scholarly journals Leukotriene D4 induces stress-fibre formation in intestinal epithelial cells via activation of RhoA and PKCδ

2002 ◽  
Vol 115 (17) ◽  
pp. 3509-3515 ◽  
Author(s):  
Ramin Massoumi ◽  
Christer Larsson ◽  
Anita Sjölander
Keyword(s):  
Intestinal Epithelial Cells ◽  
Dominant Negative ◽  
Intestinal Epithelial ◽  
Regulatory Domain ◽  
Leukotriene D4 ◽  
Stress Fibre ◽  
Fibre Formation ◽  
Induced Stress ◽  
C3 Exoenzyme ◽  
Stress Fibre Formation

The intestinal epithelial barrier, which is regulated by the actin cytoskeleton, exhibits permeability changes during inflammation. Here we show that activation of the CysLT1 receptor by the inflammatory mediator leukotriene D4 (LTD4) causes a rapid increase in stress-fibre formation in intestinal epithelial cells. This effect was mimicked by cytotoxic necrotising factor-1 (CNF-1)-induced activation of RhoA,overexpression of constitutively active RhoA (L63-RhoA) and phorbol-ester-induced activation of protein kinase C (PKC). In accordance,inhibition of RhoA, by C3 exoenzyme or by dominant-negative RhoA (N19-RhoA),as well as GF109203X-induced inhibition of PKC, suppressed the LTD4-induced stress-fibre formation. Introduction of the dominant-negative regulatory domain of PKCδ, but not the corresponding structures from PKCα, βII or ϵ, blocked the LTD4-induced stress-fibre formation. Evaluating the relationship between PKCδ and RhoA in LTD4-induced stress-fibre formation,we found that C3 exoenzyme inhibited the rapid LTD4-elicited translocation of PKCδ to the plasma membrane. Furthermore, CNF-1-induced stress-fibre formation was blocked by GF109203X and by overexpression of the regulatory domain of PKC-δ, whereas PKC-induced stress-fibre production was not affected by N19-RhoA. We conclude that PKC-δ is located downstream of RhoA and that active RhoA and PKCδ are both necessary for LTD4-induced stress-fibre formation.

Leukotriene D4 activates MAPK through a Ras-independent but PKCϵ-dependent pathway in intestinal epithelial cells

2002 ◽  
Vol 115 (9) ◽  
pp. 1883-1893
Author(s):  
Sailaja Paruchuri ◽  
Bengt Hallberg ◽  
Maria Juhas ◽  
Christer Larsson ◽  
Anita Sjölander
Keyword(s):  
Protein Kinase ◽  
Epithelial Cells ◽  
Proliferative Response ◽  
Egf Receptor ◽  
Intestinal Epithelial Cells ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Intestinal Epithelial ◽  
Leukotriene D4 ◽  
Mitogen Activated Protein

We have recently shown that leukotriene D4 (LTD4)increases cell survival in intestinal epithelial cells. Here we report and explore the complementary finding that LTD4 also enhances proliferation in these cells. This proliferative response was approximately half of that induced by epidermal growth factor (EGF) and its required activation of protein kinase C (PKC), Ras and the mitogen-activated protein kinase (MAPK) Erk-1/2. EGF also activated Erk-1/2 in these cells; however the EGF-receptor inhibitor PD153035 did not affect the LTD4-induced activation of Erk-1/2. In addition, LTD4 did not induce phosphorylation of the EGF receptor, nor did pertussis toxin (PTX) block EGF-induced activation of Erk-1/2, thus refuting a possible crosstalk between the receptors. Furthermore, LTD4-induced, but not EGF-induced,activation of Erk-1/2 was sensitive to PTX, PKC inhibitors and downregulation of PKCϵ. A definite role for PKCϵ in LTD4-induced stimulation of Erk-1/2 was documented by the inability of LTD4 to activate Erk-1/2 in cells transfected with either the regulatory domain of PKCϵ (an isoform specific dominant-negative inhibitor) or a kinase-dead PKCϵ. Although Ras and Raf-1 were both transiently activated by LTD4, only Raf-1 activation was abolished by abrogation of the PKC signal. Furthermore, the LTD4-induced activation of Erk-1/2 was unaffected by transfection with dominant-negative N17 Ras but blocked by transfection with kinase-dead Raf-1. Consequently, LTD4 regulates the proliferative response by a distinct Ras-independent, PKCϵ-dependent activation of Erk-1/2 and a parallel Ras-dependent signaling pathway.

