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ABSTRACT: Raf-1 is a serine/threonine protein kinase that has an essential role in cell proliferation. The mechanisms that regulate Raf-1 in airway smooth muscle are not well understood. In this study, treatment with platelet-derived growth factor (PDGF) induced spatial redistribution of Raf-1 from the cytoplasm to the periphery of human airway smooth muscle cells. Moreover, a pool of Raf-1 was found in F-actin of human airway smooth muscle cells. Activation with PDGF led to an increase in the association of Raf-1 with cytoskeletal actin. Treatment of cells with the actin polymerization inhibitor latrunculin A, but not the microtubule depolymerizer nocodazole, inhibited the interaction of Raf-1 with actin in response to PDGF activation. Because Abl is known to specifically regulate actin dynamics in smooth muscle, the role of Abl in modulating the coupling of Raf-1 with actin was also evaluated. Abl knockdown by RNA interference attenuated the association of Raf-1 with actin, which is recovered by Abl rescue. Treatment with latrunculin A, but not nocodazole, inhibited the spatial redistribution of Raf-1 during PDGF activation. However, treatment with both latrunculin A and nocodazole attenuated smooth muscle cell proliferation. Finally, Abl knockdown attenuated the redistribution of Raf-1 and cell proliferation, which were restored by Abl re-expression. The results suggest a novel mechanism that the interaction of Raf-1 with cytoskeletal actin is critical for Raf-1 redistribution and airway smooth muscle cell proliferation during activation with the growth factor.
American Journal of Respiratory Cell and Molecular Biology 10/2012; · 5.13 Impact Factor
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ABSTRACT: Abl is a nonreceptor tyrosine kinase that has a role in regulating migration and adhesion of nonmuscle cells as well as smooth muscle contraction. The role of Abl in smooth muscle cell proliferation has not been investigated. In this study, treatment with endothelin-1 (ET-1) and platelet-derived growth factor (PDGF) increased Abl phosphorylation at Tyr(412) (an indication of Abl activation) in vascular smooth muscle cells. To assess the role of Abl in smooth muscle cell proliferation, we generated stable Abl knockdown cells by using lentivirus-mediated RNA interference. ET-1- and PDGF-induced cell proliferation was attenuated in Abl knockdown cells compared with cells expressing control shRNA and uninfected cells. Abl silencing also arrested cell cycle progression from G(0)/G(1) to S phase. Furthermore, activation of smooth muscle cells with ET-1 and PDGF induced phosphorylation of ERK1/2 and Akt. Abl knockdown attenuated ERK1/2 phosphorylation in smooth muscle cells stimulated with ET-1 and PDGF. However, Akt phosphorylation upon stimulation with ET-1 and PDGF was not reduced. Because Abl is known to regulate actin polymerization in smooth muscle, we also evaluated the effects of inhibition of actin polymerization on phosphorylation of ERK1/2. Pretreatment with the actin polymerization inhibitor latrunculin-A also blocked ERK1/2 phosphorylation during activation with ET-1 and PDGF. The results suggest that Abl may regulate smooth muscle cell proliferation by modulating actin dynamics and ERK1/2 phosphorylation during mitogenic activation.
