H2O2-mediated permeability: role of MAPK and occludin

Christopher G. Kevil, Tadayuki Oshima, Brett Alexander, Laura L. Coe, and J. Steven Alexander

Department of Molecular and Cellular Physiology, Louisiana State University Medical Center Shreveport, 1501 Kings Highway, Shreveport, Louisiana 71130


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

H2O2-mediated elevation in endothelial solute permeability is associated with pathological events such as ischemia-reperfusion and inflammation. To understand how H2O2 mediates increased permeability, we investigated the effects of H2O2 administration on vascular endothelial barrier properties and tight junction organization and function. We report that H2O2 exposure caused an increase in endothelial solute permeability in a time-dependent manner through extracellularly regulated kinase 1 and 2 (ERK1/ERK2) signal pathways. H2O2 exposure caused the tight junctional protein occludin to be rearranged from endothelial cell-cell junctions. Occludin rearrangement involved redistribution of occludin on the cell surface and dissociation of occludin from ZO-1. Occludin also was heavily phosphorylated on serine residues upon H2O2 administration. H2O2 mediates changes in ERK1/ERK2 phosphorylation, increases endothelial solute permeability, and alters occludin localization and phosphorylation were all blocked by PD-98059, a specific mitogen-activated protein (MAP) or ERK kinase 1 inhibitor. These data strongly suggest that H2O2-mediated increased endothelial solute permeability involves the loss of endothelial tight junction integrity through increased ERK1/ERK2 activation.

oxidants; hydrogen peroxide; tight junctions; extracellularly regulated kinase 1 and 2; mitogen-activated protein kinase


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IN VIVO, H2o2 has been shown to mediate significant increases in endothelial solute permeability during pathological events such as inflammation and ischemia-reperfusion injury (14, 15, 20, 33). Unfortunately, the mechanisms responsible for increased endothelial permeability are poorly understood. Many investigators have begun to address these questions using in vitro endothelial cell models (4, 35, 36, 37). Human endothelial cell cultures have been used to study mechanisms of endothelial permeability in response to various agents, yet the precise mechanisms involved in H2O2-mediated permeability are still unknown (7, 24).

The endothelial solute barrier is regulated and maintained by endothelial cell-cell junction proteins. Investigators have reported that endothelial solute barrier properties correlate with the amount and integrity of tight junctional complexes between endothelial cells (2, 3, 23, 24, 38, 39, 40, 44). We have demonstrated that the endothelial tight junctional protein occludin is significantly involved in maintaining endothelial solute barrier properties (23, 24, 44). With the use of antisense oligonucleotides directed at occludin mRNA, we have previously shown that a reduction in occludin expression in either human arterial or venous endothelial cells causes a loss of normal solute barrier, indicating that occludin is necessary for maintaining normal endothelial solute barrier. Hirase et al. (18) also has demonstrated that occludin is more abundant in vascular beds where the endothelial solute barrier is tight (e.g., blood-brain barrier). We have also shown that treatment of human umbilical vein endothelial cell (HUVEC) monolayers with vascular endothelial growth factor (VEGF) disorganized endothelial occludin junction organization, which involved the activation of extracellularly regulated kinase 1 and 2 (ERK1/ERK2) (24). The objective of this study was to determine the effects of H2O2 exposure on occludin organization and endothelial tight junction function.

We report that H2O2 administration caused an increase in HUVEC solute permeability that was accompanied by a loss of occludin localization from endothelial cell-cell junctions. Loss of occludin localization appeared to occur through increased occludin phosphorylation and decreased ZO-1 association. All of these events were blocked by inhibition of mitogen-activated protein (MAP) or ERK kinase 1 (MEK1), suggesting that ERK1/ERK2 signaling pathways are important for H2O2-mediated endothelial permeability. These data strongly suggest that H2O2-mediated increased endothelial permeability occurs through ERK1/ERK2-mediated loss of endothelial tight junction integrity.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Tissue culture. HUVEC were cultured as previously reported (23, 24). Cells were maintained in endothelial growth medium (EGM; Clonetics, San Diego, CA) and cultured on fibronectin-coated (Sigma, St. Louis, MO) tissue culture plates. Endothelial cells were used at passages 1 and 2 for all experiments reported.

Cell-column permeability assay. We used a previously reported bioassay for measurement of permeability across endothelial cell monolayers (22, 24). Briefly, HUVEC were grown on fibronectin-coated cytodex-3 beads (Sigma). Cell-covered beads were poured into a water-jacketed glass column (maintained at 37°C) and perfused with a normal perfusate solution of Hanks' balanced salt solution containing 0.5% BSA (fraction V; Sigma) and 15 mM HEPES, pH 7.4 (Sigma). Permeability measurements were made by injecting 50 µl of tracer dye (blue dextran mol mass 2,000 kDa, cyanocobalamin mol mass 1.355 kDa, and sodium fluorescein mol mass 0.376 kDa) onto the cell column and measuring the resultant elution profile. Data are expressed as percent permeability to sodium fluorescein. Permeability measurements were made under baseline, 1 mM H2O2, or 1 mM H2O2 plus one of the following protein kinase inhibitors: tyrphostin 23 (50 µM; Biomol, Plymouth, MA), chelerythrine chloride (750 nM; Biomol), or PD-98059 (20 µM; Calbiochem, San Diego, CA). Cells were pretreated with inhibitor for 30 min and then exposed to the same inhibitor plus H2O2. Incubations were carried out for 90 min with measurements taken once after the addition of inhibitors and every 15 min thereafter. Four separate experiments were performed for each treatment group.

