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Regulation of VE-Cadherin Linkage to the Cytoskeleton in Endothelial Cells Exposed to Fluid Shear Stress
Experimental Cell Research 273, 240 –247 (2002) doi:10.1006/excr.2001.5453, available online at http://www.idealibrary.com on
Regulation of VE-Cadherin Linkage to the Cytoskeleton in Endothelial Cells Exposed to Fluid Shear Stress Jon A. Ukropec, M. Katherine Hollinger, and Marilyn J. Woolkalis 1 Department of Physiology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
Endothelial cells exposed to shear stress realigned and elongated in the direction of flow through the coordinated remodeling of their adherens junctions and actin cytoskeleton. The elaborate networks of VEcadherin complexes in static cultures became more uniform and compact in response to shear. In contrast, the cortical actin present in static cultures was reorganized into numerous stress fiber bundles distributed parallel to the direction of flow. Exposure to shear did not significantly alter the expression of the junctional proteins VE-cadherin, -catenin, and ␣-catenin, but the composition of the junctional complexes did change. We detected a marked decrease in the ␣-catenin associated with VE-cadherin complexes in endothelial monolayers subjected to shear. This loss of ␣-catenin, the protein that links -catenin-bound cadherin to the actin cytoskeleton, was not due to decreased quantities of -catenin associated with VEcadherin. Instead, the loss of ␣-catenin from the junctional complexes coincided with the increased tyrosine phosphorylation of -catenin associated with VE-cadherin. The change in -catenin phosphorylation closely correlated with the shear-induced loss of the protein tyrosine phosphatase SHP-2 from VE-cadherin complexes. Thus, the functional interaction of ␣-catenin with VE-cadherin-bound -catenin is regulated by the extent of tyrosine phosphorylation of -catenin. This, concomitantly, is regulated by SHP-2 associated with VE-cadherin complexes. © 2002 Elsevier Science (USA)
Key Words: VE-cadherin; -catenin; ␣-catenin; SHP-2; shear stress; tyrosine phosphorylation.
INTRODUCTION
Blood flowing through the vasculature is in direct contact with the endothelium, generating a frictional force or shear stress parallel to the apical surface of the endothelium. In nonpathological states, endothelial 1 To whom correspondence and reprint requests should be addressed at Department of Physiology, Thomas Jefferson University, 411 Jefferson Alumni Hall, 1020 Locust Street, Philadelphia, PA 19107. Fax: (215) 503-2073. E-mail: [email protected].
0014-4827/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.
cells align the major axis of their cell bodies parallel to the direction of laminar flow [1]. The in situ organization of endothelial cell filamentous actin ranges from cortical bands to stress fibers depending upon the direction and magnitude of the fluid dynamic forces [2, 3]. When confluent, quiescent cultured endothelial cells are exposed to shearing forces, the cortical actin is rapidly restructured into bundles of actin filaments that are incorporated into stress fibers running parallel to the direction of flow [4, 5]. Microtubules and intermediate filaments also align in the direction of laminar flow [6, 7]. Such cellular remodeling in response to unidirectional flow is regulated by numerous signaling events, including changes in protein tyrosine phosphorylation. Recent studies have begun to examine how shear affects cell– cell junctions and their interaction with the actin cytoskeleton [8 –11]. The adherens junctions between endothelial cells are critical for maintaining the barrier between circulating components of blood and subendothelial tissues. The predominant cadherins expressed in endothelial cells, vascular endothelial (VE) 2 cadherin (cadherin 5), and neural (N) cadherin, are single-span transmembrane proteins with five extracellular cadherin repeats and a cytoplasmic tail. Only VE-cadherin is found at endothelial cell– cell junctions due to its capacity to exclude N-cadherin from the junctions [12, 13]. Cadherin dimers are formed by calcium-dependent, homophilic associations between the extracellular domains of the cadherin molecules on adjacent cells, which “zipper” the neighboring cells together [14, 15]. The cadherin cytoplasmic domain contains a binding site at the carboxy terminus for either -catenin or ␥-catenin (plakoglobin) and a juxtamembrane site for binding p120catenin (p120 ctn) [16 –19]. These catenins contain 10 –13 homologous armadillo repeats (⬃42 amino acid sequences originally described in Drosophila) which mediate binding to the cadherin cytoplasmic tail [14, 20]. -Catenin and ␥-catenin can bind ␣-catenin, thereby linking the cadherin complex to the actin cy2 Abbreviations used: VE-cadherin, vascular endothelial cadherin; N-cadherin, neural cadherin; HUVEC, human umbilical vein endothelial cells; PBS⫹Ca,Mg, PBS containing CaCl 2 and MgCl 2; TX, Triton X-100.
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toskeleton and strengthening the adhesivity of the adherens junctions [19, 21–23]. p120-catenin does not bind ␣-catenin and does not appear to link cadherin complexes to the actin cytoskeleton [24]. Tyrosine phosphorylation of the catenins can modulate the association of -catenin and ␥-catenin with both cadherin and ␣-catenin [25–28]. To examine how shear stress signals the remodeling of adherens junctions, we subjected confluent, quiescent human umbilical vein endothelial cells (HUVEC) to laminar flow for 6 – 48 h. In cells exposed to shear, the relative expression of the junctional proteins remained fairly constant but the composition of VE-cadherin complexes changed. The tyrosine phosphorylation of -catenin increased within these complexes and this correlated with both the diminished association of the protein tyrosine phosphatase SHP-2 and the loss of ␣-catenin. This has led us to propose that shear-stress-induced reorganization of endothelial cell– cell junctions occurs by eliciting the dissociation of SHP-2 from VE-cadherin complexes. This results in the increased tyrosine phosphorylation of VE-cadherin-associated -catenin, which appears to affect the binding of ␣-catenin to the complexes. The dissociation of ␣-catenin from VE-cadherin complexes results in the detachment of the cadherin complexes from the actin cytoskeleton, allowing for shear-mediated remodeling of the adherens junctions within the endothelial monolayer. EXPERIMENTAL PROCEDURES Cell culture. HUVEC were isolated and characterized as described [29]. Cells were grown in complete medium (Medium 199, 10 mM Hepes, pH 7.4, 10% fetal calf serum, 1 mM glutamine, 12 U/ml heparin, 100 g/ml crude endothelial cell growth supplement, 100 U/ml penicillin, and 100 g/ml streptomycin) at 37°C on fibronectincoated tissue culture dishes. Cells were used at passage 1 or 2. Antibodies. The following primary antibodies were used: VE-cadherin, clone 75 (Transduction Laboratories, Lexington, KY), clone TEA1/31 (Biodesign International, Kennebunk, ME), and polyclonal antibody (Santa Cruz Biotechnologies, Inc, Santa Cruz, CA); ␣-catenin, polyclonal antibody (Sigma, St. Louis, MO); -catenin, clone 5H10 (Dr. M. Wheelock, University of Toledo, OH), clone 6F9 and polyclonal antibody (Sigma); phosphotyrosine, clone 4G10 (Upstate Biotechnology Inc., Lake Placid, NY); and SHP-2, polyclonal antibody (Santa Cruz Biotechnologies, Inc.). All secondary antibodies were obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Flow system. HUVEC were plated on fibronectin-coated 35- or 60-mm culture dishes and were used 2–3 days postconfluence. Gasketed inserts were clamped onto the monolayers in tissue culture dishes, forming parallel-plate flow chambers (modified from the design of McIntire and colleagues [30, 31]). The exposed areas of the monolayers were subjected to laminar flow by circulating 75 ml of 25 mM Hepes-buffered complete medium using a Rainin Dynamax 10roller peristaltic pump to generate 10 dyn/cm 2 fluid shear stress. All components of the flow systems were maintained at 37°C throughout the experiment.
