Intracellular signal transduction for migration and actin remodeling in vascular smooth muscle cells after sphingosylphosphorylcholine stimulation

Li, Sheng; Tanaka, Hideyuki; Wang, Hong‐Hui; Yoshiyama, Shinji; Kumagai, Hidehiko; Nakamura, Akio; Brown, D.; Thatcher, Sean E.; Wright, Gary L.; Kohama, Kazuhiro

doi:10.1152/ajpheart.00901.2005

Cited by 21 publications

(33 citation statements)

References 47 publications

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“…In addition, the current study shows that p38 plays an important role in HB-EGF-induced actin stress fiber disassembly. In accordance with our results, studies conducted in VSM cells also showed that p38 is involved in cell migration by remodeling the actin cytoskeleton (25). Pichon et al (34) showed that the p38/MK2/Hsp27 cascade controls actin polymerization and lamellipodium formation in PDGF-induced VSM cell migration.…”

Section: Discussionsupporting

confidence: 91%

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HB-EGF–Promoted Airway Smooth Muscle Cells and Their Progenitor Migration Contribute to Airway Smooth Muscle Remodeling in Asthmatic Mouse

Wang

Yao

et al. 2016

The Journal of Immunology

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The airway smooth muscle (ASM) cells’ proliferation, migration, and their progenitor’s migration are currently regarded as causative factors for ASM remodeling in asthma. Heparin-binding epidermal growth factor (HB-EGF), a potent mitogen and chemotactic factor, could promote ASM cell proliferation through MAPK pathways. In this study, we obtained primary ASM cells and their progenitors from C57BL/6 mice and went on to explore the role of HB-EGF in these cells migration and the underlying mechanisms. We found that recombinant HB-EGF (rHB-EGF) intratracheal instillation accelerated ASM layer thickening in an OVA-induced asthmatic mouse. Modified Boyden chamber assay revealed that rHB-EGF facilitate ASM cell migration in a dose-dependent manner and ASM cells from asthmatic mice had a greater migration ability than that from normal counterparts. rHB-EGF could stimulate the phosphorylation of ERK1/2 and p38 in ASM cells but further migration assay showed that only epidermal growth factor receptor inhibitor (AG1478) or p38 inhibitor (SB203580), but not ERK1/2 inhibitor (PD98059), could inhibit rHB-EGF–mediated ASM cells migration. Actin cytoskeleton experiments exhibited that rHB-EGF could cause actin stress fibers disassembly and focal adhesions formation of ASM cells through the activation of p38. Finally, airway instillation of rHB-EGF promoted the recruitment of bone marrow–derived smooth muscle progenitor cells, which were transferred via caudal vein, migrating into the airway from the circulation. These observations demonstrated that ASM remodeling in asthma might have resulted from HB-EGF–mediated ASM cells and their progenitor cells migration, via p38 MAPK-dependent actin cytoskeleton remodeling.

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Section: Discussionsupporting

confidence: 91%

“…Studies included four independent experiments. induced by CXCL2 (24) or in the process of VSM cell migration (25,26), suggesting an important role for p38 rather than ERK in cell migration. In contrast, there are also some studies that reported the involvement of ERK in ASM and VSM cell migration (27)(28)(29).…”

Section: Discussionmentioning

confidence: 99%

HB-EGF–Promoted Airway Smooth Muscle Cells and Their Progenitor Migration Contribute to Airway Smooth Muscle Remodeling in Asthmatic Mouse

Wang

Yao

et al. 2016

The Journal of Immunology

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“…To determine the smooth muscle myosin II content of total myosin II in VSMCs, GbaSM-4 and A7r5 cells were extracted with SDS sample buffer as previously described (28). The heavy chain of myosin II isoforms was separated by SDS-PAGE and transferred to a PVDF membrane.…”

