TGF-b1 evokes human airway smooth muscle cell shortening and hyperresponsiveness via Smad3

Abstract

Transforming growth factor b1 (TGF-b1), a cytokine whose levels are elevated in the airways of patients with asthma, perpetuates airway inflammation and modulates airway structural cell remodeling. However, the role of TGF-b1 in excessive airway narrowing in asthma, or airway hyperresponsiveness (AHR), remains unclear. In this study, we set out to investigate the direct effects of TGF-b1 on human airway smooth muscle (HASM) cell shortening and hyperresponsiveness. The dynamics of AHR and single-cell excitation-contraction coupling were measured in human precision-cut lung slices and in isolated HASM cells using supravital microscopy and magnetic twisting cytometry, respectively. In human precision-cut lung slices, overnight treatment with TGF-b1 significantly augmented basal and carbachol-induced bronchoconstriction. In isolated HASM cells, TGF-b1 increased basal and methacholine-induced cytoskeletal stiffness in a dose- and time-dependent manner. TGF-b1–induced single-cell contraction was corroborated by concomitant increases in myosin light chain and myosin phosphatase target subunit 1 phosphorylation levels, which were attenuated by small interfering RNA–mediated knockdown of Smad3 and pharmacological inhibition of Rho kinase. Strikingly, these physiological effects of TGF-b1 occurred through a RhoA-independent mechanism, with little effect on HASM cell [Ca 21 ] i levels. Together, our data suggest that TGF-b1 enhances HASM excitation-contraction coupling pathways to induce HASM cell shortening and hyperresponsiveness. These findings reveal a potential link between airway injury–repair responses and bronchial hyperreactivity in asthma, and define TGF-b1 signaling as a potential target to reduce AHR in asthma.