Non-muscle (NM) myosin heavy chain phosphorylation regulates the formation of NM myosin filaments, adhesome assembly and smooth muscle contraction

KEY POINTS: Non-muscle (NM) and smooth muscle (SM) myosin II are both expressed in smooth muscle tissues, however the role of NM myosin in SM contraction is unknown. Contractile stimulation of tracheal smooth muscle tissues stimulates phosphorylation of the NM myosin heavy chain on Ser1943 and causes NM myosin filament assembly at the SM cell cortex. Expression of a non-phosphorylatable NM myosin mutant, NM myosin S1943A, in SM tissues inhibits ACh-induced NM myosin filament assembly and SM contraction, and also inhibits the assembly of membrane adhesome complexes during contractile stimulation. NM myosin regulatory light chain (RLC) phosphorylation but not SM myosin RLC phosphorylation is regulated by RhoA GTPase during ACh stimulation, and NM RLC phosphorylation is required for NM myosin filament assembly and SM contraction. NM myosin II plays a critical role in airway SM contraction that is independent and distinct from the function of SM myosin. ABSTRACT: The molecular function of non-muscle (NM) isoforms of myosin II in smooth muscle (SM) tissues and their possible role in contraction are largely unknown. We evaluated the function of NM myosin during contractile stimulation of canine tracheal SM tissues. Stimulation with ACh caused NM myosin filament assembly, as assessed by a Triton solubility assay and a proximity ligation assay aiming to measure interactions between NM myosin monomers. ACh stimulated the phosphorylation of NM myosin heavy chain on Ser1943 in tracheal SM tissues, which can regulate NM myosin IIA filament assembly in vitro. Expression of the non-phosphorylatable mutant NM myosin S1943A in SM tissues inhibited ACh-induced endogenous NM myosin Ser1943 phosphorylation, NM myosin filament formation, the assembly of membrane adhesome complexes and tension development. The NM myosin cross-bridge cycling inhibitor blebbistatin suppressed adhesome complex assembly and SM contraction without inhibiting NM myosin Ser1943 phosphorylation or NM myosin filament assembly. RhoA inactivation selectively inhibited phosphorylation of the NM myosin regulatory light chain (RLC), NM myosin filament assembly and contraction, although it did not inhibit SM RLC phosphorylation. We conclude that the assembly and activation of NM myosin II is regulated during contractile stimulation of airway SM tissues by RhoA-mediated NM myosin RLC phosphorylation and by NM myosin heavy chain Ser1943 phosphorylation. NM myosin II actomyosin cross-bridge cycling regulates the assembly of membrane adhesome complexes that mediate the cytoskeletal processes required for tension generation. NM myosin II plays a critical role in airway SM contraction that is independent and distinct from the function of SM myosin

Molecular Mechanisms for the Mechanical Modulation of Airway Responsiveness

This article is made available for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.The smooth muscle of the airways is exposed to continuously changing mechanical forces during normal breathing. The mechanical oscillations that occur during breathing have profound effects on airway tone and airway responsiveness both in experimental animals and humans in vivo and in isolated airway tissues in vitro. Experimental evidence suggests that alterations in the contractile and mechanical properties of airway smooth muscle tissues caused by mechanical perturbations result from adaptive changes in the organization of the cytoskeletal architecture of the smooth muscle cell. The cytoskeleton is a dynamic structure that undergoes rapid reorganization in response to external mechanical and pharmacologic stimuli. Contractile stimulation initiates the assembly of cytoskeletal/extracellular matrix adhesion complex proteins into large macromolecular signaling complexes (adhesomes) that undergo activation to mediate the polymerization and reorganization of a submembranous network of actin filaments at the cortex of the cell. Cortical actin polymerization is catalyzed by Neuronal-Wiskott–Aldrich syndrome protein (N-WASP) and the Arp2/3 complex, which are activated by pathways regulated by paxillin and the small GTPase, cdc42. These processes create a strong and rigid cytoskeletal framework that may serve to strengthen the membrane for the transmission of force generated by the contractile apparatus to the extracellular matrix, and to enable the adaptation of smooth muscle cells to mechanical stresses. This model for the regulation of airway smooth muscle function can provide novel perspectives to explain the normal physiologic behavior of the airways and pathophysiologic properties of the airways in asthma

Vasodilator Stimulated Phosphoprotein (VASP) Regulates Actin Polymerization and Contraction in Airway Smooth Muscle by a Vinculin-dependent Mechanism