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Immunofluorescence. Cell monolayers were fixed at room temperature for 20 min in 4% methanol-free formalin buffered with 0.1 M Pipes, pH 6.8, 2 mM EGTA, 2 mM MgCl 2, and 155 mM NaCl. Fixed monolayers were permeabilized with 0.2% Triton X-100 (TX) in PBS containing 0.7 mM CaCl 2 and 0.5 mM MgCl 2 (PBS⫹Ca,Mg) for 10 min, washed with PBS⫹Ca,Mg, and blocked with 10% goat serum in PBS⫹Ca,Mg. Primary and secondary antibodies, as well as FITC– phalloidin (Molecular Probes, Inc., Eugene, OR) for staining filamentous actin, were added sequentially. Monolayers were examined by fluorescence microscopy with a Nikon photomicroscope equipped with appropriate filters. Extraction of cells. Cells were washed in PBS⫹Ca,Mg and then incubated with 1 mM Na 3VO 4 and 0.2 mM H 2O 2 in M199 containing 25 mM Hepes at 37°C for 5 min prior to extraction [25]. Monolayers were washed with ice-cold PBS⫹Ca,Mg and extracted in ice-cold TX buffer: 1% TX, 10 mM Tris–HCl, pH 7.6, 50 mM NaCl, 3 mM Na 4P 2O 7, 50 mM NaF, 1 mM Na 3VO 4, 2 mM CaCl 2, 0.2 mM H 2O 2, 2 g/ml pepstatin, 1 g/ml trans-epoxysuccinyl-leucylamido(4-guanidino)-butane, 1 g/ml aprotinin, 1 g/ml leupeptin, and 0.1 g/ml 4-(2-aminoethyl)-benzenesulfonyl fluoride on ice for 15 min. The TX extracts were microcentrifuged for 10 min and the supernatants recovered. Immunoblotting. For all time points, equal amounts of protein from the TX extracts were examined. All samples were solubilized in Laemmli sample buffer [32], separated by SDS–PAGE (8% polyacrylamide), and transferred to PVDF membrane (Millipore, Bedford, MA). Blots probed with antibody 4G10 were blocked and probed in the presence of 3% BSA (ICN Biomedicals, Inc., Aurora, OH). All other blots were blocked with 5% milk in Tris-buffered saline, pH 7.5, and probed sequentially with primary and secondary antibodies diluted in 0.5% milk in Tris-buffered saline, pH 7.5. Detection was by enhanced chemiluminescence (NEN Life Science Products, Beverly, MA). Films were scanned and then quantified by NIH Image V1.61. Immunoprecipitation. For all time points, equal amounts of protein from the TX extracts were analyzed. Each sample was brought up to 1 ml with TX buffer and rotated for 1 h at 4°C with antibody and then for 2 h with protein G–agarose (Sigma). The immunoprecipitated proteins were collected by centrifugation and the pellets washed three times with PBS⫹Ca,Mg containing 1 mM Na 3VO 4 and 0.2 mM H 2O 2. Pellets were resuspended in sample buffer and analyzed as described above for immunoblotting. Sequential immunoprecipitations for analysis of the tyrosine phosphorylation of -catenin associated with VE-cadherin were performed as described above but the washed pellet was resuspended in 1% SDS, 20 mM Tris–HCl, pH 7.6, 100 mM NaCl, 1 mM Na 3VO 4, and 0.2 mM H 2O 2 and boiled for 3 min. These supernatants were recovered and subjected to immunoprecipitation as described above with a -catenin-specific antibody, followed by immunoblot analysis with phosphotyrosine antibody.
RESULTS
Shear Stress Elicited the Concurrent Reorganization of Adherens Junction Complexes and Filamentous Actin in HUVEC Monolayers When postconfluent HUVEC cultured under static conditions were immunostained for adherens junction proteins, the antibodies against VE-cadherin and the associated catenins did not label thin, uniform junctions bridging neighboring cells as are normally observed in epithelia [33, 34]. Instead, the intercellular junctions were organized in complex lattice-like structures (Fig. 1A, no shear), probably
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FIG. 1. VE-cadherin and actin localization in HUVEC monolayers. (A) HUVEC monolayers were maintained in static culture (no shear) or exposed to fluid shear stress at 10 dyn/cm 2 for 6, 20, or 47 h. The monolayers were fixed, permeabilized, and stained with antibody against VE-cadherin. VE-cadherin in the adherens junctions of static-cultured monolayers exhibited an intricate, lattice-like architecture. In monolayers exposed to laminar flow, the VE-cadherin reassembled over time into fairly uniform, circumferential bands. A1 and A2 are two-fold magnifications of the boxed areas of the VE-cadherin immunostained monolayers maintained in static culture or exposed to shear for 47 h. The results shown are representative of three independent experiments. Direction of flow, left to right. Bar, 25 m. (B) Postconfluent HUVEC monolayers were maintained in static culture (no shear) or exposed to fluid shear for 6, 24, or 48 h. The monolayers were fixed, permeabilized, and stained with FITC–phalloidin. The filamentous actin in static-cultured cells was organized in cortical bands with some thin stress fibers. Shear stress induced the development of numerous stress fibers exhibiting no preferred orientation (6 –24 h). As the cells in the monolayer reoriented and elongated, the stress fibers redistributed into actin cables aligned in the direction of flow (48 h). The results shown are representative of five independent experiments. Direction of flow, left to right. Bar, 25 m.
