for vinculin (green) and actin (red). Bar, 10 μm. B, 400 cells/condition were scored for focal adhesion formation. Total focal adhesion area (C) or total focal adhesion integrated density (D) of 25-35 syndecan-4^-/- cells/condition was measured using image J software. Error bars, S.E.; significance values are as follows: ^*, p < 0.05; ^**, p < 0.01; ^***, p < 0.001. Images and analyses are representative of experiments performed on at least three separate occasions.

FIGURE 3.

Ultrasound does not elicit its effect through integrin activation. A, flow cytometry of K562 cells following ultrasound stimulation using monoclonal antibody 12G10, which recognizes an activation epitope of human β₁ integrin. 1 mm manganese was used as a positive control to drive integrin activation. Green, IgG control; black, 12G10 with calcium/magnesium; blue, 12G10 with calcium/magnesium plus ultrasound; red, 12G10 with manganese. B, binding of biotinylated 50K to isolated, purified α₅β₁ integrin in a solid phase assay either with (closed bars) or without (open bars) a 20-min ultrasound stimulation or the addition of the activating antibody 12G10. C, rate of integrin-mediated spreading of control (open circles) or ultrasound-stimulated (closed circles) MEFs. The area of 150 cells was measured using Image J software. Error bars, S.E. D and E, the areas of MEFs plated onto poly-l-lysine, H/0 or 50K for 120 min were unaffected by stimulation with ultrasound. Bar, 50 μm. F, fibroblasts spread on 50K or the inhibitory β₁ monoclonal antibody, mab13, and stimulated with ultrasound were stained for vinculin (green) and actin (red). Bar, 5 μm. Error bars, S.E. Each result is representative of three independent experiments.

FIGURE 4.

Ultrasound causes syndecan-4-independent Rac1 regulation. Rac1 activity was measured by effector pull-down assays in combination with quantitative Western blotting using fluorophore-conjugated antibodies. Wild-type MEFs (A-C) and syndecan-4-null MEFs (D) were prespread on 50K for 2 h and stimulated with H/0 for 0-60 min (A) and ultrasound for up to 20 min, followed by an appropriate delay (B and D) or by ultrasound for 0-60 min (C). In all cases, lysates were prepared 0-60 min after the start of stimulation. Equivalent loading between experiments was confirmed by blotting crude lysates for total vinculin. Graphs are representative of 5-10 individual experiments. Error bars, S.E. Significance values are as follows: ^*, p < 0.05.

FIGURE 5.

Ultrasound stimulates a modest increase in RhoA activation. RhoA activity was measured using a G-LISA™ RhoA activation assay. Wild-type MEFs were prespread on 50K for 2 h before stimulating with ultrasound (A) or H/0 (B) and preparing lysates over a 90-min time course. Error bars, S.E. Significance values are as follows: ^*, p < 0.05; ^**, p < 0.01. Graphs are representative of 12 and six individual experiments, respectively.

FIGURE 6.

Ultrasound-stimulated Rac1 regulation occurs independently of PKCα. Rac1 activity was measured by effector pull-down assays in combination with quantitative Western blotting using fluorophore-conjugated antibodies. Wild-type MEFs transfected with a nontargeting control siRNA (A), an siRNA specific to PKCα (C and D), or treated with 200 nm BIM for 30 min before and throughout stimulation (E and F) were prespread on 50K for 2 h and stimulated with ultrasound (A, C, and E) or H/0 (D and F) over a 60-min time course, before preparing lysates. Equivalent loading between experiments was confirmed by blotting crude lysates for total vinculin. Error bars, S.E. Significance values are as follows: ^*, p < 0.05. B, expression levels of PKCα, PKCδ, or PKCε following RNAi. Analyses are representative of 5-10 independent experiments.

FIGURE 7.

Ultrasound-induced focal adhesion formation is independent of the syndecan-4-PKCα signaling axis. MEFs transfected with a nontargeting control siRNA (A) or an siRNA specific to PKCα (C) or treated with 200 nm BIM throughout (E) were prespread on 50K for 2 h prior to stimulation with H/0 or ultrasound, followed by fixing and staining cells with vinculin (green) and actin (red). Bar, 5 μm. Focal adhesion area was quantified for 20 cells/condition using Image J software (B, D, F, and G). Error bars, S.E. RNAi and BIM results are representative of two and three independent experiments, respectively.

FIGURE 8.

Ultrasound and H/0 stimuli increase the persistence of cell migration. MEFs were spread in the absence of serum on 50K for 1.5 h before the addition of H/0 or stimulation with ultrasound for 20 min. Migration was filmed for 12 h and tracked using ImageJ software. A, speed of migration. B, persistence of migration calculated by dividing linear displacement of a cell by total distance migrated. C, representative migration tracks. Bar, 200 μm. Error bars, S.E. of 50 different cells. Asterisks indicate a significant difference in persistence (^*) (p < 0.05).