Rho-kinase-mediated pathway induces enhanced myosin light chain phosphorylations in a swine model of coronary artery spasm

Coronary artery spasm (CAS) is an intense vasoconstriction of coronary arteries that cause total or subtotal vessel occlusion. The cardioprotective effect of sirtuin-1 (SIRT1) has been extensively highlighted in coronary artery diseases. The aims within this study include the investigation of the molecular mechanism by which SIRT1 alleviates CAS. SIRT1 expression was first determined by RT-qPCR and Western blot analysis in an endothelin-1 (ET-1)-induced rat CAS model. Interaction among SIRT1, nuclear factor-kappaB (NF-κB), myosin light chain kinase/myosin light chain-2 (MLCK/MLC2), and ET-1 was analyzed using luciferase reporter assay, RT-qPCR and Western blot analysis. After ectopic expression and depletion experiments in vascular smooth muscle cells (VSMCs), contraction and proliferation VSMCs, and expression of contraction-related proteins (α-SMA, calponin, and SM22α) were measured by collagen gel contraction, EdU assay, RT-qPCR and Western blot analysis. The obtained results showed that SIRT1 expression was reduced in rat CAS models. However, overexpression of SIRT1 inhibited the contraction and proliferation of VSMCs in vitro. Mechanistic investigation indicated that SIRT1 inhibited NF-κB expression through deacetylation. Moreover, NF-κB could activate the MLCK/MLC2 pathway and up-regulate ET-1 expression by binding to their promoter regions, thus inducing VSMC contraction and proliferation in vitro. In vivo experimental results also revealed that SIRT1 alleviated CAS through regulation of the NF-κB/MLCK/MLC2/ET-1 signaling axis. Collectively, our data suggested that SIRT1 could mediate the deacetylation of NF-κB, disrupt the MLCK/MLC2 pathway and inhibit the expression of ET-1 to relieve CAS, providing a theoretical basis for the prospect of CAS treatment and prevention.

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Phosphorylated Myosin Light Chain 2 (p-MLC2) as a Molecular Marker of Antemortem Coronary Artery Spasm

Medical Science Monitor ◽

10.12659/msm.900152 ◽

2016 ◽

Vol 22 ◽

pp. 3316-3327 ◽

Cited By ~ 2

Author(s):

Liliang Li ◽

Yuhua Li ◽

Junyi Lin ◽

Jieqing Jiang ◽

Meng He ◽

...

Keyword(s):

Coronary Artery ◽

Molecular Marker ◽

Light Chain ◽

Myosin Light Chain ◽

Coronary Artery Spasm ◽

Myosin Light Chain 2

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A Novel Regulatory Mechanism of Myosin Light Chain Phosphorylation via Binding of 14-3-3 to Myosin Phosphatase

Molecular Biology of the Cell ◽

10.1091/mbc.e07-07-0668 ◽

2008 ◽

Vol 19 (3) ◽

pp. 1062-1071 ◽

Cited By ~ 25

Author(s):

Yasuhiko Koga ◽

Mitsuo Ikebe

Keyword(s):

Light Chain ◽

Myosin Light Chain ◽

Regulatory Mechanism ◽

Kinase Inhibitor ◽

Myosin Ii ◽

Critical Role ◽

Rho Kinase ◽

Myosin Phosphatase ◽

Dependent Cell ◽

Direct Binding

Myosin II phosphorylation–dependent cell motile events are regulated by myosin light-chain (MLC) kinase and MLC phosphatase (MLCP). Recent studies have revealed myosin phosphatase targeting subunit (MYPT1), a myosin-binding subunit of MLCP, plays a critical role in MLCP regulation. Here we report the new regulatory mechanism of MLCP via the interaction between 14-3-3 and MYPT1. The binding of 14-3-3β to MYPT1 diminished the direct binding between MYPT1 and myosin II, and 14-3-3β overexpression abolished MYPT1 localization at stress fiber. Furthermore, 14-3-3β inhibited MLCP holoenzyme activity via the interaction with MYPT1. Consistently, 14-3-3β overexpression increased myosin II phosphorylation in cells. We found that MYPT1 phosphorylation at Ser472 was critical for the binding to 14-3-3. Epidermal growth factor (EGF) stimulation increased both Ser472 phosphorylation and the binding of MYPT1-14-3-3. Rho-kinase inhibitor inhibited the EGF-induced Ser472 phosphorylation and the binding of MYPT1-14-3-3. Rho-kinase specific siRNA also decreased EGF-induced Ser472 phosphorylation correlated with the decrease in MLC phosphorylation. The present study revealed a new RhoA/Rho-kinase–dependent regulatory mechanism of myosin II phosphorylation by 14-3-3 that dissociates MLCP from myosin II and attenuates MLCP activity.

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Rho kinase inhibitors stimulate the migration of human cultured osteoblastic cells by regulating actomyosin activity

Cellular & Molecular Biology Letters ◽

10.2478/s11658-011-0006-z ◽

2011 ◽

Vol 16 (2) ◽

Cited By ~ 13

Author(s):

Xuejiao Zhang ◽

Cheng Li ◽

Huiling Gao ◽

Hiroaki Nabeka ◽

Tetsuya Shimokawa ◽

...