AJP Cell Physiology 02/2012; 302(7):C1026-34. · 3.54 Impact Factor
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ABSTRACT: Abl is a nonreceptor tyrosine kinase that is required for smooth muscle contraction. However, the mechanism by which Abl regulates smooth muscle contraction is not completely understood. In the present study, Abl underwent phosphorylation at Tyr412 (an index of Abl activation) in smooth muscle in response to contractile activation. Treatment with a cell-permeable decoy peptide, but not the control peptide, attenuated Abl phosphorylation during contractile stimulation. Treatment with the decoy peptide did not affect the association of Abl with the cytoskeletal protein vinculin and the spatial location of vinculin in smooth muscle. Inhibition of Abl phosphorylation by the decoy peptide attenuated the agonist-induced phosphorylation of Crk-associated substrate (CAS), an adapter protein participating in the signaling processes that regulate force development in smooth muscle. Additionally, previous studies have shown that contractile stimulation triggers the dissociation of CAS from the vimentin network, which is important for cytoskeletal signaling and contraction in smooth muscle. In this report, the decrease in the amount of CAS in cytoskeletal vimentin in response to contractile activation was reversed by the Abl inhibition with the decoy peptide. Moreover, force development and the enhancement of F-actin-to-G-actin ratios (an indication of actin polymerization) upon contractile activation were also attenuated by the Abl inhibition. However, myosin phosphorylation induced by contractile activation was not affected by the inhibition of Abl. These results suggest that Abl regulates the dissociation of CAS from the vimentin network, actin polymerization, and contraction by modulating CAS phosphorylation in smooth muscle.
AJP Cell Physiology 09/2010; 299(3):C630-7. · 3.54 Impact Factor
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ABSTRACT: Cdc42GAP (GTPase activating protein) has been shown to regulate smooth muscle contraction as well as cell motility, adhesion, proliferation, and apoptosis. We have recently shown that Cdc42GAP activity is suppressed in smooth muscle cells during contractile activation, which is reversed by inhibitors of reactive oxygen species (ROS). Because p47(phox), a regulatory subunit of NAD(P)H oxidase, has been implicated in smooth muscle signaling, we determined whether this subunit modulates Cdc42GAP activity in response to contractile stimulation. Transfection of smooth muscle cells with plasmids encoding short hairpin RNA (shRNA) against p47(phox), but not plasmids for luciferase shRNA, inhibited the expression of p47(phox). ROS production and the suppression of Cdc42GAP activity in response to stimulation with 5-hydroxytryptamine (5-HT) were attenuated in cells producing p47(phox) shRNA compared with cells producing luciferase shRNA. In contrast, the addition of hydrogen peroxide to p47(phox)-deficient cells suppressed the activity of Cdc42GAP. Furthermore, exposure to hydrogen peroxide led to a decrease in Cdc42GAP activity in an in vitro assay. Cdc42 activation, p21-activated kinase 1 (PAK1) phosphorylation at Thr-423 (an indication of PAK activation), and vimentin phosphorylation at Ser-56 in response to 5-HT activation were also attenuated in smooth muscle cells producing shRNA against p47(phox). The knockdown of p47(phox) inhibited smooth muscle contraction during stimulation with 5-HT but not hydrogen peroxide. These results suggest that the p47(phox) subunit of NAD(P)H oxidase may mediate the agonist-induced GAP suppression by controlling ROS generation in smooth muscle cells during agonist stimulation. p47(phox)-regulated GAP affects smooth muscle contraction likely through the Cdc42/PAK1/vimentin pathway.
AJP Cell Physiology 10/2009; 297(6):C1424-33. · 3.54 Impact Factor
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ABSTRACT: Cdc42GAP (GTPase-activating protein) has been implicated in the regulation of cell motility, adhesion, proliferation, and apoptosis. In this study, Cdc42GAP was cloned from smooth muscle tissues. Cdc42GAP, but not inactive R282A Cdc42GAP (alanine substitution at arginine-282), enhanced the GTP hydrolysis of Cdc42 in an in vitro assay. Furthermore, we developed an assay to evaluate the activity of Cdc42GAP in vivo. Stimulation of smooth muscle cells with 5-hydroxytryptamine (5-HT) resulted in the decrease in Cdc42GAP activity. The agonist-induced GAP suppression was reversed by reactive oxygen species inhibitors. Treatment with hydrogen peroxide also inhibited GAP activity in smooth muscle cells. Because the vimentin cytoskeleton undergoes dynamic changes in response to contractile activation, we evaluated the role of Cdc42GAP in regulating vimentin filaments. Smooth muscle cells were infected with retroviruses encoding wild-type Cdc42GAP or its R282A mutant. Expression of wild-type Cdc42GAP, but not mutant R282A GAP, inhibited the increase in the activation of Cdc42 upon agonist stimulation. Phosphorylation of p21-activated kinase (PAK) at Thr-423 (an indication of PAK activation), vimentin phosphorylation (Ser-56), partial disassembly and spatial remodeling, and contraction were also attenuated in smooth muscle cells expressing Cdc42GAP. Our results suggest that the activity of Cdc42GAP is regulated upon contractile activation, which is mediated by intracellular ROS. Cdc42GAP regulates the vimentin network through the Cdc42-PAK pathway in smooth muscle cells during 5-HT stimulation.
