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American Journal of Respiratory Cell and Molecular Biology. Vol. 28, pp. 626-636, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0085OC

Role of Protein Kinase C Isoforms in Rat Epididymal Microvascular Endothelial Barrier Function

Elizabeth O. Harrington, Jodi L. Brunelle, Christopher J. Shannon, Eric S. Kim, Kirstin Mennella and Sharon Rounds

Pulmonary Vascular Biology Research Laboratory, Providence Veterans Affairs Medical Center, Department of Medicine, Brown Medical School, Providence, Rhode Island

Address correspondence to: Elizabeth O. Harrington, Ph.D., Providence VA Medical Center, Research Services, 151, 830 Chalkstone Avenue, Providence, RI 02908. E-mail: Elizabeth_Harrington{at}brown.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Endothelial barrier dysfunction is involved in a variety of diseased states. We investigated the role of protein kinase C (PKC) in monolayer permeability using endothelial cells (EC) overexpressing PKC{alpha} (PKC{alpha}EC), PKC{delta} (PKC{delta}EC) or vector (vector control EC) cDNAs. Thrombin induced permeability changes in all EC, and induced significantly elevated rates of monolayer permeability in PKC{alpha}EC. Conversely, the basal level of permeability was significantly blunted in PKC{delta}EC, resulting in diminished thrombin-induced changes in permeability. PKC inhibitors, Gö6976 and rottlerin, reversed the effects of PKC{alpha} and PKC{delta} overexpression on permeability, respectively. Immunoblot analyses demonstrated significantly less ß-catenin associated with the cytoskeletal subcellular fraction in thrombin-treated PKC{alpha}EC, an effect blocked by pretreatment with Gö6976. PKC{delta}EC contained significantly greater numbers of focal contacts per cell. Thrombin enhanced RhoA GTPase activity in all EC; with a 3-fold greater level of activity in PKC{delta}EC. Rottlerin significantly blunted RhoA GTPase activity in all EC. Overexpression of RhoA dominant-negative cDNA diminished the size and number of focal contacts in EC, and significantly enhanced the basal rate of PKC{delta}EC monolayer permeability. These findings demonstrate that monolayer permeability changes are differentially regulated by PKC isoenzymes, suggesting that PKC{alpha} promotes endothelial barrier dysfunction and PKC{delta} enhances basal endothelial barrier function.

Abbreviations: endothelial cell(s), EC • focal adhesion kinase, FAK • glycogen synthase kinase 3ß, GSK-3ß • horseradish peroxidase, HRP • protein kinase C, PKC • sodium dodecyl sulfate–polyacrylamide gel electrophoresis, SDS-PAGE


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In blood vessels, endothelial cells (EC) form a continuous monolayer selectively restricting the passage of solutes and macromolecules into the surrounding tissue. Much of the transport occurs paracellularly by means of the interendothelial junctions, tight junctions, and adherens junctions (1, 2). The release of inflammatory and thrombogenic mediators or ischemia/reperfusion injury causes endothelial barrier dysfunction leading to (i) tissue edema and inflammatory cell infiltration, (ii) vasodysregulation, and (iii) disruption of oxygen transport and use by surrounding tissue (3–6). Interendothelial cell gaps formed during endothelial barrier dysfunction result from endothelial cell contraction and disruption of endothelial cell–cell contacts (1, 2, 7).

Several thrombogenic and inflammatory mediators activate protein kinase C (PKC) (8, 9). Additionally, direct activation of PKC by phorbol esters has been shown to increase endothelial cell monolayer permeability (8, 9), disrupt cell–cell junction protein complexes (10), and promote actin cytoskeleton reorganization (11). Nonspecific pharmacologic inhibitors of PKC block thrombin- (12, 13) and glucose-induced (14) endothelial cell monolayer permeability and actin stress fiber formation (15, 16). It is unclear how PKC alters endothelial cell monolayer permeability; but it is possible that PKC modulation of endothelial cell–cell junctions or cell–extracellular interactions results in altered endothelial monolayer permeability.

PKC is a family of serine-threonine kinases. To date, twelve PKC isoenzymes have been identified and are classified as the conventional PKCs (cPKCs), novel PKCs (nPKCs), and atypical PKCs (aPKCs). Each PKC isoenzyme is thought to have its own discrete activators, cofactors, and substrates; however, few have been identified. Interpretation of pharmacologic studies has been limited by lack of specificity of the available chemical reagents for PKC. For example, PKC-activating phorbol esters also bind to and regulate other intracellular targets (17). Also, many of the chemical inhibitors that have been studied in intact cells also interfere with the function of other protein kinases (18). Thus, we have used the method of overexpression of selective PKC isoenzymes to study their role in endothelial cell function. Previously, we had established and characterized microvascular endothelial cells which stably overexpressed PKC{alpha} (PKC{alpha} EC), PKC{delta} (PKC{delta} EC), or vector (vector control EC) cDNAs (19). Analysis of endothelial cell function demonstrated that overexpression of PKC{alpha} elevated rates of basal and agonist-stimulated endothelial cell migration. In contrast, overexpression of PKC{delta} diminished the rate of endothelial cell proliferation and increased endothelial cell adhesion to extracellular matrix proteins. In this study, we investigated the role of these PKC isoenzymes in microvascular endothelial monolayer permeability. We found that PKC{alpha} and -{delta} differentially regulate endothelial monolayer permeability. Futhermore, we present data that suggests that PKC{alpha} overexpression may alter endothelial barrier function by destabilizing adherens junction complexes. Conversely, PKC{delta} may enhance basal endothelial barrier function by enhancing focal adhesion contact formation by a RhoA GTPase-mediated event.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Lines and Reagents
Rat epididymal microvascular endothelial cells were isolated and characterized previously (20) (a generous gift from R. D. Rosenberg, MIT, Cambridge, MA). Rat microvascular endothelial cells stably overexpressing vector control cDNA (control EC), PKC{alpha} cDNA (PKC{alpha} EC), or PKC{delta} cDNA (PKC{delta} EC) were previously created and characterized (19).

