Activation of the Arp2/3 complex by N-WASp is required for actin polymerization and contraction in smooth muscle

2005 ◽  
Vol 288 (5) ◽  
pp. C1145-C1160 ◽  
Author(s):  
Wenwu Zhang ◽  
Yidi Wu ◽  
Liping Du ◽  
Dale D. Tang ◽  
Susan J. Gunst
Keyword(s):  
Smooth Muscle ◽  
Actin Polymerization ◽  
Fluorescent Protein ◽  
Tracheal Smooth Muscle ◽  
Wild Type ◽  
Tension Development ◽  
Muscle Tissues ◽  
Muscarinic Stimulation ◽  
Mlc Phosphorylation

Contractile stimulation has been shown to initiate actin polymerization in smooth muscle tissues, and this actin polymerization is required for active tension development. We evaluated whether neuronal Wiskott-Aldrich syndrome protein (N-WASp)-mediated activation of the actin-related proteins 2 and 3 (Arp2/3) complex regulates actin polymerization and tension development initiated by muscarinic stimulation in canine tracheal smooth muscle tissues. In vitro, the COOH-terminal CA domain of N-WASp acts as an inhibitor of N-WASp-mediated actin polymerization; whereas the COOH-terminal VCA domain of N-WASp is constitutively active and is sufficient by itself to catalyze actin polymerization. Plasmids encoding EGFP-tagged wild-type N-WASp, the N-WASp VCA and CA domains, or enhanced green fluorescent protein (EGFP) were introduced into tracheal smooth muscle strips by reversible permeabilization, and the tissues were incubated for 2 days to allow for expression of the proteins. Expression of the CA domain inhibited actin polymerization and tension development in response to ACh, whereas expression of the wild-type N-WASp, the VCA domain, or EGFP did not. The increase in myosin light-chain (MLC) phosphorylation in response to contractile stimulation was not affected by expression of either the CA or VCA domain of N-WASp. Stimulation of the tissues with ACh increased the association of the Arp2/3 complex with N-WASp, and this association was inhibited by expression of the CA domain. The results demonstrate that 1) N-WASp-mediated activation of the Arp2/3 complex is necessary for actin polymerization and tension development in response to muscarinic stimulation in tracheal smooth muscle and 2) these effects are independent of the regulation of MLC phosphorylation.

scholarly journals The Small GTPase Cdc42 Regulates Actin Polymerization and Tension Development during Contractile Stimulation of Smooth Muscle

2004 ◽  
Vol 279 (50) ◽  
pp. 51722-51728 ◽  
Author(s):  
Dale D. Tang ◽  
Susan J. Gunst
Keyword(s):  
Smooth Muscle ◽  
Actin Polymerization ◽  
Myosin Light Chain ◽  
Dominant Negative ◽  
Related Protein ◽  
Wild Type ◽  
Tension Development ◽  
Muscle Tissues ◽  
Syndrome Protein ◽  
Stimulation Of

Contractile stimulation induces actin polymerization in smooth muscle tissues and cells, and the inhibition of actin polymerization depresses smooth muscle force development. In the present study, the role of Cdc42 in the regulation of actin polymerization and tension development in smooth muscle was evaluated. Acetylcholine stimulation of tracheal smooth muscle tissues increased the activation of Cdc42. Plasmids encoding wild type Cdc42 or a dominant negative Cdc42 mutant, Asn-17 Cdc42, were introduced into tracheal smooth muscle strips by reversible permeabilization, and tissues were incubated for 2 days to allow for protein expression. Expression of recombinant proteins was confirmed by immunoblot analysis. The expression of the dominant negative Cdc42 mutant inhibited contractile force and the increase in actin polymerization in response to acetylcholine stimulation but did not inhibit the increase in myosin light chain phosphorylation. The expression of wild type Cdc42 had no significant effect on force, actin polymerization, or myosin light chain phosphorylation. Contractile stimulation increased the association of neuronal Wiskott-Aldrich syndrome protein with Cdc42 and the Arp2/3 (actin-related protein) complex in smooth muscle tissues expressing wild type Cdc42. The agonist-induced increase in these protein interactions was inhibited in tissues expressing the inactive Cdc42 mutant. We conclude that Cdc42 activation regulates active tension development and actin polymerization during contractile stimulation. Cdc42 may regulate the activation of neuronal Wiskott-Aldrich syndrome protein and the actin related protein complex, which in turn regulate actin filament polymerization initiated by the contractile stimulation of smooth muscle.

