Abstract WP438: Rho Kinase-mediated Di-phosphorylation of Myosin Light Chain in the Sub-membranous Regions and Circumferential Actomyosin Contraction Mediate Thrombin-induced Barrier Disruption in Vascular Endothelial Cells

Stroke ◽  
2013 ◽  
Vol 44 (suppl_1) ◽  
Author(s):  
Mayumi Hirano ◽  
Katsuya Hirano
Keyword(s):  
Endothelial Cells ◽  
Myosin Light Chain ◽  
Critical Role ◽  
Rho Kinase ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Cell Contact ◽  
Barrier Disruption ◽  
Stress Fiber Formation ◽  
Cell Cell

The disruption of blood-brain barrier plays a critical role in the pathophysiology of cerebrovascular diseases. Thrombin is one of the major factors which cause barrier disruption. The phosphorylation of myosin light chain (MLC) is a key signal of barrier disruption. MLC is thought to be di-phosphorylated sequentially at Ser19 and then Thr18, thereby inducing stress fiber formation to generate traction force to disrupt cell-cell contact. However, it is unclear how the phosphorylation at two sites contributes to barrier disruption. The present study investigated the role of mono- and di-phosphorylation of MLC (MLC-P and MLC-PP) in thrombin-induced barrier disruption. Thrombin (1 u/mL) decreased the transendothelial electrical resistance (TEER) with a peak at 3-5 min in porcine aortic endothelial cells (PAEC). A Phos-tag SDS-PAGE method was used to quantify the amount of MLC-P and MLC-PP. PAEC at confluence contained 25% MLC-P and 2% MLC-PP before stimulation. Upon thrombin stimulation, MLC-P marginally increased, while MLC-PP transiently increased to a peak of 35% at 3-5 min. MLC-P was localized mainly in the peri-nuclear region, while MLC-PP was localized mainly in the sub-membranous region of cell-cell contact. MLC-PP was also co-localized with the peripheral actin bundles. In contrast, thrombin induced stress fiber formation and localization of MLC-P and MLC-PP on the stress fibers when the cell-cell contact was loosed by removing extracellular Ca 2+ or in the cells at the growing phase with sparse cell-cell contact. Two different Rho kinase inhibitors, Y27632 and H1152, inhibited the thrombin-induced increase in MLC-PP, sub-membranous localization of MLC-PP and decrease in TEER, while having no effect on the level of MLC-P. Inhibition of myosin ATPase activity by 100 μmol/L blebbistatin inhibited the thrombin-induced decrease in TEER. The present study suggests that MLC-P and MLC-PP are independently regulated in endothelial cells, and that Rho kinase-mediated MLC-PP in the sub-membranous regions and the circumferential, but not radial, contraction plays a critical role in the thrombin-induced barrier disruption. Inhibition of MLC-PP thus provides a crucial strategy for restoring normal function of the blood-brain barrier.

GEF-H1 is involved in agonist-induced human pulmonary endothelial barrier dysfunction

2006 ◽  
Vol 290 (3) ◽  
pp. L540-L548 ◽  
Author(s):  
Anna A. Birukova ◽  
Djanybek Adyshev ◽  
Boris Gorshkov ◽  
Gary M. Bokoch ◽  
Konstantin G. Birukov ◽  
...  
Keyword(s):  
Critical Role ◽  
Focal Adhesions ◽  
Stress Fiber ◽  
Small Gtpase ◽  
Fiber Formation ◽  
Cell Contact ◽  
Actin Stress Fiber ◽  
Barrier Dysfunction ◽  
Actin Remodeling ◽  
Stress Fiber Formation

