Integrin α2β1 regulates collagen I tethering to modulate hyperresponsiveness in reactive airway disease models
Sean Liu, Uyen Ngo, Xin-Zi Tang, Xin Ren, Wenli Qiu, Xiaozhu Huang, William DeGrado, Christopher D.C. Allen, Hyunil Jo, Dean Sheppard, Aparna B. Sundaram
Sean Liu, Uyen Ngo, Xin-Zi Tang, Xin Ren, Wenli Qiu, Xiaozhu Huang, William DeGrado, Christopher D.C. Allen, Hyunil Jo, Dean Sheppard, Aparna B. Sundaram
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Research Article Muscle biology Pulmonology

Integrin α2β1 regulates collagen I tethering to modulate hyperresponsiveness in reactive airway disease models

Abstract

Severe asthma remains challenging to manage and has limited treatment options. We have previously shown that targeting smooth muscle integrin α5β1 interaction with fibronectin can mitigate the effects of airway hyperresponsiveness by impairing force transmission. In this study, we show that another member of the integrin superfamily, integrin α2β1, is present in airway smooth muscle and capable of regulating force transmission via cellular tethering to the matrix protein collagen I and, to a lesser degree, laminin-111. The addition of an inhibitor of integrin α2β1 impaired IL-13–enhanced contraction in mouse tracheal rings and human bronchial rings and abrogated the exaggerated bronchoconstriction induced by allergen sensitization and challenge. We confirmed that this effect was not due to alterations in classic intracellular myosin light chain phosphorylation regulating muscle shortening. Although IL-13 did not affect surface expression of α2β1, it did increase α2β1-mediated adhesion and the level of expression of an activation-specific epitope on the β1 subunit. We developed a method to simultaneously quantify airway narrowing and muscle shortening using 2-photon microscopy and demonstrated that inhibition of α2β1 mitigated IL-13–enhanced airway narrowing without altering muscle shortening by impairing the tethering of muscle to the surrounding matrix. Our data identified cell matrix tethering as an attractive therapeutic target to mitigate the severity of airway contraction in asthma.

Authors

Sean Liu, Uyen Ngo, Xin-Zi Tang, Xin Ren, Wenli Qiu, Xiaozhu Huang, William DeGrado, Christopher D.C. Allen, Hyunil Jo, Dean Sheppard, Aparna B. Sundaram

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Figure 4

IL-13 activates β1 integrins to promote adhesion without altering intracellular signaling.

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IL-13 activates β1 integrins to promote adhesion without altering intrac...
(A) Adhesion (measured by absorbance of crystal violet at 595 nm) of human airway smooth muscle cells to the indicated concentrations of collagen I after incubation for 12 hours with IL-13 (100 ng/mL) or saline. NS = not significant, **P < 0.01, ***P < 0.001 compared with vehicle; 2-way ANOVA with Tukey’s multiple-comparison test. Experiment performed in triplicate with 3 biological replicates. (B) Human airway smooth muscle cells were incubated for 12 hours with IL-13 (100 ng/mL), 20 minutes with Mn2+ (1 mM), or saline, then suspended and labeled with a primary antibody specific for activated integrin β1 and a secondary antibody conjugated to allophycocyanin (APC). Cells were then analyzed by flow cytometry and gated for live cells. The resultant population was analyzed for APC expression. Human airway smooth muscle cells labeled with secondary antibody alone served as an unstained control (gray short dashed line). Vehicle is shown by a long, gray dashed line. Vehicle/Mn2+ is shown by a long, black dashed line. IL-13 is shown by a solid black line. Representative histogram of APC expression (MFI) versus cell count scaled to mode (percentage of maximum) is shown. Results verified with 2 biological replicates. (C) Representative Western blot of phosphorylation of myosin light chain (p-MLC) and total myosin light chain (MLC) after incubation for 12 hours with IL-13 (100 ng/mL) or saline, and then for 1 hour with c15 (10 μg/mL) or vehicle followed by a single dose of methacholine (10–4 M). GAPDH used as a loading control. Densitometric data are from n = 3–5 independent experiments. *P < 0.05, **P < 0.01 compared with vehicle; 2-way ANOVA with Tukey’s multiple-comparison test. Data are the mean ± SEM for all panels.