Section: Methodsmentioning

confidence: 99%

“…However, the details of the regulatory mechanism of myosin II in cell migration remain to be established. The general understanding is that cell migration is regulated not only by pathways of signal transduction involving myosin light chain (MLC) phosphorylation, but also by MLC phosphorylation-independent pathways (2,18,28,30). The exact role of myosin II in cell migration and the mechanism whereby myosin II contributes force generation for cell migration remain unanswered.…”

mentioning

confidence: 99%

Blebbistatin inhibits the chemotaxis of vascular smooth muscle cells by disrupting the myosin II-actin interaction

Wang¹,

Tanaka²,

Qin³

et al. 2008

American Journal of Physiology-Heart and Circulatory Physiology

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Blebbistatin is a myosin II-specific inhibitor. However, the mechanism and tissue specificity of the drug are not well understood. Blebbistatin blocked the chemotaxis of vascular smooth muscle cells (VSMCs) toward sphingosylphosphorylcholine (IC50 ϭ 26.1 Ϯ 0.2 and 27.5 Ϯ 0.5 M for GbaSM-4 and A7r5 cells, respectively) and platelet-derived growth factor BB (IC 50 ϭ 32.3 Ϯ 0.9 and 31.6 Ϯ 1.3 M for GbaSM-4 and A7r5 cells, respectively) at similar concentrations. Immunofluorescence and fluorescent resonance energy transfer analysis indicated a blebbistatin-induced disruption of the actin-myosin interaction in VSMCs. Subsequent experiments indicated that blebbistatin inhibited the Mg 2ϩ -ATPase activity of the unphosphorylated (IC 50 ϭ 12.6 Ϯ 1.6 and 4.3 Ϯ 0.5 M for gizzard and bovine stomach, respectively) and phosphorylated (IC 50 ϭ 15.0 Ϯ 0.6 M for gizzard) forms of purified smooth muscle myosin II, suggesting a direct effect on myosin II motor activity. It was further observed that the Mg 2ϩ -ATPase activities of gizzard myosin II fragments, heavy meromyosin (IC 50 ϭ 14.4 Ϯ 1.6 M) and subfragment 1 (IC50 ϭ 5.5 Ϯ 0.4 M), were also inhibited by blebbistatin. Assay by in vitro motility indicated that the inhibitory effect of blebbistatin was reversible. Electron-microscopic evaluation showed that blebbistatin induced a distinct conformational change (i.e., swelling) of the myosin II head. The results suggest that the site of blebbistatin action is within the S1 portion of smooth muscle myosin II. Boyden chamber; fluorescence resonance energy transfer; ATPase; in vitro motility assay; electron microscopy CELL MIGRATION IS DRIVEN through complex processes, such as extension of the leading membrane edge, with formation of adhesive contacts and stress fibers (26, 52). Myosin II is widely believed to be one of the main components producing the forces required in this process (12). However, the details of the regulatory mechanism of myosin II in cell migration remain to be established. The general understanding is that cell migration is regulated not only by pathways of signal transduction involving myosin light chain (MLC) phosphorylation, but also by MLC phosphorylation-independent pathways (2,18,28,30). The exact role of myosin II in cell migration and the mechanism whereby myosin II contributes force generation for cell migration remain unanswered.Blebbistatin was recently identified as a specific inhibitor of myosin II-dependent cell processes (49). Its membrane-permeable characteristic and effect on various myosin II isoforms make this agent an invaluable tool in research of myosin II-involved cellular events, including cell motility (5, 23), cell shape maintenance (50), muscle contraction (7, 15), and cytokinesis (49). Blebbistatin has been shown to preferentially bind to the myosin-ADP-phosphate complex with high affinity and prevent phosphate release (24, 37), resulting in inhibition of the actin-myosin interaction. Blebbistatin also inhibited the ability of skeletal muscle and nonmuscle myosin II to move actin f...