Immunofluorescent staining of occludin and ZO-1. HUVEC were cultured on fibronectin-coated glass coverslips. Upon confluency, coverslips were treated, fixed, and stained as previously reported (23, 24). HUVEC coverslips were either pretreated with protein kinase inhibitors and then exposed to both the inhibitor plus H2O2, or exposed to H2O2 alone. H2O2 incubation periods, with or without inhibitors, were for 30 min. Treatments were removed, and coverslips were incubated with ice-cold 95% ethanol for 30 min at -20°C, then incubated with acetone for 1 min and left to air dry at room temperature. Primary antibodies of mouse anti-occludin (1:250) and rabbit anti-ZO-1 (1:250; Zymed, San Francisco, CA) were added for 1 h at 37°C. Coverslips were then washed with 0.1% milk/PBS solution for 5 min three times each. Fluorescently labeled secondary antibodies (1:250) donkey anti-mouse FITC and donkey anti-rabbit Cy3 (Jackson Immunoresearch Laboratories, Westgrove, PA) were incubated at 37°C for 1 h. Coverslips were again washed with 0.1% milk/PBS solution for 5 min three times each. Coverslips were mounted with an antiquenching solution [1 mg/ml phenylenediamine (Sigma) in 50% glycerol, 50% PBS]. All photomicrographs were taken at ×100 with exposure times of 8 s. Three separate experiments were performed for all treatment groups.

Western blotting. Western blot analysis was performed as previously described (23, 44). Cell lysates were prepared by adding 500 µl of boiling sample buffer [125 mM Tris · HCl (pH 6.8), 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride (PMSF), 25 µg/ml aprotinin, 2 µg/ml leupeptin, and 0.025% bromphenol blue] directly to a P60 culture dish. Ten percent SDS polyacrylamide gels were run with 120,000 cells of HUVEC per lane (50 µg total protein). Gels were electrically transferred onto nitrocellulose membranes (Sigma) and blocked with blotto (5% milk powder in PBS) overnight. Membranes were cut in half and incubated in either mouse anti-occludin or rabbit anti-ZO-1 antibodies at a dilution of 1:2,000 for 2 h. Membranes were then washed in 0.1% milk/PBS three times for 5 min each wash. Secondary antibodies of goat anti-rabbit IgG-alkaline phosphatase and sheep anti-mouse IgG-alkaline phosphatase (Sigma) were incubated with the membranes for 2 h at a 1:2,000 dilution. Membranes were then washed three times with 0.1%/PBS for 5 min each and developed using nitro blue tetrazolium/bromochloroindolyl phosphate (NBT/BCIP; Sigma) colorimetric reagents. Three separate experiments were performed for each treatment group.

ERK1/ERK2 kinase phosphorylation. ERK1/ERK2 phosphorylation was determined by probing HUVEC cell lysates with an anti-active MAP kinase antibody (Promega, Madison, WI). Treatment protocols were as previously stated. Equal amounts of total protein (50 µg) were electrophoresed on 12.5% SDS polyacrylamide gels. Gels were transferred and blotted using the anti-active MAP kinase antibody (1:20,000) followed by goat anti-mouse horseradish peroxidase (1:5,000) secondary antibody (Sigma). Blots were developed using the enhanced chemiluminescence (ECL) detection system (Amersham, La Jolla, CA). Four separate experiments were performed for each treatment group.

Surface biotinylation of occludin. Endothelial monolayers were pretreated with PD-98059 (20 µM) for 30 min. Cells were then incubated with H2O2 + PD-98059 (20 µM), H2O2, or PD-98059 (20 µM) alone for 30 min. After the 30-min treatment period, cells were placed on ice and washed twice with ice-cold PBS. Cell monolayers were then labeled with 200 µg/ml biotin in PBS (Sigma) for 20 min on ice. The biotin solution was removed, and the reaction was quenched with serum-free DMEM. Cell monolayers were washed twice with ice-cold PBS and lysed in 1 ml of RIPA buffer [150 mM NaCl, 50 mM Tris (pH 8.0), 1% Triton X-100, 0.1% SDS, 1 µg/ml leupeptin, 5 µg/ml aprotinin, 1 mM PMSF, 500 µM sodium metavanadate, and 100 µM okadaic acid]. Immunoprecipitation of occludin was done as previously described (11, 21). Occludin protein was immunoprecipitated by adding 4 µg of rabbit anti-occludin polyclonal antibody (Zymed) and rotating at 4°C for 2 h. Thirty microliters of protein A-Sepharose beads (Sigma) were added and samples rotated at 4°C for 1 h. Protein A beads were pelleted by centrifugation, and the supernatant was removed. Pellets were washed four times with RIPA buffer. Sample buffer was added to each tube, boiled for 3 min, and loaded on 10% SDS polyacrylamide gels. Gels were electrophoresed and transferred to nitrocellulose. Blots were blocked with 5% milk/PBS solution overnight at 4°C. The blots were then cut in half, and the upper portion was reacted with rabbit anti ZO-1 antibody as previously described while the lower portion was reacted with avidin alkaline phosphatase (1:500) for 2 h at room temperature. The blots were developed using NBT/BCIP colorimetric reagents (Sigma). Normalization to immunoprecipitated occludin was performed using a monoclonal mouse anti-occludin antibody (1:1,000; Zymed) followed by incubation with goat anti-mouse horseradish peroxidase secondary antibody (1:15,000; Bio-Rad, Hercules, CA) and developed using the ECL detection system (Amersham). Four separate experiments were performed for each treatment group.

Phosphoserine detection of occludin. HUVEC were exposed to H2O2, H2O2 + PD-98059, or PD-98059 treatment protocols. Four micrograms of rabbit anti-occludin was added to the cell lysates and immunoprecipitated as described. Pelleted samples were electrophoresed using SDS polyacrylamide gels. Gels were transferred to nitrocellulose and blocked with 1% BSA/PBS solution. Blots were incubated with mouse anti-phosphoserine monoclonal antibody (1:1,000; Sigma) for 2 h at room temperature. Blots were washed three times with 0.1% BSA/PBS solution and incubated with goat anti-mouse horseradish peroxidase conjugated secondary antibody (1:15,000; Bio-Rad) for 2 h at room temperature. Blots were washed again and developed using the ECL detection system (Amersham). Normalization of the blots was performed using mouse anti-occludin or rabbit anti-ZO-1 antibodies and developed using NBT/BCIP. Four separate experiments were performed for each treatment group.