Endothelial cell (EC) permeability is precisely controlled by cytoskeletal elements [actin filaments, microtubules (MT), intermediate filaments] and cell contact protein complexes (focal adhesions, adherens junctions, tight junctions). We have recently shown that the edemagenic agonist thrombin caused partial MT disassembly, which was linked to activation of small GTPase Rho, Rho-mediated actin remodeling, cell contraction, and dysfunction of lung EC barrier. GEF-H1 is an MT-associated Rho-specific guanosine nucleotide (GDP/GTP) exchange factor, which in MT-unbound state stimulates Rho activity. In this study we tested hypothesis that GEF-H1 may be a key molecule involved in Rho activation, myosin light chain phosphorylation, actin remodeling, and EC barrier dysfunction associated with partial MT disassembly. Our results show that depletion of GEF-H1 or expression of dominant negative GEF-H1 mutant significantly attenuated permeability increase, actin stress fiber formation, and increased MLC and MYPT1 phosphorylation induced by thrombin or MT-depolymerizing agent nocodazole. In contrast, expression of wild-type or activated GEF-H1 mutants dramatically enhanced thrombin and nocodazole effects on stress fiber formation and cell retraction. These results show a critical role for the GEF-H1 in the Rho activation caused by MT disassembly and suggest GEF-H1 as a key molecule involved in cross talk between MT and actin cytoskeleton in agonist-induced Rho-dependent EC barrier regulation.

scholarly journals Delayed stress fiber formation mediates pulmonary myofibroblast differentiation in response to TGF-β

2011 ◽  
Vol 301 (5) ◽  
pp. L656-L666 ◽  
Author(s):  
Nathan Sandbo ◽  
Andrew Lau ◽  
Jacob Kach ◽  
Caitlyn Ngam ◽  
Douglas Yau ◽  
...  
Keyword(s):  
Rho Kinase ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Stress Fibers ◽  
Nuclear Accumulation ◽  
Myofibroblast Differentiation ◽  
Latrunculin B ◽  
Stress Fiber Formation ◽  
Actin Expression ◽  
Actin Stress Fibers

Myofibroblast differentiation induced by transforming growth factor-β (TGF-β) and characterized by de novo expression of smooth muscle (SM)-specific proteins is a key process in wound healing and in the pathogenesis of fibrosis. We have previously shown that TGF-β-induced expression and activation of serum response factor (SRF) is required for this process. In this study, we examined the signaling mechanism for SRF activation by TGF-β as it relates to pulmonary myofibroblast differentiation. TGF-β stimulated a profound, but delayed (18–24 h), activation of Rho kinase and formation of actin stress fibers, which paralleled SM α-actin expression. The translational inhibitor cycloheximide blocked these processes without affecting Smad-dependent gene transcription. Inhibition of Rho kinase by Y-27632 or depolymerization of actin by latrunculin B resulted in inhibition TGF-β-induced SRF activation and SM α-actin expression, having no effect on Smad signaling. Conversely, stabilization of actin stress fibers by jasplakinolide was sufficient to drive these processes in the absence of TGF-β. TGF-β promoted a delayed nuclear accumulation of the SRF coactivator megakaryoblastic leukemia-1 (MKL1)/myocardin-related transcription factor-A, which was inhibited by latrunculin B. Furthermore, TGF-β also induced MKL1 expression, which was inhibited by latrunculin B, by SRF inhibitor CCG-1423, or by SRF knockdown. Together, these data suggest a triphasic model for myofibroblast differentiation in response to TGF-β that involves 1) initial Smad-dependent expression of intermediate signaling molecules driving Rho activation and stress fiber formation, 2) nuclear accumulation of MKL1 and activation of SRF as a result of actin polymerization, and 3) SRF-dependent expression of MKL1, driving further myofibroblast differentiation.

Endothelial cytoskeletal reorganization in response to PAR1 stimulation is mediated by membrane rafts but not caveolae

2007 ◽  
Vol 293 (1) ◽  
pp. H366-H375 ◽  
Author(s):  
MaryEllen Carlile-Klusacek ◽  
Victor Rizzo
Keyword(s):  
Endothelial Cells ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Signaling Cascade ◽  
Membrane Rafts ◽  
Actin Stress Fiber ◽  
Caveolin 1 ◽  
Stress Fiber Formation ◽  
Mlc Phosphorylation ◽  
Actin Stress Fiber Formation