the result of overlapping and interdigitating regions of membrane on apposed cells [9, 11, 35–38]. When quiescent HUVEC cultures were exposed to a fluid shear stress of 10 dyn/cm 2 , the VE-cadherin junctional architecture changed dramatically. In monolayers subjected to shear stress for 6 h, the intricate latticeworks of the intercellular cadherin complexes appeared to broaden and stain more intensely (Fig. 1A, 6 h shear), consistent with increased lamellar membrane activity at the cell– cell junctions that has been observed with other activating agents prior to junctional disassembly [37]. After 20 h of exposure to shear, the intensity of the VE-cadherin staining diminished. By 47 h, the VE-cadherin complexes were reorganized into fairly compact junctions at the endothelial cell interfaces (Fig. 1A, 47 h; also compare the enlarged areas A1, no shear, to A2, 47 h). Similar transitions in junctional organization were
detected with -catenin (data not shown). Gaps between endothelial cells were rarely detected, even during the first 6 –24 h exposure of monolayers to laminar flow when the most extensive remodeling occurred. Shear stress also elicited major changes in actin organization. In quiescent, static cultures of HUVEC, the filamentous actin was distributed in cortical bands with some fine stress fibers stretching across the cell bodies (Fig. 1B, no shear). The cortical actin bands appeared contiguous with the adherens junctions. When HUVEC were subjected to shear for 6 to 24 h, a time when their adherens junctions were undergoing dramatic remodeling, the filamentous actin was remodeled into numerous disordered arrays of stress fibers (Fig. 1B, 6 or 24 h). Many actin bundles appeared shorter or fragmented at 24 h. By 48 h, the majority of the stress fibers extended across the length of the cells
VE-CADHERIN COMPLEX REGULATION BY SHEAR STRESS
FIG. 2. Expression of VE-cadherin, -catenin, and ␣-catenin did not change in HUVEC monolayers exposed to fluid shear stress. HUVEC monolayers were maintained in static culture (0) or exposed to laminar flow for 6, 24, or 48 h. Extracts were subjected to SDS– PAGE, transferred to PVDF membranes, and probed for VE-cadherin, -catenin, or ␣-catenin. No significant differences were detected in the expression of VE-cadherin, -catenin, or ␣-catenin upon exposure of the endothelial cells to fluid shear for times up to 48 h. The results shown are representative of five to six independent experiments.
parallel to the direction of flow (Fig. 1B, 48 h shear). Thus, laminar flow has a profound influence on the organization of endothelial cell– cell junctions as well as the underlying actin cytoskeleton. Cadherin Complex Protein Expression Is Unaltered in HUVEC Exposed to Fluid Shear Stress The thin, uniform bands of adherens junction proteins detected by indirect immunofluorescence microscopy in HUVEC monolayers after 48 h of laminar flow contrasted markedly with the broad and intensely stained latticeworks of cadherin complexes observed in static-cultured HUVEC monolayers. We questioned whether the apparent reduction in fluorescence signal was due to decreased protein expression of cadherin complex components in HUVEC subjected to shear. We found no statistically significant differences in the quantities of extracted VE-cadherin, -catenin, or ␣-catenin between HUVEC monolayers cultured under static conditions and those sheared at 10 dyn/cm 2 for different times up to 48 h (Fig. 2).
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ers exposed to fluid shear. The association of ␣-catenin with VE-cadherin complexes decreased markedly within 6 h of laminar flow and remained low for 48 h (Fig. 3). The loss from the VE-cadherin complexes of the actin linker ␣-catenin could have been due either to its direct dissociation from -catenin or by the dissociation of ␣-catenin/-catenin complexes from VE-cadherin. When the same VE-cadherin immunoprecipitates were probed for -catenin, no decrease in the quantity of -catenin associated with VE-cadherin was detected in extracts from HUVEC subjected to laminar flow (Fig. 3). Increased Tyrosine Phosphorylation of VE-CadherinAssociated -Catenin in Cells Exposed to Fluid Shear Since exposure to fluid shear resulted in the decreased binding of ␣-catenin to cadherin-bound -catenin, we investigated whether this was due to posttranslational modifications of the -catenin. Tyrosine phosphorylation of -catenin has frequently been implicated as a means of modulating cadherin adhesive functions [28, 39, 40]. To examine -catenin tyrosine phosphorylation within VE-cadherin complexes only and to exclude -catenin associated with N-cadherin (the other major cadherin present in endothelial cells), extracts from static-cultured and fluid-sheared HUVEC were first immunoprecipitated with antibodies against VE-cadherin. The isolated VE-cadherin complexes were then dissociated by boiling in 1% SDS. -Catenin from the dissociated VE-cadherin complexes was immunoprecipitated and analyzed by immunoblotting for phosphotyrosine. A significant increase in the
Regulation of VE-Cadherin Complex Linkage to the Cytoskeleton during Junctional Remodeling For HUVEC to accommodate the extensive shear stress-induced cellular realignment in the direction of flow, the VE-cadherin complexes likely detach from the actin cytoskeleton in a regulated manner to permit localized junctional plasticity. Therefore, we examined possible mechanisms by which the linkage of VE-cadherin complexes to the actin cytoskeleton might be altered upon exposure of HUVEC to fluid shear. It is well established that ␣-catenin associated with cadherin-bound -catenin tethers cadherin complexes to filamentous actin [21, 22]. We analyzed how much ␣-catenin coprecipitated with VE-cadherin from static cultured endothelial monolayers compared to monolay-
FIG. 3. ␣-Catenin association with the VE-cadherin complexes decreased in HUVEC exposed to shear stress. HUVEC monolayers were cultured under static conditions (0) or exposed to fluid shear for 6, 24, or 48 h. Extracts were immunoprecipitated using a polyclonal antibody against VE-cadherin and then subjected to immunoblot analysis with antibodies against VE-cadherin, ␣-catenin, or -catenin. Immunoblots were probed for VE-cadherin to demonstrate that equal quantities of VE-cadherin complexes were immunoprecipitated from each sample. A decrease in the quantity of ␣-catenin associated with VE-cadherin complexes was detected in cells exposed to laminar flow for 6 – 48 h. The quantity of -catenin associated with VE-cadherin was unaltered by fluid shear. These results are representative of three independent experiments.
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the actin cytoskeleton to allow cellular remodeling in response to fluid shear. DISCUSSION
FIG. 4. Shear stress stimulated increased tyrosine phosphorylation of -catenin associated with VE-cadherin. Extracts from HUVEC monolayers cultured under static conditions (0) or exposed to fluid shear for 6, 24, or 48 h were first immunoprecipitated with a polyclonal antibody against VE-cadherin. The washed precipitates were boiled in 1% SDS buffer and the supernatants recovered. The supernatants were subjected to a second immunoprecipitation using a polyclonal antibody against -catenin and processed for immunoblot analysis with the phosphotyrosine antibody 4G10. The immunoblots were then reprobed for -catenin to confirm that equivalent amounts of -catenin were immunoprecipitated from the dissociated VE-cadherin complexes. The immunoblots are representative of two independent experiments.