Densitometric and statistical analysis. Densitometry was performed using a Hewlett-Packard Scanjet IIcx flatbed scanner and Image Pro Plus image analysis software (Media Cybernetics, Silver Spring, MD). Statistical analysis was performed using Instat statistical software (Graphpad Software, San Diego, CA). One-way ANOVA with Bonferroni posttesting was used to compare control vs. treatment groups for all experiments reported. Data are reported as mean ± SD.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

H2O2-mediated permeability occurs through MAP kinase pathways. HUVEC solute permeability was measured in response to 1 mM H2O2 administration using the cell-column method of determining monolayer permeability. Figure 1 illustrates that H2O2 initially caused significant increases in HUVEC solute permeability after 30 min and a maximal 2.5-fold increase in HUVEC solute permeability by 90 min. Importantly, we have previously shown that this increase in permeability is not due to loss of endothelial cells or cell death (22, 28).


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Fig. 1.   Role of protein kinase C (PKC), tyrosine kinase, and extracellularly regulated kinase 1 and 2 (ERK1/ERK2) activity in H2O2-mediated permeability. Human umbilical vein endothelial cell (HUVEC) solute permeability was measured using the cell-column method. Treatment of HUVEC with 1 mM H2O2 () shows a significant increase in permeability at 30 min compared with baseline levels (). Treatment with the PKC inhibitor chelerythrine chloride (Chele Cl, black-down-triangle , 750 nM) or the tyrosine kinase inhibitor tyrphostin 23 (Tyr23, open circle , 50 µM) + H2O2 failed to prevent H2O2-mediated increased endothelial solute permeability. Treatment with PD-98059 (20 µM) prevented H2O2-mediated permeability (down-triangle). Pretreatment with the blockers alone did not significantly alter basal solute permeability. Data are shown as mean ± SD with * P < 0.05, ** P < 0.01.

Based on the observation that H2O2 can cause the activation of various signal transduction cascades in bovine pulmonary artery endothelial cells and other nonendothelial cell types, we sought to determine whether similar signal transduction pathways were involved in H2O2-mediated HUVEC permeability (4, 16, 22, 26, 27, 36, 45). Figure 1 demonstrates that H2O2-mediated permeability was not prevented by the protein kinase C (PKC) inhibitor chelerythrine chloride (750 nM) or the tyrosine kinase inhibitor tyrphostin 23 (50 µM). These observations are in contrast to reports using other nonhuman endothelial cell models (4, 22).

Several reports have shown that H2O2 administration results in activation of ERK1/ERK2 (16, 19, 27). We also have recently reported that HUVEC solute permeability due to VEGF administration involves ERK1/ERK2 activation (24). Therefore, we examined the contribution of ERK1/ERK2 activity in H2O2-mediated permeability by inhibiting MEK1, the upstream activator of ERK1/ERK2. Administration of the MEK1 inhibitor PD-98059 (20 µM) prevented H2O2-mediated increased permeability (Fig. 1). Moreover, we have previously reported that PD-98059 itself does not alter solute permeability (24). These data support our previous finding that MAP kinase activity is involved in solute barrier regulation of human endothelial cells and suggests that H2O2 increases solute permeability through similar mechanisms.

H2O2-mediated phosphorylation of ERK1/ERK2. Previous studies have shown that exogenous H2O2 administration can stimulate ERK1/ERK2 activity in endothelial and nonendothelial cell types (16, 19, 27). We observed that a 30-min exposure to H2O2 caused a significant increase in ERK1/ERK2 phosphorylation that was blocked by PD-98059 (Fig. 2A). Densitometric analysis of both ERK1 and ERK2 revealed that ERK1/ERK2 phosphorylation was five times greater in H2O2-treated cells compared with control (Fig. 2B). ERK1/ERK2 activation by H2O2 + PD-98059 was not significantly different from control. These data suggest that H2O2 administration results in increased HUVEC ERK1/ERK2 phosphorylation through MEK1-dependent mechanisms.


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Fig. 2.   H2O2 stimulates ERK1/ERK2 activation through MAP or ERK kinase 1. A: 30-min exposure of H2O2 causes an increase in ERK1/ERK2 phosphorylation that is blocked by PD-98059. B: densitometric analysis revealed a significant 5-fold increase in ERK1/ERK2 phosphorylation. Data are shown as mean ± SD with *P < 0.01.

Immunofluorescent localization of the tight junctional proteins occludin and ZO-1. The normal basal solute permeability level of human venous and arterial endothelial cells are governed in part by the expression and localization of the tight junctional protein occludin (23, 24, 44). These observations prompted us to examine the effects of H2O2 administration on endothelial junctional localization of the tight junctional protein occludin and an associated linker protein ZO-1. Figure 3, A and D shows control staining of HUVEC occludin and ZO-1, respectively. Control occludin staining (Fig. 3A) demonstrates that the protein is localized junctionally although more discontinuously compared with ZO-1 staining. Control ZO-1 is distributed continuously around the periphery of the endothelial cells (Fig. 3D). After a 30-min exposure to H2O2, the endothelial cell junctions showed a loss of occludin junctional localization with subsequent punctate occludin staining (Fig. 3B). However, H2O2 treatment did not cause a dramatic loss of ZO-1 from the junction except for where gaps are observed (Fig. 3E, arrows). This suggests that occludin and ZO-1 may functionally dissociate from one another during H2O2 administration. H2O2 + PD-98059 treatment showed occludin and ZO-1 localization to be maintained and apparently enhanced at the endothelial cell-cell junction compared with control endothelial monolayers (Fig. 3, C and F). We have previously reported that PD-98059 itself does not alter endothelial junctional protein organization (24). These data suggest that the MEK1/ERK1/ERK2 signal pathway is involved in H2O2-mediated loss of occludin junctional organization.