Vasodilator-stimulated phosphoprotein (VASP) can catalyze actin polymerization by elongating actin filaments. The elongation mechanism involves VASP oligomerization and its binding to profilin, a G-actin chaperone. Actin polymerization is required for tension generation during the contraction of airway smooth muscle (ASM); however, the role of VASP in regulating actin dynamics in ASM is not known. We stimulated ASM cells and tissues with the contractile agonist acetylcholine (ACh) or the adenylyl cyclase activator, forskolin (FSK), a dilatory agent. ACh and FSK stimulated VASP Ser157 phosphorylation by different kinases. Inhibition of VASP Ser157 phosphorylation by expression of the mutant VASP S157A in ASM tissues suppressed VASP phosphorylation and membrane localization in response to ACh, and also inhibited contraction and actin polymerization. ACh but not FSK triggered the formation of VASP-VASP complexes as well as VASP-vinculin and VASP-profilin complexes at membrane sites. VASP-VASP complex formation and the interaction of VASP with vinculin and profilin were inhibited by expression of the inactive vinculin mutant, vinculin Y1065F, but VASP phosphorylation and membrane localization were unaffected. We conclude that VASP phosphorylation at Ser157 mediates its localization at the membrane, but that VASP Ser157 phosphorylation and membrane localization are not sufficient to activate its actin catalytic activity. The interaction of VASP with activated vinculin at membrane adhesion sites is a necessary prerequisite for VASP-mediated molecular processes necessary for actin polymerization. Our results show that VASP is a critical regulator of actin dynamics and tension generation during the contractile activation of ASM

Rho Kinase (ROCK) collaborates with Pak to Regulate Actin Polymerization and Contraction in Airway Smooth Muscle

Rho kinase (ROCK), a RhoA GTPase effector, can regulate the contraction of airway and other smooth muscle tissues. In some tissues, ROCK can inhibit myosin regulatory light chain (RLC) phosphatase, which increases the phosphorylation of myosin RLC and promotes smooth muscle contraction. ROCK can also regulate cell motility and migration by affecting F‐actin dynamics. Actin polymerization is stimulated by contractile agonists in airway smooth muscle tissues and is required for contractile tension development in addition to myosin RLC phosphorylation. We investigated the mechanisms by which ROCK regulates the contractility of tracheal smooth muscle tissues by expressing a kinase inactive mutant of ROCK, ROCK‐K121G, in the tissues or by treating them with the ROCK inhibitor, H‐1152P. Our results show no role for ROCK in the regulation of non‐muscle or smooth muscle myosin RLC phosphorylation during contractile stimulation in this tissue. We find that ROCK regulates airway smooth muscle contraction by mediating activation of the serine‐threonine kinase, Pak, to promote actin polymerization. Pak catalyzes paxillin phosphorylation on Ser273 and coupling of the GIT1‐βPIX‐Pak signaling module to paxillin, which activates the GEF activity βPIX towards cdc42. Cdc42 is required for the activation of Neuronal Wiskott‐Aldrich Syndrome protein (N‐WASp), which transmits signals from cdc42 to the Arp2/3 complex for the nucleation of actin filaments. Our results demonstrate a novel molecular function for ROCK in the regulation of Pak and cdc42 activation that is critical for the processes of actin polymerization and contractility in airway smooth muscle

A novel role for RhoA GTPase in the regulation of airway smooth muscle contraction

Recent studies have demonstrated a novel molecular mechanism for the regulation of airway smooth muscle (ASM) contraction by RhoA GTPase. In ASM tissues, both myosin light chain (MLC) phosphorylation and actin polymerization are required for active tension generation. RhoA inactivation dramatically suppresses agonist-induced tension development and completely inhibits agonist-induced actin polymerization, but only slightly reduces MLC phosphorylation. The inhibition of MLC phosphatase does not reverse the effects of RhoA inactivation on contraction or actin polymerization. Thus, RhoA regulates ASM contraction through its effects on actin polymerization rather than MLC phosphorylation. Contractile stimulation of ASM induces the recruitment and assembly of paxillin, vinculin, and focal adhesion kinase (FAK) into membrane adhesion complexes (adhesomes) that regulate actin polymerization by catalyzing the activation of cdc42 GTPase by the G-protein-coupled receptor kinase-interacting target (GIT) - p21-activated kinase (PAK) - PAK-interacting exchange factor (PIX) complex. Cdc42 is a necessary and specific activator of the actin filament nucleation activator, N-WASp. The recruitment and activation of paxillin, vinculin, and FAK is prevented by RhoA inactivation, thus preventing cdc42 and N-WASp activation. We conclude that RhoA regulates ASM contraction by catalyzing the assembly and activation of membrane adhesome signaling modules that regulate actin polymerization, and that the RhoA-mediated assembly of adhesome complexes is a fundamental step in the signal transduction process in response to a contractile agonist

S100A4 is activated by RhoA and catalyses the polymerization of non-muscle myosin, adhesion complex assembly and contraction in airway smooth muscle

S100A4 binds to the heavy chain of non-muscle (NM) myosin II and can regulate the motility of crawling cells. S100A4 is widely expressed in many tissues including smooth muscle (SM), although its role in the regulation of their physiologic function is not known. We hypothesized that S100A4 contributes to the regulation of contraction in airway SM by regulating a pool of NM myosin II at the cell cortex. NM myosin II undergoes polymerization in airway SM and regulates contraction by catalysing the assembly of integrin-associated adhesome complexes that activate pathways that catalyse actin polymerization. ACh stimulated the interaction of S100A4 with NM myosin II in airway SM at the cell cortex and catalysed NM myosin filament assembly. RhoA GTPase regulated the activation of S100A4 via rhotekin, which facilitated the formation of a complex between RhoA, S100A4 and NM myosin II. The depletion of S100A4, RhoA or rhotekin from airway SM tissues using short hairpin RNA or small interfering RNA prevented NM myosin II polymerization as well as the recruitment of vinculin and paxillin to adhesome signalling complexes in response to ACh, and inhibited actin polymerization and tension development. S100A4 depletion did not affect ACh-stimulated SMmyosin regulatory light chain phosphorylation. The results show that S100A4 plays a critical role in tension development in airway SM tissue by catalysing NM myosin filament assembly, and that the interaction of S100A4 with NM myosin in response to contractile stimulation is activated by RhoA GTPase. These results may be broadly relevant to the physiologic function of S100A4 in other cell and tissue types