The PKC inhibitors, rottlerin and Gö6976, were purchased from Sigma Chemicals (St. Louis, MO) and Biomol Research Laboratories (Plymouth Meeting, PA), respectively. Thrombin, horseradish peroxidase (HRP), and O-phenylenediamine HCl were supplied by Sigma Chemicals. ß-Catenin, {gamma}-catenin, and glycogen synthase kinase-3ß (GSK-3ß)-specific antibodies were purchased from Transduction Laboratories (San Diego, CA). Antibodies directed against {alpha}-catenin were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA). Phospho-specific antibodies to ß-catenin (serine33/serine37/threonine41) and GSK-3ß (serine9) were purchased from Cell Signaling, Inc (Beverly, MA). Antibodies directed against vinculin were obtained from Sigma Chemicals.

The pGST-C21 construct was a generous gift from Dr. John G. Collard, The Netherlands Cancer Institute (21). Antibodies directed against RhoA were purchased from Santa Cruz Biotechnologies. The pGST-PBD construct was a generous gift from Dr. Richard A. Cerione, Cornell University (Ithaca, NY) (22). Cdc42 and Rac1-specific antibodies were purchased from Transduction Laboratories (San Diego, CA). Y-27632 and tautomycin were obtained from Biomol Research Laboratories. The Ad5.CMV GFP adenovirus was purchased from Q-BIOgene (Carlsbad, CA). The Ad CMV RhoA dn adenovirus (referred to as AdRhoAdn) was a generous gift from Dr. T. Hirase, Kobe University (23).

Permeability Assay
Permeability assays were performed using a two-compartment chamber system obtained from Costar, Inc. (Acton, MA). This system contained 0.4-µm pore polycarbonate Transwell supports. The endothelial cells were plated to confluence in the rehydrated Transwell supports at a density of 8 x 104 cells per well and permitted to adhere overnight in complete medium. The following day, the medium was changed in both chambers. Thrombin and HRP were then placed in the upper chamber. The appearance of HRP in the lower chamber was determined by retrieving 20-µl aliquots of medium from the lower compartment every 30 min, over 3 h. The HRP concentration was determined using a spectrophotometric assay. The assay was carried out by incubating the sample in a solution containing 0.4 mg ml-1 O-phenylenediamine HCl and 0.012% H2O2 in 0.5M phosphate-citrate buffer, pH 5.0, for 30 min at 25°C. The reaction was stopped with HCl, a final concentration of 0.55 M, and the absorbance was taken at 492 nm. The data are presented as the number of moles of HRP that have diffused to the lower chamber over time.

In some experiments, the endothelial cells were preincubated with a vehicle, Gö6976, rottlerin, tautomycin, or Y-27632 for 1 h. The vehicle or inhibitor was then replenished in the upper chamber upon addition of thrombin and HRP. In other experiments, EC were infected with Ad GFP or Ad RhoA dn virus particles at optimal concentrations (24) to achieve overexpression in the entire monolayer and incubated for 16 h. Permeability experiments were performed as described above. Parallel wells of infected EC were collected, and overexpression of the protein of interest was confirmed by both immunofluorescence and immunoblot analysis.

Immunofluorescence Analysis
EC grown on coverslips were stimulated as described. The cells were fixed with 4% paraformaldehyde, and rendered permeable with Triton X-100. The cells were immunofluorescently stained for ß-catenin or vinculin, as previously described (19). Images were viewed at x630 magnification with a laser scanning confocal microscope and recorded.