Keyword(s):

Light Chain ◽

Myosin Light Chain ◽

Kinase Inhibitors ◽

Rho Kinase ◽

Cell Types ◽

Osteosarcoma Cells ◽

Human Osteoblasts ◽

Protein Levels ◽

Human Osteosarcoma Cells ◽

Joint Prostheses

AbstractWe investigated the effects of Rho-associated kinase (ROCK) on migration and cytoskeletal organization in primary human osteoblasts and Saos-2 human osteosarcoma cells. Both cell types were exposed to two different ROCK inhibitors, Y-27632 and HA-1077. In the improved motility assay used in the present study, Y-27632 and HA-1077 significantly increased the migration of both osteoblasts and osteosarcoma cells on plastic in a dose-dependent and reversible manner. Fluorescent images showed that cells of both types cultured with Y-27632 or HA-1077 exhibited a stellate appearance, with poor assembly of stress fibers and focal contacts. Western blotting showed that ROCK inhibitors reduced myosin light chain (MLC) phosphorylation within 5 min without affecting overall myosin light-chain protein levels. Inhibition of ROCK activity is thought to enhance the migration of human osteoblasts through reorganization of the actin cytoskeleton and regulation of myosin activity. ROCK inhibitors may be potentially useful as anabolic agents to enhance the biocompatibility of bone and joint prostheses.

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Maturation and the role of PKC-mediated contractility in ovine cerebral arteries

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00681.2009 ◽

2009 ◽

Vol 297 (6) ◽

pp. H2242-H2252 ◽

Cited By ~ 22

Author(s):

Ravi Goyal ◽

Ashwani Mittal ◽

Nina Chu ◽

Lijun Shi ◽

Lubo Zhang ◽

...

Keyword(s):

Light Chain ◽

Myosin Light Chain ◽

Contractile Response ◽

Cerebral Arteries ◽

Age Groups ◽

Rho Kinase ◽

Immunoblot Analysis ◽

Mek Inhibitor ◽

Inhibitory Protein ◽

Regulatory Myosin Light Chain

Ca2+-independent pathways such as protein kinase C (PKC), extracellular-regulated kinases 1 and 2 (ERK1/2), and Rho kinase 1 and 2 (ROCK1/2) play important roles in modulating cerebral vascular tone. Because the roles of these kinases vary with maturational age, we tested the hypothesis that PKC differentially regulates the Ca2+-independent pathways and their effects on cerebral arterial contractility with development. We simultaneously examined the responses of arterial tension and intracellular Ca2+ concentration and used Western immunoblot analysis to measure ERK1/2, RhoA, 20 kDa regulatory myosin light chain (MLC20), PKC-potentiated inhibitory protein of 17 kDa (CPI-17), and caldesmon. Phorbol 12,13-dibutyrate (PDBu)-mediated PKC activation produced a robust contractile response, which was increased a further 20 to 30% by U-0126 (MEK inhibitor) in cerebral arteries of both age groups. Of interest, in the fetal cerebral arteries, PDBu leads to an increased phosphorylation of ERK2 compared with ERK1, whereas in adult arteries, we observed an increased phosphorylation of ERK1 compared with ERK2. Also, in the present study, RhoA/ROCK played a significant role in the PDBu-mediated contractility of fetal cerebral arteries, whereas in adult cerebral arteries, CPI-17 and caldesmon had a significantly greater role compared with the fetus. PDBu also led to an increased MLC20 phosphorylation, a response blunted by the inhibition of myosin light chain kinase only in the fetus. Overall, the present study demonstrates an important maturational shift from RhoA/ROCK-mediated to CPI-17/caldesmon-mediated PKC-induced contractile response in ovine cerebral arteries.

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Involvement of Rho-Kinase–Mediated Phosphorylation of Myosin Light Chain in Enhancement of Cerebral Vasospasm

Circulation Research ◽

10.1161/01.res.87.3.195 ◽

2000 ◽

Vol 87 (3) ◽

pp. 195-200 ◽

Cited By ~ 175

Author(s):

Motohiko Sato ◽

Eiichi Tani ◽

Hirokazu Fujikawa ◽

Kozo Kaibuchi

Keyword(s):

Cerebral Vasospasm ◽

Light Chain ◽

Myosin Light Chain ◽

Rho Kinase

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Endothelium-independent vasorelaxant effect of Asphodelus tenuifolius Cav. via inhibition of myosin light chain kinase activity in the porcine coronary artery

Journal of Ethnopharmacology ◽

10.1016/j.jep.2020.113693 ◽

2021 ◽

Vol 269 ◽

pp. 113693

Author(s):

Waqas Younis ◽

Alamgeer ◽

V.B. Schini-Kerth ◽

Arquimedes Gasparotto Junior ◽

Samara Requena Nocchi ◽

...

Keyword(s):

Coronary Artery ◽

Light Chain ◽

Myosin Light Chain Kinase ◽

Myosin Light Chain ◽

Kinase Activity ◽

Porcine Coronary Artery ◽

Vasorelaxant Effect

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Lanthanum chloride causes blood–brain barrier disruption through intracellular calcium-mediated RhoA/Rho kinase signaling and myosin light chain kinase

Metallomics ◽

10.1039/d0mt00187b ◽

2020 ◽

Author(s):

Jie Wu ◽

Jinghua Yang ◽

Miao Yu ◽

Wenchang Sun ◽

Yarao Han ◽

...

Keyword(s):

Actin Cytoskeleton ◽

Intracellular Calcium ◽

Light Chain ◽

Myosin Light Chain Kinase ◽

Myosin Light Chain ◽

Rho Kinase ◽

Endothelial Barrier ◽

Blood Brain Barrier Disruption ◽

Intracellular Ca ◽

Brain Barrier Disruption

Lanthanum caused endothelial barrier hyperpermeability, loss of VE-cadherin and rearrangement of the actin cytoskeleton, though intracellular Ca2+-mediated RhoA/ROCK and MLCK pathways.

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