The vasoactive protease thrombin is a known activator of the protease-activated receptor-1 (PAR1) via cleavage of its NH2 terminus. PAR1 activation stimulates the RhoA/Rho kinase signaling cascade, leading to myosin light chain (MLC) phosphorylation, actin stress fiber formation, and changes in endothelial monolayer integrity. Previous studies suggest that some elements of this signaling pathway are localized to caveolin-containing cholesterol-rich membrane domains. Here we show that PAR1 and key components of the PAR-associated signaling cascade localize to membrane rafts and caveolae in bovine aortic endothelial cells (BAEC). To investigate the functional significance of this localization, BAEC were pretreated with filipin (5 μg/ml, 5 min) to ablate lipid rafts before thrombin (100 nM) or PAR agonist stimulation. We found that diphosphorylation of MLC and the actin stress fiber formation normally induced by PAR activation were attenuated after lipid raft disruption. To target caveolae specifically, we used a small interferring RNA approach to knockdown caveolin-1 expression. Thrombin-induced MLC phosphorylation and stress fiber formation were not altered in caveolin-1-depleted cells, suggesting that lipid rafts, but not necessarily caveolae, modulate thrombin-activated signaling pathways leading to alteration of the actin cytoskeleton in endothelial cells.

Regulation of the RhoA pathway in human endothelial cell spreading on type IV collagen: role of calcium influx

1999 ◽  
Vol 112 (19) ◽  
pp. 3205-3213 ◽  
Author(s):  
L. Masiero ◽  
K.A. Lapidos ◽  
I. Ambudkar ◽  
E.C. Kohn
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Focal Adhesion ◽  
Type Iv Collagen ◽  
Cell Spreading ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Stress Fibers ◽  
Type Iv ◽  
Stress Fiber Formation

We have shown that nonvoltage-operated Ca(2+) entry regulates human umbilical vein endothelial cell adhesion, migration, and proliferation on type IV collagen. We now demonstrate a requirement for Ca(2+) influx for activation of the RhoA pathway during endothelial cell spreading on type IV collagen. Reorganization of actin into stress fibers was complete when the cells where fully spread at 90 minutes. No actin organization into stress fibers was seen in endothelial cells plated on type I collagen, indicating a permissive effect of type IV collagen. CAI, a blocker of nonvoltage-operated Ca(2+) channels, prevented development of stress fiber formation in endothelial cells on type IV collagen. This permissive effect was augmented by Ca(2+) influx, as stimulated by 0. 5 microM thapsigargin or 0.1 microM ionomycin, yielding faster development of actin stress fibers. Ca(2+) influx and actin rearrangement in response to thapsigargin and ionomycin were abrogated by CAI. Activated, membrane-bound RhoA is a substrate for C3 exoenzyme which ADP-ribosylates and inactivates RhoA, preventing actin stress fiber formation. Pretreatment of endothelial cells with C3 exoenzyme prevented basal and thapsigargin-augmented stress fiber formation. While regulation of Ca(2+) influx did not alter RhoA translocation, it reduced in vitro ADP-ribosylation of RhoA (P(2)<0. 05), suggesting Ca(2+) influx is needed for RhoA activation during spreading on type IV collagen; no Ca(2+) regulated change in RhoA was seen in HUVECs spreading on type I collagen matrix. Blockade of Ca(2+) influx of HUVEC spread on type IV collagen also reduced tyrosine phosphorylation of p190Rho-GAP and blocked thapsigargin-enhanced binding of p190Rho-GAP to focal adhesion kinase. Thus, Ca(2+) influx is necessary for RhoA activation and for linkage of the RhoA/stress fiber cascade to the focal adhesion/focal adhesion kinase pathway during human umbilical vein endothelial cell spreading on type IV collagen.

scholarly journals Activation of ROCK and MLCK tunes regional stress fiber formation and mechanics via preferential myosin light chain phosphorylation