Immunoprecipitations and Immunoblot Analyses
For the catenin translocation experiments, the endothelial cells were grown to confluence and treated with or without thrombin for the indicated times. Endothelial cells were washed once with phosphate-buffered saline and lysed in 500 µl NP40 buffer (25 mM Hepes, pH 7.4, 150 mM NaCl, 4 mM ethylenediamine tetraacetic acid, 25 mM NaF, 1% NP40, 1 mM Na3VO4, 1 mM phenylmethanesulfonyl fluoride, 10 µg ml-1 leupeptin, and 10 µg ml-1 aprotinin) as previously described (25). The cell lysates were incubated on ice for 30 min and centrifuged at 10,000 x g for 30 min at 4°C. The soluble supernatant represented the cytosolic proteins. The pellet was solubilized by sonication in 50 µl of a solution containing 25 mM Hepes, pH 7.5, 4 mM ethylenediamine tetraacetic acid, 25 mM NaF, 1% sodium dodecyl sulfate (SDS), and 1 mM Na3VO4. This cellular fraction then was diluted with 450 µl of NP40 buffer and incubated at 4°C for 30 min, shaking. Any remaining insoluble material was removed by centrifuging at 10,000 x g for 30 min at 4°C. The soluble supernatant contained the cytoskeletal/membrane associated proteins. The protein was quantitated using Bio-Rad DC protein assay kit (Bio-Rad, Hercules, CA), and equivalent protein quantities were resolved on 10% SDS–polyacrylamide gel electrophoresis (SDS-PAGE).

For analyses assessing the phosphorylation state of ß-catenin or GSK-3ß, endothelial cells were lysed directly in Laemmli buffer and equivalent volumes of protein were resolved by SDS-PAGE. For analyses assessing the tyrosine phosphorylation state of FAK, paxillin, and p130Cas, cells were isolated and proteins were processed as previously described (26).

Proteins were transferred to Immobilon-P membranes supplied by Millipore, Inc. (Bedford, MA) according to manufacturer's recommendations. Immunoblot analyses were performed as previously described (19).

RhoA GTPase Activation Assays
The endothelial cells were grown to confluence and treated with or without thrombin for the indicated times. Endothelial cells were washed once with phosphate-buffered saline and lysed in FISH buffer (10% glycerol, 50 mM Tris, pH 7.4, 100 mM NaCl, 1% NP 40, and 2 mM MgCl2) as previously described (21, 27). Cell lysates were incubated on ice for 10 min, and insoluble debris was removed by centrifuging at 13000 x g for 5 min at 4°C. Equivalent volumes of supernatants were incubated with 50 µg of bacterially produced GST-C21 or GST-PBD bound to glutathione agarose beads for 1 h at 4°C. The beads were washed three times with FISH buffer and suspended in Laemmli buffer. Protein complexes bound to the beads were resolved on 15% SDS-PAGE. Parallel gels were run with corresponding crude cell lysates. All gels were transferred to Immobilon-P.

In some experiments, the endothelial cells were preincubated with vehicle or rottlerin for 1 h. The vehicle or inhibitor was then added to the cells in the presence or absence of thrombin, and cell lysates were collected and analyzed as described above.

Statistical Analyses
For permeability experiments, linear regression analysis was performed to determine the rate of HRP diffusion in individual wells using Statview 4.0 (SAS Institute, Cary, NC). The mean and standard error were determined from these slopes. For three or more groups, differences among the means were tested for significance in all experiments, using ANOVA with Fisher's least significance difference test. Significance was reached when P < 0.05. All data are presented as mean ± SE. n is indicated for each set of data.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Modulation of Endothelial Monolayer Permeability by Selective PKC Isoenzymes
In previous studies, overexpression of PKC{delta} cDNA was shown to significantly decrease the rate of endothelial cell proliferation, as compared with overexpression of vector or PKC{alpha} cDNAs (19). Thus, comparison of macromolecular permeability differences between stably transfected vector control EC, PKC{delta} EC, and PKC{alpha} EC was determined on equivalent cell numbers of transfected endothelial monolayers, which were plated on polycarbonate membranes and permitted to adhere overnight. Under these experimental conditions, confluent endothelial monolayers were established as demonstrated by the low level of macromolecular diffusion of HRP in unstimulated vector control EC monolayers, as compared with membranes lacking cell monolayers and treated under the same conditions (Figure 1A). Additionally, thrombin stimulated an increase in HRP diffusion across the vector control EC monolayers. Similar results were demonstrated for PKC{delta} and PKC{alpha} EC (data not shown).




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  		Figure 1. Effects of PKC{alpha} and PKC{delta} overexpression on endothelial monolayer permeability. EC overexpressing vector control, PKC{delta}, or PKC{alpha} cDNAs were grown to confluence on Transwell supports. The endothelial monolayers were stimulated with 1.5 U thrombin ml-1 media and the amount of HRP that diffused through the monolayer was determined over 3 h. (A) Diffusion of HRP across vector control EC monolayer over time in the presence and absence of thrombin stimulation. Circles, no cells; open squares, no stimulant; filled squares, 1.5 U/ml thrombin. In B, the rate of monolayer permeability was determined for unstimulated or thrombin-stimulated vector control EC (solid bars), PKC{delta} EC (open bars), and PKC{alpha} EC (striped bars) monolayers. Data presented as the mean ± SE from 10–14 independent experiments. *P < 0.0001 versus basal permeability for each respective cell type. {ddagger}P < 0.0001 versus thrombin-treated vector control EC and PKC{alpha} EC. {dagger}P < 0.001 versus thrombin-stimulated control EC and PKC{delta} EC. *P < 0.01 versus unstimulated vector control EC and PKC{alpha} EC.