tyrosine phosphorylation of -catenin associated with VE-cadherin was observed in extracts from endothelial cells sheared for 6 – 48 h (Fig. 4, blot: phosphotyrosine). The shear-induced changes in -catenin tyrosine phosphorylation were not due to unequal immunoprecipitation of -catenin (Fig. 4, Blot: -catenin). Tyrosine phosphorylation of ␣-catenin was not detected, irrespective of whether the cells were exposed to fluid shear or not (data not shown), an observation that is consistent with the studies of many others [25, 28, 41]. Fluid Shear Promoted the Dissociation of the Protein Tyrosine Phosphatase SHP-2 from VE-Cadherin Complexes Investigations of thrombin-initiated changes in HUVEC adherens junctions by our laboratory have demonstrated a correlation between increased tyrosine phosphorylation of VE-cadherin-associated catenins and the loss of the protein tyrosine phosphatase SHP-2 from VE-cadherin complexes [42]. We also showed in these studies that SHP-2 appears to bind selectively to -catenin in the VE-cadherin complexes. Based on this precedent, the next series of experiments tested whether subjecting HUVEC to fluid shear stress prompted changes in the association of SHP-2 with VE-cadherin complexes. The data in Fig. 5 clearly demonstrate the loss of SHP-2 from VE-cadherin complexes in cells exposed to fluid shear for 6 – 48 h. As was demonstrated in Fig. 3, the quantity of -catenin associated with VE-cadherin did not change with shear. Thus, the shear-induced dissociation of SHP-2 from VE-cadherin/-catenin complexes correlated both with the increased tyrosine phosphorylation of -catenin and the loss of ␣-catenin. This provides a potential mechanism for releasing VE-cadherin complexes from
Confluent quiescent endothelial cells respond to the mechanical stimulus of fluid shear by reorienting and elongating. Endothelial cells adapting to shear stress strictly maintain monolayer integrity. This was apparent from the few intercellular gaps that we and others have observed and was consistent with the very small shear-induced decrease in transendothelial electrical resistance detected in porcine endothelial cells exposed to shear stress of up to 50 dyn/cm 2 [11]. The shearmediated reorganization of the cells within the monolayer—a form of “constrained motility” described by others [7, 43]—requires the controlled remodeling of the adherens junctions between neighboring cells. This likely is accomplished by the coordinated release and reattachment of subpopulations of VE-cadherin complexes with the dynamically changing actin cytoskeleton. Transient uncoupling of the VE-cadherin complexes from the actin cytoskeleton could occur by disruption at the binding sites of cadherin to -catenin, -catenin to ␣-catenin, or ␣-catenin to actin. Our studies are consistent with a regulated disruption of the linkage between cadherin-bound -catenin and ␣-catenin. The tyrosine phosphorylation of -catenin associated with VE-cadherin markedly increased in HUVEC exposed to fluid shear stress. Elevated levels of tyrosinephosphorylated catenins characteristically are detected in subconfluent, actively migrating cells, or oncogene-transformed cells and correlate with weak cell– cell adhesiveness [25, 44 – 48]. In contrast, the catenins isolated from cells in confluent monolayers with stable intercellular junctions exhibit minimal tyrosine phosphorylation (Fig. 4, No shear, and Ref. [25, 42, 49]). In contact-inhibited cells, the stringent regu-
FIG. 5. SHP-2 association with VE-cadherin complexes decreased in HUVEC exposed to shear stress. Extracts prepared from HUVEC monolayers cultured under static conditions (0) or exposed to fluid shear for 6, 24, or 48 h were immunoprecipitated using a polyclonal antibody against VE-cadherin and then subjected to immunoblot analysis with an antibody against SHP-2. Immunoblots were probed for VE-cadherin to demonstrate that equal quantities of VE-cadherin were immunoprecipitated from the samples. These results are representative of three independent experiments.
VE-CADHERIN COMPLEX REGULATION BY SHEAR STRESS
lation of the tyrosine phosphorylation of the catenins by closely associated protein tyrosine kinases [10] appears to be due to an array of protein tyrosine phosphatases, some in association with cadherin complexes, that localize at the cell– cell junctions when the cells attain confluence. Inactivation of tyrosine phosphatases by inhibitors, proteolysis, or overexpression of catalytically inactive protein tyrosine phosphatases is accompanied by increased tyrosine phosphorylation of -catenin and junctional weakening [50 –54]. It is interesting that overexpression of phosphatase PTPLAR ablated not only the tyrosine phosphorylation of -catenin but also epithelial cell migration [48]. We have recently demonstrated that the protein tyrosine phosphatase SHP-2 is associated with -catenin in VEcadherin complexes in confluent, quiescent HUVEC [42]. Thrombin stimulation of HUVEC results in the loss of SHP-2 from VE-cadherin complexes, an event that correlates with the increased tyrosine phosphorylation of the catenins bound to VE-cadherin. Fluid shear stress, as thrombin, causes the loss of SHP-2 from VE-cadherin complexes. This is likely due to Srcmediated tyrosine phosphorylation of SHP-2. Shear stress elicits the increased activity of Src family kinases in endothelial cells [55, 56], observations that are consistent with the increased tyrosine phosphorylation of SHP-2 that we observe in HUVEC exposed to fluid shear stress (data not shown). SHP-2 tyrosine phosphorylation also increases in thrombin-stimulated HUVEC. Such SHP-2 tyrosine phosphorylation as well as the dissociation of SHP-2 from the VE-cadherin complexes can be prevented by blocking Src activity with the Src family kinase-specific inhibitor PP1 [42]. Thus, the regulated dissociation of the protein tyrosine phosphatase SHP-2 from the adherens junctions provides a plausible mechanism for the increased tyrosine phosphorylation of -catenin associated with VE-cadherin and the resulting dissociation of ␣-catenin from VE-cadherin complexes. The role of ␣-catenin in linking cadherin complexes to the actin cytoskeleton is well documented [21, 22, 57]. How the linkage can be regulated has been less clear. Others also have demonstrated the diminished association of ␣-catenin with cadherin complexes and the weakening of intercellular junctions when the tyrosine phosphorylation of -catenin increases [28, 39, 48]. The modulatory role of -catenin in junctional regulation was made apparent in a study where E-cadherin/␣-catenin fusion proteins, which bypass the cadherin to ␣-catenin linkage function of -catenin, were expressed in cadherin-deficient L cells [58]. The cells expressing the E-cadherin/␣-catenin fusion proteins developed stable cell– cell junctions while cell motility within the monolayer was greatly suppressed. Thus, the malleability of adherens junctions appears to require the intermediary role of -catenin which,
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through posttranslational modifications, can regulate the linkage of cadherins to ␣-catenin and the actin cytoskeleton. In summary, during endothelial cell adaptation to fluid shear, transient dissociation of a population of VE-cadherin complexes from the actin cytoskeleton is necessary to allow reorientation of the cells within the monolayer. The diminished presence of the protein tyrosine phosphatase SHP-2 in VE-cadherin complexes serves to promote the increased tyrosine phosphorylation of -catenin. This modification of -catenin appears to decrease its capacity for binding ␣-catenin, thereby releasing VE-cadherin/-catenin complexes from the actin cytoskeleton. The transient nature of regulating -catenin by tyrosine phosphorylation allows rapid, well-controlled cadherin complex dissociation and reassociation with the actin cytoskeleton with minimal gap formation as the endothelial cells within the monolayer elongate and align in the direction of laminar flow. The authors thank Drs. A. S. Menko and G. L. Gerton for valuable discussions and critical reading of the manuscript. We thank Dr. Margaret Wheelock for providing antiserum against -catenin. We acknowledge the assistance of JoAnne Kenney. This work was supported in part by NIH Grant HL52132 and a grant from the American Heart Association, Mid-Atlantic Affiliate.