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“…6 in ref. 10) may phosphorylate myosin, as discussed previously (13). Although the studies are in vitro, the actin-binding domain of MLCK exerts an inhibitory effect on the phosphorylated myosin (2,12).…”

Section: Down-regulation Of Myosin Light Chain Kinase Expression In Vmentioning

confidence: 80%

Down-Regulation of Myosin Light Chain Kinase Expression in Vascular Smooth Muscle Cells Accelerates Cell Proliferation: Requirement of Its Actin-binding Domain for Reversion to Normal Rates

et al. 2012

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J Pharmacol Sci 119, 91 -96 (2012) Myosin light-chain kinase (MLCK) is a key regulatory protein of smooth muscle contraction, non-muscle migration, and cytokinesis. MLCK is a calmodulin-dependent kinase that phosphorylates smooth muscle myosin regulatory light-chain (reviewed in ref. 1). MLCK is a multi-domain protein composed of an actin-binding domain at its N-terminal and a myosin-binding domain at its C-terminal, in addition to a kinase domain in its central core. We have been investigating the function of MLCK under conditions where the kinase activity has been deleted (reviewed in ref. 2).Many investigations have shown the importance of the kinase activity of MLCK in cytokinesis (reviewed in ref.3). The contractile ring is formed during cytokinesis by the assembly of myosin with actin filaments. When the myosin light chain is phosphorylated by MLCK, cell division is induced by actin-myosin interaction to originate daughter cells. On the other hand, contrary views that do not support a role for MLCK in cytokinesis, like in the case of C. elegans (4), were also reported. Moreover, in our literature survey, we found no reports regarding the roles of MLCK other than its kinase activity in the cytokinesis. Therefore, in order to elucidate the functions of MLCK, we knocked-out the MLCK expression in vascular smooth muscle (VSM) cells of vertebrate origin (5, 6). The cultured VSM cells were able to proliferate rapidly in spite of the lack of MLCK. In the above reports, we described the functions of MLCK other than its kinase activity in smooth muscle contraction, but on that occasion we did not address its role in pro liferation.Recently, the role of the long isoform of MLCK was identified, and its regulatory role in the cytokinesis was elucidated (7). Because our previous studies have examined only the short isoform of MLCK, we decided herein to down-regulate the expressions of both MLCK isoforms, to readdress the roles of both MLCKs in cytokinesis, by using cell proliferation as a marker.We examined two types of vascular smooth muscle cell lines, GbaSM-4 cells of guinea pig basilar artery origin (8) and SM3 cells of rabbit aorta origin (9). We Nishitokyo, Tokyo 202-8585, Japan Received November 10, 2011; Accepted March 2, 2012 Abstract. Myosin light-chain kinase (MLCK) is a multi-domain protein with kinase and actinbinding domains, among others. Deficiency of MLCK expression in GBaSM-4 vascular smooth muscle cells enhanced cell proliferation rate and shortened cell doubling time. Transient transfection of the MLCK-deficient cells with cDNA constructs of either wild-type MLCK or its mutant lacking the kinase activity reverted the cell proliferation rate to that of wild-type cells, whereas that of MLCK lacking the actin-binding domain maintained cell proliferation at an elevated rate similar to the MLCK-deficient cells. Thus, the actin-binding domain of MLCK seems to play a role in regulating cell proliferation.

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Intracellular signal transduction for migration and actin remodeling in vascular smooth muscle cells after sphingosylphosphorylcholine stimulation

Cited by 21 publications

References 47 publications

HB-EGF–Promoted Airway Smooth Muscle Cells and Their Progenitor Migration Contribute to Airway Smooth Muscle Remodeling in Asthmatic Mouse

HB-EGF–Promoted Airway Smooth Muscle Cells and Their Progenitor Migration Contribute to Airway Smooth Muscle Remodeling in Asthmatic Mouse

Blebbistatin inhibits the chemotaxis of vascular smooth muscle cells by disrupting the myosin II-actin interaction

Down-Regulation of Myosin Light Chain Kinase Expression in Vascular Smooth Muscle Cells Accelerates Cell Proliferation: Requirement of Its Actin-binding Domain for Reversion to Normal Rates

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