 
Comparisons of rates of monolayer permeability between the stably transfected EC demonstrated thrombin to significantly elevate rates of monolayer permeability in all endothelial cell types, as compared with the basal permeability rates (Figure 1B). PKC{alpha} ECs demonstrated significantly elevated rate of thrombin-stimulated permeability, as compared with vector control EC and PKC{delta} EC. However, endothelial monolayers overexpressing PKC{delta} cDNA had a significantly reduced basal rate of permeability, as compared with vector control EC and PKC{alpha} EC. Additionally, the rate of thrombin-induced permeability was significantly lower in PKC{delta} EC, as compared with the vector control EC and PKC{alpha} EC. Although the fold change in the rate of HRP diffusion between unstimulated and thrombin-stimulated monolayers was significantly different between vector control EC and PKC{alpha}EC (2.52- versus 3.2-fold, respectively), it was not significantly different between vector control EC and PKC{delta} EC (2.52- versus 2.67-fold, respectively).

Next, we tested the ability to reverse the effects of PKC{alpha} and PKC{delta} overexpression on endothelial monolayer permeability using commercially available PKC{alpha} and -{delta} inhibitors, Gö6976 and rottlerin, respectively. The PKC{alpha} inhibitor, Gö6976, blunted the effect of PKC{alpha} overexpression on endothelial monolayer permeability by reducing the rate of thrombin-induced PKC{alpha} EC monolayer permeability to a level not significantly enhanced above vector control EC (Figure 2A). The addition of Gö6976 had no significant effect on the rates of monolayer permeability in vector control EC or PKC{delta} EC exposed to thrombin. The PKC{delta} inhibitor, rottlerin, blunted the effect of PKC{delta} overexpression by significantly increasing the rates of basal monolayer permeability in PKC{delta} EC to a level not significantly different from vector control EC (Figure 2B). These data suggest selective effects of PKC{alpha} and PKC{delta} on endothelial monolayer permeability, with PKC{alpha} enhancing thrombin-induced barrier dysfunction and PKC{delta} enhancing basal endothelial barrier function.




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  		Figure 2. Effects of Gö6976 and rottlerin on PKC{alpha} EC and PKC{delta} EC barrier function. Equivalent numbers of control EC (solid bars), PKC{alpha} EC (striped bars), and PKC{delta} EC (open bars) were grown to confluence on Transwell supports. The endothelial monolayers were preincubated with vehicle, 10 nM Gö6976 (A), or 10 µM rottlerin (B) for 1 h. The endothelial cells were then either not stimulated (B) or stimulated with 1.5 U thrombin ml-1 media in the presence of vehicle or PKC inhibitor (A), and the changes in monolayer permeability were determined (n = 6; *P < 0.05; ns = not significant).

 
PKC{alpha} Overexpression Enhances {alpha}-Catenin and ß-Catenin Subcellular Relocalization in Endothelial Cells following Thrombin Exposure
We next hypothesized that the differential effects of PKC{alpha} or PKC{delta} overexpression on endothelial barrier function were due to alterations in cell–cell structures. {alpha}-, ß-, and {gamma}-catenin (plakoglobin) are localized to the adherens junction structures in confluent endothelial cells. Upon disruption of adherens junction complexes, the catenins can relocalize from the cytoskeleton to the cytosol (7, 12, 28, 29). Immunofluorescence analysis of ß-catenin protein localization in thrombin-stimulated endothelial cells demonstrated visible gaps at cell–cell contacts in all endothelial cells, as early as 10 min after thrombin exposure (Figures 3B, 3E, and 3H), as compared with unstimulated endothelial cells (Figures 3A, 3D, and 3G). Larger gaps at cell–cell junctions were noted in confluent endothelial monolayers following 30 min of exposure to thrombin (Figures 3C, 3F, and 3I). To determine if there were any quantitatable differences in the amounts of the catenins associated with the cytoskeletal subcellular fraction, we assessed vector control EC, PKC{delta} EC, and PKC{alpha} EC following exposure to thrombin by immunoblot analysis (25). Significantly less {alpha}-catenin was associated with the cytoskeleton in vector control EC and PKC{delta} EC by 3 h of thrombin treatment (Figures 4A and 4B), as compared with the {alpha}-catenin protein content in unstimulated endothelial cells. However, in PKC{alpha} EC, a significant amount of {alpha}-catenin relocalized from the cytoskeleton earlier, after just 1 h of thrombin exposure. No significant changes in the cytoskeleton-associated ß-catenin protein content were noted in vector control EC or PKC{delta} EC following exposure to thrombin. Again, the cytoskeleton-associated ß-catenin protein content was significantly decreased in PKC{alpha} EC following thrombin after a 1-h exposure (Figures 4A and 4C). Finally, analysis of the {gamma}-catenin protein content in the cytoskeleton subcellular fraction revealed no significant changes in any of the endothelial cells following exposure to thrombin (Figures 4A and 4D). Analysis of equivalent numbers of unstimulated vector control EC, PKC{delta} EC, and PKC{alpha} EC demonstrated equivalent amounts of {alpha}-, ß-, and {gamma}-catenin protein in all EC types (data not shown), suggesting that the differential rates of catenin subcellular relocalization were not due to differences in the basal expression level of {alpha}-, ß-, or {gamma}-catenin. Preincubation of PKC{alpha} EC with Gö6976 blunted the relocalization of ß-catenin from the cytoskeleton following exposure to thrombin (Figure 4E); however, it had no significant effect on the {alpha}-catenin subcellular relocalization (data not shown).