Use of continuous positive airway pressure reduces airway reactivity in adults with asthma

Asthma is characterised by airway hyperreactivity, which is primarily treated with β-adrenergic bronchodilators and anti-inflammatory agents. However, mechanical strain during breathing is an important modulator of airway responsiveness and we have previously demonstrated in animal models that continuous positive airway pressure (CPAP) resulted in lower in vivo airway reactivity. We now evaluated whether using nocturnal CPAP decreased airway reactivity in clinically-stable adults with asthma. Adults with stable asthma and normal spirometry used nocturnal CPAP (8-10 cmH(2)O) or sham treatment (0-2 cmH(2)O) for 7 days. Spirometry and bronchial challenges were obtained before and after treatment. The primary outcome was the provocative concentration of methacholine causing a 20% fall in forced expiratory volume in 1 s (PC(20)). The CPAP group (n=16) had a significant decrease in airway reactivity (change in (Δ)logPC(20) 0.406, p<0.0017) while the sham group (n=9) had no significant change in airway reactivity (ΔlogPC(20) 0.003, p=0.9850). There was a significant difference in the change in airway reactivity for the CPAP versus the sham group (ΔlogPC(20) 0.41, p<0.043). Our findings indicate that chronic mechanical strain of the lungs produced using nocturnal CPAP for 7 days reduced airway reactivity in clinically stable asthmatics. Future studies of longer duration are required to determine whether CPAP can also decrease asthma symptoms and/or medication usage

Effect of CPAP on airway reactivity and airway inflammation in children with moderate-severe asthma

BACKGROUND AND OBJECTIVE: Asthma is characterized by airway hyperreactivity and airway inflammation. We previously demonstrated that adults with mild well-controlled asthma exhibited a marked decrease in airway reactivity (PC20 increased >2-fold) after using nocturnal continuous positive airway pressure (CPAP) for 1 week. If CPAP produces a similar suppression of airway reactivity in children with moderate-severe asthma, who require chronic use of corticosteroids, then this non-pharmacological therapy might provide a beneficial alternative or supplemental therapy in these subjects. METHODS: Children aged 8-17 years with moderate-severe asthma were treated with 4 weeks of nocturnal CPAP (8-10 cm H2 O) or sham CPAP (<2 cm H2 O). Adherence was monitored with a modem installed in the equipment or by memory cards. Airway reactivity, assessed by methacholine bronchial challenge, was measured prior to and following treatment. RESULTS: The percentage of subjects adherent to treatment was similar in both groups (19/27 CPAP vs 19/28 sham, ~70%). There was a tendency for PC20 to increase with treatment in both groups (3.0-5.3 mg/mL CPAP vs 3.2 to 4.3 mg/mL sham, P = 0.083); however, the change did not differ significantly between groups (P = 0.569). CONCLUSION: We found that the 4-week treatment with nocturnal CPAP did not produce a twofold suppression of airway reactivity in children with moderate-severe asthma

Regulation of 130kDa smooth muscle myosin light chain kinase expression by an intronic CArG element

The mylk1 gene encodes a 220-kDa nonmuscle myosin light chain kinase (MLCK), a 130-kDa smooth muscle MLCK (smMLCK), as well as the non-catalytic product telokin. Together, these proteins play critical roles in regulating smooth muscle contractility. Changes in their expression are associated with many pathological conditions; thus, it is important to understand the mechanisms regulating expression of mylk1 gene transcripts. Previously, we reported a highly conserved CArG box, which binds serum response factor, in intron 15 of mylk1. Because this CArG element is near the promoter that drives transcription of the 130-kDa smMLCK, we examined its role in regulating expression of this transcript. Results show that deletion of the intronic CArG region from a β-galactosidase reporter gene abolished transgene expression in mice in vivo. Deletion of the CArG region from the endogenous mylk1 gene, specifically in smooth muscle cells, decreased expression of the 130-kDa smMLCK by 40% without affecting expression of the 220-kDa MLCK or telokin. This reduction in 130-kDa smMLCK expression resulted in decreased phosphorylation of myosin light chains, attenuated smooth muscle contractility, and a 24% decrease in small intestine length that was associated with a significant reduction of Ki67-positive smooth muscle cells. Overall, these data show that the CArG element in intron 15 of the mylk1 gene is necessary for maximal expression of the 130-kDa smMLCK and that the 130-kDa smMLCK isoform is specifically required to regulate smooth muscle contractility and small intestine smooth muscle cell proliferation