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Regulation of VE-Cadherin Linkage to the Cytoskeleton in Endothelial Cells Exposed to Fluid Shear Stress Jon A. Ukropec, M. Katherine Hollinger, and Marilyn J. Woolkalis 1 Department of Physiology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
Endothelial cells exposed to shear stress realigned and elongated in the direction of flow through the coordinated remodeling of their adherens junctions and actin cytoskeleton. The elaborate networks of VEcadherin complexes in static cultures became more uniform and compact in response to shear. In contrast, the cortical actin present in static cultures was reorganized into numerous stress fiber bundles distributed parallel to the direction of flow. Exposure to shear did not significantly alter the expression of the junctional proteins VE-cadherin, -catenin, and ␣-catenin, but the composition of the junctional complexes did change. We detected a marked decrease in the ␣-catenin associated with VE-cadherin complexes in endothelial monolayers subjected to shear. This loss of ␣-catenin, the protein that links -catenin-bound cadherin to the actin cytoskeleton, was not due to decreased quantities of -catenin associated with VEcadherin. Instead, the loss of ␣-catenin from the junctional complexes coincided with the increased tyrosine phosphorylation of -catenin associated with VE-cadherin. The change in -catenin phosphorylation closely correlated with the shear-induced loss of the protein tyrosine phosphatase SHP-2 from VE-cadherin complexes. Thus, the functional interaction of ␣-catenin with VE-cadherin-bound -catenin is regulated by the extent of tyrosine phosphorylation of -catenin. This, concomitantly, is regulated by SHP-2 associated with VE-cadherin complexes. © 2002 Elsevier Science (USA)
Key Words: VE-cadherin; -catenin; ␣-catenin; SHP-2; shear stress; tyrosine phosphorylation.
INTRODUCTION
Blood flowing through the vasculature is in direct contact with the endothelium, generating a frictional force or shear stress parallel to the apical surface of the endothelium. In nonpathological states, endothelial 1 To whom correspondence and reprint requests should be addressed at Department of Physiology, Thomas Jefferson University, 411 Jefferson Alumni Hall, 1020 Locust Street, Philadelphia, PA 19107. Fax: (215) 503-2073. E-mail: [email protected].
0014-4827/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.
cells align the major axis of their cell bodies parallel to the direction of laminar flow [1]. The in situ organization of endothelial cell filamentous actin ranges from cortical bands to stress fibers depending upon the direction and magnitude of the fluid dynamic forces [2, 3]. When confluent, quiescent cultured endothelial cells are exposed to shearing forces, the cortical actin is rapidly restructured into bundles of actin filaments that are incorporated into stress fibers running parallel to the direction of flow [4, 5]. Microtubules and intermediate filaments also align in the direction of laminar flow [6, 7]. Such cellular remodeling in response to unidirectional flow is regulated by numerous signaling events, including changes in protein tyrosine phosphorylation. Recent studies have begun to examine how shear affects cell– cell junctions and their interaction with the actin cytoskeleton [8 –11]. The adherens junctions between endothelial cells are critical for maintaining the barrier between circulating components of blood and subendothelial tissues. The predominant cadherins expressed in endothelial cells, vascular endothelial (VE) 2 cadherin (cadherin 5), and neural (N) cadherin, are single-span transmembrane proteins with five extracellular cadherin repeats and a cytoplasmic tail. Only VE-cadherin is found at endothelial cell– cell junctions due to its capacity to exclude N-cadherin from the junctions [12, 13]. Cadherin dimers are formed by calcium-dependent, homophilic associations between the extracellular domains of the cadherin molecules on adjacent cells, which “zipper” the neighboring cells together [14, 15]. The cadherin cytoplasmic domain contains a binding site at the carboxy terminus for either -catenin or ␥-catenin (plakoglobin) and a juxtamembrane site for binding p120catenin (p120 ctn) [16 –19]. These catenins contain 10 –13 homologous armadillo repeats (⬃42 amino acid sequences originally described in Drosophila) which mediate binding to the cadherin cytoplasmic tail [14, 20]. -Catenin and ␥-catenin can bind ␣-catenin, thereby linking the cadherin complex to the actin cy2 Abbreviations used: VE-cadherin, vascular endothelial cadherin; N-cadherin, neural cadherin; HUVEC, human umbilical vein endothelial cells; PBS⫹Ca,Mg, PBS containing CaCl 2 and MgCl 2; TX, Triton X-100.
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toskeleton and strengthening the adhesivity of the adherens junctions [19, 21–23]. p120-catenin does not bind ␣-catenin and does not appear to link cadherin complexes to the actin cytoskeleton [24]. Tyrosine phosphorylation of the catenins can modulate the association of -catenin and ␥-catenin with both cadherin and ␣-catenin [25–28]. To examine how shear stress signals the remodeling of adherens junctions, we subjected confluent, quiescent human umbilical vein endothelial cells (HUVEC) to laminar flow for 6 – 48 h. In cells exposed to shear, the relative expression of the junctional proteins remained fairly constant but the composition of VE-cadherin complexes changed. The tyrosine phosphorylation of -catenin increased within these complexes and this correlated with both the diminished association of the protein tyrosine phosphatase SHP-2 and the loss of ␣-catenin. This has led us to propose that shear-stress-induced reorganization of endothelial cell– cell junctions occurs by eliciting the dissociation of SHP-2 from VE-cadherin complexes. This results in the increased tyrosine phosphorylation of VE-cadherin-associated -catenin, which appears to affect the binding of ␣-catenin to the complexes. The dissociation of ␣-catenin from VE-cadherin complexes results in the detachment of the cadherin complexes from the actin cytoskeleton, allowing for shear-mediated remodeling of the adherens junctions within the endothelial monolayer. EXPERIMENTAL PROCEDURES Cell culture. HUVEC were isolated and characterized as described [29]. Cells were grown in complete medium (Medium 199, 10 mM Hepes, pH 7.4, 10% fetal calf serum, 1 mM glutamine, 12 U/ml heparin, 100 g/ml crude endothelial cell growth supplement, 100 U/ml penicillin, and 100 g/ml streptomycin) at 37°C on fibronectincoated tissue culture dishes. Cells were used at passage 1 or 2. Antibodies. The following primary antibodies were used: VE-cadherin, clone 75 (Transduction Laboratories, Lexington, KY), clone TEA1/31 (Biodesign International, Kennebunk, ME), and polyclonal antibody (Santa Cruz Biotechnologies, Inc, Santa Cruz, CA); ␣-catenin, polyclonal antibody (Sigma, St. Louis, MO); -catenin, clone 5H10 (Dr. M. Wheelock, University of Toledo, OH), clone 6F9 and polyclonal antibody (Sigma); phosphotyrosine, clone 4G10 (Upstate Biotechnology Inc., Lake Placid, NY); and SHP-2, polyclonal antibody (Santa Cruz Biotechnologies, Inc.). All secondary antibodies were obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Flow system. HUVEC were plated on fibronectin-coated 35- or 60-mm culture dishes and were used 2–3 days postconfluence. Gasketed inserts were clamped onto the monolayers in tissue culture dishes, forming parallel-plate flow chambers (modified from the design of McIntire and colleagues [30, 31]). The exposed areas of the monolayers were subjected to laminar flow by circulating 75 ml of 25 mM Hepes-buffered complete medium using a Rainin Dynamax 10roller peristaltic pump to generate 10 dyn/cm 2 fluid shear stress. All components of the flow systems were maintained at 37°C throughout the experiment.