AJP Cell Physiology 07/2009; 297(2):C299-309. · 3.54 Impact Factor
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ABSTRACT: Actin polymerization has recently emerged as an important cellular process that regulates smooth muscle contraction. Abelson tyrosine kinase (Abl) has been implicated in the regulation of actin dynamics and force development in vascular smooth muscle. In the present study, the systolic blood pressure was lower in Abl(-/-) knockout mice compared with wild-type mice. The knockout of Abl diminished the tyrosine phosphorylation of p130 Crk-associated substrate (CAS, an adapter protein associated with smooth muscle contraction) in resistance arteries upon stimulation with phenylephrine or angiotensin II. The agonist-elicited enhancement of F-actin-to-G-actin ratios in arteries assessed by fluorescent microscopy was also reduced in Abl(-/-) mice. It has been known that vinculin is a structural protein that links actin filaments to extracellular matrix via transmembrane integrins, whereas paxillin is a signaling protein associated with focal contacts mediating actin cytoskeleton remodeling. The expression of vinculin and paxillin at protein and messenger levels was lower in arterial vessels from Abl knockout mice. However, the agonist-induced increase in myosin phosphorylation was not attenuated in arteries from Abl knockout mice. These results indicate that Abl differentially regulates Crk-associated substrate, vinculin, and paxillin in arterial vessels. The Abl-regulated cellular process and blood pressure are independent of myosin activation in vascular smooth muscle.
AJP Heart and Circulatory Physiology 07/2009; 297(2):H533-9. · 3.71 Impact Factor
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Dale D Tang
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ABSTRACT: Vascular smooth muscle is a key effector in the wall of blood vessels during the pathogenesis of hypertension. Various factors directly elicit smooth muscle cell contraction, migration, growth, and hypertrophy, which lead to the progression of hypertension. Crk-associated substrate (CAS), the first discovered member of the adapter protein CAS family, has recently emerged as a critical cellular component that regulates smooth muscle functions. In this review, the molecular structure and protein interactions of the CAS family members are summarized. Evidence for the role of CAS in the regulation of vascular smooth muscle contractility, cell migration, hypertrophy, and growth is presented. Regulation of CAS by novel tyrosine kinases/phosphatases and unique downstream signaling partners of CAS are also discussed. These new findings establish the important role for CAS in regulating vascular smooth muscle functions. The CAS-associated processes may be new biological targets for the development of new treatment of cardiovascular diseases such as hypertension.
Journal of Cardiovascular Pharmacology and Therapeutics 04/2009; 14(2):89-98. · 1.75 Impact Factor
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ABSTRACT: Vascular smooth muscle tone plays a fundamental role in regulating blood pressure, blood flow, microcirculation, and other cardiovascular functions. The cellular and molecular mechanisms by which vascular smooth muscle contractility is regulated are not completely elucidated. Recent studies show that the actin cytoskeleton in smooth muscle is dynamic, which regulates force development. In this review, evidence for actin polymerization in smooth muscle upon external stimulation is summarized. Protein kinases such as Abelson tyrosine kinase, focal adhesion kinase, Src, and mitogen-activated protein kinase have been documented to coordinate actin polymerization in smooth muscle. Transmembrane integrins have also been reported to link to signaling pathways modulating actin dynamics. The roles of Rho family of the small proteins that bind to guanosine triphosphate (GTP), also known as GTPases, and the actin-regulatory proteins, including Crk-associated substrate, neuronal Wiskott-Aldrich Syndrome protein, the Arp2/3 complex, and profilin, and heat shock proteins in regulating actin assembly are discussed. These new findings promote our understanding on how smooth muscle contraction is regulated at cellular and molecular levels.