9Rgr; and Rac but not Cdc42 regulate endothelial cell permeability

2001 ◽  
Vol 114 (7) ◽  
pp. 1343-1355 ◽  
Author(s):  
B. Wojciak-Stothard ◽  
S. Potempa ◽  
T. Eichholtz ◽  
A.J. Ridley
Keyword(s):  
Barrier Function ◽  
Endothelial Permeability ◽  
Dominant Negative ◽  
Cell Permeability ◽  
Endothelial Barrier ◽  
Endothelial Barrier Function ◽  
Stress Fibre ◽  
Fibre Formation ◽  
Endothelial Cell Permeability ◽  
Stress Fibre Formation

Endothelial permeability induced by thrombin and histamine is accompanied by actin stress fibre assembly and intercellular gap formation. Here, we investigate the roles of the Ρ family GTPases Rho1, Rac1 and Cdc42 in regulating endothelial barrier function, and correlate this with their effects on F-actin organization and intercellular junctions. RhoA, Rac1 and Cdc42 proteins were expressed efficiently in human umbilical vein endothelial cells by adenovirus-mediated gene transfer. We show that inhibition of Ρ prevents both thrombin- and histamine-induced increases in endothelial permeability and decreases in transendothelial resistance. Dominant-negative RhoA and a Ρ kinase inhibitor, Y-27632, not only inhibit stress fibre assembly and contractility but also prevent thrombin- and histamine-induced disassembly of adherens and tight junctions in endothelial cells, providing an explanation for their effects on permeability. In contrast, dominant-negative Rac1 induces permeability in unstimulated cells and enhances thrombin-induced permeability, yet inhibits stress fibre assembly, indicating that increased stress fibre formation is not essential for endothelial permeability. Dominant-negative Cdc42 reduces thrombin-induced stress fibre formation and contractility but does not affect endothelial cell permeability or responses to histamine. These results demonstrate that Ρ and Rac act in different ways to alter endothelial barrier function, whereas Cdc42 does not affect barrier function.

scholarly journals Novel role for the Lu/BCAM–spectrin interaction in actin cytoskeleton reorganization

2011 ◽  
Vol 436 (3) ◽  
pp. 699-708 ◽  
Author(s):  
Emmanuel Collec ◽  
Marie-Christine Lecomte ◽  
Wassim El Nemer ◽  
Yves Colin ◽  
Caroline Le Van Kim
Keyword(s):  
Cell Adhesion ◽  
Membrane Skeleton ◽  
Actin Dynamics ◽  
Actin Reorganization ◽  
Stress Fibre ◽  
Fibre Formation ◽  
Cytoskeleton Reorganization ◽  
Main Component ◽  
Darby Canine Kidney ◽  
Stress Fibre Formation

Lu/BCAM (Lutheran/basal cell-adhesion molecule) is a laminin 511/521 receptor expressed in erythroid and endothelial cells, and in epithelial tissues. The RK573–574 (Arg573-Lys574) motif of the Lu/BCAM cytoplasmic domain interacts with αI-spectrin, the main component of the membrane skeleton in red blood cells. In the present paper we report that Lu/BCAM binds to the non-erythroid αII-spectrin via the RK573–574 motif. Alanine substitution of this motif abolished the Lu/BCAM–spectrin interaction, enhanced the half-life of Lu/BCAM at the MDCK (Madin–Darby canine kidney) cell surface, and increased Lu/BCAM-mediated cell adhesion and spreading on laminin 511/521. We have shown that the Lu/BCAM–spectrin interaction mediated actin reorganization during cell adhesion and spreading on laminin 511/521. This interaction was involved in a laminin 511/521-to-actin signalling pathway leading to stress fibre formation. This skeletal rearrangement was associated with an activation of the small GTP-binding protein RhoA, which depended on the integrity of the Lu/BCAM laminin 511/521-binding site. It also required a Lu/BCAM–αII-spectrin interaction, since its disruption decreased stress fibre formation and RhoA activation. We conclude that the Lu/BCAM–spectrin interaction is required for stress fibre formation during cell spreading on laminin 511/521, and that spectrin acts as a signal relay between laminin 511/521 and actin that is involved in actin dynamics.

scholarly journals Enteropathogenic Escherichia coli Activates Ezrin, Which Participates in Disruption of Tight Junction Barrier Function

2001 ◽  
Vol 69 (9) ◽  
pp. 5679-5688 ◽  
Author(s):  
Ivana Simonovic ◽  
Monique Arpin ◽  
Athanasia Koutsouris ◽  
Holly J. Falk-Krzesinski ◽  
Gail Hecht
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Cytoskeletal Proteins ◽  
Dominant Negative ◽  
Intestinal Epithelial ◽  
E Coli ◽  
Membrane Cytoskeleton ◽  
Intestinal Pathogen ◽  
Response Expression

ABSTRACT Enteropathogenic Escherichia coli (EPEC) is an important human intestinal pathogen, especially in infants. EPEC adherence to intestinal epithelial cells induces the accumulation of a number of cytoskeletal proteins beneath the bacteria, including the membrane-cytoskeleton linker ezrin. Evidence suggests that ezrin can participate in signal transduction. The aim of this study was to determine whether ezrin is activated following EPEC infection and if it is involved in the cross talk with host intestinal epithelial cells. We show here that following EPEC attachment to intestinal epithelial cells there was significant phosphorylation of ezrin, first on threonine and later on tyrosine residues. A significant increase in cytoskeleton-associated ezrin occurred following phosphorylation, suggesting activation of this molecule. Nonpathogenic E. coli and EPEC strains harboring mutations in type III secretion failed to elicit this response. Expression of dominant-negative ezrin significantly decreased the EPEC-elicited association of ezrin with the cytoskeleton and attenuated the disruption of intestinal epithelial tight junctions. These results suggest that ezrin is involved in transducing EPEC-initiated signals that ultimately affect host physiological functions.