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  		Figure 3. Thrombin promotes the disruption of cell–cell junctions. Vector control EC (A, B, C), PKC{delta} EC (D, E, F), and PKC{alpha} EC (G, H, I) were untreated (A, D, G) or stimulated with 1.5 U thrombin ml-1 media for 10 min (B, E, H) or 30 min (C, F, I). EC were immunofluorescently stained for ß-catenin and visualized with immunofluorescence microscopy. Images are representative of three independent experiments. Arrows indicate intact cell–cell junctions. Arrowheads indicate gaps appearing at interendothelial junctions.

 






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  		Figure 4. {alpha}-Catenin and ß-catenin translocation from the cytoskeletal fraction is enhanced in PKC{alpha} overexpressing EC. EC were incubated with 1.5 U thrombin ml-1 media and harvested after indicated times of treatment. Equivalent quantities of cytoskeleton-associated proteins were resolved on SDS-PAGE and transferred for immunoblot analysis. The membranes were immunoblotted for {alpha}-catenin and subsequently stripped and reprobed for ß-catenin and then {gamma}-catenin. Immunoblots are representative images of at least four experiments (A). The amount of {alpha}-catenin (B), ß-catenin (C), and {gamma}-catenin (D) associated with the cytoskeletal subcellular fraction was quantitated by densitometry. The data are presented as the mean ± SE of the catenin protein content, normalized to the unstimulated ß-catenin protein amount. *P < 0.005 versus catenin protein level of respective cell type at 0 h. {dagger}P < 0.05 versus catenin protein level of respective cell type at 0 h. (E) EC were incubated with vehicle or 10 nM Gö6976 for 1 h. The cells were then collected after a 1-h incubation with thrombin in the presence of vehicle or 10 nM Gö6976, and ß-catenin was isolated and analyzed as described above. **P < 0.05 versus vector control EC treated with respective treatment. (B, C, and D) Circles, control EC; squares, PKC{delta} EC; triangles, PKC{alpha} EC. (E) Solid bars, control EC; striped bars, PKC{alpha} EC.

 
Several PKC isoenzymes have been implicated as signaling regulators of GSK-3ß, possibly by phosphorylating and inactivating GSK-3ß. Furthermore, GSK-3ß phosphorylation of ß-catenin promotes its degradation by the ubiquitin–proteasome system, regulating the accumulation of cytosolic ß-catenin. Thus, we investigated whether thrombin-induced ß-catenin relocalization resulted in the subsequent degradation of the protein by the GSK-3ß pathway and whether this was the mechanism by which PKC{alpha} overexpression enhances endothelial monolayer permeability. Using phospho-specific antibodies against the NH2 terminal GSK-3ß consensus phosphorylation site (serine33/serine37/threonine41) of ß-catenin, we were unable to detect any significant changes in the ß-catenin phosphorylation state in vector control EC, PKC{delta} EC, or PKC{alpha} EC following thrombin stimulation (data not shown). Additionally, cell lysates from equivalent numbers of vector control EC, PKC{delta} EC, and PKC{alpha} EC demonstrated no significant changes in total ß-catenin protein content at any time, up to 3 h, following exposure to thrombin (data not shown). Finally, no significant differences were noted in the amount of active GSK-3ß in any thrombin-stimulated endothelial cells (data not shown). These results suggest that overexpression of PKC{alpha} may enhance the rate of monolayer permeability upon exposure to thrombin by destabilizing ß-catenin–mediated protein–protein interactions, independent of the GSK-3ß signaling pathway.

Enhanced Focal Contact Formation and RhoA Activity in PKC{delta} EC
Previously, we demonstrated an enhanced ability of PKC{delta} EC to adhere to the extracellular matrix protein, vitronectin, as compared with the vector control EC or PKC{alpha} EC (19). Thus, we hypothesized that the enhanced basal barrier function noted in PKC{delta} EC was due to changes in focal contact formation. Quantitation of cell–extracellular matrix interactions in untreated transfected endothelial cells immunofluorescently stained for vinculin demonstrated a significantly greater number of focal contacts in the PKC{delta} EC, as compared with vector control EC or PKC{alpha} EC (Figure 5A). The number of focal contacts was significantly diminished in vector control EC and PKC{delta} EC by 1 min of thrombin exposure and by 10 min of thrombin treatment in PKC{alpha} EC (Figure 5B), with an increase in the number of focal contacts following 30 min of thrombin exposure. The total number of focal contacts in PKC{delta} EC was enhanced by 10 min after thrombin treatment, as compared with control EC and PKC{alpha} EC. No significant differences were noted between cell types with regards to several morphologic parameters, including area, perimeter, or breadth of the focal contacts. Pretreatment of the EC with rottlerin blunted the number and size of the focal contacts present in all EC types (Figure 5C), with the resulting smaller protein structures resembling focal adhesion complexes. Comparison of the level of FAK, paxillin, vinculin, or p130Cas protein content in equivalent numbers of vector control EC, PKC{delta} EC, and PKC{alpha} EC demonstrated similar protein levels, indicating that the enhanced focal contacts seen in PKC{delta} EC was not due to elevated focal contact protein synthesis. Similarly, no significant differences were noted between vector control EC, PKC{delta} EC, and PKC{alpha} EC in changes in the tyrosine phosphorylation level of FAK, paxillin, or p130CAS. Finally, rottlerin did not significantly alter the overall protein content of FAK, paxillin, p130Cas, or vinculin in EC. Thus, PKC{delta} modulation of focal contacts may be at the point of formation or stability of the cell–extracellular structures.