Journal of Cardiovascular Pharmacology and Therapeutics 07/2008; 13(2):130-40. · 1.75 Impact Factor
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Dale D Tang
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ABSTRACT: The intermediate filament (IF) network is one of the three cytoskeletal systems in smooth muscle. The type III IF proteins vimentin and desmin are major constituents of the network in smooth muscle cells and tissues. Lack of vimentin or desmin impairs contractile ability of various smooth muscle preparations, implying their important role for smooth muscle force development. The IF framework has long been viewed as a fixed cytostructure that solely provides mechanical integrity for the cell. However, recent studies suggest that the IF cytoskeleton is dynamic in mammalian cells in response to various external stimulation. In this review, the structure and biological properties of IF proteins in smooth muscle are summarized. The role of IF proteins in the modulation of smooth muscle force development and redistribution/translocation of signaling partners (such as p130 Crk-associated substrate, CAS) is depicted. This review also summarizes our latest understanding on how the IF network may be regulated in smooth muscle.
AJP Cell Physiology 05/2008; 294(4):C869-78. · 3.54 Impact Factor
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ABSTRACT: The tyrosine phosphorylated protein Crk-associated substrate (CAS) has previously been shown to participate in the cellular processes regulating dynamic changes in the actin architecture and arterial constriction. In the present study, treatment of rat mesenteric arteries with phenylephrine (PE) led to the increase in CAS tyrosine phosphorylation and the association of CAS with the adapter protein CrkII. CAS phosphorylation was catalyzed by Abl in an in vitro study. To determine the role of Abl tyrosine kinase in arterial vessels, plasmids encoding Abl short hairpin RNA (shRNA) were transduced into mesenteric arteries by chemical loading plus liposomes. Abl silencing diminished increases in CAS phosphorylation on PE stimulation. Previous studies have shown that assembly of the multiprotein compound containing CrkII, neuronal Wiskott-Aldrich Syndrome Protein (N-WASP) and the Arp2/3 (Actin Related Protein) complex triggers actin polymerization in smooth muscle as well as in nonmuscle cells. In this study, Abl silencing attenuated the assembly of the multiprotein compound in resistance arteries on contractile stimulation. Furthermore, the increase in F/G-actin ratios (an index of actin assembly) and constriction on contractile stimulation were reduced in Abl-deficient arterial segments compared with control arteries. However, myosin regulatory light chain phosphorylation (MRLCP) elicited by contractile activation was not inhibited in Abl-deficient arteries. These results suggest that Abl may play a pivotal role in mediating CAS phosphorylation, the assembly of the multiprotein complex, actin assembly, and constriction in resistance arteries. Abl does not participate in the regulation of myosin activation in arterial vessels during contractile stimulation.