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Fig. 3.   H2O2 stimulates occludin disorganization through ERK1/ERK2 activation. A and D show control dual staining for HUVEC occludin and ZO-1, respectively. B and E demonstrate occludin and ZO-1 dual staining after a 30-min exposure of H2O2. Note that peroxide causes a loss of junctional occludin organization with the appearance of punctate occludin staining, whereas ZO-1 junctional localization was better maintained except for areas demonstrating gap formation (arrows). C and F illustrate the effects of combined administration of H2O2 and PD-98059 (20 µM) on occludin and ZO-1 dual junction localization, respectively. Note that junctional localization of both occludin and ZO-1 was maintained with an apparent enhancement of occludin organization. All fields were shot at ×100. Bar, 2.5 µm.

Levels of occludin and ZO-1 in H2O2-treated HUVEC monolayers. Levels of occludin protein expression can affect endothelial solute barrier properties. We have reported that a reduction of occludin protein levels through antisense oligonucleotide treatment decreases the level of barrier formation in arterial and venous endothelial monolayers (23). Moreover, we have also shown that a model of transplantation injury using HUVEC monolayers resulted in a loss of occludin protein that was accompanied by an increase in solute permeability (44). Figure 4, A and B, illustrates the steady-state levels of ZO-1 and occludin after H2O2 and H2O2 + PD-98059 exposures. Densitometric analysis of both the occludin (Fig. 4B) and ZO-1 (Fig. 4A) bands failed to show significant differences of these proteins between treatment groups. These data suggest that H2O2 does not stimulate the loss or degradation of these proteins.


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Fig. 4.   Effects of H2O2 on occludin and ZO-1 protein levels. HUVEC monolayers were treated either with H2O2 or H2O2 + PD-98059 (20 µM) as described in MATERIALS AND METHODS. Western blot analysis shows the steady-state level of ZO-1 (A) or occludin (B) among the various treatment groups. Densitometric analysis of the blots showed no statistical difference between the control and treatment groups.

Surface organization of occludin and ZO-1 association. We have previously observed that endothelial adherens junctional proteins are endocytosed upon exposure to H2O2 (22). Therefore, we sought to determine whether occludin would also be endocytosed upon H2O2 exposure. To determine the amount of occludin at the cell surface, cells were biotinylated after a 30-min incubation period with H2O2, H2O2 + PD-98059, or PD-98059 alone treatments. These cells were lysed, and a coimmunoprecipitation was performed for occludin and ZO-1 using a polyclonal occludin antibody. To determine the amount of biotinylated occludin, the immunoprecipitates were run on SDS polyacrylamide gels, and blots were probed with avidin alkaline phosphatase (Fig. 5A). The upper portions of the blots were also probed for ZO-1 to determine the amount of occludin-ZO-1 interactions (Fig. 5B). Densitometric analysis of the blots showed no significant difference in the amount of biotinylated occludin between control and H2O2-treated monolayers. This suggested that H2O2 did not cause occludin endocytosis and that occludin was reorganized on the cell surface. Interestingly, the H2O2 + PD-98059 treatment revealed a threefold greater amount of biotinylated occludin, suggesting that this combination enhanced occludin surface localization. This observation supports our immunofluorescence data showing enhanced occludin junctional localization with H2O2 + PD-98059 (Fig. 3C).


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Fig. 5.   Effects of H2O2 on occludin cellular localization and ZO-1 associations. Biotinylation of the cell surface and immunoprecipitation of occludin was used to determine HUVEC occludin surface localization. A: the amount of biotinylated occludin located on the endothelial cell surface after various treatments. There was no significant difference between control and H2O2-treated monolayers, yet the combination of H2O2 and PD-98059 (20 µM) enhanced occludin surface localization 3-fold as determined by densitometric analysis. PD-98059 control treatments did not demonstrate differences in surface occludin localization compared with control. B: the amount of ZO-1 associated with the biotinylated occludin. Treatment of HUVEC with H2O2 caused a 60% decrease in ZO-1 association compared with control as determined by densitometric analysis. Densitometric analysis of the combined treatments of H2O2 + PD-98059 demonstrated a 3-fold significant increase in ZO-1 association compared with control. PD-98059 control treatments failed to show any significant differences in ZO-1 association compared with control.

Densitometric analysis of coimmunoprecipitated ZO-1 revealed that H2O2 exposure significantly decreased the occludin-ZO-1 associations by 60%. The combined treatment of H2O2 + PD-98059 showed that ZO-1 association with occludin increased similarly to the amount of occludin found at the cell surface (i.e., 3-fold greater than control). Control experiments with the PD-98059 compound alone did not demonstrate any significant difference in either occludin biotinylation or ZO-1 association compared with control. These data demonstrate that occludin-ZO-1 interactions are reduced in response to H2O2 treatment and that occludin is redistributed across the endothelial cell surface. These data also further illustrate that the ERK1/ERK2 signal pathway is involved in maintaining occludin localization to the cell-cell junction and occludin-ZO-1 association.

Serine phosphorylation of occludin. Occludin has been reported to be phosphorylated on serine residues (34). We sought to determine what effects H2O2 had on serine phosphorylation of occludin. Treatment with H2O2 resulted in a significant increase in occludin phosphoserine content (Fig. 6A). Normalizing the phosphoserine signal to the amounts of immunoprecipitated occludin showed a significant threefold increase in serine phosphorylation, which was prevented by PD-98059 treatment (Fig. 6, A and B). This is the first report directly demonstrating that occludin phosphorylation is associated with a loss of endothelial solute barrier properties. Moreover, our data suggest that occludin is not normally phosphorylated on serine residues under basal conditions.