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  		Figure 5. PKC{delta} EC display enhanced focal contacts. Vector control EC (circles), PKC{delta} EC (squares), and PKC{alpha} EC (triangles) were untreated (A and B) or stimulated (B) with 1.5 U thrombin ml-1 media for 1, 10, or 30 min. EC were immunofluorescently stained for vinculin and visualized by immunofluorescence microscopy. Focal contacts were analyzed as described in MATERIALS AND METHODS. (C) PKC{delta} EC incubated with vehicle or 10 µM rottlerin for 30 min. EC were immunofluorescently stained for vinculin and visualized by immunofluorescence microscopy. Images are representative of three independent experiments. For A and B, the data are presented as mean ± SE of three to eight independent samples. *P < 0.01 versus vector control EC. {dagger}P < 0.05 versus 0 min.

 
Focal adhesion complex and focal contact formation and stability depend upon the family of Rho GTPases. Thus, we examined the activities of RhoA, Cdc42, and Rac1 to determine if there were any significant differences between vector control EC, PKC{alpha} EC, or PKC{delta} EC. Thrombin enhanced RhoA GTPase activity in all EC types following a 1-min thrombin exposure (Figures 6A and 6B). However, PKC{delta} EC demonstrated an approximately 3-fold greater level of RhoA GTPase activity, as compared with vector control EC or PKC{alpha} EC. Pretreatment with rottlerin significantly blunted RhoA GTPase activity in all EC types (Figure 6C), suggesting that PKC{delta} activity may modulate RhoA GTPase. The GTPase activity levels of Cdc42 or Rac1 were not changed significantly following thrombin stimulation in any of the EC (data not shown). Finally, preincubation with the Rho kinase inhibitor, 5–10 µM Y27932, or the myosin light chain phosphatase inhibitor, 6–30 nM tautomycin, had no significant effect on thrombin-induced barrier dysfunction of vector control EC, PKC{alpha} EC, or PKC{delta} EC, as measured by changes in the rates of macromolecular permeability. To confirm that the enhanced basal barrier function noted in PKC{delta} EC was mediated by RhoA GTPase-dependent pathway, we overexpressed a dominant-negative RhoA cDNA in vector control EC or PKC{delta} EC and assessed the effects on monolayer permeability and focal contact formation. Inhibition of RhoA GTPase significantly enhanced the basal level of permeability in PKC{delta} EC, but not in vector control EC (Figure 7B). Additionally, overexpression of RhoA dominant-negative construct diminished the number and size of focal contacts (Figure 7C). The data suggests that thrombin-induced barrier dysfunction in these microvascular endothelial cells is mediated through a RhoA GTPase pathway, independent of the Rho kinase/myosin light chain phosphatase pathway. These data further suggest that PKC{delta}-enhanced barrier function may be mediated by a RhoA GTPase-mediated promotion of focal contact formation or stabilization.





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  		Figure 6. Effects of PKC{alpha} and PKC{delta} on thrombin-induced RhoA GTPase activation. EC were incubated with 1.5 U thrombin ml-1 media for indicated times. Cell lysates were harvested and active RhoA GTPase was purified with bacterially produced glutathione-S-transferase (GST) fused Rho-binding protein, rhotekin (GST-C21), bound to glutathione agarose beads. (A) GST-C21–bound protein complexes were resolved on SDS-PAGE. Parallel gels were run with corresponding crude lysates. All gels were immunoblotted for RhoA GTPase. Immunoblots are representative of six experiments. (B) Immunoblot signals were quantitated by densitometry and are presented as the mean ± SE of the ratio of GST-C21–bound RhoA GTPase to total RhoA GTPase present in crude lysate and normalized to time 0 h (n = 6; *P < 0.05 versus time 0 h). Circles, control EC; squares, PKC{delta} EC; triangles, PKC{alpha} EC. (C) Control EC (solid circles, open circles) and PKC{delta} EC (solid squares, open squares) were pretreated with either vehicle (solid circles, solid squares) or 10 µM rottlerin (open circles, open squares) for 1 h. The cells were then incubated with 1.5 U thrombin ml-1 media for indicated times and the cell lysates were processed for active RhoA GTPase, as described above (n = 3; {dagger}P < 0.001 versus time 0 h).