Circulation Research 09/2007; 101(4):420-8. · 9.49 Impact Factor
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ABSTRACT: The intermediate filament protein vimentin has been shown to be required for smooth muscle contraction. The adapter protein p130 Crk-associated substrate (CAS) participates in the signaling processes that regulate force development in smooth muscle. However, the interaction of vimentin filaments with CAS has not been well elucidated. In the present study, ACh stimulation of tracheal smooth muscle strips increased the ratio of soluble to insoluble vimentin (an index of vimentin disassembly) in association with force development. ACh activation also induced vimentin phosphorylation at Ser(56) as assessed by immunoblot analysis. More importantly, CAS was found in the cytoskeletal vimentin fraction, and the amount of CAS in cytoskeletal vimentin was reduced in smooth muscle strips on contractile stimulation. CAS redistributed from the myoplasm to the periphery during ACh activation of smooth muscle cells. The ACh-elicited decrease in CAS distribution in cytoskeletal vimentin was attenuated by the downregulation of p21-activated kinase (PAK) 1 with antisense oligodeoxynucleotides. Vimentin phosphorylation at this residue, the ratio of soluble to insoluble vimentin, and active force in smooth muscle strips induced by ACh were also reduced in PAK-depleted tissues. These results suggest that PAK may regulate CAS release from the vimentin intermediate filaments by mediating vimentin phosphorylation at Ser(56) and the transition of cytoskeletal vimentin to soluble vimentin. The PAK-mediated dissociation of CAS from the vimentin network may participate in the cellular processes that affect active force development during ACh activation of tracheal smooth muscle tissues.
AJP Lung Cellular and Molecular Physiology 02/2007; 292(1):L240-8. · 3.66 Impact Factor
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ABSTRACT: Phosphorylation and spatial reorganization of the vimentin network have been implicated in mediating smooth muscle contraction, cell migration, and mitosis. In this study, stimulation of cultured smooth muscle cells with 5-hydroxytryptamine (5-HT) induced PAK1 phosphorylation at Thr-423 (an indication of p21-activated kinase (PAK) activation). Treatment with PAK led to disassembly of wild-type (but not mutant S56A) vimentin filaments as assessed by an in vitro filament assembly assay. Furthermore, stimulation with 5-HT resulted in the dissociation of Crk-associated substrate (CAS; an adapter protein associated with smooth muscle force development) from cytoskeletal vimentin. Expression of mutant S56A vimentin in cells inhibited the increase in phosphorylation at Ser-56 and in the ratios of soluble to insoluble vimentin (an index of vimentin disassembly) and the dissociation of CAS from cytoskeletal vimentin in response to 5-HT activation compared with cells expressing wild-type vimentin. Because CAS may be involved in PAK activation, PAK phosphorylation was evaluated in cells expressing the S56A mutant. Expression of mutant S56A vimentin depressed PAK phosphorylation at Thr-423 induced by 5-HT. Expression of the S56A mutant also inhibited the spatial reorientation of vimentin filaments in cells in response to 5-HT stimulation. Our results suggest that vimentin phosphorylation at Ser-56 may inversely regulate PAK activation possibly via the increase in the amount of soluble CAS upon agonist stimulation of smooth muscle cells. Additionally, vimentin phosphorylation at this position is critical for vimentin filament spatial rearrangement elicited by agonists.
Journal of Biological Chemistry 12/2006; 281(45):34716-24. · 4.77 Impact Factor
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ABSTRACT: Vimentin intermediate filaments undergo spatial reorganization in cultured smooth muscle cells in response to contractile activation; however, the role of vimentin in the physiological properties of smooth muscle has not been well elucidated. Tracheal smooth muscle strips were loaded with antisense oligonucleotides (ODNs) against vimentin and then cultured for 2 days to allow for protein degradation. Treatment with vimentin antisense, but not sense, ODNs suppressed vimentin protein expression; neither vimentin antisense nor sense ODNs affected protein levels of desmin and actin. Force development in response to ACh stimulation or KCl depolarization was lower in vimentin-deficient tissues than in vimentin sense ODN- or non-ODN-treated muscle strips. Passive tension was also depressed in vimentin-depleted muscle tissues. Vimentin downregulation did not attenuate increases in myosin light chain (MLC) phosphorylation in response to contractile stimulation or basal MLC phosphorylation. In vimentin sense ODN-treated or non-ODN-treated smooth muscle strips, the desmosomal protein plakoglobin was primarily localized in the cell periphery. The membrane-associated localization of plakoglobin was reduced in vimentin-depleted muscle tissues. These studies suggest that vimentin filaments play an important role in mediating active force development and passive tension, which are not regulated by MLC phosphorylation. Vimentin downregulation impairs the structural organization of desmosomes, which may be associated with the decrease in force development.
