Thrombin Inactivates Myosin Light Chain Phosphatase via Rho and Its Target Rho Kinase in Human Endothelial Cells

Essler, Markus; Amano, Mutsuki; Kruse, H.J.; Kaibuchi, Kozo; Weber, Peter; Aepfelbacher, Martin

doi:10.1074/jbc.273.34.21867

Cited by 333 publications

(320 citation statements)

References 57 publications

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“…These effects were reported to be mediated through enhanced myosin phosphatase activity. Essler et al (72) confirmed these observations by showing that agents that elevate cAMP increase myosin phosphatase activity to a similar degree as occurs by direct inhibition of RhoA or suppression of Rho kinase activity with Y27632. BPAE monolayers treated to elevate cAMP then stimulated with thrombin fail to maintain maximal tension development and RLC phosphorylation.…”

Section: Discussionsupporting

confidence: 68%

“…This increase in myosin phosphatase activity correlates with the decline in basal BPAE RLC phosphorylation and isometric tension, strongly suggesting that the decline in RLC phosphorylation mediates the drop in tension. Studies have shown that agents that interfere with RhoA function block lysophosphatidic acidinduced RLC phosphorylation in fibroblasts (45,71) and prevent thrombin-induced increases in human umbilical vein endothelial cell permeability and RLC phosphorylation (72). These effects were reported to be mediated through enhanced myosin phosphatase activity.…”

Section: Discussionmentioning

confidence: 97%

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Myosin Phosphatase and Cofilin Mediate cAMP/cAMP-dependent Protein Kinase-induced Decline in Endothelial Cell Isometric Tension and Myosin II Regulatory Light Chain Phosphorylation

Goeckeler

Wysolmerski

2005

Journal of Biological Chemistry

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This study determined the effects of increased intracellular cAMP and cAMP-dependent protein kinase activation on endothelial cell basal and thrombin-induced isometric tension development. Elevation of cAMP and maximal cAMP-dependent protein kinase activation induced by 10 M forskolin, 40 M 3-isobutyl-1-methylxanthine caused a 50% reduction in myosin II regulatory light chain (RLC) phosphorylation and a 35% drop in isometric tension, but it did not inhibit thrombin-stimulated increases in RLC phosphorylation and isometric tension. Elevation of cAMP did not alter myosin light chain kinase catalytic activity. However, direct inhibition of myosin light chain kinase with KT5926 resulted in a 90% decrease in RLC phosphorylation and only a minimal decrease in isometric tension, but it prevented thrombin-induced increases in RLC phosphorylation and isometric tension development. We showed that elevated cAMP increases phosphorylation of RhoA 10-fold, and this is accompanied by a 60% decrease in RhoA activity and a 78% increase in RLC phosphatase activity. Evidence is presented that it is this inactivation of RhoA that regulates the decrease in isometric tension through a pathway involving cofilin. Activated cofilin correlates with increased F-actin severing activity in cell extracts from monolayers treated with forskolin/3-isobutyl-1-methylxanthine. Pretreatment of cultures with tautomycin, a protein phosphatase type 1 inhibitor, blocked the effect of cAMP on 1) the dephosphorylation of cofilin, 2) the decrease in RLC phosphorylation, and 3) the decrease in isometric tension. Together, these data provide in vivo evidence that elevated intracellular cAMP regulates endothelial cell isometric tension and RLC phosphorylation through inhibition of RhoA signaling and its downstream pathways that regulate myosin II activity and actin reorganization.Endothelial cells lining blood vessels form a continuous layer that constrains proteins and blood elements to the vascular lumen. Disruption of the continuous endothelial barrier leads to an increase in permeability and development of edema, a hallmark of acute and chronic inflammation. Chemical or inflammatory mediators activate signaling pathways that cause endothelial cells to contract forming gaps between adjacent cells leading to an increase in vascular permeability. Several laboratories studying methods to inhibit endothelial cell contraction and vascular permeability have demonstrated that elevation of intracellular cAMP augments barrier function and prevents increases in permeability to a wide range of inflammatory agonists. The primary pathway implicated in preventing increases in permeability is believed to occur through the activation of adenylate cyclase, accumulation of intracellular cAMP, and activation of PKA.2 Even though there are numerous reports (1-5) implicating cAMP/PKA in enhancing barrier function and inhibiting endothelial cell contraction, the mechanism by which cAMP/PKA protects vascular integrity is poorly understood.cAMP is a primary mediator of the biolo...