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Fig. 6.   Effects of H2O2 on occludin phosphoserine content. HUVEC monolayers were exposed to H2O2, H2O2 + PD-98059 (20 µM), or PD-98059 alone. Occludin was immunoprecipitated and probed for phosphoserine content. A: occludin is phosphorylated on serine residues in response to H2O2 administration. Combined treatment with H2O2 + PD-98059 shows that phosphorylation of occludin serine residues was prevented. B: the normalization of phosphoserine data to immunoprecipitated occludin as determined by densitometric analysis revealing a significant 3-fold increase in occludin phosphorylation that was attenuated by PD-98059. Data are shown as the mean ± SD with * P < 0.05.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Endothelial cell tight junctions have been shown to regulate and maintain endothelial solute barrier properties (3, 38, 39). Recent reports have shown that the tight junctional protein occludin is intimately involved in maintaining endothelial solute barriers (23, 24, 44). With the use of antisense oligonucleotides directed at occludin mRNA, we have shown that decreasing HUVEC occludin expression to less than one-half of control levels results in a significant increase in solute permeability (44). We also have reported that substances such as VEGF and organ transplantation preservation solutions cause a loss of junctional occludin organization, decreased occludin expression, and increased endothelial solute permeability (24, 44). These studies demonstrate that occludin is involved in the regulation and maintenance of endothelial solute barrier function. However, precise identification of the molecular mechanisms involved in the loss of endothelial solute barrier and occludin organization are lacking. This report correlates the molecular mechanisms and signal transduction pathways that may be involved in the loss of normal endothelial solute barrier.

Several studies have shown that pathological conditions such as ischemia-reperfusion and leukocyte-mediated inflammation are accompanied by H2O2 formation and microvascular permeability (14, 15, 33). Kurose et al. (25) showed that infusion of platelet-activating factor (PAF) to mesenteric microvessels caused increased leukocyte adhesion, H2O2 formation, and microvascular permeability, which were all attenuated by catalase. Similarly, Suematsu et al. (42) demonstrated that PAF administration to mesenteric microvessels resulted in oxidant formation similar to treatment with 800 µM tert-butyl hydroperoxide. These observations indicate that H2O2 is intimately involved in PAF-mediated microvascular dysfunction, yet the mechanisms involved are unknown. Because most studies have used nonhuman endothelial cell lines to examine permeability changes due to H2O2, we evaluated H2O2-mediated permeability in human endothelial monolayers (22, 30, 35, 36, 37). In addition, we sought to identify the molecular targets and mechanisms that were involved in H2O2-mediated increased endothelial solute permeability.

Our data indicate that H2O2 increased HUVEC permeability to a similar magnitude and time course as seen with endothelial cells of other species (22, 30, 35, 37). In bovine pulmonary artery and aortic endothelial cells, PKC activation contributes to H2O2-mediated permeability (22, 36). Studies also have shown that tyrosine kinase activity affects endothelial and epithelial solute properties (4, 41). However, treatment of HUVEC with the PKC inhibitor chelerythrine chloride or the tyrosine kinase inhibitor tyrphostin 23 failed to prevent H2O2-mediated permeability. These observations suggested that tyrosine kinase and PKC activity are not involved in H2O2-mediated increased permeability of human endothelial monolayers and that not all endothelial cell types regulate their solute barrier properties the same way.

Recently, activation of other signal transduction pathways have been identified in H2O2-treated HUVEC. Huot et al. (19) published data indicating that H2O2 administration causes an increase in ERK1/ERK2 activity. Additional studies using nonendothelial cell types also have reported that exogenous H2O2 administration causes increased ERK1/ERK2 activity that peaks within 10-15 min (16, 45). Guyton et al. (16) further demonstrated that H2O2-mediated increased ERK2 activity was abrogated by expression of a dominant-negative mutant of p21Ras. Lander et al. (26, 27) has also shown that H2O2 increases p21Ras activity by oxidizing the protein. Therefore, we determined the role of ERK1/ERK2 kinase activity in H2O2-mediated endothelial permeability.

Combined treatment of HUVEC with H2O2 plus the specific MEK1 inhibitor PD-98059 completely prevented H2O2-mediated permeability. These results support our previous finding that ERK1/ERK2 activity is involved in the regulation of HUVEC solute permeability (24). Determination of ERK1/ERK2 phosphorylation using an anti-active MAP kinase antibody further suggested that H2O2 significantly increased HUVEC ERK1/ERK2 activation. Moreover, the H2O2-mediated increase in ERK1/ERK2 phosphorylation was completely prevented by PD-98059. These data suggest that H2O2-mediated permeability involves MEK1/ERK1/ERK2 signal pathways, or that by blocking MEK1 with PD-98059 activation, other kinases such as ras, rac, rho, or p38 MAP kinase may be involved in the observed cellular events. We feel that this possibility is unlikely because we have previously investigated the effects of PD-98059 alone and found that this agent did not change basal endothelial permeability nor substantially alter endothelial occludin or vascular endothelial cadherin localization (24). However, we are currently investigating the contribution of p38 MAP kinase in H2O2-mediated permeability to address these possibilities.

Several groups, including ours, have previously demonstrated that H2O2-mediated permeability causes gap formation between endothelial cells and the loss of endothelial junctional protein organization (i.e., actin and cadherin) (4, 22, 30, 35, 36). Because occludin participates in normal endothelial solute barrier, and the direct examination of occludin and ZO-1 has not been reported using this model system, we determined the effect of H2O2 administration on the organization of these tight junctional proteins.

Immunofluorescent staining for these two proteins showed that H2O2 caused a loss of occludin from the endothelial cell junctions and that ZO-1 junctional localization was not altered except for regions where gap formation occurred. Reports have demonstrated that occludin binds directly to the NH2-terminal region of ZO-1, which is bound to the actin cytoskeleton (8, 12). Together, the immunofluorescence and immunoprecipitation data suggested that occludin-ZO-1 binding was reduced by H2O2 administration. Our ZO-1 findings corroborate two previous reports in the literature that show that: 1) ZO-1 remains localized in junctions despite a loss of tight junction protein organization, and 2) ZO-1 junctional localization is no longer observed at regions where endothelial cells are not in contact with one another (29, 31).