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Immunofluorescence. Cell monolayers were fixed at room temperature for 20 min in 4% methanol-free formalin buffered with 0.1 M Pipes, pH 6.8, 2 mM EGTA, 2 mM MgCl 2, and 155 mM NaCl. Fixed monolayers were permeabilized with 0.2% Triton X-100 (TX) in PBS containing 0.7 mM CaCl 2 and 0.5 mM MgCl 2 (PBS⫹Ca,Mg) for 10 min, washed with PBS⫹Ca,Mg, and blocked with 10% goat serum in PBS⫹Ca,Mg. Primary and secondary antibodies, as well as FITC– phalloidin (Molecular Probes, Inc., Eugene, OR) for staining filamentous actin, were added sequentially. Monolayers were examined by fluorescence microscopy with a Nikon photomicroscope equipped with appropriate filters. Extraction of cells. Cells were washed in PBS⫹Ca,Mg and then incubated with 1 mM Na 3VO 4 and 0.2 mM H 2O 2 in M199 containing 25 mM Hepes at 37°C for 5 min prior to extraction [25]. Monolayers were washed with ice-cold PBS⫹Ca,Mg and extracted in ice-cold TX buffer: 1% TX, 10 mM Tris–HCl, pH 7.6, 50 mM NaCl, 3 mM Na 4P 2O 7, 50 mM NaF, 1 mM Na 3VO 4, 2 mM CaCl 2, 0.2 mM H 2O 2, 2 g/ml pepstatin, 1 g/ml trans-epoxysuccinyl-leucylamido(4-guanidino)-butane, 1 g/ml aprotinin, 1 g/ml leupeptin, and 0.1 g/ml 4-(2-aminoethyl)-benzenesulfonyl fluoride on ice for 15 min. The TX extracts were microcentrifuged for 10 min and the supernatants recovered. Immunoblotting. For all time points, equal amounts of protein from the TX extracts were examined. All samples were solubilized in Laemmli sample buffer [32], separated by SDS–PAGE (8% polyacrylamide), and transferred to PVDF membrane (Millipore, Bedford, MA). Blots probed with antibody 4G10 were blocked and probed in the presence of 3% BSA (ICN Biomedicals, Inc., Aurora, OH). All other blots were blocked with 5% milk in Tris-buffered saline, pH 7.5, and probed sequentially with primary and secondary antibodies diluted in 0.5% milk in Tris-buffered saline, pH 7.5. Detection was by enhanced chemiluminescence (NEN Life Science Products, Beverly, MA). Films were scanned and then quantified by NIH Image V1.61. Immunoprecipitation. For all time points, equal amounts of protein from the TX extracts were analyzed. Each sample was brought up to 1 ml with TX buffer and rotated for 1 h at 4°C with antibody and then for 2 h with protein G–agarose (Sigma). The immunoprecipitated proteins were collected by centrifugation and the pellets washed three times with PBS⫹Ca,Mg containing 1 mM Na 3VO 4 and 0.2 mM H 2O 2. Pellets were resuspended in sample buffer and analyzed as described above for immunoblotting. Sequential immunoprecipitations for analysis of the tyrosine phosphorylation of -catenin associated with VE-cadherin were performed as described above but the washed pellet was resuspended in 1% SDS, 20 mM Tris–HCl, pH 7.6, 100 mM NaCl, 1 mM Na 3VO 4, and 0.2 mM H 2O 2 and boiled for 3 min. These supernatants were recovered and subjected to immunoprecipitation as described above with a -catenin-specific antibody, followed by immunoblot analysis with phosphotyrosine antibody.
RESULTS
Shear Stress Elicited the Concurrent Reorganization of Adherens Junction Complexes and Filamentous Actin in HUVEC Monolayers When postconfluent HUVEC cultured under static conditions were immunostained for adherens junction proteins, the antibodies against VE-cadherin and the associated catenins did not label thin, uniform junctions bridging neighboring cells as are normally observed in epithelia [33, 34]. Instead, the intercellular junctions were organized in complex lattice-like structures (Fig. 1A, no shear), probably
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FIG. 1. VE-cadherin and actin localization in HUVEC monolayers. (A) HUVEC monolayers were maintained in static culture (no shear) or exposed to fluid shear stress at 10 dyn/cm 2 for 6, 20, or 47 h. The monolayers were fixed, permeabilized, and stained with antibody against VE-cadherin. VE-cadherin in the adherens junctions of static-cultured monolayers exhibited an intricate, lattice-like architecture. In monolayers exposed to laminar flow, the VE-cadherin reassembled over time into fairly uniform, circumferential bands. A1 and A2 are two-fold magnifications of the boxed areas of the VE-cadherin immunostained monolayers maintained in static culture or exposed to shear for 47 h. The results shown are representative of three independent experiments. Direction of flow, left to right. Bar, 25 m. (B) Postconfluent HUVEC monolayers were maintained in static culture (no shear) or exposed to fluid shear for 6, 24, or 48 h. The monolayers were fixed, permeabilized, and stained with FITC–phalloidin. The filamentous actin in static-cultured cells was organized in cortical bands with some thin stress fibers. Shear stress induced the development of numerous stress fibers exhibiting no preferred orientation (6 –24 h). As the cells in the monolayer reoriented and elongated, the stress fibers redistributed into actin cables aligned in the direction of flow (48 h). The results shown are representative of five independent experiments. Direction of flow, left to right. Bar, 25 m.