AJP Cell Physiology 10/2006; 291(3):C483-9. · 3.54 Impact Factor
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ABSTRACT: Actin polymerization has been shown to occur in tracheal smooth muscle tissues and cells in response to contractile stimulation, and there is evidence that the polymerization of actin is required for contraction. In tracheal smooth muscle, agonist-induced actin polymerization is mediated by activation of neuronal Wiskott-Aldrich syndrome protein (N-WASp) and the Arp (actin-related protein) 2/3 complex, and activation of the small GTPase Cdc42 regulates the activation of N-WASp. In the present study, the role of the adapter protein CrkII in the regulation of N-WASp and Cdc42 activation, actin polymerization, and tension development in smooth muscle tissues was evaluated. Stimulation of tracheal smooth muscle tissues with acetylcholine increased the association of CrkII with N-WASp. Plasmids encoding wild type CrkII or a CrkII mutant lacking the SH3 effector-binding ability, CrkII SH3N, were introduced into tracheal smooth muscle tissues, and the tissues were incubated for 2 days to allow for protein expression. Expression of the CrkII SH3N mutant in smooth muscle tissues inhibited the association of CrkII with N-WASp and the activation of Cdc42. The CrkII SH3N mutant also inhibited the increase in the association of N-WASp with Arp2, a major component of the Arp2/3 complex, in response to contractile stimulation, indicating inhibition of N-WASp activation. Expression of the CrkII SH3N mutant also inhibited tension generation and actin polymerization in response to contractile stimulation; however, it did not inhibit myosin light chain phosphorylation. These results suggest that CrkII plays a critical role in the regulation of N-WASp activation, perhaps by regulating the activation of Cdc42, and that it thereby regulates actin polymerization and active tension generation in tracheal smooth muscle. These studies suggest a novel signaling pathway for the regulation of N-WASp activation and active contraction in smooth muscle tissues.
Journal of Biological Chemistry 07/2005; 280(24):23380-9. · 4.77 Impact Factor
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ABSTRACT: Vimentin intermediate filaments undergo spatial reorganization in endothelial cells and fibroblasts in response to stimulation with platelet-derived growth factor and epidermal growth factor. In the present study, the vimentin network exhibited a curved filamentous structure in unstimulated smooth muscle cells. Vimentin filaments became straight and were arranged along the long axis of cells upon stimulation with 5-hydroxytryptamine (5-HT; serotonin). Stimulation of smooth muscle cells with 5-HT also induced phosphorylation of vimentin on Ser-56. Treatment of cells with small interfering RNA selectively down-regulated the expression of PAK1 (p21-activated kinase 1) without affecting the content of smooth muscle alpha-actin. The silencing of PAK1 inhibited the site-specific phosphorylation and spatial rearrangement of the vimentin network in response to stimulation with 5-HT. Neither the disruption of stress fibres by cytochalasin D nor the inhibition of protein tyrosine phosphorylation affects the spatial reorganization of vimentin intermediate filaments in response to stimulation with 5-HT. In addition, stimulation of smooth muscle cells with 5-HT increased the ratio of soluble to insoluble vimentin. PAK1 silencing attenuated increases in the ratio of soluble to insoluble vimentin upon stimulation with 5-HT. These results suggest that the PAK-mediated site-specific phosphorylation of vimentin may play a role in regulating the reorganization of vimentin intermediate filaments during stimulation of smooth muscle cells with 5-HT.
Biochemical Journal 07/2005; 388(Pt 3):773-83. · 4.90 Impact Factor
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ABSTRACT: 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.
AJP Cell Physiology 06/2005; 288(5):C1145-60. · 3.54 Impact Factor
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ABSTRACT: 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.
