Current therapeutic approaches to avoid or reverse bronchoconstriction rely primarily on β2 adrenoceptor agonists (β‐agonists) that regulate pharmacomechanical coupling/cross bridge cycling in airway smooth muscle (ASM). Targeting actin cytoskeleton polymerization in ASM represents an alternative means to regulate ASM contraction. Herein we report the cooperative effects of targeting these distinct pathways with β‐agonists and inhibitors of the mammalian Abelson tyrosine kinase (Abl1 or c‐Abl). The cooperative effect of β‐agonists (isoproterenol) and c‐Abl inhibitors (GNF‐5, or imatinib) on contractile agonist (methacholine, or histamine) ‐induced ASM contraction was assessed in cultured human ASM cells (using Fourier Transfer Traction Microscopy), in murine precision cut lung slices, and in vivo (flexiVent in mice). Regulation of intracellular signaling that regulates contraction (pMLC20, pMYPT1, pHSP20), and actin polymerization state (F:G actin ratio) were assessed in cultured primary human ASM cells. In each (cell, tissue, in vivo) model, c‐Abl inhibitors and β‐agonist exhibited additive effects in either preventing or reversing ASM contraction. Treatment of contracted ASM cells with c‐Abl inhibitors and β‐agonist cooperatively increased actin disassembly as evidenced by a significant reduction in the F:G actin ratio. Mechanistic studies indicated that the inhibition of pharmacomechanical coupling by β‐agonists is near optimal and is not increased by c‐Abl inhibitors, and the cooperative effect on ASM relaxation resides in further relaxation of ASM tension development caused by actin cytoskeleton depolymerization, which is regulated by both β‐agonists and c‐Abl inhibitors. Thus, targeting actin cytoskeleton polymerization represents an untapped therapeutic reserve for managing airway resistance.
Airway smooth muscle cell migration plays an essential role in airway development, repair, and remodeling. Smooth muscle myosin II has been traditionally thought to localize in the cytoplasm solely and regulates cell migration by affecting stress fiber formation and focal adhesion assembly. In this study, we unexpectedly found that 20-kDa myosin light chain (MLC20) and myosin-11 (MYH11), important components of smooth muscle myosin, were present at the edge of lamellipodia. The knockdown of MLC20 or MYH11 attenuated the recruitment of c-Abl, cortactinProfilin-1 (Pfn-1), and Abi1 to the cell edge. Moreover, myosin light chain kinase (MLCK) colocalized with integrin β1 at the tip of protrusion. The inhibition of MLCK attenuated the recruitment of c-Abl, cortactin, Pfn-1, and Abi1 to the cell edge. Furthermore, MLCK localization at the leading edge was reduced by integrin β1 knockdown. Taken together, our results demonstrate that smooth muscle myosin localizes at the leading edge and orchestrates the recruitment of actin-regulatory proteins to the tip of lamellipodia. Mechanistically, integrin β1 recruits MLCK to the leading edge, which catalyzes MLC20 phosphorylation. Activated myosin regulates the recruitment of actin-regulatory proteins to the leading edge, and promotes lamellipodial formation and migration.
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