Treatment of cells with either the PKC or tyrosine kinase inhibitor did not prevent this loss of occludin staining at junctions nor did it block gap formation between endothelial cells (data not shown). Interestingly, treatment with the MEK inhibitor PD-98059 + H2O2 actually appeared to enhance occludin junction organization and prevent gap formation, indicating that ERK1/ERK2 pathways are involved in occludin organization at cell-cell junctions. This agrees with our previous finding that VEGF reorganizes occludin junctional localization via the ERK1/ERK2 pathways (24). These data strongly suggest that occludin disorganization and subsequent gap formation involves ERK1/ERK2 pathways.

Having observed the dissociation of occludin from the junction, we wanted to determine exactly what was happening to the steady-state levels of tight junctional proteins during H2O2 exposure. As stated, we reported that the amount of occludin protein in the cell regulates the barrier properties of endothelial monolayers (23). Similarly, we recently have shown that treatment of HUVEC monolayers with organ preservation solutions results in shedding and loss of occludin with increased endothelial permeability (44). Western analysis showed that H2O2 alone or H2O2 + PD-98059 slightly, yet not significantly, increased occludin steady-state protein levels compared with control monolayers. This observation suggested that the occludin protein was not lost but was reorganized in the endothelial cell.

We next considered how occludin was being reorganized in H2O2-treated endothelial cells. We have previously reported that H2O2 administration causes bovine pulmonary artery endothelial cells to internalize junctional cadherins (22). Moreover, earlier reports suggested that tight junction proteins in epithelial cells were endocytosed when exposed to low-extracellular calcium environments (5, 13). Surface biotinylation followed by occludin immunoprecipitation data showed that occludin was not endocytosed in response to H2O2. We found that occludin remains on the endothelial cell surface and is dissociated from ZO-1. The combined treatment of H2O2 + PD-98059 showed a significantly greater amount of biotinylated occludin (3-fold) that was accompanied by a significantly greater association with ZO-1. This observation suggests that this combined treatment dramatically enhanced the amount of occludin present on the cell surface. Clearly, the slight and statistically insignificant increase in occludin protein levels are not completely responsible for such a large increase in occludin surface localization, especially because there was no increase in ZO-1 protein levels; however, the contribution of protein synthesis cannot be completely discounted without the use of protein synthesis inhibitors. One possible explanation as to why the combination of H2O2 and PD-98059 causes increased surface localization of occludin is that inhibition of MEK1 activity may affect proteins involved in occludin and tight junction organization, like the recently described tight junctional proteins of the claudin family and junctional adhesion molecule, which might enhance or alter occludin localization (11, 32). Moreover, occludin could possibly be recruited from intracellular pools. We and others have reported that occludin staining in HUVEC or Madin-Darby canine kidney (MDCK) epithelium occurs in a less continuous, punctate manner with localization at the cell-cell junction and within the cytoplasm, compared with that of human umbilical artery endothelial cells and during extracellular low-calcium treatments, respectively (23, 34). However, the precise identification of cytoplasmic structures containing occludin is not well known.

Another possibility that could explain the differences in occludin surface or intracellular localization could be due to different states of occludin phosphorylation. Sakakibara et al. (34) has reported that in MDCK epithelial cells exposed to low-calcium treatments, occludin phosphorylation is decreased and that occludin no longer associates with the cytoskeleton (34). The authors surmised that in MDCK epithelial cells, phosphorylation of occludin is necessary for binding to the cytoskeleton and functional integration into the tight junction. Another study by Wong (46) demonstrated that occludin phosphorylation was different in the two MDCK strains I and II. The author hypothesized that differences in occludin phosphorylation could possibly explain the differences in transepithelial resistance between the two cell types. In contrast, a study by Cordenonsi et al. (6) reported that in Xenopus development, occludin becomes progressively dephosphorylated after fertilization. This study further demonstrated that the protein kinases casein kinase 2 and p34 cdc2 could, but that MAP kinase and p38 syk tyrosine kinase could not, phosphorylate recombinant chicken occludin in cell-free phosphorylation assays. Unfortunately, the physiologically active kinase responsible for occludin phosphorylation was not identified in this study.

We found that endothelial occludin was only weakly phosphorylated on serine residues basally. Treatment of HUVEC monolayers with H2O2 significantly increased the amount of phosphoserine residues of occludin. Treatment of endothelial cells with H2O2 + PD-98059 showed no such increase in occludin phosphoserine content, strongly suggesting that ERK1/ERK2 promotes occludin phosphorylation either directly or through an unidentified serine/threonine kinase in the ERK1/ERK2 pathway. In support of our findings, Hirase (17) has recently shown that treatment of HUVEC with histamine causes an increase in solute permeability combined with an increase in occludin phosphoserine content. Pyrilamine, an H1 antagonist, was reported to prevent phosphorylation of occludin serine residues and blocked histamine-mediated increased permeability. Moreover, Antonetti et al. (1) has recently reported that VEGF induces phosphorylation of occludin on serine residues in vivo and in vitro. Together with our findings, these observations suggest that HUVEC occludin is not normally phosphorylated on serine residues, and agents that increase solute permeability increase occludin phosphoserine content, which disorganizes occludin localization from the cell junction. Our data demonstrate for the first time that increased occludin phosphorylation is associated with increased endothelial permeability in response to H2O2.

In conclusion, we report that H2O2-mediated permeability of HUVEC monolayers involves occludin disorganization on the endothelial surface with loss of ZO-1 association and increased phosphorylation of occludin serine residues mediated by ERK1/ERK2 signal pathways. Future studies are needed to elucidate how the combination of H2O2 and PD-98059 increases the amount of occludin on the cell surface.


    ACKNOWLEDGEMENTS

We thank Drs. Steve Trocha, Seth Berney, Robert Specian, and Paul Berger for helpful discussions. We thank April Carpenter for technical assistance.


    FOOTNOTES

This work was funded by National Institutes of Health Grants HL-47615 and PO1-DK-43785.