 




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  		Figure 7. Inhibition of RhoA GTPase alters PKC{delta} EC basal monolayer permeability and FAC formation. In A and B, EC were grown to confluence on Transwell supports and infected with Ad GFP or Ad RhoA dn virus particles for 24 h. The rate of diffusion of HRP through the monolayer was subsequently measured. Lysates obtained from uninfected EC or adenovirus infected EC were harvested, resolved on SDS-PAGE, and immunoblotted with indicated antibody. The data are presented as mean ± SE (n = 3–14; *P < 0.05; ns = not significant). Solid bars, control EC; open bars, PKC{delta} EC. (C) PKC{delta} EC were infected with Ad GFP or Ad RhoA dn adenovirus particles for 24 h, immunofluorescently stained for vinculin, and visualized by immunofluorescence microscopy. Arrows indicate representative focal contacts.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We present data showing selective effects of PKC{alpha} and PKC{delta} overexpression on microvascular endothelial barrier function. Exposure to thrombin increased monolayer permeability of all overexpressing EC, as compared with basal rates of permeability. The permeability changes seen in PKC{alpha} EC treated with thrombin were significantly greater than vector control EC or PKC{delta} EC monolayers. Conversely, PKC{delta} EC monolayers demonstrated significantly lower basal rate of permeability, which resulted in a diminished rate of macromolecular permeability as compared with vector control EC or PKC{alpha} EC. Inhibitor studies of both the vector control EC and the PKC-overexpressing EC further showed that PKC{alpha} and PKC{delta} are required to modulate the response of the endothelial monolayers permeability changes. This study suggests that changes in microvascular endothelial barrier function are regulated by selective PKC isoenzymes; with PKC{alpha} promoting, and PKC{delta} protecting against, endothelial barrier dysfunction.

Similar to our study, overexpression of PKC{alpha} in epithelial cells or PKCßI in ECs elevated basal and phorbol ester–stimulated monolayer permeability (30, 31). Additionally, inhibition of PKC{alpha} or PKCß prevented thrombin-, tumor necrosis factor-{alpha}–, or glucose-induced endothelial barrier dysfunction (14, 32–34). EC overexpressing PKC{delta} also exhibited elevated phorbol ester–stimulated transepithelial permeability (35). In contrast, we have demonstrated that PKC{delta} overexpression enhanced basal barrier function in endothelial cells. The reason for the discrepancy between cell types is not clear. It is possible that the conventional PKCs (PKC{alpha}, ßI, ßII, and {gamma}) modulate cellular monolayer permeability by targeting similar intracellular targets in epithelial and endothelial cell types, such as cell–cell junction structures. On the other hand, PKC{delta} may produce disparate responses in cellular monolayer permeability depending upon the cell type and agonist. For example, Mullin and coworkers suggested that PKC{delta} was modulating transepithelial permeability by disrupting tight junctions within the overexpressing cells (35). We did not observe any notable differences in changes in adherens junctions when comparing PKC{delta} EC with vector control EC. Further study is needed to determine if PKC{delta} modulates tight junctions in EC.

This study demonstrated that EC overexpressing PKC{delta} have significantly enhanced number of focal contacts. Inhibition of either PKC{delta} kinase or RhoA GTPase activities diminished the number and size of focal contacts in these cells, and significantly enhanced the rates of basal monolayer permeability in PKC{delta} EC to levels no longer different from vector control EC. Previously, we have shown PKC{delta} EC to have an enhanced ability to adhere to the extracellular matrix protein, vitronectin (19). Increased stress fiber formation and focal contact stability have been shown to correlate with enhanced RhoA GTPase activation in fibroblasts (36). Other studies suggested actin polymerization and focal adhesion assembly to be parallel pathways downstream from RhoA GTPase (37). Ren and colleagues demonstrated enhanced focal contact stability in FAK null fibroblasts; this change correlated with increased RhoA GTPase activity (38). Likewise, microinjection of wild-type RhoA GTPase into fibroblasts enhanced stress fiber and focal contact formation (36). Our data suggest that PKC{delta}-mediated activation of RhoA GTPase may promote microvascular endothelial basal barrier function by increasing focal contact formation. Similar to our findings, Clark and coworkers noted small, vinculin-containing focal contacts in fibroblasts overexpressing dominant-negative RhoA GTPase (39). These investigators suggested that RhoA GTPase functioned to cluster focal adhesion complexes and that this is a precursor to RhoA-dependent cell contraction.