the result of overlapping and interdigitating regions of membrane on apposed cells [9, 11, 35–38]. When quiescent HUVEC cultures were exposed to a fluid shear stress of 10 dyn/cm 2 , the VE-cadherin junctional architecture changed dramatically. In monolayers subjected to shear stress for 6 h, the intricate latticeworks of the intercellular cadherin complexes appeared to broaden and stain more intensely (Fig. 1A, 6 h shear), consistent with increased lamellar membrane activity at the cell– cell junctions that has been observed with other activating agents prior to junctional disassembly [37]. After 20 h of exposure to shear, the intensity of the VE-cadherin staining diminished. By 47 h, the VE-cadherin complexes were reorganized into fairly compact junctions at the endothelial cell interfaces (Fig. 1A, 47 h; also compare the enlarged areas A1, no shear, to A2, 47 h). Similar transitions in junctional organization were
detected with -catenin (data not shown). Gaps between endothelial cells were rarely detected, even during the first 6 –24 h exposure of monolayers to laminar flow when the most extensive remodeling occurred. Shear stress also elicited major changes in actin organization. In quiescent, static cultures of HUVEC, the filamentous actin was distributed in cortical bands with some fine stress fibers stretching across the cell bodies (Fig. 1B, no shear). The cortical actin bands appeared contiguous with the adherens junctions. When HUVEC were subjected to shear for 6 to 24 h, a time when their adherens junctions were undergoing dramatic remodeling, the filamentous actin was remodeled into numerous disordered arrays of stress fibers (Fig. 1B, 6 or 24 h). Many actin bundles appeared shorter or fragmented at 24 h. By 48 h, the majority of the stress fibers extended across the length of the cells
VE-CADHERIN COMPLEX REGULATION BY SHEAR STRESS
FIG. 2. Expression of VE-cadherin, -catenin, and ␣-catenin did not change in HUVEC monolayers exposed to fluid shear stress. HUVEC monolayers were maintained in static culture (0) or exposed to laminar flow for 6, 24, or 48 h. Extracts were subjected to SDS– PAGE, transferred to PVDF membranes, and probed for VE-cadherin, -catenin, or ␣-catenin. No significant differences were detected in the expression of VE-cadherin, -catenin, or ␣-catenin upon exposure of the endothelial cells to fluid shear for times up to 48 h. The results shown are representative of five to six independent experiments.
parallel to the direction of flow (Fig. 1B, 48 h shear). Thus, laminar flow has a profound influence on the organization of endothelial cell– cell junctions as well as the underlying actin cytoskeleton. Cadherin Complex Protein Expression Is Unaltered in HUVEC Exposed to Fluid Shear Stress The thin, uniform bands of adherens junction proteins detected by indirect immunofluorescence microscopy in HUVEC monolayers after 48 h of laminar flow contrasted markedly with the broad and intensely stained latticeworks of cadherin complexes observed in static-cultured HUVEC monolayers. We questioned whether the apparent reduction in fluorescence signal was due to decreased protein expression of cadherin complex components in HUVEC subjected to shear. We found no statistically significant differences in the quantities of extracted VE-cadherin, -catenin, or ␣-catenin between HUVEC monolayers cultured under static conditions and those sheared at 10 dyn/cm 2 for different times up to 48 h (Fig. 2).
243
ers exposed to fluid shear. The association of ␣-catenin with VE-cadherin complexes decreased markedly within 6 h of laminar flow and remained low for 48 h (Fig. 3). The loss from the VE-cadherin complexes of the actin linker ␣-catenin could have been due either to its direct dissociation from -catenin or by the dissociation of ␣-catenin/-catenin complexes from VE-cadherin. When the same VE-cadherin immunoprecipitates were probed for -catenin, no decrease in the quantity of -catenin associated with VE-cadherin was detected in extracts from HUVEC subjected to laminar flow (Fig. 3). Increased Tyrosine Phosphorylation of VE-CadherinAssociated -Catenin in Cells Exposed to Fluid Shear Since exposure to fluid shear resulted in the decreased binding of ␣-catenin to cadherin-bound -catenin, we investigated whether this was due to posttranslational modifications of the -catenin. Tyrosine phosphorylation of -catenin has frequently been implicated as a means of modulating cadherin adhesive functions [28, 39, 40]. To examine -catenin tyrosine phosphorylation within VE-cadherin complexes only and to exclude -catenin associated with N-cadherin (the other major cadherin present in endothelial cells), extracts from static-cultured and fluid-sheared HUVEC were first immunoprecipitated with antibodies against VE-cadherin. The isolated VE-cadherin complexes were then dissociated by boiling in 1% SDS. -Catenin from the dissociated VE-cadherin complexes was immunoprecipitated and analyzed by immunoblotting for phosphotyrosine. A significant increase in the
Regulation of VE-Cadherin Complex Linkage to the Cytoskeleton during Junctional Remodeling For HUVEC to accommodate the extensive shear stress-induced cellular realignment in the direction of flow, the VE-cadherin complexes likely detach from the actin cytoskeleton in a regulated manner to permit localized junctional plasticity. Therefore, we examined possible mechanisms by which the linkage of VE-cadherin complexes to the actin cytoskeleton might be altered upon exposure of HUVEC to fluid shear. It is well established that ␣-catenin associated with cadherin-bound -catenin tethers cadherin complexes to filamentous actin [21, 22]. We analyzed how much ␣-catenin coprecipitated with VE-cadherin from static cultured endothelial monolayers compared to monolay-
FIG. 3. ␣-Catenin association with the VE-cadherin complexes decreased in HUVEC exposed to shear stress. HUVEC monolayers were cultured under static conditions (0) or exposed to fluid shear for 6, 24, or 48 h. Extracts were immunoprecipitated using a polyclonal antibody against VE-cadherin and then subjected to immunoblot analysis with antibodies against VE-cadherin, ␣-catenin, or -catenin. Immunoblots were probed for VE-cadherin to demonstrate that equal quantities of VE-cadherin complexes were immunoprecipitated from each sample. A decrease in the quantity of ␣-catenin associated with VE-cadherin complexes was detected in cells exposed to laminar flow for 6 – 48 h. The quantity of -catenin associated with VE-cadherin was unaltered by fluid shear. These results are representative of three independent experiments.
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the actin cytoskeleton to allow cellular remodeling in response to fluid shear. DISCUSSION
FIG. 4. Shear stress stimulated increased tyrosine phosphorylation of -catenin associated with VE-cadherin. Extracts from HUVEC monolayers cultured under static conditions (0) or exposed to fluid shear for 6, 24, or 48 h were first immunoprecipitated with a polyclonal antibody against VE-cadherin. The washed precipitates were boiled in 1% SDS buffer and the supernatants recovered. The supernatants were subjected to a second immunoprecipitation using a polyclonal antibody against -catenin and processed for immunoblot analysis with the phosphotyrosine antibody 4G10. The immunoblots were then reprobed for -catenin to confirm that equivalent amounts of -catenin were immunoprecipitated from the dissociated VE-cadherin complexes. The immunoblots are representative of two independent experiments.