Address for reprint requests and other correspondence: J. S. Alexander, Dept. of Molecular and Cellular Physiology, LSU Medical Center Shreveport, 1501 Kings Hwy, Shreveport, LA 71130 (E-mail: jalexa{at}lsumc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 13 October 1999; accepted in final form 14 January 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Antonetti, DA, Barber AJ, Hollinger LA, Wolpert EB, and Gardner TW. Vascular endothelial growth factor induces rapid phosphorylation of tight junction proteins occludin and zonula occludens 1. A potential mechanism for vascular permeability in diabetic retinopathy and tumors. J Biol Chem 274: 23463-23467, 1999[Abstract/Free Full Text].

2.   Blum, MS, Toninelli E, Anderson JM, Balda MS, Zhou J, O'Donnell L, Pardi R, and Bender JR. Cytoskeletal rearrangement mediates human microvascular endothelial tight junction modulation by cytokines. Am J Physiol Heart Circ Physiol 273: H286-H294, 1997[Abstract/Free Full Text].

3.   Bottaro, D, Shepro D, and Hechtman HH. Heterogeneity of intimal and microvessel endothelial barriers in vitro. Microvasc Res 32: 389-398, 1986[ISI][Medline].

4.   Carbajal, JM, and Schaeffer RC. H2O2 and genistein differentially modulate protein tyrosine phosphorylation, endothelial morphology, and monolayer barrier function. Biochem Biophys Res Commun 249: 461-466, 1998[ISI][Medline].

5.   Contreras, RG, Miller JH, Zamora M, Gonzalez-Mariscal L, and Cereijido M. Interaction of calcium with plasma membrane of epithelial (MDCK) cells during junction formation. Am J Physiol Cell Physiol 263: C313-C318, 1992[Abstract/Free Full Text].

6.   Cordenonsi, M, Mazzon E, Rigo LD, Baraldo S, Meggio F, and Citi S. Occludin dephosphorylation in early development of Xenopus laevis. J Cell Sci 110: 3131-3139, 1997[Abstract/Free Full Text].

7.   Eaton, BM, Toothill VJ, Davies HA, Pearson JD, and Mann GE. Permeability of human venous endothelial cell monolayers perfused in microcarrier cultures: effects of flow rate, thrombin, and cytochalasin D. J Cell Physiol 149: 88-99, 1991[ISI][Medline].

8.   Fanning, AS, Jameson BJ, Jesaitis LA, and Anderson JM. The tight junction protein ZO-1 establishes a link between the transmembrane protein occludin and the actin cytoskeleton. J Biol Chem 273: 29745-29753, 1998[Abstract/Free Full Text].

9.   Farshori, P, and Kachar B. Redistribution and phosphorylation of occludin during opening and resealing of tight junctions in cultured epithelial cells. J Membr Biol 170: 147-156, 1999[ISI][Medline].

10.   Firth, JA, Bauman KF, and Sibley CP. The intercellular junctions of guinea pig placental capillaries: a possible structural basis for endothelial solute permeability. J Ultrastruct Res 85: 45-57, 1983[ISI][Medline].

11.   Furuse, M, Fujita K, Hiiragi T, Fujimoto K, and Tsukita S. Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin. J Cell Biol 141: 1539-1550, 1998[Abstract/Free Full Text].

12.   Furuse, M, Itoh M, Hirase T, Nagafuchi A, Yonemura S, Tsukita S, and Tsukita S. Direct association of occludin with ZO-1 and its possible involvement in the localization of occludin at tight junctions. J Cell Biol 127: 1617-1626, 1994[Abstract].

13.   Gonzalez-Mariscal, L, Contreras RG, Bolivar JJ, Ponce A, Ramirez BCD, and Cereijido M. Role of calcium in tight junction formation between epithelial cells. Am J Physiol Cell Physiol 259: C978-C986, 1990[Abstract/Free Full Text].

14.   Granger, DN, Grisham MB, and Kvietys PR. Physiology of the Gastrointestinal Tract. New York: Raven, 1994, p. 1693-1722.

15.   Granger, DN, Höllwarth ME, and Parks DA. Ischemia-reperfusion injury: role of oxygen-derived free radicals. Acta Physiol Scand 548: 47-63, 1986.

16.   Guyton, KZ, Liu Y, Gorospe M, Xu Q, and Holbrook NJ. Activation of mitogen-activated protein kinase by H2O2. J Biol Chem 271: 4138-4142, 1996[Abstract/Free Full Text].

17.  Hirase T. Occludin in Endothelial Cells. Kobe, Japan: First Japan-United States Joint Meeting on Vascular Biology, Session 5, 1998.

18.   Hirase, T, Staddon JM, Saitou M, Ando-Akatsuka Y, Itoh M, Furuse M, Fujimoto K, Tsukita S, and Rubin LL. Occludin as a possible determinant of tight junction permeability in endothelial cells. J Cell Sci 110: 1603-1613, 1997[Abstract/Free Full Text].

19.   Huot, J, Huole F, Marceau F, and Landry J. Oxidative stress-induced actin reorganization mediated by the p38 mitogen-activated protein kinase/heat shock protein 27 pathway in vascular endothelial cells. Circ Res 80: 383-392, 1997[Abstract/Free Full Text].

20.   Kennedy, TP, Rao NV, Hopkins C, Pennington L, Tolley E, and Hoidal JR. Role of reactive oxygen species in reperfusion injury of the rabbit lung. J Clin Invest 83: 1326-1335, 1989[ISI][Medline].

21.   Kevil, C, Carter P, Hu B, and DeBenedetti A. Translational enhancement of FGF-2 by eIF-4 factors, and alternative utilization of CUG and AUG codons for translation initiation. Oncogene 11: 2339-2348, 1995[ISI][Medline].

22.   Kevil, CG, Ohno N, Gute DC, Okayama N, Robinson SA, Chaney E, and Alexander JS. Role of cadherin internalization in H2O2 mediated endothelial permeability. Free Radic Biol Med 24: 1015-1022, 1998[ISI][Medline].