Journal of Biological Chemistry 01/2005; 279(50):51722-8. · 4.77 Impact Factor
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Tony R Bai,
Jason H T Bates,
Vito Brusasco,
Blanca Camoretti-Mercado,
Pasquale Chitano,
Lin Hong Deng,
Maria Dowell,
Ben Fabry,
Lincoln E Ford,
Jeffrey J Fredberg, [......],
R Robert Schellenberg,
Chun Y Seow,
Gary C Sieck,
Paul G Smith,
Alex V Smolensky,
Julian Solway,
Newman L Stephens,
Alastair G Stewart, Dale D Tang,
Lu Wang
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ABSTRACT: The observation that the length-force relationship in airway smooth muscle can be shifted along the length axis by accommodating the muscle at different lengths has stimulated great interest. In light of the recent understanding of the dynamic nature of length-force relationship, many of our concepts regarding smooth muscle mechanical properties, including the notion that the muscle possesses a unique optimal length that correlates to maximal force generation, are likely to be incorrect. To facilitate accurate and efficient communication among scientists interested in the function of airway smooth muscle, a revised and collectively accepted nomenclature describing the adaptive and dynamic nature of the length-force relationship will be invaluable. Setting aside the issue of underlying mechanism, the purpose of this article is to define terminology that will aid investigators in describing observed phenomena. In particular, we recommend that the term "optimal length" (or any other term implying a unique length that correlates with maximal force generation) for airway smooth muscle be avoided. Instead, the in situ length or an arbitrary but clearly defined reference length should be used. We propose the usage of "length adaptation" to describe the phenomenon whereby the length-force curve of a muscle shifts along the length axis due to accommodation of the muscle at different lengths. We also discuss frequently used terms that do not have commonly accepted definitions that should be used cautiously.
Journal of Applied Physiology 01/2005; 97(6):2029-34. · 3.75 Impact Factor
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ABSTRACT: 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.
AJP Cell Physiology 03/2004; 286(2):C433-47. · 3.54 Impact Factor
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ABSTRACT: The adapter protein paxillin has been implicated in the regulation of cytoskeletal organization and cell motility. Paxillin undergoes tyrosine phosphorylation in response to the contractile stimulation of smooth muscle, and the depletion of paxillin by antisense inhibits smooth muscle contraction. In the present study, acetylcholine (ACh)-stimulation of tracheal smooth muscle tissues increased paxillin phosphorylation at tyr-31 and tyr-118 by three- to fourfold. The role of tyr-31 and tyr-118 phosphorylation of paxillin in smooth muscle was evaluated by introducing plasmids encoding wild type paxillin or paxillin mutants F31, F118 or F31/118 (phenylalanine substitution at tyrosine sites 31, 118) into tracheal smooth muscle strips by reversible permeabilization, and incubating the tissues for 2 days. The expression of recombinant proteins was confirmed by immunoblot and immunofluorescence analysis. Expression of the paxillin mutants F31, F118 or F31/118 inhibited the contractile response to ACh stimulation but did not inhibit the increase in myosin light chain phosphorylation. The expression of wild type paxillin had no significant affect on force or myosin light chain phosphorylation. ACh stimulation reduced G-actin/F-actin ratio in tissues expressing wild type paxillin; whereas the agonist-induced decrease in G-actin/F-actin was inhibited in strips expressing paxillin mutant F31/118. The paxillin mutant F31/118 showed a marked decrease in their interaction with the SH2/SH3 adaptor protein CrkII but not with vinculin or focal adhesion kinase. We conclude that paxillin phosphorylation at tyr-31 and tyr-118 regulates active tension development during contractile stimulation. Paxillin phosphorylation at these two sites may be important in regulating actin filament dynamics and organization during smooth muscle contraction.
The Journal of Physiology 12/2003; 553(Pt 1):21-35. · 4.72 Impact Factor