scholarly journals Sphingosine 1-phosphate stimulates Rho-mediated tyrosine phosphorylation of focal adhesion kinase and paxillin in Swiss 3T3 fibroblasts

1997 ◽  
Vol 324 (2) ◽  
pp. 481-488 ◽  
Author(s):  
Fang WANG ◽  
Catherine D. NOBES ◽  
Alan HALL ◽  
Sarah SPIEGEL
Keyword(s):  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Stress Fibre ◽  
3T3 Fibroblasts ◽  
Fibre Formation ◽  
Sphingosine 1 Phosphate ◽  
Swiss 3T3 Fibroblasts ◽  
Stress Fibre Formation ◽  
Stimulation Of

Sphingosine 1-phosphate (SPP), a sphingolipid second messenger implicated in the mitogenic action of platelet-derived growth factor [Olivera, A. and Spiegel, S. (1993) Nature (London) 365, 557–560], induced rapid reorganization of the actin cytoskeleton resulting in stress-fibre formation. SPP also induced transient tyrosine phosphorylation of focal adhesion kinase (p125FAK), a cytosolic tyrosine kinase that localizes in focal adhesions, and of the cytoskeleton-associated protein paxillin. Exoenzyme C3 transferase, which ADP-ribosylates Rho (a Ras-related small GTP binding protein) on asparagine-41 and renders it biologically inactive, inhibited both stress-fibre formation and protein tyrosine phosphorylation induced by SPP. Thus Rho may be an upstream regulator of both stress-fibre formation and tyrosine phosphorylation of p125FAK and paxillin. Pretreatment with PMA, an activator of protein kinase C (PKC), inhibited the stimulation of stress-fibre formation induced by 1-oleoyl-lysophosphatidic acid (LPA) but not that by SPP. Similarly, PMA also decreased LPA-induced tyrosine phosphorylation of p125FAK and paxillin without abrogating the response to SPP. Thus PKC is involved in LPA- but not SPP-dependent signalling. The polyanionic drug suramin, a broad-specificity inhibitor of ligand–receptor interactions, did not inhibit either the mitogenic effect of SPP or its stimulation of tyrosine phosphorylation of p125FAK. However, suramin markedly inhibited these responses induced by LPA. These results suggest that in contrast with LPA, SPP may be acting intracellularly in Swiss 3T3 fibroblasts to stimulate tyrosine phosphorylation of p125FAK and paxillin and cell growth.

Expression of BRG1, a human SWI/SNF component, affects the organisation of actin filaments through the RhoA signalling pathway

2002 ◽  
Vol 115 (13) ◽  
pp. 2735-2746 ◽  
Author(s):  
Patrik Asp ◽  
Margareta Wihlborg ◽  
Mattias Karlén ◽  
Ann-Kristin Östlund Farrants
Keyword(s):  
Protein Level ◽  
Actin Filaments ◽  
Rho Kinase ◽  
Stress Fibre ◽  
Fibre Formation ◽  
Downstream Effectors ◽  
Rhoa Gtpase ◽  
Filament Bundles ◽  
Activity State ◽  
Stress Fibre Formation

The human BRG1 (brahma-related gene 1) protein is a component of the SWI/SNF family of the ATP-dependent chromatin remodelling complexes. We show here that expression of the BRG1 protein, but not of an ATPase-deficient BRG1 protein, in BRG1-deficient SW13 cells alters the organisation of actin filaments. BRG1 expression induces the formation of thick actin filament bundles resembling stress-fibres, structures that are rarely seen in native SW13 cells. BRG1 expression does not influence the activity state of the RhoA-GTPase, which is involved in stress-fibre formation. We find that RhoA is equally activated by stimuli, such as serum, in BRG1-expressing cells,ATPase-deficient BRG1-expressing cells and native SW13 cells. However, the activation of RhoA by lysophosphatidic acid and serum does not trigger the formation of stress-fibre-like structures in SW13 cells. Activation of the RhoA-GTPase in BRG1-expressing cells induces stress-fibre-like structures,indicating that the BRG1 can couple RhoA activation to stress-fibre formation. At least two downstream effectors are involved in stress-fibre formation,Rho-kinase/ROCK and Dia. BRG1 expression, but not the expression of the ATP-deficient BRG1, increases the protein level of ROCK1, one form of the Rho-kinase/ROCK. That this is of importance is supported by the findings that an increased Rho-kinase/ROCK activity in SW13 cells, obtained by overexpressing wild-type ROCK1 and ROCK2, induces stress-fibre formation. No specificity between the two Rho-kinase/ROCK forms exists. Our results suggest that the BRG1 protein affects the RhoA pathway by increasing the protein level of ROCK1, which allows stress-fibre-like structures to form.

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