Role of Rho in Ca2+-insensitive contraction and paxillin tyrosine phosphorylation in smooth muscle

2000 ◽  
Vol 279 (2) ◽  
pp. C308-C318 ◽  
Author(s):  
Dolly Mehta ◽  
Dale D. Tang ◽  
Ming-Fang Wu ◽  
Simon Atkinson ◽  
Susan J. Gunst
Keyword(s):  
Smooth Muscle ◽  
Tyrosine Phosphorylation ◽  
Actin Polymerization ◽  
Kinase Inhibitor ◽  
Rho Kinase ◽  
Cytochalasin D ◽  
Cytoskeletal Proteins ◽  
Tracheal Smooth Muscle ◽  
Contractile Activation ◽  
Mlc Phosphorylation

We investigated whether Rho activation is required for Ca2+-insensitive paxillin phosphorylation, myosin light chain (MLC) phosphorylation, and contraction in tracheal muscle. Tyrosine-phosphorylated proteins have been implicated in the Ca2+-insensitive contractile activation of smooth muscle tissues. The contractile activation of tracheal smooth muscle increases tyrosine phosphorylation of the cytoskeletal proteins paxillin and focal adhesion kinase. Paxillin is implicated in integrin-mediated signal transduction pathways that regulate cytoskeletal organization and cell motility. In fibroblasts and other nonmuscle cells, paxillin tyrosine phosphorylation depends on the activation of Rho and is inhibited by cytochalasin, an inhibitor of actin polymerization. In permeabilized muscle strips, we found that ACh induced Ca2+-insensitive contraction, MLC phosphorylation, and paxillin tyrosine phosphorylation. Ca2+-insensitive contraction and MLC phosphorylation induced by ACh were inhibited by C3 transferase, an inhibitor of Rho activation; however, C3 transferase did not inhibit paxillin tyrosine phosphorylation. Ca2+-insensitive paxillin tyrosine phosphorylation was also not inhibited by the Rho kinase inhibitor Y-27632, by cytochalasin D, or by the inhibition of MLC phosphorylation. We conclude that, in tracheal smooth muscle, Rho mediates Ca2+-insensitive contraction and MLC phosphorylation but that Rho is not required for Ca2+-insensitive paxillin tyrosine phosphorylation. Paxillin phosphorylation also does not require actomyosin activation, nor is it inhibited by the actin filament capping agent cytochalasin D.

scholarly journals Mice that overexpress Cu/Zn superoxide dismutase are resistant to allergen-induced changes in airway control

2000 ◽  
Vol 279 (2) ◽  
pp. L350-L359 ◽  
Author(s):  
Gary L. Larsen ◽  
Carl W. White ◽  
Katsuyuki Takeda ◽  
Joan E. Loader ◽  
Dee Dee H. Nguyen ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Smooth Muscle ◽  
Transgenic Mice ◽  
Neural Control ◽  
Electrical Field Stimulation ◽  
Tracheal Smooth Muscle ◽  
Wild Type ◽  
Airway Control ◽  
Induced Changes