tyrosine phosphorylation of -catenin associated with VE-cadherin was observed in extracts from endothelial cells sheared for 6 – 48 h (Fig. 4, blot: phosphotyrosine). The shear-induced changes in -catenin tyrosine phosphorylation were not due to unequal immunoprecipitation of -catenin (Fig. 4, Blot: -catenin). Tyrosine phosphorylation of ␣-catenin was not detected, irrespective of whether the cells were exposed to fluid shear or not (data not shown), an observation that is consistent with the studies of many others [25, 28, 41]. Fluid Shear Promoted the Dissociation of the Protein Tyrosine Phosphatase SHP-2 from VE-Cadherin Complexes Investigations of thrombin-initiated changes in HUVEC adherens junctions by our laboratory have demonstrated a correlation between increased tyrosine phosphorylation of VE-cadherin-associated catenins and the loss of the protein tyrosine phosphatase SHP-2 from VE-cadherin complexes [42]. We also showed in these studies that SHP-2 appears to bind selectively to -catenin in the VE-cadherin complexes. Based on this precedent, the next series of experiments tested whether subjecting HUVEC to fluid shear stress prompted changes in the association of SHP-2 with VE-cadherin complexes. The data in Fig. 5 clearly demonstrate the loss of SHP-2 from VE-cadherin complexes in cells exposed to fluid shear for 6 – 48 h. As was demonstrated in Fig. 3, the quantity of -catenin associated with VE-cadherin did not change with shear. Thus, the shear-induced dissociation of SHP-2 from VE-cadherin/-catenin complexes correlated both with the increased tyrosine phosphorylation of -catenin and the loss of ␣-catenin. This provides a potential mechanism for releasing VE-cadherin complexes from
Confluent quiescent endothelial cells respond to the mechanical stimulus of fluid shear by reorienting and elongating. Endothelial cells adapting to shear stress strictly maintain monolayer integrity. This was apparent from the few intercellular gaps that we and others have observed and was consistent with the very small shear-induced decrease in transendothelial electrical resistance detected in porcine endothelial cells exposed to shear stress of up to 50 dyn/cm 2 [11]. The shearmediated reorganization of the cells within the monolayer—a form of “constrained motility” described by others [7, 43]—requires the controlled remodeling of the adherens junctions between neighboring cells. This likely is accomplished by the coordinated release and reattachment of subpopulations of VE-cadherin complexes with the dynamically changing actin cytoskeleton. Transient uncoupling of the VE-cadherin complexes from the actin cytoskeleton could occur by disruption at the binding sites of cadherin to -catenin, -catenin to ␣-catenin, or ␣-catenin to actin. Our studies are consistent with a regulated disruption of the linkage between cadherin-bound -catenin and ␣-catenin. The tyrosine phosphorylation of -catenin associated with VE-cadherin markedly increased in HUVEC exposed to fluid shear stress. Elevated levels of tyrosinephosphorylated catenins characteristically are detected in subconfluent, actively migrating cells, or oncogene-transformed cells and correlate with weak cell– cell adhesiveness [25, 44 – 48]. In contrast, the catenins isolated from cells in confluent monolayers with stable intercellular junctions exhibit minimal tyrosine phosphorylation (Fig. 4, No shear, and Ref. [25, 42, 49]). In contact-inhibited cells, the stringent regu-
FIG. 5. SHP-2 association with VE-cadherin complexes decreased in HUVEC exposed to shear stress. Extracts prepared from HUVEC monolayers cultured under static conditions (0) or exposed to fluid shear for 6, 24, or 48 h were immunoprecipitated using a polyclonal antibody against VE-cadherin and then subjected to immunoblot analysis with an antibody against SHP-2. Immunoblots were probed for VE-cadherin to demonstrate that equal quantities of VE-cadherin were immunoprecipitated from the samples. These results are representative of three independent experiments.
VE-CADHERIN COMPLEX REGULATION BY SHEAR STRESS
lation of the tyrosine phosphorylation of the catenins by closely associated protein tyrosine kinases [10] appears to be due to an array of protein tyrosine phosphatases, some in association with cadherin complexes, that localize at the cell– cell junctions when the cells attain confluence. Inactivation of tyrosine phosphatases by inhibitors, proteolysis, or overexpression of catalytically inactive protein tyrosine phosphatases is accompanied by increased tyrosine phosphorylation of -catenin and junctional weakening [50 –54]. It is interesting that overexpression of phosphatase PTPLAR ablated not only the tyrosine phosphorylation of -catenin but also epithelial cell migration [48]. We have recently demonstrated that the protein tyrosine phosphatase SHP-2 is associated with -catenin in VEcadherin complexes in confluent, quiescent HUVEC [42]. Thrombin stimulation of HUVEC results in the loss of SHP-2 from VE-cadherin complexes, an event that correlates with the increased tyrosine phosphorylation of the catenins bound to VE-cadherin. Fluid shear stress, as thrombin, causes the loss of SHP-2 from VE-cadherin complexes. This is likely due to Srcmediated tyrosine phosphorylation of SHP-2. Shear stress elicits the increased activity of Src family kinases in endothelial cells [55, 56], observations that are consistent with the increased tyrosine phosphorylation of SHP-2 that we observe in HUVEC exposed to fluid shear stress (data not shown). SHP-2 tyrosine phosphorylation also increases in thrombin-stimulated HUVEC. Such SHP-2 tyrosine phosphorylation as well as the dissociation of SHP-2 from the VE-cadherin complexes can be prevented by blocking Src activity with the Src family kinase-specific inhibitor PP1 [42]. Thus, the regulated dissociation of the protein tyrosine phosphatase SHP-2 from the adherens junctions provides a plausible mechanism for the increased tyrosine phosphorylation of -catenin associated with VE-cadherin and the resulting dissociation of ␣-catenin from VE-cadherin complexes. The role of ␣-catenin in linking cadherin complexes to the actin cytoskeleton is well documented [21, 22, 57]. How the linkage can be regulated has been less clear. Others also have demonstrated the diminished association of ␣-catenin with cadherin complexes and the weakening of intercellular junctions when the tyrosine phosphorylation of -catenin increases [28, 39, 48]. The modulatory role of -catenin in junctional regulation was made apparent in a study where E-cadherin/␣-catenin fusion proteins, which bypass the cadherin to ␣-catenin linkage function of -catenin, were expressed in cadherin-deficient L cells [58]. The cells expressing the E-cadherin/␣-catenin fusion proteins developed stable cell– cell junctions while cell motility within the monolayer was greatly suppressed. Thus, the malleability of adherens junctions appears to require the intermediary role of -catenin which,
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through posttranslational modifications, can regulate the linkage of cadherins to ␣-catenin and the actin cytoskeleton. In summary, during endothelial cell adaptation to fluid shear, transient dissociation of a population of VE-cadherin complexes from the actin cytoskeleton is necessary to allow reorientation of the cells within the monolayer. The diminished presence of the protein tyrosine phosphatase SHP-2 in VE-cadherin complexes serves to promote the increased tyrosine phosphorylation of -catenin. This modification of -catenin appears to decrease its capacity for binding ␣-catenin, thereby releasing VE-cadherin/-catenin complexes from the actin cytoskeleton. The transient nature of regulating -catenin by tyrosine phosphorylation allows rapid, well-controlled cadherin complex dissociation and reassociation with the actin cytoskeleton with minimal gap formation as the endothelial cells within the monolayer elongate and align in the direction of laminar flow. The authors thank Drs. A. S. Menko and G. L. Gerton for valuable discussions and critical reading of the manuscript. We thank Dr. Margaret Wheelock for providing antiserum against -catenin. We acknowledge the assistance of JoAnne Kenney. This work was supported in part by NIH Grant HL52132 and a grant from the American Heart Association, Mid-Atlantic Affiliate.
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