2017 ◽  
Vol 28 (26) ◽  
pp. 3832-3843 ◽  
Author(s):  
Elena Kassianidou ◽  
Jasmine H. Hughes ◽  
Sanjay Kumar
Keyword(s):  
Light Chain ◽  
Viscoelastic Properties ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Kinase Activity ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Myosin Regulatory Light Chain ◽  
Regional Stress ◽  
Stress Fiber Formation

The assembly and mechanics of actomyosin stress fibers (SFs) depend on myosin regulatory light chain (RLC) phosphorylation, which is driven by myosin light chain kinase (MLCK) and Rho-associated kinase (ROCK). Although previous work suggests that MLCK and ROCK control distinct pools of cellular SFs, it remains unclear how these kinases differ in their regulation of RLC phosphorylation or how phosphorylation influences individual SF mechanics. Here, we combine genetic approaches with biophysical tools to explore relationships between kinase activity, RLC phosphorylation, SF localization, and SF mechanics. We show that graded MLCK overexpression increases RLC monophosphorylation (p-RLC) in a graded manner and that this p-RLC localizes to peripheral SFs. Conversely, graded ROCK overexpression preferentially increases RLC diphosphorylation (pp-RLC), with pp-RLC localizing to central SFs. Interrogation of single SFs with subcellular laser ablation reveals that MLCK and ROCK quantitatively regulate the viscoelastic properties of peripheral and central SFs, respectively. The effects of MLCK and ROCK on single-SF mechanics may be correspondingly phenocopied by overexpression of mono- and diphosphomimetic RLC mutants. Our results point to a model in which MLCK and ROCK regulate peripheral and central SF viscoelastic properties through mono- and diphosphorylation of RLC, offering new quantitative connections between kinase activity, RLC phosphorylation, and SF viscoelasticity.

Tumor necrosis factor-α-induced TRPC1 expression amplifies store-operated Ca2+ influx and endothelial permeability

2004 ◽  
Vol 287 (6) ◽  
pp. L1303-L1313 ◽  
Author(s):  
Biman C. Paria ◽  
Stephen M. Vogel ◽  
Gias U. Ahmmed ◽  
Setara Alamgir ◽  
Jennifer Shroff ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Transient Receptor Potential ◽  
Endothelial Permeability ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Actin Stress Fiber ◽  
Cationic Current ◽  
Stress Fiber Formation ◽  
Tnf Α ◽  
Actin Stress Fiber Formation

We determined the effects of TNF-α on the expression of transient receptor potential channel (TRPC) homologues in human vascular endothelial cells and the consequences of TRPC expression on the endothelial permeability response. We observed that TNF-α exposure increased TRPC1 expression without significantly altering expression of other TRPC isoforms in human pulmonary artery endothelial cells (HPAEC). Because TRPC1 belongs to the store-operated cation channel family, we measured the Ca2+ store depletion-mediated Ca2+ influx in response to thrombin exposure. We observed that thrombin-induced Ca2+ influx in TNF-α-stimulated HPAEC was twofold greater than in control cells. To address the relationship between store-operated Ca2+ influx and TRPC1 expression, we overexpressed TRPC1 by three- to fourfold in the human dermal microvascular endothelial cell line (HMEC) using the TRPC1 cDNA. Thrombin-induced store Ca2+ depletion in these cells caused approximately twofold greater increase in Ca2+ influx than in control cells. Furthermore, the inositol 1,4,5-trisphosphate-sensitive store-operated cationic current was increased greater than twofold in TRPC1-transfected cells compared with control. To address the role of Ca2+ influx via TRPC1 in signaling endothelial permeability, we measured actin-stress fiber formation and transendothelial monolayer electrical resistance (TER) in the TRPC1 cDNA-transfected HMEC and TNF-α-challenged HPAEC. Both thrombin-induced actin-stress fiber formation and a decrease in TER were augmented in TRPC1-overexpressing HMEC compared with control cells. TNF-α-induced increased TRPC1 expression in HPAEC also resulted in marked endothelial barrier dysfunction in response to thrombin. These findings indicate the expression level of TRPC1 in endothelial cells is a critical determinant of Ca2+ influx and signaling of the increase in endothelial permeability.

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