Within the respiratory epithelium of asthmatic patients, copper/zinc-containing superoxide dismutase (Cu/Zn SOD) is decreased. To address the hypothesis that lung Cu/Zn SOD protects against allergen-induced injury, wild-type and transgenic mice that overexpress human Cu/Zn SOD were either passively sensitized to ovalbumin (OVA) or actively sensitized by repeated airway exposure to OVA. Controls included nonsensitized wild-type and transgenic mice given intravenous saline or airway exposure to saline. After aerosol challenge to saline or OVA, segments of tracheal smooth muscle were obtained for in vitro analysis of neural control. In response to electrical field stimulation, wild-type sensitized mice challenged with OVA had significant increases in cholinergic reactivity. Conversely, sensitized transgenic mice challenged with OVA were resistant to changes in neural control. Stimulation of tracheal smooth muscle to elicit acetylcholine release showed that passively sensitized wild-type but not transgenic mice released more acetylcholine after OVA challenge. Function of the M2 muscarinic autoreceptor was preserved in transgenic mice. These results demonstrate that murine airways with elevated Cu/Zn SOD were resistant to allergen-induced changes in neural control.

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

2015 ◽  
Vol 93 (2) ◽  
pp. 129-136 ◽  
Author(s):  
Wenwu Zhang ◽  
Youliang Huang ◽  
Yidi Wu ◽  
Susan J. Gunst
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Actin Polymerization ◽  
Smooth Muscle Contraction ◽  
Tension Development ◽  
Rhoa Gtpase ◽  
Tension Generation ◽  
P21 Activated Kinase ◽  
Signaling Modules ◽  
Mlc Phosphorylation

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.

Tension development during contractile stimulation of smooth muscle requires recruitment of paxillin and vinculin to the membrane

2004 ◽  
Vol 286 (2) ◽  
pp. C433-C447 ◽  
Author(s):  
Anabelle Opazo Saez ◽  
Wenwu Zhang ◽  
Yidi Wu ◽  
Christopher E. Turner ◽  
Dale D. Tang ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Focal Adhesions ◽  
Smooth Muscle Contraction ◽  
Cytoskeletal Proteins ◽  
Tracheal Smooth Muscle ◽  
Muscle Membrane ◽  
Tension Development ◽  
Muscle Tissues ◽  
Primary Determinant ◽  
Smooth Muscle Membrane

Cytoskeletal reorganization of the smooth muscle cell in response to contractile stimulation may be an important fundamental process in regulation of tension development. We used confocal microscopy to analyze the effects of cholinergic stimulation on localization of the cytoskeletal proteins vinculin, paxillin, talin and focal adhesion kinase (FAK) in freshly dissociated tracheal smooth muscle cells. All four proteins were localized at the membrane and throughout the cytoplasm of unstimulated cells, but their concentration at the membrane was greater in acetylcholine (ACh)-stimulated cells. Antisense oligonucleotides were introduced into tracheal smooth muscle tissues to deplete paxillin protein, which also inhibited contraction in response to ACh. In cells dissociated from paxillin-depleted muscle tissues, redistribution of vinculin to the membrane in response to ACh was prevented, but redistribution of FAK and talin was not inhibited. Muscle tissues were transfected with plasmids encoding a paxillin mutant containing a deletion of the LIM3 domain (paxillin LIM3 dl 444–494), the primary determinant for targeting paxillin to focal adhesions. Expression of paxillin LIM3 dl in muscle tissues also inhibited contractile force and prevented cellular redistribution of paxillin and vinculin to the membrane in response to ACh, but paxillin LIM3 dl did not inhibit increases in intracellular Ca2+ or myosin light chain phosphorylation. Our results demonstrate that recruitment of paxillin and vinculin to smooth muscle membrane is necessary for tension development and that recruitment of vinculin to the membrane is regulated by paxillin. Vinculin and paxillin may participate in regulating the formation of linkages between the cytoskeleton and integrin proteins that mediate tension transmission between the contractile apparatus and the extracellular matrix during smooth muscle contraction.