Many studies have shown thrombin-induced increase in endothelial monolayer permeability to be mediated by stress fiber formation and actomyosin-mediated cell contraction (1, 2, 40), changes shown to require RhoA GTPase activity (40–42). Our current study demonstrated a marked increase in RhoA GTPase activity (3-fold) in PKC{delta} EC in response to thrombin, as compared with vector control EC and PKC{alpha} EC, observations which were blunted by inhibition of PKC{delta}. The enhanced RhoA GTPase activation correlated with changes in monolayer permeability in all cell types and with the reappearance of focal contacts following thrombin exposure. Although it is probable that enhanced RhoA GTPase activity mediated stress fiber formation and cellular retraction in response to thrombin exposure in these microvascular EC, this has not yet been examined. It is possible that enhanced RhoA GTPase activity noted in thrombin exposed PKC{delta} EC was required, in part, to reassemble the elevated number of focal contacts observed in these cells. Further study is needed to determine if PKC{delta}-enhanced basal endothelial barrier function is mediated at focal contacts, and to determine the roles of RhoA GTPase and stress fiber formation in this process.

This study further suggests that thrombin-induced increases in microvascular monolayer permeability occurred by a RhoA GTPase–dependent, Rho kinase/myosin light chain phosphatase–independent pathway. Adamson and colleagues also reported enhanced basal barrier dysfunction in perfused rat mesenteric venules upon RhoA GTPase inhibition, and no attenuation of agonist-induced endothelial permeability changes upon Rho kinase inhibition (43). In contrast, other investigators have shown a requirement for the RhoA GTPase/Rho kinase signaling pathway in thrombin-stimulated endothelial barrier dysfunction (40, 44, 45). It is possible that the discrepancy regarding the importance of the Rho kinase signaling pathway in endothelial barrier integrity may lie in the differences between EC subtypes (46), with macrovascular isolated EC relying on the Rho kinase signaling pathway, and microvascular endothelial cells using an alternative pathway to regulate barrier function.

Endothelial cell–cell contacts are mediated, in part, by adherens junction complexes. {alpha}-, ß-, and {gamma}-catenin are localized at cell–cell contacts of confluent unstimulated EC (7, 12). In subconfluent, proliferating, and migrating EC, the catenins redistribute away from cell–cell junctions (7). Additionally, transient disruption of adherens junctions occurs during leukocyte and monocyte transmigration (28, 29). Previously, thrombin was shown to reversibly promote the disruption of {alpha}-, ß-, and {gamma}-catenin from adherens junctions in EC in a PKC-dependent manner (12). In the current study, PKC{alpha}-overexpressing EC displayed enhanced monolayer permeability to macromolecules upon thrombin exposure. Similar to previous studies, we noted thrombin-induced dissociation of ß-catenin from cell–cell junctions in all cell types as assessed by immunofluorescence. We also noted significant quantitative differences between PKC{alpha} EC and vector control EC or PKC{delta} EC with respect to the rates at which {alpha}-catenin and ß-catenin redistributed away from the cytoskeleton subcellular fraction. PKC{alpha} EC demonstrated significant {alpha}-catenin and ß-catenin relocalization from the cytoskeletal subcellular fraction following 1 h thrombin stimulation. Yet, only {alpha}-catenin relocalized from the cytoskeletal subcellular fraction in thrombin-stimulated vector control EC and PKC{delta} EC. Finally, thrombin did not stimulate the disassociation of {gamma}-catenin from the cytoskeletal fraction in any cell type. Thus, it is possible that overexpression of PKC{alpha} augments thrombin-induced permeability changes by weakening {alpha}-catenin and/or ß-catenin protein–protein interactions with other adherens junction–associated protein components. How PKC{alpha} augments the endothelial monolayer permeability changes is unknown. It is possible that thrombin enhances endothelial barrier dysfunction by promoting weakened interactions between the actin cytoskeleton and adherens junction complex. This, in turn, promotes the disruption of {alpha}-catenin and/or ß-catenin association with the cell–cell junctions and increasing macromolecular monolayer permeability. Thus, PKC{alpha} may promote changes in adherens junction complex stability indirectly by modulating other cytoskeleton components, such as F-actin or vinculin.

In summary, our results show that overexpression of PKC{alpha} and PKC{delta} produce differential effects on the integrity of the endothelial monolayer. Overexpression of PKC{alpha} promotes elevated thrombin-induced endothelial monolayer permeability. Conversely, PKC{delta} overexpresssion lowered the basal rate of endothelial monolayer changes. The data suggests that PKC{alpha} alters the stability of interendothelial junctions. It is possible that PKC{alpha} overexpression enhances endothelial barrier dysfunction and migration (19) by altering the stability of adherens junction complexes by promoting the dissociation of {alpha}-catenin and ß-catenin from cell–cell junctions. Conversely, PKC{delta} promotes endothelial barrier function, possibly by enhancing the formation or stabilization of focal contacts by a RhoA GTPase–mediated pathway. Understanding the molecular mechanisms by which PKC{alpha} promotes changes in endothelial monolayer permeability and by which PKC{delta} enhances basal endothelial barrier function may lead to the development of agents therapeutic in endothelial barrier dysfunction.


   Acknowledgments
 
This work was supported by grants from the Department of Veterans Affairs, Merit Review Type II, American Heart Association, grant 9860025T, American Cancer Society, grant IN-45-39, and National Heart, Lung, and Blood Institute Grant HL67795 (to E.O.H.) and National Heart, Lung, and Blood Institute Grant HL64936 (to S.R.).

Received in original form June 13, 2002

Received in final form November 27, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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