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Section: Discussionsupporting

confidence: 68%

Section: Discussionmentioning

confidence: 97%

Myosin Phosphatase and Cofilin Mediate cAMP/cAMP-dependent Protein Kinase-induced Decline in Endothelial Cell Isometric Tension and Myosin II Regulatory Light Chain Phosphorylation

Goeckeler

Wysolmerski

2005

Journal of Biological Chemistry

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“…Changes in cytoskeletal reorganization and activation of the RhoA pathway precede these functional responses (6,8,46,47). Hsp90 is also expressed in fibroblasts, platelets, and endothelial cells, and our preliminary experiments 2 suggest a role for Hsp90 in the regulation of PAR-1-mediated morphological changes in these cells.…”

Section: Discussionmentioning

confidence: 89%

Thrombin Receptor Signaling to Cytoskeleton Requires Hsp90

Pai

Mahajan

Lau

et al. 2001

Journal of Biological Chemistry

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Thrombin is a serine protease that evokes various cellular responses involved in injury and repair of the nervous system through the activation of protease-activated receptor-1 (PAR-1). Signals that modulate cell morphology precede most PAR-1 effects, but the initial signal transduction molecules are not known. Using the yeast two-hybrid system, we identified Hsp90, a chaperone with known signaling properties, as a binding partner of PAR-1. The interaction was confirmed by glutathione Stransferase pull-down, overlay, and co-immunoprecipitation assays. Morphological assays in mouse astrocytes were carried out to evaluate the importance of Hsp90 during cytoskeletal signaling. Reducing Hsp90 levels by antisense treatment or disruption of the Hsp90⅐PAR-1 complex by the Hsp90-specific drug geldanamycin attenuated thrombinmediated astrocyte shape changes. Furthermore, overexpression of the PAR-1 cytoplasmic tail abrogated thrombininduced cytoskeletal changes in neuronal cells. Treatment with geldanamycin specifically inhibited activation of RhoA without affecting thrombin-mediated intracellular calcium release, revealing the regulation of a distinct signaling pathway by Hsp90. Taken together, these studies demonstrate that Hsp90 may be essential for PAR-1-mediated signaling to the cytoskeleton.Thrombin is a serine protease involved in a number of pathophysiological processes that include blood clotting, inflammation, repair processes, and tumor metastasis (1-4). In brain, thrombin regulates the viability of neurons and astrocytes by increasing survival under conditions of hypoglycemia and oxidative stress and inducing apoptosis under other conditions (5-8). Thrombin is also chemotactic for macrophages and mitogenic for smooth muscle cells, fibroblasts, and astrocytes and induces secretion of growth factors and cytokines from fibroblasts and smooth muscle cells (9). Most of the thrombinmediated effects are preceded by morphological changes in cells that follow activation of a seven-transmembrane G proteincoupled receptor called protease-activated receptor-1 (PAR-1)

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“…Inflammatory mediators are known to inactivate myosinassociated phosphatases, which further promoted MLC phosphorylation (111,112). Phosphorylation of MLC phosphatase subunit (MYPT1) was increased by ROS followed by cell contraction (199).…”

Section: B Myosin Light Chain Kinasementioning

confidence: 99%

Reactive Oxygen Species in Inflammation and Tissue Injury

Mittal

Siddiqui

Tran

et al. 2014

Antioxidants & Redox Signaling

3,471

2,424

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Reactive oxygen species (ROS) are key signaling molecules that play an important role in the progression of inflammatory disorders. An enhanced ROS generation by polymorphonuclear neutrophils (PMNs) at the site of inflammation causes endothelial dysfunction and tissue injury. The vascular endothelium plays an important role in passage of macromolecules and inflammatory cells from the blood to tissue. Under the inflammatory conditions, oxidative stress produced by PMNs leads to the opening of inter-endothelial junctions and promotes the migration of inflammatory cells across the endothelial barrier. The migrated inflammatory cells not only help in the clearance of pathogens and foreign particles but also lead to tissue injury. The current review compiles the past and current research in the area of inflammation with particular emphasis on oxidative stress-mediated signaling mechanisms that are involved in inflammation and tissue injury. Antioxid. Redox Signal. 20, 1126-1167.

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Thrombin Inactivates Myosin Light Chain Phosphatase via Rho and Its Target Rho Kinase in Human Endothelial Cells

Cited by 333 publications

References 57 publications

Myosin Phosphatase and Cofilin Mediate cAMP/cAMP-dependent Protein Kinase-induced Decline in Endothelial Cell Isometric Tension and Myosin II Regulatory Light Chain Phosphorylation

Myosin Phosphatase and Cofilin Mediate cAMP/cAMP-dependent Protein Kinase-induced Decline in Endothelial Cell Isometric Tension and Myosin II Regulatory Light Chain Phosphorylation

Thrombin Receptor Signaling to Cytoskeleton Requires Hsp90

Reactive Oxygen Species in Inflammation and Tissue Injury

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