Regulation of isotonic shortening velocity by second messengers in tracheal smooth muscle

1994 ◽  
Vol 266 (3) ◽  
pp. C684-C691 ◽  
Author(s):  
S. J. Gunst ◽  
M. H. al-Hassani ◽  
L. P. Adam
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Second Messengers ◽  
Time Dependent ◽  
Tracheal Smooth Muscle ◽  
Shortening Velocity ◽  
Active Stress ◽  
Muscle Tissues ◽  
Mlc Phosphorylation ◽  
Isotonic Shortening

Evidence suggests that the mechanical behavior of smooth muscle tissues is regulated by Ca(2+)-dependent changes in the phosphorylation of the 20,000-Da light chain of myosin (MLC). However, alternative mechanisms activated by specific kinases may be involved in regulating the shortening velocity in some smooth muscle tissues. To determine how the activation of protein kinases A or C affects the regulation of the shortening velocity in canine tracheal smooth muscle, we evaluated the effects of forskolin (10(-5) M) and phorbol 12,13-dibutyrate (PDBu, 3 x 10(-6) M) on active stress, intracellular Ca2+ ([Ca2+]i), MLC phosphorylation, and isotonic shortening velocity during contractions elicited by 60 mM KCl. Forskolin depressed and PDBu increased active stress, [Ca2+]i, MLC phosphorylation, and shortening velocity; thus the effects of these agents on the shortening velocity may result from changes in Ca(2+)-dependent MLC phosphorylation. In contrast, the decline in velocity that occurred with time during tonic contractions elicited by K+ depolarization was not associated with significant changes in MLC phosphorylation; thus the time-dependent changes in shortening velocity may be regulated by a mechanism other than MLC phosphorylation.

scholarly journals FFAR1 activation attenuates histamine-induced myosin light chain phosphorylation and cortical tension development in human airway smooth muscle cells

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Shengjie Xu ◽  
Anthony Schwab ◽  
Nikhil Karmacharya ◽  
Gaoyuan Cao ◽  
Joanna Woo ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Airway Hyperreactivity ◽  
Human Airway Smooth Muscle ◽  
Tension Development ◽  
Cortical Tension ◽  
Human Airway ◽  
Mlc Phosphorylation

Abstract Background Activation of free fatty acid receptors (FFAR1 and FFAR4) which are G protein-coupled receptors (GPCRs) with established (patho)physiological roles in a variety of obesity-related disorders, induce human airway smooth muscle (HASM) cell proliferation and shortening. We reported amplified agonist-induced cell shortening in HASM cells obtained from obese lung donors. We hypothesized that FFAR1 modulate excitation–contraction (EC) coupling in HASM cells and play a role in obesity-associated airway hyperresponsiveness. Methods In HASM cells pre-treated (30 min) with FFAR1 agonists TAK875 and GW9508, we measured histamine-induced Ca2+ mobilization, myosin light chain (MLC) phosphorylation, and cortical tension development with magnetic twisting cytometry (MTC). Phosphorylation of MLC phosphatase and Akt also were determined in the presence of the FFAR1 agonists or vehicle. In addition, the effects of TAK875 on MLC phosphorylation were measured in HASM cells desensitized to β2AR agonists by overnight salmeterol treatment. The inhibitory effect of TAK875 on MLC phosphorylation was compared between HASM cells from age and sex-matched non-obese and obese human lung donors. The mean measurements were compared using One-Way ANOVA with Dunnett’s test for multiple group comparisons or Student’s t-test two-group comparison. For cortical tension measurements by magnetic twisted cytometry, mixed effect model using SAS V.9.2 was applied. Means were considered significant when p ≤ 0.05. Results Unexpectedly, we found that TAK875, a synthetic FFAR1 agonist, attenuated histamine-induced MLC phosphorylation and cortical tension development in HASM cells. These physiological outcomes were unassociated with changes in histamine-evoked Ca2+ flux, protein kinase B (AKT) activation, or MLC phosphatase inhibition. Of note, TAK875-mediated inhibition of MLC phosphorylation was maintained in β2AR-desensitized HASM cells and across obese and non-obese donor-derived HASM cells. Conclusions Taken together, our findings identified the FFAR1 agonist TAK875 as a novel bronchoprotective agent that warrants further investigation to treat difficult-to-control asthma and/or airway hyperreactivity in obesity.

Sign in / Sign up

Export Citation Format

Share Document