23.   Kevil, CG, Okayama N, Trocha SD, Kalogeris TJ, Coe LL, Specian RD, Davis CP, and Alexander JS. Expression of zonula occludens and adherens junctional proteins in human venous and arterial endothelial cells: role of occludin in endothelial solute barriers. Microcirculation 5: 197-210, 1998[Medline].

24.   Kevil, CG, Payne DK, Mire E, and Alexander JS. Vascular permeability factor/vascular endothelial cell growth factor-mediated permeability occurs through disorganization of endothelial junctional proteins. J Biol Chem 273: 15099-15103, 1998[Abstract/Free Full Text].

25.   Kurose, I, Argenbright LW, Wolf R, and Granger DN. Oxidative stress during platelet-activating factor-induced microvascular dysfunction. Microcirculation 3: 401-410, 1996[Medline].

26.   Lander, HM. An essential role of free radicals and derived species in signal transduction. FASEB J 11: 118-124, 1997[Abstract/Free Full Text].

27.   Lander, HM, Ogiste JS, Teng KK, and Novogrodsky A. p21 ras as a common signaling target of reactive free radicals and cellular redox status. J Biol Chem 270: 21195-21198, 1995[Abstract/Free Full Text].

28.   Lewis, MS, Whatley RE, Cain P, McIntyre TM, Prescott SM, and Zimmerman GA. H2O2 stimulates the synthesis of platelet-activating factor by endothelium and induces endothelial cell-dependent neutrophil adhesion. J Clin Invest 82: 2045-2055, 1988[ISI][Medline].

29.   Li, C, and Poznansky MJ. Characterization of the ZO-1 protein in endothelial and other cell lines. J Cell Sci 97: 231-237, 1990[Abstract].

30.   Lum, H, Barr DA, Shaffer JR, Gordon RJ, Ezrin AM, and Malik AB. Reoxygenation of endothelial cells increases permeability by oxidation-dependent mechanisms. Circ Res 70: 991-998, 1992[Abstract].

31.   Madara, JL, Carlson S, and Anderson JM. ZO-1 maintains its spatial distribution but dissociates from junctional fibrils during tight junction regulation. Am J Physiol Cell Physiol 264: C1096-C1101, 1993[Abstract/Free Full Text].

32.   Martin-Padura, I, Lostaglio S, Schneemann M, Williams L, Romano M, Fruscella P, Panzeri C, Stoppacciaro A, Ruco L, Villa A, Simmons D, and Dejana E. Junctional adhesion molecule, a novel member of the immunoglobulin superfamily that distributes at intercellular junctions and modulates monocyte transmigration. J Cell Biol 142: 117-127, 1998[Abstract/Free Full Text].

33.   Parks, DA, Shah AK, and Granger DN. Oxygen radicals: effects on intestinal vascular permeability. Am J Physiol Gastrointest Liver Physiol 247: G167-G170, 1984[Abstract/Free Full Text].

34.   Sakakibara, A, Furuse M, Saitou M, Ando-Akatsuka Y, and Tsukita S. Possible involvement of phosphorylation of occludin in tight junction formation. J Cell Biol 137: 1393-1401, 1997[Abstract/Free Full Text].

35.   Shasby, DM, Lind SE, Shasby SS, Goldsmith JC, and Hunninghake GW. Reversible oxidant-induced increases in albumin transfer across cultured endothelium: alterations in cell shape and calcium homeostasis. Blood 65: 605-614, 1985[Abstract].

36.   Siflinger-Birnboim, A, Goligorsky MS, Vecchio PJD, and Malik AB. Activation of protein kinase C pathway contributes to H2O2-induced increase in endothelial permeability. Lab Invest 67: 24-30, 1992[ISI][Medline].

37.   Siflinger-Birnboim, A, Lum H, Vecchio PJD, and Malik AB. Involvement of Ca2+ in the H2O2-induced increase in endothelial permeability. Am J Physiol Lung Cell Mol Physiol 270: L973-L978, 1996[Abstract/Free Full Text].

38.   Simionescu, M, Simionescu N, and Palade GE. Segmental differentiations of cell junctions in the vascular endothelium: the microvasculature. J Cell Biol 67: 863-885, 1975[Abstract].

39.   Simionescu, M, Simionescu N, and Palade GE. Segmental differentiations of cell junctions in the vascular endothelium: arteries and veins. J Cell Biol 68: 705-723, 1976[Abstract].

40.   Simionescu, N, Simionescu M, and Palade GE. Open junctions in the endothelium of the post capillary venules of the diaphragm. J Cell Biol 79: 27-44, 1978[Abstract].

41.   Staddon, JM, Herrenknecht K, Smales C, and Rubin LL. Evidence that tyrosine phosphorylation may increase tight junction permeability. J Cell Sci 108: 609-619, 1995[Abstract/Free Full Text].

42.   Suematsu, M, Schmid-Schönbein GW, Chavez-Chavez RH, Yee TT, Tamatani T, Miyasaka M, Delano FA, and Zweifach BW. In vivo visualization of oxidative changes in microvessels during neutrophil activation. Am J Physiol Heart Circ Physiol 264: H881-H891, 1993[Abstract/Free Full Text].

43.   Suzuki, YJ, Forman HJ, and Sevanian A. Oxidants as stimulators of signal transduction. Free Radic Biol Med 22: 269-285, 1997[ISI][Medline].

44.   Trocha, SD, Kevil CG, Mancini M, and Alexander JS. Organ preservation solutions increase endothelial permeability and promote loss of junctional proteins. Ann Surg 230: 105-113, 1999[ISI][Medline].

45.   Whisler, RL, Goyette MA, Grants IS, and Newhouse YG. Sub lethal levels of oxidant stress stimulate multiple serine/threonine kinases and suppress protein phosphatases in jurkat T cells. Arch Biochem Biophys 319: 23-35, 1995[ISI][Medline].

46.   Wong, V. Phosphorylation of occludin correlates with occludin localization and function at the tight junction. Am J Physiol Cell Physiol 273: C1859-C1867, 1997[Abstract/Free Full Text].


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