Regulation of the actin cytoskeleton by thrombin in human endothelial cells: role of Rho proteins in endothelial barrier function - PubMed (original) (raw)

Regulation of the actin cytoskeleton by thrombin in human endothelial cells: role of Rho proteins in endothelial barrier function

V Vouret-Craviari et al. Mol Biol Cell. 1998 Sep.

Free PMC article

Abstract

Endothelial barrier function is regulated at the cellular level by cytoskeletal-dependent anchoring and retracting forces. In the present study we have examined the signal transduction pathways underlying agonist-stimulated reorganization of the actin cytoskeleton in human umbilical vein endothelial cells. Receptor activation by thrombin, or the thrombin receptor (proteinase-activated receptor 1) agonist peptide, leads to an early increase in stress fiber formation followed by cortical actin accumulation and cell rounding. Selective inhibition of thrombin-stimulated signaling systems, including Gi/o (pertussis toxin sensitive), p42/p44, and p38 MAP kinase cascades, Src family kinases, PI-3 kinase, or S6 kinase pathways had no effect on the thrombin response. In contrast, staurosporine and KT5926, an inhibitor of myosin light chain kinase, effectively blocked thrombin-induced cell rounding and retraction. The contribution of Rho to these effects was analyzed by using bacterial toxins that either activate or inhibit the GTPase. Escherichia coli cytotoxic necrotizing factor 1, an activator of Rho, induced the appearance of dense actin cables across cells without perturbing monolayer integrity. Accordingly, lysophosphatidic acid, an activator of Rho-dependent stress fiber formation in fibroblasts, led to reorganization of polymerized actin into stress fibers but failed to induce cell rounding. Inhibition of Rho with Clostridium botulinum exoenzyme C3 fused to the B fragment of diphtheria toxin caused loss of stress fibers with only partial attenuation of thrombin-induced cell rounding. The implication of Rac and Cdc42 was analyzed in transient transfection experiments using either constitutively active (V12) or dominant-interfering (N17) mutants. Expression of RacV12 mimicked the effect of thrombin on cell rounding, and RacN17 blocked the response to thrombin, whereas Cdc42 mutants were without effect. These observations suggest that Rho is involved in the maintenance of endothelial barrier function and Rac participates in cytoskeletal remodeling by thrombin in human umbilical vein endothelial cells.

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Figures

Figure 1

Thrombin receptor (PAR-1) activation induces cytoskeletal reorganization in human endothelial cell monolayers. Primary cultured HUVECs (left panel) or the HUVEC-derived cell line EA.hy926 (right panel) were grown to confluence and treated with medium alone (A and D), 10 nM thrombin (B and E), or 100 μM TRP (C and F) for 15 min at 37°C. Staining of the actin cytoskeleton was performed as described in MATERIALS AND METHODS. Arrowheads indicate the formation of a ring of peripheral actin, whereas arrows indicate the blebs that are present in some round cells. Photographs (magnification, 1000×) are representative of results from at least four independent experiments.

Figure 2

Thrombin-induced rounding of endothelial cells is preceded by stress fiber formation. Confluent EA.hy926 monolayers were stained with FITC-phalloidin after treatment with medium alone (A), 10 nM thrombin for 2 min (B) or 10 nM thrombin for 15 min (C). Photographs (magnification, 1000×) are representative of three different experiments. Arrow indicates cell surface blebbing.

Figure 3

Thrombin-induced rounding of endothelial cells is linked to an increase in monolayer permeability. (A) Medium alone (a), 100 nM PdBu (b), 1 nM PAF (c), 100 μM PAR-2 agonist peptide (d), 10−4 M histamine (e), or 20 ng/ml LPA (f) was added to confluent monolayers of EA.hy926 cells for 15 min (or 5 min in the case of PAF), and the actin cytoskeleton was visualized as described in MATERIALS AND METHODS. Arrows indicate membrane ruffling (b and c), and stress fibers (d–f). Photographs (magnification, 1000×) are representative of results from three independent experiments. (B) 10 nM thrombin, 100 μM TRP, 100 nM PdBu, 1 nM PAF, 100 μM PAR2, 10−4 M histamine, and 20 ng/ml LPA was added to confluent EA.hy926 cells grown on transwell filters, and the increase of transmonolayer permeability was determined after 15 min, as described in MATERIALS AND METHODS. Mean values ± SE obtained from four separate filters are represented.

Figure 4

PKC inhibitor GF 109203X has no effect on thrombin-induced endothelial cell rounding. Confluent monolayers of EA.hy926 cells were pretreated for 30 min with the PKC inhibitor GF 109203X (D–F), and then medium (A and D), 10 nM thrombin (B and E), or 100 nM PdBu (C and F) was added to the cells for 15 min. F-actin staining was then performed as described in MATERIALS AND METHODS. Representative results from two independent experiments are shown (magnification, 400×).

Figure 5

Staurosporine and KT5926 pretreatment prevent the rounding of endothelial cells. Confluent EA.hy926 cells were pretreated for 30 min with 50 nM staurosporine (B and E), 1 μM KT5926 (C and F), or DMSO alone (A) before the addition of 10 nM thrombin (D–F) for an additional 15 min. Staining of the actin cytoskeleton was then performed as described in MATERIALS AND METHODS. Photographs (magnification, 1000×) are representative of four independent experiments.

Figure 6

Implication of Rho in control of the endothelial cell actin cytoskeleton. Confluent HUVEC monolayers were pretreated with 10−9 M CNF1 for 16 h (A) or 10−7 M DC3B for 50 h (D and E). Thrombin (10 nM) was added during the last 15 min of the experiment in C and E. The actin cytoskeleton was stained with FITC-phalloidin as described in MATERIALS AND METHODS. Photos (magnification, 1000×) are representative of at least four independent experiments. Arrows indicate gap formation between bordering cells.

Figure 7

Implication of Rac and Cdc42 in thrombin-induced cell rounding. (A) EA.hy926 cells were transfected with plasmids encoding green fluorescent protein (GFP) or myc-tagged GTPase mutants, as described in MATERIALS AND METHODS. The morphology of transfected cells, treated or not for 15 min with 10 nM thrombin, was observed, and 100 positive cells were scored (flat or round) per transfection condition. Data represent the mean of two independent experiments. (B) EA.hy926 cells were transfected with pEXVRac1V12 (a and c) or pEXVRac1N17 (b and d). Thrombin was added (10 nM) to cells shown in B and D 15 min before staining. Expression of Rac mutants was detected using anti-Myc antibody, and F-actin was stained with FITC-phalloidin.

Figure 7

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Regulation of the actin cytoskeleton by thrombin in human endothelial cells: role of Rho proteins in endothelial barrier function - PubMed (original) (raw)